JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1992, p. 2730-2732
Vol. 30, No. 10
0095-1137/92/102730-03$02.00/0 Copyright © 1992, American Society for Microbiology
Vibrio-Associated Gastroenteritis in the Lower Cross-River Basin of Nigeria JOHN A. NDON,l* SUNDE M. UDO,2 AND WILLIAM B. WEHRENBERG' Department of Health Sciences, University of Wisconsin, Milwaukee, Wisconsin 53201,1 and Department ofPathology, University of Calabar School ofMedicine, Calabar, Nigeria2 Received 7 May 1992/Accepted 24 July 1992
A total of 120 Vibrio species were isolated from 588 patients with acute diarrheal disease during an outbreak of gastrointestinal tract infections at different locations in the lower Cross River Basin of Nigeria. Vibrio cholerae 01, biotype El Tor, serotype Ogawa, was the prominent organism isolated from the Vibrio-associated diarrheal cases. During the 3 months of study, V. cholerae non-Ol was recovered from 10 patients while Vbrio parahaemolyticus was recovered from 19 patients. The significance of this study is the recognition that there is an ecological niche which supports V. cholerae non-Ol and V. parahaemolyticus in the Cross River Basin of Nigeria.
Cholera has become endemic in certain parts of West Africa following the worldwide cholera epidemic of 1970 (8, 17). In 1991 a cholera epidemic occurred in Nigeria (17). It resulted in 56,352 confirmed cases of cholera and 7,289 deaths. The initial report of the epidemic in the Cross River Basin can be traced to the January 1991 outbreak of gastroenteritis in Calabar and Uyo, Nigeria. These two cities are located approximately 160 km apart on the Cross River. On 19 January 1991, an abnormal increase in the number of cases of acute gastroenteritis in the two cities and reports of cholera in Kano, Kano State, prompted Nigerian health authorities to alert the communities of a possible cholera epidemic. Two days later, first cases of cholera were confirmed simultaneously in Uyo, Akwa Ibom State, and Calabar, Cross River State. This study examines the incidence of Vibrio-associated gastroenteritis from 21 January to 19 April 1991 in this geographical region and examines some of the characteristics of the isolates. Fecal specimens were obtained from 588 patients who visited the outpatient clinic of the University of Calabar Teaching Hospital, Calabar, and the Diarrhea Clinic of St. Luke's Hospital, Uyo. All patients had watery diarrheal stools when they visited the clinics and stated that they had not received any antibiotics and had not travelled out of their states during the preceding week. All fecal specimens were processed in the Microbiology Laboratory of the University of Calabar Teaching Hospital. Specimens obtained from the Diarrhea Clinic of St. Luke's Hospital were transported to Calabar in Carey-Blair semisolid transport medium. The specimens were inoculated directly onto several standard commercial enteric media, including Hektoen agar, xylose-lysine-deoxycholate agar, thiosulfate-citrate-bile-sucrose agar (TCBS), MacConkey agar, Campy blood agar plate, and salmonella-shigella agar. In addition, other samples from each specimen were placed into alkaline peptone water which contained 1% peptone and 1% NaCl at pH 8.6 and into broth for gram-negative organisms. Subcultures from alkaline peptone water to TCBS were performed after 6 h. All primary cultures and the alkaline peptone water subculture were inspected for colo-
nies of Vibrio species and other common bacterial enteric pathogens. Yellow and green colonies from TCBS were subsequently subcultured for determination of glucose fermentation, oxidase reaction, salt tolerance, and sugar fermentation. Since some strains of Vibrio cholerae and most strains of many other Vibrio species grow poorly or not at all in the absence of salts, the media used for fermentation of carbohydrates and for most other tests were supplemented with 0.5% NaCl (indole, methyl red, Voges-Proskauer, nitrate reduction, gelatin, esculin, lysine, arginine, and ornithine tests). Salt tolerance was determined by the ability of strains to grow in nutrient broth containing 0 to 10% NaCl. Other enteric pathogens were identified by using the API system (Analytab Products, Inc., Plainview, N.Y.). Fecal specimens were also checked for ova and parasites. The frequency of enteric pathogens isolated from the 588 patients is shown in Table 1. Numerous enteric bacteria and parasites were recovered from the stools of these patients. Similar high incidence of these enteric pathogens had been reported by Osisanya and coworkers (13). However, these authors did not observe cases of Vibrio species during a 1988 outbreak of gastroenteritis, while we report an incidence of over 20%. Of the 120 Vibrio specimens isolated, 101 were TABLE 1. Enteropathogens isolated from 588 patients with diarrhea Organism
V. cholerae 01 Classical biotype ........................................ El Tor biotype ........................................ V. cholerae non-Ol ....................................... V. parahaemolyticus ........................................
