ORIGINAL PAPER

Acaricidal effect of herbal extracts against cattle tickRhipicephalus (Boophilus) microplus using in vitro studies

K. P. Shyma & J. P. Gupta & S. Ghosh & K. K. Patel &Veer Singh

Received: 10 February 2014 /Accepted: 24 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The crude methanolic extract of Daturastramonium, Azadirachta indica, and Calotropis proceraleaves, Allium sativum (AS) cloves, and Carica papaya (CP)seeds collected from Banaskanta, Gujarat (India) was testedfor its acaricidal properties against Rhipicephalus (Boophilus)microplus. The percent adult mortality within 15 days, repro-ductive index, percentage inhibition of oviposition, hatchingof laid ova, and percentage larval mortality were studied atconcentrations of 12.5, 25, 50, and 100 mg/ml. At the highestconcentration (100 mg/ml), the adult tick mortality was 66.67,73.33, 80.00, and 93.33 % for C. procera, D. stramonium,A. sativum, and C. papaya extracts, respectively, and it wasstatistically significant (P<0.001). However, for A. indica,mortality was low and estimated to be 33.33 %. Inhibition ofoviposition at the highest concentration of A. indica,C. procera, D. stramonium, A. sativum, and C. papayaextract-treated ticks was 20.73, 71.34, 77.17, 85.83, and100.00 %, respectively. Inhibition of fecundity of treatedgroups differed significantly from the control and was

concentration dependent. Larvae treated with all the test-ed concen t r a t i ons o f A. ind ica , C. procera ,D. stramonium, A. sativum, and C. papaya extracts bylarval packet test showed significant mortali ty(P<0.001) than that of control tick larvae, and at thehighest concentration, it was 55.2, 63.2, 71.8, 69.0, and82.2 %, respectively. Garlic cloves and papaya seedextract produced complete failure of eclosion of eggsfrom the treated ticks even at lower concentrations;however, neem, calotropis, and datura was capable ofreducing hatchability by 20, 50, and 70 %, respectively.The results pointed that the crude extracts of A. sativumcloves and C. papaya seeds have very good acaricidalproperties and could be a potential component of alter-native R. (B.) microplus tick control strategy.

Keywords Allium sativum .Carica papaya .Daturastramonium . Azadirachta indica .Calotropis procera .

Acaricidal activities . Eclosion . Rhipicephalus (Boophilus)microplus

Introduction

Ticks and tick-borne diseases (TTBD) of cattle poseserious threats on the growth of dairy industry (FAO1996) and cause a significant reduction in profit bysevere loss in lactation (McLeod and Kristjanson1999). The problem of TTBD has been ranked high interms of their adverse impact on the livelihood of poorfarming communities in developing countries includingIndia. Rhipicephalus (Boophilus) microplus is the mostwidely distributed ticks and serves as the major vectorof economically important pathogens like Babesiabigemina and Anaplasma marginale (Ghosh et al.2007a, b). The epidemiology of tick infestations and

K. P. Shyma :V. Singh (*)Department of Parasitology, Sardar Krushinagar DantiwadaAgricultural University, Sardarkrushinagar 385506, Indiae-mail: veersinghgau@yahoo.com

K. P. Shymae-mail: dr.shymakpvet@gmail.com

J. P. GuptaDepartment of Animal Genetics and Breeding, Sardar KrushinagarDantiwada Agricultural University, Sardarkrushinagar 385506, India

S. GhoshDivision of Parasitology, Indian Veterinary Research Institute,Izatnagar, Uttar Pradesh 243122, India

K. K. PatelBanas Dairy, Banaskantha, Gujarat, India

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their vectorial potential are likely to change with thechanging environmental conditions due to globalwarming. In this scenario, the control of tick infestationis considered a major factor for sustainable livestockproduction world wide and especially in tropical countrylike India.

At present, tick control in India is based on largescale repeated use of synthetic acaricides of three majorclasses, namely organophosphates, carbamates, and py-rethroids. Besides various indirect effects like environ-mental pollution due to large-scale use of syntheticacaricides, there is an emerging problem of developmentof resistance in the ticks to these acaricides. The devel-opment of acaricidal resistance in ticks arrests the prog-ress of tick control programs in a country (Michaelet al. 2001). In Indian subcontinent, the incidence ofacaricide resistance has been widely reported in onehost ticks of the genus Boophilus (Singh et al. 2012;Kumar et al. 2013; Shyma et al. 2013) and in multihosttick Hyalomma anatolicum (Shyma et al. 2012). This isbecause a much larger fraction of the total population ofsuch species remains under chemical challenge.

