East African Journal of Veterinary and Animal Sciences (2018) Volume 2 (2): 85-90

In Vitro Acaricidal Activity of Crude Methanolic Extract of Leaves of Parthenium

hysterophorus
Anteneh Wondimu1*, Kedir Mehadi2, Yimer Muktar1, and Yehualashet Bayu1

1College of Veterinary Medicine, Haramaya University, P. O. Box 138, Dire Dawa, Ethiopia
2Field veterinarian, Gursum livestock office, P. O. Box 002 Gursum district, Ethiopia

Abstract: A study was conducted to investigate in vitro acaricidal activity of crude methanolic extract
of leaves of Parthenium hysterophorus against three ixodid cattle ticks, namely Amblyomma variegatum,
Amblyomma cohaerens, and Rhipecephalus decoloratus. The ticks were collected from cattle kept at
Haramaya university beef farm for fattening, which were purchased from different markets of Eastern
Hararghe zone. Standard physical and direct stereomicroscopy techniques were employed for
identification of ticks. The adult ticks were exposed to six graded concentrations of the extract in
triplicates; 200mg/ml, 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, and 6.25mg/ml for 24hrs using in
vitro adult immersion test. Mortality rates were recorded at 30min, 1hr, 2hrs, 3hrs, 6hrs, 12hrs and
24hrs post exposure. Diazinon (0.1%), and distilled water were used as positive and negative controls,
respectively. Acaricidal activitiy of the extract concentrations was measured by mean number of ticks
died and acaricidal efficacy (%) relative to the negative control. The result showed time and
concentration dependent acaricidal activity of the extract. Mean mortality showed significant
difference between higher dose (200mg/ml) and lower doses (p<0.05). It was also noted that ticks
exposed to higher concentration of extract showed greater mean mortality compared to diazinon.
Extract diluted to 200mg/ml caused 100%, 100%, and 86.7% while diazinon 91.6%, 95.8%, and
71.1% deaths of A. variegatum, A. cohaerens, and R. decoloratus during 24 hrs, respectively. This
preliminary work suggests that leaves of P. hysterophorus possess a promising acaricidal potential
warranting a follow up work encompassing in vivo toxicity and efficacy tests.

Keywords: Acaricide, Diazinon, In vitro test, Parthenium hysterophorus, Tick

Introduction reports indicated the development of drug resistance by

Ticks are harmful blood sucking external parasites of different tick species that create difficulty of
livestock (Rajput et al., 2006) and potentially pose ectoparasites control by chemicals available in the
serious economic loses to the livestock industry market (Nolan, 1987). These challenges led scholars to
(Worku et al., 2011). They are effective disease vectors, work on alternative and safe ways of tick control.
transmitting several economically important infectious Ethiopia is the home of remarkably diverse flora,
diseases (Bars, 2009). In Ethiopia, ticks and tick-borne including numerous endemic species used by vast
diseases (TBDs) are economically very important and majority of the rural populations for different
cause substantial loses to the livestock economy, traditional medical practices (Dawit, 1986; Shiferaw,
ranking third among the major parasitic disasters, next 1996). Plants are rich sources of natural ingredients
to trypanosomes and endoparasites (Abebaw 2005; which are safe and cost effective and possess acaricidal,
Belew and Mekonnen, 2011). growth inhibiting, anti-molting, and repellent activities.
Ticks of veterinary importance in the country belong Antimicrobial and anti-parasitic compounds of plant
to genus Amblyomma, Haemaphysalis, Rhipicephalus origin are found in plant stems, roots, leaves, bark,
(Boophilus), and Hyalomma (Walker et al., 2007; Nejash, flowers, or fruits (Beuchat et al., 1994). Plant derived
2016). Different authors reported the presence of compounds such as saponins, tannins, polyphenols,
various tick species in different parts of Ethiopia. The and essential oils were reported to have acaricidal
most abundant species found were A. variegatum, A. properties against ticks (Deore and Khadabadi, 2009;
cohaerens, Rh. decoloratus, Rh. Purchelus, A. gemma, and Magadum et al., 2009). These can act on parasites by
Rhipecephalus evertsi evertsi (Semagn and Aregawi, 2012; reducing their development, survival, and reproductive
Tsegaw et al., 2015). rate (Habluetzel et al., 2007). Therefore, alternative
Tick infestation in domestic animals is most acaricides can be developed from many compounds
commonly controlled by using conventional synthetic sequestered in different parts of plants, especially the
acaricides. Currently, several factors deter the use of leaves.
acarcides, which includes cost, its non-biodegradability, The use of extracts from various plants has been
its toxicity to the environment, and retention as a studied since they are potentially less toxic to the
residue in animal body (Jonsson et al., 2007). Moreover, animals and environmentally safer. Different parts of
________________________________________________________________
*Corresponding Author. E-mail: [email protected] Published by Haramaya University, 2018

