ARTICLE 1 : Effets de la consommation d’une plante riche en
tannins (Lysiloma latisiliquum) sur l’installation larvaire des
nématodes gastro-intestinaux chez les chèvres.
BRUNET, S., MARTINEZ-ORTIZ DE MONTELLANO, C., TORRES-ACOSTA, J.F.J.,
SANDOVAL-CASTRO, C.A., AGUILAR-CABALLERO, A.J., CAPETILLO-LEAL, C. et HOSTE, H.
RESUME - ARTICLE 1
Introduction
La consommation de plantes riches en tannins a été associée à des effets néfastes sur les nématodes gastro-intestinaux (GIs) et à une amélioration de la résilience de l’hôte vis-à-vis du parasitisme GI. Il a été suggéré que les tannins affecteraient la capacité des larves infestantes (L3s) de s’installer chez l’hôte. Dans cette étude, nous avons testé cette hypothèse en utilisant Lysiloma latisiliquum, une plante d’Amérique Centrale, comme modèle de plante riche en tannins.
Cette étude avait pour objectifs de : i) évaluer l’effet de la consommation de L.latisiliquum sur l’installation des L3s ; ii) définir le rôle des tannins dans les effets observés en utilisant un inhibiteur de tannins, le polyéthylène glycol (PEG); iii) examiner un possible effet indirect des tannins sur la réponse inflammatoire des muqueuses digestives.
Matériel et méthodes
18 chèvres Criollo, exemptes de nématodes GIs, ont été réparties en 3 groupes expérimentaux selon leur poids. Les animaux témoins ont reçu ad libitum des feuilles fraîches de Brosimum allicastrum, une plante à faible teneur en tannins (Groupe B.A.). Les animaux des 2 autres groupes ont reçu ad libitum des feuilles fraîches de L.latisiliquum (Groupes L.L. et L.L.+PEG) et une dose quotidienne de 25g de PEG a été administrée aux animaux du groupe L.L.+PEG. Après une phase d’adaptation de 7 jours au régime alimentaire, les chèvres ont été infestées par 3000 L3s d’Haemonchus contortus et 3000 L3s de Trichostrongylus colubriformis. Les animaux ont été sacrifiés 5 jours après l’infestation. Le comptage de larves présentes dans l’abomasum et l’intestin grêle, ainsi que des analyses histologiques ont été réalisés pour chaque animal.
Résultats et discussion
La consommation quotidienne de la ration a été comparable dans les trois groupes pendant la période expérimentale.
La consommation de L.latisiliquum a induit une réduction significative du taux d’installation des L3s pour les deux espèces de nématodes GIs par rapport au groupe
B.A.. De plus, l’administration de PEG a été associée à une restauration des taux d’installation d’H.contortus et de T.colubriformis comparables à ceux observées pour les animaux témoins, suggérant ainsi le rôle des tannins dans les effets observés. Enfin, seules des différences mineures ont été détectées entre les trois groupes concernant le nombre de cellules inflammatoires dans les muqueuses digestives.
En conclusion, notre étude a confirmé que la consommation d’une plante riche en tannins interférait avec la phase précoce de l’installation larvaire des nématodes GIs chez l’hôte. Ce phénomène serait fondé a priori sur un mécanisme d’action direct des tannins sur les L3s.
ELSEVIER
veterinary
parasitology
www.elsevier.com/locate/vetparEffect of the consumption of Lysiloma latisiliquum on the larval
establishment of gastrointestinal nematodes in goats
Abstract
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. © 2008 Elsevier B.V. All rights reserved.
Keywords: Haemonchus contortus; Trichostrongylus colubriformis; Larval establishment; Tannins; Lysiloma latisiliquum; Goat
1. Introduction
* Corresponding author at: UMR1225 INRA/DGER IHAP, ENVT, 23 chemin des Capelles, F31076 Toulouse, France. Tel.: +33 5 61 19 38 75; fax: +33 5 61 19 32 43.
