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Full Research Article

Detection of Ivermectin Resistance Status in Rhipicephalus (Boophilus) microplus Ticks Infesting Cattle from Different Agro-climatic Zones of Gujarat, India

D
D.B. Bhinsara1,*
https://orcid.org/0009-0006-1900-9672
J
J.B. Solanki2
N
Niranjan Kumar1
H
H.R. Parsani1
G
Gopal Puri3
D
D.C. Patel4
Y
Y.P. Variya1
1Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari-396 450, Gujarat, India.
2Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
3Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Bhuj (Kutch)-370 001, Gujarat, India.
4Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Junagadh-362 001, Gujarat, India.

ABSTRACT

Background: Ivermectin is widely used against endo and ectoparasites of cattle, but repeated exposure can select resistant populations of the cattle tick Rhipicephalus (Boophilus) microplus. Information on the geographical distribution of ivermectin resistance in Gujarat is limited. This study evaluated ivermectin susceptibility in R. (B.) microplus collected from seven agro-climatic zones of Gujarat, India.

Methods: Engorged female ticks were collected from Southern Hills, Southern Gujarat, Middle Gujarat, North Gujarat, North-West Arid, North Saurashtra and South Saurashtra. Technical-grade ivermectin at 10, 20, 40, 80 and 160 ppm was evaluated by adult immersion test (AIT) and larval packet test (LPT). Adult mortality, egg mass, reproductive index and inhibition of oviposition were assessed in AIT, while larval mortality was recorded in LPT. LC50 values were estimated from concentration-response data and resistance factor (RF) was calculated relative to the susceptible IVRI-1 reference LC50 of 21.68 ppm.

Result: Both assays showed the same regional resistance pattern. Southern Hills and Southern Gujarat were susceptible, with AIT LC50 values of 27.63 and 28.90 ppm and LPT LC50 values of 27.03 and 29.21 ppm, respectively. North-West Arid, North Saurashtra and South Saurashtra showed Level I resistance, whereas Middle Gujarat and North Gujarat showed Level II resistance. Middle Gujarat had the highest LC50 in AIT (120.01 ppm; RF 5.53) and LPT (119.91 ppm; RF 5.53). At 160 ppm, adult mortality in Middle and North Gujarat remained 56.67% and 63.33%, while larval mortality was 56.96% and 59.47%, respectively. The concordance between adult and larval assays demonstrates marked regional variation and supports resistance-guided acaricide use in Gujarat.

INTRODUCTION

Ticks are among the most important ectoparasites affecting cattle in tropical and subtropical production systems. Infestation reduces productivity through irritation, blood loss, hide damage, impaired growth and fertility and transmission of tick-borne pathogens. Although biological control, pasture management, host resistance and vaccination can contribute to control, chemical acaricides remain the principal method used against Rhipicephalus (Boophilus) microplus because of their rapid action and practical field application. Continued dependence on chemical control, however, has created strong selection pressure for acaricide resistance, making integrated tick management increasingly important (FAO, 2004; Mondal et al., 2013; Patel et al., 2019; Willadsen, 2006).
       
Macrocyclic lactones, particularly ivermectin, are extensively used in cattle for their broad-spectrum activity against endoparasites and ectoparasites. Ivermectin acts primarily on ligand-gated chloride channels of invertebrates, producing neuromuscular paralysis and death (Cully et al., 1994; Geary et al., 1993; Omura, 2008). Repeated use, under-dosing and frequent exposure of tick populations to the same active compound can reduce susceptibility over time. Resistance in R. (B.) microplus is of particular concern because its one-host life cycle exposes several parasitic stages on the same animal to treatment and facilitates selection within repeatedly treated herds.
       
Acaricide resistance may involve altered target sensitivity, enhanced detoxification and altered drug transport (Le Gall et al., 2018; Pohl et al., 2011). Phenotypic bioassays remain important because they directly measure field-population responses to acaricide exposure and adult and larval assays provide complementary information for resistance surveillance (FAO, 2004; Shyma et al., 2019; Dalei et al., 2024; Raja et al., 2024; Klafke et al., 2012).
       
Ivermectin resistance in R. (B.) microplus has been documented internationally and in India. The first field report from Punjab demonstrated reduced ivermectin susceptibility in Indian cattle ticks (Singh et al., 2015). Nandi et al., (2018) subsequently standardized an Adult Immersion Test approach for ivermectin resistance monitoring and reported a discriminating concentration using the susceptible IVRI-I reference strain. More recently, Nazim et al., (2022) reported widespread ivermectin resistance in R. (B.) microplus populations from the north-western Himalayas. In Gujarat, ivermectin resistance has also been detected in Hyalomma anatolicum, indicating substantial selection pressure from macrocyclic lactone use in the state (Shyma et al., 2021).
       
