J Arthropod-Borne Dis, September 2020, 14(3): 286–292 M Salehi-Vaziri et al.: Molecular Assay on … 286 http://jad.tums.ac.ir Published Online: September 30, 2020 Original Article Molecular Assay on Detection of Crimean Congo Hemorrhagic Fever (CCHF) Virus in Ixodid Ticks Collected from Livestock in Slaughterhouse from South of Iran Mostafa Salehi-Vaziri1,2; Hassan Vatandoost3,4; Alireza Sanei-Dehkordi5,6; Mehdi Fazlalipour2; Mohammad Hassan Pouriayevali2; Tahmineh Jalali2; Tahereh Mohammadi2; Mahsa Tavakoli2; Azim Paksa7; *Yaser Salim Abadi8 1Research Centre for Emerging and Reemerrging Infectious Diseases, Pasteur Institute of Iran, Tehran, Iran 2Department of Arboviruses and Viral Hemorrhagic Fevers (National Ref Lab), Pasteur Institute of Iran, Tehran, Iran 3Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 5Department of Medical Entomology and Vector Control, Faculty of Health, Hormozgan University of Medical Sciences, Bandar Abbas, Iran 6Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran 7Department of Medical Entomology and Vector Control, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran 8Department of Health Services and Health Promotion, School of Health, Rafsanjan University of Medical Sciences, Rafsanjan, Iran *Corresponding author: Dr Yaser Salim Abadi, E-mail: yasernls@gmail.com (Received 08 Sep 2020; accepted 29 Sep 2020) Abstract Background: Ticks are vectors of a wide variety of pathogens that can be transmitted to humans, and tick-borne diseas- es are a significant public health issue worldwide. The present study was carried out on the hard tick infestation of live- stock transported to Rafsanjan slaughter house in the southeast of Iran. Methods: A cross-sectional survey was carried out biweekly from April to September 2016 to determine tick infesta - tion of the meat-producing animals. All the livestock included in our study were thoroughly inspected for the presence of hard ticks on different parts of their bodies. Results: A total of 258 hard ticks were collected from the body of livestock hosts. The ticks that were sampled were classified into two genera and five species: Hyalomma marginatum, Hy. anatolicum, Hy. asiaticum, Hy. dromedarii, and Rhipicephalus sanguineus. Hyalomma dromedarii was the most abundant species in the study area. More than 50 per- cent of the sampled ticks were collected from the body of camels brought to the slaughter house however molecular analysis showed no Crimean Congo Hemorrhagic Fever (CCHF) virus infection in tick specimens. The Sex ratio of the sampled hard ticks shows that female tick infestation was more common among the study livestock. Conclusion: Due to the crucial role of hard ticks in the transmission of different pathogens to humans, additional inves- tigations are necessary to determine the risk of consumption of infested meat-producing animals in the study area. Keywords: Ticks; Livestock; Abattoir; Rafsanjan; Iran Introduction Deply (1, 2) introduced hard Ticks (Ixodi- dae) and two genera of these arthropods (Hy- alomma and Haemaphysalis) for the first time in Iran. Filipova et al. (3) reported the collec- tion of 642 ixodid ticks from small-size mam- mals, mainly rodents, in Iran. Later, Hoogstral Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://www.google.com/search?client=firefox-b-d&q=Slaughterhouse&spell=1&sa=X&ved=2ahUKEwivl6b6iK_rAhWoBWMBHUz8DpsQkeECKAB6BAgUECQ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, September 2020, 14(3): 286–292 M Salehi-Vaziri et al.: Molecular Assay on … 287 http://jad.tums.ac.ir Published Online: September 30, 2020 and Valdez (4) investigated and explained the medical and veterinary implications of Ixodoi- dea ticks from wild sheep and goats in Iran. During the past quarter-century, many studies were conducted on fauna, biology, and geo- graphical distribution of both soft and hard ticks in different parts of Iran (5-13). In a study conducted by Nabian et al. (7) 1720 tick spec- imens belonging to fourteen tick species were collected from cattle, sheep, and goats in dif- ferent localities of Caspian Sea areas in the north of Iran. Seven species of hard ticks were similarly sampled from cattle and sheep in the southeast part of Iran (5). The distribution of ticks and tick infestation rate of sheep in the West of Iran was studied by Nasiri et al. (8) who identified 864 hard ticks, which were clas- sified into two genera and five species. In a sur- vey conducted in the eastern part of Iran on species diversity and distribution of ticks in sheep, goats, cattle, and camels, 469 adult ticks belonging to 9 species were collected (6). Hard tick infestation of Livestock and seasonal pop- ulation dynamics of these ticks