Avaliação das reações de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) na detecção de anticorpos IgG anti-T. gondii em exsudatos cárneos bovinos / Evaluation of indirect Hemagglutination (HI) and Modified Agglutination (MAT) reactions in the detection of anti-T. gondii IgG antibodies in bovine meat exudates

Toxoplasmose é uma zoonose parasitária com ampla distribuição mundial provocada pelo Toxoplasma gondii, considerado um dos protozoários mais bem sucedidos do planeta, pois infecta cerca de um terço da população mundial. Dentre as formas de transmissão, o consumo de carne mal cozida, contendo cistos, tem sido considerado um fator de risco para aquisição desta zoonose. Uma abordagem alternativa para o controle da toxoplasmose pela ingestão de carne bovina seria a sorologia dos bovinos, já que animais soropositivos albergam cistos teciduais. Contudo, a obtenção de soro para esta avaliação, nem sempre é factível, dada a dificuldade de coleta de sangue durante a linha de abate e sua ausência em cortes comerciais. O exsudato cárneo é uma alternativa para detecção de anticorpos anti - T. gondii em cortes comerciais de carne, que foi a proposta deste estudo para avaliar o desempenho dos testes de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) quando comparados ao ELISA usando exsudato cárneo. Este estudo mostrou que a acurácia dos testes de aglutinação não foi viável devido aos baixos índices de sensibilidade e especificidade quando comparados ao ELISA. Estes dados demonstram a importância da escolha de testes eficientes como ELISA para aplicação no controle da qualidade e inocuidade de cortes comerciais de carne bovina.

Toxoplasmosis is a parasitic zoonosis with a wide worldwide distribution caused by Toxoplasma gondii, which is considered one of the most successful protozoa on the planet, since it can infect a third of the world population. Among the forms of transmission, consumption of undercooked meat has been considered as a risk factor for the acquisition of this zoonosis. An alternative approach to toxoplasmosis control by beef ingestion could be the serological diagnosis in cattle, since seropositives animals harbor tissue cysts. However, the use of serum for this evaluation is not always feasible due to the difficulty of blood collection during slaughter and its absence in commercial beef cuts. Meat exudate is an alternative for the detection of anti-T. gondii antibodies in commercial beef cuts, which was the propose of this study to evaluate the performance of Indirect Hemagglutination (HI) and Agglutination Modified (MAT) tests compared to ELISA using meat exudates. This study showed that the agglutination tests accuracy was not viable due to low sensitivity and specificity indexes when compared to ELISA. These data demonstrate the importance of choosing accurate tests such as ELISA for application in quality control and safety of commercial beef cuts.

Avaliação da adesão às boas práticas de fabricação em estabelecimentos que manipulam carne no município de Uberlândia/MG / Evaluation of the adhesion to good manufacturing practices in establishments that handle meat in Uberlandia municipality / MG

O objetivo do trabalho foi avaliar a utilização de boas práticas de fabricação em açougues localizados em Uberlândia (MG). Foram feitas visitas à seis açougues (A-F) para avaliação dos itens: edificações e instalações; equipamentos, móveis e utensílios; e manipuladores. Os dados foram comparados pelo Teste Exato de Fischer (p<0,05). As não conformidades do estabelecimento D foram maiores que A e B (p<0,05) e dentre as categorias avaliadas a maior frequência de inconformidades foi observada nas edificações e instalações (45,5%). Nesta categoria, a maior frequência de inconformidades apresentou-se na área interna (72,2%), com presença de produtos de limpeza; ventiladores e garrafas d’água. Os resultados indicam a necessidade de maior monitoramento por parte dos estabelecimentos para que possam oferecer produtos com melhor qualidade aos consumidores.

