Research in VeterinaryScience, 1992, 53, 267-268
Culture of sheep chlamydia in a sheep fibroblast cell culture H. L. PHILIPS, M. J. CLARKSON, University of Liverpool, Department of Veterinary Clinical Science, Leahurst, Neston, Wirral, L64 7TE
Abortion and enteric isolates of Chlamydiapsittacifrom sheep differed in their growth in a fibroblastic cell culture derived from the small intestine of a lamb. Twenty abortion isolates, each from a different farm, produced large inclusions which could be passaged several times whereas 10 enteric isolates each from different farms (but from some of the farms of origin of the abortion isolates) produced sparse inclusions which could not be passaged. This appears to be a rapid method of distinguishing abortion and enteric isolates and may indicate different nutritional requirements or be related to the invasiveness of the isolates.
E N Z O O T I C abortion of ewes (EAE), caused by Chlarnydiapsittaci, is an important and increasing disease in Britain. Large numbers of organisms are found in the placenta. Cpsittaci is commonly isolated from the faeces of sheep, especially lambs, in flocks with and without EAE (Dungworth and Cordy 1962). Abortion and enteric isolates may be distinguished in a number of ways including differences in inclusion morphology in cell culture (Anderson and Baxter 1986), antigenic differences (Perez-Martinez and Storz 1985, Griffiths et al 1992) and differences in the D N A (McClenaghan et al 1984, A. Herring and I. E. Anderson, personal communication). In addition, abortion isolates are much more invasive than enteric isolates in mice and sheep (Buzoni-Gatel and Rodolakis 1983, Anderson and Baxter 1986, Rodolakis and Souriau 1989) and their protein patterns in sospolyacrylamide gel electrophoresis have been linked to their invasiveness (Rodolakis et al 1989). There is some confusion regarding the biology of these two types of Cpsittaci in sheep in that abortiontype chlamydia have been isolated from the faeces of recently aborted ewes (Anderson and Baxter 1986) and may be shed in the faeces of ewes throughout the year (A. Herring and S. Baxter, personal communication). This communication describes a further difference in that abortion isolates grew in fibroblasts derived from the intestine of a dead lamb whereas enteric isolates did not. A small piece of duodenum was taken aseptically from a neonatal dead lamb which died during partu-
rition, minced with scissors, washed in phosphate buffered saline and trypsinised. The cells were resuspended in 199 medium (Gibco) containing 15 per cent fetal calf serum (Flow Laboratories). Chlortetracycline (Sigma) was incorporated into the medium at a concentration of 10 gg ml I for three weeks. The cells were split in medium containing vancomycin and streptomycin at a concentration of 100 gg m1-1, but no chlortetracycline, before they were used for growing chlamydia. Monolayers for infecting with chlamydia were made by seeding pathology laboratory bottles (Lab Sales) containing a coverslip with 30,000 cells in 1 ml of medium. Chlamydia were centrifuged on to the monolayers the following day at 3000 g for one hour and the monolayers were fixed after 24 hours to 72 hours in methanol and stained with methylene blue. About 10,000 inclusion forming units of 20 abortion isolates (each from a different farm), the conjunctival isolate, 15R (Andrews et al 1987) and 10 enteric isolates, from farms with and without EAE (each from a different farm but some from the same farms as the abortion isolates), were inoculated on to the monolayers. Chlamydia were passaged by sonicating the monolayer in a waterbath and inoculating 100 ~tl of suspension on to a monolayer and centrifuging. All the abortion isolates and the conjunctival isolate produced inclusions which filled nearly all of the cytoplasm of the cell and could be passaged about five times whereas the enteric isolates produced either no inclusions at all or low numbers of small, sparse inclusions which disappeared at the f r s t passage. Buzoni-Gatel and Rodolakis (1983) described chlamydial virulence as the capacity to cross mucous barriers, to spread beyond lymph nodes, to multiply in the reticuloendothelial system and to reach and colonise target organs. Since they found that abortion isolates were virulent whereas enteric isolates were not, it is possible that the ability of abortion isolates to grow in the sheep fibroblast culture correlates with their invasiveness. Alternatively, it may reflect different nutritional requirements. This difference of growth in a cell culture appears to be a rapid and simple method for distinguishing abortion from enteric isolates.
