J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 268 http://jad.tums.ac.ir Published Online: September 30, 2019 Original Article Effect of Meteorological Factors on Hyalomma Species Composition and Their Host Preference, Seasonal Prevalence and Infection Status to Crimean-Congo Haemorrhagic Fever in Iran Nayyereh Choubdar1; *Mohammad Ali Oshaghi1; Javad Rafinejad1; Mohammad Reza Pourmand2; Naseh Maleki-Ravasan3; Mostafa Salehi-Vaziri4; Zakkyeh Telmadarraiy1; Fateh Karimian1; Mona Koosha1; Abbas Rahimi-Foroushani5; Safdar Masoomi5; Kourosh Arzamani6; Jalil Nejati7; Mohsen Karami8; Ehsan Mozaffari1; Yaser Salim-Abadi9; Eslam Moradi-Asl10; Behrooz Taghilou11; Manouchehr Shirani12 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran 4Department of Arboviruses and Viral Hemorrhagic Fevers, Pasteur Institute of Iran, Tehran, Iran 5Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 6Vector-Borne Diseases Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran 7Department of Public Health, School of Public Health, Zahedan Unversity of Medical Sciences, Zahedan, Iran 8Department of Parasitology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran 9Department of Health Services and Health Promotion, School of Public Health, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 10Department of Public Health, School of Public Health, Ardabil University of Medical Sciences, Ardabil, Iran 11Zanjan Health Center, Zanjan University of Medical Sciences, Zanjan, Iran 12Mamasani Health Center, Shiraz University of Medical Sciences, Shiraz, Iran (Received 26 Jan 2019; accepted 30 Apr 2019) Abstract Background: The impact of environmental factors and host on Hyalomma spp. community structure and abundance in the main Crimean-Congo haemorrhagic fever (CCHF) foci of Iran is largely unknown. Methods: Biotic and abiotic factors, including host, temperature, humidity, altitude, Köppen-Geiger climate types, season, and precipitation on Hyalomma spp. community structure and abundances in 11 provinces of Iran were investigated. Additionally, the possible infection of ticks with CCHF virus was evaluated using reverse transcription PCR technique. Results: Species analyses demonstrated the presence of Hyalomma anatolicum, H. marginatum, H. dromedarii, H. asiaticum, H. detritum and H. schulzei in the study area. Hyalomma anatolicum was the dominant species in the southern and northern parts, whereas H. dromedarii was distributed mostly in central parts of the country. The highest tick infestation was recog- nized in hot season. Spatial variation in tick relative density was observed between habitat types where more ticks were col- lected in deserts, semi-deserts, and Mediterranean habitats. Except for H. dromedarii, which was more prevalent on camel (P= 0.044), there were no significant variations in the frequencies of other Hyalomma species on different hosts. Hyalomma ana- tolicum, H. dromedarii frequencies had significant positive and negative association with temperature and precipitation re- spectively. Also humidity has positive impact on H. asiaticum frequency. Conclusion: Data presented here will help improve ecological models to forecast the distribution of Hyalomma spp. ticks, to evaluate the risk of CCHF and other tick-borne diseases, and to design proper vector control measures to suppress Hyalomma populations in Iran. Keywords: Hyalomma; Ticks; Climate; Host; Environment Introduction Ticks are among the major vectors of path- ogens for animals and humans in the world. They can play a crucial role in the transmis- sion of a wider range of pathogens including *Corresponding author: Dr Mohammad Ali Oshaghi, E-mail: moshaghi@tums.ac.ir http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=Choubdar%20N%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=Oshaghi%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=Maleki-Ravasan%20N%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=Karimian%20F%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=Koosha%20M%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=Arzamani%20K%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=Moradi-Asl%20E%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=Shirani%20M%5BAuthor%5D&cauthor=true&cauthor_uid=29745300 https://scholar.google.com/citations?view_op=view_org&hl=en&org=16806557757143553874 J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 269 http://jad.tums.ac.ir Published Online: September 30, 2019 protozoans, viruses and bacteria than other ar- thropod vectors (1). The main public health prob- lem and the largest geographical