Immunological characterization of diphtheria toxin recovered from Corynebacterium pseudotuberculosis

Abstract

Diphtheria toxin (DT) is a potent toxin produced by the so-called diphtheria group which includes Corynebacterium diphtheriae (C. diphtheriae), Corynebacterium ulcerans (C. ulcerans), and Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The present investigation is aimed to study in detail the production of DT by C. pseudotuberculosis. Twenty isolates were obtained from sheep diseased with caseous lymphadenitis (CLA) and twenty-six isolates were obtained from 26 buffaloes diseased with oedematous skin disease (OSD). All isolates were identified by standard microbiological and DT production was assayed serologically by modified Elek test and immunoblotting. All sheep isolates were nitrate negative, failed to hydrolyze starch and could not produce DT, while all buffalo isolates (biotype II) revealed positive results and a specific band of 62 kDa, specific to DT, was resulted in all concentrated cell fractions (CF), but was absent from non-toxigenic biotype I isolates. At the same time, another band of 31 kDa specific to the PLD gene was obtained with all isolates of biotype I and II. Moreover, all isolates showed positive synergistic hemolytic activity and antagonistic hemolysis with β-hemolytic Staphylococci. The obtained results also indicated that C. pseudotuberculosis could be classified into two strains; non-toxigenic biotype I strain, which failed to produce DT as well as being negative to nitrate and starch hydrolysis, and toxigenic biotype II strain, which can reduce nitrate, hydrolyze starch as well as produce DT.

Keywords: C. pseudotuberculosis; Diphtheria toxin; Immuno-blotting technique; Immunological characterization; Modified Elek test.

Figure 1

AIW test with a 5 mm diameter center well containing 9 μl of anti DT surrounded by 6 different Corynebacteria. Streaks 1 and 6 are sheep isolates, streak 2 is standard PW8 C. diphtheriae and streaks 3, 4 and 5 are buffalo isolates showing lines of identity with the PW8 strain. Sheep isolates (1 and 6) failed to produce DT toxins.

Figure 2

Elek plate with a central well contains positive serum collected from diseased buffaloes with OSD. Streak A is PW8 and streaks F, E and D are buffalo isolates showing an outer line of identity with PW8 producing DT. Streaks F, E, D, C and B show inner lines due to the production of PLD by all isolates. No inner line was seen with PW8.

Figure 3

Immunoblotting of concentrated culture filtrates from 4 sheep isolates (lane 2–5) and of 9 buffalo isolates in lane (6–14) and lane 1 contain standard anti-DT serum and lane 15 a broad range protein marker. Bands were detected by positive serum obtained from buffaloes with OSD. All buffalo isolates show a band at 62.0 KDa at the specific region of standard anti DT. Know DT bands appeared with sheep isolates. All sheep and buffalo isolates develop a band at 31 KDa at the specific region of PLD protein.