Phylogenetic and functional characterization of the distal intestinal microbiome of rainbow trout Oncorhynchus mykiss from both farm and aquarium settings.

AIMS: This study focused on comparing the phylogenetic composition and functional potential of the intestinal microbiome of rainbow trout sourced from both farm and aquarium settings. METHODS AND RESULTS: Samples of distal intestinal contents were collected from fish and subjected to high throughput 16S rRNA sequencing, to accurately determine the composition of the intestinal microbiome. The predominant phyla identified from both groups were Tenericutes, Firmicutes, Proteobacteria, Spirochaetae and Bacteroidetes. A novel metagenomic tool, PICRUSt, was used to determine the functional potential of the bacterial communities present in the rainbow trout intestine. Pathways concerning membrane transport activity were dominant in the intestinal microbiome of all fish samples. Furthermore, this analysis revealed that gene pathways relating to metabolism, and in particular amino acid and carbohydrate metabolism, were upregulated in the rainbow trout intestinal microbiome. CONCLUSIONS: The results suggest that the structure of the intestinal microbiome in farmed rainbow trout may be similar regardless of where the fish are located and hence could be shaped by host factors. Differences were, however, noted in the microbial community membership within the intestine of both fish populations, suggesting that more sporadic taxa could be unique to each environment and may have the ability to colonize the rainbow trout gastrointestinal tract. Finally, the functional analysis provides evidence that the microbiome of rainbow trout contains genes that could contribute to the metabolism of dietary ingredients and therefore may actively influence the digestive process in these fish. SIGNIFICANCE AND IMPACT OF THE STUDY: To better understand and exploit the intestinal microbiome and its impact on fish health, it is vital to determine its structure, diversity and potential functional capacity. This study improves our knowledge of these areas and suggests that the intestinal microbiome of rainbow trout may play an important role in the digestive physiology of these fish.

Comparative imaging of European eels (Anguilla anguilla) for the evaluation of swimbladder nematode (Anguillicoloides crassus) infestation.

This study compares diagnostic imaging tools in detecting the parasitic swimbladder nematode Anguillicoloides crassus in Anguilla anguilla (L.) and focuses on ultrasound in an attempt to develop a non-destructive, field diagnostic test. Ultrasound use could allow the parasite to be diagnosed without decreasing the number of critically endangered European eels through post-mortem. In the preliminary study, eels were examined with computed radiography, computed tomography, magnetic resonance imaging, 14 MHz high-end ultrasound and 5 MHz low-end portable ultrasound, and the results were compared with post-mortem findings. This ultrasound scanning technique did not produce any promising results. A second batch of eels was examined using the same high-end and low-end ultrasounds, but employing a different scanning technique and comparing the results with post-mortem. This second study, scanning along the midline from below, allowed for the detection of anomalies associated with moderately infected animals. None of the eels used in this study were severely infected; thus, no conclusions can be made regarding the use of ultrasound in those animals. Overall, it was found that none of the techniques were useful in diagnosing mildly infected individuals; therefore, no single diagnostic imaging tool is sensitive enough to replace post-mortem for definite diagnosis.

Achieving consensus on current and future priorities for farmed fish welfare: a case study from the UK.

