Parasite load and genotype are associated with clinical outcome of piroplasm-infected equines in Israel

Background: Equine piroplasmosis is a highly endemic protozoan disease of horses worldwide, caused by Theileria equi and Babesia caballi. While most horses in endemic areas are subclinically infected, the mechanisms leading to clinical outcome are vastly unknown. Moreover, since clinical signs of disease are not specific, and the prevalence in endemic areas is high, it is difficult to determine if equine piroplasmosis is the cause of disease. To identify possible mechanisms leading to the clinical outcome in an endemic area, we compared parasite loads and genotypes in clinically and subclinically infected horses. Methods: Blood was collected from horses with clinical signs consistent with equine piroplasmosis, and from apparently healthy horses in Israel. Packed cell volume and total solids were measured. Quantitative and diagnostic polymerase chain reaction were used to identify, quantify and classify equine piroplasmosis infection. Phylogenetic analyses were used to determine the genotype of both parasites. Results: For both parasites, clinical cases were associated with low mean packed cell volume and high mean parasite load (P < 0.001), enabling the determination of a cut-off value to distinguish between clinically and subclinically infected horses. Samples of Theileria equi from subclinical horses were classified into three different 18S rRNA genotypes, D (n = 23), A (n = 12) and C (n = 5), while samples from all clinical cases (n = 6) were classified as genotype A. The sequences of T. equi equi merozoite antigens 1 (ema-1, n = 9) and 2 (ema-2, n = 11) genes were fairly conserved and did not differ between clinical and subclinical cases. Babesia caballi rhoptry associated protein-1 (rap-1) was classified into sub-genotypes A1 (n = 14) and A2 (n = 5) with no association to clinical outcome. Classification of the 18S rRNA gene (sub-genotypes B1 and B2) agreed with the rap-1 classification. Conclusions: The results of this study suggest that quantification of parasite loads of infected horses may be used to distinguish between infections resulting in disease and subclinical cases. Although number of clinical cases is limited, we identified T. equi 18S rRNA genotype A to be associated with clinical disease. This finding emphasizes the importance of in-depth genetic characterization of T. equi genotypes to identify possible markers for virulence.[Figure not available: See fulltext.]