奶牛源产气荚膜梭菌分离鉴定和生物信息学分析

doi:10.11843/j.issn.0366-6964.2025.10.035

摘要/Abstract

摘要：

旨在探究我国河北、河南等10个地区奶牛场牛源产气荚膜梭菌分离鉴定类型及5株分离株全基因测序信息。利用PCR、测序等技术对全国规模化奶牛场收集的89份疑似患梭菌病的牛病料进行分离鉴定，结合从NCBI公共数据库中获取的17株牛产气荚膜梭菌基因组，运用生物信息学技术对分离株进行泛基因组，系统发育、毒力基因及耐药基因分析。结果显示：经全基因组测序分析显示，分离菌株基因组大小为2.98~3.19 MB，平均每个基因组含有60个tRNA和2 873个基因编码区(CDS)。MLST(多位点序列分型)显示：本研究分离株与从NCBI下载的17株牛源产气荚膜梭菌菌株信息均属于不同类型，除PAN2309B1CP属于ST777型外，其余4株均属于ST72型。22株牛源产气荚膜梭菌共检测到13种耐药基因，30个毒力因子，其中四环素类耐药基因tetA(P) 携带率最高，为86.3%。泛基因组结果分析显示，本研究5株分离株共含有7 927个基因，核心基因数量为1647(占比21.0%)。核心基因组和plc基因进化树分析显示，PAN2305I2CP、PAN2304V2CP、PAN2404F3CP、PAN2404F4CP均在同一分支，PAN2309B1CP在另一分支。本研究为全国首例对奶牛源产气荚膜梭菌分离鉴定和生物信息学分析，进一步丰富了国内奶牛源产气荚膜梭菌流行株菌株库以及其基因组结构、组成和进化信息。同时对产气荚膜梭菌疾病预防治疗和基因组进一步研究提供参考价值。

关键词: 奶牛产气荚膜梭菌, 分离纯化, 全基因组测序, MLST分型, 毒力因子, 耐药基因, 遗传进化

Abstract:

This study aimed to investigate the types of Clostridium perfringens isolated from bovine sources in 10 dairy farms in Hebei and Henan, China, and to investigate the information of five isolates by whole gene sequencing. The isolates were identified using PCR and sequencing techniques from 89 bovine disease cases suspected of having C. perfringens disease collected from large-scale dairy farms nationwide. Combined with the genomes of 17 strains of bovine C. perfringens obtained from NCBI public databases, bioinformatics techniques were used to analyze the isolates for pangenomic, phylogenetic, virulence, and drug-resistance genes. The whole genome sequencing analysis showed that the genome size of the isolates ranged from 2.98 to 3.19 MB, with an average of 60 tRNAs and 2 873 coding regions of genes (CDS) per genome. MLST (multilocus sequence typing) showed that the isolates in this study belonged to a different type from the 17 C. perfringens strains of bovine origin downloaded from NCBI, with the exception of PAN2309B1CP belonged to ST777 type, and the remaining 4 strains belonged to ST72 type. A total of 13 drug resistance genes and 30 virulence factors were detected in 22 strains of C. perfringens of bovine origin, among which the tetracycline resistance gene, tetA(P), had the highest carrier rate of 86.3%. Analysis of the pan-genomic results showed that the 5 isolates in this study contained a total of 7 927 genes, of which the number of core genes was 1647 (21.0%). The analysis of the core genome and plc gene evolution tree showed that PAN2305I2CP, PAN2304V2CP, PAN2404F3CP, and PAN2404F4CP were all in the same branch, and PAN2309B1CP was in another branch. This study is the first case of isolation, identification and bioinformatics analysis of Clostridium perfringens from dairy cows, which further enriches the domestic database of epidemic strains of Clostridium perfringens from dairy cows, as well as its genomic structure, composition and evolutionary information. It also provides reference value for the prevention and treatment of C. perfringens disease and further genomic research.

Key words: Clostridium perfringens in cows, isolation and purification, whole genome sequencing, MLST typing, virulence factors, resistance genes, genetic evolution

中图分类号:

S852.61

王梦瑶, 张志, 吴春琳, 施志玉, 孙学强, 姚火春, 潘子豪. 奶牛源产气荚膜梭菌分离鉴定和生物信息学分析[J]. 畜牧兽医学报, 2025, 56(10): 5159-5169.

WANG Mengyao, ZHANG Zhi, WU Chunlin, SHI Zhiyu, SUN Xueqiang, YAO Huochun, PAN Zihao. Isolation, Characterization and Bioinformatics Analysis of Clostridium perfringens of Bovine Origin[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5159-5169.

