INTRODUCTION

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Until recently, it was believed that all the chlamydial diseases in sheep, including abortion, polyarthritis, and conjunctivitis, were caused by Chlamydia psittaci and that the organism could also be found in the intestines of sheep with no clinical signs of disease. Fukushi and Hirai (5) differentiated a new species, C. pecorum, from C. psittaci, as the species responsible for diseases other than enzootic abortion. Both species have been isolated from the feces of sheep, although the majority of these isolates were probably C. pecorum. Prior to their separation as independent species, C. psittaci and C. pecorum were frequently referred to as abortion or invasive types or strains and enteric, intestinal, or noninvasive types of C. psittaci, respectively.

Clarkson and Philips (4) isolated C. pecorum from the feces of a high proportion of lambs on all 26 farms sampled in Britain, and some of these isolates were shown to be antigenically heterogeneous in contrast to the homogeneity seen in abortion isolates of C. psittaci (2, 6). Jones (9) has recently referred to subtypes of C. pecorum, depending on host and clinical significance, but these subtypes have not yet been differentiated by precise techniques. Examples of subtypes found in sheep are the arthritis/conjunctivitis subtype and the enteric subtype although the intestine may act as a reservoir for other invasive subtypes.

Rodolakis et al. (12) used two mouse models to compare the invasiveness of 10 intestinal isolates and 27 pathogenic isolates of C. psittaci, the majority of which were derived from abortion matter. Although the intestinal isolates were noninvasive when they were injected subcutaneously, they were capable of becoming established in the placentas of pregnant mice after intravenous (i.v.) injection; however, these isolates produced fewer chlamydiae than did the invasive abortion strains.

Little experimental work has been done to investigate whether C. pecorum can invade the placentas of pregnant sheep. Wilson and Dungworth (15) injected 12 pregnant ewes subcutaneously with an isolate from sheep feces, but none developed placental infection. Anderson and Baxter (1) injected four pregnant ewes subcutaneously with an isolate from the feces of a 3-month-old lamb, which also failed to infect the placentas. The circumstances of the isolation of these chlamydiae strongly suggest that they were C. pecorum whereas work in which isolates from the feces of sheep have resulted in placental infection (1, 14) strongly suggests that the species involved was C. psittaci.

The work described here was undertaken to clarify the virulence of several enteric subtypes of C. pecorum isolated from the feces of healthy sheep by their administration to pregnant ewes by oral and i.v. routes. Buzoni-Gatel and Rodolakis (3) suggested that intestinal lesions induced by other bacteria or parasites could promote penetration of the intestinal barrier by intestinal chlamydiae and thus provide the chlamydiae with circumstances for reaching the placenta. If this scenario were possible, abortion could result or, at least, it could be responsible for the cross-reactions which occur during testing for antibody to C. psittaci in sheep health schemes (9, 10). These possibilities were investigated by infecting pregnant ewes with Fasciola hepatica at the same time as C. pecorum since this helminth parasite penetrates the intestinal mucosa for a few days after infection.

	MATERIALS AND METHODS

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Chlamydial isolates. Three isolates were obtained from the feces of lambs on three different farms in Britain and were designated T22, T23, and T25. Isolates T22 and T23 were from farms where it was known that enzootic abortion did not occur, but T25 originated from a farm on which enzootic abortion occurred and C. psittaci had been isolated from fetal membranes. In all three cases, cultures derived from several infected lambs were pooled to establish the isolates.

Cultural methods. The methods used to isolate chlamydiae from rectal fecal samples from lambs have been described previously (4). In order to produce sufficient material to infect sheep, chlamydiae were grown in specific-pathogen-free fertile hens' eggs. Harvested yolk sacs were mixed with an equal volume of transport medium and frozen at 80°C. A small amount was inoculated onto a monolayer in order to calculate the titer (8). For use, the egg-grown material was thawed, sonicated, and centrifuged at 1,000 × g for 10 min, and the sediment and the upper fatty layer were discarded.

Experimental procedure. Ewes, purchased from farms where enzootic abortion did not occur, were bled, and their sera were tested by the complement fixation test for chlamydial antibodies at the Edinburgh Veterinary Investigation Centre. The majority of ewes had titers of 1 in 8, and the remainder had titers of 1 in 16, titers which are considered negative under the regulations of the official Sheep and Goat Health Scheme in Britain. The majority of the ewes were the Welsh Mountain breed, and a few were Welsh Mountain crosses. The ewes were estrus synchronized with intravaginal medroxyprogesterone sponges (Veramix; Upjohn Ltd.), and pregnancy was confirmed by ultrasound scanning 70 days after tupping.

Identification of chlamydiae. Chlamydiae were identified as C. psittaci or C. pecorum initially by their rate of growth, by the type of inclusion in primary culture in McCoy cells (1, 13), and by their ability to multiply in a sheep fibroblast cell culture (11). Further identification of selected isolates was done by restriction endonuclease profiles of DNA amplified by PCR as described previously (7).

	RESULTS

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Lambing results. The lambing results are summarized in Table 1. The 11 uninfected ewes produced 13 lambs, one of which was dead at birth, with a gestation time of 147 days. The 18 ewes which were infected i.v. with C. pecorum produced 21 lambs, 2 of which were regarded as abortions, since they were born dead at gestation times of 137 and 138 days, respectively, and 1 was dead at birth, with a gestation time of 141 days. The 10 ewes which were infected orally produced 13 lambs, 1 dead at birth with a gestation time of 143 days. The six ewes infected orally and also with F. hepatica produced nine live lambs.

