ABSTRACT
The Asian longhorned tick (Haemaphysalis longicornis) was first reported in the United States in 2017 and has since been detected in at least 17 states. This tick infests cattle and can produce large populations quickly due to its parthenogenetic nature, leading to significant livestock mortalities and economic losses. While H. longicornis has not been detected in Texas, species distribution models have identified southern Texas as a possible hospitable region for this tick. Southern Texas is currently home to the southern cattle tick (Rhipicephalus microplus), which can transmit the causative agent of cattle fever (Babesia bovis). With the potential for H. longicornis and B. bovis to overlap in southern Texas and their potential to negatively impact the national and global livestock industry, it is imperative to identify the role H. longicornis may play in the cattle fever disease system. A controlled acquisition and transmission experiment tested whether H. longicornis is a vector for B. bovis, with the R. microplus-B. bovis system used as a positive control. Transstadial (nymphs to adults) and transovarial (adults to larvae) transmission and subsequent transstadial maintenance (nymphs and adults) routes were tested in this study. Acquisition-fed, splenectomized animals were used to increase the probability of tick infection. Acquisition nymphs were macerated whole and acquisition adults were dissected to remove midguts and ovaries at five time points (4, 6, 8, 10, and 12 days post-repletion), with 40 ticks processed per time point and life stage. The greatest percentage of nymphs with detectable B. bovis DNA occurred six days post-repletion (20.0 %). For adults, the percentage of positive midguts and ovaries increased as days post-repletion progressed, with day 12 having the highest percentage of positive samples (67.5 % and 60.0 %, respectively). When egg batches were tested in triplicate, all H. longicornis egg batches were negative for B. bovis, while all R. microplus egg batches were positive for B. bovis. During the transmission phase, the subsequent life stages for transstadial (adults) and transovarial transmission/transstadial maintenance (larvae, nymphs, and adults) were fed on naïve, splenectomized calves. All life stages of H. longicornis ticks tested during transmission were negative for B. bovis. Furthermore, the transmission fed animals were also negative for B. bovis and did not show signs of bovine babesiosis during the 45-day post tick transmission period. Given the lack of successful transstadial or transovarial transmission, it is unlikely that H. longicornis is a vector for B. bovis.
ABSTRACT
BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.
Subject(s)
Antelopes , Babesia bovis , Babesiosis , Animals , Babesia bovis/genetics , Babesia bovis/pathogenicity , Babesia bovis/isolation & purification , Babesia bovis/immunology , Babesiosis/parasitology , Cattle , Antelopes/parasitology , Cattle Diseases/parasitology , Erythrocytes/parasitology , Texas , Virulence , Rhipicephalus/parasitology , Female , Polymerase Chain ReactionABSTRACT
Vector-borne diseases pose a significant threat to human and animal health worldwide. Their emergence is influenced by various factors such as environmental changes, host characteristics, and human behavior. The One Health approach is necessary to thoroughly investigate tick-borne diseases and understand the complex interactions between environmental, animal, and human health. Anthropogenic changes have impacted predators, leading to cascading effects on wildlife prey species and the emergence of vector-borne diseases. The increase in global trade and travel has led to the introduction of several invasive vector species, increasing the risk of zoonotic pathogen spillover. Tick and tick-borne disease research requires an interdisciplinary approach to address challenges in a One Health paradigm.
