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Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus
Veterinary Parasitology 181 (2011) 360–364
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus M.A. Alonso-Díaz a,∗ , J.F.J. Torres-Acosta b,∗ , C.A. Sandoval-Castro b , H. Hoste c a Centro de Ense˜ nanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km. 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600, Veracruz, Mexico b Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Km 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, Mexico c UMR IHAP 1225 INRA/ENVT, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse Cedex, France
a r t i c l e
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Article history: Received 8 July 2010 Received in revised form 28 January 2011 Accepted 29 March 2011 Keywords: Haemonchus contortus In vitro Larval exsheatment inhibition assay Larval migration inhibition assay Tannins Polyphenols Anthelmintic activity
a b s t r a c t The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 g/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P < 0.001) and A. gaumeri (P < 0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L3 larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Some tannin-rich (TR) plants can have direct anthelmintic (AH) effects against the main nematode species of sheep and goats (Athanasiadou et al., 2001; Paolini et al., 2004). The AH effect has been related to
∗ Corresponding authors. Tel.: +52 2323243941; fax: +52 2323243943. E-mail addresses: [email protected], [email protected] (M.A. Alonso-Díaz), [email protected] (J.F.J. Torres-Acosta). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.03.052
the tannins and their ability to form complexes with parasite proteins. Tannins seem to affect the biological processes of nematodes depending on where and how the tannins bind with various nematode structures (e.g. cuticle, digestive or reproductive tract) (Hoste et al., 2006). Previous in vitro studies reported discrepancies between the conclusions drawn from results obtained with the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), using the same TR source (Alonso-Díaz et al., 2008a,b). In particular, Piscidia piscipula extracts (a plant with low quantity of tannins and
M.A. Alonso-Díaz et al. / Veterinary Parasitology 181 (2011) 360–364
low biological activity) at the concentration of 1200 g/ml PBS did not show any AH effect based on LMIA results but it significantly inhibited the exsheathment process of both Haemonchus contortus and Trichostrongylus colubriformis (Alonso-Díaz et al., 2008a,b). It was hypothesized that tropical TR plant extracts were more potent inhibitors of the exsheathment process than the motility of the infective larvae. Thus, the LMIA could be less sensitive than LEIA to investigate the in vitro AH effect of TR plant extracts against infective larvae. However, because only a single high dose level (1200 g/ml) was used in the LEIA, it was difficult to verify this assumption based on those results. To assess differences in sensitivity between assays, the same tannin source and a dose–response design for both tests must be used. Some authors have pointed out the necessity to compare the methodology used for the in vitro evaluation of plant AH effects (Ketzis et al., 2006; Athanasiadou et al., 2007). The present study aimed at comparing the sensitivity of two in vitro methods (LMIA and LEIA) for the evaluation of the AH activity of TR plant extracts against H. contortus infective larvae.
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2.4. Larval migration inhibition assay (LMIA) The mobility of ensheathed H. contortus L3 larvae was performed as described by Wagland et al. (1992), modified by Rabel et al. (1994). Live ensheated L3 (c. 1000) were added to centrifuge tubes containing either the negative control (PBS; pH 7.2) (BioMerieux® ) or a commercial anthelmintic control (levamisole at 1% concentration) or each solution to be tested (75, 150, 300, 600 and 1200 g of extract/ml). All incubations were carried out for 3 h at 20 ◦ C. Thereafter, the L3 from each tube were washed with PBS and centrifuged (3500 rpm) three times. The larvae were then transferred to sieves (inserts equipped with a 20 m mesh positioned in a conical tube). After 3 h at room temperature, the number of larvae that migrated through the mesh was counted at a 40× magnification using a 15% aliquot technique. The percentage of migration was calculated as M/T × 100 (where T is the total number of L3 deposited on the sieve and M the number of L3 that had successfully migrated through the sieve). Four replicates were run for each plant extract and for the negative and the positive (levamisol) controls.
