Detection of Listeria spp. in cattle and environment of pasture-based dairy farms / Detecção de Listeria spp. em gados leiteiros no meio ambiente com base na pastagem

The aim of the study was to detect Listeria spp., particularly Listeria monocytogenes, in cattle and environment of pasture based dairy farms in Paysandú, Uruguay. A two-stage sampling was conducted, 10 farms were selected by probability proportional to size. A single visit was made to each farm. Samples from bovine faeces, feedstuffs, bulk tank milk, drinking water and soil from the entry and exit pens of the milking parlour were collected for bacteriological studies. PCR assays were used to confirm species and determine the serotype profile of L. monocytogenes isolates. AscI-pulsed-field gel electrophoresis was done to genetically compare them. Listeria spp. were isolated from eight of ten dairy farms, whereas L. monocytogenes in three of them. Serotype distribution in L. monocytogenes was as follows: 1/2a, three isolates; 4b, one isolate. L. monocytogenes or L. innocua excreted from clinically healthy milking cows was detected via faeces. In feedstuffs, only one L. monocytogenes 1/2a isolate from a pasture was obtained. The strain was identical by PFGE to an isolate 1/2a obtained from a pool of milking cow feces that grazed on this farm. No isolation of Listeria spp. was retrieved from the bulk tank milk or drinking water from any of the farms. Listeria innocua was detected in 13 feedstuffs and seven samples of soil from the entry and exit pens of the milking parlour. This is a first local study that confirms the presence of Listeria spp. including L. monocytogenes in healthy cattle and environment of pasture-based dairy farms. These results suggest the potential role that healthy cattle and their sub-products would play as a source of these agents for humans and/or others animals. More detailed studies that include genetic comparison of human and animal isolates are required in order to clearly establish the epidemiological relationship.(AU)

O objetivo deste trabalho foi detectar a presença de bactérias do gênero Listeria e particularmente Listeria monocytogenes, em bovinos leiteiros no ambiente de Paysandú, Uruguai. Foi realizada uma amostragem em duas etapas, dez estabelecimentos foram selecionados por probabilidade proporcional ao tamanho. Foi realizada uma única visita a cada propriedade. Foram coletadas amostras para cultura bacteriológica de matéria fecal bovina, além de alimentos, leite do tanque de resfriamento, água e solo na entrada e saída da sala de ordenha. Com os isolados de L. monocytogenes foi realizado PCR para a confirmação da espécie e determinação do perfil do serotipo. AscI-elctroforese em gel de campo pulsado foi realizado para compará-los geneticamente. Listeria spp. foram isoladas de oito de dez estabelecimentos, enquanto L. monocytogenes foram detectadas em três deles. A distribuição dos serotipos nos isolados de L. monocytogenes recuperados foi: 1/2a três isolados, 4b um isolado. Foram detectadas vacas leiteras clinicamente sadias ​​que excretaram L. monocytogenes ou L. innocua nas fezes. Dos alimentos do gado houve só um isolamento de L. monocytogenes 1/2a em uma pastagem. Esta estirpe foi idêntica no PFGE a um isolado 1/2a obtido de uma "piscina" de fezes de vacas leiteiras do mesmo estabelecimento. Não houve isolamento de Listeria no leite do tanque de resfriamento ou na água de nenhum dos estabelecimentos. Listeria innocua foi detectada em 13 alimentos para o gado e sete amostras de solo na entrada e saída da sala de ordenha. Este parece ser o primeiro estudo local que confirma a presença de Listeria spp. incluindo L. monocytogenes em vacas leiteiras sadias e no meio ambiente de propriedades leiteiras com base alimentícia na pastagem. Esses resultados sugerem o potencial de vacas sadias e seus subprodutos como possível fonte desses agentes para humanos e/ou outros animais. São necessários estudos mais detalhados que incluem a comparação genética de isolados humanos e de animais para estabelecer claramente seu relacionamento epidemiológico.(AU)

Avaliação da qualidade microbiológica de queijos do tipo Minas padrão de produção industrial, artesanal e informal / Microbiological evaluation of Minas type cheeses from industrial, artisanal and informal manufacturing

