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.

Detection of pigeon circovirus in cloacal swabs: implications for diagnosis, epidemiology and control.

Pigeon circovirus (picv) was detected in cloacal swab samples by means of a newly-developed, sensitive pcr. An initial investigation of 17 Belgian racing pigeons aged up to eight months showed that rates of detection of 88 per cent and above were achieved using samples of cloacal swab, blood and bursa of Fabricius. The sampling of 15 caged pigeons six times when they were from three to 31 weeks of age indicated that picv infections were more readily detected in cloacal swabs than in blood, and that the virus could be detected in cloacal swabs for longer periods after infection than in blood. picv infections were detected in cloacal swabs from 38 of 47 young pigeons aged from two to 31 weeks, from 12 racing lofts, which had clinical signs including diarrhoea and weight loss, regurgitation and respiratory signs. Samples from birds from two infected lofts indicated that picv could be detected in some birds for at least 27 weeks. Although nine of 14 pigeons aged from 32 to 45 weeks were virus-positive, picv was detected in only one of 18 adult pigeons that originated from four infected lofts.

Molecular analyses of mycobacteria other than the M. tuberculosis complex isolated from Northern Ireland cattle.

Mycobacteria other than the Mycobacterium tuberculosis complex (MOTT), isolated from Northern Ireland cattle, were identified by PCR amplification of the 16S rRNA gene, and subsequent reverse cross blot hybridisation and sequence analyses. Elucidation of the MOTT species was to facilitate specificity testing of new and existing diagnostic test reagents for bovine tuberculosis. The presence of the genes for potential diagnostic antigens: MPB70, MPB64, ESAT-6 and CFP-10 in the isolated MOTT species was investigated. Molecular analyses of cultured isolates from bovine lymph node specimens of 48 cattle identified a wide variety of mycobacterial species including Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium bohemicum, Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium holsaticum, Mycobacterium palustre, Mycobacterium sp. IWGMT 90210, Mycobacterium sp. LIV-2129, a potentially novel mycobacterial species (EMBL/GenBank/DDBJ Accession Number AJ617495) and Rhodococcus equi. Apart from M. kansasii, the results of traditional (standard phenotypic and biochemical) and molecular identification methods did not correlate well, with traditional methods identifying fewer species. Most of the species identified were either recognised pathogenic or potential pathogenic species. The genes for ESAT-6, CFP-10 and, unusually, MPB64 were detected in M. kansasii only. The MPB70 gene was not detected in any of the species. This study supported restricted species distribution of these genes as well as identifying a different range of MOTT species that could be included in specificity testing of new diagnostic reagents for bovine tuberculosis.

Isolation and preliminary characterization of chicken anemia virus from chickens in Nigeria.

Chicken anemia virus (CAV) was isolated for the first time from the Nigerian chicken population. The virus was recovered from necropsied birds from broiler and pullet flocks that suffered disease outbreaks tentatively diagnosed as infectious bursal disease. A sensitive polymerase chain reaction (PCR) assay detected CAV DNA in tissues of necropsied birds. Restriction endonuclease analysis performed with the 733-bp PCR product and the Cfo I enzyme indicated at least two different CAVs were circulating among the Nigerian chicken population. Four isolates were obtained from pooled liver and thymus tissues using the MDCC-MSB1 cell line. These isolates were found to be antigenically closely related to the Cuxhaven-1 (Cux-1) reference strain of CAV when reacted with four monoclonal antibodies prepared against the Cux-1 virus. One of the isolates (isolate A) induced thymus atrophy, bone marrow aplasia, and low hematocrit values when inoculated into 1-day-old specific-pathogen-free chickens. These findings not only demonstrate that CAV is present in Nigeria, but they also likely represent the first cell culture isolation of the virus in Africa.

Virological and serological evidence of bovine herpesvirus type 4 in cattle in Northern Ireland.

Bovine herpesvirus type 4 (BHV-4), a member of the genus Rhadinovirus, subfamily Gammaherpesvirinae, within the family Herpesviridae, was isolated in fetal bovine lung cells from samples of vaginal discharge taken from a dairy herd in which approximately 50 per cent of the cattle developed metritis after calving. The identity of the isolate was confirmed by immunofluorescent staining with a BHV-4-specific monoclonal antibody and partial sequencing of a portion of the glycoprotein B gene. Serological testing failed to demonstrate a significant association between the exposure of the cattle to BHV-4 and the metritis, but several cattle seroconverted during the periparturient period, consistent with the recrudescence and shedding of virus associated with the stresses of parturition and the onset of lactation. Despite the previous failure to detect BHV-4 in Northern Ireland, a serological survey of 999 cattle in 49 dairy herds and 51 beef herds found widespread evidence of exposure: 29 of the dairy herds and 35 of the beef herds contained one or more seropositive cattle, and 33.3 per cent of the dairy cattle and 23.3 per cent of the beef cattle were positive.

Nucleotide sequence-based identification of a novel circovirus of canaries.

