INFECTION AND IMMUNITY, June 1975, p. 1222-1225 Copyright 0 1975 American Society for Microbiology
Vol. 11, No. 6 Printed in U.S.A.
Bacteremia in Suckling Rabbits After Oral Challenge with Vibrio parahaemolyticus FRANK M. CALIA* AND DAVID E. JOHNSON
Baltimore Veterans Administration Hospital, Baltimore, Maryland 21218,* and University of Maryland School of Medicine, Baltimore, Maryland 21201 Received for publication 10 February 1975
Vibrio parahaemolyticus, a halophilic marine enteropathogen, produces diarrhea in man after ingestion of contaminated seafood. Only strains capable of producing beta-hemolysis (Kanagawa-positive strains) are enteropathogenic. Yet the majority of marine isolants are nonhemolytic and Kanagawa negative. Studies were initiated in suckling rabbits in an attempt to elucidate pathogenetic mechanisms. Fasting animals were infected orally with Kanagawa-positive and Kanagawa-negative strains of V. parahaemolyticus, V. cholerae, two enteropathogenic strains of Escherichia coli, Shigella flexneri, and strains of salmonellae. Seven hours postchallenge, cardiac blood, liver, and spleen cultures were obtained. V. parahaemolyticus strains failed to induce intestinal fluid accumulation during this study period. Bacteremia occurred in animals challenged with Kanagawa-positive V. parahaemolyticus, S. flexneri, and salmonella strains only. Animal passage increased the ability of V. parahaemolyticus to cause bacteremia. Liver and spleen cultures were positive in approximately 50% of animals challenged with the Kanagawa-positive strains of V. parphaemolyticus. The ability of this organism to penetrate the intestinal epithelium of suckling rabbits may represent a specific property that plays a role in pathogenesis. Vibrio parahaemolyticus, a halophilic marine gram-negative bacillus, has been established as an enteric pathogen in man (8, 11). Infection usually follows ingestion of contaminated seafood. This organism is found commonly in coastal and estuarine waters in the summer months and has been incriminated as a major cause of summer diarrhea in Japan (6, 7). Outbreaks have been reported in India and recently in the United Stages. Not all strains appear to be pathogenic. The ability to produce beta-hemolysis under certain conditions, the Kanagawa phenomenon, has been associated with pathogenicity (8). Ninetysix percent of strains recovered from stools of patients ill with diarrhea have been found to be Kanagawa positive, whereas 99% of the strains isolated from the sea are Kanagawa negative (8). Attempts to identify pathogenetic mechanisms thus far have been unsuccessful. Several laboratories have attempted to demonstrate an enterotoxin and have reported conflicting results (1, 9, 10, 13). Observations made in the infant rabbit during a study to elucidate pathogenetic mechanisms of V. parahaemolyticus are reported. MATERIALS AND METHODS Strains of organisms included in the study are listed in Table 1. All strains of V. parahaemolyticus
were grown in 3% peptone (Difco) with 3% sodium chloride (pH 7.3). Vibrio cholerae was grown in 2% peptone with 0.5% sodium chloride (pH 7.8). Shigella flexneri and strains of Escherichia coli and salmonellae were grown in Trypticase soy broth. The broth, after inoculation from a stock culture, was incubated ovemight at 35 C without shaking. This overnight culture (0.1 ml) was used to inoculate 50 ml of the appropriate broth in a 250-ml Erlenmeyer flask. The flask was incubated overnight at 35 C at 100 oscillations/min at a stroke length of 2 inches (ca. 5 cm) in an incubation shaker. The concentration of organisms ranged from 1.4 x 108 to 4.3 x 1010/ml. Experimental animals. A modification of the technique of Gorbach and Khurana using infant New Zealand white rabbits weighing from 46 to 173 g was used (5). Animals, starved for 24 h before the experiment, were anesthetized lightly with Metofane (Pittman Moore, Inc.). A no. 5 French polyethylene catheter was inserted into the stomach through the mouth, and 2 ml of an overnight shake culture was injected into the stomach. The quantitation of the challenge dose is listed in Table 2. The tubing was removed, and the animals were held in separate cages. Seven hours postchallenge, as the animals were being sacrificed with chloroform, their anterior thoracic and abdominal walls were cleansed with 70% ethyl alcohol. This was accomplished by vigorous scrubbing with alcohol-saturated gauze pads (4 by 4). Transthoracic needle puncture was performed, and 1 ml of cardiac blood was removed and inoculated into 10 ml of broth. The blood culture medium selected was identical to the one used to grow the challenge
