Skin biopsy sample collection
Skin biopsy samples were opportunistically collected from euthanatized 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 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. Carcasses were stored at 4°C until sample collection. Higher frequency of C. bainae microfilariae in interscapular and head regions has been previously described , 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 . The skin was removed and remaining PBS was centrifuged at 388 x g for 5 minutes to concentrate microfilariae. Supernatants were decanted and resulting pellets were stained with 0.1% methylene blue for microscopic examination.
Stained sediment was transferred to microscope slides and covered with 22x60 mm glass cover slips; all sedimentation material from each skin sample was scanned under 100X total magnification. When observed, microfilariae on slides were enumerated, and up to 10 microfilariae were measured using an ocular micrometer (length and width) under 400X total magnification. Microfilariae measurements were compared to those available in the literature identifying filarioid species including Acanthocheilonema reconditum (215–288 x 4.5–5.8 µm), Cercopithifilaria bainae (173.8–200 x 5.6–6.9 µm), and Dirofilaria immitis (280–325 x 5–7.5 µm) [13–15]. 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 (approximately 1–3 minutes) 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 . Identified ticks were then individually dissected and internal contents removed and digested in Proteinase K and lysis buffer solution at ambient temperature .
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 negative water controls were used for DNA extractions and for PCR. A sample containing DNA of D. immitis was used as a positive control.
Nucleic acid was extracted from approximately 30 mg sections of skin biopsy samples using the QIAamp® Fast DNA Tissue Kit (Qiagen, Valencia, CA, USA). Refrigerated microfilariae (washed with PBS from glass microscope slides) were extracted for DNA using the IllustraTM blood genomicPrep Mini Spin Kit (GE Healthcare, Piscataway, NJ, USA). After tissue digestion, individual tick samples were extracted for DNA using the QIAamp® DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). DNA extractions were carried out according to the manufacturer’s instructions specific to each kit.
PCR amplifying a ~ 330 base pair region of the filarioid 12S rRNA mitochondrial gene was performed on DNA extractions from skin, microfilariae, and ticks using previously described primers Fila12SF and Fila12SR . Individual reactions were carried out in a total volume of 25 µl containing 1X AmpliTaq Gold 360 (Applied Biosystems, Carlsbad, CA), 0.8 µM of each primer, and 2 µl of DNA. Thermocycler conditions were as follows: 94°C for 10 min, followed by 40 cycles of 94°C for 45 sec, 52°C for 45 sec, and 72°C for 90 sec, and ending with a final extension step of 72°C for 7 min.
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 [17, 18].
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 (QIAGEN, Valencia, CA, USA) or from PCR reactions using the QIAquick® PCR Purification Kit (QIAGEN, Valencia, CA, USA).
Purified amplicons were bi-directionally sequenced (Sanger method) by Eurofins Genomics (Louisville, KY) or the Oklahoma State University Molecular Core Facility (Stillwater, OK). Sequences from skin samples and ticks were compared to those available in the National Center for Biotechnology Information database (GenBankTM) to determine filarioid species identity and R. sanguineus s.l. genetic lineage. Sequence alignments were constructed using ClustalW to determine percent similarities of Oklahoma filarioid 12S rRNA mitochondrial gene sequences to each other and to additional filarioid sequences previously contributed to the GenBankTM repository, as well as to determine R. sanguineus s.l. genetic lineage.