Veterinary Biosciences - Theses
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Characterising the prevalence of Coxiella burnetii in Victorian wildlife
Coxiella burnetii (C. burnetii) is an intracellular gram-negative bacterium, which is the causative agent of the widespread zoonotic disease Q fever. Q fever infection in humans can cause flu-like symptoms and sometimes develop into chronic Q fever, possibly leading to endocarditis. Animals infected by C. burnetii often show no clinical signs, although some may develop reproductive problems such as stillbirth and abortion in cattle, sheep, and goats. Worldwide, investigations into the prevalence of C. burnetii in wildlife have been conducted, and results indicate that free-ranging animals could be potential reservoirs of this bacterium, although the role of wildlife in C. burnetii transmission is unclear. Australian marsupials, such as kangaroos, wallabies and bandicoots have also been found to be carriers of C. burnetii. Nevertheless, there has not been a study investigating the prevalence of C. burnetii in Victorian wildlife. Immunodetection and molecular detection are commonly used to diagnose infection with C. burnetii. However, with the discovery and study of Coxiella-like bacteria (CLB), many PCR genes targeted in routine molecular detection assays were found to also be present in CLB. Thus, it is not possible to distinguish between C. burnetii and CLB based on assays targeting these genes. Immunogenicity tests are commonly used for detection of antibodies against C. burnetii in wildlife species. ELISA is a preferred test due to its performance, objective interpretation, and capacity in high-throughput screening. There is, however, no validated ELISA test for detection of C. burnetii antibodies across Australian wildlife species. This project therefore aimed to: a) differentiate C. burnetii from CLB; b) study the prevalence of C. burnetii in Victorian wildlife using PCR/qPCR methods; and c) investigate the possibility of developing an ELISA to detect serum antibodies in a wide range of wildlife species. An overall prevalence of 3.4% (95% CI 1.7 - 5.2%) of C. burnetii was detected in 406 Victorian wildlife samples by qPCR, indicating that Victorian wildlife may act as potential reservoirs of C. burnetii. The highest prevalence of C. burnetii was found in eastern grey kangaroos (Macropus giganteus) at 7.9% (95% CI 3.2 - 12.6%). Other wildlife samples were found to be positive for Coxiella, but the species remained undetermined. These samples may represent samples that are positive for CLB. Three out of the four C. burnetii proteins selected (CBU0109, CBU0612, CBU0891, and CBU1910) as antigen candidates for ELISA development were expressed and purified. Immunogenicity tests were applied to three antigen candidates using C. burnetii positive and negative goat serum samples by Western blotting and indirect ELISA. The results revealed that none of the antigen candidates were suitable for ELISA development as all reacted with C. burnetii negative goat sera. Findings from this project address the importance of appropriate molecular methods used for the detection of C. burnetii. Future investigations into C. burnetii in wildlife should aim to perform the tests on a larger population of animals and wider range of species. The cross-reactivity between antigen candidates and C. burnetii negative goat sera indicates the necessity of validation for immunological methods when they are applied to a new species.
Discovery of new chemicals with anthelmintic activity against the barber’s pole worm and other parasitic nematodes
Parasitic nematodes cause a substantial disease burden on humans and animals worldwide. A review of the literature (Chapter 1) showed that, on one hand, neglected tropical diseases caused by parasitic nematodes have a devastating, long-term impact on human health; on the other hand, gastrointestinal nematodes are a major constraint to the livestock industries, causing subclinical infections and diseases in animals and leading to a substantial reduction in meat, milk and fibre production. Currently, anthelmintic treatment remains the mainstay of controlling parasitic nematode infections. However, the massive and widespread resistance to the limited number of commercial anthelmintics, particularly in the veterinary and agricultural contexts, demonstrates an urgency to discover new and effective anthelmintics to sustain the economic and health benefits from the application of anthelmintics. Thus, the key focus of this thesis was to discover new chemical entities and/or known drugs with anthelmintic activities against Haemonchus contortus and/or other socioeconomically important parasitic nematodes for subsequent development. Whole worm-based phenotypic screening assays were employed, compound collections were obtained via product-development-partnerships and/or collaborators, and active compounds were assessed for their potential as anthelmintic candidates. In this thesis, one new chemical entity (designated SN00797439), two human kinase inhibitors (SNS-032 and AG-1295), 14 AG-1295 (tetrahydroquinoxaline) analogues, one insecticide (tolfenpyrad) and two tolfenpyrad (pyrazole-5-carboxamide) derivatives (a-15 and a-17) with anthelmintic activity in vitro were discovered following the screening of a total of 15,333 chemicals from five distinct compound collections against H. contortus. In Chapter 2, a new chemical entity, SN00797439, was identified with activity against a range of parasitic nematodes, including H. contortus, Ancylostoma ceylanicum, Brugia malayi, Dirofilaria immitis and/or Trichuris muris in vitro, offering a novel, lead-like scaffold for the development of a relatively broad-spectrum anthelmintic. In Chapter 3, two human kinase inhibitors under pharmaceutical development, SNS-032 (piperidinecarboxamide) and AG-1295 (quinoxaline), were identified to have inhibitory activity on the motility and development of parasitic larvae of H. contortus in vitro. AG-1295 had limited cytotoxicity against a normal mammalian epithelial cell line (designated MCF10A). In Chapters 4 and 5, three pyrazole-5-carboxamides (tolfenpyrad, a-15 and a-17) were shown to possess significant inhibitory effects on H. contortus without detectable toxicity on a human neonatal foreskin fibroblast (NFF) cell line in vitro. All three of these chemicals were shown to inhibit the oxygen consumption in H. contortus larvae, a finding that was consistent with the known, specific inhibition of complex I of the respiratory electron transport chain by selected pyrazole-5-carboxamides in arthropods. The evaluation of these hit compounds using various technologies employed in parasitology, drug discovery, chemistry, histology, toxicology, molecular biology and bioinformatics should offer data to support their potential as leads for future drug development and to facilitate the exploration of their mode(s) of action in this and related nematodes. Encouraged by the findings in Chapter 3 and the detection of a non-wildtype phenotype in treated worms in vitro, Chapter 6 investigated the activities of 14 additional tetrahydroquinoxaline (AG-1295) analogues on H. contortus. Qualitative and quantitative assessments of larval motility, development and morphological alterations showed that these 14 chemicals all affected the viability of parasitic larvae and, interestingly, induced an eviscerated larval phenotype and led to cuticular damage and/or stunted growth in in vitro H. contortus. Taken together, Chapters 2 to 6 identified a series of 20 hit compounds, some of which have selectivity against H. contortus compared with selected human cell lines tested. In Chapter 7, the research achievements are summarised, and the next steps to be pursued in future research are outlined, including (i) the chemical optimisation of representative chemicals via structure-activity relationship (SAR) evaluations; (ii) assessment of the breadth of spectrum of anthelmintic activity on other parasitic nematodes, such as other strongyloids, ascaridoids, enoplids and filarioids; (iii) detailed investigations of the absorption, distribution, metabolism, excretion and toxicity (ADMET) of optimised chemicals with broad nematocidal or nematostatic activity; (iv) establishment of the modes of action of lead candidates. The findings from the thesis are then put into a broad context and discussed. In conclusion, the present thesis contributes to the fields of parasitology and anthelmintic discovery by identifying compounds with in vitro anthelmintic activity that represent sound starting points for ‘lead’ discovery.
The epidemiology of pork-borne parasitic zoonoses in rural communities in the central highlands of Vietnam
Background Food-borne parasitic zoonoses impact negatively on humans in terms of health and loss of productivity as well as economically, through loss of market access and trade. Taenia and Trichinella are two of the most important meat-borne zoonoses globally. In the Central Highlands of Vietnam, living standards are poor; open defaecation via use of outdoor latrines is commonly practiced and livestock access to these latrine areas is, for the most part, unrestricted. Moreover, the consumption of undercooked or raw pork dishes is common traditional practice. These factors combined are highly conducive for the transmission of several pork-borne parasitic zoonoses, including taeniasis, Taenia solium cysticercosis and trichinellosis. In this context, developing and validating diagnostic assays to study the epidemiology of these pork-borne parasites within these communities allows risk factors for exposure and infection to be ascertained and this, in turn, allows community-based control programs to be better targeted. Methodology/Principle findings This project was conducted as a cross-sectional study in Buon Don, Krong Nang and M’Drak district in Dak Lak province in the Central Highlands of Vietnam. A combination of parasitological, serological and molecular diagnostic methods were utilised to determine the levels of infection of Taenia spp. in humans and Trichinella spp. in pigs, and seroexposure to T. solium in humans and pigs. The data were analysed in relation to host, management and environmental risk factors using frequentist and Bayesian multivariable analyses. In Chapter 2, a systematic review on previous research on taeniasis, T. solium cysticercosis and trichinellosis was performed and showed that little to no data are available on the prevalence of these food-borne zoonoses in the central and southern areas of the country. Moreover, utilisation of tests with either low sensitivity or poor specificity for the diagnosis of taeniasis in humans and T. solium cysticercosis in humans and pigs produced either an over- or underestimation of their TP. To address this, a Bayesian model was used to estimate the TP of taeniasis and Taenia solium cysticercosis and demonstrated a true prevalences of up to 25% and 24%, respectively for specific rural ‘hotspots’. In Chapter 3, a multiplex Taq-Man probe-based real-time PCR for the species-specific detection of three Taenia species in human stool was developed and its diagnostic parameters compared with the Kato-Katz thick smear and coproantigen ELISA assay using field samples collected as part of a cross-sectional survey in Dak Lak province. The overall AP of human taeniasis by the multiplex qPCR was 6.7% (95% CI 4.4 to 10). The sympatric existence of Taenia solium, Taenia asiatica and Taenia saginata in Dak Lak province was confirmed for the first time, as was the extension of the known distribution of T. asiatica to southern Vietnam. Risk factors associated with Taenia spp. infection included the routine consumption of undercooked beef and