Shigella species ....................................... Campylobacterjejuni ........................................
Ascaris lumbricoides ........................................ Aeromonas hydrophila ....................................... Enterotoxigenic Escherichia coli ............... .......... Entamoeba histolytica ....................................... Salmonella species ....................................... Giardia lamblia ........................................
Multiple pathogens .......................................
*
No pathogen isolated ........................................
Corresponding author. 2730
No. of cases (%)
6 (1.0) 85 (14.5) 10 (1.7) 19 (3.2) 62 (10.5) 60 (10.2) 50 (8.5) 29 (5.0) 25 (4.4) 25 (4.4) 15 (2.6) 12 (2.0) 30 (5.0) 160 (27)
NOTES
VOL. 30, 1992
2731
TABLE 2. Biochemical reactions of Vibrio species isolated from 588 patients with diarrhea Reactiona of: Parameter
V. cholerae 01 (n = 91)
V. cholerae non-01 (n = 10)
V. parahaemwlyticus (n = 19)
Colonial appearance on TCBS at 37'C in 24 h Oxidase (Kovac's) reaction String test Indole + 1% NaCib Urease production Lysine decarboxylase + 1% NaClb Ornithine decarboxylase + 1% NaCib Arginine dihydrolase production Fermentation of: Sucrose Lactose
Yellow
Yellow
Green
+ + +
+ + +
+ +
+ +
+ +
+ + +
+
+
+
L-Arabfinose Gas from glucose Growth in nutrient broth + 0% NaCl + 3% NaCl + 6% NaCl + 8% NaCl + 10% NaCl
+
+ +
+ + d
d
+ + +
a +, 90% or more strains are positive; -, 90% or more strains are negative; d, delayed reaction. b Production of the indicated enzyme in medium containing 1% NaCl.
identified as V. cholerae on the basis of the properties presented in Table 2. Nineteen isolates were identified as Vibrio parahaemolyticus on the basis of the properties presented in Table 2 and their ability to produce hemolysin on high-salt-mannitol agar containing human erythrocytes (7, 9). Of the 101 V. cholerae specimens isolated, 91 were grouped as V. cholerae 01 and 10 were grouped as V. cholerae non-Ol on the basis of their ability to agglutinate in polyvalent 01 antiserum (Oxoid, Inc., London, England). The 91 V. cholerae 01 specimens were further tested with absorbed, monospecific Ogawa and Inaba antisera (Oxoid, Inc.) to determine their serotype. All were found to be of the Ogawa subtype. Biotyping of the 91 V. cholerae 01 specimens indicated that 6 of the isolates belonged to the classical biotype and 85 belonged to the El Tor biotype. This was based on the following characteristics: the classical biotype did not lyse or agglutinate sheep erythrocytes, was VogesProskauer negative, and was susceptible to a 50-IU disk of polymyxin B; the El Tor biotype was Voges-Proskauer positive, was resistant to polymyxin B and Murkejee's group 1V bacteriophage, and agglutinated chicken erythrocytes (3, 5, 12). Beta-hemolysis on blood agar plates was revealed by 50 of the 85 strains of the El Tor biotype. Our study focusing on the 1991 outbreak of gastroenteritis in Nigeria shows that 20.4% of the cases were Vibrioassociated gastroenteritis. While V. cholerae non-Ol and V. parahaemolyticus have not been associated with epidemic cholera (1, 7, 11), the recovery of these two Vibrio species during the outbreaks of gastrointestinal tract infections points to the fact that there must be some ecological niche which supports V. cholerae non-Ol and V. parahaemolyticus in this region. Locations may include sewage, estuarine waters, and seafoods. Studies of sporadic cases of diarrheal illness associated with isolation of either V. cholerae non-O1 or V. parahaemolyticus have reported that the patients frequently have a history of seafood consumption (2, 11). Indeed, the two communities where this study was conducted depend on the Cross River for their seafood. Only
