Abortion and Subsequent Excretion of Chlamydiae from the Reproductive Tract of Sheep during Estrus

Transcription

1 INFECrION AND IMMUNITY, Sept. 1994, p Vol. 62, No /94/$ Copyright X 1994, American Society for Microbiology Abortion and Subsequent Excretion of Chlamydiae from the Reproductive Tract of Sheep during Estrus JOHN R. PAPP,1* PATRICIA E. SHEWEN,1 AND CATHY J. GARTLEY2 Department of Veterinary Microbiology and Immunology, 1 and Department of Population Medicine,2 Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 18 April 1994/Returned for modification 30 May 1994/Accepted 21 June 1994 Chiamydia psittaci serovar 1 infection in pregnant sheep typically causes abortion or the birth of weak lambs. Eight sheep that experienced chlamydia-induced abortion during their first pregnancy were successfully rebred yearly for the past 2 years. Chiamydia-specific lipopolysaccharide was detectable for approximately 3 weeks in vaginal swabs taken from the experimentally infected sheep following abortion. There was no evidence of chlamydiae in vaginal, placental, or neonatal samples obtained immediately after each subsequent successful pregnancy. Sera collected from the experimentall infected sheep had persistent, high antibody levels to C. psittaci, suggesting continued exposure of the immune system to the organism. Examination of vaginal specimens obtained during various stages of the estrus cycle revealed detectable levels of chlamydiae only when the animal was near ovulation. Chlamydiae were not detected in swabs from sheep that did not experience abortion. Enhanced chlamydial excretion during the periovulation period of sheep may provide sufficient stimulation of the immune system to account for the persistent antibody response. Furthermore, the association between estrus and chlamydial shedding has important implications for transmission of infection to other ewes during breeding. Ovine abortion induced by Chlamydia psittaci serovar 1 is known commonly as enzootic abortion and is a major cause of reproductive failure in most sheep-producing countries, with the exception of Australia and New Zealand. Pregnant sheep initially infected with C. psittaci either abort late in gestation or give birth to weak or stillborn lambs as a result of placental pathology associated with infection (16). The infection may become established first at a mucosal site, from which it may disseminate by blood or lymph to the placenta. Experimental infection of tonsillar or vaginal mucosae resulted in chlamydial abortion in susceptible sheep (3, 9). Although placental infection may be detected as early as 15 days postinfection (13), pathological changes do not become apparent until the third trimester (6). In the final stages of pregnancy, the placenta is heavily infected with C. psittaci, and damage to the maternalfetal junctions may lead to fetal death and/or infection. However, sheep are seldom affected clinically following abortion and remain fertile (24). Environmental contamination resulting from diseased placentas or vaginal discharge is considered as the primary source of infection to other sheep. Ingestion of infected tissue or contaminated feed may result in intestinal colonization and persistence of C. psittaci serovar 1 within the flock. Although chlamydiae may be detected in the feces of clinically healthy sheep (15), the degree of intestinal infection with C. psittaci serovar 1 is not known. It is believed that susceptible sheep initially become infected via oropharnygeal exposure during the previous or current lambing season (9). Since abortion is recognized to induce immunity to subsequent abortion, sheep management strategies have been directed towards limiting the exposure of naive animals to infected material by physical separation, antibiotic therapy, or vaccination. Although such * Corresponding author. Mailing address: Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada NlG 2W1. Phone: (519) , ext Fax: (519) Electronic mail address: jpapp@uoguelph.ca practices reduce the level of abortion within a flock, they are not successful in eliminating the disease. In the present report, we demonstrate that pregnant sheep experimentally infected with an ovine abortion isolate of C. psittaci either abort or give birth to weak lambs and maintain a persistent systemic antibody response to the organism for up to 2.5 years postinfection. Furthermore, sheep that experienced an abortive episode excreted detectable amounts of chlamydial antigen from the reproductive tract during subsequent estrus cycles. MATERIALS AND METHODS Chlamydia culture and purification. C. psittaci