Distribution of additional virulence factors related to adhesion and toxicity in Shiga toxin-producing Escherichia coli isolated from raw products in Argentina.

UNLABELLED: A total of 73 Shiga toxin-producing Escherichia coli (STEC) isolates, belonging to 25 serotypes and isolated from raw products in Argentina, were examined for the occurrence of genes responsible for bacterial adhesions to intestine, ehaA (EHEC autotransporter), lpfAO113 (long polar fimbriae), sab (STEC autotransporter [AT] contributing to biofilm formation), ecpA (E. coli common pilus), hcpA (haemorrhagic coli pilus), elfA (E. coli laminin-binding fimbriae), sfpA (sorbitol-fermenting EHEC O157 fimbriae plasmid-encoded) and of the toxigenic gene cdt-V (cytolethal distending toxin). Our study showed different adhesin profiles that are not linked to one specific serotype and that all analysed isolates possess, besides stx genes, some adherence genes. Several of the isolates contained also multiple toxin genes. The results of the present work alert the presence of genes coding for additional adhesins and cdt-V toxin in LEE-negative STEC strains that occur in foods, and this traits could increase their pathogenic potential. SIGNIFICANCE AND IMPACT OF THE STUDY: Meat products are one of the main vehicles of Shiga toxin-producing E. coli, and the presence of genes coding for additional adhesins and toxins could increase their pathogenic potential. There is a need for a more detailed characterization of the strains in regard to these extra virulence factors.

Seasonal variation of HUS occurrence and VTEC infection in children with acute diarrhoea from Argentina.

In order to study the seasonality of haemolytic uraemic syndrome (HUS) and verotoxigenic Escherichia coli (VTEC) infection in children, 437 patients under 6 years of age with acute diarrhoea were studied, 8% of whom progressed to HUS. VTEC was found in 10% of all of the stool samples analysed and seasonal occurrence of HUS (p < 0.01) was confirmed. VTEC infection was more prevalent in warm months, although the differences were not statistically significant. Moreover, a significant difference in the detection of O157:H7 serotype and in the vt profile between cold and warm months (autumn and winter; spring and summer, respectively) was established. The O157:H7 serotype was isolated more frequently during warm months. Moreover, a predominance of vt (2) was noted, which was partially replaced by the combination of vt (1) with vt (2) in the cold season. The results of this study indicate the seasonal variation of the disease and the presence of serotype O157:H7 and the vt types. They also reinforce the need to develop prevention programmes considering the seasonal pattern of the disease, which would generate an impact on public health. Control strategies of the pathogen in cattle in the most risky season of the year would also be of benefit.

Short communication: characterization of Shiga toxin-producing Escherichia coli isolated from newborn, milk-fed, and growing calves in Argentina.

Shiga toxin-producing Escherichia coli (STEC) cause foodborne pathogenic disease that is shed in the feces of cattle. The aim of this study was to evaluate how early young calves are colonized by STEC strains, potentially pathogenic for humans, and the prevalence in different calf categories. From 808 rectal swabs analyzed by PCR, 38% were stx positive. The prevalence in newborn (<24 h from birth), milk-fed (<2-mo-old), and growing calves (2-8 mo old) were 25, 43, and 58%, respectively. Forty different STEC serotypes were found among isolates from newborn, milk-fed, and growing calves that shed STEC strains potentially pathogenic for humans. The STEC strains could be acquired early from mothers, enabling the infection of other animal categories and confirming the risk to public health.

Multiplex PCR assay for the detection of five putative virulence genes encoded in verotoxigenic Escherichia coli plasmids.

The aim was to perform a pentavalent PCR assay for the detection of putative virulence genes encoded in VTEC plasmids, katP, espP, subA, stcE, and ehxA. The five-specific primer pairs used in the assay do not interfere with each other and generate amplification products of 914, 774, 556, 399, and 262 bp. It was selected at random 39 strains belonged to 20 serotypes in order to evaluate the multiplex in a wide variety of strains. The results of this study indicate that it is possible to perform simultaneous amplification and search for recognized plasmid-encoded virulence markers from different E. coli serotypes and apply this technique to the genetic characterization of E. coli strains isolated from reservoirs, foods or patients. This complementary technique is a useful tool to detect interstrain differences for epidemiological studies and to provide information that could be related to the risk of human infection.

Enteropathogenic Escherichia coli contamination at different stages of the chicken slaughtering process.

