Acaricidal Properties of Herbal Extracts against Deltamethrin Resistant Multi-host Cattle Tick Hyalomma anatolicum

Hyalomma anatolicum, a multi-host tick is widely distributed in tropical and subtropical areas including the Indian subcontinent mainly in arid and semi-arid regions with extremes of temperature. H. anatolicum serves as the major vector of economically important pathogens like Theileria annulata in cattle and buffaloes. Moreover, these ticks act as vector of Crimean-Congo hemorrhagic fever (CCHF) virus, which is widespread in Africa, Asia, Southeast Europe and the Middle East. The CCHF was first confirmed in India during 2010-2011, in Ahmedabad, Gujarat (Maurya et al., 2012). Subsequently few more cases were reported within short span of time; mostly from Gujarat (The New Indian Express, 2015). Tick infestation has emerged as a major threat to dairy industry (Kumar, 2015). Several methods have been employed for the control of ticks, viz. use of chemical acaricides, biological control, tick vaccines and very recently fluorescent dyes like safranin. Presently, chemical method of control with acaricides is the most widely used approach (Li et al., 2007). This has however been complicated by the emergence of drug resistance (Shyma et al., 2019). There is dearth of information on acaricidal resistance in multi-host ticks; however, studies have shown presence of resistance in H. anatolicum in India (Shyma et al., 2012, 2013, Sharma et al., 2018) and in other multi-host ticks elsewhere in the world (Mohammed et al., 2003). In order to tackle the problem of resistance, there is need to assess botanical extracts to offer an ecofriendly acaricide (Singh et al., 2017). Compounds of plant origin constitute an important segment of such effort and have been used in recent past against all the stages (adult, nymph and larva) of economically important tick species with encouraging results (Ghosh et al., 2011). Based on indigenous traditional knowledge of farmers of Gujarat state of India, number of botanicals with acaricidal effect have been identified (Shyma et al., 2014) and such botanicals need a scientific validation by using standard parasitological procedures.
       
Cymbopogon genus is indigenous in tropical and semi-tropical areas of Asia. These are tufted perennial C₄ grasses with numerous stiff stems arising from a short, rhizomatous root stock (Kumar et al., 2000). The insecticidal and repellent property of Cymbopogon citrates has been reported in literature. Citrus aurantiifolia, is a thorny shrub or small tree in the Rutaceae (citrus family) that originated in South East Asia and distributed throughout tropics and sub tropics. Along with delicacy in food the plants are used as insect and mosquito repellents. Annona squamosa, commonly known as custard apple is a native of West Indies and is cultivated throughout India. The extracts of leaves were reported to have larvicidal effect and have shown some acaricidal activity (Ghosh et al., 2007). Carica papaya is considered as an effective and well-known antiparasitic remedy (Rawani et al., 2012). Pioneer work on acaricidal properties of this plant in Rhipicephalus (Boophilus) microplus was done by Shyma et al., (2014). Catharanthus roseus (Vinca rosea) belongs to family Apocynaceae present all over India and many parts of world with insecticidal properties (Velayutham et al., 2012). Although acaricidal properties of some of these plants have been reported earlier but these products are either not evaluated against H. anatolicum or limited information is available in this regard. Keeping in view these perspectives, the present study was undertaken to investigate an alternate and effective control measure against ticks, which have developed resistance against commonly used acaricides by assessing the acaricidal properties of C. citratus, C. aurantiifolia, A. squamosa, C. papaya and C. roseus against larvae of H. anatolicum.

Collection of ticks
 
Fully engorged female ticks were collected from cattle sheds located in Vav Taluka of Banaskantha district, Gujarat (India). Ticks were identified and after washing and drying, kept individually in labeled tubes. Tubes were covered with muslin cloth and kept in BOD incubator (28±1°C and 85±5% relative humidity) for oviposition. The eggs laid were allowed to hatch to larvae.
 
Acaricides
 
Commercially available deltamethrin (1.25%) was used for the experimental bioassay. Various concentrations of deltamethrin (12.5, 25, 50, 100 and 200 ppm) were prepared in distilled water and tested against larvae of H. anatolicum.
 
Larval packet test (LPT)
 
The LPT was conducted as prescribed by FAO with some minor modifications (FAO, 2004). For each active ingredient, a dilution series was set up as described earlier to obtain a concentration gradient resulting in 0 to 100% larval mortality. For each dilution series, a negative control was used. For each concentration, three replicates were made. A volume of approximately 0.6 ml of each solution was applied to Whatman No. 1 (3.75×8.5 cm) filter paper. After saturation, the compound was dried by keeping the filter paper for 30 minutes in incubator at 37°C. Treated and dried parallelograms of paper were folded in half forming equilateral triangular packets and sealed on the sides with adhesive tapes forming an open-ended packet. After insertion of approximately 100 larvae, the open side of each packet was sealed with adhesive tape and the packets were placed in a desiccator kept in BOD incubator maintained at 28°C and 80-85% RH. After 24 hours, the packets were opened and both the live and the dead larvae were counted. The ability of the larvae to walk on the surface of the filter paper was used as the criterion for determining whether larvae were dead or alive. The dose-response data of each acaricide using reference susceptible lines were analyzed.
 
