Veterinary Microbiology (2ed-opt)

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Second Edition

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Second Edition

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Bibliotf!ca I C A P

Second Edition

Dwight C. Hirsh N. James Maclachlan Richard L. Walker

Blackwell Publishing

Dwight C. Hirsh, DVM, PhD, is cmeritus professor, Department of Patl1ology, Microl.Jiulogy a11d Im mu nology, l Jnivcrsity of California-Davis.

N. Jan1es MacLachla11, DVM. PhD, is protcssor, Dcpart1ncnt of Pathology, Microbiology and l1nmunology, Universíty of California-Davis. Richard L. Walker, DVM, Pl1D, MPVM, is profe:;sor of clinical cliagnostic bacteriology, California Animal Health and Food Safcty Laboratory, Univcrsity of CaliforrúaDavis.

Authori7.le 2.1). Ir1 a :c-ll3,. eosinophils and macrophages can also beco me in\·o,-ec o ADCC. ADCC is an effective u1ethu:; are treated ir1 the same 1nanner. Do not refrigerate sampl gel immunodiffuSion, Cf = Eomplement tixation, EOFAl = complement.fixatioll for avían Jeukosis, CPE= cyto11athic efféct. ECE = embryonating chicken eggs, EM = électron microscopy, FA = imm11nofloorescenr.e. HA =hem,ag9l11:tinin. HAO = hema~orption. RI = hemagglutination inhioitiori, RJA = radioiinmunoassaY., VI =virus isolation,VN =J1irus neutrali:ration, MI = molewlar/ irnmunofogic.

'lleportable ;'1!$eaie or a foreign animal dísease in l!níted States. ~VEE

=Venezuelan equíne enc~flalomyelltis, EEE= eastem equine encephalomyel.itis, WEE =western equtne encépl\alomyelitis.

lsolation of Virus from Clinical Specimens Cultivation in Tissue Culture. Viruses are isolaled fro111 cli11ical specimens hy inoculating susce.ptihlP primary or contin11-

lular monol.ayers and absorbed 1 hour or longer at 3.SºC to 37ºC, and tl1e inoculun1 is left 011 or renioved and fresh media added. Ino(u!ated and uninfected cell cultures are

ous cell cultures derived fro m the host or related species, embryonated eggs. o r laboratory animals. Specime11s submitted for viral isolation should be placed in virus transport media (e.g., a balancect salt solution containing antibiotics) in sealed containers for safet y in handling. They shoulcl tJe clearly itlent ifiecl l>y appropriate labeling, an!cwcastlc diocosc.

22

PART I

Introduction

F 1G U RE 3 . 1 . Cytopathic effect (syncyti;i/) nn hnvinP fpt;¡/ kídney ce/Is by the herpes virus of malignant catarrhal fever (X200).

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The chicken embryo (10 to 12 days olú) iny >e1 u111 obldined before and after natural infection with BTV serotype 17. Virus proteins identified are given in the right margin; LMW represents three low molecular weight virus proteins that have not been previously defined; X represents an additiona/ noncharacterized virus protein; P designates specific BTV protein; NS is the nonstructural virus protein. 1 anPs A and R rPpresent prP.- anrl postinfection serum, respective/y. Jmmune complexes were detected in lane B using a biorin-avidin-enzyme probe, e.g., biotin-tabeled rabbit antisheep /gG in association with peroxidase-/abe/ed avidin. (Reproduccd with pcrmission from MA Adkison pt>nicillin -hincling protcins (PBPs), of which there are three to eight in bacteria; many of these PBPs are transpeptidase enzymes. They are rcsponsiblc for thc formation and rc1nodeling of the cell wall during gro'vth and division. Dúfercnt PBPs have different affinities for drugs, explaining the variation in the spectrum of acrton of dlffcrcn t beta-lactarn antibiotics. L1egradative rnechanisms are also involvPc1 in y arP active only against gran1-positive bacteria. Penicillins. Sir Alexandcr f.lcming's observation that colonies ot staphy!ococci lysed on a plate that had become contaminated ~''ith a Penicillurn fu 11gus was the discovery that led to the development of antiblotlcs. In 1940, Chain,

Chapter 4

Florey, and tl1eir associates succeeded in producing therapeutic quantities of perlicillin from Penicilliu111 notaturn. .Almost a decade Iater, penicillin G becan1e widely availnblc for clinicnl use. In thc ycnrs that followcd, this antibiOtic was found to have certain limitations: its relative instability to stomach acid, its susceptibility to inactivation by penicillinase, and lts relatlve lnactivlty agalnst most gram-ncgative bacteria . Tsolat.ion of the active moiety, 6a111inopenicillanic acid, in the penicillin molecule has resulted in the design and development of se1nisynthetic penicillins that overcome so1ne of these limitations. ·rne deve1op1nent ot the cep11aJosporin tamily, which shares with penicillin the beta-lactam ring, has led to a remarkable array of drugs with in1proved ability to penetrate rliffP.rt>nt gram-nP.gativt> haria 1 spP.cit>s anrl to rt>sist ht>tillactamase enzymes. ln recent years, other naturally occurring beta-lactam antibiotics have been describcd that lack the bicyclic ring of the classical beta lactam penicillins and cephalosporins. Many oftl1ese new drugs have potent antibacterial activity and are highly resistant to beta-lactamase el tly1ne:;. C.l"in ically i n1 portant pen icill íns c.an hP. cliviclt>cl i nto six groups: l. Beuzyl pe1licilli11s a11d it:; lu11g-acti11g fur1ns. Injec-

2.

3.

4.

5. 6.

tahle penicillins, highest activity against grampositive organisms, but susceptible to acid hydrolysis a11d beta-lactamase inactivatio11 (e.g., penicillin G). Orally absorbed penicillins, spectrum similar to benzyl penicillins (e.g., pen.icillin V). Antistaphylococcal isoxazolyl pe11icillins. Relatively reslstant to staphylococcal beta-lacta.n1ases (e.g., cloxacillin, methicillin) . Exlended-spectrun1 penicilli11s: An1inopenicillins (P..g., ntration r;i tios (0.3) . The rel attri buted to enterohepatic circulation. Penetration to cerebrospinal fluid (CSF) is usually poot bul is e11 hanced by int1ammat.ion. In addition, active removal of

30

.PART l

lntroduction

penicillin from CSF is diminishcd by inflammation. The pcnicillins are eJiminated almost entirely by renal excre tion, which results in very high Ievels in the urine. The renal excretion mechanisms include glomerular filtration and mainly proximal tubular secretion. Adverse Effects. Penicillins are remarkably free of toxic cffcc..:I~, 1::vc11 al doses grossly in excess of 1l1ose recon1me n liPs hPnf>ilth the c:ell \"lall, enc:losing the cytoplasm . lt controls the passage of materia]s into or out of the cell. lf its function is damaged, cellular contents (proteins, nucleotides, ions) can leak from the cell and result in cell damage and deatl1. Polymyxins. Tl1t: strui.:turt: oí ll1e polyinyxillS is such LltaL they have well-clefined separate hydrophilic and hydrophobic sectors. Polyn1yxins act by binding to n1embrane phospholipids, which results in strltctural disorganization, pcrmcability damagc, a11d ccll lysis. The polymyxins are scJect1ve1y tox1c to gran1-negat1ve bacteria because of the presence of certain phospholipids in the cell membrax1e and because the outer surface uf tlu: uuter 1uet111Jra11e u[ grarn-negative b;ictPria consists rna in ly of 1ipopolysacch.aride. Parenteral use is assocíated wíth nephrotoxíc, neurotoxic, and neurornuscular blocking effects. 1'he major clinical applications are lirnítcd to thc oral trcatrnent of gram-negative bacteria! infections. Polyenes. The polyenes are selectively active against fun6'1 since t11ey only affect m.embranes containíng ergosteroL Polyenes inllilJit tl11:: furruation of 1nen1brane lipids, forn1ing porPs through which the vital contents of the cytoplasn1 are lost. See the section on antifungal therapy, below: lmidazoles. lmidazoles interfere with the biosynthesis of sterols and bind cell 1nelnbrane phospholipids to cause leakage of cell i.:outeuts. They are aclive againsl Lhe fungal cPll mt>mhr;:ine. See the section on antifungal t herapy, below. lnhibition of Nucleic Acid Function Examples of drugs that inhibit nucleic acíd function are nitroimidazoles, nltrofurans, nalldiXlc acld, the fluoroq11inolonc; (ri rroflox;iri n, rl;inoflox;irin, rliflnx;icin, PnroflOX3CÍil, orbifloxacin, sarafloxacin), novobiocin, rifampin, sulfonamides, trimethoprim, and 5-flucytosine. Because the mechanisms of nucleic acid synthesis, replica7 tion, and transcription are similar in ali cells, drugs affecting nucleic acid functío11 have peor selective toxicity. Most act IJy IJiudiug tu DNA tu inlrilJit it:; replication or transcription. Drugs with greater st>lectivt> toxicity ilrt> the sulfonamides and trimethoprim, which inhibit the syntl1esis offolic. acid.

Antimicrobial Chemotherapy

31

Nitroimidazoles. Nitroimiclazoles, such ;is mf>troni and din1etridazole, possess antiprotozoal and antibacterial properties. Activity within bacterial cells is due to the unidentified, rcduccd products of thc drug, which are only seen in anaerobes or microaerophiles. Nitroimidazoles cause extensive DNA strand breakage either by inlúbiting the DNA repair enzyme, UN ase 1, or by forming complexes 1Aríth the nucleotide bases th.at the enzyme ratio in thi> pl11sm;.i. This variation not important since the synergistic interaction occur~ "'a wide range of conccntration ratios of the two drugs. Resistance. Resistance to sulfona1nides is due to struc·al alteration in the dihydrofolic acid synthesizing en-me (dihydropteroate synthetase), whereas resistance to .....1ellluprirr1 usually results frorn plasmid-encoded syn• oCStS of a resistan t ciihyci rofolatf' ri>rl11rtil~f' Pnryme Rae- 31 resistance to the combination has progressively deped with use of these preparations in animals. ,bsorption, Distribution, and Elimination. Trimethoprim - :ipid-soluble organic base that is approx1mately 60o/o ..;nd to plasma proteins and 60ºAi ionized in the plasma. .:. cun1l>ination of physicochem!cal properties enables """ ci n1g to distribute widely, to penetra te cellular barriers nonionic diffusio11, aJ1d to atlain effeclivc co11ce11lrat.. ns in most body fluids and tissucs, including brain and (tPtr11ryclines, aminoglycosidcs, aminocyclitols) and those affecting the 505 ribosome (chloramphenicol, macrolides, lincosamides).

Antimicrohial Chemotherapy

33

Tetracydines. ·retracyclines interfere with protein synthesis 1Jy i11llilJitiug tl1t: lJir1ding of aminoacyl tRNA to the recognition site. The various tetraryrlinPs havt> simi111r ;intimicrobial activity but differ in pl1armacologic characteristics. Antinzicrobial Activity. Tetracyclines are broad-spectrum drugs active against gram-positive and gram-ncgativc bactena, 1ncluding ricketts1a aod chlamydia, sorne mycoplasmas, and protozoa such as Theilcria. Tetracyclines have guod activity against rnany gram-positive bacteria, the more fastidious nonentPrir h;irti>riil ~11rh ilS Actinohacil111s, Dordetella, Drucella, Ilaen1ophilus, sorne Pasteurella, and many anaerobic bacteria, but thcir activity against these bacteria and against membcrs of thc family Entcrobactcriaceae are l1m1ted by acquircd resistance. I'seudomonas 1 aeruginosa is resistant, except in urinary tract infectiolls whcrc, uecause of their t1igh concenttattons, tetracyclines may he drugs of c:hoirt>. Resistance. Widespread resistance to the tetracyclines l has considerably reduccd their usefulness. Such resistancc is high leve! and usually plasmid- and transposon-mcdi- · UJ ated. Cross-resistance between tctracyclines is complete. Adverse Effects. Tetracyclines are generally safe antibi iu lit:)) witl1 a reasonably high therapeutlc index. The main W adverse effects art> as~ori11ti>rl with thcir severely irritant CJ) nature, with disturbances in gastrointestinal flora, wilh u Ll1aL faulLy prolclns are produced; b) it hinct~ to "initiating" ribosomes to prcvcnt the formation of 70S ribosomes; and c) it inhibits thc clongation reaction of protein synthesis. ·rhe othcr aminoglycosides act SJ1n1larly to streptomycin in causiog mistranslatioo of the gcnctic code and in irreversible inhibition of initiation, although thc cxtent and type often differ. They have multiple blndlng sites on the rilJoso1nc, wl1erca:> :>Lreplo1nycin has only one, ;-incl can also inhihit the translocation step in protein synthesis. Spectinomycin is a bacteriostatic arninocyclitol antibiotic that is believed to inhibit polypeptidc chuin clongation at thc tra11slocation step. ·r11e aminoglycoside antib1otics are polar organic bases. Their polarity largely accounts for the similar pharmacoklnetlc properties that are shared by ali members of tl1e group. Chemically, they con~i~t of a hPxosP nuclPus to wl1ich an1ino sugars are attached by glycosidic linkages. All are potentially ototoxic and nephrotoxic. The newer uminoglycosidcs are more resistant lo plasmid mediated enzymat1c degradation and are less toxic than the older compouods. Amikacio > tobramycin > gentamicin > neomycln = kanamyc.in > strcptomycln in potency, spectrum of activity, and stability to plria are 1esislanl lo older drugs (strcptomycin, neornycin) but may be inhibited by newer drugs (gentamicin, ami.kaci n ). A particularly useful property is the activity of newer arninoglycos1cles against P. aeruginosa. 'rhcir bacteridclal action on aerobic gram-negative bacilli is markedly influenced by pH; they are most active in an alkall11e environment. lncrea:seu local acidity secondary to tissu(' damag may account for ll1e failu1e of an a111i11oglycoside to kili usually susceptible microorganisms at infection sites or in abscess cavities. Combinations of aminoglycosidcs with penicillins are oltcn synergistic; the concurrent admrn1stration of the oewcr beta-lactam antibiotics with gentamicin or tobramycln has been usecl to trcat serlous gram-negativc iufections, for example, those caused by P. ru n1ginnsa. 1