The welfare of farmed fish has attracted attention in recent years, which has resulted in notable changes within the aquaculture industry. However, a lack of communication between stakeholders and opposing ethical views are perceived as barriers to achieving consensus on how to improve farmed fish welfare. To address these issues, we developed an interactive approach that could be used during stakeholder meetings to (1) improve communication between different stakeholder groups, (2) build consensus on priorities for farmed fish welfare and (3) establish mechanisms to address welfare priorities. We then applied this approach during a meeting of stakeholders to identify current and future priorities for farmed fish welfare in the UK. During the meeting in the UK, stakeholders initially identified 32 areas that they felt were in need of development for future improvements in farmed fish welfare. These were further refined via peer review and discussion to the seven most important "priority" areas. Establishing a "better understanding of what good fish welfare is" emerged as the highest priority area for farmed fish welfare. The second highest priority area was "the need for welfare monitoring and documentation systems", with mortality recording proposed as an example. The other five priority areas were "[improved understanding of] the role of genetic selection in producing fish suited to the farming environment", "a need for integration and application of behavioural and physiological measures", "the need for a more liberal regime in Europe for introducing new medicines", "a need to address the issues of training existing and new workers within the industry", and "ensuring best practise in aquaculture is followed by individual businesses". Feedback from attendees, and the meeting outputs, indicated that the approach had been successful in improving communication between stakeholders and in achieving consensus on the priorities for farmed fish welfare. The approach therefore proved highly beneficial for future improvements in fish welfare in the UK.

Seasonal development and pathological changes associated with the parasitic nematode Philometroides sanguineus in wild crucian carp Carassius carassius (L.) in England.

Pathological changes associated with the parasitic nematode Philometroides sanguineus (Rudolphi, 1819) are described for the first time from observations of infections in wild crucian carp Carassius carassius (L.) in England. The damage caused by P. sanguineus was influenced strongly by host size, parasite development and the seasonal migrations of female nematodes within host tissues. Male and unfertilized female nematodes located on the swim-bladder wall caused only minor, localized changes. In contrast, the migration of gravid female nematodes into the fins during autumn provoked an acute inflammatory response comprising neutrophils, eosinophils and lymphocytes. This was accompanied by fin distortion, swelling of the dorsal and caudal tissues, degenerative changes and localized oedema. The encapsulation of female nematodes in connective tissue throughout winter limited additional tissue damage. The emergence of gravid nematodes from the fins in spring to facilitate larval dispersal caused necrosis, hyperplasia and loss of skin integrity. This activity was again accompanied by acute inflammatory reactions. Pathological changes were more severe in crucian carp measuring less than 60 mm in length, with no signs of debilitation in larger fish. These observations suggest that any impact of P. sanguineus is strictly seasonal and may be limited to fry. Lesions caused by this parasite, only recently recorded in Britain, may represent an additional pressure upon wild crucian carp populations already threatened by hybridization, competition and habitat loss.

Histopathological and ultrastructural studies of the tapeworm Monobothrium wageneri (Caryophyllidea) in the intestinal tract of tench Tinca tinca.

Monobothrium wageneri is a monozoic caryophyllidean tapeworm of tench Tinca tinca. The pathological changes caused by this parasite within the intestinal tract of wild tench are described for the first time. Parasites were found attached to the anterior third of the intestine in tight clusters comprising up to 109 tapeworms. Infection was associated with the formation of raised inflammatory swellings surrounding the parasites. This host response, combined with the deep penetration of the scolex into the gut wall, formed a very firm seat of parasite attachment. Histopathological changes were characterised by a pronounced fibrogranulomatous lesion that extended through all layers of the intestine. This was accompanied by haemorrhage, oedema, necrosis and degeneration of the muscularis. A marked eosinophilic interface layer between the scolex of the tapeworm and gut wall indicated intimate host-parasite contact. Ultrastructural examinations revealed coniform spinitriches covering the neck and lateral sides of the scolex and capilliform filitriches present on the apical end of the scolex. Numerous glandular cytons (tegumental glands) were recorded throughout the scolex tegument. Large numbers of secretory granules discharged from the glands through a network of processes onto the scolex surface were consistent with distancing the cellular responses of the host. Observations of severe inflammatory lesions, partial intestinal occlusion and the potential for intestinal perforation represent important pathological changes that are consistent with loss of normal gut function. The lesions associated with the attachment of M. wageneri are more severe than those recorded for any other tapeworm of British freshwater fish.

Seasonality and heterogeneity of live fish movements in Scottish fish farms.

