J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 297 http://jad.tums.ac.ir Published Online: September 30, 2019 Original Article Molecular Detection and Differentiation of Theileria lestoquardi, T. ovis and T. annulata in Blood of Goats and Ticks in Kermanshah Province, Iran Mozhgan Rahmani-Varmale; *Mousa Tavassoli; Bijan Esmaeilnejad Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran (Received 29 Nov 2016; accepted 1 Jul 2019) Abstract Background: This study was carried out to identify Theileria spp. infections in goats and ticksin Kermanshah Prov- ince, western Iran from May–Sep 2015. Methods: For differentiation of different Theileria spp. both blood and tick samples were examined by nested PCR- RFLP. Results: Light microscopy of blood smears revealed Theileria spp. infection in 22 (5.5%), while 68 (17%) of blood samples were positive using nested PCR. Out of 68 positive samples, 85.3% (58/68) and 11.7% (8/68) were respec- tively positive for Theileria ovis and T. lestoquardi. Mixed infection was detected in 3% (2/68) cases. Overall, 420 ixodid ticks belong to seven different hard ticks species were collected from goats. Rhipicephalus turanicus 112 (26.7%), R. sanguineus 95 (22.6%), R. bursa, 91(21.7%), Hyalomma anatolicum, 55(13.1%), H. excavatum 27(6.4%), H. marginatum, 22(5.3%) and Dermacentor marginatus, 18(4.2%) were the main tick species infesting goats. The PCR products obtained from ticks were subjected to the differentiation of Theileria species. Respectively, 2 and 8 pools of H. marginatum and R. turanicus salivary glands were infected with T. ovis and T. lestoquardi. In addition, T. annulata and T. lestoquardi infection weredetected in three pools of H. anatolicum. Conclusion: This is the first report of goats and collected ticks to Theileria spp infection in Iran. The results suggest that T. ovis has a higher prevalence than T. lestoquardi. It is also postulated H. marginatum, R. turanicus and H. ana- tolicum might play an important role in the field as a vector of Theileria spp in this area. Keywords: Theileria; Tick; Goat; Nested PCR-RFLP; Iran Introduction Theileriosis is a hemoparasitic disease of domestic and wild ruminants caused by spe- cies of the genus Theileria that transmitted by species of Ixodid ticks. It is one of fatal dis- eases of sheep and goats in tropical and sub- tropical regions, where it causes significant eco- nomic losses as well as reduced production (1). The disease occurs due to at least six species of Theileria spp. including T. ovis, T. separata, T. recondita, T. lestoquardi (T. hirci), and Theil- eria sp. (China 1) and Theileria sp. (China 2) that recently was reported from north of Chi- na (1). Among Theileria parasites, T. lestoquar- di, Theileria sp. (China 1) and Theileria sp. (China 2) are considered highly pathogenic (2). Two species, T. lestoquardi and T. ovis, cause ovine/caprine theileriosis in Iran (3). The presence of these two species was also con- firmed by sequencing analysis and nested PCR-RLFP in Iran (4). Theileria annulata, which is a causal agent of tropical malignant theileriosis in cattle, can also infect sheep (3). Hyalomma species have been implicated in the transmission of T. lestoquardi and T. annulata, namely H. ana- tolicum, H. savignyi, H. aegyptium, and H. im- peltatum (5). Rhipicephalus bursa, R. evretsi, and R. sanguineus are considered as the main field vectors of T. ovis (6). Gold standard for diagnosis of theileriosis is usually based on microscopic examination of Giemsa-stained blood smears, clinical signs and the species of *Corresponding author: Dr Mousa Tavassoli, E-mail: mtavassoli2000@yahoo.com http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 298 http://jad.tums.ac.ir Published Online: September 30, 2019 Theileria specified purely based on the ani- mal host, which the sample was taken. On the other hand, since in most endemic areas, T. lestoquardi and T. annulata infections can oc- cur naturally in both cross host and vectors (4, 7). The exact identification and differentiation between Theileria spp. parasites are important for understanding their epidemiology (8). Achieving to identify Theileria species and vector ticks in goat is not feasible by using conventional Theileria diagnostic methods based on morphological features, especially during mixed infections and low parasitemias (4). Serological assays e.g. indirect immuno- fluorescence and ELISA have been devel- oped for the laboratory diagnosis of theiler- iosis. However, the antibodies cannot always be detected in long-term as well as