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Replication of Australian porcine isolates of Ileal symbiont intracellularis in tissue culture
veterinary microbiology
ELSEWER
Veterinary
Microbiology
49 (1996) 249-255
Replication of Australian porcine isolates of IZeaL symbiont intracellularis in tissue culture A.M. Collins av*,I. Swift a, R.P. Monckton b aL.uTrobe University Be&go, Biological and Chemical Sciences, PO Box 199, Bendigo 3550. Australia b Cyanamid Websters, PO Box 234, Baulkham Hills 2153, Australia
Received 12 December 1994; accepted 24 October 1995
Abstract Ileal sytnbiont intracellularis (ISI) isolated from Australian cases of PIA and PHE was replicated in the rat ileum enterocyte cell line IEC 18. The number of IS1 within cells varied, as did the number of IS1 infected within the monolayer. At 24 h post infection a large number of cells were infected with approximately 100 IS1 per cell. At the termination of infection, fewer IEC 18 cells were infected but IS1 had replicated to fill the cell cytoplasmic space. Numerous foci of infected cells were visible in the monolayer, containing as many as 15 densely infected cells. Division of IS1 infected cells indicated the transmission of IS1 in the cytoplasm to daughter cells. This suggests that the replication of IS1 in culture appears to be reasonably cell dependent. No cytopathic effects were observed in the infected cultures. Keywords: Ileal symbiont intracellularis (ISI); Cell culture; 16s ribosomal RNA; Porcine matosis (PIA); Proliferative haemorrhagic enteropathy (PHE); Proliferative enteritis (PE)
intestinal
adeno-
1. Introduction Zleal syrnbionf inrrucelluluris (ISI) is an obligate intracellular bacterium associated with the conditions porcine intestinal adenomatosis (PIA) and proliferative haemorrhagic enteropathy (PHE) (Rowland and Lawson, 1992; Gebhart et al., 1993). PIA is an economically significant enteric disease of intensively reared, weaned pigs. Clinical signs in.clude ill thrift, diarrhoea and a retarded growth rate. More acute symptoms occur
l
Corresponding
author. Tel: 61 54 447222; Fax: 61 54 447777.
0378-I 135/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0378-l 135(95)00194-8
250
A.M. Collins et al./ Veterinary Microbiology
49 (1996) 249-255
in the related condition PHE, where intestinal haemorrhage leads to sudden death in grower pigs. Both conditions are presently controlled using costly in-feed antibiotics. Lesions of PIA and PHE commonly occur in the terminal ileum and proximal large intestine. Histologically lesions appear as adenomatous thickening of the mucosa, composed of enlarged, branching glands of poorly differentiated columnar epithelium. Secretory goblet cells are absent from the adenomatous tissue (Rowland and Lawson, 1974; Rowland and Lawson, 1992). IS1 are spiral or curved rods which lie freely within the apical cytoplasm of proliferating enterocytes in the porcine ileum. They are nonflagellate, Gram-negative bacteria, approximately 2 pm in length and 0.3 pm in diameter (Rowland and Lawson, 1974). IS1 has been most closely related to the sulfate-reducing bacterium Desuljiuvibtio desulfuricuns (91% similarity) using 16s ribosomal RNA (rRNA) sequence homology. It is also relatively similar to other members of the Desulfovibrionaceae (greater than 86%) (Gebhart et al., 1993). This degree of sequence similarity suggests a relationship with the sulfate-reducing bacteria, but DNA/DNA homology studies are required for further classification. Attempts to culture IS1 in media designed for the isolation of Desulfovibrios are currently unsuccessful (Collins, unpublished). Prior to our culture attempts, two isolates of the bacterium had been cultured in the rat ileum enterocyte cell line IEC 18 (Lawson et al., 1993). IS1 isolates passaged several times in IEC 18 cells prior to oral inoculation successfully reproduced lesions of proliferative enteropathy in the ileum of conventional pigs (McOrist et al., 1993). In addition, the same cultures of IS1 were able to reproduce lesions of PIA in gnotobiotic pigs previously challenged with known gut microflora, demonstrated to be non-pathogenic for gnotobiotic piglets (McOrist et al., 1994). This communication describes the in vitro replication of IS1 isolated from an Australian case of PHE by the method of Lawson et al. ( 1993). Detection of intracellular IS1 in the rat ileum enterocyte cell culture was by indirect immunogold staining of infected monolayers.
