Open Veterinary Journal, (2026), Vol. 16(4): 2203-2211

Research Article

10.5455/OVJ.2026.v16.i4.22

Molecular and serological survey on Theileria equi and Babesia caballi infecting Arabian horses in the northern provinces of Syria

Mohamed Abdaljwad^1*, Mohammad N. S. Al-Sabi², Turke Shawaf³, Ahmed Al Mokahwi³, Walid Alfares⁴ and Ayham Abdalkader⁵

¹Department of Microbiology, College of Veterinary Medicine, Idlib University, Idlib, Syria

²Department of Basic Medical Veterinary Sciences, Jordan University of Science & Technology, Irbid, Jordan

³Department of Clinical Sciences, College of Veterinary Medicine, King Faisal University, Al-Ahsa, Saudi Arabia

⁴Department of Field Crops, Faculty of Agricultural Engineering, Idlib University, Idlib, Syria

⁵Department of Nutrition, College of Veterinary Medicine, Idlib University, Idlib, Syria

*Corresponding Author: Mohamed E. Abdaljwad. Department of Microbiology, College of Veterinary Medicine, Idlib University, Idlib, Syria. Email: mohamed_abdaljwad [at] idlib.edu.sy

Submitted: 19/10/2025 Revised: 22/02/2026 Accepted: 12/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Equine babesiosis is one of the prevalent diseases in horses worldwide, but its presence in Syria generally and in the northern region specifically is not known. In Syria, the civil war has severely disrupted veterinary services, hence disease control measures, creating a fertile ground for the spread of tick-borne diseases.

Aim: This study aimed to conduct a comprehensive molecular and serological survey of Theileria. equi and Babesia. caballi in Arabian horses from the northern provinces of Syria, regions heavily impacted by the war.

Methods: A total of 110 Arabian horses, aged between 2 and 10 years, were included in the study. Each horse underwent a thorough clinical examination and blood analysis. The blood sample was taken from each animal for hematological and immunological analysis, including competitive enzyme-linked immunosorbent assay (cELISA) and polymerase chain reaction (PCR).

Results: Clinical signs of piroplasmosis, such as fever, jaundice, anorexia, and lethargy, were observed in only a small fraction of the horses (4/110). Hematological analysis of these symptomatic horses revealed a significant decrease in lymphocyte, hematocrit, and platelet counts compared to their healthy counterparts. The cELISA detected antibodies against T. equi in 47% (52/110) of the horses, while no antibodies for B. caballi were detected. In the T. equi-positive group, a significant increase in eosinophil percentage and a decrease in red blood cell distribution width were noted. Molecular diagnosis using a nested PCR approach targeting the EMA-1 gene completes the presence of T. equi Deoxyribonucleic Acid (DNA) in 9 out of 10 samples that were initially positive in a screening PCR, as evidenced by the expected specific amplicon at 274 bp. In contrast, no B. caballi DNA was detected in any examined sample, as no specific 566 bp fragment corresponding to the RAP-1 gene was observed. The PCR-positive horses also showed a significant increase in eosinophil percentage and a decrease in platelet counts.

Conclusion: The current results confirm the widespread exposure of horses in Northern Syria to T. equi but not B. caballi, which should be taken into account when planning effective control measures in the post-war time.

Keywords: ELISA, Horse, PCR, Piroplasmosis, Syria.

