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It is our opinion that Hamazaki et al. (2013; Dis Aquat Org 105:21-25) overstate the usefulness of PCR as a field diagnostic technique and underestimate the accuracy and utility of in vitro explant culture. In order for field diagnostic studies to be meaningful they should accurately and dependably identify the infected individuals within a population, both subclinical and clinical cases. Although explant culture, like most techniques, can miss some infected individuals, 'false positives' are impossible, unlike for cPCR based methodologies.

Skin homeostasis is critical to preserve animal integrity. Although the skin of most vertebrates is known to contain a skin-associated lymphoid tissue (SALT), very little is known about skin B-cell responses as well as their evolutionary origins. Teleost fish represent the most ancient bony vertebrates containing a SALT. Due to its lack of keratinization, teleost skin possesses living epithelial cells in direct contact with the water medium. Interestingly, teleost SALT structurally resembles that of the gut-associated lymphoid tissue, and it possesses a diverse microbiota. Thus, we hypothesized that, because teleost SALT and gut-associated lymphoid tissue have probably been subjected to similar evolutionary selective forces, their B-cell responses would be analogous. Confirming this hypothesis, we show that IgT, a teleost immunoglobulin specialized in gut immunity, plays the prevailing role in skin mucosal immunity. We found that IgT(+) B cells represent the major B-cell subset in the skin epidermis and that IgT is mainly present in polymeric form in the skin mucus. Critically, we found that the majority of the skin microbiota are coated with IgT. Moreover, IgT responses against a skin parasite were mainly limited to the skin whereas IgM responses were almost exclusively detected in the serum. Strikingly, we found that the teleost skin mucosa showed key features of mammalian mucosal surfaces exhibiting a mucosa-associated lymphoid tissue. Thus, from an evolutionary viewpoint, our findings suggest that, regardless of their phylogenetic origin and tissue localization, the chief immunoglobulins of all mucosa-associated lymphoid tissue operate under the guidance of primordially conserved principles.

Small amoeboid cells, believed to be the infectious stage of Ichthyophonus sp., were observed in the bolus (stomach contents) and tunica propria (stomach wall) of Pacific staghorn sculpins and rainbow trout shortly after they ingested Ichthyophonus sp.-infected tissues. By 24-48 hr post-exposure (PE) the parasite morphed from the classically reported multinucleate thick walled schizonts to 2 distinct cell types, i.e., a larger multinucleate amoeboid cell surrounded by a narrow translucent zone and a smaller spherical cell surrounded by a "halo" and resembling a small schizont. Both cell types also appeared in the tunica propria, indicating that they had recently penetrated the columnar epithelium of the stomach. No Ichthyophonus sp. pseudo-hyphae ("germination tubes") were observed in the bolus or penetrating the stomach wall. Simultaneously, Ichthyophonus sp. was isolated in vitro from aortic blood, which was consistently positive from 6 to 144 hr PE, then only intermittently for the next 4 wk. Small PAS-positive cells observed in blood cultures grew into colonies consisting of non-septate tubules (pseudo-hyphae) terminating in multinucleated knob-like apices similar to those seen in organ explant cultures. Organ explants were culture positive every day; however, typical Ichthyophonus sp. schizonts were not observed histologically until 20-25 days PE. From 20 to 60 days PE, schizont diameter increased from ≤ 25 μm to ≥ 82 μm. Based on the data presented herein, we are confident that we have resolved the life cycle of Ichthyophonus sp. within the piscivorous host.

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease and has significant economic impacts on aquaculture production worldwide. Molecular analyses have demonstrated that there is genetic diversity among F. columnare isolates. A review of the published literature that used restriction fragment length polymorphism analysis of the 16S rRNA gene revealed that all isolates typed from salmonids were Genomovar I. Our objective was to develop a laboratory challenge model for F. columnare in rainbow trout Oncorhynchus mykiss (Walbaum) and use the model to determine the virulence of Genomovar I and II isolates. Six F. columnare isolates were obtained from rainbow trout experiencing losses due to columnaris disease and were determined to be Genomovar I. Three of these were chosen for a preliminary assessment of virulence, and isolate 051-10-S5 was chosen for additional experiments to determine the reproducibility of the waterborne challenge model. In 2 independent experiments, cumulative percent mortalities (CPM) were 49 ± 10% and 50 ± 19%. Challenge of rainbow trout with Genomovar I and II isolates demonstrated a difference in the CPM, with the Genomovar II isolates inducing significantly higher CPM. This reproducible waterborne challenge model for columnaris disease in rainbow trout will be useful to investigate host-pathogen interactions, vaccine development, and other potential control strategies. This research also provides a basis for further defining the molecular diversity and virulence associated with F. columnare genomovars in rainbow trout and other salmonid species.

