Abstract: A series of tests to examine the utility of Neem leaves as anthelmintic effect on Haemonchus contortus in goats was conducted. In vitro test revealed that the pure azadirachtin tested at graded level on motility of Haemonchus contortus L3 larvae had the maximum anti larval activity at 1000 microgram concentration. The motility of Haemonchus contortus L3 larvae was not reduced up to 400 μg. The concentration of azadirachtin content in the Neem leaves was determined to be 0.0244%. In yet another experiment involving twelve weaned nondescript male kids for a duration of 63 days, the efficacy of feeding Neem leaves as an anthelmintic source was studied by infecting with 5000 larvae of Haemonchus contortus (L3) on animals fed with or without Neem leaves. It was inferred that feeding Neem leaves reduced the Egg per Gram (EPG) count from 42nd day and worm count was also significantly reduced compared to infected groups fed with complete diet that did not contain Neem leaves. Hence, it is hypothecated that a continuous feeding of Neem leaves for more than 6-9 weeks would result in reducing the EPG count as well as the worm count. It is suggested that at least during the epizootics of worm infection, Neem leaves could play a role as an alternate non-chemical organic source of anthelmintic. Thus Neem can be considered as a potential candidate for organic farming as apart from delivering nutrient and organic source of anthelmintic.
INTRODUCTION
Neem (Azadirachta indica), a tree in the mahogany family Meliaceae, is native to India, Pakistan and Burma, growing in tropical and semi-tropical regions. The roundworm, Haemonchus contortus, is responsible for 80% of worm infections in small ruminants causing heavy economic losses especially to poor farmers in India. Presently, this infection is treated by giving synthetic chemical compounds (anthelmintics), which are costly, causing drug residues in food products. Thus they indirectly affect the health of the human beings. Further, overdependence on these anthelmintics has resulted in drug resistance in animals. To overcome these situations, several plants have been investigated for their anthelmintic properties. Assis et al. (2003) observed that maximum inhibition of Haemonchus contortus larvae was observed with hexane and methanol extracts of Spigelia anthelmia. Significant decrease in faecal egg counts of Haemonchus contortus was recorded in Boer goats fed with Acacia karoo diets (Kahiya et al., 2003). Literature is very scanty about feeding Neem leaves to goats and its effect in reducing the worm load especially Haemonchus contortus. Though Costa et al. (2006) reported no anthelmintic activity at lower level of feeding Neem leaves to sheep, the neutraceutical properties of neem and the level of feeding to derive beneficial effects are yet to be documented. It is in this context, a study was planned to evaluate the anthelmintic efficacy of Neem leaves in goats.
MATERIALS AND METHODS
The experiment involved a series of tests carried out at the Department of Animal Nutrition, Madras Veterinary College, Tamilnadu, India during 2006, to examine the utility of Neem leaves as anthelmintic which include screening azadirachtin for its efficacy against intestinal worms, assessing the concentration of azadirachtin in Neem leaves and evaluating the anthelmintic effect of Neem leaves in complete diet on Haemonchus contortus in goats.
Efficacy of Azadirachtin
The efficacy of azadirachtin as anthelmintic was tested against intestinal
worms. The abomasal worms, Haemonchus contortus, were collected from
goats slaughtered at perambur slaughterhouse Chennai, Tamilnadu. The coprocultures
of Haemonchus contortus were prepared as per Gordon-Whitlock jar method.
The culture jars were incubated at 28°C in a dark place for a week (Gathuma
et al., 2004). The larvae of Haemonchus contortus were harvested
and aliquoted in 1.5 mL Eppendorf tubes. The larval counts were adjusted to
have 100 larvae per 100 μL of distilled water. These larvae were bioassayed
in 24 well plates with pure azadirachtin (Sigma, USA A7430) at different concentrations
viz., 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mg in triplicate.
The mortality was observed once in six hours for a period of 24 h. The activity
of the larvae was graded as per Boisvenue et al. (1983).
Azadirachtin Analysis
The Neem leaves samples collected from six trees were shade dried and ground
to pass through 1 mm sieve and the samples were stored for analysis. The azadirachtin
content in the Neem leaf samples were analysed as per BIS (1995) and the equipment
operating manual for HPLC system used for analyzing the azadirachtin content
was EOM/PSP/ACY/-001-00.
