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.

Observations on polymerase chain reaction amplification of infectious bursal disease virus dsRNA.

Two methods for denaturing double stranded (ds)RNA of infectious bursal disease virus for the purpose of reverse transcribing it were compared: Heat denaturation at 65 degrees C in the presence of DMSO and in the absence of DMSO. As part of the analysis, the nature of cDNA in the two preparations was examined by polymerase chain reaction (PCR) amplification, firstly by varying the number of cycles of PCR, and secondly by re-amplification of serial dilutions of the reaction products. The results show that denaturation of dsRNA in the presence of DMSO (method 1) is superior to denaturation without DMSO (method 2) judging by the yield of a specific PCR fragment after 30 cycles, and that the products of method 2 can be re-amplified, albeit poorly, with the generation of heterologous products.

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.

Comparison of eight different procedures for harvesting avian reoviruses grown in Vero cells.

14 avian reovirus isolates adapted to replicate in an African green monkey (Vero) cell line were studied for the nature of their replication. The growth curves of 5 viruses showed them to be highly cell-associated in Vero cells. Different procedures were examined for releasing the cell-associated virus following propagation in Vero cells, including several freeze-thaw cycles, treatment with sterile distilled deionized water (ddH2O), freon extraction, and trypsin treatment. Treatment of virus infected cultures with ddH2O was the most effective, and trypsin treatment was the least effective procedure for dissociation of virus from cells. Treatment of virus infected cultures with ddH2O is a simple and effective procedure which can be used where large amounts of virus are required for experimental purposes.

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.

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.

Staphylococcus aureus isolated from poultry in Australia. I. Phage typing and cultural characteristics.

The phage typing and cultural characteristics of 574 strains of S. aureus of poultry origin in Australia were examined. With the avian phage set of Shimizu (1979) it was possible to type 74.2% of strains. A number of significant variations in the phage typing patterns of Australian strains compared to those reported from Japan and Europe were observed. A lower proportion of Australian strains were of avian phage group I and a higher proportion of group III. A high proportion of strains were of mixed lytic groups. No locally isolated phages were able to increase significantly the percentage of typeable strains, although four local phages appeared to be of greater value for phage typing poultry strains of S. aureus than some other phages of the avian phage set. The international (human) phage set was of limited value in typing Australian strains of poultry origin although four strains were identified which were indistinguishable from strains of human origin. Using cultural characteristics of the strains in conjunction with phage typing, the Australian strains of S. aureus were assigned to one of three major groups and nine subgroups. A list of typing phages considered to be valuable for use on Australian poultry strains of S. aureus is given.

Lysogeny and other characteristics of Staphylococcus hyicus isolated from chickens.

Lysogeny was readily demonstrated among strains of Staphylococcus hyicus that were isolated from chickens. Susceptibility to phage lysis was affected by prophage immunity, but lipase activity and erythromycin resistance were not affected by the presence of temperate phage. In contrast to previously published results, lipase-negative strains of S. hyicus were relatively common and the use of selective media based on lipase activity would have been unsuitable for detection of the S. hyicus strains examined.

Staphylococcus aureus isolated from poultry in Australia. II. Epidemiology of strains associated with tenosynovitis.

Bacteriophage typing of Staphylococcus aureus from different outbreaks of tenosynovitis in broiler breeder replacement chickens showed that, although a mixture of phage types was present on affected farms, there was a predominant phage type isolated from lesions of affected chickens. The predominant phage type isolated from chickens in different outbreaks was variable. The source of S. aureus associated with tenosynovitis appeared to be a resident population present on the skin.

Spatial and temporal distribution of lamprin mRNA during chondrogenesis of trabecular cartilage in the sea lamprey.

The temporal and spatial expression patterns of lamprin, the principle structural protein in lamprey cartilages, were examined by in situ hybridization during chondrogenesis of trabecular cartilage in day 17-33 post-fertilization prolarval lampreys. Lamprin mRNA transcripts were first detected during day 19, concomitant with the end of the condensation phase of chondrogenesis and the initiation of matrix synthesis as indicated by light microscopic examination. In the stages which followed, the hybridization signal increased with progressive intensity, paralleling matrix synthesis, suggesting transcriptional control of lamprin gene expression. Spatially, lamprin expression patterns mirrored the rostrocaudal development of the trabecular cartilage rudiment. No signal was detected over adjacent tissues or control sections. Some similarities exist between the temporal patterns of lamprin expression and the expression of matrix proteins such as elastin and collagen of higher vertebrates. It is concluded that certain aspects of chondrogenesis are critical to the normal development of a functional cartilaginous matrix and are conserved throughout the vertebrate taxa.

