THE ROLE OF MUSCID FLIES IN BOVINE PATHOGEN DISSEMINATION

<b>Project Methods</b><br> Obj. 1: Characterize the prevalence of clinically relevant bacterial taxa in flies and their breeding environmentsMethods. Here, we will use 16S rRNA gene amplicon sequencing and high-throughput quantitative PCR (qPCR) to determine the abundance of clinically relevant bacterial taxa and their associated ARGs in fly and environmental samples collected weekly from the UW Dairy Cattle Center and Emmons Blaine Dairy Cattle Center over a single fly breeding season (July-September). Flies will be collected using paired alsynite and baited fly traps placed at the four compass points of each facility, while pools of soiled bedding and manure, the preferred breeding sites for synanthropic flies, will be collected from the surrounding areas. Upon retrieval, all samples will be stored on ice and brought back to the lab for processing. In brief, flies will be sorted by species, washed by agitation in sterile phosphate-buffered saline solution (PBS) for recovery of external bacteria, and then surface-sterilized. Individual surface-sterilized fly and manure samples will then be homogenized in PBS and half of this homogenate will be used to generate a glycerol stock for long-term cryopreservation of any viable bacteria. The other half will be frozen at -80°C prior to DNA isolation and high-throughput sequencing of 16S rRNA gene amplicons to characterize bacterial diversity. DNA will also be used as template in massively parallel qPCR reactions using the Takura SmartChip technology and established ARG arrays to characterize ARG diversity and abundance across all samples.We will also generate collections of bacterial strains isolated from the glycerol stocks obtained from fly and manure samples. Strains will be isolated by plating on media selective for bacterial taxa of interest. Each fly- or manure-derived strain will then be whole genome sequenced along with known clinical isolates to compare ARGs and phylogenetic relatedness among strains.Expected Outcomes. Results from this objective will help to (i) determine seasonal trends in the presence and abundance of clinically relevant bacterial taxa in dairy barn flies and their breeding environments, and (ii) address if flies are able to attenuate or exacerbate the dissemination of ARGs. By collecting samples from farms between which cows are periodically transported, results from this objective will also strongly position us to generate preliminary insights into the consistency of observed patterns and the potential for large-scale transmission of both fly- and environment-associated bacteria and their ARGs between different facilities.Obj. 2: Experimentally assess the ability of bovine pathogens to be disseminated by fly hostsMethods. Here, we will conduct controlled experiments to assess the ability of known mastitis-causing bacterial isolates to colonize, persist, and be disseminated by fly hosts using the housefly, M. domestica, as a model system. In brief, we will screen for the ability of E. coli, Klebsiella, Enterococcus, Staphylococcus, and/or Salmonella spp. to colonize and persist within the M. domestica gastrointestinal tract. Experiments will be performed both with known clinical strains (previously isolated from cows) and field strains isolated from samples collected in Obj. 1, each of which will be genetically modified to express a fluorescent protein. In brief, newly emerged M. domestica will be transferred to sterile containers containing autoclaved manure inoculated with defined quantities of individual bacterial isolates. Following 24 hours of exposure, flies will be moved to sterile 7.5-liter enclosed cages with continuous access to sterile water and a standard, sterilized diet. The presence and abundance of fluorescently labeled bacteria in individual fly guts, feces, and diet will then be examined at 1-, 3- and 5- days post-inoculation. Egg samples will also be collected five days post adult emergence. Diet, fecal, and egg samples will be homogenized, serially diluted, and plated on a permissive medium to compare the number of fluorescent bacteria to total bacterial abundance. Flies will first be vortexed in 1-ml of PBS to recover any external bacteria, followed by dissection of the entire gut prior to being subjected to either (i) homogenization and plating for bacterial colony counts, or (ii) fluorescent microscopy and qPCR using marker-specific primers to visualize/quantify the abundance/localization of fluorescent bacteria in the digestive tract. PBS homogenates containing any external bacteria will also be plated and subjected to qPCR.We will also characterize transstadial transmission of bacteria from the larval stages through adulthood. Larvae will be reared with fluorescent bacterial strains until pupation, when they will be surface sterilized and transferred to individual sterile vials. Upon immediate emergence (<6 hours), a cohort of adult flies will be homogenized and plated to determine abundance of each bacterium initially persisting through metamorphosis. Remaining adults will be transferred to individual vials containing sterile diet inoculated with defined microbial communities or PBS as a control. Counts of fly-associated bacteria will be collected at 1-, 3- and 5- days post transfer to determine if initially low abundance transstadial transmitted bacteria can proliferate in the fly host. Overall, this will help elucidate the role in transstadial transmission versus ingestion of microbes in shaping the fly gut microbiota.Expected Outcomes. Results from these experiments will provide novel insights into the survival and fate of known dairy cow pathogens upon contact and ingestion by flies. They will also establish infection models for follow-up work to identify bacterial genes involved in environmental persistence, survival, and transmission.