内蒙古地区羊源副结核分枝杆菌的分离与基因分型

doi:10.3864/j.issn.0578-1752.2023.06.015

Abstract

Abstract:

【Objective】 Paratuberculosis is listed in the must-report at the list of “World Organization for Animal Health (OIE) diseases, infections and Invasion” by OIE. It is classified as the second kind of animal disease in China. It causes chronic and proliferative enteritis in many ruminants. The infected animals become a continuous source of infection in farms through intestinal intermittent excretion, which has brought great economic losses to aquaculture. The pathogen of Mycobacterium avium subsp. paratuberculosis (MAP) belongs to intracellular parasitic Gram-positive bacteria, and is a third group of zoological pathogenic microorganisms, including type C (also designated as type II) and type S. Type C also includes type B. Type S can be further subdivided into sub-group types I and III. And sub-lineages of camelid isolates Studies have shown that each subtype of MAP has no host specificity, but is regional. Inner Mongolia is the first region of the disease in China. It is of great significance to obtain and accurately identify the subtype and genetic characteristics of MAP strains in Inner Mongolia for the prevention and control of paratuberculosis. 【Method】 28 MAP-positive sheep disease samples collected in Inner Mongolia were isolated and cultured by MAP, and the colonies were stained with Ziehl-Neelsen. The positive bacteria were propagated and the genomic DNA was extracted. IS900 gene, IS1311 gene and DMC gene were amplified, sequenced, and analyzed. The PCR products of IS1311 gene were identified by Hinf I and Mse I double digestion. 【Result】 28 samples were cultured for 7- 2 weeks, a total of 9 mediums grew colonies, and the colonies were translucent milky white smooth surface. Single colonies were selected for acid-fast staining, and irregular (single or branched), red-stained Brevibacterium was observed under the microscope, which was consistent with the morphological characteristics and acid-fast staining characteristics of Mycobacterium. The PCR products of IS900, IS1311 and DMC genes of 9 isolates were consistent with the expected size of the target gene fragment. 9 isolates were identified as MAP strains, named MAP-NM1 to MAP-NM9. DMC gene amplification product size of 310bp, which was consistent with type II MAP characteristics. IS1311 gene amplification products were digested by Hinf I and Mse I restriction endonucleases, and 4 target bands were obtained in 9 strains of MAP, which were consistent with type II MAP. The sequencing results of IS1311 gene and the analysis of MAP representative strains of type I, type II, type III, Indian Buffalo and American Buffalo showed that the nucleotide sites at positions 64, 65, 68, 223, 236, 422, 527 and 628 of the nine MAP IS1311 gene fragments were conformed to the characteristics of type C and type B MAP. Sequence analysis of IS900 gene sequencing results showed that the 169th and 216th nucleotides of the nine MAP IS900 gene fragments were C (cytosine) and A (adenine), and accorded with Type II and type III MAP. The phylogenetic tree of 17 MAP IS900 gene reference sequences from GenBank database with 9 isolates in this study showed that the 9 isolates in this study were all in the type II MAP branch. Blast online analysis was performed on the sequencing results of the three genes. The reference sequences with the highest homology with the isolates obtained in this study were all type II MAP, and the homology was higher than 98%. In conclusion, all the 9 MAP isolates were type II MAP. 【Conclusion】 To the best of our knowledge, this was the first isolate of the MAP type Ⅱ strains in sheep in Inner Mongolia.

Key words: Mycobacterium avium subsp. paratuberculosis, isolated, typing, sheep, Inner Mongolia

ZHAO WeiHong, HAN WenXiong, YANG Bo, MENG WeiKang, CHAI HaiLiang, MA YiMin, ZHANG ZhanSheng, WANG LiFeng, WANG Yan, WANG MingYuan, ZHANG Shan, DING YuLin, WANG JinLing, JIRINTAI Sulijid, WANG FengLong, ZHAO Li, LIU YongHong. Isolation and Genotyping of Mycobacterium avium subsp. paratuberculosis from Sheep in Inner Mongolia[J].Scientia Agricultura Sinica, 2023, 56(6): 1204-1214.

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编号 No.	采集地点 Place of collection	采集时间 Time of collection	物种 Species
1	呼和浩特Hohhot	2018.5	绵羊Sheep
2-4	呼和浩特Hohhot	2016.12	山羊Gost
5-7	锡林郭勒Xilingol	2015.4	绵羊Sheep
8-10	锡林郭勒Xilingol	2015.5	山羊Gost
11-12	阿拉善Alxa	2021.4	山羊Gost
A1-A16	阿拉善Alxa	2020.6	山羊Gost

引物 Primer	引物序列 Primer Sequence (5'→3')	大小Size (bp)	参考文献 References
IS900-F	GTTCGGGGCCGTCGCTTAGG	400	[17]
IS900-R	GAGGTCGATCGCCCACGTGA	400	[17]
IS1311-F	GCGTGAGGCTCTGTGGTGAA	608	[18]
IS1311-R	ATGACGACCGCTTGGGAGAC	608	[18]
DMC529	TTGACAACGTCATTGAGAATCC	146（Ⅰ型Type Ⅰ） 310（Ⅱ型Type Ⅱ）	[19]
DMC531	TCTTATCGGACTTCTTCTGGC
DMC533	CGGATTGACCTGCGTTTCAC