2. Materials and methods 2.5. Larval exsheathment inhibition assay (LEIA) 2.1. Plant materials and extraction procedure Fresh leaves of Acacia gaumeri, H. albicans, Brosimum alicastrum and Leucaena leucocephala were used. A. gaumeri was chosen for its high content of tannins. On the other hand, B. alicastrum is a plant that has normally negligible levels of condensed tannins (Alonso-Díaz et al., 2008c) and was included as a negative control for the bioassays. The H. albicans and L. leucocephala plant extracts were included as a positive control because previous studies showed AH effect against H. contortus (Alonso-Díaz et al., ˜ et al., 2008) or T. colubriformis 2008a; Hernández-Orduno (Alonso-Díaz et al., 2008b). Five hundred grams of fresh leaves of each plant species were chopped to obtain the extracts. The chopped material was extracted with acetone:water (70:30) containing ascorbic acid. Then, the acetone was evaporated and the extract was washed four times with methylene chloride in order to eliminate pigments. Finally, each plant extract was lyophilized.
2.2. Quantification of condensed tannins The condensed tannin (CT) content of the extracts was quantified using the Butanol–HCl assay (Makkar, 2003) reading with a spectophotometer at 550 nm. The CT contents were expressed as leucocyanidin equivalent.
Ensheathed H. contortus L3 larvae (c. 1000), from the same batch used for the LMIA, were incubated for 3 h with each plant extract at concentrations of 75, 150, 300, 600 and 1200 g/ml in PBS before being re-suspended. After incubation, the larvae were washed and centrifuged (1000 rpm) three times in PBS (pH 7.2). The larvae were then submitted to an artificial exsheathment process by contact with a solution of sodium hypochloride (2%, w/v) and sodium chloride (16.5%, w/v) diluted in 1 to 300 in PBS (pH 7.2) as described by Bahuaud et al. (2006). The kinetics of larval exsheathment in the different experimental treatments was then monitored by microscopic observation (40×). The percentages of exsheathed larvae were identified at 0, 10, 20, 30, 40, 50 and 60 min intervals. Four replicates were run for each plant extract to examine the changes in proportion of exsheated larvae with time.
2.6. Statistical analyses A General Lineal Model (GLM) test was used to determine the dose effect of each plant extract in the LMIA and LEIA (SAS, 1991). Calculation of the lethal dose 50 (LD50), 90 (LD90 and LD99) were performed using a probit analysis (LeOra, 2003).
3. Results 2.3. Infective larvae
3.1. Condensed tannins in plant extracts
The third stage larvae (L3 ) were obtained from a donor goat with a monospecific infection of H. contortus susceptible to anthelmintics (INRA strain, France). The larvae were stored at 4 ◦ C for two months before use.
The highest levels of CT were found in H. albicans followed by A. gaumeri and L. leucocephala (16.45, 12.2 and 7.75 g/kg respectively). B. alicastrum extract had low levels of CT (0.81 g/kg).
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Fig. 1. Effect of tropical tannin rich plant extracts on the larval migration inhibition of infective L3 larvae of Haemonchus contortus (**P < 0.001, *P < 0.05).
