Epidemiological Investigation And Molecular Characteristics Of Diarrheal Pathogens In Pigs In Yunnan Province

Diarrhea is one of the important factors that threaten the development of pig industry,which can cause the death of many newborn pigs.Porcine circovirus,Porcine teschovirus,Cryptosporidium spp.,Enterocytozoon bieneusi,Giardia lamblia and Blastocystis are important pathogens,which can cause diarrhea in pigs.Infection with these pathogens can seriously affect the normal growth of pigs,causing different degrees of diarrhea,even death.They have a serious negative impact on the pig industry and the development of human public health.However,there are few reports of the prevalence of these pathogens in pigs in Yunnan Province,particular the species or genotype distribution of the four enteric protozoans.Therefore,in order to assess the risks and prevalence of these pathogens in pigs in Yunnan Province,the present study aimed to determine the prevalence and species of P.circovirus,P.teschovirus by ELISA and PCR,and to investigate the prevalence and species/genotypes of Cryptosporidium spp.,E.bieneusi,G.lamblia and Blastocystis in pigs in Yunnan province.Objectives: In this study,serum samples were collected from 68 pig farms in 9regions of yuxi city,Yunnan Province and feces samples were collected from 11 regions of Yunnan Province and were tested by serological testing and molecular biology to analyze the prevalence,species and genotype of the 6 pathogens that cause diarrhea in pigs in Yunnan Province.These findings will help us to understand the spread of its pathways,identify the source of pollution,and investigate outbreaks,so as to effectively prevent and control these pathogens that cause diarrhea in pigs.Methods: In the present study,P.circovirus type 2(PCV2)and P.teschovirus(PTV)were detected by enzyme-linked immunosorbent assay(ELISA);the prevalence was determined by statistical analysis,and the risk factors were analyzed under the condition of single factor.The odds ratio(OR)and its confidence interval(95% CI)were calculated.Virus species were identified by PCR amplification and and sequencing.In this study,the feces were collected from pigs in 11 regions of Yunnan Province,and the prevalence and species/genotypes of Cryptosporidium,E.bieneusi,G.lamblia and Blastocystis were determined.The PCR was used to amplify the 18 S r RNA gene of Cryptosporidium,the tpi gene of G.lamblia,the ITS gene of E.bieneusi and the SSU r RNA gene of Blastocystis.The prevalence was determined by statistical analysis and the risk factors were analyzed.The species and genotypes were identified by sequence comparison,and the genetic and evolutionary relationships were analyzed by constructing an evolutionary tree.Results:(I)The infection rate of P.circovirus was exmained by enzyme-linked immunosorptive assay(ELISA)in 561 pig serum samples from 49 pig farms(not immunized with commercial P.circovirus type 2(PCV2)vaccine)in 9 districts of Yuxi city in Yunnan province.The overall P.circovirus prevalence was 27.45%(154/561;95%CI: 23.76-31.14).Statistical analysis found that age was the risk factor affecting the P.circovirus prevalence.In addition,220 serum samples of pigs from 19 farms(immunized with PCV2 vaccine)were examined for P.circovirus prevalence by ELISA.The overall prevalence was 71.82%(158/220;95% CI: 65.88-77.76).Statistical analysis showed that age was also a risk factor for porcine circovirus infection.P.circovirus prevalence and genotype were examined by DNA extraction of 142 samples with suspected clinical symptoms of P.circovirus from some farms(which were not immunized with PCV2vaccine)in nine regions of Yuxi city,Yunnan Province.The overall prevalence was32.39%(46/142;95% CI: 24.69-40.09).The P.circovirus species was confirmed by aligning the obtained sequences with reference sequences in Gen Bank.The results of sequencing and comparative analysis of the amplified sequences showed that the amplified sequences came from the PCV2 virus genome,which further confirmed previous results of our serological detection results.