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1-1. L. Philips, M. J. Clarkson
Acknowledgement We should like to thank the Agricultural and Food Research Council for a grant for this study. References ANDERSON, I. E. & BAXTER, T. A. (1986) Chlamydia psittaci: Inclusion morphology in cell culture and virulence in mice of ovine isolates. Veterinary Record 119, 453-455 ANDREWS, A. H., GODDARD, P. C., WILSMORE, A. J. & DAGNALL, G. J. R. (1987) A ehlamydial keratoconjunctivitis in a British sheep flock. Veterinary Record 120, 238-239 BUZONI-GATEL, D. & RODOLAKIS, A. (1983) A mouse model to compare virulence of abortion and intestinal ovine strains of Chlamydia psittaci.- influence of the route of inoculation. Annales de Mierobiologie (Institut Pasteur) 134A, 91-99 DUNGWORTH, D. L. & CORDY, D. R. (1962) The pathogenesis of ovine pneumonia. II. Isolation of virus from faeces; Comparison of pneumonia caused by faecal, enzootic abortion and pneumonia
viruses. Journal of Comparative Pathology 72, 71-79 GRIFFITHS, P. C., PHILIPS, H. L , DAWSON, M & CLARKSON, M. J. (1992) Antigenic and morphological differentiation of placental and intestinal isolates of Chlarnydia psittaci. Veterinary Microbiology 30, 165-177 McCLENAGHAN, M., HERRING, A. J. & AITKEN, I. D. (1984) Comparison of Chlamydia psittaci isolates by ONA restriction endonuclease activity. Infection and lmmunity 45, 384-389 PEREZ-MARTINEZ, J. A. & STORZ, J. (1985) Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence. Infection and Immunity 50, 905-910 RODOLAKIS, A., BERNARD, F., SOURIAU, A., LAYACHI, K. & BUZONI-GATEL, D. (1989) Relationship between virulence of Chlamydia psittaci strains and establishment of persistent infections of McCoy cells. Veterinary Microbiology 19, 65-73 RODOLAKIS, A. & SOURIAU, A. (1989) Variation in the virulence of strains of Chlamydiapsittaci for pregnant ewes. Veterinary Record 125, 87-90
Received February 11, 1992 Accepted May 11, 1992
Culture of sheep chlamydia in a sheep fibroblast cell culture H. L. PHILIPS, M. J. CLARKSON, University of Liverpool, Department of Veterinary Clinical Science, Leahurst, Neston, Wirral, L64 7TE
Abortion and enteric isolates of Chlamydiapsittacifrom sheep differed in their growth in a fibroblastic cell culture derived from the small intestine of a lamb. Twenty abortion isolates, each from a different farm, produced large inclusions which could be passaged several times whereas 10 enteric isolates each from different farms (but from some of the farms of origin of the abortion isolates) produced sparse inclusions which could not be passaged. This appears to be a rapid method of distinguishing abortion and enteric isolates and may indicate different nutritional requirements or be related to the invasiveness of the isolates.
E N Z O O T I C abortion of ewes (EAE), caused by Chlarnydiapsittaci, is an important and increasing disease in Britain. Large numbers of organisms are found in the placenta. Cpsittaci is commonly isolated from the faeces of sheep, especially lambs, in flocks with and without EAE (Dungworth and Cordy 1962). Abortion and enteric isolates may be distinguished in a number of ways including differences in inclusion morphology in cell culture (Anderson and Baxter 1986), antigenic differences (Perez-Martinez and Storz 1985, Griffiths et al 1992) and differences in the D N A (McClenaghan et al 1984, A. Herring and I. E. Anderson, personal communication). In addition, abortion isolates are much more invasive than enteric isolates in mice and sheep (Buzoni-Gatel and Rodolakis 1983, Anderson and Baxter 1986, Rodolakis and Souriau 1989) and their protein patterns in sospolyacrylamide gel electrophoresis have been linked to their invasiveness (Rodolakis et al 1989). There is some confusion regarding the biology of these two types of Cpsittaci in sheep in that abortiontype chlamydia have been isolated from the faeces of recently aborted ewes (Anderson and Baxter 1986) and may be shed in the faeces of ewes throughout the year (A. Herring and S. Baxter, personal communication). This communication describes a further difference in that abortion isolates grew in fibroblasts derived from the intestine of a dead lamb whereas enteric isolates did not. A small piece of duodenum was taken aseptically from a neonatal dead lamb which died during partu-