Enteropathogenic Escherichia coli is a foodborne pathogen that produces potentially fatal infant diarrhea, noticeably in developing countries. The aim of this study was to detect EPEC contamination by PCR at different stages of the chicken slaughtering process. We collected swabs from chicken cloacae and washed carcasses (external and visceral cavity) during the slaughtering process in 3 sampling occasions. Unwashed eviscerated carcasses were also sampled (at the visceral cavity) in the second and third sampling occasions. Enteropathogenic Escherichia coli was detected in 6 to 28% of cloacal samples, 39 and 56% of unwashed eviscerated carcasses, and 4 to 58% of washed carcasses. None of the samples were positive for bfpA, suggesting contamination with atypical EPEC. The detection of EPEC at different stages of the chicken slaughtering process showed that the proportion of contaminated samples remained or even increased during processing. In addition, the high proportion of contaminated carcasses during chicken processing represents a risk for the consumers and a challenge to improve procedures for those working in the sanitary control service.

Characterization of Shiga toxin-producing Escherichia coli isolated from dairy cows in Argentina.

AIMS: To feno-genotypically characterize the Shiga toxin-producing Escherichia coli (STEC) population in Argentinean dairy cows. METHODS AND RESULTS: From 540 STEC positive samples, 170 isolates were analyzed by multiplex PCR and serotyping. Of these, 11% carried stx1, 52% stx2 and 37% stx1/stx2. The ehxA, saa and eae were detected in 77%, 66% and 3%, respectively. Thirty-five per cent of strains harboured the profile stx1, stx2, saa, ehxA and 29% stx2, saa, ehxA. One hundred and fifty-six strains were associated with 29 different O serogroups, and 19 H antigens were distributed among 157 strains. STEC O113:H21, O130:H11 and O178:H19 were the most frequently found serotypes. The STEC O157:H7 were detected in low rate and corresponded to the stx2(+) , eae(+) , ehxA(+) virulence pattern. CONCLUSIONS: We detected a diversity of STEC strains in dairy cattle from Argentina, most of them carrying genes linked to human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The non-O157 STEC serotypes described in this study are associated worldwide with disease in humans and represent a risk for the public health. For this, any microbiological control in dairy farms should be targeted not only to the search of O157:H7 serotype.

Seasonal variation of Shiga toxin-encoding genes (stx) and detection of E. coli O157 in dairy cattle from Argentina.

AIMS: To study the seasonal variation of Shiga toxin-encoding genes (stx) and to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) O157 in cattle belonging to five dairy farms from Argentina. METHODS AND RESULTS: Rectal swab samples were collected from 360 dairy cows in each season and 115 and 137 calves in autumn and in spring, respectively. The stx were investigated by multiplex PCR and it was used as the indicator for STEC. Samples positives for stx were tested by PCR for eae-gamma1 of E. coli O157 and then subjected to IMS (immunomagnetic separation). In positive animals significant differences in the prevalence of stx between warm and cold seasons were detected. In warm seasons, stx1 + stx2 increased and stx1 decreased, independently of the animal category. The prevalence of STEC O157 in cows and calves were 0.2% and 0.8%, respectively. CONCLUSIONS: This work provides new data about the occurrence of stx and STEC O157 in dairy herds from Argentina and suggests a relationship between the type of stx and season of year. SIGNIFICANCE AND IMPACT OF STUDY: The detection of STEC O157 and the seasonality of stx and its types provide an opportunity to improve control strategies designed to prevent contamination of food products and transmission animal-person.

Isolation of cDNA clones encoding proteins of complex structures: analysis of the Trypanosoma brucei cytoskeleton.

We have adapted a group of well-known procedures in order to devise a simple method that allows the isolation of specific cDNAs encoding proteins located in different regions of the Trypanosoma brucei cytoskeleton. cDNA clones were isolated by screening a lambda gt11 expression library with a polyspecific, polyclonal antiserum against a complex immunogen, in this case the complete cytoskeleton. The fusion proteins produced by the clones were then used as an affinity immunoadsorbant to select monospecific polyclonals. The monospecific antisera isolated were used as probes to identify and localize different cytoskeleton proteins by Western blotting and immunofluorescence. This method proved particularly useful for the molecular identification of minor components in a complex structure. It should prove applicable to the molecular analysis of other organelles or protein complexes.

Analysis and in vivo assay of B. abortus agglutinating and non-agglutinating antibodies.

Studies were made of physicochemical and immunochemical characteristics of Brucella abortus agglutinating and non-agglutinating antibodies in the sera of cattle repeatedly injected with living B. abortus (Strain 1119). Both agglutinating and non-agglutinating antibody were shown to be IgG1, and by immunodiffusion against rabbit anti-cattle gamma-globulin, agglutinating antibody gave a precipitation line of identity with that given by non-agglutinating antibody. Whilst agglutinating antibody increased clearance of antigen from the blood of passively protected mice, non-agglutinating antibody did not enhance clearance. Determination of the spleen infection index in mice pre-treated with agglutinating and non-agglutinating antibody showed that in animals passively immunized with non-agglutinating antibody the number of living (infecting) bacteria was approximately 4 times higher than in the case of agglutinating antibody. The possible potentiation of chronic B. abortus infection by non-agglutinating antibody is discussed.

