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ARS Home Plains Area Manhattan, Kansas Center for Grain and Animal Health Research ABADRU Research





- Nayduch, Dana
- Rusch, Travis
- Shults, Phillip
- Molik, David
- Drolet, Barbara
- McGregor, Bethany
- 2023
- 2022
- 2021
- 2020
- Publications
- Animal Health (103)
- Veterinary, Medical and Urban Entomology (104)
- Food Safety, (Animal and Plant Products) (108)

Research Project: Biology and Management of Dipteran Pests of Livestock and Other Animals **Location:
                            Arthropod-borne Animal Diseases Research** 2020 Annual Report **Objectives**
                                

Objective 1: Conduct surveillance and evaluate the effect of nocturnal dipteran pests on dairy cattle and estimate their impact on production by quantifying defensive behaviors. These studies are intended to inform development of insect management strategies.

Objective 2: Develop new and improved strategies to reduce transmission risk between livestock and biting midges that are vectors of Bluetongue and Epizootic Hemorrhagic Disease.

Objective 3: Determine the risk of bacterial pathogen transmission by house flies and develop strategies to mitigate pathogen transmission. **Approach**
                                

Among insects, Dipteran species that significantly impact livestock and human health. The studies presented here focus on three key dipteran pests: mosquitoes, biting midges and house flies. Hematophagous mosquitoes and biting midges cause direct damage to the host during blood feeding, while vector species transmit disease agents that cause morbidity and mortality. House flies are nuisance pests to humans and livestock, and annoyance is exacerbated when animals are confined in high density. Being filth-associated, house flies also disseminate and transmit a wide variety of microbes, including pathogenic or antimicrobial-resistant species, especially in operations with poor waste management. The common purpose of the proposed project is to understand key components of the host-pathogen-vector cycle to: (1) estimate pest impact on livestock and/or human health, (2) inform mitigation and management strategies for reducing host contact and pest populations and (3) ultimately reduce or prevent pathogen transmission. The mosquito projects will quantify fitness and economic impacts using wearable technology while also evaluating efficacy of novel management strategies. The biting midge research uses transcriptomics to explore how virus infection alters sensory perception and neurological function in midges, providing information key to developing or modifying control methods. The house fly studies utilize both next-generation sequencing and culture-based approaches to characterize the bacterial microbiome in flies collected from cattle operations across four US climate zones. Data will be used to perform risk assessment, pathogen and antimicrobial-resistance surveillance and to identify biotic and management variables associated with changes in the fly associated microbial community. **Progress Report**
                                

