四川省奶牛犊牛隐孢子虫流行情况及其分子特性分析

目的了解四川部分地区断奶前奶牛犊隐孢子虫感染情况及其分子特征。方法2016年6月至2017年3月在四川省10个地区的11个奶牛场(规模化养殖7个,散养4个)采集1月龄内断奶前牛犊的新鲜粪样。采用改良饱和蔗糖溶液漂浮法处理粪样,提取粪样DNA,巢式PCR扩增隐抱子虫小亚基核糖体核苷酸(SSU rRNA)基因,以扩增阳性数计算隐孢子虫感染率,分析不同养殖模式感染率差异。感染率间的比较采用卡方检验。SSU rRNA基因测序后提交GenBank中进行BLAST比对,鉴定扩增序列所属的虫种,采用MEGA7邻接法构建基于SSUrRNA基因的系统发育树。对SSUrRNA序列比对确定为微小隐孢子虫的样品,扩增相对分子质量(Mr)为60000的糖蛋白基因(gp60),鉴定基因亚型。结果共调查牛犊278头,采集粪样278份。SSU rRNA基因检测隐抱子虫阳性40份,总感染率为14.4%。11个养殖场中10个存在隐抱子虫感染,其中感染率最高为绵阳1场,为35.7%(10/28),其次为阿坝(35%,7/20)和资阳(30.8%,8/26),最低为眉山(0)。10个检测阳性的养殖场牛犊的感染率差异有统计学意义(P<0.05)。规模化和散养养殖场的牛犊感染率分别为17.4%(34/195)和7.2%(6/83)(P<0.05)。隐抱子虫感染阳性的共40头牛犊中,感染牛隐抱子虫、微小隐抱子虫、瑞氏隐抱子虫分别为28、7、5头。40条隐孢子虫序列剔除相同的序列后共获得10条序列(ABC881、AYC6953,AYC6969,CDC16111,CDC16117,DYC014,MYC116、MYC117、MYC126、ZYC6874,GenBank登录号:MF671870至MF671879)。序列比对结果显示,AYC6953,AYC6969.CDC16117,ZYC6874序列与牛隐抱子虫(JX515546)—致性均为99%;DYC014序列与瑞氏隐孢子虫(HQ179574)一致性为99%;MYC126与牛隐抱子虫(JX416366)—致性为99%;ABC881与微小隐抱子虫(AH006572),CDC16111与瑞氏隐孢子虫(HQ179574),MYC116与牛隐抱子虫(HQ179573),MYC117与牛隐抱子虫(MH166335)的一致性均为100%,在系统进化树上显示为同一分支。7份鉴定为微小隐孢子虫样品的gp60基因亚型分析结果均为DdA15G1,与已报道的宁夏分离株(KM067092)—致性为100%结论四川省部分地区断奶前牛犊存在隐孢子虫感染,感染虫种为牛隐孢子虫、微小隐孢子虫、瑞氏隐孢子虫。

Prevalence and molecular characteristics of Cryptosporidium infection in pre-weaned dairy calves in Sichuan Province

Objective To understand the infection and molecular characteristics of Cryptosporidium spp. in pre-weaned dairy calves in Sichuan Province. Methods The fecal samples were collected from 278 pre-weaned dairy calves with age less than one month in 11 dairy farms (7 in-house farming and 4 free-range farming) located in 10 different regions of Sichuan Province from June 2016 to March 2017. The parasites in the collected fecal samples were concentrated using a modified floatation method with saturated sucrose solution and the fecal parasite DNA was extracted. The small subunit ribosomal RNA (SSU rRNA) of Cryptosporidium was amplified from the fecal DNAs by nested-PCR. The PCR positive fecal samples were used to calculate Cryptosporidium infection rate in these examined caves. Chi-square test was used to compare the difference of infection rates between in-house farming and free-range farming calves. The amplified SSU rRNA PCR products were sequenced and the obtained sequences were BLAST searched against Cryptosporidium sequences deposited in GenBank to identify the infected species. The phylogenetic tree was constructed based on the SSU rRNA sequences by MEGA 7 adjacency method. The Mr 60 000 glycoprotein (gp60) gene was further amplified from the samples identified as C. parvum by SSU rRNA sequencing to further determine their genotypes. Results A total of 278 calves were investigated and the fecal samples were collected from each of them. The SSU rRNA amplification results identified the total infection rate of Cryptosporidium was 14.4%(40/278) in the examined calves. Cryptosporidium infection was found in 10 of the 11 examined farms, among which the highest infection rate was 35.7%(10/28) in Mianyang followed by Aba (35%, 7/20) and Ziyang (30.8%, 8/26). No Cryptosporidium infection was found in Meishan. The infection rate was statistically different among the 10 positive farms (P < 0.05). The Cryptosporidium infection rate in calves in in-house farms was 17.4%(34/195) which is significantly higher than that in free-range farms (7.2%, 6/83)(P < 0.05). Among the 40 Cryptosporidium-positive samples, 28 were identified as C. bovis, 7 as C. parvum and 5 as C. ryanae. After removing the identical sequences, total 10 unique sequences were obtained (ABC881, AYC6953, AYC6969, CDC16111, CDC16117, DYC014, MYC116, MYC117, MYC126, ZYC6874, GenBank login number: MF671870- MF671879). Among them AYC6953, AYC6969, CDC16117 and ZYC6874 were 99% identical to C. bovis (JX515546), DYC014 is 99% identical to C. ryanae (HQ179574), MYC126 is 99% identical to C. bovis (JX416366). ABC881, CDC16111, MYC116 and MYC117 is 100% identical to C. parvum (AH006572), C. ryanae (HQ 179574), C. bovis (HQ179573) and C. bovis (MH166335), respectively. The 7 C. parvum-positive samples were further determined as IIdA15Gl subtype which is 100% identical to the Ningxia isolate of KM067092, based on gp60 gene sequencing results. Conclusion The Cryptosporidium infection was prevalent in pre-weaned dairy calves in Sichuan Province and the major infected species were C. bovis, C. parvum. and C. ryanae.