Bacterial communities and prevalence of antibiotic resistance genes carried within house flies (Diptera: Muscidae) associated with beef and dairy cattle farms.

House flies (Musca domestica Linnaeus) are vectors of human and animal pathogens at livestock operations. Microbial communities in flies are acquired from, and correlate with, their local environment. However, variation among microbial communities carried by flies from farms in different geographical areas is not well understood. We characterized bacterial communities of female house flies collected from beef and dairy farms in Oklahoma, Kansas, and Nebraska using 16S rDNA amplicon sequencing and PCR. Bacterial community composition in house flies was affected by farm type and location. While the shared number of taxa between flies from beef or dairy farms was low, those taxa accounted >97% of the total bacterial community abundance. Bacterial species richness was 4% greater in flies collected from beef than in those collected from dairy farms and varied by farm type within states. Several potential pathogenic taxa were highly prevalent, comprising a core bacterial community in house flies from cattle farms. Prevalence of the pathogens Moraxella bovis and Moraxella bovoculi was greater in flies from beef farms relative to those collected on dairy cattle farms. House flies also carried bacteria with multiple tetracycline and florfenicol resistance genes. This study suggests that the house flies are significant reservoirs and disseminators of microbial threats to human and cattle health.

Effects of woody plant encroachment by eastern redcedar on mosquito communities in Oklahoma.

Woody plant encroachment into grasslands is occurring worldwide, affecting ecosystems in ways that likely influence mosquito-borne disease transmission. In the U.S. Great Plains, encroachment by eastern redcedar (Juniperus virginiana) (ERC) may be expanding conducive habitat for mosquitoes and their hosts, but few studies have evaluated associations between ERC encroachment and West Nile virus (WNV). To test the hypotheses that mosquito abundance and WNV-infected mosquitoes increase with increasing ERC cover, we collected mosquitoes in 32 sites in Oklahoma reflecting various ERC encroachment stages. We found support for our first hypothesis, as mean abundance of Aedes albopictus increased significantly with ERC cover. However, Psorophora columbiae and Anopheles quadrimaculatus abundance decreased with increasing ERC. There was no significant association with ERC for other mosquito species. We could not test our second hypothesis due to low WNV prevalence, but the only detected WNV-infected pool of mosquitoes (Cx. tarsalis) was collected in ERC. Our results suggest ERC encroachment increases abundance of at least one medically important mosquito species, but further research is needed to clarify how encroachment affects ecology of the entire WNV disease system through changes to vector and host communities, vector-host interactions, and thus disease transmission and prevalence. Understanding relationships between woody plant encroachment and the nidus of infection for mosquito-borne diseases will be crucial for targeting public health efforts, including land management activities that limit and/or eradicate woody plant encroachment, particularly in areas with high levels of disease risk.

Seasonality of Ticks and Prevalence of Rickettsiae Species in Dermacentor variabilis and Amblyomma maculatum Across Oklahoma Pastures.

Tick-borne diseases are an increasing concern for people and companion animals in the United States, but there is a need for continued vigilance regarding livestock in pasture systems. The south-central United States has some of the highest incidences of tick-borne diseases, and there is a need to re-examine the ecology of tick vectors in relation to pasture systems and livestock. The objective of this study was to establish a baseline of seasonal activity for tick species in diverse regional Oklahoma pastures and screen for important pathogens in Dermacentor variabilis (Say) and Amblyomma maculatum Koch group that may impact livestock and human health. Between 2015 and 2017, transects in five pastures across Oklahoma were visited each month. DNA extracted from adult D. variabilis and A. maculatum group was tested for the presence of bacterial pathogens. We found that tick communities in pastures across Oklahoma differ by season, abundance, and bacterial presence and prevalence. The peak abundance of Amblyomma americanum (L.) adults and nymphs occurred a month earlier over the 2 yr of the study compared with historical studies in the same regions. Additionally, we observed notable differences in peak activity between A. americanum adults and nymphs collected in pastures in central Oklahoma (April) versus pastures in northern part of the state (May). We detected Rickettsia parkeri, R. bellii, and Anaplasma sp. DNA in D. variabilis from pastures across the state. These results potentially have important ramifications for human and livestock risk of encountering infected ticks in pastures across the southern Great Plains.