Frecuencia, caracterización y análisis genotípico de Escherichia coli productor de toxina Shiga en frigoríficos de carne bovina de Argentina / Frequency, characterization and genotypic analysis of Shiga toxin-producing Escherichia coli in beef slaughterhouses of Argentina

The objectives of this study were: (1) to estimate STEC frequency in hide and carcass samples taken from beef slaughterhouses supplying the domestic market in Argentina, (2) to establish the pheno-genotypic characteristics of STEC and non-toxigenic Escherichia coli of serogroups O26, O45, O103, O121, O111, O145 or O157 isolated from the analyzed samples and, (3) to study their clonal relatedness. Sixty hides and 60 carcasses were analyzed. At the screening step, 48% of hide and 80% of carcass samples tested positive for the stx gene by endpoint PCR. The STEC isolation rate was 5% for hides and 8% for carcasses. The isolation rate of STEC-positive for O26, O45, O103, O111, O145 or O157 serogroups was 0% for hides and 2% for carcasses. With the purpose of studying the clonal relatedness of isolates, macrorestriction fragment analysis by pulsed-field gel electrophoresis was performed. The results indicated cross-contamination between hides and between carcasses of animals in the same lot and, that the origin of carcass contamination was their own hide, or the hides of other animals in the same lot. The high detection rate at the screening step, especially in carcasses, and the evidence of cross-contamination show the need to apply additional in-plant intervention strategies aimed at preventing carcass contamination.

Los objetivos del presente estudio fueron tres: 1) estimar la frecuencia de Escherichia coli productor de toxina Shiga (STEC) en muestras de cuero y carcasa de bovinos en frigoríficos de consumo interno de Argentina; 2) realizar la caracterización feno-genotípica de las cepas STEC y de Escherichia coli no toxigénicas pertenecientes a los serogrupos O26, O45, 0103, O121, O145 u O157 aisladas a partir de las muestras analizadas; 3) establecer la relación clonal de ese conjunto de cepas. Se analizaron 60 cueros y 60 carcasas. En la etapa de tamizaje, el gen stx se detectó en el 48% de las muestras de cuero y en el 80% de las muestras de carcasa por una PCR de punto final. La frecuencia de recuperación de cepas STEC fue del 5% en cueros y del 8% en carcasas, y la de cepas STEC positivas para los serogrupos O26, O45, O103, O121, O111, O145 u O157 fue del 0% en los cueros y del 2% en las carcasas. La relación clonal de las cepas aisladas se investigó a través de electroforesis de campo pulsado y análisis de los patrones de macrorrestricción generados. Los resultados demostraron la existencia de contaminación cruzada entre cueros y carcasas de animales pertenecientes a un mismo lote, y también que el origen de la contaminación fue el propio cuero del animal o el cuero de otros animales pertenecientes al mismo lote. Los altos porcentajes de detección en la etapa de tamizaje, especialmente en carcasas, y la evidencia de contaminación cruzada ponen de manifiesto la necesidad de evaluar la implementación de estrategias de intervención tendientes a evitar la contaminación de carcasas.

Detection of Diarrheagenic Escherichia coli in Bovine Meat in the Northern Region of Paraná State, Brazil

Abstract Ground bovine meat is commonly consumed by the population of Brazil. However, it constitutes an excellent medium for the multiplication of microorganisms due to available nutrients and handling practices prior to consumption. Here, we examined 100 samples of ground beef for the presence of diarrheagenic Escherichia coli (DEC) pathotypes by PCR, and characterized isolates by analyzing their adherence to HEp-2 cells, serotype, antimicrobial susceptibility, and phylogeny. Enteroaggregative E. coli was detected in five (5%) meat samples, Shiga toxin-producing E. coli in three (3%), and atypical enteropathogenic E. coli in two (2%). According to the phylogeny, six isolates (60%) were classified in group A, two (20%) in group B1, and two (20%) in group E. The detected serotypes were O3:H2, O93:H9, O93:H46, O105ab:H7, O152:H8, O156:H10, and O175:H7. The antimicrobial susceptibility testing showed that one sample (10%) was resistant to ampicillin, two (20%) to sulfamethoxazole-trimethoprim, and two (20%) to cephalothin. Based on these results, bovine ground meat for human consumption can serve as a reservoir of DEC, which emphasizes the importance of appropriate hygienic-sanitary conditions during handling at every stage from slaughter to table.

