Prevalence, species composition, and patterns of ixodid tick infestations in theileriosis suspected cattle in Southern Tamil Nadu

Introduction

In India theileriosis is an important tick-borne haemoprotozoan disease of cattle, caused by intracellular apicomplexan parasites of the genus Theileria. While Theileria annulata is well known for its high pathogenicity, Theileria orientalis, traditionally considered a benign species, has recently emerged as a major pathogen in several regions. Field outbreaks have documented that clinical Oriental theileriosis causes substantial economic losses due to reduced milk production in dairy herds, with an average loss of 624 litres per animal at 305 days of lactation compared to uninfected cows in Victoria, Australia (Perera et al., 2014) [1] . Lew-Tabor and Rodriguez Valle (2016) [2] reported that worldwide tick and tick borne diseases (TTDS) caused an estimated loss of 22-30 billion USD/annum. Singh et al. (2022) [3] evaluated the economic impact of TTDS, including theileriosis, on Indian dairy production systems and reported a significantly high cumulative loss of 787.63 million USD due to TTDS in India. This shift in disease significance is largely attributed to evolving parasite genotypes and expanding tick vector populations across diverse agro-climatic zones (Watts et al., 2016; Jacob et al., 2024) [4,5] . Globally, for T. orientalis, Haemaphysalis longicornis is the principal vector, while in India, Rhipicephalus microplus and Haemaphysalis bispinosa have been reported as vectors under natural field conditions, especially in crossbred cattle with lower innate tick resistance (Aparna et al., 2011; Kakati et al., 2015; Vinodkumar et al., 2016) [6][7][8] . Transmission of T. orientalis occurs mainly through transstadial passage, but recent studies demonstrated parasite DNA in eggs supporting the possibility of limited transovarial transmission (Kakati et al., 2015) [7] . These mechanisms contribute to persistent environmental contamination and silent spread of pathogenic genotypes.

Several ixodid tick genera, including Haemaphysalis, Rhipicephalus, and Amblyomma, are implicated in the epidemiology of bovine theileriosis worldwide (Baghel et al., 2023) [9] . India remains a major endemic region, with R. microplus, H. bispinosa, and H. anatolicum playing key roles in bovine Theileria transmission. Notably, R. microplus in northern India was found to harbor T. orientalis in 21.7% of examined ticks, confirming its vector competence. Similarly, the invasive H. longicornis in the United States has been shown to carry and transmit the highly pathogenic Ikeda genotype (Dinkel et al., 2021) [10] . Studies from Vietnam and Myanmar also reported frequent detection of T. orientalis in Haemaphysalis and Rhipicephalus spp., indicating active cocirculation among ticks and livestock (Khukhuu et al., 2011; Bawm et al., 2014) [11,12] . In India, repeated identification of the H. bispinosa and R. microplus from T. orientalis affected animals accentuates the risk of genotype driven pathogenicity (Aparna et al., 2011; Baghel et al., 2023) [6,9] . Furthermore, the detection of T. orientalis DNA in ticks collected from wildlife-livestock interfaces in Kerala highlights the broader ecological involvement of reservoir hosts and the potential for silent enzootic maintenance (Nimisha et al., 2019) [13] . The rapid ecological expansion of H. longicornis, aided by parthenogenetic reproduction and climatic adaptability, further increases the risk of dissemination in new geographical zones (Thompson et al., 2020; Dinkel et al., 2021) [14,10] . Collectively, global evidence indicates that multiple tick genera are capable vectors of T. orientalis, although the dominant species vary regionally, H. longicornis in Asia-Pacific and Australasia, R. microplus and H. bispinosa in the Indian subcontinent, and the emerging role of invasive H. longicornis in the United States. This regional dependence reinforces the need for localized vector surveillance to understand transmission dynamics more accurately. In this context, the present study was undertaken to assess the prevalence and species composition of hard ticks infesting theileriosis suspected cattle in the southern districts of Tamil Nadu and to generate baseline data essential for regional tick control strategies and molecular epidemiology of T. orientalis.

Materials and Methods

Study Area and Experimental Design

The study was conducted in the southern agro-climatic belt of Tamil Nadu, covering four districts, which represent diverse ecological zones ranging from semi-arid plains (Tirunelveli), high rainfall foothills (Tenkasi), dry coastal belts (Thoothukudi), to humid coastal cum highland regions (Kanyakumari). Mean annual temperatures range from 25-34 °C, with relative humidity often exceeding 70% in coastal localities. The study spanned from January 2024 to August 2025. Cattle from organized farms, smallholder units, and free grazing rural herds were included, representing varied management systems with differential tick exposure. Animals showing clinical signs such as pyrexia, anaemia, jaundice, lymphadenopathy, reduced milk yield, and visible tick infestation were sampled as theileriosis suspected cases. The sample size of cattle was determined by power analysis using a 95% confidence level and 5% allowable error based on an expected prevalence of 20% as reported in previous studies from India (Velusamy et al., 2014; Krishnamoorthy et al., 2021) [15,16] .

