Abstract
Objectives
The objectives of the present study were to determine the duration of infestation by Amblyomma americanum necessary for transmission of Cytauxzoon felis to domestic cats and to determine if ingestion of C felis-infected A americanum by cats is a route of transmission.
Methods
Forty-nine cats were assigned to one of seven groups, with seven cats per group. Cats were infested with A americanum adults, acquisition fed as nymphs on a cytauxzoonosis survivor cat, for 12 h (group 1), 18 h (group 2), 24 h (group 3), 36 h (group 4), 48 h (group 5) and to repletion (group 7; control). Cats in group 6 were fed C felis-infected ticks. Thumb counts were performed at the end of the duration of infestation for groups 1–5 and at 48 h for the control group. For group 6, 50 live C felis-infected adult A americanum were mixed with food and fed to each of the cats. Transmission of C felis was determined by examining blood of cats by DNA extraction followed by PCR.
Results
Of 50 ticks placed on each cat (groups 1–5 and 7), the arithmetic mean attachment ± SEM ranged from 46.9 ± 1.9 in group 3 to 49.3 ± 0.3 in group 1. In group 6, the average number ± SEM of ticks ingested was 46.5 ± 2.3. One cat in group 5 that had been infested for 48 h became infected with C felis. None of the cats in group 6 (ingestion) became infected with C felis. Six of 7 (85.7%) cats in group 7, the control group, became infected with C felis.
Conclusions and relevance
Our results indicate that transmission of C felis to domestic cats can happen as quickly as >36 h but ⩽48 h of exposure to A americanum infected with C felis and that ingestion of C felis-infected A americanum is not a likely route of transmission.
Introduction
Cytauxzoon felis is an emerging tick-transmitted apicomplexan that causes severe disease in domestic cats. Cats with cytauxzoonosis are often febrile, dehydrated, emaciated, anorexic and icteric.1,2 Clinical signs begin approximately 11 days after being bitten by an infected tick. 2 As clinical signs appear after ticks have had enough time to feed to repletion, vectors may no longer be present on ill cats when they present to veterinary clinics. The absence of ticks on ill cats should not limit or reduce a practitioner’s index of suspicion of cytauxzoonosis.
Both Dermacentor variabilis (American dog tick)3,4 and Amblyomma americanum (lone star tick)2,5 have been shown to be competent vectors of C felis to domestic cats. However, A americanum has shown to be a more reliable vector under laboratory conditions.2,5 In the south-central USA most cases of cytauxzoonosis are observed in late spring or early summer, followed by a second but smaller uptick in cases during the fall months. 6 The bimodal pattern of cytauxzoonosis cases throughout the year likely reflects a slight delay in the questing activity and host-seeking behaviors of tick vectors. Cats living in low-density residential areas, urban edge habitat, wooded cover and in closer proximity to natural or unmanaged areas (ie, areas likely to harbor ticks and vertebrate reservoir hosts) were at higher risk of C felis infection. 6
Transmission of C felis is dependent upon infected ticks taking a blood meal from a feline host. Control strategies have focused on limiting cats’ access to tick-infested areas,7,8 or application of an acaricide to prevent ticks from feeding on cats. 9 Currently, four acaricides are approved for use on cats in the USA: etofenprox, fipronil, fluralaner and flumethrin. Delineating the transmission time of C felis from A americanum to cats is important as acaricides have different mechanisms of action, 10 speed of kill and repellency. Indeed, anecdotal reports suggest that C felis may be transmitted to ticks, despite routine application of fipronil,7,8 whereas application of an imidacloprid/flumethrin collar prevented the transmission of C felis to cats by A americanum. 9
A previous study demonstrated the duration of infestation (DOI) necessary for transmission of C felis by A americanum was 72 h. 11 However, we hypothesized that transmission of C felis occurred in less than 72 h, considering the reports of cats contracting cytauxzoonosis, despite prophylactic treatment with fipronil, a compound with a labeled 48 h speed of kill for ticks. 12 Additionally, as most cats are fastidious groomers and are thought to ingest ticks, we speculated that cats could become infected with C felis through ingesting infected A americanum. The objectives of the present study were to determine if transmission of C felis occurred before 72 h of infestation with A americanum and to ascertain if ingestion of C felis-infected A americanum by cats is a route of transmission.