Koi herpesvirus (KHV) has recently been classified as a member of the family of Alloherpesviridae within the order of Herpesvirales. One of the unique features of Herpesviridae is latent infection following a primary infection. However, KHV latency has not been recognized. To determine if latency occurs in clinically normal fish from facilities with a history of KHV infection or exposure, the presence of the KHV genome was investigated in healthy koi by PCR and Southern blotting. KHV DNA, but not infectious virus or mRNAs from lytic infection, was detected in white blood cells from investigated koi. Virus shedding was examined via tissue culture and reverse transcription-PCR (RT-PCR) testing of gill mucus and feces from six koi every other day for 1 month. No infectious virus or KHV DNA was detected in fecal secretion or gill swabs, suggesting that neither acute nor persistent infection was present. To determine if KHV latent infections can be reactivated, six koi were subjected to a temperature stress regime. KHV DNA and infectious virus were detected in both gill and fecal swabs by day 8 following temperature stress. KHV DNA was also detectable in brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas in euthanized koi 1 month post-temperature stress. Our study suggests that KHV may become latent in leukocytes and other tissues, that it can be reactivated from latency by temperature stress, and that it may be more widespread in the koi population than previously suspected.

DNA vaccines encoding the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV are very efficient in eliciting protective immune responses against their respective diseases in rainbow trout (Oncorhynchus mykiss). The early anti-viral response (EAVR) provides protection by 4 days post vaccination and is non-specific and transient while the specific anti-viral response (SAVR) is long lasting and highly specific. Since both VHSV and IHNV are endemic in rainbow trout in several geographical regions of Europe and Atlantic salmon (Salmo salar) on the Pacific coast of North America, co-vaccination against the two diseases would be a preferable option. In the present study we demonstrated that a single injection of mixed DNA vaccines induced long-lasting protection against both individual and a simultaneous virus challenge 80 days post vaccination. Transfected muscle cells at the injection site expressed both G proteins. This study confirms the applied potential of using a combined DNA vaccination for protection of fish against two different rhabdoviral diseases.

The present paradigm dictates that phagocytosis is accomplished mainly by 'professional' phagocytes (such as macrophages and monocytes), whereas B cells lack phagocytic capabilities. Here we demonstrate that B cells from teleost fish have potent in vitro and in vivo phagocytic activities. Particle uptake by B cells induced activation of 'downstream' degradative pathways, leading to 'phagolysosome' formation and intracellular killing of ingested microbes. Those results indicate a previously unknown function for B cells in the innate immunity of these primitive animals. A considerable proportion of Xenopus laevis B cells were also phagocytic. Our findings support the idea that B cells evolved from an ancestral phagocytic cell type and provide an evolutionary framework for understanding the close relationship between mammalian B lymphocytes and macrophages.

To determine the transmission dynamics of furunculosis, Chinook salmon, Oncorhynchus tshawytscha held at various densities were challenged with a single infectious fish. Data from the in vivo experiments were compared to predicted values from a susceptible--infected--removed (SIR) model. Seven different densities were tested including 9.13, 4.56, 0.72, 0.36, 0.19, 0.06 and 0.03 fish/L and each produced different transmission coefficients (beta) of 0.01, 0.019, 0.0051, 0.0076, 0.0001, 0.0005 and 0.00, respectively. Furunculosis related mortality rates decreased as density of the host decreased. Mortality rates at the highest fish densities where disease specific mortality (DSM) was observed were 0.42, 0.44, 0.18 and 0.02 infected animals per day. The natural mortality rate also followed a similar pattern with 0.005, 0.005, 0.001, 0.005, 0.0001, 0.000 and 0.000 surviving animals per day. The results indicated that the transmission coefficient and the DSM of furunculosis was dependent on host density. It is possible that fish behavioral changes at the lower densities caused the reproductive rate of disease to be persistent even at very low densities. In summary, the simple SIR model suggested that low mortality does not always mean low prevalence of furunculosis in a given population. This is the first study evaluating host density as a risk factor for fish diseases using experimental furunculosis data.

