In vitro anthelmintic efficacy of some indigenous medicinal plants against gastrointestinal nematodes of cattle

The prevalence of natural gastrointestinal nematodes was observed in cattle during the period from June, 2004 to May, 2005 in Sadar upazila of Mymensingh district. The prevalence of gastrointestinal nematodes was 84.1% (rainy seasons-97%, summer-85.5% and winter seasons-69.8%). The prevalence of strongyles (Haemonchus sp., Trichostrongylus sp., Oesophagostomum sp. and Mecistocirrus sp.), Bunostomum sp., Strongyloides sp., Trichuris sp. and Capillaria sp. were 63.9%, 26.3%, 21.5%, 17.3% and 24.5%, respectively. Water extracts of 20 indigenous plants(neem, tobacco plant, barbados lilac, betel leaf, pineapple, jute, turmeric, garlic, devil's tree, papaya, lime tree, dodder, white teak, conessi tree, bitter gourd, sweet basil, white verticillia, pomegranate, sage, chaste tree) showed potential in vitro activities against adult parasites. Out of these, 20 plant extracts, 10 plants (neem, tobacco, barbados lilac, betel leaf, pineapple, jute, turmeric, garlic, dodder and bitter gourd) showed 100% efficacy against adult worms, 4 plants (devil's tree, papaya, white verticillia and chaste tree) showed 9098% and others (lime tree, white teak, conessi tree, sweet basil, pomegranate and sage) showed below 90%.


Introduction
Parasitism is an important limiting factor that responsible for deteriorating the health and productivity of livestock.The agro-ecological and geo-climatic conditions of Bangladesh are highly favorable for the growth and multiplication of parasites.As a result about 50% apparently healthy cattle populations are affected with different species of parasites (Garrels, 1975).Infections by gastrointestinal helminth parasites of livestock are among the most common, which are considered as economically important diseases of grazing livestock (Perry et al., 2002).They are characterized by lower outputs of animal products (meat, milk, hides and skins), manure and traction, which all have impact of the livelihood of small holder farmers (Perry and Randolph, 1999).The production performances of these cattle are low, because of wide spread occurrence of pathogenic parasites.Parasitic diseases are considered important causing enormous economic losses through morbidity and mortality in livestock.Among the parasitic diseases, gastrointestinal nematodes such as Haemonchus spp., Trichostrongylus spp., Cooperia spp., Oesophagostomum spp., Trichuris spp.and Strongyloides spp.are most common (Qadir, 1981;Rahman and Mondal, 1983).Imported synthetic anthelmintics are considered the only effective way of controlling parasitic infection.However, as these are expensive and unavailable, livestock producers are not interested to use these anthelmintics.Furthermore, some serious disadvantages of using those anthelmintics, notably the development of resistance to helminth parasites (Waller and Prichard, 1985) against various anthelmintic compounds and classes, as well as their residues and toxicity problems (Kaemmerer and Butenkotter, 1973) poses hazards to livestock development and public health.For these reasons, interest in the screening of medicinal plants for their anthelmintic activity has remained of great scientific interest despite extensive use of synthetic chemicals in clinical practices (Akhtar et al., 2000).Until today very little works are performed in our country to investigate the anthelmintic properties of indigenous medicinal plants in cattle.Considering all of these constraints, this work was undertaken with following objectives: i) screening of medicinal plants having in vitro anthelmintic activity, ii) prevalence of gastrointestinal nematodes in cattle of Sadar Upazila of Mymensingh district.

Collection of fecal samples
Fecal samples were collected from 1200 randomly selected cattle of Sadar upazila of Mymensingh district (100 month) were examined at various seasons (rainy seasons, summer and winter).Fecal samples were collected from the rectum of cattle by hand and kept in polythene bags and examined by Floatation method (Rahman et al., 1996).The simple test tube flotation method is a qualitative test for the detection of nematode and cestode eggs and coccidia oocysts in the faeces.It is based on the separating of eggs from faecal material and concentrating them by means of a flotation fluid with an appropriate specific gravity.

Collection of indigenous medicinal plants
Indigenous medicinal plants (Table -3) from different location of Bangladesh were collected and the plants material were dried in shade and then dried off in the hot air oven at 55-60 0 C to gain constant weight.

Preparation of plants powder
Powders were prepared by pulverizing the dried indigenous medicinal plants with the help of electric grinder.A 25-mesh diameter sieve was used to obtain fine dust and preserved them into airtight plastic container, till their use for extract preparation.Previously prepared plants powders were used for preparation of plants extract.Ten grams of each powder were taken in a 500 ml beaker and separately mixed with 100 ml of distilled water.Then the mixtures were stirred for 30 min by a magnetic stirrer (6000 rpm) and left as such for next 24 hrs.The extracts were then filtered through a fine cloth and again through filter paper (Whatman No. 1).The filtered material was taken into round bottom flask and then concentrated by evaporation of water from filtrate in a water bath at 50 0 C till it reached the final volume of 10 ml.After the evaporation of water from filtrate, the condensed extracts were preserved in tightly corked-labelled bottle and stored in a refrigerator until used for screening of anthelmintic activity.

