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A salmonelose é uma das mais importantes causas de toxinfecção alimentar em humanos, provocando sérios problemas à saúde pública, além de ser uma das doenças de maior impacto na produção avícola. Em vista disso, este estudo objetivou avaliar o uso de ácido fórmico no controle de Salmonella Enteritidis (SE). Assim, foram utilizados 360 pintinhos de um dia de idade: 50% das aves foram inoculadas via inglúvio com 3x107 UFC/0,1mL de SE e 50% não (aves contactantes). As aves foram divididas em cinco (05) grupos com seis (06) repetições cada: T1) controle sem o aditivo; T2) ácido fórmico 0,3% na ração; T3) ácido fórmico 0,2% na ração; T4) ácido fórmico diluído em água de beber até que a mesma atingisse um pH 4; T5) ácido fórmico 0,2% protegido na ração. As aves receberam ácido do 1º ao 7º dia e do 35º ao 42º dia. A cada sete dias, amostras de fezes foram colhidas da cama em todos os grupos para se avaliar o isolamento de SE. Aos 42 dias foram colhidas à necropsia amostras de inglúvio, fígado/baço e ceco para o isolamento de SE e sangue para a avaliação sorológica. Nas aves do T1, do T2 e do T5 SE foi isolada do inglúvio, fígado/baço e ceco das aves inoculadas e das contactantes. Houve isolamento de SE no T3 apenas em inglúvio e ceco das aves inoculadas e, no T4, apenas do ceco das contactantes. Em todos os grupos SE foi reisolada do material da cama, embora não em todas as semanas. A avaliação sorológica realizada com teste comercial de ELISA revelou aves sororeagentes no T1 (inoculadas e contactantes), no T2 (inoculadas) e no T3 (contactantes). Nenhuma ave foi sororeagente no T4 e T5. A transmissão horizontal foi evidenciada em todos os grupos estudados. Os dados foram comprovados por ANOVA e teste LCD. Nos grupos T3 e T4 o ácido fórmico foi eficaz em reduzir a presença e a transmissão horizontal de SE. / Salmonellosis is one of the most common and widely distributed foodborne diseases, with tens of millions of human cases occurring worldwide every year. Furthermore, this disease is responsible to significant economic losses to the poultry industry. Endemic infection of broiler chickens with Salmonella. Enteritidis (SE) and consequential human illness has been well documented. For these reasons the aim of this study was to evaluate the use of formic acid to control SE in broilers experimentally infected. Thus 360 day-old chicks were used in the experiment: 50% (180) of birds were inoculated in crop with 3x107CFU/0.1 mL of SE and 50% (180) were used as controls and kept in contact with the infected chicks. For the formic acid treatment the birds were divided into five (05) groups with six (06) replicates as follows: T1) control without additive; T2) 0.3% formic acid in the diet ; T3) 0.2% formic acid in the diet ; T4) formic acid diluted in drinking until it reached pH 4 water; T5) 0.2% formic acid microencapsulated in the food. The birds received the formic acid from the 1st to 7th day and from the 35th to 42nd days. Every seven days faeces samples were collected from the chick\'s litter and SE isolation was attempted. In the 42 day blood samples were collected for serological evaluation by means of Elisa. Additionally samples from the crop, liver/spleen and cecum were collected at necropsy and submitted to culture for SE detection. SE was isolated from crop, liver/spleen and cecum of the inoculated and contacts birds belonging to the T1, T2 and T5. Regarding the T3, SE was isolated in the crop and cecum of the inoculated birds and in the T4 only in the contacts chicks cecum. In all groups SE was isolated from the litter, although not in every week. Serological evaluation revealed serum positive birds in T1 (inoculated and contacts), T2 (inoculated) and T3 (contacts). Antibodies anti-SE were not detected in the serum from the birds of T4 and T5. Horizontal transmission was observed in all groups. The data were confirmed by ANOVA and LCD test. In groups T3 and T4 formic acid was effective in reducing the presence and horizontal transmission of SE.

A salmonelose é uma das mais importantes causas de toxinfecção alimentar em humanos, provocando sérios problemas à saúde pública, além de ser uma das doenças de maior impacto na produção avícola. Em vista disso, este estudo objetivou avaliar o uso de ácido fórmico no controle de Salmonella Enteritidis (SE). Assim, foram utilizados 360 pintinhos de um dia de idade: 50% das aves foram inoculadas via inglúvio com 3x107 UFC/0,1mL de SE e 50% não (aves contactantes). As aves foram divididas em cinco (05) grupos com seis (06) repetições cada: T1) controle sem o aditivo; T2) ácido fórmico 0,3% na ração; T3) ácido fórmico 0,2% na ração; T4) ácido fórmico diluído em água de beber até que a mesma atingisse um pH 4; T5) ácido fórmico 0,2% protegido na ração. As aves receberam ácido do 1º ao 7º dia e do 35º ao 42º dia. A cada sete dias, amostras de fezes foram colhidas da cama em todos os grupos para se avaliar o isolamento de SE. Aos 42 dias foram colhidas à necropsia amostras de inglúvio, fígado/baço e ceco para o isolamento de SE e sangue para a avaliação sorológica. Nas aves do T1, do T2 e do T5 SE foi isolada do inglúvio, fígado/baço e ceco das aves inoculadas e das contactantes. Houve isolamento de SE no T3 apenas em inglúvio e ceco das aves inoculadas e, no T4, apenas do ceco das contactantes. Em todos os grupos SE foi reisolada do material da cama, embora não em todas as semanas. A avaliação sorológica realizada com teste comercial de ELISA revelou aves sororeagentes no T1 (inoculadas e contactantes), no T2 (inoculadas) e no T3 (contactantes). Nenhuma ave foi sororeagente no T4 e T5. A transmissão horizontal foi evidenciada em todos os grupos estudados. Os dados foram comprovados por ANOVA e teste LCD. Nos grupos T3 e T4 o ácido fórmico foi eficaz em reduzir a presença