Phenology of five tick species in the central Great Plains.

The states of Kansas and Oklahoma, in the central Great Plains, lie at the western periphery of the geographic distributions of several tick species. As the focus of most research on ticks and tick-borne diseases has been on Lyme disease which commonly occurs in areas to the north and east, the ticks of this region have seen little research attention. Here, we report on the phenology and activity patterns shown by tick species observed at 10 sites across the two states and explore factors associated with abundance of all and life specific individuals of the dominant species. Ticks were collected in 2020-2022 using dragging, flagging and carbon-dioxide trapping techniques, designed to detect questing ticks. The dominant species was A. americanum (24098, 97%) followed by Dermacentor variabilis (370, 2%), D. albipictus (271, 1%), Ixodes scapularis (91, <1%) and A. maculatum (38, <1%). Amblyomma americanum, A. maculatum and D. variabilis were active in Spring and Summer, while D. albipictus and I. scapularis were active in Fall and Winter. Factors associated with numbers of individuals of A. americanum included day of year, habitat, and latitude. Similar associations were observed when abundance was examined by life-stage. Overall, the picture is one of broadly distributed tick species that shows seasonal limitations in the timing of their questing activity.

New hard tick (Acari: Ixodidae) reports and detection of Rickettsia in ticks from Sierra Nevada de Santa Marta, Colombia.

The Sierra Nevada de Santa Marta (SNSM), located in northern Colombia, is considered a geographical island with high levels of biodiversity and endemism. However, little is known about tick species and their associated microorganisms at the SNSM. In this study we sampled host-seeking ticks in areas of the town of Minca within the SNSM. We collected 47 ticks identified as Amblyomma pacae, Amblyomma longirostre, Amblyomma ovale, Amblyomma mixtum, Haemaphysalis juxtakochi, Ixodes sp. cf. Ixodes affinis and Ixodes sp. Of these ticks, we tested for Rickettsia spp. by amplifying the gltA, SCA1, and 16S rRNA genes via PCR. Rickettsia amblyommatis was detected in one pool of 3 larvae and in a female of A. pacae. Additonally, we isolated Rickettsia sp. belonging to the group of spotted fevers in larvae of A. longirostre. This study reports new findings of six species of ticks and two species of Rickettsia within the SNSM.

High-resolution prediction models for Rhipicephalus microplus and Amblyomma cajennense s.l. ticks affecting cattle and their spatial distribution in continental Ecuador using bioclimatic factors.

In Ecuador, the main tick species affecting cattle are Rhipicephalus microplus and Amblyomma cajennense sensu lato. Understanding their spatial distribution is crucial. To assess their distribution, data from 2895 farms visited between 2012 and 2017 were utilized. Ticks were collected during animal inspections, with each farm's location georeferenced. Bioclimatic variables and vapor pressure deficit data were obtained from Climatologies at High resolution for the Earth´s Land Surface Areas (CHELSA) dataset. They were overlaid to develop predictive maps for each species using Random Forest (RF) models. The cross-validation results for RF prediction models showed high accuracy for both R. microplus and A. cajennense s.l. presence with values of accuracy = 0.97 and 0.98, sensitivity = 0.96 and 0.99, and specificity = 0.96 and 0.93, respectively. A carefully selected subset of bioclimatic variables was used to describe the presence of each tick species. Higher levels of precipitation had positive effect on the presence of R. microplus but a negative effect on A. cajennense s.l. In contrast, isothermality (BIO3) was more important for the presence of A. cajennense s.l. compared to R. microplus. As a result, R. microplus had a broader distribution across the country, while A. cajennense s.l. was mainly found in coastal areas with evident seasonality. The coexistence of both species in some regions could be attributed to transitional zones, whereas high altitudes limited tick presence. This information can aid in developing appropriate tick management plans, particularly considering A. cajennense s.l.'s broad host range species and R. microplus's specificity for cattle. Moreover, the predictive models can identify areas at risk of associated challenging hemoparasite, requiring special attention and mitigation measures.

Repellent activity of the non-host semiochemical (E)-2-octenal against Amblyomma sculptum and Amblyomma dubitatum ticks under field conditions.

