Development of a PCR assay to detect cyprinid herpesvirus 1 in koi and common carp.

Cyprinid herpesvirus 1 (CyHV1) infects all scaled and color varieties of common carp Cyprinus carpio, including koi. While it is most often associated with unsightly growths known as 'carp pox,' the underlying lesion (epidermal hyperplasia) can arise from a variety of disease processes. CyHV1-induced epidermal hyperplasia may occur transiently in response to water temperature, and thus histopathology cannot be used in isolation to assess CyHV1 infection status. To address this problem, here we describe a PCR assay targeted to the putative thymidine kinase gene of CyHV1. The PCR assay generates a 141 bp amplicon and reliably detects down to 10 copies of control plasmid DNA sequence (analytic sensitivity). The PCR does not cross-detect genomic DNA from cyprinid herpesvirus 2 and 3 (analytic specificity). The CyHV1 PCR effectively detected viral DNA in koi and common carp sampled from various locations in the UK, USA, Brazil, and Japan. Viral DNA was detected in both normal appearing and grossly affected epidermal tissues from koi experiencing natural epizootics. The new CyHV1 PCR provides an additional approach to histopathology for the rapid detection of CyHV1. Analysis of the thymidine kinase gene sequences determined for 7 PCR-positive carp originating from disparate geographical regions identified 3 sequence types, with 1 type occurring in both koi and common carp.

Whirling disease revisited: pathogenesis, parasite biology and disease intervention.

Whirling disease (WD) is an ecologically and economically debilitating disease of rainbow trout Oncorhynchus mykiss caused by the actinosporean spores of the parasite Myxobolus cerebralis. M. cerebralis has a complex, 2-host life cycle alternating between salmonid fish and the oligochaete host Tubifex tubifex. The parasite alternates between 2 spore forms as transmission stages: an actinosporean triactinomyxon spore that is produced in the oligochaete host and a myxosporean spore that develops in the salmonid host. Waterborne triactinomyxon spores released from infected T. tubifex oligochaetes attach to the salmonid host by polar filament extrusion elicited by chemical (nucleoside) and mechanical (thigmotropy) stimuli-a process which is rapidly followed by active penetration of the sporoplasms into the fish epidermis. Upon penetration, sporoplasms multiply and migrate via peripheral nerves and the central nervous system to reach the cartilage where they form trophozoites which undergo further multiplication and subsequent sporogenesis. M. cerebralis myxospores are released into the aquatic environment when infected fish die and autolyse, or when they are consumed and excreted by predators. Myxospores released into the water are ingested by susceptible T. tubifex where they develop intercellularly in the intestine over a period of 3 mo through 4 developmental stages to give rise to mature actinospores. In this article, we review our current understanding of WD-the parasite and its alternate hosts, life cycle and development of the parasite in either host, disease distribution, susceptibility and resistance mechanisms in salmonid host and strategies involved in diagnosis, prevention and control of WD.

Comparative genomics of carp herpesviruses.

Three alloherpesviruses are known to cause disease in cyprinid fish: cyprinid herpesviruses 1 and 3 (CyHV1 and CyHV3) in common carp and koi and cyprinid herpesvirus 2 (CyHV2) in goldfish. We have determined the genome sequences of CyHV1 and CyHV2 and compared them with the published CyHV3 sequence. The CyHV1 and CyHV2 genomes are 291,144 and 290,304 bp, respectively, in size, and thus the CyHV3 genome, at 295,146 bp, remains the largest recorded among the herpesviruses. Each of the three genomes consists of a unique region flanked at each terminus by a sizeable direct repeat. The CyHV1, CyHV2, and CyHV3 genomes are predicted to contain 137, 150, and 155 unique, functional protein-coding genes, respectively, of which six, four, and eight, respectively, are duplicated in the terminal repeat. The three viruses share 120 orthologous genes in a largely colinear arrangement, of which up to 55 are also conserved in the other member of the genus Cyprinivirus, anguillid herpesvirus 1. Twelve genes are conserved convincingly in all sequenced alloherpesviruses, and two others are conserved marginally. The reference CyHV3 strain has been reported to contain five fragmented genes that are presumably nonfunctional. The CyHV2 strain has two fragmented genes, and the CyHV1 strain has none. CyHV1, CyHV2, and CyHV3 have five, six, and five families of paralogous genes, respectively. One family unique to CyHV1 is related to cellular JUNB, which encodes a transcription factor involved in oncogenesis. To our knowledge, this is the first time that JUNB-related sequences have been reported in a herpesvirus.

