In vitro acaricidal effect of the medicinal plant Tinospora cordifolia on pathogenic ticks of domestic cows in the Sikar district, Rajasthan, India

Introduction

Rajasthan's economy is driven by agricultural production, and animal husbandry is an important part of agriculture. Agriculture and animal husbandry engage the people of Rajasthan. Rajasthan ranks second in India in terms of livestock (10.60%) and second in milk production (14.51%). Rajasthan has a healthy livestock population. Ticks are pathogenic ectoparasites that are often found on cows. Ticks transfer many disease-causing germs and make milk dirty. When cows have infections of ticks, they lose blood, which can cause anemia, weight loss, frailty, less productivity, and less milk output. Because of this, the animal gets weak, and productivity goes down a lot. Ticks caused discomfort, sores, and secondary infections that hurt the animal's skin and hide quality. Ticks are known to spread infections that can kill cows, and they often have unforeseen effects. Different acaricidal synthetic drugs are currently used for the control of ticks. Synthetic drugs are not easily available in some of the remotest rural areas and have some serious disadvantages, such as the development of drug resistance with continuous use, adverse drug reactions, or residual effects. The emergence of several problems related to synthetic drugs has encouraged researchers to search for new compounds that can improve the effectiveness of existing medications. The most commonly used in vitro acaricidal synthetic drug is cypermethrin emulsifiable concentrate (Cypermethrin-EC) against ticks (FAO, 2002; Kaplan, 2004; Rahmann and Seip, 2007) [23,29,37] . The Indian Institute of Agricultural Research and the Indian National Animal Health Research Center have also discovered the benefits and efficacy of herbal medicines in eliminating worms, pathogens, and parasites. Most of the published screening investigations are based on in vitro tests of different medicinal plants on species like Ascaris, Ascardia galli, trematodes, and cestodes. Other researchers also observed effects of medicinal plants for controlling cow tick infestations, along with 28 plant species that may have acaricidal properties. A range of medicinal plants produce a number of bioactive phytochemicals with effective acaricidal actions. Many scientists screened the in vitro acaricidal effects of different indigenous medicinal plants against ticks to develop safer, cheaper, eco-friendly, and effective remedies for ruminants. The herbal plant Tinospora cordifolia (giloy) can be used to treat a multitude of ailments, such as viral infections, dengue fever, allergic rhinitis, and a fever that won't go away. The Indian system of medicine (ISM) uses the giloy plant to treat a wide range of diseases, such as diabetes, leprosy, skin problems, fever, diarrhea, and urinary problems. [4,6,19,27,31,33,34,35,40,41,45,49,50,53,54,55,56,57] . However, no research work has been carried out so far on the in vitro acaricidal effects of the medicinal plant Tinospora cordifolia (Neem Giloy) on pathogenic ticks. Therefore, it was decided to undertake the work on "In vitro acaricidal effect of medicinal plant Tinospora cordifolia on pathogenic ticks of domestic cows of the Sikar district." The current research work would be significant because it will help to prepare affordable, environment-friendly, acaricidal, and veterinary herbal drugs to replace the synthetic drug cypermethrin-EC that is currently in use. The findings of the study will improve the socio-economic condition of the livestock farmers of the Sikar district. and2).

Materials and

Collection and identification of ticks:

Live ticks were collected from infected domestic cows from different tehsils in randomly selected villages in the Sikar district of Rajasthan. After removing the ticks carefully from the infected cows, they were again washed several times in physiological saline. These ticks were kept in 0.7% physiological saline for identification and investigations. Species of the ticks were identified with the help of a stereomicroscope according to the description and key mentioned in the reference book (Soulsby, 1982 and Walker et al., 2003) [47,58] (Plate 1; Figs.

Results

Tick infestations were observed in domestic cows of all randomly selected villages in all nine tehsils of Sikar district in the period of March 2022 to February 2025. The tick species were identified by comparing important morphological characters such as scutum pattern, spiracle plate, coxa structure, adanal plates, and capitulum shape of different species. Two species of ticks, such as Hyalomma anatolicum and Rhipicephalus microplus (Boophilus microplus) (Plate 1; Figs. 5 and6), were identified. The present research work has studied the effect of cumulative concentrations of both aqueous and alcoholic extracts from the most effective plant, Tinospora cordifolia stem, on pathogenic ticks in domestic cows.

