BioMed Research International
Volume 2016 (2016), Article ID 7078029, 8 pages
http://dx.doi.org/10.1155/2016/7078029

Research Article

Acute Oral Mammalian Toxicity and Effect of Solvents on Efficacy of Maerua edulis (Gilg. & Ben.) De Wolf against Rhipicephalus (Boophilus) decoloratus Koch, 1844 (Acarina: Ixodidae), Tick Larvae

Emmanuel T. Nyahangare,¹ Brighton M. Mvumi,² and Tatenda Maramba¹

¹Department of Animal Science, University of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe
²Department of Soil Science & Agricultural Engineering, University of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe

Received 7 April 2016; Revised 6 July 2016; Accepted 21 July 2016

Academic Editor: Carlos A. N. Ramos

Copyright © 2016 Emmanuel T. Nyahangare et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Efficacy and toxicity of aqueous and organic solvents extracts of Maerua edulis against ticks and mice, respectively, were determined. Ground leaves were extracted separately using cold water, cold water plus surfactant (1% v/v liquid soap), hot water plus surfactant, hexane, or methanol to make 25% w/v stock solutions from which serial dilutions of 5, 10, 20, and 25% were made. For each concentration, 20 Rhipicephalus decoloratus tick larvae were put in filter papers impregnated with extracts and incubated for 48 h at 27°C and 85–90% RH for mortality observation after 24 h and 48 h. In the toxicity experiment, hot water plus surfactant treatments of 5, 10, 20, and 25% (w/v) M. edulis were administered in suspension per os to sexually mature Balb/C mice and observed for clinical signs and mortality for 72 h. Larvae mortality was highest (>98%) in methanol-extracted M. edulis treatments (20 and 25%), which was not different from the amitraz-based control (Tickbuster®). Mortality was also higher in the hot water than in cold water plus surfactant treatments (). No postadministration adverse health effects were observed in the mice. These results suggest that M. edulis is an effective tick remedy best extracted using methanol or hot water plus surfactant.

1. Introduction

The control of ticks remains prohibitively costly with global estimates for cost of conventional tick control in excess of USD7 billion per year [1]. It is also estimated that losses associated with ticks and tick-borne diseases (TTBD) range between USD13.9 and USD18.7 billion [2]. As a result, there is continued demand for new and improved methods of controlling ticks with the general consensus being that current conventional tick control methods are inadequate for several reasons [3, 4] or inaccessible, especially in the context of smallholder farmers. Intensive and/or indiscriminate use of arsenic, organophosphorus, and pyrethroid acaricides has led to serious problems of development of resistance in ticks [5–7]. In most parts of the world, chlorinated hydrocarbon acaricides are no longer used or marketed due to high toxicity and long residual effect on meat and other animal products [4]. There are also concerns on effective disposal means of these chemicals and the negative effects on the environment. Meanwhile, the threat of ticks and tick-borne diseases (TTBD) to livestock productivity and farmers’ livelihoods remains high, especially for resource-poor and marginalised farmers [8, 9]. Livestock, and especially cattle, have a direct correlation with livelihoods issues in developing countries such that when the productivity of animals is poor, the quality of life of many people also suffers [3].

The use of locally available plants that have acaricidal properties has been hypothesized as a potentially effective method for holistic management of TTBD, especially in developing countries with limited financial resources to purchase the synthetic acaricides [10]. However, Stevenson and coworkers [11] noted that the scientific chemical structures of such plants and their associated activities are still poorly understood, which prevents their effective use and gaining such knowledge will enhance their usefulness and optimal use. Examples of plants that possess acaricidal properties against cattle ticks include Tephrosia vogelii Hook.f. [12, 13], Azadirachta indica A Juss. [14–16], Origanum minutiflorum O. Schwarz et P.H. Davis [17], Euphorbia ovalifolia Klotzsch & Garcke, Ficus brachypoda (Miq.) Miq. [18], Petiveria alliacea L. [19], and Copaifera reticulata Ducke [20]. In Zimbabwe, Madzimure et al. in [21] showed that simple aqueous leaf crude extracts of Lippia javanica were effective against cattle ticks at 10% w/v application level. In laboratory assays and on-station cattle experiments, Strychnos spinosa Lam and Solanum incanum L. also demonstrated that they have antitick properties [22]. The confirmation of acaricidal properties in these plants has opened up interest in other plants that possess similar properties and in finding ways to increase the efficacy. Previous on-station studies showed that some plant-based treatments were not as effective as commercial synthetic acaricides [21, 22].