e a transmissão horizontal de SE. / Salmonellosis is one of the most common and widely distributed foodborne diseases, with tens of millions of human cases occurring worldwide every year. Furthermore, this disease is responsible to significant economic losses to the poultry industry. Endemic infection of broiler chickens with Salmonella. Enteritidis (SE) and consequential human illness has been well documented. For these reasons the aim of this study was to evaluate the use of formic acid to control SE in broilers experimentally infected. Thus 360 day-old chicks were used in the experiment: 50% (180) of birds were inoculated in crop with 3x107CFU/0.1 mL of SE and 50% (180) were used as controls and kept in contact with the infected chicks. For the formic acid treatment the birds were divided into five (05) groups with six (06) replicates as follows: T1) control without additive; T2) 0.3% formic acid in the diet ; T3) 0.2% formic acid in the diet ; T4) formic acid diluted in drinking until it reached pH 4 water; T5) 0.2% formic acid microencapsulated in the food. The birds received the formic acid from the 1st to 7th day and from the 35th to 42nd days. Every seven days faeces samples were collected from the chick\'s litter and SE isolation was attempted. In the 42 day blood samples were collected for serological evaluation by means of Elisa. Additionally samples from the crop, liver/spleen and cecum were collected at necropsy and submitted to culture for SE detection. SE was isolated from crop, liver/spleen and cecum of the inoculated and contacts birds belonging to the T1, T2 and T5. Regarding the T3, SE was isolated in the crop and cecum of the inoculated birds and in the T4 only in the contacts chicks cecum. In all groups SE was isolated from the litter, although not in every week. Serological evaluation revealed serum positive birds in T1 (inoculated and contacts), T2 (inoculated) and T3 (contacts). Antibodies anti-SE were not detected in the serum from the birds of T4 and T5. Horizontal transmission was observed in all groups. The data were confirmed by ANOVA and LCD test. In groups T3 and T4 formic acid was effective in reducing the presence and horizontal transmission of SE.

A doença decorrente da infecção por Salmonella é um dos maiores problemas de saúde no mundo em termos de morbidade e mortalidade. Entre as sorovariedades de Salmonella, a sorovariedade Enteritidis é a de maior ocorrência mundial e compreende linhagens que tem seu nicho biológico relacionado a frangos e ovos. Várias metodologias de tipagem fenotípicas e genotípicas foram desenvolvidas a fim de se delinear a epidemiologia das infecções por S. Enteritidis. Entretanto a tipagem fenotípica usualmente falha em discriminar linhagens relacionadas das nãorelacionadas epidemiologicamente e apresenta problemas de reprodutibilidade que foram minimizados com a utilização de métodos genotípicos. No Brasil, poucos estudos que utilizaram técnicas moleculares na tipagem de linhagens dessa sorovariedade foram realizados. Os objetivos desse estudo foram investigar o potencial patogênico, a resistência a antimicrobianos e realizar a tipagem molecular de linhagens de Salmonella Enteritidis isoladas de humanos, de alimentos e de frangos no Brasil. Para isso foram estudadas 188 linhagens de Salmonella Enteritidis isoladas de surtos e de casos esporádicos, de humanos (67) de alimentos (61) e de frangos (60), durante o período de 1986 a 2010, de vários locais do Brasil. A susceptibilidade frente a 14 antimicrobianos foi analisada através da técnica de disco difusão e a presença de 13 genes de virulência das ilhas de patogenicidade de Salmonella I e II e do plasmídio pSEV foram pesquisados por PCR. Os mecanismos de resistência a quinolonas foram verificados através da pesquisa de genes de resistência plasmidiais e cromossomais e também através da verificação de mutações no gene gyrA por High resolution melting analysis (HRMA) seguida de sequenciamento de algumas linhagens. As linhagens também foram tipadas molecularmente pelas metodologias Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) com a enzima XbaI, Multilocus variable-number tandem repeat analysis (MLVA) e por Multilocus sequence typing (MLST). Das 188 linhagens estudadas, 42,5% foi resistente ao ácido nalidíxico e somente 0,5% foi resistente a sulfametoxazol-trimetoprima e estreptomicina. A resistência a quinolonas foi relacionada principalmente a mutações no gene gyrA. A maioria das linhagens estudadas (98,4%) apresentou todos os genes de virulência pesquisados, sendo uma linhagem negativa para o gene sipA e duas linhagens negativas para o gene prot6E. ERIC-PCR dividiu as 128 linhagens isoladas de humanos e alimentos em 55 perfis diferentes com similaridade >79,7%. PFGE dividiu essas mesmas linhagens em 68 perfis diferentes com uma similaridade >73,1%. Para as linhagens isoladas de frango, o dendrograma concatenado de ERIC-PCR e PFGE dividiu as 60 linhagens em dois grandes grupos com 73,3% de similaridade. O grupo A consistiu de linhagens isoladas tanto de material clínico de frangos (23) quanto do ambiente da granja (5) com 81,2% de similaridade. O grupo B também consistiu de linhagens isoladas tanto de casos clínicos de frangos (21) quanto do ambiente da granja (11) com 81,1% de similaridade. MLVA dividiu as 188 linhagens isoladas no Brasil e outras 100 linhagens isoladas na América do Norte em dois grandes grupos. O grupo MLVA-A apresentou 71 linhagens isoladas na América do Norte e somente três linhagens isoladas no Brasil. Essas linhagens do ii Brasil incluíram as isoladas antes do início da pandemia de S. Enteritidis se iniciar no país. Em contraste, o grupo MLVA-B agrupou 185 linhagens isoladas no Brasil e 29 linhagens isoladas na América do Norte. As linhagens presentes no grupo A, foram divididas em 34 tipos genéticos diferentes