Despite these reports, comparative information on ivermectin susceptibility of R. (B.) microplus across the major agro-climatic zones of Gujarat is limited. Regional surveillance is important because acaricide use, farm management, climate and treatment frequency may differ between zones. Accordingly, this study evaluated field isolates of R. (B.) microplus from seven agro-climatic zones of Gujarat using the Adult Immersion Test (AIT) and Larval Packet Test (LPT), estimated LC50 and resistance factors and classified regional resistance status to provide baseline information for evidence-based resistance management.

MATERIALS AND METHODS

Collection and identification of ticks
 
The research work was carried out at the Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari, Gujarat, India. Research period: July 2023 to July 2026. Engorged adult female ticks were collected randomly from organized and unorganized cattle farms and individual cattle sheds in seven agro-climatic zones of Gujarat: Southern Hills (Dangs and Valsad), Southern Gujarat (Navsari, Surat and Tapi), Middle Gujarat (Anand, Vadodara and Bharuch), North Gujarat (Banaskantha and Mehsana), North-West Arid (Patan and Kutch), North Saurashtra (Botad and Surendranagar) and South Saurashtra (Junagadh, Porbandar and Amreli). Ticks were transported in clean ventilated vials to the Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Navsari. Fully engorged females were washed, dried on absorbent paper and identified using standard morphological descriptions and taxonomic keys (Miranpuri and Gill, 1983; Walker, 2003).
 
Rearing and preparation of ivermectin
 
Engorged females selected for rearing were maintained in a BOD incubator at 28±1°C and 85 ± 5% relative humidity. Egg masses were incubated under the same conditions and unfed larvae approximately 12-21 days old were used for larval bioassays. Technical-grade ivermectin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in absolute ethanol. Working concentrations of 10, 20, 40, 80 and 160 ppm were prepared in 1% ethanol-Triton X-100 solution, with a corresponding ivermectin-free control.
 
Adult immersion test
 
The AIT was performed with suitable modifications of Drummond et al., (1973) and FAO (2004). Ten randomly selected engorged females were immersed for 2 min in 10 mL of each ivermectin concentration with gentle agitation. Control ticks were treated similarly without ivermectin. After drying on Whatman No. 1 filter paper, ticks were incubated at approximately 28±2°C and 85±5% relative humidity. Mortality was assessed on day 14 after treatment and egg mass was weighed. Reproductive index (RI) was calculated as egg mass weight/adult female weight. Inhibition of oviposition (IO%) was calculated as:

 
Concentration-response data were used to estimate LC50 values.
 
Larval packet test
 
The LPT was conducted according to FAO (2004) with suitable modifications. Approximately 0.6 mL of each ivermectin dilution was applied to Whatman No. 1 filter paper (3.75 × 8.5 cm). Papers were dried at 37°C for 30 min, folded into packets and sealed. Approximately 100 larvae were introduced into each packet and maintained at about 28°C and 75-85% relative humidity. After 24 h, live and dead larvae were counted; larvae unable to walk on the filter-paper surface were considered dead.
 
Statistical analysis and resistance classification
 
Concentration-mortality data were subjected to linear regression analysis using Microsoft Excel. The slope and coefficient of determination (R2) of each regression equation were recorded. The median lethal concentration (LC50) of ivermectin for each field isolate was estimated from the concentration-mortality regression equation at 50% mortality, following the analytical approach described by Finney (1962). The concentration-mortality relationships for the seven agro-climatic zones are presented in the regression curves for the Adult Immersion Test (AIT) and Larval Packet Test (LPT) (Fig 1 to 14). Resistance factor (RF) was calculated as LC50 of the field isolate divided by the susceptible IVRI-1 reference LC50 of 21.68 ppm, as used in the source dataset (Nandi et al., 2018). Resistance was categorized as susceptible (RF<1.4), Level I (RF 1.5-5.0), Level II (RF 5.1-25.0), Level III (RF 25.1-40) and Level IV (>40.1), following the classification used in the source study (Kumar et al., 2011; Sharma et al., 2012; Shyma et al., 2021).

Fig 1: Regression curve showing mortality of adult ticks due to ivermectin in AIT_Southern hills.



Fig 2: Regression curve showing mortality of adult ticks due to ivermectin in AIT_Southern Gujarat.



Fig 3: Regression curve showing mortality of adult ticks due to ivermectin in AIT_Middle Gujarat.



