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Full Research Article

In-vitro Activity of Moxifloxacin against Theileria annulata - A Preliminary Study

A
Ankit S. Prajapati1,*
0000-0002-8940-8840
A
Abhinav N. Suthar2
H
Harshad H. Panchasara3
B
Bhupamani Das4
P
Pankaj A. Patel5
1Veterinary Clinical Complex Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Anand-388 001, Gujarat, India.
2Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
3Research Scientist, Livestock Research Station, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
4Department of Clinics Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
5Department of Veterinary Physiology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.

ABSTRACT

Background: Theileriosis is a tick-borne disease that has a maximum impact on the health and economy of developing countries. A number of clinical manifestations have been found in animals affected with Theileria annulata. A similar trend has been observed in the therapeutic management of theileriosis, especially in cattle. Resistance to available treatment options is one of the significant concerns with theileriosis.

Methods: The present study evaluated the in-vitro activity of moxifloxacin, the fluoroquinolone compound, against T. annulata in 96 well culture plates using resazurin dye. The histopaque method was used for the separation of infected and healthy PBMCs (Polymorph Blood Mononuclear Cells) for growth inhibition assay of T. annulata and cytotoxicity assay measurement, respectively. The hemolytic assay was also evaluated against normal bovine RBCs (red blood cells). The 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50) values were measured and analyzed using a correlation and regression method.

Result: The IC50 value of moxifloxacin was observed at 5,559 µM against in-vitro T. annulata. The CC50 value of cytotoxicity on PMBCs was found to be 3,984 µM. However, the HC50 value of hemolytic assay on bovine red blood cells was observed at 2,59,202 µM. The moxifloxacin showed a wide safety margin on red blood cells. Moxifloxacin can be further investigated in-vivo to determine its actual effect on T. annulata.

INTRODUCTION

Theileria annulata in livestock is one of the major health concerns. As T. annulata is a vector-borne disease, it covers large groups of animals, including sheep and goats (Verma et al., 2016; Shahedi et al., 2022; Prajapati et al., 2023; Ntesang et al., 2024; Sinha et al., 2024; Alnahari et al., 2026). According to estimates, bovine tropical theileriosis causes an annual loss of about US$ 384.3 million to the Indian livestock sector (Seeber and Steinfelder, 2016; Keroack et al., 2019). The clinical signs exhibited by the animals also vary (Prajapati et al., 2019). T. annulata outbreaks have been reported from India (Sinha et al., 2021; Velusamy et al., 2023). Buparvaquone is the veterinarians’ choice of drug. However, resistance against buparvaquone has been reported for a while (Salim et al., 2019; Yousef et al., 2020).
       
Fluoroquinolones are drugs indicated primarily as anti-bacterial agents (Pathania and Sharma, 2010). They work on microorganisms by inhibiting the topoisomerase II pathway (Pietrusinski and Staczek, 2006; Fabrega et al., 2009). DNA topoisomerases are essential to the survival of a large number of deadly protozoan parasites. Since the topoisomerase structures of protozoan parasites differ from those of humans, DNA topoisomerase serves as a crucial target for the creation of effective medications for parasitic illnesses in animals (Lamba and Roy, 2022). The exact pathway was evaluated in T. annulata and it was suggested that it could be used for new drug discovery (Lizundia et al., 2009).
       
Fluoroquinolone compounds have been tested against apicomplexan organisms (Batiha et al., 2020; Fontinha et al., 2020).  Among them, moxifloxacin was highly effective against apicomplexan organisms such as Toxoplasma gondii and Plasmodium falciparum (Gozalbes et al., 2000; Mahmoudi et al., 2003). The present study was executed to assess the in-vitro activity of moxifloxacin, a fluoroquinolone compound, against T. annulata. Further, cytotoxicity and hemolytic activity were also evaluated on healthy PBMCs and red blood cells.

MATERIALS AND METHODS

Ethical approval
 
The Institutional Animal Ethics Committee of the College approved the current study under protocol number IAEC No. VETCOLL/IAEC/2022/20/PROTOCOL-16.
 
Study period and location
 
The study was conducted from December 2023 to August 2024 at the Department of Veterinary Medicine, Kamdhenu University, Sardarkrushinagar, Gujarat, India, in collaboration with the Department of Veterinary Biotechnology, Veterinary Microbiology and Veterinary Physiology.
 