3.2. Larval migration inhibition assay For the negative controls (PBS), the percentage of migration ranged from 68.75% to 83.65%. In the positive controls (levamisol groups), the percentage was less than 1% for each assay. Havardia albicans, showed a significant (P < 0.001) dosedependent anthelmintic effect against H. contortus (Fig. 1). The A. gaumeri extract, also showed significant dose dependent AH effect (P < 0.05). At the highest dose (1200 g/ml in PBS) the inhibition of larval migration was respectively 48.5% and 20.9% for H. albicans and A. gaumeri. However, significant effects were not found either with B. alicastrum or L. leucocephala extracts (Fig. 1). 3.3. Larval exsheathment inhibition assay The percentage of exsheathment of H. contortus L3 in the control groups was similar in the four assays and ranged respectively from 84.9%, 100%, 100% and 98.3%, after 60 min in contact with the solution of sodium hypochlorid and sodium chloride for A. gaumeri, H. albicans, B. alicastrum and L. leucocephala, respectively (Table 1). The four plant extracts inhibited (P < 0.001) the exsheathment process after 3 h contact. However, depending of the dose used, variations were found between plants. For example, with A. gaumeri and H. albicans, the plant extracts with the highest levels of CT, a severe delay or a total inhibition of exsheathment was found even at the lowest dose (75 g/ml in PBS) (P < 0.001) (Table 1). In contrast, for the B. alicastrum and L. leucocephala extracts, the exsheathment
process was dependent of the concentrations. The L. leucocephala extract showed a significant dose-dependent effect from 75 to 1200 g/ml. For B. alicastrum, a significant effect was evident from 300 to 1200 (P < 0.01). 3.4. Lethal dose values (50%, 90% and 99%) The calculation of lethal dose (LD) values was not possible for the LMIA tests as none of the plant extracts and doses tested reached a 50% reduction in motility compared to control values. On the other hand, extracts of H. albicans and A. pennatula reached more than 99% reductions in LEIA even at the lowest concentrations tested. Thus, calculations of LD 50, 90 or 99 were not feasible. However, it was feasible to make the LD calculation with B. alicastrum and L. leucocephala extracts for LEIA. The values of LD50, LD90 and LD99 for B. alicastrum extract were 291.6, 666.6 and 1308.2 g/ml respectively. For L. leucocephala extract the LD50, LD90 and LD99 values were 212.9, 846.5, 2608.4 g/ml, respectively. 4. Discussion This is the first report, using the same tannin-rich sources and nematode stage, aiming at comparing the sensitivity of the LMIA and LEIA to evaluate the AH effect of tropical TR plant extracts against H. contortus infective larvae. The LMIA is widely used to detect AH effects of tannin rich plants against GIN (Paolini et al., 2004; Alonso-Díaz ˜ et al., 2008). The prinet al., 2008a,b; Hernández-Orduno
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Table 1 Mean larval exsheathment (±SD) of Haemonchus contortus infective L3 larvae after 60 min of incubation with sodium hypochloride (2%, w/v) and sodium chloride (16.5%, w/v) solution. Larvae were previously exposed during 3 h to different concentrations (75, 150, 300, 600 or 1200 g/ml in PBS) of tropical tannin rich plant extracts. Plant extracts
Mean larval exsheathment (±SD) 75 g/ml
PBS Acacia gaumeri Brosimum alicastrum Havardia albicans Leucaena leucocephala *
84.9 100.0 100.0 98.3
± ± ± ±
17. 3 0.0 0.0 3.3
28.6 97.5 10.4 93.3
± ± ± ±
40.4* 5.0 8.1* 7.8
150 g/ml 13.7 95.0 5.9 62.1
± ± ± ±
11.1* 10.0 7.4* 34.7*
300 g/ml 6.9 40.8 1.9 31.2
± ± ± ±
4.8* 25.1* 3.8* 46.3*
600 g/ml 6.5 14.0 1.9 21.1
± ± ± ±
7.5* 12.7* 3.8* 23.6*
1200 g/ml 4.8 2.5 1.9 8.8
± ± ± ±
6.3* 5.0* 3.8* 7.5*
Significantly different compared to the respective PBS control (P < 0.05).