Amblyomma ticks pose a significant public health threat due to their potential to transmit pathogens associated with rickettsial diseases. (E)-2-octenal, a compound found in donkeys (Equus asinus), exhibits strong repellent properties against Amblyomma sculptum nymphs under laboratory conditions. This study assessed the effectiveness of the (E)-2-octenal in wearable slow-release devices for personal human protection against Amblyomma ticks under natural conditions. Slow-release devices treated with (E)-2-octenal and untreated controls were prepared and tested on two volunteers walking through a tick-infested area in Goiania, Brazil. The experiment was conducted twice daily for three series of 10 days, with each volunteer wearing two devices attached to each leg, one on the ankle and one just above the thigh. Volunteers with control and treated devices exchanged them between rounds. Also, the daily release rate of (E)-2-octenal from the slow-release devices was determined in the laboratory, increasing significantly from 0.77 ± 0.14 µg/day on the first day to 9.93 ± 1.92 µg/day on the 4th day and remaining constant until the 16th day. A total of 5409 ticks were collected from both volunteers. Treated devices resulted in recovering fewer ticks (n = 1,666; 31%) compared to untreated devices (control: n = 3,743; 69%). (E)-2-octenal effectively repelled Amblyomma spp. larvae, A. sculptum adults, and exhibited pronounced repellency against A. dubitatum nymphs and adults. These findings suggest the potential of (E)-2-octenal delivered by wearable slow-release devices as a green-based repellent. Further improvements, however, are necessary to provide better protection for humans against A. sculptum and A. dubitatum in field conditions.

Responses of ticks to immersion in hot bathing water: Effect of surface type, water temperature, and soap on tick motor control.

Preventing bites from undetected ticks through bathing practices would benefit public health, but the effects of these practices have been researched minimally. We immersed nymphal and adult hard ticks of species common in the eastern United States in tap water, using temperatures and durations that are realistic for human hot bathing. The effect of (a) different skin-equivalent surfaces (silicone and pig skin), and (b) water temperature was tested on Amblyomma americanum, Dermacentor variabilis and Ixodes scapularis nymphs. Overall, the type of surface had a much larger effect on the nymphs' tendency to stay in contact with the surface than water temperature did. Most nymphs that separated from the surface did so within the first 10 s of immersion, with the majority losing contact due to the formation of an air bubble between their ventral side and the test surface. In addition, adult Ixodes scapularis were tested for the effect of immersion time, temperature, and soap on tick responsiveness. Some individual adults moved abnormally or stopped moving as a result of longer or hotter immersion, but soap had little effect on responsiveness. Taken together, our results suggest that the surface plays a role in ticks' tendency to stay in contact; the use of different bath additives warrants further research. While water temperature did not have a significant short-term effect on tick separation, ticks that have not attached by their mouth parts may be rendered unresponsive and eventually lose contact with a person's skin in a hot bath. It should be noted that our research did not consider potential temperature effects on the pathogens themselves, as previous research suggests that some tickborne pathogens may become less hazardous even if the tick harboring them survives hot-water exposures and later bites the bather after remaining undetected.

Molecular detection of dugbe orthonairovirus in cattle and their infesting ticks (Amblyomma and Rhipicephalus (Boophilus)) in Nigeria.

Dugbe orthonairovirus (DUGV), a tick-borne zoonotic arbovirus, was first isolated in 1964 in Nigeria. For over four decades, no active surveillance was conducted to monitor the spread and genetic variation of DUGV. This study detected and genetically characterized DUGV circulating in cattle and their infesting ticks (Amblyomma and Rhipicephalus (Boophilus)) in Kwara State, North-Central Nigeria. Blood and or ticks were collected from 1051 cattle at 31 sampling sites (abattoirs and farms) across 10 local government areas of the State. DUGV detection was carried out by RT-qPCR, and positive samples sequenced and phylogenetically analysed. A total of 11824 ticks, mostly A. variegatum (36.0%) and R. (B.) microplus (63.9%), were obtained with mean tick burden of 12 ticks/cattle. Thirty-four (32 A. variegatum and two R. (B.) microplus) of 4644 examined ticks were DUGV-positive, whereas all of the cattle sera tested negative for DUGV genome. Whole genome sequence (S, M and L segments) and phylogenetic analyses indicate that the positive samples shared up to 99.88% nucleotide identity with and clustered around the Nigerian DUGV prototype strain IbAr 1792. Hence, DUGV with high similarity to the previously characterised strain has been detected in Nigeria. To our knowledge, this is the first report of DUGV in North-Central Nigeria and the most recent information after its last surveillance in 1974.