Subject(s)
One Health , Tick-Borne Diseases , Ticks , Animals , Humans , Animals, Wild , Tick-Borne Diseases/prevention & controlABSTRACT
In the United States, there has been a steady increase in diagnosed cases of tick-borne diseases in people, most notably Lyme disease. The pathogen that causes Lyme disease, Borrelia burgdorferi, is transmitted by the blacklegged tick (Ixodes scapularis). Several small mammals are considered key reservoirs of this pathogen and are frequently-used hosts by blacklegged ticks. However, limited studies have evaluated between-species host use by ticks. This study compared I. scapularis burdens and tick-associated pathogen presence in wild-caught Clethrionomys gapperi (southern red-backed voles) and Peromyscus spp. (white-footed mice) in forested areas where the habitat of both species overlapped. Rodent trapping data collected over two summers showed a significant difference in the average tick burden between species. Adult Peromyscus spp. had an overall mean of 4.03 ticks per capture, while adult C. gapperi had a mean of 0.47 ticks per capture. There was a significant association between B. burgdorferi infection and host species with more Peromyscus spp. positive samples than C. gapperi (65.8% and 10.2%, respectively). This work confirms significant differences in tick-host use and pathogen presence between sympatric rodent species. It is critical to understand tick-host interactions and tick distributions to develop effective and efficient tick control methods.
Subject(s)
Ixodes , Lyme Disease , Humans , Animals , Adult , Rodentia , Peromyscus , ArvicolinaeABSTRACT
Theileria orientalis Ikeda has caused an epidemic of bovine anemia and abortion across several U.S. states. This apicomplexan hemoparasite is transmitted by Haemaphysalis longicornis ticks; however, it is unknown if other North American ticks are competent vectors. Since the disease movement is largely determined by the host tick range(s), the prediction of the T. orientalis spread among U.S. cattle populations requires determination of additional competent tick vectors. Although Rhipicephalus microplus has mostly been eradicated from the U.S., outbreaks in populations occur frequently, and the U.S. remains at risk for reintroduction. Since R. microplus is a vector of Theileria equi and T. orientalis DNA has been detected in R. microplus, the goal of this study was to determine whether R. microplus is a competent vector of T. orientalis. Larval R. microplus were applied to a splenectomized, T. orientalis Ikeda-infected calf for parasite acquisition, removed as molted adults, and applied to two T. orientalis naïve, splenectomized calves for transmission. After 60 days, the naïve calves remained negative for T. orientalis by PCR and cytology. Additionally, T. orientalis was not detected in the salivary glands or larval progeny of acquisition-fed adults. These data suggest that R. microplus is not a competent vector of the U.S. T. orientalis Ikeda isolate.
ABSTRACT
OBJECTIVE: To evaluate the safety of repeated applications of permethrin concentrations (0% control, 1.5%, 5%, and 10%) to the necks and faces of horses and assess the efficacy and longevity of permethrin as an equine tick repellent. ANIMALS: 5 healthy adult Quarter Horses. PROCEDURES: Each treatment was applied to the neck of each horse (0.01 m2) 4 times a day, for up to 10 days. An 8-mm biopsy was taken to evaluate postexposure dermal responses. Any treatments that were not withdrawn were applied to a quadrant of the horse's face 4 times a day, for up to 5 days. For tick bioassays, a treatment was applied to 1 leg of a horse and 5 female blacklegged ticks (Ixodes scapularis) were evaluated as "repelled" or "not repelled" by the treatment. The bioassays were repeated up to 5 days, but treatment application took place only on the first day of the experiment. RESULTS: Histological results of neck biopsies indicated that more repeated exposures or higher concentrations resulted in more dermal damage. Tick bioassays showed that 5% and 10% permethrin had the greatest efficacy and longevity as a tick repellent, but the differences in tick repellency were not significant overall. CLINICAL RELEVANCE: While there was a nonsignificant trend of higher permethrin concentrations repelling more ticks with longer-lasting residual repellent effects, higher concentrations also produced greater skin damage after repeated exposures. These opposing findings emphasize the need for better tick prevention and control methods that balance safety and efficacy for the equine community.