Isolation of chlamydiae from reproductive tracts. No chlamydiae were isolated from the placentas or vaginal swabs from the uninfected ewes or from any of those infected orally. Placentas were obtained from 13 of the 18 ewes infected i.v., and vaginal swabs were obtained from the remaining 5. Twelve placentas and four vaginal swabs were positive for chlamydiae by culture.

Identification of chlamydiae. The chlamydiae possessed cultural characteristics of C. pecorum in that they produced mature diffuse inclusions within 48 h and failed to infect a sheep fibroblast cell line. The genomic DNA of isolates from the placentas of sheep 3 and 4 infected i.v. with isolate T22, from placentas or vaginal swabs from sheep 5, 6, 7, 8, 10, 11, and 12 infected i.v. with T23, and from placentas or vaginal swabs from sheep 15 and 17 infected i.v. with T25 were analyzed as described previously (7). Amplified DNA fragments were digested with the enzyme AluI, and the fragments were separated by polyacrylamide gel electrophoresis. All 11 isolates showed bands characteristic of C. pecorum, which were distinct from the pattern seen for C. psittaci (2). The isolated chlamydiae were similar to those used to infect the ewes.

Isolation of chlamydiae from feces. No chlamydiae were isolated from the feces of four ewes infected orally, four ewes infected i.v., or two uninfected ewes sampled twice weekly from 1 week before infection to 30 days after lambing. Chlamydiae were isolated from the feces of two of seven lambs sampled over the first 70 days of life. These lambs were twins from sheep 1, which had been infected i.v. with T22. One lamb had chlamydiae in its feces 30 and 38 days after birth, and the other was positive for chlamydiae 38, 42, and 52 days after birth. The chlamydiae were identified as C. pecorum by their growth characteristics in McCoy cells and their inability to grow in lamb fibroblast cells.

Serology. All the ewes had reciprocal titers of chlamydial complement-fixing antibody of 8 or 16 prior to infection. The majority showed no increase in titer after lambing, even when the ewe had a placental infection. After lambing, one i.v. infected ewe had a reciprocal titer of 32 and two i.v. infected ewes, one of which aborted, had titers of 64.

	DISCUSSION

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This study of the infection of pregnant ewes with C. pecorum produced clear-cut results. i.v. infection resulted in placental colonization whereas oral infection, even with high numbers of chlamydiae, did not. The ewes showed no evidence that the chlamydiae had become established in the alimentary tracts or in any other tissues. Since Wilson and Dungworth (15) and Anderson and Baxter (1) were unable to infect pregnant ewes with fecal isolates of C. psittaci (probably C. pecorum) by subcutaneous injection, enteric isolates of C. pecorum do not appear to be normally invasive in sheep. Similar results have been seen for subcutaneous and i.v. infections in mice (12).

It has been suggested that concurrent infection with other organisms may cause enteric chlamydiae to be able to colonize the placenta (3, 12); Jones et al. (10) speculated that serologically false-positive reactions for C. psittaci may be caused by intestinal infections with parasites, such as coccidia or helminths, which allow the enteric subtype of C. pecorum to change from a localized to a systemic infection. F. hepatica is a helminth which penetrates the small intestine and migrates through the peritoneal cavity to reach the liver, but concurrent infection with this parasite and oral infection with C. pecorum did not result in placental colonization. These results strongly suggest that the enteric subtypes of C. pecorum are unlikely to be invasive. Since most sheep which were infected i.v. did not show a serological response, even though placental invasion occurred, it also seems unlikely that enteric isolates of C. pecorum are responsible for cross-reactions in serological tests for enzootic abortion. Of 18 ewes infected i.v., only 2 showed a reciprocal titer of 64 and another had a titer of 32, which would be considered positive and questionable, respectively, by the official Sheep and Goat Health Scheme in Britain. It should be noted that the ewes were infected i.v. with chlamydiae grown in eggs and that egg proteins contribute to these titers, since the chlamydial group antigen used in the complement fixation test is also egg derived. Jones et al. (10) did not detect cross-reactions to enzootic abortion antigens in specific-pathogen-free lambs which had been vaccinated with an enteric subtype of C. pecorum. They suggested that false seropositivity in field samples for enzootic abortion may be associated with the arthritogenic/conjunctival subtype, since cross-reactions did occur with sera from specific-pathogen-free lambs vaccinated with this subtype.

Although 16 of 18 ewes injected i.v. showed placental infection, only 2 aborted whereas in other experiments with much lower doses of C. psittaci derived from aborting ewes, 70% of pregnant ewes aborted (unpublished observations).

Although no systemic infection resulted from oral infection, probably because these isolates are not invasive, the ewes were from farms on which enteric chlamydiae were common (4). It is possible that the inability to cross the intestinal barrier was a manifestation of immunity in the ewes rather than an intrinsic property of C. pecorum.

Despite frequent examination of the feces of the ewes which had been infected with large numbers of C. pecorum, no chlamydiae were isolated from the feces of any of the ewes, indicating that the bacteria must have been destroyed rapidly. This result may be evidence of a strong immune response, supporting the finding that chlamydiae could not be isolated from adult ewes from farms on which chlamydiae were detected in fecal samples from a high proportion of the lambs (4). No conclusions can be drawn from the finding that twin lambs from an infected ewe tested positive for chlamydiae in their feces on a few occasions since the infection may have been acquired from chlamydiae in the environment and not in the uterus. There is a need for further work on the natural history of C. pecorum in lambs and on the relationship between the subtypes in the intestine and those responsible for diseases such as conjunctivitis and arthritis.