Morphological and Molecular Characterization of Paragonimus Species Isolated from Freshwater Crabs in Southern Yunnan, China

Paragonimus species are highly prevalent in various regions of China. The study’s objective is to isolate and identify Paragonimus from natural habitats and compare the phylogenetic diversity of Paragonimus in southern Yunnan province, China. 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 114 days, adult worms and eggs were isolated from multiple organs. Morphologic identification confirmed the initial identification. DNA was extracted from 5 adult worms, and molecular characterization was performed by amplification and sequencing of CO1 and ITS2 regions, followed by phylogenetic analysis. Out of 447 crabs captured, 186 crabs were found to be infected. A total of 4 species of Paragonimus was observed from naturally infected crabs. Paragonimus microrchis (2), Paragonimus heterotremus (1), Paragonimus proliferus (1), and Paragonimus skrjabini (1) were isolated and identified. A total of 32 sequences were downloaded from the National Center for Biotechnology Information, and 5 sequences generated in the study were used for phylogenetic analysis. In the phylogenetic tree of the CO1 gene, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini were clustered with the same species, and the confidence values of their branches were >95%. A congruent phylogenetic relationship was observed with the ITS2 phylogenetic tree. In the phylogenetic tree constructed with the combined dataset of CO1 and ITS2 datasets, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini clustered with the same species, and their branch confidence values were >94%. Paragonimus microrchis clustered with Paragonimus bangkokensis in both datasets. Phylogenetic analysis revealed robustness of the double loci method as against the single-locus method with either CO1 or ITS2 alone. Paragonimus species isolated from the southern Yunnan province, China, was phylogenetically diverse, and the analysis revealed the clustering of multiple species of Paragonimus isolated from different geographic locations.

Morphological and Molecular Characterization of Paragonimus skrjabini Complex from Yunnan, China: A Brief Report

Purpose To perform environmental sampling and molecular identification of Paragonimus in endemic regions, which may help in minimizing transmission among humans. Methods Mountain crabs from the genus Potamiscus were collected and the encysted metacercariae were extracted and subjected to morphological identification, followed by animal inoculation in Sprague–Dawley (SD) rats. After 112 days of infection, animals were killed and adult worms were extracted from lungs and muscles. The morphology of adult worms was characterized by microscopy and molecular identification was done by polymerase chain reaction, followed by sequencing of cox1 and ITS2 genes. Phylogenetic analysis was done by maximum parsimony method. Results A total of 447 crabs were captured from the streams of Tongchang Town, Jinping County, Yunnan Province, China. The infection rate was found to be 41% (186 out of 447 crabs). The metacercariae of Paragonimus skrjabini was identified by the characteristics round or spherical encysted form measuring 410 to 460 × 400 to 460 µm. After animal infection in SD rats, adults were presumptively confirmed to be P. skrjabini, which was also confirmed by gene amplification and sequence analysis of cox1 and ITS2 regions. Paragonimus skrjabini clustered with previously reported P. skrjabini from Yunnan and Vietnam. The confidence values of their branches were > 95%. Phylogenetic analysis of the ITS2 region revealed two distinct clusters with distinct geographical grouping. Phylogenetic analysis with the combined data sets reiterated the geographical grouping with P. skrjabini from Yunnan clustering with strains from Vietnam. Conclusion Metacercariae of P. skrjabini was discovered in freshwater crabs in Yunnan province, China, and the strains were phylogenetically related to P. skrjabini from Vietnam.

Discovery of Paragonimus skrjabini in Vietnam and its phylogenetic status in the Paragonimus skrjabini complex

Two members of the Paragonimus skrjabini complex, P. skrjabini and P. miyazakii, are now considered as two sub-species, P. skrjabini skrjabini and P. skrjabini miyazakii. They are well known as important pathogens for human paragonimiasis in China and Japan. Recently, members of this species complex have been reported from India. Here we report the first discovery of P. skrjabini from freshwater crab hosts in Thanh Hoa province, Vietnam. For morphological and molecular phylogenetic studies, adult worms were obtained by experimental infection in cats and dogs. Molecular analyses of metacercariae and adults revealed that the P. skrjabini population from Thanh Hoa, Vietnam was almost completely identical with that from Yunnan province, China. Those populations from Thanh Hoa, Vietnam and Yunnan, China and those from Manipur, India were significantly different from P. skrjabini populations reported from other localities of China in cytochrome oxidase subunit 1 (CO1) gene sequences, indicating considerable genetic variation within the P. skrjabini complex. Moreover, low bootstrap values in the CO1 tree suggested that more variant genotypes belonging to P. skrjabini complex may be found in other Asian countries in between Vietnam and India, such as Myanmar, Laos and Thailand. Since P. skrjabini is known as a pathogen for humans, paragonimiasis cases caused by P. skrjabini might be found in Vietnam and other Asian countries.