J. Med. Microbiol. - Vol. 37 (1992), 382-384 0 1992 The Pathological Society of Great Britain and Ireland
Protection from Shigella sonnei i nfection by immun isat ion of rabbits with Plesiornonas shigelloides (SVC 01) S. SAYEED*, D. A. SACK? and F. QADRlf Department of Microbiology, University of Dhaka and t International Centre for Diarrhoeal Disease Research, Bangladesh
Summary. Plesiornonas shigelloides, an organism commonly found in water, is only rarely associated with diarrhoea in man. P . shigelloides serotype 0 :17 (SVC 01), which is antigenically similar to Shigella sonnei, was found to be neither virulent nor toxic for rabbits. Rabbits immunised by feeding with P . shigelloides (SVC 01) were completely protected against an oral challenge with 1O1Ocells of S. sonnei but non-immunised rabbits were not. P . shigelloides (SVC 01) may be a useful vaccine strain for shigellosis.
International Centre for Diarrhoeal Disease Research, Bangladesh.
Introduction Plesiornonas shigelloides is commonly found in surface water and has also been isolated sporadically from patients' faeces during episodes of diarrhoea.'I It is rarely found in the faeces of healthy individual^.^ Investigations of the enteropathogenicity of P . shigelloides by several workers have not demonstrated it to be a cause of diarrhoea.*v6P . shigelloides has been reported as a causative agent of various extra-intestinal diseases, such as septicaemia, neonatal meningitis, cellulitis, septic arthritis, endophthalmitis and acute cholecystitis.6 A few serotypes of Plesiornonas spp. have antigenic similarity with Shigella sonnei phase I O-antigen,'q * and this study was undertaken to find out if one such serotype, P . shigelloides 0:17 (SVC Ol),' could give cross-protection to S. sonnei infection. As contaminated water is a frequent source of infection for man, this type of natural infection by P. shigelloides might provide immune protection against S. sonnei infection. This may be the reason for the relative absence of S. sonnei infection in developing countries compared to that in developed countries, where people usually drink boiled or chemically-treated drinking water.
Sereny test
This test was carried out according to the method described by Sereny.l0 The bacterial cultures were inoculated on to brain heart infusion agar (BHIA) and incubated at 37°C for 24 h. The cells were harvested and suspended in physiological saline to a density of lolo cells/ml. The cell suspension (20 pl) of P . shigelloides (SVC 01) was applied to one eye of a guinea-pig. An equal inoculum of the S . sonnei suspension was used as a positive control and sterile physiological saline as a negative control. The animals were inspected over a period of 72 h for keratoconjunctivitis. HeLa cell cytotoxicity tests
Cytotoxicity tests with HeLa cells were done by the method of Herrington et al.ll P . shigelloides (SVC 0 l), and a strain of S. dysenteriae as a positive control, were grown on BHIA and incubated at 37°C for 24 h. The cells were suspended in brain heart infusion broth (BHIB) containing polymyxin B (0.2 mg/ml) and incubated for 30min at 37°C. The suspensions were centrifuged at 6000 g for 10 min in a micro-centrifuge and the supernates were removed and filtered through Whatman filter paper (pore size 0.45 pn). A series of 10-fold dilutions of the filtratesin BHIB was made and 20 pl of each dilution was added in duplicate to HeLa cell monolayers in modified Eagles' minimum essential medium. Saline was used as a negative control. The cells were examined after incubation overnight at 37°C in a moist chamber. Toxin formation by the positive control caused the cells to shrink and detach from the well surface.
Materials and methods Bacterial strains P . shigelloides (SVC 01) was obtained from Professor A. Lindberg, Department of Clinical Bacteriology, Huddinge University Hospital, Sweden. S. sonnei strain AB3998 was isolated from a patient with diarrhoea at the Clinical Research Centre of the
Preparation of immunogen
Received 1 1 Nov. 1991 ; revised version accepted 5 March 1992. * Present address: Department of Genetics and Microbiology, University of Liverpool, PO Box 147, Liverpool L69 3BX.
