Fasciola hepatica and Fasciola hybrid form co-existence in yak from Tibet of China: application of rDNA internal transcribed spacer.

Fasciolosis is a parasitic disease affecting humans and livestock, caused by digenean trematodes of the genus Fasciola, primarily F. hepatica and F. gigantica. This study investigates the coexistence of these species and their hybrids in yaks from Tibet, China. We analyzed the nuclear rDNA internal transcribed spacer (ITS) regions, including ITS1 and ITS2, through Sanger sequencing and Next-Generation Sequencing (NGS) to assess single-nucleotide polymorphisms (SNPs). Our results reveal that one specimen (NM008B) is identical to pure F. hepatica, while another (NM008A) contains genetic information from both F. hepatica and F. gigantica, indicating potential hybridization or introgression. The morphological analysis reveals that the collected adult F. hepatica specimens exhibit distinct characteristics, while the hybrid specimens display "intermediate" features of F. hepatica and F. gigantica. This study is the first to document the coexistence of F. hepatica and hybrid Fasciola forms in a single yak. The findings underscore the complexities of hybridization dynamics and the necessity for advanced molecular techniques in accurately identifying Fasciola species. Future research should focus on mitochondrial DNA and other nuclear gene analysis to further elucidate the nature of these hybrids and their ecological implications.

Optimization of CRISPR/Cas12a detection assay and its application in the detection of Echinococcus granulosus.

Cystic echinococcosis, resulting from infection with Echinococcus granulosus, poses a significant challenge as a neglected tropical disease owing to the lack of any known effective treatment. Primarily affecting under-resourced, remote, and conflict-ridden regions, the disease is compounded by the limitations of current detection techniques, such as microscopy, physical imaging, ELISA, and qPCR, which are unsuitable for application in these areas. The emergence of CRISPR/Cas12a as a promising tool for nucleic acid detection, characterized by its unparalleled specificity, heightened sensitivity, and rapid detection time, offers a potential solution. In this study, we present a one-pot CRISPR/Cas12a detection method for E. granulosus (genotype G1, sheep strain) integrating recombinase polymerase amplification (RPA) with suboptimal protospacer adjacent motif (PAM) and structured CRISPR RNA (crRNA) to enhance reaction efficiency. The evaluation of the assay's performance using hydatid cyst spiked dog feces and the examination of 62 dog fecal samples collected from various regions of Western China demonstrate its efficacy. The assay permits visual observation of test results about 15 minutes under blue light and displays superior portability and reaction speed relative to qPCR, achieving a sensitivity level of 10 copies of standard plasmids of the target gene. Analytic specificity was verified against four tapeworm species (E. multilocularis, H. taeniaeformis, M. benedeni, and D. caninum) and two other helminths (T. canis and F. hepatica), with negative results also noted for Mesocestoides sp. This study presents a rapid, sensitive, and time-efficient DNA detection method for E. granulosus of hydatid cyst spiked and clinical dog feces, potential serving as an alternative tool for field detection. This novel assay is primarily used to diagnose the definitive host of E. granulosus. Further validation using a larger set of clinical fecal samples is warranted, along with additional exploration of more effective approaches for nucleic acid release.

Characterization of the Complete Mitochondrial Genome and Phylogenetic Analyses of Eurytrema coelomaticum (Trematoda: Dicrocoeliidae).

Eurytrema coelomaticum, a pancreatic fluke, is recognized as a causative agent of substantial economic losses in ruminants. This infection, commonly referred to as eurytrematosis, is a significant concern due to its detrimental impact on livestock production. However, there is a paucity of knowledge regarding the mitochondrial genome of E. coelomaticum. In this study, we performed the initial sequencing of the complete mitochondrial genome of E. coelomaticum. Our findings unveiled that the mitochondrial genome of E. coelomaticum spans a length of 15,831 bp and consists of 12 protein-coding genes, 22 tRNA genes, two rRNA genes, and two noncoding regions. The A+T content constituted 62.49% of the genome. Moreover, all 12 protein-coding genes of E. coelomaticum exhibit the same arrangement as those of E. pancreaticum and other published species belonging to the family Dicrocoeliidae. The presence of a short string of additional amino acids (approximately 20~23 aa) at the N-terminal of the cox1 protein in both E. coelomaticum and E. pancreaticum mitochondrial genomes has contributed to the elongation of the cox1 gene in genus Eurytrema, surpassing that of all previously sequenced Dicrocoeliidae. The phylogenetic analysis displayed a close relationship between E. coelomaticum and E. pancreaticum, along with a genus-level association between Eurytrema and Lyperosomum. These findings underscore the importance of mitochondrial genomic data for comparative studies of Dicrocoeliidae and even Digenea, offering valuable DNA markers for future investigations in the systematic, epidemiological, and population genetic studies of this parasite and other digenean trematodes.