Mycoplasma Infections of Food and Fiber Animals: Development of Nonvertebrate Insect Models for Assessment of Mycoplasma Virulence Potential

Non-Technical Summary:<br/>
Mycoplasmas belong to the class Mollicutes, are true bacteria which lack cell walls, and cause important economic diseases in food and fiber animals, including ruminants, poultry, swine, and alligators/crocodiles. Although respiratory disease is the most common clinical manifestation of mycoplasmosis, systemic disease (arthritis, CNS involvement, otitis, urogenital infection) can occur when the pathogen disseminates from the respiratory surfaces to other sites. In addition, M bovis outbreaks have been observed in bison beef herds and this emerging disease is now the leading cause of death in bison, with mortality rates as high as 30%. Although antimicrobial sensitivity testing standards and susceptibilities are available, mycoplasmal disease is often chronic and responds poorly to therapy. Although vaccines have been developed, protection from experimental challenge does not always translate to protection in under field conditions and in some cases may actually exacerbate disease. Despite the availability of full genome sequences on all the major mycoplasma animal pathogens, there are critical gaps in our current knowledge with regard to the molecular basis for pathogenicity, particularly those factors contributing to the development of clinical disease. Our long-term goal is to determine the host:parasite interactions involved in mycoplasmal infections of food and fiber animals, with particular emphasis on ruminants (cows and goats) and reptiles (alligators). We also have a special interest in the epidemiology of disease transmission and survival in the environment. Improved understanding of pathogenic mechanisms and epidemiology will advance the development of preventative and interventional strategies for control of disease associated with mycoplasmas.
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Approach:<br/>
Development of nonvertebrate models for virulence testing. We will establish insect models for virulence testing, as insects can be maintained at both 30 and 35 degrees C and are thus amenable to pathogens of both reptiles and mammals. These systems are relatively new tools that have been used to study virulence in a wide range of bacterial, viral and fungal pathogens, but to date have not been used for mycoplasmas. The dose response survival curves in insects will be compared to test virulence potential of selected mutants as compared with the wild type of selected animal mycoplasmas. Role of mechanosensitive channel proteins. Mechanosensitive channel proteins are membrane pore-forming channel proteins that are involved in protection from osmotic shock. Based on in silico analysis of mycoplasma genomes, we identified the MscL gene in a number of mycoplasmal species, including both M bovis and M alligatoris. We will compare mutants in which the mscL gene has been disrupted with the wild types for the ability to withstand osmotic shock. We will also screen the mutants in the insect models for decreased fitness. We will use proteome profiling to determine if the mutants have an altered secretome when compared with wild type.