Experimentally-induced Placentitis in Late Gestation Ewes and Mares: Evaluation of Pathogen Progression Using Lux-modified Bacteria and Bioluminescence Imaging

Authors: RYAN, P - MISS. STATE UNIV.; MOULTON, K - MISS. STATE UNIV.; WILLARD, S - MISS. STATE UNIV.; CROUCH, J - MISS. STATE UNIV.; SYKES, D - MISS. STATE UNIV.; CHRISTIANSEN, D - MISS. STATE UNIV.; BENNETT, W - U. MISS. MEDICAL CTR.

Submitted to: Uterine Infections in Mares and Women: A Comparative Study II
Publication Type: Proceedings
Publication Acceptance Date: 9/20/2005
Publication Date: 1/1/2008
Citation: Ryan, P.L., Moulton, K., Willard, S., Crouch, J., Sykes, D., Christiansen, D., Bennett, W. 2008. Experimentally-induced Placentitis in Late Gestation Ewes and Mares: Evaluation of Pathogen Progression Using Lux-modified Bacteria and Bioluminescence Imaging. Uterine Infections in Mares and Women: A Comparative Study II, eds M. LeBlanc, J.F. Wade and L. Foster, Havemeyer Foundation Monograph Series #19, R and W Communications, Newmarket, UK, pp 35-36. (peer-reviewed)

Interpretive Summary: Placental infection is one of the primary causes of late term abortion or pre-term delivery in women. Understanding the mechanism of infection is important in developing therapeutic strategies to prevent pre-term labor. Tracking pathogen invasion of fetal tissue using biophotonic imaging we identified organisms in the fetal lungs, digestive tract and bladder, but not the brain. These studies demonstrate that biophotonics and real time imaging provide a novel but valuable means of understanding the pathogenesis of bacteria associated with placentitis and pre-term birth in living mammalian species. Development of the pregnant ewe or mare as a model for monitoring pathogen progression during placentitis in women has real potential.

Technical Abstract: Placental infection due to opportunistic pathogens such as Escherichia coli (E. coli) is the single most common cause of abortion, still birth and premature delivery in women and domestic animal species. The consequence of placentitis during late pregnancy increases the incidence of abortion, preterm delivery or still-birth. Thus, the objectives of this pilot study were 1) to develop a model for pre-term delivery using late term pregnant ewes; and 2) to determine pathogen progression and invasion of the fetal environment by experimentally-induced uterine infection with a lux gene-modified E. coli using real time biophotonics imaging technology in the ewe and mare. In Experiment 1 (Exp. 1), 30 pregnant ewes (~124 days gestation) were assigned to one of three experimental groups. Ewes were inoculated trans-abdominally (ultrasound-guided either intra-cotyledon or intra-amniotic fluid) with 1 ml sterile broth alone (Control), or with either a low (Low, 1.2-4.0 x106 CFU) or high (High; 5.6-20x106 CFU) dose of E. coli transformed with the pAK1-lux plasmid (E. coli-lux). Following inoculation, ewes were continuously monitored for signs of pre-term delivery. Trans-abdominal ultrasonography was performed every 24 h for confirmation of fetal viability over a 7-day interval post-inoculation. Rectal temperatures were recorded twice daily and blood collected by venipuncture daily for cortisol (C) and progesterone (P4) analysis. Lambs from ewes that pre-term delivered within the 7-day interval were imaged immediately. If ewes did not pre-term within the 7-day interval, euthanasia was performed on the 7th day and lambs recovered. Following euthanasia of ewes, the uterus and lambs or pre-term lambs were subjected to biophotonic imaging using a Berthold/NightOwl camera for detection of lux-expressing bacteria (photon emission) over a 5 min period and single frame accumulation to determine pathogen tissue localization. Subsequent to intact uteri and whole animal imaging, lambs were dissected and specific organs imaged including heart, lungs, liver, gastro-intestinal (GI) tract and brain. Uterine and fetal fluids and fluids from GI tract, stomach and bladder were imaged for presence of emitting bacteria. All fluid samples were analyzed to determine total bacteria counts (CFU/ml). Pre-term delivery in ewes occurred between 48 and 120 h post inoculation. Of the 10 control ewes, two (20%) pre-term delivered and 8 carried normal pregnancies to day-7 post inoculation (sterile broth) when lambs were recovered. While a mixed bacterial growth was found in fetal fluid from one of the pre-term fetuses, no photon-emitting bacteria were detected in any tissue or fluids from control lambs. Of the Low and High infected ewes, 6 (60%) and 7 (70%) of the ewes pre-term delivered, respectively. Imaging revealed in most lambs that pre-term delivered photon-emitting bacteria were observed in the lungs, stomach, GI tract, bladder, and in uterine fluids, but not in the heart or liver. No photon-emitting bacteria were detected in brain tissue from any of the lambs delivered or recovered from E. coli-lux-infected ewes. Cultures of fetal stomach, bladder and uterine fluids confirmed presence of photon-emitting bacteria. In Experiment 2 (Exp. 2), two pony mares (late gestation) were inoculated trans-amniotically with E. coli-lux (1x106 CFU) to determine pathogen progression and localization in fetal foal tissues using the same imaging approach as in Exp. I. Fetuses were collected 24 and 40 h post-inoculation for imaging. Lux-expressing bacteria were found at 24 and 40 h post-inoculation in the lungs, GI tract, nares and sinuses but not in the brain, heart or liver. These data clearly demonstrate the power of bioluminescence imaging technology in detecting the progression and localization of pathogens involved in placentitis and pre-term delivery in domestic species. In conc