distribution re- lated to ticks is Crimean-Congo haemorrhagic fever (CCHF), a viral hemorrhagic fever, for which ticks serve as reservoirs of the virus. Humans are infected either through tick bites or due to direct contact with infected blood and tissues of a mammalian host (2). Infected do- mestic animals are known to act as amplifying hosts in the absence of any clinical symptoms (3). The main mode of CCHFV transmission to humans in Iran is exposure to the blood or vis- cera of infected livestock. Moreover, direct ex- posure to ticks (tick bites) accounts for note- worthy transmission in humans (4-7). CCHF's case mortality rate ranging from 5–80% (8, 9). CCHF virus is endemic to Africa, the Balkans and Ukraine, the Middle East and Central Asia (10). It is prevalent in most of Iran’s neighbor- ing countries, including Pakistan and Afghan- istan (11). Iran is known as one of the main foci of CCHF in western Asia where the first outbreak of the disease was reported in 1999 and has since become the main public health concern in the country (5, 12-14). CCHF virus exists in an enzootic cycle be- tween ticks and mammals, and geographic dis- tribution of the virus mirrors the distribution of the primary tick vector species (15). In ad- dition, other factors such as environment, cli- mate, geographical features, socioeconomic pa- rameters, grazing system, and livestock’s age can affect the distribution and transmission of the disease (16-18). Seasonal pattern, temper- ature, relative humidity, and lower altitude pos- itively affect the occurrence of the disease (15). Moreover, the main foci of this disease in hu- mans were in the eastern, northeastern, and cen- tral regions of Iran (5, 12-14). Currently, CCHF is prevalent in 23 provinces (Ps) of Iran, par- ticularly the ones having a long border with three high-risk countries, Turkey, Afghanistan and Pakistan (9, 11, 18, 19). Since 2000, the most CCHF cases have been reported from Sistan and Baluchistan Province in southeast corner of Iran where Hyalomma spp. ticks are present and CCHFV is endemic (4, 15, 20). The CCHFV has been isolated from 30 spe- cies of hard ticks; however, the main group of vectors appears to be ticks of the genus Hy- alomma in most parts of the world (21). Hy- alomma marginatum Koch, 1844, and H. asi- aticum Schulze and Schlottke, 1930, are the main CCHFV vector in Europe and Asia respectively (9, 21). In Iran, the virus has been isolated from several species of hard ticks (Ixodidae) includes; Hyalomma, Rhipicephalus, Haemaphysalis, and Dermacentor genera. The most frequent spe- cies of this genus reported in Iran include; H. marginatum, H. asiaticum, H. anatolicum Koch, 1844, H. detritum Schulze, 1919, H. drome- darii Koch, 1844, H. rufipes Koch, 1844 and H. schulzei Olenev, 1931 (22-24). Climate may play an essential role in the distribution and seasonal abundance of ticks (25, 26). Iran is composed of nine climate types out of 31 possible Köppen-Geiger climate types (27). Most parts of central, eastern and south- ern Iran are characterized by Köppen BWh (Hot desert climate) and BWk (Cold desert climate) climate types. The coastal areas of the Caspi- an Sea and most parts of mountainous areas of Zagros and Alborz in the west and north of Iran have moderate climate type Csa (temper- ate with hot and dry summer=hot summer cli- mate). However, the eastern slope of Zagros and southern slope of Alborz connected to the central arid and semi-arid climate of central Iran are distinguished with BSk climate (Cold semi-arid climate= cold steppe). The southern parts of Zagros region are mostly dominated by BSh climate (Hot semi-arid). Dsa (Hot-sum- mer Mediterranean) and Dsb (warm-summer Mediterranean) climate types are found in some parts of mountainous areas of Zagros and Al- borz, while Csb (Warm-summer Mediterrane- an) and Cfa (humid subtropical) are the local- ized climate types found in coastal areas of the Caspian Sea. Nevertheless, very few studies on Hyalomma spp. community have incorporated biotic and abiotic environmental factors such http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 270 http://jad.tums.ac.ir Published Online: September 30, 2019 as host, season, altitude, latitude, temperature, humidity, precipitation, and climate features in Iran. In this study, we have tried to determine Hyalomma species community structure on livestock in different regions of Iran to assess their spatial and temporal distribution, and to test overall impact of the environmental fac- tors on Hyalomma species density and diver- sity, and possible association with CCHF dis- tribution