Subject(s)
Dog Diseases , Horse Diseases , Ixodes , Horses , Animals , Female , Dogs , Ixodes/physiology , Permethrin/pharmacology , Permethrin/therapeutic use , Dog Diseases/drug therapyABSTRACT
The New World screwworm, Cochliomyia hominivorax, is a major parasite that causes myiasis in livestock, humans, and other warm-blooded animals in the western hemisphere. There is a permanent biological border that is maintained between Panama and Colombia, as it has been eradicated from North and Central America. However, it still exists in much of the Caribbean and South America causing an estimated annual loss of $3.6 billion dollars in South America alone. Less information is available for C. hominivorax in the Caribbean. Thus, here we examined its presence and genetic landscape in order to gain insights into this fly's distribution in this region. First, through sampling efforts, novel GPS (Global Positioning System) coordinates were collected. Second, the environmental correlates of those presence points were examined. Next, samples were sequenced in order to obtain a pairwise ΦIT genetic distance matrix. And lastly, this matrix was used to create a genetic landscape of divergence. The results of the genetic landscape show flies as more diverse in Trinidad and Tobago and less diverse in the Dominican Republic. This is perhaps due to the proximity of Trinidad to Venezuela and gene flow may be occurring between these two areas. This information will aid in screwworm surveillance and control programs by providing environmental correlates and a view into the distribution of these flies.
Subject(s)
Diptera , Myiasis , Animals , Humans , Diptera/genetics , Animals, Domestic/parasitology , Calliphoridae , Caribbean Region , Trinidad and TobagoABSTRACT
Deer keds, such as Lipoptena cervi Linnaeus (Diptera: Hippoboscidae), are blood-feeding flies from which several human and animal pathogens have been detected, including Borrelia burgdorferi sensu lato Johnson (Spirochaetales: Borreliaceae), the causative agent of Lyme disease. Cervids (Artiodactyla: Cervidae), which are the primary hosts of deer keds, are not natural reservoirs of B. burgdorferi sl, and it has been suggested that deer keds may acquire bacterial pathogens via co-feeding near infected ticks. We screened L. cervi (n = 306) and Ixodes scapularis Say (Ixodida: Ixodidae) (n = 315) collected from 38 white-tailed deer in Pennsylvania for the family Anaplasmataceae, Bartonella spp. (Hyphomicrobiales: Bartonellaceae), Borrelia spp., and Rickettsia spp. (Rickettsiales: Rickettsiaceae). Limited similarity in the bacterial DNA detected between these ectoparasites per host suggested that co-feeding may not be a mechanism by which deer keds acquire these bacteria. The feeding biology and life history of deer keds may impact the observed results, as could the season when specimens were collected. We separately screened L. cervi (n = 410), L. mazamae Róndani (n = 13), L. depressa Say (n = 10), and Neolipoptena ferrisi Bequaert (n = 14) collections from locations within the United States and Canada for the same pathogens. These results highlight the need to further study deer ked-host and deer ked-tick relationships.
Subject(s)
Deer , Diptera , Ixodes , Ixodidae , Lyme Disease , United States , Animals , Humans , Deer/parasitology , Lyme Disease/veterinary , Ixodidae/microbiology , Diptera/microbiologyABSTRACT
Many vector-borne diseases that affect humans are zoonotic, often involving some animal host amplifying the pathogen and infecting an arthropod vector, followed by pathogen spillover into the human population via the bite of the infected vector. As urbanization, globalization, travel, and trade continue to increase, so does the risk posed by vector-borne diseases and spillover events. With the introduction of new vectors and potential pathogens as well as range expansions of native vectors, it is vital to conduct vector and vector-borne disease surveillance. Traditional surveillance methods can be time-consuming and labor-intensive, especially when surveillance involves sampling from animals. In order to monitor for potential vector-borne disease threats, researchers have turned to the public to help with data collection. To address vector-borne disease and animal conservation needs, we conducted a literature review of studies from the United States and Canada utilizing citizen science efforts to collect arthropods of public health and veterinary interest from animals. We identified common stakeholder groups, the types of surveillance that are common with each group, and the literature gaps on understudied vectors and populations. From this review, we synthesized considerations for future research projects involving citizen scientist collection of arthropods that affect humans and animals.
