Molecular prevalence of Theileria annulata in apparently healthy cattle using nested polymerase chain reaction

Introduction

What fraction of outwardly healthy cattle harbour Theileria annulata without showing any clinical signs? This seemingly simple query carries enormous weight for livestock economies across the Mediterranean basin, the Middle East, and parts of Central Asia, where tropical theileriosis remains a persistent threat [1] . The disease, transmitted mainly by ticks of the genus Hyalomma, triggers fever, anaemia, lymph node enlargement, and, in severe cases, death in naive cattle [2] . While acute outbreaks draw immediate attention, the carrier state-where animals recover clinically yet retain low-level parasitaemia-receives far less scrutiny despite its equally damaging long-term consequences [3] . Carrier animals pose a dual problem. On one hand, they supply a ready blood meal for feeding ticks, maintaining the parasite's life cycle within the herd. On the other, they may suffer from reduced milk yield, slower weight gain, and impaired immune function, translating into hidden economic losses that accumulate over years [4] . Traditional diagnostic approaches such as Giemsa-stained blood smears can detect piroplasms only when parasitaemia exceeds a threshold of roughly 0.01%, a level that many carriers fall well below [5] . Serological tests, including the indirect fluorescent antibody test (IFAT), detect exposure rather than active infection and cannot differentiate current carriers from animals that cleared the parasite long ago [6] . Molecular techniques, particularly the polymerase chain reaction (PCR), have transformed the detection landscape. By amplifying species-specific DNA, PCR achieves sensitivity several orders of magnitude above microscopy [7] . Nested PCR refines this further: a first amplification with outer primers is followed by a second round using inner primers situated within the first product, boosting both sensitivity and specificity [8] . The Tams1 (T. annulata merozoite surface antigen 1) gene has proven a reliable target for nPCR-based detection, producing a characteristic 721-bp amplicon that distinguishes T. annulata from closely related species [9] . Turkey occupies a geographic zone where Hyalomma ticks thrive, and earlier serological surveys have placed the seroprevalence of T. annulata between 15% and 38% depending on the region and season [10] . Yet molecular data on apparently healthy cattle remain limited, with most published work focusing on clinically suspect animals. The central Anatolian plateau, characterised by semi-arid summers and dense cattle populations, presents conditions that favour tick activity from April through October [11] . Despite this, no large-scale nPCR survey has targeted the carrier population across multiple provinces simultaneously. The present research aimed to fill this gap by determining the molecular prevalence of T. annulata in apparently healthy cattle from five provinces-Ankara, Konya, Sivas, Kayseri, and Yozgat-using a Tams1-based nested PCR. Secondary objectives included evaluating the influence of age, sex, and geographical location on carrier rates. The data generated here are intended to support evidence-based tick control strategies and inform vaccination planning in the region [12] .

Materials and Methods

Materials

The research was carried out from March 2022 to February 2023 at the Molecular Parasitology Laboratory, Department of Parasitology, Faculty of Veterinary Medicine, Ankara Institute of Veterinary Biosciences, Turkey. Ethical clearance was granted by the Institutional Animal Ethics Committee (Protocol No. AIVB/IAEC/2022-017, dated 14 January 2022). A cross-sectional sampling design was adopted, covering five provinces of central Turkey: Ankara, Konya, Sivas, Kayseri, and Yozgat. These provinces were chosen because of their large cattle populations and reported tick burdens in prior veterinary records [13] . A total of 384 blood samples were collected from apparently healthy crossbred cattle (Holstein-Friesian × local breeds) presented for routine veterinary checks at government livestock centres. Animals displaying any clinical signs of theileriosis-fever, lymphadenopathy, petechiae-were excluded. Approximately 5 mL of jugular blood was drawn into EDTA-coated vacutainer tubes and transported on ice to the laboratory within 6 hours of collection. Samples were stored at -20 °C until DNA extraction. Animal age ranged from 1 to 9 years, and sex was recorded for each individual.

