Location: Arthropod-borne Animal Diseases Research
2024 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. The overall goal was to quantify the impact of mosquito blood feeding on cattle production. To identify cattle defensive behaviors and amount of time spent with each behavior, ear tags that identified resting, feeding, ruminating, and other behaviors were placed on dairy cattle and the weekly insect burden was quantified using an insect trap. Preliminary data using trail cameras indicated significant reductions in resting and ruminating behaviors when the trap collected more than 20 mosquitoes. On nights where traps collected few mosquitoes (less than 5), cattle exhibit typical behaviors. In 2021, ear tag monitoring of dairy cattle behavior continued along with seasonal collections during insect-free times (early spring, late fall, and winter) to provide patterns of behaviors to compare to spring, summer, and fall when biting insects are present. A clear change in cattle behavior was present during weeks of high insect abundance, compared to weeks when weather limited insect interaction with traps or cattle. Nocturnal biting insects affected time spent sleeping and ruminating (low energy activities) at night and increased the mid and high energy activities. Research under this objective was transferred to another ARS location in 2021 and did not continue.
Objective 2. The aim was to examine the effect of orbivirus infection on gene expression and behavior in biting midges. Over the course of the project, methodology was perfected to optimize microinjection efficacy, to perfect tissue separation techniques, to optimize RNA extraction techniques, and to identify optimal conditions, controls and reference genes for gene expression analysis. Research on the effect of Bluetongue virus (BTV) or vesicular stomatitis virus (VSV) on gene expression in female midges (Culicoides sonorensis) was performed. Female midges were either fed a virus treatment bloodmeal, a control blood meal with just virus media, or sugar. RNA was sequenced (RNAseq) and the counts of RNA were compared across conditions and normalized across 3 replicates. Informatic analysis revealed a great amount of variability between midge pools, and ongoing work is being modified to use single midges for transcriptomes. Genes, gene families, and networks were differentially expressed across the three treatments. Single-midge RNA isolation protocols were developed in order to assess transcriptome-level responses of individual female biting midges to vesicular stomatitis virus or bluetongue virus infection. From this same sample, assessment of infection status, i.e., via detection of viral RNA is also accomplished. A new bioinformatic pipeline was developed which will facilitate and improve differential expression analyses. In addition, transcriptomes allowed for selection and downstream testing of candidate housekeeping/reference genes to be used in qRTPCR, and at least two candidate genes were identified. As in vivo study optimization continues, the in vitro transcriptome response of C. sonorensis cell lines to VSV infection (vs. controls) was characterized. The transcriptomic response of VSV-infected cells at 1 h exposure was significant, but by 8 h there were no detectable differences between the transcriptome profiles of VSV-infected and mock infected cells although VSV was highly replicating in the treated cells. Genes involved in the insect immune response were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 h. VSV infection in midge cells therefore elicited an early immune response that quickly waned, indicating that midge cells may be tolerant to VSV infection.
Objective 2A. The aim of this objective, which was added in 2021, was to investigate and describe the species abundance, diversity, and habitat associations of larval and adult Culicoides at two field sites in northeastern Kansas: The Konza Prairie Biological Station (KPBS) and the Kansas State University (KSU) Animal Science Units. At each site, light traps were used to collect adult midges and mud was collected from larval habitats to investigate immature midge populations. In total, 19 Culicoides species and two subgenera complexes were collected in adult light traps. While both sites had a high abundance of C. crepuscularis, C. haematopotus, and C. stellifer, there were significantly more C. sonorensis collected at the KSU sites (10,212) with relatively few at KPBS (78). This finding shows that this species has a strong association with animal agriculture but is not prevalent in sylvatic environments, even within a short distance. This strengthens the evidence that other species likely act as bridge vectors in sylvatic environments, moving viruses between farms where the primary vector, C. sonorensis, then spreads the viruses amongst livestock. For this reason, targeting sylvatic midge communities and bridge vectors surrounding animal agriculture lands may be a more effective method of preventing outbreaks preemptively. Fewer species were found in larval collections from the two sites (including twelve species and one subgenus complex). Culicoides sonorensis was found predominantly in the dairy wastewater ponds at the KSU Dairy Teaching and Research Center. Interestingly, this species was not found in great numbers in other cattle-associated pond habitats at either KPBS or KSU indicating their extremely specialized larval habitat. Springs were the most common larval habitat sampled due to their availability throughout the prairie ecosystem, and species such as C. stellifer and C. variipennis, two suspected vector species for midge-borne viruses, were found to use these habitats more than other habitats on average. Other species were found more in streams (C. haematopotus) and pond habitats (C. crepuscularis), on average, although both species are believed to primarily feed on birds.