2. Materials
and methods
2.1. ISI preparation Ileal sections of intensively reared pigs, diagnosed with PIA or PHE by histopathology, were frozen at - 80°C. IS1 bacteria were isolated by scraping the thawed ileal mucosa, separating detached enterocytes from extracellular contaminants by repeated washing and low speed centrifugation, homogenising enterocytes in an omnimixer, and treating with trypsin to release the intracellular bacteria. IS1 were then partially purified by filtration through 0.65um filters. The filtrate was stained by modified Ziehl-Neelsen to identify the number of acid fast IS1 isolated. One relatively abundant preparation of IS1 was chosen for the culture inoculum in this study. The inoculum was isolated from a PHE outbreak in 22 week old non-medicated pigs. Four other Australian isolates have
A.M. Collins et al./ Veterinary Microbiology
49 (1996) 249-255
251
also been cultured in the IEC 18 cell line. Post filtration the IS1 inocula were frozen at - 80°C in Dulbeccos Modified Eagles Media (DMEM) containing 7% v/v fetal calf serum (FCS), and 10% v/v dimethyl sulphoxide (DMSO) or in sucrose potassium glutamate with 10% FCS. 2.2. CelE culture The rat ileum enterocyte cell line IEC 18 (American Type Culture Collection CRL1589) was routinely grown in DMEM with L-glutamine and S%FCS (Cytosystems, Castle Hill, Australia), without antibiotics, at 37°C in 5% CO,. IEC 18 cells were infected with IS1 as described in detail by Lawson et al. (1993). Briefly, IEC 18 cells were trypsinised, then 7 X 10’ cells were seeded into 75 cm* flasks. Frozen aliquots of the IS1 inocula were thawed rapidly, diluted in DMEM with 7% FCS before being added to recently subcultured IEC 18 cells. The IEC 18 cells and IS1 inoculum were centrifuged at 4000 g for 20 min, then incubated in a microaerophillic atmosphere of 8% O,, 8.8% CO, and 82% N, at 37°C. Three hours post infection (pi) fresh growth medium was added with 100 pg/ml vancomycin and 50 pg/l gentamicin and reincubated as above. The medium was replaced 2-3 days pi, and the infe’ction terminated at 5 days pi. 2.3. Indirect immunogold assay Intact monolayers of IEC 18 cells infected with IS1 were washed with PBS, fixed in 85% acetone for 20 min at - 2O”C, then incubated with a monoclonal antibody directed against an outer membrane protein (25-27 kDa) specific to IS1 (McOrist et al., 1987; McOrist et al., 1989). Specimens were washed in PBS/Tween 20, then incubated with colloidal gold (10 nm particles) conjugated goat-anti-mouse IgG (Sigma, Castle Hill, Australia). The colour reaction was developed with quinol(0.5M), citrate buffer (0.02 M trisodium citrate, 0.0295 M citric acid, pH 4.0) and silver lactate (0.037 M). Cells were counterstained with eosin, and viewed by light microscopy.
3. Results The rat ileum enterocyte cells IEC 18 have been infected with preparations of IS1 bacteria isolated from Australian cases of PIA and PHE. Indirect immunogold staining of ISI infected IEC 18 monolayers revealed curved bacteria in the cytoplasm of IEC 18 cells. Cultures fixed and stained at 24 h pi indicated that a large percentage of IEC 18 cells were infected with ISI. The number of IS1 within cells varied. Some cells contained approxkmately one hundred IS1 in the cytoplasm forming a ring surrounding the nucleus (Fig. la). It is important to note however that at the termination of infection only a small percentage of cells remained infected with ISI. In these densely infected cells the IS1 had replicated to fill most of the cytoplasmic space of the IEC 18 cell (Fig. lb).