Resistance. Most clinically import;-int rt>sistanct> is caused by a variety of R plasn1id-specified degradatíve cnzymes locatcd in the periplasmic space. Certain of these cnzymc:; inactivatc only thc oldcr •uninoglycosides (strep tomycin, or neomycin and kanan1ycin), but others are broader spectrum. 'fh.e remarkablc property of arnikacin is its resistance to many of thc c11zyn1es t hat inactivéltc other aminoglycosides. Plasmid-mecliatt>d rPsistance to :>lreµ1u111yci11 is v•>'idespread and con1monly linked to sulfonamides, tetracyclines, and ampicillin. Chromosomal resistance to streptomycin, but not to the other aminogly cosides, develops tairly readily during treatrnent. Absorption, Distribution, and E:.:cretion. Aminoglycosides are poorly absorbed from the gastrointestinal tral.t, 1.Ji11u Lu a low extcnt to plasma proteins, and havt> limited capacity to 1;;:11tcr cclls a11d penelrale ccll u lar barriers. ·r11ey do not r0.adily attain therapeutic concentrations in transcellular fluids, particularly cerebrospinal and ocular fluid. Poor dif fusibility can be attributed to their low degree of lipid solubility. ·rheir apparent volumes of distribution are relatively small, and their half-llves are short (2 l1uurs) i11 domestic animals. Even though these dn1gs have a small volume uf uistril.Juliu11, seleclive binding to renal tissue (kiclney cortPx) occurs. Elimination takes place entirely by renal excretion (glomcrular filtration), and uncha.ngcd drug is rapidly excreted in the urine. ln1pairecl renal function dccrcascs tl1cir rate of excretion and makes adjustrnent of the maintenance ctosage necessary to p revent accumulation with attendant toxicity. Major changes are taklng place 1n recornrueu tlatiuus fur in tra111uscular dosage with ami noglycosiciPs, wh ich is r11uvi11g frorn ll1ree lin1es daily to a single daily dosagc. This has the effect of increasing therapeutic efficacy, since antibacterial activity dcpcnds oo both peak concentra tions and total conccntration, and of reducing toxicity, since the oephrotoxic effects depend oo a threshold effect, concentrations above which have no further action. ·rhis dran1atically changed understandíng of aminoglyco~icit> uu:>agt! 111ay increase Lhe use of tl1e less toxic members. Adverse Effects. All aminoglycosides can cause varying dc.:grccs of otot oxicity ond ncphrotoxicity. "fhc tcndency to produce vestibular or cochlear damage varies with the drug: neo rnycin is the most likely to cause cochlear damage ancl streptomycin to cause vestibular clarnélge. Ncpl 1rutoxicity (aCL1te tubular necrosi~) occ11r~ in association with µrulu11ged Lherapy and excessive trough conccntrutions of the aminoglycoside (particularly gentamicin) in plasma. The arninoglycosidcs can produce neuromuscular blockage ot the nondepo1anz1ng typc, wh1ch causes flacc10 paralysis and apnea. This is most likely to occur in associatlon with anesthesia.

A1ninocyclitols. Spcctinon1ycin is an a111in ocyclitol antibiotic with a spectrum of activity and mechanism ot action similar to that of kanamycin but without the toxic effects of the aminoglycosides. lt is normally bacteriostatic and Is not particularly active on a wcight basis. Its activity against gram-oegative bacteria Is unpretlil.tabll.! 1.Jecau:>e uf naturally resistant strail1s. Chromosomal resistance dcvelops readily bul does oot cross-react ""ith arninoglycosidcs. Plasmid resistance is uncommon but often extcncls to

Chapter 4 r--:.-- ->myrin The clrt1g h¡¡s most of the ph;1rm;1coldnetic

of aminoglycosides but appears to penetrate ü:!"E~.::rospinal flujd better. It has been used in agricultmaJ ~ - ce to trcnt snlmoncllosis nnd mycoplnsmn infections. '."'.X"~es

.;;,::::: des. Macrolide antibiotics are bacteriostatic with ac- partlcularly agatnst gram-posltlve bacteria and my..... -~ 'TTI" Thf'y bind to SOS ribosome in competition with ""'-""-ramphenicol and inhibit the translocation step of ;:ii:e·n synthesis. ·rhe precise mechanism of action is un- ·-n. Mncrolidc nntibiotics (azithromycin, clnrithromy- ~ue roncpntr;:itionnts decrease colonization resistan ce ~spí talized dogs were 38 times n1ore likely lo have ac- ..rl:ed resistant Safmonella if they were first given an anL:ñotic). Within 21to18 hours after administration of an -:.rtmicrobial, the normal flora is depressed to the degree ~ -..a.t resistant strains start to recolonize since those mioorganis111s Llial rt::plact:: tl1e 11orrnal flora will be reslst-¡ to the antin1icrohial drug arlministPrPrl. 1'hpsp ri>si~t­ ~t strains may be a part of the normal flora (e.g., norn1al, .~:nedicated dogs pcriodically shed large num bers of R - asmid containing E. coli i.n their fcces-thc rcason for -;:;sis unknown) or from the animal's environment. ln ei~-:cr case, the replacement strain wíll be resistant to the an-=:!!icrot>lal belng used. Having rcslstant bacteria occupy~g ;i ~11rface is not harmful in itself, unless the resistant .:::ain has the ability to il1vadc Lhc hosl cell Lo which il has ':'.'ai-'1ed access. But as mentioned above, if a normally ster~ sitc contiguous to the recolonized surface becomes mpromised, bacteria (now resistant bacteria) ~.ll coni2minate, and the resulting disease will be more d1ff1cult to :.:eat. This recolonization effect is the reason beh1nd the -~ommendation that prophylactic use of antimicrobial __.:ul::> t::xL1;:11tl 110 lu11ger tl1C1u 24 to 48 liour~. Colonization resistance is rcduced hy thP. strPss of ill'.€5-S and the stress of nevv social/envi.ronmcntal experie:ces. ·rhese changes occur secondary to changes in the - ormal flora. What appears to transpire are changes in thc =.en1ents responsible for the maintenance of t l1e stable, ::wrmal flora. Although these changes have been defined ~osl p11::cisely i11 Liie orc1l cavity, tl1ere are uata that suggest ~at they al so occur in the inte.d of the infectious ageut il~t:lf (t:ilher livc or killed), a portion of the agcnt that induces a protcctive immune response (subunit vac· cine), or a product of thc agent. Products containing a killcd bacteria! agcnt are more propcrly called baccerins. Products that have toxic activities are called toxins, and toxrns that have bccn inactivated are calleo tuxuids. To be effective, vaccines must eliclt rc~­ sponse that ir1tcrfcre~ vvill1 Lhe "lifc style" ofthe infectious

persensitivity.

s. ·rhus, extracellu· lar bacteria (live or killed), inactivated viral particlcs, por· tions (s11h11nit'\) of virus, and products are processed by the exogenous pathway. Epi topes are presented to thc immune system in context of MHC-11 by an ailtigen·presenting ccll that secretes IL· 1 and little, if any, !L 12. T helper cells (fHz subset of C:U4 lymphocytes) respond to this stimulus by sccreting cytokincs that trigger an antibody response (U.-4,

Humoral Immunity

Antibodies function im1nunologically by binding to epi· topes on the surface of the infectious agent and/or one of its products. By binding to the surface of an infectlous agcnt, antibodies interfere with attachn1ent to host tareí•t cells by stearic iutl'.rfcre1 Jlt: a11d/01 by cha!1gü1g the charge or hydrophohiciLy of the surface of the agent, and trigger the complement cascade generating products that are op· sonic and products that are damaging to agents that have surface membranes. Antibodies that bind to products of infectious agents can block tl1e attachment of the product to receptors on cell u lar targets and/or change the co11fie11ratlon of the protlu1.;l rt:::.u!Liug in a change in binding ;.i ffi ni ty. Cell·Mediated lmmunity Cell-mediated immunity is an immune response that re· sults in the gcnt~r•1tion of "activated" macrophages a11d/or specific cytotoxic 1· lyinphocytes. Th!s aspect of the im· mune response concerns agents that live insic1t> of cPlls, vvhich are tl1us prolt:1.:Lt:d fro111 interaction with the ele· mt>nts of the humoral components of the system. Activatcd macrophages are mononuclear phagocytic cells that havc come in contact with lnterleukin 1 (IL· I) unc.tions that he.gin at m ucosa] s11rfac.es l1ut then cause a systernic i1lfection with viremia, and subsequent infection of distant target tissues. In these infcctions, botb immunity at the mucosa! surface as well as system1c 11nmunity are important. 3. Infections that gain direct entry into the host's circulation via lnsect bite (arthropod-born.e viruses), inadvertent inoculation, ora traumatic break in an epllhelial surfacc. Jn sucll. infections, systemic immunity is the primary line of defense. l.

Adjuvants Adjuvants are used to influence the nature of t he immune response elicited by an antigen. The response is influenced at various stngcs, dcpc11ding upon the adjuvant. Sorne adjuvants function as depots, so that antigen is slowly released over an extended period of time to maximize the immune response. Examplcs lnclude water/oil emulsions, .minerals/salts (bentonite, aluminu1n), and inert p;irtic:les 1n icrospheres). OLher adjuvants direct activity to t he processing step in the initiation of t11e im1nune response. Examples in.elude "imrnune stimulating complexes" ncP thP hPlpPr ·r lymphoc.ytP

'fhese mechanisrns of viral il1fection and subsequent llisse111i11ation 111ust IJe consideretl iu vacciue tlevelopment. Modified live (attenuated) and inactivated (killed)

subset selcction, an,d gra.nulocyte macrophage colony-

virus vaccines dominate thc vcterinary vaccinc markct

stimulating factors activate macrophages and increase efficiency of antigen processing.

fiable 6.1).

Live Attenuated Viral Vaccines

Viral Vaccines Immunization of animals with viral vaccines is critica! to t he prevention of many viral diseases. 'fhe basis of an effective vaccine is its ability to induce an imrnune response or responses capable of eliciting protection to subsequent field exposure to patl1ogenic viruses. A multitude of vacciu1;: preparatio11s l 1ave 1Jee11 tlevelupetl a11tl us1.:d ovt::r tlie years. with variable success. The success of a potential vaccine hinges primarily on safety and efficacy; hptor) resuJting in deregulatiu11ufl1u::.l cell fui1ction without undue harm to the ccll, intt>rference of thc normal physiology of the host cell(s), o r death of the host ccll. Antibodies elicited to various cp1topes on toxins that rcsult in neutralization are sometimes called antitoxins. As mentioned previously, an antibody may block intcn:11.:Liuu betwee11 a toxin and its cOn ,.v hether the source of the sample is intestinal or ex-:alntestinal. When the source is extraintestinal, isolating "ly of the family from normally sterile sites is significant. _..,_ culLurt: u1t:mol)ISins 1 and 2"). l.

lrun llcquisiliun. lron is an alJsolute growth requirement r most, if not ali, living things. Sic1Prophores (e.e., aerbacti n) that remove iron from host iron-binding proteins .-re necessary if a microbc is to have invasive capabilities.

Enterobacteriaceae: Escllerichía

63

protein, 1'ir (translocated intimin receptor) inserted into the host cell membrane. 3 . Type III secretion system. ·rhe genes encod1ng a Type ITI secretion system (an assemblage of proLei11~-1uore thntial Lauses of diarrhea. 'fhe diarrhea is usually watery (though blood and leukocytes may be obscrvcd in sorne cases). Histologically, sheets ot bacteria (entrapped in mucus) will be seen covering the small intestinal epithelia. lnvasíve Dísease. Associatlon of susceptible animals (usually a neonate that has received inadequatf' i1rno11nts uf t.:ulu~lrur11 vr coloslrun1 of inadcquate quality) with invasive strains of E. coli may occur by way of the conjunctivuc, inadcquatcly treatcd un1bilicus, or ingestion. If the invasive strain associatcs via ingestion, it first adheres to target cells in the distal small bowel. Adherence is probably related to expression of any number of acthesins, L>ut c:S31A is one that is comrnonly associated with inv;isivP F.. coli. Likewi~e, tlre adhc=>i11 Fl 7 01iginally described on a pl:ilootlstream follovvs. Extensive multiplication within thP intPsLinal epill1elial cell probably docs not occur. The mechanism by which the invasive strain gains access to lyrnphatics aftcr uptakc by thc cpithclial ccll is unknown. l.ikewise, the mechanism of entry into lymphatics after association with conjunctivae or thc un1bilicus is unknown. Once the epithelial surface is traversect, expression of adhesins is repressed (otl1erwisc adhesi n-expressing hartPria could adhe1e Lo l1osl pl1agocylic cclls with disastrous consequences for the bacteriu1n). Thc infccting strain 1nulti plic~ in thc ly1nphatics and blooclstream ano endotoxcmla develops (Fig 8.1). Death of the host occurs if antibacterial therapy, the iinmune systc111, ur !Jull1 Ju uuLrcJ1tOVt: Lhe n1icroorga11ism. lnvasive sttains h;ivP athogenicity Islands, a cluster of genes encoding virulcncc deter1ninant(s), an integrase protcin, a spccific inscrtlon sitc, and mobility. There are at least five ~almonella Pathogenicity lslands (SPis). While SPI-1 is found in both spt:!cit:s uf Sulmunella, SPI-2 (annesium lransport syste1n1 e11codedby a number of mgt genes located within SPI-3). These genes are induced by low concentration of magnesium ions (as occurs \·vithin rnaLTuphages), aud the encotled proteins ctppear to be important for survival inside of macrophages. The effector proteins associated with SPl-4 and SPl-5 are associated with intracellular survival, and as yet are not clearly defined. Enterotoxin. Members of the génus Salmonella secretean enterotoxin, Stn (Salmonella enterotoxin) associated with vvater and elec:trolyte secietion by t1ost target cells. Stn differs fro·m cholera toxi n and 1:r of l!sr.herichia r.nli (see Chaptcr 8) by being a peptide rather than being composed of subunits. 'I'he role (if any) of Stn in the production of diarrhea is unclear. Jron Acquisition. Salmonellae produce siderophores (e11terobactin) when growing in iron-lin1iting conditions. Stress Proteins. Stress proteins are definell as proteins macle when the mirroorganism is placed 11nder conditions of stress (e.g., 11eat1 cold, low pIJ, high pJI). RNA polyn1erase containing RpoS preferentially transcribes genes responsible for acid tolexance (survival at pH 5) and regu lates genes found on Spv plasmids (see belo\-v). RNA poly-

merase containing Rpo.E is involved with survival \.Vithin phagocytic cells. Virulence Plasmids. Salmonellae possess plasmids ofvarious sizes, son1e of which have been associated with vi.rulence. 'l'l1e n1osl 11olable is a fanuly of large (approximatelr SO to 100 kilobases [kb]) plasmids, termed "Salrnonella virulence plasmids" (Spv plasn1ids) that are found within those species of salmonellae with potential to produce disse.nlinated disease. Sorne of the genes (spv genes) carried by tl1ese plasmids are necessary for intracellular growth and are regulated in part by RNA polymerase conty one or more of the adhesins Agf, Pef, Lpf, o r by others :et to be determined. Follo\.ving adhesion, salmonellae are :nrernallzed following the induction of membrane ruffles !U the target cells t riggered by Ssps and Sops subsequent to ~l)eir "iujecliou" by L11e 'I'ype III secretion systern . 1'he lar5t:t cell is irreversibly damaged by t his interaction, under5oing apoptosis. Sahn.o nellae are now found "vithin the :arget cells, the lymph nodule, and submucosal tissue. An ...c"lflammatory response is initlated by release of various c!:temokines from affected host cells, as well as release of ?roinflarnmatory cytokines following host int eraction '\'ith cell >vall LPS- activities that result in an i11flux of ?Qlymorphonuclear neutrophil leukocytes (P1vfNs) and :nacrophages. Tl1e influx uf PMN~ i11ay !Je reflected i11 a ::;ansient peripheral neutropenia. PMNs are highly effi. dent iI1 phagocytosing and destroying salmonellae, the macrophage less so. If the immune status of t he host and the charactcristics of the salmonellae are such, the infectious process is arrested at t his stage. Diarrhea is thought to :-esult from prostaglandin synthesis by the recruited PMNs and perhaps by the affected host cells), as well as activa. don ofvarious inositol-signalingpathways within affected hosl cells. The nel resull is the secretion of cl1loride ions and water. ·rhe role of Stn in the production of diarrhea is w1clear. lt the infecting strain ot Salrnonella 11as properties that allow disscmination (posscssion of SPI-2, 3, 4, and 5associ.ated gene products that allow growtl1 within :nacrophages; Spv plasmid encoding ability to gro~" intra.