(II)455 serum samples of pigs were collected from three areas in Yuxi city in Yunnan Province and the PTV prevalence was examined by ELISA.The results showed that the prevalence was 82.42%(95% CI: 78.92～-85.92).The region factor was significantly correlated with the PTV prevalence.DNA extraction was conducted on 8 samples of pig tissues which were suspected symptoms of jieshen virus(PTV)infection)collected from Yuxi city in Yunnan Province,and species of P.teschovirus were determined using the nested RT-PCR method combined with sequencing.The homology between the obtained sequences and the P.teschovirus gene sequences available in Gen Bank were 90% ～ 99%.PTV infection was confirmed in the pig samples tested,which is the first time that the PTV infection has been confirmed in pigs in Yunnan province.(III)994 fresh fecal samples were collected from 11 pig farms in 11 regions of Yunnan Province and the prevalence and genotype distribution of Cryptosporidium spp.,E.bieneusi,G.lamblia and Blastocystis were examined.The Cryptosporidium prevalence was2.82%(28/994;95% CI: 1.79-3.85),the age and region were significantly correlated with the Cryptosporidium prevalence.The gene sequence alignment and phylogenetic tree analysis showed that two species,C.scrofarum(proporine gene type II)and C.suis(porcine Cryptosporidium),were identified in the present study.(IV)The E.bieneusi prevalence and geotypes were examined based on sequence analysis of the ribosomal internal transcribed spacer(ITS)region.The prevalence was 15.80%(157/994;95% CI:13.53 18.06).The region and age were significantly correlated with the E.bieneusi prevalence.A total of 12 genotypes were identified by sequence analysis,including 6known genotypes(CHC5,CHG19,Ebp D,Ebp A,Ebp C,CHS12),and 6 novel genotypes(YNZ1,YNZ2,YNZ3,YNZ4,YNZ5,and YNZ6).Phylogenetic analysis found that all 12 genotypes belonged to the group 1,which infect humans and animals.(V)The G.lamblia prevalence and geotypes were examined based on sequence analysis of the tpi gene.The G.lamblia prevalence was 0.3%(3/994;95% CI: 0.00-0.64).No factors were significantly correlated with the G.lamblia prevalence.Sequence and phylogenetic analysis showed that all the G.lamblia-positive samples represented assemblage B.(VI)A total of 570 fecal samples of pigs were Blastocystis-positive by amplification of the SSU r RNA gene with an overall prevalence of 57.34%(570/994;95% CI: 54.27-60.42).The region and age were significantly correlated with the Blastocystis prevalence.Sequence and phylogenetic analysis showed that all the Blastocystis positive samples represented the ST5 subtype.Conclusions: The serological detection was used to detected the P.circovirus and P.teschovirus infection in the pigs of yuxi city,Yunnan Province.The results showed that the antibody positive rate was high in the tested areas,which suggested that we should pay attention to the prevention and control of the two viruses in pigs.In addition,statistical analysis showed that age and region were significantly correlated with the infection rate of the two viruses,so we could develop corresponding control strategies for pigs of different ages and regions.Through molecular detection of the pathogens,we confirmed that the pigs in the investigated areas were infected with P.circovirus type 2 and P.teschovirus,which was the firstly found in pigs in Yunnan Province.Pigs in Yunnan Province were infected with the four diarrhea-related intestinal protozoans.Statistical analysis revealed that region and age were the main risk factors for prevalence of the these intestinal protozoans,suggesting us to take some control measures.Comparison of gene sequences suggested that all the species or genotypes were zoonotic,which suggested that we should strengthen the control of the pig farm environment to avoid human infection.Pig industry is the most important industry in the agricultural development of Yunnan province.Infection of pigs with these pathogens will cause abnormal growth,which causes significant economic losses to the animal husbandry.Moreover,due to poor breeding conditions and weak awareness of diseases,farmers are easily infected with these pathogens to endanger their health.So the present study examined the prevalence and subtypes distribution of P.circovirus,P.teschovirus,Cryptosporidium spp.,E.bieneusi,G.lamblia and Blastocystis in pigs in Yunnan province.The present study assessed risk factors of pathogen infections in pigs in Yunan province and provided foundation data for controlling and preventing these pathogens in pigs in Yunnan province.