Health and fitness effects of Anaplasma species infection in African buffalo (Syncerus caffer) Danielle Rae Sisson Thesis submitted for the degree of Master of Philosophy Melbourne Veterinary School Faculty of Veterinary and Agricultural Sciences ORCID ID: https://orcid.org/0000-0002-1111-9635 September 2017 Abstract Anaplasma marginale and A. centrale are intra-erythrocytic bacteria of domestic and wild ruminants and are mainly transmitted by ixodid ticks. Most of the work on anaplasmosis has been done on A. marginale infections in cattle, as it can cause disease with varying levels of severity, from icterus and anaemia, to abortions and death. However, wildlife, such as African buffalo (Syncerus caffer), appear to be only subclinically infected with A. marginale and A. centrale. This thesis aimed to characterise A. marginale and A. centrale in African buffalo from Kruger National Park (KNP), South Africa, and investigate the effects of the burden of Anaplasma species on the health and fitness of their host. Firstly, the major surface protein 1α (msp1α) and heat-shock protein (groEL) genes were used to characterise A. marginale and A. centrale, respectively, from African buffalo. Sequence variation and phylogenetic analyses revealed that sequences of Anaplasma spp. from African buffalo were unique and that they grouped separately when compared with previously published sequences of both species. Sequencing the same species in cattle from the same area in the future will allow for more conclusive evidence as to whether African buffalo are a reservoir for anaplasmosis, thereby providing insights into the interface of domestic and wild ruminants. Secondly, the burdens of A. marginale and A. centrale in blood samples from African buffalo were determined, using an established quantitative PCR, and then various statistical models were run to investigate associations between Anaplasma burden, co-infection dynamics and health outcomes for African buffalo. There appeared to be a time-lag between infection and host response, or co-infection response, for some of the parameters examined, showing the i importance of considering such delays in studies of disease. Despite finding a positive association between the concurrent burdens of infection with the two Anaplasma species examined, once the time-lag was accounted for, there was a negative association between the species, possibly indicating resource competition or the development of cross-immunity. African buffalo did not have an anaemic response to infection with either A. marginale or A. centrale; in contrast, for animals infected with A. marginale, there was an increase in haematocrit levels in response to infection. On the other hand, there were higher serum total protein levels associated with increased burdens of A. centrale, which may be due to the development of an immune response. Host responses to infection were also affected by external factors, including season and resource availability, and host factors such as gender. Younger buffalo appeared to be infected with higher burdens of A. marginale and A. centrale. In calves, infection with A. marginale appeared to occur before infection with A. centrale, and more frequently, which could be the result of different invasion and evasion techniques of the two species in this host. This study provides an insight into the effects of a subclinical infection on a wildlife host, caused by a pathogen which may cause severe clinical disease in domesticated animals. ii Declaration This is to certify that: i) the thesis comprises only my original work towards the Master of Philosophy (Veterinary Science), except where indicated in the preface; ii) due acknowledgement has been made in the text to all other material used; and iii) the thesis is fewer than the 40,000 words in length, exclusive of tables, maps, bibliographies and appendices ………………….. Danielle Sisson BSc(Hons)/BA, GradCertJour iii Preface All work presented in this thesis was completed during the MPhil candidature and was completed solely by myself, Danielle Rae Sisson, excluding the following: In chapters 2 and 3, Dr Anna Jolles and Vanessa Ezenwa are the principal investigators on the NSF EEID (#DEB-1102493/EF-0723928, EF-0723918) grant which financially supported the field captures for longitudinal buffalo sampling from a free-ranging herd from 2008-2012. In chapters 2 and 3, Anna Jolles was the principal investigator on the USDA-NIFA-AFRI grant (#2013-67015-21291), and Bryan Charleston the principal investigator of the UK Biotechnology and Biological Sciences Research Council grant (#BB/L011085/1) that funded the managed herd project as part of the joint USDA-NSF-BBSRC Ecology and Evolution of Infectious Diseases program. Anna Jolles’ USDA-NIFA-AFRI grant was also used to fund the laboratory work for this thesis. In chapter 3, Brian Henrichs completed Reverse-line Blot Hybridisation for Anaplasma marginale and A. centrale from African buffalo serum from the