Os objetivos deste estudo foram de avaliar a qualidade microbiológica de queijos do tipo Minas padrão,produzidos de forma industrial com inspeção federal, artesanal com inspeções estadual e municipal (a partir de leite não pasteurizado) e informal (produção caseira), bem como de analisar os hábitos de consumo desse tipo de queijo no Distrito Federal, Brasil. As amostras (n = 21) foram submetidas a análises para a pesquisa de coliformes a 30 °C e 45 °C, Staphylococcus coagulase positiva, Listeria monocytogenes e Salmonella spp. Os resultados foram avaliados de acordo com a RDC 12/2001 da ANVISA; 57,14% das amostras de queijos industriais, 100% das informais e 100% das artesanais estavam em desacordo quanto às contagens de Staphylococcus coagulase positiva. As contagens de coliformes a 45°C estavam em desacordo em 71,42% das amostras de queijos informais e 14,28% das industrializadas e artesanais. Nenhuma amostra foi positiva para L. monocytogenes ou Salmonella spp. Um questionário simplificado sobre o consumo de queijo Minas foi aplicado a 50 pessoas no momento da compra e houve indicação de 47% de preferência ao sabor de queijos informais. Em virtude destes queijos não serem inspecionados e não seguirem padrões de produção, representam um risco à saúde pública...

Vasculitis séptica por Listeria monocytogenes / Septic vasculitis caused by Listeria monocytogenes

Septic vasculitis is a medium and small-vessel vasculitis caused by direct action of pathogens, associated with bacteremia. It is an uncommon condition; clinical manifestations include purpura, ulcers and vesicles-pustules. Most cases of septic vasculitis are related to meningococcemia. There are no cases reported in medical literature associated to Listeria spp. We report a case of a 71 year-old man who presented sepsis by Listeria monocytogenes, and then evolved with purpuric skin lesions. Skin biopsy revealed a septic vasculitis.

La vasculitis séptica es una inflamación de los vasos sanguíneos de pequeño y mediano calibre causada por la acción directa de agentes patógenos en el contexto de una sepsis. Es una condición infrecuente y se manifiesta clínicamente por lesiones cutáneas como púrpura, vesículo-pústulas e incluso úlceras. La mayoría de los casos de vasculitis séptica se asocian a una meningococcemia. No se han reportado casos en la literatura médica de vasculitis séptica secundaria a Listeria spp. Se presenta el caso de un hombre de 71 años, con cuadro de sepsis por Listeria monocytogenes y que presentó lesiones purpúricas con una biopsia compatible con una vasculitis séptica.

First Isolation and Identification of Listeria monocytogenes Isolated from Frozen and Shock Frozen Dressed Broiler Chicken in Sudan.

Aim: To isolate and identify Listeria monocytogenes from frozen and shock frozen raw dressed broiler chicken in Khartoum State, Sudan. Place and Duration of Study: Department of Pathology, Microbiology and Parasitology, Sudan University of Science and Technology, Khartoum-North, Sudan, between July 2011 and June 2012. Methodology: Eight hundred samples were used in this the study. Five hundred frozen (-18º) raw dressed broiler chickens samples were collected from five station chicken abattoirs in Khartoum State. Three hundred samples were collected as fresh, -18ºC and shock frozen (-40ºC) raw dressed broiler chickens. Detection and isolation of L. monocytogenes was carried out using the conventional International Organization for Standardization method. Results: Out of the 500 samples, 195(39%) were found to be contaminated with Iisteria spp.; L. monocytogenes 64(12.8%), Listeria ivanovii 97(19.4%), Listeria grayi 20(4%), Listeria seeligeri 5(1%), Listeria welshimeri 9(1.8%). Out of the 300 samples, 111(37%) were found to be contaminated with Iisteria spp.; L. monocytogenes 39(13%), Listeria ivanovii 54(18%), Listeria grayi 11(3.6%), Listeria seeligeri 3(1%) and Listeria welshimeri 4(1.3%). Conclusion: The results presented in this study indicated that L. monocytogenes was found in frozen (-18ºC) raw dressed broiler chicken and shock frozen (-40ºC) raw dressed broiler chickens.