Deoxyribonucleic acid (DNA) vaccination is based on the administration of the gene encoding the vaccine antigen, rather than the antigen itself. Subsequent expression of the antigen by cells in the vaccinated hosts triggers the host immune system. Among the many experimental DNA vaccines tested in various animal species as well as in humans, the vaccines against rhabdovirus diseases in fish have given some of the most promising results. A single intramuscular (IM) injection of microgram amounts of DNA induces rapid and long-lasting protection in farmed salmonids against economically important viruses such as infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). DNA vaccines against other types of fish pathogens, however, have so far had limited success. The most efficient delivery route at present is IM injection, and suitable delivery strategies for mass vaccination of small fish have yet to be developed. In terms of safety, no adverse effects in the vaccinated fish have been observed to date. As DNA vaccination is a relatively new technology, various theoretical and long-term safety issues related to the environment and the consumer remain to be fully addressed, although inherently the risks should not be any greater than with the commercial fish vaccines that are currently used. Present classification systems lack clarity in distinguishing DNA-vaccinated animals from genetically modified organisms (GMOs), which could raise issues in terms of licensing and public acceptance of the technology. The potential benefits of DNA vaccines for farmed fish include improved animal welfare, reduced environmental impacts of aquaculture activities, increased food quality and quantity, and more sustainable production. Testing under commercial production conditions has recently been initiated in Canada and Denmark.

Three cell lines derived from fin (WSF), head soft tissue (WSHST) and body muscle (WSBM) were established from white sturgeon (Acipenser transmontanus). Characterization included determination of optimal growth kinetics, karyotyping, and mitochondrial ribosomal RNA (rRNA) genotyping. The primary cultures of these cells were generated by the explant technique using the L-15 medium supplemented with 20% fetal bovine serum and epidermal/fibroblast growth factors. The cells grew between 15-30 degrees C, but optimal growth occurred at 25 degrees C with a doubling time of 48 hours. The cell lines can be readily maintained in-vitro and have been subcultured over 35 times. Following cryopreservation in liquid nitrogen, thawed cells exhibit a viability of > 90% after a 16-month storage period. Chromosomal typing of these cell lines at their 17th passage revealed a chromosomal distribution of 242 to 278 with an apparent peak ranging from 250 to 260. Polymerase chain reaction amplification of mitochondrial 16S ribosomal RNA and sequence analysis indicated 100% identity of the sequences found in the cell lines with those found in the source animal, confirming that the cell lines were of A. transmontanus origin.

Atlantic salmon Salmo salar naturally and experimentally exposed to infectious hematopoietic necrosis virus (IHNV) in British Columbia, Canada, developed antibodies against the virus. More than 50% of the fish exposed to IHNV remained seropositive for several months after the IHN epizootic had subsided. The virus itself could not be detected in asymptomatic fish once the fish had recovered from IHN. The persistence of IHNV-specific antibodies in a large percentage of Atlantic salmon, from 4 different populations that survived an outbreak of IHN, and the lack of IHNV-specific antibodies in fish with no history of the disease, suggests that serology may be a useful tool for determining previous exposure to the virus. It may be important to determine whether Atlantic salmon have been infected with IHNV because, although the virus is difficult to detect in asymptomatic fish, an incidental finding suggests it may persist in a small number of fish after the outbreak has subsided. Furthermore, the presence of seropositive fish would be an indication that the virus may be enzootic at a farm, and such information would thus aid producers with stocking decisions.

A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout (Oncorhynchus mykiss) held at 15 degrees C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 microg.

