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In VitroAcaricidal Efficacy of Neem (Azadirachta indica) Oil against Ear Mites

(Otodectes cynotis)

Article · December 2017

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Journal of Agriculture and Technology Management (JATM)
19(2)

Original Article

In VitroAcaricidal Efficacy of Neem (Azadirachta indica) Oil against Ear Mites

(Otodectes cynotis)
Ali-Akbar M. Kusin, Melvin A. Bagot*, John Philip Lou M. Lumain

College of Veterinary Medicine, Visayas State University, Baybay, Leyte, 6525 Philippines

ABSTRACT unavailable (Huang et al., 2000). Furthermore, the wide

spread use of synthetic pesticide has commonly lead to
Neem tree (Azadirachta indica) is an herbal plant multiple resistance (Coles and Stafford, 1999). These,
that has unique properties against insects. This study in turn, lead to the development of alternative strategies
aimed to assess the in vitro acaricidal efficacy of Neem for ear mite treatment and control. Hence, preparations
seed oil against the ear mite (Otodectes cynotis). A from the plant have been widely used and developed
preliminary test was conducted to determine the LC50 of (Traina et al., 2005).
Neem seed oil immersed in distilled water in DMSO. Neem tree (Azadirachta indica) is a herbal plant
Mites were exposed with approximately 2ml of varying that has many potential uses, including its medicinal
concentrations (0.39%, 0.44%, 0.50%, 0.56%, and applications and its unique properties against insects
0.62%) of Neem seed extract and were observed at 12 (Nisbet, 2000). Its main component is a complex
and 24 hours post-exposure. Results of the study tetranortriterpenoid limonoid called Azadirachtin,
showed that the highest concentration (0.62%) of Neem which is responsible for both anti-feedant and toxic
seed oil mixture is considered highly effective against effects in insects (Subrahmanyam et al., 1989). In this
O. cynotis with 86% mortality rate at 12 hours study, the potential of Neem seed oil against ear mites
exposure. At 24 hours exposure, the mortality was 98% was determined.
and was comparable to 2% Ivermectin at 100%. The
LC50 of the Neem seed oil against O. cynotis were
0.54% and 0.49% at 12 hours and 24 hours, 2 MATERIALS AND METHODS
respectively. The acaricidal activity of the Neem seed
oil may be an effect of the secondary metabolites as Preparation of Neem Seed Oil. A commercially
bioactive compounds that were found during qualitative cold pressed 100% Neem seed oil preparation was
phytochemical analysis (alkaloids, flavonoids and utilized. DMSO (Dimethyl sulfoxide) and distilled
saponins). The present study showed the acaricidal water were utilized to create different concentrations of
efficacy of the Neem seed oil in O. cynotis. Its the extract. The different concentrations were
application to other external parasites can be explored. determined after the LC50 was established. The initial
concentrations for the LC50 of Neem seed oil were
Keywords: in vitro, Otodectes cynotis, Neem oil, 0.50% and 1.0%. After establishing the LC50, which
acaricidal, secondary metabolites was at 0.50% concentration, two higher and two lower
concentrations were computed. The different
concentrations (0.39%, 0.44%, 0.50%, 0.56% and
1 INTRODUCTION 0.62%) were computed as previously described
(Guevara and Recio, 2005).
Ear mite infestation or otocariosis is caused by Collection of Ear Mites. Ear mites were collected
Otodectes cynotis, an obligate parasite that inhabits the from a naturally infested dog and cat using previously
vertical and horizontal ear canals of dogs, cats and wild described procedures (Soulsby, 1982). Briefly, samples
carnivores (Scott and Horn, 1987). It induces various were collected through ear swabbing using cotton buds
degrees of pruritus and symptoms such as scratching, and transferred into a glass slide with a mineral oil. At
rubbing of the ears or violent shaking of the head least ten adult ear mites were picked individually using
leading to hematoma and serious complication on the a single brush strand and transferred into a clean, dry
host’s health (Baker, 1999). Infested animals develop Petri plate. Samples were examined using a microscope
otitis externa, characterized by vertical and horizontal (at 100×) and were identified based on its
canal erythema and a dark brown, ceruminous otic characteristics.
exudates (Herwick, 1978). Assay of Acaricidal activity. The experiment was
The use of synthetic pesticides is the most conducted as previously described (Seddiek et al.,
common control and treatment of otocariosis (Paradis, 2013). Different concentrations of the Neem seed oil
1998). However, these drugs are expensive and mixture were applied (approximately 2 ml) to Petri
sometimes plates with at least ten ear mites and were covered with

