Cohabitation Trial Research Articles

Decreased water temperature enhance Piscine orthoreovirus genotype 3 replication and severe heart pathology in experimentally infected rainbow trout.

Piscine orthoreovirus genotype 3 (PRV-3) was first discovered in Denmark in 2017 in relation to disease outbreaks in rainbow trout (Oncorhynchus mykiss). While the virus appears to be widespread in farmed rainbow trout, disease outbreaks associated with detection of PRV-3 have only occurred in recirculating aquaculture systems, and has predominantly been observed during the winter months. To explore the possible effects of water temperature on PRV-3 infection in rainbow trout, an in vivo cohabitation trial was conducted at 5, 12, and 18°C. For each water temperature, a control tank containing mock-injected shedder fish and a tank with PRV-3 exposed fish were included. Samples were collected from all experimental groups every 2nd week post challenge (WPC) up until trial termination at 12 WPC. PRV-3 RNA load measured in heart tissue of cohabitants peaked at 6 WPC for animals maintained at 12 and 18°C, while it reached its peak at 12 WPC in fish maintained at 5°C. In addition to the time shift, significantly more virus was detected at the peak in fish maintained at 5°C compared to 12 and 18°C. In shedders, fish at 12 and 18°C cleared the infection considerably faster than the fish at 5°C: while shedders at 18 and 12°C had cleared most of the virus at 4 and 6 WPC, respectively, high virus load persisted in the shedders at 5°C until 12 WPC. Furthermore, a significant reduction in the hematocrit levels was observed in the cohabitants at 12°C in correlation with the peak in viremia at 6 WPC; no changes in hematocrit was observed at 18°C, while a non-significant reduction (due to large individual variation) trend was observed at cohabitants held at 5°C. Importantly, isg15 expression was positively correlated with PRV-3 virus load in all PRV-3 exposed groups. Immune gene expression analysis showed a distinct gene profile in PRV-3 exposed fish maintained at 5°C compared to 12 and 18°C. The immune markers mostly differentially expressed in the group at 5°C were important antiviral genes including rigi, ifit5 and rsad2 (viperin). In conclusion, these data show that low water temperature allow for significantly higher PRV-3 replication in rainbow trout, and a tendency for more severe heart pathology development in PRV-3 injected fish. Increased viral replication was mirrored by increased expression of important antiviral genes. Despite no mortality being observed in the experimental trial, the data comply with field observations of clinical disease outbreaks during winter and cold months.

Temperature-associated virulence, species susceptibility and interspecies transmission of a Lactococcus petauri strain from rainbow trout.

Lactococcus petauri is an important emergent aquaculture pathogen in the USA. To better understand environmental conditions conducive to piscine lactococcosis and the susceptibility of fish species, laboratory-controlled challenges were used as models of infection. Rainbow trout Oncorhynchus mykiss maintained at 13 or 18°C were challenged by intracoelomic (ICe) injection with 101, 103 or 105 colony-forming units per fish (CFU fish-1) and monitored for 21 d. At 13°C, trout experienced mortalities of 7, 7 and 0%, and bacterial persistence of 0, 20 and 0% in survivors, respectively. When exposed to the same bacterial doses, trout maintained at 18°C experienced mortalities of 59, 84 and 91%, and bacterial persistence of 60, 66 and 0% in survivors, confirming a significant role of temperature in the pathogenesis of lactococcosis. Additionally, the susceptibility of rainbow trout, Chinook salmon Oncorhynchus tshawytscha, white sturgeon Acipenser transmontanus, Nile tilapia Oreochromis niloticus, and koi Cyprinus carpio to infection by L. petauri was compared using ICe challenges at 18°C. Trout and salmon experienced 96 and 56% cumulative mortality, respectively, and 17% of surviving salmon remained persistently infected. There were no mortalities in the other fish species, and no culturable bacteria recovered at the end of the challenge. However, when surviving fish were used in further cohabitation trials, naïve trout housed with previously exposed tilapia exhibited 6% mortality, demonstrating that non-salmonids can become sub-clinical carriers of this pathogen. The data obtained provide useful information regarding temperature-associated virulence, fish species susceptibility, and potential carrier transmission of L. petauri that can be used in the development of better management practices to protect against piscine lactococcosis.

