Y1 Adrenal Cell Assay Research Articles

Prevalent Phenotypic and Genotypic Profile of Enterotoxigenic Escherichia coli among Iranian Children

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea among children. ETEC strains express colonization factors (CFs), which mediate adherence to the small intestinal epithelium and produce entrotoxins that induce diarrhea. Here, we characterized the phenotypes and genotypes of ETEC strains from 261 diarrheal stool samples from Iranian children. The prevalence of ETEC was 8.04%. Most of the isolates were positive for heat-labile and heat-stable toxins. CFA/I, CS3, CS2, and CS5 were detected from some of the clinical isolates. 33.3% of the isolates did not express CFs. The majority of ETEC isolates were identified as O127 and O128 serotypes, and 57% of the strains were resistant to more than 1 antimicrobial agent. Heat-labile enterotoxin activity was confirmed using the Y1 adrenal cell assay, rabbit ileal loop and adenylate cyclase activation tests. Regional phenotypic and genotypic characterization could help to elucidate the ecology and pathogenicity of ETEC to efficiently reduce the burden of illness brought about by ETEC. This study may lead to development of effective prophylactic measures.

Seasonal, nontoxigenic Vibrio cholerae 01 Ogawa infections in the Eastern Region of Saudi Arabia

Background: Surveillance for Vibrio cholerae in the Eastern Region of Saudi Arabia has been ongoing since 1985 to detect and prevent local proliferation of imported cholera. In 1996 and 1997 the authors performed additional microbiologic and epidemiologic assessment of V. cholerae surveillance to better characterize a recurrent summertime pattern of V. cholerae infections in the Eastern Region of Saudi Arabia. Methods: All health facilities routinely submitted stool or rectal swab specimens for isolation of V. cholerae from patients with gastroenteritis. In addition, specimens were taken from expatriate workers and household contacts of persons with confirmed V. cholerae infection. Forty-two isolates were evaluated for cholera enterotoxin by enzyme-linked immunosorbent assay, cholera toxin polymerase chain reaction, and Y1 adrenal cell assay; 12 isolates also were characterized by pulsed-field gel electrophoresis (PFGE). Interviews about potential exposures were done for all V. cholerae infections. Results: Vibrio cholerae O1 serotype Ogawa biotype El Tor was identified in q113 gastroenteritis patients (6.0 per 100,00 population per year), 28 asymptomatic expatriate workers, and 16 of 982 household contacts of index patients. All symptomatic infected persons had mild illness that was not typical of cholera, and all 42 isolates evaluated were nontoxigenic. All 12 isolates evaluated by PFGE had an indistinguishable pattern (pattern 81). Infections appeared in late May, decreased in mid-July through August, increased again in September, and disappeared from December through April. Infections had a uniform geographic distribution and affected all ages. No linkage was identified between affected households, or between community cases and food-handlers or domestic servants. Discussion: Surveillance in the Eastern Region of Saudi Arabia has identified a novel strain of nontoxigenic V. cholera 01 Ogawa. This strain probably has a local environmental reservoir. Since cholera toxin is the primary virulence factor involved in the cause of cholera, assays for cholera toxin should be included in cholera surveillance.

Search for LT-II and STb DNA Sequences Among Escherichia coli Isolated from Bovine Meat Products by Colony Hybridization

A total of 1,066 Escherichia coli colonies isolated from 105 raw bovine meat samples purchased at supermarkets in Rio de Janeiro were submitted to hybridization assays with gene probes for LT-II and STb enterotoxins. Five colonies comprising four different E. coli strains isolated from four pieces of beef, two samples of ground beef (5.7%) and two hamburger patties (5.7%) hybridized with the LT-II probe, while no hybridization occurred with the STb probe. Expression of LT-II enterotoxin using the Y1 adrenal cell assay was verified in two of four E. coli strains. A serotype diversity existed among LT-II E. coli strains.

A comparison of alkaline phosphatase and radiolabelled gene probes with bioassays for enterotoxigenic Escherichia coli

Alkaline phosphatase-conjugated oligonucleotide probes (APO), 32P-labelled oligonucleotide (RO) and cloned polynucleotide (RP) probes were evaluated for their ability to detect enterotoxigenic Escherichia coli (ETEC) as defined by bioassay. These three sets of probes were applied to 301 E. coli strains that had previously been defined by the Y1 adrenal cell assay for heat-labile enterotoxin (LT) and the infant mouse assay for heat-stable enterotoxin (ST). The correlation of the APO probe for LT with the bioassay was 98% with five discrepancies and a positive predictive value (PPV) of 100%. For the APO/ST probe the correlation with the bioassay was 98% with seven discrepancies and a PPV of 96%. The correlation of the RO probe for LT was 99% with four discrepancies and a PPV of 100%, while the overall correlation for the two RO/ST probes was 97% with eight discrepancies and a PPV of 97%. For the RP probes, the correlation for LT was 99% with four discrepancies and a PPV of 100% and for ST was 98% with seven discrepancies and a PPV of 98%. These findings suggest that the APO probes were as sensitive as the RO and RP probes in detecting ETEC by colony hybridization and could be a practical alternative to bioassays and radiolabelled probes for ETEC since they do not require expensive equipment or extensive technical training.

Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.

Variation in chemical properties and antigenic determinants among type II heat-labile enterotoxins of Escherichia coli

Type II heat-labile enterotoxin (LT-II) from Escherichia coli 41 was purified and compared with prototype LT-II encoded by genes from E. coli SA53. Both toxins were oligomeric proteins consisting of polypeptides A (Mr, 28,000) and B (Mr, 11,800). The A polypeptides were cleaved by trypsin into fragments A1 (Mr, 21,000) and A2 (Mr, about 7,000). These two toxins were shown to belong to two different subclasses of LT-II. We propose to designate the prototype toxin LT-IIa and the new variant LT-IIb. The pI of LT-IIb was between 5.2 and 5.6, significantly lower than the pI of 6.8 for LT-IIa, and the behavior of LT-IIb during purification differed significantly from that of LT-IIa. The toxic dose of unnicked LT-IIb in the Y1 adrenal-cell assay was 94 pg, but trypsin-treated, nicked LT-IIb was toxic at about 3 pg. In contrast, the toxic dose of LT-IIa was previously shown to be 0.5 to 1 pg for several preparations that varied from unnicked to partially nicked, and treatment with trypsin was not required for full toxicity. The titer of LT-II antiserum in neutralization tests was 100-fold greater against LT-IIa than against LT-IIb. In immunodiffusion tests, LT-IIa and LT-IIb gave a reaction of partial identity. In a radioimmunobinding assay, the titer of LT-IIa antiserum against homologous LT-IIa was approximately 10-fold greater than against LT-IIb. The cholera-E. coli family of heat-labile enterotoxins has been divided into serogroup I, which includes cholera toxin and the antigenic variants of E. coli heat-labile toxin designated LTh-I and LTp-I, and serogroup II, which includes LT-IIa and LT-IIb. The type I and type II toxins do not cross-react in neutralization or immunodiffusion tests. By using very sensitive radioimmunobinding assays, it was possible to demonstrate common antigenic determinants between the type I and type II toxins. However, the titers of antibodies in hyperimmune sera that recognized these common determinants were very low.

Purification and characterization of type II heat-labile enterotoxin of Escherichia coli.

Type II heat-labile enterotoxin (LT-II) of Escherichia coli has several biologic activities similar to cholera toxin (CT) and E. coli type I heat-labile enterotoxin (LT-I), but it is not neutralized by antiserum prepared against CT or LT-I. LT-II was purified from E. coli SA53 and from E. coli HB101(pCP3837), a strain that contains the cloned LT-II genes in a hybrid plasmid and produces up to 600 times more LT-II than does SA53. Purification involved sonic disruption of bacterial cells, ammonium sulfate fractionation, chromatography on Affi-Gel Blue, chromatofocusing, and gel filtration on Sephadex G-100. The LT-II purified to apparent homogeneity from HB101(pCP3837) had an isoelectric point of 6.8, induced increased vascular permeability in rabbit intracutaneous tests, caused rounding of cultured Y1 adrenal cells accompanied by increased intracellular cyclic AMP, and was 25 to 50 times more potent than CT or LT-I in the Y1 adrenal-cell assay. In contrast, purified LT-II did not cause secretion in ligated rabbit ileal segments at doses corresponding to CT controls that gave strongly positive reactions. LT-II was composed of two different polypeptides with MrS of 28,000 (A) and 11,800 (B); treatment of LT-II with trypsin cleaved the A polypeptide to fragments A1 (Mr, 21,000) and A2 (Mr, 7,000). The activity of LT-II was not blocked by ganglioside GM1 at concentrations that inactivated LT-I or CT. Antiserum against the LT-II from E. coli HB101(pCP3837) completely neutralized purified LT-II and the LT-II in crude extracts of SA53, but it did not neutralize purified LT-I or CT.

Comparison of methods to detect Escherichia coli heat-labile enterotoxin in stool and cell-free culture supernatants.

We compared the standard Y-1 adrenal cell (YAC) assay for heat-labile enterotoxin (LT) with newer rapid and economical immunological methods of detection. Stool samples were collected from 164 acutely ill American students in Guadalajara, Mexico. Supernatants were prepared from each stool. Stools were cultured for Escherichia coli by standard techniques. Individual E. coli-like colonies were examined for LT production by the Biken assay. Culture supernatants and stool supernatants were assayed for the presence of LT by the YAC assay, counterimmunoelectrophoresis, and enzyme-linked immunosorbent assay (ELISA). Standard YAC assays of culture supernatants revealed that 40 of the 164 specimens (24%) were LT positive. Counterimmunoelectrophoresis detected 60% and ELISA detected 65% of the 40 known positives when culture supernatants were used. The Biken assay detected 35% of the 40 known positives. With stool supernatants, the YAC assay detected only 18% of the known positives, counterimmunoelectrophoresis detected 60%, and ELISA detected 90%. In addition, ELISA detected 13 LT-positive stool supernatants not detected by the YAC assay of culture supernatants. The ELISA in which stool supernatants are used may be a useful method to detect LT.