cellularly and serun1 resislance; PhoQ/PhoP syste1n alluwing resistance to defensins; SlyA allowing resistance to oA-ygen-dependent by-products; arcA), septicemia may result. 'fhe likelihood ot th.is occurring is increased if imm une status of the host is diminished. Salmonellae disseminate and multiply within phagocytic cells (macro-

tnterobacteriaceae: Salmonella

71

pbages mainly) witl1in. phagoso1nes. Not only are the invasivc strains bcttcr ablc to withstand thc action of lysosom al contents, sorne "sort" to phagosomes that do not fuse with lysosomes. The presenting signs are usually, but not always, septicemia and shock (see Fig 8.1). Strains producing thi host :incl n1ultip ly within macrophages of the liver and spleen, as well as intravascularly. During the dissemination process, salmonellae are occasionally outside of the intracellular environment and therefore at risk from the formation of complement membrane attack complexes on their surfaces. 'fhis occu rrence is cliscouraged by at least two n1echi"lnisms: a proc'h 1ct of thP Spv p l:isrnicl ;inci t he IPngth of the 0 -repeat unit of the LPS (there is a direct correlat ion between 0 -repeat length and viru lence). Invasive salrnonellae are capablc oí sccrcting a sidcrophore, enterobactin that ren1oves iron from the iron-binding proteins of the host, although it is doubtful whether tllis i~ 11eeded within the cells of the host. Multiplic.;ition of thP organism res11lts in endotoxemia (see Fig 8.1), which accou nts for most signs and t he course of illness. Pathology. If the infcctious proccss is limitcd to thc intestinal tract, the Jesions vvill consist of a hemorrhagic inflammation of the distal small intestine and large bowel. ·r11ere 1nay l.Je superficial necrosis. In the septicemic form of the diseasP., thP.tP. ilrf> inflamm:ito ry c.h:ingt>s in livPr, spleen, and intestinal tissue. 'fhere may be hemorrhages 011 pericardium, peritoneal surfaces, and adrenal cortices.

Disease Patterns Ruminants. Salmonellosis 1s a slg111flcant dlsease of ruminan.ts, mainly cattle. Th.e d isease affects young (usualJy 4 to 6 weeks of age) as well as adult anin1als. Anin1als in feedlots are commo11ly affected. The disease may be a septicemia orbe lirn ited to the enteric tract. Pneurnonia, he1natoge11ously acquired, is a comrnon presenting sign in calves with septicemia due to S. enterica serotype Dublin (S. Dublin). Abortion may follow septicemia. S. enterica serotype Typhimurium, S. Dublin, and S. enterica serotype Newporl are Lhe sero Lypes con11nonly isola led íron1 callle, S. Typhimurium the serotype from sheep. Swine. Salmonellosis in swine can p resentas an acute, fulminat ing septicemia oras a chronic debilitat ing intestinal disease. The form depends upon the strain of Salmonella, the dose, and the colonization resistance of the infected a rLiu 1aJ. The wport, afff'c.t a wide !Ju~L

ra11ge a1nong whicl1 fcral birds and rodents play important roles in interspecific dissemi.J.1ation of infection. Long periods of asymptomatic and convalcsccnt shcdding ensure widespread, unchccked distribution of the organisms. Clinical outbreaks are correlated with depressed immune states, as in newborn animals (calves, foals) and :.l1e:.:>eu duulls, f.ld1 lu1i1:11l 1..vvv:., eY.uiue swgical palie11Ls,

and swinc with systcmic viral diseases. Ali animals are at increased risk of developing discasc if thcir normal flora is disrupted (stress, antibiotics). These circumstances render anima Is susceptible to exogenous exposure or activation of silent infecttons. Humans appear to be susceptible to all Salmonella :-.e1ulypes, lhe n1osl in1porla11l sourcc for wl1icl1 are anim;ils ancrotype to anothcr is undcr thc rcgulution of thc = ...:es::::i con tained within a l'athogenicity Jsland (Shi-0 for ~a lsland) a cluster of genes encodingvirule11ce deter--r2,..l\S), an integrase protein, a specific insertion site, ::rrobility. ln t he case of Shi-0, the genes are contained .na defeclive (in1n1obile) lysogenic bacleriophage, so - crological traits are stable.

•,._

ar Products of Medical lnterest ".!SinS. A su rface protein, IpaD (for invasion protein anti-

·s responsible for adherence of shigellae to g integrins ~e surface of M ceUs, and lhe basolaLeral surface of ~ -e ~ ntestinal epitl1elial cells. ::dJ Wall. The cell wall of the 1nembers of this genus is ~-ypical ot gram negatives. ·rhe lipopolysaccharide fS in the outer membrane is an important virulence de ""'"..:;,,nant. Not only is the lipid A com.ponent toxic (endo•: x1a,, b ut the length of the side chain i:n the 0 -repeat ~ - hin de1s Lhe aLLacl1111.eul of Llte 111e1111Jraut'. attilck cuu1- _ of the complement systen1 to the outer memhrane.

LPS binds to lipopolysar.r.haride-binding protein (a serurn

protein), which in turn trans(e.r:s il lo tl1e blood phase of CD14. "l'he CD14-LPS complex binds to Toll-like (eceptor protcins (scc Chaptcr 2) on thc surfacc of n1acrophagc cclls triggering tl1e release of proinflammatory cytokines "lnvasion" Proteins. Virulence factors (invasion proteins) produced by members of this genus are mainly encocti:>ct on l;:irge p lasmi.ds (terrn.ed invasion plasmids). 'fhese genes, for the n1ost parl, are regulaled by al leasl si.Ji. chromosomal genes. The plasmid gene products cncode proteins that are rcsponsiblc for a 'fypc III cxcrction apparatus whereby son1e i11vasion proteins (lpa for invasion plasmict antigen) are "in jected" in to host cells, namely lpaB,C. 'fhe ·rype III secretion system con sists of an assemblage of protf'ins (morf> than ?.O) that form a hollow tube-likc structure through vvhich effector protein.s are "il1jected" into hosl "target" cells: l . lpal3. "Injection" of lpal3 (like IpaC) into the host

target cell leacts to activation of small CiTP-binding proteins of tl1e Rl10 family resulting in cytoskeletal changes anct the for1nation of "ruffles." IpaB also is responsible for lysis of the membrane of t he pl1agoso1ne contalnlng entrapped shigellae. Wlthln macrophages, IpaB activates caspase-1 resulting in a popto:;is.

2. lpaC. "lnjection" of lpaC (like lpaB) leads to activation of small GTP-binding protcins of thc Rho family resultíng in cytoskeletal changes and the formation of "ruffles." 3. IcsA. The surface prot ein IcsA (for intracel.Jular spre;id) is required for intracellular sp.read . lcsAis required and is sufficien.t for actin deposition and motility \\7hen shigellae are within host epithelial cclls. 4. lcsB. 'l he surface protein lcsH (tor intercellular spread) is for intercellular spread. Enterotoxins. Two e11terotoxins have been described, ShETl (Shigella entcrotoxin, encoded within the chromosor ual Pa Lhugeuicily Isla11in, a specific insertion si.te, an.d mobility) and a 63kL)a product of the sen gene (Shigella enterotoxin, encoded on the invasion plasmid). How these proteins elicit fluid sccrction is not known. Exotoxins. Shigella clysenteriae is the only member of the gruup that has t he genes necessary for production of shiga toxin . Shiga toxin is chron1oso1nally encoded . 1'he toxin is 81

82

PART

Table 11.1.

11

Bacteria and fungi

Species of Shigella

Ecology

Species

Group {Types)

Reservo ir

S. dysenteri.ie S. ffexneri S. boydii S. sonnei

A (1-10)

The reservoir for members of the genus Shigella is thc largc bowel of clinically ill, recovered, or asympton1atic primates

8 (1-6)

e c1-1 s¡ D (1)

Transmission

a prolein of 70,000MW composed of an A subunit (32,000MW) and íivc ident ical B subunits (each about 7700MW). The targct cclls fo r thc toxin are thc cndothclial cells that linc blood vcsscls. lleceptors on these cells are recognizcd by thc B suhunit. The toxin, by vvay of the A subunlt, inhlblts peptlde ct1a1n elongation at the level of the rihosome by affecting elongation factor 1-clf'pf'ndf>nt processes. 'l'his actlon rcsults in the death of the cell. ·rhe production of toxln is iron-regulated (by way of Fur. see bclow), more bcing produced in conditions of low iron concentration. 1he v1rulcncc of a particular strain or isolate is directly related to the amo unt of toxin produced. Other Exotoxins. Shigcllac produce at Ieast two other exotoxins that, at least theoretically, play a role in pathogenesis. 'l'!Je~c twu ¡.¡rulciu:> i:1fl.'. SigA a11Ll PiL: 1. SigA (for ShigeUa lgA protease). SigA is postulated ro play a role by inactivating Shigella-specific lgA in the

intestinal tract, therelJy i:1lluwiug tl1e Llisease ¡.¡ro to progress. SigA is encoded withln the chromosomal Pathogenicity Island, Shi-1. 2. Pie (lor protcin involved in intestinal colorúzation). Pie is a scri ne pro tease, v•lhich is thought to digest intestinal mucus that overlays the intestinal epithelial cells so that shigellae have access to host "target" ccll~. 'J'l1c gcuc:1 c11ponsible for acid tolerance (surviva1 at pH 5), pern1itting safe transit t hrough thc stomach. Variability Slúgellae are "typPr thP Pxprf>ssion and excrction of virulence protcins. Ingested shigellae safely traverse the stomach (RpoS dircctcd acid tolerance response). 'fhe targct cclls are thc apical surface of M cells in the large íntes ti11c to wruch sh1gellae attach by way of IpaD after digestion of thc ovcrlying mucus !ayer (Pie) . Cell-associated bacteria are rrappe more concentraled along actin "stress fibers" and shigellac with their actin "tails" move toward cadhcrin proteins marking intcrccllular bridgcs. Thus, shigellae n1ove laterally. IcsB lyses the aouble membrane resultmg úom movement between cells. Affected host cells together with dead and dying macrophages secrete varlous chemokines (i11iarrhea is brought about by the activation of phos-::-;olipase e (perhaps dueto "ruffle" formation) leading to -creases in intracellular calcium io11s, activation of pro~::::: : kinase e and subsequent pnosphorylation ot proteins -= rhe chloride ion cha11nels a11d those of tl1e membrane~d ated ion transport proteins involved In NaCl absorpti. Thf'Sf' activitif's Jp;id to cii;irrhf'a . The role of enterotoxin in this disease is unclear. The di::.dlea, sometimes watery, may be caused by interactions "'e11terotoxin \vith small intestin.al epithelial cells and in - -'-r b e due to changes in t he colonic epithelium brought mout by tl1e inv asion/ir1flammatory process. Stti¿;c:llu dy~c:nleriue prolia l ct>lls (ht>morrhage) and ,....ay cause t h e development of hen1olytic u ren1ic syn..::om e (HUS) (see Chapter 8). Shigella flexneri 1 has been en.c ountered i n periodontal sease of monls not work at ali. Por enrichmen t, GN bro th is preferred. Selenite or tetrathionate broths do not enrich for shigellae. Shigellae appear as lactose-nonfermenting colonies on lactose-containing media. Although sorne species (S. sonnei and S. boydii 9) ferment this sugar, n ot enough is fern1e11ted will1in lhe 24- to 48-h o u r incu1Jalio11 µeriotl to affect the selection of appropriate colonies for further testing. Suspicious colonics are tested directly with shigellaespecific antisera or inoculated into ditferential media and then tested with antisera. Primers have been desigoed to amplify (by the polymerase chain reaction) D N A specific for shigellae. This assay 11as bee11 used lo detecL aud i1u fru111 vhagocylosis.

Growth Characteristics Pasteurell11 and Ma1111llei111ia grow best in the prcscncc of scrum or blood . After overnight incubation (35- 3/"C), colonics are about 11nm in diameter, clear, and smooth or mucoid. Mannl1eirnia flae"1olytica, P. trehalosi, and P. glucosida produce hemolysis on ruminant blood agar. Ali are gram-negatJvc, nonmotíle i:.t:. uf five capsular (A, B, D, E, F) and 11 ~om;itic (1-11) serotypes occurring in 20 different

con1binations. Serotypes are often related to host spccificity and pathogcnicity. The serotype is designated with a lcttcr dcsignnting the capsule type anda number designating the soma tic type, e.g., A: l. Mannheirnia haetnolytica consists of 12 capsular types (1, 2, 5-9, 12-14, 16, 17), P. lrehulu:;i, fuur. (3, 4, 10, 15).

Ecology Reservoir

Pasteurclla and Mannheimia are carried on mucous mf'mbranes (most commonly in the uruvl1a1yngeal region) of susceptible host species (;iciherence to these surfaces is due lo adheslns). Carri11ge m11y be widcsprcad, a:; with P. multo

cida in carnivores, or exceptional, as \-Vith aVJan cholera-

producing strains in birds or hemorrhagic septicemiaproducing stra1ns 1n ruminants. In avian cholera, one ho:>t species may serve as reservoi r for another. Transmission

Infection is by inh;il;ition, ingestion, or bites and scratch wounds. Many infections are probably cndogcnous. In bovine hemorrhagic septicemia and avian cholera, environmcntal contamination contributes to indirect transmission.

r.ltapter 12

In general, there are thrcc manifcstations of - ~teurella/ Mannheimia-inctuced disease: respiratory tract -;;oJvement, septicemia, and trauma-associatcd condi - ::>ns: cc1zanisn1s.

