宏转录组测序技术在一起仔猪病毒性腹泻疾病诊断中的运用及分析

doi:10.11843/j.issn.0366-6964.2024.08.029

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (8): 3579-3589.doi: 10.11843/j.issn.0366-6964.2024.08.029

宏转录组测序技术在一起仔猪病毒性腹泻疾病诊断中的运用及分析

李跃¹^,²(), 张长春¹^,², 刘光裕¹^,², 高梦源¹^,², 符超俊¹^,², 邢家宝¹^,², 徐思佳¹^,², 邝麒元¹^,², 刘静¹^,², 高校鹏¹^,⁴, 王衡¹^,², 龚浪², 张桂红²^,³, 孙彦阔¹^,²^,³^,*()

1. 华南农业大学兽医学院/广东省动物源性人兽共患病预防与控制重点实验室, 广州 510642
2. 岭南现代农业科学与技术广东省实验室茂名分中心, 茂名 525000
3. 华南农业大学国家生猪种业工程技术研究中心, 广州 510642
4. 温氏食品集团股份有限公司广东省畜禽健康养殖与环境控制企业重点实验室, 云浮 527400

收稿日期:2023-09-25 出版日期:2024-08-23 发布日期:2024-08-28
通讯作者: 孙彦阔 E-mail:liyue20222028024@stu.scau.edu.cn;yankuosun@scau.edu.cn
作者简介:李跃(2000-)，女，河南南阳人，硕士生，主要从事兽医传染病和生物信息学研究, E-mail: liyue20222028024@stu.scau.edu.cn
基金资助:
“十四五”国家重点研发计划项目(2022YFD1800804);国家自然科学基金项目(32102704);茂名实验室科研启动项目(2021TDQD002);财政部和农业农村部：国家现代农业产业技术体系资助(CARS-35)

Application and Analysis of Meta-transcriptomics Sequencing Technology in the Diagnosis of Viral Diarrhea Diseases in Piglets

Yue LI¹^,²(), Changchun ZHANG¹^,², Guangyu LIU¹^,², Mengyuan GAO¹^,², Chaojun FU¹^,², Jiabao XING¹^,², Sijia XU¹^,², Qiyuan KUANG¹^,², Jing LIU¹^,², Xiaopeng GAO¹^,⁴, Heng WANG¹^,², Lang GONG², Guihong ZHANG²^,³, Yankuo SUN¹^,²^,³^,*()

1. College of Veterinary Medicine, South China Agricultural University/Key Laboratory of Prevention and Control of Animal-borne Zoonoses in Guangdong Province, Guangzhou 510642, China
2. Maoming Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Maoming 525000, China
3. National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China
4. Guangdong Provincial Key Laboratory of Livestock and Poultry Health Breeding and Environmental Control, Wens Food Group Co., Ltd, Yunfu 527400, China

Received:2023-09-25 Online:2024-08-23 Published:2024-08-28
Contact: Yankuo SUN E-mail:liyue20222028024@stu.scau.edu.cn;yankuosun@scau.edu.cn

摘要/Abstract

摘要：

为了确定2023年春季广西贺州地区猪场仔猪腹泻病原，采用宏转录组测序技术筛查该地区猪群发病和感染情况，为当地仔猪腹泻疾病的防控提供依据。(方法)采集2023年3月广西贺州地区10 km范围内6个相邻并同时暴发腹泻的猪场仔猪的肛拭子和粪便，运用MGISEQ 200测序平台进行宏转录组测序开展猪病原感染组学研究，系统性地筛查该区域仔猪腹泻的病原谱，使用实时荧光定量RT-PCR对最主要病毒进行确认，定量分析各病原的丰度并选取鉴定到的病毒基因序列进行系统发育分析，Pearson分析解析病原混合感染的相关性。结果显示：通过宏转录组测序技术在这些样品中共检测到17种致病性细菌、12种病毒和3种寄生虫。病毒以猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)代表的腹泻相关病毒为主，包括猪札幌病毒(porcine sapovirus，SaV)、猪嵴病毒(porcine kobuvirus，PKV)、猪星状病毒(porcine astrovirus，PAstV)、猪猴禽猪肠病毒(porcine sapelovirus，PSV)、轮状病毒A(rotavirus，RVA)等；细菌主要包括3种肠杆菌科(Enterobacteriaceae)、3种乳杆菌科(Lactobacillaceae)、2种芽胞杆菌科(Bacillaceae)等；寄生虫包括两种滴虫和碘阿米巴原虫。来自5个场区的PEDV刺突基因(spike gene，S基因)系统发育分析显示, 毒株均属于G2c簇群，毒株之间核苷酸相似性为99.99%，毒株S基因存在重组情况；其中, 一发病猪场还检测到P[6]基因型的rotavirus A部分VP4序列，与人源轮状病毒的核苷酸相似性最高。此外，在4个猪场检测到2种以上的仔猪腹泻相关病毒，系统发育分析结果提示, 此区域存在腹泻病原多谱系共同流行的情况。病原相关性分析显示, PEDV/PSV(P < 0.001)、PEDV/猪环曲病毒(porcine torovirus，PToV)(P < 0.001)和PEDV/PAstV(P < 0.05)有显著的负相关，而PEDV/PKV(P < 0.05)有显著的正相关。以上结果提示, 在该地区相邻养殖区域中，仔猪腹泻的病原谱具有多样性，其中, 直接致病病原是PEDV，同时，PEDV与样品中其他腹泻相关病毒之间存在关联。本研究较为全面地展示该地区仔猪腹泻病原的感染谱，并鉴定出导致腹泻发生最直接的病原体；同时也能分析小范围养殖区域内腹泻相关病原的相关性，为该区域仔猪腹泻疾病防控提供更加精准的参考和指导。

