云南省大理白族自治州家畜片形吸虫感染情况调查及分子鉴定

doi:10.12140/j.issn.1000-7423.2024.06.004

中国寄生虫学与寄生虫病杂志 ›› 2024, Vol. 42 ›› Issue (6): 715-720.doi: 10.12140/j.issn.1000-7423.2024.06.004

云南省大理白族自治州家畜片形吸虫感染情况调查及分子鉴定

李诗韵¹(), 李慈怀², 陈凤¹^,^*(), 郝明明¹, 李新娣¹, 赵银娇¹, 张冬琦¹, 张全富¹, 李芹翠¹, 张剑平¹, 陈绍荣¹, 罗天鹏¹, 刘榆华¹

1 大理白族自治州血吸虫病防治研究所，云南大理 671000
2 大理大学公共卫生学院，云南大理 671000

收稿日期:2024-06-18 修回日期:2024-08-16 出版日期:2024-12-30 发布日期:2025-01-14
通讯作者: *陈凤（1978—），女，本科，主任医师，从事血吸虫病与寄生虫病防治工作。E-mail：yndlcf@126.com
作者简介:李诗韵（1996—），女，硕士，医师，从事血吸虫病与寄生虫病防治工作。E-mail：yndlxflsy@126.com
基金资助:
国家寄生虫资源库资助(NPRC-2019-194-30)

Prevalence of Fasciola infections and molecular identification of Fasciola in livestock in Dali Bai Autonomous Prefecture, Yunnan Province

LI Shiyun¹(), LI Cihuai², CHEN Feng¹^,^*(), HAO Mingming¹, LI Xindi¹, ZHAO Yinjiao¹, ZHANG Dongqi¹, ZHANG Quanfu¹, LI Qincui¹, ZHANG Jianping¹, CHEN Shaorong¹, LUO Tianpeng¹, LIU Yuhua¹

1 Institute of Schistosomiasis Control in Dali Bai Autonomous Prefecture, Dali 671000, Yunnan Province, China
2 School of Public Health, Dali University, Dali 671000, Yunnan Province, China

Received:2024-06-18 Revised:2024-08-16 Online:2024-12-30 Published:2025-01-14
Contact: *E-mail: yndlcf@126.com
Supported by:
National Parasitic Resources Center, and the Ministry of Science and Technology fund(NPRC-2019-194-30)

摘要/Abstract

摘要：

目的了解云南省大理白族自治州（以下简称大理州）家畜片形吸虫感染情况并鉴定片形吸虫种类。方法 2023年8月至2024年3月期间在大理州12个县（市）采集牛羊等家畜的粪样检查片形吸虫虫卵，每个县选取3份阳性样本的虫卵，利用显微镜成像系统拍照、测量后提取基因组DNA，PCR扩增核糖体内转录间隔区（ITS）序列，鉴定片形吸虫种类。测序序列在NCBI 中进行BLAST比对分析，用MEGA11.0软件以邻接法构建系统进化树。使用SPSS 19.0软件进行统计分析，片形吸虫阳性率的比较采用χ²检验。结果共收集家畜粪样1 302份，其中牛粪样1 228份（占94.3%）、羊粪样45份（占3.5%）、马粪样18份（占1.4%）、猪粪样11份（占0.8%）。检出疑似片形吸虫虫卵阳性粪样212份，阳性率为16.3%（212/1 302）。牛、羊粪样的阳性率分别为17.2%（211/1 228）、2.2%（1/45），未检出马和猪的阳性粪样。家畜粪样阳性率由高到低依次为巍山县（45.0%，49/109）、洱源县（42.7%，44/103）、云龙县（28.2%，29/103）、弥渡县（25.4%，30/118）、宾川县（18.0%，18/100）、鹤庆县（16.5%，17/103）、祥云县（8.7%，9/103）、剑川县（6.9%，8/116）、南涧县（4.7%，5/107）、漾濞县（2.2%，3/137），大理市和永平县未检出阳性粪样。不同地区家畜片形吸虫阳性率差异有统计学意义（χ² = 222.975，P < 0.05）；雌性家畜阳性率（19.0%，165/873）高于雄性（11.0%，47/429）（χ² = 13.319，P < 0.05）。大理州巨片形吸虫的虫卵大小为（182.30 ± 4.35）μm ×（97.80 ± 5.11）μm，肝片形吸虫的虫卵大小为（149.49 ± 5.90）μm ×（80.46 ± 4.37）μm。共扩增获得7条ITS序列，2条为肝片形吸虫，5条为巨片形吸虫。这7条序列均已上传至国家微生物科学数据中心数据库，获得的登录号为：宾川县A1（NMDCN0005QTQ）、巍山县A32（NMDCN0005TN）、剑川县A70（NMDCN0005QTO）、漾濞县A19（NMDCN0005ELF）、鹤庆县A7（NMDCN0005QTR）、弥渡县A35（NMDCN0005ELH）和祥云县A18（NMDCN0005QTP）。其中4条序列（宾川县A1、巍山县A32、剑川县A70、鹤庆县A7）分别与巨片形吸虫序列（GenBank：JF496711、OQ064778、MN608171、MN97009）同源性为100%，1条序列（祥云县A18）与巨片形吸虫的序列（GenBank：MN97009）同源性为93%，2条序列（漾濞县A19、巍山县A35）与肝片形吸虫的序列（GenBank：JF432071、JF496716）同源性为100%。系统进化树分析结果显示，漾濞县A19与来自澳大利亚（GenBank：MN970007）、伊朗（GenBank：JF432071），弥渡县A35与中国大理（GenBank：JF496716）的肝片形吸虫聚在同一分支上，亲缘关系较近；其余序列与来自中国德宏（GenBank：JF496711）、孟加拉国（GenBank：OQ064778）、尼日利亚（GenBank：MN608171）和越南（GenBank：MN970009、MN970008）的巨片形吸虫聚在同一分支上，亲缘关系较近。结论云南省大理白族自治州家畜片形吸虫阳性率高，感染的片形吸虫种类以巨片形吸虫为主。