Commercial Vaccines Do Not Confer Protection against Two Genogroups of Piscirickettsia salmonis, LF-89 and EM-90, in Atlantic Salmon.

Simple SummaryVaccination represents one of the most relevant strategies to prevent and control infectious diseases in aquaculture. However, vaccines have failed to control and prevent Piscirickettsia salmonis, a bacterium that causes large economic losses to the industry. Therefore, we evaluated the performance of two commercial vaccines in Atlantic salmon through a cohabitation challenge (healthy fish were challenged by cohabitation with infected fish) of the two most prevalent and ubiquitous Piscirickettsia genetic variants in Chile. We found no evidence that vaccines confer protection against the LF-89 or EM-90 genogroups in Atlantic salmon.In Atlantic salmon, vaccines have failed to control and prevent Piscirickettsiosis, for reasons that remain elusive. In this study, we report the efficacy of two commercial vaccines developed with the Piscirickettsia salmonis isolates AL100005 and AL 20542 against another two genogroups which are considered highly and ubiquitously prevalent in Chile: LF-89 and EM-90. Two cohabitation trials were performed to mimic field conditions and vaccine performance: (1) post-smolt fish were challenged with a single infection of LF-89, (2) adults were coinfected with EM-90, and a low level coinfection of sea lice. In the first trial, the vaccine delayed smolt mortalities by two days; however, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 60.3%, vaccinated: 56.7%; p = 0.28). In the second trial, mortality started three days later for vaccinated fish than unvaccinated fish. However, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 64.6%, vaccinated: 60.2%, p = 0.58). Thus, we found no evidence that the evaluated vaccines confer effective protection against the genogroups LF-89 and EM-90 of P. salmonis with estimated relative survival proportions (RPSs) of −9% and −12%, respectively. More studies are necessary to evaluate whether pathogen heterogeneity is a key determinant of the lack of vaccine efficacy against P. salmonis.

Mass mortality of pearl oyster (Pinctada fucata (Gould)) in Japan in 2019 and 2020 is caused by an unidentified infectious agent.

Mass mortality of 0-year-old pearl oysters, Pinctada fucata (Gould), and anomalies in adults were observed in Japan’s major pearl farming areas in the summer of 2019 and 2020. Although adult oyster mortality was low, both adult and juvenile oysters underwent atrophy of the soft body, detachment of the mantle from nacre (the shiny inner surface of the valves), deposition of brownish material on the nacre, and loss of nacre luster. Infection trials were conducted to verify the involvement of pathogens in this phenomenon. Healthy adult pearl oysters were obtained from areas where this disease had not occurred to use as the recipients. The sources of infection were either affected adult oysters with atrophied soft bodies or batches of juveniles in which mortality had reached conspicuous levels. Transmission of the disease to the healthy oysters were tested either by cohabitation with affected oysters or by injections of the hemolymph of affected animals. The injection infection test examined the effects of filtration and chloroform exposure on the pathogen. Occurrence of the disease was confirmed by the appearance of brown deposits on the nacre and loss of nacre luster. The abnormalities of nacre were clearly reproduced in recipient shells in three out of four cohabitation trials with affected oysters. The disease was also reproduced in six out of six injection trails either with hemolymph filtered through 100 nm filter or with hemolymph treated with chloroform. In a serial passage with hemolymph injections, the disease was successfully transmitted through eight passages. These results suggest that the etiology of the disease is a non-enveloped virus with a diameter ≤100 nm.