New disease records for hatchery-reared sturgeon. I. Expansion of frog virus 3 host range into Scaphirhynchus albus.

In 2009, juvenile pallid sturgeon Scaphirhynchus albus, reared at the Blind Pony State Fish Hatchery (Missouri, USA) to replenish dwindling wild stocks, experienced mass mortality. Histological examination revealed extensive necrosis of the haematopoietic tissues, and a virus was isolated from affected organs in cell culture and then observed by electron microscopy. Experimental infection studies revealed that the virus is highly pathogenic to juvenile pallid sturgeon, one of several species of sturgeon currently listed as Endangered. The DNA sequence of the full length major capsid protein gene of the virus was identical to that of the species Frog virus 3 (FV3), the type species for the genus Ranavirus, originally isolated from northern leopard frog Lithobates pipiens. Although FV3 infections and epizootics in amphibians and reptiles are well documented, there is only 1 prior report of a natural infection of FV3 in fish. Our results illustrate the broad potential host range for FV3, with the known potential to cause significant mortality in poikilothermic vertebrates across 3 taxonomic classes including bony fishes, anuran and caudate amphibians, and squamate and testudine reptiles.

Systemic iridovirus from threespine stickleback Gasterosteus aculeatus represents a new megalocytivirus species (family Iridoviridae).

Megalocytiviruses have been associated with epizootics resulting in significant economic losses in public aquaria and food-fish and ornamental fish industries, as well as threatening wild fish stocks. The present report describes characteristics of the first megalocytivirus from a wild temperate North American fish, the threespine stickleback Gasterosteus aculeatus. Moribund and dead fish sampled after transfer to quarantine for an aquarium exhibit had amphophilic to basophilic intracytoplasmic inclusions (histopathology) and icosahedral virions (transmission electron microscopy) consistent with an iridovirus infection. Phylogenetic analyses of the major capsid, ATPase, and DNA polymerase genes confirmed the virus as the first known member of the genus Megalocytivirus (family Iridoviridae) from a gasterosteid fish. The unique biologic and genetic properties of this virus are sufficient to establish a new Megalocytivirus species to be formally known as the threespine stickleback iridovirus (TSIV). The threespine stickleback is widely distributed throughout the northern hemisphere in both freshwater and estuarine environments. The presence of megalocytiviruses with broad host specificity and detrimental economic and ecologic impacts among such a widely dispersed fish species indicates the need for sampling of other stickleback populations as well as other North American sympatric marine and freshwater ichthyofauna.

Comparative Susceptibilities of Selected California Chinook Salmon and Steelhead Populations to Isolates of L Genogroup Infectious Hematopoietic Necrosis Virus (IHNV).

Salmonid species demonstrate varied susceptibility to the viral pathogen infectious hematopoietic necrosis virus (IHNV). In California conservation hatcheries, juvenile Chinook salmon (Oncorhynchus tshawytscha) have experienced disease outbreaks due to L genogroup IHNV since the 1940s, while indigenous steelhead (anadromous O. mykiss) appear relatively resistant. To characterize factors contributing to the losses of California salmonid fish due to IHNV, three populations of Chinook salmon and two populations of steelhead native to California watersheds were compared in controlled waterborne challenges with California L genogroup IHNV isolates at viral doses of 104-106 pfu mL-1. Chinook salmon fry were moderately to highly susceptible (CPM = 47-87%) when exposed to subgroup LI and LII IHNV. Susceptibility to mortality decreased with increasing age and also with a higher temperature. Mortality for steelhead fry exposed to two IHNV isolates was low (CPM = 1.3-33%). There was little intraspecies variation in susceptibility among populations of Chinook salmon and no differences in virulence between viruses strains. Viral persistence was demonstrated by the isolation of low levels of infectious IHNV from the skin of two juvenile Chinook salmon at 215 d post exposure. The persistence of the virus among Chinook salmon used for stocking into Lake Oroville may be an explanation for the severe epidemics of IHN at the Feather River hatchery in 1998-2002.

Systemic iridovirus infection in the Banggai cardinalfish (Pterapogon kauderni Koumans 1933).

