Development and evaluation of real-time TaqMan® RT-PCR assays for the detection of avian nephritis virus and chicken astrovirus in chickens.

The development and preliminary evaluations of two TaqMan®-based, real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assays for the quantitative detection of avian nephritis virus (ANV) and chicken astrovirus (CAstV) RNAs are described. The assays used amplicons generated from the 3' untranslated region of the ANV genome and a conserved region of CAstV open reading frame 1b including its junction with open reading frame 2. High virus RNA levels (>10(5.99) viral copies) were detected for ANV and CAstV in 81% and 67% gut content samples from growth-retarded broiler flocks. Results from longitudinal surveys of two broiler flocks showed that ANV and CAstV RNAs were detected in most gut content and kidney samples collected at all time points from day 0 to day 35, with RNA levels of both astroviruses being higher in the gut contents than in the kidneys, and with the ANV RNA levels being greater than those of CAstV especially at early (days 7 and 14) time points. When the results obtained for the days 4/5 time-point samples from four broiler flocks with varying growth performances were compared, the two better-performing flocks had 100-fold to 1000-fold less ANV viral copies than the flocks that performed least well. Application of the rRT-PCR tests to samples collected from broiler chicks, which were experimentally infected with a crude gut content inoculum, demonstrated that ANV RNA could be detected in gut content and kidney samples at levels similar to those found at corresponding time points in longitudinal survey samples, whereas CAstV RNA was detected at lower levels than in the longitudinal survey samples, especially in kidney samples.

Development and application of an RT-PCR test for detecting avian nephritis virus.

The development of a reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting avian nephritis virus (ANV) is described. Primers, which amplified a fragment of 182 base pairs (bp), were located in the conserved 3' untranslated region (UTR) of the genome. The limit of detection of the test was estimated to be approximately 18 viral copies using a 10-fold dilution series of in vitro transcribed RNA. Positive signals were produced with representative ANV samples, some of which were not detected by previously described RT-PCR tests for detecting ANV, but other avian astroviruses including chicken astrovirus isolates and duck hepatitis virus types 2 and 3 tested negative. When applied to gut content samples from UK, German and US broiler flocks with enteritis/growth problems, ANVs were detected by RT-PCR in 82/82 (100%) samples. ANVs were also detected in 80/96 (83%) pooled gut content samples from longitudinal surveys of four broiler flocks displaying below-average performance. Whereas all samples collected on day 0 from the surveys were negative for ANV, all samples collected at days 4/5, 7, 10, 14, 21 and 28 tested positive. Sequence determinations performed with amplicons produced with 14 field samples confirmed the ANV specificity of the test, while comparative and phylogenetic analyses based on 109-nucleotide 3'-UTR sequences demonstrated that the majority of ANVs investigated were more closely related to the serotype 2 ANV (accession number AB 046864) than to the serotype 1 ANV (accession number NC 003790).

Detection of chicken astrovirus by reverse transcriptase-polymerase chain reaction.

The development of a reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting chicken astroviruses (CAstV) is described. Primers, which amplified a fragment of 510 base pairs, were located in conserved regions within the ORF 1b (RNA polymerase) gene. The limit of detection of the test was estimated to be approximately 60 viral copies using a 10-fold dilution series of in vitro transcribed RNA. Positive signals were produced with representative CAstV samples, some of which were not detected by a previously described RT-PCR test for detecting CAstV, but other avian astroviruses including avian nephritis virus and duck hepatitis virus types 2 and 3 tested negative. When applied to gut content samples and swabs from UK and German broiler flocks with growth problems, CAstVs were detected by RT-PCR in 50/52 (96%) samples. CAstVs were detected in between 30% and 72.5% pooled gut content samples from longitudinal surveys of four broiler flocks displaying below-average performances. Whereas all day 0 samples were CAstV-negative, high detection rates were observed when the surveyed birds were aged 4, 5 and 7 days. Based on partial ORF 1b sequences, a phylogenetic analysis of 20 CAstVs indicated the existence of two groups. One group comprised four CAstV isolates, including FP3 and 11672, and two field CAstVs, which shared >94% nucleotide identity. The remaining 14 CAstVs, comprising the first characterized CAstV and 612 isolates and 12 field CAstVs, shared 85% to 99% nucleotide identity and displayed 76% to 79% nucleotide identity with the 11672-like and FP3-like CAstVs.

