Show us your ticks: a survey of ticks infesting dogs and cats across the USA

Background A variety of tick species infest dogs and cats in North America. Although most of these species also readily feed on people, national data regarding the species and abundance of ticks on dogs and cats are lacking. Here we report a large-scale study of ticks from dogs and cats in the USA over a 12-month period. Methods Tick submissions were invited from veterinary practices in all 50 states. Ticks were submitted with information about the pet and the attachment sites of each tick marked on a biopsy chart. Upon receipt, ticks were identified to species and stage using morphologic keys; when necessary, species identification was confirmed molecularly. Results From February 2018 through January 2019, 10,978 ticks were submitted from 1494 dogs and 336 cats in 49 states and ticks were collected in every month. Dog and cat infestation intensities ranged from 1 to 4765 and from 1 to 38 (median = 1, mean = 6.7 and 2.6), respectively. Dogs were primarily infested with Dermacentor variabilis (532/1494; 35.6%), Ixodes scapularis (409/1494; 27.4%), Amblyomma americanum (345/1494; 23.1%) and Rhipicephalus sanguineus (172/1494; 11.5%). Cats were primarily infested with I. scapularis (156/336; 46.4%), A. americanum (99/336; 29.5%) and D. variabilis (60/336; 17.9%). Other submitted ticks included A. maculatum, Haemaphysalis longicornis, Otobius megnini, and less common Dermacentor spp. and Ixodes spp. Co-infestations were documented in 93 dogs and 14 cats. Reported attachment sites of common tick species differed. In dogs, A. americanum was most commonly attached to the abdomen, axillary, and inguinal regions; D. variabilis and I. scapularis to the head, neck, and back; and R. sanguineus to the head, neck, abdomen, legs, and feet. In cats, I. scapularis was most commonly attached to the head and A. americanum was most commonly attached to the tail and perianal region. Conclusions These data confirm that dogs and cats in the USA are at risk of tick infestation throughout the year and that tick species present in the region have apparent attachment site preferences.

Pet dogs presented to veterinarians (562; nr) R. sanguineus (sensu lato) (1058) China [44] Haemaphysalis longicornis (286) Rhipicephalus haemaphysaloides (195) 11 too damaged for identification (A/N/L; species nos. for each stage not specified) Pet dogs presented to veterinarians (180; nr) R. sanguineus (s.l.) (1242) USA (Florida) [9] Amblyomma americanum (36) Ixodes scapularis (24) Dermacentor variabilis (10) Amblyomma maculatum (4) Pet dogs presented to veterinarians and individual submissions (643; nr) R. sanguineus (s.l.) (3069; F/M/N/L) Australia [33] "citizen-science" survey detailed tick infestations on people and animals across the USA, but did not report which tick species were found on dogs and cats or in the different geographical regions [5]. Several detailed reports of ticks on pets in limited geographical areas of the USA are available (Tables 1, 2) [2, [6][7][8][9][10][11][12][13][14], but none are national in scope. Compiling current, comprehensive data about ticks infesting dogs and cats in the USA is time-and resource-intensive but critically important for both veterinary and human health [15]. Improved knowledge of the tick species that pets encounter across the USA can provide valuable information about the geographical distribution of ticks throughout the country and thus the risk posed to humans that share the same environment. Because tick removal was documented at veterinary practices in the present study, we were also able to gain insight into attachment site preferences. The purpose of the present study was to determine the species and stages of ticks infesting dogs and cats throughout the USA and determine tick-host attachment site preferences.

Tick collections
Ticks submissions were invited from 190 enrolled veterinary practices in all 50 states to ensure broad geographical representation and were supplemented by submissions from other veterinary practices interested in supporting the study. Each practice was provided with instructions and submission kits containing forceps, tick containers, prepaid mailing envelopes and submission forms. Instructions for tick submissions were also made available on a study website [16]. Ticks identified on a dog or cat were removed and placed in a hard-plastic container with a tightly fitting lid which was then sealed in a plastic bag with a completed submission form and shipped to Oklahoma State University; occasionally ticks were submitted in serum tubes or similar hard, tightly sealed containers. The submission and a diagram to indicate the tick attachment location(s) on the dog or cat. When multiple ticks were present we requested that all ticks be collected and submitted.

Tick identification
Ticks were immediately examined upon receipt, the stage (female, male, nymph, larva) of each tick recorded and tick genus and species determined using standard keys [17][18][19][20][21][22][23]. After identification, specimens were held in 70% ethanol at − 20 °C. An e-mail was sent to the submitting veterinarian with the initial morphologic identification and a list of pathogens that species/stage is known to transmit, if any. When damage to the specimen precluded identification by morphology, or if the species identification was unusual or uncertain due to morphologic similarity between congeners, ticks were bisected to retain anterior morphologic features and nucleic acid extracted from the posterior half with a commercial kit (Illustra GenomicPrep Kit, GE Healthcare, Marlborough, MA, USA) for molecular identification. Briefly, PCR amplification and direct sequencing of a 16S rRNA gene fragment [24,25] was utilized for Ixodes, Haemaphysalis and Amblyomma, a cox1 gene fragment [26] was also utilized for Ixodes and Haemaphysalis and an ITS2 gene fragment [27] was used for Dermacentor. Amplicons were visualized in GelRedstained (Biotium, Inc., Freemont, CA, USA) agarose gels to confirm expected size and purified using a commercial kit according to manufacturer's instructions (Wizard ® SV Gel and PCR Clean-Up System, Promega, Madison, WI, USA). Sequence analysis and alignment were performed using MacVector software (MacVector, Inc., Cary, NC, USA) and were compared with available sequences using the nucleotide Basic Local Alignment Search Tool (BLASTn, National Center for Biotechnology Information, Bethesda, MD, USA). Sequence identity was confirmed via visual inspection of the chromatogram and identity to available sequences. Anterior halves of bisected ticks were retained in 70% ethanol at − 20 °C.