Effect of bovine non-agglutinating antibodies on the blood clearance of 131I-labelled Brucella abortus strain 45/20.

Non-agglutinating anti-Brucella abortus S45/20 antibodies were isolated and purified from sera of immunized cattle by means of immunoadsorption and ion-exchange chromatography (DEAE-Sephadex A-50). They corresponded to the IgG1 isotype as shown by immunoelectrophoresis using monospecific anti-IgG1 and anti-bovine gamma globulin sera. These antibodies failed to agglutinate the antigen. They were detected by the anti-bovine gamma globulin test, showing higher titres than those of agglutinating antibodies during the whole period of the experiment. Blood clearance of 131I-S45/20 in mice, was slower in those groups which had received non-agglutinating antibodies than in the control group.

Detection of antigenic fractions from Brucella abortus S45/20 which bind to non-agglutinating antibodies using electroblotting and enzyme-linked antibody probes.

Outer membrane antigens which bind to non-agglutinating antibodies (NAAb) elicited by smooth (S19) and rough (S45/20) Brucella abortus strains, were extracted from S45/20 by stirring in cold 2.5% NaCl and then analyzed by SDS-PAGE, electroblotting and enzyme-linked antibody test. Eight bands were observed in the gel stained with Coomassie blue. Seven antigenic fractions were transferred to nitrocellulose by blotting. A 27-kd band was recognized by bovine anti-S45/20 non-agglutinating serum and not by purified NAAb against surface antigens. Bands 10 kd and 14.3 kd bound to bovine anti-S45/20 NAAb from calves immunized with either S19 or S45/20. A 12.0-kd band was recognized by the serum and NAAb from calves immunized with S45/20 but not by those injected with S19. There are thus antigenic fractions shared by S19 and S45/20 which bind in vitro to NAAb.

Kinetic analysis of cattle anti-Brucella abortus S45/20 non-agglutinating antibodies in antibody-dependent cell-mediated cytotoxicity.

Calves inoculated with Brucella abortus S45/20 produced, against surface antigens, non-agglutinating antibodies (NAAb) which were isolated and purified. A kinetic analysis was carried out of NAAb in antibody-dependent cell-mediated cytotoxicity (ADCC) using sheep red blood cells labelled with surface antigen from B. abortus S45/20 as target cells. Three parameters were examined: time of incubation, effector cell:target cell (E:T) ratio and NAAb dose. It was found that the NAAb were not able to mediate ADCC with bovine spleen cells.

Toxigenic Escherichia coli isolated from pigs in Argentina.

The presence of porcine toxigenic E. coli (ETEC, VTEC) in 28 piggeries (5% of total) of the central and northeast region of Argentina was studied for a better understanding of the epidemiology of porcine strains. Samples were taken by rectal swabs from healthy piglets and from those with diarrhoea, in addition to their dams. Between 5-10 colonies were isolated from each one of 223 animals sampled from 1992 to 1997. By using specific primers each strain was screened by PCR for VT1, VT2all, VT2e, STIa, and LTI toxin genes. Only strains positive for any of the toxins mentioned above were screened for STb. Their O serogroups were determined by agglutination. All of the above enterotoxins and verocytotoxins were found in E. coli isolated from the animals. The STIa gene was detected in E. coli isolated from 27/127 piglets with diarrhoea, in comparison with LTI (4/127 pigs). No toxin gene was amplified from E. coli isolated from either healthy piglets or their dams. When strains isolated from 48 piglets without diarrhoea but showing delayed growth were analysed by PCR, their toxin profile was determined to be VT1 (1/48 piglets), VT2all (5/48), STIa (1/48), LTI (3/48) and VT2e (3/48). Serogroup O64 prevailed among ETEC; O138 prevailed for ETEC/VTEC strains. This is the first extensive study regarding porcine toxigenic E. coli in Argentina and constitutes an important database for the implementation of prevention measures.

A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea.

Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans serovar pomona genomic lambda gt11 library. Although there are references of transcription of leptospiral genes in E. coli from their own leptospiral promoters, in this recombinant construction the leptospiral DNA was located under the control of lacZ promoter since no expression could be detected in the absence of IPTG. This clone, isolated by expression screening with polyclonal serum raised against equine corneal proteins, encodes a 90 kDa protein of L. interrogans which crossreacts with equine cornea as proved Western-blotting. Antibodies directed against this leptospiral protein strongly recognised a 66 kDa equine corneal protein, one of those recognised by an anti-equine cornea serum. Our findings suggest that an immune response to 90 kDa protein participates in pathogenesis of equine uveitis.