1222
1223
V. PARAHAEMOLYTICUS BACTEREMIA
VOL. 11, 1975
TABLE 1. Strains of challenge organisms Source Serotype
Organism
Escherichia coli HS
Comment Nonpathogenic
334A
015
Healthy, adult lab technician Human diarrhea in India
C1272
0124K72:H-
Human diarrhea in Hungary
(+) Rabbit ileal loop (toxigenic) (+) Sereny test (penetrating)
Vibrio cholerae 569B
Inaba
Human cholera in Calcutta
(+) Rabbit ileal loop (toxigenic)
Vibrio parahaemolyticus P-1 P-5 P-25a
04:K11
Human diarrhea in Maryland Crab isolant in Maryland P-1 passed once in suckling rabbit P-1 passed twice in suckling rabbit
Kanagawa positive Kanagawa negative Kanagawa positive
P_27a P-31a
P_32a
Kanagawa positive
Shigella flexneri
2a
Human dysentery
(+) Sereny test (penetrating)
Salmonella typhimurium
1,4,5,12:i:1,2
Human food poisoning
ATCC 13311
Salmonella typhosa
9,12,Vi:d
Human carrier
Pathogenic to man
a
Obtained from blood culture after intragastric challenge.
organism. The anterior abdominal wall was reflected with sterile instruments and technique. The instruments were alcohol and flame sterilized before removing the spleen and again before removing the liver. The liver and spleen were removed into separate, sterile petri dishes, slices were made with sterile scissors, and a portion of each tissue was cultured in the broth medium appropriate for the infecting organism.
Before cardiac bleeding, the animals were weighed. After the removal of the liver and spleen, the gut from the pylorus to the rectum was removed and weighed. The gut weight divided by the rest of the carcass weight was calculated as an index of the relative quantity of fluid within the lumen of the gut. Bacteriology. Blood cultures were incubated at 35 C for up to 7 days. As cultures became turbid, they were subcultured on solid media. All clear tuhes were subcultured after day 7 of incubation. Subcultures were performed on sheep blood agar (BBL), Levine eosin-methylene blue agar (BBL), and thiosulfate citrate bile salt sucrose agar (BBL). Liver and spleen cultures were processed in a similar manner. All organisms recovered were identified by conventional biochemical methods.
RESULTS
The results of the intragastric challenge of 187 infant rabbits with test organisms are shown in Table 2. Animals given the following yielded negative blood cultures: broth alone; various strains of E. coli including a nonpathogenic
strain, a toxigenic strain, and a penetrating strain; a strain of V. cholerae; and a Kanagawanegative strain of V. parahaemolyticus. However, positive blood cultures were obtained in 9 out of 36 animals given the Kanagawa-positive strain of V. parahaemolyticus. Additionally, the percentage of positive blood cultures was increased (50%) when strains that had been passed in suckling rabbits were used. Bacteremia was also observed in animals challenged with shigella and salmonella (Table 2). Seventy-four rabbits infected with Kanagawa-positive V. parahaemolyticus had liver and spleen cultures performed; liver cultures were positive in 45 animals, and spleen cultures were positive in 37 (Table 3). Similarly liver and spleen cultures were positive with animals challenged with shigella and salmonella strains. Of 40 animals infected with the remaining organisms, including Kanagawa-negative V. parahaemolyticus, only a single positive liver and spleen culture was obtained in a V. cholerae-infected rabbit. The gut fluid index, a measure of intestinal fluid response to infection, was calculated (Table 2). V. cholerae and toxigenic E. coli produced a significantly greater quantity of fluid in the gut during the period of study when compared to Kanagawa-positive and -negative V. parahaemolyticus or other E. coli, salmo-
CALIA AND JOHNSON
1224
TABLE 2. Response of infant rabbits to intragastric challenge Organism
Range of inoculum count
positive
gut
blood cultures
fluid index
6
0
0.0666
(x 109/ml)
Broth control Escherichia coli HS 334A C1272
2.8-7.7 4.0-9.1 1.7-16.0
14 8 11
0 0 0
0.0516 0.0795a 0.0571
Vibrio cholerae 569B
0.14-27.0
11
0
0.0858a
Vibrio parahaemolyticus P-1 P-25,27,31,32 P-5
0.8-8.2 2.9-10.7
36 51 12
9 26 0
0.0675 0.0646 0.0595
1.6-43.0
Shigella flexneri
2a
3.9-6.3
14
1
0.0512
Salmonella typhimurium 13311
Salmonella typhosa Quailes ap
TABLE 3. Spleen and liver cultural results
No. of Average No. of animals
1.4-1.7
13
3
0.0522
2.3-4.6
11
1
0.0492
< 0.01.
