Anthelmintic activity of Leucaena leucocephala seed extract on Haemonchus contortus-infective larvae

IN most countries, the profitability of sheep breeding is low and farmers look for convenient, safe, efficient, broad-spectrum drugs to control parasites. It is desirable to limit the cost of treatment in parasite control initiatives in order to improve the overall profitability. Farmers in Nigeria commonly use fresh seeds of Leucaena leucocephala to deworm their animals. A paste made by pounding 50 to 100 seeds is administered orally, with 200 ml of water. In Senegal, the seed is also reported to be useful in expelling Ascaris species worms (Adebowale 1993). The seeds of L leucocephala are popular for their nutritive value, albeit limited by the toxicity of mimosine, a non-nutrient component of the seed. Chicken fed on meal containing 15 per cent L leucocephala seed powder for 12 days at one to five weeks of age show a reduction in feed intake and bodyweight gain regardless of their growing stage (Okonkwo and others 1995). This has been attributed to mimosine toxicity as evidenced by experimentation with male broilers. Reverting to feeding on a normal diet rapidly reduced the stored mimosine in the birds’ tissue (Kamada and others 1997). A feed containing 15 per cent L leucocephala seed given to crossbred cows for 240 days was responsible for the maintenance of good health and these cows were free from reproductive disorders. Nutrient intake, digestibility of nutrients and nitrogen, phosphorus and calcium levels were not affected by the treatment. The rumen pH, total volatile fatty acids and the concentration of total nitrogen fractions in strained rumen liquor of an experimental cow were unaffected (Talpada and others 1997). Documentation of the true value of the claimed use of the extract of L leucocephala is of interest and practical relevance, because of the well-known toxicity of the mimosine component of the seed. This short communication describes an assessment of the anthelmintic effect of an aqueous extract of L leucocephala seed on the infective larvae of strongyles from sheep with a naturally acquired infection using a larval development viability assay (LDVA). The larvae were identified as predominantly Haemonchus contortus. L leucocephala is a shrub that is freely available in Ibadan, south-west Nigeria. Dry pods and seeds of the shrub were collected in the morning from near the University of Ibadan. Voucher specimens, that is, samples of the plant used for reference at the herbarium after taxonomical identification, were deposited at the herbarium of the Department of Botany and Microbiology, University of Ibadan. A sample of dried, powdered seed (10 g) was dispersed in 100 ml distilled water. The dispersion was stirred with a glass rod for 10 minutes and filtered using a previously weighed filter paper. A dark brown filtrate was obtained. The residue was dried in an oven at 125°C for one hour, weighed again, and the difference in weight gave the amount of powdered seed dissolved in water (0·58 per cent w/v). Appropriate aliquots of the aqueous extract was taken and diluted to prepare graded doses of the extract for use in the assay. A stock solution of the aqueous extract (2·9 mg/ml) was prepared from the filtrate obtained from the seed extract. Appropriate aliquots were taken to produce 0·25, 0·5, 0·75, 1·00, 1·25, 1·50, 1·75 and 2·00 mg/ml in a final volume of 2·0 ml of the culture medium. A control experiment was set up by replacing the test extract with distilled water. The technique used for nematode egg recovery was that previously described by Hubert and Kerboeuf (1992). A sample of faeces (10 to 15 g) from sheep with a naturally acquired strongyle infection was suspended in water and cleared of organic debris by filtration through 1 mm and 100 μm sieves. Eggs were collected on a 20 μm sieve and further cleared of organic debris by centrifugation in magnesium sulphate (density 1·10) for five minutes at 1000 g. The supernatant was filtered through 100 μm and 60 μm sieves and the eggs were washed in water and collected on a 20 μm sieve. The concentration of eggs was estimated in 50 μl samples and adjusted to 1200 to 1300/ml. The concentration of eggs was then estimated in 50 μl samples and adjusted to 100 to 120 eggs/ml. The egg suspension was diluted with the filtrate from the first step of the egg extraction (described above) in order to provide the rumen bacteria necessary for nematode larvae development. To avoid the proliferation of fungi, 5 μg of amphotericin B (Fungizone ND; Squibb) was added per ml of egg suspension. The nutritive medium used was that described by Hubert and Kerboeuf (1992), and comprised of Earle’s balanced salt solution (Eurobio) plus yeast extract (Difco Laboratories) in saline solution (1 g of yeast extract/90 ml of saline solution) at a ratio of 1:9 v/v. The LDVA was carried out in 5 ml tubes. Twenty microlitres of nutritive medium was added to 80 μl of egg suspension containing approximately 100 eggs. Three replicates per concentration of extract or water (control tubes) were made. The tubes were covered and placed in an incubator at 27°C for 48 hours. At this stage the parasite had developed into first-stage larvae (L1) and the plant extract aliquots were added. The third-stage larvae (L3) were obtained seven days later. At this time, the parasites were counted by separating the larvae into two classes: living L3 larvae and dead larvae and living larvae of other stages. The lethal concentration of the extract of L leucocephala required to kill 50 per cent of the helminths (LC50) for the