Iridoviruses infect food and ornamental fish species from a wide range of freshwater to marine habitats across the globe. The objective of the current study was to characterize an iridovirus causing systemic infection of wild-caught Pterapogon kauderni Koumans 1933 (Banggai cardinalfish). Freshly frozen and fixed specimens were processed for histopathologic evaluation, transmission electron microscopic examination, virus culture, molecular virologic testing, microbiology, and in situ hybridization (ISH) using riboprobes. Basophilic granular cytoplasmic inclusions were identified in cytomegalic cells often found beneath endothelium, and hexagonal virus particles typical of iridovirus were identified in the cytoplasm of enlarged cells by transmission electron microscopy. Attempts at virus isolation in cell culture were unsuccessful; however, polymerase chain reaction (PCR)-based molecular testing resulted in amplification and sequencing of regions of the DNA polymerase and major capsid protein genes, along with the full-length ATPase gene of the putative iridovirus. Virus gene sequences were then used to infer phylogenetic relationships of the P. kauderni agent to other known systemic iridoviruses from fishes. Riboprobes, which were transcribed from a cloned PCR amplification product from the viral genome generated hybridization signals from inclusions within cytomegalic cells in histologic sections tested in ISH experiments. To the authors' knowledge, this is the first report of a systemic iridovirus from P. kauderni. The pathologic changes induced and the genomic sequence data confirm placement of the Banggai cardinalfish iridovirus in the genus Megalocytivirus family Iridoviridae. The ISH provides an additional molecular diagnostic technique for confirmation of presumptive infections detected in histologic sections from infected fish.

Phylogenetic relationships in the family Alloherpesviridae.

Phylogenetic relationships among herpesviruses (HVs) of mammals, birds, and reptiles have been studied extensively, whereas those among other HVs are relatively unexplored. We have reconstructed the phylogenetic relationships among 13 fish and amphibian HVs using maximum likelihood and Bayesian analyses of amino acid sequences predicted from parts of the DNA polymerase and terminase genes. The relationships among 6 of these viruses were confirmed using the partial DNA polymerase data plus the complete sequences of the terminase, helicase, and triplex protein genes; the position of these viruses among all other sequenced HVs was also investigated using the complete terminase gene. The results established the monophyly of the fish and amphibian HVs (Alloherpesviridae) separate from the HVs of mammals, birds, and reptiles (Herpesviridae) and the single recognized HV of bivalve mollusks (Malacoherpesviridae) in the order Herpesvirales. Two major clades in the family Alloherpesviridae were recognized: one consisting of viruses from cyprinid and anguillid hosts and the other of viruses from ictalurid, salmonid, acipenserid, and ranid hosts. A comparison of virus and host phylogenies suggested that closely related HVs in this family may have coevolved with their hosts, whereas significant codiversification was not apparent for the more distantly related viruses.

Discovery of genes implicated in whirling disease infection and resistance in rainbow trout using genome-wide expression profiling.

BACKGROUND: Whirling disease, caused by the pathogen Myxobolus cerebralis, afflicts several salmonid species. Rainbow trout are particularly susceptible and may suffer high mortality rates. The disease is persistent and spreading in hatcheries and natural waters of several countries, including the U.S.A., and the economic losses attributed to whirling disease are substantial. In this study, genome-wide expression profiling using cDNA microarrays was conducted for resistant Hofer and susceptible Trout Lodge rainbow trout strains following pathogen exposure with the primary objective of identifying specific genes implicated in whirling disease resistance. RESULTS: Several genes were significantly up-regulated in skin following pathogen exposure for both the resistant and susceptible rainbow trout strains. For both strains, response to infection appears to be linked with the interferon system. Expression profiles for three genes identified with microarrays were confirmed with qRT-PCR. Ubiquitin-like protein 1 was up-regulated over 100 fold and interferon regulating factor 1 was up-regulated over 15 fold following pathogen exposure for both strains. Expression of metallothionein B, which has known roles in inflammation and immune response, was up-regulated over 5 fold in the resistant Hofer strain but was unchanged in the susceptible Trout Lodge strain following pathogen exposure. CONCLUSION: The present study has provided an initial view into the genetic basis underlying immune response and resistance of rainbow trout to the whirling disease parasite. The identified genes have allowed us to gain insight into the molecular mechanisms implicated in salmonid immune response and resistance to whirling disease infection.

Risk of introducing viral hemorrhagic septicemia virus (VHSV) to the Chilean South Pacific via sardine imports from Europe.