isolate V287 or strain B577 (ATCC VR-656) was used to experimentally infect pregnant sheep. Isolate V287 was recovered from an aborted ovine fetus in The organism was originally isolated by inoculation of infected fetal tissue into the yolk sac of 7-day-old embryonated hen eggs as described previously (18). Organisms were subsequently grown in HeLa 229 cell (ATCC CCL 2.1) monolayers in 175-cm2 polystyrene culture flasks (Fisher Scientific, Unionville, Ontario, Canada) in 50 ml of Eagle minimum essential medium (EMEM; ICN Flow, Mississauga, Ontario, Canada) supplemented with 10% fetal bovine serum (GIBCO BRL, Burlington, Ontario, Canada), 100,ug of vancomycin hydrochloride per ml (Sigma Chemical Co., St. Louis, Mo.), and 25 U of nystatin per ml (Sigma). Confluent monolayers were infected with yolk sac-propagated C. psittaci isolate V287 or strain B577 suspended in sucrosephosphate-glutamic acid (SPG; ph 7.2) by shaking on a rocking platform at room temperature for 4 h. Fresh EMEM containing 0.5,ug of cycloheximide per ml (Sigma) was added to the cells, and the flasks were incubated at 37 C under 5% CO2 for 3 to 4 days. Infected monolayers were gently dislodged with glass beads and centrifuged at 500 x g for 10 min at 4 C. The supernatant was collected, and the pellet was resuspended in SPG. Following brief sonication, the SPG suspension was centrifuged again as described above. The supernatants were pooled and centrifuged at 30,000 x g for 30 min at 4 C (50.2 Ti

2 VOL. 62, 1994 CHLAMYDIAL EXCRETION IN SHEEP AFTER ABORTION 3787 Group A Four ewes infected with C. psittaci isolate V287 at day 60 of gestation Group B Four ewes infected with C. psittaci isolate V287 at day 60 ofgestation Monitored to parturtion. Rebred the following year. All four ewes were infected wiffi C. psittaci isolate V287 at day 60 of gestation. Monitored to parturition. Rebred the following year. to parturition. Rebred approximately four months following parturition. All four ewes were infected with C. psittaci strain B577 at 60 days gestation. Monitored to parturition. All four ewes were rebred approximately four months following parturition. Group D Four ewes infcted with C. psittaci strain B577 at 60 days gestation. (Monitored to parturition.l FIG. 1. Experimental design for challenge of sheep with C. psittaci. Groups A, B, and C were maintained for approximately 2.5 years. Group D was added to the study during the second year. rotor; Beckman Instruments, Inc., Mississauga, Ontario, Canada). The pellets containing infectious chlamydiae were resuspended in SPG to approximately 1/10 the original volume and stored at C. Propagation of the B577 strain was carried out similarly in HeLa 229 cells. Preparations were confirmed to be free of viruses, mycoplasmas, or other bacteria by conventional culture (4). The inoculum used to experimentally infect sheep contained an estimated % egg-lethal dose (18). Chlamydial antigen required for enzyme-linked immunosorbent assay (ELISA) was prepared from McCoy cell (ATCC CRL 1696) monolayers, grown in EMEM supplemented with 10% fetal bovine serum and 200,ug of streptomycin (Sigma), and inoculated with C. psittaci V287 infectious stock. After 4 days of incubation at 37 C under 5% C02, chlamydiae were recovered as described above. The elementary bodies (EBs) were then partially purified by being layered over 10 ml of 35% (vol/vol) Renografin solution (diatrizoate melgumine and diatrizoate sodium, 76% for injection; E. R. Squibb & Sons, Princeton, N.J.) in 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Sigma) containing 0.15 M NaCl (Fisher Scientific) and centrifuged at 43,000 x g for 1 h at 4 C (SW27 rotor; Beckman). The pellet was resuspended in approximately 5 ml of SPG and centrifuged at 30,000 x g for 30 min at 4 C. One to 2 ml of SPG was used to resuspend the pellet in each centrifuge tube, and the EB suspension was stored at -135 C. The protein concentration of the partially purified chlamydial EBs (7) was determined by the method of Lowry et al. (10). Isolation of chlamydiae from vaginal swabs was attempted in L929 cells (ATCC CCL 1) grown in 25-cm2 plastic tissue culture flasks (GIBCO BRL) containing EMEM supplemented with 10% fetal bovine serum and 200,ug of streptomycin (Sigma). Immediately following collection, the swabs were placed in transport medium, i.e., 2 ml of EMEM containing 100,ug of vancomycin hydrochloride (Sigma) per ml and 25 U of nystatin (Sigma) per ml. Inoculation of L929 cells with the transport medium and subsequent treatment were similar to that described with infected HeLa cells. The pellet obtained after ultracentrifugation