Reagents and Chemicals

Phenol (Tris-saturated, pH 8.0), chloroform, isoamyl alcohol, sodium dodecyl sulphate (SDS), and proteinase K were sourced from Sigma-Aldrich (St. Louis, MO, USA). Taq DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), MgCl2, and 10× PCR buffer were obtained from Thermo Fisher Scientific (Vilnius, Lithuania). Agarose (molecular biology grade) and ethidium bromide were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Oligonucleotide primers were custom-synthesised by Integrated DNA Technologies (Leuven, Belgium). A 100-bp DNA ladder (GeneRuler, Thermo Fisher) served as the molecular weight marker. All reagents were analytical grade and used before their expiry dates.

Methods

Genomic DNA was isolated from 200 μL of each blood sample using the conventional phenol-chloroform-isoamyl alcohol (25:24:1) extraction protocol. Blood was first lysed with SDS (1%) and digested overnight at 56 °C with proteinase K (20 mg/mL). After two rounds of organic extraction and ethanol precipitation, DNA pellets were resuspended in 50 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher); samples with A260/A280 ratios between 1.7 and 2.0 were accepted [7] . The nested PCR targeted the Tams1 gene. In the primary reaction, outer primers Tams1-F (5′-ATGCTGCAAATGAGGAT-3′) and Tams1-R (5′-GGACTGATGAGAAGATCGATG-3′) amplified a ~930-bp fragment in a 25-μL volume containing 1× buffer, 1.5 mM MgCl 2 , 200 μM each dNTP, 0.4 μM each primer, 1 U Taq polymerase, and 2 μL template DNA. Cycling conditions were: 94 °C for 5 min; 35 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min; and a final extension at 72 °C for 7 min. One microlitre of the primary product served as the template for the nested reaction, which used inner primers Tams1-NF (5′-CAGAAACTTGAACGACCG-3′) and Tams1-NR (5′-AAGGTAACCTTCCGATACG-3′) under identical conditions except for an annealing temperature of 58 °C, yielding a 721-bp product [9] . Amplicons were resolved on 1.5% agarose gels stained with ethidium bromide and visualised under UV transillumination.

Quality Control

Each PCR batch included a positive control (DNA from a microscopically confirmed T. annulata-infected blood sample), a negative extraction control (reagents processed without blood), and a no-template control (PCR master mix with nuclease-free water). To guard against crosscontamination, DNA extraction, PCR setup, and gel electrophoresis were performed in separate rooms with dedicated pipettes. Ten percent of positive samples were randomly selected, re-extracted, and re-amplified to confirm reproducibility. Representative amplicons (n = 8) were purified and submitted for bidirectional Sanger sequencing. Resulting sequences were compared against the NCBI GenBank database using BLASTn, and identity scores above 97% were accepted as confirmation of T. annulata [14] . Statistical analysis was performed in SPSS v26.0 (IBM, Armonk, NY, USA). The chi-squared (χ²) test assessed differences in prevalence by region, age group, and sex. A pvalue below 0.05 was treated as statistically significant.

Results: Of the 384 blood samples subjected to nested PCR, 67 (17.4%) produced the expected 721-bp amplicon on agarose gel electrophoresis, confirming the presence of T. annulata DNA. No amplification occurred in any negative or no-template control, and all positive controls yielded the correct band. Sequencing of eight representative amplicons returned identity scores ranging from 98.2% to 99.6% against T. annulata Tams1 gene entries in GenBank (accession numbers KY078166, MH560283, and MK432923). The highest prevalence was recorded in Ankara Province (21.3%), and the lowest in Kayseri Province (12.5%). The inter-regional difference did not reach statistical significance (χ² = 6.12, df = 4, p = 0.187), though a clear gradient from west to east was evident across the sampled area. Age exerted a marked effect on carrier status. Cattle older than four years were significantly more likely to test positive (27.3%) than animals aged one to two years (8.7%) (χ² = 14.38, df = 2, p = 0.001). Female animals showed a slightly higher prevalence (19.1%) relative to males (15.1%), yet this gap did not reach significance (χ² = 1.03, df = 1, p = 0.311).

The asterisk (*) marks statistical significance at p<0.05.