ciple of the assay relied on the ability of various natural substances to paralyze L3 and to inhibit their active passage through a 20 m nylon mesh sieves (Rabel et al., 1994). The LEIA has been developed to measure the ability of plant extracts to delay or inhibit an artificially induced exsheathment process of infective larvae (Bahuaud et al., 2006). By preventing exsheathment, the larvae may not be able to infect the host (Hertzberg et al., 2002). Both in vitro and in vivo results support the hypothesis that the interference of tannins and/or flavonoids with larval exsheathment as possible mechanisms of action against trichostrongyles (Hoste et al., 2006; Brunet and Hoste, 2006; Brunet et al., 2007, 2008a,b). In this experiment both assays were performed in parallel using the same batch of H. contortus larvae and the same plant extracts. Also, the same range of extract concentrations was used. The results showed that the LEIA was more sensitive than the LMIA for the detection of the AH activity against H. contortus. The latter might suggest that the exsheathment process is more affected than the motility process when exposed to the same tropical TR plants extracts. The B. alicastrum and L. leucocephala extracts did not inhibit significantly the larval migration but they did affect the exsheathment in a dose-dependent manner. The H. albicans extract provoked a nearly total inhibition of the exsheathment even at the lowest concentration (75 g/ml in PBS) whereas the highest reduction observed in the inhibition of migration was less than 50% with the same plant extract. The fact that such reduction in LMIA never exceeded 50% also explained why it was not possible to calculate a LD50 with the LMIA values. On the other hand, with the LEIA the calculation of LD50, LD90 and LD99 values was only feasible for B. alicastrum and L. leucocephala. For both in vitro assays the plant extracts with the highest concentration of CT (H. albicans and A. gaumeri) were more potent inhibitors of both the larval migration and exsheathment. Unfortunately the design of the study did not include the confirmation of the role of tannins in the AH effect observed (using polivynil–pirrolidone or polyethylene glycol as tannin blocking agents). Thus, it is not possible to rule out the participation of other plant secondary metabolites in the AH effect detected in the present trial. However, it is possible to speculate that the more specific and strong actions of the tannin rich extracts on the exsheathment process is related to the presence of proline and hydroxiproline rich proteins in the nematode larval sheath, cuticle and exsheathing fluid and the high affinity of tannins to those proteins (Fetterer, 1989; Fetterer and
Rhoads, 1993; Ozerol and Silverman, 1969; Page, 2001). Thus, tannins could bind to proteins in these structures hence affecting their exsheatment. Most studies reporting on the possible AH properties of TR plant extracts on nematode infective larvae have related the observed antiparasitic effects with the concentration of CT (Athanasiadou et al., 2001; Molan et al., 2000; Paolini et al., 2004; Ademola and Idowu, 2006; Alonso-Díaz et al., 2008a). Comparatively, only a few studies have explored, other polyphenolic or flavonoid compounds which might contribute to the AH activity (Barrau et al., 2005; Ademola et al., 2005; Brunet and Hoste, 2006). The moderate content of total phenols and total tannins reported earlier in B. alicastrum (Alonso-Díaz et al., 2008c) may help to explain the significant effects on the LEIA found with this extract despite its negligible content of CT. The LEIA seem to present several advantages compared to LMIA to evaluate the in vitro AH properties of plant extracts containing polyphenolic compounds: (I) It is more sensitive. Due to this highest sensitivity and in contrast with the LMI assay, it enables the calculations of LD. This facilitates the comparison/ranking of results between plants or between nematode species in the same study or between studies in various laboratories. (II) In contrast to most of the current in vitro assays which were derived from methodologies developed for chemical AH (Wood et al., 1995), the LEIA has been directly designed based on results describing specific interactions of tannins or flavonoids with the nematode infective larvae. (III) The conditions of application are simple and request only limited material. This study may help other groups working in similar areas to define what test they want to use: (a) a sensitive test like LEIA (i.e. screening purposes) or (b) a less sensitive test like LMIA that can be used for purposes beyond screening. Calderón-Quintal et al. (2010), for example, tested the variability in the in vitro AH effect of TR extracts against different H. contortus strains obtained in different geographical locations. They used the LMIA to compare the different strains against TR extracts with known in vitro AH effect. In that study a sensitive test such as LEIA could have not allowed the comparison. Conflict of interest statement The authors of this manuscript have no financial or personal relationships with other people or organizations that could inappropriately influence or bias the content of the paper.