This is an open access article under the CC BY-NC license ISSN: 2616-8804 (Print)
(https://creativecommons.org/licenses/by-nc/4.0/).
Anteneh et al. East African Journal of Veterinary and Animal Sciences 2 (2): 85-90

plants are reported to be used as traditional medicine palps and others as described by Soulsby (1992) and
for treatment of wounds, ulcerated sores, anemia, fever, Walker et al. (2003).
and heart troubles (Kumar et al., 2013). Study by Deka
et al. (2013) reported methanol extract of P. hysterophorus Plant Collection and Extraction
to have good acaricidal activity and may be utilized in P. hysterophorus leaves were collected from Haramaya
integrated pest management system. Phytochemical University campus. Plant specimen was identified and
study revealed that methanol extract of Parthenium voucher specimen was deposited at the School of Plant
hysterophorus contains carbohydrates, alkaloids, Sciences, Haramaya University. The leaves were
glycosides, flavonoids, tannins, saponins, and phenols washed with distilled water and dried in the laboratory
(Sesha et al., 2013). Different research indicated the for two weeks. The dried leaves were milled finely by
presence of variation in the concentration of secondary electric grinder (GM1001Retsh, Germany). About 150
metabolites in different parts of plants; for example, gram of the dry powder of P. hysterophorous leaves were
alkaloids, tannins, and glycosides are the highest in soaked in 750 ml of 100% methanol in flask and
leaves than other parts (Eloff, 1998). Another study on shaked for 2 days by automatic orbital shaker (GFL
P. hysterophorus extracts showed leaves to contain better 3006, Germany) at 80 rpm (Magano et al., 2011). The
anthelmintic activity compared with other parts (Min et mixture was later strained using a muslin cloth and
al., 2005). This could be attributed to the accumulation filtered using Whatman® filter paper No. 1. Then, the
of effective metabolites at high concentration in the filtrate was concentrated in a vacuum rotary evaporator
leaves than the other parts. Although all parts of P. (RE-5299 Henan, China) at 40°C. The resulting
hysterophorus are rich source of useful bioactive concentrated extracts of plant material was kept in vials
ingredients, no study was reported for the acaricidal with tight lids and stored at 4°C until used (Bagavan et
activities of P. hysterophorous against ticks (Shashank and al., 2009).
Abhay, 2014). Therefore, the present study was
conducted with the objective of evaluating in vitro Phytochemical Screening
acaricidal activitiy of crude methanolic extract of leaves Phytochemical screening was done using standard
of P. hysterophorus. screening methods to detect the presence of secondary
metabolites such as saponins, tannins, phenolics,
Materials and Methods alkaloids, steroids, flavonoids, glycosides and
Study Design phlobatannins (Harborne, 1998).
The study employed an experimental design involving
in vitro adult immersion test described by Sharma et al. Bioassay
(2012) with modifications. For each extract The ticks were identified to species and divided into
concentration, 90 ticks were used. Three groups of 10 triplicate groups containing 10 ticks per species for
ticks were created for six extract dilutions (200, 100, 50, evaluation of in vitro acaricidal activity of the various
25, 12.5, 6.25 mg/ml) and two controls (distilled water concentrations of extracts by adult immersion test
and diazinon) with three replications each. Treated (AIT) (Sharma et al., 2012). The extracts were dissolved
groups were immersed for 30 seconds in one of the in distilled water and diluted in to six concentrations of
extract dilutions, in Petri-dishes (5.5 cm diameter, 1.5 200, 100, 50, 25, 12.5, 6.25 mg/ml of serial dilution.
cm high) containing 10.0 mL of the respective dilutions Diazinon (60%) was diluted in water according to the
of 100% methanol extract from leaves part of P. manufacturer recommendation (1:1000) (Heukelbach
hysterophorous and control immersed in distilled water and Canyon, 2006). The mortality of ticks in every
and diazinon. After treatment, ticks were removed group was recorded after 30 mint, 1hr, 2hr, 3hr, 6hr,
from solution, dried on filter paper and gently allocated 12hr and 24hr of exposure (Nanaa et al., 2010). The
individually in sterile Petri dishes, incubated at 28°C viability of ticks was checked regularly with a blunt
and 75% in a biological oxygen demand (BOD) needle and ticks were recorded as dead if no reaction
incubator. was shown.

