High throughput pyrosequencing technology for molecular differential detection of Babesia vogeli, Hepatozoon canis, Ehrlichia canis and Anaplasma platys in canine blood samples.
Canine babesiosis, hepatozoonosis, ehrlichiosis, and anaplasmosis are tick-borne diseases caused by different hemopathogens. These diseases are causes of morbidity and mortality in dogs. The classic method for parasite detection and differentiation is based on microscopic observation of blood smears. The limitations of the microscopic method are that its performance requires a specially qualified person with professional competence, and it is ineffective in differentiating closely related species. This study applied PCR amplification with high throughput pyrosequencing for molecular differential detection of the following 4 hemoparasites common to tropical areas in dog blood samples: Babesia vogeli, Hepatozoon canis, Ehrlichia canis, and Anaplasma platys. PCR was initially used to amplify specific target regions of the ribosomal RNA genes of each parasite using 2 primer pairs that included 18S rRNA for protozoa (B. vogeli and H. canis) and 16S rRNA for rickettsia (E. canis and A. platys). Babesia vogeli and H. canis were discriminated using 9 nucleotide positions out of 30 base pairs, whereas E. canis and A. platys were differentiated using 15 nucleotide positions out of 34 base pairs that were determined from regions adjacent to 3' ends of the sequencing primers. This method provides a challenging alternative for a rapid diagnosis and surveillance of these tick-borne diseases in canines.
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand.
Worasak
W
Kaewkong
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Pewpan M
PM
Intapan
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Oranuch
O
Sanpool
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Penchom
P
Janwan
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Faculty of Medicine, Mahasarakham University, Mahasarakham 44000, Thailand.
Tongjit
T
Thanchomnang
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Amornmas
A
Kongklieng
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Division of Cell Biology, Department of Preclinical Sciences, Thammasat University, Rangsit Campus, Pathum Thani 12121, Thailand.
Chairat
C
Tantrawatpan
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Thidarut
T
Boonmars
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Viraphong
V
Lulitanond
Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
Piyanan
P
Taweethavonsawat
Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
Sudchit
S
Chungpivat
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Electronic address: wanch_ma@kku.ac.th.
Wanchai
W
Maleewong
D000709:Anaplasma / Q000235:genetics / Q000302:isolation & purification; D000712:Anaplasmosis / Q000175:diagnosis* / Q000382:microbiology; D000818:Animals; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification; D001404:Babesiosis / Q000175:diagnosis* / Q000469:parasitology; D001483:Base Sequence; D003044:Coccidia / Q000235:genetics / Q000302:isolation & purification; D003048:Coccidiosis / Q000175:diagnosis* / Q000469:parasitology; D004269:DNA, Bacterial / Q000097:blood; D016054:DNA, Protozoan / Q000097:blood; D004283:Dog Diseases / Q000175:diagnosis* / Q000382:microbiology / Q000469:parasitology; D004285:Dogs; D041061:Ehrlichia canis / Q000235:genetics / Q000302:isolation & purification; D016873:Ehrlichiosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D059014:High-Throughput Nucleotide Sequencing / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013045:Species Specificity
D004269:DNA, Bacterial; D016054:DNA, Protozoan
Anaplasma platys; Babesia vogeli; Differential detection; Ehrlichia canis; Hepatozoon canis; Pyrosequencing