Estimation of resistance status
 
Dose response data were analyzed by probit method (Finney, 1952) using GraphPad Prism software. The lethal concentration for 50% (LC₅₀) and 95% (LC₉₅) values of deltamethrin against H. anatolicum were determined by applying regression equation analysis to the probit transformed data of mortality. Resistance factor (RF) was worked out by the quotient between LC₅₀ of field ticks and LC₅₀ of susceptible line of H. anatolicum (Castro-Janer et al., 2009). The LC₅₀ values of deltamethrin against acaricides susceptible reference IVRI-II line of H. anatolicum were used as per Shyma et al., (2012). On the basis of RF, the resistance status was classified 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 (RF>40.1) resistance.
 
Preparation of plant extracts
 
The leaves of Cymbopogon citratus, Citrus aurantiifolia, Annona squamosa, Carica papaya and Catharanthus roseus were collected from the gardens of university campus, Sardarkrushinagar Dantiwada Agricultural University (SDAU), Gujarat, India. The plant material was identified and authenticated by the Department of Agronomy, SDAU, Sardarkrushinagar, India. The leaves were dried for 7-10 days in the shade at the environmental temperatures (25-35°C) during the daytime. The dried leaves were powdered mechanically using commercial electrical stainless steel blender and the powdered leaves (50 g) were extracted with ethanol in a Soxhlet apparatus (boiling point ranged between 60-80°C) for 8 h. The extract was concentrated and the residue obtained was stored at 4°C. Solvents were removed using evaporator. Crude extracts were dried (at room temperature), weighed and dissolved in methanol for making the dilutions of 12.5 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml which were used for testing the acaricidal potential against deltamethrin resistant H. anatolicum larvae.
 
Bioassay with plant extracts
 
For evaluation of anti-tick activity of plant extracts, LPT as described earlier was adopted. Various concentrations (12.5, 25, 50 and 100 mg/ml) of ethanolic extracts of leaves of C. citratus, C. aurantiifolia, A. squamosa, C. papaya and C. roseus were used and the larval mortality was determined against each concentration after exposure of 24 h.
 
Statistical analysis
 
All the data were expressed as mean±SE. Dose-response data were analysed by probit method (Finney, 1952). Various concentration groups for each botanical were compared using one-way ANOVA. Duncan’s test was used for post hoc analysis. A value of P<0.05 was considered as significant.

Resistance status against chemical acaricides
 
The slope, LC₅₀, R², RF values and the level of resistance to deltamethrin are shown in Table 1. The regression graph of mean mortality of larval ticks plotted against log values of progressively increasing concentrations of deltamethrin has been depicted in Fig 1. From the regression equation, LC₅₀ (95% CL) value of deltamethrin was calculated as 18.22 (14.75-22.50) ppm, thus revealing slow establishment of resistance of level I against deltamethrin (RF=1.60).
 

 

 
In vitro efficacy of various plants extracts
 
In vitro efficacy of leaf extracts of different plants against deltamethrin resistant H. anatolicum were assessed by determining the mortality of larvae, slope, coefficient of determination and LC₅₀ (95% CL). The LC₉₅ values of ethanolic extracts of leaves of C. citratus, C. aurantiifolia, A. squamosa, C. papaya and C. roseus have been shown in Table 2. Larval mortality caused by crude extracts of different herbs, showed concentration dependent increase and it varied from 37.43 to 93.01%, when tested at concentrations ranging from 12.5 to 100 mg/ml. All the concentrations of the herbs, C. aurantiifolia, A. squamosa and C. citratus were found to have significant acaricidal properties with LC₉₅ values of 1.07×10², 6.09×10² and 3.62×10³ mg/ml, respectively (Fig 2). These were followed by C. roseus (3.59×10⁹ mg/ml) and C. papaya (1.52×10¹⁰ mg/ml) (Fig 3).
 

 

 

       
Ticks and tick-borne diseases are the major limitations for sustainable dairy industry in Gujarat region because of the presence of highly favorable environmental conditions for development and propagation of tick stages. Moreover, the increased number of susceptible cross-bred population is further aggravating the problem. The problem of ticks is widely prevalent in India. Under-dosing or over-dosing and repeated use of chemical compounds has led to the development of resistance even in the multi-host tick like H. anatolicum (Shyma et al., 2012, 2013). The H. anatolicum is one of the important ticks infesting dairy animals of India including Gujarat (Ghosh et al., 2007).
       
