Modifications ultra-structurales des larves infestantes de nématodes parasites

de sainfoin (Onobrychis viciifolia).

BRUNET, S., FOURQUAUX, I., SANCHEZ, M.E. et HOSTE, H.

RESUME - ARTICLE 5

Introduction

Les plantes riches en tannins représentent une alternative à l’emploi d’anthelminthiques de synthèse (AHs) pour la maîtrise du parasitisme gastro- intestinal (GI). Des études précédentes menées in vitro ont montré que les fonctions larvaires sont affectées par un contact avec un extrait de sainfoin (Onobrychis viciifolia). Néanmoins, le mode d’action des plantes riches en tannins sur les nématodes GIs reste mal connu.

Dans cette étude, nous avons voulu examiner les lésions des larves infestantes (L3s) des nématodes digestifs après un contact avec un extrait de sainfoin. Les objectifs étaient de : i) déterminer les effets d’une incubation avec un extrait de sainfoin sur l’ultra-structure des L3s par observations en microscopie électronique à transmission (MET) ; ii) comparer ces modifications chez des L3s engainées ou dégainées ; ii) évaluer la spécificité des effets de l’extrait du sainfoin en comparant les observations sur Haemonchus contortus et Trichostrongylus colubriformis.

Matériel et méthodes

Les L3s d’H.contortus et de T.colubriformis ont été artificiellement dégainées par un bullage de CO2 selon la technique de Conder et Johnson (1996).

Les L3s engainées ou dégainées ont été ensuite incubées à 20°C pendant 3 heures dans du PBS (témoin négatif) ou avec un extrait de sainfoin (1200µg/ml). Après lavage, les L3s ont été soumises immédiatement à un protocole de fixation avec une solution du glutaraldéhyde, puis d’immobilisation en agarose, de déshydratation et enfin d’inclusion en résine ‘London Resin white’.

Les coupes ultrafines (90nm) des L3s ont été observées à l’aide d’un MET HITACHI.

Résultats et discussion

Notre étude a montré pour la première fois que l’incubation des L3s avec un extrait de sainfoin induit des modifications ultra-structurales des L3s d’H.contortus et de T.colubriformis.

Les principales modifications observées étaient une altération de l’hypoderme, la présence de nombreux lysosomes et des signes d’autophagie et de mort cellulaire par nécrose. Dans l’ensemble, ces altérations étaient similaires chez les deux espèces suggérant un effet non-spécifique du sainfoin quelque soit l’espèce de nématode GI.

A l’inverse, la comparaison des lésions entre les L3s engainées et dégainées montre des différences selon le statut larvaire. Chez les L3s engainées, les lésions étaient essentiellement localisées au niveau de l’hypoderme et de la couche musculaire alors que chez les L3s dégainées, les modifications étaient surtout bornées aux cellules intestinales. Ces différences semblent associées à la présence de la gaine chez les L3s engainées et pourraient s’expliquer par des sites d’action des TCs différents selon le statut larvaire.

ULTRASTRUCTURAL CHANGES IN THIRD-STAGE LARVAE OF PARASITIC

NEMATODES OF RUMINANTS TREATED WITH A SAINFOIN (Onobrychis viciifolia)

EXTRACT

BRUNET S.

(1)(2)

, FOURQUAUX I.

(3)

, SANCHEZ M.E.

(1)(2)

, and HOSTE H.

(1)(2)*

(1) INRA, UMR 1225, F-31076 Toulouse, France.

(2) Université de Toulouse ; ENVT ; UMR 1225 ; F-31076 Toulouse, France.

(3) Centre de Microscopie Électronique Appliquée à la Biologie. Université de Toulouse,

Faculté de Médecine Rangueil, 133 route de Narbonne, F31062 Toulouse Cedex. France.

*

ABSTRACT

Condensed tannin (CT)-rich plants represent one alternative to chemical anthelmintics

to control gastrointestinal nematodes (GINs) in ruminants. Previous functional studies

have shown that sainfoin extracts affect the two forms of infective larvae (L3), i.e.

ensheathed and exsheathed L3s. However, the mechanisms of action remain unknown.

