Bath Challenge Research Articles

Detection and persistence of Lymphocystis disease virus (LCDV) in Artemia sp

Lymphocystis disease virus (LCDV) was detected by PCR-hybridisation in Artemia cysts and metanauplii, and the infectivity of these viral particles was confirmed by inoculation on SAF-1 cells. Viral genome and antigens have been detected by whole-mount in situ hybridisation (ISH) and immunofluorescence assay (IFA) in the digestive tract of nauplii hatched from LCDV-contaminated cysts, but not at the umbrella and instar I stages. Instar II nauplii also became LCDV-contaminated after bath challenge. LCDV was detected by ISH and immunohistochemistry (IHC) in the digestive tract and in some cells in the ovisac of adults reared from LCDV-positive instar II nauplii. Reproductive cysts arising from LCDV-contaminated Artemia breeders, as well as their offspring nauplii, were also LCDV-positive. No viral genome and antigens were detected in the eggs, which may indicate an external cyst contamination during spawning. Moreover, viral DNA on cysts disappears by the decapsulation treatment, which may be applied to prevent the transmission of LCDV in Artemia. Infective LCDV particles persist along crustacean life cycle, as demonstrated by cell culture. These findings suggest that Artemia might act as a reservoir of LCDV, although further studies are necessary to establish its role as a vector of this virus to cultured fish.

Susceptibility of pike Esox lucius to a panel of Ranavirus isolates

In order to study the pathogenicity of ranaviruses to a wild European freshwater fish species, pike Esox lucius fry were challenged with the following Ranavirus isolates: epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV), European catfish virus (ECV), pike-perch iridovirus (PPIV), New Zealand eel virus (NZeelV) and frog virus 3 (FV3). The fry were infected using bath challenge at 12 and 22 degrees C. Significant mortalities were observed at 12 degrees C for EHNV, ESV, PPIV and NZeelV. Background mortality was too high in the experiments performed at 22 degrees C for any conclusions about viral pathogenicity at this temperature to be drawn. Viruses could be re-isolated from samples from all challenged groups, and their presence in infected tissue was demonstrated using immunohistochemistry. The findings suggest that pike fry are susceptible to EHNV, ESV, PPIV and NZeelV and can be a vector for ECV and FV3. Statistical analysis of the factors associated with positive virus re-isolation showed that the number of fish in the sample influenced the outcome of virus re-isolation. Moreover, the likelihood of positive virus re-isolation significantly differed among the 6 viral isolates. The temperature from where the sample was taken and the number of days after infection were not associated with the probability of a positive virus re-isolation.

Estimation of infectious dose and viral shedding rates for infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post‐smolts

Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post-smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 10(2) to 10(-4) 50% end-point tissue culture infectious dose (TCID(50)) mL(-1) sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post-smolts was <10(-1) TCID(50) mL(-1) by bath immersion (4 h at 10 degrees C). The peak shedding rate for IPNV following intraperitoneal challenge using post-smolts was estimated to be 6.8 x 10(3) TCID(50) h(-1) kg(-1) and occurred 11 days post-challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.

Specific and natural antibody response of cod juveniles vaccinated against Vibrio anguillarum

The purpose of the present study was to study specific and natural antibody levels in individual cod juveniles before and after being vaccinated against Vibrio anguillarum. Different vaccine preparations and vaccination regimes, i.e. bathing, dipping, i.p. injection or combination of treatments were employed and the performance of different groups to bath challenge by the bacterium tested. Antibody responses to V. anguillarum antigens in groups vaccinated by bathing and/or dipping were negligible, while responses were observed in i.p. injected fish. Fish receiving i.p. injection in addition to bathing, showed significant antibody response. Both groups showed increased levels of natural antibodies while levels were low in other groups. Fish bathed or dipped showed higher mortality when challenged than untreated fish, while fish that received a second vaccination showed the best protection. It was not ascertained whether there is a long term difference between the effects of immersion versus i.p. injection as a booster method. Levels of antibodies against V. anguillarum antigens or natural antibodies in groups with the lowest mortalities show that neither could have been used to predict protection given by the vaccines tested.