Equine attachment site preferences and seasonality of common North American ticks: Amblyomma americanum, Dermacentor albipictus, and Ixodes scapularis.

BACKGROUND: Ticks are common on horses, but recent publications characterizing equine tick infestations in North America are lacking. METHODS: To further understand attachment site preferences of common ticks of horses, and to document the seasonality of equine tick infestation in northeastern Oklahoma, horses from eight farms were evaluated twice a month over a 1-year period. Each horse was systematically inspected beginning at the head and moving caudally to the tail. Attachment sites of ticks were recorded and all ticks collected were identified to species and stage. RESULTS: Horses (26 males and 62 females) enrolled in the study ranged in age from 1 to 23 years (mean = 12, 95% CI 11-13). A total of 2731 ticks were collected; 84.1% (74/88) of the horses were infested (median = 3 ticks) at one or more examinations. Five tick species were identified, including Amblyomma americanum (78.2%; 2136/2731), Ixodes scapularis (18.2%; 497/2731), Dermacentor albipictus brown variant (2.6%; 71/2731), Dermacentor variabilis (0.7%; 20/2731), and Amblyomma maculatum (0.3%; 7/231). Most ticks were adults (83.6%; 2282/2731), but immature A. americanum (436/2136; 20.4%), D. albipictus (12/71; 16.9%), and A. maculatum (n = 1) were occasionally recovered. Amblyomma americanum were most often attached to the inguinal area, and I. scapularis and D. albipictus were most commonly found on the chest and axillary region (P < 0.0001). Ticks were found on horses in every month of the year. The largest number of ticks (638/2731; 23.4%) were collected in May (P < 0.0001). Amblyomma americanum, primarily immature, was the only tick recovered in September, I. scapularis and D. albipictus predominated October through February, and both A. americanum and I. scapularis were common in March. In the warmer months, April through August, A. americanum was the most common tick, followed by D. variabilis and A. maculatum. CONCLUSIONS: This research confirms that ticks common on horses in North America have attachment site preferences and that ticks infest horses in Oklahoma throughout the year, including during the winter. Additional research is warranted to fully understand the risk these infestations pose to equine health.

Presence of two species of the Amblyomma cajennense complex (Acari: Ixodidae) and probable zones of sympatry in northwestern Colombia.

The A. cajennense tick complex has been thought to be the main vector of Rickettsia rickettsii in Central and South America. Studies in Colombia have determined the presence of species A. patinoi and A. mixtum of the A. cajennense complex, but it is unknown which species of this complex exist in northwestern Colombia. Our aim was to identify the species of the A. cajennense complex that are present in northwestern Colombia. We sampled ticks of A. cajennense sensu lato infesting equids. Females identified according to the morphology of their genital pore were selected for genetic confirmation. Specimens from each locality were selected to perform molecular and genetic analysis. Specimens were analyzed from five departments (Antioquia, Bolívar, Córdoba, Magdalena, and Sucre). Morphologically 65 specimens were identified as A. patinoi and 5 as A. mixtum. Molecular analysis allowed to confirm the morphological identification of 27 specimens. In this study A. patinoi was widely distributed in the departments of Antioquia, Bolívar, and Córdoba with allopatric and sympatric distribution in some places. These two species in the region could have unexpected effects on the epidemiology of rickettsiosis.

Potential distribution of Amblyomma mixtum (Koch, 1844) in climate change scenarios in the Americas.

Amblyomma mixtum is a Neotropical generalist tick of medical and veterinary importance which is widely distributed from United States of America to Ecuador. The aim of this study was to evaluate changes in the geographic projections of the ecological niche models of A. mixtum in climate change scenarios in America. We constructed a database of published scientific publications, personal collections, personal communications, and online databases. Ecological niche modelling was performed with 15 Bioclimatic variables using kuenm in R and was projected to three time periods (Last Glacial Maximum, Current and 2050) for America. Our model indicated a wide distribution for A. mixtum, with higher probability of occurrence along the Gulf of Mexico and occurring in a lesser proportion in the Pacific states, Central America, and the northern part of South America. The areas of new invasion are located mainly on the border of Mexico with Guatemala and Belize, some regions of Central America and Colombia. We conclude that the ecological niche modelling are effective tools to infer the potential distribution of A. mixtum in America, in addition to helping to propose future measures of epidemiological control and surveillance in the new potential areas of invasion.

