Molecular epidemiology of Crimean–Congo hemorrhagic fever virus in ticks collected from western Iran
Article Category: Brief communication (Original)
Published Online: Mar 31, 2017
Page range: 603 - 607
DOI: https://doi.org/10.5372/1905-7415.1006.530
Keywords
© 2016 Taher Mehran, Dayer Mohammad Saaid, Jalali Tahmineh, Khakifirouz Sahar, Telmadarraiy Zakkyeh, Salehi-Vaziri Mostafa
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Crimean–Congo hemorrhagic fever (CCHF) is a potentially severe viral disease with a case–fatality rate of 30%–50% [1]. The etiology of the disease was first described in the Crimea in 1944 and currently is endemic in Europe, Africa, Central Asia, and Middle East [2, 3, 4, 5, 6]. The causative agent of the disease is the Crimean-Congo hemorrhagic fever virus (CCHFV) belonging to the genus Nairovirus of the family Bunyaviridae [7]. Because of its epidemic potential, high mortality rate, nosocomial outbreaks, and difficulties in treatment and prevention, CCHF outbreaks are considered to be a serious threat to public health. Iran is recognized as a major focus of CCHF in the Middle East where the disease was reported from different Iranian provinces including Fars, Khorasan, and Yazd provinces [1, 8, 9, 10].
The CCHF virus is transmitted to humans either by the bite of an infected tick or through direct contact with blood or tissues of viremic livestock animals or humans [11, 12]. Given that ticks are both the reservoir and the vector of the CCHFV, they play an important role in CCHF epidemiology. In nature, the CCHF virus is maintained by hard ticks (Ixodidae). Despite the notion that
Determining the infection rate of the tick population in different geographical regions and identification of tick fauna are critical for designing public health strategies to avoid CCHF outbreaks. Therefore, this study was designed to collect hard ticks from livestock and to detect CCHFV infection within the collected ticks in 3 counties namely Asadabad, Nahavand, and Famnin in Hamadan province, west of Iran.
Hamadan province is located in the west of Iran, placed between 45°32ˊ and 48Έ and 34°47ˊ and 35° 1ˊ N. Despite its high altitude and cold climate, Hamadan is one of the main animal farming centers in Iran. Therefore, a large number of its residents are in direct contact with livestock and exposed to an increased risk of CCHFV infection.
After approval by the Ethics Committee of Tarbiat Modares University (reference No. 52/6855) observing the national guidelines on animal care, including the wellbeing of the animals during sampling, this study was conducted during June 2013 to May 2014. Sampling was performed in 5% of villages randomly selected in Asadabad, Nahavand, and Famnin counties after documented informed consent from the property/farm owners for collecting ticks from the live host animals. Provincial authority approval from the Health Center of Assadabad County under the Hamadan University of Medical Sciences (reference No. 16/41/144) was issued for the researchers to refer to local veterinary and health care offices for assistance and coordination. In total, 881 ticks were collected from plain (Asadabad and Famnin) and mountain (Nahavand) region in Hamadan province. Sample collection was conducted according to Tahmasebi et al. [14]. Collected ticks were kept alive in separate labeled vials. After species determination, 100 randomly selected hard ticks were sent to the Laboratory of Arboviruses and Viral Hemorrhagic Fevers (National Reference Laboratory) Pasteur Institute of Iran, to detect CCHFV infection.
Ticks were separately washed twice with phosphate-buffered saline, pH 7.4 (PBS) and then crushed using a mortar and pestle in 200 μ1 of PBS. Total RNA of ticks was extracted using RN easy plus Mini Kits (Cat. No. 74136, Qiagen, Venlo, The Netherlands) according to the manufacturer’s protocol. The extracted total RNA was stored at –70°C until further analysis [16].