Screening and characterisation of potentially pathogenic bacteria associated with Atlantic codGadus morhualarvae: bath challenge trials using a multidish system

In intensive aquaculture systems, high concentrations of nutrients and high densities of fish larvae provide favorable conditions for opportunistic pathogenic bacteria to flourish. We screened potentially pathogenic bacterial strains isolated from moribund Atlantic cod Gadus morhua larvae, pollack Pollachius pollachius, coalfish Pollachius virens, Atlantic halibut Hippoglossus hippoglossus, rotifers, algae and water samples from different hatcheries. Three identical challenge experiments tested a total of 53 strains. A multidish system was used: cod eggs were placed in single wells, together with 2 ml of sterile seawater, and exposed to the bacterial cultures. Final bacterial concentrations in the wells were 10(6) and 10(4) CFU ml(-1). Eggs and larvae not exposed to bacteria were used as unchallenged controls. Challenged controls were exposed to Vibrio anguillarum strain 610. Eggs were challenged approximately 48 h prior to hatching and mortality was recorded daily throughout the yolk-sac period. In spite of the high challenge dose of 106 CFU ml(-1), only 5 bacterial strains tested caused higher mortality than the unchallenged controls. Four of these strains were identified by 16S rDNA and gyrase B gene (GyrB) sequencing as resembling V. anguillarum and 1 strain resembled Carnobacterium sp. Most of the larvae exposed to these strains died within 10 d of challenge. Serotyping of the strains resembling V. anguillarum gave inconclusive results. This indicates differences in serology compared to the serotypes O1, O2 and O3, associated with disease. Three bacterial strains seemed to have a slower infection rate, indicating a longer incubation period. The remaining 45 strains did not seem to have a negative effect on larval survival, suggesting that these are not primary pathogens.

Strong genetic influence on IPN vaccination‐and‐challenge trials in Atlantic salmon, Salmo salar L.

Two series of experimental challenge trials were performed for evaluation of multivalent oil-adjuvanted vaccines with and without an infectious pancreatic necrosis virus (IPNV) antigen component. In both the trial series, Atlantic salmon were hatched, reared, vaccinated and subjected to temperature and light manipulation to induce smoltification. When ready for sea the fish were transported to the VESO Vikan experimental laboratory for bath or cohabitant challenge with IPNV. In the first series, four vaccination and bath challenge trials involving 2-year classes of experimental fish were conducted. In the second series, three groups of eyed eggs of Atlantic salmon allegedly differing in their innate resistance to IPNV were used (Storset, Strand, Wetten, Kjøglum & Ramstad 2007). Hatching, rearing and smoltification were synchronized for each group, and fish from each genetic group were randomly allocated IPN vaccine, reference vaccine or saline before being placed into parallel tanks for bath or cohabitant challenge. In the first series of trials, IPN-specific mortality commenced on day 10-12 after bath challenge. Replicates showed similar results. In trials 1 and 2 belonging to the same experimental fish year class, the average cumulative control mortality reached 60.6% and 79.5%, respectively, whereas in trials 3 and 4 belonging to the following year class the control mortality was consistently below 50%. In the second series of trials, the experimental fish originating from allegedly IPN susceptible parents consistently showed the highest cumulative mortality among the unvaccinated controls (>75%) whereas smolts derived from allegedly IPNV resistant parents showed only 26-35% control mortality. The IPN-vaccinated fish experienced significantly improved survival vs. the fish immunized with reference vaccine, with RPS values above 75% in the IPN susceptible strain. In the IPN resistant strain, the protection outcomes were variable and in part non-significant. The outcome of both the trial series suggests that control mortalities above 50% are necessary to reliably demonstrate specific protection with IPN vaccines.

