Establishment and application of natural hapten agar diffusion test for brucella

doi:10.6048/j.issn.1001-4330.2024.08.027

Xinjiang Agricultural Sciences ›› 2024, Vol. 61 ›› Issue (8): 2063-2070.DOI: 10.6048/j.issn.1001-4330.2024.08.027

• Plant Protection · Facility Agriculture · Animal Husbandry Veterinarian · Agricultural Eeconomy • Previous Articles Next Articles

Establishment and application of natural hapten agar diffusion test for brucella

LIU Liya¹(), YAN Wenliang², CAO Rui³, YE Feng¹, MA Xiaojing¹, GU Wenxi¹, ZHAO Jiangshan⁴, ZHANG Zirong⁵, SONG Jie⁶, LI Yan²(), YI Xinping¹()

1. Xinjiang Key Laboratory of Animal Infectious Diseases, Institute of Veterinary Medicine, Xinjang Academy of Animal Sciences, Urumqi 830011,China
2. The General Station of Animal Husbandry and Veterinary of Xinjiang Production and Construction Corps, Urumqi 830021, China
3. Qingdao Real Bio-Technology Co., Ltd, Qingdao Shandong 266000, China
4. Centre for Disease Control and Prevention of Xinjiang Uygur Autonomous Region, Urumqi 830000,China
5. Animal Husbandry and Veterinary Work Station of the Sixth Division, Xinjiang Production and Construction Corps, Wujiaqu Xinjiang 831304,China
6. Xinjiang Tianrun Dairy limited Co.,Ltd, Urumqi 830000, China

Received:2024-01-18 Online:2024-08-20 Published:2024-09-19
Supported by:
Key R&D Special Project of Xinjiang Uygur Autonomous Region(2022B03013-1);The Project of Tackling Hard-Nut Problems in Science and Technology of XPCC(2020AB015)

牛羊布鲁氏菌天然半抗原琼脂扩散试验的建立与应用

刘丽娅¹(), 剡文亮², 曹瑞³, 叶锋¹, 马晓菁¹, 谷文喜¹, 赵江山⁴, 张子荣⁵, 宋洁⁶, 李岩²(), 易新萍¹()

1.新疆动物疫病研究重点实验室/新疆畜牧科学院兽医研究所,乌鲁木齐 830011
2.新疆生产建设兵团畜牧兽医工作总站,乌鲁木齐 830021
3.青岛瑞尔唯特生物技术有限公司,山东青岛 266000
4.新疆维吾尔自治区疾病预防控制中心,乌鲁木齐 830000
5.新疆生产建设兵团第六师畜牧兽医工作站,新疆五家渠 831304
6.新疆天润乳业股份有限公司,乌鲁木齐 830000

作者简介:刘丽娅(1984-),女,安徽阜阳人,副研究员,硕士,研究方向为动物疫病防控,(E-mail)286238083@qq.com
基金资助:
新疆维吾尔自治区重点研发专项(2022B03013-1);新疆生产建设兵团重点领域科技攻关计划项目(2020AB015)

Abstract

Abstract:

【Objective】 To establish and optimize the natural hapten agar gel immuno-diffusion test (NH-AGID) for Brucella abortus in clinical samples.【Methods】 The NH-AGID method was established to evaluate the specificity, sensitivity and reproducibility of the method; 2,287 bovine and sheep sera from immunized areas and 252 from non-immunized areas were tested. 【Results】 The NH-AGID method with stable and reproducible assay and high specificity was successfully established; The sensitivity of the assay for naturally infected positive animals was lower than 80%; The sensitivity of the assay for releasing animals was higher than 90%; The threshold for detecting bruclosis-positive antibodies was higher than RBT. The average detection rate of serum NH antibodies in immunized areas was 17.8%, and the average detection rate of LPS antibodies was 52.3%; the average detection rate of serum NH antibodies in non-immunized areas was 40.9%, and the average detection rate of LPS antibodies was 54.8%. 【Conclusion】 The NH-AGID method can be applied to identify and distinguish bovine and sheep brucellosis-infected animals from immunized animals in immunized areas. Animals detected with NH antibodies in the active or excretory phase of Brucella abortus are brucellosis-infected animals and should be removed from the herd in time.

