TaqMan real-time RT-PCR detection of infectious salmon anaemia virus (ISAV) from formalin-fixed paraffin-embedded Atlantic salmon Salmo salar tissues.

The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.

Identification and localization of a putative ATP-binding cassette transporter in sea lice (Lepeophtheirus salmonis) and host Atlantic salmon (Salmo salar).

Some members of the ABC-transporter superfamily, such as P-glycoprotein and the multidrug resistance associated protein, may confer resistance to the avermectin subclass of macrocyclic lactones. The aim of this study was to examine the presence of ABC transporters in both sea lice (Lepeophtheirus salmonis) and its Atlantic salmon host (Salmo salar) using monoclonal antibodies (C219 and JSB-1, with high selectivity for P-gp) and a new polyclonal antibody (SL0525) generated against a putative sea louse ABC transporter. The antibody raised to SL0525 did not react with rat P-gp, suggesting that an ABC transporter, not necessarily P-gp, was isolated. C219 was the only antibody to localize P-gp in all 3 salmon tissues (intestine, kidney and liver). American lobster (Homarus americanus) was used as a reference crustacean for L. salmonis immunostaining reactions and showed positive staining in the hepatopancreatic and intestinal tissues with all 3 antibodies. The L. salmonis showed positive staining in the intestinal epithelial lining with all antibodies. This report represents the first documented evidence for the expression of ABC transporters in L. salmonis, its Atlantic salmon host, and the American lobster.

Correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with infectious salmon anemia virus.

We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U5575-1, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P < 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R = 0.81, 0.70, and 0.78, respectively; P < 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.

Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss).

The intracellular microsporidian parasite Loma salmonae affects salmonids of the genus Oncorhynchus and is a significant cause of economic losses in pen-reared Chinook salmon (O. tshawytscha) in British Columbia. Loma salmonae infection is easily recognized by the xenomas that form in the gills, but early stages of infection are difficult to detect in histologic sections. In situ hybridization (ISH), using an L. salmonae-specific digoxigenin-labeled single-stranded DNA probe, was used to detect the parasite during the early stages of infection. Loma salmonae was detected in the gut mucosal epithelium as early as 24 hours postexposure (PE), and it localized in the lamina propria of the intestine within 24 hours of infection. After the parasite was detected in the lamina propria, dividing merogonic stages in infected cells in the heart were detected by ISH as early as 2 days PE, providing the first evidence of parasitaemia and hematogenous distribution of this parasite in infected blood cells. The parasites inside the infected cells appeared to be undergoing merogony as they passed through the heart, indicating that proliferation may start at the site of infection, before the parasite arrives to the gills for their final developmental phase. This is the first time that L. salmonae passage through the intestinal wall and migration to the heart has been visualized; however, the identity of the cells harboring the parasite has yet to be determined.

Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile.

The isolation of infectious salmon anaemia virus (ISAV) from asymptomatic wild fish species including wild salmon, sea trout and eel established that wild fish can be a reservoir of ISAV for farmed Atlantic salmon. This report characterizes the biological properties of ISAV isolated from a disease outbreak in farmed Coho salmon in Chile and compares it with ISAV isolated from farmed Atlantic salmon in Canada and Europe. The virus that was isolated from Coho salmon tissues was initially detected with ISAV-specific RT-PCR (reverse transcription-polymerase chain reaction). The ability of the virus to grow in cell culture was poor, as cytopathology was not always conspicuous and isolation required passage in the presence of trypsin. Virus replication in cell culture was detected by RT-PCR and IFAT (indirect fluorescent antibody test), and the virus morphology was confirmed by positive staining electron microscopy. Further analysis of the Chilean virus revealed similarities to Canadian ISAV isolates in their ability to grow in the CHSE-214 cell line and in viral protein profile. Sequence analysis of genome segment 2, which encodes the viral RNA polymerase PB1, and segment 8, which encodes the nonstructural proteins NS1 and NS2, showed the Chilean virus to be very similar to Canadian strains of ISAV. This high sequence similarity of ISAV strains of geographically distinct origins illustrates the highly conserved nature of ISAV proteins PB1, NS1 and NS2 of ISAV. It is noteworthy that ISAV was associated with disease outbreaks in farmed Coho salmon in Chile without corresponding clinical disease in farmed Atlantic salmon. This outbreak, which produced high mortality in Coho salmon due to ISAV, is unique and may represent the introduction of the virus to a native wild fish population or a new strain of ISAV.