252
A.M. Collins et al./ Veterinary Microbiology 49 (1996) 249-255
IS1 within the cytoplasm of a dividing cell were transferred to the daughter cells in the final stages of cell division (Fig. 1~). Foci of infected cells were visible in the monolayer (Fig. Id) containing up to 15 densely infected cells.
Fig. 1. a. Immunogold stained monolayer of ISI infected IEC 18 cells, using an anti-IS1 monoclonal antibody supplied by Dr S. McOrist. Cells were counterstained with eosin. The darkly stained curved bodies in a ring around the nucleus within the IEC 18 cells are intracellular ISI. Darkly stained extracellular aggregates of antigen may be due to suboptimal growth of the intracellular bacterium. Magnification X 1000. b. Focus of three densely infected IEC 18 cells where IS1 have replicated to fill most of the cell cytoplasm space. Magnification X 1000. c. IS1 infected parent IEC 18 cell in the late stages of cell division where nuclear and cytoplasmic contents, including the intracellular ISI, have separated to the two daughter cells, just prior to the formation of a new cell membrane. Magnification X 1000. d. Part of a focus of infection visible in the monolayer containing 15 densely infected cells. Intracellular ISI, stained by immunogold, form a ring around the nucleus. Magnification X 1000.
A.M. Collins et al. / Veterinary Microbiolo,qy 49 (1996) 249-2.55
253
Fig. 1(continued).
No cytopathic effects were observed in IS1 infected IEC 18 cells. In addition, cultures of IEC 18 cells infected with IS1 appeared to cover the tissue culture flask surface area in the same number of days as non-infected IEC 18 cells.
4. Discussion We have demonstrated the in vitro replication of the bacterium ISI, isolated from an Australian case of PHE, in the rat ileum enterocyte cell line IEC 18. The bacterium we
254
A.M. Collins et al./ Veterinary Microbiology
49 (1996) 249-255
have cultured appears to be related to IS intracellularis strains deposited with the National Collection of Type Cultures (NCTC 12656 and NCTC 12657) by Lawson et al. (1993). This relationship can be verified by the ability of the monoclonal antibody directed against an outer membrane protein of IS1 (supplied by McOrist), to detect our intracellular curved bacterium in IEC 18 cells. The reactive protein of this antibody is absent from the Campylobacter sp. commonly found in porcine intestines affected by proliferative enteritis (McOrist et al., 1987; McOrist et al., 1989). The presence of extracellular aggregates staining with the IS1 specific monoclonal antibody was also observed by Lawson et al. (1993) and explained as suboptimal growth of the intracellular organism. The similarity of our Australian IS1 isolate to the above mentioned isolates deposited with NCTC, is further verified by the independent sequencing of the IS1 16s rRNA gene (Morrow, unpublished). This sequence is identical to that published by Gebhart et al. (19931, which places IS1 in a distinct new genus within the delta group of Proteobacteria. IS1 infected IEC 18 cells (Fig. lc) observed during the final stages of cell division indicate that the intracellular bacteria in the cytoplasm are transferred to daughter cells. Lawson et al. (1993) also observed that division of infected IEC 18 cells was responsible for much of the spread of infection in IEC 18 cell monolayers. There seems enough evidence to suggest that IS1 multiplies best in dividing cells and may rely on host cell division for continued replication. The presence of foci of IS1 infected IEC 18 cells appears to mimic the suggested naturally occurring in-vivo IS1 infection of porcine enterocytes, where IS1 initially infect crypt enterocytes and spread to the surrounding epithelium by cell division (Lawson