CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

Adults of Opisthorchis viverrini reside in the biliary system, inducing inflammation of bile ducts and cholangitis, leading to hepatobiliary disease (HBD) including cholangiocarcinoma. O. viverrini infection also has major implications for the bacterial community in bile ducts and liver. To investigate this in chronic O. viverrini infection (≥ 8 months p.i.), bacterial genomic DNA from livers of hamsters and from worms was investigated using culture techniques, PCR for Helicobacter spp. and high-throughput next-generation sequencing targeting the V3-V4 hypervariable regions of prokaryotic 16S rRNA gene. Of a total of 855,046 DNA sequence reads, 417,953 were useable after filtering. Metagenomic analyses assigned these to 93 operational taxonomic units (OTUs) consisting of 80 OTUs of bacteria, including 6 phyla and 42 genera. In the chronic O. viverrini-infected group, bacterial community composition and diversity were significantly increased compared to controls. Sequences of Fusobacterium spp. were the most common (13.81%), followed by Streptococcus luteciae (10.76%), Escherichia coli (10.18%), and Bifidobacterium spp. (0.58%). In addition, Helicobacter pylori (0.17% of sequences) was also identified in the liver of chronic O. viverrini infections, but not in normal liver. The presence of H. pylori was confirmed by PCR and by use of an antibody against bacterial antigen, supporting the metagenomics data. The identities of bacteria cultured for enrichment suggested that chronic O. viverrini infection changes the liver microbiome and promotes Helicobacter spp. growth. There may be synergy between O. viverrini and the liver microbiome in enhancing immune response-mediated hepatobiliary diseases.

To increase public health awareness for prevention of opisthorchiasis caused by eating raw freshwater fish, the distribution and abundance of Opisthorchis viverrini metacercariae (OV MC) was investigated in freshwater fish obtained from 20 provinces in northeastern Thailand between April 2011 and February 2012. A cross-sectional survey was conducted on 12,890 fish consisting of 13 species randomly caught from 26 rivers, 10 dams, and 38 ponds/lakes. Fish, were collected in each of the rainy and winter seasons from each province. Fish were identified, counted, weighed, and digested using pepsin-HCl. Samples were examined for OV MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. OV MC were found in 6 species of fish; i.e., Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti, and Puntioplites proctozysron from localities in 13 provinces. Among the sites where OV MC-infected fish were found, 70.0% were dams, 23.7% were ponds/lakes, and 7.7% were rivers. The mean intensity of OV MC ranged from 0.01 to 6.5 cysts per fish (or 1.3-287.5 cysts per kg of fish). A high mean intensity of OV MC per fish (>3 cysts) was found in 5 provinces: Amnat Charoen (6.5 cysts), Nakhon Phanom (4.3), Mukdahan (4.1), Khon Kaen, (3.5) and Si Sa Ket (3.4). In conclusion, OV MC are prevalent in natural cyprinid fish, with the infection rate varying according to fish species and habitats.

Persistent infection with Opisthorchis viverrini causes hepatobiliary abnormalities, predisposing infected individuals to cholangiocarcinoma (CCA). In addition, Helicobacter pylori is highly prevalent in most countries and is a possible risk factor for CCA; however, its role in enhancing hepatobiliary abnormality is unclear. Here, we investigated the effects of coinfection with H. pylori and O. viverrini on hepatobiliary abnormality. Hamsters were divided into four groups: (i) normal, (ii) H. pylori infected (HP), (iii) O. viverrini infected (OV), and (iv) O. viverrini and H. pylori infected (OVϩHP). At 6 months postinfection, PCR and immunohistochemistry were used to test for the presence of H. pylori in the stomach, gallbladder, and liver. In the liver, H. pylori was detected in the following order: OVϩHP, 5 of 8 (62.5%); HP, 2 of 5 (40%); OV, 2 of 8 (25%). H. pylori was not detected in normal (control) liver tissues. Coinfection induced the most severe hepatobiliary abnormalities, including periductal fibrosis, cholangitis, and bile duct hyperplasia, leading to a significantly decreased survival rate of experimental animals. The greatest thickness of periductal fibrosis was associated with a significant increase in fibrogenesis markers (expression of alpha smooth muscle actin and transforming growth factor beta). Quantitative reverse transcription-PCR revealed that the highest expression levels of genes for proinflammatory cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) were also observed in the OVϩHP group. These results suggest that coinfection with H. pylori and O. viverrini increased the severity of hepatobiliary abnormalities to a greater extent than either single infection did.