Survey of Salmonella spp. in beef meat for export at slaughterhouses in Brazil / Pesquisa de Salmonella spp. em carcaças bovinas durante o processamento em abatedouros-frigoríficos exportadores

The aim of the present study was to investigate the presence of Salmonella spp. in samples collected from beef meat at three points of the slaughter line (after skinning, washing and cooling) at three slaughterhouses in Brazil that export meat. Detection was based on ISO 6579:2002 and confirmed by PCR and qPCR. The isolates were typified using slide agglutination tests and PFGE. The antibiotic sensitivity profile was determined using the disk diffusion method. Contamination was detected in only one slaughterhouse. The overall frequency of contamination by Salmonella spp. was 6.7% of carcasses (6/90) and 2.6% of carcass surface samples (7/270). All isolates were confirmed by PCR and qPCR. The serological analysis and the PFGE showed a single profile: Typhimurium. The strains demonstrated 100% susceptibility to ampicillin, cefotaxime, ciprofloxacin, chloramphenicol, gentamicin and tetracycline. Positive carcasses after cooling pose a direct risk to consumers, since the meat is considered ready to be marketed after this process.(AU)

O objetivo deste trabalho foi investigar a presença de Salmonella spp. em amostras coletadas de carcaças de bovinos, em três pontos da linha de abate (após a esfola, lavagem e refrigeração) de três frigoríficos exportadores no Brasil. A detecção foi realizada pela ISO 6579:2002, e confirmada por PCR e qPCR. Os isolados foram tipificados por testes de soroaglutinação e PFGE e avaliado o perfil de sensibilidade aos antibióticos pelo método de difusão em disco. A contaminação foi detectada em apenas um abatedouro-frigorífico. As contaminações das carcaças (n=90) e amostras de carne (n=270) por Salmonella spp. foram 6 (6,7%) e 7 (2,6%), respectivamente. Todos os isolados foram confirmados por PCR e qPCR. A análise sorológica e o PFGE mostraram um único perfil: Typhimurium. As cepas apresentaram 100% de suscetibilidade à ampicilina, cefotaxima, ciprofloxacina, cloranfenicol, gentamicina e tetraciclina. As carcaças positivas após a refrigeração apresentam um risco direto para o consumidor, uma vez que, após este processo, a carne está pronta para ser comercializada.(AU)

Frecuencia, susceptibilidad antimicrobiana y patrón de adherencia de Salmonella enterica aislada de carne de pollo, res y cerdo de la Ciudad de México / Frequency, antimicrobial susceptibility and adherence patterns of Salmonella enterica isolated from chicken meat, beef and pork from Mexico City

Resumen Introducción: Los alimentos de origen animal frecuentemente están implicados en brotes de salmonelosis. Objetivo: Evaluar la frecuencia de Salmonella enterica en carnes molidas de pollo, res y cerdo (un total de 2.592 muestras) obtenidas de mercados sobre ruedas y supermercados de la Delegación Iztapalapa en la Ciudad de México, determinar la susceptibilidad antimicrobiana y efectuar ensayos de adherencia en las cepas aisladas. Métodos: El aislamiento de S. enterica se hizo de acuerdo a la BAM-FDA, la susceptibilidad antimicrobiana de acuerdo con CLSI y el ensayo de adherencia en células HEp-2 conforme a Baffone y cols., 2001. Resultados: Salmonella enterica fue aislada en 511 del total de muestras analizadas (19,7%), de las cuales 244 (47,7%), 152 (29,7%) y 115 (22,5%) correspondieron a carne molida de pollo, res y cerdo, respectivamente. La mayor frecuencia de resistencia de S. enterica a antimicrobianos fue a ampicilina y cloranfenicol en pollo, perfloxacina y ampicilina en res y carbenicilina, ampicilina, cloranfenicol, cefotaxima y perfloxacina en cerdo. Noventa por ciento de las cepas mostraron un patrón de adherencia agregativo. Conclusión: La frecuencia de S. enterica en productos cárnicos es alta, por lo que es importante la adecuada cocción de la carne para disminuir el riesgo de una salmonelosis.

Background: Food of animal origin is often involved in salmonellosis outbreaks. Aim: To evaluate the frequency of Salmonella enterica in chicken, beef and pork ground meat (a total of 2,592 samples) obtained from travelling markets and supermarkets at the Iztapalapa area of Mexico City, in order to determine the antimicrobial susceptibility and adherence capacity of isolated strains. Methods: Isolation of S. enterica was carried out according to the BAM-FDA, the microbial susceptibility according with CLSI and adherence assay on HEp-2 cell line according with Baffone et al., 2001. Results: S. enterica was isolated from 511 of all the analyzed samples (19.7%), from which 244 (47.7%), 152 (29.7%) and 115 (22.5%) corresponded to chicken, beef and pork ground meat, respectively. The highest frequency of resistance of S. enterica to antimicrobials was to ampicillin and chloramphenicol in chicken, perfloxacin and ampicillin in beef and carbenicillin, ampicillin, chloramphenicol, cefotaxime and perfloxacin in pork. Ninety percent of the strains showed an aggregative adherence pattern on HEp-2 cells. Conclusion: The frequency of S. enterica on meat products is high, which is the reason why a proper cooking of these ground meats is important in order to reduce the risk of acquiring salmonellosis.