Materials and methods
Study design
This study was an unmasked, randomized, controlled block study with an individual cat as the experimental unit. There were seven groups each, comprised of seven principal cats (Table 1). Cats were either infested or fed 25 pairs (50 total) of C felis-acquisition-fed A americanum. To determine the duration of infestation necessary for C felis transmission to cats, ticks on infested cats were allowed to feed for 12 h ± 30 mins (group 1), 18 h ± 30 mins (group 2), 24 h ± 30 mins (group 3), 36 h ± 30 mins (group 4) or 48 h ± 30 mins (group 5), after which they were removed from infested cats. To determine if cats could become infected through ingestion of infected ticks, ticks were fed with canned food to cats (group 6). In the control group, ticks were allowed to feed until repletion (group 7) and detached/fell off on their own. Infection of C felis was determined by monitoring the cats for clinical signs suggestive of cytauxzoonosis and by PCR analysis of blood samples collected throughout the study.
Table 1.
Grouping and treatment of principal cats
| Group | Tick exposure | Number of cats |
|---|---|---|
| Group 1 | 12 h infestation | 7 |
| Group 2 | 18 h infestation | 7 |
| Group 3 | 24 h infestation | 7 |
| Group 4 | 36 h infestation | 7 |
| Group 5 | 48 h infestation | 7 |
| Group 6 | Ingestion of ticks | 7 |
| Group 7 | Fed to repletion | 7 |
Donor and principal cats
All procedures were reviewed and approved by the Institution Animal Care and Use Committee at Oklahoma State University. Two C felis donor cats maintained in the Animal Resources unit at Oklahoma State University (Stillwater, OK, USA) were used to acquisition feed A americanum nymphs. The 49 principal cats used for transmission feeding of ticks and fed C felis-infected ticks were purpose-bred Class A research cats purchased through a commercial supplier (Liberty Research). All cats used were domestic shorthairs. All principal cats tested negative for C felis infection by PCR (described below) prior to study initiation.
Cat infestations, cat ingestion of ticks and tick counts
A americanum nymphs were purchased from the Tick Rearing Facility at Oklahoma State University. These nymphs were acquisition fed on two C felis donor cats. The donor cats were anesthetized via intramuscular injection with tiletamine HCl and zolazepam HCl (Telazol; Zoetis) at 4.0–12 mg/kg and their torsos shorn. Nymphs were placed between the shoulder blades of the cats. Knit cotton tubes that encompassed the cats’ thorax and abdomen were used to help confine and minimize escape of the ticks during feeding, as well as prevent donor cats from grooming ticks during acquisition feeding. Engorged nymphs were collected daily, placed into paper cartons and stored in a humidity chamber maintained with 90–98% relative humidity at 20–25°C with a 14 h light/10 h dark photoperiod while they molt to adults. Adult A americanum acquisition fed with C felis were enumerated and transmission fed on the 49 principal cats.
Transmission trials were performed using principal cats infested with 25 pairs (50 total) of adult A americanum that were acquisition fed as nymphs on the C felis donor cat. Infestations on principal cats were performed in a similar manner as described for the C felis donor. Permitted DOIs by A americanum on the principal study cats were 12 h ± 30 mins (group 1), 18 h ± 30 mins (group 2), 24 h ± 30 mins (group 3), 36 h ± 30 mins (group 4), 48 h ± 30 mins (group 5) and to repletion (group 7, control). At each of these respective time points, cats in groups 1–5 were anesthetized with Telazol and all ticks, attached and non-attached, were removed from cats, enumerated and placed in 70% ethanol. Group 7 cats were sedated with Telazol at 48 h ± 60 mins and ticks were enumerated but not removed (‘thumb counts’), after which these group 7 cats were returned to standard housing and ticks were allowed to feed until repletion.
