Sea Bass and Sea Bream : A Practical Approach to Disease Control and Health Management 9781912178643, 9781910455791

Citation preview

SEA BASS AND SEA BREAM

VENDRAMIN PRINT.indd 1

26/05/2017 08:33

VENDRAMIN PRINT.indd 2

26/05/2017 08:33

SEA BASS AND SEA BREAM A Practical Approach to Disease Control and Health Management Pierpaolo Patarnello Niccolò Vendramin

VENDRAMIN PRINT.indd 3

26/05/2017 08:33

First published 2017 Copyright © Pierpaolo Patarnello, Niccolò Vendramin 2017 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior permission of the copyright holder. Published by 5m Aquaculture, An imprint of 5M Publishing Ltd, Benchmark House, 8 Smithy Wood Drive, Sheffield, S35 1QN, UK Tel: +44 (0) 1234 81 81 80 www.5mpublishing.com A Catalogue record for this book is available from the British Library ISBN 9781910455791

Book layout by Servis Filmsetting Ltd, Stockport, Cheshire Printed by Replika Press Ltd, Pvt India Photos by Niccolò Vendramin and Pierpaolo Patarnello unless otherwise indicated A special thanks to Dr Matthijs Metselaar DVM Ph.D. MRCVS CertAqV MIFM, for his time and consideration in reviewing this material. Important note: Medicine is an ever-changing science, so the contents of this publication, especially recommendations concerning diagnostic and therapeutic procedures, can only give an account of the knowledge at the time of publication. While utmost care has been taken to ensure that all specifications regarding drug selection and dosage and treatment options are accurate, readers are urged to review the production information sheet and any relevant material supplied by the manufacturer, and, in case of doubt, to consult a specialist. The publisher will appreciate being informed of possible inconsistences. The ultimate responsibility for any diagnostic or therapeutic application lies with the reader.   No special reference is made to registered names, proprietary names trademarks, etc. in this publication.   This publication is subject to copyright, all rights are reserved, whether the whole or part of the material is concerned. Any use of this publication outside the limits set by copyright legislation, without the prior written permission of the publisher, is liable to prosecution.

VENDRAMIN PRINT.indd 4

26/05/2017 08:33

Contents

Prefacevii PART I THE MEDITERRANEAN PRODUCTION SYSTEM Farmed Species Main Biosecurity Problems

4 6

8 Phases of the diagnostic approach:  1. Equipment 9   2. Signaling – Case description 11   3. Anamnesis – Clinical history 11   4. Site inspection and clinical examination 14   5. General Clinical Examination (GCE) 17   6. Necropsy technique and Internal pathological examination20   7. Parasitological examination 25   8. Bacteriological examination 28   9. Virological examination 31 PART II THE MAIN INFECTIOUS DISEASES IN MODERN MEDITERRANEAN AQUACULTURE BACTERIAL DISEASES 37 Vibriosis (Vibrio anguillarum)40 Photobacteriosis (previously known as Pasteurellosis) 46 Flexibacteriosis (Tenacibaculum maritimum)53

VENDRAMIN PRINT.indd 5

26/05/2017 08:33

vi  ♦  Contents Streptococcosis59 Septicemia by Aeromonas sp. 64 VIRAL DISEASES Viral Encephalopathy and Retinopathy (VER, also known   as Viral Nervous Necrosis – VNN) Lymphocystis disease

67 70 77

81 PARASITIC INFESTATIONS Myxosporid Diseases Enteromyxum leei99 Isopod Infestations (Ceratothoa, Nerocilia, Anilocra)104 110 Mongenean Parasitic Infestations SUMMARY115 115 BACTERIAL DISEASES Typical Vibriosis 115 Photobacteriosis116 Flexibacteriosis117 Streptococcosis118 Septicemia by Aeromonas119 VIRAL DISEASES Viral Encephalopathy and Retinopathy (VER, also known as   Viral Nervous Necrosis – VNN) Lymphocystis disease

120

PARASITIC INFESTATIONS Diseases caused by Myxozoa Isopod and copepod crustaceans Infestation by Trematoda

122 122 124 125

REFERENCES FOR FURTHER READING

126

Index

133

VENDRAMIN PRINT.indd 6

120 121

26/05/2017 08:33

Preface

T

he prevention and control of the main pathologies affecting intensively cultured marine fish species are becoming of greater importance in the field of modern zootechnical aquaculture, where the health of the reared animal represents the main expectation by the market. For this reason, in every advanced form of industrial farming, biosecurity becomes the cornerstone of a production process that develops according to increasingly precise and defined common regulations. The aim is to reduce as much as possible the use of chemotherapeutic drugs and disinfectants in order to obtain a fish product with safe and healthy characteristics, allowing it to be distinguished from other aquaculture products. In this sense, a diagnostic center able to provide not only the prevention and prophylaxis measures essential for successful fish farming, but also early detection of the most common diseases is now a need for every modern industrial facility. In this manual the experience in the diagnosing of disease in cultured marine fish resulting from more than 15 years of field activity, is combined with the knowledge and activities carried out in a fish disease reference laboratory. The aim is to provide practical and easily applicable advice, with particular reference to the speed of diagnostic response for some of the most dangerous pathogenic diseases that often cause severe ­economic losses in Mediterranean farms.

VENDRAMIN PRINT.indd 7

26/05/2017 08:33

VENDRAMIN PRINT.indd 8

26/05/2017 08:33

PA R T I

The Mediterranean Production System

M

editerranean marine aquaculture, intended as intensive aquaculture production systems, has recently experienced extremely rapid and ongoing development. The implementation of standardized breeding protocols, the availability of more efficient fish feed and some principles related to disease control have been the basic elements for the development of an industrial production system. When considering the biosecurity approach to the complex productive situation in the Mediterranean two different types of companies that represent two categories of operating systems quite different from each other and characterized by different and peculiar hygiene aspects are distinguished. On one hand consider the centers for breeding and larval rearing (hatcheries). These are facilities characterized by the major use of technology and water “control”, which usually develop in confined environments controlled with high biosecurity standards and ­monitoring of every biophysical parameter. In this context biosecurity problems are extremely complex and articulated, so this will not be covered in this book. On the other hand, consider the large number of companies involved in the on-growing and fattening phase of marine fish and the topics covered are particularly relevant to this area. Once the juveniles have been bought, these farms carry out the growing phase until market size is achieved. The farms use various production systems that are very different from each other, but which are increasingly oriented towards the use of off-shore sea floating cages (both for ­production cost reasons and for technical–environmental reasons).

VENDRAMIN PRINT.indd 1

26/05/2017 08:33

2  ♦  The Mediterranean Production System

In these varied productive scenarios the biosecurity problems to be considered, their development, the control strategies and the analytical tools are very different from each other, but all of them depend on one constant common factor: the acquisition of intimate knowledge about the specific production system to be analyzed. This kind of knowledge is essential for the fish veterinarian or the ­biosecurity expert: without it, every consideration, every forecast and every therapeutic or prophylactic suggestion risks becoming ineffective or inapplicable, thus nullifying all the efforts linked to this delicate aspect of the modern production system.

Fig. 1.1: Tanks for larval stages rearing in a hatchery. Note the high level of automation in a closed environment.

VENDRAMIN PRINT.indd 2

26/05/2017 08:33

The Mediterranean Production System  ♦  3

Fig. 1.2: Square floating cage typical of sheltered sites or inland waters.

VENDRAMIN PRINT.indd 3

26/05/2017 08:33

Farmed Species

I

n the Mediterranean, the species involved in modern aquaculture considered in this work are mainly represented by gilthead sea bream (Sparus aurata family Sparidae) and European sea bass (Dicentrarchus labrax family Moronidae). Several other species have been tried in intensive rearing without, however, obtaining production levels comparable to that obtained with the two main species. Nowadays the contribution of

Fig. 1.3: Gilthead sea bream specimens. (S. aurata)

VENDRAMIN PRINT.indd 4

26/05/2017 08:33

Farmed Species  ♦  5

Fig. 1.4: European sea bass specimens. (D. labrax)

these other species remain marginal, but expect future production increases for “alternative” species as a consequence of both a greater know-how about the feeding and the management of the stock, and of the increasing demand for diversification of supply by consumers. Among the new commercially important marine species there are several sparids: sharpsnout sea bream (Diplodus puntazzo), white bream (Diplodus sargus), striped seabream (Lithognathus mormyrus), red porgy (Pagrus pagrus), common pandora (Pagellus erythrinus) and the common dentex (Dentex dentex). Recently, some attempts at industrial farming have been carried out also for sole (Solea solea and Solea senegalensis), meagre (Argyrosomus regius), shi drum (Umbrina cirrosa), and the first results have been obtained in the fattening and breeding of bluefin tuna (Thunnus thynnus).

VENDRAMIN PRINT.indd 5

26/05/2017 08:33

Main Biosecurity Problems

I

n modern mariculture, and in particular in the newly reared species, biosecurity represents a crucial point of the production, both for legislative and commercial reasons (fish products are increasingly subject to food safety checks) and for economic and production reasons (farming without diseases is economically vital). In a complex scenario where infectious and non infectious diseases are involved, infectious diseases represent a group of greater importance and often are the main cause of severe economic losses in ongoing farms. In addition to infectious diseases, equipment failure (breaks in the cages at various levels with partial or total loss of the farmed fish stock) should be considered as risk factors with the greatest economic and production impact. Main infectious diseases affecting farmed marine fish are clustered in three major groups according to their aetiology (viral, bacterial, parasitic); such groups include both primary aetiological agents and others defined as secondary that are less frequent and often less studied. The main viral diseases to be considered are viral encephalopathy and retinopathy (VER, known in the past as VNN) and ­lypmhocystis infections. Among the main bacterial diseases are the group of vibriosis (the main one caused by vibrio anguillarum and several other secondary vibriosis) and photobacteriosis (known in the past as pasteurellosis, caused by Photobacterium damselae subspecies piscicida). Several other bacterial infections, such as tenacibaculosis (Tenacibaculum spp.), streptococcosis (Streptococcus iniae and others)

VENDRAMIN PRINT.indd 6

26/05/2017 08:33

Main Biosecurity Problems  ♦  7 Table 1.1  A  etiological agents responsible for the main infectious diseases in warm water marine fish species

Virus

AETIOLOGIC AGENT Bacteria

Parasites

Betanodavirus

Vibrio anguillarum, Enteromyxum leei V.ordalii, V.vulnificus, Lymphocystis disease Photobacterium damselae Crustacea Isopoda subspecies piscicida Tenacibaculum maritimum Mongenean parasitic Streptococcus iniae, infestations S. agalactiae, Streptococcus Sparicotyle chrysophrii parauberis, Enterococcus seriolicida Aeromonas sobria complex

are named and the septicemic forms caused by Aeromonas sp., which, although less frequent, must be considered as emerging diseases. Finally, with respect to parasitic diseases, there are considerations made about the evolution of these diseases in relation to the different farming system that developed together with the use of inshore and offshore floating cages. In these modern farms, among the parasitic diseases of primary importance enteromixidiosis (particularly Enteromyxum leei), infestations by crustacean isopoda (i.e. Ceratothoa oestrides, Anylocra, etc.) and gill infestation with monogeneans (i.e. Sparicotyle chrysophrii) are found: these are extremely important problems given the high difficulty in the treatment. With respect to land-based farms using tanks, the economic/­ productive impact of more traditional ectoparasite infestations such as Oodiniasis is still relevant. In addition, these diseases are often an indication of more general para-physiological or pathological phenomena such as repeated feeding errors, sub-clinical viral infections or adverse environmental conditions. These circumstances can induce different levels of

VENDRAMIN PRINT.indd 7

26/05/2017 08:33

8  ♦  Main Biosecurity Problems

immune depression in the farmed stock, which could result in severe parasitic infestations. For this reason, such diseases are often important indicators, linked to factors of physiological, pathological or management unbalance, and must always get the maximum attention by the pathologist. Besides infectious diseases, other causes should be considered. These pathological conditions are generated by a series of factors arising, at least at the beginning, not from primary pathogens but from wrong zootechnical procedures. These could be management, nutritional, feeding or structural factors that force the fish to live in inadequate farming conditions. The direct consequence of this condition, defined as “dysmetabolic”, when it does not cause mortality per se, is the predisposition of farmed fish stocks towards various kinds of pathogens, with easily conceivable biosecurity consequences. The range of possible diseases needs a dynamic approach in its evaluation and continuous updating about the role and the importance of each considered pathogen. The farming system, the species involved, the environment and everything considered relevant in such a complex activity should be taken into account in every phase of the study of a disease.

Phases of the Diagnostic Approach 1. 2. 3. 4. 5. 6. 7. 8. 9.

VENDRAMIN PRINT.indd 8

Equipment Signaling – Case description Anamnesis – Clinical history Site inspection and clinical examination General Clinical Examination Autopsy technique and Internal Pathological Examination Parasitological examination Bacteriological examination Virological examination

26/05/2017 08:33

Phases of the Diagnostic Approach  ♦  9

1. Equipment In order to carry out a correct analysis of fish samples, it is ­necessary to have a facility with the following equipment:

U An isolated room, if possible not adjacent to production departments, possibly ventilated, adequately covered (walls floor) with easily washable and disinfectable materials. U and A source of fresh water with a sink, preferably made of stainless steel, equipped with a shelf, to store cleaning products and surgical tools. U A table, preferably made of stainless steel, equipped with a waste water collection system (like a dissection table for pathological anatomy). U One or more light sources to give an effective illumination of the examined samples. U A fridge-freezer. U A light microscope (Fig. 1.5) with at least four lenses (5×, 20×, 40×, 100×) (preferably with for a digital camera).

Fig. 1.5: A light microscope.

VENDRAMIN PRINT.indd 9

26/05/2017 08:33

10  ♦  Phases of the Diagnostic Approach

U A stereo microscope (Fig. 1.6) for parasitological investigations.

Fig. 1.6: A stereo microscope.

U An electric heated incubator. U A balance with minimum capacity 1kg and 0.1g increments. U A fixed or mobile heat source (Fig. 1.7: portable Bunsen).

Fig. 1.7: A portable Bunsen.

VENDRAMIN PRINT.indd 10

26/05/2017 08:33

Phases of the Diagnostic Approach  ♦  11

U Possibly a sterile hood. U An autoclave. U Glassware and adequate surgical equipment (Fig. 1.8: pathological anatomy instruments, bacteriology loops).

Fig. 1.8: Clinical instruments.

2.  Case description In this phase the particular characteristics of the farm must be reported on a special form and include: the produced species, if the plant is open-cycle or closed-cycle, the facilities utilized (ponds, concrete tanks, floating cages) the kind of feed, the quantities of fish produced, the source of water supply and so on. 3.  Anamnesis – Clinical history This section of the diagnostic approach is among the most important and delicate. The primary goal is to gather as much information as possible, with the best level of detail about the condition of the

VENDRAMIN PRINT.indd 11

26/05/2017 08:33

12  ♦  Main Biosecurity Problems

farm. It must include the characteristics of the farm, the environmental conditions and the local geography. All the data should be gathered in a written document: the farm dossier, which, because of its importance, should be compiled by the farmer himself or herself in collaboration with the health manager of the company. The investigation involving clinical history if based on the f­ armer’s memory, often leaves out some important details and significantly alters the events and the timetable. With a well-organized dossier it is possible to set out, both for the veterinarian and for the production manager a detailed and recent examination of the fish stocks present. Along with the diagnostic activity carried out by the veterinary aquaculture expert, this is a solid base from which to make daily decisions on the farm. The main points to be considered in the ­dossier are the following: 1.

Seed quality The analysis and report of seeding procedures must include all the problems and the abnormalities that could occur in the transportation after the delivery of the batches, during the initial transfer of the juveniles, in their acclimatisation process to the new farming environment (tank or cage) and to a different diet. In the first life phases it is important to monitor and report data on the growth performance of the fry (ponderal increase, density per productive unit, etc). Alterations in these parameters can significantly influence the ratio between the maximum potential growth (which is the farmer’s primary goal) and the real progression of the production cycle.

2.

Handling and traceability All handling including the selection procedures, net changes, vaccination, movements from other cages and the creation of stock with fish having similar size but different origin, must be recorded.

VENDRAMIN PRINT.indd 12

26/05/2017 08:33

Main Biosecurity Problems  ♦  13

Traceability, namely the ability to recover at any time a stock of a particular origin, is a quality requirement of the product. Also, accidents in the manipulations must be adequately reported. 3.

Weather conditions Weather conditions, particularly if bad, must be recorded and described. General symptoms widespread in the stock, inadvertently attributed to an outbreak of an infectious disease, could be caused by climatic events (for example injuries caused by lightning or by fish becoming trapped in the nets by powerful waves).

4.

Water parameters The systematic collection of the main physico–chemical parameters (temperature and dissolved oxygen) should be made regularly. These data, organized into archives, should be kept in a database so that they can be correlated to the growth rate and the feeding. This important analytical tool should also include occasional phenomena such as algal blooms or environmental contaminations (oil spills or the dumping of agro–industrial wastewater that is accidentally introduced into the environment).

5.

Recent diseases and treatment Everything that has been diagnosed in the farm and every associated treatment must be reported, together with the ­veterinarian prescriptions.

6.

Epizoological archive In this chapter of the report, the pathological archive and the environmental conditions should be correlated. Using this data, a veterinarian aquaculture expert will be able to evaluate

VENDRAMIN PRINT.indd 13

26/05/2017 08:33

14  ♦  Main Biosecurity Problems

the recurrence of some diseases, and the association between some outbreaks and particular environmental conditions. On the basis of this data he or she will also be able to suggest preventive treatments, organize a correct vaccination strategy, and schedule and evaluate the deadlines for the delivery of the seed. By consultation of an up-to-date and accurate archive, it is also possible to evaluate the rules for an adequate insurance policy. The more precise, accurate and complete this report is, the better will be the likelihood that the veterinarian will be able to provide a good diagnosis in a very short time, especially when consulted about complex and rapidly worsening cases for which the time to determine a diagnosis through laboratory tests is very short. 4.  Site inspection and clinical examination Once the clinical history has been obtained it will be possible to start the inspection, which will not just be focused on the sector affected by a problem, but will evaluate the entire farm. The main topics to be considered are:

U Observation of fish behavior U Presence of specimens with color abnormalities U Variations in appetite U Presence of mortality The observation of the fish stock must be done for an adequate timeframe. Fish behavior can be evaluated through the analysis of the reactivity to external stimuli and the reaction to feeding. In order to avoid important evaluation errors, the reaction to stimuli must be judged before feeding because during the cold season the stimulus to feed is generally diminished in relation to the lower temperature. Attention must be paid also to aggression; i.e. cannibalism, and the average body condition of the entire stock in the productive unit should also be evaluated.

VENDRAMIN PRINT.indd 14

26/05/2017 08:33

Main Biosecurity Problems  ♦  15

In regards to the presence of mortality, it should never be considered a “normal” report. Although in intensive farming a certain amount of daily mortality is widely accepted as paraphysiological, it should always receive the maximum attention by the pathologist. This data, together with some particular features of the mortality event, should be considered the starting point of a correct diagnostic approach. It is important to collect all the data related to the beginning of the mortality event, to evaluate daily losses, to describe the trend through a graph, and to identify correlations with other reports (the presence of moribund specimens, appetite variations, symptoms and clinical findings in surrounding productive units, etc.). Another factor to be evaluated is the location in the water column of dead or moribund specimens. Usually they can be found moribund near the surface or on the bottom (typically a white spot on the bottom). The location of such specimens can provide guidance for a diagnosis. In fact, some infectious diseases can induce, during the evolution of the pathogenetic process, the hyperinflation of the swim bladder (viral encephalopathy and retinopathy) while, on the contrary, other infectious diseases (photobacteriosis) are able to induce acute mortality, providing, as the only diagnostic observation, the presence of a considerable quantity of dead specimens on the bottom. These considerations can be decisive only if adequately contextualized. It is important to remember that, particularly in the context of farming with floating cages, environmental conditions can suddenly change and become an important factor in the development of some pathologies.

Sampling procedures Methods for the choice and the collection of the sample In order to obtain a correct diagnosis, the sample must be representative of the identified problem. Hence it is necessary to collect a significant number of individuals, from five to fifteen in the case of intensive fattening.