Comprehensive Interpretation

Taken as a whole, the molecular data reveal a carrier prevalence of 17.4% across five provinces of central Turkey, with regional rates spanning from 12.5% (Kayseri) to 21.3% (Ankara). Age emerged as the sole statistically significant risk factor-cattle beyond four years of age were roughly three times more likely to harbour the parasite than those under two years. Sequence confirmation removed any doubt about assay accuracy, with identity scores above 98% across all tested amplicons. The combination of a consistent nPCR protocol, broad geographic coverage, and sequencing validation gives confidence that these figures reflect the true carrier burden in the surveyed population.

Discussion

The overall molecular prevalence of 17.4% recorded here falls within the range reported by earlier PCR-based surveys in the wider region. A research conducted in Tunisia using a Tams1-targeted conventional PCR found 22.8% positivity among asymptomatic cattle [1] , while investigations in Iran documented rates between 12% and 26% depending on the province and sampling season [3] . The somewhat lower figure in the current work may reflect the timing of sampling, which spanned a full year and therefore diluted the peak prevalence expected during the summer tick season. Alternatively, the crossbred genetic background of the sampled cattle-which often carry partial resistance inherited from local Anatolian breeds could have moderated infection levels [4] . Ankara Province recording the highest rate (21.3%) aligns with its denser cattle population and greater movement of livestock through weekly markets, both of which facilitate tick transfer between herds [10] . Kayseri's lower figure (12.5%) might be linked to its higher elevation and cooler microclimate, conditions less favourable for Hyalomma tick activity [11] . But geography alone doesn't explain everything; herd management practices such as acaricide application frequency and pasture rotation also vary across provinces and likely contribute to the observed gradient.

The strong age effect 27.3% in cattle over four years versus 8.7% in those under two echoes findings from Egypt and Iraq where older cattle consistently showed higher carrier rates [5,14] . The likely mechanism involves cumulative exposure: each successive tick season adds another opportunity for infection, and while many animals mount an immune response strong enough to suppress clinical disease, they seldom clear the parasite entirely. This persistent low-grade parasitaemia is precisely what makes older animals dangerous reservoirs. From a practical standpoint, targeting acaricide treatments and serological monitoring toward older cows in the herd could yield a disproportionately large reduction in transmission pressure. The absence of a significant sex difference (19.1% female vs 15.1% male, p = 0.311) contrasts with some reports that attributed higher rates in females to immunosuppression during pregnancy and lactation [6] . Our sample, however, did not distinguish between pregnant and non-pregnant females, which may have masked an effect. Future investigations that record reproductive status alongside infection data would help clarify this point.

Conclusion

This research set out to measure how commonly apparently healthy cattle in central Turkey carry T. annulata at the molecular level. The nested PCR approach, targeting the Tams1 gene, proved both sensitive and specific, detecting parasite DNA in 17.4% of 384 animals that showed no outward signs of disease. Regional prevalence ranged from 12.5% in Kayseri to 21.3% in Ankara, painting a picture of widespread but uneven carrier distribution across the central Anatolian plateau. Among the risk factors examined, age stood out as the strongest predictor of carrier status. Cattle older than four years were about three times as likely to test positive as those younger than two years, pointing to cumulative tick exposure over successive grazing seasons as the main driver. Sex had no meaningful impact once the data were adjusted for herd size and sampling location. Sequence analysis of representative amplicons confirmed the identity of the parasite beyond any reasonable doubt, with identity scores ranging from 98.2% to 99.6% against published T. annulata sequences. From a field perspective, these numbers carry a clear message: nearly one in five apparently healthy cattle is silently harbouring the parasite and feeding the transmission cycle. Routine molecular screening especially of older animals and herds in high-traffic provinces could help veterinary services identify and manage carriers before they seed new outbreaks. Integrating nPCR-based surveillance into existing animal health programmes would be a cost-effective step toward reducing the economic burden of tropical theileriosis in Turkey. Looking ahead, several avenues deserve attention. Longitudinal sampling across two or three consecutive years would clarify whether carrier rates fluctuate with seasonal tick activity or remain stable. Genotyping of the Tams1 amplicons could reveal whether certain parasite strains predominate in specific provinces, information that would be directly useful for vaccine candidate selection. And expanding the geographic scope to include the Aegean and Mediterranean coastal regions where Hyalomma diversity is even greater would complete the national picture and support a coordinated, evidence-based control strategy.

