An Attempt to Establish Experimental Dysenteric Bacilli Cystitis

Transcription

1 Japan. J. Microbiol. Vol. 13 (4), , 1969 An Attempt to Establish Experimental Dysenteric Bacilli Cystitis Shigemi AWATAGUCHI, Yoshishige KAWANO, Akihiro KOJIMA, and Sadashige SAKUMA Biological Research Laboratory, Tanabe Seiyaku Co., Ltd., Toda, Saitama (Received for publication, January 8, 1969) ABSTRACT It was demonstrated that transurethral inoculation of the freshly isolated strains of Shigella into guinea pigs led to an acute severe dysenteric cystitis, resulting in the death of a majority of them, whereas stock cultures of the same species did not. Investigations concerning shigellosis have shown that man, the chimpanzee and the monkey are the only species susceptible to natural Shigella infection, small laboratory animals being more or less resistant. Attempts to develop experimental models of shigellosis, in recent years, have been oriented to modify the methodology used in experimental infection in small laboratory animals. Sereny [8] and Mackel et al. [4] reported that they could establish infection in conjunctival sac of guinea pigs. Bingel [1] and Manolov and Kosturkov [5] also noted that cystitis in guinea pigs could be caused by Shigella. This paper deals with the establishment of dysenteric cystitis in guinea pigs inoculated with freshly isolated strains of Shigella. MATERIALS AND METHODS Test organisms. Freshly isolated strains of Shigella from the feces of shigellosis patients were employed: Sh. flexneri 2a, strain TMH-1; Sh. flexneri 2a, strain TMH- 2; Sh. flexneri 3a, strain TMH-3; Sh. flexneri variant Y, strain TMH-4; Sh. sonnei I, strain TMH-5; and Sh. sonnei I, strain TMH-6; and were supplied through the courtesy of Dr. T. Iimura (Toshima Municipal Hospital, Tokyo). These were maintained in lyophilized state during experiments in order to prevent reduction of virulence. Stock strains of Shigella employed were kindly supplied from Department of Microbiology, Faculty of Medicine, University of Tokyo: Sh. flexneri 2a, strain DMT-1; Sh. flexneri 3a, strain MZ and Sh. sonnei, strain DMT-2. These had been preserved by periodic transfer on nutrient agar (Difco) in our laboratory for about three years. Strains were reconfirmed by the authors to be biochemically and serologically typical Shigella species. As controls Escherichia coli, strain FE1422 (type O-124, K-72), strain FE1193 (type O-126) and strain FE1912 (type O-89), which were stock cultures and enteropathogenic strains, were used. They were kindly given by Department of Bacteriology, Tokyo-to- 325

2 326 S. AWATAGUCHI, Y. KAWANO, A. KOJIMA AND S. SAKUMA Laboratories for Medical Sciences, Tokyo. Experimental animals. Healthy female guinea pigs of age 6-8 weeks weighing g were used. Culture medium. For cultivation of Shigella for inoculation into guinea pigs, heart infusion broth (Difco) was used, and for isolation of the inoculated bacilli from the organs, MacConkey's agar and Salmonella-Shigella agar were used. Infection of guinea pigs. The method described by Manolov and Kosturkov [5] was used. After urination, through a vinyl urethral catheter, 20 gauge size, each guinea pig was inoculated with 0.5 ml of a Shigella or Escherichia culture into the bladder via the urethra using the same catheter and then the general state was observed for 7 days. Dead guinea pigs were immediately autopsied and examined macroscopically and histopathologically. Cultures were done from urine, bladder and kidney homogenates. Keratoconjunctivitis test. Employing a modified method described by Mackel et al. [4], 0.05 ml of a hour-shigella or Escherichia broth cultures were dropped onto the conjunctival sac of guinea pigs, and observation was done for 3 days. Intra-allantoic chick embryo test. 0.1 ml of serial 10-fold dilutions of Shigella cultures was injected through the air sac into the allantoic cavities of 10 day old chick embryos and observation was done for 2 days. Histological examinations. Organs and tissues excised were, as routine procedures, fixed in 10% formalin solution, embedded in paraffin, and sections were cut and stained with hematoxylin and eosin solution. RESULTS Dysenteric Cystitis in Guinea Pigs Guinea pigs inoculated with the freshly isolated strains of Shigella died between 3 and 6 days after infection. All of them evidenced acute severe dysenteric cystitis. On the other hand, no deaths were observed in guinea pigs inoculated with the stock strains of Shigella or Escherichia coli. These results are summarized in Table 1. Table 1. Dysenteric cystitis in guinea pigs Note. +: Positive in establishment of infection. -: Negative. Abbreviation: Sh. flexneri VY... Sh. flexneri variant Y It was also noted that at 6 days after inoculation, Shigella bacilli were excreted in the urine of guinea pigs inoculated with the freshly isolated strains of Shigella, but none were from those animals inoculated with the stock strains of Shigella. Susceptibility of Guinea Pigs by Transurethral Administration to Shigella The results of susceptibility of guinea pigs to Sh. flexneri 2a, strain TMH-1, transurethral administration are shown in Table 2. As seen in Table 2, guinea pigs challenged with 1.25 ~108 cells of Shigella

