In-vivo acaricidal efficacy of Tephrosia vogelii leaf extract against tick infestation in cattle

doi:10.21203/rs.3.rs-1743094/v1

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In-vivo acaricidal efficacy of Tephrosia vogelii leaf extract against tick infestation in cattle

https://doi.org/10.21203/rs.3.rs-1743094/v1

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A study was conducted with the objective of evaluating the in-vivo acaricidal efficacy of Tephrosia vogelii leaf extract in controlling ticks on cattle. For this experiment, forty five naturally tick infested cattle were selected and equally divided into five treatment groups of nine animals in completely randomized design. The first three groups were topically treated with 20%, 25% and 30% w/v extract, the forth group was sprayed with diazinon as positive control at a recommended dose of 1:1000 while the fifth group was left untreated as negative control. Tick counts were conducted on day0 pre-treatment and on days4 and 7 post treatment on each animal. Post-treatment quantitative assessment of tick burden revealed a significant (P < 0.05) reduction in tick load in all treated groups compared to untreated ones. The percentage tick count reduction was higher at day 7 than day 4 for all treated groups with their respective efficacy of 80.33%, 85.42% and 90% for 20%, 25% and 30% extracts and 97.67% for diazinon at day 7 post treatment. The observed tick count reductions were found to be statistically significant (P < 0.05) at all dilution levels of the extract but without significant difference (P > 0.05) among themselves. The results also revealed both time and dose dependent efficacy of the extract and its comparable effectiveness with diazinon which was an evidence suggestive of its significant anti-tick effect. Therefore Tephrosia vogelii could be recommended as effective natural tick control alternative, especially for poorly-resourced small-scale farmers in rural areas where commercial acaricides are unavailable or unaffordable.

Acaricide

Leaf extract

Tephrosia vogelii

Tick count

Ethiopia is among the countries naturally endowed with a huge livestock resource. This subsector of agriculture has an enormous contribution to the livelihoods of many Ethiopians and to the national economy of the country at large (Leta and Mesele 2014). But the productivity of this huge resource remained relatively low due to the challenges of high livestock disease incidences, poor management of diseases, poor economy, shortage of feeds, low genetic potential of the animals and unorganized marketing system of the country (Berhe 2010). Among the major constraints to productivity and improvement of the livestock industry is the transmission of economically important diseases by external and internal parasites (Belay et al. 2013). From the external parasites of livestock, tick infestation is the most important whose impact of feeding and the diseases they transmit are responsible for high morbidity and mortality of cattle, reduced meat and milk productivity; high economic losses from downgraded hide and skin; loss of draught power and increased cost of control (Biswas 2003; Jongejan and Uilenberg 2004). Hence, ticks and the diseases they transmit are posing serious impacts on the individual and national economy of especially developing countries. In the view of the aforementioned problems, the control of this ectoparasite is highly desirable if better economic gain is to be obtained from the industry (Bansal 2005). The usual and most effective control method of ticks and subsequently the diseases they transmit has mainly relied on the direct applications of commercial acaricides on the host animals (Willadsen 2006). However, the repeated and inappropriate use of these chemicals resulted in a number of constraints like the development of acaricide resistance, poisoning of treated animals and humans, hazard of residues in meat, milk and its products and environmental contamination (Rajput et al. 2006).

This issue therefore, calls for the need to come up with the adoption of alternative methods that are both safe and effective and able avoid or minimize such problems. The use of plant derived natural compounds is considered as a viable organic alternative to the use of synthetic chemicals and thus would be suitable candidates for the control of livestock tick infestations (Moyo and Masika 2009). Tephrosia is a genus of leguminous plant which belongs to the family Leguminosae and subfamily Papilionaceae. Within this genus, there are approximately 400 different species included which are widely distributed in tropical, sub-tropical and arid regions of the world (Atchison 1951). Tephrosia vogelii (T. vogelii), an important species of the plant genus is used traditionally by many African farmers for different purposes such as to control ticks and worms in animal production systems; cultivated as green manure because of its known nitrogen-fixing ability; for control larval stages of mosquitoes; for protection of grains against weevils, and as fish poison (Mwaura et al. 2012). Phytochemical investigations have revealed the principal bio-active compounds of this plant to be rotenoids, particularly rotenone and deguelin (Gaskins et al. 1972; Al-Hazimi et al. 2005) and these chemicals are well known for their anti-arthropodal effects (Gaskins et al. 1972; Kumar 1984; Lambert et al. 1993).

