INnCTION AND IMMUNITY, Feb. 1976, p. 457-463 Copyright C 1976 American Society for Microbiology
Vol. 13, No. 2 Printed in U.S.A.
Virulence Factors Involved in the Intraperitoneal Infection of Adult Mice with Vibrio cholerae ELIZABETH R. EUBANKS,' M. NEAL GUENTZEL,2 and L. JOE BERRY* Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received for publication 24 September 1975
Nonmotile mutants of Vibrio cholerae isolated from Ogawa, Inaba, and El Tor strains were less virulent than parent wild types when administered to adult mice intraperitoneally. The cells were suspended in 5% hog gastric mucin. Antitoxic immunity did not protect mice against this type of challenge, but a ribosomally derived vaccine did. Intraperitoneal injection of 10 50% lethal doses of enterotoxin (based on intravenous doses) was without toxic manifestations as were 1010 heat-killed vibrios similarly administered, regardless of strain. Virulent organisms killed with formalin or ultraviolet irradiation were significantly lethal at a dose of 1010 cells. Mice made tolerant to endotoxin were protected from death caused by an injection of 3 x 10"' boiled cells, but they did not survive an injection of formalin-killed cells. It is believed that the cause of death in this animal model of cholera is dependent, at least in part, on a toxic heat-labile moiety closely associated with the vibrios.
Although it is well known that choleragen is the primary factor responsible for the pathogenesis of cholera, there appear to be other factors involved in the virulence of Vibrio cholerae. Guentzel and Berry (5) have provided convincing evidence that the organism must be motile in order for it to produce disease in mice. Nonmotile but toxigenic mutants were markedly less virulent as measured by death of orally challenged baby mice than the motile parental wild types. We report here the results obtained when adult mice were challenged with these mutants intraperitoneally (i.p.). Even though this type of infection provides an artificial model of classical cholera, it has been used successfully in the mouse protection test described by Feeley and Pittman (3) for measuring the efficacy of cholera vaccines. The level of protection against infection seems to correlate well with the titer of vibriocidal antibody (12), and Watanabe et al. (13) describe the test as "a measure of anti-infective immunity." It is not known, however, how vibrios, which are not invasive, cause death when introduced into the peritoneal cavity. From the data reported here, death does not seem to be attributable to choleragen nor to the heat-stable cell wall components (endotoxin) present in the bacteria. There is, however, a heat-labile component in-
volved, possibly related to the one described by Pike and Chandler (8).
MATERIALS AND METHODS Mice. Outbred ICR mice purchased from Texas Inbred, Houston, CFW mice, originally obtained from Carworth Division, Charles River Farms, and HaICR specific-pathogen-free mice raised in departmental facilities were used in these studies. The animals were housed and fed as previously described (4) and used when 6 weeks old. Bacterial strains. Lyophilized cultures of V. cholerae strains CA401 (Inaba), CA411 (Ogawa), NIH41 (Ogawa), and 8233 (El Tor Inaba) were kindly provided by Charles E. Lankford of our department. Strain 569B (Inaba) was a gift of Richard Finkelstein (Southwestern Medical School, Dallas, Tex.). The nonmotile mutants were those selected and characterized previously (5). All cultures were maintained in the lyophilized state and restored as needed. Vaccines. Choleragen (National Institutes of Health [NIH] lot 0172) was reconstituted shortly before use and administered i.p. or subcutaneously. Choleragenoid (NIH lot 11101) was reconstituted and diluted in the protamine sulfate diluent provided by NIH and administered subcutaneously. HS222, an Ogawa-derived ribosomal vaccine, the purification and properties of which have already been described (6, 9), was a gift of Paul Actor of Smith, Kline, and French Laboratories, Philadelphia, Pa. HS222 was administered subcutaneously. Unless otherwise stated, vaccines were diluted in nonpyrogenic saline (Travenol, Inc.) immediately before use. ' Present address: Department of Botany and MicrobiolPreparation of materials for intraperitoneal Ariz. 85281. State University, Tempe, ogy, Arizona challenge. Choleragen was diluted in nonpyrogenic 2 Present address: Department of Allied Health and Life Sciences, The University of Texas at San Antonio, San saline or 5% hog gastric mucin (Wilson Laboratories). V. cholerae cultures were grown at 37 C on Antonio, Tex. 78285. 457
458
INFECT. IMMUN.