Cats in group 6 were fed 50 adult A americanum that had been acquisition fed on the C felis donor cats. Briefly, 25 male and 25 female ticks were mixed with canned cat food and fed to principal cats in group 6. Cats were monitored for 1 h after feeding, to determine if cats vomited any of the canned food or ticks. After 1 h the remaining cat food was examined and ticks that were not ingested were quantified. Ticks fed to cats in group 6 came from the same batch of acquisition-fed A americanum adults used for transmission studies in groups 1–5 and 7, and overlapped in time with C felis transmission trials. As feeding of infected A americanum is considered the primary route of C felis transmission to domestic cats, cats in group 7 were used as controls for the group 6 cats that were fed C felis-infected ticks, as well as groups 1–5.
Determination of C felis infection
Cats were observed daily for clinical signs of cytauxzoonosis until 30 days postinfestation. Blood samples were collected periodically throughout the study. Up to 1 ml of blood was collected from the jugular vein by sterile collection. Blood was stored at 4°C until processing for analysis of C felis infection. In addition to observing for clinical signs consistent with cytauxzoonosis, C felis infection was determined by DNA extraction and PCR amplification. DNA was extracted from 200 µl whole blood, using a commercially available genomic DNA extraction kit (GeneJET Whole Blood Genomic DNA Purification; Thermo Scientific) according to the manufacturer’s instructions. Infection with C felis in the donor cat was confirmed through primary PCR amplification performed on an aliquot of purified DNA, using previously described methods. 13 A nested PCR amplification was also performed using previously described methods. 2
Statistical methods
Descriptive statistics, including the geometric mean, arithmetic mean, SEM and range, were used to quantify tick attachment and cat ingestion of ticks. The DOI necessary for acquisition-fed A americanum transmission of C felis to domestic cats was modeled using binary response regression. The analysis of the proportion of cats found positive from multiple blood draws among study groups was analyzed using Fisher’s exact tests. An alpha value of 0.05 was assumed and SAS 9.2 statistical software was used. Data for tick attachment (secondary variable) were transformed using an angular or arcsine square root transformation to alleviate issues associated with percentage data and heterogeneity of variance.
Results
Tick attachment and ingestion
The number of ticks attached to cats in the treatment and control groups was excellent (Table 2). The lowest number of ticks attached was in group 3, with a geometric mean of 46.6. The highest number of ticks attached was in group 1, with a geometric mean of 49.3. A significant difference in the number of ticks attached to cats among groups was not detected (F-value = 0.58, df = 5, P = 0.7113). The number of ticks ingested by cats in group 6 was also high. The geometric mean, arithmetic mean ± SEM and range of ticks ingested by cats in group 6 were 45.0, 45.5 ± 2.3 and 36–50, respectively.
Table 2.