pork, routine consumption of pork tongue, and personal observation of Taenia proglottids in stool. Chapter 4 describes a cross-sectional study on the seroprevalence of human T. solium cysticercosis in Dak Lak determined by two diagnostic assays, the apDia-ELISA, a commercial antigen detection assay and the LLGP-EITB antibody detection assay. The seroprevalence of exposure to T. solium in humans was 5% (95% CI 3 to 8), while the apDia-ELISA, detected circulating T. solium antigens in 7.3% (95% CI 4.9 to 10.8) of the sampled population. Consuming raw vegetables, drinking water sourced from lakes, streams or ponds, and outdoor defaecation were associated with increased T. solium exposure risk in humans. In Chapter 5, the seroexposure of pigs to T. solium cysticercosis in Dak Lak was determined using rT24H and native LLGP antigens in series, in an EITB format. The seroprevalence of exposure to T. solium was low at 0.94 (95% CI 0.51 to 1.68). Coprophagy of human faeces and scavenging for food were factors significantly associated with T. solium exposure in pigs. In Chapter 6, the contribution of all modifiable risk factors for taeniasis, T. solium exposure in humans and pigs is explored and this was estimated to range from 24% to 77%. Spatial analyses demonstrated that human- or pig-T. solium exposure-positive households were more likely to be surrounded by other exposure-positive households in some study locations. This spatial aggregation of human exposure-positive households was hypothesised to be due to a combination of demographic, anthropological and micro environmental factors, whilst scavenging for food and coprophagy of human faeces are likely to drive the aggregation of pig exposure-positive households. Finally, no Trichinella larvae were detected in the confined and free-roaming pig populations using the artificial digestion technique on tongue, diaphragm and masseter muscle. The estimated TP of Trichinella was low, ranging from 0.09 to 0.80% in domestic pigs. Further diagnostic screening using serology was beyond the scope of this current project but advocated for future research. Conclusions This thesis has contributed significantly to a better understanding of the epidemiological characteristics of Taenia spp. infection, and seroexposure to T. solium and Trichinella spp. infection among humans and pigs in the Central Highlands of Vietnam. This information will assist local government and residents to develop appropriate methods and strategies to reduce or mitigate the burden of these diseases in Dak Lak.
The effect of tonB deletion on the expression of the genes encoding Shiga toxin, TonB-dependent receptors and fimbriae in the 16001 oedema disease strain of E. coli
Oedema disease is caused by Shiga toxin producing Escherichia coli (STEC) and generally affects piglets at one to two weeks after weaning. The stress related to dietary change and loss of lactogenic immunity after weaning are believed to contribute to the onset of the disease. Affected piglets typically have oedematous lesions in multiple organs, including the stomach, colon and eyelids, and also neurological signs, such as ataxia and paralysis. Occasionally, the disease can result in sudden death without any apparent clinical signs. The Shiga toxin and fimbriae are the two key virulence factors and they play an essential role in the pathogenesis of the disease. Multiple attempts have been made to develop active, passive and maternal approaches to immunization. These studies have mainly focussed on attenuating the organism by manipulating these virulence factors at the molecular level. Although the disease is endemic and has a considerable economic impact, there is no commercial vaccine available in Australia. Control measures mainly focus on antibiotic treatment but this has been challenged by the emergence of drug resistance in the strain. Iron is one of the most important micronutrients for the growth of bacteria and its availability to pathogens is restricted both inside and outside the host. Pathogenic bacteria respond by elaboration of siderophores in order to transport iron across the outer membrane, and the TonB protein supplies the necessary energy for this transport. The aims of this research project were to develop tonB mutants of an Australian STEC strain (16001) isolated from pigs with oedema disease by deleting the tonB gene using the lambda Red recombination system and to investigate whether the transcription of the virulence factors Shiga toxin 2e (stx2e) and the F18 fimbriae and TonB-dependent transporter genes was influenced by the tonB deletion. The lambda Red recombination system was used to replace the target gene with an antibiotic resistance cassette, and successful mutagenesis was confirmed by molecular and phenotypic characterisation of the mutant. Sequencing studies showed that the tonB gene was replaced with a kanamycin resistance gene. The mutant had a slower growth rate and produced greater concentrations of siderophores on chrome azurol S (CAS) agar. In general, phenotypic characterization of the tonB mutant indicated that it had reduced intracellular iron levels as a result of the tonB deletion. The aim of deleting tonB was to reduce intracellular iron levels, which was anticipated to attenuate the organism and also upregulate the iron regulon to increase the production of protective antigens. During iron shortage, siderophore synthesis and expression of siderophore transporters is increased, thus eliciting an enhanced immune response against these antigens. Upregulation of the genes for some outer membrane receptors, as well as those for the key virulence factors Stx2e and F18 fimbriae, was demonstrated in this study. In conclusion, the deletion mutant generated in this study may be able to be used as the basis for development of a vaccine candidate for control of this important disease of pigs.
