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

doi:10.12140/j.issn.1000-7423.2019.04.009

Abstract

Abstract:

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 M_r 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 (HQ179574), C. bovis (HQ179573) and C. bovis (MH166335), respectively. The 7 C. parvum-positive samples were further determined as IIdA15G1 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.

Key words: Cryptosporidium, Small subunit ribosomal RNA, Nested PCR, Pre-weaned dairy calves, Sichuan Province

CLC Number:

R382.3

Ding JIA, Jia-ming DAN, Yan SUN, Jing-yi LUO, Zi-qi ZHU, Xue-ping ZHANG, Hai-feng LIU, Zi-yao ZHOU, Xiao-ping MA, Guang-neng PENG, Zhi-jun ZHONG. Prevalence and molecular characteristics of Cryptosporidium infection in pre-weaned dairy calves in Sichuan Province[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(4): 422-427.

Figures/Tables 2

Table 1

Fig. 1

References 35

[1]	Xiao LH.Molecular epidemiology of cryptosporidiosis: an update[J]. Exp Parasitol, 2010, 124(1): 80-89.
[2]	Gong C, Cao XF, Deng L, et al. Epidemiology of Cryptosporidium infection in cattle in China: a review[J]. Parasite, 2017, 24: 1.
[3]	Abeywardena H, Jex AR, Gasser RB.A perspective on Cryptosporidium and Giardia, with an emphasis on bovines and recent epidemiological findings[J]. Adv Parasitol, 2015, 88: 243-301.
[4]	王雪婷. 陕西部分地区犊牛隐孢子虫和蓝氏贾第虫种群结构研究[D]. 杨凌: 西北农林科技大学, 2017.
[5]	Zahedi A, Paparini A, Jian FC, et al. Public health significance of zoonotic Cryptosporidium species in wildlife: critical insights into better drinking water management[J]. Int J Parasitol Parasites Wildl, 2016, 5(1): 88-109.
[6]	刘珍珍, 胡苏辉, 张璐, 等. 基于凝集素基因对部分隐孢子虫种类的分子鉴定[J]. 中国人兽共患病学报, 2018, 34(4): 290-295.
[7]	杨子吟, 曹建平, 沈玉娟, 等. 几种新现人体隐孢子虫种和基因型流行现状[J]. 中国人兽共患病学报, 2017, 33(2): 148-155.
[8]	Ryan U, Fayer R, Xiao LH.Cryptosporidium species in humans and animals: current understanding and research needs[J]. Parasitology, 2014, 141(13): 1667-1685.
[9]	Qi MZ, Fang YQ, Wang XT, et al. Molecular characterization of Cryptosporidium spp. in pre-weaned calves in Shaanxi Province, north-Western China[J]. J Med Microbiol, 2015, 64(Pt 1): 111-116.
[10]	齐萌, 罗楠楠, 黄建营, 等. 河南省奶牛安氏隐孢子虫感染的流行病学调查[J]. 畜牧与兽医, 2015, 47(8): 109-112.
[11]	Hu SH, Liu ZZ, Yan FB, et al. Zoonotic and host-adapted genotypes of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in dairy cattle in Hebei and Tianjin, China[J]. Vet Parasitol, 2017, 248: 68-73.
[12]	但佳明, 欧红萍, 严光文, 等. 四川部分地区断奶前犊牛贾第虫感染调查和基因分型研究[J]. 浙江农业学报, 2018, 30(7): 1116-1121.
[13]	Li JQ, Qi M, Chang YK, et al. Molecular characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in captive wildlife at Zhengzhou Zoo, China[J]. J Eukaryot Microbiol, 2015, 62(6): 833-839.
[14]	Alves M, Xiao LH, Sulaiman I, et al. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal[J]. J Clin Microbiol, 2003, 41(6): 2744-2747.
[15]	Qi M, Wang HY, Jing B, et al. Occurrence and molecular identification of Cryptosporidium spp. in dairy calves in Xinjiang, Northwestern China[J]. Vet Parasitol, 2015, 212(3/4): 404-407.
[16]	孙涛, 刘维, 王菊花, 等. 合肥地区奶牛源隐孢子虫的分离与鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(6): 447-452.
[17]	Cai M, Guo YQ, Pan BL, et al. Longitudinal monitoring of Cryptosporidium species in pre-weaned dairy calves on five farms in Shanghai, China[J]. Vet Parasitol, 2017, 241: 14-19.
[18]	Benhouda D, Hakem A, Sannella AR, et al. First molecular investigation of Cryptosporidium spp. in young calves in Algeria[J]. Parasite, 2017, 24: 15.
[19]	Al Mawly J, Grinberg A, Velathanthiri N, et al. Cross sectional study of prevalence, genetic diversity and zoonotic potential of Cryptosporidium parvum cycling in New Zealand dairy farms[J]. Parasit Vectors, 2015, 8: 240.
[20]	Avendaño C, Ramo A, Vergara-Castiblanco C, et al. Genetic uniqueness of Cryptosporidium parvum from dairy calves in Colombia[J]. Parasitol Res, 2018, 117(5): 1317-1323.
[21]	Inpankaew T, Jiyipong T, Sunanta C, et al. Prevalence and molecular characterization of bovine Cryptosporidium from dairy cows in Northern Thailand[J]. Acta Parasitol, 2017, 62(4): 772-774.
[22]	Hingole AC, Gudewar JG, Pednekar RP, et al. Prevalence and molecular characterization of Cryptosporidium species in cattle and buffalo calves in Mumbai region of India[J]. J Parasit Dis, 2017, 41(1): 131-136.
[23]	Aquino MC, Widmer G, Zucatto AS, et al. First molecular characterization of Cryptosporidium spp. infecting buffalo calves in Brazil[J]. J Eukaryot Microbiol, 2015, 62(5): 657-661.
[24]	杨佳冰, 周海宁, 米荣升, 等. 宁夏牛隐孢子虫感染情况调查研究[J]. 动物医学进展, 2018, 39(8): 121-125.
[25]	Inpankaew T, Jiyipong T, Wongpanit K, et al. Molecular detection of Cryptosporidium spp. infections in water buffaloes from northeast Thailand[J]. Trop Anim Health Prod, 2014, 46(2): 487-490.
[26]	王昆. 牛隐孢子虫分子鉴定及防治抗体研究[D]. 郑州: 河南农业大学, 2009.
[27]	魏晓瑞. 广西奶牛隐孢子虫的流行病学调查[D]. 南宁: 广西大学, 2013.
[28]	张君. 青海省牛隐孢子虫感染状况的调查[J]. 中国兽医杂志, 2007, 43(3): 36.
[29]	Feng YY, Gong XQ, Zhu KX, et al. Prevalence and genotypic identification of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in pre-weaned dairy calves in Guangdong, China[J]. Parasit Vectors, 2019, 12(1): 41.
[30]	Feng YY, Xiao LH.Molecular epidemiology of cryptosporidiosis in China[J]. Front Microbiol, 2017, 8: 1701.
[31]	Wang RJ, Zhao GH, Gong YY, et al. Advances and perspectives on the epidemiology of bovine Cryptosporidium in China in the past 30 years[J]. Front Microbiol, 2017, 8: 1823.
[32]	Huang JY, Yue DY, Qi M, et al. Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Ningxia, northwestern China[J]. BMC Vet Res, 2014, 10: 292.
[33]	Zhang XX, Tan QD, Zhou DH, et al. Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle, northwest China[J]. Parasitol Res, 2015, 114(7): 2781-2787.
[34]	Wang RJ, Wang HL, Sun YR, et al. Characteristics of Cryptosporidium transmission in preweaned dairy cattle in Henan, China[J]. J Clin Microbiol, 2011, 49(3): 1077-1082.
[35]	徐宁, 尹建海, 沈玉娟, 等. 隐孢子虫和蓝氏贾第鞭毛虫分子流行病学研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(6): 661-665, 672.

