Molecular Detection of Theileria annulata among Dairy Cattle and Vector Ticks in the Herat Area, Afghanistan

Introduction

Theileriosis is an important tick-borne protozoan disease associated with Theileria species (spp) among cattle and buffalo in the world. Thus far, nine species of Theileria have been recognized in dairy cattle ( Perston, 2001 ), four of which, including Theileria parva, Theileria mutans, Theileria taurotragi, and Theileria velifera, are geographically distributed in Sub-Saharan Africa, and the other ones, including Theileria annulata, Theileria orientalis, Theileria orientalis.sergenti and Theileria buffeli, are found in Asia, Southern Europe, Northern Africa, North America, and Australia. Among Theileria spp, T. parva and T. annulata are considered highly pathogenic among cattle, while other spp are non-pathogenic or mildly pathogenic ( Uilenberg, 1995 ). Theileria annulata is the causative of tropical theileriosis in cattle in Asia and Hyalomma spp are known as vectors of T. annulata. ( Perston, 2001 ). Afghanistan has a subarctic mountainous climate with dry and cold winters, except for the lowlands, which have arid and semiarid climates. Cattle and small ruminants are important agricultural animals in Afghanistan. This country still imports dairy products despite there are adequate cattle and dairy farmers to produce the amount needed in this country. The increased rates of dairy products were accomplished through the control of infectious diseases (The Food and Agriculture Organization ( FAO, 2018 ). Among infectious diseases, Transboundary Animal Diseases and Tick and tick-borne diseases have the most significant negative impacts on animal production and national economies related to dairy cattle in Afghanistan ( FAO, 2018 ). Although, this country is located in endemic areas and tropical theileriosis has been reported in dairy cattle ( Bulman et al., 1979 ), the epidemiological aspect of bovine theileriosis is poorly understood. This study aimed to identify T. annulata and vector ticks in dairy cattle by using microscopic examination and Polymerase chain reaction (PCR) in the Herat area, Afghanistan.

Material and Methods

Field Study Area. The study was carried out in the Herat area from 2015-16. The Herat area is located next to the western border of Afghanistan (34°13'N, 62°13'E) with a local steppe climate. The average annual temperature and rainfall in this region are reported as 16.1°C and 265 mm, respectively (http://www.herat.climatemps.com/).

Blood Sample Collection. The population of this study consisted of 100 dairy cattle randomly selected from different parts of the Herat area during the seasonal tick activity. Every dairy cattle was clinically examined and data, including age and breed, were recorded. Blood smears were taken from the capillary vein of the ear. The whole body of the infected animal was inspected for the presence of ticks, which were then removed and kept in a labeled flask. Furthermore, the blood samples of cattle were collected by syringe from the jugular vein and stored in lavender-top tubes (EDTA). The collected samples were transferred to the parasitology laboratory in cold conditions (4°C).

Blood Smears Examination. The smears were fixed in methanol and stained in 10% Giemsa solution in phosphate-buffered saline (PBS) pH 7.2. The slides were examined with an oil immersion lens at a total magnification of 1,000x. The parasitemia of Theileria spp infection was determined by counting parasites in 100 microscopic fields in the blood smears.

Tick Examination. The ticks were counted and underwent speciation using the identification keys ( Estrada-Pena et al., 2004 ). The ticks were divided into pools with five ticks according to tick species. Subsequently, their salivary glands of each tick in the pools were dissected out in 0.85% saline solution under a stereomicroscope. The salivary gland samples were kept at -20°C until they were used for DNA extraction and PCR.

DNA Extraction and PCR. Total DNA was extracted from EDTA blood and tick samples using a DNA isolation kit (Molecular Biological System Transfer, Tehran, Iran), followed by a semi-nested PCR being performed according to the described methods ( d'Oliveira et al., 1995 ). Briefly, in amplification of PCR, two oligonucleotide PCR primers, namely forward primer (N 516): 5′- GTAACCTTTAAAAACGT -3′, and the reverse (p2): 5′- GTTACGAACATGGGTT -3', were used to detect T. annulata. Amplification was conducted in 20 μl reaction volumes (Accupower PCR premix kit, Bioneer®, South Korea) with a final concentration of each dNTP of 250 μM in 10 mM Tris-HCl pH 9.0, 30 mM KCl and 1.5 mM MgCl2, 1U Taq DNA polymerase, and 10 pmol of each PCR primer (Takapouzist Co., Iran). Subsequently, 1 μl of DNA template was added to each reaction and the remaining 20 μl reaction volume was filled with sterile distilled water. The reactions were subjected to the following cycling conditions using a Bio-Rad thermocycler: 95°C for 10 min, 30 cycles with denaturing at 94°C for 1 min, primer annealing at 55°C for 1 min, and extension step at 72°C for 1 min, followed by a final extension at 72°C for 10 min. The products were then chilled to 4°C. The PCR products were electrophoresed in a 1.7% agarose gel with Tris/Borate/EDTA buffer and visualized using ethidium bromide and ultraviolet-eliminator. A visible band at 721 bp for T .annulata was produced in the PCR. The positive controls were prepared from the blood of infected cattle according to a previously conducted study ( Khodabandeh and Razmi, 2015 ), and the blood of healthy cattle was considered a negative control for each PCR amplification.