Viruses of the other mammals: genomics and epidemiology of marsupial herpesviruses
Improving wildlife population health requires an understanding of the infectious agents within those populations. Historical accounts of herpesviruses in marsupials indicate that they can have a significant impact on animal health. This evidence is strongest for the macropodid alphaherpesviruses, but with improvements in molecular diagnostics the discovery of novel viruses has outpaced our understanding of their impact and significance. This thesis aimed to expand our knowledge of marsupial herpesviruses by examining relationships between marsupial herpesviruses and other herpesviruses, and by describing the clinical significance of infection. This thesis also aimed to improve diagnostic tools for detecting herpesvirus infection in marsupials. The core genomes of three marsupial herpesviruses were determined; macropodid alphaherpesvirus 1 (MaHV1, infecting wallabies), phascolarctid gammaherpesvirus 1 (PhaHV1, infecting koalas) and vombatid gammaherpesvirus 1 (VoHV1, infecting wombats). MaHV1 had a similar genome arrangement to other simplexviruses, but contained gene clusters that may be unique to the macropodid simplexviruses. PhaHV1 and VoHV1 had a shared gene arrangement and were likely to have speciated from a common ancestor. Over 30 new ORFs were identified within the genomes. Functional enzymatic characterisation was performed on two viral NTPDase homologs encoded within the two gammaherpesviruses. NTPDase activity was confirmed for the PhaHV1 homolog but not the VoHV1 homolog. Koalas are host to two divergent gammaherpesvirus species, PhaHV1 and -2. To understand the clinical significance of each individual virus a large molecular epidemiological study of 810 koalas from 7 separate geographic regions was conducted. Samples were tested using a rapid and differential PCR-HRM assay. Available signalment and clinical observation data was analysed in comparison to infection status through univariable and multivariable logistic regression analysis. Additional factors considered were location, year, body condition, fecundity in females, as well as the presence of other infectious agents (Chlamydia pecorum and koala retrovirus). PhaHV1 and -2 were present in 17% and 22% of koalas tested (state-wide), although some variation from the state average was observed in particular populations. Neither virus was associated with a particular sex. PhaHV1 detection was uniquely associated with the presence of koala retrovirus as well as increasing age. PhaHV2 detection did not change with age, which may indicate differences in how these two viruses are acquired over the life of the animal. Both viruses were positively associated with genital tract abnormalities, lowered fertility in females, emaciated body condition, urinary tract infection (wet bottom) and detection of C. pecorum, although the strength of these associations varied by sex and herpesvirus species. To further the development of herpesviruses serological tools, this thesis examined the ability of four commercially-available immunoglobulin-binding reagents to bind serum antibodies from 17 species within the Marsupialia and Monotremata. Serum samples were assessed for binding using immunoblots and ELISAs to three microbially-derived proteins; staphylococcal protein A, streptococcal protein G and peptostreptococcal protein L, and to an anti-kangaroo antibody. The inter- and intra-familial binding patterns of the reagents to serum immunoglobulins varied and evolutionary distance between animal species was not an accurate predictor of the ability of reagents to bind immunoglobulins.
Investigations into bacterial causes of disease in swine with emphasis on airborne pathogens
Pig farming is a major agricultural production system globally. Over the years, many aspects of pig farming have been investigated. However, controlling infectious diseases remains an area of ongoing research, with new technologies allowing the development of improved management strategies and vaccines. The family Pasteurellaceae is mainly composed of commensal species, but includes notable pathogens of livestock. The species of major concern in pigs include Actinobacillus pleuropneumoniae and Glaesserella parasuis. Although publication of the genomes of both species has advanced understanding of their virulence existing vaccines for these pathogens have limited efficacy. Therefore, control of A. pleuropneumoniae and G. parasuis currently relies heavily on farm management. The studies in this thesis sought to develop novel non-invasive methods for detecting A. pleuropneumoniae on farms. The Coriolis µ air sampler, with a qPCR designed for the detection of the apxIV gene, which is specific for A. pleuropneumoniae, were used for this purpose. Application of the assay on two commercial farms revealed that one farm had circulating levels of A. pleuropneumoniae in houses containing pigs aged between 8 and 21. This method could be used for the detection of A. pleuropneumoniae in clinically healthy populations. A novel member of the Pasteurellaceae family isolated from pigs with severe respiratory infections was characterised at the pathological, biochemical, genomic and phylogenetic levels. This bacterium was identified as a member of the genus Glaesserella, and found to be most closely related to Glaesserella parasuis. Analysis of the predicted proteins encoded by its genome showed that it had a mosaic structure, with 60% of the coding sequences sharing closest identity with G. parasuis, while 20% had closest identity with other members of Pasteurellaceae and 20% were related to proteins encoded by other bacterial genomes. A gene homologous to A. pleuropneumoniae apxIII was identified in the genome of this new species and is proposed to have been acquired by horizontal gene transfer. The isolation and identification of this novel Glaesserella species highlights the need for large scale epidemiological studies to understand the impact of this pathogen on pig health. Genome analysis of this organism identified other putative virulence factors, leading to studying its pathogenicity. As many pathogenic species in the Pasteurellaceae family have been explored using mouse models, one was sought for this novel pathogen. The organism was compared to A. pleuropneumoniae in BALB/c mice inoculated either intraperitoneally or intranasally. While no bacteria were recovered from the infected mice, intranasal inoculation resulted in formation of significant lung lesions, and intranasal and intraperitoneal inoculation resulted in significantly increased levels of cytokines in the lungs. These experiments demonstrated that the pathological response to infection with the novel Glaesserella sp. significantly differed from that seen with A. pleuropneumoniae and suggest that the mouse model is a promising method for studying this organism. Finally, assays were developed to detect changes in the dependence of A. pleuropneumoniae on environmental iron concentrations. Growth in presence or absence of streptonigrin and 2,2′dipyridyl was measured by monitoring the optical density of the cultures over time. There was a clear and consistent difference in growth rate between groups, allowing the differentiation of a clone’s ability to grow under iron restricted conditions. These assays were designed to help assist with the identification of a live attenuated vaccine candidate with an impaired iron uptake capacity. The studies in this thesis further understanding of respiratory disease caused by members of the family Pasteurellaceae and highlight areas needing further research.