E-mail addresses:h.hoste@envt.fr,s.brunet@envt.fr (H. Hoste). 0304-4017/$ - see front matter © 2008 Elsevier B.V. All rights
The use of tannin-rich (TR) plants has been proposed as an alternative strategy for the control of gastrointestinal nematode (GIN) infections in sheep and goats in order to reduce the dependence on chemical anthelmintic (AH) treatments and to delay the selection and the transmission of AH resistances in worm
S. Brunet et al. /Veterinary Parasitology 157 (2008) 81-88
populations (Kahn and Diaz-Hernandez, 2000; Hoste et al., 2006; Ketzis et al., 2006).
It has been suggested that the effects related to the consumption of TR plants varied depending on the parasitic stages exposed to tannins. Several studies in the two small ruminant species have shown that the consumption of a TR feed was associated with a modulation of the biology of adult worm populations, affecting particularly the egg excretion (Paolini et al., 2005a; Tzamaloukas et al., 2005; Heckendorn et al., 2006, 2007; Lange et al., 2006; Shaik et al., 2006). On the other hand, some recent in vitro evidence has shown that a contact with TR extracts affects the establishment of third-stage larvae, either by disturbing the exsheath-ment (Brunet et al., 2007) or the association with the mucosae (Brunet et al., 2008). However, only a few in
vivo studies support the hypothesis that the consumption of
TR resource provoked a significant decrease in the establishment of infective larvae (L3) of nematodes (Paolini et al., 2003a, 2005b; Tzamaloukas et al., 2005). However, in these studies, the consequences of tannins on the incoming L3 were measured after a delay of 2-3 weeks which makes it difficult to interpret the results and to differentiate between any possible direct vs indirect (possible stimulation of the mucosal response) effects of tannins in the infected host.
In tropical regions, the native vegetation provides a large range of browsing legume forages, used to feed animal.
Lysiloma latisiliquum (Fabaceae) is a common tree species
of the tropical forest vegetation of Central America. Its leaves contain high levels of tannins (Sandoval-Castro et al., 2005; Alonso-Diaz et al., 2008a, in press). Short-term preference studies have confirmed that goats and sheep can achieve high voluntary feed intake (VFI) of L. latisiliquum fodder (Alonso-Diaz et al., 2008a, in press). Moreover, it has been demonstrated that the in vitro incubation of L3 with a
L. latisiliquum extract affected the mobility and the
exsheathment process of both H. contortus (Alonso-Diaz et al., 2008b) and T. colubriformis L3 larvae (Alonso-Diaz et al., 2008c). Therefore, L. latisiliquum appears as a suitable candidate to test if tannins affect the in vivo establishment of GIN larvae and whether this process is due either to a direct or to an indirect mechanism.
Therefore, the current study aimed at: (i) evaluating the effect of L. latisiliquum consumption on the establishment of L3 in goats; (ii) defining the role of tannins in these in vivo effects by using an inhibitor of tannins (Polyethylene Glycol, PEG); and (iii) examining the consequences on the inflammatory response of the digestive mucosae.
2. Materials and methods
2.1. Plants
The leaves of Brosimum alicastrum and L. latisiliquum, two native tree species of Central America, were used in the experiment. L. latisiliquum is a TR tree whereas B.
alicastrum is a plant with very low levels of tannins (Alonso-
Diaz et al., 2008a, in press).
2.2. Infective larvae
The infective larvae (L3) of H. contortus (CENID- INIFAP sheep strain, Mexico) and T. colubriformis (INRA goat strain, France) were obtained respectively from donor sheep and goats infected with a pure strain of nematode. For the two species, batches of 3-month-old larvae were used in the assays.
2.3. Experimental design and diets
Eighteen, 6-month-old, nematode free, female Criollo goats, with no browsing experience, were involved in the study. They were divided into three balanced groups (n = 6), according to the bodyweight (13.6 ± 2.3 kg live weight). They were raised indoors, in individual concrete floor pens, to avoid any natural nematode infection. The goats were fed daily with a concentrate feed (10 g/kg live weight) and had free access to water and chopped Cynodon spp. hay (African star grass). The grass was suspended from the diet when the trial started.
