Location: Arthropod-borne Animal Diseases Research
2019 Annual Report
Objectives
Objective 1: Perform risk assessment of bacterial pathogen transmission by house flies.
Sub-objective 1.A: Develop more effective larval control techniques by understanding the role of microbes in larval development and fitness.
Sub-objective 1.B: Evaluate the role of fly-bacteria and bacteria-bacteria interactions in house fly pathogen transmission.
Objective 2: Determine biological characteristics of mosquito vectors influencing animal health in a changing climate.
Sub-objective 2.A: Model mosquito ecological niches and impact of climate change.
Sub-objective 2.B: Characterize the biology of discrete mosquito populations.
Objective 3: Develop methods to reduce biting midge transmission of animal pathogens.
Sub-objective 3.A: Identify and characterize the salivary protein components of Culicoides sonorensis.
Sub-objective 3.B: Identify potential Culicoides vectors of epizootic hemorrhagic disease and bluetongue.
Sub-objective 3.C: Determine breeding site characteristics of Culicoides spp..
Sub-objective 3.D: Evaluate efficacy of candidate pesticides against C. sonorensis.
Approach
An extremely small percentage of insect species transmit disease-causing pathogens to animals and humans. Specific biological and behavioral characteristics allow these vector insect species to be efficient means of pathogen propagation and transmission; however these same characteristics may be targeted by control measures to limit pathogen spread or disease vector abundance. The common purpose of these projects is to understand key components of the host-pathogen-vector cycle to reduce or prevent pathogen transmission by the most common disease vectors: house flies, mosquitoes, and biting midges (Fig. 1). House flies associate with bacteria-rich environments due to the nutritional requirements of their larvae. This research defines the role of bacteria in fly development, bacterial persistence during microbe and insect interactions, and pathogen dissemination. Natural selection for increased Culex tarsalis mosquito fitness for various habitats and animal hosts has left genetic markers (single nucleotide polymorphisms) throughout the genome. These markers can be associated with traits and used to predict regional entomological risk in a changing climate throughout the mosquito’s large geographic range. The identification of biting midges or Culicoides saliva components that facilitate pathogen transmission will lead to improved transmission and pathogenesis models. This information will enhance development of vaccines and other countermeasures to reduce disease transmission. Lastly, not all Culicoides are competent vectors and this study will determine vector species and their habitats to help estimate risk in specific geographic regions. This plan aims to limit pathogen transmission by targeting the connections between hosts, vectors, and their environments via the insects’ unique characteristics using novel disease control methods.
Progress Report
Objective 1, Subobjective 1a. In this subobjective, transstadial carriage of viable bacteria in house flies was studied, which is important in understanding the role of this phenomenon in bacterial carriage and dissemination potential. Additionally, using a molecular (MiSeq) approach, housefly larval modification of the microbial community of dairy cattle manure during development (egg to pupae) was described. Results revealed that fly larvae greatly utilize rumen ciliates for nutrition, and modify some bacterial species over time. This is the first description of housefly larval utilization of the microbial community in manure and identified possible targets that could be modified to hinder development.
Objective 1, Subobjective 1b. Interactions between bacteria and the housefly ultimately determine microbe fate and transmission potential. In a series of studies, it was determined that variables including bacterial species, bacterial dose and fly sex all impact acquisition, persistence, excretion and transmission of bacteria by house flies. Some studies had particular focus on the livestock and human pathogen Salmonella, and indicated that female flies acquire and harbor a greater abundance of this pathogen, while male flies excrete bacteria more frequently. This knowledge helps in assessing risk flies pose in vectoring pathogens that impact human and animal health.
Objective 2 was to understand the relationships between mosquito species and their environment and how this effects mosquito movement, the epidemiology of disease vectored pathogens, and the entomological risk to humans and livestock. In subobjective 2a, Genetic, mathematical, epidemiological, and network models were used to define mosquito sub-populations and the movement between areas throughout the continental United States. The past five years have focused on the genetics of two major disease vectors Culex tarsalis and Aedes vexans and their transmission of endemic and exotic viruses. Significant findings include the putative origins of the mosquito species and the subsequent geographic spread throughout the continent. Subobjective 2b, used this disease vector distribution and movement information in epidemiological models, ARS scientists and Kansas State University researchers estimated the risk of foreign mosquito transmitted pathogens such as Japanese encephalitis, Rift Valley fever, and Zika viruses. In the event of a foreign virus introduction, intentionally or accidently, mathematical and network models were used to optimize the possible mosquito or livestock management measures to limit the size of the outbreak. The predictive epidemiological and mathematical models were then used to forecast outbreaks based on historic case data of endemic West Nile virus and the other previously mentioned exotic viruses. Overall, this research has elucidated the role of mosquito vector movement and the importance of reducing contact with reservoirs (cattle, pigs, migratory birds, and humans). The research has identified geographic areas of highest risk to exotic virus introduction and the likely routes of spread to new areas within the United States. Together both subobjectives provide response planners, mosquito control districts, and public health agencies basic information about the movement of mosquitoes and pathogens on a continental scale, which allows for better mosquito mitigation.