was used to inoculate fresh L929 cells. After three inoculation cycles in fresh cells, the media and cell pellet were tested for chlamydia-specific lipopolysaccharide (LPS) by using a Clearview Chlamydiae test kit (Unipath, Inc., Nepean, Ontario, Canada). Sheep, experimental challenge, and design. Twelve 1-yearold Arcott ewes, confirmed pregnant by ultrasound examination at 50 days of gestation, were obtained from a flock free of C. psittaci serovar 1 and housed in a disease containment facility. The sheep were randomly separated into three groups of four (Fig. 1), and each group was housed in separate rooms. At 60 days of gestation, two groups (groups A and B) were injected subcutaneously in the left axilla with 1 ml of infectious C. psittaci V287 (2). The third group (group C) was similarly injected with a control inoculum prepared from uninfected HeLa cells. Rectal temperatures, appetite, and attitude were recorded daily for 5 days preinfection and 10 days postinfection. The sheep were retained and rebred the following year. Four ewes and one ram were grouped together after synchronization of the estrus cycles of the ewes by using vaginally applied progesterone sponges (Veramix; The Upjohn Co., Orangeville, Ontario, Canada) and injectable pregnant mare serum gonadotrophin (PMSG; Equinex; Ayerst Laboratories, Montreal, Quebec, Canada). The ewes were mated with a ram obtained from the C. psittaci-free source flock. Pregnancy diagnosis was completed by ultrasound examination at 45 days after ovulation. The ram was mated first with ewes of the control group and then the two groups of ewes that had been experimentally infected with chlamydiae during the previous pregnancy. Only one control ewe failed to conceive. This ewe was not rebred and was removed from the remainder of the study. Group A

3 3788 PAPP ET AL. sheep were reinfected at 60 days of gestation with a subcutaneous injection of C. psittaci V287 into the left axilla while group B sheep were not reinfected. Group C animals were again injected with the control inoculum. The animals were monitored for clinical signs as described before. One of the three remaining control ewes developed clinical signs consistent with acute copper toxicity late in gestation and was euthanized by intravenous infusion with sodium pentobarbital (Euthanyl Forte; M.T.C. Pharmaceuticals, Cambridge, Ontario, Canada). Approximately 4 months after parturition, the ewes were synchronized for breeding as described before. The ewes of the two experimentally infected groups were mated with one ram, and the control ewes were bred with a different ram obtained from the same C. psittaci-free sheep flock. The four ewes in group A were experimentally infected with C. psittaci B577 at 60 days of gestation as described previously with isolate V287. During this phase of the study, a fourth group (group D) of four pregnant 2-year-old Arcott ewes were obtained from the C. psittaci-free flock, and the left axilla of each animal was subcutaneously injected with C. psittaci B577 at 60 days of gestation (Fig. 1). The sheep in group B were not infected, and the two remaining ewes in group C received control inoculum. The sheep were monitored as described before. All sheep were bled by jugular venipuncture at weekly intervals when they were discovered to be pregnant and monthly when they were not confirmed pregnant. Tissues associated with abortion or parturition were examined for macroscopic lesions, bacterial contamination, and the presence of chlamydial infection by using a Clearview Chlamydiae test kit. Vaginal swabs were collected from each postpartum ewe at 3- to 4-day intervals until three consecutive swabs were negative for chlamydial antigen. The animal experimentation was in accordance with the guidelines of the Canadian Council on Animal Care and approved by the University of Guelph's Animal Care Committee ĖLISA. Ovine sera were tested for the presence of chlamydial-specific immunoglobulin G (IgG) antibodies by an ELISA. Polystyrene microtitration plates (GIBCO BRL) were inoculated with partially purified C. psittaci V287 EBs, diluted to 2,ug of protein per 100 RI of phosphate-buffered saline (PBS), and incubated for 4 h at 37 C. The V287 EB antigen did not dry on the microtiter plates during the 4-h incubation period. The plates were washed with block buffer, i.e., PBS containing 0.05% Tween 20 (Fisher Scientific) and 0.5% fish skin gelatin (Fisher Scientific) warmed to 37 C. Uncoated sites in the microtitration plate wells were then blocked by incubation with block buffer for 30 min at 37 C in a humidified incubator. Test sera were diluted