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基因 Gene	引物序列(5′→3′) Sequence	产物长度/bp Size	参考文献 Reference
16S rRNA	AGAGTTTGATCCTGGCTCAG GGTTACCTTGTTACGACTT	1 465	Sipos等，2007^[11]
cpa	ATGAGCTTCAATTAGGTTCTACT ATCAGCATAAAAATCCTCATT	398	张曼，2010^[12]
cpb	GCGAATATGCTGAATCATCTA GCAGGAACATTAGTATATCTTC	196	Meer和Songer，1997^[13]
cpe	GGAGATGGTTGGATATTAGG GGACCAGCAGTTGTAGATA	233	Meer和Songer，1997^[13]
netB	CTTCTAGTGATACCGCTTCAC CGTTATATTCACTTGTTGACGAAAG	738	Rood等，2018^[14]
etx	CCACTTACTTGTCCTACTAAC GCGGTGATATCCATCTATTC	656	Meer和Songer，1997^[13]
ia	GGAAAAGAAAATTATAGTGATTGG CCTGCATAACCTGGAATGGC	461	Rood等，2018^[14]

牧场 Ranch	来源地 Source location	样品数量 Number of samples	阳性数 Number of positives	检出率 Detection rate
A牧场Ranch A	安徽蚌埠Bengbu, Anhui	23	13	13/23
B牧场Ranch B	甘肃武威Wuwei, Gansu	7	0	0/7
C牧场Ranch C	河北廊坊Langfang, Hebei	6	3	3/6
D牧场Ranch D	河北唐山Tangshan, Hebei	6	6	6/6
E牧场Ranch E	河南驻马店Zhumadian, Henan	4	0	0/4
F牧场Ranch F	河南驻马店Zhumadian, Henan	10	9	9/10
G牧场Ranch G	黑龙江绥化Suihua, Heilongjiang	2	2	2/2
H牧场Ranch H	湖北黄冈Huanggang, Hubei	10	0	0/10
I牧场I Ranch	山东烟台Yantai, Shandong	2	2	2/2
J牧场Ranch J	山西大同Datong, Shanxi	12	1	1/12
K牧场Ranch K	云南丽江Lijiang, Yunnan	4	4	4/4
L牧场Ranch L	浙江温州Wenzhou, Zhejiang	3	2	2/3
合计Total		89	42	42/89

菌株 Strains	登录号 Accession No.	分离时间 Separation time	基因组大小/Mb Genome size	转运核糖核酸 tRNA	蛋白质编码区 CDS	核糖体核糖核酸 rRNA
PAN2304V2CP	JBIEKN000000000	2024年4月	3.19	72	2 933	4
PAN2309B1CP	JBIELU000000000	2024年5月	2.98	65	2 767	3
PAN2305I2CP	JBIEKM000000000	2024年4月	3.10	53	2 857	2
PAN2404F3CP	JBIELT000000000	2024年5月	3.15	54	2 895	3
PAN2404F4CP	JBIELS000000000	2024年5月	3.17	54	2 912	3

菌株名称 Strain name	基因登录号 Accession No.	分离地 Isolation place	分离时间 Separation time	基因组大小/Mb Genome size	序列型 ST
CP-22	GCF 011062975	Beijing, China	2020.3.3	3.6	181
CP-02	GCF 011063325	Beijing, China	2020.3.3	3.0	237
CPN17a	GCF 020138355	Finland	2021.9.24	3.3	174
JXNC-B4	GCF 024652865	Jiangxi, China	2022.8.14	3.3	558
ZWCP172	GCF 029846815	Nanjing, China	2023.4.19	3.3	27
ZWCP168	GCF 029846835	Nanjing, China	2023.4.19	3.5	102
ZWCP165	GCF 029846845	Nanjing, China	2023.4.19	3.4	181
ZWCP162	GCF 029846875	Nanjing, China	2023.4.19	3.4	282
ZWCP079	GCF 029846895	Nanjing, China	2023.4.19	3.4	279
ZWCP067	GCF 029846915	Nanjing, China	2023.4.19	3.5	282
ZWCP209	GCF 030434295	Nanjing, China	2023.4.19	3.6	700
ZWCP210	GCF 030434355	Nanjing, China	2023.4.19	3.6	700
ZWCP036	GCA 030766735	Nanjing, China	2023.4.19	3.3	69
17SBCL85	GCA 015932165	French	2020.12.10	3.5	5
17SBCL79	GCA 015932215	French	2020.12.10	3.5	5
C.perfringens C4-5	GCA 033187995	Denmark	2023.11.1	3.3	485
C.perfringens C17-3	GCA 033187955	Denmark	2023.11.1	3.4	268

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