nella, and shigella strains. The distended bowel of animals infected with toxigenic organisms was grossly apparent, and only these animals were grossly soiled with stool._This was not the case with any of the other pathogens. Despite this soiling, enterotoxic E. coli and V. cholerae were not recovered in cultures of blood. DISCUSSION During studies to determine the pathogenicity of V. parahaemolyticus in various animals, infant rabbits were challenged intragestrically. This model and time of sacrifice were selected in an attempt to utilize a sensitive indicator of enterotoxin activity as described (5). Challenge with sterile broth, nonpathogenic E. coli, two strains of pathogenic E. coli including a toxigenic and penetrating strain, V. cholerae, S. flexneri, and salmonella strains were compared with two strains of V. parahaemolyticus, a Kanagawa-negative and Kanagawa-positive strain. Enterotoxin activity was not identified in animals challenged with V. parahaemolyticus strains. Consequently, other pathogenetic mechanisms were suspected. In an at-
INFECT. IMMUN.
Organism
Total no. of animals
No. of animals with
No. of animals with
positive
positive
spleen cultures
liver cultures
Escherichia coli HS 334A C1272
6 8 7
0 0 0
0 0 0
Vibrio cholerae 569B
7
1
1
21
Vibrio parahaemolvticus P-1 P-25,27,31,32 P-5
53 12
11 26 0
31 0
Shigella flexneri 2a
14
4
4
Salmonella typhimurium 13311
13
5
5
Salmonella typhosa Quailes
11
3
3
14
tempt to detect invasion, blood, liver, and spleen were cultured. Within 7 h only the Kanagawa-positive strain of V. parahaemolyticus, S. flexneri, and salmonella strains produced bacteremia in the rabbits. Positive liver and spleen cultures were also found after challenge with these organisms. These findings suggest that V. parahaemolyticus, like salmonella and shigella, is capable of penetrating intestinal epithelium of the infant rabbit. Trauma from the gastric tube is an unlikely explanation for the bacteremia because control organisms were not recovered from blood cultures. Bacteremia could not be explained on the basis of differences in challenge dose since the dose for V'. parahaemolyticus fell within the range of all other test organisms. The exact site of penetration is unknown. Gross pathologic changes of the gut were not noted in these animals. Histologic studies are currently being performed and will be the subject of a future communication. V. parahaemolyticus is Sereny test negative; it does not penetrate the corneal epithelium of guinea pigs and thus differs from shigella and penetrating E. coli strains (4). All strains of V. parahaemolyticus failed to induce the accumulation of gut fluid during the study period. In addition, culture filtrates and whole cell lysates of these strains do not produce a positive adult
VOL. 11, 1975
V. PARAHAEMOLYTICUS BACTEREMIA
rabbit ileal loop response (unpublished data). This suggests that the strains of V. parahaemolyticus we tested do not produce an enterotoxin. The activity of an organism in animals and in man may differ considerably. Whether or not bacteremia occurs during clinical enteric infection with V. parahaemolyticus in man has not been established. Several investigators have reported cases of bacteremia due to this organism and other halophilic noncholera vibrios in patients with diarrhea (12, 14). Therefore, the ability to penetrate the intestinal epithelium of infant rabbits may represent a fundamental property of the organism that plays a role in pathogenesis. ACKNOWLEDGMENTS We would like to express our gratitude to Merrill J. Snyder for reviewing the manuscript and for many helpful suggestions. This study was supported by the Veterans Adminstration Research and Education Services. LITERATURE CITED 1. Bhattacharya, S., A. K. Bose, and A. K. Ghosh. 1971. Permeability and enterotoxic factors of nonagglutinable vibrios. Vibrio alcalignes and Vibrio parahaemolyticus. Appl. Microbiol. 22:1159-1161. 2. Chatterjee, B. D., S. L. Gorbach, and K. N. Neogy. 1970. Vibrio parahaemolyticus and diarrhea associated with non-cholera Vibrios. Bull. W.H.O. 42:460-463. 3. Dadisman, J. A., R. Nelson, J. R. Molenda, and H. J. Barber. 1973. Vibrio parahaemolyticus in Maryland. 1. Clinical and epidemiologic aspects. Am. J. Epidemiol. 96:414-426. 4. Ghosh, A. K., and R. N. Majuneder. 1974. Studies on mechanism of pathogenicity of Vibrio parahaemolyticus, p. 219-226. In T. Fujino, G. Sakaguchi, R.