It is quite difficult and expensive for resource poor rural farmers to manage ticks using the usual conventional acaricides because of their inaccessibility or unaffordability and unsatisfactory dipping services besides the aforementioned chemical problems (Gunjal et al. 2009). It is therefore imperative for them to use T. vogelii as potential source of non-residual botanical acaricide in mitigating the impact of this ectoparasite. Despite its wide traditional use against ticks, there is little scientific evidence demonstrating the effectiveness of T. vogelii in tick control. The objective of this study was therefore to evaluate the in-vivo acaricidal efficacy of T. vogelii leaf extract on cattle tick.

Study area

The present study was conducted in the Worabe agricultural research center found in Worabe town, which is the administrative center of Siltie zone. The town has latitude and longitude of 8°1′N 38°20′E with an elevation of 2,113 meters above sea level. Siltie zone is found in northern part of Southern Nations, Nationalities and Peoples' Region (SNNPR) of Ethiopia at 173km away from Addis Ababa to south west direction and 107km from Hawassa, capital city of SNNPR. The zone covers an area of 3047.83 sq. km and accounts for 2.63% of the total area of the region. The zone zone has varied ecological zones that range from lowland to mountains and can be classified into three major climatic zones on the basis of altitude, rainfall and temperature: 20.6% Dega, 5% Kola and 74.4% Woina-Dega of the total area of the region. The mean anual temperature range from 12 to 26°C and the average annual rainfall ranges from 780 to 1818 mm.

Collection and extraction of T. vogelii leaf

Mature fresh leaves of the plant were harvested from Areka agricultural research center, rinsed with tap water, spread on tarpaulin sheet under well aerated room for a period of two weeks for appropriate air drying to take place at room temperature. The air dried leaves were then finely powdered using electric grinding mill and kept in airtight plastic bags in the dark until sent to laboratory. The extraction process was undertaken at Wondogenet agricultural research center by maceration technique using methanol 95% as solvent. After soaking the powder in methanol overnight, it was strained using double layered gauze to remove the large pieces of leaf material and the resulting extract was filtered using whatman filter paper No.3. The resulting filtrate was then concentrated by allowing the solvent to evaporate under reduced pressure with a rotary evaporator. The final extract was then kept in tightly closed bottle in refrigerator until used for the efficacy study.

Experimental animals

Fort five naturally tick infested cattle of either sex aged between 8 months and 3 years from Werabe agricultural research center were used for this study. The animals were equally divided into five treatment groups of nine cattle each in a completely randomized design. On the day of the treatment (day0), ticks of the genus Amblyoma (the species present on the animals) were counted and recorded in situ along both sides of tick predilection sites of ear, head, neck, dewlap, axilla, perineum and scrotal/udder regions on individual animal through visual inspection before the application of test materials and also on days 4 and 7 post treatment. Fully engorged female ticks were not counted on the first day of the experiment as they are expected to drop off any time. The identities of the selected animals were recorded at the beginning of the study for easy identiﬁcation and participant animals are allowed to graze together with the rest of the herd at the center’s pasture.

In-vivo application procedure of T. vogelii leaf extract

To evaluate the acaricidal activity of test material, the leaf extract was topically applied directly on tick infested body areas of cattle at three different dilution levels of 20%, 25% and 15% w/v after reconstituting using distilled water. The application was done by hand dressing using a paint brush. Positive control cattle were sprayed with commercial acaricide, diazinon as per the manufacturer’s recommended dilution rate of 1:1000 while animals in negative control group were left untreated. All the treatments were applied only once and all the participating animals were monitored daily for any signs of toxicity or any adverse reaction on skin. The efficacy of the leaf extract was calculated based on the following formula as stated by Holdsworth et al. 2006:

% Tick reduction)*100, Where: N0 = Pre- treatment tick count at day 0 and N = post- treatment tick count (i.e. at days 4 and 7).

Data management and statistical analysis

The raw data generated were first entered in to Microsoft Excel 2010 spreadsheet and then transferred to Statistical software package called STATA for windows version 14 for analysis. All analyses were performed at 95% CI and statistical significance level of P<0.05. Descriptive statistics such as mean, standard deviation, percentage and graph were used to express the results of analysis. Analysis of variance (one-way ANOVA-Tukey test) was also used to compare the means of different treatments and control in the in-vivo efficacy study.

