Tropical theileriosis, which is caused by Theileria annulata and transmitted by Hyalomma ticks, is among the most significant tick-borne disease affecting cattle widespread between latitudes 15°N–60°N and longitudes 30°W–150°E, resulting in substantial economic losses. (Robinson, 1982; Brown, 1990; Norval et al., 1992; Bilgic et al., 2019). Among disease control methods, treatment of infected animals with buparvaquone (BPQ) serves as the most common treatment of acute infections within the vertebrate host (Hashemi-Fesharki, 1991). Besides its widespread use in endemic regions, recent reports indicate the emergence of resistance to BPQ. Studies on BPQ resistance in certain T. annulata populations have identified mutations in putative ubiquinone-binding sites (Q_O) (Korsinczky et al., 2000; Birth et al., 2014) of the cytochrome b (Cyto b) gene of T. annulata (Mhadhbi et al., 2010, 2015; Sharifiyazdi et al., 2012; Chatanga et al., 2019; Yousef et al., 2020; Ali et al., 2022; Hacilarlioglu et al., 2023) as prevalent resistance-associated alterations. However, the underlying mechanism of such BPQ resistance has not been fully investigated so far.

Biological fitness refers to a parasite’s ability to survive and reproduce within its host, while also successfully transmitting to new hosts (Segovia et al., 2025). Thus, it is critically important to limit the spread of mutant parasites and delay the emergence of drug resistance in new areas (WHO, 2024). The efficacy of the disease control strategies is largely dependent on the fitness cost associated with the drug resistance (Segovia et al., 2025). In the absence of BPQ pressure, the drug resistance in a particular parasite population often results in a loss of fitness compared with wild-type parasite populations (Walliker et al., 2005). However, 3 critical conditions must be met: (1) drug-susceptible parasites still need to be present within the parasite population; (2) the lower fitness nature of the resistant parasite population compared with the susceptible ones; and (3) a sufficient transmission to allow the formation of mixed parasite infections (Peters et al., 2002; Bell et al., 2012). Experimental studies on Plasmodium chabaudi both in humans (Smith et al., 1999; Bousema et al., 2008; Harrington et al., 2009) and in laboratory mice (Taylor et al., 1997; de Roode et al., 2004; Huijben et al., 2010, 2011) demonstrated that the presence of different genotypes in mixed infections can slow the evolutionary rate of competitively resistant populations in the absence of drug treatment. This effect may lead to suppression by competitors in the absence of drug treatment. Conversely, it has been reported that elimination of drug-susceptible genotypes from the population under excessive drug pressure can significantly increase the spread of resistant genotypes (Hastings, 1997; Mackinnon and Hastings, 1998; Felger and Beck, 2008; Read et al., 2011). The fitness of drug-resistant Plasmodium spp. populations, such as Plasmodium berghei, P. chabaudi, and Plasmodium falciparum, exhibited variable reproductive success or lower fitness when compared with drug-sensitive populations (Rosario et al., 1978; Shinondo et al., 1994; Peters et al., 2002; Preechapornkul et al., 2009). In animal models, treatment with a drug will inevitably alter the rate of drug-resistant populations within the host and vectors compared with before treatment (Bell et al., 2012). Fitness is highly dependent on genotype (e.g., resistant or susceptible) and environmental conditions (e.g., the presence or absence of a drug pressure). Competition between different parasite strains may lower the fitness of drug-resistant ones (Huijben et al., 2018; Dhingra et al., 2019; Tirrell et al., 2019; Mathieu et al., 2020). Recent findings indicate that the proliferation rate of an in vitro–generated drug-resistant clone of the mutated (M128I) T. annulata population was comparable to that of the wild type in the absence of BPQ pressure (Tajeri et al., 2024). This suggests that there is no fitness cost associated with either population. However, the fitness of mutated subpopulations or clones cocultivated with wild-types of T. annulata remains undocumented.

Programmed cell death (apoptosis) is used by organisms as a defense mechanism against intracellular infectious agents such as viruses, bacteria, and parasites (Vaux et al., 1994). In response, these pathogens have developed various strategies to inhibit apoptosis for proliferation (Moss et al., 1999). Intracellular apicomplexan parasites have evolved strategies to inhibit apoptosis and control host cell survival pathways (Heussler et al., 2001). Among them, Theileria parasites have the unique ability to induce uncontrolled but fully reversible proliferation of the host lymphocytes following invasion (Irvin, 1985) in a manner reminiscent of cancer cells (Chaussepied and Langsley, 1996; Dobbelaere and Heussler, 1999; Dobbelaere and Rottenberg, 2003). However, Theileria-induced transformation can be reversed by in vivo or in vitro or both with BPQ treatment. In vitro–treated cells survive for a few more days and undergo apoptosis (Heussler et al., 1999, 2001; Guergnon et al., 2003). Besides, the capacity of drug-resistant T. annulata populations to inhibit apoptosis is still questionable.

