Relationship between the excretion of eggs of parasitic helminths in roe deer and local livestock density.

Because of their continuing expansion, wildlife ruminant species that prosper in rural landscapes may be increasingly affected by and/or contribute to the circulation of certain generalist pathogens also infecting domestic ruminants, when they share common spaces or resources. In this study, we aimed to test the hypothesis that parasitism with gastrointestinal nematodes (GINs) of wild roe deer inhabiting different rural landscapes is correlated with livestock density. We used faecal egg counts of GINs and spatial data of 74 GPS-collared roe deer, inhabiting various landscapes from closed forests to open fields, together with weekly records of livestock abundances on pasture. We tested whether the excretion of GIN eggs in roe deer was influenced by the density of livestock in their home range over the grazing season. Our results showed that all of the roe deer home ranges, except four, contained pastures occupied by livestock. Excretion of GIN eggs occurred in 77% of the roe deer. The excretion of GIN eggs in roe deer tended to increase with livestock density in their home range. This result suggests, but does not prove, a higher risk of ingesting GIN larvae originating from livestock dung. In the context of increasing overlap between roe deer and livestock ranges, the exchange of pathogens between both hosts is plausible, although species identity of the parasites present was not determined. Assessing which GIN species are shared between wild and domestic ruminants, and how this may affect the health of both hosts, is a central question for future research in the context of interspecific pathogen circulation.

Coccidiostatic effects of tannin-rich diets in rabbit production.

The potential anti-eimerial effect of tannin containing resources such as sainfoin and carob in rabbits was tested on does at pre-weaning and to growing rabbits in their feed. The trial began at parturition (D0), when 24 does and their litters were assigned into three groups. They were fed either with a control (Group CO), a carob (containing 10% carob pods meal) (Group CP) or a sainfoin diet (containing 34% dehydrated sainfoin pellets) (Group SA). All diets were made isoproteic and isoenergetic and also balanced for crude fibre but differed by their tannin content. Weaning occurred at D37, and growing rabbits remained in the same cage until D51. Then, they were transferred to fattening cages until the end of the trial (D104) and slaughtering. Weight gain of young rabbits among the three groups (mean = 31.2 g/day) did not differ statistically. The mortality rates were 10% (SA), 15% (CP) and 20% (CO), respectively, but the differences were not statistically significant. Post-weaning economical feed conversion ratio (FCR) was reduced between rabbits of group SA compared to CO and CP groups. Faecal oocyst count (FOC) in group SA was 60% lower than in CO and CP groups. Areas under the curve (AUCs) calculated between sampling days and FOC, after transfer to fattening cages, was 62% lower in group SA than in CO and CP groups. The main Eimeria species identified (from D59 to D83) was Eimeria magna (53% of oocysts). AUCs for E. magna did not differ according to diet. In conclusion, the diet containing sainfoin reduced oocyst excretion of Eimeria spp. by 60%, and improved the economical FCR.

In vitro assessment of the anthelmintic activity of Hedysarum carnosum Desf. at different phenological stages and from six locations in Tunisia.

Gastrointestinal nematodes are compromising productivity of grazing sheep and goats. Therefore, scientists have been looking for cost-effective alternative options. Forage legumes (Fabacea Family) contain tannins that could improve livestock performance and their health as well. The present study aimed to (i) determine the in vitro anthelmintic (AH) activity of 19 acetonic extracts of Hedysarum carnosum Desf on Haemonchus contortus by a larval exsheathment assay (LEA); (ii) test the anthelmintic activity of condensed tannins using a deactivating reagent, polyvinylpolypyrrolidone (PVPP); (iii) study the effect of location and the phenological stage on the percentage of exsheathment. The LEA was used at different concentrations (150, 300, 600, 1200 µg mL-1 of acetonic extract/mL of purified buffer solution (PBS)). The larval exsheathment is concentration, location, phenological stage dependent. All extracts, caused a delay of the percentage of exsheathment over 50% so the AH activity of H. carnosum was confirmed. After addition of PVPP, the % exsheathment was similar to the 150 µg mL-1 concentration. The biplot showed that Loc1(S), Loc4(B), Loc 5(PF), Loc 6(BM) and Loc 6(PF) were isolated from other plant extract sample. Our in vitro study showed that H. carnosum seems to be a promising alternative to AH drugs.

Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions.

