南昌市动物园圈养动物3种肠道原虫的分子检测及基因型鉴定

Molecular Detection and Genotyping of Three Enteric Protozoa in Captive Animals from Nanchang Zoo

【目的】隐孢子虫、毕氏肠微孢子虫和芽囊原虫是3种常见的寄生于人、家养动物和野生动物等肠道的寄生原虫,主要引起人和动物的腹泻。试验旨在调查南昌市动物园部分圈养动物3种原虫的感染情况并鉴定其基因型,为制订动物园人兽共患病传播防控措施提供参考依据。【方法】从南昌市动物园采集非人灵长类、偶蹄类、奇蹄类、象类、有袋类、鸟类和食肉类等圈养动物粪便样品共42份,提取粪样总DNA;采用PCR技术扩增隐孢子虫小亚单位核糖体RNA(SSU rRNA)、毕氏肠微孢子虫核糖体RNA内转录间隔区(ITS)和芽囊原虫SSU rRNA基因序列;并对阳性产物进行测序、序列比对及系统进化分析,确定3种肠道原虫的感染情况和基因型。【结果】42份粪便样本中有17份样品原虫为阳性,总检出率40.5%(17/42);其中隐孢子虫、毕氏肠微孢子虫和芽囊原虫感染率分别为2.4%(1/42)、14.3%(6/42)和23.8%(10/42)。检出动物阳性占比分别为:非人灵长类64.7%(11/17)、偶蹄类23.5%(4/17)、奇蹄类5.9%(1/17)、有袋类5.9%(1/17);且非人灵长类动物存在毕氏肠微孢子虫和芽囊原虫混合感染。通过序列比对及系统进化分析,在矮马(奇蹄类)鉴定出1种隐孢子虫基因型(姬鼠基因型II),在长臂猿、金丝猴、博士猴、红尾长尾猴和羊驼中共鉴定到2种毕氏肠微孢子虫基因型(D,ALP1)。在松鼠猴、黑白疣猴、博士猴、黑叶猴、金丝猴、羚羊和袋鼠中共鉴定到5种芽囊原虫基因型(ST1、ST2、ST5、ST13和ST14);其中芽囊原虫(ST1、ST5)和毕氏肠微孢子虫(D、ALP1)均属人兽共患基因型。【结论】南昌市动物园圈养动物存在毕氏肠微孢子虫、芽囊原虫和隐孢子虫感染;从毕氏肠微孢子虫和芽囊原虫中鉴定出人兽共患基因型,有潜在的人兽共患传播风险。动物园应进一步加强圈养动物肠道寄生原虫的监测,做好驱虫、消毒及预防人-兽传播等预防工作。

[Objective] Cryptosporidium,Enterocytozoon bieneusi and Blastocystis sp.are three common parasites,which inhabit in the intestines of humans,domestic animals,and wildlife,primarily causing diarrhea in human and animals.This experiment aimed to investigate the epidemiological status and identify their genotypes of three parasites among captive animals in Nanchang Zoo,and provide references for the prevention and treatment of intestinal parasitic diseases in wild animals.[Method] A total of 42 fecal samples were collected from captive animals including non-human primates,ungulates,perissodactyls,elephants,marsupials,birds,and carnivores in Nanchang Zoo.Total DNA was extracted from the fecal samples,and a PCR assay was employed to amplify the small subunit ribosomal RNA(SSU rRNA) gene of Cryptosporidium,the internal transcribed spacer(ITS) region of E.bieneusi,and the SSU r RNA gene of Blastocystis sp..Positive amplicons were sequenced,aligned,and subjected to phylogenetic analysis to determine the infection status and genotypes/subtypes of these three intestinal parasites.[Result] The results indicated that 17 out of 42 fecal samples were positive by PCR,with a total infection rate of 40.5%(17/42).The infection rates of Cryptosporidium,E.bieneusi,and Blastocystis sp.were 2.4%(1/42),14.3%(6/42),and 23.8%(10/42),respectively.Among the detected animals,the positivity rates were as follows:non-human primates 64.7%(11/17),ungulates 23.5%(4/17),perissodactyls 5.9%(1/17),and marsupials 5.9%(1/17).Furthermore,mixed infections of E.bieneusi and Blastocystis sp.were observed in non-human primates.Through sequence alignment and phylogenetic analysis,one Cryptosporidium genotype(Cryptosporidium sp.apodemus genotype II) was identified in ponies,and two E.bieneusi genotypes(D,ALP1)were identified in gibbons,golden snub-nosed monkeys,rhesus monkeys,red-tailed langurs,and alpacas.Five Blastocystis sp.subtypes(ST1,ST2,ST5,ST13,and ST14) were identified in squirrel monkeys,black-and-white colobus monkeys,rhesus monkeys,black leaf monkeys,golden snub-nosed monkeys,antelopes,and kangaroos.Notably,the genotypes ST1,ST5,D,and ALP1 are known as zoonotic genotypes.[Conclusion] Captive animals in Nanchang Zoo were infected with Cryptosporidium,E.bieneusi and Blastocystis sp..The zoonotic genotypes are identified from E.bieneusi and Blastocystis sp.,which have a potential transmission risk.This underscores the need for enhanced monitoring of intestinal protozoa in captive animals,and the implementation of deworming,disinfection,and preventive measures against zoonotic transmission within the zoo setting.