A next generation sequencing assay combining Ixodes species identification with pathogen detection to support tick surveillance efforts in the United States.

The burden of tick-borne diseases continues to increase in the United States. Tick surveillance has been implemented to monitor changes in the distribution and prevalence of human disease-causing pathogens in ticks that frequently bite humans. Such efforts require accurate identification of ticks to species and highly sensitive and specific assays that can detect and differentiate pathogens from genetically similar microbes in ticks that have not been demonstrated to be pathogenic in humans. We describe a modification to a next generation sequencing pathogen detection assay that includes a target that accurately identifies Ixodes ticks to species. We show that the replacement of internal control primers used to ensure assay performance with primers that also act as an internal control and can additionally differentiate tick species, retains high sensitivity and specificity, improves efficiency, and reduces costs by eliminating the need to run separate assays to screen for pathogens and for tick identification.

Corrigendum: Development of a quadruplex PCR amplicon next generation sequencing assay for detection and differentiation of Bartonella spp.

[This corrects the article DOI: 10.3389/fmicb.2023.1243471.].

Detection of Borrelia burgdorferi sensu lato species in host-seeking Ixodes species ticks in the United States.

Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Ixodes scapularis in the eastern US and I. pacificus in the west. The causative agents, Borrelia burgdorferi sensu stricto (Bbss) and B. mayonii belong to the B. burgdorferi sensu lato (Bbsl) species complex. An additional eight species of Bbsl have been identified in Ixodes species ticks in the US, but their geographic distribution, vector associations, human encounter rates and pathogenicity in humans are poorly defined. To better understand the geographic distribution and vector associations of Bbsl spirochetes in frequent and infrequent human-biting Ixodes species ticks in the US, we previously screened 29,517 host-seeking I. scapularis or I. pacificus ticks and 692 ticks belonging to eight other Ixodes species for Borrelia spirochetes using a previously described tick testing algorithm that utilizes a combination of real-time PCR and Sanger sequencing for Borrelia species identification. The assay was designed to detect known human pathogens spread by Ixodes species ticks, but it was not optimized to detect Bbsl co-infections. To determine if such co-infections were overlooked particularly in ticks infected with Bbss, we retested and analyzed a subsample of 845 Borrelia infected ticks using a next generation sequencing multiplex PCR amplicon sequencing (MPAS) assay that can identify Borrelia species and Bbsl co-infections. The assay also includes targets that can molecularly confirm identifications of Ixodes species ticks to better inform pathogen-vector associations. We show that Bbss is the most prevalent species in I. scapularis and I. pacificus; other Bbsl species were rarely detected in I. scapularis and the only Bbsl co-infections identified in I. scapularis were with Bbss and B. mayonii. We detected B. andersonii in I. dentatus in the Mid-Atlantic and Upper Midwest regions, B. kurtenbachii in I. scapularis in the Upper Midwest, B. bissettiae in I. pacificus and I. spinipalpis in the Northwest, and B. carolinensis in I. affinis in the Mid-Atlantic and Southeast, and B. lanei in I. spinipalpis in the Northwest. Twelve of 62 (19.4%) Borrelia-infected I. affinis from the Mid-Atlantic region were co-infected with Bbss and B. carolinensis. Our data support the notion that Bbsl species are maintained in largely independent enzootic cycles, with occasional spill-over resulting in multiple Bbsl species detected in Ixodes species ticks.

Development of a quadruplex PCR amplicon next generation sequencing assay for detection and differentiation of Bartonella spp.

The genus Bartonella includes a group of species that are associated with a wide range of mammalian species, including human. It is challenging to detect all Bartonella species using a single molecular target due to its high genetic diversity. To solve this issue, we developed a quadruplex PCR amplicon sequencing assay using next-generation sequencing (NGS) technology for the detection and differentiation of Bartonella species. Our objective was to obtain the specific sequences of a minimum of two of the four target genes as confirmation of the identity of a particular Bartonella species using the assay. Four pairs of primers targeting specific regions on gltA, groEL, rpoB, and ssrA were evaluated for their capability of differentiating Bartonella species individually and collectively by performing singular PCR amplicon sequencing and quadruplex PCR amplicon sequencing. Using the quadruplex PCR amplicon sequencing, 24 Bartonella reference species were tested, all of which were successfully differentiated by at least two targets. Bartonella species were accurately identified from the artificially mixed DNA templates developed to simulate coinfections. The limit of detection was determined to be 1 fg based on testing a series of 10-fold dilutions of DNA from the Bartonella species. Testing of high DNA concentrations of 19 non-Bartonella species showed high specificity with none of the non-Bartonella species misclassified as Bartonella. Finally, the assay was evaluated by testing DNA extracts from field-collected body lice (Pediculus humanus humanus) and Norway rats (Rattus norvegicus): Bartonella quintana was detected and confirmed by three targets in the lice and Bartonella tribocorum was detected and confirmed by two targets in the rats. These results demonstrated that Bartonella species could be accurately and rapidly detected and differentiated into different tissue types using the quadruplex sequencing assay.