Tick Collection and Preservation

Ticks were collected from infested cattle during clinical examinations across the study districts. Ticks were carefully removed using blunt forceps by grasping near the mouthparts to avoid damage to diagnostic structures. Individual ticks were placed into sterile, labelled screw cap vials containing 70% ethanol and transported to the Department of Veterinary Parasitology, Veterinary College and Research Institute, Tirunelveli, Tamil Nadu.

Gross and Microscopic identification of tick species

Collected ticks were briefly air dried to remove excess ethanol and subjected to detailed gross and microscopic examination (Lynx LM-52-3611) following ixodid tick identification guides (Walker et al., 2003) [17] .

Statistical Analysis

Tick infestation prevalence was calculated as the proportion of tick infested cattle out of total examined and the confidence intervals (95%) were calculated using the Wilson score method (OpenEpi Version 3.01), as described in standard epidemiological methods (Thrusfield, 2018) [18] . Pearson's Chi-square (χ²) test was applied to compare overall prevalence among districts, and to assess district wise variation in single species infestations, mixed species infestations, and the distribution of individual tick species and their combinations (Ghosh et al., 2019; Yenew et al., 2025) [19, 20] . Statistical significance was set at p<0.05.

Results

District wise Prevalence of Tick Infestation in Cattle

A total of 410 out of 525 cattle examined across the four southern districts of Tamil Nadu were found infested with ixodid ticks, yielding an overall prevalence of 78.10% (95% CI: 74.36-81.42). District wise, the highest prevalence of tick infestation was recorded in Kanyakumari district (80.00%; 95% CI: 71.77-86.29), while Thoothukudi district showed the lowest prevalence (74.67%; 95% CI: 63.79-83.14). However, a comparable prevalence was observed in Tenkasi (78.47%; 95% CI: 71.07-84.40) and Tirunelveli (78.01%; 95% CI: 71.62-83.30) (Table 1). The Chi-square test revealed that there was no statistically significant association between district and tick infestation status in cattle (χ²=0.77, P=0.856), indicating a uniformly high prevalence of tick infestation across all study districts (Table 2).

Pattern of single and mixed tick species infestations across districts

Out of 525 cattle examined, 324 cattle (61.71%) harboured single species tick infestations, while 86 animals (16.38%) exhibited mixed species infestations (Table 3). District wise prevalence of single infestations ranged between 58.67% (Thoothukudi) and 63.48% (Kanyakumari), whereas mixed infestations remained consistently uniform across districts (16.00-16.67%). Chi-square analysis revealed no statistically significant association between district and tick infestation pattern (single vs mixed) (χ²=0.02, P=0.999), shows that the distribution of single and mixed tick infestations was similar across all districts. Indeed, single tick infestation was more common than mixed infestation in all districts.

Single Species Tick Infestations

Single species tick infestations varied significantly across districts (χ²=61.87, p<0.001) (Table 4). Rhipicephalus microplus was the predominant species in all districts, with highest prevalence in Tenkasi (45.83%) and Thoothukudi (40.00%). Haemaphysalis bispinosa showed marked focal distribution, with high prevalence in Kanyakumari (31.30%) compared to other districts (4.86-10.47%). Hyalomma marginatum was most prevalent in Tirunelveli (13.09%), occurred at moderate levels in Thoothukudi (9.33%) and Tenkasi (9.03%), and was absent in Kanyakumari. Rhipicephalus haemaphysaloides was rare, detected only in Tirunelveli (0.52%). Notably, Rhipicephalus decoloratus was recorded for the first time in the study area as a single species infestation, occurring at low prevalence across districts (0.87-2.09%), with higher occurrence in Tirunelveli and Tenkasi. 6). Similarly, R. microplus + H. marginatum mixed infestation showed significant district wise variation (χ²=13.78, P=0.003), occurring in Tirunelveli (10.47%), Thoothukudi (10.67%), and Tenkasi (6.25%), but was absent in Kanyakumari (Table 7). [19,13] . Such consistently high prevalence indicates that tick infestation remains a persistent constraint in tropical cattle production systems. The absence of significant spatial variation suggests that ecological and management factors governing tick survival are broadly similar across southern Tamil Nadu. Climatic conditions characterised by sustained warmth, high humidity, and moderate rainfall provide favourable microenvironments for tick development and off host survival throughout the year. Comparable uniform prevalence patterns have been reported from Ethiopia, where similar agro-ecological conditions and grazing practices were implicated (Yenew et al., 2025) [20] . In the present region, extensive or semiintensive rearing systems, communal grazing, and limited adoption of systematic acaricidal control likely contribute to sustained tick exposure across districts. The high prevalence observed also parallels findings from Assam and other endemic regions, where tick infestation persists at elevated levels once enzootic stability is established (Khukhuu et [21,9] . The findings of the present study emphasize that tick infestation in southern Tamil Nadu represents a region wide challenge, necessitating coordinated, area based tick surveillance and control strategies rather than district specific interventions.