in block buffer at a concentration of 1:400 in volumes of 100,u per well, and the samples were replicated four times on each plate as described by Wright (26). The plates were incubated at 37 C for 2 h in a humidified incubator and then washed three times with block buffer as described before. Rabbit anti-sheep IgG (heavy and light chain)-alkaline phosphatase conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was added at a concentration of 1:5,000, and the plates were incubated for 1 h as described before. The plates were washed three times, and color was developed by adding p-nitrophenylphosphate (Sigma). The positive control serum was obtained from a ewe experimentally infected with C. psittaci and used at a dilution of 1:400. The negative control was serum collected from a noninfected ewe. The optical density (OD) at 405 nm was recorded with a microplate autoreader (Biotechnology Instruments, Winooski, Vt.) when the positive control gave an OD reading of approximately 1. Results were expressed as a ratio of the OD reading of the test sample to the OD reading of the positive control. Determination of 1gM reactivity to C. psittaci in the ovine sera was performed similarly except that serum samples were diluted 1/50 prior to testing. The concentration of partially purified C. psittaci V287 was increased to 4,ug of protein per 100 RI of PBS, and rabbit anti-sheep IgM(,) alkaline phosphatase conjugate (Kirkegaard & Perry) was used at a concentration of 1:100. The positive control serum was a pool of ovine sera collected approximately 1 week after experimental infection with C. psittaci V287. The negative control serum was the same as that for the IgG ELISA, and both sera were used at a dilution of 1/50. Vaginal detection of chlamydiae. Approximately 2.5 years after the primary experimental infection of groups A and B, the estrus cycles of groups A, B, and C were synchronized with progesterone sponges (Veramix) and injectable gonadotrophin (PMSG). A sponge was inserted into the vagina of each ewe for a period of 14 days, and PMSG was administered intramuscularly at the time of sponge removal. Ovulation is expected to occur 60 to 70 h after PMSG treatment (20). Prior to sponge application, vaginal swabs were collected from all of the ewes and, following removal of the sponge, at 2- to 3-day intervals for up to 10 days postovulation. The external os of the vagina was cleaned with 4% chlorhexidine gluconate skin cleanser, and the left lateral wall of the vaginal mucosa was sampled, approximately 9 cm distal to the cervix, with the aid of a vaginal speculum. Each swab was tested for chlamydiaspecific LPS by using the Clearview Chlamydiae test kit. Serum samples were collected at 2-day intervals during the course of the experiment and tested for presence of anti-c. psittaci IgG and IgM antibodies as described previously. Approximately 2 months after estrus synchronization with progesterone sponges and injectable gonadotrophin, the sheep in groups A, B, and C were randomly allocated to two subgroups. The estrus cycle of subgroup 1 was synchronized as described before, while estrus synchronization of subgroup 2 was achieved by using injectable progesterone and then PMSG. Subgroup 2 received 1 ml of Centra Progestin (Dispar, Joliette, Quebec, Canada) given intramuscularly at 3-day intervals for 9 days and then four daily intramuscular injections of 0.1 ml of Gesterol in Oil (E. L. Stickley and Co., Brantford, Ontario, Canada). One day after the last progesterone injection, the sheep were given PMSG, and ovulation occurred 60 to 70 h later. Vaginal and serum samples were collected and processed as described previously. Statistical analysis. Stringency of the ELISA was assigned by using the coefficient of variation between quadruplicate positive control wells on each plate. Data obtained from a plate were used only if the coefficient of variation was less than or equal to 5%. The antibody responses to C. psittaci in experimental groups A, B, and C were compared by using the Student t test (17). RESULTS INFECT. IMMUN. Experimental infection. Primary infection of pregnant sheep with C. psittaci V287 resulted in a febrile response 24 to 48 h postinfection. The rise in rectal temperature varied from 1 to 2 C above preinoculation means and lasted for 5 to 7 days. During this time, the sheep were mildly depressed and slightly anorexic, but they recovered rapidly and remained clinically normal until abortion. A similar but less prolonged elevation in rectal temperature was observed in sheep reinfected (group A) with C. psittaci V287 or B577 during subsequent pregnancies.

