Chinese Hamster Ovary Cell Assay Research Articles

Enterotoxigenic bacteria in food and water from an Ethiopian community.

Food and water samples from an Ethiopian community were screened for the presence of enterotoxin-producing bacteria. Using the Chinese hamster ovary cell assay, 40 of 213 isolates (18.8%) produced heat-labile (LT) enterotoxin. These LT-producing isolates comprised 33 of 177 (18.6%) strains from 24 of 68 food samples (35.3%) and 7 of 36 (19.4%) isolates of 4 of 17 water samples (23.5%). One LT-producing strain each of Salmonella emek and of Shigella dysenteriae was found. Three pseudomonads, all LT producers, produced heat-stable enterotoxin as gauged by the suckling mouse test. Two strains of LT-enterotoxigenic Escherichia coli O68 were found in water samples. No enterotoxigenic E. coli were isolated from food samples, but 13 of the LT-producing strains were Enterobacter, Klebsiella, Serratia, and Proteus species, and 7 food samples yielded more than one species of enterotoxigenic bacterium. Of the enterotoxigenic isolates from food, 15 were oxidase-positive strains of the genera Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Vibrio. LT-enterotoxigenic Enterobacter, Acinetobacter, Klebsiella, Proteus, Providencia, and Serratia species represented 20 of the food and water isolates. Culture supernatant fluids of representative strains of oxidase-positive and oxidase-negative species giving positive reactions in Chinese hamster ovary cell tests induced fluid accumulation in rabbit ileal loops. Eight of the food samples and two of the water samples contained more than one isolate or species of enterotoxigenic bacterium. The stability of the LT production by oxidase-positive bacteria and non-E. coli strains was assessed by the rabbit skin and adrenal cell tests after 9 months and 1 year of storage, respectively, in Trypticase soy broth with glycerol at -70 degrees C. Only 33% of the oxidase-positive strains were still LT enterotoxigenic. Of the oxidase-negative strains, 50 and 33% were LT producing at 9 months and 1 year, respectively. None of the E. coli isolates, both enterotoxigenic and nonenterotoxigenic, possessed K88, K99, or colonization factor antigen. The survey demonstrates the presence in food and water of enterotoxigenic bacteria of the same species as those isolated from cases of infantile diarrhea in the same community, although a correlation between these sources and infantile diarrhea remains to be established.

Modified Elek test for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli.

The Elek test was modified for detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli. A total of 164 strains of E. coli were tested by the modified Elek test, and the results correlated well with those of the Chinese hamster ovary cell assay and passive immune hemolysis. It is concluded that the modified Elek test is a simple and reproducible assay method for identification of E. coli which produce heat-labile enterotoxin, and is suitable for use in clinical laboratories.

Heat-stable enterotoxin produced by Yersinia enterocolitica isolated from patients.

Twenty-three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15-fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 micrograms of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C X 20 min or 100 C X 60 min). These observations indicated that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat-stable enterotoxin of Escherichia coli. However its physiochemical properties seem to be different from those of E. coli.

Comparison of Assay of Coliform Enterotoxins by Conventional Techniques Versus In Vivo Intestinal Perfusion

Thirty-six strains of coliform bacteria were tested for enterotoxigenicity both by conventional assays, including the Y-1 adrenal and Chinese hamster ovary cell assays for heat-labile toxin and the suckling mouse assay for heat-stable toxin, and by determining the ability of graded concentrations of ultrafiltrate high- or low-molecular-weight toxin preparations to induce water secretion during in vivo perfusion in the rat jejunum. The ultrafiltrates of all 18 strains isolated from persons with infectious diarrheal disease, including seven of Escherichia coli, seven of Klebsiella pneumoniae, and four of Enterobacter cloacae, contained one (nine strains) or two (nine strains) potent toxin fractions (resembling either heat-labile or heat-stable toxin in terms of apparent molecular weight and heat lability characteristics) that induced water secretion at perfusion concentrations of 10 ng/ml or less. Unconcentrated broth filtrates of five of the E. coli strains and two of Klebsiella reacted positively in one or more of the conventional assay systems. Concentrated ultrafiltrates from two strains that were negative in the in vitro assays for heat-labile toxin were tested and also proved to be inactive in these test systems. None of 18 strains isolated from control sources produced, in the ultrafiltrates, enterotoxins capable of inducing water secretion at low concentrations, and none reacted positively in the conventional assays. These results indicate that some strains of coliform bacteria elaborate potent toxin materials that are capable of inducing water secretion and can be detected by perfusion of concentrated ultrafiltrates but not by conventional assay systems for enterotoxigenicity. Whether this represents quantitative or qualitative differences between the toxin materials that stimulate these different test systems remains to be established.

Adaptation of the staphylococcal coagglutination technique for detection of heat-labile enterotoxin of Escherichia coli

Protein A-containing staphylococci coated with specific antiserum were tested for heat-labile enterotoxin of Escherichia coli. The immunological cross-reactivity of E. coli heat-labile enterotoxin with Vibrio cholerae toxin (choleragen) was the basis for sensitizing stabilized suspensions of the Cowan I strain of Staphylococcus aureus with anticholeragen. Unconcentrated culture supernatant fluid containing E. coli heat-labile enterotoxin produced macroscopic agglutination when mixed with sensitized staphylococci in capillary tubes. A total of 15 toxigenic and 61 nontoxigenic isolates were tested by the staphylococcal coagglutination technique in a coded fashion and found to be in agreement with previous results of the Chinese hamster ovary cell assay and the passive immune hemolysis test. The staphylococcal coagglutination technique is simple, relatively inexpensive to perform, and requires the immunoglobulin fraction of anticholeragen as the only specific reagent. The staphylococcal coagglutination technique appears to have potential for routine use in diagnostic microbiology laboratories.