Caracterização molecular de Listeria monocytogenes oriundas de cortes cárneos bovinos e de abatedouros frigoríficos de bovinos localizados no Distrito Federal, Brasil / Molecular characterization of Listeria monocytogenes from beef samples and cattle slaughterhouses located in the Federal District, Brazil

Este trabalho teve como objetivo realizar a detecção de cepas de Listeria monocytogenes de cortes cárneos bovinos bem como no ambiente de abatedouros frigoríficos localizados no Distrito Federal, promover a sorotipificação pela reação em cadeia da polimerase (PCR), realizar antibiograma e submeter às cepas à eletroforese de campo pulsado (Pulsed-field gel electrophoresis - PFGE). Foram analisados um total de 125 cortes cárneos bovinos, 45 amostras de swabs de carcaças e 43 amostras de swabs em que foram detectados 13 cepas de Listeria monocytogenes, sendo 11 em cortes cárneos bovinos e 2 swabs de ambiente em um abatedouro frigorifico. Não foram isoladas cepas de swabs de carcaça. Dentre as 13 cepas de Listeria monocytogenes foram encontradas seis cepas do sorotipo 4b, cinco do sorotipo 1/2c e duas cepas do sorotipo 1/2a. Dentre as 11 cepas de L. monocytogenes encontradas em cortes cárneos bovino, uma (9,1%) cepa apresentou resistência a eritromicina, outra (9,1%) cepa a gentamicina e outra a ciprofloxacina (9,1%) e todas as cepas (100%) apresentaram resistência ao Ác. Nalidíxico. Das duas (2) cepas oriundas de ralos de abatedouro frigorífico, todas (100%) apresentaram resistência ao Ác. Nalidíxico e a sulfonamidas. A análise por eletroforese de campo pulsante (PFGE) demonstrou 13 diferentes pulsotipos, em que foram agrupados em 3 diferentes grupos clonais, que coincidentemente se correlacionavam com os 3 diferentes sorotipos encontrados sugerindo uma ampla disseminação desses perfis no Distrito Federal.(AU)

The aim of the study was the analysis of Listeria monocytogenes strains in beef samples as well as slaughterhouse environment, located in the Federal District, promote serotyping by polymerase chain reaction (PCR), perform antibiotic susceptibility and submit the strains to Pulsed-field gel electrophoresis (PFGE). A total of 125 beef samples were analyzed, 45 samples of carcasses swabs and 43 swab samples. It detected 13 strains of Listeria monocytogenes, 11 in beef samples. and 2 in slaughterhouse environment. No carcass swabs strains were isolated. Among the 13 strains of L. monocytogenes six strains of serotype 4b were found, five serotype 1/2c and two strains of serotype 1/2a. Among the 11 strains of L. monocytogenes found in beef, one (9.1%) strain showed resistance to erythromycin, one (9.1%) strain to gentamicin, one to ciprofloxacin (9.1%) and all strains (100%) were resistant to nalidixic acid. The two strains coming from the slaughterhouse drains, all (100%) were resistant to nalidixic acid and Sulfonamides. The analysis by pulsed field gel electrophoresis (PFGE) showed 13 different pulsotypes; they were grouped into three different clonal groups, coincidentally correlated with the three different serotypes found, what suggests a widespread dissemination of these profiles in the Federal District, Brazil.(AU)

Perfil de resistencia a antibióticos de serotipos de Salmonella spp. aislados de carne de res molida en la Ciudad de México / Antibiotic of resistence profile of Salmonella spp. serotypes isolated from retail beef in Mexico City