The failure of selenium supplementation to prevent copper-induced liver damage in Fischer 344 rats.

This study evaluates the ability of selenium (Se) supplementation to prevent experimental copper (Cu)-induced hepatocellular damage. Weanling male Fischer 344 rats were randomly assigned to groups of 15, 3 groups (A,B,C) were fed Cu-loaded diets (containing 2000 microg/g copper, added as CuSO4) and different levels of Se (added as Na2SeO3 x 5H2O) as follows: A) Cu-loaded/Se adequate diet (0.4 microg/g Se, fed basis); B) Cu-loaded/Se-supplemented diet (2 microg/g Se); and C) Cu-loaded/Se-deficient diet (< 0.2 microg/g). Three additional groups (D,E,F) were fed diets containing adequate levels of Cu (14 microg/g Cu, fed basis) and different levels of Se as follows: D) Cu-adequate/Se-adequate diet; E) Cu-adequate/Se-supplemented diet (2 microg/g Se); and F) Cu-adequate/Se-deficient (< 0.2 microg/g) diet. After 4, 8, and 12 weeks on the experimental diets, liver samples were processed for histology, histochemistry, metal analysis, glutathione peroxidase (GSH-Px) measurement, and quantification of malondialdehyde (MDA). Morphologic changes characteristic of Cu-associated hepatitis, without an increase in hepatic MDA levels, were seen in all Cu-loaded rats in each sampling. Similar changes occurred in rats fed Se-adequate, Se-supplemented and Se-deficient diets. This study demonstrates that Fischer 344 rats fed 2000 microg/g Cu develop morphologic changes due to Cu toxicity without evidence of lipid peroxidation. Furthermore, Se supplementation does not result in protection against Cu-induced liver injury.

Selection of an infectious bursal disease virus mutant with increased immunogenicity following passage under humoral immune pressure.

It is generally known that the pathogenicity of infectious bursal disease virus (IBDV) strains decreases following passage in cell culture. However, there is no information about the effect of passage under immune pressure on the phenotypic and molecular properties of IBDV. In the present study, a small plaque mutant virus with poor neutralization capability, but showing similar growth characteristics as the parental virus strain, QC2, was isolated after serial passage in Vero cells in presence of IBDV serotype 1 chicken polyclonal antiserum. This mutant virus showed reduced pathogenicity but enhanced immunogenicity compared to the parental virus. Sequence analysis of the non-coding regions of the genome revealed 4 and 3 nucleotide changes in the 3' non-coding regions of segments A and B, respectively, and none in the 5' non-coding regions. Restriction enzyme analysis of selected coding regions of the IBDV genome in both viruses revealed a loss of the PstI site in the VP2 region of the mutant virus. Selection of such mutant viruses by passaging under immune pressure may offer an improved method for developing safer and more effective attenuated vaccine strains against infectious bursal disease of chickens.

Morphological and biochemical assessment of the liver response to excess dietary copper in Fischer 344 rats.

