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. 2015 Spring;16(2):202–204.

Molecular detection of equine piroplasms in donkeys (Equus asinus) in North Khorasan province, Iran

V Abedi 1, Gh Razmi 1,*, H Seifi 2, A Naghibi 1
PMCID: PMC4827686  PMID: 27175176

Abstract

Equine piroplasmosis is a tickborne disease of equids with worldwide distribution, caused by Theileria equi and Babesia caballi. The aim of this study was molecular detection of T. equi and B. caballi in donkeys in northeastern Iran and investigate the association between positivity of piroplasm infection and host-related factors. In the present study, Blood samples were collected from 106 apparently healthy donkeys (Equus asinus) in North Khorasan province, Iran. Blood smears were prepared and stained by giemsa method. DNA was extracted from blood and then multiplex-PCR was done for detection of any piroplasms infection. According to the results, four donkeys showed T. equi in blood smears but B. caballi was not found. Also, fifty four donkeys (50.94%) showed T. equi infection using multiplex-PCR. No siginificant difference was observed between the frequency of T. equi infection with host-related factors in donkeys. This is the first report on the molecular detection of eqiune piroplamosis in donkeys in Iran. Also, no significant association was found between the rate of T. equi infected animals.

Key Words: Babesia caballi, Donkey, PCR, Theileria equi

Introduction

Equine piroplasmosis is an intraerythrocytic parasitic disease of equids that is caused by Theileria equi and Babesia caballi (Wise et al., 2013 ▶). Equine piro-plasmosis has world wide distribution and is endemic throughout Asia except Siberia and Japan and infection rates in the Middle East are high (Friedhoff et al., 1990 ▶).

Direct and indirect methods have been used for the diagnosis of equine piroplasmosis. The microscopical examination and molecular methods such as PCR derivatives should be considered as a direct method. The microscopy is suitable in acute cases with clinical signs while PCR is applicable in chronic with low parasitemia. Indirect detection of infection is accomplished by antibody detection via several tests such as complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA) and indircet fluorescent antibody test (IFAT) (Reiter and Weiland, 1989 ▶; Bruning, 1996 ▶).

Among the ixodid ticks, Hyalomma, Rhipicephalus, Haemaphysalis and Dermacentor have been identified as vectors of T. equi and B. caballi (De waal, 1992 ▶). Iatrogenic transmission of the parasites can occur via blood-contaminated instruments and through blood transfusion. Theileria equi are transmitted to offspring during pregnancy via placenta. The infected foal may be born healthy but remain a carrier for life (Allsopp et al., 2007 ▶; Georges et al., 2011 ▶).

Donkey is an important member of equidae family with a population of more than 40 million. Donkey has been used as working domestic animal in rural areas. Equine piroplasmosis affects health status and work performance of donkeys. The anti-babesial drugs can not completetly eliminate infection in donkey and the infected animal becomes carriers without clinical signs (Kumar et al., 2009 ▶).

In Iran, equnie piroplasmosis were reported for the first time in 2000 (Aslani, 2000 ▶; Seifi et al., 2000 ▶); however, it seems that there are no other reports about piroplasm infection in donkey in this country. The goal of this study was molecular detection of the T. equi and B. caballi infection in donkeys and to determine the importance of host-related factors.

Materials and Methods

Field study area

The study was conducted between June and August 2011, in the North Khorasan province. North Khorasan province is level with the southern Caspian sea, and is southern of Turkmenistan, between latitudes 36°37′-38°17′ N and longitudes 55°53′-58°20′ E. The annual rainfall in the province is approximately 250 mm. Also, more than 18000 equids breed in this area (approximately 0.64/km2).

Blood collection

One hundred and six apparently healthy donkeys from 10 villages were randomly selected. Blood samples were collected from the jugular vein and placed into EDTA tubes. In addition, blood smears were prepared for each blood sample. Data of each donkey including age, gender, activity, and any grazing in pastures were recorded. The samples were maintained under cool conditions and immediately transferred to the laboratory. The blood EDTA-tubes were stored at -20°C until the time of molecular examination.

Microscopical examination

The smears were fixed in methanol, stained with 10% giemsa solution in phosphate-buffered saline (PBS), pH = 7.2 and examined with an oil immersion lens at a magnification of ×1000.

To identify Theileria and Babesia species, the full length of the intraerythrocytic mature piroplasm organism was measured using a graded ocular microscope. Parasitemia was assessed by counting the number of infected red blood cells on examination of 50 microscopic fields (approximately 50.000 cells). The number of the infected cells was then expressed as a percentage.

