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.

Isolamento de Salmonella Heidelberg em diferentes pontos da tecnologia de abate de frangos de corte

Bacteria of the genus Salmonella are part of the normal flora of poultry, and its occurrence in poultry products varies with the production management methods and slaughter technology, representing risks to the consumer and difficulties in exports. S. Heidelberg appears to be more invasive and to cause more severe disease than other non-typhoidal serovars. The objective of the present study was to search for Salmonella at different points of the slaughterhouse and to report on the isolation of S. Heidelberg in a slaughterhouse under federal inspection. S. Heidelberg was isolated in poultry soon after feathering-out and in the chiller water, indicating that, although it was not isolated at another sampled points, the bacteria was present in the environment and could contaminate carcasses ready for consumption, with an impact on public health. This concern is underscored by the fact that S. Heidelberg is a proven causative agent of foodborne infections.

Bactérias do gênero Salmonella fazem parte da flora normal das aves e sua ocorrência em produtos avícolas varia com o manejo na criação e tecnologia de abate, representando riscos para o consumidor final e dificuldades nas exportações. A S. Heidelberg parece ser mais invasiva e causar doenças com maior gravidade que outros sorovares paratíficos. Este trabalho teve por objetivos pesquisar Salmonella em diferentes pontos da tecnologia de abate de frangos e relatar o isolamento de S. Heidelberg em um abatedouro sob inspeção federal. S. Heidelberg foi isolada em frangos logo após a depenagem e na água do chiller indicando que, embora não tenha sido realizado o isolamento em outros pontos amostrados, a bactéria estava presente no ambiente e poderia contaminar carcaças prontas para consumo, com reflexos na saúde pública. O isolamento de S. Heidelberg reforça esta preocupação uma vez que este sorovar tem se destacado como causador de doenças transmitidas por alimentos.

Isolamento de Salmonella Heidelberg em diferentes pontos da tecnologia de abate de frangos de corte

Bacteria of the genus Salmonella are part of the normal flora of poultry, and its occurrence in poultry products varies with the production management methods and slaughter technology, representing risks to the consumer and difficulties in exports. S. Heidelberg appears to be more invasive and to cause more severe disease than other non-typhoidal serovars. The objective of the present study was to search for Salmonella at different points of the slaughterhouse and to report on the isolation of S. Heidelberg in a slaughterhouse under federal inspection. S. Heidelberg was isolated in poultry soon after feathering-out and in the chiller water, indicating that, although it was not isolated at another sampled points, the bacteria was present in the environment and could contaminate carcasses ready for consumption, with an impact on public health. This concern is underscored by the fact that S. Heidelberg is a proven causative agent of foodborne infections.

Bactérias do gênero Salmonella fazem parte da flora normal das aves e sua ocorrência em produtos avícolas varia com o manejo na criação e tecnologia de abate, representando riscos para o consumidor final e dificuldades nas exportações. A S. Heidelberg parece ser mais invasiva e causar doenças com maior gravidade que outros sorovares paratíficos. Este trabalho teve por objetivos pesquisar Salmonella em diferentes pontos da tecnologia de abate de frangos e relatar o isolamento de S. Heidelberg em um abatedouro sob inspeção federal. S. Heidelberg foi isolada em frangos logo após a depenagem e na água do chiller indicando que, embora não tenha sido realizado o isolamento em outros pontos amostrados, a bactéria estava presente no ambiente e poderia contaminar carcaças prontas para consumo, com reflexos na saúde pública. O isolamento de S. Heidelberg reforça esta preocupação uma vez que este sorovar tem se destacado como causador de doenças transmitidas por alimentos.

Sistema api campy para caracterização de amostras de campylobacter isoladas de descarga cecal, fezes, swabs cloacais e carcaças de frangos de corte

ABSTRACT The aim of the present study was to assess the API Campy system for characterization of Campylobacter strains. Forty-eight Campylobacter strains were isolated from 22 three-week-old broiler flocks: 15 from cecal droppings, 5 from feces, 3 from cloacal swabs, and 21 from carcasses. The strains were presumptively analyzed by phase contrast microscopy, Gram staining, catalase/oxidase activity, and latex agglutination test, and were then inoculated into the API Campy system, which consists of enzyme-linked and conventional assays under aerobic conditions and assimilation or inhibition tests under microaerophilic conditions, both incubated at 37º C for 2448 hours, the readings made with a computerized system. A total of 43 strains (89.58%) could be identified, whereas 5 (10.41%) yielded an unacceptable profile. The following species were identified: C. jejuni subsp. jejuni (68.8%), C. coli (8.3%), C. jejuni subsp doylei (6.3%), C. upsaliensis (4.2%) and C. fetus subsp. fetus (2.1%). An 81.8% prevalence was found for Campylobacter in broiler flocks, mainly Campylobacter jejuni subsp. jejuni, identified by the API Campy system, showing a larger number of species, subspecies and biotypes of Campylobacter among the strains isolated from carcasses than among the clinical strains obtained from poultry farms.