Circovirus-like, spherical particles measuring 16 to 18 nm in diameter were detected in organ homogenates from adult canaries that had died after a short illness characterized by dullness, anorexia, lethargy and feather disorder. A polymerase chain reaction method, based on degenerate primers specific to conserved amino acid sequences in the circovirus replication-associated protein, was used to amplify DNA specific to a novel circovirus, tentatively named canary circovirus (CCV). Sequence analysis of a 510 nucleotide genomic fragment indicated that CCV exhibited 67.4, 64.9, 53.7 and 53.9% nucleotide identities and 70.0, 61.8, 40.4 and 40.1% amino acid identities with columbid (pigeon) circovirus (CoCV), beak and feather disease virus (BFDV), porcine circovirus type 1 and porcine circovirus type 2, respectively. CCV therefore represents a new avian virus of the genus Circovirus of the family Circoviridae, and is more closely related to CoCV than BFDV.The availability of nucleotide sequence data will facilitate the development of DNA-detecting diagnostics with which the prevalence of CCV infections can be assessed.

Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections.

Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.

Disseminated Mycobacterium genavense infection in a FIV-positive cat.

An 8-year-old FIV-positive Australian cat was presented with coughing, periocular alopecia, pyrexia and inappetence. Skin scrapings demonstrated Demodex cati mites. Antibiotics were administered and it was treated successfully for periocular demodectic mange, but the cat continued to exhibit respiratory signs and lose weight. Further investigation revealed an ascarid infection and active chronic inflammation of undetected cause affecting the lower airways. Repetitive treatment with pyrantel failed to eradicate the ascarid infection. The cat became cachectic and developed moist ulcerative dermatitis of the neck, severe non-regenerative anaemia, leucopenia and thrombocytopenia. Necropsy and histopathology revealed mycobacteriosis affecting skin, lungs, spleen, lymph nodes, liver and kidney. Attempted culture of frozen tissues at a mycobacteria reference laboratory was unsuccessful. Paraffin-embedded, formalin-fixed tissue was retrieved and examined using PCR to amplify part of the 16S rRNA gene. A diagnosis of disseminated Mycobacterium genavense infection was made based on the presence of acid fast bacteria in many tissues and partial sequence of the 16S rRNA gene. Although M genavense has been identified previously as a cause of disseminated disease in AIDS patients, this is the first report of infection in a cat. It was suspected that the demodecosis, recurrent ascarid infections and disseminated M genavense infection resulted from an immune deficiency syndrome consequent to longstanding FIV infection.

Determination of the etiology of presumptive feline leprosy by 16S rRNA gene analysis.

PCR-amplified 16S rRNA gene sequences were obtained directly from tissue specimens from eight cats with presumptive feline leprosy. Acid-fast bacilli were observed in sections from all eight specimens, but culture for mycobacteria was successful for one specimen only. Analysis of the V2 variable region of each 16S rRNA PCR product identified a sequence with 100% nucleotide identity to the sequences of Mycobacterium lepraemurium, Mycobacterium avium, and Mycobacterium paratuberculosis in four of the specimens from cats with feline leprosy. Separate M. paratuberculosis- and M. avium-specific PCR amplifications of the four specimens were negative, thus substantiating the identification of M. lepraemurium in these specimens from cats with feline leprosy. Further sequence analysis of the V3 variable region of one of the four specimens provided conclusive evidence of the presence of M. lepraemurium. This is the first report of the definitive identification of M. lepraemurium in cats with feline leprosy by molecular biology-based analyses. M. avium, which is rarely reported in cats, and Mycobacterium chitae, a reported nonpathogenic, rapidly growing mycobacterial species found in the environment, were identified in the specimen from which acid-fast bacilli were cultured. Two of the specimens from cats were infected with a potentially novel species of mycobacteria which had a 16S rRNA gene sequence sharing the closest nucleotide sequence identity with that of Mycobacterium malmoense. Molecular biology-based analyses provided for the accurate and rapid diagnosis of mycobacterial infections in cats and circumvented the problems of culture and misdiagnosis of feline leprosy associated with traditional methods.

Diagnosis of goose circovirus infection in Hungarian geese samples using polymerase chain reaction and dot blot hybridization tests.

A polymerase chain reaction (PCR) and dot blot hybridization (DBH) test have been developed for the diagnosis of infection by a novel circovirus of geese (GoCV). These tests were applied to samples of bursae of Fabricius from sick and dead birds from commercial goose farms in Hungary. In this second report of the occurrence of circovirus infection in diseased geese, 103 of 214 (48.1%) and 37 of 150 (24.6%) birds, and 49 of 76 (64.5%) and 18 of 76 (23.7%) flocks were positive by PCR and DBH respectively. The sensitivity of the PCR test was such that 0.10 fg of virus DNA was detectable. The DBH test was less sensitive, only detecting larger amounts (40 pg) of DNA, but was used as a semi-quantitative method for detecting the presence of virus. The incidence of infection was affected by factors such as the age of the birds and rearing methods.