VENDRAMIN PRINT.indd 15

26/05/2017 08:33

16  ♦  Main Biosecurity Problems

The collected individuals must represent the various “clinical” categories of the productive unit (cage or tank) examined: moribund, apparently healthy, and freshly dead. This allows an evaluation of the various stages of the disease. Sample preparation The sample must be examined as soon as possible; in the case of a laboratory internal to the farm this procedure is particularly easy: alive and moribund samples must be transported into a container filled with water. The container must be perfectly clean, and the water must possibly be taken from the original tank. Dead samples must be stored in ice if necessary, in relation to the external temperature. The proximate transportation of moribund and dead specimens in the same container must be avoided. On the other hand, in the case of an external laboratory, mori­ bund specimens must be collected and shipped so that they can ­possibly arrive alive. In order to achieve such a goal specimens can be inserted in plastic bags containing water for a third of the total volume and air for the remaining part. The best solution is to use a portable fridge, possibly equipped with an oxygenator. Moribund specimens, with clinical signs, must be sent to the laboratory. For relatively small specimens (up to 80g) with variable symptoms, at least twenty samples must be collected in order to obtain solid conclusions. If it is necessary to carry out histological examinations, tissues or entire organs must be dissected immediately after the collection of the fish and stored in a special fixative (buffered formalin, or Bouin’s fixative). If the laboratory is not reachable in a short time, the fish vet can carry out the clinical and parasitological examination directly on the farm and subsequently prepare the samples destined for the laboratory. The preparation of such samples is based on the plating of the sample an agar plate with a medium suitable for bacterial growth. Adequate media for the growth of pathogens affecting marine-cultured fish are mainly blood agar or tryptic soy agar (TSA) and marine agar.

VENDRAMIN PRINT.indd 16

26/05/2017 08:33

Main Biosecurity Problems  ♦  17

5.  General Clinical Examination (GCE) Once in the laboratory, the first step is the evaluation of the sample through an initial General Clinical Examination, which allows the identification of any possible abnormalities in one or more specimens belonging to the sampled batch. It is important to carefully record particular characteristics related to the skeletal development (spinal cord deformity), nutritive condition, presence of particular symtoms (unilateral or bilateral exophthalmos), external lesions (hyperemic areas, skin and fin erosion, ulcers), malformations (alterations in the jaw or mandible), color alterations (melanosis) and shape alterations (abdominal distension). This phase includes the external parasitological examination of skin and gills. Sampling and analysis procedures are described in a specific paragraph.

Fig. 1.9: Lateral view of D. labrax.

VENDRAMIN PRINT.indd 17

26/05/2017 08:33

18  ♦  Main Biosecurity Problems

Fig. 1.10: Hemorrhagic-ulcerative dorsal lesion probably caused by fish-eating birds.

Fig. 1.11: Severe skeletal deformity on the upper part of the skull in specimen of D. labrax.

VENDRAMIN PRINT.indd 18

26/05/2017 08:33

Main Biosecurity Problems  ♦  19

Fig. 1.12: Specimen of D. labrax with evident unilateral exophthalmos.

Fig. 1.13: Opercular deformity in D. labrax.

VENDRAMIN PRINT.indd 19

26/05/2017 08:33

20  ♦  Main Biosecurity Problems

6. Necropsy technique and Internal Pathological Examination (IPE) Once the General Clinical Examination has been completed, the second step involves the anatomo-pathological examination. After the evaluation of the external appearance of the sample and the recording of unusual characteristics, proceed with the inspection of the organs contained in the coelomic cavity. The sample must be placed on its right side, with the head on the left and the caudal fin on the right of the operator, in order to present the entrails correctly for the inspection. With the help of a scalpel and forceps, make an incision in the caudo-ventral part of the animal, laterally to the anus. Subsequently, with the help of scissors, cut the abdominal wall, continuing in the side region, dorsally along the swim bladder until the gill operculum is reached and ventrally along the mid-line.

VENDRAMIN PRINT.indd 20

26/05/2017 08:33

Main Biosecurity Problems  ♦  21

Photo Table 1 How to Open the Coelomic Cavity and Perform an Organ Inspection

VENDRAMIN PRINT.indd 21

26/05/2017 08:33

22  ♦  Main Biosecurity Problems

The goal of the pathological examination is to analyze in detail the different organs of the sampled individuals, with particular reference to the gills and the organs contained in the coelomic cavity: liver, spleen, kidney, intestine, heart, gall bladder, swim bladder, gonads. Also, the brain must be carefully inspected because during ­septicemic forms it can often show different grades of alteration (vascular congestion, edema, hemorrhage). For all these organs, an Internal Pathological Examination (IPE) is essential for the detection of any possible anomaly in the following characteristics:

U Size U Edge U Color U U Consistency Odour

Fig. 1.14: Tympanism of the swim bladder, color alteration of the liver parenchyma and peritoneal lipid accumulation in specimen of D. labrax.

VENDRAMIN PRINT.indd 22

26/05/2017 08:33

Main Biosecurity Problems  ♦  23

Fig. 1.15: Skin erosion with pervasive hemorrhages due to overcrowding in the tank.

Fig. 1.16: Skin lesions with absence of scales and fin hemorrhages, abdominal distension and pathological condition of the liver, characterized by color and vascular alterations in specimen of S. aurata.

VENDRAMIN PRINT.indd 23

26/05/2017 08:33

24  ♦  Main Biosecurity Problems

Field necropsy The collection of the specimens for the post-mortem examination is an aspect that must be considered carefully. It is important to remember that the sample significance is also linked to the health status of the fish stock in the farming unit. It is often necessary to operate in uncomfortable conditions, but it is important to disturb the fish’s welfare as little as possible. During the incubation phase of a VER event, for example, collection of fish through nets could cause stress that can enhance clinical outbreak of the disease, while a less invasive sampling method (using a fishing line or other methods), will surely cause a smaller impact on the productive unit. Most farms do not have suitable rooms in which to carry out an adequate post-mortem examination. Furthermore, the fish vet could also have to face adverse conditions (heat, cold, wind, insects, insistent questions by the farm staff). In these cases, the sample must be taken and stored in ice, and then transported to the laboratory, where adequate analyses can be conducted. The basic post-mortem examination includes:

U The external appearance (development of the skeletal muscle, fins, skin, eyes, mouth and buccal cavity, gills and gill chamber) U Presence of visible parasites U  peritoneal cavity inspection (presence of ascitic fluid, signs of peritonitis) U Internal organ description (liver, gall bladder, spleen, i­ ntestine, swim bladder, kidney)

All the observations must be written down. If a room with a microscope is available, portions of gill tissue, and scraped material from skin and fin lesions must be examined in order to evaluate the presence of parasites (trematodes, crustaceans, protozoans) and bacterial load. Impression smears of the intestinal content and of the kidney must be examined in order to detect the presence of parasites (coccidia and Myxosporea). It is also possible to prepare blood smear stained with Giemsa, fixed with ethanol and air-dried.

VENDRAMIN PRINT.indd 24

26/05/2017 08:33

Main Biosecurity Problems  ♦  25

A more detailed description on impression smear procedure of spleen or kidney tissue for bacterial observation is described in the specific chapters of bacterial diseases. 7.  Parasitological examination After the routine analysis of skin and gills, carried out before the opening of the coelomic cavity, the next step is a specific parasitological examination. Such an examination could become the subject of a specific investigation if it is suspected that there are parasitic diseases affecting internal organs (myxosporidiosis, microsporidiosis, coccidiosis, etc.). In the evaluation of the impact of an internal parasitic infestation (number of parasites for each investigation unit), it is recommended that a standardized procedure is followed, which allows the infestation level of the examined specimen to be quantified correctly. For example, in evaluating the level of gill infestation it is recommended that the same portion of branchial tissue is analyzed for each individual in order to obtain additional information about the evolution of the disease during the treatment. The gill examination is made by removing the branchial operculum with suitable scissors so the gill chamber can be inspected fully. Subsequently, a portion of branchial lamellae is selected to be placed on a microscope slide. In this phase it is important to gather a moderate quantity of sample in order to observe the slide while avoiding the excessive presence of mucus or debris. It is also possible to dilute the sample slightly with sterile water. With regard to the skin examination, this is conducted by scraping the mucus and the external surface with a coverslip to be placed on the slide, focusing the attention on the fins and the adjacent skin regions. For the internal parasitological examination, one can take an impression of the crosswise-dissected intestine or put some material scraped from the intestinal mucosa on a slide in the case of a suspected infection by Enteromyxum leei or other digestive system parasites. In the case of other suspected myxozoan infestations (Ceratomyxa spp) or other digestive system microparasites, small quantities of bile fluids can be sampled and put on a slide to be analyzed.

VENDRAMIN PRINT.indd 25

26/05/2017 08:33

26  ♦  Main Biosecurity Problems

For macroparasite investigations, the use of a stereo microscope is essential, while for microparasites investigations and evaluation of possible morphological damage, the use of a light microscope is required.

Photo Table 2 How to Collect a Gill Sample for Parasitological Survey

VENDRAMIN PRINT.indd 26

26/05/2017 08:33

Main Biosecurity Problems  ♦  27

Main Necropsy Findings Associated with Organs and Pathogens Table 1.2 Examined organ/ system

Parasitic lesions

Skin

Erosions and ulcers Macroparasites (crustaceans like Anilocra, Ceratothoa and other)

Viral lesions

Hemorrhagic Erosions of the suffusions and head (VER) hemorrhages secondary to vibriosis, blindness and Aeromonas septicema incoordination Fin fraying and skin erosion Flexibacteriosis

Other

Lesions caused by fish-eating birds

Proliferative lesions (Lymphocystis disease)

Hyperemia Hypermucosity

Presence of gas bubbles in the vascular lumen

Liver

Petechiae, hemorragic soffusion (vibriosis)

Steatosis

Spleen

Splenomegaly vibriosis, photobacteriosis

Gills

Hyperemia Hypermucosity

Bacterial lesions

Pseudo tubercular nodules (chronic photobacteriosis) Presence of crystals (dietary imbalances)

Kidney

Digestive system

Intestinal ectasia with pouring of intraluminal whitish fluid (enteromyxidiosis) Hyperinflation (VER)

Swim bladder Central Nervous System (CNS)

VENDRAMIN PRINT.indd 27

hemorrages (Streptococcosis)

congestion (VER)

26/05/2017 08:33

28  ♦  Main Biosecurity Problems

8.  Bacteriological examination Once the parasitological examination has been completed, the fourth step is represented by the bacteriological examination. Under suitable conditions, this is carried out by inoculating on an agar plate, initially with a non-selective medium, samples taken from the spleen, liver, kidney and/or brain with the help of a sterile loop. Once the microorganism has been isolated, it is possible to deepen the analysis and proceed with the identification of the bacterium. For most bacterial diseases, plates can be incubated at room temperature and away from contamination sources. Each plate must clearly report the stock, the sample origin, the species and the organ from which the sample was taken. In the case of particular suspicion about more “difficult” bacteria, the use of a special incubator at a controlled temperature is recommended. Considerations leading to this examination could be the suspicion about a particular disease or the completion of a general diagnostic investigation. Besides the

Fig. 1.17: Example of tool kit for conducting necropsy and bacteriological analysis.

VENDRAMIN PRINT.indd 28

26/05/2017 08:33

Main Biosecurity Problems  ♦  29

typical clinical symptoms, the suspicion of a bacterial disease usually also arises from specific pathological surveys, which are generally considered pathognomonic of particular diseases. In this particular situation, a bacteriological investigation is of primary importance and is focused on what is considered the target organ of the suspected disease. Also, the medium must be chosen specifically to promote the growth and isolation of the suspected pathogen. In contrast, in the case of a general bacteriological investigation, the target organs for the examination are, in order of priority, the kidney, the spleen, the brain and the liver, which generally represent the target organs of bacterial infections. Also, the medium is chosen from among the most generic to allow the growth of a wide range of bacteria. Once the investigated bacterial strain, pure or mixed, has been isolated, the next two steps are the typing and the antibiogram. For the typing of the two main bacterial diseases affecting the sea bass and the sea bream, a first response can be obtained using a rapid agglutination kit, currently available on the market for:

U Vibrio anguillarum U Photobacterium damselae subsp. piscicida For all the other kinds of typing and for a greater diagnostic certainty, it is better to use biochemical typing systems (API Galleries), some of which can also be applied in a field laboratory or to more advanced analyses in specialized laboratories. On the other hand, the antibiogram must be set with the active principles usable in aquaculture (Oxytetracycline, Amoxicillin, Flumequin, Sulphamidics-Trimethoprim); this facilitates the achievement of a more targeted response when an antibiotic therapy becomes necessary.

VENDRAMIN PRINT.indd 29

26/05/2017 08:33

30  ♦  Main Biosecurity Problems

Photo Table 3 Sterile sampling for bacteriological examination

Organs and Specific Media for the Main Pathogens Investigated Table 1.3 Microorganism

Organ

Medium

Vibriosis Vibrio anguillarum

Kidney

Blood agar, TCBS

Photobacterium damselae subsp. Piscicida

Kidney/Spleen

Blood agar

Tenacibaculum maritimum Cutaneous form

Skin and fins

Marine agar, Anacker and Ordal agar

Tenacibaculum maritimum Systemic form

Kidney/Spleen

Marine agar, Anacker and Ordal agar

Streptococcosis

Kidney/Spleen

Blood agar, TCBS

Aeromonas spp.

Kidney

Blood agar, TCBS

VENDRAMIN PRINT.indd 30

26/05/2017 08:33

Main Biosecurity Problems  ♦  31

9.  Virological examination Finally, the fifth step concerns the complex chapter of viral diseases, which still represent one of the less known pathological aspects, and are difficult to investigate and manage for our marine fish species. In the diagnosis of the two main viral diseases affecting the sea bass and the sea bream, respectively viral encephalopathy and retinopathy (VER) and Lymphocystis disease, a crucial role in the diagnosis is played by clinical symptoms (VER) and by pathological reports (Lymphocystis disease) as well as the epidemiological data of the outbreak. The diagnostic verification of a viral disease remains the exclusive task of a specialized laboratory since it involves classical virological investigations carried out on cell cultures, histological and immunohistochemical examinations, or the use of molecular biology techniques. The aim of any classical virological investigation is the isolation of the virus through the highlighting of the cytopathic effect (CPE) in sensitive cell monolayers and subsequently the identification of the virus through serological or molecular techniques. Both the aetiological agents, Betanodavirus and Lymphocystis disease, show a cytopathic effect on specific cell cultures: it is currently possible to isolate Betanodavirus on specific cell monolayers (i.e. SSN-1 and E-11) as well as for Lymphocystis virus (i.e. GHSBL, GHSBF e SAF-1). This technique is able to identify the presence of the living and infectious virus in the investigated sample, but is unfortunately time-consuming before reaching a conclusive negative result (almost twenty days). For this reason, this examination is highly recommended in the case of an official confirmation subsequent to the collection of other diagnostic clues. Histological and immunohistochemical analyses show the lesions caused by the virus and aim to stain the viral antigen. In the case of Betanodavirus infection, such lesions occur in the brain and retina, but are not always present or detectable, and this is especially true for larger specimens. In the case of Lymphocystis disease, such lesions are proliferative and occur on the skin, already identifiable at a macroscopic level in the acute phase of the disease.

VENDRAMIN PRINT.indd 31

26/05/2017 08:33

32  ♦  Main Biosecurity Problems

Molecular techniques are extremely sensitive, fast and relatively inexpensive. They can also be carried out on materials that can be sampled without being lethal to the fish, and do not require the presence of the living and infectious virus. For this reason, the limit of such techniques is that they cannot distinguish between living and infectious virus and traces of viral genetic material. It is important to highlight that when structuring an adequate protocol for the processing of the sample, modern diagnostic techniques can also enable a quantitative evaluation of some pathogens. With the use of Quantitative Polymerase Chain Reaction (qPCR) techniques it is possible to quantify the sequence product and in some cases to provide useful precautionary and prognostic indications about the ongoing infection.

Fig. 1.18: Sea bass juveniles with pathognomonic nervous symptoms.

VENDRAMIN PRINT.indd 32

26/05/2017 08:33

Main Biosecurity Problems  ♦  33

Fig. 1.19: Sea bream juvenile affected by Lymphocystis disease with typical proliferative skin lesions.

VENDRAMIN PRINT.indd 33

26/05/2017 08:33

34  ♦  Main Biosecurity Problems

Photo Table 4 Technique for the Sampling of the Encephalon

With the help of sterile scissors, the eye-socket is penetrated dorsally. The first incision is carried out by caudo-medially directing the scissors. After repeating the same procedure symmetrically proceed with the opening of the cranium with the exposure of the encephalon.

VENDRAMIN PRINT.indd 34

26/05/2017 08:33

PA R T I I

The Main Infectious Diseases in Modern Mediterranean Aquaculture

VENDRAMIN PRINT.indd 35

26/05/2017 08:33

VENDRAMIN PRINT.indd 36

26/05/2017 08:33

Bacterial Diseases

B

acterial diseases represent the first topic specifically explored more deeply in this second part of the book. The broad chapter dedicated to this group of diseases represents the pathological condition with the greatest incidence of disease for the sector. Despite the fact that the treatment of this kind of disease benefits from the few registered chemotherapeutic agents in aquaculture, and that strategies of vaccinal prophylaxis have been developed for some of them so far, bacterial diseases represent, also for cage aquaculture, one of the most dreadful pathological phenomena that can occur in a production cycle. The great spread of bacterial pathogens, the fact that they are ubiquitous, and the way that they are frequently carried asymptomatically by many wild species that usually live around the cages, make this group a significant threat for this sector. Thus, good epidemiological knowledge of the environment operated within and a wise policy of precautionary and diagnostic biosecurity are the basis for an effective control and containment policy of such events. The main symptom that characterizes this heterogeneous group of diseases is, indeed, the sudden anorexia shown by marine populations affected by the most common pathogenic bacteria. This phenomenon also represents the main obstacle to therapeutic intervention. The therapy is normally carried out through the administration of medicated feed containing active substance more or less specifically selected for the considered pathogen. Another general but serious problem is the time gap needed to obtained such medicated feed, which, in the best case, requires three to five days since the ­diagnostic suspicion.

VENDRAMIN PRINT.indd 37

26/05/2017 08:33

38  ♦  Bacterial Diseases

This time frame is often sufficient for the establishment, in the affected population, of anorexic forms that in many cases nullify the late administration of the medicated feed. Another problem linked to this kind of infectious diseases is represented by the environmental and zootechnical consequences induced by an orally administered antibiotic therapy on a farmed fish population. Besides the potential risk for the dangerous phenomena of antibiotic resistance and residual contamination, often linked to the continuous and repeated use of antibiotics and chemotherapies in a given site, the economic consequences arising from the respect of “withdrawal times”, which often represent heavy damage for the farm affected by a bacteriosis, especially for fish at the end of their cycle cannot be disregarded. From a practical point of view, the success of the management and control of a bacterial pathological event in an off-shore farm with floating cages depends on two factors:

U diagnosis U AA prompt correct management strategy of the event. It is important to highlight that both these characteristics must be managed only by an expert specialized veterinarian who, together with field experience and his or her constant presence on the productive sites, should lead the fish vet to detect the first signs of a bacterial disease in its initial stage. In this sense, a detailed epidemiological knowledge of the environment is necessary and, possibly, the availability of a disease record of the farm and the farmed fish stocks, can help the pathologist considerably in detecting the typical symptoms that usually precede a bacterial disease outbreak. Once a diagnostic suspicion has been determined it is necessary to plan an operative strategy leading to the prompt finding of the therapeutic treatment. Then proceed with diagnostic investigation procedures that require the cultivation of the bacterial strain in a plate and the subsequent antibiogram. In the most complex and serious cases, an antibiogram will be essential in order to confirm the in vivo efficacy of the active

VENDRAMIN PRINT.indd 38

26/05/2017 08:33

Bacterial Diseases  ♦  39

substance used to allow a possible rapid change of the therapeutic strategy before anorexia occurs. Increasing pressure from the public, and particularly from consumers, for production without the use of antibiotics and disinfectants, mean that it is important to highlight the importance of preventing this kind of disease through adequate prophylactic campaigns. This should be done both directly through a careful and severe analysis of the newly introduced seeds, and indirectly through adequate vaccination and immunostimulant strategies for the most vulnerable populations. Regarding the field use of medicated feed, the therapeutic treatment must be carried out following some reservations:

U Starvation before the administration U Reduction of the food load U Manual administration of medical feed. The administration frequency and the duration of the treatment will vary according to the different species, the age of the affected batch, the evolution of the disease and the kind of farm considered. All these factors are crucial for the success of the treatment and can be evaluated correctly only by an expert consultant who has a specific knowledge of the anatomy and physiology of the treated species and about the pharmacokinetics of the active substances used. The selected drug and the therapeutic strategy (intended as dosage) must be compatible with the technical management of the considered farm in order to be included in the daily routine with the least possible distortion. Finally, here are some of the main mistakes frequently made in the treatment of bacterial diseases:

U Late start of the therapy U Wrong choice or wrong dosage of the administered drug U Inadequate treatment duration U of antibiotics during viral disease outbreaks U Use Repeated use of the same drugs at ineffective dosages.

