Development of diagnostic tests for the detection of Neospora caninum infected cattle
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The protozoan parasite Neospora caninum is among the most frequently diagnosed infectious causes of bovine abortion thus causing significant economic losses, production inefficiency and welfare concern to the cattle sector worldwide. The control of bovine neosporosis relies on management techniques within which the identification of infected animals by serological testing plays a key role. However, the reliable diagnosis of the disease is hindered by the complex host-parasite interactions and the intrinsic limitations of the serological diagnostic tools currently available; as a result, some infected animals may not be detected. At the herd, regional and national levels, this can potentially undermine efforts for the control of the disease. The work presented in this thesis was undertaken to further investigate avenues to improve the diagnosis of N. caninum infection in cattle. It has been shown previously that recombinant antigens expressed by the rapidly proliferating tachyzoite stage and the slowly multiplying bradyzoite stage of N. caninum can be successfully employed for the detection of specific antibody responses during acute and persistent infections respectively. Following the establishment of persistent infections, sustained by the bradyzoite stage, the antibody levels against the tachyzoite stage may decline below the detection limits of currently available diagnostic tests which are exclusively based on antigens expressed by the tachyzoite. Consequently, the use of bradyzoite antigens for the development of serological diagnostic tests, may enhance the identification of infected animals. Novel antigens putatively expressed by the quiescent bradyzoite stage of N. caninum have been identified, expressed as recombinant proteins and assayed for the detection of specific antibodies. The recognition of recombinant tNcSRS12A-B and tNcSRS44- A by specific antibodies in sera from persistently N. caninum-infected cattle suggested that these proteins could be used for the detection of persistently infected animals. Indirect ELISAs based on previously characterised N. caninum antigens, such as the tachyzoite surface protein rNcSRS2, the immunodominant dense granule protein rNcGRA7 and the bradyzoite specific surface antigens rNcSAG4, rNcBSR4 and rNcSRS9, as well as a commercial test using whole tachyzoite lysate as antigenic preparation, were evaluated within a cross-sectional study to estimate the seroprevalence of bovine neosporosis in British dairy cattle. Moderate, but not high, agreement was found amongst the tests based on whole tachyzoite lysate, rNcSRS2 and rNcGRA7, and amongst the bradyzoite-specific antigen-based iELISAs. In contrast, only slight agreement was observed when each test detecting antibody responses indicative of acute infection (whole tachyzoite lysate, rNcSRS2 and rNcGRA7) was compared with each test detecting antibody responses indicative of persistent infection (bradyzoite-specific antigen-based iELISAs). Most N. caninum antibody-positive cattle samples showed detectable antibodies only against either antigens predominantly expressed by the tachyzoite or bradyzoite antigens thus suggesting that the exclusive use of one type of test may result in the misclassification of a proportion of animals, which test negative despite harbouring the parasite. This may result in the underestimation of the seroprevalence. Consequently, the combination of multiple tests in parallel, both tachyzoite and bradyzoite antigen-based, would improve the diagnosis of bovine neosporosis. Molecular tools for the genetic discrimination of different N. caninum isolates were also investigated. A novel multilocus fragment typing (MLFT) tool based on twelve highly polymorphic microsatellite markers was developed and applied to the analysis of DNA samples obtained from laboratory-maintained N. caninum isolates and tissues collected from bovine foetuses aborted due to N. caninum. The locus-specific nested PCRs associated with automated fragment analysis by capillary electrophoresis enabled to evaluate the markers in terms of typeability and discriminatory power. Overall, the typing tool was characterised by good typeability and discriminatory power and enabled to provide information on the genetic diversity amongst the laboratory-maintained and clinical N. caninum isolates studied. The MLFT tool may help to investigate the likely source of infection within abortion outbreaks and aid the study of the association between the genetic heterogeneity of N. caninum and the diverse biological features in vitro and in vivo. Furthermore, the loci characterised by the highest discriminatory power and typeability may be used alongside already established microsatellite markers for the development of an improved typing tool which could be proposed at the inter-laboratory level. Finally, current perceptions and common veterinary practice related to the diagnosis and control of bovine neosporosis were studied by developing a questionnaire for cattle practitioners in the United Kingdom. The survey highlighted the awareness of the limitations of current serological techniques and the demand for additional tools in terms of diagnostics and vaccines to tackle the economic losses and animal welfare implications related to N. caninum in cattle.