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Introduction

The horse industry is a large and important part of Syrian economy, with diversity in terms of horse uses, including agriculture, business, sport, entertainment, and recreation (Almarzook et al., 2017). The Syrian civil war (2011–2024) has severely impacted horse populations, threatening their preservation due to a lack of secure health management in general (AlFraj, 2024). Conflicts clearly hinder tick control programs and disease surveillance, increasing equine exposure to vectors and facilitating pathogen spread through unregulated animal movements (Dzemo et al., 2024; Jamra et al., 2024). Consequently, tick-borne diseases such as Piroplasmosis are now considered one of the most prevalent diseases in the country with marked economic losses (Alanazi et al., 2018; Ghafar and Amer, 2019). While Syria-specific data on equine piroplasmosis (EP) remain limited, regional epidemiological trends and the impact of socio-political disruptions assist in its spread locally (Mohammed, 2021). Equine piroplasmosis is a significant tick-borne disease in Syria's neighboring countries. Studies indicate its prevalence in Jordan (Abutarbush et al., 2012) and Turkey (Karatepe et al., 2009; Guven et al., 2017), highlighting its regional importance. EP is an infectious tick-borne disease caused by Theileria equi (formerly Babesia equi) and Babesia caballi in most tropical, subtropical, and some temperate zones of the world (Leblond, 2019; Onyiche et al., 2020). EP causes significant economic losses in the equine industry globally (Tirosh-Levy et al., 2020). Clinical signs are not specific, making the diagnosis of babesiosis challenging (Wise et al., 2013). Presently, diagnosis can be obtained by microscopic examination, indirect immunofluorescent antibody test, enzyme-linked immunoassays, Enzyme-Linked Immunosorbent Assay (ELISA), and polymerase chain reaction (PCR) (Camino et al., 2020; Dorrego et al., 2023). Hematological parameters, such as anemia, thrombocytopenia, leukocytosis, and hyperbilirubinemia, provide supportive evidence of hemolysis, though they lack specificity in identifying the exact causative pathogen (Wise et al., 2013; Osman, 2017). Serological methods, particularly competitive ELISA (cELISA), are gold standards for detecting chronic or carrier states (Terkawi et al., 2012; Bravo-Barriga et al., 2022). cELISA demonstrates high sensitivity for antibodies against B. caballi and T. equi, identifying infections even when parasites are undetectable microscopically (Abutarbush et al., 2012; Facile et al., 2025). However, ELISA cannot distinguish active infections from past exposure, necessitating complementary molecular methods (Terkawi et al., 2012). PCR’s high specificity and sensitivity enable early diagnosis and differentiation between T. equi and B. caballi. Combining blood exams, ELISA, and PCR enhances diagnostic accuracy (Wise et al., 2013; Zeng et al., 2025). For acute cases, blood smears paired with PCR optimize detection, while cELISA and PCR together effectively identify carriers (Abutarbush et al., 2012; Osman, 2017). This multi-method strategy is vital for managing EP’s economic and health impacts, particularly in endemic regions (Abutarbush et al., 2012). The current study was designed to investigate the clinical, hematological, and molecular characterization of the EP in Arabian horses in the northern province of Syria.

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Materials and Methods

Animals and clinical examination

Arabian horses (N=110) were 22 stallion and 88 mares, to the correct age 2–25 years, and were sampled in the period between October 2022 and October 2023 from the northern province of Syria (Edlib and Aleppo provinces) (Fig. 1). A questionnaire to collect data was filled for each horse from the owner, containing the animal's basic data and results of clinical examination. Each horse was examined clinically for the presence of fever, oedema of the ventral abdomen, icterus, mucous membrane color, and hemoglobinuria (Abutarbush et al., 2012). Any other abnormal signs were recorded. After the clinical examination was completed and data were recorded for each horse, a blood sample was taken from each animal for hematological and immunological analysis.

Fig. 1. The map shows the country of Syria, the borders of neighboring countries, and the two regions of Idlib and Aleppo in northwestern Syria, colored green, which are the location of collecting samples.