et al., 1993). The foci pattern of IS1 infection in IEC 18 monolayers may further correlate with the contrast observed in-vivo between diseased proliferating tissue and adjacent areas of healthy epithelium (Lawson and McOrist, 1993). Comparison of non-infected IEC 18 monolayers with IS1 infected monolayers indicated that no cytopathic effects were observed in IS1 infected IEC 18 cells. We have not as yet been able to eliminate the possibility of non-cytopathic viruses in the IS1 culture. Lawson et al. (1993) observed that in the first 48 h of infection, some IS1 infected IEC 18 cells rounded up and appeared to be detaching from the monolayer. This may in part explain why in our cultures fewer IEC 18 cells were infected at the termination of infection compared with the number of cells infected at 24 h post infection. Further studies may indicate whether it is possible that certain IS1 infected IEC 18 cells may provide a more suitable environment for bacterial replication in the first 24 h of infection. The immunogold histochemical method of detection of IS1 in cell culture has enabled the quantification of cells infected with ISI per unit area of monolayer. However further culture experiments need to be undertaken to determine how much of a role the dose and the isolate of ISI play in the replication of IS1 in culture. Jasni et al. (1994) suggested in his disease transmission experiments with IS1 in hamsters that the severity of PE may be influenced by the number of infective bacteria in the inoculum.
A.M. Collins et al./ Veterinary Microbiology 49 (1996) 249-255
255
Acknowledgements The authors would like to acknowledge the financial support of the Pig Research and Development Corporation and the advice of Dr Frank Allison.
References Gebhart, C.J., Barnes, SM., McOrist, S.. Lin, G.F. and Lawson, G.H.K., 1993. Ileal symbiont intracellularis, an oblil:ate intracellular bacterium of porcine intestines showing a relationship to Desulfouibrio species. Int. J. Syst. Bacterial., 43: 533-538. Jasni, S., McOrist, S. and Lawson, G.H.K., 1994. Reproduction of proliferative enteritis in hamsters with a pure culture of porcine ileal symbiont intracellularis. Vet. Microbial., 41: l-9. Lawson, G.H.K. and McOrist, S., 1993. The enigma of the Proliferative Enteropathies: a review. J. Comp. Path., 108: 41-46. Lawson, G.H.K., McOrist, S., Jasni, S. and Mackie, R.A., 1993. Intracellular bacteria of proliferative enteropathy: cultivation and maintenance in vitro. J. Clin. Microbial., 3 1: 1136-l 142. McOrist, S., Boid, R., Lawson, G.H.K. and McConnell, I., 1987. Monoclonal antibodies to intracellular campylobacter-like organisms of the porcine proliferative enteropathies. Vet. Rec., 12 1: 42 l-422. McOrist, S., Boid, R. and Lawson, G.H.K., 1989. Antigenic analysis of Campylobacter species and an intracellular Campylobacter-like organism associated with porcine proliferative enteropathies. Infect. lmmun., 57: 957-962. McOrist, S , Jasni, S., Mackie, R.A., Macintyte, N., Neef, N. and Lawson, G.H.K., 1993. Reproduction of porcine proliferative enteropathy with pure cultures of ileal symbiont intracellularis. Infect. Immun., 62: 4210-4215. McOrist, S., Mackie, R.A.. Neef, N., Aitken, I. and Lawson, G.H.K., 1994. Synergism of IS1 and gut bacteria in the reproduction of porcine proliferative enteropathy. Vet. Rec., 134: 33 l-332. Rowland, AC. and Lawson, G.H.K.. 1974. Intestinal adenomatosis in the pig: immunofluorescent and electron microscopic studies. Res. Vet. Sci., 17: 323-330. Rowland, P,.C. and Lawson, G.H.K., 1992. Porcine proliferative enteropathies, In: A.D. Leman. B.E. Straw, W.L. Mengeling, S. D’Allaire and D.J. Taylor (Eds.), Diseases of swine, 7th ed., Iowa State University Press, Ames, pp 560-569.