Crowdsourced Tick Image-Informed Updates to U.S. County Records of Three Medically Important Tick Species.

Burgeoning cases of tick-borne disease present a significant public health problem in the United States. Passive tick surveillance gained traction as an effective way to collect epidemiologic data, and in particular, photograph-based tick surveillance can complement in-hand tick specimen identification to amass distribution data and related encounter demographics. We compared the Federal Information Processing Standards (FIPS) code of tick photos submitted to a free public identification service (TickSpotters) from 2014 to 2019 to published nationwide county reports for three tick species of medical concern: Ixodes scapularis Say (Ixodida: Ixodidae), Ixodes pacificus Cooley and Kohls (Ixodida: Ixodidae), and Amblyomma americanum Linneaus (Ixodida: Ixodidae). We tallied the number of TickSpotters submissions for each tick species according to "Reported" or "Established" criteria per county, and found that TickSpotters submissions represented more than half of the reported counties of documented occurrence, and potentially identified hundreds of new counties with the occurrence of these species. We detected the largest number of new county reports of I. scapularis presence in Michigan, North Carolina, and Texas. Tick image submissions revealed potentially nine new counties of occurrence for I. pacificus, and we documented the largest increase in new county reports of A. americanum in Kentucky, Illinois, Indiana, and Ohio. These findings demonstrate the utility of crowdsourced photograph-based tick surveillance as a complement to other tick surveillance strategies in documenting tick distributions on a nationwide scale, its potential for identifying new foci, and its ability to highlight at-risk localities that might benefit from tick-bite prevention education.

Diversity of Rickettsia in ticks collected from wild animals in Panama.

This paper presents new data about Rickettsia species detected in ticks collected from wild animals, using 16S rRNA, gltA and ompA. Rickettsia DNA was found in 66 of 101 ticks. Using EZ BioCloud libraries were produced reads that identified Rickettsia aeschlimannii, and Illumina BaseSpace produced reads of Rickettsia rickettsii group, Rickettsia bellii group, and unclassified Rickettsia. Using gltA and ompA gene-specific primers, R. aeschlimannii could not be confirmed, but detection of Rickettsia amblyommatis was achieved in Amblyomma auricularium, Amblyomma geayi, Amblyomma mixtum, and Amblyomma pacae; R. bellii from Amblyomma dissimile, "Candidatus Rickettsia colombianensi" from A. dissimile, Rickettsia spp. closely related to R. raoultii from A. geayi, Rickettsia tamurae from A. dissimile, and Rickettsia endosymbionts of Ixodes from Ixodes affinis. There were no databases available specifically for 16S rRNA of Neotropical Rickettsia, highlighting the need to use species primers over only 16S rRNA primers to achieve more accurate interpretations and identifications. These findings increase the number of Rickettsia species detected in Panama and highlight the need to establish isolates to further characterize the nature of Rickettsia in the area.

Validating Species Distribution Models With Standardized Surveys for Ixodid Ticks in Mainland Florida.

Tick-borne pathogens are of growing concern. The U.S. Centers for Disease Control and Prevention (CDC) developed guidelines standardizing surveys of tick vectors to better monitor the changes in their occurrences. Unbiased surveillance data, from standardized surveys, are presumed critical to generate valid species distribution models (SDMs). We tested previously generated SDMs from standardized protocols for three medically important ticks [Amblyomma americanum (Linnaeus, Ixodida, Ixodidae), Ixodes scapularis (Say, Ixodida, Ixodidae), and Dermacentor variabilis (Say, Ixodida, Ixodidae)]. These previous models ruled out a quarter to half of the state as having these species, with consensus occurrence in about a quarter of the state. New surveys performed throughout 2019 on 250 transects at 43 sites indicated the rule-out functions were 100% accurate for I. scapularis and D. variabilis and 91.9% for A. americanum. As SDM concordance increased, the proportion of transects yielding ticks increased. Independent surveys of SDMs provide external validation-an aspect missing from many SDM studies.

Low Abundance of Three Tick Species in the Piedmont of North Carolina.