Blood collection and hematological analysis

Whole blood was collected into plain and EDETA.K3 tubes from the jugular vein for hematological parameters (Jiangsu kangjian medical apparatus Co, Ltd). The plain tubes were transferred to the research lab at college of Veterinary Medicine, Idlib University. The tubes were centrifuged at 3,000 rpm for 7 minutes for separating serum, which stored at −20°C until performing cELISA. The analyzed hematological parameters included total and differential leukocytes count lymphocyte white blood cell (WBC, Lym%, Mon%, Gran% and Eso%), erythrocyte counts red blood cell (RBC), hemoglobin concentration, packed cell volume (PCV), the mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and thrombocytes count, determined using an automated hematological analyzer (Spincell3® Hematology Analyzer, Girona, Spain).

cELISA test

Serum samples were evaluated for the detection of antibodies to T. equi and B. caballi using a commercial cELISA test kit, following the manufacturer’s instructions (Babesia caballi Antibody Test Kit, cELISA, catalogue number: 273–2, and T. equi Antibody Test Kit, cELISA, catalogue number: 274–2; VMRD, Inc., Pullman, USA). The optical density of the controls and samples were measured at 630 nm wavelength using an automatic microplate reader (ChroMate® 4300, Awareness Technology, Palm City, FL, USA) and the percentage of inhibition (%) was calculated from three positive controls in the same plate. Serum samples with ≥ 40% inhibition were considered as positive and samples otherwise negative, according to the manufacturer’s guidelines.

Molecular diagnosis

Only 96 samples were tested for PCR, the rest of the samples (n=14) were not tested due to reduced Deoxyribonucleic Acid (DNA) quality/quantity, which may be due to hemolysis in these samples. Total DNA extraction from Ethylenediaminetetraacetic Acid blood samples was performed using a commercial kit following the manufacturer’s instructions (GeneAll® Exgene^TM DNA Mini Kit, Seoul, Korea). Total genomic DNA samples were initially tested using the primer pair RLB-F2 (5′-GACACAGGGAGGTAGTGACAAG-3′) and RLB-R2 (5′-CTAAGAATTTCACCTCTGACAGT-3′) to amplify the hypervariable V4 region of the 18S rRNA gene of Theileria and Babesia (Gubbels et al., 1999; Nagore et al., 2004). The PCR was carried out in a final reaction volume of 20 μl, containing 10 μl of PCR Master Mix (Hibrigen, Turkey), 5 μl of DNA template, 1 μl of each primer (10 pmol; final concentration 0.5 μM), and nuclease-free water to reach the final volume. PCR amplification was performed under the following cycling conditions: initial denaturation at 95°C for 5 minutes; followed by 35 cycles of 94°C for 30seconds, 51°C for 30seconds, and 72°C for 90seconds; with a final extension at 72°C for 5 minutes. PCR-positive samples identified in the initial assay were subsequently re-examined using a multiplex nested-PCR (n-PCR) to differentiate between Babesia and Theileria infections. In the first n-PCR, two primer pairs were simultaneously included in the same reaction mixture: EMA1-outer-F (5′-ATGATTTCCAAATCCTTTGC-3′) and EMA1-outer-R (5′-TGTCGTCACTTAGTAAAATAGAG-3′), targeting the merozoite antigen 1 gene of T. equi; and RAP1-outer-F (5′-GCGCCCTCTTGCTYGTAG-3′) and RAP1-outer-R (5′-GCTTCATGTACCACTTCTTATAC-3′), targeting the rhoptry-associated protein 1 gene of B. caballi (Montes Cortes et al., 2019). The PCR mixture was prepared in a final volume of 20 μl as described above. Thermal cycling conditions for the first n-PCR were as follows: initial denaturation at 94°C for 5 minutes; followed by 35 cycles of 94°C for 45 seconds, 52°C for 60 seconds, and 72°C for 60 seconds; with a final extension at 72°C for 7 minutes. In the second-round n-PCR, separate 20 μl reaction mixtures were prepared, each targeting one parasite, using 1 μl of the first-round n-PCR product diluted 1:4 with distilled water. For the detection of T. equi, the primer EMA1-outer-F was paired with EMA1-inner-R (5′-TGTCCTTGATGTGCCTGAC-3′), while for B. caballi, the primer RAP1-outer-R was paired with RAP1-inner-F (5′-GTACCAACCGCTGACCCTTC-3′). Each primer was used at a final concentration of 0.5 μM. The thermal cycling conditions for the second-round n-PCR were identical to those applied in the first-round assay. PCR products were analyzed by electrophoresis on agarose gels pre-stained with CYBR safe and visualized using a gel documentation system.