A seroprevalence investigation of chicken astrovirus infections.

Two genetically different isolates of chicken astrovirus (CAstV), named CAstV612 and CAstV11672, which share low levels of antigenic relatedness in cross-indirect immunofluorescence (IIF) tests, have been identified recently. In the present study, separate IIF tests for detecting antibodies to the CAstV612 and CAstV11672 isolates have been used to determine the seroprevalences of CAstV infections in four generations of flocks involved in broiler chicken production. CAstV antibodies were detected in 78% (73% CAstV612; 46% CAstV11672) of serum samples from UK broiler flocks and in all 10 flocks tested, indicating that infections were very common. Twenty-three (96%) out of 24 and 26 (93%) out of 28 broiler parent flocks, aged 23 to 26 weeks from three UK organizations, were positive for antibody to CAstV612 and CAstV11672, respectively. Of 718 samples tested from these parent flocks, 415 (53%) were positive for either CAstV612 or CAstV11672 antibody. CAstV infections were also widespread in parent flocks, with screening of pooled serum samples showing that antibodies to both CAstVs were detected in flocks from seven other UK poultry organizations and in flocks from eight other European countries. The seropositivities for CAstVs were substantially less in grandparent (28%) and great grandparent (21%) flocks. Overall, higher seropositivities were observed for CAstV612 than for CAstV11672 in broiler, parent, grandparent and great-grandparent flocks. A limited study of 99 sera from 10 turkey breeder flocks showed low-level seropositivities for CAstV612 (9%) and CAstV11672 (2%), indicating that turkeys were infected with CAstVs or antigenically related viruses.

Identification of chicken enterovirus-like viruses, duck hepatitis virus type 2 and duck hepatitis virus type 3 as astroviruses.

Earlier work identified and biologically characterized antigenically distinct enterovirus-like viruses (ELVs) of chickens. Three of these ELVs can now be identified as astroviruses. Characterization involved the use of a hitherto undescribed, degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) to amplify astrovirus open reading frame (ORF) 1b-specific cDNA fragments followed by nucleotide sequence determination and analysis of the amplified fragments. ELV-1 was confirmed as an isolate of the astrovirus avian nephritis virus (ANV). ELV-4 (isolate 612) and ELV-3 (isolates FP3 and 11672) were antigenically and genetically related to the second characterized astrovirus of chickens, namely chicken astrovirus (CAstV). Using indirect immunofluorescence, the FP3 and 11672 ELV-3 isolates were very closely related to one another, and less closely related to ELV-4 and the previously described CAstV (P22 18.8.00 reference isolate). Comparative analyses based on the ORF 1b amplicon sequences showed that the FP3 and 11672 ELV-3 isolates shared high nucleotide (95%) and amino acid (98%) identities with one another, and lower nucleotide (76% to 79%) and amino acid (84% to 85%) identity levels with ELV-4 and the reference CAstV P22 18.8.00 isolates. The combined degenerate primer RT-PCR and sequencing methods also provided a nucleotide sequence specific to duck hepatitis virus type 2 (DHV-2) (renamed duck astrovirus) and duck hepatitis virus type 3 (DHV-3), which, for the first time, can also be identified as an astrovirus. Phylogenetic analyses based on the amplified ORF 1b sequences showed that ANV was the most distantly related avian astrovirus, with DHV-3 being more closely related to turkey astrovirus type 2 than DHV-2.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water.

The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites have unique toxicities and relatively greater potency compared to their pentavalent counterparts for many endpoints. In this study, dose-dependency in tissue distribution and urinary excretion for inorganic arsenic and its methylated metabolites was assessed in female C57Bl/6 mice exposed to 0, 0.5, 2, 10 or 50 ppm arsenic (as arsenate, AsV) in their drinking water for 12 weeks. No adverse effects were observed and body weight gain did not differ significantly among groups. Urinary excretion of arsenite monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), dimethylarsinic acid (DMAV), and trimethylarsine oxide (TMAO) increased linearly with dose, whereas AsV and monomethylarsonic acid (MMAV) excretion was non-linear with respect to dose. Total tissue arsenic accumulation was greatest in kidney > lung > urinary bladder >>> skin > blood > liver. Monomethyl arsenic (MMA, i.e. MMA(III)+MMAV) was the predominant metabolite in kidney, whereas dimethylarsenic (DMA, i.e., DMA(III)+DMAV) was the predominant metabolite in lung. Urinary bladder tissue had roughly equivalent levels of inorganic arsenic and dimethylarsenic, as did skin. These data indicate that pharmacokinetic models for arsenic metabolism and disposition need to include mechanisms for organ-specific accumulation of some arsenicals and that urinary metabolite profiles are not necessarily reflective of target tissue dosimetry.

