Effects of Spigelia anthelmia decoction on sheep gastrointestinal nematodes

Accepted Manuscript Title: Effects of Spigelia anthelmia decoction on sheep gastrointestinal nematodes Authors: Wesley L.C. Ribeiro, Weibson P.P. Andre, G´essica S. Cavalcante, Jos´e V. de Ara´ujo-Filho, Jessica M.L. Santos, Iara T.F. Macedo, Janaina V. de Melo, Selene M. de Morais, Claudia M.L. Bevilaqua PII: DOI: Reference:

S0921-4488(17)30160-8 http://dx.doi.org/doi:10.1016/j.smallrumres.2017.06.001 RUMIN 5493

To appear in:

Small Ruminant Research

Received date: Revised date: Accepted date:

9-12-2016 24-5-2017 1-6-2017

Please cite this article as: Ribeiro, Wesley L.C., Andre, Weibson P.P., Cavalcante, G´essica S., de Ara´ujo-Filho, Jos´e V., Santos, Jessica M.L., Macedo, Iara T.F., de Melo, Janaina V., de Morais, Selene M., Bevilaqua, Claudia M.L., Effects of Spigelia anthelmia decoction on sheep gastrointestinal nematodes.Small Ruminant Research http://dx.doi.org/10.1016/j.smallrumres.2017.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Effects of Spigelia anthelmia decoction on sheep gastrointestinal nematodes Wesley L. C. Ribeiro1, Weibson P. P. Andre1, Géssica S. Cavalcante1, José V. de AraújoFilho1, Jessica M. L. Santos1, Iara T. F. Macedo1, Janaina V. de Melo2, Selene M. de Morais1, Claudia M. L. Bevilaqua1* 1

Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Ceará, Brazil.

2

Laboratório de Microscopia e Microanálise, Centro de Tecnologias Estratégicas do Nordeste, Brazil.

* Corresponding author: Claudia Maria Leal Bevilaqua Programa de Pós-graduação em Ciências Veterinárias/FAVET/UECE Av. Dr. Silas Munguba, 1700, Campus do Itaperi CEP: 60.714-903 Fortaleza, Ceará, Brazil Phone: + 55 85 31019853 Fax: + 55 85 31019840 E-mail: [email protected]

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

2

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.

Keywords: small ruminants; Haemonchus contortus; phytotherapic; tannins.

1 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 (Martinez-Valladares et al., 2015). GIN control programs are primarily based on a combination of animal management practices and the use of anti-parasitic

3

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 et al., 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 et al., 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

4

(2.5- 3), there is a dissociation of the tannin-protein complexes formed in the rumen (Hagerman et al., 1982, 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. (2008). 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

5

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.

2 Materials and methods

2.1 Animal ethics approval

The experimental protocol was approved by the ethics committee for animal use of the Universidade Estadual do Ceará (Approval number: 5166759/14).

2.2 S. anthelmia decoction

The aerial parts of S. anthelmia were collected on the campus of Universidade Estadual do Ceará, Brazil, between January and March 2015. The samples of the plant were identified and authenticated by botanists at the Prisco Bezerra Herbarium of the Universidade Federal do Ceará, Brazil (Voucher specimen number: 55223). In total, 1,650 g of the aerial parts of S. anthelmia was crushed in an electric crusher. Subsequently, 4 l of distilled water was added to the resulting material. The mixture was heated at 85 °C (decoction method) for 30 minutes. The decoction was filtered with gauze and then with Whatman® No. 1 filter paper (diameter: 110 mm; pore size: 11 µm). The resulting decoction was lyophilized.

6

2.3 Phytochemistry Phytochemical screening to identify the major classes of secondary metabolites of SaDec was performed according to the methodology described by Matos (2009). The chemical characterization was based on the addition of specific reagents to aliquots of the decoction and observation of the changes in the solution color or precipitate formation (Macedo et al., 2015). The total phenolics (TP) in the SaDec were measured using the Folin-Ciocalteu method according to Makkar (2003). Polyvinyl polypyrrolidone (PVPP) was added, and then the total tannin was calculated as the difference in the TP when measured with and without the addition of PVPP. Then, a tannic acid standard curve was plotted, with the results expressed as tannic acid equivalents (Oliveira et al., 2013). The TP content was converted to milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW) using a calibration curve for gallic acid (0–400 µg/ml) (Vermerris and Nicholson, 2006). The condensed tannins were measured using the butanol–HCl assay (Makkar, 2003).