VENDRAMIN PRINT.indd 39

26/05/2017 08:33

Vibriosis (Vibrio anguillarum)

Aetiology The infection is caused by the bacterium Vibrio anguillarum, belonging to the family Vibrionaceae. It is a Gram-negative bacterium, with a characteristic comma shape, for which several serotypes have been described. Also consider the so-called atypical vibriosis, caused by other species belonging to the genus Vibrio, such as V. harveyi, V. ordalii, V. vulnificus, V. alginolyticus. Epidemiology This important bacterial disease affects, among farmed species, mainly the sea bass (D. labrax). From an economic point of view, such a disease represents the main bacterial problem for mariculture farms. Besides the severe damage caused in the first phases of the rearing process, both in hatcheries and in nursery, the disease could also represent a serious threat for Mediterranean farmed fish during the fattening and finishing phases. Despite several kinds of vaccine being available on the market and the vaccine prophylaxis being quite a common practice, treat reinfection phenomena in the last phase of the cycle. Such episodes could be caused by a lack of sufficient protection that, in the absence of an effective booster, is not provided by the first vaccination. This disease is generally characterized by high mortality. Despite the heaviest losses being caused by acute and peracute forms during juvenile rearing stages, the disease represents an economic problem

VENDRAMIN PRINT.indd 40

26/05/2017 08:33

Vibriosis ( Vibrio anguillarum)  ♦  41

mainly during the final stages of the cycle. There, besides the direct damage caused by mortality, several previously defined aspects should be considered such as indirect damage, which represent the bigger proportion in the economic impact evaluation. Among these aspects please consider: the costs for medicated feed, the frequent relapses after antibiotic treatments, the severe gastroenteric consequences of bacteriosis in affected specimens with the unavoidable drastic drop in the feed conversion rate, the costs for the collection and the disposal of dead fish, and finally the long withdrawal periods imposed by legislation for the units subjected to therapeutic treatment. For all these considerations, the disease is still considered the main economic problem in modern mariculture. As for every other infectious disease, a vibriosis outbreak is often related to immunosuppression as a consequence of stress. For this particular disease, sudden temperature fluctuations in the aquatic environment are among the main triggering factors. For this reason, historically, this problem was related to a spring syndrome and a fall syndrome, establishing a strict connection with the appearance of outbreaks in these two seasons when temperature fluctuations are more frequent. Areas where there is a high density of farms are subjected to continuous reinfection or subclinical infection ready to become clinical as a consequence of every stressful event. Pathological lesions and symptoms The pathological feature of this disease is widespread hemorrhagic lesions. During the external observation characterizing the GCE (General Clinical Examination) it is possible to see:

U Diffused rash all over the ventral part of the specimen and, in particular, hemorrhages, often triangular, at the base of the fins, rash and anal protrusion in the ventral part U ventral Hyperemia together with petechiae and hemorrhagic suffuin the oral part affecting the tongue and the palate U sions In the internal pathological examination observe: hepatic hemorrhages, splenomegaly with congestion and darkening of the organ, hemorrhagic enteritis.

VENDRAMIN PRINT.indd 41

26/05/2017 08:33

42  ♦  Vibriosis ( Vibrio anguillarum)

Most common clinical signs observed by the fish vet at the beginning of a vibriosis outbreak in a cage are often represented by the presence of specimens swimming apart from the group with darkened skin (skin melanosis) and lethargy. The observation of some specimens in the described condition must rapidly lead to a diagnostic deepening through an immediate examination of the collected samples, involving firstly GCE, secondly a IPE, and then a laboratory confirmation. Diagnosis Granted that a prompt diagnosis is the first step of a successful intervention for every bacterial disease, one of the elements favoring a rapid intervention is usually represented by the knowledge of the remote clinical history of the disease in the farm. As a matter of fact, the observation of seasonal recurrence of the problem in historically affected sites is quite common. A first field confirmation derives from the results of the pathological examination, which in the classical forms are quite peculiar. The easiness of isolation on plate with the most common available media (blood agar and TSA) and the rapid growth of the microorganism allow the use of ELISA kit and rapid agglutination tests for the diagnostic confirmation. During incubation at room temperature (20°C) circular colonies with a diameter of 1–1,5mm can develop in twenty-four to thirty-six hours. If blood agar is used, it is possible to observe a hemolytic zone. The observation of atypical forms with different symptomatic and pathological characteristics is increasingly common. For this reason, a laboratory diagnostic confirmation with microorganism typing and antibiogram is always recommended, despite the additional time required. Therapeutic recommendations and control strategies The therapeutic strategy to act against this disease is certainly represented by the use of antibiotics. If correctly employed, all the main molecules currently available and registered for fish (penicillins, tetracyclines, quinolones, sulphonamides) are generally effective for the treatment of this disease.

VENDRAMIN PRINT.indd 42

26/05/2017 08:33

Vibriosis ( Vibrio anguillarum)  ♦  43

For this purpose, it is essential to act promptly before anorexia, which is a typical symptom of this and other bacteriosis, nullifies any attempt at the oral administration of medical feed. For the success of the therapy, it is equally important to follow a correct administration protocol of the medicated feed depending on the food and therapeutic requirements of the affected stock. A best practice during the administration of the medical feed is to reduce the food ratio and to administer the drug for at least five to six days (in the case of quinolones) and, often, also for longer periods in the case of other active substance such as tetracyclines or sulphonamides. Authorized and registered vaccines against vibriosis are available, such prophylactic tools have proven their efficacy when correctly employed. It is possible to vaccinate both through immersion and through intraperitoneal injection. The two vaccination techniques require different application ­protocols and are often used for different sized animals. In bath vaccination, generally used for animals weighing less than 5g, the operational procedures are simpler and easily applicable. However, the young age of vaccinated specimens often limits their immunity to a period of four to six months. The intraperitoneal administration of the vaccine, even if characterized by greater operational difficulties and greater manipulation risks, ensures a major efficacy and a more durable covering. The combination of the two techniques, bathing as a first vaccine and inoculation as a booster, represents the ideal protocol of vaccinal prophylaxis for this disease, but often the overall costs for the entire protocol operations are so high that the single farmer cannot afford them. It is to be hoped that in the future these prophylactic procedures will be increasingly preferred as opposed to the therapeutic ones, and innovative technologies will reduce their costs increasing the use in the aquaculture sector.

VENDRAMIN PRINT.indd 43

26/05/2017 08:33

44  ♦  Vibriosis ( Vibrio anguillarum)

Fig. 2.1: Hemorrhagic lesions and suffusions in D. labrax operculum, lateral and ventral fins.

Fig. 2.2: Severe vascular congestion of the viscera in D. labrax.

VENDRAMIN PRINT.indd 44

26/05/2017 08:33

Vibriosis ( Vibrio anguillarum)  ♦  45

Fig. 2.3: Splenomegaly and spleen darkening in adult D. labrax affected by vibriosis.

Fig. 2.4: Gram-negative vibriosis in a microscopic spleen sample. Picture kindly provided by Dr. Carlos Zarza.

VENDRAMIN PRINT.indd 45

26/05/2017 08:33

Photobacteriosis (previously known as Pasteurellosis)

Aetiology Photobacterium damselae subsp. piscicida, halophilic, Gram-negative, rod-shaped bacterium belonging to the family Vibrionaceae. Epidemiology The second most severe bacterial disease affecting our farmed marine fish is represented by photobacteriosis (previously known as pasteurellosis). This disease can also affect the entire farming cycle; unlike vibriosis, it equally affects the two main farmed species in the Mediterranean: the sea bass and the sea bream. Photobacteriosis has significantly modified its “epidemiological behavior” in relation to changes in the sector of marine farming. For a long time, the disease was considered a seasonal disease because, due to the prevalent temperatures around 20°C, it typically occurred in late spring and during all the summer, and then disappeared in late fall. After the spread of cage farming, which replaced the more traditional forms of fattening in inland based farms and is characterized by a minor biomass density and better general condition of the farmed stocks, an increase of sub-acute or even chronic and asymptomatic forms was observed, while the classical acute and peracute forms described in the past mainly for juvenile stages have strongly decreased. The most severe forms usually occur when water temperature is

VENDRAMIN PRINT.indd 46

26/05/2017 08:33

Photobacteriosis (Photobacterium damselae subsp. piscicida)  ♦  47

higher than 22–23°C and salinity is lower. When favorable conditions for pathogen replication occur together with especially adverse conditions for farmed fish in the productive unit, it is possible to observe the most severe disease forms. Among the conditions that can more frequently weaken the farmed fish immune system are high density, low oxygen levels, food stress and adverse weather conditions (storms, rain, etc.). All the sparids experimentally farmed with intensive methods were susceptible, at different levels, to this pathogen. In particular, sea bream is susceptible to infection during the first stages of the farming cycle, starting from the post-larval phase to the size of 5/6g. In fact, such first stages can be affected by devastating disease outbreaks in the hatchery with mortality rates often higher than half of the affected stocks. The occurrence of bacteriosis at such early stages, usually characterized by high densities and temperatures higher than 18°C, together with an incomplete development of the immune system, cause the occurrence of the disease in acute or peracute forms. During the subsequent stages, starting from the pre-fattening stage, with sizes higher than 10g, the sparids’ resistance to infection increases significantly and, in the case of both first infection and relapse, the reported forms are mainly sub-acute or chronic with definitely lower mortalities (not higher than 3–4%). For sea bass, susceptibility to this disease is quite high during all the juvenile phase (but not as early as in the sea bream) and the most frequent disease events occur at a size between 0.8–1g and 40–50g. For this species, the most frequent disease outbreaks occur from the late spring to the fall, depending on the latitude and temperature. Predisposing factors play a more crucial role than in the sea bream, including the stress caused by environmental and management factors, especially by transportation and stocking. In fact, most of these bacterial disease outbreaks in the fattening sector occur, usually, during the first weeks after stocking. Within this time frame a significant drop in immunity due to manipulations carried out during handling operations (loading, transportation and unloading) and to changes in the environment (often also

VENDRAMIN PRINT.indd 47

26/05/2017 08:33

48  ♦  Photobacteriosis (Photobacterium damselae subsp. piscicida)

of temperature) occur simultaneously and severely challenge this species’ resistance, triggering clinical disease outbreak. A crucial role in maintaining the pathogen in the farming site is played by some wild fish species. These natural reservoirs, such as some mullet species that are often common around the farms, play the role of asymptomatic carriers, dangerously active throughout the summer and the fall. Every time that particularly stressful conditions occur for the farmed stocks, the carriers cause new disease outbreaks. The presence of several sources of infection (natural reservoirs, new stock, etc.) and the little immune memory of the already affected stocks ensure that, in areas characterized by high densities of farms, the disease becomes endemic and causes several outbreaks during every production cycle. Such epidemiological behavior promoted the habit of carrying out an antibiotic treatment or metaphylaxis (defined as “preventive”) after every stressful event (such as a net change or a new introduction). The important effects of such practice on the treated stocks, on the ecosystem and on wild populations close to the farm can be easily imagined. Last but not least, consider the impact of the antibiotic resistance induced in microbial populations. Pathological lesions and symptoms The peracute forms occur asymptomatic; sea bream larvae, juveniles or fry are often found dead in great quantities on the bottom of the tank, with a few melanotic fish gasping on the surface. Fish behavior usually does not show abnormalities; the response to feeding is usually good until a few hours before the disease occurs. Dead fish often have the stomach and the intestine filled with feed. In acute forms, when disease symptoms occur in sea bream and sea bass fry, observe inflammatory responses on the mouth and the jaw, and necrotic areas on the skin near the pectoral or the dorsal fin, or on the caudal peduncle. Fins, particularly the lateral, dorsal and caudal ones, can be eroded (GCE). The hemorrhagic appearance, which is typical of vibriosis, is not present. Skin and fin erosions are covered with mucus and in the water appear as whitish spots. On the

VENDRAMIN PRINT.indd 48

26/05/2017 08:33

Photobacteriosis (Photobacterium damselae subsp. piscicida)  ♦  49

gills there is inflammation together with hypersecretion of mucus, and often areas with necrotic tissue. The liver is often congested, the spleen is noticeably enlarged and the kidney is pale and edematous. In the chronic forms there are typical pseudo tubercular lesions in the splenic and renal parenchyma. In the intestine there is a certain amount of transparent fluid. The swim bladder is typically uninflated, and for this reason the few dead specimens can usually be found on the bottom (IPE). In fattening sites, the severity of the losses depends on the fish age and on the beginning of the outbreak. The greatest problems occur when the infection begins in the early spring since it will probably last throughout the warm season. In this case, the production could be impaired by repeated outbreaks, despite the therapies, for several months until the fall, or until the fish have reached a less susceptible size. The worst situation occurs when the early infection affects very small sized fish stocks. In this case losses could be very high. Losses higher than 20% for the sea bream and 40% for the sea bass are not uncommon. The overall effects on fish growth are hardly quantifiable. Additional resources are required for the removal, the transportation and the disposal of the dead fish, and for the daily preparation of the medical feed. Diagnosis The diagnostic suspicion is based on the remote clinical history, on the symptoms and on characteristic post-mortem examinations. The confirmation of the suspicion can be obtained through ELISA diagnostic kits, pathogen isolation in TSA or blood agar. API systems can be used for the biochemical identification. Colonies develop with a typical morphology after twenty-four to thirty-six hours of incubation at room temperature. Colonies are whitish and small (0.5mm in diameter), if observed with the stereomicroscope they appear semi-translucent with irregular edges. Spleen stained with Gram proved to be quite effective for diagnosing. In this case in acute or peracute forms, where the spleen does not show the typical granulomatous formations yet, typical cellular and bacterial clusters characterized by bipolar bluish refractivity can be observed.

VENDRAMIN PRINT.indd 49

26/05/2017 08:33

50  ♦  Photobacteriosis (Photobacterium damselae subsp. piscicida)

Therapeutic recommendations and control strategies Prophylactic and preventive tools are available for this disease. Where possible, vaccination in juvenile stages shows objective difficulties. Bath administration (the only one currently applicable for small sizes) requires long administration times with high stress levels and short duration of effective protection. Such procedures can cause high mortalities. On the other hand, the possibility to vaccinate bigger specimens through injection is not usually taken into account since adult specimens are less susceptible. From a logistical point of view, oral vaccination could be a good solution, however it does not seem to provide an adequate protection. Prevention is further complicated by the fact that, in Italy, registered vaccines to prevent this disease are not available. In pre-fattening and fattening units the strategy for the containment of the clinical outbreak considers the use of antibiotics with the related problems already discussed for vibriosis. Also, the actives normally used belong to the same limited number of authorized substances, with a preference, in this case, for tetracycline or enhanced sulphonamides rather than quinolones. Relapses and recurrent outbreaks often occurs for this disease, to the point that its endemization in areas having a long farming tradition is quite widespread. A best practice is to prevent photobacteriosis in fattening units by screening of the seed. Stocking during the warm months should be avoided, if possible from a practical point of view. In the case of a suspected outbreak, the veterinarian must be contacted as soon as possible in order to confirm the suspicion and start the most adequate therapy. It is crucial to minimize every form of stress on the affected stock (in this sense, the shading of the tank or the cage during the summer is quite effective), and to remove and dispose of the dead specimens daily. Also, for this severe bacterial disease it is desirable that there will be the rapid development of a more effective vaccine.

VENDRAMIN PRINT.indd 50

26/05/2017 08:33

Photobacteriosis (Photobacterium damselae subsp. piscicida)  ♦  51

Fig. 2.5: D. labrax juveniles with evident splenomegaly.

Fig. 2.6: Splenic nodules in a case of chronic Photobacteriosis.

VENDRAMIN PRINT.indd 51

26/05/2017 08:33

52  ♦  Photobacteriosis (Photobacterium damselae subsp. piscicida)

Photo Table 5 Impression smear of spleen aspiration biopsy, colored with Gram staining

After opening of the coelomic cavity and exposing the spleen, the organ is aspirated using a sterile needle with an insulin syringe. Spleen tissue is smeared on a slide and color with Gram staining. In the picture on the bottom right observe the positive sample at 100× with maximum presence of Gram-negative bacteria. Microscopic picture kindly provided by Dr. Carlos Zarza.

VENDRAMIN PRINT.indd 52

26/05/2017 08:33

Flexibacteriosis (Tenacibaculum maritimum)

Aetiology Tenacibaculum maritimum (previously known as Cytophaga marina, Flexibacter marinus), Gram-negative mobile bacterium, is the aetiologic agent of flexibacteriosis in marine fish. In the past the disease has been identified with many other names, including filamentous bacteria disease of marine fish, eroded mouth syndrome and myxobacteriosis. Marine flexibacteriosis is widespread among farmed and wild fish in Europe, Japan, North America and Australia. Epidemiology This important bacterial disease affects several species of farmed fish. With regard to the intensively farmed species in the Mediterranean, which is the main topic of this work, sea bass (Dicentrarchus labrax) is cited as the main host for this pathogen and, in a subordinate position, sea bream (Sparus aurata). Scientific literature also reports disease events for other species, such as the turbot (Scophtalmus maximus) and the sole (Solea solea and Solea senegalensis), which have been subjected to experimental farming for a long time but in the Mediterranean have not reached the level of importance and the productive and commercial significance of the two euryhaline species cited above. This disease occurs also in some farmed exotic species, such as Pagrus major in Asia and Australia and Limanda in the north European Atlantic. Although flexibacteriosis can affect all biological stages, the most severe disease events occur during juvenile stages. The disease is

VENDRAMIN PRINT.indd 53

26/05/2017 08:33

54  ♦  Flexibacteriosis ( Tenacibaculum maritimum)

strongly influenced by the host and the environment. Regarding environmental factors, the prevalence and the severity of the disease increases when water temperature exceeds 15–16°C. Furthermore, the disease is influenced by several factors such as the microbial load of the aquatic medium, pH and salinity. As regards the host, its resistance and the general condition of the affected specimens must be evaluated. Finally, always consider the “technical” factors, which are closely linked to the productive activity, such as all the manipulations typical of the first farming phases of most fish species cited above. Particularly important are the operations of selection, transfer and transport that occur during the first months of the farming process of such fish species. These frequently cause mechanical lesions, which represent the “ideal” point of entry for this disease. The transport phase, with its delicate loading and unloading operations of the juveniles represents one of the most risky moments, particularly for the sea bass because of the occurrence of skin lesions and the consequent appearance of bacteriosis during a period of three to six days after the arrival in the cage. Pathological lesions and symptoms The most evident and typical aspect of this disease is represented by external lesions, which are erosive and ulcerative, having different characteristics depending on the affected species and on its size. The appearance of such lesions, which can often be complicated by the presence of hemorrhages, is very frequent around the mouth. On the skin, the disease is often evident near the caudal peduncle (sea bass) or the dorsal fin (sea bream), and in every species it shows the typical fin “fraying” (GCE). Sometimes a form of systemic disease involving several internal organs can be observed, particularly in the sea bass. Such a form can reach remarkable levels, often with extremely complicated prognosis and in absence of visible lesions on the organ (IPE). Skin lesions, which are typical of this disease, frequently represent the point of entry for other bacterial and parasitic pathogens that will considerably complicate the case history.

VENDRAMIN PRINT.indd 54

26/05/2017 08:33

Flexibacteriosis ( Tenacibaculum maritimum)  ♦  55

Diagnosis A flexibacteriosis diagnostic suspicion is based on GCE pathological verifications (typical lesions), together with microscope observations of impression smears that highlight the presence of long filaments or of Gram-stained smears (Gram-negative) obtained from gills or external lesions. The preliminary diagnosis can be supported by pathogen isolation in a suitable medium or by verifications through molecular investigations applied directly to the damaged tissue. This pathogen grows up only in a specific medium and needs sea water and low nutrient concentration; recommended media for the isolation of this microorganism are, among others, Anacker and Ordal agar, marine agar and Flexibacter maritimus medium (FMM), although the isolation is often difficult and requires a long incubation time. Typical T. maritimum colonies are pale yellow, low and with discontinuous margins. Also, the exact “role” of this bacterium in the complex clinical presentation of an affected batch is of great importance: the report of an infection by generic “myxobacteria/filamentous bacteria” is typical of almost every disease event (particularly those that last for some days) since all the factors cited above as predisposing, such as stress and weakening, often lead the affected specimens to abnormal swimming behavior and consequent skin lesions that are quickly colonized by these bacterial forms. The expert fish vet will assign a correct diagnosis to each report and will decide whether to focus on deepening the report of presence of filamentous bacteria or searching for the triggering cause of this secondary infection. Therapeutic recommendations and control strategies It is a common practice to keep these external bacterial forms under control through the use of “disinfecting baths” made with many substances, among which the most “famous” and commonly used is formalin. This is an improper and illegal treatment system, as is the use of those substances such as hydrogen peroxide or quaternary ammonium salts, which are registered for ambient sanitation. Both direct

VENDRAMIN PRINT.indd 55

26/05/2017 08:33

56  ♦  Flexibacteriosis ( Tenacibaculum maritimum)

and indirect prophylaxis (vaccine) are surely the preferred ways to control this kind of problem. Water treatment, with the use of sterilizing methods and the development of more efficient and less traumatic farming techniques, together with the application of codified hygienic rules, contributed to remarkably reduce the incidence of these bacterial diseases in the pre-fattening units of the most modern hatcheries. The occurrence of episodes consequent to transportation and unloading operations in fattening units characterized by high contamination and high summer temperatures is still quite frequent. In these cases the disease appears some days after the unloading operation, and the lesions (particularly in the sea bass) are mainly concentrated on the caudal peduncle and could result in the complete erosion of the entire tail. For such specimens, any therapeutic intervention is useless and counterproductive (expensive and polluting), while the continuous removal of moribund and dead specimens is very useful in order to avoid the spread of the infection to specimens free from traumatic skin lesions. In the rare cases in which the disease occurs in a systemic form, antibiotic therapy is complex and not always effective. The reasons for this lack of efficacy in treatment are due to the fact that any of the available active substances will hardly reach effective concentrations in the affected organs because, in the most severe form (which is always the case for systemic myxobacteriosis affecting sea bass and bream) the involvement of the mouth seriously compromises the feeding and thus hampers the intake of medical feed. This makes the therapy useless and expensive. As regards vaccine prophylaxis, several experiments on marine cold water species, particularly the turbot, are being conducted. The development of effective vaccine prophylaxis could also soon be available for the most common warm water-reared species. Currently the most effective form of prevention is represented by a correct and “gentle” manipulation of the fish in order to avoid the formation of dangerous traumatic lesions – particularly during the most delicate phases of transportation and unloading – which is the first predisposing factor for the colonization by this pathogen.