1225
Sakazaki, and Y. Takeda (ed.), International symposium on Vibrio parahaemolvticus. Saikon Publishing Co., Ltd., Tokyo. 5. Gorbach, S. L., and C. H. Khurana. 1972. Toxigenic Escherichia coli. A cause of infantile diarrhea in Chicago. N. Engl. J. Med. 287:791-795. 6. Kaneko, T., and R. Colwell. 1973. Ecology of Vibrio parahaemolyticus in Chesapeake Bay. J. Bacteriol. 113:24-32. 7. Sakazaki, R., S. Iwanami, and H. Fukumi. 1963. Studies on the enteropathogenic facultatively halophilic bacteria, Vibrio parahaemolyticus. I. Morphological, cultural and biochemical properties and its taxonomical position. Jpn. J. Med. Sci. Biol. 16:161-188. 8. Sakazaki, R., K. Tamura, T. Kato, Y. Abara, S. Yamai, and K. Hobo. 1968. Studies on the enteropathogenic facultatively halophilic bacteria, Vibrio parahaemolyticus. III. Enteropathogenicity. Jpn. J. Med. Sci. Biol. 21:325-331. 9. Sakazaki, R., K. Tamura, A. Nakamura, T. Kurata, A. Ghoda, and Y. Kazuno. 1974. Enteropathogenic activity of Vibrio parahaemolyticus, p. 231-235. In T. Fujino, G. Sakaguchi, R. Sagazaki, and Y. Takeda (ed.), International symposium on Vibrio parahaemolyticus. Saikon Publishing Co. Ltd., Tokyo. 10. Sasaki, S., A. Ghoda, and H. Yahagi, 1967. Early features of infection in ligated loops of the rabbit small intestine inoculated with Shigella flexneri 3a, enteropathogenic E. coli, Escherichia coli and Vibrio parahaemolvticus. Keio J. Med. 16:101-116. 11. Takikawa, I. 1958. Studies on pathogenic halophilic bacteria. Yokohama Med. Bull. 2:313-322. 12. Torsteinsson, S. B., J. N. Minuth, and D. M. Musher. 1974. Clinical manifestations of halophilic non-cholera Vibrio infections. Lancet ii:1283-1284. 13. Twedt, R. M., and D. F. Brown. 1974. Studies on the enteropathogenicity of Vibrio parahaemolyticus in the ligated rabbit ilium, p. 211-217. In T. Fujino, G. Sakaguchi, R. Sakazaki, and Y. Takeda (ed.), International symposium on Vibrio parahaemolyticus. Saikon Publishing Co., Ltd. Tokyo. 14. Zide, N., J. Davis, and N. J. Ehrenkranz. 1974. Fulminating Vibrio parahaemolyticus septicemia. Arch. Intern. Med. 133:479-481.
Vol. 11, No. 6 Printed in U.S.A.
