J Arthropod-Borne Dis, June 2020, 14(2): 126–133 R Ranjbar et al.: Anaplasma Infection in … 126 http://jad.tums.ac.ir Published Online: June 30, 2020 Original Article Anaplasma Infection in Ticks in Southeastern Region of Iran Reza Ranjbar 1 ; Mehdi Anjomruz 2 ; Ahmad Ali Enayati 3 ; Mehdi Khoobdel 4 ; Atiyeh Rafinejad 2 ; *Javad Rafinejad 2 1 Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran 2 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3 Department of Medical Entomology, School of Public Health and Health Sciences Research Centre, Mazandaran University of Medical Sciences, Sari, Iran 4 Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran (Received 02 Jul 2016; accepted 12 Nov 2016) Abstract Background: Anaplasmosis and Ehrlichiosis are the most important tick-borne diseases. This study was conducted in three cities of Kerman Province in Iran to investigate the circulation of the bacteria in ticks collected from sheep. Methods: Ticks were collected from animals using Srkj forceps and transferred to the Entomology lab in cold chain. After specimen’s identification, they kept at -70 ºC. Tick DNA was extracted using Bioneers DNA extraction kits followed by Nested PCR technique to amplify ribosomal 16S rRNA gene to detect Anaplasma infection in ticks. Results: 472 sheep were examined from which 349 ticks were collected and identified in laboratory using valid keys. Tick specimens belonged to two genera and four species; Hyalomma marginatum (62.47%) was the most frequent and Hylomma asiaticum (5.73%) showed the least abundance. The infestation rate to different tick species was different in three regions of Kerman Province. Observation revealed that 24 specimens (58.3%) were positive for Anaplasma. There is a significant difference between male and female infection rate. However, there is no sig- nificant difference between these variables in each of these cities. Conclusion: This study shows high infection rates to Anaplasma in hard ticks. It is essential for health and veteri- nary authorities and farmers to use appropriate strategies to control ticks to reduce the infestation. Keywords: Anaplasma; Molecular; Ticks; Iran Introduction Ticks are mandatory blood-sucking ecto- parasites of vertebrate’s especially wild ani- mals and only about 10 percent of its spe- cies feed from humans and livestock, partic- ularly sheep and cow. Anaplasmosis usually occurs in tropical and subtropical regions and is reported from Africa, the Middle East, Asia, Australia, USA, South and Central America and southern Europe (1, 2, 3). Ticks directly affect their host by causing irritation, allergic reactions, anemia, weight loss, paralysis and even death. Spotted fever, Rocky Mountains fever, Siberia tick typhus, tularemia, Lyme disease, relapsing fever, Cri- mean-Congo hemorrhagic fever, Anaplasmosis and Ehrlichiosis are among the most important tick-borne diseases transmitted to humans by ticks (4, 5, 6, 7). Ticks are also involved in transmitting certain diseases to livestock the most important of which include swine fever in Asia, viral hemorrhagic fever, anaplasmosis, theileriosis and babesiosis (8, 9, 10). Certain pathogens transmitted by ticks can be spread by contaminated airborne particles, hence, they can be used for military purposes (Bio- terrorism), many diseases having bioterror- ism potential are among diseases transmit- ted by arthropods including plague, yellow *Corresponding author: Dr Javad Rafinejad, E- mail: jrafinejad@tums.ac.ir mailto:jrafinejad@tums.ac.ir J Arthropod-Borne Dis, June 2020, 14(2): 126–133 R Ranjbar et al.: Anaplasma Infection in … 127 http://jad.tums.ac.ir Published Online: June 30, 2020 fever, tularemia, Rift Valley fever, Q fever, viral encephalitis, etc. The first practical use of insects was by the Japanese during World War II using ticks infected with plague agent (Yersinia pestis), Musca domestica with chol- erae bacteria (vibrio cholerae). During World War II, the Russians used lice infected with typhus agent (Rickttesia typhi) against the Germans (11, 12, 13, 14, 15). Development of Genetics and production of arthropods with the ability to transmit life-threatening diseases like Human immunodeficiency virus infec- tion and acquired