com similaridade maior do que 46%, enquanto no grupo B as linhagens se diferenciaram em 15 tipos genéticos diferentes com mais de 66% de similaridade. MLST caracterizou 44 das 46 linhagens estudadas como pertencentes ao ST 11. As outras duas linhagens apresentaram alelos que não existiam no banco de dados e caracterizaram dois novos STs, o 1632 e o 1633. Os resultados de tipagem molecular obtidos por ERICPCR, PFGE e MLVA no presente estudo, demonstraram uma alta similaridade genotípica entre linhagens de S. Enteritidis isoladas no Brasil, o que sugere que as linhagens estudadas descendem de um precursor comum que pouco se diferenciou genotipicamente ao longo de 24 anos no país. Ademais, os resultados de MLVA sugerem que um novo e prevalente subtipo foi introduzido no Brasil após 1993 e tem contaminado alimentos e infectado humanos e animais. O grande número de genes de virulência encontrados reforça o potencial das mesmas causarem doenças em humanos e animais, bem como, os riscos de sua presença em alimentos. Ademais, a grande porcentagem de linhagens resistentes ao ácido nalidíxico observadas a partir de 1996 sugere o uso de quinolonas no tratamento de infecções em animais causadas por S. Enteritidis no Brasil. / The disease caused of the infection by Salmonella is one of the major health problem worldwide in terms of morbid and mortality. Among the Salmonella serovars, the Enteritidis is the most frequent isolated one and comprises strains that have their biological niche related to chickens and eggs. Several phenotypic and genotypic methodologies were developed to trace epidemiologically the infections by S. Enteritidis. However, the phenotypic typing usually fail to discriminate related from unrelated epidemiologicaly strains and presents problems of reproducibility that were minimized with the introduction of genotypic methods. In Brazil, few studies that used molecular typing techniques to type strains of this serovar were conducted. The aims of this study were to investigate the pathogenic potential, the antimicrobial resistance and to molecularly type of Salmonella Enteritidis strains isolated from humans, food and chickens in Brazil. For this, it was studied 188 strains of Salmonella Enteritidis isolated from outbreaks and sporadic cases, from humans (67), food (61) and chickens (60), during the period of 1986 to 2010, from various places of Brazil. The susceptibility to 14 antimicrobials were analyzed by the disc diffusion technique and the presence of 13 virulence genes of the Salmonella pathogenicity islands I and II and from the pSEV plasmid were searched by PCR. The mechanisms of resistance to quinolones were verified by the search of plasmidial and cromossomal resistance genes and also by the verification of mutations in the gyrA gene by High resolution melting analysis (HRMA) followed by sequencing of some strains. The strains were also molecularly typed by the methodologies Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) using the enzyme XbaI, Multilocus variable-number tandem repeat analysis (MLVA) and by Multilocus sequence typing (MLST). From the 188 strains studied, 42.5% were resistant to nalidixic acid and only 0.5% were resistant to sulfamethoxazoletrimethoprim and streptomycin. Resistance to quinolones was related mainly to mutations in the gyrA gene. The majority of the strains studied (98.4%) harbored all the virulence genes searched, being only one strain negative for the sipA gene and two strains negative for the prot6E gene. ERIC-PCR divided the 128 strains isolated from humans and food in 55 different profiles with >79.7% of similarity. PFGE divided the same strains in 68 different profiles with a similarity of >73.1%. Regarding the strains isolated from chickens, the concatenated dendrogram of ERIC-PCR and PFGE divided the 60 strains in two major groups with a similarity of 73.3%. Group A consisted of strains isolated either from chicken\'s clinical samples (23) or from the farm environment (5) with a similarity of 81.2%. Group B also consisted of strains isolated either from chicken\'s clinical samples (21) or from the environment (11) with a similarity of 81.1%. MLVA divided the 188 strains isolated in Brazil and other 100 strains isolated from North America in two major groups. MLVA-A group consisted of 71 strains isolated in North America and only three strains isolated in Brazil. These strains from Brazil included the ones isolated before the beginning of the pandemic of S. Enteritidis in this country. In contrast, MLVA-B group clustered 185 strains isolated in Brazil and 29 strains isolated in North America. The strains in the MLVA-A group were divided in 34 different genotypic types with a similarity of 46%, while strains in iv the group B were divided in 15 different genotypic types with a similarity of 66%. MLST characterized 44 of the 46 strains studied as belonging to ST 11. The other two strains presented new alleles that characterized two new STs, the 1632 and the 1633. The results of molecular typing obtained by ERIC-PCR, PFGE and MLVA in this study showed a high genotypic similarity among S. Enteritidis strains isolated in Brazil, which suggests that the strains studied descend from a common ancestor that differed little genotypically during 24 years in the country. Moreover, the results of MLVA suggest that a new and prevalent subtype was introduced in Brazil after 1993 and has been contaminating food and infecting humans and animals. The high prevalence of virulence genes found in the strains studied reinforce their potential to cause disease in humans and animals, as well as the risks of their presence in food. Moreover, the high percentage of strains resistant to nalidixic acid observed after 1996 suggests the use of quinolones in the treatment of animal infections by S. Enteritidis in Brazil.