The structural changes induced in adult Haemonchus contortus after in vitro and in vivo contact with a tannin-rich (TR) plant, either tzalam (Lysiloma latisiliquum) or sainfoin (Onobrychis viciifolia), were assessed using scanning electron microscopy (SEM). All the worms used in the study were adult females. The Haemonchus adult worms were obtained from the abomasum of infected donor goats. Adult H. contortus were kept in contact with the extracts of TR plants for 24h for the in vitro assays and were compared to worms maintained in PBS (control). For the in vivo studies, the adult H. contortus parasites were obtained from artificially infected goats. All the goats were fed a tannin-free diet until D27 post-infection when infection was patent. On D28 some goats were fed fodder of one of the TR plants for seven consecutive days. Thus, their H. contortus were in contact with TR fodder within the gastrointestinal tract. The control worms were obtained from goats fed only a tannin-free diet. In the in vitro assays and in vivo studies, the SEM observations revealed structural alterations in the worms after contact with TR plants when compared to the control worms (i.e.: longitudinal and transversal folds and thicker cuticular ridges, material aggregates around the buccal capsule and/or vulva or anus). The main changes concerned the cuticle and the buccal area. The structural changes found in the worms exposed to TR plants might affect their motility and nutrition with possible consequences on their reproduction, as suggested by previous in vivo trials.

In vitro anthelmintic activity of the essential oils of Zanthoxylum zanthoxyloides and Newbouldia laevis against Strongyloides ratti.

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: ß-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC(50) and IC(90)) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC(50) values (19.5 and 18.2 µg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC(50) = 2.5 µg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC(50) = 46 µg/ml), N. laevis (IC(50) = 51 µg/ml), and the control [levamisole (IC(50) = 36 µg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC(50) values higher than 50 µg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.

Anthelmintic resistance in ruminants: challenges and solutions.

Anthelmintic resistance (AR) is a growing concern for effective parasite control in farmed ruminants globally. Combatting AR will require intensified and integrated research efforts in the development of innovative diagnostic tests to detect helminth infections and AR, sustainable anthelmintic treatment strategies and the development of complementary control approaches such as vaccination and plant-based control. It will also require a better understanding of socio-economic drivers of anthelmintic treatment decisions, in order to support a behavioural shift and develop targeted communication strategies that promote the uptake of evidence-based sustainable solutions. Here, we review the state-of-the-art in these different fields of research activity related to AR in helminths of livestock ruminants in Europe and beyond. We conclude that in the advent of new challenges and solutions emerging from continuing spread of AR and intensified research efforts, respectively, there is a strong need for transnational multi-actor initiatives. These should involve all key stakeholders to develop indicators of infection and sustainable control, set targets and promote good practices to achieve them.

Anthelmintic activity of some Mediterranean browse plants against parasitic nematodes.

The anthelmintic properties of tannin-rich plants are being explored as an alternative to chemical drugs. Most data have been acquired on legume forages, but only few on browse plants. The present study aimed to (i) screen the in vitro effects of extracts from 7 Mediterranean plants on Haemonchus contortus, (ii) verify the role of tannins using an inhibitor, polyvinyl polypyrrolidone (PVPP) and (iii) verify the in vivo effects of extracts from 4 plants. Significant inhibition was shown in vitro using a larval migration inhibition (LMI) assay for all extracts except that from Olea europaea var. koroneiki. After adding PVPP, the LMI values were restored to control levels for all plants except Pistacia lentiscus and Ceratonia siliqua, confirming a role for tannins in the activity. In the in vivo experiment, 48 lambs composed 6 groups, depending on diet. On Day 0, groups G1-G5 received H. contortus and Trichostrongylus colubriformis larvae and G6 remained uninfected. The various diets were distributed from Days 14 to 45: P. lentiscus (G1), Quercus coccifera (G2), C. siliqua (G3), Onobrychis viciifolia (G4), or Medicago sativa for the 2 control groups (G5, G6). Egg excretion, packed cell volumes (PCVs) and inorganic phosphate were measured weekly throughout the entire experimental period. At slaughter, the worms were enumerated and their fecundity assessed. Consumption of the 4 browser plants did not provoke differences in pathophysiological measurements but there were significant decreases in egg excretion, mainly explained by significant decreases in worm fecundity for both species, without any statistical difference in worm numbers.

