Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

Analysis of colostrum IgA against bacteria involved in neonatal infections / Análise de IgA do colostro contra bactérias envolvidas em infecções neonatais

ABSTRACT Objective To describe e compare the specificity of IgA antibodies against bacteria extract of Klebsiella pneumoniae , Staphylococcus aureus , Escherichia coli , and Salmonella enteritidis . Methods Colostrum samples were aseptically collected in the first 12 hours after C-section delivery. The specificity of IgA against bacteria extracts was analyzed by the Western blot. Results The findings showed proteins of high molecular weight frequently detectable in the samples. S. aureus was the most frequently found bacterium in the samples (p<0.05). Approximately 93.8, 56.3, 62.5 and 60.4% of samples presented IgA reactive to S. aureus , K. pneumoniae , S. enteritidis, and E. coli, respectively. Roughly 40% of samples showed no IgA reactive to K. pneumoniae, S. enteritidis and E. coli . Conclusion Clinical evidence of the importance of breastfeeding for the immune protection of neonates was consistent with the observed immunological findings, since most samples showed IgA reactive against the species tested. The application and development of immunotherapies during pregnancy, focused on frequently detected antigens, could be an important tool to enhance the presence of IgA in colostrum.

RESUMO Objetivo Descrever e comparar a especificidade de anticorpos IgA de amostras de colostro contra extratos bacterianos de Klebsiella pneumoniae , Staphylococcus aureus , Escherichia coli e Salmonella enteritidis . Métodos As amostras de colostro foram coletadas assepticamente nas primeiras 12 horas após o nascimento por cesariana. A especificidade de IgA contra extratos de bactérias foi analisada por Western blot. Resultados Os achados mostraram proteínas de alto peso molecular frequentemente detectáveis nas amostras. S. aureus foi a bactéria mais encontrada nas amostras (p<0,05). Cerca de 93,8, 56,3, 62,5 e 60,4% das amostras apresentaram IgA reativa a S. aureus , K. pneumoniae , S. enteritidis e E. coli , respectivamente. Aproximadamente 40% das amostras não apresentaram IgA reativa contra K. pneumoniae , S. enteritidis e E. coli. Conclusão A evidência clínica da importância da amamentação para proteção imunológica ao recém-nascido foi consistente com os achados imunológicos observados, uma vez que a maioria das amostras mostrou IgA reativa contra as espécies testadas. A aplicação e o desenvolvimento de imunoterapias durante a gestação, focada nos antígenos frequentemente detectados, poderiam ser importantes instrumentos para aumentar a presença de IgA no colostro.

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens.

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with nonmotile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens. Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin. Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

Atividade antibacteriana in vitro de inflorescências de Achyrocline satureioides (Lam.) DC. - Asteraceae ("macela", "marcela") sobre agentes bacterianos de interesse em alimentos / In vitro antibacterial activity of inflorescences of Achyroclines satureioides (Lam.) DC. - Asteraceae ("macela", "marcela") on bacterial agents of interest in food

Através de Testes de diluição em sistema de tubos múltiplos determinou-se in vitro, atividade antibacteriana em inflorescências de Achyrocline satureioides (Lam.) DC. - Asteraceae ("macela", "marcela"), expressa como Intensidade de Atividade de Inibição Bacteriana (IINIB/bacteriostasia) e Intensidade de Atividade de Inativação Bacteriana (IINAB/bactericidia), a partir de formas de extração etanólica (hidroalcoolaturas) e hídrica (decoctos), sobre inóculos padronizados de Enterococcus faecalis (ATCC 19433), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 11229) e Salmonella enteritidis (ATCC 11076). E. faecalis apresentou a maior sensibilidade, seguido por Staphylococcus aureus, enquanto S. enteritidis e E. coli apresentaram-se mais resistentes. Dentre as formas de extração, a hidroalcoolatura apresentou capacidade de inibição e/ou inativação intensa e seletiva frente aos quatro inóculos bacterianos. Os decoctos mostraram-se completamente ineficazes frente às bactérias Gram-negativas, enquanto que as Gram-positivas apresentaram somente bacteriostasia/inibição.

Dilution test in multiple tube system was used to determine the in vitro antibacterial activity in inflorescences of Achyrocline satureioides (Lam.) DC. - Asteraceae ("macela", "marcela"), expressed as intensity of bacterial inhibition activity (IINIB/bacteriostasis) and intensity of bacterial inactivation activity (IINAB/bactericidie), from ethanol (hydroalcoholic) and water (decoction) extraction forms on standardized inocula of Enterococcus faecalis (ATCC 19433), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 11229) and Salmonella enteritidis (ATCC 11076). E. faecalis had the highest sensitivity, followed by S. aureus, while S. enteritidis and E. coli were more resistant. Of the extraction forms, the hydroalcoholic one showed intense and selective inhibition and/or inactivation capacity against four bacterial inocula. Decoctions were completely ineffective against the Gram-negative bacteria, whereas Gram-positive bacteria showed only bacteriostasis/inhibition.

