J.F.J. Torres-Acosta

Non chemical control of helminths in ruminants: adapting solutions for changing worms in a changing world.

Infections with gastrointestinal nematodes (GIN) remain a major threat for ruminant production, health and welfare associated with outdoor breeding. The control of these helminth parasites has relied on the strategic or tactical use of chemical anthelmintic (AH) drugs. However, the expanding development and diffusion of anthelmintic resistance in nematode populations imposes the need to explore and validate novel solutions (or to re-discover old knowledge) for a more sustainable control of GIN. The different solutions refer to three main principles of action. The first one is to limit the contact between the hosts and the infective larvae in the field through grazing management methods. The latter were described since the 1970s and, at present, they benefit from innovations based on computer models. Several biological control agents have also been studied in the last three decades as potential tools to reduce the infective larvae in the field. The second principle aims at improving the host response against GIN infections relying on the genetic selection between or within breeds of sheep or goats, crossbreeding of resistant and susceptible breeds and/or the manipulation of nutrition. These approaches may benefit from a better understanding of the potential underlying mechanisms, in particular in regard of the host immune response against the worms. The third principle is the control of GIN based on non-conventional AH materials (plant or mineral compounds). Worldwide studies show that non conventional AH materials can eliminate worms and/or negatively affect the parasites biology. The recent developments and pros and cons concerning these various options are discussed. Last, some results are presented which illustrate how the integration of these different solutions can be efficient and applicable in different systems of production and/or epidemiological conditions. The integration of different control tools seems to be a pre-requisite for the sustainable management of GIN infections. This new era of GIN management requires a new paradigm: to achieve enough control to reduce the negative impact of GIN infections enabling an optimum level of production, health and welfare.

Direct and indirect effects of bioactive tannin-rich tropical and temperate legumes against nematode infections

Parasitic infections with gastrointestinal nematodes (GINs) represent a major pathological threat associated with the outdoor production of various livestock species. Up to now, the control of these parasitic diseases essentially relied on the use of commercial anthelmintic drugs. However, resistance to anthelmintics is nowadays widespread in worm populations. Recent results indicate that bioactive tanniniferous plants represent a valuable option as an alternative to commercial drugs for the control of GINs. The pertinent use of tannin-containing fodders as nutraceuticals supposes a clear understanding of the mode of action against the worms. The objectives of this paper are: (1) to discuss the nature and quantity of the active molecules involved in the anthelmintic activity; and (2) to review and analyze the changes provoked to the various parasitic stages. The possible involvement of some main polyphenols to explain the bioactivity of some tannin-rich plants will be discussed as well as the possible effects on the various nematode stages, relying on data obtained either with the temperate forage, sainfoin (Onobrychis viciifoliae) or with the tropical legume tree (Lysiloma latisiliquum). The information on the mode of action will be related to the potential consequences for better field applications under entirely different environmental and ecological conditions of productions.

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.

Nutrition-parasite interactions in goats: is immunoregulation involved in the control of gastrointestinal nematodes?

Compared to sheep, goats seem to develop a low immune response against the parasitic nematodes of the gastrointestinal tract. Nevertheless, some variability in the host response has been observed either at the individual level or depending on internal (genetic) or external (physiological status, nutrition) factors suggesting the possibility to exploit and manipulate this response. There is good evidence from field studies to suggest that a better plane of nutrition might contribute to improve goat resilience. However, the effects on immunoregulation and host resistance remain less clear. Due to their peculiarities in feeding behaviour (‘intermediate browser’), goats represent a valuable model to explore the relationships between the three possible strategies to control nematode infection through nutrition: (i) by increasing the immune response; (ii) by avoiding the infective larvae; and (iii) by selecting plants with direct anthelmintic properties (self medication).

Effect of a tropical tannin-rich plant Lysiloma latisiliquum on adult populations of Haemonchus contortus in sheep.

