Barry J. Rodwell

A protein immunoblot test for detection of bovine leukemia virus p24 antibody in cattle and experimentally infected sheep

A protein immunoblot test for detecting antibody to the bovine leukemia virus p24 antigen is described. The test employs a crude antigen preparation derived from concentrated cell culture fluid, and an optimised biotin-streptavidin-peroxidase amplification system for immunodetection. The test is highly specific and is more sensitive than the gp51 agar gel immunodiffusion (AGID) test for detection of BLV antibody in cattle and experimentally infected sheep. In a selected set of 30 field sera from cattle which had given equivocal results in the gp51-AGID test, 21 were positive, 4 were negative, and 5 gave an uncertain result in the p24-immunoblot test.

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.

An enzyme-linked immunosorbent assay for detection of bovine leukaemia virus p24 antibody in cattle

An ELISA for detecting antibody to the bovine leukaemia virus (BLV) core protein p24 is described. The test uses p24 antigen purified from concentrated cell culture supernate by lectin-affinity chromatography and gel filtration. The sensitivity and specificity of the p24-ELISA for diagnosing BLV infection relative to the gp51 agar gel immunodiffusion test, were 98.1 and 96.7%, respectively. In the event of widespread use of gp51 based vaccines, the p24-ELISA should differentiate effectively between naturally infected and vaccinated animals.

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.

Babesia rodhaini, Babesia bovis, and Babesia bigemina: Analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst

Abstract The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.

Comparison of DNA-spot hybridization, cell culture and direct immunofluorescence staining for the diagnosis of avian chlamydiae

DNA-spot hybridization, cell culture and direct immunofluorescence staining were compared for the detection of avian Chlamydia psittaci strains in cell culture dilutions and in routine samples submitted for diagnosis. With dilutions of infected cell culture material, growth in BGM cells was by far the most sensitive technique, detecting 0.01 infected cells (20 elementary bodies) ml-1. DNA-spot hybridization and direct immunofluorescence staining were of approximately equal sensitivity, both detecting 16 infected cells (3.2 x 10(4) elementary bodies) per ml-1. When 27 avian liver and spleen samples were assayed, all 3 tests performed similarly (13 positive and 12 negative by all 3 tests). This suggests that in most avian samples presented for diagnosis, sufficient numbers of chlamydiae are present to allow any of the test to the be used. Thus, the direct immunofluorescence staining method is currently the test of choice for routine diagnosis since it is available in kit form, is relatively simple and quick to perform, and like DNA-spot hybridization, detects non-viable as well as viable organisms. However, if low levels of chlamydiae are to be effectively detected, such as in carrier birds or birds with recently acquired infections, then cell culture should be used.

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.