Objective 3. The main goal was to characterize bacterial communities associated with female house flies collected from confined cattle operations in several U.S. climate zones using molecular (16S bacterial community analysis via next-gen sequencing) or culture (selective culture, antimicrobial susceptibility testing; AST) approaches. For the molecular studies, the following was accomplished. Microbial communities of female house flies from dairy and beef cattle operations in Kansas, Nebraska and Oklahoma in September 2019 was performed. House fly bacterial community composition significantly varied between farm types and states. Flies carried abundant cattle and human pathogens. Bacterial species richness was significantly greater in flies from feedlots than dairy cattle farms. Both farm type and geographic location significantly influenced bacterial community composition and diversity. Female house flies and manure samples also were collected monthly from Oklahoma, Kansas and Texas dairy farms and beef feedlots. At both operations, house flies carried a high abundance and prevalence of human and animal pathogenic bacterial taxa, of note were Staphylococcus, Escherichia, and Moraxella, along with several dozen others. Bacterial species richness varied across geographic regions and dates. Location and month significantly affected bacteria phyla in flies. Bacterial community compositions of individual flies from the same date and location were most similar indicating that fly communities represent that of their surrounding environment. Bacterial carriage by house flies in U.S. dairy cattle operations was performed in three climate zones (Florida, North Carolina and Tennessee) once monthly May-August, 2021. Individual flies carried diverse bacterial communities, which encompassed 100% of the bacterial taxa also found in co-located cattle manure. Bacterial community assemblage within a farm was influenced by month. Bacterial diversity in individual flies varied greatly. Several potential pathogenic bacterial taxa were highly prevalent and abundant in both house flies and manure samples and some taxa were differentially associated with sample type. For the culture-based studies of fly microbial communities, the following was accomplished. The effects of fly sex, location and collection date on total bacteria and coliform abundance in house flies collected from Kansas feedlots and antimicrobial resistance (AMR) in a subset of coliforms was performed. Male and female flies were collected from feedlots in Riley, Washington, and Marion counties in Kansas (5 dates; August-October 2019). Individual flies were cultured to enumerate bacteria including total coliforms (TC). Female flies harbored more bacteria than males. Collection date, location and the interaction affected TC abundance in flies from Marion and Washington. Distinct colonies were selected from cultures for AST to tetracycline, florfenicol, enrofloxacin, ampicillin, and ceftiofur. The majority of AMR and multidrug resistant (MDR) coliforms were resistant to tetracycline, ampicillin and/or florfenicol. AMR isolates were identified by 16S sanger sequencing. Most AMR and MDR coliforms were Escherichia/Shigella sp. (commonly tetracycline-resistant) or Klebsiella sp. (more often ampicillin-resistant). Whole genome sequencing of isolates revealed resistance genes, and many were carried on plasmids, indicating transfer potential. Male and female flies, manure, feed and water also were collected from beef cattle operations in four Kansas counties. Flies and environmental samples were cultured on MacConkey agar to enumerate enteric bacteria and AST was performed. Fly sex and collection date significantly affected bacterial abundance. Within each site, females carried more bacteria than males. Bacterial abundance in environmental samples varied among operations. Flies carried the greatest variability in bacterial morphotypes, and many were similar to those in other samples. AST on these isolates is underway and will determine whether AMR and MDR isolates are shared across flies and other samples.
Accomplishments
1. Stage-specific usage of house fly defensin genes reflects distinct aspects of their filthy lifestyle. House flies have evolved an immune system full of redundant copies of genes for antimicrobial effectors, molecules that target and kill microbes like bacteria. These effectors are key players that protect flies from environmental microbes across all life stages (larvae, pupae, adults) and are also co-opted to help them eat bacteria as larvae. One family of antimicrobial effectors, the defensins, consist of twelve slightly different copies in the house fly genome and many show broad spectrum activity against a variety of microbes. ARS scientists in Manhattan, Kansas, in collaboration with researchers from the University of Houston, Texas, discovered that the genes coding for the different defensins are activated in a pattern that reflects aspects the fly’s biology. A subset of defensin genes were mostly turned on by larvae and pupae, who live immersed in a sea of microbes during their development in substrates like cattle manure. In contrast, another subset of defensins, that have distinct features compared to the larval/pupal copies of the gene, was activated by adult house flies, who only have ephemeral encounters with microbes during feeding or breeding activities. The unique patterns of defensin usage by house flies could reveal a vulnerability that could be exploited for controlling flies.