cross-re- activity of the antibodies against other Theil- eria species has limited the specificity of se- rological tests (7). The use of alternative tech- niques, such as nested PCR-RFLP molecular tool based on 18S rRNA has become neces- sary to provide complementary diagnostic in- formation for low-costly and effectively iden- tification of Theileria or Babesia species in the common host (4, 9-11). To date, earlier investigations about molecular epidemiology of ovine theileriosis in Iran restricted to east- ern geographical areas and none so far has ad- dressed the question of the Theileria infection in goats and vector ticks in Iran (4, 12-15). Considering the importance of goat farming in Iran and paucity of data about species of Theileria, which can infect goats and field- collected Ixodid ticks, we aimed to accurate, identify Theileria species in goats and ticks by using microscopic examination and nested PCR-restriction fragment length polymorphism (RFLP) screening assay. Materials and Methods Field study area The study was carried out during tick active season, from early May through late Sep 2015, in Kermanshah Province (an area of more than 24640km2), located in an important livestock production region and tropical area in western Iran between 33°37'–35°17' N latitudes and 45°20'–48°1' E longitudes and 1420 above sea level. Ecologically, this area is classified as a semi-arid zone. Goat rising is a very important economically occupations in this province. Animals, blood sampling, collection and ex- amination of blood smears and ticks Forty goat flocks were randomly selected by the local veterinary service of Kangavar, Songhor, Kermanshah, Gilan-e Gharb and Qasr-e Shirin areas and 400 peripheral blood samples were collected and then immediate- ly thin blood smears prepared from ear capil- laries were fixed in methanol for 5min and stained in 10% Giemsa solution in phosphate buffer solution (PBS), pH 7.2, for 20min and examined under a magnification of 1000x for the presence of intracellular forms of the par- asite with morphology similar to Theileria spp. The percentage of infected red cell per 100 red blood cells was calculated. For estimating par- asitemia, 100 microscopic fields containing ap- proximate 1000 red blood cells per field were reviewed and the number of parasites per 100,000 red blood cells was enumerated. Even the presence of a single piroplasm was con- sidered as positive. From each flock, at least 8 animals were randomly chosen. During the sampling, data on the variable parameters of the flock and animals were recorded. Based on size, the flocks divided into flocks with 15– 50 goats and flocks with more than 50 goats. Age was the individual-level factor. In each flock, goats were categorized into two age clas- ses (<1yr old versus ≥1yr old). Flocks were divided into two categorized: flock with tick infestation and no tick infestation. At the same location where blood samples were collected, ticks were collected by inspecting the whole body of each goat for the presence of ticks, mainly on their ears, along their nape of neck, http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 299 http://jad.tums.ac.ir Published Online: September 30, 2019 perineum, and udder/orchid, inner thighs, shoulder region and tail base. Ixodid ticks were manually collected from the body surface of examined animals by rubbing alcohol pads surrounding the skin to remove embedded liv- ing ticks (16). The hard ticks were placed into labeled glass vials with 70% ethanol (Merck, Germany) and transferred to the parasitology laboratory. Tick species were identified using taxonomic keys (17, 18). Whole tick were washed once in Roccal (1% benzalkonium chloride) and three times in 70% ethanol and then dried. The ticks’ salivary glands were dis- sected out and placed into 200µl lysis buffer containing 20µl proteinase K, incubated for 10min at 55 °C. After adding of 360µl bind- ing buffer and incubation for 10min at 70 °C, 270µl ethanol (100%) was added to solution, and after vortexing, complete volume was transferred to the MBST column. The MBST column was first centrifuged and then washed twice with 500µl washing buffer. Finally, DNA was eluted from the carrier with 50µl elution buffer and stored at -70 °C until use. They were divided into 55 pools comprising 4–10 tick specimens according to the source species and bioclimatic zones. DNA isolation and nested PCR Total DNA was extracted from blood and tick samples using molecular biological system transfer kit (MBST Iran). Briefly, 200µl blood and tick samples were first