Source: Google Earth Maps, (2016)
| Livestock Research for Rural Development 31 (6) 2019 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
The cattle tick, Rhipicephalus appendiculatus is one of the most important ectoparasites of cattle. Traditionally, the control measure for this kind of tick is mainly with synthetic chemicals, which are expensive, toxic to the environment and applicators, and they require skills to administer. Indigenous knowledge (IKs) are eco-friendly and best alternative interventions to sustain and improve livestock productivity in resource-challenged smallholder farming areas. Hence, the study aimed at assessing the potential acaricidal properties of the Tobacco (Nicotiana tabacum) in combination with laundry soap (UfreshTM tablet) formulation against Rhipicephalus appendiculatus larvae and ticks. The in vitro study was carried at the Central Veterinary Laboratory in Lilongwe, Central Region of Malawi from November 2016 to February 2017. The ticks and the larvae were exposed to five concentrations (0.04ml/ml, 0.05ml/ ml, 0.1ml/ml, 0.2ml/ml and 0.13g/ml) of seven different prepared solutions (Tobacco-water extract, Tobacco-ethanol extract, Soap, Soap tobacco-water extract, Soap tobacco-ethanol extract, Ashtraz 12.5% (Amitraz Group) and Cyba-Dip, 15% (Cypermethrine) using the Adult Immersion Test (AIT) and laying ability tests. Results from the in-vitro study showed that all the tobacco remains extract concentrations including the control chemicals induced mortality and anti-laying ability in line with the increased concentration levels of the exposed solution, (P<0.05). Tobacco-water extracts, tobacco ethanol extracts and the two control solutions (Amitraz and Cyba-dip) recorded the lowest LC50 in all the tests conducted. The average LC50 concentration on mortality test was observed at 0.11ml/ml while in laying ability test it was recorded at 0.062ml/ml. However, the study showed a negative interaction of soap when used in combination with these extracts. In conclusion, the study opens a potential avenue in dealing with ticks. Making it possible to make a simple, effective, inexpensive and eco-friendly acaricidal that eliminates the need for the commercial acaricides, which are expensive.
Keywords: Adult Immersion Test, climate change, ethno veterinary practices, Nicotiana tabacum, Rhipicephalus appendiculatus
In tropical Africa and subtropical regions of the world, tick and tick borne diseases (TBDs) are economically important diseases that attack bovines next to trypanosomiasis (Mukhebi, Perry, and Kruska, 1992; Belew and Mekonnen, 2011). Tick are voracious bloodsuckers; loss of blood for their rapid development impoverishes the hosts. The hematophagic (feeding on blood) living habit of ticks causes annoyance to the host, besides triggering anaemia, reducing milk production, transmitting hemoparasitic infections, and predisposing the host to dermatophilus congolensis an agent of dermatophilosis. For example in Tanzania, total annual national loss due to TBD was estimated to be 364 million USD, including an estimated mortality of 1.3 million cattle (Kivaria, 2006). This challenge did not spare Malawi, of which East Coast Fever (ECF) is the most predominant TBD among exotic and crossbred cattle. East Coast Fever occurs where the vector tick, Rhipicephalus appendiculatus, is established is estimated at one cow death every 30 seconds, putting the lives of more than 25 million cattle at risk globally (Castro-Janer et al 2009). Hence creating a significant economic burden for poor livestock keepers, particularly in tropical African and subtropical regions of the World. Although, economic losses due to ticks are mainly due to the diseases which they transmit, (Garcia, 2003) financial losses associated with the decreasing of the value of skins and hides (up to 20-30%) are significant (Biswas, 2003). In Malawi, the highest number of cases of ECF occurs between January and March and the peak is markedly influenced by rainfall distribution over these months. Transmission at that time of the year is by the adult tick. A small peak occurring between May and July is due to nymphal transmission. This peak depends on the climatic conditions in this period, particularly moisture and temperature. Currently controlling ticks and tick-borne diseases with acaricides is of paramount importance in Malawi. However, this proves not working as they fail to produce promising results, because many species of ticks have developed resistance due to continuous and indiscriminate use of them. Although no study has yet assessed the status of acaricidal resistance in ticks, as such resistance has been recorded from various other countries. In contrast, herbal acaricides have diverse advantages over chemical ones, as they are environment friendly, ticks do not develop resistance against these substances, they are nontoxic, and there is no residual effect of the drug, (Pirali-Kheirabadi et al, 2009). In addition, treatment costs are high, the use of chemical acaricides increases the risk of leaving drug residues in meat, milk as well as the environment (Miller, Davey, and George, 2002). According to Kambewa et al (1997) diseases and parasites like ticks remains one of the major problems hindering livestock production in Malawi despite the efforts by Department of Animal Health and Livestock Development (DAHLD), the main government body responsible for implementing the animal health strategy in control and irradication of ticks in the country.