On the trail of detecting genetic (co)variation between resistance to parasite infections (Diplectanum aequans and Lernanthropus kroyeri) and growth in European seabass (Dicentrarchus labrax)

The European seabass is one of the main commercial fish produced in Mediterranean marine aquaculture. Recently, its production has been negatively affected by losses due to frequent and recurring outbreaks of parasitic, bacterial and viral diseases. In recent years, the gill parasites Diplectanum aequans and Lernanthropus kroyeri are increasingly becoming more dominant and contributing more significantly to the observed losses. Genetic improvement for disease resistance represents an important strategy for controlling infectious diseases in farmed fish by increasing their robustness. In order to determine the possibility of including such trait in selective breeding programs, we need to comprehend whether additive genetic variation for resistance against Diplectanum aequans and Lernanthropus kroyeri exists. For this purpose, two open-sea parasite cohabitation trials (for two consecutive years) were performed in the commercial production sites of a private company (Nireus S.A), that had high infestation with Diplectanum aequans and Lernanthropus kroyeri. Juvenile European seabass (9425 offspring from 91 full-sib and half-sib families per year), originating from the company's breeding program were equally divided into two groups and transferred to two commercial farming sites located in the areas of Nafpactos and Sagiada, in western Greece, for the intended cohabitation studies. The parasite numbers on all the gill arches were counted and recorded at the end of the trials for the infestation levels. A third site (Palairos) without any parasite infestation was used as a control site. A multi-trait animal model was used to estimate the variance-covariance components and to evaluate the genetic parameters for Parasite Counts, recorded from all the gill arches of the fish, and their corresponding growth in sea cages. The estimated heritabilities for parasite count, using untransformed data, were 0.20 (D. aequans) and 0.28 (L. kroyeri) and for transformed data 0.29 and 0.26, respectively. The heritability estimates for body weight were 0.42−0.51 for D. aequans and 0.28−0.51 for L. kroyeri trials. Similarly, estimated heritabilities for the growth in sea cages were 0.43 and 0.29, respectively. Although parasite count has a low to medium unfavorable genetic correlation with body weight and growth (0.09−0.37), it seems that it is not significantly impairing selection for growth. Furthermore, the results of this study are very promising in terms of the existing potential for genetic improvement of parasite resistance, and provides a good basis for further genetic analysis using molecular markers.

Transmission pattern of the parasitic dinoflagellate Hematodinium perezi in polyculture ponds of coastal China

Since 2012, Hematodinium perezi epizootics have occurred frequently in cultured swimming crabs Portunus trituberculatus in polyculture ponds along the coast of Shandong Province, China. These outbreaks have resulted in significant economic losses to local farmers. The cryptic transmission pattern of H. perezi has hampered the prevention and control of the pathogen in affected farms. The mudflat crab Helice tientsinensis is one of the dominant wild crabs in the region with considerable prevalence of the parasitic infection covering the full culture period of P. trituberculatus. Mudflat crabs can migrate in and out of polyculture ponds during water exchange and may act as an important reservoir host and source of H. perezi for newly seeded P. trituberculatus. During 2019, we carried out continuous field investigations and sentinel experiments in the polyculture pond system, as well as a series of laboratory infection trials, to study whether waterborne transmission of H. perezi can occur in polyculture ponds, and to determine whether H. perezi can be transmitted from H. tientsinensis to cultured P. trituberculatus. The sentinel experiment verified waterborne transmission of H. perezi to cultured P. trituberculatus in the polyculture pond. The cohabitation trial further indicated that H. perezi was transmittable from H. tientsinensis to P. trituberculatus through waterborne transmission, whereas P. trituberculatus was not able to acquire H. perezi infection by feeding on tissues of infected H. tientsinensis. The results highlighted the transmission pattern of the parasitic dinoflagellate H. perezi in the polyculture pond systems widely used along the coast line of China. In addition, the mudflat crab H. tientsinensis was confirmed as a reservoir host of H. perezi in the epidemic area, and confirmed as the likely source of the parasite during the intensive culture of P. trituberculatus.