Untreated pathogenic ticks

Pathogenic ticks were collected carefully from different body parts of domestic cows and subsequently washed multiple times under tap water. After washing, they were transferred into saline solution having dissolved glucose so that ticks would be alive during the experimental procedure of about 08-10 hours. Pathogenic ticks remained actively in mobile condition with whole-body movement through the study period of 1 hr to 5 hr.

In vitro acaricidal effect of different concentrations of the synthetic drug cypermethrin-EC on ticks

Different concentrations of the synthetic drug cypermethrin-EC show an acaricidal effect and healing effect against ticks, as shown in Table 1 and Graph 1. Cypermethrin-EC was most effective against ticks, where it exhibited a 75% mortality rate, and a coefficient of variation of 0% was revealed at a high concentration of 50 mg/ml in 5 hours of exposure time. The lowest mortality rate was 2.78%, and a coefficient of variation of 174.84% was observed in the 20 mg/ml concentration at 1 hour's exposure time. A lower concentration of permethrin was allowed to survive the ticks (Table 1 and Graph 1).

In vitro acaricidal effect and healing effect of different concentrations of aqueous Tinospora cordifolia against pathogenic ticks

The acaricidal effect of the aqueous concentration of Tinospora cordifolia has been shown in Table 2 and Graph 1. In this experiment, aqueous extracts of T. cordifolia resulted in a high mortality rate of 100%, and a 0% coefficient of variation was achieved with these extracts at concentrations of 40 mg/ml and 50 mg/ml after 5 hours of exposure. The aqueous extract concentrations of 10 mg/ml, 20 mg/ml, and 30 mg/ml resulted in mortality rates of 80.56%, 86.11%, and 91.67%, respectively, after 5 hours of exposure; this mortality also induced paralysis in the experimental ticks, bringing them close to death. A mortality rate of 75% was observed at a 50 mg/ml concentration after 4 hours of exposure. The lowest mortality rate was 2.78%, and a coefficient of variation of 174.84% was observed in the 10 mg/ml concentration at 1 hour's exposure time (Table 2 and Graph 1).

In vitro acaricidal effect of different concentrations of alcoholic stem extracts of Tinospora cordifolia on ticks

The acaricidal and healing effects of Tinospora cordifolia alcoholic concentration are represented in Table 3 and Graph 1. The complete mortality rate of 100% is observed from dose concentrations of 20 mg/ml after 4 hours of exposure time onwards. The 10 mg/ml dose concentration also resulted in a significant mortality rate of 94.44% after 4 hours of exposure. All the dose concentrations resulted in a 100% mortality rate at a 5-hour exposure period. The lowest mortality of 5.56% appears at the concentration of 10 mg/ml after 1 hour (Table 3 and Graph 1). The extract of Tinospora cordifolia killed 100% of pathogenic ectoparasite ticks at dose concentrations of 20 mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml after 4 hours of in vitro exposure time. The alcoholic extracts of Tinospora cordifolia have higher acaricidal and healing effectiveness than the aqueous extracts and synthetic drug cypermethrin-EC. The cypermethrin-EC drug was observed to be least effective in comparison with plant extracts, particularly in terms of its acaricidal properties and overall efficacy against ticks (Graph 1).