Selection of positive animal
 
The cattle showed clinical signs of theileriosis and were used for further in-vitro evaluation. T. annulata organism in the blood was confirmed by Giemsa-stained blood smear examination and polymerase chain reaction. T. annulata was explicitly detected in cows using PCR with a 430 bp amplicon using the forward primer CCAGGACCACCCTCA AGTTC and the reverse primer GCATCTAGTTCCTTGG CGGA (Kundave et al., 2015).
 
In-vitro growth inhibition assay
 
In-vitro activity of moxifloxacin against T. annulata was evaluated in 96 well culture plate (Gopalakrishnan et al., 2016). Histopaque was used to separate the infected lymphocytes from blood collected from the infected animals with Theileria. Three times, PBS wash was given to lymphocytes and 100 µL (equivalent to 2.5 × 105 cells/ml) seeded onto the plates. A complete medium consisting of RPMI-1640 supplemented with L-glutamine, 100 IU/ml penicillin and 10% fetal bovine serum was used. Incubation conditions for the culture plates were 37oC, 95% relative humidity and 5% CO2. Different concentrations of moxifloxacin from 16000 μM to 250 μM were evaluated against T. annulata. The in-vitro growth inhibition assay was conducted, with resazurin added as 10% of the total volume. After four hours, the optical density (OD) was measured at 570 and 650 nm. Moxifloxacin was purchased from Merck (Merck KGaA, Darmstadt, Germany) company. Triton ×-100 (T×100) was used as a positive control agent and DMSO was used for dilution control. The IC50 value of moxifloxacin was measured using the formula mentioned (Suthar et al., 2021).
 
In-vitro cytotoxicity assay on PBMCs
 
The cytotoxicity assay of moxifloxacin was evaluated from healthy PBMCs to assess the detrimental effect of the drug on cells described earlier (Araveti and Srivastava, 2019). Healthy PBMCs were separated by the Histopaque method and mixed with complete media. 100 µl PBMCs were seeded into the 96 well plate. The well culture plates were incubated at 37oC in 5 % CO2 for the next 48 hours. Moxifloxacin concentration was prepared from 8000 μM to 62.5 μM and added into wells. Again, incubation was done for another 24 hours, followed by the addition of 25 μl of resazurin dye (150 μg/ml) was done. The culture plate was again incubated for another 4 hours. The change of dye colour was monitored by measuring optical density (OD) at 570 nm and 650 nm. The effective OD value for each well was calculated by deducting the OD570 value from its respective OD650 value. The CC50 (50% Cytotoxic Concentration) value of moxifloxacin was measured using the mentioned formula (Suthar et al., 2021).
 
In-Vitro hemolytic assay
 
The hemolytic activity of moxifloxacin on bovine RBCs was evaluated and calculated using the method described earlier (Suthar et al., 2021). The study involved adding 20 μl of RBC suspension to 96 well culture plates, followed by 180 μl of different drug concentrations. The culture plates were kept at 37oC for 90 minutes, then transferred to a microcentrifuge tube and centrifuged for five minutes. The optical density was determined to be 543 nm. DMSO was used in the dilution control well and water was used in the positive control well. HC50 (50% Hemolytic Concentration) value was calculated using the formula mentioned (Suthar et al., 2021).
 
Statistical analysis
 
Regression analysis and correlation were used to calculate the efficacy of moxifloxacin compounds against T. annulata. The differences between the drug-treated wells and the control wells were considered to be statistically significant when the P values were less than 0.05. GraphPad Prism version 10.0 software (GraphPad Software, San Diego, California, USA) was used to facilitate regression analysis and correlation between the drug concentration and its cytotoxicity and hemolytic activity.

RESULTS AND DISCUSSION

In-vitro growth inhibition assay
 
The in-vitro growth inhibition assay of different concentrations of moxifloxacin was mentioned and shown in Table 1. A negative correlation was observed (R=-0.9262; P<0.004) between cell viability and drug concentration. IC50 was calculated and observed at 5559 μM.  Meanwhile, dilution control (DC), positive control (PC) and negative control (NC) showed 94.47%, 0.77% and 100% viability, respectively.

Table 1: Per cent cell viability in different concentrations of Moxifloxacin against in-vitro infected lymphocytes.