An elution independent collection device (EICD) for rapid collection of Anaplasma marginale DNA from blood samples.

Currently utilized molecular detection methods are based mainly on nucleic acid extraction, amplification, and detection procedures that may require costly equipment, numerous reagents, and highly trained personnel. These requirements make diagnostic tests expensive, time-consuming, and not suitable for point-of-care applications. There is an increasing demand for simple, low-cost portable technologies. To overcome these challenges, a paper-based elution independent collection device (EICD) was designed to collect microorganisms and recover nucleic acids for molecular biology applications with minimal steps. In this study, we demonstrate a simpler Anaplasma marginale detection that uses an EICD for nucleic acid collection combined with recombinase polymerase amplification (RPA), and a lateral flow dipstick for detection of the specified target. A pre-lysis blood treatment was optimized that uses Triton X-100 lysis buffer and bovine serum album in wash buffer. Blood samples were incubated for 5 min at room temperature and run through the EICD. Four 1-mm diameter discs excised from EICD were used as template in basic RPA and lateral flow (nfo) (endonuclease IV) RPA assays. Each disc of soluble central membrane (SCM) carried circa 0.249 pg/µl of Anaplasma DNA. The percentage of nucleic acid recoverable from the SCM ranged between 60% - 70%. Blood samples infected with A. marginale were treated with Triton X-100 pre-lysis protocol. All samples tested positive by PCR and RPA methods. EICD-driven collection of blood samples is a practical method successfully adapted to detect Anaplasma spp. or blood-borne pathogen DNA and has potential for point-of-care detection in resource-limited settings.

Invasive Plants as Foci of Mosquito-Borne Pathogens: Red Cedar in the Southern Great Plains of the USA.

West Nile virus (WNV) is the most significant mosquito-borne disease affecting humans in the United States. Eastern redcedar (ERC) is a native encroaching plant in the southern Great Plains that greatly alters abiotic conditions and bird and mosquito populations. This study tested the hypotheses that mosquito communities and their likelihood of WNV infection differ between ERC and other habitats in the southern Great Plains of the United States. We found support for our first hypothesis, with significantly more Culex tarsalis and Culex erraticus in ERC than deciduous and grass habitats. Mosquito communities in Central Oklahoma were more diverse (21 species) than western Oklahoma (11 species) but this difference was not associated with vegetation. Our second hypothesis was also supported, with significantly more WNV-infected Culex from ERC in both regions, as was our third hypothesis, with significantly more Culex tarsalis and Culex pipiens collected in ERC than other habitats in urban areas. The connection of mosquito-borne disease with invasive plants suggests that land management initiatives can affect human health and should be considered in light of public health impact. Evidence from other vector-borne disease suggests invasive plants, both in the Great Plains and globally, may facilitate the transmission of vector-borne pathogens.

College Students' Knowledge of Ticks in Oklahoma: Assessment and Insights.

Ticks (Arachnida: Acari) are common in Oklahoma and may transmit tick-borne diseases (TBDs) to people. Due to the difficulty in reducing tick populations, awareness of tick bite prevention, proper tick removal, and knowledge of when to seek medical treatment are critical. However, outreach and extension programs are hampered by a lack of knowledge of what community members know about ticks. To address this limitation, we surveyed college students enrolled in three non-major Entomology courses at Oklahoma State University in 2018. Of the 483 students invited to take a survey, 224 (46.4%) students took both surveys. Pre-survey responses indicated lower levels of knowledge of tick biology compared to post-survey responses. For both pre- and post-survey respondents, "ticks can jump" and "ticks reside up in trees" received the fewest correct responses. A majority of survey respondents considered Lyme disease to be the predominant TBD in Oklahoma, although it is not established in Oklahoma. Supplemental education overcame these knowledge gaps, with the exception of knowledge of Lyme disease which was still considered to be the predominant TBD in the post-survey. Our results can be used to develop assessment tools to improve extension programs and enhance protection from TBDs.

Recombinase polymerase amplification (RPA) with lateral flow detection for three Anaplasma species of importance to livestock health.

Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited.

Landscape and Anthropogenic Factors Associated with Adult Aedes aegypti and Aedes albopictus in Small Cities in the Southern Great Plains.