in the country. We also tested their in- fection to CCHFV to evaluate the abundance of potential CCHFV vectors in the region. This would provide data on the relative prevalence of Hyalomma spp., which might facilitate the control of ticks and tick-borne diseases in the study areas. Materials and Methods Study area Iran lies between latitudes 24° and 40° N and longitudes 44° and 64° E and locates in Eurasia and Western Asia. It is bordered to the northwest by Armenia and the Republic of Azer- baijan; to the north by the Caspian Sea; to the northeast by Turkmenistan; to the east by Af- ghanistan and Pakistan; to the south by the Persian Gulf and Oman Sea, and to the west by Turkey and Iraq (Fig. 1). Iran is considered as an area with wide ranges of altitude (below sea-level in shores of the Caspian Sea to 5,770m of the Damavand Mountain), climate (humid and nearly jungle-like forests in the north to arid places in Dasht-e Lut with less than 100 mm annual rainfall) and temperature (from -35 °C in the northwest to 70 °C in the deserts of Dasht-e Lut). Tick specimens were collected from elev- en Ps in Iran as follows: North Khorasan, East Azerbaijan, Ardabil (Pars Abad), Kerman and Fars with BSk climate, Yazd, Sistan and Ba- luchistan with BWh climate, Isfahan of BWk climate, Illam, Zanjan, and Golestan of Csa climate and Meshkin Shahr of Ardabil of Csb climate (Fig. 1, Table 1). Tick Collection Samplings were carried out in both hot and cold seasons in 2016 and 2017. Since infected livestock is the main mode of CCHFV trans- mission to humans in Iran, ticks were collect- ed from livestock during three to five visits in per season from eight farms/stables in each district. Thirteen districts were tested for the presence of ticks (Table 1). The cattle were ran- domly selected from farms/stables (nomadic grazing system) and whole body of 20 ani- mals (cow, sheep, goat and or camel, based on availability) were carefully examined, just af- ter whipping, the ticks were manually collect- ed. Ticks were carefully removed from ani- mals by forceps. Details of sample collection including location, type of animal, and date were recorded. Ticks were stored in tubes and covered on top with small piece of muslin cloth and carefully labeled at the collection site. All of the collected specimens were iden- tified to species level by using a stereomicro- scope (Olympus SZ51-Japanese) and a taxo- nomic key (28). Environmental factors Environmental data were obtained from various data providers (Table 1). Elevation (al- titude) and latitude/longitude data were sup- plied by www.gps-coordinates.org. Mean an- nual precipitation, relative humidity, tempera- ture, and climate data were obtained from http://www.irimo.ir. The human CCHF cases were provided by Ministry of Health and Med- ical Education of Iran. RNA extraction and RT-PCR Alive tick specimens were separately washed with Phosphate Buffered Saline (PBS) and crushed with pestle in 200–300µl of PBS. Total RNA was extracted from each sample homogenate using the QIAamp RNA Mini kit (Qiagen, Germany) according to the manufac- turer's instructions. The extracted RNA was dis- solved in 50µl of RNase-free water and stored at -70 ºC until analysis. Reverse transcription http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/Eurasia http://www.gps-coordinates.org/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 271 http://jad.tums.ac.ir Published Online: September 30, 2019 of RNA was performed individually using one- step RT-PCR kit and specific primers F2 (5ʹ- TGGACACCTTCACAAACTC -3ʹ) and R3 (5ʹ- GACAAATTCCCTGCACCA -3ʹ) (29), which amplify a 536bp fragment of the S segment of the CCHF viral genome. The total CCHFV ge- nome, extracted from a previously confirmed RT-PCR-positive serum was used as the posi- tive control, and no template control (NTC) was used as the negative control in the RT-PCR tests. For gel-based analysis, 5µl of the PCR products were mixed with 1µl loading buffer and electrophoresis was performed on 2% Aga- rose gel and visualized on a UV Transillumi- nator. Statistical analysis The numbers of ticks per season in each district were counted. Data for each individual Hyalomma tick were used to determine its spe- cies, and its association with factors such as season, host, and location. Associations be- tween each specific tick species and categori- cal parameters were analyzed using χ2 test. Because the data were not normally distrib- uted, Mann-Whitney and Kruskal-Wallis tests were used to assess the statistical significance of the variables. Spearman’s correlation coef- ficient