Respiratory tract involven1ent is e ither pneumonia or uppcr tract disease (Atrophic Rltinitis in swinc). P11~u111u11ia is seen wu:st frequently in ruminants and is usually associated wi th M. liaP111nlytir.a, P. rnultocida, or I'. trehalosi. Environmcntal stress (e.g., shipping, weaning), virus infection, or other bacteria! infections (e.g., Mycoplas111a) usually precede pncumonia, and are thought to decrease host defenses of the tract allowing commensal bacteria living in Lile up¡;er LradonoviruC$VÍ. 1nultocida, develops under stress of pregnancy, lactation , or n1isn1anagement. Complicat ions include bronchopneumonia, m iddle and iru1er car infection, conjunctivitis, and septicemia. Genilul Truc;L Diseuse. Ill Lhe genital lracl, J>. rrzultocida may cause orchitis, halanoposthitis, and pyometra.

dagmatis).

Human Beings. Pasteurella rnultocída (a11d rarely other pasteurellae) causes wound infections in hunians resulting fron1 a11ilnal bites or scratches. A second form, especially Íll tl1c resviralury lracl, is usually 110l directly traceable to animal sources. Epidemiology ·rabie 12.3 shows the relative roles of exogenous reservoir, dissemination, a11d stress in various conditions associated with Pasteurella and Mannheimia. In avian cbolera, feral sources are sometimes implicated.

lmmunologic Aspects Basis of lln1nunity (;irculating antibody is significant in protection against hemorrhagic septicemia and fowl cholera. The typespecific capsular antigens are esse11ti(:ll i111111u11ogens in hen1orrhagic septicemia . With other forms of disease associated with Pasteurella and Mannhcinúa, the picture is lcss clear. Both antitoxíc and antibacterial antibodies are important in protection . Artificial lmmunization Pastcurella adjuvant bacterins are effective in preventing bovlne hemorrhagic septice111ia, coufe1ring p rolection for up to 2 yi>ars. Antiserum is useful for short-term protection. The essential attributes of an effective avian cholcra vaccine are not k.nown. Field perfor1nance of l)acterü1s has

Chapter 12

Pasteurellaceae: Pasteurella, Mannhcimia

89

Ta b 1e 1 2 . 3 . The Relative Roles of Stress, Agent lntroduction, and Agent Disscminiltion in Various Types of Pasteurel/a lnfections lmportance of: Obsemlnatton of Agent Wrthin Population

fnVlronmental or Individual Stress or lnjury

+++ +++

++++

?

Bovine )hiµµi11y fever, uvine enzootic

+

++

pneumonia. avine septicemic pasteurellosis, rabbit pasteurellosis Human pasteurellosis Sporadic pneumonia in ruminants, Pasreurella infection in dogs and cats

+?

l11l1U\lui.llu11 ur

Agent into Population Avian cholera BovinP hPmorrhagic septicemia

++++

+++ ++++ +++·+

l"!Jeml. • =irrelevant; + =contributory; ++ = Slgnifleant; +++ =criúcal; ++++ =paramount

- inconsistent, even with autogenous preparations. ~ pro1nising havt: l.Jt:t:u live vaccines contalning atten-...o..~= " orga nisms (e.g., CU or M-9). Attf'n11;ition appears ine;- rclatcd to immunogcnicity. •ost shipping fever vaccincs are bacterins of mixtures . haen10/ytica, P. rnultocida, and P. trehalosi combined s>..ISpect viruses and other bovine bacteria! pathogens. o::::J:ts are inconsistent. Vaccination of calves with bac, prepared from M . haernolytica reduces the degree of :-ization of the upper respiratory tract by this organ::he effectiveness of n1odified live vac1.:i 11 1::~ await.s fiel ti ~ation. Recombinant Lkt alone does not protf'rt e from 1\lf. haemolytica-induced pneumonia. ·rhe gene :c.., g Lkt has bee11 cloned into thc genome of- and ;:-s¿quently cxprcsscd by-white clover, the strategy - : m 1mmun1ze by teed1ng. No data are available on the ---..r-1mess ofthis method. =er1rellu rr1ull1u..iúu ;111u B. brunchiseptica bacterins with t:X::::'._C. are val uable in the control of atrophir rhinitis. _::~ratory ;e-

Diagnosis

:.xamination

:..:._.:...-..=. ~te,

tissuc imprcssions, scdiments of transtracheal -..-~es. and, 1n birds, bloocl smears can be stained with -:-chrome stain (e.g., Giemsa, W right 's, Wayson's) cé - ""111li11eu for bipolar stalning microorganisms. The1r ":'."3é'!1Ce is s11ggf'sti ve but not unique to Pasteurella/ 1eirnia . On Gran1 staln, Pasteu.rellu/lvfunnheimia clo o;:i~ disti nctive.

ampli fy spccific rcgions of the bactcrial chromoso1nc by the polymcrase chain reaction are available for demonstration and identification of Pasteurella/ Mannheimia.

Treatment and Control Disea.;pasteurellaceae, require for propn one or both of two 6>TOwth tactors: porphyrins :==- or nicotinamide adenine dinucleotide (Nl\D, - • originally called X (heat-stable), and V (heat'actor, respectively. Sorne members of the family _ _...~_llaceae showi11g Lht::>t: 11eed:; are genetically unre-v t11e type species T-1. influmzaP. (a~~ociated with :!ryngeal, middle car, and respiratory tract co11di~ hurnan patients) . .cussed in this ch•tpter are Haemophilus paragallina· 'ie cause of infectious coryza in chickens), H. para·he cause of a septicemic disease called Glasser's dis 'polyserositls, and secondary respiratory disease of ;ind Histophilus so1nni (the cause of septicemic, res.=:.~~. and genilal Lracl disease iu calUt'. a11d :;l1e::ep). .:!us somni is the name now given to those n1icro- sms prcviously dcnotcd as "Hae1nophilus sornnus," -npllilus agni," "Histophilus ovis." Hrietly discussed is _.._. b.icteriu111 rhinotracheale, a bacterium that is not a ::r:=: ~: of the famlly Pasreurellaceae, but phcnotypically 'inically) resembles some strains of H. paragalli-'1e members of the family PasteurP.llar.eae ;irp gr;im~ coccobacilli. Th cy are facultative anaerobes, and -~ ox1dase-positive (setting them apart from mem:he family Enterobacteriaceae). Most are commensal ---- o uf IDimal:s.

.. -~= ..·ptive Features .._.,...._- : ogy and Staining ·~-

e-:. uf tl1e genera Haetnophílus and Histophilus are :cegative roct.~. IPSS than a micrometer wide and 1 to 3 -::; . but sometimes forn1 longe1 fila.rut:.ut:>. Sorne !>-pecles - :.1/linarum and H. infiuenzae) are encars11l;itPrl.

EKNs·r L. B1BERSTETN

Cellular Constituents and Products Capsules are polysaccharide. The ccll wall is typical of gram-negative microorgan1sms consisting mainly of lipopolysaccharides and proteins. Sorne of the latter are iron-regulalcd (i.e., Llit:y are expressed under iron-poor conditions).

Cellular Products of Medica! lnterest Adhesins. The role of adhesins, as in othcr microorganisms, Is to allow the bactenum expressing them to adhere to cells lining a particular niche, as well as to the surface of so called "Largel" acl R. hronr:hiseptir:a. The ultimate source are B. afJortus, B. carlis, B. 1nelirensis, B. neoro1·i~, and B. suis. . spccies of Brucella are capable of causing disease -ans. Infections are chronic and debilitating. The brucellosis in humans are relatively nonspecific ~ :vidua ls wíth brucellosis are somctimes labeled _ ,......, o ndriacs hecause of the va¡,rue prcsenting clinical

.t:=: :igy and Stainlng

rs of the genus Brucella are small, gram-negative -~ cilli measuring 0.6 to 1.5 µm by 0.5 to 0.7 µm in ..s are fa1rly un1form and can casily be mistaken tor Tlley are typically arranged singly but also occur in clu~ters. No capsu les, flagella, or spores are pronowever, an PxtPrn;:il f'nvf'lc.ipe has been demonii.:::::3! ny clcctron m icroscopy around R. abortus, B. rnelind B. suis. Brucella stain red with Macchiavello and _ ,...,, d Ziehl Neelsen stains. -----~--'"·

Structure and Composition

po:.pectively. lnfectio11:. i11 liu1:.e:. u:.ually resull íro111 ce. t.ict with infPcte.c1 cattlP. J3ruccllosis i..11 l1un1ans is primarily a disease of the ret ulocndothelial system. A mild lyrnphadcnopathy, spler mcgaly, and hcpatomcgaly may be dctected. Onset of si;occurs within z to 3 weeks of cxposurc. Clinical signs;: nonspecific and include alternating fever and chills \\~ night sweats, fatigue, musclc and joint pains, anlla 1nelite11sis is considered t 1nost virulent species for humans followed by B. suis abortus, and B. canis. Bruce/la avis and B. neotornae do :-: infcct humans. Common sources for infection are abor fetuscs, placentas, and postabortion uterine flu ids, a!: which con tain large n u mbers oí organi s111s. Veterinaria::ranchers, an d slaugttterl!uu:>c 1.vurker:. a1e par liculail. r isk For gs io suburban areas are least likely to be infected ~ - B. can is. Prcvalcncc of infcction is grcatcst il1 eco110111.......:-- depressed arcas. Closc confincment settings such as ":::cls incrcasc thc likclihood for transmitting infections.

-:nunologic Aspects - - :ine Mechanisms in Pathogenesis dencc indicates that antibodies against Bruce/la play ::i a protectlve and detrlmental role. IgM antibodies,

Bruce/la

109

which appear initially after infection, and low Jevels of lgG will cause complement-mediatcd lysis of Brucella. Elcvatcd levels of IgG antlbodlcs, howcvcr, appcar to actas blocklng antibodies that m odulate the ability of the complement n1e111brane attack con1plcx to lyse cclls. This n1ay account for resistance to complement-mediated lysis in the tace of high spccific antibody lcvcls and thc lack of corrclation bctween protection and high antibody titers. ·rhe blocking antibodies are opsonizing and promote uptake by phagocytes where Brucella have developed mechanisms for survival and proliferation. Phagocytic cclls unablc to elin1i11ate Brucella play a role in dissemination of organisms to other parts of tbe body and in pcrsistcncc of infcction. lgA autoantibody has been demonstrated in dogs infected with B. can is and may explain sorne of the observed effect on fertll!ty. Mechanisms of Resistance and Recovery Effcctivc immunity is primarily ccllulur in oature. Specifically sensitized ·r lymphocytes release cytokines that activate macrophages, which in turn control Brucella by reactive oxygen intermediates. Bn1cella can actívate NK cells by induci ne antigen-prcsenting cells to secrete IL-12. NK cells can then kili infected target cells. A n1orc cffeclive ü11111u11ily develops "''hen animals are infccted prior to sexual maturity. Artificial lmmunization canle are 1mmun1zed w1th e1ther nonVlable (B. abortus 45/ 20) or attenuated live (B. abortus strains 19 and RB51) vaccines. These p1oducts p1ovide p1otection úo111 abo1tion, the major mode of dissemination, but not from infection. A single dose at 3 to 7 and 4 to 12 months of age is required with B. aborh's strain 19 and strain RBSl, respectively. Two doses 6 weeks apart in animals over 6 months of age are requ1red w1th B. abortus 45/20. Adult cow vaccination is sometimes performed as a regulatory effort to control inft!ction In a hen.l. Strain 19 is uccasionally sl1eu in thP mi lk ;:incl c;:in c;:i11SP (lhortions in c:attlP. Aclult vac:c:ination with D. abortus strain RD51 only rarely causes abortion. Bulls should not be vaccinatcd becausc orchitis can dcvclop. Thc typc of vnccinc uscd is gcncrnlly cstablishcd by the particular country's regulatory agency in charge ot brucellosis control. Recently, in a number of regulatory programs 13. atJorrus straln RB.51 has replaced stra1n 19 as the approved calfhood vaccinc because it does not interfere with serologic evaluation. Bruce/la melitensis Rev 1, a partially attenuated vaccine, is uscd to control bruccllosis in goats and shccp causcd by H. melitensis. A killed product, Hn,cella melitensis H38, is also available. Bacterins for control of B. ovis are availablc, but their efficacy is limited. Bruce/la n1elitensis Rev l has been used to prolecl againsl B. ovis infeclions in sheep; however, il caunot be used in countrics free of B. tnelitensis because the antibody litcrs intcrfcrc with serologic evaluations for D. n-1elitensis intection. Vaccination is not practiced for control of disease caused by B. suis or B. canis.

110

PARr 11

Bacteria and Fungi

Laboratory Diagnosis Specimens Great care should be employed when working with infected tissues and cultures in the laboratory. Ali Brucella cultures should be handled following biosafety leve\ 3 pra1...tices l.Jecause uf tl1t: 9utt:ut.ial fur laborátory infecLion. All lahoratory proc:edures should he performed in a manner that prevents aerosolizatio11, and all work should be conducted in a biological safety cabinet. Appropriate san1ples for diagnosis ofbrucellosis depe11d on the aniJnal species affected, species of Brucella involved, and clinical presentation.. Abscess material, semen, and vag1r1al flulus assuclateu wiLll rece11L auv1 liuu::. a10:: u::.o::ful for rt>covt>ring organisms anten1ortem. Milk samples from cattlc and goats are uses in mi 1k. ·rhP test is p erformed •.uk tan k Tn ilk samples as a means of scrce11ing dairy - .)tained Bruce/la an tigcn is added to milk . If an tibod-e prescnt, agglutinated antigcn is buoycd to thc top '" rising cream and a purple r1ng develops at the top ot be (Fig 16.4). Iogit. Le:;L:; i:lrc 1.:ornrnonly used to Identlfy infected ~--- he rds and monitor hrcJ tilh1 in mammals is virtually unlirnited, and avian cases are rcported. Human intections range trom the rapidly fatal to the subclinical. .A. wet environment, such as a swampy terraln or rice paddies, is related to exposure.

nresence of 2% sodium chloridc, and at 42°C.

lmmunologic Aspects

---f'e =--~- '

and • survive chilling and freezing in biologic speci;s generally susceptible in vitro to fluoroquinoctracyclines, chloramphcnicol, trimcthoprim_z=:~ hoxa2ole, and novobiocin. --'--~,.,:-ria

pseudomallPi is kil1Pc1 hy

c1i~infectants

Complement-fi:xing and indircct hemagglutinating antibodies are produced duri11g i11fcctiur1s. Cell-mediated hypersensitivity has been demonstratPcl in infPrtP mi>thoc.ific flnorescent antibody conjugatcs can be applied dircctly to suspect colonies on p lates tor idcnlilication even o f dissociant colonies (epifluorescence). Poly1nerase chain reaction-based assays utilizing M. bovis-spccific primers are available for detection and idcntification.