Ocorrência de Listeria spp e de Listeria monocytogenes, em um matadouro-frigorífico de bovinos do estado de São Paulo / Occurrence of Listeria spp and Listeria monocytogenes, at a slaughterhouse bovine of the state of São Paulo

A ocorrência de Listeria spp. e de Listeria monocytogenes, foi avaliada em amostras ambientais por meio de suabes colhidos em matadouro-frigorífico de bovinos habilitado à exportação, localizado no Estado de São Paulo, Brasil. Após o pré-enriquecimento a 30 ± 1oC por 22 à 26h as amostras foram analisadas empregando o BAX® System Listeria. As amostras positivas para Listeria spp. foram submetidas à uma nova reação de PCR para a confirmação da presença de Listeria monocytogenes. Das 411 amostras ambientais analisadas, 62 (15,1%) foram positivas para Listeria spp. e 21 (5,1%) para Listeria monocytogenes (5,1%), o que mostrou sua persistência na planta de abate. Não foi detectada diferença estatística entre os períodos seco e chuvoso e entre as superfícies amostradas, porém diferença estatística foi encontrada entre setores. A superfície do piso e o setor de cortes apresentaram os maiores índices de positividade.

We evaluated the presence of Listeria spp. and Listeria monocytogenes in environmental samples by means of swabs collected the bovine slaughter plant enabled to export, located in the state of Sao Paulo, Brazil. After the pre-enrichment at 30±1oC for 22 to 26h the samples were analyzed using the BAX System Listeria. Those positive for Listeria spp. were submitted a second PCR reaction to confirm the presence of Listeria monocytogenes. From 411 environmental samples analyzed, 62 (15.1%) were positive for Listeria spp. and 21 (5.1%) for Listeria monocytogenes, which showed their persistence in the slaughter plant. There were no statistical differences between the rainy and dry

Investigação de Salmonella spp., Listeria spp., Campylobacter spp. e Pseudomonas spp. no trato intestinal de avestruzes (Struthio camelus) criados no estado do Rio de Janeiro, Brasil / Investigation of Salmonella spp., Listeria spp., Campylobacter spp. and Pseudomonas spp. in ostriches (Struthio camelus) intestine from Rio de Janeiro State, Brazil

Na estrutiocultura, pesquisas relacionadas com a prevalência de micro-organismos patogênicos para esses animais e de importância em saúde pública ainda são escassas. O objetivo deste trabalho foi investigar a presença de Salmonella spp., Listeria spp., Campylobacter spp. e Pseudomonas spp. a partir de 60 amostras de suabes cloacais, provenientes de quatro criatórios de avestruzes e avaliar o perfil de susceptibilidade destes micro-organismos frente aos antimicrobianos. Os micro-organismos dos gêneros Salmonella, Listeria e Campylobacter não foram encontrados nas amostras analisadas. Pseudomonas aeruginosa foi isolado a partir de 17 amostras (28,3,%), provenientes de animais de diferentes faixas etárias, oriundos dos quatro criatórios investigados. Adicionalmente, Escherichia coli foi isolado de 57 amostras (95%), Klebsiella spp., de cinco amostras (8,33%), Proteus mirabilis, Proteus vulgaris e Enterobacter spp. de uma amostra (1,66%). Das 17 cepas de P. aeruginosa submetidas ao teste de susceptibilidade aos antimicrobianos, todas (100%) apresentaram sensibilidade à Amicacina e à Ciprofloxacina e todas (100%) foram resistentes ao Sulfametoxazol/Trimetoprim e à Tetraciclina. Foram encontrados cinco perfis diferentes de resistência aos antimicrobianos, indicando uma variação da resistência entre as cepas de Pseudomonas isoladas, inclusive em animais do mesmo criatório e mantidos no mesmo piquete.

There has been limited research on the prevalence of ostrich intestinal pathogens and on the role of these animals as foodborne pathogens source. This study was conducted to estimate the frequence of Salmonella spp., Listeria spp., Campylobacter spp. and Pseudomonas spp. from intestinal swabs of ostriches and to investigate the antibiotic resistance of the isolates. Sixty samples collected from four ostrich farms were examined. Salmonella, Listeria and Campylobacter were not isolated from the samples investigated and Pseudomonas aeruginosa was isolated from 17 (28.3%) samples. Additionally, Escherichia coli was isolated from 57 samples, (95%), Klebsiella spp. from five (8.33%), and Proteus mirabilis, Proteus vulgaris and Enterobacter spp. from one sample (1,66%). All Pseudomonas isolates were susceptible to Amicacin and Ciprofloxacin, and all of them (100%) were resistant to Trimethoprin/Sulfametox and to Tetracicline. Five antimicrobial resistance profiles were found, showing a resistance diversity among the Pseudomonas isolates, even in the same farm and raised at the same pen.

