Identification of the capsule type of Pasteurella multocida isolates from cases of fowl cholera by multiplex PCR and comparison with phenotypic methods

The ability of Pasteurella multocida to invade and multiply in its host is enhanced by the presence of the capsule, one of the most important virulence factors for this bacterium. Capsular typing methods are often used in epidemiological and pathogenesis studies of this agent. Five different serogroups have been identified based on serological typing. However, such tests are laborious, and agglutination of homologous antiserum may fail. The aim of this study was to develop a multiplex PCR protocol for the identification of the hyaD-hyaC and dcbF genes specific to serogroups A and D, respectively, and to compare these results with those of phenotypic tests for 54 strains isolated from fowl cholera cases in southern Brazil. The kappa coefficient and chisquare statistics were calculated to assess the agreement between the diagnostic methods and to determine the significance of the results, respectively. The multiplex PCR was able to detect the evaluated genes. Forty-nine strains (90.74%) were classified into serogroup A, and only two isolates (3.7%) were not identified as belonging to any of the serogroups analyzed. In contrast, with the phenotypic tests, only 41 strains (75.93%) were classified into serogroup A and 11 samples (20.37%) were unidentifiable. Of the strains analyzed, 70.37% were classified into the same serogroup (A) by both methods, and the kappa coefficient (k = 0.017) indicated poor agreement between the tests. Thus, multiplex PCR is an alternative for P. multocida capsular typing, as it allows the simultaneous and rapid detection of genes and also provides a greater strain-typing capacity.

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)

Interference of Salmonella typhimurium lipopolysaccharide on performance and biological parameters of broiler chickens

This study was conducted to determine the interference of Salmonella typhimurium lipopolysaccharide (sLPS) on the performance, biological parameters, and histological evaluations of 198 one-day-old male broiler chickens divided into three treatments according to sLPS dose (0, 250, or 500 µg/application/bird) that was applied to the birds every other day, from 15 to 27 days of age. At the end of the experiment (28 days), significant effects were observed on body weight (R= -0.17 and P=0.05), total cholesterol serum levels(R=0.43 and p 0.01), phosphorus (R=0.53 and P 0.01), uric acid (R= -0.38 and P 0.01), C-reactive protein (R=0.68 and p 0.01), serum activity of aspartate aminotransferase (R=0.39 and p 0.01) and alkaline phosphatase (R= -0.39 and p 0.01). According to these results, sLPS mainly affect broiler biological parameters, but also their live performance.(AU)

Identification of the capsule type of Pasteurella multocida isolates from cases of fowl cholera by multiplex PCR and comparison with phenotypic methods

The ability of Pasteurella multocida to invade and multiply in its host is enhanced by the presence of the capsule, one of the most important virulence factors for this bacterium. Capsular typing methods are often used in epidemiological and pathogenesis studies of this agent. Five different serogroups have been identified based on serological typing. However, such tests are laborious, and agglutination of homologous antiserum may fail. The aim of this study was to develop a multiplex PCR protocol for the identification of the hyaD-hyaC and dcbF genes specific to serogroups A and D, respectively, and to compare these results with those of phenotypic tests for 54 strains isolated from fowl cholera cases in southern Brazil. The kappa coefficient and chisquare statistics were calculated to assess the agreement between the diagnostic methods and to determine the significance of the results, respectively. The multiplex PCR was able to detect the evaluated genes. Forty-nine strains (90.74%) were classified into serogroup A, and only two isolates (3.7%) were not identified as belonging to any of the serogroups analyzed. In contrast, with the phenotypic tests, only 41 strains (75.93%) were classified into serogroup A and 11 samples (20.37%) were unidentifiable. Of the strains analyzed, 70.37% were classified into the same serogroup (A) by both methods, and the kappa coefficient (k = 0.017) indicated poor agreement between the tests. Thus, multiplex PCR is an alternative for P. multocida capsular typing, as it allows the simultaneous and rapid detection of genes and also provides a greater strain-typing capacity.(AU)

Isolamento de Salmonella Heidelberg em diferentes pontos da tecnologia de abate de frangos de corte / Salmonella Heidelberg isolated at different points of the broiler slaughterhouse

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. (AU)

SALMONELLA HEIDELBERG ISOLATED AT DIFFERENT POINTS OF THE BROILER SLAUGHTERHOUSE. 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. (AU)

Isolamento de Salmonella Heidelberg em diferentes pontos da tecnologia de abate de frangos de corte / Salmonella Heidelberg isolated at different points of the broiler slaughterhouse

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.

SALMONELLA HEIDELBERG ISOLATED AT DIFFERENT POINTS OF THE BROILER SLAUGHTERHOUSE. 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.

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.

Resistência antimicrobiana em salmonella enterica subsp. enterica sorovar hadar isoladas de carcaças de frango

ABSTRACT Antimicrobial resistance is a widely studied issue in all bacterial genera, but in the case of those responsible for zoonotic illnesses, like Salmonella, they merit special attention, because it can be transmitted via foodstuffs to human beings. The present study was carried out to verify the occurrence of antimicrobial resistance in 22 samples of Salmonella Hadar isolated from broiler chicken carcasses in the State of Rio Grande do Sul, Brazil, from May 1995 to April 1996. In the present study, the results indicated that 100% of the Salmonella Hadar isolates were resistant to tetracycline, streptomycin and sulfazotrim, while intermediate resistance was observed at different levels, to nalidixic acid (86.36%), nitrofurantoin (18.18%) and chloramphenicol (4.54%). All isolates were resistant to three or more antimicorbials agents, with five different patterns of resistance. These levels of resistance highlight the need for responsible use of antimicrobial agents in animal production.

RESUMO A resistência antimicrobiana é um assunto amplamente estudado em todos os gêneros bacterianos, sobretudo em relação aos responsáveis por zoonoses, como o caso da Salmonella, a qual merece especial atenção, já que pode ser transmitida para os seres humanos. O presente estudo foi conduzido para verificar a ocorrência de resistência a agentes antimicrobianos em 22 cepas de Salmonella Hadar isoladas de carcaças congeladas de frango no Estado do Rio Grande do Sul, no período de maio de 1995 a abril de 1996. No presente estudo, os resultados indicaram que 100% das cepas de Salmonella Hadar apresentaram resistência a tetraciclina, estreptomicina e sulfazotrim, tendo também apresentado resistência em diferentes níveis ao ácido nalidíxico (86,36%), nitrofurantoína (18,18%) e cloranfenicol (4,54%). Todas as cepas de Salmonella Hadar apresentaram resistência a três ou mais agentes antimicrobianos, com cinco diferentes padrões de resistência. Os níveis de resistência observados enfatizam a necessidade do uso responsável dos agentes antimicrobianos na produção animal.