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Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus M.A. Alonso-Díaz a,∗ , J.F.J. Torres-Acosta b,∗ , C.A. Sandoval-Castro b , H. Hoste c a Centro de Ense˜ nanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km. 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600, Veracruz, Mexico b Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Km 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, Mexico c UMR IHAP 1225 INRA/ENVT, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse Cedex, France
a r t i c l e
i n f o
Article history: Received 8 July 2010 Received in revised form 28 January 2011 Accepted 29 March 2011 Keywords: Haemonchus contortus In vitro Larval exsheatment inhibition assay Larval migration inhibition assay Tannins Polyphenols Anthelmintic activity
a b s t r a c t The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 g/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P < 0.001) and A. gaumeri (P < 0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L3 larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Some tannin-rich (TR) plants can have direct anthelmintic (AH) effects against the main nematode species of sheep and goats (Athanasiadou et al., 2001; Paolini et al., 2004). The AH effect has been related to
∗ Corresponding authors. Tel.: +52 2323243941; fax: +52 2323243943. E-mail addresses: [email protected], [email protected] (M.A. Alonso-Díaz), [email protected] (J.F.J. Torres-Acosta). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.03.052
the tannins and their ability to form complexes with parasite proteins. Tannins seem to affect the biological processes of nematodes depending on where and how the tannins bind with various nematode structures (e.g. cuticle, digestive or reproductive tract) (Hoste et al., 2006). Previous in vitro studies reported discrepancies between the conclusions drawn from results obtained with the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), using the same TR source (Alonso-Díaz et al., 2008a,b). In particular, Piscidia piscipula extracts (a plant with low quantity of tannins and
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low biological activity) at the concentration of 1200 g/ml PBS did not show any AH effect based on LMIA results but it significantly inhibited the exsheathment process of both Haemonchus contortus and Trichostrongylus colubriformis (Alonso-Díaz et al., 2008a,b). It was hypothesized that tropical TR plant extracts were more potent inhibitors of the exsheathment process than the motility of the infective larvae. Thus, the LMIA could be less sensitive than LEIA to investigate the in vitro AH effect of TR plant extracts against infective larvae. However, because only a single high dose level (1200 g/ml) was used in the LEIA, it was difficult to verify this assumption based on those results. To assess differences in sensitivity between assays, the same tannin source and a dose–response design for both tests must be used. Some authors have pointed out the necessity to compare the methodology used for the in vitro evaluation of plant AH effects (Ketzis et al., 2006; Athanasiadou et al., 2007). The present study aimed at comparing the sensitivity of two in vitro methods (LMIA and LEIA) for the evaluation of the AH activity of TR plant extracts against H. contortus infective larvae.
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2.4. Larval migration inhibition assay (LMIA) The mobility of ensheathed H. contortus L3 larvae was performed as described by Wagland et al. (1992), modified by Rabel et al. (1994). Live ensheated L3 (c. 1000) were added to centrifuge tubes containing either the negative control (PBS; pH 7.2) (BioMerieux® ) or a commercial anthelmintic control (levamisole at 1% concentration) or each solution to be tested (75, 150, 300, 600 and 1200 g of extract/ml). All incubations were carried out for 3 h at 20 ◦ C. Thereafter, the L3 from each tube were washed with PBS and centrifuged (3500 rpm) three times. The larvae were then transferred to sieves (inserts equipped with a 20 m mesh positioned in a conical tube). After 3 h at room temperature, the number of larvae that migrated through the mesh was counted at a 40× magnification using a 15% aliquot technique. The percentage of migration was calculated as M/T × 100 (where T is the total number of L3 deposited on the sieve and M the number of L3 that had successfully migrated through the sieve). Four replicates were run for each plant extract and for the negative and the positive (levamisol) controls.