lysed in 180μl ly- sis buffer and the proteins were degraded with 20μl proteinase K for 10min at 55 °C. After addition of 360μl bindings’ buffer and incuba- tion for 10min at 70 °C, 270μl ethanol (100 %) was added to the solution and after vor- texing, the complete volume was transferred to the MBST column. The MBST column was first centrifuged and then washed twice with 500μl washing buffer. Finally, DNA was elut- ed from the carrier with elution buffer. Purity of DNA was tested spectrophotometrically at wavelength of 260 and 280nm. Extracted DNA of Theileria was diluted to give a final con- centration of 50ng. A nested PCR was used to detect Theil- eria spp. DNAs. The homologous and varia- ble regions of 18S rRNA gene were ampli- fied by employing two-pair primers. Outer pri- mers for the primary PCR were forward strand primer Thei F1 5'-AAC CTG GTT GAT CCT GCC AG-3' and reverse strand primer Thei R1 5'-AAA CCT TGT TAC GAC TTC TC-3'. The amplicon size of the primary PCR was 1700bp. The nested inner primers were for- ward strand primer Thei F2 5'-TGA TGT TCG TTT YTA CAT GG-3’, and reverse strand pri- mer Thei R2 5'-CTA GGC ATT CCT CGT TCA CG-3'. After the second PCR, the size of nested PCR products for different species ranged from 1417 to 1426bp. The primer's specificity and sensitivity was assessed (11). Primary PCR was performed in a 30μl total reaction volume containing 3μl DNA(45–150 ng), outer primer (20pg), dNTP (250μM of each deoxynucleotide triphosphates), 10X PCR buffer (100mM Tris-HCl (pH 9), 500mM KCl, 1% Triton X-100), Taq polymerase (1.25U, Promega Madison, WI, USA), and MgCl2 (1.5 mM) in an automated thermocycler (Corbet Re- search, Australia) under following program: initial denaturation stage (5min at 94 °C), 25 cycles (denaturation step, 30 sec at 94 °C, an- nealing step, 30sec at 51 °C, extension step, 30sec at 72 °C) and final extension of 5min at 72 °C. Fifty ng of PCR products were used as template in nested PCR. In this stage, the amplification mixture was the same as that used in primary PCR, except that the inner pri- mers were used. Nested PCR conditions were at 94 °C for 2min followed by 30 cycles of 94 °C for 30sec, 52 °C for 30sec 72 °C for 30sec, and final extension, 5min at 72 °C. Then, 10μl aliquots of the PCR products were stained with cyber green solution and electrophoresed through a 1.5% gel. After electrophoresis, am- plified samples were visualized by UV trans- illuminator (BTS-20M, Japan). Expected PCR products for the different Theileria species are shown in Table 1. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 300 http://jad.tums.ac.ir Published Online: September 30, 2019 The extracted DNA from salivary glands of ticks were amplified according to the pro- tocol was previously described for blood sam- ples. The positive DNA from Theileria spp. schizont was kindly provided by Razi Vaccine and Serum Research Institute (Tehran branch, Iran) (Accession numbers: KF429799, MG 208059, KP019206). Sterile water was served as negative control. Restriction enzyme analysis The PCR product was purified using the MBST PCR product purification kit. 10–15µl of purified PCR product were then digested with 1ml (10U) of HpaII and HaeII restriction enzymes in 3µl 10× buffer and 5µl H2O at 37 °C for 2h according to supplier recommen- dations (Jena Bioscience, Jena, Germany), and analyzed using 2% agarose gel(Jena Biosci- ence, Jena, Germany), and analyzed using 2% agarose gel. The restriction analysis patterns for Theileria spp. are listed in Table 2. Statistical analysis The Fisher’s exact test and Mantel-Haenszel test were used to show association between the presence (positive and negative blood samples) of Theileria and the various parameters i.e. flock size, gender and age of animal, tick in- festation of goats and presence of ticks in the flock. McNemar’s chi-square test was used to compare the data of blood smears with blood PCR method. Results were displayed as P-val- ues as well as relative risk values (with 95% confidence intervals). P< 0.05 was accepted to be statistically significant. Results Microscopic examination of thin blood smears showed parasitemia in infected animals ranging from 0.011% to 0.012% piroplasms. Low numbers of highly polymorphous parasites inside the red blood cells were detected in most of the blood smears. Samples with round, oval, ring and anaplasmoid forms were tentatively classified as Theileria spp. Microscopic ex- amination of 400 blood smears obtained from five different areas of Kermanshah Province revealed that 22 (5.5%) goats were