Fig 4: Regression curve showing mortality of adult ticks due to ivermectin in AIT_North Gujarat.



Fig 5: Regression curve showing mortality of adult ticks due to ivermectin in AIT_North-West Arid.



Fig 6: Regression curve showing mortality of adult ticks due to ivermectin in AIT_North Saurashtra.



Fig 7: Regression curve showing mortality of adult ticks due to ivermectin in AIT_South Saurashtra.



Fig 8: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_Southern Hills.



Fig 9: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_Southern Gujarat.



Fig 10: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_Middle Gujarat.



Fig 11: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_North Gujarat.



Fig 12: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_North-West Arid.



Fig 13: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_North Saurashtra.



Fig 14: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_South Saurashtra.


 
Ethical statement
 
The study involved collection of naturally infesting R. (B.) microplus ticks from cattle followed by in vitro AIT and LPT. No experimental infection, invasive intervention or administration of ivermectin to live animals was performed. Therefore, formal Institutional Animal Ethics Committee approval was not required for these in vitro bioassays. Tick collection was undertaken with permission and informed consent of cattle owners while minimizing animal handling and discomfort.

RESULTS AND DISCUSSION

The summarized AIT findings are presented in Table 1. Southern Hills and Southern Gujarat were the most susceptible populations. Their AIT LC50 values were 27.63 and 28.90 ppm, corresponding to RF values of 1.27 and 1.33, respectively. Both were classified as susceptible. Adult mortality reached 100% at 160 ppm and inhibition of oviposition exceeded 96% in both populations. These findings indicate that ivermectin retained substantial activity against adult R. (B.) microplus in the southern zones included in the study. In contrast, Middle Gujarat and North Gujarat showed the highest AIT resistance. Middle Gujarat recorded an LC50 of 120.01 ppm (RF 5.53), while North Gujarat recorded 113.68 ppm (RF 5.24); both were classified as Level II resistant. Importantly, increasing the concentration to 160 ppm did not produce complete mortality. Adult mortality remained 56.67% in Middle Gujarat and 63.33% in North Gujarat. The persistence of surviving females at the highest tested concentration, together with continued egg production, demonstrates a marked reduction in susceptibility. North-West Arid, North Saurashtra and South Saurashtra showed intermediate responses and were classified as Level I resistant, with LC50 values of 45.62, 51.69 and 55.69 ppm and RF values of 2.10, 2.38 and 2.57, respectively.

Table 1: Adult immersion test-derived ivermectin resistance status of R. (B.) microplus from seven agro-climatic zones of Gujarat.


       
The LPT produced a closely comparable geographical pattern (Table 2). Larval mortality increased progressively with ivermectin concentration in all isolates. Southern Hills had the lowest LPT LC50 (27.03 ppm; RF 1.25), followed by Southern Gujarat (29.21 ppm; RF 1.34); both were susceptible and reached 100% larval mortality at 160 ppm. Middle Gujarat again showed the greatest resistance, with an LC50 of 119.91 ppm and RF of 5.53, followed by North Gujarat with an LC50 of 112.39 ppm and RF of 5.18. Both were Level II resistant. At 160 ppm, larval mortality remained only 56.96% in Middle Gujarat and 59.47% in North Gujarat. North-West Arid, North Saurashtra and South Saurashtra were Level I resistant, with LC50 values of 43.06, 52.53 and 58.54 ppm and RF values of 1.98, 2.42 and 2.70, respectively.

Table 2: Larval packet test-derived ivermectin resistance status of R. (B.) microplus from seven agro-climatic zones of Gujarat.


       
AIT LC50 values ranged from 27.63 to 120.01 ppm, while LPT values ranged from 27.03 to 119.91 ppm, giving an approximately fourfold difference between the least and most susceptible populations. The similar response of adults and larvae indicates that the regional differences were consistent across developmental stages. Middle Gujarat and North Gujarat deserve particular attention from a field-control perspective. In both zones, the AIT LC50 was above the discriminating concentration of 93.54 ppm reported by Nandi et al., (2018). Even at 160 ppm, mortality remained incomplete in adults and larvae, whereas the same concentration produced complete mortality in the susceptible southern populations. Surviving adult females also retained some reproductive activity, which could allow less-susceptible ticks to contribute offspring to the next generation.
       
The pattern observed in Gujarat is comparable with reports from other parts of India, In vitro studies have similarly demonstrated variation in tick responses to acaricides and the utility of immersion and larval assays for susceptibility assessment (Pradeep et al., 2012; Shyma et al., 2019). Singh et al., (2015) documented ivermectin resistance in field populations of R. (B.) microplus from Punjab. Nandi et al., (2018) also found marked variation among field isolates and stressed the value of standardized surveillance. In the north-western Himalayas, Nazim et al., (2022) recorded both Level I and Level II resistance. Finding Level I or Level II resistance in five of the seven Gujarat zones therefore suggests that reduced ivermectin susceptibility is not an isolated local occurrence.
       
