Integrated control of gastrointestinal nematodes of sheep using plant extracts. and bicontrol agents.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Infection of small ruminants by gastrointestinal nematodes (GIN) is a major health concern because they cause substantial economic losses, especially in the tropics and sub-tropics. For many years, control of GIN has been based upon use of anthelmintics. However, there is now a global challange because mutant GIN individuals can tolerate most of the widely used anthelmintics. Therefore, alternative control measures are needed. The objective of the study was to screen a number of plant species for their anthelmintic effects, and to evaluate selected strains of Bacillus thuringiensis (Berliner) and Clonostachys rosea (Schroers) for activity against sheep GIN. Subsequently, the combined treatments would test a dual control strategy for nematodes by using a combination of plant extracts with biocontrol agents. Ethanol extracts of 25 plant species were screened for their anthelmintic effects against Haemonchus contortus (Rudolphi 1803). Extracts of each plant were used in vitro at various concentrations (10, 20 and 30%) to treat 10 day faecal cultures. Five plants with high efficacies (Ananas comosus L. Merr., Aloe ferox Mill., Allium sativum Linn., Lespedeza cuneata Dum. Cours. and Warburgia salutaris Bertol.f. Chiov) were selected for further investigation, using ethanol, dichloromethane and water extracts at four concentrations (2.5, 5, 10 and 20%). Ethanol was the most effective solvent. Larval counts decreased as a result of increasing extract concentrations. An ethanolic extract of Lespedeza cuneata caused more than 70% mortality at all concentrations. In an in vivo study, the five plants A. comosus, A. ferox, A. sativum, L. cuneata and W. salutaris extracts were compared to a positive Control (Equimax®, a modern anthelmintic based on abamectin and praziquantel). Gender, eggs count (EPG₀) and initial body weights were used in assigning sheep (24 females and 24 males) to six groups. Each group was randomly assigned a treatment. Plant extracts were applied as an oral dose (100 mg kg⁻¹ BW), one dose per week per animal for 42 days (Phase 1). Subsequently, the same sheep were dosed for three consecutive days with the same treatments, keeping them in the same groups (Phase 2). Rectal faecal samples were taken for counting of eggs per gram of faeces (EPG) and L₃ larvae per gram (LPG) in faecal cultures. With application of plant extracts, the EPG count decreased with time (P<0.001), and the impact of the plant extracts increased (P<0.001) with time. Two extracts, from A. comosus and L. cuneata, were the most effective in Phase 1 (58% and 61% reduction of EPG, respectively,), and in Phase 2 (77% and 81% reduction of EPG, respectively). In a study on potential biocontrol agents, two strains of Bacillus thuringiensis (Bt) and one of Clonostachys rosea f. rosea (C. rosea), and compared with a diatomaceous earth (DE) product for their anthelmintic activity in sheep. Bacillus thuringiensis and C. rosea were fed to sheep at a rate of 1g kg⁻¹ BW, and DE was fed at 2% of sheep diet. The biocontrol treatments had no effect on EPG (P>0.05), but reduced GIN larvae per gram (LPG) (P<0.001) in faecal culture. Efficacy varied with time (P<0.001). By Day 7 Bt, C. rosea and DE had caused mortalities of GIN of 75.7, 86.9 and 60.6%, respectively. In addition, the efficacy of feeding 1g kg⁻¹ BW of C. rosea chlamydospores to sheep every day, every second day and every third day was tested. Daily feeding of fungal chlamydospores reduced LPG (a count of 12±1.67 GIN larvae) (P<0.001) more than feeding them the biocontrol agent every second day (39±0.77) or third day (58±1.77). By Day 12, feeding the biocontrol agent to sheep every day, every second day, or every third day caused mortality of GIN larvae of 90, 63 and 49%, respectively. Four dietary levels (treatments) of C. rosea (0.25g (F1), 0.5g (F2), 1g (F3) and control (C) of C. rosea product kg-1 BW) were tested. Treatments were each mixed with a complete diet and fed to sheep once daily for 10 weeks, according to body weights. Increased doses of the biocontrol agent reduced LPG (P<0.001), larval development (LD) (P<0.001), and increased efficacy (P<0.001). On Day 70, F1, F2, F3 and the Control controlled LD by 33.3, 72.3, 89.4 and 2.6%, respectively. Clonostachys rosea was effective in reducing third stage larvae (L₃) on pastures significantly (P<0.001) by Day 63 and Day 70. Ethanolic extracts of A. comosus, A. ferox, A. sativum, L. cuneata and W. salutaris all reduced egg production by GIN parasites of sheep. Feeding sheep cultured chlamydospores of a biocontrol fungus, Clonostachys rosea, reduced counts of nematode larvae in sheep; and 1g C. rosea chlamydospores kg⁻¹ BW daily was enough to reduce nematode infective larvae, therefore reducing the degree of pasture contamination. An initial trial showed that the combination of the two treatments of an A. comosus extract and C. rosea chlamydospores was more effective than either treatment on its own in controlling gastrointestinal nematodes in sheep. A long-term trial is being undertaken currently to confirm this finding.