RESUMO O presente estudo verificou a aplicação do sistema API Campy para identificação de bactérias do gênero Campylobacter. Foram utilizadas 48 amostras, sendo 15 oriundas de descarga cecal, cinco de fezes, três de swabs cloacais e 21 de carcaças, isoladas de 22 lotes de frangos de corte com três semanas de idade. As amostras caracterizadas presuntivamente por microscopia em contraste de fase, coloração de Gram, catalase/oxidase e aglutinação em látex foram inoculadas no sistema API Campy, que consta de testes enzimáticos e convencionais em aerobiose e testes de assimilação ou inibição em microaerofilia, ambos incubados a 37º C por 24-48 horas, sendo a leitura realizada em sistema informatizado. Foi possível identificar 43 amostras (89,58%), enquanto cinco (10,41%) tiveram perfil inaceitável. Identificou-se as espécies C. jejuni subsp. jejuni (68,8%); C. coli (8,3%); C. jejuni subsp. doylei (6,3%); C. upsaliensis (4,2%) e C. fetus subsp. fetus (2,1%). A ocorrência de Campylobacter nos lotes de frango de corte estudados foi de 81,8% e as espécies identificadas principalmente como Campylobacter jejuni subesp. jejuni pelo sistema API Campy, apresentando um maior número de espécies, subespécies e biotipos deCampylobacter dentre as amostras isoladas de carcaças de frango do que dentre as amostras clínicas isoladas nas granjas.

Rapd/pcr and phage typing of salmonella enteritidis isolated from poultry and food poisoning outbreaks

ABSTRACT Salmonella Enteritidis (SE) is an important pathogen, causing both food poisoning outbreaks in humans and economic losses to the poultry industry, being also widely spread in the environment. This work aimed to identify SE phage types and to standardize the Random Amplified Polymorphic DNA (RAPD) for evaluating SE isolates obtained from different origins. To do so, 238 SE strains were selected, of which 104 were isolated from broiler carcasses, 106 from food samples and human biological materials involved in food poisoning outbreaks and 28 from different poultry materials. Among these 238 SE isolates, 111 were phage typed, and 57.7% (64/ 111) corresponded to phage type (PT) 4, 32.4% (36/111) to PT 4a, 3.6% (4/111) to PT 6a and 0.9% (1/111) to PT 7, whereas 5.4% .6/111) of the strains were not typeable (RDNC, reacts but does not conform). After the standardization of amplification conditions, all 238 SE isolates were submitted to RAPD/PCR. Among these, 91.8% (217/238) were classified as pattern A. Twenty-one isolates were differentiated into four patterns and into seven subtypes with the use of primer 1254, and into four patterns and ten subtypes using primer OPB 17. The combination of phage typing and RAPD/PCR proved to be a useful tool in epidemiological investigations. RAPD/PCR can be easily used as a routine laboratory method, thus helping with the monitoring of SE isolates and contributing to the establishment of effective Salmonella Enteritidis control and preventive programs.

RESUMO Salmonella Enteritidis (SE) é um patógeno de importância destacada como causa de toxinfecções alimentares em humanos, prejuízos ao setor produtivo e ampla disseminação no ambiente. Este trabalho objetivou identificar fagotipos e padronizar a RAPD/PCR (DNA polimórfico amplificado ao acaso) para a avaliação de isolados de SE. Foram selecionadas 238 amostras, sendo 104 oriundas de carcaças de frango, 106 de alimentos e material biológico de humanos isolados em episódios de toxinfecções alimentares e 28 de materiais de origem avícola. Foram fagotipadas 111 amostras sendo 57,7% (64/111) do fagotipo 4, 32,4% (36/111) fagotipo 4a, 3,6% (4/111) fagotipo 6a e 0,9% (1/111) fagotipo 7, enquanto 5,4% (6/111) não foram fagotipáveis (RDNC, reagent do not conform). Para a RAPD/PCR foram utilizados 238 isolados de SE. Destes, 91,8% (217/238) foram enquadrados no padrão A e 21 isolados (8,8%) foram diferenciados em quatro padrões e sete subtipos com o primer 1254 e em quatro padrões e dez subtipos com o primer OPB 17. A facilidade de execução da RAPD/PCR, após padronizada, habilita a sua implantação em uma rotina laboratorial, auxiliando na monitoria dos isolados de SE e, conseqüentemente, contribuindo para a elaboração de programas efetivos de controle e prevenção de S. Enteritidis.