4 VOL. 62, 1994 CHLAMYDIAL EXCRETION IN SHEEP AFTER ABORTION ~~0.8 ~ Infection with Abortion or birth heterologous of weak lambs strain > Primary ilinfection Parturition teneto ~~~~~Parturition Months post-primary infection * Group A -all-groupb FIG. 2. Average IgM antibody response of sheep to C. psittaci. The sheep were initially infected with C. psittaci V287. Group A ewes were reinfected with isolate V287 in the second pregnancy and C. psittaci B577 during the third pregnancy. Sheep in Group B were not reinfected. Both groups aborted or gave birth to weak lambs following primary infection. There were no clinical signs associated with the administration of control inoculum to group C sheep. Seven of eight experimentally infected ewes aborted their first pregnancies between 115 and 139 days of gestation. One infected ewe delivered two weak and one dead lamb at 141 days of gestation. Placentas obtained from the infected animals contained numerous discolored necrotic cotyledons and a thickened edematous intercotyledonary chorion with adherent clay-colored exudate. Both placental and vaginal swabs collected within 8 h of abortion were positive for chlamydial antigen by the Clearview Chlamydiae test kit. Other than chlamydiae, there were no significant bacterial findings. Vaginal excretion of chlamydiae was apparent for approximately 3 weeks postabortion. All subsequent pregnancies resulted in the birth of healthy lambs without evidence of chlamydial infection from placental and vaginal swabs taken postpartum. Sheep that previously experienced abortion induced by C. psittaci V287 had normal pregnancies and delivered healthy lambs after experimental infection with strain B577 (group A). However, primary infection of chlamydia-negative pregnant sheep with C. psittaci B577 (group D) resulted in two animals aborting at 115 and 128 days of gestation. The other two ewes gave birth to weak full-term lambs. Placental lesions and bacteriological findings from group D were similar to those obtained from groups A and B following primary infection with isolate V287. Vaginal swabs collected from sheep in group D were positive for chlamydial antigen up to 3 weeks postpartum, while similar swabs from group A were negative. The first pregnancy in the control group ended in the birth of healthy lambs from three ewes at approximately 142 days of gestation. Parturition in the remaining ewe resulted in two healthy lambs and one dead lamb. Necropsy of the dead lamb revealed a lacerated liver and an abdominal cavity filled with approximately 15 ml of blood. There were no significant bacterial findings in the fetal liver, lung, spleen, and kidney. Placental tissues obtained from all control sheep were free of gross pathologic lesions and chlamydial antigen. Vaginal swabs from these animals were negative for chlamydial antigen. One of the three control sheep that conceived for a second pregnancy aborted four fetuses at 130 days of gestation. The body condition of the lambs was normal, and there were no significant postmortem findings. Approximately 36 h postabortion, the ewe underwent a hemolytic crisis and was euthanized. Ante- and postmortem findings were consistent with acute copper toxicosis. The two remaining ewes gave birth to healthy lambs at approximately 143 days of gestation. The third pregnancies in these two animals were uneventful and terminated normally. In all cases, chlamydial LPS was not detected in placental or vaginal swabs obtained from the control group. Systemic antibody response. Chlamydia-specific IgM antibodies were detected only in postinoculation sera obtained from the experimentally infected groups (Fig. 2). The response increased 1 week postinfection and then steadily declined until the sheep aborted or gave birth. Subsequent infection of group A with isolate V287 was associated with increased IgM antibody reactivity to C. psittaci approximately 2 weeks postinfection. However, when the sheep were infected with the heterologous strain B577, a much stronger IgM response was detected and yet the ewes gave birth to healthy lambs. The IgG response to C. psittaci continued to increase for 2

