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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2023 May 15;47(3):513–519. doi: 10.1007/s12639-023-01593-8

Acaricidal activity of paste of methanolic extract of leaf of Azadirachta indica, Eucalyptus hybrid, Saraca asoca and Murraya koenigii against the bovine ticks in in-vitro condition

Niranjan Kumar 1,✉, Jayesh B Solanki 1, Dharmesh C Patel 1, Nabanita Thakuria 1, Nitin Varshney 2
PMCID: PMC10382387  PMID: 37520197

Abstract

The objective of this study was to evaluate the acaricidal activity of a paste made from freeze-dried methanolic extracts of air-dried leaf powders of Azadirachta indica, Eucalyptus hybrid, Saraca asoca, and Murraya koenigii against Rhipicephalus (Boophilus) microplus on cellulose paper. The extracts were tested in both single form (100% and 50% concentration) and dual combination (prepared by mixing equal proportions of the extracts in 100% concentration). The results showed a direct proportional relationship (p < 0.05) between the concentration of the extracts and the mortality percentage at 72 h post-treatment, as well as the inhibition of oviposition (I.O.) percentage. The highest mortality rate of 98.75 ± 1.25% and I.O. of 44.47 ± 0.87% was observed with the A. indica extract at 100% concentration, followed by E. hybrid, S. asoca, and M. koenigii. The combination of A. indica and E. hybrid extracts had a mortality rate of 87.5 ± 5.59% and I.O. of 42.91 ± 0.44%, followed by the combinations of S. asoca: E. hybrid, A. indica: S. asoca, E. hybrid: M. koenigii, and A. indica: M. koenigii. The extracts of A. indica and E. hybrid demonstrated the highest mortality and inhibition of oviposition percentages compared to the other extracts in both single and dual combinations. These extracts required 72 h to reach their maximum mortality.

Keywords: Acaricides, Extract, Herbal, Inhibition of oviposition, Mortality

Introduction

Ticks are external parasites that depend on the blood of domestic and wild animals worldwide, with a greater prevalence in tropical and subtropical regions (Guglielmone et al. 2020). These parasites can cause skin damage and anemia, which can reduce the productivity of the animals. Furthermore, tick-borne diseases remain a significant animal health issue and cause significant economic losses for farmers (Gerem et al. 2016). Rhipicephalus (Boophilus) microplus is the most economically important tick species and widely prevalent in dairy cattle in India, including South Gujarat (Ghosh et al. 2007; Patel et al. 2019). Chemical acaricides are commonly used to control ticks, but their overuse can lead to tick resistance, environmental pollution, food residues, and harm to workers (Obaid et al. 2022). To address these issues, an eco-friendly, biodegradable, safe, effective, and economical plant-based acaricide strategy has been proposed. Plants have been used as medicine for centuries, and their extracts contain chemically active ingredients that can interfere with tick biology, including their life cycle and dispersal (Habeeb 2010; Zaman et al. 2012). Furthermore, botanical compounds can prevent blood feeding, chitin formation, moulting, fecundity, hatching, and egg development and act as antagonists of growth regulatory hormones, disrupt sexual communication, and repel ticks (Young et al. 1988; Patel et al. 2019). The development of resistance to plant-based acaricides is unlikely since the chemical constituents of these extracts act via different mechanisms (de Souza Chagas et al. 2012; Pavela et al. 2016). To find new sources of natural acaricides, the potential of four locally available medicinal plants from south Gujarat region (Azadirachta indica, Eucalyptus hybrid, Saraca asoca, and Murraya koenigii) was evaluated in this study. These plants have been reported to have acaricidal properties in various works (de Souza Chagas et al. 2002; Srivastava et al. 2008; Magadum et al. 2009; Pirali-Kheirabadi et al. 2009; Broglio-Micheletti et al. 2010; Borges et al. 2011; Singh et al. 2015; Sabuj et al. 2017). The aim of the study was to assess the potential of methanol extracts of the leaves of these four plants as herbal acaricides against adult Rhipicephalus (Boophilus) microplus ticks in vitro.

