Bulletin of Insectology 68 (1): 39-44, 2015 ISSN 1721-8861 Experimental inoculation of house flies Musca domestica with Corynebacterium pseudotuberculosis biovar equi Marta BARBA 1 , Allison J. STEWART 1 , Thomas PASSLER 1 , Terri HATHCOCK 2 , Anne A. WOOLDRIDGE 1 , E. VAN SANTEN 3 , Manuel F. CHAMORRO 1 , Russell C. CATTLEY 2 , Jerome A. HOGSETTE 4 , Xing Ping HU 5 1 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, AL, USA 2 Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL, USA 3 Department of Crop, Soil and Environmental Sciences, College of Agriculture, Auburn University, AL, USA 4 USDA/ARS/CMAVE, Gainesville, FL, USA 5 Department of Entomology and Plant Pathology, College of Agriculture, Auburn University, AL, USA Abstract Corynebacterium pseudotuberculosis (Actinomycetales Corynebacteriaceae) infection in horses causes three different disease syndromes: external abscesses, infection of internal organs and ulcerative lymphangitis. The exact mechanism of infection in horses remains undetermined, but transmission by insect vectors is suspected. The present study first determined the optimal cul- ture media for inoculation of house flies (Musca domestica L.) (Diptera Muscidae), with C. pseudotuberculosis biovar equi and the time required for fly inoculation. A second experiment determined the duration of bacterial survival on flies. Exposure of house flies to 3 different preparations of blood agar supplemented with dextrose and colonized with C. pseudotuberculosis deter- mined that a 10 minute exposure to the bacteria was enough to inoculate the flies. C. pseudotuberculosis could be recovered for up to 24 hours after house flies were exposed for 30 minutes to a blood agar plate colonized with the bacteria and moistened with 10% dextrose. These findings support the hypothesis that the house fly is a potential vector of pigeon fever and aid in establishing a protocol for a future experimental model to demonstrate the role of house flies as mechanical vectors in C. pseudotuberculosis infection. Key words: Horse, pigeon fever, vector, equine pathogen, arthropod, transmission. Introduction Corynebacterium pseudotuberculosis (Actinomycetales Corynebacteriaceae) is a pleomorphic, facultative intra- cellular, Gram-positive rod with a worldwide distribu- tion (Quinn et al., 2011). Two biotypes of C. pseudotu- berculosis are described and are distinguished by genet- ic characteristics, including restriction fragment length polymorphisms and the ability of cultured organisms to reduce nitrate to nitrite. Nitrate-positive biovar equi is commonly isolated from horses and cattle but nitrate- negative biovar ovis is typically recovered from small ruminants and cattle (Biberstein et al., 1971; Sutherland et al., 1996). In ruminants, C. pseudotuberculosis causes caseous lymphadenitis, abscesses, and occasionally mastitis (Yeruham et al., 1996). In horses, C. pseudotu- berculosis biovar equi causes three clinical forms of disease (Aleman et al., 1996). The most common form, known as “pigeon fever” or “dryland distemper”, is cha- racterized by subcutaneous abscessation of the pectoral region; the second form causes abscesses of internal or- gans; and the third causes ulcerative lymphangitis (Aleman et al., 1996). Natural cross-species infection by specific biotypes is not known to occur (Foley et al., 2004). Human infection, although rare, is considered an occupational zoonosis (Dorella et al., 2006). C. pseudotuberculosis biovar equi infection is preva- lent in the western United States (California, Utah, Col- orado and Texas) and Brazil, but a recent increase in case numbers and spread to regions previously consi- dered non-endemic have been reported (Foley et al., 2004; Pratt et al., 2005; Kilyone et al., 2014). The routes of C. pseudotuberculosis infection in horses remain undetermined (Pratt et al., 2005; Dorella et al., 2006). Transmission by insects is suspected be- cause of the seasonal occurrence patterns during fall and early winter in the western United States (Miers et al., 1980). The highest annual incidences in horses have been observed during dry months of the year following winters with above average rainfall, which provides op- timal breeding conditions for insects in the subsequent summer and fall (Miers et al., 1980; Aleman et al., 1996; Kilyone et al., 2014; Szonyi et al., 2014). Three fly species, including the horn fly, Haematobia irritans L., the stable fly, Stomoxys calcitrans L. and the house fly, Musca domestica L., were reported as potential vec- tors by PCR detection of the C. pseudotuberculosis bio- var equi phospholipase D (PLD) exotoxin gene in field samples of fly homogenates (Spier et al., 2002). In house flies contaminated with C. pseudotuberculosis biovar ovis from cattle, bacteria were isolated from the flies’ intestine and feces, and saliva for 1-4 and 1-3 h, respectively, post exposure (Yeruham et al., 1996; Braverman et al., 1999). The objectives of the present study were to determine the optimal inoculation time of C. pseudotuberculosis biovar equi, the most appropriate inoculation media, the duration of bacterial retention in house flies after expo- sure and the survival of flies after inoculation. The overall goal of this project was to develop a house fly inoculation system which could be used subsequently in a controlled experimental model to evaluate the role of house flies as mechanical vectors of C. pseudotubercu- losis in horses.