Validation of Alternative Methods of Detection of Salmonella Spp. in Experimentally Contaminated Poultry Environmental Samples

The analysis of Salmonella in the feces and the birds environment is a way of monitoring the colonization in the flocks and verifying the need for the introduction of stricter controls, in such a way that the results of the tests should be known before being sent for slaughter. The polymerase chain reaction (PCR), as well as other rapid methods represent alternatives increasingly used to detect enteric pathogens, but they need proof of effectiveness for their wide use. The aim of this study was to evaluate the equivalence between the results obtained by the methods: real-time PCR (BAX® System), Modified Rappaport-Vassiliadis Semi-solid Medium (MSRV) (ISO 6579) and the traditional method of official reference in Brazil for research of S. Typhimurium and S. Enteritidis in poultry samples. Two hundred and fifty-two samples of disposable shoe covers (DSC) and 252 samples of feces were infected with an average of 2 to 3 log CFU/g of each serovar, and the same samples without fortification were evaluated by the three methods. Five hundred and four diagnoses were obtained with satisfactory results in terms of repeatability (greater than 80%), reproducibility (mean 83,1%), sensitivity (81% to 100%), specificity (95% to 100%), and accuracy (90% to 100%). The compliance test verified that there was not a significant difference between the alternative and the official methods, allowing us to state that the methodologies have had equivalent performances.(AU)

Validation of Alternative Methods of Detection of Salmonella Spp. in Experimentally Contaminated Poultry Environmental Samples

The analysis of Salmonella in the feces and the birds environment is a way of monitoring the colonization in the flocks and verifying the need for the introduction of stricter controls, in such a way that the results of the tests should be known before being sent for slaughter. The polymerase chain reaction (PCR), as well as other rapid methods represent alternatives increasingly used to detect enteric pathogens, but they need proof of effectiveness for their wide use. The aim of this study was to evaluate the equivalence between the results obtained by the methods: real-time PCR (BAX® System), Modified Rappaport-Vassiliadis Semi-solid Medium (MSRV) (ISO 6579) and the traditional method of official reference in Brazil for research of S. Typhimurium and S. Enteritidis in poultry samples. Two hundred and fifty-two samples of disposable shoe covers (DSC) and 252 samples of feces were infected with an average of 2 to 3 log CFU/g of each serovar, and the same samples without fortification were evaluated by the three methods. Five hundred and four diagnoses were obtained with satisfactory results in terms of repeatability (greater than 80%), reproducibility (mean 83,1%), sensitivity (81% to 100%), specificity (95% to 100%), and accuracy (90% to 100%). The compliance test verified that there was not a significant difference between the alternative and the official methods, allowing us to state that the methodologies have had equivalent performances.

Miniaturized most probable number for the enumeration of Salmonella sp in artificially contaminated chicken meat

Salmonella is traditionally identified by conventional microbiological tests, but the enumeration of this bacterium is not used on a routine basis. Methods such as the most probable number (MPN), which utilize an array of multiple tubes, are time-consuming and expensive, whereas miniaturized most probable number (mMPN) methods, which use microplates, can be adapted for the enumeration of bacteria, saving up time and materials. The aim of the present paper is to assess two mMPN methods for the enumeration of Salmonella sp in artificially-contaminated chicken meat samples. Microplates containing 24 wells (method A) and 96 wells (method B), both with peptone water as pre-enrichment medium and modified semi-solid Rappaport-Vassiliadis (MSRV) as selective enrichment medium, were used. The meat matrix consisted of 25g of autoclaved ground chicken breast contaminated with dilutions of up to 10(6) of Salmonella Typhimurium (ST) and Escherichia coli (EC). In method A, the dilution 10-5 of Salmonella Typhimurium corresponded to >57 MPN/mL and the dilution 10-6 was equal to 30 MPN/mL. There was a correlation between the counts used for the artificial contamination of the samples and those recovered by mMPN, indicating that the method A was sensitive for the enumeration of different levels of contamination of the meat matrix. In method B, there was no correlation between the inoculated dilutions and the mMPN results.