Movement of live animals is a key contributor to disease spread. Farmed Atlantic salmon Salmo salar, rainbow trout Onchorynchus mykiss and brown/sea trout Salmo trutta are initially raised in freshwater (FW) farms; all the salmon and some of the trout are subsequently moved to seawater (SW) farms. Frequently, fish are moved between farms during their FW stage and sometimes during their SW stage. Seasonality and differences in contact patterns across production phases have been shown to influence the course of an epidemic in livestock; however, these parameters have not been included in previous network models studying disease transmission in salmonids. In Scotland, farmers are required to register fish movements onto and off their farms; these records were used in the present study to investigate seasonality and heterogeneity of movements for each production phase separately for farmed salmon, rainbow trout and brown/sea trout. Salmon FW-FW and FW-SW movements showed a higher degree of heterogeneity in number of contacts and different seasonal patterns compared with SW-SW movements. FW-FW movements peaked from May to July and FW-SW movements peaked from March to April and from October to November. Salmon SW-SW movements occurred more consistently over the year and showed fewer connections and number of repeated connections between farms. Therefore, the salmon SW-SW network might be treated as homogeneous regarding the number of connections between farms and without seasonality. However, seasonality and production phase should be included in simulation models concerning FW-FW and FW-SW movements specifically. The number of rainbow trout FW-FW and brown/sea trout FW-FW movements were different from random. However, movements from other production phases were too low to discern a seasonal pattern or differences in contact pattern.

Histopathology and ultrastructure of segmented filamentous bacteria-associated rainbow trout gastroenteritis.

Rainbow trout gastroenteritis (RTGE) is an emerging syndrome linked to the presence of large numbers of the segmented filamentous bacterium "Candidatus arthromitus" within the intestine. The present study examined the histopathological changes of the digestive tract of 152 trout with gross lesions typical of RTGE. Histopathology showed that 129 of 152 fish (85%) affected with RTGE had segmented filamentous bacteria in the distal intestine and/or pyloric caeca. The presence and number of segmented filamentous bacteria were always significantly higher (P < .001) in pyloric caeca, thereby suggesting the preferred site for these bacteria. Histopathological changes included enterocyte detachment and congestion of the lamina propria and adventitial layers. Samples from 6 RTGE-affected trout were examined using scanning and transmission electron microscopy, revealing a close interaction of segmented filamentous bacteria with the mucosa of distal intestine and pyloric caeca, with the presence of bacterial attachment sites, and with associated morphological changes of the apical membrane of enterocytes. Despite these interactions, segmented filamentous bacteria were not always adjacent to the areas with pathological changes, suggesting that if these organisms play a role in the pathogenesis of RTGE, extracellular products may be involved. Ultrastructural changes included loss of microvillar structure, membrane blebbing, hydropic mitochondrial damage, and basal hydropic degeneration of enterocytes, which frequently resulted in disruption of tight junctions and enterocyte detachment. The resulting exposure of large areas of lamina propria probably resulted in the compromise of the host osmotic balance and the facilitation of the entry of secondary pathogens.

A study of gross, histological and blood biochemical changes in rainbow trout, Oncorhynchus mykiss (Walbaum), with rainbow trout gastroenteritis (RTGE).

The mechanisms behind the pathogenesis of rainbow trout gastroenteritis (RTGE) are still unknown. This study examined the macroscopic and microscopic changes in trout with RTGE (RTGE+), as well as the blood chemistry. A total of 464 rainbow trout were sampled from 11 sites in the UK, comprising 152 RTGE+ fish and 330 random, apparently healthy fish. A case definition for RTGE was assessed by the analysis of its agreement with three laboratory tests: histopathology, packed cell volume and kidney bacteriology. Cluster analysis indicated the presence of three distinct presentations within the population of RTGE+ fish. Cluster A included gross signs associated with moribund RTGE+ fish, and clusters B and C identified gross signs consistent with concurrent diseases, notably furunculosis, enteric redmouth and proliferative kidney disease. The information gained was used to select RTGE+ fish without concurrent disease for the analysis of RTGE pathogenesis with blood biochemistry. This analysis revealed a severe osmotic imbalance and a reduced albumin/globulin ratio as indicatives of selective loss of albumin. These findings are compatible with a protein losing enteropathy.