Treatment and Control Affected animals should be placed in a dark stall, free from dust and fl1es. Topical corticosteroids may relieve the inflamrnation, while antimicrobial drugs, given topically or systemically, may be beneficia!. Long-ac.'ting tetracycline or florfPnicol ar(' considered the drugs of choice. Fimbrial vaccines a1e Lile 111osl ¡.irulllisir1g specific prophylactics.

Pseudomonas DwrGH'f C. HrRsH Members of the genus Pseudornonas are gram-negat ive, aerobic rods. Of the rnany recognized species of I'seudomonas, only P. aeru,'{inosa is of veterinary importance. Previously named pseud.omonads of veterinary importance, P. mallei and P. pseudornallei, have been moved to the genus Burkholderia (see Cl1apter 17). · Pseudornunus ueruginosu is very rarely involved wilh primary di.sease, although it is extremely important in clinical medicine. Most strains are resistant to the commonly used anti.luicrobial agents and are therefore sometimes dLfficult to eliminate when they contaminate a compro mísed site.

Descriptive Features Morphology and Staining

The organisms are gram-negative rods, O.Sto 1.0 ¡1m hy 1.S to 5 .() prn .

Cellular Anatomy and Composition

Pseudomonads produce a typical gram-negative cell wall, surrounded by a carbohydrale-conlai1ú11g cavsule. All members of t he genus are motile by n1eans of polar flagella. Pili (fimbria! adhesins) are produced. Cellular Products of Medica! lnterest Adhesíns. Pseudomonas aer11gínosa produces several prod-

u adl1t:siu;:,. Tlit:st:. iucludt: a JlJulJrial adhesin that has affinity for certain glycoproteins on epithelial cells. In addition, t here are non-fimbria ad.hesins, an outer memhrane protein witl1 affinity for mucin, and another, the lipopolysacchatide of the cell wall that has affinity for chloride channeJ proteins. Capsule. The capsule prot ects the outer membrane from the membrane attack complex of the complement cascade. The capsule also inhibits attachment to, and ingestion by, phagocytic host cells. Cell Wall. The cPll wall of the memhers of this genus is 011e typical of g.ran1-negative bacteria. The lipopolysaccharidc (LPS) in the outer 111embra11e is an important virulcncc dctcrminant. Not ouly is the lipid A compone11t toxic (endotoxin), but the length of the side chain in the 0 -repeat unit h inders the attachment of the me.m brane attack complex of the complement syste111 tu lile ouLer membrane, a11J ¡1 uu1ube1 of oll1er proteins with hiologic.al activity (proteases, phospholipases) . .E.xotoxins S, 1', U , and Y art~ "injected" into host cells by way of a Type 111 secretion apparatus (an assemblagc of protcins- morc than 20- tl1at form a holloi;.v tube-like structure through which effector proteins are "injected" into host "target" cells): l. Exotoxtn A. Exotoxin A i.nhibits protein synthes1s

hy rihosylation of Plongation fartor-2 (F.F-2) followh1g receplor-111edialed endocylosis. 2. Exotoxins S and T. Exotoxins S and T ribosylate host cell G1'f'-binding proteins, intcrrupting cell íun.ctions relying on the actin cytoskeleton, e.g., phagocytosis. 3. Exotoxin U . Exotoxin U is cytotoxic, but the mechanism is undefined. 4. Exoloxh1 Y. Exoloxin Y is a11 adc11ylyl cyclase that raises tbe amount of intracellular cAMP to damagi11g Jcvcls.

Miscellaneous Products. Pseudomonas aeruginosa produces bactcriocins (pyocins) and pigments (pyocyanins). Pyocins are useful epidemiologically for tracing epidemics within the hospital environn1ent. Pyocyanin has toxic acti\1ity and is used asan aid in the laburatury iJl:'.11LificalioL of P. aeruginosa. Pyocyanin rearts with oxygen to forro reactive oxygen rad icals t hat are toxic to eukaryotic and prokaryotic organisms. Pseudomonas aeruginosa protects i~­ self from thc toxic cffccts of pyocyanin by increasing syr.. thesis of catalase and superoxide dismutase. Product Regulation. Regulation of the expression and e...:cretio11 of cellular proclucts invulveJ i11 t!JI:'. palhoge11esh of disease producen hy P. aeruginnsa is complex. Secretio:: of products that are secreted by way of the 'fype III secretion apparatus (exotoxins S, 1', U, and Y) is initiated iollo•"-

Chapter 20

·,g hacteria-host cell interaction. 'fhe remaining bacteria] cell products are under the control of thc "quorum scns.ng" system of P. aeruginosa. ·rhe genes that encode these !J'FOducts are expressed when concentrations of bacterially ?IOduced homoserine lactones reach a threshold leve! (a -~uo.rum"). All P. aeruginosa cells excrete homoserine lac.011t::., IJuL Liie couce11 L1alion is Loo low lo lrigger virule11ce -zene expression until a critica! number of bacteria] cells is :eached. Finally, exotox.in A and a11 c11doprotcasc are also :eguJated by levels ot pyoverdin. When free iron concen:ration.s are low (as wo1lld be the case in vivo), these two protei ns are expresse:ising spread of the disease is attributed to increased deer populations, i11creasin.g hun1an movement into ru ral areas, and t he dissemination of infectcd ticks by migratory birds. 'rhe agent is harborcd by several ixodid ticks (.primarily Jxodes scapularis and J. pacificus in North America). The tick has a 2-year life cycle comprlsed of a larval, nymph, and adult stage, requiring a blood meal at each molt. Deer mice, white-footed mice, and oll1er sn1all rodenls serve as reservoirs for Lile spirochete. Borrelia burgdorferi has been isolated from the u rine of dogs and cows as well as milk from infected cows, potentially serving as aJternate routes for exposure and infection. Hu man Lyme borreliosis, caused by B. burgdorferi, typically begins with a skin lesion (erythema migrans) often followed vveeks or u1011ll1s laler !Jy ut:ural, cartliac, anu :irthritic complications. F.ndotoxin, hemolysin, immune complexes, and in1n1u11osuppressio11 n1ay be i11volved in pathogenesis. In othcr animals, dogs are most ftcn affected, witl1 manifestations of polyarthritis, fever, and anorexia the most coxnmon signs. 1vfalaise, lymphadenopathy, carditis,

auu rt:11al c.lisease lklVt: also lJeen noted in dogs. S11n1lar sih'Tls have also heen rt>pnrtf'rl in clomesti.c cats, thougl1 feline b orreliosis is rare. Borreliosis also occurs in horses and cattle . T11 horses, polyart hritis, ocular a11d ncural involvcmcnt, and foal mortality have been reported. Diagnosis Diag11osis involves den1onstratlo11 of Lhe agenL in lissues and fluids (darkfield, immunofluorescence microscopy),

130

PART 11

Bacteria and fungí

antibody in serum or other fluids (indirect immunofluorescent test, enzyme linked immunosorbcnt assay, ELISA), or DNA amplification of tissue or fluid samples using genus specific DNA primers and the polyrnerase chain re~ a.:lion.

Culture is laborious and often unrewarding. However, culturing cnr punch biopsies from infected dogs and mice has proven to be reliable. Culture of synovial fluid from affected joints is also possiblc. 13SK is n goorugs shown to be cffective in treating swine dysentery and intestinal spirochetosis in swinc include o rganic arsenicals, tylosin, gentamicin, nitroturazone, virginiamycin, and lincomycin. These have been used at low pro phylaclic lt::vcls, lJut it should be kept in mind that drugs used routinely to prPvPnt the disease will ultimately lose their effectivcness. Metronidazole is Lile 11:atl er felu ~ ~ulJ~peUes fetus in the ;-omach fluid of an aborted /amb. Gram stain, 1000X.

135

tein that increascs intraccllular cAMP follo,.ved by cell death (a cytolethal dlstcnding toxin, see Chapter 8); a protcin that has hemolytic activity (hemolysin); anda protci n tltat was shuwn tu Induce hepatitis in mice (hepatotoxin). All of these toxic compounds have a tenuous association wilh Lhe disease 111ucess lu l1Ulllaus and other ani:mals. Miscellaneous Products. Campylnhartt>r jejuni posscss a Typc TTT sccrction system (an assemblage of proteinsmore tnan LU-tnat torm a hollow tu bc-like st ructure th rough which effector proteins are "injected" lnto host "target" cells). ·rhe proteins involved wlth this syste1n1 Cía (for Campylnh;i1tPr inv;ision antigens) are responsiblc for triggering lhe uplake uf C. jejuni after adherence to intestinal epithelial cells. Growth Characteristics

...1psule. 'fhe glycoprotein capsule protccts thc outer -brane from tl1e mcmbrane attack complex ot the plement cascade. Thc capsule also inhibits attach ~to, and ingesuon by, phagocyrtc host cells. !l Wall. The cell wall of the members of this genus is :··pi cal uf grau1-11cgati ve bacteria. The l!popolysacchaLPS) in the outC'r memhr;ine is an impnrt;int viru~ determina11t. Not only is the lipid A con1po11ent -:: endotoxin). but the length of the side chain in the ~at unit hinders the attachment of the membranc at.:omplex of the complement systcm to the outer ...,rane. Lipopolysaccharide binds to the serum pro.:popolysaccharldc-binding protein, "vhtch transfers thP hloorl-phase of CD14. 'fhe CL)14-LPS complex '--'• to Toll-like receptor protcins (sce Chaplc1 2) on Llie ..e of macrophage cells triggering the rclease of proin...._.-:1atory cytokincs. _Tocoxin. campylobacter ¡e¡un1 1nvo1vcd with enteric e secrete a toxin "vith similar activity to cholera toxin .1e heat-lauilc tuxin (LT) uf Eschericl1ia coli (see •er 8) by increasing intr;ic.ellul;ir IPvPls of cAMP and ~ · ... letal rearrangements. Iloth toxins are in1n1unologi: Eolated and bind to the sanie ganglioside (GMl ) on _:face of tl1e target cell. Can1pylobactcr coli and C. lari -·'-ro tissue culture cells; a pro-

Can1pylobacter spp. a re m icroaerophilic (with the exception of C. hormnis which appears to be obligatcly anaero bic), re4ulriug an atmosphere contaln!ng 3o/o to 15º/o oxygen and 3% to :'l% 1arbon dioxide fo r growth. Sorne, such as C. ¡ejuni, grow at 42°C, a charactc1islic Lhal i~ useful fur its selectivity in isolation from intestinal sources. Unlike mcmbers of the fam ily Entcrobactcriaccac, thcy are oxidasepositive. They do not fer1nent or oxidize carbohydrates, generating cnergy from oxidation of amino acids or tricar boxylic acitl iuterrnetliares through the respiratory pathway. Though thPy poilheIial cells, and the c.:hemistry of thf' ce ll wall lead~ to the develop111c111 uf tliairl1ea. Diarrhea follows prostaglandin sy11thesis hy the recruitcd PMNs (and perhaps by the altccted host ce lis), as well as activation of vnrious inositol signaling path>vays within atlccted host cells. The net result is the secrction of chloride ions and water. The bactericidal effects of serun1 PntirP -act of the agent if they are not rcinfcctcd. Clearance rarely ...ies more than one year. The mechanism responsible for ra ring is unknown but is p robably due to the fact that tl1c ~:npylobacle1s have tu deal with an immune response as ·¡as the normal flora of thP vagina. Thc disease can be controlled by vaccil1atlng heifers or \"'S vvith bacterins o r by eliminating carrier an imals, iniding the bull. Bulls do not carry the organism effi::::!tly untll they are older than about tive years. The most 'nmonly held explanation is that the preputial crypts of _:er bulls a1e dee¡;cr and more hospitablc than the shal-er crypts found in yo11ngt>r h11 ll~ Reproductivc Discase (Sheep and Goats). Shccp aud guats = illlmune following abortion. The basis for this irnmu- is mainly antibody of the IgM and IgC types. These ....bollit!S btnd to the surface of the agent while it is in t he :'.ldstrpam, resulting in removal by phagocytic cells in -= '.~ ,-er and spleen. A11Liliud y lJound to the surface also ~tes the complement c.asraclf' leading to lysis of the --=:::r. Thc imrnune response is also effeclive i11 killing or- .sms that had reachcd the placenta but hacl not yet ~uced enough damagc to tcrminate pregnan cy. :':iertc Dlsease. Circulating antibody develops as a result -íection. The disease is self-limiting due to the com-=K~ effe::cts uf secretory antibody and the normal flora .

...:: ::ratory Diagnosis -- ¿

Collection

d11ctivc Disease (Catl'le). Samplcs for culture or obser-~~ are best taken trom the prepuce ofthe hull. SmPgma .:eted by aspiration into thc tip of an insemination -...,-""'- If the female is to be cultured, samples are col-- from the anterior vagina. With either sex, 101Jfi of = d 01 20 arürnals (whlchever is greater) are sampled -;nostic testi ng. ~-.iuctive Dísease (Shecp a11d Gouts). Sa111µl~s from the a.::a thc abomasum of the aborted fetus are most rP-~-." ~- The placenta and fluids of thc abortus are usually . :amlnated. :;:;;r;_ Disease. Fecal samples are taken for thc diagnosis ..111i infecliou~.

~-~

::.... amination

-'-'--·i·e Disease (Cattle). T11t: luw numbers of campy-

together with the high n11mbers of organisms of - ~al flora m akc it diffi.cult to observe Lhe carnpy--=:::::¡:¡~~ small curved rods) in stained s1nears (Gram or ::;;;:~;.