Blue bush-berry, Maerua edulis (Gilg. & Ben.) De Wolf (Figure 1), is a shrub that can be added to the database of plants with antitick properties. The plant is of the Capparaceae family found in some African countries including Democratic Republic of Congo, Kenya, Tanzania, Malawi, South Africa, Uganda, Zambia, and Zimbabwe in well drained soils [23].

Figure 1: Maerua edulis plant in flower; picture courtesy of Aluka (http://www.bihrmann.com/caudiciforms/subs/mae-edu-sub.asp).

Early efforts to validate the efficacy claims of M. edulis against cattle ticks were reported by Kaposhi et al. in [24] where results showed some antitick properties in the aqueous extracts of the plant. Apart from uses in tick control, the plant has other uses in grain protection during storage in some areas of Zimbabwe, particularly in and around Gokwe and Binga districts [25]. Despite the pesticidal properties, there have not been follow-up studies to further investigate and optimize the reported activity. In the current study, M. edulis was tested for efficacy against ticks using different extraction solvents as an optimization strategy. Nontarget safety was also evaluated in laboratory tests by administering extracts in single dose acute oral toxicity studies using Balb/C mice.

2. Materials and Methods

Laboratory experiments were conducted to determine the effect of different extraction solvents on the efficacy of M. edulis against R. decoloratus tick larvae (Experiment I) and to determine the relative safety of M. edulis on Balb/c mice (Experiment II).

2.1. Experiment I: The Laboratory Efficacy of M. edulis Crude Extracts against R. decoloratus Tick Larvae

2.1.1. Study Site

The experiments were conducted at the Central Veterinary Laboratory (CVL) of Zimbabwe in Harare.

2.1.2. Tick Collection and Rearing

Adult fully engorged ticks with no history of acaricidal resistance were collected from cattle in Mutorashanga (17.1488°S, 30.6761°E), in Makonde district and Mashonaland West province. The area is in Natural farming region II, characterized by annual rainfall of values between 700 and 1050 mm and a mean maximum temperature range of 16–19°C [26]. The ticks were prepared as described by Madzimure et al. in [22] by experts at the Central Veterinary Laboratory (Department of Veterinary Services in the Ministry of Agriculture, Mechanisation and Irrigation Development, Zimbabwe). Initially, the ticks were cleaned of all possible eggs laid during transportation from the field to the laboratory using distilled water. Subsequently, the ticks were put in plastic rearing tubes firmly closed with a ventilated stopper for egg laying in an incubator set 27-28°C and 85–95% relative humidity. All eggs laid were collected within 7 days from commencement of incubation and hatched. Tick larvae between 17 and 21 days were used for the larval package test.

2.1.3. Plant Material Collection and Preparation

Leaves of the M. edulis plant were collected from a remote district called Binga (17.6241°S, 27.3411°E) found in the northern low-veld of Zimbabwe with an altitude of about 500 m. The district is in natural farming region V, characterized by very low rainfall and high temperatures (<450 mm per annum and 30°C, resp.). The collected fresh M. edulis leaves were shade-dried under ambient mean temperatures ranging from 25 to 32°C after which they were ground to powder and stored in a freezer at temperatures below 5°C until point of use.

2.1.4. Treatments

For all the treatments, 5 g of the M. edulis powder was weighed and soaked separately in 20 mL of each of the following solvents: cold water, hot water (at 70°C), hexane, and methanol for 24 h to produce a stock solution of 25% w/v. The mixture was filtered through a filter paper and serial dilutions of 5, 10, 20, and 25% v/v made with each solvent. 1% v/v of liquid soap was added to the initial extraction mixture producing the stock solution, where the effect of adding a surfactant was to be determined. The aqueous extraction experiments used distilled water and distilled water plus surfactant as the negative controls and the organic solvents used for extraction as the negative controls (hexane or methanol). A commercial amitraz-based acaricide (Tickbuster) was prepared as per manufacturer’s specifications (0.2% v/v) and used as the positive control. A summary on how the treatments were prepared is presented in Table 1.

Table 1: A summary of experimental treatments used in the study.

Type of solvent used
Description of treatments

Cold water only
5, 10, 20, 25% v/v serial dilutions of cold water extracted M. edulis

Cold water plus surfactant
5, 10, 20, 25% v/v serial dilutions of cold water extracted M. edulis and 1% v/v surfactant

Hot water plus surfactant
5, 10, 20, 25 v/v serial dilutions of hot water (70°C) extracted M. edulis and 1% v/v surfactant

Hexane
5, 10, 20, 25 v/v serial dilutions of hexane extracted M. edulis

Methanol
5, 10, 20, 25% v/v serial dilutions of methanol extracted M. edulis

Positive control: Tickbuster prepared at 0.2% v/v with water.

Negative controls: Either distilled water, distilled water plus 1% surfactant, methanol, or hexane.