immune deficiency syn- drome (HIV/AIDS), high potency sting, re- sistance to pesticides and also production of biological agents with more virulence, drug resistance and stability at ambient conditions have greatly contributed to the development of bioterrorism (13) a fact that increases their importance (16). Anaplasmosis, a rickettsia zoonosis, is one such disease, among 30 new ones that have emerged since 1973. The rick- ettsia is a mandatory intracellular parasite of red blood cells. Infected red blood cells lose their functionality in circulatory system and are destroyed. This causes diseases in live- stock and wild ruminants like cow, sheep, goats, and deer although this disease causes clinical symptoms only in cow and giraffes. The disease is transmitted through red blood cells of infected animals. When an animal becomes ill, despite its recovery, it remains as a source of infection forever. Anaplasmo- sis is transmitted to others hosts by ticks that have bitten infected animals. The pathogen will also multiply in infected ticks. Then in- fected ticks transmit the disease to healthy animals. The pathogen can be transmitted to healthy animals by mosquito bites or con- taminated equipment with infected animal blood such as infected syringe or infected equipment of cutting horn as well (17, 18, 19, 20, 21). Anaplasmosis begins with a mild fever, but it gradually becomes more seri- ous. Treatment is usually effective if it is pro- vided early. Tetracycline and Amido propio- nate are two first line drugs used for treat- ment (18, 19, 22, 23). In Iran, the first case of this disease was reported in 2003 from north of the country. Anaplasma bacteria are detected in animal blood samples in prov- inces of Khorasan, Esfahan, Fars, Mazanda- ran and Sistan and Baluchistan (24, 25, 26, 27, 28). Considering the relatively high tick burden of animals and importance of Ana- plasmosis and other tick-borne diseases in livestock as well as humans, determining the species of infecting bacteria and tick vectors and their infection rate are very important. This could be an effective step in diagnosis of diseases transmitted by ticks and devel- opment of strategies to preventing diseases by controlling ticks and patient treatment. Materials and Methods Study area This study is undertaken in three cities of Kerman Province including Kerman, Koh- banan and Sirjan. Kerman is located in the south-east of Iran central plateau, between 53º 26' to 59º 29' east longitude and 25º 55' to 32º north latitude. Encompassing an area of more than 183/285 square kilometers and approximately eleven percent of the coun- try’s area, Kerman is the largest province in Iran located in southeast of the country with a history of human settlement since four mil- lennium BC. Kohbanan is located at 31º.42' 36" North and 56º.28' 75" East in the far northern parts of Kerman Province, south- east of Iran with a population in 2011 of 21721 people. Sirjan is another city of Ker- man Province (29°.45' 55°.67') is located in the West of this province and has a popula- tion in 2011 of about 269000 (Fig. 1). Sample Collection Ticks were separated from body of sheep, goat and cow (ear, groin, tail, back and neck) using Srkj forceps so as not to harm the ticks and animals. They were then put in appro- priately labeled tubes and transferred to the Entomology lab in cold chain. After speci- J Arthropod-Borne Dis, June 2020, 14(2): 126–133 R Ranjbar et al.: Anaplasma Infection in … 128 http://jad.tums.ac.ir Published Online: June 30, 2020 mens identification, selected ticks were kept at -70 ºC in micro tubes for PCR (29, 30). DNA extraction Bioneers DNA extraction Kits (iNtRON Bi- otechnology, Korea) was used to extract DNA according to the instructions of the manu- facturer. Nested PCR technique with primers designed by Rar et al. was used to amplify ribosomal 16S rRNA gene in order to detect Anaplasma infection in ticks (31). The pri- mers are designed based on conserved and highly changeable region V1 of ribosomal gene 16S (16S rRNA) of Anaplasma sp. and Ehrlichia sp. producing DNA fragments of about 500bp (Table 1). Polymerase Chain Re- action (PCR) protocol consisted of 35 cycles of a preheating phase at 94 ºC for 