A bioinformatics pipeline for a tick pathogen surveillance multiplex amplicon sequencing assay.

The Centers for Disease Control and Prevention's national tick and tick-borne pathogen surveillance program collects information to better understand the regional distribution, prevalence, and exposure risk of host-seeking medically important ticks in the United States. A recently developed next generation sequencing (NGS) targeted multiplex PCR amplicon sequencing (MPAS) assay has enhanced the detection capabilities for Ixodes-associated human pathogens found in Ixodes scapularis and Ixodes pacificus ticks compared to the routinely used real-time PCR assay. To operationalize the MPAS assay for the large number of tick surveillance submissions processed each year, a reproducible high throughput bioinformatics pipeline is needed. We describe the development and validation of the MPAS pipeline, a bioinformatics pipeline that identifies and summarizes amplicon sequences produced by the MPAS assay. This pipeline is portable and reproducible across different computing environments, and flexible by allowing modifications to input parameters, assay primer and reference sequences. The automation of the summary report, BLAST report, and phylogenetic analysis reduces the amount of time needed for downstream analysis. To validate this pipeline, we compared the analysis of a MPAS assay dataset consisting of 175 I. scapularis nymphs with the MPAS pipeline and previously published results analyzed with a CLC Genomic Workbench workflow. The MPAS pipeline identified the same number of positive ticks for Anaplasma phagocytophilum and Babesia species as the original analysis, but the MPAS pipeline provided enhanced sequencing resolution of Borrelia burgdorferi sensu lato co-infected samples. The reproducibility, flexibility, analysis automation, and improved sequence resolution of the MPAS pipeline make it well suited for a high throughput tick pathogen surveillance program.

Using next generation sequencing for molecular detection and differentiation of Anaplasma phagocytophilum variants from host seeking Ixodes scapularis ticks in the United States.

Anaplasmosis is increasingly common in the United States, with cases being reported over an expanding geographic area. To monitor for changes in risk of human infection, the U.S. Centers for Disease Control and Prevention monitors the distribution and abundance of host-seeking vector ticks (Ixodes scapularis and Ixodes pacificus) and their infection with Anaplasma phagocytophilum. While several variants of A. phagocytophilum circulate in I. scapularis, only the human-active variant (Ap-ha) appears to be pathogenic in humans. Failure to differentiate between human and non-human variants may artificially inflate estimates of the risk of human infection. Efforts to differentiate the Ap-ha variant from the deer variant (Ap-V1) in ticks typically rely on traditional PCR assays coupled with sequencing of PCR products. However, laboratories are increasingly turning to Next Generation Sequencing (NGS) to increase testing efficiency, retain high sensitivity, and increase specificity compared with traditional PCR assays. We describe a new NGS assay with novel targets that accurately segregate the Ap-ha variant from other non-human variants and further identify unique clades within the human and non-human variants. Recognizing that not all investigators have access to NGS technology, we also developed a PCR assay based on one of the novel targets so that variants can be visualized using agarose gel electrophoresis without the need for subsequent sequencing. Such an assay may be used to improve estimates of human risk of developing anaplasmosis in North America.

Detection of Genetic Variability in Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) Between and Within the Eastern and Western United States.

Borrelia miyamotoi is a hard tick-associated relapsing fever spirochete that is geographically widespread in Ixodes spp. (Acari: Ixodidae) ticks, but typically occurs at low prevalence. Genetic variability has been described among strains derived from Asia, Europe, and North America, and among tick species that carry the infection, but little variability has been described within foci or tick species. Capitalizing on access to B. miyamotoi nucleic acid extracted from host-seeking Ixodes scapularis Say or Ixodes pacificus Cooley & Kohls from 16 states, we explored genetic variability based on sequence analysis of four amplicons described herein. Consistent with previous studies, we detected significant genetic differences between strains derived from I. scapularis (eastern United States) and I. pacificus (western United States) and identified two distinct sequences in the western United States (Am-West-1 and Am-West-2). Unique to this study, we identified two distinct sequences in the eastern United States (Am-East-1 and Am-East-2). Based on the 161 samples we analyzed, Am-East-1 was the only type represented in 50 B. miyamotoi-infected ticks collected from the Northeast (Vermont, Maine, New York, Connecticut, and Rhode Island), whereas ticks collected from the North-Central and Mid-Atlantic states harbored B. miyamotoi comprised of both Am-East-1 and Am-East-2. Further studies are needed to better characterize the phylogeography of B. miyamotoi and to discern if there are biologically meaningful differences among sequence types. To facilitate further exploration, we developed a polymerase chain reaction (PCR) assay designed to differentiate Am-East-1, Am-East-2, and Am-West sequence types without having to sequence the amplicon.