C3 fixed in vivo to cornea from horses inoculated with Leptospira interrogans.

C3 was detected bound in vivo to the opaque cornea of horses inoculated with killed Leptospira interrogans. Employing epithelial corneal cells isolated from a monolayer in tissue culture, we proved that C3 is fixed in vitro to the intact cell surface after incubation with a fresh equine anti-Leptospira serum. These findings, in addition to the infiltration of cornea with neutrophils and lymphocytes, may explain the mechanisms of tissue damage in recurrent uveitis of horses with leptospirosis.

Biochemical analysis by SDS-PAGE and western blotting of the antigenic relationship between Leptospira and equine ocular tissues.

The antigenic relationship between Leptospira interrogans, equine cornea and lens was previously noted in our studies. Serum antibodies from horses inoculated with serovars wolffi, pomona, icterohaemorrhagiae, and tarassovi, were able to bind to five antigenic fractions from both cornea and lens, as demonstrated by immunoblotting. These antigens seem to be made up of protein and carbohydrates. After treatment with periodate for cleavage of glycoside ring structures, those fractions kept their condition of target for anti-Leptospira antibodies. Nevertheless, all fractions lost that condition after being subjected to peptide hydrolysis.

Tears and aqueous humor from horses inoculated with Leptospira contain antibodies which bind to cornea.

An antigenic relationship between Leptospira interrogans and equine cornea was previously described by us. An enzyme-linked immunosorbent assay was employed in the present work to investigate the existence of anti-leptospira and anti-cornea antibodies in tears, aqueous humor and serum from horses inoculated i.m. with those antigens. Ten days after a booster by the same route, antibodies that bind to microtiter plates, coated with an homogenate of either equine cornea or leptospira, were detected in those fluids and in the sera. At the same time, the corneas of the horses began to develop a diffuse opacity. This finding of anti-leptospira antibodies in equine tears and aqueous humor shows the pathway along which they arrive at the cornea and bind to it.

Experimental demonstration of an antigenic relationship between Leptospira and equine cornea.

Horses inoculated with either equine cornea or killed Leptospira interrogans serovars pomona, tarassovi, icterohaemorrhagiae, wolffi and hardjo, developed corneal opacity and produced antibodies which made it possible to demonstrate partial antigenic identity between equine cornea and four of those serovars employed. These antibodies were isolated by means of immunoadsorptions, purified by ion-exchange chromatography (DEAE-Sephadex A-50) and run by immuno-electrophoresis in agar gel. Both antibodies, anti-equine cornea and anti-leptospira, showed that they corresponded to the IgGb subclass. They bound themselves to equine cornea in vivo and in vitro as was proved by immunofluorescence. This antigenic relationship may be in part responsible for pathogenesis of corneal opacity in leptospirosis of horses.

The immune response in cattle infected with Tritrichomonas foetus.

Holando-Argentina calves (males and females) were experimentally infected with Tritrichomonas foetus var. Belfast (T. foetus) by introducing 10(7) protozoa into the preputial and vaginal cavities, in order to analyse the course of the immune response to infection. Samples of serum, vaginal mucus and preputial secretion were taken periodically and assayed by means of microagglutination of living protozoa. The serum antibody titre, which averaged 32 before infection and was equivalent to titres in a non-infected group, increased to 512 in the heifers 11 weeks later and to 128 in the bulls 4 months post-infection. Agglutinating antibodies were not detected in the preputial cavity, but heifers showed antibodies in the vaginal mucus and became trichomoniasis free after 4 months. Conversely, genital secretions from the bulls gave rise to positive cultures during the whole period of experimentation. The intradermal sensitivity was checked using a soluble antigen from T. foetus. The diameter of the papula increased up to three times in heifers, while in bulls the results were no different than those from the non-infected group. Serum antibodies were of the IgG2 subclass, while those isolated from vaginal mucus were characterized as IgG1, an opsonizing antibody. Heifers were refractory to challenge infection after 1 year. The poor immune response in bulls is consistent with their role as carriers of T. foetus.

Detection of an antigenic protein of Leptospira interrogans which shares epitopes with the equine cornea and lens.

A protein epitope which is involved in an antigenic relationship between equine ocular tissues and Leptospira interrogans was detected in homogenates of the bacterium. The antigenic determinant was harboured on a peptide structure which was shown to be sensitive to the action of denaturing and reducing agents by means of Western blotting. The outer surface of the leptospires appeared to be free of this epitope as was proved by dot-blot and electron microscopic studies.