From the experiment the extract was found to be safe for the in-vivo application as there were no observed signs of toxicity or any adverse skin reaction in all the groups throughout the study period. The herbal extract showed both time and dose dependent in-vivo anti-tick activity. Diazinon and the leaf extract exhibited a general positive in vivo acaricidal activity against ticks compared to untreated group. The study result indicated a reduction in tick burden in all the treated groups and that there was no significant difference (P > 0.05) in mean tick counts among different dilution levels as shown in Table 1.

All treatments including diazinon caused a significant (P < 0.05) mean tick load reduction with respect to both time and concentration compared with untreated control group but without significant difference (P > 0.05) among themselves. Furthermore, all the three dilution levels of T. vogelii leaf extract had comparable efficacy with the reference drug, diazinon. The overall tick count reduction pattern after treatment indicated a progressive increase in reduction of tick loads on cattle with time since the reduction is higher on day 7 than day 4 (Fig. 1, Table 1).

Diazinon has resulted in maximum percentage mean tick count reduction of 83.16% at day 4 and 97.67% at day 7 post treatment while the 20%, 25% and 30% extracts had 71.04%, 80.83% and 72.17% at day 4 and 80.33%, 85.42% and 90% at day 7 post treatment, respectively. Diazinon had the maximum percentage mean tick count reduction of 97.67% on day 7 followed by 30% extract which caused percentage mean tick count reduction of 90% on the same day. The percentage tick count reduction or percent mortality was higher at day 7 than day 4 for all treated groups as indicated in Table 1.

Table 1

Effect of single topical administration of *T.vogelii* leaf extract on tick count (Mean ± SD) and percentage reduction in naturally tick infested cattle
Treatment group	n	Time			% reduction
Treatment group	n	Day 0	Day 4	Day 7	At day 4	At day 7
Control	9	25.33 ± 9.11	24.33 ± 8.51	24.33 ± 8.94	3.95	3.95
Diazinon	9	42.89 ± 15.91	7.22 ± 5.65^rc	1 ± 1.12^rc	83.17	97.67
20% extract	9	20.33 ± 11	5.89 ± 3.89^rc	4 ± 2.96^rc	71.04	80.33
25% extract	9	26.67 ± 9.79	5.11 ± 3.18^rc	3.89 ± 2.52^rc	80.83	85.42
30% extract	9	45.11 ± 30.19	12.56 ± 7.38^rc	4.56 ± 2.83^rc	72.17	90
r = significant row wise compared to day 0, c = significant column wise compared to control

The overall effect of T. vogelii caused percent mortality of 86.48% at day 7 next to diazinon which caused 97.67% percent mortality on same day without significant difference between them (p > 0.05) as shown in Table 2.

Table 2

The overall acaricidal effect of *T. vogelii* leaf extract against ticks compared with diazinon and control
Treatment group		Tick count (mean ± SD)			% Mortality
Treatment group	n	Day 0	Day 4	Day7	At day 4	At day 7
Control	9	25.33 ± 9.11	24.33 ± 8.51	24.33 ± 8.94	3.95	3.95
Diazinon	9	42.89 ± 15.91	7.22 ± 5.65^rc	1 ± 1.12^rc	83.17	97.67
T. vogelii	27	30.7 ± 21.49	7.85 ± 6.01^rc	4.15 ± 2.68^rc	74.23	86.48
r = significant row wise compared to day 0, c = significant column wise compared to control

The present study was aimed to evaluate the efficacy of different concentrations of T. vogelii leaf extract as a natural acaricide to control ticks on naturally infested cattle. The results of the study demonstrated the reductions in tick counts after application of the T. vogelii leaf extract which reveals its anti-tick effect. These reductions were found to be statistically significant (p < 0.05) at all dilution levels despite a slight difference in efficacy among themselves. T. vogelii is traditionally used by farmers in different parts of Africa to control ticks on livestock. As a result of its interest, several African authors have conducted in-vitro and in-vivo evaluational studies on the acaricidal effect of this plant against various tick species. In these investigational studies, the mortality induced by the plant extracts was often reported to be very high and therefore highly effective against ticks as in the case of the present finding.