Key words: brucellosis; native hapten-agar gel immuno diffusion; diagnosis

摘要：

【目的】建立并优化布鲁氏菌天然半抗原琼脂扩散试验(Native hapten agar gel immuno-diffusion test,NH-AGID),检测临床样品。【方法】建立NH-AGID方法,评价方法特异性、敏感性及重复性;检测免疫地区2 287份、非免疫地区252份牛、羊血清。【结果】建立了NH-AGID方法,检测稳定,重复性好,具有较高的特异性;对自然感染阳性动物检测敏感性低于80%,对排菌动物的检测敏感性高于90%,检出布病阳性抗体的阈值高于RBT。免疫地区血清NH抗体平均检出率17.8%,LPS抗体平均检出率52.3%;非免疫地区血清NH抗体平均检出率40.9%,LPS抗体平均检出率54.8%。【结论】应用NH-AGID方法可鉴别区分免疫地区牛、羊布病感染动物和免疫动物。检出NH抗体动物处于布鲁氏菌活动期或排菌期为布病感染动物,应及时剔除出群。

关键词: 布鲁氏菌病, 半抗原-琼脂扩散试验, 诊断

CLC Number:

S855

LIU Liya, YAN Wenliang, CAO Rui, YE Feng, MA Xiaojing, GU Wenxi, ZHAO Jiangshan, ZHANG Zirong, SONG Jie, LI Yan, YI Xinping. Establishment and application of natural hapten agar diffusion test for brucella[J]. Xinjiang Agricultural Sciences, 2024, 61(8): 2063-2070.

刘丽娅, 剡文亮, 曹瑞, 叶锋, 马晓菁, 谷文喜, 赵江山, 张子荣, 宋洁, 李岩, 易新萍. 牛羊布鲁氏菌天然半抗原琼脂扩散试验的建立与应用[J]. 新疆农业科学, 2024, 61(8): 2063-2070.

Figures/Tables 10

Fig.1 Result display diagram of NH-AGID method

Tab.1 Best antigen concentration test of Brucella NH-AGID

血清 Serum	不同抗原浓度 Different antigen concentrations
	0.5 (mg/mL)		1.0 (mg/mL)		1.5 (mg/mL)		2.0 (mg/mL)		2.5 (mg/mL)		3.0 (mg/mL)
	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS
牛强阳 Cattle strong positive serum	+	+	+	+	+	+	+	+	+	+	+	+
羊强阳 Sheep strong positive serum	+	+	+	+	+	+	+	+	-	+	+	+
牛中阳 Positive serum in cattle	+	+	+	+	+	+	+	+	+	+	-	-
羊中阳 Positive serum in sheep	+	+	+	+	+	+	-	+	+	+	-	-
牛弱阳 Cattle weakly positive serum	+	+	+	+	+	+	+	+	-	-	-	-
羊弱阳 Sheep weakly positive serum	-	+	+	+	+	+	-	+	-	+	+	+

Tab.2 The best agar plate concentration test of Brucella NH-AGID

血清 Serum	不同琼脂糖浓度 Different agarose concentrations
	0.6%		1.0%		1.2%		1.5%		1.7%		2.0%
	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS
牛强阳 Cattle strong positive serum	+	+	+	+	+	+	+	+	+	+	+	+
羊强阳 Sheep strong positive serum	+	+	+	+	+	+	+	+	+	+	+	+
牛中阳 Positive serum in cattle	+	+	+	+	+	+	+	+	+	+	+	+
羊中阳 Positive serum in sheep	+	+	+	+	+	+	+	+	+	-	+	-
牛弱阳 Cattle weakly positive serum	-	+	+	+	+	+	+	-	-	-	-	-
羊弱阳 Sheep weakly positive serum	-	-	-	+	+	+	-	-	-	-	-	-

Tab.3 Experiment on the best incubation temperature and time of Brucella NH-AGID

血清 Serum	孵育时间 Incubation time (h)	不同孵育温度 Different incubation temperatures
		18℃		22℃		25℃		30℃		37℃
		NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS
牛强阳 Cattle strong positive serum	24	-	+	+	+	+	+	+	+	+	+
羊强阳 Sheep strong positive serum	24	+	+	+	+	+	+	+	+	+	+
牛中阳 Positive serum in cattle	24	-	+	+	+	+	+	+	+	+	+
羊中阳 Positive serum in sheep	24	+	+	+	+	+	+	+	+	+	+
牛弱阳 Cattle weakly positive serum	24	-	-	+	+	+	+	+	+	+	+
羊弱阳 Sheep weakly positive serum	24	-	-	-	+	+	+	+	+	+	+

Tab.4 Sensitivity difference test of Brucella NH-AGID and RBT detection

强阳性血清稀释倍数 Strongly positive serum dilution multiplier	RBT结果 RBT Result	检测结果 Test results
强阳性血清稀释倍数 Strongly positive serum dilution multiplier	RBT结果 RBT Result	NH	LPS
1:2	++++	+	+
1:4	++++	+	+
1:8	+++	+	+
1:16	+++	-	+
1:32	++	-	+
1:64	++	-	-
1:128	-	-	-
1:256	-	-	-

