title: Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand.
abstract: Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol-chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol-chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.
authors: Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: Sittiporn.P@Chula.ac.th.
authors: Sittiporn
authors: S
authors: Pattaradilokrat
authors: Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Wisawa
authors: W
authors: Tiyamanee
authors: Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Phumin
authors: P
authors: Simpalipan
authors: Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Morakot
authors: M
authors: Kaewthamasorn
authors: Department of Parasitology and Entomology, Faculty of Public Health Science, Mahidol University, Bangkok 10400, Thailand.
authors: Tawee
authors: T
authors: Saiwichai
authors: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People's Republic of China.
authors: Jian
authors: J
authors: Li
authors: Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Pongchai
authors: P
authors: Harnyuttanakorn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000835:Animals, Wild; D001717:Birds; D002645:Chickens; D016054:DNA, Protozoan / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D008289:Malaria, Avian / Q000175:diagnosis / Q000453:epidemiology / Q000469:parasitology*; D008969:Molecular Sequence Data; D010964:Plasmodium gallinaceum / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013785:Thailand / Q000453:epidemiology
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins
keywords: Avian malaria; Chelex-100 resin; Cytochrome B; Cytochrome oxidase subunit I; DNA sequencing; Polymerase chain reaction