宁夏银川地区奶牛毕氏肠微孢子虫的感染情况与基因型鉴定

doi:10.12140/j.issn.1000-7423.2023.04.013

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (4): 476-479.doi: 10.12140/j.issn.1000-7423.2023.04.013

宁夏银川地区奶牛毕氏肠微孢子虫的感染情况与基因型鉴定

黎玉琼¹(), 于有利¹^,^*(), 郜军荣², 刘芸芸³, 李红兵³, 牛晓昊¹

1 宁夏农林科学院动物科学研究所，银川 750002
2 固原市原州区畜牧技术推广服务中心，宁夏固原 756001
3 宁夏农垦贺兰山奶业有限公司，石嘴山 753600

收稿日期:2022-11-22 修回日期:2023-03-13 出版日期:2023-08-30 发布日期:2023-09-06
通讯作者: *于有利（1991-），女，博士，副研究员，从事犊牛腹泻相关病原致病机制研究。E-mail：yyl06010323@163.com
作者简介:黎玉琼（1973-），女，本科，副研究员，从事寄生虫病防控研究。E-mail：1463805069@qq.com
基金资助:
宁夏回族自治区农业科技自主创新基金(NGSB-2021-12-06);宁夏回族自治区重点研发计划(2022BBF02019)

Enterocytozoon bieneusi infection in dairy cows and its genotype identification in Yinchuan area of Ningxia Province

LI Yuqiong¹(), YU Youli¹^,^*(), GAO Junrong², LIU Yunyun³, LI Hongbing³, NIU Xiaohao¹

1 Institute of Animal Science, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750002, China
2 Guyuan Yuanzhou District Animal Husbandry Technology Promotion Service Center, Guyuan 756001, Ningxia, China
3 Ningxia Agricultural Reclamation Helen Mountain Dairy Co. LTD, Shizuishan 753600, China

Received:2022-11-22 Revised:2023-03-13 Online:2023-08-30 Published:2023-09-06
Contact: *E-mail: yyl06010323@163.com
Supported by:
Ningxia Hui Autonomous Region Agricultural Technology Independent Innovation Fund(NGSB-2021-12-06);Key Research and Development Plan of Ningxia Hui Autonomous Region(2022BBF02019)

摘要/Abstract

摘要：

目的明确宁夏银川地区奶牛毕氏肠微孢子虫的感染情况及其主要基因型。方法 2021年8月—2022年8月，在银川市及周边县6个规模化奶牛场采集奶牛新鲜粪样（包括腹泻粪样和非腹泻粪样），提取粪样基因组DNA，用毕氏肠微孢子虫内部转录间隔区（ITS）引物进行巢式PCR，回收PCR产物后送测序并进行BLAST比对，鉴定毕氏肠微孢子虫基因型。采用SPSS 25.0软件进行分析，不同月龄奶牛毕氏肠微孢子虫阳性率差异的比较采用χ²检验。结果共采集奶牛粪样772份，巢式PCR扩增结果显示，奶牛毕氏肠微孢子虫阳性率为8.7％（67/772），目的条带大小约为400 bp。不同牧场的奶牛毕氏肠微孢子虫阳性率为4.8%（6/124）~11.5%（18/156），差异有统计学意义（χ² = 30.828，P < 0.05）。5~7月龄犊牛的阳性率最高（15.6%，23/147），12月龄以上育成牛的最低（3.4%，5/149），不同月龄的奶牛阳性率差异有统计学意义（χ² = 59.529，P < 0.05）。腹泻粪样中毕氏肠微孢子虫的阳性率为10.6%（42/398），高于非腹泻粪样（6.7%，25/374）（χ² = 3.419，P < 0.05）。测序共检出66条毕氏肠微孢子虫序列，分为CHN4、BEB4、J、I型等4种基因型，提交GenBank获得的登录号分别为OR352930、OR352929、OR352932和OR352931。其中CHN4型3条（占4.5%），与G1亚群CHN4基因型（HM992511）的序列一致性为98.4%；BEB4型18条（占27.3%），与G2亚群BEB4基因型（AY331008）的序列一致性为96.3%；J型22条（占33.3%），与G2亚群J基因型（AF135837）的序列一致性为93.1%；I型23条（占34.9%），与G2亚群I基因型（AF135836）的序列一致性为91.1%。不同月龄奶牛感染的毕氏肠微孢子虫的优势基因型不同，BEB4型、J型和I型毕氏肠微孢子虫在奶牛的各个月龄段均存在，3种基因型的总和分别占阳性腹泻粪样和阳性非腹泻粪样的88.1%（37/42）和60.0%（15/25），二者差异有统计学意义（χ² = 3.419，P < 0.05）。结论宁夏银川地区奶牛存在毕氏肠微孢子虫感染，主要基因型为BEB4、J和I基因型。

关键词: 毕氏肠微孢子虫, 感染, 内部转录间隔区, 巢式PCR, 银川

Abstract:

