Contents - Scientific Sessions - Plenary Abstracts - Oral Abstracts - Special Session Abstracts - Author Index






Molecular Analysis of Enterococcus seriolicida KG-Strain-Specific Antigen Genes

T Aoki*, H Yamashita and I Hirono

Laboratory of Genetics and Biochemistry, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108-8477, Japan.

Enterococcus seriolicida is the causative agent of an enterococcal infection in yellowtail Seriola quinqueradiata and is antigenically classified into two phenotypes, KG- and KG+. The KG- phenotype strain demonstrates a higher virulence in yellowtail than the KG+ phenotype. This study details the cloning and molecular analysis of the Enterococcus seriolicida KG- strain-specific antigen genes. We studied the relationship between these antigen genes and the virulence factors of the KG- phenotype. Five clones were isolated that corresponded to these antigen genes; SA1B05, SA1B10, SA2F01, SA8A11, SA9H10. The first four clones were identified as Bacillus subtilis hypothetical processing protease, Neisseria meningitidis dihydropteroatesynthase, Bacillus subtilis trigger factor, Haemophilus influenzae N-acetylglucosamine-6-phosphate deacetylase, respectively. However, SA9H10 was not identified. We constructed different primer sets from knowledge of these clones and performed RT-PCR for two KG- phenotype and three KG+ phenotype in order to compare expressed and unexpressed KG- strain-specific antigen genes. The results indicated that all of these antigen genes were expressed in the two KG- phenotype strains, and were unexpressed in two of the KG+ phenotype strains. However, SA9H10 was expressed in one of the KG+ phenotype strain.



Systemic Aeromonas hydrophila Infections in Rainbow Trout (Oncorhynchus mykiss Walbaum)

S Aydyn (1) * and Z Erman (2)

1 Çanakkale Onsekiz Mart University, Fisheries Faculty, Karacaören, 17100 Çanakkale, Turkey
2 Atatürk University, Faculty of Medicine, Department of Pathology, 25240 Erzurum, Turkey

Aeromonas hydrophila infections appeared during July, September and October after about 15-20 days from torrents in juvenile cultured rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Infections caused abnormal movements, anorexia, darkening of skin, pale and swollen gills, yellowish liver and cherry-red spleen, haemorrhage in kidney and serous exudate in the intestine. Histopathological examination demonstrated pathological changes in gill, heart, kidney and liver. The mean activity of glutamate oxalacetate transaminase (GOT) and level of biluribine were significantly increased in serum of diseased fish. There were no significant increases in the mean serum levels of L-lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine. While decreases in the mean amount of glucose were observed in naturally infected fish. Sensitivities of bacterial isolates identified from each farm against 16 chemotherapeutants were determined. Minimum bactericidal concentrations (MBC) of formalin for isolates were between 6.4 - 8 µL/mL for a 60-min exposure. A 23.27 µg/mL dosage of potassium permanganate (KMnO4) for 10-min was effective on only one isolate, but chloramine-T did not effect any isolate. Oral applications of ofloxacin after disinfections with formalin controlled the natural infections. Pathogenicities of three isolates were tested by intramuscular injection using healthy 1-year-old rainbow trout, and their LD50s were calculated 4 x 104, 2 x 105 and 5 x 105.



One Year Survey of the Main Infectious Problems in Cultured Seabream and Seabass from the South of Portugal

TM Baptista, F Soares, J Costa, A Albuquerque, M Almeida and MT Dinis

Universidade do Algarve, U.C.T.R.A. - Campus de Gambelas, 8000 Faro, PORTUGAL

The Laboratory of Applied Pathology in Aquaculture was set up at the University of Algarve in February 1997. Among the activities which were developed the most important was: an epidemiological study on the main diseases affecting fishfarms in the region. In this survey we provide data on the occurrence of bacterial and parasitological fish diseases in seabream and seabass reared in the South coast of Portugal during one-year period. The most threatening disease for juveniles was pasteurellosis caused by Pasteurella piscicida which produced epizootic outbreaks specially in seabass. This bacteria was responsible for causing high mortalities and important economic losses. Ectoparasitic infections caused by the ciliate Trichodina sp. and the monogenean Diplectanum sp. were generally sporadic and insignificant, however, on one occasion the crustacean Caligus sp. caused morbidity and mortality of epizootic proportion in seabass. The information achieved during this year is essential for the establishment of appropriate guidelines on fish health, concerning the prevention and control of the main infectious diseases affecting the fish farms in Portugal.



Susceptibility of Photobacterium damsela subsp. piscicida to Reactive Oxygen Species

AC Barnes(1)*, MC Balebona(2), MT Horne(3) and AE Ellis(1)

1 FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, Scotland.
2 Dept. Microbiologia, Facultad de Ciencias, Univ. Malaga, Campus Teatinos s/n, 29071-Malaga, Spain.
3 Aqua Health (Europe) Ltd., Unit 31 Enterprise House, Springkerse Business Park, Stirling FK7 7UF, Scotland.

Photobacterium damsela subsp. piscicida (formerly Pasteurella piscicida) is the causative agent of pasteurellosis or pseudotuberculosis in warm water marine fish. Enzymes which neutralize reactive oxygen species, produced during aerobic metabolism or during respiratory burst in fish macrophages are important virulence factors in many pathogens. The present study characterises a periplasmic iron-cofactored superoxide dismutase (SOD) and a cytoplasmic catalase in Ph. damsela. The SOD was common to all strains analysed in terms of type, location and activity, whilst the catalase varied in activity between strains. The catalase was constitutively expressed, but the SOD appeared to be repressed under poorly aerobic conditions. In spite of the presence of a periplasmic SOD, Ph. damsela was susceptible to killing by exogenous superoxide anion generated in a cell free system. Addition of exogenous SOD to this system did not abolish the bactericidal effect, however addition of catalase was protective. These results suggest that lack of periplasmic catalase may be implicated in susceptiblity to killing by reactive oxygen species.



Cohabitation of Atlantic Salmon (Salmo salar L.) and Scallop (Pecten maximus) - Challenge with Infectious Salmon Anemia (ISA) Virus and Aeromonas salmonicida subs. salmonicida

B Bjorshol (1)*, R Nordmo (2), K Falk (1) and S Mortensen (3)

1 National Veterinary Institute, P.O.Box 8156 Dep., 0033 Oslo, Norway,,
2 VESO Vikan AkvaVet, Alhusstrand, 7800 Namsos, Norway,
3 Institute of Marine Research, Department of Aquaculture, P.O.Box 1870, 5042 Bergen, Nordnes,

Cohabitation of Atlantic salmon and scallop is desirable in order to obtain the most favourable conditions, both in terms of production and economics. By challenge experiments, the risk of transfer of ISA virus and Aeromonas salmonicida subs. salmonicida from scallop to Atlantic salmon has been investigated. A model of challenge has been designed for this purpose based on bath challenge with the organisms in question. The risk of transferring the infectious agents from scallop to Atlantic salmon were investigated either by injecting organ homogenate from scallop into healthy Atlantic salmon or by keeping healthy Atlantic salmon in the same tank as the infected scallop according to a fixed schedule. The work has shown that scallop accumulates and excretes Aeromonas salmonicida subs. salmonicida for 14 days after challenge resulting in an increased mortality rate both among Atlantic salmon injected with scallop organ homogenate and among Atlantic salmon kept as cohabitants with scallop. When challenging scallop with ISA virus, ISA virus was not detected in the scallop nor was it possible to transmit ISA virus from scallop to Atlantic salmon in the experiments carried out. However, the possibility of this being possible cannot be ruled out.



Induction of Superoxide Dismutase in Aeromonas salmonicida subsp. salmonicida Under Changing Iron Concentrations: Possible Interpretation for Pathogen-host Interaction

TJ Bowden* and AC Barnes

FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB, Scotland.;

The process of pathogenesis for a bacterium such as Aeromonas salmonicida ssp. salmonicida is believed to involve initial contact with an environment that contains very little free iron, forcing the cell to scavenge for iron from the host's own sources. Until such time that the pathogen can release iron for its own use, it is placed under iron stress. It has previously been shown that under normal or iron-replete in vitro culture conditions A. salmonicida produces an iron-associated superoxide dismutase, but when placed under iron-depletion it produces a manganese-associated superoxide dismutase. We were interested in the effect of transferring a culture grown in iron-depleted conditons to iron-replete conditions and how this would affect the induction of superoxide dismutase. Assays were carried out using chloramphenicol to inhibit protein synthesis. Analysis of the proteins present in the culture using native-PAGE followed by staining for total protein and superoxide dismutase activity clearly showed that as the pathogen is moved from an iron poor to an iron rich environment the cell induces the ferric superoxide dismutase and slowly switches off production of the manganese superoxide dismutase. The results are considered in respect of the in vivo pathogenic process and how this species survives such iron and oxygen radical stress.



Streptococcus iniae Infection of Tilapia Oreochromis niloticus in a Recirculation Production Facility: a Case Report

PR Bowser (1), GA Wooster (1)*, RG Getchell (1) and MB Timmons (2)

1 Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401 USA.,, rgg4@cornell
2 Department of Agricultural and Biological Engineering, Reiley-Robb Hall, Cornell University, Ithaca, New York 14853-5701.

Streptococcus iniae was isolated from moribund tilapia reared in a water recirculation aqaculture facility. The extent of the infection in a number of separate culture systems within the facility was determined. Loop samples were taken from both brain and kidney tissues. Literature suggests that S. iniae infects the nervous system and is likely to be best isolated from the brain. In this study there was no significant difference between the two tissues with success of isolating S. iniae. Histological observations were similar to those reported for streptococcal infections of fish, including tilapia. Additionally, a pilot study was conducted to gain information on potential stressors that may play a role in spread of the infection within such a reuse system. Cohabitation with moribund fish, crowding, low dissolved oxygen concentrations and elevated ammonia concentrations did not produce the expected outcome of high morbidity or mortality of the stressed fish or an outbreak of S. iniae. However, the unique characteristics of this reuse system and other variables need to be further considered or determined.



Antibacterial Action of the Mucus of Rainbow Trout Against Typical Bacterial Fish Pathogens

A Castillo, CS Gallardo, D Abad, JA Gonzalez and LA Rodríguez *

Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.

The presence of compounds that inhibit the growth of pathogenic agents in the mucus has been demonstrated by several authors in different fishes. The skin mucus of turbot possesses a very wide spectrum of activity against many important Gram negative and Gram positive pathogenic bacteria. The objective of this work has been to evaluate the possible antibacterial activity of the mucus of rainbow trout (Oncorhynchus mikiss), the major fish species cultured in fresh water in the North West of Spain against typical fish pathogens (Aeromonas, Yersinia, Vibrio, Pseudomonas). The mucus was collected from the skin of healthy rainbow trout by scraping with a glass slide, resuspended in sterilized fresh water (0,1 ml mucus/2 ml freshwater), and stored at 4C & 30C. Strains were cultured on Tripticase Soy Agar (TSA) and bacterial cells were resuspended in PBS solution to obtain an initial concentration of 109 cells/ml. The assay was carried out using the classical method of diffusion on plates applying sterilized 6 mm diameter dishes impregnated with 20 5l of the mucus suspension on seeded bacterial lawns containing about 104-105 cells/ml. The effect of temperature (56:C/30 min and 80:C/20 min) and Proteinase K (1 mg/mL) on the antibacterial activity of the mucus was evaluated. Discs containing proteinase K alone were used as a control.



Influence of the Physical Conditions in Proteolytic Activity of ECP in Bacterial Fish Pathogens (Yersinia ruckeri, Aeromonas hydrophila and Hafnia alvei)

A Castillo (1), CS Gallardo (1), E Sinde (1), F Acosta (2), F Real (2) and LA Rodríguez (1)*.

1 Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.
2 Departamento de Patologma Animal. Facultad de Veterinaria. Las Palmas de Gran Canaria. Spain.

Extracellular products of Yersinia ruckeri, Aeromonas hydrophila and Hafnia alvei plays a principal role in the virulence for fish. Generally, the quantity of protein obtained in the ECP is very low and consequently the enzymatic activities are also very low, too. In our study, different fish pathogens (Yersinia ruckeri, Aeromonas hydrophila and Hafnia alvei) were incubated in TSB for 24-48 h in four different conditions to obtained ECP: In presence of magnetic field and agitation; magnetic field without agitation; agitation without magnetic field and without both (magnetic field and agitation). Cells were obtained using filtration membranes, harvested in PBS, removed by centrifugation and the supernatant was sterilized by passing through 0.225m filters for obtained the ECP. The parameters studied in the different conditions were: a. - Quantity of protein. b. - Proteolitic activity (casein). Detection of metallo- and serine proteases. Results showed that magnetic field and the agitation plays an important role in the production of the ECP and their activities showing different % in the type of proteolitic activity.



Utilization of Haem Compounds by Pasteurella piscicida

A Do Vale (1,2)*, B Magariños (1), ML Lemos (1), AE Toranzo (1) and JL Romalde (1)

1 Departamento de Microbiología y Parasitología, e Instituto de Acuicultura.Universidad de Santiago de Compostela. 15706 Spain.
2 Instituto de Biologia Molecular e Celular. Universidade do Porto. 4050 Portugal.

Pasteurella piscicida (now reclassified as Photobacterium damselae subsp. piscicida) is the ethiological agent of fish pasteurellosis. This bacterium is able to acquire iron bound to human transferrin by a siderophore-mediated mechanism. Furthermore, it can use haemin and haemoglobin as the sole iron source in vitro. However, the mechanisms by which this fish pathogen can acquire iron from the host are not clearly understood. The ability of virulent and avirulent strains of P. piscicida to utilise haem sources of iron was evaluated. Whole cells as well as membrane extracts from all the strains could clearly bind haem compounds which seems to indicate that this binding activity is associated with the outer membrane. Moreover, we detected significant differences between virulent and avirulent strains when they were cultured under iron-restricted conditions. We demonstrated that in this situation, the pathogenic strains had much more affinity for haem-compounds. These results suggest that the virulence of P. piscicida could be associated with the ability of this pathogen to acquire iron from host haem compounds.



Purification of Leucine Arylamidase from Extracellular Products of a Pathogenic VAR Strain

R Farto, MM Montes, MJ Pérez and TP Nieto*

Area de Microbiologma, Universidad de Vigo, Departamento de Biologma Fundamental y Ciencias de la Salud, E-36200 Vigo,;;;

In this work we analyzed the extracellular products (ECP) of a Vibrio VAR strain isolated as causal agent of epizootic in a turbot farm located in Galicia (Northwest, Spain). The ECP was lethal for 3g brown trout (Salmo trutta L.) and 10g turbot (Scophthalmus maximus L.) causing death between 2 and 24 h after inoculation. It showed a DL50 1.3 mg/fish and 2.25 mg/fish, respectively. It possesed a high hydrolytic activity with production of caseinase, gelatinase and a hemolysin active against turbot erythrocytes. Analysis of ECP from gel filtration cromatography showed six peaks whose a major peak had high casein proteolytic and leucine arylamidase activities. The inhibition of these activities were assayed with different concentrations of EDTA and PMSF. Proteolytic activity was inhibited by EDTA (5mM) and not by PMSF. Leucine arylamidase activity was not inhibited. Thermostability was studied, too. The proteolytic activity was lost completely heating at 70C for 3 minutes, but not leucine arylamidase activity. This fraction was stable at 60C for 3 minutes. It demonstrated the same DL50 that crude ECP. The role of this fraction in pathogenicity of this virulent strain would be evaluated.



Antimicrobial susceptibility of Bacterial Fish Pathogen Hafnia alvei

CS Gallardo (1), J Vivas (1), F Acosta (2), F Real (2) and LA Rodríguez *(1)

1 Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.
2 Departamento de Patologma Animal. Facultad de Veterinaria. Las Palmas de Gran Canaria. Spain.

In order to obtain useful information for controlling twenty-one Hafnia alvei isolated of differents sources and countries we have evaluated a total of 26 antimicrobial compounds including drugs commonly used in fisheries and other chemotherapeutans which efficacy against bacterial fish pathogens had not been determined until present. In this work the susceptibility of Hafnia alvei strains bacteria fish pathogen to different antimicrobial compounds was studied in order to obtain useful information for therapeutic purpose for each country studied. Drug resistence patterns of the isolates were determined by the disc diffusion method on Müller-Hinton agar (MHA, Difco) using the chemoterapeutics agents. The minimum inhibitory concentrations (MICs) were determined according to the agar plate dilution technique.



Dormancy Effect of Hafnia alvei in Fresh and Sea Water

CS Gallardo (1), F Real (2), E Sinde (1), F Acosta (2) and LA Rodríguez *(1)

1 Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.
2 Departamento de Patologma Animal. Facultad de Veterinaria. Universidad de Las Palmas de Gran Canaria. Las Palmas de G Canaria.

Hafnia alvei is a normal habitant of surface water, soil, grains and intestinal tract of animals. This organism has been reported to cause epizootic haemorrhagic septicaemia in rainbow trout and a causative agent of kidney pathology in cherry salmon. The ability of pathogens to survive and remain infective in the external environment is considered a major determinant of disease. The survival capacity was studied at different temperature and two types of water (sea and fresh water). Seven strains of Hafnia alvei were used: C/34, ATCC 9760, ATCC 13337, F4319, X1, OR-1 and 187/95.The bacteria were enumerated on normal and ten fold diluted medium. Using normal TSA to recover viable cells, the survival time of Hafnia alvei was less than 90 days. However a constant number of viable cells were detected on TSA diluted 1:10 for 120 days and did not full to zero until 180 days. From these data we can conclude that the presence of a dormant state of this pathogen was due to the determination of survival time using a standard plate count procedure on normal TSA. Normal medium therefore is not the most sensitive method to measure survival values for this pathogen. Thus, for accurate monitoring of the spread of Hafnia alvei in the aquatic environment the ten fold diluted TSA technique is necessary to determine the presence of the dormant state.


Extracellular Activities in the ECP of Different Isolates of Hafnia alvei

CS Gallardo (1), MJ Pérez *(1), F Acosta (2), F Real (2) and LA Rodríguez (1)

1 Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo. Spain.
2 Departamento de Patologma Animal. Facultad de Veterinaria. Universidad de Las Palmas de Gran Canaria. Las Palmas de Gran Canaria. Spain.

A Gram-, oxidase- coccobacillus was isolated from rainbow trout (Oncorhynchus mikiss) in Bulgaria with epizootic disease characterized by haemorrhagic septicaemia. It was readily distinguishable from Yersinia ruckeri, Aeromonas salmonicida, Vibrio anguillarum and other species associated with haemorrhagic disease in trout. In spite of the importance of Hafnia alvei as a pathogen of cultivated fishes its pathogenicity is poorly understood. A number of extracellular products (ECP) have been associated with virulence of other fish pathogenic bacteria and it may be assumed that similar virulent determinants could be expected to be found associated this organism. In previous studies Hafnia alvei caused mortality by i.p. experimental inoculation in brown trout with DL50 1.3x104 and 2.5x107 bacteria/fish. These values are considered to represent a high to moderate degree of virulence. The present work describes the enzymatic activity of ECPs of several strains isolated from differents sources. In order to more fully understand the production and role of Hafnia alvei ECPs, the ability to degrade gelatin, casein, DNAse, Tween 80 and haemolytic activity was assessed. Changes in proteolytic activity (metallo and serines proteases) were detected between ECP of different strains. Results suggest that the excretion of extracellular products may be a significant virulence factor for this bacterium.



Adhesive Properties of Flavobacterium psychrophilum

S García-Márquez*, I Bandín, C Cepeda, AE Toranzo and Y Santos

Departamento de Microbiología y Parasitología e Instituto de Acuicultura. Universidad de Santiago de Compostela. 15706 Spain.,,

In the present study we have evaluated the adhesive capacities of strains of Flavobacterium psychrophilum and their relationship with the virulence. With this aim, we have determined the degree of virulence for fish, the cell surface hydrophobicity, and the ability to adhere to fish tissues of strains isolated from different fish species in distinct geographic areas. The hydrophobic capacities were determined using Salt Aggregation Test (SAT), Phase partitioning with hydrocarbon solvents and adherence to nitrocellulose filters (NCF). The results of these assays varied depending on the method employed. So, while in the NCF test the strains showed to be highly hydrophobic, the SAT and the Phase partitioning assays gave similar results, indicating that, in general, the F. psychrophilum cells possess a low hydrophobicity. In addition, we have found that, regardless of their degree of virulence for fish, all the strains were unable to agglutinate erythrocytes from human and fish.



Microflora Associated with Black Bass (Micropterus salmoides L.) in Spanish Rivers

P Gil, CS Gallardo, M Torres, AI Saa and LA Rodríguez *

Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.

The microflora associated with aquatic animals appears to be restricted in terms of the range of bacteria ocurring in diets and in the surrounding waters. It remains unanswered what biological factors play the most critical role in the selection of microorganisms. Black-bass (Micropterus salmoides) is not a typical fish into the Spanish rivers. Five years ago, it was introduce in different Spanish rivers colonized by brown trout. The aim of this work was to establish which kind of microflora could be associated with black-bass and the asintomatic carrier role that black-bass could have with typical brown trout bacterial pathogens (Aeromonas, Vibrio, Pseudomonas).



"Cytophaga johnsonae-like" Infection in Young Sturgeon Reared Under Intensive Condition

NV Guseva (1), PP Golovin (1), NA Golovina (1) and JF Bernardet (2)

1 All-Russian Research Institute of Freshwater Fisheries, Rybnoe, Dmitrov region, Moscow province, 141 821, Russia,
2 Laboratoire de Virologie-Immunologie Moleculaires, Centre de Recherches INRA, F-78352 Jouy-en-Josas Cedex, France,

High mortality (>70%) in the young sturgeon (3-20 g) occurred at the Konakov hatchery, Russia. All sick and moribund fish had the following clinical symptoms: high external mucus production, fin necrosis, light spots on the body surface and hemorrhages on the abdomen and at the base of the fins. The first mortalities occurred in spring when water temperatures were 18C and higher . Bacteriological study of the water indicated a high level of bacterial contamination, including yellow pigmented bacteria in concentration 1.6 ­ 5.4 x 102 CFU/ml. These bacteria accounted for 30% of gill microflora from diseased fish. At that time only single colonies of yellow pigmented bacteria could be isolated from internal organs of affected fish. As the disease progressed, pure cultures of bacteria were isolated from internal organs and their percent in gill microflora increased to 50%. All isolates seem to belong to a group of gliding bacteria closely related to Cytophaga johnsonae. In challenge experiments, broth cultures of one isolate was added to water of two tanks to give final concentrations of 1.2x105 CFU/ml and 4.4 x 10 6 CFU/ml. Sturgeon were exposed to bacteria for 1 hour under constant aeration. During 21 days of the experiment, cumulative mortalities were 39.4 and 47.5 %, respectively, in the challenged juvenile sturgeon. From each mortality, a pure culture of the test isolate was re-isolated from gills and internal organs. This study confirmed the etiological role of the Cytophaga johnsonae-like bacteria in the case of mass mortality at the Konakov sturgeon hatchery.