Therefore, this study examined the effects of sainfoin extracts on L3 ultra-structure of two

GIN species using transmission electron microscopy. The main changes found were an

alteration of the hypodermis, the presence of numerous lysosomes in the cytoplasm and

features of degenerative cell death of muscular and intestinal cells. The changes were

similar for the two nematode species. Comparison of effects between ensheathed and

exsheathed L3s suggests different susceptibility to sainfoin extract depending on L3 form,

which might be related to the presence of the sheath. Overall, our observations confirmed

that CT-rich extracts affect GIN L3s via a direct mechanism by inducing paralysis and/or

asphyxia.

KEY WORDS: Haemonchus contortus; Trichostrongylus colubriformis; Tannins;

Sainfoin (Onobrychis viciifolia); Transmission electron microscopy; Ultra-structure.

INTRODUCTION

The use of tanniniferous plants has recently been suggested as an alternative to

synthetic anthelmintics (AHs) for the control of gastrointestinal nematodes (GINs) (1-3).

Some studies have focused on the AH properties of sainfoin (Onobrychis viciifolia), a

legume forage containing condensed tannins (CTs). In in vitro studies, sainfoin extracts

showed an inhibitory effect on adult worm viability (4). Similarly, an perturbation of various

functions of infective larvae (L3s) by sainfoin extract has been observed on both

Haemonchus contortus and Trichostrongylus colubriformis, like the mobility (4, 5), the

exsheathment (6), and the association-penetration of exsheathed L3s into the digestive

mucosae (7). Overall, these in vitro results revealed that both ensheathed and exsheathed

L3s of GINs were affected by sainfoin extracts. In vivo studies have shown that the

consumption of sainfoin was associated with changes in the biology of the adult worm

populations in goats (8) or in sheep (9), or with a substantial decreased in the L3

establishment in sheep (10).

Based on these previous results, it was suggested that CTs could have direct AH

effects on GINs by interacting with proteins of the worm cuticle (2, 6, 7). This hypothesis is

supported by cuticular changes observed by scanning electron microscopic in adults T.

colubriformis after incubation with CT-containing extracts (2). However, studies on the

ultra-structural changes to nematodes provoked by tanniniferous plants have never been

performed.

Using H.contortus as parasite model, the objectives of the current study were : i) to

determine the effects of sainfoin extracts on the L3 ultra-structure by transmission electron

microscopic (TEM) observations; ii) to compare the alterations observed on the

ensheathed and the exsheathed L3s; iii) to evaluate the specificity of the effects by

comparing data acquired on H. contortus, an abomasal species, with those acquired on

MATERIALS AND METHODS

Sainfoin

Sainfoin (Onobrychis viciifolia) hay was collected in the South-East of France in June

2005. The method of extraction has been previously described by Brunet et al. (6, 7).

Briefly, sainfoin hay was extracted with water:acetone (3:7, v/v). After removing acetone,

the aqueous solution was washed with methylene chloride. Finally, the aqueous extract

was freeze-dried. CT content was analysed using the Butanol-HCl assay according to

Makkar (11). The sainfoin CT content was estimated at 2.45% of the dry matter.

Infective larvae

Larval culture : The L3s were obtained from donor goats infected with pure strains of

either H. contortus or T. colubriformis. The same batches of 1-to-2-month-old L3s were

used in the assays.

Larval exsheathment : The exsheathment process of H. contortus or T. colubriformis

L3s was performed using a CO2-bubbling technique (12). First, ensheathed L3s were

washed with a phosphate buffer solution (PBS; 0;1 M phosphate, 0.05 M NaCl, pH 7,2)

and concentrated at a density of 2,000 L3s per ml. 2ml of larval solution were placed in a

15ml screw-top centrifuge tube. CO

2

was then bubbled into the parafilm®-closed tubes

containing the L3s at room temperature respectively for 5 and 15 minutes for H.contortus

and T.colubriformis. Thereafter, the tops were rapidly screwed on to each tube to maintain

a CO

2

rich environment and the tubes were then incubated overnight at 37°C. Finally, the

L3s were washed 3 times in PBS and used for the incubation with sainfoin extracts. Before

incubation, the percentages of exsheathed L3s were determined by microscopy. They

were respectively 95% and 45% for H.contortus and T.colubriformis.

Larval incubation : For both nematode species, a batch of 2,000 L3s (ensheathed or

1200µg/ml in PBS. After incubation, the L3s were washed 3 times in PBS. Negative

controls (incubation in PBS) were run in parallel. Larval incubations were run in duplicates.