This study evaluated the fitness costs in both mutated drug-resistant and wild-type clonal cell lines under long-term in vitro conditions in the absence of BPQ pressure. Also, the recovery of apoptotic features in both mutated drug-resistant and wild-type clones was investigated. This approach provided insights into the dynamics between drug exposure, genetic mutation, and apoptotic responses in T. annulata populations under varying BPQ selection pressures.

MATERIALS AND METHODS

RESULTS

In vitro proliferation index of mixed cell lines during 50 passages in the absence of BPQ pressure

Mixed cell lines (M1–M3) harboring both the P253S mutated drug-resistant and unmutated drug-sensitive clones were cultivated in vitro for up to 50 passages without any BPQ pressure (Fig. 1). Although 2 replicates (M3b and M3c) of the M3 line failed to grow beyond the initial passage during routine maintenance of the mixed cell lines, the other replicates continued to grow successfully up to passage 50. In every 5 passages, the degree of drug resistance in each mixed cell line (M1–M3) was evaluated using an MTT colorimetric assay. The IC₅₀ values of mixed cell lines from the initial (p0) to passage 50 are given in Table I. When analyzing the passages as a group, no statistically significant difference was found between the compared groups (P = 0.114; F = 1.641). A closer examination of each mixed cell line indicated that the IC₅₀ values displayed a varying trend, except for some unexpected fluctuations noted in the replicates of M1 and lines across different passages. Besides this, a decrease in IC₅₀ values was notable after passage 30 (Table I). When replicates of mixed cell lines were compared using independent group t-tests in every 5 passages, no statistically significant difference was found overall between groups. However, significant differences were observed between replicates of M1 and M2 mixed cell lines, particularly at the initial (p0) passage (P = 0.006) and passage 50 (P = 0.016).

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Table I.The half-maximal inhibitory concentration (IC₅₀) values of mixed cell lines (M1–M3) calculated every 5 passages during the in vitro growth.

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Apoptotic activity of drug-resistant and sensitive cell lines following BPQ treatment

Apoptotic activity in drug-resistant and drug-sensitive cell lines of T. annulata following BPQ treatment was compared by the TUNEL assay using Apoptag® Plus Peroxidase In Situ Apoptosis Detection Kit. The percentage of apoptotic cells observed in drug-resistant and drug-sensitive groups between 0 and 72 hr after BPQ treatment is given in Figure 2. No significant difference in the percentage of apoptotic cells was observed in the BPQ-resistant Is-4 clonal cell line between 0 and 72 hr after BPQ treatment (Fig. 2). In contrast, the percentage of apoptotic cells was significantly increased in the drug-sensitive clonal cell line (Is-5) between 0 and 72 hr after treatment (Fig. 2).

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DISCUSSION

Tropical theileriosis hampers livestock production in endemic countries and has a particularly strong impact on the most productive cattle breeds (Bilgic et al., 2019). The field populations of T. annulata are very dynamic and genetically diverse within and between geographic areas (Weir et al., 2007; Al-Hamidhi et al., 2015; Bilgic et al., 2019). The high genotypic diversity and coinfection with multiple genotypes observed in T. annulata infections (Bilgic et al., 2019) are influenced by various selective pressures, including vaccination, the intensity of transmission, the infection rate of ticks, and drug treatment. Treatment of infected animals with the only effective drug BPQ (Coombs and Croft, 1997) has been used worldwide. However, the emergence of treatment failure cases and the development of resistance were reported in certain T. annulata populations (Sharifiyazdi et al., 2012; Mhadhbi et al., 2015; Chatanga et al., 2019; Yousef et al., 2020; Hacilarlioglu et al., 2023).

The effectiveness of resistance management strategies largely depends on whether drug-resistant parasites incur a fitness cost (Segovia et al., 2025). One of the major challenges in this context is the limited understanding of how mutated subpopulations behave within mixed parasite populations, particularly in terms of the ability to persist and spread. In within-host competition, understanding the relationship between parasite population growth rate is critical for determining whether mixed infections result in lower or higher adaptation than single infections. Within mixed infections, those with higher growth rates tend to stand out due to natural selection in the population (Wargo et al., 2007). Assessing the competitive fitness of resistant versus sensitive parasite populations will provide insights into whether resistance affects the biological efficiency or growth capacity (O’Brien et al., 2011; Wilairat et al., 2016).

