Serological and molecular survey of Toxoplasma gondii infection in wild carnivores of the Iberian Peninsula

Within the One Health framework, knowledge gaps regarding the circulation of Toxoplasma gondii in wild animals still need to be uncovered. Due to the lack of validated diagnostic techniques in such host species, the use of different serological and molecular assays could hinder the reliable comparison of epidemiological data regarding T. gondii. Herein, we propose a workflow to investigate parasite exposure and circulating genotypes in the Iberian Peninsula, with a focus on two species of wild carnivores (red fox -Vulpes vulpes- and Iberian lynx -Lynx pardinus-). A total of 377 serum samples from lynxes and 211 from foxes were analyzed by two serological techniques (lynx: modified agglutination test -MAT- and western blot -WB-; fox: enzyme-linked immunoassay -ELISA- and WB). For molecular assays and genotyping, we followed the protocols described by Calero-Bernal et al. (2022) and Joeres et al. (2023), respectively. Briefly, pools of artificially digested tissues from 123 foxes and 143 lynxes were used. After DNA extraction using an automated system, a nested PCR targeting ITS-1 was performed, and those testing positives were subjected to quantitative PCR targeting 529 RE fragment. An attempt to genotype positive samples with a high quantity of parasite DNA (Ct values ≤32) was performed using microsatellite (MS) fragments analysis to discriminate the parasite strain. If Ct>32, GRA6 and SAG3 genotyping markers were analyzed by Sanger sequencing. Considering the coincident positive results in both serological tests, the prevalence of T. gondii antibodies was 28.9% (61/211) in foxes and 44.3% (167/377) in lynxes. Toxoplasma gondii DNA was detected in 7/123 (5.7%) foxes and 27/143 (18.9%) lynxes. Only a complete genotyping profile by MS was achieved for 3/5 lynxes samples with Ct T. gondii sylvatic life cycle. We propose the use of this exhaustive workflow (involving several serological techniques, PCR-based procedures and DNA sequencing confirmation) to provide reliable data for epidemiological and genotyping studies of T. gondii infection in wildlife.

References

Joeres et al. (2023). Eur J Clin Microbiol Infect Dis, 42, 803-818. Calero-Bernal et al. (2022). 6th ApicoWplexa 2022. Bern, Switzerland.