Establishment Of A Novel Molecular Detection Method Forsarcocystisin Venison

Recently, horse meat (basashi) contaminated withSarcocystisspp. caused food poisoning in Japan. An official detection method provided by the Ministry of Health, Labour and Welfare (MHLW), Japan, was designed to detectSarcocystis fayerito diagnose and control outbreaks ofbasashifood poisoning. In 2011,Sarcocystis-contaminated venison also caused food poisoning. However, the official MHLW detection method was not adequate for detectingSarcocystisspp. in venison. In this study, we established a novel PCR-based detection method that amplifies 18S rRNA gene based on the conserved region of the sequence in 32 species ofSarcocystisfor screening and quantification. Fifty venison samples from three areas in Hokkaido were examined by the MHLW method and the novel detection method. All samples wereSarcocystisspp.-positive. A sequence analysis indicated the presence of a species ofSarcocystisspecific to sika deer (Cervus nippon), and not to horses. Another primer pair was designed for a quantitative real-time PCR assay to determine the copy number of the Sarcocystis-18S rRNA gene in parasitized venison. The melting curve analysis revealed high specificity of this assay. The calculated curve demonstrated that this quantitative PCR assay showedR(2)value of 0.993 with 10-10(6)copies. Using this quantitative real-time PCR assay, the gene copy numbers were determined in 50 venison samples. The copy numbers of each sample ranged from 10(4)to 10(7)per gram. The copy numbers differed according to the area in Hokkaido. This indicates that the density ofSarcocystisspp. that infect Sika deer in Hokkaido is affected by the area. The novel screening and quantitative PCR method forSarcocystisin venison was useful for collecting epidemiological information onSarcocystisin wild Japanese sika deer, which will contribute to improve the safety of venison products in Japan.