Leukotoxin of Bibersteinia trehalosi Contains a Unique Neutralizing Epitope, and a Non-Neutralizing Epitope Shared with Mannheimia haemolytica Leukotoxin

Abstract

Bibersteinia trehalosi and Mannheimia haemolytica, originally classified as Pasteurella haemolytica biotype T and biotype A, respectively, under Genus Pasteurella has now been placed under two different Genera, Bibersteinia and Mannheimia, based on DNA-DNA hybridization and 16S RNA studies. While M. haemolytica has been the predominant pathogen of pneumonia in ruminants, B. trehalosi is emerging as an important pathogen of ruminant pneumonia. Leukotoxin is the critical virulence factor of these two pathogens. While the leukotoxin of M. haemolytica has been well studied, the characterization of B. trehalosi leukotoxin has lagged behind. As the first step towards addressing this problem, we developed monoclonal antibodies (mAbs) against B. trehalosi leukotoxin and used them to characterize the leukotoxin epitopes. Two mAbs that recognized sequential epitopes on the leukotoxin were developed. One of them, AM113, neutralized B. trehalosi leukotoxin while the other, AM321, did not. The mAb AM113 revealed the existence of a neutralizing epitope on B. trehalosi leukotoxin that is not present on M. haemolytica leukotoxin. A previously developed mAb, MM601, revealed the presence of a neutralizing epitope on M. haemolytica leukotoxin that is not present on B. trehalosi leukotoxin. The mAb AM321 recognized a non-neutralizing epitope shared by the leukotoxins of B. trehalosi and M. haemolytica. The mAb AM113 should pave the way for mapping the leukotoxin-neutralizing epitope on B. trehalosi leukotoxin and the development of subunit vaccines and/or virus-vectored vaccines against this economically important respiratory pathogen of ruminants.

Keywords: Bibersteinia trehalosi; Mannheimia haemolytica; leukotoxin; neutralizing epitope.

Figure 1

Previous and current taxonomical classification of Bibersteinia trehalosi and Mannheimia haemolytica.

Figure 2

Western blot analysis confirms the specificity of mAbs AM113 and AM321. Lkts of B. trehalosi and M. haemolytica serotypes A1 and A2 were subjected to SDS-PAGE followed by western blot analysis with culture fluids from the hybridomas AM113 (Panel 1) and AM321 (Panel 2). Lanes M, A1, A2, and Bt represent the molecular weight standards, Lkts of M. haemolytica serotype A1, serotype A2, and B. trehalosi, respectively.

Figure 3

Lkt neutralizing activity of mAb AM113. Serial dilutions of mAb AM113 in the form of culture fluid were incubated with B. trehalosi Lkt dilution that gives 50% cytotoxicity. Following incubation, the target cells were added to the Lkt-mAb mixture and the cytotoxicity assay was completed. Culture fluid containing the mAb AM113 exhibited a neutralizing antibody titer of 1:128. Error bars indicate standard deviations of the means.