2.1.5. Experimental Procedure

Different concentrations of M. edulis extracts and the negative and positive controls were tested on approximately 20, 2-3-week-old tick larvae, using an adaptation of the Soberanes bioassay technique described by Miller et al. in [27]. For each treatment 10 mL of the solution was placed into a 10 cm diameter Petri dish containing a 9 cm diameter Whatman number 1 filter paper for 5 minutes. The tick larvae were then placed onto the wet filter paper which was then folded and the open end was sealed with steel paper clips. The packets were put in an incubator set at 27°C, 85–90% RH, and at photoperiod of 12 : 12 (L : D). Larval mortality was recorded after 24 and 48 h after incubation. Each treatment was replicated five times. In previous experiments with M. edulis described by Kaposhi and coworkers in [24], the efficacy was tested using the larval immersion technique (LIT) and the exposure to treated surface method was described, respectively, in [28, 29]. Aspects of the Soberanes technique are not very different from the LIT.

2.1.6. Statistical Analysis

Mortality data for the tick bioassays was converted to ranks through the Proc. Frequency and Proc. Rank procedures of [30] and analysed using the Proc. GLM procedures of SAS.

2.2. Experiment II: Single Dose Acute Oral Toxicity of M. edulis on Balb/C Mice

2.2.1. Study Site and Plant Material Preparation

The acute oral toxicity experiment was carried out in the Department of Animal Science, Bioassay Laboratory, at the University of Zimbabwe. Hot water plus surfactant-extracted M. edulis was used in the toxicity study and the treatments (5, 10, 20, and 25% v/v) were prepared as in the efficacy experiments (Experiment I; Treatment 3 under Table 1). Hot water with a surfactant was used as the control.

2.2.2. Animals and Experimental Design

A total of 30 BALB/c mice, reared in individual cages from time of weaning until sexual maturity at six weeks, were tested in a completely randomized design experiment. The mice were randomly allocated to the five treatments, in which each mouse represented an experimental unit and replicated sixfold. The animals were fed commercial mouse comproids procured from National Foods (Pvt. Ltd., Harare, Zimbabwe) and water ad libitum until one day before administration of the plant extracts. All feed and water were removed and reintroduced shortly after treatment administration.

The mice husbandry and the toxicity tests were adapted from the Organization for Economic Cooperation and Materials (OECD) guidelines for assessing acute oral toxicity. The Department of Animal Science is licensed by the Veterinary Services Unit in the Ministry of Agriculture, Mechanisation and Irrigation Development in Zimbabwe under the Scientific Animal Experiments Act (License Number L624) to carry out such experiments with mice. The Ethics Committee of the University of Zimbabwe approved the experimental protocols.

2.2.3. Administration of Plant Extracts

The M. edulis plant extracts (5, 10, 20, and 25% v/v) and the control (distilled water) were administered to mice in suspension per os using a 10 mL plastic syringe with a 16 mm long 22-inch gavage needle connected to it as described in [31]. For normal physiological functions, the minimum water requirements of a mouse are on average 4 mL/day depending on weight [32] and this was used as the basis for the quantity administered to the mice.

2.2.4. Monitoring and Measurements

The mice were observed daily for 3 days, checking for mortality, behavioural changes, and development of any clinical signs.

3. Results

3.1. Experiment I: Efficacy of Differently Extracted Maerua edulis against R. decoloratus Tick Larvae

3.1.1. Aqueous Solvents

Tick larvae mortality was significantly low (<40%) () at both recording times in the M. edulis cold water only extractant and the negative control compared to the positive control (Tickbuster) which recorded 71% and 100% after 24 h and 48 h, respectively (Figure 2). In the cold water and surfactant treatments, larvae mortality was also generally low and below 80% for both sampling periods (Figure 3). The least square mean (LSM) mortalities were all significantly different to the Tickbuster positive control after 24 h and 48 h ().

Figure 2: Effect of cold water extracts of M. edulis on R. (B) decoloratus tick larvae.

Figure 3: The effect of water plus 1% surfactant of M. edulis extracts on R. (B) decoloratus tick larvae.

Larval mortalities in the M. edulis extract that used hot water and 1% surfactant were lower after 24 h but increased significantly after 48 h (Figure 4). There was no significant difference between the positive control and the plant extracts and between mortalities in the different plant concentrations (5, 10, 20, and 25%). Hot water extraction and the addition of a surfactant caused higher mortalities in the tick larvae compared to cold water treatments (Table 2).

Table 2: Comparison of least square mean (LSM) mortalities of tick larvae from cold water plus surfactant and hot water plus surfactant extracted M. edulis treatments.

