Introduction
One of the most pathogenic parasitic nematodes affecting small ruminants worldwide is the species Haemonchus contortus (Waller et al., Reference Waller2006). This is considered to be one of the most severe parasites, causing anaemia, weight loss, hypoproteinemia and even death in young animals (Risso et al., Reference Risso, Keesler and Sousa-Soriano2015). Haemonchosis and other nematodiases in ruminants cause significant economic losses worldwide. Weight loss due to haemonchosis and other nematodiases has been estimated at 27% of the total production, and milk production losses are estimated to be 29% of the total volume (Qamar et al., Reference Qamar, Maqbool and Ahmad2011).
Nematodiasis in small ruminants has been addressed from different perspectives. The most commonly used practice to control ruminant parasitic nematodes is through chemical anthelmintic drugs. This strategy helps to reduce the parasitic burden in the infected animals; however, the efficacy of these drugs is rapidly diminishing due to the imminent presence of anthelmintic resistance (Peña-Espinoza et al., Reference Peña-Espinoza2014). A number of alternatives for controlling and preventing nematodiasis, including the use of nutritional improvement, grazing management, copper particles, immunization, and biological control, have been proposed (Torres et al., Reference Torres, Molento and Mendoza-de Gives2012). In recent decades, the potential use of plants and their extracts as natural de-wormers against ruminant parasitic nematodes has gained attention (Rochfort et al., Reference Rochfort, Parker and Dunshea2008; Chan-Pérez et al., Reference Chan-Pérez2016). Similarly, the use of medicinal plants with nematicidal activity is now considered an important area of research, playing a crucial role in the treatment of parasitic diseases that affect ruminants, specifically sheep and goats, from a sustainable perspective. This alternative is considered a promising tool for controlling parasites while also reducing the harmful effects that occur with the overuse of synthetic chemical anthelmintic drugs (Squires et al., Reference Squires2010a, Reference Squiresb; Mendoza-de Gives et al., Reference Mendoza-de Gives and Ishwaran2012), i.e. anthelmintic resistance, noxious environmental effects and even public health risks (Beyene, Reference Beyene2016). Chenopodium ambrosioides and Castela tortuosa are two Mexican plants that are being investigated for medicinal properties. Chenopodium ambrosioides is an edible plant commonly consumed by Mexican people as a seasoning in traditional cuisine, and C. tortuosa is used in traditional medicine for antidiarrhoeic, anti-amoebiasis and other treatments (Galicia-Aguilar et al., Reference Galicia-Aguilar2008). Other important medicinal properties have been identified in these plants in traditional Mexican medicine, such as antioxidant (Mercado-Mercado et al., Reference Mercado-Mercado2013), anticancer (Gawlik-Dziki et al., Reference Gawlik-Dziki2013) and anti-parasitic activities (Jabbar et al., Reference Jabbar2007; Anderson, Reference Anderson2014). Clean technologies for livestock and agriculture production require the use of traditional alternatives to control a number of diseases. The use of plants or plant extracts with anti-parasitic effects to control animal parasitic nematodes has gained interest among researchers worldwide during the last few decades (Ugwuoke et al., Reference Ugwuoke, Ukwueze and Ogwulumba2011). The present study was designed to (1) assess the in vitro nematicidal effect of C. ambrosioides and C. tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) against H. contortus infective larvae (L3), and (2) evaluate the in vivo anthelmintic effect with either a single or combined use of the extracts in gerbils as a study model.
Materials and methods
Location
This research was conducted at the Laboratory of Helminthology at the National Centre for Disciplinary Research in Veterinary Parasitology (CENID-PAVET) of the National Institute of Agricultural, Forestry and Livestock Research (INIFAP) in Jiutepec Municipality, Morelos State, Mexico. Plant extractions were performed at CIByC-UAEM in Cuernavaca Municipality, Morelos, Mexico.