Infectious haematopoietic necrosis virus (IHNV) is the most significant virus pathogen of salmon and trout in North America. Previous studies have shown relatively low genetic diversity of IHNV within large geographical regions. In this study, the genetic heterogeneity of 84 IHNV isolates sampled from rainbow trout (Oncorhynchus mykiss) over a 20 year period at four aquaculture facilities within a 12 mile stretch of the Snake River in Idaho, USA was investigated. The virus isolates were characterized using an RNase protection assay (RPA) and nucleotide sequence analyses. Among the 84 isolates analysed, 46 RPA haplotypes were found and analyses revealed a high level of genetic heterogeneity relative to that detected in other regions. Sequence analyses revealed up to 7.6% nucleotide divergence, which is the highest level of diversity reported for IHNV to date. Phylogenetic analyses identified four distinct monophyletic clades representing four virus lineages. These lineages were distributed across facilities, and individual facilities contained multiple lineages. These results suggest that co-circulating IHNV lineages of relatively high genetic diversity are present in the IHNV populations in this rainbow trout culture study site. Three of the four lineages exhibited temporal trends consistent with rapid evolution.

The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.

The zebrafish Danio rerio is a new model system for studying the genetics of hematopoiesis. To define naturally occurring viruses which could infect and replicate within hematopoietic precursors of the zebrafish, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were studied. Infection of whole fish with viral supernatants demonstrated infectious replicants for both viruses, indicating that the virus host range includes the zebrafish. In other species, infection with these viruses leads to prominent hematopoietic necrosis of the head kidney, the major site of adult hematopoiesis. We detected a transient toxicity of the virus to hematopoietic precursors and terminally differentiated red cells after viral infections. The kinetics of hematopoietic defects between IHNV and IPNV infection differed; fish infected with either virus, however, recovered by 6 days postinfection. In contrast to other fish infected with the virus, hematocrit did not change appreciably during this time. These studies are the first to demonstrate IHNV and IPNV infection of the zebrafish and reveal the potential for use of such viruses for gene transfer experiments to infect zebrafish hematopoietic cells., (Copyright 2000 Academic Press.)

The objective of this work was to examine the response of homozygous clones of rainbow trout to vaccination by an attenuated strain (Nan Scott Lake; NSL) of infectious hematopoietic necrosis virus (IHNV). Adult rainbow trout of the Hot Creek Strain (YY males maintained in a recirculating system at 12 degrees C) were injected 3 times with 10(5) to 10(7) plaque forming units (pfu) of NSL. Intraperitoneal injections were given at Day 0 and at 2 and 4 mo post-infection. All fish were nonlethally bled at monthly intervals for 18 mo. Serum from each fish was analyzed by the complement-dependent neutralization assay and by western blot against purified NSL virus. The highest virus neutralization titers were detected 4 mo after the first injection, and peaked at 1280. When sera were analyzed by western blot, the predominating responses of the serum from immunized fish on the reduced western blot were against M1, a matrix protein of the virus and to a 90 kDa stress protein. The 90 kDa protein was identified by a monoclonal antibody as a stress protein derived from the CHSE-214 cells in which the purified IHN virus was grown and which associates with the virus during purification.

The efficacy of a DNA vaccine containing the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV), a rhabdovirus affecting trout and salmon, was investigated. The minimal dose of vaccine required, the protection against heterologous strains, and the titers of neutralizing antibodies produced were used to evaluate the potential of the vaccine as a control pharmaceutical. Results indicated that a single dose of as little as 1-10 ng of vaccine protected rainbow trout fry against waterborne challenge by IHNV. An optimal dose of 100 ng per fish was selected to assure strong protection under various conditions. Neutralizing antibody titers were detected in fish vaccinated with concentrations of DNA ranging from 5 to 0.01 microg. Furthermore, the DNA vaccine protected fish against a broad range of viral strains from different geographic locations, including isolates from France and Japan, suggesting that the vaccine could be used worldwide. A single dose of this DNA vaccine induced protection in fish at a lower dose than is usually reported in mammalian DNA vaccine studies.

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.

The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow trout Oncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naive fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.