Statistical analysis

Data were analysed using commercial software (GraphPad Prism^TM version 8.0.0 for Windows, Boston, Massachusetts USA). The correlations between the hematological parameters and the presence of piroplasmosis were analyzed using one-way analysis of variance and Bonferroni’s test for multiple comparisons with significance set at p < 0.05. The mean, standard division, range and p values of hematological parameters were compared based on the groups of healthy and affected horses regarding clinical, cELISA, and PCR results.

Ethical approval

All experimental procedures used in this study were approved by the Ethics Committee at Idlib University, Syria (Permission number IU-REC 2022008) and 1.12.2022.

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Results

The clinical examination of the horses revealed that only four of the horses studied showed signs of blood parasites including fever, jaundice, anorexia, and lethargy. All parameters of complete blood count (CBC) were not significantly associated with the clinical signs, except for lymphocyte (LYM%), hematocrit (HTC%) and platelet counts Platelets (PLTs), which were significantly decreased in diseased horses compared to the healthy ones (Table 1). Serological testing using cELISA detected Theileria spp. infection in 52 horses (47%), while no cases of Babesia spp. were identified. According to the cELISA results, there was a significant association between horse infections with Theileria spp. and increased (ESO%), while the (RDWc%) decreased in affected horses showed positive for ELISA results (Table 2). Out of 96 blood samples analyzed by PCR, 10 were found positive using the primer pair RLB-F2/RLB-R2 employed on total extracted DNA. The amplified PCR products were of the expected sizes, nearly 430 bp for Theileria and 390 bp for Babesia (Nagore et al., 2004). However, agarose gel electrophoresis did not allow for species-level differentiation, as band intensity varied among samples, most likely reflecting differences in parasitemia levels (Gubbels et al., 1999). The first-round multiplex n-PCR confirmed the presence of PCR products in the 10 positive samples detected above, with expected amplicon sizes of 828 bp using EMA1-outer-F/EMA1-outer-R (Theileria) and/or 891 bp using RAP1-outer-F/RAP1-outer-R (Babesia), without the ability to discriminate between the two products (Fig. 2). In addition, non-specific PCR products of larger sizes (~3,000–5,500 bp) were observed, which have not been previously reported (Montes Cortes et al., 2019). The second-round n-PCR was expected to yield PCR products of 274 bp using EMA1-outer-F/EMA1-inner-R to detect T. equi, and 566 bp using RAP1-inner-F/RAP1-outer-R to detect B. caballi (Fig. 3). Nine out of the 10 samples, described above, were positive for T. equi, with a faint non-specific band observed at approximately 760 bp. One sample (S-80) exhibited an atypical pattern, with the absence of the expected specific band and the presence of multiple non-specific bands, one of which was highly intense (~340 bp), as well as additional bands of approximately 450, 550, 1,200, and 1,500 bp. Notably, the ~760 bp non-specific band seen in the other nine T. equi-positive samples was absent in S-80, raising questions regarding the parasite species present in this sample.

The correlation between the hematological results and positive PCR showed a significant association with increased ESO% and decreased PLT counts (Table 3).

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Discussion

Conducting the current study in northern Syria provided critical insights into the clinical, hematological, and molecular dynamics of EP in a region profoundly affected by socio-political instability. The clinical manifestation of EP, partially supported by the current results, were fever, severe anemia, icterus (yellowish mucous membranes), malaise, and decreased appetite (Wise et al., 2013; Osman, 2017). Hematological examination of blood is frequently used to support clinical diagnosis of babesiosis, evaluate the disease´s severity, and assess treatment response (Akel and Mobarakai, 2017). The decade-long war has disrupted veterinary infrastructure, tick control programs, and disease surveillance, which subsequently created ideal conditions for EP transmission (AlFraj, 2024).