Tetracycline-sensitiveP . shigelloides (SVC 0 1) and 382
PROTECTION AGAINST SH. SONNEI BY P. SHIGELLOIDES
383
S. sonnei strain AB3998 were cultured overnight on BHIA plates at 37°C. The cells were harvested and suspended to a density of 1O1Ocells/ml in physiological saline; 1 ml of the suspensions was added to 14 ml BHIB and fed to rabbits.
and all remained healthy and symptomless after oral challenge with 1O1O cells of S. sonnei. After 1 month they were killed. However, all of the rabbits of the nonimmunised control group challenged with S. sonnei AB3998 died within 24 h.
Animal challenge (oral immunisation and challenge)
Discussion
The test group comprised 10 healthy albino rabbits each weighing 20-2-5 kg. They were starved for 36 h. During ths period, 10 similar control rabbits were fed with tetracycline solution (25 mg/ml) to minimise the number of existing micro-organisms in the stomach. After 36 h, cimetidine (50 mg/kg body weight) was administered intravenously to rabbits of the test group followed by two doses of NaHCO, 5 % solution by gastric tube at intervals of 15 min. Immediately afterwards, 10'O cells of P . shigelloides (SVC 01) in 15 ml of BHIB were given by gastric tube and followed by intraperitonealinjection of 2 ml of tincture of opium.12 A second dose of P . shigelloides (SVC 0 1) (1010 cells in BHIB) was given on the sixth day. The control group (non-immunised) was similarly treated but fed with sterile BHIB without any bacteria. On the sixteenth day, all the rabbits were challenged by feeding with lolo cells of S. sonnei in BHIB. The fatality rate of the test and control groups was measured after 24 h. The experiment was performed twice.
The toxicity and Sereny invasiveness tests were performed to ensure that the test strain of P . shigelloides (SVC 01) was safe for the immunisation programme. Although P . shigelloides may be isolated in higher numbers from faeces of patients with diarrhoea than from healthy controls,2 there is little evidence in the literature to support the idea that P . shigelloides is a causative agent of diarrhoea. It is possible that this organism may proliferate after some other infection has taken place and that it acts as an opportunist pathogen. There is evidence2 that P. shigelloides is neither able to cause diarrhoea in an animal model nor produce toxins. There is only one report of plesiomonas toxin accumulation in rabbit gut? In this study, diarrhoeal symptoms were not induced in rabbits through oral administration of 1O1O P. shigelloides (SVC 01) cells. As P . shigelloides (SVC the 01) shares common antigens with S. sor~nei,~ immunological response to the former antigen may give protection against infection by S. sonnei. This study was carried out to investigate this relationship experimentally. All 10 rabbits immunised by feeding two doses of lolo cells of P . shigelloides (SVC 01) at intervals of 5 days and challenged on the sixteenth day with lolo cells of S. sonnei survived. However, all 10 rabbits not immunised with P. shigelloides (SVC 01) died within 24 h when challenged with S. sonnei. This indicated that oral immunisation with P . shigelloides (SVC 01) induced an immune response that protected rabbits against the virulent S. sonnei strain. The antigen responsible for the cross-protectionis believed to be the lipopolysaccharide 0-antigen! These results suggest that avirulent P . shigelloides strains that have an 0-antigen identical to that of S. sonnei should be considered as potential new immunising agents against shigellosis caused by S. sonnei. Since P. shigelloides (SVC 01) gave complete protection against S. sonnei infection in the animal model, further studies are now required to establish whether this organism is an acceptable vaccine candidate for man in protection against shigellosis caused by S. sonnei.
Results Sereny test Guinea-pig eyes infected with P . shigelloides (SVC 01) did not develop keratoconjunctivitis but those infected with S. sonnei AB3998 did. HeLa cell cytotoxicity tests Culture filtrates of P . shigelloides (SVC 01) and the saline control did not cause any cytotoxic effect on HeLa cells. However, degenerative changes were seen after 24 h in HeLa cells treated with S. dysenteriae culture filtrates. Animal challenge No diarrhoea was observed in any of the test rabbits immunised with 1O'O cells of P.' shigelloides (SVC 01)
References 1. Sakazaki R, Tamura K, Prescott LM, Bencic Z, Sanyal SC,
Sinha R. Bacteriological examination of diarrheal stools in Calcutta. Indian J Med Res 1971; 59: 1025-1034. 2. Tsukamota T, Kinoshita Y, Shimada T, Sakazaki R. Two
epidemics of diarrhoea1 disease possibly caused by Plesiomonas shigelloides. J Hyg 1978; 80: 275-280. 3. Holmberg SD,Wachsmuth IK, Hickman-Brenner FW, Blake PA, Farmer JJ. Plesiomonas enteric infections in the United States. Ann Intern Med 1986; 105: 690-694. 4. Sanyal SC, Saraswathi B, Sharma P. Enteropathogenicity of
384
5. 6. 7. 8.