 
In-vitro cytotoxicity assay on PBMCs
 
The percent PBMC cytotoxicity of moxifloxacin concentration is shown in Table 2. A positive correlation (R = 0.9673; P<0.001) was observed between moxifloxacin concentration and cell cytotoxicity. The CC50 value for moxifloxacin was calculated and found to be 3,984 μM. At the same time, 100% cytotoxicity was found in well with positive control. However, 30.79% cytotoxicity was observed in well-labelled with dilution control.

Table 2: Per cent cytotoxicity in different concentrations of moxifloxacin on peripheral blood mononuclear cell (PBMC).


 
In-vitro hemolytic assay
 
The per cent hemolysis of moxifloxacin concentration on bovine red blood cells is shown in Table 3. A positive correlation (R = 0.9900; P<0.001) was observed between moxifloxacin concentration and hemolysis. On the other hand, 100% hemolysis was observed in a positive control well with water. The HC50 value of moxifloxacin hemolytic activity was estimated at 2,59,202 μM.

Table 3: Per cent hemolysis in different concentrations of Moxifloxacin on red blood cells (RBCs).


       
Fluoroquinolones (FQs) are very well known to inhibit replication and transcription of bacterial DNA either by targeting DNA gyrase or by targeting topoisomerase-II. The imperative presence of both enzymes precedes bacterial growth (Yadav and Talwar, 2019). Topoisomerases are ubiquitous and found in all living organisms (Deweese and Osheroff, 2009). Many eukaryotic organisms, including the protozoan parasite T. annulata, which causes tropical theileriosis in cattle, depend on the topoisomerase II pathway. The enzyme Topoisomerase II is in charge of controlling DNA topology during transcription, replication and chromosome segregation. It accomplishes this by causing brief double-strand breaks in DNA, which permit one strand of DNA to cross through another and release supercoils produced during these processes. It is essential for the distinct life cycle of T. annulata, especially during the schizont stage when the parasite infects host leukocytes and changes them into cancerous, proliferating cells. Since Theileria species must divide their hosts in order to reproduce, DNA replication and chromosome segregation by Topoisomerase II is essential for their intracellular survival and growth.
       
Resazurin, a blue dye, is converted into resorufin by cells, releasing a pink compound. Microbiologists use intracellular diaphorase enzymes to reduce resazurin, producing a fluorescent signal that can be measured using a spectrophotometer. This method is cost-effective and cell-friendly, allowing researchers to measure cell proliferation kinetics indirectly. It is safe for cells when used at low concentrations and for short incubation periods, making it valuable for studying cell growth. The resazurin reduction assay is a useful tool in studying cell growth (Zalata et al., 1998; O’Brien et al., 2000; Gloeckner et al., 2001).
       
The IC50 value (5559 μM) of moxifloxacin observed in this study was high compared to the CC50 (3984 μM)value on PBMCs. Moxifloxacin can damage normal cells and infected cells in more or less the same manner.  However, based on the selective index (CC50/IC50), further exclusive focused trials on moxifloxacin can be suggested to obtain more accurate data against T. annulata. Limited research work has been documented for moxifloxacin against apicomplexan organisms. Gozalbes et al., (2000) assessed the in-vitro activity of moxifloxacin against T. gondii and reported that moxifloxacin was the potent drug against T. gondii, with IC50 of 5.1 mg/litre. Mahmoudi et al., (2003) evaluated the antimalarial activity of moxifloxacin and observed an IC50 value of 44.8±1.7 μg/ml. The study data can be further used as a baseline value for evaluating moxifloxacin against apicomplexan organisms.

CONCLUSION

The in-vitro T. annulata growth inhibitory efficacy of a Topoisomerase II inhibitor drug, moxifloxacin, was investigated using resazurin. Moxifloxacin inhibited the in-vitro growth of T. annulata with a respective IC50 value of 5,559 μM. The drug was found toxic on the host PBMCs with a CC50 value of 3,984 μM. Moxifloxacin was safe on RBCs with HC50 of 2,59,202 μM. It can be inferred that the Theileria topoisomerase II family includes potential drug targets worthy of further investigation.

ACKNOWLEDGEMENT

The authors acknowledge the Department of Microbiology, the Department of Biotechnology and the Department of Physiology, Sardarkrushinagar, Gujarat, India, for providing the necessary facilities for the study. The author also acknowledges the Principal Veterinary College, Sardar-krushinagar, for giving the required funds for the study.
 