Table 1. Mean ± SD, range and p value of hematological parameters in apparently healthy and horses showed symptoms of piroplasmosis in Arabian horses.

Table 2. The Correlation between the Mean ± SD, range and p value of hematological parameters in horses that showed positive and negative ELISA results.

Fig. 2. Agarose gel (1%) electrophoresis of first-round n-PCR products using the primer pairs EMA1-outer-F/ EMA1-outer-R and RAP1-outer-F/ RAP1-outer-R for the 10 positive samples. M: 1 kb DNA Ladder.

Previous studies on the prevalence of T. equi and B. caballi in Arabian horses noticed decrease in the PCV, RBC, Hemoglobin (HGB), and PLT, along with eosinophilia in clinical babesiosis cases compared to apparently healthy animals (Ahmadi Afshar et al., 2020; Tirosh-Levy et al., 2020; Dorrego et al., 2023; Mendoza et al., 2024). Our study was a survey and we were not unable to make a valid comparison based on hematological blood parameters. However, few horses showed signs of parasitemia, which some differences in blood values were observed comparing to apparently healthy horses (Table. 1). The decreases in red blood cell count, hemoglobin, and hematocrit in the present study in horses exhibiting clinical signs could be explained by the hemolytic anemia characteristic of EP (Wise et al., 2013). The reduction in RBC parameters reflects the intra-erythrocytic destruction caused by T. equi and B. caballi, which disrupt erythrocyte membranes during replication (Osman, 2017). However, the values of RBC in the present study were decreased but not significant in horses showed clinical signs of EP. Notably, the high mean corpuscular hemoglobin (MCH) and MCV suggest regenerative anemia, a compensatory response to hemolysis, as bone marrow releases larger, immature erythrocytes into circulation (Alanazi et al., 2018). Our results confirmed the presence of anemia in most affected horses according to the clinical signs and CBC parameters, but the type of anemia was not determined in this study. We found slight increase in WBC and neutrophils with decrease lymphocytes which was similarly reported previously (Onyiche et al., 2020). The comparison between control horses and infected horses regarding their Eosinophils (ESO) percentage in the present study revealed higher ESO percentage in infected horses than in control horses due to hemo-protozoans exposure, which was in agreements with previous studies as well (de Waal, 1992; Dorrego et al., 2023). However, the elevated eosinophil count in the horses observed in this study can be attributed to the prevalence of both external and internal parasitic infections resulting from poor management practices, coupled with the spread of allergens in stables and the lack of regular antiparasitic programs (Onyiche et al., 2022; Dorrego et al., 2023).

Fig. 3. Agarose gel (1%) electrophoresis of second-round n-PCR products. (A): PCR using the primer pairs EMA1-outer-F/ EMA1-outer-R to detect Theileria equi with the expected specific band at 274 bp. (B): PCR using RAP1-outer-F/ RAP1-outer-R to dtetect Babesia caballi with the expected specific band at 566 bp (not detected) in the 10 positive samples. M: 100 bp DNA Ladder.

Table 3. The correlation between the mean ± SD, range and p value of hematological parameters in in horses that showed positive and negative PCR results.

The current cELISA results indicated a high prevalence rate (47%) of Theileria spp. infection among the 110 tested horses, with no cases of Babesia infections identified, which provides important results about the endemic nature of EP in northern Syria, consistent with reports from other regions where infections with T. equi were widespread (Tirosh-Levy et al., 2020). However, the high prevalence of infection in this geographical area may be due to the decline in animal health care during the war years, as well as the fact that climatic weather and drought waves during the past years have contributed to the spread of infectious agents (Leblond, 2019; Onyiche et al., 2020). A significant correlation was observed between Theileria spp. infection, as determined by cELISA, and eosinophilia (ESO%), which could associated with parasitic infections, including hemoparasites, as the immune system mounts a response against the invading organisms (Radostits et al., 2007). It is important to assess the eosinophil count in the blood and compare it with antiparasitic treatment programs and the prevalence of piroplasmosis infection (Dorrego et al., 2023). Furthermore, the observed decrease in RDWc% in infected horses, which typically reflects the variability in red blood cell size, could indicate a less uniform population of RBCs, potentially due to specific erythropoietic responses to T. equi (Mahmoud et al., 2016; Onyiche et al., 2022).