A doença decorrente da infecção por Salmonella é um dos maiores problemas de saúde no mundo em termos de morbidade e mortalidade. Entre as sorovariedades de Salmonella, a sorovariedade Enteritidis é a de maior ocorrência mundial e compreende linhagens que tem seu nicho biológico relacionado a frangos e ovos. Várias metodologias de tipagem fenotípicas e genotípicas foram desenvolvidas a fim de se delinear a epidemiologia das infecções por S. Enteritidis. Entretanto a tipagem fenotípica usualmente falha em discriminar linhagens relacionadas das nãorelacionadas epidemiologicamente e apresenta problemas de reprodutibilidade que foram minimizados com a utilização de métodos genotípicos. No Brasil, poucos estudos que utilizaram técnicas moleculares na tipagem de linhagens dessa sorovariedade foram realizados. Os objetivos desse estudo foram investigar o potencial patogênico, a resistência a antimicrobianos e realizar a tipagem molecular de linhagens de Salmonella Enteritidis isoladas de humanos, de alimentos e de frangos no Brasil. Para isso foram estudadas 188 linhagens de Salmonella Enteritidis isoladas de surtos e de casos esporádicos, de humanos (67) de alimentos (61) e de frangos (60), durante o período de 1986 a 2010, de vários locais do Brasil. A susceptibilidade frente a 14 antimicrobianos foi analisada através da técnica de disco difusão e a presença de 13 genes de virulência das ilhas de patogenicidade de Salmonella I e II e do plasmídio pSEV foram pesquisados por PCR. Os mecanismos de resistência a quinolonas foram verificados através da pesquisa de genes de resistência plasmidiais e cromossomais e também através da verificação de mutações no gene gyrA por High resolution melting analysis (HRMA) seguida de sequenciamento de algumas linhagens. As linhagens também foram tipadas molecularmente pelas metodologias Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) com a enzima XbaI, Multilocus variable-number tandem repeat analysis (MLVA) e por Multilocus sequence typing (MLST). Das 188 linhagens estudadas, 42,5% foi resistente ao ácido nalidíxico e somente 0,5% foi resistente a sulfametoxazol-trimetoprima e estreptomicina. A resistência a quinolonas foi relacionada principalmente a mutações no gene gyrA. A maioria das linhagens estudadas (98,4%) apresentou todos os genes de virulência pesquisados, sendo uma linhagem negativa para o gene sipA e duas linhagens negativas para o gene prot6E. ERIC-PCR dividiu as 128 linhagens isoladas de humanos e alimentos em 55 perfis diferentes com similaridade >79,7%. PFGE dividiu essas mesmas linhagens em 68 perfis diferentes com uma similaridade >73,1%. Para as linhagens isoladas de frango, o dendrograma concatenado de ERIC-PCR e PFGE dividiu as 60 linhagens em dois grandes grupos com 73,3% de similaridade. O grupo A consistiu de linhagens isoladas tanto de material clínico de frangos (23) quanto do ambiente da granja (5) com 81,2% de similaridade. O grupo B também consistiu de linhagens isoladas tanto de casos clínicos de frangos (21) quanto do ambiente da granja (11) com 81,1% de similaridade. MLVA dividiu as 188 linhagens isoladas no Brasil e outras 100 linhagens isoladas na América do Norte em dois grandes grupos. O grupo MLVA-A apresentou 71 linhagens isoladas na América do Norte e somente três linhagens isoladas no Brasil. Essas linhagens do ii Brasil incluíram as isoladas antes do início da pandemia de S. Enteritidis se iniciar no país. Em contraste, o grupo MLVA-B agrupou 185 linhagens isoladas no Brasil e 29 linhagens isoladas na América do Norte. As linhagens presentes no grupo A, foram divididas em 34 tipos genéticos diferentes com similaridade maior do que 46%, enquanto no grupo B as linhagens se diferenciaram em 15 tipos genéticos diferentes com mais de 66% de similaridade. MLST caracterizou 44 das 46 linhagens estudadas como pertencentes ao ST 11. As outras duas linhagens apresentaram alelos que não existiam no banco de dados e caracterizaram dois novos STs, o 1632 e o 1633. Os resultados de tipagem molecular obtidos por ERICPCR, PFGE e MLVA no presente estudo, demonstraram uma alta similaridade genotípica entre linhagens de S. Enteritidis isoladas no Brasil, o que sugere que as linhagens estudadas descendem de um precursor comum que pouco se diferenciou genotipicamente ao longo de 24 anos no país. Ademais, os resultados de MLVA sugerem que um novo e prevalente subtipo foi introduzido no Brasil após 1993 e tem contaminado alimentos e infectado humanos e animais. O grande número de genes de virulência encontrados reforça o potencial das mesmas causarem doenças em humanos e animais, bem como, os riscos de sua presença em alimentos. Ademais, a grande porcentagem de linhagens resistentes ao ácido nalidíxico observadas a partir de 1996 sugere o uso de quinolonas no tratamento de infecções em animais causadas por S. Enteritidis no Brasil. / The disease caused of the infection by Salmonella is one of the major health problem worldwide in terms of morbid and mortality. Among the Salmonella serovars, the Enteritidis is the most frequent isolated one and comprises strains that have their biological niche related to chickens and eggs. Several phenotypic and genotypic methodologies were developed to trace epidemiologically the infections by S. Enteritidis. However, the phenotypic typing usually fail to discriminate related from unrelated epidemiologicaly strains and presents problems of reproducibility that were minimized with the introduction of genotypic methods. In Brazil, few studies that used molecular typing techniques to type strains of this serovar were conducted. The aims of this study were to investigate the pathogenic potential, the antimicrobial resistance and to molecularly type of Salmonella Enteritidis strains isolated from humans, food and chickens in Brazil. For this, it was studied 188 strains of Salmonella Enteritidis isolated from outbreaks and sporadic cases, from humans (67), food (61) and chickens (60), during the period of 1986 to 2010, from various places of Brazil. The susceptibility to 14 antimicrobials were analyzed by the disc diffusion technique and the presence of 13 virulence genes of the Salmonella pathogenicity islands I and II and from the pSEV plasmid were searched by PCR. The mechanisms of resistance to quinolones were verified by the search of plasmidial and cromossomal resistance genes and also by the verification of mutations in the gyrA gene by High resolution melting analysis (HRMA) followed by sequencing of some strains. The strains were also molecularly typed by the methodologies Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) using the enzyme XbaI, Multilocus variable-number tandem repeat analysis (MLVA) and by Multilocus sequence typing (MLST). From the 188 strains studied, 42.5% were resistant to nalidixic acid and only 0.5% were resistant to sulfamethoxazoletrimethoprim and streptomycin. Resistance to quinolones was related mainly to mutations in the gyrA gene. The majority of the strains studied (98.4%) harbored all the virulence genes searched, being only one strain negative for the sipA gene and two strains negative for the prot6E gene. ERIC-PCR divided the 128 strains isolated from humans and food in 55 different profiles with >79.7% of similarity. PFGE divided the same strains in 68 different profiles with a similarity of >73.1%. Regarding the strains isolated from chickens, the concatenated dendrogram of ERIC-PCR and PFGE divided the 60 strains in two major groups with a similarity of 73.3%. Group A consisted of strains isolated either from chicken\'s clinical samples (23) or from the farm environment (5) with a similarity of 81.2%. Group B also consisted of strains isolated either from chicken\'s clinical samples (21) or from the environment (11) with a similarity of 81.1%. MLVA divided the 188 strains isolated in Brazil and other 100 strains isolated from North America in two major groups. MLVA-A group consisted of 71 strains isolated in North America and only three strains isolated in Brazil. These strains from Brazil included the ones isolated before the beginning of the pandemic of S. Enteritidis in this country. In contrast, MLVA-B group clustered 185 strains isolated in Brazil and 29 strains isolated in North America. The strains in the MLVA-A group were divided in 34 different genotypic types with a similarity of 46%, while strains in iv the group B were divided in 15 different genotypic types with a similarity of 66%. MLST characterized 44 of the 46 strains studied as belonging to ST 11. The other two strains presented new alleles that characterized two new STs, the 1632 and the 1633. The results of molecular typing obtained by ERIC-PCR, PFGE and MLVA in this study showed a high genotypic similarity among S. Enteritidis strains isolated in Brazil, which suggests that the strains studied descend from a common ancestor that differed little genotypically during 24 years in the country. Moreover, the results of MLVA suggest that a new and prevalent subtype was introduced in Brazil after 1993 and has been contaminating food and infecting humans and animals. The high prevalence of virulence genes found in the strains studied reinforce their potential to cause disease in humans and animals, as well as the risks of their presence in food. Moreover, the high percentage of strains resistant to nalidixic acid observed after 1996 suggests the use of quinolones in the treatment of animal infections by S. Enteritidis in Brazil.