A similar concern has already been reported in another cattle tick from Gujarat. Shyma et al., (2021) found ivermectin responses in Hyalomma anatolicum ranging from susceptible to high resistance levels and also documented acaricidal evaluation against resistant cattle ticks and emphasized the need for alternatives to repeated chemical control (Shyma et al., 2022). Although direct comparison between species is not appropriate, reduced susceptibility in both H. anatolicum and R. (B.) microplus points to the need for closer monitoring of ivermectin use and efficacy across the state. Recent work from South Gujarat further demonstrates the continuing epidemiological importance of R. (B.) microplus in cattle (Patel et al., 2024).
       
Reports from outside India show the same general problem. Martins and Furlong (2001) described avermectin resistance in R. microplus in Brazil and subsequent selection experiments showed that repeated ivermectin exposure could increase resistance (Klafke et al., 2006, 2010). Klafke et al., (2012) later assessed in vitro methods for diagnosing this resistance. Ivermectin-resistant populations have also been reported from Mexico (Fernández-Salas et al., 2012). These observations underline the risk of relying repeatedly on one acaricide class.
       
Ivermectin resistance can arise through more than one biological pathway. Besides changes affecting drug targets, detoxification systems and drug transport may influence susceptibility. Pohl et al., (2011) implicated ATP-binding cassette transporters in ivermectin defense in R. microplus, while Le Gall et al. (2018) reported roles for cytochrome P450 monooxygenases, glutathione-S-transferases, esterases and ABC transporters. These mechanisms were not examined in the present work, so the bioassay results demonstrate phenotypic resistance but do not identify its underlying molecular mechanism.
       
Regional differences may reflect the frequency of treatments, repeated use of ivermectin, instances of under-dosing and various farm-management practices, although these factors were not quantified in this study. The reproducible AIT and LPT pattern nevertheless has practical relevance: Level I and Level II areas should not depend on ivermectin alone. Resistance-guided treatment, correct dosing, rotation among acaricides with different modes of action and integrated non-chemical measures are appropriate components of regional control programmes (FAO, 2004; Nandi et al., 2018). Additionally, studies that evaluate plant-derived acaricides, nanoparticles and entomopathogenic fungi support the need for diversified tick-control strategies, particularly in areas where resistance threatens the long-term effectiveness of chemical treatments (Bisen et al., 2011; Mares et al., 2023; Dalei et al., 2024; Raja et al., 2024; Jumade et al., 2025). The study was designed to characterize phenotypic resistance and did not examine molecular markers or quantify ivermectin use at individual farms. Linking future bioassay results with treatment histories and molecular or biochemical markers would help explain why resistance differs among regions. Nevertheless, the close agreement between AIT and LPT provides a useful baseline picture of ivermectin susceptibility across Gujarat.

CONCLUSION

Ivermectin susceptibility of R. (B.) microplus varied markedly across the seven agro-climatic zones of Gujarat. Southern Hills and Southern Gujarat remained susceptible in both AIT and LPT; North-West Arid, North Saurashtra and South Saurashtra showed Level I resistance; and Middle Gujarat and North Gujarat showed Level II resistance. The highest LC50 and RF values occurred in Middle Gujarat, closely followed by North Gujarat and substantial adult and larval survival persisted at 160 ppm in both zones. The close agreement between adult and larval assays provides an important baseline for regional acaricide resistance surveillance. Routine susceptibility monitoring, correct dosing, avoidance of indiscriminate ivermectin use, rotation of acaricides with different modes of action and integrated tick-management practices are recommended to slow further loss of ivermectin efficacy.

ACKNOWLEDGEMENT

The authors are grateful to the Vice Chancellor and Director of Research, Kamdhenu University, Gandhinagar and the Principal, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari, for providing facilities, infrastructure and financial support.
 
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
 
The study involved collection of naturally infesting Rhipicephalus (Boophilus) microplus ticks from cattle for in vitro bioassays. Tick collection was undertaken with permission and informed consent of cattle owners, while minimizing animal handling and discomfort. No experimental infection or administration of ivermectin to live animals was performed.

