Veterinary Biosciences - Theses
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ItemEvaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis( 2023-08)Background: Intestinal mucus barrier disruption occurs with chronic inflammatory enteropathies. The lack of studies evaluating changes in mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced expression of goblet cells and/or mucin 2 (MUC2), which are key players in mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is also unknown if Ki-67 expression, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to severity of colonic inflammation in dogs with GC and LPC. Objectives: Study objectives included assessing if Ki-67 expression is upregulated in dogs with GC compared to LPC and dogs without colitis; comparing GBC and MUC2 expression among GC, LPC and non-inflamed colon; and exploring the use of RNAscope in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Methods: Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs between January 2015 and March 2022. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colonic signs (NC) (n=10) group based on the final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per slide were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using computer image analysis. Pearson correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic (WH) score and measured variables. Linear regression models were used to compare the relationships between WH scores with KI67%, GBC%, and MUC2%; and between GBC% and MUC2% across groups. Results: Median WH scores were highest in dogs with GC. Median KI67% was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation in all groups. Median MUC2% normalised to WH score (MUC2%*) was highest in the NC group (10.02%; range, 3.05-39.09%), which was two and three-fold higher compared to the GC and LPC groups respectively. Mucin 2 expression had a strongly positive correlation with GBC% in the NC group (5.71% increase in MUC2%; 95%-CI, 0.503–0.987, p =.12), while no significant trends were observed in the GC and LPC groups. With increased severity of colonic inflammation, MUC2 expression sharply declined in the LPC group (-8.90%; 95%-CI, -17.98-0.19, p =.05) but remained static in the GC group. Conclusion and relevance: Dogs with GC had the most severe histologic colonic inflammation. Dogs without colitis had the highest Ki-67 expression. Goblet cell expression did not correlate with histologic severity of colonic inflammation overall. Dogs with GC and LPC likely have differences in pathways regulating MUC2 biosynthesis and secretion. Changes in MUC2 expression appear influenced by pathways regulating GBC activity rather than quantity in GC and LPC. Development of therapeutic strategies aimed at modifying GBC function and MUC2 expression may help improve mucus barrier integrity and mucosal healing in dogs with chronic colitis.
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ItemD-Dimer in dogs, cats and horses: Investigations into immunoreactivity and structure( 2023)Background: D-dimer is a degradation product of cross-linked fibrin generated during thrombolysis. D-dimer is measured in human medicine for the early detection and exclusion of thrombotic syndromes. Commercially available D-dimer assays use monoclonal antibodies against human D-dimer, and there is limited sensitivity and specificity data for these antibodies in companion animals, thus the accuracy of human assays in veterinary species remains uncertain. Little is known about the sequence and structure of D-dimer in companion animals. Objectives: To evaluate the immunoreactivity of D-dimer in dogs, horses and cats with commercially available antibodies, and investigate potential differences in sequence and structure between species. Methods: A cross-linked fibrin lysate was prepared from canine, feline and equine plasma samples, and immunoblotting performed with a variety of commercially available D-dimer antibodies. Amino acid sequences of fibrinogen fragments involved in D-dimer were compared between species (cat, horse, dog and human), using available sequences for fibrinogen chains, and these were used for multiple sequence alignment, estimation of physiochemical properties, analysis of conserved structural domains, and prediction of three-dimensional structure by homology modelling. Results: The antibodies evaluated demonstrated variable reactivity with D-dimer in each species. The monoclonal antibody DD44 bound canine D-dimer with good specificity and sensitivity, but this antibody did not react with feline or equine D-dimer. The polyclonal antibody D2D bound putative D-dimer in dogs, cats and horses with good specificity, and increased sensitivity compared to human D-dimer. Analysis of sequence and structure revealed high inter-species homology, and three-dimensional models were prepared using a human template. Conclusions: Commercially available antibodies to human D-dimer have variable immunoreactivity with D-dimer in companion animals, suggesting that the use of human assays is inappropriate until analytical and/or clinical validation of the antibody used in that assay has been performed for each species. There are no overt structural variations to explain the variation in immunoreactivity between species, and this could reflect minor variations in structure at the site of binding for each antibody, which is largely unknown.
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ItemCharacterising the prevalence of Coxiella burnetii in Victorian wildlife( 2018)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.
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ItemThe 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( 2018)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.
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ItemImmunological studies on the protective immune response against Taeniid Cestode parasites( 2017)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.