Tab.5 Detection results of Brucella NH-AGID natural infection animals

动物种类 Animal species	检测数 Number of tests (copies)	RBT 检测 RBT Detection	SAT 检测 SAT Detection	NH-AGID
				NH & LPS		LPS
				检出数 Number of detections (copies)	检出率 Detection rate (%)	检出数 Number of detections (copies)	检出率 Detection rate (%)
牛 Cattle	50	+	+	34	68.0	44	88.0
羊 Sheep	50	+	+	38	76.0	47	94.0
合计 Total	100	+	+	72	72.0	91	91.0

Tab.6 Detection results of NH-AGID for Brucella in animals with positive PCR detection

动物种类 Animal species	检测数 Number of tests (copies)	RBT 检测 RBT Detection	PCR 检测 PCR Detection	NH-AGID
				NH & LPS		LPS
				检出数 Number of detections (copies)	检出率 Detection rate (%)	检出数 Number of detections (copies)	检出率 Detection rate (%)
牛 Cattle	24	+	+	22	91.7	24	100.0
羊 Sheep	24	+	+	23	95.8	24	100.0
合计 Total	48	+	+	45	93.8	48	100.0

Tab.7 Specificity test of Brucella NH-AGID

血清 Serum	检测结果 Test results
血清 Serum	检测数 Number of tests (copies)	NH NH (copies)	LPS LPS(copies)
口蹄疫O型抗体阳性血清 Foot-and-mouth disease O antibody-positive serum	3	0	0
口蹄疫A型抗体阳性血清 Foot-and-mouth disease A antibody-positive serum	4	0	0
牛支原体抗体阳性血清 Bovine mycoplasma antibody-positive serum	5	0	0
牛分枝杆菌抗体阳性血清 Bovine Mycobacterium antibodies positive serum	5	0	0
大肠杆菌抗体阳性血清 E. coli antibody-positive serum	10	0	0
牛布病阴性血清 Bovine brucellosis negative serum	20	0	0
羊布病阴性血清 Sheep brucellosis negative serum	20	0	0

Tab.8 Detection results of Brucella in serum samples from immune areas by NH-AGID

采集地区 Collection area	血清类型 Serum Type	检测数 Number of tests (copies)	RBT 检测 RBT Detection	NH-AGID
				NH & LPS		LPS
				阳性数 Number of positive (copies)	阳性率 Positive rate (%)	阳性数 Number of positive (copies)	阳性率 Positive rate (%)
免疫地区 Immunized areas	A19- $△$ VirB12株免疫牛血清	342	+	19	5.6	269	78.7
	A19株免疫牛血清	1688	+	313	18.5	784	46.4
	M5株免疫羊血清	257	+	74	28.8	142	55.3
合计 Total		2287	+	406	17.8	1195	52.3

Tab.8 Detection results of Brucella in serum samples from immune areas by NH-AGID

采集地区 Collection area	血清类型 Serum Type	检测数 Number of tests (copies)	RBT 检测 RBT Detection	NH-AGID
				NH & LPS		LPS
				阳性数 Number of positive (copies)	阳性率 Positive rate (%)	阳性数 Number of positive (copies)	阳性率 Positive rate (%)
免疫地区 Immunized areas	A19- $△$ VirB12株免疫牛血清	342	+	19	5.6	269	78.7
	A19株免疫牛血清	1688	+	313	18.5	784	46.4
	M5株免疫羊血清	257	+	74	28.8	142	55.3
合计 Total		2287	+	406	17.8	1195	52.3

Tab.9 Detection results of Brucella in serum samples from non immune areas by NH-AGID

采集地区 Collection area	血清类型 Serum Type	检测数 Number of tests (copies)	SAT 检测 SAT Detection	NH-AGID
				NH & LPS		LPS
				阳性数 Number of positive (copies)	阳性率 Positive rate (%)	阳性数 Number of positive (copies)	阳性率 Positive rate (%)
非免疫地区 Non-immunized areas	牛	55	+	19	34.5	33	60.0
非免疫地区 Non-immunized areas	羊	197	+	84	42.6	105	53.3
合计 Total		252	+	103	40.9	138	54.8

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Establishment and application of natural hapten agar diffusion test for brucella

牛羊布鲁氏菌天然半抗原琼脂扩散试验的建立与应用

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