In vitro effects of Cucurbita moschata seed extracts on Haemonchus contortus.

This study was carried out to evaluate the in vitro effect of Cucurbita moschata seed against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and dichloromethane) of C. moschata seed were tested in vitro on four developmental stages of H. contortus using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition (LMI) assay and adult worm motility (AWM) assay. The highly significant (P<0.001) ability to stop larval development (inhibition>90% for each extract) and the negative effect of the dichloromethane and methanolic extracts on adult worm motility (inhibition of motility >59.2% after 24h of incubation) compared to the negative controls, suggest anthelmintic properties of C. moschata seed against H. contortus. The active principles responsible for the activity could be secondary metabolites such as amino acid compounds or terpenoid compounds present in the extracts.

Relative abilities of young sheep and goats to self-medicate with tannin-rich sainfoin when infected with gastrointestinal nematodes.

Plant secondary metabolites (PSM) are one of the promising options to control gastrointestinal nematodes in sheep and goats. The objective of this study was to assess the abilities of sheep and goats to self-medicate with tannin-rich sainfoin (Onobrychis viciifolia) (SF) when infected with gastrointestinal nematodes, using a cafeteria and an operant conditioning trial. Hypotheses were that parasitized (P) lambs and goat kids would show greater intake and preference for SF than their non-parasitized (NP) counterparts, that kids would eat more SF than lambs (due to their lower resistance against parasites and their greater ability to consume PSM), and that SF intake would increase over time for P animals. We used 20 female kids and 20 ewe lambs aged 3 months. Half of the animals per species (n = 10) were experimentally infected with 170 L3 larvae of Haemonchus contortus/kg of BW (P). The other half were free from parasites throughout the study (NP). Five weeks after infection, animals were exposed to a 24-day cafeteria trial (three 8-day periods) offering a free choice between two legume pellets: SF (3.8% condensed tannins) and alfalfa (ALF, Medicago sativa; no tannin). Subsequently, animals were involved in an operant conditioning trial of two 4-day long sessions, to assess in short-term tests their motivation to walk for a SF reward when offered in choice with freely available ALF. In the cafeteria trial, SF preference was greater in kids than in lambs, particularly in the first two periods. We did not observe a greater preference for SF in P animals, which was even greater in NP animals for periods 1 and 2. Sainfoin intake increased through periods for P animals, which led to similar SF preferences for all groups during period 3. In the operant-conditioning trial, motivation to get the SF reward was similar between P and NP animals. These results support the hypotheses that goats are more willing to consume tanniferous feeds than sheep, and that P animals increased SF intake through time. However, the emergence of a curative self-medicative behaviour was not supported, as P individuals did not show greater SF intake, preference, nor a greater motivation to get SF than NP animals, regardless of animal species. These findings are discussed with previous results and some explanations are presented.

Effects of sainfoin (Onobrychis viciifolia) extract and monomers of condensed tannins on the association of abomasal nematode larvae with fundic explants.

Tannin-rich forages offer an alternative to anthelmintic chemicals to control gastrointestinal nematodes. However, the mode of action of such bioactive plants still needs to be assessed. Previous studies have shown that extracts of tannin-rich plants interfere with the first phase of host invasion, i.e., the exsheathment of infective larvae (L3s). In the current study, we examined the hypothesis that exposure to tannins could also affect the second phase of larval establishment, i.e., the tissue association/penetration of the exsheathed L3s into the digestive mucosae. An in vitro direct challenge technique using fundic explants was applied in this study. The main parasite model was Haemonchus contortus. The objectives were to verify: (i) whether a modification of the association/penetration of L3s with the mucosae occurred after contact with sainfoin extract; (ii) whether this is a dose-dependent phenomenon; (iii) whether tannins were responsible for these effects; (iv) whether these effects were dependent on the parasite species; and (v) how the biochemical structure of tannins might influence these effects. Following 3h contact with sainfoin extract at 1,200 microg/ml, the penetration of exsheathed L3s of H. contortus and Teladorsagia circumcincta into fundic explants was significantly reduced. Moreover, a dose-response relationship was found for H. contortus. For both nematodes, the changes were totally alleviated after addition of polyvinyl polypyrrolidone, an inhibitor of tannins, to the sainfoin extract, suggesting that tannins play a major role in the observed effects. Comparison of results obtained with different monomers of condensed tannins confirms a relationship between structure and activity, the prodelphinidin monomers and galloyl-derivatives being more effective than the procyanidin monomers. Combined with the delay or the inhibition of larval exsheathment previously shown, these effects could explain how tanniniferous plants reduce the establishment of infective larvae in small ruminants.

