Research in Veterinary Science 1995, 59, 136-138 In vitro survival of the BS isolate of Chlamydia psittaci (ovis) in ruminal and abomasal contents J. D. AMIN*, A. J. WILSMORE?, Department of Animal Health, The Royal Veterinary College, Boltons Park, Potters Bar, Hertfordshire EN6 1NB SUMMARY The ability of the pathogenic BS isolate of Chlamydia psittaci (ovis) to survive after inoculation into ruminal or abomasal con- tents, and ehlamydialtransport medium held at 39°C, was assessed by taking hourly samples which were cultured in mitomycin- treated McCoy cells. The chlamydiaesurvived for nine hours in the ruminal contents, eight hours in the abomasal contents and for 12 hours in transport medium, when the experimentwas concluded.There was a steady decrease in the numbers of the organism in the ruminal and abomasal contents as their pH decreased, but the numbers in the transport medium also decreased without a corresponding change in pH. It is thereforepossible that ewes may become infected with Cpsittaci (ovis) orally via the gastroin- testinal tract. THE infection of sheep with chlamydia by the oral route has been described by McEwen et al (1951) and by Wilsmore et al (1984). The infective process could there- fore involve the passage of the organism either through the rumen and abomasum or through the oropharynx and lungs. Jones and Anderson (1988) reproduced ovine enzootic abortion by inoculating C psittaci into the tonsillar crypts of ewes but could not produce the disease by intraruminal inoculation. This failure might have been due to the inabil- ity of the organism to survive in the runaen or other parts of the gastrointestinal tract, especially in view of the low dose used (1 ml of 104.5 egg-lethal dose [ELD]50m1-1 of chlamy- diae titrated by inoculation of fertile eggs). However, chlamydiae have been isolated from the faeces of sheep (Kawakami et al 1958, Dungworth and Cordy 1962) and have been demonstrated in epithelial cells of the abomasum and small intestine, especially the terminal ileum, of neonatal calves (Doughri et al 1973) indicating that the organism can colonise the gastrointestinal tract. These organisms were probably Cpsittaci immuno/biotype 2, now known as C pecorum (Fuk~shi and Hirai 1992). Amin and Wilsmore (1995) demonstrated chlamydial anti- gen in the epithelial cells &the abomasum and jejunum of sheep infected experimentally with the BS isolate of C psittaci, indicating that abortifacient isolates (immuno/ biotype I) may also colonise the gut. Although there have been no reported infections of the wall of the rumen, the chlamydiae must survive in the ruminal contents for them to pass on to other parts of the gastrointestinal tract, unless they get there haemato- genously. The present study of the in vitro survival of Cpsittaci in the ruminal and abomasal contents of sheep was designed to discover whether the elementary bodies can survive in these environments, so that they can enter the body through the gastrointestinal wall. MATERIALS AND METHODS A ewe was taken from a flock fed on hay and grass cubes. It was seronegative for chlamydial antibodies by an ELISA (Lombard et al 1987) and chlamydiae could not be isolated from its faeces. It was killed and 9 ml samples of ruminal contents (pH 6-6) and abomasal contents (pH 4.3) were collected and stored at -70°C. One of the samples of each fluid was later thawed quick- ly in warm water and placed in a container in a water bath at 39°C. Nine ml of 0.2 M sucrose phosphate chlamydia transport medium (2SP) with additional fetal bovine serum (4 per cent v/v) and antibiotics, and a pH of 7.1 (Gordon et al 1969) was also placed in a container in the bath. One ml of 2SP containing 1.45 x 104 inclusion-forming units (IFU) m1-1 of the BS isolate of C psittaei (ovis) from a case of ovine abortion, which had been passaged 14 times in fertile eggs and was grown in McCoy cells, was added to each of the three containers which were stirred with glass rods and maintained at 39°C. The pH of the mixtures was measured immediately and then hourly for 12 hours. Samples (0.1 ml) of the contents of each container were taken immedi- ately before the addition of the chlamydiae, immediately after, and then hourly for 12 hours, when the study was concluded. These specimens were placed in 0.9 ml of 2SP and stored at -70°C. Subsequently, they were thawed and whirlmixed for 20 seconds. Each sample was placed in a trac tube (Redhill Surgical) containing a monolayer of mitomycin-treated McCoy cells on a coverslip and cultured as described by Woodland et al (1987). After incubation for 48 hours, the cells on the coverslips were fixed, stained with methylene blue by the method of Johnson et al (1978), mounted on slides with DPX and examined microscopically by direct and dark ground illumination. Chlamydial inclu- sions in the McCoy cells were counted and recorded as IFU m1-1 of the original sample. * Present address: Department of Veterinary Surgery and Reproduction, Faculty of Veterinary Medicine, University of Maiduguri, PMB 1069, Maiduguri, Nigeria ? Present address: Veterinary Epidemiology and Economics Research Unit, Department of Agriculture, University of Reading, Earley Gate, PO Box 236, Reading RG6 2AT RESULTS No chlamydiae were demonstrable in the samples of ruminal or abomasal contents taken before the chlamydiae 136