and univariate and multivariate regres- sion analysis were used to find possible asso- ciations between tick density and environmen- tal factors using SPSS software (ver. 24, Chi- cago, IL, USA). For statistical data analysis, we used Pois- son regression analysis to test if there is an as- sociation between climate factors such as hu- midity, precipitation, altitude, and temperature as predictors and count of ticks and dependent variable. Differences in tick species propor- tions were considered as statistically signifi- cant if P< 0.05. All graphs and statistics were performed using GraphPad Prism ver. 5.00 for Windows (GraphPad Software). Results Tick composition Overall, 3929 hard ticks were collected from the study areas including; 76 nymphs, 451 non- Hyalomma, and 3402 Hyalomma ticks. The non- Hyalomma ticks comprised specimens of Rhip- icephalus (n= 274, 54.76%), Dermacentor (n= 168, 37.26%), Haemaphysalis (n= 11, 2.44%), Boophilus (n= 17, 3.77%), and Ixodes (n=8, 1.77 %) genera. Two season investigations resulted in col- lection of hard ticks of the genus Hyalomma from different infested cattle in the study area. Details of the collected Hyalomma spp. tick specimens are shown in Table 2. From Mar 2016 to Dec 2017, a total of 1536 cattle from 128 farms from 13 districts were inspected and 3402 Hyalomma spp. ticks were collected. More than half (53.8%) of specimens were females. The cattle infestation level ranged from zero to 20 Hyalomma spp. ticks per animal. The number of Hyalomma spp. ticks in hot season were higher than cold season (58.3 vs. 41.7 %), however, the distribution of Hyalomma spp. was the same for two seasons (Mann˗Whitney U= 57.5, P= 0.1091). Hyalomma spp tick infes- tation was observed in all study areas of Iran. Six species of genus Hyalomma includes; H. anatolicum, H. marginatum, H. dromedarii, H. asiaticum, H. detritum, and H. schulzei were found to infest the animals in the study area. Hyalomma anatolicum and H. detritum with 31.9% and 1.59% were the most and the least prevalence species in the study area. The dis- tribution of the six tick species among the prov- inces were not similar (Kruskal˗Wallis test: P= 0.006, Kruskal˗Wallis static: 16.33). Sistan and Baluchistan with six and Yazd and Fars with one Hyalomma species showed the most and the least diversity among eleven P’s (Table 1). Except for H. detritum found only in hot season, all the other Hyalomma species were collected throughout the year (Table 1). When the distributions of six species were compared in pairs, results showed that distribution of H. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 272 http://jad.tums.ac.ir Published Online: September 30, 2019 detritum and H. schulzei in the collection sites were significantly different (Mann˗Whitney, U= 18.5 and 28, P= 0.0042 and 0.0247 respectively). Tick species found in different host species Data were analyzed for the suitability and the specificity of the tick species to the hosts from which they were collected. Statistical anal- ysis showed that except for H. dromedarii, which was significantly more prevalent on camel (P= 0.044), there were no significant variations in the frequencies of other Hyalomma spp on dif- ferent hosts (Fig. 2). Hyalomma anatolicum the major tick species were found on camel, cow, sheep and goat but the number of ticks were not significantly different between the hosts (χ2= 4.623, df= 3, P= 0.202). Hyalomma drom- edarii being the second most prevalent species on the hosts was significantly more abundant on camel (χ2= 8.104, df= 3, P= 0.044). The fre- quencies of H. marginatum and H. asiaticum ticks were not significantly varied (H. margina- tum: χ2 = 2.008, df= 3, P= 0.544, H. asiaticum: χ2= 3.209, df= 3, P= 0.361) between the hosts, although both species were more established on cow. Hyalomma schulzei and H. detritum were rarely found and were almost exclusive on cow and cow/camel respectively. Impact of latitude/longitude on Hyalomma distribution To assess the impact of geographical loca- tion on Hyalomma species occurrence and com- munity, the country was divided into three di- visions of south (5 districts), center (2 districts), and north (6 districts). This analysis showed however, the differences were not significant (P> 0.05) among different parts of the country (Fig. 3). Impact of season on Hyalomma species abun- dance and community Seasonal analysis of tick abundances showed that seasonality positively affects Hyalomma spp tick abundances in the study area. Tick abun- dances in hot season were 1.4 times of cold sea- son. When Hyalomma species were