Acknowledgement

Acknowledgements Funding SourcesThis research was supported by an internal competitive research grant from the Ankara Institute of Veterinary Biosciences. The funding body had no role in the design, execution, analysis, or reporting of the research.

References

1. Rjeibi MR, Gharbi M, Mhadhbi M, Mabrouk W, Ayari B, Nasfi I, Jedidi M, Sassi L, Rekik M, Darghouth MA. Prevalence of piroplasms in small ruminants and cattle in North Africa and advances in molecular tools. Veterinary Parasitology. 2016;225:1-7.
2. Boulter N, Hall R. Immunity and vaccine development in the bovine theilerioses. Advances in Parasitology. 1999;44:41-97.
3. Durrani AZ, Kamal N, Khan MS. Incidence of theileriosis and estimation of packed cell volume, total erythrocyte count, and haemoglobin in infected animals. Journal of Animal and Plant Sciences. 2008;18(2-3):5357.
4. Morrison WI. Progress towards understanding the immunobiology of Theileria parasites. Parasitology. 2009;136(12):1415-1426.
5. Al-Hosary AAT, Ahmed LS, Ahmed JS, Gohary AHE, Rabie SA, Igarashi I, Terkawi MA. Epidemiological survey of Theileria annulata in Upper Egypt by nPCR and ELISA. Korean Journal of Parasitology. 2021;59(3):253-260.
6. Gubbels JM, de Vos AP, van der Weide M, Viseras J, Schouls LM, de Vries E, Jongejan F. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. Journal of Clinical Microbiology. 1999;37(6):1782-1789.
7. Aktas M, Altay K, Dumanli N. A molecular survey of bovine Theileria parasites among apparently healthy cattle and with a note on the distribution of ticks in eastern Turkey. Veterinary Parasitology. 2006;138(34):179-185.
8. 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. Journal of Clinical Microbiology. 1995;33(10):2665-2669.
9. Kirvar E, Ilhan T, Katzer F, Hooshmand-Rad P, Wilkie G, Preston PM, Shiels BR. Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences. Parasitology. 2000;120(3):245254.
10. Inci A, Ica A, Yildirim A, Vatansever Z, Cakmak A, Albasan H, Cam Y, Atasever A, Sari B, Duzlu O. Epidemiology of tropical theileriosis in the Cappadocia region. Turkish Journal of Veterinary and Animal Sciences. 2008;32(1):57-64.
11. Bilgic HB, Karagenc T, Simuunza M, Shiels B, Tait A, Eren H, Weir W. Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Experimental Parasitology. 2013;133(2):222-229.
12. Gharbi M, Hayouni ME, Sassi L, Dridi W, Darghouth MA. Hyalomma scupense (Acari, Ixodidae) in northeast Tunisia: seasonal population dynamics of immature instars and host range. Parasite. 2013;20:47.
13. Aydin MF, Aktas M, Dumanli N. Molecular identification of Theileria and Babesia in ticks collected from cattle in the Black Sea region of Turkey. Parasitology Research. 2015;114(1):65-69.
14. Mahmoud MS, Kandil OM, Nasr SM, Hendawy SHM, Habeeb SM, Mabrouk DM, Silva MG, Suarez CE. Serological and molecular diagnostic surveys combined with examining hematological profiles suggest increased levels of infection and hemoparasite co-infections of cattle in Egypt. Parasites & Vectors. 2015;8:647.
15. Gao J, Luo J, Fan R, Guan G, Yin H, Li Y. Molecular characterisation of a Theileria annulata isolate from China. Ticks and Tick-Borne Diseases. 2017;8(4):546549.
16. Oura CAL, Tait A, Shiels B, Weir W. Theileria annulata and the epidemiology and control of tropical theileriosis. Research in Veterinary Science. 2023;157:88-98.