2.4 Egg hatch test (EHT)

The assay was based on a modification of the EHT, which is performed to measure anthelmintic resistance (Coles et al., 1992). One sheep was experimentally infected with the Inbred-susceptible-Edinburgh (ISE) isolate of H. contortus, and it was used as a source of eggs, which were recovered according to the method described by Hubert and Kerboeuf (1992). Aliquots of a suspension containing approximately 100 fresh H. contortus eggs were incubated with lyophilized SaDec at concentrations of 0.31, 0.62, 1.25, 2.5, 5 and 10 mg/ml for 48 h at 25 °C. The hatching of eggs was stopped by adding Lugol’s iodine solution. The eggs and firststage larvae (L1) were counted under a light microscope. The negative control was distilled water, and the positive control was 0.025 mg/ml thiabendazole. Three completely replicated assays with five repetitions of each concentration were performed. 2.4 Larval development test (LDT)

The LDT was based on the method of Hubert and Kerboeuf (1992). Aliquots of 250 µl of a suspension containing approximately 100 fresh eggs obtained by fecal washing were added to each well of a 24-well microplate. Then, 80 µl of a nutrient solution containing yeast extract (Sigma-Aldrich®, USA), Escherichia coli (Sigma-Aldrich®, USA), amphotericin B (A9538,

7

Sigma-Aldrich®, USA), 0.9% saline solution and Earle's balanced salt solution (ATCC E9637, Sigma-Aldrich®, USA) was added to each well, and the microplate was incubated at 27 ± 1 °C. After 24 h, the L1 hatching and viability were checked based on sinusoidal larval movement, and subsequently, aliquots of SaDeC in concentrations from 0.31, 0.62, 1.25, 2.5 and 5 mg/ml were added to the wells that already contained the nutrient solution. SaDec was diluted in distilled water, which was also used as the negative control, and the positive control was 100 µg/ml ivermectin. The plate was again incubated at 27 ± 1 °C. After six days, the larvae were killed with Lugol's iodine solution. H. contortus in the first three larval stages (L1, L2, and L3) were counted in each well under a light microscope. Three completely replicated assays with five repetitions of each concentration were performed. 2.5 Adult worm motility (AWM)

The AWM assay was conducted according to the methodology described by Hounzangbe-Adote et al. (2005). A sheep was experimentally infected with a single dose of 4,000 larvae of the ISE isolate of H. contortus. After confirmed infection over 5,000 eggs per gram (epg) of feces, the sheep was euthanized by the use of a captive bolt pistol according to the Brazilian legislation for animal welfare (CONCEA, 2015), and it was used as source of adult worms. Adult female H. contortus were recovered from the sheep’s abomasum and washed in 0.9% saline solution at 37 °C. After the worms were assessed for viability, they were distributed in 24-well plates at a ratio of 3 worms per well in a medium containing 1 ml of phosphate buffered saline (PBS) and 4% penicillin/streptomycin (Sigma-Aldrich®, USA). The nematodes were incubated at 37 °C in a gas phase of 5% CO2/95% air mixture for 1 h. Then, 1 ml SaDeC at concentrations of 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml was added to the wells. After 6, 12, 18 and 24 h of incubation, the motility and survival of the adult worms were observed under an inverted light microscope at a magnification of 40×. The worms that showed no motility during 2 minutes of observation were considered dead. The negative control was 1 ml of PBS with 4% penicillin/streptomycin, and the positive control was 100 µg/ml ivermectin (Ivomec®, Merial Saúde Animal, Brazil). Eight replicates for each treatment and for each control were performed.

2.6 Scanning electron microscopy (SEM)

8

Adult female H. contortus in a medium containing 1 ml PBS and 4% penicillin/streptomycin (Sigma-Aldrich®, USA) were treated with 0.8 mg/ml SaDec under the same conditions as in the AWM assay for 24 h and used to evaluate the ultrastructural changes in the worm cuticle by scanning electron microscopy (SEM). Untreated worms were used as the negative control. The cuticle and cephalic region of ten parasites were evaluated for each group. Initially, the specimens were preserved according to the methodology described by Karnovsky (1965), in a medium composed of 5% glutaraldehyde in a phosphate emulsifier buffer containing 0.1 M sodium cacodylate (Sigma-Aldrich®, USA) at pH 7.4 and 4 °C for 72 h. Next, they were post-fixed in 1% osmium tetroxide in a phosphate emulsifier at pH 7.4 for 1 h. Then, the specimens were washed twice with 0.2 M sodium cacodylate buffer/distilled water and dehydrated in a series of acetone solutions of increasing concentration. The specimens were then critical point dried using a Critical Point Dryer (CPD 030, BAL-TEC, Liechtenstein), mounted on slides, covered in a 10 nm layer of gold-palladium using a Leica SCD 500 (Leica Microsystems, Germany) and analyzed using a scanning electron microscope FEI Quanta 200 FEG ESEM (FEI Company, USA) at an accelerating voltage of 20 kV.

