云南地区猪毕氏微孢子虫的分子检测及其基因型鉴定
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摘要
【目的】从寄生虫角度明确猪场发生猪腹泻的主要病原体,为防控云南规模化猪场毕氏微孢子虫感染提供理论依据。【方法】通过巢式PCR对云南玉溪和保山地区4个猪场的129份猪粪样本进行猪毕氏微孢子虫检测,扩增微孢子虫的ITS序列,经测序分析和构建系统发育进化树确定其基因型。【结果】129份猪粪样中有30份感染毕氏微孢子虫,感染率为23.26%;其中,云南保山地区的猪毕氏微孢子虫感染率为56.82%(25/44),玉溪地区的感染率为5.88%(5/85),二者差异极显著(P<0.01,下同)。按不同发育阶段划分,发现以仔猪的毕氏微孢子虫感染率最高(76.92%),成年猪的感染率相对较低(11.39%),感染率在不同发育阶段间差异极显著。经测序分析发现共有6个基因型,包括5个已知基因型[CHC5(n=3)、CHG19(n=7)、EbpD(n=9)、EbpA(n=2)和EbpC(n=4)],1个新的基因型YNZ1(n=5);从基于微孢子虫ITS序列同源性构建的系统发育进化树可知,检测出的6种基因型均属于Group 1,即具有人兽共患的可能性。【结论】云南猪场普遍存在毕氏微孢子虫感染,且均属于具有人兽共患可能性的基因型。因此,要重点加强猪场微孢子虫的防控工作,养殖过程中产生的粪便等养殖污染物必须经过堆肥发酵或消毒处理后才能作为农家肥使用或排入污水中。
【Objective】The major pathogens of porcine diarrhea in pig farms from the perspective of parasitic diseases were identified,which provided theoretical basis for the prevention and control of Enterocytozoon bieneusi infection in pig farms of Yunnan.【Method】A total of 129 fresh fecal specimens were collected from four pig farms from Yuxi and Baoshan,Yunnan by nested PCR,then test on E. bieneusi was conducted,and ITS sequence was amplified. The genotypes of E. bieneusi was determined by sequence comparison and phylogenetic tree analysis.【Result】Results showed that30 samples were infection positive out of 129 samples,the overall infection rate of E. bieneusi was 23.26%. The infection rate of E. bieneusi in Baoshan and Yuxi were 56.82%(25/44)and 5.88%(5/85)respectively. There was extremely significant difference between Baoshan and Yuxi(P<0.01,the same below). Dividing based on growth periods,the infection rate of piglets(76.92%)was the highest,that of adult pigs(11.39%)was low.There was extremely significant different in infection rate between the growth periods.Six genotypes of E. bieneusi were found by sequencing analysis,including five known genotypes namely CHC5(n=3),CHG19(n=7),EbpD(n=9),EbpA(n=2),EbpC(n=4)and one novel genotype which was YNZ1(n=5). Phylogenetic tree base on ITS sequence similarity indicated that all of the six genotypes belonged to group 1,which was zoonosis.【Conclusion】The present survey indicates that infection of pigs with E. bieneusiis widespreads in Yunnan Province,and it contains zoonosis genotypes. Therefore,it is necessary to strengthen the prevention and control of microsporidian in pig farms,the feces and other pollutants produced in the process of farming must be fermented by compost or treated with disinfection before being used as farm manure or discharged into sewage.
【Objective】The major pathogens of porcine diarrhea in pig farms from the perspective of parasitic diseases were identified,which provided theoretical basis for the prevention and control of Enterocytozoon bieneusi infection in pig farms of Yunnan.【Method】A total of 129 fresh fecal specimens were collected from four pig farms from Yuxi and Baoshan,Yunnan by nested PCR,then test on E. bieneusi was conducted,and ITS sequence was amplified. The genotypes of E. bieneusi was determined by sequence comparison and phylogenetic tree analysis.【Result】Results showed that30 samples were infection positive out of 129 samples,the overall infection rate of E. bieneusi was 23.26%. The infection rate of E. bieneusi in Baoshan and Yuxi were 56.82%(25/44)and 5.88%(5/85)respectively. There was extremely significant difference between Baoshan and Yuxi(P<0.01,the same below). Dividing based on growth periods,the infection rate of piglets(76.92%)was the highest,that of adult pigs(11.39%)was low.There was extremely significant different in infection rate between the growth periods.Six genotypes of E. bieneusi were found by sequencing analysis,including five known genotypes namely CHC5(n=3),CHG19(n=7),EbpD(n=9),EbpA(n=2),EbpC(n=4)and one novel genotype which was YNZ1(n=5). Phylogenetic tree base on ITS sequence similarity indicated that all of the six genotypes belonged to group 1,which was zoonosis.【Conclusion】The present survey indicates that infection of pigs with E. bieneusiis widespreads in Yunnan Province,and it contains zoonosis genotypes. Therefore,it is necessary to strengthen the prevention and control of microsporidian in pig farms,the feces and other pollutants produced in the process of farming must be fermented by compost or treated with disinfection before being used as farm manure or discharged into sewage.
