Proteomic analysis and immunogenicity of Mannheimia haemolytica vesicles

Abstract

Mannheimia haemolytica, a major causative agent in bovine respiratory disease, inflicts extensive losses each year on cattle producers. Commercially available vaccines are only partially efficacious. Immunity to M. haemolytica requires antibodies to secreted toxins and outer membrane proteins (OMPs) of the bacterium. Gram-negative bacteria produce membrane blebs or vesicles, the membrane components of which are primarily derived from OMPs. Accordingly, vesicles have been used as immunogens with various degrees of success. This study characterized components of M. haemolytica vesicles and determined their immunogenicity in mice and cattle. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vesicles from this bacterium identified 226 proteins, of which 58 (25.6%) were OMPs and periplasmic and one (0.44%) was extracellular. Vesicles were used to vaccinate dairy calves and BALB/c mice. Analyses of sera from calves and mice by enzyme-linked immunosorbent assay (ELISA) showed that circulating antibodies against M. haemolytica whole cells and leukotoxin were significantly higher on days 21 and 28 (P < 0.05) than on day 0. For control calves and mice, there were no significant differences in serum anti-whole-cell and leukotoxin antibody levels from days 0 and 21 or 28, respectively. Lesion scores of lungs from vaccinated calves (15.95%) were significantly (P < 0.05) lower than those from nonvaccinated calves (42.65%). Sera from mice on day 28 and calves on day 21 showed 100% serum bactericidal activity. Sera from vesicle-vaccinated mice neutralized leukotoxin.

Fig 1

Electron micrographs of M. haemolytica cells (A) magnified ×6,000 and vesicles (B) with a magnification of ×50,000.

Fig 2

Chart showing subcellular locations of proteins identified in M. haemolytica vesicles as determined by PSORTb. Of particular interest are the 58 (25.66%) proteins that constitute periplasmic and outer membrane proteins and the single extracellular protein, which happens to be leukotoxin.

Fig 3

(A) Mean ELISA showing anti-whole-cell (Anti-WC) and antileukotoxin (Anti-LKT) antibody levels in day 28 sera collected from 8 groups of mice (n = 10/group) vaccinated with MHV or Pulmo-Guard PHM-1 (PMG) and nonvaccinates (CNTRL). (B) Circulating anti-whole-cell and anti-LKT antibodies in day 0 and 21 sera collected from 4 calves vaccinated with MHV and 4 nonvaccinates (Neg. Cntrl).

Fig 4

Mean complement-mediated bacterial killing of sera from mice vaccinated with M. haemolytica MHV and Pulmo-Guard PHM-1 (PMG) and nonvaccinated control mice (Neg. Cntrl). Serum from a calf vaccinated with rPlpE, a highly immunogenic outer membrane protein from M. haemolytica , was used as a positive control. C+ and C− designate the presence and absence, respectively, of complement in the assays.

Fig 5

Mean bactericidal activity of sera from Holstein calves that were vaccinated with M. haemolytica MHV and nonvaccinated control calves (Neg. Cntrl). C+ and C− designate the presence and absence, respectively, of complement in each assay. The positive control is serum from a calf vaccinated with the recombinant form of the highly immunogenic M. haemolytica outer membrane protein PlpE. Note that there is no killing activity in the absence of complement in all cases.