CONFLICT OF INTEREST

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

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    Detection of Ivermectin Resistance Status in Rhipicephalus (Boophilus) microplus Ticks Infesting Cattle from Different Agro-climatic Zones of Gujarat, India

    D
    D.B. Bhinsara1,*
    https://orcid.org/0009-0006-1900-9672
    J
    J.B. Solanki2
    N
    Niranjan Kumar1
    H
    H.R. Parsani1
    G
    Gopal Puri3
    D
    D.C. Patel4
    Y
    Y.P. Variya1
    1Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari-396 450, Gujarat, India.
    2Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar-385 506, Gujarat, India.
    3Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Bhuj (Kutch)-370 001, Gujarat, India.
    4Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Junagadh-362 001, Gujarat, India.

    ABSTRACT

    Background: Ivermectin is widely used against endo and ectoparasites of cattle, but repeated exposure can select resistant populations of the cattle tick Rhipicephalus (Boophilus) microplus. Information on the geographical distribution of ivermectin resistance in Gujarat is limited. This study evaluated ivermectin susceptibility in R. (B.) microplus collected from seven agro-climatic zones of Gujarat, India.

    Methods: Engorged female ticks were collected from Southern Hills, Southern Gujarat, Middle Gujarat, North Gujarat, North-West Arid, North Saurashtra and South Saurashtra. Technical-grade ivermectin at 10, 20, 40, 80 and 160 ppm was evaluated by adult immersion test (AIT) and larval packet test (LPT). Adult mortality, egg mass, reproductive index and inhibition of oviposition were assessed in AIT, while larval mortality was recorded in LPT. LC50 values were estimated from concentration-response data and resistance factor (RF) was calculated relative to the susceptible IVRI-1 reference LC50 of 21.68 ppm.

    Result: Both assays showed the same regional resistance pattern. Southern Hills and Southern Gujarat were susceptible, with AIT LC50 values of 27.63 and 28.90 ppm and LPT LC50 values of 27.03 and 29.21 ppm, respectively. North-West Arid, North Saurashtra and South Saurashtra showed Level I resistance, whereas Middle Gujarat and North Gujarat showed Level II resistance. Middle Gujarat had the highest LC50 in AIT (120.01 ppm; RF 5.53) and LPT (119.91 ppm; RF 5.53). At 160 ppm, adult mortality in Middle and North Gujarat remained 56.67% and 63.33%, while larval mortality was 56.96% and 59.47%, respectively. The concordance between adult and larval assays demonstrates marked regional variation and supports resistance-guided acaricide use in Gujarat.

    INTRODUCTION

    Ticks are among the most important ectoparasites affecting cattle in tropical and subtropical production systems. Infestation reduces productivity through irritation, blood loss, hide damage, impaired growth and fertility and transmission of tick-borne pathogens. Although biological control, pasture management, host resistance and vaccination can contribute to control, chemical acaricides remain the principal method used against Rhipicephalus (Boophilus) microplus because of their rapid action and practical field application. Continued dependence on chemical control, however, has created strong selection pressure for acaricide resistance, making integrated tick management increasingly important (FAO, 2004; Mondal et al., 2013; Patel et al., 2019; Willadsen, 2006).
           
    Macrocyclic lactones, particularly ivermectin, are extensively used in cattle for their broad-spectrum activity against endoparasites and ectoparasites. Ivermectin acts primarily on ligand-gated chloride channels of invertebrates, producing neuromuscular paralysis and death (Cully et al., 1994; Geary et al., 1993; Omura, 2008). Repeated use, under-dosing and frequent exposure of tick populations to the same active compound can reduce susceptibility over time. Resistance in R. (B.) microplus is of particular concern because its one-host life cycle exposes several parasitic stages on the same animal to treatment and facilitates selection within repeatedly treated herds.
           
    Acaricide resistance may involve altered target sensitivity, enhanced detoxification and altered drug transport (Le Gall et al., 2018; Pohl et al., 2011). Phenotypic bioassays remain important because they directly measure field-population responses to acaricide exposure and adult and larval assays provide complementary information for resistance surveillance (FAO, 2004; Shyma et al., 2019; Dalei et al., 2024; Raja et al., 2024; Klafke et al., 2012).
           
    Ivermectin resistance in R. (B.) microplus has been documented internationally and in India. The first field report from Punjab demonstrated reduced ivermectin susceptibility in Indian cattle ticks (Singh et al., 2015). Nandi et al., (2018) subsequently standardized an Adult Immersion Test approach for ivermectin resistance monitoring and reported a discriminating concentration using the susceptible IVRI-I reference strain. More recently, Nazim et al., (2022) reported widespread ivermectin resistance in R. (B.) microplus populations from the north-western Himalayas. In Gujarat, ivermectin resistance has also been detected in Hyalomma anatolicum, indicating substantial selection pressure from macrocyclic lactone use in the state (Shyma et al., 2021).
           