3 EXPERIMENTAL DYSENTERIC BACILLI CYSTITIS 327 Table 2. Susceptibility of guinea pigs by transurethral administration to Sh. flexneri 2a, strain TMH-1 strain died, those challenged 1.25 ~106 cells survived, and there was a 75% mortality of the ones challenged with 1.25 ~ 107 cells. All dead animals were demonstrated to have developed acute severe dysenteric cystitis. Survivors were also observed to have dysenteric cystitis. Distribution of Shigella Organisms in the Bladder and Kidneys of Guinea Pigs Developing Dysenteric Cystitis The distribution of the Shigella organisms in the bladder and kidneys at various intervals following inoculation was determined as follows. The guinea pigs inoculated with Sh. flexneri 2a, strain TMH-1, were sacrificed after 4 days and the bladder and kidneys excised, weighed, homogenated in sterile physiological saline, and the number of Shigella organisms counted by plating on MacConkey's or Salmonella-Shigella agar. Guinea pigs challenged with Sh. flexneri 3a, strain TMH-3, which died 6 days after inoculation and were examined as mentioned above. Results of this experiment are shown in Table 3. Table 3. Number of Shigella bacilli in the bladder and kidneys of guinea pigs developing dysenteric cystitis Susceptibility of the Bladder and Conjunctival Sac to Shigella Bladder susceptibility to Shigella infection was compared with that of the keratoconjunctival sac. Infection was observed in the bladder and conjunctival sac of guinea pigs inoculated with the freshly isolated strains of Shigella and the results were shown in Table 4. We confirmed that both bladder and conjunctival sac of guinea pig were susceptible to Shigella infection. It was also noticed that the stock strains of Shigella and E. coli employed did not produce keratoconjunctivitis. Table 4. Susceptibility of bladder and conjunctival sac to Shigella Note: +: Positive in establishment of infection. Abbreviation: Sh. flexneri VY... Sh. flexneri variant Y Virulence of the Freshly Isolated and the Stock Strain of Shigella to Chick Embryo Toxicities of the freshly isolated strain TMH-1 of Sh. flexneri 2a and the stock strain DMT-1 of Sh. flexneri 2a to chick embryo were tested. The chick embryos challenged with 2.9 ~102 viable cells of the freshly isolated strain died after hours, while those challenged with 2.4 ~ 105 viable cells of the stock strain survived for more than 2 days. The results were shown in Table 5.

