Department of Clinical Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing 100193, P.R. China.
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Canada, T2N 4N1.
J Dairy Sci. 2020 Apr;103(4):3493-3504. doi: 10.3168/jds.2019-17458. Epub 2020 Feb 7.
Klebsiella pneumoniae, a common cause of clinical mastitis (CM) in dairy cows, can cause severe clinical symptoms. However, its pathogenicity in the bovine mammary gland is not well understood. Our objectives were to establish an in vitro infection model of K. pneumoniae on bovine mammary epithelial cells (bMEC) to assess (1) cytopathogenicity (adhesive and invasive ability, damage and apoptosis, pro-inflammatory effects) of K. pneumoniae on bMEC and (2) the role of hypermucoviscous (HMV) phenotype on cytopathogenicity. Two K. pneumoniae isolates from CM cows, 1 HMV and 1 non-HMV, were used to infect bMEC. Adhesion and invasion ability, release of lactate dehydrogenase (LDH), ultrastructural morphology, apoptosis, transcriptional expression of pro-inflammatory genes and production of pro-inflammatory cytokines were characterized at various intervals. Both K. pneumoniae isolates rapidly adhered to and invaded bMEC within 1 h post infection (pi), causing ultrastructural damage (swelling of mitochondria and vesicle formation on cell surface) after 3 h pi and apoptotic death after 9 h pi. In addition, K. pneumoniae promoted transcriptional expression of pro-inflammatory genes IL-6, IL-8, IL-1β, and tumor necrosis factor (TNF)-α and production of IL-8, IL-1β, and TNF-α cytokines. Compared with non-HMV K. pneumoniae, the HMV isolate had lower adhesive and invasive abilities but caused more serious cellular damage. In conclusion, K. pneumoniae was cytopathogenic on bMEC and induced a pro-inflammatory response; however, the HMV phenotype did not have a key role in pathogenicity. Therefore, more attention should be paid to milk loss, and targeted prevention and treatment strategies should be implemented in Klebsiella mastitis episodes.
肺炎克雷伯菌是奶牛临床乳腺炎（CM）的常见病原体，可引起严重的临床症状。然而，其在牛乳腺中的致病性尚不清楚。我们的目的是建立肺炎克雷伯菌在奶牛乳腺上皮细胞（bMEC）上的体外感染模型，以评估（1）肺炎克雷伯菌对 bMEC 的细胞病变作用（黏附与侵袭能力、损伤与凋亡、促炎作用）和（2）高黏液表型（HMV）对细胞病变作用的影响。我们使用 2 株来自 CM 奶牛的肺炎克雷伯菌分离株（1 株 HMV 和 1 株非 HMV）感染 bMEC。在不同时间点，我们检测了黏附和侵袭能力、乳酸脱氢酶（LDH）释放、超微结构形态、凋亡、促炎基因转录表达和促炎细胞因子产生情况。在感染后 1 小时内，两种肺炎克雷伯菌分离株均迅速黏附和侵袭 bMEC，在感染后 3 小时引起超微结构损伤（线粒体肿胀和细胞表面形成小泡），在感染后 9 小时引起凋亡死亡。此外，肺炎克雷伯菌促进了促炎基因 IL-6、IL-8、IL-1β 和肿瘤坏死因子（TNF）-α的转录表达以及 IL-8、IL-1β 和 TNF-α细胞因子的产生。与非 HMV 肺炎克雷伯菌相比，HMV 分离株的黏附和侵袭能力较低，但引起的细胞损伤更严重。总之，肺炎克雷伯菌对 bMEC 具有细胞病变作用，并诱导促炎反应；然而，HMV 表型在致病性方面并非关键因素。因此，在出现克雷伯氏乳腺炎时，应更加关注产奶量下降的问题，并采取有针对性的预防和治疗策略。