Tick Collection and Identification Statistical Analysis

The ticks were collected from cattle kept at Haramaya Data were entered into Microsoft Excel and analyzed
university beef farm for fattening, which were using SPSS Statistics for Windows (SPSS Version 20,
purchased from different markets of Eastern Hararghe 2011). Comparison of mean percentage of tick
Zone. The ticks were collected from different body mortality, at different concentrations was performed by
parts of the animal by hand picking and placed into a one-way ANOVA. Statistical significance was
test tube and immediately transported to Haramaya determined by one way analysis of variance (ANOVA)
University Veterinary Parasitology Laboratory where with multiple comparison tests (Post Hoc/Tukey’s
identification of ticks species were done within few test/HSD) to compare parameter between groups. The
hours of collection under stereomicroscope using results were expressed as mean and the difference
standard identification keys based on their between the means were considered significant when P
morphological features such as ornamentation of value is <0.05.
scutum, festoons, eye and shape of basis capitulum,

86
Anteneh et al. In Vitro Acaricidal Activity of Parthenium hysterophorus
Results and Discussion egg development, disruption of mating, and sexual
The P. hysterophorus leaves crude extract showed dark communication, and inhibition of chitin formation
green color and semi-liquid consistency with (Katoch et al., 2007; Wink, 2012; Chagas et al., 2014).
percentage yield of 11.92%. Phytochemical analysis of The result of the present study indicated that A.
crude extract showed the presence of different variegatum, Rh. decoloratus, and A. cohaerens exposed to
bioactive metabolites such as phenolic compounds, higher doses of crude extract (100 and 200mg/ml)
alkaloids, steroids, tannin, saponins, and glycosides showed greater mean mortality percentage than
(Table 1), which could be related to the medicinal roles diazinon (Table 2). The mean percentage mortality of
of this plant. Many scholars reported that these and ticks species were also significant when compared
other plant derived compounds have acaricidal among the different concentrations, especially between
properties against different species of ticks (Hanus et higher dose (200mg/ml) and the lower doses (6-
al., 2005; Ghosh et al., 2007; Anbazhagi et al., 2009; 12.5mg/ml) (p<0.05) (Table 2). Similarly, earlier studies
Deore and Khadabadi, 2009; Magadum et al., 2009; (Mech et al., 2013; Verge et al., 2016) noted that higher
Mkangara et al., 2014; Srinivas et al., 2014). Studies concentrations of crude extracts displayed promising
reported different mechanism by which phytochemicals acaricidal activities which are an attribute of greater
may act on ectoparasites such as counteraction of amounts of active ingredients at higher level of
growth regulatory hormones, neurotoxic, inhibition of concentration.

