基于荧光定量PCR检测和原位杂交技术分析湖北省蛋鸡滑液支原体的感染情况

doi:10.11843/j.issn.0366-6964.2025.03.038

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (3): 1396-1407.doi: 10.11843/j.issn.0366-6964.2025.03.038

基于荧光定量PCR检测和原位杂交技术分析湖北省蛋鸡滑液支原体的感染情况

王纯¹(), 王晴¹, 吴晓倩¹, 胡婷¹, 崔卫涛¹, 裴洁², 宋铁平³, 胡思顺¹, 张万坡¹, 李自力¹, 周祖涛¹^,*()

1. 华中农业大学动物医学院/湖北省预防兽医学重点实验室, 武汉 430070
2. 湖北省动物疫病预防控制中心, 武汉 430070
3. 宜昌天佑华牧科技有限公司, 宜昌 443000

收稿日期:2024-04-11 出版日期:2025-03-23 发布日期:2025-04-02
通讯作者: 周祖涛 E-mail:wchun1101@webmail.hzau.edu.cn;ztzhou@mail.hzau.edu.cn
作者简介:王纯(1996-)，女，江苏东台人，博士生，主要从事兽医微生物学与免疫学研究，E-mail: wchun1101@webmail.hzau.edu.cn
基金资助:
国家肉鸡产业技术体系(CARS-41)；湖北省重点研发计划项目(2021BBA252)

Analysis of Mycoplasma synoviae Infection in the Laying Hens in Hubei Province based on Fluorescence Quantitative PCR Detection and in situ Hybridization Technology

WANG Chun¹(), WANG Qing¹, WU Xiaoqian¹, HU Ting¹, CUI Weitao¹, PEI Jie², SONG Tieping³, HU Sishun¹, ZHANG Wanpo¹, LI Zili¹, ZHOU Zutao¹^,*()

1. College of Veterinary Medicine, Huazhong Agricultural University/Hubei Key Laboratory of Preventive Veterinary Medicine, Wuhan 430070, China
2. Hubei Provincial Center for Animal Disease Control and Prevention, Wuhan 430070, China
3. Yichang Tianyou Huamu Technology Co., Ltd, Yichang 443000, China

Received:2024-04-11 Online:2025-03-23 Published:2025-04-02
Contact: ZHOU Zutao E-mail:wchun1101@webmail.hzau.edu.cn;ztzhou@mail.hzau.edu.cn

摘要/Abstract

摘要：

近年来，滑液支原体(Mycoplasma synoviae, MS)在鸡群中的感染率日益上升。湖北省蛋鸡存栏规模较大，各蛋鸡品种均有饲养，目前湖北省蛋鸡群MS感染情况尚不清楚。因此，本研究开展湖北省规模蛋鸡养殖场鸡群MS感染情况的调查并探究MS在产蛋壳顶端异常(eggshell apex abnormality，EAA)蛋鸡输卵管中的分布。采集湖北省36个规模蛋鸡养殖场82个鸡群血清和气管棉拭子各1 637份，利用建立的荧光定量PCR(real-time qPCR)方法检测MS载量，ELISA试剂盒检测免疫鸡群和非免疫鸡群血清抗体，并对产EAA蛋的蛋鸡输卵管进行MS载量、病理切片和MS定位分析。结果显示，临床样本中，喉气管拭子MS阳性率9.41%；血清抗体阳性率92.24%，其中非免疫鸡群抗体阳性率89.04%，且6月龄抗体达100%阳性，免疫鸡群抗体阳性率95%；36个规模场中有26个(72.22%)MS感染阳性场，82个鸡群中有61个(74.39%)感染阳性群；10个蛋鸡品种均有MS感染。产EAA蛋的鸡输卵管部位检出MS且膨大部的载量极显著高于子宫部、峡部和漏斗部(P＜0.01)，子宫部显著高于峡部和漏斗部(P＜0.05)；在产EAA蛋的蛋鸡输卵管中，峡部和子宫部出现黏膜上皮细胞变性和腺体肿大以及膨大部出现腺体上皮细胞脱落和坏死，子宫部和峡部的黏膜上皮以及膨大部的黏膜上皮和管状腺周围都有MS阳性信号检出，其中膨大部和子宫部阳性信号最强。综上，湖北省蛋鸡场MS感染严重，不同品种均有感染，MS主要定植在蛋鸡输卵管子宫部和膨大部中并造成病理损伤。本研究为湖北省蛋鸡MS感染情况的认识和MS引起EAA蛋的研究提供了数据支撑。

关键词: 滑液支原体, 蛋壳顶端异常, 荧光定量PCR, 原位杂交, 组织分布

Abstract:

In recent years, the infection rate of Mycoplasma synoviae (MS) in chicken flocks has been increasing. The inventory of laying hens in Hubei Province is relatively large, and all commodity breeds are raised. Currently, the situation of MS infection in laying hens in Hubei Province is not clear. Therefore, this study conducted an investigation into the infection status of MS in chicken flocks of large-scale laying hen farms in Hubei and explored the impact of MS infection on clinical eggshell apex abnormality (EAA). One thousand six hundred and thirty-seven serum samples and tracheal cotton swabs were collected from 82 chicken flocks in 36 large-scale laying hen farms in Hubei Province. The MS load was detected using the established real-time qPCR method, and the serum antibodies of vaccinated and non-vaccinated chicken flocks were detected using ELISA kits. Perform MS loading, pathological sectioning, and MS localization analysis on the oviduct of EAA laying hens. The results showed that in clinical samples, the MS positive rate of tracheal swabs was 9.41%; The positivity rate of serum antibodies was 92.24%, among which the positivity rate of antibodies in non-vaccinated chicken flocks was 89.04%, and the positivity rate of antibodies in 6-month-old chickens was 100%, while the positivity rate of antibodies in vaccinated chicken flocks was 95%; Out of 36 large-scale farms, 26 (72.22%) were infected with MS, and out of 82 chicken flocks, 61 (74.39%) were infected; All 10 breeds of laying hens were infected with MS. MS was detected in the oviduct of EAA laying hens, and the load in the magnum was significantly higher than that in the uterus, isthmus and infundibulum (P < 0.01), the uterus was higher than that in the isthmus and infundibulum (P < 0.05). In the oviduct of EAA laying hens, mucosal epithelial cell degeneration and glandular enlargement occur in the isthmus and uterus, as well as shedding and necrosis of glandular epithelial cells in the magnum. Moreover, MS positive signals were detected in the mucosal epithelium of the uterus and isthmus, as well as in the mucosal epithelium and surrounding tubular glands of the magnum, and among them, the magnum and uterus had the strongest positive signals. The infection of MS in laying hens in Hubei Province is severe, and different breeds are infected. MS mainly colonizes in the uterus and magnum of the laying hens' oviduct, causing pathological damage. This study provides data support for understanding the situation of MS infection in laying hens in Hubei Province and the study of MS induced EAA eggs.

Key words: Mycoplasma synoviae, eggshell apex abnormality, real-time qPCR, in situ hybridization, tissue distribution

中图分类号:

S858.31

王纯, 王晴, 吴晓倩, 胡婷, 崔卫涛, 裴洁, 宋铁平, 胡思顺, 张万坡, 李自力, 周祖涛. 基于荧光定量PCR检测和原位杂交技术分析湖北省蛋鸡滑液支原体的感染情况[J]. 畜牧兽医学报, 2025, 56(3): 1396-1407.

WANG Chun, WANG Qing, WU Xiaoqian, HU Ting, CUI Weitao, PEI Jie, SONG Tieping, HU Sishun, ZHANG Wanpo, LI Zili, ZHOU Zutao. Analysis of Mycoplasma synoviae Infection in the Laying Hens in Hubei Province based on Fluorescence Quantitative PCR Detection and in situ Hybridization Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1396-1407.

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标准浓度/(copies·μL^-1) Standard concentration	批内重复试验 Intra-assay repeatability			批间重复试验 Inter-assay repeatability
标准浓度/(copies·μL^-1) Standard concentration	AVG	SD	CV/%	AVG	SD	CV/%
1.91×10⁶	16.76	0.026	0.16	17.4	0.714	4.10
1.91×10⁵	20.11	0.123	0.61	20.51	0.633	3.09
1.91×10⁴	24.35	0.130	0.53	24.47	0.125	0.51

群体 Flocks	总数 Count	核酸阳性数 Nucleic acid-positive count	阳性率/% Positive rate	抗体阳性数 Antibody-positive count	阳性率/% Positive rate
免疫鸡群	880	94	10.68^a	836	95.00^A
Vaccinated chicken
非免疫鸡群	757	60	7.93^a	674	89.04^B
Non-vaccinated chicken

品种 Breeds	日龄 Days	产蛋率/% Laying rate	破损率/% Damage rate	EAA	核酸阳性率/% Nucleic acid-positive rate	抗体阳性率/% Antibody-positive rate	每克黏液MS载量(×10³拷贝)(${\bar x}$±s) MS load per gram of mucus (×10³ copies) (${\bar x}$±s)
罗曼粉	360	70	12.0	√	54.55	100	3.15±1.29
海兰灰	359	86	6.0	√	30	100	4.79±3.44
罗曼粉	200	83.5	7.8	√	30	100	2.00±0.54
罗曼灰	170	87	1.2		25	100	18.15±29.90
罗曼灰	355	85	10.0	√	25	100	6.39±10.27
罗曼粉	450	82	3.7	√	25	100	5.69±4.74
罗曼粉	491	81	4.5	√	25	100	4.18±2.34
罗曼灰	264	89	3.0		25	100	8.31±4.93
罗曼粉	220	87	0.8		20	100	3.17±1.51
京粉6号	442	83	6.0		20	100	14.95±10.63

部位 Location	Ct值 Ct value	每克黏膜MS载量(×10⁴拷贝) MS load per gram of mucosa (×10⁴ copies)
子宫部Uterus	27.22±0.67^Aa	3.31±1.68^Aa
峡部Isthmus	28.53±0.13^AbC	1.24±0.11^Ab
膨大部magnum	25.64±0.16^Bc	9.26±1.08^Bc
漏斗部Infundibulum	29.24±0.81^bC	0.60±0.14^Ab

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基于荧光定量PCR检测和原位杂交技术分析湖北省蛋鸡滑液支原体的感染情况

Analysis of Mycoplasma synoviae Infection in the Laying Hens in Hubei Province based on Fluorescence Quantitative PCR Detection and in situ Hybridization Technology

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