Transmission electron microscopy (TEM)

For TEM analysis, the L3s were fixed immediately after washing in 2% glutaraldehyde in

Sorensen buffer (v/v) (pH 7.4; 0.1M) at 4°C for 4h (13). After fixation, L3s were washed

overnight in Sorensen buffer (4°C) and immobilized in 2% low-melting point agar (SIGMA,

St Louis, Mo, USA) at 4°C. The solidified blocks were cut into little cubes for a better resin

penetration. Then, the L3s were post-fixed at room temperature in buffered osmium

tetraoxide (1%; w/v) for 1h. Thereafter, the L3s were dehydrated through a series of

graded ethanol solution (30, 50, 70 and 95%; 10 minutes each) and 3 washes in 100%

ethanol (15 minutes).

The L3s were then embedded in a London Resins White resin (LRW) (EMS-

Euromedex, France) through successive washes of a 1:2 LRW:ethanol solution (v/v), a 2:1

LRW:ethanol solution (v/v) and in pure LRW (respectively for 30, 60 and twice 60 minutes;

at 4°C). The final inclusion was achieved by the resin polymerisation which was performed

using a LRW UV accelerator (EMS-Euromedex, France) (overnight; at room temperature).

Ultra-thin sections (90nm) of the embedded L3s were obtained using an ultra-microtome

(ULTRACUT, Reichert; Austria). Then, the sections were collected on Colladion-coated

copper grids (100mesh; EMS-Euromedex, France) and were stained with a 2% water:

uranyl acetate solution (w/v) for 2 minutes followed by Reynold’s lead citrate for 7minutes

(14). Last, the ultra-thin sections of L3s were examined with a HITACHI HU 12A

RESULTS

A minimum of 50 L3 cross-sections were examined per treatment (PBS vs sainfoin), per

nematode species (H.contortus vs T.colubriformis) and per larval status (ensheathed vs

exsheathed). Since no more than 45% of the T.colubriformis L3s were exsheathed, special

care was taken to observe only the sections of exsheathed L3s which were easily

identified by the lack of sheath (Figure 4).

The frequencies of the various ultra-structural changes observed in treated vs untreated

L3s are summarized in Table 1 according to the larval status and to the GIN species.

Sections of control larvae :

The general structure of H.contortus (Figures 1 and 3) and T.colubriformis (Figures 2

and 4) L3s was similar. From the external to the internal layers, it was possible to

distinguish a multi-layer cuticle, a syncitial hypodermis, a single longitudinally-oriented

layer of somatic muscle cells showing striated and non-striated regions and finally,

intestinal cells boarding the lumen of the digestive tract. In the case of ensheathed L3s, an

additional thick homogeneous sheath was observed externally (Figures 1 and 2).

Effects of sainfoin extract on the ensheathed larvae :

The changes in H.contortus (Figure 1) and T.colubriformis (Figure 2) ensheathed L3s

caused by sainfoin treatment were comparable, and are therefore described together.

No differences in the larval size were observed between the control and the treated L3s

of both H.contortus and T.colubriformis.

No alteration of either the sheath or the cuticle was observed. The thickness and the

structure of the sheath of treated L3s were similar to those of control L3s of H.contortus

(Figures 1A vs 1D) and T.colubriformis (Figures 2B vs 2D). The different layers of the

and the basal fibrillar layer, were similar to the untreated L3s of H.contortus (Figures 1C vs

1F) and of T.colubriformis (Figures 2B vs 2D).

Overall, the sections of treated L3s showed a loss of electron density compared to the

untreated L3s for both H.contortus (Figures 1A vs 1D, 1B vs 1E) and T.colubriformis

(Figures 2A vs 2C). Moreover, the internal morphology appeared more preserved in the

sections of control L3s than in those of treated L3s. Moreover, several ultra-structural

internal modifications were observed in treated L3s of both H.contortus and T.

colubriformis (Table 1).

First, for both nematode species, ultra-thin sections of treated L3s revealed an alteration

of the hypodermis, the layer underlying the cuticle. A local or general separation of the

cuticle from the hypodermis was observed in most of the treated L3s in comparison to the

control L3s of H.contortus (Figures 1B vs 1E and 1F) and of T.colubriformis (Figures 2B vs

2C and 2D). In fact, the observed rupture was within the hypodermis layer itself which

appeared to be torn from the basal layer of the cuticle.