Within Objective 2, a national mosquito monitoring program called the Invasive Mosquito Project (IMP) was founded to monitor invasive mosquitoes throughout the U.S. The citizen science-based network received over 800 mosquito submissions from schools during the five-year period. The submissions indicate the IMP has successfully been integrated into educational curricula around the country, which will enable niche mosquito sampling from any location within the continental U.S. This education program helps participants understand the risk of mosquitoes and the role of the individuals in protecting their families and communities.
For Objective 3a, ARS scientists in Manhattan, Kansas, identified 45 proteins in midge saliva, which are deposited into the dermis of animals during midge feeding and are critical for successful acquisition of a bloodmeal. Orbivirus-infected midges deposit virus along with the salivary proteins. Prior to this research, the extreme efficiency with which midges can transmit some viruses was not clearly understood, as much was still unknown about the physiological trauma of the bite and immune responses to saliva deposited during feeding. Of interest were the first few hours and days after the bite; a critical time for orbiviruses to quickly establish a localized infection and disseminate, while avoiding the hosts’ immune responses. A mouse model was used to demonstrate midge feeding elicits a potent pro-inflammatory Th-mediated cellular response with significant mast cell degranulation, subcutaneous hematomas, hypodermal edema and dermal capillary vasodilation, and rapid infiltration of leukocytes to the bite sites. Mast cell degranulation was key to physiological and immunological responses and the ability of midges to feed to repletion. These responses were highly favorable for rapid infection and systemic dissemination midge-transmitted orbiviruses. Virus-susceptible cells are recruited directly to bite sites, drain to lymph nodes, which become hyperplastic, and results in robust viral replication and dissemination via the lymph system. Additionally, saliva-induced vasodilation and direct breaches in dermal capillaries by biting mouthparts exposes susceptible vascular endothelial cells, thereby providing immediate sites of virus replication and dissemination via the circulatory system. This research has advanced our understanding of the complex myriad of proteins in Culicoides midge saliva and provided insights into their functional role in blood feeding, arbovirus transmission, and arboviral disease pathogenesis.
Subobjective 3b, bottle bioassays have been used to assess the efficacy (mortality) of various pyrethroids on colony biting midges. Biting midges are incredibly susceptible to pyrethroids and are “knocked down” and rendered immobile almost immediately from contact with miniscule amounts of active ingredient. However, the resulting immobilization reduced contact with the insecticides and less insecticide entered the insects, which resulted in low mortality uncommon with pyrethroids pesticides. After a 24-hour recovery period almost all biting midges will recover post exposure, therefore higher doses than originally calculated are necessary. This important research established baseline levels of time and pesticide concentration necessary to detect the evolution of pesticide resistance in field populations.
Accomplishments
1. House fly larvae change the microbial community in manure. Being microbe-rich, cattle manure serves as a developmental substrate for house fly larvae. The larvae feed on microbes in the manure, but little is known about the influence that larval grazing has on manure bacterial communities over time. ARS scientists in Manhattan, Kansas, collaborated with Kansas State University, to determine changes in the manure bacterial community that occur after housefly larval feeding and development. The study compared composition, structure and bacterial diversity between manure that had larvae developing within and to age-matched manure without larvae using a molecular approach (sequencing 16S rDNA on MiSeq platform). These sequences, which represent all the bacteria in the sample, were examined and bacteria species were reduced as well as other taxonomic levels such as Phyla. Statistical analyses revealed that the composition (e.g. species present) of the bacterial community in manure that supported larval development (which lasted 10 days) was significantly different from the control manure (day 0 of the experiment) and the age-matched (day 10) manure without larvae. Bacterial species richness (e.g. the number of species in a community) and diversity of these species were both significantly lower in manure that contained larvae at day 10 compared to both control groups of manure. Relative abundances of the phyla Bacteroidetes and Proteobacteria significantly increased after larval feeding, whereas relative abundance of the phylum Firmicutes decreased, when larval treatment day 10 was compared to both control day 0 and control day 10. These results demonstrate that housefly larval feeding substantially alters bacterial community composition and diversity in cattle manure. Further analyses of this dataset are aimed at determining how larval grazing influenced the presence of pathogenic bacteria in cattle manure, in order to understand whether housefly presence affects the risk of manure-associated pathogen transmission to humans.