five of our patients with V. parahaemolyticus-associated gastroenteritis denied having eaten fish in the previous 7 days. Patients with confirmed V. cholerae 01, V. cholerae non-O1, and V. parahaemolyticus had no other enteric pathogen isolated from their stools. Patients with V. cholerae 01 presented with a severe form of acute gastroenteritis, and they were adequately treated with oral administration of glucose-electrolyte solution and tetracycline. Patients with V. cholerae non-O1-associated gastroenteritis presented with a mild form of diarrhea and were treated successfully with fluid replacement and in some cases with tetracycline. The major manifestation of the V. parahaemolyticus-associated gastroenteritis was the presence of blood and mucus in the stools of 15 of our 19 patients. These patients were successfully treated with fluid replacement and in some cases tetracycline. The precise mechanisms by which V. cholerae non-O1 and V. parahaemolyticus cause the gastroenteritis are poorly understood; both cholera toxin-producing and non-toxin-producing strains have been isolated from patients with gastrointestinal (6, 7, 9-11) and extraintestinal (14-16) infections. To our knowledge there have been no previous reports of the association of V. cholerae non-O1 and V. parahaemolyticus in acute diarrheal diseases in these two coastal states of Nigeria. While it may be possible that the 1991 outbreak of gastroenteritis was the first in which V. cholerae non-O1 and V. parahaemolyticus were involved, it is more likely that our observations reflect a detailed evaluation of the Vibrio species in this area. REFERENCES 1. Blake, P. A., R. E. Weaver, and D. G. Hollis. 1980. Disease of humans (other than cholera) caused by Vibrios. Annu. Rev. Microbiol. 34:341-367. 2. Bonner, J. R., A. S. Coker, C. R. Berryman, and H. M. Pollock. 1983. Spectrum of Vibrio infections in a Gulf Coast community. Ann. Intern. Med. 99:464-469. 3. Finkelstein, R. A., and S. Mukerjee. 1963. Haemagglutination: a rapid method for differentiating V. cholerae and El Tor vibrios.
2732
NOTES
Proc. Soc. Expt. Biol. Med. 112:355-359. 4. Gelbart, S. M., and M. M. Prabhudesai. 1986. Vibrio cholerae non-Ol cellulitis. Acta Pathol. Lab. Med. 110:1182-1183. 5. Ichinose, Y., M. Ehara, S. Watanabe, S. Shimodori, P. G. Waiyaki, A. M. Kibue, F. C. Sang, J. Ngugi, and J. N. Kaviti. 1986. The characterization of Vibrio cholerae isolated in Kenya in 1983. J. Trop. Med. Hyg. 89:269-276. 6. Janda, J. M., C. Powers, R. G. Bryant, and S. L. Abbott. 1988. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin. Microbiol. Rev. 1:245267. 7. Joseph, S., R. R. Colwell, and J. P. Kaper. 1982. Vibrio parahaemolyticus and related halophilic vibrios. Crit. Rev. Microbiol. 10:77-124. 8. Kelly, M. T. 1986. Cholera: a worldwide perspective. Pediatr. Infect. Dis. 5:S101-S105. 9. Miyamoto, Y., T. Kato, Y. Obara, S. Akinyama, T. Kinjiro, K. Takizawa, and S. Yamai. 1969. In vitro hemolytic characteristic of Vibrio parahaemolyticus: its close correlation with human pathogenicity. J. Bacteriol. 100:1147-1149. 10. Morris, J. G., T. Takeda, B. D. Tall, G. A. Losonsky, S. K. Bhattacharya, B. D. Forrest, B. A. Kay, and M. Nishibuchi. 1990. Experimental non-O group 1 Vibno cholerae gastroenteri-