Isolation of bovine adenovirus serotype 6 from a calf in the United Kingdom.

Two viruses, designated 99-8130(C) and 99-8130(I), were isolated in calf testis cells from the colon and ileum, respectively, of a suckled beef calf which had developed dysentery and died. Electron microscopy indicated that the mean (sd) size of the viral particles, 83 (2.5) nm, and their morphology were consistent with their being members of the family Adenoviridae. They were confirmed as adenoviruses by PCR when products of the expected size (608 bp) were amplified from both isolates by using a primer pair specific for members of the genus Atadenovirus. A comparison of the sequence of a 567 bp segment of the 99-8130(C) amplicon with that of other prototype bovine adenovirus (BAdV) strains of atadenoviruses identified the isolate as BAdV serotype 6 (BAdV-6), which had 99.3 per cent and 100 per cent identities at the nucleotide and amino acid levels, respectively, with the prototype BAdV-6 strain 671130. A virus neutralisation test was developed and indicated a high prevalence of antibody to BAdV-6 in Northern Irish cattle. There was no evidence of adenoviral inclusions in tissues from the affected calf and no antigen was detected when the tissues were stained by an immunoperoxidase technique, using a homologous antiserum raised in rabbits. The two viruses were the third reported isolation of BAdV-6, and the first from a clinically ill bovine animal.

Peptide motif of a cattle MHC class I molecule.

A consensus motif for a bovine major histocompatibility complex (MHC) class I molecule, A20, was derived from parainfluenza type-3 (PI-3) virus-infected muscle-derived fibroblast cells and peripheral blood leukocytes by extraction of the naturally processed peptides from MHC class I molecules by treatment with TFA and peptide sequencing of the complex mixture. The results showed that the majority of peptides were 9 amino acids long with position 2 occupied by lysine and position 9 occupied by arginine. The arginine at position 9 suggests that cattle, like humans, but unlike the mouse have permissive TAP transporter molecules accepting peptides with positively charged amino acids at their C-terminus. This is the first report of a MHC ligand motif in cattle.

Immunopathogenesis of chicken anemia virus infection.

The immunopathogenesis of chicken anemia virus (CAV) infection is reviewed. The virus causes a disease in young chicks which is characterised by generalised lymphoid atrophy, increased mortality and severe anemia. The virus appears to target erythroid and lymphoid progenitor cells in the bone marrow and thymus respectively. The B cells in the chicken are not susceptible to CAV infection and are not directly affected by the virus. Destruction of erythroid progenitors in bone marrow results in severe anemia, and depletion of granulocytes and thrombocytes. Destruction of precursor T cells results in depletion of mature cytotoxic and helper T cells with consequent effects on susceptibility to, and enhancement of, the pathogenicity of secondary infectious agents, and sub-optimal antibody responses. Apoptosis appears to be a feature of the lymphocyte depletion in the thymic cortex, which may be mediated by one of the non-structural viral proteins, VP3 (apoptin).

A comparison of in situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (PMWS).

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified condition affecting pigs in North America and Europe. Porcine circovirus antigen and nucleic acid have been demonstrated associated with lesions, and a new porcine circovirus designated PCV2 has been recovered from tissues of these animals. In this study, in situ hybridisation and immunohistochemical protocols were developed, optimized and compared for their relative sensitivity in detecting PCV2 antigens and nucleic acid in tissues from cases of PMWS that had been fixed for up to 6 months in formalin. For both immunohistochemistry and in situ hybridization, an increase in specific signal was observed following increased exposure to both protease XIV and proteinase K. Maximum signal and minimal loss of tissue morphology was seen after 40 min treatment with protease XIV (0.5 mg/ml). After optimisation, a comparison of these techniques on sequential sections demonstrated that both techniques successfully detected antigen or nucleic acid in all of the tissues examined. More positive cells, with increased signal intensity, were detected following immunohistochemistry.

Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera.

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1/4), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.

Studies on the antigenic relationship between bovine subgroup 2 and conventional mammalian adenoviruses using immunofluorescence.