EUBANKS, GUENTZEL, AND BERRY
heart infusion agar slants for 18 h. The cells were suspended in saline and diluted in 5% gastric mucin. Viable cell numbers were determined by plate counts on brain heart infusion agar. Killed vibrios were prepared in several ways. (i) Cells suspended in saline at a density of 6 x 10"' per ml were boiled in a water bath for 30 min and then diluted appropriately in saline. (ii) Cells suspended in saline at a density of 2 x 1010 per ml were heated at 60 C for 60 min. (iii) A similar suspension of cells at a density of 2 x 1010 per ml was exposed for 8 h to a 75-W G.E. germicidal lamp. The bacteria were in petri dishes gently agitated on a rotary shaker. The lamp was at a distance of 15 cm. The original volume of the suspensions was restored by adding saline. Vibrios killed by methods (i), (ii), or (iii) were not washed before injection. (iv) Cells were suspended at a density of 2 x 1010 per ml in 1% phenol in saline and incubated at ambient temperature for 2 h. (v) Cells were suspended at a density of 2 x 1010 per ml in 0.5% formalin and incubated for 4 h at ambient temperature. Cells killed by methods (iv) or (v) were washed four times in saline before injection. Attempts to culture vibrios treated by any of these methods failed. Culture filtrates. Strain 8233 was cultured in casein hydrolysate yeast extract medium under conditions for optimal choleragen production (1). The cells were removed by centrifugation, and the supernatant fluid was passed through a 0.45-,um membrane filter (Millipore Corp.). Filtrate of peritoneal exudate. Mice in groups of 10 were given an i.p. injection of 105 colony-forming units (CFU) of strain 8233. After 8 h, the mice were cyanotic and moribund. They were sacrificed and the peritoneal cavity was washed with 0.5 ml of saline. The washes were combined and passed through a 0.45-iAm membrane filter. Bacterial counts in peritoneal wash. At 5 and 8 h postinfection, six mice were sacrificed. A 1.0-ml amount of saline was injected into the peritoneal cavity. The abdomen was massaged and the fluid was withdrawn with a syringe fitted with a 22-gauge needle. The washes were pooled and the number of viable vibrios (CFU) was determined by plate counts on brain heart infusion agar. Tolerant mice. Endotoxin was prepared from Salmonella typhimurium, strain SR11, by the method of Staub (11). CFW mice were made tolerant to 3 50% lethal doses (LD50) (420 lLg) of the endotoxin by injecting them with 22 ,ug of the same material 72 h previously.