A naked plasmid DNA encoding the glycoprotein (pCMV4-G) of a 1976 isolate of infectious hematopoietic necrosis virus (IHNV) obtained from steelhead Oncorhynchus mykiss was used to vaccinate Atlantic salmon Salmo salar against IHNV. Eight weeks post-vaccination the fish were challenged with a strain of IHNV originally isolated from farmed Atlantic salmon undergoing an epizootic. Fish injected with the glycoprotein-encoding plasmid were significantly (p < 0.05) protected against IHNV by both immersion and cohabitation challenge. Survivors of the first challenges were pooled and re-challenged by immersion 12 wk after the initial challenge. Significant (p < 0.05) protection was observed in all of the previously challenged groups including those receiving the complete vaccine. Fish injected with the glycoprotein-encoding plasmid produced low levels of virus-neutralizing antibodies prior to the first challenge. Neutralizing antibodies increased in all groups after exposure to the IHNV. Passive transfer of pooled sera from pCMV4-G vaccinates and IHN survivors provided relative survivals of 40 to 100% compared to fish injected with sera collected from fish immunized with control vaccines or left unhandled. In this study, DNA vaccination effectively protected Atlantic salmon smolts against challenges with IHNV.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein) was produced in insect cells using a baculovirus vector (Autographa californica nuclear polyhedrosis virus). Characteristics of this protein were evaluated in relation to native viral G protein. A full-length (1.6 kb) cDNA copy of the glycoprotein gene of IHNV was inserted into the baculovirus vector under control of the polyhedrin promoter. High levels of G protein (approximately 0.5 microgram/1 x 10(5) cells) were produced in Spodoptera frugiperda (Sf9) cells following recombinant baculovirus infection. Analysis of cell lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot revealed a recombinant IHNV G of slightly higher mobility on the gel than the viral G protein. Differences in mobility were abrogated by endoglycosidase treatment. When the recombinant G protein was produced in insect cells at 20 degrees C (RecGlow), immunostaining and cell fusion activity demonstrated surface localization of the protein. In contrast, when recombinant protein was produced at 27 degrees C (RecGhigh), G protein was sequestered within the cell, suggesting that at the 2 different temperatures processing differences may exist. Eleven monoclonal antibodies (MAbs) were tested by immunoblotting for reactivity to the recombinant G protein. All 11 MAbs reacted to the reduced proteins. Four MAbs recognized both RecGhigh and RecGlow under non-reducing conditions; however, 1 neutralizing MAb (92A) recognized RecGlow but failed to react to RecGhigh under non-reducing conditions. This suggests that differences exist between RecGlow and RecGhigh which may have implications in the development of a properly folded recombinant G protein with the ability to elicit protective immunity in fish.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein), produced in Spodoptera frugiperda (Sf9) cells following infection with a baculovirus vector containing the full-length (1.6 kb) glycoprotein gene, provided very limited protection in rainbow trout Oncorhynchus mykiss challenged with IHNV. Fish were injected intraperitoneally (i.p.) with Sf9 cells grown at 20 degrees C (RecGlow) or 27 degrees C (RecGhigh) expressing the glycoprotein gene. Various antigen (Ag) preparations were administered to adult rainbow trout or rainbow trout fry. Sera collected from adult fish were evaluated for IHNV neutralization activity by a complement-dependent neutralization assay. Anti-IHNV neutralizing activity was observed in sera, but the percent of fish responding was significantly lower (p < 0.05) in comparison to fish immunized with a low virulence strain of IHNV (LV-IHNV). A small number of fish immunized with RecGlow or RecGhigh possessed IHNV G protein specific antibodies (Abs) in their serum. Cumulative mortality (CM) of rainbow trout fry (mean weight, 1 g) vaccinated by i.p. injection of freeze/thawed Sf9 cells producing RecGlow was 18% in initial trials following IHNV challenge. This level of protection was significant (p < 0.05) but was not long lasting, and neutralizing Abs were not detected in pooled serum samples. When trout fry (mean weight, 0.6 g) were vaccinated with supernatant collected from sonicated Sf9 cells, Sf9 cells producing RecGlow, or Sf9 cells producing RecGhigh, CM averaged 46%. Protection was enhanced over negative controls, but not the positive controls (2% CM), suggesting that in the first trial soluble cellular proteins may have provided some level of non-specific protection, regardless of recombinant protein expression. Although some immunity was elicited in fish, and RecGlow provided short-term protection from IHNV, Ab-mediated protection could not be demonstrated. The results suggest that recombinant G proteins produced in insect cells lack the immunogenicity associated with vaccination of fish with an attenuated strain of IHNV.