Disclaimers

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsor- ship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    In-vitro Activity of Moxifloxacin against Theileria annulata - A Preliminary Study

    A
    Ankit S. Prajapati1,*
    0000-0002-8940-8840
    A
    Abhinav N. Suthar2
    H
    Harshad H. Panchasara3
    B
    Bhupamani Das4
    P
    Pankaj A. Patel5
    1Veterinary Clinical Complex Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Anand-388 001, Gujarat, India.
    2Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
    3Research Scientist, Livestock Research Station, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
    4Department of Clinics Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
    5Department of Veterinary Physiology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.

    ABSTRACT

    Background: Theileriosis is a tick-borne disease that has a maximum impact on the health and economy of developing countries. A number of clinical manifestations have been found in animals affected with Theileria annulata. A similar trend has been observed in the therapeutic management of theileriosis, especially in cattle. Resistance to available treatment options is one of the significant concerns with theileriosis.

    Methods: The present study evaluated the in-vitro activity of moxifloxacin, the fluoroquinolone compound, against T. annulata in 96 well culture plates using resazurin dye. The histopaque method was used for the separation of infected and healthy PBMCs (Polymorph Blood Mononuclear Cells) for growth inhibition assay of T. annulata and cytotoxicity assay measurement, respectively. The hemolytic assay was also evaluated against normal bovine RBCs (red blood cells). The 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50) values were measured and analyzed using a correlation and regression method.

    Result: The IC50 value of moxifloxacin was observed at 5,559 µM against in-vitro T. annulata. The CC50 value of cytotoxicity on PMBCs was found to be 3,984 µM. However, the HC50 value of hemolytic assay on bovine red blood cells was observed at 2,59,202 µM. The moxifloxacin showed a wide safety margin on red blood cells. Moxifloxacin can be further investigated in-vivo to determine its actual effect on T. annulata.

    INTRODUCTION

    Theileria annulata in livestock is one of the major health concerns. As T. annulata is a vector-borne disease, it covers large groups of animals, including sheep and goats (Verma et al., 2016; Shahedi et al., 2022; Prajapati et al., 2023; Ntesang et al., 2024; Sinha et al., 2024; Alnahari et al., 2026). According to estimates, bovine tropical theileriosis causes an annual loss of about US$ 384.3 million to the Indian livestock sector (Seeber and Steinfelder, 2016; Keroack et al., 2019). The clinical signs exhibited by the animals also vary (Prajapati et al., 2019). T. annulata outbreaks have been reported from India (Sinha et al., 2021; Velusamy et al., 2023). Buparvaquone is the veterinarians’ choice of drug. However, resistance against buparvaquone has been reported for a while (Salim et al., 2019; Yousef et al., 2020).
           
    Fluoroquinolones are drugs indicated primarily as anti-bacterial agents (Pathania and Sharma, 2010). They work on microorganisms by inhibiting the topoisomerase II pathway (Pietrusinski and Staczek, 2006; Fabrega et al., 2009). DNA topoisomerases are essential to the survival of a large number of deadly protozoan parasites. Since the topoisomerase structures of protozoan parasites differ from those of humans, DNA topoisomerase serves as a crucial target for the creation of effective medications for parasitic illnesses in animals (Lamba and Roy, 2022). The exact pathway was evaluated in T. annulata and it was suggested that it could be used for new drug discovery (Lizundia et al., 2009).
           
    Fluoroquinolone compounds have been tested against apicomplexan organisms (Batiha et al., 2020; Fontinha et al., 2020).  Among them, moxifloxacin was highly effective against apicomplexan organisms such as Toxoplasma gondii and Plasmodium falciparum (Gozalbes et al., 2000; Mahmoudi et al., 2003). The present study was executed to assess the in-vitro activity of moxifloxacin, a fluoroquinolone compound, against T. annulata. Further, cytotoxicity and hemolytic activity were also evaluated on healthy PBMCs and red blood cells.

    MATERIALS AND METHODS

    Ethical approval
     
    The Institutional Animal Ethics Committee of the College approved the current study under protocol number IAEC No. VETCOLL/IAEC/2022/20/PROTOCOL-16.
     
    Study period and location
     
    The study was conducted from December 2023 to August 2024 at the Department of Veterinary Medicine, Kamdhenu University, Sardarkrushinagar, Gujarat, India, in collaboration with the Department of Veterinary Biotechnology, Veterinary Microbiology and Veterinary Physiology.
     