引文
杜海利.2013.河南省猪、羊毕氏微孢子虫病分子流行病学调查及遗传特征初步研究[D].郑州:河南农业大学.[Du H L.2013.Studies on molecular epidemiology and genetic characters of Enterocvtozoon bieneusi in pig,sheep and goat in Henan Province[D].Zhengzhou:Henan Agricultural University.]
杜帅之,潘广林,邵俊峰,田格如,王会宝,张敏,赵光辉,于三科.2016.秦岭地区几种珍稀动物毕氏肠微孢子虫基因分型[J].中国兽医学报,36(5):790-794.[Du S Z,Pan G L,Shao J F,Tian G R,Wang H B,Zhang M,Zhao GH,Yu S K.2016.Genotyping of Enterocytozoon bieneusi in some rare animals in Qinling Mountains[J].Chinese Journal of Veterinary Science,36(5):790-794.]
李威,王利勤,钟志军,刘学涵,邓磊,谢娜,弓超,余建秋,杨俊,李奇,杨平,赵波,彭广能.2016.一株金丝猴源毕氏肠微孢子虫的基因鉴定[J].中国兽医科学,46(8):986-990.[Li W,Wang L Q,Zhong Z J,Liu X H,Deng L,Xie N,Gong C,Yu J Q,Yang J,Li Q,Yang P,Zhao B,Peng G N.2016.Genetic identification of a golden monkey-derived Enterocytozoon bieneusi[J].Chinese Veterinary Science,46(8):986-990.]
刘婷丽,彭先启,宋军科,胡瑞思,王莎莎,潘广林,赵光辉.2017.陕西部分地区山羊毕氏肠微孢子虫多位点基因分型研究[J].畜牧兽医学报,48(9):1730-1736.[Liu T L,Peng X Q,Song J K,Hu R S,Wang S S,Pan G L,Zhao G H.2017.Multi-locus sequence genotyping study of Enterocytozoon bieneusi in goats form partial areas of Shaanxi Province[J].Acta Veterinaria et Zootechnica Sinica,48(9):1730-1736.]
万强.2016.中国东北地区猪两种肠道原虫遗传特征分析及猪源毕氏肠微孢子虫群体遗传学研究[D].哈尔滨:东北农业大学.[Wan Q.2016.Genetic features of enteric protozoans and population genetics study of Enterocytozoon bieneusi in pigs from Northeast China[D].Harbin:Northeast Agricultural University.]
王利勤,李威,余星明,吴孔菊,刘俊卿,余建秋,赵波,谢瑶,邓磊,刘学涵,谢娜,弓超,杨俊,李奇,杨大洪,彭广能.2017.圈养野生动物隐孢子虫、贾第虫、微孢子虫感染情况调查[J].黑龙江畜牧兽医,(4):193-195.[Wang L Q,Li W,Yu X M,Wu K J,Liu J Q,Yu J Q,Zhao B,Xie Y,Deng L,Liu X H,Xie N,Gong C,Yang J,Li Q,Yang DH,Peng G N.2017.Investigation of Cryptosporidium spp.,giardia and Enterocytozoon bieneusi in captive wild animals[J].Heilongjiang Animal Science and Veterinary Medicine,(4):193-195.]
徐海玲.2016.上海伴侣动物中隐孢子虫、贾第虫和微孢子虫的感染现状及基因型分布[D].上海:华东理工大学.[Xu H L.2016.Infection and genotype distribution of Cryptosporidium spp.,Giardia duodenalis and Enterocytozoon bieneusi in companion animals in Shanghai,China[D].Shanghai:East China University of Science and Technology.]
Deplazes P,Mathis A,Müller C,Weber R.1996.Molecular epidemiology of Encephalitozoon cuniculi and first detection of Enterocytozoon bieneusi in faecal samples of pigs[J].Journal of Eukaryotic Microbiology,43(5):93S.
Franzen C,Müller A.1999.Molecular techniques for detection,species differentiation,and phylogenetic analysis of microsporidia[J].Clinical Microbiology Reviews,12(2):243-285.