关键词: 宏转录组测序技术, 仔猪腹泻疾病, 病原识别, PEDV, 遗传进化分析, 相关性分析

Abstract:

The purpose of this research is to determine the pathogen of piglet diarrhea in pig farms in Hezhou area of Guangxi in the spring of 2023 by using the Meta-transcriptomics sequencing technology to screen the incidence and infection of pigs in this area, so as to provide theoretical basis for the prevention and control of piglet diarrhea diseases in local pig farms. In March 2023, swabs and feces of six adjacent pig farms with simultaneous outbreak of diarrhea within a range of 10 kilometers were collected in Hezhou, Guangxi, and Meta-transcriptomics sequencing was performed by MGISEQ 200 sequencing platform for porcine pathogenic infectomes research. The pathogenic spectrum of piglet diarrhea in this area was systematically screened and the abundance of each pathogen was quantitatively analyzed. The most important viruses were confirmed by Real-time RT-PCR, after which the identified viral gene sequences were selected for phylogenetic analysis.Pearson analysis was used to analyze the correlation of pathogenic co-infection. The results showed that a total of 17 pathogenic bacteria, 12 viruses, and 3 parasites were detected in these samples by Meta-transcriptomics sequencing. The predominant viruses were diarrhea-associated viruses represented by the porcine epidemic diarrhea virus (PEDV), including porcine sapovirus (SaV), porcine kobuvirus (PKV), porcine astrovirus (PAstV), porcine sapelovirus (PSV), and rotavirus A (RVA). The bacteria mainly consisted of three kinds of Enterobacteriaceae, three kinds of Lactobacillaceae, and two kinds of Bacillaceae. The parasites included two kinds of Trichomonads and Iodamoeba. The phylogenetic analysis of spike gene (S gene) of PEDV from five farms showed that the strains all belonged to the G2c cluster group, that the nucleotide similarity between the strains was 99.99%, and that S gene was recombined. Additionally, partial VP4 gene of rotavirus A, of the P[6] genotype, was detected in one of the farms, which had the highest nucleotide similarity with human rotavirus. In addition, more than 2 piglet diarrhea-related viral infections were detected in 4 pig farms. Phylogenetic analysis showed that there was a multi-lineage co-epidemic of diarrheal pathogens in this region, and even different lineages of the same virus were circulating in the same farm. Pathogen correlation analysis showed that there was a significant negative correlation between PEDV/PSV (P < 0.001), PEDV/porcine torovirus (PToV) (P < 0.001) and PEDV/PAstV (P < 0.05), while PEDV/PKV had a significant positive correlation (P < 0.05). The above findings indicate a diverse pathogenic spectrum of piglet diarrhea in adjacent breeding regions of this area, with PEDV identified as the primary pathogen. Furthermore, a correlation between PEDV and other diarrhea-related viruses was observed in the samples. This study offers a comprehensive overview of the infection spectrum of diarrhea pathogens in piglets in this region, pinpointing the most direct causes of diarrhea. Additionally, it enables the analysis of the relationship between diarrhea-associated pathogens in small-scale breeding areas, providing precise guidance and reference for the prevention and control of piglet diarrhea in this region.

Key words: meta-transcriptomics, piglet diarrhea disease, pathogen identification, PEDV, genetic evolution analysis, correlation analysis

中图分类号:

S852.65

李跃, 张长春, 刘光裕, 高梦源, 符超俊, 邢家宝, 徐思佳, 邝麒元, 刘静, 高校鹏, 王衡, 龚浪, 张桂红, 孙彦阔. 宏转录组测序技术在一起仔猪病毒性腹泻疾病诊断中的运用及分析[J]. 畜牧兽医学报, 2024, 55(8): 3579-3589.

Yue LI, Changchun ZHANG, Guangyu LIU, Mengyuan GAO, Chaojun FU, Jiabao XING, Sijia XU, Qiyuan KUANG, Jing LIU, Xiaopeng GAO, Heng WANG, Lang GONG, Guihong ZHANG, Yankuo SUN. Application and Analysis of Meta-transcriptomics Sequencing Technology in the Diagnosis of Viral Diarrhea Diseases in Piglets[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3579-3589.

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名称 Name	Ct值 Cycle threshold
Farm 1	24.02
Farm 2	18.72
Farm 3	24.29
Farm 4	16.57
Farm 5	20.86
Farm 6	—

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宏转录组测序技术在一起仔猪病毒性腹泻疾病诊断中的运用及分析

Application and Analysis of Meta-transcriptomics Sequencing Technology in the Diagnosis of Viral Diarrhea Diseases in Piglets

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