Bacteremia in Suckling Rabbits After Oral Challenge with Vibrio parahaemolyticus FRANK M. CALIA* AND DAVID E. JOHNSON
Baltimore Veterans Administration Hospital, Baltimore, Maryland 21218,* and University of Maryland School of Medicine, Baltimore, Maryland 21201 Received for publication 10 February 1975
Vibrio parahaemolyticus, a halophilic marine enteropathogen, produces diarrhea in man after ingestion of contaminated seafood. Only strains capable of producing beta-hemolysis (Kanagawa-positive strains) are enteropathogenic. Yet the majority of marine isolants are nonhemolytic and Kanagawa negative. Studies were initiated in suckling rabbits in an attempt to elucidate pathogenetic mechanisms. Fasting animals were infected orally with Kanagawa-positive and Kanagawa-negative strains of V. parahaemolyticus, V. cholerae, two enteropathogenic strains of Escherichia coli, Shigella flexneri, and strains of salmonellae. Seven hours postchallenge, cardiac blood, liver, and spleen cultures were obtained. V. parahaemolyticus strains failed to induce intestinal fluid accumulation during this study period. Bacteremia occurred in animals challenged with Kanagawa-positive V. parahaemolyticus, S. flexneri, and salmonella strains only. Animal passage increased the ability of V. parahaemolyticus to cause bacteremia. Liver and spleen cultures were positive in approximately 50% of animals challenged with the Kanagawa-positive strains of V. parphaemolyticus. The ability of this organism to penetrate the intestinal epithelium of suckling rabbits may represent a specific property that plays a role in pathogenesis. Vibrio parahaemolyticus, a halophilic marine gram-negative bacillus, has been established as an enteric pathogen in man (8, 11). Infection usually follows ingestion of contaminated seafood. This organism is found commonly in coastal and estuarine waters in the summer months and has been incriminated as a major cause of summer diarrhea in Japan (6, 7). Outbreaks have been reported in India and recently in the United Stages. Not all strains appear to be pathogenic. The ability to produce beta-hemolysis under certain conditions, the Kanagawa phenomenon, has been associated with pathogenicity (8). Ninetysix percent of strains recovered from stools of patients ill with diarrhea have been found to be Kanagawa positive, whereas 99% of the strains isolated from the sea are Kanagawa negative (8). Attempts to identify pathogenetic mechanisms thus far have been unsuccessful. Several laboratories have attempted to demonstrate an enterotoxin and have reported conflicting results (1, 9, 10, 13). Observations made in the infant rabbit during a study to elucidate pathogenetic mechanisms of V. parahaemolyticus are reported. MATERIALS AND METHODS Strains of organisms included in the study are listed in Table 1. All strains of V. parahaemolyticus
were grown in 3% peptone (Difco) with 3% sodium chloride (pH 7.3). Vibrio cholerae was grown in 2% peptone with 0.5% sodium chloride (pH 7.8). Shigella flexneri and strains of Escherichia coli and salmonellae were grown in Trypticase soy broth. The broth, after inoculation from a stock culture, was incubated ovemight at 35 C without shaking. This overnight culture (0.1 ml) was used to inoculate 50 ml of the appropriate broth in a 250-ml Erlenmeyer flask. The flask was incubated overnight at 35 C at 100 oscillations/min at a stroke length of 2 inches (ca. 5 cm) in an incubation shaker. The concentration of organisms ranged from 1.4 x 108 to 4.3 x 1010/ml. Experimental animals. A modification of the technique of Gorbach and Khurana using infant New Zealand white rabbits weighing from 46 to 173 g was used (5). Animals, starved for 24 h before the experiment, were anesthetized lightly with Metofane (Pittman Moore, Inc.). A no. 5 French polyethylene catheter was inserted into the stomach through the mouth, and 2 ml of an overnight shake culture was injected into the stomach. The quantitation of the challenge dose is listed in Table 2. The tubing was removed, and the animals were held in separate cages. Seven hours postchallenge, as the animals were being sacrificed with chloroform, their anterior thoracic and abdominal walls were cleansed with 70% ethyl alcohol. This was accomplished by vigorous scrubbing with alcohol-saturated gauze pads (4 by 4). Transthoracic needle puncture was performed, and 1 ml of cardiac blood was removed and inoculated into 10 ml of broth. The blood culture medium selected was identical to the one used to grow the challenge
1222
1223
V. PARAHAEMOLYTICUS BACTEREMIA
VOL. 11, 1975
TABLE 1. Strains of challenge organisms Source Serotype
Organism
Escherichia coli HS
Comment Nonpathogenic
334A
015
Healthy, adult lab technician Human diarrhea in India
C1272
0124K72:H-
Human diarrhea in Hungary
(+) Rabbit ileal loop (toxigenic) (+) Sereny test (penetrating)
Vibrio cholerae 569B
Inaba
Human cholera in Calcutta
(+) Rabbit ileal loop (toxigenic)
Vibrio parahaemolyticus P-1 P-5 P-25a
04:K11
Human diarrhea in Maryland Crab isolant in Maryland P-1 passed once in suckling rabbit P-1 passed twice in suckling rabbit
Kanagawa positive Kanagawa negative Kanagawa positive
P_27a P-31a
P_32a
Kanagawa positive
Shigella flexneri
2a
Human dysentery
(+) Sereny test (penetrating)
Salmonella typhimurium
1,4,5,12:i:1,2
Human food poisoning
ATCC 13311
Salmonella typhosa
9,12,Vi:d
Human carrier
Pathogenic to man
a
Obtained from blood culture after intragastric challenge.
