Detection and prevalence of Cercopithifilaria bainae infection in shelter dogs in Oklahoma, USA

Cercopithifilaria bainae is a filarioid nematode of dogs. Infection with the parasite was not reported in the USA until 2017, when a dog with skin lesions in Florida was diagnosed. Brown dog ticks, Rhipicephalus sanguineus sensu lato , are the purported tick vectors, and are widespread in the USA. Therefore, C. bainae is likely present in additional states. Here, we tested dogs and ticks in Oklahoma for evidence of C. bainae infection.


3
(99-100% identity). One attached R. sanguineus s.l. male on the same dog harbored filarioid DNA sequence which was difficult to interpret at numerous base-pair locations, but was closest in identity (~80%) to C. bainae.

Conclusions
The distribution of C. bainae is more widespread than previously known. Here, we document C. bainae infections in dogs and DNA in brown dog ticks in Oklahoma for the first time. As brown dog ticks are commonly found throughout the USA, veterinarians in this region should consider C. bainae infection as a differential diagnosis in canine patients with dermatitis or polyarthritis.

Background
Cercopithifilaria bainae is a tick-borne filarial nematode of dogs that was first described in Brazil in 1984 [1]. Adults of C. bainae parasitize the subcutaneous tissue of canine hosts, and microfilariae remain sequestered in the dermis, making detection of the parasite in infected dogs challenging [2]. Cercopithifilaria bainae is considered primarily non-pathogenic, but erythematous, papular and pruritic dermatitis, non-healing and ulcerative skin lesions, and subcutaneous nodules associated with infection have been reported [3][4][5]. One case of polyarthritis has also been documented [3].
Cercopithifilaria bainae infections in dogs occur predominantly in the Mediterranean region, and DNA of the parasite has been reported in the suspected tick vector, Rhipicephalus sanguineus sensu lato (s.l.), collected in Australia, Brazil, Malaysia, and South Africa [3,4,6]. Rhipicephalus sanguineus s.l., commonly called brown dog ticks, are thought to be important natural vectors of C. bainae based on the development of third-stage larvae in adult ticks acquisition fed as nymphs on a 4 naturally infected dog [7]. Although C. bainae has been molecularly detected in other ticks, including Dermacentor reticulatus and Ixodes ricinus, parasite development within these tick species has not been experimentally demonstrated [7,8].
Despite the cosmopolitan distribution of brown dog ticks, C. bainae had not been documented in the USA until 2017. A dog from Florida with no travel history was presented with dermatitis, with plaques on the dorsal head, and alopecia, erythema, and ulceration of both medial canthi. Microfilariae isolated from skin biopsy samples via saline sedimentation were identified as C. bainae by PCR and microscopy [5].
Brown dog ticks are widespread in the USA, with all stages preferentially feeding on dogs, and it is likely that C. bainae is present in dogs in states in addition to Florida [9,10]. To the authors' knowledge, however, no studies investigating geographic distribution and infection prevalence of C. bainae in dogs in the USA have been conducted. To determine if C. bainae is present in dogs in Oklahoma, multiple dermal punch biopsy samples were evaluated by saline sedimentation and PCR.
Additionally, ticks observed on dogs were tested for filarioid DNA.

Skin biopsy sample collection
Skin biopsy samples were opportunistically collected from dogs in Oklahoma, USA, over a 10-month period (January-October 2018). Shelter dogs were temporarily housed at animal control facilities prior to euthanasia following standard approved shelter protocols and client-owned dogs were submitted for necropsy at the Oklahoma Animal Disease Diagnostic Laboratory (Payne County, Stillwater, OK).
When possible, sex and estimated age were documented. Travel histories were not 5 available for the majority of animals, nor was information regarding prior treatment with parasiticides.
Multiple skin samples were collected from individual animals using sterile 6 mm biopsy punches within hours, but sometimes up to four days, after death. Increased frequency of C. bainae microfilariae in interscapular and head regions has been previously described [11], and therefore these focal regions were sampled; up to four interscapular and up to three head samples were collected from each animal.
At times of skin biopsy sample collection, the skin was briefly examined for cutaneous nodules and other lesions.
Single interscapular biopsy samples were placed in microcentrifuge tubes containing phosphate buffered saline (PBS) and transported to the laboratory for storage at -20 °C and later DNA extraction and molecular analyses. Additional biopsy samples were placed in PBS-filled, sterile 15 ml conical tubes and, upon transport to the laboratory, processed to recover microfilariae as described below. After processing, the majority of skin samples were stored at -20 °C for subsequent DNA extraction and PCR.