Table 1. Phytochemical screening of crude extract of leaves of P. hysterophorus

Chemical Methods used Result
Phenolic compounds Frothing test +
Alkaloid Ferric chloride +
Steroid Ferric chloride test +
Flavonoids Mayer’s test −
Tannin Libermann Burchardt test +
Saponnin Shinoda test +
Glycoside Ferric chloride test +
+= Present; −= Absent.

Greater post exposure mortality of A. variegatum and hysterophorus contain higher bioactive components than
A. cohaerens was recorded at all concentrations of the other parts of the plant which make it more effective
extract showing relative susceptibility of these tick acaricide (Eloff, 1998; Habluetzel et al., 2007; Deka et
species to P. hysterophorus as compared to Rh. decoloratus al., 2013). In addition, P. hysterophorus shows various
(Table 2). In general, increased number of tick medicinal values including anti-inflammatory,
mortality across concentration gradient and time was antimicrobial, anthelmintic, antifungal, anti-cancerous,
observed (Figure 1). This indicates that ticks exposed pesticide, and thrombolytic activities (Kuhn and
to low dose take relatively longer time to die than at Winston, 2007; Kumar et al., 2013). It was also reported
higher doses. The overall mortality showed that the that methanol extraction of P. hysterophorus are effective
crude methanol extract of P. hysterophorous leaves against certain gram negative and gram positive
resulted in greater deaths of ticks than the standard pathogenic bacteria (Rastogi and Mehrotra, 1991;
acaricide (diazinon). Comparable results were reported Sukanya et al., 2009; Kamarapu et al., 2015).
by several scholars who revealed that leaves of P.

Table 2. Mean mortality percentage of ticks exposed to different concentration of crude extract of leaves of P.
hysterophorus at 24hrs of exposure time
Treatments (mg/ml)
Species Water 6.25 12.5 25 50 100 200 Diazinon
A. variegatum 0(0.0)c 66.7(3.3)b 80.0(5.7)b 86.7(6.7)ab 100(0.0)a 100(0.0)a 100(0.0)a 91.6(8.6)a
Rh. Decoloratus 0(0.0)bc 43(6.4)ab 56.6(8.9)ad 72.3(11.7)ad 77.5(11.4)ad 78.9(10.6)ad 86.7(6.7)d 71(9.2)ad
A. cohaerens 0(0.0)c 75.6(7.2)b 81.5(5.1)ab 86.6(6.6)ab 92.9(3.5)ab 100(0.0)a 100(0.0)a 95.8(4.1)ab
a-d
Means with similar superscript across rows are not different (P≥0.05); A. variegatum= Amblyomma variegatum; A. cohaerens=
Amblyomma cohaerens, Rh. Decoloratus= Rhipecephalus decoloratus; Value are Mean ± SEM (standard error of the mean); a, b, c, d=
Mean difference at different concentration.

87
Anteneh et al. East African Journal of Veterinary and Animal Sciences 2 (2): 85-90

10
24hr

8
12hr

6hr
6
Mean Mortality

3hr
4
2hr

2
1hr

30min
0

Crude Extract Concentration

Figure 1. Mean mortality of ticks exposed to graded concentrations of crude extracts of leaves of P. hysterophorus across
time

Conclusion Anbazhagi, T., Kaduval, K., Suguna, G. & Petrus, A. J.

