Anthelmintic effects of tropical shrub legumes in ruminant animals

Gastrointestinal (GI) nematode parasitism causes major economic losses and animal health problems in farmed livestock and thus poses a serious challenge to livestock production worldwide. Parasite control strategies historically have relied heavily on anthelmintic drenches, but with the emergence of anthelmintic resistance, new strategies are required. A number of forage species containing high concentrations of condensed tannins have been shown to have anthelmintic properties. The aim of the current study was to determine whether selected tropical legumes used as protein supplements for livestock, and in particular Calliandra calothyrsus, might also be useful for the control of GI nematode parasites. A pilot study was undertaken initially to determine the effects of Calliandra, when fed to lambs as a sole diet, on burdens of Haemonchus contortus or Trichostrongylus colubriformis and on egg production by adult worms. Lucerne (Medicago sativa) pellets were used as a high protein control diet and Mitchell grass (Astrebla species) hay was used as a low protein control diet. The effects of diet on the worms were assessed by faecal egg counts and post-mortem worm counts. Worm burdens of each species and egg production by H. contortus were similar in lambs on all three diets. However, egg production of T. colubriformis was reduced by 85-90 % by feeding Calliandra to the host lambs and the number of eggs in utero of female T. colubriformis was significantly increased. These results were confirmed and extended in a larger-scale feeding experiment, in which the Mitchell grass diet was omitted. A number of haematological parameters were measured to examine the nutritional status, resistance and resilience of the host lambs in response to the worm burdens. Because pH was expected to affect the ability of condensed tannins to bind to worms, the pH was recorded immediately after slaughter in each segment of the GI tract to examine the relationship between pH and faecal egg counts. Worm burdens of H. contortus were similar in lambs fed Calliandra (2237 ± 395 worms) and lucerne (1861 ± 230 worms). Worm burdens of T. colubriformis were also similar in lambs fed Calliandra (5718 ± 339 worms) and vii lucerne (4861 ± 452 worms). However, egg production by both H. contortus and T. colubriformis were reduced by 64-84 % and 24-68 % respectively. There were no improvements in any of the haematological parameters measured attributable to the different diets, indicating that reduced egg counts were due to a direct toxic effect of the Calliandra diet and not due to improved resistance or resilience to the worms. The variation in pH in each of the GI segments was too small to identify any relationship between pH and reduced egg output by female worms. A study was then conducted to confirm that the anthelmintic effects of Calliandra were due to the condensed tannins in the plant. Condensed tannins were extracted and purified from Calliandra leaves and incubated with H. contortus and T. colubriformis eggs and larvae in in vitro assays. Egg hatch and larval development rates were determined. Calliandra tannins delayed hatching and reduced egg hatch and larval development in both species. Development of infective larvae was almost completely inhibited at tannin concentrations above 300 μg/mL. In vitro assays were also used to screen a number of other tropical legumes for anthelmintic activity. Crude extracts of nine legume species, including Calliandra and lucerne pellets, and two different fractions of purified Calliandra were incubated with H. contortus and T. colubriformis eggs and larvae. In addition to Calliandra, Leucaena leucocephala and Desmanthus virgatus were identified as having possible anthelmintic activity against nematode eggs and larvae. The two Calliandra fractions both had similar anthelmintic activity to the Calliandra crude extract. In an attempt to determine the mode of action of Calliandra condensed tannins against GI nematodes, a staining technique was developed to identify tannins histologically in mammalian and nematode tissues. Haematoxylin & Eosin (H&E), ferric chloride, butanol-H2SO4 and vanillin-HCl techniques were examined, initially using plant tissues, and then optimised for use in mammalian tissues. The H &E and vanillin-HCl techniques were then used to stain GI tissues and nematodes obtained from Calliandra-fed lambs. In the ovine GI tissue, condensed tannins were present in the lumen and in macrophages and giant cells in the lamina propria, particularly in the abomasum. Condensed tannins were observed on the outside of the cuticle of intact adult H. contortus and T. colubriformis, mainly in adherent digesta. Condensed tannins were also observed in the pharynx and intestine of sectioned H. contortus, but no condensed tannins were observed in the reproductive tract. A final experiment was conducted to determine the effects of Calliandra on the development, establishment and reproductive capacity of nematodes derived from eggs exposed to the legume in the host diet but with no subsequent exposure as adults. Eggs of H. contortus and T. colubriformis obtained from Calliandra-fed or lucerne-fed donor lambs were cultured in vitro to obtain infective larvae, which were used to infect groups of recipient lambs. Worm egg and larval production from the donor lambs, and adult worm egg output and worm burdens of the recipient lambs, were monitored. Although worm egg production and larval production from the donor lambs was reduced by the Calliandra diet, the ratio of eggs to larvae was also reduced. It was not clear whether this was due to problems with the experimental technique or to selection pressure exerted on the worms by the Calliandra diet. Worm burdens were higher in the recipient lambs receiving larvae from the Calliandra-fed donors. Egg production by the female worms derived from Calliandra-fed donors may have been reduced, but the results were not clear. Calliandra, in addition to its value as a source of protein for tropical livestock, may be useful in reducing egg production by GI parasitic nematodes, thus reducing larval contamination of tropical pastures and infection rates of livestock. Calliandra therefore has potential as an anthelmintic alternative in tropical regions