Assessment of FTA card employment for Pasteurella multocida DNA transport and detection of virulence-associated genes in strains isolated from fowl cholera in the United States / Avaliação do emprego de cartões FTA para o transporte do DNA de Pasteurella multocida e pesquisa de genes associados à virulência em cepas isoladas de cólera aviária nos Estados Unidos

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)

Antimicrobial resistance and virulence gene profiles in P. multocida strains isolated from cats

Cats are often described as carriers of Pasteurella multocida in their oral microbiota. This agent is thought to cause pneumonia, conjunctivitis, rhinitis, gingivostomatitis, abscess and osteonecrosis in cats. Human infection with P. multocida has been described in several cases affecting cat owners or after cat bites. In Brazil, the cat population is approximately 21 million animals and is increasing, but there are no studies of the presence of P. multocida in the feline population or of human cases of infection associated with cats. In this study, one hundred and ninety-one healthy cats from owners and shelters in São Paulo State, Brazil, were evaluated for the presence of P. multocida in their oral cavities. Twenty animals were positive for P. multocida, and forty-one strains were selected and characterized by means of biochemical tests and PCR. The P. multocida strains were tested for capsular type, virulence genes and resistance profile. A total of 75.6% (31/41) of isolates belonged to capsular type A, and 24.4% (10/41) of the isolates were untypeable. None of the strains harboured toxA, tbpA or pfhA genes. The frequencies of the other genes tested were variable, and the data generated were used to build a dendrogram showing the relatedness of strains, which were clustered according to origin. The most common resistance profile observed was against sulfizoxazole and trimethoprim-sulphamethoxazole.

Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR / Pesquisa de genes associados à virulência em cepas de Pasteurella multocida isoladas em casos de cólera aviária através da técnica de multiplex-PCR

The current systems of breeding poultry, based on high population density, increase the risk of spreading pathogens, especially those causing respiratory diseases and those that have more than one host. Fowl Cholera (FC) is one such pathogen, and even though it represents one of several avian diseases that should be considered in the differential diagnosis of notifiable diseases that present with sudden death, the pathogenesis and virulence factors involved in FC are still poorly understood. The objective of this study was to investigate twelve genes related to virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. The protocols developed were capable of detecting all of the proposed genes. The ompH, oma87, sodC, hgbA, hgbB, exBD-tonB and nanB genes were present in 100% of the samples (25/25), the sodA and nanH genes were present in 96% (24/25), ptfA was present in 92% (23/25), and pfhA was present in 60% (15/25). Gene toxA was not identified in any of the samples studied (0/25). Five different genetic profiles were obtained, of which P1 (negative to toxA) was the most common. We concluded that the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and the fact that all samples belonged to the same subspecies of P. multocida, five genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

Os atuais sistemas de criação na avicultura, baseados na alta densidade populacional, aumentam os riscos de disseminação de patógenos, especialmente das doenças respiratórias e daquelas cujos agentes etiológicos possuam mais de um hospedeiro. A Cólera Aviária (CA) apresenta estas características e apesar de representar uma das patologias aviárias que deve ser considerada para o diagnóstico diferencial de enfermidades com notificação obrigatória que cursam com morte súbita, a patogenia e os fatores de virulência envolvidos na CA ainda estão pouco elucidados. O objetivo deste trabalho foi pesquisar doze genes associados à virulência em 25 amostras de Pasteurella multocida isoladas de casos de CA na região sul do Brasil através do desenvolvimento de protocolos de multiplex-PCR. Os protocolos de multiplex-PCR desenvolvidos foram capazes de detectar todos os genes propostos. Os genes ompH, oma87, sodC, hgbA, hgbB, exBD-tonB, nanB estiveram presentes em 100% das amostras (25/25). Os genes sodA e nanH em 96% (24/25), o gene ptfA em 92% (23/25) e o gene pfhA em 60% (15/25). O gene toxA não foi identificado em nenhuma das amostras pesquisadas (0/25). Foram obtidos cinco diferentes perfis genéticos, sendo P1 (negativo para o gene toxA) o mais comum. Com este trabalho, concluiu-se que os protocolos de multiplex-PCR desenvolvidos tornam-se uma ferramenta bastante útil e rápida para a detecção simultânea dos genes de virulência. Apesar da alta frequência dos genes estudados e de todas as amostras pertencerem à mesma subespécie de P. multocida, foram observados cinco perfis genéticos, os quais devem ser confirmados em um estudo com um maior número de amostras.

Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.

Evaluación de dos técnicas de subtipificación molecular para el estudio de Pasteurella multocida / Evaluation of two techniques of molecular subtyping to study Pasteurella multocida

Se determinó la tipibilidad, la reproducibilidad y el poder discriminatorio de ERIC-PCR y ApaI-PFGE para establecer la relación genética de cepas de Pasteurella multocida. Se estudiaron 49 cepas de diferente origen, subespecie, biotipo, grupo capsular, serotipo somático y perfil de resistencia antimicrobiana. Por ERIC-PCR se establecieron 31 patrones, los que presentaron entre 10 y 14 bandas en un rango comprendido entre 0,2 y 1,2 kb. Por ApaI-PFGE se detectaron 37 patrones de restricción, los cuales presentaron entre 7 y 15 bandas bien definidas de 34 a 450 kb. La tipibilidad de ERIC-PCR fue del 100% (T=1) y la de ApaI-PFGE del 94% (T=0,94). La reproducibilidad de ambas técnicas fue del 100% (R=1); sin embargo, el poder discriminatorio de ERIC-PCR fue 93% (D=0,93) y el de ApaI-PFGE 98% (D=0,98). Mediante ambas técnicas fue posible agrupar las cepas con relación epidemiológica y diferenciar claramente las cepas no relacionadas. Se demostró el valor de ERIC-PCR y ApaI-PFGE para complementar estudios epidemiológicos, principalmente si las cepas en estudio son analizadas por ambas técnicas.

Typeability, reproducibility, and discriminatory power of ERIC-PCR and ApaI-PFGE to establish the genetic relation of P. multocida strains were determined. Forty-nine strains of different source, biotype, capsular group, somatic serotype, and resistance to antimicrobials were studied. By ERIC-PCR, 31 patterns were defined with 10 to 14 bands in a rank of 0.2 and 1.2 kb. By ApaI-PFGE, 37 restriction patterns were established with 7 to 15 bands of 34 to 450 kb. Typeability was 100% (T=1) for ERIC-PCR, and 94% (T=0.94) for ApaI-PFGE. Reproducibility of both techniques was 100% (R=1). Discriminatory power was 93% (D=0.93) for ERIC-PCR, and 98% (D=0.98) for ApaI-PFGE. By using both techniques, epidemiologically related strains were grouped, and unrelated strains were clearly differentiated. The value of ERIC-PCR and ApaI-PFGE as complements to epidemiologic studies was demonstrated, especially when both techniques were used to analyze the strains.

Detection of Pasteurella multocida in experimentally infected embryonated chicken eggs by PCR assay.

Applicability of polymerase chain reaction (PCR) assay to detect Pasteurella multocida in experimentally infected embryonated chicken egg was assessed in the present study. PCR assay rapidly and specifically detected the genome of P. multocida in amniotic fluid, allantoic fluid and homogenates of infected embryo and its membranes. The sensitivity of detection was as low as 20 bacterial cells/ml of allantoic or amniotic fluids. Detection of P. multocida in dead embryos by PCR was possible up to 6 and 30 days or more following storage of dead embryos at 37 degrees C, and at 4 degrees C as well as at -20 degrees C, respectively. The study revealed that PCR assays could be employed directly for detection and confirmation of P. multocida infection in experimentally infected chicken embryos.