rition, minced with scissors, washed in phosphate buffered saline and trypsinised. The cells were resuspended in 199 medium (Gibco) containing 15 per cent fetal calf serum (Flow Laboratories). Chlortetracycline (Sigma) was incorporated into the medium at a concentration of 10 gg ml I for three weeks. The cells were split in medium containing vancomycin and streptomycin at a concentration of 100 gg m1-1, but no chlortetracycline, before they were used for growing chlamydia. Monolayers for infecting with chlamydia were made by seeding pathology laboratory bottles (Lab Sales) containing a coverslip with 30,000 cells in 1 ml of medium. Chlamydia were centrifuged on to the monolayers the following day at 3000 g for one hour and the monolayers were fixed after 24 hours to 72 hours in methanol and stained with methylene blue. About 10,000 inclusion forming units of 20 abortion isolates (each from a different farm), the conjunctival isolate, 15R (Andrews et al 1987) and 10 enteric isolates, from farms with and without EAE (each from a different farm but some from the same farms as the abortion isolates), were inoculated on to the monolayers. Chlamydia were passaged by sonicating the monolayer in a waterbath and inoculating 100 ~tl of suspension on to a monolayer and centrifuging. All the abortion isolates and the conjunctival isolate produced inclusions which filled nearly all of the cytoplasm of the cell and could be passaged about five times whereas the enteric isolates produced either no inclusions at all or low numbers of small, sparse inclusions which disappeared at the f r s t passage. Buzoni-Gatel and Rodolakis (1983) described chlamydial virulence as the capacity to cross mucous barriers, to spread beyond lymph nodes, to multiply in the reticuloendothelial system and to reach and colonise target organs. Since they found that abortion isolates were virulent whereas enteric isolates were not, it is possible that the ability of abortion isolates to grow in the sheep fibroblast culture correlates with their invasiveness. Alternatively, it may reflect different nutritional requirements. This difference of growth in a cell culture appears to be a rapid and simple method for distinguishing abortion from enteric isolates.
267
268
1-1. L. Philips, M. J. Clarkson
Acknowledgement We should like to thank the Agricultural and Food Research Council for a grant for this study. References ANDERSON, I. E. & BAXTER, T. A. (1986) Chlamydia psittaci: Inclusion morphology in cell culture and virulence in mice of ovine isolates. Veterinary Record 119, 453-455 ANDREWS, A. H., GODDARD, P. C., WILSMORE, A. J. & DAGNALL, G. J. R. (1987) A ehlamydial keratoconjunctivitis in a British sheep flock. Veterinary Record 120, 238-239 BUZONI-GATEL, D. & RODOLAKIS, A. (1983) A mouse model to compare virulence of abortion and intestinal ovine strains of Chlamydia psittaci.- influence of the route of inoculation. Annales de Mierobiologie (Institut Pasteur) 134A, 91-99 DUNGWORTH, D. L. & CORDY, D. R. (1962) The pathogenesis of ovine pneumonia. II. Isolation of virus from faeces; Comparison of pneumonia caused by faecal, enzootic abortion and pneumonia
viruses. Journal of Comparative Pathology 72, 71-79 GRIFFITHS, P. C., PHILIPS, H. L , DAWSON, M & CLARKSON, M. J. (1992) Antigenic and morphological differentiation of placental and intestinal isolates of Chlarnydia psittaci. Veterinary Microbiology 30, 165-177 McCLENAGHAN, M., HERRING, A. J. & AITKEN, I. D. (1984) Comparison of Chlamydia psittaci isolates by ONA restriction endonuclease activity. Infection and lmmunity 45, 384-389 PEREZ-MARTINEZ, J. A. & STORZ, J. (1985) Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence. Infection and Immunity 50, 905-910 RODOLAKIS, A., BERNARD, F., SOURIAU, A., LAYACHI, K. & BUZONI-GATEL, D. (1989) Relationship between virulence of Chlamydia psittaci strains and establishment of persistent infections of McCoy cells. Veterinary Microbiology 19, 65-73 RODOLAKIS, A. & SOURIAU, A. (1989) Variation in the virulence of strains of Chlamydiapsittaci for pregnant ewes. Veterinary Record 125, 87-90
Received February 11, 1992 Accepted May 11, 1992