5 3790 PAPP ET AL. INFECT. IMMUN. 1.4 Infection with 1.2 heterologous Reinfection strain *n0.8 CL. 0.6 Abortion or birth 0.4 of weak lambs Parturition i Primary Infection 0.2 rtuntiontion Months post-primary infection -4--Group A - Group B N Group C FIG. 3. Average IgG antibody response of sheep to C. psittaci. The sheep were initially infected with C. psittaci isolate V287. Group A ewes were reinfected with isolate V287 in the second pregnancy and C. psittaci B577 during the third pregnancy. Sheep in Group B were not reinfected. Group C animals were not infected. Groups A and B aborted or gave birth to weak lambs following primary infection. Downloaded from weeks postinfection and then slightly decreased until parturition (Fig. 3). Both groups A and B maintained a persistent IgG response to the organism for up to 2.5 years postinfection. However, the baseline response in group A was moderately higher, compared with that of group B, following reexposure to isolate V287. Infection with strain B577 increased the response approximately 3 to 4 weeks postinfection. The mean IgG reactivity of the serum from the control sheep to C. psittaci was significantly less (P < 0.05) than any of the experimentally infected groups. Estrus synchronization of sheep in groups A and B resulted in a slight increase in IgM and IgG antibody reactivity to C. psittaci. Sequential serum samples collected from groups A and B at 2- to 3-day intervals during the estrus cycle revealed a slight increase in antichlamydial reactivity during the periovulation period in three of eight sheep. However, this response was not statistically different when compared with that of sheep in other periods of the estrus cycle. Detection of chlamydiae. Two and a half years after primary infection, estrus synchronization in groups A and B resulted in vaginal excretion of chlamydiae during the periovulation period (Tables 1 and 2). Vaginal shedding of chlamydiae became apparent approximately 3 days prior to ovulation and lasted up to 4 days postovulation. There was no detection of chlamydial LPS on vaginal swabs obtained prior to estrus synchronization or in the noninfected control sheep. L929 cell monolayers inoculated with material from vaginal swabs collected during ovulation were also positive for chlamydial LPS after the third passage. Chlamydiae could not be detected, either by direct examination of the swab or by cell culture inoculation, in the noninfected control sheep. DISCUSSION As expected, infection of pregnant sheep with C. psittaci V287 resulted in abortion or the birth of weak lambs (2). With the exception of a slight increase in rectal temperature after experimental infection, the poor outcome of the first pregnancy was the only clinical indication of chlamydial infection. This mimics the clinical situation in natural infection, and therefore, sheep producers are usually unaware of impending chlamydia-induced abortions. Epidemiological data suggest that 30% of ewes may abort or give birth to weak lambs in flocks newly infected with C. psittaci, and once infection is established, rates of 5 to 10% are common (1). Because of the lack of clinical disease, the significant loss of potential income for the sheep producer may be apparent only at the end of the lambing season and unrecoverable until the next season. Once the sheep experienced abortion or the birth of weak lambs, they remained refractory to subsequent C. psittaciinduced abortion. Chlamydiae were not detected in placentas or postpartum vaginal secretions obtained at the conclusion of the second and third pregnancies. However, Wilsmore and associates (24) were able to identify one ewe that previously aborted and then excreted chlamydiae in the products of the next parturition. The authors noted that the lambs from this ewe were healthy and did not appear to be infected with C. psittaci. This suggests that although sheep do not experience further chlamydia-induced abortions, they may be chronically infected. The IgM antibody response to C. psittaci, as determined by ELISA, quickly dropped after primary infection and occasionally was not detectable. Traditionally, the complement fixation on October 1, 2018 by guest

6 VOL. 62, 1994 CHLAMYDLAL EXCRETION IN SHEEP AFTER ABORTION 3791 TABLE 1. Presence of chlamydial antigen on vaginal swabs collected from sheep after estrus synchronization with progesterone sponges Treatment Stage of estrus cycle Presence of chlamydial antigen' on vaginal swabs' (no. of positive samples/total no. tested) Experimentally infected sheep Noninfected sheep Preinsertion of progesterone spongec NDd 0/8 0/2 Sponge removal and injection of PMSG 3 days preovulation 8/8 0/2 NYt Ovulation 8/8 0/2 NT 2 days postovulation 8/8 0/2 NT 4 days postovulation 5/8 0/2 NT 6 days postovulation 0/8 0/2 NT 8 days postovulation 0/8 0/2 NT 10 days postovulation 0/8 0/2 Chlamydia-specific LPS was detected by using a Clearview Chlamydiae test kit (Unipath, Inc.). "Vaginal swabs were obtained from a site approximately 9 cm distal to the cervix. Veramix (Upjohn Co.) sponges were used. dnd, not determined. ent, no treatment. test (CFT) was employed for the serologic monitoring of sheep for C. psittaci infection. Since the CFT has an IgM bias, our results might indicate why the CFT has been less than successful in identifying seropositive animals. The lack of sensitivity of the CFT when compared with the ELISA has been demonstrated previously (11). The IgG antibody response to C. psittaci remained elevated for up to 2.5 years post-primary infection. This appears to correlate with immunity to subsequent abortion