Emerging Foodborne Pathogens 9781855739635, 1855739631

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Part I Identifying emerging pathogens

1 How bacterial pathogens evolve B. Wren, London School of Hygiene and Tropical Medicine, UK

1.1

Introduction

Despite advances in hygiene, consumer knowledge, food treatment and food processing, foodborne pathogens still represent a significant threat to human health worldwide. Many foodborne pathogens have never been adequately controlled, while others have re-emerged due to factors related to lifestyle, political, economic, and ecological changes. Bacterial pathogens are particularly adept at continual change due to their short generation time combined with multiple mechanisms to alter their genetic repertoire. Foodborne pathogens have a further level of potential complexity as the evolutionary pressures that shape their existence are compounded by changes in human behaviour and practices such as changes in eating habits, agriculture and food manufacturing processes. The task of keeping pace with the emergence of pathogens seems daunting. However, the recent availability of whole genome sequences of virtually all pathogens, coupled with the development of complementary high-throughput genomics technologies such as DNA microarrays, means that we are now in a position to monitor and determine the underlying genetic mechanisms responsible for the emergence of fitter pathogens. Using case studies and examples from recent post-genome analyses, this chapter will describe the genetic mechanisms by which selected bacterial pathogens have evolved and will describe likely trends that could be used to reduce the emergence of foodborne pathogens.

1.2

Evolution and diversification of bacterial pathogens

Bacteria are the most successful organisms on the planet. Over a billion

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years ago they were the first living organisms and inhabited a diverse range of inhospitable niches ranging from boiling hot vents in the oceans to highly acidic springs. Bacterial pathogens, which are the best-understood group of bacteria, have chosen as their niche animals or plants and either deliberately, or inadvertently, cause damage to the host, resulting in disease. This has resulted in a constant ‘arms race’ between the pathogen and host, where the stakes, often death, are high. Thus, it is not surprising that both pathogen and host responses have developed finely tuned genetic mechanisms as part of their survival mechanisms. In contrast to eukaryotes, most bacteria have a rapid generation time; 30 minutes in Escherichia coli, compared with 30 years in humans. This means that bacteria are particularly adept at responding and adapting to different evolutionary pressures allowing the fittest to survive. For example, this may include the need to survive in an inhospitable environment, or for pathogens, in particular, the necessity to increase transmissibility to the next host or to establish themselves in a new host species. When new selection pressures appear, these are the individuals that survive, at the expense of the general population, and forge new populations. Depending on the severity and uniqueness of the selection pressure, this could lead to new speciation. The evolutionary success of bacteria is due to their versatility and diversity, which are largely reflected in their DNA content. Genome sequence data from bacterial pathogens have confirmed the genetic diversity between species, (e.g. the genome sequence of most members of the enterobacteriaceae are 30% different) and even within species (e.g. most strains of E. coli that have been sequenced are at least 10% different). To put this diversity into context, the difference in the genome content between man and mouse is 1%. The diversity of microbial gene content is also exemplified by the observation that approximately half of the predicted genes from bacterial genome sequences are of unknown biological function, and around a half of these appear to be unique to the individual species. For example, over 30% of the genes from E. coli, the best studied of all microorganisms, have no known function. These statistics underscore our limited knowledge of bacteria and microbial pathogens in general. However, it is not just the presence or absence of genes in the genome that contribute to the diversity of micro-organisms. Bacterial pathogens, in particular, have evolved many mechanisms for altering their genetic repertoire that provide a further level of genetic diversification.

1.3

Genetic mechanisms of bacterial evolution

1.3.1 Recombination and gene duplication Most bacteria can undergo DNA recombination at sites of similar nucleotide identity, which often results in an increase in the genetic content of the organism. This allows bacteria to extend or shuffle their genetic repertoire

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during reproduction. This can occur at several levels including duplication of open reading frames (formation of paralogous genes), cassettes of genes (genetic islands) or even entire chromosomes. This has advantages for the bacterium because it increases specific gene families that may increase the functional processes of the bacterium in subsequent generations (e.g., enable it to survive in an additional niche). The potential disadvantage of increasing the genetic load is that in terms of energy provision for the additional genetic elements this may be a burden on the efficient functioning of the cell. One example of the use of gene duplication is for generation of diverse surface structures. The genome sequence of the gastric pathogen Helicobacter pylori encodes a family of over 30 paralogous (homologous proteins from the same family that have been expanded by gene duplication) outer membrane proteins that may play a role in generating antigenic variation (Tomb et al., 1997). These are absent in the genome sequence of the foodborne pathogen Campylobacter jejuni and represent one of the few major differences between these closely related species (Parkhill et al., 2000). Similarly, genome analysis of Mycobacterium tuberculosis revealed two novel families of glycine-rich proteins with repetitive structure which constitute 10% of the genome. These gene families may represent a source of antigenic variation for M. tuberculosis (Cole et al., 1998). 1.3.2 Lateral gene transfer – the driving force for diversification Several mechanisms could be responsible for the differences evident amongst bacterial species. Point mutations (single nucleotide changes) leading to the modification, inactivation or differential regulation of existing genes have certainly contributed to the diversification of micro-organisms on an evolutionary timescale. However, it is difficult to account for the ability of bacteria to exploit new environments and become pathogenic based on the accumulation of point mutations alone. In fact, none of the phenotypic traits that are typically used to distinguish E. coli from Salmonella enterica can be attributed to the point mutational evolution of genes common to both species (Lawrence and Ochman, 1998). Instead there is growing evidence that lateral gene transfer (the acquisition of regions of DNA, largely from other microorganisms) in terms of gain of trait functions, has played an integral role in the evolution of bacterial genomes, and in particular the diversification and speciation of the enteric bacteria. Established vehicles for DNA transfer among bacteria include transpositions, conjugative plasmids, phages and natural transformation (innate ability to uptake DNA from other micro-organisms). One characteristic of loci acquired by lateral gene transfer is an atypical G+C content, relative to the rest of the genome (Fleischmann et al., 1995). In contrast to most eukaryotes, bacteria have variable whole G+C contents ranging from 23% to 78%. The availability of a complete genome sequence allows genome-wide screening for ‘spikes’ of G+C variation, offering the opportunity to measure and compare the

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cumulative effect of lateral gene transfer among pathogens. Comparison of sequenced genomes confirms that bacteria have undergone frequent gene transfer events, many of which act as markers for possible virulence determinants. Salmonella Typhimurium has over 30 G+C spikes and a number of these contain telltale remnants of portable transposons or insertion sequences (Parkhill et al., 2001a, McClelland et al., 2001). This contrasts with another foodborne pathogen C. jejuni (strain NCTC 11168) in which there is little evidence of lateral gene transfer (Parkhill et al., 2000). It appears that the similarity between these pathogens is mainly restricted to housekeeping genes and that genes required for most functions related to survival, transmission and pathogenesis are remarkably dissimilar. Thus lateral gene transfer is largely responsible for the genotypic and phenotypic differences between these pathogens and it suggests that selective pressures have driven profound evolutionary changes to create two very different foodborne pathogens from a relatively recent common ancestor. Lateral gene transfer often involves linked blocks of genes ranging in size from 5 to 100 kb and, if they contribute to virulence, such cassettes have been termed pathogenicity islands or pathogenicity loci (Hacker et al., 1997). Upon incorporation into a recipient bacterium these DNA regions can convert a benign organism into a pathogen. Examples of the products of pathogenicity islands include many type III and type IV secretion systems from bacterial species including foodborne pathogens such as E. coli and Salmonellae and Yersiniae. Both systems encode specialised organelles that act as molecular syringes to export effector molecules (generally toxins) across the bacterial membrane of Gram-negative bacteria into the host cell to modulate host cellular functions (Cheng and Schneewind, 2000, Galan and Collmer, 1999; Covacci et al., 1999). S. Typhimurium has two well-characterised type III secretion systems (Spi-1 and Spi-2) that play an integral role in their invasion and survival within host cells (Kingsley and Baumler, 2002). Other examples of pathogenicity islands include cluster of genes required for the fitness and survival of an organism such as the iron uptake system in Yersinia species. Many are situated at tRNA loci which thus represent a readily identifiable target to identify putative pathogenicity islands (Hacker et al., 1997). For some bacteria the pathogenicity cassette is not located near tRNA and these are often referred to as pathogenicity loci (PaLocs). Examples include the listeriolysin toxin and phospolipase virulence cassette from the foodborne pathogen Listeria monocytogenes and the cytotoxin and enterotoxin from the enteric pathogen Clostridium difficile (Portnoy et al., 1992; Hundsberger et al., 1997). Several further studies have demonstrated that many virulenceassociated as well as antibiotic resistance genes are located on mobile accessory DNA elements. Phage infection of bacteria has frequently been implicated in the horizontal gene transfer of virulence determinants. Mirold et al. have demonstrated that S. Typhimurium has evolved to a more pathogenic state by bacteriophage lysogenic conversion of SopE, an effector toxic protein transferred by the

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Spi-2 type III secretion system (Mirold et al., 1999). Strikingly, most of the isolates harbouring SopE belong to the small group of epidemic strains that have been responsible for a large percentage of human and animal salmonellosis (Mirold et al., 1999). Recent outbreaks of S. Typhimurium have been attributed to a multiply antibiotic resistant phage type, DT104 that appears to have increased virulence in humans and cattle (Wight et al., 1996; Cloeckaert and Schwarz, 2001). This example indicates that differences exist between highly similar salmonellae within animal populations and that more virulent organisms may be selected and expanded. In Vibrio cholerae the VPIf bacteriophage has been shown to encode a pilus that functions as a colonisation factor for the human intestine as well as the receptor for the cholera-toxin encoding CTXφ bacteriophage (Karaolis et al., 1999). Another example of the recent emergence of a pathogen with a bacteriophage-encoded toxin is E. coli O157:H7 which is an identified virulence factor (Park et al., 2001; Reid et al., 2000). Additionally for V. cholerae, it has been suggested that the smaller of the two chromosomes can be considered as a mega-plasmid that was captured by the micro-organism in its ancestral past and which provides it with a protective advantage (Heidelberg et al., 2000). This selective advantage may relate to the marine part of the V. cholerae life cycle (the probable natural habitat of V. cholerae) rather than to adaptation to the human host. Lateral gene transfer has and will continue to change the pathogenic character of bacterial species. 1.3.3 Point mutations and slipped-strand mispairing Phase variation, the reversible, high-frequency, gain or loss of a phenotype, resulting from changes of expression of single or multiple genes, is a common survival strategy employed by bacterial pathogens (Henderson et al., 1999). Variation of surface structure by pathogens, frequently referred to as antigenic variation, is often used to avoid detection or to outwit a host’s immune system. In some pathogens, such variation can occur by the slipped-strand mispairing of repeat sequences of DNA during replication. Alteration of the length of these tracts within, or immediately upstream of genes causes the translation of the respective protein to move in and out of the correct frame, affecting the synthesis of the protein. The reversible on/off feature of these genes can be seen as recombinational changes of a binary system resulting in an exponential increase of gene variation. Therefore, the phenotype of an individual bacterium within a growing population can change, leading to a stable sub-population with altered properties. If this sub-population has gained a significant survival advantage then it can persist more readily than the original ‘parent’ strain. But this is a reversible process allowing the subpopulation to switch back should a new environment prove more favourable. Genes with variation in simple repeat sequences have been termed contingency genes and the repeating unit can vary from single nucleotides (homopolymeric tracts) to penta nucleotides (Moxon et al., 1994).

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Close scrutiny of whole-genome sequences can identify such repeats. Such analysis simplifies the identification and investigation of potential contingency genes that are often involved in host adaptation and pathogenesis. Prior to sequencing of the H. influenzae genome, only two examples of contingency genes were known in the organism. The search for simple nucleotide repeats in the H. influenzae genome sequence identified at least a further dozen potential contingency genes, four of which are involved in lipopolysaccharide biosynthesis and four more involved in iron uptake (Fleischmann et al., 1995; Hood et al., 1996). Analysis of the H. pylori genome sequence suggested the presence of 27 putative phase variable genes based upon the presence of simple repeats (Tomb et al., 1997; Alm et al., 1999). Two of these repeats were found in independent alpha-3fucosyltransferase genes, which have been shown to be responsible for the variable expression of the Lewis X and Lewis Y antigens on the surface of H. pylori (Wang et al., 1999; Appelmelk et al., 1999; Wang et al., 2000). A striking observation from the C. jejuni genome sequence was the apparently high level of genetic variation affecting translation of over 25 contingency genes (Parkhill et al., 2000). During sequencing of the eightfold redundant shotgun library of clones, regions were identified where the sequences of otherwise identical reads varied at a single point (Parkhill et al., 2000). These were mainly on runs of G and C that varied in length by one or more base pairs. Most of the 25-hypervariable regions group into three clusters in the genome, and these are coincident with loci responsible for lipooligosaccharide (LOS) biosynthesis, capsule biosynthesis and flagellar modification (Parkhill et al., 2000). One of these genes encodes a beta-1,3 galactosyltransferase responsible for expression of a GM1 ganglioside mimic on C. jejuni LOS. An assay to detect the presence of GM1 is its ability to bind cholera toxin (Linton et al., 2000b). This assay has been used to clearly demonstrate the on/off reversible switching of this determinant on the surface of C. jejuni cells (Linton et al., 2000a). Figure 1.1 demonstrates the analysis of six independent colonies with the terminal transferase in frame and out of frame. The ‘in frame’ colonies clearly bind to cholera toxin and when sequenced have the run of 8Gs in the beta-1,3 galactosyl terminal transferase gene. By contrast the ‘out of frame’ colonies barely bind cholera toxin (note that a small sub-population will revert and therefore bind cholera toxin) and when sequenced have a run of 9Gs (Fig. 1.1). This study demonstrates the power of genome sequence data in identifying genes likely to be important in cell surface structures and host-pathogen interactions. 1.3.4 Genome decay and the potential for increased virulence Loss of gene function, or genome decay, occurs as a bacterium adapts to its host. For example, many pseudogenes (DNA sequences that may have once encoded a functional protein), often ignored as sequencing artefacts, may in fact be remnants of functional genes from a pathogen in the process of

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6.5 kDa A

B

C

D

E

F

(b) GGGTGGGGGGGGTA

GGGTGGGGGGGGGTA

B

A GGGTGGGGGGGGTA

GGGTGGGGGGGGGTA

D

C GGGTGGGGGGGGTA

GGGTGGGGGGGGGTA

F

E

(a)

(c)

ATG GGT GGG GGG GGT AAA ATT GAT … … … . . . M G G G G K I D Binds cholera toxin

ATG GGT GGG GGG GGG TAA AAT TGA … … … M

G

G

G

G

*

Fails to bind cholera toxin (d)

Fig. 1.1 Demonstration of antigenic variation in C. jejuni β−1,3 galactosyltransferase gene WlaN. (a) Colony immunoblot of a natural population C. jejuni NCTC 11168 cells probed with cholera toxin. The minority of colonies, three of which are boxed, show little binding to cholera toxin, the majority of colonies, three of which are circled, show strong binding to cholera toxin. (b) Analysis of lipooligosaccharide from C. jejuni NCTC 11168 wild type cholera toxin binding variants. SDS-PAGE gel probed with cholera toxin. (c) Sequencing profiles of wlaN intragenic homopolymeric tracts from C. jejuni NCTC 11168 cholera toxin binding variants. Panels A, C and E were obtained from C. jejuni NCTC 11168 colonies circled, whilst panels B, D and F were obtained from C. jejuni NCTC 11168 colonies boxed. (d) Nucleotide and derived amino acid sequence of the region around the homopolymeric tract of the wlaN gene for sequences with eight and nine G residues. Eight G residues allows translational read through and full-length product formation whilst nine G residues leads to translational termination at a now in frame stop codon (TAA) (*) immediately following the homopolymeric tract.

downsizing its genome content as the organism adapts in response to evolutionary pressures. Comparison of the genome sequence of the broad host range foodborne pathogen S. Typhimurium with that of the human host adapted, closely related pathogen S. Typhi has revealed that the latter genome is in the early stages of genome decay with 5% of the predicted genes being non-functional pseudogenes. Most of the pseudogenes appear in genes that formerly encoded for cell surface functions, host interaction or pathogenesis functions, reflecting the evolutionary pressures to streamline the genome in its new human restricted

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host. Most pseudogenes are caused by single mutations rather than multiple mutations, again indicative that the observed genome downsizing is a recent event in evolutionary terms. An extreme of genome downsizing can been seen from the genome sequence of the obligate intracellular pathogen Rickettsia prowazekii (Andersson et al., 1998). The 1.11 Mb R. prowazekii genome is packed with pseudogenes, and has the highest proportion of non-coding DNA in any prokaryote – over 24%. The DNA between genes may represent the scattered remnants of genes that are no longer required (or harmful to the existence of the organism) – lost in a step-wise process as the organism acquires an obligate intracellular lifestyle. Analysis of the genome of the leprosy bacillus Mycobacterium leprae paints a similar picture – numerous pseudogenes and extensive genetic downsizing not found in other mycobacterial species that tend to have a more free-living existence (Brosch et al., 2000). Although counterintuitive, it is becoming increasingly evident that some genes actually increase the organism’s virulence when they are inactivated in the process of genome downsizing. When pathogenic Shigella strains arose from a non-pathogenic E. coli ancestor, the loss of ompT and cadA genes may have contributed to their virulence and evolution (so-called ‘black holes’) (Maurelli et al., 1998; Nakata et al., 1993). More recently, this phenomenon has been demonstrated in M. tuberculosis, where several experimentally designed knockout mutants appear more virulent than the wild type strain (Parish et al., 2003). This may also be a contributory factor to the evolution of the highly virulent plague bacillus Yersinia pestis (see below). 1.3.5 Modulation of the frequency of genetic variation and fidelity of proof reading enzymes The genetic diversity of bacteria results not only from horizontal exchange and recombination of DNA sequences from similar and disparate species, but also from errors in DNA replication and repair as well (Radman et al., 2000a, b; Brown et al., 2001). Most bacteria have dedicated genetic systems such as DNA replication and mismatch repair systems that ensure the faithful replication of genetic material in the cell. Occasionally some of these systems may be defective due to genetic mutations altering the fidelity of DNA replication or mismatch repair. This may result in progeny that have a higher mutation rate, so called mutator strains (Radman et al., 2000a, b; Brown et al., 2001). These mutation-prone strains can profoundly affect the evolution rates of bacteria. Potentially a beneficial mutation may allow the rapid emergence of a more fit strain that may have extended its host range as a result of one of the mutational events. A well-characterised system is MutS, which is involved in methyl-directed mismatch repair (Radman et al., 2000a, b). MutS mutants generate a mutator phenotype typified by high mutation rates and promiscuous recombination.

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An example of how mutator strains can dominate in selected environments is in the lungs of cystic fibrosis patients who are chronically infected for years by one or a few lineages of Pseudomonas aeruginosa. These bacterial populations adapt to the highly compartmentalised and anatomically deteriorating lung environment of cystic fibrosis patients, as well as to the challenges of the immune defences and antibiotic therapy. In P. aeruginosa a high proportion of isolates (20%) from the lungs of cystic fibrosis patients have an increased mutation frequency (mutators) (Oliver et al., 2000, 2002); In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1 (Oliver et al., 2002). Further studies have shown seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD) (Oliver et al., 2000). The results show that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype. Mutator strains were not found in 75 non-CF patients acutely infected with P. aeruginosa (Oliver et al., 2000, 2002). These studies also reveal a link between high mutation rates in vivo and the evolution of antibiotic resistance by P. aeruginosa (Blazquez, 2003). For foodborne pathogens such as E. coli and Salmonella mutator strains have been characterised and undoubtedly contribute to the frequency of the mutation rate and evolution of the species. However, inspection of the C. jejuni and H. pylori genomes reveal a lack of mut genes, suggesting that such mechanisms do not apply in these organisms (Tomb et al., 1997; Parkhill et al., 2000).

1.4

Case studies and the evolution of pathogenic Yersinia

1.4.1 Recently emerged pathogens and the Yersinia pestis genome recipe Examples of recently emerged pathogens include V. cholerae non-O1/nonO139 cholera toxin positive strains responsible for the current cholera pandemic (Faruque et al., 1998; Dziejman et al., 2002). E. coli O157 that sporadically appears in the food chain in several parts of the world (Park et al., 2001; Reid et al., 2000) and S. enteritidis phage type 4 replacing a previous niche occupied by pathogenic Salmonella species (Wight et al., 1996; Cloeckaert and Schwarz, 2001). However, the most striking example of the emergence of a highly virulent pathogen is the evolution of the plague bacillus Y. pestis, which evolved from Yersinia pseudotuberculosis, a mild foodborne pathogen in about 2,000 to 20,000 years – an eye blink of evolutionary time (Achtman et al., 1999). By contrast the other Yersinia foodborne pathogen Yersinia enterocolitica, is distantly related to Y. pseudotuberculosis and Y. pestis. This has been referred to as the Yersinia paradox – the two closely related species,

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Y. pseudotuberculosis and Y. pestis cause vastly different diseases and are the most closely related (97% at the DNA level), yet the least related, the enteropathogenic yersiniae, are foodborne pathogens causing similar disease. An understanding of how one species evolved from the other can now be gained through genome sequence and microarray analyses. The Y. pestis genome seems to be particularly flexible and in an intermediate stage of genome flux and genome decay. This paradigm of bacterial pathogen evolution can be embodied as the Yersinia genome recipe – add DNA, stir and reduce. Add DNA As the nucleotide sequences of more bacterial genomes become available it is evident that they are mosaics of DNA sequences from different origins, due to the lateral exchange of large mobile genetic elements such as plasmids, phage or transposons. The genetic material acquired often contributes to an organism’s virulence – broadening its host range, for example, or improving its ability to overcome host defences or to cause tissue damage. This lateral gene transfer allows microbial pathogens to evolve extremely rapidly, in ‘quantum leaps’. For Y. pestis, plasmid acquisition certainly seems to be a key element in its evolutionary jump from enteric pathogen to flea-transmitted systemic pathogen. In addition to the virulence plasmid pYV that is also common to the enteropathogenic Yersinia, virtually all Y. pestis strains have two further plasmids – pPla that encodes the plasminogen activator Pla, (Brubaker, 1991) and pMT1 that encodes the putative murine toxin Ymt, as well as the F1 capsule. The precise role of these determinants in host adaptation and virulence is uncertain, but there are several hints that they are involved in transmission. Pla, for example, is important for dissemination of Y. pestis after subcutaneous injection into a mammalian host (Sodeinde et al., 1988) and strains that lack the entire pMT1 plasmid are unable to colonise fleas (Hinnebusch et al., 1998). However, also required for fleaborne transmission is the unstable chromosomally located haemin storage locus (hms), which encodes outer surface proteins. Thus, overall, the acquisition of two plasmid (pPla and pMT1) by horizontal gene transfer, along with the pre-existing chromosomal hms locus, help to explain the rapid evolutionary transition of Y. pestis to fleaborne vector transmitted pathogen (Wren, 2003). The pPla and pMT1 plasmids and the hms locus were known before the Y. pestis strain CO92 genome sequence became available. But what about the rest of the 4.65 Mb chromosome? G+C analysis of the Y. pestis CO92 genome identified at least 21 G+C ‘spike’ regions characteristic of lateral gene transfer, including the 102 kb unstable element that contains hms. (Parkhill et al., 2001b). Among these regions were several genes that appear to have come from other insect pathogens. Sequences related to the parasitism of insects include homologues of insecticidal toxin complexes (Tcs) from Photorhabdus luminescens, Serratia entomophila and Xenorhabdus nematophilus (Waterfield et al., 2001). In addition, a predicted coding sequence

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showing similarity to an insect virus-like enhancin protein, a proteolytic enzyme that can damage insect gut membranes was also identified in a region of low G+C content (Parkhill et al., 2001b). The sequence is flanked by transposase fragments, suggesting horizontal acquisition. Other apparent acquisitions include a chromosomally encoded type-III secretion system, similar in gene content and order to the Spi2 type-III system of S. typhimurium (Shea et al., 1996), and several adhesins and iron-scavenging systems. However, subsequent DNA microarray analysis has shown that virtually all these determinants are also present in Y. pseudotuberculosis (Hinchliffe et al., 2003), suggesting that they have been acquired in the Y. pestis genome for some time and that Y. pseudotuberculosis probably has some association with insects, hitherto unknown. Stir A most striking feature of the Y. pestis CO92 genome sequence was the large number of insertion sequence (IS) elements. IS elements consist of perfectly repeated sequences, and are likely sites for homologous recombination events that can rearrange the genome. The total of 140 IS elements in the CO92 genome exceeds that described in most other bacterial genomes and comprises 3.7% of the genome. All bacterial genomes sequenced to date have a small but detectable bias towards G on the leading strand of the bi-directional replication fork (Lobry, 1996). So the G/C skew in different parts of the genome highlights any irregularities in its composition. The G/C skew plot of Y. pestis CO92 shows three anomalies (two inversions and one translocation) (Parkhill et al., 2001b). Each is bounded by IS elements, suggesting that they could be the result of recent recombination. It seems several different chromosomal configurations can exist in the same population, suggesting that genomic rearrangements occur during growth of the organism. This is a particularly unusual feature for a bacterial chromosome and it is unknown how these events affect the biology of the organism. However, because the expression of bacterial genes is influenced by their orientation with respect to the direction of DNA replication, it seems reasonable to conclude that such rearrangements could alter pathogenesis, and be a rapid mechanism to switch on and off virulence. Reduce Close inspection of the CO92 nucleotide sequence identified at least 149 pseudogenes, representing 4% of the genome. Several mechanisms account for the accumulation of pseudogenes in Y. pestis, including IS element expansion, deletion, point mutation and slipped strand mispairing. As Y. pestis has changed its lifestyle from that of the ancestral Y. pseudotuberculosis, it would not be expected to use genes required for enteropathogenicity as the newly evolved Y. pestis would no longer be transmitted by the faecal-oral route. Enteropathogens, particularly those transmitted in the food chain, specifically adhere to surfaces of the gut and

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invade cells lining it. Proteins important for this process in Y. pseudotuberculosis include YadA, and Inv, both of which are represented by pseudogenes in Y. pestis (Simonet et al., 1996; Rosqvist et al., 1988). Many of the other pseudogenes reported, for example a putative intimin adhesion protein, may have encoded adhesin molecules that potentially played a role in enteropathogenesis. Additionally many surface exposed features appear to have been lost in the rapid transition from Y. pseudotuberculosis to Y. pestis such as fimbrial-usher systems. This may represent an example of Y. pestis shutting down opportunities for the mammalian host immune system to recognise Y. pestis during systemic disease. Some pseudogenes may be able to regain their function. As outlined above, several pathogens have been shown to switch surface-expressed antigens on or off by slipped-strand mispairing of repeat sequences during replication (Henderson et al., 1999) and a similar process has been demonstrated in Y. pestis in the ureD gene. The organism is characteristically urease negative, but activity can be restored in vitro by the spontaneous deletion of a single base pair in a homopolymeric tract. This type of reversible mutation would free Y. pestis from the metabolic burden of producing proteins that are not required in its new flea/mammal life cycle, yet still allow the potential to express them should a subsequent need arise. But some enteropathogen virulence traits seem to be irreversible in Y. pestis, because the gene pathways encoding them have been inactivated by multiple mutations. Examples here include motility and LPS biosynthesis, where at least five genes in each pathway appear to no longer function in Y. pestis CO92 (Skurnik et al., 2000; Parkhill et al., 2001b). There are also several pseudogenes of unknown function. Given that many of the familiar pseudogenes appear to be associated with a redundant enteric life cycle, identifying these sequences in Y. pestis may reveal potential virulence determinants for investigation in the enteropathogenic yersiniae. Not all of the lost genes relate to putative virulence determinants. In Y. pestis, many pseudogenes relate to physiological functions, particularly with respect to the loss of bioenergetic functions such as dicarboxylic amino acid metabolism. For some time it had already been known that all Y. pestis strains tested lack glucose 6-phosphate dehydrogenase and aspartase among other enzymes that alter the catabolic flow of carbon (Brubaker, 2000; Dreyfus and Brubaker, 1978; Mortlock and Brubaker, 1962). The reduction of unnecessary metabolic load may enable the organism to conserve energy. The newly evolved streamlined organism may then contribute to the development of acute disease. Thus rather like gene acquisition being important in the evolution of highly pathogenic bacteria, genome downsizing may be at least equally important. This loss appears to have been triggered by the extensive expansion of IS elements, which caused major genome rearrangements. Once Y. pseudotuberculosis had acquired certain critical genes, the instability introduced by the IS elements was the major force to release its potential – as Y. pestis.

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The question remains why would Y. pestis want to evolve from a common foodborne pathogen to one that kills its host by bacteraemia? The answer may lie in the respective life cycles of the organisms. For the enteropathogenic Yersinia, shedding and spreading by inducing diarrhoea in the host is the most efficient mechanism to transfer to the next host. By contrast Y. pestis is transmitted by a flea vector, which because it is a recent evolutionary event is relatively inefficient, therefore a high bacterial load, i.e., severe bacteraemia in the host, would be required for the organism to be transmitted and ensure an efficient life cycle. Thus there is a very strong selective pressure to cause severe disease. The factors influencing the rise and fall of plague pandemics also remain obscure. Undoubtedly there will be many factors involved, but the genetic make up of Y. pestis is likely to be important. It is possible that during the spread of an epidemic, passage through humans may allow Y. pestis to become more transmissible or more pathogenic, particularly during pneumonic transfer where close human contact may aid transmission. Such a ‘hypervirulent’ strain of V. cholerae was recently demonstrated to have arisen during passage through humans in a cholera epidemic (Merrell et al., 2002). The flexible genome of Y. pestis makes it a likely candidate for such a mechanism. Indeed there is evidence the rapid emergence of the three Yersinia pestis biovars Antiqua, Mediaevalis and Orientalis, arose through parallel micro-evolution as a result of a flexible genome. As Orientalis biovar is glycerol negative and nitrate positive and Mediaevalis is glycerol positive and nitrate negative, it is likely that these biovars arose independently from the glycerol and nitrate positive Antiqua progenitor (Fig. 1.2). Further analysis using subtractive hybridisation and microarray analysis have confirmed that Mediaevalis and Orientalis evolved independently from Antiqua (Radnedge et al., 2002, Hinchliffe et al., 2003). Very recently a fourth biovar, biovar microtus, has been proposed, confirming the process of parallel microevolution in natural plague foci (Zhou et al., 2004). 1.4.2 Evolution of enteropathogenic Yersinia The genome sequences of Y. pseudotuberculosis IP 32953 (serotype I) (Chain et al., 2004) and Y. enterocolitica 8081 (biogroup1B, serotype O8) (http:// www.sanger.ac.uk/Projects/Y_enterocolitica/) have been recently made available. Insights from genome analysis allow us to piece together a picture of how the three pathogenic Yersinia species may have arisen. It seems clear that Y. enterocolitica has evolved independently. It can be separated into three lineages – the mostly avirulent biogroup 1A strains that lack the virulence plasmid, the mouse-virulent Old World strains (biogroups 2 to 5) and mouselethal New World strains (biogroup 1B) (Fig. 1.2). The New World strains appear to have acquired several elements by lateral gene transfer that contribute to their increased virulence compared to Old World strains. In particular, the New World strains contain a ‘high pathogenicity

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Emerging foodborne pathogens Non-pathogenic environmental Yersinia +pYV Predecessor of pathogenic Yersinia +Yst

Y. enterocolitica

Hms & HPI*

–pYV

Y. enterocolitica IA

HPI & type II secretion

Y. enterocolitica Old-world

Y. enterocolitica New-world

insect toxins

Y. pseudotb non-biofilm

Y. pseudotb biofilm O1b/O3 strains pPla pMT1

Add IS elements, stir and reduce

Y. pestis Antiqua Y. pestis Mediaevalis

Y. pestis Orientalis

Fig. 1.2 Proposed evolution of Yersinia species. The non-pathogenic Yersinia gain the virulence plasmid that contain the prototype type III secretion system to form the predecessor of pathogenic Yersinia. Y. enterocolitica diverges from Y. pseudotuberculosis and forms three lineages. 1A, Old-world and New-world. Y. pseudotuberculosis gains ability to parasitise insects and form biofilms in hosts before evolving into Y. pestis through adding DNA (pPla and pMT1), stirring and reducing. For Y. pestis ensuing microevolution results in three lineages given the biovar designations, Antiqua, Mediaevalis and Orientalis. Note, the high pathogenicity island (HPI) was independently acquired to high pathogenicity island (HPI)*.

island’ (HPI) which encodes the synthesis of the siderophore yersiniabactin, an iron-sequestering low-molecular-weight compound that is invaluable in the iron-limiting environment of the host (Pelludat et al., 1998). The importance of the HPI region to mouse virulence has been demonstrated by transferring it from a New World strain into an Old World strain, whereupon the modified strain was lethal in mice (Pelludat et al., 2002). The HPI has also been found in other enterobacteriaceae, (Schubert et al., 1998) some of which might be candidates for donating the HPI to the Y. enterocolitica New World strains and Y. pseudotuberculosis (Y. pestis). The region is also present in Y. pseudotuberculosis and Y. pestis, but sequence analysis reveals that it is significantly different to the HPI in Y. enterocolitica, suggesting that it may have been acquired independently (Schubert et al., 1998). More recently, another element has been identified that appears to occur exclusively in New World strains – a further type II secretion gene cluster (Iwobi et al., 2003). Genome sequence analysis of the genomes of Y. enterocolitica and Y. pseudotuberculosis have confirmed that they have far fewer IS elements and pseudogenes than Y. pestis CO92 or a more recently sequenced strain of Y. pestis KIM10 (Parkhill and Thompson, 2002; Garcia, 2002; Chain et al., 2004). This suggests that the Y. pseudotuberculosis and Y. enterocolitica genomes are far more stable than Y. pestis. Genome sequence data also

How bacterial pathogens evolve

17

confirms that Y. pestis and Y. pseudotuberculosis are closely related, with gene homology of nearly 97% and largely co-linear gene organisation (Garcia, 2002; Chain et al., 2004). In contrast, Y. enterocolitica is more distantly related, about the same evolutionary distance away from Y. pseudotuberculosis and Y. pestis as Escherichia coli is from Salmonella species. Closer inspection of the disease syndromes of Y. enterocolitica and Y. pseudotuberculosis suggests that although they appear similar, the two species do, in fact, cause different infections. Although both pathogens invade through M cells, Y. enterocolitica colonises the Peyer’s patches, while Y. pseudotuberculosis is more widely disseminated and typically causes acute abdominal pain with mesenteric lymphadenitis of the small intestine. One distinguishing feature of Y. enterocolitica disease compared to Y. pseudotuberculosis is that it causes a more severe diarrhoea (pronounced watery diarrhoea and occasionally bloody diarrhoea with fever in children). The heat stable toxin (Yst) has been identified in all enteropathogenic Y. enterocolitica, but is absent in Y. pseudotuberculosis (Delor and Cornelis, 1992). This could be one of the distinguishing genetic features responsible for this difference in symptoms. Thus, although diarrhoea is a common outcome, the diseases are different. This partly explains the ‘Yersinia paradox’, although it does not shed light on why Y. pestis causes such a different disease.

1.5

Sources of further information

The UK represents a special case in terms of emerging foodborne pathogens because of the heightened awareness of foodborne disease following high profile BSE, E. coli, Listeria and Salmonella outbreaks and the ever increasing reported incidence of campylobacteriosis. Most microbial surveillance in the UK is carried out at the Health Protection Agency (formerly the Central Public Health Laboratories) at Colindale, London. This centralised facility maintains national records of the different species, strains and strain types responsible for foodborne diseases. Through a process of monitoring and reporting the Centre can detect trends, trace the sources and route of transmission of foodborne pathogens, and act to prevent the further spread of disease. In the USA, the Communicable Disease Centre in Atlanta performs a similar role, and most countries have similar disease surveillance centres.

1.6

Future studies

The bedrock of infectious disease prevention and control is high-quality microbiology and surveillance that allows outbreaks to be anticipated and prevented. Traditionally, this has relied on phenotypic markers such as

18

Emerging foodborne pathogens

serotyping and phage typing and limited genotypic methods such as pulsed field gel electrophoresis. More recently, multi locus sequence typing (MLST) has been used effectively for retrospective population genetic studies and for determining clonality of strains of diverse origin. All of these methods suffer from providing limited information. In an ideal world the complete genome sequence of every problem pathogen would be determined as and when outbreaks occurred. Such information would enable us to determine if the genetic information from a given pathogen is changing both in terms of gene acquisition and genome decay. However, even if this were ever possible, we are several years away from being able to do this on a routine basis. In terms of surveillance and prevention of the spread of infectious disease, if a putative virulence determinant(s) from one species were to be identified unexpectedly in another bacterial species (through lateral gene transfer) this would raise concern. If this were identified rapidly, appropriate measures could be taken. There are numerous precedents of virulence determinants being present in different species including several families of toxins (e.g. thiol activating toxins, ADP ribosylating toxins such as cholera toxin and E. coli LT toxins,) type III and IV secretion systems, autotransporters and the Yersinia high-pathogenicity island which is widely distributed among different enterobacteria such as E. coli, Klebsiella and Salmonella (Bach et al., 2000). Specifically designed microarrays may offer a new dimension to microbial surveillance. We are currently developing an active surveillance pathogen (ASP) DNA microarray that contains elements that would not only identify specific foodborne pathogens, but would also contain elements of the known genome flux including pathogenicity islands, pathogenicity loci, antibiotic resistance genes, transposons, plasmids and phages that carry known virulence determinants, many of which have been described in this chapter. The ASP array would be useful for supplying information on tracing the sources and routes of transmission of a given pathogen, active surveillance may allow the identification of the emergence of highly transmissible or virulent strains that could, for example, be traced back to an individual flock or herd, eliminated, thus averting the spread of an emerging virulent strain. The long term potential of the ASP array could be applied to provide information on the • • • • • •

emergence of new or more virulent foodborne pathogens spread of antibiotic resistance and associated virulence determinants. (For example, is the practice of adding antibiotics to animal feed contributing to antibiotic resistance in foodborne pathogens?) change in bacterial populations before and after vaccine trials. (For example, does the introduction of new Salmonella poultry vaccine influence the emergence of other foodborne pathogens?) any unusual isolates from patients with traveller’s diarrhoea emergence of nosocomial pathogens whether the genome of a pathogen has been tampered with, either through deliberate or accidental release.

How bacterial pathogens evolve

1.7

19

Conclusion

Common themes to emerge from the genome analysis of over a hundred bacterial pathogens sequenced so far include extensive lateral gene transfer (particularly among enteric pathogens), genome decay (among obligate intracellular pathogens) and extensive antigenic variation by gene shuffling or slipped-strand mispairing. Y. pestis, perhaps the most feared of all recently evolved bacterial pathogens, appears to have all these characteristics, it is an organism in an intermediate stage of genetic flux, where the acquisition of novel sequences by lateral gene transfer appears to be counterbalanced by ongoing genome decay. If we are to continue to outwit pathogens and avoid future outbreaks, there can be no substitute for continuing to undertake basic research against our old adversaries and maintaining active microbial surveillance.

1.8

Acknowledgements

The author wishes to acknowledge Manu Davies for assistance with preparing the manuscript and Val Curtis for useful discussions. The author acknowledges the BBSRC, MRC and The Leverhulme Trust for funding research on foodborne pathogens in his laboratory.

1.9

References

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2 Surveillance for emerging pathogens in the United States C. R. Braden and R. V. Tauxe, Centers for Disease Control and Prevention, USA

2.1

Introduction

Public health surveillance is the ongoing systematic collection, analysis, interpretation, and dissemination of health outcome specific data for use in public health action to reduce morbidity and mortality and to improve health (Thacker and Berkelman, 1988). Surveillance of many infections and intoxications, including those that are frequently foodborne, is a fundamental public health activity in many countries. Human foodborne disease surveillance is conducted for three principal reasons: (i) to identify, control and prevent outbreaks of foodborne disease; (ii) to monitor trends and determine the targets for and efficacy of control measures; and (iii) to determine the burden of specific diseases on the public’s health (Potter et al., 2000). The information collected through surveillance systems is essential for conducting microbiological risk assessment studies, making risk management decisions and designing processes used to determine and control potential danger points for microbial contamination in food production, known as hazard analysis and critical control point (HACCP) systems (Borgdorff and Motarjemi, 1997). Surveillance information is used to prioritize and design food safety educational programs and materials for policy makers, public health officials, medical care providers, food industry workers, consumers and others. These activities, based largely on information provided by public health surveillance systems, constitute effective mitigation and prevention programs in food safety. Public health surveillance typically starts with information that already exists for another purpose, and then adds to it. The information useful to clinicians is somewhat different from what is needed for public health surveillance. The clinic makes the diagnosis of salmonellosis, and the clinical

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Emerging foodborne pathogens

laboratory may determine the antimicrobial resistance of the organism to facilitate treatment. However, neither a detailed food history nor determination of serotype will influence the management of the case, so it is up to public health authorities to interview the patient and to further characterize the infecting organism. In countries without nationalized health systems, the cost of reporting and further public health activities is separate from the cost of patient care, and is borne as one of the core functions of government. Surveillance is a keystone in the effort to define, control and prevent foodborne diseases as depicted in the cycle of surveillance and prevention (Fig. 2.1). Through surveillance activities, emerging pathogens and outbreaks can be identified. Epidemiologic investigation, applied research and interventions may then be applied in prevention efforts. The cycle of surveillance and prevention is exemplified by the description of E. coli O157 in apple cider below. Although the specific targets chosen for surveillance vary from nation to nation, depending on local history, concern and resources, the general goals remain the same. Surveillance is a major source of information for the detection of foodborne outbreaks. Efficient surveillance can detect outbreaks quickly, and subsequent outbreak investigations can lead to rapid interventions such as the removal of contaminated products from the market or temporary closure of a food business. Outbreak investigations are also important opportunities to identify critical gaps in knowledge, leading to applied research and ultimately to better long-term prevention, as unsafe processes are corrected, or new food hazards are identified and controlled. Once prevention measures are in place, continued surveillance will document their success, or indicate the need for further investigation, research, knowledge dissemination and prevention. The information gathered by surveillance and by investigations of sporadic cases or outbreaks can reveal the magnitude and general trends of foodborne disease, helping policy makers target prevention strategies and providing information critical to risk assessment studies.

Surveillance

Epidemiologic investigation

Intervention

Applied research

Fig. 2.1 Cycle of surveillance and prevention. Surveillance serves as the basis for epidemiologic investigation and applied research, each of which determines modes of intervention. As interventions take effect, surveillance changes and the iterative cycle begins again.

Surveillance for emerging pathogens in the United States

25

Foodborne disease surveillance in the United States is conducted primarily by local and state public health agencies. Some form of local surveillance for diseases of public health concern has been conducted for centuries in many countries. In the 19th century, reporting of cholera ushered in the modern concept of the reportable disease in the United States (Rosenberg, 1987). Reporting of typhoid fever cases and deaths drove many improvements in water and food safety at the beginning of the 20th century. More recently, the increase in concern following the large E. coli O157:H7 outbreak in 1993 stimulated enhancements in surveillance for foodborne infections in the United States, as well as other changes in the food safety system (Anon., 1998). The foodborne mode of transmission for many pathogens was discovered in the course of outbreak investigations, as was much of the knowledge we have about specific hazards and how they enter the food supply. As new foodborne disease sources and agents emerge, the strategies used to control them must also evolve. Thus, surveillance for foodborne and other infections is an ever changing arena. The most dramatic recent example of this progress concerns the evolving understanding of the modes of transmission for E. coli O157:H7. In 1982, two outbreaks of severe bloody diarrhea and hemolytic uremic syndrome in the United States were linked to the same fast food restaurant chain (Griffin et al., 2002). The bacterium Escherichia coli was isolated for the stools of ill persons. The specific serotype, O157:H7, had not previously been recognized to cause human illness. Soon thereafter, multiple outbreaks of similar severe illness in the U.S. and Canada were associated with this newly discovered pathogen, E. coli O157:H7. In these outbreaks, the vehicle was determined to be foods of bovine origin, mainly ground beef. Subsequently, national surveillance for E. coli O157:H7 was established, which has been greatly enhanced with the advent of laboratory methods to characterize the pathogen, such as Shiga toxin typing and genotyping. Outbreaks recognized and investigated as a result of national surveillance have led to a much greater understanding of the ecology of E. coli O157:H7 and its modes of transmission. By the late 1990s, multiple foods and water sources were recognized as potential vehicles of transmission, including raw milk, sprouts, juices, lettuce, and contaminated recreational and well water (Griffin et al., 2002). Direct animal to human transmission has also been recognized as an important mode of transmission, responsible for multiple outbreaks at agricultural fairs and petting zoos. Figure 2.2 depicts the evolving understanding of the modes of E. coli O157:H7 transmission. As a result, food and water safety and animal exposure guidelines and regulations have been advanced to control and prevent this important infection.

2.2

Detecting new and emerging pathogens

The identification of new foodborne pathogens may occur outside the purview of public health. This remains a critical aspect of foodborne illness research,

26

Emerging foodborne pathogens

Meat Cow

Cow

Human

Human

Milk (a) Sheep Caribou, other ungulates Cow

Cow

Meat Contact Milk

Water

Human

Human

Water Manure Fruits and vegetables Deer (b)

Fig. 2.2 Transmission of E. coli O157:H7 (a) 1988 model, (b) 1998 model. Through surveillance and outbreak investigation, the modes of E. coli O157:H7 transmission were discovered. E. coli O157:H7 can be transmitted through multiple food vehicles, from contaminated water, and from direct contact with cattle and other ungulates.

as the vast majority of foodborne illnesses remain unexplained (Mead et al., 1999). Microbiological research into the etiology of previously unexplained human foodborne illness is accomplished by the intensive investigation of individual cases of illness in research settings. Putative pathogens may be first identified in veterinary or plant pathological studies. Identification of new pathogens relies on specialized testing often unavailable in clinical laboratories. The history of the identification and characterization of campylobacteriosis is a prime example. Bacteria of the genus eventually called Campylobacter were first identified in 1909 (Holmberg and Feldman, 1984), but garnered little attention outside the veterinary literature. It was not until 1942 that extraintestinal Campylobacter isolates were recovered from humans. Laboratory methodologies advanced to facilitate the isolation of Campylobacter from faeces, first using filtration techniques in 1972, and finally antibiotic-containing selective media in 1977 (Butzler et al., 1973; Skirrow, 1977). After a span of six decades from its first identification, Campylobacter could be routinely isolated from human faeces in the clinical laboratory. This set the stage for the implementation of public health surveillance and the recognition of campylobacteriosis as the most frequent identified human foodborne bacterial pathogen. With the advent of a greater repertoire of diagnostic methodologies, especially molecular diagnostics, the time from first identification to routine

Surveillance for emerging pathogens in the United States

27

isolation has been much reduced. Over the past decade, diagnosis of foodborne illness due to norovirus has advanced from a purely research endeavour using electron microscopy to the routine application of reverse transcriptase polymerase chain reaction in public health laboratories across the United States. When new or emerging pathogens are discovered, specialized laboratories and investigations may be required to determine the associated clinical manifestations and the potential burden in select populations. These studies aim to collect detailed clinical information, and potential exposures, whether they be foods consumed or other routes of transmission. The inclusion of well persons as controls may allow the statistical association with illness in order to determine the true pathogenic nature of the potential pathogen. These special studies may also be used to evaluate the best detection methods for the new or emerging pathogen. If the burden is determined to be significant, and the methods of detection are transferable to clinical or public health laboratories, then general surveillance may be instituted. The recognition of new and emerging pathogens is not limited to the discovery, characterization and diagnosis of new genera and species of pathogens. Existing methods of characterization may be used to identify emerging strains within pathogen groups. This is especially true for the recognition of antibiotic resistant strains. A multidrug-resistant strain of Salmonella enterica, serotype Newport has recently been identified as an emerging foodborne threat in the United States, for example (see below). In any case, the routine application of methodologies to isolate and identify foodborne pathogens (or their variants) must be implemented in order for public health surveillance to take place in an effective manner. Public health surveillance may then determine the general burden of illness, recognize the emergence or re-emergence of disease, characterize risk factors for illness, and monitor prevention and control policies and practices. One surveillance method may be more appropriate than another, depending on the purpose. Surveillance conducted primarily to detect outbreaks and protect the public should cover the whole population, and should include conditions most likely to appear in outbreak form, for which an agreement has been reached that cases will be routinely reported to public health authorities. While syndromic surveillance for cases of acute dehydrating diarrhoeal illness may provide warning of the advent of a cholera epidemic in the developing world, or of a sudden large local outbreak in the industrialized world, this approach is not specific enough for most surveillance needs, which depend on the report of specific infections. In general, the methods of surveillance are becoming more specific, and more uniform, and are increasingly likely to combine both epidemiological data about the ill person and microbiological information about the infecting organism. Surveillance can be characterized as passive or active, reflecting the level of activity at the public health department. Passive surveillance depends on the clinics and laboratories remembering to report case of specific illness

28

Emerging foodborne pathogens

with more or less reminding. Active surveillance depends on the health department contacting clinics and laboratories, gathering reports directly; this is more expensive and intrusive. The concept of automated surveillance, depending on clinical and laboratory computers to report conditions as soon as they are entered, remains attractive but elusive. When local case surveillance is linked together into regional or national networks, it becomes possible to detect dispersed outbreaks, which would otherwise be missed. Because the modern food supply is itself dispersed, so that persons across a broad geographic area may be exposed to foods from the same source, this geographic linkage is critical. To detect dispersed outbreaks, it can be critical to compare specific markers of the infecting organisms, such as genetic ‘fingerprints’, across many jurisdictions (Swaminathan et al., 2001). Such comparison of subtypes may reveal an unusual clustering of infections with a single strain of a pathogen, which can then be further investigated. Public health laboratories have used a variety of methods to subtype pathogens, and can be linked in regional and national networks to permit rapid comparison of results to provide warning of dispersed outbreaks. Foodborne outbreaks are clusters of the same illness that follow consumption of the same food. Outbreaks can also be the unit of surveillance, which may be an important source of information about the recurrent linkage between specific pathogens and specific foods, as well as drawing attention to the foods that are most frequently associated with illness. However, sporadic individual cases are typically far more common than are recognized outbreaks. Extrapolation from observations made in outbreaks to the entire burden of disease caused by a pathogen should be cautious. For surveillance that is conducted primarily to measure the public health burden of disease or to track long-term trends, collecting detailed data from a representative sample of sites around the country can provide useful information (Angulo and Group, 1997). This so-called ‘sentinel-site’ approach can provide data on important illnesses that are not well represented in national surveillance, because they are not reportable in many jurisdictions, or because they rarely cause outbreaks. Also, in the more controlled sentinel site approach, more active surveillance may be instituted. For the purpose of determining the food source of infections, surveillance based on outbreak investigations provides answers for those illnesses that frequently appear in outbreak form. A sentinel site system can provide a platform for more detailed research efforts to better understand the burden and sources of sporadic infections. Beyond the reports of human disease, systematic collection of data about the prevalence of pathogens in foods or food animals, and about the prevalence of practices and behaviours in the food producers and in the general population may be useful to guide and assess prevention measures. To construct a detailed attribution of the burden of a specific illness such as salmonellosis to specific animal or food sources, systematic monitoring of the pathogen in food and

Surveillance for emerging pathogens in the United States

29

animal reservoirs, with molecular subtyping and comparison of strains with human infections, can be very helpful.

2.3 Range of methods used for surveillance in the United States 2.3.1 Surveillance for nationally notifiable diseases In the United States reports of notifiable diseases have been collected for more than a century, using an ever-expanding list of illnesses. Since 1961, these reports have been voluntarily submitted to the Centers for Disease Control and Prevention, which publishes them weekly and in annual summaries (Thacker, 1994). At an annual meeting, the Council of State and Territorial Epidemiologists decides on which specific illnesses should be nationally notifiable, and agrees on specific case definitions. This general umbrella of reporting covers all parts of the United States, provides information useful to local, state and national authorities, and is relatively inexpensive. Most disease reporting is passive from the standpoint of the public health system, which means that clinicians and laboratories are expected to report cases, but public health agencies do not directly monitor diagnoses. States and local jurisdictions typically institute public health reporting laws for some pathogens to increase the completeness of reporting. Basic case surveillance has been further enhanced for some infections by further characterization of the infecting pathogen in public health laboratories. This began with Salmonella. Following large multistate outbreaks of salmonellosis early in the 1960s, state and large city health department laboratories began to routinely serotype strains of Salmonella isolated from humans; the results of this subtyping were shared with CDC as well, in order to detect outbreaks affecting more than one state (CDC, 1964). Since 1962, national Salmonella surveillance has depended on this serotypebased reporting (Olsen et al., 2001). These data are critical to the detection and investigation of many outbreaks of salmonellosis each year. Since the 1980s, these data have been relayed electronically from states to CDC via the Public Health Laboratory Information System (Bean et al., 1992). Since 1995, these data have been routinely examined using an automated statistical outbreak detection algorithm that compares current reports with the preceding five-year mean number of cases for the same geographic area and week of the year to look for unusual clusters of infection (Hutwagner et al., 1997). The usefulness of this outbreak detection algorithm is limited by timeliness of reporting and high background rates of reporting for common serotypes, such as Salmonella serotypes Typhimurium and Enteritidis. The greatest sensitivity for Salmonella serotyping to detect meaningful clusters is for the rare serotypes; further differentiation is needed for the most common serotypes. The utility of serotyping as an international language for Salmonella subtypes has led to its widespread adoption: in a recent survey, 61 countries reported

30

Emerging foodborne pathogens

that they used Salmonella serotyping for public health surveillance (Herikstad et al., 2002a). A collaborative WHO program called Global SalmSurv promotes the use of Salmonella serotyping internationally, among countries that wish to upgrade their national capacity for foodborne disease surveillance (WHO, 2001). Molecular subtyping is now expanding the power of surveillance to detect outbreaks in the background of sporadic cases and improving the ability to investigate them by distinguishing the molecular ‘fingerprint’, or genotype, of an outbreak strain. These new techniques can define subtypes within a single pathogen and serotype, and provide useful strain differentiation for a growing number of pathogens (Swaminathan et al., 2001). In the United States, state public health laboratories began using a standardized genotyping method, called pulsed-field gel electrophoresis (PFGE), for E. coli O157:H7, after it proved useful in the 1993 West Coast outbreak (Bell et al., 1994), and have now expanded the use of this technique to common serotypes of Salmonella such as Typhimurium and Enteritidis, and Listeria monocytogenes (Swaminathan et al., 2001). Developing this capacity at the state level also enhances rapid detection of multi-county clusters within the state (Bender et al., 1997, 2001). Standardized subtyping protocols have now been developed for seven pathogens, and next-generation, gene-based technologies are under development for the future. PulseNet is the national network formed by linking all state public health laboratories performing PFGE via Internet to the national database of PFGE subtypes maintained by CDC. If clinical isolates are referred to the public health laboratories and rapidly typed, PulseNet can rapidly identify multistate clusters of infections due to specific strains of typed pathogens. Once a cluster of infections is identified, rapid epidemiological investigation can determine whether the cluster is a true outbreak with a common source. Laboratories at the Food and Drug Administration (FDA) and at the U.S. Department of Agriculture (USDA) also participate, so isolates from foods and animals can also be compared in the system. Canada has adopted a compatible system, Coordinated by the National Microbiology Laboratory in Winnipeg. The European network for laboratory based surveillance of foodborne infections, EnterNet, is adopting a compatible system, and discussions are rapidly advancing for PulseNet Asia Pacific, and PulseNet Latin America. As with Salmonella serotyping itself, the global use of standard genotyping will facilitate the detection of multi-continental clusters. Monitoring levels of antimicrobial resistance in foodborne pathogens is another form of subtype-based surveillance. Since 1996 in the United States, the National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS-EB), a collaborative effort of the CDC, USDA and FDA has been monitoring the prevalence of antibiotic resistance in Salmonella, Campylobacter and other foodborne bacterial pathogens isolated from humans, animals and foods (Marano et al., 2000). This provides information about the trends in resistance to specific drugs, identifies the emergence of new resistance threats, and permits the comparison of strains identified in the different locations.

Surveillance for emerging pathogens in the United States

31

Notifiable disease surveillance, amplified by pathogen subtyping, is a powerful and useful tool for public health, but it depends critically on the clinical laboratory infrastructure, and on the capacity and authority of public health institutions. Obviously, if a condition is not diagnosed by clinicians or microbiologists, it will not be possible to conduct surveillance on it by this means. There must be agreement and resources to inform clinicians of reporting requirements, for laboratories to conduct subtyping, and for public authorities to link reports from clinicians with the additional information from the laboratory. One jurisdiction may vary substantially from another in resources, requirements, and interest, making differences in reported incidence difficult to interpret. Limited case information may need to be supplemented by follow-up interviews once a cluster is detected, which can delay identification and control of the food vehicle. As with virtually all surveillance, notifiable disease surveillance captures only a fraction of the actual cases that occur, because many illnesses may not result in a visit to a clinic, a microbiological diagnosis, or a report to a public health authority. 2.3.2 Sentinel site surveillance Separate from the national umbrella of routine notifiable disease surveillance, a sentinel-site surveillance system provides more detailed information about specific illnesses in a representative sample of jurisdictions. With additional resources, that surveillance can be active, and can gather more information about sporadic cases than is usually possible. In the United States this strategy was first developed for monitoring cases of hepatitis, providing detailed laboratory and epidemiological data on cases (Bell et al., 1998). In 1996, the Foodborne Disease Active Surveillance Network (FoodNet), a collaborative program of the CDC, ten sites, the USDA and the FDA, began under the aegis of CDC’s Emerging Infections Program (Angulo and Swerdlow, 1999). FoodNet conducts active case finding for a panel of foodborne infections, as well as epidemiological studies to better understand the trends and sources of foodborne diseases in the United States. FoodNet began with an initial five sites in 1996, and expanded to ten sites by 2004. The surveillance area that year covered 36 million persons, or approximately 13% of the U.S. population (CDC, 2002c). Because case ascertainment is active, reporting is more uniform and complete, and data are better than in passive reporting systems. However, it is also more expensive and limited in geographic scope. FoodNet surveys laboratory, physician, and patient practices that cause an individual case to be diagnosed so that trends in the reported incidence can be interpreted in the light of possible changes in diagnostic practices. In addition, FoodNet has been a platform for conducting case-control studies of sporadic infections to identify general risk factors for infection that distinguish the persons who become ill from those who stay healthy. Similar active surveillance programs have been developed in other countries, including OzFoodNet in Australia, and the Food Safety Authority of Ireland.

32

Emerging foodborne pathogens

2.3.3 Foodborne outbreak reporting A foodborne outbreak is a cluster of two or more similar infections that are shown by investigation to result from ingestion of the same food (Olsen et al., 2000). Most foodborne outbreak investigations are conducted by local and state or provincial health departments. Since 1967, CDC has collected reports of outbreaks of foodborne illnesses investigated by local, state, and national public health authorities (Olsen et al., 2000). Many countries find it useful to conduct similar surveillance. Reports of outbreaks include the nature of the pathogen or toxin, the type of food that caused the outbreak, and limited information about factors that contributed to the outbreak. In the United States, before 1998, these reports were collected on paper and slowly reviewed and compiled. The foodborne outbreak surveillance system has been recently overhauled with an improved form, active solicitation of reports from states, and web-based reporting (CDC, 2002a). The foodborne outbreak surveillance system has provided useful information on long-term trends in many pathogens for which surveillance otherwise does not exist and summaries of the outbreaks caused by a particular pathogen, hazard, or food (Bean and Griffin, 1990). In the future, as the speed of reporting and analysis increases, it may provide more systematic detection of clusters of unusual outbreaks, based both on laboratory testing and epidemiological assessment of the outbreak presentation (Hall et al., 2001). Because outbreak investigations often take some time to reach the final conclusions about sources and contributing factors, the need for speed in reporting must be balanced with the value of complete information. Systematic analysis of reported outbreaks can be used to allocate the burden of many infections and other hazards across broad food categories. Surveillance based on outbreaks has the virtue of covering many pathogens for which there is no individual surveillance, and can even provide helpful information about illnesses for which no pathogen is identified (Hall et al., 2001). However, it will not provide information about pathogens that rarely or never cause recognized outbreaks, such as Campylobacter jejuni or Vibrio vulnificus. 2.3.4 Limitations of surveillance Surveillance of any sort has limitations. One is underreporting. Many cases and even many outbreaks go unrecognized. Individual cases may not be detected because people who are ill do not seek medical care due to the selflimited nature of many foodborne diseases, physicians and laboratories may not make a specific diagnosis, cases may not be reported to authorities, and authorities with stretched resources may not investigate or report them. Thus, the actual number of cases that occur is likely to be substantially greater than the number of cases that are reported. For example, it has been estimated that 38 cases of salmonellosis occur for every one that is reported (Voetsch et al., 2004). A common source outbreak in a restaurant may not be recognized because patrons were exposed in small groups that were unknown to each

Surveillance for emerging pathogens in the United States

33

other. For some foodborne infections, the incubation period may have been long enough to obscure the relationship with the meal, unless persons attending a large gathering, such as a banquet or wedding reception, have some reason to compare their experiences afterwards. As long as it is recognized that underreporting occurs, this limitation does not diminish the utility of the surveillance. A limited amount of systematically collected and well characterized data is far more useful than a larger volume of data from many non-comparable sources. A second limitation is the difficulty in attributing a specific case to a specific source. E. coli O157:H7, Shigella ssp., Salmonella and many other pathogens can be transmitted by a variety of different food and non-food exposures. It is often difficult to determine in the individual case of illness, which of numerous possible exposures in the days preceding illness was the actual source. In the outbreak setting, where careful comparison of food consumption patterns of ill persons with those who remained well can often identify the immediate food vehicle, it is still sometimes difficult to determine among the various inputs which was the potential source: was it the ham, turkey or lettuce on the club sandwiches; did raw meat drip on other food items in the cooler; or did an infected food handler contaminate items on the salad bar? However, many outbreak investigations yield clear answers, and comparison of patterns observed among groups of outbreaks can help define patterns. Finally, case-based surveillance (that is surveillance based on the reports of diagnosed cases of an illness) can count only that which is measurable and known. Because laboratory diagnosis of norovirus infections is not routinely performed in clinical laboratories, this extremely common illness cannot be monitored with the case-based surveillance used for infections caused by Salmonella or Campylobacter. The importance of norovirus infections can be defined in outbreaks where the typical combination of signs, symptoms, incubation period, and duration of illness can be documented and when specimens reach public health laboratories that can make the specific diagnosis. Similarly, enterotoxigenic E. coli (ETEC), the cause of much travellers’ diarrhea, is increasingly recognized as a cause of outbreaks in the United States, and may also be a common cause of sporadic cases, yet the specialized tests to detect it are rarely applied (Dalton et al., 1999). In a recent survey in Minnesota, ETEC was identified in 1.5% of diarrheal stools, more frequently than Salmonella or E. coli O157:H7 (Gahr et al., 2001). It is likely that there are many foodborne agents yet to be discovered (Tauxe, 1997). 2.3.5 Behavioral surveillance and surveys of foods Surveillance efforts also provide systematic data on behavior and exposure of the population to specific risks. Studies conducted through CDC’s Behavioral Risk Factor Surveillance System (BRFSS) documented the high frequency of risky food behavior (Yang et al., 1998). More recently, the FoodNet

34

Emerging foodborne pathogens

population surveys have provided population-based data on the incidence of diarrhoeal illness and the likelihood that someone would seek medical care for a diarrhoeal illness; these surveys were critical to develop a general estimate of the burden of foodborne disease (Herikstad et al., 2002b; Mead et al., 1999). They also provide general population based data on the frequency of exposure to a wide variety of foods and other potential sources of intestinal infection (CDC, 1998). Such surveys depend on what persons can and will report about themselves, and thus may overestimate such desirable behaviors as hand washing and underestimate known risk behaviors such as eating undercooked ground beef. Another potential source of information is the complaint systems maintained by local and state health departments for persons to report illnesses or hazardous conditions they believe may be related to food (Samuel et al., 2001). While such systems are far less specific than systems built on diagnosed cases of illness, they may provide early warning of problems. Public health and regulatory agencies, as well as members of food production industry, have increased the frequency and completeness of microbiological monitoring of foods as food safety programs have become enhanced and strengthened. Microbiological monitoring programs for foods are not intended to test the safety of any particular product, but rather they assess the efficacy of the entire food safety control programs at the farm, production plant, or even national level, as is the case with microbiological testing of imported foods. The pathogens identified in these food surveys can be useful markers for what may be transmitted in the food supply. Some countries (e.g. Denmark) have been able to systematically subtype isolates identified from food surveys at multiple points along the farm to table continuum, and compare these subtypes with pathogens isolated from people, enabling an attribution of a pathogen causing human illness to specific foods. Trends in the emergence of pathogens or pathogen subtypes can be monitored, starting in foods at certain points in production, through illness in humans. In this way, infections in the United States with strains of Campylobacter resistant to the fluoroquinolone class of antibiotics have been shown to be associated with poultry consumption (Kassenborg et al., 2004). Information about pathogen isolates from food and humans exists among several agencies responsible for food safety in the United States, and subtyping and comparison of isolates is now becoming more routine. 2.3.6 Standardizing the methods of surveillance and monitoring A necessary attribute of any successful surveillance system is standardization of information. Comparing information emanating from different tests conducted in different circumstances for different reasons can become so riddled with limitations as to be futile. For example, this is a challenge in the monitoring of antibiotic resistant pathogens. Clinical laboratory data may be biased because susceptibility testing is often done because of some suspicion

Surveillance for emerging pathogens in the United States

35

that a resistant organism is present. The number of different types of susceptibility tests available also makes the data difficult to combine and compare. For these reasons, the systematic collection and testing of isolates has been established in the U.S. called the National Antimicrobial Resistance Monitoring System, Enteric Bacteria (NARMS). In this system, a systematic collection of E. coli O157:H7, Shigella, Salmonella and Campylobacter isolates from across the United States is sent to the CDC for testing to the same panel of 17 antimicrobials in the same laboratory using highly standardized tests. In this way, results are both representative and comparable.

