Location: Arthropod-borne Animal Diseases Research
2022 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 2A: Describe species abundance, diversity, and habitat associations of larval and adult Culicoides communities collected on agricultural and wild sites in Northeastern Kansas which will facilitate improved, more targeted control strategies for midges.
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. Research under this objective was transferred to another ARS location in late 2021.
Objective 2. Research on the effect of Vesicular Stomatitis Virus (VSV) on gene expression in female midges (Culicoides sonorensis) is being performed in summer-fall 2022. Female midges were fed one of three treatments: blood spiked with VSV, control blood containing media without virus, or sugar, with a target of 3-6 biological replicates per treatment. RNA (e.g. genes that are expressed) will be sequenced from individual or whole midges (this process is currently being optimized). The aim is to identify genes whose expression (up or down) changes across treatments and controls, in order to elucidate their function in the midge during virus infection and to extrapolate the effect the virus imparts on the midge.
Objective 2. Collections of adult and larval Culicoides biting midges at agricultural sites (Kansas State University animal science units) and wild sites (Konza Prairie Biological Station) have continued for a second field season. Adult midges are being collected monthly at the wild sites and twice monthly on the agricultural sites using Centers for Disease Control and Prevention (CDC) miniature light traps. Species composition, abundance, and age structure of the midge community are being evaluated. To date, the wild sites are predominantly dominated by Culicoides haematopotus, Culicoides stellifer, and Culicoides crepuscularis while the agricultural sites see high numbers of those three species in addition to Culicoides variipennis and Culicoides sonorensis. The low abundance of Culicoides sonorensis at the wild sites could provide further evidence that this species is not the primary vector for Culicoides-borne pathogens in more natural habitats. In addition to adult sampling, larval midges are being collected monthly from the same sites, with larval species composition and abundance patterns mirroring adult collections except for greater abundance of C. variipennis being collected on wild rather than agricultural sites. Variation in preferred larval habitats is becoming apparent with species like C. crepuscularis and C. sonorensis preferring to use pond habitats while species like C. haematopotus, C. stellifer, and C. variipennis are using spring habitats. Collections for these datasets will be complete around the beginning of fiscal year (FY) 2023 and formal analyses of the species composition and abundance, habitat associations, and phenology of populations will be completed at that time.
Objective 3. Molecular investigations of bacterial carriage by house flies in U.S. cattle operations continued in FY 22. Female house flies were collected from two dairy farms in three climate zones (Florida, North Carolina, Tennessee) across 4 months and shipped to ARS for extraction and analysis of microbial communities. Climate and other explanatory data, such as operation size and management practices, were also recorded. DNA was extracted from whole flies and 16S rRNA genes were sequenced to categorize and quantify bacterial communities. At the time of this report, community sequencing and annotation is complete, but comparative analyses are ongoing (e.g. across date, site).
A separate study used culture-based analyses to determine the prevalence of antimicrobial-resistant (AMR) and multi-drug resistant (MDR) coliforms carried by house flies from Kansas feedlot and dairy operations in 2019. Bacteria cultured from flies were tested for resistance to five antibiotics: tetracycline, florfenicol, enrofloxacin, ampicillin, and ceftiofur. We previously found that the majority of AMR and MDR coliforms were resistant to tetracycline, ampicillin and/or florfenicol. In FY 2022, the identity of the AMR isolates carried by flies was determined by 16S sanger sequencing. Most AMR and MDR coliforms were identified as Escherichia/Shigella sp. or Klebsiella sp., regardless of farm type or fly sex. However, coliform identity was associated with resistance phenotype, where Escherichia/Shigella sp. were commonly tetracycline-resistant, while Klebsiella sp. were more often ampicillin-resistant. Whole genome sequencing of MDR isolates from this study also has been completed and informatic analyses that will help us understand the genetic underpinnings of AMR and MDR in these isolates is underway.
Accomplishments
1. House fly sex-specific feeding behavior and disease transmission and control. House flies carry and transmit disease-causing microorganisms, making them important to animal and human health. Females need dietary protein for egg production while males do not have this requirement and can subsist on sugar. ARS researchers in Manhattan, Kansas, in collaboration with researchers at Kansas State University, assessed whether fly sex influenced their preference for foods with varying nutrients such as those rich in carbohydrates, proteins, fats or without nutrients (water). Females preferred protein-rich foods and males preferred carbohydrate rich foods. Furthermore, females were 3 times more likely to forage on multiple food types than males. These findings indicate that pest-management companies or workers should consider sex-specific food preferences when designing bait-based fly control products aimed at targeting and killing both male and female house flies. Additionally, these results indicate that females may be more important than males in acquiring and spreading microbes as they were more likely to feed on multiple food types and sources, which is important in assessing risk of fly-transmitted diseases.
2. House flies have adapted their immune systems to live and thrive in filth. Flies have evolved unique strategies for flourishing in these microbe-rich environments, including many copies of genes coding for immune molecules, such as antimicrobial peptides (AMPs). Across insects, AMPs provide protection from harmful microbes and disease. ARS scientists in Manhattan, Kansas and Kerrville, Texas, in collaboration with researchers at Kansas State University determined which immune defenses house flies used during each of their life stages (egg, larva, pupa, and adult). The research revealed that over the course of evolution, house flies have adapted their immune system to live within filth environments and that they use these defense genes to help eat and digest bacteria that they encounter in their septic lifestyle. These molecules potentially can be targeted for fly control or explored as novel alternatives to antimicrobials.