were investi- gated in the central part of Iran, and 583 hard ticks belonging to three genera and seven spe- cies were identified (12). Due to the crucial role of ticks in the transmission of pathogens such as the Crimean Congo Hemorrhagic fever vi- rus (CCHFV) to humans, they are considered as a significant problem in public health. Crime- an Congo Hemorrhagic Fever Virus CCHFV has been isolated from at least 31 species of ticks (14-16). Crimean Congo Hemorrhagic Fe- ver Virus CCHFV was also isolated from many genera and species of hard ticks and soft ticks in a study conducted on the distribution of ticks. Hard ticks of the genus Hyalomma were iden- tified as numerically dominant hard tick vectors for the transmission of CCHFV in different climatic zones of the country (14, 16). Crimean Congo Hemorrhagic Fever is currently present in most areas of Iran (17). The aim of the pre- sent study was to identify species composition of hard ticks (Acari: Ixodidae) parasitizing live- stock transported to Rafsanjan abattoir for the purpose of meat production and to investigate CCHFV infection rate in collected ticks. Materials and Methods Sample Collection In the present study, a cross-sectional study was carried out biweekly from April to Sep- tember 2016 in Rafsanjan City of Kerman Prov- ince. The cities that livestock brought to Raf- sanjan’s abattoir have been shown in figure 1 using Arc-GIS10.2 software (Redlands, CA). Animals included in our study were livestock considered as hosts of hard ticks, including cat- tle, sheep, and camels, which were brought to Rafsanjan’s abattoir for the purpose of meat production. All the livestock included in our study were surveyed for the presence of hard ticks on their body. Tick specimens collected from each host were kept alive in separate la- beled holding tubes, and the information relat- ed to each tube was recorded. Using the key identification guide (18), all collected hard ticks were identified according to their morphologi- cal characteristics until the species determination. Molecular analysis of Crimean Congo Hem- orrhagic Fever (CCHF) Tick specimens were homogenized indi- vidually in PBS using the mortar and pestle. Total RNA extraction was purified by the use of the RNeasy mini kit (QIAGEN) according to the manufacturer’s kit and stored at -70 °C freezer until needed for CCHFV detection. We performed RT-PCR for a 536bp fragment in S segment of CCHFV as previously described (19). Briefly, RT-PCR reaction was carried out in a final 50µl volume with 10µl of 5x QI- AGEN OneStep RT-PCR Buffer, 2µl of dNTP mixture (containing 10mM of each dNTP), 2µl of QIAGEN OneStep RT-PCR Enzyme Mix, 0.2µM of primer F2 (5'-TGGACACCTT CACAAACTC-3), 0.2µM of primer R3 (5'- GACAATTCCCTACACC-3'), 1µl of QIAGEN RNase Inhibitor (4 units/µl), and 5µl of ex- tracted RNA as a template. The RT-PCR con- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 286–292 M Salehi-Vaziri et al.: Molecular Assay on … 288 http://jad.tums.ac.ir Published Online: September 30, 2020 ditions began with 30min at 50 °C for reverse transcription reaction step and then 15min at 95 °C for initial denaturation, hot start activa- tion and inactivation of reverse transcriptase enzyme followed by 40 cycles of 95 °C for 30s, 50 °C for 30s, 72 °C for 45s, and a final extension at 72 °C for 10min. Polymerase Chain Reaction (PCR) products were ana- lyzed into 1.5% W/V electrophorese gel aga- rose. Results During the study period, a total of 258 hard ticks were collected from different live- stock hosts, including cattle, sheep, and cam- el. The number and type of hard tick species collected from each livestock have been sum- marized in Table 1. The sampled ticks were classified into two genera, including Hyalom- ma and Rhipicephalus. These two genera of ticks were further classified into five species based on their morphological characteristics. The tick species comprised of Hyalomma mar- ginatum, Hy. anatolicum, Hy. asiaticum, Hy. dromedarii and Rhipicephalus sanguineus (Ta- ble 1). Hyalomma dromedarii was the most abundant species among the collected ticks, followed by Hy. marginatum, as seen in Table 1. Rhipicephalus sanguineus was the least abun- dant tick species collected from the livestock in the study area. Between the different live- stock hosts, the occurrence of hard ticks was more common on the body of camels; more than 50 percent (N= 137) of all collected sam- ples were related to this host (Table 1). The sex ratio of the collected hard ticks shows that female tick infestation (60%) was more prev- alent among the study animals. After a thor- ough inspection, ticks were collected from dif- ferent body parts of the livestock. The rump region