Plant material
Both C. ambrosioides and C. tortuosa plants were acquired at a local market in the town centre of Cuernavaca City, Morelos, Mexico. Dr Gabriel Flores, an expert in botanical taxonomy from the Autonomous University of the State of Morelos, Mexico, identified the two plants. One sample of each plant was deposited in the Herbarium of the Research Centre in Biodiversity and Conservation at the same institution. The key registration numbers of the plants are C. ambrosioides (HUMO 2900) and C. tortuosa (HUMO 7019).
Plant extracts
The plant material was processed through organic maceration (Azwanida, Reference Azwanida2015; Ngaha-Njila et al., Reference Ngaha-Njila2017). The aerial parts of the fresh plants, including the leaves and stems, were dried in a closed chamber with electrical heating at 52°C for two days. Once the plants were completely dried, 1 kg of each dried plant was placed in a 10-l glass container, and 5 l of n-hexane was added to completely cover the plant with the organic solvent. The plant material remained under these conditions for 3 days, and the process was repeated two consecutive times. The solvent was removed by distillation under reduced pressure with a rotary evaporator (BUCHI mark 205). The dried extracts were compared by thin layer chromatography (TLC). The samples were gathered based on their similar chemical patterns and were subsequently mixed, generating a 2.22% total yield. Different concentrations of these extracts, 0.6, 1.25, 2.50, 5, 10, 20 and 40 mg/ml, were prepared with 3% Tween 20 in distilled water (Palacios-Landín et al., Reference Palacios-Landín2015). The final extracts were referred to as E-Cham (from C. ambrosioides) and E-Cato (from C. tortuosa).
Experimental animals
A population of male and female gerbils 3 months of age, weighing 35 g, were obtained from a commercial pet store. The gerbils were de-wormed with Albendazole (Valbazen) administered orally at 50 μl per day for 4 days. In a preliminary test, the optimal oral dose of L3 to infect gerbils was determined to be 20,000 L3 (exsheathed larvae) to establish a good infection. Similarly, the optimal date after infection to sacrifice gerbils to evaluate the potential reduction in parasitic burden was 12–13 days (unpublished data).
Parasitological techniques
Production of H. contortus L3
A 3-month-old male hair sheep, weighing 35 kg, was artificially infected with 350 (L3) per kg of body weight. Faecal samples were collected directly from the rectum of the sheep after the 20-day pre-patent period. The faecal samples were processed through a faecal culture technique in plastic bowls to obtain as many infective larvae as possible (Liébano-Hernández, Reference Liébano-Hernández, Campos Ruelas and Bautista Garfias1998). The faecal cultures were incubated for 6 days at room temperature (18–25°C) to obtain infective larvae. The larvae were recovered from faecal cultures using the Baermann funnel technique (OEPP/EPPO Bulletin, 2013). The recovered larvae were filtered through lens paper using the Baermann technique. The larvae were washed via gravity density centrifugation with a 40% sucrose solution at 3500 rpm for 5 minutes; a pellet formed in the white ring interphase corresponding to the larvae. The larvae were recovered and re-suspended in sterile distilled water to eliminate sucrose residues (Figueroa Castillo et al., Reference Figueroa Castillo and Rodríguez Vivas2015). The L3 were exsheathed using 0.18% sodium hypochlorite for 6–8 minutes at room temperature (18–25°C); the larvae were then rinsed and subsequently re-suspended in sterile distilled water (Ramírez et al., Reference Ramírez2006). Counting of larvae was performed using five 10-μl aliquot drops, and the total number of larvae present in a known volume was estimated.
In vitro bioassays to determine the 50% and 90% lethal concentrations against infective larvae of H. contortus
Different concentrations of E-Cham, E-Cato and the combination of both extracts (0.6, 1.25, 2.50, 5, 10, 20 and 40 mg/ml) were evaluated to determine the 50% (LC50) and 90% (LC90) lethal concentrations (López et al., Reference López2015). Each concentration was assessed considering three replicates. A concentrated solution, ‘stock solution’ non-diluted extract (40 mg/ml), was used initially; fold dilutions were prepared from this concentration. The test was conducted in 24-well cell culture plates (Nunc, InterMed, Denmark), with 500 μl of each corresponding extract deposited into each well of the three replicates. Tween 20 (3%) and distilled water were used as controls, as was fenbendazole. Subsequently, 500 exsheathed L3 were added to each well of the different treatments and incubated at room temperature (18–25°C). The results were considered by counting the number of live and dead larvae in ten 5-μl drops at 24 and 72 h post confrontation, as previously described (López-Arellano et al., Reference López-Arellano2006).