J. CLIN. MICROBIOL.
tis in humans. J. Clin. Invest. 85:697-705. 11. Morris, J. G., R. Wilson, B. R Davis, K. Wachsmuth, C. F. Riddle, H. G. Wathen, R. A. Pollard, and P. A. Blake. 1981. Non-O group 1 Vibrio cholerae gastroenteritis in the United States. Ann. Intern. Med. 94:656-658. 12. Murkerjee, S. 1978. Principles and practice of typing Vibrio cholerae. Methods Microbiol. 12:51-115. 13. Osisanya, J. O., S. 0. Daniel, S. C. Sehgal, A. Afigbo, A. Iyanda, F. I. Okoro, and N. Mbelu. 1988. Acute diarrhoeal disease in Nigeria: detection of enteropathogens in a rural sub-Saharan population. Trans. R. Soc. Trop. Med. Hyg. 82:773-777. 14. Pitrak, D. L., and J. D. Gindorf. 1989. Bacteremic cellulitis caused by non-serogroup 01 Vibrio cholerae acquired in a freshwater inland lake. J. Clin. Microbiol. 27:2874-2876. 15. Robson, R. A., R. Gulting, and J. J. Joubert. 1989. Isolation of Vibrio cholerae non-Ol from cerebrospinal fluid. Trans. R. Soc. Trop. Med. Hyg. 83:136. 16. Safrin, S., J. G. Morris, M. Adams, V. Pons, R. Jacobs, and J. E. Conte. 1988. Non-O:1 Vibrio cholerae bacteremia: case report and review. Rev. Infect. Dis. 10:1012-1017. 17. World Health Organization. 1991. Cholera in Africa. Weekly Epidemiol. Rec. 66:305-311.
Vol. 30, No. 10
0095-1137/92/102730-03$02.00/0 Copyright © 1992, American Society for Microbiology
Vibrio-Associated Gastroenteritis in the Lower Cross-River Basin of Nigeria JOHN A. NDON,l* SUNDE M. UDO,2 AND WILLIAM B. WEHRENBERG' Department of Health Sciences, University of Wisconsin, Milwaukee, Wisconsin 53201,1 and Department ofPathology, University of Calabar School ofMedicine, Calabar, Nigeria2 Received 7 May 1992/Accepted 24 July 1992
A total of 120 Vibrio species were isolated from 588 patients with acute diarrheal disease during an outbreak of gastrointestinal tract infections at different locations in the lower Cross River Basin of Nigeria. Vibrio cholerae 01, biotype El Tor, serotype Ogawa, was the prominent organism isolated from the Vibrio-associated diarrheal cases. During the 3 months of study, V. cholerae non-Ol was recovered from 10 patients while Vbrio parahaemolyticus was recovered from 19 patients. The significance of this study is the recognition that there is an ecological niche which supports V. cholerae non-Ol and V. parahaemolyticus in the Cross River Basin of Nigeria.