2.4

Use of surveillance data

2.4.1 Assessing the burden of disease Routine public health surveillance records only a fraction of the total number of cases of foodborne diseases which occur in the United States. If that fraction is stable, and the factors that contribute to a case being included are unchanged, then the analyses of associated trends are relevant. However, if a particular pathogen or subtype exhibits a change in pathogenicity or the microbiologic tests used to identify it change, then the representation of that disease by surveillance may be compromised. The emergence of E. coli O157:H7 and other Shiga toxin-producing E. coli in the United States is a prime example of the complexities of surveillance in a changing situation. Trends in laboratory-based surveillance for E. coli O157:H7 showed a marked and rapid increase in cases in the early 1990s, but this increase in cases followed closely the increase in the number of states making this a reportable illness, and the number of laboratories performing stool culture using SorbitolMacConkey agar to identify it (Fig. 2.3). In 2000, surveillance for illness due to Shiga toxin-producing E. coli expanded from just one serotype, O157:H7, to all associated serotypes, though a growing body of evidence indicates that some serotypes of Shiga toxin-producing E. coli are less virulent than others. In addition, methods to test for the Shiga toxin directly were more widely used. For these reasons, the surveillance trends for Shiga toxin-producing E. coli are difficult to interpret. It is thus important to characterize and quantify the various factors involved in the report of an illness, which may be described as a series of events. For any specific illness, a large number of people may be afflicted. Some persons will seek medical care, while others will not, often depending on the severity of illness. Upon visiting a medical care facility, diagnosis and treatment may be empirical, or specimens may be taken for microbiological testing. Within the laboratory, some tests may be applied to the specimen routinely, others not. Even when a reportable pathogen is identified in the clinical laboratory, not all isolates are reported to public health authorities. One can construct a ‘surveillance pyramid’ (Fig. 2.4) of successively smaller layers with the reported illnesses represented by the small tip over a much larger foundation

Emerging foodborne pathogens 50 45

550 States

Isolates

500 450

States reporting

40

400

35

350

30

300 25 250 20

200

15

150

10

100

5

50

0

1993

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1995

1996 Year

1997

1998

1999

Isolates reported

36

0

Fig. 2.3 E. coli O157 isolates reported to CDC, 1993–99. National reporting for E. coli O157:H7 started in 1992 in the United States. The number of isolates reported increased steadily over the ensuing years, commensurate with the increase in the number of states submitting reports.

Reported to health dept./CDC Culture-confirmed case Lab tests for organism Specimen obtained Person seeks care Person becomes ill Population exposures

Fig. 2.4 Surveillance pyramid. The number of illnesses reported to public health surveillance systems is a small fraction of the total number of illnesses. Of all persons exposed and ill, only a portion will seek care, and still fewer will have a specimen obtained and tested and finally reported to public health surveillance.

of all illnesses. Public health agencies in several counties have conducted special short-term surveys of their population to determine the number of gastrointestinal illnesses, of physicians to determine the proportion of visits whereby patient samples are obtained for microbiological testing, of laboratories to determine the methods applied to samples to identify various pathogens, and of reporting sources to identify the number of unreported cases. By quantifying each step in the diagnosis and reporting path, an overall multiplier may be determined, as exemplified by the estimate of 38 unrecognized

Surveillance for emerging pathogens in the United States

37

salmonellosis cases for every case reported in the United States (Voetsch et al., 2004). Information from surveillance has recently been integrated into a general estimate of the overall burden of foodborne disease in the United States (Mead et al., 1999). This included the number of cases, hospitalizations, and deaths that were attributed to specific pathogens and to the large number of illnesses that remain unaccounted for. These pathogen-based point estimates provide a benchmark for assessing the economic impact of foodborne diseases, such as the estimate of $6.9 billion dollars for the major bacterial pathogens (Buzby and Roberts, 1996). Some foodborne infections can also cause chronic complications in a small percentage of cases, such as kidney failure related to E. coli O157:H7, reported to occur in 4% to 8% of cases (Griffin et al., 2002), and Guillain Barre Syndrome paralysis, which may complicate 1 in 1000 Campylobacter infections (Nachamkin et al., 2000). Death rates in the months following some infections may be higher than among uninfected but otherwise similar persons (Helms et al., 2002). The full impact of illnesses includes both acute morbidity and mortality, as well as the impact of subsequent complications and of long-term effects such as lifelong impairments from congenital toxoplasmosis or early childhood diarrheal illnesses in impoverished areas (Guerrant et al., 2002). With more information about the frequency, duration, and disability caused by these complications, the burden of foodborne illness could be re-estimated to include Disability Adjusted Life Years (DALYs), a measure used to characterize the burden of many other public health problems (Murray and Lopez, 1997). Surveillance data can subdivide the burden of a specific infection, such as salmonellosis. The contribution of specific Salmonella serotypes to this burden can be derived from their frequency. For example, the three most common serotypes of Salmonella, Typhimurium, Enteritidis, and Newport, together account for over half of all reported cases of salmonellosis in 2002 (Table 2.1).

Table 2.1 Rank of Salmonella serotypes, United States, 2002. Top ten Salmonella serotypes isolated from humans, number of percent of total for 2002 Rank

Serotype

Number

Percent

1 2 3 4 5 6 7 8 9 10

Typhimurium Enteritidis Newport Heidelberg Javiana Montevideo Muenchen Oranienburg Saintpaul Infantis

7062 5116 4204 1957 1188 717 591 585 535 463

21.9 15.8 13.0 6.1 3.7 2.2 1.8 1.8 1.7 1.4

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Emerging foodborne pathogens

The burden of reported foodborne outbreaks can also be measured. National foodborne outbreak reporting in 1998 through 2000 gave a combined annual incidence of 4.8 outbreaks per 1,000,000 population (CDC). Considering just outbreaks affecting at least ten persons, the rate in the FoodNet coverage area was 3.3 outbreaks per million population per year in 2000 (CDC, 2001a). It is likely that outbreak surveillance undercounts the true frequency of events for the reasons noted above. 2.4.2 Associating human diseases with specific reservoirs For many foodborne pathogens, the association with a specific reservoir, either in a food animal or a human, means that the illnesses they cause are often associated with a characteristic food or group of foods. The data that link a pathogen to a specific food and reservoir often come from outbreak investigations. For many pathogens, a series of investigated outbreaks is the best information available to define the association of the illness with specific foods. For example, the first investigation of E. coli O157:H7 infections identified the pathogen and linked the distinctive illness it caused to eating undercooked hamburgers (Riley et al., 1983). Trace back from an outbreak caused by ground beef, and from sporadic cases caused by drinking raw milk, led to identification of the bovine reservoir for E. coli O157:H7, noteworthy because infected cows are asymptomatic (Wells et al., 1991; Martin et al., 1986). More recently, outbreaks of this infection have been associated with an expanding array of foods such as lettuce, sprouts, and unpasteurized apple cider (Griffin et al., 2002). Early investigations of Campylobacter outbreaks identified raw milk, undercooked poultry, and contaminated water as common sources (Blaser et al., 1979; Vogt et al., 1982; Deming et al., 1987). Pathogens having human reservoirs can also be linked to specific foods, depending on the most characteristic mechanisms of contamination. In 1924, a large epidemic of typhoid fever was linked to raw oysters that were harvested and held near sewage sources (Lumsden et al., 1925). More recently, outbreaks of norovirus infection, which has a human reservoir, have been linked to shellfish (and to direct contamination from ill fishermen), and to foods such as cold salads and sandwiches that are handled extensively in the kitchen, (and to direct contamination from ill food handlers) (Kohn et al., 1995; Parashar and Monroe, 2001). For pathogens that rarely cause outbreaks, studies of sporadic cases and comparison with healthy controls can define associations with particular foods. For example, Vibrio vulnificus was definitively associated with consumption of raw oysters soon after it was first described (Blake et al., 1979). Studies of E. coli O157:H7 infections linked sporadic cases of infection with this pathogen to eating undercooked ground beef, thus supplementing the data from outbreaks (Slutsker et al., 1998; Kassenborg et al., 1998; Mead et al., 1997). Studies of sporadic Campylobacter infection link it to eating poultry and other meats outside the home, as well as to drinking untreated water and to other sources; around the

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world, poultry remains the dominant reservoir for this pathogen (Friedman et al., 2000; Anon., 2000). Allocating the burden of infections across specific food groups is a complex challenge, which has been approached in several ways. The first depends on epidemiological and public health investigations. Outbreak data on association with foods, supplemented with data from sporadic cases, provides the most readily available public health information for allocating the burden of specific infections across food groups. For example, between 1992 and 1997, 1,153 foodborne outbreaks involving 46,453 illnesses were reported in the United States, for which a food vehicle was determined (Olsen et al., 2000) (CDC, unpublished data). For the 714 outbreaks in which the implicated food could be assigned to a single group, 28% of the illnesses were associated with produce, 21% with meat, 15% with seafood, 11% with poultry and 26% with other foods. This indicates that food safety concerns exist for all major food groups. For those illnesses that rarely appear in outbreak form, data from individual case series or from case-control studies can be used to allocate the burden. Other ways of allocating the burden depend on using systematic sampling data from many foods. For example, the patterns of molecular subtypes in strains of Salmonella isolated from people can be compared to those isolated from a variety of different foods, and those patterns which are unique to one food can be allocated to that food. To be successful, this depends on extensive and systematic sampling of many foods, and use of standardized subtyping methods on a large number of strains. It has been done routinely in Denmark to track the burden of salmonellosis associated with different foods (Anon., 2000) (see below). Finally, if data on pathogen prevalence are available for a large number of foods, a risk allocation can be constructed using the methods of risk analysis, which has been attempted for Listeria monocytogenes (FDA 2003). This approach depends on the assumption that all strains are equally likely to cause disease, and that the distribution of the pathogen in foods can be reliably estimated from studies using a broad range of methods, and conducted over a substantial time span. These assumptions are limitations that significantly impact attribution modeling. Once a food is implicated as a common source of a pathogen, detailed review of its production process may reveal the likely points in the process where the food became contaminated. In an outbreak investigation, this most often happens by tracing back along the production process from the implicated food the ill persons ate. Such a review may identify where the contamination was likely to have originated and where it may have been further amplified or controlled. This information, of particular interest to risk assessors, is only gathered in a minority of foodborne outbreak investigations, and requires a multi-disciplinary approach. Epidemiological investigations of cases can also provide important insight into the precise mechanisms of exposure and the variations in human behaviour that contribute to it. For example, in an outbreak of salmonellosis in Wisconsin

40

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in 1998, illness was associated with eating raw ground beef, a common practice among some ethnic groups (CDC, 1995). In another outbreak, illness was particularly associated with tasting raw ground beef in the process of seasoning and cooking it (Fontaine et al., 1978). In an investigation of Campylobacter infections in Colorado, illness was associated particularly with handling and preparing chicken, not with eating it (Hopkins and Scott, 1983). In an assessment of sporadic ground beef-associated E. coli O157:H7 infections in New Jersey, ill persons were no less likely to have noticed the new meat handling recommendations on the meat wrapper than were those who were well, but were less likely to have washed their hands after handling raw beef (Mead et al., 1997). 2.4.3 Developing control strategies and measuring their effectiveness Preventing foodborne disease is complex, requiring attention and intervention from farm or fishery to table (Anon., 1998). There are no vaccines for the pathogens most commonly transmitted through foods, and while education of the consumer provides an important final safety barrier, it is not by itself sufficient. Making food safer before it reaches the final consumer is critical to maintain confidence in the food supply. The consumer eats many foods without cooking them at all, prepares raw foods of animal origin with the same hands as the uncooked salads, is instructed by tradition and by cookery texts to prepare many meat, poultry, egg and seafood dishes with more concern about overcooking than undercooking, and is told routinely to season them ‘to taste’ during the preparation process before cooking is complete. When new foodborne hazards are identified, the knowledge base for defining such preventions may be quite limited (Holmberg and Feldman, 1984). Public health surveillance, with detailed investigations of outbreaks, can identify new and emerging hazards, define the likely points of control, identify questions in need of further research, and track the effectiveness of control measures. For some hazards, the control measures are obvious and immediate, and do not require extensive risk assessment or other deliberations. For example, requiring toilets with holding tanks on oyster boats makes it less likely that oyster gatherers will contaminate the oyster beds (Kohn et al., 1995). Similarly, restaurant designers who wish to reduce the risk of making their customers ill can install a foot-operated hand washing station near the salad preparation area. For other hazards, the relative merits of potential strategies are not obvious at the outset, and control proceeds by an iterative process. As more is learned, refinements in prevention strategies are progressively refined. Five examples will illustrate how this process can lead to improved prevention. Salmonella and pre-cooked roast beef From 1975 to 1977, Salmonella surveillance detected repeated outbreaks of Salmonella infection associated with pre-cooked deli roast beef (Parham,

Surveillance for emerging pathogens in the United States

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1984). Evaluation of cooking temperatures revealed that they were sometimes insufficient to kill Salmonella, and an improved approach using specific temperature requirements was applied as an emergency regulation in 1977. In 1981, outbreaks of salmonellosis were again traced to pre-cooked roast beef prepared under these new regulations, showing that these measures were insufficient (CDC, 1981). Re-evaluation clarified that the humidity inside the oven was as critical as the time and temperature of cooking (Parham, 1984). After further regulations were promulgated, outbreaks traced to precooked roast beef have become extremely rare. E. coli O157:H7 and apple cider In 1992, investigation of an outbreak of E. coli O157:H7 infections in Massachusetts linked this pathogen for the first time to apple cider (Besser et al., 1993). This traditional beverage was often pressed from fallen apples, with minimal cleaning, but was long believed to be sufficiently acidic to be safe. Investigators thought that the apples were probably contaminated before they were pressed, possibly in the orchard, which was visited by deer. The first control measures adopted by the industry were simply to wash and brush the apples before pressing them. However, assessment of the survival of the organism in apple cider revealed that E. coli O157:H7 was acid tolerant and could easily survive in cider more acidic than the defined limit of safety of pH 4.5 (Zhao et al., 1993). Recurrent outbreaks of E. coli O157:H7 and Cryptosporidia infections traced to cider mills where apples had been brushed and washed showed that even with cleansing of the apples, cider could be hazardous (Millard et al., 1994; CDC, 1997; Cody et al., 1999). It was also shown that E. coli O157:H7 could, under some circumstances, be internalized into apples, and thus be protected from washing, brushing, or external disinfection (Buchanan et al., 1999; Burnett et al., 2000). Continued outbreaks and research led to the promulgation of juice regulations requiring a pathogen reduction step such as pasteurization (FDA, 2001). To date, no further cider associated outbreaks have occurred in apple cider produced in accordance with this regulation. Salmonella Enteritidis and shell eggs In the 1980s, dramatic outbreaks of Salmonella serotype Enteritidis (SE) infections were traced to Grade A shell eggs (St. Louis et al., 1988). This was surprising, as the egg grading and disinfection process instituted in the 1960s as a result of egg-associated salmonellosis had appeared to be effective. The earlier problem was related to Salmonella on the outside of the shell. It was suggested that the new problem might reflect internal contamination of eggs, possibly as a result of infection of the hen’s reproductive tissues themselves. Sporadic cases of SE infections were also increasing, first in the Northeast, and later over most of the country (Fig. 2.5) (CDC, 1993). These were also shown to be related to eggs, and it was even possible to show a gradient of risk according to the degree of cooking, from hard boiled and hard cooked

Emerging foodborne pathogens 12 10 8 6 4

02

98

00

20

20

96

92

94

90

88

86

84

82

76

78

80

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0

74

2

70

Number /100,000 pop.

42

Year Total

New England

Mid Atlantic

Pacific

Other

Mountain

Fig. 2.5 Salmonella Enteritidis rates, United States, 1970–2002. The rate of reported Salmonella serotype Enteritidis (SE) per 100,000 population varied by region in the United States. The New England region was the first to experience increases in salmonellosis due to SE, followed by Mid Atlantic and then Pacific states. The epidemic has been linked to internally contaminated table eggs. Rates have fallen since 1996 in most regions with the implementation of control measures.

through over easy, to soft boiled and sunny side up (Passaro et al., 1996; Hedberg et al., 1993). Comparison of the strains found in the birds on farms implicated by trace back as the source of contaminated eggs demonstrated the same strains of Salmonella that were found in the affected humans, proving that the source of contamination was the birds themselves (Mishu et al., 1991; Altekruse et al., 1993). Feeding experimental birds SE showed that they did develop silent ovarian infection and then laid normal-looking eggs with contaminated contents (Gast, 1999). A pilot project to develop flock-based screening and control measures was begun, the Pennsylvania Egg Quality Assurance Program (Schlosser et al., 1999). This project was the model for other state egg quality assurance programs (EQAPs). The incidence of SE infections in the mid-Atlantic states, for which Pennsylvania was the main egg source, began decreasing, followed later by the incidence in other states (CDC, 2000). Microbiological screening of farms for SE is an integral part of the EQAPs, with voluntary diversion of the eggs to liquid egg pasteurization if they are found positive. Thus, many potentially tainted eggs are sent for safe processing before they enter the shell egg market. As the epidemic among egg-laying flocks spread from the Northeast to virtually the entire country, outbreak investigations and the attendant trace backs have defined the spread of this problem into new areas, stimulating them to develop their own EQAPs (CDC, 1993; Burr et al., 1999). The slow decline in incidence prompted further measures, such as the refrigeration requirement for eggs in 1999 and the commercialization of a new in-shell pasteurization process. A risk assessment was completed in 1998 (Baker et al., 1998). Current control policies of egg-associated SE appear to be having an impact. By 2002, the incidence of SE had decreased to less than two per 100,000,

Surveillance for emerging pathogens in the United States

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down from the peak of nearly four per 100,000 in 1995, but it remains above the pre-epidemic incidence of one per 100,000 (Fig. 2.5). Also in 2002, 29 outbreaks of SE infection were reported, a significant decrease from 54 outbreaks in 1995, but still above the Healthy People 2010 objective of 25. The surveillance data clearly show that progress is being made in slowing the Salmonella Enteritidis problems in eggs, but that further efforts are needed to completely control it. Alfalfa sprouts Like SE in eggs, this food safety challenge is not an emerging pathogen, but rather the emergence of well-known pathogens in a new food vehicle. In 1995, shortly after the statistical outbreak detection algorithm was developed for the Salmonella surveillance system, a large 22-state outbreak of infections caused by Salmonella serotype Stanley was detected in the United States (Mahon et al., 1997). This serotype is usually quite rare. Simultaneously, public health officials in Finland identified an outbreak caused by the same organism. Both outbreaks were linked to the consumption of alfalfa sprouts, sprouted from the same batch of seeds (Mahon et al., 1997). Research showed that the sprouting process could greatly amplify the number of Salmonella originally present in the seed, and that the pathogen can be inside the sprout, where it could not be affected by washing or disinfecting (Jaquette et al., 1996; Itoh et al., 1998). The next three years witnessed at least seven outbreaks caused by several serotypes of Salmonella and E. coli O157:H7 in sprouts in the United States, often from contaminated seeds (Taormina et al.). Japan experienced a devastating outbreak that affected 6,000 schoolchildren traced to radish sprouts (Michino et al., 1999; Watanabe et al., 1999). Alfalfa and other seeds for sprouting are produced as raw agricultural commodities and may be easily contaminated in the field or warehouse, where they may be held for years before being sprouted (Breuer et al., 2001). After researchers determined that disinfecting seeds with chlorine could reduce contamination and preserve the ability of seeds to germinate, the FDA promulgated guidelines on seed disinfection and the major seed distributors put these instructions on the package (FDA, 1999). Since then, outbreaks of salmonellosis have been linked to a sprouter that reported disinfecting the seeds following those guidelines (Proctor et al., 2000), as well as to a sprouter using less chlorine than recommended (Winthrop et al., 2002). One recent outbreak involved a single lot of clover seed shipped to two sprouters in Colorado (Brooks et al., 2001). The first did not disinfect the seed before sprouting, and caused 1.13 documented infections per 50 pound bag of seed sprouted, while the second did disinfect, and caused only 0.29 infections per bag of seed. This outbreak showed that the disinfection strategy works partially, but is by itself insufficient to completely protect the public. In addition to disinfection, FDA also recommended lot by lot testing of the irrigation water for Salmonella (FDA, 1999). One outbreak has occurred linked to sprouts that had passed such a test, suggesting that false negative tests may occur (Winthrop et al., 2002).

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Thus, continued surveillance and investigation indicate that the challenge of preventing outbreaks of salmonellosis from sprouts has been partially met, but also that a complete solution has still not been implemented. Multi-drug resistant Salmonella Newport and foods of bovine origin One of the latest food hazards to emerge in the United States is a new and highly resistant strain of Salmonella serotype Newport (Gupta et al., 2004). This strain was first identified through the National Antimicrobial Resistance Monitoring System surveillance in 1998, and its detection increased rapidly in 1999 and 2000. The strain is resistant to at least nine antibiotics because it possesses a large plasmid bearing several resistance genes, including an unusual gene, the AmpC cmy2 gene, which confers resistance to most cephalosporins. In 2001, a retrospective study of these strains in Massachusetts identified the same strains in ill and dying dairy cattle, and showed that visiting or working on dairy farms was a risk factor for illness (Zansky et al., 2002). Later in 2001, an outbreak in Connecticut was traced to traditional cheese made from insufficiently pasteurized milk from Massachusetts dairy farms (McCarthy et al., 2002). In 2002, an investigation of a multistate cluster of cases in the Northeast linked the illness to eating ground beef traced to meat from a single slaughter plant (Zansky et al., 2002). Surveillance of human infections indicates a sharp increase in Salmonella Newport infections, which in 2000 represented 9% of human salmonellosis (CDC, 2001b). Many of the Newport strains were multidrug resistant (CDC, 2002b). The same organism has been detected since 1998 among isolates from animals, including bovines (Fedorka-Cray et al., 2002). Among Salmonella Newport isolated from cattle in 2000, 74% had the AmpC multi-drug resistance profile (USDA-ARS, 2002). The evidence to date indicates that this strain has spread in epidemic fashion among cattle herds, that it affects the animals themselves, persons in contact with the animals, and consumers of bovine products, including meat and cheese. Once control measures are identified and implemented, their success can be measured by monitoring animals and meat for this strain, by trends in human illness and by outbreak surveillance. Surveillance activities in animals, meat and poultry can also provide early warning of the spread of this strain or its plasmid to other food animal populations.

2.5

Future trends

In the future, we should expect that new pathogens and new food vehicles will continue to be recognized. New diagnostic strategies will identify some pathogens that currently are often or completely missed. Globalization of the food supply and concentration of its production will create new challenges for detection, investigation, control and prevention. Enhanced public health surveillance for human illness will be vital to identify and investigate these

Surveillance for emerging pathogens in the United States

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new challenges. In addition, a flexible monitoring system that permits comparison of information from multiple points in the food supply is needed. Just as monitoring cattle at slaughter is an important strategy for documenting the continuing absence of bovine spongiform encephalopathy, so could a system for documenting the frequency of other foodborne hazards at point of slaughter or processing be critical to assessing and controlling other hazards in the future. Minimizing and preventing contamination early in the chain as well as identifying foods at higher risk of being contaminated so that they can be diverted out of the usual food chain to safer processing may become the norm. Increasingly, preventing foodborne disease will mean preventing contamination before food reaches the consumer. Farm management policy, as well as slaughter and processing plant policy, and kitchen practices are all key parts of food safety.

2.6

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MICHINO, H., ARAKI, K., MINAMI, S., TAKAYA, S., SAKAI, N., MIYAZALI, M., ONO, A.

and YANAGAWA, (1999) American Journal of Epidemiology, 150, 797–803. MILLARD, P., GENSHEIMER, K., ADDISS, D., SOSIN, D., BECKETT, G., HOUCK-JANKOWSKI, A. and HUDSON, A. (1994) Journal of the American Medical Association, 272, 1592–1596. MISHU, B., GRIFFIN, P. M., TAUXE, R. V., CAMERON, D. N., HUTCHESON, R. H. and SCHAFFNER, W. (1991) Annals of Internal Medicine, 115, 190–194. MURRAY, C. and LOPEZ, A. (1997) The global burden of disease: A comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in 1900 and projected to 2020, Harvard University Press, Cambridge, MA. NACHAMKIN, I., ALLOS, B. and HO, T. (2000) In Campylobacter, 2nd edition (eds, Nachamkin, I. and Blaser, M.) ASM Press, Washington, DC, pp. 155–175. OLSEN, S., MACKINNON, L., GOULDING, J., BEAN, N. and SLUTSKER, L. (2000) Morbidity and Mortality Weekly Report, Supplemement 01, 1–51. OLSEN, S., BISHOP, R., BRENNER, F., ROELS, T., BEAN, N., TAUXE, R. and SLUTSKER, L. (2001) Journal of Infectious Diseases, 183, 753–761. PARASHAR, U. and MONROE, S. (2001) Reviews in Medical Virology, 11, 243–252. PARHAM, G. (1984) In International Symposium on Salmonella (ed., Snoeyenbos, G.) American Association of Avian Pathologists. University of Pennsylvania. Kennett Square, PA, New Orleans, Louisiana, pp. 275–280. PASSARO, D., REPORTER, R., MASCOLA, L., KILMAN, L., MALCOLM, G., ROLKA, H., WERNER, S. and VUGIA, D. (1996) Western Journal of Medicine, 165, 126–130. POTTER, M., ARCHER, D., BENSON, A., BUSTA, F. and DICKSON, J. (2000) Emerging microbiological food safety issues; Implications for control in the 21st century, Institute of Food Technology, Chicago. PROCTOR, M., HAMACHER, M., TORTORELLO, M., ARCHER, J ., BARNETT, M., YING, M., SLUTSKER, L. and DAVIS, J. (2000) In 2nd International Conference on Emerging Infectious Diseases CDC, Atlanta, Atlanta, Georgia, pp. 98. RILEY, L. W., REMIS, R., HELGERSON, S., MCGEE, H. B., WELLS, J. G. DAVIS, B. R., HERBERT, R. J ., OLCOTT, E. S., JOHNSON, L. M., HARGRETT, N. T., BLAKE, P. A. and COHEN, M. L. (1983) New England Journal of Medicine, 308, 681–685. ROSENBERG, C. (1987) The Cholera Years: The United States in 1832, 1849, and 1866, University of Chicago Press, Chicago, Illinois. SAMUEL, M., PORTNOY, D., TAUXE, R., ANGULO, F. and VUGIA, D. (2001) Journal of Food Protection, 64, 1261–1264. SCHLOSSER, W., HENZLER, D., MASON, J., KRADEL, D., SHIPMAN, L., TROCK, S., HURD, S., HOGUE, A., SISCHO, W. and EBEL, E. (1999) In Salmonella enterica serovar Enteritidis in humans and animals: Epidemiology, pathogenesis and control (eds, Saeed, A., Gast, R., Potter, M. and Wall, P.) Iowa City University Press, Ames, Iowa, pp. 353–365. SKIRROW, M. (1977) British Medical Journal, 2, 9–11. SLUTSKER, L., RIES, A., MALONEY, K., WELLS, J., GREENE, K. and GRIFFIN, P. (1998) Journal of Infectious Diseases, 177, 962–966. ST. LOUIS, M. E., MORSE, D. L., POTTER, M. E., DEMELFI, T. M., GUZEWICH, J. J., TAUXE, R. V. and BLAKE, P. A. (1988) Journal of the American Medical Association, 259, 2103–2107. SWAMINATHAN, B., BARRETT, T., HUNTER, S., TAUXE, R. and FORCE, C. P. T. (2001) Emerging Infectious Diseases, 7, 382–389. TAORMINA, P., BEUCHAT, L. and SLUTSKER, L. (1999) Emerging Infectious Diseases, 5, 626– 634. TAUXE, R. V. (1997) Emerging Infectious Diseases, 3, 425–434. THACKER, S. (1994) In Principles and practice of public health surveillance (eds, Teutsch, S. and Churchill, R.) Oxford University Press, New York, pp. 3–17. THACKER, S. and BERKELMAN, R. (1988) Epidemiologic Reviews, 10, 164–90. USDA–ARS (2002), http://www.arru.saa.ars.usda.gov. VOETSCH, A., VAN GILDER, T., ANGULO, F., FARLEY, M., SHALLOW, S., MARCUS, R., CIESLAK, P., DENEEN, V., TAUXE, R. and THE EMERGING INFECTIONS PROGRAM FOODNET WORKING GROUP (2004) Clinical Infectious Diseases, 38, S127–S134. H.

Surveillance for emerging pathogens in the United States VOGT, R. L., SOURS, H. E., BARRETT, T., FELDMAN, R. A., DICKINSON, R. J.

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and WITHERELL, L. (1982) Annals of Internal Medicine, 96, 292–296. WATANABE, Y., OZASA, K., MERMIN, J., GRIFFIN, P., MASUDA, K., IMASHUKU, S. and SAWADA, T. (1999) Emerging Infectious Diseases, 5, 424–428. WELLS, J. G., SHIPMAN, L. D., GREENE, K. D., SOWERS, E. G., GREEN, J. H., CAMERON, D. N., DOWNES, F. P., MARTIN, M. L., GRIFFIN, P. M., OSTROFF, S. M., POTTER, M. E., TAUXE, R. V. and WACHSMUTH, I. K. (1991) Journal of Clinical Microbiology, 29, 985–989. WHO (2001) http://www.who.int/salmsurv/en/. WINTHROP, K., PALUMBO, M., FARRAR, J., MOHLE-BOETANI, J., ABBOTT, S., INAMI, G. and WERNER, S. (2002) In 89th Annual Meeting of the International Association for Food Protection International Association for Food Protection, San Diego, California June 30–July 3, 2002, pp. 81. YANG, S., LEFF, M. G., MCTAGUE, D., HORVATH, K., JACKSON-THOMPSON, J., MURAYI, T., BOESELAGER, G., MELNIK, T., GILDEMASTER, M., RIDINGS, D., ALTEKRUSE, S. and ANGULO, F. (1998) Morbidity and Mortality Weekly Report: CDC Surveillance Summaries, 47, 33–57. ZANSKY, S., WALLACE, B., SCHOONMAKER-BOPP, D., SMITH, P., RAMSEY, F., PAINTER, J., GUPTA, A., KALLURI, P. and NOVIELLO, S. (2002) Morbidity and Mortality Weekly Report, 51, 545– 548. ZHAO, T., DOYLE, M. and BESSER, R. (1993) Applied and Environmental Microbiology, 59, 2526–2530.

3 Surveillance of emerging pathogens in Europe S. J. O’Brien and I. S. T. Fisher, Health Protection Agency Centre for Infections, UK

3.1

Introduction

3.1.1 The origins of communicable disease control in Europe Communicable disease control in Europe has a long and proud history. In the fifteenth century the Venetians introduced quarantine at their ports in an effort to control the spread of infectious diseases, in particular plague, a tactic that was subsequently adopted throughout Western Europe (MacLehose et al., 2002). By the 19th Century it was clear that quarantine for cholera control, by then the main concern, was inadequate (MacLehose et al., 2002). In 1851, the first International Sanitary Conference was held in Paris leading to the subsequent establishment of a permanent International Committee on Epidemics and the adoption of an International Sanitary Convention (ISC). By 1903, the ISC agreed that states would ‘immediately notify the other governments of the first appearance in its territory of authentic cases of plague or cholera’ (MacLehose et al., 2002). This eventually paved the way for a set of International Health Regulations (IHRs), adopted by the World Health Assembly (WHA) in 1969. In a proposed revision of the IHRs, scheduled for adoption by the WHA in 2005, core requirements for surveillance and response are laid out (World Health Organization (WHO), 2004). 3.1.2 Surveillance for food safety Epidemiological surveillance is essential for any food safety programme. Wisely interpreted surveillance data are used to determine the burden of foodborne illness, the causative organisms and their epidemiology and to detect foodborne disease outbreaks. Surveillance data are thus fundamental for planning, implementing and evaluating food safety programmes (Borgdorff

Surveillance of emerging pathogens in Europe

51

and Motarjemi, 1997). Routine data sources include death registration, hospital discharge data, statutory notifications, laboratory surveillance and outbreak investigation. Borgdorff and Motarjemi (1997) have reviewed comprehensively the strengths and weaknesses of these various data sources. Briefly, death certificates and hospital discharge data include only those with fatal and/or severe disease. Coding errors and a tendency to use ‘catch-all’ diagnostic categories, for example, ‘diarrhoea of unknown aetiology’, are limiting features. Nevertheless, they provide a minimum estimate of the burden of serious disease. Statutory notifications of foodborne disease have the advantage that they are a legal requirement. However, in order to be captured by this system a patient has to feel sufficiently unwell to seek medical advice. Then their medical practitioner not only has to decide that the cause of the patient’s gastroenteritis is foodborne, as opposed to other routes of transmission, but he has to remember his legal obligation to notify the case. Furthermore, the definition of what constitutes food poisoning or food-related illness varies. There is good evidence that notifications are a poor reflection of the burden of foodborne disease (Wall et al., 1996; Atkinson and Maguire, 1998; Simmons et al., 2002). Laboratory report surveillance has the advantage over notifications that a diagnosis is attached to the patient’s symptoms. However, as with notification, the patient must present to medical attention before appropriate investigations can be undertaken. The physician may decide that laboratory investigation is not required to help guide patient management, in which case valuable epidemiological information is lost. Even if investigations are undertaken variations in laboratory protocols and in case definitions influence what is found and reported centrally. For national collation of surveillance data there needs to be a robust system for capturing positive results. Attrition of information at various stages throughout the surveillance process is often expressed as a pyramid (Fig. 3.1). To overcome shortcomings in routinely collected data some countries have embarked on burden of illness studies. These studies have permitted an understanding of the relationship between disease in the community and the data that appear in national statistics (Wheeler et al., 1999; de Wit et al., 2001). Routinely collected surveillance data may be supplemented by the use of sentinel studies and by research to determine the sources and food vehicles for sporadic infection as well as for outbreaks. During the last three decades, foodborne diseases have received increased recognition as an important public health issue. Subsequently, several surveillance programmes for foodborne diseases and pathogens have been established in Europe. These are described in detail below.

3.2 The WHO surveillance programme for control of foodborne infections and intoxications in Europe This is, perhaps, the most comprehensive scheme in terms of its geographical

52

Emerging foodborne pathogens Appears in national statistics Positive result

Specimen submitted

Specimen requested

Presenting to a physician

Disease in the community

Fig. 3.1

Surveillance reporting pyramid showing the stages at which data are lost.

coverage, its longevity and the range of organisms covered under one umbrella. The WHO European region stretches from the Republic of Ireland in the west to the Russian Federation in the east, and as far as Israel to the south (Fig. 3.2). The surveillance programme was launched by WHO/Europe in 1980. Since that time the number of participating countries has grown from just eight at the inception of the programme to 51 by 2000 (Tirado and Schmidt, 2001). The programme is co-ordinated by the Federal Institute for Risk Assessment (BfR) in Berlin, and overall management is from the WHO Centre for Environment and Health in Rome.

Source: http://www.who.int/about/regions/euro/en/index.html

Fig. 3.2 Map showing the countries in the WHO European region that report to the WHO Programme for foodborne infections and intoxications in Europe.

Surveillance of emerging pathogens in Europe

53

Participation in the programme is voluntary and the scheme is based on surveillance activities at national level. Specifically its objectives are to: • • • •

evaluate trends in incidence of foodborne diseases and outbreaks identify the causes and epidemiology of foodborne diseases in Europe disseminate relevant information on surveillance collaborate with national authorities to identify approaches to reinforce their surveillance systems (Tirado and Schmidt, 2001).

There is a designated national contact point in each participating country, usually at the health ministry, responsible for collecting and reporting official data on foodborne outbreaks, along with other relevant information. The programme office compiles and reports the data, issuing quarterly newsletters and annual reports both in printed form and published on the worldwide web. Great efforts have been made to standardize data collection and coding, through the use of harmonized definitions and creation of standardized reporting forms and coding systems (Tirado and Schmidt, 2001). One of the prime data sources concerns the epidemiological investigation of outbreaks. The dataset to be reported to WHO comprises the number of people affected, causative agent(s), the implicated food(s), the location at which the food was contaminated or mishandled, the place of purchase or consumption and any contributory factors leading to the outbreak. Figure 3.3 shows the contributory factors leading to foodborne outbreaks in 2000 for six of the countries that report to the WHO Programme. Two general points are illustrated with respect to all countries reporting data to the programme. The first is that the numbers of outbreaks reported vary considerably. Secondly the proportion of outbreaks for which contributory factors cannot be identified is fairly sizeable. In addition to information derived from epidemiological investigation of outbreaks, the WHO also requests statutory notification data, laboratory-report surveillance data and findings from special surveys. Table 3.1 shows campylobacter infection rates from 1993 to 2000. A major strength of the programme is the standardisation which has been brought to bear to improve reporting. Despite this, however, it is still difficult to draw direct comparisons between participating countries. Until recently, surveillance in Europe was primarily seen as a national responsibility and, perhaps not surprisingly, different approaches emerged (MacLehose et al., 2002; Tirado and Schmidt, 2001). The robustness of the data compiled by the WHO is heavily dependent upon that of the national surveillance systems that feed into the system and influenced by timeliness and completeness of reporting. Added to this, diagnostic and reference laboratory methods for detecting pathogens and/or their toxins vary, further complicating inter-country comparisons. The creation in 2000 of WHO Global Salm-surv (GSS) should help to alleviate this latter problem in due course. The foundation for GSS was laid in the late 1990s through a survey conducted by WHO to increase understanding of the worldwide epidemiology of human salmonellosis and the national surveillance systems in place to record disease (Herikstad et al.,

Belgium (N = 74)

Finland (N = 69)

Lithuania (N = 8) Contamination by infected equipment Contamination by infected person Inadequate cooking Inadequate refrigeration Other Unknown

Croatia (N = 71)

Sweden (N = 75)

Ireland (N = 64) Contamination by infected equipment Contamination by infected person Inadequate cooking Inadequate refrigeration Other Unknown

Fig. 3.3

Contributory faults reported in outbreaks in selected countries reporting to the WHO programme for foodborne infections and intoxications in Europe.

Table 3.1 Incidence rates of campylobacteriosis in European countries, 1993–2000 Country

1993 No. of cases

Northern Europe Denmark N/A Finland 1600 Iceland 59 Norway 877 Sweden 4485 Western Europe Belgium 4394 Netherlands N/A U.K. 39477 England & Wales U.K 4011 Scotland Southern Europe Greece N/A Israel 1067 Malta 49 Spain 2387

1994

1995

1996

1997

1998

1999

Incidence rate

No. of cases

Incidence rate

No. of cases

Incidence rate

No. of cases

Incidence rate

No. of cases

Incidence rate

No. of cases

Incidence rate

33 31 21 20 21

2 1804 48 1050 5529

41 35 17 24 29

N/A 2197 41 1046 5580

49 42 15 24 29

3 2629 85 1145 5081

56 51 31 26 21

2 2404 93 1178 5306

50 46 4 27 21

N/A 2851 220 1700 6543

44 N/A 74

4879 N/A 45207

48 N/A 84

4779 2871 43449

47 18 83

4991 3741 43978

49 24 82

5417 3646 51360

54 23 95

78

4152

80

4381

86

5107

102

5533

N/A 18 13 6

N/A 1455 16 2943

N/A 25 4 7

N/A 2014 8 3235

N/A 34 2 8

N/A 1314 28 3687

N/A 22 8 9

N/A 1461 21 3755

2000

No. of cases

Incidence rate

No. of cases

Incidence rate

54 46 80 39 29

N/A 3303 435 2032 7137

N/A 64 156 46 81

N/A 3527 245 2331 7646

N/A 68 87 52 86

6610 3398 56852

65 22 110

6514 3160 54987

64 N/A 104

7473 3362 55887

73 N/A 106

108

6381

125

5865

115

6482

127

N/A 25 6 9

136 2446 21 4389

1 41 6 11

306 N/A 11 N/A

3 N/A 3 N/A

261 N/A 30 N/A

3 N/A 8 N/A

Table 3.1 Continued Country

1993 No. of cases

1994

1995

Incidence rate

No. of cases

Incidence rate

South-Eastern Europe Croatia N/A The F.Y.R. N/A of Macedonia

N/A 0

N/A 20

N/A 1

14 37

Eastern Europe Armenia Republic of Moldovia

N/A N/A

N/A N/A

N/A N/A

N/A N/A

N/A N/A

N/A 22

N/A N/A

7 1045 N/A 5058

2/4 d < 1/2 d; < 2/4 d; > 3/7 d

Mbithi et al., 1991

Fingertips Paper Aluminium, china Latex Sea water

River water Drinking water Groundwater Bottled mineral water Lettuce Carrot Fennel

4 °C, 90% humidity 20 °C, 50–85% humidity 4–20 °C, 50–90% humidity 4–20 °C, 50–90% humidity 5 °C 25 °C 20 °C 4 °C 19 °C 25 °C 2–28 °C 4 °C 23 °C 5 °C, 25 °C 4 °C 23 °C 4 °C 20–25 °C 4–25 °C, modified atmosphere packaging* 4 °C 4 °C

* Lettuce was stored in plastic bags with various mixtures of carbon dioxide and nitrogen gas

Mbithi et al., 1992 Abad et al., 1994

Bosch, 1995 Callahan et al., 1995 Crance et al., 1998 Springthorpe et al., 1993 Enriquez et al., 1995 Sobsey et al., 1989 Biziagos et al., 1988 Croci et al., 2002 Bidawid et al., 2001 Croci et al., 2002

298 Emerging foodborne pathogens Table 11.3

HAV inactivation by various treatments

Treatment

Liquid/food item

Heat

Milk Skim milk Cream Cockles

Chlorination Gamma irradiation High pressure

Well/tap water Lettuce/ strawberries Clams/oysters Cell culture medium

Reduction in infectivity (log10/time)

Reference

< < < > < > >

Parry and Mortimer, 1984 Bidawid et al., 2000b

62.8 °Ca 71.6 °Ca 65 °C 71 °C 65 °C 71 °C 100/101 °Cb 95 °Cc 1 mg/L FCd 2.7–3.0 kGy

< 3/2 h < 1 log10

Abad et al., 1997 Bidawid et al., 2000c

2.0 kGy 450 MPa

> 6/5 min

Kingsley et al., 2002b

3/30 min 2/15 sec 3/16 min 5/14 min 2/16 min 4/16 min 4/2 min

Millard et al., 1987

a – pasteurisation conditions, b – steaming (steam temperature), c – immersion (water temperature), d – free chlorine

by the treatment. For some processes, such as boiling of liquid food at 100 °C and ultra heat treatment of dairy products, the resulting risk was considered to be negligible (i.e., it was highly unlikely that the product would contain infectious viruses); however, for others such as pasteurisation of liquid and solid foods, and acidification of fruit juices, the risk was deemed medium (the product might contain infectious viruses in numbers that could cause disease). High-risk processes were, e.g., freezing or chilling of frozen desserts, where viruses could survive with little or no loss of infectivity. The evaluation was made on the basis of a study which showed that 1,000 virus particles can be transferred from contaminated fingertips to food surfaces (Bidawid et al., 2000a); this would then require at least a 3 log10 inactivation of infectious viruses by the food treatment process to reduce any risk to negligible proportions. Although somewhat prosaic, the evaluation does provide a good basic overview of the hazard of virus resistance and the consequent risk to consumers, and it should be useful to food manufactures when formulating risk assessments and hazard analysis/critical control points (HACCP) plans. There are various governmental regulations in place which specify sanitary controls for shellfish production, for example European Directive 91/492/ EEC (Anonymous, 1991), and the US Food and Drug Administration National Shellfish Sanitation Program (Anonymous, 1993a). These controls cover several areas, such as the quality of the shellfish-growing waters, processing, and marketing of the food products (Lees, 2000). Waters are classified according to their level of pollution, indicated by the presence of faecal bacteria, and appropriate post-harvest treatment is specified according to the classification.

Hepatitis viruses

299

In Europe, when heating is applied to process shellfish, it should be performed so that the internal temperature of the shellfish is at 90 °C for 1.5 minutes (Anonymous, 1993b); this is based on studies on the inactivation of HAV in cockles (Millard et al., 1987). A large amount of shellfish, particularly oysters, are sold live for consumption, and therefore heating cannot be used to process them. Depuration is often used to reduce microbial contamination, and in many countries the process is subject to legal control (Lees, 2000); however it is suspected that it is not entirely effective. Many of these controls have been based upon data obtained with bacterial indicators of contamination, but these may not always be appropriate as indicators of viral contamination of shellfish; new research has been aimed at direct detection of viruses, or detection of bacteriophage as indicators of potential virus presence (Lees, 2000). Bacteriophages may have applications as indicators in other food types (Cliver, 1995); however, they would be useful only to identify foods which may have been contaminated by sewage, but not for foods contaminated by handling (Cliver, 1997b). With regard to agricultural industries, such as those involved in the production of soft fruit and fresh vegetables, Koopmans and Duizer (2004) have pointed out that good agricultural practice must be adhered to, to minimise the risk of transmission of enteric viruses. An important control point is water used for irrigation or washing which must be of good sanitary quality (Richards, 2001). Food handlers should be educated about microbial safety guidelines and hygiene rules (Koopmans and Duizer, 2004), and this should, particularly for HAV, include information about the risk of sick children. This is particularly important in countries where hepatitis A is highly endemic. Many of these countries grow and export fresh produce, and food production on farms is a large source of employment. It is likely that most adult workers are immune to infection with HAV, having been infected as children (Hadler, 1991). However, adults working on farms could bring their children with them, or may not adequately wash their hands after caring for children, most of whom could be asymptomatically infected (WHO, 2000b) and shedding virus. A key measure in the prevention of foodborne transmission of viral disease in any setting, either domestic or industrial, is good hygienic practice. Industrial premises must have adequate sanitary and hand washing facilitates to allow staff to observe good hygienic practice.* Food handlers should be properly trained in food hygiene (WHO, 2000a) and be aware of the risks of low hygienic standards. The World Health Organization has issued a number of educational and training manuals providing guidance on hygienic handling of foods (WHO, 2000a). Amongst other WHO recommendations, individual *Thorough hand washing is very important. The study of Bidawid et al. (2000a) indicated that inadequate hand washing may not be sufficient to remove all HAV particles from contaminated hands, or prevent the transfer of virus from fingers to foods.

300 Emerging foodborne pathogens food handlers suffering from any disease or infection which may be transmitted by food should report this to their supervisor, who should then use discretion as to whether or not to exclude the person from food-handling duties. Vaccination is available for HAV, and in the US the use of hepatitis A vaccine has resulted in an overall decline in the number of cases being reported annually (Acheson and Fiore, 2004). Vaccination of all food handlers, from primary production through to retail and service, would be an effective means of preventing foodborne hepatitis A (Cliver 1997a), and the WHO has recommended (WHO, 1993) that it should be considered if resources are available.† However, the number of reported handler-associated outbreaks of hepatitis A may not be sufficient to justify the routine vaccination of food handlers (Anonymous, 1996; Jacobs et al. 2000), although it could be worthwhile to keep this issue under consideration (ACMSF, 1998; Keeffe, 2004). During an outbreak, immunisation of at-risk individuals can be used to control the spread of infection (D’Argenio et al., 2003; Sanchez et al., 2002). Immunoprophylaxis for HEV is not yet available. Although it is not certain that pets could harbour or transmit hepatitis E viruses, the presence of antibodies in pet cats found by Okamoto et al. (2004) emphasises the need to always apply good hygienic practices in household settings.

11.8

Areas for further research

There are several general areas where there are knowledge gaps about HAV, for example, what is the infectious dose? With specific regard to the foodborne transmission of hepatitis A, Fiore (2004) has pointed out that it is not clearly known how fresh produce becomes contaminated, and why items such as green onions and strawberries appear to be more susceptible to contamination. In the outbreaks which have occurred where fresh produce was the vehicle of transmission, the extent of the contamination of the foodstuff was not fully ascertained. For example, in the Michigan 1997 hepatitis A outbreak attributed to strawberries, was all the fruit contaminated or only a portion? Was one infected handler responsible or could it have been several? How much handling is required, and how contaminated must the handler’s or handlers’ hands be, to result in several hundred kilos of produce harbouring sufficient HAV to infect several hundred people? Or was the contamination due to washing in contaminated water, and if so, how contaminated must the water have been? etc. There is a need for information on these factors which would facilitate a realistic risk assessment of these issues. Therefore, as well as acquiring more detailed information from outbreak investigations, practical research will be necessary to acquire information on, e.g., how much †

It would be expensive (Jacobs et al., 2000; Meltzer et al., 2001)

Hepatitis viruses

301

contamination (how many strawberries/onions, etc.) results from handling by a person whose hands are contaminated by HAV or, e.g., how much contamination results from washing with water containing, for example, 102 infectious particles per litre? To this end it is important that standard virus detection methods are produced, with comprehensive input from the various groups of researchers who have experience in this area. Such methods will support surveillance programs, epidemiological investigations, and, to some extent, routine monitoring as part of food companies’ food hazard management systems. Considerably more knowledge of HAV and HEV survival in the environment and food, and the factors which influence it, is required for a realistic appraisal of the risks these pathogens pose, and to control their transmission. Specific questions include whether HAV can survive in sewage-amended soils, and whether it can be transferred to crops, and just how long is it able to survive on fresh or frozen produce? The discovery in pigs of HEV strains related to human strains (van der Poel et al., 2001) is potentially significant as regards the possibility of interspecies transfer and zoonotic infection. Pork products are usually thoroughly cooked* at temperatures which should inactivate viruses, but a potential for zoonotic foodborne transfer of HEV may lie in environmental contamination via manure from infected pigs. HEV may be widespread in the general pig population (Banks et al., 2004; Meng, 2000a; van der Poel et al., 2001), and if so, it is possible that much of the pig manure which is stored on farms and subsequently spread onto agricultural land as fertiliser could contain infectious HEV particles. This could result in exposure of the human population (Cook et al., 2004). It may be informative to study prevalence and survival of HEV in the environment and on crops and foods, and also to develop methods to detect interspecies transfer at an early stage (van der Poel et al., 2001).

11.9

Sources of further information

The reader who wishes to learn in greater detail about the historical background of hepatitis infections and the elucidation of their etiology will be well *The studies of Yazaki et al. (2003) and Tamada et al. (2004) suggest that consumption of undercooked pig liver, and undercooked wild boar meat, may have been the cause of some cases of hepatitis E in Japan. Wild boar liver is also often eaten raw in Japan, and this has also been linked to some hepatitis E cases (Matsuda et al., 2003). In Bali, raw pig meat or fresh pig blood can be consumed, and seropositivity to HEV is relatively high in the human population (Wibawa et al., 2004). In a case of hepatitis E in the UK which was caused by an HEV strain very similar to pig strains, the patient had admitted to eating raw pork products, although this was not conclusively the cause of the infection (Banks et al., 2004).

302 Emerging foodborne pathogens served by consulting an appropriate review such as Zuckerman and Howard (1979), and a good narrative account of how hepatitis viruses were discovered can be found in The Invisible Invaders (Radestky, 1994). The main handbook for information about the general characteristics of hepatitis viruses is Fields Virology, which in its several editions provides comprehensive details regarding virus morphology, replication, pathogenesis, etc. The websites of the Centers for Disease Control (www.cdc.gov), and the World Health Organisation (www.who.int) are up-to-date sources of statistics concerning the prevalence of disease, and other epidemiological information. The WHO book Foodborne Disease: a focus for health education (WHO, 2000a) is an excellent source of information on food hygiene guidelines, which emphasises the importance of training and education of food handlers for the prevention of foodborne disease. The latest research on the prevalence of hepatitis viruses in foods and the environment, the development of methods to mediate their detection, and development of methods to reduce the risk of contamination of foods can be obtained in scientific journals such as the Journal of Food Protection, Applied and Environmental Microbiology, and the International Journal of Food Microbiology. A continuous reporting service providing day-by-day details of disease outbreaks around the world is provided by ProMED-mail (www.promedmail.org), under the International Society for Infectious Diseases (www.isid.org). Currently, there are plans under way to establish formal networks of virologists in both Europe and North America, with the ultimate intention of forming a global network for research and dissemination of information concerning viruses including HAV and HEV in food and the environment.

11.10

Acknowledgement

Artur Rzeżutka was supported by a European Commission Marie Curie Fellowship (QLKI-CT-2002-51453).

11.11

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12 Prion diseases C. J. Sigurdson and A. Aguzzi, Universitätsspital Zürich, Switzerland

12.1

Introduction

Prion infections ultimately result in fatal neurodegenerative diseases in humans and a wide variety of animals (Aguzzi, Montrasio et al. 2001). Although prion diseases may present with certain pathophysiological parallels to other progressive encephalopathies, such as Alzheimer’s and Parkinson’s disease (Aguzzi and Raeber 1998), they are unique in being transmissible. Homogenization of brain tissue from affected individuals and intracerebral inoculation into another individual (same species) typically reproduces the disease. This important fact was recognized more than half a century ago in the case of scrapie (Cuille and Chelle 1939), a prototypic prion disease that affects sheep and goats. Inspired by a suggestion of Hadlow that kuru in humans might be an infectious disease similar to scrapie (Hadlow 1959), Gajdusek showed that kuru and Creutzfeldt-Jakob disease (CJD) were transmissible to primates and mice (Gajdusek, Gibbs et al. 1966; Gibbs, Gajdusek et al. 1968), and as later discovered from accidental iatrogenic transmissions, also to other humans (Brown et al. 2000). Therefore, prion diseases are also called transmissible spongiform encephalopathies (TSEs), a term that emphasizes their infectious nature. When Stanley Prusiner started his first attempts at tackling the cause of TSEs (Prusiner, Hadlow et al. 1977), this group of diseases was not in the public limelight. However, bovine spongiform encephalopathy (BSE) was recognized a few years later (Wells, Scott et al. 1987) – an event that would dramatically change the public perception of prion diseases. CJD in humans was, and fortunately continues to be, exceedingly rare; its incidence is typically 1/106 inhabitants/year, but reaches 3/106 inhabitants/year in Switzerland,

310 Emerging foodborne pathogens which is currently reporting the highest number of cases (Glatzel, Rogivue et al. 2002; Glatzel, Ott et al. 2003). While only less than 1% of all reported cases of Creutzfeldt-Jakob disease (CJD) can be traced to a defined infectious source, the identification of bovine spongiform encephalopathy (BSE) (Wells, Scott et al. 1987) and its subsequent epizootic spread has highlighted prion-contaminated meat-andbone meal as an efficient vector for bovine prion diseases (Weissmann and Aguzzi 1997). Infectious prions do not completely lose their infectious potential even after extensive autoclaving (Taylor 2000).When transmitted to primates, BSE produces a pathology strikingly similar to that of vCJD (Aguzzi and Weissmann 1996b; Lasmezas, Deslys et al. 1996). BSE is most likely transmissible to humans, too, and strong circumstantial evidence (Aguzzi 1996; Aguzzi and Weissmann 1996b; Collinge, Sidle et al. 1996; Bruce, Will et al. 1997; Hill, Desbruslais et al. 1997) suggests that BSE is the cause of variant Creutzfeldt-Jakob disease (vCJD) which has claimed more than 150 lives in the United Kingdom (Will, Ironside et al. 1996; http:// www.cjd.ed.ac.uk) and a much smaller number in some other countries (Chazot, Broussolle et al. 1996). Several aspects of CJD epidemiology continue to be enigmatic. For example, CJD incidence in Switzerland increased twofold in 2001, and appears to be increasing even further in the year 2002 (Glatzel, Pekarik et al. 2002). A screen for recognized or hypothetical risk factors for CJD has, to date, not exposed any causal factors. Several scenarios may account for the increase in incidence, including improved reporting, iatrogenic transmission, and transmission of a prion zoonosis. Prion diseases typically exhibit a very long latency period between the time of infection and the clinical manifestation; this is the reason why these diseases were originally thought to be caused by ‘slow viruses’. From the viewpoint of interventional approaches, this peculiarity may be exploitable, since it opens a possible window of intervention after infection has occurred, but before brain damage is being initiated. Prions spend much of this latency time executing neuroinvasion, which is the process of reaching the central nervous system after entering the body from peripheral sites (Aguzzi 1997; Nicotera 2001). During this process, little or no damage occurs to the brain, and one might hope that its interruption may prevent neurodegeneration. Indeed, the incubation period of prion diseases can be long, ranging from two years to 50 years in some cases of kuru in humans. Once neurodegeneration begins, the disease progresses and is ultimately fatal. Brain lesions are characterized by spongiform degeneration, astrocytic gliosis, occasionally amyloid plaques, and neuronal loss without an inflammatory infiltrate. 12.1.1 What is a prion? Stanley Prusiner’s protein-only hypothesis The most widely accepted hypothesis on the nature of the infectious agent causing TSEs (which was termed prion by Stanley B. Prusiner) (Prusiner

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1982) predicates that it consists essentially of PrPSc, an abnormally folded, protease-resistant, beta-sheet rich isoform of a normal cellular protein termed PrPC. According to this theory, the prion does not contain any informational nucleic acids, and its infectivity propagates simply by recruitment and ‘autocatalytic’ conformational conversion of cellular prion protein into diseaseassociated PrPSc (Aguzzi and Weissmann 1997). A large body of experimental and epidemiological evidence is compatible with the protein-only hypothesis, and very stringently designed experiments have failed to disprove it. It would go well beyond the scope of this chapter to review all efforts that have been undertaken in this respect. Perhaps most impressively, knockout mice carrying a homozygous deletion of the Prnp gene that encodes PrPC, fail to develop disease upon inoculation with infectious brain homogenate (Büeler, Aguzzi et al. 1993), nor does their brain carry prion infectivity (Sailer, Büeler et al. 1994). Reintroduction of Prnp by transgenesis – even in a shortened, redacted form – restores infectibility and prion replication in Prnp% mice (Fischer, Rülicke et al. 1996; Shmerling, Hegyi et al. 1998; Supattapone, Bosque et al. 1999; Flechsig, Shmerling et al. 2000). In addition, all familial cases of human TSEs are characterized by Prnp mutations (Aguzzi and Weissmann 1996a; Prusiner, Scott et al. 1998).