In vivo bioassays to assess the nematicidal effect of plant extracts against H. contortus in gerbils
Thirty gerbils (previously described) were used. All animals were treated with dexamethasone (Azium, Schering Plough Laboratories) using an intra-muscular route at a total dose of 100 μl per day for 2 consecutive days prior to artificial infection to improve L3 establishment. Every animal was infected orally with 3 ml of an aqueous suspension containing 20,000 exsheathed L3. At 9 days post infection, six groups of five gerbils each were assigned randomly as follows: groups 1 and 2 were treated intraperitoneally (IP) with E-Cham and E-Cato extracts, respectively, at 40 mg/kg BW. Group 3 comprised gerbils treated with a combination of extracts in a 1 : 1 relationship at the same dose and route of administration. Group 4 comprised gerbils IP treated with 100 μl of 3% Tween 20; this was considered a negative control group. Group 5 comprised animals treated with only water (100 μl) as another negative control (placebo) to indicate larval viability. Finally, group 6 included animals IP treated with fenbendazole (Sigma-Aldrich) at a unique dose of 10 mg/kg BW (Villar et al., Reference Villar2007) as a positive control. All animals were sacrificed by cervical dislocation at 13 days post infection, according to the Official Mexican Norm 062-ZOO 1999 regarding technical specifications for the production, care and use of laboratory animals. The animals were necropsied, and the total parasites were recovered from their stomachs. The recovered parasites were eventually counted, and the mean numbers per group were estimated for comparison. Similarly, the gastric mucosa of each animal was placed in artificial gastric juice to simulate stomach digestion to obtain larvae in hypobiosis from mucosa (Campos & Bautista, Reference Campos and Bautista1989). Two evaluation criteria were considered: the mean number of nematodes recovered from the treated group with respect to the mean number of nematodes recovered from group 4 (negative control 3% Tween 20), and the mean number of nematodes recovered compared with the mean number of nematodes recovered from group 5 (control: water). In both cases, the mean number of nematodes recovered in the control groups was considered as 100% larval viability.
The reduction percentage was estimated based on the following formula (Mendoza-de Gives et al., Reference Mendoza-de Gives1998):
Percentage reduction of parasites = (
$\bar {\rm x}$ control − 
$\bar {\rm x}$ treatment / 
$\bar {\rm x}$ control) × 100
where
$\bar {\rm x}$control = mean number of nematodes recovered from the negative control groups
$\bar {\rm x}$treatment = mean number of nematodes recovered from treated group
Preparation of plant extracts
The aerial parts of fresh plants, including the leaves and stems, were dried in a closed chamber with electrical heating at 52°C for 2 days. Once the plants were completely dried, 1 kg of dried individual plant material was placed into a 10-l glass container, and 5 l of n-hexane was added, with the organic solvent completely covering the plant. The plant materials were maintained under these conditions for 3 days, and the process was repeated two consecutive times. The solvent was removed by distillation under reduced pressure with a rotary evaporator, BUCHI mark 205 (von Son de Fernex et al., Reference von Son de Fernex2016). The dried extracts were compared by thin layer chromatography (TLC). The plant yields were estimated as follows: (E-Cham = 2.22%; E-Cato = 2.5%). Different concentrations of these extracts, 0.6, 1.25, 2.50, 5, 10, 20 and 40 mg/ml, were prepared with 3% Tween 20 in distilled water (Palacios-Landín et al., Reference Palacios-Landín2015).
Statistical analysis
The 50% and 90% lethal concentrations (LC50 and LC90) were determined using the Polo statistical programme (LeOra Software). In the in vivo assay, the dependent variable was calculated as the mean number of recovered nematodes at necropsy. A comparison among the means of the different groups was conducted through analysis of variance (ANOVA). A complementary Tukey's test was also conducted to determine the difference between treatments, using SAS statistical software version 2002 (Zar, Reference Zar1999).