In double immunodiffusion tests between a bovine subgroup 2 adenovirus (serotype 8) and other mammalian adenoviruses, no group-specific crossing was demonstrated. However, in cross-fluorescent antibody tests (FAT) between bovine subgroup 2 viruses (serotypes 5, 7 and 8) and conventional (non-subgroup 2) adenoviruses from several species (bovine adenovirus serotypes 1 and 2, ovine serotypes 1, 4 and 5, porcine serotype 1, and human serotypes 2 and 5) sharing of antigens was demonstrated. The FAT titres observed when rabbit antisera to conventional adenoviruses were used to stain bovine subgroup 2 viruses were, however, much lower than titres with other non-subgroup 2 viruses. The converse was also true. The crossing was also predominantly one-sided. The low level cross was confirmed using antisera to selected viruses prepared in chickens to exclude interference by possible natural adenovirus infections in the rabbits used to prepare the antisera in the initial experiments.

Infection of leucocyte cell cultures derived from different species with pig circovirus.

Cultures of leucocyte cells were prepared from pig bone marrow, peripheral blood, lung washings, thymus and lymph nodes. Cell cultures were also prepared from peripheral blood from sheep, cattle and a human. Immunofluorescent (IF) staining of all these cultures, following inoculation with pig circovirus (PCV), detected virus replication in all the cell cultures derived from pigs and in the cell cultures derived from cattle. Virus replication in pig leucocyte cell cultures was confirmed by demonstrating the production of infectious virus. Double immunostaining of PCV infected cells using monoclonal antibodies specific for cell membrane markers indicated infection was confined to monocyte/macrophage cell types. No PCV antigen was detected in T or B cells in infected cell cultures.

Characterisation of Australian ovine adenovirus isolates.

We have characterised two groups of adenoviruses isolated from sheep in Australia. Restriction endonuclease maps for enzymes BamHI, ClaI, SalI, SmaI and SphI have been determined for the genome of ovine adenoviruses related to bovine adenovirus serotype 7 (BAV 7) from sheep in Western Australia. Although previously serotyped as BAV 7 these isolates are different from bovine isolates of BAV 7 based on comparison with published restriction endonuclease profiles and maps of BAV 7 cattle isolates. Additional adenovirus isolates obtained from Victorian sheep have been serotyped as ovine adenovirus type 5 (OAV 5). On the basis of restriction endonuclease analysis these viruses are different from the sheep BAV 7 isolates. Following infection of sheep with ovine BAV 7 and OAV 5 isolates, virus was recovered from nasal and rectal swabs for several days. Antibodies detected by ELISA and serum neutralisation tests (SN) developed by 15 days after infection. Virus also spread from the infected sheep to an incontact control and one of ten sheep purchased for infection studies had SN antibodies to BAV 7 suggesting that BAV 7-like viruses naturally infect sheep in Victoria and Western Australia. With further development, these ovine adenoviruses may be suitable as vectors for the delivery of vaccine antigens to sheep and cattle.

The reverse transcription polymerase chain reaction for the diagnosis of porcine reproductive and respiratory syndrome: comparison with virus isolation and serology.

A single-tube reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of porcine reproductive and respiratory syndrome (PRRS) virus in blood samples from infected pigs was developed. This test was assessed for sensitivity and application as a rapid diagnostic tool by comparison with virus isolation and detection of PRRS virus antibody in blood. The RT-PCR test was slightly more sensitive than virus isolation for detection of virus in serum and markedly more sensitive than virus isolation from plasma from experimentally infected pigs. The RT-PCR test was also applicable when using whole blood-impregnated filter paper discs, with 94% of the specimens taken by this procedure being positive when compared to RT-PCR performed on serum. PRRS viral nucleic acid was detected in blood samples as early as 24 h after infection and persisted for some time, whereas circulating antibody to PRRS virus was not detected in the same animals until 9 days after infection. These results indicate that the RT-PCR may be an useful technique for the early identification of PRRS viral nucleic acid in blood samples of infected pigs.