organism. The anterior abdominal wall was reflected with sterile instruments and technique. The instruments were alcohol and flame sterilized before removing the spleen and again before removing the liver. The liver and spleen were removed into separate, sterile petri dishes, slices were made with sterile scissors, and a portion of each tissue was cultured in the broth medium appropriate for the infecting organism.
Before cardiac bleeding, the animals were weighed. After the removal of the liver and spleen, the gut from the pylorus to the rectum was removed and weighed. The gut weight divided by the rest of the carcass weight was calculated as an index of the relative quantity of fluid within the lumen of the gut. Bacteriology. Blood cultures were incubated at 35 C for up to 7 days. As cultures became turbid, they were subcultured on solid media. All clear tuhes were subcultured after day 7 of incubation. Subcultures were performed on sheep blood agar (BBL), Levine eosin-methylene blue agar (BBL), and thiosulfate citrate bile salt sucrose agar (BBL). Liver and spleen cultures were processed in a similar manner. All organisms recovered were identified by conventional biochemical methods.
RESULTS
The results of the intragastric challenge of 187 infant rabbits with test organisms are shown in Table 2. Animals given the following yielded negative blood cultures: broth alone; various strains of E. coli including a nonpathogenic
strain, a toxigenic strain, and a penetrating strain; a strain of V. cholerae; and a Kanagawanegative strain of V. parahaemolyticus. However, positive blood cultures were obtained in 9 out of 36 animals given the Kanagawa-positive strain of V. parahaemolyticus. Additionally, the percentage of positive blood cultures was increased (50%) when strains that had been passed in suckling rabbits were used. Bacteremia was also observed in animals challenged with shigella and salmonella (Table 2). Seventy-four rabbits infected with Kanagawa-positive V. parahaemolyticus had liver and spleen cultures performed; liver cultures were positive in 45 animals, and spleen cultures were positive in 37 (Table 3). Similarly liver and spleen cultures were positive with animals challenged with shigella and salmonella strains. Of 40 animals infected with the remaining organisms, including Kanagawa-negative V. parahaemolyticus, only a single positive liver and spleen culture was obtained in a V. cholerae-infected rabbit. The gut fluid index, a measure of intestinal fluid response to infection, was calculated (Table 2). V. cholerae and toxigenic E. coli produced a significantly greater quantity of fluid in the gut during the period of study when compared to Kanagawa-positive and -negative V. parahaemolyticus or other E. coli, salmo-
CALIA AND JOHNSON
1224
TABLE 2. Response of infant rabbits to intragastric challenge Organism
Range of inoculum count
positive
gut
blood cultures
fluid index
6
0
0.0666
(x 109/ml)
Broth control Escherichia coli HS 334A C1272
2.8-7.7 4.0-9.1 1.7-16.0
14 8 11
0 0 0
0.0516 0.0795a 0.0571
Vibrio cholerae 569B
0.14-27.0
11
0
0.0858a
Vibrio parahaemolyticus P-1 P-25,27,31,32 P-5
0.8-8.2 2.9-10.7
36 51 12
9 26 0
0.0675 0.0646 0.0595
1.6-43.0
Shigella flexneri
2a
3.9-6.3
14
1
0.0512
Salmonella typhimurium 13311
Salmonella typhosa Quailes ap
TABLE 3. Spleen and liver cultural results
No. of Average No. of animals
1.4-1.7
13
3
0.0522
2.3-4.6
11
1
0.0492
< 0.01.
