内蒙古乌审旗地区牛、羊片形吸虫感染情况及分子鉴定

doi:10.12140/j.issn.1000-7423.2024.01.005

中国寄生虫学与寄生虫病杂志 ›› 2024, Vol. 42 ›› Issue (1): 36-41.doi: 10.12140/j.issn.1000-7423.2024.01.005

内蒙古乌审旗地区牛、羊片形吸虫感染情况及分子鉴定

李娜¹(), 藏达热², 乌日力格¹, 阿拉腾布日古德², 海鹰², 哈斯苏荣¹^,^*()

1 内蒙古农业大学兽医学院，农业部动物疾病临床诊疗技术重点实验室，国家级动物医学实验教学中心，呼和浩特 010018
2 乌审旗动物疫病预防控制中心，内蒙古鄂尔多斯 017300

收稿日期:2023-07-05 修回日期:2023-09-04 出版日期:2024-02-28 发布日期:2024-03-12
通讯作者: *哈斯苏荣（1965—），男，博士，教授，从事兽医药理与毒理学专业教学工作。E-mail：surong@imau.edu.cn
作者简介:李娜（1998—），女，硕士研究生，从事寄生虫研究。E-mail：2638427149@qq.com
基金资助:
鄂尔多斯市科技重大专项(2022EEDSKJZDZX026)

Fasciola spp. infection and molecular identification in cattle and sheep in Wushen Banner, Inner Mongolia

LI Na¹(), ZANG Dare², WURI Lige¹, ALATENG Burigude², HAI Ying², HASI Surong¹^,^*()

1 College of Veterinary Medicine, Inner Mongolia Agricultural University; Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Diseases, Ministry of Agriculture; National Animal Medicine Experimental Teaching Center, Hohhot 010018, China
2 Wushen Animal Disease Prevention and Control Center, Ordos 017300, Inner Mongolia, China

Received:2023-07-05 Revised:2023-09-04 Online:2024-02-28 Published:2024-03-12
Contact: *E-mail: surong@imau.edu.cn
Supported by:
Ordos Science and Technology Major Project(2022EEDSKJZDZX026)

摘要/Abstract

摘要：

目的了解内蒙古乌审旗地区牛、羊片形吸虫感染情况。方法 2022年8月至2023年3月在乌审旗嘎鲁图、苏力德苏木、乌兰陶勒盖、乌审召和图克镇采集牛、羊血样和粪样，间接ELISA检测血清抗肝片形吸虫抗体，随机抽取部分血清抗体检测样品对应的牛、羊粪样，采用虫卵沉淀法定性检查虫卵，对血清抗体检测和粪样虫卵检查结果进行一致性检验。2023年4月，在嘎鲁图、苏力德苏木和图克镇因感染片形吸虫死亡牛、羊尸首的肝脏和胆汁中采集片形吸虫成虫和虫卵，提取虫体和虫卵DNA，PCR扩增片形吸虫核糖体内转录间隔区2（ITS2）并测序，在NCBI数据库中进行BLAST比对分析，采用邻接法构建系统进化树。采用SPSS 25.0软件进行统计学分析，不同地区的阳性率的比较采用χ²检验。结果共采集825份牛、羊血样，其中295份血清肝片形吸虫抗体阳性，总阳性率为35.8%（295/825），羊、牛阳性率分别为34.7%（227/655）、40.0%（68/170）。羊血清阳性率以苏力德苏木最高[81.8%（45/55）]，乌审召镇最低[22.0%（27/123）]，不同地区血清抗体阳性率差异有统计学意义（χ² = 73.93，P < 0.05）。牛血清抗体阳性率以嘎鲁图镇最高[65.1%（28/43）]，图克镇最低[19.2%（5/26）]，不同地区血清抗体阳性率差异有统计学意义（χ² = 21.50，P < 0.05）。共抽取88份牛、羊粪样（羊粪样59份，牛粪样29份）检查，其中1份羊粪样、5份牛粪样检出肝片形吸虫虫卵，4份牛粪样检出同盘吸虫虫卵。88份牛、羊血样、粪样样品中，血清肝片形吸虫抗体的阳性率为29.5%（26/88），粪样肝片形吸虫卵的阳性率为6.8%（6/88），kappa值为0.086，两种方法的一致性为微弱。从2头牛体内收集到成虫和虫卵，从2头羊体内收集到成虫。共取5条成虫和2份虫卵提取DNA，PCR扩增共获得7条约550 bp的条带。BLAST比对分析结果显示，4条序列与来自中国的巨片形吸虫ITS2（GenBank：HQ700438）的序列一致性分别96.62%、95.91%、95.74%和92.97%，鉴定为巨片形吸虫；3条序列与来自中国的肝片形吸虫ITS2（GenBank：JF496717）的序列一致性分别为95.69%、99.80%和99.23%，鉴定为肝片形吸虫。系统进化树分析结果显示，本研究鉴定的肝片形吸虫与来自中国（GenBank： JF496717）、肯尼亚（GenBank：MZ396926）、埃及（GenBank：MW620063）的肝片形吸虫聚在同一分支上；巨片形吸虫与来自中国（GenBank：HQ700438）和印度（GenBank：OL691113）的巨片形吸虫聚在同一分支上。结论内蒙古乌审旗地区牛、羊均存在肝片形吸虫和巨片形吸虫感染，片形吸虫的感染率较高。

关键词: 片形吸虫, 肝片形吸虫, 巨片形吸虫, 血清抗体, ELISA, 分子鉴定, 牛羊, 内蒙古

Abstract:

