河北省圈养貉子隐孢子虫流行现状调查

Prevalence of Cryptosporidium spp. in captive-bred raccoon dogs in Hebei Province

目的:确定河北省圈养貉子隐孢子虫的流行和虫种/基因亚型分布情况。方法:从河北省多地共采集389份新鲜圈养貉子粪便样品,采用基于隐孢子虫核糖体小亚基rRNA(SSU rRNA)基因的巢式PCR方法进行检测,并对获得的隐孢子虫SSU rRNA基因阳性样本进行隐孢子虫60 kDa糖蛋白(gp 60)基因的扩增,对获得的隐孢子虫SSU rRNA和gp 60基因进行测序和分析,并分别在Mega中构建基于这2个基因的进化树(最大似然法),进一步分析所获隐孢子虫的分子特性。结果:基于隐孢子虫SSU rRNA基因的巢式PCR检测显示,河北省圈养貉子中共检测出6份隐孢子虫阳性样本,隐孢子虫感染率为1.5%(6/389)。其中,6月龄以上貉子的隐孢子虫感染率为1.2%(1/85),6月龄以下貉子的隐孢子虫感染率为1.6%(5/304),二者无显著差异(χ^(2)=0.096,P=0.384)。粪便非正常的貉子隐孢子虫感染率(11.1%,4/36)显著高于粪便正常的貉子(0.6%,2/353)(χ^(2)=23.18,P=0.001)。序列及进化树分析显示,本次从河北省貉子中分离的隐孢子虫虫种均为C.canis,共鉴定出2种基因亚型,即XXa2和XXa4。结论:河北省圈养貉子中存在2种人兽共患的C.canis基因亚型XXa2和XXa4,提示貉子可能成为人隐孢子虫感染的潜在来源。

Objective:To determine the prevalence and species/genotype distribution of Cryptosporidium spp.in captive raccoon dogs in Hebei Province.Methods:A total of 389 fresh fecal samples were collected from captive raccoon dog farms in Hebei Province.Nested PCR based on the small subunit ribosomal RNA(SSU rRNA)gene of Cryptosporidium spp.was used to detect all samples.The 60 kDa glycoprotein(gp 60)gene from the positive samples of Cryptosporidium SSU rRNA gene were amplified,and the obtained SSU rRNA and gp 60 gene of Cryptosporidium spp.in this study were sequenced and analyzed.The constructed evolutionary trees based on these two genes separately in Mega(maximum-likelihood method)were used to analyze the molecular characteristics of the obtained Cryptosporidium isolates in this study.Results:A total of 6 positive samples of Cryptosporidium were detected by nested PCR in captive raccoon dogs in Hebei Province,with a Cryptosporidium infection rate of 1.5%(6/389).The infection rate of Cryptosporidium in>6-month-old raccoon dogs was 1.2%(1/85),while the infection rate of Cryptosporidium was 1.6%(5/304)in<6-month-old raccoon dogs,and no significant difference was seen(χ^(2)=0.096,P=0.384).The infection rate of Cryptosporidium in raccoon dogs with abnormal feces(11.1%,4/36)was significantly higher than that in raccoon dogs with normal feces(0.6%,2/353)(χ^(2)=23.18,P=0.001).Sequence and evolutionary tree analysis showed that all Cryptosporidium isolates in this study were identified as C.canis,and all isolates belonged to the XXa subtype family,including two known subtypes XXa2 and XXa4.Conclusion:The presence of two known zoonotic C.canis subtypes in captive raccoon dogs in Hebei Province implied that that raccoon dogs might be potential source of human Cryptosporidium infection.