Statistical Analysis. Dairy cattle were grouped by age and breed to determine whether these factors were associated with theileriosis by the Chi-square test. Statistical comparisons were carried out using SPSS software (version, 21). P-values of <0.05 were considered significant.

Results

Theileria spp. infection was microscopically detected in 22% (37) of blood smears. The parasitemia of Theieleria spp infection was estimated at 0.001% to 1% among infected dairy cattle. Based on the results of PCR, 74% (74) of blood samples were positive for T. annulata (Figure1). The frequency of Theileria infection was significant in dairy cattle by age (P<0.05) and not significant by breed (P>0.05) (Table 1).

Figure 1. Polymerase chain reaction amplification products of Theileria annulata in blood samples Lane M = molecular weight marker (between 50 and 1,000 bp); Lane 1= positive control; Lane 2 = negative control; lanes 3, 4, 5= positive samples

In this study, 349 ixodid ticks were collected from dairy cattle of the Herat area. The most common tick species was found to be Hyalomma anatolicum (73.9%), followed by H. excavatum (22.3%), H. asiaticum (1.1%), H. marginatum (1.7%), H. rufipes (0.85%), and Hyalomma nymph spp (12%) (Table 2). Among a total of 70 tick pools, T. annulata DNA was detected in 7 (7.4%) pools, which belonged to the salivary glands of H. anatolicum (Table 2). All infected ticks were separately collected from seven infected dairy cattle.

Discussion

Based on the microscopic examination of blood smears, Theileria spp infection was observed in 22% of blood smears of dairy cattle. According to the results of a recent study, the prevalence of Theileria infection has been microscopically reported between 4% and 37% in cattle in the Herat area ( Tookhy et al., 2018 ).Similar studies were conducted in the neighboring countries of Afghanistan reporting the frequency of T. annulata infection from 4% to 20 % in Iran ( Razmi et al., 2009 ; Khodabandeh and Razmi, 2015 ; Arjmand Yamchi and Tavassoli, 2016 ; Majidiani et al., 2016 ), from 3% to 8% in Pakistan ( Shahnawaz et al., 2011 ; Khattak et al., 2012 ), from 8% to 16% in India ( Nair et al., 2011 ; Tuli et al., 2015 ), and from 7% to 17% in China ( Guo et al., 2018 ). The highest frequency of Theileria infection was observed in less than 1-year-old group animals in this study. This result was in line with those of other studies finding a high frequency of T. annulata infection among calves, compared to adult cattle ( Razmi et al., 2009 ; Shahnawaz et al., 2011 ). Nevertheless, such finding was inconsistent with those of other studies indicating that the frequency of T. annulata infection was significantly higher among cattle aging more than 1 year old ( Khattak et al., 2012 ). In the present study, significant differences were observed between the frequencies of Theileria infection among different cattle breeds. In contrast, the results of a study showed a high prevalence of T. annulata in Friesian cattle, compared to native breeds ( Rizk et al., 2017 ). They concluded that the reason for this result may be attributed to the susceptibility of the foreign breed to Theileria infection. In the present study, T. annulata was detected in 74% of dairy cattle by PCR. Our result was in agreement with those of a similar study that showed Theileria infection was not detectable in many carrier animals due to the low diagnosis of parasitemia by microscopy ( Noaman, 2014 ). The frequency of T. annulata infection in this study was higher than that in other similar molecular studies reported in neighbored countries, such as Iran ( Majidiani et al., 2016 ), Pakistan ( Hassan et al., 2018 ), India ( George et al., 2015 ; Tuli et al., 2015 ), and China ( Guo et al., 2018 ). The frequency difference of Theileria infection may be related to climatic conditions and control measures against bovine theileriosis in each country.