Objective To investigate the endemic status of Fasciola spp. infection in cattle and sheep in Wushen Banner of Inner Mongolia. Methods From August 2022 to March 2023, blood and fecal samples from cattle and sheep were collected from Galutu, Sulide Sumu, Wulantaolegai, Wushenzhao and Tuke towns in Wushen Banner to detect serum Fasciola hepatica antibodies by using indirect ELISA. Cattle and sheep feces samples corresponding to some serum antibody detection samples were randomly selected, in which the parasite eggs were qualitatively examined by egg sedimentation method. The consistency test was performed for the findings from serum and fecal examination. In April 2023, Fasciola spp. adult worms and eggs were collected from the carcasses livers and bile of cattle and sheep that died of Fasciola spp. infection in Galutu, Sulidesumu and Tuke towns. The DNA of the worms and eggs was extracted to amplify ribosomal transcriptional spacer sequence 2 (ITS2) by PCR and sequenced. Sequence alignment was then performed by BLAST with NCBI database. The phylogenetic tree was constructed by the neighbour-joining method. SPSS 25.0 software was used for statistical analysis, and the chi-square test was used to compare the positive rates in different regions. Results A total of 825 serum samples were collected from cattle and sheep, in which 295 samples were positive for serological antibodies, with a total sero-positive rate of 35.8% (295/835), of which the sero-positive rates of sheep and cattle were 34.7% (227/655) and 40.0% (68/170), respectively. The sero-positive rate of sheep was the highest at 81.8% (45/55) in Sulide Sumu, and the lowest at 22.0% (27/123) in Wushenzhao. There was a significant difference in the sero-positive rate of antibodies in different regions (χ² = 73.93, P < 0.05). The sero-positive in bovine was the highest at 65.1% (28/43) in Garutu and lowest at 19.2% (5/26) in Tuke. There was a significant difference in the sero-positive rate of antibodies in different regions (χ² = 21.50, P < 0.05). A total of 88 fecal samples (59 sheep fecal samples and 29 bovine fecal samples) were detected, among which 1 sheep and 5 bovine fecal samples were positive for F. hepatica eggs, and 4 bovine fecal samples were positive for Paramphistomum eggs. Among the 88 samples, the positive rate of anti-F. hepatica antibody was 29.5% (26/88), and the positive rate of F. hepatica eggs was 6.8% (6/88). The kappa value was 0.086, indicating weak consistency between the two methods. Adult worms and eggs were collected from 2 dead cattle and adult worms from 2 dead sheep. The DNA were extracted from 5 adult worms and 2 eggs samples. A total of 7 bands of approximately 550 bp were obtained by PCR amplification for sequencing. BLAST alignment analysis showed that the identities of 4 obtained sequences were 92.97%, 96.62%, 95.74% and 95.91% with the ITS2 sequences (GenBank: HQ700438) of F. gigantica from China, respectively, and identified as F. gigantica. The identities of the other three obtained sequences were 95.69%, 99.80% and 99.23% with the ITS2 sequences (GenBank: JF496717) of F. hepatica from China, respectively, and identified as F. hepatica. The phylogenetic tree showed that the F. hepatica identified in this study clustered on the same branch with the F. hepatica from China (GenBank: JF496717), Kenya (GenBank: MZ396926) and Egypt (GenBank: MW620063). The F. gigantica identified in this study clustered in the same branch with the F. gigantica from China (GenBank: HQ700438) and India （GenBank: OL691113）. Conclusion F. hepatica and F. gigantica infections are present in both cattle and sheep in Wushenqi area of Inner Mongolia, and the infection rate of Fasciola spp. was relatively high.

Key words: Fasciola spp., Fasciola hepatica, Fasciola gigantica, Serum antibody, ELISA, Molecular identification, Cattle and sheep, Inner Mongolia

中图分类号:

R383.26

李娜, 藏达热, 乌日力格, 阿拉腾布日古德, 海鹰, 哈斯苏荣. 内蒙古乌审旗地区牛、羊片形吸虫感染情况及分子鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 36-41.

LI Na, ZANG Dare, WURI Lige, ALATENG Burigude, HAI Ying, HASI Surong. Fasciola spp. infection and molecular identification in cattle and sheep in Wushen Banner, Inner Mongolia[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(1): 36-41.

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采样点 Site of collection	羊 Sheep			牛 Bovine			合计 Total
采样点 Site of collection	血样数/份 No. blood sample	阳性数/份 No. positive	阳性率/% Positive rate/%	血样数/份 No. blood sample	阳性数/份 No. positive	阳性率/% Positive rate/%	血样数/份 No. blood sample	阳性数/份 No. positive	阳性率/% Positive rate/%
嘎鲁图 Galutu	244	82	33.6	43	28	65.1	287	110	38.3
苏力德苏木 Sulidesumu	55	45	81.8	35	17	48.6	90	62	68.9
乌兰陶勒盖 Wulantaolegai	116	38	32.8	33	9	27.3	149	47	31.5
乌审召 Wushenzhao	123	27	22.0	33	9	27.3	156	36	23.1
图克 Tuke	117	35	29.9	26	5	19.2	143	40	28.0
合计 Total	655	227	34.7	170	68	40.0	825	295	35.8

粪便虫卵检查 Fecal egg examination	血清抗体检测 Serum antibody detection		合计 Total
粪便虫卵检查 Fecal egg examination	阳性 Positive	阴性 Negative	合计 Total
阳性 Positive	3	3	6
阴性 Negative	23	59	82
合计 Total	26	62	88

内蒙古乌审旗地区牛、羊片形吸虫感染情况及分子鉴定

Fasciola spp. infection and molecular identification in cattle and sheep in Wushen Banner, Inner Mongolia

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