12.2

Epidemiology

12.2.1 Natural scrapie in sheep and goats Scrapie is the most common natural prion disease of animals. Even though scrapie was recognized as a distinct disorder of sheep with respect to its clinical manifestations as early as 1738, the disease remained an enigma, even with respect to its pathology, for more than two centuries (Parry 1983). Some veterinarians thought that scrapie was a disease of muscle caused by parasites, whilst others thought that it was a dystrophic process (M’Gowan 1914). An investigation into the etiology of scrapie followed the vaccination of sheep for louping-ill virus with formalin-treated extracts of ovine lymphoid tissue unknowingly contaminated with scrapie prions (Gordon 1946). Two years later, more than 1500 sheep developed scrapie from this vaccine. Scrapie of sheep and goats shares with chronic wasting disease (CWD) of deer and elk a unique property among prion diseases: they seem to be readily communicable within flocks. Although the transmissibility of scrapie seems to be well established, the mechanism of the natural spread of scrapie among sheep is puzzling. The placenta has been implicated as one source of prions accounting for the horizontal spread of scrapie within flocks (via ingestion or environmental contamination) (Pattison and Millson 1961; Pattison 1964; Pattison, Hoare et al. 1972; Onodera, Ikeda et al. 1993), although it is unlikely to be the sole source of infectivity. In Iceland, scrapie-infected flocks of sheep were destroyed and the pastures left vacant for several years; however,

312 Emerging foodborne pathogens reintroduction of sheep from flocks known to be free of scrapie for many years eventually resulted in scrapie (Palsson 1979). The source of the scrapie prions that attacked the sheep from flocks without a history of scrapie is unknown. Transmission through mites has been advocated (Wisniewski, Sigurdarson et al. 1996), but its significance on the field remains somewhat anecdotal. Tissues that accumulate infectious prions in sheep include, besides the central and peripheral nervous systems, the spleen, lymph nodes (Hadlow, Kennedy et al. 1980) and some non-lymphoid organs. In various experimental animal models, these non-lymphoid organs include the pituitary gland (Pattison and Millson 1962), adrenal gland (Pattison and Millson 1962), pancreas (Pattison and Millson 1960), nasal mucosa (Hadlow, Eklund et al. 1974), intestine (Hadlow, Eklund et al. 1974), muscle (Pattison and Millson 1962), and the eye (retina) (Buyukmihci, Rorvik et al. 1980). How do prions reach these distant sites? Although nerves (Pattison and Millson 1962) and blood (Houston, Foster et al. 2000) may contain infectivity, the precise carrier mechanisms are unclear. 12.2.2 Feline spongiform encephalopathy (FSE) In non-domestic cats, the prion diseases were likely due to ingestion of BSEinfected cattle carcasses. Feline spongiform encephalopathy has been described in a captive cheetah, puma, an ocelot, and a tiger from zoological collections in Great Britain (Kirkwood and Cunningham 1994) (Williams, Kirkwood et al. 2001). In addition to the non-domestic felids, 87 domestic cats in Great Britain and sporadic cases in Norway, Northern Ireland and Liechtenstein have been diagnosed with FSE, all cats were > 2 years old (Ryder, Wells et al. 2001). Clinically affected cats initially demonstrated behavior changes (more timid or aggressive), with subsequent ataxia, hypermetria, and hyperesthesia to sound and touch (Wyatt, Pearson et al. 1991; Bratberg, Ueland et al. 1995). Histopathology revealed spongiform degeneration in the neuropil of the brain and spinal cord with the most severe lesions localized to the medial geniculate nucleus of the thalamus and the basal nuclei (Ryder, Wells et al. 2001). A ban on bovine spleen and CNS tissue in pet foods was initiated in 1990, and all but one of the FSE cases to date occurred in cats born prior to the ban (Nathanson, Wilesmith et al. 1999). 12.2.3 Transmissible mink encephalopathy (TME) Transmissible mink encephalopathy (TME), initially recognized in Wisconsin and Minnesota in 1947, has appeared sporadically in farmed mink in several countries, including the United States, Canada, Finland, Russia, and East Germany (Marsh and Hadlow 1992). Nonetheless, TME outbreaks are rare with the most recent occurrence in the United States in 1985. Epidemiological studies indicate that the disease is causally linked to the ingestion of prion-

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contaminated meat, potentially scrapie sheep (Marsh and Bessen 1993). However, in the 1985 TME outbreak in Stetsonville, Wisconsin, the mink rancher stated with certainty that sheep were not fed to mink. Instead, downer (ill) cattle were the primary source of mink food – a discovery which has led to much speculation on a potentially unrecognized BSE-like disease of American cattle (Marsh and Bessen 1993). Despite such speculation, the ultimate origins of TME epidemics remain uncertain. To further investigate potential foodborne sources of TME, mink were intracerebrally (IC) exposed to United Kingdom- and North American-derived sheep scrapie brain homogenates. Mink were highly susceptible to the Suffolk sheep scrapie from the United States, but only after IC inoculation (Hanson, Eckroade et al. 1971). Mink did not develop disease from ingesting scrapie brain (Marsh 1979). These studies suggested, at minimum, that mink are susceptible to scrapie. However, further experiments demonstrated that TME could pass into cattle, and moreover, that brain from these cattle could transmit the TME agent efficiently to mink by either the IC or the oral route, with an incubation period of only four and seven months, respectively. This indicates that TSEs can be transmitted efficiently between cattle and mink (Marsh, Bessen et al. 1991) although the epidemiological significance of these findings are less clear. 12.2.4 Bovine spongiform encephalopathy In 1986, an epidemic of a previously unknown neurological disease appeared in cattle in Great Britain (Wells, Scott et al. 1987): bovine spongiform encephalopathy (BSE) or ‘mad cow’ disease. BSE was shown to be a prion disease by demonstrating protease-resistant PrP in brains of ill cattle (Hope, Reekie et al. 1988; Prusiner, Groth et al. 1993). Based mainly on epidemiological evidence, it has been proposed that BSE represents a massive common source epidemic which has caused more than 180,000 cases to date (Weissmann and Aguzzi 1997). In Britain, cattle, particularly dairy cows, were routinely fed meat and bone meal (MBM) as a nutritional supplement (Wilesmith, Wells et al. 1988; Wilesmith and Wells 1991; Wilesmith, Hoinville et al. 1992; Wilesmith, Ryan et al. 1992). The MBM was prepared by rendering the offal of sheep and cattle using a process that involved steam treatment and hydrocarbon solvent extraction. The extraction process produced protein and fat-rich fractions; the protein or greaves fraction contained about 1% fat from which the MBM was prepared. In the late 1970s, the price of tallow prepared from the fat fraction fell, making it no longer profitable to use hydrocarbons in the rendering process. The resulting MBM contained about 14% fat; maybe the high lipid content protected scrapie prions in the sheep offal from being completely inactivated by steam. Since 1988, the practice of using dietary protein supplements for domestic animals derived from rendered sheep or cattle offal has been forbidden in the UK. Curiously, almost half of the BSE cases have occurred in herds where

314 Emerging foodborne pathogens only a single affected animal has been found; several cases of BSE in a single herd are infrequent (Wilesmith, Wells et al. 1988; Dealler and Lacey 1990; Wilesmith and Wells 1991; Weissmann and Aguzzi 1997). In 1992, the BSE epidemic reached a peak, with over 35,000 cattle afflicted. In 1993, fewer than 32,000 cattle were diagnosed with BSE and in 1994 the number was approximately 22,000. In 2004, BSE had become a rare disease in British and European cattle, but regrettably (and quite inexplicably) it still had not completely disappeared. Indeed, there are currently concerns over whether BSE could be transmitted to sheep and goats and present a new threat to human health. Experimental studies have shown that sheep are susceptible to oral BSE infections (Jeffrey et al. 2001), and recently, a natural case of BSE in a goat was discovered (Eloit, 2005). Transmission of BSE to experimental animals Brain extracts from BSE cattle have transmitted disease to mice, cattle, sheep and pigs after intracerebral inoculation (Fraser, McConnell et al. 1988; Dawson, Wells et al. 1990a, b; Bruce, Chree et al. 1993). Transmissions to mice and sheep suggest that cattle preferentially propagate a single ‘strain’ of prions; seven BSE brains all produced similar incubation times as measured in each of three strains of inbred mice (Bruce, Chree et al. 1993). However, this notion was recently challenged on the basis of transgenetic studies (Asante, Linehan et al. 2002). Incontrovertible evidence for the existence of several BSE strains, in the opinion of the authors, has yet to be provided. Of particular importance to the BSE epidemic is the transmission of BSE to the non-human primate marmoset after intracerebral inoculation following a prolonged incubation period (Baker, Ridley et al. 1993). The potential parallels with kuru of humans, confined to the Fore region of New Guinea (Gajdusek, Gibbs et al. 1966; Gajdusek 1977), are worthy of consideration. Once the most common cause of death among women and children in that region, kuru has almost disappeared with the cessation of ritualistic cannibalism (Alpers 1987). Although it seems likely that kuru was transmitted orally, as proposed for BSE among cattle, some investigators argue that other routes of transmission were important because oral transmission of kuru to apes and monkeys has been difficult to demonstrate (Gajdusek 1977; Gibbs, Amyx et al. 1980). Oral interspecies prion transmission Besides BSE, four other animal diseases seem to have arisen from ingestion of prions. It has been suggested that an outbreak of transmissible mink encephalopathy in 1985 arose from feeding BSE-contaminated foodstuffs to mink (Marsh, Bessen et al. 1991). The prion-contaminated MBM thought to be the cause of BSE as well as BSE-contaminated pet foods are also most likely the cause of exotic ungulate encephalopathy and feline spongiform encephalopathy (FSE) respectively. FSE has been found in domestic cats throughout Europe, as well as in a puma and a cheetah (Willoughby, Kelly

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et al. 1992). Three cases of FSE in domestic cats have been transmitted to laboratory mice and PrPSc has been identified in their brains by immunoblotting (Pearson, Wyatt et al. 1992). Whether FSE may have transmitted to a human (Zanusso, Nardelli et al. 1998) remains anectodal and unproven. Prion disease has been found in the brains of the nyala, greater kudu, eland, gembok and Arabian oryx in British zoos; all of these animals are exotic ungulates. Of eight greater kudu born into a herd maintained in a London zoo since 1987, five have developed prion disease. Except for the first case, none of the other four kudu was exposed to feeds containing ruminant-derived MBM (Kirkwood, Cunningham et al. 1993). Brain extracts prepared from a nyala and a greater kudu have been transmitted to mice (Kirkwood, Wells et al. 1990; Cunningham, Wells et al. 1993). PrP of the greater kudu differs from the bovine protein at four residues; Arabian oryx PrP differs from the sheep PrP at only one residue (Poidinger, Kirkwood et al. 1993). 12.2.5 Chronic wasting disease of deer and elk As the only prion disease of wildlife, chronic wasting disease of deer and elk (CWD) was initially reported in 1980 as a TSE in captive research deer in Colorado and Wyoming, although the disease origin remains obscure (Williams and Young 1980). Since 1980, free-ranging mule deer (Odocoileus hemionus), white-tailed deer (O. virginanus), and Rocky Mountain elk (Cervus elaphus nelsoni) have been detected in the same region of Colorado and Wyoming (Spraker, Miller et al. 1997), but recently increased surveillance efforts across the U.S. and Canada have startlingly revealed CWD not only in adjacent states (Nebraska, New Mexico, and Utah) but also distant (Wisconsin, Illinois, Canada) from this original endemic region. Additionally, game ranched elk have not escaped the disease, with CWD being first detected in 1997. The staggeringly high transmission apparent in captive deer (up to 90% infected in one facility; (Williams and Young 1992)) and in free-ranging deer (up to 15%) (Miller, Williams et al. 2000), occurs by unknown mechanisms, but it is likely by a horizontal route; saliva or feces could potentially harbor infectious prions and contaminate grazing areas. Recently it was shown that environmental transmission can play a role in CWD transmission (Miller,Williams et al. 2004). Clinical signs of CWD are remarkably subtle and nonspecific, characterized by lethargy, weight loss, flaccid hypotonic facial muscles, polydipsia/polyuria, excessive salivation, and behavioral changes, such as loss of fear of humans (Williams and Young 1980). Of the TSEs, histologic brain lesions are most similar to BSE and scrapie, and include large single or septate intraneuronal vacuoles prominent in the parasympathetic vagal nucleus of the medulla oblongata, and also evident in the thalamus, hypothalamus, pons, midbrain, and olfactory cortex (Williams and Young 1993; Spraker, Miller et al. 1997). Similar to scrapie, PrPCWD is abundant in secondary lymphoid tissues, and can be detected in tonsil, Peyer’s patches, and ileocecal lymph node by IHC

316 Emerging foodborne pathogens as early as 12 weeks after experimental oral CWD inoculation (Sigurdson, Williams et al. 1999). The capacity for CWD transmission to other species is clearly an area of great concern. Unfortunately very little is known about the risk for other wildlife species, domestic ruminants, or humans contracting the disease. Cattle have been inoculated intracerebrally with CWD, and by six years post-inoculation (pi), five of 13 had developed PrPSc in the brain (Hamir, Cutlip et al. 2001; Hamir, et al. 2005). No cattle which have been orally inoculated have showed clinical signs of a TSE at 63 months pi (M.W. Miller and E.S. Williams, unpublished findings). Thus cattle appear to be highly resistant to CWD by a natural route of exposure. The ability of PrPCWD to convert human PrPc in vitro was determined to be inefficient, but similar to the efficiency of PrPBSE to convert human PrPc (Raymond, Bossers et al. 2000). 12.2.6 Human prion diseases The human prion diseases are manifest as infectious, inherited and sporadic disorders, and are often referred to as kuru, sporadic CJD, familial CJD, variant CJD (vCJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI), depending upon the clinical, genetic, and neuropathological findings. Familial CJD, GSS, and FFI are inherited prion diseases due to genetic mutations in the PRNP gene. Sporadic CJD (sCJD) occurs worldwide, yet the cause of sCJD remains unknown. Less than 300 iatrogenic CJD cases have occurred from human growth hormones and dura mater transplants. 12.2.7 Variant Creutzfeldt-Jakob disease (vCJD) The most recently recognized form of CJD in humans, variant CJD (vCJD), was first described in 1996 and has been linked to BSE (Will and Zeidler 1996). vCJD represents a distinct clinico-pathological entity that is characterized at onset by psychiatric abnormalities, sensory symptoms and ataxia, and eventually leads to dementia along with other features usually observed in sporadic CJD. vCJD is distinguishable from sporadic cases in that the patients are very young (vCJD: 19–39 yr.; sporadic CJD: 55–70 yr.) and duration of the illness is rather long (vCJD: 7.5–22 months; sporadic CJD: 2.5–6.5 months). Moreover, vCJD displays a distinct pathology within the brain characterized by abundant ‘florid plaques’, decorated by a daisy-like pattern of vacuolation. Most cases of vCJD have been observed in the United Kingdom (Will, Zeidler et al. 2000). For several years, it has been thought that prions are distributed much more broadly within the body of vCJD victims than of sCJD patients (Wadsworth, Joiner et al. 2001). However, this view has recently been challenged by the finding of PrPSc in muscle and spleen of sCJD patients (Glatzel, Abela et al. 2003).

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Given that a large fraction of the European population may have been exposed to BSE prions, yet only a minute minority developed vCJD, there can be hardly any doubt that additional genetic modifiers exist, other than PRNP polymorphisms. It was originally claimed that specific allelotypes of the major histocompatibility complex may represent such modifiers (Jackson, Beck et al. 2001), but this was not confirmed by later studies (Pepys, Bybee et al. 2003). So what will the numbers of vCJD victims be in the future? Fortunately, we have not yet seen a large-scale epidemic of this terrible disease. Although many mathematical models have been generated (Ghani, Ferguson et al. 1998; Ghani, Donnelly et al. 2000), the number of cases is still too small to predict future developments with any certainty. Since the year 2001, the incidence of vCJD in the UK appears to be stabilizing and may actually even be falling. One may argue that it is too early to draw any far-reaching conclusions, but each month passing without any dramatic rise in the number of cases increases the hope that perhaps the total number of vCJD victims will be limited (Valleron, Boelle et al. 2001).

12.3

Detection

As in any other disease, early diagnosis would significantly advance the chances of success of an interventional approach. But when compared to other fields of microbiological diagnostics, the tools for prion diagnosis appear to be depressingly unsophisticated. Presymptomatic diagnosis is virtually impossible, and the earliest possible diagnosis is based on clinical signs and symptoms. Hence, prion infection is typically diagnosed after the disease has progressed considerably. 12.3.1 Diagnosis of animal prion diseases Animal TSEs vary considerably in the PrPSc tissue distribution as well as clinical signs. Scrapie and CWD can be diagnosed preclinically by tonsil or conjunctival biopsy and PrP immunohistochemistry, however, even in these diseases not all cases have a lymphoid phase of PrP replication (Spraker, Balachandran et al. 2004). With regard to animals destined for the human food chain, there is the additional crucial need to determine presence of the prion agent in tissues in asymptomatic carriers, well before the appearance of any clinical symptoms. This applies equally to the detection of subclinically prion-infected humans, who may participate in tissue donation programs. To be optimally useful, prion diagnostics should identify ‘suspect’ cases as early during pathogenesis as possible. However, the currently available methods are quite insensitive when compared to those available for other infectious diseases. PrPSc represents the only disease-specific macromolecule identified to date, and all approved commercial testing procedures are based

318 Emerging foodborne pathogens on the immunological detection of PrPSc. While around fifty companies are reported to be developing prion diagnostic assays, all commercial test kits validated for use by the European Union rely on proteolytic removal of endogenous PrPC prior to detection of PrPSc. Circumvention of the protease digestion step might theoretically yield increased sensitivity of PrPSc-based detection methods and make these methods more amenable to high-throughput technologies. However, it has proved difficult to discriminate between PrPC and PrPSc with antibodies, despite some early reports (Korth, Stierli et al. 1997). Interestingly, tyrosine-tyrosinearginine (YYR) motifs (Paramithiotis, Pinard et al. 2003) were believed to be more solvent-accessible in the pathological isoform of PrP and a monoclonal antibody directed against these motifs was reported to be capable of selectively detecting PrPSc across a variety of platforms. However, YYR motifs are certainly not unique to pathological prion proteins and it remains to be determined whether this reagent can really improve the sensitivity of detection of prion infections. A significant advance in prion diagnostics was achieved in 1997 by the discovery that protease-resistant PrPSc can be detected in tonsillar tissue of vCJD patients (Hill, Zeidler et al. 1997). It was hence proposed that tonsil biopsy may be the method of choice for diagnosis of vCJD (Hill, Butterworth et al. 1999). Furthermore, there have been reports of individual cases showing detection of PrPSc at preclinical stages of the disease in tonsil (Schreuder, Vankeulen et al. 1996) as well as in the appendix (Hilton, Fathers et al. 1998), indicating that lymphoid tissue biopsy may be useful for diagnosing presymptomatic individuals. These observations triggered large screenings of human populations for subclinical vCJD prevalence using appendectomy and tonsillectomy specimens (Glatzel, Ott et al. 2003). PrPSc-positive lymphoid tissue was long considered to be a vCJD-specific feature which would not apply to any other forms of human prion diseases (Hill, Butterworth et al. 1999). However, a recent survey of peripheral tissues of patients with sporadic CJD has identified PrPSc in as many as one-third of skeletal muscle and spleen samples (Glatzel, Abela et al. 2003), as well as the olfactory epithelium of patients suffering from sCJD (Zanusso, Ferrari et al. 2003). These unexpected findings raise the hope that minimally invasive diagnostic procedures may take the place of brain biopsies in intravital CJD diagnostics. The sensitivity of PrPSc detection was significantly improved by the sodium phosphotungstic (NaPTA) precipitation method (Safar, Wille et al. 1998). Concentration of PrPSc prior to Western blot analysis improves the sensitivity of diagnostic assays by as much as four orders of magnitude (Wadsworth, Joiner et al. 2001). Another interesting development was brought about by the conformation-dependent immunoassay (CDI), in which conformational differences of PrP isoforms are mapped by quantitating the relative binding of antibodies to denatured and native protein (Safar, Wille et al. 1998). Rather than relying on protease resistance, the CDI measures a variety of misfolded PrP isoforms, which may increase its sensitivity (Safar, Scott et al. 2002; Bellon, Seyfert-Brandt et al. 2003).

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Be that as it may, all techniques described above suffer from the fact that PrPSc continues to represent a surrogate marker for prion infectivity – since (i) PrPSc has not been incontrovertibly shown to be congruent with the prion, and (ii) several manipulations in vitro and in vivo can render PrPC proteaseresistant without bestowing infectivity on it (Jackson, Hosszu et al. 1999). Therefore, determination of prion infectivity by bioassay remains the gold standard. As in Pasteur’s day, the concentration of the infectious agent is determined by inoculating serial dilutions of the test material into experimental animals, and the dilution at which 50% of the animals contract the disease (termed ID 50 ) is determined. Naturally, this system is riddled with inconveniences; scores of animals need to be sacrificed, and the incubation times are lengthy (transgenetic overexpression of PrPC can help, but only to some extent). Also the method tends to be very inaccurate. The inoculation schemes used in most studies typically suffer from standard errors of ±1 order of magnitude! A radical improvement of this situation is likely to be brought about by the use of prion-susceptible cell lines (Race, Fadness et al. 1987; Bosque and Prusiner 2000). The determination of prion infectivity endpoints in cultures of highly susceptible cells combines the sensitivity and intrinsic biological validity of the bioassay (i.e. direct measurement of the infectivity) with the speed and convenience of an in vitro methodology amenable to mediumthroughput automation (Klohn, Stoltze et al. 2003). 12.3.2 Diagnosis of human prion diseases Human prion disease should be considered in any patient who develops a progressive subacute or chronic decline in cognitive or motor function. Typically adults between 40 and 70 (or more) years of age, patients often exhibit clinical features helpful in providing a premorbid diagnosis of prion disease, particularly sporadic CJD (Roos, Gajdusek et al. 1973; Brown, Cathala et al. 1986). There is as yet no specific diagnostic test for prion disease in the cerebrospinal fluid; the concentration of the proteins 14-3-3 (Hsich, Kinney et al. 1996), S-100 and neuron-specific enolase is often increased, but these represent general markers of neuronal breakdown and do not allow for definite diagnosis of prion encephalopathy. A definitive diagnosis of human prion disease, which is invariably fatal, can often be made from the examination of brain tissue. Since 1992, knowledge of the molecular genetics of prion diseases has made it possible, using peripheral tissues, to diagnose inherited prion disease in living patients by sequencing the prion alleles. A broad spectrum of neuropathological features in human prion diseases precludes a precise neuropathological definition. The classic neuropathological features of human prion disease include spongiform degeneration, gliosis, and neuronal loss in the absence of any significant inflammatory reaction (Budka, Aguzzi et al. 1995). When present, amyloid plaques that stain with α-PrP antibodies are diagnostic.

320 Emerging foodborne pathogens Horizontal transmission of inherited prion diseases from humans to experimental animals is frequently negative when using rodents, despite the presence of a pathogenic mutation in the PRNP gene (Tateishi, Kitamoto et al. 1992). New lines of transgenic mice with enhanced susceptibility to human or animal prions (Telling, Scott et al. 1994; Weissmann, Fischer et al. 1998) are starting to enable transmission studies that were not practical in apes and monkeys (Brown, Gibbs et al. 1994). In practical terms, however, a definitive diagnosis of human prion disease can be rapidly accomplished if PrPSc can be detected by Western immunoblot analysis of brain homogenates in which samples are subjected to limited proteolysis to remove PrPC before immunostaining (Bockman, Kingsbury et al. 1985; Brown, Coker Vann et al. 1986; Bockman, Prusiner et al. 1987; Serban, Taraboulos et al. 1990). Because of regional variations in PrPSc concentration, methods using homogenates prepared from small brain regions may give false negative results. Alternatively, PrPSc may be detected in situ in cryostat sections bound to nitrocellulose membranes followed by limited proteolysis to remove PrPC, and guanidinium salt treatment to denature PrPSc, and thus enhance its accessibility to α-PrP antibodies (Taraboulos, Jendroska et al. 1992). Denaturation of PrPSc in situ prior to immunostaining has also been accomplished by autoclaving fixed tissue sections (Kitamoto, Shin et al. 1992). In the familial forms of the prion diseases, molecular genetic analyses of PrP can be diagnostic and can be performed on DNA extracted from blood leucocytes ante mortem. Unfortunately, such testing is of little value in the diagnosis of the sporadic or infectious forms of prion disease. In summary, the diagnosis of prion disease may be made in patients on the basis of (i) the presence of PrPSc, (ii) mutant PrP genotype or (iii) appropriate immunohistology, and should not be excluded in patients with atypical neurodegenerative diseases until one, or preferably two, of these examinations have been performed.

12.4

Transmission

There is no example of zoonotic transmission of prions from sheep and goats to humans: many epidemiological studies have failed to implicate scrapie prions from sheep as a cause of CJD (Malmgren, Kurland et al. 1979; Harries Jones, Knight et al. 1988; Cousens, Harries Jones et al. 1990). However, there is uncertainty about whether chonic wasting disease of deer and elk represents a threat to humans who ingest the meat. Actually, even transmissibility of BSE to humans relies on circumstantial evidence. Epidemiology and biochemistry favour the link between BSE and vCJD, but the evidence is not ultimately conclusive. The Koch postulates (which would unambiguously assign an infectious agent to a disease) have never been fulfilled, i.e., experimental inoculation of humans was never performed to

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prove this link. Also, accidental oral exposure to BSE infectivity of a sizable group of people at a precisely defined time point has never occurred, or did not result in disease. For these reasons, speculation that vCJD disease may not be due to BSE have never completely subsided. Likewise, we do not know whether scrapie affects only sheep and goats, or whether it can cross species barriers and infect humans. Finally, it is unknown whether BSE, upon transmission to sheep, remains as dangerous for humans as cow-derived BSE, or whether it becomes attenuated and acquires the (allegedly) innocuous properties of bona fide sheep scrapie. Another question relates to the possibility of chronic sub-clinical disease or a permanent ‘carrier’ status in cows as well as in humans. Evidence that such a carrier status may be produced by the passage of the infectious agent across species was first reported by Race and Chesebro (Aguzzi and Weissmann 1998; Race and Chesebro 1998), and has been confirmed by others (Hill, Joiner et al. 2000) – at least for the passage between hamsters and mice. Immune deficiency can also lead to a similar situation in which prions replicate silently in the body, even when there is no species barrier (Frigg, Klein et al. 1999). So the problem of animal transmissible spongiform encephalopathies could be more widespread than is assumed, and may call for drastic prion surveillance measures in farm animals. Moreover, people carrying the infectious agent may transmit it horizontally (Aguzzi 2000), and the risks associated with this possibility can be met only if we know more about how the agent is transmitted and how prions reach the brain from peripheral sites.

12.5 Prevention and control Several substances may alter prion infection in mammals; a non-exhaustive list includes compounds as diverse as Congo red (Caughey and Race 1992), amphotericin B (Pocchiari, Schmittinger et al. 1987), anthracyclin derivatives (Tagliavini, McArthur et al. 1997), sulfated polyanions (Caughey and Raymond 1993), pentosan polysulphate (Farquhar, Dickinson et al. 1999), soluble lymphotoxin-ß receptors (Montrasio, Frigg et al. 2000), porphyrins (Priola, Raines et al. 2000), branched polyamines (Supattapone, Wille et al. 2001), and beta-sheet breaker peptides (Soto, Kascsak et al. 2000). However, it is sobering that none of the substances have yet made it to any validated clinical use; quinacrine appears to represent the most recent unfulfilled promise (Collins, Lewis et al. 2002). On the other hand, the tremendous interest in this field has attracted researchers from various neighbouring disciplines, including immunology, genetics, and pharmacology, and therefore it is to be hoped that rational and efficient methods for managing prion infections will be developed in the future.

322 Emerging foodborne pathogens 12.5.1 Targeting follicular dendritic cells PrPSc typically accumulates in secondary follicles of the spleen, lymph node, tonsils, and Peyer’s patches on cells known as follicular dendritic cells (FDCs). This phenomenon appears to occur, to a variable extent, in prion diseases including vCJD, scrapie, and CWD, as well as in mouse scrapie and CJD models. Would ablation of FDCs impair formation of PrPSc depots? This question was addressed by inhibiting the LTα/β signalling pathway with soluble LTβR immunoglobulin fusion protein (LTβR-Ig ‘immunoadhesin’), which effectively disbands mature FDC networks. Soluble LTβR-Ig administered before prion inoculation abolished splenic prion replication and delayed neuroinvasion. Moreover, post-prion treatment with soluble LTβR also led to a modest delay in disease development, although splenic prion infectivity was detectable at eight weeks post-inoculation. Therefore, post-exposure treatment of humans with LTβR-Ig could only be considered as a prophylaxis in cases of known prion exposure at well defined early time points. This might include researchers, pathologists, neurologists, neurosurgeons and technicians after accidental CJD injection, or recipients of blood transfusions from patients with CJD. In these instances, we think that a case could be made for experimental use of post-exposure prophylaxis with LTβR-Ig. Prophylaxis should be administered as soon as possible, and it should be clearly kept in mind that LTβR-Ig will not offer any relief to patients with overt CJD, for whom neural entry has already taken place. 12.5.2 Immunization against prion disease Antibodies against PrPSc are not generated during the course of prion infections, but artificial induction of humoral immune responses to PrPC and/or PrPSc might be protective. However, vaccination against the prion protein has proven exceedingly challenging. First, wild-type mice are highly tolerant to PrP as an immunogen. This is not surprising, since PrPC is expressed nearly ubiquitously and is found on the surface of both B and T cells. Second, it was generally believed – with good reason – that antibodies against PrPC, if they can be elicited at all, might lead to severe systemic immune mediated diseases, since PrP is expressed on many cell types. Finally, PrP-specific antibodies would be unlikely to cross the blood brain barrier in therapeutic concentrations. In 2001, it was reported that a transgenic mouse model expressing antiprion µ chain antibodies experienced complete protection against prion disease after a peripheral route of exposure (intraperitoneal). In parallel, many other publications addressed prion immunoprophylaxis, both in vitro and in vivo. White et al. (2003) showed that passive anti-PrP immunization delivered after prion exposure delayed disease, and markedly increased the incubation period compared with non-immunized mice. However, active immunization efforts have so far not led to a high anti-PrP titer, owing to the immune tolerance. Moreover, intracranial delivery of PrPC-specific antibodies has

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been recently shown to result in neuronal apoptosis in the cerebellum and hippocampus, most likely through clustering of PrPC, which is thought to trigger an abnormal signalling pathway. These results are alarming and certainly reinforce the concept that adequate in vivo safety studies must be carried out before prion immunoprophylaxis trials in humans. On the other hand, it should be noted that neuronal loss occurred only with antibodies targeting a subset of PrP epitopes (within amino acids 95–105 of the PrP sequence), and only at extremely high concentrations of antibody. Hence, we do not believe that Solforosi’s data entirely rules out any prospect for antiprion immunization. In terms of food safety, surveillance of farm animals for BSE and new atypical TSEs will continue to be important. Equally key for human health is the exclusion of specified risk materials (SRM), such as brain and spinal cord of ruminants, from human and animal food.

12.6 Future trends The extent of exposure of the European population to BSE is unknown. It could well be that millions of people have been exposed to the BSE agent, considering that the prevalence of subclinical BSE in slaughtered cattle may have peaked at about 20% in the UK and was certainly high in other European countries. Fortunately, only approximately 147 cases of vCJD have been recorded so far (as of July 2004) (CJD Surveillance Unit, University of Edinburough; http://www.cjd.ed.ac.uk/figures.htm) Since BSE, in the meantime, has been drastically reduced among cattle, and multiple safety measures have been put into place throughout the human food chain, the danger of contracting a prion infection from cattle is, nowadays, arguably minimal. However, it must be assumed that an unknown proportion of humans are currently subclinically infected with BSE prions. While some of these people may progress to overt disease (vCJD or other, hitherto unrecognized phenotypic manifestations of BSE infection), an unknown proportion of infected individuals may establish a permanent subclinical carrier state. It will be a high public health priority to find out whether – and how – these people may inadvertently pass on the agent to others. For example, the possibility of prion transmission through blood transfusion from preclinical vCJD patients has incited radical measures to prevent this scenario. As a consequence, the United Kingdom and many other countries have introduced universal leukodepletion of blood units. However, it is unknown whether this very costly measure is adequate or necessary, since the partitioning of prion infectivity in blood is unclear. In addition, the United Kingdom is sourcing its entire supply of plasma and plasma-derived products from abroad. Most recently, recipients of blood transfusions have been banned from donating further. By contrast, labile blood products, such as thrombocytes and erythrocytes, are too scarce to be outsourced. Nonetheless, a vCJD case has recently emerged in the UK that might have resulted from

324 Emerging foodborne pathogens transfusing a blood unit from a preclinical vCJD-infected donor. Unfortunately, records show that 48 people in the UK have received blood transfusions from donors who later developed vCJD. In the long run, one would hope that this accrued knowledge will be put into useful practice. What surrogate markers might be useful to improve sensitivity and specificity of prion disease diagnosis? What prevention strategy could be used in people with known exposure to prions or in animals in high risk environments? What treatment strategy could be attempted to curb prion diseases during the clinical phase? The tremendous interest in the prion field has attracted researchers from various neighboring disciplines, including immunology, genetics, and pharmacology, and therefore it is to be hoped that rational and efficient methods for managing prion infections will be developed in the future. Additionally, with the spread of BSE to goats, potential consequences to food safety should be considered and thus an EU Committee has formed to address this potential risk.

12.7 Prion terminology Prion: agent of transmissible spongiform encephalopathy (TSE), with unconventional properties. The term does not have structural implications other than that a protein is an essential component. ‘Protein-only’ hypothesis: maintains that the prion is devoid of informational nucleic acid, and that the essential pathogenic component is protein (or glycoprotein). Genetic evidence indicates that the protein is an abnormal form of PrP (perhaps identical with PrPSc). The association with other ‘noninformational’ molecules (such as lipids, glycosamino glycans, or maybe even short nucleic acids) is not excluded. PrPC: the naturally occurring form of the mature Prnp gene product. Its presence in a given cell type is necessary, but not sufficient, for replication of the prion. PrPSc: an ‘abnormal’ form of the mature Prnp gene product found in tissue of TSE sufferers, defined as being partly resistant to digestion by proteinase K under standardized conditions. It is believed to differ from PrPC only (or mainly) conformationally, and is often considered to be the transmissible agent or prion. Encephalopathy: a general term for diseases affecting the brain, including metabolic, toxic, traumatic, infectious, and neoplastic disorders. Although used to describe the lesions of prion diseases, the transmissible spongiform encephalopathies also affect the spinal cord. Follicular dendritic cells (FDCs): cells with a dendritic morphology that are present in the lymphoid germinal centers, where they present intact antigens

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held in immune complexes or associated with complement receptors to B cells. FDCs are of non-haematopoietic origin, and are not related to dendritic cells. These definitions (Aguzzi and Weissmann 1997) describe our terminology which is, however, not agreed on by convention and is not necessarily used by others.

12.8

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331

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13 Vibrios G. B. Nair, S. M. Faruque and D. A. Sack, ICDDR,B – Centre for Health and Population Research, Bangladesh

13.1

Introduction

Foodborne diseases are increasing in the industrialized as well as in developing countries. The best approximation of the impact of foodborne diseases is available from the United States where an estimated 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths are attributed to foodborne diseases with known pathogens accounting for an estimated 14 million illnesses, 60,000 hospitalizations, and 1,800 deaths (Mead et al. 1999). Foodborne illness can be caused by a variety of etiologies and these include viruses, bacteria, parasites, and can also be induced by chemical and pesticide pollutants. Listeriosis, salmonellosis, campylobacteriosis, Vibrio infections and haemorrhagic colitis are some of the important foodborne diseases of bacterial origin with salmonellosis being the economically most significant disease (Todd 1989). Vibrio species cause a majority of the illnesses among the various human diseases attributed to the natural bacterial flora of seafoods (Food and Nutrition Board, Institute of Medicine 1991). Recently, the Centers for Disease Control and Prevention (CDC) has estimated a 126% increase in the incidence of Vibrio infections in the US between 1996 and 2002 despite efforts directed at seafood consumers to warn them of the potential hazards of eating raw shellfish (Centres for Disease Control and Prevention 2004b). The foodborne illness statistics in Japan also showed unusual changes with the doubling in the number of food poisoning cases of V. parahaemolyticus in 1998 exceeding the number of Salmonella cases, which was the dominant cause of foodborne illness in Japan the previous two years (World Health Organization 1999). Globally, more than 63.5 million tons of seafood are caught and consumed

Vibrios 333 each year (Lipp and Rose 1997) and there has been an overall increase in seafood consumption and a corresponding increase in seafood-related outbreaks of diseases (Butt et al. 2004). Further, the international food trade, especially trade of warm water shrimps, has shown a quantum leap in recent years. The global production of warm water shrimp was about four million tons, of which 1.3 million tons was traded internationally (Food and Agriculture Organization 1998). With the increase in seafood consumption, expansion in aquaculture practices and rapidly expanding international food trade, medical practitioners can expect to see more infections caused by vibrios. Climatic changes associated with global warming resulting in increased ocean surface temperatures may make conditions conducive for growth of halophilic vibrios and thereby enhance the risk of Vibrio foodborne infections. Among the vibrios, Vibrio cholerae, V. parahaemolyticus and V. vulnificus are responsible for most cases of foodborne illness. To a much lesser extent, other vibrios associated with foodborne illness due to consumption of contaminated seafood include V. alginolyticus (Ji 1989), V. mimicus (Campos et al. 1996), V. damsela (Perez-Tirse et al. 1993; Shin et al. 1996), V. hollisae (Abbot and Janda 1994), V. cholerae non-O1 non-O139 (Morris et al. 1981) and V. fluvialis (Thekdi et al. 1990). The epidemiology of V. cholerae and V. parahaemolyticus/vulnificus are somewhat different in the sense for V. cholerae, the contamination through faecal-oral route is particularly important and seafood may not play the same degree of importance in transmission of cholera as it does for Vibrio parahaemolyticus or V. vulnificus infections. In other words for V. cholerae, when food has been the vehicle, a variety of foods have been implicated including, but not exclusively, seafood; whereas for other vibrios the vehicle is primarily seafood. Of all foodborne infectious diseases in the United States, V. vulnificus has the highest (0.39) case fatality rate (Mead et al. 1999). Several recent events underscore the increasing importance of vibrios as foodborne pathogen of marine origin. Notable among these was the sudden appearance of specific serotypes of V. parahaemolyticus that have lately caused a pandemic of gastroenteritis and the escalation of V. vulnificus infection in the US and Taiwan in recent years.

13.2 Taxonomy and brief historical background The family Vibrionaceae includes seven genera (Vibrio, Photobacterium, Allomonas, Listonella, Enhydrobacter, Salinivibrio and Enterovibrio) of which the genus Vibrio has the largest number of species. Vibrios are gram-negative γ-proteobacteria that are ubiquitous in marine, estuarine and freshwater environments and encompass a diverse group of bacteria including many facultative symbiotic and pathogenic strains. Some of the vibrios have multiple life styles that could include a free-swimming planktonic state, a sessile existence attached to zooplankton or shellfish in a commensal association or

334 Emerging foodborne pathogens to other surfaces in the ocean and few of them have the capacity to infect humans, causing intestinal or extra intestinal diseases. Of the 48 currently recognized species in the genus Vibrio, ten are recognized as human pathogens as shown in Table 13.1 (http://www.theicsp.org/taxa/vibriolist.htm#vibrio). V. parahaemolyticus, V. cholerae and V. vulnificus are the most important among the vibrios from a foodborne infection standpoint, and will be dealt with in detail in this chapter. V. cholerae O1 and O139 are causative agents of the disease cholera and are associated with devastating epidemics and pandemics. Koch and co-workers discovered what is now known as V. cholerae O1 in Egypt in 1883 (Barua and Burrows 1974) while O139 emerged in the Indian subcontinent in 1992 (Albert et al. 1993; Nair et al. 1994b; Ramamurthy et al. 1993). V. parahaemolyticus is currently recognized as a major, worldwide cause of gastroenteritis, particularly in the Far East where seafood consumption is high (Miwatani and Takeda 1975). The halophile was first identified as a cause of foodborne illness in Japan in 1950 when 272 individuals became ill, and 20 died after consumption of semidried juvenile sardines (Fujino et al. 1953). V. vulnificus, first identified and described by CDC in 1976 (Hollis et al. 1976), is one of the leading causes of seafood-related illness in the United States and is responsible for more than 95% of all seafood related deaths in this country (Oliver and Kaper 1997).

13.3 Clinical signs and symptoms Infection due to V. cholerae O1 or O139 begins with the ingestion of contaminated food or water containing the organism. After passage through Table 13.1

Vibrio species associated with human infections

Species

V. cholerae O1/O139 V. cholerae non O1 non O139 V. mimicus V. parahaemolyticus V. vulnificus V. alginolyticus V. fluvialis V. furnissii V. hollisae V. metschnikovii

Type strain

Clinical syndrome Diarrhoea

Wound infection

Bacteraemia

ATCC14035/ ATCC51394 N/A

+

–

R

+

+

+

ATCC33653 ATCC17802 ACTC27562 ATC617749 ATC633809 ATC635016 ATC633564 NCT68443

+ + + + + + + R

+ + + + R – R –

+ + + – R – R R

R = rare; N/A = not available; + = prescence of the clinical syndrome; – = absence of the clinical syndrome

Vibrios 335 the acid barrier of the stomach, V. cholerae colonizes the small intestine, and produces cholera toxin (CT) which is mainly responsible for the manifestation of the disease (Kaper et al. 1995). CT acts as a typical A–B type toxin, leading to ADP-ribosylation of a small G protein, and constitutive activation of adenylate cyclase, thus giving rise to increased levels of cyclic AMP within the host cell. This results in the rapid efflux of chloride ions and water from host intestinal cells. The subsequent loss of water and electrolytes leads to the severe diarrhoea and vomiting characteristic of cholera. Massive outpouring of fluid and electrolytes leads to severe dehydration, electrolyte abnormalities, and metabolic acidosis (Rabbani and Greenough 1992). Volunteer challenge studies with V. cholerae have shown that generally a high dose of the organism is required for pathogenesis. Approximately 1011 V. cholerae organisms are required to induce diarrhoea in fasting North American volunteers, unless NaHCO3 was administered to neutralize gastric acid (Cash et al. 1974). When 2.0 g of NaHCO3 is concomitantly administered, 105 or 6 vibrios can induce diarrhoea in 90% of volunteers (Cash et al. 1974; Levine et al. 1981; Sack et al. 1998). Further studies demonstrated that most volunteers who receive as few as 103 to 104 organisms with buffer develop diarrhoea, although lower inocula correlated with a longer incubation period and diminished severity of the disease (Levine et al. 1981). The incubation period generally varies between 25 and 33 hours. In severe disease, death may occur in as high as 50 to 70% of cases if they are not adequately rehydrated (Rabbani and Greenough 1992). V. parahaemolyticus causes three major syndromes of clinical illness that includes gastroenteritis, wound infections, and septicemia. The most common syndrome is gastroenteritis; the symptoms include watery diarrhoea with abdominal cramps, nausea, vomiting, headache and low-grade fever (Honda and Iida 1993). Sometimes the diarrhoea is bloody with stools described as ‘meat washed’ since the stool is a reddish, watery stool (Qadri et al. 2003). The illness is self-limiting and lasts an average of three days in immunocompetent patients. Rarely, sudden cardiac arythmia has been reported (Honda et al. 1976). The mean incubation period for V. parahaemolyticus infection is 15 hours (range 4–96 hours). Under appropriate conditions, this halophile has an extremely short generation time ranging from 8 to 12 minutes and doubling times of 27 minutes have been reported in crabmeat at both 20 and 30 °C (Liston 1974). Infection can cause serious illness in persons with underlying disease (Hlady and Klontz, 1996), especially in immunocompromised individuals, e.g. leukemia, liver disease, diabetes and those infected with HIV-AIDS where it can cause serious systemic infections (Hsu et al. 1993; Hally et al. 1995; Ng et al. 1999). Infection with V. parahaemolyticus results in B cell responses and an acute inflammatory response that is self limiting with features similar to those seen in disease caused by Shigella species (Raqib et al. 2000) and is more severe than those seen in disease caused by V. cholerae O1 or O139 (Qadri et al. 2003). Although V. parahaemolyticus and V. cholerae

336 Emerging foodborne pathogens causes similar diseases, the two vibrios use distinct mechanisms to establish infection (Makino et al. 2003). Diseases associated with V. vulnificus infection present in two patterns that includes localized wound infections acquired through exposure of a wound to salt water or shellfish and primary septicemia acquired through oral ingestion of the organisms with raw oysters as the most common vehicle (Blake et al. 1979). Most patients suffering from V. vulnificus primary septicemia have an underlying chronic disorder such as chronic cirrhosis, haemochromatosis, thalassemia, elevated serum iron level, immune function abnormalities and chronic renal insufficiency and HIV-AIDS (Johnston et al. 1985; Oliver and Kaper 1997; Tacket et al. 1984). Opportunistic infections in susceptible individuals typically cause mortality within 24 to 48 h of the exposure. Wound infection can occur in the absence of predisposing conditions but progresses more frequently to septicemia and has a higher mortality rate in predisposed people (Cerveny et al. 2002). A hallmark of V. vulnificus infection is the fulminant reaction caused by the invading bacteria in connective tissues, displayed as blisters and haemorrhagic necrosis (Chuang et al. 1992). Even in non-fatal cases, V. vulnificus infection evokes intensive tissue damage and occasionally results in amputation and disability (Chen et al. 2003).

13.4 Virulence factors V. parahaemolyticus strains that are isolated from diarrhoeal patients produce either the thermostable direct haemolysin (TDH) or the TDH-related haemolysin (TRH), or both, while hardly any isolates from the environment have these properties (Honda and Iida 1993; Shirai et al. 1990; Takeda, 1983). A strain producing TDH is referred to as Kanagawa positive (KP) and can be identified by β haemolysis on Wagatsuma blood agar (Takeda 1983; Wagatsuma 1974). TDH has been shown to have haemolytic, enterotoxic, cardiotoxic and cytotoxic activities (Honda and Iida 1993; Nishibuchi et al. 1992; Shirai et al. 1990; Takeda, 1983). Biological, immunological, and physicochemical characteristics of TRH are similar but not identical to those of TDH (Honda et al. 1988). TDH and TRH are each composed of 165-amino acid residues and show approximately 67% identity in the amino acid sequences (Honda and Iida 1993; Park et al. 2000). A strong correlation between urease production (unusual phenotype for V. parahaemolyticus) and trh gene exists (Okuda et al. 1997). Enteroinvasiveness of the bacteria has been reported in a rabbit model, in which the organism invaded, colonized, and produced inflammation in the small intestine (Chatterjee et al. 1984). The overall mechanism of pathogenesis by V. parahaemolyticus, however, remains unclear. Among the 206 currently recognized O serogroups of V. cholerae (Yamai et al. 1997), only O1 and O139 serogroups are associated with the disease cholera. This is related to the observation that more than 95% of the strains belonging to O1 and O139 serogroups produce cholera toxin (CT). In contrast,

Vibrios 337 more than 95% of the strains belonging to non-O1 non-O139 serogroups do not produce CT (Kaper et al. 1995). Another important virulence factor of V. cholerae is the toxin-coregulated pilus (TCP), which is an adhesin that is coordinately regulated with CT production (Taylor et al. 1987). TCP is the only V. cholerae pilus that has been demonstrated to date to have a role in colonization of the gut mucosa of humans (Herrington et al. 1988) and of infant mice (Taylor et al. 1987) the latter being an experimental cholera model. The genes encoding CT form part of the genome of a lysogenic filamentous bacteriophage, designated as CTXφ (Waldor and Mekalanos et al. 1996). The pilus colonization factor TCP is also known to act as a receptor for CTXφ, which can infect nontoxigenic V. cholerae, leading to the emergence of new toxigenic strains. The tcpA gene is part of a pathogenicity island of about 39.5 kb size known as V. cholerae pathogenicity island (VPI) (Karolis et al. 1998). V. vulnificus is a highly invasive pathogen, being able to reach the bloodstream and cause septicemia via translocation across the intact intestinal wall but little is known about the virulence mechanism of this organism. V. vulnificus is divided into three biotypes based on differences in biochemical and biological properties (Linkous and Oliver 1999). Among the three biotypes, strains belonging to biotype 1 (indole-negative) are most frequently isolated from clinical specimens. Biotype 2 strains have been reported to cause disease mainly among eels and rarely infect humans (Amaro and Biosca, 1996). Biotype 3 was associated with a major outbreak of systemic V. vulnificus infections started among Israeli fish market workers and fish consumers (Bisharat and Raz, 1996; Bisharat et al. 1999). Recent multilocus genotype data and modern molecular evolutionary analysis have shown that biotype 3 is a hybrid organism that evolved by the hybridization of the genomes from two distinct and independent populations of V. vulnificus (Bisharat et al. 2005). Strains of V. vulnificus secrete a variety of products that have been implicated in bacterial virulence and pathogenesis, including capsular polysaccharide (Wright et al. 1990), cytolysin (Gray and Kreger 1985; Kreger and Lockwood, 1981), metalloprotease (protease) (Kothary and Kreger 1985, 1987; Miyoshi et al. 1987), phospholipases (Testa et al. 1984) and siderophores (Okujo and Yamamoto, 1994). Collectively, the cytolysin and the protease are thought to be important for the pathogenesis of V. vulnificus. The primary virulence factor is the polysaccharide capsule, which prevents phagocytosis and activation of complement (Gray and Kreger 1985; Shinoda et al. 1987; Tamplin et al. 1985; Yoshida et al. 1985). The ability to acquire iron from the host via siderophore production is also an essential virulence attribute (Litwin et al. 1996). Biochemical and genetic studies suggest that extracellular proteins released by the invading bacteria mediate the pathogenesis process of penetrating cellular barriers, vascular dissemination, and local destruction of affected tissues (Chen et al. 2003). Multifactor interaction in bacterial virulence is likely to produce the dramatic infection caused by V. vulnificus

338 Emerging foodborne pathogens (Hsueh et al. 2004). What host aspects are essential to infection are yet to be elucidated.

13.5 Epidemiology of Vibrio infections 13.5.1 Vibrio cholerae Cholera is a water and foodborne disease. The importance of water ecology is suggested by the close association of V. cholerae with surface water and the population interacting with the water (Sack et al. 2004). The faecal-oral transmission of cholera usually occurs by the ingestion of faecally contaminated water by susceptible individuals. Besides drinking water, food has also been recognized as an important vehicle of transmission of cholera (Table 13.2). The disease is endemic in Southern Asia and parts of Africa and Latin America, where outbreaks occur regularly and is particularly associated with poverty and poor sanitation (Fig. 13.1). A distinctive epidemiological feature of cholera is its appearance with seasonal regularity in endemic areas, and in explosive outbreaks often starting simultaneously in several distinct foci (Glass and Black 1992; Kaper et al. 1995) indicating a possible role of environmental factors in triggering the epidemic process. However, the underlying mechanisms are still not well understood. It is possible that an undefined environmental signal triggers a rapid increase in the concentration of V. cholerae in the environment. As human cases start to occur, they, in turn, amplify the number of organisms present, leading to even more cases. Colwell and Spira (1992) have hypothesized that V. cholerae colonize copepods and other zooplankton, and zooplankton blooms play a key role in this process. However, since infection due to V. cholerae occurs exclusively through the oral route, contaminated food and water are the direct sources of human infection, while environmental factors are likely to influence the seasonal prevalence of V. cholerae in the aquatic environment, as well as their survival and epidemic spread. Numbers of culturable V. cholerae isolated from environmental waters are usually far less than the required dose for a severe infection, and the majority of natural infections due to V. cholerae are asymptomatic or lead to mild disease (McCormack et al. 1969). However, it seems likely that a pre-enrichment of the organism in contaminated food may be important in the epidemiology of cholera, particularly the occurrence of the first case of cholera, which subsequently leads to an epidemic, fostered by contaminated water, and poor sanitation. In developing countries, where both poverty and poor sanitation are common, faecal contamination of domestic and commercial food is likely to occur, and in many outbreaks the infection has been traced to consumption of faecally contaminated foods. Persons with acute cholera excrete 107 to 108 V. cholerae organisms per gram of stool (Levine et al. 1988), and total output of V. cholerae by a patient can be in the range of 1011 to 1013 CFU. This large number of organisms can contaminate environmental waters, and people

Table 13.2

Examples of foodborne outbreaks of cholera reported in the literature*

Year

Place

Food

No. of cases

Reference

1962 1963 1970 1972 1973 1974 1974 1974–1986 1977 1978 1978 1978 1978 1979 1981 1982 1982 1984 1984 1986 1986 1986 1987 1988

Philippines Hong Kong Israel Australia Italy Portugal Portugal Guam Gilbert Island Singapore USA Bahrain Japan Spain USA Micronesia Singapore India Mali Guinea USA USA Thailand Thailand

Raw shrimp Cold cooked meat Raw vegetables Mixed hors d’oeuvre Raw mussels, clams Bottled mineral water Seafood Raw seafoods Raw & salted fish and clams Steamed prawn, chicken, rice Crabs Bottle-feeding of infants Rock lobster Raw fish Cooked rice Shellfish/leftover rice Cooked squids Ice candies Milted gruel Peanut sauce/cooked rice Crabs/shrimps Raw oysters Raw pork Raw beef

– 5 258 25 278 136 2467 19–46 572 12 11 42 18 267 15 509 22 22 1793 35 18 2 130 52

Felsenfeld (1972) Teng (1965) Fattal et al. (1986) Sutton (1974) Baine et al. (1974) Blake et al. (1977) Blake et al. (1977) Haddock (1987) McIntyre et al. (1979) Khan et al. (1987) Blake (1980) Gunn et al. (1979) Fukumi (1980) World Health Organization (1980) Johnston et al. (1983) Holmberg et al. (1984) Goh et al. (1984) Patnaik et al. (1989) Tauxe et al. (1988) St Louis et al. (1990) Lowry et al. (1989) Klontz et al. (1987) Swaddiwudhipong et al. (1990) Swaddiwudhipong et al. (1992)

Table 13.2 Continued Year

Place

Food

1989 1991 1991 1991 1992

Philippines USA Ecuador USA South America to Los Angeles, USA Guatemala USA Thailand Hong Kong Lusaka, Zambia

Street food Imported crabs Seafood Imported frozen coconut milk Cold seafood salad (international flight)

1993 1994 1994 1994 2004

Leftover rice Imported food (palm fruit) Yellow rice Seafood Raw vegetables

No. of cases – 4 – 3 75 26 2 6 12 2529

Reference Lim-Quizon et al. (1994) Centers for Disease Control and Prevention (1991) Weber et al. (1994) Lacey et al. (1991) Eberhart-Phillips et al (1997) Koo et al. (1996) Centers for Disease Control and Prevention (1995) Boyce et al. (1995) Kam et al. (1995) Centers for Disease Control and Prevention (2004a)

This table has been taken from the book, Food Borne Disease: A focus for Health Education (World Health Organization 2000). Information for this Table was obtained from Quevedo (1993) and Albert et al. (1997). The table has been updated with some recent foodborne outbreaks caused by Vibrio cholerae O1/O139

Vibrios 341 Cholera 2004–2005

Countries with imported cholera cases Countries with reported cholera cases

Fig. 13.1 Global incidence of cholera (this map was provided by the kind courtesy of L. Olsson and PA Parment, SBL Vaccin AB, Stockholm, Sweden). Information from different sources was used to create the updated map and an updated list of cholera in the world. http://www.sblvaccines.se/uploads/Cholera-2004-20052.jpg)

dependent on such water for household purposes are likely to contaminate food. Even after cessation of symptoms, patients who have not been treated with antibiotics may continue to excrete vibrios for one to two weeks (Levine et al. 1988), and a small minority of patients may continue to excrete the organism for even longer periods of time. Asymptomatic carriers are most commonly identified among household members of persons with acute illness. Volunteer studies have shown that when stomach acidity is neutralized with sodium bicarbonate, administration of an inoculum of 106 V. cholerae cells can result in an attack rate of 90% (Levine et al. 1981). Food has a buffering capacity comparable to that seen with sodium bicarbonate. Ingestion of 106 vibrios with food such as fish and rice resulted in the same high attack rate (100%) (Levine et al. 1981). One important epidemiological finding is that an initial infection with V. cholerae provides protection against subsequent disease. In endemic areas such as Bangladesh, cases of cholera are highest in children aged two to nine years (Glass et al. 1982). The decreased rates in children under the age of one year may relate to decreased exposure, and to the protective effect of breast-feeding or breast milk (Clemens et al. 1990). In contrast to the above findings, cholera, when introduced into populations lacking prior exposure to the disease, tends to occur with equal frequency in all age groups (Glass and Black 1992, Holmberg et al. 1984), and this was clearly observed in the South American epidemics (Swerdlow et al. 1992). A similar pattern was seen in the initial epidemics with V. cholerae O139 strains in Bangladesh. In contrast to findings with endemic O1 strains, the majority of O139 cases

342 Emerging foodborne pathogens occurred in adults (Albert et al. 1993). Susceptibility to cholera depends also on largely unknown host factors. Individuals of blood group O are at increased risk of more severe cholera, which has been shown for natural infection (Clemens et al. 1989; Glass et al. 1985) as well as with experimental infection (Levine et al. 1979). In cholera endemic regions of Asia, including Bangladesh, contamination of food is likely to be an important factor in the transmission of cholera (Spira et al. 1980). Water may serve as a source of secondary contamination of food during its preparation. In endemic areas, transmission of cholera through contaminated foods served by street vendors and restaurants should be considered. In the developed countries, foodborne outbreaks of cholera have on many occasions occurred due to consumption of contaminated seafood. It is clear that CT-producing V. cholerae O1 or O139 can persist in the environment in the absence of known human disease. Periodic introduction of such environmental isolates into the human population through ingestion of uncooked or undercooked shellfish appears to be responsible for isolated foci of endemic disease along the U.S. Gulf Coast and in Australia (Blake et al. 1979, Lowry et al. 1989; Tacket et al. 1984). Environmental isolates contaminating seafood may also have been responsible for the initial case clusters in the South American epidemic. Food has been frequently implicated as a vehicle responsible for introduction of cholera into a new area (Kaper et al. 1995; Glass and Black 1992). In studies in Piura, Peru, drinking unboiled water, eating food from a street vendor, and eating rice after three hours without reheating were all independently associated with illness (Ries et al. 1992). The survival and growth of V. cholerae in foods depend on the physico-chemical properties of the particular foods that have been contaminated. Food characteristics, which enhance the growth of V. cholerae, are low temperature, high-organic content, neutral or alkaline pH, high-moisture content, and absence of other competing microorganisms in the food (Depaola 1981; Pan American Health Organization 1991; Singleton et al. 1982). Of particular concern from a public health perspective is that V. cholerae can survive in water and contaminate foods where it can grow in enough numbers to cause illness in people consuming the food or drink. Food can also provide an ideal culture medium: cooked rice, for example, has been shown to support rapid growth of V. cholerae (Kolvin and Roberts 1982). V. cholerae can survive for more than two weeks in different dairy products, including milk, milk products, soft desserts, and cakes. Addition of sugar and eggs enhances bacterial survival. Although V. cholerae is killed by pasteurization of milk, the organisms can persist in raw milk for as long as four weeks, even if refrigerated. Contamination of meat of animal origin occurs exogenously during processing, cooking, storage, or consumption. It has been shown that V. cholerae can live and grow on cooked chicken; an increase in numbers of V. cholerae from 103 to 106 within 16 hours has been demonstrated (Kolvin and Roberts 1982). There are many other types of

Vibrios 343 food that may be contaminated with V. cholerae, for example V. cholerae can survive on cooked potatoes, eggs, and pasta for up to five days, and can also survive in spices, including pepper and cinnamon, for up to several days. V. cholerae is very sensitive to heat, and is rapidly killed when exposed to a temperature of 100 °C. Drying and exposure to sunlight is also an effective means of killing V. cholerae (Kaper et al. 1982; Pan American Health Organization 1991). V. cholerae can survive domestic freezing and can be found after a long period in a frozen state. Leftover rice eaten with tomato sauce, having an acidic pH, unfavourable for V. cholerae, was not associated with cholera cases (St. Louis et al. 1990). The pH of a specific fruit is an important factor that influences contamination by V. cholerae. Sour fruits, such as lemons and oranges, with lower pH (below 4.5) do not support the growth of V. cholerae, and, thus, do not pose risk of cholera transmission. Fruit pulp and concentrate preserved in cans are also less likely to be contaminated if they have an acidic pH. Spices, including raw onions and garlic, can support the survival of V. cholerae for two to three days at ambient temperature (Pan American Health Organization 1991). During the course of the seventh cholera pandemic, contaminated seafoods have been identified as the source of infection in several outbreaks. Seafoods and seafood products most frequently incriminated are shellfish. These foods have been identified as a source of repeated outbreaks in the Unites States and elsewhere (Centers for Disease Control 1991; Gergatz and McFarland 1989; Lowry et al. 1989). Fish becomes infected with V. cholerae either due to sewage contamination of water or by ingestion of aquatic vegetation and zooplankton infested with V. cholerae (Huq et al. 1983). Zooplankton secretes a self-protective coat of chitin that can be dissolved by chitinase, an enzyme produced by V. cholerae O1. Seafoods, including molluscs, crustaceans, crabs, and oysters, feed on plankton and can become infected with V. cholerae (Huq et al. 1983). Once infected, particularly clams and oysters can harbour V. cholerae for weeks, even if refrigerated (DePaola 1981). In crabs, the organisms can rapidly multiply at ambient temperature, and boiling for less than ten minutes or steaming for less than 30 minutes does not completely kill V. cholerae (DePaola 1981). In a food survey in Taiwan, 1,088 vibrios, including V. cholerae and other species, were isolated from seafoods and aquacultured foods (Wong et al. 1992). In many countries, fish is eaten raw or undercooked (Klontz et al. 1987, Wong et al. 1992). Outbreaks of cholera due to consumption of raw fish have been reported from Japan as early as 1886 (Pavia et al. 1987). Fish may serve as an important vehicle of transmission of cholera in the endemic areas of Asia, where it is a major food item and is likely to be contaminated by V. cholerae due to both poor environmental sanitation and poverty. Several other examples of foodborne outbreaks are as follows. In 1978, Singapore experienced a cholera outbreak, which was traced to consumption of prawns and squid that were likely to be contaminated by infected food handlers (Goh 1979). In 1979, an outbreak of cholera occurred in Sardinia;

344 Emerging foodborne pathogens the source of infection was traced to eating of bivalves from which V. cholerae O1 were isolated (Salmaso et al. 1980). A statistically significant association of cholera with shellfish consumption in Italy in 1973 has shown the importance of shellfish as a vehicle of cholera transmission (Baine et al. 1974; De Lorenzo et al. 1974). In the autumn of 1991, a single cholera case was identified in an oil rig barge in Texas, which was followed by 13 secondary cases of cholera and one asymptomatic infection (Weissman et al. 1974). The source of infection in the index case was traced to consumption of infected seafoods from local water. The secondary cases were infected by consuming rice prepared with water contaminated by the faeces of the index case through cross-connection between a sewer drain and the drinking water supply. Since 1973, a total of 65 cholera cases have been associated with the Gulf Coast reservoir in the United States. In 1971 cholera reappeared in Africa, after an absence of 70 years, and 30 of the 46 countries started reporting cholera. During a cholera outbreak in Mali in 1984, a case-control study showed that eating leftover millet gruel by villagers in an arid region was associated with cholera (St. Louis et al. 1990). In another outbreak in Guinea, consumption of leftover rice with peanut sauce was incriminated as the vehicle of transmission of cholera. Consumption of improperly cooked horsemeat was incriminated in a small outbreak of cholera in Berlin in 1918 (Kolvin and Roberts 1982). An infected butcher who succumbed to cholera the next day had prepared the meat. The importance of contaminated vegetables as a vehicle of cholera transmission is indicated by an outbreak in Jerusalem (Johnston et al. 1983) and some of these cases was shown to be infected by secondary spread of V. cholerae through consumption of vegetables contaminated by faeces. In many countries, the practice of fertilizing gardens with untreated night soil and the habit of consuming uncooked vegetables have often resulted in cholera outbreaks. Vegetables may be contaminated during washing with polluted water. This can also occur when contaminated water is injected into fruits, such as watermelons, to preserve their weight and taste (Feachem 1981). 13.5.2 Vibrio parahaemolyticus V. parahaemolyticus is widely disseminated in estuarine, marine and coastal environments throughout the world (Joseph et al. 1982) and has been detected as far north as in Alaska (Vasconcelos et al. 1975). It has also been reported either as a source of human disease or in the environment along the North American, African, and Mediterranean coasts (Barbieri et al. 1999, Eko et al. 1994, Daniels et al. 2000). The organism accounts for nearly half or more of the bacterial foodborne illness cases in Japan (Zen-Yoji et al. 1965) and in Taiwan (Pan et al. 1996). Surprisingly, V. parahaemolyticus is also an important etiological agent of diarrhoea in inland areas like Calcutta, India.

Vibrios 345 Etiological studies on acute diarrhoeal diseases have shown that gastroenteritis caused by V. parahaemolyticus ranks second to cholera in terms of incidence in Calcutta (Chatterjee et al. 1970; Sakazaki et al. 1971). Epidemiological studies have further revealed the high incidence of human carriers of V. parahaemolyticus in this metropolis (Pal et al. 1984). V. parahaemolyticus was the most commonly isolated Vibrio species isolated during a year-long surveillance along the Gulf Coast of the United States, as well as over a 13-year period in Florida. In a population-based study that relied on passive surveillance in the Khanh Hoa province of Vietnam, a surprising risk factor for V. parahaemolyticus infection was high socioeconomic status (Tuyet et al. 2002). The explanation for this finding was that only the more affluent members of the community could afford seafood thereby attesting the importance of seafood in acquiring infection caused by V. parahaemolyticus. Water temperature, salinity, zooplankton blooms, tidal flushing and dissolved oxygen play an important role in dictating the spatial and temporal distribution of V. parahaemolyticus (Kaneko and Colwell 1978). This pathogen is typically not recovered from estuarine waters during winter months in temperate zones when water temperature is too low for its existence. Water temperatures have been shown to influence the growth of V. parahaemolyticus (Kaneko and Colwell 1975; Kaper et al. 1981; Kelly and Stroh, 1981; Thompson et al. 1976) and the importance of water temperature in the epidemiology of infections is reflected by the fact that most outbreaks occur during the warmer months. Outbreaks of V. parahaemolyticus infections are most common in Japan and Southeast Asia where the consumption of raw or undercooked fish and shellfish is high; they occur occasionally in North and South America and rarely in Europe. Foodborne outbreaks of V. parahaemolyticus have been reported from several countries including Bangladesh, Canada, Guam, India, Thailand, Taiwan Russia, Peru, Senegal and Nigeria. This pathogen is one of the most important foodborne pathogen in Taiwan, Japan and other coastal regions. Most V. parahaemolyticus outbreaks that occurred between 1973 and 1998 in the US occurred during the warmer months were attributed to seafood, particularly shellfish, and had a median attack rate among persons who consumed the implicated seafood of 56% (Abbot and Janda 1994). In tropical countries, in contrast, the seasonality of V. parahaemolyticus is less defined with infection occurring throughout the year. Studies in Calcutta have shown that both marine and freshwater fish provide an ideal substrate for the survival and proliferation of V. parahaemolyticus. They attributed the isolation of V. parahaemolyticus in market samples of freshwater fish to the cross-contamination due to mishandling at the fishmongers’ stalls (Sarkar et al. 1985). 13.5.3 Vibrio vulnificus V. vulnificus is present in tropical and temperate estuarine ecosystems throughout the world. Infection due to this organism has been reported from

346 Emerging foodborne pathogens USA, Europe, Korea, Taiwan and other countries (Chuang et al. 1992; Dalsgaard et al. 1995; Hlady and Klontz 1996; Park et al. 1991). One factor that influences the incidence of disease caused by V. vulnificus is the prevalence of this organism in the environment. The incidence of V. vulnificus disease parallels its concentration in oyster tissues, with the greatest number of infections occurring during the summer months when seawater temperature ranges between 20 and 30 °C (Hlady et al. 1993; Howard et al. 1988). The relationship between environmental factors and V. vulnificus densities in oysters collected monthly in 14 states in the US showed that the levels ranged from none detected to 1,100,000 per gram (Tamplin 1994). The concentration of V. vulnificus in oysters across the northern gulf coast was influenced primarily by water temperature and salinities below 25 parts per thousand (Motes et al. 1998). Variations in surface water temperature above 26 °C have little effect on densities, but the densities decline rapidly as temperature declines below 26 °C (Motes et al. 1998). The relationship between disease and high infective dose is also supported by data showing that V. vulnificus infections do not occur during cold months when the numbers of organisms are very low, even though greater numbers of oysters are consumed in winter months than in summer months (Tamplin et al. 1996). In Taiwan, V. vulnificus infections are rising and one of the factors associated with this increase is the high prevalence of hepatitis B or C virus infection-related hepatic diseases (liver cirrhosis and hepatoma), the environment, and the popularity of preparing and eating raw or under-cooked seafood (Chuang et al. 1992; Chiang and Chuang 2003). Infection due to V. vulnificus is mainly due to consumption of raw molluscan shellfish particularly filter-feeding oysters. Estimates of the prevalence of V. vulnificus in oysters from the Gulf of Mexico during the summer months have been as high as nearly 100% (Motes et al. 1998). Examination of oysters from three geographically distinct estuaries on the northern Gulf Coast showed that MPN counts were usually 103 to 104 per gram during warm weather months when >85% of the shellfish-associated V. vulnificus cases occur as compared to 105 per gram were observed in oysters during the summer of 1991 in Apalachicola Bay (Jackson et al. 1997). The multiplication of V. vulnificus in summer harvest oyster shell stock held without refrigeration has been shown to be rapid (Cook, 1997). However, the numbers of V. vulnificus organisms do not differ significantly from those at the time of harvest through 30 hours of storage if shell stock are chilled immediately after harvest and stored at temperatures of 90% positive, +/–, variable; >50% positive, –/+, variable; 32 parts per thousand) was shown to reduce V. vulnificus numbers by 3–4 logs (10 tdh- and/or trh-positive V. parahaemolyticus per g in environmental oysters should be considered extraordinary (DePaola et al. 2000). Bacteriological monitoring at harvest sites in the Galveston Bay, however, did not prevent the outbreak in Texas suggesting that current policy and regulations regarding the safety of raw oysters require reevaluation (Daniels et al. 2000). Regulations of the US Food and Drug Administration therefore require additional specific bacteriologic monitoring in shellfish, with a requirement that shellfish have less than 10,000 V. parahaemolyticus organisms per gram of meat. Following the Texas outbreak, Daniels et al. (2000) were of the opinion that V. parahaemolyticus prevention strategies should be based on environmental trigger points, sampling schemes, public education, and the use of new technologies (e.g., pasteurization or radiation) to reduce or eliminate contamination (Daniels et al. 2000).