nella, and shigella strains. The distended bowel of animals infected with toxigenic organisms was grossly apparent, and only these animals were grossly soiled with stool._This was not the case with any of the other pathogens. Despite this soiling, enterotoxic E. coli and V. cholerae were not recovered in cultures of blood. DISCUSSION During studies to determine the pathogenicity of V. parahaemolyticus in various animals, infant rabbits were challenged intragestrically. This model and time of sacrifice were selected in an attempt to utilize a sensitive indicator of enterotoxin activity as described (5). Challenge with sterile broth, nonpathogenic E. coli, two strains of pathogenic E. coli including a toxigenic and penetrating strain, V. cholerae, S. flexneri, and salmonella strains were compared with two strains of V. parahaemolyticus, a Kanagawa-negative and Kanagawa-positive strain. Enterotoxin activity was not identified in animals challenged with V. parahaemolyticus strains. Consequently, other pathogenetic mechanisms were suspected. In an at-
INFECT. IMMUN.
Organism
Total no. of animals
No. of animals with
No. of animals with
positive
positive
spleen cultures
liver cultures
Escherichia coli HS 334A C1272
6 8 7
0 0 0
0 0 0
Vibrio cholerae 569B
7
1
1
21
Vibrio parahaemolvticus P-1 P-25,27,31,32 P-5
53 12
11 26 0
31 0
Shigella flexneri 2a
14
4
4
Salmonella typhimurium 13311
13
5
5
Salmonella typhosa Quailes
11
3
3
14
tempt to detect invasion, blood, liver, and spleen were cultured. Within 7 h only the Kanagawa-positive strain of V. parahaemolyticus, S. flexneri, and salmonella strains produced bacteremia in the rabbits. Positive liver and spleen cultures were also found after challenge with these organisms. These findings suggest that V. parahaemolyticus, like salmonella and shigella, is capable of penetrating intestinal epithelium of the infant rabbit. Trauma from the gastric tube is an unlikely explanation for the bacteremia because control organisms were not recovered from blood cultures. Bacteremia could not be explained on the basis of differences in challenge dose since the dose for V'. parahaemolyticus fell within the range of all other test organisms. The exact site of penetration is unknown. Gross pathologic changes of the gut were not noted in these animals. Histologic studies are currently being performed and will be the subject of a future communication. V. parahaemolyticus is Sereny test negative; it does not penetrate the corneal epithelium of guinea pigs and thus differs from shigella and penetrating E. coli strains (4). All strains of V. parahaemolyticus failed to induce the accumulation of gut fluid during the study period. In addition, culture filtrates and whole cell lysates of these strains do not produce a positive adult
VOL. 11, 1975
V. PARAHAEMOLYTICUS BACTEREMIA
rabbit ileal loop response (unpublished data). This suggests that the strains of V. parahaemolyticus we tested do not produce an enterotoxin. The activity of an organism in animals and in man may differ considerably. Whether or not bacteremia occurs during clinical enteric infection with V. parahaemolyticus in man has not been established. Several investigators have reported cases of bacteremia due to this organism and other halophilic noncholera vibrios in patients with diarrhea (12, 14). Therefore, the ability to penetrate the intestinal epithelium of infant rabbits may represent a fundamental property of the organism that plays a role in pathogenesis. ACKNOWLEDGMENTS We would like to express our gratitude to Merrill J. Snyder for reviewing the manuscript and for many helpful suggestions. This study was supported by the Veterans Adminstration Research and Education Services. LITERATURE CITED 1. Bhattacharya, S., A. K. Bose, and A. K. Ghosh. 1971. Permeability and enterotoxic factors of nonagglutinable vibrios. Vibrio alcalignes and Vibrio parahaemolyticus. Appl. Microbiol. 22:1159-1161. 2. Chatterjee, B. D., S. L. Gorbach, and K. N. Neogy. 1970. Vibrio parahaemolyticus and diarrhea associated with non-cholera Vibrios. Bull. W.H.O. 42:460-463. 3. Dadisman, J. A., R. Nelson, J. R. Molenda, and H. J. Barber. 1973. Vibrio parahaemolyticus in Maryland. 1. Clinical and epidemiologic aspects. Am. J. Epidemiol. 96:414-426. 4. Ghosh, A. K., and R. N. Majuneder. 1974. Studies on mechanism of pathogenicity of Vibrio parahaemolyticus, p. 219-226. In T. Fujino, G. Sakaguchi, R.
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