2. Habitat shapes the bacterial communities in the gut of female house flies. Adult house flies feed and breed in wide range of microbe–rich habitats and harbor and transmit bacteria including many human and animal pathogens. This study aimed to evaluate the bacterial communities in wild-caught flies and assessed the influence of habitat on community composition in order to understand the role house flies play as a risk to humans and other animals. ARS scientists in Manhattan, Kansas, in collaboration with Kansas State University, characterized the bacterial communities in the gut of female house flies collected from three different environments in Kansas: agricultural (beef cattle feedlot), urban (business area dumpsters) and mixed environment (business located near agriculture) using next-generation sequencing of the bacterial 16S rRNA gene. The taxonomic types and specific species of bacteria associated with flies differed between flies collected from different sites but very little variation occurred among flies collected from the same site. We found potential human pathogenic bacteria associated with flies from the mixed environment and feces-associated bacteria in flies from both the agricultural and urban environment. Our results show that the housefly gut harbors complex bacterial communities, including potential human and animal pathogens, and that community composition is strongly influenced by the habitat within which the flies are located.
3. The role of Culicoides midge salivary proteins in blood feeding and orbivirus transmission. ARS scientists in Manhattan, Kansas, identified 45 proteins in midge saliva which are deposited into the dermis of animals during midge feeding and are critical for successful acquisition of a bloodmeal. Orbivirus-infected midges deposit virus along with the salivary proteins. Prior to this research, the extreme efficiency with which midges can transmit some viruses was not clearly understood, as much was still unknown about the physiological trauma of the bite and immune responses to saliva deposited during feeding. Of interest were the first few hours and days after the bite; a critical time for orbiviruses to quickly establish a localized infection and disseminate, while avoiding the hosts’ immune responses. Using a mouse model, we showed midge feeding elicits a potent pro-inflammatory Th-mediated cellular response with significant mast cell degranulation, subcutaneous hematomas, hypodermal edema and dermal capillary vasodilation, and rapid infiltration of leukocytes to the bite sites. Mast cell degranulation was key to physiological and immunological responses and the ability of midges to feed to repletion. These responses were highly favorable for rapid infection and systemic dissemination midge-transmitted orbiviruses. Virus-susceptible cells are recruited directly to bite sites, drain to lymph nodes, which become hyperplastic, and results in robust viral replication and dissemination via the lymph system. Additionally, saliva-induced vasodilation and direct breaches in dermal capillaries by biting mouthparts exposes susceptible vascular endothelial cells, thereby providing immediate sites of virus replication and dissemination via the circulatory system. This research has advanced our understanding of the complex myriad of proteins in Culicoides midge saliva and provided insights into their functional role in blood feeding, arbovirus transmission, and arboviral disease pathogenesis.
Review Publications
Swanson, D.A., Kapaldo, N., Maki, E.C., Carpenter, J., Cohnstaedt, L.W. 2018. Diversity and abundance of non-culicid biting flies (Diptera) in a zoo environment. Journal of the American Mosquito Control Association. 34:4.
Pages, N., Cohnstaedt, L.W. 2018. Mosquito-borne diseases in the livestock industry. In: Garros, C., Bouyer, J., Takken, W. and Smallegange, R.C., editors. Pests and vector-borne diseases in the livestock industry. Ecology and control of vector-borne diseases. 5th Edition. Wageningen Academic Publishers. Pages: 195-219. https://doi.org/10.3920/978-90-8686-863-6_8.
Riad, M.H., Scoglio, C., McVey, D.S., Cohnstaedt, L.W. 2017. An individual-level network model for a hypothetical outbreak of Japanese Encephalitis in the USA. Stochastic Environmental Research and Risk Assessment (SERRA). 31:2.
Snyder, D., Cernicchiaro, N., Cohnstaedt, L.W. 2015. Sugar-feeding status alters biting midge photoattraction. Medical and Veterinary Entomology. 30:31-38.
Xue, L., Scoglio, C., McVey, D.S., Cohnstaedt, L.W. 2015. Two introductions of Lyme disease into Connecticut: A geo-spatial analysis of human cases from 1984 to 2012. Journal of the American Medical Association. http://doi.org/10.1089/vbz.2015.1791.
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.
