A systematic surveillance programme for infectious salmon anaemia virus supports its absence in the Pacific Northwest of the United States.

In response to reported findings of infectious salmon anaemia virus (ISAV) in British Columbia (BC), Canada, in 2011, U.S. national, state and tribal fisheries managers and fish health specialists developed and implemented a collaborative ISAV surveillance plan for the Pacific Northwest region of the United States. Accordingly, over a 3-1/2-year period, 4,962 salmonids were sampled and successfully tested by real-time reverse-transcription PCR. The sample set included multiple tissues from free-ranging Pacific salmonids from coastal regions of Alaska and Washington and farmed Atlantic salmon (Salmo salar L.) from Washington, all representing fish exposed to marine environments. The survey design targeted physiologically compromised or moribund animals more vulnerable to infection as well as species considered susceptible to ISAV. Samples were handled with a documented chain of custody and testing protocols, and criteria for interpretation of test results were defined in advance. All 4,962 completed tests were negative for ISAV RNA. Results of this surveillance effort provide sound evidence to support the absence of ISAV in represented populations of free-ranging and marine-farmed salmonids on the northwest coast of the United States.

Description and characterization of IS994, a putative IS3 family insertion sequence from the salmon pathogen, Renibacterium salmoninarum.

Renibacterium salmoninarum, a slowly growing, Gram-positive bacterium, is responsible for bacterial kidney disease in salmonid fishes world-wide. To date, no mobile genetic elements have been reported for this pathogen. Here, we describe the first insertion sequence (IS) identified from R. salmoninarum. This element, IS994, has a significant predicted amino acid sequence homology (64.8 and 71.9%) to the two open reading frames encoding the transposase of IS6110 of Mycobacterium tuberculosis. Protein parsimony and protein distance matrix analyses show that IS994 is a member of group IS51 of the IS3 family. From a conservative estimate, there are at least 17 chromosomal insertions of IS994 or closely related elements. Sequence analysis of seven of these loci reveals single nucleotide polymorphisms throughout the element (including the terminal inverted repeats), a 15bp insertion in three of the seven loci, and an absence of flanking direct repeats or conserved insertion site. Restriction fragment length polymorphism analysis of XbaI-digested chromosomal DNA shows variations among European and North American isolates, indicating that IS994 may be a useful molecular marker for epizootiological studies.

Sensitive detection of Renibacterium salmoninarum in whole fry, blood, and other tissues of pacific salmon by reverse transcription-polymerase chain reaction.

A sensitive, reproducible assay for detecting Renibacterium salmoninarum in a variety of tissues, including blood, has been developed. This assay, based on reverse transcription-polymerase chain reaction (RT-PCR) of 16S ribosomal RNA, exhibited sensitivity to

Isolation of the cDNA and characterization of mRNA expression of ribosomal protein S19 from the soft-shell clam, Mya arenaria.

Ribosomal proteins contribute to the regulation and activity of ribosomes, and hence, the translational activity of the cell. Aberrant expression of ribosomal proteins has been linked to certain pathological conditions such as neoplasms. We have isolated and characterized a cDNA for the ribosomal protein (rp) S19 from a marine bivalve, the soft-shell clam (Mya arenaria), and we have examined its pattern of mRNA expression in the ovary and testis. The S19 cDNA contains a 450 nucleotide (nt) open reading frame (ORF), flanked by 89 nt and 26 nt of 5' and 3' untranslated regions, respectively. Probes synthesized from the S19 cDNA recognize a single transcript of approximately 550 nt in four different tissues. The predicted amino acid sequence from the ORF exhibits 58% identity with human and rat S19. Southern analysis of genomic DNA suggests that M. arenaria may have multiple copies of S19, a feature that is more similar to vertebrate than invertebrate rp genes. Expression of S19 mRNA in both ovary and testis was elevated throughout gametogenesis until after spawning, when a decrease in S19 message was observed. A comparison of S19 mRNA levels in post-spawn animals revealed a trend of elevated expression in ovaries and testes affected by a gonadal neoplasm, indicating that S19 may be a useful molecular marker for the pathological condition.

Cloning of the p53 tumor suppressor gene from the Japanese medaka (Oryzias latipes) and evaluation of mutational hotspots in MNNG-exposed fish.