The loss of acaricidal effectiveness as a sequel to thedevelopment of resistance entails increased applicationfrequencies and dosage of the acaricides, and finally,adoption of more expensive replacement compound asnew acaricides becomes increasingly more cost intensiveand difficult to discover (NRC 1986). Emphasis should bemade for a reliable compound against the ticks which areeffective against all the stages of the ticks and does notleave any residues in the environment as per requirementof World Trade Organization. Compounds of plant originconstitute an important segment of such effort and havebeen used in recent past against all the stages (adult,nymph, and larva) of economically important tick specieswith encouraging results (Mehlhorn et al. 2005; Mehlhorn2008; Kamaraj et al. 2010; Ghosh et al. 2011; Semmleret al. 2011; Singh et al. 2013). Keeping in view of theabove, present study was undertaken to evaluate thepotential of crude extracts of garlic cloves (Alliumsativum), papaya seeds (Carica papaya), datura leaf(Datura stramonium), neem leaves (Azadirachta indica),and calotropis leaves (Calotropis procera) as an herbalacaricide against R. (B.) microplus.

Materials and methods

Preparation of plant extract

The plant materials viz. garlic cloves (GC), papaya seeds(PS), datura leaves (DL), neem leaves (NL), andcalotropis leaves (CL) were collected from Dantiwada

Taluk of Banaskanta district, Gujarat, India. The plantmaterials were identified, cleaned, and dried in shadeat room temperature. Dried plant materials were finelypulverized using a grinder. Extraction was carried outby maceration wherein 100 g of the powdered materialwas taken and dissolved in methanol for making thefollowing dilutions of 6.25, 12.5, 25, 50, and100 mg/ml that were used for the testing of ticks byadult immersion test (AIT) and larval packet test(LPT).

Collection of field isolates of R. (B.) microplus ticks

The engorged R. (B.) microplus ticks were collected fromdifferent areas of northern Gujarat. Both organized andunorganized farms were selected to collect the samples.The farms in these areas have dairy herd of mainly crossbred population. In organized farms, the animals are keptin zero grazing system whereas unorganized farms aremainly reared by farmers in villages. Engorged femalesof R. (B.) microplus ticks were handpicked during postmonsoon months, from the body of cattle and buffaloes ofvarying age and in the vicinity of cattle pens. The tickswere washed in tap water and dried on an absorbentpaper. A total of 465 adult engorged female ticks wereused for the present study. Out of this, 15 ticks wereseparated and were held individually at 28±1 °C and 85±5 % relative humidity in labeled glass bottle with the mouthcovered by muslin cloth for oviposition. The eggs wereallowed to hatch to larvae in 18–25 days under similar condi-tions of incubation. The larvae were used for performing“larval packet test” (LPT). The remaining 450 ticks weregathered in to five groups (one for each plant extract) eachof 90 ticks. Again, these 90 ticks were divided into six groups(five treatments and one control) each comprising of 15 ticksas five ticks each in three replicates. Each group of ticks wasused to estimate the acaricidal effects of respective concentra-tion of plant extract by AIT.

Adult immersion test

The AIT was conducted according to FAO (1984) guide-lines. The engorged female ticks from the field werewashed thoroughly thrice with distilled water and keptfor drying on filter paper and weighed. The ticks wereimmersed in crude extract of plant material for 5 min. Thecontrol group was immersed in methanol. The ticks werethen placed on Petri dishes over Whatman filter paperno.1. All the Petri dishes with treated ticks were kept atroom temperature for 24 h. After 24 h, ticks were trans-ferred to glass vials covered with muslin cloth and kept indesiccators having 85±2 % relative humidity and placedin BOD incubator at 28±2 °C. These ticks were observed

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for oviposition and death up to 15 days. The percent adulttick mortality and the weight of the eggs laid by thetreated ticks were recorded in comparison with the con-trol. The eggs were incubated at the same condition, andthe percentage of hatched eggs was estimated visually.The index of egg laying and percentage inhibition of

fecundity were calculated using formulae 1 and 2, respec-tively (Goncalves et al. 2007).