关键词: 片形吸虫, 分子鉴定, 家畜, 大理

Abstract:

Objective To investigate the prevalence of Fasciola infections and to characterize the Fasciola species in livestock in Dali Bai Autonomous Prefecture. Methods Cattle, sheep, horse and pig stool samples were collected from 12 counties (city) in Dali Bai Autonomous Prefecture during the period from August 2023 to March 2024 for detection of Fasciola eggs. Parasite eggs were sampled from three Fasciola-infected stool specimens in each county, and photographed and measured with a microscopic imaging system. Then, genomic DNA was extracted from Fasciola eggs, and the ribosomal internal transcribed spacer (ITS) gene was amplified using a PCR assay for characterization of Fasciola species. The sequence of the ITS gene was aligned using the BLAST program in NCBI, and a neighbor-joining phylogenetic tree of Fasciola spp. was built based on the ITS gene sequence using the software MEGA 11.0. All statistical analyses were performed using the software SPSS 19.0, and differences in the prevalence of Fasciola infections were compared with chi-square test. Results A total of 1 302 livestock stool samples were collected, including 1 228 cattle stool samples (94.3%), 45 sheep stool samples (3.5%), 18 horse stool samples (1.4%), and 11 pig stool samples (0.8%), and there were 212 stool samples detected with suspected Fasciola eggs (16.3%). The detection of Fasciola eggs was 17.2% in cattle stool samples (211/1 228) and 2.2% in sheep stool samples (1/45), and no Fasciola eggs were detected in horse or pig stool samples. The highest detection of Fasciola eggs was seen in livestock stool samples from Weishan County (45.0%, 49/109), followed by from Eryuan County (42.7%, 44/103), Yunlong County (28.2%, 29/103), Midu County (25.4%, 30/118), Binchuan County (18.0%, 18/100), Heqing County (16.5%, 17/103), Xiangyun County (8.7%, 9/103), Jianchuan County (6.9%, 8/116), Nanjian County (4.7%, 5/107), Yangbi County (2.2%, 3/137), and no Fasciola eggs were detected in livestock stool samples from Dali City or Yongping County. There was a region-specific prevalence of Fasciola infections in livestock in Dali Bai Autonomous Prefecture (χ² = 222.975, P < 0.05), and the prevalence of Fasciola infections was higher in female livestock (19.0%, 165/873) than in males (11.0%, 47/429) (χ² = 13.319, P < 0.05). The eggs of F. gigantica and F. hepatica measured (182.30 ± 4.35) μm × (97.80 ± 5.11) μm and (149.49 ± 5.90) μm × (80.46 ± 4.37) μm, respectively. A total of 7 ITS gene sequences were obtained, including 2 from F. hepatica and 5 from F. gigantica, and these 7 sequences had been uploaded to the National Microbiology Data Center (NMDC), with NMDC numbers of NMDCN0005QTQ (sequence number: Binchuan County A1), NMDCN0005TN (sequence number: Weishan County A32), NMDCN0005QTO (sequence number: Jianchuan County A70), NMDCN0005ELF (sequence number: Yangbi County A19), NMDCN0005QTR (sequence number: Heqing County A7), NMDCN0005ELH (sequence number: Midu County A35) and NMDCN0005QTP (sequence number: Xiangyun County A18). Four sequences numbered Binchuan County A1, Weishan County A32, Jianchuan County A70 and Heqing County A7 had 100% sequence homology to those from F. gigantica (GenBank accession numbers: JF496711, OQ064778, MN608171 and MN97009), and the sequence numbered Xiangyun County A18 had 93% sequence homology to that from F. gigantica (GenBank accession number: MN97009), while two sequences numbered Yangbi County A19 and Weishan County A35 had 100% sequence homology to those from F. hepatica (GenBank accession number: JF432071 and JF496716). Phylogenetic analysis showed that the sequence numbered Yangbi A19 and sequences of F. hepatica from Australia (GenBank accession number: MN970007) and Iran (GenBank accession number: JF432071) were clustered into the same clade, and the sequence numbered Midu A35 and that of F. hepatica from Dali, China (GenBank accession number: JF496716) was clustered into the same clade, indicating a close genetic relationship. In addition, the remaining five sequences and those of F. gigantica from Dehong, China (GenBank accession number: JF496711), Bangladesh (GenBank accession number: OQ064778), Nigeria (GenBank accession number: MN608171), and Vietnam (GenBank accession numbers: MN970009 and MN970008) were clustered into the same clade, indicating a close genetic relationship. Conclusions The prevalence of Fasciola infections is high in livestock Dali Bai Autonomous Prefecture, Yunnan Province, and F. gigantica is the predominant species of Fasciola.