In the present study, five Hyalomma species were found among the cattle, among which H. anatolicum and H. rufipes exhibited the highest and lowest frequencies of infestation. In a previous study, H. anatolicum was reported as a common tick among other Hyalomma spp in Afghanistan ( Kaiser and Hoogstraal, 1963 ). Hyalomma anatolicum is adapted to the Mediterranean and steppe climates of North Africa. Although large and small ruminants and the Equidae family can be hosts for this tick, a heavy infestation is found among cattle ( Estrada-Pena et al., 2004 ). Hyalomma anatolicum was also reported as the dominant tick spp infesting cattle in neighboring countries of Afghanistan ( Rasulov, 2007 ; Nabian et al., 2009 ; Noaman et al., 2017 ; Rehman et al., 2017 ; Biglari et al., 2018 ). In this study, T. annulata infection was detected in the salivary glands of H. anatolicum by PCR. The obtained result was predictable since H. anatolicum is known as the main vector of T. annulata in dairy cattle in the world ( Estrada-Pena et al., 2004 ). Based on the findings of molecular studies performed in other countries, where theileriosis is endemic, a high prevalence of Theileria infection was reported in H. anatolicum ( Aktas et al., 2004 ; Tavassoli et al., 2011 ; Khodabandeh and Razmi, 2015 ). To the best of our knowledge, this is the first molecular study conducted on Theileria infection in dairy cattle and ixodid tick in Afghanistan. Our results showed that T. annulata and H. anatolicum were important agents and vector ticks for tropical theileriosis in dairy cattle in the Herat area.

Authors' Contribution

Gh. R. was the supervisor of the project and analyzed the data and was a major contributor in writing the manuscript. M. S. A. collected samples and performed all experiments. S. Y. helped to identify tick species and examine the PCR. All authors read and approved the final manuscript.

Ethics

Study protocols and methodologies were revised and approved by the Ethical Committee of the Ferdowsi University of Mashhad.

Grant Support

This study was supported by the grant No: 9423605011 from the vice president Research and Technology of Ferdowsi University of Mashhad.