It is believed that some plant extracts mixed with other known ingredients acts as tick repellents and used to control ticks in cattle. Study by Odugbemi, (2008) in East Africa, showed that a mixture of dried leaves of Nicotiana tabacum and Securinega virosa paste could be used externally to destroy worms in animal sores. While in Iran, infusion of the dried tobacco leaf (Nicotiana tabacum) was applied externally as an insect repellent; ointments made from crushed leaves for treating infectious ulceration and as a pediculicide (Cheraghi Niroumand et al 2016). Nicotine a product from tobacco has a proven in vitro lethal effect on Rhipicephalus haemophysaloides (Choudhary et al 2004). According to a survey study carried by Trustees of Agricultural Promotion Programmes (TAPP) in 2015, livestock farmers around Bolero Extension Planning Area (EPA), Rumphi district in Malawi claimed that a combination of tobacco (Nicotiana tabacum) remains and laundry soap ((UfreshTM) solution is effective in control of ticks in cattle. This is not particularly surprising given tobacco and its extracts, nicotine were used for the control of insects long before it was known that nicotine was the toxic agent (Palmer et al 1971). However, there is no scientific paper that has proven true to this claim and there is no study on the synergistic effect of these two phytochemicals.
The present study was undertaken to evaluate the in vitro efficacy of a mixture of tobacco (Nicotiana tabacum) extracts with laundry soap ((UfreshTM) on Rhipicephalus appendiculatus from Malawi Zebu herds in Northern part of Malawi, Rumphi district and to assess smallholder livestock farmer’s knowledge on the management, ways of acaricide usage and delivery and methods of tick control practiced in the area.
The present study evaluates the effectiveness of a mixture of tobacco extracts with laundry soap ((UfreshTM), through in vitro laboratory bioassays against Rhipicephalus appendiculatus using Adult Immersion Test (AIT) and laying ability test.
The specific objectives include;
The hypothesis tested
Hoi: Tobacco (Nicotiana tabacum) extracts through in vitro laboratory bioassays has acaricidal effect against Rhipicephalus appendiculatus.
Hoii: Combination of Tobacco (Nicotiana tabacum) extracts with laundry soap ((UfreshTM) through in vitro laboratory bioassays has acaricidal effect against Rhipicephalus appendiculatus.
This study was carried out among livestock farmers of Bolero Extension Planning Area (EPA) in Rumphi District, Northern Region of Malawi. Bolero is a populated place, located in Rumphi District. The area lies at Latitude: -10°58'16.64" and Longitude: 33°44'17.59" with an estimated terrain elevation above sea level of 1108 meters. The wide variety of landscapes and climates in Rumphi allows for a wide variety of plants. Droughts and erratic rains resulting in crop failure and perpetual food shortages characterize the area. Average annual rainfall range from 300mm in bad seasons to far less than 800mm in good seasons. With the majority of the inhabitants being rain fed agrarians. The area presented itself suitable for this nature of study mainly because of its vulnerability to climate variability and change besides being deeply rich in livestock especially large ruminants.
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| Figure 1. Map of the Northern Region of Malawi, Rumphi District-Bolero EPA Source: Google Earth Maps, (2016) |
A total of 900 fully engorged adult female R. appendiculatus were collected from naturally infested Malawi Zebu cattle herds within the four sampled sections (Chikwawa, Chirambo, Bolero A and Njuma) with a history of no prior exposure to any conventional acaricides. This was accomplished through the collection of ticks from herds, which have not be administered with any conventional acaricides in the previous 30 or more days.
