Toxigenic Escherichia coli in calves with diarrhea in Minas Gerais, Brazil

Foram estudadas 165 amostras de Escherichia coli isoladas de 41 bezerros provenientes de 11 rebanhos leiteiros do Estado de Minas Gerais. Sete amostras produziram toxina termoestável. Destas, duas aglutinaram hemácias de cavalo na presença de manose e foram sorologicamente positivas para K99 (FS), uma apresentou aglutinaçäo de hemácias de cavalo e sorologia negativa para K99 (FS) e duas näo aglutinaram hemácias de cavalo e foram sorologicamente positivas para K99 (FS). Vinte e cinco amostras, isoladas de 18 bezerros com idade entre sete e 51 dias, foram produtoras de citotoxinas. Estes resultados indicam maior importância das amostras citotoxigênicas de E. coli sobre as enterotoxigênicas, no Estado de Minas Gerais

Use of an indirect haemagglutination test for the detection of Clostridium perfringens type A enterotoxin.

An indirect haemagglutination (IH) test is described for the detection of Clostridium perfringens type A enterotoxin, produced by strains isolated from human cases of food poisoning and from contaminated food. Though no strict relationship could be observed between titers in the IH test and the time it took mice to die from the intravenous inoculation of mice (IIM test), results of the supernatants examined by both methods demonstrated that the IH test was more sensitive than the IIM one. No unspecific reaction was obtained in the IH with a negative control and the inhibitions of the IH and IIM tests by specific antiserum against C. perfringens enterotoxin showed that the IH test is very specific. The IH assay is recommended for its sensitivity and easy performance by less-equipped laboratories, by these and other data.

[The incidence of enterotoxigenic Escherichia coli, rotavirus and Clostridium perfringens from cases of diarrhea in children, in the region of Campinas, SP, Brazil]. / Incidência de Escherichia coli enterotoxigênica (ETEC), rotavírus e Clostridium perfringens de casos de diarréia em crianças, na região de Campinas, SP, Brasil.

A survey for the detection of enterotoxigenic Escherichia coli (ETEC), rotavirus and enterotoxigenic Clostridium perfringens in diarrheic stools of children up to 2 years old was carried out in the region of Campinas, SP, Brazil. Twenty-seven (20.45%) faecal specimens were positive for ETEC. From these samples 41 strains of ETEC were isolated from which 40 produced only thermolabile (LT) enterotoxin, as detected by a modified radial immune haemolysis test. Among the 183 faecal specimens examined for the detection of rotavirus, 29 (15.84%) were positive when examined by polyacrilamide gel electrophoresis (PAGE) and immunoenzymatic assay (EIA) being 15 (51.7%), derived from stools collected from winter months. All strains of rotavirus belonged to group A and through the PAGE technique, it was observed that the most frequent (9 strains) electrophoretype, according to the adopted classification, was Ib, IIc, IIIb, IVa. Only 113 fecal specimens were examined for the presence of enterotoxigenic C. perfringens. For the detection of enterotoxin in culture supernatants the reverse passive haemagglutination and intravenous inoculation of mice were used. Twelve (10.61%) enterotoxigenic C. perfringens strains were found. Taking into consideration these findings the authors call the attention of the relative value of conventional coprocultures for diagnostic purposes, pointing out the important of establishing simplified methods which would render easier, the detection and identification of the groups of enteropathogenic agents studied in this research.

Evaluation of the Biken test to detect heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains.

Fifty-seven strains of enterotoxigenic Escherichia coli isolated from humans and pigs and producing thermolabile (LT) enterotoxin were used to ascertain the efficiency of the Biken test compared to the passive immune haemolysis test (PIH), considered as very sensitive for detecting that enterotoxin. The two assays were carried out using anti-porcine (anti-LTp), anti-human (anti-LTh), anti-cholera toxin (anti-CT) and anti-choleragenoid (anti-Cg) antisera. Our results showed that the Biken test was very irregular, with many false-negative results. Positive results (ranging from 78.9 to 22.8) were dependent upon the antiserum used. Conversely, the PIH test was much more efficient in the detection of LT, since 100% of the LT+ strains were positive in this test whatever the antiserum used.