The contact with sainfoin extract induced also changes in the muscle layer. In

transverse sections of treated L3s, the contractile region of muscle cells showed a

degradation of the muscular fibrils (Figures 1F and 2D). Moreover, the non-striated region

of the muscle cells revealed signs of lysis, i.e. a disruption of organelles and a swollen

cytoplasm, in both H.contorrtus (Figures 1D and 1F) and T.colubriformis (Figures 2C and

2D) L3s.

The intestinal cells were also affected by the sainfoin treatment since their cytoplasm

appeared less electron-dense. The cellular components were distorted in treated L3s

compared to control L3s of H.contortus (Figures 1B vs 1E) and of T.colubriformis (Figures

1C). However, these changes were less frequently observed than the lesions to the

In most of the ultra-thin sections of treated L3s (Table 1), a high number of electron-

dense vesicles could be seen in the intestinal and muscular cells in both GIN species

(Figures 1D, 1E and 2C). These vesicles have a diameter of 0.25µm to 0.35µm. Based on

their size and morphology, these micro-organelles could be lysosomes (15).

Changes to the nuclei were only rarely observed. Some cells showed nuclear

disorganization with amorphous chromatin (data not shown) without changes of the

nucleus size (Figures 1B vs 1E). In ultra-thin sections of treated L3s, a separation of the

plasma membranes of adjacent cells was also detectable (Figure 1E and 2D).

Effects of sainfoin extract on the exsheathed larvae :

The CO

2

bubbling technique did not seem to have affected the L3s since the TEM

analysis of control exsheathed L3s did not show ultra-structural changes in comparison

with control ensheathed L3s for both H.contortus (Figures 1A vs 3A; Table 1) and

T.colubriformis (Figures 2B vs 4B; Table 1).

For both nematode species, the changes induced to the exsheathed L3s by sainfoin

extracts were overall similar to those observed in the ensheathed L3s. However, the ultra-

structural lesions were less frequently detected in the exsheathed L3s than in the

ensheathed L3s and presented differences in repartition (Table 1).

Ultra-thin sections of treated exsheathed L3s of H.contortus (Figures 3D and 3E) and

T.colubriformis (Figure 4C) showed numerous lysosomes and features of cell lysis in both

intestinal and muscular cells. An alteration of the hypodermis layer was observed

hypodermis was torn only locally (Figures 3F and 4D). The contractile region of muscle

cells appeared unaffected by sainfoin treatment since no alteration was observed in

comparison to untreated L3s for both H.contortus (Figures 3C vs 3F) and T.colubriformis

(Figures 4B vs 4D). However, signs of lysis in intestinal cells were more frequently

DISCUSSION

To our knowledge, this is the first description of the ultra-structural changes in the

nematode L3s caused by incubation with sainfoin extracts.

Our general objective of this study was to understand the mode of action of sainfoin on

nematode L3s. So, preliminary trials were performed to adapt methods described

previously (16) to embed H.contortus and T.colubriformis L3s in order to limit possible

artefacts. The general organization and the cuticle structure of L3s were similar to those

observed in other nematode larvae (16-18). Similarly, the question of the physiological

significance of concentration of plant extracts tested in vitro is also relevant. An extract

concentration of 1200µg/ml was used for the larval incubation because it corresponds to a

concentration previously applied in in vitro assays and for which AH effects on L3s have

been demonstrated (6, 7). Moreover, total tannin concentrations in the abomasal and

duodenal digesta of sheep fed with tanniniferous diet ranged from 1100 to 2800µg/ml with

extractable tannin concentrations ranging between 350 and 900µg/ml (19-21). Therefore,

the concentration applied in the present study was considered as being close to the

physiological conditions. In addition, we used a CO

2

-bubbling exsheathment technique to

obtain exsheathed L3s because it was described as the less disturbing method of L3

viability (12).

After a 3 hour contact with sainfoin extract, two main effects on L3 ultra-structure of both

H.contortus and T.colubriformis were observed.