Listeria spp. associated to different levels of autochthonous microbiota in meat, meat products and processing plants

High levels of microbial contamination, commonly found in animal origin foods and food processing environments, are able to hinder the growth of pathogens in these products and interfere in the results of laboratory analyses for detection of these pathogens. With the aim of verifying the possible interference of the autochthonous microbiota encountered in meat and meat products and processing plants over the presence of Listeria spp., 443 samples, collected from 11 meat retail establishments, were submitted to microbiological analysis to determine the levels of mesophilic aerobes, total coliforms and Escherichia coli and the presence of Listeria spp., according to the methodology proposed by the USDA. The results did not show evident interference of the autochthonous microbiota over Listeria spp., once the genus was detected even in the meat, meat products and environmental samples with high levels of contamination by mesophilic aerobes and coliforms.

Altos níveis de contaminação microbiana, usualmente encontrados em alimentos de origem animal e nos ambientes de processamento, podem inibir a multiplicação de microrganismos patogênicos nesses produtos e interferir nos resultados das análises laboratoriais para o isolamento desses patógenos. Com o objetivo de verificar as possíveis interferências da microbiota autóctone encontrada na carne, produtos cárneos e plantas de processamento sobre a presença de Listeria spp., 443 amostras, coletadas em 11 estabelecimentos processadores, foram submetidas a análises microbiológicas para determinação dos níveis de contaminação por aeróbios mesófilos, coliformes totais e Escherichia coli e para verificação da presença de Listeria spp., de acordo com a metodologia proposta pelo USDA. Os resultados obtidos não mostraram uma interferência evidente da microbiota autóctone sobre Listeria spp., uma vez que esse gênero foi detectado mesmo nas amostras de carne e produtos cárneos e amostras ambientais e de superfície de equipamentos que apresentaram altos níveis de contaminação por aeróbios mesófilos e coliformes.

Determinación Microbiológica y Molecular de Listeria sp. y Listeria monocytogenes en Cerdas a nivel de una planta beneficiadora en EUA

Con el objeto de comparar la técnica de PCR múltiple con el método microbiológico comercial para la determinación de la presencia de Listeria spp. y Listeria monocytogenes en alimentos, se llevó a cabo un muestreo en una planta procesadora de cerdas de descarte ubicada en Estados Unidos de Norteamérica (EUA). A 160 cerdas sacrificadas, se le tomaron muestras de ganglios sub-ilíacos, ganglios ileocecales, contenido cecal e hisopado de la superficie de las canales. Adicionalmente se tomaron muestras del ambiente de la planta y de cortes de carne. Un total de 708 muestras se procesaron con ambas técnicas. Mediante técnicas microbiológicas, el 5,2% resultaron positivas a Listeria spp. y el 0,14% a L. monocytogenes. Mediante la técnica de PCR 4,1% resultaron positivas a Listeria spp. y 0,85% positivas a L. monocytogenes. No se observó diferencias significativas (P > o = 0,05) entre estos valores. Se determinó una incidencia del 1,9% de Listeria spp. en los hisopados de la superficie de las canales de las cerdas evaluadas. Por otro lado, se identificó L. monocytogenes, en el 5% de las muestras de cortes de carne. En relación a los ganglios, los sub-ilíacos presentaron 6,3% de incidencia a Listeria spp. y de 1,3% a L. monocytogenes no encontrándose en ganglios ileocecales. Para el contenido cecal la incidencia a Listeria spp. fue de 19% y para L. monocytogenes fue de 2,5%. En el ambiente el 4,2% de las muestras resultaron positivas a Listeria spp. y ninguna a L. monocytogenes. Al comparar los resultados logrados entre ambas técnicas no hubo diferencia significativa entre los valores obtenidos con las muestras de los hisopados de las canales y la de los ganglios ileocecales. Si hubo diferencia significativa entre los resultados de contenido cecal P = 0,0168 < 0,05 y de los ganglios sub-ilíacos P = 0,0038 < 0,05. La utilización de la técnica de PCR múltiple, permitió que los resultados se obtuvieran en 8 h, luego del segundo período de enriquecimiento de cada muestra, y con el método microbiológico convencional el procedimiento tomó 8 días. La incorporación de la metodología molecular en el proceso de verificación del status microbiológico en la industria de alimentos, resulta una mejora para la efectividad y la dinámica en los sistemas de seguridad alimentaria.

