Phylogenetic relationship of Prohemistomum vivax to other trematodes based on the internal transcribed spacer region and mitochondrial genes.

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax. The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3-0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus. This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax, including cytochrome c oxidase subunit III (cox3) partial sequence; tRNA-His, cytochrome b (cytb), and NADH dehydrogenase subunit 4L (nad4L) complete sequences; and NADH dehydrogenase subunit 4 (nad4), cytochrome c oxidase I (cox1), and NADH dehydrogenase subunit 5 (nad5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox1, cox3, and cytb genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.

Immune pathogenesis in pigeons during experimental Prohemistomum vivax infection.

Prohemistomum vivax is a small trematode belonging to the family Cyathocotylidae, infecting fish-eating birds and mammals, including humans. However, no data on molecular identification and immune pathogenesis are available, challenging effective diagnostic and therapeutic interventions. Here, we identified P. vivax based on combined morphological and molecular data and examined histopathological lesions and the differential cytokines expression in experimentally infected pigeons. Pigeons were orally infected with 500 prohemistomid metacercariae. Intestinal and spleen tissues were harvested 2, 4, 7, 14, 21, and 28 days post-infection (dpi). Gene expression levels of eleven cytokines (IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-18, IFN-γ, and TGF-ß3) were assessed using quantitative reverse-transcription PCR (RT-qPCR). We identified the recovered flukes as Prohemistomum vivax based on morphological features and the sequence and phylogenetic analysis of the internal transcribed spacer 1 (ITS1), 5.8 ribosomal RNA, and ITS2 region. Histopathological lesions were induced as early as 2 dpi, with the intensity of villi atrophy and inflammatory cell infiltration increasing as the infection progressed. An early immunosuppressive state (2 and 4 dpi), with TGF-ß3 overexpression, developed to allow parasite colonization. A mixed Th1/Th2 immune response (overexpressed IFN-γ, IL-12, IL-2, IL-4, and IL-5) was activated as the infection progressed from 7 to 28 dpi. Inflammatory cytokines (IL-1, IL-6, IL-18, and IL-15) were generally overexpressed at 7-28 dpi, peaking at 7 or 14 dpi. The upregulated Treg IL-10 expression peaking between 21 and 28 dpi might promote the Th1/Th2 balance and immune homeostasis to protect the host from excessive tissue pathology and inflammation. The intestine and spleen expressed a significantly different relative quantity of cytokines throughout the infection. To conclude, our results presented distinct cytokine alteration throughout P. vivax infection in pigeons, which may aid in understanding the immune pathogenesis and host defense mechanism against this infection.