analyzed in- dividually, tick abundances’ for all species were higher in hot season than cold season, howev- er, differences were significant only for three species of H. dromedarii, H. marginatum and H. detritum (P< 0.05) (Fig. 4). Except for one lo- cation, Hyalomma species communities were higher in hot season than cold season (Table 1). Impact of temperature on Hyalomma species abundance and community Analysis of data showed a positive associ- ation between the annual mean temperature and total tick abundances (Fig. 5). When Hyalom- ma species were analyzed individually, tick abundances’ were significantly higher for two species of H. dromedarii (P= 0.03) and H. an- atolicum (P= 0.018) (Table 3). Impact of altitude on Hyalomma species abun- dance and community Analysis of the data revealed a weak neg- ative association between the altitude (meter above sea level) and tick abundances (Fig. 5). Chah˗bahar District in southeastern part of the country with the lowest altitude comprised the highest Hyalomma spp tick abundance and spe- cies community (Table 1). When Hyalomma species were analyzed individually, tick abun- dances’ differences were not significant (P= 0.647–0.947) for the species studied (Table 3). Impact of relative humidity on Hyalomma species distribution and community Analysis of data showed there is a general positive association between the annual mean relative humidity (RH) and either the tick abun- dances (Fig. 5) or the tick species community. However, when each Hyalomma species were analyzed, tick abundances’ differences were not significant (P= 0.143–0.968) for the species stud- ied (Table 3) suggesting no or, at best, a very weakly significant association between RH and Hyalomma spp present. Impact of precipitation on Hyalomma spe- cies abundance and community On general, analysis of data showed there is http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 273 http://jad.tums.ac.ir Published Online: September 30, 2019 a negative association between the annual mean precipitation and either the tick abundances (Fig. 5) or the tick species community. Reduc- tion in precipitation is favored to Hyalomma spp tick abundance and community. However, when individual Hyalomma species were ana- lyzed, tick abundances’ differences were sig- nificant only for H. dromedarii (P< 0.001) and H. anatolicum (P= 0.05) (Table 3). Impact of climate type on Hyalomma species abundance and community Analysis of data revealed that 46% of Hy- alomma spp. tick specimens were collected from desert climate (BWh) in Yazd and Sistan and Baluchistan. Total tick abundance rates were significantly much lower in other climate types of the country including warm-summer Med- iterranean climate (Csb) in Meshkin Shahr of Ardabil, hot-summer Mediterranean climate (Csa) in Illam, Zanjan, and Golestan, cold de- sert climate (BWk) in Isfahan, cold semi-arid climate (BSk) in North Khorasan, East Azer- baijan, Ardabil (Pars Abad), Kerman and Fars (Fig. 6). Except for Csb climate with two Hy- alomma species, other climate norms had sim- ilar number (5) of Hyalomma species. Statisti- cal analysis showed that abundances’ frequen- cy of four more prevalent Hyalomma species was not significantly different in various cli- mate types (P= 0.392). CCHFV infection RT-PCR was used to detect the CCHF vi- ral infection in less than 10% (390 out of 3929 ticks) of the Hyalomma spp ticks collected in the study areas. Among this sample (390), in- cluding all of the six Hyalomma species, no CCHFV positive sample was found. Table 1. Details of meteorological variables in the study areas Province District Climate norm Coordinates AASL (m) MAP (mm) MAT (ºC) MARH (%) Ardabil Meshgin shahr Csb 38°24 ʹ 50.8ʹʹ N 47°38ʹ 27.3ʹʹ E 1338 356 9.7 63 Pars-abad BSk 39°26 ʹ 44.2 ʹʹN 47°25ʹ 07.1ʹʹ E 32 382 12.1 63 East Azerbai- jan Khoda- afarin BSk 39°08’26.5”N 46°57’27.5”E 1144 403 13.6 82 Fars Marvdasht BSk 30°01’19.7”N 52°38’22.9”E 1620 263 16.7 47 Ilam Pardeh Csa 33°46’48.2”N 46°04’26.9”E 1378 442 16.2 35 Golestan Gonbad-e kavus Csa 37°15’50.7”N 55°12’18.5”E 52 363 18.6 93 Isfahan Nain BWk 32°50’38.1”N 53°04’29.2”E 1571 76 16.6 50 Kerman Rayen BSk 29°35’00.8”N 57°28’01.2”E 1756 176 14.2 60 North Khora- san Raz and Jargaland BSk 37°56’32.4”N 57°06’16.3”E 1086 262 11.5 42 Sistan and Baluchestan Chabahar BWh 26°14’27.5”N 61°24’10.0”E 7 106 25.8 70 Sarbaz BWh 26°37’35.5”N 61°15’42.9”E 915 134 23.5 70 Yazd Mehriz BWh 31°35’33.7”N 54°30’25.3”E 1230 55 18.9 32 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 274 http://jad.tums.ac.ir Published Online: September 30, 2019 Zanjan Ijrud