Listeria spp. and Listeria monocytogenes presence in a cull sows processor plant in USA, was evaluated by and PCR multiplex method. 160 cull sows were surveyed after slaughter. Samples were collected from sub-iliac node, iliocecal node, cecal content and carcass swabs. Additionally samples were taken from environment plant and from raw meat ready to be processed. A total of 708 samples were processed. Using traditional microbiology method were found 5.2% of samples positive to Listeria spp. and 0.14% to L. monocytogenes. With PCR multiplex 4.1% were positive to Listeria spp. and 0.85% to L. monocytogenes. There was not significant difference (P > or = 0.05) in the results obtained with PCR multiplex and traditional microbiology procedures. In relation with the raw material that leaves the slaughter area to be processed inside the same plant, Listeria spp. was observed in 1.9% swabs carcass, and 5% of raw sow meat sampled. Listeria spp. was identified in 6.3% and L. monocytogenes in 1.3% of subiliac nodes. It was not any iliocecal node positive to Listeria. Samples supposedly related with the infection source in the process plant, cecal content sample were 19% positive to Listeria spp. and 2.5% to L. monocytogenes. Environmental samples were 4.2% positive to Listeria spp. There were not differences between the conventional microbiology procedure and PCR multiplex technique for this pathogen when carcass swabs and ilicecal node were evaluated with both techniques. Differences were observed between the samples from cecal content and sub-iliac node P = 0.0168 < 0.05 and P = 0.0038 < 0.05 respectively. With the PCR multiplex technique, results were obtained in 8 hours after the second enrichment culture period of the each sample. With traditional microbiological it took 8 days. The incorporation of molecular methodology in the verification process for microbiological controls in the food industry, would allow an important improvement of the effectiveness and dynamics in the food safety systems implanted at the food industries.

DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis: Argentina / Caracterización molecular por ERIC-PCR de cepas de Listeria spp. aisladas de diversos orígenes en San Luis: Argentina

In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina) from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR). Repetitive intergenic consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.

En este estudio se analizaron 24 cepas de Listeria spp. De ellas, 22 fueron obtenidas en San Luis (Argentina), a partir de muestras humanas, de animales y alimentos. Se incluyeron 2 cepas de referencia Listeria monocytogenes CLIP 22762 y Listeria innocua CLIP 74915. Todos los aislamientos fueron identificados bioquímicamente y caracterizados por serotipificación, fagotipificación y detección del gen flaA por reacción en cadena de la polimerasa (PCR). Se generaron perfiles de bandas de ADN mediante la amplificación de secuencias repetitivas de consenso intergénico de enterobacterias (ERIC-PCR). De acuerdo a los resultados obtenidos por ERIC-PCR, las cepas de Listeria spp. fueron divididas en 3 grupos según su origen. Los perfiles de los aislamientos humanos y animales fueron distintos de los correspondientes a alimentos. Por otra parte, dentro de los grupos I y II se incluyeron 10 cepas de L. monocytogenes y solamente una de Listeria seeligeri. Dentro del grupo III no sólo estuvieron incluidas las 9 cepas de L. innocua sino también 4 de L. monocytogenes. La evaluación de los perfiles de bandas obtenidos por ERIC-PCR permitió la discriminación entre los serotipos ensayados 1/2b, 4b, 6a y 6b dentro de cada grupo. El índice de discriminación calculado para ERIC-PCR fue de 0,94. Los resultados sugieren que la técnica de ERIC-PCR provee un método alternativo válido para la identificación de especies de Listeria y, asimismo, permite la diferenciación de cepas dentro de una misma especie.

Development of a sandwich ELISA for the detection of Listeria spp. using specific flagella antibodies

Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x105cells/0.1 ml at 22degrees C and 1x106 cells/0.1 ml at 30degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 104cells/0.1 ml at 22degrees C and 30degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.