Reproductive index RIð Þ ¼ Weight of eggs laid mgð ÞWeight of adult females mgð Þ

ð1Þ

Percentage inhibition of oviposition IO%ð Þ ¼ RI control groupð Þ‐RI treated groupð ÞRI control groupð Þ � 100

Larval packet test

The larval packet test (LPT) was conducted according to FAO(1984) guidelines with minor modifications. Briefly, 0.6 ml ofcrude plant extract in methanol was used to impregnate 3.75-cm by 8.5-cmWhatman filter paper rectangles. The compoundwas dried by keeping the filter paper for 30 min in incubator at37 °C. The rectangles were folded in half and sealed on thesides with adhesive tapes, forming an open-ended packet toplace tick larvae. After the insertion of approximately 200larvae (12 days old), the top of each packet was sealed withadhesive tape, and the packets were placed in a desiccatorsplaced in BOD incubator maintained at 28±1 °C and 85±5 %RH. The packets were removed after 24 h, and larval mortalitywas calculated. Control group was treated with 0.6 ml ofmethanol similar to the abovementioned procedure.

Statistical analysis

Data were expressed as the mean±SEM. Groups were com-pared using one-way ANOVA for repeated measurementsusing SPSS software. Duncan’s test was used for post hocanalysis. A value of P<0.05 was considered significant.

Results

The results of adult immersion test using the crude methanolicextracts of NL, CL, DL, GC, and PS are shown in Table 1. Theefficacy of crude extract of different herbs against R. (B.)microplus was assessed by estimating the percent adult mor-tality (cut off date for mortality observation was fixed at15 days post-treatment and eggs mass laid up to this day wasconsidered under the study), reproductive index, inhibition ofoviposition, hatching rate, and larval mortality. The percent

mortality caused by the methanolic extracts of different plantsvaried from 6.67 to 93.33 %, when tested at concentrationsranging from 12.5 to 100 %. It is noteworthy that the live ticks,treated with different concentrations of the various extracts,laid egg masses which were significantly (P<0.001) lower inweight than the egg masses of control ticks except the tickstreated with NL extract, in spite of the fact that the percentageof adult mortality was low in the treated ticks (Table 1).Visually hatching of the eggs was completely blocked onlyby GC and PS extracts; however, other extracts were onlypartially able to block the hatching. Further, larva treated withdifferent concentrations of the various extracts showed signifi-cant mortality (p<0.001) than that of control ticks.

The percent adult tick mortality caused by the crude meth-anolic extract of the NL varied from 6.67 to 33.33 %, whentested at concentrations ranging from 12.5 to 100 mg/ml. Onlyhigher concentrations (50 and 100 mg/ml) produced low butsignificant (P<0.05 and P<0.01) mortality of adult engorgedticks and reduction in mass of eggs laid, when compared tocontrol; consequently, there was small but non-significant re-duction in fecundity index. Calotropis leaf extracts in AITshowed mortality ranging from 13.33 to 66.67 % but signifi-cantly different from the control (P<0.01), only at higherconcentrations. Mean egg mass and fecundity index of thetreated ticks and LPT result further enunciate the significant(P<0.001) effect at all concentrations. Extract of DL, amongthe concentrations tested, 50–100 mg/ml concentrations werehighly efficacious, and significant (P<0.01) mortality of ticksin the range of 73.33±6.67 to 13.33±6.67%was recorded. Thehighest percentage of ticks died when treated at 100 mg/mlconcentration, and the surviving ticks laid fewer eggs andfecundity was inhibited by 0.11869±0.0086 (77.17 %) signif-icantly (P<0.001) lower when compared with control.

Adult immersion test showed that GC extract were impartedmortality ranging from 13.33 to 80 % (significant P<0.01 at

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higher concentration) and inhibited fecundity index up to0.07366±0.0014 (85.83 %) significantly (P<0.001) differentwhen compared to the control ticks. Papaya seed extractshowed significant (P<0.05) adult mortality except at concen-tration 12.5 mg/ml ranging from 26.67 to 93.33 %.Mortality athigher concentrations (100 and 50 mg/ml) was significantly(P<0.01) higher than at the lower concentrations. At thehighest concentration, egg laying was completely blocked,and percentage inhibition of fecundity was more than 90.Also when LPT was performed, mortality of tick larvaewas registered up to 82 %. A dose-dependent anti-tickeffect was observed in all the tests carried out in this study(Figs. 1 and 2).

