RESEARCH WORK
In vitro effect of the aqueous extract of Dichrostachys cinerea (L.) Wight & Arn. on the development of exogenous stages of gastrointestinal strongyles in sheep
Estación Experimental de Pastos y Forrajes "Indio Hatuey". Central España Republicana, CP 44280, Matanzas, Cuba
Email: javier.arece@indio.atenas.inf.cu
ABSTRACT
The in vitro effect of aqueous extract of leaves from Dichrostachys cinerea on egg hatching, larval development and migration of third-stage larvae of gastrointestinal strongyles, was evaluated. The treatments were three concentrations of aqueous extracts of D. cinerea leaves (500, 250 and 125 mg/mL), albendazole and levamisole solutions, as well as Phosphate Buffer Saline (PBS) and dimethyl sulfoxide (DMSO) as controls; the design was completely randomized. The hatching percentages showed significant differences among post-treatments. PBS caused the highest hatching rate (96,68%); 30,51% hatched with albendazole and the D. cinerea leaf extracts showed moderate hatching rates (between 49,75 and 66,54%), seemingly dose-dependent. The development of L1/L2 larvae to L3 treated with aqueous D. cinerea extract also showed dose-dependent effects and significant differences with regards to the positive and negative control groups. The dose of 500 mg/mL inhibited larval development in similar percentages as those of albendazole (8,89 and 1,28%, respectively). The PBS medium did not interfere in the larval migration capacity, while with levamisole the values were reduced from 86,45 to 93,92%; the aqueous extracts also significantly reduced such migration, with values between 77,67% and 48,97%. The aqueous D. cinerea extracts showed in vitro anthelmintic activity in the three development stages of the exogenous cycle of gastrointestinal strongyles in sheep; this activity was more evident in the larval development and migration of these nematodes.
Key words: Dichrostachys cinerea, parasites, sheep.
The search for alternatives to minimize the use of anthelmintics for parasite control in small ruminants constitutes a challenge for animal production worldwide, because their indiscriminate use has caused generalized resistance to the main antiparasitic drugs (Arece et al., 2004; Wolstenholme et al., 2004), which is stressed by the slow short term- development of antiparasitic products.
At present, there are different strategies aimed at solving the problem of gastrointestinal parasitism in sheep, among which the ones related to the use of plants with antiparasitic potential stand out (Athanasiadou et al., 2001; Githiori et al., 2006; Marie Magdeleine et al., 2010).
In the in vivo studies with native plants from different countries a better response of the animals to parasite infestation has been reported, which is ascribed to: 1) the direct effects, related to the influence of some plant secondary metabolite on the parasites (mainly condensed tannins), and 2) the secondary effects, linked to an increase of the resilience status of the animals, due to an improvement of the diet, when consuming this type of plants (Kyriazakis et al., 2010).
On the other hand, in vitro studies have also been conducted to determine the activity of plant extracts on the inhibition of egg hatching, and also on the development of larval migration and the motility of adult parasites (Marie-Magdeleine, 2009). These studies have allowed the selection of a large number of plants, of which there is some evidence of antiparasitic activity.
In Cuba there are experiences regarding the use of phytopharmaceuticals in parasite control; the result with the highest impact was generated with Bromelia pinguin, which was effective against Haemonchus spp. in calves (Marrero et al., 1994). In Mexico it was used against Oesophagostomum columbianum in sheep (Olivares, 2001).
Among the main secondary metabolites to which antiparasitic effects are ascribed are condensed tannins CT- (Wolstenholme et al., 2004). Dichrostachys cinerea (L.) Wight & Arn is a shrub with high contents of this metabolite (Pedraza et al., 2008), for which it has potential for parasite control, because its CTs do not need to be deactivated (addition of polyethylene glycol or NaOH) before supplementing small ruminants with the foliage fresh or as meal (Mlambo et al., 2004).
In Cuba this plant constitutes an important feed source in many sheep and goat productive systems, mainly during the dry season. For such reason, the objective of this work was to evaluate in vitro the antiparasitic properties of aqueous D. cinerea extracts, in three stages of the biological cycle of gastrointestinal strongyles in sheep.
The study was conducted in the parasitology laboratory of the EEPF "Indio Hatuey". The D. cinerea leaves were harvested in the grazing area of the institution, in the morning. An aqueous extraction was made with Phosphate Buffer Saline (PBS) solution in fresh, using liquid nitrogen, through the method described by Díaz et al. (2010). A mother solution of 500 mg/mL was prepared, from which three concentrations were prepared to be used in the trials: 500, 250 and 125 mg/mL, identified as MAR-500, MAR-250 and MAR-125, respectively.
The assays were conducted from the modifications of or made to the techniques described by Hubert and Kerboeuf (1984) and Marie-Magdeleine et al. (2010). Six treatments were evaluated, which corresponded to the three concentrations of the D. cinerea leaf extract (MAR-500, MAR-250 and MAR-125), PBS and dimethyl sulfoxide (DMSO) as positive controls, and albendazole 0,5% (ABZ), as negative control. Strongyle eggs were collected from infested animals (Hubert and Kerboeuf, 1992) and they were deposited in 24-well cell culture plates, to be faced with the different solutions or experimental treatments. The eggs were incubated during 48 h and, after that time, the hatching was stopped with 100 µL of Lugol's solution. The larvae and eggs were counted in 20 aliquots of 10 µL and the hatching percentage was determined. The design was completely randomized, with six replications per treatment.