Resumen: Objetivo: Determinar el serotipo y perfil de resistencia a antibióticos de cepas de Salmonella spp. presentes en la carne de res que se expende en la capital mexicana. Material y métodos: Se analizaron 100 muestras de carne molida. Se aisló el patógeno por métodos convencionales y se confirmó por PCR (gen InvA, 284 pb). El perfil de resistencia a antibióticos se determinó por el método de Kirby-Bauer y la serotipificación por el esquema de Kauffman-White. Resultados: Se detectaron los serotipos Lomita (6), Derby (4), Senftenberg (2), Javiana y Cannsttat (1). Se observó alta resistencia a ampicilina (18/19), carbenicilina (16/19), tetraciclina (13/19) y trimetoprim-sulfametoxasol (13/19). Cinco cepas fueron no tipificables y 14 mostraron multirresistencia. Conclusiones: La carne de res que se vende en el principal centro de consumo del país está contaminada con serotipos de Salmonella spp. relevantes para la salud pública. Una importante proporción de éstos es resistente a múltiples antibióticos.

Abstract: Objective: To determine the serotype and antibiotic resistance profile of Salmonella spp. isolated from retail ground beef in Mexico City. Materials and methods: A total of 100 samples of ground beef were analyzed. The pathogen was isolated by conventional methods and confirmed by PCR (invA gene, 284 bp).The antibiotic resistance profile was determined by the Kirby-Bauer method while serotyping was performed according to the Kauffman-White scheme. Results: We isolated a total of 19 strains of Lomita (6), Derby (4), Senftenberg (2), Javiana and Cannsttat (1) and undetermined (5) serotypes. The strains showed a high resistance rate to ampicillin (18/19), carbenicillin (16/19), tetracyclin (13/19), and trimethoprim-sulfamethoxazole (13/19). Multidrug resistance was observed in 14 isolates. Conclusions: Several Salmonella spp. serotypes of public health significance are circulating in ground beef sold in the major Mexican city. Some of these strains are multi-drug resistance.

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2⁴ factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R² = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L⁻¹ and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL⁻¹) occurred in the MRS broth supplemented with 5.5 g L⁻¹ glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL⁻¹.

Avaliação das condições higienicossanitárias de produção de carne moída em açougues

A carne é a principal fonte de proteína do mundo contemporâneo. A carne bovina moída comercializada em açougues está frequentemente relacionada a casos de intoxicação alimentar. Uma ferramenta utilizada pelos serviços de inspeção municipais para fiscalizar esses locais é a lista de verificação de boas práticas. O objetivo dessa pesquisa foi avaliar as condições higienicossanitárias de produção de carne bovina moída, através da aplicação de uma lista de verificação de boas práticas de fabricação em açougues de um município da região sudeste do Estado de São Paulo. Pesquisa descritiva transversal de intervenção observacional com amostra constituída por 27 açougues, nos quais foram analisadas as não conformidades cometidas relativas à fabricação de carne moída, durante o período de agosto a setembro de 2009. As inadequações de ordem higienicossanitária mais frequentemente encontradas foram: risco de contaminação cruzada, falhas na manipulação e no armazenamento da carne moída, acúmulo de resíduos de carnes nos moedores, temperatura abusiva das carnes durante o processo de produção; não armazenamento dos canhões dos moedores sob refrigeração; falta de higiene dos equipamentos e utensílios; e, falha no procedimento de higienização das mãos. Concluiu-se que, a lista de verificação possibilitou identificar as principais falhas na fabricação de carne moída por açougues, as quais devem ser corrigidas e monitoradas para que se garanta a oferta de um produto seguro ao consumidor.

Meat is the main source of protein of the contemporaneous world. The minced meat commercialized in butcheries is considered to be related to foodbourne diseases and a tool used by the districtal sanitary surveillance service to inspect these places is the good manufacturing practices checklist. The objective of this research was to evaluate the hygienic-sanitary condition of minced meat production through the application of the good manufacturing practice checklist in the butcheries of a district located in the south-east of the State of São Paulo. Transversal descriptive research with observational intervention and sample represented by 27 butcheries in which were analyzed the non-compliances acts related to the production of minced meat in the course of August and September 2009. The most common non-compliances found were: cross-contamination risk; inadequate handling and storage conditions, concentration of minced meat residue on the grinder, high temperatures during the production process, non storage of grinder plates under refrigeration, lack of hygiene of equipment and utensils; and, failure on handwashing methods. In conclusion, the situations observed by the inspection instrument made possible to identify the main failures of the production of minced meat in butcheries, which must be corrected and monitorized to ensure a safe product to consumers.