2.7 Acute toxicity in mice

The acute toxicity test in mice was designed to define the safe dose of SaDec for administration in sheep. Therefore, female Swiss albino mice (n= 36) with an average weight of 25.6 ± 2.2 g were allowed to acclimate to the experimental conditions (cycle of 12 h light/dark and a temperature of 22 ± 2 °C) for seven days, during which they were kept in polypropylene boxes. Ionized commercial feed (Nuvilab®, Brazil) and filtered water were provided ad libitum to the rodents. The mice were randomly divided into the following 6 groups: G1 to G5, in which they received 1,000, 2,000, 3,000, 4,000 and 5,000 mg/kg SaDec, respectively; and G6 (the negative control), which received distilled water. The treatments were administered in a single oral dose of 0.2 ml. The animals were carefully observed individually for any signs of toxicity at least once during the first 30 minutes after dosing, periodically during the first 24 h (with special attention given during the first 4 h), and daily thereafter for a period of 14 days (OECD, 2001).

2.8 Fecal egg count reduction test (FECRT)

9

Thirty sheep from 6 to 18 months old and with an average weight of 21 ± 6.5 kg that were naturally infected with GIN at an epg greater than 1,000 were selected and randomly divided (n=10) into three groups: G1, receiving 350 mg/kg SaDec; G2, receiving 2.5 mg/kg monepantel (Zolvix®, Novartis Animal Health, New Zealand) and G3, receiving distilled water. The treatments were administered orally in a single dose. Fecal samples were collected on days 0, 7 and 14 post-treatment to estimate the epg. A larval culture using a pool of feces from each group was performed to identify the nematode genera. The identification of L3 was based on Ueno and Gonçalves (1998).

2.9 Statistical analysis The larval hatching percentage was determined according to the following formula: (number of hatched larvae / number of hatched larvae + number of eggs) × 100. The percent inhibition of larval development was calculated as follows: (Number of L3/total number of larvae per well) × 100. The larval hatching percentage in the EHT and the percent inhibition of larval development in the LDT were analyzed using analysis of variance (ANOVA), and the mean effects for each concentration were compared by Tukey’s test using GraphPad Prism® 5.0 software (GraphPad Software Inc., USA). The dose–response relationships in the EHT and LDT were determined considering the statistical level of significance as P<0.05. The effective concentrations to inhibit 50% (EC50) of larval hatching or to cause 50% inhibition (EC50) of larval development were determined by the probit method using IBM SPSS Statistics version 22 for Windows (IBM, USA). The adult worm motility was evaluated as the number of motile worms/total number of worms per well. The survival of worms was analyzed with the non-parametric stratified Cox regression test for evaluation of dose–response relationship using Minitab® Release 17 software (Minitab Inc., USA). The results of the worm motility inhibition are expressed as the mean ± standard deviation (SD) (P<0.05). The anthelmintic efficacy of SaDec was interpreted through the FECRT based on each group arithmetic mean faecal egg counts using the following formula: FECRT = 100×(1[T2/T1] [C1/C2]), in which the arithmetic faecal egg count means in controls (C) and treated (T) animals before (T1 and C1) and 7 or 14 days after (T2 and C2) deworming were compared (Dash et al., 1988), and 95% confidence intervals were estimated using BootStreat 1.0 software (Cabaret, 2014). The epg were log transformed (log10[x+1]), submitted to ANOVA and compared using Tukey’s test using Graph Pad Prism® 5.0 software (Ribeiro et al., 2014).

10

3 Results

3.1 Phytochemistry The yield of the SaDec after lyophilization was 10.4% of the initial weight of the aerial parts of S. anthelmia. The phytochemical test showed the presence of condensed tannins, flavonoids, flavones, saponins, alkaloids and xanthones. The weights of the total phenolics and condensed tannins were 96.56 and 51.25 mg GAE/g DW, respectively.