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| Figure 2. Pictorial view of activities during tick collection and transportation: (a) During tick collection in the field, (b) Engorged female ticks,
Rhipicephalus appendiculatus in petri dish, (c) Plastic bottles that was used in transportation and incubation of the ticks, the top covered with a sieve cloth to allow air circulation and (d) Packing and transpotartion mode of the ticks from field to the laboratory, collecting bottle sourrounded by a wet cotton wool |
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Collected ticks were placed in perforated plastic containers (Figure 2 c-d) and sent immediately to Central Veterinary Laboratory (CVL) in Lilongwe, where the tests (adult immersion test - AIT and laying ability tests) were conducted. This was done within the 24 hours period.
Plant Material, tobacco (Nicotiana tabacum) leaves remains used in the present study were collected from Kanengo Tobacco Auction Floor in Lilongwe, Malawi. The collected leave remains were powdered using a plant grinder and 50g of the powdered tobacco leaves remains were soaked in 400ml of two different solutions (70% Ethanol and boiling water) for a period of 3 days. Then the mixture were carefully filtered using a tea strainer up to when all the liquid were extracted. The volume obtained from each mixture were recorded with the aim of calculating the effective solvent of the extraction.
To make soap ((UfreshTM Tablet) solution, two tablets (120g each) were sliced (Figure 3.c) and boiled in 400ml of water until all the soap dissolved. The final solutions which contained soap solution were prepared in the following concentrations; 200ml of tobacco extracts (water and ethanol) were mixed in separate 200ml of soap solution to come up with a 400ml of Soap Tobacco Water extracts (STW) and another 400ml Soap Tobacco Ethanol extracts (STE). These final prepared solutions (Figure 3.f) were then diluted based on the proposed dilutions rates (0.99ml/ml (72%), 0.2ml/ml (14.5%) 0.1ml/ml (7.2%), 0.05ml/ml (3.6%) and 0.03ml/ml (2.2%)). In addition to the five prepared solution, two commercial acaricides, Amitraz and Cyba-Dip were also used as control solutions making seven solutions. These two acaricides were diluted based on the manufacture recommended dilution rate.
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| Figure 3. Solution preparation procedures: (a) Powdered tobacco, (b) UfreshTM tablet laundry soap, (c) Sliced UfreshTM laundry soap, (d) Soap solution preparation procedure, (e) Powdered tobacco leaves remains immersed in Water and 70% Ethanol, (f) Final solution made from Soap-Tobacco Water extracts, Soap Tobacco Ethanol extracts, Tobacco Water extracts, Tobacco Ethanol extracts and Soap respectively |
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Stock solution of desired concentration (v1/v2) from each extract was prepared and subsequently five dilutions of stock solution using distilled water were prepared at the following dilution percent; 72%, 14.5%, 7.2%, 3.6% and 2.2%
The fully engorged adult R. appendiculatus ticks were collected from different livestock farmer herds. Ticks were handpicked from the body of cattle of varying age and from the vicinity of their pens. Engorged female ticks were washed in water after collection and were stored at 28°C before being used. A total of 900 adult engorged female ticks were used for the present study. Out of this, 450 ticks were separated and were held individually at 28°C and 75–85% relative humidity in labeled glass bottle with the mouth covered by muslin cloth for oviposition. The remaining 450 ticks were gathered into seven groups each of 60 ticks (one for each concentration of chemical acaricides and two as a control; one for Amitraz 12.5% and Cyba-dip 15%). Out of 60 ticks in each group, three replicates were used to estimate the acaricidal effects of respective concentration of chemical acaricide by AIT.
The test were conducted as per protocol described by Drummond et al (1973) modified technique by FAO, (2004) and the South Africa Bureau of Standards (SABS) in East London and South Africa. Four ticks were immersed in seven different prepared solutions (soap tobacco ethanol-extracts, soap tobacco water-extracts, tobacco water-extracts, tobacco ethanol-extracts, soap, cyba-dip 15% and ashtraz -12.5%). Each concentration was tested using three replicates. Each replicate contained cleaned, healthy, engorged female ticks that were placed in 30 ml of each of five dilutions (0.99ml/ml (72%), 0.2ml/ml (14.5%), 0.1ml/ml (7.2%), 0.05ml/ml (3.6%) and 0.03ml/ml (2.2%)) of solution in Petri dishes. Each replicate contained cleaned, healthy, engorged female ticks that were placed in 30 ml of each dilution of solution in Petri dishes. The ticks were held in the solution, with occasional gentle agitation, at room temperature (approximately 25°C) for 3 min before being removed and gently dried on absorbent paper. These ticks were observed for death at 30, 60 and 90 minutes interval. The mortalities were recorded in control with the control.
