Development the detection method of Sarcocystis DNA from stool specimen as the proof for foodborne cases

Abstract

Purpose: Sarcocystosis is parasitic infectious disease caused by protozoan apicomplexan parasite of the genus Sarcocystis. Sarcocystis are globally distributed, and have a two-host cycle, generally with carnivores or omnivores as definitive hosts (DH) and herbivores as intermediate hosts (IH). Humans can serve as definitive hosts for S. hominis and S. suihominis after eating raw meat from cattle and pig, respectively. In Japanese cuisine, raw horse meat is known as “Basashi”, and it is popular in some regions of Japan. To date, “Basashi” has been known as safe food from the risk of contamination by parasite compared with raw beef meat or raw pork meat. However, between 2009, June and 2014, Jan., 48 cases of gastroenteritis with unknown sources, but ate raw horse meat was reported. Since Sarcocystis fayeri was reported as possibly the causative agent, the issue on food safety for consumption of raw horse meat has been concerned. To prove the food poisoning, the evidence from both of food and patient is important, but suspicious meat sample is not always remained, thus testing meat if it contain infectious agents is difficult. In this study, to develop an alternative tool is focused on to investigate parasitic DNA that might be contained in stool of patients when the leftover raw horse meat is not available. Methods: Using spike experiment with genomic DNA of Sarcocystis sp. and bradyzoites originally obtained from sarcocyst in horse meat, try to detect the parasitic DNA from stool specimens derived from a patient who is obviously not associated with any food poisoning cases. Recombinant plasmid DNA contained target region of S. fayeri. 18S rRNA gene was used to spike experiment and to detect parasitic DNA from stool samples, nested PCR was carried out. Results: Specific bands of spiked 10 copies of plasmid DNA containing 18S rRNA genes of S. fayeri was identified with gel electrophoresis under the UVtransilluminator. Conclusions: When the leftover meat is not available, detection of specific DNA for Sarcocystis sp. from stool specimens derived from patients who is involved with gastroenteritis can be useful. This method can be used as diagnostic tool to investigate foodborne cases that is responsible for eating raw horse meat.