Effects of Spigelia anthelmia decoction on sheep gastrointestinal nematodes

Highlights

• Spigelia anthelmia decoction (SaDec) is rich in tannins.

• SaDec had ovicidal, larvicidal and adulticidal effects against Haemonchus contortus.

• SaDec caused ultrastructural changes in the cuticle of adult H. contortus.

• SaDec was not toxic in mice.

• A dose of 350 mg/kg SaDec reduced sheep epg by 47% at 14 days post-treatment.

Abstract

The use of herbal medicines either in combination with or instead of synthetic anthelmintics is an approach to reducing the exposure of parasites to synthetic chemicals. The present study aimed to assess the in vitro and in vivo effects of Spigelia anthelmia decoction (SaDec) on sheep gastrointestinal nematodes. SaDec was obtained by extracting active constituents of the plant in boiling water. The condensed tannins present in SaDec were quantified and subjected to phytochemical analysis. The egg hatch test (EHT), the larval development test (LDT), and an adult worm motility (AWM) assay were performed. Ultrastructural changes in the cuticle of the adult Haemonchus contortus were evaluated by scanning electron microscopy (SEM). Acute toxicity tests in mice were performed to define the safe dose of SaDec to be administered in the fecal egg count reduction test (FECRT) In the FECRT, fecal samples were collected at days 0, 7 and 14 post-treatment to estimate the eggs per gram (epg) and to identify the most prevalent nematode genera. The results of the EHT and LDT were analyzed using analysis of variance (ANOVA) and compared using Tukey’s test (P < 0.05). The effective concentration to inhibit 50% (EC₅₀) of egg hatching or larval development was determined by the probit method. In the AWM assay, worm survival was analyzed with the non-parametric stratified Cox regression test. The efficacy in the FECRT was calculated using BootStreat 1.0 software. The phytochemical screening detected high concentrations of condensed tannins, flavonoids, flavones, saponins, alkaloids and xanthones. The weights of the total phenolics and the condensed tannins were 96.56 and 51.25 mg gallic acid equivalents (GAE)/g dry weight (DW), respectively. The EC₅₀ ± 95% confidence interval values of SaDec for the EHT and LDT were 1.4 (1.2–1.6) and 1.2 (1–1.3) mg/ml, respectively. Treatment with SaDec at 1.6 mg/ml produced 100% inhibition of worm motility after 12 h of exposure. SEM revealed ultrastructural changes in the cephalic region and cuticle of H. contortus females. In the acute toxicity test, there was no mortality in mice. SaDec at 350 mg/ml reduced the sheep epg by 47% at 14 days post-treatment. Haemonchus was the most prevalent nematode genus. This study demonstrated that SaDec shows promising efficacy against gastrointestinal nematodes in small ruminants.

Introduction

The impact of gastrointestinal nematode (GIN) infection in small ruminants is linked to clinical signs associated with infection and also to subclinical economic losses due to decreased growth and milk production (Martínez-Valladares et al., 2015). GIN control programs are primarily based on a combination of animal management practices and the use of anti-parasitic drugs (Lifschitz et al., 2014). The intensive use of synthetic chemical anthelmintics in small ruminant grazing farms has resulted in the widespread development of resistance to these products (Jackson et al., 2012). Furthermore, the residue of some persistent chemicals in the environment disrupts the ecosystem and poses a threat to human health (Qi et al., 2015). Therefore, anthelmintic resistance in parasitic nematodes is a global threat to sustainable livestock production (Kaplan, 2004, Dos Santos et al., 2017).

The development of sustainable and environmentally acceptable methods of nematode control has become a necessity (Ribeiro et al., 2015). The use of phytotherapics has been considered a suitable approach to nematode control in small ruminants (Sandoval-Castro et al., 2012, Macedo et al., 2015, Ribeiro et al., 2015). The anthelmintic effects of phytotherapics have generally been associated with the presence of one or more plant secondary metabolites, such as condensed tannins (CTs) (Hoste and Torres-Acosta, 2011). CTs of different plants have different physical and chemical properties, and CT composition may vary between organs within the same plant species (Mangan, 1988; Salminen and Karonen, 2011). The mechanism of action of differing subgroups of CTs on small ruminant gastrointestinal nematodes has not been clearly described (Kommuru et al., 2015). For example, tannin-rich plants may act through direct antiparasitic activity but might also act indirectly by increasing host resistance (Hoste et al., 2006).

Reduced nematode egg laying, impaired development of eggs into third-stage larvae (L₃), and lower establishment of L₃ in the host can be considered direct effects of tanniniferous plants (Hoste et al., 2012). Alternatively, the tannins may act indirectly, by improving the interactions of proteins in the host and consequently improving the immune response to parasites (Hoste et al., 2006).

Physicochemical conditions such as the pH of the gastrointestinal tract organs of small ruminants may influence the biological effect of CTs; for example at the pH of the abomasum (2.5–3), there is a dissociation of the tannin-protein complexes formed in the rumen (Hagerman et al., 1992, Min et al., 2003). However, at the beginning of the small intestine, where the pH is approximately 5.5, these complexes can be reconstituted (McNabb et al., 1998). This condition can cause CTs to have a greater effect on Haemonchus contortus than on Trichostrongylus colubriformis as reported by Minho et al. (2010).

Tannin-rich plants can be used as nutraceuticals for small ruminants (Hoste et al., 2015), especially in situations of feed scarcity during dry periods (Oliveira et al., 2013), and their use can be considered for combined treatment with synthetic anthelmintics (Gaudin et al., 2016). However, the excessive consumption of tannins can detrimentally affect the parasitized host (Hoste et al., 2006). Possible anti-nutritional consequences of these compounds have been reported and should be considered in the use of tanniniferous plants as nutraceutical products (Athanasiadou et al., 2001). In particular, disturbances of digestive physiology and decreases in nutrient digestibility can occur in small ruminants (Min et al., 2003).

Spigelia anthelmia (Loganiaceae) is a plant native to Asia and tropical South America and is widely used as an anthelmintic in Brazilian folk medicine (Braga, 2001). Phytochemical studies have revealed that the alkaloid spiganthine is the major component of S. anthelmia (Achenbach et al., 1995, Morais et al., 2002). Other minority compounds linked to the alkaloid spiganthine exhibit insect antifeedant activities (Hübner et al., 2001).

The nematicidal effect of S. anthelmia ethanolic extract against sheep GIN has been described previously (Ademola et al., 2007). S. anthelmia ethyl acetate extract exhibited ovicidal and larvicidal effects on H. contortus (Assis et al., 2003) and can cause tonic paralysis at the level of acetylcholine neurotransmission (Camurça-Vasconcelos et al., 2004). However, decoction, a method of extracting plant material in boiling water, is the one most commonly used by small farmers and traditional communities in the empirical treatment of animals and humans in North and Northeast Brazil (Monteiro et al., 2011, Paulino et al., 2012). These ethnoveterinary descriptions thus prompted the present study to evaluate the anthelmintic effect of S. anthelmia decoction (SaDec).

Few studies have tried to explain the mode of action of the herbal and/or isolated products from a vegetable against small ruminant GINs. Recently, a scanning electron microscopy (SEM) technique has been used to demonstrate potential anthelmintic effects on the H. contortus cuticle in an attempt to predict the direct effect of these products on the cuticle of sheep nematodes (Martínez-Ortíz-de-Montellano et al., 2013, Kommuru et al., 2015, Andre et al., 2016).

The objective of the present study was to assess the in vitro and in vivo effects of SaDec on sheep GIN.