Results
In vitro lethal concentration (LC50) and (LC90) and nematicidal efficacy percentages against H. contortus L3
The 50% and 90% in vitro lethal concentrations against L3 attributed to the effect of E-Cham and E-Cato extracts used either individually or combined at 24 and 72 h post confrontation are shown in table 1. Concentration-dependent lethal activity was identified in most of the treatments, reaching the maximum activity at 40 mg/ml either at 24 or 72 h post confrontation. Notably, the Polo statistical programme predicted 90% LC at concentrations higher than 40 mg/ml. The highest lethal effect against L3 was observed when E-Cham was used individually. An increase in the lethal activity of both combined extracts was identified after a 72-h interaction at 20 and 40 mg/ml concentrations.
Individual and combined in vitro nematicidal activity of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) against H. contortus L3, expressed as % efficacy, with 50% and 90% lethal concentrations after 24 and 72 h.
24 h: *P < 0.0001; CV = 19.8 ** P = 0.3236; CV = 85.1 *** P < 0.0267; CV = 48.4
72 h: * P < 0.0002; CV = 17.4 ** P < 0.0171; CV = 30.4 *** P < 0.0467; CV = 60
Means with different letters are significantly different according to Tukey's test.
Apart from this finding, no significant increase in the in vitro lethal activity was identified when the two extracts were used in a combined manner at all assessed concentrations. The highest in vitro lethal activity (close to 100%) was obtained with the combined use of both extracts at 72 h post confrontation at 40 mg/ml concentrations.
In vivo nematicidal activity of plant extracts in gerbils
The in vivo nematicidal activity of E-Cham and E-Cato extracts either individually or combined at different concentrations in gerbils is shown in table 2. When administered individually in gerbils, the E-Cato and E-Cham extracts reduced the parasitic burden by 27.1% and 45.8%, respectively, when compared with the control group in water. Moreover, the anthelmintic efficacy increased to 57.3% (P > 0.05) when both plant extracts were used as a combined inoculum.
Individual and combined nematicidal activity of C. ambrosioides and C. tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) against H. contortus in artificially infected gerbils, expressed as the percentage reduction of H. contortus larvae.
Different letters indicate significant differences between treatments.
Dose of n-extracts of both plants was 40 mg/kg BW; ANOVA: P < 0.0005; CV = 45.5.
* Percentage of population reduction considering the water control group as 100%.
** Percentage reduction of the population in the control group prepared with Tween 20 as 100%.
Discussion
The results of the present study provide evidence that the organic extracts E-Cham and E-Cato from C. ambrosioides and C. tortuosa, respectively, possess an in vitro nematicidal effect when used individually. A clear synergistic effect was also recorded when the two extracts were used at the two highest concentrations after a 72-h interaction. The combination of other plant/plant extracts has demonstrated better results than using individual plant materials alone. For example, better results were obtained with the combined use of methanolic extracts obtained from Caesalpinia crista and Chenopodium album flowers than when the extracts were used individually against the larvae and eggs of trichostrongyle nematodes (Jabbar et al., Reference Jabbar2007). However, the activity of both organic extracts was dose dependent, because the in vitro nematicidal effect increased proportionally with increasing plant concentration. The in vitro dose effect has been reported previously for other plant extracts. For example, the larval migration inhibition (LMI) test showed a dose-dependent anthelmintic effect for Acacia pennatula, Lysiloma latisiliquum and Leucaena leucocephala extracts (Alonso et al., Reference Alonso2008). In a review of the available literature, the authors of the present study found only one reference describing the results of C. ambrosioides and C. tortuosa in vitro nematicidal activity of an n-hexane extract of these plants. The results in this reference showed that C. ambrosioides possesses a high in vitro nematicidal effect (99.7%) against H. contortus fourth instar larvae after a 96-h interaction at the same concentration shown in the present study (Galicia-Aguilar et al., Reference Galicia-Aguilar2008). These authors found that the n-hexane extract of C. tortuosa achieved the highest in vitro nematicidal effect (of 85.8%) after a 96-h interaction at the same larval stage. These results are similar to those obtained in the present study using L3 stage larvae. Furthermore, the in vitro nematicidal effect using a combination of both extracts was higher than the individual effects of either plant extract after a 72-h confrontation. However, in the present study, for both in vitro and in vivo assays, we used two control groups: L3 larvae in a Tween 20 suspension and L3 larvae contained in only water. Tween 20 was used because it was not possible to dissolve the extracts in water due to the immiscibility of these compounds. Tween 20 has been used in similar research studies as a suitable plant extract dissolvent because most L3 are typically not affected either in vitro or in vivo (De Jesús Gabino et al., Reference De Jesús-Gabino2010). However, the results of the present study show that this dissolvent had certain lethal effects against nematodes, which were considered when we estimated the efficiency of the extracts.