Jiang Y,Tao W,Wan Q,Li Q,Yang Y Q,Lin Y C,Zhang SW,Li W.2015.Zoonotic and potentially host-adapted Enterocytozoon bieneusi genotypes in sheep and cattle in Northeast China and an increasing concern about the zoonotic importance of previously considered ruminantadapted genotypes[J].Applied&Environmental Microbiology,81(10):3326-3335.
Karim M R,Dong H,Li T,Yu F,Li D,Zhang L,Li J,Wang R,Li S,Li X,Rume F I,Ning C.2015.Predomination and new genotypes of Enterocytozoon bieneusi in captive nonhuman primates in zoos in China:High genetic diversity and zoonotic significance[J].PLoS One,10(2):e0117991.
Li W,Diao R,Yang J,Xiao L,Lu Y,Li Y,Song M.2014a.High diversity of human-pathogenic Enterocytozoon bieneusi genotypes in swine in northeast China[J].Parasitology Research,113(3):1147-1153.
Li W,Li Y,Li W,Yang J,Song M,Diao R,Jia H,Lu Y,Zheng J,Zhang X,Xiao L.2014b.Genotypes of Enterocytozoon bieneusi in livestock in China:High prevalence and zoonotic potential[J].PLoS One,9(5):e97623.
Ma J G,Zhang N Z,Hou J L,Zou Y,Hu G X,Zhu X Q,Zhou D H.2017.Detection of Enterocytozoon bieneusi in white yaks in Gansu Province,China[J].BioMed Research International,2017:5790181.doi:10.1155/2017/5790181.
Mathis A,Weber R,Deplazes P.2005.Zoonotic potential of the microsporidia[J].Clinical Microbiology Reviews,18(3):423-445.
Santín M,Fayer R.2009.Enterocytozoon bieneusi genotype nomenclature based on the internal transcribed spacer sequence:A consensus[J].The Journal of Eukaryotic Microbiology,56(1):34-38.
Yue D M,Ma J G,Li F C,Hou J L,Zheng W B,Zhao Q,Zhang X X,Zhu X Q.2017.Occurrence of Enterocytozoon bieneusi in donkeys(Equus asinus)in China:A public health concern[J].Frontiers in Microbiology,8:565.doi:10.3389/fmicb.2017.00565.
Zhang X,Wang Z X,Su Y,Liang X Y,Sun X J,Peng S,Lu H J,Jiang N,Yin J G,Xiang M W,Chen Q J.2011.Identification and genotyping of Enterocytozoon bieneusi in China[J].Journal of Clinical Microbiology,49(5):2006-2008.
Zhang X X,Cong W,Liu G H,Ni X T,Ma J G,Zheng W B,Zhao Q,Zhu X Q.2016.Prevalence and genotypes of Enterocytozoon bieneusi in sika deer in Jilin Province,Northeastern China[J].Acta Parasitologica,61(2):382-388.
杜帅之,潘广林,邵俊峰,田格如,王会宝,张敏,赵光辉,于三科.2016.秦岭地区几种珍稀动物毕氏肠微孢子虫基因分型[J].中国兽医学报,36(5):790-794.[Du S Z,Pan G L,Shao J F,Tian G R,Wang H B,Zhang M,Zhao GH,Yu S K.2016.Genotyping of Enterocytozoon bieneusi in some rare animals in Qinling Mountains[J].Chinese Journal of Veterinary Science,36(5):790-794.]
李威,王利勤,钟志军,刘学涵,邓磊,谢娜,弓超,余建秋,杨俊,李奇,杨平,赵波,彭广能.2016.一株金丝猴源毕氏肠微孢子虫的基因鉴定[J].中国兽医科学,46(8):986-990.[Li W,Wang L Q,Zhong Z J,Liu X H,Deng L,Xie N,Gong C,Yu J Q,Yang J,Li Q,Yang P,Zhao B,Peng G N.2016.Genetic identification of a golden monkey-derived Enterocytozoon bieneusi[J].Chinese Veterinary Science,46(8):986-990.]
刘婷丽,彭先启,宋军科,胡瑞思,王莎莎,潘广林,赵光辉.2017.陕西部分地区山羊毕氏肠微孢子虫多位点基因分型研究[J].畜牧兽医学报,48(9):1730-1736.[Liu T L,Peng X Q,Song J K,Hu R S,Wang S S,Pan G L,Zhao G H.2017.Multi-locus sequence genotyping study of Enterocytozoon bieneusi in goats form partial areas of Shaanxi Province[J].Acta Veterinaria et Zootechnica Sinica,48(9):1730-1736.]
万强.2016.中国东北地区猪两种肠道原虫遗传特征分析及猪源毕氏肠微孢子虫群体遗传学研究[D].哈尔滨:东北农业大学.[Wan Q.2016.Genetic features of enteric protozoans and population genetics study of Enterocytozoon bieneusi in pigs from Northeast China[D].Harbin:Northeast Agricultural University.]
