Clostrídios entéricos de leitões neonatos, desenvolvimento e avaliação de uma vacina experimental

Abstract

The involvement of clostridia as agents causing diarrhea in neonatal piglets has grown worldwide. In Brazil there are few reports of the occurrence of Clostridium difficile, Clostridium perfringens types A and C causing diarrhea in piglets due to the lack of laboratory diagnosis of these agents. Given this situation, the aims of this study were to determine the frequency of enteric clostridia affecting newborn piglets with up to seven days, on commercial farms over 500 sows, located in the Triangulo Mineiro and Alto Paranaíba, state of Minas Gerais, Brazil; and develop and evaluate a polyvalent vaccine containing the alpha toxoid of C. perfringens type A, beta toxoid of C. perfringens type C and A/B toxoid of C. difficile. Of the 60 animals used in this work, it was possible to isolate C. difficile from the feces of 12 (20%), five (41.7%) of the diarrheal group and seven (58.3%) of the group without diarrhea. When interpreting the data from the qualitative detection of toxins A/B of C. difficile by ELISA, it showed that of the 60 animals analyzed, 10 (16.7%) allowed the identification of toxins. Of these 10 piglets, seven (70%) were from the diarrheal group and the other three (30%) from the group without diarrhea. At necropsy, 13 piglets (21.67%) had mesocolon edema, the main macroscopic change associated with infection of C. difficile. Of the 13 animals that had mesocolon edema, eight (61.54%) were from the diarrheal group and the other five (38.46%) without diarrhea. Microscopically, 16 animals showed histopathological lesions characterized by inflammatory lesions. Of these 16 piglets, seven (43.75%) had clinical cases of diarrhea and other nine (56.25%) were non-diarrheal. Of the 60 animals used in this study, it was possible to isolate characteristic colonies of C. perfringens from the feces of 50 (83.3%). With the characterization of these samples, we found that all were C. perfringens type A. Of these 50 strains of C. perfringens type A, 23 (46%) were isolated from animals with diarrhea and 27 (54%) isolated from apparently healthy animals. In 31 (62%) strains of C. perfringens type A isolated, we detected the presence of cpb2 gene, encoding the beta-2 toxin. The result of the sequencing of the cpb2 gene revealed that only 10 samples, eight from the diarrheic group and two from the non-diarrheic group, have a mutation in the gene cpb2 that generated a truncated protein without effective action. The other 21 samples, 12 from the diarrheal group and nine from the group without diarrhea, didnt have the mutation, thereby allowing normal translation of beta-2 toxin. There was not a single sample of C. perfringens type C. The minimal inhibitory concentration (MIC) of C. perfringens type A strains has shown that amoxicillin, ceftiofur and narasin were the antimicrobials which had the best inhibitory activity. The polyvalent vaccine containing recombinant toxoids recombinant alpha, beta and native A/B was effective in stimulating neutralizing antibodies in animal studies. Therefore this vaccine proved to be an important tool for controlling diarrhea caused by clostridia