A full-length cDNA clone of the medaka (Oryzias latipes) p53 tumor suppressor gene was isolated from a cDNA library from adult liver tissue, sequenced and characterized. Sequence analysis revealed a high degree of homology between putative functional domains of medaka p53 and p53 genes from other vertebrate taxa including rainbow trout (Oncorhynchus mykiss), frog (Xenopus laevis), chicken (Gallus gallus), rat (Rattus norvegicus), mouse (Mus musculus), hamster (Mesocricetus auratus), green monkey (Ceropithecus aethiops) and human (Homo sapiens). A single 1.9-kb p53 mRNA is expressed at a very low level in normal adult liver tissue. This transcript is similar in size to transcripts of p53 genes from other species. Preliminary screening of six MNNG-induced tumors in four adult medaka revealed no mutations within characteristic mutational hotspots encompassing conserved domains IV and V.

Two different IFN-gamma nonresponsive variants derived from the B-cell lymphoma 70Z/3.

The kappa immunoglobulin (Igk) light chain locus is transcriptionally silent in the mouse B-cell lymphoma 70Z/3. However, exposure to lipopolysaccharide (LPS) or interferon-gamma (IFN) causes a marked increase in Igk transcription. By immunoselection, we isolated two variants that are nonresponsive to IFN. One variant, AT7.2, has retained its response to LPS (IFN-LPS+), whereas the other, AT3.3, is also nonresponsive to LPS (IFN-LPS-). Stable transfection of an intact Igk gene does not rescue the phenotype of either variant. Both variants have intact Igk genes and neither is deficient in the binding or uptake of IFN. Nuclear extracts from LPS-treated wild-type 70Z/3 cells show strong increases in three transcription factors: OTF-2, NF-kappa B, and kBF-A. Remarkably, when the IFN-LPS- variant is treated with LPS, all three transcription factors are still observed in the nuclear extracts. Treatment of wild-type cells with either LPS or IFN also causes a decrease in nuclear complexes that bind to two other regions of the Igk intron enhancer, the octenh and the E kappa MHCIC regions. Both of these changes are also observed after LPS or IFN treatment of the IFN-LPS- variant. Thus, this variant transduces the IFN and LPS signals at least into the nuclear compartment, but still fails to activate Igk transcription. In contrast, the IFN-LPS+ variant decreases neither the octenh nor the E kappa MHCIC binding complexes in response to IFN. This variant may be defective in transducing the IFN signal to the nucleus. These variants will be useful in studying the activation of Igk transcription and the IFN signaling pathway in B cells.

Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

Consistent associations between hepatic lesions in English sole (Parophrys vetulus) and polycyclic aromatic hydrocarbons in bottom sediment.

A consistent and statistically significant association between prevalence of hepatic neoplasms in free-living sole (Parophrys vetulus) and levels of anthropogenic polycyclic aromatic hydrocarbons in bottom sediment from sites of fish capture was documented in a series of studies conducted over a period of 7 years in Puget Sound, Washington. This result strengthens the evidence supporting a causal relationship between exposure to sediment-associated hydrocarbons and development of hepatic neoplasms in this bottom-dwelling marine fish species. Prevalences of two other distinct categories of idiopathic hepatic lesions-megalocytic hepatosis and steatosis/hemosiderosis-also showed consistent, statistically significant associations with polycylic aromatic hydrocarbon concentrations in bottom sediment, and association with prevalence of a third category (putatively preneoplastic foci of cellular alteration) approached statistical significance. On the basis of other studies, megalocytic hepatosis and foci of cellular alteration are both considered to be important precursor lesions in the stepwise histogenesis of hepatic neoplasms.

Differential response among cells in the chick embryo myogenic lineage to photosensitization by Merocyanine 540.

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10-day embryos (E10 cells), concentrations of MC540 greater than or equal to 60 micrograms/ml resulted in death of nearly all myogenic cells upon exposure to light, but non-myogenic cells survived and replicated. Below 60 micrograms/ml, there was a dose-dependent reduction in muscle differentiation. At concentrations less than 40 micrograms/ml, there was no effect on myogenesis. Cultures of cells from 18-day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub-populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540-resistant myogenic population is present in low numbers in 10-day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.

The ultrastructure and histology of hepatocellular carcinomas of English sole (Parophrys vetulus) from Puget Sound, Washington.

The cellular and subcellular morphology of hepatocellular carcinomas (HCs) from feral English sole (Parophrys vetulus) living in contaminated waterways of Puget Sound was compared with normal-appearing liver from English sole inhabiting reference sites in Puget Sound. The most distinctive feature of English sole HCs was the occurrence of large dilations of RER containing densely packed microfilamentous-like material that was often arranged in complex branching arrays that nearly filled the cytoplasm of affected hepatocytes. These arrays have not previously been reported in HCs of any other fishes. Several other characteristics also distinguished HCs from reference liver, including: the apparent proliferation of Golgi complexes and mitochondria; occurrence of torus-shaped mitochondria; elongation and/or proliferation of perisinusoidal cells; and inclusion of large intraheptocellular vacuoles.