Treatment
Concentration (%)
Mortality ranks

24 h
48 h

Cold water + surfactant
5
54.1
73.1

10
62.0
46.7

20
73.9
47.0

25
63.9
72.1

Hot water + surfactant
5
128.9
107.4

10
132.7
120.8

20
132.0
113.5

25
138.9
113.0

Note: within a column, means with different superscripts differ significantly ().

Figure 4: The effect of hot water and 1% surfactant-extracted M. edulis on R. (B) decoloratus tick larvae.

3.1.2. Organic Solvents

There was no significant difference in mortality between the hexane control and hexane extracted M. edulis after 24 h. Even though the tick larval populations were reduced after 48 h, there was still no significant difference between mortality in the hexane control and hexane extracted plant extracts up to the 20% treatment (Figure 5). The mortality of the ticks was highest in the positive control (Tickbuster) than in all the other treatments ().

Figure 5: Effect of hexane extracted M. edulis on R. (B) decoloratus tick larvae.

In the methanol treatment, after 24 h, larvae mortality was low except in the 25% treatment, which recorded 80% mortality (Figure 6). There was dose-dependent increase in larval mortality after 48 h from 5–25% w/v M. edulis treatments. After 48 h, all M. edulis treatments, except the 5% treatment, showed significant mortality differences compared to the negative methanol control (), but it was only the 20 and 25% w/v treatments that had insignificant rank compared to the Tickbuster positive control.

Figure 6: Effect of methanolic extracts of M. edulis against R. (B) decoloratus tick larvae.

3.2. Experiment II: Acute Oral Toxicity of M. edulis Extracts against Mice

The mice in all the treatments only appeared lethargic soon after oral administration but regained normal activity after 30–60 minutes. Neither obvious clinical signs nor mortality was recorded during the observation period.

4. Discussion

The study provides evidence that M. edulis does have antitick properties which are better expressed under hot water and organic solvent extraction. This is confirmation of work done earlier by Kaposhi reported in [24] wherein a modest efficacy of 51% was reported for aqueous extracts of this plant against Rhipicephalus appendiculatus tick larvae. Combined with other insecticidal properties against postharvest grain pests reported by Stathers et al. in [25], M. edulis can also control a wide array of agricultural pests. There is, however, more documented information on the medicinal properties compared to the pesticidal properties of the plant. This is synonymous with most plant materials used in ethnoveterinary practices where initial interest is for treatment of particular diseases, but because of the multifunctional nature of the plants, other uses are also discovered. The tubers of M. edulis have anthelminthic properties against livestock nematodes and have been used successfully against the faecal worm (Heligmosomoides polygyrus) eggs in sheep [33]. In Tanzania, Komwihangilo et al. in [34] reported the use of M. edulis and Boscia grandiflora leaves in the treatment of poultry diseases. In another study by [35], hexane extracts of M. edulis were observed to possess notable activity against Mycobacterium bovis and tuberculosis. Despite all this, there has not been many studies on the phytochemistry of M. edulis but hexane extracts of this plant have been reported to consist of linear chain unsaturated fatty acids which have been attributed to the observed biological activity [35].

The hot water with 1% surfactant extract of M. edulis gave better mortality results compared to cold water with or without a surfactant extract. Extraction of bioactive compounds is dependent on several factors including type of solvent and extraction method used [36]. While water is generally regarded as a safe universal solvent used for preparing traditional remedies, several authors have noted its limitation as a solvent because of high polarity [37–39]. Hot water however provides better results in the preparation of ethnomedicines and is widely used in countries where traditional medicines are commonly used like China and India [40, 41]. In South Africa and many other countries, L. javanica leaves are immersed in hot water for the efficient extraction of essential oils in the preparation of herbal tea for treatment of colds [42]. Increasing the temperature to a threshold level breaks the bonds tightly holding the water molecules together making it less polar. Surfactants are also known to increase the efficacy of traditional remedies [43, 44]. However, some surfactants can also cause toxicity to the ticks especially if they are in high concentrations as observed by Gonçalves et al. in [45]. The use of a detergent like liquid soap has been recommended and used by several authors as they work both as a surfactant and as a spreader at 1% v/v inclusion level [13, 39, 46].