Field validation of experimental challenge models for IPN vaccines

Atlantic salmon S1/2 pre-smolts from the VESO Vikan hatchery were assigned to study groups, i.p. immunized with commercially available, multivalent oil-adjuvanted vaccines with (Norvax Compact 6 - NC-6) or without (Norvax Compact 4 - NC-4) recombinant infectious pancreatic necrosis virus (IPNV) antigen. A control group received saline solution. When ready for sea, the fish were transported to the VESO Vikan experimental laboratory, where two identical tanks were stocked with 75 fish per group before being transferred to 10 degrees C sea water and exposed by bath to first passage IPNV grown in CHSE-214 cells. The third tank containing 40 fish from each group was challenged by the introduction of 116 fish that had received an i.p injection of IPNV-challenge material. The remaining vaccinated fish were transported to the VESO Vikan marine field trial site and placed in two identical pens, each containing approximately 53 000 fish from the NC-6 group and 9000 fish from the NC-4 group. In the experimental bath challenge trial, the cumulative mortality was 75% and 78% in the control groups, and the relative percentage survival (RPS) of the NC-6-immunized fish vs. the reference vaccine groups was 60% and 82%, respectively. In the cohabitation challenge, the control mortality reached 74% and the IPNV-specific vaccine RPS was 72%. In both models, the reference vaccine lacking IPNV antigen gave a moderate but statistically significant non-specific protection. In the field, a natural outbreak of infectious pancreatic necrosis (IPN) occurred after 7 weeks lasting for approximately 3.5 months before problems due to winter ulcers became dominating. During this outbreak, mortality in the NC-4 groups were 33.5% and 31.6%, respectively, whereas mortality in the NC-6 groups were 6.9% and 5.3%, respectively, amounting to 81% IPNV-specific protection. In conclusion, the IPN protection estimates obtained by experimental challenges were consistent between tanks, and were confirmed by the field results.

Variability among Vibrio anguillarum O2 isolates from Atlantic cod ( Gadus morhua L.): Characterisation and vaccination studies

Farmed Atlantic cod, Gadus morhua L., in Norway are vaccinated against Vibrio anguillarum serotypes O1, O2a and O2b, but outbreaks of classical vibriosis remain relatively common. In the present study, V. anguillarum isolated from vaccinated and unvaccinated cod between 2002 and 2005, were shown to differ in biochemical, serological and genetic characteristics compared to V. anguillarum serotypes O2a and O2b. They did not utilize sorbitol and some were shown to be lysine decarboxylase positive or did not produce gelatinase. Furthermore, they displayed varying reaction patterns with monoclonal antibodies, and appeared in a separate cluster in a similarity dendrogram based on amplified fragment length polymorphisms (AFLP) analysis, distant from the cluster containing both serotypes O2a and O2b. The results indicate that these V. anguillarum isolates belong to a sero-subtype other than O2a or O2b. The efficacy of ALPHA MARINE™ Vibrio vaccine in cod varied depending on the V. anguillarum O2 isolate used for challenge. The vaccine elicited good protection against bath challenge with isolates of V. anguillarum O2b and O2a (RPS > 80) comparable to the vaccine strains, but protected poorly (RPS of 32) or not at all when challenged with two recent and differing isolates from diseased vaccinated cod.

Isolation ofVibrio parahaemolyticus andVibrio cholerae (Non-O1 and O139) from moribund shrimp (Penaeus monodon) and experimental challenge study against post larvae and juveniles

Outbreak ofVibrio infection was reported from a shrimp farm near Chennai, south India. Both green and yellowVibrio were isolated from disease outbreak ofPenaeus monodon (Milne Edwards) culture farm and biochemically confirmed asVibrio parahaemolyticus andVibrio cholerae. Randomly selectedV. parahaemolyticus (VP1) andV. cholerae (VC1) were reconfirmed by partial 16S rRNA gene analysis. Both were tested against post larvae and juveniles ofP. monodon by bath challenge test and intramuscular injection respectively. The study revealed that VP1 is more virulent than VC1 isolated from same source againstP. monodon post larvae and juveniles. Phylogenetic tree based on comparison of partial 16S rRNA gene analysis revealed close relationship of VP1 with other shrimp pathogens likeVibrio harveyi andVibrio alginolyticus. There might be some close relationship for disease development among all these strains.

Cytokine expression in the intestine of rainbow trout ( Oncorhynchus mykiss) during infection with Aeromonas salmonicida

Gene expression of a number of cytokines in the intestine of rainbow trout ( Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1β, IL-8, TNF-α and IFN-γ was increased in the proximal intestine. TGF-β was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1β, IL-8, TNF-α and TGF-β. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection.

Response to selection for resistance against infectious pancreatic necrosis in Atlantic salmon (Salmo salar L.)