Materials and methods

Extract preparation

The leaves of A. indica, E. hybrid, S. asoca, and M. koenigii were collected from Jalalpore taluka of Navsari district, South Gujarat, India. After air-drying for 3 weeks in shade at room temperature, the leaves were powdered using a mixer-grinder machine. The powdered leaves were macerated in a ratio of 100 g in 1000 ml methanol (as extractant) in an amber glass, which was incubated in a refrigerator (35–38°F) for 2 weeks to complete the extraction process. The extract was initially filtered through a double layer of muslin cloth and then twice through Whatman filter paper 4 on a flat tray. The filtered extract was allowed to stand in the air for evaporation at room temperature, and the resulting extract was collected in a glass bottle from the tray. It was then incubated in the refrigerator for freeze-drying to obtain its powder form and stored in the refrigerator for future use. For this experiment, Tween-20 detergent in 5% was used as a vehicle for these extracts. Extract strengths of 100% (100 gm extract in 100 ml of 5% Tween-20) and 50% (50 gm extract in 100 ml of 5% Tween-20) were evaluated for their acaricidal activities.

Collection and identification of ticks

Fully engorged female adult ticks were manually collected using forceps from the bodies of cattle and from the crevices and gaps of the animal housing area. The collected ticks were placed in vials and covered with dry cotton cloth to allow for oxygen supply during transport to the laboratory. The ticks were examined under a microscope and identified based on their morphology as Rhipicephalus (Boophilus) microplus.

Acaricidal activity of extracts soaked in cellulose filter paper

The leaves of A. indica, E. hybrid, S. asoca, and M. koenigii were air-dried for 3 weeks in shade and then powdered in a mixer-grinder machine. The resulting macerated leaf powder was mixed with methanol (in a ratio of 100 g powder to 1000 ml methanol), placed in an amber glass, and incubated in the refrigerator (35–38°F) for 2 weeks for extraction. The resulting extract was filtered twice through Whatman filter paper 4 and allowed to stand for evaporation at room temperature. The dried extract was collected in a glass bottle, stored in the refrigerator, and later mixed with Tween-20 detergent in a 5% solution. Extracts of 100% and 50% strength were prepared and applied to Whatman filter paper 4. Similarly, extracts in dual combinations of A. indica: E. hybrid, A. indica: S. asoca, A. indica: M. koenigii, S. asoca: E. hybrid, and E. hybrid: M. koenigii were prepared by mixing the extracts in a 1:1 ratio. Positive and negative controls were created using Fipronil and 5% Tween-20, respectively. The filter papers were air-dried and 10 adult ticks were released onto them. The percent adult tick mortality was observed at 24, 48, and 72 h after treatment. Dead ticks were identified by observing loss of motility and pedal reflex. The ticks were observed for oviposition for up to 15 days, and the weight of ticks before treatment and the egg weight were measured to calculate the reproductive index and inhibition of oviposition in the treated group.

Statistical analysis

Data comparison and initial descriptive analyses, including column graphs, were performed using Microsoft Excel (MS Office, 2010). Mortality was converted to percent mortality. The data was analyzed using a single factor ANOVA, and the subsequent Duncan Multiple Range Test (DMRT) was applied to determine the differences between means. Values with p < 0.05 were considered significant. The statistical analysis program OPSTAT software (Sheoran et al. 1998) was utilized for the analysis. The acaricidal status was assessed based on three parameters: Mortality % {[Number of dead ticks/Number of treated ticks at the start of experiment] × 100}, reproductive index (R.I.) (Weight of egg mass/Weight of tick before treatment), and inhibition of oviposition (I.O.) % {[(R.I. of control group − R.I. of treated group)/R.I. of control group] × 100}.

Results

Extract preparation and identification of ticks

The percentage of yield obtained from A. indica, E. hybrid, S. asoca, and M. koenigii were 25.2%, 28.4%, 21.3%, and 19.5%, respectively. The weight of freeze-dried extracted material from the leaves of A. indica, E. hybrid, S. asoca, and M. koenigii were 54 gm, 45 gm, 38 gm, and 30 gm, respectively (Fig. 1).

