Bruce G. Corney

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland.

Aims:u2002 To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent‐treated soil.

The first five days: field and laboratory investigations during the early stages of the equine influenza outbreak in Australia, 2007.

Until August 2007, Australia was one of only three countries internationally recognised to be free of equine influenza (EI). This report documents the diagnosis of the first cases of EI in Australian horses and summarises the investigations that took place over the next 5 days. During that time, a multifocal outbreak was identified across eastern New South Wales and south-eastern Queensland. The use of an influenza type A pan-reactive real-time reverse transcription polymerase chain reaction allowed rapid confirmation of suspect cases of EI.

Equine herpesvirus infections in yearlings in South-East Queensland

Twelve nasal swabs were collected from yearling horses with respiratory distress and tested for equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4) by real-time PCR targeting the glycoprotein B gene. All samples were negative for EHV-1; however, 3 were positive for EHV-4. When these samples were tested for EHV-2 and EHV-5 by PCR, all samples were negative for EHV-2 and 11 were positive for EHV-5. All three samples that were positive for EHV-4 were also positive for EHV-5. These three samples gave a limited CPE in ED cells reminiscent of EHV-4 CPE. EHV-4 CPE was obvious after 3xa0days and was characterised by syncytia. None of the samples produced cytopathic effect (CPE) on African green monkey kidney (Vero) cells or hamster kidney (BSR) cells. Four of the samples, which were positive in the EHV-5 PCR, produced CPE on rabbit kidney (RK13) cells and equine dermis (ED) cells. EHV-5 CPE on both cell lines was slow and was apparent after four 7-day passages. On RK13 cells, the CPE was characteristic of equid herpesvirus, with the formation of syncytia. However, in ED cells, the CPE was characterised by ring-shaped syncytia. For the first time, a case of equine respiratory disease involving dual infection with EHV-4 and EHV-5 has been reported in Queensland (Australia). This was shown by simultaneously isolating EHV-4 and EHV-5 from clinical samples. EHV5 was recovered from all samples except one, suggesting that EHV5 was more prevalent in young horses than EHV2.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

Isolation of bovine herpesvirus type 5 from the semen of a healthy bull in Australia

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.

Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus)

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5′ Taq nuclease assay: a new tool for diagnosing infectious coryza

A 5′ Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5′ Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5′ Taq nuclease assay was demonstrated.

Detection and quantitation of gallid herpesvirus 1 in avian samples by 5′ Taq nuclease assay utilizing Minor Groove Binder technology

A 5′ Taq nuclease assay utilizing Minor Groove Binder technology and targeting the thymidine kinase gene of gallid herpesvirus 1 (GaHV-1) was designed and optimized for use in diagnosing avian infectious laryngotracheitis. The assay was specific for GaHV-1 in that it did not react with other avian viral or bacterial pathogens. The detection limit was 1.0×10−2 median tissue culture infectious dose per reaction or 90 target copies per reaction. Fifteen out of 41 diagnostic samples from sick birds reacted in the assay, five of which produced a typical alphaherpesvirus cytopathic effect (CPE) on chicken kidney (CK) cells. Sequencing, using amplicons generated by a polymerase chain reaction with primers flanking the 5′ Taq nuclease amplicon, confirmed the presence of GaHV-1 in six samples (two producing alphaherpesvirus CPE on CK cells, three not producing alphaherpesvirus CPE, and one that was not inoculated onto CK cells). Tracheal swabs taken from 18 healthy broilers did not react in the assay. The ability of the assay to determine viral load in samples was demonstrated. Overall the assay is suitable for the rapid diagnosis of infectious laryngotracheitis.

Validation of an influenza virus A 5'Taq nuclease assay for the detection of equine influenza virus A RNA in nasal swab samples.

OBJECTIVEnDescribe the in-house validation of a previously reported influenza virus type A 5Taq nuclease assay for detecting equine influenza virus A RNA in nasal swab material.nnnMETHODSnThe validation compares the 5Taq nuclease assay with a gel-based reverse transcription nested polymerase chain reaction (PCR) previously reported by the Irish Equine Centre for detection of H3N8 and H7N7 equine influenza viruses. This test was chosen because it targets a different region of the viral genome to the real-time test, so it is not merely a repeat of the same test in a different format. Moreover, nested PCRs are commonly considered to have similar sensitivity to real-time PCRs and are therefore ideal for evaluation comparisons.nnnRESULTSnThe sensitivity of the nested PCR was comparable to the 5Taq nuclease test. Known positive samples and known negative samples reacted with both tests with 100% correlation. Parallel testing of 276 nasal swab samples showed 98% agreement.nnnCONCLUSIONnThe specificity of the nested amplicons was confirmed by nucleotide sequencing and showed >99.5% identity with the same region of previously published equine influenza virus A sequences. The results of this work are appropriate validation for the acceptance of the real-time PCR for equine influenza A virus in equine nasal swabs.