Discussion

In the present study, the acaricidal effects of the cypermethrin-EC drug and alcoholic and aqueous extracts of Tinospora cordifolia stems were evaluated on the pathogenic ticks. In vitro studies to evaluate the acaricidal and healing effects of plant extracts exhibited dose-dependent mortality, paralyzing or causing death within the incubation period on ticks. Results were compared with those of the cypermethrin drug at different concentrations. Cypermethrin-EC drugs cause low motility, and the alcoholic extract from medicinal plants is stronger than the water extract. Ticks are involved in the transmission of disease-causing pathogens affecting humans, animals, and domestic cows. From the early days to combat tick species, various groups of synthetic acaricides have been used from time to time. The enhanced accessibility, visible efficacy, and easy administration of these acaricides via handheld sprayers, immersion tanks, spot-on, pour-on, injection, and chemicalladen collars are the primary preference of livestock owners across agricultural and livestock sectors (Holdsworth et [11,20,26] . [2,7,10,22,30] . Organic solvents may be more effective in acaricidal bioassays (Adenubi et al., 2016) [2] as the tick's cuticle, which is primarily composed of wax externally and proteins internally, can be better penetrated by non-polar chemical compounds (Chagas et al., 2002) [18] . Similar findings were demonstrated by Ravindran et al. (2011) [38] , who concluded that the polarity of the solvent is inversely related to the acaricidal effects. An effective sustainable tick management strategy involves targeting the juvenile stages of ticks to disrupt their life cycle. This facilitates the management of infestations during favorable weather conditions. Adenubi et al. (2018) [1] say that some of the best medicinal plants are used for keeping pests away. Their eggs only hatch 90% of the time, which kills many ticks. Azadirachtin is a bioactive chemical made from the fruits of Azadirachta indica, commonly known as the neem tree, and Melia azedarach, also known as the chinaberry tree. Azadirachta indica (commonly known as neem) and Melia azedarach (also known as chinaberry) fruit extracts are the sources of the bioactive compound azadirachtin. It prevents tick eggs from developing and molting (Gareh et al., 2022) [24] . This indicates that A. indica extracts, utilized as food additives, may have practical implications in tick management for both public health and veterinary objectives (Pohlit et al., 2011) [36] . One significant limitation in the research on the development of antitick products is the absence of a standardized testing protocol (Adenubi et al., 2018) [1] . A significant variation in testing methodologies, evaluation time, the tick species, and the concentrations of active ingredients in crude extracts, fractions, or the solvents utilized was reported. The disparities complicate the comparison of results across different studies, and the issue was previously highlighted (Bissinger & Roe, 2010; Benelli et al., 2017; Adenubi et al., 2018 and Benelli et al., 2018) [1,9,10,15] . As in many other areas of drug development research, translating laboratory efficacy into field conditions is one of the main obstacles in natural product development research (Borges et al., 2011) [16] . Achieving significant efficacy and adequate residual activity in field conditions presents new challenges, as most of the studies on the acaricidal activity of various plant species are conducted in vitro (De et al., 2012). Additionally, the literature reviewed primarily focused on determining the mechanism of action of the plant extracts or isolated compounds. The development of herbal medicinal products: Quality, safety, and efficacy as key factors are important properties and sources of acaricidal and healing effects in domestic animals (Canigueral et al., 2008 and Bisht et al., 2025) [14,17] . The plant is also a beneficial source of micronutrients such as calcium, iron, phosphorus, manganese, zinc, and copper. T. cordifolia will help in promoting and popularizing the health benefits of this plant and has the potential to control various ailments, such as inflammation, diabetes, and respiratory issues. T. cordifolia has been used to treat various diseases like inflammation, chronic cough, and respiratory distress. The present findings align with other observations of scientists regarding the in vitro acaricidal properties of herbal plants (Sharma et [3,5,8,12,14,21,28,32,39,42,43,44,46,48,52] . The present studies on the in vitro effects of Tinospora cordifolia against ticks elucidated the acaricidal and healing effects based on visual and physical observations of tick mortality; it was found that T. cordifolia is effective against ticks. The study indicated that aqueous and alcoholic extracts of T. cordifolia caused more mortality than the synthetic drug cypermethrin-EC, and higher concentrations were more effective than lower concentrations. It was also noted that aqueous extracts were less strong than alcoholic extracts.

Conclusion

The present study is highly significant for the medicoveterinary field. Findings may help to initiate pharmacological aspects of the extracts of Tinospora cordifolia herbal plants to prepare eco-friendly, less costly, acaricidal, healing, and synthetic veterinary drugs. The current study will improve the socio-economic condition of the domestic cow farmers and livestock holders of the Sikar district by removing the pathogenic ticks with the treatment of the most effective medicinal plant, T. cordifolia. The plant extract of T. cordifolia causes higher mortality in ticks compared to the synthetic drug cypermethrin-EC.

Acknowledgement

AcknowledgmentThe authors would like to acknowledge the veterinary hospital in Nangal, Sikar district, for providing the laboratory facilities, lab technicians, and a senior veterinary doctor, as well as the farmers in the study area for their cordial support during this work.

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