Prevalencia serológica de Salmonellaenteritidis en la población canina del Municipio de Tunja, Colombia / The serological prevalence of Salmonella enteritidis amongst Tunja's canine population (a large Colombian city)

Objetivo Determinar la prevalencia serológica de Salmonella enteritidis en la población canina de Tunja (Boyacá), durante julio y octubre del 2006 y establecer la relación entre los factores predisponentes y la serorreactividad en la población canina. Materiales y Métodos Sobre la población canina estimada en 9 623 animales (censo de 2002), y asumiendo una prevalencia critica preestablecida de 3 por ciento y un nivel de confianza del 95 por ciento, se recolectaron 72 muestras sanguíneas caninas distribuidas en cinco zonas de la ciudad, se aplicó una encuesta para cada animal para determinar la presencia de factores predisponentes y un examen clínico completo. Con los sueros obtenidos, se tituló el nivel de anticuerpos mediante la prueba de microaglutinación en placa para Salmonella enteritidis (MAG). Resultados La prevalencia serológica a Salmonella enteritidis fue del 41,7 por ciento, las prevalencias zonales fueron: 10 por ciento Norte, 10 por ciento occidente, 16,7 por ciento Oriente, 36,7 por ciento sur y 26,6 por ciento centro. Se determinó una correlación directa con la presentación de problemas entéricos anteriores (p<0,05) (OR 3,5) y en menor grado con el acceso a desechos orgánicos (p<0,05) (OR 0,4); otros factores como la convivencia con otros animales y la salud mostraron niveles de riesgo positivo (OR 1,4 Y OR 2). Conclusiones Existe una alta prevalencia serológica a Salmonella enteritidis en la ciudad de Tunja, con mayor incidencia en la zona sur, siendo el principal factor predisponente la presentación de problemas entéricos anteriores, el cual es un factor de alto riesgo en la diseminación de esta entidad.

Objective Determining the serological prevalence of Salmonella enteritidis in the city of Tunja's canine population (in Boyacá) from July to October 2006 and establishing the relationship between predisposed factors and seroreactivity within this canine population. Materials and Methods 72 canine blood samples were gathered from all of the city's five areas from an estimated 9 623 canine population (2002 census), assuming a 3 percent pre-established critical prevalence and 95 percent confidence level. A survey was made of each animal to determine the presence of predisposed factors and they were all given a complete clinical examination. The sera so obtained were used for titering antibody levels by means of the microagglutination plate test for Salmonella enteritidis (MAG). Results Salmonella enteritidis serological prevalence was 41,7 percent. Area prevalence within the city was 10 percent for the north, 10 percent for the west, 16,7 percent for the east, 36,7 percent the south and 26,6 percent for the city-center. A direct correlation was established with the presentation of previous enteric problems (p<0.05) (OR 3.5) and, to a lesser extent, with access to organic waste (p<0.05) (OR 0.4). Other factors presented positive risk levels, such as coexistence with other animals and health (OR 1.4 and OR 2) Conclusions Salmonella enteritidis had a high serological prevalence in the city of Tunja, most incidents occurring in the southern area. The presentation of previous enteric problems was the main predisposed factor, this being a high risk factor in this entity's dissemination.

Aglutinación de inmunoglobulinas de yema de huevo de gallina (IgY) contra Salmonella enterica serovariedad Enteritidis / Agglutination of chicken egg yolk immunoglobulins (IgY) against Salmonella enterica serovar Enteriditis

Para producir extractos de IgY se emplearon dos lotes de 6 gallinas ponedoras cada uno. En el lote vacunado (V) las aves se inocularon por vía intramuscular con dos dosis de una bacterina contra Salmonella enterica serovariedad Enteritidis. En el lote testigo (T) las aves no se vacunaron. Con las yemas de ambos lotes se efectuaron 4 extracciones de IgY. Las dos primeras se realizaron con las yemas de los huevos producidos durante la 4§ y 5§ semana post-vacunación (extractos 1V y 1T) y las otras dos con las de la 6§, 7§ y 8§ semana (2V y 2T). Los extractos 1V y 1T se congelaton y descongelaron (1V-A y 1T-A) y se concentraron por diálisis simple (1V-B y 1T-B) o doble (1V-C y 1T-C). Mediante una prueba de ELISA para detectar antígenos flagelares de S. Enteritidis, los extractos V fueron positivos y los T negativos. El extracto 1V-C fue el más positivo y se seleccionó para realizar las aglutinaciones. Este extracto contenía 1,9 g/dl de proteínas totales y presentó bandas electroforéticas características de IgY. El extracto 1T-C fue usado como control negativo de aglutinación. Emplenado ambos extractos IgY y antisueros policlonales de conejo (IgG) se efectuaron aglutinaciones somáticas en placa y flagelares en tubo. Se estudiaron 357 cepas de S. enterica, 10 cepas de diferentes especies de la familia Enterobacteriaceae y dos cepas de la familia Vibrionaceae. El extracto 1V-C aglutinó a distintas cepas de Salmonella con estructura antigénica somática o flagelar relacionada con la cepa vacunal, mientras que las salmonelas con antígenos diferentes y otras especies de la familia Enterobacteriaceae y Vibrionaceae fueron negativas. Este trabajo demuestra que es posible emplear las IgY para identificar serovariedades de S. enterica