In der vorliegenden Arbeit wurde einerseits der Verlauf der Schutzwirkung der zurzeit in Sachsens Legehennenbeständen überwiegend angewandten Impfschemata gegen Salmonella Enteritidis (SE) untersucht. Andererseits wurden die Impfschemata, die ausschließlich modifizierte Lebendimpfstoffe (MLV) umfassen, und Impfschemata, die aus einer Kombination von MLV und Inaktivatimpfstoffen (KV) bestehen, auf Unterschiede in der Wirksamkeit geprüft. Um die Wirksamkeit der Impfschemata im Verlauf der Legeperiode und im Vergleich miteinander untersuchen zu können, wurden zu drei verschiedenen Zeitpunkten der Legeperiode (39., 54. und 69. Lebenswoche (LW)) Infektionsversuche mit einem Nalidixinsäure-resistenten SE-Stamm durchgeführt. Es wurden insgesamt 180 Legehennen verwendet, die in fünf verschiedenen Herkunftsbetrieben etwa gleicher Größe aufgezogen und geimpft worden waren. In jedem der Herkunftsbetriebe wurde eines von fünf Impfschemata (A bis E) angewendet. Die Impfschemata A und C beinhalteten ausschließlich MLV, die Impfschemata B, D und E eine Kombination aus MLV und KV. Zu den genannten Zeitpunkten (39., 54. und 69. LW) wurden jeweils zwölf Tiere aus den Betrieben in den Infektionsstall des Instituts für Tierhygiene und Öffentliches Veterinärwesen verbracht und eingestallt. Nach der Adaptionsphase von einer Woche und der Prüfung der Tiere auf Salmonellenfreiheit wurde jedes Tier mit 1,0 x109 KbE SE oral infiziert. Zwei und sieben Tage post infectionem (p.inf). wurden jeweils sechs Hennen euthanasiert und seziert. Caeca, Leber-, Ovar- und Oviduktproben wurden entnommen und gemäß anerkannter quantitativer und qualitativer Untersuchungsmethoden auf den Infektionsstamm untersucht. Zur Kontrolle der Erregerausscheidung wurden ein, drei und fünf Tage p.inf. Kloakentupferproben von jedem Tier entnommen und entsprechend auf SE untersucht. Die Ergebnisse dieser Untersuchungen variierten in Abhängigkeit von Versuchs- und Sektions- bzw. Kloakentupferentnahmezeitpunkt, untersuchtem Organ sowie quantitativer und qualitativer Untersuchung. Die ausschließlich mit MLV geimpften Gruppen A und C wiesen im Vergleich zu den Gruppen D und E, die mit demselben MLV und zusätzlich mit einem KV geimpft worden waren, in den Kloakentupfer- und Caecumproben in der Regel qualitativ und quantitativ weniger Salmonellen auf. Der Salmonellennachweis in den Leberproben und den vereinzelt besiedelten Reproduktionsorganen variierte nur geringfügig zwischen den Impfgruppen. Da die Tiere der vier Impfgruppen aus jeweils anderen Herkunftsbetrieben stammten, sind haltungsbedingte Unterschiede anzunehmen. Das äußere Erscheinungsbild (Befiederung, Bemuskelung, makroskopische pathologische Veränderungen) und das Sozialverhalten der Tiere variierten, was mit Unterschieden in der Immunität einhergehen kann. Bei den Tieren der Gruppe A bzw. C waren in der 69. LW und in 54. LW, respektive, ein ausgeprägtes kannibalistisches Verhalten und dessen Konsequenzen (reduzierte Wasser- und Futteraufnahme der gepickten Tiere, Hackverletzungen) zu beobachten. Tendenziell waren die Tiere der Gruppen B bis E in der 54. LW stärker mit SE belastet als in der 39. und 69. LW. Ein Einfluss der unter Umständen erhöhten Umgebungstemperaturen in den Betrieben sowie des hohen Leistungsstresses während der mittleren Phase der Legeperiode auf die Immunität der 54 LW alten Tiere, die im August 2010 infiziert wurden, ist nicht auszuschließen. Auch eine sich ausbildende Altersresistenz könnte die bessere Salmonellenabwehr der 69 LW alten Tiere erklären. Tiere der Gruppe A waren jedoch in der 69. LW am stärksten mit SE belastet, was auf ein Nachlassen der durch die Impfung induzierten Immunität und möglicherweise auf den im Vergleich zu den jüngeren Tieren allgemein schwächeren Zustand der Tiere zurückzuführen ist. Die Ergebnisse einer Kontrollgruppe zur Beurteilung der von der Impfung unabhängigen Faktoren fehlen. Da in den sächsischen Legehennenhaltungen aufgrund der hohen Tierzahlen entsprechend der Hühner-Salmonellen Verordnung (Impfpflicht für Betriebe mit mehr als 350 Tieren) gegen SE geimpft wird, hätten zur Bildung