positive for piroplasms. The percentage of positive smears for theileriosis was determined be- tween 1.25% and 10% in this region. The highest cases of Theileria piroplasmosis was seen in Qasr Shirin (10%), followed by Gi- lan Gharb (8.75%), Kermanshah (5%), Kan- gavar (2.5%) and Songhor (1.25%) (Table 3). The results of nested PCR assay showed that 17% (68/400) of the goats were infected with Theileria spp. (Table 3, Fig. 1). The high- est rate of infection was observed in Qasr-e Shirin, 26.25% (21/68), that was followed by Gilan Gharb, Kermanshah, Kangavar, and Son- gor, 22.5% (18/68), 17.5% (14/68), 12.5% (10/ 68), and 6.25% (5/68), respectively. RFLP anal- ysis proved presence of T. ovis in majority (85.3%) of Theileria positive samples, while T. lestoquardi and mixed infection was de- tected in 11.7% (8/68) and 3% (2/68) of the samples, respectively. Moreover, enzymatic di- gestion of PCR products of mixed infections revealed that 1 sample was infected with T. lestoquardi and T. ovis, and one sample with T. lestoquardi and T. annulata. All microscop- ically positive samples were confirmed by nest- ed PCR. No Theileria piroplasms were seen on blood smears of samples that were negative in nested PCR. However, there were 46 PCR positive samples, which were negative in mi- croscopic examination (Table 4). The prevalence of Theileria spp. infection in age groups and different gender were not significantly different (Table 5). In this study, 420 ticks of seven different species, R. turanicus 112 (26.7%), R. san- guineus 95 (22.6%), R. bursa 91 (21.7%), H. anatolicum 55(13.1%), H. excavatum 27 (6.4 %), H. marginatum 22 (5.3%) and D. mar- ginatus, 18(4.2%) (Table 6) were isolated and then analyzed using nested PCR in terms of the presence of Theileria spp. infection. There are 45 tick pools, of which, 13 pools http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 301 http://jad.tums.ac.ir Published Online: September 30, 2019 that belong to the salivary glands of H. mar- ginatum, R. turanicusand H. anatolicumwere positive for Theileria spp. infection (Table 7) (Fig. 1). The frequency of Theileria spp. in- fection was higher in flocks with tick infes- tation than no tick infestation (P< 0.05). Fre- quency of Theileria spp. infection was sig- nificantly (P< 0.05) higher in female ticks. Molecular identification results by nested PCR-RFLP screening technique demonstrat- ed that at least three genetically distinct Theil- eria spp. are found in both blood and ticks samples in surveyed-area. Digestion of PCR product sobtained from goats and ticks, which were positive for Theileria spp. using HpaII and HaeII, was shown in Table 2. Out of 68 positive samples, 85.3% (59) and 11.7% (8) were positive for T. ovis and T. lestoquardi, respectively. Mixed infection was detected in 3% (2) cases. Digestion of PCR products ob- tained from animals revealed that one sample with T. ovis and T. lestoquardi, as well as one sample with T. lestoquardi and T. annulata mixed infections were detected (Table 3, Fig. 2). Respectively, 2 and 8 pools of H. mar- ginatumand R. turanicussalivary glands were infected with T. ovisand T. lestoquardi (Ta- ble 7). Table 1. Expected PCR products for different Theileia spp (11) Species The size of nested-PCR product T. lestoquardi 1417bp T. annulata 1420bp T. ovis 1426bp Table 2. The pattern of RFLP of PCR products of different Theileria spp by using of HpaII and HaeII (13) Species HpaII HaeII T. lestoquardi 900-, 278-, 106-, 94- and 39bp No digestion T. annulata 1178-, 106-, 94- and 39bp N digestion T. ovis 856-, 326-, 204-and 39bp 295 and 1131bp Table 3. Results of microscopic examination and nested PCR and RFLP for Theileria spp in different areas of the Kermanshah Province Area MEa Nested PCR T. ovis T. lestoquardi T. annulata Mixed NSb PC % Parasitemia (%) P % p % p % p % p % Kangavar 80 2 2.5 0.011 10 12.5 10 100 0 0 0 0 0 0 Songar 80 1 1.25 0.011 5 6.25 5 100 0 0 0 0 0 0 Kermanshah 80 4 5 0.011 14 17.5 14 100 0 0 0 0 0 0 Gilan-e-gharb 80 7 8.75 0.011 18 22.5 14 77.8 3 16.7 0 0 1 5.5 Qasr-e-shirin 80 8 10 0.012 21 26.25 15 71.4 5 23.9 0 0 1 4.7 Total 400 22 5.5 0.011 68 17 59 85.3 8 11.7 0 0 2 3 aMicroscopic examination, bNumber of sample, cPositive sample http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 302 http://jad.tums.ac.ir Published Online: September 30, 2019 Table 4. Comparison of microscopic examination and nested PCR analysis results in Theileria infection diagnosis in goats Result Test Thin blood smear Nested PCR Positive 22 68 Negative 378 332 Total 400 400 Table 