First, a severe alteration of the hypodermis layer since a local or a general rupture of

the hypodermis from the cuticle was detected in the ultra-thin sections of both the

ensheathed and exsheathed L3s. A study performed on C.elegans has demonstrated that

a separation of the muscle layer from the cuticle, through a rupture within the hypodermis,

So, these hypodermis alterations observed in the current study could be compatible with a

partial or total reduction of L3 mobility, impairing the biological functions.

Second, both H. contortus and T. colubriformis treated L3s showed severe signs of a

loss of internal integrity and many signs of cell suffering such as the presence of increased

number of lysosomes, the degradation of the cytoplasm, the local rupture of plasma

membranes, the cellular swelling and, in few case, some abnormal condensation of the

nuclear chromatin (15, 23, 24). In some treated L3s, these lesions were more severe than

in others since a total lysis of the intestinal and muscular cells, indicative of dying cells. We

can thus suggest that the incubation of GIN L3s with sainfoin extracts caused features of

degenerative cell death in muscle and intestinal cells, resulting in the death of GIN L3s.

The hypotheses of paralysis and cellular death of L3s provoked by sainfoin extracts are

in agreement with previous in vitro results obtained with both ensheathed and exsheathed

L3s. In general, the incubation of H. contortus and T. colubriformis L3s with CT-containing

extracts was associated with a failure of larval functions. For example, sainfoin treatment

led to an inhibition of mobility (4, 5) and of the exsheathment process (6) of ensheathed

L3s, as well as an inhibition of the capacity of exsheathed L3s to penetrate into the

digestive mucosae (7).

The comparison of observations on H. contortus and T.colubriformis aims at evaluating

the specificity of sainfoin effects on the L3 ultra-structure. Our TEM analysis showed

strong similarities since comparable changes were detected in L3s of both H. contortus

and T.colubriformis after sainfoin treatment. This suggests that there are no differences in

susceptibility between the GIN species and confirms previous in vitro conclusions (4, 6, 7).

In this study, we have examined whether differences in effects exist between the

ensheathed L3s and the exsheathed L3s. Overall, sainfoin extracts produced similar

changes in the L3 ultra-structure, i.e. alteration of the hypodermis, presence of numerous

nematode species. However, there were differences between the two L3 forms in the

frequency of the lesions and their localization in treated L3s. In the ensheathed L3s,

lesions were mainly located in the L3 external periphery, the hypodermis layer and the

muscle cells whereas, in the exsheathed L3s, major damages concerned the intestinal

cells. In particular, they showed features of cell death. In contrast, changes in the

hypodermis and the muscular layers were less pronounced than in the ensheathed L3s.

Obviously, the presence of the sheath represents the main difference between the two L3

forms and might explain these differences. Because of the sheath, the ensheathed L3s are

unable to feed in contrast of the exsheathed ones (25, 26). Moreover, the sheath is

composed in majority of cross-linked collagens and which acts as a semi-permeable

barrier to the external environment (17, 26) and permits exchanges between the

surroundings and the L3 tissues. Consequently, we hypothesise that sainfoin extracts will

interact on L3s at different sites. For ensheathed L3s, the lesions could be due to a

blocking of the larval exchange with its environment, which may result in asphyxia or

toxicity due to accumulation of cellular waste products. For exsheathed L3s, the major

damage observed in intestinal cells might be due to the ingestion of toxic molecules

present in sainfoin extracts, as proposed by Mori et al. (27) to explain the ellagitannin

toxicity in C.elegans intestine.

Previous studies, using CT-containing fractions of sainfoin (5) or strong binding agent of

tannins (4, 6, 7), have suggested that CTs were the major components responsible for the

observed effects on GIN larvae. Because of the ability of CTs to complex with

macromolecules, in particular with proteins (28-30), it has been proposed that CTs might

interact with nematode proteins (2, 6, 7). Therefore, it can be hypothesized that the ultra-

structural changes observed in L3s after sainfoin treatment were caused by the ability of

process might modify the biological proprieties of the sheath and the cuticle, such as their

flexibility and their permeability.

In conclusion, the current study has shown that the ultra-structure of GIN larvae was

altered by sainfoin extracts. This study complements previous functional ones supporting

the hypothesis of a direct mode of action of tanniniferous plant extract on nematodes (6, 7)

It also underline that sainfoin may affect L3s through a non-specific way since similar

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