In vitro larval migration and kinetics of exsheathment of Haemonchus contortus larvae exposed to four tropical tanniniferous plant extracts.

As for some temperate forage, some tropical tanniniferous plants (TTP) from browsing might represent an alternative to chemical anthelmintic. The anthelmintic effect of four TTP (Acacia pennatula, Lysiloma latisiliquum, Piscidia piscipula, Leucaena leucocephala) on Haemonchus contortus was measured using two in vitro assays. First, the effects of increasing concentrations of lyophilized extracts (150, 300, 600, 1200 microg/ml PBS) were tested on H. contortus larvae (L(3)) using the larval migration inhibition (LMI) test. An inhibitor of tannin, polyvinyl polypyrrolidone (PVPP), was used to verify whether tannins were responsible for the AH effect. Secondly, the effects of extracts on larval exsheathment were examined. Larvae (L(3)) were in contact with extracts (1200 microg/ml) for 3h, and then were exposed to an artificial exsheathment procedure with observations of the process at 10 min intervals. A general lineal model (GLM) test was used to determine the dose effect in the LMI test and the difference of the percentage of exsheathed larvae between the control and the treatment groups. A Kruskal Wallis test was used to determine the effect of PVPP on LMI results. The LMI test showed a dose-dependent anthelmintic effect for A. pennatula, L. latisiliquum and L. leucocephala (P<0.01), which disappeared after PVPP addition, confirming the role of tannins. No effect was found for P. piscipula on H. contortus in the LMI test. However, all four plant extracts interfered with the process of L(3) exsheathment which might be involved as a mechanism of action of tannins on H. contortus larvae. A. pennatula, L. latisiliquum and L. leucocephala could be used as an anthelmintic for the control of H. contortus after confirmation based on in vivo studies.

Effects of four tropical tanniniferous plant extracts on the inhibition of larval migration and the exsheathment process of Trichostrongylus colubriformis infective stage.

The anthelmintic (AH) effect of Acacia pennatula, Leucaena leucocephala, Lisyloma latisiliquum and Piscidia piscipula was evaluated in the infective larvae (L(3)) of Trichostrongylus colubriformis. Different concentrations of lyophilized extracts were tested using the larval migration inhibition (LMI) test. An inhibitor of tannins (the polyvinyl polypyrrolidone [PVPP]) was used to verify whether these compounds were responsible for the AH effects. Then, the effect of extracts on larval exsheathment was examined by observing the exsheathment process at 10-min intervals for 70 min. The LMI test showed a dose-dependant AH effect for A. pennatula, L. leucocephala and L. latisiliquum (P<0.01), but not for P. piscipula. The restoration of L(3) migration to values similar to those of controls after the addition of PVPP, indicates that tannins are involved in AH effects. Trichostrongylus colubriformis exsheathment was partially or totally blocked by the four plants extracts. Tropical tanniniferous plants evaluated in the current study may have potential as AH for the control of T. colubriformis if in vivo investigations indicate useful effects.

Effect of the consumption of Lysiloma latisiliquum on the larval establishment of gastrointestinal nematodes in goats.

The consumption of tannin-rich (TR) forages has been associated with negative effects against gastrointestinal nematodes and with an improved host resilience. It has been hypothesized that tannins affect the capacity of infective larvae to establish in the mucosae of the host. In this study, we aimed at testing this hypothesis using Lysiloma latisiliquum, a tropical TR tree. The objectives were: (i) to evaluate the effect of the consumption of L. latisiliquum on the establishment of nematode third-stage larvae (L3) in goats; (ii) to define the role of tannins in these effects in vivo by using an inhibitor (polyethylene glycol, PEG); and (iii) to examine a possible indirect effect of tannins on the inflammatory response in the digestive mucosa. Eighteen Criollo goats composed three experimental groups. The control group received fresh leaves of Brosimum alicastrum, a plant with a low level of tannins. Two groups received L. latisiliquum leaves either with (L.L.+PEG) or without (L.L.) daily addition of 25 g PEG. After a 7-day adaptation period, each goat was infected with both Haemonchus contortus and Trichostrongylus colubriformis (3000 L3 per species). The goats were slaughtered 5 days after infection and worm counts and histological analyses were performed. No difference in the voluntary feed intake of foliage was observed between the 3 groups. The consumption of L. latisiliquum significantly reduced the larval establishment of both nematode species compared to the control (P<0.01). For both worm species, the effects were totally alleviated with PEG (L.L.+PEG group), suggesting a major role of tannins in the observed effects. Only minor differences in the mucosal cellular response were observed between the 3 groups. These results confirm that the consumption of TR plants reduces the establishment of nematode larvae in the host and that a direct effect is principally involved.

Mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs.

Gastrointestinal (GI) nematode control has an important role to play in increasing livestock production from a limited natural resource base and to improve animal health and welfare. In this synthetic review, we identify key research priorities for GI nematode control in farmed ruminants and pigs, to support the development of roadmaps and strategic research agendas by governments, industry and policymakers. These priorities were derived from the DISCONTOOLS gap analysis for nematodes and follow-up discussions within the recently formed Livestock Helminth Research Alliance (LiHRA). In the face of ongoing spread of anthelmintic resistance (AR), we are increasingly faced with a failure of existing control methods against GI nematodes. Effective vaccines against GI nematodes are generally not available, and anthelmintic treatment will therefore remain a cornerstone for their effective control. At the same time, consumers and producers are increasingly concerned with environmental issues associated with chemical parasite control. To address current challenges in GI nematode control, it is crucial to deepen our insights into diverse aspects of epidemiology, AR, host immune mechanisms and the socio-psychological aspects of nematode control. This will enhance the development, and subsequent uptake, of the new diagnostics, vaccines, pharma-/nutraceuticals, control methods and decision support tools required to respond to the spread of AR and the shifting epidemiology of GI nematodes in response to climatic, land-use and farm husbandry changes. More emphasis needs to be placed on the upfront evaluation of the economic value of these innovations as well as the socio-psychological aspects to prioritize research and facilitate uptake of innovations in practice. Finally, targeted regulatory guidance is needed to create an innovation-supportive environment for industries and to accelerate the access to market of new control tools.

Combining the effects of supplementary feeding and copper oxide needles for the control of gastrointestinal nematodes in browsing goats.

The aim was to assess the benefits obtained from combining supplementary feeding and copper needles (COWP), compared to the use of both approaches independently, for the control of gastrointestinal nematode (GIN) infections in browsing kids. Forty-four nematode free Criollo kids were exposed to natural parasite infection. The kids were divided into six experimental groups: not treated, supplemented (NT-S), not treated, not supplemented (NT-NS), moxidectin treated, supplemented (M-S), moxidectin treated not supplemented (M-NS), copper treated, supplemented (COWP-S) and copper treated, non-supplemented (COWP-NS). Copper treated groups received Copinox (2 g capsules) on day 0 and on day 60 of the trial. Moxidectin treated groups received Cydectin (0.2 mg/kg of body weight s.c.) every 28 days. Three of the groups received individual supplementation (100 g of feed/day fresh basis; 74% sorghum: 26% soybean meal; NT-S, M-S and COWP-S) and the other three groups were not supplemented (NT-NS, M-NS and COWP-NS). Animals browsed native vegetation (6.5 h/day) during the wet season (154 days). Kids were weighed every 14 days to determine live weight gain (LWG) and blood and faecal samples were obtained to determine packed cell volume (PCV), haemoglobin (Hb), peripheral eosinophil counts (PEC) and faecal egg counts (FEC). At the end of the trial, four kids of each group were euthanatized (six kids in each COWP treated group). Worm burdens, female worm lengths and prolificacy were determined. Liver samples were used to determine copper concentration and were stained with haematoxylin-eosin to determine microscopic lesions. Animals receiving the combination of supplementary feeding and COWP improved their LWG, PCV and Hb to similar levels of animals with suppressive AH treatment. This was not the case when COWP was used without supplementation. Liver copper concentration in COWP treated groups increased significantly especially in the COWP-NS kids but this was not associated with liver lesions or clinical signs. Post-mortem Haemonchus contortus and Trichostrongylus colubriformis worm counts had a tendency to be reduced in the different groups (66-35% reduction) compared to NT-NS group at the end of the trial (P>0.05). Also, COWP treatment and/or supplementation reduced female worm length of T. colubriformis and prolificacy of H. contortus and T. colubriformis. This study, confirmed the value of nutritional supplementation in the control of GIN in growing kids. The use of COWP in addition to supplementation had a limited contribution on the kids' resilience against GIN. This may be due to the reduced infection of H. contortus during this trial.