Fig. 1.

Fig. 1

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Freeze dried methanolic extract of air dried leaf powder of A. indica (Neem) (a), E. hybrid (Nilgiri) (b), S. asoca (Ashok) (c) and M. koenigii (Curry) (d)

Acaricidal activity of extracts soaked in cellulose filter paper

Recently introduced acaricidal drug, fipronil as positive control had shown high level of effectiveness with 100% mortality of adult ticks following 24 h of post-treatment. Mortality recorded in the negative control (5% Tween-20 detergent) was nil at 24–48 h of post-treatment while it was 0–10% at 72 h of post treatment.

Acaricidal activity of the soaked extracts in the filter paper in singlet form in term of mortality is depicted in Table 1. The crude extracts of A. indica and E. hybrid showed a maximum mortality rate of 100% in 7 out of 8 and 5 out of 8 replicates, respectively, after 72 h of treatment. These extracts required a time lag of 72 h to exhibit their full potential in terms of mortality. Acaricidal activity in terms of % I.O. of the extract in the singlet form is depicted in Table 2. The mortality and I.O. were significantly higher in A. indica (mortality of 98.75 ± 1.25% in 100% and 77.5 ± 3.66% in 50%; I.O. of 44.47 ± 0.87% in 100% and 27.27 ± 1.26% in 50%) and E. hybrid (mortality of 96.25 ± 1.83% in 100% and 66.25 ± 6.53% in 50%; I.O. of 42.39 ± 0.48% in 100% and 25.05 ± 0.69% in 50%) compared to the extracts of S. asoca (mortality of 63.75 ± 6.25% in 100% and 47.5 ± 3.13% in 50%; I.O. of 29.02 ± 1.127% in 100% and 16.15 ± 0.58% in 50%) and M. koenigii (mortality of 68.75 ± 5.49% in 100% and 52.5 ± 5.26% in 50%; I.O. of 18.73 ± 0.67% in 100% and 8.53 ± 0.69% in 50%). The acaricidal activity in term of % mortality of the extract in dual combination is presented in Table 3. Mortality was 100% at 72 h of treatment in 4 out of 8 replicates of crude extract of A. indica and E. hybrid mixed in 1:1 ratio. Acaricidal activity in term of % I.O. of extract in the dual combination is depicted in Table 4. Extract in the dual combination of A. indica: E. hybrid, S. asoca: E. hybrid, A. indica: S. asoca, E. hybrid: M. koenigii and A. indica: M. koenigii noted mortality of 87.5 ± 5.59%, 77.5 ± 7.01%, 71.25 ± 4.40%, 68.75 ± 6.11% and 66.25 ± 6.53%/ I.O. of 42.91 ± 0.44%, 38.47 ± 0.97%, 23.88 ± 1.75%, 15.41 ± 0.49% and 9.85 ± 0.61%, respectively.

Table 1.

Acaricidal activity in terms of % mortality (Mean ± S.E.) of the extract soaked in the cellulose filter paper in the singlet form