王利勤,李威,余星明,吴孔菊,刘俊卿,余建秋,赵波,谢瑶,邓磊,刘学涵,谢娜,弓超,杨俊,李奇,杨大洪,彭广能.2017.圈养野生动物隐孢子虫、贾第虫、微孢子虫感染情况调查[J].黑龙江畜牧兽医,(4):193-195.[Wang L Q,Li W,Yu X M,Wu K J,Liu J Q,Yu J Q,Zhao B,Xie Y,Deng L,Liu X H,Xie N,Gong C,Yang J,Li Q,Yang DH,Peng G N.2017.Investigation of Cryptosporidium spp.,giardia and Enterocytozoon bieneusi in captive wild animals[J].Heilongjiang Animal Science and Veterinary Medicine,(4):193-195.]
徐海玲.2016.上海伴侣动物中隐孢子虫、贾第虫和微孢子虫的感染现状及基因型分布[D].上海:华东理工大学.[Xu H L.2016.Infection and genotype distribution of Cryptosporidium spp.,Giardia duodenalis and Enterocytozoon bieneusi in companion animals in Shanghai,China[D].Shanghai:East China University of Science and Technology.]
Deplazes P,Mathis A,Müller C,Weber R.1996.Molecular epidemiology of Encephalitozoon cuniculi and first detection of Enterocytozoon bieneusi in faecal samples of pigs[J].Journal of Eukaryotic Microbiology,43(5):93S.
Franzen C,Müller A.1999.Molecular techniques for detection,species differentiation,and phylogenetic analysis of microsporidia[J].Clinical Microbiology Reviews,12(2):243-285.
Jiang Y,Tao W,Wan Q,Li Q,Yang Y Q,Lin Y C,Zhang SW,Li W.2015.Zoonotic and potentially host-adapted Enterocytozoon bieneusi genotypes in sheep and cattle in Northeast China and an increasing concern about the zoonotic importance of previously considered ruminantadapted genotypes[J].Applied&Environmental Microbiology,81(10):3326-3335.
Karim M R,Dong H,Li T,Yu F,Li D,Zhang L,Li J,Wang R,Li S,Li X,Rume F I,Ning C.2015.Predomination and new genotypes of Enterocytozoon bieneusi in captive nonhuman primates in zoos in China:High genetic diversity and zoonotic significance[J].PLoS One,10(2):e0117991.
Li W,Diao R,Yang J,Xiao L,Lu Y,Li Y,Song M.2014a.High diversity of human-pathogenic Enterocytozoon bieneusi genotypes in swine in northeast China[J].Parasitology Research,113(3):1147-1153.
Li W,Li Y,Li W,Yang J,Song M,Diao R,Jia H,Lu Y,Zheng J,Zhang X,Xiao L.2014b.Genotypes of Enterocytozoon bieneusi in livestock in China:High prevalence and zoonotic potential[J].PLoS One,9(5):e97623.
Ma J G,Zhang N Z,Hou J L,Zou Y,Hu G X,Zhu X Q,Zhou D H.2017.Detection of Enterocytozoon bieneusi in white yaks in Gansu Province,China[J].BioMed Research International,2017:5790181.doi:10.1155/2017/5790181.
Mathis A,Weber R,Deplazes P.2005.Zoonotic potential of the microsporidia[J].Clinical Microbiology Reviews,18(3):423-445.
Santín M,Fayer R.2009.Enterocytozoon bieneusi genotype nomenclature based on the internal transcribed spacer sequence:A consensus[J].The Journal of Eukaryotic Microbiology,56(1):34-38.
Yue D M,Ma J G,Li F C,Hou J L,Zheng W B,Zhao Q,Zhang X X,Zhu X Q.2017.Occurrence of Enterocytozoon bieneusi in donkeys(Equus asinus)in China:A public health concern[J].Frontiers in Microbiology,8:565.doi:10.3389/fmicb.2017.00565.
Zhang X,Wang Z X,Su Y,Liang X Y,Sun X J,Peng S,Lu H J,Jiang N,Yin J G,Xiang M W,Chen Q J.2011.Identification and genotyping of Enterocytozoon bieneusi in China[J].Journal of Clinical Microbiology,49(5):2006-2008.
Zhang X X,Cong W,Liu G H,Ni X T,Ma J G,Zheng W B,Zhao Q,Zhu X Q.2016.Prevalence and genotypes of Enterocytozoon bieneusi in sika deer in Jilin Province,Northeastern China[J].Acta Parasitologica,61(2):382-388.