ELSEWER
Veterinary
Microbiology
49 (1996) 249-255
Replication of Australian porcine isolates of IZeaL symbiont intracellularis in tissue culture A.M. Collins av*,I. Swift a, R.P. Monckton b aL.uTrobe University Be&go, Biological and Chemical Sciences, PO Box 199, Bendigo 3550. Australia b Cyanamid Websters, PO Box 234, Baulkham Hills 2153, Australia
Received 12 December 1994; accepted 24 October 1995
Abstract Ileal sytnbiont intracellularis (ISI) isolated from Australian cases of PIA and PHE was replicated in the rat ileum enterocyte cell line IEC 18. The number of IS1 within cells varied, as did the number of IS1 infected within the monolayer. At 24 h post infection a large number of cells were infected with approximately 100 IS1 per cell. At the termination of infection, fewer IEC 18 cells were infected but IS1 had replicated to fill the cell cytoplasmic space. Numerous foci of infected cells were visible in the monolayer, containing as many as 15 densely infected cells. Division of IS1 infected cells indicated the transmission of IS1 in the cytoplasm to daughter cells. This suggests that the replication of IS1 in culture appears to be reasonably cell dependent. No cytopathic effects were observed in the infected cultures. Keywords: Ileal symbiont intracellularis (ISI); Cell culture; 16s ribosomal RNA; Porcine matosis (PIA); Proliferative haemorrhagic enteropathy (PHE); Proliferative enteritis (PE)
intestinal
adeno-
1. Introduction Zleal syrnbionf inrrucelluluris (ISI) is an obligate intracellular bacterium associated with the conditions porcine intestinal adenomatosis (PIA) and proliferative haemorrhagic enteropathy (PHE) (Rowland and Lawson, 1992; Gebhart et al., 1993). PIA is an economically significant enteric disease of intensively reared, weaned pigs. Clinical signs in.clude ill thrift, diarrhoea and a retarded growth rate. More acute symptoms occur
l
Corresponding
author. Tel: 61 54 447222; Fax: 61 54 447777.
0378-I 135/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0378-l 135(95)00194-8
250
A.M. Collins et al./ Veterinary Microbiology
49 (1996) 249-255
in the related condition PHE, where intestinal haemorrhage leads to sudden death in grower pigs. Both conditions are presently controlled using costly in-feed antibiotics. Lesions of PIA and PHE commonly occur in the terminal ileum and proximal large intestine. Histologically lesions appear as adenomatous thickening of the mucosa, composed of enlarged, branching glands of poorly differentiated columnar epithelium. Secretory goblet cells are absent from the adenomatous tissue (Rowland and Lawson, 1974; Rowland and Lawson, 1992). IS1 are spiral or curved rods which lie freely within the apical cytoplasm of proliferating enterocytes in the porcine ileum. They are nonflagellate, Gram-negative bacteria, approximately 2 pm in length and 0.3 pm in diameter (Rowland and Lawson, 1974). IS1 has been most closely related to the sulfate-reducing bacterium Desuljiuvibtio desulfuricuns (91% similarity) using 16s ribosomal RNA (rRNA) sequence homology. It is also relatively similar to other members of the Desulfovibrionaceae (greater than 86%) (Gebhart et al., 1993). This degree of sequence similarity suggests a relationship with the sulfate-reducing bacteria, but DNA/DNA homology studies are required for further classification. Attempts to culture IS1 in media designed for the isolation of Desulfovibrios are currently unsuccessful (Collins, unpublished). Prior to our culture attempts, two isolates of the bacterium had been cultured in the rat ileum enterocyte cell line IEC 18 (Lawson et al., 1993). IS1 isolates passaged several times in IEC 18 cells prior to oral inoculation successfully reproduced lesions of proliferative enteropathy in the ileum of conventional pigs (McOrist et al., 1993). In addition, the same cultures of IS1 were able to reproduce lesions of PIA in gnotobiotic pigs previously challenged with known gut microflora, demonstrated to be non-pathogenic for gnotobiotic piglets (McOrist et al., 1994). This communication describes the in vitro replication of IS1 isolated from an Australian case of PHE by the method of Lawson et al. ( 1993). Detection of intracellular IS1 in the rat ileum enterocyte cell culture was by indirect immunogold staining of infected monolayers.