The role of invasive tunicates as reservoirs of molluscan pathogens

Ascidian tunicates frequently display rapid expansion when introduced beyond their native range and are considered successful invaders. This invasive potential may be exacerbated by a warming climate, allowing for the occupation of environmental niches previously held by native species. Research into tunicate invasion ecology is prevalent, but less is known about their role in pathogen maintenance. This study investigated the impact of invasive tunicates on the maintenance of pathogens that affect commercial bivalves, including the cultured species Ostrea edulis (European flat oyster) and Crassostrea gigas (Pacific oyster), and the fished species Cerastoderma edule (Common cockle). Focal pathogens included ostreid herpesvirus OsHV-1 μVar, Vibrio aestuarianus, Bonamia ostreae and Minchinia spp. The range of pathogens in their molluscan hosts was determined and the tunicates Botrylloides violaceus, Didemnum vexillum and Styela clava were then screened for these same pathogens, using both field samples from oyster culture sites and marinas and a series of laboratory cohabitation trials. Sample sites reflected areas close to and further away from known pathogen sources. PCR, Sanger sequencing and histology confirmed the presence of B. ostreae and Minchinia mercenariae-like in S. clava, and V. aestuarianus was confirmed by qPCR in B. violaceus and D. vexillum. Furthermore, histology confirmed Minchinia mercenariae-like sporonts in S. clava suggesting that the tunicate can facilitate replication of this species. S. clava also maintained B. ostreae in tanks with no oysters present. The results indicate that tunicates can act as reservoirs of infection in areas where disease occurs and potentially transport diseases to uninfected sites.

Genome wide analysis of infectious salmon anemia resistance in commercial Saint John River Atlantic salmon

Infectious salmon anemia (ISA) is an economically costly viral disease to the global Atlantic salmon aquaculture industry due to the high virulence and high mortality rate. Selective breeding for disease resistance traits using genomic information has been suggested to increase the resistance to various diseases in the breeding population. The aim of this study was to perform a genome wide association (GWA) analysis to identify regions of the genome associated with ISA resistance in the commercial Saint John River population. A total of 2233 parr from 72 families from the 2015 year class were challenged with ISA using a cohabitation trial. The heritability of ISA resistance was 0.16 ± 0.04 on the observed scale for the binary trait of ISA survival at 50% mortality of all cohabitating fish. Binary phenotypes were recorded for all fish with those that survived the challenge being classified as resistant and those that died being classified as susceptible. Selective genotyping using a custom North American 50 K SNP chip was performed by choosing, where possible, four resistant fish and four susceptible fish from each challenged family (n = 572). After quality control a total of 547 fish and 38,954 SNP markers remained for GWA analysis. Two survival traits were analyzed for the genotyped fish: ‘survival to day 37’ when mortality had reached 50% and ‘survival at trial termination’ on day 54. Two SNPs on Ssa03 and on Ssa07 were significant associated with ‘survival to day 37’ but only at the chromosome wide level. More notably nine significant SNPs on nine different chromosomes, were significantly associated with ‘survival at trial termination’ at the genome wide level. The most significant SNP, on Ssa13, explained 8.6% of the phenotypic variation, while SNPs on Ssa12 and Ssa11 explained 7.6% and 6.4% respectively. The remaining six SNPs explained between 4 and 6% of the phenotypic variation, indicating oligogenic architecture of the ‘survival at trial termination’ trait. A total of 33 genes known to be differentially expressed during infection with ISA were located downstream on the same chromosome arm of the nine significant SNPs. Identification of causal mutations is not necessary to implement genomic information into selective breeding programs but improves understanding of the genetic basis of ISA resistance.

Contrasting outcomes of Vibrio harveyi pathogenicity in gilthead seabream, Sparus aurata and European seabass, Dicentrachus labrax

Vibrio harveyi has been reported as the dominant heterotrophic bacterial species in western Mediterranean coastal areas during warm seasons, and is recognized as an economically significant pathogen for the aquaculture industry. The present work aimed to evaluate the pathogenicity of a V. harveyi strain isolated from ascitic fluid collected from cultured gilthead seabream and then used in a challenge experiment involving the two most important fish species in Mediterranean aquaculture: gilthead seabream, Sparus aurata and European seabass, Dicentrarchus labrax. The ascitic fluid from diseased juvenile seabreams, previously vaccinated against Photobacterium damselae and Vibrio anguillarum, was extracted and bacteria cultivated for isolation and characterization. Additionally, different tissues were sampled for histological evaluation and description. Significant histopathological responses were observed in hepatic and mucosal tissues. One of the strains isolated from ascitic fluid, IRTA 17-43, was selected for a bacterial challenge. Additionally, the attenuation of virulence through sequential passage of the strain on solid media was also assessed. In parallel, a co-habitation trial was performed in order to evaluate the possible transfer of the bacteria between injected and healthy individuals. Pathogenicity trials in gilthead seabream resulted in only 25% mortality when injected with 107 CFU mL−1, whereas, for European seabass, a mortality of 95% was recorded, with clear signs of vibriosis. When passed sequentially on solid media, the strain IRTA-17-43 showed a decrease of 35% in cumulative mortality for European seabass. No apparent transmission of the pathogen occurred during the co-habitation trial for both species. In conclusion, although few external signs of V. harveyi are observed in vaccinated carriers, internal effects of the infection were clear and severe. Although no horizontal transfer of infection was observed, the risk of occurrence between carriers and immunosuppressed individuals or between different species should be considered. This further validates that the establishment of a good health management system within fish farms is of major importance in order to avoid the onset of disease outbreaks.

