Veterinary Biosciences - Research Publications
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ItemNo Preview AvailableBayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum(AMER SOC MICROBIOLOGY, 2016-06)Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants.
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ItemViral enteritis in domestic animals(CSIRO PUBLISHING, 2012-05)Viral enteritis is a major cause of morbidity and mortality in neonatal domestic animals, but the most significant pathogens responsible vary considerably between animal species. The viral pathogens currently recognised as significant concerns in animal health were all identified over 20 years ago, and there has been limited recent investigation of the aetiology of viral enteritis in domestic animals using newer pathogen discovery techniques. While effective vaccines are available to control some of these enteric pathogens in some animal species, comprehensive and specific control measures for viral enteritis are lacking in most domestic species. Further research is needed to identify all the major viral pathogens responsible and to develop vaccines to facilitate more effective control.
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ItemAnthropogenic Factors Are the Major Cause of Hospital Admission of a Threatened Species, the Grey-Headed Flying Fox (Pteropus poliocephalus), in Victoria, Australia(PUBLIC LIBRARY SCIENCE, 2015-07-24)To determine the reasons for presentation and outcomes of hospitalised grey-headed flying foxes (Pteropus poliocephalus) in Victoria, Australia, a retrospective analysis was performed on 532 records from two wildlife hospitals. Cases were categorised based on presenting signs and outcomes determined. Anthropogenic factors (63.7%) were a major cause of flying fox admissions with entanglement in fruit netting the most significant risk for bats (36.8%). Overall the mortality rate for flying fox admissions was 59.3%. This study highlights the effects of urbanisation on wild animal populations and a need for continued public education in order to reduce morbidity and mortality of wildlife, especially threatened species.
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ItemEquine rhinitis A virus infection and cytokine expression in primary tracheobronchial epithelial cell culture(Elsevier BV, 2016-04)Primary bronchial cells and ex-vivo explants are commonly used as a model for respiratory viral infections and lung diseases in humans. Similarly, these systems can be used to mimic and study respiratory conditions in the horse. Therefore, equine tracheobronchial epithelial cells (TBEC) could be used to investigate viral respiratory infections and their immune/inflammatory responses in this species. The aims of the present study were to investigate the cytopathic characteristics of equine rhinitis A virus (ERAV) in primary TBECs and the gene expression of IFN-gamma, IFN-Beta, IL-4, and IL-8. TBECs were obtained from the lower trachea, carina and primary bronchi of five euthanized horses. After dissection, the tracheal mucosa was removed, washed and kept overnight on pronase. Twelve to eighteen hours later, the airway mucosa was scraped and cells were mechanically separated. TBECs were then washed, plated on collagen coated flasks/plates using modified DMEM F12 cell culture medium and allowed to propagate to confluency. In order to investigate the cytopathic characteristics, confluent TBEC monolayers were exposed to ERAV and observed daily for 5 consecutive days. Additionally, to investigate cytokine expression by qPCR, TBEC monolayers were exposed to either ERAV or equine influenza virus (EIV) and supernatant samples were collected at 0, 2, 4, 6, 8, 10, 12, 16, 20 and 24 hours post-infection. EIV was used as a comparison for cytokine expression. Interestingly, in this study ERAV infected TBECs developed syncytial cell formations and/or cell clumping commonly observed during other in-vitro viral infections, such as syncytial or herpesviruses. Contrary to our expectations, significant changes in the expression of interferons and IL-4 were not detectable by qPCR after exposure of TBECs to ERAV or EIV within 24 hours. However, up-regulation of IL-8 after exposure to these two viruses was identified from 2 up to 24 hours post-infection in a similar magnitude. IL-8 is consistently detected in respiratory secretions during viral infections in humans and has been shown to induce chemotaxis and phagocytosis of inflammatory cells in the airways. Therefore, changes in the expression of IL-8 indicate that this chemokine might play an important role during early infections, perhaps as a chemotactic factor and/or phagocytic inductor.