Mean attachment of Amblyomma americanum adults, acquisition fed as nymphs on Cytauxzoon felis survivor donor cats, on principal cats in groups 1–5 and 7
| Tick attachment | Statistic | Experimental group |
|||||
|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 7 | ||
| Not attached | Geometric mean | NA | NA | NA | NA | NA | 1.1 |
| Arithmetic mean ± SEM | 0.6 ± 0.2 | 1.6 ± 0.9 | 1.6 ± 0.8 | 2.6 ± 1.3 | 1.6 ± 0.8 | 1.1 ± 0.1 | |
| Minimum–maximum | 0–1 | 0–7 | 0–6 | 0–9 | 0–6 | 1–2 | |
| Attached | Geometric mean | 49.3 | 48.0 | 46.6 | 46.7 | 47.7 | 48.1 |
| Arithmetic mean ± SEM | 49.3 ± 0.3 | 48.0 ± 1.1 | 46.9 ± 1.9 | 46.9 ± 1.3 | 47.9 ± 1.1 | 48.1 ± 0.3 | |
| Minimum–maximum | 48–50 | 42–50 | 36–50 | 41–50 | 42–50 | 47–49 | |
NA = not applicable
Infection of C felis
None of the cats in groups 1, 2, 3, 4 or 6 became infected with C felis (Table 3). One cat (14%) in group 5 (48 h) became infected with C felis at 12 days after infestation. Six cats (86%) in group 7 (control group) became infected with C felis (Table 3) at 8, 8, 10, 12, 12 and 12 days after infestation, respectively. Significantly more cats in the control group (P = 0.0047) became infected with C felis than cats in groups 1, 2, 3, 4 and 6. Similarly, more cats (P = 0.0291) in the control group became infected with C felis than in group 5. All cats infected with C felis were determined by PCR before the onset of clinical signs cytauxzoonosis. Cats infected with C felis were transferred to another protocol where they were eligible to be treated for cytauxzoonosis.
Table 3.
Number of cats infected with Cytauxzoon felis
| Group | Number of cats infected with C felis | Number of cats not infected with C felis | Total number of cats |
|---|---|---|---|
| 1 (12 h infestation) | 0 | 7 | 7 |
| 2 (18 h infestation) | 0 | 7 | 7 |
| 3 (24 h infestation) | 0 | 7 | 7 |
| 4 (36 h infestation) | 0 | 7 | 7 |
| 5 (48 h infestation) | 1 | 6 | 7 |
| 6 (ingestion of ticks) | 0 | 7 | 7 |
| 7 (control group) | 6 | 1 | 7 |
| Total | 7 | 42 | 49 |
Discussion
The life cycle of C felis in A americanum is not known. Generally speaking, a short period of time is usually necessary from when ticks attach to hosts and begin to feed to when pathogens are transmitted. During this delay it is thought that pathogens move from a state of dormancy or are initiated to continue their development and progress through stages of their life cycle.14–20 Transfer of pathogens from ticks to hosts is thought to occur across a continuum whereby a few pathogens are transmitted early with the probability of transmission increasing over the DOI.14,18 A notable exception to the reactivation paradigm is Powassan virus. Ebel and Kramer demonstrated transmission of Powassan virus from Ixodes scapularis to mice after 15 mins of attachment. 15
Theileria parva, a piroplasm closely related to C felis, 21 is transmitted to cattle by Rhipicephalus appendiculatus. Ochanda et al demonstrated that the time necessary for transmission of T parva was affected, in part, by the ambient temperatures in which the ticks where held prior to infestation on cattle. 19 R appendiculatus infected with T parva held at 18°C (64.4°F) for 6 days and 85% relative humidity prior to infestation transmitted the pathogen in ⩽72 h. Ticks held at 37°C (98.6°F) for 6 days and 85% relative humidity prior to infestation transmitted T parva in ⩽24 h. In the current study, A americanum acquistion fed as nymphs on a cytauxzoonosis survivor cat were incubated at 20–25°C (60–77°F) and 90–98% relative humidity prior to transmission feeding on principal cats. None of the principal cats infested for 12, 18, 24 or 36 h became infected, whereas 1/7 (14%) cats became infected with C felis after 48 h of infestation, and 6/7 (86%) control cats in which A americanum were allowed to feed until repletion became infected. Our results indicated that the minimum amount of time for transmission of C felis with A americanum adults maintained at room temperature is >36 h to ⩽48 h. In Oklahoma where C felis is enzootic and the current study was performed, the average high temperature between April and October ranges from 22.4°C to 34.4°C (72–94°F) and average low temperature from 9.8°C to 22.3°C (50–72°F). 22 Moreover, in July and August the average low and high temperatures range from 21.6–34.4°C (71–94°F). That is, for 2 months of the year, ambient temperatures in an area enzootic for C felis are at or above the temperature at which the current study was conducted. It has yet to be determined if increasing the ambient temperature shortens the transmission time of C felis other than what was demonstrated in the current study.