Determination of Corynebacterium diphtheriae toxigenicity by a colorimetric tissue culture assay

Chinese hamster ovary (CHO) cell cultures in microtiter wells are sensitive to growth inhibition and killing by picogram quantities of diphtheria toxin. In the absence of biologically active toxin, the CHO cell culture produces sufficient acidic metabolites to change the phenol red pH indicator from pink to yellow within 56 h. In the presence of 10 pg of toxin per well, growth inhibition can be observed microscopically within 24 h. Diphtheria toxin can be qualitatively assayed from culture supernatants of Corynebacterium diphtheriae or from beta-phage agar plaque plugs. The colorimetric CHO cell assay method for determining toxigenicity allows for the large-scale screening of either diphtheria toxigenicity or antitoxin titration of sera.

Characterization of the Chinese Hamster Ovary Cell Assay for the Enterotoxins of Vibrio cholerae and Escherichia coli and for Antitoxin: Differential Inhibition by Gangliosides, Specific Antisera, and Toxoid

The morphologic change in Chinese hamster ovary (CHO) cells that is associated with increased intracellular concentrations of adenosine 3':5'-cyclic phosphate (cyclic AMP) provided a highly sensitive assay for the heat-liable enterotoxins of Vibrio cholerae and Escherichia coli. The similarity between the responses of CHO cells and those of intestinal mucosa to enterotoxins was demonstrated by the inhibition of these effects by the ganglioside galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide (GM1), as well as by specific antisera. The antisera against either enterotoxin were more effective in inhibiting the effects of homologous toxin than those of heterologous toxin, but cross-neutralization also occurred in both cases. When choleragenoid, the natural toxoid of cholera toxin, was incubated with the CHO cells, the subsequent effects of the toxin on the morphology of the cells were prevented as were the effects of the toxin on intestinal secretion. However, choleragenoid did not prevent the effects of E. coli toxin. The similarity between the action of enterotoxins on CHO cell morphology and on secretion by the small intestine suggests that there are similar binding sites in these tissues. The potential usefulness of the CHO cell assay in quantitative measurement of antitoxic immunity is thus demonstrated.

Diarrhea Due to Escherichia coli in the Rabbit: A Novel Mechanism

A strain of Escherichia coli O15 (RDEC-1) isolated from several rabbits with diarrhea was examined to determine (1) whether the strain could produce diarrhea when administered by the orogastric route to other rabbits and (2) whether this strain was invasive or enterotoxigenic. Strain RDEC-1 produced diarrhea in 48 of 62 rabbits when given by the orogastric route in doses that ranged from 1.5 X 10(2) to 4 X 10(10) bacteria. The organism did not give a positive result in the 18-hr ileal loop or Serény tests and did not invade HeLa cells. Culture supernatants of strain RDEC-1 did not give positive results in the 6- or 18-hr rabbit ileal loop, suckling mouse, Y-1 adrenal cell, or Chinese hamster ovary cell assays. Fluorescent antibody staining of sections of intestine prepared in a cryostat revealed great numbers of E. coli strain RDEC-1 that adhered to the epithelial surface. It is evident that E. coli can produce diarrhea without being able to invade the mucosa or synthesize enterotoxin. Strains of E. coli similar to RDEC-1 may account for some of the E. coli-associated diarrhea that occurs in humans.

Laboratory investigation of diarrhea in travelers to Mexico: evaluation of methods for detecting enterotoxigenic Echerichia coli

A laboratory investigation was conducted on cultures collected from travelers before, during, and after a trip to Mexico to characterize the etiology of traveler's diarrhea. Four laboratory methods for detecting enterotoxigenicity of Escherichia coli were evaluated: the infant mouse assay, the Chinese hamster ovary (CHO) cell assay, the Y1 adrenal cell assay, and the rabbit ileal loop. Although a number of common enteric pathogens were identified as a cause of traveler's diarrhea, including six serotypes of Salmonella, two serotypes of Shigella, Vibrio parahaemolyticus, Giardia lamblia, and Entamoeba histolytica, enterotoxigenic Escherichia coli was most commonly isolated. Strains were identified that produced only heat-labile enterotoxin (LT), only heat-stable enterotoxin (ST), or both LT and ST. The infant mouse assay yielded results falling into two distinct groups, providing a clear separation of positive and negative cultures. The CHO assay also formed two groups, with positive cultures producing 11% or more of the elongated cells. There was good agreement between the CHO and the Y1 adrenal cell assays for detection of LT. The adrenal cell system for detection of LT was more suitable than the CHO assay for processing large numbers of specimens because of the miniculture modification of this method utilized in this study. The infant mouse method was a simple and reliable method for detecting ST.

ENTEROTOXIGENIC ESCHERICHIA COLI AND IDIOPATHIC DIARRHŒA IN BANGLADESH

Fæcal Escherichia coli from Bangalee patients with idiopathic diarrhœa were tested in the Chinese hamster ovary cell (C.H.O.) assay to detect production of thermolabile enterotoxin (L.T.). C.H.O.-positive E. coli produce both L.T. and a thermostable toxin (S.T.). C.H.O.-positive strains were found in 19.2% of all cases and in 70% of the most severely ill patients with non-vibrio cholera. E. coli which produce S.T. alone were identified in a third of the C.H.O.-negative cases. Enterotoxigenic E. coli were found in 55% of inpatients with idiopathic diarrhœa and, if an ætiological role is confirmed, may be one of the commonest causes of tropical diarrhœa.