Safeguarding the many guises of farmed fish welfare.

There has been a great deal of debate and sometimes open hostility between people with differing approaches to the welfare of farmed animals, but relatively little progress towards compromise or consensus. It has been suggested that progress has been inhibited by a fundamental lack of common ground; people are debating different questions. Compromise or consensus can only be achieved through understanding and this in turn requires effective presentation of information and constructive dialogue. In this paper we adapt a previously published framework to present and evaluate information relevant to a wide range of definitions of fish welfare. Through improved understanding we will increase our capacity to safeguard many aspects of welfare of farmed fish, satisfying the demands of more but not all stakeholders.

Fin erosion on rainbow trout on commercial trout farms in the United Kingdom.

Fish weighing less than 30 g and more than 100 g were sampled from 38 rearing units on 10 commercial farms growing rainbow trout for the table market. A fin index was calculated for each of the eight rayed fins on 949 trout by dividing their length by the standard length of the fish. There was a large range in the indices of all eight fins. The fin indices of the small and large fish were compared, controlling for farm effect. With the exception of the dorsal fin, all the indices were larger for the small fish than for the large fish, but the magnitude of the difference was greater for some fins than others. In comparison with the fins of wild fish, the pectoral and dorsal fins appeared to be most eroded and the damage to these fins was evident even in the small fish. The erosion of the caudal, anal and ventral (or pelvic) fins was more prominent in the larger fish. Variations in the fin indices of the caudal, anal and ventral fins suggested that there was little variation between rearing units on the same farm, but that there was significant variation between individual fish in the same rearing units, and between fish on different farms.

Problems and solutions with the design and execution of an epidemiological study of white spot disease in black tiger shrimp (Penaeus monodon) in Vietnam.

White spot disease (WSD) is caused by white spot syndrome virus (WSSV) and is an acutely fatal pandemic disease of crustaceans. It has resulted in massive losses to the shrimp-farming industry in Asia and has now spread to the Americas. This paper reports the problems and solutions associated with the design and execution of a longitudinal epidemiological study of shrimp (Penaeus monodon) health on farms practising a crop rotation of rice and shrimp in the Mekong Delta of Vietnam. The pre-sampling phase of the project involved selecting an appropriate site and sampling variables, obtaining permission and establishing the necessary laboratory and logistic facilities. At the start of the sampling phase, 40 farmers were selected and 32 of these were visited and interviewed. This resulted in the enrolment of only 17 farmers. A further seven had to be enrolled to obtain the maximum number of farmers that could be sampled by the study team. Compliance was enhanced through meetings, regular visits by senior members of the project team and ensuring that visits were punctual and that all information was treated confidentially. The production cycle began in January 1998 and lasted for approximately 5 months. An attempt was made to collect 500 post larvae (PL) before each pond was stocked to assess the health of the batch and to test for the presence of WSSV by one-step PCR. After stocking, the wild crustaceans also were sampled from the pond for PCR analyses. Information was collected on the management practices and samples of water, pond bottom, feed and shrimp collected throughout the production cycle. Water quality variables with predictable diurnal variation were sampled in the morning and afternoon, twice a week. Two months after stocking, the first outbreak of WSD occurred; subsequently, 18 farms conducted a complete emergency harvest due to the actual or perceived presence of a WSD outbreak. Detectable mortalities were reported from 19 farms, and moribund shrimps were collected from four of these for PCR and histological analyses.

Clearing mechanisms of Vibrio vulnificus biotype I in the black tiger shrimp Penaeus monodon.