    Selection of positive animal
     
    The cattle showed clinical signs of theileriosis and were used for further in-vitro evaluation. T. annulata organism in the blood was confirmed by Giemsa-stained blood smear examination and polymerase chain reaction. T. annulata was explicitly detected in cows using PCR with a 430 bp amplicon using the forward primer CCAGGACCACCCTCA AGTTC and the reverse primer GCATCTAGTTCCTTGG CGGA (Kundave et al., 2015).
     
    In-vitro growth inhibition assay
     
    In-vitro activity of moxifloxacin against T. annulata was evaluated in 96 well culture plate (Gopalakrishnan et al., 2016). Histopaque was used to separate the infected lymphocytes from blood collected from the infected animals with Theileria. Three times, PBS wash was given to lymphocytes and 100 µL (equivalent to 2.5 × 105 cells/ml) seeded onto the plates. A complete medium consisting of RPMI-1640 supplemented with L-glutamine, 100 IU/ml penicillin and 10% fetal bovine serum was used. Incubation conditions for the culture plates were 37oC, 95% relative humidity and 5% CO2. Different concentrations of moxifloxacin from 16000 μM to 250 μM were evaluated against T. annulata. The in-vitro growth inhibition assay was conducted, with resazurin added as 10% of the total volume. After four hours, the optical density (OD) was measured at 570 and 650 nm. Moxifloxacin was purchased from Merck (Merck KGaA, Darmstadt, Germany) company. Triton ×-100 (T×100) was used as a positive control agent and DMSO was used for dilution control. The IC50 value of moxifloxacin was measured using the formula mentioned (Suthar et al., 2021).
     
    In-vitro cytotoxicity assay on PBMCs
     
    The cytotoxicity assay of moxifloxacin was evaluated from healthy PBMCs to assess the detrimental effect of the drug on cells described earlier (Araveti and Srivastava, 2019). Healthy PBMCs were separated by the Histopaque method and mixed with complete media. 100 µl PBMCs were seeded into the 96 well plate. The well culture plates were incubated at 37oC in 5 % CO2 for the next 48 hours. Moxifloxacin concentration was prepared from 8000 μM to 62.5 μM and added into wells. Again, incubation was done for another 24 hours, followed by the addition of 25 μl of resazurin dye (150 μg/ml) was done. The culture plate was again incubated for another 4 hours. The change of dye colour was monitored by measuring optical density (OD) at 570 nm and 650 nm. The effective OD value for each well was calculated by deducting the OD570 value from its respective OD650 value. The CC50 (50% Cytotoxic Concentration) value of moxifloxacin was measured using the mentioned formula (Suthar et al., 2021).
     
    In-Vitro hemolytic assay
     
    The hemolytic activity of moxifloxacin on bovine RBCs was evaluated and calculated using the method described earlier (Suthar et al., 2021). The study involved adding 20 μl of RBC suspension to 96 well culture plates, followed by 180 μl of different drug concentrations. The culture plates were kept at 37oC for 90 minutes, then transferred to a microcentrifuge tube and centrifuged for five minutes. The optical density was determined to be 543 nm. DMSO was used in the dilution control well and water was used in the positive control well. HC50 (50% Hemolytic Concentration) value was calculated using the formula mentioned (Suthar et al., 2021).
     
    Statistical analysis
     
    Regression analysis and correlation were used to calculate the efficacy of moxifloxacin compounds against T. annulata. The differences between the drug-treated wells and the control wells were considered to be statistically significant when the P values were less than 0.05. GraphPad Prism version 10.0 software (GraphPad Software, San Diego, California, USA) was used to facilitate regression analysis and correlation between the drug concentration and its cytotoxicity and hemolytic activity.

    RESULTS AND DISCUSSION

    In-vitro growth inhibition assay
     
    The in-vitro growth inhibition assay of different concentrations of moxifloxacin was mentioned and shown in Table 1. A negative correlation was observed (R=-0.9262; P<0.004) between cell viability and drug concentration. IC50 was calculated and observed at 5559 μM.  Meanwhile, dilution control (DC), positive control (PC) and negative control (NC) showed 94.47%, 0.77% and 100% viability, respectively.

    Table 1: Per cent cell viability in different concentrations of Moxifloxacin against in-vitro infected lymphocytes.