After 48 h postincubation, hot water plant extracts performed as well as methanol extracts. This indicates that the yield of bioactive compounds extracted from the plant material by using hot water approaches that obtained by using methanol. Methanol-extracted M. edulis showed high acaricidal activity against tick larvae because the hydroxyl (-OH) on the methanol formula (CH₃OH) contains a greater negative charge than the methane structure, making it a very effective solvent [47]. Elsewhere Afify et al. in [48] extracted Syzygium cumini L. using methanol, hexane, and ethyl acetate in experiments to determine acaricidal activity of the plant against the two-spotted spider mite, Tetranychus urticae Koch. The methanol extracts showed the highest acaricidal activity of 95.5% mortality, while hexane and ethyl acetate had 94% and 90% mortalities, respectively. The insignificant mortalities obtained by hexane only and hexane-extracted M. edulis are an indication that the mortality of the ticks could be coming from the hexane and not the active compounds from the M. edulis. The use of hexane and other solvents like acetone, chloroform, and butanol is not recommended in efficacy experiments because they have been shown to be very toxic especially to adult engorged ticks [49]. In this trial, hexane was however because tick larvae are not as susceptible to the toxicity of solvents compared to adult ticks as reported by Gonçalves et al. and Ravindran et al. in [45, 49]. Acetone, for example, one of the most toxic solvents in Adult Immersion Techniques (AIT) experiments, could only cause 10% mortality in LPTs [45]. Resende in [50] also shows that methanol, acetone, ethanol, and xylol cause no larval mortality against Amblyomma cajennense Fabricius, 1787 ticks. The difference in susceptibility of the different age groups of ticks to the solvents is attributed to the fact that adult ticks have a protective cuticle layer that is supposed to protect the tick from the effects of dehydration and other physical and chemical effects. This cuticle is also important in the reproduction processes of the tick because it is the site for pheromone production [51, 52]. The challenge is that, despite some organic solvents like acetone and chloroform being very good solvents to active components of plants, they can also dissolve the cuticle layer of ticks with fatal consequences of mortality and reduced reproductive capacity of the ticks [45]. The same can be said for other nonpolar solvents, for example, hexane and petroleum ether, but it must be noted that all solvents have differential effects on ticks depending on the capacity and concentrations of the solvents used [49, 53]. Tick larvae however do not have the cuticle layer and therefore do not suffer the same fate as adult ticks.

While organic solvents generally yield high active compounds, hexane in this study was ineffective. To confirm this assertion, there is need to compare the chemical yield extracted using the different organic solvents.

Organic solvents are generally superior for extraction of bioactive compounds [37, 54]. However, in the search for least cost animal health products for use by marginalised smallholder farmers, the use of organic solvents is limited by affordability and availability. There are also environmental and human health concerns worldwide over using organic solvents [55].

The absence of adverse clinical signs and behavioural change in the mice that received the different hot water leaf extract concentrations is a clear demonstration that use of this plant can be relatively safe. It does not, however, rule out health problems after repeated exposure to higher concentrations or misuse. Findings by Gakuya et al. in [33] showed five mortalities out of eight sampled mice in toxicity experiments that investigated the effect of aqueous M. edulis root-tuber extracts on nematodes in livestock using a 20 g/kg body mass concentration. Differences in the current experiment and the one conducted by Gakuya et al. may be attributed to the different plant parts used in the experiments. Bioactive compounds are more likely to be more concentrated in the roots than in the leaves.

5. Conclusion

Maerua edulis possesses acaricidal properties that potentially match those of amitraz-based Tickbuster when extracted with the suitable solvents and when applied at the correct concentrations. Preparing the plant material by soaking in hot water and adding 1% v/v surfactant improves effectiveness and is an easier way of optimizing activity of this plant for controlling ticks. The method is suitable for use by resource-poor farmers in developing countries because it promises to be both effective and much more economical than using organic solvent extraction. Further work is required to determine the efficacy of the same treatments against adult ticks.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

The authors would like to acknowledge the European Union, 9th European Development Fund, through the African, Caribbean and Pacific (ACP) Science and Technology Programme and the African Dryland Alliance for Pesticidal-Plant Technologies (ADAPPT) Project, for providing the financial support towards this project.

References

F. Jongejan and G. Uilenberg, “The global importance of ticks,” Parasitology, vol. 129, no. 7, pp. S3–S14, 2004.

J. J. de Castro, “Sustainable tick and tickborne disease control in livestock improvement in developing countries,” Veterinary Parasitology, vol. 71, no. 2-3, pp. 77–97, 1997.

B. Perry, T. Randolph, A. Omore, O. Perera, and A. Vatta, “Improving the health of livestock kept by the resource-poor in developing countries,” in Livestock and Wealth Creation: Improving the Husbandry of Animals Kept by Resource-Poor People in Developing Countries, E. Owen, A. Kitalyi, N. Jayasuriya, and T. Smith, Eds., pp. 233–262, Nottingham University Press, Nottingham, UK, 2005.

B. Moyo and P. J. Masika, “Tick control methods used by resource-limited farmers and the effect of ticks on cattle in rural areas of the Eastern Cape Province, South Africa,” Tropical Animal Health and Production, vol. 41, no. 4, pp. 517–523, 2009.