2. Materials
and methods
2.1. ISI preparation Ileal sections of intensively reared pigs, diagnosed with PIA or PHE by histopathology, were frozen at - 80°C. IS1 bacteria were isolated by scraping the thawed ileal mucosa, separating detached enterocytes from extracellular contaminants by repeated washing and low speed centrifugation, homogenising enterocytes in an omnimixer, and treating with trypsin to release the intracellular bacteria. IS1 were then partially purified by filtration through 0.65um filters. The filtrate was stained by modified Ziehl-Neelsen to identify the number of acid fast IS1 isolated. One relatively abundant preparation of IS1 was chosen for the culture inoculum in this study. The inoculum was isolated from a PHE outbreak in 22 week old non-medicated pigs. Four other Australian isolates have
A.M. Collins et al./ Veterinary Microbiology
49 (1996) 249-255
251
also been cultured in the IEC 18 cell line. Post filtration the IS1 inocula were frozen at - 80°C in Dulbeccos Modified Eagles Media (DMEM) containing 7% v/v fetal calf serum (FCS), and 10% v/v dimethyl sulphoxide (DMSO) or in sucrose potassium glutamate with 10% FCS. 2.2. CelE culture The rat ileum enterocyte cell line IEC 18 (American Type Culture Collection CRL1589) was routinely grown in DMEM with L-glutamine and S%FCS (Cytosystems, Castle Hill, Australia), without antibiotics, at 37°C in 5% CO,. IEC 18 cells were infected with IS1 as described in detail by Lawson et al. (1993). Briefly, IEC 18 cells were trypsinised, then 7 X 10’ cells were seeded into 75 cm* flasks. Frozen aliquots of the IS1 inocula were thawed rapidly, diluted in DMEM with 7% FCS before being added to recently subcultured IEC 18 cells. The IEC 18 cells and IS1 inoculum were centrifuged at 4000 g for 20 min, then incubated in a microaerophillic atmosphere of 8% O,, 8.8% CO, and 82% N, at 37°C. Three hours post infection (pi) fresh growth medium was added with 100 pg/ml vancomycin and 50 pg/l gentamicin and reincubated as above. The medium was replaced 2-3 days pi, and the infe’ction terminated at 5 days pi. 2.3. Indirect immunogold assay Intact monolayers of IEC 18 cells infected with IS1 were washed with PBS, fixed in 85% acetone for 20 min at - 2O”C, then incubated with a monoclonal antibody directed against an outer membrane protein (25-27 kDa) specific to IS1 (McOrist et al., 1987; McOrist et al., 1989). Specimens were washed in PBS/Tween 20, then incubated with colloidal gold (10 nm particles) conjugated goat-anti-mouse IgG (Sigma, Castle Hill, Australia). The colour reaction was developed with quinol(0.5M), citrate buffer (0.02 M trisodium citrate, 0.0295 M citric acid, pH 4.0) and silver lactate (0.037 M). Cells were counterstained with eosin, and viewed by light microscopy.
3. Results The rat ileum enterocyte cells IEC 18 have been infected with preparations of IS1 bacteria isolated from Australian cases of PIA and PHE. Indirect immunogold staining of ISI infected IEC 18 monolayers revealed curved bacteria in the cytoplasm of IEC 18 cells. Cultures fixed and stained at 24 h pi indicated that a large percentage of IEC 18 cells were infected with ISI. The number of IS1 within cells varied. Some cells contained approxkmately one hundred IS1 in the cytoplasm forming a ring surrounding the nucleus (Fig. la). It is important to note however that at the termination of infection only a small percentage of cells remained infected with ISI. In these densely infected cells the IS1 had replicated to fill most of the cytoplasmic space of the IEC 18 cell (Fig. lb).