Antibacterial efficacy of commercial disinfectants on dirt floor used in poultry breeder houses

Dirt floors are used on most Brazilian poultry farms since the construction of concrete floors is very expensive. In vitro tests carried out to verify the effectiveness of disinfectants do not consider the adverse conditions found in poultry farms. Therefore, the present study aimed at evaluating the effect of six commercial disinfectants on the reduction of total and fecal coliforms on the dirt floor of breeder houses. The amount of disinfectant solution to be used per square meter was defined by counting total and fecal coliforms at different soil depths and by analyzing soil physical properties. Coliforms were detected at 0.5 cm, and one liter of disinfectant solution was sufficient for soil saturation at this depth. After that, the efficacy of six commercial products (caustic soda, hydrated lime, phenols 1 and 2, iodine, glutaraldehyde, and quaternary ammonium) in reducing the number of coliforms, after six hours of contact with the dirt floor, was assessed using the most probable number (MPN) method. Escherichia coli specimens isolated from the dirt floor were used to evaluate in vitro effectiveness of disinfectants. Products that yielded the best results in the MPN method were also effective in the in vitro tests. Among the tested disinfectants, hydrated lime was the most efficient, reducing the initial contamination by 2.9 log after six hours of contact with the dirt floor.

Antibacterial efficacy of commercial disinfectants on dirt floor used in poultry breeder houses

Dirt floors are used on most Brazilian poultry farms since the construction of concrete floors is very expensive. In vitro tests carried out to verify the effectiveness of disinfectants do not consider the adverse conditions found in poultry farms. Therefore, the present study aimed at evaluating the effect of six commercial disinfectants on the reduction of total and fecal coliforms on the dirt floor of breeder houses. The amount of disinfectant solution to be used per square meter was defined by counting total and fecal coliforms at different soil depths and by analyzing soil physical properties. Coliforms were detected at 0.5 cm, and one liter of disinfectant solution was sufficient for soil saturation at this depth. After that, the efficacy of six commercial products (caustic soda, hydrated lime, phenols 1 and 2, iodine, glutaraldehyde, and quaternary ammonium) in reducing the number of coliforms, after six hours of contact with the dirt floor, was assessed using the most probable number (MPN) method. Escherichia coli specimens isolated from the dirt floor were used to evaluate in vitro effectiveness of disinfectants. Products that yielded the best results in the MPN method were also effective in the in vitro tests. Among the tested disinfectants, hydrated lime was the most efficient, reducing the initial contamination by 2.9 log after six hours of contact with the dirt floor.

Risk factors and likelihood of Campylobacter colonization in broiler flocks

Campylobacter was investigated in cecal droppings, feces, and cloacal swabs of 22 flocks of 3 to 5 week-old broilers. Risk factors and the likelihood of the presence of this agent in these flocks were determined. Management practices, such as cleaning and disinfection, feeding, drinkers, and litter treatments, were assessed. Results were evaluated using Odds Ratio (OR) test, and their significance was tested by Fisher's test (p 0.05). A Campylobacter prevalence of 81.8% was found in the broiler flocks (18/22), and within positive flocks, it varied between 85 and 100%. Campylobacter incidence among sample types was homogenous, being 81.8% in cecal droppings, 80.9% in feces, and 80.4% in cloacal swabs (230). Flocks fed by automatic feeding systems presented higher incidence of Campylobacter as compared to those fed by tube feeders. Litter was reused in 63.6% of the farm, and, despite the lack of statistical significance, there was higher likelihood of Campylobacter incidence when litter was reused. Foot bath was not used in 45.5% of the flocks, whereas the use of foot bath associated to deficient lime management increased the number of positive flocks, although with no statiscal significance. The evaluated parameters were not significantly associated with Campylobacter colonization in the assessed broiler flocks.

Risk factors and likelihood of Campylobacter colonization in broiler flocks

Campylobacter was investigated in cecal droppings, feces, and cloacal swabs of 22 flocks of 3 to 5 week-old broilers. Risk factors and the likelihood of the presence of this agent in these flocks were determined. Management practices, such as cleaning and disinfection, feeding, drinkers, and litter treatments, were assessed. Results were evaluated using Odds Ratio (OR) test, and their significance was tested by Fisher's test (p 0.05). A Campylobacter prevalence of 81.8% was found in the broiler flocks (18/22), and within positive flocks, it varied between 85 and 100%. Campylobacter incidence among sample types was homogenous, being 81.8% in cecal droppings, 80.9% in feces, and 80.4% in cloacal swabs (230). Flocks fed by automatic feeding systems presented higher incidence of Campylobacter as compared to those fed by tube feeders. Litter was reused in 63.6% of the farm, and, despite the lack of statistical significance, there was higher likelihood of Campylobacter incidence when litter was reused. Foot bath was not used in 45.5% of the flocks, whereas the use of foot bath associated to deficient lime management increased the number of positive flocks, although with no statiscal significance. The evaluated parameters were not significantly associated with Campylobacter colonization in the assessed broiler flocks.