The trial lasted for 13 days and was divided into two successive periods: a 7-day period for adaptation to the diet (D-7 to D-l) and a 6-day experimental period (DO to D5). DO corresponded to the day when the 18 goats were experimentally infected with 3000 H. contortus L3 and 3000
T. colubriformis L3. All the goats were humanely
slaughtered 5 days after infection (D5) complying with local regulations on animal welfare.
The control group (B.A. group) received fresh leaves of
B. alicastrum ad libitum. The L.L. and L.L. + PEG groups
received fresh leaves of L. latisiliquum ad libitum. In addition, 25 g per day of polyethylene glycol (PEG Wt 3350; SIGMA®), dissolved in water, were orally administered to goats from the L.L. + PEG group at two times: before and 30 min after offering fresh leaves (Silanikove et al., 2001). PEG is an inhibitor of tannins whose in vivo administration to small ruminants fed with TR plants has been used to asses the role of tannins in the observed effects in nutrition studies (Makkar, 2003b).
S. Brunet et al. /Veterinary Parasitology 157 (2008) 81-88 2.4. Measurements of the plant consumption
Fresh leaves of B. alicastrum and L. latisiliquum were collected each day to feed the goats ad libitum for a 13-day period. Animals were fed individually in concrete floor cages. The individual refusals were measured daily in order to estimate the consumption of fresh leaves of B. alicastrum or L. latisiliquum per group.
2.5. Plant analysis
During the experimental period (DO to D5), samples of fresh leaves of both B. alicastrum and L. latisiliquum were collected daily and were oven dried separately at 50 °C for 72 h. Pooled samples of either B. alicastrum (PS-B.A.) or L.
latisiliquum (PS-L.L.) were obtained respectively by mixing
the same proportion of the 6 daily dried samples. Samples were kept in airtight containers until analyses. Dry matter (DM; 7.007), ash (As; 7.009), crude proteins (CP; 2.057), neutral detergent fibers (NDF), acid detergent fibers (ADF), lignin and fat contents were analyzed on the PS-B.A. and PS-L.L. according to the AOAC procedures (1980). In addition, on two replicates per daily dried sample, total phenol (TP) and total tannin (TT) contents were determined using the Folin-Ciocalteau assay and the condensed tannin (CT) contents were measured using the Butanol-HCl assay (Makkar, 2003a).
For both PS-B.A. and PS-L.L., the biological activity was measured using the radial diffusion assay developed by Hagerman (1987) and modified by Makkar (2003a). The results were expressed as protein precipitation activities (PPA; cm /g dry matter) relative to a standard (Resorcinol). Each sample was measured with four replicates.
2.6. Parasitological techniques and worm count procedures
On D5, the goats were slaughtered humanely and the digestive tracts were collected immediately. The abomasums and the first 4 m of the small intestines were rapidly opened and washed in order to recover the late L3 and the early L4 larvae in the luminal contents. In addition, the mucosae from abomasums and small intestines were submitted to a pepsic digestion in order to collect the larvae. For both the abomasum and the small intestine of each goat, the total numbers of larvae were estimated from a 10% aliquot technique, with separate countings on the luminal contents and the mucosal digestions.
The rates of larval establishment were calculated for both
H. contortus and T. colubriformis according to the results of
the worm counting as the total number of worms recovered divided by the total number of L3 given, multiplied by 100.
2.7. Histological analysis
At necropsy, histological samples were collected from fundus, pylorus and small intestine (duodenum) for the determination of the number of eosinophils (EOS), globule leukocytes (GL) and goblet cells (GC) in the mucosae according to the technique described by Larsen et al. (1994) and Huntley et al. (1995).
The mucosal samples were fixed in 10% buffered formalin and embedded in paraffin. 5 \x,m sections were obtained and stained with Haematoxilin-Eosin (H-E) for counting of EOS and GL, or with Periodic Acid-Schiff (PAS) reagents for counting of GC. The stained cells were enumerated at a x400 magnification using a calibrated reticule encompassing an area of 0.25 mm2. Mean cell densities for each tissue and each cellular type were obtained from counts on 10 histological fields, randomly selected. The results were expressed as the mean number of cells per mm2 of mucosae.