Use of multiplex-tandem polymerase chain reaction in an operational environment to assess cyanotoxins and taste and odour metabolites of cyanobacterial blooms in Victorian waters
Cyanobacterial blooms represent a major water quality and public health issue due to their potential to produce taste and odour (T/O) compounds, and a variety of toxic metabolites. Monitoring and management of cyanobacterial blooms in various water resources is in turn dependent on the availability of accurate and sensitive diagnostic tools, and contemporary or prior knowledge of the regional distribution of toxin and T/O types. For example, in Victoria, Australia, there is limited knowledge of the distribution/ecology of geosmin producers, such that prioritising taxa for routine monitoring is difficult. Additionally, knowledge about the prevalence of many yet to be identified or emerging toxic cyanobacteria (eg., anatoxin producers) in Australia, remains poorly understood. Due to the many limitations of commonly used detection methods (i.e., microscopy and direct detection techniques), to identify toxic and T/O producing cyanobacteria, there is a need to develop sensitive and accurate diagnostic tools. PCR-based tools have shown potential to accurately identify toxic or T/O producing cyanobacteria; however, they have important limitations for routine monitoring. Although multiplex-tandem PCR (MT-PCR) tool has shown significant potential to accurately detect four major cyanotoxins (i.e., MC, NOD, STX and CYN) in Australia, it requires additional field testing and further, this tool is not presently suitable to detect anatoxin and geosmin-producing cyanobacteria. The work presented in this study used PCR-based genetic screening tools (i.e., conventional PCR, nested-PCR or MT-PCR) for accurate diagnosis of cyanotoxins and geosmin-producing cyanobacteria on environmental cyanobacterial bloom samples collected from various parts of Victoria, Australia, over a period of eight years (i.e., from 2010 to 2018). The study also employed metagenomic approaches (eg., Illumina MiSeq and PacBio) using biomarker genes [eg., 16S ribosomal RNA (16S rRNA) and phycocyanin intergenic spacer sequence (PC-IGS or cpcBA-IGS)] to elucidate the community structure of a recent (i.e., 2016) cyanobacterial bloom in the Murray River, Victoria, Australia. Based on conventional PCR-based genetic screening (using three different primer pairs) of more than 250 cyanobacteria bloom samples collected over a period of six years (i.e., 2010-2016), we detected Dolichospermum ucrainicum as the major geosmin producer in 87% of sequenced samples. Using these data, we developed a novel, small amplicon PCR primer pair capable to broadly identify all geosmin-producing cyanobacteria identified in the current study using a single standardised protocol. Additionally, genetic screening using nested-PCR on samples (n=226) collected from 2010-2017 revealed the presence and distribution of several anatoxin-producing cyanobacteria, including Cuspidothrix issatschenkoi, Aphanizomenon sp., Dolichospermum sp., at an overall sample prevalence of 30.1%. An overwhelming majority (86.8%) of anatoxin positive nested-PCR detections were from samples collected from 2016 to 2017, as compared to only 6% of samples collected prior to 2016. Using this data obtained, novel, short amplicon, PCR primers were designed for better PCR-based detection of geosmin primers. This study is the first confirming the presence of anatoxin producers in Australia. Further, field evaluation of the efficiency of MT-PCR to assess toxic cyanobacterial blooms and analysis of total community composition on samples (n=194) collected from the Murray River bloom (March 2016 – May 2016) have shown to greatly supplement microscopic examination, and highlights the potential utility of combining targeted qPCR with metagenomic amplicon sequencing methods. Based on these findings, the MT-PCR assay was expanded to detect anatoxin and major geosmin-producing cyanobacteria. The expanded MT-PCR demonstrated superior performance in comparison to conventional or nested PCRs for all but one (anatoxin) gene target in all samples (i.e., n=91 from March 2016 – February 2018) tested. Here, we report a diagnostic specificity of 100% and diagnostic sensitivity of ≥ 81% for all gene targets, and the MT-PCR platform may provide a much-needed tool for routine monitoring of toxic and geosmin-producing cyanobacterial blooms of global relevance.