Multiple species of ticks, including Ixodes scapularis (Say, Ixodida:Ixodidae), Amblyomma americanum (L., Ixodida:Ixodidae), and Dermacentor variabilis (Say, Ixodida:Ixodidae), occur in high and increasing abundance in both the northeast and southeast United States. North Carolina is at the nexus of spread of these species, with high occurrence and abundance of I. scapularis to the north and A. americanum to the south. Despite this, there are few records of these species in the Piedmont of North Carolina, including the greater Charlotte metropolitan area. Here, we update the known occurrence and abundance of these species in the North Carolina Piedmont. We surveyed for ticks using cloth drags, CO2 traps, and leaf litter samples at a total of 79 sites within five locations: Mecklenburg County, South Mountains State Park, Stone Mountain State Park, Duke Forest, and Morrow Mountain State Park, all in North Carolina, during the late spring, summer, and fall seasons of 2019. From these surveys, we had only 20 tick captures, illuminating the surprisingly low abundance of ticks in this region of North Carolina. Our results indicate the possibility of underlying habitat and host factors limiting tick distribution and abundance in the North Carolina Piedmont.

Distribution modeling of Amblyomma rotundatum and Amblyomma dissimile in Brazil: estimates of environmental suitability.

The number of reports of tick parasitism in amphibians and reptiles has increased over the past few years, including new host and location records for Amblyomma rotundatum and Amblyomma dissimile. However, knowledge of the geographical distribution remains incomplete, and in many regions of Brazil, the presence of these vectors has not been investigated. Several candidate models were built using a correlative maximum entropy approach, and best-fitting models were selected based on statistical significance, predictive ability, and complexity based on current climatic trends and future projected climate changes. Final models showed a good ability to discriminate A. rotundatum and A. dissimile current and future potential distributions. The entire country had higher predicted suitability for A. rotundatum while A. dissimile was mainly restricted to the Amazon and Pantanal biomes. A. rotundatum is a species with enormous potential for dissemination in the next decades, potentially through the legal and illegal transport of reptiles and amphibians. The proposed model is useful for targeting surveillance efforts increasing the efficiency and accuracy of future ecological research and tick management efforts.

Amblyomma testudinarium infestation on a brown bear (Ursus arctos yesoensis) captured in Hokkaido, a northern island of Japan.

The tick Amblyomma testudinarium Koch, 1844 (Acari: Ixodidae) is known as a vector of several pathogens such as Rickettsia tamurae and severe fever with thrombocytopenia syndrome (SFTS) virus. This tick species is present in many Asian countries, including Japan, where its distribution is limited to the warm areas of Kanto region and the southwestern region. The present study reports the recovery of a partially engorged A. testudinarium from a wild brown bear captured in Shari town, Hokkaido. In addition to morphological identification, the specimen was genetically characterized by the complete mitochondrial genome sequencing. The results showed that the length of the obtained mitogenome is 14,835 bp that encodes 13 protein-coding, two ribosomal RNA (rRNA) (12S and 16S), and 22 transfer RNA genes with two non-coding control regions. The phylogenetic analysis indicated that our sample clustered with A. testudinarium from Nara, Japan, but separated from A. testudinarium from China. Although the introduction of the tick through livestock transportation cannot be ruled out, the detection of A. testudinarium in Hokkaido prefecture, which is separated from the main island where A. testudinarium is present in the south, may suggest the introduction by migratory birds. This study provides important insights on the distribution and host range of A. testudinarium. This will be useful for the future taxonomic analysis of ticks based on the complete mitogenome sequencing. To our knowledge, this is the northernmost detection point of the tropical tick A. testudinarium.

Locomotion activity and its effects on the survival of Amblyomma sculptum (Acari: Ixodidae) nymphs under laboratory conditions.

The active locomotion of ticks is directly associated with the epidemiology of tick-borne diseases, as it has important implications for the interaction of ticks with their hosts and their dispersion in the environment. In an attempt to elucidate the factors involved in the dispersion of Amblyomma sculptum, the present work aimed to characterize different aspects of the active locomotion of A. sculptum nymphs under laboratory conditions. To this end, nymphs were placed on a string at a 70° inclination and their walking activity was recorded daily along with their survival period. During their lifetime, ticks walked an average of 110 m. Their locomotion was not in a straight line and nymphs changed direction 142 times throughout their lifetimes. The mean distance walked per experimental day was 1.8 m, while the average walking distance before changing direction was 52 cm. The distance walked per experimental day reduced over time. The survival of ticks was affected by walking; resting nymphs survived for over 6 months, while the survival of those that walked daily was reduced to approximately 62 days. The results showed that A. sculptum nymphs were able to cover distances of over 100 m throughout their lifetimes, but they walked short distances at a time and constantly changed direction. This behavior indicates that ticks are not able to disperse over long distances by means of active locomotion.