    Despite these reports, comparative information on ivermectin susceptibility of R. (B.) microplus across the major agro-climatic zones of Gujarat is limited. Regional surveillance is important because acaricide use, farm management, climate and treatment frequency may differ between zones. Accordingly, this study evaluated field isolates of R. (B.) microplus from seven agro-climatic zones of Gujarat using the Adult Immersion Test (AIT) and Larval Packet Test (LPT), estimated LC50 and resistance factors and classified regional resistance status to provide baseline information for evidence-based resistance management.

    MATERIALS AND METHODS

    Collection and identification of ticks
     
    The research work was carried out at the Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari, Gujarat, India. Research period: July 2023 to July 2026. Engorged adult female ticks were collected randomly from organized and unorganized cattle farms and individual cattle sheds in seven agro-climatic zones of Gujarat: Southern Hills (Dangs and Valsad), Southern Gujarat (Navsari, Surat and Tapi), Middle Gujarat (Anand, Vadodara and Bharuch), North Gujarat (Banaskantha and Mehsana), North-West Arid (Patan and Kutch), North Saurashtra (Botad and Surendranagar) and South Saurashtra (Junagadh, Porbandar and Amreli). Ticks were transported in clean ventilated vials to the Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Navsari. Fully engorged females were washed, dried on absorbent paper and identified using standard morphological descriptions and taxonomic keys (Miranpuri and Gill, 1983; Walker, 2003).
     
    Rearing and preparation of ivermectin
     
    Engorged females selected for rearing were maintained in a BOD incubator at 28±1°C and 85 ± 5% relative humidity. Egg masses were incubated under the same conditions and unfed larvae approximately 12-21 days old were used for larval bioassays. Technical-grade ivermectin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in absolute ethanol. Working concentrations of 10, 20, 40, 80 and 160 ppm were prepared in 1% ethanol-Triton X-100 solution, with a corresponding ivermectin-free control.
     
    Adult immersion test
     
    The AIT was performed with suitable modifications of Drummond et al., (1973) and FAO (2004). Ten randomly selected engorged females were immersed for 2 min in 10 mL of each ivermectin concentration with gentle agitation. Control ticks were treated similarly without ivermectin. After drying on Whatman No. 1 filter paper, ticks were incubated at approximately 28±2°C and 85±5% relative humidity. Mortality was assessed on day 14 after treatment and egg mass was weighed. Reproductive index (RI) was calculated as egg mass weight/adult female weight. Inhibition of oviposition (IO%) was calculated as:

     
    Concentration-response data were used to estimate LC50 values.
     
    Larval packet test
     
    The LPT was conducted according to FAO (2004) with suitable modifications. Approximately 0.6 mL of each ivermectin dilution was applied to Whatman No. 1 filter paper (3.75 × 8.5 cm). Papers were dried at 37°C for 30 min, folded into packets and sealed. Approximately 100 larvae were introduced into each packet and maintained at about 28°C and 75-85% relative humidity. After 24 h, live and dead larvae were counted; larvae unable to walk on the filter-paper surface were considered dead.
     
    Statistical analysis and resistance classification
     
    Concentration-mortality data were subjected to linear regression analysis using Microsoft Excel. The slope and coefficient of determination (R2) of each regression equation were recorded. The median lethal concentration (LC50) of ivermectin for each field isolate was estimated from the concentration-mortality regression equation at 50% mortality, following the analytical approach described by Finney (1962). The concentration-mortality relationships for the seven agro-climatic zones are presented in the regression curves for the Adult Immersion Test (AIT) and Larval Packet Test (LPT) (Fig 1 to 14). Resistance factor (RF) was calculated as LC50 of the field isolate divided by the susceptible IVRI-1 reference LC50 of 21.68 ppm, as used in the source dataset (Nandi et al., 2018). Resistance was categorized as susceptible (RF<1.4), Level I (RF 1.5-5.0), Level II (RF 5.1-25.0), Level III (RF 25.1-40) and Level IV (>40.1), following the classification used in the source study (Kumar et al., 2011; Sharma et al., 2012; Shyma et al., 2021).

    Fig 1: Regression curve showing mortality of adult ticks due to ivermectin in AIT_Southern hills.



    Fig 2: Regression curve showing mortality of adult ticks due to ivermectin in AIT_Southern Gujarat.



    Fig 3: Regression curve showing mortality of adult ticks due to ivermectin in AIT_Middle Gujarat.