3. Multidrug-resistant bacteria widespread in house flies and their environment. Livestock operations are the largest consumer of antibiotics, which are used to promote animal health and increase production. House flies are persistent pests in livestock operations and acquire bacteria when they feed and reproduce in production waste. ARS researchers in Manhattan, Kansas, in collaboration with Kansas State University, determined the prevalence of AMR and multi-drug resistant (MDR) bacteria in house flies and in environmental samples (manure, feed, water) that were collected from four different Kansas beef cattle operations in summer 2020. Bacteria were counted and tested for resistance to three different antibiotics commonly used in beef production: tetracycline, florfenicol, and enrofloxacin. Of the 246 house flies tested, 98.8% carried at least one AMR bacteria and 97.6% carried at least one MDR bacteria. Female house flies carried more bacteria than males, but the presence of MDR bacteria was similar between the sexes. At least 96% of manure compost and feed samples carried AMR bacteria and at least 91% carried MDR bacteria, while 100% of manure patty and water samples carried AMR and MDR bacteria. Resistance to all three antibiotics was observed within all sample types and locations. Considering the ability of flies to acquire and spread drug-resistant bacteria, as well as their frequent interactions with animals and their manure, managing house flies at confined cattle operations should be prioritized by producers in order to mitigate AMR and to protect human and animal health.
4. House flies at beef cattle operations carry bacterial communities. House flies are nuisance pests that flourish where animals are concentrated, such as feedlot operations, due to easy access to food and reproductive sites. Flies not only disturb animals but also serve as reservoirs and transmitters of bacteria, bridging the gap between sick and healthy animals as well as manure and feed. ARS researchers in Manhattan, Kansas, characterized bacteria carried by female house flies collected July-October 2020 from beef cattle operations in Kansas, Oklahoma and Texas. Across all sites, house flies carried foodborne pathogens of humans and cattle pathogens associated with pink eye and bovine respiratory disease. Bacterial communities found in individual flies at the same time and location were most similar to each other, indicating that microbes within the flies represent a snapshot of their environment. Therefore, female house flies not only pose a risk as reservoirs and spreaders of human and animal pathogens, but also can be used as sentinels by producers and scientists for monitoring pathogens in the locations where they are collected.
5. Biting midges prefer breeding habitats that are grazed by cattle or bison. Biting midges are blood-feeding pests that cause annoyance via painful bites and transmit deadly viral diseases to wild and domestic animals. Knowing key components that comprise the larval habitats midges prefer, including the chemical characteristics of the soil and the microbes therein, may help us predict where midges will emerge and take actions to suppress midge populations. ARS researchers in Manhattan, Kansas, and collaborators from Kansas State University characterized microbes and soil properties from potential midge habitats in both disturbed soils that had been grazed by cattle or bison, and undisturbed, non-grazed soils. The types of microbes in the habitat soil were significantly influenced by grazing type, and more midge larvae were found in grazed sites than in non-grazed sites. Midges were more likely to be present when a rich community of protists (single celled microbes) and fungi were present. The amount of carbon, nitrogen and organic matter was negatively correlated with midge presence. The increased prevalence of midges in grazed sites may indicate a preference for habitat with abundant hosts, who both provide microbes for the larvae with their manure while also serving as a source of food for blood-feeding adults. By improving our ability to identify larval midge habitats, more specific guidance can be provided to help ranchers target control and mitigation strategies to decrease disease transmission and improve herd health outcomes.
Review Publications
Neupane, S., Saski, C.A., Nayduch, D. 2021. House fly larval grazing alters dairy cattle manure microbial communities. BMC Microbiology. 21:1-14. https://doi.org/10.1186/s12866-021-02418-5.
Burgess, E.T., Taylor, E.E., Acevedo, A., Tworek, M., Nayduch, D., Khurana, N., Miller, J.S., Geden, C.J. 2021. Diets of Erythritol, Xylitol and Sucrose affect the digestive activity and gut bacterial community in adult house flies. Entomologia Experimentalis et Applicata. https://doi.org/10.1111/eea.13088.
Neupane, S., Nayduch, D. 2022. Effects of habitat and sampling time on bacterial community composition and diversity in the gut of the female house fly, Musca domestica Linnaeus (Diptera:Muscidae). Medical and Veterinary Entomology. https://doi.org/10.1111/mve.12581.
Geden, C.J., Nayduch, D., Scott, J.G., Burgess, E.R., Gerry, A.C., Kaufman, P.E., Thomson, J., Pickens, V., Machtinger, E.T. 2021. House fly (Diptera: Muscidae): biology, pest status, current management prospects, and research needs. Journal of Integrated Pest Management. 12(1):39, 1-38. https://doi.org/10.1093/jipm/pmaa021.
Cetinkaya, T., Mendes, A.C., Jacobsen, C., Ceylan, Z., Chronakis, I.S., Bean, S.R., Garcia-Moreno, P.J. 2020. Development of kafirin-based nanocapsules by electrospraying for encapsulation of fish oil. LWT - Food Science and Technology. https://doi.org/10.1016/j.lwt.2020.110297.