*corresponding author: [email protected] 25

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©Cebu Technological University, R. Palma St. corner M.J. Cuenco Ave., Cebu City, 6000 Philippines
Journal of Agriculture and Technology Management (JATM) 19(2)

a nylon mesh. The ear mites were checked under the that are used for the attachment of the male for
microscope at 12 and 24 hours after exposure. copulation.
The mortality and efficacy were determined (Reik After twelve hours of exposure, variation in the
and Keitz, 1954) as follows: number of dead mites was observed and recorded. The
different treatments were dose-dependent. The efficacy
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑚𝑖𝑡𝑒𝑠 of the Neem seed oil mixture according to percent mean
Percent Mortality = 𝑥 100
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑡𝑒𝑠𝑡𝑒𝑑 𝑚𝑖𝑡𝑒𝑠 mortality rate and its equivalent percent efficacy were:
0.62% (T5) with the highest mortality rate (86%) and
Wherein: 70% mortality is ineffective;
71-80% mortality is effective;
was highly effective; 0.56% (T4) with 54% mortality;
81-100% mortality is said to be highly effective. 0.50% (T3) with 22% mortality; 0.44% (T2) with 15%
mortality; and 0.39% (T1) with 11% mortality. The last
Experimental Design Trial. The experiment was four treatments were considered ineffective.
laid out using completely randomized design (CRD) The mean percent efficacy of the Neem seed oil
with eight treatment groups (representing the five mixture at 24 hours exposure of the ear mites was
concentrations and the controls) with five replicates similarly dose-dependent. T5 had the highest mean
each were used. Each replicate had at least ten ear mites mortality (98%) and was considered highly effective
and was impregnated uniformly with the varying (Reik and Keitz, 1954). This was followed by 77%
concentrations of Neem seed oil mixture. Also, two (T4), which was effective. The remaining Neem seed
negative controls, the T01 (-) 10% DMSO, and T02 (-) oil mixtures (T3, T2, and T1) were considered
distilled water; and T0 (+) as a positive control with 2% ineffective. The efficacy of T5 was statistically
Ivermectin were used. comparable with 2% Ivermectin (Table 1). The LC50 of
Phytochemical Analysis of the Plant Oil. A Neem oil mixture were 0.538 and 0.486 based on Probit
qualitative phytochemical analysis of the Neem seed oil analysis at 95% confidence level.
was conducted to test the following secondary The Neem seed oil mixture exhibited potential
metabolites: alkaloids (Mayer’s test, Wagner’s test), acaricidal activity against O. cynotis at 12 and 24 hours
tannins (Tannins test), saponins (Froth test), flavonoids exposure. The efficacy based on mite’s mortality of T5
(Alkaline Reagent test, Lead acetate test, Ferric in 24 hours exposure was found to be comparable with
Chloride test), and terpenoids (Chloroform and sulfuric the commercially available acaricide (2% Ivermectin).
acid test). The qualitative grading system of the The efficacy based on the mortality may be an effect of
phytochemicals was described in the following manner: the secondary metabolites that were identified during
(+) positive, (-) traces and (0) undetected (Senguttuvan the qualitative grading system of the phytochemicals
et al., 2014). tests, wherein flavonoids, alkaloids and saponins were
Statistical Analysis. The mean differences detected (Table 2). Figure 2 shows the results of the
between treatments were analyzed using the One- way qualitative phytochemical tests (flavonoids as indicated
Analysis of Variance (ANOVA). Tukey’s HSD test with intense yellow color; alkaloids with reddish brown
(Honest Significant Difference) was used to evaluate and cream-colored precipitate for Mayer’s and
for the significant differences among treatment groups. Wagner’s tests, respectively; and the presence of froth
Probit analysis was performed to estimate the lethal for saponins).
concentration 50 (LC50) and the 95% confidence limit Furthermore, the main component of Neem oil is a
of the Neem seed oil mixture. complex tetranortriterpenoid limonoid called
Ethical Considerations.The procedures performed Azadirachtin, which is responsible for both anti-feedant
in this study were guided by the principles of animal and toxic effects to organisms (Nisbet, 2000;
welfare, Animal Welfare Act of the Philippines (RA Subrahmanyam et al., 1989). The effects of other
8485) and Administrative Order No. 45 of the Bureau secondary metabolites were: 1) For flavonoids, it
of the Animal Industry of the Philippines. affects insects and bacterial microorganisms (Havsteen,
2002). Its insecticidal activity is mainly inhibition to
enzymatic pathways, such as the action of a cytochrome
3RESULTS AND DISCUSSION P-450 dependent oxidase (Kumar and Pandey, 2013). In
another study, the acaricidal activity of flavonoids was
The characteristics of the ear mites (Figure 1) demonstrated against Psoroptescunicula (Macchioni et
were depicted by their sexual differences. As cited by al., 2004). 2) Alkaloids can act as defense compounds
Souza et al. (2008) and described by Soulsby (1982), of plants, which can be efficient against pathogens due
the adult male is 274 to 362 μm while the ovigerous to their toxicity (Anitha Sri, 2016). It produces multiple
female is between 345 to 451 μm in length. In the distal toxic effects on insects by inhibiting choline
portions, pretarsi of the anterior pairs of the legs reveal acetyltransferase, affecting neurotransmission and
a wine-glass shaped caruncle on a short pedicel. The several other neuroreceptors and DNA synthesis (Wink
male mites have a caruncle on all four pairs of legs, et al., 1998; Wink, 2000). Also, in a review of
while the third and fourth pairs of legs on the female Chowański et al., (2016), alkaloids were reported to be
terminate in long hairs or setae. The posterior body of effective against insects. 3) Saponins have biological
the male also possesses two ventrally situated suckers and pharmacological activities (Shibata, 1977). It has a
pesticidal and insecticidal potential (Chaieb, 2010). The