in the four ewes reinfected with C. psittaci during each pregnancy. However, the importance of other components of the immune reaction, such as T lymphocytes and various cytokines, for the control and resolution of chlamydial infections has been documented (8, 12). Although specific antibodies are known to neutralize in vitro cellular infectivity by C. psittaci (12), it is unknown if the antibody response observed in the present study was merely an indication of immune response or an actual contributor to protection. The four ewes that were challenged only once with C. psittaci maintained a relatively high IgG antibody response to the organism throughout the course of the study. This was somewhat surprising since the sheep were segregated from potential reexposure to infective material. Therefore, the possibility of a persistent chlamydial infection in all of the experimentally TABLE 2. infected sheep was explored. We noticed slight incremental increases in the antibody response of individual ewes when they were prepared for breeding by synchronization of the estrus cycles. Since abortion is accompanied by the passage of an infected placenta and the excretion of the organism for at least 3 weeks, we targeted the reproductive tract as a likely site of chlamydial colonization and persistence. By careful examination of vaginal swabs taken at timed intervals during the 17-day estrus cycle, we discovered that excretion of chlamydiae occurred only during the periovulation period. This narrow window of detection was observed in all of the previously experimentally infected sheep and was consistent regardless of the method of estrus synchronization. That is, chlamydial shedding could not be accounted for simply by mechanical irritation due to the presence of a vaginal sponge. Since chlamydial shedding from the reproductive tract coincided with ovulation and therefore the period these animals would normally be bred, the potential for venereal transmission is significant. Appleyard and associates (3) demonstrated that dose-dependent venereal transmission of C. psittaci in sheep can occur but concluded that it was unlikely to significantly contribute to infection of sheep in Great Britain. However, the observation that detectable amounts of chlamydiae are shed during estrus may be relevant to transmission and Presence of chlamydial antigen on vaginal swabs collected from sheep after estrus synchronization with either injectable progesterone or progesterone sponges Presence of chlamydial antigena on vaginal swabsb obtained from sheep after estrus synchronization with: Stage of estrus cycle Injectable progesteronec Progesterone spongesd Experimentally Noninfected Experimentally Noninfected infected sheep sheep infected sheep sheep Prior to estrus synchronization 0/4e 0/1 0/4 0/1 3 days preovulation 4/4 0/1 3/4 0/1 Ovulation 4/4 0/1 4/4 0/1 2 days postovulation 4/4 0/1 4/4 0/1 4 days postovulation 0/4 0/1 1/4 0/1 6 days postovulation 0/4 0/1 0/4 0/1 8 days postovulation 0/4 0/1 0/4 0/1 10 days postovulation 0/4 0/1 0/4 0/1 Chlamydia-specific LPS was detected by using the Clearview Chlamydiae kit (Unipath, Inc.). b Vaginal swabs were obtained from a site approximately 9 cm distal to the cervix. Centra Progestin (Dispar) and Gesterol in Oil (E. L. Stickley and Co.) were the progesterones used. d Veramix (Upjohn Co.) sponges were used. Number of positive samples per total number of samples tested.

7 3792 PAPP ET AL. persistence of the organism within a flock. Furthermore, it may be possible to improve management of infected flocks through detection of carrier ewes at estrus. In humans, Chlamydia trachomatis is an important reproductive tract pathogen with the potential to cause tubal infertility in females. The risk of tubal occlusion increases with the frequency and intensity of salpingitis (22, 23). In monkeys, repeated chlamydial infection of the fallopian tubes produces extensive scarring, with peritubal adhesions, chronic salpingitis, and distal tubal obstruction (14). Although the pathogenesis is unclear, there is a growing body of evidence supporting an immunopathogenic mechanism (5, 19, 21). If the augmented immune response is due to persistent or repeated infection, then the sheep described in this study may also be prone to developing reproductive pathology. In the United Kingdom, Winter and Dobson (25) examined the genital tracts of ewes that were sent for slaughter. Although the history of the cull ewes was unknown, they were sampled during the spring when sheep are usually slaughtered because they are barren. Of the 133 cull ewes examined, 16.8% had macroscopic abnormalities likely to interfere with pregnancy, including damage to the fallopian tubes and fibrinous adhesions of the mucosal folds. Conclusive evidence for reproductive tract pathology developing in sheep with cryptic chlamydial infections has not