Concentration- and time-dependent genomic changes in the mouse urinary bladder following exposure to arsenate in drinking water for up to 12 weeks.

Inorganic arsenic (As(i)) is a known human bladder carcinogen. The objective of this study was to examine the concentration dependence of the genomic response to As(i) in the urinary bladders of mice. C57BL/6J mice were exposed for 1 or 12 weeks to arsenate in drinking water at concentrations of 0.5, 2, 10, and 50 mg As/l. Urinary bladders were analyzed using gene expression microarrays. A consistent reversal was observed in the direction of gene expression change: from predominantly decreased expression at 1 week to predominantly increased expression at 12 weeks. These results are consistent with evidence from in vitro studies of an acute adaptive response that is suppressed on longer exposure due to downregulation of Fos. Pathways with the highest enrichment in gene expression changes were associated with epithelial-to-mesenchymal transition, inflammation, and proliferation. Benchmark dose (BMD) analysis determined that the lowest median BMD values for pathways were above 5 mg As/l, despite the fact that pathway enrichment was observed at the 0.5 mg As/l exposure concentration. This disparity may result from the nonmonotonic nature of the concentration-responses for the expression changes of a number of genes, as evidenced by the much fewer gene expression changes at 2 mg As/l compared with lower or higher concentrations. Pathway categories with concentration-related gene expression changes included cellular morphogenesis, inflammation, apoptosis/survival, cell cycle control, and DNA damage response. The results of this study provide evidence of a concentration-dependent transition in the mode of action for the subchronic effects of As(i) in mouse bladder cells in the vicinity of 2 mg As(i)/l.

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.

Mercury and methylmercury accumulation and excretion in prairie voles (Microtus ochrogaster) receiving chronic doses of methylmercury.

Methylmercury cation (MeHg) and divalent mercury (Hg++) accumulation in liver, kidney, and brain were quantified in prairie voles (Microtus ochrogaster) at 0, 3, 6, and 12 weeks during chronic exposure to aqueous MeHg. Dose groups received deionized water or aqueous solutions containing 9, 103, or 920 ng MeHg/ml. Our study presents temporal patterns of Hg++ and MeHg concentrations in organ tissues and makes inter-tissue comparisons at each time point to illustrate the accumulation and distribution of Hg species during the study. MeHg was accumulated in tissues for 3 weeks and then concentrations plateaued. Mercury accumulated in brain, liver, and kidney to average concentrations of 510 ng/g, 180 ng/g, and 3400 ng/g, respectively. MeHg and Hg++ concentrations were roughly equivalent in liver, kidney, and urine. MeHg concentrations in brain tissue were 2 to 20 times the concentrations of Hg++. Regression analysis was also used to demonstrate the utility of urinalysis as an indicator of Hg++ and MeHg concentrations in organ tissue (p < 0.001).

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.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system.

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication.

Immunopathologic investigations with an attenuated chicken anemia virus in day-old chickens.

The immunopathologic effects induced by two attenuated chicken anemia virus (CAV) isolates, known as cloned isolate 34 (CI 34) and cloned revertant isolate 18 (CRI 18), that were derived from highly passaged pools of Cux-1 CAV isolate, were compared with those induced by a pathogenic, molecularly cloned, low-passage Cux-1 isolate (CI Cux). This comparison involved the intramuscular inoculation of 1-day-old specific-pathogen-free chicks with each of the viruses and investigation of birds at selected days postinoculation for gross pathology and depletions in the thymic T-cell populations as determined by flow cytometry. Whereas infection with the pathogenic CI Cux produced severe anemia and pronounced bone marrow and thymus lesions, infections with the attenuated CRI 18 and CI 34 isolates produced no anemia, no or mild lesions, respectively, and moderate T-cell depletion. The results suggest that, with CAV, reduced pathogenicity for 1-day-old chicks correlates with reduced depletion of T-cell populations in the thymus and with reduced severity of lesions in the thymus and bone marrow.

Mercury in Morelet's crocodile eggs from northern Belize.

Recent studies have examined mercury accumulation in crocodilians. However, though most researchers have focused on tissue concentrations, few have examined mercury levels in crocodilian eggs. In July 1995, we analyzed mercury in 31 nonviable Morelet's crocodile ( Crocodylus moreletii) eggs collected from eight nests across three localities in northern Belize. All eggs were found to contain mercury. Based on an individual egg basis, mean concentration of mercury for all three localities was among the lowest reported for any crocodilian species. When localities were examined separately, mean concentrations for Laguna Seca and Gold Button Lagoon were comparable to those observed in other studies, and the mean for Sapote Lagoon was the lowest ever reported. Based on mean nest concentrations, mercury in eggs from Laguna Seca was approximately two- and tenfold higher than for Gold Button Lagoon and Sapote Lagoon, respectively. Variability in mercury concentrations among localities is likely the result of site-specific differences in mercury input, bioavailabilty, and bioaccumulation. Mercury concentrations were relatively uniform in eggs from the same nest and among nests from the same localities. The presence of mercury in Morelet's crocodile eggs suggests exposure in adult females, developing embryos, and neonates. However, crocodiles in these areas show no overt signs of mercury toxicity, and no indication of population decline is evident. A paucity of data on the effects of mercury on crocodilians precludes meaningful speculation as to the biological significance of tissue and egg concentrations. Controlled laboratory studies and long-term population monitoring are needed to address these questions.