The overall 86.48% mortality effect of the extract is comparable with 91.16% against A. variegatum reported from Makoni district in Zimbabwe (Ndava et al. 2018). This overall finding is lower than the report of Kalume et al. (2012) from Congo who revealed mortality of 95% and 100% using 10 and 20 mg/ml of leaves of two varieties of T. vogelii against R. appendiculatus even though it is slightly in agreement with 90% efficacy of 30% extract at day 7. In favor of the current finding similar finding was reported by Dougnon et al. (2014) in Benin in which ethanolic extract of T. vogelii leaf resulted in 98.51% mortality of A. variegatum. The finding of this research also supports the observations made by Matovu and Olila (2007) who investigated high acaricidal activity of T. vogelii methanol extracts on nymph and adult ticks in Uganda in their in-vitro study in which 25% extract has killed 100% exposed ticks. In addition, the study of Kaposhi (1993) in Zambia supported the ant-tick effect of this plant by indicating that a 10% w/v concentration of aqueous leaf extract can adequately protect cattle from Boophilus decoloratus. The present observation further revealed the absence of significant difference between the effectiveness of T. vogelii and diazinon which is in agreement with the works of Gadzirayi et al. (2009) who tried to compare the effectiveness of T. vogelii with conventional Triatix dip in the control of ticks on dairy animals among small scale dairy farmers in Zimbabwe. The result of their study demonstrated that there was no significant difference in the effectiveness of T. vogelii and Triatix dip in controlling ticks. A similar observation was made in Congo by kalume et al. (2012) in which no significant differences were observed between the positive control (amitraz) and the plant extract. Furthermore, Siame et al. (2019) from Zambia also evidenced that the application of aqueous leaf extract at concentration of 5 to 40% w/v significantly reduced tick numbers without significant difference in the observed efficacies between low and high concentrations which is in line with the current finding that showed statistically similar effectiveness at all levels of the tested concentrations. This research also demonstrated that the pattern of post-treatment tick count reduction was in such a manner that there is a progressive increase in reduction of tick load on cattle with time and concentration and this is consistent with the finding of Siame et al. (2019) who revealed similar pattern of tick reduction in Zambia. This is suggestive of the impression that the extract efficacy increases from the day of treatment with subsequent drop in tick burden on treated animals. It was remarkable that in the present study no significant differences were observed between the positive control (diazinon) and the leaf extract. This is an indicative that T. vogelii could be used as natural alternative to the use of synthetic products which are usually unavailable and unaffordable especially in rural areas.

Generally, the herbal extract from this experiment exhibited a significant in-vivo acaricidal effect against ticks indicating the effectiveness of T. vogelii as botanical acaricide. This is probably attributed to the presence of phytochemicals with anti-tick effect in the plant. The findings of previously conducted phytochemical investigations have revealed the principal active components of this plant to be rotenoid compounds, particularly rotenone and deguelin (Gaskins et al. 1972; Al-Hazimi et al. 2005), compounds which are well known for their anti-arthropodal effects (Gaskins et al. 1972; Kumar 1984; Lambert et al. 1993) even though their exact mode of action is poorly elucidated which needs further investigational studies.

The leaf extract was observed to exhibit significant acaricidal activities and was effective in reducing tick burden on cattle. In view of present finding, it could be concluded that T. vogelii leaf extract is effective against ticks and thus could be used as a promising natural alternative for tick control especially by resource-limited rural livestock farmers where commercial veterinary drugs are not easily accessible and unaffordable. From the promising result of this study, it can be recommended that T. vogelii leaf extract could effectively control tick on cattle and therefore be considered for use as botanical acaricide. Further studies should also be conducted to investigate the phytochemical constituents and their exact mechanisms of action behind the acaricidal properties so as to develop herbal based drug formulations.

Acknowledgments

I am grateful to sincerely acknowledge Southern Agricultural Research Institute for funding the research and Werabe Agricultural Research Center for the success of the work through monitoring and evaluating the activities.

Author contributions

The study conception and design, material preparation, data collection and analysis and write-up of the final manuscript were performed by Tajebe Jerjero.

Compliance with ethical standards

In this research work all the procedures including data collection were conducted in accordance with the guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP).

Conﬂict of interest

The author declares that there are no conflicts of interest in the study.

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In-vivo acaricidal efficacy of Tephrosia vogelii leaf extract against tick infestation in cattle

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Abstract

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Materials And Methods

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Conclusion And Recommendation

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