VENDRAMIN PRINT.indd 56

26/05/2017 08:33

Flexibacteriosis ( Tenacibaculum maritimum)  ♦  57

Fig. 2.7: D. labrax specimens showing evident erosions and caudal fin fraying.

VENDRAMIN PRINT.indd 57

26/05/2017 08:33

58  ♦  Flexibacteriosis ( Tenacibaculum maritimum)

Fig. 2.8: D. labrax juveniles showing erosions and skin ulcers caused by T. maritimum.

Fig. 2.9: T. maritimum stained with gram 100×. Picture kindly provided by Dr. Carlos Zarza.

VENDRAMIN PRINT.indd 58

26/05/2017 08:33

Streptococcosis

Aetiology Some systemic bacterial infections found in marine fish are classified, after the isolation of the aetiologic agent, as Streptococcosis or Enterococcosis. The disease is also known as “pop-eye” since one of the typical symptoms is exophthalmos, which could be characterized by the accumulation of mucopurulent exudate (particularly in the turbot). The Streptococcosis reported for marine fish can be divided into at least three groups of streptococci according to their hemolytic ability. The reported disease events have been associated to Alpha-hemolytic, Beta-hemolytic and non-hemolytic bacteria. Among them, the most frequent are Alpha-hemolytic streptococci. The most commonly found species are: Lactococcus garvieae (also known as Enterococcus seriolicida), Lactococcus piscium, Streptococcus iniae, Streptococcus agalactiae, Streptococcus parauberis and Vagococcus salmoninarum. They are Gram-positive cocci. Oxidase and catalase negative, asporigenous, unmoving. Epidemiology Streptococcosis has been found in several farmed and wild marine fish species. As regards Mediterranean populations, affected species are the sea bass (D. labrax) and the sea bream (S. aurata). In addition, some species that have been considered for production diversification such as the Sharpsnout seabream (Diplodus puntazzo) the

VENDRAMIN PRINT.indd 59

26/05/2017 08:33

60  ♦  Streptococcosis

shi drum (Umbrina cirrosa), the amberjack (Seriola dumerilii) and the turbot (Scophthalmus maximus) are susceptible. Infections get more severe during the summer; in addition to the increased water temperature, a series of stressors and factors linked to the host could influence the occurrence of the disease. All age groups are susceptible to the infection, which must then be prevented throughout the whole productive cycle. The main reservoir of these pathogens is the environment, with particular respect to the water and the sediments near the farms. Particular attention should be paid also to the feeding strategy since streptococci can live for up to six months in the frozen food (breeders fed with fish). Horizontal transmission has been proven and occurs through water, especially in the presence of microlesions or abrasions that alter the integrity of fish mucous membranes. The fecal–oral cycle has been hypothesized, while the condition of the carrier has been clearly shown; such confirmation came with both sensitive and non-sensitive species. This disease is today one of the major factors limiting the development of turbot (S. maximus) farming in Spain and yellowtail (S. quinqueradiata) farming in Japan. Pathological lesions and symptoms The common necropsy findings of this kind of disease is a diffused hemorrhagic syndrome, which appears in the affected specimens, regardless of the species. During an outbreak, despite an increase of daily mortality and of melanotic specimens swimming in an uncoordinated way near the surface, the overall population seems not to lose appetite and the behavior remains physiological. Fish with clinical disease show skin lesions covered with whitish mucus. Removing the necrotic mucous exudates from the surface reveals deep cutaneous ulcers. The mouth, the oral cavity, the jaw and the fins are inflamed and show necrotic areas. Exophthalmos, ophthalmitis and eye necrosis are frequent. In adult sea bass, the anus is inflamed and protrudes. The necropsy shows an empty stomach, and the intestine is distended and filled with fluid. The spleen is often enlarged, the swim bladder is distended, and the cholecyst is replete, especially in the sparids. The gills are anemic with widespread necrotic areas. The

VENDRAMIN PRINT.indd 60

26/05/2017 08:33

Streptococcosis  ♦  61

liver appears pale, often inflamed with darker areas. In the juveniles, mainly in the sea bass, meningitis and hemorrhages in the central nervous system are often evident. The infection could also derive from an intestinal dismicrobism, with consequent enteritis and diarrhea, from which bacteria spread into the fish’s organs. Diagnosis Fish streptococci are routinely isolated from internal organs (spleen, kidney, brain) using media such as blood agar, TSA (with addition of 1% yeast and 0.5% glucose) or BHIA. The incubation time is usually two days at 25–30°C. The small isolated colonies (1mm diameter) are white with regular edges. Bacteria are spherical or egg-shaped. Therapeutic recommendations and control strategies Despite the fact several vaccination protocols that proved to be effective for freshwater species (lactococcosis in the trout) are being studied and are under advanced experimentation, a registered vaccine for marine species is still not available. However, in some areas characterized by particularly high water temperatures in the southern Mediterranean, huge experimental tests are being carried out for the sea bass. Good management practices, cleanliness and hygiene in the farming environment, together with the use of high quality feed, are crucial conditions for an effective direct prophylaxis. Antibiotic treatments are often ineffective because of both a precocious lack of appetite, which characterize affected fish, and bacterial resistance.

VENDRAMIN PRINT.indd 61

26/05/2017 08:33

62  ♦  Streptococcosis

Fig. 2.10: Severe unilateral exophthalmos in D. labrax in case of streptoccocosis.

Fig. 2.11: Severe meningitis in D. labrax in case of streptococcosis.

VENDRAMIN PRINT.indd 62

26/05/2017 08:33

Streptococcosis  ♦  63

Fig. 2.12: Gram-positive streptococci stained with Gram on impression smear. Picture kindly provided by Dr. Carlos Zarza.

VENDRAMIN PRINT.indd 63

26/05/2017 08:33

Septicemia by Aeromonas sp.

Aetiology The aetiological agents of these pathological septicemic diseases are Gram-negative bacteria belonging to Aeromonas genus, with specific reports of Aeromonas complex sobria and Aeromonas veronii. Epidemiology The disease appeared recently in Mediterranean mariculture, affecting sea bass (D. labrax), the sea bream (S. aurata) and also the sharpsnout seabream (D. puntazzo). The disease has usually an insidious behavior, with underlying mortalities, which can unfortunately last for a long time and is often difficult to control with antibiotics. The infection has occurred under a wide range of environmental conditions; data show the adaptability of this group of pathogens and its ability to cause the disease under several different conditions. With regard to sea bass, hemorrhagic septicemia caused by Aeromonas has been described for both adult specimens (fish with an average weight of 200g) kept in ponds with well water (salinity of 3–4‰) at high temperatures (25°C), and in adult specimens farmed in sea cages with 33‰ salinity and lower temperatures (13–15°C). Adverse environmental conditions aggravate the disease, significantly increasing the mortality. Sea bream, as well as sharpsnout seabream, seemingly show lower susceptibility to the infection.

VENDRAMIN PRINT.indd 64

26/05/2017 08:33

Septicemia by Aeromonas sp.  ♦  65

Pathological lesions Aeromonas infections are usually characterized by hemorrhagic septicemia. The typical lesions are represented by anal protrusion, rash and hemorrhage at the base of the fins and in the mouth. Fish necropsy shows the presence of petechiae on the serous membranes in the coelomic cavity and on the organs. The intestine shows catarrhal enteritis. Diagnosis After the observation of pathological lesions, samples taken from the anterior kidney are smeared on plates with blood agar or TCBS. Colonies usually appear after twenty-four to forty-eight hours of incubation at ambient temperature. The subsequent identification of the strains requires biochemical and/or molecular methods. Therapeutic and prophylactic recommendations One of the main problems in the reported episodes is the difficulty in controlling the infection through the use of medical feed because of the rapid onset of antibiotic resistance. For the currently described cases, general rules of direct prophylaxis are allowed to reduce the incidence of mortality and to avoid the recurrence of outbreaks. The control of the environmental conditions, and in particular of water physico-chemical quality, seems to play a key role in fighting these emerging forms of disease. In clinical outbreaks the essential use of antibiotic therapy is complicated by the high resistance of these microorganisms towards the most common therapeutic substances.

VENDRAMIN PRINT.indd 65

26/05/2017 08:33

66  ♦  Septicemia by Aeromonas sp.

Fig. 2.13: Severe hemorrhagic skin lesions on D. labrax operculum.

VENDRAMIN PRINT.indd 66

26/05/2017 08:33

Viral Diseases

As already mentioned in the general overview, modern Mediterranean mariculture suffers from two main viral diseases, Viral Encephalopathy and Retinopathy (VER) and Lymphocystis disease; The chapter of viral infectious diseases is extremely complex and represents a problem of great importance. Due to the level of danger, the direct and indirect damage characterizing these diseases (especially VER) and the important role of such diseases in the interaction between farmed and wild populations, there is a specific section to consider this particular group of pathogens. The two considered diseases, Lymphocystis disease and VER, currently affect the main Mediterranean fish species subjected to intensive cage farming. Despite the fact that Lymphocystis disease mainly affect sea bream and shows low pathogenicity and a self-limiting course, it is a relevant problem due to organizational issues and the important ­consequences linked to indirect damage. Viral encephalopathy and retinopathy is currently characterized by severe mortalities with important economical and zootechnical consequences. It repeatedly affects sea bass, as a target species, but seems to quickly adapt to other fish species with different farming conditions and at different temperatures, representing, in our opinion, the main emerging issue for the whole sector. Some severe VER episodes that recently occurred in the Mediterranean also make this disease one of the most urgent issues for wild fish fauna and highlight the importance to direct studies on

VENDRAMIN PRINT.indd 67

26/05/2017 08:33

68  ♦  Viral Diseases

fish health towards the entire aquatic system in which aquaculture activities are set. Viral diseases can spread rapidly and are difficult to contain, even more so than bacterial and parasitic ones. Due to such characteristics and to the complex diagnosis, which always involves the intervention of a specialized laboratory, this group of diseases is one of the most interesting topics for marine ichthyopathologists. For these reasons, the approach involving the containment of viral diseases must be focused on the adoption of biosecurity regulations and the preservation of high biosafety standards in order to keep the various productive units separated as much as possible. In Mediterranean aquaculture farms such severe episodes are often considered accidental events, to which the farmer is subjected without having the opportunity to prevent or to contain them. On the contrary, through the collaboration between expert technicians and specialized laboratories operating with modern diagnostic techniques, it is possible to rapidly exclude or confirm viral diseases, allowing for the application or the elaboration of management and precautionary protocols so as to reduce their incidence and their economic impact. Among the necessary precautionary measures aimed at reducing the possibility of pathogen introduction in the farming environment, the knowledge about the supplier and the health condition of the received stock becomes crucial. Therefore, it is essential to perform a specific screening to discover the health status of the fish introduced to the farm. These examinations allow an evaluation of several important aspects for a successful transfer, such as the presence of pathogens, also in latent or asymptomatic forms, which, after a stressing ­transportation, could develop into severe disease outbreaks. The management of an outbreak is a particularly complex issue, which a health technician would never like to face. Measures to be adopted, which are described in the dedicated section for each disease, differ significantly according to the farm productive system. Once again, the importance of the general farming conditions should be emphasised, which is being able to significantly influence the level of stress, and consequently the natural resistance

VENDRAMIN PRINT.indd 68

26/05/2017 08:33

Viral Diseases  ♦  69

of farmed populations, in the development of such diseases defined as “stress-related”. For this reason, husbandry of the stock represents the essential base for a correct prophylactic and precautionary management policy.

VENDRAMIN PRINT.indd 69

26/05/2017 08:33

Viral Encephalopathy and Retinopathy (VER, also known as Viral Nervous Necrosis – VNN)

Aetiology Betanodavirus is the aetiologic agent responsible for viral encephalopathy and retinopathy (VER). The Nodaviridae family includes two genera: Alphanodavirus, which causes disease in insects, and Betanodavirus, which is pathogenetic for several farmed and wild aquatic species throughout the world. It is a small virus, having an icosahedral capsid with a diameter ranging from 25 to 30nm and with single-stranded, bi-segmented RNA. Genetic analyses of viral RNA allowed the classification of the aetiologic agent into four parental genotypes (RGNNV, SJNNV, TPNNV and BFNNV) and two reassortants (RG/SJNNV; SJ/ RGNNV). Several other genotypes have been proposed for the classification (for example AHNNV – Atlantic Halibut Nervous Nodavirus) but currently they are not officially recognized. Serological analyses showed three different serotypes: A serotype characterizing the SJNNV genotype, B serotype significantly correlated with TPNNV genotype, while C serotype show correlations with RGNNV and BFNNV genotypes. In the Mediterranean virus belonging to RGNNV and SJNNV, and the related reassortants are found. One of the main features of this pathogen is the extremely high resistance in the environment; experimental trials showed how only particularly aggressive disinfection treatments, through ozone or high irradiation with UV rays, can neutralize the virus.

VENDRAMIN PRINT.indd 70

26/05/2017 08:33

Viral Encephalopathy and Retinopathy  ♦  71

Epidemiology This important disease is currently the most important viral problem in the entire marine Mediterranean aquaculture industry and surely represents a serious issue all over the world. Almost fifty species, mainly saltwater but also freshwater species, are susceptible to this pathogen. Since the description of the first episodes at the beginning of the 1990s, the sea bass has become the most affected farmed species in modern Mediterranean mariculture. Initially the disease affected mainly larval and juvenile stages; subsequently VER episodes affected all biological stages. Today the two more critical phases in the entire productive unit are:

U Larval rearing phases, where the infection is characterized by extremely high mortality (up to 100%) U Pre-fattening phases (particularly the period immediately

subsequent to the transfer into cages). During this second phase animals with no antibody coverage can encounter the pathogen present in the farm or in the wild.

Exposure usually occurs after particularly stressful events (manipulation, loading, unloading, transfer into the cage, etc.) and under high temperature conditions (above 24°C); in this kind of situation the possibilities of successful infection resulting in clinical outbreak increase significantly. For a long time, the Gilthead sea bream has been considered to be an asymptomatic carrier, however severe mortalities during larval stages of this species have recently been reported. For several species (including the farmed sea bream) the role of asymptomatic carrier in marine farms is still to be clarified. Several other Mediterranean species, some of which have been subjected to farming trials, are susceptible:

U Among the sparids, in addition to the sea bream also the sharpsnout seabream (Diplodus puntazzo)

VENDRAMIN PRINT.indd 71

26/05/2017 08:33

72  ♦  Viral Encephalopathy and Retinopathy

U Among flat fish, the turbot (Scophthalmus maximus) and the two species of sole subjected to experimental farming (Solea solea, Solea senegalensis) U Among the sciaenids, the shi drum (Umbrina cirrosa), the meagre (Argyrosomus regius) and the brown meagre (Sciaena umbra) U Among the carangids, the amberjack (Seriola dumerilii). Finally, the following Mediterranean species also tested positive in epidemiological investigations: eel (Anguilla anguilla), grouper (Epinephelus aeneus, Epinephelus marginatus, Epinephelus alexandrinus), striped mullet (Mullus barbatus), striped red mullet (Mullus surmuletus), thin-lipped grey mullet (Liza ramada), black goby (Gobius niger), common Pandora (Pagellus erythrinus), sardine (Sardina pilchardus) and poor cod (Trisopterus minutus). The hosts list also includes typical freshwater fish (Micropterus salmoides, Sander lucioperca, Morone saxatilis and Salaria fluviatilis). This long list indicates it is extremely important to carry out epidemiological evaluations during an outbreak. In fact, when a disease event occurs it presents a double problem: the aspect of primary importance is to find the source of infection, which usually starts from infected asymptomatic juveniles. Secondly, evaluate the environmental impact consequent to viral shedding in the environment, which can maintain the pathogen on the farm and pass it to the subsequent batches. The infection mainly spreads horizontally, through water, from an infected to a susceptible animal; vertical transmission is also highly suspected and could be due to an external contamination of the eggs rather than to the intra ova presence of the virus. One of the particular characteristics of this disease is that it is highly influenced (“stress-related”) by several factors. The conditions that mainly influence the clinical evolution of the disease are the temperature (with some exceptions most of the outbreaks occur when the temperature is above 25°C), the stress, the infected specimens’ size (bigger specimens seem to be more resistant), and all of those factors considered in the general part (water quality, oxygenation, stocking-density for each productive unit, food ration,

VENDRAMIN PRINT.indd 72

26/05/2017 08:33

Viral Encephalopathy and Retinopathy  ♦  73

concomitant infections, etc.) which each contribute to weaken the hosts’ immune defense. Pathological lesions and symptoms Sea bass is the species for which the highest number of clinical case studies is available. There are three different forms of the disease, often correlated to the biological stage:

U Peracute form, generally occurring during the larval stages. The only symptom is represented by mass mortalities in the tanks without any clinical signs U affected Acute form, occurs in fry and juveniles. It shows the classical

nervous symptoms, abnormal swimming at the surface, difficulty in controlling depth, surfing, hypermelanosis, lack of appetite, blindness Chronic form, characteristic of already affected stock or bigger specimens. Animals usually do not show the nervous symptoms but frequently show bladder tympany and clouding of the cornea.

U

The pathology of the affected specimens often shows hyperinflation of the swim bladder, vessel congestion in the central nervous system, and erosive-ulcerative lesions of the skin over the head and over the most exposed contact surfaces, produced by repeated injuries consequent to collisions with the net (typical occipital and buccal lesion). The intestine is usually empty. As a consequence of the blindness that characterizes the fish during the phase of clinical symptoms, a marked clouding of the cornea due to self-induced traumatic lesions is often observed. In other susceptible species, such as the shi drum, overlapping scenarios, aggravated by the extremely high sensitivity of this species to environmental stress (O2 variations, manipulations, etc.) is seen. At an histological level, in the peracute and acute phase observe, especially in larvae and juvenile specimens, cytoplasmic vacuolations in neural cells and in neuroglial cells of the brain parenchyma, in

VENDRAMIN PRINT.indd 73

26/05/2017 08:33

74  ♦  Viral Encephalopathy and Retinopathy

the medulla oblongata, in the spinal medulla and in the retina, all characterized by a modest inflammatory infiltrate. In sea bream, the available information is related to mass mortality events in larval stages (peracute phase), with histological evidence of neural and retinal vacuolation. Diagnosis Typical clinical symptoms for the most frequent forms of the disease, in the fattening unit in floating cages or in tanks, help the pathologist to advance a diagnostic hypothesis. An expert pathologist could issue a diagnostic suspicion by analyzing data of environmental parameters (particularly water temperature) and by collecting some clinical history data about the presence of the disease in the site under consideration, or in epidemiologically correlated sites. He or she could use this together with information about the seed origin and the health documents, with specific reference to the disease. The analysis of the sample collected during the outbreak could confirm the suspicion through the observation of some typical lesions, among which are skin erosions in the occipital region, in the mouth and in the eye (unilateral and bilateral), the typical skin melanosis and the fraying of the lower lobe in the caudal fin (GCE). Cerebral congestion and tympanism of the bladder are the most frequent pathological report found in affected fish during the internal pathological examination. The confirmation of the diagnosis by the laboratory is extremely important for some strategic and epidemiological considerations. Although the symptomatic and pathological framework could provide sufficient reliability to the field diagnosis, the opportunity to confirm and subsequently deepen the genetic characteristics of the virus should, in our opinion, always be pursued. With the help of modern molecular methods it is indeed possible to characterize the gene pool of the isolated viral strain in order to formulate important epidemiological hypotheses. In a specialized fish virology laboratory a first screening analysis will be carried out through molecular methods (PCR or quantitative

VENDRAMIN PRINT.indd 74

26/05/2017 08:33

Viral Encephalopathy and Retinopathy  ♦  75

PCR) and a contemporary viral strain isolation on cell monolayers will be performed in order to confirm the presence of the virus in the analyzed sample. Therapeutic recommendations and control strategies Since this disease represents the main viral problem for our farmed marine fish, the main points at the base of any health plan are represented by prevention and monitoring. Through the use of modern diagnostic tools for the screening of the seeds before their introduction in the farm and through the periodical health screening of the farmed stock, important information about the specific infective condition of fish that allow the application of adequate management procedures should be obtained. In the case of a VER outbreak, the actions to be taken are aimed at limiting the level of stress in the productive unit as much as possible in order to reduce the mortality and the intensity of an outbreak. All handling procedures must be limited or avoided, and it is also important to remove and to regularly dispose of the dead fish. Through the development of modern molecular methods it is now possible to monitor the course of the asymptomatic phases of this disease, highlighting the presence of the virus before the clinical form, accompanied by mortality, occurs. This opportunity must be taken by the farmer because it allows the application of some important zootechnical and management regulations, which will significantly reduce the incidence of mortality in the farm. In recent years, increased interest of farmers to have more prophylactic tools available for such diseases have been met with the introduction of vaccines. To date in the Mediterranean, there are some commercial prototypes that mainly rely on the concept of an autologous vaccines.