S. SAYEED, D. A. SACK AND F. QADRI Plesiomonas shigelloides. J Med Microbiol 1980; 13: 40149. Manorama T, Vaneja V, Agarwal RK, Sanyal SC. Enterotoxins of Plesiomonasshigelloides :partial purification and characterization. Toxicon 1983; 3, Suppl: 269-272. Reinhardt JF, George WL. Plesiomonas shigelloides-associated diarrhoea. JAMA 1985; 253: 3294-3295. Schmid E, Valaudapillai T, Niles GR. Study of paracolon organisms with the major antigen of Shigella sonnei, form I. J Bacterioll954; 68: 50-52. Aldova E. Experiences with serology of Plesiomonas shigelloides 1. O-antigenic structure. J Hyg Epidemiol Microbiol Immunoll985; 29: 201-210.
9. Sayeed S, Sack DA, Qadri F. Cross-reacting antigens between Plesiomonas shigelloides 0 : 17 and Shigella sonnei. Bangladesh J Microbioll989; 6: 7-12. 10. Sereny B. Experimental Shigella keratoconjunctivitis : a preliminary report. Acta Microbiol Acad Sci Hung 1955; 2: 293-296. 11. Herrington DA, Tzipori S, Robins-Browne RM, Tall BD, Levine MM. In vitro and in vivo pathogenicity of Plesiomonas shigelloides. Infect Immun 1987; 55: 979-985. 12. Cray WC, Tokunaga E, Pierce NF. Successful colonization and immunization of adult rabbits by oral inoculation with Vibrio cholerae 01. Infect Immun 1983; 41: 735-741.
Protection from Shigella sonnei i nfection by immun isat ion of rabbits with Plesiornonas shigelloides (SVC 01) S. SAYEED*, D. A. SACK? and F. QADRlf Department of Microbiology, University of Dhaka and t International Centre for Diarrhoeal Disease Research, Bangladesh
Summary. Plesiornonas shigelloides, an organism commonly found in water, is only rarely associated with diarrhoea in man. P . shigelloides serotype 0 :17 (SVC 01), which is antigenically similar to Shigella sonnei, was found to be neither virulent nor toxic for rabbits. Rabbits immunised by feeding with P . shigelloides (SVC 01) were completely protected against an oral challenge with 1O1Ocells of S. sonnei but non-immunised rabbits were not. P . shigelloides (SVC 01) may be a useful vaccine strain for shigellosis.
International Centre for Diarrhoeal Disease Research, Bangladesh.
Introduction Plesiornonas shigelloides is commonly found in surface water and has also been isolated sporadically from patients' faeces during episodes of diarrhoea.'I It is rarely found in the faeces of healthy individual^.^ Investigations of the enteropathogenicity of P . shigelloides by several workers have not demonstrated it to be a cause of diarrhoea.*v6P . shigelloides has been reported as a causative agent of various extra-intestinal diseases, such as septicaemia, neonatal meningitis, cellulitis, septic arthritis, endophthalmitis and acute cholecystitis.6 A few serotypes of Plesiornonas spp. have antigenic similarity with Shigella sonnei phase I O-antigen,'q * and this study was undertaken to find out if one such serotype, P . shigelloides 0:17 (SVC Ol),' could give cross-protection to S. sonnei infection. As contaminated water is a frequent source of infection for man, this type of natural infection by P. shigelloides might provide immune protection against S. sonnei infection. This may be the reason for the relative absence of S. sonnei infection in developing countries compared to that in developed countries, where people usually drink boiled or chemically-treated drinking water.