Crude methanolic extracts of P. hysterophorus leaves A., (2009). Studies on the pharmacological and in
displayed high acaricidal potential against A. variegatum, vitro antioxidant potential of Cleome gynandra
Rh. decoloratus, and A. cohaerens ticks species from cattle. Linn. Leaves. Natural Product Radiance, 8 (2): 151-
Higher concentration of the crude extract showed 157.
greater acaricidal effects. Therefore, we suggest future Bagavan, A., Kamaraj, C., Elango, G., Zahir, A. &
research that encompasses larval and nymphal stages, in Rahuman, A. A. (2009). Adulticidal and larvicidal
vivo protocols, and toxicological evaluation of the plant efficacy of some medicinal plant extracts against
for possible validation of the leaf extract for future ticks, flukes and mosquitoes. Veterinary Parasitology,
alternative development of medicine. 166 (3): 286-292.
Bars, C. L. (2009). Tick-borne disease management.
Acknowledgements Veterinary Times, 18th May.
The researchers acknowledge Haramaya University for Belew Tiki & Mekonnen Addis (2011). Distribution of
allowing the use of laboratory facilities for this study. Ixodid Ticks on Cattle in and Around Holeta Town,
Ethiopia. Global Veterinaria, 7 (6): 527-531.
Beuchat, L. R., Brackett, R. W. & Doyle, M. P. (1994).
Conflict of Interests Lethality of carrot juice to L. monocytogenes as
The authors declare that they have no competing affected by pH, sodium chloride, and temperature.
interests. Journal of Food Protection, 57: 470-474.
Chagas, A. C. S., Domingues, L. F., Fantatto, R. R.,
References Giglioti, R., Oliveira, M. C., Oliveira, D. H., Mano,
Abebaw Gashaw (2005). Host preference and seasonal R. A. & Jacob, R. G. (2014). In vitro and in vivo
variation of tick (Amblyomma) on naturally infested acaricide action of juvenoid analogs produced from
cattle in Jimma zone, south western Ethiopia. the chemical modification of Cymbopogon spp. and
Journal of Agriculture and Rural Development in the Corymbia citriodora essential oil on the cattle tick
Tropics and Subtropics, 106 (1): 49-57.

