Establishment of a rapid diagnostic method to detect the presence of intracellular infection with Staphylococcus aureus
10.16303/j.cnki.1005-4545.2025.09.15
- VernacularTitle:奶牛乳腺上皮细胞内金黄色葡萄球菌快速检测方法的建立
- Author:
Luyao WEN
1
;
Minghang LI
1
;
Qijun ZHOU
1
;
Jianhui WANG
1
;
Shuai LIAN
1
;
Rui WU
1
;
Jianfa WANG
1
Author Information
1. 黑龙江八一农垦大学农业农村部东北寒区牛病防治重点实验室/黑龙江省牛病防制重点实验室,黑龙江大庆 163319
- Publication Type:Journal Article
- Keywords:
Staphylococcus aureus;
mastitis in dairy cows;
intracellular infection;
soluble staphylo-coccal enzymes;
accuracy
- From:
Chinese Journal of Veterinary Science
2025;45(9):1937-1943
- CountryChina
- Language:Chinese
-
Abstract:
Staphylococcus aureus has the ability to invade bovine mammary epithelial cells and cause persistent intracellular infections.For diagnosing mastitis in cows caused by the bacterial in-fections,it is necessary to determine whether there is a persistent intracellular infection by S.au-reus within mammary epithelial cells,and to establish scientific treatment strategies and imple-ment proactive management decisions.The content of this study is the establishment of a brand new diagnostic method aimed at confirming this type of bovine disease efficiently and cost-effec-tively.The diagnostic procedure is as follows:Add 100 μL S.aureus liquid culture with a concentra-tion of 8.5 ×1010 CFU/mL to the well-grown bovine mammary epithelial cell line and co-culture at 37 degrees Celsius for 2 h to establish an intracellular infection model;After the co-cultivation is completed,add 100 μL of lysostaphin to the dish to thoroughly eliminate extracellular S.aureus.It can be confirmed that the model has been successfully established that black spherical bacteria with a diameter of approximately 1μm are observed within the sample cells using a transmission e-lectron microscope.Intracellular infection is evidenced by the observation of DNA-staining positive material within the cytoplasm of DAPI-stained sample cells,as observed through a laser confocal microscope.The sample is subjected to gram staining after being treated with 200 μL of 0.5％Tri-tonX-100 for 15 min,and intracellular infection is observed within the cytoplasm of the sample cells with Gram-positive results under oil immersion microscopy.The results of the novel"Tri-tonX-100 & gram staining method"validate that the presence of Staphylococcus aureus individu-als within bovine mammary epithelial cells is consistent with images observed under transmission electron microscopy and laser confocal microscopy.The verification results indicate that the"Tri-tonX-100 & gram staining method"is suitable for the diagnosis of S.aureus persistent intracellu-lar infection in bovine mastitis.Later,somatic cells from milk were extracted by gradient centrifu-gation and purified to obtain bovine mammary epithelial cells for clinical testing of 70 S.aureus positive mastitis cattle,which showed a positive rate of 82.9％and a negative rate of 17.1％.