137

Romanovsky). Fluorcsccnt antibody-stained prepa1alio11s have preven useful in detection. Campylobacters exhibit a characteristic "tumbling" motility when observed in wct n1ounls of affectcd rnaterial. Reproductive Dí~P.ase (Sheep and Goats). Gram (carbol fuchsin as counterstain) or Ro1nauuv:.ky-~1:ained preparations of stomach contents from ahortPcl fPtuses often demo11stratc thc age11t (see Pig 24.1). Such fi11Lliug:;, in conj unction with doughnut-shaped necrotic foci somctimes found on the liver, help support thc diagnosis (scc Fig 74.5). Exau1ination of wet mounts of affected material is sometimes uscf111. Rnteric Disease. Stained (G1a1n sLaiu with carbol fuchsin as counterstain; Romanovsky-type stain) ~mears of fecal material will revea! numcrous slcnder, curved rods in n1osl cases ot diarrhea produced by C. jejuni. lsolation

Reprod11ctive Dísease (Cutlle). Smegma, vaginal fluid, or stomach contents are plateo onto media that contain antimicrobial agcnt:; (vonco rnycin, polyn1y:ti11 B V! e, a1IU trimerhopri1n are common Jy added to decreasc t he growth of noncampylobacters; amphoterici n Bis included in son1e forwulatlons to lnhibit growth of fungí). ·rhe plate is incubated at ~7°C in an atmosphere containing 6% oxygcn and 5% to IO'Xi carbon dioxiue. Piares are examined in 48 hours. Reproductive Díscasc (Sheep and Goats). Abomasal contenrs and liver (aborted fetus) are plated onto blood agar plates (with o r without antimicrobials, dcpcndin g upon lhe de::gree uf contamlnation). The plates are incubated at 37ºC in an atmo-;phere containing 6% oxygen and SO/o to 10% carbon dioxide. Plales a1t! exa1nin~d In 48 hours. Enteric Disease. Campylobacter jejuni and C. coli are best isolated from affccted intestinal samples on selecl.ivt! m edia containing antimicrobial agents (e.g., Campy-CVA containing cefoperazone, vancomycin, and ampl1otericin B). 'fhe plates are Incubated at 37ºC, or at 4zvc when isolation of r.. jeiuni or C. coli from feces is attempted , in an at mosphere of 6% oxygeu aud So/o to 10o/o carbon dioxide. ldentification

Tsolation of a gram-ncgativc, curved rod that is oxidasepositive is presumptive evidcnce that a rnember of the genus Campylohacter (or "Arcobacter," see bclow) h as been isolalt:Ll. Tl1uugh thcre are a number of fermentation reactions that havP hl'Pn rlP~rrihed far the identification of Carnpylobacter, thesc are tedious and Li111e consumlng. Methods based on detection of specific DNA sequc~nces may be used for identification (e.g., gencration of fJagments of a certain s1ze following amplification of specific DNA sequences by polymerase chain reaction, or dctcrmining the seque::11 s::i mP ::itmos.ric environment as do campylobactcrs. They wilJ, how~ grow in a microaerophiliic atmospherc.

Descriptive Features Cellular Products of Medica! lnterest intrace1l11/aris exprcsses a surface protcin, LsaA (for Lawsonia surface anlige11) that is responsihle Adhesin~. T.aw~nnia

for the attacl1ment to and entry into target cells (the ;ipical surface of in1mature intestinal epithelial cells). c·eu Wall. ·rhc cell wall of the mcmbers of this gcnus is one typical of gram negative bacteria. 1'he lipopolysacchartue (LPS) tn rhc ourer memorane 1s an 1mportant virulencP c1PtPrminant ot only is thc lipid A component toxic (cndotoxin), but tl1e lc11glh of L11t: sille cl1t cells in the gastrointestinal tract and to cells comprising the niche for thc strain. Helitubuc.ler pyluri expresses at lcast two adhesins with spt>c:ifirity for eiistric epithelia:

l. Sialic acid-blnding adhesin (SabA). SabA (for sialic acid-úi11ui 1tg udhesin) binr mi>mbrane. LPS binds to lipopolysaccharide-binding protein (a seru111 pr0Ltd11), which in turn transfers it to the blood phase of CD14. The CDl4-LPS complex binds to Toll-likc receptor proteins (see Chapter 2) on thc surface of macrophage cells triggering thc release of proinflammatory cytokines cag Pulftu8eriic1y lsland. cag (for cytotoxin-associated gene product) Pathogf'nicity Island (a cluster of genes en141

142

PART

11

Bacteria and Fungi Table 25 .1.

Habitats of Helicobacrer species

Helícobacter taxon

Source(s)

Primary Site

Human, cat, dog, cheetah, primates. wild rats Human, cat, dog Human. wild geese Human, hall15ter, macaquc, dog Human Human. dlicken Human, m;;raque. cat Human, dog, sheep, mouse

Stomach

Human

lntc1tinc

Hamster Cheetah Mouse, dog, rat cat Dolphins, whales Hamster Cat. dog Mouse Mouse Cat woodchuck Hamster

Stomach. intestine Stomach lntestine Stomach Gallbladder Stomach lntestine lntestine lntestine lntestine lntcstinc Stomach lntestine lntestine Stomarh Stomach lntestine lntcstinc

Secondary Site

HUMAN H. bízzozeron/F' H. canís H. canadensis H. cinaedi

H. fennel/iae H. pullorom H. pylori Flexispitil (Helkoúdth!r) taxon gb H. winghamensis

lntestine lntestine lntC$tinc lntestine lntestine Stomach lntestine

liver {dog) Blood, brain, joint (human)

Liver (ú1itke11)

Placentalfetus (sheep) Blood (humans)

NONHUMAN H. aurati H. acinonychis

H. bilis H. cetorom H. cholecystus H. fe/is H. ganmani H. hepatictl( H.

llldllllUld~

H. mesocricetorum N. muridarum H. mustelae /-/. pametensis H. rodentium H. salomonis H. 5UÍ~ H. typhlonius H. trogontum

Mouse, rat

Ferret, mirik Birds, swine Mouse Dog Piy~

Mouse Rat

liver {mouse)

Liver/woodchuck

• t ikely the same as •H. hei/maMií.• "flelicobaeter heilmannii" (form¡rly Gastrospirillum hominis) has the same phenotype as listed here for H. biuozeronii. Only a single "H. heilmannii" strain has been isolate, Vac, and orease, together with certain proteins exp.ressed by genes located on the cag Pathogenicity Island), which ln tl1e persiste11tly infected host, initiate sustalned productlon of proilúlan1111all)ry cyloki11es. Pathology Helicobacter

fe/is and "H. bizzozeronii" ("H. heilmannii")

llave been associated vvith gastric h istopathology in laboratory-reared beagle dogs. When these b acteria were ob~erveu il1 lovv 110111\Jers e.g., i11 lhe fundus o f lhe slo1nach, the organisn1 was considered innocuous; ho,.vever, in large numbers, as seen in the cardia and fundic pyloric junctio11, thc organisn1 1nay induce ly1nphoreticular hyperp lasia and n1ay cause premature se11escence of parietal cells. The presence of gastric Helicobacter-l i ke organisms (GI-ILOs) is often accompanied by reduction in m ucus content of sur-

face epithelia, occasional intracpit hclial lcukoc;son1e degenerating glands. Of the glandular epitl:1e .: only t h e parietal cells are markedly altered ...\b- -. findings include vacuolation, enlarged size, and :- ~---­ degeneration consisting of hoth karyolysis ancl - ¿~­ rhexis. The presence of large nun1bers of !l. pyl~ gastric n1ucosa of commercially reared cats is ass. ~~ with a lymphofollicular gastritis, characterizcd tr phoid aggregates and diffuse int1ammation in th.: 1n.ucosa and lamina propria. Helicobacter pylori colonizes the gastrointestinal ="'gnotohiotic ciogs o rally rhallenged with this mirrr>---ism. Such dogs are colonized with JI pylori in all pa.tl1e stomach examined: cardia, fu11dus, antrum, an:. loric ant rum; thc fundus is tl1e 1nost heavHy colon .. Focal to diffuse Iymphoplasmacytic infiltrates with f.,,-~-­ formation a11d focal infil tration of neutrophils eoslnophils ln the gastric lamina propria are olJ5e:~"'"~· Also, gnotobiotir ciogs orally inoculated with H. fe/i.; the organisn1 recovered from all areas of the stomacl1, colonization being 11eaviest in the body and an~ Occasionally, H . fe/is is obscrvcd vvithi11 the canalicu.. gastric p ariet al cells. Ex peri m entally, "H. bizzozeronii" ("H. heílrnan--; causes m ucosa-associated Iymphoma in rnice and is ü :1, with the saine d iseases in h u mans. The hisLopalhological changes occurrh1g in ll1e sl, _ ach closely coincided in topography with the presenet H . 1nustclae. A superficial gastritis p resent in the bod;· t he ston1ach shows tha t H. rnustelae is located on the sl.4face of the mucosa b ut 11ot in the crypts. In the dw._ antru m, infla1n mation occupies t he full thickness of t::,, mucosa, t he so-called d iffuse antral gastritis described ~ humans. In t his locatio n H . mustelae is seen at the surfaC>.: in the pits, and on the superficial portion of the glancis. ·tl1e proxin1al an trum and thc transitional mucosa, a prtcancerous lesion, focal gland ular atropl1y, and regeneratio11 is present, in addition to those lesio11s seen 111 the d istal antru1n . The liver lesion present in naturally H. hepaticusir1fecteu rnice prugressively ir1lii lUUI::>:>, Vi JJt:liVUÍL vpl1ll1al1uia) ale lht' lUU:>l

Cellular Anatomy and Composition

common manifestations in horses. California sea lions are susceptible to acutc, scpticcn1ic le.ptospiral infections. Other host species. though susceptible to infection, develop clinical signs less frequently. Leptospirosis in humans is typically an acute febri le disease. 'l'axonomy studies, based on DNA analyses, have led to the description of eight pathogenic species: Leptospira

Leptospiral cells consist of an outer sheat h, axial fib~ ("endoflageJJa"), and a cytoplasrnic cylinder. '!he oute: sheath combines features of a capsule and outer m ee: brane. A cell mernt)rane and t he peptidoglycan !ayer of tt..:: cell \vall cover the cytoplasm ic i>ssentially rahhit sPn1m (10ºAi), in sr...lutions of peptones, vitamins, electrolytes, anct bufft:.!' Sorne newer media have substituted polysorbates a~ bovine albumin substituted for rabbit serum. Protein is r. required. Unlikc most prokaryotes, lcptospi rae are not ab•.:: to synthesize pyrimidines and th us 5-fluorouracil is addE:to growth media as an inhibitory agent to contarrlinati::~ bacteria and fungí .

Chapter 26

Spiral-Curved Organisms V: Leptospira

149

F 1G U RE 2 6. 1 . Leptospira interroga ns, serovar pomona in the renal tubules of a pig. Levaditi si/ver stain, 1000X.

media are fluid or semisolid (0.1 % agar). In fluid __ dia , little turl.Jillity Llevelups. Iu seruisulid u1edia, :::::;:::-,\· th is concentrated in a disc- called a "dinger zone".....:cut 0 .5 cm below the surface. ~-íost

· .;{hemical Reactions '"""-ptospirae are oxidase and catalase-positive; many have .:tase activity. Sorne produce u rease. Identification be-o:Jd genus is based on serology. Species-specific L)NA pri_ ers in conjunclion vvilh Lhe polyn1erase chain reaclion :CR) have also heen developed for a more accurate char1CTerization of pathogenic leptospirae.

;:>istance :_¿ptospirae are killed by drying, freezing, heat (SOºC for 10 °!liJ1uLe:s), soav, bile salLs, deLeigenLs, acidic environrnenls, .!..:d putrefaction. They persist in a moist, temperate envi:;Jnment at neutral to slightly alkaline pH (see "Epidemiol::-gy," below). .ari¡ibility _.!o re Lha11 200 servvars o í varasi lic leIJLOSIJirae exi:sL Tl1t:y ira serovar pomona in cattle causes intravascular ben1olysis due Lo a hen1olylic exoloxin. Autoimmune phenomena may also contribute to this condition. Sccondary changes include icterus due to liver damage an.d blood destruction, and acute, subacute, or chronic nephritis dueto renal tubular injury. 'l'he cellular exudates contain predominantly lymphocytes and plasma

150

PA!tT 11

Bacteria and Fu11gi

cells. In surviving ani rnals, leptospirae are ren1oved from circulation with the appearance of antibolow). l .Pptn~pira sprovar pnrnnna and an as yet unidentified leptospira havc bccn cuJtured from the aqueous humor of horses with clinical signs of this disease. Miscellaneous Species. In small ruminants, Jeptospirosis, usually dueto serovar pornona, resembles that seen in cattle. Tnfectlons wlth seruv ci>ss;i::>n of septicemia and the appcarancc of circulating anti- ....~P proc111rts, ~nrh ;:is endotoxin, may be re-:-onsible for the adverse reactions reported in dogs. ln North America, cattle and swine are vaccinated with pentavalcnt bactcrin containing thc most common :Jrth American serovars (fzardjo, grippotypfzosa, pomona, -.aohae1norrhagiae, and canica/a), with thc addition of ~:o\·ar bratislava anda second scrovar f1ardjo component .:: sorne vaccines. Humans who are at risk may opt for vac.naliuu as we;:IL ProLecLiu11 i::. ::.eruvctr-:.pecifispi>cially for serovar hardjo, the slowest growing of the com rnon scrovars. Replicate inoculations are 1nade into EMJH medium with and without sclcctivc inhibitors (5-fluorouracil, neomycin, cyclohex1m1de). Cultures are exam ined microscopically at intervals during incubation fui up lo :.everal 1noutl1s. Animal inoculation (hamsti>r' or gninP;:i pigs) eliminates minor contaminants from the prirnary inoculun1, which is injccted intraperitoneally. Blood is drawn periodically for culture starting a fcw days aftcr inoculation. Aftcr 3 to 4 weeks, the animals are killed and their kidneys examined and cultured for leptospirae. Jf irlfected with lepto.splrae, they will have dcvclopcd antlbody. Any isolate ri>rovi>ri>c1 hy thi>si> methods can bC' identified morphologically as a 1nen1be1 of Lhe ge11u::. Lcplu:;piru. Defi1litive ider1tification is carried out hy refercnc:e J;ihoratories.

le(hanisms of Resistance and Recovery

1

_aboratory Diagnosis iagnosis of leptospirosis must be established by labora"•Y co11fi1111aLio11.

!:mple Collection

.,. uu1 1iviI1g sulJjects, t;lood, urinnt;:i I cotylc~dons are examined. ~lood is usually negative after the first febri le pl1ase. Milk destructive to leptospirae and nota pron1ising source for _-.iltures. Urine should always be testcd. From cadavers, including aborted fetuscs, kidneys are ±e most likely organs to harbor leptospi rae. In septic fatal..:t::. (i11yP, may contain the agent. Culturing is done promptly after san1plc colleclion.

Serology

Dircct examination is often unreli;ihlP 11nc1 r11lt11re l11borious, cxpensive, and slo'"'· Serology is the most common diagnostic method. ·rhe microscopic agglutination test employing live antigen is most widely uscd. Othcrs include macroscopic plate and tube agglutination tests, complemcnt fixation tests, and enzyme-linked antibody a:-.say:.. Paircll sa1nples are preferred: onc collected at first presentation and one 2 wi>Pk.~ lllti>r. Tf leptospirosis was the problern, a fourfüld or greater rise in titer should have occurrcd in the interval. In bovine abortion, these relations may not hold. This is duc to thc fact that scrovar /1ardjo infections ot cattle clicit a very weak immune response that is probably dueto their adaptation to this an hual species.