Granulomatous Dermatitis of Moray Eels

LH Herbst (1)*, L Johnson (1), M Levi (1), L Weiss (1), M Walsh (2), R Davis (2) and Y Kress (1)

1 Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, 10461 USA
2 Sea World, Orlando, Florida, USA

Cases of granulomatous dermatitis and cellulitis in captive moray eels, Gymnothorax funebris, have occurred sporadically at an aquarium. Similar gross lesions were observed in free-ranging eels found in Florida waters. The following report is of an aquarium maintained Green Moray Eel, with widely distributed ulcerative dermatitis and cellulitis. The most severe lesions were found near the nares and dorsolateral to the vent. Light microscopy reveals random, multifocal granulomatous cellulitis characterized by large multinucleate giant cells containing non-acid fast, PAS positive, Gram positive organisms. Transmission electron microscopy of these organisms suggests they are bacteria, with an irregular three-dimensional pattern of cell division. Similar organisms were observed in a series of earlier cases but bacterial isolation was unsuccessful. In the present case, bacterial cultures isolated Streptococcus spp from one lesion and two species of Vibrio from all cultured lesions. However, none of these isolates appear to be the bacterial forms identified in giant cells. PCR assays of 16S ribosomal RNA from an earlier case identified Streptococcus iniae from cultured monocytes. Additional PCR studies are ongoing in the present eel case and transmission experiments are planned to determine the role of Vibrio in this disease.



The Role of Indigenous Bacteria in Early Larval Growth of Summer Flounder (Paralichthys dentatus)

SH Jones (1)*, SD Eddy (1), BW Summer-Brason (1) and G Nardi (2)

1 Jackson Estuarine Laboratory, University of New Hampshire, Durham, NH 03824 USA;
2 Great Bay Aquafarms, Inc., Portsmouth, NH 03801 USA.

Finfish in early larval stages of growth can suffer high mortality in aquacultural facilities because of diseases and nutritional problems. Bacteria associated with the live feed and hatchery environments can have either beneficial or detrimental effects on fish health. At Great Bay AquaFarms, Inc., the first marine finfish hatchery dedicated to the culture of summer flounder in the U.S., the microbiology of the larval flounder is not well characterized. Samples of fish, tank water and live feed collected during an actual production run at times of change in feeding regime, metamorphosis and the incidence of disease/high mortality were analyzed for bacteria, targeting total heterotrophs, total vibrios and Vibrio anguillarum. Isolates were identified to species and/or genus. Differences in microbial communities at different life stages and between feed, fish and water were observed that were related to fish growth stage. Total heterotroph and vibrios were detected at high concentrations (105-106/g DW fish) during metamorphosis and later during elevated mortality events. In separate experiments, larval fish were challenged with potentially pathogenic Vibrio sp. isolated from sick larval fish. Strain H12, a hemolytic, putative Vibrio damsela isolate, gave >40% mortality during a 6 day challenge experiment, compared to <10% mortality for strain NH16 and the uninoculated control. The results provide an initial database for determining the role of bacteria in the onset of disease and the health of early stages of summer flounder growth.



Epidemiology of Renibacterium salmoninarum in Wild Arctic Charr and Brown Trout in Iceland

H Jónsdóttir (1)*, HJ Malmquist (2), SS Snorrason (3), G Guobergsson (4) and S Guomundsdóttir (1)

1 Institute for Experimental Pathology, University of Iceland, Keldur, IS-112, Reykjavík, Iceland;
2 Natural History Museum of Kópavogur, Digranesvegur 12, 200 Kópavogur, Iceland.
3 Institute of Biology, University of Iceland, Grensásvegur 11, IS-108 Reykjavík, Iceland
4 Institute of Freshwater Fisheries, Vagnhöf_a 7, IS-112 Reykjavík Iceland,

Renibacterium salmoninarum (Rs) is common in Arctic charr (Salvelinus alpinus) and brown trout (Salmo trutta) in Iceland. In two out of 22 populations of Arctic charr, only one fish exceeded the Rs antigen detection limit and in one of these cases the ELISA-value was within uncertainty limits of the infection criterion. Mean prevalence of infection was 46% for Arctic charr (range: 3-100%) and 35% for brown trout (range: 6-81%). No infected fish showed gross pathological signs of bacterial kidney disease (BKD). The ubiquity and high prevalences of infection indicated that the bacterium has been endemic for a long time and is probably a normal, low density resident in the fish. A lack of correlation in mean intensity of Rs antigens and prevalence of infection between sympatric populations of Arctic charr and brown trout suggests that the dynamics of infection and internal proliferation of bacteria can be quite independent in the two species even if they live in the same lake. Rs intensity and its coefficient of variation decreased with age in older fish, suggesting a connection between Rs-intensity and host mortality. However, other ecological factors, especially increased food availability lower the susceptibility to Rs, in Arctic charr.



Numerical Taxonomy of Heterotrophic Bacteria Associated with Reared Turbot and Surrounding Water

MM Montes, A Freire, SP Armada and TP Nieto*

Area de Microbiología, Universidad de Vigo, Departamento de Biología Fundamental y Ciencias de la Salud, E-36200 Vigo, España.;;;

Numerical taxonomy, used to characterise 498 strains of gram-negative, motil, heterotrophic bacteria isolated from skin of turbot and surrounding water. The study included 65 reference strains belonging to the genera Aeromonas and Vibrio. The strains were characterized using 90 biochemical, physiological and nutritional test and data were examined by Simple Matching (SSM) and Jaccard (SJ) coefficients. Clustering was achieved by the unweighted pair group mathematical averaging method (UPGMA). The correlation coefficients (r) between the similarity matrix and the corresponding dendrogram derived from the SSM and SJ coefficients were 0,75 and 0,82, respectively. A similarity value of > 85% or above, as defined by the SSM, was used to cluster the strains. The 84% of the isolates were clustered in 88 phena including eleven major groups that contained ten or more isolates. Two of these groups contained more than 40 strains and were identified as V. ordalii and V. fisheri (they included the reference strains DF3K and LGM4414, respectively). The percentage of clustered strains based on SJ/UPGMA was lower than for SSM (63% of total) but similar grouping were obtained. At similarity value of SJ > 75%, 88 groups were defined. The utility of both coefficients will be discussed.



Study of Iron-uptake Mechanisms in Strains of Vibrio anguillarum of Serotype O3

L Muiño, F Pazos, ML Lemos, JL Barja and Y Santos*

Departamento de Microbiología y Parasitología e Instituto de Acuicultur a. Universidad de Santiago de Compostela. 15706 Spain.,,

Vibrio anguillarum is the main causative agent of vibriosis throughout the world. Many works about virulence characteristics of serotypes O1 and O2 of this species are avaliable. Nevertheless, few data exists about serotype O3 strains. This fact justifies the necessity for a better characterization of this serotype, including the study of the iron-uptake mechanisms, which have been probed to be involved in the pathogenesis of the vibriosis caused by serotypes O1 and O2. The present study demonstrates that minimum inhibitory concentrations of EDDHA (MIC) for strains of serotype O3 are similar to that displayed by serotype O1 strains and ranged between 75 and 150Fm. The assay in Schwyn and Neilands medium shows that all strains produce some kind of siderophore. The electrophoretic analysis of membrane proteins from strains of serotype O3 grown under iron restriction conditions revealed the presence of at least two induced proteins with molecular weights different to that found in strains of serotypes O1 and O2. Heterogeneity between subgroups O3A and O3B and also between strains of the same subgroup was found.



Rapid Identification of Fish Pathogens Using RFLP Analysis of Bacterial 16S Ribosomal RNA

WB Nilsson(1)*, PJ Noonan(2) and MS Strom(1)

1 Northwest Fisheries Science Center/REUTD 2725 Montlake Blvd. E. Seattle, WA 98112
2 Department of Botany, University of Washington Seattle, WA

Rapid and accurate identification of fish pathogens is essential towards selection of appropriate therapies. In many instances, it is also desirable to have a technique for nonlethal pathogen identification, e.g., in captive broodstock management. Here we demonstrate a rapid, sensitive technique for identification of pathogens such as Renibacterium salmoninarum, Flexibacter columnaris, F. psychrophilus, Piscirickettsia salmonis, and Aeromonas salmonicida. The method targets two highly conserved regions in the gene encoding the 16S subunit of ribosomal RNA (rRNA). These highly conserved regions, which are shared by virtually all eubacteria, flank a region of variable sequence. Using fluorescently tagged primers which target these conserved regions, the PCR is performed to amplify approximately 500 base pairs (bp) of the 16S rRNA gene. The amplified products are then digested with a series of restriction endonucleases which recognize four and six bp sequences of DNA. The size of fragments from the resultant digests is determined using an ABI PRISM 310 capillary electrophoresis with laser induced fluorescent detection and GeneScanTM analysis software. Only those fragments containing the fluorescent tag are detected. Due to restriction fragment length polymorphisms (RFLPs) in the region flanked by the primers, the sizes of labeled fragments are unique for each bacterial species.



Occurrence of Flavobacterium-like Bacteria in Skin Lesions of Atlantic Salmon (Salmo salar L.) with "Winter Ulcer" in Norway

AB Olsen* and HK Nilsen

National Veterinary Institute Bergen, PO Box 40, N-5032 Minde, Norway,

Skin lesions of Atlantic salmon (Salmo salar L.) in seawater in the winter season, so called "winter ulcer", is a serious problem for the salmon farming industry in Norway. A large proportion of the fish on a farm may be affected. Mortality is usually below 10%. There is a considerable economical loss due to reduced quality at slaughter. The etiology and the patogenesis of the condition is not clear. A bacterium prelimenary called Vibrio "viscosus" is considered to be an important factor in the development of "winter ulcer". In 18 out of 20 cases of "winter ulcer" examined in 1996, histopathology of skin lesions revealed few to numerous Flavobacterium-like bacteria. The bacteria were most often superficially located, but could also be found deep in the ulcers. The histological examination also revealed vibrio-like bacteria as a prominent finding. The results of microbiological, histopathological and immunohistochemical examination of skin ulcers and characterisation of the Flavobacterium-like bacteria isolated will be presented. Data from a transmission experiment will also be given.



The Most Prevalent Diseases in Cultivated Sea Bass (Dicentrarchus labrax) and Sea Bream (Sparus aurata) in Fish Farms Along the Croatian Coast

D Oraic (1)*, S Zrncic and B Sostaric

Croatian Veterinary Institute, Department for Fish Diseases, Savska 143, Zagreb, Croatia

Basically, there are two main marine fish species which are cultivated on the Croatian coast; sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). At the present along the almost 6000 km of Croatian Adriatic coast about 20 more significant fish farms are in production. This prosperous production is in permanent progress and the total amount of produced fish is now about 1700 tons per year. This report derives from sistematic clinical, parasitological, pathoanatomical, histopathological and bacteriological monitoring of cultivated marine fish on larger part of production during last five years. Among pathological conditions recognized on our farms, some specific viruses, bacteria and parasites represent frequently established causative nosologic agents. Viral diseases are represented by lymphocistis (Iridoviridae) and epitelocystis due to rickettia in sea bream but their economical impact is not so large as of those caused by bacteria. The most frequent bacterial problems are in both cultivated species mainly due to Vibrio anguillarum, Pasteurella piscicida and Myxobacteria spp. Although isolated bacteria are somewhat specific pathogens, it has to be emphasized that they are basically in ethiological dependance with technology applied. Parasitological examinations often reveal protozoan parasite Trichodina sp and monogenean gill flukes, namely Microcotyle sp. in sea bream and Diplectanum sp. in sea bass as well as crustacean parasites Caligus sp. and Lernanthropus sp. in sea bass. The importance of diagnosed parasitic infections is not entirely clear and that topic remains to be studied thorougly in further research.



Identification and Characterization of a Transposable Element in the Fish Pathogen, Renibacterium salmoninarum

LD Rhodes* and MS Strom

Northwest Fisheries Science Center, Resource Enhancement and Utilization Technologies Division, 2725 Montlake Boulevard East, Seattle, WA 98112 USA.;

Transposable genetic elements have been found in a range of prokaryotic and eukaryotic organisms. The simplest mobile element, the insertion sequence (IS), consists of a gene encoding a transposase (which catalyzes DNA movement) flanked by terminal inverted repeats. Mobilization of an IS can result in mutation, and IS elements that are sufficiently active can be used as population or strain markers. In a molecular study of Renibacterium salmoninarum, the causative agent of bacterial kidney disease, we isolated a mobile element that has strong similarity to IS986/IS6110, an element found in Mycobacterium tuberculosis. Sequence conservation was high in both the inverted repeats (80% nucleic acid identity), and in the transposase gene (>60% nucleic acid and predicted amino acid identity). Southern analysis revealed multiple chromosomal insertions, suggesting this element had been, and may still be, active. Since this is the first report of a transposable element in R. salmoninarum, we have evaluated its utility as a strain marker for epizootiological surveys and as a tool in mutagenesis studies.



Bacterial Environment in Shrimp Hatcheries, Two Case Studies: People's Republic of China and Ecuador

R Robles (1)*, J Vandenberghe (2), J Calderon (3), HS Xu (4), P Sorgeloos (1) and J Swings (2)

1 Laboratory of Aquaculture and Artemia Reference Center, University of Ghent, Rozier, 44, B- 9000 Gent, Belgium.;
2 Laboratory of Microbiology, University of Ghent, Ledeganckstraat, 35, B-9000 Gent, Belgium;
3 Centro Nacional de Acuicultura e Investigaciones Marinas "Edgar Arellano", CENAIM, P.O. Box 09-01-4519, Campus Politecnico (ESPOL), Guayaquil, Ecuador.
4 Department of Marine Biology, College of Marine Life Sciences, Ocean University of Qingdao, 5 Yushan Road, Quingdao 266003, People's Republic of China.

In the framework of the international cooperation project "Diagnosis and control of bacterial diseases in penaeid shrimp hatcheries", several bacteriological surveys were performed during 1995 and 1996 in three shrimp hatcheries (Penaeus chinensis) in People's Republic of China (P.R.C.) and in four hatcheries in Ecuador (P. vannamei). Samples were taken from larvae, post-larval and adult stages. The dominant bacteria flora were characterized by Biolog metabolic fingerprinting and identified by comparison with a database of 850 Vibrio type- and reference strains. In P.R.C. survey, an overwhelming predominance of V. alginolyticus and V. harveyi was observed in the larval developmental stages from zoea stage on. The microbiota associated with larvae are not very stable and highly influenced by the bacteria present in the administered feed and the environment. During 1995, the microflora of successful P. chinensis larvae production were mainly dominated by V. alginolyticus and unidentified Gram negative strains, while V. harveyi was absent. In Ecuador, V. alginolyticus was associated with healthy nauplii and zoea, but might be involved in "bolitas" syndrome and other diseases. Because V. alginolyticus is commonly used as a probiotic in commercial hatcheries, selection criteria for probiotics, which are now largely empirical, should be revised. V. harveyi was not recovered from nauplii and zoea stages. Also V. parahaehemolyticus, V. damsela and other Vibrio strains were found. These results suggest that many presumed pathogens might be associated with specific developmental stage of the shrimp. Careful challenge testing may clarify the role of the different Vibrio strains.



Lethal Effect of Hafnia alvei Isolates with Experimental Infections in Salmo trutta

LA Rodríguez *(1), CS Gallardo (1), A Castillo (1), F Acosta (2) and F Real (2)

1 Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.
2 Departamento de Patologma Animal. Facultad de Veterinaria. Universidad de Las Palmas de Gran Canaria. Spain.

Hafnia alvei is a worldwide Gram negative bacterium, classified as a causative agent of intestinal disorders, distributed in animals, birds and natural enviroments such as soil, sewage and water. This organism has been isolated from differents foods and has been reported to cause epizootic haemorrhagic septicaemia in rainbow trout and is a causative agent of kidney pathology in cherry salmon with clinical signs in affected moribund fish as follows: abnormal swimming, haemorrhagic septicaemia, dark skin and a swollen abdomen. Experimental infections using seven Hafnia alvei strains were carried out to verify pathogenicity and lethality in brown trout. The present study reports the isolation of Hafnia alvei from samples of the spleen, liver, kidney and intestine after i.p. inoculation showing this bacterium to be a causal agent of mortalities in brown trout. Preliminary results shown that there are differences in relation to the degree of pathogenicity of various isolates of Hafnia alvei. LD50 values ranging between 1.3x104 and 2.5x107 bacteria/fish were obtained, values considered virulent for fish.



Differences in Biochemical Tests Carried out at 37ºC and 22ºC of Genus Salmonella Isolated from Environmental Sources

AI Sáa, M Torres, P Gil, S Rodríguez Orbán and LA Rodríguez *

Área de Microbiología. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.

The presence of Salmonella as a causative agent in fish diseases is controversial, as it has only been isolated from ornamental fish and its possible role as a fish pathogen is unknown. The aim of this work was to determine the utility of using conventional laboratory biochemical tests to taxonomically identify Salmonella spp. when incubated at environmental temperature (22ºC). This is important because misleading false positive or negative results could have major management implications.



Infectivity of Genus Salmonella from Environmental Sources in Rainbow Trout

AI Saa, M Torres, A Castillo, J Vivas and LA Rodríguez *

Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.

Gram negative represented the highest percentage of bacterial fish disease. However many of these pathogens such as Vibrio anguillarum, Aeromonas hydrophila and Pseudomonas fluorescens are considered opportunistic pathogens, since other species such as Aeromonas salmonicida or Yersinia ruckeri have been nominated as obligate pathogens. Members of the genus Salmonella, are known to produce gastrointestinal diseases in vertebrates. However, species of Salmonella just only have been associated with ornamental fish diseases. The objective of this work has been to evaluate the infectivity capacity of different species and serotypes of Salmonella from enviromental sources to healthy and diseased rainbow trout.



Toxic Cyanobacterium Microcystis aeruginosa and Summer Kills of Tilapia

R Santhanam, B Manimaran and D Manikandavelu

Fisheries College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Tuticorin-628 008, India.

Summer kills of tilapia fish, Orochromis mossamticus, were recorded during May 1997 in a segment of Tambirabarani river system, 20km away from Tuticorin, Southern India. Prior to these fish kills, hydrobiological studies showed the appearance of monospecific blooms belonging to the toxic cyanobacterium Microcystis aeruginosa the density of which was 3.1 x 105 cells/mL. The dry weight of the seston was approximately 22.5 mg/L. During fish kills, the cells were in a state of decay. The fish kills were associated with high temperature (31.2ºC), deep blue-green color of the water column, high turbidity (k=6.07), high dissolved oxygen content (6.2 mg/L), and high COD (230 ppm). The mass mortality of these omnivorous tilapia fish in this riverine system could be due to their feeding on the microcystin toxin containing M. aeruginosa cells which was evident by the presence of these cells in the gut contents of the dead fishes. The exogenous nutrient loadings and related hydrobiological parameters which triggered the formation of M. aeruginosa blooms and affected the health of the above riverine ecosystem have been assessed. Water quality management measures in preventing the formation of similar toxic algal blooms in the future will also been discussed.



Comparison of Biochemical Tests at Different Temperature and Growth in Different Culture Medium of Hafnia alvei

E Sinde (1), CS Gallardo (1), F Acosta (2), F Real (2) and LA Rodríguez* (1)

1 Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.
2 Departamento de Patologma Animal. Facultad de Veterinaria. Las Palmas de Gran Canaria. Spain.

The organism Hafnia alvei is a normal inhabitant of surface water, soil, grains and intestinal tract of animals. This organism has been reported to cause epizootic haemorrhagic septicaemia in rainbow trout and isa causative agent of kidney pathologenesis in cherry salmon. The absence of a selective specific medium for Hafnia alvei makes difficult their identification at initial isolation due to their similarity to other taxonomically related microorganisms. The aim of this study was to compare the biochemical tests results with incubation at different temperatures (22 and 37C), the Hafnia alvei growth in several commercially selective medium and the growth capacity in Tripticase Soya Agar (TSA) supplemented with different salt concentrations.



Survival in Sea and Fresh Water of Different Environmental Salmonella Strains Compared with Members of F.Vibrionacea.

M Torres, AI Saa, M Vendrell, P Gil and LA Rodríguez *

Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.

Members of F. Vibrionaceae are considered to be obligate and opportunistic pathogens of both freshwater and sea water fish, while members of F. Enterobacteriaceae have been isolated from a great variety of sources such as the gastrointestinal tract of different animals, as well as water, soil, etc. A few cases about Salmonella spp. related with fish have been reported. The majority of them about ornamental fish isolation. The aim of this work was to compare the survival in sea and fresh water of different enviromental Salmonella strains and members of F. Vibrionaceae considered typical pathogens of fish. Experiments were carried out at two temperatures (10C and 22C) using seawater and fresh water, previously sterilized by filtration and the survival time was determined by the direct counts plate method. Results showed that Salmonella strains were able to survive long times in both types of water at different temperatures, and could be a risk for outbreak of epizooties.


Survival Comparison of Different Fish Pathogens in Two Aquatic Ecosystems

MC Vendrell, D Abad, M Torres, A Andris and LA Rodríguez *

Area de Microbiologma. Facultad de Ciencias. Universidad de Vigo, Campus Orense. Spain.

In the Northwest of Spain there are a lot of hydrothermal emissions that emerge to the surface at high to moderate temperature (20-45C). The localization of these emissions generally are quite near to the border of the rivers, causing a mixture of both types of water, and subsequently an increase of temperature in the microcosm is produced. This temperature effect on the water implies a development of the fish population around it. The objective of this work was to evaluate the survival time of different typical fish pathogens able to induce diseases at two differents temperatures (normal fresh water and warm fresh water).


Evaluation of the Microscan System for Determining Antimicrobial Susceptibility of Gram Negative Fish Pathogens

J Vivas, AI Sáa, E Sinde, MJ Pérez * and LA Rodríguez

Área de Microbiología. Facultad de Ciencias. Universidad de Vigo. Spain.