The aim of this study was to determine the amount of excess dietary copper (Cu) necessary to experimentally induce liver lesions characteristic of Cu-associated disease in Fischer 344 rats. Male weanling Fischer 344 rats of uniform age were divided into 6 groups (n = 5) and fed a rodent diet containing 18 (control), 750, 1000, 1250, 1500, and 2000 microg/g Cu added as CuSO4. Rats were euthanized after 3 months on the experimental diets and their livers processed for histology, histochemistry, Cu analysis (by atomic absorption spectrophotometry), and quantification of malondialdehyde (MDA) by the thiobarbituric acid reaction. Hepatic Cu levels were significantly higher (P < 0.01) in rats receiving over 1000 microg/g Cu compared to the controls (means for each diet: control = 4.8 microg/g, 750 microg/g Cu = 39.6 microg/g, 1000 microg/g Cu = 111.2 microg/g, 1250 microg/g Cu = 389 microg/g, 1500 microg/g Cu = 509.4 microg/g, and 2000 microg/g Cu = 766 microg/g). Histological lesions increased gradually according to the level of dietary Cu. Significant morphologic changes (necrosis, portal inflammation, hyaline remnants) and reduced growth rate occurred in rats receiving over 1250 microg/g Cu. However, no significant differences were found for MDA levels between groups. The present study demonstrates that compared to other species, very high levels of excess dietary Cu are needed to induce significant liver injury in Fischer 344 rats. Increased MDA content was not detected in rats with morphologic evidence of liver damage, suggesting that lipid peroxidation may not play a major role in this model of Cu toxicity.

Pathology associated with an aquareovirus in captive juvenile Atlantic halibut Hippoglossus hippoglossus and an experimental treatment strategy for a concurrent bacterial infection.

A large-scale mortality of larval and juvenile halibut Hippoglossus hippoglossus occurred at a semi-commercial halibut farm in Atlantic Canada. Investigation of the cause revealed aquareovirus particles in necrotic liver tissue of affected fish. Cytopathic effect on CHSE-214 cell lines occurred from all fish cultured for viruses, and the viral morphology of the particles in culture was consistent with that observed in necrotic host tissue. The virus was placed in the family of Reoviridae, genus Aquareovirus based on morphology and RT-PCR results. Multifocal hepatocellular necrosis was a consistent finding in all fish as well as acute necrosis of proximal renal tubules. Concurrent bacterial infections were present in some specimens. Fish experimentally treated with oxytetracycline or a combination of oxytetracycline and chloramine-T had a significantly lower mortality rate than untreated fish. Fish treated with chloramine-T alone had a significantly elevated mortality rate compared to controls. Despite supportive medical therapy, mortality levels in treated and untreated groups remained elevated, supporting the hypothesis that the primary pathogen was of viral origin. This is the first report of elevated mortalities in Atlantic halibut associated with an aquareovirus.

Characterization of Salmonella isolates from beef cattle, broiler chickens and human sources on Prince Edward Island.

Non-typhoid Salmonella serovars remain a potential threat to human health, and beef cattle and broiler chickens are possible sources of these organisms on Prince Edward Island (PEI). In this study, the ceca of beef cattle belonging to fasted and non-fasted groups, and broiler chickens were examined for Salmonella at the time of slaughter. The characteristics of the isolates, including antimicrobial resistance patterns and virulence genes, were studied along with the isolates obtained from cases of human salmonellosis on PEI during the study period (1996-97). The prevalence of Salmonella in beef cattle was 4.6% (11/240). The rate was significantly higher in fasted cattle (7.46%), than in non-fasted cattle (0.94%). The prevalence rate in chickens was 32.5% (39/120). In beef cattle, Salmonella typhimurium phage type (PT) or definitive type (DT) 104 which was resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline, was the most predominant type (64%). In chickens, S. heidelberg, with resistance to gentamicin, streptomycin and sulfisoxazole, predominated. Of 26 isolates from humans, the most common serovar was S. typhimurium, including a multidrug-resistant strain of DT104. Examination by PCR revealed presence of the virulence gene invA in all serovars, and the spvC gene in all S. typhimurium isolates, of both beef cattle and human origin. Among the other serovars the latter gene was found in 7 human isolates, but in none of the chicken or beef isolates. All but 3 of the spvC-positive isolates possessed a 90 kilobasepair (kbp) plasmid suggesting that the 3 isolates had the spvC gene on their chromosome. These findings were confirmed by plasmid DNA isolation using 3 different protocols and by sequence analysis of the spvC-PCR product.

A dual infection of infectious salmon anaemia (ISA) virus and a togavirus-like virus in ISA of Atlantic salmon Salmo salar in New Brunswick, Canada.