Exposure of Domestic Cats to Three Zoonotic Bartonella Species in the United States.

Cat-associated Bartonella species, which include B. henselae, B. koehlerae, and B. clarridgeiae, can cause mild to severe illness in humans. In the present study, we evaluated 1362 serum samples obtained from domestic cats across the U.S. for seroreactivity against three species and two strain types of Bartonella associated with cats (B. henselae type 1, B. henselae type 2, B. koehlerae, and B. clarridgeiae) using an indirect immunofluorescent assay (IFA). Overall, the seroprevalence at the cutoff titer level of ≥1:64 was 23.1%. Seroreactivity was 11.1% and 3.7% at the titer level cutoff of ≥1:128 and at the cutoff of ≥1:256, respectively. The highest observation of seroreactivity occurred in the East South-Central, South Atlantic, West North-Central, and West South-Central regions. The lowest seroreactivity was detected in the East North-Central, Middle Atlantic, Mountain, New England, and Pacific regions. We observed reactivity against all four Bartonella spp. antigens in samples from eight out of the nine U.S. geographic regions.

Evaluation of a novel multiplex PCR amplicon sequencing assay for detection of human pathogens in Ixodes ticks.

Tickborne diseases are an increasing public health concern in the United States, where the majority of notifiable cases are caused by pathogens vectored by Ixodes ticks. To better monitor changes in acarological risk of human encounters with these ticks and their associated pathogens, the Centers for Disease Control and Prevention (CDC) recently established a national tick and tickborne pathogen surveillance program. Here, we describe and evaluate a new Multiplex PCR Amplicon Sequencing (MPAS) assay for potential use in surveillance programs targeting two common human-biting vector ticks, Ixodes scapularis and Ixodes pacificus. The ability of the MPAS assay to detect five Ixodes-associated human pathogens (Borrelia burgdorferi sensu stricto, Borrelia mayonii, Borrelia miyamotoi, Anaplasma phagocytophilum and Babesia microti) was compared to that of a previously published and routinely used probe-based (TaqMan) PCR testing algorithm for pathogen detection in Ixodes ticks. Assay performance comparisons included a set of 175 host-seeking Ixodes nymphs collected in Connecticut as well as DNA from our pathogen reference collection. The MPAS assay and the CDC standard TaqMan PCR pathogen testing algorithm were found to have equivalent detection sensitivity for Ixodes-associated human pathogens. However, the MPAS assay was able to detect a broader range of tick-associated microorganisms, more effectively detected co-infections of multiple pathogens in a single tick (including different species within the Borrelia burgdorferi sensu lato complex), and required a smaller volume of test sample (thus preserving more sample for future testing).

Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics.

Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real-time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis-specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis-specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis-specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis-spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die-off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis-specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent.

Bartonella species in medically important mosquitoes, Central Europe.

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.

Detection of 'Candidatus Ehrlichia khabarensis' in rodents and ticks removed from rodents in British Columbia, Canada.

'Candidatus Ehrlichia khabarensis' was first described from rodents and insectivores in the Far East territory of Khabarovsk on the Russian Pacific Coast. Here we report the detection of DNA from this microorganism in rodents and fed ticks collected from rodents in British Columbia, Canada in 2013-2014. 'Candidatus Ehrlichia khabarensis' was detected in (i) a female Ixodes angustus tick collected from a Peromyscus maniculatus; (ii) a female Dermacentor andersoni tick collected from a Perognathus parvus; (iii) a pool of 2 larval Ixodes pacificus ticks collected from a single P. maniculatus; and (iv) a pool of 3 nymphal I. pacificus ticks collected from a single P. maniculatus. Three of these four rodents (2 P. maniculatus and 1 P. parvus) with infected ticks also had evidence of 'Candidatus Ehrlichia khabarensis' in at least one tissue type. The infected P. maniculatus and Ixodes ticks came from the Vancouver area in western British Columbia and the P. parvus and Dermacentor tick from an inland site in central British Columbia. Although it remains to be determined whether 'Candidatus Ehrlichia khabarensis' has any negative impacts on wildlife, domestic animals or humans, we note that all three tick species found to contain the DNA of this microorganism are known to bite humans. Future detection of this microorganism either in ticks collected from rodents and allowed to molt to the next life stage prior to being tested, or from host-seeking ticks, is required to determine if it can survive the tick's molt after being ingested via an infectious blood meal.