einer ungeimpften Kontrollgruppe Tiere aus kleineren Betrieben oder Zuchttierhaltungen mit völlig anderen Haltungsstrukturen verwendet werden müssen. Damit wären die wissenschaftlichen Ansprüche an eine Kontrollgruppe jedoch nicht erfüllt worden. Entsprechend der Ergebnisse und Umstände dieser Studie scheint eine Zusatzimpfung mit einem KV im Vergleich zu einer Impfung mit ausschließlich MLV keinen Vorteil im Schutz vor SE zu bieten. Es konnte gezeigt werden, dass die Immunität der gemäß der Impfschemata B bis E geimpften Legehennen gegen SE am Ende der Legeperiode nicht nachlässt. Die Impfung allein kann den Erreger nicht eliminieren und muss daher stets in ein verantwortungsvolles und vielseitiges Bekämpfungsprogramm integriert werden.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study work was to evaluate the effect of two organic acids (formic acid and propionic acid) and of one mannanoligosaccharide added to the diet to control the intestinal infection caused by Salmonella Enteritidis in broilers. In these 39 days of study it was used 150 birds, 1-day-old, of the Cobb lineage, both sexes and free of Salmonella Enteritidis. They were divided in 6 different treatments (T) with 25 birds each, where: T1 (diet and no infection), T2 (diet + organic acids + no infection), T3 (diet + organic acids + mannanoligosaccharide and no infection), T4 (diet + organic acids and infection with Salmonella Enteritidis) T5 (diet + organic acids + mannanoligosaccharide + and infection with Salmonella Enteritidis) T6 (diet and infection with Salmonella Enteritidis). After housing, the chicken litter was instilled on the 4th day with Salmonella Enteritidis and every seven days, five birds from each group were killed through cervical dislocation. The necropsy was performed and also the bacteriological exams to detect Salmonella Enteritidis using the feces collected over the chicken litter of the groups bacteriological analysis of the cecal tonsils was done as well. On the 18th day only 60% of birds were infected in treatments T4 and T5; on the 25th day, 40% of birds in T4 and 20% in the T5 were infected; on the 32nd day, 100% of tested samples were negative in both treatments. The T6 group was 100% positive until the 32nd day, but on the 39th day, it got reduced in 20% of the number of infected animals. In the experimental conditions of this study, the organic acids and the mannanoligosaccharide added to the diet possibly contributted to control the infection caused by Salmonella Enteritidis on tested birds. / O objetivo deste estudo foi avaliar a ação de dois ácidos orgânicos (ácido fórmico e ácido propiônico) e de um mananoligossacarídeo (MOS) adicionados à dieta no controle da infecção intestinal por Salmonella Enteritidis (SE) em frangos de corte. Neste estudo de 39 dias foram utilizadas 150 aves, de um dia de idade, da linhagem Cobb, lote misto, livre de SE, divididos em seis tratamentos (T) com 25 animais cada, onde: T1 (dieta e ausência de infecção), T2 (dieta + ácidos orgânicos e ausência de infecção), T3 (dieta + ácidos orgânicos + MOS e ausência de infecção), T4 (dieta + ácidos orgânicos e infecção com SE), T5 (dieta + ácidos orgânicos + MOS e infecção com SE) e T6 (dieta e infecção com SE). No 4º dia após o alojamento, a cama foi instilada, com SE e, a cada sete dias, cinco aves por grupo foram submetidas à eutanásia por deslocamento cervical, necropsiadas e realizados os exames bacteriológicos para SE, utilizando-se fezes coletadas sobre a cama de maravalha dos grupos, e das tonsilas cecais dos animais necropsiados. No 18º dia, somente 60% das aves estavam infectadas nos tratamentos T4 e T5; no 25º dia, 40% das aves no T4 e 20% no T5 estavam infectadas; no 32º dia 100% das amostras testadas foram negativos em ambos os tratamentos (T4 e T5). Constatou-se que o T6 foi 100% positivo até o 32º dia, e no 39º dia reduziu em 20% o número de animais infectados. Dentro dos parâmetros de avaliação deste experimento, os ácidos orgânicos e o mananoligossacarídeo adicionados à dieta, possivelmente contribuíram no controle da infecção por SE nas aves testadas.