According to Drummond et al (1973) nicotine, the active ingredient in tobacco is a neurotoxic, upon exposure; the tick nerve system is disturbed. The tick become paralyzed and this paralysis is characterized by an acute ascending flaccid motor paralysis and probably become less active which is noticed through reduction in movements followed by coiling of the legs which is a clear sign of death. The mortality percentages were calculated using the following equation:
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Immediately upon arrival at the laboratory, engorged female ticks were washed with distilled water mainly to remove any eggs laid during transport. Four ticks were immersed in seven different prepared solutions (soap tobacco ethanol-extracts, soap tobacco water-extracts, tobacco water-extracts, tobacco ethanol-extracts, soap, cyba-dip 15% and ashtraz -12.5%). Each concentration was tested using three replicates. Each replicate contained cleaned, healthy, engorged female ticks that were placed in 30 ml of each of five dilutions (0.99ml/ml (72%), 0.2ml/ml (14.5%), 0.1ml/ml (7.2%), 0.05ml/ml (3.6%) and 0.03ml/ml (2.2%)) of solution in Petri dishes. After exposure to different solution concentration level, the females ticks were dried on paper towels and placed in a separate plastic specimen tube closed with cotton plugs and incubated at 28 °C and 85 % relative humidity in a biological oxygen demand (BOD) incubator for oviposition after fourteen days.
Data were analysed using General Linear Model (GLM). The model included the fixed effects of treatments (dip type), life cycle stage of ticks and the random effect of concentration nested within treatment. Fisher and Turkey Test was used for post hoc analysis in order to understand significance levels for the difference between the groups of means. A value of <0.05 was considered significant. The data were expressed as the standard mean ± standard error of mean (SEM). The groups were compared using one-way analysis of variance (ANOVA) for repeated measurements using Minitab Statistical Software Version 17 and Genstat 18th Edition.
No noticeable effect on the mortality of the ticks was observed after a time interval of 1 - 30 minutes post-exposure; however, there was some changes in the activeness of the ticks. Most of the ticks were seen to reduce movements as a sign of less activeness with less number of dead ticks. The highest mortalities were recorded at 60 - 90 minutes after exposure. It was noted that in petri dishes treated with soap, STE and STW specifically the highest concentration level solutions, dead ticks were found glued to the base of the petri dish, owing to the sticky nature of the soap solution.
The detailed mortality curve results of various dose concentrations of different prepared solution individually as well as in combination has been depicted in figure 4. Mortality figures shown were based on tick deaths that occurred before completion of egg laying. In vitro test for TE, TW, STE and the two control solutions, Ashtraz and Cyba-Dip demonstrated better killing effects.
There was 16.7% difference (p=0.002) in the mortality rate of ticks treated with a Standard dilution rate of TE, STE and TW as against Control group (Ashtraz and Cyba-Dip). The mortalities rate of ticks treated with TE, STE and TW were significantly higher than the control (table 1), while there was a decline in mortalities for ticks treated with soap and STW as low as 25% below the control group.
| Table 1. Means (± SE) of mortality percentage of standard concentration (0.99ml/ml) as affected by the treatment |
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| Principal solution - 0.99ml/ml | Mean ± SE |
| Ashtraz | 83.3±8.33a |
| Cyba-Dip | 83.3±8.33a |
| Soap | 25.0±0.00b |
| STE | 100±0.00a |
| STW | 75.0+25.0ab |
| TE | 100±0.00a |
| TW | 100±0.00a |
| Pooled Standard error (SEM) = 4.25 | |
| p = 0.002 | |
| Means that do not share a superscript letter are significantly different (p<0.05) | |
In the present study N. tabacum water and ethanol extracts showed an increasing mortality rate against the R. appendiculatus compared to the control group (commercial acaricides).
