Khemgaew Rathanon, Omachi Mari, Takesada Tomoe, Vetchapitak Torrung, Sato Hiroyuki, Taniguchi Takako, Misawa Naoaki
Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, 5200 Kihara-Kiyotakecho, Miyazaki 889-1692, Japan.
Laboratory of Veterinary Public Health, Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuenkibanadai-nishi, Miyazaki 889-2192, Japan.
J Vet Med Sci. 2021 Jun 9;83(6):889-897. doi: 10.1292/jvms.21-0034. Epub 2021 Apr 14.
Papillomatous digital dermatitis (PDD) is a polymicrobial infection causing lameness in dairy cattle. Culture-independent analysis has shown that Treponema phagedenis is present consistently and predominantly in the lesions. However, the pathogenesis of PDD, especially the tissue penetration pathway, has not been examined. In the present study, we investigated whether T. phagedenis strains isolated from PDD produce proteolytic enzyme (s) for disruption of the epithelial cell barrier and have the ability to translocate in polarized normal human epidermal keratinocytes (NHEK) in vitro. Ten strains of T. phagedenis isolated from lesions did not show proteolytic activity on modified skim milk agar, although a human strain of T. denticola used as a control showed such activity. The integrity of tight junctions was monitored by measurement of transepithelial electrical resistance (TER). The TER values after inoculation of the T. phagedenis strains examined did not change during the experimental period; however, apical to basolateral translocation of T. phagedenis was confirmed after 24 hr by microscopy and Treponema-specific PCR. We further confirmed that translocation of T. phagedenis was accelerated by co-inoculation with live T. denticola, but not with heat-killed organisms. Furthermore, tight junction ZO-1 protein was not lost intensity after inoculation with T. phagedenis and the organism was observed in NHEK cells using a florescence microscope. These results suggest that T. phagedenis strains may translocate via a transcellular route in vitro and that the invasion is accelerated by other bacteria, such as T. denticola, producing proteolytic activity.
乳头瘤状指皮炎（PDD）是一种导致奶牛跛行的多微生物感染。非培养分析表明，噬菌密螺旋体始终且主要存在于病变中。然而，PDD的发病机制，尤其是组织穿透途径，尚未得到研究。在本研究中，我们调查了从PDD分离出的噬菌密螺旋体菌株是否产生用于破坏上皮细胞屏障的蛋白水解酶，以及是否具有在体外极化的正常人表皮角质形成细胞（NHEK）中转运的能力。从病变中分离出的10株噬菌密螺旋体在改良脱脂乳琼脂上未显示蛋白水解活性，尽管用作对照的一株人齿垢密螺旋体显示出这种活性。通过测量跨上皮电阻（TER）来监测紧密连接的完整性。在所检测的噬菌密螺旋体菌株接种后，TER值在实验期间没有变化；然而，24小时后通过显微镜检查和密螺旋体特异性PCR证实了噬菌密螺旋体从顶端向基底外侧的转运。我们进一步证实，与活的齿垢密螺旋体共同接种可加速噬菌密螺旋体的转运，但与热灭活的生物体共同接种则不会。此外，接种噬菌密螺旋体后紧密连接的ZO-1蛋白强度没有降低，并且使用荧光显微镜在NHEK细胞中观察到了该生物体。这些结果表明，噬菌密螺旋体菌株可能在体外通过跨细胞途径转运，并且其他细菌（如齿垢密螺旋体）产生的蛋白水解活性可加速其侵袭。