and Muench (10). Fully motile strains were virulent (Table 1). Only 33% of mice given 102 CFU of strain 8233, the most virulent, survived. In contrast, 83% of mice challenged with 10; CFU of 569B survived. To determine whether motility contributes to the virulence of vibrios in an i.p. infection, mice were challenged with either a highly motile strain or a nonmotile mutant derived from it. Survival was recorded for 48 h postchallenge. Other groups of mice similarly challenged but with only the largest dose were used to obtain peritoneal wash-outs for enumeration of vibrios (Table 2). All of the nonmotile derivatives were significantly less virulent than the respective motile strains as judged either by survival data or by viable counts in the peritoneal washes. The latter show that motile vibrios multiplied faster in the peritoneal cavity than nonmotile mutants. Thus, the ability of the organisms to proliferate correlated with their virulence. Even though there was a marked difference in growth rates in vivo, essentially no difference between motile and nonmotile mutants appeared when the strains were cultured in tubes of Trypticase soy broth with or without 5% gastric mucin. The reduced virulence of the nonmotile strains cannot be attributed to reduced toxin production since Guentzel and Berry (5) demonstrated that wild types and mutants were comparably toxigenic. Except for 82336B3R2, which is a rough strain, none of the mutants has an alteration in cell surface as determined by susceptibility to a variety of cholera phages (5). Since ICR mice are resistant to i.p. infection with strain CA401 or CA411, additional experiments were done with HaICR mice. Nonmotile mutant vibrios were less virulent than the motile parental strains, and they also failed to multiply in the peritoneal cavity as rapidly (Table 3). Role of choleragen in death from i.p. infection. The preceding data establish that motile vibrios are more virulent than nonmotile strains, and associated with this greater virulence is an enhanced ability of the cells to
RESULTS
TABLE 1. Estimated LD50 for CFW mice infectd i.p. with motile strains of V. cholerae
Virulence of motile and nonmotile strains. The virulence of motile strains of vibrios, both classical and El Tor, was compared with that of the highly toxigenic but weakly motile Inaba strain 569B. For this purpose, mice were challenged i.p. in doses varying in 10-fold increments from 102 to 105 CFU. The LD50 for each strain was determined by the method of Reed
V. cholerae strain
LD50 (CFU)a
8233 (El Tor Inaba) ............. 1 x 10" a
Deaths recorded for 48 h.
VOL. 13, 1976
FACTORS IN I.P. INFECTION WITH V. CHOLERAE
459
TABLE 2. Survival and colony counts ofperitoneal washings of ICR mice infected i.p. with motile and nonmotile strains of V. cholerae cholerae train V. V. cholerae strain
Challenge dose Motile Motile(CFU)
Survivorsttotal (48 h)
CFU in peritoneal wash obtained after: 5h
8233
+
8233, M-7
8233, 6B3
8233, 6B3R2
CA411
+
CA411, M-5
CA411, M-6
CA401
CA401, M-5
CA401, M-6
+
1 X 102 1 X 103 1 X 104
8h
4/12 0/12 0/12
3.8
x
10.
1.2 x 108
2/6 4/6 2/6
1.2
x
106
1.7
x
107
5/6 2/6 4/6
Vol. 13, No. 2 Printed in U.S.A.
Virulence Factors Involved in the Intraperitoneal Infection of Adult Mice with Vibrio cholerae ELIZABETH R. EUBANKS,' M. NEAL GUENTZEL,2 and L. JOE BERRY* Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received for publication 24 September 1975
Nonmotile mutants of Vibrio cholerae isolated from Ogawa, Inaba, and El Tor strains were less virulent than parent wild types when administered to adult mice intraperitoneally. The cells were suspended in 5% hog gastric mucin. Antitoxic immunity did not protect mice against this type of challenge, but a ribosomally derived vaccine did. Intraperitoneal injection of 10 50% lethal doses of enterotoxin (based on intravenous doses) was without toxic manifestations as were 1010 heat-killed vibrios similarly administered, regardless of strain. Virulent organisms killed with formalin or ultraviolet irradiation were significantly lethal at a dose of 1010 cells. Mice made tolerant to endotoxin were protected from death caused by an injection of 3 x 10"' boiled cells, but they did not survive an injection of formalin-killed cells. It is believed that the cause of death in this animal model of cholera is dependent, at least in part, on a toxic heat-labile moiety closely associated with the vibrios.
Although it is well known that choleragen is the primary factor responsible for the pathogenesis of cholera, there appear to be other factors involved in the virulence of Vibrio cholerae. Guentzel and Berry (5) have provided convincing evidence that the organism must be motile in order for it to produce disease in mice. Nonmotile but toxigenic mutants were markedly less virulent as measured by death of orally challenged baby mice than the motile parental wild types. We report here the results obtained when adult mice were challenged with these mutants intraperitoneally (i.p.). Even though this type of infection provides an artificial model of classical cholera, it has been used successfully in the mouse protection test described by Feeley and Pittman (3) for measuring the efficacy of cholera vaccines. The level of protection against infection seems to correlate well with the titer of vibriocidal antibody (12), and Watanabe et al. (13) describe the test as "a measure of anti-infective immunity." It is not known, however, how vibrios, which are not invasive, cause death when introduced into the peritoneal cavity. From the data reported here, death does not seem to be attributable to choleragen nor to the heat-stable cell wall components (endotoxin) present in the bacteria. There is, however, a heat-labile component in-
volved, possibly related to the one described by Pike and Chandler (8).