Key words: Fasciola spp., Molecular identification, Livestock, Dali

中图分类号:

R383.26

李诗韵, 李慈怀, 陈凤, 郝明明, 李新娣, 赵银娇, 张冬琦, 张全富, 李芹翠, 张剑平, 陈绍荣, 罗天鹏, 刘榆华. 云南省大理白族自治州家畜片形吸虫感染情况调查及分子鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(6): 715-720.

LI Shiyun, LI Cihuai, CHEN Feng, HAO Mingming, LI Xindi, ZHAO Yinjiao, ZHANG Dongqi, ZHANG Quanfu, LI Qincui, ZHANG Jianping, CHEN Shaorong, LUO Tianpeng, LIU Yuhua. Prevalence of Fasciola infections and molecular identification of Fasciola in livestock in Dali Bai Autonomous Prefecture, Yunnan Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(6): 715-720.

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参考文献 21

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云南省大理白族自治州家畜片形吸虫感染情况调查及分子鉴定

Prevalence of Fasciola infections and molecular identification of Fasciola in livestock in Dali Bai Autonomous Prefecture, Yunnan Province

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特征 Characteristic	牛 Cattle			合计^aTotal^a
特征 Characteristic	检查数/份 No. investigated	阳性数/份 No. positive	阳性率/% Positive rate/%	检查数/份 No. investigated	阳性数/份 No. positive	阳性率/% Positive rate/%	χ² χ² value	P值 P value
地区 Area							222.975	< 0.05
巍山县 Weishan county	109	49	45	109	49	45
洱源县 Eryuan county	103	44	42.7	103	44	42.7
云龙县 Yunlong county	103	29	28.2	103	29	28.2
弥渡县 Midu county	118	30	25.4	118	30	25.4
宾川县 Binchuan county	100	18	18	100	18	18
鹤庆县 Heqing county	103	17	16.5	103	17	16.5
祥云县 Xiangyun county	103	9	8.7	103	9	8.7
剑川县 Jianchuan county	112	7	6.3	116	8	6.9
南涧县 Nanjiancounty	107	5	4.7	107	5	4.7
漾濞县 Yangbi county	137	3	2.2	137	3	2.2
大理市^aDali City^a	42	0	0	101	0	0
永平县 Yongping county	91	0	0	102	0	0
性别 Gender							13.319	< 0.05
雄 Male	411	47	11.4	429	47	11
雌 Female	846	164	19.9	873	165	18.9
年龄 Age							0.498	0.48
≤ 2	717	116	16.1	741	116	15.7
> 2	540	95	17.6	561	96	17.1

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