Development of a vaccine of cross-linked heat-stable and heat-labile enterotoxins that protects against Escherichia coli producing either enterotoxin.

A vaccine of cross-linked heat-stable (ST) and heat-labile (LT) toxins that protects against heterologous serotypes of strains of Escherichia coli which produce either the LT or ST enterotoxin was developed by conjugating ST to LT by the carbodiimide reaction. Three interrelated factors were found to affect the composition and properties of the final conjugate: (i) the amount of carbodiimide added to the toxins, (ii) the initial ratio of ST to LT, and (iii) the duration of the conjugation reaction. Optimal conjugation conditions were identified as a carbodiimide-to-toxin ratio of 10:1 by weight, an initial molar ratio of ST to LT of 100:1, and a conjugation reaction time of 96 h. This approach yielded a conjugate that contained 96% by moles and 36% by weight pure ST, determined with radioiodinated pure ST, and 34% by weight semi-pure ST, determined by the Lowry protein method. The retained antigenicities of the conjugated toxins, as determined by enzyme-linked immunosorbent assays, was greater than or equal to 82%, and their toxicities, as determined by the Y1 adrenal cell assay for LT and by the suckling mouse assay for ST, were reduced to less than or equal to 0.15%. Immunization of rats with this cross-linked ST-LT vaccine provided strong protection against challenge with either the LT or the ST toxin or with viable heterologous strains which produce these toxins, either singly or together. These observations indicate that conjugation of ST to LT results in a unique new immunogen in that ST acquires immunogenicity as a function of the reaction, LT retains most of its antigenicity, and the toxic properties of each individual toxin are greatly reduced.

GM1 ganglioside enzyme-linked immunosorbent assay for detection of heat-labile enterotoxin produced by human and porcine Escherichia coli strains.

Human and porcine enterotoxigenic strains of Escherichia coli were cultivated in tryptone-yeast extract medium or brain heart infusion broth and tested for production of heat-labile enterotoxin by the GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) and the Y1 adrenal cell assay. When testing for enterotoxigenicity by the GM1-ELISA technique, homologous antisera for human and porcine heat-labile enterotoxins had to be used to detect enterotoxigenic strains of human and porcine origin, respectively. This observation indicates a serological difference between the heat-labile enterotoxins produced by human and porcine strains. Furthermore, brain heart infusion broth was found to have an inhibitory effect on detection of enterotoxin both in the GM1-ELISA and in a toxin-binding modification of the Y1 adrenal cell test, but not in the conventional adrenal cell assay.

Microtiter ganglioside enzyme-linked immunosorbent assay for vibrio and Escherichia coli heat-labile enterotoxins and antitoxin.

We have developed a microtiter enzyme-linked immunosorbent assay method for detecting the heat-labile enterotoxins of Vibrio cholerae and Escherichia coli using GM1 ganglioside as the base coat. This method compares favorably with a similar assay using anticholera toxin as the base coat, and with the Y1 adrenal cell assay. The assay should be useful in detecting enterotoxin production in E. coli and vibrios (including non-agglutinating Vibrio), in quantitating the toxin, and in determining binding properties of enterotoxins to ganglioside. The assay can also be used to quantitate antibodies which block the attachment of the toxin to the ganglioside.

Sensitivity, precision, and accuracy of the Y1 adrenal cell enterotoxin assay

The Y1 adrenal cell assay for heat-labile enterotoxin (LT) was found to be relatively insensitive for filtrates of toxigenic Escherichia coli H10407. When observations were made blindly and subjected to rigorous controls, reliable detection of LT occured only at filtrate dilutions from 1:4 to 1:10. Detection of LT was unreliable when E. coli H10407 was mixed with another enteric organism. Overall, 7.1% of observers' reports from a single assay were imprecise, and 2.6% would have resulted in errors in detection of LT; however, triplicate assays prevented false positive reports of LT detection. Routine testing for LT production in the clinical diagnostic laboratory awaits a simpler, more sensitive, and more direct method of testing stool specimens.

Detection of heat-labile enterotoxin-like activity in stools of patients with cholera and Escherichia coli diarrhea

The Y1 adrenal cell tissue culture assay was used to detect heat-labile enterotoxin-like activity in the stools of 14 of 74 patients with diarrhea. A positive effect of the stool on the adrenal cells was heat-labile and neutralized by cholera antitoxin. Enterotoxin-like activity was detected in the stools of 10 of 30 patients with cholera and in those of 2 of 4 from whom heat-labile Escherichia coli were isolated. None of the stools from nine individuals with Vibrio parahaemolyticus, Salmonella, or Shigella infections were positive. Two of 31 individuals from whom no pathogens were isolated had detectable toxin-like activity in their stools. The Y1 adrenal cell assay provides a rapid method of diagnosing heat-labile enterotoxigenic diarrhea and could be an adjunct in epidemiological studies of gastroenteritis.

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.