MATERIALS AND METHODS Mice. Outbred ICR mice purchased from Texas Inbred, Houston, CFW mice, originally obtained from Carworth Division, Charles River Farms, and HaICR specific-pathogen-free mice raised in departmental facilities were used in these studies. The animals were housed and fed as previously described (4) and used when 6 weeks old. Bacterial strains. Lyophilized cultures of V. cholerae strains CA401 (Inaba), CA411 (Ogawa), NIH41 (Ogawa), and 8233 (El Tor Inaba) were kindly provided by Charles E. Lankford of our department. Strain 569B (Inaba) was a gift of Richard Finkelstein (Southwestern Medical School, Dallas, Tex.). The nonmotile mutants were those selected and characterized previously (5). All cultures were maintained in the lyophilized state and restored as needed. Vaccines. Choleragen (National Institutes of Health [NIH] lot 0172) was reconstituted shortly before use and administered i.p. or subcutaneously. Choleragenoid (NIH lot 11101) was reconstituted and diluted in the protamine sulfate diluent provided by NIH and administered subcutaneously. HS222, an Ogawa-derived ribosomal vaccine, the purification and properties of which have already been described (6, 9), was a gift of Paul Actor of Smith, Kline, and French Laboratories, Philadelphia, Pa. HS222 was administered subcutaneously. Unless otherwise stated, vaccines were diluted in nonpyrogenic saline (Travenol, Inc.) immediately before use. ' Present address: Department of Botany and MicrobiolPreparation of materials for intraperitoneal Ariz. 85281. State University, Tempe, ogy, Arizona challenge. Choleragen was diluted in nonpyrogenic 2 Present address: Department of Allied Health and Life Sciences, The University of Texas at San Antonio, San saline or 5% hog gastric mucin (Wilson Laboratories). V. cholerae cultures were grown at 37 C on Antonio, Tex. 78285. 457
458
INFECT. IMMUN.
EUBANKS, GUENTZEL, AND BERRY
heart infusion agar slants for 18 h. The cells were suspended in saline and diluted in 5% gastric mucin. Viable cell numbers were determined by plate counts on brain heart infusion agar. Killed vibrios were prepared in several ways. (i) Cells suspended in saline at a density of 6 x 10"' per ml were boiled in a water bath for 30 min and then diluted appropriately in saline. (ii) Cells suspended in saline at a density of 2 x 1010 per ml were heated at 60 C for 60 min. (iii) A similar suspension of cells at a density of 2 x 1010 per ml was exposed for 8 h to a 75-W G.E. germicidal lamp. The bacteria were in petri dishes gently agitated on a rotary shaker. The lamp was at a distance of 15 cm. The original volume of the suspensions was restored by adding saline. Vibrios killed by methods (i), (ii), or (iii) were not washed before injection. (iv) Cells were suspended at a density of 2 x 1010 per ml in 1% phenol in saline and incubated at ambient temperature for 2 h. (v) Cells were suspended at a density of 2 x 1010 per ml in 0.5% formalin and incubated for 4 h at ambient temperature. Cells killed by methods (iv) or (v) were washed four times in saline before injection. Attempts to culture vibrios treated by any of these methods failed. Culture filtrates. Strain 8233 was cultured in casein hydrolysate yeast extract medium under conditions for optimal choleragen production (1). The cells were removed by centrifugation, and the supernatant fluid was passed through a 0.45-,um membrane filter (Millipore Corp.). Filtrate of peritoneal exudate. Mice in groups of 10 were given an i.p. injection of 105 colony-forming units (CFU) of strain 8233. After 8 h, the mice were cyanotic and moribund. They were sacrificed and the peritoneal cavity was washed with 0.5 ml of saline. The washes were combined and passed through a 0.45-iAm membrane filter. Bacterial counts in peritoneal wash. At 5 and 8 h postinfection, six mice were sacrificed. A 1.0-ml amount of saline was injected into the peritoneal cavity. The abdomen was massaged and the fluid was withdrawn with a syringe fitted with a 22-gauge needle. The washes were pooled and the number of viable vibrios (CFU) was determined by plate counts on brain heart infusion agar. Tolerant mice. Endotoxin was prepared from Salmonella typhimurium, strain SR11, by the method of Staub (11). CFW mice were made tolerant to 3 50% lethal doses (LD50) (420 lLg) of the endotoxin by injecting them with 22 ,ug of the same material 72 h previously.