Pattern of single and mixed tick species infestations across districts

The present study showed that single tick species infestations predominated among cattle across southern Tamil Nadu (61.71%), whereas mixed infestations were less frequent (16.38%) and uniformly distributed across districts, with no significant district-wise association. Similar dominance of single-species infestations has been reported from other parts of Tamil Nadu and southern India, where environmental homogeneity and stable host-tick interactions favour infestation by a single dominant species at a given time (Nimisha et al., 2019; Ranganathan et al., 2025) [13,22] . The predominance of single infestations is likely driven by ecological niche specialization and competitive exclusion among ixodid ticks. In particular, Rhipicephalus (Boophilus) microplus, with its strong host preference, rapid life cycle and efficient attachment behaviour, often achieves numerical dominance, limiting opportunities for co-infestation by other species. Seasonal and microhabitat influences further reinforce this dominance under favourable climatic and management conditions (Ranganathan et al., 2025) [22] . Although less common, mixed tick infestations were consistently detected across all districts, suggesting stable ecological overlap rather than sporadic occurrence. Comparable low but persistent mixed infestations have been documented from endemic regions where repeated exposure through communal grazing facilitates contact with multiple tick species (Kakati et al., 2015; Ghosh et al., 2019) [7,19] . Epidemiologically, such mixed infestations are important, as concurrent feeding by different tick species may increase transmission complexity and support the maintenance of multiple haemoprotozoan species within cattle populations. From a disease transmission perspective, the coexistence of single and mixed infestations may influence the epidemiology of Theileria orientalis. [23,1,24,5] . Overall, the observed pattern reflects a stable tick host equilibrium in southern Tamil Nadu and supports region wide integrated tick management strategies targeting dominant species while accounting for secondary vectors involved in pathogen persistence. [19,13,25] . A pronounced focal distribution of Haemaphysalis bispinosa was observed in Kanyakumari (31.30%), contrasting with its lower prevalence elsewhere. This pattern likely reflects the district's humid coastal climate, dense vegetation, and wildlife-livestock interface, conditions favourable for Haemaphysalis spp., as reported from Kerala and Western Ghats regions (Nimisha et al., 2019; Ranganathan et al., 2025) [13,22] . In contrast, Hyalomma marginatum predominated in Tirunelveli and was absent in Kanyakumari, consistent with its preference for drier, open grazing ecosystems (Aktas et al., 2006; Ghosh et al., 2019) [21,19] . Rhipicephalus haemaphysaloides occurred rarely and was restricted to Tirunelveli, indicating its limited role in cattle infestation. Importantly, Rhipicephalus decoloratus was recorded for the first time as a single species infestation in the study area, albeit at low prevalence, suggesting possible range expansion linked to livestock movement or climatic shifts, as reported by Bawm et al. (2014) [12] and Baghel et al. (2023) [9] . These findings highlight the strong influence of local ecological and husbandry factors on tick species composition. The dominance of R. microplus signify its priority in control programmes, while the focal distribution of secondary species and emergence of R. decoloratus emphasize the need for region specific surveillance and adaptive tick management strategies. [13,23] . The detection of rare mixed infestations involving H. hussaini and Amblyomma spp., though sporadic, is epidemiologically important, indicating the presence of low abundance or emerging tick species that may contribute to pathogen introduction and maintenance. Mixed infestations are of particular concern as they can facilitate co-transmission of haemoparasites and enhance the circulation of T. orientalis genotypes, as reported in India and other endemic regions (Perera et al., 2014; Oakes et al., 2019; Jacob et al., 2024) [1,24,5] .

Tick species composition in cattle across

Mixed Species Tick Infestations

The district specific distribution of mixed tick infestations accentuates the influence of local ecological and climatic factors on tick community structure and highlights the need for integrated tick control strategies that address both dominant and secondary species across agro-ecological zones.

Conclusion

The present study elucidates the epidemiology of ixodid tick infestations in cattle across southern Tamil Nadu, revealing a uniformly high tick burden with marked district wise variation in species composition. Rhipicephalus microplus predominated across all districts, confirming its central role in sustaining tick populations and facilitating endemic transmission of tick-borne haemoprotozoa, including Theileria orientalis. The spatial distribution of Haemaphysalis bispinosa and Hyalomma marginatum reflected their ecological preferences for humid and xeric environments, respectively, while the first record of Rhipicephalus decoloratus highlights the dynamic expansion of tick fauna in the region. Although single species infestations were most frequent, the consistent occurrence of mixed infestations particularly involving R. microplus with H. marginatum and H. bispinosa indicates complex tick-host-environment interactions that may enhance pathogen transmission and genetic diversity. These findings demonstrate that tick infestation is a region wide challenge rather than a district specific phenomenon and emphasize the necessity for integrated, area wide tick control strategies that prioritize dominant vectors while accounting for secondary and emerging tick species to effectively mitigate tick borne disease risks in southern India.

Acknowledgement

AcknowledgmentsThe authors sincerely thank the Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai for providing the necessary facilities to conduct this study.

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