Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.

Toxinas e perfil protéico de amostras de Actinobacillus suis provenientes de plantéis suínos norte-americanos / Toxins and proteic profile of Actinobacillus suis samples from North American hog herds

Actinobacillus suis (A.suis) surgiu como uma grande ameaça aos plantéis suínos norte-americanos. Os sinais clínicos e as lesões são particularmente variáveis e podem lembrar aquelas causadas por outros organismos, como o Actinobacillus pleuropneumoniae (App), podendo ter como causa a similaridade na produção das toxinas ApxI e ApxII. Os objetivos do estudo foram confirmar a produção das toxinas ApxI e ApxII, investigar a produção de toxina geneticamente semelhante à Apx III e analisar as proteínas totais, verificando se existe similaridade entre os isolados provenientes de diferentes plantéis de suínos norte-americanos. Neste estudo, todas as cepas de A. suis foram positivas para os genes codificadores das toxinas ApxI e ApxII, usando o método de reação em cadeia de polimerase - multiplex (PCR-multiplex); e as proteínas totais de 70 amostras de A. suis, oriundos de diferentes plantéis suínos norte-americanos, foram analisadas por meio de eletroforese em gel de poliacrilaminda desnaturante (SDS-PAGE) e foram idênticas. A similaridade eletroforética observada entre as proteínas totais das bactérias analisadas indica a possibilidade de haver uma proteção cruzada a partir de uma provável vacina universal desenvolvida com esses antígenos para A. suis.

Actinobacillus suis (A. suis) has arisen as a great threat to the North American hog herds. The clinical symptoms and lesions are particularly variable and may resemble the same caused by other pathogenic organisms, such as Actinobacillus pleuropneumoniae (App), which can similarly lead to the production of the toxins ApxI and ApxII. This study aimed to confirm the production of the toxins ApxI and ApxII, as well as, to investigate the production of toxins that are genetically similar to ApxIII, and analyze total protein to verify whether there is any similarity among the isolated samples obtained from different North American hog herds. In this study, all the strains of A. suis were positive for the genes that codify the toxins ApxI and ApxII using the multiplex polymerase chain reaction (PCR-multiplex) method; and total protein from 70 samples of A. suis, obtained from different North American hog herds, were analyzed through denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and were identical. The electrophoretic similarity observed among total protein of the analyzed bacteria indicates that there is the possibility of existing a cross protection in case of developing a probable universal vaccine with the antigens of A. suis.