Growth of Mannheimia haemolytica:Inhibitory agents and putative mechanism of inhibition

Thesis (Ph.D.), College of Veterinary Medicine, Washington State UniversityPneumonia is the most important disease of bighorn sheep (BHS). Pathogens detected in pneumonic BHS lungs include Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida and Mycoplasma ovipneumoniae. Although leukotoxin-producing M. haemolytica consistently causes fatal pneumonia in BHS under experimental conditions, B. trehalosi and P. multocida are isolated more frequently than M. haemolytica from pneumonic BHS lungs by culture. In this study, I extended the previous findings from our laboratory that B. trehalosi and P. multocida inhibit the growth of M. haemolytica. I hypothesized that the inhibitory phenotype of B. trehalosi is conserved across B. trehalosi strains. Fifty-five B. trehalosi isolates were tested by bacterial competition assays. All of them inhibited M. haemolytica suggesting that the inhibitory phenotype is conserved. No plasmids were isolated from any of the 30 B. trehalosi isolates tested, suggesting that the effectors are chromosomally-encoded. The observation that M. haemolytica was not isolated frequently even from pneumonic BHS lungs that did not carry B. trehalosi or P. multocida led to my second hypothesis that bacteria other than B. trehalosi and P. multocida inhibit the growth of M. haemolytica. Bacterial competition assays showed that Escherichia coli also inhibits the growth of M. haemolytica via a proximity-dependent mechanism. Streptococcus spp. and Staphylococcus spp. did not have any inhibitory effect. Fatal pneumonia in BHS often results from acquisition of leukotoxin-positive M. haemolytica from domestic sheep (DS). I hypothesized that intranasal inoculation of B. trehalosi will eliminate or reduce shedding of M. haemolytica by DS. Domestic sheep were oro-nasally inoculated with B. trehalosi following antibiotic treatment. These DS shed reduced numbers of M. haemolytica than un-treated DS. However, healthy BHS commingled with B. trehalosi-treated DS acquired M. haemolytica around day 14 post-commingling which resulted in the development of leukotoxin-neutralizing antibodies. They did not develop pneumonia for 211 days. However, they died of pneumonia when commingled with DS that carried leukotoxin-positive M. haemolytica, B. trehalosi and M. ovipneumoniae. These findings suggest that B. trehalosi can be used to reduce the shedding of M. haemolytica by DS. However, infection with leukotoxin-positive M. haemolytica, B. trehalosi and M. ovipneumoniae synergistically causes fatal pneumonia in BHS.College of Veterinary Medicine, Washington State Universit