Resistance to infectious pancreatic necrosis virus (IPNV) has been included in a commercial selection program for Atlantic salmon since 2001. The IPN selection is based on bath challenge tests of several hundred families of first feeding fry, and the breeding value for each family is estimated from the resultant cumulative mortality rate. Since IPN selection is based on breeding values estimated from the results of a challenge test, it is difficult to evaluate the selection response in the population from generation to generation. Although a heritability of 0.31 has been estimated (unpublished results), we wanted to confirm the effect of selection for IPN resistance. The trial was carried out in two experiments where two groups of first feeding Atlantic salmon fry and postsmolts were challenged with IPNV:A)Families selected for high resistance to IPN, denoted as HR.B)Families from the same breeding population as A) selected for low resistance to IPN, denoted as LR.Both groups were challenged in two different tests: i) in fresh water in seven replicate tanks (7×100 fry). ii) in sea water after smoltification in five replicate tanks. Cumulative mortalities in the groups were significantly different. Group B) had more than double the mortality than group A), both in fresh water and sea water. The average cumulative mortalities in the A)/B) groups were 29.3%/66.6% and 32.0%/79.0% in fresh water and sea water, respectively.

A comparative study of susceptibility and induced pathology of cod, Gadus morhua (L.), and halibut, Hippoglossus hippoglossus (L.), following experimental infection with Moritella viscosa

In this study experimental challenges with Moritella viscosa, the causative agent of winter ulcers in salmonids, were performed on juvenile Atlantic cod and Atlantic halibut. The challenges involved both intramuscular (i.m.) and intraperitoneal (i.p.) injections and bath with a strain originally isolated from Atlantic salmon. Cod was found to be significantly more sensitive than halibut to the infection. Both fish species were found to be more sensitive to i.m. than i.p. challenges. Both challenges induced a systemic disease in cod and halibut, but only cod was infected with an experimental bath challenge. Pathognomonic signs were found to be comparable with those described in M. viscosa-infected salmon and turbot. The main distinguishing pathological sign was that the cod showed host response to M. viscosa infection resulting in granuloma formation in infected tissues, which is a known response of cod to a infection with another Gram-negative bacterium, Aeromonas salmonicida. Re-isolation of M. viscosa from kidneys of cod and halibut with clear disease signs was problematic and optimization of isolation measures is needed. The results of this study indicate that M. viscosa infection may be a risk factor in cod farming, but that halibut is more resistant.

Susceptibility of corkwing wrasse Symphodus melops, goldsinny wrasse Ctenolabrus rupestis, and Atlantic salmon Salmo salar smolt, to experimental challenge with Vibrio tapetis and Vibrio splendidus isolated from corkwing wrasse

The virulence of two Vibrio strains, previously isolated from diseased corkwing wrasse Symphodus melops and identified as V. tapetis and V. splendidus, to corkwing and goldsinny wrasse Ctenolabrus rupestris and to Atlantic salmon Salmo salar, was studied under laboratory conditions. Both bacteria were shown to be opportunistically pathogenic to corkwing wrasse, causing significantly higher mortality in the challenged groups than in the controls. Bacterial cultivation of kidney samples and re-isolation of V. tapetis and V. splendidus from most mortalities confirmed the two strains as the probable cause of mortality in the challenged groups. The control group also suffered relatively high mortality, but no specific pathogens that were suspected to be the main cause of death were isolated, other than a mixture of Vibrio spp. and, in the case of one individual, atypical Aeromonas salmonicida. Following injection challenge with both bacterial strains, no mortality was recorded in Atlantic salmon. In bath challenge trials with goldsinny wrasse, only slight mortality was observed in the challenged groups and the unchallenged control group. Bacterial examination showed that atypical Aeromonas salmonicida was the probable cause of death in both bath challenged and control groups of goldsinny wrasse, and no indication of infection by any Vibrio sp. was found.

First description of non‐motile Yersinia ruckeri serovar I strains causing disease in rainbow trout, Oncorhynchus mykiss (Walbaum), cultured in Spain

Yersinia ruckeri, the causal agent of enteric redmouth (ERM) disease, was isolated from epizootics that occurred in different Spanish rainbow trout, Oncorhynchus mykiss (Walbaum), farms in which vaccination against ERM had been performed. In all episodes, the most pronounced clinical signs exhibited by affected fish were severe haemorrhages in the mouth, eyes and around the vent. The isolates were identified as Y. ruckeri serovar I by 16S rRNA sequencing together with serological tests. They lacked motility and lipase activity and thus belonged to biotype 2, and were highly virulent for juvenile rainbow trout, both by intraperitoneal injection (from 3.1 x 10(2) to 6.3 x 10(3) cfu per fish) and bath challenge (5.1-7.3 x 10(6) cfu mL(-1)). This is the first description of Y. ruckeri serovar I biotype 2 causing disease in cultured trout in Spain vaccinated with commercial ERM vaccines. The occurrence of this emergent pathogen in Spanish continental aquaculture from its first isolation in 2001 to date is also documented.

