RESUMO

Fowl Cholera (FC) is a disease caused by Pasteurella multocida. The severity of this disease is partly caused by virulence factors. Genes encoding fimbriae, capsule, sialidases and proteins for iron metabolism may be related to P. multocida's ability to infect the host. Besides to examining DNA for the presence of virulence genes, DNA is essential for the diagnostic and FTA cards are an alternative for genetic material transport. The study aims to evaluate the viability of P. multocida DNA transport using the cards and to detect 14 virulence genes in 27 strains isolated from FC cases in the United States by multiplex-PCR. No growth was observed in any of the FTA cards, which was essential to assess the security. Furthermore, DNA detection was possible in 100% of the samples, independent of the storage period (7 to 35 days) and temperature (4°C and 37°C). ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH and pfhA genes were detected in more than 80% of the samples. FTA cards have proven to be a viable and safe tool for DNA transport of P. multocida. A majority of genes showed a high frequency, which was similar to strains isolated from FC cases.(AU)

Cólera aviária (CA) é uma doença causada pela bactéria Pasteurella multocida e a severidade dos casos é em parte justificada por fatores de virulência. Genes codificando fímbrias, cápsulas, sialidases, dismutases e proteínas do metabolismo férrico podem ser relacionados à capacidade do agente em infectar o hospedeiro. Além da obtenção do DNA para pesquisa de genes de virulência, o material genético é fundamental para o diagnóstico, e os cartões FTA seriam uma alternativa no transporte de microrganismos. Os objetivos da presente pesquisa foram avaliar a viabilidade do transporte de DNA de P. multocida através dos cartões e detectar 14 genes de virulência em 27 cepas isoladas de CA nos Estados Unidos, por meio de multiplex-PCR. Nenhuma das amostras para análise microbiológica da segurança dos cartões apresentou crescimento. Foi possível a detecção do DNA em 100% das amostras, independentemente do tempo de estocagem (sete a 35 dias) e das temperaturas (4°C e 37°C) avaliadas. Genes ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH e pfhA foram detectados em mais de 80% das amostras. Os cartões FTA demonstraram ser uma ferramenta viável e segura para o transporte do DNA de P. multocida. A maioria dos genes apresentou uma alta frequência, compatível com isolados de CA.(AU)

Assuntos

RESUMO

Fowl Cholera (FC) is a disease caused by Pasteurella multocida. The severity of this disease is partly caused by virulence factors. Genes encoding fimbriae, capsule, sialidases and proteins for iron metabolism may be related to P. multocida's ability to infect the host. Besides to examining DNA for the presence of virulence genes, DNA is essential for the diagnostic and FTA cards are an alternative for genetic material transport. The study aims to evaluate the viability of P. multocida DNA transport using the cards and to detect 14 virulence genes in 27 strains isolated from FC cases in the United States by multiplex-PCR. No growth was observed in any of the FTA cards, which was essential to assess the security. Furthermore, DNA detection was possible in 100% of the samples, independent of the storage period (7 to 35 days) and temperature (4°C and 37°C). ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH and pfhA genes were detected in more than 80% of the samples. FTA cards have proven to be a viable and safe tool for DNA transport of P. multocida. A majority of genes showed a high frequency, which was similar to strains isolated from FC cases.(AU)

Cólera aviária (CA) é uma doença causada pela bactéria Pasteurella multocida e a severidade dos casos é em parte justificada por fatores de virulência. Genes codificando fímbrias, cápsulas, sialidases, dismutases e proteínas do metabolismo férrico podem ser relacionados à capacidade do agente em infectar o hospedeiro. Além da obtenção do DNA para pesquisa de genes de virulência, o material genético é fundamental para o diagnóstico, e os cartões FTA seriam uma alternativa no transporte de microrganismos. Os objetivos da presente pesquisa foram avaliar a viabilidade do transporte de DNA de P. multocida através dos cartões e detectar 14 genes de virulência em 27 cepas isoladas de CA nos Estados Unidos, por meio de multiplex-PCR. Nenhuma das amostras para análise microbiológica da segurança dos cartões apresentou crescimento. Foi possível a detecção do DNA em 100% das amostras, independentemente do tempo de estocagem (sete a 35 dias) e das temperaturas (4°C e 37°C) avaliadas. Genes ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH e pfhA foram detectados em mais de 80% das amostras. Os cartões FTA demonstraram ser uma ferramenta viável e segura para o transporte do DNA de P. multocida. A maioria dos genes apresentou uma alta frequência, compatível com isolados de CA.(AU)

