Detection of Cryptosporidium-specific serum immunoglobulins in captive snakes by a polyclonal antibody in the indirect ELISA

Summary -An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of systemic Cryptosporidium-specific antibodies (Ab) in the serum of captive snakes. An optimal concentration of Cryptosporidium soluble protein-cocktail antigen was 20 μg/mL. In a direct ELISA, alkaline phosphatase-labeled rabbit-anti-snake polyvalent Ab detected snake immunoglobulins at a dilution of 1:12 800. It was possible to detect Cryptosporidium-specific Ab at a serum dilution of 1:1 600. Nineteen of 26 (73%) captive snakes (eight species) were Cryptosporidium-seroconverted; however, the differences in Ab titer among species were not significant. The distribution of absorbance values did not conform to a normal distribution (P < 0.05); mean Ab titer of nine snakes with cryptosporidiosis was significantly higher than mean Ab titer of the remaining ten snakes (P < 0.01). The monoclonal Ab test showed that the stools of 17 (89%) of 19 seropositive snakes contained Cryptosporidium oocysts; nine of those 19 (47%) stools were positive by acid-fast-stained (AFS) direct wet smear (DWS). A significant positive relationship between Cryptosporidium-seroconversion and snake age was observed (P < 0.03); the mean age of seropositive snakes was significantly higher than the mean age of seronegative animals (P < 0.01). The proposed assay is fast, reproducible, could be used for retrospective and prospective seroepizootiological studies, and assist ophidian centers in establishing Cryptosporidium-free collections. After standardization, the assay can be used for large-scale screening of captive snakes.