VENDRAMIN PRINT.indd 75

26/05/2017 08:33

76  ♦  Viral Encephalopathy and Retinopathy

VER: symptomatological, pathological and histological reports

Fig. 2.14 Top left: Adult specimen of D. labrax dying on the surface during the neurological phase of VER. Fig. 2.15 Top right: Adult specimen of D. labrax with skin erosions in the head portion. Fig. 2.16 Bottom left: Juvenile of D. labrax with bladder tympanism. Fig. 2.17 Bottom right: Positive histological report of D. labrax juvenile, diffused vacuolations in the encephalon and retina.

VENDRAMIN PRINT.indd 76

26/05/2017 08:33

Lymphocystis disease

Aetiology The aetiologic agent responsible for Lymphocystis disease is a virus that, according to international nomenclature, is classified as Lymphocystisvirus, belonging to the genus Iridoviridae. This systematic group includes several other fish pathogens. This pathogen is a DNA virus with ichosahedric symmetry, and it is quite big, generally with a diameter ranging from 120 to 200nm. According to the genetic analysis of the gene of MCP (Major Capsid Protein), seven different genotypes, each one linked to a particular host or geographic area can be distinguished. Epidemiology In modern Mediterranean mariculture, the main target species is sea bream (S. aurata), followed by sharpsnout seabream (D. puntazzo) and by the dentex (D. dentex). This important disease is considered self-limiting: affected individuals show no recurrence of the clinical once they have recovered from the disease. The virus can spread horizontally between infected and susceptible fish. As regards vertical transmission, it has never been scientifically proven but it is hypothesized that infected breeders under certain stressful environmental conditions could release infectious viral particles, contaminating the water and the gametes during the emission. The development of the clinical signs, the severity of lesions, the rapidity of their formation and the time required for the remission

VENDRAMIN PRINT.indd 77

26/05/2017 08:33

78  ♦  Lymphocystis disease

of the symptoms are strongly influenced by both environmental and zootechnical factors (water temperature, exposure to solar radiations, stocking density, food ration, fish handling, transportation stress, etc.) and by factors linked to the size. For the sea bream the highest susceptibility seems to range between 2 and 10g. The morbidity is extremely variable and is influenced by all the factors cited above. The mortality is considered to be marginal in the absence of other complications. Pathological lesions and symptoms The disease is characterized by the appearance of cauliflower-like growths on the fish’s external surface, which are nodular agglomerates of single hypertrophic cells (GCE). The pathogenetic mechanism generating the single lesion seems to be a block of the cellular mitosis caused by the virus, which determines an abnormal hypertrophy in the affected cells. Lesions sometimes occur also in the mouth, determining severe feeding impairment. The benign nature of this disease is due to the possible complete remission of the symptoms after the clinical phase; despite this the economic importance of such a disease is often high for the farmer. Considering the extreme sensitivity shown on the skin by affected specimens, especially in the acute phase, all the forms of zootechnical manipulations required for a correct management of a juvenile sea bream stock become practically inopportune. The need to delay operations such as grading, transfer and transportation inevitably leads to the creation of great disparity among the stock themselves, with consequent cannibalism, or, in the best-case scenario, difficulties in evaluating the correct feed ration and nutrition. If the disease occurs in a hatchery or pre-fattening unit, another limiting factor is represented by the loading and transportation of the affected stock, which, if done during the latent but asymptomatic phase of the disease, will typically manifest as lesions a few days after stocking in the cage, with severe market consequences.

VENDRAMIN PRINT.indd 78

26/05/2017 08:33

Lymphocystis disease  ♦  79

Diagnosis The clinical diagnosis of Lymphocystis disease is made through the macroscopic observation of growths. Other techniques such as histology and virus isolation in cell cultures for the pathogen identification have also begun taking root. The great limit of these techniques is their low sensitivity, since they can confirm the pathogen’s presence only during the occurrence of an outbreak. Recent advances in the field of biotechnology allowed the implementation of molecular protocols (mainly PCR) able to detect the pathogen’s presence in non-symptomatic specimens and by the collection of non-invasive samples (fin clip). Therapeutic recommendations and control strategies Due to the absence of vaccines and therapeutic devices, the most effective solution for the management of this disease is represented by bio-security measures, health screening protocols and management of stressful factors. The rapid confirmation times obtained through modern molecular techniques can provide a technical–scientific support for the selection of the stock, identifying with great reliability the ones carrying the virus.

VENDRAMIN PRINT.indd 79

26/05/2017 08:33

80  ♦  Lymphocystis disease

Lymphocystis disease: necropsy and histological findings

Fig. 2.18 Top: Lymphocystis disease clinical form in S. aurata juveniles, with involvement of the fin surface. Fig. 2.19 Bottom left: Positive histological report of skin in clinical specimens of S. aurata with evident hypertrophic cells in the nodular formation (10×). Fig. 2.20 Bottom right: Detail of an hypertrophic cell (25×).

VENDRAMIN PRINT.indd 80

26/05/2017 08:33

Parasitic Infestations

T

he recent rapid development of marine cage fish farming has produced appreciable changes in the importance and severity of the impact represented by parasitic diseases. Regarding parasitic diseases, a general increase in their incidence and importance is observed, also for the zootechnical and environmental reasons. Currently, the three main groups of emerging pathogens in cage farming are represented by isopods, myxosporids and monogenean trematodes, which differ considerably in their size, biological cycle and type of infestation. Isopods are ectoparasites, macroscopically observable and with a direct biological cycle. Monogenea are microscopic, require a stereomicroscope or a microscope (depending on the species involved) too see, and have a direct life cycle. Finally, myxosporids are endoparasites that can only be observed microscopically and are characterized by an indirect biological cycle. When approaching marine aquaculture, it has to be taken into consideration that farmed and confined stocks in a sea cage and all wild animals in the surrounding environment establish a series of complex biological relationships defined as a biocoenosis. The production unit i.e the sea cage is characterized by the high biomass typical of modern productive units, the continuous introduction of new hosts, the homogeneity of farmed populations, the rapid growth and a potential reduction in genetic diversity. This system represents a very interesting observatory in which to study the relationship between pathogens and host, and specifically parasites, as the setting can provide all the required favorable ­conditions for the establishment of parasitic infestations.

VENDRAMIN PRINT.indd 81

26/05/2017 08:33

82  ♦  Parasitic Infestations

For these reasons, the approach to parasitic diseases in modern Mediterranean mariculture is extremely important and will focus on identifying all the different characters (primary hosts, intermediate hosts, etc) and their role in the disease dynamics. The simple observation of a macroparasite during the pathological examination, or a microparasite during the internal pathological examination, must not lead to the diagnosis of a ­parasitic disease. Paradoxically, it is common to claim that “there is no healthy fish without parasites”. In the study of farmed and wild marine populations the presence of parasites belonging to different classes and species must be considered absolutely normal. Many wild fish, particularly the big ones, are subjected to different levels of infestation, although they live, in most cases, in good balance with their parasite. A different situation occurs when farmed specimens are subjected to the particular conditions typical of an intensive production cycle. This consideration is also important in relation to specific aspects of diagnostics. Specific and rather sensitive molecular assays based on PCR and qPCR techniques have also been developed for parasites. Such methods provide a useful tool for the screening of the presence, and to some extent to the quantification, of the parasite in a specific batch. They can also be used for corroborating a diagnosis, which has to be based on clinical, necropsy findings, and epidemiological evidences. The kind of parasite, the relative parasitic load, the host resistance and the farming conditions represent a series of elements that, although they are frequently in equilibrium with the fish, in most cases they greatly limit its zootechnical performances. The negative consequences of parasitic infestations produce the already cited indirect damage, which are often more important than the direct ones for the farm economy. Although the importance of all diseases, which will be described briefly and which represent, according to the different situations, relevant health problems, it is necessary to look in more depth at three particular parasitic diseases. The first is caused by myxosporids,

VENDRAMIN PRINT.indd 82

26/05/2017 08:33

Parasitic Infestations  ♦  83

the second by some species of isopod crustaceans and the third by monogenean trematodes. Such diseases correspond perfectly to the concept ­outlined above. All three parasitic diseases are indeed characterized by an apparent low pathogenicity, and only rarely lead the affected stock to a mortality outbreak. Despite this, due to their biological characteristics and to their interaction with the farmed population, they are becoming three of the most economically relevant parasitic diseases in the new offshore mariculture system. Another reason for which parasitic diseases in general, and particularly the ones reported here, must be regarded as emerging health problems, is the extreme difficulty in the application of therapeutic procedures. The organisms involved in a parasitic episode belong to a high zoological level. For this reason, the therapy for the control or the eradication of such a type of pathogens is extremely difficult and complex. Similarly to what happens for salmon culture, where the main parasitic problem (the crustacean ectoparasite Lepeophtheirus salmonis or sea lice) poses some serious challenges when it comes to antiparasitic treatment, isopod infestations (Ceratothoa, Nerocilia and Anilocra) and myxosporids infestations are almost impossible to treat. The considerations expressed above, together with those given for other infectious diseases, highlight the importance of a concrete knowledge of the biology and the environmental conditions around a marine fish farm, which, together with strong commercial motivations, represent the basis for the development of a production strategy, for the choice of the site and of the farmed species. An environmental survey, which possibly considers all the different organisms in the selected area, a deep epidemiological knowledge of the health status of wild species and of other nearby productive activities, and an accurate planning of the proposed productive cycles are only a few essential components of a correct production strategy.

VENDRAMIN PRINT.indd 83

26/05/2017 08:33

84  ♦  Parasitic Infestations

Among the countless species of parasites described for our marine fish, the following groups are cited: 1. 2. 3. 4. 5. 6. 7. 8.

Flagellated protozoans (Amilodinium ocellatum, Cryptobia branchialis) Ciliate protozoans (Cryptocarion irritans, Trichodina sp. and Trichodinella sp.) Coccids (Eimeria sp.) Microsporids (Microsporidium sp., Glugea sp.) Myxozoa (Enteromyxum leei, Sphaerospora dicentrarchi, Sphaerospora testicularis, Ceratomyxa) Monogenean trematodes (Girodactylus sp., Diplectanum ­aequans, Sparicotyle chrysophrii) Digenean trematodes (Sanguinicolidae sp.) Crustaceans (copepods and isopods).

Each of these groups often plays an important pathological role in some particular environmental and zootechnical conditions. Dont’ aim to specifically treat the possible pathogenetic role assumed by each form of parasitic disease when the equilibrium among the host, the environment and the parasite is altered. Every group of parasites can become a problem with serious disease consequences for our mariculture, and all of them pose complex management and prophylactic issues. For some fattening units in ponds, some of these parasites still represent the main infectious disease problem and are the main cause of economic losses for direct (mortality) and most of all ­indirect (treatment expenses, etc.) damage. 1 – Flagellated protozoans Marine velvet disease, one of the main parasitic disease problems for the inland-based farm is caused by an ectoparasite: Amyloodinium ocellatum. From a morphological point of view, the adult parasite, the trophont, is different from the infectious free living stage, the dinospore.

VENDRAMIN PRINT.indd 84

26/05/2017 08:33

Parasitic Infestations  ♦  85

The trophont has no pigment, has a vaguely oval shape, and can be as long as 350µm. The infectious form attaches through a disc provided with rhizoids. The dinospore is smaller at 10–12 × 8–13µm. This important parasitic disease affects all marine and euryhaline fish species, and is widespread in marine and brackish environments of warm and temperate areas. The sea bass seems to be particularly susceptible to this parasite. Amyloodinium has a direct biological cycle, characterized by a parasitic phase, located on the skin or the gills (the trophont) and by two environmental phases: the tomont and the dinospore. A mature trophont detaches from the host surface and becomes a tomont (the reproductive form); the tomont produces the dinospores that, moving in the water column, search for new hosts. The biological cycle, which under optimal salinity and temperature conditions lasts for about one week, can be completed under a wide temperature range and this factor considerably complicates the choice of management strategies towards this disease. Such a disease is still one of the main disease problems for an on-growing inland unit. The gills usually represent the primary site of infection. The trophont attaches to epithelial cells through the rhizoids, causing hyperplastic and inflammatory reaction. Such lesions could become aggravated when alterations in the osmoregulatory system and ­secondary bacterial and fungal infections occur. The most severe symptoms generally occur in the fry, where it is possible to observe the creation of a velvet panniculus on the skin, hyperproduction of mucus, hypermelanosis, dyspnea, slow and superficial swimming, and, sometimes, skin ulcers. The diagnostic suspicion, which can be expressed observing the clinical signs, must be confirmed through the microscopic observation at low magnification of skin and gill scrapes. In the light of the high pathogenicity of Amyloodinium ocellatum, it is necessary to act as soon as the main symptoms of the disease appear. Treatments against this pathogen through chemotherapeutics, mainly directed against dinospores, make use of substances such as copper sulphate, which are not authorized by EU countries. .

VENDRAMIN PRINT.indd 85

26/05/2017 08:33

86  ♦  Parasitic Infestations

Fig. 2.21: Biological cycle of Amyloodinium ocellatum.

Fig. 2.22: Massive infestation of Amyloodinium in fresh gill preparation.

VENDRAMIN PRINT.indd 86

26/05/2017 08:33

Parasitic Infestations  ♦  87

Fig. 2.23: Detail of the parasite.

In some inland-based farms it is possible to regulate water salinity with the effect of delaying the infection. Also, a lower temperature could stop the disease. Luckily, in an offshore floating cage farm the disease does not find the ideal conditions for its development and generally does not represent a problem. Cryptobia branchialis, another ectoparasite belonging to the flagellated protozoans, is an organism with a flat shape with a pointed anterior apex and an enlarged posterior apex. Body length ranges between 5 and 12µm and shows two flagella, both of them originating from the anterior pole. The anterior flagellum is projected forward; the posterior one forms a wavy membrane around the surface and is projected behind. At the centre of the body there is an elongated nucleus, surrounded by granules of condensed chromatin, and an endosome is also present. This pathogen is usually located on the gills, destroying the epithelium and branchial lamellae and producing a thrombosis in the blood vessels of the lamella. The state of inflammation caused by the

VENDRAMIN PRINT.indd 87

26/05/2017 08:33

88  ♦  Parasitic Infestations

parasite alters the branchial blood circulation; the mucus produced as an innate response to irritation covers the undamaged lamella; gas exchange is slowed down. The branchial mucosa is predisposed to the occurrence of secondary infections. From a clinical point of view, the fish shows dyspnea. With the help of a microscope, it is possible to observe this parasite in skin and gill scrapes. With cage farming, the disease could occasionally become a serious threat under some particular conditions characterized by shallow waters and light currents. Some epidemiological studies carried out in the Mediterranean basin reported this parasite in off-shore farms. 2 – Ciliate protozoans Cryptocaryon irritans: this ectoparasite spends both the theront phase (infective) and the trophont phase (adult parasite) on the host. This dangerous protozoan shows a complex cycle characterized by several biological stages, some of which occur on the host, others as a free-living organism in the environment. The theront is pear-shaped, covered with cilia and is 25–60µm long. The trophont is rounded, shows an internal polylobated macronucleus, and has a diameter ranging from 60 to 450µm. Parasite establishment in the gills causes hyperplasia with an increase in muciparous cells and hyperproduction of mucus, edema and inflammation, atrophy and necrosis. The skin can show ulcers that harbor secondary bacterial and fungal infections. The first sign of this parasitic disease is the presence of “white spots”, often visible to the naked eye, on the body surface, mainly on the dorsal part. Affected hosts show, particularly during the initial phase, a scratching and “flashing” behavior; subsequently, lethargy, dyspnea and anorexia occur. The parasitological diagnosis is made through the observation of skin and gill scrape preparations at low magnification. When positive it is possible to take preventive and therapeutic actions by relocating the cage in a place where the depth and the current are able to interrupt the parasite biological cycle.

VENDRAMIN PRINT.indd 88

26/05/2017 08:33

Parasitic Infestations  ♦  89

Due to the complexity of its biological cycle, the parasite does not seem to represent a problem for cage farms. Trichodina sp. and Trichodinella sp. are among the ciliate ­protozoans characterized by their classical disc shape; these parasites are not restricted in their development by the need for specific water temperatures. The pathogen can cause hyperproduction of mucus and hyperplasia on the gills in the branchial respiratory epithelium. From a clinical point of view, some signs linked to respiratory problems and changes of the coloration in some parts of the body can be observed. Trichodina usually invades the juvenile skin, feeding on tissues and damaging the skin. Trichodinella sp, mainly affects the gills, concentrating on the edge of the lamellae or between filaments. During a massive infestation, branchial tissue becomes necrotic; consequently, observe a reduction of respiratory exchanges. These ciliated protozoans do not appear to be a problem for ­floating cage farms. 3 – Coccids Coccids are endocellular parasites, characterized by a complex biological cycle, specific for each species. Coccids can have a monoxenous (developing in one single host) and heteroxenous (requiring more than one host for its development) life cycle. Several genera of marine coccids have been described: Eimeria, Goussia, Calyptospra, Cristallospora. The Eimeria biological cycle includes a gametogony phase and a schizogony phase. The oocyst is released with feces and is non-sporulated. Depending on physical and chemical conditions, when released into the environment sporulation produces an infectious oocyst. Once ingested, it enters the intestinal lumen, releasing the sporocysts, which in turn release the sporozoites. This biological stage invades the cells of the intestinal mucosa, producing firstly a trophozoite and, subsequently, a schizont that

VENDRAMIN PRINT.indd 89

26/05/2017 08:33

90  ♦  Parasitic Infestations

grows up until it breaks the enterocyte, releasing merozoites that represent the sexual phases of the life cycle. Merozoites release male and female gametes, which produce an oocyst that will be released with feces. Symptoms vary according to the parasite location. In the case of intestinal coccidiosis, observe a decline in the productive indexes, progressive weight loss, and production of feces with lower consistency. In some production sites in off-shore floating cages, some severe episodes of intestinal coccidiosis have been described for farmed sea bass, with strong weight loss. 4 – Microsporids Microsporids are endocellular parasites; they are small (3–8µm) and ovoid-shaped, with a characteristic vacuole and sporoplasm. The biological cycle consists of merogony and sporogony phases. During merogony, adult microsporidium inject the sporoplasm into the affected cell; the sporoplasm form into meronts. During sporogony, meronts mature, becoming sporoblasts, and then spores. Spores damage the cell with the subsequent release of infectious material. These parasites are extremely species-specific. Their location is strictly related to the species; some microsporids are located in the muscle (Katabana sp.), others in the intestine (Glugea sp.). The microsporids’ pathogenetic action is carried out through the formation of cysts or xenoms due to hypertrophy of the parasitized cells, which could reach a size of several millimetres. Such hypertrophy can produce a compression on the tissue to the point that it determines a functional damage. Particularly in the case of a Katabana sp. infestation, the presence of several cysts in the muscle can cause severe dystrophy and the unmarketability of the product. In the case of Glugea sp., observe a degeneration of the intestinal mucosa with progressive weight loss leading to cachexia. Wild hosts can play an important role in the development of this infestation, which is therefore difficult to control. To date,

VENDRAMIN PRINT.indd 90

26/05/2017 08:33

Parasitic Infestations  ♦  91

experimental trials have been carried out for some therapeutic devices with various levels of effectiveness, but there is no available treatment. This is an emerging group of parasites for off-shore farms. 5 – Myxozoa Besides Enteromyxum leei, which will be discussed in a specific section, the group of Myxozoa includes several other parasites that are pathogenetic for marine fish farmed in the Mediterranean. Cited are Sphaerospora dicentrarchi, Sphaerospora testicularis, Polysporoplasma sparis, Leptotheca sparidarum, Henneguya sp., Ceratomyxa sp. Such parasites can establish in cavities of internal organs (coelozoic forms) or in tissues (histozoic forms); they present a very complex biological cycle, with a free-living phase in which the parasite is represented by binucleate spores, whose nuclei merge in the host producing an amoeboid stage. From this originates, through schizogony, a plurinucleate plasmodium, in which somatic and reproductive nuclei differentiates, and from a reproductive unit the free-living spores finally develop. Myxozoa are extremely important emerging pathogens for cage farming, where they can caush much damage to the farmed population. Sphaerospora dicentrarchi is a round-shaped myxozoan (3.5–6 × 4.6–8 × 3.5–4µm), containing two pear-shaped polar capsules. The most frequently affected host is sea bass; it is a histozoic species with a special tropism for the connective tissue. Sphaerospora testicularis is a small (15µm), round-shaped myxozoan that affects the sea bass too, but, unlike Sphaerospora dicentrarchi, it has a tropism for seminiferous tubules. It plays its pathogenetic role by strongly limiting the development of gonads and by considerably decreasing performances during the reproductive season. Affected gonads become brownish with the presence of yellowish nodules and an increase in consistency. Polysporoplasma sparis affects sea bream. It is a subspherical parasite (19.5 × 21µm) with two little posterior protuberances; it is

VENDRAMIN PRINT.indd 91

26/05/2017 08:33

92  ♦  Parasitic Infestations

Fig. 2.24: Ceratomyxa spp. in impression smear.