13.11

Vibrios: the genomic era

The whole genome sequences of V. cholerae, V. parahaemolyticus and V. vulnificus have become available in the past four years reflecting their significance as important human pathogens. A distinctive feature of the genome of the genus Vibrio is that most, if not all, species have two circular chromosomes, a large and a small chromosome. It has been proposed that the genes on the large and small chromosomes of V. cholerae function differently depending on the environments encountered by the organism and the organism adapts to different situation by varying the copy number of the chromosomes (Heidelberg et al. 2000; Trucksis et al. 1998; Yamaichi et al. 1999). Sequencing of the whole genome of a strain each of V. cholerae (Heidelberg et al. 2000), V. parahaemolyticus (Makino et al. 2003) and V. vulnificus (Chen et al. 2003) has shown the presence of super integrons that provide additional sources of genetic variability by their ability to incorporate ORFs and convert exogenous sequences into functional genes (Hall and Collis 1995; RoweMagnus et al. 2001). Based on genome sequence information, it has been postulated that the small chromosome of V. cholerae may have originally been a mega plasmid that was captured by an ancestral Vibrio species (Heidelberg et al. 2000). Comparison of the V. parahaemolyticus genome with that of V. cholerae showed many rearrangements within and between the two chromosomes and

Vibrios 359 the presence of genes for the type III secretion system in V. parahaemolyticus which was not identified in V. cholerae. Genome sequencing has shown that V. parahaemolyticus may have a characteristic common among diarrhoeacausing pathogens such as Shigella, Salmonella and enteropathogenic Escherichia coli, which cause inflammatory diarrhoea by invading or intimately interacting with host intestinal epithelial cells (Makino et al. 2003). The genomic constitution and organization of the three vibrios show how rapid genome evolution has enabled Vibrio species to survive frequently changing conditions in aquatic environments.

13.12 Acknowledgement We acknowledge with gratitude core donors to the ICDDR,B who support our work. Current donors providing unrestricted support include the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Japan, Kingdom of Saudi Arabia, the Netherlands, Sweden, Sri Lanka, Switzerland and the United States of America.

13.13

References

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14 Yersinia enterocolitica T. Nesbakken, Norwegian School of Veterinary Science, Norway

14.1

Introduction

The chapter starts with a description of the agent, the emergence of yersiniosis and the historical aspects related not only to Yersinia enterocolitica, but also to Yersinia pestis and Yersinia pseudotuberculosis. Characteristics and taxonomy are important since Yersinia enterocolitica is the only foodborne agent, but has close relations to Y. enterocolitica-like bacteria, and correlations between biovars, serovars, ecology and pathogenicity mean that phenotypic characterisation is relevant. In many instances, attempts to isolate Y. enterocolitica from foods implicated in cases of disease in humans have been unsuccessful. Accordingly, efficient isolation and detection methods are described. Under the heading of epidemiology, clinical symptoms of Y. enterocolitica infection, pathogenesis and immunity, sporadic cases, outbreaks and sources of infection like pigs and pork are discussed. Risk factors connected to the agent (plasmid and chromosomal), the host (infection and immunity) and the agent’s ability to survive and grow under different conditions in foods are presented. An important property of the bacterium is its ability to multiply at temperatures near to 0 °C, and therefore in many chilled foods. Evidence from large outbreaks of yersiniosis in the USA, Canada and Japan (Cover and Amber, 1989) and from epidemiological studies of sporadic cases (Ostroff et al., 1994; Tauxe et al., 1987) has shown that Y. enterocolitica is a foodborne pathogen, and that in many cases pork is implicated as the source of infection (Hurvell, 1981; Ostroff et al., 1994; Tauxe et al., 1987). Preventive measures and control in the food chain are possible, and some examples of successful control measures in the abattoir are described. Possible interventions at different stages of the food chain are discussed.

374 Emerging foodborne pathogens 14.1.1 Historical aspects The genus Yersinia of the family Enterobacteriaceae includes three wellestablished pathogens (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica) and several non-pathogens (Mollaret et al., 1979). Y. pestis was isolated by Alexandre Yersin in 1894 (Yersin, 1894). The most important Yersinia infection, plague, caused by Y. pestis, is one of the oldest recognised human diseases. Historically, Y. pestis evolved from Y. pseudotuberculosis (Achtman et al., 1999) while Y. enterocolitica is distantly related to Y. pseudotuberculosis and Y. pestis (Chapter 1). Although infections caused by Y. pseudotuberculosis and Y. enterocolitica have been reported regularly only during the last 30 years, it is nevertheless likely that these infections have also occurred for many years. Disease due to Y. pseudotuberculosis (first described in 1884) has been recognised since the beginning of the 20th century, and Y. enterocolitica was first shown to be associated with human disease in 1939 (Mollaret, 1995). The current interest in Y. enterocolitica started in 1958 following a number of epizootics among chinchillas and hares (Hurvell, 1981; Mollaret et al., 1979), and after the establishment of a causal relationship with abscedising lymphadenitis in man. The similarity between the human and animal isolates was established in 1963, and in 1964 the species name Y. enterocolitica was formally proposed by Frederiksen (1964). During the past thirty years, the bacterium has been found with increasing frequency as a cause of human disease, and from animals and inanimate sources.

14.2 Taxonomy and characteristics of Yersinia enterocolitica A general numerical taxonomic study from 1958 placed Yersinia between Klebsiella and Escherichia coli (Sneath and Cowan 1958). The allocation of Yersinia to the family Enterobacteriaceae was further supported by Frederiksen (1964). Y. enterocolitica is a Gram-negative, oxidase-negative, catalase-positive, nitrate reductase-positive, facultative anaerobic rod (occasionally coccoid), 0.5–0.8 × 1–3 µm in size (Bercovier and Mollaret, 1984). It does not form a capsule or spores. It is non-motile at 35–37 °C, but motile at 22–25 °C with relatively few, peritrichous flagellae. Some human pathogenic strains of serovar O:3 are, however, non-motile at both temperatures. In addition, the bacterium is urease-positive, H2S-negative, ferments mannitol, and produces acid, but not gas, from glucose (Bercovier and Mollaret, 1984). Aspects of survival and growth are described in Section 14.8 ‘Risk factors in connection with survival and growth in foods’. 14.2.1 Differentiation of Y. enterocolitica from other Yersinia spp. A range of strains of Yersinia variants have been isolated from animals, water and food (Hurvell, 1981; Lee et al., 1981; Mollaret et al., 1979). Many

Yersinia enterocolitica

375

of these bacteria have characteristics that deviate considerably from the typical pattern shown by Y. enterocolitica, but can be classified as belonging to the genus Yersinia (Mollaret et al., 1979). Such Y. enterocolitica-like bacteria have been divided on a genetic basis into seven species (Aleksic et al., 1987; Bercovier et al., 1980a,b, 1984; Brenner, 1981; Brenner et al., 1980a, b, Ursing et al., 1980; Wauters et al., 1988b): Yersinia frederiksenii, Yersinia kristensenii, Yersinia intermedia, Yersinia aldovae, Yersinia rohdei, Yersinia mollaretii and Yersinia bercovieri.

14.3 Phenotypic characterisation 14.3.1 Biotyping The bacteria that are currently classified as Y. enterocolitica do not constitute a homogeneous group. Within the species there is a wide spectrum of biochemical variants. Such variations form the basis for dividing Y. enterocolitica into biovars. Wauters (1991) described a biotyping scheme that differentiates between pathogenic (biovars 1B, 2, 3, 4, 5) and nonpathogenic (only biovar 1A) variants.

14.3.2 Serotyping by using O-antigens Y. enterocolitica can be divided into serovars using O-antigens. So far, 76 different O-factors have been described in both Y. enterocolitica and Y. enterocolitica-like bacteria (Wauters, 1991). A few strains, however, cannot be typed by this system, and the number of described antigen factors is, therefore, likely to increase in the future.

14.3.3 Correlation between biovars, serovars, ecology and pathogenicity Although some antigenic factors are linked to pathogenic strains, for example O:3, O:9, O:8 etc., serotyping alone cannot be used to indicate pathogenicity because these antigenic factors also occur in non-pathogenic species or biovars. For instance, factor O:3 is common in Y. frederiksenii, and may also be encountered in other species and in biovar 1A of Y. enterocolitica. Factor O:8 occurs in biovar 1A and in Y. bercovieri, and factor O:9 in Y. frederiksenii. Hence, antigenic typing of the strains should always be done after appropriate biochemical characterisation. The relationships between the biovars, the Oantigens and the ecology of Y. enterocolitica and related species are presented in the list below (Wauters, 1991). In addition, geographic distributions are presented in Table 14.1. Biovar 1A includes a large number of serovars which are found in the environment, in food and occasionally in the digestive tract of animals and

376 Emerging foodborne pathogens Table 14.1

Correlation between biovars, serovars, sources and geographic distribution

Serovar

Biovar

Main source(s)

Geographic distribution

O:3 O:3 O:5,27 O:8 O:9 O:13a and O:13b O:21

4 3 2 1B 2 1B 1B

Pig Pig Several Several Pig Several Several

Originally, Europe – now global Japan, East Asia Mainly North America Mainly North America Originally, Europe – also found global Mainly North America Mainly North America

humans. They do not possess virulence properties and are of little or no clinical significance for man. Biovars 1B, 2, 3, 4 and 5 are potential pathogens for man or animals. They exhibit pathogenic properties and, when freshly isolated, usually harbour the virulence plasmid. Strains of biovar 1B belong to a small number of pathogenic serovars, the most frequent being O:8, O:21, O:13a and O:13b, whereas O:4, O:18 and O:20 occur less frequently. They have been isolated mainly in the United States and were therefore called ‘American strains’. Since the beginning of the 1990s, a few strains have been isolated outside North-America; in Europe, Japan, India and Chile (Wauters, 1991). Biovar 2 includes only two serovars, O:9 and O:5,27, which are pathogens for man. O:9 is important in Belgium, Holland and France, while O:5,27 is quite common in the United States (Mollaret et al., 1979; World Health Organization, 1983). Biovar 3 includes serovar O:1,2,3 that has been isolated mainly from chinchilla and rodents and rarely from man (Hurvell, 1981; Mollaret et al., 1979). There are also a few serovars O:5,27 in this biovar (Wauters, 1991). In the beginning of the 1990s, a Voges-Proskauer negative variant of biovar 3, belonging to serovar O:3 was isolated in Japan and East Asia and has become a prominent pathogen for man in these countries (Wauters, 1991). Biovar 4 contains only one serovar, O:3, which is the main pathogenic serovar for man, distributed worldwide. It is also isolated regularly from healthy pigs (Hurvell, 1981; Schiemann, 1989). Biovar 5 is of no importance for humans. It includes serovar O:2,3 that is found in hares and some other animals (Hurvell, 1981; Krogstad, 1974; Lanada, 1990; Mollaret et al., 1979; Slee and Button, 1990; Wauters, 1991).

14.4 Methods of detection 14.4.1 Principles of detection The analytical methods available today for the isolation of pathogenic Y. enterocolitica suffer from limitations such as insufficient selectivity, and, in particular inadequate differentiation between pathogenic and non-pathogenic strains.

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14.4.2 Specific principles for isolation A three-step method based on a combination of cold enrichment in a nonselective medium with subsequent inoculation onto a highly selective medium, has been developed for the Nordic Committee on Food Analysis (1987). Wauters et al. (1988a) developed a method for isolation of serovar O:3 from meat and meat products. The procedure is based on a two to three-day selective enrichment period in irgasan-ticarcillin-potassium chlorate (ITC) enrichment broth at room temperature, and is therefore very timesaving compared with the method described above. Both Y. enterocolitica and Y. pseudotuberculosis seem to be more tolerant of alkaline conditions than most other Enterobacteriaceae, and treatment of food enrichments with potassium hydroxide (KOH) may be used to selectively reduce the level of background flora (Aulisio et al., 1980). Elements of the methods from the Nordic Committee on Food Analysis (1987), Wauters et al. (1988a), Schiemann (1982), and KOH treatment (Schiemann, 1983) are incorporated into the International Organization for Standardization (ISO) method (ISO 10273) (Figs. 14.1 and 14.2) (International Organization for Standardization, 1994). 14.4.3 Detection by DNA colony hybridization Genetic probes can also be used in DNA colony hybridisation to demonstrate virulent Y. enterocolitica strains (Kapperud et al., 1990a; Tenover, 1988; Wachsmuth, 1985). Isolation plus hybridisation increased the detection rate Test portion (x g) Dilution 1:100

2 days, 25 °C

ITC

SSDC (CIN)

(Some researchers are also using CIN though it is not recommended by ISO)

Fig. 14.1 Method for recovery of Y. enterocolitica from foods according to the International Organization for Standardization (1994): first element. This element of the method is recommended for serovar O:3 in particular (ITC irgasan-ticarcillinpotassium chlorate enrichment; SSDC = Salmonella-Shigella + sodium deoxycholate, CaCl2 agar.

378 Emerging foodborne pathogens

Test portion (x g) Dilution 1 : 10

PSB

Five days (or three days with agitation), 22–25 °C 0.5 ml PSB + 4.5 ml KOH for 20 s CIN

CIN

Fig. 14.2 Method for recovery of Y. enterocolitica from foods according to the International Organization for Standardization (1994): second element. This element of the method is recommended for all pathogenic serovars (PSB = phosphate-buffered saline, sorbitol, and bile salts; KOH = KOH and NaCl; CIN (cefsulodin-irgasannovobiocin agar).

from 16% to 38% for the method according to Wauters et al., (1988b) and from 10% to 48% for the Nordic method. The results of this investigation (Nesbakken et al., 1991a) support the supposition that conventional culture methods lead to underestimation of virulent Y. enterocolitica in pork products. 14.4.4 Detection by polymerase chain reaction (PCR) These methods often use primers targeting the virF (Thisted-Lambertz et al., 1996; Weynants et al., 1996a) or the yadA (Kapperud et al., 1993) genes, but also the IcrE (Viitanen et al., 1991) and the yopT genes (Arnold et al., 2001) from the virulence plasmid have been used. Y. enterocolitica may lose the virulence plasmid during culture, subculture or storage (Blais and Philippe, 1995). Accordingly, PCR methods based on chromosomal virulence genes, often the ail gene, have been developed. Often a combination of genes from the virulence plasmid and the chromosome are used. A common gene combination in such a multiplex PCR assay is the virF and ail genes (Kaneko et al., 1995; Nilsson et al., 1998). Rasmussen et al. (1995) detected Y. enterocolitica O:3 in faecal samples and tonsil swabs from pigs using immunomagnetic separation (IMS) and PCR based on the inv gene. O:3 cells were detected after pre-enrichment, but direct detection needed further optimisation of the sample preparation procedures. By combining inv, virF and ail genes in a multiplex-PCR assay, Weynants et al. (1996a) could differentiate between Y. pseudotuberculosis, virulent Y. enterocolitica, and Y. enterocolitica O:3.

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Epidemiology

Our understanding of the epidemiology of yersiniosis is still incomplete. Worldwide surveillance data show that great changes have occurred over the last two decades. The importance of Y. enterocolitica as the cause of a number of clinical syndromes is unclear in many areas of the world. Standardised disease surveillance is needed within and across national boundaries so that data from each location are comparable. Improved screening of stools and other specimens for Y. enterocolitica is necessary to further elucidate the epidemiology of the disease. 14.5.1 Clinical symptoms of Y. enterocolitica infection Yersinia enterocolitica is an important cause of gastroenteritis in humans, particularly in temperate countries (Mollaret et al., 1979; World Health Organization, 1983). The consequences of yersiniosis may be severe, and include prolonged acute infections, pseudoappendicitis, and long-term sequelae such as reactive arthritis and erythema nodosum, particularly in northern Europe where the prevalence of the HLA-B27 histocompatibility type is high (Ahvonen, 1972; Cover and Aber, 1989; Winblad, 1975). These consequences make Y. enterocolitica infection a public health and economic problem of greater magnitude than the actual number of recorded cases would suggest (Ostroff et al., 1994). The incubation period is uncertain, but has been estimated as being between two and 11 days (Szita et al., 1973). Gastroenteritis is by far the most common symptom of Y. enterocolitica infection (yersiniosis) in humans (Cover and Aber, 1989; Mollaret et al., 1979). The clinical picture is usually one of a self-limiting diarrhoea associated with mild fever and abdominal pain (Wormser and Keusch, 1981). Nausea and vomiting occur, but less frequently. The portion of the gastrointestinal tract usually involved is the ileocaecal region (Sandler et al., 1982). The colon may also be affected and the infection may simulate Crohn’s disease, which has a different prognosis (Vantrappen et al., 1977). The illness typically lasts from a few days to three weeks, although some patients develop chronic enterocolitis, that may persist for several months (Saebø and Lassen, 1992). Occasionally the infection is limited to the right fossa iliaca in the form of terminal ileitis or mesenteriel lymphadenitis, with symptoms that can be confused with those of acute appendicitis. In several studies of patients with the appendicitis-like syndrome, Y. enterocolitica has been found in up to 9% of patients (Attwood et al., 1987; Niléhn and Sjöström, 1967; Pai et al., 1982; Samadi et al., 1982). Infections with serovars O:3 or O:9 are, in some patients, followed by reactive arthritis (Aho et al., 1981) which is most common in patients possessing the tissue type HLA-B27. Often, although not always, the patient has shown prior gastrointestinal symptoms. Other complications seen with Y. enterocolitica infection are reactive skin complaints, erythema nodosum being the most common. Many such patients have no

380 Emerging foodborne pathogens history of prior gastrointestinal involvement. Septicaemia due to Y. enterocolitica is seen almost exclusively in individuals with underlying disease (Bottone, 1977), while those with cirrhosis and disorders associated with excess iron are particularly predisposed to infection and increased mortality. Clinical symptoms are influenced by the age of the patient (Bottone, 1977; Wormser and Keusch, 1981). Gastroenteritis dominates in children and young people, while various forms of reactive arthritis are most common in young adults, and most patients with skin manifestations are adult females (Wormser and Keusch, 1981). In Scandinavia, there is a relatively high incidence of both reactive arthritis (10–30% of infections) (Winblad, 1975) and erythema nodosum (30% of infections) (Ahvonen, 1972), caused by serovars O:3 and O:9. These forms of the disease have been almost totally absent in the USA, where O:8, historically, has been the most common cause of yersiniosis. This situation may change, however, as serovar O:3 is on the increase in the USA (Bissett et al., 1990). Transient carriage and excretion of both pathogenic and non-pathogenic Y. enterocolitica may occur following exposure to the bacteria. High rates of asymptomatic carriage have been reported in connection with outbreaks (Tacket et al., 1985), although in surveys unrelated to outbreaks, Y. enterocolitica has been detected in the stools of less than 1% of individuals (Niléhn and Sjöström, 1967). In patients with Y. enterocolitica enteritis, the organism may be excreted in the stools for lengthy periods after symptoms have resolved. In a study of Norwegian patients, convalescent carriage of Y. enterocolitica O:3 was detected in the stools of 47% of 57 patients for a median period of 40 days (range 17–116 days) (Ostroff et al., 1992). 14.5.2 Sporadic cases Y. enterocolitica has been isolated from humans in many countries, but it seems to be found most frequently in cooler climates (North America, the western coast of South America, Europe, northern-, central- and eastern Asia, Australia, New Zealand and South Africa) (Mollaret et al., 1979; Tauxe, 2002; World Health Organization, 1983). The widespread nature of Y. enterocolitica has been well documented; by the mid-1970s, Mollaret et al. (1979) had compiled reports of isolates from 35 countries on six continents. Y. enterocolitica infections are an important cause of gastroenteritis in the developed world, occurring particularly as sporadic cases in northern Europe (Cover and Aber, 1989), where a clustering of cases during autumn and winter has been reported (World Health Organization, 1983). There are appreciable geographic differences in the distribution of the different phenotypes of Y. enterocolitica isolated from man (Mollaret et al., 1979; Wauters, 1991). There is also a strong correlation between the serovars isolated from humans and pigs in the same geographical area (Schiemann, 1989; Tauxe, 2002; Wauters, 1991). Serovar O:3 is widespread in Europe, Japan, Canada, Africa and Latin America. Sometimes, but not always, phage typing makes it possible to distinguish between European, Canadian and

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Japanese strains (Kapperud et al., 1990b; Mollaret et al., 1979). Serovar O:3 seem to be responsible for more than 90% of the cases in Denmark, Norway, Sweden and New Zealand, and as many as 78.8% of the cases in Belgium (Table 14.2). In general, the data in Table 14.2 originating from different surveillance programs, national statistics and even estimates are not directly comparable. Serovar O:9/ biovar 2 is the second most common in Europe, but its distribution is uneven; while it still accounts for a relatively high percentage of the strains isolated in France, Belgium and the Netherlands, only a few strains have been isolated in Scandinavia (World Health Organization, 1983). However, recent data shows that serovar O:9 is on the decrease in Belgium. In 1979–1981 this serovar was implicated in 23.3% of the cases, while in 1994 it was responsible for only 5.9% of the cases (Ministere Table 14.2 Verified and estimated cases of yersiniosis in some countries. Adapted from Nesbakken T (2005), ‘Yersinia enterocolitica’, in Fratamico P M, Bhunia A K and Smith J L, Foodborne pathogens: Microbiology and Molecular Biology, Norwich, UK, Caister Academic Press, 228–249. With kind permission of Horizon Scientific Press/Caister Academic Press. Country

Total number of cases (year)

Cases per 100,000 inh.

Verified cases Belgium

8291 (1994)

8.5

Denmark

2452 (2003)

4.5

Finland

647 (2003)

12.4

Germany Norway

7113 (2001) 862 (2003)

8.7 1.9

Sweden

7142 (2003)

8.0

Switzerland

51 (1998)

0.7

The European Union

73853 (2000)

Estimated cases New Zealand 3,0002 (1994) United States 87,000 (1997) 1 2 3

84 33.4

Serovar O:3: 78.8%; serovar O:9: 5.9% Serovar O:3: >90% Figures from nine countries in the European Union

References

Ministere des Affaires Sociales, de la Sante Publique et de l’Environnement. Institut d’Hygiene et d’Epidemiologie (1995) Danish Zoonosis Centre, Copenhagen (www.dfvf.dk) National Public Health Institute, Helsinki, Finland (www.ktl.fi) RKI, 2002 National Institute of Public Health, Oslo (www.fhi.no) Swedish Institute for Infectious Disease Control (www.smittskyddsinstitutet.se Swiss National Reference Laboratory for Foodborne Diseases, Berne www.btr.bund.de/ internet/7threport/CRs/swi.pdf European Commission, Health & Consumer Protection DirectorateGeneral www.euro.who.int/nen/ resources/eusanco/20030723 1 Wright et al., 1995 Mead et al., 1999

382 Emerging foodborne pathogens des Affaires Sociales, de la Sante Publique et de l’Environnement. Institut d’Hygiene et d’Epidemiologie, 1995). A similar reduction has also been seen in France and The Netherlands (L. de Zutter, personal communication, 1996). Worldwide, infection with Y. enterocolitica in humans seems to reflect the serovars in pigs (Nesbakken, 1992). Maybe Y. enterocolitica O:3 has become the dominating serovar in pig herds at the expense of serovar O:9 in this region? Until recently, the most frequently reported serovars in the United States were O:8 followed by O:5,27 (Bisset et al., 1990; Mollaret et al., 1979; World Health Organization, 1995). In recent years, serovar O:3 has been on the increase in the United States; O:3 now accounts for the majority of sporadic Y. enterocolitica isolates in California (Bisset et al., 1990). In 1989, the estimated cost of yersiniosis in the United States was 138 millions of dollars (World Health Organization, 1995). Principal foodborne infections, as estimated for 1997, are ranked by estimated number of cases caused by foodborne transmission each year in the United States. Y. enterocolitica is number ten in the list (among the bacteria in the list, Y. enterocolitica is number seven) (Mead et al., 1999). The appearance of strains of serovars O:3 and O:9 in Europe, Japan in the 1970s, and in North America by the end of the 1980s, is an example of a global pandemic (Tauxe, 2002). The first Japanese case of Y. enterocolitica O:8 infection was linked to consumption of imported raw pork (Ichinohe et al., 1991). The incidence of Y. enterocolitica infection in patients with acute enterocolitis ranges from 0 to 4%, depending on the geographic location, study method, and population: in Australia, Canada, Denmark, Germany, New Zealand, Norway and Sweden (Aleksic and Bockemühl, 1990; Swedish Institute for Infectious Disease Control; Wright et al., 1995; Danish Zoonosis Centre, Copenhagen; National Institute of Public Health, Oslo), Y. enterocolitica has surpassed Shigella, and now rivals Salmonella and Campylobacter as a cause of acute bacterial gastroenteritis. Worldwide surveillance data show great changes over the last two decades. There appears to have been a real and generalised increase in incidence (World Health Organization, 1983, 1995), even though there is still much under-reporting. Nevertheless, improvements in detection and reporting systems may have contributed a great deal to the observed increase.

14.5.3 Outbreaks In the United States, chocolate milk (Black et al., 1978), pasteurised milk (Tacket et al., 1984), soybean curd (tofu) (Tacket et al., 1985), and bean sprouts (Aber et al., 1982) have been implicated as sources in outbreaks of Y. enterocolitica infection. These outbreaks, all of which occurred before 1983, were caused by Y. enterocolitica serovars which have been infrequently associated with human disease (serovars O:13, O:18) or which no longer predominate in the United States (serovar O:8). The milk-borne outbreak in Sweden in 1988 (Alsterlund et al., 1995) was probably caused by

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recontamination of pasteurised milk because of lack of chlorination of the water supply. In the multistate outbreak in 1982 (Tacket et al. 1984), milk cartons were contaminated with mud from a pig farm (Aulisio et al., 1982). In the case of the outbreak described by Greenwood and Hooper (1990), post-pasteurisation contamination may have occurred from bottles. Previous studies have shown that milk-associated Y. enterocolitica outbreaks have been linked to the addition of ingredients after pasteurisation (Black et al., 1978; Morse et al., 1984). The preparation of raw pork intestines (chitterlings) was associated with an outbreak of Y. enterocolitica O:3 infections among black American infants in Georgia (Lee et al., 1990); the organism was isolated from samples of the pork intestines. Also, in outbreaks in Buffalo, New York, 1994–1996, (Kondracki et al., 1996) chitterlings were the vehicle. In 1981, an outbreak of infection due to Y. enterocolitica O:8 in Washington State occurred in association with the consumption of tofu packed in untreated spring water (Tacket et al., 1985). The outbreak serovar was isolated from the spring water samples. Another outbreak caused by serovar O:8 was traced to ingestion of contaminated water used in manufacturing or preparation of food (Schiemann, 1989). Two other Yersinia outbreaks have been associated with well water. One occurred among members of a Pennsylvania girl scout troop after they ate bean sprouts grown in contaminated well water (Aber et al., 1982); the other was a familial outbreak of yersiniosis in Canada (Thompson and Gravel, 1986). Y. enterocolitica O:3 was isolated from members of a family as well as from the well used as a source of their drinking water. The epidemiology of yersiniosis in the United States seems to have evolved into a pattern similar to the picture in Europe (Bisset et al., 1990; Tauxe, 2002), where foodborne Yersinia outbreaks are rare, and where serovar 3 predominates (Mollaret et al., 1979). Although yersiniosis appears to be more common in Europe than in the United States, only a few foodborne outbreaks have been reported in Europe (Greenwood and Hooper, 1990; Olsovsky et al., 1975; Toivanen et al., 1973; Swedish Institute for Infectious Disease Control). The number of cases in the USA presented in Table 14.2 is only an estimate and should not be directly compared to reported cases in countries in Europe. 14.5.4

Reservoirs for Yersinia

The pig Healthy pigs are often carriers of strains of Y. enterocolitica that are pathogenic to humans, in particular strains of serovar O:3/biovar 4 and serovar O:9/ biovar 2) (Hurvell, 1981; Schiemann, 1989). The organisms are present in the oral cavity, especially the tongue and tonsils, submaxillar lymph nodes, in the intestine and faeces (Nesbakken et al., 2003a,b) (Fig. 14.3). Shiozawa et al. (1991) reported that O:3 strains were isolated from 85% of oral swabs

384 Emerging foodborne pathogens from 40 freshly slaughtered, healthy pigs and presented evidence that the organism colonized the pigs’ tonsils. In this study 24.3% of 140 pigs were carriers of the organism in the caecum, with counts ranging from fewer than 300 to 110,000 Y. enterocolitica/g of caecal contents. Strains of O:3 have been found frequently on the surface of freshly slaughtered pig carcasses (in frequencies up to 63.3%) (Nesbakken, 1988). This is probably the result of spread of the organism via faeces and intestinal contents during slaughter and dressing operations (Fig. 14.3). The association between yersiniosis in humans and the consumption of raw pork in Belgium (Tauxe et al., 1987) and the apparently rare incidence of the infection in Moslem countries (Samadi et al., 1982), where consumption of pork is restricted, point to pork as a source of infection with Y. enterocolitica. Other pathogenic strains do not appear to be as closely associated with pigs, and may have a different ecology. In western Canada, O:8 and O:5,27 strains have been found most commonly in humans, but only O:5,27 strains were found in the throats of slaughter-age pigs (Schiemann, 1989). In the USA, O:5,27 strains were isolated from the caecal contents and faeces of two out of 50 pigs at slaughter (Kotula and Sharar, 1993). Serovar O:8/ biovar 1B, until recently considered to be the most common human pathogenic strain of Y. enterocolitica in the USA (Tauxe, 2002) and in western Canada (Toma and Lafleur, 1981), has seldom been reported in pigs. Healthy pigs have been found to be infected with Y. enterocolitica O:3 in frequencies up to 85% (Hurvell, 1981; Nesbakken, 1988; Schiemann, 1989; Shiozawa et al., 1991) and in numbers up to 1720/cm2 (Nesbakken, 1988).

24 19 14 9

. ln eck dia

reg

Me

m

vis

ney Kid

Pel

Ha

ces Fae

lon Co

m

m

ecu Ca

lleu

Ser olo gy

–1

Ton sils Su lym b-ma ph xill, no de lym Mes ph ent. no de Sto ma ch

4

Fig. 14.3 Antibodies against Y. enterocolitica O:3 in blood, and Y. enterocolitica O:3 in lymphoid tissues, intestinal contents and on pig carcasses (n = 24). Taken from: Nesbakken et al. (2003b), ‘Occurrence of Yersinia enterocolitica in slaughter pigs and consequences for meat inspection, slaughtering, and dressing procedures’, in Skurnik M, Bengoechea J A and Granfors K, The Genus Yersinia. Entering the Functional Genomic Area. Advances in Experimental Medicine and Biology vol. 529, New York, Kluwer Academic/Plenum Publishers, 303–308. With kind permission of Kluwer Academic Publishers.

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Cattle Positive tests in serological control programs for brucellosis in cattle, have in some cases proved to be cross-reactions against Y. enterocolitica serovar O:9 (Danish Zoonosis Centre, Copenhagen; Wauters, 1981; Weynants et al., 1996b). The existence of cross-reactions between Y. enterocolitica O:9 and Brucella is described in section 14.7.1 ‘Pathogenesis and immunity’. With a few exceptions (Danish Zoonosis Centre, Copenhagen; Wauters, 1981; Weynants et al., 1996b), cattle are generally not considered to be carriers of human pathogenic Y. enterocolitica. Sheep and goats In Norway, Krogstad (1974) demonstrated outbreaks of Y. enterocolitica infection in goatherds in which serovar O:2/biovar 5 was implicated. He also described a case in which an animal attendant was infected by the same serovar. Biovar 5 has also been isolated from goats in New Zealand (Lanada, 1990). Enteritis in sheep and goats due to infection of Y. enterocolitica O:2,3, biovar 5 is also seen in Australia (Slee and Button, 1990). Serovar O:3 was isolated from the rectal contents in two (3.0%) of 66 lambs in New Zealand (Bullians, 1987). Sheep and goats are generally not considered to be carriers of human pathogenic Y. enterocolitica (Hurvell, 1981). Poultry Stengel (1985) isolated Y. enterocolitica serovars O:3 (n=3), O:9 (n=3), and non-virulent Y. enterocolitica (n=13) from 130 samples of poultry. This is probably the first time these virulent serovars have been isolated from poultry, and there was no obvious opportunity for cross-contamination from pigs or pork. Nevertheless, according to other investigations, poultry has not been proven to be carrier of pathogenic Y. enterocolitica (Nesbakken et al. 1991b). Deer Surveys in New Zealand have found deer to carry both O:5,27/biovar 2 and O:9/biovar 2 (S. Fenwick, personal communication, 1996). 14.5.5

Vehicles for transmission

Pork In contrast to the frequent occurrence of the bacterium in pigs and on freshly slaughtered carcasses, pathogenic Y. enterocolitica have only exceptionally been found from pork products at the retail sale stage (Nesbakken et al., 1991a; Schiemann, 1989; Wauters et al., 1988a), with the exception of fresh tongues. This phenomenon might be explained by the lack of proper selective methodology for the isolation of pathogenic strains. However, Wauters et al. (1988a) described a method which allowed Y. enterocolitica O:3 to be recovered from as many as 12 (24%) of 50 ground pork samples. Genetic probes can

386 Emerging foodborne pathogens also be used in DNA colony hybridisation to demonstrate virulent Y. enterocolitica strains. The results of one such investigation support the supposition that traditional culture methods lead to underestimation of the presence of virulent Y. enterocolitica in pork products. A significantly higher detection rate (6%) was achieved when two isolation procedures were combined with colony hybridisation, than when the isolation procedures were employed alone (18%) (Nesbakken et al., 1991a). In this study, counts of virulent yersiniae in pork sausage meat varied from 50 to 2,500/g, and in pork cuts from 50 to 300/g. Ostroff et al. (1994) showed in a case-control study that persons with Y. enterocolitica infection reported having eaten significantly more pork or sausages than their matched controls. This phenomenon might be explained by recontamination of sausages after pasteurisation, bacterial multiplication during storage, and insufficient heat treatment in the kitchen. Beef The possibility exists of cross-contamination to beef from pig carcasses and pork in abattoir, cutting plants, meat processing establishments, and butchers’ shops. Beef is sometimes consumed after little or no heat treatment, and some concern has therefore been expressed about Y. enterocolitica contamination in beef. According to the case-control study of Ostroff et al. (1994), persons with yersiniosis were also more likely than controls to report a preference for eating meat prepared raw or rare. Milk and dairy products Worldwide studies indicate that non-pathogenic Y. enterocolitica is fairly common in raw milk (Lee et al., 1981). Non-pathogenic variants of Y. enterocolitica have also been isolated from ice cream (Mollaret et al., 1972) and pasteurised milk (Sarrouy, 1972; Zen-Yoji et al., 1973) as early as 1970. However, it is almost solely in connection with outbreaks caused by contaminated pasteurised milk (Alsterlund et al., 1995; Greenwood and Hooper, 1990; Tacket et al., 1984), reconstituted powdered milk (Morse et al., 1984), and contaminated chocolate milk (Black et al., 1978) that one has been able to find the pathogenic strains. In section 14.5.3, second paragraph, possible pathways of contamination of milk and milk products with Y. enterocolitica are discussed. Water Shallow wells in particular, and also rivers and lakes, are susceptible to contamination with by surface runoff from rain or snowmelt. Such runoff may become contaminated by faeces from wild or domestic animals, or by leakage from septic tanks or open latrines in the surrounding areas. Water is a significant reservoir of Y. enterocolitica (Brennhovd, 1991; Harvey et al., 1976; Kapperud and Jonsson, 1978; Langeland, 1983; Lassen, 1972; Saari and Jansen, 1979). However, most isolates of Y. enterocolitica and Y.

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enterocolitica-like bacteria obtained from water are variants with no known pathogenic significance to man.

14.6 Risk factors connected to the agent 14.6.1 The virulence plasmid Human pathogenic strains of Y. enterocolitica possess a special plasmid, 40– 50 megadaltons in size (Portnoy and Martinez, 1985). The presence of this plasmid is an essential, though not sufficient, prerequisite for the bacterium to be able to induce disease. Properties associated with the chromosome are also necessary for virulence. Corresponding plasmids are found in Y. pseudotuberculosis and Y. pestis, and there is thus a family of related virulence plasmids within the genus Yersinia (Portnoy and Martinez, 1985). The way in which the plasmid contributes to virulence has not been fully elucidated. The presence of this virulence plasmid has been associated with several properties, most of which are phenotypically expressed only at elevated growth temperatures of 35–37 °C (Portnoy and Martinez, 1985). The list of such plasmid-mediated and temperature-regulated properties includes: Ca++dependent growth (Perry and Brubaker, 1983), production of V and W antigens (Perry and Brubaker, 1983), spontaneous autoagglutination (Laird and Cavanaugh, 1980), mannose-resistant haemagglutination (Kapperud et al., 1987), serum resistance (Pai and DeStephano, 1982), binding of Congo red dye (Prpic et al., 1985), hydrophobicity (Lachica et al., 1984), mouse virulence (Nesbakken et al., 1987; Pai and DeStephano, 1982), and production of a number of proteins (Portnoy and Martinez, 1985), of which one is a true outer membrane protein (YadA, previously termed Yop1) (Michiels et al., 1990). This true outer membrane protein forms a fibrillar matrix on the bacterial surface and mediates cellular attachment and entry (Bliska and Falkow, 1994). It also confers resistance to the bactericidal effect of normal human serum and inhibition of the anti-invasive effect of interferon. The mechanism of virulence is known to vary even between different serovars within Yersinia enterocolitica. Plasmid-containing strains of O:8, O:13a, O:13b, and O:21 are lethal to orally infected mice, whereas O:3, O:9 and O:5,27 are not (Nesbakken et al., 1987; Pai and DeStephano, 1982). These latter serovars, however, are capable of maintaining intestinal colonisation for at least one week after orogastric challenge (Nesbakken et al., 1987). 14.6.2 The chromosome Elements encoded by the chromosome are also necessary for maximum virulence. The pathogenic yersiniae share at least two chromosomal loci, inv and ail, that play a role in their entry into eukaryotic cells (Miller et al., 1988). The inv and ail gene products can be classified as adhesins since they

388 Emerging foodborne pathogens mediate adherence to the eukaryotic surface. Unlike other previously characterised bacterial adhesins, they also mediate entry into a variety of mammalian cells. A high pathogenicity island in pathogenic species of Yersinia encodes genes for three yersiniabactin (Ybt) transport proteins, six Ybt biosynthetic enzymes, one transcriptional regulator (YbtA), and one protein of unknown function (YbtX) (Perry et al., 2001). See also Chapter 1. 14.6.3 Enterotoxin production Many strains of Y. enterocolitica and related species produce a heat-stable enterotoxin (YEST) when the bacteria are cultured at 20–30 °C (Pai et al., 1978). Certain strains, especially within the species Y. kristensenii, are also able to produce YEST at 4 and 37 °C (Pai et al., 1978). This property is regulated by chromosomal genes and is independent of the virulence plasmid. Although there is no evidence to support the involvement of YEST in the pathogenesis of Y. enterocolitica enteritis, the possibility still remains that enterotoxigenic strains may produce foodborne intoxication by means of preformed enterotoxins. This assumption is based on the fact that YEST is able to resist gastric acidity as well as temperatures used in food processing and storage, without losing activity (Boyce et al., 1979). The ability to produce YEST at room and refrigeration temperature may give the ‘environmental’ yersiniae a new significance in food hygiene as potential agents of foodborne intoxication.

14.7 Risk factors in connection with the host 14.7.1 Pathogenesis and immunity Human infection due to Y. enterocolitica is most often acquired by the oral route. The minimal infectious dose required to cause disease is unknown. In one volunteer, ingestion of 3.5 × 109 organisms was sufficient to produce illness (Szita et al., 1973). Enteric infection leads to proliferation of Y. enterocolitica in the lumen of the bowel and in the lymphoid tissue of the intestine. Adherence to and penetration into the epithelial cells of the intestinal mucosa are essential factors in the pathogenesis of Y. enterocolitica infection (Bliska and Falkow, 1994; Cornelis et al., 1987; Miller et al., 1988; Portnoy and Martinez, 1985). When the bacteria reach the lymphoid tissues in the terminal ileum, a massive multiplication of bacteria and inflammatory response takes place in the Peyer’s patches. Reactive arthritis and erythema nodosum appear to be delayed immunological sequelae of the original intestinal infection. In humans, infection with pathogenic strains of Y. enterocolitica stimulates development of specific antibodies. It is not known whether specific serum antibody protects against reinfection with Y. enterocolitica organisms of the same or different serovars. The immunological response can be measured by a variety of techniques, including tube agglutination, indirect

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haemagglutination, enzyme-linked immunosorbent assay (ELISA), and solidphase radioimmunoassay (Attwood et al., 1987). An indirect immunofluorescent-antibody assay has also been used (Cafferkey and Buckley, 1987). However, ELISA is probably the most suitable and extensively applied method in the world today. Agglutinating antibodies appear soon after the onset of illness and persist for from two to six months. Some serovars may be associated with illness without eliciting a detectable serological response (Toma and Lafleur, 1981). The serological diagnosis of Y. enterocolitica infection may be complicated by the existence of cross-reactions between Y. enterocolitica, most notably serovar O:9, and such organisms as Y. pseudotuberculosis, Brucella, Vibrio, Salmonella, Proteus and Escherichia coli (Wauters, 1981). The interpretation may also be confounded by high prevalence of seropositive individuals in the healthy population. Of 813 Norwegian military recruits selected at random, 67 (8.2%) had antibodies against Y. enterocolitica O:3 (Nesbakken et al., 1991b). Patients with thyroiditis of an immunological aetiology have an unexplained increased frequency of cross-reacting antibodies to Y. enterocolitica (Shenkman and Bottone, 1976). Detection of antibodies to plasmid-encoded proteins, Yersinia outer membrane proteins (Yops), by immunoblots, has been suggested as a highly specific means of demonstrating previous Y. enterocolitica infection (Ståhlberg et al., 1989). Demonstration of specific circulating IgA to the Yops is indicative of recent or persistent infection and is strongly correlated with the presence of virulent Y. enterocolitica in the intestinal lymphatic tissue of patients with reactive arthritis.

14.8 Risk factors in connection with survival and growth in foods Y. enterocolitica is a facultative organism able to multiply in both aerobic and anaerobic conditions (Bercovier and Mollaret, 1984). 14.8.1 Temperature The ability of Y. enterocolitica to multiply at low temperatures is of considerable concern to food producers. The reported growth range is –2 to 42 °C (Bercovier and Mollaret, 1984). Optimum temperature is 28–29 °C (Bercovier and Mollaret, 1984). Y. enterocolitica can multiply in foods such as meat and milk at temperatures approaching and even below 0 °C (Lee et al., 1981; Stern et al., 1980). It is important to recognise the rate at which Y. enterocolitica can multiply, which is considerably greater than that for L. monocytogenes (Bhaduri et al., 1994). Results show that, in a food with a neutral pH stored at 5 °C, Y. enterocolitica counts may increase from, e.g., 10/ml to 2.8 × 107/ ml in five days (Bhaduri et al., 1994).

390 Emerging foodborne pathogens 14.8.2 pH The minimum pH for growth has been reported as being between 4.2 and 4.4 (Kendall and Gilbert, 1980), while in a medium in which the pH had been adjusted with HCl, growth occurred at pH 4.18 and 22 °C (Brocklehurst and Lund, 1990). The presence of organic acids will reduce the ability of Y. enterocolitica to multiply at low pH, acetic acid being more inhibitory on a molar basis at a given pH than lactic and citric acids (Brocklehurst and Lund, 1990). 14.8.3 Growth and survival in food The ability to propagate at refrigeration temperature in vacuum-packed foods with a prolonged shelf-life (Bercovier and Mollaret, 1984) is of considerable significance in food hygiene Y. enterocolitica may survive in frozen foods for long periods (Schiemann, 1989). Y. enterocolitica is not able to grow at pH 9.0 (Kendall and Gilbert, 1980; Stern et al., 1980) or at salt concentrations higher than 7% (aw < 0.945) (Stern et al., 1980). The organism does not survive pasteurisation or normal cooking, boiling, baking, and frying temperatures. Heat treatment of raw milk operated at 60 to 72 °C for a minimum holding time of 16.2 s rapidly inactivated Yersinia enterocolitica when the bacterium was inoculated at a level of approximately 1.0 × 105 cfu/ml (D’Aoust et al., 1988). Yersinia enterocolitica showed a 4-log reduction in counts at 60 °C and absence of viable cells at greater than or equal to 63 °C. Heat-treatment of meat products at 60 °C for 1–3 minutes effectively inactivates Y. enterocolitica (Lee et al., 1981) indicating that normal pasteurisation treatment or cooking to a core temperature of 70 °C would be sufficient for killing the organism. D-values determined in scalding water were 96, 27 and 11 seconds at 58, 60 and 62 °C, respectively (Sörqvist and Danielsson-Tham, 1990). The literature is contradictory regarding the multiplication of Y. enterocolitica in meat during conventional cold storage (Bredholt et al., 1999; Fukushima and Gomyoda, 1986; Kleinlein and Untermann, 1990; Lee et al., 1981; Lindberg and Borch, 1994; Schiemann, 1989; Stern et al., 1980). A comparison of published (Hanna et al., 1977) and predicted generation times (GT) (Sutherland and Bayliss, 1994) for Y. enterocolitica in raw pork at 7 °C, 0.5% NaCl (w/v) and pH 5.5–6.5 shows GTs of 8.4 – 12.4 hours (published) and 8.15–5.05 hours (predicted). However, according to many reports, the ability of Y. enterocolitica to compete with other psychotrophic organisms normally present in food may be poor (Fukushima and Gomyoda, 1986; Kleinlein and Untermann, 1990; Schiemann, 1989). In contrast, a number of studies have shown that Y. enterocolitica is able to multiply in foods kept under chill storage and might even compete successfully (Bredholt et al., 1999; Gill and Reichel, 1989; Grau, 1981; Lee et al., 1981; Lindberg and Borch, 1994; Stern et al., 1980).

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Pig carcasses are often held in chilling rooms for two to four days after slaughter prior to cutting. Pre-packaged raw meat products may remain in retail chill cabinets for more than a week, depending on the product, packaging, package atmosphere, and rate of turnover. Pathogenic variants of Y. enterocolitica might propagate considerably during the course of this relatively long storage period. 14.8.4 Fermentation Use of fermentation and starter cultures could prevent growth of Y. enterocolitica. Examples are Leuconostoc spp. or Lactobacillus plantarum in fish (Jeppesen and Huss, 1993). Antagonistic effect of chosen lactic acid bacteria (LAB) strains on Y. enterocolitica species were demonstrated in model set-ups, meat and fermented sausages (Gomolka-Pawlicka and Uradzinski, 2003). The results show that all the LAB strains used within the framework of the model set-ups had antagonistic effect on all the Y. enterocolitica strains. However, this ability was not observed with respect to the tested LAB strains in meat and fermented sausage. This ability was possessed by one of the strains investigated, a L. helveticus strain. There are also examples of survival and growth of Y. enterocolitica in Feta cheese with Streptococcus cremoris (Erkmen, 1996). During fermentation by various lactobacilli, for instance L. bulgarcus and L. acidophilus in skim milk (Ozbas and Aytac, 1996) Y. enterocolitica survived. The study of Bredholt et al. (1999) indicates that 104 cfu/g of Y. enterocolitica is able to grow well at 8 °C in vacuum-packaged cooked ham and servelat sausage in the presence of 104–5 cfu/g LAB. These LAB cultures, for instance L. sakei, inhibited growth of Listeria monocytogenes and Escherichia coli O157:H7 in the same experiment. The effect of lactic acid (concentration range of 0.1 to 1.1% v/v within a pH range of 3.9 to 5.8 at 4 °C) on growth of Y. enterocolitica O:9 is greater under anaerobic than aerobic conditions, although the bacterium has proved to be more tolerant of low pH conditions under anaerobic atmosphere than under an aerobic atmosphere in the absence of lactic acid (El-Ziney et al., 1995). 14.8.5 Radiation of food Y. enterocolitica is among the most sensitive bacteria needing the lowest radiation doses for elimination (D10~0.20 kGy) (Molins et al., 2001). 14.8.6 Packaging As a facultative organism, the gaseous atmosphere drastically affects the growth of Y. enterocolitica. Under anaerobic conditions, Y. enterocolitica is unable to grow in beef at pH 5.4–5.8, whereas growth occurs at pH 6.0 (Grau, 1981). 100% CO2 is reported to inhibit the growth of Y. enterocolitica

392 Emerging foodborne pathogens (Gill and Reichel, 1989). In the study of Gill and Reichel (1989), Y. enterocolitica was inoculated into high pH (>6.0) beef DFD (dark firm dry)meat. Samples were packaged under vacuum or in an oxygen-free CO2 atmosphere maintained at atmospheric pressure after the meat had been saturated with the gas and stored at –2, 0, 2, 5 or 10 °C. In vacuum packs, Y. enterocolitica grew at all storage temperatures at rates similar or faster than those of the spoilage flora. In CO2 packs, the bacterium grew at both 5 and 10 °C, but not at lower temperatures. Growth of Y. enterocolitica was nearly totally inhibited both at 4 and 10 °C in a 60% CO2/0.4% CO mixture, while the bacterial numbers in samples packed in high O2 mixture (70% O2/30% CO2) increased from about 5 × 102 bacteria/g at day 0 to about 104 at day 5 at 4 °C and to 105 at 10 °C. Growth in chub packs (stuffed in plastic casings) was even higher (Nissen et al., 2000).

14.9 Risk factors based on epidemiological studies Only a few epidemiological studies have been performed to investigate the sources of sporadic human infections. A 1985 study of Y. enterocolitica in Belgium identified consumption of raw pork as a risk factor for disease (Tauxe et al., 1987). The following variables were found to be independently related to an increased risk of yersiniosis in a case-control study conducted in Norway: drinking untreated water, general preference for meat to be prepared raw or rare, and frequency of consumption of pork and sausages (Ostroff et al., 1994). The infrequent occurrence of infections with Y. enterocolitica in some areas of the world may be due in part to avoidance of certain risk factors, such as lack of consumption of pork in Muslim countries (Samadi et al., 1982). A seroepidemiological study has indicated that occupational exposure to pigs may be a risk factor (Nesbakken et al., 1991b), however, confounders could not be excluded.

14.10 Prevention and control at different steps of the food chain Possibility for preventive action and reduction in the food chain is shown in Table 14.3. In addition to the specific prevention measures described in this section, World Health Organization (2000) provides a guide, Foodborne disease: A focus for health education, to the education of food handlers, consumers, food safety managers in public and private sectors as well as policy makers as an effective strategy for reducing the illness and economic losses caused by foodborne disease.

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Table 14.3 The putative effects of preventive action on occurrence of Y. enterocolitica in the food chain (+++ = great effect, ++ = good effect, + = limited effect, – = probably no effect) Herd level

Slaughter

Meat inspection

Cutting and de-boning

Processing

Preparation and consumption

+++

++

–

–

+++*

+++*

* Reduction/elimination by heat treatment. However, the main problem at these stages is crosscontamination

14.10.1 At the farm level New-born piglets are easily colonised and become long-term healthy carriers of Y. enterocolitica in the oral cavity and intestines (Schiemann, 1989). In a recent study (Skjerve et al., 1998), an enzyme-linked immunosorbent assay (ELISA) was used to detect IgG antibodies against Y. enterocolitica O:3 in sera from 1,605 slaughter pigs from 321 different herds. Positive titres were found in 869 (54.1%) of the samples. In the final epidemiological study 182 (63.4%) of 287 herds were defined as positive. Among the positive herds, there were significantly fewer combined herds of piglets and fatteners than fattening herds. Among the risk factors were using an own-farm vehicle for transport of slaughter pigs to abattoirs, daily observations of a cat with kittens at the farm, and using straw bedding for slaughter pigs. In conclusion, the epidemiological data suggest that it is possible to reduce the herd prevalence of Y. enterocolitica O:3 by minimising contact between infected and noninfected herds. Further, attempts to reduce the prevalence at the top levels of the breeding pyramids may be beneficial for the industry as a whole. The meat industry may use serological tests as a tool to lower the prevalence in the pig population by limiting the contact between seropositive and seronegative herds. 14.10.2 Slaughter Because of the high prevalence of Y. enterocolitica in pig herds, strict slaughter hygiene will remain an important means to reduce carcass contamination with Y. enterocolitica as well as other pathogenic microorganisms (Skjerve et al., 1998). However, it is not possible to sort out pigs contaminated with Y. enterocolitica at post-mortem meat inspection. Pig slaughter is an open process with many opportunities for the contamination of the pork carcass with Y. enterocolitica, and it does not contain any point where hazards are completely eliminated (Borch et al., 1996). Contamination of the carcass with Y. enterocolitica during pig slaughter is most likely to arise from faecal and pharyngeal sources (Nesbakken et al., 2003a). HACCP (Hazard Analysis Critical Control Point) and GMP (Good Manufacturing Practice) in pig slaughter must be focused on limiting this spread. As a guide, attention should be given to the establishment of control measures and identification

394 Emerging foodborne pathogens of critical control points considering different steps during slaughter and dressing including: lairage, killing, scalding, dehairing, singeing/flaming, scraping, circum-anal incision and removal of the intestines, excision of the tongue, pharynx, and in particular the tonsils, splitting, post-mortem meat inspection procedures, and deboning of the head (Borch et al., 1996). Some of the above-mentioned critical control points will be discussed in more detail below. During transport and lairage, pathogenic Y. enterocolitica may spread from infected to non-infected pigs. If possible, herds should be handled separately, and cleaning and disinfection of the lairage facilities should be performed between herds, since some herds are free from this pathogen (Skjerve et al., 1998). A working procedure that is employed in many abattoirs, and that can be introduced immediately in all abattoirs, is the two-knife method. This can be used to interrupt the path of infection from oral cavity and intestine to other parts of the carcass. The two-knife method involves the installation in the slaughter hall of knife decontaminators, with running water held at a temperature of approximately 82 °C. When an unclean working operation has been performed, for example in the region around the rectum or oral cavity, the knife is rinsed before being placed in the decontaminator. The operator should then wash his hands before the other knife is used for clean working operations. The two-knife method should be used both by operators and meat inspection personnel. The possibility of decapitation early on in the carcass dressing procedure has been considered, with the head, including tongue and tonsils, then being removed on a separate line for heat-treatment and cutting. The results presented by Nesbakken et al., (1994) indicate that it is important to modify procedures for removal of the guts, in order to avoid contamination of the carcass from the rectum. Technological solutions have already been found which allow removal of the rectum without soiling of the carcass. This can be done, inter alia, by insertion of a pre-frozen plug into the anus prior to rectum-loosening and gut removal. The sealing off of the rectum with a plastic bag immediately after it has been freed can significantly reduce the spread of Y. enterocolitica to pig carcasses (Nesbakken et al., 1994). According to data from the Norwegian National Institute of Public Health (2005), the occurrence of human yersiniosis has dropped after the introduction of the plastic bag technique in the main abattoirs slaughtering more than 90% of the pigs in Norway (Fig. 14.4). 14.10.3 Meat inspection Meat inspection procedures concerning the head also seem to represent a cross-contamination risk; incision of the sub-maxillary lymph nodes (Fig. 14.3) is a compulsory procedure according to the EU regulations (European Commission, 1995). In a screening of 97 animals, 5.2% of samples from the sub-maxillary lymph nodes were positive and by the sampling of 24 these lymph nodes in a follow-up study, 12.5% of the samples were positive

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Number of human cases 300

200

1994: Introduction of the plastic bag

100

2

4

200

200

0 200

98

96

94

92

90

88

86

84

82

0

Fig. 14.4 Number of reported human cases of yersiniosis in Norway, 1982–2004. In the middle of 1994, the slaughterhouses in Norway implemented the procedure of sealing off the rectum with a plastic bag (Source: Norwegian National Institute of Public Health (2005), http://www.msis.no, 3/2/2005).

(Fig. 14.3) (Nesbakken et al., 2003a, b). This may, however, result in the bacterium being transported from the medial neck region to other parts of the carcass by the knives and hands of the meat inspection personnel (Nesbakken, 1988; Nesbakken et al., 2003a). In view of the fact that the incidence of tuberculosis in pigs and humans has been reduced to a very low level in many parts of the world, it may be possible to re-consider regulations that require incision of the sub-maxillary lymph nodes by meat inspectors. 14.10.4 Cutting and de-boning Cutting and removal of head-meat in pigs should be carried out on a separate worktable, preferably in a separate room (European Commission, 1995). This room should be considered to be an unclean area. Knives and equipment must not be used for cutting and deboning other parts of the carcass without prior cleaning and disinfection. Current EU regulations that require removal of pig head-meat to be carried out in a separate department are, unfortunately, not complied with in all abattoirs. 14.10.5 Processing of meat products In addition to the slaughter hall, and the cutting and de-boning departments, the sausage-making department must also be considered to be a contaminated area. It must be assumed that raw materials such as pig head-meat and pork cuts, and consequently also sausage meat, are likely to be contaminated with pathogenic Y. enterocolitica. Strict cleaning and disinfecting requirements must therefore also apply here. It is important to maintain an effective separation

396 Emerging foodborne pathogens between sausage preparation and packing, so as to avoid recontamination after heat-treatment. 14.10.6 Refrigeration Y. enterocolitica is able to propagate at temperatures approaching 0 °C. While refrigeration of food does not prevent the multiplication of Y. enterocolitica, the rate at which this takes place will be reduced. 14.10.7 Cross-contamination Raw meats (in particular pork) should be separated from other foods. Crosscontamination from raw meat to heat-treated end products must be avoided in meat-processing establishments, butchers’ shops, meat departments in retail food stores, and in kitchens in institutions, restaurants and homes. 14.10.8 Adequate cooking of meat A case-control study carried out in Norway revealed that inadequate heattreatment of meat is a risk factor for human yersiniosis (Ostroff et al., 1994). Consumption of undercooked pork should be discouraged. 14.10.9 Personal hygiene Precautions aimed at preventing faecal-oral spread of the pathogens should be taken. Hand-washing and proper stool disposal must be employed in household, day-care, and hospital settings. 14.10.10 Cleaning and disinfection Knives, equipment, and machines used to cut or process raw meat products must be cleaned and disinfected before being used for handling other foods. All surfaces that have been in contact with raw meat must be cleaned and disinfected with appropriate and effective agents.

14.11

Future trends

The emergence of yersiniosis may be related to changes that have occurred in livestock farming, food technology and the food industry. Of greatest importance are probably changes in the meat industry, where meat production has shifted from small-scale slaughterhouses with limited distribution patterns, to large facilities that process thousands of pigs each day and distribute their products nationally and internationally. Farm sizes have increased and animal husbandry methods have also become more intensive. Intensive husbandry

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in the porcine industries creates difficulties in maintaining adequate hygienic conditions in rearing pens, and in limiting cross-contamination between animals. While many modern slaughter techniques reduce the risk of meat contamination, opportunities for animal-to-animal transmission of the organism during transport and lairage, and for cross-contamination of carcasses and meat products, exist on a scale that was unthinkable decades ago. In addition, advances in packaging and refrigeration now allow industry and consumers to store foods for much longer periods, a significant factor with regard to a cold-adapted pathogen such as Y. enterocolitica. However, if a successful reduction of Y. enterocolitica could be accomplished on the top levels of the breeding pyramid, lowering of prevalence of Y. enterocolitica might be obtained in the general pig population. Analysing herds for antibodies might be an easy way to assess if a herd is infected or not. If negative herds only buy animals from certified, negative herds, a closed circle without carriers of Y. enterocolitica could be obtained.

14.12 Sources of further information and advice Some relevant articles and books: Ostroff S M (1995), ‘Yersinia as an emerging infection: epidemiologic aspects of yersiniosis’, Contrib Microbiol Immunol, 13, 5–10. Mollaret H H (1995), ‘Fifteen centuries of yersiniosis’, Contrib Microbiol Immunol, 13, 1–4. Skurnik M, Bengoechea J A and Granfors K (2003) The Genus Yersinia. Entering the Functional Genomic Area. Advances in Experimental Medicine and Biology vol. 529, New York: Kluwer Academic/Plenum Publishers. Tauxe R V (2002), ‘Emerging foodborne pathogens’, Int J Food Microbiol, 78, 31–42.

14.13

References

ABER R C, MCCARTHY M A, BERMAN R, DEMELFI T

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and DANIELSSON-THAM M L (1996), ‘A comparison between a PCR method and a conventional culture method for detection of pathogenic Yersinia enterocolitica in foods’, J Appl Bacteriol, 81, 303–8. THOMPSON J S and GRAVEL M J (1986), ‘Family outbreak of gastroenteritis due to Yersinia enterocolitica serotype O:3 from well water’, Can J Microbiol, 32, 700–1. TOIVANEN P, TOIVANEN A, OLKKONEN L and AANTAA S (1973), ‘Hospital outbreak of Yersinia enterocolitica infection’, Lancet, I, 1801–3. TOMA S and LAFLEUR L (1981), ‘Yersinia enterocolitica infections in Canada 1966 to August 1978’, in E J Bottone, Yersinia enterocolitica, Boca Raton, Fla: CRC Press, Inc, 183– 91. URSING J, BRENNER D J , BERCOVIER H, FANNING G R, STEIGERWALT A G, BRAULT J and MOLLARET H H (1980), ‘Yersinia frederiksenii: a new species of Enterobacteriaceae composed of rhamnose-positive strains (formerly called atypical Yersinia enterocolitica or Yersinia enterocolitica-like)’, Curr Microbiol, 4, 213–17. VANTRAPPEN G, AGG H O, PONETTE E, GEBOES K and BERTRAND P (1977), ‘Yersinia enteritis and enterocolitis: Gastroenterological aspects’, Gastroenterology, 72, 220–7. VIITANEN A M, ARSTILA P, LAHESMAA R, GRANFORS K, SKURNIK M, and TOIVONEN, P (1991), ‘Application of the polymerase chain reaction and immunofluorescence techniques to the detection of bacteria in Yersinia-triggered reactive arthritis’, Arthritis Rheum, 34, 89–96. WACHSMUTH K (1985), ‘Genotypic approaches to the diagnosis of bacterial infections: Plasmid analyses and gene probes’, Infect Control, 6, 100–9. WAUTERS G (1981), ‘Antigens of Yersinia enterocolitica’, in Bottone E J, Yersinia enterocolitica, Boca Raton, Fla, CRC Press, Inc, 41–53. WAUTERS G (1991), ‘Taxonomy, identification and epidemiology of Yersinia enterocolitica and related species’, in Grimme H, Landi E and S Dumontet. I Problemi della Moderna Biologia: Ecologia Microbica, Analitica di Laboratorio, Biotecnologia, Vol. 1. Atti del IV Convegno Internazionale, Sorrento, 93–101. WAUTERS G, GOOSSENS V, JANSSENS M and VANDEPITTE J (1988a), ‘New enrichment method for isolation of pathogenic Yersinia enterocolitica O:3 from pork’, Appl Environ Microbiol, 54, 851–4. WAUTERS G, JANSSENS M, STEIGERWALT A G and BRENNER D J (1988b), ‘Yersinia mollaretii sp. nov., formerly called Yersinia enterocolitica biogroups 3A and 3B’, Int J Syst Bacteriol, 38, 424–9. WEYNANTS V, JADOT V, DENOEL P A, TIBOR A and LETESSON J-J (1996a), ‘Detection of Yersinia enterocolitica serogroup O:3 by a PCR method’, Journal of Clinical Microbiology, 34, 1224–7. WEYNANTS V, TIBOR A, DENOEL P A, SAEGERMAN C, GODFROID J, THIANGE P and LETESSON J -J (1996b), ‘Infection of cattle with Yersinia enterocolitica O:9 a cause of the false positive serological reactions in bovine brucellosis diagnostic tests’, Vet Microbiol, 48, 101–112. WINBLAD S. (1975), ‘Arthritis associated with Yersinia enterocolitica infections’, Scand J Infect Dis, 7, 191–5. WORLD HEALTH ORGANIZATION (1983), Yersiniosis: report on a WHO meeting, Paris, 1981, WHO Regional Office for Europe, Euro reports and studies 60, Copenhagen, 31pp. WORLD HEALTH ORGANIZATION (1995), Report of the WHO Consultation on Emerging Foodborne Diseases, Berlin. WORLD HEALTH ORGANIZATION (2000), Foodborne disease: A focus for health education, Geneva, Switzerland WORMSER G P and KEUSCH G T (1981), ‘Yersinia enterocolitica: clinical observations’, in Bottone E J, Yersinia enterocolitica, Boca Raton, Fla, CRC Press, Inc, 83–93. WRIGHT J, FENWICK S and MCCARTHY M (1995), ‘Yersiniosis: an emerging problem in New Zealand’, N Z Public Health Rep, 2, 65–6.

