Identification of potential reservoirs of Q fever in Queensland, Australia

Q fever is a zoonotic disease caused by the bacterium, Coxiella burnetii. The bacterium has a wide host range and human infections are most commonly contracted following contact with infected livestock. Australian surveys have shown an increased prevalence of human disease in recent years, with an increase in cases involving patients with no known contact with the typical reservoir species. The aim of this project was to determine the seroprevalence of C. burnetii in livestock, companion animals, feral animals and native wildlife and their ability to act as reservoirs of Q fever in Queensland, Australia. Due to the unavailability of secondary antibodies for native Australian marsupials, this project also aimed to develop a phage display library of chicken recombinant antibodies (CRAbs) for a variety of Australian native marsupials and determine their effectiveness as secondary antibodies for epidemiological studies and pathogen surveillance in wildlife populations. This project also aimed to determine the prevalence of C. burnetii in the ticks and blood of Australian native marsupials to determine their potential capacity to act as reservoirs of Q fever. Prior to the development of diagnostic tools for the detection of antibodies to C. burnetii in animals, an appropriate C. burnetii isolate needed to be selected for the purpose of antigen production. A comparison of virulence and ability to induce seroconversion was performed in mice and guinea pigs, which resulted in the selection of the Australian Cumberland Q fever isolate. A series of quantitative reverse transcriptase PCRs were developed to determine the antigenic phase of the isolate and were validated against traditional methods for determining antigenic phase. ELISAs were then developed for the detection of antibodies to both C. burnetii antigenic phases and validated using sera from infected and uninfected mice and guinea pigs. The ELISAs developed in this study are the first known use of an Australian Q fever isolate as antigen. The ELISAs developed were then modified for use with bovine, canine, feline, porcine and human sera. A total of 1,835 bovine, 1,522 human, 127 dingo, 201 domestic dog, 49 domestic cat, 31 feral cat, 19 feral pig and 16 feral fox samples were tested for antibodies to C. burnetii. Seroprevalence was found to be highest in foxes (43.8%; 95% CI 42.5-48.1%) and feral cats (38.7%; 38.7-40.6%). Similar seroprevalence was found in beef cattle (16.8%; 16.78-16.80%), domestic dogs (both currently (21.8%; 21.6-22.1%) and retrospectively (16.0%; 15.9-16.2%) and wild dogs/dingoes (17.3%; 17.2-17.5%). Seroprevalence was relatively low in domestic cats (6.1%; 6.1-6.5%) and the human population (3.5%; 3.48-3.50%). No significant difference was found between seroprevalence in domestic dogs and dingoes. However, the difference between seroprevalence in domestic cats and feral cats was statistically significant (P<0.05). In order to produce recombinant secondary phage-displayed antibodies for Australian native marsupials, the technique was initially optimised and validated using a murine model. Purified murine IgG was used to immunise domestic chickens and reverse transcriptase PCR was used to amplify genes encoding the heavy and light chain immunoglobulin. These were then inserted into a phage-display vector and used to create libraries of chicken recombinant antibody (CRAb). This library was then screened for phage-displayed antibodies binding to murine IgG. Selected CRAbs were then characterised in ELISA and DNA sequences obtained. The selected CRAbs were then validated in ELISA using the sera of mice infected with C. burnetti and uninfected mice. Using the optimised method for the production CRAbs against IgG, further libraries were produced for macropods (Macropus sp.), common northern bandicoot (Isoodon macrourus) and brushtail possum (Trichosurus vulpecula). Selected CRAbs were also characterised in ELISA and DNA sequences obtained. Each CRAb was tested for cross-reactivity against the IgG of the other species. The CRAb raised against murine IgG in the initial optimisation experiments was found to bind to the IgG of all species tested. This CRAb was used in subsequent serological testing to simplify development of ELISAs as only this CRAb would need to be amplified for the production of ELISA conjugate. The phage-displayed CRAb was compared to competitive ELISA, standard indirect ELISA and complement fixation in serological testing on serum samples from a variety of Australian native marsupials. A total of 500 macropod, 56 brushtail possum and 52 common northern bandicoot samples were tested for antibodies to C. burnetii. Seroprevalence was found to vary significantly between sites (P<0.01) and regions (P<0.01). Seroprevalence was highest in bandicoots (26.9%; 95% CI 26.6-27.7%), followed by macropods (25.6%; 25.6-25.7%) and possums (19.6%; 19.5-20.2%). Agreement between the ELISA methods used was poor and it is thought that this was due to a combination of immunoglobulin isotype subclass and antigen epitope specificity. The heterogeneity of serological responses in native marsupials made the ultimate validation of phage-displayed CRAbs in ELISA difficult, as they could not be directly compared to another test in field surveys. A qPCR was successfully developed for the detection of the Coxiella-specific com1 gene in tick extracts and whole blood collected from Australian native marsupials. A total of 323 ticks were collected from 34 bandicoots, 14 macropods and one human. Whole blood was collected from 35 bandicoots, 31 macropods and two possums. The detection of the com1 gene indicated the presence of C. burnetii in both the ticks (15.5% pools) and whole blood (24.2%) of bandicoots and a variety of macropods in northern Queensland. Coxiella burnetii was also detected in the whole blood of one of the two possums tested. The additional detection of a PCR product with regions of DNA homologous with the com1 gene in the ticks and whole blood of these species indicated the presence of an, as yet, unidentified tick-borne agent. This project demonstrated the prevalence of antibodies to C. burnetii in the serum of a variety of animals, including livestock, domestic animals, feral animals and native Australian marsupials. In addition, PCR assays detected the presence of C. burnetii in both ticks and whole blood of native Australian marsupials. The serological and molecular assays performed in this study demonstrated the potential for a wide variety of animals to act as reservoirs of Q fever in Queensland, Australia.