Abstract
Fascioliasis is a globally distributed and zoonotically important parasitic disease caused by liver fluke species of the genus Fasciola. It has always been well recognized because of its high veterinary impact but remains neglected for decades with regard to human infection. Worldwide devastating effect of it is estimated to be US$ 3.2 billion per annum, 2.4 million people is infected and 180 million are at risk of infection. Consequently, WHO recognized it as emerging human disease and included the disease in the agenda of food borne trematode zoonoses priority. Fasciola hepatica (Fh) and Fasciola gigantica (Fg) are sister species of Fasciola, while Fg occurs mainly in tropical and Fh in temperate areas, both species overlap in subtropical zones. Most of the Asian countries are abundant with intermediate form of Fasciola that are inflicting profound problems in their identification, notably in India and Myanmar. So far, no comprehensive investigation of the disease took place in Bangladesh yet. The species differentiation can only be made by either a morphometric study or by molecular tools. Taking these into account, present research was undertaken to characterize Fasciola based on morphometric and molecular approaches; and to screen reported molecular marker for anthelmintic sensitivity of Fasciola present in Bangladesh. In order to accomplish these, adult flukes (n=2295) were collected from naturally infected 10 buffaloes (n=472), 27 cattle (n=774), 34 goats (n=920) and 11 sheep (n=129) at slaughter during April through November, 2012 and 2013. For phenotypic characterization of flukes, whole mount staining (n=222); observation of spermatogenic status (n=28), ovarian position (n=30), body shape and cone arch (n=9), and branching pattern of caeca and testes of Fasciola was done. CIAS using standard biometric measurements and development of factor map by multivariate data and principal component analysis using SPSS, Imagepro and CLIC package software were done for morphometric measurement. Concerning genotypic characterization, Genomic DNA was extracted from Fasciola (n=28) using WizardTM genomic DNA purification kit and PCR was done using GoTaq® Flexi DNA reagent. Complete ITS1 and ITS2, ITSs and partial 28S rDNA were amplified by PCR and documented through UV Trans illuminator; and cleaned products were subjected to direct sequencing. Raw sequences (abi text) were edited chromatographically using Finch TV and aligned with published sequences using MEGA6 software. Phenotypically, study of spermatogenesis of Fasciola (n=28) represented both spermic (n=25) (Fg) and aspermic (n=3) (Fsp.) flukes. Furthermore, mostly right sided ovarian position; elongated shape and indistinct cone arch; much complex branching of caeca and testes indicated presence of Fg in Bangladesh. However, morphometrically, present study demonstrated existence of Fg and intermediate Fasciola (Fsp.) in Bangladesh which are globally unique dwarf in size. Genotypically, in case of ITS1 and ITS2, heterozygous nucleotides at five variable sites from alignment comparison segregated liver flukes of Bangladesh into Fg and hybrid intermediate form of Fasciola (Fsp.). Each of both the gene represented two haplotypes. In case of ITSs and 28S, alignment comparison revealed single haplotype in each gene fragments. Phylogenetic study revealed haplotype similarity with Asian fasciolids and sequence homogeneity demonstrated recent introduction in Bangladesh. In terms of screening for reported marker of anthelmintic sensitivity, absence of heterozygous nucleotide at position 23 in alignment comparison of 28S rDNA indicated that anthelmintic sensitive fasciolids exists in Bangladesh. Phylogeny of ITS1, ITS2, ITSS and 28S sequences of this research also revealed that Fg of Bangladesh belongs to Asian clades. In conclusion, present research demonstrated the existence of globally dwarf sized, recently introduced Fg and Fsp.; and anthelmintic sensitive Fg in Bangladesh. Further studies are suggested to analyze complete mtDNA including more comprehensive investigation of Fsp.; epidemiology and molecular study of lymnaeids; development of Fasciola risk model; and investigation of human infection.