Cholera has become endemic in certain parts of West Africa following the worldwide cholera epidemic of 1970 (8, 17). In 1991 a cholera epidemic occurred in Nigeria (17). It resulted in 56,352 confirmed cases of cholera and 7,289 deaths. The initial report of the epidemic in the Cross River Basin can be traced to the January 1991 outbreak of gastroenteritis in Calabar and Uyo, Nigeria. These two cities are located approximately 160 km apart on the Cross River. On 19 January 1991, an abnormal increase in the number of cases of acute gastroenteritis in the two cities and reports of cholera in Kano, Kano State, prompted Nigerian health authorities to alert the communities of a possible cholera epidemic. Two days later, first cases of cholera were confirmed simultaneously in Uyo, Akwa Ibom State, and Calabar, Cross River State. This study examines the incidence of Vibrio-associated gastroenteritis from 21 January to 19 April 1991 in this geographical region and examines some of the characteristics of the isolates. Fecal specimens were obtained from 588 patients who visited the outpatient clinic of the University of Calabar Teaching Hospital, Calabar, and the Diarrhea Clinic of St. Luke's Hospital, Uyo. All patients had watery diarrheal stools when they visited the clinics and stated that they had not received any antibiotics and had not travelled out of their states during the preceding week. All fecal specimens were processed in the Microbiology Laboratory of the University of Calabar Teaching Hospital. Specimens obtained from the Diarrhea Clinic of St. Luke's Hospital were transported to Calabar in Carey-Blair semisolid transport medium. The specimens were inoculated directly onto several standard commercial enteric media, including Hektoen agar, xylose-lysine-deoxycholate agar, thiosulfate-citrate-bile-sucrose agar (TCBS), MacConkey agar, Campy blood agar plate, and salmonella-shigella agar. In addition, other samples from each specimen were placed into alkaline peptone water which contained 1% peptone and 1% NaCl at pH 8.6 and into broth for gram-negative organisms. Subcultures from alkaline peptone water to TCBS were performed after 6 h. All primary cultures and the alkaline peptone water subculture were inspected for colo-
nies of Vibrio species and other common bacterial enteric pathogens. Yellow and green colonies from TCBS were subsequently subcultured for determination of glucose fermentation, oxidase reaction, salt tolerance, and sugar fermentation. Since some strains of Vibrio cholerae and most strains of many other Vibrio species grow poorly or not at all in the absence of salts, the media used for fermentation of carbohydrates and for most other tests were supplemented with 0.5% NaCl (indole, methyl red, Voges-Proskauer, nitrate reduction, gelatin, esculin, lysine, arginine, and ornithine tests). Salt tolerance was determined by the ability of strains to grow in nutrient broth containing 0 to 10% NaCl. Other enteric pathogens were identified by using the API system (Analytab Products, Inc., Plainview, N.Y.). Fecal specimens were also checked for ova and parasites. The frequency of enteric pathogens isolated from the 588 patients is shown in Table 1. Numerous enteric bacteria and parasites were recovered from the stools of these patients. Similar high incidence of these enteric pathogens had been reported by Osisanya and coworkers (13). However, these authors did not observe cases of Vibrio species during a 1988 outbreak of gastroenteritis, while we report an incidence of over 20%. Of the 120 Vibrio specimens isolated, 101 were TABLE 1. Enteropathogens isolated from 588 patients with diarrhea Organism
V. cholerae 01 Classical biotype ........................................ El Tor biotype ........................................ V. cholerae non-Ol ....................................... V. parahaemolyticus ........................................
Shigella species ....................................... Campylobacterjejuni ........................................
Ascaris lumbricoides ........................................ Aeromonas hydrophila ....................................... Enterotoxigenic Escherichia coli ............... .......... Entamoeba histolytica ....................................... Salmonella species ....................................... Giardia lamblia ........................................
Multiple pathogens .......................................
*
No pathogen isolated ........................................
Corresponding author. 2730
No. of cases (%)
6 (1.0) 85 (14.5) 10 (1.7) 19 (3.2) 62 (10.5) 60 (10.2) 50 (8.5) 29 (5.0) 25 (4.4) 25 (4.4) 15 (2.6) 12 (2.0) 30 (5.0) 160 (27)
NOTES
VOL. 30, 1992
2731
TABLE 2. Biochemical reactions of Vibrio species isolated from 588 patients with diarrhea Reactiona of: Parameter
V. cholerae 01 (n = 91)
V. cholerae non-01 (n = 10)
V. parahaemwlyticus (n = 19)
Colonial appearance on TCBS at 37'C in 24 h Oxidase (Kovac's) reaction String test Indole + 1% NaCib Urease production Lysine decarboxylase + 1% NaClb Ornithine decarboxylase + 1% NaCib Arginine dihydrolase production Fermentation of: Sucrose Lactose
Yellow
Yellow
Green
+ + +
+ + +
+ +
+ +
+ +
+ + +
+
+
+
L-Arabfinose Gas from glucose Growth in nutrient broth + 0% NaCl + 3% NaCl + 6% NaCl + 8% NaCl + 10% NaCl
+
+ +
+ + d
d
+ + +
a +, 90% or more strains are positive; -, 90% or more strains are negative; d, delayed reaction. b Production of the indicated enzyme in medium containing 1% NaCl.