Concentration Time (h) Extract Negative control
A. indica E. hybrid S. asoca M. koenigii
100% 24 41.25fgh ± 2.95 28.75ijkl ± 2.95 20 lm ± 2.67 20lmn ± 2.67 0o ± 0.00
48 60 cd ± 2.67 50def ± 2.67 37.5ghij ± 3.66 42.5efg ± 4.12 0o ± 0.00
72 98.75a ± 1.25 96.25a ± 1.83 63.75c ± 6.25 68.75bc ± 5.49 2.5o ± 1.64
50% 24 31.25hijk ± 2.95 20lmn ± 2.67 10mo ± 3.78 8.75o ± 2.95 0o ± 0.00
48 52.5de ± 2.50 38.75ghi ± 2.95 25kl ± 4.63 27.5jkl ± 4.12 0o ± 0.00
72 77.5b ± 3.66 66.25c ± 6.53 47.5efg ± 3.13 52.5de ± 5.26 3.75o ± 1.83
Source of variation D.F Sum of squares Mean squares F-calculated Significance C.D S.E.D S.E.M
Analysis of variance table
Concentration (C) 1 7593.75 7593.75 83.22 0.00 2.43 1.23 0.87
Extract (E) 4 96,385.83 24,096.46 264.07 0.00 3.84 1.95 1.38
Time of mortality (T) 2 64,282.50 32,141.25 352.23 0.00 2.98 1.51 1.07
Interaction C × E 4 2154.17 538.542 5.90 0.00 5.44 2.76 1.95
Interaction C × T 2 842.5 421.25 4.62 0.01 4.21 2.14 1.51
Interaction E × T 8 14,921.67 1865.21 20,441 0.00 6.66 3.38 2.39
Interaction C × E × T 8 803.33 100.42 1.1 0.36 N/A 4.78 3.38
Error 210 19,162.50
Total 239 206,146.25
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D.F. Degree of freedom, C.D. Critical difference, S.E. Standard error, S.E.D. Standard error of deviation, S.E.M. Standard error of mean

a–oDifferent superscript in the same column showed significant difference (DMRT: p < 0.05)

Table 2.

Acaricidal activity in term of % inhibition of oviposition (Mean ± S.E.) of extract soaked in the cellulose filter paper in the singlet form

Extract/control Weight of tick before treatment (mg) Egg weight (mg) Reproductive index % Inhibition of oviposition
100% 50% 100% 50% 100% 50% 100% 50%
A. indica 138.63 ± 0.50 138.75 ± 0.45 37.88 ± 0.61 50.38 ± 0.91 0.27 ± 0.00 0.36 ± 0.01 44.47a ± 0.87 27.27a ± 1.26
E. hybrid 139.38 ± 0.50 139 ± 0.46 39.5 ± 0.42 52 ± 0.54 0.28 ± 0.00 0.37 ± 0.00 42.39a ± 0.48 25.05a ± 0.69
M. koenigii 139.25 ± 0.37 139.5 ± 0.42 48.63 ± 0.82 58.38 ± 0.38 0.35 ± 0.00 0.42 ± 0.00 29.02b ± 1.12 16.15b ± 0.58
S. asoca 139.13 ± 0.52 139.38 ± 0.42 55.63 ± 0.78 63.63 ± 0.42 0.40 ± 0.00 0.46 ± 0.00 18.73c ± 0.67 8.53c ± 0.69
Negative control 139 ± 0.57 138.75 ± 0.53 68.38 ± 0.65 69.25 ± 0.59 0.49 ± 0.00 0.50 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Concentration Source of Variation D.F Sum of Squares Mean Squares F-Calculated Significance C.D S.E.D S.E.M
Analysis of variance table
100% Extract 3 3494.87 1164.96 216.47 0.00 2.39 0.82 1.16
Error 28 150.69 5.38
Total 31 3645.56
50% Extract 3 1779.38 593.13 103.57 0.00 2.46 0.85 1.20
Error 28 160.35 5.73
Total 31 1939.72
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a–cDifferent superscript in the same column showed significant difference (DMRT: p < 0.05)

Table 3.

Acaricidal activity in terms of % mortality (Mean ± S.E.) of the extract soaked in the cellulose filter paper in the dual combination

Time (h) Extract Negative control
A. indica: E. hybrid A. indica: S. asoca A. indica: M. koenigii S. asoca: E. hybrid E. hybrid: M. koenigii
24 35de ± 4.23 22.5e ± 3.13 22.5c ± 3.66 28.75bc ± 4.41 25c ± 3.27 0d ± 0.00
48 53.75c ± 5.96 40d ± 3.27 42.5b ± 5.90 42.5b ± 3.66 48.75b ± 6.67 0d ± 0.00
72 87.5a ± 5.59 71.25b ± 4.40 66.25a ± 6.53 77.5a ± 7.01 68.75a ± 6.11 3.75d ± 1.83
Time (h) Source of variation D.F Sum of squares Mean squares F-calculated Significance C.D S.E.D S.E.M
Analysis of variance table
24 Extract 5 23,491.67 4698.33 28.291 0.000 13.049 4.556 6.443
Error 42 6975.00 166.071
Total 47 30,466.67
48 Extract 5 18,191.67 3638.33 15.794 0.000 15.369 5.366 7.589
Error 42 9675.00 230.357
Total 47 27,866.67
72 Extract 5 33,418.75 6683.75 52.348 0.000 11.44 3.995 5.65
Error 42 5362.50 127.679
Total 47 38,781.25
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a–eDifferent superscript in the same column showed significant difference (DMRT: p < 0.05)