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(1894), ‘La peste bubonique a Hong Kong’, Ann Inst Pasteur, Paris, 8, 662–7. (1973), ‘An outbreak of enteritis due to Yersinia enterocolitica occurring at a junior high school’, Jpn J Microbiol, 17, 220–2.

YERSIN A

ZEN-YOJI H, MARUYAMA T, SAKAI S, KIMURA S, MIZUNO T, AND MOMOSE T

15 Listeria J. McLauchlin, Health Protection Agency Food Safety Microbiology Laboratory, UK

15.1

Introduction

The bacterium Listeria monocytogenes was first identified as responsible for illness in animals in 1925, and the first suggestion that this was predominantly a foodborne illness was published in 1927. However, it was not until a series of large outbreaks occurred in the 1980s that it was realised that this bacterium is amongst the most important agents responsible for a serious foodborne infection in humans. The properties of the bacterium favour transmission through food and listeriosis most often affects the unborn, newly delivered, neonates, elderly and immuno-compromised individuals. Changes in eating habits to consumption of more ready-to-eat foods with extended refrigerated shelf lives also favours this disease. During the 1980s and 1990s much information was amassed on the distribution, behaviour and susceptibility to environmental challenges of L. monocytogenes. In addition, better recognition of this pathogen and vastly improved diagnostic procedures were developed. The application of control measures throughout the food chain (including the application of HACCP) have now resulted in a dramatic reduction in the levels of contamination of foods on retail sale. Changes in food processing technology together with ever increasing global food sourcing (both of raw materials and distribution of finished products) will present different and changing challenges for the control of this bacterium. Demographic changes will inevitably result in an increased at risk and susceptible population, e.g. the immunocompromised and elderly. Constant attention to food processes and control of L. monocytogenes contamination is an essential on-going responsibility of the food industry.

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15.2 Historical summary and emergence of listeriosis as a major foodborne disease In 1925, Murray, Webb and Swann in Cambridge (UK) described a series of spontaneous severe infections amongst laboratory rabbits and guinea pigs characterised by a marked mononuclear leukocytosis (Murray et al. 1926). The disease was caused by a Gram-positive bacterium, which they named Bacterium monocytogenes. In the following year, Pirie (1927) also isolated a Gram-positive bacterium, in this instance from infected wild gerbils in South Africa; he proposed the generic name Listerella in honour of the surgeon Lord Lister. Murray and Pirie realised that they were dealing with the same bacterium, and combined the names to form Listerella monocytogenes. This was later changed for taxonomic reasons to Listeria monocytogenes (Pirie 1940). In 1929 in Denmark, Nyfeldt isolated L. monocytogenes from the blood cultures of patients with a mononucleosis-like infection (a rare manifestation of the disease), and in 1936, Burn in the USA established listeriosis as a cause of both sepsis amongst newborn infants as well as a cause of meningitis in adults (Gray and Killinger 1966). Prior to 1926 there were published descriptions of disease likely to have been listeriosis; indeed, a ‘diphtheroid’ isolated from the cerebrospinal fluid of a soldier in Paris in 1919 was later identified as L. monocytogenes (Gray and Killinger 1966). The concept that listeriosis is a foodborne disease is not new, indeed Murray and colleagues (1926) commented: ‘The seasonal incidence of the natural disease seems to have been in the early spring and autumn. At these times the fresh food upon which the breeding establishment largely depended either became scarce or rank.’ Pirie (1927) went even further, producing the prophetic, but somewhat forgotten, statements: ‘... infection can be produced by subcutaneous inoculation or by feeding, and it is thought that by feeding that the disease is spread in nature.’ However, human listeriosis remained a relatively obscure disease until the 1980s, attracting limited attention, although large outbreaks of considerable morbidity and mortality but of unknown transmission occurred. For example, 279 and 166 human listeriosis cases respectively occurred in Halle (Germany) during 1966, and in the Anjou region (France) between 1975–76. During the early to mid-1980s, there was a rise in the total numbers of human and animal listeriosis cases in Europe and North America and a series of human foodborne outbreaks (Table 15.1). There was considerable interest in the disease, the causative organism and its interaction and behaviour, together with methods for its detection and isolation, and resulted in the emergence of L. monocytogenes as one of the most important foodborne pathogens. The numbers of reported human listeriosis cases has subsequently declined in most of Europe and North America, although local upsurges in sporadic cases together with large foodborne outbreaks continue to occur. Recent estimates ranked listeriosis as the second and fourth most common cause of death from foodborne infectious diseases in the USA and in England and Wales respectively (Table 15.2).

408 Emerging foodborne pathogens Table 15.1 Foodborne outbreaks of human listeriosis which led to the emergence of this disease during the 1980s Year

Place

Country

Cases

Deaths

Food vehicle

1981 1983 1985 1983–87 1987–89

Nova Scotia Boston Los Angeles Vaud National

Canada USA USA Switzerland UK

41 49 142 122 337

18 14 30 34 94

Coleslaw Milk Soft cheese Soft cheese Pâté

Table 15.2 Estimation of the five most common causes of death from foodborne pathogens in the USA and England and Wales Annual total of cases of foodborne illness Total numbers of cases

Deaths

USA (data adapted from Mead et al., 1999) All cases 76,000,000 Salmonella 1,412,498 Listeria 2,518 Toxoplasma 225,000 Norovirus 23,000,000 Campylobacter 2,453,926

5,194 843 761 571 190 136

England and Wales* (adapted from Adak et al., 2002) All cases 1,338,772 Salmonella 41,616 Clostridium perfringens 84,081 Campylobacter 358,466 Listeria 194 VTEC O157 995

480 119 89 86 68 22

* Endogenous foodborne disease

15.3 Listeria taxonomy, properties, occurrence and pathogenicity The genus Listeria was originally described as monotypic, containing only L. monocytogenes. However DNA base composition studies by Rocourt and colleagues in the 1982, followed by 16S and 23S rRNA sequence studies a decade later show that six species occur. All species within the genus Listeria form a homogeneous group together with the non-pathogenic species Brochothrix thermospacta and Brochothrix campestris, and justify family status as the Listeriaceae (Collins et al. 1991). 16S rRNA sequence analyses show relationships with other Gram-positive genera of low G + C ratio, including members of the genera Bacillus (Collins et al. 1991). Indeed, the completed genome sequences of L. monocytogenes and Listeria innocua

409

Listeria

which are now available show a close relationship to that from Bacillus subtilis and suggest a common albeit distant origin (Glaser et al. 2001). Listeria are coccobacillary- to bacillus-shaped Gram-positive bacteria. They are non-sporing and motile by peritrichate flagella, aerobic and microaerophilic organisms that grow between 103 organisms per g) with the implicated strain; processed with an extended (refrigerated) shelf life; consumed without further cooking. Outbreaks of human listeriosis involving >100 individuals have occurred, some lasting for several years. This is likely to represent a long-term colonisation of a single site in the food manufacturing environment as well as the long incubation periods shown by some patients. Sites of contamination within food processing facilities involved in human infection have included wooden manufacturing equipment, slicing machinery, wooden and metallic shelving, porous conveyor belts, food residues, cool-room condensates and floor drains; one outbreak of listeriosis was also associated with reconstruction within the food manufacturing environment. L. monocytogenes survives well in moist environments with organic material, and it is from such sites that contamination of food occurs during processing.

15.6 Growth and isolation of Listeria Listeria spp. grow well on a wide variety of non-selective laboratory media, hence culture from normally sterile sites such as blood or cerebrospinal fluid does not require special media. For specimens such as faeces, vaginal secretions, food and environmental samples, special selective media are necessary. Prior to the mid-1980s, ‘cold enrichment’, utilising the ability of Listeria to outgrow competing organisms at refrigeration temperatures in non-selective broths, was the main method used for selective isolation (Gray and Killinger 1966). When growing on transparent media illuminated by oblique transmitted light and viewed at low magnification (‘Henry’ illumination technique) all Listeria colonies have a characteristic blue colour with a central ‘ground glass’ appearance. However, because of the degree of skill required in recognising characteristic colonies, the lack of specificity and the slowness of these methods (some workers subcultured broths for up to six months), the emergence of listeriosis in 1980s resulted in much improved methodologies. Media have been developed that rely on a number of selective agents, these include: acriflavin, lithium chloride, colistin, ceftazidime, cefotetan, fosfomycin, moxolactam, nalidixic acid, cycloheximide and polymyxin. Such

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media have resulted in the widespread ability of microbiology laboratories (especially those involved with the examination of foods) to selectively isolate Listeria. Numerous enrichment and selective isolation media have now been developed. Those mentioned here (or modifications of these) are used most frequently for the examination of foods. For selective broths: US Food and Drugs Administration (FDA) method (Lovett et al. 1987), the US Department of Agriculture (USDA) method (McClain and Lee 1988), or the Netherlands Government Food Inspection Service (NGFIS) method described by Van Netten et al. (1989) are most often used. Selective agars most frequently used are those of Curtis et al. (1989; ‘Oxford’ formulation) or the PALCAM agar (named after an acronym of the ingredients, polymyxin B, acriflavine, lithium chloride, ceftazidime, aesculin and mannitol) of Van Netten et al. (1989). These media are listed in internationally agreed standard methods (Anon. 1996b), which can also be use for quantification of the levels of Listeria contamination in an individual food (Anon. 1998). All Listeria species are isolated by these methods and are morphologically indistinguishable from each other. To differentiate L. monocytogenes from other Listeria species on selective agars, substrates have been added to selective media to detect phospholipase (Notermans et al.,1991) or β-glucosidase and enhanced haemolysis (Beumer et al. 1997). Selective media, based on lipase and β-glucosidase activity, which successfully differentiates L.monocytogenes from populations of other Listeria species, are now commercially available (Vlaemynck et al. 2000). Non-cultural techniques such as those based upon immunoassays and the polymerase chain reaction are used increasingly for the detection of Listeria in enrichment broths for the examination of foods. L. monocytogenes has been isolated from numerous types of raw, processed, cooked and ready-to-eat foods, usually at levels below 10 organisms per g. As outlined in section 15.3, the properties favour transmission through food and a wide variety of food and food matrices will support the growth of this bacterium, which, especially towards the end of an extended shelf-life can become very heavily contaminated. Such ‘problem’ foods types which support the growth of L. monocytogenes include soft cheese, milk, pâté, frankfurters and other sausages, cooked meat and poultry, smoked fish and shellfish, processed vegetables and some cut fruit including melon. Examples of rates of contamination for two of these ‘problem’ food types examined in the UK are given in Table 15.6, and these are further discussed in Section 15.7. Growth can be localised within specific areas of an individual food, either because of the source of contamination (i.e. within cut or contact surfaces or where raw herbs and spices have been added) or because of the physicochemical properties of the foods such as in the areas of higher pH associated with the rind or with mould growth within a soft cheese. The unusual tolerance of the bacterium to sodium chloride and sodium nitrite, and the ability to multiply (albeit slowly) at refrigeration temperatures makes L. monocytogenes of particular concern as a post-processing contaminant in

422 Emerging foodborne pathogens long-shelf-life refrigerated foods. The widespread distribution of L. monocytogenes and the ability to survive on dry and moist surfaces favours post-processing contamination of foods from both raw product and factory sites.

15.7 Prevention and control Control measures for human listeriosis principally rely on the successful exclusion of L. monocytogenes from the food chain. However, since not all foods undergo a Listeria-cidal process this is not practicable, hence all reasonable steps should be implemented to prevent contamination and reduce the multiplication of the organism by adequate temperature and shelf-life control at all stages, hygiene of environmental and factory sites, and quality of raw materials. In addition, a strong education programme should be implemented covering the properties of Listeria and consequences of listeriosis to all those involved with the food production distribution and retailing. During the past two decades, the food industry has been active in investigating Listeria in foods and the factory environment, and implementing control measures (especially HACCP and pathogen monitoring) for this bacterium. The success of these measures is reflected in reductions in the extent of contamination of foods on retail sale in the England and Wales (Table 15.6). In the USA, reductions in the incidence of human listeriosis have also been attributed to industry ‘clean up’ (Tappero et al. 1995). A consideration of food regulation and Listeria contamination is outside the scope of this chapter. However, national regulatory bodies have come to quite different positions (even for different food types within a single country) ranging from an overall zero tolerance, to accommodation of certain levels of contamination together with considerations on shelf-life and the ability of the food to support the growth of the organism. Considerable efforts have been directed towards quantitative risk assessments (Anon. 1999, 2001) and it is envisaged that these will provide a better foundation for identifying the most appropriate interventions, to better inform food regulators, and provide a more rational basis for the formulation of food safety objectives. The final strategy for control of listeriosis is by advice to vulnerable groups. Dietary advice has been given out to vulnerable groups in the UK and USA (Table 15.7). Similar advice has been give in other countries including France, Australia and New Zealand. This advice is certainly prudent, although efforts must be made to continually reinforce it. It is clearly not possible to warn vulnerable groups against all food types associated with infection, and a balance must be made between providing sensible guidance allowing individuals to make informed choices, and scare mongering. Targeting of advice to the general public and attitudes to risk are outside the scope of this chapter, however dietary advice is no substitute for controlling the organism in the food chain. To date there has been limited investigation on the assessment of how effective dietary advice is for controlling listeriosis.

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Table 15.7 Dietary advice for the prevention of listeriosis. (Advice from Anon. 1992, 1994, 1996a) USA Advice to the general public Cook thoroughly raw food from animal sources such as beef, pork and poultry Wash raw vegetables thoroughly before eating. Keep uncooked meats separate from vegetables and from cooked foods and ready-to-eat foods. Avoid raw (unpasteurised) milk or foods made from raw milk. Wash hands, knives and cutting boards after handling uncooked foods.

Advice for at-risk groups Cook until steaming hot left-over foods or ready-to-eat foods such as hot dogs before eating. Avoid soft cheese such as feta, brie, camembert, blue-veined and Mexican style cheese. Hard cheeses, processed cheese, cottage cheese or yogurt need not be avoided. Raw vegetables should be thoroughly washed before eating. Although the risk of listeriosis associated with foods from deli counters is relatively low, pregnant women and immunosuppressed persons may choose to avoid these foods or thoroughly reheat cold cuts before serving.

UK Keep foods for as short a time as possible, follow the storage instructions carefully and observe the ‘best by’ and ‘eat by’ dates on the label. Do not eat undercooked poultry or meat products. Make sure you reheat cookedchilled meals thoroughly and according to the instructions on the label. Wash salad, fruit and vegetables that will be eaten raw. Make sure that your refrigerator is working properly and keep foods stored in it really cold. When reheating food, make sure that it is piping hot all the way through and do not reheat more than once. When using a microwave oven to cook or reheat food, observe the standing times recommended by the oven manufacturer to ensure that food attains an even temperature before it is eaten. Throw away left-over food. Cooked food which is not eaten straight away should be cooled as rapidly as possible and stored in the refrigerator. Pregnant women and anyone with low resistance to infection should not eat soft ripened cheeses of the brie, camembert or blue veined types. Nor should they eat pâté. Any bought cooked-chilled meals or readyto-eat poultry should be reheated until piping hot. Do not eat them cold.

15.8 Future trends The bacterium L. monocytogenes continues to demonstrate its ability to cause considerable morbidity, and is now recognised as one of the major foodborne pathogens. Not only do the properties of the organism favour

424 Emerging foodborne pathogens transmission through foods, but changes in eating habits to consumption of more ready-to-eat foods which are less well preserved but highly processed with extended refrigerated shelf-lives also favours this disease. Demographic changes will inevitably result in increased at risk and susceptible populations (e.g. the immunocompromised and elderly) and although dietary advice can be given to these groups, this is no substitute for controlling the organism in the food chain. Better recognition of this pathogen and vastly improved diagnostic procedures have allowed a huge body of information to be amassed on the distribution, behaviour and susceptibility to environmental challenges of L. monocytogenes. As has been demonstrated in the UK, application of control measures throughout the food chain (including the application of HACCP) have resulted in a dramatic reduction in the levels of contamination of foods on retail sale, although constant attention to food processes and control of L. monocytogenes contamination is an essential on-going responsibility of the food industry. Changes in food processing technology together with ever increasing food globalisation (both of raw materials and distribution of finished products) will present different and changing challenges for the control of this bacterium. It is likely that improvements in quantitative risk assessments will result in more harmonised food regulation with respect to L. monocytogenes contamination, which will also allow freer international trade. Better awareness of food safety and continued education of all involved with the food chain concerning the properties of L. monocytogenes are essential for the continued and improved control of listeriosis.

15.9 Sources of information and advice 15.9.1 General review articles on Listeria Bille J, Rocourt J (1996) WHO International Multicenter Listeria monocytogenes subtyping study: rationale and set-up of the study. Int J Food Microbiol, 32, 251–262. Buchrieser C, Rusniok C, Kunst F, Cossart P, Glaser P; Listeria Consortium. (2003) Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity. FEMS Immunol Med Microbiol, 35, 207–213. Charpentier E, Courvalin P (1999) Antibiotic resistance in Listeria spp. Antimicrobial Agents Chemoth, 43, 2103–2108. Collins MD, Wallbanks S, Lane DJ, Shah J, Nietupski R, Smida J, Dorsch M, Stackebrandt E (1991) Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA. Int J Syst Bacteriol 41, 240–246. Donnelly CW (2001) Listeria monocytogenes: a continuing challenge. Nutr Rev, 59, 183–194.

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Doyle ME, Mazzotta AS, Wang T, Wiseman DW, Scott VN (2001) Heat resistance of Listeria monocytogenes. J Food Prot 64, 410–429. Farber JM, Peterkin PI (1991) Listeria monocytogenes, a food-borne pathogen. Microbiol Rev 55, 476–511. Gray ML, Killinger AH (1966) Listeria monocytogenes and listeric infections. Bacteriol Rev 30, 309–382. Huss HH, Jorgensen LV, Vogel BF (2000) Control options for Listeria monocytogenes in seafoods. Int J Food Microbiol, 62: 267–274. Kathariou S (2002) Listeria monocytogenes virulence and pathogenicity, a food safety perspective. J Food Prot, 65: 1811–1829. Low JC, Donachie W (1997) A review of Listeria monocytogenes and listeriosis. Vet J 153, 9–29. McLauchlin J (1996) The relationship between Listeria and listeriosis. Food Control 7, 187–193. McLauchlin J (1997) The pathogenicity of Listeria monocytogenes: a public health perspective. Rev Med Microbiol, 8, 1–14. Rocourt J, BenEmbarek P, Toyofuku H, Schlundt J (2003) Quantitative risk assessment of Listeria monocytogenes in ready-to-eat foods: the FAO/ WHO approach. FEMS Immunol Med Microbiol, 35, 263–267. Ryser ET, Marth EH eds (1998) Listeria, Listeriosis, and Food Safety, second edition, Marcel Dekker, New York. Schuchat A, Deaver KA, Wenger JD, Plikaytis BD, Mascola L, Pinner RW, Reingold AL, Broome CV (1992) Role of foods in sporadic listeriosis. I. Case-control study of dietary risk factors. The Listeria Study Group. JAMA, 267: 2041–2045. Schuchat A, Swaminathan B, Broome CV (1991) Epidemiology of human listeriosis. Clin Microbiol Rev, 4, 169–183. Schlech WF. (1991) Listeriosis: epidemiology, virulence and significance of contaminated foodstuffs. J Hosp Infect 19, 211–224. Schlech WF (1997) Listeria gastroenteritis – old syndrome, new pathogen. N Engl J Med, 336: 130–132. Tappero JW, Schuchat A, Deaver KA, Mascola L, Wenger JD (1995) Reduction in the incidence of human listeriosis in the United States. Effectiveness of prevention efforts? J Am Med Assoc 273, 1118–1122. Tompkin RB (2002) Control of Listeria monocytogenes in the food-processing environment. J Food Prot, 65, 709–725. Vazquez-Boland JA, Kuhn M, Berche P, Chakraborty T, Dominguez-Bernal G, Goebel W, Gonzalez-Zorn B, Wehland J, Kreft J (2001) Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev, 14, 584–640. WHO. Foodborne disease: a focus for health. 2000, WHO Geneva. 15.9.2 General information and public health data Centres for Disease Control and Prevention, USA

426 Emerging foodborne pathogens http://www.cdc.gov/ncidod/dbmd/diseaseinfo/listeriosis_g.htm U.S. Food & Drug Administration, Center for Food Safety & Applied Nutrition, USA http://vm.cfsan.fda.gov/~mow/chap6.html Health protection Agency, UK http://www.hpa.org.uk/infections/topics_az/listeria/gen_inf.htm World Health Organisation, risk assessment. http://www.who.int/foodsafety/micro/jemra/assessment/listeria/en/print.html FAO Expert consultation on the trade impact of Listeria in fish products http://www.fao.org/DOCREP/003/X3018E/X3018E00.HTM 15.9.3 Microbiological media, identification and detection systems http://www.chromagar.com/products/listeria.html http://www.laboratorytalk.com/news/oxo/oxo157.html http://www.qualicon.com/pressreleases/pr_baxomalm.html http://service.merck.de/microbiology/ http://industry.biomerieux-usa.com/industry/food/api/apiproducts.htm http://www.800ezmicro.com/productPubs.asp?mb=01&ez=61

15.10

References

ADAK GK, LONG SM, O’BRIEN SJ

(2002) Trends in indigenous foodborne disease and deaths, England and Wales: 1992 to 2000. Gut, 51, 832–841. ANON. (1992) Centers for Disease Control/National Center for Infectious Disease. Preventing foodborne listeriosis. USDHHS PHS May 1992, Atlanta, Georgia, USA. ANON. (1994) Ministry of Agriculture, Fisheries and Foods. Food Sense No 1. Food Safety. PB0551 London. ANON. (1996a) Department of Health. While you are pregnant: how to avoid infection from food and from contact with animals. H15/005 812 1P Sept 96. ANON. (1996b) International standard Microbiology of food and animal feeding stuffs: horizontal method for the detection and enumeration of Listeria monocytogenes, part 1 detection method ISO 11290-1 1996 (E). British Standards Institute, London. ANON. (1998) International Standard Microbiology of food and animal feeding stuffs: horizontal method for the detection and enumeration of Listeria monocytogenes, part 2 enumeration method ISO 11290-2 1998 (E). British Standards Institute, London. ANON. (1999). Codex Alimentarium Commission. Principals and guidelines for the conduct of microbial risk assessment (CAC/GL-30, 1999). http://www.fao.org/docrep/004/y1579e/y1579e05.htm ANON. (2001) Draft Assessment of the Relative Risk to Public Health for Food-borne Listeria monocytogenes among selected categories of ready to eat foods. http://www.foodsafety.gov/~dms/lmrisk.html BEUMER RR, TE GIFFEL MC, COX LJ (1997) Optimization of haemolysis in enhanced haemolysis agar (EHA) – a selective medium for the isolation of Listeria monocytogenes. Lett Appl Microbiol 24, 421–425. BOURRY A, POUTREL B, ROCOURT J (1995) Bovine mastitis caused by Listeria monocytogenes: characteristics of natural and experimental infections. J Med Microbiol 43, 125–132.

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COLLINS MD, WALLBANKS S, LANE DJ, SHAH J, NIETUPSKI R, SMIDA J, DORSCH M, STACKEBRANDT E

(1991) Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA. Int J Syst Bacteriol 41, 240–246. CUMMINS AJ, FIELDING AK, MCLAUCHLIN J (1994) Listeria ivanovii infection in a patient with AIDS. J Infect, 28, 89–91. CURTIS GDW, MITCHELL RG, KING AF GRIFFIN EJ (1989) A selective differential medium for the isolation of Listeria monocytogenes. Lett Appl Microbiol 8, 95–98. ELSON R, BURGESS F, LITTLE CL, MITCHELL RT (2004) Microbiological examination of readyto-eat cold sliced meats and pâté from catering and retail premises in the United Kingdom. J Appl Microbiol, 96: 499–509. FARBER JM, PETERKIN PI (1991) Listeria monocytogenes, a food-borne pathogen. Microbiol Rev 55, 476–511. GLASER P, FRANGEUL L, BUCHRIESER C, et al. (2001) Comparative genomics of Listeria species. Science 294, 849–852. GRAY ML, KILLINGER AH (1966) Listeria monocytogenes and listeric infections. Bacteriol Rev 30, 309–382. LOVETT J, FRANCIS DW, HUNT JM (1987) Listeria monocytogenes in raw milk: detection, incidence and pathogenicity. J Food Prot, 50, 188–192. LOW JC, DONACHIE W (1997) A review of Listeria monocytogenes and listeriosis. Vet J 153, 9–29. MACKEY BM, BRATCHELL N (1989) The heat resistance of Listeria monocytogenes. Lett Appl Microbiol 9, 89–94. McCLAIN D, LEE WH (1988) Development of the ‘USDA-FSIS’ method for isolation of Listeria monocytogenes from raw meat and poultry. J Assoc Off Anal Chem 71, 660– 664. McLAUCHLIN J (1996) The role of the PHLS in the investigation of listeriosis during the 1980s and 1990s. Food Control 7, 235–239. Mc LAUCHLIN J (1997) The pathogenicity of Listeria monocytogenes: a public health perspective. Rev Med Microbiol 8, 1–14. McLAUCHLIN J, LOW C (1994) Primary cutaneous listeriosis in adults: An occupational disease of veterinarians and farmers. Vet Rec, 135, 615–617. MEAD PS, SLUTSKER L, DIETZ V, MCCAIG LF, BRESEE JS, SHAPIRO C, GRIFFIN PM, TAUXE RV (1999) Food-related illness and death in the United States. Emerg Infect Dis, 5, 607–625. MURRAY EGD, WEBB RA, SWANN MBR (1926) A disease of rabbits characterised by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n.sp.). J Pathol Bacteriol 29, 407–439. NOTERMANS SH, DUFRENNE J, LEIMEISTER-WACHTER M, DOMANN E, CHAKRABORTY T (1991). Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species. Appl Environ Microbiol 57, 2666– 2670. PIRIE JHH (1927) ‘A new disease of veld rodents, “Tiger River Disease” ’ Publ S Afr Inst Med Res, 3, 163-186. PIRIE JHH (1940) Listeria: change of name for a genus of bacteria. Nature (London) 145, 264. ROCOURT J, SCHRETTENBRUNNER A, HOF H, ESPAZE EP (1987) Une nouvelle espèce du genre Listeria: Listeria seeligeri. Pathol Biol 35, 1075–1080. RYSER ET, MARTH EH eds (1998) Listeria, Listeriosis, and Food Safety, second edition, Marcel Dekker, New York. SWAMINATHAN B, BARRETT TJ, HUNTER SB, TAUXE RV and The CDC PulseNet Task Force (2001) PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis, 7, 382–389. TAPPERO JW, SCHUCHAT A, DEAVER KA, MASCOLA L, WENGER JD (1995) Reduction in the incidence of human listeriosis in the United States. Effectiveness of prevention efforts? J Am Med Assoc 273, 1118–1122.

428 Emerging foodborne pathogens DE VALK H, JACQUET C, GOULET V, et al. (2003) Feasibility study for a collaborative surveillance

of Listeria infections in Europe. Report to the European Commission, DGSANCO, Paris. VAN NETTEN P, PERALES I, VAN DE MOOSDIJK A, CURTIS GD, MOSSEL DA (1989) Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int J Food Microbiol, 8, 299–316. VAZQUEZ-BOLAND JA, KUHN M, BERCHE P, CHAKRABORTY T, DOMINGUEZ-BERNAL G, GOEBEL W, GONZALEZ-ZORN B, WEHLAND J, KREFT J (2001) Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev, 14, 584–640. VLAEMYNCK G, LAFARGE V, SCOTTER S (2000) Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium. J Appl Microbiol, 88, 430–441. WALKER JK, MORGAN JH, McLAUCHLIN J, GRANT K, SHALLCROSS JA (1994) Listeria innocua isolated from a case of ovine meningoencephalitis. Vet Microbiol 42, 245–253.

16 Helicobacter pylori S. F. Park, University of Surrey, UK

16.1

Introduction

The genus Helicobacter was created in 1989 with Helicobacter pylori as the type species. Since this time, the genus has expanded to include about 18 species (Owen, 1998). An essential diagnostic feature of nearly all helicobacters is the possession of a sheathed flagellum with most also being urease positive and microaerophilic. It seems likely that a majority, if not all, mammals carry Helicobacter species as part of the indigenous biota of their gastric contents (Lecoindre et al., 2000; Fox and Lee, 1997). Whilst helicobacters are traditionally associated with the stomach, increasingly they are also being isolated from extragastric niches within the mammalian body including the intestinal tracts of humans, animals, and birds (On et al., 2002). In addition, certain species have been isolated from diseased livers in mice, and one of these H. hepaticus, has been linked to the formation of liver tumours in these animals (Fox, 1997). H. pylori, however, is primarily associated with the human stomach. Indeed, much evidence suggests that it may actually be part of indigenous microflora of this environment and that this has been the case for at least tens of thousands of years (Blaser, 1998; Ghose et al., 2002; Falush et al., 2003). It is remarkable then, that it was not until 1982, when Marshall and Warren (1984) isolated H. pylori (then designated Campylobacter pyloridis) as a novel Gram negative spiral shaped bacterium from humans with gastric ulcers, that the significance of this species became apparent. Today the pathogenicity of H. pylori disease is well proven and the scope of infection and illness around the world known to be vast, it being the most prevalent bacterial infection among humans. Estimates suggest that as many as 50% of the world’s population may be infected (Goodwin et al., 1997; Lambert et

430 Emerging foodborne pathogens al., 1995). In countries with low socio-economic status the prevalence of carriage may even be as high as 60% in childhood and 80–90% in elderly people (Brown, 2000). In most infected individuals (~80%), H. pylori does not generate clinical symptoms yet can remain as a persistent infection over a lifetime. However, some 10–20% of infected persons subsequently develop gastric hyperacidity and peptic ulcers. In the United States alone some five million people are diagnosed with ulcers (Sonnenberg and Everhart, 1997). An even smaller, yet significant, proportion of persons (0.1–0.4%) develop distal gastric adenocarcinoma and as a consequence of this H. pylori infection is designated as a class I carcinogen in human gastric cancer (Anon., 1994). The human gastrointestinal tract is thought to be the principal reservoir for infection by H. pylori (Axon, 1996). The mode of transmission, however, has not yet been fully elucidated as epidemiological studies have been impeded by the difficulty of isolating this fastidious organism from food, water and other environmental sources. The finding of helicobacters in nonhuman sources such as livestock (Vaira et al., 1992), vegetables (Hopkins et al., 1993), milk (Fujimura et al., 2002) and water (Hegarty et al., 1999) suggests that food and water may play at least some role in transmission. Consequently, the aim of this chapter is to consider the likelihood that H. pylori is an emerging foodborne pathogen and its main focus, therefore, will be a consideration of actual reports of its isolation from food and its physiology, in terms of its ability to survive in food and water. In addition, because of its association with food and water, the review will also consider the protocols available for the culture of H. pylori from food and water samples, and the use of alternative strategies for its detection in these environments.

16.2 Physiology and growth requirements H. pylori is a member of the epsilon sub group of the proteobacteria, and as such, is closely related to Campylobacter jejuni a leading cause of foodborne illness (Park, 2002). One characteristic it shares with C. jejuni, and one which hampered its initial isolation, is its fastidious growth requirements. Notably, this species will grow well only in microaerobic environments (5– 10% oxygen) and in carbon dioxide enriched atmospheres (Goodwin and Armstrong, 1990; Xia et al., 1993). Even when provided with these environments, and an optimal growth incubation temperature (35–37 °C), growth of the bacterium is very slow and generally from its first isolation 3– 4 days are required before colonies become apparent. Other environmental factors also place restrictions on the growth of the organism and are likely to limit its growth in food. For example, growth of H. pylori can occur only between 33 to 40.5 °C (Goodwin and Armstrong, 1990) and at pH4.5 or above (Jiang and Doyle, 1998). The organism is sensitive to sodium chloride,

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failing to grow in 2% NaCl, and the minimum aw for growth is between 0.96 and 0.98 (Jiang and Doyle, 1998). H. pylori strains are auxotrophic for several amino acids with some diversity existing in these requirements (Nedenskov, 1994; Reynolds and Penn, 1994). A genome scale metabolic model, which takes into account the genome sequence annotation (Tomb et al., 1997) and physiological data, calculates that of a set 47 metabolites are necessary for growth. Eight of these are amino acids with L-arginine and alanine thought to provide the major sources of carbon (Schilling et al., 2002). However, the requirement for this element can also be met by other compounds including glucose, pyruvate, lactate, malate, fumarate, succinate, α-ketoglutarate and several other amino acids. Notably, of the amino acids that are required for growth, six are also essential amino acids in the human diet. It seems H. pylori may have evolved to selectively utilise these since the host’s nutritional needs, and the subsequent proteolysis of food sources, would generally guarantee their presence in the human gastric environment (Schilling et al., 2002).

16.3 Disease associations and mechanisms of virulence Whilst many individuals infected with H. pylori remain asymptomatic over a lifetime, in a significant number of cases H. pylori causes gastritis, gastric and duodenal ulcers (Blaser, 1995). Infection is also a significant risk factor for gastric malignancies (Nomura et al., 1991; Uemura et al., 2001). As a consequence of the severe nature of these disease syndromes, and the high incidence of H. pylori infection, this pathogen, its virulence factors and its interaction with the human host have been the focus of intensive research for over two decades. Many recent reviews (Moran et al., 2002; Boquet et al., 2003; Sachs et al., 2003; Prinz et al., 2003; Blaser and Atherton, 2004) have focused in detail on this area, and since a detailed assessment of this is beyond the scope of this review, only the major virulence mechanisms will be considered here. 16.3.1 Mechanisms of acid resistance required for survival in the stomach Because of its specialised niche, one of the major challenges faced by H. pylori is survival and growth in the acidic environment of the human stomach. In the laboratory, however, the pathogen behaves as a neutralophile and accordingly, does not survive well below pH4.0. It is now clear that in order to grow at low pH, H. pylori has evolved, not a general acid resistance mechanism, but one that operates specifically in the unique niche of the human stomach. This mechanism centres on the Ni2+ containing cytoplasmic enzyme urease that converts urea to ammonia and carbon dioxide. H. pylori in fact produces higher activities of urease than any other microbe known

432 Emerging foodborne pathogens (Mobley et al., 1995) and therefore it is not surprising that this was one the first virulence factors to be identified (Sachs et al., 2003). The role of this cytoplasmic enzyme in acid tolerance is to generate ammonia, which subsequently diffuses into the periplasm, buffering it against the low pH and increasing the membrane potential to allow growth (Scott et al., 1998). This is one of the primary acid tolerance mechanisms of H. pylori and consequently urease activity is essential for survival in the stomach of animal models (Tsuda et al., 1994). However, while this constitutes a highly effective acid tolerance mechanism, it can also be detrimental since urease activity is toxic to H. pylori at neutral pH (Clyne et al., 1995). As a defence against this, H. pylori urease is active only at low pH where its expression is beneficial. Uniquely, control of urease activity occurs at the level of substrate access to the enzyme and urea specific channels in the inner membrane only open at pH values below 6.5 (Weeks et al., 2000). Accordingly, the substrate is only delivered to the enzyme when its activity will not be toxic to the cell. A number of other acid tolerance mechanisms do exist but these are of less importance and are outlined in detail in Sachs et al., (2003).

16.3.2 Toxins and interaction with host epithelial cells The virulence of many pathogens is associated with the production of toxins which interact with host cells to potentiate disease. The observation that H. pylori culture supernatants induce the formation of large vacuoles in eukaryotic cells, led to the discovery of the vacuolating toxin, VacA (Leunk et al., 1988). This high molecular weight pore-forming toxin, which causes massive vacuolation in susceptible cells (Papini et al., 1994), is one of the primary virulence factors for H. pylori. The gene encoding VacA is present in all strains, yet production of the toxin varies significantly (Atherton et al., 1995). This is one of the most fully characterised virulence factors, and while many activities have been proposed for VacA (Papini et al., 2001; Boquet et al., 2003), its role in vivo has not yet been fully elucidated. Recently, however, two independent groups have shown that VacA induces epithelial cell apoptosis (Kuck et al., 2001; Cover et al., 2003) The role of this toxin in virulence may, therefore, be in the destruction of parietal cells, which are the acid producing cells in the stomach. The death of these cells, and the concomitant reduction in stomach HCl production may facilitate enhanced colonisation (Salama et al., 2001; Boquet et al., 2003). Another common paradigm of bacterial pathogenicity is the interaction of bacterium with host cells via the elaboration of specific effector proteins. These generally bring about directed changes in the biology of the host that are of benefit to the pathogen. For H. pylori, the identification of a strain specific gene termed cagA (Cover et al., 1990), which later became recognised as a marker for strains that have increased risk of generating peptic ulcer disease (Nomora et al., 2002) and gastric cancer (Blaser et al., 1995), eventually led to the discovery of such a mechanism. The cagA gene was later found to

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be part of a larger pathogenicity island (a 40 kbp DNA insertion into the chromosomal glutamate racemase gene) (Censini et al., 1996) which also encodes a type IV secretion system (Tummuru et al., 1995). It is now known that H. pylori uses the type IV secretion system to inject CagA, and probably other macromolecules, into gastric epithelial cells. Once inside the host cell, the translocated CagA becomes phosphorylated and initiates a number of changes that affect spreading migration and adhesion (Segal et al., 1999). 16.3.3 Adhesion to host tissue Adhesion to host tissue plays an important role in the initial colonisation process for a variety of bacterial pathogens. For H. pylori such contact with host gastric epithelium is beneficial as it prevents removal by mucosal shedding and also gives the bacterium access to nutrients derived from the damaged host epithelium. Not surprisingly, H. pylori possesses a number of proteins that facilitate adhesion. BabA, for example is an adhesion factor that enables this pathogen to bind specifically to Lewis blood group antigens (Ilver et al., 1998). Notably, the target for BabA seems to differ depending on the predominance of blood group in a particular population since South American Indian strains bind blood group O antigens best and this specialisation coincides with the unique predominance of blood group O in these people (AspholmHurtig et al., 2004). Another adhesin, SabA, enables the bacterial cell to bind to inflamed, but not healthy mucosa or Lewis antigens. Since such tissue is normally encountered only during chronic inflammation, and this is often a hallmark of persistent H. pylori infection, this protein might contribute extraordinary chronicity of infection (Mahdavi et al., 2002). One of the emerging aspects of H. pylori virulence, that separates it from other well characterised bacterial pathogens, is the fact that isolates possess an extremely high degree of phenotypic and genotypic diversity. As a consequence of this, different strains induce varying host inflammatory responses, and these in turn, influence the clinical outcome (Lamarque and Peek, 2003; Blaser and Atherton, 2004). In fact, the extent of this variation is so extreme that in effect, each host is colonised by a fluid bacterial gene pool that is, not a single clone, but a mixture of closely related strains (Kuipers et al., 2000). This may be an adaptation to the fact that each individual human host offers distinct and different gastric microenvironments. Since these will be differentially selective and manifold, conventional phenotypic adaptation may be impossible, and in this situation only highly plastic cell populations may be able give rise to individual cell-types suited to some of these environments. This mechanism of extreme genetic variation, in concert with the heterogeneous selection imposed by the host, may enable H. pylori to persist in multiple niches in the stomach of one individual, and to colonise essentially all members of the human race despite its heterogeneous nature (Blaser and Atherton, 2004).

434 Emerging foodborne pathogens

16.4 Epidemiology and routes of transmission 16.4.1 Person to person transmission H. pylori infection is thought to be acquired mainly in childhood and persists throughout life unless specific treatment is applied (Jones and Sherman, 1998; Rowland, 2000). Although definitive routes of transmission have not yet been established, intra-familial infection, with mother to child or sibling to sibling contact, is considered significant (Rothenbacher et al., 2002; RomaGiannikou et al., 2003). Indeed, in experimental systems where Rhesus Macaques have been used to examine natural acquisition, H. pylori infection is most likely acquired from the mother, and since infants from infected dams are more commonly infected, close contact may facilitate infection (Solnick et al., 2003). Outside of the close-knit family situation, however, there is little evidence to suggest that child-child transmission occurs (Tindberg et al., 2001). Numerous studies have suggested that low socio-economic status, including overcrowding and poor sanitation in childhood (Mendall et al., 1992), is a major risk factor for infection. In these environments, H. pylori infection has been correlated with reduced susceptibility to gastro-enteritis. Here the strong negative association between H. pylori infection and this illness is most likely caused by prior exposure to, and thus acquired immunity to other enteric pathogens (Perry et al., 2004). H. pylori has been isolated from faeces (Thomas et al., 1992: Kelly et al., 1994) and also dental plaque (Krajden et al., 1989; Majmudar et al., 1990) though whether faecal-oral or oral-oral transmission occurs has not yet been clearly established. However, there is some evidence that this organism may be transmitted by saliva since a study by Megraud et al., (1995) demonstrated that a risk factor for H. pylori infection is the eating of premasticated foods. Also, if this route is important then the risk of infection may be higher in individuals where occupational exposure to saliva and dental plaque is frequent. In the context, a number of studies have examined the prevalence of H. pylori in dentists but with conflicting outcomes (Honda et al., 2001; Matsuda et al., 2002). H. pylori can be readily cultured from induced vomitus and air contaminated following its emission. The organism can also be isolated from induced stools (Parsonnet et al., 1999). However, since the organism is difficult to isolate from normal stools (Parsonnet et al., 1999), but readily isolated from naturally produced vomitus (Leung et al., 1999), the organism is potentially transmissible during episodes of gastrointestinal tract illness, particularly where vomiting is apparent. Endoscopes are frequently contaminated with H. pylori immediately after gastroduodenal endoscopy and since the pathogen can survive manual cleaning (Nurnberg et al., 2003) the potential for iatrogenic transmission is high. Whilst transmission by contaminated endoscopes or instruments has been reported (Graham et al., 1988; Langenberg et al., 1990) this is preventable

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if effective disinfection regimes are followed (Cronmiller et al., 1999). Whilst iatrogenic transmission is the only proven mode of transmission (Fantry et al., 1995), given the high rate of infection in individuals who have not undergone endoscopy and that the rate of infection by this route may approximate to four per one thousand endoscopies (Tytgat, 1995), this pathway is unlikely to constitute a numerically significant mode of transmission. 16.4.2 The potential for zoonotic or vector borne transmission Gastric Helicobacter species, other than H. pylori, are widespread in mammals and have been reported in various wild and domestic mammals of different dietary habits such as cats (Paster et al., 1991; Lecoindre et al., 2000), small mammals (Goto et al.,1998) pigs (De Groote et al., 1999), dogs (Hanninen et al., 1996) and avian hosts (Waldenstrom et al., 2003) including poultry (Atabay et al., 1998). In contrast, members of this genus have not yet been isolated from goats which has led to the suggestion that these animals are naturally resistant to Helicobacter infection (Gueneau et al., 2002). H. pylori is primarily a human-specific inhabitant but there are a number of reports that have associated this bacterium with non-human sources including livestock (Vaira et al.,1992). H. pylori has also been associated with domestic cats (Handt et al., 1994) and since the organism has been cultured from salivary and gastric secretions (Fox et al., 1996), the possibility exists that it could be transmitted from cats to humans. The existence of such a route of infection, however, remains contentious since there is no evidence of H. pylori carriage in stray cats which have little contact with humans. Consequently, it has been suggested that infection in domesticated cats may be an anthroponosis, an animal infection with a human pathogen (El-Zaatari et al., 1997). More recently, the organism has been isolated from sheep tissue and therefore sheep may be another potential zoonotic source for H. pylori (Dore et al., 2001). The close association of certain insects with human habitats has led to the suggestion that insects, infected with H. pylori, are a possible route of transmission. Two factors in particular appear to make house flies an ideal vector. Firstly, the mid-mid gut is acidic, like the human stomach, and could potentially select for H. pylori. Secondly, flies must regurgitate their gastric contents to facilitate feeding. H. pylori DNA has been detected in wild house flies (Grubel et al., 1998), and the organism can be cultured from experimentally infected flies for at least 30 hours after inoculation (Grubel et al., 1997). However, since house flies do not appear to acquire H. pylori readily from fresh human faeces (Osato et al.,1998) their role in transmission of this pathogen is believed to be limited. Similarly, cockroaches are also a common insect within the home and environment and since they also habitually infiltrate food-keeping areas, they have also been implicated in H. pylori transmission. Indeed, whilst transmission by contaminated external body parts does not occur, experimentally infected cockroaches can contaminate the environment

436 Emerging foodborne pathogens through the spreading of contaminated excreta (Imamura et al., 2003) suggesting that cockroaches are also a potential vector. 16.4.3 The potential for foodborne transmission From the above it can be seen that H. pylori has been associated with various animals, and since these include livestock, there is a possibility that it can enter the food chain via this route. For example, DNA from H. pylori has been detected in cow faeces (Sasaki et al., 1999) and thus contaminated beef could potentially serve as a vector. However, a study which assayed over one hundred beef samples did not isolate H. pylori (Stevenson et al., 2000a), suggesting that transmission from beef and beef products is not likely to be a primary factor for the high prevalence of human infection. Similarly, pigs have the potential to carry the organism since gnotobiotic pigs were the first animals to be experimentally infected with H. pylori (Krakowka et al., 1987). However, H. pylori was not isolated from pigs or pork in an extensive survey (Stevenson and Acuff, 1999) and epidemiological evidence does not support the role of pork as a vehicle. One strict Muslim country, that does not farm pigs, has a very high prevalence of H. pylori infection (Megraud et al., 1989). In contrast, epidemiological evidence does suggest a role for sheep in H. pylori transmission since shepherds in Sardinia have an unusually high carriage rate of 98% (Dore et al., 1999a). Freshly collected non-pasteurised sheep’s milk has been implicated, therefore, as a vehicle particularly since both H. pylori DNA and culturable cells have been detected in this food (Dore et al., 1999b; Dore et al., 2001). Whether sheep or raw sheep’s milk do represent a significant and widespread vector, however, is not yet clear since a similar study, in which 440 raw sheep’s milk samples from the Burdur region of Turkey were assessed, did not isolate the organism (Turutoglu and Mudul, 2002). Raw, but not pasteurised cows’ milk, has also been implicated as a possible vehicle following the culture of H. pylori from this food in a Japanese study (Fujimura et al., 2002). However, since a separate study from the US failed to detect either DNA or viable cells in milk (Jiang and Doyle, 2002) the role of raw milk as a major vehicle is unclear. In countries of low socio-economic status, contamination of irrigation water by raw sewage, and the subsequent contamination of vegetables that are eaten uncooked, is thought to be a key factor in the transmission of enteric pathogens. Since H. pylori has been isolated from faeces (Thomas et al., 1992: Kelly et al.,1994) it may be transmitted by a faecally contaminated foods. However, whilst the consumption of uncooked vegetables has been correlated with an increased risk of infection in Chile (Hopkins et al., 1993), the organism has not yet been cultured from any raw vegetable. 16.4.4 The potential for waterborne transmission The presence of H. pylori DNA in water raises the suspicion that water might be one of the vehicles of infection. It has, for example, been detected in river

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water (Fujimura et al., 2004), drinking water (Hulten et al., 1996), surface water (Benson et al., 2004) and seawater (Cellini et al., 2004). In addition, fluorescent in situ hybridisation has been used to detect H. pylori cells in river and wastewater (Moreno et al., 2003) and actively respiring microorganisms binding monoclonal anti-H. pylori antibody appear to be common in surface and shallow groundwater samples (Hegarty et al., 1999). To date, however, there is only one report concerning the culture of H. pylori from water and this followed the specific isolation of the organism from raw sewage using immunomagnetic separation (Lu et al., 2002). Accordingly, the contamination of environmental water by sewage is a possible route of transmission. H. pylori DNA appears to be present in poor quality potable waters (Hulten et al., 1996; Bunn et al., 2002), and well waters (Horiuchi et al., 2001; Mazari-Hiriart et al., 2001). As a consequence, drinking water may be a reservoir for H. pylori in areas of the developing world where water quality is poor but whether or not H. pylori can survive in treated water systems is a matter of debate. An early study suggested that this micro-organism is very sensitive to the chlorine used in water treatment plants and implied that H. pylori can be controlled by disinfection practices normally employed in the treatment of drinking water (Johnson et al., 1997). This finding is supported by the fact that H. pylori does not survive well in tap water (Fan et al., 1998), and by a number of recent studies that have failed to detect H. pylori DNA in treated waters (Horiuchi et al., 2001; Mazari-Hiriart et al., 2001). More recently though, it has been suggested that H. pylori can tolerate chlorinebased disinfectants better than the classical faecal indicator Escherichia coli, a finding that increases the likelihood of waterborne transmission in developed countries (Baker et al., 2002). Indeed one study has detected DNA from Helicobacter species in water associated biofilms in pipes used to carry treated drinking water (Park et al., 2001). However, since the identity of this DNA has not been confirmed by DNA sequencing the species may not be H. pylori.

16.5 Detection methods and culture from clinical samples, food and water Discrete strategies for the detection of H. pylori have been developed for environmental and clinical samples. Since the focus of this chapter is the potential for foodborne transmission, the detection and isolation of H. pylori from food will be considered in detail and its clinical detection will be considered only briefly. 16.5.1 Diagnostic tests and detection in clinical samples At present endoscopy represents the gold standard for the diagnosis of

438 Emerging foodborne pathogens H. pylori disease in the clinical situation but the invasive nature of this procedure limits its routine use. In response to this, a number of non-invasive methods such as stool antigen tests and DNA detection in saliva samples are increasingly used (Versalovic, 2003; Bonamico et al., 2004). The specific use of PCR for detecting H. pylori in saliva or faeces is reviewed by Kabir (2004). While the culture of H. pylori is desirable for diagnosis in patients with suspected infection, again it is not generally suitable for routine use because samples from gastric biopsies are most commonly used. Consequently, because they are more easily obtainable and less invasive, alternative sampling procedures using stools, vomitus, saliva, and dental plaque are being explored. A timely review of the methods used to culture H. pylori from such clinical samples has been compiled by (Ndip et al., 2003). The natural habitat of H. pylori is the human stomach where the secretion of hydrochloric acid serves to eliminate many bacteria. Since H. pylori has the unique ability to grow here, its isolation from samples from this environment generally only requires media containing minimal selective agents because of the lack of competing bacterial species. Solid plating media commonly used are based on Columbia agar, brain heart infusion agar or charcoal based agar. These are invariably supplemented with various forms of blood or serum to enhance growth (Goodwin et al., 1985; Dent and McNulty, 1988; Henriksen et al., 1995; Jiang and Doyle, 2000). In addition, numerous broths have been used to culture H. pylori but there is no convincing evidence that any particular one provides a superior growth environment (Stevenson et al., 2000b). 16.5.2 Detection in food and water The isolation of H. pylori from food, water and environmental samples is complicated by the presence of mixed microbial populations. Several selective media have been specifically developed for the recovery of H. pylori from such samples (Stevenson et al., 2000c; Degnan et al., 2003). These are based on H. pylori special peptone agar (Stevenson et al., 2000b) in which the inclusion of special peptone and calf serum with iron, promotes growth (Degnan et al., 2003). As a consequence, colony size is increased (Stevenson et al., 2000b) and this in turn, leads to faster visual identification. While H. pylori is able to thrive in the stomach at low pH and in the presence of urea, these conditions do not provide appropriate selection because they allow the growth of other bacteria (Stevenson et al., 2000c). Consequently, antibiotics are commonly used for selection. In this context, a combination consisting of vancomycin amphotericin B, cefsulodin, polymyxin B sulfate, trimethoprim and sulfamethoxazole is highly selective but still allows H. pylori to grow (Stevenson et al., 2000c). A selective media developed for the isolation of H. pylori from water also contains amphotericin B and polymixin B as selective agents, and has the additional benefit of a colour indicator system, which

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detects urease activity, and which reduces the time required to detect growth of colonies (Degnan et al., 2003). Whilst H. pylori DNA has been detected frequently in environmental waters (Hulten et al., 1996; Benson et al., 2004; Cellini et al., 2004 Fujimura et al., 2004), recovery of the bacterium from these samples has remained elusive. This might be due to the fact that the bacterium is in fact not able to survive in such environments or because recovery parameters, such as the media composition and the incubation atmosphere, are not adequate for colony formation. Furthermore, because of the fastidious nature of this pathogen, and its sensitivity to stress, it is possible that cells from such environments are sub-lethally damaged, and while still viable, are unable to recover on unspecialised laboratory media. In this context, the finding that half-strength media gives higher recoveries than standard media suggests that nutrient shock is an important factor hampering the isolation of H. pylori from dilute environments (Azevedo et al., 2004).

16.6 Survival in food and water Whether food acts as a significant vehicle for H. pylori infection is not clear at present. What is more certain is that given its fastidious growth requirements (see Section 16.2), including limits imposed by growth temperature requirements and its microaerophilic nature, H. pylori is unlikely to grow in food (Jiang and Doyle, 1998). Nevertheless, if the organism is introduced into foods, it can survive for extended periods in low acid, high moisture environments under refrigerated storage (Jiang and Doyle, 1998) and is likely therefore, to remain infective. In experimentally contaminated foods including milk and tofu it can be recovered for up to five days at 4 °C (Poms and Tatini, 2001). In contrast, it appears to survive less well in raw chicken and lettuce and in these environments it is possible that the lack of protection against oxidation and desiccation, potentiates the death of H. pylori. A similar mechanism may explain the limited survival seen in experimentally contaminated ground beef (Stevenson et al., 2000a). While the organism has been shown to grow in ground beef this occurs only in experimental situations, where a microaerobic atmosphere and enrichment broth are provided (Jiang and Doyle, 2002). An additional factor that may limit the survival of H. pylori in certain types of food, is that the organism does not survive freezing well (Ohkusa et al., 2004). 16.6.1 The role of viable non-culturable states in transmission In aquatic and food environments pathogenic bacteria, which most often grow optimally at the temperature of their host, and which also generally have fastidious requirements for nutrients, often encounter stress in the form of starvation, and osmotic and temperature stresses. As a consequence of

440 Emerging foodborne pathogens these stresses certain bacteria may enter a viable non-culturable (VNBC) state. This concept, of a bacterium that remains infectious but that can no longer be cultured by conventional means, was first proposed by Colwell following a study on the survival of Salmonella in aquatic systems (Roszak et al., 1984). It has been proposed that in this state, bacteria may retain metabolic activity, yet are unable of undergoing the cellular division under the prevailing environmental conditions. Many bacterial species also alter morphology as they enter this state (Nilsson et al., 1991). Clearly, the presence of a VNBC form of H. pylori would have significant ramifications for the detection and epidemiology of this pathogen and it may explain the difficulty encountered during attempts to isolate the organism from environmental samples. During infection and recovery on laboratory media, the majority of H. pylori cells present are actively dividing spiral shaped cells (Warren and Marshall, 1983). Under certain environmental conditions, however, such as exposure to air (Catrenich and Makin, 1991), prolonged incubation (Shahamat et al., 1993) and oxygen limitation (Donelli et al., 1998), H. pylori cells undergo transition from rods to cocci, similar to the conversion to the VNC form seen in other bacteria. These coccoid forms have also been observed in the human stomach where they are closely associated with damaged gastric mucous cells (Janas et al., 1995). The significance of the these forms, however, remains obscure and whilst various studies indicate viability on the basis of the LIVE/DEAD BacLight viability assay (Adams et al., 2003), the reduction of tetrazolium salts by oxidative metabolism (Gribbon and Barer, 1995; Cellini et al., 1998) and the ability to take up tritium labelled thymidine (Shahamat et al., 1993), others suggest that the coccoids merely represent a morphological manifestation of cellular degeneration and death (Kusters et al., 1997). A recent study has suggested that whilst the coccoid form represents the VBNC state generated as cells age in laboratory media, when the VBNC form is induced by exposure to natural fresh water environments, it occurs as rod-shaped cells (Adams et al., 2003). The existence of a VBNC form may be controversial but cells, which are not recoverable on media, rapidly produce ATP and synthesise mRNA in response to a stimulus provided by lysed human erythrocytes (Nilsson et al., 2002) and thus at least seem capable of responding to external stimuli and inducing gene expression accordingly.

16.7 Conclusions and future trends H. pylori, being the most common bacterial infection world-wide, has globally profound social and economic consequences. However, even today, over 15 years since its discovery, we are only just beginning to understand this pathogen and its mechanisms of transmission. Our understanding of how H. pylori cause illness is far from complete and consequently, this aspect of their biology will remain an intense area of

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future research (the likely direction of this research has been considered by Moran et al., (2002) and Prinz et al., (2003)) since it has the potential to generate improved therapeutic treatments. Emerging research, however, suggests that the decision to use such agents to eradicate this organism in an infected but symptomless individual to prevent progression of the illness may be complicated. In this context, whilst it is well known that the presence of H. pylori can lead to diseases such as peptic ulcers and distal gastric cancer, there is accumulating evidence that its absence is associated with increased risk for cancers of the oesophagus (Chow et al., 1998). These findings pose important questions related to the removal of this pathogen from infected, but otherwise healthy individuals, and the resolution of this question is likely to form the focus of future research. The availability of the complete genome sequence for H. pylori (Tomb et al., 1997), and the availability of microarrays will allow the response of this pathogen to environmental stresses, such as those encountered in food and water to be mapped in intricate detail and this may lead to a better understanding of its ability to survive in these environments. Already, for example, the global responses of H. pylori to acid stress (Ang et al., 2001) and iron limitation (Merrell et al., 2003) have been mapped using this technique. Understanding the route of H. pylori transmission is important if measures are to be implemented to prevent its spread. For the general population the most likely route of transmission seems to be from person to person either through oral to oral or faecal-oral mechanisms. Although there is some debate which of these is significant most researchers agree that infection is acquired by close contact with infected people in early childhood. However, it has been suggested that H. pylori infection is the consequence of a multiple pathway phenomenon and accordingly, the possibility of other transmission routes cannot be excluded. Thus, at present, food and water have to be considered as potential vehicles. As a consequence of its fastidious nature, H. pylori is not likely to grow in foods. Nevertheless, a number of reports have suggested that it does survive in certain foods and if it exists in a VNC state in these environments this might lead to an underestimation of its prevalence in food and water. Given the failure of many studies to detect H. pylori DNA or isolate the organism from common foods such as meats and vegetables, though, it is unlikely that any food type represents a primary vector. Furthermore, given the possible vectors of infection, such as saliva, faeces and insects, the measures which are already used by food establishments and industry to control the spread of other foodborne bacterial pathogens are likely to be highly effective against H. pylori.

16.8 Sources of further information Two of the most comprehensive and detailed accounts of H. pylori biology are Achtman and Suerbaum (2001) and Mobley et al., (2001). Obviously,

442 Emerging foodborne pathogens these contain far more information than could reasonably be considered here, and as standard references for this fascinating pathogen, these are an excellent source for further study. Readers will also find many smaller but useful reviews that focus on individual aspects of the biology of H. pylori such as pathogenicity and virulence (Moran et al., 2002; Boquet et al., 2003; Sachs et al., 2003; Prinz et al., 2003; Blaser and Atherton, 2004), and their gastric biology and mechanisms of acid resistance (Sachs et al., 2003). A general collection of protocols for detection, molecular epidemiology and molecular manipulation of H. pylori can be found in Clayton and Mobley (1997). More specifically, non-invasive methods for detection such as stool antigen tests and DNA detection in saliva samples are reviewed in Versalovic (2003), Bonamico et al., (2004), and Kabir (2004). A recent review of the methods used to culture H. pylori from clinical samples has been compiled by Ndip et al., (2003). Finally, Helicobacter (www.blackwellpublishing.com/ journal.asp?ref = 1083-438) is a scientific journal dedicated to these intriguing organisms.

16.9

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17 Enterobacteriaceae J-L. Cordier, Nestlé Nutrition, Switzerland

17.1

Introduction

The name Enterobacteriaceae was proposed by Rahn (1937) for a phenotypic group comprising the single genus ‘Enterobacter’ and species of several genera which are still recognised such as Erwinia, Escherichia, Klebsiella, Proteus, Salmonella, Serratia and Shigella. The type genus is Escherichia. Enterobacteriaceae are Gram-negative, oxidase-negative, non-spore forming, straight, rod-shaped bacteria, 0.3–1.0 × 1.0–6.0 µm (Brenner, 1984). They are facultatively anaerobic, ferment glucose and produce acid and gas. They contain, with few exceptions (Erwinia chrysanthemi), the enterobacterial common antigen (ECA) (Ramia et al., 1982; Kuhn et al., 1988). Over the last 20–30 years the number of genera and species have been increasing and reorganisations of the taxonomic groups have occurred frequently. The family currently encompasses 41 genera (Garrity, 2001) and an exhaustive review of new members of the family has been performed by Janda (2004). Modern biochemical and molecular identification and typing methods have caused a constant evolution of the nomenclature but in the case of Enterobacteriaceae, the correspondence between the molecular and the original phenotypic classification schemes is rather good. Details on the phylogenetic relation between strains based on the analysis of small subunit (SSU) rRNA have been published by Francino et al. (2004). The name Enterobacteriaceae is derived from the Latin ‘enterobacterium’ meaning intestinal bacterium. The origin of this name and the reference as ‘enteric bacteria’ provides, however, a misleading impression of their ecological niches and occurrence. Although certain members of this family are found in the intestines of humans and animals, in reality they are very widely distributed

Enterobacteriaceae 451 in soil and water as well as in plants. Many species are responsible for diseases of plants thus causing important economic losses. They also play an important role as endosymbionts of insects or of the parasites that feed on insect larvae (Moran and Baumann, 2000). Different Enterobacteriaceae have been implicated or involved in spoilage of food products such as meat and cured meat products (Borch et al., 1996; Garcia et al., 2000) or fish and fish products (Gram and Huss, 1996). K. pneumoniae and K. oxytoca have been identified as the cause of blowing in Mozzarella cheese (Massa et al., 1992) while E. agglomerans caused spoilage of cottage cheese (Brocklehurst and Lund, 1988). K. pneumoniae present in numbers as high as 106 cfu/ml have been the cause of off-odours and offcolours in orange juice concentrate (Fuentes et al., 1985) and different species of Enterobacteriaceae were able to spoil fruit nectars (Vicini et al., 1999). Different species of Serratia and Rahnella aquatilis have been described by Whitfield (2003) as producers of metabolites responsible for off-flavours in different types of foods and Jensen et al. (2001) studied the formation of guaiacol in chocolate milk by a psychrotrophic Rahnella aquatilis. Enterobacteriaceae are also frequently used as hygiene indicators by food processors to demonstrate adherence to Good Hygiene and Good Manufacturing Practices during processing. This is done to detect as early as possible deviations and thus the potential presence of pathogens such as Salmonella (Cox et al., 1988). Considering the widespread occurrence of Enterobacteriaceae in our environment, use of the term ‘faecal indicator’ as done by several authors (e.g. Gauthier and Archibald, 2001) is misleading. It is applicable to some extent only to untreated raw water or foods such as produce or fresh milk but not in processed foods. Up to the 1940s only Salmonella spp. (including S. Arizonae) and Shigella spp. were considered to be food or waterborne pathogens. They were thus divided into pathogens and non-pathogens according to their ability to cause diarrhoeal disease in humans. However, in the years that followed, other members of the family such as certain pathogenic Escherichia coli or Yersinia enterocolitica have been identified as causative agents of gastrointestinal diseases. They are now well-known and well-established pathogens. Numerous Enterobacteriaceae have been associated with extraintestinal infections and are a major cause of nosocomial infections, i.e., hospital acquired infections, in particular in neonatal and paediatric units (Horan et al., 1986; Andresen et al., 1994; Hervas et al., 2001; Parodi et al., 2003). The role and importance of Enterobacteriaceae have been increasing over the last 10–20 years and they are more and more frequently associated with antibiotic resistant strains. In the following sections discussion of nosocomial infections is limited, with few exceptions, to those where exposure occurred through oral or parenteral routes. Several species causing extraintestinal infections such as Enterobacter, Citrobacter, Serratia, Klebsiella, Proteus and others have been involved on occasion with food or waterborne outbreaks and they will be reviewed in the

452 Emerging foodborne pathogens following sections. Particular attention will be given to Enterobacter sakazakii to acknowledge the increasing importance of this species. The Enterobacteriaceae discussed below can be considered as opportunistic pathogens. Indeed many of the cases reported, including the ones due to E. sakazakii, are the consequence of some parameters and conditions or combinations of them leading to outbreaks. Such parameters are the health status of the patients, the type of food, specific contamination and inappropriate preparation or handling of the foods involved. The symptoms of these diseases range from diarrhoea to severe meningitis, in particular in infants. The mortality rate due to such infections can be high and this is not observed only for E. sakazakii.