Assuntos

RESUMO

Background: The contamination of products with Salmonella is a major threat to the poultry industry because the possible transmission to humans and animals can produce a huge negative impact. The diversity of Salmonella enterica serotypes complicates the diagnostic systems and the transport of live cultures to the diagnostic labs may represent a biohazard. Current methods for serotyping using antibodies do not work well for many Salmonella serotypes and reagents are not often available. For these reasons, methods that assign serotype by the analysis of DNA are preferred. One step that is currently in development is streamlining methods for DNA submission to the laboratories for sequencing. For this purpose, we investigated fi lter papers commercially available (Flinders Technology Associates - FTA) to ship DNA samples. Filter papers are impregnated with a chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. The objective of this study was to assess the feasibility of the FTA cards for transporting Salmonella DNA samples in order to reduce biohazards and if they would yield enough DNA in quantity and quality for molecular analyses.Material, Methods & Results: In this study 156 samples of Salmonella enterica serotypes Enteritidis, Heidelberg, Hadar, Gallinarum, Typhimurium, Agona and Pullorum were isolated from poultry products and environ

Background: The contamination of products with Salmonella is a major threat to the poultry industry because the possible transmission to humans and animals can produce a huge negative impact. The diversity of Salmonella enterica serotypes complicates the diagnostic systems and the transport of live cultures to the diagnostic labs may represent a biohazard. Current methods for serotyping using antibodies do not work well for many Salmonella serotypes and reagents are not often available. For these reasons, methods that assign serotype by the analysis of DNA are preferred. One step that is currently in development is streamlining methods for DNA submission to the laboratories for sequencing. For this purpose, we investigated fi lter papers commercially available (Flinders Technology Associates - FTA) to ship DNA samples. Filter papers are impregnated with a chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. The objective of this study was to assess the feasibility of the FTA cards for transporting Salmonella DNA samples in order to reduce biohazards and if they would yield enough DNA in quantity and quality for molecular analyses.Material, Methods & Results: In this study 156 samples of Salmonella enterica serotypes Enteritidis, Heidelberg, Hadar, Gallinarum, Typhimurium, Agona and Pullorum were isolated from poultry products and environ

RESUMO

Background: The contamination of products with Salmonella is a major threat to the poultry industry because the possible transmission to humans and animals can produce a huge negative impact. The diversity of Salmonella enterica serotypes complicates the diagnostic systems and the transport of live cultures to the diagnostic labs may represent a biohazard. Current methods for serotyping using antibodies do not work well for many Salmonella serotypes and reagents are not often available. For these reasons, methods that assign serotype by the analysis of DNA are preferred. One step that is currently in development is streamlining methods for DNA submission to the laboratories for sequencing. For this purpose, we investigated fi lter papers commercially available (Flinders Technology Associates - FTA) to ship DNA samples. Filter papers are impregnated with a chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. The objective of this study was to assess the feasibility of the FTA cards for transporting Salmonella DNA samples in order to reduce biohazards and if they would yield enough DNA in quantity and quality for molecular analyses.Material, Methods & Results: In this study 156 samples of Salmonella enterica serotypes Enteritidis, Heidelberg, Hadar, Gallinarum, Typhimurium, Agona and Pullorum were isolated from poultry products and environ

