Abstract

Background: The coinfection process of Escherichia coli, an etiological agent of clinical mastitis and Mycobacterium avium subsp. paratuberculosis (MAP), a non-mastitic etiological agent in the bovine mammary gland is not fully known.

Objective: Verify the ability of MAP to interfere with the invasion and translocation of E. coli in bovine mammary epithelial cell line (MAC-T).

Methods: For the invasion assay, MAC-T cells were challenged with MAP K10 for 2 h and then challenged with E. coli for 10, 30 and 120 min. For the translocation assay, the trans well plates were used and the challenge sequence was repeated as previously described. The amount of E. coli in the assays was determined by counting colony forming units (CFU) in Luria-Bertani medium. Quantitative real-time PCR was used to quantify MAP in MAC-T cells. To verify the viability of the MAC-T cells, the MTT assay was performed. MAP culture supernatant was also evaluated at different percentages for E. coli growth.

Results: Previous MAP infection in MAC-T cells inhibited E. coli invasion in 10, 30 and 120 min. No significant interference of MAP in the translocation of E. coli from the apical-basal direction was verified. Quantity of MAP DNA inside the MAC-T cells was statistically similar. Neither reduction in MAC-T cells viability was detected during the experiment nor MAP-released factor in the supernatant inhibited E. coli invasion.

Conclusion: These findings suggest that MAP-positive cows could be more resistant to E. coli infection, but when infected, could rapidly translocate E. coli to the subepithelial region.

摘要

背景：临床乳腺炎和鸟分枝杆菌亚种的病原体大肠杆菌的合并感染过程。副结核病（MAP），在牛乳腺非乳腺炎病原体腺尚不完全清楚。

目的：验证MAP干扰牛乳腺上皮细胞系（MAC-T）中大肠杆菌的侵袭和转运的能力。

方法：对于侵袭测定，将MAC-T细胞用MAP K10攻击2小时，然后用大肠杆菌攻击10、30和120分钟。对于易位测定，使用了trans孔板，并且如前所述重复攻击序列。通过计数Luria-Bertani培养基中的菌落形成单位（CFU）来确定测定中的大肠杆菌量。实时定量PCR用于定量MAC-T细胞中的MAP。为了验证MAC-T细胞的活力，进行了MTT测定。还以不同百分比评估了MAP培养上清液的大肠杆菌生长。

结果： MAC-T细胞中先前的MAP感染在10、30和120分钟内抑制了大肠杆菌的入侵。没有验证MAP从根基部方向对大肠杆菌的转运产生显着干扰。MAC-T细胞内部的MAP DNA数量在统计学上相似。实验期间既未检测到MAC-T细胞活力的降低，也未检测到上清液中的MAP释放因子抑制了大肠杆菌的入侵。

结论：这些发现表明，MAP阳性奶牛对大肠杆菌的感染可能更具抵抗力，但是当被感染时，它们可以迅速将大肠杆菌转移到上皮下区域。