identified as V. cholerae on the basis of the properties presented in Table 2. Nineteen isolates were identified as Vibrio parahaemolyticus on the basis of the properties presented in Table 2 and their ability to produce hemolysin on high-salt-mannitol agar containing human erythrocytes (7, 9). Of the 101 V. cholerae specimens isolated, 91 were grouped as V. cholerae 01 and 10 were grouped as V. cholerae non-Ol on the basis of their ability to agglutinate in polyvalent 01 antiserum (Oxoid, Inc., London, England). The 91 V. cholerae 01 specimens were further tested with absorbed, monospecific Ogawa and Inaba antisera (Oxoid, Inc.) to determine their serotype. All were found to be of the Ogawa subtype. Biotyping of the 91 V. cholerae 01 specimens indicated that 6 of the isolates belonged to the classical biotype and 85 belonged to the El Tor biotype. This was based on the following characteristics: the classical biotype did not lyse or agglutinate sheep erythrocytes, was VogesProskauer negative, and was susceptible to a 50-IU disk of polymyxin B; the El Tor biotype was Voges-Proskauer positive, was resistant to polymyxin B and Murkejee's group 1V bacteriophage, and agglutinated chicken erythrocytes (3, 5, 12). Beta-hemolysis on blood agar plates was revealed by 50 of the 85 strains of the El Tor biotype. Our study focusing on the 1991 outbreak of gastroenteritis in Nigeria shows that 20.4% of the cases were Vibrioassociated gastroenteritis. While V. cholerae non-Ol and V. parahaemolyticus have not been associated with epidemic cholera (1, 7, 11), the recovery of these two Vibrio species during the outbreaks of gastrointestinal tract infections points to the fact that there must be some ecological niche which supports V. cholerae non-Ol and V. parahaemolyticus in this region. Locations may include sewage, estuarine waters, and seafoods. Studies of sporadic cases of diarrheal illness associated with isolation of either V. cholerae non-O1 or V. parahaemolyticus have reported that the patients frequently have a history of seafood consumption (2, 11). Indeed, the two communities where this study was conducted depend on the Cross River for their seafood. Only
five of our patients with V. parahaemolyticus-associated gastroenteritis denied having eaten fish in the previous 7 days. Patients with confirmed V. cholerae 01, V. cholerae non-O1, and V. parahaemolyticus had no other enteric pathogen isolated from their stools. Patients with V. cholerae 01 presented with a severe form of acute gastroenteritis, and they were adequately treated with oral administration of glucose-electrolyte solution and tetracycline. Patients with V. cholerae non-O1-associated gastroenteritis presented with a mild form of diarrhea and were treated successfully with fluid replacement and in some cases with tetracycline. The major manifestation of the V. parahaemolyticus-associated gastroenteritis was the presence of blood and mucus in the stools of 15 of our 19 patients. These patients were successfully treated with fluid replacement and in some cases tetracycline. The precise mechanisms by which V. cholerae non-O1 and V. parahaemolyticus cause the gastroenteritis are poorly understood; both cholera toxin-producing and non-toxin-producing strains have been isolated from patients with gastrointestinal (6, 7, 9-11) and extraintestinal (14-16) infections. To our knowledge there have been no previous reports of the association of V. cholerae non-O1 and V. parahaemolyticus in acute diarrheal diseases in these two coastal states of Nigeria. While it may be possible that the 1991 outbreak of gastroenteritis was the first in which V. cholerae non-O1 and V. parahaemolyticus were involved, it is more likely that our observations reflect a detailed evaluation of the Vibrio species in this area. REFERENCES 1. Blake, P. A., R. E. Weaver, and D. G. Hollis. 1980. Disease of humans (other than cholera) caused by Vibrios. Annu. Rev. Microbiol. 34:341-367. 2. Bonner, J. R., A. S. Coker, C. R. Berryman, and H. M. Pollock. 1983. Spectrum of Vibrio infections in a Gulf Coast community. Ann. Intern. Med. 99:464-469. 3. Finkelstein, R. A., and S. Mukerjee. 1963. Haemagglutination: a rapid method for differentiating V. cholerae and El Tor vibrios.