Made available in DSpace on 2014-07-29T15:07:29Z (GMT). No. of bitstreams: 1 Dissertacao_Dunya_Moraes.pdf: 432826 bytes, checksum: 63ffc1c5c83c7f760eb90cca2350b35f (MD5) Previous issue date: 2010-03-26 / The objective of this research to investigate the presence of Salmonella sp. in raw materials of animal origin used in the manufacture of feed for broilers, in diets collected directly from bird feeders, and in organs ceca contents and liners carry case for newly hatched chicks, in environmental samples, in samples from swabs of the hands of officials of the farm and slaughterhouse samples and classify and determine the resistance of strains of Salmonella sp. found, before the action of chemotherapeutic nine. For data analysis was descriptive frequency results. 1200 samples were collected from flour and Salmonella sp. was found in 10.5% of samples with a predominance of serovar Enteritidis. The frequency of bacteria in meat meal was 12%, 6.8% in blood, the feathers of 4.3% and 14.6% in the viscera. Were also isolated Salmonella Cerro, Salmonella Montevideo, Salmonella anatum, Salmonella Tennessee, and Salmonella typhimurium among others. Regarding the resistance of strains found in the various categories of flour was observed resistance to sulfonamides, neomycin, tetracycline, sulphamethoxazole-to trimetopim and florfenicol. Of the three strains of bacteria isolated from two diets were of a Salmonella enteritidis and S. Anatum, showing resistance to sulfonamides and neomycin. Of the 32 batches of newly hatched chicks 9.4% were positive for Salmonella and 32 batches of liners carrying case 9.4%. Environmental samples, before bed accommodation, swabs of feeder and drinker and the drinking water of birds tested negative for Salmonella sp .. Drag swabs of poultry manure, and samples Alphitobius diaperinus swabs from the hands of officials of the farms had a frequency of 12.5%, 12.5% and 6.5% respectively. In samples from swabs of drag serovar Enteritidis was the most frequent and catfishes of the samples and swabs of hands was the only serovar isolated. In samples from slaughterhouse 26.7% of the lots from crop and 33.3% of batches of ceca were positive for Salmonella sp .. Regarding the resistance of strains, there was resistance sulfonamides, the amoxicillin and enrofloxacin in samples of newly hatched chicks and amoxicillin in the samples of liners carrying case. In drag swabs and samples of catfishes, the bacteria were resistant to sulfonamides. In samples from crop, were resistant to sulfonamide and enrofloxacin and the caeca to sulfonamides, the trimetopim-sulphamethoxazole, tetracycline, amoxicillin and ampicillin to. / Objetivou-se com esta pesquisa investigar a presença de Salmonella sp. em matérias primas de origem animal utilizadas na fabricação de rações de frangos de corte, em rações coletadas diretamente dos comedouros das aves, em órgãos e conteúdos de cecos e forros de caixa de transporte de pintos de um dia, em amostras ambientais, em amostras de suabes de mãos de funcionários de granjas e em amostras de abatedouro bem como tipificar e determinar o perfil de resistência das cepas de Salmonella sp. encontradas, frente à ação de nove quimioterápicos. Para análise dos dados foi feita freqüência descritiva dos resultados encontrados. Coletou-se 1200 amostras de farinhas e Salmonella sp. foi encontrada em 10,5% das amostras com predominância do sorovar Enteritidis. A freqüência da bactéria em farinhas de carne foi de 12%, nas de sangue 6,8%, nas de penas 4,3% e nas de vísceras 14,6%. Foram isoladas também Salmonella Cerro, Salmonella Montevideo, Salmonella Anatum, Salmonella Tennessee e Salmonella Typhimurium entre outras. Em relação ao perfil de resistência das cepas encontradas nas diversas categorias de farinhas foi observado resistência à sulfonamidas, à neomicina, à tetraciclina, ao trimetopim-sulfametoxasol e ao florfenicol. Das três cepas da bactéria isoladas de rações duas eram de Salmonella Enteritidis e uma de S. Anatum, apresentando resistência à sulfonamidas e à neomicina. Dos 32 lotes de pintos de um dia 9,4% apresentaram positividade para Salmonella e dos 32 lotes de forros de caixa de transporte 9,4%. As amostras ambientais, cama antes do alojamento, suabes de comedouro e de bebedouro e água de bebida das aves apresentaram resultados negativos para Salmonella sp.. Suabes de arrasto de cama de aviários, amostras de Alphitobius diaperinus e suabes de mãos de funcionários das granjas apresentaram freqüência de 12,5%, 12,5% e 6,5% respectivamente. Nas amostras de suabes de arrasto o sorovar Enteritidis foi o mais freqüente e nas amostras de cascudinhos e suabes de mãos foi o único sorovar isolado. Nas amostras de abatedouro 26,7% dos lotes de inglúvio e 33,3% dos lotes de cecos foram positivos para Salmonella sp.. Com relação ao perfil de resistência das cepas isoladas, observou-se resistência à sulfonamidas, à amoxacilina e à enrofloxacina nas amostras de pintos de um dia e à amoxacilina nas amostras de forros de caixa de transporte. Nos suabes de arrasto e amostras de cascudinhos, as bactérias foram resistentes à sulfonamidas. Nas amostras de inglúvios, foi observada resistência à sulfonamidas e à enrofloxacina e nos cecos à sulfonamidas, ao trimetopim-sulfametoxasol, à tetraciclina, à amoxacilina e à ampicilina.

In der vorliegenden Arbeit wurde einerseits der Verlauf der Schutzwirkung der zurzeit in Sachsens Legehennenbeständen überwiegend angewandten Impfschemata gegen Salmonella Enteritidis (SE) untersucht. Andererseits wurden die Impfschemata, die ausschließlich modifizierte Lebendimpfstoffe (MLV) umfassen, und Impfschemata, die aus einer Kombination von MLV und Inaktivatimpfstoffen (KV) bestehen, auf Unterschiede in der Wirksamkeit geprüft. Um die Wirksamkeit der Impfschemata im Verlauf der Legeperiode und im Vergleich miteinander untersuchen zu können, wurden zu drei verschiedenen Zeitpunkten der Legeperiode (39., 54. und 69. Lebenswoche (LW)) Infektionsversuche mit einem Nalidixinsäure-resistenten SE-Stamm durchgeführt. Es wurden insgesamt 180 Legehennen verwendet, die in fünf verschiedenen Herkunftsbetrieben etwa gleicher Größe aufgezogen und geimpft worden waren. In jedem der Herkunftsbetriebe wurde eines von fünf Impfschemata (A bis E) angewendet. Die Impfschemata A und C beinhalteten ausschließlich MLV, die Impfschemata B, D und E eine Kombination aus MLV und KV. Zu den genannten Zeitpunkten (39., 54. und 69. LW) wurden jeweils zwölf Tiere aus den Betrieben in den Infektionsstall des Instituts für Tierhygiene und Öffentliches