A. F. Pestana de Castro

Genes coding for Shiga-like toxins in bovine verotoxin-producing Escherichia coli (VTEC) strains belonging to different O:K:H serotypes

Forty-six verotoxin-producing Escherichia coli (VTEC) strains isolated from diarrhoeic and healthy calves in Spain were examined for DNA sequences homologous to genes for verotoxins (VT1 and VT2) and enterotoxins (LT-I, LT-II, STaH, STaP and STb). Hybridisation showed that 26 (57%) of VTEC strains carried VT1 genes, 13 (28%) possessed VT2 genes, and 7 (15%) carried both VT1 and VT2 genes. No VTEC strains hybridised with DNA probes for enterotoxins. A correlation was found between the serotype and type of VT produced. Thus, all strains of serotypes O26:K-:H11 (13 strains), O103:K-:H2 (3 strains) and O128:K?:H- (4 strains) hybridised with the VT1 probe only, whereas all strains of serotypes O4:K-:H4 (3 strains) and O113:K-:H21 (4 strains) were positive with the VT2 probe only. By contrast, O81:K?:H28 (2 strains) and O157:K-:H- (2 strains) strains hybridised with both VT1 and VT2 probes. One strain of serotype O157:K-:H7 was VT2 positive.

Production, purification and partial characterization of a new adhesive factor (F42) produced by enterotoxigenic Escherichia coli isolated from pigs.

Production of the F42 adhesive factor by porcine enterotoxigenic Escherichia coli (ETEC) grown on minimal solid medium was glucose-dependent. The addition of alanine and sodium acetate to this medium repressed the expression of this antigen whose production was also inhibited when the pH of the growing medium was lower than 7.4. The antigen was extracted from F42-positive ETEC grown in minimal liquid medium supplemented with 0.5% glucose. The cells were suspended in buffered 1 M NaCl and heated at 60 degrees C. The supernatant was then treated with ammonium sulphate and the resulting precipitate treated with deoxycholate followed by chromatography of the deoxycholate-soluble material on Sepharose-4B. The molecular weight of F42 purified antigen was near 31,000 daltons and its pI 3.2, as determined by polyacrylamide gel electrophoresis and isoelectric focusing, respectively. Immunoelectrophoretic studies showed that the purified F42 antigen presented a slight anodic migration and was recognized only by its homologous antiserum.

Presence of group A and non-A rotaviruses in neonatal piglets in Campinas, SP, Brazil

Rotaviruses were detected by polyacrylamide gel electrophoresis (PAGE) in 11 (84,6%) of 13 faecal specimens from neonatal piglets with acute diarrhoea in a piggery near the city of Campinas, State of São Paulo, Brazil. An immunoenzimatic assay for group A rotavirus (IEA-A) was positive in ten of the samples, all of which showed a PAGE profile typical of that group. Another sample was showed a group B profile in PAGE. An immunoenzimatic assay specific for group B (IEA-B) for this faecal sample was positive, confirming the PAGE results.

Modulating action of Escherichia coli heat-stable enterotoxin (STa) on the humoral immune response

The effect of Escherichia coli enterotoxin STa on the primary and secondary immune response in F1 (CBA × C57 B1/10) mice immunized against sheep red blood cells (SRBC) was investigated. Modulating action on the IgM and IgG response was found to be dependent on the dose-time administration of the toxin. Immunosuppression of the primary response on the 4th day after immunization was observed when the toxin was injected 15 min before the SRBC, followed by immunostimulation on the 6th day after antigen (Ag) injection. Moreover, toxin administration 48 h before SRBC caused immunosuppression of the primary immune response on the 4th and 6th days. On the other hand, the IgM and IgG secondary immune response, determined 6 days after boosting, was greatly enhanced by toxin administration 15 min before priming (day 0) or boosting (day 26) and 48 h before priming. The same response was suppressed by toxin administration 48 h before booster antigen injection.

Indirect haemagglutination test for the detection of thermolabile (LT) enterotoxin fromEscherichia coli

An indirect haemagglutination test (IH) for the detection of enterotoxigenicE. coli (LT) was developed. Twenty-five enterotoxigenicE. coli (ETEC) from human and porcine diarrhoca and from river water were examined. The described IH test was more specific and sensitive than the passive immune haemolysis test (PIH).

Evaluation of a defined medium for the production of both thermolabile (LT) and thermostable (ST) enterotoxins ofEscherichia coli

The production of thermolabile (LT) enterotoxin was compared in a defined medium reported by Staples et al. (SAG medium) for the production of thermostable (ST) enterotoxin and the Casamino acids-Yeast extract (CAYE) medium. Aliquots were drawn from cultures of an enterotoxigenic (LT+, ST+)E. coli in both media at different times, growth curves were plotted, and culture filtrates tested for toxin activity. Levels of LT and ST in the SAG showed that it is as suitable as CAYE for the production of LT.The addition of either glucose (1%) or lincomycin (90 ug/ml) to SAG medium increased LT levels, but no synergistic effect could be observed if both substances were added concomitantly. Cultures in SAG medium incubated stationarily for 72 h at 37‡C yielded more LT than shaking cultures incubated similarly.

Storage ofEscherichia coli thermolabile enterotoxin on filter papers for assay by immune haemolysis reactions

Thermolabile (LT) enterotoxin was prepared from stationary and shaken cultures of enterotoxigenicEscherichia coli (ETEC) grown in casamino acids-yeast extract medium and dried onto filter discs. These were then examined by a modification of the single radial immune haemolysis (SRIH) test. It was observed that LT antigenicity, as detected by this test, remained unaltered for as long as 30 days at room temperature.

Erythrocyte receptors for cholera and heat-labile enterotoxins of Escherichia coli

Many serological reactions using red blood cells (RBC) such as radial immune haemolysis (RIH) and indirect haemagglutination (IH) tests have often been used for the detection of cholera toxin (CT) and heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains. In these tests, the enterotoxins bind to sheep, bovine and guinea-pig RBC without any ligand. We studied several factors which might interfere with such binding, as well as the nature of the receptors involved. Treatment of erythrocytes with different enzymes revealed that proteolytic enzymes had no effect on the adsorption of enterotoxins to RBC. Conversely, treatment with neuraminidase increased the adsorption. Experiments carried out with delipidized RBC revealed that none of the enterotoxins under study bound to the cells thus treated. Pre-incubation of ganglioside fractions with the enterotoxins blocked RIH and IH reactions and the biological effect of them on Vero cells. Assaying RBC ganglioside fractions by thin-layer chromatography revealed the presence of GM1. Our results suggest that the receptors for GT and LT enterotoxins in sheep, bovine and guinea pig RBC are gangliosides: mainly GM1.

Bluetongue virus: production and study of viral antigen for serological diagnosis

A soluble antigen, produced from the culture supernatant of VERO cells infected with bluetongue virus serotype 4 (BTV-S4) and concentrated by sequential ultrafiltration with membranes with cut-off values 10(3) and 25 x 10(3) NMWP, showed complete identity to standard antigens when compared by agar gel immunodiffusion (AGID) and SDS-PAGE profiles, revealing that the main protein component responsible for the AGID reaction has a molecular weight of about 60 kDa corresponding probably to the NS1 protein.

Stability of thermolabile (LT) enterotoxin produced from enterotoxigenicEscherichia coli strains maintained in vitro

The stability of thermolabile (LT) enterotoxin in 26 strains of porcine enterotoxigenicEscherichia 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°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−.