free-ranging herd to determine presence of infection for his Masters dissertation. These results were used in analyses for this thesis, and to determine samples to be used in further laboratory work. Anna Jolles, Brianna Beechler, Jasmin Hufschmid and Abdul Jabbar provided help with the conceptualisation of each chapter. All laboratory work was completed by Danielle Sisson. Chapter 2 is a published paper (Sisson, D., Hufschmid, J., Jolles, A., Beechler, B. and Jabbar, A. (2017). "Molecular characterisation of Anaplasma species from African buffalo (Syncerus iv caffer) in Kruger National Park, South Africa." Ticks and Tick-borne Diseases 8(3): 400-406). Phylogenetic analyses for this paper was completed by Danielle Sisson under the guidance of Abdul Jabbar. The paper was written by Danielle Sisson with the assistance of Abdul Jabbar, and Jasmin Hufschmid, Anna Jolles and Brianna Beechler assisted in editing the manuscript. Brianna Beechler and Mark Stevenson assisted with the statistical analyses, with all analyses being completed by Danielle Sisson for Chapter 3. All thesis chapters were written by Danielle Sisson. Brianna Beechler, Jasmin Hufschmid and Abdul Jabbar provided help in editing of this thesis. Brian Henrichs, Caroline Glidden, Katherine Potgieter-Forssman, Henri Combrink, Hannah Tavalire, Claire Couch, Brian Dugovich, Robert Spaan, Johannie Spaan, Julie Rushmore, Courtney Coon, Erin Gorsich, Morgan Movius, Becca Sullivan, Emma Devereux, George Meleleu, Abby Sage, Daniel Trovillion, Juliana Masseleux and Danielle Sisson were part of the buffalo research team, working on buffalo captures and sample processing which provided data on explanatory and predictive variables used in analyses. South African National Parks (SANParks) granted permission to conduct this study in Kruger National Park, and Markus Hofmeyr, Peter Buss and the entire SANParks Veterinary Wildlife Services Department assisted with animal captures and project logistics, as well as Kruger National Park DAFF veterinarians, including Lin Mari De-Klerk Lorist and Louis van Schalkwyk. Collaborators from the Pirbright Institute, including Eva Perez, Bryan Charleston and Fuquan Zhang were also involved in data and sample collection from buffalo captures. v Danielle Sisson was supported by an Australian Government Research Training Program Scholarship. vi Acknowledgements Firstly, thank you to my supervisors, Jasmin Hufschmid and Abdul Jabbar. I appreciate the time you made to go over and over and over the methods, and then over and over writing it up. I have learnt so much working on this project in the past couple of years. Thanks to the Uni of Melbourne for funding a trip back to South Africa and the U.S. as well, to collaborate with the other researchers working with some of this data. I do not think I could have pulled any of it together if I wasn’t able to sit in a room and pick the brains of people who designed the overall project and organised all the tests we ran on samples from the buffalo. A huge thank you to the Jolles lab from Oregon State University, and in particular, Anna Jolles and Brianna Beechler. Thank you, Anna, for letting me a part of this project. Though we haven’t had too many cross-overs in person since I started invading your lab group, whenever I have gotten to meet up with you, your advice and assistance has had an enormously greater impact than the time it took to impart it upon me. Bree, you’ve been an absolute champ throughout my degree, and you have more patience with annoying little wannabe researchers like me than I have or will have with anyone (which is why I’m still leaning towards the hermit life). Thank you for answering my constant flood of questions, helping me with setting up my database, cracking on with the stats, reading over drafts and giving me reliable and accurate feedback about my abilities and my work. I would have been much more lost than my usual base level of disorientation if it was not for your input. Even though none of them will probably ever come across this paragraph, I’m extremely grateful to my hockey and Krav Maga clubs with which I was involved throughout my time in Melbourne for keeping me fit, sane and socialised. Having to run out with you MUHC ladies vii twice a week in freezing (sometimes wet) Melbourne winter nights, and then spend a fair chunk of each Saturday playing and watching hockey, helped me get through months and months of lab work that could have been finished in a couple of weeks if it was working, but, as goes with most people’s research, it mostly wasn’t… Thank you to my friends (both in Aus and in the second home of my heart, South Africa) and family for keeping me happy throughout this Masters. Mum, Dad, Jay, Jem, Britt and my relatively fresh niece, Ella, thanks for having my back and checking in on me. Ella, you’ve been pretty useless at that actually, but I’ll forgive you for it because you’ve just turned one, and you’re an extremely entertaining time-absorber.