152

PA1rr TI

Rs. ~ protein ("clumping factor," "bound coagulase") is y present in S. aureus and S. interrnedius. Clumping - intcracts in vitro with fibrinogcn to produce an --.,,...-~-nation-lilcapsule," a loosely associated carbol1ydrate strucp1oclucec.1 lJy :strains staphylococci are internalized by nonprofessional

phagocytes (endothelial cells, sorne epithelial cells), but c:;cupc th.c cn.do:>oroc to multiply within t hc cy

toplasm. Endoso1nal escape is thought to be associated vvith alpha hemolysin-mediated lysis of the en-

dosomal membrane. 2 . Beta tox.i11. Beta toxi11, a phospholipase C is prt>valent i11 a11in1al strains. Beta toxin p roduces broad

zones of "hot-cold lysis" on sheep or cattle blood

F1G U RE 2 7 . 2 .

Staphylococcus beta toxin activity on bovine

b/ood agar. For explanation, see text.

Chapter 27 flammatory responses and tissue damage. Gamma toxin is not observed surrounding colonies growing 0111.JlouLl agar plate::; ::;iuce il i::; i11hibitecl l.Jy agar, l.Jut viTtually ali strains of coagulase-positive staphylococci produce the toxin. _ Delta toxin. Delta toxin lyses cells of various species by a detergent like action but is inh ibited by seru·m. Like gamma tox.in, almost ali strains of coagu lasepositive straios produce this toxin. Its role in disease, however, is undefined.

Acquisition. Staphylococci with pathogenic poten~e., coahTUlase-positive strains) grow bett er in iron-

- ;;n

~~-":cted

Staphylococcus

155

Peptide Pheromone or AIP, wbich is the modified product encoded by the agrD gene) produced by other staphylocucci iI1 tl1e irrnnertion of vancomycin-rcsistant strains by the gro,'\'th promoter

166

PAKT

11

Bacteria and Fungi

avoparcin is discussed below (see below, "Epidemiology," and Chapter 4).

Ecology Reservoir Enterococci live in the intestil1al tra1.:1. uf 111a1nrnals and hirds as part of the normal flora of these species. Whether the enterococci associated with "primary" disease, H. durans, E. hirae, E. víllorurn, as opposed to opportunistic-type discase, are inembers of the normal flora, is unknoiv11. Transmission

Enterococci that are associated vvith opportunistic disease are part of tl1e norn1al flora of the 11ost. It is unknown whether this is true for E. durans, E. hirae, and E. villorum. Pathogenesis Except for E. durans, E. hirae, and E. villorum- associated disease, endogenous enterococci infect a comprornised site (e.g., urinary bladder, moist externa! ear canal, catl1eter). The cell wall peptidoglycan and Upoteichoic acids initlate an infla1nmatory response. Capsule, cytolysin, and superoxide puteutxternal ear canal. 'fhe bacteria involved are usually an environmental species (e.g., Pseudon1011as and Proteu_s, see Chapters 20 and 7, rcspectively} ora member ot the patient's normal flora (e.g., Enterococcus, Staph}'lococcus interrnedius, see Chapter 27). Enterococcus is a common isolate frorn do8S with lower uri11ary tract infections (see Fig 74.2B). F.ntf!ror:ot:t:11s spp. (P. durans, E. hirae, E. villorum) are associated with diarrhea in puppies, kittens, and adult dogs and cats. Aln1ost any compro1niscd site may be contaminated \-vith an enterococcus. Horse. Enterococcus spp. (H. durans, E. hirae, E. villorun1) are associated with diarrhea in foals. Entcrococcus spp. should be expected in any co11dltloa th.at resutts from co11tamination of a compronused s1re witl1 fecal material (e.g., street nail/sole abscess, ~.vound) . Cattle. Enterococc;us spp. (E. durans, E. hirae, J::. villorurn} are associated with diarrhea in calves. Enterococcus spp. should be expecteú in any conditlon that results from contamination of a comprornised site with fecal material (e.g., wuu1H.l).

Swine. cnterococcus spp. (E. durans, E. fzirae, E. villon1m associated with diarrhea in piglets. Enterococcus spp. should be expected in any 1.:unvere discovered as sn1all colonies g iowing as saLelliLes arou1 1Ll other bacteria] coloni.es when samples obtained from normal human m ucosa! surfaccs (eycs, genital tract, mouth, respiratory tract) were inoculated onto blood agar plates. 'fhe bacteria comprising t hese colo11ies were gram]JU:;itiv«::, robes (set> C haptt>r 35). Epidemiology

Arcanobacteriurn pyogenes causes suppuratlve

- _:cesses, usually complicatecl by other potentially pa tho~.Jc con1111ensals, especially non-spore-forn1ing anaer-.e-; ( Rar.tr.rnidr.s, Pusnhar.teriurn, Porphyrnrnonas, Prevotella, -.ostreptococcus, see Chapter 35). Pyolysin O (PLO) is a ~ or virulence determinant (supported by the finding -~- antibodies to PLO are protective, and mutants defi--:t in PLO are avirulent). Deposition of A. pyogenes (fro1n ~ ;itiguous surface or from the iin1nediate environn1ent) .;._,- a normally sterile ~ite initiate~ au iuflauunatury re--~,p (arlhPrPnc.P., hy way of Nans, cell wall constituents, ::;,on of PLO). The exudate (pus) consists of bacteria, neu... ~il s (live or dead), and host cell debris. Patl1olo3y. The lesions are abscesses, empyemas, or pyo---.-_uJomas. Abscesses are often heavily enea psulated. ~ offensive odors are contributions of anaerobic parti-

Because ,4. pyogenes is a permanent resident of susceptible species, disease prevalence is sporadic and governed by precipilali11g slress or trau1na. "Sum1nf~r mastitis" is most prevalent in northern Europe. Jt .is spread by flies attracted to traumatized teats.

lmmunologic Aspects Itnmune r~~sponses to A. pyogenes are not '"'ell understood . Infection or vaccination confers no useful resistance. The usefulness of pyolysin as a vaccine remains to be demon strated, but toxoids produced from PLO are protective in laboratory anima1s.

Laboratory Diagnosis

~t:; .

- -:-;se Patterns

Arcanobacteriun1 pyogenes is involved in most puru - 'nfections of traumatic or opportunistic origins; -~~-.:i may be local, regional, or metastatic. Common lo__,.___~· ~iu11~ CJre the luug, pericCJrtliurn, entlocCJrtliurn, ___,_:ti. p eritoneum, liver, joints, uterus, renal cortex, =::::.. bones, and subcutaneous tissues. In other suscepti;pecies (sheep, goats, wild rurninants, S>vine) similar le.:;s may be found. --~mobacterium pyogenes causes abortion and mastitis in. _ ::.e. "Summer mastitis" is a communicable disease '--""'ng pdsturells. Pat11ology. In lissue, spores genninale and lhe vegetali\-. form prolifera tes, prodúcing gelatinous edema. Inflarrunatory rcactions are minimal. lnfection disseminates to reticutoenc1othelial sites. When these are saturated, a termina. bacteremia occurs, with enormous numbers of organism... in circulation. Postmorten1 findings are widespread llemorrhage.s; a hlack, e.ngorgecl, friahle spleen; tarry, nonclocting blood¡ and abse11ce of rigor n1ortis. Bleedi11g at bod orífices is common. Disease Pattern

·rhe process described above is typical for rfit: n1ost susceptible species- cattle and sl1eep. 'fhe coUJ:k. following an incubation period of 1 to 5 days, ranges fro!"'" a few hours to 2 days. Sorne animals d ie without overt cTI:ical signs. Othcrs dcvclop high fcvcr, agalactia, and t b .... n1ay abort. ·rhere is congestion of mucous men1bran~ hematuria, hemorrhagic diarrhea, and often-regior_ edema. 'l'hese forms are regularly fatal. Occasio11al arli.ma... show just localized edema oran ulcerative sk.Ln lesionar;.~

Ruminants.

recover.

Horses. Horscs dcvclop colic and diarrhea¡ edema also e~­ curs, particularly of dependent parts and at the point of w-

Chapter 30

Bacillus

173

"ection (e.g., the intestine or thc throat) where it may cause death by asphyxiation. Altern;:itively, thP course m;:iy -.e sepliccn1ic, as in run1inants.

produced by microorganisms lnto which the encoding 8PnP h;:iPl1 placP~ate precedes death. Inhalation anthrax (e.g., "wool ner's dlsease") produces pulmonary edema, hemor:nagic pnP11monia, ano somi>timPs mi>ningitis. Mort;:ility :zces approach 100%. :oidemiology -. soil rich in calcium and nitra te, with a pH range of 5.0 to :..O, favors sporulation and bacteria] prollferation at te1n:;-ieratures above 15.SºC (60ºF), especially after flooding. ~e geogiaphy a11d seasonalily of oulb1caks rellecl such :ucumstances. ln cattle, sheep, and possibly horses, out=:eaks begin with a few cases contractcd from thc soil. ..!ter excretions and postmortem discharges seed the area, ;econdary cases occur. Floods and industrial effluents ::om rendering works, tanneries, carpet milis, brush facto-::es, or wherever else carcasses are salvaged may contami--:3te areas. Bone mea!, an animal feeh ir lP in noni>nc1Pmir ilrPilS. \.;:irnivores (m ink) ?.;e usually exposed via infccted n1cat. Human exposures are contracted in occupations deal..::g with animals and animal derived material such as im X>rted h1des, wool, and bone. Anthrax occurring under in.::.istrial conditions is often the lethal airborne version.

11munologic Aspects ::~i>erimmune

sera can prevent and alleviate disease. -_'1tibactenal and antitoxic factors are thou¡;ht to be in"'Olved . In most species, immunity is directed against the -:otecuve anti.gen. Capsular polypeptlde falls to stimulate :-· otPctivP ;intihnn y

Artificial immunization of livestock has utilizcd 1nostly ~dified live spore vaccines. Currently these are derived -om avirulent (noncapsulated) mutants. The most widely -Sed is the Sterne vaccine (a strain of B. anthrads that lacks ·~e pX02 plasmid). A cell-free vaccine consisting of con_culrateu l:ulture filtrate has l.Jeen used on humans ex- sed to industrial anthrax. lt prod11cP~ tPmporary p rotPC'-'ln against cutaneous infection. Protective antigen,

Sample Collection During sample collection, precautions against contaminatlon ofthe environment are important. Blood maybe aspiratcd from a superficial vessel. Aqueous humor has the added advanlage oí ren1oleness f1on1 sou1ces of early poslmortem contamination. Por direct examination, bloody dischargcs fron1 orilices are samplcd. lf the carcass has been opened, spteen nlaterial may be collected . Direct Examination Blood and organ smears are stained by Gram stain and a capsule stain such as McFadyean's methylene blue. Chains of encapsulated, gram-positive, non-spore-torming rods (see Figs 30.l and 67.1) suggest B. anthracis. Contaminant Baclllus spp. are usually not encapsulated and lack the clipped, squared-off appearancc of anthrax bacilli. Fluorescent antibody helps in. the diffcrenlialion. lsolation and ldentification Bacillus anthracis grows on all common media. Presumptive ictentitication can be made by the characteristics outli ncd in Table 30.1 and the "stri ng of pearls" test (the characteristic blebbing that occurs when B. anthracis contacts penicillin). Definitive identification is by specific bal:lcriu¡Jhagt: (ga11Lllla ¡Jhagc). Fluu11:::.cc11l a11libody a11d IPctin of appropriate specific:ities are helpful. Bacillus cereus is a commonly cncountcre of horc:PmhP.rs of the gen us r:oryneb..;.;:;;. riu111 of C 22- C: 38) . Cellular Products of Medical lnterest Adhesin. Members of tl1e C. renal e Group express a fibr> prutel11 atil1e~l11 (pili, fi1111Jriae) uu Lheil :>u1fc11..e;:,.

\,e/l Wall. The gram-positive cell wall contains

pol~

charides, and lipids that are of medica! interest. The lir ichoic acids and peptidoglycan of the gran1-positi,-e

Chapter 31

Corynebacterium

179

\Vall interact witl1 macrophage cells resulting in the release

and pyelonephritis. Rectal palpation reveals thicke11ed

of proinflammatory cytoldnes.

bladder and ureteral walls, distended u.reters, an.d enla.rged

Urease. Meruuer:; uf tht: C. renule C;roup p1oduce urease, ;.;h ic.h is assoc.iated with the virulence o f this group.

kidneys wilh obscured lobulatio11s. Early cases show pollakiuria, hematuria, and increasing degrees of abdominal pain. Chronic infections progress to debilitation and death due to ure1nia.

~Iowth

Characteristics

"'1embers of the C. renale Group are facultative anaerobes i::apable of gro,"ling on. most common laboratory media as -LOnl1e111ulytic, opaque, off-·wI1ile colo11ies ll1al develop i thin 48 hours at 37ºC= on blood agar. .: ~ochemical

Activities

• ~embers of tl1e C. renale Group have impressive urease ;ctivity, which is demonstrable in most strains v.rithin din ules of conlacl wilh its subst1ate. Glucose is slowly _ddified, otber carbohydrates variably so. All strains are :::a:alasc-1Josjtive. Mcmbcrs of thc C. renalc Group produce :::. protein, renalin, which results in a positive CAM1' test see St-reptococcu.~ agalactiae, Chapter 28). ~ ;.sista nce

agents are not particularly resistan.t to heat, disinfec-:=_.-¡ts, or a11tin1icrobial age11ts. -"lt'

Small Ruminants. Ovine posthitis ("pizzle rot"), the more common form of infection in sheep, is a necrotizing inflammation of the prepuce and adíacent tissues in wethers or rams. Disease clevelops in the prese11ce of the urealytic age11l il1 a11 area constantly irrigated witl1 urine. An1n1onia is thougl1t to initiate the il1fla1nmatory process. A sirnilar condition. occurs in goats. Only C. renale and C. pilosurn have been tound in ovine posthitis. Epiden1iology Bovine pyelonephritis is found rnostly in cows near parturition, appearing asan opportunlstic infection by a comn1ensal organlstn. Bulls are rarely affected, but are commi>nsast> is rt>cognized prima.ri.ly in Brita.i n a.n d continen~ Europe but also in North America, Australia, and Ho::;

Kong. 'f'reatment is rarely successful.