Objective 1 is to quantify the impact of mosquito blood feeding on cattle production. Cattle defensive behaviors to daytime biting flies is well documented and fewer than 10 biting flies can have a significant impact on cattle weight gain. However correlating cattle behaviors with difficult to identify nocturnal biting insects such as mosquitoes that can number in the hundreds to thousands each night has not been demonstrated. To identify behaviors and amount of time used for each behavior, cattle ear tags that identified resting, feeding, ruminating, and other behaviors were placed and the weekly insect exposure was quantified using an insect trap. Preliminary data with trail cameras suggests significant reductions in resting and ruminating behaviors on calm nights when the trap collects more than 20 mosquitoes. On nights that are windy or rainy when the trap collects few mosquitoes (less than five), the cattle exhibit typical behaviors (eight hours of feeding, ruminating, and sleeping). 
Objective 2 aims to examine the effect of orbivirus infection on gene expression and behavior in biting midges. Optimization of procedures is paramount before the experiments commence. To determine microinjection efficacy, approximately 300 female midges were microinjected with phosphate buffered saline (PBS) and techniques were refined to optimize survival rates. To perfect tissue separation techniques, over 200 midges were dissected to remove heads with salivary glands from the body. To optimize RNA extraction techniques, pools of whole midges, bodies or heads with salivary glands were tested for RNA yields using a variety of protocols and procedures. Employing these optimized techniques, a preliminary experiment was performed. Given that febrile responses frequently accompany the period where bluetongue virus (BTV) is present in the blood of infected animals, blood meal temperature preferences of BTV-infected and uninfected Culicoides sonorensis female midges was evaluated. Midges were fed a blood meal, held for 2 days, then microinjected with saline (control) or BTV. After 5 days, midges were offered a blood meal at two temperatures: 37C or 42C. Midges, irrespective of treatment, preferred to feed on the warmer blood meal (42C), indicating that infection status with BTV did not affect blood meal temperature preference. 
In collaboration with Texas A&M researchers, the population genetics of the Culicoides variipennis biting midge species complex (a group of closely related species that are physically similar looking) was performed. The research group collected biting midges throughout the continental United States for genetic analysis. Results from this analysis revealed that instead of just three species, there are five. Despite being physically identical, not all the species transmit hemorrhagic disease viruses such as bluetongue or epizootic hemorrhagic disease virus; in fact, the geographic distribution of the species suggests only two are disease vectors.  Distinguishing these species is important to understand their distributions and the at-risk areas for viral transmission of vesicular stomatitis, epizootic hemohragic disease, and bluetongue.  
The main goal of Objective 3 is to characterize bacterial communities associated with female house flies collected from confined cattle operations. A preliminary analysis of female flies that were collected from dairy and cattle operations in Kansas, Nebraska and Oklahoma in September 2019 was performed. Female house flies were collected by sweep net, total DNA was extracted from individual flies, and high-throughput sequencing of bacterial 16S rRNA genes was performed. The bacterial community composition in house flies significantly varied between farm type and across states. Interestingly, bacterial species richness was significantly greater in flies from feedlots compared to dairy cattle farms, and both farm type and geographic location significantly influenced bacterial diversity. Both dairy and feedlot flies predominantly carried bacteria from the phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Significant effects of farm type and geographic location on the abundance of Proteobacteria and Bacteroidetes were observed. Only geographic location significantly influenced the abundance of Firmicutes and Actinobacteria. Bacterial communities and diversity within female house flies was significantly influenced by farm type and geographic location. Further analyses are being performed in order to determine the risk adult female house flies from feedlot and dairy farms pose as reservoirs of potential human and animal pathogens.
A separate study was conducted to assess the effects of fly sex, location and collection date on coliform abundance in house flies collected from Kansas feedlots as well as tetracycline resistance in a subset of coliforms. Male and female house flies were collected from three feedlot operations in Riley, Washington and Marion counties in Kansas across 5 dates (August-October 2019). Individual flies were processed and cultured on violet-red bile agar (VRBA) to enumerate colony forming units (CFUs) of total coliforms (TC). A general linear model was implemented to determine fly sex, date, feedlot location (fixed effects) on TC abundance in flies. Overall, female flies harbored more TC than male flies. Collection date, feedlot location and the interaction of both variables affected TC abundance in flies from Marion and Washington county feedlots, but not Riley. The interaction of sex and collection date was significant only in flies from Riley county. Distinct bacterial colonies (morphotypes) were selected from house fly cultures from two dates (August 27, September 10) and tested for resistance to tetracycline. Irrespective of fly sex, location and date, 113/186 morphotypes (61%) were tetracycline resistant. 89% of male and 92% of female flies harbored at least one tetracycline-resistant morphotype. In addition, 16/28 (57%) male and 13/24 (54%) female flies harbored more than one tetracycline-resistant morphotype. Although there was no apparent sex effect on carriage of tetracycline-resistant coliforms by flies, females overall harbored more coliforms across locations, which implies that female flies may serve as more significant reservoirs of antimicrobial-resistant coliforms. Isolates from the rest of the collection dates as well as dairy farm flies are being tested for tetracycline resistance. Together these analyses will help determine the role house flies play as reservoirs for antimicrobial-resistant bacteria at dairy and feedlot operations, which also has potential impacts on food safety. **Accomplishments**
                                