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ItemNo Preview AvailableInfectious Laryngotracheitis Virus Viral Chemokine-Binding Protein Glycoprotein G Alters Transcription of Key Inflammatory Mediators In Vitro and In Vivo(AMER SOC MICROBIOLOGY, 2018-01)Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad-range viral chemokine-binding protein conserved among most alphaherpesviruses, including ILTV. A number of studies comparing the immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and the composition of the inflammatory response, and modulation of the immune responses toward antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. In vitro infection studies using tracheal organ tissue specimen cultures and blood-derived monocytes and in vivo infection studies in specific-pathogen-free chickens showed that leukocyte recruitment to the site of infection is an important component of the induced pathology and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXC chemokine ligand 8 (CXCL8), chicken CXCLi1 and chicken CXCLi2, among other cytokines and chemokines. The results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence, and the balance of the immune response to infection.IMPORTANCE Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad-range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signaling cascade associated with infection. Here, data from in vitro and in vivo infection studies are presented. These data show that gG has a direct impact on the transcription of cytokines and chemokine ligands in vitro (such as chicken CXCL8 orthologues, among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with the dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.
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ItemNo Preview AvailableThe major membrane nuclease MnuA degrades neutrophil extracellular traps induced by Mycoplasma bovis(ELSEVIER SCIENCE BV, 2018-05)Mycoplasma bovis has been increasingly recognised worldwide as an economically important pathogen of cattle, causing a range of diseases, including pneumonia, mastitis, polyarthritis and otitis media. It is believed that M. bovis utilises a range of cell surface proteins, including nucleases, to evade the host immune response and survive. However, despite the importance of neutrophils in controlling pathogenic bacteria, the interaction between these cells and M. bovis is not well-characterised. In addition to phagocytosis, neutrophils combat pathogens through the release of neutrophil extracellular traps (NETs), which are composed of their nuclear and granular components, including DNA. Here we investigated the effect of the major membrane nuclease MnuA of M. bovis, which in vitro is responsible for the majority of the nuclease activity of M. bovis, on NET formation. We quantified NET formation by bovine neutrophils 4 h after stimulation with wild-type M. bovis, an mnuA mutant and a mnuA-pIRR45 complemented mnuA mutant. NETs were detected following stimulation of neutrophils with the mnuA mutant but not after exposure to either the wild-type or the mnuA-pIRR45 complemented mutant, and NETs were degraded in the presence of even low concentrations of wild type M. bovis. Surprisingly, there was no increase in levels of intracellular reactive oxygen species (ROS) production in neutrophils stimulated with M. bovis, even though these neutrophils produced NETs. These results clearly demonstrate that M. bovis can induce NET formation in bovine neutrophils, but that the major membrane nuclease MnuA is able to rapidly degrade NETs, and thus is likely to play a significant role in virulence. In addition, M. bovis appears to induce NETs even though ROS production seems to be suppressed.
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ItemNo Preview AvailableReplication-independent reduction in the number and diversity of recombinant progeny viruses in chickens vaccinated with an attenuated infectious laryngotracheitis vaccine(ELSEVIER SCI LTD, 2018-09-11)Recombination is closely linked with virus replication and is an important mechanism that contributes to genome diversification and evolution in alphaherpesviruses. Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) is an alphaherpesvirus that causes respiratory disease in poultry. In the past, natural (field) recombination events between different strains of ILTV generated virulent recombinant viruses that have caused severe disease and economic loss in poultry industries. In this study, chickens were vaccinated with attenuated ILTV vaccines to examine the effect of vaccination on viral recombination and diversity following subsequent co-inoculation with two field strains of ILTV. Two of the vaccines (SA2 and A20) prevented ILTV replication in the trachea after challenge, but the level of viral replication after co-infection in birds that received the Serva ILTV vaccine strain did not differ from that of the mock-vaccinated (control) birds. Even though the levels of viral replication were similar in the two groups, the number of recombinant progeny viruses and the level of viral diversity were significantly lower in the Serva-vaccinated birds than in mock-vaccinated birds. In both the mock-vaccinated and Serva-vaccinated groups, a high proportion of recombinant viruses were detected in naïve in-contact chickens that were housed with the co-inoculated birds. Our results indicate that vaccination can limit the number and diversity of recombinant progeny viruses in a manner that is independent of the level of virus replication. It is possible that immune responses induced by vaccination can select for virus genotypes that replicate well under the pressure of the host immune response.