Predicting the ecological impacts of an alien invader: Experimental approaches reveal the trophic consequences of competition

Ecological theory on the trophic impacts of invasive fauna on native competitors is equivocal. Whilst increased interspecific competition can result in coexisting species having constricted and diverged trophic niches, the competing species might instead increase their niche sizes to maintain energy intakes. Empirical experiments can test invasion theory on competitive interactions and niche sizes across different spatial scales and complexity. The consequences of increased interspecific competition from a model alien fish Leuciscus idus were tested on two taxonomically and trophically similar native fishes, Squalius cephalus and Barbus barbus. Competitive interactions were tested in tank aquaria using comparative functional responses (CFRs) and cohabitation trials. The consequences of these competitive interactions for the trophic niche sizes and positions of the fishes were tested in pond mesocosms. Comparative functional responses revealed that compared to B.barbus, L.idus had significantly higher attack and consumption rates; cohabitation trials revealed B.barbus growth rates were depressed in sympatry with L.idus. For L.idus and S.cephalus, differences in their functional response parameters and growth rates were not significant. Pond mesocosms used stable isotope metrics to quantify shifts in the trophic niche sizes of the fishes between allopatry and sympatry using a substitutive experimental design. Isotopic niches were smaller and more divergent in sympatric paired species than predicted by their allopatric treatments, suggesting trophic impacts from interspecific competition. However, an all-species sympatric treatment revealed similar niche sizes with allopatry. This maintenance of niche sizes in the presence of all species potentially resulted from the buffering of direct competitive effects of the species pairs by indirect effects. Experimental predictions from tank aquaria assisted the interpretation of the constricted and diverged trophic niches detected in the paired-species sympatric treatments of the pond mesocosms. However, the all-species sympatric treatment of this experiment revealed greater complexity in the outcomes of the competitive interactions within and between the species. These results have important implications for understanding how alien species integrate into food webs and influence the trophic relationships between native species.

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV.

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land‐based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real‐time RT‐PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re‐isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%–50% of the cohabiting lumpfish survived after 4 weeks. 80%–92% of the Atlantic salmon survived, but no viral RNA was detected by real‐time RT‐PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.

Hypoxia tolerance and responses to hypoxic stress during heart and skeletal muscle inflammation in Atlantic salmon (Salmo salar).

Heart and skeletal muscle inflammation (HSMI) is associated with Piscine orthoreovirus (PRV) infection and is an important disease in Atlantic salmon (Salmo salar) aquaculture. Since PRV infects erythrocytes and farmed salmon frequently experience environmental hypoxia, the current study examined mutual effects of PRV infection and hypoxia on pathogenesis and fish performance. Furthermore, effects of HSMI on hypoxia tolerance, cardiorespiratory performance and blood oxygen transport were studied. A cohabitation trial including PRV-infected post-smolts exposed to periodic hypoxic stress (4 h of 40% O2; PRV-H) at 4, 7 and 10 weeks post-infection (WPI) and infected fish reared under normoxic conditions (PRV) was conducted. Periodic hypoxic stress did not influence infection levels or histopathological changes in the heart. Individual incipient lethal oxygen saturation (ILOS) was examined using a standardized hypoxia challenge test (HCT). At 7 WPI, i.e. peak level of infection, both PRV and PRV-H groups exhibited reduced hypoxia tolerance compared to non-infected fish. Three weeks later (10 WPI), during peak levels of pathological changes, reduced hypoxia tolerance was still observed for the PRV group while PRV-H performed equal to non-infected fish, implying a positive effect of the repeated exposure to hypoxic stress. This was in line with maximum heart rate (fHmax) measurements, showing equal performance of PRV-H and non-infected groups, but lower fHmax above 19°C as well as lower temperature optimum (Topt) for aerobic scope for PRV, suggesting reduced cardiac performance and thermal tolerance. In contrast, the PRV-H group had reduced hemoglobin-oxygen affinity compared to non-infected fish. In conclusion, Atlantic salmon suffering from HSMI have reduced hypoxia tolerance and cardiac performance, which can be improved by preconditioning fish to transient hypoxic stress episodes.