Vibrio species' infections are a common sequelae to environmental stress or other disease processes in shrimp, but the mechanism by which the shrimp eliminate the bacteria is poorly understood. In this study, the penetration, fate and the clearing of V. vulnificus were investigated in Penaeus monodon. A bacterial disease isolate from a shrimp farm was identified as V. vulnificus biotype I. Polyclonal antiserum was raised in rabbits against the bacterium and the specificity was verified by ELISA and immunoblot against a range of Vibrio spp. and other gram-negative bacteria. The bacteria were then administered to P. monodon juveniles by injection, immersion and oral intubation. An indirect immunoperoxidase technique was employed in a time course study to follow the bacteria and bacterial antigens in the tissue of the shrimp. Bacteria were cleared by a common route, regardless of the method of administration. Observations in immersion challenge were similar to a combination of those for oral and injection challenges. With immersion, bacteria entered the shrimp through damaged cuticle or via insertion points of cuticular setae. Shortly after entry, whole bacterial cells were observed in the haemolymph and connective tissue. They were either phagocytosed by haemocytes, or broken down outside host cells. Haemocytes containing bacterial cells or antigens (HCB) were observed in the connective tissue and haemolymph. HCB accumulated around the hepatopancreas, midgut, midgut-caecum, gills, heart and lymphoid organ. Free bacterial antigens also accumulated in the heart and lymphoid organ. Bacteria entering through the mouth by oral intubation or immersion were broken down so that only soluble or very fine particles entered the hepatopancreas. Bacterial antigens passed through the hepatopancreas into the haemolymph. Antigens were initially observed in the haemolymph sinuses and subsequently accumulated in the heart and lymphoid organ. Bacterial antigens were released from the shrimp, initially through the gills and subsequently through hepatopancreatic B-cells, branchial podocytes and sub-cuticular podocytes.

Effect of methyl parathion on the susceptibility of shrimp Litopenaeus vannamei to experimental vibriosis.

Following increasing calls for environmental safety over the past 2 decades, persistent pesticides are being replaced by more rapidly degradable products. However, even these pesticides can affect non-target species, and may be associated with slow growth and increased susceptibility to viral and bacterial infections. In this study, juvenile white shrimp Litopenaeus vannamei (also named Penaeus vannamei) were challenged by intramuscular injection with Vibrio parahaemolyticus after 4 d prior exposure to methyl parathion in feed pellets at 0.080 microg g(-1). The bacterial injection control group consisted of shrimp fed pellets containing the methyl parathion-carrier solvent acetonitrile. Three additional control groups comprised 2 sterile saline-injection groups fed pellets containing methyl parathion or acetonitrile prior to injection, and 1 uninjected group fed normal pellets. Cumulative mortalities were recorded on the 4th and 8th days, and the presence of histological lesions was recorded on the 8th day. Cumulative mortalities were significantly higher in the group exposed to methyl parathion and bacteria on Day 8. Histological lesions, typical of vibriosis, were significantly associated with the injection of V. parahaemolyticus. The study provides strong experimental evidence that prior exposure to methyl parathion can increase the severity of Vibrio infections.

Usefulness of dead shrimp specimens in studying the epidemiology of white spot syndrome virus (WSSV) and chronic bacterial infection.