Saline sedimentation of skin biopsy samples
To detect microfilariae in skin biopsy samples, up to three skin samples from individual dogs were placed in 15 ml conical tubes containing PBS and incubated for 1-3 hours at 37 °C to allow live microfilariae to migrate out of the tissue [11]. The skin was removed and remaining PBS was centrifuged at 1,500 rpm for 5 minutes to concentrate microfilariae. Supernatants were decanted and resulting pellets were stained with 0.1% methylene blue for microscopic examination.
Microfilariae in sedimentations were gently washed from slides with PBS and stored at 4 °C for DNA extraction within 48 hours for subsequent molecular identification.

Tick collection and processing
Animals were briefly examined for ticks at the time of skin biopsy collection. When present, ticks were placed in 70% ethanol and stored at -20˚C. At the time of dissection, ticks were removed from ethanol and identified to species by microscopic examination and comparison with standard keys [15]. Identified ticks were then individually dissected and internal contents removed and digested in Proteinase K and lysis buffer solution at ambient temperature [16].
DNA extraction methods, PCR, and sequence analysis Tick dissection, DNA extraction, PCR amplification, and amplicon purification were carried out in dedicated laboratory areas to prevent DNA contamination. Separate Additionally, PCR amplifying a 340-370 base pair region of the 12S rRNA mitochondrial gene was performed on R. sanguineus s.l. testing positive for C. bainae, using previously described primers 12SF and 12SR, to determine the genetic lineage (temperate or tropical) of the ticks as previously described [16,17].
Standard gel electrophoresis in a 2% agarose matrix with GelRed® staining (Biotium, Fremont, CA) was used to detect amplicons. Correctly sized amplicons were purified either directly from the gel using the QIAquick® Gel Extraction Kit   [25].
To the authors' knowledge, the present study is the first report of Cercopithifilaria sp. DNA in ticks in the USA, and suggests R. sanguineus s.l. may serve as vector in this region, as has been reported in other areas of the world [26]. All three of the PCR positive R. sanguineus s.l. were attached to one dog that was later determined to have C. bainae microfilariae; the female ticks were engorged, but it was not apparent for how long the male tick had been attached or if a blood meal was taken.
The presence of R. sanguineus s.l. on a dog infected with C. bainae is noteworthy, and compels the authors to suspect that the parasite is cycling between this tick group and dogs in the USA. If C. bainae microfilariae had been ingested by immature R. sanguineus s.l. stages, they may have gone on to develop into infective 14 third-stage larvae within ticks during ecdysis, as has been experimentally demonstrated in this tick group in other areas of the world [26].
Alternatively, the Cercopithifilaria sp. DNA amplified from the three ticks may have occurred following incidental ingestion of dermal microfilariae from the infected dog on which they were found. This possibility is evidenced by the fact that DNA of D.
sanguineus s.l. tested. As D. immitis is adapted to mosquito intermediate hosts [12], it is extremely unlikely that developing larvae were present within ticks, but rather microfilariae were incidentally ingested in blood. Although previous studies have demonstrated molecular evidence of C. bainae in other tick species (Dermacentor reticulatus and Ixodes ricinus,), R. sanguineus s.l. is the only tick group which has been experimentally demonstrated to host developing stages of the parasite [7,8].
In this study, C. bainae, was not detected in A. americanum, A. maculatum, or D.
variabilis. However, if more specimens of each of these tick species were tested,