Experimental challenges with Renibacterium salmoninarum in Arctic charr Salvelinus alpinus.

Arctic charr Salvelinus alpinus L. is an important species in Icelandic aquaculture and the most common wild salmonid in Iceland. A study on the course of infection with the bacterium Renibacterium salmoninarum was conducted using 3 different challenge methods in brackish and fresh water. Bacterial isolation, ELISA and PCR tests were used for detection of the bacterium in multiple organ samples. In an experiment, run for 34 wk in brackish water, infection was established by intraperitoneal injection with 5 × 106 colony forming units (CFU) fish-1. There were external and internal symptoms of bacterial kidney disease (BKD) and mortalities between 6 and 13 wk after injection. A cohabitation trial was run simultaneously and infection was well established after 4 wk, as demonstrated by the detection methods applied. Symptoms of BKD were not seen and all but 1 cohabitant survived. In a separate experiment, infection was established by pumping a fixed amount of water from a tank with fingerlings infected by intraperitoneal injection into tanks with naïve fish, in fresh or brackish water, for 6 wk. Fish in the inflow tanks were reared for an additional 3 wk. There were neither macroscopic symptoms nor mortalities. ELISA and PCR tests showed that infection started to take hold after 3 wk. The challenge trials demonstrated that Arctic charr is susceptible to R. salmoninarum. Cohabitation and inflow of water from tanks with infected fish provide useful models for further studies on R. salmoninarum infection acquired in a natural way in Arctic charr.

Selective precipitation reaction: a novel diagnostic test for tissue pathology in Atlantic salmon, Salmo salar, infected with salmonid alphavirus (SAV3).

While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on‐farm qualitative test and an in‐laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (Δ340). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the Δ340 in infected fish rose from undetectable to a maximum at 6 weeks post‐infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.

Horizontal transmission dynamics of White spot syndrome virus by cohabitation trials in juvenile Penaeus monodon and P. vannamei

White spot syndrome virus (WSSV), a rod-shaped double-stranded DNA virus, is an infectious agent causing fatal disease in shrimp farming around the globe. Within shrimp populations WSSV is transmitted very fast, however, the modes and dynamics of transmission of this virus are not well understood. In the current study the dynamics of disease transmission of WSSV were investigated in small, closed populations of Penaeus monodon and Penaeus vannamei. Pair cohabitation experiments using PCR as a readout for virus infection were used to estimate transmission parameters for WSSV in these two species. The mortality rate of contact-infected shrimp in P. monodon was higher than the rate in P. vannamei. The transmission rate parameters for WSSV were not different between the two species. The relative contribution of direct and indirect transmission rates of WSSV differed between the two species. For P. vannamei the direct contact transmission rate of WSSV was significantly lower than the indirect environmental transmission rate, but for P. monodon, the opposite was found. The reproduction ratio R0 for WSSV for these two species of shrimp was estimated to be above one: 2.07 (95%CI 1.53, 2.79) for P. monodon and 1.51 (95%CI 1.12, 2.03) for P. vannamei. The difference in R0 between the two species is due to a lower host mortality and hence a longer infectious period of WSSV in P. monodon.

Persistence of Tetracapsuloides bryosalmonae (Myxozoa) in chronically infected brown trout Salmo trutta.