To amplify the genomic RNA of CCHFV, a OneStep RT-PCR Kit (Cat. No. 210212, Qiagen) was employed. All components were mixed to prepare the master mix as follows: RNase free water 28 (L), 5× Qiagen OneStep RT-PCR Buffer (10 μL), dNTP Mix (containing 10 mM of each dNTP) 2 (μL), Iran-F2 primer 5ˊ-TGGACACCTTCACAAACTC-3ˊ(10 μΜ) 1 μL, Iran-R3 5ˊ-GACAATTCCCTACACC-3ˊ primer (10 μΜ) 1 μL, enzyme mix (2 μL), and RNase inhibitor 0.5 μL. (Cat. No. 10777-019, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Finally, 5 μL of template was added to the tube and the thermal-cycler program was started. The cycling parameters were as follows: reverse transcription for 30 min at 50°C, 95°C for 15 min as hot start, 35 cycles of 30 s of denaturation at 95°C, 30 s of annealing at 50°C, 45 s of elongation at 72°C, with final elongation of 5 min at 72°C.
We examined 5 μL of the PCR amplicons on an 1.5% agarose gel using gel loading buffer with DNA stain, (Cat. No. PCR-255-bl, Jena Bioscience, Germany) and Gene ruler 100 bp ladder (Cat. No. SM0242, Thermo Scientific) as a molecular-weight size marker.
The data were analyzed using SPSS Statistics for Windows, version 20 (IBM Corp, Armonk, NY, USA) and P < 0.05 was considered significant.
A total of 881 hard ticks were collected from 141 sheep and goats in the present study. All ticks belonged to 3 genera including,
The RT-PCR amplification of the S segment of the CCHFV genome using RNA extracted from each tick showed a band of 536 bp (
Figure 1
Agarose gel (1.5%) electrophoresis of amplified S segment DNA from the Crimean-Congo hemorrhagic fever virus genome using reverse transcriptase-polymerase chain reaction of RNA in tick samples from Hamadan province. Lad, 100 bp marker ladder; NC, negative control; PC, positive control (536 bp); lanes 2, 6, 8, and 10 positive tick samples
Rate of the Crimean-Congo hemorrhagic fever virus infection in collected ticksTick species Percent of 881 ticks Reverse transcriptase–polymerase chain reaction positive cases (Of 7 infected ticks in 100 randomly selected) 65.1% 4 29.0% 2 0.9% 0 1.02% 1 1.6% 0 0.1% 0 0.1% 0 0.1% 0 0.56% 0 0.34% 0
All infected ticks were collected from sheep. The majority of CCHFV-positive ticks were collected from male animals. The infection was most common in one-year-old sheep. Four of CCHFV-positive ticks were among those collected form highland areas, whereas the remaining 3 ticks were from lowland origin.
Ticks are important vectors of several infections affecting animals and humans [17]. Determination of the tick fauna and their transmitted infectious agents are essential for controlling the tick-borne diseases [18]. In the present study, we investigated prevalence of CCHFV in hard ticks in Asadabad, Nahavand, and Famnin counties of Hamadan Province, western Iran
We found CCHFV infection in 7% of tested ticks, which is a lower rate than obtained by similar earlier studies conducted in Hamadan province. Moradi et al. [19] reported in Bahar (central part of Hamadan Province) the rate of CCHFV infection was 11.3% among collected ticks. Another study conducted in the same province showed an even higher CCHFV infection rate at 19.3% [14]. However, the reported rates of CCHFV infection in ticks showed variation in time and geographically so that the infection rate was 28% in Ardabil province (northwest of Iran) [18], 6.6% in Ilam province (south west of Iran) [9], and 5.7% in Yazd province (center of Iran) [20]. The variation of CCHFV infection has been attributed to weather and geographical diversity, animal hosts of ticks, and ecological requirements of various tick species [21, 20]. Comparing the CCHFV infection rates reported earlier from Hamadan and Ardabil provinces with those more recently obtained from Ilam, and Yazd provinces, one may conclude that the rate of infected ticks in Iran has decreased since 2009.
Although