Experimental Demonstration of Reservoir Competence of the White-Footed Mouse, Peromyscus leucopus (Rodentia: Cricetidae), for the Lyme Disease Spirochete, Borrelia mayonii (Spirochaetales: Spirochaetaceae).

The white-footed mouse, Peromyscus leucopus (Rafinesque), is a reservoir for the Lyme disease spirochete Borrelia burgdorferi sensu stricto in the eastern half of the United States, where the blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae), is the primary vector. In the Midwest, an additional Lyme disease spirochete, Borrelia mayonii, was recorded from naturally infected I. scapularis and P. leucopus. However, an experimental demonstration of reservoir competence was lacking for a natural tick host. We therefore experimentally infected P. leucopus with B. mayonii via I. scapularis nymphal bites and then fed uninfected larvae on the mice to demonstrate spirochete acquisition and passage to resulting nymphs. Of 23 mice fed on by B. mayonii-infected nymphs, 21 (91%) developed active infections. The infection prevalence for nymphs fed as larvae on these infected mice 4 wk post-infection ranged from 56 to 98%, and the overall infection prevalence for 842 nymphs across all 21 P. leucopus was 75% (95% confidence interval, 72-77%). To assess duration of infectivity, 10 of the P. leucopus were reinfested with uninfected larval ticks 12 wk after the mice were infected. The overall infection prevalence for 480 nymphs across all 10 P. leucopus at the 12-wk time point was 26% (95% confidence interval, 23-31%), when compared with 76% (95% confidence interval, 71-79%) for 474 nymphs from the same subset of 10 mice at the 4-wk time point. We conclude that P. leucopus is susceptible to infection with B. mayonii via bite by I. scapularis nymphs and an efficient reservoir for this Lyme disease spirochete.

Failure of the Asian longhorned tick, Haemaphysalis longicornis, to serve as an experimental vector of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto.

The invasive, human-biting Asian longhorned tick, Haemaphysalis longicornis, was detected in New Jersey in the eastern United States in August of 2017 and by November of 2018 this tick had been recorded from 45 counties across 9 states, primarily along the Eastern Seaboard. The establishment of H. longicornis in the United States has raised the questions of how commonly it will bite humans and which native pathogens may naturally infect this tick. There also is a need for experimental vector competence studies with native pathogens to determine if H. longicornis can acquire a given pathogen while feeding, pass it transstadially, and then transmit the pathogen in the next life stage. In this experimental study, we evaluated the vector competence of a population of H. longicornis originating from the United States (New York) for a native isolate (B31) of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.). In agreement with a previous experimental study on the vector competence of H. longicornis for Borrelia garinii, we found that uninfected H. longicornis larvae could acquire B. burgdorferi s.s. while feeding on infected Mus musculus mice (infection prevalence >50% in freshly fed larvae) but that the infection was lost during the molt to the nymphal stage. None of 520 tested molted nymphs were found to be infected, indicating that transstadial passage of B. burgdorferi s.s. is absent or rare in H. longicornis; and based on the potential error associated with the number of nymphs testing negative in this study, we estimate that the upper 95% limit for infection prevalence was 0.73%. An Ixodes scapularis process control showed both effective acquisition of B. burgdorferi s.s. from infected mice by uninfected larvae and transstadial passage to the nymphal stage (infection prevalence of 80-82% for both freshly fed larvae and molted nymphs). We also observed that although H. longicornis larvae could be compelled to feed on mice by placing the ticks within feeding capsules, attachment and feeding success was minimal (<0.5%) when larvae were placed freely on the fur of the mice. We conclude that H. longicornis is unlikely to contribute more than minimally, if at all, to transmission of Lyme disease spirochetes in the United States.

Identification of Bartonella rochalimae in Guinea Pigs (Cavia porcellus) and Fleas Collected from Rural Peruvian Households.