Nutritive value and anthelmintic effect of sainfoin pellets fed to experimentally infected growing rabbits.

Alternative strategies to synthetic chemical drugs are needed in livestock and are a key issue in organic farming today. This study aimed at examining the potentialities of sainfoin, a legume rich in condensed tannins, as a nutraceutical that combines nutritive and antiparasitic effects in rabbits. To test the effect of infection with a helminth (I: infected groups; NI: not infected groups) and the effect of substituting 40% of the alfalfa in a control diet (C) with sainfoin (diet S), four groups of 16 weaned rabbits were arranged according to a 2×2 bifactorial design. The sainfoin diet differed from the control by its tannin concentration (1.8% v. 1.0% tannic acid equivalent) and its ADL concentration (84 v. 43 g/kg). For each diet, 16 rabbits were infected with 2125 third-stage larvae of Trichostrongylus colubriformis. Growth, feed intake, feed conversion ratio and nematode faecal egg counts (FECs) were controlled for 6 weeks. A digestibility trial was performed. After necropsy, adult worms and eggs in utero per female were counted and egg-hatching rate calculated. Growth tended to be lower for S groups than for C groups (38.2 v. 39.5 g/day; P=0.06). Feed intake was higher for S groups compared with C groups (+5.2 g dry matter/day; P<0.01), as was the feed conversion ratio (3.2 v. 2.9; P<0.001), probably in relation to the dietary ADL level. Protein digestibility was reduced in S groups compared with C groups (-6.0 points; P<0.001), probably associated with the effect of the tannin concentration. Digestibility of hemicelluloses was reduced in infected rabbits compared with non-infected ones (-5 points; P=0.01). Using the substitution method, the digestible energy of dehydrated sainfoin pellets used as raw material was calculated at 11.12 MJ/kg and digestible proteins at 110 g/kg. The infection did not produce any clinical signs of digestive disorders. No differences were observed according to the diet, neither in the number of adult worms (972; P=0.50), the number of eggs in utero per female (14; P=0.95), nor FEC (400 eggs/g; P=0.57). In contrast, the rate of faecal egg hatching in the S group tended to be lower than in the control (58.3% v. 85.2%; P=0.08). In conclusion, sainfoin seems to fit nutritive requirements for rabbits, supplies a large quantity of fibre and particularly lignins, and limits the development of nematode eggs in faeces.

The in vitro anthelmintic properties of browse plant species against Haemonchus contortus is determined by the polyphenol content and composition.

The aims of the present study were to (a) evaluate the anthelmintic activity of 10 East African browse plant extracts, (b) examine their role in inhibition of Haemonchus contortus larval exsheathment, (c) establish relationship between inhibition of larval exsheathment and browse plant extract polyphenol composition. Acetone/water (70/30%) extracts of air dried leaves of Acacia etbaica, Cadaba farinosa, Capparis tomentosa, Dichrostachys cinerea, Dodonaea angustifolia, Euclea racemosa, Maerua angolensis, Maytenus senegalensis, Rhus natalensis and Senna singueana were used. The larval exsheathment inhibition assay (LEIA) was applied using H. contortus third stage larvae (L3) and browse plant extract concentrations of 0, 150, 300, 600, 1200µg/ml in phosphate buffered saline (PBS). Data were analysed using the PROC MIXED procedure of SAS. Polyvinylpolypyrrolidone (PVPP) was used to evaluate whether polyphenols were involved in L3 exsheathment inhibition. All browse plant extracts significantly (P≤0.001) inhibited larval exsheathment in a dose dependent manner. The dose required to inhibit 50% of the larvae (EC50) was highest in C. farinosa and lowest in E. racemosa and M. senegalensis. Significant differences (P<0.001) between the control and PVPP treated A. etbaica, C. tomentosa, M. angolensis, R. natalensis and D. cinerea indicates that larval inhibition was largely due to non-phenol compounds. For E. racemosa, M. senegalensis, D. angustifolia and S. singueana, PVPP treatment reversed inhibition activity and in these extracts, inhibition was mostly attributable to tannin and other polyphenols (kaempferol, quercetin and myricetin based glycosides). Overall, the browse plant extracts have anthelmintic property against H. contortus and larval inhibition resulting from the presence of phenolic and non-phenolic compounds.

Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts.

The larval exsheathment inhibition assay (LEIA) of infective larvae (L3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia pennatula leaves was used to perform respective LEIA tests with H. contortus L3 of different ages (1-7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40µg/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraíso) strain. In order to identify changes in L3 vitality on different weeks (1-7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC50, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6µg/mL) compared to older larvae (P<0.05). The EC50 and EC90 for weeks 2-5 were similar (P>0.05), while older larvae tended to show higher EC50 and EC90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥-0.83; P <0.05) and EC50 (r≥-0.80; P<0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age.

Susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of polyphenol-rich plants. Part 2: Infective L3 larvae.

This study explored the variation in susceptibility to acetone:water plant extracts between infective larvae (L3) of ten Haemonchus contortus isolates from different geographical origin. The L3 of 10 different isolates were exposed either to the acetone:water extract of a temperate plant (Onobrychis viciifolia) or a tropical plant (Acacia pennatula) and were evaluated with the larval exsheathment inhibition assay (LEIA). The L3 of each isolate were incubated with different concentrations of each extract (0, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200µg/mL of phosphate buffered saline (PBS)). After incubation, the exsheathment process of L3 was induced using a solution with sodium hypochlorite (2%) and sodium chloride (16.5%). The proportion of exsheathed L3 was determined for each concentration at 0, 20, 40 and 60min. Effective concentrations 50% (EC50) and the corresponding 95% confidence intervals (95% CI) were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated for each extract to compare isolates, using the most susceptible isolate as the respective reference for each extract. To determine the role of polyphenols on the reported effect, a second set of incubations was made for each isolate and each extract, using the extracts at a concentration of 1200µg/mL PBS with or without polyvinylpolypyrrolidone (PVPP), a polyphenol blocking agent, and controls without extract. The ten different H. contortus isolates showed variation in susceptibility for each of the 2 extracts tested (P<0.05). The EC50 values for A. pennatula extract ranged from 36 to 501µg/mL (RR: 2.11-13.68). Meanwhile, the EC50 values for O. viciifolia extract ranged from 128 to 1003µg/mL (RR: 1.25-7.82). The use of PVPP revealed that polyphenols were responsible for the anthelmintic activity recorded for both extracts. However, tested H. contortus isolates suggested that susceptibility to one polyphenol-rich extract did not determine the susceptibility to the other polyphenol rich extract. The latter result indicated that the different H. contortus isolates varied in their susceptibility to the polyphenols present in each extract evaluated.

Targeted selective treatment for worm management--how do we sell rational programs to farmers?

Seriously escalating global anthelmintic resistance in gastrointestinal nematodes of small ruminants has spawned a variety of alternatives to anthelmintics for worm management, based on the need for sustainable Integrated Parasite Management (sIPM). Pivotal to the sIPM approach is the concept of refugia, the proportion of a given parasite population that escapes exposure to control measures. By balancing drug applications with the maintenance of refugia, the accumulation of anthelmintic resistance alleles in worm populations can be considerably delayed, while still providing good levels of control. The over-dispersed nature of parasitic infections provides an opportunity to achieve this balance, by targeting treatments to the members of a flock or herd that are least tolerant to nematode infection. However, implementation of this strategy has only recently become feasible, with the development of the FAMACHA((c)) system for clinical evaluation of anaemia due to haemonchosis. Subsequently, the use of milk yields has proven an effective indicator in dairy goats infected predominantly with nematodes other than Haemonchus contortus. In addition, short-term weight changes and perhaps also body condition scoring may provide indices of parasitism, permitting the rapid identification of animals likely to benefit from treatment. However, sIPM and refugia-based approaches are more complex than whole-flock treatments in conventional programs, and adoption by farmers is most likely where the theoretical basis is understood. As close communication with informed advisors is generally limited, there is a danger that sIPM will remain a theoretical concept without alternative modes of communication. The development of computer-based decision support programs, which use epidemiological, seasonal and clinical information to provide recommendations for specific situations, should be accorded high priority in the future development of worm management systems.