Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-γ production, IgG avidity and antigen recognition by Western blotting

Abstract

Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil. The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection. Eight goats were infected intradermally with a single dose of virulent C. pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks. At the end of the follow-up, animals were slaughtered and necropsied. Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response. The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group. The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups. IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks. With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment. IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands. Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.

Genome Organization and Pathogenicity ofCorynebacterium diphtheriaeC7(−) and PW8 Strains

“…The model reflects one of the major forms of human diphtheria, cutaneous diphtheria, as sometimes associated with physical invasion, such as that due to insect bites (12). Experimental infection models for several Corynebacterium species of veterinary importance (C. kutscheri [8], C. bovis [28], and C. pseudotuberculosis [47]) also employ physical invasion mechanisms, including intradermal injection. In our rabbit experiments, we confirmed the necrotizing effect of diphtheria toxin by inoculating toxigenic strains (the reference strain and PW8).…”

Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence

“…Nevertheless, some enzyme-linked immunosorbent assay (ELISA)-based diagnostic tests have been reported to be effective in control and eradication programs [32,33,110]. Recently, ELISA tests to detect gamma interferon (IFN-γ), as a marker of cell-mediated immunity against C. pseudotuberculosis, have been developed [71,86,93]. The IFN-γ ELISA test appears to be more sensitive than the normal antibody ELISA in detecting prior infection in goats, and it does not seem to be affected by vaccination in sheep [71].…”

“…described, using immunoblot techniques [86,87], these proteins have not been identified. However, they reliably detected CLA infection in goats, and they could be used as vaccine components.…”

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

“…Consequently, the low level of specific antibodies against this enzyme in animals with no clinical signs of disease probably occurs because of the low production of exotoxin by the bacteria or poor humoral immune activation. The 70KDa antigen that was well recognized by both clinical and subclinical animals may be the 67KDa protein described by other authors (Sting et al 1998, Paule et al 2003, Moura-Costa et al 2008; it is classified as a heat shock protein (Ellis et al 1991).…”

“…Quantification of INF-gamma in blood cultures was performed for samples collected from thirty-two infected sheep, as already described (Paule et al 2003) with some modifications. Briefly, 2 h after collection, heparinized blood was incubated at 37°C for 48 h in a humidified 5% CO 2 atmosphere in a 24-well culture tray with 20 µg of antigen concentrated using TPP technique; 5 µl of pokeweed mitogen was used as a positive control and sterile PBS was used as a negative control.…”