5min, de- naturation at 94 ºC for 1min, annealing at 57 ºC for 1min, extension at 72 ºC for 1min and a final incubation at 72 ºC for 7min. Before finishing the first phase of PCR reaction, mas- ter mix of the second phase is prepared. In the second phase of the nested PCR, different pri- mers are used (Table 1) and also two micro liter of (PCR) product is used as template and annealing was set at 54 ºC. PCR products were kept in a refrigerator before electrophoresis using 1% agarose gel. Results During this study, a total of 472 sheep were examined for tick infestation in three cities of Kerman Province in spring 2015. A total of 349 ticks were collected from 154 ani- mals resulting in a tick burden of 2.26. Col- lected ticks were identified in laboratory us- ing valid diagnosis keys (32, 33). As present- ed in Fig. 2, collected Ixodidae tick specimens belonged to two genera and four species in- cluding Hy. marginatum (62.47%), Hy. ana- tolicum (9.47%), Hy. asiaticum (5.73%), Hy- alomma.sp (7.44%), Rhipicephalus sanguineus (14.33%) and unidentified specimens (0.58 %) (Fig. 2). Among identified species, Hy. margina- tum with 62.47% showed the highest frequen- cy and Hy. asiaticum (5.73%) the lowest (Fig. 2). Totally, from 349 tick specimens, 63 were collected in Kerman, 98 in Sirjan and 188 in Kohbanan. According to the results in Ker- man, Rh. sanguineus with a prevalence of 10.05% showed the highest frequency and Hy. anatolicum, Hy. marginatum and Hy. asi- aticum species with 3.43%, 2.58% and 0.29 % were in the next orders respectively. Hy- alomma marginatum, Hy. anatolicum, Rh. sanguineus and Hy. asiaticum with 20.92, 2.87, 1.72 and 0.85 percent showed the high- est frequency in Sirjan City respectively. Hy- alomma marginatum with 39.97 percent had the highest frequency and Hy. asiaticum, Hy. anatolicum and Rh. sanguineus with 4.59, 3.15 and 2.56 percent showed the highest fre- quency in Kohbanan respectively (Table 2). Fig. 1. Geographical location of Kerman Province, Iran Table 1. List and details of primers used in this study Primer Nucleotide sequences Forward: Ehr1 5′-GAACGAACGCTGGCGGCAAGC-3′ Reverse: Ehr2 Reverse: Ehr3 Reverse: Ehr4 5′-AGTATCCGAGACCAGATAGCCGC-3′ 5´-TGCATAGGAATCTACCTAGTAG-3´ 5´-CTAGGAATTCCGCTATCCTCT-3´ J Arthropod-Borne Dis, June 2020, 14(2): 126–133 R Ranjbar et al.: Anaplasma Infection in … 129 http://jad.tums.ac.ir Published Online: June 30, 2020 Table 2. Species composition in Kerman Province (A=Kohbanan and B=Sirjan and C=Kerman) Genus/Species A B C province (100%) A B C Hy. marginatum 136(38.97%) 73(20.92%) 9(2.58%) 218(62.47%) Hy. anatolicum 11(3.15%) 10(2.87%) 12(3.43%) 33(9.45%) Hy. asiaticum 16(4.59%) 3(0.85%) 1(0.29%) 20(5.73%) Hy. sp 16(4.59%) 4(1.14%) 6(1.71%) 26(7.44%) Rh. sanguineus 9(2.56%) 6(1.72%) 35(10.05%) 50(14.33%) unidentified sp. 0(0%) 2(0.58%) 0(0%) 2(0.58%) Total 188(53.86%) 98(28.08%) 63(18.06%) 349(100%) Table 3. Infection rates of Anaplasmosis in ticks collected from Kerman Province, southern Iran in 2015 City Genus/Species Nymph Female)♀( Male)♂( (100%) (100%) Negative Positive Negative Positive Negative Positive Negative City A Hy. marginatum 0(0%) 0(0%) 6(16.22%) 2(5.4%) 13(35.14%) 2(5.41%) 19(51.35%) 21(56.76%) Hy. mnatoilicum 0(0%) 0(0%) 2(5.4%) 0(0%) 4(10.82%) 0(0%) 6(16.22%) 6(16.22%) Hy. msiaticum 0(0%) 0(0%) 2(5.4%) 0(0%) 4(10.82%) 0(0%) 6(16.22%) 6(16.22%) Hy. Sp 0(0%) 0(0%) 1(2.7%) 0(0%) 0(0%) 0(%) 1(2.7%) 1(2.7%) Rh. manguineus 0(0%) 0(0%) 1(2.7%) 0(0%) 2(5.4%) 0(0%) 3(8.1%) 3(8.1%) Total A 0(0%) 0(0%) 12(32.43%) 2(5.4%) 23(62.17%) 2(5.41%) 35(94.59%) 37(100%) City B Hy. marginatum 0(0%) 2(5.71%) 5(14.29%) 5(14.29%) 10(28.57%) 7(20%) 15(42.86%) 22(62.86%) Hy. anatoilicum 0(0%) 0(0%) 2(5.71%) 0(0%) 4(11.43%) 0(0%) 6(17.14%) 6(17.14%) Hy. asiaticum 0(0%) 0(0%) 0(0%) 0(0%) 2(5.71%) 0(0%) 2(5.71%) 2(5.71%) Hy. Sp 0(0%) 0(0%) 2(5.71%) 0(0%) 0(0%) 0(0%) 2(5.71%) 2(5.71%) Rh. Ssanguineus 0(0%) 0(0%) 1(2.86%) 0(0%) 2(5.71%) 0(0%) 3(8.58%) 3(8.58%) Total B 0(0%) 2(5.71%) 10(28.57%) 5(14.29%) 18(51.42%) 7(20%) 28(80%) 35(100%) City C Hy. marginatum 0(0%) 1(4.17%) 0(0%) 1(4.17%) 3(12.5%) 2(8.33%) 3(12.5%) 5(20.83%) Hy. mnatoilicum 0(0%) 0(0%) 0(0%) 2(8.33%) 3(12.5%) 2(8.33%) 3(12.5%) 5(20.83%) Hy. msiaticum 0(0%) 0(0%) 0(0%) 