Babesia microti, another piroplasm related to C felis, 21 is transmitted to rodents and humans by the bite of infected I scapularis. Piesman and Spielman used I scapularis nymphs aquistion fed as larvae on a hamster infected with B microti, to determine the transmission time of B microti. 17 They demonstrated that 9% (1/11) of the hamsters became infected with B microti after 36 h of attachment, 17% (2/12) were infected after 48 h and 50% (6/12) were infected with B microti after 54 h of attachment. In the current study, 14% (1/7) of cats became infected with C felis after 48 h of infestation, whereas none of the cats in the 12, 18, 24 or 36 h groups became infected.
Factors that govern the transmission of pathogens from ticks to vertebrate host are not well understood. We can only speculate as to the effects that doses of pathogens in ticks, vertebrate host immune responses to ticks and to pathogens, strain variation of pathogen and extrinsic environmental variables, among a few, have on the transmission time of pathogens to veterbrate hosts through ticks. Future research should explore the effects of increasing ambient temperature on C felis transmission from A americanum to cats, to determine if the minimum transmission time can be quicker than what was demonstrated in the current study. Similarily, intrastadial transmission of infected ticks from an infested cat or other vertebrate host to a naive cat should also be explored as a mechanism for which the minimum transmission time may be shorter than what was demonstrated, as pathogen replication would already be intitiated and a reactivation period would not be necessary.
Acaricides approved for use on cats in the USA include etofenprox, fipronil, flumethrin and fluralaner. Etofenprox and flumethrin are pyrethroids that kill and repel ticks. 23 Fipronil is a phenylpyrazole and does not have repellant properties against ticks. Fluralaner is an isoxazoline and does not have repellant properties against ticks. Fourie et al compared the acaricidal effiacy of two combination products used to control ticks on cats. 24 They demonstrated that flumethrin 4.5% (w/w)/imidacloprid 10% (w/w) was 100% effective against Ixodes ricinus adults on cats at 6, 12, 24 and 48 h postinfestation, whereas fipronil 8.3% (w/v)/(S)-methoprene 10% (w/v)/eprinomectin 0.4% (w/v)/praziquantel 8.3% (w/v) was 0–16%, 26.8–50%, 31.5–81.5% and 83.2–100% at 6, 12, 24 and 48 h postinfestation, respectively. Reichard et al demonstrated that application of flumethrin 4.5% (w/w)/imidacloprid 10% (w/w) prevented the transmission of C felis by adult A americanum, 9 which demonstrated, for the first time, that transmission of a tick-borne pathogen of cats could be blocked using a repellant.
Vertebrate hosts becoming infected with tick-borne protozoa by ingestion of infected ticks is not novel. Dogs can become infected with Hepatozoon americanum through ingestion of infected Amblyomma maculatum,25,26 and Hepatozoon canis by ingestion of infected Rhipicephalus sanguineus. 27 As most cats are considered to be excellent groomers, we sought to determine if cats could become infected with C felis through ingestion of infected A americanum. We fed up to 50 A americanum acquisition fed as nymphs on a C felis donor to seven principal cats (group 6). None of the cats in this tick ingestion group became infected with C felis, whereas 6/7 cats in the control group did become infected with C felis suggesting that ingestion of C felis-infected A americanum adults is not a likely route of transmission to domestic cats.