2. Materials and methods 2.5. Larval exsheathment inhibition assay (LEIA) 2.1. Plant materials and extraction procedure Fresh leaves of Acacia gaumeri, H. albicans, Brosimum alicastrum and Leucaena leucocephala were used. A. gaumeri was chosen for its high content of tannins. On the other hand, B. alicastrum is a plant that has normally negligible levels of condensed tannins (Alonso-Díaz et al., 2008c) and was included as a negative control for the bioassays. The H. albicans and L. leucocephala plant extracts were included as a positive control because previous studies showed AH effect against H. contortus (Alonso-Díaz et al., ˜ et al., 2008) or T. colubriformis 2008a; Hernández-Orduno (Alonso-Díaz et al., 2008b). Five hundred grams of fresh leaves of each plant species were chopped to obtain the extracts. The chopped material was extracted with acetone:water (70:30) containing ascorbic acid. Then, the acetone was evaporated and the extract was washed four times with methylene chloride in order to eliminate pigments. Finally, each plant extract was lyophilized.
2.2. Quantification of condensed tannins The condensed tannin (CT) content of the extracts was quantified using the Butanol–HCl assay (Makkar, 2003) reading with a spectophotometer at 550 nm. The CT contents were expressed as leucocyanidin equivalent.
Ensheathed H. contortus L3 larvae (c. 1000), from the same batch used for the LMIA, were incubated for 3 h with each plant extract at concentrations of 75, 150, 300, 600 and 1200 g/ml in PBS before being re-suspended. After incubation, the larvae were washed and centrifuged (1000 rpm) three times in PBS (pH 7.2). The larvae were then submitted to an artificial exsheathment process by contact with a solution of sodium hypochloride (2%, w/v) and sodium chloride (16.5%, w/v) diluted in 1 to 300 in PBS (pH 7.2) as described by Bahuaud et al. (2006). The kinetics of larval exsheathment in the different experimental treatments was then monitored by microscopic observation (40×). The percentages of exsheathed larvae were identified at 0, 10, 20, 30, 40, 50 and 60 min intervals. Four replicates were run for each plant extract to examine the changes in proportion of exsheated larvae with time.
2.6. Statistical analyses A General Lineal Model (GLM) test was used to determine the dose effect of each plant extract in the LMIA and LEIA (SAS, 1991). Calculation of the lethal dose 50 (LD50), 90 (LD90 and LD99) were performed using a probit analysis (LeOra, 2003).
3. Results 2.3. Infective larvae
3.1. Condensed tannins in plant extracts
The third stage larvae (L3 ) were obtained from a donor goat with a monospecific infection of H. contortus susceptible to anthelmintics (INRA strain, France). The larvae were stored at 4 ◦ C for two months before use.
The highest levels of CT were found in H. albicans followed by A. gaumeri and L. leucocephala (16.45, 12.2 and 7.75 g/kg respectively). B. alicastrum extract had low levels of CT (0.81 g/kg).
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Fig. 1. Effect of tropical tannin rich plant extracts on the larval migration inhibition of infective L3 larvae of Haemonchus contortus (**P < 0.001, *P < 0.05).