17.2 Methods of detection The reference methods for the detection or enumeration of Enterobacteriaceae have been published by the International Organization for Standardization (ISO, 1991a and 1993). In the case of enumeration, samples or appropriate dilutions are plated on Violet Red Bile Glucose (VRBG) agar, overlaid and incubated at 37 °C. VRBG contains selective as well as elective agents such as bile salts, crystal violet and neutral red which suppress competitive organisms and detect the acidification due to the fermentation of glucose. Presumptive typical colonies are then submitted to biochemical tests such as the Gramstain, the oxidase test and the glucose fermentation test. In routine food analysis, frequently only the group of the coliforms (lactose fermenting Enterobacteriaceae) are tested for (ISO 1991b, 1991c). Alternative enumeration methods have been developed which are based on the reference method, an example being the Petrifilm (Silbernagel and Lindberg, 2002). Traditional biochemical test strips for the identification of isolated strains have been commercialised by different companies (e.g. Hayek and Willis, 1976; Staneck et al., 1983; Kronvall and Hagelberg, 2002). In the case of very low numbers of Enterobacteriaceae it is necessary to perform an enrichment and therefore either presence/absence tests in predetermined quantities of products or to determine a Most Probable Number (MPN). In such cases a non-selective enrichment in buffered peptone water is done to allow for resuscitation of injured cells followed by a selective enrichment in Enterobacter Enrichment (EE) broth containing brilliant green and bile salts as selective agents. Confirmation of positive tubes is performed on VRBG, followed by biochemical identification as described above. In the case of the analysis of coliforms, Lauryl Sulfate Broth (LST) is used as enrichment broth followed by confirmation on VRBL and using biochemical tests. For most of the Enterobacteriaceae there is no specific method available. In very few cases media have been developed in the clinical field in trying to detect specific Enterobacteriaceae, examples being P. alcalifaciens (Senior,

Enterobacteriaceae 453 1997) or S. marcescens (Giri et al., 2004). An exception is E. sakazakii for which numerous studies have been performed. Up to 1980 E. sakazakii has been classified as yellow pigmented E. cloacae on the basis of its biochemical profile. Following DNA-DNA hybridisation studies it was recognised as a separate species in 1980 (Farmer et al., 1985). Biochemical identification, including with commercial kits, did not always provide satisfactory results. The yellow pigment is, however, not a unique characteristic and it is formed by other members of the Enterobacteriaceae such as strains of E. agglomerans, Escherichia vulneris, Pantoea dispersa or different Erwinia spp. In attempts to find simple discriminatory characteristics several studies have been performed: the occurrence of different morphological types (Farmer et al., 1985; Nazarowec-White and Farber, 1997a), the enzymatic profiles showing the presence of α-glucosidase, of Tween 80 esterase and the absence of phosphoamidase have been studied showing that E. sakazakii is very different from other Enterobacter species (Aldova et al., 1983; Postupa and Aldova, 1984; Farmer et al., 1985; Muytjens et al., 1984). Today α-glucosidase is frequently used in identification schemes as a rapid discriminatory test (Khandai et al., 2004b) and as an elective parameter in several plating media including commercial ones (Iversen and Forsythe, 2004; Iversen et al., 2004a; Leuschner and Bew, 2004; Leuschner et al., 2004; Oh and Kang, 2004). With respect to the detection methods, initially and currently, detection is usually based on methods for Enterobacteriaceae or coliforms with subsequent identification of suspect colonies (FDA/CFSAN, 2002a). Recently a much more specific method using LST supplemented with 0.5 M NaCl and 10 mg/ l vancomycin (mLST) and an incubation temperature of 45 °C has been developed (Guillaume-Gentil et al., 2005). This method is being used as the basis for the development of a new international technical standard (ISO, 2005). Epidemiological studies or tracing of environmental isolates require more discriminatory methods such as molecular methods. Several studies have already been dedicated to the typing of E. sakazakii. In a study of several cases Muytjens et al. (1983) used plasmid profiles to compare isolates from patients and from infant formulae. Plasmid profiles were different between the two groups and no clear link could be demonstrated. Clark et al. (1990) on their side investigated two unrelated hospital outbreaks. They compared strains isolated from patients and from infant formulae using antibiograms, plasmid analysis, DNA restriction fragment length polymorphism (DNARFLP), ribotyping and multilocus enzyme electrophoresis. Although the latter technique provided the best discrimination, the other molecular techniques were also effective typing tools, while the antibiograms were not sufficiently reliable. Similar conclusions were drawn by Nazarowec-White and Farber (1999) who found RAPD and PFGE to be the more discriminatory methods followed by ribotyping. Van Acker et al. (2001) used arbitrarily primed PCR to investigate the relationship between isolates from patients and infant formulae. Recent phylogenetic studies using 16S ribosomal DNA and hsp60 sequencing indicated substantial heterogeneity among isolates of E. sakazakii

454 Emerging foodborne pathogens and similarities with both Citrobacter koseri and E. cloacae (Iversen et al., 2004c; Lehner et al., 2004). PCR methods have been developed in several laboratories both for the specific detection or identification of E. sakazakii (Hamilton et al., 2003; Seo and Brackett, 2004; Lehner et al., 2004). A commercial PCR, the BAX system developed by Dupont Qualicon, has already been included in a methods collection of Health Canada (Anonymous, 2003).

17.3

Epidemiology

17.3.1 Citrobacter spp. Different species of the genus Citrobacter and in particular C. freundii, C. amalonaticus, C. diversus or C. koseri have been associated to human disease (Drelichman and Band, 1985; Gupta et al., 2003c). Enterotoxigenic strains may cause diarrhoea as shown by different authors (Popovici et al., 1964; Guerrant et al., 1976; Farmer et al., 1985; Guarino et al., 1987; Jertborn and Svennerholm, 1991; Doran, 1999). These studies have however focused on the characterisation of the isolates from patients but have often not investigated the type of food involved and the origin of the strains. In the case of a severe outbreak at a nursery school and kindergarten, however, sandwiches prepared with green butter made with parsley were identified as the likely source of infection with a verotoxinogenic strain of C. freundii. Identical strains were isolated from patients suffering from gastroenteritis, from haemolytic uremic syndrome and thrombocytopenic purpura as well as from the parsley which had been grown in an organic garden where pig manure was used as fertiliser (Tschäpe et al., 1995). In another outbreak an antibiotic resistant C. freundii was associated with infant formula as the probable vehicle. This case involved a premature baby fed through a contaminated enteral feeding tube used to deliver the reconstituted infant formula. Overall the plasmid carrying multiple resistance genes persisted in the hospital environment for over seven years and caused other nosocomial outbreaks (Gericke et al., 1993; Thurm and Gericke, 1994). Carneiro et al. (2003) isolated C. freundii as well as other Enterobacteriaceae from reconstituted formulae in a teaching hospital contamination and high counts were attributed to poor hygiene practices and inadequate handling of the reconstituted formula allowing growth to high levels. 17.3.2 Klebsiella spp. K. pneumoniae usually occurs in low numbers in faeces and colonisation to high levels of 108cfu/g and more are considered to be a condition for infections (Selden et al., 1971). Direct transmission from person to person is the most common route of infection (Holzman et al., 1974; Jarvis et al., 1985; FDA/ CFSAN, 2002b) and Klebsiella spp. are common nosocomial pathogens in

Enterobacteriaceae 455 hospitals causing different types of infections (Podschun and Ullmann, 1998; Gupta, 2002). Infants, elderly and immunocompromised individuals are the groups of patients at highest risk (Highsmith and Jarvis, 1985; Sahly et al., 2000). The role of K. pneumoniae as a cause of gastroenteritis was discussed almost 40 years ago (Despres et al., 1969) and has been confirmed recently particularly in infants and children by several authors (Panigrahi et al., 1991; Ananthan-Raju and Alavandi, 1999). Klebsiella spp. have been isolated from different types of produce such as salads, vegetables and fruits or sprouts (Viswanathan and Kaur, 2001; Robertson et al., 2002). Foods, in particular prepared in hospitals, have been implicated on different occasions and related to the intestinal carriage of Klebsiella in patients (Montgomerie et al., 1970; Casewell and Phillip, 1978; Cooke et al., 1980). A foodborne outbreak due to a particular strain of K. pneumoniae has been associated with the consumption of contaminated turkey (Rennie et al., 1990). However, due to the concomitant presence of Clostridium perfringens in the samples this finding was questioned by Hatheway and Farmer (1991). Sabota et al. (1998) reported a case of gastroenteritis followed by multiple organ failure following the consumption of a hamburger contaminated with an enteroinvasive strain of K. pneumoniae. Contamination during food handling seems to have also been the cause of this case. Poor hygienic practices in hospitals have been at the origin of several outbreaks due to contaminated milk-based drink, mothers’ milk or enteral feed. In two cases K. aerogenes has been traced back to a food blender used to prepare the drinks or enteral solutions (Kiddy et al., 1987; Thurn et al., 1990), in other cases poor hygiene during collection and handling of the mothers’ milk, i.e., the use of a contaminated breast pump tubing and safety trap, or of enteral solutions have led to contamination with coliforms and in particular with K. pneumoniae (Donowitz et al., 1981; Novak et al., 2001; Arias et al., 2003). In another case caused by breast milk, the precise source of contamination was not identified (MacRae et al., 1991). Several other similar cases have been reported by different authors and the role of nasogastric feeding tubes in the transmission of antibiotic resistant K. pneumoniae was confirmed by Mayhall et al. (1980). A comparative study on the quality of enteral feeds showed that the frequency in contamination was higher in home-prepared solutions than in those prepared in hospitals, indicating poor hygienic practices at home (Anderton et al., 1993). K. oxytoca has been associated with diarrhoea ranging from mild forms to hemorrhagic colitis (Beaugerie et al., 2003; Beaugerie and Petit, 2004) but no information was provided as to the possible source or vehicle. In another study Berthelot et al. (2001) showed that the nosocomial colonisation of premature babies by K. oxytoca was due to contaminated enteral feeding and readjustment of the hygiene practices of health care workers stopped the outbreak. Different species of Klebsiella, including an enterotoxigenic K. pneumoniae, have been isolated from fish and seafood (Singh and Kulshreshtha, 1992). Although to our knowledge, they have not been implicated in foodborne

456 Emerging foodborne pathogens outbreaks, K. pneumoniae seems to play a role in cases of scombroid fish poisoning due to histamine production in tuna sashimi (Taylor et al., 1979). Kanki et al., (2002) showed however that the histamine producing bacteria K. pneumoniae and K. oxytoca were misidentified, the correct designations being Raoultella planticola and Raoultella ornithinolytica, both still belonging to the Enterobacteriaceae. Marino et al. (2000) on their side showed that contamination of cheese with Enterobacteriaceae during cheese making and/ or storage led to an increase in biogenic amines. 17.3.3 Enterobacter spp. Enterobacter spp. are normally found as saprophytes in the gastrointestinal tract of humans and animals. They are, however, also very frequently found in water, in sewage and soils, in plants as well as in numerous foods such as dairy products, meat, fish products, spices and vegetables or fruits. Several species of Enterobacter have clinical significance: E. cloacae, E. aerogenes, E. agglomerans, E. gergoviae and E. sakazakii. A significant increase of these infections has been observed over recent years (Gaston, 1988; Shlaes, 1993; Andresen et al., 1994; Gupta et al., 2003a, b) and E. cloacae, along with K. pneumoniae, remains responsible for significant morbidity and mortality, especially in very-low-birth-weight infants (Cordero et al., 2004). Most of these species have also been associated with nosocomial outbreaks, leading either to sepsis due to contaminated parenteral solutions or illnesses of gastrointestinal origin with different levels of severity. These range from diarrhoea to necrotizing enterocolitis and meningitis. In most severe cases, in particular when premature babies are affected, death may ensue. Although parenteral solutions cannot be directly compared to enteral feed, both can be considered nutrients and the mechanisms of contamination compared. Transmission of E. cloacae, and E. agglomerans in one case, through contaminated parenteral solutions has been reported in several occasions. Investigations in Mexico and Brazil traced infections back to such contaminated parenteral solutions, the data indicating contamination during their preparation by hospital personnel (Goncalves et al., 2000; Tresoldi et al., 2000; Macias et al., 2004). In the same year, six premature babies died after having been fed with nutritional drips (vitamins, fats and dextrose) prepared at the hospital contaminated with E. cloacae (Anonymous, 2004a). Several case studies have in fact demonstrated that E. cloacae is frequently found in hospital environments and that poor hygiene practices lead to spread and infection through different routes such as hands, thermometers, saline solutions, dextrose solutions or distilled water (Archibald et al., 1998; Harbarth et al., 1999; Wang et al., 1991; van den Berg et al., 2000; Yu et al., 2000). In a similar nosocomial outbreak involving 11 babies, nine of whom were prematures, E. gergoviae present in a parenteral dextrose saline solution was traced back to the healthcare workers (Ganeswire et al., 2003).

Enterobacteriaceae 457 17.3.4 Enterobacter sakazakii E. sakazakii is an opportunistic pathogen and has been involved in sporadic individual cases or small outbreaks of infections. On a few occasions, the symptoms were limited to septicaemia or diarrhoea only (Monroe and Tift, 1979; El Maadani, 1996). In some outbreaks it has been at the origin of neonatal necrotizing enterocolitis (NEC) the most common gastrointestinal emergency in newborns (Muytjens et al., 1983; Van Acker et al., 2001). In most cases described, however, E. sakazakii overcomes the gastrointestinal barrier gaining access to the bloodstream and finally the cerebrospinal fluid to cause meningitis (e.g. Gallagher and Ball, 1991). It is interesting to note that recently Bar-Oz et al. (2001) have described three cases of prematures with E. sakazakii in the stool samples but showing absolutely no symptoms. Intestinal colonisation without any symptoms as known for other Enterobacteriaceae seems therefore also possible and has been confirmed in a recent outbreak in France (Anonymous, 2004b). Summaries of cases have been published by Nazarowec-White and Farber (1997a), Lai (2001) and Iverson and Forsythe (2003). Since then several other cases have been reported (CDC, 2002; Barreira et al., 2003; Anonymous, 2004b; Stoll et al., 2004; Tuohy and Jacobs, 2005). Mortality rates have been as high as 50% or more but have declined to < 20% in recent years. Even in the case of recovery, long-term neurologic sequelae have been reported in affected babies (Lai, 2001). Outbreaks due to E. sakazakii are very rare and around 30–40 cases involving about 80–100 infants have been described and reported during the last 40–45 years. In the United States, the number of infections due to E. sakazakii in infants has been estimated at 1:100,000 on the basis of a US FoodNet survey in 2002, the figure being based only on statistics of isolates from clinical specimens (IFT, 2004; Lehner and Stephan, 2004). In a threeyear survey performed by Stoll et al. (2004) in neonatal intensive care units (NICUs) in 6,825 blood or cerebrospinal fluid cultures, only one case of E. sakazakii (septicaemia) was found. When compared to E. claoacae (101 cases) and E. aerogenes (20 cases) E. sakazakii was considered by the authors to be a very rare systemic infection. Several cases of bacteremia due to E. sakazakii have been described for older children and adults suffering from different underlying diseases or having been undergoing surgery (Jimenez and Gimenez, 1982; Reina et al., 1989; Hawkins 1991; Tekkok et al., 1996; Lai, 2001; Ongradi, 2002). These cases, however, do not seem to be linked to food intake and a recent preliminary report from New Zealand indicates indeed that no other populations than infants are at risk to become infected when consuming dairy products (Anonymous, 2004c) Powdered infant formulae were identified as source and vehicle of E. sakazakii in a number of these outbreaks (Biering et al., 1989; Simmons et al., 1989; Van Acker et al., 2001; Anonymous, 2002, 2004b; Tuohy and Jacobs, 2005). In other outbreaks, however, the source of the contamination

458 Emerging foodborne pathogens was not investigated or not detected. In others, E. sakazakii was found in the hospital environment, i.e., in incubators, on blenders and utensils used to prepare the formulae (Urmenyi and Franklin, 1961; Simmons et al. 1989; Jaspar et al., 1990) In the outbreak described by Bar-Oz et al. (2001) E. sakazakii was found only on utensils such as the blender but not in the formula. It is also important to note that in several cases of E. sakazakii infections, infants have not been fed with powdered infant formulae. In one case the infected premature baby received parenteral nutrition consisting of breast milk and ready-to-use, i.e., sterile, premature infant formula (Stoll et al., 2004) and in another case starch was added to a sterile ready-to-feed formula (FAO/WHO, 2004). In another recent case the infected baby was exclusively fed with mother milk (Barreira et al., 2003). The fact that improperly handled mother milk stored in a milk bank may become contaminated with E. sakazakii was shown by Novak et al. (2001). Powdered infant formulae are not sterile and presence of low levels of E. sakazakii due to post-process (heat-treatment) contamination can presently not be excluded. E. sakazakii is readily killed by pasteurisation as demonstrated by several authors (Nazarowec-White and Farber, 1997b; Breeuwer et al., 2003; Edelson-Mammel and Buchanan, 2004). It is however quite resistant to osmotic shock and therefore survives well in dry environments and can be found in processing environments (Breeuwer et al., 2003). As a consequence it may occasionally come to post-process contamination with low levels of E. sakazakii or other Enterobacteriaceae showing similar characteristics. Quantitative determination of E. sakazakii in powdered infant formulae has been performed by several authors and all contaminated products (about 14% of the samples analysed) were found to meet the current specifications of Codex Alimentarius of 25% of the mussels and oysters collected from the Netherlands in 1993– 1994 were colonized by C. lari (Endtz et al., 1997). They also found a similar incidence of Campylobacter in mussels from Germany, Denmark, and England and oysters from Ireland; none of the strains isolated were speciated. Similarly, two other studies demonstrated C. lari incidence in shellfish of at least 33% (Wilson and Moore, 1996; Van Doorn et al., 1998). C. lari is present at higher levels than C. jejuni in sea water (Obiri-Danso and Jones, 1999; Obiri-Danso et al., 2001; Glunder and Petermann, 1989), whereas C. jejuni is often the most isolated Campylobacter from fresh water (Obiri-Danso and Jones, 1999). The presence of C. lari in sea water and the incidence of this organism in shellfish is presumably a result of the shedding of C. lari by gulls and other shore birds that are colonized by this organism (Glunder and Petermann, 1989). Also, C. lari is more halotolerant than both C. jejuni and C. coli, growing at NaCl concentrations of 1.5% (Smibert,

490 Emerging foodborne pathogens 1984). Although the salt content of seawater is 3.5%, the halotolerance of C. lari may allow it to survive longer in marine environments than other Campylobacter species (Obiri-Danso et al., 2001). C. lari, shed by seagulls, enters the sediment layer at low tide. Obiri-Danso and Jones reported that Campylobacter could be isolated from marine sediments at the highest levels during winter and autumn and at the lowest levels during the summer (ObiriDanso and Jones, 1998). They speculated that higher UV-B levels during the summer kill the Campylobacter cells on the sediments (Obiri-Danso et al., 2001). Similarly, Wilson and Moore (Wilson and Moore, 1996) reported that 81% of the shellfish sampled were positive for Campylobacter during October to January whereas only 6% of the shellfish sampled were positive during May to August. More importantly, these results are consistent with the time of year (October and November) that four Campylobacter outbreaks associated with shellfish occurred. 18.5.3 C. upsaliensis and C. helveticus C. upsaliensis and C. helveticus have been isolated frequently from domestic cats and dogs (Hald and Madsen, 1997; Shen et al., 2001; Moser et al., 2001; Engvall et al., 2003; Sandstedt et al., 1983; Stanley et al., 1992). C. upsaliensis is associated predominantly with dogs and C. helveticus is associated primarily with cats. However, domestic cats can also carry C. upsaliensis; C. helveticus is rarely isolated from dogs. C. upsaliensis has been isolated also from poultry and shellfish (Logue et al., 2003; Van Doorn et al., 1998; Waino et al., 2003). Unlike C. upsaliensis, C. helveticus has not been isolated from humans or food, or implicated in human illness. The source of C. upsaliensis infection is unknown; although it has been found occasionally in food, no known human illness has been associated with consumption of C. upsaliensiscontaminated food. Therefore, it is possible that transmission of these organisms occurs not through the food supply but, as has been reported, from animal to man (Gurgan and Diker, 1994; Goossens et al., 1991) or person-to-person (Walmsley and Karmali, 1989; Goossens et al., 1995). In one study (Labarca et al., 2002), C. upsaliensis strains were isolated from dogs living in the households of campylobacteriosis patients. The C. upsaliensis strains isolated from the patients and the canine isolates were not clonal; however, 3–6 months had elapsed between the collection of the clinical samples and collection of the canine samples. Alternatively, sporadic cases of foodborne illness may be occurring and are unidentified due to inadequate culture methods or lack of traceback in sporadic cases. 18.5.4 C. fetus, C. hyointestinalis, C. sputorum, C. mucosalis, and C. lanienae Several other non-jejuni/coli Campylobacter species have been associated with livestock or other game animals (Table 18.2). These include C. fetus, C.

Campylobacter

491

hyointestinalis, C. sputorum, C. mucosalis, and C. lanienae. C. fetus (incl. subsp. fetus and venerealis) has been isolated from cattle and sheep (Varga, 1990; Atabay and Corry, 1998; Busato et al., 1999; Wesley and Bryner, 1989; Giacoboni et al., 1993). C. hyointestinalis (incl. subsp. hyointestinalis and lawsonii) and C. sputorum (incl. subsp. sputorum, faecalis, and paraureolyticus) are isolated from cattle and swine (Gebhart et al., 1994; Atabay and Corry, 1998; Busato et al., 1999; van der Walt and van der Lugt, 1988; Wilson et al., 1986; Chang et al., 1984; Terzolo, 1988; Piazza and Lasta, 1986). C. mucosalis is predominantly isolated from swine (Lawson et al., 1975; Lomax and Glock, 1982; van der Walt et al., 1988; Wilson et al., 1986). Isolation from humans also has been reported (Soderstrom et al., 1991; Figura et al., 1993; Anderson et al., 1996), however, at least one strain was later identified as a C. concisus (On, 1994). C. lanienae was isolated from the faeces of abattoir workers in Switzerland who routinely handled cattle and pig carcasses (Logan et al., 2000). C. lanienae was also isolated from cattle faeces by Inglis et al (Inglis et al., 2003; Inglis and Kalischuk, 2004; Inglis and Kalischuk, 2003; Inglis et al., 2004); these studies also detected C. hyointestinalis and C. fetus. Detection of these species from food or water sources is infrequent and has not been reported for C. mucosalis or C. lanienae. C. fetus subsp. fetus has been isolated from processed turkey, livestock livers and raw milk (Tauxe et al., 1988; Kramer et al., 2000; Logue et al., 2003), C. sputorum subsp. faecalis from poultry and water (Atanassova and Ring, 1999; DaczkowskaKozon and Brzostek-Nowakowska, 2001), and C. hyointestinalis from shellfish and reindeer meat (Hanninen et al., 2002; Endtz et al., 1997; Van Doorn et al., 1998). As with C. upsaliensis, no human illness has been associated with consumption of C. sputorum- or C. hyointestinalis-contaminated food. The association of C. lanienae with livestock suggests that transmission may be zoonotic since this organism has not been isolated from pork or beef. Similarly, zoonotic transmission of C. hyointestinalis has been documented (Gorkiewicz et al., 2002). Also, transmission of C. upsaliensis may be via domestic dogs or cats. This suggests that a potentially large percentage of human illness caused by non-thermotolerant Campylobacter may be due to proximity or handling of pets, livestock, or livestock carcasses. 18.5.5 ECS restricted to human hosts (C. concisus, C. curvus, C. rectus, C. gracilis, C. showae, and C. hominis) These species are unique in that their isolation from animals other than human has not been reported. Additionally, none of these six species has been isolated, to date, from food or water. Although C. concisus, C. curvus, and C. gracilis have been associated with gastroenteritis (Vandamme et al., 1989; Lindblom et al., 1995; Musmanno et al., 1998; Aabenhus et al., 2002; Matsheka et al., 2002; Maher et al., 2003; Abbott et al., 2005) and C. concisus, C. rectus, C. showae, and C. gracilis have been associated with periodontal

492 Emerging foodborne pathogens disease (Macuch and Tanner, 2000; Siqueira and Rocas, 2003; Kamma et al., 2000; Tanner et al., 1998; Rams et al., 1993), the source of infection with these species has not been identified. C. hominis is apparently commensal (Lawson et al., 2001); no association with human illness has been reported. The methods for isolating these campylobacters from the oral cavity to determine incidence and association with periodontal disease must be precise. Since they represent a minor proportion of the microflora, their growth will be affected negatively by too high an oxygen concentration during sampling, and up to seven days incubation may be required for adequate growth (Macuch and Tanner, 2000). Also, methods appropriate for oral cavity samples may not be ideal for isolating these species from food, water or stool samples. It is probable that many of these fastidious Campylobacter species are being missed in surveillance or clinical studies due to inadequate culture and identification methods.

18.6 Culture and isolation of ECS from human faeces, food and water sources 18.6.1 Isolation from human faeces Many ECS illnesses are identified as a result of their isolation from nonenteric sites and are associated with serious illnesses in immunocompromised or debilitated hosts, or with periodontal disease (Tanner et al., 1981; Rams et al., 1993; Tanner et al., 1998; Macuch and Tanner, 2000). However, surveys of human diarrhoeal stool samples with culture methods conducive to isolation of ECS (e.g. filtration without antibiotics, 2–10% H2) have reported a higher incidence of ECS than with conventional thermophilic Campylobacter culture methods (Steele and McDermott, 1984; Goossens et al., 1986; 1990b; Medema et al., 1992; Engberg et al., 2000; Le Roux and Lastovica, 1998; Matsheka et al., 2001; McClurg et al., 2002). For example, Dr Albert Lastovica and colleagues at a single hospital in Cape Town, South Africa between October 1990 and April 1999, isolated 2,216 strains of non-jejuni/non-coli Campylobacter species from diarrhetic stools of pediatric patients, by filtration of diluted stool samples onto an antibiotic-free medium and 2–6 days incubation in a H2-enriched microaerophilic atmosphere (‘Cape Town Protocol’) (Lastovica and Skirrow, 2000). Ninety-seven percent of the non-jejuni/noncoli (ECS) isolates were C. concisus (911 isolates), C. upsaliensis (882 isolates) and C. jejuni subsp. doylei (358 isolates), which combined was 62.5% of the total number of Campylobacter strains isolated of any species (3,545 isolates). Fifteen Arcobacter butzleri and almost 300 Helicobacter isolates were recovered also (Lastovica and Skirrow, 2000). The difference in the incidence of ECS in South Africa compared to most other parts of the world could be explained by geographic, genetic or underlying disease factors rather than differences in methodology, however, ECS have been recovered also in European laboratories that employ the appropriate culture methods

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(Goossens et al., 1990a; Bourke et al., 1998), and recently in the United States (Abbott et al., 2005). 18.6.2 General isolation methodology for food and water The low levels of detection of the non-thermophilic Campylobacter species in the food supply appear to suggest initially that transmission of these organisms into humans is not through food or water. However, this may be an incorrect assumption. Campylobacter isolation methods used by many laboratories worldwide focus mainly on the isolation of C. jejuni subsp. jejuni since this organism is considered to be the main cause of campylobacteriosis. Isolation of C. jejuni subsp. jejuni and C. coli from food and water sources has been reviewed extensively (Uyttendaele et al., 1995; Corry et al., 1995; Uyttendaele and Debevere, 1996; Mason et al., 1999; Talibart et al., 2000; Jacobs-Reitsma, 2000; Miller and Mandrell, 2005) and will not be discussed in detail here. Essentially, Campylobacter is present on food and in water at much lower levels than in faecal samples (100 strains of known Campylobacter species, and tested with >100 additional clinical and environmental strains, yielding 100% agreement with biochemical typing methods (Klena et al., 2004). As more sequence data becomes available for the less characterized

498 Emerging foodborne pathogens Campylobacter species, e.g., C. showae and C. mucosalis, better speciation primer sets can be constructed. Additional details of PCR-based detection of C. coli and C. jejuni in water, milk and poultry samples are provided in previous reviews (Mandrell and Wachtel, 1999; Miller and Mandrell, 2005).

18.8 Comparative genomics of C. coli, C. lari, C. upsaliensis and C. jejuni The first strain of Campylobacter fully sequenced was C. jejuni NCTC 11168 reported in 2000 (Parkhill et al., 2000). Recently, the complete sequence of a second strain of C. jejuni, RM1221, isolated from chicken, and the partial sequences of a strain of each of C. coli (RM2228, isolated from chicken), C. lari (RM2100, isolated from human stool), and C. upsaliensis (RM3195, isolated from human stool) were reported (Fouts et al., 2005). Although closed genomes will provide the most definitive comparisons, preliminary analysis of the initial sequence data has revealed a number of interesting features regarding the ECS. The total chromosome sizes range from 1.52 Mb for the C. lari strain to 1.78 Mb for C. jejuni strain RM1221. The G + C content ranges from 29.64% for C. lari to 34.54% for C. upsaliensis (Fouts et al., 2005). 18.8.1 Integrated elements and phage One of the major differences between the two fully sequenced C. jejuni strains was the presence of four ‘large integrated elements’, one of which resembles a Mu-like phage (CMLP1), containing structural Mu phage head, tail, and transposase homologues (Fouts et al., 2005). Primers designed from the CMLP1 sequence revealed similar genes, and possibly functional CMLP in many ECS strains including C. jejuni subsp. doylei, C. coli, C. lari, C. upsaliensis, C. mucosalis, C. hyointestinalis, C. concisus, and C. curvus (W. Miller, unpublished data). ORFs within additional integrated elements in the C. jejuni, C. lari and C. upsaliensis genomes encode endonucleases, methylases or repressors. Thus, CMLP, and other phage, may be present in many Campylobacter strains, especially those exposed to the food and animal production environment. Regardless, the elements may be important genetic factors in lateral transfer of DNA among compatible ECS. 18.8.2 Plasmids C. coli RM2228, C. lari RM2100 and C. upsaliensis RM3195 have megaplasmids of 180 (pCC178), 46 (pCL46), and 110 kb (pCU110), respectively. pCC178 contains antibiotic resistance genes and putative mobile genetic elements (Fouts et al., 2005). However, the most interesting feature

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within these three plasmids is the presence of type IV secretion system (T4SS) genes (Fouts et al., 2005). A putative T4SS locus was first reported for the C. jejuni pVir plasmid (Bacon et al., 2000). T4SS proteins are associated with formation of conjugation pili, mobilization of DNA and secretion of virulence factors for both animal and plant pathogens (Craig et al., 2004). However, the ECS T4SS genes are more similar to those of T4SSs that mobilize DNA, suggesting they serve a similar function for the ECS and may be important in lateral transfer of DNA between species (Fouts et al., 2005). 18.8.3 Polynucleotide tracts A gene-regulation system discovered in the first C. jejuni genome project was based on variable polynucleotide tracts resulting in slipped-strand mispairing during replication (Parkhill et al., 2000). Variable poly G/C tracts were identified also in the C. coli, C. lari and C. upsaliensis sequences, with a high of at least 22 variable poly G/C tracts (range 12–19bp) in C. upsaliensis RM3195 (Fouts et al., 2005). The function of less than half of the C. upsaliensis genes in which the variable tracts occur is unknown; six of the genes have homology with sugar transferases and synthetases.

18.9 Putative and potential ECS virulence factors Compared to other well-characterized human pathogens (i.e. Salmonella spp. and E. coli), relatively little is known in C. jejuni about the molecular mechanisms of virulence. Similarly, with the exception of C. fetus, even less is known about virulence in the ECS (e.g. C. lari and C. upsaliensis). The recent genome sequencing of C. coli, C. lari, and C. upsaliensis (see above) has enhanced our ability to study the mechanisms of pathogenicity for ECS, although it is worth noting that some virulence determinants might be strainspecific and, therefore, not yet identified. Nevertheless, several genes encoding known or putative virulence determinants in C. jejuni are present in the ECS; these and other virulence determinants are discussed below. 18.9.1 Toxins The toxin of any Campylobacter species that has been best defined genetically and functionally is cytolethal distending toxin (CDT) (Pickett et al., 1996; Lara-Tejero and Galan, 2001). CDT in C. jejuni is encoded by the cdtABC genes. All three proteins are necessary for toxin activity and evidence suggests that the CDT holotoxin is a heterotrimer with a 1:1:1 stoichiometry. CDT causes cell cycle arrest in the G1 or G2 phase, with subsequent cell death (Lara-Tejero and Galan, 2001). CDT genes are present in nearly 100% of C. jejuni strains, and also in some ECS (e.g. C. coli, C. lari, C. upsaliensis,

500 Emerging foodborne pathogens C. hyointestinalis) and in several related Helicobacter species (Pickett et al., 1996). CDT in extracts of C. upsaliensis reportedly caused cell-cycle arrest of HeLa and human T-lymphocytes (Mooney et al., 2001). Each of the cdtA, B and C genes were identified also in the C. coli, C. lari and C. upsaliensis genomes reported recently (Fouts et al., 2005). Although most strains tested contain the CDT genes, expression of the toxin in Campylobacter is variable, and it is not known if this variability is due to differences in expression or allelic differences in the CDT subunits. Additional enterotoxins, cytotoxins, hemolysins, and even a shigalike toxin, have been reported as present in Campylobacter species (Wassenaar, 1997). These toxins have been identified generally by immunochemical and functional assays, and only in a subset of Campylobacter strains, (C. jejuni predominantly and C. coli infrequently). However, genes encoding toxin synthesis have been elusive. C. coli and C. jejuni strains from different geographic and animal sources were reported to express enterotoxins similar immunochemically to Vibrio cholera toxin and E. coli heat-labile toxin and of variable activity in cell culture assays (Wassenaar, 1997). Enterotoxinlike activity was reported for C. lari (Johnson and Lior, 1986) and C. hyointestinalis (Johnson and Lior, 1988) strains, but genes encoding them have not been identified and the mechanisms of activity remain unclear. 18.9.2 Adhesins and invasins Several putative adhesins described in C. jejuni are also present in C. coli, C. lari, and C. upsaliensis. The PEB proteins of C. jejuni are present in other Campylobacter species; however, not all PEB proteins are present in all campylobacters. PEB1a and PEB1b genes are present in the genome sequences of C. coli RM2228 and C. upsaliensis RM3195, but not C. lari RM2100 (Fouts et al., 2005). PEB1c is present in C. lari RM2100, but the similarities between the C. jejuni and C. lari predicted proteins (53%) are much lower than the C. jejuni and C. coli or C. upsaliensis proteins (91–95%). Also, PEB2 and PEB3 are present in both C. jejuni and C. lari, but not C. coli or C. upsaliensis. All four species contain PEB4. Other Campylobacter adhesins and invasins, such as JlpA CadF, CiaB and fibronectin binding protein (FN), and other putative virulence factors, such as proteins involved in motility and two-component sensors, are present in all four thermophilic Campylobacter species (Fouts et al., 2005). A CiaB homologue has been reported also in C. fetus. Therefore, differences in pathogenesis between C. jejuni and the other thermophilic Campylobacter species may be associated with the function of these known factors and/or hypothetical C. jejuni-specific genes in human hosts. Alternatively, perhaps ECS causing sporadic illness are not being identified. 18.9.3 Campylobacter glycome C. jejuni strains synthesize multiple surface-expressed glycoconjugates that

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also are virulence factors. These include capsular polysaccharide, lipooligosaccharide, and glycoproteins with both O- and N-linked sugars (Karlyshev et al., 2005). These molecules compose the C. jejuni glycome (Karlyshev et al., 2005). Although less is known about the glycomes of nonjejuni Campylobacter species, the ECS genomes (i.e. C. coli, C. lari, C. upsaliensis) revealed the potential for equally complex glycomes involved in biology. 18.9.4 Lipooligosaccharides and lipopolysaccharides Lipooligosaccharides are important surface structures in C. jejuni and, presumably, also in ECS. The lipooligosaccharide genetic loci in each of the three sequenced ECS are similar to C. jejuni in organization with heptosyltransferase genes waaC and waaF at opposite ends of the lipooligosaccharide locus surrounding ORFs that are different among the species and probably encode different structures (Fouts et al., 2005; Gilbert et al., 2005). (Presumably, variability exists also among strains as has been described for C. jejuni, by phase variation as a result of variable poly-NT repeats within glycosyltransferase genes (Linton et al., 2000; Gilbert et al., 2005). However, no genes with strong homology to the genes in C. jejuni that synthesize N-acetylneuraminic acid (sialic acid) or the sialyltransferases involved in synthesis of oligosaccharides that mimic ganglioseries glycosphingolipids (e.g. GM1) were evident (Gilbert et al., 2000; Gilbert et al., 2002). C. fetus subsp. fetus (Cff ) and C. fetus subsp. venerealis apparently synthesize smooth-type lipopolysaccharide with high MW repeating units, in contrast to lipooligosaccharide expressed by C. jejuni and C. coli (Perez-Perez et al., 1986; Moran et al., 1994). Lipopolysaccharide is a major serotype antigen of Cff strains and strains susceptible to normal human serum are associated with the serogroup defined by the lipopolysaccharide; serogroup A Cff strains were more resistant than serogroup AB and B strains (Perez-Perez et al., 1986). Lipopolysaccharide and the S-layer protein interactions are critical in the pathogenesis of Cff, since the diseases caused by Cff usually involve invasion into the circulation and involvement of the humoral and cellular immune systems of the host (Table 18.1). Structural analysis of Cff serotype A strain and serotype B strain lipopolysaccharides revealed they are composed of partially O-acetylated D-mannan chains (Senchenkova et al., 1997), or D-rhamnose chains with a terminal O-methyl D-rhamnose (Senchenkova et al., 1996), respectively. In a separate study, sialic acid, fucose, glucose, galactose, D-glucosamine and D-galactosamine were also identified by composition analysis of multiple Cff strains (Moran et al., 1994). It is probable that there are multiple Cff lipopolysaccharide structures that reflect serotype differences and, possibly, the types of disease caused in animal and human hosts (Table 18.1).

502 Emerging foodborne pathogens The oligosaccharide portion of the lipooligosaccharide of a heat stable (HS) serotype 30 (O:30) strain of C. coli reported by Aspinall et al. was composed of a β-D-Qui3NAc-(1→2)-β-D-Qui3NAc disaccharide at the nonreducing end (Aspinall et al., 1993b). Qui3NAc is 3-acylamino-3,6-dideoxyD-glucose, an unusual sugar that in the C. coli lipooligosaccharide is acylated with either 3-hydroxybutanoyl or 3-hydroxy-2,3-dimethyl-5-oxoproly residues (Aspinall et al., 1993b). There appear to be significant differences in the types of sugars and the organization of the lipooligosaccharide and/or lipopolysaccharide related to C. jejuni and ECS. For example, the initial characterizations of C. coli and C. jejuni heat-stable (HS) serotype antigens described enteric-like lipopolysaccharide with varying length O-repeat units resulting in ‘ladder’ patterns of lipopolysaccharide molecules by SDS-PAGE (Preston and Penner, 1987; Penner, 1988). It is now accepted generally, that the endotoxic lipid Acontaining glycolipid is not the HS antigen (Chart et al., 1996) and that the ladder patterns are due to lipid-linked capsular polysaccharide molecules, rather than a lipopolysaccharide (Karlyshev et al., 2000). Similar immunochemical, genetic and structural studies of other ECS will be important to determine whether they have lipooligosaccharide (and mimic mammalian glycosphingolipids) and/or lipopolysaccharide. 18.9.5 Capsular polysaccharide The capsular polysaccharides are important surface structures of many Gramnegative bacteria, and are involved in pathogenesis by protecting organisms from both the cellular and humoral immune systems in animals (Orskov et al., 1977). As noted above, the capsular polysaccharide of C. jejuni are the major molecules responsible for HS (Penner) serotype antigens (Karlyshev et al., 2000) and are important virulence factors (Bacon et al., 2001). Functions of capsular polysaccharides of other bacteria that have been described, in addition to resistance to both non-specific and specific host immunity, are prevention of desiccation, adherence and biofilm formation (Roberts, 1996). The only chemical information reported for capsular polysaccharides of any ECS is for a C. coli serotype O:30 strain (Aspinall et al., 1993a). The capsule was shown to be composed of a repeating 5-ribitol-1-phosphate sugar with side chains at O-2 of O-(6-deoxy-β-D-talo-heptopyranosyl)-(1→4)(2-acetylamino-2-deoxy-β-D-glucopyranosyl) units. The 6-deoxy-talo-heptose sugar has not been detected in any other Gram-negative bacteria. kps genes involved in synthesis and export of capsular polysaccharides were identified in recent C. coli (RM2228), C. lari (RM2100) and C. upsaliensis (RM3195) genome sequences (Fouts et al., 2005). The organization of the C. upsaliensis kps genes was different from that of the other two ECS strains and the two C. jejuni strains that have been sequenced (NCTC 11168 and RM1221), with multiple clusters of orthologues located outside the kps locus, and some that are unique to C. upsaliensis (Fouts et al., 2005). A variable

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poly-G tract in one of three putative GDP-fucose synthetase genes for C. upsaliensis RM3195 suggests that regulation of the concentration of this or an analogous sugar may occur. Orthologues of C. jejuni kps genes were identified, but many unique genes were present for each of the three ECS, indicating synthesis of different polysaccharide capsule structures. 18.9.6 Phosphorylcholine A putative licABCD locus was identified in C. upsaliensis strain RM3195 (Fouts et al., 2005). The locus had varying, but significant, identity to genes present in Haemophilus influenzae (Weiser et al., 1989), commensal Neisseria species (Serino and Virji, 2002) and Streptococcus pneumoniae (Zhang et al., 1999). licABCD genes in these mucosal and respiratory pathogens putatively encode proteins involved in acquisition of choline (licB), synthesis of phosphorylcholine (licA,C) and transfer of phosphorylcholine (licD) to lipooligosaccharides or teichoic/lipoteichoic acids of Gram-negative or Grampositive bacteria, respectively (Serino and Virji, 2002; Zhang et al., 1999). Preliminary studies indicate that most strains of C. upsaliensis from South Africa, and some from European countries, also have licA (REM, unpublished observations). A variable poly-G tract within the licA gene of C. upsaliensis RM3195 probably regulates synthesis of phosphorylcholine and ultimately affects decoration of lipooligosaccharides. 18.9.7 Protein glycosylation Campylobacter species possess the capability to glycosylate flagellin and other proteins with both O- (Doig et al., 1996; Parkhill et al., 2000) and Nlinked sugars (Szymanski et al., 1999; Young et al., 2002). A genetic locus thought originally to be involved in synthesis of lipooligosaccharides (Fry et al., 1998) was revealed later to be an N-linked protein glycosylation pathway of ~16 kb, designated pgl (Szymanski et al., 1999), and composed of genes putatively encoding a variety of transferases (Szymanski and Wren, 2005). Structural analysis determined the N-linked oligosaccharide to be G a l N A c α1 , 4 G a l N A c α1 , 4 ( G l c β1 , 3 - ) G a l N A c α1 , 4 G a l N A c α1 , 4 GalNAcα1,3Bacβ1,N-Asn (Bac = trideoxyglucose) (Young et al., 2002). The O-linked glycosylation pathway has been shown to be important in motility (Logan et al., 2002) and the N-linked pathway important in attachment and invasion of eukaryotic cells (Szymanski et al., 2002), and colonization of GI tracts of mice and chickens (Hendrixson and DiRita, 2004). The genome sequences of C. coli RM2228, C. lari RM2100 and C. upsaliensis RM3195 revealed that the pgl genes are conserved and organized similarly among the ECS, except for the absence of one and two pgl genes in the C. coli and C. lari genomes, respectively, and the separation of three pgl genes in the C. upsaliensis genome (Fouts et al., 2005; Szymanski and Wren, 2005). There is no information on how these differences affect O- and N-linked protein

504 Emerging foodborne pathogens glycosylation for the genome strains of C. coli, C. lari and C. upsaliensis that have homolog genes. However, it seems likely O- and N-linked glycosylation are important factors in ECS biology. 18.9.8 The S (surface) layer of C. fetus and C. rectus. C. fetus cells have been shown to contain a proteinaceous, acidic, and antigenically variable outer layer (Yang et al., 1992). This outer layer, termed surface (S) layer, is responsible for phagocytosis resistance in this species, inhibiting uptake of C. fetus cells by macrophages (Blaser et al., 1988). The S-layer also makes C. fetus cells more resistant to the complement system; it is believed that complement resistance accounts for the large number of systemic C. fetus infections relative to C. jejuni and C. coli (Table 18.1), which are both complement sensitive (Blaser et al., 1988). The S-layer proteins (SLPs) of C. fetus are encoded by alleles of the sapA or sapB genes. SapA alleles are highly conserved at the amino-terminal end; however, very little sequence conservation is present in the carboxy-terminal end of the protein (Tu et al., 2003). SapB alleles show similar organization. Antigenic variation of the SapA SLPs in C. fetus is due to the presence of an invertible segment containing both the sapA promoter and the sapDEF genes that encode a type I SLP transport system (Tu et al., 2004). Recombination at the conserved 5’ end of any two of the clustered sapA alleles results in the juxtaposition of the sapA promoter with one of the sapA alleles and subsequent expression of a new SLP antigen. The periodontal pathogen C. rectus also contains an S-layer (Kaneko, 1992); however, very little similarity exists between the SLPs, although the C. rectus SLPs may also be transported by a type I transport system. The C. rectus SLP is encoded by crsA (Wang et al., 1998). The S-layer in C. rectus is not important for binding to epithelial cells; however, CrsA has been shown to decrease expression of the proinflammatory cytokines IL-6, IL-8 and TNF-α, perhaps thereby permitting persistence of C. rectus in periodontal sites with concomitant inflammation (Wang et al., 2000). 18.9.9 Other virulence factors Additional virulence factors will be revealed as more genetic sequence data are obtained and Campylobacter species are characterized. One area of potential future research involves the characterization of the plasmids and prophage of Campylobacter species. Prophage and plasmids have been demonstrated to be reservoirs of pathogenicity functions in multiple taxa. Although no known virulence genes were present in the prophage regions of the sequenced genomes of C. jejuni and C. lari, other uncharacterized bacteriophage of this genus might contain toxins. Finally, putative virulence genes are present on the megaplasmids of C. lari and C. coli. The 46 kb C. lari RM2100 plasmid contains an S. enterica sinH/sivH adhesin/invasin homolog, and the 180 kb

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C. coli RM2228 plasmid contains a block of genes with high similarity to a gene cluster in V. cholerae. However, it is unknown whether either set of genes is important for pathogenicity in these two species. Two-component signal transduction systems (TCSTSs) mediate adaptation of bacteria to changes in their environment through a sensor (histidine kinase) and response regulator system (Albright et al., 1989). Two separate C. jejuni TCSTSs were shown previously to be important in colonization of one-dayold chicks (racR-racS, (Brás et al., 1999)), and colonization and inflammation of normal and immunodeficient mice (dccR-dccS, (Mackichan et al., 2004)). An examination of the C. coli RM2228, C. lari RM2100 and C. upsaliensis RM3195 genomes (Fouts et al., 2005) revealed for each species dccR-dccS homologues, and homologues also for each of the three genes positivelyregulated by dccR-dccS (two putative periplasmic and one integral membrane protein(s)) (Mackichan et al., 2004). Three TCSTSs in addition to the racRracS and dccR-dccS homologues were identified in the C. coli, C. lari and C. upsaliensis genomes (Fouts et al., 2005), indicating similar mechanisms among the ECS for adapting to different environmental stimuli.

18.10

Genotyping

Genotypic typing schemes, reviewed in Wassenaar and Newell (2000) and Newell et al. (2000), rely mainly on bp differences between strains that can be altered by restriction enzymes. Pulsed field gel electrophoresis (PFGE: rare cutting restriction enzymes), ribotyping (polymorphisms in the rDNA loci), PCR-restriction fragment length polymorphisms (PCR-RFLP: polymorphisms at a select locus, e.g. flagellin), and amplified fragment length polymorphisms (AFLP: amplification of digestion products flanked by specific restriction sites), have all been applied to differentiating ECS (Wassenaar and Newell, 2000; Newell et al., 2000; On and Harrington, 2000). However, these restriction-based typing methods have several drawbacks, including lack of standardization, reagent and method differences, variable digestion or fragment sizes due to methylation differences, and labour intensive procedures. AFLP is the most discriminatory of these methods, generally, but the ultimate genotypic marker is the genomic sequence. Although complete genome sequencing of multiple epidemiologically important strains (e.g. outbreak isolates) is not practical, multilocus sequence typing (MLST) (Dingle et al., 2001), which relies on sequencing short (approx. 500 bp) regions from multiple loci, has proven valuable for differentiating C. jejuni strains and assessing their relatedness (Dingle et al., 2002). The availability of three non-jejuni Campylobacter draft genome sequences (Fouts et al., 2005) facilitated the development of an expanded MLST system for genotyping four ECS: C. coli, C. lari, C. upsaliensis and C. helveticus (Miller et al., 2005). One hundred and twenty-eight sequence types were identified for the four species,

506 Emerging foodborne pathogens indicating that this expanded MLST method is a robust typing system for many ECS. For example, the expanded MLST method was used to type nearly 500 C. coli srains isolated from different geographical locations in the US over a 6 year period from cattle, chickens, swine and turkeys, resulting in 149 MLST sequence types (Miller et al., 2006). The most striking findings, however, were that many of the alleles and resulting sequence types were host-associated, and that cattle isolates diverse both spatially and temporally appeared to be highly clonal (52/63 cattle isolates were a single sequence type). The results of this MLST study indicated that source-tracking of C. coli strains may be possible, and suggested that some C. coli strains may have adapted or been selected in some hosts, i.e. cattle strains (Miller et al., 2006). Also, preliminary MLST analysis of human C. coli strains indicated that attribution of human illnesses to a food source also may be possible (unpublished observations). Other preliminary results suggest that this MLST method can be extended into many of the remaining ECS, including C. concisus, C. fetus, and C. sputorum (unpublished results). Thus, a comprehensive typing method for all the ECS may be feasible. Other potential assays for speciation of ECS include biochemical (Acuff, 1992), genetic (PCR) (Acuff, 1992; Fermer and Engvall, 1999; Wang et al., 2002; Bang et al., 2002; Klena et al., 2004), immunochemical (Kosunen et al., 1984; Lamoureux et al., 1997; Mandrell et al., 2002), chemotaxonomic fatty acid profiling (Brondz and Olsen, 1991), and protein one-dimensional gel electrophoresis methods (Vandamme et al., 1991b). Recently, an oligonucleotide-probe microarray assay was developed for identifying C. coli, C. jejuni, C. lari, and C. upsaliensis strains (Volokhov et al., 2003). MALDI-TOF mass spectrometric analyses of whole Campylobacter cells for intact proteins to identify biomarker ions have been reported previously. Protein biomarker ions in the 10–20 kilodalton (kDa) range were reported to be the most discriminatory ((Winkler et al., 1999), unpublished data). Speciation of C. coli, C. jejuni, C. lari, C. helveticus, C. sputorum subsp. faecalis and C. upsaliensis has been achieved by analyzing a portion of multiple or single colonies isolated from a variety of food and animal samples cultured on agar media (Mandrell et al., 2005). The species- and sub-species specific biomarker ions were in most cases conserved, high copy number, cytosolic proteins (unpublished data) that could be identified by analysis of public (Parkhill et al., 2000; Fouts et al., 2005) and internal (unpublished data) Campylobacter genome databases. MALDI-TOF MS may be especially useful for characterizing ECS strains, because of the minimal genetic and biochemical data available for most ECS.

18.11

Prevention and control

The broad host range and prevalence of Campylobacter species in animals and the food supply (Tables 18.2 and 18.3) reflect the urgent need to develop

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better methods for controlling them both in the pre- and post-harvest food production and processing environments. Most of the control and intervention methods that have been developed and tested have targeted C. jejuni and, in some cases, C. coli in animals and food, predominantly chickens and chicken meat (Stern, 1992; White et al., 1997; Stern and Line, 2000; Ransom et al., 2000). As noted above, C. jejuni are prevalent in poultry operations and in retail chicken (Table 18.3 and Ransom et al. (2000) and it is this production system that has had the most attention in development of prevention or control measures. Some pre-harvest control measures for minimizing or eliminating C. jejuni in poultry, but worthy of consideration also for ECS in animals or on food, are sanitation, biosecurity, vaccinations and/or antibiotics to prevent or control debilitating infectious diseases, and the use of nonpathogenic bacteria to competitively exclude Campylobacter colonization (Stern, 1992; White et al., 1997; Ransom et al., 2000; Humphrey, 2004). Post-harvest control measures that have been tested are chemicals (e.g. chlorine, trisodium phosphate, organic acids, herbal extracts) added to processing water, plus water replacements and counter-flow, steam, and irradiation (White et al., 1997; Stern and Line, 2000; Ransom et al., 2000; Humphrey, 2004). Most of these control strategies have not been reported to be highly effective at decreasing Campylobacter significantly or consistently. Decreasing stress during transportation of animals, proper temperature control during food storage, and proper handling of food by food-preparation workers and by consumers, also would probably assist in controlling human illness. Heat treatment or irradiation are logically the most effective post-harvest control methods, but will probably require increased effort, money and public acceptance for success.

18.12 Conclusions and future trends After nearly three decades of intensive research on Campylobacter species, many questions remain regarding the ecology, biology and epidemiology of ECS, and their incidence in food and role in public health. The risk of getting sick with any Campylobacter species is associated with factors like geographic location (e.g. intensive animal production, farming practices, climate, water, food processing, travel), host-susceptibility (e.g. immunity, host-genetics, underlying disease), ecology (e.g. strain differences and competitive microflora) and habits (e.g. restaurants, cooking techniques, awareness of risks, pets). The major risk factors include consumption of chicken or pork, daily contact with pets (cats and dogs) or chickens, travel abroad, drinking unpasteurized milk, involvement in water sports, and barbecuing meat (Kapperud et al., 1992; Altekruse et al., 1999; Studahl and Andersson, 2000; Rodrigues et al., 2001; Sopwith et al., 2003). However, the sources associated with outbreaks for any Campylobacter species (Miller and Mandrell, 2005), and the limited number of outbreaks documented for ECS (Table 18.4), confirm the increased

508 Emerging foodborne pathogens risk of consuming raw milk or untreated water. The prevalence of C. coli and C. jejuni in the environment (Miller and Mandrell, 2005), and the incidence of ECS documented in animals and food (Tables 18.2 and 18.3), suggests that humans are exposed frequently to low doses of Campylobacter. Immunocompetent individuals exposed to low doses may develop immunity to subsequent low doses of antigenically similar strains (Blaser et al., 1987; Black et al., 1988). The small genome size of Campylobacter species (~1.6–2.0 Mb) may be compensated by the diversity and hypervariability that occurs through genetic exchange and rearrangements (de Boer et al., 2002; Wassenaar et al., 1998; Miller and Mandrell, 2005), plasmids, and polynucleotide repeats (Fouts et al., 2005; Parkhill et al., 2000). These mechanisms may enhance survival of ECS in animal GI tracts, water, and during food processing (e.g. hot and cold water, oxidative stress, chemical sanitizers). New genotypes and phenotypes of Campylobacter could emerge in animals or food due to increased fitness characteristics that enhance survival under stress conditions (Kelly et al., 2003). Multiple mechanisms of genetic exchange and hypervariability in intensive animal or food production environments probably result in ECS with enhanced fitness for certain animal hosts (e.g. C. fetus and sheep and cattle, C. jejuni and poultry, C. coli and swine, C. upsaliensis and C. helveticus and cats and dogs, C. lari and wild birds). The emergence of strains with increased fitness for a host suggests ECS strains contain clues for determining their source by identifying specific genes and/or mutations crucial for survival. Epidemiologically relevant strains analyzed by novel molecular methods (e.g. MLST and AFLP methods) may help to identify differences between strains for source-tracking, and also clues for developing novel strategies for decreasing the prevalence and virulence of Campylobacter species in food production environments. Isolation procedures used by clinical laboratories focus on the detection of C. jejuni and C. coli since these two species are accepted by most researchers to be the primary pathogenic organisms in the family Campylobacteracae. However, improvements in the detection and isolation of ECS have indicated that these Campylobacter and Arcobacter species cause more human illness than appreciated by the public health community (Vandamme et al., 1992a; Vandenberg et al., 2004). Genetic evidence of Campylobacter species in faeces of patients with GI illness, but with no other cause identified, emphasizes the need for further studies (Maher et al., 2003). The second most isolated Campylobacter species in South Africa is C. concisus, followed closely by C. upsaliensis (Lastovica and Skirrow, 2000). Clinical isolation of ECS in some parts of the world, such as the U.S., is uncommon, although it cannot be ascertained presently whether this is a result of the isolation methods employed or is due to inherent differences in environmental reservoirs. ECS have been isolated from blood and stool samples, but there is minimal data on ECS incidence in food. The most likely explanation for this discrepancy is the difficulty in culturing ECS. Therefore, improved protocols for isolation,

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detection and identification of ECS in the food supply are needed (Lastovica and Skirrow, 2000). Because generic Campylobacter isolation methods may not be possible and because foodborne Campylobacter species are present in low levels and possibly in a viable but nonculturable state, molecular methods must be developed to increase detection sensitivity. Extensive genomic data now exist for the four thermotolerant Campylobacter species (Fouts et al., 2005), but PCR-based methods for ECS are hindered by a lack of genomic data for the non-thermotolerant ECS. The only genomic data available are 16S and/ or 23S rDNA sequences; minimal sequence data is available for C. rectus, C. concisus, C. fetus, and Arcobacter species. However, sequencing projects completed or ongoing for some of the ECS will provide genomic data invaluable for development of new and/or improved molecular detection methods (Fouts et al., 2005; Miller et al., 2005; TIGR, 2006). To address some of the issues described above, a Consortium Grant (CAMPYCHECK) was established through the European Commission Fifth Framework Programme (FP5: 1998–2002) (CAMPYCHECK, 2003). The goals of the CAMPYCHECK project are to develop new isolation and detection methods and survey multiple foods for the incidence of ECS. It is anticipated that this research will yield improved assessment of ECS in the food chain and whether new zoonotic reservoirs for these organisms exist. Improved methods will delineate also the clinical importance of ECS and their impact on public health.

18.13 Acknowledgements This work was supported by the United States Department of Agriculture, Agricultural Research Service CRIS project 5325-42000-041, and it also supports collaboration between the U.S. and the European Commission in the Fifth Framework Project QLK1-CT-2002-0220, ‘CAMPYCHECK’.

18.14

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19 Mycobacterium paratuberculosis M. W. Griffiths, University of Guelph Canada

19.1

Introduction

‘Johne’s bacillus’, which is now known as Mycobacterium avium subsp. paratuberculosis, was first isolated by Johne and Frothingham in 1895 during an investigation of the cause of chronic diarrhoea in cattle. The organism was originally thought to be a strain of Mycobacterium avium but it has also been classified as M. johnei and M. enteritidis. Based on its cell wall composition it has been confirmed to be a member of the M. avium complex and as such Mycobacterium avium subsp. paratuberculosis (MAP) is a member of the family Mycobacteriaceae (Wayne and Kubica 1986). Mycobacteriaceae are Gram-positive, strictly aerobic, non-motile, acid-fast rod-shaped bacteria with fastidious growth requirements, and are characterised by their slow growth rate and resistance to acid and alcohol. This resistance is due to a strong cell wall containing a high lipid concentration. MAP requires the presence of mycobactins, which are iron-binding hydroxamate compounds, for growth. The organism will grow in the temperature range 25 to 45 °C with an optimum of 39 °C. It will grow at salt concentrations below 5% and at a pH of 5.5 or greater (Collins et al. 2001).

19.2 Johne’s disease MAP is the causative agent of Johne’s disease, an incurable, chronic, infectious enteritis of ruminants, which results in diarrhoea, weight loss and, ultimately, death; although asymptomatic carriage may occur in cattle (Olsen et al. 2002). Johne’s disease is among the most common bacterial infections in

Mycobacterium paratuberculosis

523

domesticated animals worldwide, but is most common in cattle. It has been estimated that economic losses to the cattle industry in the United States are about $1.5 billion (approximately, ∈ 1.2 billion) annually (Harris and Barletta 2001). The organism has also been isolated from deer and wild boar (Alvarez et al. 2005) and rabbits (Raizman et al. 2005); albeit at low frequency. Results of a national survey for Johne’s disease conducted in the US in the 1980s showed that the infection was present in 1.6% of all cattle and 2.9% of cull cows (Merkal et al. 1987). The United States Department of Agriculture reports that between 20% and 40% of US dairy herds are infected with paratuberculosis (Broxmeyer 2005) with herd prevalence being strongly linked to herd size (Manning and Collins 2001). Forty percent of herds consisting of more than 300 head were infected with MAP. The prevalence of MAP in 4,579 purebred beef cattle from 115 ranches in Texas was determined using a commercial ELISA (Roussel et al. 2005). Serum samples were analysed for antibodies and faecal matter from seropositive cattle was submitted for mycobacterial culture. Three percent of cattle were seropositive and 50 of the 115 (43.8%) herds had at least one seropositive animal. Faeces of 73% of the seropositive cattle were culture positive, and these animals were found in 18% of the seropositive herds. A survey of veterinary practices and farms in south west England concluded that 1% of farms had cattle with Johne’s disease and 2% of the animals in these herds were infected (Cetinkaya et al. 1996). Similar prevalence rates have been found in other European countries (Scientific Committee on Animal Health and Animal Welfare 2000) and Canada (VanLeeuwen et al. 2005). However, other studies have shown that the prevalence of MAP in culled dairy cattle in Eastern Canada and Maine was 16.1% based on a systematic random sample of 984 abattoir cattle (McKenna et al. 2004). Herd prevalence in Europe ranges from 7 to 55% and rates in Australia range from 9 to 22% (Manning and Collins 2001). A seasonal pattern of positive cows was also detected, with the highest proportion of cows being positive in June (42.5%). The prevalence rates also vary according to the method of detection. In a study of MAP in cattle in Alberta, Canada, it was determined that the true herd-level prevalence, as determined by ELISA, was 26.8% +/– 9.6%; whereas the rate ranged from 27.6% +/– 6.5% to 57.1% +/– 8.3% when determined by MAP faecal culture, depending on the number of pooled individual faecal samples that were culture positive (Sorensen et al. 2003). There is some evidence that strains are host specific (Motiwala et al. 2004), with strains isolated from sheep and goats being more difficult to culture than the bovine strain (Juste et al. 1991). Johne’s disease usually occurs in young animals as a result of ingestion of feed contaminated with MAP. The ingested bacteria are transported across the epithelium through M-cells overlying the Peyer’s patches and are subsequently released and taken up by macrophages (Tessema et al. 2001). Unlike other intestinal epithelial cells, M-cells express integrins on their luminal faces. Efficient attachment and ingestion of MAP by cultured epithelial

524 Emerging foodborne pathogens cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by the bacterium. Targeting and invasion of M-cells by MAP in vivo is mediated primarily by the formation of a FN bridge formed between FAP-P and the integrins (Secott et al. 2002, 2004). Inside the macrophage, the mycobacteria are able to resist degradation and grow until the infected macrophage ruptures. The thick, lipid-rich cell envelope of MAP is mainly responsible for their resistance, as well as providing a physical barrier. The mycobacterial cell wall also contains several components that down-regulate the bactericidal function of macrophages. The basic intracellular survival strategy of pathogenic mycobacteria consists of the use of entry pathways that do not trigger oxidative attack, modification of the intracellular trafficking of mycobacteria-containing phagosomes, and modification of the link between the innate and specific immunity. These survival strategies, as well as their implications in the epidemiology, diagnosis, and control of Johne’s disease have been reviewed by Tessema et al. (2001).