4 328 S. AWATAGUCHI, Y. KAWANO, A. KOJIMA AND S. SAKUMA Table 5. Virulence of the freshly isolated and stock strain of Shigella to chick embryo guinea pig on the 3rd day after instillation of the freshly isolated strain of Shigella. The corneal ulceration with polymorphonuclear neutrophile infiltration and edematous swelling of the lamina propria was seen also in the cornea and conjunctiva of the guinea pigs inoculated with the freshly isolated strains of Shigella. On the other hand, no remarkable histological changes were seen in cornea and conjunctiva of the guinea pigs inoculated with the stock strains of Shigella, as seen in Fig. 8. DISCUSSION Histological Findings Histological examinations of the bladder and keratoconjunctiva of guinea pigs challenged with the freshly isolated and the stock strains of Shigella were performed. Figures 1 and 2 showed the typical ulcerative lesion in the bladder of the guinea pigs which died on the 3rd day or were killed on the 6th day after inoculation with the freshly isolated strain of Sh. flexneri 2a, strain TMH-1. These lesions were characterized by a heavy inflammatory cell infiltration, hemorrhage, edema and hyperemia in the submucosa. Figures 3 and 4 also show the desquamation, vacuolation and edematous swelling of the epithelium. Inflammatory cell infiltration into the intramusclar bundles was also seen as noted in Fig. 5. The histological picture of the guinea pigs inoculated with the stock strains of Shigella was strikingly different from that described above, as seen in Fig. 6. Here no conspicuous changes were found in the mucous membrane and submucosa. Figure 7 showed the cornea of the The present study demonstrated that the guinea pigs challenged with the freshly isolated strains of Shigella by transurethral route developed acute severe dysenteric cystitis, while those challenged with the stock strains did not. It was also confirmed that keratoconjunctival sac of guinea pig was susceptible to Shigella infection which had been reported by Sereny [8] and Mackel et al. [4], since all freshly isolated strains of Shigella used gave rise to keratoconjunctivitis. It was noted that the stock strains of Shigella did not cause the development dysenteric cystitis and keratoconjunctivitis. Testing the correlation between the intensity of virulence of freshly isolated and stock strains of Shigella, it was observed from our experimental results with chick embryos that the freshly isolated strains of Shigella were obviously higher in virulence than the stock strains. Rauss et al. [7] reported that avirulent strains of Shigella had probably lost their penetrating capacity when examined by chick embryo tests. Formal et al. [2], working with starved guinea pigs, concluded that in the pathomechanism of dysentery Shigella bacilli penetrated through the intestinal epithelium and multiplied in the lamina propria. LaBrec et al. [3], using fluorescent antibody technique, demonstrated that virulent strain of Shigella penetrated into the bowel epithelium. According to these findings and observations, it may be postulated that capacity for local invasion and multiplication is one of the predominating virulence factors of the Shigellae. Judging from the facts mentioned above, it might be considered that the patho-

5 EXPERIMENTAL DYSENTERIC BACILLI CYSTITIS 329 mechanism of dysenteric cystitis was the same mechanism demonstrated by Formal et al. [2] and LaBrec et al. [3]. It might be suggested that the stock strains of Shigella had lost some of the virulence factors that the freshly isolated strains of Shigella possessed. it was also noted that in our experiments the enteropathogenic strains of E. coli did not produce cystitis or keratoconjunctivitis in guinea pigs. Sereny [8] and Mackel et al. [4] reported that the freshly isolated strains of E. coli did not produce keratoconjunctivitis in guinea pig eyes. However, Nakamura et al. [6] recently reported that E. coli (type O-124) produced keratoconjunctivitis under their experimental conditions. This dichotomy remains to be resolved. Regarding the susceptibility of the bladder of the guinea pig to those isolates of E. coli which have the capacity of producing keratoconjunctivitis, further investigations are under way. ACKNOWLEDGEMENTS The authors are indebted to Dr. T. Iimura (Toshima Municipal Hospital, Tokyo) for supplying Shigella strains. We also wish to express our sincere thanks to Prof. H. Takayasu (Department of Urology, Faculty of Medicine, University of Tokyo) and Dr. K. Abe (Director of our Laboratory) for their kind advice and encouragement during the course of this work. REFERENCES [1] Bingel, K. F Eine tierexperimentelle Methode zum Studium der Infektion mit gramnegativen Darmbakterien, ins besondere der Ruhr. Z. Hyg. Infekt. 125: [2] Formal, S. B., Dammin, G. J., LaBrec, E. H., and Schneider, H Experimental Shigella infection: Characteristics of a fatal infection produced in guinea pigs. J. Bacteriol. 75: [3] LaBrec, E. H., Schneider, H., Magnani, T. J., and Formal, S. B Epitherial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J. Bacteriol. 88: [4] Mackel, D. C., Langley, L. F., and Venice, L. A The use of the guinea pig conjunctivae as an experimental model for the study of virulence of Shigella organisms. Amer. J. Hyg. 73: [5] Manolov, D. G., and Kosturkov, G. B The effect of dysentery bacteriophage on the course of experimental cystitis produced in guinea pigs by Shigella flexneri. J. Hyg. Epidemiol. Microbiol. Immunol. 9: [6] Nakamura, A., Sakazaki, T., and Ogawa, H Pathogenicity of Enterophathogenic Strain of E. coli. (Report of the 22nd Kanto Branch Meeting of the Japan Bacteriological Society) Japan. J. Bacteriol. 23: [7] Rauss, K., Ketyi, I., and Angyl, T Studies on the virulence of Shigella flexneri. Acta Microbiol. Acad. Sci. Hung. 14: [8] Sereny, B Experimental Shigella keratoconjunctivitis. Acta Microbiol. Acad. Hung. 2:

6 330 S. AWATAGUGHI, Y. KAWANO, A. KOJIMA AND S. SAKUMA EXPLANATION OF PLATE Plate-Fig. 1. Guinea pig bladder from animals which died on the 3rd day after transurethral inoculation with freshly isolated strain of Sh. flexneri 2a, strain TMH-1. Severe desquamation of epithelial cells and ulceration with inflammatory cell infiltration in submucosa. Hematoxylin and Eosin Stain ( ~100) Plate-Fig. 2. Guinea pig bladder from animals killed on the 6th day after transurethral inoculation with freshly isolated strain of Sh. flexneri 2a, strain TMH-1. Ulcerative lesion can be seen with desquamated epithelial cells and exudates. Severe inflammatory reaction is also seen in submucosa. Hematoxylin and Eosin Stain ( ~100) Plate-Fig. 3. Guinea pig bladder from animals which died on the 3rd day after transurethral inoculation with freshly isolated strain of Sh. flexneri, strain TMH-1. Large vacuolated and edematous swelling of epithelial cells, hemorrhage and hyperemia of mucous membrane. Ulcerative lesion can be also seen. Hematoxylin and Eosin Stain ( ~100) Plate-Fig. 4. Guinea pig bladder from animals killed on the 6th day after transurethral inoculation with freshly isolated strain of Sh. flexneri 2a, strain TMH-1. Completely desquamated epithelial cells and severe inflammatory cells infiltration in submucosa. Hematoxylin and Eosin Stain ( ~100)

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8 332 S. AWATAGUCHI, Y. KAWANO, A. KOJIMA AND S. SAKUMA EXPLANATION OF PLATE Plate-Fig. 5. Guinea pig bladder from animals which died on the 4th day after transurethral inoculation with freshly isolated strain of Sh. flexneri variant Y, strain TMH-4. Inflammatory cell infiltration intra muscle bundles. Hematoxylin and Eosin Stain ( ~100) Plate-Fig. 6. Guinea pig bladder from animals killed on the 4th day after transurethral inoculation with stock strain of Sh. flexneri 2a, strain DMT-1. The mucous membrane and submucosa were normal. Hematoxylin and Eosin Stain ( ~100) Plate-Fig. 7. Guinea pig cornea from animals killed on the 3rd day after infection of the freshly isolated strain of Sh. flexneri 2a strain TMH-1. Diffuse and severe corneal ulceration with Complete desquamation of epithelium, polymorphonuclear neutrophile infiltration and edematous swelling of lamina propria. Hematoxylin and Eosin Stain ( ~200) Plate-Fig. 8. Guinea pig cornea from animals killed on the 3rd day after infection of the stock strain of Sh. flexneri 2a, strain DMT-1. No remarkable histologic change of cornea can be seen. Hematoxylin and Eosin Stain ( ~200)

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