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ItemNo Preview AvailableSingle Nucleotide Polymorphism Genotyping Analysis Shows That Vaccination Can Limit the Number and Diversity of Recombinant Progeny of Infectious Laryngotracheitis Viruses from the United States(AMER SOC MICROBIOLOGY, 2018-12)Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) causes mild to severe respiratory disease in poultry worldwide. Recombination in this virus under natural (field) conditions was first described in 2012 and more recently has been studied under laboratory conditions. Previous studies have revealed that natural recombination is widespread in ILTV and have also demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In the United States, natural ILTV recombination has also been detected, but not as frequently as in Australia. To better understand recombination in ILTV strains originating from the United States, we developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two virulent U.S. field strains of ILTV (63140 and 1874c5) under experimental in vivo conditions. We also tested the capacity of the Innovax-ILT vaccine (a recombinant vaccine using herpesvirus of turkeys as a vector) and the Trachivax vaccine (a conventionally attenuated chicken embryo origin vaccine) to reduce recombination. The Trachivax vaccine prevented ILTV replication, and therefore recombination, in the trachea after challenge. The Innovax-ILT vaccine allowed the challenge viruses to replicate and to recombine, but at a significantly lower rate than in an unvaccinated group of birds. Our results demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination between these ILTV strains and also show that vaccination can limit the number and diversity of recombinant progeny viruses.IMPORTANCE Recombination allows alphaherpesviruses to evolve over time and become more virulent. Historically, characterization of viral vaccines in poultry have mainly focused on limiting clinical disease, rather than limiting virus replication, but such approaches can allow field viruses to persist and evolve in vaccinated populations. In this study, we vaccinated chickens with Gallid alphaherpesvirus 1 vaccines that are commercially available in the United States and then performed coinoculations with two field strains of virus to measure the ability of the vaccines to prevent field strains from replicating and recombining. We found that vaccination reduced viral replication, recombination, and diversity compared to those in unvaccinated chickens, although the extent to which this occurred differed between vaccines. We suggest that characterization of vaccines could include studies to examine the ability of vaccines to reduce viral recombination in order to limit the rise of new virulent field strains due to recombination, especially for those vaccines that are known not to prevent viral replication following challenge.
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ItemNo Preview AvailableDetection and Differentiation of Two Koala Gammaherpesviruses Using High Resolution Melt (Hrm) Analysis Reveals Differences in Viral Prevalence and Clinical Associations in a Large Study of Free-Ranging Koalas(American Society for Microbiology, 2019-01-09)The iconic koala (Phascolarctos cinereus) is host to two divergent gammaherpesviruses, phascolarctid gammaherpesviruses 1 and 2 (PhaHV-1 and -2), but the clinical significance of the individual viruses is unknown and current diagnostic methods are unsuitable for differentiating between the viruses in large-scale studies. To address this, we modified a pan-herpesvirus nested PCR to incorporate high-resolution melt analysis. We applied this assay in a molecular epidemiological study of 810 koalas from disparate populations across Victoria, Australia, including isolated island populations. Animal and clinical data recorded at sampling were analyzed and compared to infection status. Between populations, the prevalence of PhaHV-1 and -2 varied significantly, ranging from 1% to 55%. Adult and older animals were 5 to 13 times more likely to be positive for PhaHV-1 than juveniles (P < 0.001), whereas PhaHV-2 detection did not change with age, suggesting differences in how these two viruses are acquired over the life of the animal. PhaHV-1 detection was uniquely associated with the detection of koala retrovirus, particularly in females (P = 0.008). Both viruses were significantly associated (P < 0.05) with the presence of genital tract abnormalities (uterine/ovarian cysts and testicular malformation), reduced fertility in females, urinary incontinence, and detection of Chlamydia pecorum, although the strength of these associations varied by sex and virus. Understanding the clinical significance of these viruses and how they interact with other pathogens will inform future management of threatened koala populations.