Assessment of genetic markers for multilocus sequence typing (MLST) of Fasciola isolates from Iran.

BACKGROUND: Several markers have been described to characterise the population structure and genetic diversity of Fasciola species (Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica). However, sequence analysis of a single genomic locus cannot provide sufficient resolution for the genetic diversity of the Fasciola parasite whose genomes are ∼1.3 GB in size. OBJECTIVES: To gain a better understanding of the gene diversity of Fasciola isolates from western Iran and to identify the most informative markers as candidates for epidemiological studies, five housekeeping genes were evaluated using a multilocus sequence typing (MLST) approach. METHODS: MLST analysis was developed based on five genes (ND1, Pepck, Pold, Cyt b and HSP70) after genomic DNA extraction, amplification and sequencing. Nucleotide diversity and phylogeny analysis were conducted on both concatenated MLST loci and each individual locus. A median joining haplotype network was created to examine the haplotypes relationship among Fasciola isolates. RESULTS: Thirty-three Fasciola isolates (19 F. hepatica and 14 F. gigantica) were included in the study. A total of 2971 bp was analysed for each isolate and 31 sequence types (STs) were identified among the 33 isolates (19 for F. hepatica and 14 for F. gigantica isolates). The STs produced 44 and 42 polymorphic sites and 17 and 14 haplotypes for F. hepatica and F. gigantica, respectively. Haplotype diversity was 0.982 ± 0.026 and 1.000 ± 0.027 and nucleotide diversity was 0.00200 and 0.00353 ± 0.00088 for F. hepatica and F. gigantica, respectively. There was a high degree of genetic diversity with a Simpson's index of diversity of 0.98 and 1 for F. hepatica and F. gigantica, respectively. While HSP70 and Pold haplotypes from Fasciola species were separated by one to three mutational steps, the haplotype networks of ND1 and Cyt b were more complex and numerous mutational steps were found, likely due to recombination. CONCLUSIONS: Although HSP70 and Pold genes from F. gigantica were invariant over the entire region of sequence coverage, MLST was useful for investigating the phylogenetic relationship of Fasciola species. The present study also provided insight into markers more suitable for phylogenetic studies and the genetic structure of Fasciola parasites.

Identification of Fasciola Species Using Tegumental Spines in Tissue Sections.

BACKGROUND: Efforts to find a reliable non-molecular means of identification has been the main purpose of the current work that always is persuaded by researchers interested in the field of parasitology. METHODS: Adult fasciolids were obtained from the slaughterhouses in different parts of Iran in 2017, and investigated using the classical old fashion morphological appearances of the worms implementing a camera lucida equipped microscope. Histological procedure was subsequently performed for almost the entire collected adult worms followed by Hematoxylin and Eosin (H&E) staining technique. DNA extraction and RFLP-PCR technique were carried out for the entire fasciolid liver flukes. To attain more comparable morphological conclusions, Scanning Electron Micrographs were also implemented for two molecularly identified fasciolids. RESULTS: Based on spine morphology observed in worm's tissue sections two types of tegumental spines, "pointed" and "molar" shapes have been identified addressing to distinguish F. hepatica and F. gigantica species respectively. The present identification has been also supported by Molecular analysis using RFLP-PCR technique. CONCLUSION: There are some hidden morphological characters implemented in species identification for certain helminths. Meanwhile, the emergence of computer image analysis system (CIAS) on the scene of taxonomy, has revolutionized the accuracy of measurement in morphology by employing detailed parameters that have not been regarded before. The current study has illustrated the tegumental spines of two Fasciola species in tissue sections which has not been enough considered in helminthological publications so far.

Bioclimatic Analysis and Spatial Distribution of Livestock Fascioliasis in Iran.

BACKGROUND: This study aimed to investigate the prevalence of fascioliasis and to perform a climatological analysis of different regions of Iran based on the current situation of the parasite and its intermediate host using Geographical Information System (GIS). METHODS: Meteorological data were obtained from Iran Meteorological Organization. Risk map of fascioliasis transmission was prepared based on this data and using forecasting indices. Further, the number of fascioliasis cases from 31 provinces reported to the Iran Veterinary Organization were collected and prevalence maps of livestock fascioliasis were drawn. RESULTS: The main risk hotspots were found in Northern provinces like Golestan, Mazandaran and Gilan as well as some Southern provinces such as Kohgiluyeh and Boyer-Ahmad and Chaharmahal and Bakhtiari and Fars, which have ideal conditions for completion of the parasite life cycle. Moreover, Gilan Province with 10.83% had the highest rate of fascioliasis infection in slaughtered animal. CONCLUSION: Iran is one of the most important foci of fascioliasis globally. Several provinces of Iran have appropriate conditions for evolution of parasite life cycle and presence of its intermediate host. These regions require special attention and serious determination in order to control fascioliasis in human and animals.

Epidemiology of Fascioliasis in Kermanshah Province, Western Iran.

BACKGROUND: We aimed to determine the prevalence of fasciolosis in the definitive hosts (human and livestock) and intermediate (Lymnaea snails) hosts in Kermanshah Province, western Iran from 2014-2016. METHODS: The study on animals was descriptive and retrospective one. All daily records of animals slaughtered in the abattoirs were analyzed. For the study of human fascioliasis, 975 serum samples were collected from different parts of Kermanshah Province and analyzed using ELISA based on excretory-secretory antigens. Moreover, 4400 Lymnaea snails were collected from 25 habitats. The snails were identified and examined for presence of cercariae by shedding method. RESULTS: Fasciolosis was diagnosed in 1.7% of slaughtered animals, which was significantly greater than the other species (P<0.005). There was significant difference (P<0.001) between the prevalence of fasciolosis and seasonal pattern. As for human cases, five cases (0.5%) were positive for fascioliasis. Regarding the seropositivity to fasciolosis, no significant differences were found for age groups, sex, level of education and occupation. No Fasciola infection was seen in snails of the family Lymnaeidae. CONCLUSION: The prevalence of Fasciola parasite was lower compared to other provinces. This is probably due to sequential decline in rainfall and hot climate that makes conditions difficult for the snail intermediate host snails and the larval stages of fasciolid trematodes. The habitual food of people is another important point.

Construction and Identification of a Recombinant Plasmid Encoding Echinococcus granulosus Oncosphere Antigen (EG95).

BACKGROUND: Cystic echinococcosis (CE), as a zoonotic disease cause to health threat and economic losses. Despite implemented control programs, few countries have been able to decrease or eliminate this infection. Vaccination of the intermediate host offers an additional strategy to control the parasite transmission and EG95 antigen is considered more than the others in the vaccine issue. According to the high protection induced by the EG95 recombinant vaccine, this study was designed to construct recombinant plasmid formulation of EG95 antigen. METHODS: In 2015, the Echinococcus granulosus eggs were recovered from an infected dog in Parasitological laboratory of Tarbiat Modares University in Tehran, Iran. Following hatching, the oncospheres of E. granulosus were activated to increase the presence of the desired mRNA. The extracted mRNA was transcribed to the cDNA which used as template in RTPCR. Then the EG95 gene cloned into pET28a vector and the recombinant plasmids expression was investigated in prokaryotic and eukaryotic cells. RESULTS: The recombinant plasmid encoding EG95 antigen was successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. In vitro expression of the EG95 antigen was confirmed in prokaryotic and eukaryotic systems by SDS-PAGE and western blotting analysis. CONCLUSION: Because of potential advantages of DNA vaccines, including ability to induce long-term immune responses, low production cost and stability in different temperatures, this study carried out to construct the EG95 gene into a vector. This recombinant vector can be evaluated in further studies as a DNA vaccine may provide new prospects for the development of a vaccine against cystic hydatid disease.

Molecular Characterization of Animal Fasciola spp. Isolates from Kermanshah, Western Iran.

BACKGROUND: We evaluated the genetic diversity of samples identified morphologically as Fasciola spp. from sheep, cattle and goat from Kermanshah Province, western Iran using PCR-RFLP method. METHODS: We used PCR-RFLP analysis of ribosomal ITS1 fragment using RsaI restriction enzyme to investigate the genetic characteristics of Fasciola species obtained from different hosts (16 sheep, 28 cattle, 4 goats). The species of Fasciola were confirmed by sequencing the 700 bp region of ribosomal ITS1 gene. RESULTS: In Kermanshah, F. hepatica was present in 96% of the samples, F. gigantica was found only in two cattle sample. No hybrid forms were detected in the present study. CONCLUSION: Our results contribute to clarify the dark spots of Fasciola genotyping in different parts of Iran.