This paper describes the utility of dead shrimp samples in epidemiological investigations of the white spot syndrome virus (WSSV) and chronic bacterial infections. A longitudinal observational study was undertaken in shrimp farms in Kundapur, Karnataka, India, from September 1999 to April 2000 to identify risk factors associated with outbreaks of white spot disease (WSD) in cultured Penaeus monodon. As a part of the larger study, farmers were trained to collect and preserve dead and moribund shrimp (when observed) during the production cycle. At the end of the production cycle, 73 samples from 50 ponds had been collected for histopathology and 55 samples from 44 ponds for PCR. Intranuclear viral inclusion bodies diagnostic of WSSV infection were detected in dead samples from 32 ponds (64 %). Samples of dead shrimp from 18 ponds (36%) showed no histopathological evidence of WSSV infection. However, of these, samples from 13 ponds (26%) showed clear evidence of shell, oral, enteric and systemic chronic inflammatory lesions (CIL) in the form of haemocytic nodules, typical of bacterial infection. Samples from 5 ponds (10%) were negative for both WSSV and CIL. Samples from 8 ponds had dual WSSV and CIL, although both WSSV and CIL were only observed in the same shrimp from 1 pond. Useful information was obtained from these shrimp despite the presence of post-mortem changes. Samples from 19 ponds (43%) tested positive for WSSV by 1-step PCR and samples from an additional 10 ponds (22.7%) were positive by 2-step nested PCR. Samples from 15 ponds (34.1%) were negative for WSSV by 2-step nested PCR. There was moderate to substantial agreement between PCR and histopathology in the diagnosis of WSSV infection in dead shrimp. WSSV infection in dead shrimp was significantly associated with crop failures as defined by a shorter length of the production cycle (<90 d) and lower average weight at harvest (<22 g). WSSV infection was also associated with lower survival (<50%), but this was not significant. Ponds with CIL did not experience any crop failures, and the presence of CIL was significantly associated with successful crops. The study demonstrates that samples of dead shrimp can provide useful information for disease surveillance and epidemiological investigations of WSSV and chronic bacterial infections.

Estimation of prevalence of white spot syndrome virus (WSSV) by polymerase chain reaction in Penaeus monodon postlarvae at time of stocking in shrimp farms of Karnataka, India: a population-based study.

White spot disease (WSD) is at present the most serious viral disease affecting cultivated shrimp species globally. The causative agent, white spot syndrome virus (WSSV), is extremely virulent, has a wide host range and can also be transmitted from broodstock to their offspring. The shrimp postlarvae (PL) act as asymptomatic, latent carriers of the virus, and stocking of WSSV-infected PL has been reported as a risk factor for WSD outbreaks in culture ponds. However, there is no population-based study on WSSV prevalence in PL of shrimp. The present manuscript documents the approaches and the results in the estimation of prevalence of WSSV in PL populations of Penaeus monodon at the time of stocking. A maximum of 300 PL from each of the 73 batches of PL stocked at various farms in the west coast of India during September 1999 to January 2000 were tested for the presence of WSSV by 2-step nested PCR. Thirty-six (49%) of the 73 batches tested positive for WSSV either by 1-step alone (3 batches) or after 2-step nested PCR (33 batches). Sub-samples of 5 PL each or 1 PL each tested to quantify the proportion of infected PL within batches showed that WSSV prevalence was very high in 1-step PCR-positive batches and low in 2-step PCR-positive batches. The study also showed that appropriate sampling and sample size were major factors in determining the prevalence of WSSV in PL populations, underlining the need for testing large samples of PL to reduce errors from falsely negative results.

Relationship between white spot syndrome virus and indicators of quality in Penaeus monodon postlarvae in Karnataka, India.

White spot disease (WSD) is a viral disease of shrimp caused by white spot syndrome virus (WSSV). Stocking WSSV-infected seed has been implicated as a major risk factor for outbreaks of WSD. In addition, the quality of postlarvae batches has been proposed as a predictor for good crops. This paper describes the relationship between indicators of quality and WSSV in postlarvae (PL) of Penaeus monodon from Karnataka, India, over the period September 1999 to January 2000. Three outcome variables were considered: the WSSV status of the PL, as determined by PCR, and 2 subjective assessments of PL quality, namely the activity of the PL and the quality of the PL as determined by research assistants and farmers, respectively. Of the 73 batches of PL, 49.3% from a random sample of farms tested positive for WSSV. After adjusting for confounding, stocking earlier in the growing season and duration of transportation were the main risk factors for the presence of WSSV. The quality assessed by farmers and the PL activity assessed by research assistants showed only fair agreement (kappa 0.252) reaffirming the subjective nature of such techniques. The only variables consistently associated with either assessment of quality in univariate analysis were PL length, number per bag and salinity of the water in the delivery bags. After adjusting for confounding, no single variable was consistently associated with PL quality and activity. The research assistants' assessment of PL activity was also associated with the hatchery and a brown-orange hepatopancreas in univariate analysis. After adjusting for confounding, a brown-orange hepatopancreas was still significant and fitted into the model together with the salinity of the water in the PL bags. The farmers' assessment of quality was associated with PL length, date of stocking and duration of transportation in both univariate and multivariable analyses. There was no relationship between quality assessment and WSSV in PCR-positive PL.