Gram negative bacteria cause the highest percentage of bacterial fish disease. In this work, we evaluated the utility of the MicroScan system (Dade MicroScan Inc., West Sacramento, Calif.) in conjunction with the fluorometric Neg Combo panels for rapid identification and susceptibility testing of important gram negative fish pathogen bacteria. The identification patterns were analyzed using twenty-six conventional biochemical laboratory tests and the susceptibility with the agar diffusion disk and macrodilution broth methods. The number of the biochemical tests evaluated were twenty-six and the antibiotics evaluated were Amikacin (AK), Ampicillin (Am), Cephalothin (Cf), Cefotaxime (Cft), Cefuroxime (Crm), Ciprofloxacin (Cp), Fosfomicin (Fos) and Gentamicin (Gm). The level of concordance between conventional laboratory biochemical tests and the MicroScan System was high, but this system classified as rare some strains of six typical fish pathogen genus. In the susceptibility test, the MicroScan System demonstrated less than 5% of false results. The MicroScan system´s MICs demonstrated a good correlation with the macrodilution broth method test.


Survival of Flexibacter psychrophilus in Laboratory Microcosms

T Wiklund*(1) and S Nystedt

Institute of Parasitology & Department of Biology, Åbo Akademi University, BioCity, Artillerigatan 6, 20520 Åbo, Finland;
1 Present address: Fish Disease Laboratory, Danish Institute for Fisheries Research, Bülowsvej 13, DK-1870 Frederiksberg C, Denmark

The influence of different environmental factors (salinity, temperature and presence of sediment) on the survival of two strains of Flexibacter psychrophilus (syn. Flavobacterium psychrophilum, Cytophaga psychrophila) was studied in laboratory experiments. The type strain NCIMB 1947 and a Finnish strain isolated from diseased rainbow trout were included in the study. In microcosms containing sterile natural fresh and brackish water the bacteria showed a survival of at least eight weeks, without additional nutrients. In long term experiments the type strain survived in fresh water for about 300 d. Viable cells of the examined strains could not be detected in the microcosms containing sterile marine water 1 day after inoculation. Incubation of the bacterial cells in fresh water at different temperatures (5ºC, 10ºC and 15ºC) did not give any conclusive results concerning optimal survival temperature, but the examined strains were viable after 56 d incubation at all temperatures. The presence of sediment in the microcosms significantly enhanced the survival of the examined bacteria compared to the microcosms containing only water. The examined strains showed good survival in closed systems for long periods without addition of nutrients or removal of waste products. The salinity of the water influenced the survival of the examined cells significantly, whereas the temperature did not influence the survival. The results suggest that F. psychrophilus is not obligatory parasitic for fish and free living bacterial cells can survive in fresh and brackish water habitats for a long time.



Ocular Lesions in an Albino Strain of Japanese Medaka (Oryzias latipes) Exposed to Methylazoxymethanol Acetate (MAM-Ac)

DR Geter (1)*, WE Hawkins (1), JW Fournie (2) and GK Ostrander (3)

1 Gulf Coast Research Laboratory, Institute of Marine Sciences, University of Southern Mississippi, PO Box 7000, Ocean Springs, Mississippi 39566 USA.;
2 US Environmental Protection Agency, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, Florida 32561 USA.
3 Department of Biology and Department of Comparative Medicine, Johns Hopkins University, 224 Mergenthaler Hall, 3400 N. Charles Street, Baltimore, Maryland 21218-2685 USA.

Exposure of a small fish, the Japanese medaka (Oryzias latipes), to the carcinogen methylazoxymethanol acetate (MAM-Ac) results in neoplasms of the retina and other organs. Retinal lesions appeared as complexes of neoplastic cells arising from undifferentiated cells ringing the anterior margin of the retina. The complexes consist of mitotically active, heterogeneous cells with characteristics of both sensory retina and pigmented epithelium that gave rise to neoplasms diagnosed as medulloepitheliomas because of their organoid appearance. In many lesions heavy pigmentation obscured histological features. To further investigate the role of pigmented cells in the lesions, we used an amelanotic (albino) mutant strain of medaka. About 320 ten-day-old fish were exposed to 0, 10, 30 or 50 ppm MAM for two h then transferred to aquaria. Within one month, behavioral effects including loss of equilibrium and morbidity were seen in fish from the 30 and 50 ppm MAM groups. Histological analysis of 55 fish from those groups sampled as either recently dead or dying from day 0 to day 71 post exposure, revealed marked cytotoxicity of the retina and liver. Livers showed necrosis, gross and single cell hypertrophy, mitotic figures, and macrophage infiltration, along with early neoplastic changes. Retinal changes occurred mainly in the retinal growth zone and included dysplastic lesions with rosette formations, hemorrhage, retinitis and early neoplastic lesions. With no obscuring pigment in the lesions, it was clear that dysplastic and neoplastic changes involved cells of both sensory and epithelial retina and probably arose from a precursor retinoblast.



Rectal Adenocarcinoma in a Queen Trigger Fish

R Reimschuessel* (1), H Krum (2), R Cooper (2) and J Harshbarger (3)

1 Aquatic Pathobiology Center, University of Maryland, Baltimore, MD 21201.
2 New England Aquarium, Boston, Mass.
3 Registry of Tumors in Lower Animals, Department of Pathology, The George Washington University Medical Center, 2300 Eye Street, N.W., Washington DC, 20036.

We present here the first registered case of a mucinous rectal adenocarcinoma in fish. The fish, a Queen trigger fish, Balistes vetula, presented with a swelling near the anus. This fish had been on exhibit at the New England Aquarium in Boston. The tissue around the anus was surgically excised and preserved for histopathologic examination in 10% neutral buffered formalin. Microscopic examination revealed a well-differentiated mucinous rectal adenocarcinoma. Some of the ducts contained mucinous material within their lumens. Several of the ducts penetrated to the surface squamous epithelium. A moderate amount of stromal connective tissue was also present in the lesion. This case has been accessioned RTLA 6435 with the Registry for Tumors in Lower Animals. It is the first recorded tumor in this genus of fish and the only mucinous rectal adenocarcinoma in fish.



Cloning of Japanese Flounder, Paralichthys olivaceus, Tumor Necrosis Factor Alpha Gene

M Sakai and M Miyanohara

Faculty of agriculture, Miyazaki University, Miyazaki, 889-21, Japan

Tumor necrosis factor a (TNFa) is a cytokine with a wide range of effects on both lymphoid and non-lymphoid cell types. By PCR the corresponding Japanese flounder, Paralichthys olivaceus, the TNFa gene was partially isolated from cDNA obtained from kidney tissue stimulated with lipopolysaccharide (LPS). The nucleotide sequence translated in one reading frame with on stop codons to produce a partial peptide containing 191 amino acid. Based on the sequence of human TNFa, the amino terminal amino acid of mature flounder TNFa is predicted to be 165 amino acids with a molecular weight of 17,485 kd. This flounder TNFa gene exhibited conserved cysteine residues that are involved in a single disulfide bond in human TNFa, and one potential N-linked glycosylation site. The nucleotide and amino acid sequences showed about 40% homology to the equivalent human and mouse molecules.



Survival of Vibrio spp. Pathogenic for Fish

SP Armada*, R Farto, TP Nieto and MJ Pérez

Area de Microbiologma, Universidad de Vigo, Departamento de Biologma Fundamental y Ciencias de la Salud, E-36200 Vigo, Espaqa.;;;

Vibrio splendidus and Vibrio pelagius are considered to be normal microbiote in seawater, but they were reported as causal agents of epizootics in a turbot farm in the Ria de Vigo. In this work we evaluated the survival time of this pathogens in a defined medium, in seawater and in freshwater, comparing the viability of these strains at different temperatures, salt concentrations and nutrient levels. Experiments were monitored by viable count plate methods, acridine orange direct counts (AODC), direct viable counts (DVC) with nalidixic acid (Kogure4s method) and respiratory activity with a tetrazolium salt (INT). Survival times were shorter in freshwater than in any other microcosms and the nutrient concentration was a determinant in the survival of these strains.



Clinical and Pathological Effects of Serratia liquefaciens Infection in Rainbow Trout (Oncorhynchus mykiss Walbaum)

S Aydyn (1)*, AK Çiltab (2) and ÖC Bilgin (3)

1 Çanakkale Onsekiz Mart University, Fisheries Faculty, Karacaören, 17100 Çanakkale, Turkey
2 Atatürk University, Agriculture College, Department of Fishery Sciences, 25240 Erzurum, Turkey
3 Atatürk University, Agriculture College, Department of Animal Sciences, 25240 Erzurum, Turkey

This research was conducted to investigate Serratia liquefaciens infections during June and July after about 20 days from torrents in juvenile cultured rainbow trout (Oncorhynchus mykiss Walbaum). Bacterial isolates were identified from each farm and tested to determine sensitivity against 18 chemotherapeutants. The mean minimum bactericidal concentrations (MBC) of chloramine-T and potassium per manganate (KMnO4) were 17.07 mg/L for 1 h and 23.27 mg/L for a 10-min exposure, respectively. Infections caused fallen scales, bloody and swollen kidney, hyperaemic and pale regions in liver, haemorrhaging spots in gills and the presence of bloody exudate in intestine. Histopathological examination demonstrated pathological changes in liver, spleen and kidney. Naturally infected fish were examined for chemical parameters of blood and compared to healthy fish. Amylase and glutamate oxalacetate transaminase (GOT) enzymes, triglyceride, cholesterole, albumin, albumin/globulin (A/G), iron levels, total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC) and saturation in blood serum of infected fish were significantly increased. Glutamate pyruvate transaminase (GPT) and L-lactate dehydrogenase (LDH) enzymes, bilirubin, glucose, creatinine, and total protein (TP) values were not significantly different between the naturally infected and healthy fish. In the therapeutic applications, oral applications of sulphonamide after disinfections with chloramine-T controlled the infections.



Extracellular Products of the New Bacterial Fish Pathogen Vibrio viscosus (Moritella viscosa)

E Benediktsdóttir (1)*, Áp Jónsdóttir (1), S Helgason (2) and SH Bambir (2)

1 Institute of Biology, Mircobiology Laboratory, University of Iceland, Ármúli 1A, IS-108 Reykjavík, Iceland.
2 Institute for Experimental Pathology, Fish Disease Laboratory, Keldur, University of Iceland, IS-128 Reykjavík, Iceland.

Vibrio viscosus (Moritella viscosa) is a psychrotrophic bacterium that has been found in winter ulcers in salmonids in Norway, Iceland and Scotland. Strains of V. viscosus have been found to be virulent to Atlantic salmon parr. Different subgroups of V. viscosus strains have been recognized. A comparison was performed of the extracellular products (ECP) of five strains, representing different subgroups of V. viscosus: MT1887 (Scotland), 478/88 (Norway), K58 (SW-Iceland), K56 (N-Iceland), and F57 (SW-Iceland, low-virulent). When ECP of K58 were injected intraperitoneally to fish, similar internal disease symptoms were seen as when challenged with life bacteria. Cytotoxic activity against EPC and BF-2 cells was detected in the ECP from all strains, the lowest activity was seen when ECP from K56 was used. Cytotoxic activity against human fibroblasts was detected in the ECP from three strains, strain MT1887, 478/88, and K58. The highest hemolytic activity against three kinds of erythrocytes was shown by the low virulent strain F57, but lowest by K56 and K58. All strains were hemolytic against rainbow trout erythrocytes, but in different dilutions. Protease activity was detected in the ECP of all strains isolated from salmonid fish, but no protease activity was detected in the ECP of F57. Comparison of the disease symptoms produced by the different ECPs in Atlantic salmon parr is being performed and the first results will be presented.


Isolation of Infectious Salmon Anemia Virus and a Previously Undescribed "Togavirus" from the Bay of Fundy

DA Bouchard(1), HM Opitz(2), BM Nicholson(3), S Blake(3) and WR Keleher(1)

1 Micro Technologies, Inc., 41 Main Street, Richmond, Maine 04357 USA
2 Animal Disease Diagnostic Laboratory, University of Maine Cooperative Extension, 5735 Hitchner Hall, Orono, Maine 04469 USA.
3 Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, Maine 04469 USA.;

Hemorrhagic kidney syndrome/infectious salmon anemia (HKS/ISA) is a newly described disease affecting farmed Atlantic salmon in the Bay of Fundy region, New Brunswick, Canda. The causative orthomyxovirus (infectious salmon anemia virus (ISAV)) was cultured on the chinook salmon embryo (CHSE-214) cell line and it appears that pH plays an important role in isolating the virus. Evidence of a cytopathic effect (CPE) is first seen at 14-28 days. Sequence analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) amplification products confirmed this North American isolate as ISAV but indicated that it is not identical to the published Norwegian isolate. During the course of identifying the etiological agent of HKS/ISA another as yet unidentified virus was isolated. It has been described as a member of the Togaviridae family. This virus has not been shown to cause mortality or associated pathology in affected fish. This virus, however, has been found quite extensively on farms in the Bay of Fundy region with 9 of 10 farms testing positive. Of samples taken from these farms, 27% of the pools tested positive for the virus. Isolation of the virus occurred on the CHSE-214 cell line at 9 days post inoculation. Most of the observed CPE was seen at 21 days post inoculation.



Vibrio viscosus in Farmed Atlantic Salmon, Salmo salar L. in Scotland: Field and Experimental observations

Bruno DW*, Griffiths J, Petrie J and Hastings TS

FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB, Scotland

Market-sized (2-3 kg) Atlantic salmon, Salmo salar, reared in sea cages in Scottish waters with skin ulcers were examined. A Vibrio sp. was isolated on laboratory media. Biochemically this was identical to a previously described bacterium with the name Vibrio viscosus from farmed salmon in Norway. Internally a prominent dark-brown petechiae or ecchymotic haemorrhage was noted. Splenomegaly was associated histologically with congestion and widespread necrosis. An experimental study reproduced the field observations and Koch's postulates were fulfilled. This work examines the occurrence of V.viscosus in marine reared Atlantic salmon for the first time in Scottish waters. The histopathology associated with infection is compared with that reproduced in laboratory-infected fish.



Are Tasmanian Platypuses (Ornithorhynchus anatinus) Endangered as a Result of the Spread of Ulcerative Mycosis?

NJ Stewart (1), BL Munday (1) and AB Heinrich (2)

1 Department of Aquaculture, University of Tasmania, Launceston, 7250 Tasmania, Australia;
2 Division of Comparative Medicine, John Hopkins University, School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205-2196, USA.

Ulcerative mycosis is a serious fungal disease that affects platypuses (Ornithorhynchus anatinus) only in the north of the island state of Tasmania, and not elsewhere in Australia. The disease is characterised by severe ulceration of the haired and unhaired parts of the body, and invades the musculature to a depth of up to 10mm below the skin. In some instances there are also granulomas in the lungs. The presumed causative agent, Mucor amphibiorum, has been more commonly reported with mycotic infections in amphibians. The fungus is heterozygous, comprising positive and negative mating types. It may have been introduced to the state, possible by green tree frogs in crates of fruit imported from Queensland. It is also possible that the Tasmanian platypus population is in some way immunocompromised, as most known serious fungal infections do not occur in healthy hosts. To determine the prevalence and distribution of the disease, platypuses from throughout the state have been trapped and examined, and swabs taken from ulcerated animals. In areas in which the disease was found, approximately 30% of animals captured have been ulcerated. The M. amphibiorum isolates taken from affected animals have all been positive mating types. From throughout the state, 9 carcasses of accidentally killed platypuses were obtained. Tail fat samples were analysed for total DDT, lindane, and polychlorinated biphenyls. All animals tested contained DDT residues, ranging from 52 ng/g to 822 ng/g. Lindane was found in only 3 of the animals, with a maximum concentration of 3 ng/g. Traces of PCBs were found in all animals, ranging from 40 ng/g to 570 ng/g. As PCBs are known immunosuppressants, they may play a role by increasing the susceptibility of platypuses to the fungus. It is our opinion that even though the disease can be fatal and lead to temporary, localized population declines, it is not likely to cause long term decline in platypus numbers in Tasmania.



Fibrosing Pancreatitis and Myonecrosis in Cultured Yellow Perch (Perca flavescens)

JC Wolf * and SA Smith

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 USA.;

Yellow perch fingerlings were obtained from two different domestic commercial hatcheries for grow-out in a closed-recirculation aquaculture facility. Several weeks after their arrival, dramatically decreased growth and emaciation (i.e. runts) were noted in approximately 10% of perch fingerlings. Despite their debilitated appearance, most affected fish continued to feed, and overall mortality for perch within the facility remained low. Macroscopically, affected fingerlings possessed small, dark-brown livers, a paucity of intraperitoneal adipose tissue, and evidence of recent food consumption. The most significant histologic lesion was partial to complete pancreatic attenuation with replacement fibrosis, and sporadic evidence of focal pancreatic necrosis. Additional microscopic findings included occasional small foci of rhabdomyolysis within epaxial muscles, and comparative hepatocellular condensation consistent with hepatic glycogen and fat depletion. Approximately 1 month subsequent to the start of the runting problem, non-runted fish in one of nine perch tanks abruptly began to exhibit aberrant swimming patterns, apparent generalized weakness, and high mortality (up to 10% per day). Microscopic findings in these fish primarily consisted of localized to extensive epaxial muscle necrosis. Microbial cultures obtained from multiple viscera throughout the disease period were negative for bacteria, however, a cytopathic agent was eventually isolated by cell culture from hepatic tissues of the non-runted fish. This isolated agent is presently under investigation, and additional ultrastructural and transmission studies are pending.



Investigation of High Mortalities in Eyed Eggs and Fry of Rainbow Trout (Oncorhynchus mykiss Walbaum) and Brook Trout (Salmo trutta)

S Aydyn*

Çanakkale Onsekiz Mart University, Fisheries Faculty, Karaca ören, 17100 Çanakkale, Turkey

This research was conducted to investigate high mortalities of eyed-eggs and fry in trout hatcheries during the winter seasons of the years 1995 and 1996. In one of the hatcheries, the reasons of death such as low water temperature (0-6oC), clays covering eggs, direct sun-light and mechanically made faults were observed in brook trout (Salmo trutta) eyed-eggs and fry. There were reddening and haemorrhages around eyes of eggs and became hard with reddening and haemorrhages in yolk sac of fry. Vibrio parahaemolyticus infections were present in rainbow trout eyed-eggs (Oncorhynchus mykiss Walbaum) in the other hatcheries. Infections caused reddening and haemorrhages around eyes of eggs. Bacterial isolates were identified from each of the hatcheries and tested to determine sensitivity against to 12 chemotherapeutants. Also, pathogenicities of two isolates were examined. A therapeutic experiment bath with chloramine-T and norfloxacin controlled the natural V. parahaemolyticus infections.



Swimbladder Inflation by Zebrafish Larvae (Danio rerio) in the Absence of an Air-water Interface

AS Goolish (1)* and K Okutake (2)

1 Lockheed Martin Engineering and Sciences Company, Space Station Biological Research Project, NASA Ames Research Center, Moffett Field, California 94035
2 BioSource, Space Station Biological Research Project, NASA Ames Research Center, Moffett Field, California 94035

The larval stages of most fish, both physostomous and physoclistous, are believed to require access to an air-water interface for normal development. Limited published data suggests that swimbladder inflation does not occur normally when access to an air-water interface is denied, which can result in curvature of the spine, decreased growth rate, and increased mortality. Incidences of very high mortality in aquacultural systems have been attributed to this mechanism, both in indoor aquaria and outdoor production ponds, where a high diet ration can result in an oily surface film and increased surface tension. The culture of zebrafish (Danio rerio) has become widespread in recent years as it has become a major vertebrate model for the study of developmental biology, neurobiology, and molecular genetics. Here we report on swimbladder inflation success rate, growth rate, incidence of spinal curvature, and survivorship of zebrafish larvae maintained for up to 14 days in the absence of an air-water interface. Sixty larvae, 72 hours post-fertilization, were tested in replicate 250 mL chambers which were maintained either open or closed to the air. Both treatments were supplied with clean, aerated, recirculated water which could be delivered to the closed test chambers without the introduction of bubbles. A live diet and system water change-outs were provided daily. Preliminary results indicate that all of the above test variables are affected, and that access to an air-water interface is critical for the normal development of zebrafish larvae.



The Estimation Criteria of Fish Farm Epizootic State under the Conditions of Anthropogenic Pollution

AV Kazarnikova(1), EV Shestakovskaya (1), LY Katycheva (2) and GM Hoteva (3)

1 RosribNIIproekt ichthiopathological laboratory Oborona-street 49 Rostov-on-Don Russia 344008
2 Head, Central Production Station:Fish acclimatization and Diseases Control, !8a Zheltovskogo st., Rosrybhoz, Moscow, Russia
3 Head, Northen Caucasus Branch of the Central Production Station: Fish acclimatization and Diseases Control, 122, Ponomaryov st., Krasnodar, Russia

The fishery of the northen Caucasus is based on commercial fish farms. The main objects of rearing are: carp (Cyprinus carpio), bigheads (Hypophthalmichthys molitrix, Aristichthys nobilis) and grasscarp (Ctenopharyngodon idella). Under the conditions of anthropogenic pollution at fishponds, we observed several diseases, causing pathological alterations in fish: gill necrosis, aeromonosis x toxcosis, ambiphriosis x toxcosis, etc. The toxicants are likely to depress the resistance of fish to non-specific parasites, such as Ambiphrya ameiuri registered on fry and juveniles of carp, bigheads and grasscarp. Numerous factors affect the epizootic state of fish farms. Comprehensive investigations allow us to appropriate main criteria (type of fish farm, technical equipment, hydrochemical indexes, etc.) affecting the fish farm efficiency. In the final analysis, we have defined controlled criteria such as the level of invasion, the percent of invaded fish, hydrochemical, toxicological and veterinary-sanitarian indexes. We've collected the information for creating a data bank of the northen Caucasus fish farms. Using the data bank we can find nessessary information on any fish farm, its type, water supply, reared fish and their diseases. With the help of this information we can make a prognosis of the epizootic state of a fish farm, prevent the spreading of pathogenic parasites throughoutt the region and develop preventive measures not only for a local fish farm but for the region.



Health Status and Survival of American Lobster (Homarus americanus) Caught in Prince Edward Island Waters

J Lavallée (1,2)*, LK Hammell (1), ES Spangler (1) and R Cawthorn (2)

1 1 Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, Prince Edward Island, Canada, C1A 4P3.,,
2 Lobster Health Research Centre, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, Prince Edward Island, Canada, C1A 4P3.