Discussion

R. (B.) microplus is widely prevalent and the most economi-cally important tick, infesting dairy animals (Ghosh et al.

2007a, b) in India and north Gujarat in particular (Singhet al. 2013). The plants as medicine are important componentsin traditional Indian therapeutic measures since time imme-morial. Natural botanical compounds are residue less, floraand fauna friendly, biodegradable, contain a range of

Table 1 The results of adult immersion test (AIT) and larval packet test (LPT) to various methanol herbal extracts against R. (B.) microplus

Concentration (mg/ml) ATR±SE MA15±SE MMR±SE RI±SE IO (%) Hatching (%) (visual) MDL±SE (%)

Neem

Control 0.56±0.02 00.00±0.00 0.285±0.004 0.520±0.039 00.00 100 00.8±00.37

100 0.56±0.01 33.33±6.67** 0.230±0.0109* 0.412±0.016 20.73 80 55.2±1.02*

50 0.56±0.02 26.67±6.67*** 0.252±0.001** 0.440±0.013 15.42 80 51.6±1.17*

25 0.58±0.02 13.33±6.67 0.262±0.001 0.447±0.013 13.97 80 34.0±0.63*

12.5 0.54±0.02 6.67±6.67 0.270±0.003 0.492±0.027 05.37 80 10.8±1.02*

Calotropis

100 0.57±0.01 66.67±06.67* 0.085±0.0021* 0.149±0.006* 71.34 50 63.2±0.58*

50 0.56±0.02 53.33±06.67** 0.099±0.0011* 0.178±0.012* 65.75 50 54.0±0.71*

25 0.56±0.01 20.00±11.55 0.111±0.0013* 0.198±0.009* 62.00 50 41.6±0.51*

12.5 0.56±0.02 13.33±06.67 0.122±0.0009* 0.215±0.016* 58.62 50 13.6±0.60*

Datura

100 0.55±0.02 73.33±6.67* 0.064±0.002* 0.119±0.009* 77.17 30 71.8±0.58*

50 0.56±0.01 53.33±13.33** 0.070±0.001* 0.128±0.001* 75.52 30 65±1.41*

25 0.58±0.01 33.33±6.67 0.081±0.001* 0.140±0.003* 73.15 30 60.2±0.86*

12.5 0.58±0.01 13.33±6.67 0.089±0.001* 0.153±0.004* 70.61 30 22.6±1.44*

Garlic

100 0.58±0.01 80.00±11.55* 0.042±0.001* 0.074±0.001* 85.83 0 69.0±1.41*

50 0.55±0.02 60.00±11.55** 0.052±0.001* 0.100±0.003* 80.85 0 64.2±1.39*

25 0.57±0.01 40.00±11.55 0.059±0.001* 0.101±0.003* 80.53 0 62.8±1.36*

12.5 0.58±0.01 13.33±06.67 0.064±0.001* 0.112±0.001* 78.48 0 30.6±1.08*

Papaya

100 0.58±0.01 93.33±6.67* 0.000±0.000* 0.000±0.000* 100.00 0 82.2±2.31*

50 0.57±0.01 66.67±13.33** 0.012±0.001* 0.022±0.002* 95.87 0 74.2±1.39*

25 0.56±0.01 46.67±13.33*** 0.017±0.001* 0.031±0.002* 94.07 0 64.0±1.08*

12.5 0.58±0.01 26.67±6.67 0.021±0.001* 0.036±0.001* 93.07 0 43.8±1.24*

ATR average tick weight per replicate, SE standard error, MA15 mean % adult mortality within 15 days, MMR mean egg mass per replicate, RIreproductive index, IO (%) percent inhibition of oviposition, MDL (%) mean dead larva

*P<0.001; **P<0.01; ***P<0.05

Fig. 1 Dose response curve of adult R. (B.) microplus against variousmethanolic herbal extracts

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chemically active ingredients which can intervene in all bio-logical processes of the insects, thus interrupting their lifecycle and dispersal, and are accepted as an integrated part ofethno-veterinary practices (Habeeb 2010; Zaman et al. 2012).As per our knowledge, no reports are available on the effect ofselected plant from the western arid and semi-arid region ofthe Indian subcontinent. The plants materials included in thisstudy were selected on the basis of their reported acaricidalactivities, frequency of usage in traditional Indian veterinarymedicine, ease of availability, and cost of their application.Among the natural products, plant extracts have shown sig-nificant activity against all the stages of R. (B.) microplus tickspecies.