Sereny test
This test was carried out according to the method described by Sereny.l0 The bacterial cultures were inoculated on to brain heart infusion agar (BHIA) and incubated at 37°C for 24 h. The cells were harvested and suspended in physiological saline to a density of lolo cells/ml. The cell suspension (20 pl) of P . shigelloides (SVC 01) was applied to one eye of a guinea-pig. An equal inoculum of the S . sonnei suspension was used as a positive control and sterile physiological saline as a negative control. The animals were inspected over a period of 72 h for keratoconjunctivitis. HeLa cell cytotoxicity tests
Cytotoxicity tests with HeLa cells were done by the method of Herrington et al.ll P . shigelloides (SVC 0 l), and a strain of S. dysenteriae as a positive control, were grown on BHIA and incubated at 37°C for 24 h. The cells were suspended in brain heart infusion broth (BHIB) containing polymyxin B (0.2 mg/ml) and incubated for 30min at 37°C. The suspensions were centrifuged at 6000 g for 10 min in a micro-centrifuge and the supernates were removed and filtered through Whatman filter paper (pore size 0.45 pn). A series of 10-fold dilutions of the filtratesin BHIB was made and 20 pl of each dilution was added in duplicate to HeLa cell monolayers in modified Eagles' minimum essential medium. Saline was used as a negative control. The cells were examined after incubation overnight at 37°C in a moist chamber. Toxin formation by the positive control caused the cells to shrink and detach from the well surface.
Materials and methods Bacterial strains P . shigelloides (SVC 01) was obtained from Professor A. Lindberg, Department of Clinical Bacteriology, Huddinge University Hospital, Sweden. S. sonnei strain AB3998 was isolated from a patient with diarrhoea at the Clinical Research Centre of the
Preparation of immunogen
Received 1 1 Nov. 1991 ; revised version accepted 5 March 1992. * Present address: Department of Genetics and Microbiology, University of Liverpool, PO Box 147, Liverpool L69 3BX.
Tetracycline-sensitiveP . shigelloides (SVC 0 1) and 382
PROTECTION AGAINST SH. SONNEI BY P. SHIGELLOIDES
383
S. sonnei strain AB3998 were cultured overnight on BHIA plates at 37°C. The cells were harvested and suspended to a density of 1O1Ocells/ml in physiological saline; 1 ml of the suspensions was added to 14 ml BHIB and fed to rabbits.
and all remained healthy and symptomless after oral challenge with 1O1O cells of S. sonnei. After 1 month they were killed. However, all of the rabbits of the nonimmunised control group challenged with S. sonnei AB3998 died within 24 h.
Animal challenge (oral immunisation and challenge)
Discussion
The test group comprised 10 healthy albino rabbits each weighing 20-2-5 kg. They were starved for 36 h. During ths period, 10 similar control rabbits were fed with tetracycline solution (25 mg/ml) to minimise the number of existing micro-organisms in the stomach. After 36 h, cimetidine (50 mg/kg body weight) was administered intravenously to rabbits of the test group followed by two doses of NaHCO, 5 % solution by gastric tube at intervals of 15 min. Immediately afterwards, 10'O cells of P . shigelloides (SVC 01) in 15 ml of BHIB were given by gastric tube and followed by intraperitonealinjection of 2 ml of tincture of opium.12 A second dose of P . shigelloides (SVC 0 1) (1010 cells in BHIB) was given on the sixth day. The control group (non-immunised) was similarly treated but fed with sterile BHIB without any bacteria. On the sixteenth day, all the rabbits were challenged by feeding with lolo cells of S. sonnei in BHIB. The fatality rate of the test and control groups was measured after 24 h. The experiment was performed twice.