Canadian landings reached a record peak in 1992 with more than 42,000 metric tons, and were stable in 1996 at 39,000 metric tons, with an estimated value of $375 million (Can.). Prince Edward Island accounts for at least 20% of the Canadian landings annually. To supply the market with a live year-round product, the industry keeps lobsters in captivity for various periods. Mortalities can occur during that holding time, resulting in substantial losses. These losses have been estimated by the industry at 10-15%. Our study assessed lobster fishing practices on various boats from Prince Edward Island, between May and August 1997, and related these practices to lobster survival. A total of 2,197 lobsters coming from 64 boats were tagged and assessed while fishing practices were monitored on these boats. There was an increase of 6.5% in the proportion of lobsters with open wounds from the time of capture to the time of entry into the processing plant, while the increase was 18.1% from the time of capture to immediately prior to processing. Lobsters had significantly increasing serum protein levels and haemocyte counts as water temperature increased. Better health status through to processing was associated with fishing practices that prevented contact among lobsters on the boat before being measured and banded, while the use of fresh mackerel decreased overall health status. Warmer water temperatures were also associated with moderate decrease in the health status of lobsters.



Performance of Silver Carp (Hypophthalmichthys molitrix) as Affected by Level of Duck Manure and Stocking Density

FA Hafez (1), A El-Hakim (2), EA El-Helaly (2) and AA Mahmoud (1)

1 Central Laboratory for Aquaculture, Abbassa, Agriculture Research Center
2 Faculty of Agriculture, Al-Azhar University

This study was carried out to investigate the effect of duck manure level and stocking density on growth performance of silver carp. The experiment was applied at the Central Laboratory for Aquaculture, Abbassa, Abou-Hammad, Sharkia, Agriculture Research Center. Six rectangle earthen ponds each of total area of 0.25 Feddan (50 x 20m) representing six treatments (3 levels of duck manure 150, 300, and 450 kg/fed within each two stocking density 3200 and 4800/fed). The experimental period expanded 20 weeks. The statistical evaluation of results showed that differences in body weight and length at the successive growth were significantly (p<0.05) in favor of higher manuring level (450 kg/fed). Regardless of level of manuring, average body weight of silver carp decreased significantly (p<0.05) with increasing stocking density from 3200 fish/fed to 4800 fish/fed during all experimental period. Relative growth rate increased with decreasing stocking density and improved with increasing the rate of manure. Level of manure and stocking density had no influence on condition factor. Total yield increased in a linear manner with each increase in manuring level and stocking density.



How Healthy Are Shrimp with IHHN Virus in Water with Very Low Salinities?

AO Meyer-Willerer (1)* and MC Rivera-Rodriguez (2)

1 Centro Universitario de Investigaciones Oceanologicas, Universidad de Colima, Departamento de Acuacultura, km 20 Carretera Manzanillo - Barra de Navidad, Manzanillo, Colima 28860 MEXICO
2 Facultad de Ciencias Marinas, Universidad de Colima, Departamento de Acuacultura, km 20 Carretera Manzanillo - Barra de Navidad, Manzanillo, Colima 28860 MEXICO

Healthy shrimp are more tolerant to environmental changes such as temperature, salinity and sensitivity to light. The environmental conditions are interactive and dynamic and one factor may affect other variables, producing changes that may influence survival, food conversion ratio and growth rate. The white shrimp Penaeus vannamei is a robust crustacean that is produced in intensive systems supporting high densities and thus having to be fed large quantities of first quality formulated feeds. Shrimp postlarvae that carry IHHN virus seem to be apparently healthy and when adapted to brackish water, they may grow asymptomatically carrying the virus until they reach market size. This is one reason shrimp farmers tend to grow the white shrimp in brackish water between 2 and 4 ppt, although harvests are not as good as those in marine water. These waters may be eutrophic and algae blooms occur. A series of tests were conducted in aquaria under controlled conditions to determine growth ratios and survival of P. vannamei postlarvae that carried IHHN virus. Postlarvae adapted to 3.0 ppt water were purchased from a farm. Water salinity of 0.2, 0.44, 0.55, 0.70, 1.7, 11.0, 22.0 and 33.0 ppt was obtained by mixing oceanic water with clear chlorine-free municipal water and acquired postlarvae adapted gradually to the respective salinities in two weeks. In another trial, the presence or absence of light was compared in aquaria with green water at 0.2 or 1.0 ppt. The obtained results will be discussed and compared with farm productions.



Culture of Freshwater Mussel and Its Disease Control in China

J Ou*

China Wuxi Fisheries Medicine Factory

Freshwater mussel resources in the world are very rich and there are many species with important economic value. In China, there are more than 100 species. The conservation of these species, especially those which are cultured, is considered very important, particularly in light of the introduction of "invading species." Mussel culture parameters, diseases and control strategies will be discussed.



Cathepsins B and L Responsible for Defense Bacteriolysis in Anguilliform Skin

F Aranishi*

National Research Institute of Fisheries Science, MAFF, Fukuura, Kanazawa, Yokohama 236-8648, JAPAN

Fish skin proteinases have been suspected as major defense agents but are yet to be verified. This work enzymatically identifies skin proteinases in anguilliformes and demonstrates their defense abilities. By using an array of fluorescent sensitive substrates, the skin extracts of Japanese, European and American eels commonly contained four types of proteinases. They were determined to be cathepsins B and L, aminopeptidase and collagenase through the detailed enzymatic characterization. Two cathepsins and two other proteinases seemed to serve as defense and house-keeping proteinases from their enzymatic specificities, respectively. A newly established fluorescent activity staining histochemically demonstrated that these cathepsins were widespread in mucus secretory cells and club cells, where small fluorescing granules were distributed, of the epidermis of Japanese eel. This suggests the secretion of eel skin cathepsins into either the mucus or epidermis by these cells when needed. Under the experimental thermal stress and bacterial infection, both of these catheptic actions, that were accompanied by skin bacteriolytic action, increased in Japanese and European eels. The skin extracts of the both eels showed a marked ability to lyse various gram-negative pathogenic and gram-positive non-pathogenic bacteria at pH 5.0-5.5, and all of these bacteriolytic activities were powerfully inhibited by typical cysteine proteinase inhibitors. Results indicate that skin cathepsins B and L are novel bacteriolysins available for extensive bacterial species in the nonspecific defense of anguilliform eels.



Immunomodulatory Effects of Clofibrate and Retinoic Acid in Fundulus heteroclitus: Implications for the Mechanism of Toxicity

C Baier-Anderson, RS Anderson and ML Haasch

University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, Maryland, 20688 USA.;;

There is conflicting evidence on the immunomodulating potential of peroxisome proliferating agents (PPAs). Retinoids are important to immune system function and there is evidence that enzymes induced by exposure to PPAs may alter retinoid homeostasis, which, in turn, could modulate tissue macrophage activity. The purpose of this research is to (1) determine if exposure of fish (Fundulus heteroclitus) to all-trans-retinoic acid modulates tissue macrophage function; (2) to correlate immunomodulation with lauric acid hydroxylase induction; and (3) to characterize the immunomodulatory potential of clofibrate, a potent model PPA. In this preliminary study fish were exposed for 24 hours to all-trans-retinoic acid (1 uM), clofibrate (0.04 and 0.4 mM) or vehicle. Macrophages from the anterior kidneys were harvested and zymosan-stimulated reactive oxygen species (ROS) production was measured using luminol-augmented chemiluminescence. Liver microsomal fractions were probed for lauric acid hydroxylase induction and cytosolic fractions were probed for nitric oxide synthase (iNOS) induction. The results indicate that all-trans-retinoic acid significantly increases, whereas clofibrate decreases, ROS production. Both compounds are capable of inducing CYP2K1 and iNOS enzymes, although there is individual variation among fish. Taken together, these results suggest that environmental exposures to PPAs could decrease disease resistance by suppressing macrophage function. Furthermore, since all-trans-retinoic acid activates macrophages, it is possible that altered retinoid homeostasis could be the mechanism by which PPAs exert their immunomodulatory effects.



Resistance to Edwardsiella ictaluri by Channel Catfish, Ictalurus punctatus, is Associated with Enhanced Splenic Macrophage Aggregation

KL Camp (1), CD Rice (2) and WR Wolters (3)

1 KL Camp, Delta Research and Extension Center, College of Veterinary Medicine, Mississippi State University, P.O. Box 197, Stoneville, MS 38776 USA.
2 CD Rice*, College of Veterinary Medicine, Mississippi State University, P.O. Box 9825, Mississippi State, MS 39762.
3 WR Wolters, U.S. Department of Agriculture, ARS, Catfish Genetics Research Unit, P.O. Box 38, Stoneville, MS 38776 USA.

Infectious diseases are prevalent and costly in channel catfish aquaculture, especially the bacterial disease Enteric Septicemia of Catfish (ESC). Considerable research has focused on different aspects of this disease, including the biology of the causative agent, Edwardsiella ictaluri. However, efforts at controlling or managing ESC have been only moderately successful. One solution is to increase the natural disease resistance of fish through selective breeding. Recent research has demonstrated that genetic factors influence resistance to infection in both fish and mammals. Selective breeding for disease resistance in channel catfish has been undertaken for several years, however differences in defense mechanisms among Edwardsiella ictaluri challenged strains and families have been given minimal treatment. We investigated select immune responses of full-sib families of channel catfish to a laboratory challenge with Edwardsiella ictaluri. Immune function parameters included non-specific as well as specific mechanisms of defense and, from these, a profile of disease resistant and sensitive families was created. Both resistant and sensitive families exhibited a humoral response in the form of specific antibody titers but there was no difference between them. Neither family produced significant amounts of stress-related cortisol. Resistant families had a higher percentage of head kidney neutrophils and higher percentages of circulating T-cell percentages. Sensitive families, on the other hand, had higher percentages of circulating B-cells. But the most striking and significant observation was that resistant families had more splenic and trunk kidney macrophage aggregates throughout the infection than did the sensitive families.



The Production of a Lymphokine (Macrophage Activating Factor) by Rainbow Trout (Oncorhynchus mykiss) Leucocytes Stimulated with the Extracellular Products of Mycobacterium spp.

S-C Chen (1), KD Thompson (2), A Adams (2) and RH Richards (2)

1 Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, 91207,Taiwan.
2 Institute of Aquaculture, University of Stirling, Stirling FK 9 4LA, Scotland, UK,,

The production of macrophage activation factor (MAF) by rainbow trout (Oncorhynchus mykiss) head kidney leukocytes in the presence and absence of Mycobacterium spp. extracellular products (ECP) was examined in vitro. Cultures of head kidney macrophages, prepared from naive fish or fish previously vaccinated with ECP or whole cell sonicates (WSC), were incubated with supernatants from the leukocyte cultures. The respiratory burst of these macrophages was then determined as an indicator of macrophage activation by measuring their ability to reduce nitroblue tetrazolium (NBT).
Incubation of head kidney macrophages from naive fish directly with 1,10 and 100mg of ECP for 48 h significantly enhanced macrophage activation compared to macrophages not treated with ECP. Vaccination with either ECP or WSC had no significant effect on the respiratory burst of the macrophages 4 wks post-vaccination, but significant differences were observed in vaccinated fish 8 wks post-vaccination. This activity had abated by wk 12 to levels well below those observed in fish injected with PBS. It was not until wk 12 post-vaccination that re-exposure to Mycobacterium spp. antigen in vitro resulted in significant increases in NBT reduction by macrophages cultured with leukocyte supernatants from fish vaccinated with ECP and WCS. The results of this study suggest that determination of MAF production by fish leukocytes maybe useful for monitoring their immune response following vaccination.



Improved Disease Resistance in Rana rugulosa Following b-Glugan Administration

M Crumlish* and V Inglis

Institute of Aquaculture, Stirling University, Stirling FK9 4LA Scotland U.K

Farmed frogs are highly susceptible to bacterial disease, precipitated by stress-associated factors. Little is known about the functional capacity of the immune system in tropical ranid species, nor the prophylactic value of immunostimulants. This study was carried out to evaluate the efficacy of b-glucan administration in preventing bacterial disease in cultured Rana rugulosa.
Animals with a mean weight of 40g were separated into three experimental groups (n = 5) with four replicate tanks per group. All treatments were administered by intraperitoneal injection (i.p). Group A received 100 ml of sterile saline. Group B was challenged with 100 ml of an unspeciated aeromonad at 1 x 10 2 cfu ml-1 and group C received 100 ml of a 1% (w/v) suspension of the b-1,3 ­1,6 glucan (Macrogard") three days before bacterial challenge at 1 x 10 2 cfu ml-1. Animals were observed twice daily, mortalities recorded and the liver sampled for bacterial recovery using routine methods.
No more mortalites occurred five days after challenge and then the percentage cumulative mortality rate was 40% in group A, 95% in group B and 45% in group C. The increased survival rate of the b-glucan treated frogs was statistically significant compared with bacterial challenge only group.



Measurement of Apoptosis in In Vivo Chemical-exposed Fish Splenic and Pronephric Leukocytes

RM Gogal Jr, SA Smith*, JL Robertson and SD Holladay

Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 USA.,,,

Research on programmed cell death (apoptosis) is expanding rapidly. It has become recognized that chemical-induced apoptosis may contribute to toxicity, thus, certain toxic responses to chemical agents may relate, in part, to altered regulation of this phenomenon. As a result, the diagnostic tools for measuring apoptosis have increased both in sensitivity and number. The present study was part of a larger project evaluating fish (tilapia, Oreochromis niloticus) as a potential alternate animal species for preliminary chemical immunotoxicity risk assessment. Cytocentrifugation of leukocyte-enriched cell samples from the pronephros (the fish primary hematopoietic compartment) and spleen consistently demonstrated apparent chemical-related increases in apoptotic bodies. This observation was therefore further examined using two DNA-binding probes developed for detecting apoptosis, 7-Aminoactinomycin D (7-AAD) and propidium iodide (PI). The cytometric data generated using these apoptotic probes agree well with the microscopic observations, and support the increased incidence of apoptosis in fish exposed to several immunotoxic chemicals (azathioprine, benzo[a]pyrene, 7,12-dimethylbenzanthracene, 2,3,7,8-tetrachlorodibenzo-p-dioxin). These findings agree with previous reports that fish may respond immunologically in a manner similar to mammals following immunotoxicant challenge. (Supported by NIH ES 07612-01 SRC).



Assessment of Some Non-specific Immune Parameters in Atlantic Halibut, Hippoglossus hippoglossus

AL Langston*, RM Hoare, H Wergeland, R Fitzgerald and M Mulcahy

Aquaculture Development Centre, Department of Zoology and Animal Ecology, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland.;;;;

As part of a multidisciplinary investigation into the optimal growth conditions for culture of halibut, effects of temperature on immunocompetence is being assessed. Three populations (Norwegian, Icelandic and Canadian) of juvenile halibut have been reared at 4 temperatures; 8, 12, 15 and 18 oC. A range of immunological parameters (kidney macrophage phagocytosis and respiratory burst activity, serum lysozyme, iron binding capacity and antiproteinase activity) are being assessed and the fish challenged with Vibrio. Results from the disease challenge show clear strain and temperature differences with the Norwegian strain being most resistant and the Icelandic strain being least resistant. Analysis of non-specific immune parameters is continuing but preliminary results also show strain differences (e.g. intracellular superoxide anion production is greatest in the Norwegian strain but lowest in the Icelandic strain).



Investigation of Lactoferrin and Vitamin C as Dietary Immunostimulants in Atlantic Salmon (Salmo salar)

B Lygren (1)*, H Sveier (2), B Hjeltnes (3) and R Waagbø

1 Institute of Nutrition, Directorate of Fisheries, P.O. Box 185, N 5002 Bergen, Norway.;
2 Nor Aqua Innovation A/S, N-4335 Dirdal, Norway.
3 Institute of Marine Research, P.O. Box 1870, N 5024 Bergen, Norway.

Lactoferrin and vitamin C have been shown to modulate immune functions in mammals as well as in fish. In the present work, Atlantic salmon were fed commercial diets with or without supplementation of 140 mg bovine lactoferrin kg-1 feed, and with 150 or 1000 mg ascorbic acid (AA) polyphosphate kg-1 feed, in a 22 factorial design for 19 days. Following the dietary treatment, the experimental fish were challenged with Aeromonas salmonicida ssp. salmonicida or Infectious Salmon Aneamia (ISA) virus. There was a positive effect of dietary vitamin C on the concentration of vitamin C in the liver and the hepatosomatic index was slightly higher in the AA150 groups compared to the AA1000 groups. Lysozyme activity in serum and head kidney, spontaneous and antibody specific serum complement mediated hemolysis as well as phagocytic chemiluminescent responses were all unaffected by the differences in dietary composition. In the challenge experiments, there were no significant differences in cumulative mortality between the groups. Liver iron and head kidney catalase did not differ significantly between the groups. The present data do not support immunomodulatory effects of dietary lactoferrin and vitamin C.



New Expressed Sequence Tags Identify Diversity of cDNA in Leukocytes of Japanese Flounder Infected with Hirame Rhabdovirus

H Masako (1)*, N Erika (1), N Bo-Hye (1), K Takayuki (1), Y Eiichi (2), H Ikuo (1) and A Takashi (1)

1 Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108-8477. Japan.
2 Tottori Prefectural Fisheries Experimental Station, Tomari, Tottori 689-0602, Japan

Partial sequencing of cDNAs to generate expressed sequence tags (ESTs) is a rapid and efficient way to establish a detailed profile of genes expressed in a tissue or cell type. We attempted to analyze some of the genes which are induced and expressed by a virus infection in the immune system. We chose to investigate expression in leukocytes as they are important cells for the specific and nonspecific immune systems. We randomly selected and partially sequenced cDNA clones from a library of Japanese flounder (Paralichthys olivaceus) leukocytes that were infected by the Hirame rhabdovirus (HRV). A total of 197 different clones were sequenced, either from the 5' or 3' end of inserts. These sequences were compared to those registered in the GenBank database, 79 (40.1%) matched to known genes, whereas 118 (59.9%) did not. Unexpectedly, only 10 clones (5%) of the 79 known genes showed similarities with fish genes. The functional classification of putatively identified genes could be grouped into 12 categories, including those involved with biodefense. Ten of the clones (5%) which represented MHC class IIB, MHC class IA and CD45, were identified as putative biodefense genes or as associated with immune response. Our data, therefore, can supply significant information about genome expression of fish leukocytes in response to a Hirame rhabdovirus infection.



Relationship Between Degranulation of Eosinophilic Granular Cells and Exudation of Neutrophils

T Matsuyama and T Iida

Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi, Miyazaki 889-2192, Japan

There are few reports about the physiological role of the eosinophilic granular cell (EGC). We studied the relationship between degranulation of the EGC and number of the exuded neutrophils. Observations were made on stretched preparations from the swim bladder and peritoneum of tilapia, Oreochromis niloticus, after injection of formalin-killed Escherichia coli (1mg/fish) into swim bladder. The tissues were fixed in absolute ethyl alcohol and stained with Giemsa stain. The EGCs in the swim bladder were degranulated rapidly after inoculation, and the ratio of the degranulated EGCs was highest at 1 hour post-injection, while the peritoneum EGCs were not degranulated. From these facts, EGCs degranulate rapidly after inoculation, and degranulation occurs only in the inoculated site. Formalin-killed E.coli (1mg/fish, 0.1mg/fish), proteose peptone or HBSS was injected into the swim bladder respectively. There was a correlation between the ratio of the degranulated EGCs and number of the exuded neutrophils. Killed E.coli (1mg/fish) led to the highest response to degranulation of EGCs and migration of neutrophils into the swim bladder. On the other hand, the rate of the degranulation and number of the neutrophils were lowest when HBSS was injected. When EGC was degranulated in the high proportion, a large number of neutrophils exuded into the swim bladder (inflammatory site). Degranulation of EGCs is responsible for neutrophil migration.



Carp (Cyprinus carpio L) Immune Response to Aeromonas Hydrophilla

BR Mikryakov*

Institute for Biology of Inland Waters, Russian Academy of Sciences, Department of immunology Borok Nekouz, Yaroslavl, 152742, Russia.

Based on the analysis of immune system interaction with bacterial antigen during different stages of immunogenesis, a study was conducted on carp Cyprinus carpio L acquired immunity to Aeromonas hydrophilla. Experiments were carried out on 1-2 year old carps. The fish were kept in aerated aquaria at 18-20C water temperature. Immune system response to A. hydrophilla was investigated by a content of antigen-reacting and antigen-destructing cells; distribution of C14 traced bacteria in immunocompetent tissues and organs; excretion of their decomposition products; differentiation rate of immunocompetent cells transformation; and synthesis of antibodies. Material was collected every 5, 30 and 60 minutes and 1, 2, 3, 5, 10, 20, and 40 days after intraperitoneal introduction of heat-inactivated A.hydrophilla in concentration of 107-108 bacterial bodies. The study shows that acquired immunity in carp is similar to that of warm-blooded animals and is based on the process of recognition; destruction; antigen metabolism; elimination of bacteria decomposition products; increase of antibody-producing cell concentration; and antibody synthesis. It is concluded that predetermined lymphocytes are responsible for bacteria recognition. Immunization stimulates formation of antigen-recognizing immunocytes and functional activity of cell and humoral immunity factors. Using C14 traced A.hydrophilla it is shown that hydrolytic splitting into CO2 and unidentified organic matters is a biochemical base for bacteria destruction in fish. Carbon dioxide is eliminated through gills and the organic matters through excretion and skin. 65-70% of bacteria organic matter stay in the immunocompetent tissues for an indefinitely long time (more than a year). It is suggested that the carp immune system responds to intraperitoneal introduction of A.hydrophilla with modificational variability of immunity cell and humoral factors and is accompanied by an increase of antigen-reacting, antigen-destroying, antibody-synthesising cell concentrations as well as their functional activity.