As mosquito-borne diseases are a growing human health concern in the United States, the distribution and potential arbovirus risk from container-breeding Aedes mosquitoes is understudied in the southern Great Plains. The aim of the study was to assess landscape and anthropogenic factors associated with encountering adult container-breeding mosquitoes in small cities in southern Oklahoma. Collections were carried out over a 10 week period from June to August 2017 along two geographical transects, each consisting of three cities, equally distant from the Red River/Texas border. Mosquitoes were collected weekly using two trap types along with data for 13 landscape, vegetation, and anthropogenic variables. After five rounds of collection, 6628 female mosquitoes were collected over 2110 trap-nights involving 242 commercial or residential sites in six cities. Of the mosquitoes collected, 80% consisted of container-breeding species: Aedes albopictus (72%), Culex pipiens complex (16%) and Aedes aegypti (8%). Regionally, Aedes aegypti was more likely present in cities closest to the Texas border while Ae. albopictus was spread throughout the region. In general, Ae. aegypti and Ae. albopictus were significantly more present in sites featuring no or low vegetation and residential sites. Variables associated with Ae. albopictus presence and abundance varied between cities and highlighted the urban nature of the species. The study highlighted the distribution of Ae. aegypti geographically and within the urban context, indicated potential habitat preferences of container-breeding mosquito species in small towns, and demonstrated the usefulness of Gravid Aedes traps (GAT) traps for monitoring Aedes populations in urban habitats in small cities.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia.

BACKGROUND: The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. METHODOLOGY/PRINCIPAL FINDINGS: A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/µl (25µl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.

Climate Extremes, Vegetation Change, and Decoupling of Interactive Fire-Grazing Processes Exacerbate Fly Parasitism of Cattle.

We assessed local horn fly (Haematobia irritans L.) and face fly (Musca autumnalis De Geer) communities on cattle in 2012 and 2013 relative to vegetation and climate data to understand how parasitism of cattle is influenced by change in climate and vegetation structure. We compared heterogeneity management using spatially and temporally discrete fires (i.e., patch-burning one-third of a pasture annually) to homogeneity management (i.e., burning entire pasture in 2012 then no burning in 2013), with cattle grazing all years in both treatments. Predicted emergence of horn flies and face flies was 24 and 34 d earlier in 2012 associated with earlier spring warming, a significant deviation from the five-year mean. Intraannual horn fly dynamics were explained by concurrent high ambient air temperature the day of observations, but face flies were explained by low ambient air temperatures and dry conditions 3 wk before observations. Importance values of information for the theoretic models including fire treatments ranged from 0.89 to 1, indicating that both horn flies and face flies are sensitive to habitat alterations and fire-driven animal movements. Ordination indicates herds on unburned pastures were dissimilar to herds on pastures burned with patchy fires or pastures burned completely and species-specific fly responses to different vegetation structure metrics. For example, horn flies were correlated with vegetation visual obstruction, and face flies were correlated with woody plant cover. Vegetation structure may be as important as climate in driving the dynamics of fly parasites of cattle.

Transfer of Escherichia coli O157:H7 to spinach by house flies, Musca domestica (Diptera: Muscidae).

Filth flies are known mechanical vectors of pathogenic bacteria in hospital and restaurant settings, but their role as vectors for disseminating microbes to plants has not been demonstrated. Escherichia coli O157:H7 deposition by flies onto spinach was studied using molecular, microbiological, and microscopy techniques. Relative quantitative polymerase chain reaction studies showed that bacteria acquired by flies from contaminated cattle manure and deposited in regurgitation spots on leaves survived and multiplied. Scanning electron microscopy of the regurgitation spots of flies exposed to manure inoculated with E. coli suggested the multiplication of bacteria-like organisms within the spots. This finding implies that the bacteria were active and is consistent with a hypothesis that regurgitation spots serve as a nutrition source allowing E. coli O157:H7 to survive on the spinach phylloplane. E. coli O157:H7 persisted on fly body surfaces up to 13 days after exposure to acquisition sources, suggesting that fly cuticular surfaces are conducive to the growth of this pathogen. These results are consistent with the hypothesis of bioenhanced transmission of human pathogens by house flies and suggest that filth flies may affect the microbial safety of fresh produce.