Table 4.

Acaricidal activity in terms of % inhibition of oviposition (Mean ± S.E.) extract in the dual combination

Extract/control Weight of tick before treatment (mg) Egg weight (mg) Reproductive index % Inhibition of oviposition
A. indica: E. hybrid 138.88 ± 0.44 39.5 ± 0.42 0.28 ± 0.00 42.91a ± 0.44
S. asoca: E. hybrid 139 ± 0.46 42.63 ± 0.82 0.31 ± 0.01 38.47b ± 0.97
A. indica: S. asoca 138.75 ± 0.45 52.63 ± 1.28 0.38 ± 0.01 23.88c ± 1.75
E. hybrid: M. koenigii 139.13 ± 0.52 58.63 ± 0.26 0.42 ± 0.00 15.41d ± 0.49
A. indica: M. koenigii 138.88 ± 0.44 62.38 ± 0.42 0.45 ± 0.00 9.85e ± 0.61
Negative control 139.25 ± 0.56 69.38 ± 0.50 0.50 ± 0.00 0.00 ± 0.00
Source of variation D.F Sum of squares Mean squares F-calculated Significance C.D S.E.D S.E.M
Analysis of variance table
Extract 4 6556.37 1639.09 212.971 0.000 2.828 0.981 1.387
Error 35 269.372 7.696
Total 39 6825.74
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a–eDifferent superscript in the same column showed significant difference (DMRT: p < 0.05)

Discussion

Plant extracts’ population-limiting property has been utilized to create sustainable methods for addressing tick infestations in domestic animals. Certain herbal extracts have shown significant activity against all stages of the R. (B.) microplus tick (Borges et al. 2011). In an effort to identify herbal acaricides, methanolic extracts were prepared from air-dried leaves powder of A. indica, E. hybrid, S. asoca, and M. koenigii, and evaluated for their acaricidal properties against the cattle tick, R. (B.) microplus. To our knowledge, no reports are available on the effect of these selected plants from the south Gujarat region. The selection of these plants was based on their reported medicinal activities, frequency of use in traditional Indian veterinary medicine, ease of availability, and cost of their application. These plants are commonly grown in domestic backyards in India. Herbal preparation originated from these plants demonstrated to have acaricidal effect against R. (B.) microplus (Gupta et al. 2000; de Souza Chagas et al. 2002; Srivastava et al. 2008; Magadum et al. 2009; Zaman et al. 2012; Singh et al. 2015; Catherine et al. 2022). Methanol is widely used as an extractant to prepare herbal preparations from different plant parts (Nithya et al. 2013). In our study, we obtained a comparatively higher yield percentage of the methanolic extract than what has been reported in the literature (Magadum et al. 2009; Ibrahim and Kiranmai 2012; Bendigeri et al. 2019). This difference could be due to several factors, such as the plant parts used, the origin of the plants, the particle size of the powder, the solvents used, and the extraction method. We dispensed the freeze-dried form of the methanolic extracts of leaf powder from the selected plants in 5% Tween-20 detergent to form pastes of 100% and 50% concentration, which were then applied on Whatman filter paper 4 to assess their acaricidal effects under in-vitro conditions. In order to achieve maximum acaricidal effects in an in-vitro condition, a high concentration of the extracts was used. In a study by Kumar et al (2011), a higher level of in-vitro mortality percentage was observed with an increased concentration of the extract in natural tick infestations in goats. The results of the present study indicate that the tested plant extracts are effective acaricidal agents against the cattle tick, as evidenced by data on % mortality, reproductive index, and % inhibition of oviposition. The extracts also significantly reduced the egg-laying capacity of exposed ticks compared to the negative control. To obtain possible synergistic or additive effects as acaricides, extracts at 100% concentration were mixed in equal proportions of A. indica: E. hybrid, A. indica: S. asoca, A. indica: M. koenigii, S. asoca: E. hybrid, and E. hybrid: M. koenigii. It is common practice to blend plant extracts to maximize their cidal effects against animal pests (Magadum et al. 2009; Kalakumar et al. 2000; Nithya et al. 2013; Bhikane et al. 2018). The plant extracts, either in singlet form or in dual combination, required a period of 72 h time to reach their maximum mortality rate. This observation was also replicated by Jumde et al (2013) in their experiment against the R. (B.) microplus tick. The acaricidal activity of the extracts soaked in cellulose filter paper was highest in A. indica/E. hybrid, followed by M. koenigii and S. asoca, in both 100% and 50% concentration. The %I.O. was higher in the 100% concentration compared to the 50% concentration. In dual combination, the highest acaricidal activity was observed in A. indica: E. hybrid, followed by S. asoca: E. hybrid, A. indica: S. asoca, E. hybrid: M. koenigii, and A. indica: M. koenigii, with the % I.O. being highest in A. indica: E. hybrid. Maximum mortality rate was achieved in all cases after 72 h of treatment.