Sequencing the Inducible Nitric Oxide Synthase (iNOS) Gene in Channel Catfish, Ictalurus punctatus, and Designing Diagnostic Antibodies

CD Rice*(1), X Yuan (1) and CJ Secombes (2)

1 College of Veterinary Medicine, Mississippi State University, P.O. Box 9825, Mississippi State, MS 39762, USA.
2 Department of Zoology, University of Aberdeen, Tillydrone Ave., Aberdeen AB24 2TZ

A number of acute infectious diseases of fish have intense inflammation, blood coagulation, and edema as hallmark features of their presentation. While macrophages and endothelia are linked to these lesions in mammals, the role of these cells in acute inflammatory diseases of fish is unclear. Enteric Septicemia of Catfish (ESC) is a devastating disease in channel catfish aquaculture caused by Edwardsiella ictaluri. We have recently demonstrated that full-sib families of channel catfish resistant to ESC have more and larger splenic macrophage aggregations (MAs) than do sensitive families. Along with a suite of cytokines and peptides, one of the most potent products of activated macrophages is nitric oxide (NO), a by-product of inducible nitric oxide synthase (iNOS). The expression of iNOS by these MAs, and other tissues, may be an early feature of inflammation. The detection of fish iNOS would be possible if iNOS-specific antibodies and molecular probes were available. Using semi-nested RT-PCR, RACE, and by screening a cDNA library developed from lymphoid tissues of ESC-infected catfish, we have obtained two overlapping 500 base sequences showing a high degree of homology with both mammalian and rainbow trout iNOS genes. From these products a molecular probe was developed for northern blotting detection of iNOS expression. We are now developing antibodies to peptide sequences of the gene that, along with suitable molecular probes, are expected to provide a sensitive marker for iNOS in acute infectious diseases of fish.



The 3-Hydroxy-3-Methylbutyrate (HMB) and Fish Immune Responses

AK Siwicki (1)*, S Fuller Jr (2), S Nissen (2), M Morand (3) and P Ostaszewski (4)

1 Department of Fish Pathology and Immunology, IFI, 05-500 Piaseczno, Poland.
2 Metabolic Technologies Inc. 2625 M. Loop Dr Suite 2150, Ames, IA 50010, USA.
3 2 Laboratoire Departemental d'Analyses, Conseil General du Jura, 39016 Lons le Saunier, France
4 Department of Physiology, Warsaw Agricultural University, Poland

HMB-breakdown product of the amino acid leucine, has been shown to counteract many of the negative effects of modern production methods and results in increased growth and protection against diseases in animals. The hypothesis that HMB could be an effective supplement in reducing the effects of stress on the defense mechanisms was first tested in fish. In our in vitro study we analyzed for the first time the influence of different concentrations of HMB on the immunocompetence cells activity in rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio). The results showed that HMB addition increased the polymorphonuclear (PMN) and mononuclear (MN) cell activity, analyzed by phagocytic ability and potential killing activity of PMN and MN cells and lymphocyte proliferation stimulated by mitogens. HMB applicated per os increased the antibody secreting cells analyzed by ELISPOT assay after in vitro immunization of the spleen with vaccine. The in vitro and in vivo studies suggest that HMB activated the immunocompetence cell activity and cell-mediated immune response in rainbow trout and carp.



Dimerized Lysozyme (KLP-602)-influence on Cellular and Humoral Immune Responses Induced by Furogen Vaccine in Rainbow Trout (Oncorhynchus mykiss)

AK Siwicki (1)*, P Klein (2), K Goryczko (3), E Terech-Majewska (4), K Kazun (1) and E Glombski (1)

1 Department of Fish Pathology and Immunology, IFI, 05-500 Piaseczno, Poland.
2 Nika Health Products Limited, Lawrenceville, New Jersey 08648 USA
3 Department of Salmonid Research, IFI Rutki, Poland.
4 Department of Epizootiology, Faculty of Veterinary Medicine, Agricultural University in Olsztyn, 10-957 Olsztyn-Kortowo, Poland.

Immunostimulants and adjuvants have been divided into general groups of function, used to enhance the nonspecific and specific immune response. Adjuvants are usually mixed and applied with antigen preparation, activating the specific immune response. Immunostimulants can be administered before, with, or after vaccines to amplify the nonspecific defense mechanisms and cellular and humoral immune responses. In the present study we determined the influence of dimerized lysozyme (KLP-602) on the cellular and humoral immune response after immunization of rainbow trout with Furogen Immersion vaccine (Aqua Health Ltd, Canada). Dimerized lysozyme (KLP-602) was administered before or after vaccine applicated by immersion. ELISPOT assay were used for the quantification of total and specific ASC. The nonspecific immune response (total Ig level and lysozyme activity) and circulating specific antibody titres were also determined 10, 15, 20, 27, 35 and 60 days after immunization. Dimerized lysozyme (KLP602) applied before and after vaccine increased the cellular and humoral immune response, compared to control.



In vitro Effects of Human Tumor Necrosis Factor-a (TNF-a) Muteins on Macrophage and Lymphocyte Activity in Rainbow Trout (Oncorhynchus mykiss)

AK Siwicki (1)*, M Studnicka (2), B Szymañska (3), A Rymuszka (2) and A Bownik (2)

1 Department of Fish Pathology and Immunology IFI, 05-500 Piaseczno, Poland
2 Department of Bioorganic Chemistry, Centre of Molecular nad Macromolecular Studies, Polish Academy of Sciences, 90-363 £ódz, Poland
3 Department of Biology and Toxicology, Catholic University, 20-35 Lublin, Poland

TNF-a belongs to a class of hormone-like molecules termed cytokines. These polypeptyde mediators form a complex network of interactive signals that regulate their own production, and the growth, differentiation, or function of many cell types, principally cells of the hematopoietic and immune systems. Experimental studies have shown that rainbow trout lymphocytes and macrophages can respond to recombinant human TNF-a. In our study we used four TNF-a derivatives termed "muteins" which were produced using synthetic cDNA expressed in Escherichia coli. The effects of native TNF-a muteins (III, V, VI, VII) on the pronephric macrophage and lymphocyte activity in rainbow trout were studied. The results showed that native TNF-a and muteins activated the respiratory burst activity (RBA) and potential killing activity (PKA) of macrophages. The lymphocyte proliferation analysis by MTT assay indicated a similar pattern. The results showed that TNF-a muteins activated lymphocyte T and B proliferation stimulated by mitogens, and the highest immunomodulating influences were observed with muteins III and V as compared to human native TNF-a.



Viral Antigen-specific in vitro Proliferation of Leucocytes from Clonal ginbuna crucian Carp

T Somamoto(1)*, S Hasegawa(1), T Nakanishi(2) and N Okamoto(1)

1 Tokyo Univercity of Fisheres, Department of Acquatic Biosciences, Department of Acquatic Biosciences, Konan 4, Minato-ku,Tokyo 108, Japan.
2 Natinal Research Institute of Aquaculture, Tamaki, Mie 519-04, Japan.

Viral antigen-specific in vitro proliferation of leukocytes from clonal ginbuna crucian carp was induced as a result of intravenous immunization with the virus-infected syngeneic cell line. We have used EVA (eel rhabdovirus. Sano 1976) and IPNV-sp as antigens, and ginbuna crucian carp (S3N clone) as an experimental fish. Leukocytes from ginbuna crucian carp primed with the EVA- or IPNV-sp-infected syngeneic cell line were capable of in vitro proliferation when cultured in the precence of homologous stimulating cells (virus-pulsed syngeneic lymphoid cells). Most leukocytes stimulated by homologous antigen and cultured for 8 days were morphologically similar to the blast cells appearing in ConA- or PHA-stimulated ginbuna leucocyte. The present study could be applied to research on viral-antigen specific immunity using clonal fish and the syngeneic cell line.



Turbot (Scophthalmus maximus) Eicosanoids in Leucocyte-derived Supernatants Inhibit Macrophage and Lymphocyte Functions

C Tafalla, B Novoa, MC Ordás* and A Figueras

Instituto de Investigaciones Marinas. CSIC. Eduardo Cabello, 6. 36208 Vigo, Spain.

The effects of the addition of leukocytes or leukocyte supernatants on the turbot macrophage respiratory burst were determined. When macrophages and leukocytes were coincubated, the highest suppressive effect on the respiratory burst was found after 24 h. The addition of turbot cultured leukocyte's supernatants (cells were incubated for 17 h with culture media and foetal calf serum), to macrophage monolayers from the same or different animals resulted, in both cases, in the inhibition of the macrophage respiratory burst. Leukocyte supernatants also suppressed PHA-induced proliferation of blood lymphocytes. Inhibitors of the two main pathways of eicosanoid biosynthesis, cycloxygenase and lipoxygenase pathways, were also used to obtain supernatants. Although both resulting supernatants restored the macrophage respiratory burst activity, only products derived from the cycloxygenase seem to be implicated on the suppressor effect on macrophage respiratory burst. Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, was able to restore the normal levels of lymphocyte proliferation (1:2 dilution), suggesting that eicosanoids derived from lipoxygenase can be related to the inhibition of the PHA-induced proliferation. These findings could help to understand the modulation of fish immune response under culture conditions.



Differences Between Plasma and Serum Chemistry Values in Four Species of Fish: Trout, Catfish, Tilapia, and Hybrid Striped Bass

TC Hrubec and SA Smith*

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg VA 24061 USA.

Blood chemistry analytes are often determined for fish using either serum or plasma samples. Theoretically, plasma and serum are similar except for the clotting factors present in plasma. This study was conducted to determine if there were any notable differences between blood analytes when measured from plasma or serum. Ten individuals from four species, channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), tilapia (Oreochromis hybrid) and hybrid striped bass (Morone saxatilis X Morone chrysops) were used in this study. A single blood sample was collected from each fish and was divided between a serum blood tube and a heparinized blood tube. The serum tube clotted at room temperature for 1 hour and was then centrifuged to collect the serum. Plasma tubes were centrifuged immediately and the plasma removed. The blood was analyzed for the following standard biochemical analytes: total protein, albumin, globulin, creatinine, total bilirubin, ALP, AST, sodium, potassium, chloride, calcium, magnesium, phosphorus, glucose and cholesterol. The following clinically significant changes were observed. Serum values for potassium were lower while magnesium and phosphorus values were higher compared to the plasma in all four species. Serum glucose was lower for trout, hybrid striped bass, and catfish; although the change was only slight in catfish. Cholesterol was elevated in the serum of all species except hybrid striped bass. The changes observed between serum and plasma most likely represent sample degradation and biochemical changes in analyte levels during the clotting process of sample preparation. Therefore, serum should not be used for clinical analysis as plasma more accurately reflects true blood concentrations.



Cationic Peptides Protect Fish from Infectious Disease

X Jia (1)*, A Patrzykat (1), RH Devlin (2), PA Ackerman (3), G Iwama (3) and REW Hancock (1)

1 Department of Microbiology and Immunology, University of British Columbia, B.C. Canada V6T 1Z3
2 Fisheries and Oceans Canada, 4160 Marine Drive, West Vancouver, B.C. Canada V7V 1N6
3 Department of Animal Science, University of British Columbia, B.C. Canada V6T 2A2

Fish loss from disease is a significant problem in aquaculture worldwide. However, expression of natural cationic peptide genes in fish could increase disease resistance because of continual cellular production of the antimicrobial peptides. In order to choose optimal peptides for transgenic fish, the antimicrobial activities of some cationic peptides were determined by testing minimum inhibitory concentrations (MICs) in vitro. Although several cationic peptides have been shown to have antimicrobial activities, the most effective cationic peptides tested were CEME, a cecropin/mellitin hybrid peptide, and pleurocidin-CN, a C-terminal amidated form of the flounder fish peptide. The in vivo effect of CEME was examined by intraperitoneal injection of the peptide along with V. anguillarum into juvenile coho salmon. Fish in the bacteria alone control group had 60% mortality, while fish receiving peptide and bacteria injections had 82% mortality. Apparently, a single injection of CEME did not protect fish from the bacteria infection. Therefore, constant delivery of CEME and Pleurocidin-CN using intraperitoneal mini-osmotic pumps were carried out. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum. The CEME and pleurocidin-CN pump group had longer survival time and lower mortalities than the control groups (50 % vs. 13%, and 75% vs. 5%, respectively). Indolicidin transgenic cutthroat trout were made using a construct including a MT promoter, growth hormone signal, pre-region, indolicidin, and terminator. Transgenic individuals were identified by serum analysis using PCR with construct-specific oligonucleotide primers. The development of disease resistant transgenic fish will greatly contribute to fish aquaculture.



Novel Gene Expression in Neogenic Goldfish Kidney

M Liu (1)*, B Hassel (1) and R Reimschuessel (2)

1 Greenebaum Cancer Center, University of Maryland at Baltimore, Howard Hall room 348, 660 West Redwood Street Baltimore, MD 21201.,
2 Aquatic Pathobiology Center, University of Maryland, Baltimore, MD 21201

Molecular techniques are increasingly being used in fisheries science. The technique of differential display is used to examine the expression of RNA in several different groups. We have recently used differential display to which genes expressed in goldfish kidneys undergoing nephron neogenesis following toxicant- induced injury. We have found a PCR product, C40.1, which is expressed in the neogenic kidney 3-7 days following injury, but is not expressed or in neogenic kidneys 14 days after injury or in control kidneys. The product was sequenced. Searches in GENBANK were negative for known genes with this sequence. Additional studies are being conducted to determine if this gene is present in mammals and if it is a renal development gene.



The First Century of Fish Health in the United States (1797 to 1897)

AJ Mitchell*

Stuttgart National Aquaculture Research Center, Department of Agriculture, Agricultural Research Service, P. O. Box 860, Stuttgart, Arkansas 72160 USA.

Starting with the 1797 Benjamin Henry Latrobe description of a crustacean from the mouth of an alewife (Alosa psuedoharengus), caught in the York River in Virginia, 78 researchers and fisheries workers published more than 180 fish health papers on parasites, fungi, feral fish kills, hatchery diseases, diagnostic efforts, disease treatments, and stressors affecting fish health through 1897. During this period two researchers, Joseph Leidy, a physician and naturalist working with the Philadelphia Academy of Natural History and Edwin Linton, a zoologist with the Washington and Jefferson College, Washington, PA, collectively wrote about one-forth (45) of the fish health papers published in the 100 year period. Prior to 1870 most of the interest was focused on parasites infesting fish, often with little concern about the host species. Following 1870 interest in the health of the host species was given considerable attention by early fish culturists, most notably Livingston Stone (U. S. Fish Commission) and the first effort (1884) in diagnostic fish pathology (including histopatholology and bacteriology) was initiated by Stephen Alfred Forbes, a biologist from Champaign, Illinois.



A Study on Particle Uptake Mechanism in the Skin of Rainbow Trout During Bath Immersion

H Wakabayashi* and I Kiryu

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan 113-8657.;

The skin is the main site responsible for uptake of the environmental particles including particulate antigens, but little is known about the process in the uptake and clearance of inert particles attached to the skin. Fluorescent latex beads, 1 micrometer in diameter, were administered to 3-10 g rainbow trout by bath immersion, followed by time series sampling and histological examination. Examination of the skin surface under a dissection microscope revealed that the beads existed in patches not of the same density throughout. When the live fish exposed to the beads for 5 min was dipped in 0.05% trypan blue for 10 min before sampling, the patches of beads were observed at the skin area stained with trypan blue. In the skin examined histologically, there were beads adhering to the superficial epithelial cells at the edge of wounds. During healing from wounds, the damaged cells and the beads sloughed together from the surface. However, the migrating epithelial cells which would repair the wounds displayed intensive phagocytosis of the beads at the edge of the wounds. The beads were also found in the intracellular gap in the dermis sealed with the migrating epithelium. Thus, the superficial wound areas in the skin are implicated as sites of particle uptake during bath immersion.



Fathead Minnow Histological Atlas: Worldwide Web Outreach and Utilization

LT Yonkos (1)*, AS Kane (2) and R Reimschuessel (2)

1 University of Maryland, Wye Research and Education Center, P.O.Box 169, Queenstown, Maryland 21658, USA.
2 Aquatic Pathobiology Center, University of Maryland School of Medicine, Department of Pathology, 10 South Pine Street, Baltimore, Maryland 21201 USA.

The fathead minnow (Pimephales promelas), the "white rat" of freshwater aquatic toxicological studies, is used in USEPA test protocols for examining acute, shortterm chronic, early lifestage, and lifecycle toxicity. Additionally, a substantial database cataloguing fathead minnow sensitivity to toxicants is available both in the printed literature and online (AQUIRE, ECOTOX, etc.). Regulatory programs that monitor effluent discharges routinely perform bioassay tests with larval fathead minnows. Test endpoints include survival, growth and reproduction. Recent attention has been directed toward development of sensitive sublethal indicators of contaminant-induced stress in aquatic species. Histopathological identification and quantification of health effects and contaminant-induced lesions are used as such indicators. Identification of tissue lesions requires a baseline appreciation of normal tissue conditions. While histological atlases exist for several commercially valuable species (i.e., striped bass, salmonids, and catfish), such a resource has been unavailable for cyprinids, the dominant family of freshwater fish (with more than 2,000 species worldwide including approximately 300 in North America). For this reason we have generated an atlas of photomicrographs detailing normal microanatomy of the fathead minnow. Currently, the atlas is available as a work in progress on the worldwide web (WWW) ( Development of the atlas was accomplished through routine histological preparation of representative tissue samples. Photomicrographs of tissue samples were taken at various magnifications and digitized for use in the atlas. Digital images were compiled by organ system into separate sections. Sections are navigable by index and images are retrievable via "hotbuttons" and hypertext. A WWW version of the atlas will be available for hands-on demonstration. Methods of development and outreach capabilities will be reviewed.



Fatty Acid Composition of Fish Diet Influences the Fertilization Rate of Eggs of Zebrafish, Danio rerio

T Meinelt (1)*, C Schulz (2), M Wirth (1) and H Kurzinger (3)

1 Institute of Freshwater Ecology and Inland Fisheries, Department of Fish Culture and Fish Pathology, Mueggelseedamm 310, 12587 Berlin, Germany.
2 Humboldt-University at Berlin, Invalidenstrasse 42, 10115 Berlin, Germany
3 Tetra Werke, Herrenteich 78, Postfach 1580, 49304 Melle, Germany

Investigations reported in the literature show that the fatty acid (FA) composition of a fish's diet affects the FA content of internal organs, including testes and ovary, and hence the quality of eggs in the F1 generation. It is suggested, therefore, that FA composition of a fish's diet may also affect the fertilization rate of eggs. If so, this would have repercussions both for the production of the numerous fish eggs routinely used in subchronic toxicity testing, and the accurate interpretation of results (FA content of fish diet is not routinely determined in such tests). We therefore conducted a series of experiments using Zebrafish (Danio rerio), a warmwater cyprinid species increasingly used in laboratory toxicity tests. Over a period of 8 weeks parental fish were fed with diets with 5 different quotients (1.71- 0.76) of n-3/n-6 polyunsaturated fatty acids (PUFA). The fertilization rate was determined the following day by the number of developing eggs in relation to those which had failed to develop (number of developing eggs/total number of eggs). The fertilization rate of each diet group was determined and recorded daily. In the final week, the eggs of each diet group were sampled, and the FA composition in the triglyceride fraction in the testes and the eggs of each diet group was analysed. The quotient of n-3/n-6 PUFA in the diet of the fish determined the FA composition (quotient of n-3/n-6 PUFA) of the testes and eggs. Furthermore, the varying quotients of n-3/n-6 PUFA in the diets, and thus in the testes and eggs, were reflected in differences in the fertilization rate of the eggs. Low quotients of n-3/n-6 PUFA resulted in the highest fertilization rates, and demonstrated a high demand for n-6 PUFA in this fish species. Thus, in the case of Zebrafish, adequate production of eggs for toxicity testing, and correct interpretation of experimental results, can be influenced by FA composition of the diet of the parental fish.



Tilapia Fed Good Quality Protein Guarantees Growth Enhancement Through Effective Intestine Absorption

AO Meyer-Willerer (1)*and A Hernández-Hernández (2)

1  1 Centro Universitario de Investigaciones Oceanologicas, Universidad de Colima, Departamento de Acuacultura, km 20 Carretera Manzanillo - Barra de Navidad, Manzanillo, Colima 28860 MEXICO
2  2 Facultad de Ciencias Marinas, Universidad de Colima, Departamento de Acuacultura, km 20 Carretera Manzanillo - Barra de Navidad, Manzanillo, Colima 28860 MEXICO

Amino acids in plasma come from protein digestion and part of the non-essentials show no direct correlation between the serum concentrations and that of the diet. Feed protein is the only source of essential amino acids and the limiting of one of them will regulate protein synthesis, and the excess of all others will be catabolized. Absorption mostly occurs in the stomach and the foregut and least in the hindgut. Digestibility of various isoproteic and isocaloric diets for tilapia "Stirling" fry and juveniles was studied by measuring the perfusion of amino acids through stomach and intestine. The tilapias were fed diets prepared with white or brown fishmeal of third class marine fish and compared with a commercial first class feed. The perfusion of amino acids was obtained from tilapia exposed viscera. The fish was immobilized with a strike on the neck, opened from the genital pore through the pectoral fin and the still attached stomach was placed in separate isotonic solution, the first half of the intestine in another separated isotonic solution, and in a third one the last half of the intestine that continued to be attached to the breathing fish. The isotonic solutions placed in a tempered chamber at 30°C were collected and renewed each 30 min until completing 90 min, centrifuged and colored with ninhydrin reagent or analyzed for free amino acids with chromatographic techniques. The detected free amino acids at different times for each tested diet, and the respective growth curves will be discussed.



Effect of Vitamin A Supplementation on the Occurrence of Bacterial and Parasitic Diseases and Mortality in Reared Young Atlantic Salmon (Salmo salar)

P Rintamäki-Kinnunen (1)*, M Pasternack (2) and P Koivunen (1)

1  1 Department of Biology, University of Oulu, PO Box 333, FIN-90571 Oulu, Finland
2  2 Finnish Game and Fisheries Research Institute, PO Box 6, FIN-00721 Helsinki, Finland

Enhancement of disease resistance by nutritional factors would have great advantages in fish farming, as it would mean a decrease in the use of antibiotics and chemicals. Dietary modifications could have applications particularly with young fish, which are too small for successful vaccination. Young Atlantic salmon were fed a commercial diet supplemented with retinolacetate, corresponding to 10,000 IU of vitamin A/kg, during two 14 day feeding periods, the first in September, four months after hatching, and the second in late May. After the second feeding period, general resistance to diseases was assessed by monitoring parasitic and bacterial diseases weekly between June and September in 133 fish fed a normal diet and 84 fish that received the additional vitamin A. Dead fish were collected daily. Mean daily mortality was 9.1 fish in the control tanks and 6.5 in the "vitamin A" tanks. Furunculosis caused by Aeromonas salmonicida was diagnosed more often in the control fish (prevalence 16.2% vs 1.2%, p<0.001), and a similar observation was made regarding the occurrence of the flagellate Ichthyobodo necator, which causes costiasis (21.5% in the control fish and 8.3% in the vitamin A fish, p<0.05). None of the other 9 protozoan parasites found had a higher prevalence in the vitamin A fish. Likewise, dorsal fin and tail erosion, often containing Flavobacterium spp., was found predominantly in the control fish (56.4% vs 28.6%, p<0.001). The results suggest that vitamin A supplementation enhances disease resistance in salmon.