88
Anteneh et al. In Vitro Acaricidal Activity of Parthenium hysterophorus
Rhipicephalus (Boophilus) microplus. Veterinary anticancer, antioxidant and anti-HIV agents. BioMed
Parasitology, 205 (1): 277-284. Research International, 2013: 1-11.
Dawit Abebe (1986). Traditional medicine in Ethiopia: Magadum, S., Mondal, D. B. & Cehosh, S. (2009).
The attempts being made for its effective and better Comparative efficacy of Annona squamosa and
utilization. SINET, Ethiopian Journal of Science, 9 (l): Azadirachta indica extracts against Boophilus Microplus
61-69. Izatnagar isolate. Parasitology Research, 105: 1085-
Deka, B. P., Jiten, M. & Bhattacharyya, P. R. (2013). 1091.
Acaricidal Activities of Parthenium Hysterophorus L. Magano, S. R., Mkolo, M. N. & Shai, L. J. (2011).
against Red Spider Mite, Oligonychus coffeae Nietner Repellent properties of Nicotiana tabacum and
(Acarina: Tetranychidae) of Tea. International Journal of Eucalyptus globoidea against adults of Hyalomma
Science and Research, 4 (5): 2319-7064. marginatum rufipes. African Journal of Microbiology
Deore, S. L. & Khadabadi, S. S. (2009). Larvicidal Research, 5: 4508-4512.
activity of the saponin fractions of Mech, J., Bhuyan, P. D., & Bhattacharyya, P. R. (2013).
Chlorophytumborivili anumsantapau and Fernandes. Acaricidal Activities of Parthenium hysterophorus
Journal of Entomology and Nematology, 1 (5): 064-066. L. against Red Spider Mite, Oligonychus Coffeae
Eloff, J. N. (1998). Which extractant should be used for Nietner (Acarina: Tetranychidae) of Tea.
screening and isolation of antimicrobial International Journal of Science and Research, (4) 5:
components from plants. Journal of 2319-7064.
Ethnopharmacology, 60 (1): 1-8. Min, B. R., Hart, S. P., Miller, D., Tomita, G. M., Loetz,
Ghosh, S., Azhahianambi, P. & Yadav, M. P. (2007). E. & Sahlu, T. (2005). The effect of grazing forage
Upcoming and future strategies of tick control: a containing condensed tannins on gastrointestinal
review. Journal of Vector Borne Disease, 44 (2): 79-89. parasite infection and milk composition in Angora
Habluetzel, A., Carnevali, F., Lucantoni, L., Grana, L., does. Veterinary Parasitology, 130: 105-113.
Attili, A. R., Archilei, F., Antonini, M., Valbonesi, Mkangara, M., Erasto, P. & Chacha, M. (2014).
A., Abbadessa, V., Esposito, F. & Van der Esch, S. Acaricidal activity of Commiphora swynnertonii (Burtt)
A. (2007). Impact of the botanical insecticide Neem stem bark extracts against adult Rhipicephalus
Azal® on survival and reproduction of the biting Appendiculatus and Amblyomma Variegatum. American
louse Damalinia limbata on angora goats. Veterinary Journal of Research Communication, 2 (9): 82-92.
Parasitology, 144: 328–337. Nanaa, P. B., Manianiaa, N. K., Marangab, R. O.,
Hanus, L. O., Řezanka, T., Dembitsky, V. M. & Kutimab, H. L., Bogab, H. I., Nchuc, F. & Eloff, D.
Moussaieff, A. (2005). Myrrh-commiphora (2010). Attraction response of adult Rhipicephalus
chemistry. Biomedical Papers, 149 (1): 3-28. appendiculatus and Rhipicephalus pulchellus ticks to
Harborne, J. B. (1998). Phytochemical methods a guide extracts from Calpurnia aurea (Fabaceae). Veterinary
to modern techniques of plant analysis, Chapman Parasitology, 174: 124-130.
and Hall, London, pp: 182-190. Nejash Abdela (2016). Review of economically
Heukelbach, J., Speare, R. & Canyon, D. (2006). important cattle tick and its control in Ethiopia.
Natural products and their application to control Journal of Veterinary Medicine Research, 3 (1): 1044.
head lice: an evidence-based review. In: G. Nolan, J. (1987). New approaches to the development
Brahmachari (Ed.), Chemistry of Natural Products: and management of drugs used in ectoparasite
Recent Trends & Developments, Research control. Veterinary Parasitology, 25: 135-45.
Signpost, India, pp: 277-302. Rajput, Z. I., Song-hua, H., Wan-jun, C., Abdullah, G.
Jonsson, N. N. & Piper, E. K. (2007). Integrated A. & Chenwen, X. (2006). Review of Importance of
control programs for ticks on cattle, UQ Printery, tick and their chemical and immunological control
Australia, pp: 135-136. in livestock. Journal of Zhejiang University Science, 7:
Kamarapu, M., Ravulakolanu, V. & Yalavarthy, P. D. 912-921.
(2015). In vitro evaluation of antibacterial activity of Rastogi, R. P. & Mehrotra, B. N. (1991). Compendium
leaf and flower extracts of Parthenium hysterophorus L. of Indian Medicinal Plants, Vol. 2, (1970-1979),
Journal of Biology and Life Science, 3 (4): 838-842. Central Drug Research Institute, Lucknow and
Katoch, R., Katoch, M., Yadav, A. & Srivastava, A. K. Publications and Information Directorate, New
(2007). Formulation of herbal ectoparasiticidals. In: Delhi, pp: 514.
Compendium of 18th National Congress of Semagn Kassa & Aregawi Yalew (2012). Identification
Veterinary Parasitology, September 7–9, Jammu, of Ixodide ticks of cattle in and around Haramaya
India, pp: 24-31. district, eastern Ethiopia. Scientific Journal of Crop
Kuhn, M. A. & Winston, D. (2007). Winston & Kuhn’s Science, 1 (1): 32-38.
Herbal Therapy and Supplements: A Scientific and Sesha, M. S. D., Satyavathi, K., Bhojaraju, P., Preeti, P.
Traditional Approach, 2nd ed., Lippincott Williams M., Lovaraju, K. P., Priya, K. S., Kumari, Y. R.,
& Wilkins, Philadelphia, United States. Prasad, D. V. & Kanthal, L. K. (2013). Cytotoxic
Kumar, G., Chashoo, A. K. & Saxena, A. K. (2013). and anthelmintic activities of methanolic extract of
Parthenium hysterophorus: a probable source of Parthenium hysterophorus L. Pharmacophore, 4 (6): 275-
279.