Enhancing control of virulent recombinant strains of laryngotracheitis virus using vaccination
Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute upper respiratory tract disease in chickens. Attenuated live ILTV vaccines are often used to help control the disease, but these vaccines have well-documented limitations including natural recombination between different vaccine strains. Recently, two novel ILTV field strains (class 8 and 9 ILTV viruses) emerged in Australia due to natural recombination involving two distinct commercial ILTV vaccines. These recombinant field strains became dominant in important poultry producing areas and caused severe disease in commercial poultry flocks, showing that more options are needed to enable control of ILTV. The work described in this thesis investigated tools to better control disease due to ILTV. Firstly, different commercial ILTV vaccines and a developmental candidate vaccine, glycoprotein G-deficient ILTV (ΔgG ILTV, registered as Vaxsafe ILT, Bioproperties Pty Ltd) were investigated for their ability to protect commercial broiler chickens against challenge with the virulent recombinant class 9 ILTV after drinking water vaccine delivery. All vaccines induced partial protection by direct (drinking-water) and indirect (contact) exposure when birds were subsequently challenged with the virulent class 9 challenge strain. A vaccination and challenge study was then performed to determine the minimum effective dose of ∆gG ILTV that, when delivered by eye-drop to layer birds, would protect the birds from a robust challenge with class 9 ILTV. A dose of 103.8 plaque forming units per bird was the lowest dose capable of providing a high level of protection against challenge, as measured by clinical signs of disease, tracheal pathology and viral replication after challenge. Finally, attempts were made to develop suitable tools to measure the level of immunity induced by ILTV vaccination. To this end, an ELISA that measures the amount of chicken interferon gamma (IFN-γ) was developed and used to quantitate IFN-γ production from splenocytes stimulated with control mitogens, or with ILTV antigen. The assay could detect IFN-γ released from chicken splenocytes after stimulation by concanavalin A. However, when splenocytes were incubated with semi-purified ILTV antigens in vitro, there was no increase in the level of ILTV specific IFN-γ production by splenocytes from ILTV infected birds, compared to uninfected birds. A number of potential avenues for further development of this assay were identified. The work described in this thesis demonstrates that currently available vaccines and the new Vaxsafe ILT vaccine can be used to help control class 9 ILTV when delivered by drinking water. When delivered by eye-drop the Vaxsafe ILT vaccine candidate can induce a high level of protection against class 9 ILTV at a commercially feasible dose. Taken together, the results from this work lay the foundations on which a commercial vaccine may be developed, thereby offering the potential to provide producers with another important tool to help control ILTV. Future development of a tool to measure protective immunity after vaccination is needed and would be a valuable addition to disease control programmes
Investigation of the infectious causes of diarrhoea in Australian thoroughbred foals
Diarrhoea is a common disease in foals that is costly and labour intensive to manage. A large number of potential enteric pathogens have been detected in the faeces of foals, however, the role of these infectious agents in causing clinical disease is not clearly understood and their prevalence in Australia is unknown. In addition, timely methods for definitive diagnosis are not readily available for some of these agents. Therefore, this study aimed to develop rapid molecular detection assays to investigate the presence of equine rotaviruses, equine coronaviruses, Salmonella spp. and Clostridium difficile in Australian thoroughbred foals with and without diarrhoea. A prospective case control study was conducted on five thoroughbred breeding farms in the Hunter Valley (New South Wales, Australia) during the 2010 breeding season. Faecal samples were collected from age-matched foals with diarrhoea and without diarrhoea from the same farm (age-matched pair). In addition, faeces were collected from foals with diarrhoea from an equine veterinary hospital. All faecal samples were analysed for equine rotaviruses, equine coronaviruses, Salmonella spp. and Clostridium difficile by quantitative PCR (qPCR) assay. All faecal samples were also cultured for Salmonella spp. using selective growth media. Samples positive by qPCR and 15 randomly selected control foal samples negative by qPCR were cultured anaerobically for Clostridium difficile. Faecal samples were collected from 117 pairs of age-matched case control foals and 26 hospitalised foals with diarrhoea. In the age-matched case control foals, equine rotaviruses were the most frequently detected infectious agent (25% case foals, 5% control foals) and the only infectious agent significantly associated with the presence of diarrhoea. In hospitalised foals, Clostridium difficile (23%) was the most frequently detected infectious agent. In this investigation co-infections were detected in 4% of matched case foals and 4% of hospital foals, with equine rotaviruses and Salmonella spp. being the most frequent combination. Four different Clostridium difficile ribotypes were detected, including ribotype 012 and 078. Importantly, this is the first report of the detection of C. difficile ribotype 078 in Australian horses. As this ribotype has been associated with severe disease in humans, this finding may have public health implications. The availability of rapid molecular screening tests for infectious causes of foal diarrhoea enhances the veterinary practitioner’s ability to instigate appropriate therapy and control measures in foals with diarrhoea. However, the detection of pathogens in foals without diarrhoea highlights the need for more research into the role some of these pathogens play in clinical disease both individually and in combination.