序号 No.	基因 Name of gene	菌株名称 Name of strain	GenBank登录号 GenBank accession number	亚型 Subtypes
1	IS900	ATCC 53950	AF305073	Ⅱ型Type Ⅱ
2		A1	MN928512	Ⅱ型Type Ⅱ
3		FA0010	MT017597	Ⅱ型Type Ⅱ
4		JTC 1285	NZ_AGAL01000886	Ⅱ型Type Ⅱ
5		316F	AF416985	Ⅱ型Type Ⅱ
6		21P	FJ775181	Ⅰ型Type Ⅰ
7		不详Unknown	AJ250015	Ⅱ型Type Ⅱ
8		不详Unknown	AJ250022	Ⅱ型Type Ⅱ
9		Ben	X16293	Ⅱ型Type Ⅱ
10		Case-2	MW546836	Ⅱ型Type Ⅱ
11		Case-7	MW546841	Ⅲ型Type Ⅲ
12		JII-1961	CP022105	Ⅱ型Type Ⅱ
13		JIII-386	CP042454	Ⅲ型Type Ⅲ
14		MAPK_CN4-13	CP033910	Ⅱ型Type Ⅱ
15		MAPK_CN9-15	CP033427	Ⅱ型Type Ⅱ
16		S397	CP053749	Ⅲ型Type Ⅲ
17		Telford	CP033688	Ⅰ型Type Ⅰ
18	IS1311	JD97/1-2	AJ223975	S型Type S
19		316V	AJ223974	C型Type C
20		98/1010	AJ308375	美国野牛型USA Bison type
21		'Bison type' S5	EF514838	印度野牛型Indian Bison type

菌株名 Name of strain	亚型 Subtypes	IS1311位点 Position on IS1311
菌株名 Name of strain	亚型 Subtypes	64	65	68	223	236	422	527	628
JD97/1-2	S型Type S	T	G	C	C	C	C	A	C
316V	C型Type C	T	G	C	Y	C	C	A	C
98/1010	B型美国野牛型 (Type B) USA Bison type	T	G	C	T	C	C	A	C
'Bison type' S5	B型印度野牛型 (Type B) Indian Bison type	-	-	C	T	C	N	N	N
MAP-NM1至MAP-NM9 MAP-NM1 to MAP-NM9	本研究分离株 The isolates in this study	T	G	C	T	C	C	A	C

序号 No.	菌株名 Name of strain	基因名称 Name of gene	同源性最高序列名称 Name of the most homologous sequence	GenBank登录号 GenBank accession number	同源性 Nucleotide homology (%)	亚型 Subtypes
1	MAP-NM1	IS900	Case-3	MW546837.1	99.45	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.47	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	98.93	Ⅱ型Type Ⅱ
2	MAP-NM2	IS900	DSM 44135	CP053068.1	99.46	Ⅱ型Type Ⅱ
		IS1311	Case-2	MW546850.1	99.31	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546844.1	98.94	Ⅱ型Type Ⅱ
3	MAP-NM3	IS900	DSM 44135	CP053068.1	99.45	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.65	Ⅱ型Type Ⅱ
		DMC	Case-4	MW546845.1	99.29	Ⅱ型Type Ⅱ
4	MAP-NM4	IS900	DSM 44135	CP053068.1	99.72	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.64	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	98.58	Ⅱ型Type Ⅱ
5	MAP-NM5	IS900	DSM 44135	CP053068.1	99.73	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.66	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546844.1	99.28	Ⅱ型Type Ⅱ
6	MAP-NM6	IS900	DSM 44135	CP053068.1	100.00	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.64	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	99.29	Ⅱ型Type Ⅱ
7	MAP-NM7	IS900	DSM 44135	CP053068.1	99.45	Ⅱ型Type Ⅱ
		IS1311	Case-4	MW546852.1	99.48	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546844.1	99.29	Ⅱ型Type Ⅱ
8	MAP-NM8	IS900	DSM 44135	CP053068.1	98.91	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.82	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	99.29	Ⅱ型Type Ⅱ
9	MAP-NM9	IS900	DSM 44135	CP053068.1	99.18	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.65	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	98.93	Ⅱ型Type Ⅱ

Isolation and Genotyping of Mycobacterium avium subsp. paratuberculosis from Sheep in Inner Mongolia

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分离株 Isolate	PCR结果 Result of PCR	IS900序列分析 Sequence analysis of IS900	IS1311序列分析 Sequence analysis of IS1311	BLAST分析 BLAST analysis	进化树 Evolutionary tree	结论 Conclusion
MAP-NM1	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM2	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM3	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM4	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM5	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM6	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM7	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM8	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM9	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ

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