3.2. Larval migration inhibition assay For the negative controls (PBS), the percentage of migration ranged from 68.75% to 83.65%. In the positive controls (levamisol groups), the percentage was less than 1% for each assay. Havardia albicans, showed a significant (P < 0.001) dosedependent anthelmintic effect against H. contortus (Fig. 1). The A. gaumeri extract, also showed significant dose dependent AH effect (P < 0.05). At the highest dose (1200 g/ml in PBS) the inhibition of larval migration was respectively 48.5% and 20.9% for H. albicans and A. gaumeri. However, significant effects were not found either with B. alicastrum or L. leucocephala extracts (Fig. 1). 3.3. Larval exsheathment inhibition assay The percentage of exsheathment of H. contortus L3 in the control groups was similar in the four assays and ranged respectively from 84.9%, 100%, 100% and 98.3%, after 60 min in contact with the solution of sodium hypochlorid and sodium chloride for A. gaumeri, H. albicans, B. alicastrum and L. leucocephala, respectively (Table 1). The four plant extracts inhibited (P < 0.001) the exsheathment process after 3 h contact. However, depending of the dose used, variations were found between plants. For example, with A. gaumeri and H. albicans, the plant extracts with the highest levels of CT, a severe delay or a total inhibition of exsheathment was found even at the lowest dose (75 g/ml in PBS) (P < 0.001) (Table 1). In contrast, for the B. alicastrum and L. leucocephala extracts, the exsheathment
process was dependent of the concentrations. The L. leucocephala extract showed a significant dose-dependent effect from 75 to 1200 g/ml. For B. alicastrum, a significant effect was evident from 300 to 1200 (P < 0.01). 3.4. Lethal dose values (50%, 90% and 99%) The calculation of lethal dose (LD) values was not possible for the LMIA tests as none of the plant extracts and doses tested reached a 50% reduction in motility compared to control values. On the other hand, extracts of H. albicans and A. pennatula reached more than 99% reductions in LEIA even at the lowest concentrations tested. Thus, calculations of LD 50, 90 or 99 were not feasible. However, it was feasible to make the LD calculation with B. alicastrum and L. leucocephala extracts for LEIA. The values of LD50, LD90 and LD99 for B. alicastrum extract were 291.6, 666.6 and 1308.2 g/ml respectively. For L. leucocephala extract the LD50, LD90 and LD99 values were 212.9, 846.5, 2608.4 g/ml, respectively. 4. Discussion This is the first report, using the same tannin-rich sources and nematode stage, aiming at comparing the sensitivity of the LMIA and LEIA to evaluate the AH effect of tropical TR plant extracts against H. contortus infective larvae. The LMIA is widely used to detect AH effects of tannin rich plants against GIN (Paolini et al., 2004; Alonso-Díaz ˜ et al., 2008). The prinet al., 2008a,b; Hernández-Orduno
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Table 1 Mean larval exsheathment (±SD) of Haemonchus contortus infective L3 larvae after 60 min of incubation with sodium hypochloride (2%, w/v) and sodium chloride (16.5%, w/v) solution. Larvae were previously exposed during 3 h to different concentrations (75, 150, 300, 600 or 1200 g/ml in PBS) of tropical tannin rich plant extracts. Plant extracts
Mean larval exsheathment (±SD) 75 g/ml
PBS Acacia gaumeri Brosimum alicastrum Havardia albicans Leucaena leucocephala *
84.9 100.0 100.0 98.3
± ± ± ±
17. 3 0.0 0.0 3.3
28.6 97.5 10.4 93.3
± ± ± ±
40.4* 5.0 8.1* 7.8
150 g/ml 13.7 95.0 5.9 62.1
± ± ± ±
11.1* 10.0 7.4* 34.7*
300 g/ml 6.9 40.8 1.9 31.2
± ± ± ±
4.8* 25.1* 3.8* 46.3*
600 g/ml 6.5 14.0 1.9 21.1
± ± ± ±
7.5* 12.7* 3.8* 23.6*
1200 g/ml 4.8 2.5 1.9 8.8
± ± ± ±
6.3* 5.0* 3.8* 7.5*
Significantly different compared to the respective PBS control (P < 0.05).