Stability of thermolabile (LT) enterotoxin produced from enterotoxigenic Escherichia coli strains maintained in vitro.

The stability of thermolabile (LT) enterotoxin in 26 strains of porcine enterotoxigenic Escherichia coli (PETEC) belonging to serogroups 08 and 0149 was assayed by the passive immune hemolysis (PIH) test, over a period of 9 months at -70 degrees C. It was found that the percentage of LT+ colonies (% LT+) and the mean value of hemoglobin release (XHb), could predict a change from LT+ to LT-.

Factors affecting detection of Yersinia enterocolitica heat-stable enterotoxin by the infant mouse test.

With regard to the assay of heat-stable enterotoxin (ST) from Yersinia enterocolitica, we made a comparative study of the conventional infant mouse test read at 4 h and a modified infant mouse test read at 2 h. The influence of several factors, such as the medium used to prepare ST, lysing of bacterial cells from the broth cultures used to prepare ST, and the temperature at which the inoculated mice were kept during the test, was also investigated. Thus, with a few exceptions, Pai-Mors medium was more suitable than Casamino Acids-yeast extract medium, for the preparation of yersinial ST. Gut/carcass weight ratios obtained with lysed supernatants or with supernatants from whole cultures of Y. enterocolitica were similar, suggesting that most of the ST produced by this microorganism in broth cultures is extracellular. The amount of ST produced by Y. enterocolitica, as well as the ambient temperature at which inoculated mice were kept during the assay, was found to influence gut/carcass weight ratios obtained with both tests. Enterotoxigenicity and the temperature at which mice were kept were interrelated, such that for weakly enterotoxigenic strains there were no significant differences among gut/carcass weight ratios for the conventional and modified infant mouse tests carried out at 18 or 25 degrees C, but at 30 degrees C the values in the modified test were higher for most ST preparations with Pai-Mors medium. The influence of ambient temperature was more pronounced at 37 degrees C, since most strains produced negative results in the conventional test at this temperature. We conclude that the conventional infant mouse test is adequate for assaying yersinial ST, provided that the temperature at which mice are kept during the assay is fixed at around 25 degrees C.

Escherichia coli enterotoxigenicas e rotavirus detectados em criancas com gastroenterite aguda em Belem, Para. / Enterotoxigenic Escherichia coli isolated from children with acute gastroenteritis, Belem, Para

Procurou-se pesquisar, em Belem do Para, a presenca de Escherichia coli enterotoxigenica e outros enteropatogenos, incluindo agentes virais, nas fezes de 44 criancas de 0 a 5 anos de idade, com quadro diarreico agudo. A presenca de bacterias enteropatogenicas foi investigada pela coprocultura e a pesquisa de rotavirus atraves das tecnicas de ELISA e contra-imunoeletroosmoforese. O isolamento de enterovirus foi feito em cultura de celulas (Vero e HEp 2) e camundongo recem-nascido. Helmintos e protozoarios intestinais foram procurados pelo metodo direto. Trinta e tres (75%) dos casos foram positivos para um ou mais enteropatogenos, sendo 30,3% de etiologia bacteriana, 39,4% de origem viral e 30,3% de infeccao mista. A procura de enterotoxina LT e ST em 153 cepas de E.coli isoladas dos pacientes, foi feita, pelos testes de imuno-hemolise passiva (PIH) e camundongo recem-nascido (Teste de Dean), respectivamente. Cepas enterotoxigenicas de E. coli, em numero de 17 foram isoladas de sete dos 44 pacientes estudados. Em nenhuma amostra ocorreu producao simultanea de duas enterotoxinas

Permeability factor-like skin reaction in rabbits induced by endotoxin of Salmonella sp.