Chile imports from Spain 100s of metric tons of frozen sardine Sardina pilchardus fished in European oceans, which, with several other clupeids, are presumed susceptible to infection with viral hemorrhagic septicemia virus (VHSV). The frozen sardines are directly introduced into the sea as bait to catch southern hake Merluccius australis in the same areas where wild and pen-raised salmonids are present. A simulation model was therefore developed to evaluate the potential risk of infection of wild Chilean southern hake with VHSV from imported bait. The model indicated that VHSV-susceptible fish species present in Chilean waters, like southern hake, are not at immediate risk of infection. However, sensitivity analyses showed that infectious doses at lower concentrations of VHSV combined with higher VHSV-prevalence import scenarios could likely result in VHSV infections of a moderate number of indigenous southern hake (> or =54 fish yr(-1)).

Genotypes and phylogeographical relationships of infectious hematopoietic necrosis virus in California, USA.

Infectious hematopoietic necrosis virus (IHNV) contains 3 major genogroups in North America with discreet geographic ranges designated as upper (U), middle (M), and lower (L). A comprehensive genotyping of 237 IHNV isolates from hatchery and wild salmonids in California revealed 25 different sequence types (a to y) all in the L genogroup; specifically, the genogroup contained 14 sequence types that were unique to individual isolates as well as 11 sequence types representing 2 or more identical isolates. The most evident trend was the phylogenetic and geographical division of the L genogroup into 2 distinct subgroups designated as LI and LII. Isolates within Subgroup LI were primarily found within waterways linked to southern Oregon and northern California coastal rivers. Isolates in Subgroup LII were concentrated within inland valley watersheds that included the Sacramento River, San Joaquin River, and their tributaries. The temporal and spatial patterns of virus occurrence suggested that infections among adult Chinook salmon in the hatchery or that spawn in the river are a major source of virus potentially infecting other migrating or resident salmonids in California. Serum neutralization results of the California isolates of IHNV corroborated a temporal trend of sequence divergence; specifically, 2 progressive shifts in which more recent virus isolates represent new serotypes. A comparison of the estimates of divergence rates for Subgroup LI (1 x 10(-5) mutations per nucleotide site per year) indicated stasis similar to that observed in the U genogroup, while the Subgroup LII rate (1 x 10(-3) mutations per nucleotide site per year) suggested a more active evolution similar to that of the M genogroup.

Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus).

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log(10) improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in <5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.

Persistence of Piscirickettsia salmonis and detection of serum antibodies to the bacterium in white seabass Atractoscion nobilis following experimental exposure.

White seabass Atractoscion nobilis surviving experimental exposure to Piscirickettsia salmonis harbored the bacterium for periods up to at least 123 d post injection (dpi). Intraperitoneal injections of juvenile white seabass with 1.26 x 10(2) TCID50 P. salmonis fish(-1) resulted in a 29% cumulative mortality over a 27 d period. Both molecular and histologic methods provided evidence for persistence of the bacterium in fish sampled sequentially from the surviving population. Throughout the period of acute mortality, the bacterium was detected in all impression smears of liver tissue stained with Giemsa and was reisolated in cell cultures from all dead fish sampled. Polymerase chain reaction (PCR) assays detected P. salmonis-specific DNA in 13.3 to 50% of the fish sampled at time points between 28 and 123 dpi, while cell culture reisolation was largely ineffective in detecting the bacterium. An enzyme-linked immunosorbent assay (ELISA) detected serum anti-P. salmonis antibodies in 48 of 59 white seabass exposed to P. salmonis but not in fish which were not exposed to the bacterium. At the end of the 4 mo experiment, microscopic lesions consisting of single to multiple and coalescing granulomas were found in liver and kidney tissues of 9 of 10 fish examined from the exposure group, while no lesions were detected in the fish from the control group. Immunohistochemical staining with anti-P. salmonis polyclonal antibodies detected bacterial antigens in some but not all granulomas examined from the exposure group at 4 mo. This study demonstrates that P. salmonis may persist among white seabass following infection, and thus provide a potential reservoir of infection contributing to transmission both within and between fish species in the marine environment.

Effects of temperature on infectivity and of commercial freezing on survival of the North American strain of viral hemorrhagic septicemia virus (VHSV).

Temperature affected the growth of the North American strain of viral hemorrhagic septicemia virus (VHSV) in experimentally infected cell cultures and in Pacific sardine Sardinops sagax. In addition, commercial freezing significantly reduced the infectivity of VHSV in tissues of experimentally infected sardine. Isolates of VHSV representing the geographic range of North American VHSV replicated in the EPC (Epithelioma papulosum cyprini) cell line at 10, 15 and 20 degrees C, but the more northern isolates from British Columbia, Canada, demonstrated significantly reduced growth at 20 degrees C compared to VHSV from more southern locations (p <0.001). An injection challenge of Pacific sardine with VHSV from California resulted in 66.7% mortality at a seawater temperature of 13 degrees C compared to 6.7% at 20 degrees C. Commercial blast-freezing of sardine experimentally infected with VHSV reduced median concentrations of virus in the kidney and spleen from 5.25 x 10(6) to 5.5 x 10(3) pfu (plaque-forming units) g(-1). Decreased growth of the California isolate of VHSV at higher temperatures following experimental infection of the sardine and reduced virus survival following commercial freezing of infected sardine are factors that would lessen the risk of transmission of VHSV through frozen baitfishes.