Proliferative kidney disease (PKD) is a widespread disease of farmed and wild salmonid populations in Europe and North America, caused by the myxozoan parasite Tetracapsuloides bryosalmonae. Limited studies have been performed on the epidemiological role in spread of the disease played by fish that survive infection with T. bryosalmonae. The aim of the present study was to evaluate the persistence of T. bryosalmonae developmental stages in chronically infected brown trout Salmo trutta up to 2 yr after initial exposure to laboratory-infected colonies of the parasite's alternate host, the bryozoan Fredericella sultana. Kidney, liver, spleen, intestine, brain, gills and blood were sampled 24, 52, 78 and 104 wk post-exposure (wpe) and tested for T. bryosalmonae by PCR and immunohistochemistry (IHC). Cohabitation trials with specific pathogen free (SPF) F. sultana colonies were conducted to test the viability of T. bryosalmonae. PCR detected T. bryosalmonae DNA in all tissue samples collected at the 4 time points. Developmental stages of T. bryosalmonae were demonstrated by IHC in most samples at the 4 time points. Cohabitation of SPF F. sultana with chronically infected brown trout resulted in successful transmission of T. bryosalmonae to the bryozoan. This study verified the persistence of T. bryosalmonae in chronically infected brown trout and their ability to infect the bryozoan F. sultana up to 104 wpe.

Is horizontal transmission of the Ostreid herpesvirus OsHV-1 in Crassostrea gigas affected by unselected or selected survival status in adults to juveniles?

Massive mortality outbreaks, mostly affecting spat Crassostrea gigas, have been reported in France since 2008. Disease investigations revealed that most of the mortality events are related to the detection of the Ostreid herpesvirus 1 (OsHV-1 μVar). Meanwhile, selection to improve the survival in juvenile C. gigas has been successfully implemented in this context and selected oysters were found disease-resistant to OsHV-1 infection. This paper reports the first investigation of the horizontal transmission of the disease throughout cohabitation trials within batch and between batches using unselected and selected oysters for their higher survival. Two batches, unselected (AC) and selected (AR), were produced in February 2009 and deployed in the field in August 2009. Mortality associated with OsHV-1 detection occurred within the first two weeks of the deployment to reach 19% and 56% for the AR and AC batches, respectively. The remaining alive oysters were then brought into the laboratory in March 2010 for the cohabitation trials to assess the potential horizontal transmission of OsHV-1 within batch or between one of the batches and a third. The third batch (J) was produced using unselected oysters in August 2009 and it was always kept in the hatchery and the nursery to isolate it from the mortality risk factors. At the end of the cohabitation trials in July 2010, no mortality was found for the cohabitation trials between AR and J oysters as well as for the AR oysters and also for the J oysters grown alone in a tank. Conversely, high mortality was reported for the cohabitation trials using the J and AC batches as well as for the AC oysters alone. For the later, mortality occurred 3days post-spawning while the AR oysters, which also spawned the same day, did not suffer of any abnormal mortality. Disease screening revealed that OsHV-1 was not detected for the J batch in March 2010, while 4 and 46% of the AR and AC oysters were found positive seven months post-mortality, indicating a difference in potential reservoir role within the C. gigas species. Our study clearly revealed the horizontal transmission of the disease from unselected asymptomatic adult to juvenile C. gigas, inducing mortality, and that OsHV-1 resistant oysters may offer one way to limit the spread of the disease and the potential reservoir role. Mortality due to OsHV-1 and its kinetic depended of the seawater temperature, which appeared as a strong risk factor that can reactivate the virus from latent-like or sub-clinical infection in asymptomatic adults. The lower threshold of 14°C to observe mortality due to OsHV-1 was evidenced even if its replication was activated during the two previous weeks when seawater temperature was 16°C. Finally, a sub-clinical level of virus was observed in AC asymptomatic oysters, it can be reactivated and cause viral replication, suggesting latency-like infection can exist in OsHV-1.

