Development of an in vitro model and fluorescent protein expression system for the study of highly abortigenic Campylobacter jejuni

Campylobacter spp. are a leading cause of sheep abortions worldwide; Campylobacter fetus ssp. fetus has historically been the major culprit. Increasingly, however, C. jejuni has been isolated from cases of sheep abortion, and it has now replaced C. fetus ssp. fetus as the predominant cause of Campylobacter-related ovine abortion in the United States. Emergence of a single tetracycline resistant clone (clone SA) has been implicated as the primary reason for this shift. Virulence factors for clone SA have still not been completely elucidated, and it is not known how this bacterium reaches the uterine and placental tissue from the gut. Although an effective animal model has been described, development of an in vitro cell culture model would provide a cost-effective and reliable alternative. In this study, we assessed the ability of IA3902, a clinical isolate of clone SA, to invade and survive within two different cell lines, AH-1 ovine placental trophoblasts and RAW264.7 murine macrophages. Our results indicate that IA3902 is actually less invasive and has lower survival within AH-1 trophoblast cells than the nonabortigenic isolate NCTC11168; in contrast, IA3902 displayed increased invasion and survival in RAW macrophages as compared to NCTC11168. Next, we tested the hypothesis that C. jejuni\u27s invasive abilities in AH-1 cells would be enhanced in the presence of progesterone, which is present in high levels in the serum of ewes at the time that C. jejuni abortion typically occurs. Contrary to our hypothesis, progesterone treatment of AH-1 cells actually decreased invasion of C. jejuni into trophoblasts. Finally, we developed a system of mCherry fluorescent protein expression in both IA3902 and NCTC11168 to facilitate noninvasive detection of bacteria in an in vitro system. mCherry was successfully expressed in Campylobacter as observed via fluorescent microscopy and measured using a fluorospectrometer; however, low background fluorescence values and an apparent fitness cost in mCherry-expressing Campylobacter may limit its utility for certain studies. These findings indicate that C. jejuni clone SA\u27s ability to survive within immune cells may provide the driving force for its high abortigenicity, rather than an increased tropism for placental tissues. They also provide a basis for future use of in vitro systems for the study of clone SA, and identify RAW264.7 murine macrophages as a suitable cell culture model for further pathogenesis studies