2. Bacteria carried by house flies at dairy cattle operations reflect animals and manure in their local environment. House flies flourish in dairy cattle operations where they have unlimited access to cattle manure which serves as both food and developmental substrate for their larvae. Female house flies perform efficient and effective sampling of both animals and their waste through their feeding and breeding habits, respectively, and subsequently are important reservoirs and potential transmitters of bacteria. ARS researchers in Manhattan, Kansas, collaborated with researchers at University of Tennessee, North Carolina State University, and University of Florida, to assess bacterial communities carried by female house flies collected from two dairy cattle operations in each of three states (Tennessee, North Carolina, Florida), once per month for 4 months. Across all locations, house flies carried both human and cattle pathogens, including those causing foodborne illnesses and mastitis, respectively. Interestingly, house fly microbial communities captured the entire microbial community of co-located manure, and the types of bacteria within the flies represented a snapshot of the bacteria found both in animals and manure from their surrounding environment. These findings show that house flies not only are a problem in carrying and disseminating pathogens, but also that that they can help us monitor threats to both livestock and human health at dairy cattle operations.
3. Harvesting pest flies from livestock facilities show potential as a sustainable source of protein in animal feed. The growing demand for protein in agriculture has necessitated the search for alternatives due to environmental concerns and high costs associated with traditional ingredients like fishmeal. A substantial amount of biomass already exists at many livestock facilities in the form of pest flies. If this untapped source of protein can be efficiently harvested, it would turn a huge problem into a valuable resource. Using a newly designed mass-harvesting trap, ARS scientists in Manhattan, Kansas, explored the feasibility, safety, and nutritional value of using wild-caught flies in animal feed. The first year of trapping yielded 3.0 kg of flies with the second year yielding 12.0 kg. This equates to the removal of well over 1.5 million flies from a single farm. Additionally, milling and heating the harvested flies significantly reduced bacterial loads, although further testing of pathogen removal is needed. The nutritional value of wild-caught flies was comparable to that of farm-raised insects, with key nutrients remaining intact despite the processing methods. These findings suggest wild-caught pest flies can be integrated into animal feed and provide an alternative to traditional protein sources, benefiting both agricultural productivity and sustainability. Current work is investigating various disinfection methods and assessing the digestibility of house fly diets.
Review Publications
Osborne, C., Cooper, A., Hall, B., Bird, E., Nayduch, D., Silver, K. 2023. Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses. Scientific Reports. 13. Article 16729. https://doi.org/10.1038/s41598-023-43750-2.
Scroggs, S.L., Bird, E.J., Molik, D.C., Nayduch, D. 2023. Vesicular stomatitis virus elicits early transcriptome response in Culicoides sonorensis cells. Viruses. 15(10). Article 2108. https://doi.org/10.3390/v15102108.
Asgari, D., Purvis, T.J., Pickens, V., Saski, C., Meisel, R.P., Nayduch, D. 2024. Expression of defensin genes across house fly (Musca domestica) life history gives insight into immune system subfunctionalization. Genome. https://doi.org/10.1139/gen-2024-0016.
Asgari, D., Nayduch, D., Meisel, R. 2024. Defensins of the stable fly have developmental-specific regulation and evolve at different rates. Integrative & Comparative Biology. https://doi.org/10.1093/icb/icae015.
Benn, J.S., Orange, J.P., Gomez, J., Dinh, E.T., McGregor, B.L., Blosser, E., Burkett-Cadena, N.D., Wisely, S.M., Blackburn, J.K. 2024. Culicoides midge abundance across years: Modeling inter-annual variation for an avian feeder and a candidate vector of hemorrhagic diseases in farmed wildlife. Viruses. 16(5). Article 766. https://doi.org/10.3390/v16050766.
Blosser, E.M., McGregor, B.L., Burkett-Cadena, N.D. 2024. A photographic key to the adult female biting midges (Diptera: Ceratopogondiae: Culicoides) of Florida, USA. Zootaxa. 5433(2):151-182. https://doi.org/10.11646/ZOOTAXA.5433.2.1.