ABSTRACT
BACKGROUND: White-tailed deer (Odocoileus virginianus) host numerous ectoparasitic species in the eastern USA, most notably various species of ticks and two species of deer keds. Several pathogens transmitted by ticks to humans and other animal hosts have also been found in deer keds. Little is known about the acquisition and potential for transmission of these pathogens by deer keds; however, tick-deer ked co-feeding transmission is one possible scenario. On-host localization of ticks and deer keds on white-tailed deer was evaluated across several geographical regions of the eastern US to define tick-deer ked spatial relationships on host deer, which may impact the vector-borne disease ecology of these ectoparasites. METHODS: Ticks and deer keds were collected from hunter-harvested white-tailed deer from six states in the eastern US. Each deer was divided into three body sections, and each section was checked for 4 person-minutes. Differences in ectoparasite counts across body sections and/or states were evaluated using a Bayesian generalized mixed model. RESULTS: A total of 168 white-tailed deer were inspected for ticks and deer keds across the study sites. Ticks (n = 1636) were collected from all surveyed states, with Ixodes scapularis (n = 1427) being the predominant species. Counts of I. scapularis from the head and front sections were greater than from the rear section. Neotropical deer keds (Lipoptena mazamae) from Alabama and Tennessee (n = 247) were more often found on the rear body section. European deer keds from Pennsylvania (all Lipoptena cervi, n = 314) were found on all body sections of deer. CONCLUSIONS: The distributions of ticks and deer keds on white-tailed deer were significantly different from each other, providing the first evidence of possible on-host niche partitioning of ticks and two geographically distinct deer ked species (L. cervi in the northeast and L. mazamae in the southeast). These differences in spatial distributions may have implications for acquisition and/or transmission of vector-borne pathogens and therefore warrant further study over a wider geographic range and longer time frame.
Subject(s)
Deer/parasitology , Diptera/physiology , Ixodidae/physiology , Tick Infestations/epidemiology , Tick Infestations/veterinary , Alabama/epidemiology , Animals , Bayes Theorem , Pennsylvania/epidemiology , Tennessee/epidemiologyABSTRACT
Deer keds (Diptera: Hippoboscidae: Lipoptena Nitzsch, 1818 and Neolipoptena Bequaert, 1942) are blood-feeding ectoparasites that primarily attack cervids and occasionally bite humans, while ticks may be found on cervids, but are more generalized in host choice. Recent detection of pathogens such as Anaplasma and Borrelia in deer keds and historical infections of tick-borne diseases provides reason to investigate these ectoparasites as vectors. However, previous methods employed to sample deer keds and ticks vary, making it difficult to standardize and compare ectoparasite burdens on cervids. Therefore, we propose a standardized protocol to collect deer keds and ticks from hunter-harvested deer, which combines previous methods of sampling, including timing of collections, dividing sections of the deer, and materials used in the collection process. We tested a three-section and a five-section sampling scheme in 2018 and 2019, respectively, and found that dividing the deer body into five sections provided more specificity in identifying where deer keds and ticks may be found on deer. Data from 2018 suggested that deer keds and ticks were found on all three sections (head, anterior, posterior), while data from 2019 suggested that more Ixodes scapularis were found on the head and deer keds were found on all body sections (head, dorsal anterior, dorsal posterior, ventral anterior, and ventral posterior). The protocol provides an efficient way to sample deer for deer keds and ticks and allows researchers to compare ectoparasite burdens across geographical regions. Furthermore, this protocol can be used to collect other ectoparasites from deer or other cervids.