Two viruses, infectious salmon anaemia (ISA) virus and a novel togavirus-like virus, were isolated from ISA disease outbreaks that were first reported as a new syndrome, haemorrhagic kidney syndrome (HKS) affecting farmed Atlantic salmon Salmo salar L. on the East coast of Canada. Laboratory confirmation of ISA diagnosis was initially complicated by isolation of only the togavirus-like agent using the CHSE-214 cell line. Here we demonstrate that a clinical sample from a disease outbreak of ISA contained a mixture of ISA virus and togavirus-like virus. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence of both viruses during serial passage of cultures in SHK-1 and CHSE-214 cells. Virus harvested at passage level 3 in both cell lines caused high mortalities and severe gross pathology consistent with ISA virus infection in experimentally inoculated Atlantic salmon parr (approximately 35 g) in freshwater, beginning 12 d post inoculation. ISA virus was detected by virus isolation from kidney and liver tissues of all dead or moribund fish tested. A comparison of virus isolation, 1-step procedure RT-PCR and RNA dot-blot hybridization for detection of ISA virus (ISAV) in fish tissues showed virus isolation to have 100% sensitivity, followed by RT-PCR (66 and 28% sensitivity in kidney and liver, respectively), with RNA dot-blot hybridization as the least sensitive method (20 and 10% sensitivity in kidney and liver, respectively). No togavirus-like virus was detected in these samples by virus isolation. Moreover, another togavirus-like virus isolate grown in CHSE-214 cells in the absence of any other detectable pathogen was non-pathogenic in experimentally inoculated fish. This study confirms that the original ISA outbreaks in New Brunswick, Canada, were caused solely by ISAV.

Growth of infectious salmon anaemia virus in CHSE-214 cells and evidence for phenotypic differences between virus strains.

Infectious salmon anaemia virus (ISAV) is a new orthomyxovirus-like virus. Thirteen isolates of ISAV (11 from Canada, one from Norway and one from Scotland) were studied for their replication in the CHSE-214 cell line compared with that in the SHK-1 cell line. All isolates replicated in SHK-1 cells, producing CPE between 3 and 12 days post-inoculation (p.i.). Six Canadian isolates also replicated in CHSE-214 cells, with production of CPE between 4 and 17 days p.i. Analysis of a one-step growth curve of ISAV in CHSE-214 cells showed that progeny virions remained predominantly cell-associated, accounting for the focalized nature of the CPE in the cell monolayer. One isolate (HKS 36) replicated in CHSE-214 cells, as shown by positive RT-PCR results of blind passages, but was non-cytopathic. All of the isolates were analysed for genetic heterogeneity by RT-PCR and RFLP with EcoRI and XhoI in a fraction of genome segment 2. The Canadian isolates showed a different RFLP profile to those of isolates Glesvaer/2/90 from Norway and 390/98 from Scotland. Structural proteins of four isolates, 'Back Bay 98', RPC/NB-877, RPC/NB-049 and Glesvaer/2/90, were examined further by SDS-PAGE. All viruses showed four major polypeptides, designated here as VP1-VP4, in Coomassie blue-stained gels. In isolates Glesvaer/2/90 and RPC/NB-877, these viral proteins had estimated molecular masses of 74, 53, 46 and 26.5 kDa, respectively. Viral proteins in isolates 'Back Bay 98' and RPC/NB-049 were of similar sizes, except that VP3 was 43 kDa. Taken together, these results show that there are phenotypic differences among strains of ISAV.

Characterization of monoclonal antibodies against bovine herpesvirus 1 gD fusion protein expressed in E. coli.

A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E. coli expressed bovine herpesvirus-1 (BHV-1) gD fusion protein were produced following the fusion of Sp2/0 myeloma cells with splenocytes from BALB/c mice immunized previously with immunoaffinity purified BHV-1 gD fusion protein. An indirect fluorescent antibody test (IFAT) using BHV-1 infected MDBK cells was used for the selection of positive hybridomas secreting specific antibody. The monoclonal antibody isotypes were 11 IgM, six IgG2b, one IgG1 and two IgG3. All MAbs reacted positively with the E. coli expressed BHV-1 gD fusion protein, BHV-1 infected MDBK cell lysates and PCR BHV-1 gD transcription-translation polypeptide antigens by an ELISA.

