Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1) and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortium

Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines

Padronização do teste ELISA baseado em antígeno capsular purificado dos sorotipos 3, 5 e 7 de Actinobacillus pleuropneumoniae Standarization of ELISA test based on purified capsular antigen from serotypes 3, 5 and 7 of Actinobacillus pleuropneumoniae

Foram padronizados testes de ELISA (Enzyme-linked immunosorbent assay) baseados em antígeno capsular purificado de Actinobacillus pleuropneumoniae sorotipos 3, 5 e 7, prevalentes no Brasil. Para a padronização foram utilizadas amostras de soro provenientes de leitões inoculados com os três sorotipos do agente em estudo, dos quais se colheram amostras de sangue semanais, durante 15 semanas para estudo da dinâmica da síntese de anticorpos. O controle negativo dos testes constituiu-se de um mistura de 130 soros de animais livres de Actinobacillus pleuropneumoniae (App). Os antígenos também foram testados com amostras de soro de animais infectados com outros agentes causadores de doenças respiratórias e vacinados contra rinite atrófica. Os antígenos produzidos foram eficientes na detecção de animais infectados com App, permitindo determinar densidades óticas superiores à média dos soros controles negativos acrescida de quatro desvios-padrões. Os testes de ELISA para os sorotipos 3, 5 e 7 apresentaram especificidade de 100% e sensibilidade de 92, 88 e 90%, respectivamente. Não ocorreram reações cruzadas com outros sorotipos, assim como com soros de animais inoculados com outros agentes causadores de problemas respiratórios. Os resultados foram analisados através da análise discriminante de ANDERSON (1958), utilizando-se o programa Statistical Analysis System. Concluiu-se que os antígenos testados são adequados para sorotipar animais que tenham sido submetidos ao screening através de um teste de ELISA polivalente baseado em LPS-LC.<br>Three ELISA (Enzime-linked immunosorbent assay) tests based on purified capsular antigen from serotypes 3, 5 and 7 of Actinobacillus pleuropneumoniae, prevalent in Brazil, were standardized. Serum samples, collected from piglets inoculated with these three serotypes, were used to standardize the test. In order to study the dynamic of antibody synthesis, weekly blood samples were collected from these piglets. A pool of 130 sera obtained from Actinobacillus pleuropneumoniae -free pigs was used as negative control for the tests. The antigens were also tested with serum samples from animals infected with other respiratory infectious agents and vaccinated against athrofic rinitis. The antigens were efficient in detecting animals infected with App. Optical densities above the average of the negative control sera plus four standard deviation were detected. ELISA tests to serotypes 3, 5 and 7 showed specificity of 100% and sensibility of 92, 88 and 90%, respectively. No cross reaction with other serotypes or with sera of animals inoculated with other respiratory pathogens was observed. The results were analyzed using the Statistical Analysis System program. Antigens tested were adequate for serotyping animals previously screened through a polyvalent LPS-LC ELISA test