An inflammatory skin reaction similar to the permeability factor (PF) described for the thermolabile (TL) enterotoxin of Escherichia coli was induced in rabbits inoculated intradermally with supernatants from cultures of Salmonella typhimurium and S. enteritidis. This PF-like activity was observed with both crude supernatants as well as those which were submitted to gel filtration through Sephadex G-100. PF-like activity was found only in fraction 1 (F1) of the chromatographed materials. It was resistant to boiling, proteolytic enzymes and wide variations of pH. Serological studies based on agglutination and immunodiffusion tests demonstrated that F1 materials were closely related to the somatic antigen of group B Salmonella. No specific TL activity, as detected by the Y-1 adrenal cell assay and the passive immune haemolysis test, could be demonstrated. Furthermore, F1 materials were not enterotoxigenic as assayed by the rabbit ileal loop assay, and no neutralization of PF-like activity could be obtained in tests carried out using F1 preparations pre-incubated with either anti-F1 or cholera antitoxin. Based upon these findings, it seems reasonable to suppose that most PF reactions, already reported as being caused by a TL-like enterotoxin produced by Salmonella, are probably due to endotoxin. In fact, this possibility was reinforced by the Sanarelli-Shwartzman phenomenon which was produced in rabbits inoculated with F1 materials.

Pesquisa de fatores de virulencia em Yersinia enterocolitica. / Research on virulence factors in Yersinia enterocolitica

Cinquenta amostras de Y. enterocolitica (32 provenientes da colecao do Instituto Pasteur, Paris, pertencentes a diferentes sorotipos, e 18 isoladas no Brasil, pertencentes aos sorotipos 0:3 e 0:5) foram estudadas quanto a invasibilidade, pesquisada pelo teste de Sereny e em celulas HeLa, e quanto a producao de enterotoxinas termolabil (LT) e termoestavel (ST). Tambem foi pesquisada, atraves de provas de hemaglutinacao manose-resistente, a presenca dos antigenos de aderencia CFA/I, CFA/II,K88 e K99. Nenhuma amostra produziu enterotoxina LT ou apresentou hemaglutinacao manose-resistente. Em celulas HeLa o teste foi positivo para 94,4% das amostras isoladas no Brasil e para 68,7% das amostras da colecao do Instituto Pasteur. No teste de Sereny, 77,7% das amostras isoladas no Brasil e 40,6% das amostras do Instituto Pasteur provocaram intensa conjuntivite em cobaios sem, entretanto, provocar a classica ceratoconjuntivite, produzida por amostras virulentas de Shigella.Producao de enterotoxina ST foi observada em 18,0% das amostras. Tanto a invasibilidade como a producao de ST foram observadas em diferentes sorotipos

Single radial immune hemolysis test for detection of Escherichia coli thermolabile enterotoxin.

A single radial immune hemolysis test for the detection of thermolabile enterotoxin has been developed for routine purposes. Stationary cultures from enterotoxigenic Escherichia coli in Casamino Acids-yeast extract medium may be used for the detection of this enterotoxin, and under the conditions of the experiment, the single radial immune hemolysis test was as sensitive as the passive immune hemolysis test. The results obtained in the single radial immune hemolysis test agreed entirely with those obtained in the passive immune hemolysis test, and no false-positive reactions were obtained when cholera antitoxin diluted 1:80 was used. The assay is easy to perform, inexpensive, and specially designed for less-equipped laboratories.

Mannose-resistant haemagglutination and colonisation factors among Escherichia coli strains isolated from pigs.

Ten heat-labile (LT) and 23 heat-stable (ST) enterotoxin-producing strains of enterotoxigenic Escherichia coli (ETEC) and 100 non-enterotoxigenic E coli (non-ETEC) strains isolated from pigs with diarrhoea were examined for the presence of colonisation factors by means of mannose-resistant haemagglutination and serological tests. Seven of 10 LT strains, one of 23 ST and 18 of 100 non-ETEC carried K88 antigen; and four ST and four non-ETEC strains possessed CFA/I antigen. All strains carrying K88, K99 and CFA/I antigens were positive in the mannose-resistant microhaemagglutination (MRMH) test but four ST and 14 non-ETEC isolates positive in the MRMH test did not possess K88, K99 or CFA/I antigens. A considerable number of non-ETEC strains were positive in the MRMH test but lacked K88 and K99 antigens, suggesting that other unknown haemagglutinating colonisation factors may exist in porcine E coli strains.