and Muench (10). Fully motile strains were virulent (Table 1). Only 33% of mice given 102 CFU of strain 8233, the most virulent, survived. In contrast, 83% of mice challenged with 10; CFU of 569B survived. To determine whether motility contributes to the virulence of vibrios in an i.p. infection, mice were challenged with either a highly motile strain or a nonmotile mutant derived from it. Survival was recorded for 48 h postchallenge. Other groups of mice similarly challenged but with only the largest dose were used to obtain peritoneal wash-outs for enumeration of vibrios (Table 2). All of the nonmotile derivatives were significantly less virulent than the respective motile strains as judged either by survival data or by viable counts in the peritoneal washes. The latter show that motile vibrios multiplied faster in the peritoneal cavity than nonmotile mutants. Thus, the ability of the organisms to proliferate correlated with their virulence. Even though there was a marked difference in growth rates in vivo, essentially no difference between motile and nonmotile mutants appeared when the strains were cultured in tubes of Trypticase soy broth with or without 5% gastric mucin. The reduced virulence of the nonmotile strains cannot be attributed to reduced toxin production since Guentzel and Berry (5) demonstrated that wild types and mutants were comparably toxigenic. Except for 82336B3R2, which is a rough strain, none of the mutants has an alteration in cell surface as determined by susceptibility to a variety of cholera phages (5). Since ICR mice are resistant to i.p. infection with strain CA401 or CA411, additional experiments were done with HaICR mice. Nonmotile mutant vibrios were less virulent than the motile parental strains, and they also failed to multiply in the peritoneal cavity as rapidly (Table 3). Role of choleragen in death from i.p. infection. The preceding data establish that motile vibrios are more virulent than nonmotile strains, and associated with this greater virulence is an enhanced ability of the cells to
RESULTS
TABLE 1. Estimated LD50 for CFW mice infectd i.p. with motile strains of V. cholerae
Virulence of motile and nonmotile strains. The virulence of motile strains of vibrios, both classical and El Tor, was compared with that of the highly toxigenic but weakly motile Inaba strain 569B. For this purpose, mice were challenged i.p. in doses varying in 10-fold increments from 102 to 105 CFU. The LD50 for each strain was determined by the method of Reed
V. cholerae strain
LD50 (CFU)a
8233 (El Tor Inaba) ............. 1 x 10" a
Deaths recorded for 48 h.
VOL. 13, 1976
FACTORS IN I.P. INFECTION WITH V. CHOLERAE
459
TABLE 2. Survival and colony counts ofperitoneal washings of ICR mice infected i.p. with motile and nonmotile strains of V. cholerae cholerae train V. V. cholerae strain
Challenge dose Motile Motile(CFU)
Survivorsttotal (48 h)
CFU in peritoneal wash obtained after: 5h
8233
+
8233, M-7
8233, 6B3
8233, 6B3R2
CA411
+
CA411, M-5
CA411, M-6
CA401
CA401, M-5
CA401, M-6
+
1 X 102 1 X 103 1 X 104
8h
4/12 0/12 0/12
3.8
x
10.
1.2 x 108
2/6 4/6 2/6
1.2
x
106
1.7
x
107
5/6 2/6 4/6
