Usefulness of PCR–RFLP of 18S rRNA gene for rapid post-mortem diagnostics of highly pathogenic Eimeria spp. (Apicomplexa: Eimeriidae) of European bison, Bison bonasus L. with histopathological correlation

摘要

Eimeria spp. infection was investigated in 10 free-roaming European bison aged three months to 26 years by anatomopathological, histopathological, coproscopic and PCR-RFLP examination. The coproscopic study identified Eimeria oocysts in the faeces of five bison. The most prevalent morphotypes were E. bovis , present in all positive samples, and E. zuernii , in all but one. Additionally, mixed infections consisting of E. bovis , E. zuernii , E. alabamensis , E. auburnensis , E. canadensis , E. cylindrica , E. ellipsoidalis and E. subspherica were diagnosed in two bison calves. Besides being the most prevalent form, E. bovis also demonstrated the highest OPG (2,750). The presence of oocysts in the faeces was associated with those of macrogamonts, microgamonts and oocysts in the epithelium of the large intestine. Intestinal coccidiosis associated with lymphoplasmacytic enteritis was observed in many bison, not only those with positive OPG. Four animals with negative coproscopy results demonstrated early-stage gametogony in the large intestine; one case presented no endogenous stages of coccidians in the histopathological sections of the intestine, nor oocysts in the faecal samples. A 530 bp product of E. bovis 18S rDNA (GenBank: MK951685) was obtained from both the colon wall and oocysts; this was subjected to PCR-RFLP analysis based on AluI and Hin1II (NlaIII) restriction enzymes. Both samples yielded a consistent seven-band pattern, four of which (270 bp, 40 bp, 180 bp and 84 bp) were expected, and the other three represented undigested fragments. The obtained digestion pattern is indicative of Eimeria spp. infection, and can serve as a first-step diagnostic approach in detection of infection. The result of computer-based virtual digestion of the PCR product suggests that double digestion with Mval (BstNI) and KpnI restriction enzymes may be used as a second-step tool to distinguish between E. bovis , E. zuernii and E. alabamensis , all of which are highly-pathogenic species.