Competitive growth assays are important for the overall determination of fitness (Ender et al., 2004; Cong et al., 2007; Boutwell et al., 2009; Han et al., 2012; Valsecchi et al., 2015; Tirrell et al., 2019). Thus, by measuring growth rates of mutant and wild-type strains in vitro, one could compare intrinsic growth rates, as well as examine head-to-head competition (Segovia et al., 2025). In this study, the fitness of the P253S mutated populations within a mixed T. annulata population was evaluated by in vitro cultivating mixed cell lines (M1–M3) harboring both the P253S mutated drug-resistant and unmutated drug-sensitive (wild-type) clones for up to 50 passages without any BPQ pressure (Fig. 1A). During routine maintenance of the mixed cell lines, 2 replicates of the M3 line (M3b and M3c) failed to grow beyond the initial passage, suggesting an inability to adapt to in vitro conditions. The primary challenge for parasite populations in an isolate undergoing in vitro cultivation is adaptation. The varying competitive fitness levels of cultured parasites demonstrate the effects on the relative in vitro growth rates of different genotypes (O’Brien et al., 2011; Wilairat et al., 2016). Although some populations readily adapt to new environmental and nutritional conditions, others decline or disappear, potentially due to competition for resources or an inherent inability to adapt (Basco, 2023; Nkhoma et al., 2023). Thus, the frequency of the mutated (P253S) and wild-type populations and the degree of drug resistance in mixed cell lines were routinely evaluated every 5 passages throughout the trial.

When DNA samples were subjected to AS-PCR, it was found that the frequency of the wild-type parasite population was similar to or slightly higher than that of the mutated (P253S) population during the initial (p0) and passage 5 (p5) in all mixed cell lines (Fig. 1C, D). In a series of head-to-head competitions of genetically distinct Plasmodium strains, it was also found that strains showing resistance to artemisinin derivatives were the poorer competitors (Tirrell et al., 2019). Similarly, P. falciparum parasites with mutations in Cyto b (leading to atovaquone resistance) were outcompeted by the wild-type parasites (Peters et al., 2002). However, it was also reported that some P. falciparum strains showing chloroquine resistance had higher proliferation rates (Reilly et al., 2007) and were able to outcompete the susceptible ones in head-to-head competition (Wacker et al., 2012). In this study, T. annulata populations harboring the P253S mutation were not outcompeted by the wild-type, and they became more abundant than wild type in the M1 and M2 mixed cell lines from passage 10 onward (Fig. 1D). In the M3 mixed line, fluctuations in the intensity of amplicons were observed between p5 and p25, yet these variations did not consistently reflect the IC₅₀ results. Such discrepancies may indicate that band intensity alone is not always a reliable predictor of resistance dynamics. Moreover, the black of statistically significant differences in IC₅₀ values among groups suggests that both wild-type and mutant populations were able to persist under in vitro conditions without a clear fitness disadvantage. This suggests that both wild-type and mutated, drug-resistant T. annulata populations were able to survive when cocultivated in vitro for up to 50 passages for about 1 yr without a fitness cost. Although the IC₅₀ values of the mixed cell lines exhibited some fluctuations across passages, no consistent or overall statistically significant differences were evident between groups. The occasional differences observed between certain replicates, particularly at the beginning and end of the cultivation period, are likely attributable to genetic variation among the clones rather than to a systematic fitness cost. The genetic background significantly influences in vitro fitness costs (Gabryszewski et al., 2016). Thus, head-to-head competition of genetically distinct populations will inevitably either aggravate or alleviate the observed fitness costs. In this study, the significant difference observed in IC₅₀ values at the initial passage (p0) may be due to the genetic diversity among the clones of mixed cell lines (M1 and M2) used. Overall, the fluctuations observed both in the intensity of the bands and IC₅₀ values in every 5 passages suggested that the growth rate of the mutated population increased inversely proportional to the growth rate of the wild-type population among 50 passages, and this dynamic change continued during the routine in vitro maintenance. The observed decrease or increase in the frequency of a population within a mixed population over a certain time in the absence of drug pressure has an existential proposition. Mixed infections with higher growth rates tend to stand out due to natural selection in the population (Wargo et al., 2007). Observed fluctuations in this study were interpreted as part of the adaptation process during the in vitro cultivation (Oduola et al., 1992; Ochong et al., 2013). Fitness cost of mutated strains can vary within and between parasite populations. Thus, peaks in the fitness landscape represent optimal fitness, whereas valleys represent lower fitness; parasites may become trapped on a smaller peak, unable to evolve toward a higher peak without crossing a valley of reduced fitness (Ogbunugafor and Hartl., 2016). Similar dynamics have also been reported in other cultivation studies of various parasites (Oduola et al., 1992; Ochong et al., 2013). However, note that fitness costs are not fixed parameters (Huijben et al., 2018). Similar to malarial infections, diseases caused by T. annulata often consist of mixed genotypes, and the resulting within-host interactions affect the growth and thus the fitness of each coinfecting strain. It was shown that the fitness cost could range anywhere from 0 to 100%, depending on whether these costs were observed in competition or during singular infection and which metric of fitness was used (Huijben et al., 2018).