19.3 Crohn’s disease Crohn’s disease is a chronic inflammatory disease of humans that most commonly affects the distal ileum and colon, but it can occur in any part of the gastrointestinal tract (Hendrickson et al. 2002). It is a life-long debilitating illness, the symptoms of which generally first appear in people aged 15 to 24. As yet, there is no known cure for Crohn’s disease. However, management of the disease has become easier with the advent of drugs such as aminosalicylates, budesonide and immunosuppressive drugs (Achkar and Hanauer 2000; Hendrickson, et al. 2002; Hermon-Taylor 2002; Selby 2000). Revolutionary treatments involving genetically engineered anti-TNF α antibody, infliximab, may also provide ways to radically alter the course of severe Crohn’s disease by targeting a specific inflammatory mediator (Bell and Kamm 2000; Sandborn 2005). The illness is often cyclical with patients undergoing intermittent remission followed by recurrence. Surgical intervention is necessary in a large proportion of sufferers. It is primarily a disease of the industrialised world which has led to several hypotheses for this phenomenon. These include the ‘hygiene hypothesis’ which concludes that the lack of enteric parasitic infections, which have all been mainly eradicated in developed countries, causes a weakened systemic immune system, leading to the development of immunomediated diseases such as Crohn’s disease (Wells and Blennerhassett 2005) and the ‘cold chain hypothesis’ which states that cold-chain development has paralleled the outbreak of Crohn’s disease during the 20th century and suggests that psychrotrophic bacteria such as Yersinia spp. and Listeria spp. contribute to the disease as they have been found in Crohn’s disease lesions (Hugot et al. 2003). Several other causes of Crohn’s disease have been

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525

postulated such as infectious agents, including bacteria and viruses; allergic and nutritionally related causes; and microparticles, which is part of the concept behind toothpaste as a cause (Korzenik 2005). The underlying theory behind many of these ideas is that the central defect leading to Crohn’s disease is an increased intestinal permeability which may be the result of an innate immune deficiency (Korzenik 2005).

19.4 Mycobacterium paratuberculosis and Crohn’s disease Because the pathological changes that occur in the small intestines of people with Crohn’s disease are similar to those observed in cattle suffering from Johne’s disease, it has been postulated that MAP is the aetiological agent responsible for Crohn’s disease (Chiodini 1989; Chiodini and Rossiter 1996; Hermon-Taylor 1998, 2001; Hermon-Taylor et al. 2000; Hermon-Taylor and Bull 2002). However, despite the similarities between the two conditions, detailed pathological comparisons reveal important differences, including important extraintestinal manifestations (Van Kruiningen 1999). The clinical features of the two infections are shown in Table 19.1. Evidence linking MAP and Crohn’s disease is far from conclusive. Studies have shown that the organism can be cultured from about 7.5% of patients with Crohn’s disease but from only 1% of healthy individuals (Chiodini and Rossiter 1996). The results of several surveys are summarised in Table 19.2. A two-fold increase in the prevalence of the organism in Crohn’s patients compared with control groups has been reported. However, the biggest differences were noticed when a PCR method based on the IS900 sequence was used to detect the organism (Moss et al. 1992; Scientific Committee on Animal Health and Animal Welfare 2000; Wall et al. 1993). Concerns have been raised because of the difficulty in isolating DNA from mycobacteria (Hermon-Taylor 1998), the inability of PCR to differentiate between viable and dead cells and the fact that some IS900 primers may not be specific for MAP (Cousins et al. 1999; St Amand et al. 2005; Vansnick et al. 2004). The identification of MAP DNA in Crohn’s disease tissue only reveals that MAP infection and Crohn’s disease can exist at the same point in time. MAP infection may occur before or after the development of Crohn’s disease and in either case may have little influence on the natural history of the disease (Cook 2000). Other studies have shown that the prevalence of MAP in Crohn’s disease patients is no different from that in controls (Table 19.2). In a recent population-based case control study of seroprevalence of MAP in patients with Crohn’s disease and ulcerative colitis, the authors could not prove the difference between inflammatory bowel disease patients and healthy volunteers (Bernstein et al. 2004). However, the rate of positive enzymelinked immunosorbent assay results was high (approximately 35%) for all study groups, and there was no difference in rates among Crohn’s disease patients, ulcerative colitis patients, healthy controls, and unaffected siblings.

526 Emerging foodborne pathogens Table 19.1 Feature

Clinical features of Crohn’s disease and Johne’s disease Crohn’s disease

Preclinical stage Symptoms and signs Not known Incubation period Not known Clinical stage Presenting Chronic diarrhoea, symptoms and abdominal pain, signs weight loss Gastrointestinal symptoms and signs Diarrhoea Chronic (3 weeks+) Blood in stool Rare Vomiting Rare Abdominal pain Yes Obstruction Yes Extraintestinal manifestations Polyarthritis Yes, but rare Uveitis Yes, but rare Skin lesions Yes Amyloidosis Yes, but rare Hepatic Yes, but rare granulomatosis Renal involvement Yes, but rare Clinical course Remission and relapse Yes

Johne’s disease Decreased milk yield Minimum 6 months Chronic diarrhoea, dull hair, weight loss, decrease in lactation Chronica Rare No Unknown No No No No Yesb Yes Yesc Yes

a

Not seen in sheep Goats primarily Goats, deer, primates primarily, also camelids From (Board on Agriculture and Natural Resources 2003) b c

There has also been evidence linking cell-wall deficient forms of MAP with Crohn’s disease (Hulten et al. 2001; Sechi et al. 2001, 2004) but the results produced by this group have been questioned (Roholl et al. 2002). Many other bacteria have been isolated from biopsies taken from Crohn’s patients, including Helicobacter spp., Listeria monocytogenes and Escherichia coli (Tiveljung et al. 1999). Indeed, Crohn’s disease of the terminal ileum, especially if associated with a NOD2 mutation, is characterised by a reduced defensin (antimicrobial peptides) response. This could lead to increased bacterial invasion into the intestinal mucosa. Although it is uncertain that this deficient defensin response leads to a reduced antibacterial activity of the intestinal mucosa, Schmid et al. propose that the most plausible concept of pathogenesis of Crohn’s disease is a defensin deficiency syndrome (Schmid et al. 2004). Further evidence implicating MAP as the aetiological agent in Crohn’s disease was produced by El-Zaatari and colleagues (El-Zaatari et al. 1994, 1997, 1999; Naser et al. 1999). They identified two MAP proteins, p35 and p36 and, when sera of Crohn’s patients were screened for the presence of antibodies to these two proteins, 86% of patients were seropositive for the

Table 19.2

Isolation of MAP from patients with Crohn’s disease

Methodology

Crohn’s

Ulcerative colitis

Control

Reference

Serology Serum Ab by ELISA Serum Ab by ELISA

14/42a (33.3%) 107/283 (37.8%)

ndb 50/144 (34.7%)

(Barta et al. 2004) (Bernstein et al. 2004)

16/28 (57.1%) 15/28 (53.6%) 15/28 (53.6%) 77/89 (86.5%) 40/53 (75.5%) 79/89 (88.8%) 39/53 (73.6%) 9/10 (90%)

? 0/20 2/20 5/42 1/10 4/27 1/10 0/10

3/34 (8.8%) 135/402 (33.6%) 47/138 (34.1%)c ? nd nd 4/40 (10%) 3/35 (8.6%) 5/50 (10%) 0/35 (0%) 0/10 (0%)

14/28 (50%) 3/14 (21.4%) 1/30 (3.3%) 4/82 (4.9%) 1/66 (1.5%) 8/24 (33.3%) 1/5 (20%) 0/21 (0%)

2/9 (22.2%) nd nd nd nd 5/22 (22.7%) nd 0/5 (0%)

0/15 (0%) nd nd 1/55 (1.8%) nd 1/40 (2.5%) nd 0/11 (0%)

(Naser et al. 2004) (Chiodini et al. 1984) (Coloe et al. 1986) (Gitnick et al. 1989) (Haagsma et al. 1991) (Del Prete et al. 1998) (Pavlik et al. 1994) (Clarkston et al. 1998)

6/18 (33.3%) 6/17 (35.3%) 10/12 (83.3%) 8/12 (66.7%)

nd nd 2/2 (100%) nd

1/11 (9.1%) 0/13 (0%) 1/6 (16.7%) 0/6 (0%)

(Moss, et al. 1992) (Wall et al. 1993) (Romero et al. 2005)

38kD Ag 24kD Ag 18kD Ag p36 Ag p36 Ag p35 Ag p35 + p36 Ag Serum IgA binding to mycobacterial HupB protein Culture Culture from blood Culture for 18 months Culture from colonic material Culture from surgical tissue Culture Culture from faeces Culture from surgical tissue Intestinal mucosal biopsies and surgical tissue PCR & related methods PCR of IS900 sequence from culture PCR of IS900 sequence from culture PCR of IS900 sequence from tissue FISH using tissue

(0%) (10%) (11.9%) (10%) (14.8%) (10%) (0%)

(Elsaghier et al. 1992) (El-Zaatari et al. 1999) (Naser et al. 1999) (Cohavy et al. 1999)

Table 19.2 Continued Methodology

Crohn’s

Ulcerative colitis

Control

Reference

PCR of IS900 and IS1311 sequences from tissue PCR in uncultured buffy coats Nested PCR based on IS900 sequence PCR of IS900 sequence from surgical tissue PCR of IS900 sequence from surgical tissue PCR of IS900 sequence from surgical tissue PCR of IS900 sequence from surgical tissue PCR of IS900 sequence from colon biopsy PCR of IS900 sequence PCR of IS900 sequence PCR of IS900 sequence PCR-DEIA of IS900 sequence PCR of IS900 sequence from ileal mucosa PCR PCR of IS900 sequence PCR on intestinal biopsies PCR of colon specimens Multiplex PCR on ileal biopsy PCR on faeces, serum and intestinal tissue Intestinal mucosal biopsies and surgical tissue PCR of IS900 sequence from biopsies and surgical tissue PCR PCR of intestinal tissue

0/18 (0%)

nd

nd

(Baksh et al. 2004)

13/28 (46.4%) 0/13 (0%) 26/40 (65%) 11/24 (45.8%) 13/18 (72.2%) 4/31 (12.9%) 2/9 (22.2%) 10/10 (100%) 10/26 (38.5%) 17/36 (47.2%) 15/17 (88.2%) 8/8 (100%) 3/11 (27.3%) 0/68 (0%) 0/36 (0%) 0/27 (100%) 0/10 (0%) 0/21 (0%)

4/9 (44.4%) 0/14 (0%) nd nd nd nd 2/15 (13.3%) 11/18 (61.1%) nd 2/18 (11.1%) 9/18 (50%) 2/2 (100%) nd 0/49 (0%) 0/13 (0%) nd nd 0/5 (0%)

3/15 (20%) 0/13 (0%) 6/63 (9.5%) 5/38 (13.2%) 10/35 (28.6%) 0/30 (0%) 0/11 (0%) 14/16 (87.5%) 4/35 (11.4%) 3/20 (15%) 1/40 (2.5%) 0/2 (0%) 0/11 (0%) 1/26 (3.8%) 0/23 (0%) 0/11 (0%) 0/27 (0%) 0/11 (0%)

(Naser et al. 2004) (Kanazawa et al. 1999) (Sanderson et al. 1992) (Lisby et al. 1994) (Dell’Isola et al. 1994) (Fidler et al. 1994) (Murray et al. 1995) (Suenaga et al. 1995) (Erasmus et al. 1995) (Gan et al. 1997) (Del Prete et al. 1998) (Mishina et al. 1996) (Tiveljung et al. 1999) (Rowbotham et al. 1995) (Dumonceau et al. 1996) (Frank and Cook 1996) (Al Shamali et al. 1997) (Kallinowski et al. 1998)

1/21 (4.8%)

0/5 (0%)

0/11 (0%)

(Clarkston et al. 1998)

0/34 (0%)

nd

0/17 (0%)

(Chiba et al. 1998)

0/47 (0%) 0/13 (0%)

0/27 (0%) 0/14 (0%)

0/20 (0%) 0/13 (0%)

(Cellier et al. 1998) (Kanazawa et al. 1999)

a b c

No. of positive samples/total no. sampled nd = not determined Unaffected siblings of Crohn’s patients

Mycobacterium paratuberculosis

529

p36 antibody and 74% of patients were seropositive when challenged with both proteins. The corresponding values for sera from controls were 11% and 0%, respectively. However, 100% of BCG vaccinated subjects and 89% of subjects with tuberculosis or leprosy were also seropositive for the p36 antibody. Shafran et al. (2002b) have proposed that detection of p35 and p36 can form the basis of a diagnostic test for Crohn’s disease. Other research has failed to find a link between seroprevalence of MAP antibodies and Crohn’s disease (Kobayashi et al. 1989; Tanaka et al. 1991; Walmsley et al. 1996). The interpretation of data concerning seroprevalence is made difficult because many Crohn’s sufferers take immunosuppressive drugs which can interfere with immunological assays, the assays themselves can be unreliable, and by the fact that Crohn’s disease results in a ‘leaky’ intestine and so patients with the disease readily form antibodies to intestinal and foodborne microorganisms (Blaser et al. 1984). Indeed, Ryan et al. (2004) were able to isolate E. coli DNA more frequently in granulomas from Crohn’s patients than in other non-Crohn’s bowel granulomas. They suggested that the results indicated a tendency for lumenal bacteria to colonise inflamed tissue, or they may have been due to increased uptake of bacterial DNA by gut antigen presenting cells. They concluded that the nonspecific nature of the type of bacterial DNA present in granulomas was evidence against any one bacterium having a significant causative role in Crohn’s disease. Difficulties in identification of MAP in Crohn’s disease have led to the argument that infection with the organism in these patients is paucibacillary (Selby 2004). Our understanding of the pathogenesis of Crohn’s disease has progressed rapidly with the discovery of NOD-2/CARD15 variants associated with ileal involvement, and the significance of various inflammatory mediators. It is now well established that Crohn’s disease is associated with polymorphisms of NOD2 (CARD15) (Girardin, et al. 2003). Previous work has shown that NOD2 acts as an intracellular receptor for bacteria and bacterial breakdown products, and because it appears capable of both activating and inhibiting inflammatory responses, NOD2 plays an important role in the gastrointestinal tract’s response to infectious organisms. NOD2 activation leads to the modification of NEMO. (NF-κB essential modulator), a central component of the NF-κB signalling pathway controlling inflammatory responses. NOD2 mutations responsible for Crohn’s disease cause polymorphisms that prevent the NOD2 protein from properly modifying NEMO (Abbott et al. 2004). More than 30 different genetic variations have been reported so far in Crohn’s disease patients, but three major mutations account for 82% of the total NOD2 (CARD15) mutations (Girardin et al. 2003). Saleh Naser and colleagues (2004) have reported that, although MAP DNA can be detected in the circulation of patients with inflammatory bowel disease and in controls without the disease, the viable organism could only be cultured from blood of patients with Crohn’s disease or ulcerative colitis. Naser and colleagues (2000) have also reported the culture of MAP from intestinal tissue and breast milk in people with Crohn’s disease. The existing

530 Emerging foodborne pathogens evidence that MAP is one of the causes of Crohn’s disease is inconclusive, despite arguments of protagonists (Bull et al. 2003; Chamberlin et al. 2001; Greenstein 2003; Hermon-Taylor 2001; Quirke 2001). The identification of clusters of Crohn’s disease cases may indicate that it is caused by an aetiological agent, but such clusters are uncommon. In Australia, Johne’s disease is not uniformly distributed yet Crohn’s disease occurs throughout the country. The equal circulation of MAP DNA in patients with and without inflammatory bowel disease suggests that environmental exposure to MAP is widespread, possibly from water, milk, or other sources (Greenstein and Collins 2004). Nevertheless, Naser et al. (2002) were able to culture the bacterium only in patients with inflammatory bowel disease. This may be due to a defect in the mucosal barrier in patients with inflammatory bowel disease that allows passage or persistence of viable organisms taken up by circulating monocytes, or impaired killing of MAP by macrophages in these patients. The presence of MAP in blood could thus be an effect of the disease rather than its cause. Strangely, no correlation was found between the presence of circulating MAP and immunosuppressive therapy as reactivation of tuberculosis is a significant complication of infliximab therapy. However, MAP infection has not been described with this treatment and there is only one report of Crohn’s disease in a patient with AIDS (Richter et al. 2002). There have been reports of the efficacy of antimycobacterial drugs in treatment of Crohn’s patients, but the antibiotics used are active against many other bacteria (Hermon-Taylor 2002; Prantera et al. 1989, 1994, 1996; Shafran et al. 2002a). The remission in symptoms achieved with these drugs is generally short-lived and similar results can be obtained with antibiotics not known to be effective against mycobacteria (Prantera et al. 1996). However, Borody et al. (2002) have described reversal of severe Crohn’s disease in six out of 12 patients using prolonged combination anti-MAP therapy alone. Three patients achieved long-term remission with no detectable Crohn’s disease when all therapy was removed. There is conclusive evidence that hereditary and environmental factors play an important role in the aetiology of Crohn’s disease (Kornbluth et al. 1993). This, together with the conflicting results of studies aimed at confirming a link between MAP and Crohn’s disease, suggest that even if MAP is involved in the development of Crohn’s disease it is not the sole cause. Also, if bacteria are involved in Crohn’s disease then their action may be the result of a dysfunctional immune response and not due to the virulence of the organism per se (Griffiths 2002). Ghadiali et al. (2004) identified two alleles among short sequence repeats of MAP isolated from Crohn’s disease patients, and these clustered with strains derived from animals with Johne’s disease. The authors concluded that the identification of a limited number of genotypes among human strains suggests the existence of human disease-associated genotypes and strain sharing with animals.

Mycobacterium paratuberculosis

531

19.5 Prevalence of Mycobacterium paratuberculosis in foods Because of the widespread occurrence of Johne’s disease it seems likely that MAP would be present in raw meats from ruminants as well as in raw vegetables and water due to environmental contamination by faeces. However, no data appear to exist in the literature on the prevalence of this organism from these sources, although MAP has been isolated from municipal potable water (Mishina et al. 1996) where it appears to be resistant to chlorination (Greenstein 2003). Possible routes of transmission to humans are milk, undercooked beef, water and animal contact (Greenstein 2003; Greenstein and Collins 2004). Several studies have shown that MAP can be cultured from the milk of cows clinically infected with paratuberculosis (Doyle 1954; Smith 1960; Taylor et al. 1981). MAP has been cultured from the faeces of 28.6% of cows in a single herd with high prevalence of infection. Of the faecal culturepositive cows, MAP was isolated from the colostrum of 22.2% and from the milk of 8.3%. Cows that were heavy faecal shedders were more likely to shed the organism in the colostrum than were light faecal shedders (Sweeney et al. 1992). Levels of the organism in nine culture-positive raw milks from clinically normal, faecal culture-positive cows were between 2–8 CFU/ml (Sweeney et al. 1992). However, other studies have suggested that faecal contamination was the most important contributor to MAP contamination of milk and levels as high as 104 CFU/ml could be attained (Nauta and van der Giessen 1998). Several surveys of pasteurized milk for the presence of MAP have now been carried out and these are summarised in Table 19.3. Millar et al. (1996) examined cream, whey and pellet fractions of centrifuged whole cow milk for MAP by IS900 PCR and found that the PCR assay gave the expected results for spiked milk and for native milk samples obtained directly from MAP-free, sub-clinically and clinically infected cows. These researchers also tested individual cartons and bottles of whole pasteurized cows’ milk obtained from retail outlets throughout Central and Southern England and South Wales from September 1991 to March 1993 by PCR (Millar et al. 1996) and found that 7.1% (22 of 312) tested positive for MAP. After 13–40 months of culture, 50% of the PCR-positive milk samples and 16.7% of the PCR-negative milk samples contained MAP, but the plates were overgrown by other organisms. More positive samples were found in winter and autumn using the PCR assay. During the period March 1999 to July 2000, a survey was undertaken to determine the prevalence of MAP in raw and pasteurized milks in the UK (Grant et al. (2002a). The organism was detected in milk using an initial rapid screening procedure involving immunomagnetic separation coupled to PCR (IMS-PCR). Conventional culture was used to confirm viability of the isolates and the organism was determined to be MAP if it met the following criteria: (i) acid fast, (ii) slow growth and typical colony morphology on Herrold’s egg yolk medium, (iii) presence of IS900 insertion element confirmed

Table 19.3

Prevalence of MAP in milk

Sample

Method

Commercially pasteurized milk Raw milk Commercially pasteurized milk Raw milk Commercially pasteurized milk Raw milk Raw bulk tank milk Commercially pasteurized milk Raw bulk tank milk Commercially pasteurized milk Commercially pasteurized milk

Culture for 32 weeks

No. of samples tested

No. +ve for MAP (% +ve)

Country

Reference

244

4 (1.6)

Czech Republic

(Ayele et al. 2005)

IMS-PCR of IS900

389 357

50 (12.9) 35 (9.8)

Ireland

(O’Reilly et al. 2004)

Culture

389 357

1 (0.3) 0 (0) Switzerland United Kingdom

(Corti and Stephan 2002) (Grant et al. 2002a)

Canada

(Gao et al. 2002)

PCR of IS900 IMS-PCR of IS900

1384 244 567

273 (19.3) 19 (7.8) 67 (11.8)

Culture

244 567

4 (1.6) 10 (1.8)

Nested PCR of IS900

710

110 (15.5)

Culture on Middlebrook medium

244 (including 44 PCR +ve)

0 (0)

Mycobacterium paratuberculosis

533

by PCR, and (iv) dependent on mycobactin J for growth. MAP DNA was detected by IMS-PCR in 7.8% (95% confidence interval, 4.3 to 10.8%) and 11.8% (95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. When culture, following chemical decontamination with 0.75% cetylpyridinium chloride for 5 h, was used to confirm the presence of MAP, 1.6% (95% confidence interval, 0.04 to 3.1%) and 1.8% (95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, tested positive for the organism. The ten culture-positive pasteurized milk samples were from only eight of the 241 milk processing establishments that participated in the survey. Seven of these culture-positive pasteurized milks had been heat treated at 72 to 74 °C for 15 s; whereas the remaining three had been treated at 72 to 75 °C for an extended holding time of 25 s. Gao et al. (2002) collected 710 retail milks from retail stores and dairy plants in southwest Ontario, and these were tested for the presence of MAP by nested IS900 PCR. The PCR reaction was positive for 110 samples (15.5%); however, no survivors were isolated on Middlebrook 7H9 culture broth nor Middlebrook 7H11 agar slants from 44 PCR positive and 200 PCR negative retail milks. The absence of viable MAP in the retail milks tested may have been a true result or may have been due to the presence of low numbers of viable cells below the detection limit of the culture method (Gao et al. 2002). As well as cows’ milk, MAP DNA has been detected in goats’ milk in Norway (Djonne et al. 2003; Grant et al. 2001) and the UK (Grant et al. 2001) but the UK study failed to detect the organism in a limited number (14) of sheep milk samples.

19.6 Survival in food Very little work has been published on the ability of MAP to survive in foods. To determine the ability of MAP to survive in cheese, Sung and Collins (2000) investigated the effect of pH, salt and heat treatment on viability of the organism. They showed faster rates of inactivation of MAP at lower pH. It was also concluded that NaCl concentrations between 2 and 6% had little effect on the ability of the organism to survive regardless of pH. However, the inactivation rates were higher in acetate buffer (pH 6, 2% NaCl) than in Queso Fresco cheese (pH 6.06, 2% NaCl). It was concluded that heat treatment of milk together with a 60-day curing period will reduce numbers of MAP in cheese by about 103 CFU/g. Donaghy et al. (2004) studied the survival of MAP over a 27-week ripening period in model Cheddar cheeses prepared from pasteurized milk artificially contaminated with high (104 to 105 CFU/ml) and low (101 to 102 CFU/ml) levels of three different MAP strains. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. For all manufactured cheeses, a slow gradual decrease in the count of MAP in cheese was observed

534 Emerging foodborne pathogens over the ripening period. In all cases where high levels (>104 CFU/g) of MAP were present in one-day-old cheeses, the organism could be recovered after the 27-week ripening period. At low levels of contamination, only one of the three strains of MAP used was recovered from the 27-week-old cheese. Similar research has been carried out in model hard (Swiss Emmentaler) and semi-hard (Swiss Tilsiter) cheese made from raw milk artificially contaminated with declumped cells of two strains of MAP at a concentration of 104 to 105 CFU/ml (Spahr and Schafroth 2001). As seen with the Cheddar cheeses, MAP counts decreased gradually in both the hard and the semi-hard cheeses during ripening. However, viable cells could still be detected in 120day cheese. The temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening were the greatest contributing factors to the death of MAP in cheese. The authors concluded that counts of MAP in these cheeses would be reduced by 103 to 104 CFU/g during the ripening period which lasts at least 90 to 120 days (Spahr and Schafroth 2001). It is interesting to note that the acid resistance of MAP has been shown to vary with growth conditions (Sung and Collins 2003). Since the revelation that MAP can be isolated from pasteurized milk, there have been numerous studies to ascertain its heat stability in milk. Using a holder method, Chiodini and Hermon-Taylor (1993) observed that heat treatments simulating a batch pasteurization (63 °C for 30 min) and a HTST treatment (72 °C for 15 s) resulted in over 91% and 95% destruction of MAP, respectively. When MAP was heated in milk in a sealed vial, Dvalues of 229, 48, 22 and 12 s were reported at 62, 65, 68 and 71 °C, respectively (Collins et al. 2001; Sung and Collins 1998). When a holder method (63.5 °C for up to 40 min) and a laboratory scale pasteurizer (72 °C for 15 s) were used, numbers declined rapidly during heating, but approximately 1% of the initial population remained after heating (Grant et al. 1996). This has been attributed to clumping of the bacterial cells rather than due to the presence of a more heat-resistant sub-population of cells (Klijn et al. 2001; Rowe et al. 2000). Other experiments led to the conclusion that laboratory heat treatments simulating pasteurization did not effectively eliminate MAP from the milk unless initial numbers were below 10 CFU/ml (Grant et al. 1998a) or the holding time at 72 °C was extended to 25 s (Grant et al. 1999). Using similar methods to heat treat raw milk inoculated with 103 to 107 CFU/ml of MAP, Gao et al. (2002) found no survivors on cultures of seven milks treated by a batch process at 63 °C for 30 min, but MAP cells were detected in two of the 11 HTST (72 °C for 15 s) treated milks. The positive samples were obtained from raw milks containing 105 CFU/ml and 107 CFU/ml MAP. Lund et al. (2002b) pointed out that the laboratory pasteurizing apparatus used by Grant and her colleagues (Grant et al. 1996, 1998a, 1999) may be liable to error due to condensate and splashed cells which may be able to reach the portions of the inlet and outlet tubes of the apparatus that are above the heating liquid. These cells would receive less than the full heat treatment and may drip back into the heating medium.

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Also there is a significant heat-up time of about 50 s for the milk to reach approximately 72 °C. The importance of clumping of MAP cells on heat resistance was studied by Keswani and Frank (1998). These workers used clumped and de-clumped suspensions of cultures to determine the rate of heat inactivation and survival at pasteurization temperatures in sealed capillary tubes. At 55 °C, minimal thermal inactivation was observed for both clumped and declumped cells. At 58 °C, thermal inactivation ranging from 0.3 to 0.7 log cycles was observed for both clumped and de-clumped suspensions. D values at 60 °C ranged from 8.6 to 11 min and 8.2 to 14.1 min for clumped and declumped cells, respectively, and the respective values at 63 °C ranged from 2.7 to 2.9 and 1.6 to 2.5 min. Keswani and Frank (1998) also studied the survival of MAP at initial levels ranging from 44 to 105 CFU/ml at 63 °C for 30 min and 72 °C for 15 s. No survivors were observed after incubating plates for up to four months on Middlebrook 7H11 agar and up to two months on Herrold’s egg yolk medium. This led to the conclusion that low levels of MAP, as might be found in raw milk, will not survive pasteurization treatments. A laboratory method, in which milk inoculated with MAP was heated in sealed tubes at temperatures ranging from 65 to 72 °C for up to 30 min, was compared to results obtained using a small-scale pasteurizer designed to simulate HTST units used in processing plants (Stabel et al. 1997). About 10 CFU/ml of MAP (from an original population of about 1 × 106 CFU/ml) survived heating for 30 min at 72 °C in the sealed tubes. However, results obtained using the laboratory scale pasteurizer showed that there were no detectable cells of MAP after heating for 15 s at 65, 70 or 75 °C. Thus, results obtained using the holder method could not be extrapolated to a commercial HTST unit, and that continuous flow is essential for effective killing of MAP in milk. Further studies using a continuous flow system were described by Hope et al. (1997). However, because a linear holding tube was used which failed to generate turbulent flow, this study did not simulate exactly the conditions that would exist in a commercial pasteuriser. Seventeen batches of raw milk were inoculated with 102–105 CFU/ml of MAP and pasteurised at temperatures ranging from 72–90 °C for 15–35 s. The organism could not be isolated from 96% (275/286) of pasteurised milk samples, representing at least a 4 logcycle reduction in count. Viable mycobacteria were not recovered from the heat-treated milk when raw whole milk was loaded with less than 104 mycobacteria per ml, and were not cultured in any of five batches of milk pasteurized at 72–73 °C for 25–35 s, which are the minimum conditions applied when this machine is used commercially to correct for laminar flow in the holding tube. An adequate holding time appeared to be more effective in killing MAP than higher temperatures in the small number of batches treated, and this is similar to results reported by Grant et al. (1999). The effectiveness of the holder and high-temperature short-time pasteurisation standards on the destruction of MAP were conducted using a

536 Emerging foodborne pathogens slug-flow pasteuriser and a laboratory scale pasteurizer; both of which were used to treat UHT milk inoculated with 105 and 108 CFU/ml of three different strains of MAP (Stabel and Lambertz 2004). Five different time-temperature combinations were evaluated: 62.7 °C for 30 min, 65.5 °C for 16 s, 71.7 °C for 15 s, 71.7 °C for 20 s, and 74.4 °C for 15 s. Regardless of bacterial strain or method of heating, the heat treatments resulted in an average 5.0 and 7.7 log cycle reduction in MAP count for milk inoculated with the low and high inoculum levels, respectively. The survival of MAP in inoculated batches of milk in a small-scale commercial unit cannot be directly extrapolated to commercial pasteurization of naturally infected milk because of the artificially high mycobacterial loads used in these experiments, possible differences between the thermoresistance of laboratory cultured mycobacteria, features of the smallscale unit (Hope et al. 1997) and the variation in heat resistance of MAP inocula grown under different culture conditions (Sung et al. 2004). However, pasteurization in the continuous flow small-scale unit was more efficient at killing MAP than batch experiments performed in the laboratory. The inaccuracies in heat resistance data produced by laboratory scale experiments were pointed out by Cerf and Griffiths (2000) who also pointed out that it was thermodynamically unfeasible to expect that extending the holding time would have a greater effect on survival of MAP than increasing the heating temperature as proposed by Grant et al. (1999). Several studies have now been completed to determine the effect of commercial pasteurization on the survival of MAP in milk with conflicting results. Pearce et al. (2001) used a pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) to study the heat sensitivity of five strains of MAP (ATCC 19698 type strain, the human isolate designated Linda, and three bovine isolates) in raw whole milk for 15 s at 63, 66, 69, and 72 °C. No strains survived at 72 °C for 15 s; and only one strain survived at 69 °C. Means of pooled D values (decimal reduction times) at 63 and 66 °C were 15.0 ± 2.8 s and 5.9 ± 0.7 s, respectively. The mean extrapolated D72°C was < 2.03 s, which was equivalent to a > 7 log10 kill at 72 °C for 15 s. The mean Z value (the temperature increase required for a 1 log10 cycle reduction in D value) was 8.6 °C. The five strains behaved similarly when recovery was performed on Herrold’s egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). In an additional experiment, milk was inoculated with fresh faecal material from a high-level faecal shedder with clinical Johne’s disease and heated at 72 °C for 15 s (Pearce et al. 2001). Under these conditions, a minimum inactivation of >4 log10 CFU/ml of MAP kill was achieved, indicating that properly maintained and operated pasteurizers should ensure the absence of viable MAP in retail milk and other pasteurized dairy products. In another study undertaken using a pasteurizer with a validated Reynolds number (62,112) and a flow rate of 3,000 l/h, 20 batches of milk inoculated with 103 to 104 CFU/ml MAP were homogenised and then processed with

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combinations of three temperatures of 72, 75, and 78 °C and three time intervals of 15, 20, and 25 s (McDonald et al. 2005). Surviving cells were assayed using a culture technique capable of detecting one organism per 10 ml of milk. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, whereas for 3 of the 20 runs of milk, pasteurized at 72 °C for 15 s, 75 °C for 25 s and 78 °C for 15 s, a small number of viable cells (corresponding to 0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing MAP, resulting in a reduction of > 6 log10 in 85% of runs and > 4 log10 in 14% of runs. Both the studies by Pearce et al. (2001) and McDonald et al. (2005) involved milk which had been artificially contaminated with MAP. A similar study was undertaken using raw cows’ milk naturally infected with MAP and pasteurized using an APV HXP commercial-scale pasteurizer (capacity 2,000 l/h) (Grant et al. 2002b). The milk was pasteurized at 73 °C for 15 s or 25 s, with and without prior homogenisation (2,500 psi in two stages) in an APV Manton Gaulin KF6 homogeniser. Raw and pasteurized milk samples were tested for MAP by IMS-PCR and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. On 10 of the 12 processing occasions, MAP was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk and viable cells were cultured from 4 of 60 (6.7%) raw and 10 of 144 (6.9%) pasteurized milks. Results suggested that survival was related to the initial load of MAP in the raw milk and that homogenisation increased the lethality of subsequent heat treatments. Unlike previous work conducted using a laboratory pasteurizer (Grant et al. 1999), extending the holding time at 73 °C to 25 s was no more effective at killing MAP than the 15 s holding time. In contrast to the work of Pearce et al. (2001) and McDonald et al. (2005), this study provides evidence that MAP present in naturally contaminated milk is capable of surviving commercial HTST pasteurization if present in sufficient numbers in the raw milk (Grant et al. 2002b). The reasons for the differences reported in heat resistance studies of MAP have been reviewed by Lund et al. (2002a, b). An alternative processing treatment involving high voltage electric pulses, pulsed electric field (PEF), in combination with heat treatment has been explored to determine its effect on the viability of MAP cells suspended in peptone water and in sterilised cow’s milk (Rowan et al. 2001). PEF treatment at 50 °C (2,500 pulses at 30 kV/cm) reduced the level of viable MAP cells by about 5 to 6 log cycles in peptone water and in cows’ milk. Heating at 50 °C for 25 min or at 72 °C for 25 s (extended HTST pasteurization) resulted in reductions of MAP counts of approximately 0.01 and 2.4 log cycles, respectively. Electron microscopy revealed that exposure to PEF treatment caused substantial damage to MAP cell membranes.

538 Emerging foodborne pathogens

19.7 Survival in the environment One possible route of transmission of MAP from cattle to humans is through contaminated water. MAP has been isolated from water runoff from cattle farms (Raizman et al. 2004). A study of the River Taff in South Wales, United Kingdom, running from hill pastures grazed by livestock in which MAP is endemic to a populated coastal region showed that the organism could be detected in 31 of 96 daily samples (32.3%) by IS900 PCR, and 12 of the samples by culture (Pickup et al. 2005). Sequencing of the isolates obtained by culture and from river water DNA extracts revealed that 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214 suggesting the presence of a different, unculturable strain of MAP in the river. The strains that were isolated remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, MAP was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Epidemiological studies have indicated a highly significant increase of Crohn’s disease in districts bordering the river. MAP can also survive in faecal material for up to 55 weeks in a dry, fully shaded environment, but for much shorter periods in sunny locations (Whittington et al. 2004). The organism survived for up to 24 weeks on grass that germinated through infected faecal material applied to the soil surface in completely shaded boxes and for up to nine weeks on grass in 70% shade. The results obtained indicated that the cells were able to enter a dormant state and sequences homologous to genes involved in dormancy responses in other mycobacteria were present in the MAP genome sequence. When MAP was present in water at high numbers (106 CFU/ml), chlorine treatment at concentrations of 2 µg/ml for up to 30 min was insufficient to completely eradicate the organism and < 3 log cycle reduction in counts was observed (Whan et al. 2001).

19.8 Detection, enumeration and typing Diagnostic tests for MAP have been reviewed (Grant and Rowe 2001; Nielsen et al. 2001). Culture methods are laborious and time consuming, taking 8 to 16 weeks to obtain results. Because of this long incubation period, contamination is often a problem and samples have to be treated with selective agents to reduce numbers of non-mycobacterial organisms, although care must be taken in choosing the correct decontaminant to avoid a detrimental effect on the recovery rate in different sample matrices (Donaghy et al. 2003; Dundee et al. 2001; Grant and Rowe 2004). The low numbers present in a sample often necessitate a concentration step, such as centrifugation, filtration, or, more recently, immunomagnetic separation (Djonne, et al. 2003; Grant et al. 1998b 2000). Usually an enrichment step in liquid medium, such

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as Dubos broth, is used prior to plating onto a solid medium. Of these, one of the most commonly used is Herrold’s egg yolk medium. Confirmation of the identity of the isolate involves meeting the following criteria: (i) it must be acid-fast; (ii) it must exhibit slow growth with a typical colony morphology (i.e. colonies are 1 to 2 mm in diameter, entire and white); (iii) it must be IS900 PCR-positive; (iv) it must require mycobactin J for growth. To decrease the time to obtain results, spiral plating in combination with microscopic colony counting has been investigated (Smith et al. 2003). This technique allowed counts to be obtained in 8 to 14 days. A modification of the culture method has been used successfully in which radioisotope-labelled substrates are incorporated in the growth medium. The assimilation of these substrates can be detected using the BACTEC system. Radiometric culture is faster and more sensitive than traditional culture techniques (Nielsen et al. 2001). The radiometric method has also been combined with PCR to rapidly confirm the MAP status of the sample. Other automated methods are available such as the ESP II Culture System with para-JEM liquid culture reagents that uses pressure sensing instead of radioisotopes to detect growth (Kim et al. 2004), the MB/BacT system in which CO2 produced during growth in a modified Middlebrook 7H9 broth is monitored based on changes in reflection of light from a sensor in the bottom of the culture vessel (Stich et al. 2004) and the Mycobacteria Growth Indicator Tube (MGIT) which has an O2 sensitive fluorescent sensor embedded in its base that fluoresces when O2 becomes depleted in the medium due to growth (Grant et al. 2003). Because of its slow growth there has been considerable interest in the development of more rapid detection tests. These have mainly focused on PCR of the IS900 insertion element which is unique to MAP and is present in multiple copies in the genome of the bacterium. However, recent work has shown that this cross reacts with environmental Mycobacteria sp. in ruminant faeces, which share 71 to 79% (Cousins et al. 1999) and 94% in sequence homology (Englund et al. 2002) to IS900. The limitations of the IS900 PCR assay have been discussed by Nielsen et al. (2001), but the main problem seems to be the high number of false-negative reactions generated by PCR as compared to conventional culture. Because of these limitations work has also been carried out to develop PCR assays based on other genomic loci including 251 (Rajeev et al. 2005); ISMAV2 (Shin et al. 2004); hspX (Ellingson et al. 2000); dnaA (Rodriguez-Lazaro et al. 2004); and f57 (Vansnick et al. 2004). With the availability of the complete genome sequence of MAP identification of unique open reading frames has been facilitated (Paustian et al. 2004, 2005). To improve the sensitivity of PCR assays and to assist in the removal of inhibitors present in the sample that interfere with the polymerase enzyme, methods have also been developed that combine immunomagnetic capture of the organism with PCR (Djonne et al. 2003; Grant et al. 2000). Using this approach it was possible to detect 10 CFU/ml of MAP in water (Whan et al.

540 Emerging foodborne pathogens 2005). Other techniques have been investigated to capture MAP from suspension. For example, Halldorsdottir and colleagues (Halldorsdottir et al. 2002) used buoyant density in a Percoll gradient to remove cells prior to IS900 sequence capture PCR and dot blot assay to detect MAP in faeces; whereas Stratmann et al. (2002) have developed a peptide-mediated magnetic separation technique based on phage display technology to aid selective isolation of MAP from milk. Nine recombinant bacteriophages binding to MAP were isolated from a commercial phage-peptide library encoding random 12-mer peptides. Paramagnetic beads coated with the phage or with a peptide, aMP3, allowed capture of MAP from milk, and when this was combined with an ISMav2-based PCR the bacterium could be detected at levels of 100 CFU/ml in artificially spiked milk. Experiments using milk from naturally infected cows and bulk milk samples from infected herds demonstrated that the peptide-mediated capture PCR was able to detect single strong shedders of MAP in pooled milk samples. Several commercial PCR assays for MAP are now available and the performance of three of these has been evaluated by Taddei et al. (2004). There is also growing interest in the development of real-time PCR methods. Many approaches have been used including primers and fluorescent probes targeting the 251 genomic locus (Rajeev et al. 2005); fluorescence energy transfer probes targeting IS900 (O’Mahony and Hill 2004); SYBR Green in combination with the LightCycler (O’Mahony and Hill 2002); molecular beacons (Fang et al. 2002; Rodriguez-Lazaro et al. 2004); and TaqMan probes (Kim et al. 2002). Studies comparing real-time PCR with molecular beacons to a commercial PCR/Southern blot technique, nested PCR and culture for the detection of MAP have shown that real-time PCR assays are valid alternatives to culture (Christopher-Hennings et al. 2003; Fang et al. 2002). Real-time PCR has also been combined with IMS to produce a method that was capable of detecting ≤10 cells of MAP in 2 ml of milk or 200 mg of faeces (Khare et al. 2004). PCR methods, when used alone, cannot distinguish between viable and non-viable states of the organism, and to this end PCR has been combined with culture methods including the ESP II system (Ellingson et al. 2004) and an agar culture enrichment step (Secott et al. 1999). To overcome the problem of distinguishing live and dead cells, RT-PCR and an isothermal RNA amplification method, nucleic acid sequence-based amplification (NASBA), have also been investigated (Grant and Rowe 2001; Rodriguez-Lazaro et al. 2004). Another isothermal amplification method, loop-mediated isothermal amplification (LAMP), has been successfully used to detect MAP (Enosawa et al. 2003). LAMP relies on a DNA polymerase with high strand displacement activity to catalyse auto-cycling strand displacement DNA synthesis. A specially designed set of two inner and two outer primers is used, but later during the cycling reaction only the inner primers are used for strand displacement DNA synthesis. The reaction is highly specific because the target sequence is recognised by six independent sequences in the initial stage and by four

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independent sequences during the later stages of the LAMP reaction. A large amount of DNA is synthesised during the LAMP reaction with the concomitant production of a large concentration of pyrophosphate ion which can be detected as a white precipitate of magnesium pyrophosphate in the reaction mixture. Work on optimisation of DNA extraction protocols has also been carried out (Christopher-Hennings et al. 2003; Stabel et al. 2004). O’Mahony and Hill (2004) found that a milk sample treatment involving a combination of centrifugation, harsh lysis, physical grinding, boiling, nucleic acid purification, and real-time PCR allowed detection of 40 CFU/ml of milk, within 3 h. The method was also able to enumerate the initial titer of MAP in milk by using predetermined standards. Detection of 10 CFU/ml of MAP in milk has been achieved using a bead beater (a device that breaks up bacterial cell wall mechanically by vibrating bacteria with microbeads at high speed) and lysis buffer to lyze MAP cells, followed by boiling and isopropanol precipitation to extract DNA, and inclusion of 0.0037% bovine serum albumin in the PCR reaction mixtures. The improved assay was 10- to 10,000-fold more sensitive than PCR assays that used template DNA prepared by other lysis procedures including boiling alone, freeze-thaw plus boiling, or use of commercial kits for lysis (Odumeru et al. 2001). Because detection is difficult when mycobacteria are intracellularly located or embedded within mammalian tissues, a culture-independent, in situ hybridisation (ISH) assay for the detection of members of the Mycobacterium avium complex in culture, sputum, and tissue has been developed based on a set of rRNA-based oligonucleotides (St Amand et al. 2005). Immunoassays are widely used for screening cattle for Johne’s disease but the use of ELISA assays for the detection of MAP in bulk tank milks has met with little success (Nielsen et al. 2000). However, more recent work using a commercially available ELISA for antibodies against MAP and preserved milk samples in an indirect ELISA assay format may be a convenient tool for the detection of the organism in dairy herds (Hendrick et al. 2005). Other commercial assays have also been adapted for the detection of MAP in milk (Winterhoff et al. 2002). Commercial ELISA and immunodiagnostic kits are available from a number of companies, although concern has been raised about variability between kit lots (Dargatz et al. 2004). To aid in epidemiological investigations molecular typing methods have been studied to allow differentiation of MAP isolates from human and animal reservoirs. Among the methods studied are multilocus variable-number tandemrepeat analysis (MLVA) and IS900 restriction fragment length polymorphism (RFLP) typing. When the techniques were compared, MLVA typing subdivided the most predominant RFLP type, R01, into six subtypes and, thus, showed improved discriminatory ability (Overduin et al. 2004). Good discrimination has also been obtained using a multilocus short sequence repeat sequencing approach (Amonsin et al. 2004), IS900/ ERIC-PCR using primers targeting the enterobacterial intergenic concensus (ERIC) sequence and the IS900

542 Emerging foodborne pathogens insertion sequence (Englund 2003), a multiplex PCR of IS900 loci (MPIL) (Bull et al. 2000) and randomly amplified polymorphic DNA (RAPD) using the OPE-20 primer (5’-AACGGTGACC-3’) (Pillai et al. 2001).

19.9

Control

Guidelines for minimising transmission of MAP have been published (Green 2002). Arguably the most effective way of controlling MAP is by adopting strategies that will eliminate Johne’s disease from cattle on the farm. However, this is easier said than done because of the organism’s ability to survive in the environment, the long incubation period of the disease and the lack of sensitive diagnostic tests to identify infected animals during this time (Kennedy et al. 2001). It is worth noting that test-and-cull strategies alone may not reduce the prevalence of paratuberculosis in cattle and are costly for producers to pursue but improved calf-hygiene strategies were found to be critically important in every paratuberculosis control program (Groenendaal and Galligan 2003). Risk factors for seropositivity included water source, use of dairy-type nurse cows, previous clinical signs of paratuberculosis, species of cattle and location (Roussel et al. 2005). The risk management strategies used to contain Johne’s disease include prevention of infection of the herd through maintaining a closed herd whenever possible and when new animals have to be introduced they should be from herds free from the disease. Cattle should also be kept from pastures and other environments that may be heavily contaminated due to prior exposure to animals with the disease. It has been demonstrated that MAP can survive in faeces kept outdoors for up to 246 days, depending on the conditions (Collins et al. 2001). Environmental contamination can also be reduced by good management of water and effluent flows from neighbouring high risk farms and by good manure management. The survival of MAP on pasture after destocking of all cattle infected with paratuberculosis was found to be four months during winter. Non-vertebrates, such as cockroaches (Fischer et al. 2003), beetles (Fischer et al. 2004b) and blowflies (Fischer et al. 2004a), as well as wild ruminants or non-ruminant wildlife can be vectors and potentially become a risk factor in the spread of MAP infection. (Machackova et al. 2004). Vaccines have been developed that are effective in reducing the number of clinically affected animals but vaccination does not appear to reduce the total number of infected animals in a herd. Current vaccines are not used in many countries because they interfere with subsequent tests for tuberculosis (Muskens et al. 2002). Work is being carried out on new targets for vaccine development such as a recombinant heat shock protein, rHsp-70 (Langelaar et al. 2002, 2005) and the rMPT 85B antigen (Mullerad et al. 2002). Exposure of individual calves to paratuberculosis should be minimised by rearing in clean environments free from adult cattle, coupled with the adoption of good

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hygienic practices by farm personnel. The animals should be fed milk and water that are free from contamination, and ideally the calf should be provided with an adequate colostrum intake from a paratuberculosis-negative cow. The most important source of MAP within the herd is the cow with advanced infection, as the rate of excretion of bacteria in the faeces increases as these animals approach clinical disease. Thus, infected animals should be identified as early as possible through testing and applying knowledge of history of the disease in the herd. Control is best achieved by culling calves of infected cows, early culling of suspect cows and test reactors along with animals that have been in contact with these cattle. Recent work has suggested that calves infected during the first weeks of life can be a frequent and important source of infection and in herds where poor control measures were in place MAP was shed in faeces of 7.8 to 80% of calves aged between four to six months (Pavlas 2005). However, early separation of newborn calves from cows and grazing calves under 12 months of age in areas free of adult cattle were not found to be protective against Johne’s disease by other workers (Ridge et al. 2005). Several countries have adopted national programmes to control paratuberculosis in dairy herds and/or to accredit MAP-negative herds as low risk sources of replacement cows. To reduce the level of MAP in milk the UK has recommended increasing the holding time at the minimum pasteurization temperature of 72 °C from 15 to 25 s. The control of MAP in animal populations has been reviewed (Kennedy and Benedictus 2001; Whittington and Sergeant 2001).

19.10 Further sources of information Several recent reviews on the role of MAP in Crohn’s disease have been published (Chacon et al. 2004; Collins 2003, 2004; Grant 2003; Griffiths 2002; Hermon-Taylor and Bull 2002; Manning 2001; Manning and Collins 2001; Stabel 2000).

19.11

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20 Enterococci C. M. A. P. Franz and W. H. Holzapfel, Institute for Hygiene and Toxicology, Germany

20.1

Introduction

Enterococci are typical lactic acid bacteria (LAB) and are of importance in food and clinical microbiology. They are ubiquitous microorganisms, but have a predominant habitat in the gastrointestinal tracts of humans and animals (Giraffa, 2002). Representatives of the genus Streptococcus, formerly grouped as ‘faecal streptococci’ or Lancefield’s group D streptococci, were separated from this genus on the basis of modern classification techniques and serological studies in the 1980s. The large conglomeration of streptococci was thus subdivided into three separate genera: Streptococcus, Lactococcus and Enterococcus (Schleifer and Kilpper-Bälz, 1984; Devriese et al., 1993; Devriese and Pot, 1995). The typical pathogenic species, with the exception of S. thermophilus, remained in the genus Streptococcus, and were separated from the non-pathogenic and technically important species of the new genus Lactococcus (Devriese and Pot, 1995). The ‘faecal streptococci’ associated with the gastrointestinal tract of man and animals, with some fermented foods and a range of other habitats, constituted the new genus Enterococcus. Although c. 30 Enterococcus species are currently recognised, E. faecium and E. faecalis are still the two most prominent representatives and are the species which play the most important roles both in human disease and in fermented foods and in probiotics (Franz et al., 1999). 20.1.1 Taxonomy and identification Since the description of the genus Enterococcus in the 1980s, many taxonomic investigations have resulted in assignment of about 30 species to this genus (for reviews, see Devriese et al., 1993, 2003; Devriese and Pot, 1995; Hardie

558 Emerging foodborne pathogens and Whiley, 1997; Franz et al., 2003), but the actual number fluctuates from time to time as individual species are re-classified or new taxa are discovered. For example, E. pallens, E. gilvus, E. canis, E. phoeniculicola, E. ratti, E. villorum, E. haemoperoxidus, E. moraviensis, E. hermanniensis, E. phoeniculicola, E. saccharominimus, E. canintestini and E. aquimarinus were only described in 2001 or later (Svec et al., 2001; Teixeira et al., 2001; Vancanneyt et al., 2001, 2004; Tyrrell et al., 2002; Law-Brown and Meyers, 2003; De Graef et al., 2003, Fortina et al., 2004; Koort et al., 2004; Naser et al., 2005; Svec et al., 2005a,b). The species Enterococcus flavescens (Pompei et al., 1992) appears to be identical to E. casseliflavus, which has nomenclatural priority, and Descheemaeker et al. (1997) could not distinguish between the two using either protein analysis or PCR-based typing (Devriese et al., 2003). Enterococcus solitarius (Collins et al., 1989) was shown to be more closely related to the genus Tetragenococcus (Collins et al., 1990; Williams et al., 1991) and was recently reclassified as T. solitarius (Ennahar and Cai, 2005). De Graef et al. (2003) showed that E. porcinus is a junior synonym of E. villorum. The phylogenetic relationship of the different species within the genus Enterococcus has been determined by comparative sequence analysis of their 16S rRNA genes. Based on these data the following species groups can be distinguished: E. faecium-group: E. faecium, E. durans, E. hirae, E. mundtii, E. villorum, E. canis E. avium-group: E. avium, E. malodoratus, E. pseudoavium, E. raffinosus, E. gilvus E. gallinarum-group: E. gallinarum E. casseliflavus, E. flavescens E. dispar-group: E. asini, E. dispar, E. pallens, E. hermanniensis, E. canintestini E. saccharolyticus-group: E. saccharolyticus, E. sulfureus, E. saccharominimus, E. italicus, E. aquimarinus E. cecorum-group: E. cecorum, E. columbae E. faecalis-group: E. faecalis, E. haemoperoxidus, E. moraviensis, E. ratti Members of the genus Enterococcus, like those of the genera Streptococcus and Lactococcus, are catalase-negative, Gram-positive cocci which are arranged in pairs or short chains. Within the chains, the cells are frequently arranged in pairs and are elongated in the direction of the chain. Endospores are absent. E. gallinarum and E. casseliflavus are motile, all others are nonmotile. E. casseliflavus, E. mundtii, E. sulfureus, E. pallens and E. gilvus are yellow-pigmented. All enterococci are facultatively aerobic chemoorganotrophs with a fermentative metabolism. They have a homofermentative lactic acid fermentation with L(+)-lactic acid as the predominant end product of glucose fermentation. Many of the ‘typical’ species of enterococci (E. faecalis, E. durans, E. faecium, E. gallinarum, E. hirae, and E. mundtii) can be easily distinguished from other Gram-positive, catalase-negative,

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homofermentative cocci such as streptococci and lactococci, in that they are able to grow at 10 and 45 °C, in 6.5% NaCl, in the presence of 40% bile and at pH 9.6. However, many of the more recently described Enterococcus species vary in their physiological properties from those of the typical enterococci (Franz et al., 2003). Reliable identification of the genus Enterococcus and its species thus ultimately relies on the use of a combination of phenotypic, genotypic and phylogenetic information in a polyphasic taxonomy approach as described by Vandamme et al. (1996). A variety of genotypic methods have been used successfully to identify enterococci to genus or species level and these are reviewed by Domig et al. (2003). For differentiation between enterococci and lactococci on the genus level, Deasy et al. (2000) described a rapid PCR-based method based on amplification of a region of the 16S rDNA gene. They showed that using this method they could accurately separate enterococci from streptococci, lactococci, pediococci and lactobacilli. Ozawa et al. (2000) were able to accurately identify Enterococcus species by PCR amplification and sequencing of a conserved internal fragment of the Dalanine:D-alanine ligase genes (ddl). Tyrrell et al. (1997) used restriction fragment length polymorphism of the 16S/23S intergenic spacer region to distinguish the Enterococcus species, although with some species e.g., E. avium and E. pseudoavium, such a differentiation was not possible. Baele et al. (2000) used tRNA intergenic spacer PCR for the identification of enterococci species. Williams et al. (1991), Descheemaeker et al. (1997), Quednau et al. (1998), Andrighetto et al. (2001), Gelsomino et al. (2001a) and Vancanneyt et al. (2002) showed that Enterococcus species can be differentiated quite well by RAPD-PCR. Rep-PCR using primer GTG5 also showed excellent identification possibilities (Svec et al., 2005b), while sequencing of the 16S rRNA gene also yields accurate species identification and can aid in the description of new Enterococcus species (see above). Recently, Lehner et al. (2005) reported on the development and use of an oligonucleotide microarray for the identification of Enterococcus spp. This microarray called the ‘ECCPhylochip’ consisted of 41 hierarchically nested 16S or 23S rRNA genetargeted probes and was able to differentiate between 19 tested Enterococcus species originating from pure culture. In addition, the microarray was successfully used in artificially contaminated milk to identify E. faecium and E. faecalis directly in the food sample. The enterococci are important in environmental, food and clinical microbiology. Because of their intestinal habitat in food animals, they can contaminate milk and the dairy environment or meat at the time of slaughter. Nevertheless, enterococci are of technological importance in the production of various European fermented foods such as sausages and cheeses, where they are either purposefully added to the product as starter cultures (Giraffa et al., 1997), or where their presence results from environmental contamination. As a result of their natural association with the gastrointestinal tract, as well as functional and technologically desirable properties, some strains are also used successfully as probiotics (Franz et al., 1999, 2003).

560 Emerging foodborne pathogens The detrimental activities of enterococci are related to spoilage of foods, especially meats and, more importantly, the fact that certain Enterococcus strains can cause human disease. Enterococci are typical opportunistic pathogens that may cause infections especially in the nosocomial setting in patients which have underlying disease. Over the last two decades, enterococci have emerged as important nosocomial pathogens, and this rise in their association with human disease can in part be explained by their increasing resistance to antibiotics as well as their promiscuity regarding transfer of genetic material (Franz et al., 1999, 2003; Giraffa, 2002). This ‘ambiguous’ nature of enterococci makes them, on the one hand, desirable for use as starter cultures in food production or as probiotics, while, on the other hand, they give rise to concern because of the potential transfer of antibiotic resistances, the possible presence of virulence factors and their role in human disease.

20.2

Habitat

20.2.1 Environment Enterococci occur in a wide variety of environmental niches, including soil, surface waters, waste waters and municipal water treatment plants, on plants, in the gastrointestinal tract of warm blooded animals (including humans) and, as a result of association with plants and animals, in human foods (Franz et al., 1999). On plants, enterococci occur in a truly epiphytic relationship (Mundt et al., 1962) and Enterococcus species typically associated with plants include the yellow-pigmented E. mundtii and E. casseliflavus (Martin and Mundt, 1972). The early studies on enterococci (‘faecal streptococci’) occurring on plants by Mundt et al. (1962) were performed before the genus Enterococcus was re-defined by Schleifer and Kilpper-Bälz (1984). Modern, taxonomic studies based on molecular biological techniques for classification and species identification by Ott et al. (2001) and Müller et al. (2001) validated this epiphytic relationship and enterococci occurring on plants were identified as E. faecium, E. faecalis, E. casseliflavus, E. mundtii and E. sulfureus. The majority of the isolates in the study of Müller et al. (2001), however, possessed a 16S rDNA genotype uncommon to Enterococcus species described at the time of the study. Enterococci also occur on fresh produce and possibly originate from the use of untreated irrigation water or manure slurry for crop production (Johnston and Jaykus, 2004). Interestingly, in this context Johnston and Jaykus (2004) isolated mainly E. faecalis and E. faecium strains, but also other Enterococcus spp. from fresh produce such as celery, cilantro, mustard greens, spinach, collards, parsley, dill, cabbage and cantaluope, and showed that many strains harboured antibiotic resistances. Similarly Ronconi et al. (2002) isolated predominantly E. faecium and E. faecalis strains from lettuce and many strains were also antibiotic resistant. This may

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561

have important implications for dissemination of antibiotic resistance genes and thus impact on human health (see below). Enterococci occur in surface waters, sea water as well as municipal and hospital waste waters. Water is the source of a variety of novel Enterococcus spp. that have been described more recently. For example, Svec et al. (2001) described E. haemoperoxidus and E. moraviensis from surface waters and Svec et al. (2005a) described E. aquimarinus which was isolated from seawater. Enterococci are recognised by the U.S. Environmental Protection Agency as indicator organisms for bacteriological water quality in fresh and saline waters (U.S. Environmental Protection Agency, 1986; Anonymous, 1998) and their presence, especially at elevated levels, indicates that faecal pollution from animal or human sources has occurred (Harwood et al., 2004). However, there are many possible sources for enterococci apart from sewage, including animal waste, invertebrates, plants and soils (Harwood et al., 2004). Among the enterococci, particularly E. faecalis, E. faecium, E. durans and E. hirae are considered to be of faecal origin (Godfree et al., 1997) and thus water quality studies were suggested to focus on this subset of enterococcal species that is consistently associated with faecal pollution (Harwood et al., 2004). In both lake and sea water, enterococci were described to either associate with zooplankton, or to occur in the unbound state, depending on the presence or absence of plankton (Maugeri et al., 2004; Signoretto et al., 2004). The binding of E. faecalis in the non-culturable state to plankton was considered as the main mechanism responsible for enterococci to persist in both lake and sea water (Signoretto et al., 2004). Blanch et al. (2003) studied the occurrence of enterococci in raw and treated waste water, surface waters receiving the treated waste water and hospital wastewater in three European countries (Sweden, Spain and United Kingdom). In all three countries, the levels of enterococci in these different waters were quite similar and ranged from ca. log 6 CFU/ml in raw sewage, ca. log 5-6 CFU/ml in hospital wastewater, ca. log. 3-4 CFU/ml in treated sewage to log ca. 1-4 CFU/ml in surface waters (Blanch et al., 2003). Most of the enterococci strains isolated from any and all of the different water sources were identified as E. faecalis and E. faecium, together representing more than 60% of the enterococcal population. Vancomycin and erythromycin resistant enterococci could be isolated from waters of all sources from the three countries, although at differing incidences. Isolation of such antibiotic resistant bacteria from treated waste waters and surface waters showed that the enterococci can pass through different treatments in wastewater plants and can be transferred to surface waters (Blanch et al., 2003). Moreover, Vilanova et al. (2004) followed vancomycin and erythromycin-resistant enterococci during the sewage treatment process and found that although a significant reduction in bacterial populations was observed, the persistence of such antibiotic resistant bacteria in the same proportions in sewage suggested that there was no selective elimination of antibiotic resistant populations during the treatment process. Thus, antibiotic resistant strains survive sewage treatment and this increases

562 Emerging foodborne pathogens the chance of being transmitted to the food chain especially when water reuse programmes are in place (Blanch et al., 2003; Vilanova et al., 2004). 20.2.2 Gastrointestinal tract Enterococci are well known to occur as part of the natural microflora of the intestinal tract of warm-blooded animals and man and constitute a large proportion of the autochthonous bacteria associated with this ecosystem. E. faecalis is often the predominating Enterococcus sp. in the human bowel, although in some individuals and in some countries, E. faecium outnumbers E. faecalis (Ruoff, 1990; Devriese and Pot, 1995). Numbers of E. faecalis in human faeces range from 105 to 107/g compared with 104 to 105/g for E. faecium (Noble, 1978; Chenoweth and Schaberg, 1990). In livestock such as pigs, cattle and sheep, E. faecalis, E. faecium and E. durans are less frequently isolated when compared to human faeces (Leclerc et al., 1996). E. faecalis, E. faecium, E. hirae and E. cecorum were the enterococci most frequently isolated from pig intestines, while E. faecium predominated in faecal samples (Devriese et al., 1994; Leclerc et al., 1996). The intestinal microflora of young poultry contained principally E. faecalis and E. faecium, but E. cecorum predominated in the intestine of chickens over 12 weeks old (Devriese et al., 1991). E. columbae is an important member of the gut flora of pigeons, while E. hirae frequently occurs in the intestine of pigs, but may also occur in the gut of poultry, cattle, cats and dogs (Devriese et al., 1987). E. durans has been isolated from humans, chickens and calves and E. malodoratus is often found in the tonsils of cats. The habitat of the members of the E. avium species group (E. avium, E. malodoratus, E. raffinosus and E. pseudoavium) otherwise is largely unknown (Devriese et al., 1992; Devriese et al. 2003). Various Enterococcus spp. from intestinal origin have been newly described. These include E. ratti and E. villorum which are associated with enteric disorders in animals (Teixeira et al., 2001; Vancanneyt et al., 2001), and E. canintestini which is associated with the gastrointestinal tract of healthy dogs (Naser et al., 2005).