Experiment to Evaluate the Anthelmintic Effect of Neem Leaves in Complete
Diet on Haemonchus Contortus in Goats
A trial was conducted for 63 days in non descript kids to assess the efficacy
of feeding Neem leaves as an anthelmintic source. Twelve weaned nondescript
male kids (approximately 8-9 months old) were used in the study. The animals
were dewormed and dipped for ecto parasites. Four kids were randomly allotted
to each of the following three treatments that were fed with complete diet having
iso nitrogenous and iso calorific value. The animals were experimentally infected
on day one of the experiment with 5000 larvae of Haemonchus contortus
(L3) cultured as mentioned earlier in the following manner.
| Control group (T1 ) | - | Complete rations without Neem leaves and without infection with Haemonchus contortus (L3) |
| Infected group (T2) | - | Complete rations without Neem leaves and infected with Haemonchus contortus (L3) |
| Infected and Neem leaves fed group (T3) | - | Complete rations with Neem leaves and challenged with Haemonchus contortus (L3) |
Egg per Gram (EPG) Counts in the Dung
The animals were weighed at fortnightly intervals in an electronic balance
to monitor their growth pattern. The EPG was assessed by modified McMaster procedure
(Hanson and Perry, 1994) every 7 days from 21st day post infection in dung samples
collected per rectum from experimental animals. In this technique, 3 g of dung
material was mixed thoroughly with 42 mL of floatation solution (saturated salt
solution in water). This suspension was sieved over a beaker with a 250 micron
mesh sieve and the filtrate was agitated and decanted into a 15 mL test tube.
The decanted solution was centrifuged at 2000 rpm for 2 min and 0.15 mL of the
fluid was removed from the top layer of the test tube and transferred to both
chambers of McMaster slide. Number of eggs in etched area of both chambers of
the slides was counted and multiplied by 100 to arrive at EPG.
Haemonchus contortus Worms Count in the Abomasum
At the end of ten weeks, the animals were sacrificed to assess the establishment
of infection in treated and control group of animals. The worm count was done
following the procedure mentioned in the Anonymous (1977). As per this procedure,
the abomasums were ligated and removed from the animals. The abomasums was washed
thoroughly with stream of water from tap. The mucous membrane being carefully
rubbed to remove dry worms adhering to it. The contents of the bowel were passed
through a wire mesh screen of aperture of 0.15 mm and washed with stream of
water. When all the materials have been washed in this way, the screen was inverted
and the worms collected on the screen were washed into a trough which had 4
L of water with 10% formalin. Forty milliliter of this solution were placed
in a petridish with worms counted under a dissecting microscope. Total no of
worms counted in this sample was multiplied by 100 to get the no of worms present
in the abomasums. The worms were also counted manually which were adhering to
the contents.
Statistical Analysis
The statistical analysis of the data collected during in vivo trial
was carried out as per the procedure of Snedecor and Cochran (1989).
RESULTS
Efficacy of Azadirachtin
The efficacy of azadirachtin at different concentrations is given in Table
1. The azadirachtin concentration from 25 to 400 μg had no effect on
the motility of L3 larvae of Haemonchus contortus even after 24 h. The
activity at 500, 600 and 700 μg was also nil though there was some reduction
in motility of L3 larvae after 24 h. The azadirachtin concentration at 800 μg
had a borderline anti larval activity of L3. At 1000 μg concentration of
azadirachtin, there was a reduction in motility up to 92.5% at 24 h suggesting
that azadirachtin was able to reduce motility at increased concentrations and
it had an activity level 3 while hundred percent of the larvae in the non-treated
well (control) were motile.
| Table 1: | Efficacy of azadirachtin on the motility of Haemonchus contortus L3 larvae in an in vitro study |
| Activity 0 = 50% larvae affected; Activity 1 = 50-75% larvae affected; Activity 2 = 76-90% larvae affected; Activity 3 = 91-97% larvae affected; Activity 4 = 100% larvae affected |
|
| Table 2: | Evaluation of anthelmintic efficacy of Neem leaves in goats (Mean±SE) |
| Each value is a mean of four observations, Values in rows with different superscripts vary significantly (p<0.01) | |
Azadirachtin Analysis
The concentration of azadirachtin content in the Neem leaves were 0.0244%.
