The DNA hybridization assay employing a 460-base-pair fragment of DNA encoding for the methanol-insoluble form of heat-stable toxin (ST-II) was used to determine the prevalence of ST-II enterotoxigenic Escherichia coli (ETEC) in pigs, people, and water at 57 farms in Sri Racha, Thailand. ST-II ETEC was found in 62 (3%) of 2,110 suckling, 181 (32%) of 560 weaned, and 4 (1%) of 457 adult pigs examined. Of 62 suckling pigs with ST-II ETEC infections 21% had diarrhea, but none of 185 infected older pigs had diarrhea. ST-II ETEC was found more frequently in suckling pigs with diarrhea than without diarrhea (13 of 146 versus 49 of 1,964) (P less than 0.001). ST-II ETEC was detected in water collected from 3 of 57 clay jars containing water used to bathe at three pig farms, in 1 jar used to bathe immediately after working in the barn, and from one farmer who did not have a recent history of diarrhea. Evidence of this organism was not found in 245 other individuals living on the pig farms or in 220 inhabitants and 114 water specimens collected at tapioca farms nearby. In Thailand ST-II ETEC was found in suckling pigs with diarrhea but was infrequently found in humans.

The DNA hybridization assay for genes encoding for Escherichia coli enterotoxins was used to examine water specimens in Thailand. In a reconstruction experiment, the DNA hybridization assay was 10(4) times more sensitive than testing random E. coli in the Y-1 adrenal and suckling mouse assays in identifying enterotoxigenic E. coli (ETEC) in water. Drinking and bathing water collected from 2 of 10 different homes of individuals with ETEC-associated diarrhea and 6% (5 of 78) and 11% (11 of 78) of drinking and bathing water samples collected from homes of individuals with diarrhea without ETEC infections, as well as 6% (5 of 77) and 8% (6 of 77) of drinking and bathing water collected from homes in which no inhabitants had diarrhea, were homologous with the DNA probes. Ten E. coli from each of the 31 water specimens which contained bacteria which were homologous with the DNA probes were tested in the Y-1 adrenal and suckling mouse assay. In only 2 of these 31 specimens could ETEC be identified with the standard assays. The DNA hybridization assay is a much more sensitive means of detecting organisms carrying genes coding for enterotoxin production than testing 10 individual colonies in the Y-1 adrenal and suckling mouse assays. This novel application of recombinant DNA technology provides a sensitive method of detecting organisms carrying genes coding for enterotoxin, and this method will be useful in defining the epidemiology of ETEC.

Heat-stable enterotoxigenic Escherichia coli was identified by nucleotide hybridization with RNA transcripts ot the gene encoding heat-stable A-2 enterotoxin. Radiolabeled enterotoxin gene RNA transcripts are easier to prepare and avoid the preparation of cloned DNA probes that can be nonspecific if they contain cloning vector DNA. Enterotoxigenic Escherichia coli (ETEC) is usually identified by testing isolates for enterotoxin production in bioassays or serological tests (2, 7, 11, 14, 15). Alternatively, ETEC can be identified by detecting the genes encoding these enterotoxins by DNA hybridization, a method which has been particularly useful in detecting ETEC in large numbers of specimens (3-6). Specific DNA fragments of plasmids containing the cloned enterotoxin genes have been used in DNA hybridization assays to identify ETEC (4, 5). Probes produced by endonuclease digestion must be electrophoretically separated from plasmid-cloning vector DNA to obtain a specific probe and must also be nick translated (9). Nick translation is relatively easy to perform with large DNA fragments, such as the 850-base-pair fragment used as the heat-labile enterotoxin (LT) gene probe, but is more difficult to perform on small DNA probes used to detect the genes encoding heatstable enterotoxin (STA-1 [154 base pairs] or STA-2 [216 base pairs]). Ninety-nine percent of E. coli that hybridized with the LT probe produced LT, as measured by the Y-1 adrenal cell assay, but only 72% of E. coli that hybridized with the STA-2 probe produced ST, as measured by the suckling mouse assay (5). The lack of specificity of the cloned enterotoxin gene probes was due to probes that contained cloning vector DNA. Niriety-eight percent of E. coli that hybridized with the ST oligo probe produced ST (6). To develop a more convenient probe to identify genes encoding STA-2, we clotied the genes encoding STA-2 into pSP64 and used RNA transcripts to detect ST+ ETEC by colony hybridization. Plasmid DNA was isolated from E. coli C600(pSLM004) and was digested with EcoRI, HindIII, and HpaII (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) as previously described (10). The endontclease digestion fragments were separated by electrophoresis on a 1.5% low-meltingtemperature agarose gel (FMC Corp., Marine Colloids Div., Rockland, Maine) in Tris-borate buffer, and a 216-base-pair DNA fragment encoding STA-2 was éfluted by heat, phenol extracted, and ethanol precipitated (8). This DNA fragment was blunt-end ligated with pSP64 (Promega Biotec, Madison, Wis.