地区 Area	粪样数 No. sample	阳性数 No. positive	感染率/% Infection rate/%	隐孢子虫虫种（感染牛犊数） Cryptosporidium species (No. infected calve)
成都^a Chengdu ^a	39	3	7.7	牛隐孢子虫（2）、瑞氏隐孢子虫（1） C. bovis (2), C. ryanae (1)
洪雅^a Hongya ^a	24	1	4.2	牛隐孢子虫（1） C. bovis (1)
阿坝^a Aba ^a	20	7	35.0	微小隐孢子虫（7） C. parvum (7)
眉山^b Meishan ^b	20	0	0	0
绵阳1^a Mianyang1^a	28	10	35.7	牛隐孢子虫（7）、瑞氏隐孢子虫（3） C. bovis (7), C. ryanae (3)
绵阳2^a Mianyang2^a	30	1	3.3	牛隐孢子虫（1） C. bovis (1)
资阳^a Ziyang^a	26	8	30.8	牛隐孢子虫（8） C. bovis (8)
安岳^b Anyue^b	22	2	9.1	牛隐孢子虫（2） C. bovis (2)
邛崃^a Qionglai ^a	28	4	14.3	牛隐孢子虫（4） C. bovis (4)
青白江^b Qingbaijiang ^b	20	2	10.0	牛隐孢子虫（2） C. bovis (2)
德阳^b Deyang ^b	21	2	9.5	牛隐孢子虫（1）、瑞氏隐孢子虫（1） C. bovis (1),C. ryanae (1)
总计 Total	278	40	14.4	牛隐孢子虫（28）、瑞氏隐孢子虫（5）、微小隐孢子虫（7） C. bovis (28), C. ryanae (5), C. parvum (7)