Neem (A. indica) is one of commonly grown indigenousplants in domestic backyards in India. It has been demonstrat-ed to have effect against mange (Singh 1980; Hirudkar et al.1997) and mosquito larvae (Pandian et al. 1989; Vatandoostand Vaziri 2004). Kalakumar et al (2000) also evaluated theeffect of neem oil and have shown 60–75 % efficacy againstbuffalo ticks but failed to inhibit oviposition in female ticks;however, the present finding, in contrary to this, reported up to20 % inhibition in oviposition. Ndumu et al. (1999) reportedthat undiluted neem oil (100 % concentration) was found tokill all the Amblyomma variegatum larvae 48 h post-exposure.The extracts of Melia azedarach (China berry containsazadirictine as active principle) have shown to inhibit eggproduction of immersed R. (B.) microplus ticks (Borges et al.2003). Effect of neem seed extract on larvae of R. (B.)microplus was also reported by Sindhu (2007, unpublisheddata) where he observed very slow activity and its effect onsurvival of larvae could not be determined after 24 h post-exposure. However, with leaf extract, we could determine upto 55 % mortality particularly at higher concentration.Azadirachtin (active principle in neem), a tetranortriterpenoidplant limonoid, has been reported to act as a growth-disruptingagent having an anti-feedant effect and causing delay in theproduction of ecdysone (Rembold et al. 1984; Radcliffe et al.1990). Generally, the precision of the effects of various typesof neem extracts to control pests is hard to specify, since the

complexity of the compounds and their various modes ofaction make it hard to discern the mechanisms involved(Mossini and Kemmelmeier 2005).

Calotropis procera, a desert shrub, is extensively scatteredin the semi-arid region of western Indian state of Gujarat.Leaves of Calotropis species had been proven to be anti-anopheles and anti-culex mosquito activity (Elimam et al.2009), anti-mite activity, and anti-acaricidal activity againstR. (B.) microplus (Chungsamarnyart and Jansawan 1996;Chungsamarnyart et al. 1994). Ghosh et al. 2011) and reportedup to 20 % mortality 72 h post-treatment in adult R. (B.)microplus ticks when used hydroethanolic root extracts. Theacaricidal properties of C. procera attribute to digitoxin (Jainet al. 1996) and a cardiac glycoside, cardenolide (Al-Rajhyet al. 2003). The contact and dipping LC50 values ofcardenolide against adult Hyalomma dromedarii found incamel were recorded as 1,185 and 1,096 mg/l, respectively,and it acts through inhibition of Na+, K+-ATPase of ticks (Al-Rajhy et al. 2003).

Datura stramonium is a wild-growing herb, used in tradi-tional systems of medicines, and possesses remarkable bio-logical activities. Ethanolic extracts obtained from both leafand seed in the thorn apple of Datura (Datura spp.) wereinvestigated for acaricidal, repellent, and oviposition deterrentproperties against adult two-spotted spider mites, Tetranychusurticae (Acari: Tetranychidae) in vitro. Leaf and seed extracts,which were applied in 167, 250 and 145, 750 mg/l concentra-tions, respectively, caused 98 and 25 % mortality amongspider mite adults after 2 days (Nabi et al. 2009; Kumralet al. 2010). Whole part of Datura Stramonium has got manyuses and toxicity to a greater extent. The alkaloids isolatedfrom the plants are also found to be toxic to the animals.Datura leaves are having good adulticidal as well as larvicidaleffect against R. (B.) microplus; however, the toxic effect ofthe alkaloids of the plant needs to be studied specificallybefore using it as acaricides. Careful consideration of thetoxicity of the plant is required before its use (Soni et al.2012). However, the extract of D. stramonium is composedof certain alkaloids, as anticholinergics (scopolamine, hyoscy-amine, meteloidine, atropine, etc.), terpenoids, and flavonoidsthat are believed to be responsible for many of this plant’sinsecticidal properties (Pavel 2004; Berko et al. 2006;Aronson and Jeffrey 2009). It is believed that the mechanismof action of D. stramonium may be similar to that of theorganophosphate compounds; however, the presence of otherassociated alkaloids further augments its acaricidal activities.

