Virulence exaltation of Clostridium perfringens strains from bovines

Journal of Venomous Animals and Toxins including Tropical Diseases, Feb 2019

Ten out of eighty-nine strains biochemically identified as Clostridium perfringens, isolated from bovine organs, were selected by their different results showed in toxigenicity test on mice. Those and the standard strains, ATCC types A, B, C, and D, had their virulence exalted through serial intramuscular inoculation into guinea pigs. Results showed that, for toxigenic strains (6), one or two passages were enough to cause exaltation, while for the atoxigenic (4), five or six inoculations were needed. Esterase electrophoresis of standard and isolated strains, with and without exaltation, was performed. Electrophoresis analysis permits the following conclusions: strains that do not show any clinical symptoms in mice, when exalted demonstrate decreased esterase activity; on the contrary, it is increased when correlated with animal symptoms.

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Virulence exaltation of Clostridium perfringens strains from bovines

ORIGINAL PAPER    Virulence exaltation of Clostridium perfringens strains from bovines     Baldassi L.I; Barbosa M.L.II; Bach E.E.III; Iaria S.T.IV ISeção de Bacteriologia Geral e Micobacterioses, Instituto Biológico IIInstituto Adolfo Lutz IIIInstituto Biológico - UNICASTELO IVDepartamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo (USP), São Paulo, Brasil Correspondence     ABSTRACT Ten out of eighty-nine strains biochemically identified as Clostridium perfringens, isolated from bovine organs, were selected by their different results showed in toxigenicity test on mice. Those and the standard strains, ATCC types A, B, C, and D, had their virulence exalted through serial intramuscular inoculation into guinea pigs. Results showed that, for toxigenic strains (6), one or two passages were enough to cause exaltation, while for the atoxigenic (4), five or six inoculations were needed. Esterase electrophoresis of standard and isolated strains, with and without exaltation, was performed. Electrophoresis analysis permits the following conclusions: strains that do not show any clinical symptoms in mice, when exalted demonstrate decreased esterase activity; on the contrary, it is increased when correlated with animal symptoms. Key words: Clostridium perfringens, bovine, electrophoresis, virulence exaltation.     INTRODUCTION Clostridia have little low invasive ability, and the pathogenicity of these bacteria is mainly determined by toxin production. From the species of this genus, C. perfringens is the most widely spread (13,15,30). According to Willis (38), the predominance of the several types (A, B, C, and D) is not uniform, and type A is the most prevalent one; soil is believed to be its natural environment (33,35,38). C. perfringens is also found in the feces of some animal species (35). The other types are predominantly related to the intestinal tract of animals, and their presence in soil is due to fecal contamination (33,38). C. perfringens produces several soluble antigens (toxins), and most of the studies focuses on the pathogenic effects they produce. However, the role each of the toxins has in the production of lesions and symptoms caused by this agent is not totally established, neither in men nor in animals (24,25). Different cases of acute enteritis or fatal enterotoxaemia have been reported in several animal species, such as sheep, cattle (20,21,31), pigs (12), dogs (9), and horses (26). Sudden death in ovine, bovines and caprines has also been attributed to C. perfringens (3,27,34). In the present trial, standard and isolated C. perfringens strains, from samples obtained in post-mortem examination of bovines, were submitted to virulence exaltation. Esterase-electrophoretic profile of strains with and without exalted virulence was compared.   MATERIAL AND METHODS Sampling Ten from eighty-nine strains biochemically identified as C. perfringens, isolated from post-mortem examination of 71 bovines, were selected by their different results showed in the toxigenicity test on mice (4). As a positive control, four other C. perfringens strains from American Type Culture Collection (ATCC), ATCC 3624 (type A), ATCC 3626 (type B), ATCC 3628 (type C), and ATCC 3629 (type D), were used. Isolation and biochemical identification of C. perfringens in field samples Isolated and standard strains were cultured in Cooked Meat Medium (CMM) at 37ºC for 18-24 hours and then kept at 4ºC. Three m l of this culture was streaked in a plate containing Brain Heart Infusion (BHI) agar, with 5% of defibrinated sheep blood and incubated in anaerobiosis in McIntosh and Fields jars at 37ºC for 18-24 hours. After incubation, colonies were analyzed according to the shape, color, production and type of hemolysis. Bacterial morphology was microscopically assessed in Gram-stained smears. Colonies presenting C. perfringens characteristics were isolated, cultured in CMM, and incubated at 37ºC for 18-24 hours. These cultures were submitted to the following biochemical tests for species identification: production of catalase, lecithinase and gelatinase, fermentation of glucose, lactose and skim milk. Interpretation was performed according to Holdeman and Moore (16); Mahony and Swantee (22); Mahony et al. (23). All the strains of the present trial were incubated in CMM, and after 18-24 hours of incubation at 37ºC, cultures were stored at 4ºC. Toxigenicity of isolated strains Each bacterial isolate and standard strain was cultured in plates containing Brain Heart Infusion Blood (BHIB) agar, incubated in MacIntosh and Fields jars at 37ºC for 18-24 hours. Five colonies were transferred from these cultures (23) to tubes containing 10 mL of Tryptose Yeast Extract (TYE) broth, followed by incubation in the same conditions as described above. After incubation, all the contents of each tube was transferred to a flask containing 90 mL of the sam (...truncated)


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L. Baldassi, M.L. Barbosa, E.E. Bach, S.T. Iaria. Virulence exaltation of Clostridium perfringens strains from bovines, Journal of Venomous Animals and Toxins including Tropical Diseases, pp. 280-292, Volume 10, Issue 3, DOI: 10.1590/S1678-91992004000300007