In the present study, we tested 391 fleas collected from guinea pigs (Cavia porcellus) (241 Pulex species, 110 Ctenocephalides felis, and 40 Tiamastus cavicola) and 194 fleas collected from human bedding and clothing (142 Pulex species, 43 C. felis, five T. cavicola, and four Ctenocephalides canis) for the presence of Bartonella DNA. We also tested 83 blood spots collected on Flinders Technology Associates (FTA) cards from guinea pigs inhabiting 338 Peruvian households. Bartonella DNA was detected in 81 (20.7%) of 391 guinea pig fleas, in five (2.6%) of 194 human fleas, and in 16 (19.3%) of 83 guinea pig blood spots. Among identified Bartonella species, B. rochalimae was the most prevalent in fleas (89.5%) and the only species found in the blood spots from guinea pigs. Other Bartonella species detected in fleas included B. henselae (3.5%), B. clarridgeiae (2.3%), and an undescribed Bartonella species (4.7%). Our results demonstrated a high prevalence of zoonotic B. rochalimae in households in rural areas where the research was conducted and suggested a potential role of guinea pigs as a reservoir of this bacterium.

Human Exposure to Novel Bartonella Species from Contact with Fruit Bats.

Twice a year in southwestern Nigeria, during a traditional bat festival, community participants enter designated caves to capture bats, which are then consumed for food or traded. We investigated the presence of Bartonella species in Egyptian fruit bats (Rousettus aegyptiacus) and bat flies (Eucampsipoda africana) from these caves and assessed whether Bartonella infections had occurred in persons from the surrounding communities. Our results indicate that these bats and flies harbor Bartonella strains, which multilocus sequence typing indicated probably represent a novel Bartonella species, proposed as Bartonella rousetti. In serum from 8 of 204 persons, we detected antibodies to B. rousetti without cross-reactivity to other Bartonella species. This work suggests that bat-associated Bartonella strains might be capable of infecting humans.

Acquisition of Bartonella elizabethae by Experimentally Exposed Oriental Rat Fleas (Xenopsylla cheopis; Siphonaptera, Pulicidae) and Excretion of Bartonella DNA in Flea Feces.

Few studies have been able to provide experimental evidence of the ability of fleas to maintain rodent-associated Bartonella infections and excrete these bacteria. These data are important for understanding the transmission cycles and prevalence of these bacteria in hosts and vectors. We used an artificial feeding approach to expose groups of the oriental rat flea (Xenopsylla cheopis Rothschild; Siphonaptera, Pulicidae) to rat blood inoculated with varying concentrations of Bartonella elizabethae Daly (Bartonellaceae: Rhizobiales). Flea populations were maintained by membrane feeding on pathogen-free bloodmeals for up to 13 d post infection. Individual fleas and pools of flea feces were tested for the presence of Bartonella DNA using molecular methods (quantitative and conventional polymerase chain reaction [PCR]). The threshold number of Bartonellae required in the infectious bloodmeal for fleas to be detected as positive was 106 colony-forming units per milliliter (CFU/ml). Individual fleas were capable of harboring infections for at least 13 d post infection and continuously excreted Bartonella DNA in their feces over the same period. This experiment demonstrated that X. cheopis are capable of acquiring and excreting B. elizabethae over several days. These results will guide future work to model and understand the role of X. cheopis in the natural transmission cycle of rodent-borne Bartonella species. Future experiments using this artificial feeding approach will be useful for examining the horizontal transmission of B. elizabethae or other rodent-associated Bartonella species to naïve hosts and for determining the viability of excreted bacteria.

Survey of Parasitic Bacteria in Bat Bugs, Colorado.

Bat bugs (Cimex adjunctus Barber) (Hemiptera: Cimicidae) collected from big brown bats (Eptesicus fuscus Palisot de Beauvoir) in Colorado, United States were assessed for the presence of Bartonella, Brucella, and Yersinia spp. using molecular techniques. No evidence of Brucella or Yersinia infection was found in the 55 specimens collected; however, 4/55 (7.3%) of the specimens were positive for Bartonella DNA. Multi-locus characterization of Bartonella DNA shows that sequences in bat bugs are phylogenetically related to other Bartonella isolates and sequences from European bats.

MOLECULAR SURVEILLANCE FOR BARTONELLA, BORRELIA, AND RICKETTSIA SPECIES IN TICKS FROM DESERT BIGHORN SHEEP ( OVIS CANADENSIS) AND MULE DEER ( ODOCOILEUS HEMIONUS) IN SOUTHERN CALIFORNIA, USA.

: Ticks (Acari: Ixodidae) were collected from 44 desert bighorn sheep ( Ovis canadensis) and 10 mule deer ( Odocoileus hemionus) in southern California, US during health inspections in 2015-16. Specimens were identified and screened by PCR analysis to determine the presence and prevalence of Bartonella, Borrelia, and Rickettsia species in ticks associated with these wild ruminants. None of the 60 Dermacentor hunteri and 15 Dermacentor albipictus ticks tested yielded positive PCR results. Additional tick specimens should be collected and tested to determine the prevalence of these confirmed or suspected tickborne pathogens within ruminant populations.