Veterinärwesen verbracht und eingestallt. Nach der Adaptionsphase von einer Woche und der Prüfung der Tiere auf Salmonellenfreiheit wurde jedes Tier mit 1,0 x109 KbE SE oral infiziert. Zwei und sieben Tage post infectionem (p.inf). wurden jeweils sechs Hennen euthanasiert und seziert. Caeca, Leber-, Ovar- und Oviduktproben wurden entnommen und gemäß anerkannter quantitativer und qualitativer Untersuchungsmethoden auf den Infektionsstamm untersucht. Zur Kontrolle der Erregerausscheidung wurden ein, drei und fünf Tage p.inf. Kloakentupferproben von jedem Tier entnommen und entsprechend auf SE untersucht. Die Ergebnisse dieser Untersuchungen variierten in Abhängigkeit von Versuchs- und Sektions- bzw. Kloakentupferentnahmezeitpunkt, untersuchtem Organ sowie quantitativer und qualitativer Untersuchung. Die ausschließlich mit MLV geimpften Gruppen A und C wiesen im Vergleich zu den Gruppen D und E, die mit demselben MLV und zusätzlich mit einem KV geimpft worden waren, in den Kloakentupfer- und Caecumproben in der Regel qualitativ und quantitativ weniger Salmonellen auf. Der Salmonellennachweis in den Leberproben und den vereinzelt besiedelten Reproduktionsorganen variierte nur geringfügig zwischen den Impfgruppen. Da die Tiere der vier Impfgruppen aus jeweils anderen Herkunftsbetrieben stammten, sind haltungsbedingte Unterschiede anzunehmen. Das äußere Erscheinungsbild (Befiederung, Bemuskelung, makroskopische pathologische Veränderungen) und das Sozialverhalten der Tiere variierten, was mit Unterschieden in der Immunität einhergehen kann. Bei den Tieren der Gruppe A bzw. C waren in der 69. LW und in 54. LW, respektive, ein ausgeprägtes kannibalistisches Verhalten und dessen Konsequenzen (reduzierte Wasser- und Futteraufnahme der gepickten Tiere, Hackverletzungen) zu beobachten. Tendenziell waren die Tiere der Gruppen B bis E in der 54. LW stärker mit SE belastet als in der 39. und 69. LW. Ein Einfluss der unter Umständen erhöhten Umgebungstemperaturen in den Betrieben sowie des hohen Leistungsstresses während der mittleren Phase der Legeperiode auf die Immunität der 54 LW alten Tiere, die im August 2010 infiziert wurden, ist nicht auszuschließen. Auch eine sich ausbildende Altersresistenz könnte die bessere Salmonellenabwehr der 69 LW alten Tiere erklären. Tiere der Gruppe A waren jedoch in der 69. LW am stärksten mit SE belastet, was auf ein Nachlassen der durch die Impfung induzierten Immunität und möglicherweise auf den im Vergleich zu den jüngeren Tieren allgemein schwächeren Zustand der Tiere zurückzuführen ist. Die Ergebnisse einer Kontrollgruppe zur Beurteilung der von der Impfung unabhängigen Faktoren fehlen. Da in den sächsischen Legehennenhaltungen aufgrund der hohen Tierzahlen entsprechend der Hühner-Salmonellen Verordnung (Impfpflicht für Betriebe mit mehr als 350 Tieren) gegen SE geimpft wird, hätten zur Bildung einer ungeimpften Kontrollgruppe Tiere aus kleineren Betrieben oder Zuchttierhaltungen mit völlig anderen Haltungsstrukturen verwendet werden müssen. Damit wären die wissenschaftlichen Ansprüche an eine Kontrollgruppe jedoch nicht erfüllt worden. Entsprechend der Ergebnisse und Umstände dieser Studie scheint eine Zusatzimpfung mit einem KV im Vergleich zu einer Impfung mit ausschließlich MLV keinen Vorteil im Schutz vor SE zu bieten. Es konnte gezeigt werden, dass die Immunität der gemäß der Impfschemata B bis E geimpften Legehennen gegen SE am Ende der Legeperiode nicht nachlässt. Die Impfung allein kann den Erreger nicht eliminieren und muss daher stets in ein verantwortungsvolles und vielseitiges Bekämpfungsprogramm integriert werden.

Salmonella enteritidis (S. enteritidis) is a bacterial pathogen of chickens, and is currently one of the leading causes of human food poisoning in the world. It is believed that contaminated poultry products, especially eggs and egg products, have been responsible for the dramatic increase in the incidence of this Salmonella serotype. Detection of S. entertidis has conventionally involved bacteriological examination of samples, yet these procedures are time-consuming which could lead to the rapid spread of S. enteritidis through commercial flocks and potentially cause a human health risk. A number of alternative detection techniques, mostly based on serological methods, have been reported as effective diagnostic assays. However, some of these reports have not been supported by representations of SDS-PAGE gels or Western blots. The objective of this study was the evaluation of these serological techniques as well as a PCR amplification technique, which has been reported to show promising results as a diagnostic method. The techniques discussed in these reports were evaluated with regards to how rapid they were, their specificity and their potential for use in local diagnostic laboratories. Antigens from the outer surface of S. enteritidis were purified by several methods and their antigenicity was tested by separating the antigens by means of SDS-PAGE, followed by Western blotting using sera of chickens infected with S. enteritidis. A high degree of cross reactivity was observed with many of the antigens tested, especially the lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) which had previously been reported as containing antigens which could be used for specific detection of S. enteritidis. This cross-reactivity could be explained by the conserved nature of many of the LPS and OMP antigens among the Salmonella serotypes tested. A fimbrial antigen, SEF14, which has been reported as a novel antigen, was seen as a prominent band at 14.3 kDa and was found to react with antibodies against S. enteritidis, yet not to the specificity levels described in previous reports. PCR amplification of the sefA gene sequence, which encodes for the SEF14 fimbrial antigen, was found to give a predicted product of 310 bp when using a previously described oligonucleotide primer pair. This amplified product was found to be specific for S. enteritidis and other serogroup D Salmonella serotypes that are not poultry pathogens The cross-reactivity observed with many of the serological techniques used in this study, meant that detection of S. enteritidis infection in chickens was considerably hindered. However, the identification of further novel antigens by serological means, could result in the development of new vaccines. The specificity and speed afforded by PCR amplification indicated that this technique showed excellent potential for use in local diagnostic laboratories. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.