The anthelmintic effectiveness reported in the present study could likely be improved by increasing the dose, using other routes of administration, assessing different vehicles to increase the bioavailability of bioactive compounds, or perhaps obtaining plant extracts using other organic solvents and purifying the active compounds in these extracts.
This work contributes to the scientific knowledge on the nematicidal activity of n-hexane extracts from medicinal plants against H. contortus using gerbils as an in vivo model of study. The plant extracts assessed in the present study prompted a phytochemical analysis to elucidate the compounds responsible for the consideration of nematicidal activity as viable and sustainable potential tools for controlling nematodiasis in small ruminants and other species of economic impact in the livestock industry. The present study is the first to establish a synergistic anthelmintic effect of two n-hexane plant extracts as a potential de-wormer administered in gerbils against nematodes, and this effect can be considered a foundation for future research focused on a potential natural de-wormer in sheep and other economically important animal species. The purification of these extracts, followed by the elucidation of the bioactive compounds responsible for the anthelmintic effect, is currently in preparation for publication by our research group. Exploring the potential use of these plant extracts in sheep as a potential de-wormer implies the performance of some important steps, including obtaining and identifying the molecules responsible for the anthelmintic activity and establishing the concentration of the active compound to be administered to sheep, which depends on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the molecules once administered to the animals. PK/PD modelling can be used to identify the relationship based on the plasma concentration profile to eventually estimate a proper dosage regime (Toutain & Lees, Reference Toutain and Lees2004). Other alternative strategies for using these plants as natural de-wormers could be explored; for example, identifying the content of bioactive compounds in the foliar system of these plants as well as their palatability in sheep and lack of toxicity could be important to assess a potential beneficial effect of the consumption of these plants in reducing the parasitic burden in sheep flocks maintained under grazing conditions.
Acknowledgements
The present study was conducted as part of the MSc thesis of Miss Claudia García Alanís from the Faculty of Agricultural Sciences, Autonomous University of the State of Morelos, under the direction of Dr Pedro Mendoza de Gives and Dr David O. Salinas Sánchez. The study was performed as part of the scientific activities of Dr David O. Salinas-Sánchez in collaboration with INIFAP during a sabbatical period. The authors would like to thank the RED FARMOQUÍMICOS – CONACYT – MEXICO network for promoting interdisciplinary and inter-institutional studies between CIBIS-IMSS and INIFAP. Dr A Zamilpa would like to thank the IMSS AC Foundation for supporting this research.
Financial support
The present study was financially supported through grants from the CONACYT Mexico PROJECT: CONACYT-SAGARPA – 2004-CO1-78.
Conflict of interest
None.
Ethical standards
The sheep were strictly maintained under the Norma Oficial Mexicana (Official Rule Number) NOM-051-ZOO-1995 (http://www.senasica.gob.mx) and the LEY Federal de Sanidad Animal (Federal law for animal health) DOF 07-06-2012 (http://diputados.gob.mx/LeyesBibliop/ref/lfsa.htm). These guidelines specify that all procedures performed in studies involving animals must follow the Federal Law and Official Rule strictly in accordance with the ethical standards of INIFAP. Furthermore, the guidelines are based, in part, on the Guide for the Care and Use of Laboratory Animals published by the Institute of Laboratory Animals Resources Commission on Life Sciences, National Research Council, 1996.
