Aim
The aim of this study was to survey kenneled coyote-hunting dogs for B. conradae and other apicomplexan parasites and to establish efficacy of published atovaquone and azithromycin therapy in infected dogs.
Study Population
A total of 40 dogs from four separate kennels in Oklahoma were included in this study. Group 1 consisted of 6 Greyhounds and 4 Treeing Walker Coonhounds from Crescent, OK sampled in March of 2014; Group 2 consisted of 16 Greyhounds from Kingfisher, OK also sampled in March of 2014; Group 3 consisted of 10 Greyhounds from Vinita, OK sampled in February of 2019; and Group 4 consisted of 4 Greyhounds from Hobart, OK sampled in May of 2020 (Figure 1). One dog from Group 1 displayed clinical signs of lethargy, fever, and anemia while the remaining dogs were subclinical. Clinical signs were not apparent in any of the dogs in Group 2. In Group 3, clinical signs including vomiting, lethargy, anorexia, and anemia were observed in 2 dogs, while the remaining dogs were subclinical. Group 4 had one clinical dog, which died prior to diagnosis and treatment (Dog 37) and one subclinical dog. At initial presentation, whole blood (1–5 mL) was collected in EDTA from all animals (n=40) and transported on ice overnight to the Oklahoma State University College of Veterinary Medicine (OSU-CVM). Samples were stored at 4°C prior to processing.
DNA Extraction and PCR
For Groups 1 and 2, DNA was extracted from whole blood using the DNeasy Blood and Tissue kit (QIAGEN, Valencia, CA). For Groups 3 and 4, DNA was extracted using the IllustraTM blood genomic Prep Mini Spin Kit (GE Healthcare, Piscataway, New Jersey). All extractions were performed according to kit manufacturer instructions. Dedicated laboratory areas were utilized for DNA extractions, primary and secondary PCR amplifications, and PCR product purifications to prevent contamination events. Separate ultra-purified water samples (NTC) were included as negative controls in DNA extractions and PCR amplifications. DNA extracts from Groups 1–4 were analyzed by previously described nested PCR methods which amplify a 460- to 520-bp hypervariable region of the 18S rRNA gene of Babesia spp. and some other apicomplexans (Table 1) (11, 12), except for Dog 27 who’s PCR and sequencing was carried out by the North Carolina State Vector Borne Disease Diagnostics Lab (NCSVBDDL) per their standard operating procedures. NCSVBDDL utilizes primers that also amplify the 18S rRNA gene. DNA extracts from known piroplasm positive blood samples by microscopy were included as positive controls for each sample set.
Table 1. PCR primers used to amplify partial 18S rRNA gene of Babesia spp. and other apicomplexans.
Primer
|
Primer Sequence (5ˊ→3ˊ)
|
Reference
|
BABA-F
|
CCG AAT TCG ACA ACC TGG TTG ATC CTG CCA GT
|
11
|
BABA-R
|
CCC GGA TCC AAG CTT GAT CCT TCT GCA GGT TCA CCT AC
|
11
|
3.1
|
CTC CTT CCT TTA AGT GAT AAG
|
12
|
5.1
|
CCT GGT TGA TCC TGC CAG TAG T
|
12
|
RLB-F
|
GAG GTA GTG ACA AGA AAT AAC AAT A
|
12
|
RLB-R
|
TCT TCG ATC CCC TAA CTT TC
|
12
|
For Groups 1 and 2, primary PCR reactions were performed in 25 µL volumes containing 0.25 U Taq polymerase (Promega, Madison, WI), 10× Taq buffer (Promega), 1.5 mM MgCl2, 0.8 mM dNTP mixture (Promega), 0.8 µM each primer BABA-F and BABA-R, and 5 µL template DNA. Primary reaction conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 56.6°C for 1 min, 72°C for 2 min, and a final extension step of 72°C for 5 min. Nested PCR was carried out using 1 µl of the primary product and primers RLB-F and RLB-R. Nested reaction conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 2 min, and a final extension step of 72°C for 5 min.
For Groups 3 and 4, primary PCR reactions were prepared in 25 µl volumes containing 0.075 U AccuprimeTM Taq HIFI (ThermoFisher, Waltham, MA), 1X AccuPrimeTM PCR Buffer II (ThermoFisher), 1.5 mM MgSO4 (ThermoFisher), 0.2 µM each primer 3.1 and 5.1, and 5 µl of DNA extract. Primary reaction conditions were as follows: 94°C for 2 min followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, 68°C for 1.5 min, and a final extension step of 72°C for 10 min. Nested PCR was again carried out using 1 µl of primary product and primers RLB-F and RLB-R, but cycling conditions were different than above. For Group 3 and 4 dogs, nested PCR reaction conditions were as follows: 94°C for 2 min followed by 40 cycles of 94°C for 1 minute, 50°C for 1 minute, 68°C for 1.5 minutes, and a final extension step of 72°C for 10 minutes.
PCR Product Purification and Sequencing
PCR products were electrophoresed in a 2% agarose matrix containing either GelRed® QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA) or the Wizard® SV Gel and PCR Clean-Up System (Promega) according to manufacturer’s instructions. DNA sequencing was performed at the Oklahoma State University Molecular Core Facility (Stillwater, OK) with an ABI 3730 DNA Analyzer, except for Dog 27. Forward and reverse sequences were aligned with ClustalW (Bioinformatics Center, Kyoto, Japan) and compared with sequence data available in the National Center for Biotechnology Information database (GenBank) for B. conradae (MK256976), B. canis (AY272047), and B. gibsoni (KC461261) to determine percent homology. Samples were considered positive if they were 98% or higher homologous to B. conradae (GenBank MK256976). The sequences from Dogs 1, 28, 29, 33, 34, 35, 37 and 38 have been deposited in GenBank under the accession numbers MW1470222, MT430944, MW145168, MW145196, MW145199, MW145504, MW145505 and MW145506, respectively.
Phylogenetic Tree and Percent Identity Matrix Construction
All partial 18S sequences obtained from canines in the current study were entered into MacVector, aligned and trimmed along with various 18S piroplasm reference sequences available in GenBank which were used in previous analyses (1, 7, 13). A maximum likelihood phylogenetic tree was constructed using unweighted pair group method with arithmetic mean (UPGMA) analysis. Bootstrap values are based on 1000 replicates and only bootstraps >50% are indicated. The percent homology matrix was also produced in MacVector using the same 18S full and partial sequences.
Treatment Protocol
Upon owner consent, surviving B. conradae PCR positive dogs from each cohort were treated with a previously described treatment regimen shown to eliminate infection in vivo (9). Atovaquone (GlaxoSmithKline, Research Triangle Park, NC) and azithromycin (Pfizer, New York, NY) was compounded in an oral suspension and administered to all surviving PCR-positive dogs at a dose of 13.5 mg/kg TID (atovaquone) and 10 mg/kg SID (azithromycin) for 10 days. Whole blood (1–5 mL) was collected in EDTA prior to treatment (day 0) and at 30 and 60 days post-treatment. DNA was extracted from each blood sample and tested by PCR to detect Babesia sp. infection as previously described.
Statistics
The prevalence of B. conradae infection was calculated according to Bush et al. (14) and 95% confidence intervals were calculated using QuickCalcs (15). The prevalence of B. conradae in hunting dogs among kennels was compared using chi-square tests (16).