Molecular Phylogenetic Variability of Fasciola gigantica in Iran.

Background
Fascioliasis is one of important zoonotic disease caused by Fasciola gigantica and F. hepatica. The final hosts of this parasite are ruminants and humans. Iran is one of the endemic areas in the world, about six million people at risk of infection. The aim of this study was to identify and determine the genetic diversity of Fasciola species in cattle after distinguish of their species.


Methods
One hundred and seventeen liver specimens collected from naturally infected cattle in 5 geographical regions in 2014-2017. Flukes stained with Hematoxylin-Carmine dye to examine for the existence of sperm within seminal vesicles. DNA was extracted from each individual, and ITS1, ND1and CO1 genes were amplified using specific primers. For discrimination of Fasciola species, ITS1 PCR-RFLP was used based on digestion pattern of RsaI enzyme. Genetic analyses and diversity and neutrality indices estimated by Dnasp5 based on NDI.


Results
Six nonspermic and 111 spermic flukes were diagnosed. All of nonspermic specimens were F. gigantica and collected from South East, South West and North West of Iran. Genetic haplotype diversity has been observed in F. gigantica based on ND1. Fst value analysis showed that minimum and maximum genetic difference between Iranian F. gigantica with Bangladesh (F st = 0.01414) and Egypt (F st = 0.36653) respectively.


Conclusion
It is the first report of existing of nonspermic Fasciola. High haplotype and nucleotide diversity could be due to ecological factors in life cycle, animal migration and coexisting of the final host of this parasite. Haplotype and nucleotide diversity of spermic F. gigantica in Iran and other countries in the world led to creating a variety of haplogroups.


Introduction
Fasciola gigantica and F. hepatica are among the most crucial zoonotic parasites that found in all 5 continents especially where cattle and sheep are reared. Animals or people usually become infected by eating raw watercress or other water plants contaminated with immature parasite larvae named metacercariae (1). F. gigantica is reported mainly from Asia and Africa and causes economic losses of US$3 billion annually due to its impact on livestock production, thereby affecting the food industry worldwide (2,3). In Iran, human fascioliasis has been accounted for from var-ious areas and two episodes of human fascioliasis in 1987 and 1997, influencing more than a few a huge number of individuals in Gilan Province, Bandar Anzali region (4,5). Animal fascioliasis is quite frequent and occurs generally in most areas of the state and their prevalence reaches around 50% in certain provinces (6). Moreover, climate change affected on number of Fascioliasis in recent years (7). Body size and shape are one the conventional, customary and essential strategy to recognize of Fasciola species as morphological criteria, however, these prerequisites are not, for the most part, confided in light of the variable scope of the species (8). Fasciola is meiotically functional diploid and can produce sperm and temporary store produced sperm in the seminal vesicles that named as nonpermic fluke. Male reproductive organ is the common predominant characteristic of both species that intermediate Fasciola flukes, which have morphological characteristic intermediate between F. hepatica and F. gigantica with no sperm (nonspermic fluke) in their seminal vesicles and maybe some F. gigantica, have been found in Asian countries as nonspermic (9,10). However, DNA sequences of nuclear ribosomal transcribed spacers (ITS) and RFLP methods and sequences analyses of CO1 and ND1 as mitochondrial genes appear the intraspecific phylogenetic relations of Fasciola spp. (11)(12)(13). Population genetic analyses are a way to find the origin, evolution in populations and helpful to mitigate against their spread (14). The husbandry and management of different farms have the potential to affect the population structure of parasites by influencing the movement of the definitive host and, therefore, F. gigantica (15). There are some reports from Iran ruminant fascioliasis particularly in cattle and buffaloes based on geography, climate variability and characterization of them according to molecular and phylogenetic methods (4,16,17). Likewise, there have been no rich and important overviews on molecular and spermatogenetic ability of Fasciola. This study aimed to distinguish of F. gigantica spermatogenesis ability and, and in addition investigate their phylogenetic and diversity network and association with haplotypes from different parts of the world using mitochondrial (ND1) marker.

Study Population
This cross-sectional study was performed on Fasciola spp. isolated from naturally hosts (cattle) in five regions from abattoirs in Iran from Jan 2015 to Dec 2017. Iran has different climates in different regions. The climate is influenced by Iran's location between the subtropical aridity of the Arabian Desert areas and the subtropical humidity of the eastern Mediterranean area. About 70% percent of the average rainfall in the country falls between November and March ( Fig. 1).

Sampling of Fasciola and morphological analysis for spermatogenesis status
One hundred and seventeen liver samples from cow that were naturally infected were collected from different slaughterhouses in Iran. This study was ethically approved by Shahid Beheshti University of Medical Sciences (SBMU). One fluke of them isolated and the washed in 0.9% saline solution and fixed in 70% ethanol between two glass slides, and then measurement morphological criteria such as body length and width were carried out. The whole body of Fasciola including seminal vesicle in the anterior part of the worm was stained with haematoxylin carmine solution and observed under an optical microscope to examine for the existence of sperm (18). Prior to staining, a small posterior part of the fluke was used for DNA extraction.

DNA extraction and amplification
Total DNA was extracted from individual Fasciola sample using High Pure PCR Template Preparation Kit (Dynabio®, Takapouzist, Iran), according to the manufacturer's instructions and stored at -20 °C until use. ITS1 region as a nuclear marker was amplified with primers named ITS1-F and ITS1-R and fragments of each mitochondrial target region (ND1 & and CO1) were amplified by polymerase chain . Total volume of the reaction was 40µl containing 4µl DNA template, 14 µl distilled water, 10 pmol of each primer, and 10 µl master mix (am-plicon®). Reaction cycles consisted of an initial denaturing step at 94 °C for 90 sec, followed by 35 cycles at 94 °C for 90 sec, 53 °C (ITS1) or 55 °C (ND1 & CO1) for 90 sec and 72 °C for 120 sec, with a final extension at 72 °C for 10 min using a gradient thermocycler. DNA fragments were analyzed by 1.5% agarose gel electrophoresis PCR-RFLP method ITS1 marker was used to identify different genus of Fasciola in this study. Briefly, the reaction level of 10 μL contained 5 μL of PCR products with approximately 680-bp fragments, 1 U of the RsaI restriction enzyme, and 1 μL of manufacturersupplied reaction buffer (Cinagen®, Iran). After incubation at 37 °C for 3 h and heat inactivation of RsaI at 65 °C for 15 min, the digested DNA samples were analyzed by gel electrophoresis (20).

Sequences and phylogenetic analysis and genetic diversity indices
Products of ITS1, ND1 & CO1 of isolates sequenced by Bioneer Company using the same primers, used in the PCR. The sequences were aligned and compared with those of existing sequences from the region, related to Fasciola spp. available data from Iran and other countries deposited in GenBank using the bioinformatics multiple alignments. All characters equally weighted and alignment gaps were treated as missing data. Mitochondrial sequences (ND1) haplotype networks designed by popART-1.7 software. Diversity indices (Haplotype diversity; Hd and Nucleotide diversity: π) and neutrality indices (Tajima's D and Fu's Fs tests) were estimated by DnaSP software package version 5.10 (21). The degree of gene flow (gene migration) among the populations was evaluated using a pairwise fixation index (Fst) (22).

Microscopic observation
Both spermic and nonspermic Fasciola were detected in Iran. Six nonspermic flukes from South East, South west and Northwest of Iran obtained. Remaining Fasciola was spermic and detected from all five regions. Length to width ratio in the nonspermic Fasciola was as a morphological criterion of F. gigantica.

Molecular findings
The amplicons of ITS1 (approximately 680 bp) that obtained from all of the spermic and nonspermic flukes cut using RsaI endonuclease digestion. RFLP pattern for nonspermic and spermic F. gigantica are 360,170 and 60. ND1 fragments (approximately 535 bp) and CO1 fragments (approximately 438 bp) were amplified for all specimens. We lost DNA of one nonspermic Fasciola because of staining haematoxylin before extracting DNA.

Network and genetic diversity
Haplotype diversity, nucleotide diversity and Neutrality indices of F. gigantica flukes in this study compared with other countries based on ND1 gene shown in Table 1. The nucleotide sequences for each haplotype were deposited in GenBank. Mitochondrial sequences (ND1) haplotype networks in spermic and nonsermic haplotypes of F. gigantica from 5 different geographical regions of Iran showed in Fig. 2

. Pairwise fixation index (Fst values) between different F. gigantica
populations in five geographical regions calculated by DnaSP software package with the nucleotide data set of ND1 gene (Table 2).

Discussion
Fascioliasis is one of the most imperative worry for both general wellbeing angles and veterinary issues. The separation of Fasciola species is essential on the grounds that their epidemiological patterns as far as both species of Fasciola existent in Iran in various hosts (23). The aim of this study was to identify and determine the genetic diversity of Fasciola species in cattle after distinguish of their species. All of one hundred and seventeen flukes recognized as F. gigantica by described morphometric items (8), but 6 of them observed as nonspermic by microscopy observation after staining, that obtained from South East of Iran, near the border of Pakistan and South West of Iran that traditionally nurtured.   Nonspermic Fasciola has been reported already from many Asian countries and there is genetic similarity of these flukes with spermic F. gigantica.
Although some hypothesis demonstrated that nonspermic flukes had probably retained or lost their spermatogenetic ability or capacity (9,18). Fasciola sp. taxa in Southwest of Asia in India and Bangladesh near of this region of Iran, however gigantica type of these flukes detected in nonspermic Fasciola sp. using ITS-RFLP, and also phylogenetic study in both ND1 and CO1 genes showed that they placed in F. gigantica complex (9,10). In this study, diversity indices of F. gigantica are high in both spermic and nonspermic flukes.

Conclusion
Nonspermic F. gigantica found in Iran. Different genetic structures through the other Fasciola population in the world according to genetic indices seen, but to complete and find genetic diversity and history, other molecular studies with large sample size from other regions of Iran with different climates is necessary.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.