Subject(s)
Deer/parasitology , Diptera/physiology , Entomology/methods , Ixodidae/physiology , Specimen Handling/veterinary , Animals , Ectoparasitic Infestations/parasitology , Indiana , Maryland , Pennsylvania , Specimen Handling/methods , Tick Infestations/parasitology , VirginiaABSTRACT
Deer keds (Diptera: Hippoboscidae: Lipoptena Nitzsch, 1818 and Neolipoptena Bequaert, 1942) are hematophagous ectoparasites of cervids that occasionally bite other mammals, including humans. In recent years, a number of arthropod-borne pathogens have been sequenced from deer keds. However, it is unclear if the pathogens are just present in host blood in the gut or if the pathogens are present in other organs (e.g., salivary glands) that would suggest that keds are competent vectors. Like other hippoboscoid flies, deer keds have extensive salivary glands that extend through the thorax and into the abdomen, so simply disarticulating and sequencing the thorax and abdomen separately does not circumvent the issues surrounding whole-body sequencing. Herein, we describe a technique for dissecting the terminal portion of the salivary glands from the abdomen in order to screen the thorax and salivary glands separately from the abdomen for arthropod-borne pathogens.
Subject(s)
Diptera , Dissection/veterinary , Abdomen , Animals , Deer/parasitology , Dissection/methods , Female , Male , Salivary GlandsABSTRACT
The number of recognized flea-borne pathogens has increased over the past decade. However, the true number of infections related to all flea-borne pathogens remains unknown. To better understand the enzootic cycle of flea-borne pathogens, fleas were sampled from small mammals trapped in central Pennsylvania. A total of 541 small mammals were trapped, with white-footed mice (Peromyscus leucopus) and southern red-backed voles (Myodes gapperi) accounting for over 94% of the captures. Only P. leucopus were positive for examined blood-borne pathogens, with 47 (18.1%) and ten (4.8%) positive for Anaplasma phagocytophilum and Babesia microti, respectively. In addition, 61 fleas were collected from small mammals and tested for pathogens. Orchopeas leucopus was the most common flea and Bartonella vinsonii subspecies arupensis, B. microti, and a Rickettsia felis-like bacterium were detected in various flea samples. To the best of our knowledge, this is the first report of B. microti DNA detected from a flea and the first report of a R. felis-like bacterium from rodent fleas in eastern North America. This study provides evidence of emerging pathogens found in fleas, but further investigation is required to resolve the ecology of flea-borne disease transmission cycles.
Subject(s)
Bartonella/pathogenicity , Siphonaptera/parasitology , Animals , Arvicolinae/parasitology , Babesia microti/parasitology , Babesia microti/pathogenicity , Male , Mammals/parasitology , Pennsylvania , Peromyscus/parasitology , Rickettsia felis/pathogenicity , Sciuridae/parasitologyABSTRACT
In 2012, the United States experienced one of the largest outbreaks of West Nile virus (WNV)-associated deaths, with the majority occurring in Dallas County (Co.), Texas (TX) and surrounding areas. In this study, logistic mixed models were used to identify associations between the landscape, human population, and WNV-infected Culex quinquefasciatus mosquitoes during the 2012 WNV epidemic in Dallas Co. We found increased probabilities for WNV-positive mosquitoes in north and central Dallas Co. The most significant predictors of the presence of WNV in Cx. quinquefasciatus pools were increased urbanization (based on an index composed of greater population density, lower normalized difference vegetation index, higher coverage of urban land types, and more impervious surfaces), older human populations, and lower elevation. These relationships between the landscape, sociodemographics, and risk of enzootic transmission identified regions of Dallas Co., TX with highest risk of spillover to human disease during the 2012 WNV epidemic.
Subject(s)
Culex , Spatial Analysis , West Nile Fever/epidemiology , Animals , Epidemics/statistics & numerical data , Humans , Mosquito Vectors , Population Density , Socioeconomic Factors , Texas/epidemiology , West Nile virusABSTRACT
As the threat of arboviral diseases continues to escalate worldwide, the question of, "What types of human communities are at the greatest risk of infection?" persists as a key gap in the existing knowledge of arboviral diseases transmission dynamics. Here, we comprehensively review the existing literature on the socioeconomic drivers of the most common Aedes mosquito-borne diseases and Aedes mosquito presence/abundance. We reviewed a total of 182 studies on dengue viruses (DENV), chikungunya virus (CHIKV), yellow fever virus (YFVV), Zika virus (ZIKV), and presence of Aedes mosquito vectors. In general, associations between socioeconomic conditions and both Aedes-borne diseases and Aedes mosquitoes are highly variable and often location-specific. Although 50% to 60% of studies found greater presence or prevalence of disease or vectors in areas with lower socioeconomic status, approximately half of the remaining studies found either positive or null associations. We discuss the possible causes of this lack of conclusiveness as well as the implications it holds for future research and prevention efforts.
