Development and application of a novel multiplex PCR assay for the differentiation of four haemosporidian parasites in the chicken Gallus gallus domesticus.

Haemosporidian infections in domestic chickens (Gallus gallus domesticus) are not only widely prevalent but also cause economic loss. Diagnosis is usually made by microscopic examination; however, the method has several drawbacks such as requiring an experienced microscopist, being unreliable when parasitemia is low and being unable to accurately differentiate between co-infections from multiple parasite species. Therefore, the current extent of haemosporidian infections might be underestimated and neglected. We have developed a novel multiplex PCR assay to simultaneously detect and differentiate between four haemosporidian parasites: Leucocytozoon caulleryi, Leucocytozoon sabrazesi, Plasmodium juxtanucleare and Plasmodium gallinaceum. Primers in the present study specifically amplified the corresponding targets with no cross-species amplification detected. The multiplex PCR exhibited a significantly greater detection rate when compared with microscopic examination (p =  0.0001). The results demonstrate that the detection rate of multiplex PCR for L. sabrazesi, P. juxtanucleare, and P. gallinaceum are all greater than that of microscopic examination with p =  0.002, 0.0001 and 0.004, respectively. Co-infections were also detected more effectively by multiplex PCR. We applied the current method to field samples originating from Nan, Prachinburi, and Chachoengsao Provinces. The current study revealed that positive rates of haemosporidian parasites in chickens in the three study sites ranging from 39.5%-93.8%. The present assay offers a timesaving option for molecular diagnosis instead of using singleplex PCRs for detecting the parasites individually. Within a single reaction, this assay would be a useful tool for the detection of avian haemosporidian parasites either single or under co-infection conditions and for large-scale epidemiology studies.