Development of serological tools to diagnse Babesia divergens infections in animals and humans

Aim: Babesioses are haematic tick-borne zoonoses that induce malaria-like disorders in domestic, wild animals humans, Although IFI and ELISA commercial kits are available to test the presence of antibodies against most of Babesia species, no kits exist to serologically diagnose the infections due to B.divergens, one of the most important zoonotic species in Europe (Vannier and Krause, 2009, Interdiscip Perspect Infect Dis). Aim of this work was to fill this gap and develop assays useful in routine large-scale testing to detect animal and human infections. Materials and Methods: B. divergens (Rouen strain, 1987) was used for the infection of 30 gerbils and the production of B. divergens positive sera. The same strain was microinjected in bovine and ovine erhythrocytes to obtain in vitro cultures of the parasite. Infected erhythrocytes were used to infect 2 calves and to prepare slides for IFAT and microwells for ELISA (corpuscular antigen). The surnatant medium of the cultures was collected and used to arrange an ELISA (metabolic soluble antigen) and a WB assay. All serological assays were set up with sera from experimentally infected gerbils and calves, and with heterologous sera from cattle infected by B. bovis and B. bigemina to check any cross reactions. Finally ELISA, IFAT and WB test were applied to 100 sera from domestic and wild animals proven infected by PCR, to 100 sera from animals serologically positive to B.bovis and B.bigemina, to 50 negative control sera, to 167 humans\u2019 sera from people at risk of infection. Results ELISA and IFAT seem in general agreement, even if the first one proved more sensitive. The evaluation of soluble vs. corpuscular antigen in ELISA showed that the first one provides more specific results. All sera from experimentally infected animals in WB showed 2 major protein bands at 33 and 37 kDa. Heterologous sera displayed a different proteins pattern, so suggesting the specificity of the test. As regards humans, 2/167 sera showed the specific bands for B.divergens. Conclusions These researches, aimed to set up serological tests to diagnose B. divergens in humans and animals, gave encouraging results. The ELISA tests seem sensible, however, in areas where many Babesia species are sympatric, it could suffer in specificity. The developed WB assay could overcome this problem. In fact this test specifically confirms positive results obtained with IFAT and ELISA tests. Further researches are in progress to produce monoclonal antibodies to immunodominant antigens identified in strains of B. divergens coming from different geographical areas and to develop a competitive inhibition ELISA test