Ibrahim S. Diallo

Field isolates of fowlpox virus contaminated with reticuloendotheliosis virus

The polymerase chain reaction (PCR) method was used to examine samples from field cases of fowlpox for the presence of reticuloendotheliosis virus (REV). The S-strain fowlpox vaccine, known to be contaminated with REV, served as a positive control. Fowlpox virus was grown from field samples and vaccines by inoculation of embryonated hen eggs by the chorioallantoic membrane (CAM) route. DNA was extracted from the CAM lesions and examined for REV proviral sequences using primers specific for the long terminal repeats of REV. Amplicons of the expected length were detected in all the 45 field samples from poultry and in the S strain vaccine. Two other vaccines and two isolates from wild birds contained no detectable REV sequences. The PCR products from the vaccine and one field isolate were sequenced and were identical. These products showed 81 to 87.5% homology with the published sequences for the long terminal repeats of REV. It was not determined whether the REV proviral DNA was integrated with cellular DNA, fowlpox DNA or both. Inoculation of day-old chickens with the S-strain vaccine resulted not only in the production of fowlpox lesions but also feathering defects and proventriculitis. This suggests that the REV present in the vaccine is replication competent. Problems being encountered with protection from fowlpox following vaccination in Australia might be attributed to simultaneous challenge with fowlpox virus and REV.

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.

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.

Molecular studies on avian strains of Pasteurella multocida in Australia.

A collection of 45 strains of Pasteurella multocida was assembled. The strains had been isolated from cases of fowl cholera in eastern Australia over 8 years, and included mainly type A strains. All the strains were examined for plasmids and resistance to 10 antimicrobial agents and most of the strains were examined for restriction fragment length polymorphism. Nine strains were assayed for pathogenicity for mice. Twenty strains yielded no plasmid. Seven contained a single plasmid of 1.3 kbp and 18 contained 2 plasmids, of 2.4 and 7.5 kbp. All the strains were resistant to streptomycin, trimethoprim and lincomycin while one strain was resistant to tetracycline. There was no correlation between plasmid content and resistance to antimicrobial agents. Three strains that lacked plasmids were highly virulent for mice, 6 strains containing plasmids were not. Restriction fragment length polymorphism generated by HpaII allowed the 39 strains that were tested to be divided into 10 groups.

Characteristics of a haemolytic extract from avian Pasteurella multocida.

In experimental fowl cholera, the intramuscular inoculation of Pasteurella multocida induces tissue damage that implies proteolytic or cytolytic activity of the bacteria. Such activity could not be demonstrated by conventional in vitro tests. The treatment of P. multocida strain VP21 with Tween-80 yielded an extract that lysed washed chicken red cells. Extracts were active to a maximum titre of 64. Haemolytic activity of the extract was neither affected by boiling nor by extremes of pH, indicating the active component was not a simple protein. Treatment with trypsin had no effect, but it was inactivated by Proteinase K. Yields were highest from bacteria grown in dextrose starch- or casein sucrose-yeast broths; were similar if cultured in air or anaerobically, but were reduced if the bacteria were grown in 5% CO(2). Haemolytic activity was eliminated on exposure to serum or serum albumen. The extract from strain VP21 haemolysed red cells from the chicken, rabbit, sheep, horse, bovine and human, with the highest titres observed on chicken cells. Six other avian strains and seven out of 10 strains of P. multocida from other species yielded an extract which haemolysed chicken red cells. The elaboration of this cytotoxic substance in vivo and its role in pathogenesis remains to be determined.

Australian bat lyssavirus infection in two horses

In May 2013, the first cases of Australian bat lyssavirus infections in domestic animals were identified in Australia. Two horses (filly-H1 and gelding-H2) were infected with the Yellow-bellied sheathtail bat (YBST) variant of Australian bat lyssavirus (ABLV). The horses presented with neurological signs, pyrexia and progressing ataxia. Intra-cytoplasmic inclusion bodies (Negri bodies) were detected in some Purkinje neurons in haematoxylin and eosin (H&E) stained sections from the brain of one of the two infected horses (H2) by histological examination. A morphological diagnosis of sub-acute moderate non-suppurative, predominantly angiocentric, meningo-encephalomyelitis of viral aetiology was made. The presumptive diagnosis of ABLV infection was confirmed by the positive testing of the affected brain tissue from (H2) in a range of laboratory tests including fluorescent antibody test (FAT) and real-time PCR targeting the nucleocapsid (N) gene. Retrospective testing of the oral swab from (H1) in the real-time PCR also returned a positive result. The FAT and immunohistochemistry (IHC) revealed an abundance of ABLV antigen throughout the examined brain sections. ABLV was isolated from the brain (H2) and oral swab/saliva (H1) in the neuroblastoma cell line (MNA). Alignment of the genome sequence revealed a 97.7% identity with the YBST ABLV strain.

Survival of avian strains of Pasteurella multocida in chicken serum.

The ability of bacteria to survive in serum is considered a likely virulence determinant in diseases where the infective bacteria become septicaemic. Optimal conditions were established to test the survival of Pasteurella multocida in chicken serum. Serum was used at 90%, the inoculum was 10(3)-10(4)cfu in phosphate buffered saline pH 7.4. Survival was measured after incubation for 2-4 h; if survival was <50% the strain was considered serum susceptible. Susceptible strains were either killed or their growth was inhibited. Some resistant strains not only survived but grew rapidly in unheated serum. Thirty-five strains, all originally isolated from clinical fowl cholera, were tested; eight were susceptible, of which three were killed and five inhibited, and the remainder (27) were resistant. Ten serum-resistant P. multocida serogroup A strains were grown in hyaluronidase to remove the capsule and survival in chicken serum was re-tested. Three strains became susceptible, while seven strains remained resistant. Three serum susceptible strains were then tested in the presence of cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA). This substance is present in the human serum, and is known to mask the effect of complement on Neisseria gonorrhoeae rendering susceptible strains resistant. Two of the three serum susceptible strains became resistant in the presence of CMP-NANA. Serum susceptibility/resistance was more complex than that of Escherichia coli, and the role of resistance to avian complement in the pathogenesis of fowl cholera remains to be determined.

A probable outbreak of rabies in a group of camels in Niger

Seven camels in a herd of approximately 40 camels belonging to nomadic people in Niger died over a short period. No diagnostic samples were obtained. Brain was obtained from the eighth camel in the group, which died after a clinical illness manifested by unusual behaviour, aggression, pica, ptyalism and terminal paralysis. Rabies virus was demonstrated in the brain of this camel by immunofluorescent staining. Before the deaths of the camels a feral dog had joined the nomadic encampment and died there, and a domestic dog belonging to the group had then died after displaying aggressive behaviour. It is postulated that canine rabies had been transmitted to the camels in the herd.

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus

We evaluated a fluorogenic probe–based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21–4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.