Bioactive plants with anthelmintic (AH) properties represent a promising alternative solution to chemical treatments. The AH effect of several Mexican tannin-rich (TR) plants has been screened in vitro. The in vivo AH effect of one TR legume, Lysiloma latisiliquum (Tzalam) on nematode larval establishment was confirmed. The present trial aimed at evaluating the direct and indirect effects of L. latisiliquum fodder consumption on adult Haemonchus contortus. Twenty-two parasite-naïve hair sheep lambs were allocated to an infected group (I) (400H. contortus L(3)/kg BW on D0) and a non-infected group (NI). From D0 to D28 post infection (PI), all the lambs were fed a complete diet. On D28, the two groups were sub-divided into four groups. Two control (C) groups maintained on the original basal diet (CI: 6 infected lambs and CNI: 5 non-infected lambs). The two treatment groups (T) received L. latisiliquum fodder ad libitum up to D36 when lambs were humanely slaughtered (TI: 6 infected lambs and TNI: 5 non-infected lambs). From D28 to D36 PI, individual fodder consumption and nematode egg excretion were measured daily. At necropsy, abomasal contents were recovered to obtain worm burdens and measure the female worm length and fecundity. Histological samples were taken from the respective abomasums and small intestines to count mucosal inflammatory cells. An increased consumption of TR fodder was observed in the TI vs. the TNI group (P<0.01). Before L. latisiliquum distribution, faecal egg excretion was similar in TI and CI groups. From D29 PI the TI group showed lower faecal egg counts compared to CI group (P<0.02). Although no differences in worm burdens were observed, worms of the TI group were smaller and, according to their size, contained fewer eggs in utero than worms from the CI group (P<0.05). Only minor differences in mucosal inflammatory cells were observed between groups, indicating that the indirect effect was not evident. Thus, a short-term consumption of L. latisiliquum can modulate directly the biology of adult H. contortus affecting the worm size and female fecundity while the worm burdens were not affected. Infected animals ate more L. latisiliquum fodder than non-infected animals.

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.

Challenges of nematode control in ruminants: Focus on Latin America

Gastrointestinal nematodes (GIN) of ruminants (cattle, sheep and goats) are ubiquitous and can cause severe injuries to infected animals and significant losses in farming revenues. GIN are able to survive severe environmental and host conditions, but mankind has developed a number of ingenious methods for parasite control. The commerce and use of modern anthelmintic drugs with a broad spectrum of activity has been a solid tool for nearly 40 years, however the continuous use of these drugs, has led to the selection of populations of drug-resistant worms worldwide. At present, the ever-growing agricultural systems in Latin America are facing many challenges and cannot rely on the far-reaching objective of parasitic elimination from the host or the environment. The lack of extensive programs for monitoring drug resistance exacerbates the negative consequences of reduced efficacy, which is evident in some areas with the increase in mortality rate even after treatment. Experts agree that new schemes of parasitic control are needed and should be based on the strategy of targeted selective treatment where affected hosts are identified and treated accordingly. In this article, we will focus our discussion on the challenges for the control of GIN in Latin America by 2020 imposed by reduced drug efficacy. We will evaluate phenotypic and molecular markers, methods for single-animal evaluation, and the implementation of schemes for anthelmintic treatment that address parasites in refugia.

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.

Prevalence of benzimidazole resistant nematodes in sheep flocks in Yucatan, Mexico

Thirty-eight sheep flocks, located in three municipalities in the Eastern Yucatan, Mexico, were surveyed for gastrointestinal nematodes resistant to benzimidazole (BZD) anthelmintics (AH). On each flock, 30 sheep were randomly distributed into two groups of 15 animals: albendazole group (5mg/kg BW) and untreated control group. Animals were refrained from any food (either browsing/grazing or supplement) for a period of 16 h prior to treatment. Faecal egg counts (FEC) and larval cultures were performed 10 days after anthelmintic treatment. Percentage reduction and 95% confidence intervals were determined. Anthelmintic resistance (AR) was declared when the percentage reduction in FEC was <95% and the 95% confidence interval was <90%. AR was suspected when only one of the two criteria was met. The survey indicated that AR occurred in 15.8% (n=6) (95% confidence interval=+/-11.6%) and was suspected in 23.7% of the farms (n=9) (95% confidence interval=+/-13.3%). Post-treatment larval cultures indicated that Haemonchus was the only resistant genus. The questionnaire survey showed that most farmers (92%) considered their sheep a secondary activity to cattle production. The majority of farmers (97.4%) treat their animals according to visual appreciation of weight. However, most farmers (79%) treat their flocks at very low frequencies (<3 times per year). Drug rotation was performed every 12 months or more by 84.2% of farmers. Anthelmintics used were: macrociclic lactones (47.4%), BZD (39.5%), levamisol (10.5%) and 1 farmer used closantel.

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.