Data management and quality assurance
Tick identification including number of ticks submitted, species and stage was recorded in a log along with the patient information. All data were entered into spreadsheets (Microsoft Excel version 16.16.8). Prior to summary and statistical analyses, quality assurance was performed by reviewing both individual identifications and data entry. Attachment site data were recorded from marked biopsy charts on original submission cards.
Regions of the body were divided into 5 areas for analysis: head, ears and neck; abdomen, axillary and inguinal; legs and feet; back; and tail and perianal region. Attachment site was only assessed for dogs and cats infested with a single species of adult tick.

Statistical analyses
Statistical analyses were performed using JMP (Version 12. SAS Institute Inc., Cary, NC, 1989-2019). Confidence intervals (CI 95%) were calculated for average reported weight and age. Chi-square tests, with significance levels below α = 0.05, were performed to evaluate differences in sex and altered status of dogs and cats with ticks compared to that reported from the general pet population in the USA and to evaluate differences in tick attachment site on dogs and cats among the most common tick species received. Percent ranked quintiles were established for tick attachment site data to depict attachment site preferences graphically.

Discussion
Our data confirm that tick infestations on dogs and cats in the USA are widespread. In the present study, ticks were identified from pets from a larger geographical area than has been reported in the USA [2,[4][5][6][7][8][9][10][11][12][13][14]. The primary tick species identified (R. sanguineus, A. americanum, D. variabilis and I. scapularis) constituted more than 95% of the ticks submitted from dogs, as was seen in earlier regional reports [6,9]. Similarly, more than 80% of the ticks found on cats were A. americanum, I. scapularis, or D. variabilis, as has been described in previous surveys [2,[8][9][10]. Most pets with ticks had outdoor access, but a variety of tick species were submitted from a few dogs and cats that were reported to rarely or never go outside, suggesting ticks carried into the home on clothing or other pets may create a risk to indoor pets [2]. Less common tick species were also submitted from dogs and cats in the present study. Gulf Coast ticks, A. maculatum, were submitted from 98 dogs and 5 cats and have been previously reported from pets, but the geographical distribution appears to be expanding [6,7,9,29]. Nymphs of O. megnini were submitted from the ear canals of 6 dogs and 13 cats. Although relatively uncommon, some spinose ear tick infestations in the present study were intense, with 26 nymphs from a single dog and 16 nymphs from a single cat, supporting the assertion that clinically relevant infestations with O. megnini occur in dogs and cats [1,30,31]. The Asian longhorned tick, H. longicornis, a species recently recognized in the USA [32], was submitted from 2 dogs and 1 cat. Longhorned ticks are commonly found on dogs and cats in other areas of the world where the species has long been present [33][34][35] and we expect to continue to identify this tick from pets in the USA in the future. The present study also confirmed that immature stages of some common tick species readily infest dogs and cats. Larvae and nymphs constituted the majority of A. americanum and R. sanguineus submissions from dogs, corroborating on a national scale findings from a large, regional survey of ticks infesting dogs [6]. A majority of the A. americanum submitted from cats in the present study were also larvae or nymphs, an observation that has been described in earlier reports [2,8]. Two cats harbored nymphs of R. sanguineus (sensu lato), a finding not previously reported in North America; adults of this species have been identified from cats in the USA and nymphs are reported from cats from other areas of the world [2,9,36]. Immature tick stages are important for pet health and may be overlooked due to their small size, an issue that can contribute to failing to recognize the complete tick risk faced by pets [2,8,9].
Host attachment site preferences also were evident among adult ticks in the present study. Adult A. americanum were more commonly attached ventrally and adult D. variabilis, R. sanguineus and I. scapularis were more commonly attached dorsally, as has been previously noted [2,6]. In dogs in the present study, D. variabilis and I. scapularis were found more commonly attached to the head, ears, neck and back. In a survey of ticks removed from dogs in Europe, I. ricinus and I. hexagonus preferred the head and D. reticulatus the back [37,38]. In dogs in the present study, R. sanguineus was more commonly attached to the head, ears and neck, as well as the legs and feet. This finding agrees with earlier reports in both the USA and Europe, where R. sanguineus was commonly found attached between the toes [6,39].
Limitations with the present study include sample bias, incomplete data from all pets with ticks and the broad geography from which ticks were submitted. Even when outdoor access was indicated, we do not have precise habitat information for each pet. Cats also appear to be under-represented as hosts for ticks. Estimates suggest that cats outnumber dogs as pets in the USA [40], but less than 20% of submissions were from cats. However, we relied on ticks collected from veterinary visits and cats are not taken to the veterinarian as often as dogs [41]. Complete data on factors such as attachment site were not provided for every submission and attachment sites from co-infestations with multiple species or stages were not included in the analysis as the original location of each tick on the pet could not be determined. Omitting these co-infested pets from the attachment site analysis was necessary but limited the power of our results. Finally, phenology of ticks varies with geography, precluding complete analysis of seasonality in the present paper.

Conclusions
This study revealed that a diverse array of ticks infest dogs and cats across the USA and throughout the year. Attachment site predilections were also confirmed, targeting key anatomic areas to examine when attempting to evaluate pets for active tick-infestation. This study also highlights the importance of broad-spectrum tick control in pets. Given the continued increase and geographical spread of tick populations in the USA [29] routine use of tick control is increasingly important for protecting pets from ticks. Surveillance of pets for ticks provides a valuable resource for understanding the tick risk faced by dogs, cats and people.