Csa 36°06’00.2”N 48°35’54.4”E 2050 356 10.2 53 AASL: altitude above sea level, MAP: mean annual precipitation, MAT: mean annual temperature, MARH: mean annual relative humidity, BWh: Hot desert climate, BWk: Cold desert climate, Csa: Hot sum- mer Mediterranean climate, Csb: Warm-summer Mediterranean climate, BSk: Cold semi-arid climate Table 2. Details of the collected ticks from 11 provinces of Iran Cold season Hot se Hot season District, Province (Cli- mate) Species F M Total Host Species F M Total Host Chabahar, Sistan and Baluchestan (BWh) H. anatolicum H. asiaticum H. marginatum 182 23 31 11 19 46 312 Goat Cow H. asiaticum H. dromedarii H. detritum H. marginatum H. anatolicum 45 0 0 7 11 115 150 10 70 76 484 Goat Cow Camel Sarbaz, Sistan and Baluchestan (BWh) H. anatolicum H. marginatum H. dromedarii H. schulzei 32 18 46 10 16 13 52 7 194 Goat Cow H. dromedarii H. detritum H. marginatum H. asiaticum H. anatolicum 76 0 34 16 132 80 4 14 8 43 407 Goat Cow Camel Rayen, Kerman (BSk) H. dromedarii H. asiaticum 32 0 9 31 72 Camel H. marginatum H. anatolicum H. dromedarii 44 13 0 0 0 12 69 Camel Marvdasht, Fars (BSk) H. marginatum 8 1 9 Goat Sheep H. marginatum 51 8 59 Goat Pardeh, Illam (Csa) H. anatolicum H. dromedarii H. asiaticum H. schulzei 39 8 8 0 0 4 0 6 65 Cow H. anatolicum H. dromedarii H. asiaticum H. schulzei 54 13 11 0 0 6 0 7 91 Goat Sheep Mehriz, Yazd (BWh) H. dromedarii 47 12 0 69 Goat Camel H. dromedarii 103 0 103 Camel Nain, Isfahan (BWk) H. dromedarii 48 8 56 Camel H. dromedarii H. detritum H. marginatum H. asiaticum H. anatolicum 62 0 0 14 31 53 13 11 40 47 271 Camel Cow Ijrud, Zanjan (Csa) H. asiaticum H. anatolicum H. dromedarii 102 0 3 53 7 0 165 Cow Sheep H. anatolicum H. detritum H. marginatum 69 0 22 31 27 51 200 Cow Khoda-afarin, East Azerbaijan (BSk) H. anatolicum H. asiaticum H. dromedarii 24 19 2 7 7 3 62 Cow Goat H. anatolicum H. asiaticum H. dromedarii 98 74 7 25 27 10 241 Cow Meshgin-shahr, Ardabil (Csb) H. marginatum H. asiaticum 15 0 42 9 66 Cow H. marginatum H. asiaticum 24 0 65 7 96 Cow Sheep Pars Abad, Ardabil(BSk) H. marginatum H. anatolicum 24 0 17 19 60 Cow Goat H. marginatum H. anatolicum 15 0 11 13 39 Cow Gonbad-e kavus, Golestan(Csa) H. dromedarii H. marginatum 0 0 7 5 12 Camel H. anatolicum H. dromedarii H. marginatum 9 0 0 6 7 4 26 Sheep Raz and Jargaland, North Khorasan (BSk) H. anatolicum H. dromedarii 46 0 23 9 78 Sheep Cow H. schulzei H. anatolicum H. dromedarii H. marginatum 28 13 5 0 7 9 0 26 96 Sheep Goat Table 1. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 275 http://jad.tums.ac.ir Published Online: September 30, 2019 H. asiaticum 0 8 Total 911 509 1220 959 1023 2128 F: female, M: male, BWh: Hot desert climate, BWk: Cold desert climate, Csa: Hot summer Mediterranean climate, Csb: Warm-summer Mediterranean climate, BSk: Cold semi-arid climate Table 3. Univariate regression analysis between environmental parameters and abundance of each tick species in Iran. Bold-red represents significant correlations Species Climate parameter R Square Sig Parameter Constant Estimates b1 H. anatolicum Humidity 0.082 0.222 11.125 0.772 Temperature 0.274 0.018 -20.157 4.671 Precipitation 0.197 0.050 100.784 -0.157 Altitude 0.011 0.667 63.813 -0.007 H. asiaticum Humidity 0.122 0.143 -16.804 0.845 Temperature 0.015 0.619 21.216 0.727 Precipitation 0.000 0.999 33.496 -9.155E-5 Altitude 0.013 0.647 40.882 -0.006 H. dromedarii Humidity 0.012 0.641 57.447 -0.259 Temperature 0.225 0.030 -33.177 4.264 Precipitation 0.549 < 0.001 102.277 -0.253 Altitude 0.000 0.942 40.866 0.001 H. marginatum Humidity 0.000 0.968 33.180 -0.015 Temperature 0.003 0.801 36.294 -0.240 Precipitation 0.010 0.667 27.467 0.020 Altitude 0.001 0.916 31.504 0.001 Fig. 1. Map of Iran, tick collection sites, and climate norms of the study area. 1–2: Chabahar and Sarbaz, of Sistan and Baluchestan, 3: Raven of Kerman, 4: Marvdasht of Fars, 5: Pardeh of Ilam, 6: Mehriz of Yazd, 7: Nain of Isfahan, 8: Ijrud of Zanjan, 9: Khoda-Afarin of East Azerbaijan, 10–11: Meshgin Shahr and Pars-Abad of Ardabil, 12: Gonbad-e- Kavus of Golestan, and 13: Raz and Jargaland of North Khorasan Table 2. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 276 http://jad.tums.ac.ir Published Online: September 30, 2019 Fig. 2. Tick species and their hosts from which they were collected. H. dromedarii was significantly more prevalent on camel (P= 0.044), but there were no significant variations in the frequencies of other Hyalomma spp on different hosts Fig. 3. Hyalomma abundance (A) and species numbers (B) among northern (n= 6), central (n= 2), and southern (n= 5) districts of Iran, 2016–2017. Bars indicate mean ±SEM http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 277 http://jad.tums.ac.ir Published Online: September 30, 2019 Fig. 4. Hyalomma species abundances in hot and cold seasons in 13 districts of 11 provinces of Iran, 2016–2017. Bars indicate mean ±SEM, (P< 0.05). Tick abundances were significantly different in hot season for three species of H. dromedarii, H. marginatum and H. detritum (P< 0.05) Fig. 5. Association between environmental factors and Hyalomma abundances in Iran. Red squares refer to significant relationship between environmental factors and the tick species. MAT: mean annual temperature, MARH: mean annual relative humidity, MAP: mean annual precipitation, ALT: altitude above sea level http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 278 http://jad.tums.ac.ir Published Online: September 30, 2019 Fig. 6. Relative abundance of Hyalomma ticks in five different climate norms of Iran. BSk: North Khorasan, East Azerbaijan, Ardabil (Pars Abad region), Kerman and Fars, BWh: Yazd and Sistan and Baluchistan, BWk: Isfahan, Csa: Illam, Zanjan, and Golestan, and Csb: Ardabil P (Meshkin Shahr region) Discussion This study is one of the first that documents the spatial-temporal as well as impact of envi- ronmental factors on abundance and community of Hyalomma spp. ticks in eleven provinces of Iran that include hot spots of CCHFV infection. The present study revealed the presence of six species of genus Hyalomma in the study area. H. anatolicum was the dominant species in the southern and northern parts while H. drome- darii distributed mostly in the central parts of the country. The genus of Hyalomma has known the most important tick species associated with livestock in Iran (22). Due to the importance of ticks as vector, several studies on the distribu- tion and fauna of ticks have been performed in the country. Findings of most previous studies (30-36) are in accordance with the tick fauna and distribution reported in the present study. In the present study we have found no speci- men of H. rufipes, H. aegyptium, H. excavatum, H. impeltatum which may be reflections of the sample collection method used, time and place of sampling, and ecological changes occurred in the study areas during decades. Our results pointed out that Hyalomma spp. abundance in hot season was higher than cold season. This observation may be due to other or a combination of factors: greater tick activi- ty under higher temperatures in summer, since adult Hyalomma spp. ticks are highly motile in seeking out vertebrate hosts (questing); hu- man hosts are more likely to spend time out- doors during summer (longer daylight hours); increased agriculture and natural grazing for livestock during summer months leading to increased opportunity for bringing humans into contact with ticks. These factors may explain the higher incidence of the disease in the summer- time in the country as stated by other research- ers (12). These authors analyzed monthly CCHF passive data for 15 years (from 2000 to 2014) at the county level and showed that almost 70 % cases occurred in hot seasons (May–Sep) peaked in June with more than 180 patients out http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 279 http://jad.tums.ac.ir Published Online: September 30, 2019 of 1027 cases (17.5%). Our results also pointed out that temperature influences generally on Hyalomma spp. tick abundance where this impact was significant for H. dromedarii and H. anatolicum, the two most prevalent species in the study areas. In- creasing tick vector populations directly in- crease the risk of disease transmission to human and hence tick abundance followed by rising temperature can be considered as an important predictor for CCHF incidence. This finding is in agreement with the epidemiological evidence in- dicating a significant positive correlation with CCHF disease and temperature in Iran (12, 13, 37) and other countries such as Turkey, Bulgaria (38, 39). In the present study, we found a positive but not or weakly significant association between tick abundance and the annual mean relative humidity. This weather variable had significant positive impact on the incidence of CCHF in Iran (13, 40). Our results also indicated that average an- nual precipitations affect negatively tick abun- dances of the two most prevalent species H. dromedarii and H. anatolicum in the study areas. These variables had reverse impact on the in- cidence of CCHF in Iran (14, 37, 40) and Sen- egal (41) Higher rainfall provide unfavorable conditions for