Conclusions
Cytauxzoonosis is a severe and often fatal disease of domestic cats. Cats become infected via the bite of an infected A americanum or possibly Dermacentor variabilis. Results from the current study suggest that ingestion of C felis-infected A americanum is not a likely route of transmission to naive cats. Conversely, transmission of C felis can happen as quickly as 48 h, with A americanum adults held at room temperature for at least 6 days before infesting cats. It is not known what effect increasing the ambient temperature of ticks or intrastradial transmission of C felis has on the transmission time to domestic cats. The rapid transmission of C felis to cats in >36 to ⩽48 h emphasizes the need of having cats on approved and effective acaricides to help prevent ticks from attaching and feeding.
Footnotes
Accepted: 5 January 2017
CMO and JAH were full-time employees of Bayer HealthCare Animal Health at the time of the study. In the past 5 years, JET and MVR have received honoraria and research support from multiple veterinary pharmaceutical companies for related and unrelated activities.
Funding: This research was funded through a grant from Bayer HealthCare Animal Health.
References
- 1. Wagner JE. A fatal cytauxzoonosis-like disease in cats. J Am Vet Med Assoc 1976; 168: 585–588. [PubMed] [Google Scholar]
- 2. Reichard MV, Edwards AC, Meinkoth JH, et al. Confirmation of Amblyomma americanum (Acari: Ixodidae) as a vector for Cytauxzoon felis (Piroplasmorida: Theileriidae) to domestic cats. J Med Entomol 2010; 47: 890–896. [DOI] [PubMed] [Google Scholar]
- 3. Blouin EF, Kocan AA, Glenn BL, et al. Transmission of Cytauxzoon felis Kier, 1979 from bobcats, Felis rufus (Schreber), to domestic cats by Dermacentor variabilis (Say). J Wildl Dis 1984; 20: 241–242. [DOI] [PubMed] [Google Scholar]
- 4. Blouin EF, Kocan AA, Kocan KM, et al. Evidence of a limited schizogonous cycle for Cytauxzoon felis in bobcats following exposure to infected ticks. J Wildl Dis 1987; 23: 499–501. [DOI] [PubMed] [Google Scholar]
- 5. Reichard MV, Meinkoth JH, Edwards AC, et al. Transmission of Cytauxzoon felis to a domestic cat by Amblyomma americanum. Vet Parasitol 2009; 161: 110–115. [DOI] [PubMed] [Google Scholar]
- 6. Reichard MV, Baum KA, Cadenhead SC, et al. Temporal occurrence and environmental risk factors associated with cytauxzoonosis in domestic cats. Vet Parasitol 2008; 152: 314–320. [DOI] [PubMed] [Google Scholar]
- 7. Meinkoth JH, Kocan AA. Feline cytauxzoonosis. Vet Clin North Am Small Anim Pract 2005; 35: 89–101. [DOI] [PubMed] [Google Scholar]
- 8. Sherrill MK, Cohn LA. Cytauxzoonosis: diagnosis and treatment of an emerging disease. J Feline Med Surg 2015; 17: 940–948. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Reichard MV, Thomas JE, Arther RG, et al. Efficacy of an imidacloprid 10%/flumethrin 4.5% collar (Seresto, Bayer) for preventing the transmission of Cytauxzoon felis to domestic cats by Amblyomma americanum. Parasitol Res 2013; 112: S11–S20. [DOI] [PubMed] [Google Scholar]
- 10. Plumb DC. Plumb’s veterinary drug handbook. 7th ed. Ames, IA: Wiley-Blackwell, 2011. [Google Scholar]
- 11. Nagamori Y. Transmission of Cytauxzoon felis by Amblyomma americanum: engorgement weight of nymphs and attachment time of adults for transmission to domestic cats. MS Thesis, Oklahoma State University, 2016. [Google Scholar]