A method to diagnose the carrier state of Vibrio vulnificus serovar E in eels: Development and field studies

The pathogen Vibrio vulnificus serovar E (VSE) has been related to both human infections and to epizootics causing high mortality in brackish water eel farms. To control the spread of the eel vibriosis and prevent VSE transmission to humans we designed and tested a protocol to detect carriers, which involves isolating the pathogen. To identify the organs where VSE persists in survivors we infected eels with different degrees of immunity against the pathogen (non-immune [NI], immune [I, eels vaccinated 1 year before] and freshly vaccinated [V]) by bath challenge. Then, we followed the pathogen survival in selected external and internal organs for 72 h post-infection. VSE was isolated by using previously described protocols [Sanjuan, E., Amaro, C., 2004. Protocol for specific isolation of virulent strains of V. vulnificus serovar E (biotype 2) from environmental samples. Appl. Environ. Microbiol. 70, 7024–7032.] and identified by a new specific PCR methodology [Lee C.-T., Amaro, C., Sanjuan, E., Hor, L-I., 2005. Identification of DNA sequences specific for V. vulnificus biotype 2 strains by suppression subtractive hybridization. Appl. Environ. Microbiol. 71: 5593–5597.]. The pathogen survival depended on the immunological status of the animal. Thus, VSE persisted in the gills and spleen of NI and I eels but was eliminated by the immune system of V eels in less than 9 h post-infection. We selected gills as the organ to be sampled and designed a whole protocol for carrier detection. The feasibility of the protocol was demonstrated in field studies after the positive isolation of the pathogen from asymptomatic carriers without killing the animals.

Immunohistochemistry of great scallop Pecten maximus larvae experimentally challenged with pathogenic bacteria

Three challenge experiments were carried out on larvae of the great scallop Pecten maximus. Larvae were bath-challenged with Vibrio pectenicida and 5 strains resembling Vibrio splendidus and one Pseudoalteromonas sp. Unchallenged larvae were used as negative controls. The challenge protocol was based on the use of a multidish system, where the scallop larvae (10, 13 and 15 d post-hatching in the 3 experiments, respectively) were distributed to 2 ml wells with stagnant seawater and exposed to the bacterial cultures by bath challenge. Presence of the challenge bacteria in the wells was verified by polymerase chain reaction (PCR). A significantly increased mortality was found between 24 and 48 h in most groups challenged with V. pectenicida or V. splendidus-like strains. The exception was found in larval groups challenged with a Pseudoalteromonas sp. LT 13, in which the mortality rate fell in 2 of the 3 challenge experiments. Larvae from the challenge experiments were studied by immunohistochemistry protocol. Examinations of larval groups challenged with V. pectenicida revealed no bacterial cells, despite a high degree of positive immunostaining. In contrast, intact bacterial cells were found in larvae challenged with V. splendidus. In the case of larvae challenged with the Pseudoalteromonas sp., positive immuno-staining was limited to visible bacteria inside the digestive area and cells of the mucosa. The experiments confirm that V. splendidus and V. pectenicida are pathogenic to scallop larvae, and that the Pseudoalteromonas strain is probably not a primary pathogen, although it cannot be ruled out as a secondary pathogen.

Killing Naked Soldiers: Distinguishing between Combatants and Noncombatants

Just war theorists contended that weapons are illegitimate unless they can be used in such a way so as to distinguish combatants from noncombatants. Contemporary international legal theory also draws heavily on the principle of discrimination. The Geneva Convention (IV), as interpreted in the Second Protocol of 1977, says: “The civilian population as such, as well as individual civilians, shall not be the object of attack…Indiscriminate attacks are prohibited.” In both the Just War tradition and contemporary international law, the main justification for such a principle has to do with noncombatant immunity, the idea that only those who are combatants can legitimately be attacked in war. The principle of discrimination also relies on the idea that it is possible to distinguish, in a morally significant way, those classes or groups of people who participate in wars from those who do not. The categories of “civilian” or “soldier,”“combatant” or “noncombatant,” are thought to be stable. Yet, the case of the naked soldier taking a bath challenges such stability in a way that illustrates the serious conceptual and normative problems with identifying such social groups. In this paper I argue that, because of these problems, the traditional principle of discrimination offers no clear, morally relevant, line between those who fight and those who do not. Nonetheless, I argue that a distinction of this sort should be maintained, although one that will restrict tactics in war far more than is normally recognized.

