Investigation Of Intestinal Parasites In Goats And Sheep From Partial Areas Of Jiangsu Province And Establishment Of PCR Method For Detection Of Sheep Coccidia

Goats and sheep are often infected with parasites under natural conditions.The clinical symptoms of goats and sheep with a mild infection are not obvious,and the damages caused by parasites are easy to be ignored.Goats and sheep with severe infection may present with anemia,diarrhea and malnutrition,and the production and quality of milk,mutton and leather will decline,resulting in serious economic losses.In addition,some parasites of goat and sheep are zoonotic,which can infect people and cause harm to human health.The species and numbers of parasites in goats and sheep are not only related to the life cycle of parasites,but also the feeding patterns.In recent years in China,the goat and sheep breeding industry has developed rapidly,and the breeding patterns had been changed from stocking and free range to captive and high bed breeding.The change of feeding pattern may cause the differences of parasite species and epidemic situation.The purpose of this study is to investigate the parasites in the digestive tract of goats and sheep in large-scale farms in Suqian,Lianyungang,Zhenjiang,Nantong,Xuzhou and Huai’an,know well the species and infection rate of parasites,especially analyze the genotypes of Enterocytozoon bieneusi and Cryptosporidium spp.,and evaluate the risk of zoonotic infection.At the same time,PCR methods for detecting two species of sheep coccidia were established.These results will lay the foundation and provide technical means for the control of parasitic diseases of goat and sheep in Jiangsu Province.1.Investigation of intestinal parasitic worms and coccidia infection in sheep and goatsA total of 274 goat fecal samples and 124 sheep fecal samples were collected from six large-scale farms in Suqian,Lianyungang,Zhenjiang,Nantong,Xuzhou and Huai’an,Jiangsu.The parasites were examined by the nylon sifter elutriation and sieving method and saturated floating technique.The oocysts were collected from the coccidia-positive samples,and cultured,and species of coccidia were identified microscopically.The results showed that the total infection rate was 91.71%（365/398）,in which was 91.97%（252/274）in goats and 91.13%（113/124）in sheep,respectively.Six kinds of parasites were found in goat feces,including Strongyloides spp.,Trichostrongylidae spp.,Moniezia spp.,Trichocephalus spp.,Fasciola spp.and Eimeria spp.,and the infection rates were 24.82%（68/274）,20.44%（56/274）,4.38%（12/274）,1.46%（4/274）,0.73%（2/274）and 91.97%（252/274）,respectively;Eimeria spp.was the dominant species.A total of 5 kinds of parasites were found in sheep feces,including Trichostrongylidae spp.,Moniezia spp.,Strongyloides spp.,Trichocephalus spp.and Eimeria spp.,and the infection rates were 11.29%（14/124）,11.29%（14/124）,4.84%（6/124）,1.61%（2/124）and 90.32%（112/124）,respectively;Eimeria spp.was the dominant species.Nine species of Eimeria in goats were identified,including E.arloingi,E.alijevi,E.Hirci,E.christenseni,E.caprina,E.apseronica,E.ninakohlyakimovae,E.caprovina and E.jolohijevi,and the infection rates were 74.45%（204/274）,51.09%（140/274）,37.96%（104/274）,29.2%（80/274）,26.28%（72/274）,22.63%（62/274）,21.9%（60/274）,19.71（54/274）and 13.14%（38/274）,respectively;E.arloingi was the dominant species.A total of 9 species of Eimeria in sheep were identified,including E.bakuensis,E.parva,E.ovinidalis,E.weybridgensis,E.pallida,E.furei,E.crandallis,E.ahsata and E.anticata,and the infection rates were 54.84%（68/124）,50%（62/124）,41.94%（52/124）,37.1%（46/124）,29.03%（36/124）,24.19%（30/124）,22.58%（28/124）,19.35%（24/124）and 12.9%（16/124）,respectively;E.bakuensis was the dominant species2.Investigation of Enterocytozoon bieneusi in goats and sheepThe fecal genomic DNA was extracted,and E.bieneusi was detected by nested PCR based on the ITS.The results showed that the total infection rate of E.bieneusi was 11.81%（47/398）.The infection rate in Xuzhou was as high as 21.11%（5/22）.The infection rates in Nantong,Zhenjiang,Suqian and Lianyungang were 12.5%（5/40）,11.36%（5/44）,10.32%（13/126）and 6.1%（5/82）,respectively.The infection rate of E.bieneusi in goats was 8.76%（24/274）,and the genotypes were CHG1（7）,CHG3（10）,BEB6（5）,JSX132（1）,and COS-Ⅰ（1）.The infection rate of E.bieneusi in sheep was 18.55%（23/124）,and the genotypes were CHG1（1）,BEB6（18）,and JSX164（2）,COS-I（1）,and CHC8（1）.Jsx132 and jsx164 were new genotypes found in this investigation.The phylogenetic tree showed that the genotypes detected in this research was belonged to Group 2.3.Investigation of Cryptosporidium in goats and sheepBased on the locus of SSU rRNA gene,the above fecal DNA samples were detected by nested PCR.The results showed that the total infection rate of Cryptosporidium was 0.75%（3/398）.Only 3 positive samples were detected in Nantong area,and the infection rate was 7.5%（3/40）.The infection rate of goat Cryptosporidium was 1.09%（3/274）and sheep Cryptosporidium was 0.0%（0/124）.The phylogenetic tree showed that the Cryptosporidium species were C.xiaoi.4.Establishment of PCR detection method for two species of sheep coccidiaAccording to the ITS-1 sequences of E.parva and E.ovinoidialis published in GenBank,two pairs of species-specific primers were designed.The PCR reaction conditions were optimized,and the specificity and sensitivity of the PCR system were evaluated,respectively.The results showed that each primer pair could amplify specific bands for E.parva and E.ovinoidialis with the sizes of 176 bp and 300 bp,respectively,and negative for other sheep coccidia and Toxoplasma gondii,Histomonas meleagridis and Cryptosporidium.The minimum detection concentration was 0.1 ng DNA.These results indicated that the established method had good specificity and sensitivity.Conclusion:the infection rate of intestinal parasites in goats and sheep from six large-scale farms in Jiangsu was 91.71%;Six kinds of parasites were found,and the Eimeria spp.was dominant species.The total infection rate of E.bieneusi was 11.81%,and seven genotypes were detected,two of them were new genotypes;The total infection rate of Cryptosporidium was 0.75%,and all of them were identified as C.xiaoi.The PCR method for detection of E.parva and E.ovinoidialis was established,and the minimum detection amount was 0.1 ng.