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.

Use of proteinase K in the excystation of Sarcocystis cruzi sporocysts for in vitro culture and DNA extraction.

Proteinase K was used for the cleaning of Sarcocystis cruzi (Apicomplexa) sporocysts prior to excystation. Bovine pulmonary endothelial cell cultures inoculated with the excysted sporozoites remained free of bacterial contamination for the duration of the experiment and had high yields of merozoites. The excysted sporozoites also yielded genomic DNA that could be labelled efficiently with 32P dATP by the random priming method.

Development of genomic probes to Sarcocystis cruzi (Apicomplexa).

A genomic library of Sarcocystis cruzi sporozoite DNA was constructed in bacteriophage lambda gt10. Recombinant phages containing insert DNA were selected by growth on Escherichia coli strain C600 hflA150. Of 14 clones examined, 11 contained DNA inserts ranging in size from approximately 1.45 kilobase (kb) to 6.18 kb. Insert DNA from four of these clones specifically hybridized to 32P-labelled S. cruzi merozoite DNA. One of these insert DNA, clone SL41, was selected and labelled with 32P. This probe did not hybridize with the other ten DNA inserts nor with bovine cellular DNA, but it hybridized with sporozoite, merozoite and bradyzoite DNA preparations. The SL41 probe could detect merozoite DNA in as little as 17 ng total DNA. Genomic probes detecting developmental stages of Sarcocystis spp. could provide an improved means is diagnosis of acute bovine sarcocystosis.

Use of genomic DNA probes for the diagnosis of acute sarcocystosis in experimentally infected cattle.

Two clones of 1.4 and 4.33 kilobase pairs (kbp) DNA inserts, were selected from a Sarcocystis cruzi sporozoite genomic library constructed in bacteriophage lambda gt10. These clones strongly hybridized with sporozoite and merozoite DNA and were evaluated as probes for detection of merozoite DNA in clinical samples. Of five calves in the experiment, four were each orally dosed with approximately 200,000 S. cruzi sporocysts; one calf served as non-infected control. Subsequently, blood was collected from the calves twice weekly for 3.5 months and fractionated into buffy coats, polymorphonuclear cells, and plasma. Total cellular DNA extracted from these fractions was dot blotted on nylon membranes and hybridized with the probes radiolabeled with [alpha-32P]dATP. The probes detected merozoites on Day 22 post infection in the buffy coats and intermittently from Day 25-39 in the granulocyte fraction. Parasitemia (i.e. merozoites in blood) was also detected by indirect fluorescent antibody technique (IFAT) and direct microscopy, Diagnosis of sarcocystosis in cattle using genomic DNA probes by dot blot hybridization provides an alternative method of detecting parasitemia that is more rigorous than the other two tests (IFAT, direct microscopy) which rely on morphology of the merozoite and visualization by the examiner. As probes detected merozoite DNA in the granulocyte fraction, polymorphonuclear cells may be involved in the pathogenesis of S.cruzi; however this hypothesis requires further study.

A comparison of the pathogenicity of four avian reoviruses in chickens.

Four avian reoviruses were orally inoculated into 1-day-old chickens to determine pathogenicity, virus persistence in the intestinal tract, and effects on body weight gains. Avian reoviruses Reo-25 and W3-492 belonged to two separate serotypes, and viruses TC 897 and W3-410 were antigenically related to W3-492. Isolate W3-492, which was highly pathogenic, was very rarely recovered from cloacal swabs collected 2 weeks postinoculation, but inoculated chickens gained significantly less weight (P less than or equal to 0.001) than uninoculated controls during the 5-week test study. Isolate Reo-25 persisted the longest in the intestinal tract, and isolates TC 897 and W3-410, of intermediate persistence, had no significant effect on body weights. There was no apparent correlation between serotype and pathotype of avian reoviruses.

Isolation and propagation of infectious bursal disease virus using the ovine kidney continuous cell line.