DNA extraction and PCR

DNA was extracted from 100 μL of the blood samples using a commercial kit (Molecular Biological System Transfer (MBST), Tehran, Iran), according to the manual, and kept at -20°C until use. A multiplex-PCR was conducted for the detection of T. equi and B. caballi using the method of Alhassan et al. (2005) ▶. In brief, 20 μL of a mixture containing 1 μL of template DNA, 1 μL (10 pmol) of each of the reverse primers for T. equi (EquiR: 5´-TGC CTT AAA CTT CCT TGC GAT-3´ and for B. caballi (CabR: 5´-CTC GTT CAT GAT TTA GAA TTG C-3´), 2 μL of a common forward primer (UFP: 5´-TCG AAG ACG ATC AGA TAC CGT CG-3´), and 16 μL of distilled water were added to a hotstart PCR mastermix (Accupower PCR PreMix kit, Bioneer, Seoul, South Korea) with a final concentration of 250 μM of each dNTP in 10 mM Tris-HCl, pH = 9.0, 30 mM KCl, 1.5 mM MgCl2, 1 U Taq DNA polymerase. The reactions were subjected to the following cycling conditions using a thermocycler (BioRadTM): 96°C for 10 min, 36 cycles with a denaturing step at 96°C for 1 min, annealing step at 60.5°C for 1 min, and an 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. Five μL of the PCR product was subjected to electrophoresis in a 1.5% agarose gel with the TAE buffer, submerged in an ethidium bromide tank for 15 min, and the distinct bands of T. equi (430 bp) and B. caballi (540 bp) were visualized by UV illumination. The positive controls were preparted from the blood of infected horses (Seifi et al., 2000 ▶), and water was employed as a negative control for each PCR amplification.

Sequencing

Three positive products were selected and sequenced in the facilities of Bioneer Inc. (Seoul, South Korea). Sequences were analyzed by using NCBI BLAST, National Institutes of Health, USA (http://www.ncbi.nlm. nih.gov).

Statistical analysis

The relationship between infection rate and risk factors, age and gender was analyzed by the Chi-square test. Significant association were identified when a p-value of less than 0.05 was observed.

Results

Theileria equi infections were microscopically detected in 4 (3.77%) blood smears with low parasitemia (approximately 0.001%-0.003%). In multiplex-PCR, 54 (50.94%) donkeys had predictive band at approximately 430 bp for T. equi. No significant relationship was observed between the various risk factors (age and gender) and the rate of T. equi infection (Table 1) (P>0.05). Sequenced PCR products were found 99% identical to the T. equi 18S rRNA GenBank reference (EU888902.1) by BLAST analysis. The nucleotide sequences were assembled and edited using CLC bio software and deposited in GenBank (NCBI) under access No. of KJ125440-KJ125442.

Table 1.

Frequency of T. equi PCR positive by host related factors in 106 donkeys

Risk factor Results of PCR
Number Number of positive %
Age
    <1 3 2 66.66
    1-2 11 1 9.09
    3-5 47 23 48.93
    5< 45 28 62.22
Gender
    Male 67 33 49.25
    Female 39 21 53.84
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Discussion

In this study, all donkeys were apparently healthy. The microscopical examination showed T. equi infection in 4 (3.77%) of the donkeys with low parasitemia, while 54 (50.94%) of the donkeys were T. equi-PCR positive. Our results confirmed low sensitivity of microscopy for identification of carrier animals compared to PCR methods (Bose et al., 1995 ▶). A few epidemiological studies on equine piroplasmosis have been done using different methods. The frequency of T. equi infections was detected from 0.5 to 12% of blood smears in donkeys of Ethiopia (Mekibib et al., 2010 ▶; Tefera et al., 2011 ▶; Gizachew et al., 2013 ▶). DNA of T. equi were detected in 31.81% of donkeys in Brazil using PCR method (Machado et al., 2011 ▶). Seropositivity rates of T. equi infection were in 55.7% of donkeys in Ethiopia (Gizachew et al., 2012 ▶), 4%-13.1% in Turkey (Acici et al., 2008 ▶; Balkaya et al., 2010 ▶), 47.2 in Spain (García-Bocanegra et al., 2013 ▶), 9.6% in China (Chahan et al., 2006 ▶) and 73.86% in Brazil (Machado et al., 2011 ▶). These reports indicated that donkeys are important carriers for T. equi infection in different countries. In the present study, B. caballi infection was not detected in the blood samples of donkeys. Although B. caballi infection has lower frequency and geographical distribution than T. equi worldwide (Schein, 1988; Friedhoff et al., 1990 ▶), but lack of B. caballi in blood and mollecular tests in our work was unclear and require further epidemiological studies in Iran.

For analysis of age as a risk factor, animals were divided into four groups and data was analyzed. The association between the rate of T. equi infected animals and different age and gender groups of donkeys was not significant. The management breeding system of donkeys is semiconfined and equids of all ages and with different activity can freely access pasture. Grazing in pasture, as a managenent practice, causes more tick infestation and puts animals at greater risk for occurrence of equine piroplasmosis infection (Shkap et al., 1998 ▶; Moretti et al., 2010 ▶). To the authors knowledge, this is the first reserach of molecular survey of Theileria and Babesia species in donkeys from Iran.

Taken together, the results of this study indicated that T. equi is prevalent among donkeys in North Khorasan province, Iran. No significant association was found between the rate of T. equi infected animals. All the sampled donkeys were asymptomatic, but could act as carriers to facilitate the spread of T. equi.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

We are very grateful to Mr M. Mojaradi and Dr. E. Rajabi for their help with sample collection and Mr H. Eshrati and Mr G. A. Azari for their technical assistance. This study was supported by a Grant VPRTFM-22992.2 from The Vice President for Research and Technology of Ferdowsi University of Mashhad, Iran.

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