Species-specific Nested PCR for Detecting Plasmodium gallinaceum in Fresh Chicken Blood

vian malaria, epidemic in Thailand’s chicken farms, is caused by Plasmodium gallinaceum. The standard method for detecting avian malaria is microscopic diagnosis, but parasites may go undetected in light infections. Therefore, the objective of this study was to detect P. gallinaceum DNA in infected fresh-blood samples by nested polymerase chain reaction (nested PCR). Primers were designed from small subunit ribosomal RNA (SSUrRNA) genes, with genus-specific primers for initial amplification, and species-specific primers for nested amplification. The PCR conditions were optimized using P. gallinaceum DNA as templates. The optimum conditions for the 1st and 2nd amplifications were different at the annealing stages, using 60 oC and 45 oC, respectively. The first amplification resulted in unobservable PCR products at 420 bp of the target fragment in all diluted blood samples. These products were continued in the 2nd amplification, when the species-specific 120 bp-DNA fragment of P. gallinaceum was found. This nested PCR condition was sensitive to detecting 0.0000085% parasitemia or 0.2 infected red cells/μl of the lowest blood dilution in the study. The results suggest that this assay had high sensitivity and species-specificity using whole blood samples without DNA extraction.