Hyalomma spp. tick activity and development (42). Results of this study also pointed out that climate norm may have impact on Hyalomma genus tick abundance in the study areas of Iran. Although frequencies of each species in various climate types were not significantly different, the most relative prevalence (47%) of the Hy- alomma spp. ticks were observed in hot desert climate (BWh) comprising Yazd and Sistan and Baluchistan in southeastern and south-center of the country. The combination of economic and ecological situation, host availability, and some climate variables including duration of hot sea- son and higher annual average temperature may support Hyalomma genus tick abundance and species diversity in these regions. In Sistan and Baluchistan, livestock is moved across borders and ticks could originate from distant regions. As a matter of fact, thousands of livestock (sheep, goat, and camels) are annually imported from Afghanistan and Pakistan to this province, where adult ticks carried on imported livestock from the neighboring countries is the main introduc- tory route of CCHFV into the province (37, 43). In addition to imported livestock, migratory birds can also play an important role in Hyalomma spp. tick abundance and species diversity in these regions. We should consider spread of CCHFV by ticks attached to migratory birds flying thou- sands of kilometers during their annual migra- tion from northern to southern areas to the prov- ince. The ecosystem of Oman Sea in Chabahar and Konarak coast in south and Hamoun˗e Pu- zak and Helmand river in northeast of Sistan and Baluchistan, are habitats of unique species of waterfowl and shore birds as well as migratory birds (44, 45). The infected nymphs of the ge- nus Hyalomma carried on migratory birds will molt on arrival into adults and then potentially infect their mammal hosts with CCHFV (46-49). However, the role of migratory birds carrying infected ticks has not been studied as a cause for increased CCHF in Iran particularly in southeast corner of Iran. It is suggested to investigate the role of the migratory birds in spreading CCHFV through attached ticks. The socioeconomic, en- vironmental, and ecological features of Sistan and Baluchistan caused the province is being the most CCHF endemic region of the country since 2000 (4, 12, 20). We have not found CCHF virus infection in the tick specimens collected in this study. One possible explanation for lack of CCHF infection is that although the tick specimens were collect- ed from the animals bred on nomadic grazing system, they are usually bred intensively, and grazing is mostly limited with the field around the stables. Therefore, there are limited interac- tions with wildlife and their ticks, which lead to a lower tick density and diversity. Therefore, it is necessary to test effect of real nomadic graz- ing on the tick density and diversity in the coun- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 268–283 N Choubdaret al.: Effect of Meteorological … 280 http://jad.tums.ac.ir Published Online: September 30, 2019 try. Rate of animal infection to the virus was much lower (4.5%) in the animals feeding in the stable grazing system than the ones feeding in the nomadic system (30%) (17). This situation may alter abundance and community of tick pop- ulation as well. Other possible explanation could be the lower age of animals we tested, where it was shown that rate of infection increased in older animals from 4.5% to 23.7% (17). How- ever, the epidemiology of CCHF virus in Iran reviewed and Rhipicephalus sanguineus, H. mar- ginatum, H. anatolicum, H. asiaticum and H. dromedarii were the most frequent species which were positive for CCHF virus (50). However, the infection rate was highly variable and ranged 0–16.7% for the tested tick species (48, 50, 51). In this study we had focused on Hyalomma spe- cies and did not test the virus infection in other tick species such as Rhipicephalus and Haema- physalis which also play a role in the cycle of transmission of the disease in nature in the wild cycle and in connection with humans in the do- mestic cycle and should be considered in CCHF epidemiological studies (48, 50, 51). Further studies should consider other fac- tors influencing tick life cycle and abundance over long time, including microhabitats, nomadic (natural) system, and host density, three poten- tially influent factors that were not assessed in the present study. Our study was performed in one year, but these climate factors should be assessed for several years. 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