Garlic has been used all over the world for thousands ofyears. Records indicate that garlic was in use when the Gizapyramids were built, about 5,000 years ago. The bulbs are themost frequently used parts of the plant in India. This has beentaken to relieve problems both in humans and animals such ascoughs and fevers, or applied externally to prevent the anom-alies of hairs and to improve skin conditions such as eczema

Fig. 2 Dose mortality curve of larva R. (B.) microplus against variousmethanolic herbal extracts

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and scabies. Extracts of the bulbs have been widely used infolk medicine. Garlic oil along with diatomaceous earth hastoxic effect against stored-product pests (Yang et al. 2010).Garlic is known to be an effective insecticide, acting on adult(Dhar et al. 1996) and larval stages (Amonkar and Reeves1970; Amonkar and Banerji 1971; George et al. 1973) ofmosquitoes. In sheep, maximum reduction percentage of adultR. (B.) microplus was recorded 30.02 on seventh day post-treatment of garlic clove extract (Muraleedharan et al. 2008);however, we could record up to 80 % mortality at higherconcentration in vitro. Ovipositional deterrence and ovicidaland adulticidal properties were evaluated by Roobakkumaret al. (2010) using laboratory reared mites, by directlyspraying, and the results revealed very good mortality instant-ly with garlic aqueous extract. However, all the adult miteswere killed within 96 h post-treatment. Dwivedi and Sharma(1986) applied herbal preparation consist of garlic extractsalong with extracts of lemon and onion mixed with turmericand camphor powder in Pongamia (Karanj) oil, when appliedconsecutively for 5 days eliminated Sarcoptes scabiei infesta-tion in piglets.

C. papaya is considered an effective and well-known anti-parasitic remedy (Heinrich et al. 1992). Mature leaves arewidely used to treat malaria and spleenomegaly(Adjanohoun et al. 1996). Seeds of this plants are given withhoney to humans in India to expel roundworms (Kapoor1990), and an infusion of the raw seeds is taken for intestinalparasites inMartinique (Longuefosse and Nossin 1996). Also,petroleum ether extract of the seed rind showed a considerableantimalarial activity; this may be indicative of the presence ofhighly active compounds in this plant (Bhat and Surolia2001). Stepek et al. (2007) in their study found that theextracts of this plant exhibit a potent anthelmintic effect ascystein proteinases enzymes attack on the protective cuticle ofthe worm. Calzada et al. (2007) further reported very goodanti-trichomonal activity. Pendzhiev (2002) isolated differentenzymes present in C. papaya extract including proteolyticenzymes as (papain, chymopapain, peptidases A and B, andlysozyme) having anti-inflammatory, anticoagulant, analge-sic, bactericidal, anthelmentic, and hemolytic properties.These enzymes are capable of decomposing the proteins ofpolypeptides and amino acids and dissolving necrotized cells(while not affecting intact ones). The leaves extract of Caricapapaya was found to achieve a 100 % mortality rate of theAnophelesmosquito larvae at the concentration of 0.06mg/ml.A much higher concentration (0.10 mg/ml) was required toachieve the same mortality rate for Culex larvae but the lethalstrength of the solution decreased with time of storage, losingits larvicidal property on the 18th day of storage (Okolie2006). Rawani et al. (2012) reported great anti-mosquitopotential of C. papaya, including larvicidal, pupicidal,adulticidal, smoke toxicity, and repellent activities againstCulex quinquefasciatus and Anopheles stephensi, the vectors

of filaria and malaria, respectively. Phytochemical analysis ofmethanol extract of C. papaya stems has shown the presenceof flavonoids, tannins, alkaloids, carbohydrates, andtriterpenes (Khaled Nabih Rashed et al. 2013). Meltem et al.2005 also reported high triterpenes content in the C. papayaextracts, and these compounds being Cycloartane-typetriterpene glycosides isolated from Astragalus oleifoliusshowed notable growth inhibitory activity againstLeishmania donovani.

From the results obtained, it can be concluded that extractsof A. sativum cloves and C. papaya seeds possess very goodin vitro acaricidal activities. Also, leaf extracts ofD. stramonium, A. indica, and C. procerawere shown to haveacaricidal effect at one or other life stages of ticks, thusjustifying the traditional use of these plants against insects.However, further in vivo study including these plants shouldbe undertaken to evaluate the effect of these herbs on animals.Different combination of these extracts can also be studied toexploit the synergistic and antagonistic effect of differentactive ingredients of these plants. Further, studies are indicat-ed for identification of active ingredients present in theseplants, particularly garlic and papaya that caused the mortalityof tick, decrease in egg production and inhibition of hatchingof the eggs. These studies will further be helpful in confirmingthe presence of some factors in crude extracts that augment theacaricidal property. The anti-ovipositional and other acaricidalproperties of the various extracts of these plants may makethem a valuable component of developing sustainable strategyfor integrated tick management.

Acknowledgments Authors are thankful to the Dean, College of vete-rinary Science & Animal Husbandry, SDAU, Sardarkrushinagar forproviding facilities to carry out the research work.

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