2732
NOTES
Proc. Soc. Expt. Biol. Med. 112:355-359. 4. Gelbart, S. M., and M. M. Prabhudesai. 1986. Vibrio cholerae non-Ol cellulitis. Acta Pathol. Lab. Med. 110:1182-1183. 5. Ichinose, Y., M. Ehara, S. Watanabe, S. Shimodori, P. G. Waiyaki, A. M. Kibue, F. C. Sang, J. Ngugi, and J. N. Kaviti. 1986. The characterization of Vibrio cholerae isolated in Kenya in 1983. J. Trop. Med. Hyg. 89:269-276. 6. Janda, J. M., C. Powers, R. G. Bryant, and S. L. Abbott. 1988. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin. Microbiol. Rev. 1:245267. 7. Joseph, S., R. R. Colwell, and J. P. Kaper. 1982. Vibrio parahaemolyticus and related halophilic vibrios. Crit. Rev. Microbiol. 10:77-124. 8. Kelly, M. T. 1986. Cholera: a worldwide perspective. Pediatr. Infect. Dis. 5:S101-S105. 9. Miyamoto, Y., T. Kato, Y. Obara, S. Akinyama, T. Kinjiro, K. Takizawa, and S. Yamai. 1969. In vitro hemolytic characteristic of Vibrio parahaemolyticus: its close correlation with human pathogenicity. J. Bacteriol. 100:1147-1149. 10. Morris, J. G., T. Takeda, B. D. Tall, G. A. Losonsky, S. K. Bhattacharya, B. D. Forrest, B. A. Kay, and M. Nishibuchi. 1990. Experimental non-O group 1 Vibno cholerae gastroenteri-
J. CLIN. MICROBIOL.
tis in humans. J. Clin. Invest. 85:697-705. 11. Morris, J. G., R. Wilson, B. R Davis, K. Wachsmuth, C. F. Riddle, H. G. Wathen, R. A. Pollard, and P. A. Blake. 1981. Non-O group 1 Vibrio cholerae gastroenteritis in the United States. Ann. Intern. Med. 94:656-658. 12. Murkerjee, S. 1978. Principles and practice of typing Vibrio cholerae. Methods Microbiol. 12:51-115. 13. Osisanya, J. O., S. 0. Daniel, S. C. Sehgal, A. Afigbo, A. Iyanda, F. I. Okoro, and N. Mbelu. 1988. Acute diarrhoeal disease in Nigeria: detection of enteropathogens in a rural sub-Saharan population. Trans. R. Soc. Trop. Med. Hyg. 82:773-777. 14. Pitrak, D. L., and J. D. Gindorf. 1989. Bacteremic cellulitis caused by non-serogroup 01 Vibrio cholerae acquired in a freshwater inland lake. J. Clin. Microbiol. 27:2874-2876. 15. Robson, R. A., R. Gulting, and J. J. Joubert. 1989. Isolation of Vibrio cholerae non-Ol from cerebrospinal fluid. Trans. R. Soc. Trop. Med. Hyg. 83:136. 16. Safrin, S., J. G. Morris, M. Adams, V. Pons, R. Jacobs, and J. E. Conte. 1988. Non-O:1 Vibrio cholerae bacteremia: case report and review. Rev. Infect. Dis. 10:1012-1017. 17. World Health Organization. 1991. Cholera in Africa. Weekly Epidemiol. Rec. 66:305-311.