ciple of the assay relied on the ability of various natural substances to paralyze L3 and to inhibit their active passage through a 20 m nylon mesh sieves (Rabel et al., 1994). The LEIA has been developed to measure the ability of plant extracts to delay or inhibit an artificially induced exsheathment process of infective larvae (Bahuaud et al., 2006). By preventing exsheathment, the larvae may not be able to infect the host (Hertzberg et al., 2002). Both in vitro and in vivo results support the hypothesis that the interference of tannins and/or flavonoids with larval exsheathment as possible mechanisms of action against trichostrongyles (Hoste et al., 2006; Brunet and Hoste, 2006; Brunet et al., 2007, 2008a,b). In this experiment both assays were performed in parallel using the same batch of H. contortus larvae and the same plant extracts. Also, the same range of extract concentrations was used. The results showed that the LEIA was more sensitive than the LMIA for the detection of the AH activity against H. contortus. The latter might suggest that the exsheathment process is more affected than the motility process when exposed to the same tropical TR plants extracts. The B. alicastrum and L. leucocephala extracts did not inhibit significantly the larval migration but they did affect the exsheathment in a dose-dependent manner. The H. albicans extract provoked a nearly total inhibition of the exsheathment even at the lowest concentration (75 g/ml in PBS) whereas the highest reduction observed in the inhibition of migration was less than 50% with the same plant extract. The fact that such reduction in LMIA never exceeded 50% also explained why it was not possible to calculate a LD50 with the LMIA values. On the other hand, with the LEIA the calculation of LD50, LD90 and LD99 values was only feasible for B. alicastrum and L. leucocephala. For both in vitro assays the plant extracts with the highest concentration of CT (H. albicans and A. gaumeri) were more potent inhibitors of both the larval migration and exsheathment. Unfortunately the design of the study did not include the confirmation of the role of tannins in the AH effect observed (using polivynil–pirrolidone or polyethylene glycol as tannin blocking agents). Thus, it is not possible to rule out the participation of other plant secondary metabolites in the AH effect detected in the present trial. However, it is possible to speculate that the more specific and strong actions of the tannin rich extracts on the exsheathment process is related to the presence of proline and hydroxiproline rich proteins in the nematode larval sheath, cuticle and exsheathing fluid and the high affinity of tannins to those proteins (Fetterer, 1989; Fetterer and
Rhoads, 1993; Ozerol and Silverman, 1969; Page, 2001). Thus, tannins could bind to proteins in these structures hence affecting their exsheatment. Most studies reporting on the possible AH properties of TR plant extracts on nematode infective larvae have related the observed antiparasitic effects with the concentration of CT (Athanasiadou et al., 2001; Molan et al., 2000; Paolini et al., 2004; Ademola and Idowu, 2006; Alonso-Díaz et al., 2008a). Comparatively, only a few studies have explored, other polyphenolic or flavonoid compounds which might contribute to the AH activity (Barrau et al., 2005; Ademola et al., 2005; Brunet and Hoste, 2006). The moderate content of total phenols and total tannins reported earlier in B. alicastrum (Alonso-Díaz et al., 2008c) may help to explain the significant effects on the LEIA found with this extract despite its negligible content of CT. The LEIA seem to present several advantages compared to LMIA to evaluate the in vitro AH properties of plant extracts containing polyphenolic compounds: (I) It is more sensitive. Due to this highest sensitivity and in contrast with the LMI assay, it enables the calculations of LD. This facilitates the comparison/ranking of results between plants or between nematode species in the same study or between studies in various laboratories. (II) In contrast to most of the current in vitro assays which were derived from methodologies developed for chemical AH (Wood et al., 1995), the LEIA has been directly designed based on results describing specific interactions of tannins or flavonoids with the nematode infective larvae. (III) The conditions of application are simple and request only limited material. This study may help other groups working in similar areas to define what test they want to use: (a) a sensitive test like LEIA (i.e. screening purposes) or (b) a less sensitive test like LMIA that can be used for purposes beyond screening. Calderón-Quintal et al. (2010), for example, tested the variability in the in vitro AH effect of TR extracts against different H. contortus strains obtained in different geographical locations. They used the LMIA to compare the different strains against TR extracts with known in vitro AH effect. In that study a sensitive test such as LEIA could have not allowed the comparison. Conflict of interest statement The authors of this manuscript have no financial or personal relationships with other people or organizations that could inappropriately influence or bias the content of the paper.
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