Development of a double-antibody ELISA for IgG evaluation in the pichi (Zaedyus pichiy; Xenarthra, Dasypodidae) with special interest for epidemiological surveillance

MORENO-SOSA, M.T.; JAHN, G.A.; PENNACCHIO, G.E.; SUPERINA, M.; ACTIS, E.; MACKERN-OBERTI, J.P.

Mendoza

Congreso; XXXIV Reunión Anual de la Sociedad de Biología de Cuyo; 2016

Sociedad de Biología de Cuyo

Many zoonotic pathogens have a wildlife reservoir, and hunters are especially prone to acquire a zoonotic disease while killing or consuming wildlife. It is vital that people are alerted of the risk of becoming infected with potentially zoonotic diseases while hunting and consuming wild animals. The pichi (Zaedyus pichiy; Xenarthra, Dasypodidae) is an intensely hunted armadillo species that is used as a protein source throughout its range. Although several biological features of the pichi are well studied, data about its potential role as a target or a reservoir of human pathogens, such as Trypanosoma cruzi and Brucella abortus, are scarce. Due to the lack of commercially available serological tests to detect specific IgG, the aim of this work was to develop a serological test to evaluate the presence in pichis of IgG specific to zoonotic pathogens important to public health. To this end we developed a double-antibody indirect ELISA. Gamma heavy chains (≈55kDa) from pichi serum were purified through SDS-PAGE and used to immunize BALB/c mice with Freund complete adjuvant. Unspecific control (pre-immune) sera were collected prior to immunization. Serum samples from immunized mice were collected periodically for 3 months. To confirm the specificity to pichi IgG of the collected mouse antiserum we performed conventional western blot assays in which pichi and mouse serum proteins were separated by SDS-PAGE, transferred to PVDF membranes, and blocked for 2 hours with 3% low-fat milk. Membranes were first incubated with mouse antiserum from immunized mice for 1 hour, and secondarily incubated with goat anti mouse IgG-HRP. After detection, lanes containing pichi serum displayed only a band between 50-60kDa. In addition, lanes containing mouse serum displayed a slightly smaller band between 45-55kDa corresponding to mouse gamma heavy chains. The higher molecular weight band observed in pichi lanes, corresponding to a protein larger than that observed in the mouse samples, is in accordance with published reports, highly suggesting that both, pichi and mouse, are gamma heavy chains. Similarly, we found that our mouse antiserum showed reactivity against purified pichi gamma heavy chains by ELISA. In conclusion, these data suggest that we have developed a useful tool to evaluate IgG levels from pichis. This antiserum is crucial to indirectly evaluate whether pichis have been infected or been in contact with several pathogens, including the zoonotic parasite Trypanosoma cruzi. Although complementary studies are needed to validate data from serological tests, screening for pathogen-specific antibodies could be a novel contribution to an accurate diagnosis of infection in pichis.