J. E. George, J. M. Pound, and R. B. Davey, “Chemical control of ticks on cattle and the resistance of these parasites to acaricides,” Parasitology, vol. 129, pp. S353–S366, 2004.

A. Y. Li, A. C. Chen, R. J. Miller, R. B. Davey, and J. E. George, “Acaricide resistance and synergism between permethrin and amitraz against susceptible and resistant strains of Boophilus microplus (Acari: Ixodidae),” Pest Management Science, vol. 63, no. 9, pp. 882–889, 2007.

G. Jyothimol, R. Ravindran, S. Juliet et al., “Low level deltamethrin resistance in ticks from cattle of Kerala, a south Indian state,” Veterinary Parasitology, vol. 204, pp. 433–438, 2014.

Livestock in Development, Livestock in Poverty Focused Development, Livestock in Development, Crewkerne, UK, 1999.

V. Muchenje, K. Dzama, M. Chimonyo, J. G. Raats, and P. E. Strydom, “Tick susceptibility and its effects on growth performance and carcass characteristics of Nguni, Bonsmara and Angus steers raised on natural pasture,” Animal, vol. 2, no. 2, pp. 298–304, 2008.

S. Ghosh, P. Azhahianambi, and M. P. Yadav, “Upcoming and future strategies of tick control: a review,” Journal of Vector Borne Diseases, vol. 44, no. 2, pp. 79–89, 2007.

P. C. Stevenson, S. Nyirenda, G. Sileshi et al., “Southern African Pesticidal Plants (SAPP) project. Caesalpinioid woodlands of Southern Africa: optimising the use of pesticidal plants,” Final Technical Report, Natural Resources Institute, University of Greenwich, Chatham, UK, 2010, http://projects.nri.org/sapp/docs/sapp_ftr.pdf.

C. T. Gadzirayi, E. Mutandwa, M. Mwale, and T. Chindundu, “Utilization of Tephrosia vogelii in controlling ticks in dairy cows by small-scale commercial farmers in Zimbabwe,” African Journal of Biotechnology, vol. 8, no. 17, pp. 4134–4136, 2009.

S. R. Belmain, B. A. Amoah, S. P. Nyirenda, J. F. Kamanula, and P. C. Stevenson, “Highly variable insect control efficacy of Tephrosia vogelii chemotypes,” Journal of Agricultural and Food Chemistry, vol. 60, no. 40, pp. 10055–10063, 2012.

E. C. Webb and M. David, “The efficacy of neem seed extract (Azadirachta indica) to control tick infestation in Tswana, Simmentaler and Brahman cattle,” South African Journal of Animal Sciences, vol. 32, no. 1, pp. 1–6, 2002.

R. Srivastava, S. Ghosh, D. B. Mandal et al., “Efficacy of Azadirachta indica extracts against Boophilus microplus,” Parasitology Research, vol. 104, no. 1, pp. 149–153, 2008.

S. D. Magadum, D. B. Mondal, and S. Ghosh, “Comparative efficacy of Annona squamosa and Azadirachta indica extracts against Boophilus microplus Izatnagar isolate,” Parasitology Research, vol. 105, no. 4, pp. 1085–1091, 2009.

H. Cetin, J. E. Cilek, L. Aydin, and A. Yanikoglu, “Acaricidal effects of the essential oil of Origanum minutiflorum (Lamiaceae) against Rhipicephalus turanicus (Acari: Ixodidae),” Veterinary Parasitology, vol. 160, no. 3-4, pp. 359–361, 2009.

A. Regassa, “The use of herbal preparations for tick control in western Ethiopia,” Journal of the South African Veterinary Association, vol. 71, no. 4, pp. 240–243, 2000.

J. A. Rosado-Aguilar, A. J. Aguilar-Caballero, R. I. Rodríguez-Vivas, R. Borges-Argaez, Z. García-Vázquez, and M. Méndez-González, “Screening of the acaricidal efficacy of phytochemical extracts on the cattle tick Rhipicephalus (Boophilus) microplus (Acari: ixodidae) by larval immersion test,” Tropical and Subtropical Agroecosystems, vol. 12, no. 2, pp. 417–422, 2010.

F. D. F. Fernandes and E. D. P. S. Freitas, “Acaricidal activity of an oleoresinous extract from Copaifera reticulata (Leguminosae: Caesalpinioideae) against larvae of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae),” Veterinary Parasitology, vol. 147, no. 1-2, pp. 150–154, 2007.

