Сероепизоотиологија и молекуларна дијагностика инфекције паразитом Neospora caninum код говеда / Seroepizootiology and molecular diagnosis of Neospora caninum infection in cattle in Serbia

Neospora caninum is a relatively recently recognized protozoan parasite of animals. It has been studied only since 1988, until when it was misdiagnosed as Toxoplasma gondii, due to the morphological similarity of the agents. Today, neosposoris in cattle is known as a disease of growing importance worldwide, causing abortions and large economic losses if epidemic abortions occur. The first investigations of the prevalence of N. caninum infection in cattle in Serbia were not carried out before 2008, and no data exist on the potential damaging effects of neoporosis on cattle husbandry in Serbia. In this study we examined the seroprevalence of the infection in a representative sample of cows from the entire territory of Serbia as well as the epizootiological factors that may play a role in the emergence and spread of infection. Sera originating from a total of 1496 cattle were tested by competitive ELISA assay for the presence of N. caninum-specific antibodies. It was shown that the seroprevalence was 7.2%, ranging from a low to a moderate (2.2 to 12%) prevalence accross all twelve epizootiological areas of Serbia. At least one seropositive animal was detected on 10.7% of farms. Results of the cELISA, i.e. the degree of inhibition in seropositive individuals, ranged from 40.3 to 91.8%, mean = 71.9 ± 17.1%, median = 79.9%, which means that almost one half of the seropositive cows had specific antibodies in high titres, or high values of inhibition of more than 80%. Possible association of the infection with biologically plausible risk factors including herd size, breed, husbandry methods, feeding practices (use of silage), origin of replacement stock, presence and number of dogs, occurrence of abortions in the herd, average annual rainfall and region, was analyzed by univariate analysis, and variables significant at P≤0.25 were included in multivariate logistic regression models. The results showed that the single risk factor for infection in individual animals was keeping cows in loafing barns (OR=3.31, 95% CI=1.95-5.60, P<0.001), while risk factors for the presence of infection on farms were the use of loafing barns (OR=18.49, 95% CI=5.40-63.36, P<0.001), and herd size >100 animals (OR=24.08 95% CI=3.85-150.50, P=0.001). In addition to a comprehensive insight into the current state of N. caninum infection in cattle in Serbia, the results of this research on infection risk factors both at the individual and farm level in our country suggest preventive measures that can be applied to improve animal health and reduce economic losses, thereby improving livestock production. In addition, we performed the optimization of molecular diagnosis of N. caninum infection and analyzed the possibilities of application of molecular methods in the Seroepizootiology and molecular diagnosis of Neospora caninum infection in cattle in Serbia iv diagnosis of neosporosis. Results of optimization of the real-time PCR in spiked samples of whole blood and peripheral blood mononuclear cells (PBMC) that originated from 94 cattle, showed an extremely high sensitivity of the method in detecting DNA equivalent to approximately one tachyzoite DNA in 2.5 mL of the eluate. Somewhat greater sensitivity was shown for the detection of N. caninum DNA in PBMC samples compared to whole blood samples – two times higher for the amount of 10 fg DNA, and 25% higher for 100 fg DNA. The sensitivity threshold demonstrated for DNA spiked PBMC samples was 1 fg DNA, and for whole blood samples 10 fg DNA. Since a very high overall sensitivity of the method was demonstrated (0.3 fg DNA), the introduction of real-time PCR as an extremely precise and rapid molecular diagnostic method can be recommended as an improvement of the diagnostic approach to neosporosis in specialized laboratories, which could significantly raise the level of animal health protection and improve livestock production in Serbia.