Fig. 2.25: Polysporoplasma sparis obtained by a fresh print of S. aurata kidney.

VENDRAMIN PRINT.indd 92

26/05/2017 08:33

Parasitic Infestations  ♦  93

histozoic and affects renal glomeruli. It can cause nephritis, hypertrophy of the renal corpuscles and atrophy of the parenchyma. Leptotheca sparidarum shows globular spores (5.1–8.2 × 9.4–11, 7µm), round polar capsules of the same dimensions, binucleate sporoplasm. It has been reported in sea bream. It is a coelozoic species and is found in the renal tubules and glomeruli. Henneguya sp. has complex spores that, according to their position in the space, can show a rounded, ellipsoidal or biconvex shape. The spores of this Myxozoan are characterized by caudal projections, which are joined for most of their length. It shows binucleate sporoplasm. The parasite is located in the cardiac parenchyma (aortic bulb) and on the gills. Ceratomyxa sp., found in sparids, serranids and mullets, is a parasite with elongated and curved spores, binucleate sporoplasm and round polar capsules. As already mentioned in the general section, these parasites prefer to establish in the gall bladder, where they can cause, in the case of severe infestations, deformation and necrosis of the epithelial cells, reduction and inflammation of the connective tissue. 6 – Monogenean trematodes These are the most frequently reported parasites in the gills of all the main farmed fish species. These small ectoparasites don’t usually have hosts, and have a posterior organ for the attachment (Haptor), which is equipped with hooks and/or suckers, and an anterior one. Gyrodactilus and Gyrodactiloides genera are found in marine farms and are located on the skin, gills and fins. In severe skin infestations it is possible to observe an acute dermatitis characterized by necrosis and hyperplasia; epidermal spongiosis and hydropic degeneration have also been observed. As regards the gill lamellae, a secondary bacterial infection with minor hemorrhages and cellular exfoliation can be observed. They are species-specific and reproduce very easily. Their presence in the epizootic form is a sign of bad management of the farm and is sometimes associated with protozoans.

VENDRAMIN PRINT.indd 93

26/05/2017 08:33

94  ♦  Parasitic Infestations

To date, three monogenean species have been identified in the sea bass, located in the gills. D. aequans is plausibly the most prevalent and pathogenetic. It is generally observed on the branchial lamellae. D. laubieri is very similar to D. aequans, but shows less pathogenicity, it is usually located in the central part of the lamellae. Serranocotyle labraci can be found but is not considered pathogenetic. So far, five monogenean species have been found in the sea bream, all of them located in the gills: Furnestina echeneis, which is the most prevalent and pathogenetic, Sparicotyle chrysophrii, Encotyllabe vallei and Choricotyle chrysophrii. Susceptible species are thus sea bass and sea bream. The biological cycle varies from one to two months and is influenced by water temperature. Greater prevalence and intensity are often observed during the cold season and are partially due to fish susceptibility. These parasites irritate the branchial lamellae; the host responds to the inflammation through hyperproduction of mucus and branchial

Fig. 2.26: Diplectanum aequans observed in branchial preparation of D. labrax.

VENDRAMIN PRINT.indd 94

26/05/2017 08:33

Parasitic Infestations  ♦  95

hyperplasia. Furthermore, a greater parasite density on the surface of the lamellae leads to asphyxia and related signs. Affected fish are generally dark, and could be found gasping at the surface and grouping near the entrance of the water flow, where oxygen levels are higher. They can be useful indicators of other pathological conditions (for example, chronic forms of VER in sea bass), which cause immunosuppression and thus allow anomalous parasitic p ­ roliferations on both the gills and the skin. 7 – Digenean trematodes Digeneans are parasites with an indirect biological cycle. They usually have one or two intermediate hosts; they reproduce asexually in the intermediate host and sexually in the definitive host. Among digenean trematodes described as pathogenetic for marine fish farmed in the Mediterranean Sanguinicolidae is reported. Sanguinicolidae sp.: these digenean trematodes have an elongated body and spiny cuticle, but are without suckers and pharynx. Their biological cycle is indirect: eggs are released by the adult in the bloodstream, from where they reach branchial vessels, where miracidia are released. Once escaped from branchial epithelium, they swim through cilia and reach the intermediate host represented by a gasteropod mollusk (Oxytrema spp., Fluminicola spp. Leptoxis spp.) where they become sporocysts, then daughter sporocysts, raediae and cercariae. The latter leave the mollusk and actively penetrate into the fish present in the environment through the skin and the gills (apparently mainly through the fins, where they can remain attached for almost three months). The infection seems to be ­seasonal (transmission would occur during the summer). The escape of eggs/miracidia through the branchial epithelium causes microlesions that could harbor infections is sustained by secondary pathogenic agents. Cercariae penetration during the infection can cause the formation of small wounds in the affected zones. When the level of infestation is particularly high, the adult can also disturb the circulation. In the presence of a high infestation level, generalized anemia is observed. During the escape of miracidia observe respiratory

VENDRAMIN PRINT.indd 95

26/05/2017 08:33

96  ♦  Parasitic Infestations

disorders and hemorrhages on branchial filaments, furthermore observe branchial necrosis due to secondary bacterial infections. Diagnosis is made through the microscopic observation of wet preparations of portions of branchial lamellae in order to show the eggs containing the miracidium; on the other hand, the finding of an adult in the bloodstream is quite difficult. 8 – Isopod and copepod crustaceans Among isopod crustaceans, the genera Ceratothoa, Nerocilia and Anilocra are pathogenetic for marine species. The adult stages of these species are mainly located on the caudal fin and in the caudal peduncle. The larval stage (pulli II larvae) is generally located in the buccal cavity and in the gill chamber where they cause the highest pathogenicity (obstructive action). In the wild, susceptible fish are mullets, bogues and sea bream, which belong to the marine fauna that swims around a floating cage farm. Infestation outbreaks are more frequently observed when temperatures rise, during the summer. Fish show respiratory stress with dilated opercula and group in the most oxygenated areas, becoming apathetic and anorexic. Pulli II larvae damage the lamellae, with consequent necrosis and inflammation of the gills and secondary infections sustained by T. maritimum. Mortality can be high. Diagnosis is made through direct observation of parasites in the caudal part of the body and in the oral and branchial cavity. Transmission occurs directly from fish to fish through water. The development of the early larval stages takes place in the marsupial pocket of the adult parasite. The life cycle of these isopods can be completed in the host itself. The infestation of juveniles can be prevented with the use of a small mesh around the cages or by filtering the input water in ponds. Organophosphates could be effective. The group of copepods includes some interesting pathogens of farmed marine species, such as the genera Lepeophtheirus and Caligus. Lepeophtheirus salmonis is the most important parasitic pathogen in the salmon industry and its corresponding Mediterranean ­parasite for sea bass is Caligus minimus. Adult specimens of copepod have a roundish cephalothorax,

VENDRAMIN PRINT.indd 96

26/05/2017 08:33

Parasitic Infestations  ♦  97

genital segment, abdomen and uropods. Female specimens are bigger than the males. With regard to reproduction, eggs are produced into elongated ovigerous sacs branching out from the genital segment. The genus Caligus shows frontal lunules that are absent in the genus Lepeophtheirus. The biological cycle is direct and is divided into several stages: nauplius larva, copepodid larva, chalimus, ­pre-adult, adult. Nauplii search for the host, then copepodid larva attaches through hooked antennas and, if the host is recognized as suitable, perforates the epidermis and derma, then attaches through a frontal filament to the basal membrane by means of an adhesive section. Afterwards it molts to chalimus, which remain permanently anchored to the scales, the fins or other sites through the frontal filament. Pre-adult and adult stages can move freely on the host’s surface by attaching thanks to the “suction action” of the cephalothorax and of the marginal membrane, with the help of the limbs. The duration of each phase depends on several environmental factors (temperature and salinity). The attachment and feeding activity of pre-adults and

Fig. 2.27: Liza ramada specimen parasitized by Caligus.

VENDRAMIN PRINT.indd 97

26/05/2017 08:33

98  ♦  Parasitic Infestations

adults causes edema, hyperplasia, inflammation and detachment of epidermic cells. Also, the detachment of chalimus stages can determine lesions, since the frontal filament remains stuck into the basal membrane and causes fibrotic lesions with inflammatory infiltration. Characteristic symptoms of such types of infestations vary according to the infestation site; particularly, when the parasite is located in the oral cavity, observe anorexia, weight loss, hemorrhagic areas on the palate and, if the attachment occurs on the skin surface, observe whitish areas that evolve in more or less extended ulcers, with erosion of wide areas on the skin and fins. Massive infestations can represent a serious problem for cage farming since these parasites are able to induce stress in the affected specimens, which considerably reduces their zootechnical performances, with severe losses for the farmer.

VENDRAMIN PRINT.indd 98

26/05/2017 08:33

Myxosporid Diseases (Enteromyxum leei)

In addition to the large group already described, this chapter focusses particularly on Enteromyxum leei since it causes severe infestations in several south Mediterranean farms. Aetiology This important parasitic disease is sustained by an endocellular parasite that shows lunate spores characterized by rounded apices and thin longitudinal crests; the suture line does not follow the median line regularly but forms, at every apex, a unilateral projection in which the polar capsule is leant (14.7 × 6.9 × 6µm). Two elongated polar capsules placed at the extremities of the spore form a 90 degree angle (7.4 × 3.2µm). Epidemiology In Mediterranean mariculture, Enteromyxidiosis mainly affects farmed sparids (Sparus aurata, Diplodus puntazzo and others). The disease has also been described in sea bass, but also other non-Mediterranean species affected by parasites belonging to this group. Among these Enteromyxum scophtalmi, is cited which affects turbot and sole. This disease concerns both farms with tanks and cages, and its pathogenicity seems to be highly variable. Host susceptibility appears very different according to the species and the age of the affected specimens.

VENDRAMIN PRINT.indd 99

26/05/2017 08:33

100  ♦  Myxosporid Diseases(Enteromyxum leei)

Enteromyxum leei, in its different forms, is mainly located in the gastrointestinal tract and particularly in the intestinal tract. In sea bream (Sparus aurata) the infestation is mostly chronic; specimens are usually affected during the second part of their farming cycle (above 100g) and show progressive weight loss and steady mortality. In sharpsnout seabream (Diplodus puntazzo) the disease shows a sudden and precocious course, infesting younger fish with heavy mortalities. Pathological lesions and clinical signs From the pathological point of view (GCE), enteromyxidiosis causes abdominal swelling, anal protrusion, and severe weight loss, which in some specimens shows the typical dimple in the dorsal muscles (most frequently in sharpsnout seabream). When the coelomic cavity is opened (IPE) the intestine appears ectatic and filled with catarrhal-like content. Observe ascites and intestinal congestion and the gall bladder showing the typical brownish color. With the opening of the intestinal tract observe foci with necrotic phenomena and lesions in the intestinal mucosa. In the sharpsnout seabream the damage is extremely large and spread to all the mucosa; in the sea bream necrotic areas are usually smaller. Through the microscopic observation of an intestinal smear large quantities of spores with the typical lunate shape (refractive to phase-contrast) can be seen. The histological analysis of the intestinal mucosa often highlights a widespread epithelial degeneration with more or less extended areas tending to necrosis. The submucosa is infiltrated with inflammatory cells, with several eosinophil granulocytes, which are less numerous in the muscular layer and in the subserosa layer. Diagnosis Enteromyxidiosis diagnosis is based on clinical and pathological observations, which must be confirmed through the microscopic

VENDRAMIN PRINT.indd 100

26/05/2017 08:33

Myxosporid Diseases(Enteromyxum leei)   ♦  101

observation of some organs, of an intestinal scrape and by the analysis of the gall content. Microscopic observation shows several development stages of the microorganism spread throughout all the digestive tract. Therapeutic recommendations and control strategies For this kind of microparasitic infestation, effective therapeutic devices are currently unavailable, even if some experimental trials with anticoccidics are currently performed, which, although providing some interesting results, are inapplicable for both economic and legislative reasons. Prophylactic strategies must be focused on frequent controls on both newly introduced fish and particularly sensitive fish species that are present in the farm, in order to obtain an early diagnosis.

Fig. 2.28: S. aurata affected by Enteromyxum leei, to be noted the very poor body condition of the specimen. Picture kindly provided by Dr. Carlos Zarza.

VENDRAMIN PRINT.indd 101

26/05/2017 08:33

102  ♦  Myxosporid Diseases(Enteromyxum leei)

Fig. 2.29: Intestinal scraping from Diplodus puntazzo (sharpsnout sea bream) showing E.leei spores (arrow). Picture kindly provided by Dr. Snjezana Zrncic.

Fig. 2.30: Intestinal scraping from Diplodus puntazzo (sharpsnout sea bream) showing E.leei spores (arrow). Picture kindly provided by Dr. Snjezana Zrncic.

VENDRAMIN PRINT.indd 102

26/05/2017 08:33

Myxosporid Diseases(Enteromyxum leei)   ♦  103

The only prophylactic intervention that proves to be quite ­effective is (where possible) to transfer the cages, with the intention of interrupting the parasite’s biological cycle. In most cases, the complexity of this disease forces the farmer to give up rearing the affected species in favor of less susceptible species.

VENDRAMIN PRINT.indd 103

26/05/2017 08:33

Isopod Infestations (Ceratothoa, Nerocilia, Anilocra)

Aetiology These important parasitic diseases, which are emerging in floating cage marine aquaculture, are classified among classical ectoparasitosis. The aetiologic agents responsible for these important parasitic infestations are crustaceans belonging to the order Isopoda. Crustaceans belonging to this large group are widespread in nature in several saltwater and brackish habitats and, for this reason, they can affect fish reared in many different conditions. The main representatives of this group are: Ceratothoa oestroides, Ceratothoa parallela and Nerocilia orbigny, all of them belonging to the sub-class Malacostraca. In addition, there are many other crustaceans that can often play an important role in diseases affecting floating cage farms. Almost all of them are characterized by great reproductive abilities and complex biological cycles, which often involve several larval stages that are parasitic too. The first larval stages are already able to actively search for the host, on which they perform several kinds of parasitic action, from the mechanical and hematophagous to the obstructive. Epidemiology Since this group of parasites includes a great variety of species, it represent one of the most widely spread problems throughout the Mediterranean. Among the stocks of juveniles seeded in cages, the cumulative

VENDRAMIN PRINT.indd 104

26/05/2017 08:33

Isopod Infestations  ♦  105

mortality rate can be significant, also in relation to secondary bacterial complications and obstructive problems in the oral cavity. In the wild, typical parasitized hosts are mullets (Mugil sp.), bogues (Boops boops), salema porgies (Sarpa salpa), striped sea breams (Lithognathus mormyrus) and white sea breams (Diplodus sargus). These marine species are abundant in the proximity of sea bass and sea bream cages, they feed on residual fish food and can act as carriers, favoring the spread of these pathogens among farmed stocks. These parasites show a direct biological cycle: the female puts the eggs up in her pouch, where they develop and hatch, showing the first molt. At this stage the larva leaves the mother’s pouch and begins a free-living stage in the water. This stage ends when the parasite finds a host, the parasite infests it and starts feeding, then develops and matures into subsequent biological stages. Pathological lesions and clinical signs Mechanical lesions that characterize larval parasitic stages are often complicated by inflammatory lesions around the site where the crustacean feeds: in addition to the loss of scales and superficial cutaneous layers, an accumulation of erythrocytes, eosinophils and lymphocytes can be observed. When the attachment occurs into the buccal cavity, a granulomatous inflammatory reaction can occur. Moreover, isopod cymothoid crustaceans and the praniza larvae of Gnathiidae are hematophagous. The symptoms and the functional damage are strictly related to the site and the attached organ:

U Parasites can locate in the gill chamber, causing respiratory difficulties and osmoregulatory problems. At the level of the branchial parenchyma also observe paleness, ­ erosions and progressive epithelial degeneration. In the oral cavity, they cause severe feeding difficulties, weight loss and granulomatous proliferative reactions. On the skin surface, erosions and ulcers are often evident, and can damage the muscular wall.

U U

VENDRAMIN PRINT.indd 105

26/05/2017 08:33

106  ♦  Isopod Infestations

Besides severe mortality mainly seen in sea bass during the early stages of cage farming, indirect damage caused by such diseases are also relevant. This is because the disease slows down the zootechnical performances of affected stock, inevitably causing delays in the growth and greater incidence of infectious diseases. Diagnosis The diagnosis of these parasitic diseases is made through the macroscopic observation of the specimens. Also, in these cases a correct interpretation of the diagnostic report is crucial. A deep knowledge of the epidemiological condition, the correct identification of the parasite species, seasonality and the affected marine species are all fundamental elements in order to express a correct diagnosis and start an action strategy. Therapeutic recommendation and control strategies Strategies for the control of these parasitic diseases are particularly difficult. Due to the absence of applicable therapeutic devices and

Fig. 2.31: Erosive-ulcerative lesions on the skin of L. mormyrus due to the presence of an adult parasite crustacean isopod Anilocra.

VENDRAMIN PRINT.indd 106

26/05/2017 08:33

Isopod Infestations  ♦  107

Fig. 2.32: Adult specimen of Anilocra.

Fig. 2.33: Adult of Ceratothoa in gills of D. labrax. Courtesy of Dr. Snjezana Zrncic.

VENDRAMIN PRINT.indd 107

26/05/2017 08:33

108  ♦  Isopod Infestations

Fig. 2.34: Ceratothoa in mouth of D. labrax. Courtesy of Dr. Snjezana Zrncic.

the biological complexity of this group of parasites, any kind of intervention on fish is practically ineffective. Dosages required for a therapy with the most common pesticides would be expensive and extremely near to the toxic dose for farmed fish. The only applicable forms of intervention are represented by a deep study of the crustacean parasite, an analysis of its biological cycle and a variation in the management plan that considers previously collected information.

VENDRAMIN PRINT.indd 108

26/05/2017 08:33

Isopod Infestations  ♦  109

In some cases, solutions such as delaying the stocking of the main affected species or changing the size of the stock, starting with a pre-fattened product, provides good results. This confirms the fact that a greater knowledge of the biology of our farmed species and of the organisms gravitating around them is increasingly useful and necessary for future aquaculture. Then correct productive strategies and adequate prophylaxis programs will replace expensive and ineffective treatments, which are so dangerous for the delicate marine environment.