Immunological studies on the protective immune response against Taeniid Cestode parasites
Taeniid cestodes have a lifecycle involving two mammalian hosts. Some species such as Taenia solium and Echinococcus granulosus infect humans and cause morbidity and mortality. T. solium is the aetiological agent of neurocysticercosis, the cause of substantial neurological disease in the non-Islamic regions of the developing world, including Africa, Asia and Latin America. Recombinant vaccines, derived from the larval oncosphere stage, have been developed against T. solium and several cestode parasites for use in livestock animals to prevent parasite transmission to humans. This study investigated antigenic cross-reactivity between the host-protective, recombinant oncosphere proteins with the aim of identifying a surrogate species for T. solium and E. granulosus as the target in in vitro oncosphere killing assays. Investigations into antigenic cross-reactivity were undertaken by comparative analyses of the amino acid sequence of the recombinant host-protective oncosphere antigens. Immunological assays including Indirect ELISA, Inhibition ELISA and Western blot were used to determine immunological cross-reactivity between the recombinant oncosphere antigens. Antigenic cross-reactivity between oncosphere antigens and different Taeniid species was also examined via in vitro oncosphere killing assays. Analysis of amino acid sequences of twelve recombinant oncosphere antigens identified four homology groups, To16, To18, To45 (from Taenia ovis) and EG95 (from E. granulosus) with the nomenclature based on the first recombinant antigen isolated for each group. Examination of the amino acid sequences determined high levels of sequence identity among oncosphere proteins within homology groupings, not with antigens from within a species. Limited immunological cross-reactivity was identified by Indirect ELISA and Inhibition ELISA as well as Western blot. This was in contrast to the close sequence relationship observed between several of the antigens. Detectable immunological cross-reaction was observed for the Taenia multiceps antigens TM16 and TM18 with all Taeniid proteins from within the To16 and To18 homology groups, with the exception of TSOL18 (from T. solium). Substantial antigenic cross-reactivity was detected between TM16 and TM18 in both Indirect ELISA and Inhibition ELISA, while a weak reaction was detected in Western blot, despite amino acid sequence identity being 15%. In contrast, To18 and TSOL18 share 60% amino acid sequence identity, yet no cross-reaction was detected in immunological assays or in vitro oncosphere killing assays. The lack of cross-reactivity with TSOL18 was unexpected given the high amino acid sequence identity it shares with other proteins of the To18 homology group. These studies investigated the potential to identify a surrogate Taeniid species (more amenable to laboratory manipulation) from assessment of protective immune responses raised by TSOL18 in vaccinated pigs. Oncosphere killing assays were also utilised as an in vitro method to assess antigenic cross reactivity between proteins from different Taeniid species. Oncosphere killing assays determined that anti TSOL18 sera induced significant killing of T. saginata oncospheres. None of the cross-reactions observed in immunological assays or amino acid sequence analysis was reflected in oncosphere killing assays. These studies have revealed that detectable levels of antigenic cross-reactivity exist between some of the recombinant oncosphere antigens. The successful killing of a heterologous species indicated that the use of a surrogate target Taeniid species, to monitor immune responses in vaccinated animals, is feasible. The findings here limit the potential for development of in vitro methods for assessment of host-protective immune responses induced by the vaccines. Further investigation is required in order to identify a Taeniid species with significant cross-reactive capabilities and a lifecycle that is maintainable in a laboratory setting.
Antimicrobial stewardship in Australian veterinary practices
Antimicrobial use by the veterinary profession has been coming under increasing scrutiny by medical, public health and government officials as the threat of antimicrobial resistance becomes increasingly clear. The World Health Organisation has described antimicrobial resistance as one of the major public health challenges of our time. It is clear that at least some drug-resistant pathogens have evolved under selective pressure from antimicrobial use in agriculture and may be contributing significantly to resistance in clinical setting. Antimicrobial stewardship is the selection of the most appropriate antimicrobial for a given disease in a given animal, with the aim of reducing the risk of adverse effects in that animal, and reducing the likelihood of developing resistance on an individual level, on a farm level and on a national level. Currently none of the core elements of antimicrobial stewardship are widely available for veterinarians in Australia, and there is very sparse data available on which to base an antimicrobial stewardship program. This research project aims to address this paucity of data. A range of research methods were used to assess detailed antimicrobial use by veterinarians in Australia and the enablers and barriers to antimicrobial stewardship. These included quantitative methods such as surveys and analysis of pet insurance data, and qualitative methods such as interviews and focus groups. While antimicrobials with low importance rating were predominately used in all species, under-dosing and inappropriate timing of antimicrobial therapy were common particularly in horses and cattle. Few veterinary practices in Australia had antimicrobial stewardship policies in place, or were using antimicrobial use guidelines. The key barriers to implementing antimicrobial stewardship programs were a lack of antimicrobial stewardship governance structures, client expectations and competition between practices, the cost of microbiological testing, and a lack of access to education, training and antimicrobial stewardship resources. The enablers were, firstly, concern for the role of veterinary antimicrobial use in development of antimicrobial resistance in humans, secondly , a sense of pride in the service provided, and thirdly , preparedness to change prescribing practices. This research culminated in the development of a proposed antimicrobial stewardship policy and procedure documents, to enable veterinarians to institute antimicrobial stewardship programs that suit their individual practice requirements. However, it is likely that governance changes will be necessary to compel veterinary practice owners to implement antimicrobial stewardship on a large scale.