Plasmid vectors encoding the infectious hematopoietic necrosis virus (IHNV) nucleoprotein or glycoprotein gene under the control of a cytomegalovirus promoter were used to immunize rainbow trout (Oncorhynchus mykiss) against IHNV. The plasmid DNA was injected into the skeletal muscle of rainbow trout fry, and immunization was determined by the detection of virus-neutralizing and enzyme-linked immunosorbent assay antibody activity, and by protection against live virus challenge. Fish injected with the glycoprotein-encoding plasmid pCMV4-G, either alone or in combination with the nucleoprotein-encoding plasmid pCMV4-N, generated glycoprotein-specific and virus-neutralizing antibody responses. The vaccinated fish were also protected from subsequent IHNV challenge. Fish receiving pCMV4-N alone did not produce measurable virus-specific antibody and were killed by IHNV infection. These studies show that DNA vaccination will protect rainbow trout against the lethal effects of IHNV infection.

A mild to moderate branchial epitheliocystis infection was diagnosed in subyearling (11 months old, 250-300 g) white sturgeon (Acipenser transmontanus) from a private culture facility with a 4-8% mortality in the population. Infected branchial epithelial cells contained the coccoid to coccobacillary epitheliocystis organisms, which appeared as cytoplasmic inclusions composed of a fine, homogeneous, dense, basophilic, granular material. The infected cells were variably enlarged with spherical to oval profiles and were randomly distributed throughout the branchial epithelium. The cytoplasmic inclusions stained positive with Macchiavello stain but negative with Brown and Brenn, periodic acid-Schiff, and Gimenez stains. Expression of chlamydial antigen was demonstrated within the cytoplasmic inclusions using a standard peroxidase-antiperoxidase immunohistochemical technique. Three stages of coordinated intracellular development were recognized by electron microscopy. The reticulate bodies were oval to spherical and 0.4-0.8 x 0.5-1.4 microns but often exhibited a pleomorphic and convoluted appearance because of variable membrane invaginations and evaginations suggestive of uneven fission and budding. Separate host cells contained intermediate bodies that were spherical to oval and 0.2-0.4 x 0.3-0.6 microns although often observed in the process of apparent uneven division. The presence of a cap or plaque composed of hexagonally arrayed fibrillar surface projections was initially recognized in this stage. A homogeneous population of 0.3-0.4 microns oval elementary bodies were observed separately in individual host cells. This developmental stage had a single, dense, compact, eccentrically located cytoplasmic condensation that occurred opposite to the location of the cap of hexagonally arrayed fibrillar surface projections. Morphologic characteristics of the epitheliocystis organism in these white sturgeon were similar to those previously described in other teleosts and expands the species catalogue of epitheliocystis infection. Furthermore, the ultrastructural similarities to the chalmydiae and the immunohistochemical detection of chlamydial antigen provides further evidence that the epitheliocystis agent is related to members of the Chlamydiales. Although the infection was considered mild to moderate and could not be definitively attributed to the mortality in this population, the potential adverse impact of epitheliocystis infection on sturgeon culture should be considered especially in intensive fish culture operations.

Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

Rainbow trout with a 'neurotropic' form of infectious hematopoietic necrosis (IHN) exhibited behavioral abnormalities and had high virus concentrations localized in brain tissue. Fish with 'hematopoietic' IHN exhibited typical disease signs with the highest concentrations of virus detected in kidney-spleen tissue. Infectious hematopoietic necrosis virus (IHNV) isolates obtained from brain or kidney-spleen tissue were tested for serological relatedness and virulence and pathogenicity differences in 2 sizes of rainbow trout. No major differences were detected by indirect fluorescence tests. Serum neutralization profiles showed 2 predominant patterns that differed dependent upon tissue of origin. However, virulence and pathogenicity differences were not evident.

This study investigated the stimulation of specific host defenses to IHNV that are created by prior exposure to an avirulent reovirus (chum salmon reovirus; CSV). Preexposure of rainbow trout to CSV for 1 h resulted in a relative percent survival that ranged from 68 to 100% when fish were challenged with IHNV over an 8 week period. The levels of serum neutralizing antibodies detected 28 d after the IHNV challenge were significantly lower among fish receiving prior exposures to CSV than among non-CSV-treated fish. The differences in the humoral response to IHNV in the CSV-treated fish suggested that other immune defense mechanisms may be involved.