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| Figure 4. In-vitro effect of principal solutions on survival of
Rhipicephalus appendiculatus Note: TE=Tobacco Ethanol Extracts; TW=Tobacco Water Extracts; STE=Soap Tobacco Ethanol Extracts; STW=Soap Tobacco Water Extracts |
This has been the case as the bioactive agent from the tobacco extracts is readily available, and its lethal action is immediate upon contact with an insect or tick. For centuries, gardeners have used home-made mixtures of tobacco and water as a natural pesticide to kill insect pests. When used as a contact insecticide/acaricides, nicotine acts quickly (Booker et al 2010). This explains the fast lessening of the movement as a sign of weakness and death of the ticks upon administered to the extracts compared to the control groups as observed in the present study (table 1). This is in line with the findings from Drummond et al (1973) who reported that upon being exposed to acaricides, ticks become paralyzed and this paralysis is characterized by an acute ascending flaccid motor paralysis and probably become less active, which is noticed through reduction in movements.
The results of AIT motility assay depend on; (i) minutes taken after exposure of the ticks to prepared solution and (ii) dose dependent response of ticks to different concentration of extracts of tobacco individually as well as in combination. The results of acaricidal efficacy of the prepared solution are summarised in the figure 4. Extract of ethanol and water in whole without soap combinations exhibited highest anti-tick/acaricidal activity in a dose dependent manner and all the ticks were found dead after 90 minutes post-exposure at 0.2ml/ml as well as the undiluted (standard solution).
The peak of the dose-response curve gives the effectiveness of each prepared solution; the higher the peak, the greater efficacy is. The best solution always has the lowest lethal concentration (LC50). LC50 is the concentration of the prepared solution, which causes the death of 50% (one-half) of a group of test animals.
The grouped information (table 1) using Fisher method for the tick mortality of different prepared solution at 0.99ml/ml dilution rate shows that Ashtraz, Cyba-dip, STE, STW, TE and TW shares the same superscripts which clearly explains that they are not significantly different as their means are significantly the same but different from solution prepared from Soap. Looking at their quality properties we can say that of the seven prepared solution, solution prepared from soap performed poorly, (25% mortality rate) than the rest which are above 50%.
The solutions that contained TE and TW had 100% kill rate on ticks, which was the highest, followed by the control solutions (Ashtraz and Cyba-Dip) and the other two solution made from the combination of TW and TE with Soap. These findings are in line with Booker et al (2010) who proved and concluded that tobacco extract in form of bio-oil could be very valuable as a selective acaricides. This bio-oil was found to be effective because it killed some but not all of the pesticides used in the study, which led to the conclusion. In the present study, ticks exposed to solution made from soap had the lowest mean mortality percent (16.67) out of the seven prepared solutions. With reference to the interval, at 95% confidence interval (CI) soap solution diluted at the ratio of 1ml to 5ml of water clearly shows that mortality rate of the ticks will be in the range of -19.08% to 52.41% which is the minimum mortality, which can be attained as compared to other solutions used in the present study.
Solution prepared at 0.2ml/ml dilution had similar effectiveness as to the standard solution (0.99ml/ml), where the grouped mean with Fisher method, it clearly showed that Ashtraz, Cyba-dip, STE, STW, TE and TW were not significantly different but different from solution prepared from Soap (table 2). Looking at their quality properties it can be said that of the seven prepared solution, solution prepared from soap performed poorly, (16.7% mortality rate) than the rest which are above 50%.
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Table 2. Means (± SE) of mortality percentage of 0.2ml/ml concentration level as affected by the treatment |
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| Principal Solution 0.2ml/ml | Mean ± SE |
| Ashtraz | 83.3 ± 8.33ab |
| Cyba-Dip | 75.0 ± 25.0ab |
| Soap | 16.7 ± 33.3b |
| STE | 83.3 ± 8.33ab |
| STW | 66.7 ± 33.3ab |
| TE | 100 ± 0.00a |
| TW | 100 ± 0.00a |
| Pooled Standard error (SEM) = 5.38 | |
| p = 0.001 | |
| Means that do not share a letter are significantly different (p<0.05) | |
There was a significant difference (p<0.05) associations between the principal solutions at 0.1ml/ml concentration levels on the mortality percentages of the ticks (Table 3). The highest mortalities were achieved in ticks exposed to TE (91.7%) followed by TW and STW which recorded 75.0%. The least mortalities were recorded from Soap, with mean mortality of 16.7%. The highest mortalities observed in TE and TW is attributed to the action of the active agent found in tobacco.
