José Beiro Carvalhal

Shiga toxin-producing Escherichia coli (STEC) isolated from healthy dairy cattle in southern Brazil

Over a period of 1 year, the production of verotoxin was investigated in 1127 Escherichia coli isolated from 243 dairy cattle from 60 small farms in southern Brazil. Vero cell assay was used to detect toxins in culture supernatants from E. coli isolated from bovine feces. Shiga toxin-producing E. coli (STEC) detection rates were 95% (57 of 60) for farms and 49% (119 of 243) for cattle. Prevalence of STEC-positive cattle in the farms ranged from 0 to 100%. Ninety-six percent (315 of 327) of the STEC isolates did not react in the panel of sera used for typing. Twelve isolates, all non-motile, belonged to serogroups previously associated with human diseases, and 67% (8 of 12) were of only two serotypes (O91:H- and sorbitol-fermenting O157:H-). These results indicate that dairy cattle from the region surveyed may be a source of STEC potentially pathogenic for humans.

Condições higiênico-sanitárias no comércio ambulante de alimentos em Pelotas-RS

Street-vending of ready-to-eat (RTE) foods can be a risk to the consumers health, since people usually involved in this activity does not have proper knowledge in safe handling of foods. Despite this, only a few studies have been made on the microbiological quality of these foods and places where they are prepared. In this paper we report on the higienic-sanitary quality of hot-dogs sold by street-vendors from Pelotas,RS. Samples of hot-dogs, water and work surfaces, were collected from 60 street-vending places and taken to the laboratory for analysis. Counts of aerobic mesophilic bacteria (TPC), Staphylococcus coagulase positive (SCP), total coliforms (TC) and coliforms at 45oC (FC) were made on samples of hot-dogs. SPC, TC and FC counts were made on the water and surface samples. Among the 60 samples of hot-dogs 53%, 48%, 37% and 25% were found unsatisfactory for TC, TPC, STA and FC, respectively. Only 3 (5%) water samples were found unsatisfactory according to the TPC standard used, and 27% and 23% did not read the standards for TC and FC. Regarding the surfaces, 70% were found unsatisfactory for TPC, 68% for TC and 67% for FC. Salmonella sp was not detected in any of the samples tested. These results suggest that the hygiene practices of many food street-vending places are not adequate, resulting in a high proportion of read-to-eat RTE) foods with microbiological quality unsatisfactory for consumption.

Detection of Salmonella Typhimurium in Raw Meats using In‐House Prepared Monoclonal Antibody Coated Magnetic Beads and PCR Assay of the fimA Gene

Abstract A method for detection of Salmonella Typhimurium in meat samples that uses in‐house monoclonal antibody (MAb) coated magnetic beads for immunomagnetic separation (IMS) associated with PCR amplification of the gene fimA was developed. An internal amplification control (IAC) of the PCR reaction was constructed. The fimA PCR has shown 100% sensitivity and specificity when tested with various bacteria. The detection limit of the IMS‐PCR method, using a post‐enrichment in BHI broth for 6 h between IMS and PCR, was 1–10 CFU/mL. The method proved to be rapid (27 hrs), highly sensitive (1–10 CFU/25 g), and specific for detection of S. Typhimurium from experimentally contaminated pork and chicken meat samples.

Prevalência de Salmonella em produtos de frangos e ovos de galinha comercializados em Pelotas, RS, Brasil

The prevalence of Salmonella in chicken products and hens eggs, the most frequent serovars and their antimicrobial sensitivity, were investigated in the city of Pelotas, Brazil from May 1997 through October 1998. A total of 124 samples of chicken products obtained from local supermarkets and butcher shops, and 94 samples of eggs (6 units each) obtained from supermarkets and street vendors, were examined through standard cultivation procedures. Salmonella were detected in 13 (10,48%) samples of chicken products. Among the 13 strains of salmonellae isolated, 10 were serotyped as S. enteritidis, 1 was S. anatum and 2 were S. enterica subsp. enterica sorovar 3,10:e,h:-. All strains isolated were resistant to penicillin G and susceptible to the others antimicrobial drugs tested. Hens eggs were not found contaminated with salmonellae.

Escherichia coli verotoxigênica: isolamento e prevalência em 60 propriedades de bovinos de leite da região de Pelotas, RS, Brasil

The production of verotoxin was investigated in 1127 Escherichia coli isolated from 243 dairy cattle, water for human and animal consumption, and milk samples from 60 dairy farms from Pelotas-Brazil, from December of 1999 to December of 2000, to determine the prevalence of verotoxigenic E. coli (VTEC) in farms, to detect the presence of serotypes involved in human infections and to identify potential risk factors for animal infection. Vero cell assay was used to detect toxins in culture supernatants from E. coli isolated. VTEC was isolated in 95% (57/60) from farms and in 49% (119/243) from cattle, 5% (3/60) from water of human consumption, in 8.35% (5/60) from water animal consumption and 5% (3/60) from milk samples. The prevalence of cattle infected for each farm ranged from 0 to 100%. VTEC belonging to serogroups O157, O91 and O112, which include strains responsible for cases of hemorrhagic colitis and hemolytic uremic syndrome in humans, were isolated from 7 (2.9%) out of 243 cattle. Risk factors for contamination, such as amount of rain, farm size and cattle number, influenced cattle prevalence rate. These results suggest that VTEC is widely distributed among dairy cattle in the region surveyed and includes organisms from serogroups pathogenic for humans.

Intoxicação estafilocócica em restaurante institucional

An outbreak of foodbome intoxication in an institutional restaurant was investigated. Fifty-six people, out of 88, had vomiting, diarrhea, abdominal pain, prostration, fever and headache after eating a meal that included chicken sandwich, orange drink and milk pudding. Symptoms appeared between one and a halfhour and 12 hours after eating the meal and lasted from 24 to 36 hours. Specific attack rates were 64% for chicken sandwich, 61% for orange drink and 60% for milk pudding. Enterotoxin A secreting Staphylococcus aureus count in chicken Sandwich was 2 X 108CFU.g-1. Results of the questionnaire appleied to patrons, and results of bacteriological analysis of food items, provided circunstantial evidence to conclude that an outbreak of foodbome intoxication caused by the chicken sandwich contaminated with Staphylococcus aureus enterotoxin A has ocurred.

Detection of salmonella sp in chicken cuts using immunomagnetic separation

The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100% sensitivity and 94% specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method.

Monoclonal antibodies against serogroup B salmonellae: production, characterisation and use in a sandwich ELISA

Abstract Ten monoclonal antibodies (MAbs) against serogroup B salmonellae were obtained after immunisation of BALB/c mice with outer membrane proteins (OMPs) from Salmonella enterica serovar Typhimurium. Affinity constants, measured by enzyme-linked immunosorbent assay (ELISA), ranged from 8×05 to 6×07 l mol−1. Additivity ELISA demonstrated that most MAbs recognise different epitopes. ELISA determined the antigen specificity of the MAbs. The MAbs were more reactive with live Salmonella Typhimurium than with heat-treated cells. Two MAbs were used to develop a sandwich ELISA for rapid detection of Salmonella Typhimurium in experimentally contaminated meats. Its detection limit was 105 CFU ml−1 and it was able to detect 1–10 CFU in post-enrichment broth from 25 g of beef and chicken meat samples. The sandwich ELISA developed was shown to be specific and sensitive for the detection of Salmonella Typhimurium in meat, and produced results comparable to the culture methods in 57 h.