The studies were conducted from the modifications made to the techniques described by Hubert and Kerboeuf (1984) and Assis et al. (2003). The same treatments as in the EHA were used. The assay principle consisted in exposing the hatched eggs to the different solutions, to evaluate the development of L1/L2 larvae to L3 or infective larvae, according to the technique proposed by Marie-Magdeleine et al. (2010). The L1/L2 were obtained by means of the procedure described in the EHA and were fed with a nutritional solution. After 48 h the solutions were applied and after 8 days the molt process was stopped with 100 µL of Lugol's solution. The L1/L2 and L3 were counted and the percentage of infective larvae was determined.
The principle of this test consisted in presenting the third stage larvae obtained through coprocultures (Roberts and O'Sullivan, 1952)- to the three concentrations of the aqueous extract, a positive control (PBS) and a negative control (levamisole (LV). The method described by Marie-Magdeleine et al. (2010) was used, consisting in presenting a known quantity of L3 larvae to the solution, in a conical Falcon® test tube, during two hours. After successive washings with PBS, they were put to migrate through a 20 µm sieve in a device prepared for such purpose. Afterwards, the quantity of migrated larvae was determined and the migration percentage was calculated.
The data were processed with the statistical pack SPSS®. The percentage values (X) of each test were transformed (arcsinÖX), to make a simple variance analysis. The variance homogeneity and normal data distribution was tested. The differences among means were calculated through Duncan's multiple range comparison test (Duncan, 1955).
Figure 1 shows the effect of the aqueous D. cinerea extracts and the respective control groups, on egg hatching. The hatching percentages showed significant differences (p<0,01): PBS had the highest hatching rates (96,68%), while with albendazole 30,51% of the eggs hatched, which proves the effectiveness of this drug on the viability of eggs from gastrointestinal nematodes (Martin et al., 1997).
On the other hand, the aqueous extracts of D. cinerea leaves allowed moderate hatching rates (between 49,75 and 66,54%), seemingly dose-dependent. These values were similar to the ones obtained with aqueous extracts of papaya (Carica papaya) seeds and lower than those of cassava leaves (Manihot esculenta) (12,35%) and banana leaves (Musa paradisiaca) (6,14%), reported in the Eastern Caribbean (Guadalupe) by Marie-Magdeleine (2009).
The development of L1/L2 larvae to L3, with the aqueous D. cinerea extract also showed dose-dependent effects (fig. 2) and significant differences (p<0,01) with regards to the positive and negative control groups. The dose of 500 mg/mL inhibited larval development in similar percentages as albendazole (8,89 and 1,28%, respectively; p<0,01). These values are lower than the ones reported for the aqueous extract of C. papaya seed (Marie Magdeleine, 2009) and are similar to the ones obtained with Cucurbita moschata seeds (Marie-Magdeleine et al., 2009) and Melia azedarach foliage (Cala et al., 2012). In this study, the main effects were observed in the first and third stage larvae (larval development), although they were also remarkable in L3, because their migratory activity was significantly (p<0,01) inhibited (fig. 3). Cala et al. (2012) obtained a similar performance in M. azedarach and Trichilia claussenii.
The effect of the aqueous extracts on the migration of L3 larvae is shown in figure 3. PBS did not remarkably interfered in the migration capacity of the larvae, while levamisole showed an 86,45-93,92% reduction (p<0,01) in this indicator. On the other hand, the D. cinerea extracts also significantly (p<0,01) reduced migration, with values between 77,67 and 48,97%. This could have occurred due to the direct effects of free condensed tannins present in this plant-, which interfere in the movement capacity of the larvae, or to the direct intervention in cuticle loss.
D. cinerea is considered a tanniniferous plant, with values of 178 g/kg DM of this secondary metabolite (Mlabo et al., 2004). In Cuba, Pedraza et al. (2008) reported total polyphenol contents of 120 g/kg DM.
The way in which CTs act is still being debated, and there are two hypotheses; the first one is based on the possible direct effects of these substances, which have the capacity to form complexes with the free proteins of gastrointestinal parasites (Mueller-Harvey, 2006), for which nutrient assimilation capacity is reduced. In addition, they are capable of binding to the larval cuticle (rich in glycoproteins) and cause its death (Cala et al., 2012). On the other hand, CTs have been proven to decrease the fecundation capacity of parasites (Martínez-Ortíz de Montellano et al., 2010) and have direct effects on the adult stages causing important structural lesions, which propitiates the death of the parasite (Manolaraki et al., 2010; Martínez-Ortíz de Montellano et al., 2010).
The second hypothesis is based on the fact that condensed tannins can interfere directly in the parasite biology, through the increase in the immunological response of the animals (Hoste et al., 2006). This could have been related to the increase of the flow and absorption of proteins and aminoacids directly in the small intestine, as a result of their lower degradation in the rumen, because of the presence of tannins. In this sense, Aróstica (2011) reported low parasite infestation rates in goats, in grazing areas with predominance of D. cinerea, which practically constitutes the only feeding source during the dry season under Cuban conditions.
This study proved the existence of possible dose-dependent effects on hatching and larval development, which were not so clear in the case of larval migration, because the highest doses were more effective in the two assays on the first life stages of these parasites. These effects are probably related to the presence of cuticle in L3, which makes them more resistant than the previous stages (L1 and L2).
The research showed the in vitro anthelmintic effects of the aqueous extract of D. cinerea leaves, with dose-dependent effects on hatching and larval development. The best results were obtained with the concentration of 500 mg/mL. The design of in vivo studies which allow confirming the antiparasitic activity on adult parasites is recommended.
This study was funded by the International Foundation for Science (Project B/4610) and the Experimental Station of Pastures and Forages "Indio Hatuey" (Institutional Project 9567).
