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Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a globally distributed zoonotic disease of livestock. While F. hepatica and F. gigantica have temperate and tropical distributions, respectively, parasite sympatry occurs in parts of Asia and Africa. A growing protein demand has the potential to facilitate the translocation of parasites from endemic to non-endemic areas, via associated international livestock movements. Such is the case in Southeast Asia, where livestock trade from F. hepatica-endemic countries into China and Vietnam may account for detection of F. hepatica hybrid/introgressed forms. Of particular importance is Lao People's Democratic Republic, which acts as a major livestock thoroughfare for the region. Our ability to understand the impacts of livestock-associated Fasciola spp. movements on local animal and human health is hindered by a lack of ante-mortem diagnostic tools allowing species differentiation. Molecular tools have been developed for Fasciola spp. differentiation, however those rely on access to pure DNA from adult specimens, limiting their application to post-mortem use. Our aim was to detect and differentiate F. hepatica from the endemic F. gigantica in local smallholder cattle in a region of Southeast Asia with frequent livestock trafficking. To do this we designed and validated ante-mortem molecular assays for Fasciola spp. differentiation targeting single-nucleotide polymorphisms (SNPs) within ITS1 and lsrRNA. We then deployed these SNP genotyping assays to diagnose Fasciola spp. infection in 153 local cattle from 27 villages in Northern Laos. We demonstrate the presence of F. hepatica DNA, confirmed by qualitative Sanger and quantitative Illumina amplicon sequencing of ITS1 and lsrRNA, and highlight the shortfalls of Sanger sequencing for Fasciola spp. identification due to the preferential amplification of F. gigantica nucleotides in mixed DNA samples. The outlined protocol enables rapid surveillance of faecal samples for the presence of Fasciola species eggs, their co-infection and/or infection with F. hepatica/F. gigantica hybrids., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nuclear gene markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), have been developed for precise discrimination of Fasciola flukes instead of internal transcribed spacer 1. In this study, these two genes of 730 Fasciola flukes from eight Asian countries were analyzed. The results were compared with their mitochondrial NADH dehydrogenase subunit 1 (nad1) lineages for obtaining a definitive evidence of the hybrid origin of aspermic Fasciola flukes. All the flukes categorized into the aspermic nad1 lineages possessed both the fragment patterns of F. hepatica and F. gigantica (mixed types) in pepck and/or pold. These findings provide clear evidence for the hybrid origin of aspermic Fasciola lineages and suggest that "aspermic Fasciola flukes" should hereafter be called "hybrid Fasciola flukes".

Molecular characterization is important for discriminating Fasciola specimens having the deoxyribonucleic acid (DNA) sequences of Fasciola hepatica, Fasciola gigantica, and both Fasciola species, since three Fasciola forms coexist in Asian countries. We have developed a restriction fragment length polymorphism of amplified DNA (PCR-RFLP) of the nuclear ribosomal internal transcribed spacer 1 (ITS1) region in Fasciola species. The band patterns of the fragments digested with a restriction enzyme, Rsa I, were accurately distinguished among the three forms of Fasciola. Amplicons with the sequences of F. hepatica and F. gigantica were divided into fragments of about 360, 100, and 60 bp, and 360, 170, and 60 bp, respectively, and amplicons with the sequences of both Fasciola species yielded fragments of 360, 170, 100, and 60 bp. The results of PCR-RFLP completely coincided with those of sequence analysis, and thus PCR-RFLP is a useful technique for determining the ITS1 type in Fasciola species.

Background: To address the increasing number of injuries in developing countries, the World Health Organization (WHO) encourages the establishment of hospital-based surveillance systems and systematic data collection. Although a computerized system is preferable in terms of efficiency, many developing countries have difficulty accessing the appropriate resources., Objectives: To assess the possibility of comparing and sharing data among countries, and then to discuss the possibility of establishing an international data management system through the internet., Methods: A point-by-point comparison of data directories from injury surveillance systems in Thailand, Cambodia, Sri Lanka, and Japan was conducted using guidelines published by WHO as the standard., Results: Thailand, Cambodia, and Sri Lanka used data items that are comparable to each other and to WHO guidelines, with few, readily amenable, differences. The Japanese system used quite different data items., Conclusions: Data comparability suggests the feasibility of a global data management system that can store data from various countries. Such a system, if made accessible over the internet, would benefit resource-constrained countries by providing them with a ready-made framework to implement a surveillance system at low cost.