Morphological and molecular characterization of Paragonimus species isolated from freshwater crabs in Yunnan, China.

Abstract

Background Paragonimus species are highly prevalent in ethnic minority areas of Yunnan, China. The objective of the study is to isolate and identify Paragonimus from natural habitat and compare the phylogenetic diversity of Paragonimus in Yunnan province, China. Methods Metacercariae of Paragonimus was isolated from crabs and morphologic identification was performed by microscopy. Metacercariae were injected into experimental Paragonimus free Sprague Dawley rats. After 112 days, adult worms and eggs were isolated from multiple organs. Morphologic identification confirmed the initial identification. DNA was extracted from the adult worms and molecular characterization was performed by amplification and sequencing of CO1 and ITS2 regions followed by phylogenetic analysis. Results Out of 447 crabs captured, 186 crabs were found to be infected. A total of 5 occurrences of Paragonimus was observed from naturally infected crabs. Paragonimus microrchis (2), P,heterotremus (1), P.proliferus (1) and P.skrjabini (1) were isolated and identified. A total of 32 sequences downloaded from national center for biotechnology information and 5 sequences generated in the study was used for phylogenetic analysis. Phylogenetic analysis revealed robustness of the double loci method as against the single locus method with either CO1 or ITS2 alone. Considerable phylogenetic diversity depending on the geographic location was observed. Conclusion Paragonimus species isolated from Yunnan province, China were phylogenetically diverse and the analysis revealed the clustering of multiple species of Paragonimus isolated from different geographic locations.