The expected PCR-specific product of 566 bp for B. caballi was not detected here, indicating the absence of co-infection with this species in any herein examined horses. Since sample S-80 also lacked the specific products for both B. caballi and T. equi primer sets, the identity of the parasite in that sample remains uncertain and requires further investigation. These results also suggest that the non-specific products observed in both the first- and second-round n-PCR assays predominantly originated from the primer pairs designed for the RAP1 gene. The observation of non-specific bands in some PCR reactions, particularly in sample S-80, suggests potential co-infections with other pathogens or genetic variations within T. equi strains that warrant further investigation (Allsopp et al., 1993). Genetic variations in Theileria could be mediated by changes in parasite antigenicity as a response to host immunity and resistance against the infection (Ko et al., 2008) or a consequence of persistent infection (Kubota et al., 1996). Similar challenges in species differentiation and detection of non-specific amplification have been reported in other molecular studies of equine piroplasmosis (Bashiruddin et al., 1999).

The slight disagreement between the cELISA and the PCR results in the present study highlights the complexities of diagnosing EP. cELISA detected antibodies against B. caballi and T. equi, and correlating that with elevated granulocytes, and eosinophils may indicate prolonged immune reaction to the infections (Facile et al., 2025). Granulocytosis with inflammatory responses to chronic infection, while eosinophilia are though atypical for babesiosis, and may reflect co-infections with helminths or hypersensitivity reactions exacerbated by poor tick control in conflict zones (Jamra et al., 2024). PCR’s specificity for active parasitemia likely explains the increase of eosinophils in PCR-positive horses, since carriers with low-level infections may not exhibit marked hematological shifts (Dorrego et al., 2023), which was in contrast with our study. Similar in agreement between the results ofc ELISA and PCR was previously reported, and it underlines the necessity of combining serological and molecular methods to distinguish active infections from past exposure (Tirosh-Levy et al., 2020). The combined evidence from both serological and molecular methods, correlating with specific hematological changes in our study, strengthens the diagnostic accuracy and provides a more comprehensive understanding of the disease's pathogenesis in the affected horses.

This study suffered from some limitations due to logistic reasons, including the involvement of small sample size, the lack of tick vector data, and precluding analysis of species-specific transmission risks. Additionally, focusing solely on Arabian horses may not reflect the EP dynamics in other breeds living in the same area. Future research should integrate entomological surveys, plotting the distribution map using geographic information system’s analysis, and wider longitudinal sampling to link risk factors with seasonal trends. Investigating co-infections with Anaplasma, Trypanosoma, and/or helminth infections could clarify the observed eosinophilia (Mohammed, 2021).

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Conclusion

In conclusion, this study demonstrates a high seroprevalence of T. equi among Arabian horses in northern Syria, with no evidence of B. caballi infection. The combined use of cELISA and PCR revealed widespread exposure with limited active parasitemia, highlighting the endemic nature of equine piroplasmosis and the urgent need for improved surveillance and tick control programs in conflict-affected regions.

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Acknowledgments

Many thanks to Idlib University for supporting this study. Thanks to the horse stables in northern Syria for helping collect blood samples for the research.

Conflict of interest

The author declares that they have no competing interests.

Funding

None.

Authors’ contributions

MA and AA performed the examination; MA and WA, performed the laboratory work. TS, MA, AA, and MA drafted the manuscript. The final manuscript was approved by all authors.

Data availability

All data were provided in the manuscript.

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