Twenty-six samples known to contain infectious bursal disease virus (IBDV) were examined by virus-isolation attempts on ovine kidney (OK) cell line, Vero cell line, and chicken embryo fibroblast (CEF) cultures. Virus was isolated from two of 26 samples, three of 26 samples, and three of 25 samples on OK, Vero, and CEF cultures, respectively. However, in contrast to IBDV replication in Vero and CEF cultures, isolated virus was unable to induce serially sustained cytopathic effects (CPE) during successive passages in the OK cell line, unless cell lysates were treated with chloroform between every other passage. The cytopathogenicity of the untreated virus passaged in OK cells was revived and maintained upon passage in Vero cells. An initial single passage of laboratory or field material in OK cells followed by further passages in Vero cells resulted in virus isolation from six of 26 samples, which was better virus recovery than when either cell line was used alone or when CEF cultures were used. Twenty of the 26 test samples were originally positive when examined by nucleic acid hybridization with radiolabeled IBDV cDNA, indicating that some of the samples that were negative upon virus isolation using OK and Vero cells may have contained inactivated virus.

Adenovirus infection associated with respiratory disease in commercial chickens.

The lesions and etiologic agents associated with 13 outbreaks of respiratory disease in commercial chickens were investigated. Adenoviruses were isolated from tracheal and lung tissues of affected chickens in all 13 outbreaks. Escherichia coli was isolated from the lung of an occasional bird. The tracheal specimens were consistently negative for Bordetella avium, but E. coli and occasionally Staphylococcus aureus were isolated. There was also serological evidence in one outbreak, and pathological evidence in another, of a concurrent infectious bursal disease virus (IBDV) infection of chickens affected with the disease. Gross and microscopic alterations in the tracheas and lungs of affected chickens were similar in all outbreaks and consisted of catarrhal tracheitis and occasionally multifocal pneumonia with mononuclear cell infiltrates. Hepatitis and splenitis with heterophil infiltrates occasionally were seen in birds with coliform septicemia. The tracheal and lung lesions in the present investigation were considered primarily of adenovirus etiology, complicated by secondary bacterial infection.

The use of biotin-labeled cDNA probes for the detection of infectious bursal disease viruses.

A cDNA library was prepared from the double-stranded RNA genome of the infectious bursal disease virus (IBDV) strain ST-C. The cDNA molecules were annealed into the plasmid pUC9 and used to transform Escherichia coli strain JM107. A cDNA clone that contained IBDV-specific nucleotide sequences was selected and designated STC-1. Radiolabeled probes were prepared from STC-1 and hybridized to genome segment A of ST-C in a northern blot hybridization assay. The STC-1 cDNA was 448 base pairs in length, and its nucleotide sequence indicated that it is located near the VP-2/VP-4 junction in IBDV genome segment A. Biotin-labeled probes were prepared from STC-1 and used in a dot-blot hybridization assay to detect IBDV. Under relatively low stringency conditions of hybridization, the biotinylated probes detected four subtypes of IBDV serotype 1 and a serotype 2 IBDV isolate.

Growth of serotypes I and II and variant strains of infectious bursal disease virus in Vero cells.

The growth of five strains of infectious bursal disease virus--three strains of serotype I (SAL, D-78, 2512), one of serotype II (OH), and one variant strain (Variant-A)--were compared in Vero and chicken embryo fibroblast (CEF) cell cultures in order to characterize the replication of different strains of IBDV in Vero cells. For all five virus strains, the latent period in Vero cells ranged from 12 to 18 hr, which was longer than the 4-to-6-hr latent period observed in CEF cultures for strains SAL, D-78, and OH. Virus strains SAL, D-78, and OH, which were examined in both Vero and CEF cultures, also had a more extensive maturation phase and higher yields of virus in Vero than in CEF cultures. Total titers of these viruses of 5.35 to 6.10 log10 TCID50/ml in CEFs occurred 24 to 30 hr postinoculation (PI), although the cytopathic effect (CPE) was not seen until 72 hr PI. By comparison, their total infectious virus titers of 6.85 to 8.35 log10 TCID50/ml in Vero cells occurred from 48 hr PI, coinciding with the appearance of CPE. The growth curve of Variant-A in Vero cells differed from the other viruses by showing steadily rising extracellular and cell-associated virus titers throughout the 72-hr observation period. Only very low titers of Variant-A were obtained in CEF cultures, and thus no growth curve in CEFs was performed.

Viral arthritis in fryers related to reovirus infection in breeders.

In 1983, twenty-two outbreaks of viral arthritis/tenosynovitis were diagnosed in a 6-month period on 18 fryer farms of one commercial operation located in western Washington. The main source of the reovirus infection was traced to a breeder flock that supplied progeny chicks to all of the affected farms.