Aspects of the Aetiopathogenesis and Diagnosis of Ovine Footrot

Aspects of the aetiopathogenesis and diagnosis of ovine footrot Andrew Stephen McPHERSON A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy THE UNIVERSITY OF SYDNEY Farm Animal Health Sydney School of Veterinary Science Faculty of Science June 2018 Declaration of Authorship Apart from the assistance stated in the acknowledgements section, this thesis represents the original work of the author. The results of this study have not been presented for any other degree or diploma at any other university. Andrew McPHERSON BAnVetBioSci (Hons I) February 2018 i Acknowledgements First and foremost, I would like to thank my supervisors, Dr Om P. Dhungyel and Professor Richard J. Whittington for their continual support, encouragement, and guidance throughout my candidature. There is a long history of footrot research at The University of Sydney; I am grateful for the opportunity to make my own modest contribution to the body of knowledge that has emerged from this University. The research presented in this thesis was funded by Meat and Livestock Australia (MLA) and Ian H (Peter) Wrigley; this work would not have been possible without their support. I would also like to thank Peter Wrigley and his family for supporting me personally with a generous scholarship throughout my candidature. I would like to thank the technical staff of The University of Sydney Infectious Disease Laboratory (Anna Waldron, Rebecca Maurer, Vicki Patten, Ann-Michele Whittington, Alison Tweedie, Nicole Carter, Pabitra Dhungyel, Craig Kristo, and Stuart Glover), who have all provided me with assistance and support at some stage during my candidature. In particular, I am indebted to Natalie Schiller, Nobel Toribio and James Dalton who have all gone above and beyond to provide assistance in both the laboratory and in the field. I would also like to acknowledge my office-mates and fellow postgraduate students Matt Johansen, Hannah Pooley, Kamal Acharya, Olivia Evans, Max de Kantzow, Erandi Pathirana, and Cahya Fusianto for their friendship and support. Thank you also to Dr Karren Plain, Dr Paul Hick and Ms Marion Saddington for your support and advice throughout my candidature. I am indebted to Dr Evelyn Hall and Associate Professor Navneet Dhand for their support, assistance, and patience with the statistical analyses. I am grateful to the veterinarians, animal health officers, and biosecurity officers who have provided assistance in the field: Dr Bruce Jackson, Dr Andrew Ewers, Dr Scott Ison, and Mr Neale Whitsed. I would also like to thank the farmers who allowed us onto their property to examine their flocks and collect specimens. I would like to thank my Mum Gail, Dad Steve, and Nan Thora for their support throughout my undergraduate and postgraduate studies. I would like to thank Maggie for her company whilst this thesis was written. Last, but certainly not least, I would like to thank my partner Emma for the love and support that she has provided throughout this period. This would not have been possible without you. ii Summary Ovine footrot is prevalent in most countries where sheep are reared. The essential causative agent, Dichelobacter nodosus (formerly Bacteroides nodosus, Fusiformis nodosus), is a fastidious, strictly anaerobic bacterium, and an obligate parasite of the ruminant hoof. The clinical disease is the result of a complex interplay between pathogen, environment and host. The severity of these clinical manifestations vary, from a mild interdigital dermatitis through to complete separation of the horny epidermis from the underlying dermal tissues. For descriptive purposes, two clinical forms of the disease are recognised: virulent footrot and benign footrot. The virulent form causes severe lameness, resulting in substantial, ongoing economic losses due to lost productivity, and the cost of controlling the disease. In Australia, virulent footrot is estimated to cost the sheep meat and wool industries approximately $35 million per annum. Consequently, in Australia sheep flocks diagnosed with virulent but not benign footrot may be quarantined and required to undergo a compulsory elimination program, with costs met by the farmer. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavourable pasture conditions. The most recent published genotypic virulence tests, which were developed in Europe, are based on research conducted in Australia which indicated that the expression of acidic protease isoenzyme 2 (AprV2, encoded by the gene aprV2) is the defining phenotypic characteristic of virulent strains of D. nodosus. Benign strains express the analogous protease AprB2 (encoded by the aprB2 allele). Two qPCR tests were developed that target the aprV2/B2 alleles, both of which were reported to be highly specific. However, validation data were limited and the tests were not validated for use outside of Europe. The aim of the study presented in Chapter 3 was to subject these tests to the validation pathway proposed by the World Organization for Animal Health (OIE), and assess their suitability for use in an Australian context. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions of south-eastern Australia, the analytical characteristics of the qPCR tests were evaluated, and one of the tests was compared to phenotypic protease tests (elastase test, gelatin gel test) at the flock- and isolate-levels. There was a low level of agreement between clinical diagnosis and qPCR test outcomes at both the flock- iii and sample-levels, and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests, and the diagnostic specificity of the qPCR test was low at both the flock- and individual swab-levels (31.3% and 18.8%, respectively). In contrast, agreement between the elastase test and clinical diagnosis was high at both the flock-level (DSe = 100%, DSp = 78.6%) and isolate-level (DSe =69.5%, DSp = 80.5%), which indicated that the expression of AprV2 is a good indicator of virulence. However, it was apparent that aprV2- positive D. nodosus strains with benign phenotypes are common in Australian sheep flocks. Footrot can be controlled with vaccine targeting the fimbriae of virulent strains of D. nodosus. However, the bacterium is immunologically heterogeneous, and ten distinct fimbrial serogroups have been identified. Thus in each outbreak the infecting strains must be cultured and serogrouped so that the appropriate serogroup-specific mono- or bi-valent vaccine can be administered, because multivalent vaccines lack efficacy due to antigenic competition. If clinical disease expression is suspected to be incomplete, culture-based virulence tests are also required to confirm the diagnosis, because control of benign footrot is economically unjustifiable. Both diagnosis and vaccination are conducted at flock-level. The aims of the study presented in Chapter 4 were to develop a PCR-based procedure for detecting and serogrouping D. nodosus directly from foot swabs, and to determine whether this could be done accurately from the same swab that is cultured. A total of 269 swabs from the active margins of foot lesions of 261 sheep in 12 Merino sheep flocks in south-eastern Australia were evaluated. DNA extracts from putative pure cultures of D. nodosus and directly from swabs were evaluated in PCR assays for the 16S rRNA and fimA genes of D. nodosus. Pure cultures were tested also by the slide agglutination test. Direct PCR from swabs was more sensitive than culture for detecting and serogrouping D. nodosus. Using the most sensitive sample collection method of swabs in lysis buffer, D. nodosus was more likely to be detected by PCR in active than inactive lesions, and in lesions with low levels of faecal contamination, but lesion score was not a significant factor. PCR conducted on extracts from swabs in Stuart’s transport medium that had already been used to inoculate culture plates had lower sensitivity. Despite the recognition of D. nodosus serogroup M in Australia, New Zealand, Nepal, Norway, and the United Kingdom, a serogroup M-specific PCR test has not been published. The aim of the study presented in Chapter 5 was to develop a serogroup M-specific PCR test to accompany iv existing multiplex conventional PCRs for the detection of serogroups A to I. A serogroup M PCR test was developed with a sensitivity of 250 D. nodosus cells. The test was compared with the slide agglutination test, which was previously the only means of detecting serogroup M, and was shown to be more specific. The Merino sheep is uniquely susceptible to footrot, but the basis of this susceptibility is contentious. The interdigital skin of foot of the Merino appears to be more susceptible to bacterial invasion than that of British sheep breeds; as such, it may be colonised by a greater number or diversity of opportunistic pathogens following environmental predisposition. In the study presented in Chapter 6, next-generation sequencing and analysis of the bacterial 16S rRNA gene was used to characterise the bacterial communities on the feet of a group of healthy Merino sheep, and two groups of Merino sheep with footrot. The results indicated a qualitative shift in the bacterial community of the Merino foot is triggered by infection with D. nodosus. The communities of healthy Merino feet were dominated by Gram-positive, aerobic genera such as Corynebacterium and Staphylococcus. In contrast, the communities of footrot-affected feet were dominated by Gram-negative, anaerobic genera such as Porphyromonas and Fusobacterium. In total, 15 bacterial genera were preferentially abundant on the feet of Merino sheep with footrot, only four of which were previously reported to be abundant on the feet of British breed sheep with footrot in the U.K. There was no significant difference in the composition of the bacterial communities in footrot lesions, irrespective of score, which suggests that the same bacterial genera may be of importance to both the early and latter stages of the disease process.

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