been documented. However, it is possible that enhanced chlamydial excretion during the periovulation period in sheep may eventually result in reproductive pathology not unlike that observed in women. In conclusion, sheep that experienced abortion due to experimental infection with C. psittaci became carriers for the organism. The carrier state was generally undetectable except during the periovulation period of subsequent estrus cycles. Although these animals had two healthy pregnancies following abortion, the clinically inapparent persistence of chlamydiae within the reproductive tract may be a potential source of infection to other animals. Furthermore, this infection could be identified in vaginal swabs collected a few days before and after ovulation. The possibility for similar shedding and venereal transmission under natural conditions, in which the estrus cycle is not artificially synchronized, should be examined. Also, characterization of the local and systemic immune responses in chronically infected sheep and assessment of the correlation with reproductive pathology may yield valuable insight into the mechanisms of immunopathogenesis in human females infected with C. trachomatis. ACKNOWLEDGMENT This research was supported by the Red Meat II program of the Ontario Ministry of Agriculture and Food. REFERENCES 1. Aitken, I. D Ovine chlamydial abortion, p In Z. Woldehiwet and M. Ristic (ed.), Rickettsial and chlamydial diseases of domestic animals. Pergamon Press, New York. 2. Andreani, E., A. Poli, F. Tolari, D. Cerri, R. Farina, and P. Bandecchi Experimental infection of sheep with Chlamydia psittaci. Br. Vet. J. 143: Appleyard, W. T., I. D. Aitken, and I. E. Anderson Attempted venereal transmission of Chlamydia psittaci in the sheep. Vet. Rec. 116: Balows, A., W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, INFECT. IMMUN. and H. J. Shadomy Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 5. Brunham, R. C., R. Peeling, I. MacLean, M. L. Kosseim, and M. Paraskevas Chlamydia trachomatis-associated ectopic pregnancy: serologic and histologic correlates. J. Infect. Dis. 165: Buxton, D., R. M. Barlow, J. Finlayson, I. E. Anderson, and A. MacKellar Observations on the pathogenesis of Chlamydia psittaci infection in pregnant sheep. J. Comp. Pathol. 102: Caldwell, H. D., J. Kromhout, and J. Schachter Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31: Igietseme, J. U., and R. G. Rank Susceptibility to reinfection after a primary chlamydial genital infection is associated with a decrease of antigen-specific T cells in the genital tract. Infect. Immun. 59: Jones, G. E., and I. E. Anderson Chlamydia psittaci: is tonsillar tissue the portal of entry in ovine enzootic abortion? Res. Vet. Sci. 44: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Markey, B. K., M. S. McNulty, and D. Todd Comparison of serological tests for the diagnosis of Chlamydia psittaci infection in sheep. Vet. Microbiol. 36: Moulder, J. W Interaction of chlamydiae and host cells in vitro. Microbiol. Rev. 55: Papp, J. R., P. E. Shewen, and C. J. Gartley Chlamydia psittaci infection and associated infertility in sheep. Can. J. Vet. Res. 57: Patton, D. L., C. C. Kuo, S. P. Wang, and S. A. Halbert Distal tubal obstruction induced by repeated Chiamydia trachomatis salpingeal infections in pig-tailed macaques. J. Infect. Dis. 155: Rodolakis, A., F. Bernard, and F. Lanter Mouse models for evaluation of virulence of Chlamydia psittaci isolated from ruminants. Res. Vet. Sci. 46: Shewen, P. E Chlamydial infection in animals: a review. Can. Vet. J. 21: Steel, R. G. D., and J. H. Torrie Principles and procedures of statistics. A biometrical approach. McGraw-Hill Book Co., New York. 18. Storz, J Chlamydia and chlamydia induced diseases. Charles C Thomas, Publisher, Springfield, Ill. 19. Toye, B., C. Laferriere, P. Claman, P. Jessamine, and R. Peeling Association between antibody to the chlamydial heat-shock protein and tubal infertility. J. Infect. Dis. 168: Trower, C. J Artificial control of breeding in ewes. The Compendium 15: Wagar, E. A., J. Schachter, P. Bavoil, and R. S. Stephens Differential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens. J. Infect. Dis. 162: Westrom, L Incidence, prevalence, and trends of acute pelvic inflammatory disease and its consequences in industrialized countries. Am. J. Obstet. Gynecol. 138: Westrom, L., R. Joesoef, G. Reynolds, A. Hagdu, and S. E. Thompsom Pelvic inflammatory disease and fertility. Sex. Transm. Dis. 19: Wilsmore, A. J., K. A. Izzard, B. C. Wilsmore, and G. L. R. Dagnall Breeding performance of sheep infected with Chlamydia psittaci (ovis) during their preceding pregnancy. Vet. Rec. 126: Winter, A. C., and H. Dobson Observations on the genital tract of cull ewes. Vet. Rec. 130: Wright, P Enzyme immunoassay: observations on aspects of quality control. Vet. Immunol. Immunopathol. 17: