MORPHOLOGCAL AND MOLECULAR STUDY OF HARD TICKS SPECIES THAT INFESTED SMALL RUMINANTS IN DUHOK GOVERNORATE, KURDISTAN REGION, IRAQ

Ticks are harmful ectoparasite that feed on human and animal blood and causing many diseases through the world. They infested many hosts including: mammals, reptiles and birds. Ticks are important vector and they have the ability to transmit a variety of pathogenic agent to humans and animals. Ticks are divided into two major groups which are hard tick (Ixodidae) and soft tick (Argasidae). Because there was no such study done on identification of tick species by PCR technique in Kurdistan and particularly in Duhok Governorate, therefore present study was done to identify tick species by using molecular study by using of 16S rRNA and DNA sequencing. About 1000 ticks were collected from both sheep and goat respectively (500 and 500), form Duhok Governorate including: Barwaria, Zakho, Sumeil, Mangeshik, Sersin, Shekhan and Akre, Iraqi Kurdistan, between May and June 2016, between April and June 2017. The results of present study three genera of tick were detected in small ruminants by microscopic identification including: Rhipicephalus spp., Hyalomma spp. and Boophilus spp. Distribution of tick among sheep and goat according to the gender, the rate of infection in female was higher than in male in both species Ewe and Doe was 32.6% and 31.11% respectively as compared to male in both species (Ram and Buck) was 21.15% and 15.11% respectively. The distribution of gender of tick in was higher in male ticks than female tick with ratio 2:1. Distribution of identified ticks in present study including ( Rhipicephalus , Hyalomma and Boophilus ) respectively, in Barwaria were (82.6%, 13.3%, and 4.1) respectively, in Zaxo were (48.3%, 42.5% and 10.3%), in Sumel were (47%, 42.7% and 10.3%), in Mangeshik were (73,2%, 26.8%, and 0%), in Sersink were (61.5%, 38.5% and 0%), in Shekhan were (78.8%, 11.8% and 9.4%) and in Akre were (60%, 34% and 6%). On molecular study, 60 samples from 150 were positive with size 460 bp after 16S rRNA amplification and have got clear bands on agarose gel 1% and electrophoresis and 20 PCR positive products were sent to Humanizing Genomics, Macrogen Company (Korea) using primer 16S_rRNA gene for sequencing both forward and Reverse. Six species of tick under two genera were founded including: Rhipicephalus and Hyalomma were identified which including: Hyalomma anatolicum, H. marginatum, R. annulatus, R. sanguineus and R. turanicus. H. asiaticum asiaticum for the first time was recorded in Kurdistan, and especially in Duhok city. Moreover, all sequences were submitted to NCBI using BankIt software and we obtained accession number. Phylogenetic tree was constructed based on 16S rRNA for both samples: 16S rRNA (MN594483) and (MN594490) were identical 100% to reference sequences respectively: (KU664367.1 and HM176656.1) and other sequences were identical 99% to the references sequence. In conclusion the present study is the first study for identification of tick species among sheep and goats in Duhok Governorate, Iraqi Kurdistan by sequencing analysis.


INTRODUCTION
Ticks are harmful ectoparasite that feed on human and animal blood and causing many diseases through the world. They infested many hosts including: mammals, reptiles and birds (1,2,3). Ticks are important vector and they have the ability to transmit a variety of pathogenic agent to humans and animals (4). Ticks are divided into two major groups which are hard tick (Ixodidae ) and soft tick (Argasidae) (5). Nowadays, There are about 877-878 tick species; most of these are under the two famous families including: Ixodidae and Argasidae (6). Hard ticks are distributed around the world with their hosts ranging from wild to domestic vertebrate, except for fish. Classifications and phylogenetic inferences for Ixodidae have traditionally been depend on the morphological, biological and ecological features, often suggesting host specificity as the main factor (7,8). Today, Molecular tools and DNA marker are widely used for the mix, 1 µl from both forward and reverse primers, 2µl of Template DNA, 10 pmol/μl of each of forward and reverse and complete the volume to 25μl with added of 9.5µl nuclease-free water.
According to Mangold et al., (1997), the cycler state of PCR was defined as outlined in table 1.
Eventually, for 1:40 min, 10μl of PCR products were visualized under UV on 1% agarose gel with 85 volt

Phylogenetic Tree and Analysis
During this analysis, MEGA 7 technology was used to construct phylogenetic relationships and Neighbor-Joining tree depends on the alignment of 16S rRNA sequences to evaluate the phylogenetic relationship species status of two types of ticks in this sample. Phylogenetic tree as in figure (10) studies that support all these species in Kurdistan, Iraqi region by (14,15). There was another Figure 10:.Phylogenetic Tree among Tick species infested small ruminants in Duhok Governorate, Iraqi Kurdistan article support same species that found in Yazad Province, Iran by (16). A study was disagree with this results done by AL-Fatlawi et al., (17), they recorded that Hyalomma spp. were more predominant in south of Iraq.
Hyalomma anatolicum and Rhipicephalus turanicus and Rhipicephalus sanguineus were recorded by Kadum et al., (18) in middle and south of Iraq and recorded in north of Iraq by (19) and Omer et al., (15) they reported some species of hard ticks were included anatolicum anatolicum, marginatum marginatum, and Rhipecephalus appendiculatus.
The Data showed that the infection rate of infesting with hard ticks was higher in male than in In this analysis, there was another difference was the ratio of male to female was 2:1 and the number of males was dominant, the percentage was higher in male than female as follow respectively (70.82%) and (29.17%) and these ratio were different from results were done by Kadir et al., (21) in Kurdistan, Iraq Region and this results may be due to the changes of the climate in Duhok governorate and the season of the collection of ticks. These agree with the results of Salim abadi et al., (22), they found a relative frequencies tick sex were 57% male ticks and 34% female tick.
For the first time PCR assay in Kurdistan, Iraq and in Duhok especially is used for the identification of tick species. Two markers were used in this study: the first one was ribosomal Ribonucleic acid 16S rRNA for the identification of tick species and the second one was 18S rRNA for detection of piroplasms in both tick and blood.
During this study used Ribonucleic acid 16S rRNA for the identification of tick species and sequencing of it as a good marker for identification of hard tick species to solve morphological tick identification problems and sometimes morphological identification of ticks is not sufficient to detect the species. This is study is agreed with studies done by (23,24) With regard to tick species, 16S rDNA has been used and has been successful in constructing phylogeny of species of hard tick and 16S rRNA is helpful in building of the phylogenetic tree of hard tick species, but a problem associated with 16S rDNA is that using this gene alone is not sufficient to obtain full tree resolution so that the best way to solve it is accompanied by another gene such as 12S rDNA (24,25).
Overall 150 hard ticks (male and female and engorged female) were evaluated by using S16