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Table 3. Means (± SE) of mortality percentage of 0.1ml/ml concentration level as affected by the treatment |
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| Principal Solution 0.1ml/ml | Mean ± SE |
| Ashtraz | 66.7 ± 8.33a |
| Cyba-Dip | 66.7 ± 22.1a |
| Soap | 16.7 ± 8.33b |
| STE | 50.0 ± 14.4ab |
| STW | 75.0 ± 25a |
| TE | 91.7 ± 8.33a |
| TW | 75.0 ±14.4a |
| Pooled Standard error (SEM) = 5.22 | |
| p = 0.001 | |
| Means that do not share a letter are significantly different (p<0.05) | |
The active agent is assumed to be nicotine, (Choudhary et al 2004) as based on the procedures used in the extraction and preparation of the solutions. The results on mortalities (percentage) of ticks administered with 0.03ml/ml concentration level showed no significant associations (p=0.40) on the principal solutions. However, ticks administered to solution made from soap registered no mortalities (0.00%).
Similar trend has been noticed in all the prepared solutions at different dilution rates. Where TE performed better than the rest of the solutions followed by TW, STW and the control solutions (Ashtraz and Cyba-Dip) and this shows how lethal the tobacco extracts (nicotine) to R. appendiculatus is if used alone. This findings are in line with (Choudhary et al 2004) who found out and proven that tobacco extract (nicotine) has in vitro lethal effect on Rhipicephalus haemophysaloides. However, solution made from soap had always the lowest mortality percentages at all dilution levels. The efficacy of each prepared solution is given by the peak of the dose-response curve; the higher the peak the greater the maximum effect or efficacy. Based on the available data, the prepared solutions can be ranked based on their mortality performance (efficacy) and taking in consideration their median lethal concentration (LC50), which is the concentration of the prepared solution that is estimated to be fatal to 50% of the ticks under trial as presented in figure 5.
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| Figure 5. LC50 of different prepared solutions applied in the study |
The average LC50 concentration was observed at 0.11ml/ml. Two solutions, TE and TW in addition to the two control group had mortalities within average LC50. TE recorded the lowest LC50 (0.03ml/ml) followed by TW (0.08). Similar finding were reported by Booker et al 2010, who showed that tobacco leaves have been used on a small scale, as a natural organic pesticide after undergoing a process called pyrolysis, which involves heating up the tobacco leaves at 900 degrees Fahrenheit (482.2 degrees Celsius) in a vacuum. This process end up producing unrefined substance called bio-oil, which the scientists tested as a pesticide against a wide range of insect pests, including 11 different fungi, 4 bacteria, and the Colorado potato beetle (a major agricultural pest that is very resistant to insecticides). The results from tobacco extracts (water (0.03ml/ml and ethanol, 0.08ml/ml) are comparable with the control group (the commercial used acaricides) which had their mortalities closer to the average LC50, Ashtraz (0.10ml/ml) and Cyba-Dip (0.11ml/ml). The reported LC50 for TE and TW proves to be very effective and provides a promising alternative against R. appendiculatus.
Results from the current study showed a negative interaction of soap when used in combination with these extracts. Soap affect the performance of the extracts (TE and TW) if used in combination. This negative interaction may come owing to the sticky nature of soap solution, which may act as a cover thereby coating the tick away from the extracts hence reducing the exposure. As shown in figure 6, solution made from combination of Soap (STW, 0.13ml/ml and STE, 0.18ml/ml) recorded the highest LC50, above average as compared to those without soap components (TE and TW). This indicate need for higher dose of the prepared solutions (STW and STE) to meet the 50% kill of the ticks. In addition, the finding shows an increase in mean mortalities with increase in the concentration level which simply means the concentration level had an effect in the mortalities of the ticks. Hence to eliminate larger number of ticks, higher dose of the solution need to be used. Nevertheless, when a solution solely made from soap is administered to the ticks, it does not reach even the 50% mortality. This clearly shows that soap reduces the killing effects of the extracts if used in combination.
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| Figure 6. Mortality percentage of ticks exposed to solution made from soap and a combination with the TW and TE |
The present study (Figure 6) clearly shows that, the combination of soap with the extracts has a reduced killing effect on the ticks. This was noticed in all the concentration levels of the solutions used in the present study, where it required high doses of each solution to kill half of ticks if soap is added.