252
A.M. Collins et al./ Veterinary Microbiology 49 (1996) 249-255
IS1 within the cytoplasm of a dividing cell were transferred to the daughter cells in the final stages of cell division (Fig. 1~). Foci of infected cells were visible in the monolayer (Fig. Id) containing up to 15 densely infected cells.
Fig. 1. a. Immunogold stained monolayer of ISI infected IEC 18 cells, using an anti-IS1 monoclonal antibody supplied by Dr S. McOrist. Cells were counterstained with eosin. The darkly stained curved bodies in a ring around the nucleus within the IEC 18 cells are intracellular ISI. Darkly stained extracellular aggregates of antigen may be due to suboptimal growth of the intracellular bacterium. Magnification X 1000. b. Focus of three densely infected IEC 18 cells where IS1 have replicated to fill most of the cell cytoplasm space. Magnification X 1000. c. IS1 infected parent IEC 18 cell in the late stages of cell division where nuclear and cytoplasmic contents, including the intracellular ISI, have separated to the two daughter cells, just prior to the formation of a new cell membrane. Magnification X 1000. d. Part of a focus of infection visible in the monolayer containing 15 densely infected cells. Intracellular ISI, stained by immunogold, form a ring around the nucleus. Magnification X 1000.
A.M. Collins et al. / Veterinary Microbiolo,qy 49 (1996) 249-2.55
253
Fig. 1(continued).
No cytopathic effects were observed in IS1 infected IEC 18 cells. In addition, cultures of IEC 18 cells infected with IS1 appeared to cover the tissue culture flask surface area in the same number of days as non-infected IEC 18 cells.
4. Discussion We have demonstrated the in vitro replication of the bacterium ISI, isolated from an Australian case of PHE, in the rat ileum enterocyte cell line IEC 18. The bacterium we
254
A.M. Collins et al./ Veterinary Microbiology
49 (1996) 249-255
have cultured appears to be related to IS intracellularis strains deposited with the National Collection of Type Cultures (NCTC 12656 and NCTC 12657) by Lawson et al. (1993). This relationship can be verified by the ability of the monoclonal antibody directed against an outer membrane protein of IS1 (supplied by McOrist), to detect our intracellular curved bacterium in IEC 18 cells. The reactive protein of this antibody is absent from the Campylobacter sp. commonly found in porcine intestines affected by proliferative enteritis (McOrist et al., 1987; McOrist et al., 1989). The presence of extracellular aggregates staining with the IS1 specific monoclonal antibody was also observed by Lawson et al. (1993) and explained as suboptimal growth of the intracellular organism. The similarity of our Australian IS1 isolate to the above mentioned isolates deposited with NCTC, is further verified by the independent sequencing of the IS1 16s rRNA gene (Morrow, unpublished). This sequence is identical to that published by Gebhart et al. (19931, which places IS1 in a distinct new genus within the delta group of Proteobacteria. IS1 infected IEC 18 cells (Fig. lc) observed during the final stages of cell division indicate that the intracellular bacteria in the cytoplasm are transferred to daughter cells. Lawson et al. (1993) also observed that division of infected IEC 18 cells was responsible for much of the spread of infection in IEC 18 cell monolayers. There seems enough evidence to suggest that IS1 multiplies best in dividing cells and may rely on host cell division for continued replication. The presence of foci of IS1 infected IEC 18 cells appears to mimic the suggested naturally occurring in-vivo IS1 infection of porcine enterocytes, where IS1 initially infect crypt enterocytes and spread to the surrounding epithelium by cell division (Lawson