VENDRAMIN PRINT.indd 109

26/05/2017 08:33

Mongenean Parasitic Infestations

Aetiology Among the parasitic infestations challenging Mediterranean marine aquaculture, parasitic infestations by trematodes monogeneans must be considered when reporting those characterized by the highest impact on production of the farmed spardis in the area. Two representatives, here given as key examples for farmed Gilthead sea bream, are currently Sparicotyle chrysophrii and Furnestinia echeneis. Furnestinia echeneis. This parasite is characterized by a cylindrical shape (approx. 1mm long), with both ends sharp. The parasite can attach to fish gills through an organ with adherence properties characterized by lamellae and two hooks. This parasite is localized in the gill of the affected fish. Sparicotyle chrysophrii is another trematode monogenean with an elongated body, slightly pressed from the back to the ventral part. It is approximately 3.5–4mm long and 0.4mm wide. It has also a robust organ of adherence constituted by abundant clamps situated parallel on the sides. This parasite has a direct life cycle. Once the adult has produced eggs, these hatch and release an Oncomiracidium, which will attach to the new host or the same host’s gill. The temperature seems to play an extremely important role for this parasite infestation; even though the infestation takes place almost all year around, in affected sites there is the highest prevalence during winter. The pathology of

VENDRAMIN PRINT.indd 110

26/05/2017 08:33

Mongenean Parasitic Infestations  ♦  111

this parasite takes place in the gills, where the adherence organ causes lesions in the epithelial tissue. This parasite also has hematophagic action, and the affected fish suffers different degrees of anemia in relation to the number of the parasites on the gill. As mentioned above, the nets of the cage offer an optimal substrate for the adherence of hatching eggs. The following life stages (free swimming larvae) will encounter an optimal environment (high concentration of hosts) and thereby will be able to spread the infestation rapidly. Depending on the environmental conditions, in a period of time up to a few weeks the whole fish stock in the cage can be affected by the parasite. Epidemiology In cage production, these parasites have found an optimal environment for spreading due to the large amount of susceptible hosts and temperate water temperature that characterizes the sites where cage farming is emerging (northern Africa and Turkey, among others). Both the infestation with Sparicotyle and Furnestina, which cause most of the parasitic infestations by trematodes, represent a major issue when it comes to treatment due to the characteristics of the parasites’ life cycle and the sites where therapy has eventually to be applied. As will be described later, treatment of parasites based on chemicals in offshore sea cages is difficult to implement and not very effective. Gill trematodes (including Furnestina and Sparicotyle) are spread in a large variety of wild sparids, including, among others, sharpsnout sea bream (D. puntazzo) and white bream (D. sargus). These fish are almost always present around sea cages as these last ones operate as Feed Aggregating Devices (FAD) and attract wild fish that opportunistically take advantage of aquacultural feed. These parasites induce severe gill damage to the affected fish, which compromises the performance and robustness of affected stocks. The gill damage is the result of the reaction of the fish immune response to the parasite, which is characterized by hyper-production of mucus and hyperplasia of the gill epithelium. In the case of Sparicotyle, affected fish suffer anemia due to the hematophagy of the parasite.

VENDRAMIN PRINT.indd 111

26/05/2017 08:33

112  ♦  Mongenean Parasitic Infestations

Pathological lesions and symptoms During clinical outbreaks, where generally the level of infestation is high, the diagnosis is rather simple and based on observation of the parasite and its typical structure (organ of adherence and gonads) for identification, in a wet preparation of gill tissues. In general it can already be possible to observe a reaction of the gill tissue with macroscopic observation (hyper-production of mucus). During an outbreak it is generally possible to distinguish two clinical phases of these parasitic infestations. In the first stage, the affected fish generally appears to show nervous behavior, displaying hyperactivity, and tends to scratch itself against the cage net. In the second stage, the fish shows melanosis and lethargy, and it does not feed properly. Referring to specific parasites, a single difference has to be reported in the diagnostic procedure. For Furnestina, due to its smaller dimension, the diagnosis needs to rely on the microscopic observation of the preparation; whereas, in the case of Sparicotyle, it is possible to observe the parasite using a stereomicroscope to examine fresh gill preparation (usually targeting the first gill arch, which is the most affected). The identification of the parasite relies on the features of the organs of adherence and gonads. Therapeutic recommendations and control strategies As already mentioned, treatment of parasitic infestations in sea cages is one of the most difficult challenges that the health manager of a fish farm faces during his work. It is important to remember that the difficulties are basically linked to the environment where the treatment is implemented, which does not allow proper control of different parameters (dose administered, time of administration, large volumes, etc). The use of a disinfection treatment by bath (often based on formalin or hydrogen peroxide), despite being widely employed, generally does not provide an effective response in farmed fish in the frame of sea cages where volumes are huge and the number of fish

VENDRAMIN PRINT.indd 112

26/05/2017 08:33

Mongenean Parasitic Infestations  ♦  113

to be treated rather high (normally more than 200,000 units per cage). Therapeutic treatment based on formalin (25–250ppm for 30–60 minutes for two days, once a week, repeated for three weeks) has been demonstrated to be effective in land-based facilities. Treatment has to be repeated during the weeks as eggs are not susceptible. Oral treatment based on the use of anthelmintics isoquinolone Praziquantel (PZQ) (dose 150mg/kg bw) demonstrated low efficacy and the chemical is considered to negatively affect the palatability of the feed for the fish, with zootechnical consequences on the feeding of fish batch-treated. Therefore, this option is not recommended and appears not to be applicable. It is the opinion of the authors that the control strategy to be implemented should not rely on a chemical treatment but be built on a careful analysis of the parasitic cycle in the site of interest and extensive knowledge of the environmental conditions of the specific

Fig. 2.35: Adults of Sparicotyle chrysophrii isolated observed on impression smear from skin mucus of sea bream. Courtesy of Dr. Ivona Mladineo.

VENDRAMIN PRINT.indd 113

26/05/2017 08:33

114  ♦  Mongenean Parasitic Infestations

Fig. 2.36: Sparicotyle chrysophrii detail of adhesion organ and egg. Courtesy of Dr. Ivona Mladineo.

farm. The understanding of the interaction of the parasite with the environment enables the operation of a series of management procedures, including net change and transfer of sea cages, among others, that aim to break the life cycle of the parasite and mitigate its proliferation during the production cycle until fish have reached the desired market size.

VENDRAMIN PRINT.indd 114

26/05/2017 08:33

Summary Bacterial Diseases

Typical Vibriosis Aetiological agent

Vibrio anguillarum

Species affected Symptoms Diagnosis

European sea bass (D. labrax) Hemorrhagic syndrome Evaluation of clinical signs Bacterial isolation on agar plates Bacterial identification with biochemical methods Observation of bacteria in impression smear of internal organs of affected fish

Therapeutic suggestions

Biological compound Flumequine

Typical vibriosis (Vibrio anguillarum) Dosage (mg/ Duration of kg bw) antimicrobial therapy 60mg/kg in fish up to 50g 80mg/kg in larger size fish

VENDRAMIN PRINT.indd 115

7/8 days

Specific indications In case of recurrence, antibiogram analysis is recommended to select the most effective antibiotic

26/05/2017 08:33

116  ♦  Summary

Photobacteriosis Aetiological agent

Photobacterium damselae subsp. piscicida

Species affected

European Sea bass (D. labrax) and Gilthead sea bream (S. aurata) Chronic infection with granuloma in spleen and kidney Evaluation of clinical signs Bacterial isolation on agar plates Bacterial identification with biochemical methods Observation of bacteria in impression smear of internal organs of affected fish

Symptoms Diagnosis

Therapeutic suggestions Photobacteriosis (previously known as pasteurellosis) Biological Dosage (mg/ Duration of Specific compound kg bw) antimicrobial indications therapy Flumequine

70mg/kg in fish up to 60g 90mg/kg in larger size fish

6/7 days

Preferably employed in fish weighing less than 40g

Sulphadiazine + trimetophrim

80mg/kg

9/10 days

Preferably employed in fish weighing more than 40g

VENDRAMIN PRINT.indd 116

26/05/2017 08:33

Summary  ♦  117

Flexibacteriosis Aetiological agent

Flexibacter maritimum

Species affected Symptoms Diagnosis

European sea bass (D. labrax) Skin lesions and fin erosions Evaluation of clinical signs Bacterial isolation on agar plates Bacterial identification with biochemical methods Observation of bacteria in impression smear of internal organs of affected fish

Therapeutic suggestions

Biological compound Florfenicol

VENDRAMIN PRINT.indd 117

Flexibacteriosis Dosage (mg/ Duration of kg bw) antimicrobial therapy 60mg/kg in fish up to 10g 80mg/kg in larger size fish

8/9 days

Specific indications A disinfectant bath is often used for boosting the antibiotic effect when treating fish between 4 and 10g. This treatment consists of administering formaldehyde at a concentration of 250ppm for one hour per day. Alternatively, it is used for one day with and one not for the entire length of the therapeutic treatment. Note: the application of disinfectant to live fish is forbidden in many countries

26/05/2017 08:33

118  ♦  Summary

Streptococcosis Aetiological agent

 actococcus garvieae (also known as L Enterococcus seriolicida), Lactococcus piscium, Streptococcus iniae, Streptococcus agalactiae, Streptococcus parauberis and Vagococcus salmoninarum

Species affected

European sea bass (D. labrax); shi drum (U.cirrosa); amberjack (S. dumerilii) Hemorrhagic syndrome, Exophthalmia Evaluation of clinical signs Bacterial isolation on agar plates Bacterial identification with biochemical methods Observation of bacteria in impression smear of internal organs of affected fish

Symptoms Diagnosis

Therapeutic suggestions

Biological compound Amoxicillin

VENDRAMIN PRINT.indd 118

Streptococcosis Dosage (mg/ Duration of kg bw) antimicrobial therapy 60mg/kg in fish up to 10g 80mg/kg in larger size fish

8/10 days

Specific indications Antibiogram analysis is recommended for selecting the most appropriate antibiotics and avoiding the occurrence of antibiotic resistance

26/05/2017 08:33

Summary  ♦  119

Septicemia by Aeromonas Aetiological agent

Aeromonas sp.

Species affected Symptoms

European sea bass (D. labrax) Hemorrhagic syndrome and association with skin lesions and exophthalmia Evaluation of clinical signs Bacterial isolation on agar plates Bacterial identification with biochemical methods Observation of bacteria in impression smear of internal organs of affected fish

Diagnosis

Therapeutic suggestions

Biological compound Amoxicillin

VENDRAMIN PRINT.indd 119

Septicemia by Aeromonas Dosage (mg/ Duration of kg bw) antimicrobial therapy 80mg/kg

8/9 days

Specific indications Antibiogram analysis is needed for selecting the most appropriate antibiotics and avoiding the occurrence of antibiotic resistance

26/05/2017 08:33

Viral Diseases

Viral Encephalopathy and Retinopathy (VER, also known as Viral Nervous Necrosis – VNN) Aetiological agent

Betanodavirus.

Species affected Symptoms

European sea bass (D. labrax) Neurological syndrome – abnormal swimming behavior Evaluation of clinical signs Direct PCR on relevant samples Virus isolation on cell culture

Diagnosis

Therapeutic suggestions A specific focus on Good Management Practice (GMP) aiming at stress reduction can improve the performance of the affected stock significantly. In this case, the specific care has to last throughout the whole period of the clinical outbreak. With specific reference to Viral Encephalopathy and Retinopathy, it is suggested to reduce the feeding regime and to implement some technical solutions such as shading the sea cage, avoiding transportation and handling, and taking specific care in collecting dead/moribund fish for disposal. Of great importance is the avoidance of administering of any chemical compound that could potentially cause a neurotoxical effect, such as quinolones molecules and derived antibiotics.

VENDRAMIN PRINT.indd 120

26/05/2017 08:33

Viral Diseases  ♦  121

Lymphocystis disease Aetiological agent

Lymphocystis disease virus

Species affected

Gilthead sea bream (S. aurata), Sharpsnout sea bream (D. puntazzo), other sparidae Skin proliferative lesions, resembling tumors Evaluation of clinical signs Direct PCR on relevant samples Virus isolation on cell culture

Symptoms Diagnosis

Therapeutic suggestions A specific focus on Good Management Practice (GMP) aiming at stress reduction can significantly improve the performance of the affected stock. In this case, the specific care has to last during the whole period of the clinical outbreak. With regard to Lymphocystis disease, it is suggested that handling the fish in the clinical phase (sorting, moving, vaccinating) is avoided.

Biological compound

Lymphocystis disease Dosage (mg/ Duration of kg bw) antimicrobial therapy

Vitamins Bcomplex + VitC

5g Biotin + 3g C./kg feed

Sulphadiazine + trimetophrim

80mg/kg

VENDRAMIN PRINT.indd 121

To be added to fish feed for 30 days after first appearance of skin lesions 9/10 days after appearance of skin lesions

Specific indications

Prevention of secondary bacterial infections

26/05/2017 08:33

Parasitic Infestations

Diseases caused by Myxozoa Aetiological agent

Myxozoa – Enteromyxum leei

Species affected

Gilthead sea bream (S. aurata), Sharpsnout sea bream (D. puntazzo), other sparidae Progressive thinning of the fish, with losses of muscle tissue Evaluation of clinical signs Direct observation of parasite in impression smear Histopathology

Symptoms Diagnosis

A specific focus on Good Management Practice (GMP) aiming at stress reduction can significantly improve the performance of the affected stock. In this case, the specific care has to last for the whole period of the clinical outbreak. With specific regard to infestations with myxozoan parasites, it is suggested that handling the fish in the clinical phase (sorting, moving, vaccinating) is avoided.

VENDRAMIN PRINT.indd 122

26/05/2017 08:33

Parasitic Infestations  ♦  123

Therapeutic suggestions

Biological compound

Enteromyxosis Dosage (mg/ Duration of kg bw) antimicrobial therapy

Vitamins Bcomplex + VitC

5g Biotin + 3g C./kg feed

Sulfadiazina + Trimetoprim

40mg/kg

VENDRAMIN PRINT.indd 123

To be added to fish feed for 30 days after first appearance of skin lesions 10/12 days after detection of first specimens affected and appearance of typical symptoms, such as skin discoloration and swollen abdomen

Specific indications

To prevent frequent secondary bacterial infections

26/05/2017 08:33

124  ♦  Parasitic Infestations

Isopod and copepod crustaceans Aetiological agent

I sopod crustaceans, the genera Ceratothoa, Nerocilia and Anilocra

Species affected Symptoms

Mostly European sea bass (D. labrax) Fish show reduction of feed intake and increased “respiratory” behavior due to mechanical obstructions of gill chamber and mouth Evaluation of clinical signs Direct observation of parasite in impression smear

Diagnosis

A specific focus on Good Management Practice (GMP) aiming at stress reduction can significantly improve the performance of the affected stock. In this case, the specific care has to last throughout the whole period of the clinical outbreak. With specific reference to infestations with isopod crustaceans, the planning and evaluation of seeding strategy is of primary importance. Specific focus needs to be given to the size of juvenile for the seed and the period of the year when the production cycle will start, in relation to the life stage of the parasite in the specific area. Therapeutic suggestions

Biological compound Permethrin compounds: cypermethrin or deltamethrin

VENDRAMIN PRINT.indd 124

Isopod and copepod crustaceans Dosage Duration of therapy From 2 to 10ppm in relation to the size of the affected fish

1 to 3 hours in relation to the size of the affected fish

Specific indications Hard to implement in sea cage

26/05/2017 08:33

Parasitic Infestations  ♦  125

Infestation by Trematoda Aetiological agent

Sparicotyle chrysophrii, Furnestina echeneis.

Species affected Symptoms

Mostly sea bream and other sparidae Fish show reduction of feed intake and increased “respiratory” behavior due to mechanical obstructions of gill chamber and mouth Evaluation of clinical signs Direct observation of parasite in impression smear

Diagnosis

Therapeutic suggestions A specific focus on Good Management Practice (GMP) aiming at stress reduction can significantly improve the performance of the affected stock. In this case, the specific care has to last throughout the whole period of the clinical outbreak. Specifically referring to infestations with trematodes, the planning and evaluation of seeding strategy is of primary importance. Specific focus needs to be allocated to the size of juvenile for the seed and the period of the year when the production cycle will start, in relation to the life stage of the parasite in the specific area. Infestation by Sparicotyle chrysophrii and Furnestinia echeneis sp. Biological Dosage Duration of Specific compound therapy indications Permethrin compounds: cypermethrin or deltamethrin

VENDRAMIN PRINT.indd 125

6 to 20ppm in relation to the fish size

1 to 3 hours in relation to the size of the affected fish

Hard to implement in sea cage

26/05/2017 08:33

References for Further Reading

 1 Manual on Hatchery Production of Seabass and Gilthead Seabream – Volume 1 by Alessandro Moretti, Mario Pedini Fernandez-Criado, Giancarlo Cittolin Ruggero Guidastri. ISBN 92-5-104380-9, www.fao. org/docrep/005/x3980e/x3980e00.htm  2 Manual on Hatchery Production of Seabass and Gilthead Seabream – Volume 2 by Alessandro Moretti, Mario Pedini Fernandez-Criado, René Vetillart. ISBN 92-5-105304-9, www.fao.org/docrep/008/y6018e/ y6018e00.htm   3 Sparidae: Biology and Aquaculture of Gilthead Sea Bream and other Species, Biology and Aquaculture of Gilthead Sea Bream and other Species by Michail A. Pavlidis, Constantinos C. Mylonas. Print ISBN: 9781405197724 Online ISBN: 9781444392210 DOI: 10.1002/9781444392210, http:// onlinelibrary.wiley.com/book/10.1002/9781444392210  4 Aquaculture operations in floating HDPE cages, A field handbook by Francesco Cardia and Alessandro Lovatelli, FAO Fisheries and Aquaculture Technical Paper 593. ISBN 978-92-5-108749-7, www.fao.org/3/a-i4508e. pdf   5 Fish Disease: Diagnosis and Treatment by Edward J. Noga, Wiley Blackwell. ISBN 978-0-8138-0697.  6 Fish Diseases and Disorders: Viral, Bacterial and Fungal Infections by P.T.K. Woo, David W. Bruno. ISBN 0851991947, 9780851991948.   7 Infectious Disease in Aquaculture Prevention and Control, A volume in Woodhead Publishing Series in Food Science, Technology and Nutrition, edited by B. Austin. ISBN: 978-0-85709-016-4.   8 Anon. (2007). Possible vector species and live stages of susceptible species not transmitting disease as regards certain fish diseases. EFSA Journal 584, 91–163.   9 Bovo, G.; Nishizawa, T.; Maltese C., Borghesan, F.; Mutinelli, F.; Montesi, F. and De Mas, S. (1999). Viral encephalopathy and retinopathy of farmed marine fish species in Italy. Virus Research 63, 143–146. doi: 10.1016/ S0168-1702(99)00068-4.

VENDRAMIN PRINT.indd 126

26/05/2017 08:33

References for Further Reading  ♦  127 10

11

12 13

14 15 16 17

18 19

20

21

Cano, I.; Ferro, P.; Alonso, M.C, Bergmann, S.M.; Römer-Oberdörfer, A.; Garcia-Rosado, E.; Castro, D. and Borrego, J.J. (2007). Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species. Journal of Applied Microbiology 102, 32–40. Colorni, A.; Avtalion, R.; Knibb, W.; Berger, E.; Colorni, B. and Timan, B. (1998). Histopathology of sea bass (Dicentrarchuslabrax) experimentally infected with Mycobacterium marinum and treated with streptomycin and garlic (Allium sativum) extract. Aquaculture 160, 1–17. Diamant, A.; Lom, J. and Dyková, I. (1994). Myxidium leei n. sp., a pathogenic myxosporean of cultured sea bream Sparus aurata. Diseases of Aquatic Organisms 20, 137–141. Doukas, V.; Athanassopoulou, F.; Karagouni, E. and Dotsika, E. (1998). Aeromonas hydrophila infection in cultured sea bass, Dicentrarchus labrax L., and Puntazzo Cuvier from the Aegean Sea. Journal of Fish Diseases 21, 317–320. FEAP (2015). European Aquaculture Production Report 2005–2014. Prepared by FEAP Secretariat. Munday, B.L.; Kwang, J. and Moody, N. (2002). Betanodavirus infections of teleost fish: a review. Journal of Fish Diseases 25, 127–142. doi: 10.1046/j.1365-2761.2002.00350.x. Olesen, N.J. and Vendramin, N. (2012, 2013, 2014, 2015). Report of 16th, 17th, 18th, 19th Annual Meeting of the NRL for Fish Diseases. www.eurl-fish.eu/Activities/annualmeetings Palenzuela, O.; Redondo, M.J.; Cali, A.; Takvorian, P.M.; Alonso-Naveiro, M.; Alvarez-Pellitero, P. and Sitjà-Bobadilla, A. (2014). A new intranuclearmicrosporidium, Enterosporanucleophila n. sp., causing an emaciativesyndrome in a piscine host (Sparusaurata), prompts the redescription of the family Enterocytozoonidae. International Journal of Parasitology 44, 189–203. Paperna, I. (1980). Amyloodiniumocellatum (Brown, 1931) (Dinoflagellida) infestations in cultured marine fish at Eilat, Red Sea: epizootiology and pathology. Journal of Fish Diseases 3, 363–372. Rigos, G.; Pavlidis, M.; and Divanach, P. (2001). Host susceptibility to Cryptocaryon sp. Infection of Mediterranean marine broodfish held under intensive culture conditions: a case report. Bulletin of the European Association of Fish Pathologists 21, 33–36. Sitjà-Bobadilla, A.; Redondo, M.J. and Alvarez-Pellitero, P. (2010). Occurrence of Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) in gilthead sea bream (Sparus aurata L.) from different mariculture systems in Spain. Aquaculture Research 41, 939–944. Toranzo, A.E.; Magariños, B. and Romalde, J.L. (2005). A review of the main bacterial fishdiseases in mariculture systems. Aquaculture 246, 37-61.9-944.