2.8. Statistical analysis
The data of the daily consumption of fresh leaves were subjected to a general lineal model one-way analysis of variance on repeated values with post hoc comparisons using the Tukey test. The differences in the 3 groups for the worm counts of each species were performed using the Mann and Whitney Test. The comparisons between the histological data were performed using a one-way analysis of variance with post hoc comparisons using the Bonferroni test.
3. Results
3.1. Plant analyses
The chemical composition of leaves of B. alicastrum and
L. latisiliquum did not indicate any difference in the DM
(respectively 43.6 ± 7.3 and 43.8 ± 7.8), CP (respectively 13.0 and 14.9), NDF (respectively 49.8 and 49.1), ADF (respectively 23.9 and 25.1) and fat content (respectively 4.2 and 4.6). However, differences between the 2 plants were observed for ash (respectively 11.6 and 6.4) and lignin content (respectively 5.8 and 13.2).
S. Brunet et al./Veterinary Parasitology 157 (2008) 81-88 Table 1
Compared phenol contents and biological activities (±S.D.) of leaves of B. alicastrum and L. latisiliquum
a
Expressed as g tannic acid eq./kg dry matter. Expressed as g leucocyanidin eq./kg dry matter.
As expected, L. latisiliquum presented higher contents of TP, TT and CT, than B. alicastrum (Table 1) since the ratios between L. latisiliquum and B. alicastrum were 1.96-, 3.25- and 6.68-fold respectively. Moreover, as indicated by the measurement of the biological activities, L. latisiliquum had a 2.38-fold higher capacity for precipitating proteins than B.
alicastrum (Table 1).
3.3. Worm counts and establishment rates
A significant decrease in L3 establishment was measured in the L.L. group in comparison with the B.A. group (control group) for both H. contortus and T. colubriformis (P < 0.01; Table 2) as well as for the total number of worms.
After PEG administration, the rate of L3 establishment was higher in the L.L. + PEG group compared to the L.L. group for T. colubriformis (P < 0.01; Table 2). In addition, difference in establishment rate was close to significance for
H. contortus between the L.L. + PEG group and the L.L.
group (P = 0.074; Table 2), whereas no significant difference was observed between the B.A. group (control group) and the L.L. + PEG group for each nematode species and the overall number of worms (Table 2).
3.4. Mucosal cellular response
3.2. Consumption of fresh leaves
Differences in the voluntary feed intake of leaves were observed during the adaptation period (D-7 to D-1) (P < 0.01). The control goats fed with B. alicastrum consumed significantly more fodder than the goats of the L.L. and L.L. + PEG groups fed with L. latisiliquum which showed similar consumptions. In contrast, during the experimental period, no significant difference was observed in the intake of B.
alicastrum and L. latisiliquum leaves between the 3 groups
(Fig. 1).
According to the chemical analyses and the daily consumption of fresh leaves, goats of the L.L. and L.L. + PEG groups consumed on average 1.4 mg CT/kg BW/day whereas those of the B.A. group consumed on average 0.4 mg CT/kg BW/day.
Overall, no significant differences were observed between the 3 experimental groups in regard of cell counts in the mucosae (Fig. 2) except a significant decrease in the eosinophil number in the fundus (P < 0.05) and for the globule leucocyte number in the pylorus of the L.L. group (P < 0.05).
4. Discussion
One key factor to make possible the control of nematode infections by the use of bioactive forages is the plant appetency for ruminants. Here, we confirmed a high voluntary feed intake (VFI) of L. latisiliquum by goats without browsing experience, since a constant consumption of L. latisiliquum leaves was observed after the 5-day adaptation period. Moreover, a similar intake of L.
Fig. 1. Daily consumption of fresh leaves (means ± S.D.) of B. alicastrum or L. latisiliquum according to the groups: goats fed with B. alicastrum (B.A.); with L. latisiliquum (L.L.); or with L. latisiliquum and supplied with PEG (L.L. + PEG). For the adaptation period (D-7 to D-1), the significant difference between the 3 groups is indicates by ** (P < 0.01).