Infection Intensity of Perkinsus atlanticus Disease in Clams Ruditapes decussatus Challenged from Different Doses of the Parasite

M Almeida (1,2)*, MT Dinis (1) and F Berthe (2)

1 Universidade do Algarve, UCTRA, Campus de Gambelas, 8000 Faro, Portugal.;
2 IFREMER, Laboratoire de Génétique et Pathologie, BP 133, 17390 La Tremblade, France

Perkinsus atlanticus is a protozoan parasite that has caused significant mortalities in clam culture beds along the south coast of Portugal. The disease caused by this agent is infectious and can be transmitted directly from one clam to another. The stage of zoospore (biflagellate free cell) is, probably, the most effective and the principal stage for the dissemination of the disease. Our experiments were conducted to test the response of clams to different doses of zoospores of P. atlanticus. These infective cells were obtained after isolation, purification and culture of trophozoites from heavily infected clams. Clams were inoculated by injection into the shell cavity with 0, 1x105, 1x106, 2x106 and 3x106 zoospores per clam and maintained at 22±2°C and 31±1 for 2 months. Infection intensity was measured by whole clam culture method in fluid thioglycolate medium. A significant direct positive relation was observed between infective cell dose and both prevalence (r2=0.95) and infection intensity (r2=0.99). The number of P. atlanticus cells required to determine mortalitity in clams was not verified, because there was no relevant mortality during these experiments.



Myxosporidia from Fish of Peter the Great Bay (Japan Sea)

NL Asseyeva*

Pacific Fisheries Research Centre (TINRO), 4 Shevchenko Alley, Vladivostok 690600 Russia

Myxosporidia parasites were studied from the fish caught by commercial trawl vessels in shallow (depth < 40 m) parts of Peter the Great Bay (north-western Japan Sea) in the period from May of 1987 to October of 1990. More than 500 specimens of fish from 32 species were investigated. 8 new species of parasites of genera Ceratomyxa, Leptoteca, Palliatus, and Kudoa were described from Pleuronectidae and Hexagrammidae fishes. Few parasites species were found in Peter the Great Bay for the first time: Ceratomyxa auerbachi (from Hippoglossoides elassodon and), Zchokkella hildae (from Theragra chalkogramma), Ortolinea orientalis from (Clupea harengus pallasi), Pervicapsula unicornis (from 4 species of Pleuronectidae), and Myxosoma acutum (from Mugil so-ing). The last one (parasite of freshwater fish) had unusual localization on scales. New hosts were determined for Parvicapsula unicornis (flatfish Hippoglossoides elassodon and Limanda yokohamae), for Ceratomyxa auerbachi (these two flatfish species and Myxocephalus jaok), for Myxidium japonicum (Gymnocanthus pistilliger), and for Myxosoma acutum (Mugil so-ing). Thus, 33 species of Myxosporidia of 10 genera, of 8 families are known now in Peter the Great Bay. But the list of Myxosporidia species is supposed to be wider - up to 200 species (Dogel, 1948). Fauna of Myxosporidia in Peter the Great Bay is very diverse and has both warm-water and boreal species. The majority of the species are endemics of the Japan Sea, only few species are identical with well-known species of other seas.



The Amur Catfish Parasitofauna of the Lake Khanka Basin

TY Boutorina (1)*and AV Ermolenko (2)

1 The Far Eastern State Technical Fisheries University, Lugovaya str., 52-b, 690600 GSP, Vladivostok, Russia
2 The institute os Biology and Pedology, The Far Eastern Science Branch of Russian Sciences Academy, 690022 Vladivostok, Russia,

Amur catfish is a typical inhabitant of stagnant and slow-running reservoirs of China, Japan, Korea and southern Far East of Russia. It is fed mainly on fish, but its diet may also include various other animals. The parasitofauna of this fish in different parts of the area directly reflects its way of life. There are more than 50 species of parasites in the basin of Khanka. The dominants are Monogeneans of genus Silurodiscoides. The infection of catfish by protozoans is very high: by trichodinids 100% and by myxosporeans 90%. The reason of considerable infection of Amur catfish by parasites with direct cycle of development is that it lives most of its life in the lake itself and not in the flowing streams. Catfish get infected by most of biohelminthes while eating fish or invertebrate. The first way is usual for large fish only, while for 2-3 years catfishes both ways are equally probable. Some peculiarities of parasitofauna of Khanka population of Amur catfish are: great number of species with direct cycle of development including infusorians Ichthyophthirius multifilliis (distinctive peculiarity of Khanka fish caused by anthropogenetic factor), absence of acanthocephalans, small population of Ergasilids (catfish live in the overgrown parts of reservoir). 53.6% of parasites belong to Indian plain complex (specific species for catfish), 14.3% - to Chinese plain and 28.6% - to boreal plain Such heterogenesis is typical for Far Eastern fish. The difference of Amur catfish parasitofauna of Amur basin and South Primorje result from local distribution of hydrobionts and is partly explained by historical reasons.



Losses in Cultured Turbot (Psetta maxima) Due to Myxosporean Infestation

EJ Branson (1)* and A Riaza (2)

1 Red House Farm, Llanvihangel, Monmouth, Gwent NP5 4HL, UK
2 Prodemar SA, Lira, 15292 Carnota, La CoruÀ9 Àa Spain

In the summer of 1996, and again in 1997, losses were experienced in a Spanish on-growing turbot farm. In the latter outbreak fish from 2 months to 2 years old were affected. In affected tanks, morbidity was almost 100%. Clinically affected fish were anorexic. Typically, eyes were sunken and a prominent bony ridge was seen on the skull. These symptoms, with the gut changes described below, have become almost pathognomonic of this disease. Internally there was ascites and pallor of the internal organs, the gut fluid was filled. The most obvious post-mortem finding was haemorrhage of the colon, although the entire gut could be involved. Histological examination of H&E stained tissue sections showed severe enteritis in the haemorrhagic areas of gut. Foreign cells, typical of myxosporean trophozoites, were present in the epithelium of these areas, and free within the gut lumen. Although initial infestation of the fish was probably due to water borne actinospores, development of the disease suggests the possibility of direct transmission from fish to fish. This being the case, it seems likely that the parasite is completing its life cycle within the turbot, so this species may be the definitive host. Wild turbot are found off the coast in this area. Similar pathology has been seen in gilt-head sea bream (Sparus aurata) in the Mediterranean with a Myxidium sp infestation, and on-going work suggests that the same parasite may be involved here.



Peripheral Blood Cytology of Rainbow Trout Fingerlings Exposed to Myxobolus cerebralis Spores by Parenteral Injection

CL Densmore (1)*, VS Blazer (2) and TB Waldrop (3)

National Fish Health Research Laboratory, USGS/BRD, 1700 Leetown Road, Kearneysville, WV 25430 USA

Whirling disease, caused by the myxosporidean parasite Myxobolus cerebralis, affects many species of salmonid fish. Variation in host susceptibility occurs with a number of factors such as species and age, but these relationships have not been elucidated in terms of the immune response of the host. To date, very little is known of the salmonid immune response to M. cerebralis in its different life stages. This experiment examined the effect of parenteral injection of M. cerebralis spores in fingerling rainbow trout (Oncorhynchus mykiss) on the peripheral leukocyte profile. Fingerling trout were inoculated twice with a suspension of purified M. cerebralis spores. Ten days following the second inoculation, peripheral blood samples were obtained, and total leukocyte counts with differential analyses were determined. Significantly higher numbers of circulating large lymphocytes were apparent for the fish inoculated with the spore suspension compared to control fish injected with phosphate buffered saline. No differences in total peripheral leukocyte counts or differential counts for any other type of white blood cell were noted. The elevation in numbers of large lymphocytes in circulatiom potentially represents an immunological response of the host to the M. cerebralis spore through lymphocyte recruitment and/or activation. Similar studies using other salmonid species with different levels of whirling disease susceptibility are planned.



Infections of Cryptocaryon irritans (Ciliophora) on Wild Fish from South-East Queensland, Australia

BK Diggles (1,2)* and RJG Lester (1)

1 Department of Parasitology, The University of Queensland, Brisbane, QLD 4072 Australia
2 Present address: National Institute of Water and Atmospheric Research, PO Box 14-901 Kilburnie, Wellington, New Zealand.

Wild-caught marine fish from three sites in S-E Queensland, Australia, were examined over a period of 20 months for infections of the parasitic ciliate Cryptocaryon irritans. Infections of C. irritans were found to be common on the fish sampled. Out of 397 fish (18 species), 252 (63.5%) from 15 species were infected. At site 1 at the mouth of an estuary, the prevalence of C. irritans infections was 73% and the mean intensity was 12.6 parasites / fish. At site 2, a coastal bar area, the prevalence of infection was 6% at a mean intensity of 4.8 parasites / fish, whilst at site 3 on the Great Barrier Reef, prevalence was lowest at 51% with a mean intensity of 2.3 parasites / fish. There was no apparent seasonality in prevalence or intensity of infections at sites 1 and 2 despite water temperatures ranging between 15 and 27ºC. The diameter of the tomonts varied inversely with water temperature, and was not related to host size but varied between host species. Our data indicate that C. irritans is common on wild fish, and not rare as previously thought. This may be partially due to the increased sensitivity of our tomont collection technique. Our data also suggest that C. irritans may exhibit a degree of host specificity in the wild, and that its natural distribution can be extended into estuaries and seasonally into warm temperate waters.



Salinity Responses of Red Drum Infected with Amyloodinium ocellatum

CE Harris, WH Neill and NO Dronen, Jr

Department of Wildlife and Fisheries, Texas A&M University, College Station, TX 77843-2258, 409/862-1414.

The ectoparasitic dinoflagellate Amyloodinium ocellatum is a problem of long standing in marine aquaria and recently has emerged also as a cause of mortality in marine foodfish-production facilities. The parasitic stage (trophont) attaches to the gills, skin and fins of its hosts (primarily marine teleosts). Amyloodinium ocellatum has a wide range of tolerance to salinity, but freshwater exposure is considered an effective therapy for infected fish. Red drum (Sciaenops ocellatus) is cultured intensively as a food fish in Texas and other Gulf states. Although this species is marine, it can survive in water with less than 1 ppt dissolved solids, providing sufficient hardness and chlorinity are present. Bioassays were conducted to determine the lengths of time infected red drum need to spend in freshwater to result in various degrees of parasite detachment from the gills. Preliminary results show that in infections of one day, a five-minute dip results in 80% trophont detachment; whereas, in a four-day infection, a ten-minute dip is needed to achieve the same degree of control. Additionally, a study is in progress to determine if infected red drum will preferentially select a freshwater microhabitat in order to rid themselves of the parasite. It is hypothesized that infected red drum will voluntarily expose themselves to a level of salinity much lower than normally optimal, if available--in effect giving themselves a freshwater dip.



Trematode Infections in Pomfrets of Visakhapatnam Coast, India

U Sai Jyoti and GG Mani

Department of Zoology, Andhra University, Visakhapatnam, A.P. 530003, India

Pomfrets support a lucrative and commercially important fishery both along east and west coasts of India. An investigation of trematode infections of these fish namely, Pampus argenteus, P. chinensis and Apolectis niger from Visakhapatnam Coast, Bay of Bengalwas therefore was undertaken from August 1994 to July 1996. Differences in the parasite fauna of three species of the host with respect to the species composition, diversity, prevalence as well as the abundance of individual parasite species were recorded. Altogther, nine species of parasites including two species of monogeneans and seven species of digeneans spread over six families were encountered. The study showed that the mongenan Bicotyle vellavoli is specific to Pampus argenteus. The digenetic trematodes were found to be generalists in nature.



Ecological Aspects of Trematode Infections in Carangid Fishes of Visakhapatnam Coast, India

U Sai Jyoti and GG Mani

Department of Zoology, Andhra University, Visakhapatnam, A.P. 530003, India

An investigation into ectoparasitic and endoparasitic trematode infections of twelve species of carangids collected from Visakhapatnam Coast, Bay of Bengal was undertaken. Altogether, 1069 fish were examined as monthly samples were taken during August 1994 to July 1996. Twelve species of monogeneans belonging to eight families, and twelve species of digeneans spread over seven families were encountered. Data on seasonal changes in the parasite prevalence and mean intensity of infection in all the carangids examined and two common species of carangids namely, Carangoides malabaricus and Caranx ignoblis were recorded. Similarly, data on community parameters such as mean number of parasites, parasite species per host, and diversity values were calculated. The study demonstrated that the parasite fauna of carangids is quite rich in the light of the feeding habits of the fish, local abundance and vigility.



Correlation of Body Form and Thorax Appendages of the Fish Parasitic Copepods (Crustacea: Copepoda)

VN Kazachenko*

The Far Eastern State Technical Fisheries University, Lugovaya str., 52-b, 690600 GSP, Vladivostok, Russia

We know nine body forms of the fish parasitic copepods (Kazachenko, 1989, 1991, 1994, 1996). Fewer changes are observed in natatorial appendages of copepods with cyclopoid, caligoid and eudactylinoid body forms; the swimming appendages are biramous, segmentation is as a rule well pronounced, and there are chitin thorns which take part in fixation. In copepods with lernanthropoid body forms, the first and the second pair of legs are heavily reduced, the second pair is sometimes absent, the third and the fourth are of the leaf type and the fifth is uniramous, small or absent. Copepods with sphyrioid body form have reduced legs in the first up to fourth pairs, or the legs are absent. Mature copepods with lernanthropoid body form have no thorax appendages and only several species have appendages that are distinguished under electronic scanning microscope. Copepods of chondracanthoid body form may have all pairs of appendages or none. Copepods of philichtyoid and nematodoid body form have heavily reduced appendages. The changes in thorax appendages due to the copepods transfer to a parasitic mode of life go in the following sequence: chitin thorns appear which take part in fixation; segmentation is lost; one of the branches is reduced; appendages disappear. Reduction of appendages depends on the ecology and evolution of parasites.



Lectin-Reactive Components of the Microsporidian, Glugea plecoglossi and Their Relation to Spore Phagocytosis by the Head Kidney Macrophages of Ayu, Plecoglossus altivelis

JH Kim*, K Ogawa and H Wakabayashi

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.

We have investigated the lectin reactivity with the spores of the microsporidian, Glugea plecoglossi of ayu, Plecoglossus altivelis. Smear preparations of purified spores were treated with 8 kinds of lectins. Lectin blots were conducted for the detection of glycoproteins of the spore lysates. In addition, lectin-treated spores were applied to the head kidney macrophages of ayu, percentage of phagocytosis (PP) was calculated and compared with the control. Two kinds of lectins (ConA, WGA) reacted with the surface of the spores, and a major band (55kD) and some minor bands were visualized on blots after treatment with the two lectins. PP was decreased after ConA treatment. From these results, it is suggested that lectin-mediated recognization of host cells may be a critical step for G. plecoglossi to establish infection. Ultrastructural studies must be undertaken to clarify the localization of glycoproteins in the spore.



Ecology of Diplostomum pusillum - the Pathogene of the Fishes diplostomosis in Kazakhstan

LI Kokhno, D Zhatkanbayeva and LM Pinayeva

Institute of Zoology and Genofond of Animals, Ministry of Science-Academy of Sciences of Republic of Kazakhstan, 480060 Almaty, Kazakhstan.

The trematoda D. pusillum is widely spread in North America, Europe, and Asia. The definitive hosts of this parasite are birds (Mergus merganser, M.albellus, Larus ichthyaetus, L.argentatus, L.ridibundus) and mammals (Ondatra zibethicus). The fresh-water mollusc Lymnaea pereger is widespread in water-bodies of Kazakhstan and is it's first intermediate host. Metacercaria D.pusillum affect the bottom of the fish's eye in Nemachilus dorsalis, N.strauchi, N.labiatus (Cobitidae) and Pseudogobio revularis (Cyprinidae). The disease caused by this pathogen was registered in plain and piedmont ponds of the Balkhash-Alakol basin. In the intensive diplostomosis nidi N.dorsalis is the most infected by this parasite (up to 100%) on invasion intensity 1200-1600 specimens of metacercaria per one fish. The pathogen of the acute form of the diplostomosis D.pusillum is dangerous for the young of the fishes (Cyprinus carpio, Hypophthalmichthys molitrix, Aristichthys nobilis, Ictalurus punctatus) cultured in ponds of the fish farms situated not far from the natural nidi of disease.



Mass Mortalities of Cockles, Cerastoderma edule, in Galicia (NW Spain) Associated with a Disseminated Neoplasia and an Unidentified Protistan Parasite

C López (1)*, A Villalba (1), MJ Carballal (1), L Corral (2) and C Azevedo (2)

1 Centro de Investigacións Mariñas, Aptdo. 13, E-36620 Vilanova de Arousa, Spain.
2 Departamento de Biología Celular, Instituto de Ciências Biomédicas de Abel Salazar, P-4050, Porto, Portugal

A histopathological survey was performed to search for the cause of cockle, Cerastoderma edule, mass mortality in the Ría do Barqueiro (Galicia, NW Spain). The first sampling (April, 1997) disclosed high prevalence (75% of an unidentified protistan parasite, termed cockle parasite X (CPX). A second sampling (June 1997) at the same place disclosed a prevalence of 80% for CPX and the occurrence of a disseminated neoplasia (60% of prevalence). In July 1997 samples were taken from Vicedo harbour (Ría do Barqueiro), a cockle bed with unnoticeable mortality, and from the two flanking rías, Ortigueira and Viveiro, affected by cockle high mortality. In Vicedo harbour, CPX was not found and the prevalence of disseminated neoplasia was 4%. Prevalences of CPX and the disseminated neoplasia were high in Rías de Ortigueira and Viveiro. CPX was mostly located inside haemocytes and gave rise to large granulocytomas. Disseminated neoplasia probably had haemic origin and was similar to that described in cockles from Ireland and Brittany (France). Advanced cases of both pathological conditions were associated with extensive tissue destruction. Because of the severity of the lesions and their high prevalence, both pathological conditions could be the cause of the mass mortalities. CPX and neoplastic cells were studied with TEM and their ultrastructure is described.



Evidence for the Presence of Protease Inhibitors in the Eastern (Crassostrea virginica), and the Pacific (Crassostrea gigas) Oyster

A MacIntyre(1)*, S Kaattari(2) and M Faisal(3).

Department of Environmental Science, Virginia Institute of Marine Science, School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062

The plasma of eastern oysters (Crassostrea virginica), and Pacific oysters (C. gigas) were compared for levels of inhibitory activity against a variety of proteases. Representa-tives of the serine, cysteine, metallo and aspartic protease mechanistic classes were analyzed, including extracellular proteases produced by two oyster associated pathogens; Perkinsus marinus and Vibrio vulnificus. In comparison to C. virginica, C. gigas plasma exhibited significantly higher specific inhibition levels (ng protease inhibited/µg plasma protein) for papain (P<0.001), pepsin (P<0.001), P. marinus protease (P<0.001), trypsin (P=0.015), and V. vulnificus protease (P<0.001). Plasma of C. gigas did not inhibit the metalloprotease thermolysin. Instead, a significant increase in substrate hydrolysis was seen in this case. A similar trend was noted for thermolysin with C. virginica. Our recent discovery of metalloprotease(s) present in the plasma of both C. virginica and C. gigas may explain this phenomenon. The levels of P. marinus protease inhibition, present in the plasma of C. virginica and C. gigas, were also examined during an 8-week infection study with an isolate of P. marinus, P-1. Plasma of C. gigas was found to inhibit P. marinus protease during weeks 1 and 6 of the study, while weeks 2, 4, and 8 displayed an increase in proteolytic activity. Plasma of C. virginica failed to inhibit P. marinus protease during all time points in the study, and instead displayed an increase in proteolytic activity. The cause of these increases in activity remains to be determined. These studies indicate the presence of protease inhibitors in the plasma of Crassostrea spp., which may have an impact upon host defense mechanisms, in addition to other physiological roles. Experiments to purify and characterize individual protease inhibitors present in these species, in particular serine protease inhibitors, are ongoing.



Oyster Plasma Metalloprotease(s): Possible Induction Response to Perkinsus marinus Infection in Eastern and Pacific Oysters

Alanna MacIntyre(1)*, Stephen Kaattari(2) and Mohamed Faisal(3).

Department of Environmental Science, Virginia Institute of Marine Science, School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062

The protozoan Perkinsus marinus secretes serine proteases which are believed to be the cause of extensive tissue lysis in infected eastern oysters (Crassostrea virginica). On the contrary, the Pacific oyster (C. gigas) has been found to be resistant to P. marinus infection. During our studies to detect and monitor P. marinus serine protease(s) and serine protease inhibitors in oyster plasma, we have found evidence of protease(s) in the plasma of P. marinus-free oysters. Substrate electrophoresis gels of plasma from C. virginica and C. gigas, revealed bands of protease activity with approximate molecular weights of 60 and 51 kDa in C. virginica, and 112 kDa in C. gigas. Incubation with a variety of protease inhibitors, revealed that these proteases were inactivated by the metalloprotease inhibitors 1,10 phenanthroline, captopril, EDTA, EGTA, and beta- mercapto-ethanol. No inhibition in activity was seen with serine, aspartic, or cysteine protease inhibitors. Specific activity of the plasma metalloprotease in P. marinus-free oysters was found to be approximately five fold higher in C. gigas than C. virginica. During a 10 week infection study with a P. marinus isolate, the activity of this metallo-protease increased notably in C. gigas. In C. virginica, 3 new high molecular weight bands appeared during infection, all with metalloprotease activity. No bands with serine protease activity were detected. Western blots are being performed on plasma of infected and non-infected oysters, with mono and polyclonal antibodies against P. marinus extracellular proteins, to determine whether these new metallo- protease bands are from P. marinus, and whether any P. marinus synthesized proteins are detectable in the plasma. Experiments to isolate and determine the function of metalloprotease(s) in C. virginica and C. gigas plasma are underway.



Cryptosporidium nasorum Among Mugil cephalus and Tilapia zillii from Three Different Localities in Egypt

NE Mahmoud, MM EL Bahy and MM Fahmy

Department of parasitology, faculty of veterinary medicine, Cairo university. P.B. 12211 Giza, Egypt.