Improved detection of bovine herpesvirus 1 in artificially infected bovine semen by protein amplification.

Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes serious economic losses due to loss of animals, abortions, decreased milk production, and loss of body weight. There is a real need for sensitive diagnostic procedures for detection of the presence of virus in order to achieve effective control of BHV 1-induced diseases. BHV 1 is frequently found in bovine semen and can be widely transmitted through artificial insemination. Thus the detection of BHV 1 in artificial insemination centers and semen banks is of crucial importance in the control of its dissemination to the cattle industry, worldwide. In the present study, a protein amplification assay following polymerase chain reaction (PCR) of the highly conserved BHV 1 glycoprotein D gene was used in order to improve the sensitivity of direct virus detection in bovine semen. This method of BHV 1 detection is at least 200 orders of magnitude more sensitive than traditional PCR and would have direct clinical applications in antigen-based detection tests. In this method, amplification of the BHV 1 gD gene by PCR is followed by a coupled in vitro transcription translation of a small aliquot from the reaction. When the transcription translation was carried out in the presence of [35S]methionine and the products analyzed by SDS PAGE and autoradiography, 0.0014 TCID50 of virus could be detected in raw bovine semen in contrast to 0.28 TCID50 of virus detected using traditional PCR. Given the limitations in the method used for protein detection, this 'in vitro protein amplification' has the potential of attaining superior sensitivity for direct virus detection in clinical samples.

Formation of virus-like particles when the polyprotein gene (segment A) of infectious bursal disease virus is expressed in insect cells.

The baculovirus expression vector system was used to examine the expression of the full-length infectious bursal disease virus (IBDV) segment A cDNA, which encodes the structural proteins in a polyprotein precursor that is autocatalytically cleaved to VPX, VP3, and VP4. No VP2 was observed in lysates of recombinant baculovirus infected cells indicating the lack of processing of VPX to VP2 in this system. Virus-like particles (VLP) were purified from the infected insect cells, and on negative staining electron microscopy, looked very similar to authentic IBDV particles in shape and size, suggesting that processing of VPX to VP2 is not necessary for capsid assembly.

A novel technique for in-vivo assay of viral regulatory regions in genomes of animal RNA viruses.

A novel in-vivo assay system for mapping and analyzing regulatory signals which promote transcription and expression of viral RNA genomes is described. The system was developed using infectious bursal disease virus (IBDV), a double-stranded RNA virus and Vero cells which are permissive for IBDV, as a model. The model system consisted of engineered modifications of an enhancer-less pGL3-Promoter vector such that deleted lengths of the 5' noncoding region of genome segment A of IBDV were positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the luciferase (LUC) reporter gene. Transient transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in endogenous generation of recombinant viral RNA-LUC containing the 5' terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the Vero cell lysates allowed the localization of promoter activity in the 5'-terminal 32 base pairs of genomic segment A of IBDV. Because the viral RNA transcripts produced in-vivo can be either plus-sense or minus-sense, the system can be used to assay for regulatory regions for transcription or replication for any animal RNA virus.

Evidence that virion-associated VP1 of avibirnaviruses contains viral RNA sequences.

The VP1 encoded by genomic segment B of birnaviruses is generally known to exist as a genome-linked protein (VPg) and as a "free" polypeptide of 90 kDa in virus particles. The guanylylation activity associated with infectious bursal disease virus (IBDV) was demonstrated by incubating purified virus in presence of [alpha 32P] GTP; optimum activity in the 90 kDa form of VP1 was seen in low salt concentration in the presence of 4 mM magnesium ions over a wide range of incubation temperatures. The IBDV VP1 was shown to lack guanyl transferase activity. Northwestern (RNA-protein) blot analysis of purified virus using a radiolabelled cDNA probe consisting of 3' and 5' ends of genomic segment B indicated that both forms of virion-associated VP1 contained viral RNA sequences of which those linked to VPg corresponded to the two genome segments and those linked to the 90 kDa VP1 were probably a short oligonucleotide of the terminal viral RNA sequences.