First detection of Amblyomma variegatum and molecular finding of Rickettsia africae in Sardinia, Italy.

Here we present the first detection of a male Amblyomma variegatum tick infesting a sheep on the island of Sardinia, as well as the detection of a pathogen, Rickettsia africae, in DNA extracted from this tick. The tick, the second individual of this species reported in Italy (the first one was reported in Sicily by Albanese in 1971) was collected in August 2018 from the inguinal region of an adult female sheep in a farm located near Sassari (North-West Sardinia). The tick was identified as an adult A. variegatum male under a stereomicroscope using morphological keys. A phylogenetic analysis showed that the 12S sequence clustered with that of African A. variegatum individuals and was embedded within the previously identified West African group. We tested the tick for the presence of microorganisms of the genera Ehrlichia, Rickettsia, Anaplasma, Theileria and Babesia, using published PCR protocols. The tick was found positive to Rickettsia and the obtained sequence matched at 100 % identity with R. africae. The area where the tick was detected was inspected on multiple occasions, looking for other specimens of A. variegatum, without any results. In the same period another male specimen of A. variegatum was found in Haute Corse in 2019. The authors' hypothesis is that the presence of the A. variegatum specimen is an occasional finding, probably linked to the migrating birds that cross Sardinia and Corsica from Africa during summer. Although this may have been an incidental finding, it must be considered that global warming could increase the risk of establishment of colonies of these ticks, that show a strong spreading capability. It is also important to emphasize that this tick species is a proven vector and reservoir of R. africae, an uncommon zoonotic pathogen in Italy, thus additional monitoring must be performed as the establishment of a stable population in Sardinia could represent a serious veterinary and public health issue.

Survey of Rickettsia parkeri and Amblyomma maculatum associated with small mammals in southeastern Virginia.

Small mammals are often parasitized by the immature stages of hard-bodied ticks (family Ixodidae) and may serve as reservoir hosts of tick-borne pathogens. Amblyomma maculatum, the Gulf Coast tick, is the primary vector of Rickettsia parkeri, the causative agent of R. parkeri rickettsiosis. This hard-bodied tick species is expanding its historical range from the Gulf Coast of the U.S. up the Mid-Atlantic coast. In Mid-Atlantic states, such as Virginia, R. parkeri prevalence is higher in these ticks than those found in its historical range. This high prevalence may be explained in part by small mammal populations. In this study, small mammals were trapped and checked for the presence of immature A. maculatum. The ticks as well as tissue samples from these mammals were tested for the presence of R. parkeri. This study found six rodent species acting as hosts to immature A. maculatum and three species that may play a role in the enzootic cycle of R. parkeri in Virginia.

Association between lone star tick bites and increased alpha-gal sensitization: evidence from a prospective cohort of outdoor workers.

BACKGROUND: Alpha-gal is an oligosaccharide implicated in delayed anaphylaxis following red meat consumption. Exposure to tick bites has been correlated with development of an allergic response to alpha-gal. However, evidence prospectively linking exposure to a single tick species and an immune response to alpha-gal is lacking. METHODS: We used serum samples from a prior study cohort of outdoor workers in North Carolina, USA, with high exposure to the lone star tick, Amblyomma americanum, to prospectively evaluate the relationship between tick bites and anti-alpha-gal IgE antibodies. RESULTS: Individuals who reported exposure to one or more tick bites were significantly more likely to have a positive change in anti-alpha-gal IgE compared to individuals with no reported tick bites. This relationship was not dependent on time. A trend toward increasing number of tick bites and increased anti-alpha-gal IgE levels was observed but not statistically significant. CONCLUSION: To our knowledge, this is the first study to prospectively link documented exposure to A. americanum bites and increased sensitization to alpha-gal in a cohort of outdoor workers. Our results support the role of A. americanum as likely agents for eliciting an allergic response to red meat, and highlight the importance of preventing tick bites.

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.