Miniaturized most probable number for the enumeration of Salmonella sp in artificially contaminated chicken meat

Salmonella is traditionally identified by conventional microbiological tests, but the enumeration of this bacterium is not used on a routine basis. Methods such as the most probable number (MPN), which utilize an array of multiple tubes, are time-consuming and expensive, whereas miniaturized most probable number (mMPN) methods, which use microplates, can be adapted for the enumeration of bacteria, saving up time and materials. The aim of the present paper is to assess two mMPN methods for the enumeration of Salmonella sp in artificially-contaminated chicken meat samples. Microplates containing 24 wells (method A) and 96 wells (method B), both with peptone water as pre-enrichment medium and modified semi-solid Rappaport-Vassiliadis (MSRV) as selective enrichment medium, were used. The meat matrix consisted of 25g of autoclaved ground chicken breast contaminated with dilutions of up to 10(6) of Salmonella Typhimurium (ST) and Escherichia coli (EC). In method A, the dilution 10-5 of Salmonella Typhimurium corresponded to >57 MPN/mL and the dilution 10-6 was equal to 30 MPN/mL. There was a correlation between the counts used for the artificial contamination of the samples and those recovered by mMPN, indicating that the method A was sensitive for the enumeration of different levels of contamination of the meat matrix. In method B, there was no correlation between the inoculated dilutions and the mMPN results.

Miniaturized most probable number for the enumeration of Salmonella sp in artificially contaminated chicken meat

Salmonella is traditionally identified by conventional microbiological tests, but the enumeration of this bacterium is not used on a routine basis. Methods such as the most probable number (MPN), which utilize an array of multiple tubes, are time-consuming and expensive, whereas miniaturized most probable number (mMPN) methods, which use microplates, can be adapted for the enumeration of bacteria, saving up time and materials. The aim of the present paper is to assess two mMPN methods for the enumeration of Salmonella sp in artificially-contaminated chicken meat samples. Microplates containing 24 wells (method A) and 96 wells (method B), both with peptone water as pre-enrichment medium and modified semi-solid Rappaport-Vassiliadis (MSRV) as selective enrichment medium, were used. The meat matrix consisted of 25g of autoclaved ground chicken breast contaminated with dilutions of up to 10(6) of Salmonella Typhimurium (ST) and Escherichia coli (EC). In method A, the dilution 10-5 of Salmonella Typhimurium corresponded to >57 MPN/mL and the dilution 10-6 was equal to 30 MPN/mL. There was a correlation between the counts used for the artificial contamination of the samples and those recovered by mMPN, indicating that the method A was sensitive for the enumeration of different levels of contamination of the meat matrix. In method B, there was no correlation between the inoculated dilutions and the mMPN results.(AU)

ANÁLISE COMPARATIVA ENTRE ELFA ELISA UTILIZANDO BACTERIÓFAGO RECOMBINANTE E OUTROS MÉTODOS DIAGNÓSTICOS PARA DETECÇÃO DE Salmonella spp. EM PRODUTOS DE ORIGEM ANIMAL / comparative analysis between Alfa Elisa using recombinant bacteriophage and other diagnostic methods for Salmonella Spp. detection in products of animal origin

As análises para detecção de Salmonella spp. são de grande importância no processo da agroindústria de produtos de origem animal, pois refletem diretamente na qualidade microbiológica dos alimentos, impactando no tempo de estocagem e por conseguinte na liberação do produto para venda. O presente trabalho verificou a eficiência de um novo teste ELFA ELISA utilizando proteína recombinante oriunda de bacteriófago (VIDAS UP®) para detectar a presença de Salmonella spp. em produtos de origem animal. Foram analisadas 215 amostras (15 amostras artificilamente contaminadas e 200 amostras de rotina da agroindústria), de diferentes produtos de origem animal. As amostras foram submetidas a cinco métodos de diagnóstico: ELFA ELISA, metodologia convencional de acordo com ISO 6579, reação em cadeia da polimerase - PCR, meio semi-sólido Rappaport- Vassiliadis Modificado - MSRV e meio MSRV + suplemento. Onze amostras das 215 (5,12%) foram positivas em pelo menos um dos testes, sendo quatro amostras de rotina da agroindústria e sete amostras artificialmente contaminadas. A sensibilidade e especificidade do teste ELFA ELISA com a metodologia convencional foi de 90,0% e 99,51%, respectivamente. O teste ELFA ELISA apresentou equivalência com as demais metodologias, mostrando ser eficaz e rápido na detecção de Salmonella spp. em diferentes produtos de origem animal. Palavras