Genetic Sequence Data Identifies the Cercaria of Drepanocephalus spathans (Digenea: Echinostomatidae), A Parasite of the Double-Crested Cormorant (Phalacrocorax auritus), with Notes on its Pathology in Juvenile Channel Catfish (Ictalurus punctatus)

An unidentified xiphidio-type cercaria, previously thought inconsequential to catfish health, was found to be released from marsh rams-horn snails (Planorbella trivolvis) inhabiting ponds on a commercial catfish operation in the Mississippi Delta. A preliminary challenge of cohabiting channel catfish ( Ictalurus punctatus ) with snails actively shedding the unidentified cercariae resulted in death of some fish. A second cohabitation trial yielded similar results, as did a third challenge of 250 cercariae/fish. Histopathology revealed developing metacercariae concentrated in the cranial region, especially within the branchial chamber, with several metacercariae at the base of the branchial arches within, or adjacent to, blood vessels, possibly the proximate cause of death. Genetic sequence analysis of the 18S small subunit ribosomal DNA (ssDNA), 28S large subunit rDNA (lsDNA), and cytochrome oxidase (Cox1) genes all matched the cercariae to Drepanocephalus spathans (Digenea: Echinostomatidae), a parasite of the double-crested cormorant (Phalacrocorax auritus), a piscivorous bird endemic on most catfish farms. This is the first commentary regarding pathology of D. spathans in juvenile channel catfish as well as the first report of the marsh rams-horn snail as an intermediate host in the D. spathans life cycle. The data presented here suggest this parasite could have limiting effects on catfish production, further supporting the need for adequate snail control programs to reduce trematode prevalence on commercial catfish operations.

A comparative study of marine salmonid alphavirus subtypes 1–6 using an experimental cohabitation challenge model

A comparative challenge study of six marine isolates representing subtypes 1-6 of salmonid alphavirus (salmon pancreas disease virus, Genus Alphavirus, Family Togaviridae) was conducted in Atlantic salmon in a fresh water cohabitation trial. Histopathological lesions typical of pancreas disease were observed with all subtypes, and virus was re-isolated from serum of cohabitant fish in each case. Using a virus neutralization (VN) test neutralizing salmonid alphavirus (SAV) subtype 1 strain F93-125, VN antibodies were detected in all challenge groups, consistent with serological cross-reactivity between these subtypes. Using real-time RT-PCR, SAV RNA was detected in heart tissue from 2 to 3 weeks post-challenge (wpc) in all cohabitant groups excluding controls. The results obtained suggested differences in the dynamics of infection between strains of SAV and potentially between subtypes. Results for SAV subtypes 1 and 3 suggested essentially synchronous infection of cohabitant fish. These two study groups also had the highest virus load in heart tissue as measured by quantitative RT-PCR and also had the most extensive histopathological changes. In contrast, results for SAV subtypes 2 and 6 strains were consistent with asynchronous infection in the cohabitant fish and were characterized by slow spread, low virus loads and mild histopathological changes. The SAV subtype 4 and 5 strains occupied an intermediate position in this regard. Despite the use of concentration procedures, it was not possible to detect SAV RNA in water samples from selected study tanks. However, testing of faeces from the SAV subtypes 1, 3 and 6 challenge groups found positive signals in each beginning at 1-3 wpc and remaining detectable for a further 2-3 weeks. Parallel testing of mucus samples found these became positive at 2-3 wpc and remained positive for a further 1-3 weeks. These results demonstrate for the first time that shedding and transmission of virus may occur by both these routes and suggest that dispersal in these matrices should be included in any disease transmission models.

Experimental ostreid herpesvirus 1 infection of the Pacific oyster Crassostrea gigas: Kinetics of virus DNA detection by q-PCR in seawater and in oyster samples

Herpes- and herpes-like viruses are known to infect a wide range of bivalve mollusc species throughout the world. Abnormal summer mortalities associated to the detection of ostreid herpesvirus 1 (OsHV-1) have been currently reported in France among larvae and spat of the Pacific cupped oyster Crassostrea gigas. In the present work, we have developed an experimental protocol of horizontal transmission based on the cohabitation between healthy and experimentally infected oysters. Through a cohabitation trial, the kinetics of OsHV-1 detection in different oyster organs and seawater samples were investigated and characterized for the first time using real time quantitative PCR.