| Enterocytozoon bieneusi,Blastocystis,Cryptosporidium and Giardia duodenum are four important zoonotic intestinal protozoa that can infect humans,wildlife,companion animals,birds and a variety of domestic animals.Infection with humans often causes diarrhea of varying degrees,especially in children or immunocompromised patients.Pig is an important host of these intestinal protozoan species,which can infect a variety of zoonotic genotypes,and is a potential source of infection for the intestinal protozoa species to infect humans.In order to examine the infection of the four intestinal protozoa and the zoonosis risk on pigs in Fujian,a total of 725 feces samples were collected from 6 pig farms in Fujian Province from April to October 2018 to investigate the epidemiological characteristics of four intestinal protozoa.Four species of intestinal protozoa were detected by conventional PCR/nested PCR based on different gene loci.By PCR amplification,electrophoresis,sequencing,sequence splicing and phylogenetic analysis,the main results obtained were as following:1.Based on the nested PCR amplification of the internal transcriptional spacer region of the E.bieneusi,the infection rate of E.bieneusi was 24.4%(177/725).Among which,the suckling pigs had the highest overall infection rate(36%),and the boar had the lowest infection rate(7.1%).According to the statistical analysis,the infection rate of E.bieneusi in different population groups was significantly different(P<0.05).The infection rate was highest in Nanping(43.9%)and lowest in Zhangzhou(8.6%)and the infection rate of E.bieneusi was significantly different in different areas according to the statistical analysis(P<0.05).A total of 11 known genotypes(EbpC,EbpA,CHN-RR2,KIN-1,CHG7,CHS5,CM11,CHG23,G,PigEBITS,D)and 2 new genotypes(FJF and FJS)were identified,in which EbpA and EbpC were dominant genotypes and also zoonotic genotypes,accounting for 38.8%(78/201)and 32.8%(66/201),respectively.All 11 known genotypes and 2 new genotypes belonged to Group1 with potential zoonosis.The multilocus sequence typing(MLST)method was used to analyze the polymorphism and population genetic structure of E.bieneusi positive samples by selecting gene loci MS1,MS3,MS4 and MS7.The results showed that 52 positive isolates of 177 positive samples were successfully amplified at four loci simultaneously,resulting in 48 distinct multilocus genotypes(MLGs).Strong genetic linkage disequilibria(LD)and limited genetic recombination indicated the presence of overall clonal population structure of E.bieneusi.Combined with the comparative analysis of the genetic data of pigs in northeastern China,it was found that there was no obvious geographical isolation between the E.bieneusi of pigs in different areas.2.Based on the conventional PCR amplification of the Small Subunit ribosome RNA(SSU rRNA)of the Blastocystis,the infection rate of Blastocystis was 36%(201/725).Among which,the boar had the highest infection rate(67.9%),and the nursery pigs had the lowest infection rate(12.5%)and there were significant differences in Blastocystis infection rate among pigs of different population groups using the statistical analysis(P<0.05).The infection rate was the highest(75.3%)in Zhangzhou,and the lowest(32.2%)in Fuqing.The infection rate of Blastocystis was significantly different in different areas using the statistical analysis(P<0.05).Two common zoonotic genotypes,ST1 and ST5,were identified used phylogenetic analysis.Among which,ST5(97.9%,319/326)was the dominant subtype,widely distributed in different regions and different population groups of pigs.ST1 was detected only in suckling piglets(6.9%,2/29),weaning piglets(2.8%,1/36)and nursery pigs(23.1%,3/13),accounting for 2.1%(7/326)in all positive samples.3.Based on the nested PCR amplification of the SSU rRNA of the Cryptosporidium,the infection rate of Cryptosporidium was 7.6%(55/725),and the infection rate was in the range from 0 to 12.4% in 6 regions,and the highest infection rate was found in Fuqing region(12.4%).However,no Cryptosporidium infection was detected in Zhangzhou region,and the infection rate of Cryptosporidium in different areas was significantly different using the statistical analysis(P<0.05).Among pigs of different types,the highest infection rate of Cryptosporidium was in fattening pigs(24.6%),but no Cryptosporidium infection was detected in boar.The infection rate of Cryptosporidium at different population groups was different and showed significant difference using the statistical analysis(P<0.05).Two zoonotic Cryptosporidium species were identified: C.scrofarum(72.7%,40/55)and C.suis(27.3%,15/55).There was an age-associated distribution of C.scrofarum and C.suis,with the infection rates of C.scrofarum and C.suis in weaned pigs were close to each other,while infected C.scrofarum was more commom than infected C.suis after the stage of nursery pigs.4.Based on the nested PCR amplification of the ?-giardin(BG),glutamate dehydrogenase(GDH)and triose-phosphateisomerase(TPI)of the Giardia duodenum,the total infection rate of G.duodenum was 26.9%(195/725)based on BG gene.Sanming region had the highest infection rate,which was 49.2%,and Putian region had the lowest infection rate,which was 7.5%.and the infection rate of G.duodenum in different areas was significantly different by the statistical analysis(P<0.05).The suckling piglets had the highest infection rate(35.1%)and the fattening pigs had the lowest infection rate(10.5%).The infection rate of G.duodenum at different population groups was different and showed significant difference by the statistical analysis(P<0.05).One zoonotic assemblage type was identified based on BG gene: assemblages E.Multilocus analysis of 195 BG gene positive samples based on TPI and GDH genes found that 11 and 6 positive samples were detected respectively,and the sequence analysis were all of assemblages E,and no mixed infection was found.A total of 6 samples were amplified at three gene loci simultaneously to form only one MLG.In summary,this study was investigated to assess the molecular epidemiological characteristics and zoonotic risk of four intestinal protozoa in Fujian province using molecular biology methods.The results provide a reference for the spread of four intestinal protozoa in pigs and humans,and provide important scientific data for the prevention and control of four intestinal protozoa.Research results indicate that comprehensive measures are needed to control the infection of four intestinal protozoa in the swine population and to take effective measures to prevent their spread to humans. |
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