The toxicity and Sereny invasiveness tests were performed to ensure that the test strain of P . shigelloides (SVC 01) was safe for the immunisation programme. Although P . shigelloides may be isolated in higher numbers from faeces of patients with diarrhoea than from healthy controls,2 there is little evidence in the literature to support the idea that P . shigelloides is a causative agent of diarrhoea. It is possible that this organism may proliferate after some other infection has taken place and that it acts as an opportunist pathogen. There is evidence2 that P. shigelloides is neither able to cause diarrhoea in an animal model nor produce toxins. There is only one report of plesiomonas toxin accumulation in rabbit gut? In this study, diarrhoeal symptoms were not induced in rabbits through oral administration of 1O1O P. shigelloides (SVC 01) cells. As P . shigelloides (SVC the 01) shares common antigens with S. sor~nei,~ immunological response to the former antigen may give protection against infection by S. sonnei. This study was carried out to investigate this relationship experimentally. All 10 rabbits immunised by feeding two doses of lolo cells of P . shigelloides (SVC 01) at intervals of 5 days and challenged on the sixteenth day with lolo cells of S. sonnei survived. However, all 10 rabbits not immunised with P. shigelloides (SVC 01) died within 24 h when challenged with S. sonnei. This indicated that oral immunisation with P . shigelloides (SVC 01) induced an immune response that protected rabbits against the virulent S. sonnei strain. The antigen responsible for the cross-protectionis believed to be the lipopolysaccharide 0-antigen! These results suggest that avirulent P . shigelloides strains that have an 0-antigen identical to that of S. sonnei should be considered as potential new immunising agents against shigellosis caused by S. sonnei. Since P. shigelloides (SVC 01) gave complete protection against S. sonnei infection in the animal model, further studies are now required to establish whether this organism is an acceptable vaccine candidate for man in protection against shigellosis caused by S. sonnei.
Results Sereny test Guinea-pig eyes infected with P . shigelloides (SVC 01) did not develop keratoconjunctivitis but those infected with S. sonnei AB3998 did. HeLa cell cytotoxicity tests Culture filtrates of P . shigelloides (SVC 01) and the saline control did not cause any cytotoxic effect on HeLa cells. However, degenerative changes were seen after 24 h in HeLa cells treated with S. dysenteriae culture filtrates. Animal challenge No diarrhoea was observed in any of the test rabbits immunised with 1O'O cells of P.' shigelloides (SVC 01)
References 1. Sakazaki R, Tamura K, Prescott LM, Bencic Z, Sanyal SC,
Sinha R. Bacteriological examination of diarrheal stools in Calcutta. Indian J Med Res 1971; 59: 1025-1034. 2. Tsukamota T, Kinoshita Y, Shimada T, Sakazaki R. Two
epidemics of diarrhoea1 disease possibly caused by Plesiomonas shigelloides. J Hyg 1978; 80: 275-280. 3. Holmberg SD,Wachsmuth IK, Hickman-Brenner FW, Blake PA, Farmer JJ. Plesiomonas enteric infections in the United States. Ann Intern Med 1986; 105: 690-694. 4. Sanyal SC, Saraswathi B, Sharma P. Enteropathogenicity of
384
5. 6. 7. 8.
S. SAYEED, D. A. SACK AND F. QADRI Plesiomonas shigelloides. J Med Microbiol 1980; 13: 40149. Manorama T, Vaneja V, Agarwal RK, Sanyal SC. Enterotoxins of Plesiomonasshigelloides :partial purification and characterization. Toxicon 1983; 3, Suppl: 269-272. Reinhardt JF, George WL. Plesiomonas shigelloides-associated diarrhoea. JAMA 1985; 253: 3294-3295. Schmid E, Valaudapillai T, Niles GR. Study of paracolon organisms with the major antigen of Shigella sonnei, form I. J Bacterioll954; 68: 50-52. Aldova E. Experiences with serology of Plesiomonas shigelloides 1. O-antigenic structure. J Hyg Epidemiol Microbiol Immunoll985; 29: 201-210.
9. Sayeed S, Sack DA, Qadri F. Cross-reacting antigens between Plesiomonas shigelloides 0 : 17 and Shigella sonnei. Bangladesh J Microbioll989; 6: 7-12. 10. Sereny B. Experimental Shigella keratoconjunctivitis : a preliminary report. Acta Microbiol Acad Sci Hung 1955; 2: 293-296. 11. Herrington DA, Tzipori S, Robins-Browne RM, Tall BD, Levine MM. In vitro and in vivo pathogenicity of Plesiomonas shigelloides. Infect Immun 1987; 55: 979-985. 12. Cray WC, Tokunaga E, Pierce NF. Successful colonization and immunization of adult rabbits by oral inoculation with Vibrio cholerae 01. Infect Immun 1983; 41: 735-741.