Pathologic anatomy and patterns of occurrence of hepatic neoplasms, putative preneoplastic lesions, and other idiopathic hepatic conditions in English sole (Parophrys vetulus) from Puget Sound, Washington.

A comprehensive description of the histopathologic characteristics of a spectrum of idiopathic lesions in feral English sole (Parophrys vetulus), a bottom-dwelling flatfish, from Puget Sound, Washington State, is presented. Among these lesions are unique degenerative conditions, regeneration, storage disorders, foci of hepatocellular alteration (putative preneoplastic lesions), hepatocellular and biliary neoplasms, and nonneoplastic proliferative conditions, all of which morphologically resemble the lesions induced by various hepatocarcinogens-hepatotoxins in experimental exposures of fish and/or rodents. Results from a statistical analysis of the patterns of co-occurrence of these lesions in English sole are consistent with the concept, developed from experimental studies of liver carcinogenesis in rodents, that there are morphologically identifiable steps representing progression toward hepatic neoplasms. This is the first study in which it has been possible to demonstrate a close morphological congruity between a set of idiopathic hepatic lesions in any feral population and an established series of hepatic lesions inducible in rodents by certain hepatocarcinogens under laboratory conditions. Since sediments from the habitats occupied by the fish in this study have been shown to contain multiple hepatocarcinogens, the findings strengthen cumulative evidence that English sole are useful as indicators of exposure to hepatocarcinogens in aquatic environments.

Toxic chemicals in sediments and biota from a creosote-polluted harbor: relationships with hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus).

High prevalences of idiopathic hepatic lesions, including neoplasms (e.g., hepatocellular carcinomas, cholangiocellular carcinomas) (27%, 20 of 75 fish) and foci of cellular alteration (putative 'preneoplastic' lesions) (44%, 33 of 75 fish), were found in English sole (Parophrys vetulus) exposed to creosote-contaminated sediments in Eagle Harbor, Puget Sound, WA. Sediments from the contaminated region of the harbor contained particularly high concentrations of aromatic hydrocarbons (e.g., benzo[a]pyrene and benz[a]anthracene), and a variety of nitrogen-containing aromatic compounds (e.g., carbazole and acridine). The composition of the aromatic compounds was characteristic of creosote. Dramatically lower concentrations of aromatic compounds were found in sediments from a reference site in which the bottom-dwelling fish examined were free of detectable neoplastic or 'preneoplastic' hepatic lesions. Food organisms in the stomachs of the English sole from Eagle Harbor contained substantially higher concentrations of aromatic hydrocarbons than comparable organisms from the reference site. The concentrations of individual aromatic hydrocarbons in muscle and liver from the Eagle Harbor fish were low; however, high concentrations of metabolites of aromatic compounds were present in the bile. The findings strongly suggest an association between exposure to creosote and the prevalence of hepatic lesions, including neoplasms, in the bottom-dwelling fish, and furthermore support the putative role of aromatic hydrocarbons in liver carcinogenesis in fish.

Toxic chemicals in marine sediment and biota from Mukilteo, Washington: relationships with hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus).

High prevalences of idiopathic liver lesions, including 7.5% hepatic neoplasms (e.g., hepatocellular and cholangiocellular carcinomas) and 16.7% foci of cellular alteration (putative preneoplastic hepatic lesions), were found in English sole (Parophrys vetulus) from waters near Mukilteo, a small community on Puget Sound in Washington State. Sediments from the sampling sites contained particularly high concentrations of aromatic hydrocarbons. Substantially lower concentrations of these compounds were found in sediments from another Puget Sound (reference) site in which fish showed no evidence of hepatic neoplasms or foci of cellular alteration. Stomach contents from the fish at Mukilteo contained substantially higher concentrations of the chemicals than did stomach contents of fish from the relatively uncontaminated site. High concentrations of metabolites of aromatic compounds were measured in the bile of fish from Mukilteo. These findings support previously observed relationships between sediment chemicals (e.g., aromatic hydrocarbons) and high prevalences of liver lesions in English sole from Puget Sound. In addition, a dietary route of uptake by English sole of environmental chemicals, including known carcinogens, was documented.