In order to add data on fish cryptosporidiosis in Egypt, the present work was conducted on 213 fish (Mugil cephalus and Tilapia zillii) collected from Mediterranean sea (Port Said), Suez Canal (Ismailia) and lake Quarun (Fayoum) during the period from June to November 1996. Cryptosporidium nasorum was recorded from 27.23% of them. Cross-transmission of this pathogen to a chicken host failed. Furthermore, the present work confirmed the impossible transmission of C. parvum to the lower vertebrates (fish).



On the Parasites Fauna of the Paddlefish (Polyodon spathula (WALBAUM)) from Fish Ponds in Europe

A. Moshu*

Institute of Zoology, Academy of Sciences of Moldova, Academiei str. 1, MD-2028 Chisinau, Republic of Moldova.

During parasitological surveys on Palyodon spathula (Walbaum) stocks from the rearing ponds of the Republic Moldova (reared by 1978) and Romania (1992) fish farms, 22 species of ecto-(18) and endo (4)-parasites belonging to the following systematic groups were found: Fungi (1), Protista (17) (Sarcomastigophora-2, Myxozoa - 2 and Cilophora - 12) and Metazoa (Plathelminthes - 2 and Arthropoda - 2). Saprolegnia spp., Cryptobia branchialis, Ichthyobodo necator, Sphaerospora sp., Chloromyxum sp., Chilodonella piscicola, Ch. hexasticha, Ichthyophthirius multifiliis, Ambiphrya ameiuri, Epistylis lwoffi, Apiosoma piscicola, Trichodina acuta, T. nigra, T. mutabilis, T. nobilis, T. pediculus, T. reticulata, Trichodinella epizootica, Diplostomum spathaceum, Amphilina foliacea, Lernaea cyprinacea and Argulus foliaceus. The peak values of infestation were recorded of some (with a wide range of hosts) protistian parasites (ciliata were found to be most abundant). Total fish infestation was 82%. Found in the kidney, Sphaerospora sp. seems to be a new species and probably has been introduced in our waters together with paddlefish. Paddlefish reared in ponds in polyculture with other sturgeon fishes (Acipenser ruthenus) showed infestation by Amphilina foliacea with Invasion Intensity up to 40% and Invasion Extensiveness up to 95 pieces per fish. Noted paddlefish infestation by parasites was not accompanied by any cases of fish mortality. The following parasitic groups have epizootological importance for paddlefish: Lernaea cyprinacea, Diplostomum spathaceum, Trichodinella epizootica, Amphilina foliacea, Ichthyophthirius multifiliis, Chilodonella spp., Trichodina spp. and Saprolegnia sp. So, the fauna of parasites of the paddlefish introduced in Europe is not specific (Sphaerospora sp. and Chloromyxum sp.) and was formed on the base of aborigenen/local fishes. The names, the location, the values of infestation and the epizootic importance of the revealed parasites are presented.



New Diseases of the Black Sea Hydrobionts

NN Naidenova*

Institute of Biology of Southern Seas, Department of Parasitilogy, 2 Nakhimov av. 335011 Sevastopol, Ukraine

In the ecosystem of the Black Sea, due to the influence of unfavorable environmental factors, the weakening and distruction of steady parasite-host and symbiotic connections take place. This involves the changing of species composition and quantity of parasites and strengthening of parasite pathogenicity. In the forefront of marine culture in the Black Sea at present, is a mussel Mytilus galloprovincialis. On the inside surface of nacre, numerous whitish web-like branchings were found. Electron-microscopic research of the shell sections shows that, on the phone of relatively smooth surface of ways of settlers Cliona fastifica and Polydora ciliata, we see the round ulcerations, filled with mycellia. Oysters Ostrea edulis were found ill with signs outwardly unlike the "shell diseases": through nacreous layer of valves the brown filament branchings were seen. By scanning electron microscopy, the colonies of fungus on the shell splits are presented as tangles of hyphae of different diameter. Penetrating into ways of settlers C.vastifica and P. ciliata fungi was secondary infection. Gradually they become a cause of decomposition of valves, using them for their growth and gaining ground in the pathological process. In the Black Sea three species of large shrimps inhabit: Crangon crangon, Palaemon elegans and P. adspersus. Except Terebrospira lenticularis, the shrimps have different structures and configuration settlements of protozoan organisms of uncertain systematic origin. Spreading in cuticula, organisms use it as food, eating out considerable holes: "ameoba-like" formations represent the chains of cookie-like bodies. They skirt along small cracks in the cutucula layer. "Lichen-like" formation look like round spots and is similar to leaves of lichen by the structure. The center of such formation is under a traumatized cuticular thorn of the host.



Effect of Cultured Perkinsus atlanticus Supernatants on the Mussel (Mytilus galloprovincialis) and Carpet-shell Clam (Ruditapes decussatus) Defense Mechanisms

MC Ordás*, B Novoa and A Figueras

Instituto de Investigaciones Marinas (CSIC). Eduardo Cabello, 6. 36208 Vigo, Spain.

Perkinsus atlanticus has been associated with serious losses of cultured carpet shell clam (Ruditapes decussatus) in several European countries. Little is known about the interaction of this clam species immune system with P. atlanticus. Supernatants of cultured P. atlanticus maintained in PBS for five days were collected. The highest enzymatic activity detected in this supernatant corresponded to acid phosphatase, and its protease activity was 0.255 ± 0.0275 U/ml. The effect of the Perkinsus supernatant on the phagocytic activity of two mollusc species, one susceptible (R. decussatus) and one resistant (Mytilus galloprovincialis) was determined. Three different test particles were assayed: zymosan, Escherichia coli and Vibrio tapetis, and in all cases the phagocytic rate and index of hemocytes preincubated in Perkinsus supernatant decreased. A preincubation time of 30 minutes was enough to inhibit phagocytosis, and increased preincubation times did not result in higher inhibition. The supernatant also decreased the SRBC agglutination titer of clam and mussel sera. These results suggest that the secreted products of the cultured parasite may have a negative effect on the defense response of the bivalves, and that acid phosphatase and proteases present in cultured Perkinsus supernatants could be relevant to the infection of susceptible mollusc species by Perkinsus.


Parasites of the Freshwater Fish, Silurus glanis, Captured from the River Tigris, Mosul, Iraq

ZIF Rahemo* and BS Al-Naeime

Department of Biology, College of Science, University of Mosul, Mosul, Iraq

A total of 100 specimens of freshwater fish, Silurus glanis, were captured from the River Tigris passing through Mosul City, Iraq. A total of 16 species of parasites were recovered including four species of protozoans (Trypanosoma arabica, Trichodina domerguel, Myxobolus koi and M. iranicucus), three monogeneans (Ancylodiscoides vistulensis, Paradiplozoon pavlovskii and diporpa larva), two digeneans (Orientocredium siluri and Megamonostonella rashediansis), two cestodas (Bothriocephalus ospariichthydis, Proteocephalus hemispherous), one nematode (larva of Contracaecus sp.), three crustaceans (Argulus japonicus, A. foliaceus and Erasilus mosulensis) and one mollusk (Glochidia larva). Thus, Silurus glanis is considered as a new host for 11 species of parasites recovered from this study in addition to the known one in the world.



Fish Infection by Parasites in the North Part of the Caspian Sea

GA Saparova, D Zhatkanbayeva, KS Akisheva and LV Kulkina

Institute of Zoology and Genofond of Animals, Ministry of Science-Academy of Sciences of Republic of Kazakhstan, 480060 Almaty, Kazakhstan.

The Caspian Sea is inhabited by valuable trade species offishes: Acipenseridae, Cyprinidae, Percidae, and Siluridae. Parasitic diseases influence their numbers. To clear up the contemporary epiozootic situation of invasive diseases, ichthyoparasitological investigations were carried out in August of 1997 in the North part of the Caspian Sea. 181 samples of 16 species of fish were examined. 49 species of parasites were registed: Myxosporidia (2), Monogenea (17), Amphilida (1), Cestoda (6), Trematoda (11), Nematoda (5), Acanthocephala (2), Hirudinea (2), Crustacea (3). The total infection of fish by parasites was 63.1%. Fish were infected by Monogenea (23.7%0, by Trematoda (22.1%), by Nematoda (18.1%), and were other parasites (1.1-7.2%). The Monogenea: Paradiplozoon paradoxus, P. bliccae; the Trematoda: Diplostomum spathaceum, D.volvens, D.mergi, and Tylodelphys clavata are widely distributed among the fishes: Cyprinidae, Percidae. Amphilina foliacea affects the body cavity, the gonads of Acipenser stellatus; Acanthocephala: Leptorhynchoides plagicephalus - the intestine of Huso huso, A.stellatus, A.guldenstadti. The larvae of Anisakis schupakovi which parasitize the body cavity of Cyprinidae and Percidae can be dangerous to human health.



A Polymerase Chain Reaction Method for the Routine Monitoring of Myxosporean Infection in Laboratory Populations of Tubificids and Fish

WB Schill*, TB Waldrop and VS Blazer

National Fish Health Research Laboratory, BRD/USGS, 1700 Leetown Road, Kearneysville, WV 25430 USA;;

The establishment of myxosporean-actinosporean infections such as whirling disease under laboratory conditions is a lengthy process that requires the culture and productive exposure of susceptible fish and alternate oligochaete hosts to the appropriate life stage of the parasite. We developed a polymerase chain reaction (PCR) method to monitor the infection process and thereby optimize culture conditions. Conserved sites in the 18S rDNA of myxoporeans were identified by multiple sequence alignment, and a primer set was identified by computer analysis. The method is sensitive enough to identify a single positive worm in large (up to 256) pools, and to detect positive tissues from infected fish that show clinical signs of disease. The same primers can be used for cycle sequencing of PCR product and confirmation of the infecting species.



Monitoring of the Epizootic State of the Main Commercial Fish from the Azov Sea Basin

EV Shestakovskaya (1), LY Katycheva (2), AV Kazarnikova (1) and GM Hoteva (3)

1 RosribNIIproekt ichthiopathological laboratory Oborona-street 49 Rostov-on-Don Russia 344008
2 Head, Central Production Station:Fish acclimatization and Diseases Control, !8a Zheltovskogo st., Rosrybhoz, Moscow, Russia
3 Head, Northen Caucasus Branch of the Central Production Station: Fish acclimatization and Diseases Control, 122, Ponomaryov st., Krasnodar, Russia

Parasitic, infectious and alimentarian fish diseases cause fish mortality and sometimes are not taken into consideration by ichthyologists. Twelve species of parasites have been discovered in pike perch (Luciopercae luciopercae) recently. They include 8 species in adult fish and 11 species in juveniles. Microsporidya Glugea luciopercae was one of the most epizootologically important in 1996. More numerous were the traditional gill parasites of pike perch: monogenea Ancyrocephalus paradoxus and crustacea Achtheres percarum, and also intestine trematoda Bucephalus polymorphus. Eight species of parasites were found on bream (Abramis brama). Its parasitic fauna consisted of 4 classes: monogenea (2 species), cestoda (3 species), trematoda (5 species), and nematoda (1 species). In summer, in the eastern part of the Taganrog Bay of the Azov Sea, the top level of invasion was observed for gill trematoda Dactylogyrus crucifer. In 1997 10 species of parasites of 6 classes were discovered on roach (Rutilus rutilus h.): microsporidia (1 species), monogenea (1 species), cestoda (1 species), trematoda (5 species), nematoda (1 species) and bivalvia (1 species). The most favorable parasitological status was seen in mullet (Mugil so-ing B.). The level of invasion of Russian sturgeon, Asipenser guldenstadti, by crustacea Pseudotracheliastes stellatus was lower than in previous years. Starred sturgeon, Acipenser stellatus, was not invased by P. stellatus. So, now the results of ichthyopathological investegations of the main commercial fish permit us to estimate their epizootic state as comparatively favorable.



Studies on the Thelohanelliosis (Myxosporidiosis) of Carp in China

Hsieh Shing-ren*

Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan, 430072, China

Carp is one of the major cultured fishes in China. The harms to carps by myxosporidiosis are becoming more and more serious, among them thelohanelliosis is a common epidemic disease in adult carps. Over 10 kinds of parasitic thelohanellus are found in carp organs such as the intestines, scales, gills, ureter, kidney, bladder, gall bladder, nasal cavity, etc. In China, there are two kinds of the most harmful thelohanelliosis: 1. Intestines parasitic thelohanelliosis: This kind of thelohanelliosis is widely found in the ponds, lakes, and reservoirs in the provinces of Hunan, Hubei, Henan, Hebei, Heilingjiang, etc. The pathogen discovered in the carps in the Hunan province is Thelohanellus kitauei, while in the Hubei province, the pathogen is Thelonellus sagittarius. In the system investigation on the net cage cultured carps in the Xingyang area of the Henan province in 1993, the incidence of thelohanelliosis was up to 70 percent. Thelonellus destroyed intestinal organization and absorbed the nutrition from the carps. The bodies of the sick carps lost shiness and appeared dark. The intestines appeared purplish-red and were filled with thick blood and mucus. Big ellipsoid cysts located in the middle and posterior intestines had a maximum size of 4.5 x 1.7cm, making the intestines expand and blocking the intestines totally, causeing the carps to cease eating and death. This kind of Thelohanellus was defined as Thelohanellus xinyangensis sp. nov. The author made the immunity test with the infected and uninfected blood serum and discovered that the idiosyncratic antibody existed in the body of sick carps. The pathology was also studied at the same time. 2. Skin parasitic thelohanellus: This kind of thelohanelliosis was discovered all over the country. The common pathogen is Thelohanellus rahitae, which parasites in the carp's scaled follide and forms the sphere of ellipsoid shaped cysts. In the most serious cases, under every scale there is a cyst, with the maximum cyst measuring 4 x 4 cm. It is called scale protrusion disease. They also can induce pathological changes in the carp's internal organs and can cause the carp wear and tear and finally death.


About Infection of Aquarium Fish by Capillariids Nematodes

NI Skiba*

The Far Eastern State Technical Fisheries University, Lugovaya str., 52-b, 690600 GSP, Vladivostok, Russia

The peculiarities of infection of some species of aquarium fish by Capillariids Nematodes (Ñapillaria tomentosa) were investigated. Poecilia reticulata Guppy are found out to be infected most often. 7 of 18 investigated fish were infected, with the average intensity being 38. 2 of 6 Cynolebias whitei were infected (mean intensity 1.5). Pterophyllum scalares turned out to be the most infected (intensity ­ 7.8). The mean intensity of invasion of 5 investigated fish was 1.5. Sumatra Barbs (Puntius tetrazona) were the most resistant to the infection - only 6 of 20 fish got ill (intensity ­ 1.5). At the early stages the external characteristics of the disease aren't revealed. At the late stages, fish are nervous, with ill-coordinated movement. Rocking of the body is observed, and intense exhaustion and refusal of food are observed some time later. Fins are often tightened, skin gets dark. A case of self-recovering was registered upon temperature change: 6 infected Sumatra Barbs were held for several days at +15C (minimum temperature for this species). The fish were sluggish and didn't eat. Then the temperature was increased to +25 C. On the fourth day, sick fish began to eat intensely and gradually assued healthy color and state of nourishment. Thus, viviparous toothcarps (Poecilia reticulata Guppy, Xiphophorus helleri Swordtail and etc.) turned out to be the most sensitive to nematodes among all fish.



Preliminary Studies on the Effects of Temperature and Photoperiod on Tubifex Worm Species Infected with Myxobolus cerebralis Spores

TB Waldrop*, WB Schill and VS Blazer

National Fish Health Research Laboratory, Leetown Science Center, BRD/USGS, 1700 Leetown Road, Kearneysville, West Virginia, USA.

Salmonid whirling disease is caused by the myxosporean/actinosporean parasite Myxobolus cerebralis. The parasite's complex life cycle utilizes both fish and an aquatic oligochaete host, Tubifex tubifex. Whirling disease will not develop in salmonids without the myxosporean/actinosporean conversion process that takes place within the gut epithelium of these oligocheate tubifex species. This conversion process produces actinosporean triactinomyxons (TAMs), the infective waterborn units for salmonid fish. Recently, it has been suggested that whirling disease is responsible for declines of certain wild salmonid populations in the western part of the US. Although whirling disease is endemic in many populations in the eastern US population declines have not been documented. This study is part of a collaborative project studying both "eastern" and "western" populations of worms, parasites and fish stocks. In photoperiod experiments, infected tubifex worms released higher numbers of triactinomyxons in dark conditions versus continuous light conditions. In temperature experiments at 6, 12, 15, and 24C, results showed two important trends. At water temperatures of 15C and greater, significant increases in worm mortalities were observed in uninfected and infected worms than were recorded at lower temperatures. There was also significantly higher mortalities of infected worms when compared to uninfected worms at 15 and 24C. Experiments are in progress to assess the effects of temperature and photoperiod on numbers of TAMs released and these results will be presented.



First Occurrence of Myxidium Infection in Cultivated Charp Snouted Sparus Puntazzo puntazzo in Croatia

S Zrncic (1)*, D Oraic (1), B Sostaric (1) and I Filic (2)

1 Croatian Veterinary Institute, Department for Fish Diseases, Savska 143, Zagreb, Croatia
2 Marimirna, Rovinj, G. Paliaga 4, Croatia

Although charp snouted sparus (Puntazzo puntazzo) is a native inhabitant of the Adriatic sea along the Croatian coast, representing even a part of fishermen's catch, until recently this species has not been cultivated in our fish farms. At a certain fish farm, experimental production of charp snouted sparus using imported fry was started in 1997. Two months after introduction of seven-week old fry into growing cages, high mortalities started. At the beginning disease started insidiously: affected fish swam slowly close to the water surface separated from the lot and they were emaciated. Pathoanatomically, only slight patchy skin discoloration was observed. By internal examination of recently dead and euthanized moribund fish, the stomach appeared normal, and was filled by normal amounts of ingest, while immediate proximal anatomical structure of the intestine was distended by a seromucous transparent content. The intestinal wall was thinner and more translucent than normal. Examination of gut mucosa scrapings by light microscopy (400X) showed numerous spores compatible with Myxidium leei as described by Diamant et al. (1994). By histopathological examination of intestines numerous parasitic structures in close association with enterocytes were observed. On high magnification, they were compatible with those seen on scrapings and the diagnosis of M. leei was supported. Since this is a first and mainly experimental attempt of charp snouted sparus cultivation in our mariculture, the importance of described parasitosis due to extreme losses in fish crop should be thoroughly studied.



Myxosporean Fauna from Black Sea Fish and Deterioration of the Environment

VM Yurakhno*

Institute of Biology of the Southern Seas, Department of Parasitology, 2 Nakhimov av., Sevastopol, Crimea, 335011, Ukraine

Literature and original data (1987-1991, 1997-1998) evidence that myxosporean fauna of the Black Sea has been changing, numbering today 61 species from 58 fish hosts. The author found 29 diverse myxosporeans in 34 of 66 examined host species (about 4500 fishes from coastal waters of the ex-USSR). Longterm monitoring near Sevastopol (Crimea) shows that in 1974 the diversity of myxosporean and host species were 15 and 24, respectively, while in 1998 there were 28 and 26, respectively. Species composition of fish remained similar. As for parasites, only 5 species still occur there today, with two of them in the former hosts, two species left for other locations of the sea, and 8 not found presently. Eight myxosporeans were identified as earlier unknown for the science and six for the Black Sea; six more were detected from 15 new hosts. Some of mass myxosporeans, e.g. Myxidium gadi from Merlangius merlangus, Myxobolus parvus from Mugil soiuy, display a rise of infection statistics and a growing pathogenic impact on tissues and organs of fish. Not only are myxosporeans altered in quantitative composition but parasitic worms are also, e.g. Monogenea, Cestoda and some others. Possible explanation roots in catastrophic degradation of the Black Sea due to human pressure, pollution and alien species rather than in natural evolution course.



Topical Disinfectants for Control of Providencia alcalifaciens Infection in Piranha (Serrasalmus natteren)

S Aydyn*

Çanakkale Onsekiz Mart University, Fisheries Faculty, Karacaören 17100 Çanakkale-Turkey

Providencia alcalifaciens was isolated from naturally infected piranha (Serrasalmus natteren), and its pathogenicity was tested by intramuscular injection using healthy scattered carp (Cyprinus carpio L.). Both natural infection and experimental infection caused deep lesions around the mouth, dropping of scales, cataracts in the eyes and an increase of mucus on the body surface. The sensitivity of bacteria to 15 chemotherapeutants was determined. In vitro tube dilution assays indicated that minimum inhibitory concentrations (MIC) of chloramine-T and formalin for P. alcalifaciens were 2.29 mg/L and 40 mL/L for 1 h, respectively. Copper sulphate (CuSO4) for 2-min and potassium per manganate (KMnO4) for 10-min were not effective against the pathogen. In a therapeutic application bath, chloramine-T controlled the natural infection.



A Non-Pharmacological Method for Controlling Loma salmonae Infections in Farm-Reared Pacific Salmon

HJ Beaman, DJ Speare and GJ Arsenault

Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada. C1A 4P3;

Loma salmonae, an economically important microsporidian parasite of farm-reared Pacific salmon, causes severe branchial infections. In recent years, numerous mortalities have been associated with this pathogen, creating significant financial losses for fish farmers. Because no legal pharmacological treatments currently exist in Canada to control L. salmonae infections, alternative mechanisms of control are essential. Water temperature has a regulatory effect on the development of many pathogens and it may be used to control parasitic infections with L. salmonae. Through a series of experimental infection challenges, we found that L. salmonae reaches its upper and lower lethal threshold at 21° and 7°C, respectively, when reared in rainbow trout exposed to the parasite via oral ingestion of spores. The developmental rate of the parasite was significantly faster at warmer temperatures than at colder temperatures (p=0.000). In addition, when these fish were re-challenged with infective spores at 15°C, resistance developed in almost all fish, regardless of the water temperature at which the fish were initially infected. This indicates that Loma salmonae does not need to complete its life cycle in order to induce resistance in its host. This became evident at both the low and high thresholds where no xenomas developed during the initial challenge but a high level of resistance was displayed upon reinfection. This knowledge may be used to control infections by infecting fish at low temperatures where the infection does not fully develop, but protects the fish when later exposed to the parasite, minimizing financial losses to fish farmers.