Early vaccination and protection of Atlantic cod ( Gadus morhua L.) juveniles against classical vibriosis

The effect of vaccinating Atlantic cod ( Gadus morhua L.) juveniles weighing from 1 to 20 g with a commercial vibriosis vaccine (ALPHA MARINE™ Vibrio, PHARMAQ AS) was determined by bath challenge with Vibrio anguillarum serotype O2b. Bath vaccination of cod juveniles of 1 g resulted in a poor protection when challenged 6 weeks later (RPS < 47%). In addition, the durability of the protection was poor since challenge 14 weeks post vaccination resulted in RPS < 36%. Apparently, the cod juveniles need to reach a certain size at the time of vaccination in order to obtain a good protection. A significant increase in protection was demonstrated 7 weeks post dip vaccination at 2.2 g (RPS > 75) compared to 1.7 g (RPS < 54). Moreover, dip vaccination at 5 g elicited a very good and long-lasting protection for at least 28 weeks post vaccination (RPS > 76), that was comparable to the protection obtained 17 weeks post dip or i.p. vaccination at 20 g (RPS > 74).

Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss)

Antiviral immunity in fish is not well understood. In mammals, Toll-like receptor (TLR) 3 is involved in double-stranded RNA recognition and host immune response activation. Here, we report the first identification of a rainbow trout TLR3 ortholog (rtTLR3), its genomic structure, and mRNA regulation. Six exons and five introns were identified from bacterial artificial chromosome (BAC) and expressed sequence tag (EST) sequencing, and this genomic organization is similar to mammalian and fish TLR3 genes. The putative 913 amino acid protein has a Toll/interleukin (IL)-1R (TIR) domain, a transmembrane domain, and leucine-rich repeats. In healthy trout, rtTLR3 is highly expressed in the liver, pyloric ceca, intestine, spleen, and anterior and trunk kidney tissues. To investigate whether rtTLR3 is involved in antiviral immunity, transcriptional regulation in vivo was examined by quantitative real-time polymerase chain reaction (PCR) after poly inosinic:cytidylic (I:C) and infectious hematopoietic necrosis virus (IHNV) treatments. TLR3 mRNA expression peaked 1 day after poly (I:C) injection of live animals, while the peak of gene expression after live IHNV challenge was observed on day 3. In vitro stimulation of rainbow trout anterior kidney leukocytes with poly (I:C) also enhanced rtTLR3 expression. Up-regulation was specific to viral challenge as there was no significant up-regulation of rtTLR3 mRNA levels in the spleen and a modest down-regulation in the anterior kidney after bath challenge with a gram-negative bacterial trout pathogen, Yersinia ruckeri. The sequence conservation of trout TLR3 and mRNA regulation after poly (I:C) or RNA virus exposures strongly suggest a role for trout TLR3 in antiviral immunity.

Sequential Pathology after Experimental Infection with Marine Viral Hemorrhagic Septicemia Virus Isolates of Low and High Virulence in Turbot (Scophthalmus maximus L.)

Three marine viral hemorrhagic septicemia virus isolates were used to bath challenge turbot with the purpose of studying mortality and the pathology and antigen distribution over time. Two high-virulence isolates, 860/94, 4p168 and a low-virulence isolate 1p3 from a Baltic Sea herring were used. Organ samples were collected sequentially at 2, 4, 7, 10, 15, 20, 25, and 45 days postinfection. Specimens were processed for virology, histopathology, and immunohistochemistry. Organs during the early stages of infection (from 2 to 7 days) had virus isolation from all three groups only on day 7. Virus titer in kidney and heart sampled at day 25 was higher for the two virulent isolates compared with the low-virulence isolate. The viral distribution in situ of the two more virulent isolates from turbot (860/94) and herring (4p168) resembled viral hemorrhagic septicemia in rainbow trout with regard to the target organs. Early infection of endothelial cells in both kidney and heart was observed. Accumulated mean mortality was 41.5% for the turbot isolate 860/94, 48% for the herring isolate 4p168, and 3.5% for the herring isolate 1p3. This study revealed that the isolates from turbot (860/94) and herring (4p168) induced significantly higher mortality compared with the virus-free control and the herring isolate (1p3). The onset of mortality is markedly later in turbot compared with what is seen in rainbow trout.