Experimental genital mycoplasmosis: time of infection influences pregnancy outcome
Infection and Immunity
Genital infection of rats with Mycoplasma pulmonis causes adverse pregnancy outcome and can result in in utero spread of infection to the fetus. The current study was designed to determine whether the stage of pregnancy when infection occurs influences pregnancy outcome. Rats were inoculated with 3 ؋ 10 7 CFU of M. pulmonis at 10 days prior to breeding (؊10) or at gestational day (gd) 11 or 14 and were necropsied at gd 11, 14, or 18 or within 24 h of parturition (term). Control rats received
... rile broth. M. pulmonis was isolated from the placenta, amniotic fluid, or fetal tissues only from rats infected prior to breeding (P < 0.001). All infected rats had significantly more loss of pups than did control rats (P < 0.006), but rats infected prior to breeding or at the beginning of the third trimester (gd 14) were much more likely to have fetal losses. Rats infected in the early second trimester after implantation (gd 11) did not experience severe losses. Litter sizes, total litter weight, and individual pup weight from all infected rats, regardless of gestational stage when infected, were significantly smaller than those of control rats (P < 0.001). On the basis of the results of this study, we conclude that the time of infection plays a major role in determination of pregnancy outcome and spread of infection from the genital tract to the respiratory tract. Mycoplasma pulmonis is a common pathogen of mice and rats in many conventionally maintained colonies (3) (4) (5) 14) . In addition to its role in respiratory disease, M. pulmonis is responsible for genital infections and infertility (5, 7, 17, 18) . It has been estimated that M. pulmonis infection decreases rat birth rate 50 to 100% (5, 17). We have demonstrated the adverse impact of genital infection on pregnancy outcome in experimentally infected Sprague-Dawley (SD) rats (17). Genital infection prior to breeding resulted in increased fetal resorptions and an increased number of dams with no liveborn pups (17). Furthermore, individual pup weight, litter size, and litter weight were also decreased in infected rats. In a second study, we demonstrated that M. pulmonis could invade the placenta, breach the placental barrier, and establish an amniotic fluid infection by gestational day (gd) 14 (18). M. pulmonis was isolated from the oropharynx as well as lungs of fetuses at gd 18, confirming in utero transmission (18). Histological evidence was compatible with an active infection characterized by placentitis, amnionitis, and occasional mild fetal bronchopneumonia (18). M. pulmonis is an ideal candidate for a model of intrauterine infection. First, it is a naturally occurring disease. Second, the infection can be established by intravaginal inoculation, without requiring extensive manipulation of the animal. Third, the natural course of disease in the rat is similar to that predicted for human pathogens, i.e., an ascending infection that breaches the placental barrier and establishes as an amnionitis. Finally, a strong database exists for normal reproductive physiology and pregnancy maintenance in the rat. Previous studies demonstrating the adverse effects of genital mycoplasmosis on pregnancy outcome were performed with SD rats infected prior to breeding (17, 18) . The objective of the present study was to determine the effect of stage of gestation at the time of M. pulmonis infection on pregnancy outcome. MATERIALS AND METHODS Rats. Specific-pathogen-free (SPF) SD male and female rats were purchased from a commercial vendor (Harlan Sprague-Dawley, Inc., Indianapolis, Ind.). These rats were monitored and maintained free of the following pathogens: Sendai virus, H-1 virus, rat coronavirus, sialodacryoadenitis virus, reovirus type 3, Kilham rat virus, Hantaan virus, M. pulmonis, respiratory and enteric bacterial pathogens, endoparasites, and ectoparasites. Females were either nonpregnant breeding age animals when shipped or were shipped to arrive on gd 11 or 14. These rats were shipped in filter containers to protect their SPF status. Husbandry. All rats were housed in Microisolator cages (Lab Products, Inc., Maywood, N.J.) to maintain the various infection groups in separate isolation. Rats received autoclaved food (Autoclavable Rodent Laboratory Chow 5010; Purina Mills, Richmond, Ind.) and water ad libitum. Hardwood chip bedding and cages also were autoclaved before use. The cages were opened, and rats were handled only with disinfected, gloved hands under a laminar flow hood. Rats were kept at a density of four rats or fewer per cage. Cages were changed twice per week. Approximately 3 days prior to parturition, pregnant dams were placed in individual cages. Male rats. On the day of arrival, male SPF SD rats were anesthetized with methoxyflurane inhalant anesthesia for ear notching and blood collection. Males were housed with other males when not used for breeding. Male rats were considered infected with M. pulmonis after the first exposure to an infected female. Infected males were not used to breed uninfected control female rats and were not housed with any uninfected rat at any time. Breeding and pregnancy detection. Five to 7 days prior to breeding, female rats were exposed to soiled bedding from the cages of uninfected male rats to synchronize estrous cycles. Vaginal cytology was examined every other day to detect onset of estrus. Females were determined to be ready for breeding when cornified vaginal epithelial cells were first noted. A harem breeding system was used, with two to three females at approximately the late proestrus or early estrus stage of the estrous cycle being housed with one male rat for 3 days, in which time the females would complete one entire estrous cycle. Monitoring of vaginal cytology gave good results for breeding the uninfected rats. However, vaginitis with large numbers of leukocytes in vaginal swabs of infected rats made the vaginal slides difficult to interpret. Therefore, the breeding protocol for infected rats was altered so that harems of two to three infected females were housed with a male rat continuously for 7 days. Within this period, each female was expected to go through two complete estrous cycles. Female rats were given two breeding sessions in which to become pregnant before being judged infertile. Approximately 11 to 14 days after removal of the male, abdominal palpation was performed on the female twice weekly to detect pregnancy. Rats for the 11-daypregnant groups often were chosen on the basis of breeding records alone, since small litters were difficult to palpate at this stage. By gd 14, fetuses were palpable * Corresponding author. † Journal series article R-05118 of the Florida Agricultural Experiment Station. ‡ Present address: Primate Products, Miami, FL 33166. 2315 on May 8, 2020 by guest http://iai.asm.org/ Downloaded from a Rats were infected intravaginally with 3 ϫ 10 7 CFU of M. pulmonis 10 days prior to breeding (Ϫ10) or at gd 11 or 14 or were sham inoculated with sterile broth prior to breeding (control). M. pulmonis was not isolated from any site of any control rat (data not shown). b Rats were necropsied at gd 11, 14, or 18 or within 24 h of parturition (term). c The mean CCU is the mean of the logarithm of the last dilution which showed a color change indicative of growth. The highest possible CCU was 5.0. All groups with different superscripts are statistically different; groups with the same superscript are not statistically different. For vaginal CCU at term, P ϭ 0.005; for tracheal CCU at term, P ϭ 0.0001. No other differences were significant. d Uterine lavage samples were not obtained (ND, not done) at gd 14 and 18 because of the size of the fetal units.