et al., 1993). The foci pattern of IS1 infection in IEC 18 monolayers may further correlate with the contrast observed in-vivo between diseased proliferating tissue and adjacent areas of healthy epithelium (Lawson and McOrist, 1993). Comparison of non-infected IEC 18 monolayers with IS1 infected monolayers indicated that no cytopathic effects were observed in IS1 infected IEC 18 cells. We have not as yet been able to eliminate the possibility of non-cytopathic viruses in the IS1 culture. Lawson et al. (1993) observed that in the first 48 h of infection, some IS1 infected IEC 18 cells rounded up and appeared to be detaching from the monolayer. This may in part explain why in our cultures fewer IEC 18 cells were infected at the termination of infection compared with the number of cells infected at 24 h post infection. Further studies may indicate whether it is possible that certain IS1 infected IEC 18 cells may provide a more suitable environment for bacterial replication in the first 24 h of infection. The immunogold histochemical method of detection of IS1 in cell culture has enabled the quantification of cells infected with ISI per unit area of monolayer. However further culture experiments need to be undertaken to determine how much of a role the dose and the isolate of ISI play in the replication of IS1 in culture. Jasni et al. (1994) suggested in his disease transmission experiments with IS1 in hamsters that the severity of PE may be influenced by the number of infective bacteria in the inoculum.
A.M. Collins et al./ Veterinary Microbiology 49 (1996) 249-255
255
Acknowledgements The authors would like to acknowledge the financial support of the Pig Research and Development Corporation and the advice of Dr Frank Allison.
References Gebhart, C.J., Barnes, SM., McOrist, S.. Lin, G.F. and Lawson, G.H.K., 1993. Ileal symbiont intracellularis, an oblil:ate intracellular bacterium of porcine intestines showing a relationship to Desulfouibrio species. Int. J. Syst. Bacterial., 43: 533-538. Jasni, S., McOrist, S. and Lawson, G.H.K., 1994. Reproduction of proliferative enteritis in hamsters with a pure culture of porcine ileal symbiont intracellularis. Vet. Microbial., 41: l-9. Lawson, G.H.K. and McOrist, S., 1993. The enigma of the Proliferative Enteropathies: a review. J. Comp. Path., 108: 41-46. Lawson, G.H.K., McOrist, S., Jasni, S. and Mackie, R.A., 1993. Intracellular bacteria of proliferative enteropathy: cultivation and maintenance in vitro. J. Clin. Microbial., 3 1: 1136-l 142. McOrist, S., Boid, R., Lawson, G.H.K. and McConnell, I., 1987. Monoclonal antibodies to intracellular campylobacter-like organisms of the porcine proliferative enteropathies. Vet. Rec., 12 1: 42 l-422. McOrist, S., Boid, R. and Lawson, G.H.K., 1989. Antigenic analysis of Campylobacter species and an intracellular Campylobacter-like organism associated with porcine proliferative enteropathies. Infect. lmmun., 57: 957-962. McOrist, S , Jasni, S., Mackie, R.A., Macintyte, N., Neef, N. and Lawson, G.H.K., 1993. Reproduction of porcine proliferative enteropathy with pure cultures of ileal symbiont intracellularis. Infect. Immun., 62: 4210-4215. McOrist, S., Mackie, R.A.. Neef, N., Aitken, I. and Lawson, G.H.K., 1994. Synergism of IS1 and gut bacteria in the reproduction of porcine proliferative enteropathy. Vet. Rec., 134: 33 l-332. Rowland, AC. and Lawson, G.H.K.. 1974. Intestinal adenomatosis in the pig: immunofluorescent and electron microscopic studies. Res. Vet. Sci., 17: 323-330. Rowland, P,.C. and Lawson, G.H.K., 1992. Porcine proliferative enteropathies, In: A.D. Leman. B.E. Straw, W.L. Mengeling, S. D’Allaire and D.J. Taylor (Eds.), Diseases of swine, 7th ed., Iowa State University Press, Ames, pp 560-569.