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©Cebu Technological University, R. Palma St. corner M.J. Cuenco Ave., Cebu City, 6000 Philippines
Journal of Agriculture and Technology Management (JATM) 19(2)

insecticidal activity is due to its interaction with high mortality.

cholesterol, causing a disturbance of the synthesis of However, the level of the secondary metabolites
ecdysteroids. These substances are also protease depends on the season wherein the seeds were collected
inhibitors or cytotoxic to certain insects. Furthermore, (Azmir et al., 2013). The type of soil and other factors,
Podolak et al. (2010) found that saponins are natural such as temperature, altitude, radiation, atmosphere,
glycosides that possess a wide range of light exposure, and nutrition can also influence the
pharmacological properties including cytotoxic activity. variability in plant metabolite levels which can affect
Also, Pelah et al. (2002) extracted saponins from the safety and the reliability of plant extract’s efficacy
quillaja bark and proved its biologic effects by exposing (Pavarini et al., 2012).
to Aedesaegypti and Culexpipiens that resulted in its

Table 1. Mean % mortality of Neem seed oil mixture against O. cynotis at 12 and 24 hours exposure
Treatment Mean number of Mean Number of % Mortality Mean Number of % Mortality
(concentrations) mites exposed Dead Mites at 12- Dead Mites at 24-
hour Exposure hour Exposure
T0(-)1 12 0 0%a 0 0%a
T0(-)2 11 0 0%a 0 0%a
T0(+) 14 14 100%e 14 100%f
T1 (0.39%) 11.6 1.4 11%ab 1.6 13%b
T2 (0.44%) 10.8 1.6 15%b 2.8 26%c
T3 (0.50%) 12.8 2.8 22%b 6.4 50%d
T4 (0.56%) 12.6 6.8 54%c 9.6 77%e
T5 (0.62%) 13.6 11.8 86%d 14 98%f
% Mortality with the same letters are not statistically significant

Table 2. Qualitative phytochemical tests of Neem seed oil

Secondary metabolites Phytochemical results
Alkaloids +
Tannins 0
Terpenoids 0
Flavonoids +
Saponins +

Figure 1. Images of the collected male (A) and female (B) O. cynotis showing ventral suckers (arrowhead, black), wine-glass
shaped caruncle (arrow) and hairs or setae (arrowhead, white). Magnification= 100×

Figure 2. Positive results of the qualitative phytochemical tests for flavonoids (A= Alkaline reagent test); alkaloids (B= Meyer’s
test; C= Wagner’s test); and saponins (D= Froth test)

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Journal of Agriculture and Technology Management (JATM) 19(2)

4 CONCLUSION Herwick, R. P. (1978). Lesions caused by canine ear

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ACKNOWLEDGMENT Morelli, I., and Pampiglione, S. (2004).
Acaricidal activity of aqueous extracts of
The authors are thankful to the Diagnostic camomile flowers, Matricariachamomilla, against
Laboratory of the College of Veterinary Medicine of the mite Psoroptescuniculi. Medical and
Visayas State University and Dr. Naomi Ruth Q. Veterinary Entomology, 18(2), 205–207.
Torregoza for initially postulating the study. The Available from https://doi.org/10.1111/j.0269-
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