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ABSTRACTVibrio choleraeO1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitroand in vivowas more or less equivalent to that produced by classical strains.

Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 µg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 µg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

ABSTRACTCholera toxin gene-negative Vibrio choleraenon-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

Some clinical strains of Vibrio cholerae non-O1 produce an extracellular factor that evokes a rapid and dramatic cytotoxic response which manifests as cell rounding of Chinese hamster ovary (CHO) and HeLa cells without accompanying membrane damage. This study was performed to establish the identity of the non-membrane-damaging cytotoxin (NMDCY), which was not inhibited by antitoxins against cholera toxin, heat-labile toxin of enterotoxigenic Escherichia coli, El Tor hemolysin, Shiga-like toxin I, and Shiga-like toxin II, indicating that NMDCY did not bear an apparent immunological relationship with the above toxins and hemolysin. Brain heart infusion broth and AKI medium supported the maximal production of NMDCY; culture supernatant of AKI medium was found to be free of hemolysin activity, whereas in brain heart infusion broth hemolysin was coproduced with NMDCY. Maximal production of NMDCY in AKI medium was observed at 37 degrees C under shaking conditions with the pH of the medium adjusted to 8.5. NMDCY was purified to homogeneity by a three-step purification procedure which increased the specific activity of the cytotoxin by 1.7 X 10(5)-fold. The denatured molecular weight of the purified toxin was 35,000, and the cytotoxin was heat labile and sensitive to trypsin. Purification of the cytotoxin revealed an enterotoxic activity as reflected by its ability to accumulate fluid in the rabbit ileal loop. Both the cytotoxic and enterotoxic activities of NMDCY could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified NMDCY and antiserum gave a single well-defined precipitin band which showed reactions of complete identity, while, in an immunoblot assay, a well-defined single band was observed in the 35-kDa region. Our results indicate that the cytotoxic and enterotoxic activities expressed by NMDCY appear to contribute to the pathogenesis of the disease associated with V. cholerae non-O1 strains which produce this cytotoxin.

The role of enterotoxigenic Escherichia coli (ETEC) as etiologic agents of diarrhoea in infants aged less than six months was assessed in a hospital based study in Calcutta, India. Of the 218 cases examined, ETEC strains were isolated from 26 (11.9%) cases. Among these, in 17 cases ETEC was the sole infecting pathogen (p = 0.0085). Of the 26 isolates (each isolate representing a case), 24 were distributed among seven different O:K:H serotypes and two different colonization factor antigens (CFAs) I and II. Two of the remaining isolation were untypable, non-haemagglutinating, and were nonhydrophobic as measured by the salt aggregation test (SAT). Of the 26 ETEC strains detected, 15 (57.7%) produced heat-labile toxin (LT) only, 8 (30.8%) liberated heat-stable toxin (ST) only, and the remaining 3 (11.5%) produced both LT and ST. No ETEC strain was isolated from the 102 age-matched controls included in this study. All the ETEC isolates were multiple drug resistant. The study showed that the diarrhoea due to ETEC was of brief duration, mostly within the range of 3 to 7 days.

Thirteen strains of Vibrio cholerae 01 belonging to the Inaba serotype El Tor biotype isolated from patients during an outbreak of cholera in the town of Warangal in southern India were found to be nontoxigenic (NT), since they did not produce cholera toxin or hybridize with DNA probes specific for cholera toxin, Zot, or Ace. The unheated and heated culture supernatants of the NT V. cholerae 01 evoked a rapid cell-rounding effect when introduced on confluent layers of CHO and HeLa cells which could not be inhibited by antiserum against known toxins. Culture supernatants of two representative NT V. cholerae 01 strains caused an increase in short-circuit current in rabbit ileal tissue mounted on an Ussing chamber, and the pattern of increase in short-circuit current was consistent with the presence of a quickly acting toxin like stable toxin. None of the strains of NT V. cholerae 01 hybridized with a DNA probe specific for the heat-stable enterotoxin of V. cholerae non-01, nor did the factor produced by NT V. cholerae 01 resemble the recently described heat-stable enterotoxin produced by enteroaggregative Escherichia coli as determine by a PCR assay. To our knowledge, this is the first report of NT V. cholerae 01 being associated with a cluster of cases of cholera, and it appears that a clone of NT V. cholerae 01 has the potential to cause localized outbreaks of cholera.

Some clinical strains of Vibrio cholerae non-O1 produce an extracellular factor that evokes a rapid and dramatic cytotoxic response which manifests as cell rounding of Chinese hamster ovary (CHO) and HeLa cells without accompanying membrane damage. This study was performed to establish the identity of the non-membrane-damaging cytotoxin (NMDCY), which was not inhibited by antitoxins against cholera toxin, heat-labile toxin of enterotoxigenic Escherichia coli, El Tor hemolysin, Shiga-like toxin I, and Shiga-like toxin II, indicating that NMDCY did not bear an apparent immunological relationship with the above toxins and hemolysin. Brain heart infusion broth and AKI medium supported the maximal production of NMDCY; culture supernatant of AKI medium was found to be free of hemolysin activity, whereas in brain heart infusion broth hemolysin was coproduced with NMDCY. Maximal production of NMDCY in AKI medium was observed at 37 degrees C under shaking conditions with the pH of the medium adjusted to 8.5. NMDCY was purified to homogeneity by a three-step purification procedure which increased the specific activity of the cytotoxin by 1.7 X 10(5)-fold. The denatured molecular weight of the purified toxin was 35,000, and the cytotoxin was heat labile and sensitive to trypsin. Purification of the cytotoxin revealed an enterotoxic activity as reflected by its ability to accumulate fluid in the rabbit ileal loop. Both the cytotoxic and enterotoxic activities of NMDCY could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified NMDCY and antiserum gave a single well-defined precipitin band which showed reactions of complete identity, while, in an immunoblot assay, a well-defined single band was observed in the 35-kDa region. Our results indicate that the cytotoxic and enterotoxic activities expressed by NMDCY appear to contribute to the pathogenesis of the disease associated with V. cholerae non-O1 strains which produce this cytotoxin.

In a randomized, double‐blind clinical trial, the efficacy and safety of norfloxacin were compared with nalidixic acid in the treatment of shigellosis in children. Out of 59 cases, Shigellaspp. were isolated from 8 cases in the nalidixic acid group and 14 cases in the norfloxacin group. The norfloxacin group had significantly less duration of diarrhoea and presence of blood in stool as compared to the nalidixic acid group. No joint problem was encountered in this study at up to 4 months follow‐up. Norfloxacin is safe and effective and showed no cartilage toxicity on short‐term follow‐up.