The analysis methods for Salmonella spp. detection are of great importance in the system of animal products processing industries, because they reflect directly on the microbiological quality of food, impacting the storage time and therefore the release of the product for sale. The present study verified the efficiency of a new ELFA ELISA test using a recombinant protein from bacteriophage (VIDAS UPÂ) to detect the presence of Salmonella spp. in products of animal origin. Two hundred fifteen samples (15 samples artificially contaminated and 200 samples from agribusiness routine), from different products of animal origin were analyzed. The samples were tested by five methods of diagnosis: ELFA ELISA, conventional methodology in accordance with ISO 6579, polymerase chain reaction - PCR, semi-solid Rappaport-Vassiliadis Modified - MSRV and MSRV medium + supplement. Eleven out of 215 (5.12%) samples were positive in at least one test, which four samples were from agribusiness routine and seven were from artificially contaminated samples. The sensitivity and specificity of the ELFA ELISA when compared with the conventional method was 90.0% and 99.51% respectively. The ELFA ELISA was equivalent to the other methodologies, showing to be effective and rapid to detect Salmonella spp. in different products of animal origin

Comparision of the BAX® System with an in-house MSRV method for the detection of Salmonella in chicken carcasses and pork meat

A study was performed to compare the analytical procedure of the BAX® System for Salmonella PCR assay with the Modified Semi-Solid Rappaport-Vassiliadis (MSRV) method, for the detection of Salmonella in naturally contaminated chicken carcass samples (n = 762) and raw pork meat (n = 566). The chicken carcasses samples were collected during slaughtering after defeathering or immediately after evisceration and the raw pork meat collected from the deboned head of recently slaughtered pigs and others deboned raw fresh pork meat. The BAX® System detected 134 Salmonella-positive samples in chicken carcasses and 145 samples in pork meat, while the MSRV method isolated 142 and 144 Salmonella-positive samples, respectively. No significant difference was observed between the two methods for chicken carcasses and pork meat, according to McNemar test at the 5% level.

Um estudo foi realizado com o objetivo de comparar o procedimento analítico de detecção de Salmonella com o Sistema BAX® automatizado, baseado na Reação em Cadeia da Polimerase (PCR) com o método de Rappaport-Vassiliadis em Agar Semi-Sólido modificado (MSRV) para detecção de Salmonella em amostras de carcaças de frango naturalmente contaminadas (n=762) e retalhos de carne suía (n=566). O Sistema BAX® detectou 134 amostras positivas para Salmonella em carcaças de frango e 145 amostras positivas para Salmonella em retalhos de carne suína, enquanto o MSRV detectou 142 e 144 amostras positivas respectivamente. Não houve diferença estatisticamente significativa entre os dois métodos, segundo McNemar ao nível de significância de 5%.

Equivalência de métodos alternativos oficial para determinação de Salmonella enteritidis e Typhimurium em amostras ambientais avícolas

A infecção de humanos por Salmonella, particularmente pelos sorovares Enteritidis e Typhimurium é uma preocupação para a saúde pública mundial. A análise deste patógeno nas fezes e no ambiente das aves é uma forma de monitorar a infecção nos lotes e verificar a necessidade de introdução de controles, havendo recomendação do Regulamento da Comissão Européia (CE) (n.° 646/2007) de que os lotes de aves sejam analisados para a presença destes em fases anteriores ao abate. Diferentes metodologias podem ser utilizadas para a pesquisa desses microrganismos em amostras ambientais; entretanto, a maioria demanda um longo prazo para obtenção dos resultados. Assim, é desejável que outros métodos, mais rápidos e práticos tenham seu desempenho verificado para possível implantação na rotina. Objetivou-se, avaliar a equivalência entre os resultados obtidos pelos métodos: BAX®, Meio Semi-sólido Rappaport-Vassiliadis Modificado (MSRV) (ISO 6579) e o método tradicional de referência oficial no Brasil (Portaria 126, MAPA) para pesquisa de S. Typhimurium e S. Enteritidis em amostras ambientais avícolas. Amostras de propé e fezes fortificadas com uma média de 100 a 1000 UFC/g de cada sorovar, e as mesmas amostras sem fortificação foram avaliadas pelos três métodos. Foram obtidos 504 diagnósticos e os mesmos analisados quanto à repetibilidade, reprodutibilidade, sensibilidade, especificidade e acurácia. Estes índices foram realizados individualmente e em relação ao método tradicional (índices relativos). Os resultados obtidos indicaram que os métodos analisados mostraram desempenho satisfatório e o teste de conformidade verificou correspondência entre os métodos alternativos e o método oficial, o que permite afirmar que as metodologias possuem desempenhos equivalentes