Risk factors associated with white spot syndrome virus infection in a Vietnamese rice-shrimp farming system.

White spot disease (WSD) is a pandemic disease caused by a virus commonly known as white spot syndrome virus (WSSV). Several risk factors for WSD outbreaks have been suggested. However, there have been very few studies to identify risk factors for WSD outbreaks in culture systems. This paper presents and discusses the risk factors for WSSV infection identified during a longitudinal observational study conducted in a Vietnamese rice-shrimp farming system. A total of 158 variables were measured comprising location, features of the pond, management practices, pond bottom quality, shrimp health and other animals in the pond. At the end of the study period WSSV was detected in 15 of the 24 ponds followed through the production cycle (62.5%). One hundred and thirty-nine variables were used in univariate analyses. All the variables with a p-value < or = 0.10 were used in unconditional logistic regression in a forward stepwise model. An effect of location was identified in both univariate and multivariate analyses showing that ponds located in the eastern portion of the study site, closer to the sea, were more likely to test positive for WSSV by 1-step PCR at harvest. Ponds with shrimp of a smaller average size 1 mo after stocking tended to be positive for WSSV at the end of the production cycle. Average weight at 1 mo was also highlighted in multivariate analyses when considered as either a risk factor or an outcome. Other risk factors identified in univariate analyses were earlier date of stocking and use of commercial feed. A number of variables also appeared to be associated with a reduced risk of WSSV at harvest including the presence of dead post larvae in the batch sampled at stocking, presence of Hemigrapsus spp. crabs during the first month of production, feeding vitamin premix or legumes, presence of high numbers of shrimp with bacterial infection and the presence of larger mud crabs or gobies at harvest. No associations were detected with WSSV at harvest and stocking density, presence, or number or weight of wild shrimp in the pond. The multivariate model to identify outcomes associated with WSSV infection highlighted the presence of high mortality as the main variable explaining the data. The results obtained from this study are discussed in the context of WSD control and areas requiring further investigation are suggested.

Development of a gill perfusion apparatus for studying the interaction of fish pathogens with gill tissue.

An isolated perfused gill preparation was developed for the study of the association of gill pathogens with the branchial tissue. The preparation consisted of an excised branchial arch from common carp (Cyprinus carpio L., minimum weight 300g), perfused via the afferent branchial artery. Filtered and heparinised Cortland solution was used as the perfusion fluid and infused by means of a drip (3-litre bag). The average perfusion rate was 1.5ml/minute/arch/kg body weight. The outflowing perfusate was collected from a cannula in the efferent branchial artery. The individual gill arch was suspended in a circular organ chamber filled with Ringer solution, which was aerated and kept at a constant temperature of 20°C. Unperfused gill arches maintained in Ringer solution at the same temperature served as controls. Cortland solution proved to be a satisfactory perfusion fluid, maintaining the perfused gills in a healthy condition for at least 4 hours with no, or only slight, oedema after 90 minutes, and slight or moderate oedema after 4 hours. The unperfused gill displayed excessive necrosis and loss of architecture after 4 hours. The Cortland perfused gill apparatus could thus prove to be an alternative ex vivo model of particular use in the study of the early interaction of gill associated pathogens with the branchial tissue.