Determining the gene functions of mycoplasmas
Mycoplasma gallisepticum and Mycoplasma bovis are important animal pathogens that cause considerable economic losses worldwide, due to chronic respiratory disease in poultry, and chronic pneumonia in calves, respectively. M. bovis also causes various other chronic diseases in cattle, such as mastitis, arthritis and otitis media. Effective vaccination is required to control mycoplasma infection as antibiotic resistance is a growing problem and, in the case of M. bovis, no commercial vaccine is available. Although a commercial vaccine for M. gallisepticum is available, a better vaccine is needed for protection against more virulent field strains. In order to identify potential vaccine targets, a better understanding of the fundamental biology of mycoplasmas is required, including, in the absence of toxins or many other traditional bacterial virulence factors, the metabolic pathways active in the cell, as these are likely to relate to host persistence and therefore virulence. Mycoplasmas are among the simplest-replicating bacteria, but despite this the functions of many of their genes have not been elucidated. A metabolomics approach, in which small molecules (metabolites) are detected and quantified, can be very useful in determining gene function, as these metabolites represent the end result of gene transcription and, translation and can be linked back to genome analysis to study gene function. Novel methods are described here for characterising the metabolome of both M. bovis and M. gallisepticum, firstly by using an untargeted metabolomics approach using two platforms, namely gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). Comparison of the metabolite profiles of M. gallisepticum and M. bovis suggested distinctive differences in the transport and utilisation of sugars, in particular glucose. The identified metabolites were annotated onto metabolic maps. To fill apparent gaps in metabolic pathways, bioinformatic analyses and literature mining were undertaken to assign novel annotations to genes that were likely to encode the missing enzymes. Targeted analysis using 13C stable isotope labelling in both species highlighted key differences, in particular the differing glucose utilisation in the fermentative and non-fermentative mycoplasma species. This labelling approach validated not only the connection of metabolic pathways, but also indicated that the presence of oxidative pentose phosphate pathway, which is thought to be absent in mycoplasma species. Having characterised the fundamental basic metabolism of both species, the function of several genes was then investigated by comparing the metabolomes of wild types and transposon mutants of each species. In these comparative studies, consistent differences were detected between strains in independent LC/MS analyses, although the fold differences between the wild type and the individual transposon mutants being investigated were not large. Three separate M. gallisepticum transposon mutants were studied, as previous animal studies suggested that the genes affected by transposon insertion in these mutants were required for persistence, and that one gene, MGA_1102, was required for virulence. Transposon insertion into oppD1, which encodes a putative oligopeptide transporter, resulted in consistently lower levels of intracellular amino acids and dipeptides in the mutant compared to wild type, thus validating the putative oligopeptide transport function of OppD1. MGA_1102, a hypothetical membrane protein containing a zinc-peptidase like motif, was predicted to be a membrane associated aminopeptidase from the results of metabolomic and bioinformatic analysis of the ΔMGA_1102 mutant. The function of MGA_0578, which encodes a hypothetical protein and which has multiple paralogues, could not be defined from the metabolomic data. In the M. bovis mutant study, metabolite profiling comparisons were applied to four mutants with a transposon insertion into genes encoding a putative nucleotide binding protein (MBOVPG45_0307), a putative oligopeptide transporter (MBOVPG45_0116) or one of two putative amino acid transporters (MBOVPG45_0533 and MBOVPG_0568). The consistent detection of a greater abundance of nucleotide precursors in the wild type metabolome indicated that MBOVPG45_0307 was likely to play a role in nucleotide transport. The comparison of metabolite profiles of wild type and the mutant with an insertion in the MBOVPG45_0116 gene, the putative oligopeptide ABC transporter permease gene, did not detect consistent differences in amino acids or dipeptides, in contrast to observations o the M. gallisepticum oppD1 mutant. This implies that M. bovis may be capable complementing the role of this transporter in amino acid/dipeptide uptake, as several different amino acid transporters are annotated in M. bovis, or that the substrate of this transporter differs from the bioinformatic predictions. The metabolomic profiling results predicted that the actual function of MBOVPG45_0533, currently classified as a L-type amino acid transporter, could be the uptake of UDP-sugars. Similarly, for the MBOVPG45_0568, the metabolite profile comparison suggested that biopterin transport could be the potential function, although this gene is currently annotated as encoding a decarboxylate/amino acid transporter. Targeted metabolomics using 13C-glycerol and medium footprinting for two different mutants of M. bovis with transposon insertions into putative glycerol transporters (the M. bovis glycerol uptake facilitator gene glpF and the glycerol permease gene gtsB) were performed to investigate glycerol uptake, using GC/MS as the analytical platform. No significant difference in the incorporation of 13C-glycerol into the intracellular glycerol pool was observed in either mutant. Analysis of the spent medium did not detect any significant differences in glycerol or related metabolites. These consistent results from different experimental approaches indicate that most probably each of these two transporters can compensate for the lack of the other. In conclusion, the studies described in this thesis demonstrated that metabolomics, when combined with bioinformatics, has considerable potential to assist in understanding gene function in mycoplasmas. This fundamental knowledge is crucial in developing a better understanding of the basic pathogenesis of these organisms, a critical step towards improved rational vaccine design.