References

1. ReferencesAktas M, Dumanli N, Angin M. Cattle infestation by Hyalomma ticks and prevalence of Theileria in Hyalomma species in the east of Turkey. Vet Parasitol. 2004; 119(1):1-8.
2. Arjmand Yamchi J, Tavassoli M. Survey on infection rate, vectors and molecular identification of Theileria annulata in cattle from North West, Iran. J Parasit Dis. 2016; 40(3):1071-6.
3. Biglari P, Bakhshi H, Chinikar S, Belqeiszadeh H, Ghaffari M, Javaherizadeh S, Faghihi F, Telmadarraiy Z.  Hyalomma anatolicum as the Main Infesting Tick in an Important Livestock Rearing Region, Central Area of Iran. Iran J Public Health. 2018; 47(5):742-749.
4. Bulman GM, Arzo GM, Nassimi MN. An outbreak of tropical theileriosis in cattle in Afghanistan. Trop Anim Health Prod. 1979; 11(1):17-20.
5. d'Oliveira C, van der Weide M, Habela MA, Jacquiet P, Jongejan F. Detection of Theileria annulata in blood samples of carrier cattle by PCR. J Clin Microbiol. 1995; 33(10):2665-9.
6. Estrada- Pena A, Bouattourm A, Camicas J L, Walker A R. Ticks of domestic animals in Mediterranean region, a guide to identification of specie Bioscience Reports, UK; 2004.
7. FAO. 15 Years in Afghanistan a special report: 2003-2018. Rome; 2018.
8. George N, Bhandari V, Reddy DP, Sharma P. Molecular and Phylogenetic analysis revealed new genotypes of Theileria annulata parasites from India. Parasit Vectors. 2015; 8:468.
9. Guo H, Yin C, Galon EM, Du J, Gao Y, Adjou Moumouni PF, Liu M, Efstratiou A, Lee SH, Li J, Ringo AE, Wang G, Li Y, Tumwebaze MA, Xuan X. Molecular survey and characterization of Theileria annulata and Ehrlichia ruminantium in cattle from Northwest China. Parasitol Int. 2018; 67(6):679-683.
10. Hassan MA, Liu J, Rashid M, Iqbal N, Guan G, Yin H, Luo J. Molecular survey of piroplasm species from selected areas of China and Pakistan. Parasit Vectors. 2018; 11(1):457.
11. Kaiser M N, Hoogstraal H. The Hyalomma ticks (Ixodoidea, Ixodidade) of Afghanistan. J Parasitol. 1963; 49:130-9.
12. Khattak RM, Rabib M, Khan Z, Ishaq M, Hameed H, Taqddus A, Faryal M, Durranis S, Gillani QU, Allahyar R, Shaikh RS, Khan MA, Ali M, Iqbal F. A comparison of two different techniques for the detection of blood parasite, Theileria annulata, in cattle from two districts in Khyber Pukhtoon Khwa Province (Pakistan). Parasite. 2012; 19(1):91-5.
13. Khodabandeh S, Razmi G. Molecular detection of Theileria species and its vectors in cattles in Yazd area by semi-nested PCR method. J Vet Res. 2015; 70: 249-253.
14. Majidiani H, Nabavi R, Ganjali M, Saadati D. Detection of Theileria annulata carriers in Holstein-Friesian (Bos taurus taurus) and Sistani (Bos taurus indicus) cattle breeds by polymerase chain reaction in Sistan region, Iran. J Parasit Dis. 2016; 40(4):1184-1188.
15. Nabian S, Rahbari S, Changizi A, Shayan P. The distribution of Hyalomma spp. ticks from domestic ruminants in Iran. Med Vet Entomol. 2009; 23(3):281-3.
16. Nair AS, Ravindran R, Lakshmanan B, Kumar SS, Tresamol PV, Saseendranath MR, Senthilvel K, Rao JR, Tewari AK, Ghosh S. Haemoprotozoa of cattle in Northern Kerala, India. Trop Biomed. 2011; 28(1):68-75.
17. Noaman V. Comparison of molecular and microscopic technique for detection of Theileria spp. in carrier cattle. J Parasit Dis. 2014; 38(1):64-7.
18. Noaman V, Abdigoudarzi M, Nabinejad A R. Abundance, diversity and seasonal dynamics of hard ticks infesting cattle in Isfahan province, central Iran. Arch Razi Inst. 2017; 72: 15-21.
19. Perston P M. Theileriosis In: M.W.Service (Ed.), The Encyclopedia of arthropod- transmitted infections Infections of Man and Domesticated Animals, CAB International, UK; 2001.
20. Rasulov I. Ticks status in Central Asia with a special emphasis on Uzbekistan. Parasitol Res. 2007; 101 Suppl 2:S183-6.
21. Razmi G, Barati F, Aslani M R. Prevalence of Theileria annulata in dairy cattle in Mashhad area, Iran. J Vet Parasitol. 2009; 23: 81-83.
22. Rehman A, Nijhof AM, Sauter-Louis C, Schauer B, Staubach C, Conraths FJ. Distribution of ticks infesting ruminants and risk factors associated with high tick prevalence in livestock farms in the semi-arid and arid agro-ecological zones of Pakistan. Parasit Vectors. 2017; 10(1):190.
23. Rizk MA, Salama A, El-Sayed SA, Elsify A, El-Ashkar M, Ibrahim H, Youssef M, El-Khodery S. Animal level risk factors associated with Babesia and Theileria infections in cattle in Egypt. Acta Parasitol. 2017; 62(4):796-804.
24. Shahnawaz S, Ali M, Aslam MA, Fatima R, Chaudhry ZI, Hassan MU, Ali M, Iqbal F. A study on the prevalence of a tick-transmitted pathogen, Theileria annulata, and hematological profile of cattle from Southern Punjab (Pakistan). Parasitol Res. 2011; 109(4):1155-60.
25. Tavassoli M, Tabatabaei M, Nejad B, Tabatabaei M, Najafabadi A, Pourseyed S. Detection of Theileria annulata by the PCR-RFLP in ticks (Acari, Ixodidae) collected from cattle in West and North-West Iran. Acta Parasitologica. 2011; 56: 8-13.
26. Tookhy N A, Fazly M J, Shakhes S, Mohmand N A. Prevalence Study of Bovine Theileriosis in Herat Province, Afghanistan. J Agric Vet Sci. 2018; 11: 7-9.
27. Tuli A, Singla LD, Sharma A, Bal MS, Filia G, Kaur P. Molecular epidemiology, risk factors and hematochemical alterations induced by Theileria annulata in bovines of Punjab (India). Acta Parasitol. 2015; 60(3):378-90.
28. Uilenberg G. International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health. Vet Parasitol. 1995; 57(1-3):19-41.