S. Brunet et al. /Veterinary Parasitology 157 (2008) 81-88 Table 2
Individual counts, arithmetic means and establishment rate (Est. rate) (±S.D.) of H. contortus, T. colubriformis and both species, according to the experimental groups: goats fed with B. alicastrum (B.A.); with L. latisiliquum (L.L.); or with L. latisiliquum and supplied with PEG (L.L. + PEG)
latisiliquum and B. alicastrum was maintained in the three
groups during the experimental period. These results tend to confirm the previous observations from preference studies performed with goats (Alonso-Diaz et al., 2008a) and hair sheep (Alonso-Diaz et al., in press) with previous browsing experience.
The low larval establishment rates observed in goats from the control group for both H. contortus and T.
colubriformis (less than 5%) correspond to previous values
reported with H. contortus in Criollo kids (Aguillar Caballero, 2004) or in goats infected with larvae of ovine (Perez et al., 2001) or caprine origins (Perez et al., 2008). Despite these low control values, it was possible to measure a significant decrease in the larval establishment of both H.
contortus and T. colubriformis in goats fed with L. latisiliquum fodder.
Fig. 2. Means (±S.D.) of eosinophil (EOS), globule leukocyte (GL) and goblet cell (GC) numbers in the fundic, pyloric and intestinal mucosae in the 3 experimental groups: goats fed with B. alicastrum (B.A.); with L. latisiliquum (L.L.); or with L. latisiliquum and supplied with PEG (L.L. + PEG). For cell types, values with different letters (a, b) differ at P < 0.05.
S. Brunet et al. /Veterinary Parasitology 157 (2008) 81-88
This is the first confirmation of the hypothesis that one main consequence of a TR environment on the incoming parasitic larvae is a reduction of establishment.
Many trials have shown that the administration of tannins concomitant to nematode infections can provoke a reduction in worm counts. This has been reported in experimentally infected sheep consuming Lotus pedunculatus (Niezen et al., 1998) or in goats fed on an Acacia karoo diet (Kahiya et al., 2003) or receiving quebracho (Paolini et al., 2003a,b). Also, under conditions of natural infection, Cenci et al. (2007) observed a reduced number of worms in sheep receiving leaves of Acacia mearnsii once a week. However, in these previous studies, the effects of the TR plants on worm populations were measured after several weeks of infection, making impossible to distinguish any specific action on the L3 establishment. Data on the reduction in worm burdens, explained by an effect on L3, measured only after a few days post-infection, were missing. This justifies our experimental design where animals were slaughtered on D5 post-infection.
Our second objective was to verify whether the tannins of
L. latisiliquum were responsible for the anthelmintic activity.
This was assessed by using polyethylene glycol (PEG) which bind tannins and form inert complexes with them (Silanikove et al., 2001; Waghorn, in press). In nutrition experiments, PEG is the main reagent used in order to deactivate tannins from TR forages fed to ruminants and to neutralize their potential negative effects (Makkar, 2003b; Waghorn, in press). However, previous results with PEG gave more controversial results in studies on parasitized animals. Some studies mentioned that the PEG distribution did not modify the negative effects of tannins on worm populations, either with L. pedunculatus or Acacia cyanophilla (Niezen et al., 1998; Akkari et al., 2008a). In contrast, the long-term distribution of PEG to browsing goats was associated with a significant increase in egg excretion suggesting that tannins were responsible for an AH activity (Kabasa et al., 2000). More recent results on goats fed with sainfoin (Onobrychis
viciifolia) (Hoste et al., 2007) and on lambs fed with A. cyanophylla (Akkari et al., in press) led to similar
conclusions. Our current results are in agreement with, these latter conclusions indicating that the PEG administration provoked a restoration towards control values of L3 establishment for the 2 nematode species. This suggests that tannins are largely responsible for the AH activity of L.
latisiliquum as also deduced from previous in vitro data with L. latisiliquum extracts on H. contortus and T. colubriformis
L3 (Alonso-Diaz et al., 2008b,c). These consistent in vivo
and in vitro data strongly support the hypothesis that tannins