- 12. Cruthers L, Slone RL, Guerrero AJ, et al. Evaluation of the speed of kill of fleas and ticks with Frontline Top Spot in dogs. Vet Ther 2001; 2: 170–174. [PubMed] [Google Scholar]
- 13. Bondy PJ, Cohn LA, Tyler JW, et al. Polymerase chain reaction detection of Cytauxzoon felis from field-collected ticks and sequence analysis of the small subunit and internal transcribed spacer 1 region of the ribosomal RNA gene. J Parasitol 2005; 91: 458–461. [DOI] [PubMed] [Google Scholar]
- 14. des Vignes F, Piesman J, Heffernan R, et al. Effect of tick removal on transmission of Borrelia burgdorferi and Ehrlichia phagocytophila by Ixodes scapularis nymphs. J Infect Dis 2001; 183: 773–778. [DOI] [PubMed] [Google Scholar]
- 15. Ebel GD, Kramer LD. Short report: duration of tick attachment required for transmission of powassan virus by deer ticks. Am J Trop Med Hyg 2004; 71: 268–271. [PubMed] [Google Scholar]
- 16. Katavolos P, Armstrong PM, Dawson JE, et al. Duration of tick attachment required for transmission of granulocytic ehrlichiosis. J Infect Dis 1998; 177: 1422–1425. [DOI] [PubMed] [Google Scholar]
- 17. Piesman J, Spielman A. Human babesiosis on Nantucket Island: prevalence of Babesia microti in ticks. Am J Trop Med Hyg 1980; 29: 742–746. [DOI] [PubMed] [Google Scholar]
- 18. Piesman J, Mather TN, Sinsky RJ, et al. Duration of tick attachment and Borrelia burgdorferi transmission. J Clin Microbiol 1987; 25: 557–558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Ochanda H, Young AS, Mutugi JJ, et al. The effect of temperature on the rate of transmission of Theileria parva parva infection to cattle by its tick vector, Rhipicephalus appendiculatus. Parasitol 1988; 97: 239–245. [DOI] [PubMed] [Google Scholar]
- 20. Spencer RR, Parker RR. Rocky mountain spotted fever: infectivity of fasting and recently fed ticks. Public Health Rep 1923; 38: 333–339.19314866 [Google Scholar]
- 21. Lack JB, Reichard MV, Van Den Bussche RA. Phylogeny and evolution of the Piroplasmida as inferred from 18S rRNA sequences. Int J Parasitol 2012; 42: 353–363. [DOI] [PubMed] [Google Scholar]
- 22. Data UC. Climate Oklahoma City – Oklahoma and Weather averages Oklahoma City – Weather history January 2016. http://www.usclimatedata.com/climate/oklahomacity/oklahoma/united-states/usok0400 (accessed October, 25 2016).
- 23. Bowman DD. Georgis’ parasitology for veterinarians. 10th ed. St Louis, MO: Saunders/Elsevier, 2013. [Google Scholar]
- 24. Fourie JJ, Horak IG, de Vos C, et al. Comparative speed of kill, repellent (anti-feeding) and acaricidal efficacy of an imidacloprid/flumethrin collar (Seresto) and a fipronil/(S)-methoprene/eprinomectin/praziquantel spot-on (Broadline) against Ixodes ricinus (Linne, 1758) on cats. Parasitol Res 2015; 114: S109–S116. [DOI] [PubMed] [Google Scholar]
- 25. Mathew JS, Ewing SA, Panciera RJ, et al. Experimental transmission of Hepatozoon americanum Vincent-Johnson et al., 1997 to dogs by the Gulf Coast tick, Amblyomma maculatum Koch. Vet Parasitol 1998; 80: 1–14. [DOI] [PubMed] [Google Scholar]
- 26. Garrett JJ, Kocan AA, Reichard MV, et al. Experimental infection of adult and juvenile coyotes with domestic dog and wild coyote isolates of Hepatozoon americanum (Apicomplexa: Adeleorina). J Wildl Dis 2005; 41: 588–592. [DOI] [PubMed] [Google Scholar]
- 27. Baneth G, Samish M, Shkap V. Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris). J Parasitol 2007; 93: 283–299. [DOI] [PubMed] [Google Scholar]