Improvements in the passive immune hemolysis test for assaying enterotoxigenic Escherichia coli.

The sensitivity of the passive immune hemolysis test for the detection of heat-labile enterotoxin from Escherichia coli was increased when the test was carried out with Veronal-buffered saline plus Ca2+ and Mg2+ as diluent, instead of phosphate-buffered saline. The performance of the test was further improved by using stationary cultures to which mitomycin C had been added at the end of the lag phase.

Simplification of methods for the production and storage of specimens to be tested for heat-stable enterotoxin of Escherichia coli.

Experiments with the infant mouse test demonstrated that there is no need of shaking for heat-stable Escherichia coli enterotoxin production when low volume of medium per volume of flask ratios are used in stationary cultures. Centrifugation and filtration of the cultures to be tested are not necessary either, and Merthiolate (1:10,000) used as preservative has no deleterious effect on heat-stable enterotoxin activity. Based upon these findings, some modifications of the procedures for production and storage of heat-stable enterotoxin preparations are suggested. Standardized pieces of filter papers are wetted with Merthiolated stationary cultures which are to be assayed for heat-stable enterotoxin activity by the infant mouse test. From dried filter papers, heat-stable enterotoxin can be eulted unaltered up to 2 months after specimen preparation. With the proposed modifications, even modestly equipped laboratories will be able to carry out the infant mouse test or at least to prepare specimens to be assayed by more specialized laboratories.

Passive immune hemolysis for detection of heat-labile enterotoxin produced by Escherichia coli isolated from different sources.

Fifty-one strains of Escherichia coli isolated from humans, swine, food, and water and identified as enterotoxinogenic by the Y-1 adrenal cell assay, were examined for heat-labile enterotoxin (LT) production by the passive immune hemolysis test. Cholera antitoxin, anti-choleragenoid and anti-LT were used as antisera. Cholera antitoxin was much more potent than anti-choleragenoid and LT antiserum in the detection of LT-positive strains. All strains isolated from pigs and sausage were negative in tests made with LT antiserum. A few strains isolated from humans, food, and water also gave negative results. These data showed that the passive immune hemolysis test is not as efficient as the Y-1 adrenal cell assay in the detection of enterotoxinogenic E. coli strains.

Swiss and inbred mice in the infant mouse test for the assay of Escherichia coli thermostable enterotoxin.

Swiss and inbred mice (C3H, ASn, B10A, and C57) were used to assay different thermostable enterotoxins prepared from Escherichia coli strains 3 (human), E57 (swine), B41 (cattle), and S13 (sheep). All strains of mice detected enterotoxins, but some reacted better to a given thermostable enterotoxin preparation than other inbred strains. Since the results obtained with Swiss mice were more consistent than with inbred mice, the former are apparently more suitable in the infant mouse test.

The use of 4,4'-biphenylbisdiazonium fluoroborate as a coupling agent for passive haemolysis tests.

The possibility of use a stable diazonium compound, to replace bisdiazotized benzidin (BDB) for conjugating antigens to red blood cells was investigated. Five different batches of 4,4'-biphenylbisdiazonium fluoroborate (BDF) were prepared and tested in conjugation experiments. The sensitivity to specific lysis of red cells coated with different antigens (EG cells) was assayed by both direct and indirect passive haemolysis tests. Optimal conditions for the preparation of EG cells with different protein antigens were established. With the exception of the concentration of the bifunctional reagent, these conditions were similar to those previously reported for BDB. The antibody content of different anti-protein sera could be determined, with a 10 % error, by using EG cells prepared under optimal conditions in indirect passive haemolysis tests. The amount of antibody detected by this method varied from 7 to 12 ng N Ab/ml, depending upon the nature of the antigen fixed to red cell. The content of BSA in dilute solutions could be estimated by the specific inhibition of the indirect passive haemolysis tests. Testing different samples of BDF maintained at different conditions showed that the compound could be kept at room temperature, during the least four years, without appreciable loss of the conjugating properties, provided it was kept in the dry state and in the dark.