Antibody-dependent killing of virus-infected targets by NK-like cells in bovine blood.

Natural killer (NK) cell lysis of target cells by an Fc receptor-mediated mechanism has not been conclusively demonstrated in cattle (Campos and Rossi, Vet. Immunol. Immunopathol. 8, 351-362, 1985), although it is well recognized in other species (Sulica et al., Nat. Immun. 14, 123-133, 1995). To resolve this problem, bovine peripheral blood mononuclear cells were completely depleted of adherent monocyte/macrophage type cells. The resulting enriched population of lymphocytes, was totally devoid of adherent monocytes, but contained up to 2% NK-like cells. On their own, this population had very low background levels of cytotoxicity for virus-infected target cells in 51chromium release assays, but following the addition of virus-specific antibodies, high levels of lysis were observed. This enhanced level of antibody-dependent cytotoxicity demonstrated that bovine NK-like cells can mediate killing of targets by an Fc receptor-mediated mechanism as has been demonstrated for NK cells from other species.

Accumulation of DDT and mercury in prothonotary warblers (Protonotaria citrea) foraging in a heterogeneously contaminated environment.

Foraging areas of adult prothonotary warblers (Protonotaria citrea) were determined using standard radiotelemetry techniques to determine if soil concentrations of p,p'dichlorodiphenyltrichloroethane (p,p'DDT) and mercury in foraging areas could be used to predict contaminant levels in diets and tissues of nestling warblers. Adult warblers were fitted with transmitters and monitored for approximately 2 d while foraging and feeding 6- to 8-d-old nestlings. Foraging ecology data were integrated with contaminant levels of soil, diets, and tissues into a comprehensive analysis of geographic variation in contaminant exposure and uptake using linear regression. Concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) and mercury in nestling tissues varied considerably across the study site. Mean concentration of DDE was greater in eggs than all other tissues, with individual samples ranging from 0.24 to 8.12 microgram/kg. In general, concentrations of DDT in soil were effective in describing the variation of contaminants in adipose samples. Concentrations of mercury in soils accounted for 78% of the variation in kidney samples. This was the best relationship of any of the paired variables. All other relationships showed relatively poor predictive ability.

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 parainfluenza-3 virus challenge on cell-mediated immune function in parainfluenza-3 vaccinated and non-vaccinated calves.

A group of four conventional, colostrum-fed calves was vaccinated with live parainfluenza type 3 (PI-3) virus vaccine at 1 and 5 weeks of age. A group of four control calves was treated with cell culture medium at the same time. Two weeks after the second vaccination, both groups of calves were challenged with PI-3 virus by a combined respiratory route. Blood and nasal mucus samples were collected at intervals, and alveolar macrophages were recovered before and after challenge by bronchoalveolar lavage. The results demonstrated that clearance of virus, as indicated by presence of virus antigen was more rapid in previously vaccinated calves. Several alveolar macrophage functions were markedly reduced in all calves 5 to 7 days following virus challenge, although microbicidal activity was unaffected, compared to the controls. The production of neutrophil chemotactic factors by alveolar macrophages occurred more rapidly after virus challenge in the previously vaccinated calves and this correlated with a more rapid neutrophil influx into the lungs in these animals.

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).

Detection of IgM responses to bovine respiratory syncytial virus by indirect ELISA following experimental infection and reinfection of calves: abolition of false positive and false negative results by pre-treatment of sera with protein-G agarose.

The IgM responses in three panels of sera generated by infection and reinfection of calves with bovine respiratory syncytial virus (BRSV) were measured by indirect ELISA (I-ELISA). The effect of depleting serum IgG by pre-treatment with protein G agarose (PGA) was evaluated. Following primary infection a weak IgM response was detected in the untreated sera of 3 out of 4 calves with maternally derived antibody (MDA). Both the magnitude and duration of the specific IgM responses in these calves were increased by pre-treatment with PGA. In addition, the fourth infected calf tested gave a single positive IgM result following PGA treatment. Transient or persistent IgM responses which were abolished by pre-treatment of sera with PGA were detected in 4/8 calves following reinfection. These were considered to be false positive results, consistent with the influence of IgM rheumatoid factor (IgM-RF). One of these calves and two additional calves showed transient increases in IgM which were resistant to PGA treatment. These were considered to represent specific IgM responses to reinfection. The results indicate the ability of PGA treatment to eliminate both false positive and false negative results and emphasise the necessity for controlling the influence of IgM-RF in IgM-specific indirect ELISAs.

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.