(ORYNEBACTERIUM AURICANIS •

í:nrynPhartPri11m

a11rirani~

i G +C. A ~ccharidc capsule has heen desr.riheci :ind rPl:itPcl to --ce. Little is kl10V1'l1 about thc surface proteins of ---dt:Jthrix. A protective protein, SpaA, shares similari- the e terminal regi.o n with choline-binding pro_: Srreptococcus pneumoniae. =.....::--,._,

Ptoducts of Medica! lnterest

- - ' - See "Neuraminidase," below. c'4/e. Erysipelothrix rhusiopathiae produces a polysac-

charide capsule, whicl1 protects the microorganism from phagocytosis. Ce/l l!Vall. The cell wall of the 1uembers of this genus is onc typical of gram positivc bacteria. Thc li.potcicho!c

acids and peptidoglycan of the gram-positive cell ,.vall interact wi.th macrophage cells resulting in the release of ¡.iroi11 ílau1111i:ltury cytokines. Neurarninidase. Nr.ur:im in icl:ise prociuction varies directly with virulence of E. rhusiopathiae. Cleavage of sialic acid residues on endotl1elial cells leads to thrombus formation. Neuraminidase is also responsible for adhcrcncc to cell surfaces. Miscellaneous Products. Most strains of E. rhusiopathiae produce hyaluru1üdase auu coagulase, tJut there lloes not appear to be a relationsh ip ben-ve.en vi ru lencP :inci thPsf' enzymes.

Growth Characteristics Growth is best on inedia supplemcnted with glucose. Erysipelothrix rhusiopatlliae is a facultative anaerobe preferri.ng an enviro11ment co11tainiI1g 5% to 10% COz. Optima! growth occurs at 30ºC to 37ºC and at a pH of 7.2 to 7.6; however, it is capable of growing overa temperature range of 5ºC to 42ºC anda pH range of 6.7 to 9.2.

Resistance Erysipeluthrix rhusiupathiac is resistant to drying and with.st:incls s:ilting, pickling, :incl smoking. Tt s11rvives for 11p to

6 months in swine feces and fish slin1e at cool ten1peratures. ft is killed by moist heat (SSºC) in 15 minutes, but grows in thc prcscncc of potassium tclluritc (0.05%), crystal Violet (0.001 º/o), phenol (ll.2o/o), and sodiu1n azide (0.1 %). lirysipelothrix rhusiopathiae is susceptible to penicllll n, cephalosporin, clint1amycin, and fluoroquinolones but is resistant to novobiocin, sulfonarrlides, and aminoglycosides. Resista11cc Lo eryll1ron1ycin oleando1nycin oxytetracycline, and dihydrostreptomycin has been obscrvcd. Rcsistancc is apparently not plasmid-mediated. 1

1

Variability Common heat-labile antigens account for cross reactions betwee11 strains. Heat-stab.le, somatic antigens account for the existence of at least 23 serotypes. No relationship bet1-veen 11osl species a11d :serotype has tJeen recognized. Serotypes 3, 7, 10, 14, 20, 22, and 2~ exhihit ;i highPr con!iinti>rally, or, in some countries, by aerosol. Whole-cell bacterins and soluble anligen a1e given subcuta11t:uu:sly ur intramuscularly. Most commercial vaccines are prepared from serotype 2. Ccrtoin strains havc becn rcfractory to vaccine-1nduced 1mmunity. Formalin-inactivated, aluminum-hydroxide-absorbed bactcrins appeaI to be effecUve i11 lurkt:y:..

Laborat ory Diagn osis Specimens

Specimens are collectcd from appropriate sites according tu :sign:s. Bloutl lultures from severa! affected ani.mals ate useful in d i agno.~ing media contain sodium azide (0.1 %) and crystal violet (0.001 %). Serology is ot little value in diagnosing erysipelas infections. Polymerase chain rcaction assays have becn described for diagnostic use.

Treatment, Control, an d Prevention Treatment with penicillin for at lcast S days is effective againsr the acure forms of erysipclas in swine. 'letracycline and tylosin are alternatives, although resistance to oxytet 1acycli11t: autl sorne macrolides has been reported. Antiserum (e origin) is sometimes used in conjunc-

184

PART 11

Bacteria and Fungi

F 1G U RE 3 2 . 2 . Hydrogen sulfide production by Erysipelothrix rhusiopat hiae a/ong srab fine in triple sugar iron agar slanr.

tion vvith antibiotic therapy. Treat1nent of ch..ro.n ic forros :... Jess successful. Good sanitation and nutrition are beneficial in p:-.:venting outbreaks. Infected carcasses should be dispose_~ of in a proper manner and replaceme11t aniinals isolata'.. for at least 30 days before il1troductio11 into tl1e h en:.. Vaccination is recommended in areas wlth prevlous bistory of erysipelas. In turkeys, pei.1lclllin. is the drug of choice. Subcuta:ieous in jection of penicillin and vaccination with erys:pclas bacterin are recommended, if practicable. Penicill!n in the drinking water for 4 to 5 days has been effective ~ controlling sorne outbreaks. lnjectable erythromycin is recomn1ended alternatlve treatme11t.. y cell-mediated responses. Humoral facto rs may play somf' limitf'ci rnlf' in hnst ciefense. No immunizing preparations have met vvith ~ignificant success. Killed preparations have been ineffective, while live attenuated vaccines afforded SOlne protection in shccp.

Laboratory Diagnosis Specimens Laboratory dlag11osls ls based u pon isolation of the organism. Spinal fl11id , blood, brain tissue, spleen, liver, abomasal fluid, and/or n1eco11iun1 are cultured, dependiI1g on signs, lesions, and tissues available. Direct Examination A direct smear of infected tissue may reveal numerous gram-posi.tive rods in septicen1ias and abortions; however, fewer nun1bers of organisn1s are Lypically ol>serve optim;:il. CrrO\'\'th is usually visible within 1 or 2 days. Colonies are oftcn irregular in shape and contour. Severa! clostridia S'i'Var1n ncross moist ngar media \.Yithout forming colonies. Most clostr1d1a produce hemotysis when gro~vn on blood agar. 198

ERNST T.. R rRF.RSTF.TN

Tn liquid media, clostridia often grow in air provided a reducing agent Is present (cooked ineat pieces, t hioglycoIate), though growth occi1rs only in red ur.ed p ortions of tl1t 111tc.liuu1. Biochemical Activities

Clostridial cultures typically cmit putrid odo rs d ue to products ot pcpticte catabolism, which is a comm o~ mode of energy production. Most clostridia attack carbohydrates, proteins, lipids, or nucleic acids. Biochemical reactions and their end products furui~ll a ua~i~ for ~µtcit~ idtnlificaU011. RGsistancG

1'he vegetative forro is as susceptible to environmenta. stresses and disinl:ectants as other ba111a.ll ir1te:.tiual eµitl1elial (.:ells It:!:>ultiug ir1 fluid and electrolyte ahnormalities, as vvell as providing access to tight junction proteins (specifically claudins and occuldiI1s). lnteractions of Cpe with tight junction proteins result in. further losses in control of tluid and electrolytes. 12. Perfringolysin O (also kno\.vn as theta toxin) . Perfril1golysin O (Pío íor perfrü1golysin O) is a cholesterol-binding cytolysin (see also novyilysin and chauvcolysin, below; streptococcal streptolysin O, Chapter 28; listerial listeriolysin O, Cl1apter 33; and arcanobacterial pyolysil1, Chapter 29). Pfo binds to cholesterol-containing rafts in the eukaryotic cell membrane, and forms a pore, which results in the , livf>r, anct other

U:;sues of susceplible a11d resistant species. Evidence that

soil transmits "blackleg" is circurnstantial (see under "Epidemiology," below). RouLes of infeclion aJe not kI1ow11. Ei1doge11ous an.d soil-acquired infection via ingestion or injury is assumed.

Pathogenesis ·rhe alpha (necrotizing), gamma (l1yaluro1litlase), delta (chauveolysin) toxins together with the neura1ninidase, are uelieved to be iesponsible for the i11itial lesions. Bacterial metabolism, producing gas from fermentation, mav be contributory. SeediI1g ot tissues, especial1y skeletal muscle, wit h spores from the intestine, presumably precedes disease in cattle. Co11ditions .favoring spore germination, t>acterial growth, and toxin productio11 cause formation of l?'ª.I lf>siuns llli:lfkctl uy eden1a, hen1orrh.age, and n1yohbr1llar necrosis. The centers of lesions become dry, dark, and e1nphysematous dueto bacterial fcrmentation, while the pe riphery is edematous and hemorrhagic. A ranc1d-butter odor is typical. Microscopically, one finds degenerative cha11ges i11 muscle fibers disrupted by edema, en1physem.a, an11 hPm orrt1age. Leukucytic i1Jfillralion is 111inor. (]inirally, there is high fever, anorexia, a11d depress.i on. Lan1eness is com1non. Superficial lesions cause visible swellings, which crepitate on being handled. Often lesions are cntirely internal (diaphragm, myocardium, tongue). Sorne antmals die suddenly, others within 1 or 2 days.

Epidemiology "Dlackl.eg" occurs worldwide at rates that differ betwecn and within geographic areas, which suggests a soil reservoir or climatic or scasonal factors yct to be defined. Young, well-fed cattle (r spP_~, r:. chauvoei typically causes wound infections resembling malignant edema o r gas gangrene. Other clostridia (C. septicurn, C. novyi, and C. sordcllii) may be present.

lmmunologic Aspects Circulating antibody to toxins and cellular componc11ts appare11tly determü1es resistance to(,'.. cflauvoei. Commercial formalinized adjuvant vaccines include up to six oth€.r clostrid ial components.

Laboratory Diagnosis Sporulated gra1n-positive rods can be demonstrated in s1nears of infected tissues and ide11tified with immunofluorescent reagents (see Fig 70.4). Clostridiun1 chauvoei requires strict anaerobic cony usil1g tl1e ¡;olyruerase cl1ai11 rearauou:; colitis), l>ut its significance in other anirnals is less clear. The microorganism has been isolated from syn1ptomatic as well as asymptomatic dogs and cats. Association with a "trigger event" such as use of antimicrobial agents has bccn suggcstcd but not proven. Clostridiurn difficile l1as also been isolated from normal horses; ho"l'.rever, it is more frequently isolated fl uu1 l1ur:;e:; wiLl1 lliarrliea a11ll a:s:su Pnte.rotoxic p roperty of ·roxA, in addition to the cytotoxic effect:; ju:;t de5cribed, is due to ils ability to stimulate influx of PMNs by way of the enteric nervous systcn1 (through thc rclcasc of subStance P and mast cell degranulation). J>rostaglandin synthesis by tl1e recruited PMNs (and perhaps by affected host cells), as well as activation of various inositol-signaling pathways within affected host cells, resul ts in thc secrelion of cl1loride ions and water (diarrhea). 2. ·roxin B. Clostridiuni difficílc Toxjn B (ToxB or TcdB for toxin (~. difficile), like ·ioxA, is a glycosyltransferase that glucosylates Rho GTPases (see above). However, ToxB has little enterotoxic activity. 3. ADP-ribosyltransferase. Clostridiinn difficile produces a11 ,L\DP-ril>u:;yl Lransferase (Ctlt for C. difficile transferase). Cdt is a binary toxin (see l-. perfringens iota toxin, above, and C. spirofor1ne, below), cornposed of a binding portion (C:dtb) that binds to the target intestinal epithclial cclls and an enzymatically active portion (C~dta). After the toxin binds t o specific receptors on the cell surface, Cdta gains entry into the cytoplasm. Though it is not clear precise.ly how e.ntry or.r11rs, it si>i>ms that a pore (composed of Cdtb) is formed in the cell membrane through \.Yhicl1 Cdta traverses. Cdta is an ADP ribosylating toxin that ribosylatcs actin withln the host cell resulting i n disorganization of the cellular cytoskeleton and death of th affected cell. Variability Clos1rídiun1 difr1cile is quite variable. There are l2 serogroups (A- I, K, X, and S), as well as substantial vai:iability ü1 the gene e11cotli11g tlte flagellar protei11 flagellin (9 dlfferent groups). Random amplífication of polymorbic DNA (IlAPD) analysis dernonstrates nurnerous types.

208

P,\JtT JI

Bacteria and Fungi

·r11ese di ffe1 e11Lt::> a11:: u~t:ful il1 c.leterrr1ining the epidemiology of this microorganism.

Ecology Reservoir and Transmission Clostridiun1 difficilc is found in thc intestinal canal of nor-

mal as well as clinically attcctcd animals. ·rhe spores are resistant to most environmental stresses, ''.rhich results in their widespread distributlon In Iocations where animals are l1oused. ·rhere does not seem to be an overlap behltul pathway stemming from the intense inflammatory rf't, Sacc:llarumyc:es buulardii, has been shown to be useful in prf'vPnting thf' clisease in hum;in patients. Tn human hospitals, hand washing by health care personnel is a very efficicnt mcchanism fo r curtailing spread. Disinfectants are n ot effective against the spores.

( LOSTRID/UM PILIFORME ("8AC/LLUS Pll/FORMIS") An acute fatal diarrhcal dlsease of laboratory mice with focal liver necrosis (Tyzzer's disease) is associated with a spure-forrning organlsrn, Cluslridiunz pilifurrne, w!lich uccurs in hunclles within hepatocytes. lt is unable to grow o n cell-frce inedia. Clostridiunz pilifor111e ls linked to identica1 diseascs in rabbits, harcs, gerbils, rats, hamsters, muskrats, dogs, cats, snow leopards, foals, and rhesus monkeys. Tyzzer's disease has been reported in a human patient infected with the human immunodeficiency virus, but not In lmmunocompetent persons.

Descriptive Features Clostridi11rn pilifor1ne is a large, gram -variable, spore-

Epidemiology C/ostridii11n diflici/e-assoc1ated d1arrhea appears to be

linked to administration of antibiotics, stress, chemothera¡.H::ulh.: age11l:>, ur uou~terolc.laJ anti-inflammatory drugs, although ncwhorn foi!fs of small carnivores. Tn dogs, actinomycosis may be associated (e.g., by licking) wlth foreign boclies, particularly migrating grass awns, especially of the genus Hordeu1n ("foxtails''), whicl1 so1nctilncs lodge near vertebrae, causing actinomycotic discospondylitis. Cutaneous actinomycosis in dogs is a rare nodulo ulcerative Jymphangitis.

Pathology. Mernhers of the genus Actinomyces evoke pyogra11ulon1atous reactions by unkno,-vn mechanisms. Bacteria] colonies form in tissue. triggering suppurative responses in the immediate vicinity. Peripheral to this, granulation, 1nononucJea.r infiltration, and fi brosis furnish the granulomatous element s. Sinus tracts carry exudate to the outside; the exudate often contains yellowish "sulfur gran11lt>s," which arP colonial masst>s s11rro11nrlPl1atid yl iJ1usitul u1a11 11u-

side (a pho,pholipicl) ::iiactenum, M . paratuberculosis is an obligate aerobe. Tn thc past, the growth dependency of M . paratuberculosis for mycobactin (an iron-hinciing lipi