    Fig 4: Regression curve showing mortality of adult ticks due to ivermectin in AIT_North Gujarat.



    Fig 5: Regression curve showing mortality of adult ticks due to ivermectin in AIT_North-West Arid.



    Fig 6: Regression curve showing mortality of adult ticks due to ivermectin in AIT_North Saurashtra.



    Fig 7: Regression curve showing mortality of adult ticks due to ivermectin in AIT_South Saurashtra.



    Fig 8: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_Southern Hills.



    Fig 9: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_Southern Gujarat.



    Fig 10: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_Middle Gujarat.



    Fig 11: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_North Gujarat.



    Fig 12: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_North-West Arid.



    Fig 13: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_North Saurashtra.



    Fig 14: Regression curve showing mortality of ticks larvae due to ivermectin in LPT_South Saurashtra.


     
    Ethical statement
     
    The study involved collection of naturally infesting R. (B.) microplus ticks from cattle followed by in vitro AIT and LPT. No experimental infection, invasive intervention or administration of ivermectin to live animals was performed. Therefore, formal Institutional Animal Ethics Committee approval was not required for these in vitro bioassays. Tick collection was undertaken with permission and informed consent of cattle owners while minimizing animal handling and discomfort.

    RESULTS AND DISCUSSION

    The summarized AIT findings are presented in Table 1. Southern Hills and Southern Gujarat were the most susceptible populations. Their AIT LC50 values were 27.63 and 28.90 ppm, corresponding to RF values of 1.27 and 1.33, respectively. Both were classified as susceptible. Adult mortality reached 100% at 160 ppm and inhibition of oviposition exceeded 96% in both populations. These findings indicate that ivermectin retained substantial activity against adult R. (B.) microplus in the southern zones included in the study. In contrast, Middle Gujarat and North Gujarat showed the highest AIT resistance. Middle Gujarat recorded an LC50 of 120.01 ppm (RF 5.53), while North Gujarat recorded 113.68 ppm (RF 5.24); both were classified as Level II resistant. Importantly, increasing the concentration to 160 ppm did not produce complete mortality. Adult mortality remained 56.67% in Middle Gujarat and 63.33% in North Gujarat. The persistence of surviving females at the highest tested concentration, together with continued egg production, demonstrates a marked reduction in susceptibility. North-West Arid, North Saurashtra and South Saurashtra showed intermediate responses and were classified as Level I resistant, with LC50 values of 45.62, 51.69 and 55.69 ppm and RF values of 2.10, 2.38 and 2.57, respectively.

    Table 1: Adult immersion test-derived ivermectin resistance status of R. (B.) microplus from seven agro-climatic zones of Gujarat.


           
    The LPT produced a closely comparable geographical pattern (Table 2). Larval mortality increased progressively with ivermectin concentration in all isolates. Southern Hills had the lowest LPT LC50 (27.03 ppm; RF 1.25), followed by Southern Gujarat (29.21 ppm; RF 1.34); both were susceptible and reached 100% larval mortality at 160 ppm. Middle Gujarat again showed the greatest resistance, with an LC50 of 119.91 ppm and RF of 5.53, followed by North Gujarat with an LC50 of 112.39 ppm and RF of 5.18. Both were Level II resistant. At 160 ppm, larval mortality remained only 56.96% in Middle Gujarat and 59.47% in North Gujarat. North-West Arid, North Saurashtra and South Saurashtra were Level I resistant, with LC50 values of 43.06, 52.53 and 58.54 ppm and RF values of 1.98, 2.42 and 2.70, respectively.

    Table 2: Larval packet test-derived ivermectin resistance status of R. (B.) microplus from seven agro-climatic zones of Gujarat.


           
    AIT LC50 values ranged from 27.63 to 120.01 ppm, while LPT values ranged from 27.03 to 119.91 ppm, giving an approximately fourfold difference between the least and most susceptible populations. The similar response of adults and larvae indicates that the regional differences were consistent across developmental stages. Middle Gujarat and North Gujarat deserve particular attention from a field-control perspective. In both zones, the AIT LC50 was above the discriminating concentration of 93.54 ppm reported by Nandi et al., (2018). Even at 160 ppm, mortality remained incomplete in adults and larvae, whereas the same concentration produced complete mortality in the susceptible southern populations. Surviving adult females also retained some reproductive activity, which could allow less-susceptible ticks to contribute offspring to the next generation.
           