1(4.17%) 0(0%) 1(4.17%) 0(0%) 1(4.17%) Hy. Sp 1(4.17%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 1(4.17%) 1(4.17%) Rh. manguineus 0(0%) 8(33.33%) 2(8.33%) 1(4.17%) 1(4.17) 9(37.5%) 3(12.5%) 12(50%) Total C 1(4.17%) 9(37.5%) 2(8.33%) 5(20.84%) 7(29.16%) 14(58.33%) 10(41.67%) 24(100%) Total ABC 1(1.05%) 11(11.45%) 24(25%) 12(12.5%) 48(50%) 23(23.95%) 73(76.05%) 96(100%) J Arthropod-Borne Dis, June 2020, 14(2): 126–133 R Ranjbar et al.: Anaplasma Infection in … 130 http://jad.tums.ac.ir Published Online: June 30, 2020 Fig. 2. The infestation rate to different tick species in Kerman Province, southern Iran in 2015 Discussion The results revealed that 23 out of 96 an- alyzed specimens were positives for Ana- plasma; 11.45% Hy. marginatum, 2.08% Hy. anatolicum, 1.04% Hy. asiaticum, 0% Hy. sp, and 9.37% Rh. sanguineus were positives. Nested-PCR is a sensitive molecular technique to determine infection to Anaplasma sp in ticks collected from animals. In 1910, Ana- plasma marginale was described first by Theiler in African cows’ Red Blood Cell (RBC) as a marginal spot (34); afterward, in 1911, he introduced An. centrale, a sub- species of An. marginale which is located in the center of Erythrocytes and has less pa- thogenicity (35). Lesto Kard, in 1924, de- mon-trated An. ovis in sheeps’ RBC. Dona- tien and Lestoquard observed An. Bovis in French cows’ RBC, during an experimental transmission of Theileria through Hyalomma Ticks (36). Dumler et al. (2001), after the modification of Ehrlichia equi, Ehrlichia phgocytophila species plus unknown factor of human Ehrlichia granulocytic, category- zed them under a species named An. Ph- gocytophilum (37). From the amount of 60 cows, 391 sheep, and 385 goats, through a blood smear test observed that 19.37% of cows were infected to An. marginale, and 80.3% of sheep and 38.92% of goats were infected to An. ovis, during a study between 1999 and 2002 in Khorasan province in Iran by Razmi et al. (2005), there was no a sig- nificant difference between age and gender groups regarding the infection rate of Ana- plasma and the amount of Anaplasma bac- teria was 0.005–0.5%, 0.01–3%, 0.01–3% in cows, sheep, and goats, respectively (25). Spitalska et al. in 2005 investigated 100 sam- ples of sheep blood, and 89 ticks using PCR for 16S and 18S rRNA in Shiraz City; they observed that 25% of ticks and 29% of blood samples were positive for Anaplasma sp. and Ehrlichia sp. Also, they found that 59.5% of ticks and 76% of blood samples were infec- ted with Babesia sp. and Theileria sp. (26). A study by N’aman et al. in Isfahan City J Arthropod-Borne Dis, June 2020, 14(2): 126–133 R Ranjbar et al.: Anaplasma Infection in … 131 http://jad.tums.ac.ir Published Online: June 30, 2020 (1386) revealed some Anaplasma-like bodies in 75 blood smears out of 150 cow blood sam- ples using microscopic observation. Also, they observed An. marginale in 50 blood samples using Nested-PCR (27). Another research in Qa’emshahr City in the province of Mazan- daran performed on 98 specimens of Ix. Ri- cinus using PCR on the 16S rRNA gene re- vealed that 5.1% of the ticks were infected with An. phgocytophilum. These results can be a cause for concern for the appearance of human An. granulocyte (AGE) disease in that area (28). In another study in Qa’emshahr using Nes- ted-PCR on 54 Rh. sanguineus, 1 Rh. bursa, 39 Ixodes ricinus, 4 Bo. annulatus, 2 Hae- maphysalis numidiana, and 1 Haemaphysa- lis punctaterevealed that 49.5% of the ticks were positives. At the same time, 65 samples of sheep blood, 90 samples of cow blood, and 4 samples of goat blood were studied showing 43.1%, 22.2%, 25% of sheep, cows, and goats were infected respectively. The re- searchers reported 25% of human samples (40 blood samples) of at risk individuals from different age groups and genders were in- fected (24). Conclusion Results of this study show preliminary data on relatively high infection rates in hard ticks collected from sheep in Kerman Province. Therefore, it is essential that health and vete- rinary authorities and farmers to adopt app- ropriate strategies to control ticks to reduce the infestation and infection rates in livestock and humans. 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