Preparation of stock solution
Stock solutions of plant extracts were prepared by diluting the condensed extracts with water.Different concentrations of each category of plant extracts were prepared by dissolving them in the water prior to anthelmintic screening.

Collection and maintenance of adult gastrointestinal nematodes in the laboratory
Collection of parasites from abomasum were done by following standard procedure as described by Taylor (1934) and Bell (1957).Viscera of cattle were collected from local market .The abomasums were opened through its lesser curvature with the help of scissors.The contents were emptied in glass jars containing normal saline.The abomasi were thoroughly washed and cleaned off ingesta and put in a different jar containing normal saline and left for an hour or two to release the attachments of parasites from the wall of the abomasum.The mucosal surface of the abomasum were rubbed carefully between the fingers to remove any remaining worms adhering to the abomasal wall.Finally the abomasal mucosa was examined with the help of a magnifying glass for any remaining parasites still adhering to the mucous lining of abomasum.The contents were washed several times with water and continued till the worms were free from debrises.The final wash was made with the normal saline.The sediments were examined in large petri-dishes over a black background.The parasites were collected with the help of curved needle and kept in normal saline.The parasites were cleared off debris by brushing with camel hairbrush or shaking in normal saline and identified according to the keys described by Rahman et al., (1996) and Yamaguti (1958).They were kept in petri-dishes containing normal saline incubated at 37 0 C.

In vitro screening of plant extracts for anthelmintic activity
Screening of water extracts of plant at various concentration viz. 25 mg/ml, 50 mg/ml and 100 mg/ml were performed in the petri-dishes containing adult live stomach worms of cattle collected from slaughter house in Phosphate buffer saline (PBS).PBS (100ml) containing 50 adult worms (both male and female) were pipetted in 3 petri-dishes at ratio 0.1 ml, 0.05 ml and 0.025 ml water extracts were then added, respectively.The drug-parasite petri-dishes were incubated for three hours at room temperature and the efficacy was observed by counting the dead parasites and expressed in percentages (%).

Results and Discussion
The prevalence of gastrointestinal nematodes was highest in the month of June and July.The prevalence of gastrointestinal nematodes in cattle are shown in the Table 1 and Table 2. Highest prevalence of gastrointestinal nematodes were found in rainy seasons (July-October) followed by summer (March-June) and winter seasons (November-February).Prevalence of strongyles (Haemonchus sp., Trichostrongylus sp., Oesophagostomum sp. and Mecistocirrus sp.) infection was highest followed by Bunostomum sp., Strongyloides sp., Trichuris sp. and Capillaria sp.Waruiru et al. (2001) observed the epidemiology of gastrointestinal nematodes of dairy cattle in Central Kenya.The total worm burden in the animals was highest during the rainy season (March-June and October-December) and lowest during the dry seasons (July-September and January-February).This result is in conformity with earlier reports made by Keyyu et al. (2006), Jager et al. (2005), Soca et al. (2003) and Yildirim et al. (2000).The in vitro efficacy of different concentrations of water extracts of 20 plants against adult gastrointestinal nematodes are shown in the Table 3. Water extract of 20 indigenous plants showed potential in vitro activities against adult parasites.The efficacy of water extract of these plants at the concentrations of 25 mg/ml and 50 mg/ml was much lower than that of concentration of 100 mg/ml except tobacco plants.Tobacco plants (25 mg/ml and 50 mg/ml) showed 100% in vitro efficacy against gastrointestinal nematodes of cattle.Neem, barbados lilac, betel leaf, papaya, dodder, bitter gourd and white verticillia (50 mg/ml) showed 70-80% in vitro efficacy against gastrointestinal nematodes of cattle.Turmeric (50 mg/ml) was 88% effective in vitro against gastrointestinal nematodes of cattle.The plants (100mg/ml) had highly significant activity (90-100%) against adult gastrointestinal nematodes in vitro were : neem (leaves and bark), tobacco plant (leaves), barbados lilac (leaves and bark), betel leaf (leaves), pineapple (leaves), jute (leaves), turmeric (rhizome), garlic (bulbs), devil's tree (leaves), papaya (leaves), dodder (whole plant), bitter gourd (leaves and seeds), white verticillia (leaves) and chaste tree (leaves).Iqbal et al. (2006) observed in vitro anthelmintic activity of Nicotiana tabacum leaves against gastrointestinal nematodes of sheep.The in vitro inhibitory effect was evident from the paralysis and/or mortality of worms noted at 6-hours postexposure.Raje and Jangde (2003) found in vitro anthelmintic activity of decoction of Nicotiana tabacum against Haemonchus contortus of goats.It may be concluded that the medicinal plants and/or its extracts may be used against gastrointestinal nematodes of cattle in Bangladesh.The pharmacokinetics together with its toxic effects need further studies.