    The pattern observed in Gujarat is comparable with reports from other parts of India, In vitro studies have similarly demonstrated variation in tick responses to acaricides and the utility of immersion and larval assays for susceptibility assessment (Pradeep et al., 2012; Shyma et al., 2019). Singh et al., (2015) documented ivermectin resistance in field populations of R. (B.) microplus from Punjab. Nandi et al., (2018) also found marked variation among field isolates and stressed the value of standardized surveillance. In the north-western Himalayas, Nazim et al., (2022) recorded both Level I and Level II resistance. Finding Level I or Level II resistance in five of the seven Gujarat zones therefore suggests that reduced ivermectin susceptibility is not an isolated local occurrence.
           
    A similar concern has already been reported in another cattle tick from Gujarat. Shyma et al., (2021) found ivermectin responses in Hyalomma anatolicum ranging from susceptible to high resistance levels and also documented acaricidal evaluation against resistant cattle ticks and emphasized the need for alternatives to repeated chemical control (Shyma et al., 2022). Although direct comparison between species is not appropriate, reduced susceptibility in both H. anatolicum and R. (B.) microplus points to the need for closer monitoring of ivermectin use and efficacy across the state. Recent work from South Gujarat further demonstrates the continuing epidemiological importance of R. (B.) microplus in cattle (Patel et al., 2024).
           
    Reports from outside India show the same general problem. Martins and Furlong (2001) described avermectin resistance in R. microplus in Brazil and subsequent selection experiments showed that repeated ivermectin exposure could increase resistance (Klafke et al., 2006, 2010). Klafke et al., (2012) later assessed in vitro methods for diagnosing this resistance. Ivermectin-resistant populations have also been reported from Mexico (Fernández-Salas et al., 2012). These observations underline the risk of relying repeatedly on one acaricide class.
           
    Ivermectin resistance can arise through more than one biological pathway. Besides changes affecting drug targets, detoxification systems and drug transport may influence susceptibility. Pohl et al., (2011) implicated ATP-binding cassette transporters in ivermectin defense in R. microplus, while Le Gall et al. (2018) reported roles for cytochrome P450 monooxygenases, glutathione-S-transferases, esterases and ABC transporters. These mechanisms were not examined in the present work, so the bioassay results demonstrate phenotypic resistance but do not identify its underlying molecular mechanism.
           
    Regional differences may reflect the frequency of treatments, repeated use of ivermectin, instances of under-dosing and various farm-management practices, although these factors were not quantified in this study. The reproducible AIT and LPT pattern nevertheless has practical relevance: Level I and Level II areas should not depend on ivermectin alone. Resistance-guided treatment, correct dosing, rotation among acaricides with different modes of action and integrated non-chemical measures are appropriate components of regional control programmes (FAO, 2004; Nandi et al., 2018). Additionally, studies that evaluate plant-derived acaricides, nanoparticles and entomopathogenic fungi support the need for diversified tick-control strategies, particularly in areas where resistance threatens the long-term effectiveness of chemical treatments (Bisen et al., 2011; Mares et al., 2023; Dalei et al., 2024; Raja et al., 2024; Jumade et al., 2025). The study was designed to characterize phenotypic resistance and did not examine molecular markers or quantify ivermectin use at individual farms. Linking future bioassay results with treatment histories and molecular or biochemical markers would help explain why resistance differs among regions. Nevertheless, the close agreement between AIT and LPT provides a useful baseline picture of ivermectin susceptibility across Gujarat.

    CONCLUSION

    Ivermectin susceptibility of R. (B.) microplus varied markedly across the seven agro-climatic zones of Gujarat. Southern Hills and Southern Gujarat remained susceptible in both AIT and LPT; North-West Arid, North Saurashtra and South Saurashtra showed Level I resistance; and Middle Gujarat and North Gujarat showed Level II resistance. The highest LC50 and RF values occurred in Middle Gujarat, closely followed by North Gujarat and substantial adult and larval survival persisted at 160 ppm in both zones. The close agreement between adult and larval assays provides an important baseline for regional acaricide resistance surveillance. Routine susceptibility monitoring, correct dosing, avoidance of indiscriminate ivermectin use, rotation of acaricides with different modes of action and integrated tick-management practices are recommended to slow further loss of ivermectin efficacy.

    ACKNOWLEDGEMENT

    The authors are grateful to the Vice Chancellor and Director of Research, Kamdhenu University, Gandhinagar and the Principal, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari, for providing facilities, infrastructure and financial support.
     
    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
     
    The study involved collection of naturally infesting Rhipicephalus (Boophilus) microplus ticks from cattle for in vitro bioassays. Tick collection was undertaken with permission and informed consent of cattle owners, while minimizing animal handling and discomfort. No experimental infection or administration of ivermectin to live animals was performed.

    CONFLICT OF INTEREST

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

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