The results of acaricidal efficacy of the prepared solutions are summarised in the table 4. The laying ability of the engorged female ticks were concentration dependent. The percent laying ability of TW, which recorded the lowest LC50, 0.02ml/ml, varied from 0.00 to 25% when tested at concentrations ranging 2.2 to 72%. A range of 0.00 to 58.3% and 0.00 to 66.7% were observed in the control group, Cyba-Dip and Ashtraz respectively upon tested at the same concentration levels from 2.2% to 72%. Ticks, which did not lay eggs, were swollen and black in colour prior death.
| Table 4. Laying ability LC50 of different prepared solutions applied in the study | |
| Solution | LC50 (ml/ml) |
| TW | 0.02 |
| TE | 0.035 |
| Cyba-Dip (control) | 0.035 |
| STE | 0.038 |
| Ashtraz (control) | 0.04 |
| STW | 0.045 |
| Soap | 0.15 |
| Average | 0.059 |
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Note: LC50 - lowest dilution rate of the prepared solution with 50%
laying ability of the sampled ticks |
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At 72% of the solution concentration level, (highest concentration level) only TE and Soap, 8.3 and 41.7% respectively laid their eggs. The average LC50 (<50% egg laying ability) of 0.062ml/ml was best observed in TW (at a concentration of 0.02ml/ml) followed by STE (0.035ml/ml) and the control (Cyba-Dip (0.04ml/ml) and (Ashtraz (0.04ml/ml)), and soap solution being the least of all (0.2ml/ml). All the prepared solution extracts including the control inhibited egg laying ability and thus indicated the R. appendiculatus ovicidal effects. A control of the LC50 of crude aqueous ethanol and water extracts of tobacco in combination with soap revealed that the combined effect of these extracts (Tobacco-water extracts and Tobacco-ethanol extracts) with soap were found having lowest efficacy of 14.4%. Based on the results it clearly shows that soap has a negative interaction with the extracts and it can be established that addition of soap reduce the effectiveness of the extracts. However, owing to the sticky nature of the soap solution, it can be used as a glue or a carrier of the extracts to animal hides/skin.
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| Figure 7. Laying ability LC50 of different prepared solutions applied in the study Note: LC50 - lowest dilution rate of the prepared solution with 50% laying ability of the Sampled ticks |
The best performed solution was identified as lowest solution concentration levels with ≥50% laying ability. This can be interpreted as the lowest concentration of the prepared solution with ≥50% laying ability. The average LC50 value of the extract against R. appendiculatus was 3.6%.
The activities of other plant extracts against R. appendiculatus have been reported (Kaaya et al 1995; Nawaz et al 2015). The acaricidal activity been seen in this study may be due to one or more bioactive compounds present in the leaf extract. Specifically tobacco and its extracts, nicotine were used for the control of insects long before it was known that nicotine was the toxic agent (Palmer et al 1971). The bioactive plant compounds such as nicotine and alkaloids were known to possess insecticidal, growth inhibiting, antimolting, and repellent activities.
Alkaloids in the plant extracts causes mortality and inhibition of laying ability due to their neurotoxic properties (Mtambo, 2008). However, it is indicative that an additive action of the bioactive components may be involved in the tick mortality and inhibition of the oviposition. Comparing the efficacy of the tobacco plant extracts with the controls, commercial acaricides that was used in this study, the plants extracts performed better than the commercial acaricides, Amitraz group and Cyba-dip. George et al (1998) approved the efficacy of the acaricides against B. microplus and in 1981, Stanford et al, approved the use of Amitraz group for the control of R. appendiculatus and R. evertsi. Amitraz group were effective acaricides till the issue of resistance came in (Aguirre et al 2000; Li et al 2004). Based on the above observation and as, nicotine is unique among insecticides in that it is relatively safe, because it ultimately disappears, even in the fixed forms, and leaves no residues that might be dangerous to consumers of food product sprayed with it, provides best alternative to control environmental pollution (Adehan et al 2016).
The authors would like to acknowledge the Trustees of Agriculture Promotion Programmes (TAPP) through Capacity Building for Managing Climate Change in Malawi (CABMACC) programme for financial and material support for the completion of this study, and without forgetting the Central Veterinary Laboratory (CVL) for providing with space for the study.
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Received 7 March 2019; Accepted 15 April 2019; Published 4 June 2019