of sheep, under the tail region of cattle and camels, were the most favorable location for tick attachment (Table 2). Molecular assay results indicated no CCHFV infection in col- lected ticks. Table 1. The Frequency of collected ticks in different hosts in Slaughterhouse from Rafsanjan City, Kerman Prov- ince, Iran, 2016 Species Hosts Total Cattle Camel Sheep Number Percentage Hy. marginatum 19 31 38 88 34.11 Hy. dromedarii 0 97 0 97 37.60 Rh. sanguineus 0 0 10 10 3.88 Hy. anatolicum 9 4 24 37 14.34 Hy. asiaticum 3 5 18 26 10.07 Total 31 137 90 258 100 Table 2. Distribution of ticks on different body parts of the animals' body in Slaughterhouse from Rafsanjan City, Kerman Province Iran, 2016 Hosts Body parts Under Tail/Rump Belly Thigh Neck Cattle 27 4 0 0 Camel 118 9 8 2 Sheep 52 23 15 0 Total 197 36 23 2 http://jad.tums.ac.ir/ https://www.google.com/search?client=firefox-b-d&q=Slaughterhouse&spell=1&sa=X&ved=2ahUKEwivl6b6iK_rAhWoBWMBHUz8DpsQkeECKAB6BAgUECQ J Arthropod-Borne Dis, September 2020, 14(3): 286–292 M Salehi-Vaziri et al.: Molecular Assay on … 289 http://jad.tums.ac.ir Published Online: September 30, 2020 Fig. 1. Map of Iran, sites of livestock brought to Rafsanjan’s abattoir in, Kerman Province, 2016 Discussion A total of 258 hard ticks from the differ- ent livestock hosts were collected and identi- fied. Hyalomma dromedarii was the most abun- dant among the tick species collected from study animals. On the other hand, the presence of ticks on the body of camels was more com- mon compared with the other livestock hosts. The results of the present study also show that the rump region of sheep and under the tail region of cattle and camel are the most favor- able regions for tick attachment. In our study, two genera of Ixodid ticks were identified and further classified into five species. Hyalomma dromedarii and Hy. marginatum belonging to the genus Hyalomma was the most numerical- ly dominant among sampled ticks. Similar re- sults were observed in other studies conduct- ed in different parts of Iran (5-8, 11, 12, 20- 22). The results of the present study also indi- cate that the adults of Hy. dromedarii, which were the most abundant tick species among all sampled hard ticks, prefer camels belonging to the genus Camelus dromedarius (Mammalia: Camelidae) for feeding. This finding is con- sistent with that of other studies that investi- gated the tick infestation rate of camels, but in some other studies, other species of hard ticks were collected from camels (6, 12, 22-26). In addition to camels, Hy. dromedarii has also been collected from sheep and cattle in some other studies (6, 8, 11, 12). Five tick species be- longing to two genera (Hyalomma and Rhip- icephalus) were identified in our study, in- cluding Hy. marginatum, Hy. anatolicum, Hy. asiaticum, and R. sanguineus. Rh. Sanguineus, which was the least abundant species among all collected ticks, mainly infested sheep. Sim- ilar to the results of other studies, it seems that Rh. sanguineus is one of the most common tick species that parasitize sheep and goats (5-7, 11, 27, 28). In some other studies that surveyed hard ticks which infested domestic ruminants, some other genera of hard ticks that were not found in our study were reported. For instance, http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 286–292 M Salehi-Vaziri et al.: Molecular Assay on … 290 http://jad.tums.ac.ir Published Online: September 30, 2020 in 2009, a study on tick infestation rate of sheep conducted in Abdanan County (a west area in Iran) identified Heamaphysalis sulcate, which belongs to the family Ixodidae, and oth- er species of hard ticks which infested sheep (8). Hosseini-Vasoukolaei et al. (28) reported that four genera of Ixodidae (hard ticks), in- cluding Ixodes ricinus, Boophilus annulatus, H. punctate and H. numidiana infested do- mestic ruminants in Ghaemshahr County (north of Iran), but these species were not found in the present study. Even so, we did not expect to find I. ricinus in our study area due to its habitat and bio-ecology requirements, based on the results of previous studies which mentioned that the distribution of I. ricinus is just limited to the Caspian Sea region in the northern part of Iran (7, 9, 28, 29). In the present study, we found that ticks prefer to attach to the rump and under tail (perineum) regions, compared with other body parts, and this finding is in agree- ment with the results of some other studies. The preferred site of attachment of ticks has been associated with infestation season, and it seems that during warm months of the year, hard ticks prefer perineum region for attach- ment (5, 28). 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