Experiment to evaluate the anthelmintic effect of Neem leaves in complete
diet on Haemonchus contortus in goats
The performance of kids in T1, T2 and T3 are given in Table
2. The Average Daily Gain (ADG) of the three groups was 43.33, 22.86 and
25.87 g, respectively. The ADG of kids in T1 was significantly higher (p<0.01)
compared to T2 and T3. Though the Neem leaves fed groups (T3) had a higher ADG
of 3 g compared to control challenged group, it was not statistically significant.
Egg per Gram Counts in the Dung
Average EPG count of Haemonchus contortus in the dung of goats experimentally
infected with Haemonchus contortus L3 larvae or with out infection in
a trial conducted to evaluate the anthelmintic effect of Neem leaves in complete
diet for ten weeks is presented in Table 2. The EPG count
between T2 and T3 were not statistically significant at 21st, 28th and 35th
day post infection where as it was highly significant (p<0.01) from 42nd
day onwards.
Haemonchus contortus Worms Count in the Abomasum
Neem leaves feeding was effective in lowering the worm count in the abomasums
from 262.25 to 139.25 numbers (Table 2).
DISCUSSION
Efficacy of Azadirachtin
Similar to 1000 μg concentration of azadirachtin, inhibition of motility
of Haemonchus contortus larvae was observed by Assis et al. (2003)
at higher concentrations of plant extracts of hexane and methanol indicating
the relevance of this screening test in judging anthelmintic property.
Azadirachtin Analysis
At the rate of 0.0244% of azadirachtin, about 4 g of Neem leaves is required
to provide 1000 μg of azadirachtin.
Experiment to Evaluate the Anthelmintic Effect of Neem Leaves in Complete
Diet on Haemonchus contortus in Goats
The ADG was significantly reduced in T2 and T3 compared to T1. Contrary
to the results of the current study, Niezen et al. (1998) observed no
such reduction in daily gain in lambs grazing Lotus pedunculatus and
Hedysarum coronarium plants though there was a slight gain of 3 g in
T3 compared to T2. Kukde et al. (1999) recorded an increase in total
feed intake when Neem leaves were fed to calves in contrast to the present study
where there was similar feed intake among the three treatment groups.
Egg per Gram Counts in the Dung
Similar results of highly significant (p<0.01) reduction in the EPG count
in lambs fed Neem leaves were reported by Arunachal et al. (2002). However,
Costa et al. (2006) reported no anthelmintic activity while feeding the
Neem leaves for three months to sheep. While Niezen et al. (1998) observed
reduction in EPG count of Trichostrongylus species by Sulla feeding in
ewe lambs, Hordegen et al. (2003) observed cent percent reduction in
egg counts of Haemonchus contortus with the seeds of Neem. Kahiya
et al. (2003) also observed similar decrease in EPG counts feeding Acacia
karoo diets. However, Pietrosemoli et al. (1999) did not find any
difference in EPG count by feeding Neem leaves up to 40% level as blocks to
calves.
Haemonchus contortus Worms Count in the Abomasum
Similar to the effectiveness of Neem leaves in lowering the worm count Niezen
et al. (1998) and Hordegen et al. (2003) also reported reductions
in worm burden while feeding Sulla and seeds of Neem respectively. Kahiya et
al. (2003) also reported worm count reduction more with Acacia karoo
diets (34%) than with Acacia nilotica diets (10%). Though the results
are promising, the exact mechanism of action as to how the larval activity and
worm count is reduced has to be explored. Further studies have to be performed
to ascertain the exact mode of action of the inhibitory effect of azadirachtin
on L3 larvae of Haemonchus contortus.
The anthelmintic resistance in Haemonchus contortus is an emerging problem throughout the world. Hence the need for alternative medicines is ever increasing. An effort was made to study the anthelmintic efficacy of Neem leaves in addition to being used as an organic source of feed to goats so that the farmers can derive the double benefit of Neem leaves being used as a feed source as well as an organic anthelmintic.
ACKNOWLEDGMENT
Authors are grateful to the Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai, India, for the facilities rendered to carryout this work as a partial fulfillment of Ph.D to first author.