5. Association between the presence (PCR-positive and negative blood samples) of Theileria spp infection in goats and the evaluated parameters (flock size, age, gender and tick burden of animal) Flock Size Age of animal Gender og animal Tick burden of animal 15–50 animals >50 animals <1 year >1 year Male Female No tick Tick infestation Number 210 190 100 300 85 315 305 95 Negative 17.7 (84.2%) 155 (81.5%) 84 (84%) 248 (82.4%) 70 (82.4%) 262 (83.2%) 274 (89.8%) 58 (61%) Positive 33 (15.8%) 35 (18.5%) 16 (16%) 52 (17.4%) 15 (17.6%) 53 (16.8%) 31 (10.2%) 37 (39%) P(F)* P(F)= 0.59 (NS) P(F)= 0.88 (NS) P(F)= 0.40 (NS) P(F)= 0.007 *P (F): Fisher's exact test, P value, Ns: not significant Table 6. Frequency of tick species collected on the body of goats Tick species Tick num- ber (%) Tick pool of salivary glands (No. of male) (No. of female) R. teranicus 112 (26.7%) 8 (84) 3 (28) R. sanguineus 95 (22.6%) 6 (60) 5 (48) R. bursa 91 (21.7%) 5 (54) 4 (41) H. anatoliaum 55 (13.1%) 4 (39) 2 (19) H. excavetum 27 (96.4%) 2 (17) 1 (10) H. marginatum 22 (5.3%) 2 (18) 1 (4) Dermacentormargintus 18 (4.2%) 1 (12) 1 (6) Total 420 28 (284) 17 (156) Table 7. Results of molecular methods for detection Theileria spp in salivary glands of Ixodid ticks Tick species T. ovis T. lestoquardi T. annulata Total (%) No. of male (%) No. of fe- male (%) No. of male (%) No. of female (%) No. of male (%) No. of fe- male (%) Hyalomma marginatum 1 (1) 1 (2) - - - - 2 (3) Rhipicephalus turanicus 1 (1) 3 (21) 1 (3) 3 (20) - - 8 (45) Hyalomma anatolicum - - - 1 (2) 1 (1) 1 (14) 3 (17) Total 2 (2) 4 (23) 1 (3) 4 (22) 1 (1) 1 (14) 13 (65) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 303 http://jad.tums.ac.ir Published Online: September 30, 2019 Fig. 1. Product of Theileria spp using primary PCR and nested inner primers. Lane M: Molecular marker (100bp plus ladder); lane P.Ct1and PCt2: positive controls; lane N.C: negative control, lanes 1–2 and S1–S3: PCR products from infected goats; lane 3and T1–T3: PCR products from infected adult ticks. Fig. 2. RFLP patterns of Theileria spp amplification products. (A) Lane M: molecular marker (100bp plus ladder), lane PC, undigested PCR product, Lane 1: T. lestoquardi HpaII digest, lane 2: T. ovis HpaII digest, lane 3: T. annulata HpaII digest, lane 4: mixed T. lestoquardi, T. ovis and T. annulata with HpaII. (B) Lane M: molecular marker (100bp plus ladder), lane 1: undigested PCR product, lane 2: T. ovis HaeII digest isolated from infected goats; lane 3: T. ovis HaeII digest from infected tick. Discussion The highest infection rate of Theileria in Qasr Shirin and Gilan Gharb may probably be associated to that these regions bordered with Iraq where has been known as a foci of this disease. This finding is the closest to other results (19, 20) that have reported 28.8% and 20.8% of Theileria spp. infections in Iraq, re- spectively. Moreover, several studies performed to detect Theileria infection by the microscopic method in goats in different geographical re- gions of Turkey, western neighbor of Iran (21- 23). 3.8% of sheep and goats blood samples http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 304 http://jad.tums.ac.ir Published Online: September 30, 2019 were Theileria positive in Pakistan by micros- copy observation (24). In Italy, the prevalence of Theileria infection was 1.6% (25). The re- sults concerning the lower incidence of Theil- eria infection in the goat are compatible with the results in this paper. The reason that the goat had a low prevalence of Theileria infec- tion may probably be ascribed to the ability of the goat to graze in unreachable and in- clined areas. In this situation, goat is less ex- posed to the bite of infected-ticks (25). How- ever, in contrast with the results collected in this paper regarding the low prevalence of Theileria infection in goat, the high-frequen- cy rates of Theileria spp. infection in goats were reported from various northern zones of Egypt (85.33%) and Iran (66.7%) (26).This difference is likely due to the differences be- tween surveyed-areas and sampling design as the present work sampled-goats were select- ed randomly. In the present study, the parasitemia was not exceeded by 10.012%. In similar studies, (22, 27, 28) goat infected with Theileria spp. commonly had low parasitemia. It could be attributed to first, in caprine benign theileriosis, this normally gives very low parasitaemia and second, T. lestoquardi is considered as a path- ogenic parasite that causes the death of ani- mals before the appearance of the parasite in lymphoid cells and