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

BACKGROUND: Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. RESULTS: A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. CONCLUSION: The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California.

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.

Spatial and temporal variations in sea lice (Copepoda: Caligidae) infestations of three salmonid species farmed in net pens in southern Chile.

Sea lice infestations have become a major health problem for farmed salmonids throughout the world including Chile. In southern Chile, 6 geographical areas, divided into 22 geographical zones with a total of 127 salmon farming centers and 1519 sea pens, were regularly sampled from December 1999 to April 2002. A linear mixed-effects model (LME) approach was used to describe the infestations of adult forms of sea lice on 3 salmonid species farmed in southern Chile. The variables fish species, water temperature, water salinity, fish weight, juvenile parasite count, pen shape, treatment status in previous month and the interaction of previous and current month treatments were found to be statistically significant fixed effects for the population sampled. The most susceptible species to sea lice infestation was rainbow trout Oncorhynchus mykiss, while the least susceptible species was coho salmon O. kisutch. Fishes in pens treated in the previous month with avermectins were associated with the smallest sea lice count compared to fishes in pens not treated or treated with other products. The variability in sea lice infestations in areas and zones within areas was not statistically significant when controlling for the previously mentioned fixed variables. The variability between centers, the within-pen variability, and the interaction between within-pen effect and the date of measurement were statistically significant and not explained by the fixed effects. Potential sources for this variability are discussed. We conclude that the epidemiology of sea lice infestations in farmed salmonids in southern Chile is complex and in need of further study.

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis.

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.

The severity of whirling disease among wild trout corresponds to the differences in genetic composition of Tubifex tubifex populations in central Colorado.

We analyzed the geographic distribution of Tubifex tubifex from various river drainages in central Colorado by genetic screening with specific mitochondrial 16S ribosomal DNA (mt 16S rDNA) markers. Four distinct mt 16S rDNA lineages are evident. The sites varied with respect to land- and water-use practices. All sites represented habitats presumed capable of supporting oligochaetes. At the locations where whirling disease has had the greatest impact on resident rainbow trout, T. tubifex, representing lineages I and III (genotypes known to be susceptible to Mxyobolus cerebralis), were most commonly found. In contrast, at sites less affected by whirling disease, T. tubifex of lineages V and VI that are more resistant to M. cerebralis infections were more abundant. The predominance of resistant T. tubifex worms (lineages V and VI) at low-impact sites supports the conclusion that when these genotypes are in greater abundance, the potential for more severe effects of whirling disease on wild rainbow trout populations may be diminished.

Synergistic effects of esfenvalerate and infectious hematopoietic necrosis virus on juvenile chinook salmon mortality.

Sublethal concentrations of pollutants may compromise fish, resulting in increased susceptibility to endemic pathogens. To test this hypothesis, juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed to sublethal levels of esfenvalerate or chlorpyrifos either alone or concurrently with infectious hematopoietic necrosis virus (IHNV). Three trials were performed with fish exposed to concentrations of IHNV between 0.8 x 10(2) and 2.7 x 10(6) plaque-forming units/ml and to 5.0 microg/L of chlorpyrifos or 0.1 microg/L of esfenvalerate. The presence and concentration of IHNV in dead fish were assayed by virus isolation and plaque assay techniques, respectively. Among groups exposed to both esfenvalerate and IHNV, 83% experienced highly significant (p < 0.001) mortality, ranging from 20 to 90% at 3 d post-virus exposure, and cumulatively died from 2.4 to 7.7 d sooner than fish exposed to IHNV alone. This trend was not seen in any other treatment group. Virus assays of dead fish indicate a lethal synergism of esfenvalerate and IHNV. Chlorpyrifos had no observed effect on total mortality or IHNV susceptibility. The present results suggest that accepted levels of pollutants may be seemingly nonlethal to fish but, in fact, be acting synergistically with endemic pathogens to compromise survivorship of wild fish populations through immunologic or physiologic disruption.