Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (5): 1514-1528.DOI: 10.1016/j.jia.2022.10.004

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中国和巴基斯坦奶牛场无乳链球菌的分子流行病学、毒力基因检测和耐药性分析

  

  • 收稿日期:2022-04-18 接受日期:2022-09-09 出版日期:2023-05-20 发布日期:2022-09-09

Molecular epidemiology, characterization of virulence factors and antibiotic-resistance profile of Streptococcus agalactiae isolated from dairy farms in China and Pakistan

Ambreen LEGHARI1, Shakeel Ahmed LAKHO2, Faiz Muhammad KHAND2, Khaliq ur Rehman BHUTTO3, Sameen Qayoom LONE1, Muhammad Tahir ALEEM1, Iqra BANO2, Muhammad Ali CHANDIO2, Jan Muhammad SHAH2, LIN Hui-xing1, FAN Hong-jie1, 4#   

  1. 1 MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P.R.China

    2 Shaheed Benazir Bhutto University of Veterinary and Animal Sciences, Sakrand 67210, Pakistan

    3 Central Veterinary Diagnostic Laboratory, Tando Jam 70050, Pakistan

    4 Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, P.R.China

  • Received:2022-04-18 Accepted:2022-09-09 Online:2023-05-20 Published:2022-09-09
  • About author:Ambreen LEGHARI, E-mail: 2018207075@njau.edu.cn; #Correspondence FAN Hong-jie, E-mail: fhj@njau.edu.cn
  • Supported by:

    This study was supported by the National Key Research and Development Program of China (2021YFD1800400), the National Natural Science Foundation of China (31872480), the Jiangsu Agriculture Science and Technology Innovation Fund (CX(19)2020), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD).

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摘要：

无乳链球菌是世界范围内引起奶牛乳房炎的最常见病原体之一。了解该菌的流行现状和毒力因子对于制定防治措施至关重要。本研究于2019-2021期间，从中国（n=558）和巴基斯坦（n=603）的多个奶牛场采集了1161份牛奶样本，并对其进行了无乳链球菌的分离鉴定。通过PCR检测分析了无乳链球菌的流行率、血清型、毒力基因和耐药基因。所有的分离菌株均具有溶血、生成生物被膜、细胞毒性、粘附并侵袭牛乳腺上皮细胞的特性。巴基斯坦地区由无乳链球菌引起的奶牛乳房炎的发病率显著高于中国。江苏省和信德省（Sindh）分别是中国和巴基斯坦地区无乳链球菌流行率最高的省份。血清型Ia型和II型的无乳链球菌在这两个国家的流行率均较高，而血清III型的无乳链球菌只在巴基斯坦发现。此外，无论是中国还是巴基斯坦，所有的无乳链球菌分离菌株均为PI-2b基因阳性，但PI-1和PI-2a基因均为阴性。所有的分离菌株都含有cfb、cylE、hylB和fbsB毒力基因，而大多分离菌株无bibA、rib和bca毒力基因。分离自中国的无乳链球菌均无bac和scp毒力基因，而分离自巴基斯坦的无乳链球菌均无cspA基因，同时两国的分离菌株中均未检测到spb1和lmb毒力基因。分离自巴基斯坦的无乳链球菌，尤其是血清Ia型菌株，与分离自中国的菌株相比，具有更高的生物被膜形成、溶血、细胞毒性、粘附和侵袭能力。大多数分离菌株对四环素、红霉素和克林霉素具有耐药性，而ermA、ermB、tetM和tetO等耐药基因的存在也从基因水平验证了分离菌株的耐药性。上述研究结果为由无乳链球菌引起的奶牛乳房炎特异性防治措施的制定具有重要指导意义。

Abstract:

Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. Herein, 1161 milk samples from various dairy farms in China (n=558) and Pakistan (n=603) were collected between 2019-2021 and were subjected to S. agalactiae isolation. Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S. agalactiae were evaluated by PCR assay. All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells. The prevalence of S. agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China. Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively. Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan. Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes. All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca. However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries. Pakistani isolates, particularly serotype Ia-positive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates. Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes. Our study highlights the antimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S. agalactiae in China and Pakistan. The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen. 

Key words: Streptococcus agalactiae  ,  mastitis , epidemiology , virulence characterization , antibiotic resistance