In a previous study, Shyma et al (2014) found a low level of adult tick mortality using crude methanolic extract of A. indica leaves against R. (B.) microplus tick. However, in the current study, a much higher level of mortality was observed using the same extract. Kalakumar et al (2000) reported the efficacy of neem oil against buffalo ticks, but failed to inhibit oviposition in female ticks, whereas the current study showed a significant effect on mortality and percent inhibition of oviposition. Madreseh-Ghahfarokhi et al (2019) noted the highest acaricidal activities of E. hybrid and Z. officinalis essential oils against R. bursa in the singlet form. Catherine et al. (2022) found that a combination of P. dodecandra and A. indica extracts did not show any significant difference in tick mortality compared to P. dodecandra in singlet form. Singh et al (2015) evaluated the acaricidal effects of M. koenigii extracts against R. (B.) microplus and found maximum mortality in the ethanol extract. The current study also tested the chemical acaricide fipronil, which was found to be highly effective against the R. (B.) microplus tick in the studied area, despite reports of fipronil resistance in other Indian states.

Conclusions

The paste made from a freeze-dried methanolic extract of air-dried leaf powder with 100% concentration in 5% Tween-20 of A. indica/ E. hybrid appears to be a promising acaricidal agent against the adult stage of R. (B.) microplus on cellulose filter paper. The treatment resulted in a mortality rate of 98.75 ± 1.25%/96.25 ± 1.83% after 72 h, and inhibited oviposition by 44.47 ± 0.87%/42.39 ± 0.48%. This effect was greater than that of extracts from S. asoca/ M. koenigii and the 1:1 ratio of A. indica: E. hybrid/ A. indica: S. asoca/A. indica: M. koenigii/ S. asoca: E. hybrid/ E. hybrid: M. koenigii. The highest mortality rate was observed after 72 h of treatment, indicating that the maximum effects of these plant extracts required 72 h to be demonstrated.

Acknowledgements

The authors are thankful to the Vice-chancellor, Director of Research and Principal, College of Veterinary Science and Animal Husbandry, Navsari, Navsari Agricultural/Kamdhenu University for providing necessary fund and facilities to complete the research work on time.

Author contributions

The idea of the work was conceptualized by NK and JBS. The field work was conducted by NK, DCP and NT. The statistical analysis was performed by NK and NV. NK wrote the manuscript, the revision of the data analysis and manuscript was done by NK, JBS and NV. All authors read, corrected and approved the manuscript.

Funding

The authors have not disclosed any funding.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

As the study was conducted with the cattle tick and plant extract, so ethical committee approval was not required.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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