VENDRAMIN PRINT.indd 127

26/05/2017 08:33

128  ♦  References for Further Reading 22

Office International des Epizooties (OIE) (1995): International Biosecurity Code for Aquatic Animals. Ed. by OIE Fish Diseases Commission. 23 Biology of European Sea Bass, F. Javier Sánchez Vázquez  and José A. Muñoz-Cueto, CRC Press 2014. Print ISBN: 978-1-4665-9945-1 eBook ISBN: 978-1-4665-9946-8 DOI: 10.1201/b16043-13. 24 Fish Vaccination Copyright © 2014 John Wiley & Sons Ltd, Editor(s): Roar Gudding, Atle Lillehaug, Øystein Evensen, Published online: 7 March 2014. Print ISBN: 9780470674550 Online ISBN: 9781118806913 DOI: 10.1002/9781118806913. 25 Toranzo, A.E.; Romalde, J.L.; Dopazo, C.P.; Magariños, B.; Barja, J.L. (2004) Disease trends in the primary marine fish species cultured in Spain: A 20-year study. World Aquac 35:35–38.

Vibrio anguillarum

U U

Angelidis, P.; Karagiannis, D.; Crump, E.M. Efficacy of a Listonella anguillarum (syn Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax. Dis Aquat Organ, 71 (2006), pp. 19–24. Frans, I.; Michiels, C.W.; Bossier, P.; Willems, K.A.; Lievens, B. and Rediers, H. Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention. Journal of Fish Diseases 2011, 34, 643–661

Photobacterium damselae subsp. piscicida

U U

Romalde, J.L. Photobacterium damselae subsp. piscicida: an integrated view of a bacterial fish pathogen. Int Microbiol.  2002 Mar; 5(1):3–9. Bakopoulos, V.; Volpatti, D.; Gusmani, L.; Galeotti, M.; Adams, A. and Dimitriadis, G.J. (2003). Vaccination trials of sea bass, Dicentrarchus labrax (L.), against Photobacterium damsela subsp. piscicida, using novel vaccine mixtures. Journal of Fish Diseases, 26: 77–90. doi:10.1046/j.1365-2761.2003.00438.x.

Tenacibaculum maritimum

U

VENDRAMIN PRINT.indd 128

Failde, L.D.; Losada, A.P.; Bermúdez, R.; Santos, Y.; Quiroga, M.I. Tenacibaculum maritimum infection: pathology and immunohistochem-

26/05/2017 08:33

References for Further Reading  ♦  129 istry in experimentally challenged turbot (Psetta maxima L.) Microb. Pathog., 65 (2013), pp. 82–88. Avendaño-Herrera, R.;  Toranzo, A.E.;  Magariños, B. Tenacibaculosis infection in marine fish caused by Tenacibaculum maritimum: a review. Dis Aquat Organ. 2006 Aug 30; 71(3):255–66. Avendaño-Herrera, R.; Toranzo, A.E.; Magariños, B. (2006). Tenacibaculosis infection in marine fish caused by Tenacibaculum maritimum: a review. Dis Aquat Org 71:255–266. Avendaño-Herrera, R.;  Magariños, B.;  Irgang, R.  and  Toranzo, A.E. (2006). Use of hydrogen peroxide against the fish pathogen Tenacibaculum maritimum  and its effect on infected turbot (Scophthalmus maximus). Aquaculture 257, 104–110.

U U U

Streptococcosis

U

Romalde, J.L. and Toranzo, A.E. Streptococcosis of marine fish. Leaflet No. 56 ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish. Prepared under the guidance of the ICES Working Group on Pathology and Diseases of Marine Organisms. ISSN 0109-2510.

Aeromonas

U

Suheyla Karatas*, Akin Candan, and Didem Demircan Atypical Aeromonas Infection in Cultured Sea Bass (Dicentrarchus Labrax) in the Black Sea, The Israeli Journal of Aquaculture – Bamidgeh 57(4), 2005, 255–263.

Viral Encephalopathy and Retinopathy

U U

Manual of Diagnostic Tests for Aquatic Animals OIE Chapter 2.3.12. Viral Encephalopathy and Retinopathy. Bovo G.; Gustinelli A.; Quaglio F.; Gobbo F.; Panzarin V.; Fusaro A.; Mutinelli F.; Caffara M.; Fioravanti M.L., Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy., “DISEASES OF AQUATIC ORGANISMS”, 2011, 96, pp. 45. Frerichs, G.N.; Tweedie, A.; Starkey, W.G.; Richards, R.H. Temperature, pH and electrolyte sensitivity, and heat, UV and disinfectant inactivation of

U

VENDRAMIN PRINT.indd 129

26/05/2017 08:33

130  ♦  References for Further Reading sea bass (Dicentrarchus labrax) neuropathy nodavirus (2000) Aquaculture, 185 (1-2), pp. 13-24. doi: 10.1016/S0044-8486(99)00337-3. Vendramin, N.; Patarnello, P.; Toffan, A. et al. Viral Encephalopathy and Retinopathy in groupers (Epinephelus spp.) in southern Italy: a threat for wild endangered species?  BMC Veterinary Research. 2013; 9:20. doi:10.1186/1746-6148-9-20. Thiéry, R.; Cozien, J.; Cabon, J.; Lamour, F.; Baud, M.; Schneemann, A. Induction of a Protective Immune Response against Viral Nervous Necrosis in the European Sea Bass  Dicentrarchus labrax  by Using Betanodavirus Virus-Like Particles.  Journal of Virology. 2006; 80(20):10201-10207. doi:10.1128/JVI.01098-06.

U U

Lymphocystis disease

U

Cano, I., Valverde, E. J., Garcia-Rosado, E., Alonso, M. C., LopezJimena, B., Ortiz-Delgado, J. B., Borrego, J. J., Sarasquete, C. and Castro, D. (2013). Transmission of lymphocystis disease virus to cultured gilthead seabream, Sparus aurata L., larvae. J Fish Dis, 36: 569–576. doi:10.1111/ jfd.1201. Estefania J. Valverde, Irene Cano, Alejandro Labella, Juan J. Borrego and Dolores Castro. Application of a new real-time polymerase chain reaction assay for surveillance studies of lymphocystis disease virus in farmed gilthead seabream. BMC Veterinary Research. BMC series – open, inclusive and trusted 201612:71 DOI: 10.1186/s12917-016-0696-6.

U

Parasites

U

Daudi, F. and Marques, A. (1987). Eimeria bouixi n. sp. and Eimeria dicentrarchi n. sp. (Sporozoa-Apicomplexa) parapiles from the fish Dicentrarchus labrax (Linne 1758) in Lanquedoc. Ann. Sci. Nat. Zool. Biol. Anim., 8, 237–242. Le Breton, A. and Marques, A. (1995). Occurrence of histozoic Myxidium infection in two marine cultured species: Puntazzo C. and Pagrus major. Bull. Eur. Ass. Fish Pathol., 15, 210–212. Fioravanti, M.L.; Caffara, M.; Florio, D.; Gustinelli, A.; Marcer, F.; Quaglio, F. Parasitic diseases of marine fish: epidemiological and biosecurity considerations. Parassitologia. 2006 Jun;48(1–2):15–8. An overview of the treatments for parasitic disease in Mediterranean aquaculture. Athanassopoulou, F.; Pappas, I.S.; Bitchava, K. in Rogers C. (ed.),

U U U

VENDRAMIN PRINT.indd 130

26/05/2017 08:33

References for Further Reading  ♦  131 Basurco, B. (ed.). The use of veterinary drugs and vaccines in Mediterranean aquaculture Zaragoza: CIHEAM Options Méditerranéennes: Série A. Séminaires Méditerranéens; n. 86 2009 pages 65–83. Silan, P. and Maillard, P. (1986). Mortalities of the infection of Dicentrarchus labrax by an ectoparasite Diplectanum aequans (Monogenea) in aquaculture. Epidemiological and prophylactic data. In: Pathology in marine aquaculture (Ed. by Vivares, C. P.; Bonami, J. R. and Jaspers, E.). Spec. Publ. Eur. Aqua. Soc., 9, 139–152. Sitja-Bobadilla, A. and Alvarez-Pellitero, P. (1992). Light and electron microscopic description of Sphaerospora dicentrarchi n. sp. (Myxosporea: Sphaerosporidae) from wild and cultured sea bass, Dicentrarchus labrax L. J. Protozool., 39, 273–281.

U U

Enteromyxum leei

U

Estensoro, I., Jung-Schroers, V., Álvarez-Pellitero, P. et al. (2013) Effects of Enteromyxum leei (Myxozoa) infection on gilthead seabream (Sparus aurata) (Teleostei) intestinal mucus: glycoproteinprofile and bacterial adhesion Parasitol Res 112: 567. doi:10.1007/s00436-012-3168-3. Pilar Alvarez-Pellitero, Oswaldo Palenzuela, Ariadna Sitjà-Bobadilla. Histopathology and cellular response in Enteromyxum leei (Myxozoa) infections of Diplodus puntazzo (Teleostei). Parasitology International 57 (2008) 110–120. Fleurance, R.; Sauvegrain, C.; Marques, A.; Le Breton, A.; Guereaud, C.; Cherel, Y,; Wyers, M. (2008). Histopathological changes caused by Enteromyxum leei infection in farmed sea bream Sparus aurata. Dis Aquat Org 79:219–228. Palenzuela, O. Myxozoan infections in Mediterranean mariculture. Parassitologia. 2006 Jun; 48(1–2):27–9. Review.

U U U

Isopods

U U U

Mladineo I. Life cycle of Ceratothoa oestroides, a cymothoid isopod parasite from sea bass Dicentrarchus labrax and sea bream Sparus aurata Dis Aquat Org 57:97–101 (2003) doi:10.3354/dao057097. Papapanagiotou, E.P.; Trilles, J.P. Cymothoid parasite Ceratothoa parallela inflicts great losses on cultured gilthead sea bream Sparus aurata in Greece. Dis Aquat Organ. 2001 Aug 2; 45(3):237–9. Horton, T.; Okamura, B. Cymothoid isopod parasites in aquaculture: a

VENDRAMIN PRINT.indd 131

26/05/2017 08:33

132  ♦  References for Further Reading review and case study of a Turkish sea bass  (Dicentrarchus labrax)  and sea bream  (Sparus auratus) farm Dis Aquat Organ. 46:181–188 (2001) – doi:10.3354/dao046181.

Trematodes

U

Antonelli, L.; Quilichini, Y.; Marchand, B. Sparicotyle chrysophrii (Van Beneden and Hesse 1863) (Monogenea: Polyopisthocotylea) parasite of cultured Gilthead sea bream Sparus aurata (Linnaeus 1758) (Pisces: Teleostei) from Corsica: ecological and morphological study Parasitology Research July 2010, Volume 107, Issue 2, pp 389–398. Sitjà-Bobadilla A.; Conde de Felipe, M.; Alvarez-Pellitero, P. In vivo and in vitro treatments against Sparicotyle chrysophrii (Monogenea: Microcotylidae) parasitizing the gills of gilthead sea bream (Sparus aurata L.) Aquaculture Volume 261, Issue 3, 1 December 2006, Pages 856–864. Sánchez-García, N.; Raga, J.A.; Montero, F.E. Risk assessment for parasites in cultures of Diplodus puntazzo (Sparidae) in the Western Mediterranean: prospects of cross infection with Sparus aurata. Vet Parasitol. 2014 Aug 29; 204(3-4):120-33. doi: 10.1016/j.vetpar.2014.05.013. Epub 17 May 2014.

U U

VENDRAMIN PRINT.indd 132

26/05/2017 08:33

Index

Page numbers in bold refer to illustrations abdominal distension 23 accidents 6 Aeromonas 7 aetiological agents 7 alternative species 5 amberjack (Seriola dumerilii) 60, 72 anamnesis (clinical history) 11–12 epizoological archive 13–14 manipulations and traceability 12–13 recent diseases and treatment 13 seed quality 12 water parameters 13 weather conditions 13 anatomo-pathological examination 20–4 antibiogram 29 Anylocra 7 autopsy technique 20 bacterial disease 6–7, 37, 40–66 environmental/zootechnical ­consequences 38 future direction 39 management/control 38 medical feed 39 mistakes in treatment 39 operative strategy 38–9 résumé 115–19 spread 37 symptoms 37 bacteriological examination 28–30

VENDRAMIN PRINT.indd 133

Betanodavirus 31 biosecurity inspection 14–15 biosecurity problems 1, 2, 6–8 black goby (Gobius niger) 72 bladder tympanism 76 bluefin tuna (Thunnus thynnus) 5 bogues (Boops boops) 105 brown meagre (Sciaena umbra) 72 caudal fin fraying 57 Ceratomyxa spp. 25 Ceratothoa oestrides 7 Ciliate protozoans (Cryptocarion ­irritans, Trichodina sp., Trichodinella sp.) 84, 88–9 clinical examination 14–15 Coccids (Eimeria sp.) 84, 89–90 coelomic cavity and organ inspection 21, 22 common dentex (Dentex dentex) 5 common Pandora (Pagellus erythrinus) 5, 72 Crustaceans (copepods and isopods) 7, 84, 96–8, 124 cytopathic effect (CPE) 31 dentex (D. dentex) 77 diagnostic approach, phases of 8 Digenean trematodes (Sanguinicolidae sp.) 84, 95–6 dysmetabolic 8

26/05/2017 08:33

134  ♦  Index eel (Anguilla anguilla) 72 encephalon sampling 34 enteromixidiosis 7, 99–103 Enteromixum leei 7, 25, 99–100, 101, 102 Enteromyxum scophthalmi 99 equipment 9–11 European sea bass (D. labrax) 5, 17 field necropsy 24 Flagellated protozoans (Amilodinium ocellatum, Cryptobia branchialis) 84–5, 86, 87–8 Flexibacteriosis (Tenacibaculum maritimum) aetiology 53, 117 diagnosis 55 epidemiology 53–4 pathological lesions/symptoms 54 therapeutic recommendations/control strategies 55–7, 117 floating cage 3 flow chart 8 Freshwater blenny (Salaria fluviatilis) 72 General Clinical Examination (GCE) 17–19 gill infestation with monogeneans 7 gill sample collection 26 gilthead sea bream (Sparus aurata) 4, 99, 101, 110 gram-negative vibriosis in microscopic spleen sample 45 grouper (Epinephelus aeneus, Epinephelus marginatus, Epinephelus alexandrinus) 72 hemorrhagic lesions 18, 44 histological analysis 31 hypertrophic cell 80 immune depression 8 immunohistochemical analysis 31 infectious diseases 6–7 inshore floating cages 7 Internal Pathological Examination (IPE) 22

VENDRAMIN PRINT.indd 134

Isopod infestations (Ceratothoa, Nerocilia, Anilocra) 7, 83, 106, 107, 108 aetiology 104, 124 pathological lesions/­clinical signs 105–6 diagnosis 106 epidemiology 104–5 résumé 124 therapeutic recommendation/­control strategies 106, 108–9, 124 Japanese sea bream (Pagrus major) 53 land-based farm tanks 7 largemouth bass (Micropterus salmoides) 72 Listonella anguillarum 6 liver parenchyma, color alteration 22, 28 liver, pathological condition 23 Lymphocystis disease 6, 31, 33 aetiology 77, 121 pathological lesions/­symptoms 78 diagnosis 79 epidemiology 77–8 necropsy/histological findings 80 therapeutic recommendations/control strategies 79, 121 macroparasite investigations 25 marine velvet disease 84–5 meagre (Argyrosomus regius) 5, 72 meningitis 62 microparasite investigations 25 Microsporids (Microsporidium sp., Glugea sp.) 84, 90–1 molecular techniques 32 Monogenean Parasitic Infestations (Furnestinia echeneis, Sparicotyle chrysophrii) 7 aetiology 110–11 pathological lesions/symptoms 112 epidemiology 111 therapeutic recommendations/control strategies 112–14 Monogenean trematodes (Girodactylus sp., Diplectanum aequans, Saricotyle chrysophrii) 84, 93–5 mortality 15

26/05/2017 08:33

Index  ♦  135 mullets (Mugil sp.) 105 Myxosporid diseases 99–103 Myxozoa (Enteromyxum leei, Sphaerospora dicentrarchi, Sphaerospora testicularis, Ceratomyxa) 25, 84, 91, 92, 93, 101, 102 aetiology 99, 122 pathological lesions/­clinical signs 100 diagnosis 100–1 epidemiology 99–100 therapeutic recommendations/control strategies 101, 103, 123 necropsy findings 27 offshore floating cages 7 Oodiniasis 7 opercular deformity 19 para-physiological phenomena 7 parasites 24 parasitic infestations 7, 81–2 biological/environmental conditions 83 characteristics 83 control/eradication 83 diagnostics 82 epidemiological knowledge of health condition 83–4 examination 82 negative consequences 82 résumé 122–5 species 84–98 parasitological examination 25–7 Pasteurellosis see Photobacteriosis pathognomonic nervous ­symptoms in sea bass ­juveniles 32 pathological phenomena 7 peritoneal lipid accumulation 22, 28 Photobacteriosis (Pasteurellosis) 6 aetiology 46, 116 pathological lesions/­symptoms 48–9 diagnosis 49–50 epidemiology 46–8 therapeutic recommendations/control strategies 50, 116

VENDRAMIN PRINT.indd 135

Photobacterium damselae subspecies piscicida 6, 29 Polysporoplasma sparis 92 poor cod (Trisopterus minutus) 72 post-mortem examination 24 production system 1–2 Real-time PCR techniques 32 red porgy (Pagrus pagrus) 5 righteye flounders (Limanda) 53 salema porgies (Sarpa salpa) 105 sampling procedures methods for choice and collection 15–16 sample preparation 16 sardine (Sardina pilchardus) 72 sea bass (Dicentrarchus labrax family Moronidae) 4, 5, 18, 19, 22, 32, 40, 44, 45, 51, 53, 57, 58, 59, 62, 64, 66, 76, 99, 107, 108 sea bream (Sparus aurata family Sparidae) 4, 23, 53, 59, 64, 77, 80, 99, 100, 101 sea lice (Lepeophtheirus salmonis) 83 Septicemia by Aeromonas sp. aetiology 64, 119 pathological lesions 65 diagnosis 65 epidemiology 64 therapeutic/prophylactic ­recommendations 65, 119 sharpsnout sea bream (Diplodus ­puntazzo) 5, 59, 64, 71, 77, 99, 100, 102, 111 shi drum (Umbrina cirrosa) 5, 60, 72 signaling (case description) 11 skeletal deformity 18 skin erosions 23, 57, 58, 76 skin lesions 23, 33, 66, 106 skin ulcers 58 sole (Solea solea, Solea senegalensis) 5, 53, 72, 99 Sparicotyle chrysophrii 7 spleen colored with Gram staining 52

26/05/2017 08:33

136  ♦  Index splenic nodules 51 splenomegaly 45, 51 Streptococcosis 6 aetiology 59, 118 pathological lesions/­symptoms 60–1 diagnosis 61 epidemiology 59–60 gram-positive streptococci 63 therapeutic recommendations/control strategies 61, 118 striped bass (Morone saxatilis) 72 striped mullet (Mullus barbatus) 72 striped red mullet (Mullus surmuletus) 72 striped sea bream (Lithognathus ­mormyrus) 5, 105, 106 T. maritimum 55, 58 tenacibaculosis (Tenacibaculum spp.) 6 thin-lipped grey mullet (Liza ramada) 72, 97 Trematoda infestation aetiology 125 therapeutic suggestions 125 turbot (Scophthalmus maximus) 53, 60, 71, 99 tympanism of swim bladder 22, 30 typing 29 unilateral exophthalmos 19, 62

VENDRAMIN PRINT.indd 136

vascular congestion of entrails 44 Vibriosis (Vibrio anguillarum) 6, 29 aetiology 40, 115 pathological lesions/­symptoms 41–2 diagnosis 42 epidemiology 40–1 therapeutic recommendations/­control strategies 42–3, 115 typical 115 viral disease 6, 67–8 management 68–9 precautionary measures 68 résumé 120–1 spread 68 viral encephalopathy and retinopathy (VER or VNN) 6, 24, 31, 67–8, 76 aetiology 70, 120 pathological lesions/­symptoms 73–4 diagnosis 74–5 epidemiology 71–3 therapeutic recommendations/control strategies 75, 120 virological examination 31–4 water control 1 white sea bream (Diplodus sargus) 5, 105, 111 yellowtail (S. quinqueradiata) 60 Zander (Sander lucioperca) 72

26/05/2017 08:33