An Outline for the Rational Development of Commercial Fish Vaccine Products

RA Busch*

Landolt Busch & Associates International, 2311 - 14th Avenue East, Seattle, WA 98112-2102 USA

Immunization has become a major tool in prevention of disease in aquacultured animals over the last 30 years. Technical feasibility and effectiveness are only two of the essential criteria in the product development process. In order to become a successful commercial reality in a timely and cost effective manner, the design of the vaccine product and the research and development studies must meet a variety of additional criteria including 1) regulatory acceptability, 2) potential for scale-up to commercial production and economy, 3) application and use compatible with animal production protocols, 4) adequate efficacy under typical field conditions and seasonal occurrence of the disease, 5) value to the producer and 6) reasonable economic return to the manufacturer. Based on 25 years of industry experience in developing commercial vaccines for fish, the author uses Microsoft Project '98® software to present a rational and stepwise plan, in Gantt chart format. The plan consists of a logical sequence of 92 annotated tasks and events or milestones leading to successful development, regulatory approval and commercialization of a typical product. The plan covers a three-year time line showing typical duration of each task, identifies important dichotomous decisions and emphasizes critical milestones. The commercial realization of new vaccine products for fish can benefit greatly when researchers design and conduct studies in a rational manner consistent with international regulatory requirements for licensing these products and the market forces that determine their eventual success.



Treatment of the Skin Parasite Trichodina jadranica Raabe, 1958 (Ciliophora: Trichodinidae) in Eel in Danish Aquaculture: Alternatives to Formaldehyde

HCK Lyholt (1), S Mellergaard (1) and K Buchmann (2)

1 Danish Institute for Fisheries Research, Fish Diseases Laboratory, Bülowsvej 13, DK-1870 Frederiksberg C, Denmark.
2 Royal Veterinary and Agricultural University, Bülowsvej 13, DK-1870 Frederiksberg C. Denmark.

In recent years, infestations with the skin parasite Trichodina jadranica on eels in Danish aquaculture using heated recirculated water have caused severe losses in production. Formerly, the parasite was effectively controlled with formaldehyde (50 - 120 ppm) but this treatment has now become insufficient resulting in recurrent disease problems. In addition, formaldehyde is a hazardous substance as it is suspected to be carcinogenic. In order to solve both the disease problem and the occupational hygienic problem a project has been set up to find alternatives to the traditional formaldehyde treatment. The experiments are carried out in a mobile test plant, which is a complete small-scale copy of a real eel production plant. The test system is stocked with infected eel and is connected to a production unit suffering from T. jadranica until the biological and chemical parameters are identical in the two systems. The test plant is then separated from the "mother plant" and experiments are carried out. A wide range of anti-protozoan chemicals and disinfectants were tested under laboratory conditions. The most effective substances were then tested under field conditions in the test plant. In addition to the chemical treatment, the effect of water parameters (e.g. organic matter, pH, nitrate and phosphorous) on the abundance of T. jadranica was investigated in the test plant. Preliminary results show that some disinfectants are effective against the parasite and that a relatively low load of organic matter, pH < 6 and a high concentration of nitrate are unfavorable to T. jadranica.



In Vitro and In Vivo Inhibition of Fish Pathogenic Bacteria by the Lactoperoxidase System

TF Nielsen*1, H Maas2, KD Kussendrager2, E McLean3

BioMar A/S, Mylius Erichsensvej 35, 7330 Brande, Denmark
2 DMV International, NCB-laan 80, 5460 BA Veghel, The Netherlands
3 Aalborg University, Biotechnology Laboratory, Aquaculture Division, Sohngaardsholmsvej 57, 9000 Aalborg, Denmark

Peroxidases are widely distributed enzymes, occurring in plants, animal tissues and body fluids. In animals, peroxidases participate in natural non-specific immune defense mechanisms. Many peroxidases have been termed lactoperoxidases due to their similarity to milk peroxidases, the antibacterial activity of which was recognized as early as 1924. However, lactoperoxidases themselves are not antibacterial. Rather, it is their oxidative capacity, producing hydrogen peroxide (H2O2), from substrates as diverse as halogenides (I- and Br-), thiocyanate (SCN-) and aromatic compounds, which furnish their antibacterial activity. With comparatively small quantities of H2O2 and SCN- lactoperoxidases present the body with a potent, natural antibacterial system: the so-called LP-system.
The in vitro antibacterial activity of the LP assemblage was tested against several Gram-negative fish pathogens, including Vibrio anguillarum, Yersinia ruckeri, Aeromonas salmonicida subsp. salmonicida, and Flavobacterium psychrophilum. In addition, the in vivo effect of the LP-system was examined, using dietary incorporation, with rainbow trout (Oncorhyncus mykiss). Treatments were given both prior to, and immediately following, artificial challenge with V. anguillarum and Y. ruckeri. Results demonstrated a significant (P < 0.05) inhibitory effect of the LP-system in vitro and a positive (P < 0.05) impact upon fish survival following bacterial challenge.



Flow Cytometric Evaluation of Antibiotic Effects on Refrigerated Storage of Spermatozoa from Tilapia

M Segovia (1), JA Jenkins (2)*, C Paniagua (1), RC Reigh (1) and TR Tiersch (1)

School of Forestry, Wildlife and Fisheries, Louisana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803 USA.
2 National Wetlands Research Center, Biological Resources Division, US Geological Survey, US Department of the Interior, 700 Cajundome Blvd., Lafayette, Louisiana 70506 USA.

Refrigerated storage of gametes is useful in artificial breeding and is a fundamental step in cryopreservation; however, the presence of bacteria can decrease storage time and fertility rates, and can contaminate culture systems. The objectives of this study were to test the effect of antibiotics on refrigerated storage of sperm from tilapia (Oreochromis niloticus) and to optimize flow cytometry protocols using dual staining for assessment of sperm quality. Concentrations of 1 x 109 sperm/ml were suspended in Ringer's Buffer at 325 mOsmol/kg (pH 8.0). The fluorescent stains rhodamine 123 (0.13 mM), propidium iodide (2.4 mM), and Sybr 14 (10 mM) were used to assess mitochondrial function and viability. A temperature of 70C for 5 min was used to kill sperm, and standard curves were generated from live:dead sperm mixtures (100:0, 75:25, 50:50, 25:75, and 0:100). In each case, assay values were within 10% of the predicted values. Three concentrations of ampicillin, gentamicin, and an antibiotic/antimycotic solution were added to fresh sperm, and motility estimates and flow cytometric protocols were performed daily for 7 days of refrigerated storage (4C). The greatest of each of the three antibiotic concentrations significantly reduced mitochondrial functionality. The greatest concentrations of gentamicin and antibiotic/antimycotic and all three concentrations of ampicillin significantly reduced sperm viability. This study demonstrated objective methods for assessing sperm quality that can be used in addition to motility estimates. Effects of antibiotics on sperm were determined. With minor modification, these protocols could be adapted for use with cells from other species and tissue types.



Transfer of a Transposon-Based Lytic Peptide Gene into Koi Carp Using Lipofection

BS Smith (1) *, RK Cooper (1), and TR Tiersch (2)

1 Department of Veterinary Science,; Louisiana Agricultural Center, Louisiana State University; Baton Rouge, LA 70803 USA.;
2 School of Forestry, Wildlife and Fisheries Louisiana Agricultural Center, Louisiana State University, Baton Rouge, LA 70803 USA.

In 1997, it was estimated that catfish farmers boosted Louisiana's economy by 200 million dollars. However, the biggest problem facing aquaculture is disease. Transgenic broodstock with a heritable defense mechanism for disease would be a valuable resource for the industry. The cecropin B gene encodes a lytic peptide that serves as such a defense in the giant silk moth, Hyalophora cecropia. The cecropin gene fragment contains an inducible acute phase response promoter, the native cecropin B gene and the mRNA polyadenylation signal, all within a mini Tn10 transposon. The entire transposon cassette is subcloned into a plasmid, pPC6. Lipofection was used to transfer the cecropin gene fragment into the cells of male Koi carp (Cyprinus carpio). Plasmid DNA coupled to liposomes is carried across cell membranes where the transposon directs incorporation of the gene into the fish genome. After the presence of this gene was confirmed in blood cells by the polymerase chain reaction (PCR), the fish were spawned artificially with non transfected females. PCR verified progeny that carried the lytic peptide gene after one year. PCR was also utilized to test the ability of the transposon by distinguishing incorporated gene product from the plasmid-contained gene. Stable incorporation of a transgene in fish that would defend against pathogenic invasion and be inherited by offspring would enhance the success of the aquaculture industry. This model demonstrates the feasibility of applying this technique to other fishes, including channel catfish and has potential for the success of enhancing fish survival.



A Flexibacteriosis-like Infection of Fish in Russia: Epizootiology and Treatment

VN Voronin*

Laboratory of Fish Diseases, State Research Institute for Lake and River Fisheries, Emb. Macarova 26, Saint-Petersburg 199053 Russia.

A Flexibacter/Cytophaga-like (FCL) infection has been detected on many trout and sturgeon farms in Russia. Different clinical forms of Flexibacteriosis were observed. Columnaris and Bacterial gill diseases occurred in the rainbow trout fry under tank rearing during spring, as well as Cold water disease in fingerlings under net-cage rearing during winter. Bacterial gill disease was usually observed in plastic round or square tanks and it was absent in the tanks of the canal type. Columnaris disease in the sturgeon fry appeared under tank rearing during spring and under net-cage rearing during summer. The high fish density and poor water quality especially during spring flood must be the main predisposing factors involved in the occurrence of FCL infection. The addition of Furazolidone in tanks with fish was the most simple and effective method of therapy and prophylaxy. In this case the disinfection of fish, water and tank take place simultaneously.



Effects of Waterborne Nitrite on Phase I-II Biotransformation in Channel Catfish (Ictalurus punctatus)

JF González (1)*, P Del Valle (1), S Thohan(1), and A Kane (1)

1 Aquatic Pathobiology Center, University of Maryland School of Medicine, Department of Pathology, Baltimore, MD 
2 School of Pharmacy, University of Maryland at Baltimore, Baltimore, MD.

Channel catfish exposed to 3 ppm waterborne nitrite (NO2) for 96-h were analyzed for alterations in hematological variables and phase I-II biotransformation activities in precision-cut liver slices. Nitrite-exposed fish (n=6) showed a significant increase in blood methemoglobin (35.5%) and blood nitrite (1.5 µg/ml) as compared with control fish (n=5) (0.8% and "not detectable," respectively). Hematocrit, plasma protein, hemoglobin and phase I-mediated metabolism of 7-ethoxycoumarin (EC) in liver slices was not altered in nitrite-exposed fish. Interestingly, phase II glucuronyltransferase-mediated metabolism of 7-hydroxycoumarin (HC), both as a phase I metabolite of EC and as a parent substrate, was elevated in nitrite-exposed fish (151 and 746 pmol/mg protein/hr, respectively) as compared to control fish (110 and 544 pmol/mg protein/hr). Sulfotransferase-mediated metabolism of HC, however, was not greatly altered in nitrite-exposed fish (71 and 457 pmol/mg protein/hr, respectively) as compared with control fish (91 and 427 pmol/mg protein/hr). Observed increases in glucuronyltransferase activities were significantly positively correlated with liver [NO2]. Physiological implications of nitrite exposure on biotransformation remain unclear. Studies to understand the effects of altered or suboptimal water quality on piscine metabolic capacity and homeostasis are needed.



Effects of Microcystin-LR on Zebrafish (Danio rerio) in a Range of Organismic Functions: Development, Detoxication Metabolism and Behavior

A Oberemm (1)*, C Wiegand (1), D Baganz (1), S Pflugmacher (1), GA Codd (2), CEW Steinberg (1)

1 Institute of Freshwater Ecology and Inland Fisheries P.O. Box 850123, Berlin 12561 Germany
2 Department of Biological Sciences, University of Dundee, Dundee DDI 4HN UK

In addition to contamination of waters and biota due to direct anthropogenic pollution, the effects of chemical stressors released by organisms attract increasing interest. In freshwaters, cyanobacteria have gained much attention, since potent toxins were detected in common species of this group and, supported by human eutrophication, they became the dominant group of phytoplankton in many regions of the world. In order to assess the consequences of released toxins for fish life, this study was started using zebrafish and the common cyanobacterial hepatotoxin microcystin-LR. Similar to results of studies with juvenile and adult fish, acute toxicity of dissolved microcystin-LR was low in embryonic stages, but pre-exposure of 0.5 mg/L microcystin-LR led to an inhibition of growth during larval development. In addition, 5.0 mg/L microcystin-LR decreased larval survival rate. It was detected that the toxin was absorbed quickly by the embryos and the concentration of microcystin-LR in the embryos reached the value of the test concentration. Corresponding to the low acute toxicity, it could be demonstrated that microcystin-LR led to an increase of soluble GST-activity which was already activated at 0.5 mg/L. HPLC and MS-data proved the formation of a GST-microcystin conjugate which resembles the first detoxication step. Analysis of behavior of adult zebrafish under exposure of microcystin-LR also revealed effects at a concentration of 0.5 mg/L, since there was a shift of activity showing an increase in the morning and a decrease at night. These results indicate that dissolved microcystins may adversely affect fish life in waters contaminated by cyanobacteria.



Tributyltin (TBT) Potentiates 3,3',4,4',5-Pentachlorobiphenyl (PCB-126)-Induced Hepatic CYP1A Activity in Channel Catfish, Ictalurus punctatus

CD Rice* and LE Roszell

College of Veterinary Medicine, Mississippi State University, P.O. Box 9825, Mississippi State, MS 39762, USA.

Planar halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs) are found in many aquatic systems. Their toxic effects are initiated primarily through the cytosolic aryl hydrocabon receptor (Ahr). However, only rarely are Ahr-binding contaminants found alone in the environment. Tributyltin (TBT), a common antifouling biocide, is also found in many harbor estuaries and their tributaries. Several reports indicate that TBT inhibits the cytochrome P4501A system of fish, at least in vitro, but our previous studies with rodents indicate that TBT potentiates PCB-126 (PeCB)-induced CYP1A in vivo. We exposed channel catfish, Ictalurus punctatus, to PeCB, TBT, or both in combination, with corn oil (CO) serving as the carrier control. Immunoreactive CYP1A protein and EROD activity were measured after (1) a single dose of 0.01, 0.1, or 1.0 mg/kg of each or both in combination, and (2) as six injections of 0.017, 1.7, or 17 ug/kg of each (or in combination) given over a 14 day period to yield a cumulative dose of 0.01, 0.1, or 1.0 mg/kg. PeCB, but not TBT, greatly induced these two CYP1A parameters. TBT (0.01 and 0.1 mg/kg), but not 1 mg/kg, potentiated PeCB-induced activity at these same doses. This property of TBT was even more pronounced in the fractionated exposure study. Moreover, EROD activity did not always reflect CYP1A protein induction; enzyme activity was inhibited by TBT at doses that potentiated protein induction (0.01 and 0.1 mg/kg). In summary, TBT potentiates PeCB-induced CYP1A in channel catfish at doses that may be considered environmentally relevant. (EPA-R822364010).



The Distribution of a Novel Infectious Agent in Healthy and Tumor-Bearing Bicolor Damselfish (Pomacentrus partitus) on South Florida Reefs

CE Campbell*, PDL Gibbs, S Baribeau, S Cacal, and MC Schmale

Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149.

Damselfish neurofibromatosis (DNF) is a transmissible malignant cancer that affects bicolor damselfish (Pomacentrus partitus) on South Florida reefs. DNF is characterized by the formation of plexiform neurofibromas, malignant schwannomas, and chromatophoromas. We have isolated an infectious agent that we believe to be the causative agent of DNF, which has been named DNFX (unknown damselfish neurofibromatosis agent). A series of experiments has been set up to determine the distribution of DNFX in healthy and tumored fish collected from South Florida reefs, the distribution of DNFX in a variety of fish tissues, and the distribution of DNFX in time and space in the Caribbean from 1950 to the present. Using Slot blot analysis, this agent has been shown to be present in 98% of tumors isolated from diseased fish. By the same method this agent has been shown to be present in less than 10% of apparently healthy fish. On average, ten out of twelve possible organs have been shown to have DNFX in fish with late-stage tumors, even though these organs appear healthy by gross examination. We have begun secondary experiments using PCR, nested PCR, and sequence analysis that will further characterize the nature and distribution of this agent. This stage of our research is designed to examine the possibility of sub-clinical infections in apparently healthy fish.
Supported by USPHS grants NS21997 and ES05705.



Temperature Dependent Regression of Walleye Dermal Sarcoma Tumors

RG Getchell*, GA Wooster and PR Bowser

Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 USA.,, and

Reports in the literature and field observations made by fisheries researchers have documented the seasonal occurrence and regression of skin growths on the walleye Stizostedion vitreum. The association of viruses such as walleye dermal sarcoma virus (WDSV) with these tumors, evident in the successful transmission experiments with cell-free tumor extracts, has opened up new avenues of inquiry addressing mechanisms of tumorigenesis and regression. Molecular biological techniques have provided new tools for studying the role of viruses in seasonal changes in tumor prevalence. Temperature seems to be a contributing factor in the development of many of these virally induced tumors. One study presently underway is using walleye dermal sarcoma as a model for the seasonal regression of tumors. In this experiment, tumors were induced on the left flank of each walleye by topical application of a cell-free tumor homogenate. After five months at 15C, 90% of the walleyes possessed WDS tumors. These fish were then allocated to one of the three temperature treatment groups (10, 15, 20C) and monitored for complete tumor regression. Histological sections were used to follow the regression stages. Preliminary results indicate there is an association between higher temperature and tumor regression. After two months, the incidence of tumors in fish held at 15 or 20C was significantly 0.05 lower than that observed at 10C.



Diagnosis of IPN Virus Using Nucleic Acid Hybridization with DIG-labelled cDNA Probes

JG Olveira, E Piriou, JM Cutrín, JL Barja and CP Dopazo*

Departamento de Microbioloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela. 15706, SPAIN

IPNV is one of the viral agents most frequently isolated in aquaculture in our area. Due to the rapid development of the disease, and the devastating effect it can produce in the farms, a rapid, sensitive and accurate diagnosis of this viral disease is required. For some years, and as part of our support to aquaculture facilities, our lab has employed PCR and nucleic acid hybridization with radiolabelled probes for detection of this virus in samples obtained from diseased and asymptomatic fish. Nowadays use of either of both techniques is restricted to laboratories, firstly because PCR is a relatively expensive procedure and, secondly due to the risk of manipulation of isotopes. Because fish farmers are getting interested in performing viral diagnosis directly in their facilities, our team has adapted the nucleic acid hybridization technique to be used with non-isotopic probes. For this purpose we have employed the same cDNA probes that previously proved to be effective in routine diagnosis by isotopic hybridization. Our preliminary results showed that, although sensitivity is clearly reduced, the technique seams to be suitable for diagnosis in diseased and carrier fish.



Inhibition of Channel Catfish Virus Immediate Early Transcripts Using Antisense Constracts

MP Dias, KE Nusbaum (1)*, RC Bird and P DeInnocentes

1 Auburn University, College of Veterinary Medicine, Auburn University, AL 36849

Missing abstract



Kinetics of Channel Catfish Late Gene Expression with Evidence of Gene Splicing

KE Nusbaum*, RC Bird, P DeInnocentes and BF Smith

Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849 USA

Channel catfish virus (CCV) is a herpes-virus widespread in cultivated channel catfish. CCV, in conjunction with adverse environmental conditions, causes fatal disease in the young of catfish less than 10 grams in weight and 10 cm in length. Three gene classes, immediate early (IE), early (E), and late (L), regulate CCV replication. The complete sequence of the CCV genome has been determined but the actual gene class of each open reading frame (ORF) is still largely putative. Kinetics of DNA replication in CCV were traced by 3H-thymidine incorporation to determine the characteristics of the CCV replication cycle. An early small peak of 3H-thymidine incorporation occurs at 1.5 hours, followed by a larger peak at 5 hours. Northern blots were used to determine transcription times for 2 putative CCV E or L genes (ORF 53, 59). CCO cells were infected with a multiplicity of infection of 5 CCV per cell. Flasks were harvested at one interval after infection and RNA extracted. The RNA was electrophoresed on 1.5% denaturing gel and transferred to nitrocellulose or nylon membranes, and probed with 32P-labeled cloned gene fragments. ORF 59 appeared at 3 hours of incubation and was processed to a slightly smaller transcript by 4 hours. Evidence for splicing was noted for ORF 53. A family of 4 bands was seen at 3 hours of incubation. These discrete bands disappeared by 4 hours and were replaced by a single small band. Because of their structure (according to Davison) and time of replication, both ORF 53 and 59 appear to be late genes.



Detection of Antibody against Infectious Haematopoietic Necrosis Virus (IHNV) by Enzyme Linked Immuno-Sorbent Assay (ELISA) Using the Recombinant G-Protein for Capture Antigen

M Yoshimizu (1), Y Mukai (1), T Kimura (1) and Jo-Ann Leong (2)

1 Laboratory of Microbiology, Faculty of Fisheries, Hokkaido University, Minato, Hakodate, Hokkaido 041-0821 Japan.
2 Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804 USA

Enzyme linked immuno-sorbent assay (ELISA) using rabbit serum against masu salmon (Oncorhynchus masu) or rainbow trout (O. mykiss) IgM was applied to detect the antibody against the infectious haematopoietic necrosis virus (IHNV) in the sera of masu salmon and rainbow trout. Purified IHNV and recombinant G protein were used for the capture antigen of this ELISA. The first serum was salmonid serum, the second anti-serum was rabbit anti-masu salmon or rainbow trout IgM, and the third anti-serum was peroxidase-conjugated swine anti-rabbit IgG. SPF rainbow trout and ito (Hucho perryi) were immunized with purified IHNV and recombinant G protein. ELISA absorbance was increased after the immunization and decreased when serum was mixed with IHNV and E. coli. ELISA conditions were set up using the sera from immunized fish. Purified IHNV and recombinant G protein cross-reacted with antibody in sera of fish, but the recombinant G protein gave clearer results than did IHNV. Fish sera which have a neutralization antibody showed high ELISA absorbance and the sera of recovered fish from IHNV infection showed high ELISA absorbance. When fish sera were diluted to 1:20, ELISA absorbance of sera from IHN recovered fish and from un-infected fish were distinguished. This ELISA was able to use the antibody screening for the identification of IHNV carriers in a brood stock.



Contents - Scientific Sessions - Plenary Abstracts - Oral Abstracts - Special Session Abstracts - Author Index