1. Environmental and sex effects on house fly bacterial carriage, including antimicrobial-resistant strains. House flies are primary pests of confined livestock operations such as dairies. Due to their close associations with humans, they also are nuisance pests in domestic settings. Adult flies frequent microbe-rich substrates such as garbage dumpsters and animal manure, where they encounter and ingest bacteria during feeding and reproduction activities. Due to their sex-specific nutritional needs and behaviors, male and female flies demonstrate different interactions with these substrates which ultimately affects bacterial acquisition and dissemination risk. The abundance of culturable aerobic bacteria and coliforms in male and female flies (n=107) collected from urban (restaurant dumpsters) and agricultural (dairy farm) sites was determined. Bacteria was enumerated from whole flies on nonselective media (tryptic soy agar; total culturable bacteria) and selective media (Violet-red bile agar, VRBA; coliforms). Female flies harbored more total bacteria than male flies and there was a sex by site interaction with sex effects on bacterial abundance in flies collected from the urban dumpsters. Coliform abundance did not differ among male and female flies, across sites, or between fly sex within sites. To assess human and animal health risks, unique bacterial colonies were selected from the VRBA cultures of agricultural flies, identified to species, and tested for susceptibility to 14 antimicrobials.  Both male and female flies carried antimicrobial-resistant (AMR) bacteria: 36/38 isolates (95%) were resistant to more than one antimicrobial, 33/38 were multidrug-resistant (MDR; being resistant to two or more antimicrobials), and 24/38 isolates were resistant to more than 4 antimicrobials. These results emphasize the role flies play in harboring and disseminating bacteria, including AMR and MDR strains that pose a risk to human and animal health, and add to the growing evidence implicating flies as major players in disease ecology, epidemiology and the dispersal of AMR genes. 2. House flies carry and can potentially transmit bacterial pathogens associated with bovine respiratory disease. House flies are major nuisance pests at feedlots and are of concern to animal health since they acquire, harbor and transmit numerous pathogens. Bovine respiratory disease (BRD) is an economically-important, complex illness of cattle associated with several bacterial species and viruses. The role that flies play in harboring and transmitting bacterial pathogens associated with BRD is not understood. House flies were collected from a commercial feedlot where cattle were suffering from apparent respiratory illness. Two different methods were used to examine the prevalence of the three main BRD bacterial pathogens: Mannheimia haemolytica, Pasteurella multocida and Histophilus somni in male and female flies. Using both methods, M. haemolytica was detected in 11.7% of house flies, followed by P. multocida (5.0%) and H. somni (3.3%). The presence of these BRD bacterial pathogens in house flies bolsters their role as reservoirs and disseminators of the bacteria in the feedlot environment. Further, infected flies pose a risk transmitting BRD when they acquire pathogens from sick animals and subsequently associate with healthy animals. **Review Publications**
                            *Cohnstaedt, L.W., Alfonso Parra, C. 2019. Highlights of mosquito and vector biology and control in Latin America. Journal of the American Mosquito Control Association. 35:40-46.*
                                *Neupane, S., Nayduch, D., Zurek, L. 2019. House flies (Musca domestica) pose a risk in carriage and transmission of bacterial pathogens associated with Bovine Respiratory Disease (BRD). Insects. 10:358. https://doi.org/10.3390/insects10100358.*
                                *Morales-Hojas, R., Hinsley, M., Armeam, I., Silk, R., Harrup, L., Gonzalez-Uriarte, A., Veronesi, E., Campbell, L., Nayduch, D., Saski, C., Tabachnick, W., Kersey, P., Carpenter, S., Fife, M. 2018. The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for Bluetongue virus. BMC Genomics. 19:624. https://doi.org/10.1101/249482.*
                                *Nayduch, D., Zurek, K., Thomson, J., Yeater, K.M. 2018. Effects of bacterial dose and fly sex on persistence and excretion of Salmonella enterica serovar Typhimurium from adult house flies (Diptera: Muscidae). Journal of Medical Entomology. 55:1264-1270. https://doi.org/10.1093/jme/tjy055.*
                                *Nayduch, D., Shankar, V., Mills, M., Robl, T., Drolet, B.S., Ruder, M., Scully, E.D., Saski, C. 2019. Transcriptome response of female Culicoides sonorensis biting midges (Diptera: Ceratopogonidae) to early infection with epizootic hemorrhagic disease virus (EHDV-2). Viruses. 11:473. https://doi.org/10.3390/v11050473.*
                                *Oliveira, A., Cohnstaedt, L.W., Noronha, L.E., Mitzel, D.N., McVey, D.S., Cernicchiaro, N. 2020. Perspectives regarding the risk of introduction of the Japanese encephalitis virus (JEV) in the United States. Frontiers in Veterinary Science.  https://doi.org/10.3389/fvets.2020.00048.*
                                *Neupane, S., White, K., Thomson, J.L., Zurek, L., Nayduch, D. 2020. Environmental and sex effects on bacterial carriage by adult house flies (Musca domestica L.). Insects. 11:401. https://doi.org/10.3390/insects11070401.*


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