Objective To clarify the prevalence and main genotypes of Enterocytozoon bieneusi infection in dairy cows in Yinchuan area of Ningxia Province. Methods From August 2021 to August 2022, cow fresh fecal samples (including diarrheal and non-diarrheal samples) were collected from 6 scaled dairy farms in Yinchuan City and the surrounding counties. The genomic DNA was extracted from the fecal samples, and nested PCR was performed using E. bieneusi internal transcriptional spacer (ITS) primers. The PCR amplicaons were seqeuced, and sequence alignment was conducted using BLAST to identify the genotype of E. bieneusi. SPSS 25.0 software was used for statistical analysis, and χ² test for comparing the differences in positive rates between the cows of different months of age. Results A total of 772 fresh cow fecal samples were collected. The E. bieneusi positive rate from nested PCR amplification was 8.7% (67/772). The target band size was approximately 400 bp. The E. bieneusi positive rate found in different farms ranged from 4.8% (6/124) to 11.5% (18/156), with statistically significant differences (χ² = 30.828, P < 0.05). The highest positive rate was found in cows aged 5-7 months (15.6%, 23/147) and the lowest was found in cows aged above 12 months (3.4%, 5/149). There was a statistically significant difference in the positive rate of E. bieneusi among cows of different months of age (χ² = 59.529, P < 0.05). The E. bieneusi positive rate in diarrheal samples was 10.6% (42/398), which was higher than that in non-diarrheal samples (6.7%, 25/374) (χ² = 3.419, P < 0.05). The 66 E. bieneusi sequences could be classified into 4 genotypes, including CHN4, BEB4, J and I. The accession obtained by GenBank were OR352930, OR352929, OR352932 and OR352931 respectively. 3 sequences were CHN4 (account for 4.5%), which showed a 98.4% identity to the sequence of CHN4 (HM992511) in G1. 18 sequences were BEB4 (account for 27.3%), which showed a 96.3% identity to the sequence of BEB4 (AY331008) in G2. 22 sequences were J (account for 33.3%), which showed a 93.1% identity to the sequence of J (AF135837) in G2. 23 sequences were I (account for 34.9%), which showed a 91.1% identity to the sequence of I (AF135836) in G2. The dominant E. bieneusi genotypes in cows varied with age, but BEB4, J and I presented in all age groups. The BEB4, J and I accounted for 88.1% (37/42) and 60.0% (15/25) of the total positive diarrheal samples and positive normal samples respectively, with statistically significant differences (χ² = 3.419, P < 0.05). Conclusion E. bieneusi infection was found in dariy cows in Yinchuan area of Ningxia Province, with the main genotypes BEB4, J and I.

Key words: Enterocytozoon bieneusi, Infection, Internal transcribed spacer, Nested PCR, Yinchuan

中图分类号:

R382.3

黎玉琼, 于有利, 郜军荣, 刘芸芸, 李红兵, 牛晓昊. 宁夏银川地区奶牛毕氏肠微孢子虫的感染情况与基因型鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 476-479.

LI Yuqiong, YU Youli, GAO Junrong, LIU Yunyun, LI Hongbing, NIU Xiaohao. Enterocytozoon bieneusi infection in dairy cows and its genotype identification in Yinchuan area of Ningxia Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(4): 476-479.

图/表 2

参考文献 15

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特征 Feature	粪样份数 No. sample	阳性份数 No. positive	阳性率/% Positive rate/%	不同基因型份数 No. different genotype
特征 Feature	粪样份数 No. sample	阳性份数 No. positive	阳性率/% Positive rate/%	BEB4	J	I	CHN4
粪样来源 Sample source
牧场1 Field 1	109	12	11.0	4	3	3	2
牧场2 Field 2	143	10	07.0	3	5	2	0
牧场3 Field 3	128	9	07.0	2	4	3	0
牧场4 Field 4	156	18	11.5	5	4	7	1
牧场5 Field 5	112	12	10.7	1	5	6	0
牧场6 Field 6	124	6	04.8	3	1	2	0
月龄 Month age
0~2月龄 0-2 months old	254	14	05.5	6	4	4	0
3~4月龄 3-4 months old	113	17	15.0	4	5	7	1
5~7月龄 5-7 months old	147	23	15.6	6	12	3	2
8~12月龄 8-12 months old	109	9	08.3	1	2	6	0
12月龄以上 Over 12 months old	149	5	03.4	1	1	3	0
粪便性状 Fecal property
腹泻粪样 Diarrheal sample	398	42	10.6	14	15	18	2
非腹泻粪样 Non-diarrheal sample	374	25	06.7	4	7	5	1
合计 Total	772	67	08.7	18	22	23	3

宁夏银川地区奶牛毕氏肠微孢子虫的感染情况与基因型鉴定

Enterocytozoon bieneusi infection in dairy cows and its genotype identification in Yinchuan area of Ningxia Province

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