erythrocytes (29). In Iraq, serological tests employing Indirect Fluorescent Antibody Test (IFAT) were used and the seropositivity rate of T. lestoquardiin goats varied from 7.3% to 3.8% in different regions of the country (30, 31). Moreover, using IFAT, 8.9% infection of goats by Theil- eria were reported in Turkey (22). In the pre- sent study, covering five different regions of Kermanshah Province, the molecular preva- lence ranged from 6.25% to 26.25%. Although the results of the present and previous study cannot be compared due to the different meth- ods were employed, the results clearly indicat- ed that theileriosis was not broadly dispersed in this region. In the present study, the age and sex of the animals did not show any significant associa- tion with Theileria spp. infection. In the en- demic area, due to the relatively high num- ber of infected ticks found and the fact that young and old goats are being continuously exposed to infected ticks, a stage of enzootic stability may have been developed (32). These results are in agreement with the finding of other researchers (33, 34). On the contrary, age and sex influenced the prevalence of piroplas- mosis. These two contradictory findings may be attributed to the difference in the number of examined animals, natural immunity, preg- nancy, drug administration for controlling of hemoprotozoan and recent use of acaricides, which had an effect on tick distribution (15, 35). As expected, the prevalence of Theileria spp. infection in goats detected by nested PCR was significantly (P< 0.05) higher than that ob- tained in microscopic examination of thin blood smears. Therefore, DNA amplification meth- ods had higher efficiency than microscopic ex- amination for detection of Theileria. The re- sults were in agreement with a previous re- port about ovine theileriosis (11, 36). This study also revealed that recovered animals frequent- ly sustain subclinical infections, which are mi- croscopically undetectable. Due to no report on caprine theileriosis in Iran to compare with the present study, there- fore the data was comparable to the previous studies on the prevalence of Theileria spp. in- fection in goats in other countries. In previous similar studies done in other countries in Mid- dle East e.g. Turkey (10, 21, 37) and Syria (38), T. ovis is reported as the most prevalent spe- cies. The same result was obtained from Tu- nisia as well (39). In disagreement with these findings, in Iraq, the most abundant Theileria species identified was T. lestoquardi (19, 30). The dissimilarity amongst results of the two studies may be related to the different diag- nostic methods that they used for microscop- ic examination to assess the frequency of http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 305 http://jad.tums.ac.ir Published Online: September 30, 2019 Theileria spp. infection in goats; obviously molecular methods are most reliable. The low prevalence of T. lestoquardi found in this study is in concern with those report- ed earlier (10, 21). The lower prevalence of T. lestoquardi infection found in goats might be because of H. anatolicum, which is the natural vector of this Theileria species (23). Although this species is widely distributed all over Iran, low infestation 13.1% (55/420) was recorded during this survey, probably be- cause goats may not be a preferable host for H. anatolicum anatolicum (40). Most of the malignant small ruminant theileriosis foci in Iran are situated in places with a mean annu- al temperature (MAT) between 20–25 °C that often are under latitude (L) of 30° in the south part of Iran (41). In this situation, par- asite can infect the goats but failed to reach infective numbers, subsequently, less infec- tion occurs (27). The high prevalence of T. lestoquardi was reported from Qasr Shirin as 23.9%. This result may probably be attribut- ed to that Qasr Shirin county situate in near- ly of most famous endemic foci of malignant small ruminant theileriosis in the south of Iran such as Ahvaz and Ilam with same geo- graphical and climatic features (4). Theileria annulata is transmitted by H. anatolicum in Kermanshah Province, Iran (42), and in most of these areas both cattle and goat are raised close to each other, our findings re- garding infections of T. annulata in goat are reasonable. The presence of antibodies against T. annulatawas demonstrated earlier in infect- ed goats (43). In the present study, lower rates (4.7%) of T. annulata infection in goat indi- cates that the mild to moderate susceptibility of goats to infection with