20.2.3

Foods

Meats The presence of enterococci in the gastrointestinal tract of animals clearly leads to a high potential for contamination of meats at the time of slaughter. In raw meat products, E. faecalis was shown to be the predominant isolate from beef and pork cuts in one study (Stiles et al., 1978), while in another both E. faecium and E. faecalis were the most predominant Enterococcus spp. isolated from pig carcasses (Knudtson and Hartman, 1993). These pig carcasses from three different slaughter plants contained mean counts of 104 to 108 enterococci per 100 cm2 of carcass surface throughout processing

Enterococci

563

(Knudtson and Hartman, 1993). Devriese et al. (1995) showed that E. faecium, E. faecalis and to a lesser extent E. hirae and E. durans occurred in meat and prepared meat products. In a study on poultry, E. faecalis predominated among the Gram-positive cocci isolated from chicken samples collected at abattoirs (Turtura and Lorenzelli, 1994). Capita et al. (2001) found enterococci to occur at a mean count of log 2.72 CFU/g of chicken carcasses from five retail outlets in Spain. In a study on modified-atmosphere-packaged, marinated broiler legs produced in Finland Björkroth et al. (2005) showed that enterococci dominated in the fresh product but were replaced by the spoilage LAB including carnobacteria and Lactobacillus sakei/curvatus (Björkroth et al., 2005). Interestingly, some of the isolates were identified as a novel species E. hermanniensis (Koort et al., 2004). Enterococci were also consistently isolated from beef, poultry or pig carcasses or fresh meat cuts in studies of antibiotic resistance of enterococci (Klein et al., 1998; van den Braak et al., 1998; Davies and Roberts, 1999; Robredo et al., 2000; Borgen et al., 2001; Aarestrup et al., 2002; van den Bogaard et al., 2002; Mac et al., 2002; Hayes et al., 2003; Garnier et al., 2004; Huys et al., 2004; Rizzotti et al., 2005; Wilcks et al., 2005). Enterococci may not only contaminate raw meats, but they can also be associated with processed meats. Cooking of processed meats may confer a selective advantage on enterococci as these bacteria are known to be among the most thermotolerant of the non-sporulating bacteria (Sanz Perez et al., 1982; Magnus et al., 1988). After surviving the heat-processing step, both E. faecalis and E. faecium have been implicated in spoilage of cured meat products such as canned hams and chub-packed luncheon meats (Bell and Gill, 1982; Houben, 1982; Bell and Delacey, 1984; Magnus et al., 1986). Enterococci are also isolated from certain types of fermented sausages, for example sausages known as ‘fuet’, ‘chorizo’ (Casaus et al., 1997; Cintas et al., 1997; Herranz et al., 1999; Martin et al., 2005) and ‘espetec’ (Aymerich et al., 1996) produced in Spain, in Italian sausages (Cocolin et al., 2001) or sausages such as Salami and ‘Landjäger’ produced in many European countries. Salami and Landjäger were shown to contain enterococci at numbers ranging from 102 to 105 CFU/g (Teuber et al., 1996). Because of their tolerance to sodium chloride and nitrite, enterococci can survive and even multiply in the fermenting sausages (Giraffa, 2002; Martin et al., 2005). Some enterococcal strains have the ability to produce enterocins harbouring antimicrobial activity against pathogens and spoilage microorganisms, and may thus be used as bioprotective agents. Such enterocin-producing enterococci, or their purified metabolites, may be applied as extra hurdles for preservation in sausage fermentation and in sliced vacuum-packed cooked meat products, thereby preventing the outgrowth of Listeria monocytogenes and/or slime-producing lactic acid bacteria (Hugas et al., 2003). Cheese Enterococci occur in many traditional European cheeses manufactured in

564 Emerging foodborne pathogens mostly Mediterranean countries from raw or pasteurised milk (Ordoñez et al., 1978; Coppola et al., 1988; Del Pozo et al., 1988; Litopoulou-Tzanetaki, 1990; Tzanetakis and Litopoulou-Tzanetaki, 1992; Macedo et al., 1995; Tzanetakis et al., 1995; Centeno et al., 1996; Bouton et al., 1998; Menéndez et al., 2001; Prodromou et al., 2001; Caridi et al., 2003; Manolopoulou et al., 2003; Marino et al., 2003; Cosentino et al., 2004). The source of enterococci in milk and in such cheeses is thought to be the faeces of dairy cows, contaminated water or milking equipment and bulk storage tanks (Gelsomino et al., 2001b) as well as natural milk starters (Giraffa, 2002). The isolation of enterococci from natural milk starters can be explained by their heat resistance; natural milk starters are made by pasteurising milk at 42–44 °C for 12 to 15 h, thus promoting the thermotolerant bacteria present, which include S. thermophilus strains and Enterococcus spp. (Giraffa, 2002). Strains belonging to the species E. faecalis, E. faecium and E. durans are most often isolated from such cheeses (Table 20.1) and these may contribute to ripening and product flavour (Tzanetakis and Litopoulou-Tzanetaki, 1992; Macedo et al., 1995; Freitas et al., 1995; Centeno et al., 1996; Andrighetto et al., 2001; Menéndez et al., 2001; Delgado et al., 2002; Caridi et al., 2003). Numbers of enterococci in cheese curds range from 104 to 106 CFU/g, and in the fully ripened cheeses from 105 to 107 CFU/g (Table 20.1). Enterococci can grow in the restrictive environment of high salt content and low pH of the cheese (Ordoñez et al., 1978; Litopoulou-Tzanetaki, 1990; Wessels et al., 1990; Freitas et al., 1995) and contribute to the ripening and aroma development of these products due to their proteolytic and esterolytic activities, as well as the production of diacetyl (Jensen et al., 1975; Ordoñez et al., 1978; Trovatelli and Schiesser, 1987; DeFernando et al., 1992; Tsakalidou et al., 1993; Centeno et al., 1996, 1999; Sarantinopoulous et al., 2002). Because of their role in ripening and flavour development in cheeses, enterococci with desirable technological and metabolic traits have been proposed as part of defined starter cultures or as adjunct starter cultures for different European cheeses (Litopoulou-Tzanetaki et al., 1993; Villani and Coppola, 1994; Centeno et al., 1999). Enterococci produce lactic acid as end product of metabolism and this acidifying activity can be considered a technological trait in cheese fermentations. However, the enterococci generally exhibit only low acidifying ability (Aymerich et al., 2000; Andrighetto et al., 2001; Sarantinopoulous et al., 2001; Giraffa, 2003). Proteolytic activity of enterococci for break-down of milk casein is quite important for cheese ripening. However, conflicting reports on proteolytic activity of enterococci suggest a marked strain-to-strain variation of this phenotypic trait (Arizcun et al., 1997; Durlu-Ozkaya et al., 2001; Delgado et al., 2002; Giraffa, 2003). Both esterolytic and lipolytic activity of enterococci are also considered important in the context of cheese ripening and development of flavour and texture. Esterases are arbitrarily defined as enzymes that hydrolyse substrates in solution, while lipases hydrolyse substrates in emulsion (Giraffa, 2003). Esterases have been linked to the flavour development and cheese texture by

Table 20.1

Numbers and predominant isolates of Enterococcus spp. in cheeses from Mediterranean countries

Cheese

Country of origin

Milk source

Enterococci in curd (log CFU/g)

Enterococci at end of ripening (CFU/g)

Predominant bacteria in end product (% of isolates)

Reference

White-brined cheese

Greece

Raw goat milk or mixed goat and ewes’ milk

4.0

6.7

LitopoulouTzanetaki and Tzanetakis (1992)

Kefalotyri cheese

Greece

Ewes’ milk, cow milk or mixed ewes’ and goat milk

4.9

5.8

Teleme cheese

Greece

Pasteurised ewes’ milk

n.r.a

n.r.

Orinotyri cheese La Serena ewes’ milk cheese Manchego cheese Cebreiro

Greece

Raw ewes’ milk

n.r.

6.8

L. plantarum (47%) b E. faecium (12%) L. paracasei subsp. paracasei (10%) E. faecalis (9%) E. faecium (35.6%) L. plantarum (18.4%) L. casei subsp. casei (15.8%) E. durans (9.2%) pediococci (9.2 %) Lactobacilli Leuconostocs Enterococci lactococci, enterococci, leuconostocs

Spain

Raw ewes’ milk

6.2

7.2

Spain

Raw ewes’ milk

n.r.

n.r.

Spain

Raw cow milk

n.r.

6.5

Lactobacilli Leuconostocs Enterococci Enterococci E. faecalis (30.1%) E. faecalis (var liquifaciens) (11.9%) Lact. lactis (19.0%) W. (Leuc.) paramesenteroides (7.9%) Leuc. mesenteroides subsp. mesenteroides (6.3%) E. faecium (4.8%)

LitopoulouTzanetaki (1990)

Tzanetakis and LitopoulouTzanetaki (1992) Prodromou et al. (2001) Del Pozo et al. (1988) Ordoñez et al. (1978) Centeno et al. (1996)

Table 20.1 Continued Cheese

Country of origin

Milk source

Enterococci in curd (log CFU/g)

Enterococci at end of ripening (CFU/g)

Predominant bacteria in end product (% of isolates)

Reference

San Símon cheese Tetilla cheese

Spain

Raw cow milk

5–6

6–7

Spain

Raw cow milk

n.r.

7.3

García et al. (2002) Menéndez et al. (2001)

Caprino d’ Aspromonte Montasio

Italy

4–6

5–7

4

ca. 5–7

Serra cheese

Portugal

Raw or heated goat milk Raw or heated cow milk Raw ewes’ milk

n.r.

n.r.

Picante da Beira Baixa cheese

Portugal

n.r.

n.r.

E. faecalis, E. faecium, E. durans, Staph. spp. Micrococcus spp E. faecalis, L. casei subsp. casei, Leuconostoc mesenteroides subsp. mesenteroides enterococci, lactobacilli, mesophilic and thermophilic cocci S. thermophilus, E. durans, E. faecalis, E. faecium Leuc. lactis, Lact. lactis, Leuc. mesenteroides subsp. mesenteroides/ dextranicum E. faecium E. faecium, E. faecalis, E. durans, L. plantarum, L. paracasei

a

Italy

Mixture of raw goat and ewes’ milk

Caridi et al. (2003) Marino et al. (2003) Macedo et al. (1995) Freitas et al. (1995)

n.r. = not reported; b : L. = Lactobacillus; E. = Enterococcus; Lact. = Lactococcus; Leuc. = Leuconostc; W. = Weissella, Staph. = Staphylococcus

Enterococci

567

lipolysis of milk fat and subsequent conversion of the free fatty acids produced to methylketones and thioesters, which have importance as cheese flavour compounds. Lipolysis, on the other hand, is not directly involved in cheese rheology but partial glycerides are tensio-active and influence molecular organisation, thus having an effect on cheese texture (Giraffa, 2003). Hydrolysis of triglycerides by enterococci has been reported, with E. faecalis strains appearing to be most active (Macedo and Malcata, 1997; Sarantinopoulos et al., 2001; Durlu-Ozkaya et al., 2001), while the esterolytic system of enterococci appears to be complex and more efficient than their lipolytic system (Giraffa, 2003). Because enterococci are not good acidifiers of milk and meats, and their proteolytic and esterolytic properties may not be high, it would probably be better to use enterococci in food fermentations as adjunct starter cultures in combination with established starter strains rather than using these bacteria as defined starter cultures by themselves. Nevertheless, the effect of the technological properties of the enterococci is not negligible and should not be underestimated. For example, Sarantinopoulos et al. (2002) studied the technological properties of two strains of E. faecium as adjunct starter cultures, either single or combined, on the microbiological, physicochemical and sensory characteristics of Feta cheese in a well-defined study. It was shown that the presence of the enterococcal starter strains positively affected the growth of non-starter LAB, increased the proteolytic index and free amino group concentration, enhanced the water-soluble nitrogen fractions and positively affected taste, aroma, colour, structure, and the overall sensory profile of the cheese (Sarantinopoulos et al., 2002). Clearly, the results of this study supported previous suggestions that enterococci indeed positively influence cheese fermentations. Fermented vegetables Enterococci occur in a variety of fermented vegetables, but it is often not clear whether they originate from the plant material itself or as environmental contaminants. Enterococci have been isolated also from green olive fermentations (Fernández Díez, 1983; Asehraou et al., 1992, van den Berg et al., 1993; Floriano et al., 1998; Lavermicocca et al., 1998; de Castro et al., 2002; Randazzo et al., 2004), in which E. faecalis is a frequent contaminant, and they frequently occur in retail fermented olives. De Castro et al. (2002) suggested that lactic acid bacteria growing at the beginning stages of the olive fermentation are important for improving the hygiene of the product. However, not all of the lactic acid bacteria are suited to grow at the relative high pH conditions resulting from alkaline treatment of the olive grapes to hydrolyse the bitter glucoside oleuropein. Because of their tolerance to the high pH values and salt concentration used in the olive brine, the enterococci appear to be well suited for growth at these conditions (de Castro et al., 2002). It has also been suggested that enterococci can use the antimicrobial compound oleuropein in olive grapes as a growth substrate as a result of

568 Emerging foodborne pathogens beta-glucosidase activity (Garrido-Fernandez and Vaughn, 1978; Randazzo et al., 2004), thus lowering the toxicity of the fermentation medium for growth of other LAB. In addition, the enterococci, especially E. faecalis and E. faecium strains, have also been associated with Asian and African fermented sorghum foods (Mulyowidarso et al., 1990; Mohammed et al., 1991; Hamad et al., 1997; Moreno et al., 2002; Onda et al., 2002). However, it is not always clear whether they play a constructive or detrimental role in the fermentation of such vegetable products, if they play any role at all. Moreno et al. (2002) isolated two bacteriocinogenic E. faecium strains from spoiled tempeh but did not contribute a major role of these bacteria to the spoilage process. In our own studies on traditional fermented African foods, E. faecium strains were also associated with the fermentation of products such as ‘Hussuwa’ made from sorghum in the Sudan (Yousif et al., 2005). They constituted ca. 10% of bacteria isolated from various stages of the fermentation and thus did not appear to play a significant role in the fermentation. Nevertheless, some of these E. faecium strains displayed some interesting technological properties such as degradation of indigestible sugars raffinose and stachyose, as well as bacteriocin activity, and thus may be interesting for use as adjunct cultures in the fermentation (Yousif et al., 2005). We also isolated enterococci from ‘Okpehe’ made from locust beans in Nigeria. When a selected Enterococcus strain was used together with a B. subtilis starter strain in a model Okpehe fermentation, the resulting product developed an undesirable ‘cheese-like’ aroma, which scored badly in an organoleptic taste panel evaluation. This indicated clearly that the enterococci may not play a role in flavour development in the Okpehe fermentation (Oguntoyinbo et al., in press). In the fermentation of miso-paste, the salt tolerant enterococci (belonging to the E. faeciumgroup) often predominate in the early stages of the fermentation, before they are replaced by the even more salt tolerant Tetragenococcus halophilus strains (Onda et al., 2002). The presence of enterococci to start the fermentation is considered important for two reasons: firstly, these bacteria serve to lower the acidity by production of lactic acid in the beginning stage of the fermentation, and secondly they are believed to be involved in maintaining the bright colour of the miso (the so-called ‘Sae’ effect) (Yoshii, 1995; Onda et al., 2002). From the above examples it is apparent that enterococci occur in a great variety of foods, whether they belong to dairy, meat or plant foods. This means that inevitably, foods containing high numbers of enterococci (e.g., up to 107 CFU/g cheese) are probably consumed daily or at least weekly by the average consumer. The questions which arise from this fact bear direct relevance to the safety discussion which has been going on for the last few years when considering food enterococci: can these strains harbour virulence determinants or antibiotic resistances? What are the routes of transmission? Can enterococci from foods survive gastrointestinal passage and are they able to colonise the gastrointestinal tract? Can food strains which harbour

Enterococci

569

virulence factors or antibiotic resistance determinants cause human infection or can such determinants be transferred to commensal bacteria in the gut? Are enterococci foodborne pathogens and does the presence of enterococci in foods constitute a health risk? These are the question which a tremendous amount of research was devoted to in the last five years, and the results of such investigations will be discussed in the sections below.

20.3 Use of enterococci as probiotics A few Enterococcus spp. are being used as probiotics. E. faecium SF68 has been used to treat diarrhoea and it is considered as an alternative to antibiotic treatment (Lewenstein et al., 1979; Bellomo et al., 1980). Several placebo controlled, ‘double blind’ clinical studies have shown that treatment of enteritis with E. faecium SF68 was successful for both adults and children. It decreased the duration of diarrheal symptoms and the time for normalisation of patient’s stools (Bellomo et al., 1980; D’Apuzzo and Salzberg, 1982). Another probiotic for human use that contains enterococci is the Causido® culture that consists of two strains of S. thermophilus and one of E. faecium. This probiotic has been claimed to be hypocholesterolaemic in the short-term (Agerholm-Larsen et al., 2000), but long-term reduction of LDL-cholesterol levels was not demonstrated (Richelsen et al., 1996; Sessions et al., 1997); hence, the clinical relevance of this effect is uncertain (Lund et al., 2002). Two probiotic preparations containing enterococci together with other probiotic strains (i.e., either Enterococcus, L. acidophilus and Bifidobacterium or E. faecium and B. subtilis) were used in patients with liver cirrhosis to improve their symptoms of gastrointestinal dysfunction (Zhao et al., 2004). Both types of probiotics increased the Bifidobacterium count and reduced the levels of faecal pH and faecal and blood ammonia significantly, while the preparation containing the enterococci and B. subtilis bacteria also reduced the level of endotoxin in cirrhotic patients with endotoxemia (Zhao et al., 2004). The use of enterococci as probiotics remains a controversial issue, particularly because these Enterococcus probiotics are typically administered as pharmaceutical preparations and thus are ingested in high numbers. While the probiotic benefits of some strains are well established, the emergence of antibiotic-resistant strains of enterococci and the increased association of enterococci with human disease (see below), has raised considerable concern regarding their use as probiotics. The fear that antimicrobial resistance genes or genes encoding virulence factors can be transferred to probiotic strains in the gastrointestinal tract contributes to this controversy. Whether this concern is well founded will be discussed in the sections below.

570 Emerging foodborne pathogens

20.4 Infections caused by enterococci and epidemiology 20.4.1 Enterococcal infections Enterococci are typical opportunistic pathogens and may cause infections in patients that have severe underlying disease, that have received surgery or that are immunocompromised (Morrison et al., 1997). They are commonly associated with hospital-acquired infections and cause bacteraemia, endocarditis, urinary tract and other infections (Murray, 1990; Morrison et al., 1997). Enterococci are amongst the most prevalent organisms associated with nosocomial infections, accounting for approx. 12% of nosocomial infections in the USA (Linden and Miller, 1999). They are the third most common pathogen isolated from bloodstream infections (Jones et al., 1997) and the most frequently reported pathogen in surgical site infections in intensive care units (Richards et al., 2000). In 1999, enterococci were reported as the second most common nosocomial pathogen in the USA (Richards et al., 1999). They contribute significantly to patient mortality as well as to additional hospital stay (Landry et al., 1989; Koch et al., 2004). While E. faecalis was earlier noticed to predominate (more than 80%) among enterococci from human infections and E. faecium was associated with the remainder (Jett et al., 1994), a shift towards E. faecium strains as the causative agent in enterococcal bacteraemia occurred in the last few years, probably because of the emergence of vancomycin-resistant strains (Mundy et al., 2000). Other enterococcal species are rarely associated with human disease, but strains of E. gallinarum, E. hirae, E. casseliflavus, E. durans, E. avium and E. mundtii have been reported in association with infections such as bacteraemia, endocarditis, meningitis, brain abscess and endophthalmitis (Dargere et al., 2002; Choi et al., 2004; Mirzoyev et al., 2004; Pappas et al., 2004; Stephanovic et al., 2004; Villar et al., 2004; Corso et al., 2005; Higashide et al., 2005; Iaria et al., 2005; Mohanty et al., 2005). Bacteraemia is a common form of opportunistic enterococcal infection (Lewis and Zervos, 1990; Jones et al., 1997; Morrison et al., 1997). Compared with a steady reduction in community-acquired cases of enterococcal bacteraemia, nosocomial cases may have increased threefold and reach up to 77% of cases (Shlaes et al., 1981; Maki and Agger, 1988; Morrison et al., 1997; Weinstein et al., 1997). Risk factors associated with enterococcal bacteraemia include underlying disease, presence of urethral or intravascular catheters, surgery, major burns, multiple trauma or prior antibiotic therapy (Lewis and Zervos, 1990). Sources of enterococci causing bacteraemia without endocarditis are most commonly from the urinary tract, but the gastrointestinal and hepatobiliary tracts have also been implicated (Chenoweth and Schaberg, 1990; Lewis and Zervos, 1990; Morrison et al., 1997). Mortality from enterococcal bacteraemia is generally high, most probably because of the underlying complicating factors (Murray, 1990; Kaufhold and Ferrieri, 1993). Enterococci cause an estimated 5 to 15% of cases of bacterial endocarditis with E. faecalis more commonly involved than E. faecium (Murray, 1990; Morrison et al., 1997). The enterococci usually originate from the urinary

Enterococci

571

tract (Chenoweth and Schaberg, 1990; Lewis and Zervos, 1990; Aguirre and Collins, 1993) and underlying heart disease is often present, but it is not a pre-requisite for development of this infection (Chenoweth and Schaberg, 1990; Murray, 1990; Morrison et al., 1997). Endocarditis often occurs in patients that had preceding genitourinary instrumentation or urinary tract infections (UTI), abortion, or urinary tract instrumentation (Chenoweth and Schaberg, 1990; Murray, 1990; Moellering, 1992). Urinary tract infections are commonly caused by enterococci, especially in hospitalised patients. These infections occur especially in persons who had surgery, received antibiotics, had structural abnormalities, or had recurrent enterococcal infections (Chenoweth and Schaberg, 1990; Murray, 1990; Moellering, 1992). Infections of the central nervous system by enterococci are rare and are seen primary in neonates and persons who have undergone complicated neurological procedures (Moellering, 1992; Morrison et al., 1997). Enterococci causing neonatal infection are thought to originate from the vagina, because they are detected in the vaginal microflora in 25% of healthy women (Lewis and Zervos, 1990). E. faecium and E. faecalis have been implicated in outbreaks of neonatal central nervous system infections, although infections of older children and adults have also been reported (Murray, 1990; Tailor et al., 1993). Enterococci may also cause or contribute to abdominal and pelvic abscess formation and sepsis (Murray, 1990). They were reported as a cause of spontaneous peritonitis in cirrhotics and nephrotics, and may be associated with peritonitis in patients on peritoneal dialysis (Murray, 1990; Tyrrell et al., 2002). Dialysis catheters and prior use of antibiotics are predisposing factors for intra-abdominal infections by enterococci (Chenoweth and Schaberg, 1990; Low et al., 1994). 20.4.2 Virulence factors To cause infection, enterococci must have virulence factors which allow the infecting strains to colonise host tissue, invade host tissue and translocate through epithelial cells and evade the hosts immune response. Furthermore, such virulent strains must produce pathological changes either directly by toxin production or indirectly by inflammation (Johnson, 1994). In recent years, considerable progress has been made in determining virulence traits (Table 20.2) from clinical isolates, and each of these may be associated with one or more of the stages of infection mentioned above. Moreover, in the last few years many investigations have focused on the virulence characteristics of enterococci occurring in foods in an attempt to assess the risk of foodborne enterococci for human health. Interestingly, virulence factors which occur in medical Enterococcus isolates could also be found in environmental or food Enterococcus isolates (see below). Colonisation Enterococci are normal commensals occurring in the gastrointestinal tract and thus must be able to colonise this ecological niche. A number of virulence

572 Emerging foodborne pathogens Table 20.2 Virulence factors which may be present in some Enterococcus strains, and (suggested) association with stage of virulence Virulence determinant

(Suggested) association with stage of virulence

Aggregation substance (AS)

Adhesion to eukaryotic cells (adhesin)/ promotes colonisation Invasion of eukaryotic cells (invasin) Adhesion to extracellular matrix proteins (may promote translocation) Increases survival in immune cells (evasion of host immune response) Eukaryotic cell toxin Lyses immune cells (evasion of host immune response) Can hydrolyse various biological peptides, e.g. collagens and fibrin (role in translocation?) Can hydrolyse antibacterial peptides (evasion of host innate immune response) Adhesin, promotes colonisation Exhibits characteristics of MSCRAMMs – role in evasion of immune response? Adhesion to extracellular matrix proteins (may promote translocation) Exhibits MSCRAMM characteristics: role in evasion of immune response? Adhesin: role in endocarditis

Cytolysin (Cyl) Gelatinase (Gel)

Enterococcal surface protein (Espfs and Espfm) Adhesin to collagen of E. faecalis (Ace) or E. faecium (Acm) Endocarditis antigen from E. faecalis or E. faecium (EfaAfs and EfaAfm) Hyaluronidase Pheromones E. faecium secreted antigen (Sag) Superoxide and hydrogen peroxide Capsule

Degrades hyaluronic acid, a major extracellular matrix constituent: role in translocation? Cause inflammation, induce superoxide production Adhesion to extracellular matrix proteins May cause cell/DNA damage, improves colonisation Evasion of host immune response

factors have been identified which allow the enterococci to adhere to gastrointestinal cells, the extracellular matrix and thus facilitate colonisation or the formation of vegetations. Aggregation substance (AS) Aggregation substance (AS, Table 20.2) is a glycoprotein adhesin that is encoded on pheromone-responsive plasmids. Expression of the AS gene is induced by sex pheromones which are small (7 to 8 amino acids) hydrophobic peptides and which are excreted by plasmidless, recipient E. faecalis strains. Binding of the pheromones by the donor strain leads to expression of AS on the cell surface. AS leads to clumping of donor and recipient cells by binding to a complementary receptor termed ‘binding substance’ on the recipient cell surface. This clumping of cells leads to a highly efficient transfer of the pheromone plasmids on which the AS gene is encoded (Clewell, 1993; Dunny

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et al., 1995). AS was also shown to be involved in binding to eukaryotic cells. The molecule contains two RGD (Arg-Gly-Asp) amino acid motifs that promote E. faecalis adhesion to eukaryotic cells, such as pig renal tubular cells, via integrin receptors (Kreft et al., 1992). AS was further shown to bind to a variety of cells via such β2-type integrins including human macrophages and intestinal epithelial cells (Sartingen et al., 2000; Süßmuth et al., 2000). In addition, AS can also bind to extracellular matrix (ECM) proteins such as fibronectin, thrombospondin, vitronectin and collagen type I (Rozdzinski et al., 2001). Not only was AS shown to play a role in binding to intestinal cells, it was also found to augment internalisation of enterococci cells and to promote translocation and intracellular survival (Wells et al., 1990; Kreft et al., 1992; Olmsted et al., 1994; Rakita et al., 1999; Vanek et al., 1999; Sartingen et al., 2000; Süßmuth et al., 2000; Wells et al., 2000). AS is therefore considered an important multifunction virulence factor because it acts as an adhesin and invasin; in addition it is involved in translocation as well as evasion of the immune response by intracellular survival in immune cells (Table 20.2). To underline this, AS was associated with an increased mass in valvular vegetations in an animal pathogenicity model and thus enhanced the virulence of these bacteria in these studies (Chow et al., 1993; Schlievert et al., 1998). Enterococcus surface protein from E. faecalis (Espfs) and E. faecium (Espfm) The ‘enterococcal surface protein’ (Esp) produced by E. faecalis (Espfs) or E. faecium (Esp fm ) is an adhesin (Table 20.2) which appears to be chromosomally encoded in both species. The incidence of Espfs was shown to be enriched among clinical strains of E. faecalis, indicating a role in pathogenicity (Shankar et al., 1999), although this could not be confirmed by others (Waar et al., 2002). Eaton and Gasson (2002) found Espfm to be highly conserved in infection-derived isolates and environmental isolates, but absent in food and commensal isolates, which led them also to suggest a role in pathogenicity (Eaton and Gasson, 2002). Shankar et al. (2001) used an Esp +fs strain and an isogenic mutant in a mouse model of ascending urinary tract infection to show that Espfs contributed to colonisation and persistence of E. faecalis at this site. However, the Esp +fs strain did not influence histopathological changes in the animal model (Shankar et al., 2001). The presence of Espfs also increased cell hydrophobicity, adherence to abiotic surfaces and biofilm formation in vitro (Toledo-Arana et al., 2001). Espfs was suggested to promote colonisation of host tissue by direct ligandbinding activity to the extracellular matrix in the human host because of the similarity of Esp to microbial surface components recognising adhesive matrix molecules (MSCRAMMs) (Toledo-Arana et al., 2001). However, in an animal model with clindamycin-treated mice, Pultz et al. (2005) showed that Espfs facilitated neither colonisation nor translocation of E. faecalis. Esp was also suggested to have a function in evasion of the host’s immune response (Table 20.2), based on the observation that the overall structure of both Espfs

574 Emerging foodborne pathogens and Espfm are comparable to that of MSCRAMMs for which such a ECM protein-binding role has been proposed (Rich et al., 1999; Shankar et al., 1999; Eaton and Gasson, 2002). Adhesin to collagen from E. faecalis (Ace) and from E. faecium (Acm) Ace and Acm are adhesins (Table 20.2) that also show structural similarity to MSCRAMMs of other Gram-positive bacteria, particularly to the collagen binding protein Cna of S. aureus (Rich et al., 1999; Nallapareddy et al., 2003). The structural organisation of all Ace, Acm and Cna are similar in that they contain an N-terminal signal sequence followed by the collagen-binding A domain, a B region that consists of repeat units, a cell wall domain with a characteristic LPKTS motif which is a potential target for sortase, a stretch of hydrophobic residues which are thought to stretch the membrane followed by a short cytoplasmic and charged tail (Nallapareddy et al., 2003). Because these proteins also contain repeat units and the number of repeats can be varied, these protein may also be involved in evasion of the immune response (Table 20.2) by mechanisms similar to those suggested by Shankar et al. (1999) for Esp. Ace not only binds to collagen (types I and IV) but also to laminin (Nallapareddy et al., 2000a,b). Nallapareddy et al. (2000b) showed that Ace was expressed by enterococci during human infections. Ninety percent of human sera collected from patients with E. faecalis endocarditis reacted with anti-Ace antibodies. Thus Ace may play an important role in pathogenesis of enterococci particularly during translocation, or when the intestinal epidermal layer is damaged and the underlying extracellular matrix proteins are exposed. As Enterococcus cells would become exposed to immune cells at this site, a mechanism for evading the immune system supplied by the same molecule involved in adherence may be an elegant solution for enterococci to increase their chances of survival. Acm, the collagen binding protein from E. faecium, was shown to bind collagen types I and IV by Nallapareddy et al. (2003) who also showed that particularly the clinical strains of E. faecium exhibited binding to collagen type I, while strains from the faeces of healthy human volunteers did not bind collagen I. The differences between binding capacity of clinical and community isolate strains was statistically significant, indicating that binding to collagen is a virulence factor. Interestingly, all community E. faecium isolates also contained the gene for Acm; however, this gene was in nonfunctional form as a result of nucleotide deletions or insertion of IS6770-like insertion sequence resulting in frame-shift mutations. While Ace and Acm share some (47%) amino acid sequence similarity, Acm has a far greater similarity at the primary sequence level (62%) to the collagen binding protein (Cna) of S. aureus (Nallapareddy et al., 2003). While the similarity of Acm to Ace appears to be confined to the A domain, Acm has similarity to both the A and B domains of Cna (Nallapareddy et al., 2003).

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Enterococcus endocarditis antigen from E. faecalis (EfaAfs) or E. faecium (EfaAfm) Production of the adhesin-like E. faecalis and E. faecium endocarditis antigens (EfaAfs and EfaAfm, respectively) (Table 20.2) are considered to be potential virulence determinants, and expression of the EfaA was previously shown to be induced by growth of E. faecalis in serum (Lowe et al., 1995). The EfaAfs antigen shows high homology to adhesins such as FimA, SsaB ScaA and PsaA from streptococci. EfaAfs was suggested to play a role in adhesion in endocarditis (Lowe et al., 1995). However, so far only the efaAfs gene was shown to influence pathogenicity in animal models (Singh et al., 1998). The genetic determinant for production of EfaA has been sequenced and the efa operon consists of three genes (efaC, B, A) which have homology to ABCtype metal ion transport systems (Low et al., 2003). The first gene efaC encodes an ATP binding protein, the second (efaB) a hydrophobic transmembrane protein while the third (efaA) probably functions as a solute binding protein receptor for the ABC transporter complex. Low et al. (2003) suggested that EfaCBA is a manganese-regulated operon that functions as a high affinity manganese permease in E. faecalis. It plays a role in the infection of human tissues, where the Mn2+ availability may be as low as 20 nM (Low et al., 2003). Secreted virulence factors Sex pheromones Sex pheromones can be considered as virulence determinants (Table 20.2). They are cleavage products of 21 to 22 amino acid signal peptides that are associated with surface lipoproteins of unknown function (Clewell et al., 2000). These, as well as their surface exclusion proteins, are involved in causing pathological changes such as acute inflammation (Johnson, 1994). They are chemotactic for human and rat PMNs in vitro, and induce superoxide production and secretion of lysosomal enzymes (Ember and Hugli, 1989; Sannomiya et al., 1990; Johnson, 1994). Enterococcus faecium secreted antigen (Sag) The E. faecium secreted Antigen (Sag, Table 20.2) is a 53 kDA protein which is composed of three domains being a putative coiled-coil N-terminal domain, a central domain containing direct repeats and a C-terminal domain with similarity to the P45 and P60 proteins of L. monocytogenes, the latter of which is involved in L. monocytogenes virulence (Teng et al., 2003). This extracellular protein was considered essential for growth and is presumed to play a role in cell wall metabolism. Furthermore, Sag was shown to be capable of broad-spectrum binding to extracellular matrix proteins and was antigenic during infection (Teng et al., 2003). Superoxide and hydrogen peroxide Moy et al. (2004) used E. faecium mediated killing of the nematode worm

576 Emerging foodborne pathogens Caenorhabditis elegans as an indicator of toxicity and could show that E. faecium produced hydrogen peroxide at levels sufficient to induce cellular damage. Transposon mutagenesis of the E. faecium strain studied showed that insertion mutants with altered C. elegans killing activity were altered in hydrogen peroxide production. Thus, mutation of an NADH oxidase encoding gene eliminated almost all NADH oxidase activity which resulted in reduced hydrogen peroxide production and decreased killing of C. elegans. Therefore, Moy et al. (2004) suggested that H2O2 may serve as a virulence determinant which may damage nearby host cells, although they admitted that the role of hydrogen peroxide in the pathogenesis of human disease is unclear, especially since the levels of H2O2 may be difficult to determine. Nevertheless, Huycke et al. (2002) showed that E. faecalis producing superoxide and hydrogen peroxide could damage eukaryotic cell DNA using both Chinese hamster ovary and HT-29 intestinal epithelial cells in the comet assay. In contrast, a transposon-inactivated mutant with attenuated extracellular superoxide production did not produce the same DNA-damaging effect. H2O2 arising from superoxide was identified as the actual genotoxin (Huycke et al., 2002). Furthermore, these authors showed in a rat model of intestinal colonisation that E. faecalis resulted in a significantly higher stool H2O2 concentration compared with rats colonised with a mutant strain which had decreased superoxide production. Furthermore, using the comet assay they showed that luminal cells from the colon of rats colonised with the superoxide producing E. faecalis showed significantly increased DNA damage compared with control rats colonised with the mutant (Huycke et al., 2002). Cytolysin The β-haemolysin/bacteriocin or cytolysin is a cellular toxin that enhances virulence in animal models (Ike et al., 1984; Jett et al., 1992, 1994; Chow et al., 1993; Gilmore et al., 1994) and is associated with acute mortality in humans (Huycke et al., 1991). The cytolysin gene is encoded either on pheromone-responsive plasmids or within a pathogenicity island. In addition to toxin activity, the extracellular and activated form of cytolysin (CylLS″) also induces high level expression of the cytolysin structural genes by a quorum-sensing mechanism (Haas et al., 2002; Shankar et al., 2002). In Japan, it has been shown that 60% of clinical strains involved in parenteral infection had a haemolytic phenotype, compared with only 17% of isolates from the faeces of healthy individuals (Ike et al., 1987). Similar trends were observed in a study of E. faecalis bloodstream isolates in the United States (Huycke et al., 1991). However, in a European study, only 16% E. faecalis strains isolated from blood exhibited haemolytic activity (Elsner et al., 2000). Cytolysin production can be considered as a bacterial strategy to evade the host immune response by destroying cells of the immune system, as Miyazaki et al. (1993) showed that haemolytic culture supernatants of E. faecalis lysed mouse PMNs and macrophages. Production of cytolysin appears to be a major risk factor associated with pathogenic enterococci as Huycke et al.

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(1991) determined a fivefold increased risk of death of patients within three weeks of bacteraemia caused by β-haemolytic enterococci, compared with bacteraemia caused by non-β-haemolytic strains (Huycke et al., 1991). Gelatinase Gelatinase is an extracellular Zn-metalloprotease (EC 3.4.24.30) that acts on a variety of substrates such as insulin-β chain, collagenous material in tissues, the vasoconstrictor edothelin-1, as well as sex-pheromones and their inhibitor peptides (Waters et al., 2003). Production of gelatinase increased pathogenicity in an animal model (Singh et al., 1998). Kühnen et al. (1988) reported that protease-producing E. faecalis were common (63.7%) among enterococci isolated from intensive care units in Germany, and Coque et al. (1995) showed that 54% of clinical enterococci isolates from patients with endocarditis and other nosocomial infections produced protease. The gene for gelatinase (gelE) is located in an operon together with a gene (sprE) encoding a serine protease (Qin et al., 2000). Mutants containing both defective gelE and sprE genes led to delayed time to death in a mouse peritonitis model (Singh et al., 1998; Qin et al. 2000), suggesting that both GelE and SprE are important in the infection in this animal model. However, the authors could not determine whether gelE independently influences the outcome of this enterococcal infection (Qin et al., 2000). Furthermore, while the presence of gelE and fsr (the regulatory genes for gelatinase and serine protease, see below) in entercocci may have an effect on the severity of disease in animal models, Roberts et al. (2004) compared the incidence of these genes among E. faecalis isolates from healthy individuals and from human infections, and found that neither fsr nor gelE was required for E. faecalis to cause infection. GelE was shown to cleave fibrin, which was suggested to have important implications in virulence of E. faecalis as the secreted protease can damage host tissue and thus allow bacterial migration and spread (Table 20.2). Waters et al. (2003) suggested that enterococci in blood infections and vegetations formed during endocarditis were likely to be coated with polymerised fibrin. Expression of GelE would lead to degradation of this fibrin layer surrounding the bacteria and allow further dissemination of the organism. In addition to its role in virulence, GelE was also shown to effect a variety of important housekeeping functions. For example, GelE clears the bacterial cell surface of misfolded proteins and is also responsible for activation of an autolysin. This muramidase-1 autolysin functions to reduce chain length (Waters et al., 2003). GelE also degrades sex-pheromones and their inhibitors. Waters et al. (2003) postulated that overall GelE plays a crucial role for dissemination of the organism in high cell-density environment. Accordingly, not only would degradation of fibrin aid in dissemination, but also reduction of chain length as a result of autolysin activation. Furthermore, once enterococcal growth reaches high densities, the degradation of sex-pheromones decreases aggregation of bacteria, which also increases the potential for dissemination (Waters et al., 2003).

578 Emerging foodborne pathogens The supernatant from a gelatinase expressing E. faecalis strain was also shown to inactivate the antibacterial peptide LL-37 (Schmidtchen et al., 2002). The peptide LL-37 is part of the innate immune system and has been isolated from epithelial cells, neutrophils and subpopulations of lymphocytes and monocytes. Peptide LL-37 belongs to the family of antimicrobial peptides termed cathelicidins and is activated when cathelicidin hCAP-18 is processed by proteinase 3 (Schmidtchen et al., 2002). Degradation of antimicrobial peptides which are part of the innate immune system thus is a further GelEassociated enterococcal virulence factor (Table 20.2). Hyaluronidase Enterococci may produce hyaluronidase, an enzyme that degrades hyaluronic acid which is a major component of the extracellular matrix (Table 20.2). Because production of this enzyme was linked to pathogenesis of other microorganisms, it was suggested that it may also play a role in enterococcal pathogenesis. However, there is no direct evidence for the role of hyaluronidase in disease caused by enterococci (Jett et al., 1994; Rice et al., 2003). Recently, the gene sequence for the hyaluronidase gene hylEfm from an E. faecium strain was determined (Rice et al., 2003). This gene consisted of 1659 bp which encodes a putative protein of 533 amino acids which exhibits 42% identity and 60% similarity to a hyaluronidase from S. pyogenes (Rice et al., 2003). Rice et al. (2003) screened a large number of E. faecium strains for the incidence of hylEfm and espfm genes. These strains were from stool or non-stool origin isolated from both hospitalised and community based persons. Isolates from animals, waste water and probiotic strains were also investigated. They showed that the presence of espfm was roughly twice that of hylEfm and both genotypes were found primarily in vancomycin-resistant E. faecium isolates from non-stool cultures obtained from patients hospitalised in the United States. Their data suggested that specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections (Rice et al., 2003). Capsule Koch et al. (2004) reported that about 57% of the pathogenic enterococci strains investigated possess a capsule. Huebner et al. (1999) purified a capsular polysaccharide and determined that it consisted of a repeat structure of kojibiose linked 1,2 to glycerolphosphate. By raising antibodies to this capsular polysaccharide, they used immunogold labelling to show the presence of the capsule surrounding enterococci in electron microscopic studies (Huebner et al., 1999). Hancock and Gilmore (2002) studied a different capsular polysaccharide from E. faecalis, of which the overall composition of the polymer showed some relation to the carbohydrate purified by Huebner et al. (1999). However, the capsular carbohydrate of Huebner et al. (1999) contained glucose, glycerol and phosphate in a 2:1:2 ratio, and that of Hancock and Gilmore (2002) contained glucose, galactose, glycerol and phosphate in

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a 4:1:1:2 ratio. The nature of the linkages and the structure of the capsular polysaccharide were not determined by Hancock and Gilmore (2002). Using the capsular polysaccharide producing strain and an isogenic mutant in a murine cutaneous infection model, Hancock and Gilmore (2002) were also able to show that the mutant was more readily cleared from a resulting abscess, as measured by reduction in viable microorganisms from the abdominal lymph nodes that drain this site. This clearly indicated that the production of a capsule does offer some protection to the hosts defence mechanisms (Table 20.2) and that the production of a capsule by some enterococcal strains may play an important role in evasion of the immune response. 20.4.3 Regulation of Enterococcus virulence gene expression Production of aggregation substance (AS) is a tightly regulated phenotype, because autoinduction by a plasmid-bearing donor strain must be prevented. To counteract autoinducing activity, the plasmid-bearing donor cell also excretes a competitive inhibitor, which prevents self induction and which also provides the threshold that allows recipients in the immediate environment to overcome their inhibitory activity with their secreted pheromone (Hirt et al., 2002). For Asc10, the AS of the sex-pheromone plasmid pCF10, the inhibitor iCF10 is secreted at an 80-fold excess to the pheromone cCF10 (Hirt et al., 2002). Induction by cCF10 contained in the cell wall is prevented by cell membrane associated protein PrgY. Induction occurs if neighbouring cells tip the balance in favour of cCF10, which is bound in the cell wall by the pheromonebinding protein PrgZ and consequently imported into the cytoplasm by an Opp (oligopeptide permease) system. The pheromone then interacts with regulatory protein PrgX to allow AS expression. PrgX also controls the transcription of the prgQ promoter which allows production of iCF10 (Hirt et al., 2002). Hirt et al. (2002) showed that the AS of pCF10 is actually induced in vivo, and could increase pathogenicity, as measured by size of aortic valve vegetation in a rabbit endocarditis model. In addition, they showed that the expression of AS conferred a survival advantage to cells harbouring the plasmid and led to a highly efficient transfer of plasmid. The involvement of the pheromonesensing system for in AS expression in plasma was confirmed by the absence of AS induction in a mutant lacking the pheromone-sensing protein prgZ. An interaction of plasma components with the inhibitor peptide iCF10 was proposed as affecting the mating behaviour (Hirt et al., 2002). Production of cytolysin, as mentioned before, is also a regulated phenotype. Regulation is based on autoinduction and a two-component regulatory system that responds to quorum-sensing (Haas et al., 2002). The genes necessary for cytolysin production include cylLL, cylLS (encode structural cytolysin subunits), cylM (encodes protein for intracellular modification of cytolysin), cylB (encodes ABC transporter protein), cylA (encodes protein for extracellular cytolysin activation) and cylI (encodes immunity protein) which are arranged

580 Emerging foodborne pathogens in a collinear fashion. Upstream of these biosynthesis and immunity genes on the opposite DNA strand are two ORFs (cylR2 and cylR1) which encode regulatory proteins, consisting of a non-globular, α-helical protein with a helix-turn-helix DNA-binding motif (CylR2) and an α-helical protein with three predicted transmembrane domains (CylR1). Together these were shown to repress the cytolysin operon. The inducer for expression of cytolysin was shown to be the smaller, active cytolysin subunit CylLS’’ as mentioned above. Unlike other well-known quorum-sensing systems, this two-component regulatory system did not consist of a protein histidine kinase and a response regulator, rather it depends on a small helix-turn-helix DNA-binding protein and a transmembrane protein of unknown function (Haas et al., 2002). Interestingly, Coburn et al. (2004) showed that E. faecalis can ‘sense’ target cells and in response express cytolysin. Thus, in the absence of target cells the CylLL’’ subunit of the cytolysin toxin forms a complex with the inducer CylLS’’ and blocks it from autoinducing the operon. When target cells are present, however, CylLL’’ binds preferentially to the target, allowing free CylLS’’ to accumulate above the induction threshold. Therefore, enterococci use CylLL’’ to actively probe the environment for target cells, and when these are detected, allows the bacterium to express high levels of cytolysin in response (Coburn et al., 2004). The Enterococcus faecalis endocarditis antigen EfaAfs, as mentioned above, is regulated by Mn2+. The efaCBA operon encodes a putative ABC transporter (Efa permease) of which the EfaA component forms the endocarditis antigen. Transcription of the efaCBA and EfaA production is repressed by Mn2+ by a Mn2+-responsive transcriptional regulator EfaR, which shares 27% identity with the Corynebacterium diphtheria diphtheria toxin repressor DtxR (Low et al., 2003). Low et al. (2003) suggested when Mn2+ is abundant, intracellular levels rise, resulting in EfaR-Mn2+ complexes that bind the efaCBA promoter, inhibiting transcription and hence reducing Mn2+ uptake. However, if bacteria encounter host tissues or human serum where Mn2+ availability is low, the EfaR apoprotein cannot bind the efaC promoter, de-repressing efaCBA expression and hence increasing Efa permease levels and Mn2+ scavenging. This may increase the survival of enterococci in the human environment and thus contribute to virulence. Production of gelatinase is another regulated phenotype. Upstream of the E. faecalis gelE and sprE genes, there are three genes designated fsr (for E. faecalis regulator, see above) that regulate the expression of gelE and sprE. These genes have homology with the Staphylococcus aureus agr genes. In S. aureus, the agr/hld locus contains five genes that encode a quorum-sensing system that regulates the expression of virulence factors (Recsei et al., 1985; Novick et al., 1993; 1995; Qin et al., 2000). The Agr regulatory system upregulates the expression of secreted proteins such as α-toxin, β-toxin, δtoxin, enterotoxin B, toxic shock syndrome toxin 1 and serine protease, and down-regulates surface proteins such as protein A, coagulase, and fibronectinbinding protein (Recsei et al., 1985; Qin et al., 2000). In this system, agrA

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and agrC encode a response regulator and a sensor transducer, respectively, while agrD encodes a pheromone peptide that acts as an autoinducer (Qin et al., 2000). Qin et al. (2000) demonstrated that for the Fsr system, a cyclic peptide termed gelatinase biosynthesis-activated pheromone (GBAP) is the autoinducer (Nakayama et al., 2001a,b). The amino acid sequence of this peptide corresponds to the C-terminal part of a 242 amino acid protein encoded by fsrB (Nakayama et al., 2001a). The FsrA protein has 38% similarity to the AgrA protein that encodes the response regulator in the S. aureus Agr system, while FsrC has 36% similarity to the ArgC protein that is the sensor transducer of the Agr system (Qin et al., 2000). Homology of the Fsr system of E. faecalis to the Agr system of S. aureus, which plays such an important role in global regulation of S. aureus virulence, leads to the question whether the Fsr system plays a similar role in virulence regulation. So far, the Fsr system is only known to regulate two genes in E. faecalis, the gelatinase gene, gelE, and the serine protease gene, sprE. It was demonstrated that an fsrB deletion mutant attenuated the virulence in Caennorhabditis elegans and a mouse peritonitis model, as well as a rabbit endophthalmitis model, indicating the importance of this gene in virulence (Mylonakis et al., 2002; Sifri et al., 2002). In a rabbit model of endophthalmitis, it was further shown that virulence as result a mutation in the fsr regulator was more attenuated then the attenuation of virulence obtained when either the gelatinase or serine protease genes were inactivated, indicating pleitrophic effects on other traits contributing to pathogenesis of enterococcal infection (Engelbert et al., 2004). There is conflicting evidence on the association of fsr genes with enterococci isolated from infection. In one study (Pillai et al., 2002), the fsr locus was shown to be present in all E. faecalis isolates from cases of endocarditis, whereas only 53% of stool isolates possessed these genes. In contrast, another study found the incidences of neither the fsr genes nor the gelatinase production to be more common in disease associated Enterococcus isolates than in isolates colonising healthy individuals (Roberts et al., 2004). Teng et al. (2002) studied virulence of enterococci by disrupting twocomponent regulatory systems in Enterococcus faecalis. Such two-component regulatory systems, as mentioned above for gelatinase regulation, consist of a protein histidine kinase and a response regulator protein pair. Using the genome sequence information of E. faecalis V583 obtained from The Institute of Genomic Research (TIGR), they identified eleven homologues to the PhoP-PhoS global two-component regulatory system of Bacillus subtilis (Teng et al., 2002). Seven of these pairs were disrupted in E. faecalis strain OG1RF and one mutant, disrupted in the etaR gene of the gene pair designated etaRS, showed a delayed killing and a higher lethal dose in a mouse peritonitis model. In addition, they showed that the mutant was more sensitive to low pH and high temperature than the wild-type strain, indicating that etaRS may regulate different operon(s) involved in virulence and stress response (Teng et al., 2002).

582 Emerging foodborne pathogens Oxidative stress is encountered by bacteria in many environments, but especially during the infection process as a result of the immune response. Under such conditions, many genes encoding antioxidant enzymes are induced in order to protect the microorganism against reactive oxygen species. Genes involved in the oxidative stress response of E. faecalis include ahpCF (alkyl hydroperoxide reductase), npr (NADH peroxidase), sod (superoxide dismutase and katA (catalase) genes (Verneuil et al., 2004). A mutation in the hypR gene sensitised E. faecalis to H2O2 treatment and greatly affected survival in murine peritoneal macrophages, while transcriptional analysis showed that hypR and ahpCF genes were repressed in the mutant (Verneuil et al., 2004). HypR was shown to directly regulate expression of hypR itself, as well as the ahpCF operon, and thus is a transcriptional regulator of the oxidative stress response and can be considered an E. faecalis virulence factor (Verneuil et al., 2004). Shepard and Gilmore (2002) used real-time PCR to study virulence-gene expression and show that AS, Esp, Ace, EfaA and Gel are induced in serum or urine. However, both environment and growth-phase variations were observed, demonstrating the occurrence of uncharacterised control mechanisms for gene expression that may play an important role in vivo (Shepard and Gilmore, 2002). 20.4.4 Antibiotic resistance A specific cause for concern and a contributing factor to pathogenesis of enterococci is their resistance to a wide variety of antibiotics (Murray, 1990; Landman and Quale, 1997; Leclercq, 1997). Enterococci are either intrinsically resistant and resistance genes are located on the chromosome, or they possess acquired resistance determinants which are located on plasmids or transposons (Clewell, 1990; Murray, 1990). Intrinsic antibiotic resistances include resistance to cephalosporins, β-lactams, sulphonamides and low levels of clindamycin and aminoglycosides, while acquired resistance include resistance to chloramphenicol, erythromycin, high levels of clindamycin and aminoglycosides, tetracycline, high levels of β-lactams, fluoroquinolones and glycopeptides such as vancomycin (Murray, 1990; Leclercq, 1997). Intrinsic resistance to many antibiotics suggests that treatment of infection could be difficult. However, combinations of cell-wall-active antibiotics such as penicillin or ampicillin with aminoglycosides (e.g., streptomycin, kanamycin and gentamicin) act synergistically and have been used successfully in the treatment of enterococcal infection (Moellering, 1990, 1991; Murray, 1990; Simjee and Gill, 1997). Since the early 1970s, a high level of streptomycin and gentamicin resistance was reported, and strains were also found resistant to penicillin-streptomycin or penicillin-gentamicin combinations (Moellering, 1990). In 1983, a strain of E. faecalis producing a β-lactamase identical to that produced by S. aureus was reported (Murray and Mederski-Samoraj, 1983), and it is believed that this strain of Enterococcus received the gene

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from S. aureus (Murray et al., 1986). The hitherto successful penicillinaminoglycoside treatment was no longer a viable option, resulting in a major therapeutic problem (Moellering, 1991; Morrison et al., 1997). Vancomycin resistance is of special concern because this antibiotic was considered a last resort for treatment of multiple-resistant enterococcal infections. In addition, this antibiotic was given as an alternative to ampicillin or penicillin/aminoglycoside treatment to persons with allergy against penicillin or ampicillin (Morrison et al., 1997). In the mid-1990s in Europe the source of vancomycin-resistant enterococci (VRE) was shown to be most likely from farm animals as a result of ergotropic use of avoparcin, a glycopeptide antibiotic (Klare et al., 1995a,b; Das et al., 1997). In the USA, the situation with respect to nosocomial VRE infections appears to differ considerably from that in Europe, because avoparcin has not been licensed for use (McDonald et al., 1997). A community prevalence survey failed to isolate VRE from healthy volunteers without hospital exposure and from environmental sources or probiotic preparations (Coque et al., 1996). In contrast to Europe, transmission of VRE in the USA does not appear to be from the community to the hospital, and food has not been implicated as a possible vehicle for transmission. This indicates that clinical use of vancomycin is responsible for development of VRE. Currently, six types of glycopeptide resistance (vanA, vanB, vanC, vanD, vanE and vanG) have been described in enterococci and can be distinguished on the basis of the sequence of the structural gene encoding the resistance ligase (Depardieu et al., 2004). VanA type resistance is characterised by high levels of resistance to both vancomycin and teicoplanin, VanB-type resistance is characterised by resistance to variable levels of vancomycin but the strains are susceptible to teicoplanin. VanD-type strains are resistant to moderate levels of vancomycin, while VanC, VanE, and vanG-type strains exhibit low-level resistance to vancomycin only (Depardieu et al., 2004). The emergence of vancomycin-resistant enterococci (VRE) in hospitals has led to infections that cannot be treated with conventional antibiotic therapy and thus such strains pose a serious medical concern. Aware of the vancomycin and multiple resistance problem, the US Food and Drug Administration in 1999 approved the use of the streptogramin B/ A combination quinupristin-dalfopristin for treatment of VRE. Quinupristin/ dalfopristin (Synercid®) was next considered an antibiotic of last resort following the development of VRE (Jones et al., 1998; Werner et al., 2000). However, there was some concern about the use of this antibiotic due to the use of the analogue virginiamycin in agriculture in the USA for over 25 years. Thus, while low frequencies of streptogramin resistance were detected among E. faecium from human origin in the USA and in Europe, streptogramin resistance has been detected frequently among E. faecium strains of animal origin, especially among poultry isolates (Anonymous, 1999; Welton et al., 1998; Jensen et al., 1998, 2000; Werner et al., 2000; Hayes et al., 2001; Simonsen et al., 2004). In addition, clonal spread of streptogramin A resistance from farm animals to farmers has been shown to occur (Jensen et al., 1998;

584 Emerging foodborne pathogens Werner et al., 2000), indicating that the incidence of Synercid-resistant enterococci from humans will, in all probability, rise. Linezolid is also an antibiotic use to combat VRE and was approved in 2001 by regulatory authorities in Europe and the USA. Linezolid is the first representative of a new class of antibiotics, the oxazolidinones which have a unique mode of action that blocks the assembly of a initiation complex for protein synthesis (Klos et al., 1999). In a surveillance study in Germany from November 2001 to June 2002 susceptibility data of 8,594 Gram-positive clinical isolates indicated a low prevalence of resistance for E. faecalis (2.3%) and E. faecium (1.4%) (Brauers et al., 2004). Nevertheless, first reports of development of linezolid resistance among clinical enterococci strains are emerging (Krawczyk et al., 2004; Raad et al., 2004; Liao et al., 2005) and resistance appears to be based on a single G2576U gene mutation in the 23S rRNA gene (Raad et al., 2004). Daptomycin is a novel, cyclic lipopeptide antibiotic which acts at the cytoplastic membrane of bacteria (Silverman et al., 2003). It has activity against Grampositive pathogens, particularly multiple resistant strains such as vancomycinresistant enterococci (Streit et al., 2004; Alder et al., 2005). In a study of 6,737 clinical Gram-positive organisms, enterococci showed the highest daptomycin MIC values, but almost all (797 of 798 isolates) tested were inhibited at a concentration of less than 4 µg/ml (Streit et al., 2004). Despite this apparent sensitivity of enterococci to daptomycin, recently an Enterococcus faecium strain was shown to become resistant during daptomycin therapy (Sabol et al., 2005). Clearly, although new antibiotics such as linezolid and daptomycin have become available in the post-VRE era, the development of resistance seems to be unstoppable and it will probably be only a question of time until multiple resistant strains will also become resistant to these new antibiotics. 20.4.5 Congruence of epidemiological and strain virulence profile data The involvement of a putative virulence determinant in the pathogenicity of enterococci can be difficult to determine. Undoubtedly, suitable epidemiological data which link the occurrence of a certain virulence factor at high incidence among clinical isolates appears to give a good indication of the relative contribution to virulence. Data derived from relevant cell cultures of animal models can provide valuable information about the role, and possible contribution of a putative virulence factor to virulence of the strain. But how do we know whether a virulence factor really contributes in a human infection and to what degree? Many data pertaining to incidence of virulence determinants in clinical strains, as well as cell culture and animal model data have been discussed in the section on enterococcal virulence factors above. However, together these do not constitute epidemiological data, as comparisons of virulence profile data from clinical isolates with community isolates, or data

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on virulence determined in cell cultures or animal models do not directly relate to information about their relative contribution to the infection. For many, a direct involvement in pathogenicity has not yet been shown (e.g., hyaluronidase or E. faecium endocarditis antigen) and are thus often termed ‘potential’ virulence determinants. Roberts et al. (2004) studied 215 E. faecalis isolates from human infection and showed that neither the two-component regulatory locus fsr nor gelatinase production was more common in disease-associated isolates, and thus were not probably required for E. faecalis to cause infection. However, these findings obviously did not indicate whether Fsr or gelatinase affect the severity of the disease (Roberts et al., 2004). The data were in contrast to an earlier report which showed that all endocarditis isolates of E. faecalis, vs. 53% of stool isolates carried the fsr locus and thus was suggested to support virulence in the pathogenesis of enterococci (Pillai et al., 2002). In a molecular epidemiological survey, the presence of various virulence genes such as those encoding Ace, EfaA, CylA, GelE, AS, Esp and two novel surface antigens EF0591 and EF3314 among clinical isolates, and isolates from healthy individuals and the environment were determined (Creti et al., 2004). Some genes (e.g., ace, efaA and ef3314) were present in isolates from all sources, while esp and cylA genes were never detected in endocarditis isolates. The aggregation substance gene was shown to be always present in commensal isolates, and an association was noted between the esp gene and isolates from urinary tract infection and bacteraemia (Creti et al., 2004). Vankerckhoven et al. (2004) used multiplex PCR to simultaneously detect five virulence genes (asa1, cylA, gelE, esp and hyl) among clinical and faecal E. faecium isolates from inpatients at European hospitals. These authors showed that overall, the prevalence of esp was significantly higher in clinical vancomycinresistant isolates than in faecal isolates. In addition, these authors used pulsed field gel electrophoresis typing (PFGE) to show that there was a clonal, intrahospital spread of esp-positive vancomycin-resistant E. faecium clones in Italy, and of hyaluronidase-positive, vancomycin-resistant E. faecium clones in the United Kingdom (Vankerckhoven et al., 2004). Thus, while from the above studies Esp in particular appears to be quite important as a virulence determinant, there are other studies which cannot correlate the presence of a certain virulence determinants with increased virulence or pathogenicity based on epidemiological data. Vergis et al. (2002) studied the relationship between the presence of enterococcal virulence factors gelatinase, haemolysin, and Esp and the mortality among patients with bacteraemia due to E. faecalis and could not find a significant association between 14-day mortality and any of the markers studied, either singly or in combination. Baldassari et al. (2004) investigated the presence of genes for virulence determinants among enterococci isolated from endocarditis cases. These authors found only few isolates to harbour genes for Esp and haemolysin, while genes for aggregation substance and gelatinase were more common. Nevertheless, Baldassari et al. (2004) argued that predisposing factors,

586 Emerging foodborne pathogens particularly hospitalisation and multiple antibiotic therapy, appeared to be more relevant to the development of enterococcal endocarditis. Clearly, therefore, the last word on the relevance of enterococcal virulence factors, either singly or in combination, to enterococcal pathogenesis has not been spoken. Much still needs to be revealed in connection to the relevance and relative contribution of such virulence factors to enterococcal disease. Recently, enterococci bearing so-called ‘pathogenicity islands’ have been described. This raises the question whether the presence of virulence genes clustered together on such a pathogenicity island constitutes a precondition for causing disease. Sequencing large parts of the genome of the clinical E. faecalis strains V583, V586 and MMH594 led to the discovery of a pathogenicity island of about 150 kb with a typical mol% G+C content which was different to the rest of the genome and which was flanked by two terminal repeats (Shankar et al., 2002). This pathogenicity island of E. faecalis MMH594 encoded a cytolysin operon, aggregation substance, Esp, a bile acid hydrolase, transcription regulators, transposases and other genes involved in adaptation and survival in different environments (Shankar et al., 2002). A 150 kb putative E. faecium pathogenicity island was also recently described by Leavis et al. (2004). This pathogenicity island had a lower mol% G+C content when compared to the chromosome and contained the gene for Esp, as well as other putative genes involved in virulence, transcription regulation and antibiotic resistance. Moreover, it appeared to be associated with epidemicity, since 13 of 14 clones analysed from different hospital outbreaks contained this pathogenicity island, but was absent from human surveillance and animal isolates (Leavis et al., 2004). Thus, in all possibility, the presence of virulence factors clustered on pathogenicity islands might more closely correlate with observed enterococcal pathogenicity than the presence of unlinked, single or multiple virulence determinants. However, more research would be required to investigate whether this is the case, but it should not be forgotten that predisposing factors such as multiple antibiotic use and host factors also greatly influence infection by enterococci.

20.5 Incidence of virulence factors among food enterococci In recent years, an increasing number of studies have been carried out which have investigated the occurrence of virulence traits among food enterococci. Such studies have been primarily done to assess whether enterococci from foods bear the same types of virulence determinants when compared to clinical isolates and thus to evaluate the risk of human infection associated with food enterococcal strains. Furthermore, such studies may also allow the evaluation of the safety of strains which are intended for use as probiotics or possibly as starter cultures in cheese or sausage production. From the above discussion, however, this approach seems a little too simplistic because of the lack of congruence of the virulence determinants with observed

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pathogenicity, as well as lacking information whether virulence determinants are linked and located on a pathogenicity island. Nevertheless, the importance of such investigations should also not be considered as negligible, as such first assessments of the virulence potential, however crude these may be, do offer some insight as well as an initial assessment of the perceived risk or safety of the strain, and can thus serve as selection criteria for strains which are intended for use in the food industry. In one of the first investigations on this topic, Eaton and Gasson (2001) showed that enterococcal virulence factors were present in food and medical isolates, as well as strains used as starter cultures. However, the incidence of virulence factors was highest among the medical strains, followed by food isolates and then the starter strains. Strains of E. faecalis were noted to harbour multiple virulence determinants, while E. faecium strains were generally clear of virulence determinants (Eaton and Gasson, 2001) (Table 20.3). A similarly low incidence of virulence factors was observed among E. faecium strains isolated from food in our studies, in which only a few strains produced either haemolysin (8.3%) or Esp (2.1%) (Franz et al., 2001). However, E. faecalis strains also harboured multiple virulence determinants, with a much higher incidence than in E. faecium. Further studies (Majhenic et al., 2005; Martin et al., 2005; Yousif et al., 2005) on the virulence of food enterococci strains showed similar trends in that virulence determinants are generally more commonly identified from E. faecalis strains when compared to E. faecium strains (Table 20.3) and that E. faecalis strains more often carry multiple virulence determinants. Some general trends regarding the virulence determinants were also be noted. For example, the gene encoding the E. faecalis and E. faecium endocarditis antigen (efaAfs or efaAfm) and the acm gene from E. faecium can be detected in more than 90% of food strains. Such wide distribution in food isolates may imply that these potential virulence determinants may not play a major role in enterococcal disease. Furthermore, these data (Table 20.3) clearly show that virulence factors such as gelatinase, aggregation substance and Esp generally occur at a very low incidence (