The occurrence of acaricide resistance is highest in one host ticks of the genus Boophilus (Wharton and Roulston, 1970). This is because a much larger fraction of the total population of such species remains under chemical challenge at any one time than multi host ticks. Moreover, multi-host ticks like H. anatolicum have a longer generation interval compared to Boophilus (Harley, 1966). The immature stages of the multi host ticks often feed on small wild animals, even if the adults tend to prefer large domestic animals. These could also be contributory factors, which protect these ticks from exposure to chemicals. The present study revealed that there is gradual development of resistance in H. anatolicum to deltamethrin. Though, low slope of deltamethrin curve indicates heterogeneity (both resistant and susceptible alleles) of the population in which the deltamethrin resistance seems just to appear and is in establishment phase. It might be due to the fact that the use of deltamethrin in the area was predominated (Sharma et al., 2017) and is used without the veterinarian’s advice.
       
To tackle the problem of resistance against chemical acaricides in ticks, several methods have been advocated and use of phyto-extarcts as acaricidal agents is one of the potential options (Shyma et al., 2014). Use of various phyto-extracts on ticks R. microplus is well documented (Shyma et al., 2014, Ghosh et al., 2015). Indian traditional therapeutic regimen since very beginning has emphasized the use of plants as medicine. Natural herbal extracts contain a range of chemically active ingredients, which can intervene in all biological processes of the insects, thus interrupting their life cycle as well as dispersal and are accepted as an integrated part of ethno-veterinary practices (Zaman et al., 2012). These extracts are further residue less, less toxic to animals, flora and fauna friendly, biodegradable and have meager chance of resistance development. However, studies of herbal acaricides against H. anatolicum are scanty.
       
Many species of Cymbopogon have been reported to possess very good acaricidal properties. Primarily, presence of volatile substances like monoterpenes (citronellal, eugenol, geraniol and limonene etc.) in its leaves is attributed for its acaricidal properties (Shasany et al., 2000). Findings of the present study are in line with the reports of other species of Cymbopogon and appears to be a potential herbal acaricide against deltamethrin resistant H. anatolicum larvae. Further, Citrus and Annona have also been tested for their acaricidal activities on various types of tick species. To the best of our knowledge, there is no previous information regarding the acaricidal activity of these plants against H. anatolicum, although there are enough evidences regarding their insecticidal effects (Ghosh et al., 2015). Strong toxic effect of C. sinensis essential oil on eggs of H. dromedarii, is the only available report pertaining to effect of Citrus spp. on Hyalomma ticks (Habeeb et al., 2007). Alpha-pinene, limonene, linalool, myrcene are reported to be the main components, which are responsible for acaricidal activities (Araújo-Junior et al., 2010).
       
The acaricidal properties of various parts (leaves, seeds, etc.) of A. squamosa against ixodid ticks have been reported from various parts of the world (Ilham et al., 2014). However, comparatively higher mortality of tick larvae was observed in the present study. The acaricidal property of Annona spp. may be due to number of bioactive compounds that are present in the plant as benzyl-tetrahydro-isoquinoline, borneol, camphene, camphor, sesquiterpenes, monoterpenes, phenolic compounds and various alkaloids (Vongkhamchanh et al., 2013). Similarly, in recent past C. papaya has also been evaluated for its acaricidal property against R. microplus (Shyma et al., 2014) but its efficacy against H. anatolicum has not been reported. Current study reports very low efficacy (up to 45% mortality at highest concentration) of C. papaya leaf extract on deltamethrin resistant H. anatolicum, however, authors in their previous study with seed extract of same plant recorded larval mortality of up to 82% in R. microplus.
       
Mansingh and Williams (1998) recorded mortality up to 66% by using crude ethanolic extract of leaves of C. roseus against R. microplus, however, present study recorded lower mortality in deltamethrin resistant H. anatolicum ticks. Pharmacological studies have revealed that C. roseus contains more than 70 different types of alkaloids and chemotherapeutic agents that are effective in treating various types of ailments (Sravanthi et al., 2013).
       
Results of the present study indicated that leaf extracts of C. citratus, C. aurantiifolia and A. squamosa possess very good acaricidal activity against H. anatolicum larvae. It can further be concluded that the ethanolic extracts of leaves of these plants may provide an effective eco-friendly herbal formulation for the control of tick infestation in animals. The beneficial medicinal effects of plant materials typically result from the active compounds present in the plants. These compounds exhibit their effect either as single compound or in combination thereof. The study signifies the use of these botanicals having anti tick activity, which can counter the problems associated with the chemical acaricides. However, future studies are indicated for identification of active ingredients present in these plants that caused the mortality of tick larvae. Different combinations of these extracts can also be studied to exploit the synergistic and antagonistic effects of different active ingredients of these plants. These studies will further be helpful in confirming the presence of some factors in crude extracts that augment the acaricidal property. The ethanolic extracts of these botanicals, particularly, C. aurantiifolia, A. squamosa and C. citratus could be a good source of active compound(s) that can potentially cause tick mortality; thereby making these botanicals valuable for development of sustainable strategy for integrated tick management.

We would like to express our gratitude to Director of Research, Sardarkrushinagar Dantiwada Agricultural University and Dean, College of Veterinary Science and Animal Husbandry, Sardarkrushinagar Dantiwada Agricultural University for providing all the necessary facilities for carrying out this work.