T. annulata (43) and the possibility of goat acting as a reser- voir of T. annulata under field conditions. Regarding ticks infesting goats, Rhipiceph- alus spp. (namely R. turanicus, R. sanguineu, and R. bursa respectively were found to be the most dominating in this study, as has been also recorded in Turkey (22), Iran (44), and Iraq (45). Apart from Rhipicephulus spp., the second most common species of tick infesting goats werefound to be Hyalomma spp. Similarly, the most common tick species of goat in Iraq is Rhipicephalus spp. followed by the Hyalom- ma spp. (45). In opposition to our results, Hy- alomma spp. and Dermacentor spp. existed on- ly in the mountainous parts of the countries among goats (46). In consistentwith the find- ings of other studies (44), D. marginatus ex- hibited the lowest (4.2%) distribution in this work. This is because of a different geograph- ical and metrological structure of the moun- tainous area versus semi-arid climate. In this work, nested PCR incriminated H. marginatum, R. turanicus and H. anatolicum as suspected vectors for transmission of Theil- eria spp. Based on RFLP results, H. anatol- icum was found to be infected by T. annulata and T. lestoquardi. Earlier studies, (5, 25, 42, 47) were corroborated our data. Transmission potential and infectivity of R. turanicus for T. ovis infection is in accord- ance with the other findings (13, 48). How- ever, in contrast to our results, infection of R. bursa, R. sanguineus and H. turanicum were reported by T. ovis (6, 13). A geographical dis- parity between two regions may have result- ed in a better adaptation of one tick’s species to the local conditions, thus replacing with other ones. As shown in the current study, R. turanicus was infected by T. lestoquardi. The obtained results are in conformity with the find- ing of previous investigations, which report- ed that Rhipicephalus spp. maybe has a trans- mission role in small ruminant theileriosis (12). There were no molecular evidence about the role of the R. turanicus as a vector in the trans- mission of T. lestoquardi infection in goat. In Iran, in a similar study (47), T. lestoquardi DNA was isolated from R. turanicus, obtained from unknown infested host. Moreover, R. tura- nicus could be the main vector of B. ovis (16, 49). However, further detailed experimental studies are needed to demonstrate precisely that whether R. turanicus can transmit T. lestoquardi http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 297–309 M Rahmani-Varmale et al.: Molecular Detection and … 306 http://jad.tums.ac.ir Published Online: September 30, 2019 to goats. In harmony with the findings in the pre- sent study, prevalence of theileriosis was higher in herds with tick infestation indicates the pres- ence of a positive correlation between the prev- alence of the disease and the presence of ticks (24, 25). In addition, from the results of this work, the difference between the prevalence of Theileria spp. infection in female ticks was statistically significant (P< 0.05). In agree- mentto the findings in this paper, the preva- lence of Theileria infection has been report- ed to be higher in female ticks. This may be due to female ticks had many more type III acini than male ticks and Theileria parasites were only detected in type III acini (8, 50). These statements show why female ticks have greater Theileria infection prevalence than males. Conclusion Theileria ovis is the dominant causative agent in this region but the evidence of T. lestoquardi and T. annulata infection of goats in few cases were noteworthy, as well. Theil- eria annulata was successfully transmitted from cattle to goats and vice versa by H. a. anatolicum. Taking into account the possibility of oc- currence of T. annulata infection in goats andsuccessful feeding of H. anatolicum on goat even a small portion, goats may be risk factors for theileriosis in cattle in areas where cattle and goats raised together as well as com- mon competent vector coexist in the same area. Finally, H. marginatum, R. turanicus and H. anatolicum may play an important role in transmission of different species of Theileria in this area. This work attempted to determine the prevalence, vectors and molecular identi- fication of caprine theileriosis in goats in lim- ited region of Iran, however further studies with a large number of samples from large-scale of country are recommended. Acknowledgements This study was supported financially by the Research Vice-Chancellorship of Urmia University. 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