89
Anteneh et al. East African Journal of Veterinary and Animal Sciences 2 (2): 85-90

Sharma, A. K., Kumar, R., Kumar, S., Nagar, G., Singh, medicinal plants against clinical and
N. K., Rawat, S. S., Ray, D. D. & Ghosh, S. (2012). phytopathogenic bacteria. African Journal
Deltamethrin and cypermethrin resistance status of Biotechnology, 8 (23): 6677-6682.
Rhipicephalus (Boophilus) microplus collected from six Tsegaw Desalegn, Abraham Fikru & Surafel Kasaye
agro-climatic regions of India. Veterinary Parasitology, (2015). Survey of tick infestation in domestic
188: 337-345. ruminants of Haramaya district, eastern hararghe,
Shashank, K. & Abhay, K. P. (2014). Pharmacological Ethiopia. Journal of Bacteriology and Parasitology, 6: 246.
activities of some common Indian weeds: a review. Verge, S. J. N, Coy, L. R., Caicedo, D. R. & Barrera, E.
Mintage Journal of Pharmaceutical and Medical Sciences, 3 C. (2016). Effect of acaricidal activity of Solanum
(1): 12-17. nigrum on Tetranychus urticae Koch under
Shiferaw Messeret (1996). The role of health laboratory conditions. African Journal of Biotechnology,
professionals in the development of traditional 15 (10): 363-369.
medicine in Ethiopia. In: proceedings of the Walker, A. R., Bouattour, A., Camicas, J. L., Estrada-
workshop on Development and utilization of Pena, A., Horak, I. G., Latif, A. A., Pegram, R. G. &
Herbal Remedies in Ethiopia, Ethiopian Health and Preston, P. M. (2007). Ticks of Domestic Animals
Nutrition Research Institute, Addis Ababa, in Africa: A Guide to Identification of Species.
Ethiopia, pp: 15-18. Bioscience Reports, Edinburgh Scotland, U.K., pp:
Soulsby, E. J. L. (1992). Helmithes, Arthropods and 221.
Protozoa of Domesticated Animals, Tindall, Walker, R., Butiur, A., Estrada-pen, S., Hora, A., Latif,
Bialliere, London, pp: 475-495. G., Pergam, A. & Preston, P. (2003). Tick of
SPSS (Statistical Package for the Social Sciences). domestic animal in Africa, Guide to Identification
(2011). IBM SPSS Statistics for Windows, Version Tick Species, pp: 3-210.
20.0. Armonk, NY: IBM Corp. Inc. Wink, M. (2012). Medicinal plants: a source of anti-
Srinivas, R., Jyothi Chaitanya, P., Chandrashekar, R. & parasitic secondary metabolites. Molecules, 17 (11):
Lakshmi Bhavani, N. (2014). Analysis of 12771-12791.
Preliminary phytochemicals of leaf extracts of Worku Berhanu, Haileleul Negussie, Sefinew Alemu &
Cleome gynandra L. World Journal of Pharmaceutical Hailu Mazengia (2011). Assessment on major
Science, 2 (9): 1043-1045. factors that cause skin rejection at Modjo export
Sukanya, S. L., Sudisha, J., Hariprasad, P., Niranjana, S. tannery, Ethiopia. Tropical Animal Health and
R., Prakash, H. S. & Fathima, S. K. (2009). Production, 43: 989-993.
Antimicrobial activity of leaf extracts of Indian

90