Identification Of Sarcocystis Hominis And Sarcocystis Suihominis Based On Morphological And Molecular Chacterization

Sarcocystis hominis and Sarcocystis suihominis are the pathogens of human intestinal Sarcocystis,which can cause diarrhea,abdominal pain and other clinical symptoms in human,among which the intermediate hosts are cattle and pigs.The diagnosis of human pneumocystis patients mainly depends on microscopic examination of pneumocystis oocysts or sporangia in their excreta.This method has a low sensitivity,and the species cannot be identified due to the overlapping size and similar morphology of the oocysts/sporangia of the two pathogens,thus the source of infection cannot be traced back.Therefore,based on the investigation of natural infection of intestinal carnitosporidia of cattle and pigs in farmers’ markets in Kunming and other places,the tomonts were used as materials.A variety of molecular markers（ribosome 18 S r DNA gene,ITS-1 gene,mitochondrial COX1 gene,COX3 gene,CLPC gene）were sequenced and analyzed,and a specific diagnostic method was established.The results were as follows:（1）The infection rate of S.hominis in cattle samples was 25.7%（9/35）.Under optical microscope,tomonts were fusiform,milky white,131.27μm×2024.5μm（n=6）in size,and the walls of tomonts had an average upright fingerlike process of 5.17μm（n=18）.The average similarity of 18 S r DNA gene,COX1 gene,COX3 gene,ITS-1gene,and CLPC gene among different clones were 99.3%（n=2）,98.9%（n=7）,98.9%（n=18）,95.5%（n=6）,99.6%（n=5）.（2）The infection rate of S.suihominis in pork samples was 14.7%（5/34）,and the mean size of tomonts was 142.33μm×1998.6μm（n=6）.There were thick hairy projections on the tomonts wall,and the mean length of projections was 6.44μm（n=18）.The average similarity of 18 S r DNA gene,COX1 gene,COX3 gene,ITS-1 gene and CLPC gene among different clones was 99.5%（n=3）,99.5%（n=9）,99.4%（n=12）,94.8%（n=8）,99.6%（n= 6）.（3）The average similarity of 18 S r DNA gene,COX1 gene,COX3 gene,ITS-1 gene,and CLPC gene between S.hominis and S.suihominis was 92.0%,75.4%,91.2%,57.5% and 93.4%.（4）Two pairs of primers were designed according to the 18 S r DNA markers of S.hominis and S.suihominis.F1/ Sh R5 could amplify 323 bp fragment on S.hominis.F1/ ZR1 amplified a403 bp fragment on S.suihominis,and the two primers could be used to distinguish the two species.The results showed that all of the above five molecular markers could distinguish S.hominis from S.suihominis,and the discrimination degree of ITS-1 gene and COX1 gene was better than that of 18 S r DNA gene,COX3 gene and CLPC gene.This experiment was the first time to sequence and analyze the mitochondrial COX3 gene,ITS-1 gene and CLPC gene of S.hominis,and the first time to sequence and analyze the COX3 gene and CLPC gene of S.suihominis.