Rapid detection of porcine reproductive and respiratory syndrome viral nucleic acid in blood using a fluorimeter based PCR method.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus recognised world wide as an important cause of reproductive failure and pneumonia in pigs. American and European strains of PRRSV, differentiated antigenically and genomically, have been reported. PRRSV infections are currently diagnosed using serology, virus isolation and/or immunocytochemistry. In order to overcome various drawbacks associated with these techniques, conventional, block-based RT-PCR methods for the detection of PRRSV nucleic acid in clinical samples have been described. These methods require gel electrophoresis for analysis of PCR products and present high risk of DNA carry-over contamination between the samples tested. We describe the detection of PRRSV RNA in serum samples and in blood impregnated filter disks (FDs), obtained from experimentally inoculated pigs, using a closed-tube, fluorimeter-based PCR assay. The assay eliminates the use of gel electrophoresis, and is as sensitive and specific as the conventional block-based PCR assay, detecting positive samples as early as 1 day post-inoculation. We also report a rapid fluorimeter based PCR method for differentiating American and European strains of PRRSV.

Effect of "in vitro" exposure of bovine alveolar macrophages to different strains of bovine respiratory syncytial virus.

A vaccine strain of respiratory syncytial (RS) virus and an isolate from pneumonic calves (AC2) were inoculated onto cultures of bovine alveolar macrophages recovered by lung lavage, and the functional properties of the cells observed over a period of 10 days. In most cultures no infectious virus was produced although immunofluorescence indicated the presence of virus antigens in some cells. No significant difference was noted between infected and control macrophage cultures in their capacity to phagocytose latex particles (neutral phagocytosis), although the ability to phagocytose complement-coated Candida krusei cells was affected, particularly with the AC2 strain after 6 days. Killing of C. krusei cells was slightly affected by infection of macrophages with the vaccine strain and was dramatically affected by infection with strain AC2. C3b and Fc receptor expression was adversely affected by both virus strains. Production of neutrophil chemotactic factors was increased in cultures infected with both strains, but was greater with AC2, suggesting that some properties of the cells were activated.

Testing of bovine sera by ELISA for IgG, IgM and IgA rheumatoid factors.

Sera from 19 colostrum-deprived calves less than 1 week old, 24 colostrum-supplemented calves less than 1 week old, 36 3-5-month-old calves and 200 females greater than 9 months of age were tested by ELISA for the presence of IgM, IgG and IgA rheumatoid factors (RF). An increasing level of IgM- and IgG-RF with age was found. IgG-RF levels in the colostrum-supplemented calves were significantly higher than in the non-supplemented calves (p < 0.001). Individual IgG-RF values correlated with serum IgG levels, as determined by zinc sulphate turbidity testing (r=0.59, p < 0.01). No IgA-RF was detected. The cross-reactivity of IgM-RF with heterologous IgG was found to be greatest with rabbit IgG, followed by mouse and chicken IgG. The significance of rheumatoid factors in relation to diagnostic testing is discussed.

Effect of BVD virus infection on alveolar macrophage functions.

Alveolar macrophages (AM) were recovered by bronchoalveolar lavage from a group of eight calves at various times before and after inoculation with a cytopathic respiratory isolate of bovine viral diarrhoea virus (BVDV). A second group of four calves were given tissue culture medium as a control inoculum. Macrophages were also recovered from two additional, uninoculated calves, and were exposed to BVDV in vitro. Tests were carried out on the recovered macrophages to determine the effects of the virus on several functional properties. Immunofluorescence did not indicate the AM as being readily susceptible to this isolate of BVDV, although infection did occur. Fc receptor (FcR) and complement receptor (C3R) expression, phagocytosis and microbicidal activity and the production of neutrophil chemotactic factors were all significantly reduced in macrophages recovered from BVDV infected calves, compared with pre-inoculation control levels, whereas the control inoculated calves displayed significant increases in some of the functions. With macrophages exposed to the virus in vitro however, only FcR and C3R expression and phagocytic activity were significantly reduced. The results demonstrate that BVDV can reduce local immune defences in the lung, following infection by the respiratory route, and in conjunction with the other immunosuppressive properties of BVDV would favour a pre-disposing role for the virus in the pathogenesis of respiratory disease in calves.

Primary cytotoxic response of bovine peripheral blood leukocytes to parainfluenza type 3 virus infection.

Peripheral blood mononuclear cells were isolated from calves following Parainfluenza Type-3 (PI-3) virus infection and incubated with autologous and non-autologous PI-3 virus-infected muscle cells in a 4 h chromium release assay. Peaks of cytotoxicity, which ranged from 12% to 53% were observed between Days 6 and 9 post infection, with an effector to target cell ratio of 100:1. Killing of PI-3 virus-infected or uninfected mismatched muscle cells was never more than 5%.