Data from in vitro studies of Plasmodium generally suggest that drug resistance is associated with a fitness cost (Tirrell et al., 2019; Segovia et al., 2025). However, the results of the present study indicate that parasite populations harboring the P253S mutation in the Cyto b of T. annulata do not appear to incur a fitness cost. This may help explain why T. annulata populations carrying the P253S mutation have been reported as the most geographically widespread in multiple field studies (Sharifiyazdi et al., 2012; Mhadhbi et al., 2015; Yousef et al., 2020; Ali et al., 2022; Nehra et al., 2024). A similar lack of in vitro fitness cost was observed in parasite populations with the M128I mutations in the Cyto b of T. annulata (Tajeri et al., 2024). Also, competition assays between mutant and wild-type parasites in Toxoplasma gondii did not show any difference in growth rates in vitro (Fohl and Roos, 2003). Thus, the criteria established by WHO (2015; p. 302-303) that “resistant parasite populations must be less fit than susceptible parasite populations” are not met for this strategy to be considered successful in this case. Various environmental factors within the vertebrate and invertebrate hosts and climatic conditions may likely affect the fitness cost (Segovia et al., 2025). Although it has been demonstrated that the P253S mutation can be transmitted to cattle, leading to severe disease and facilitating transmission through vector ticks (Hacilarlioglu et al., 2023), the effects of these mutations on BPQ resistance in vector ticks remain to be investigated in future studies.

Host cells use apoptosis as a defense mechanism against intracellular parasites (Vaux et al., 1994). However, one of the intracellular apicomplexan parasites, named Theileria, has evolved strategies to inhibit apoptosis and induce uncontrolled proliferation of the host cells, similar to cancer cells (Chaussepied and Langsley, 1996; Dobbelaere and Heussler, 1999; Dobbelaere and Rottenberg, 2003). Fortunately, Theileria-induced transformation can be reversed by eliminating parasites from the host cells by BPQ treatment. In vitro–treated cells regain preinfection characteristics and undergo apoptosis afterward (Guergnon et al., 2003; Wiens et al., 2014). This raises the question: What happens if the parasite population becomes drug-resistant? This study addressed the question by evaluating the effects of the P253S mutation, which confers resistance to BPQ, on the apoptotic activity of the T. annulata population in vitro. In this study, differences in the apoptotic cell rates showed that approximately 5-fold more apoptotic resistance was observed in the drug-resistant group after 72 hr (Fig. 2). This also supports the outcome of our previous report, which indicates the development of resistance to BPQ in mutant parasite populations (Hacilarlioglu et al., 2023).

CONCLUSION

This study represents a foundational step in understanding the fitness costs associated with BPQ resistance in T. annulata under in vitro conditions, with a particular focus on the effects of the P253S mutation in the Cyto b gene on the fitness of BPQ-resistant parasite populations. The findings indicate that in the absence of drug pressure, the resistant parasite population harboring the P253S mutation successfully adapted to in vitro culture alongside the drug-sensitive population. Moreover, the results demonstrate that the mutated parasite populations exhibit resistance to apoptosis, and this resistance persists even after the withdrawal of BPQ. Although these results do not directly quantify fitness costs, they indicate that drug-resistant mutations, such as P253S may persist and spread in natural populations, even in the absence of drug pressure. This persistence poses a significant challenge for future control strategies. This issue becomes even more critical in settings when only a single effective drug is available, making the development of a strategy against resistant parasites particularly difficult. Further comprehensive in vitro and in vivo studies on both vertebrate and invertebrate hosts are needed to understand the infection and transmission dynamics of parasite populations harboring other drug-resistant variants. Note that disease management strategies should incorporate comprehensive fitness cost models to provide accurate estimates of resistance evolution, including assessments of parasite fitness in both hosts.