     
    In-vitro cytotoxicity assay on PBMCs
     
    The percent PBMC cytotoxicity of moxifloxacin concentration is shown in Table 2. A positive correlation (R = 0.9673; P<0.001) was observed between moxifloxacin concentration and cell cytotoxicity. The CC50 value for moxifloxacin was calculated and found to be 3,984 μM. At the same time, 100% cytotoxicity was found in well with positive control. However, 30.79% cytotoxicity was observed in well-labelled with dilution control.

    Table 2: Per cent cytotoxicity in different concentrations of moxifloxacin on peripheral blood mononuclear cell (PBMC).


     
    In-vitro hemolytic assay
     
    The per cent hemolysis of moxifloxacin concentration on bovine red blood cells is shown in Table 3. A positive correlation (R = 0.9900; P<0.001) was observed between moxifloxacin concentration and hemolysis. On the other hand, 100% hemolysis was observed in a positive control well with water. The HC50 value of moxifloxacin hemolytic activity was estimated at 2,59,202 μM.

    Table 3: Per cent hemolysis in different concentrations of Moxifloxacin on red blood cells (RBCs).


           
    Fluoroquinolones (FQs) are very well known to inhibit replication and transcription of bacterial DNA either by targeting DNA gyrase or by targeting topoisomerase-II. The imperative presence of both enzymes precedes bacterial growth (Yadav and Talwar, 2019). Topoisomerases are ubiquitous and found in all living organisms (Deweese and Osheroff, 2009). Many eukaryotic organisms, including the protozoan parasite T. annulata, which causes tropical theileriosis in cattle, depend on the topoisomerase II pathway. The enzyme Topoisomerase II is in charge of controlling DNA topology during transcription, replication and chromosome segregation. It accomplishes this by causing brief double-strand breaks in DNA, which permit one strand of DNA to cross through another and release supercoils produced during these processes. It is essential for the distinct life cycle of T. annulata, especially during the schizont stage when the parasite infects host leukocytes and changes them into cancerous, proliferating cells. Since Theileria species must divide their hosts in order to reproduce, DNA replication and chromosome segregation by Topoisomerase II is essential for their intracellular survival and growth.
           
    Resazurin, a blue dye, is converted into resorufin by cells, releasing a pink compound. Microbiologists use intracellular diaphorase enzymes to reduce resazurin, producing a fluorescent signal that can be measured using a spectrophotometer. This method is cost-effective and cell-friendly, allowing researchers to measure cell proliferation kinetics indirectly. It is safe for cells when used at low concentrations and for short incubation periods, making it valuable for studying cell growth. The resazurin reduction assay is a useful tool in studying cell growth (Zalata et al., 1998; O’Brien et al., 2000; Gloeckner et al., 2001).
           
    The IC50 value (5559 μM) of moxifloxacin observed in this study was high compared to the CC50 (3984 μM)value on PBMCs. Moxifloxacin can damage normal cells and infected cells in more or less the same manner.  However, based on the selective index (CC50/IC50), further exclusive focused trials on moxifloxacin can be suggested to obtain more accurate data against T. annulata. Limited research work has been documented for moxifloxacin against apicomplexan organisms. Gozalbes et al., (2000) assessed the in-vitro activity of moxifloxacin against T. gondii and reported that moxifloxacin was the potent drug against T. gondii, with IC50 of 5.1 mg/litre. Mahmoudi et al., (2003) evaluated the antimalarial activity of moxifloxacin and observed an IC50 value of 44.8±1.7 μg/ml. The study data can be further used as a baseline value for evaluating moxifloxacin against apicomplexan organisms.

    CONCLUSION

    The in-vitro T. annulata growth inhibitory efficacy of a Topoisomerase II inhibitor drug, moxifloxacin, was investigated using resazurin. Moxifloxacin inhibited the in-vitro growth of T. annulata with a respective IC50 value of 5,559 μM. The drug was found toxic on the host PBMCs with a CC50 value of 3,984 μM. Moxifloxacin was safe on RBCs with HC50 of 2,59,202 μM. It can be inferred that the Theileria topoisomerase II family includes potential drug targets worthy of further investigation.

    ACKNOWLEDGEMENT

    The authors acknowledge the Department of Microbiology, the Department of Biotechnology and the Department of Physiology, Sardarkrushinagar, Gujarat, India, for providing the necessary facilities for the study. The author also acknowledges the Principal Veterinary College, Sardar-krushinagar, for giving the required funds for the study.
     
    Disclaimers

    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsor- ship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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