J. Madzimure, E. T. Nyahangare, H. Hamudikuwanda et al., “Acaricidal efficacy against cattle ticks and acute oral toxicity of Lippia javanica (Burm F.) Spreng,” Tropical Animal Health and Production, vol. 43, no. 2, pp. 481–489, 2011.

J. Madzimure, E. T. Nyahangare, H. Hamudikuwanda et al., “Efficacy of Strychnos spinosa (Lam.) and Solanum incanum L. aqueous fruit extracts against cattle ticks,” Tropical Animal Health and Production, vol. 45, no. 6, pp. 1341–1347, 2013.

M. A. Hyde, B. T. Wursten, P. Ballings, and M. Coates Palgrave, “Flora of Zimbabwe: species information: Maerua edulis,” 2014, http://www.zimbabweflora.co.zw/speciesdata/species.php?species_id=124610

C. K. M. Kaposhi, M. P. Mundia, F. S. Mwangala, R. Banda, and C. Mangoye, “Extraction and evaluation of active ingredients from selected Zambian plants with acaricidal and insecticidal properties,” in Natural Plants Products as Pesticides. Proceedings from the First National Symposium in Zambia held in Lusaka, 2nd–5th August of 1994, A. Berger and C. Mugoya, Eds., pp. 85–92, Swedish University of Agricultural Sciences, 1995.

T. E. Stathers, J. Chigariro, M. Mudiwa, B. M. Mvumi, and P. Golob, “Small-scale farmer perceptions of diatomaceous earth products as potential stored grain protectants in Zimbabwe,” Crop Protection, vol. 21, no. 10, pp. 1049–1060, 2002.

R. Mugandani, M. Wuta, A. Makarau, and B. Chipindu, “Re-classification of agro-ecological regions of Zimbabwe inconformity with climate variability and change,” African Crop Science Journal, vol. 20, no. s2, pp. 361–369, 2012.

R. J. Miller, R. B. Davey, W. H. White, and J. E. George, “A comparison of three bioassay techniques to determine amitraz susceptibility in Boophilus microplus (Acari: Ixodidae),” Journal of Medical Entomology, vol. 44, no. 2, pp. 283–294, 2007.

Plant Production and Protection Division, “Recommended methods for detection of measurements of resistance of agricultural pests and pesticides: tentative method for larvae of cattle ticks Boophilus spp. FAO method no. 7,” FAO Plant Protection Bulletin, vol. 19, no. 1, pp. 15–18, 1971.

M. D. Mathewson and G. Hughes, “The establishment of cultures of two and three host ticks in the laboratory and their use in the screening of candidate ixodicides,” in Proceedings of an International Conference, Tick Borne Diseases and their Vectors, Wilde, Ed., pp. 231–240, Lewis Reprints Ltd, Ton Bridge, UK, 1978.

Statistical Analysis System (SAS), Statistical Analysis System User's Guide, SAS Institute, Cary, NC, USA, 2004.

E. T. Nyahangare, T. Hove, B. M. Mvumi et al., “Acute mammalian toxicity of four pesticidal plants,” Journal of Medicinal Plants Research, vol. 6, no. 13, pp. 2674–2680, 2012.

J. G. Fox, S. W. Barthold, M. T. Davisson, C. E. Newcomer, F. W. Quimby, and A. L. Smith, The Mouse in Biomedical Research, Academic Press, San Jose, Calif, USA, 2nd edition, 2007.

D. W. Gakuya, P. M. Mbithi, J. M. Mugambi, T. E. Maitho, and N. K. Musimba, “Evaluation of the efficacy of aqueous extracts of Albizia antihelmintica and Maerua edulis against the nematode Heligmosomoides polgyrus infection in mice,” Kenya Veterinarian, vol. 28, no. 1, 2007.

D. M. Komwihangilo, E. H. Goromela, and J. M. N. Bwire, “Indigenous knowledge in utilization of local trees and shrubs for sustainable livestock production in central Tanzania,” Livestock Research for Rural Development, vol. 6, no. 3, 1995.

X. Luo, D. Pires, J. A. Aínsa et al., “Anti-mycobacterial evaluation and preliminary phytochemical investigation of selected medicinal plants traditionally used in Mozambique,” Journal of Ethnopharmacology, vol. 137, no. 1, pp. 114–120, 2011.

R. N. Okigbo and U. O. Ogbonnaya, “Antifungal effects of two tropical plant leaf extracts (Ocimum gratissimum and Aframomum melegueta) on postharvest yam (Dioscorea spp.) rot,” African Journal of Biotechnology, vol. 5, no. 9, pp. 727–731, 2006.

J. N. Eloff, “Which extractant should be used for the screening and isolation of antimicrobial components from plants?” Journal of Ethnopharmacology, vol. 60, no. 1, pp. 1–8, 1998.