Background: The contamination of products with Salmonella is a major threat to the poultry industry because the possible transmission to humans and animals can produce a huge negative impact. The diversity of Salmonella enterica serotypes complicates the diagnostic systems and the transport of live cultures to the diagnostic labs may represent a biohazard. Current methods for serotyping using antibodies do not work well for many Salmonella serotypes and reagents are not often available. For these reasons, methods that assign serotype by the analysis of DNA are preferred. One step that is currently in development is streamlining methods for DNA submission to the laboratories for sequencing. For this purpose, we investigated fi lter papers commercially available (Flinders Technology Associates - FTA) to ship DNA samples. Filter papers are impregnated with a chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. The objective of this study was to assess the feasibility of the FTA cards for transporting Salmonella DNA samples in order to reduce biohazards and if they would yield enough DNA in quantity and quality for molecular analyses.Material, Methods & Results: In this study 156 samples of Salmonella enterica serotypes Enteritidis, Heidelberg, Hadar, Gallinarum, Typhimurium, Agona and Pullorum were isolated from poultry products and environ

RESUMO

Background: The contamination of products with Salmonella is a major threat to the poultry industry because the possible transmission to humans and animals can produce a huge negative impact. The diversity of Salmonella enterica serotypes complicates the diagnostic systems and the transport of live cultures to the diagnostic labs may represent a biohazard. Current methods for serotyping using antibodies do not work well for many Salmonella serotypes and reagents are not often available. For these reasons, methods that assign serotype by the analysis of DNA are preferred. One step that is currently in development is streamlining methods for DNA submission to the laboratories for sequencing. For this purpose, we investigated fi lter papers commercially available (Flinders Technology Associates - FTA) to ship DNA samples. Filter papers are impregnated with a chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. The objective of this study was to assess the feasibility of the FTA cards for transporting Salmonella DNA samples in order to reduce biohazards and if they would yield enough DNA in quantity and quality for molecular analyses. Material, Methods & Results: In this study 156 samples of Salmonella enterica serotypes Enteritidis, Heidelberg, Hadar, Gallinarum, Typhimurium, Agona and Pullorum were isolated from poultry products and environments in southern Brazil. Samples were stored in the Avian Diagnostic and Research Center of the Federal University of Rio Grande do Sul. Following instructions for spotting cards with cell cultures at a density that visually matched a McFarland Turbidity Standard 0,5; they were shipped to the Agriculture Research Service of the United States Department of Agriculture (USDA-ARS, Athens, GA-USA), using FTA cards. Upon the reception of the cards, safety testing was performed by transferring one disk from each sample into 10 mL of brain heart infusion (BHI) tubes and incubated at 37°C for 24 h. The BHI tube that showed turbidity after incubation was transferred to brilliant green (BG) agar and incubated at 37°C for 24 h to 48 h. If colonies were obtained in BG, biochemical analyses were performed by using the Enterotube method. Only one sample (S. Enteritidis) showed turbidity in BHI, but any bacterial growth was observed in the BG agar. The average DNA concentration, as measured by spectrophotometry, was 42,32 (± 9,84) ng/µL and the average 280/260 ratio was 1,9 (± 0,09). All the analyzed samples were negative for live cultures of Salmonella and the DNA obtained was suitable for molecular testing. Discussion: FTA cards can be used to transport DNA samples from pathogenic bacteria, reducing biohazards associated with shipping live cultures. The possibility of shipping DNA, in an economic and safe way, for testing samples at the laboratories facilitates the identification of Salmonella enterica serotypes that are circulating in the environment of poultry. Turbidity in BHI tubes that did not result in colonies on agar media may be caused by the presence of other contaminants such as environmental saprophytic microorganisms that may occurred during the process of handling the cards. DNA samples of Salmonella enterica shipped from Brazil to the United States for this set of isolates did not show bacterial growth. Thus the FTA cards provided safe and effective inactivation of the pathogen, and the DNA obtained from the cards were adequate for downstream analyses.

Assuntos