ABSTRACT
Early warning systems for vector-borne diseases (VBDs) prediction are an ecological application where data from the interface of several environmental components can be used to predict future VBD transmission. In general, models for early warning systems only consider average environmental conditions ignoring variation in weather variables, despite the prediction from Schmalhausen's law about the importance of environmental variability for biological systems. We present results from a long-term mosquito surveillance program from Harris County, Texas, USA, where we use time series analysis techniques to study the abundance and West Nile virus (WNV) infection patterns in the local primary vector, Culex quinquefasciatus Say. We found that, as predicted by Schmalhausen's law, mosquito abundance was associated with the standard deviation and kurtosis of environmental variables. By contrast, WNV infection rates were associated with 8-month lagged temperature, suggesting environmental conditions during overwintering might be key for WNV amplification during summer outbreaks. Finally, model validation showed that seasonal autoregressive models successfully predicted mosquito WNV infection rates up to 2â¯months ahead, but did rather poorly at predicting mosquito abundance, a result that might reflect impacts of vector control for mosquito population reduction, geographic scale, and other artifacts generated by operational constraints of mosquito surveillance systems.
Subject(s)
Culicidae/growth & development , Weather , West Nile Fever/epidemiology , West Nile virus , Animals , Culicidae/virology , Humans , Mosquito Control , Mosquito Vectors , Texas/epidemiologyABSTRACT
African swine fever virus (ASFV) causes a high-consequence foreign animal disease that has emerged along international trade routes. Owing to high lethality and resulting trade sanctions, establishment of this disease in the United States would have devastating economic consequences. ASFV can be transmitted by soft ticks in the genus Ornithodoros or directly between swine, including domestic, feral, and wild swine. Consequently, the spatial risk of ASFV establishment depends on where susceptible animals, with or without competent vectors, co-occur. We synthesized county-level historical records of soft tick occurrence, current maps of feral swine distribution, and domestic swine inventory to evaluate the risk of ASFV establishment and spillover in the United States. Areas of California, Florida, and much of the southwestern United States were classified as high risk for ASFV establishment and spillover should an introduction event occur. Our analyses indicate that California, Texas, Georgia, and Florida are high-priority candidates for proactive risk reduction strategies. Domestic swine are often produced in high-biosecurity environments, mitigating health risks associated with contacting infected hosts and vectors. However, small-scale and organic pig producers in much of the southern United States remain more vulnerable to disease emergence.
Subject(s)
African Swine Fever/transmission , Arachnid Vectors , Ornithodoros , Swine , African Swine Fever/epidemiology , African Swine Fever Virus , Animal Distribution , Animals , Sus scrofa , Swine Diseases/virology , United States/epidemiologyABSTRACT
West Nile virus (WNV) and Flanders virus (FLAV) co-occur in regions of North America. Because both viruses are maintained in a transmission cycle involving Culex mosquitoes and birds, screening mosquitoes for FLAV has been suggested as an enhancement to WNV surveillance and epidemic prediction. Using samples collected in 2010 and 2012 in Chicago, IL, USA, we demonstrate the presence of FLAV in four out of 287 (1.4%) Culex pools. We estimated minimum infection rates for WNV and FLAV to be 5.66 and 1.22 in 2010 and 8.74 and 0.61 in 2012, respectively. FLAV occurred 1 and 3 wk prior to the peak of WNV transmission in 2010 and 2012, respectively. FLAV sequences from Chicago were genetically diverse and phylogenetically representative of lineage A viruses from across the United States.