S. Down, “Surfactants not organic solvents: green pressurized liquid extraction of bioactive flavonoids,” 2011, http://www.separationsnow.com/details/ezine/sepspec24906ezine/Surfactants-not-organic-solvents-Green-pressurised-liquid-extraction-of-bioactiv.html

D. Grzywacz, P. C. Stevenson, W. L. Mushobozi, S. R. Belmain, and K. Wilson, “The use of indigenous ecological resources for pest control in Africa,” Food Security, vol. 6, no. 1, pp. 71–86, 2014.

M. Martin, M. C. McCorkle, and E. Mathias, Ethnoveterinary Medicine: An Annotated Bibliography of Community Animal Health Care, ITDG, London, UK, 2001.

C. W. Huie, “A review of modern sample-preparation techniques for the extraction and analysis of medicinal plants,” Analytical and Bioanalytical Chemistry, vol. 373, no. 1-2, pp. 23–30, 2002.

B.-E. Van Wyk, “The potential of South African plants in the development of new medicinal products,” South African Journal of Botany, vol. 77, no. 4, pp. 812–829, 2011.

J. Ssenyongo and D. Kyaterekera, “Application of organic pesticide technologies for improved agriculture and rural livelihoods in Uganda,” in Agricultural Innovations for Sustainable Development, pp. 65–70, African Technology Policy Studies Network, Nairobi, Kenya, 2009.

M. Czarnota and P. A. Thomas, Using Surfactants, Wetting Agents, and Adjuvants in the Greenhouse, University of Georgia, Athens, Ga, USA, 2010.

K. Gonçalves, E. Toigo, B. Ascoli, G. Von Poser, and V. L. S. Ribeiro, “Effects of solvents and surfactant agents on the female and larvae of cattle tick Boophilus microplus,” Parasitology Research, vol. 100, no. 6, pp. 1267–1270, 2007.

P. Sola, B. M. Mvumi, J. O. Ogendo et al., “Botanical pesticide production, trade and regulatory mechanisms in sub-Saharan Africa: making a case for plant-based pesticidal products,” Food Security, vol. 6, no. 3, pp. 369–384, 2014.

A. Esteban, V. Hernandez, and K. Lunsford, “Exploit the benefits of methanol,” in Proceedings of the 79th Gas Processors Association Annual Convention (GPA '00), Atlanta, Ga, USA, March 2000.

A. E.-M. M. Afify, H. S. El-Beltagi, S. A. Fayed, and E. A. Shalaby, “Acaricidal activity of different extracts from Syzygium cumini L. Skeels (Pomposia) against Tetranychus urticae Koch,” Asian Pacific Journal of Tropical Biomedicine, vol. 1, no. 5, pp. 359–364, 2011.

R. Ravindran, S. Juliet, K. G. A. Kumar et al., “Toxic effects of various solvents against Rhipicephalus (Boophilus) annulatus,” Ticks and Tick-Borne Diseases, vol. 2, no. 3, pp. 160–162, 2011.

J. D. D. S. A. Resende, E. Daemon, C. M. D. O. Monteiro, R. Maturano, M. C. D. Azevedo Prata, and A. F. S. F. Rodrigues, “Toxicity of solvents and surfactants to Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) and Dermacentor nitens (Neumann, 1897) (Acari: Ixodidae) larvae,” Experimental Parasitology, vol. 131, no. 2, pp. 139–142, 2012.

L. M. Cherry, “The production of cuticle wax by engorged females of the cattle tick, Boophilus microplus (Canestrini),” Journal of Experimental Biology, vol. 50, no. 3, pp. 705–709, 1969.

J. G. C. Hamilton, D. E. Sonenshine, and W. R. Lusby, “Cholesteryl oleate: mounting sex pheromone of the hard tick Dermacentor variabilis (Say) (Acari: Ixodidae),” Journal of Insect Physiology, vol. 35, no. 11, pp. 873–879, 1989.

A. K. Sharma, S. Kumar, S. S. Tiwari et al., “Comparative acaricidal properties of different solvents and surfactants on Rhipicephalus (Boophilus) microplus (Acari: Ixodidae),” Indian Journal of Animal Sciences, vol. 82, no. 2, pp. 154–158, 2012.

S. E. Bizimenyera, G. E. Swan, H. Chikoto, and J. N. Eloff, “Rationale for using Peltophorum africanum (Fabaceae) extracts in veterinary medicine,” Journal of the South African Veterinary Association, vol. 76, no. 2, pp. 54–58, 2005.

J.-F. Graf, R. Gogolewski, N. Leach-Bing et al., “Tick control: an industry point of view,” Parasitology, vol. 129, pp. S427–S442, 2004.