Advances in the serological diagnosis of "Toxoplasma gondii" and "Neospora caninum" infections in the field of animal health

Abstract

Introduction: toxoplasmosis and neosporosis are two parasitic diseases caused by the Apicomplexan parasites Toxoplasma gondii and Neospora caninum, respectively, which are globally distributed. Toxoplasmosis is a zoonosis and a main cause of reproductive failure in small ruminants with the subsequent high economic impact. Neospora caninum is not a zoonotic pathogen, but it is a major cause of abortion in cattle and is also associated to reproductive failures in sheep. Currently, diagnosis of these diseases is commonly based on serological methods, but it could be improved by the application of a more sensitive technique such as Time-resolved fluorescence immunoassays (TRFIA). Material and methods: this PhD thesis comprises three main activities: (1) a systematic review and a meta-analysis on the development of new serological methods to diagnose toxoplasmosis in humans and animals, focused on their One Health approach followed and the critical analysis of the validation process; (2) a study of the seroprevalences rates and risk factors associated to T. gondii infection in dogs and cats from an anthropized area (Bangkok, Thailand); and (3) the development and validation of new TRFIAs for detection of anti-T. gondii antibodies in cat and goat sera, and anti-N. caninum antibodies in sheep serum and milk samples. In cats, several recombinant and a recombinant chimeric antigens were evaluated by this technique. Analytical validation consisted of estimating of the inter- and intra-assay precision, analytical sensitivity (Se), accuracy and cross-reactivity with closely related pathogens. Diagnostic validation was assessed by the evaluation of the positive/negative discriminative potential of the technique (Mann Whitney U test), a ROC analysis (for determination of the optimal cut- off, Se, specificity -Sp- and area under the curve), the agreement (using the kappa value) and the Spearman's correlation coefficient. Results: there is a lack of implementation of the One Health approach in the studies on serological diagnosis of toxoplasmosis, especially in those conducted in humans and by physician-based research teams. The validation process of the newly developed methods is widely heterogeneous and the exclusive evaluation of the diagnostic performance of the assays, without considering the analytical characteristics, is frequent. The seroprevalence rates of T. gondii infection in cats and dogs from Bangkok were 18.7% and 7.9%, respectively. A TRFIA was developed to detect anti-T. gondii antibodies in cats, with the chimeric antigen TgSAG1-GRA8 providing a better diagnostic performance in comparison with other antigens. In addition, a TRFIA based on the same T. gondii chimeric antigen was developed to diagnose T. gondii infection in goats with excellent results in the analytical Se, and high Se and Sp in the ROC analysis. However, the cross-reactivity detected with specific anti-N. caninum antibodies limits the use of this technique. Finally, a TRFIAs based on recombinant NcGRA7 antigen was successfully developed and validated to detect anti-N. caninum antibodies in ovine blood sera and full-cream milk samples, with high analytical and diagnostic performances. Discussion and conclusions: this thesis provides new and up-to-dated knowledge on the development of serological methods to diagnose T. gondii infection in animals and humans, highlighting the need of implementation of the One Health approach and of a close following of the different steps of the validation process recommended by the OIE. The high seroprevalence of T. gondii found in dogs and cats from Bangkok should be considered a public health concern in this anthropized area. In addition, the new TRFIA developed in this thesis increase the Se of the current diagnostic methods for the detection of T. gondii and N. caninum infections and was adapted to non-invasive samples, although future research should include the evaluation by TRFIA of new T. gondii antigens avoiding the cross-reactivity with N. caninum.