3.2 In vitro tests The effect of the SaDec in the EHT and LDT is presented in Table 1. When used at 10 mg/ml, SaDec was found to inhibit 99.3% of egg hatching. The effective concentration of SaDec that was able to inhibit 97.8% of larval development was 5 mg/ml. The effect of higher and lower concentrations of SaDec in the EHT and LDT were not significantly different from their respective positive and negative controls in both tests. (P>0.05). The EC50±95% confidence interval (CI) values were 1.4 (1.2 – 1.6) and 1.2 (1 – 1.3) mg/ml for SaDec in the EHT and LDT, respectively. The EC95±95% CI values were 6.8 (5.5 – 8.8) and 4.1 (3.4 – 5.1) mg/ml for SaDec in the EHT and LDT, respectively. The inhibition of egg hatching and larval development were dose-dependent. The results of the AWM assay are presented in Figure 1. SaDec at 1.6 mg/ml inhibited 100% of the worm motility after 12 h of exposure. This effect was not significantly different from that seen in the positive control (P<0.05). Regression analysis indicates that there is a dose-dependent effect of SaDec after 24 h of incubation. SEM revealed ultrastructural changes in the cephalic region and cuticle of female H. contortus after exposure to 0.8 mg/ml SaDec (Figure 2). Wrinkling of the cuticular ridges was observed in the cephalic region (A2) and cuticle (B2). Cuticular detachments were observed on the proximal cephalic region. Moreover, a deformation of the lancet located in the dorsal surface of the oral cavity was observed (Figure 2, A2).

3.3 In vivo tests

There was no mortality of mice in the acute toxicity test. Moreover, no change in behavior indicating a clinical condition of poisoning of the animals by SaDec was observed.

11

The FECRT results are expressed as the mean epg (± 95% CI) on days 0, 7 and 14 posttreatment (Table 2). SaDec and monepantel reduced epg by 47% and 83%, respectively, after 14 days post-treatment, indicating that both approaches presented low efficacy against the population of gastrointestinal nematodes of the evaluated sheep. Prevalence of nematode genera in the FERT is presented in Table 3. Haemonchus spp. was the most prevalent among larvae in all groups.

4. Discussion

One of the great challenges of veterinary parasitology is the search for alternative methods for controlling gastrointestinal parasites in small ruminants. In this context, the use of herbal medicine as anthelmintic has been considered for the control of GINs in small ruminants, and this alternative approach can reduce the use of anthelmintic synthetic drugs (Engströmet al., 2016). The complex composition of plant products can result in difficulties in characterizing and validating natural products as anthelmintic (Hoste et al., 2012). However, the interactions among various chemical components of a vegetable preparation may be important to slow the development of anthelmintic resistance. Plant secondary metabolites (e.g., CTs) have been considered in the control of GINs in small ruminants (Hoste et al., 2016). The CTs content can vary depending on the plant’s metabolism under the influence of biotic and abiotic factors (Pavarini et al., 2012). Furthermore, Klongsiriwet et al. (2015) demonstrated that the effect of CTs on H. contortus is associated with synergism with flavonoids. In the present study, the phytochemical analysis detected a high concentration of CTs that was greater than those described for Anadenanthera colubrina, Leucaena leucocephala and Mimosa tenuiflora leaves, which contained 40.68, 45.21 and 30.13 mg GAE/g DW, respectively (Oliveira et al., 2011). The effects of SaDec in the EHT and LDT in the present study were greater than those of the ethyl acetate extract of S. anthelmia, which inhibited 100% of the egg hatching and 81.2% of the larval development at a dose of 50 mg/ml (Assis et al., 2003). In the LDT, the LC50 of S. anthelmia aqueous and ethanolic extracts against L3 strongyles were 0.714 mg/ml and 0.628 mg/ml, respectively (Ademola et al., 2007). These values were lower than in the present study. Furthermore, a lower LC50 value in the LDT compared to that in the EHT was also shown in the present study. The possible synergistic action among the various compounds present in the S. anthelmia extracts may have been responsible for the different effects of these

12

products on H. contortus eggs and larvae, as demonstrated for Acacia pennatula and sainfoin (Onobrychis viciifolia) acetone:water extracts in the EHT (Chan-Pérez et al., 2016). In the present study, inhibition of 100% of H. contortus adult motility was achieved after incubation for 12 h at a dose of 1.6 mg/ml. The ethyl acetate extract of Calotropis procera latex at 100 µg/ml reduced H. contortus adult motility by 100% (Cavalcante et al., 2016). Crude aqueous and ethanolic extracts of Artemisia campestris at doses of 2 mg/ml reduced the in vitro motility of adult H. contortus by 71 and 100% after 24 h, respectively (Akkari et al., 2014). Although the in vitro assay was relatively brief compared to the EHT and LDT, the AWM assay provides more reliable screening results of substances with anthelmintic action in vivo. This assay is important because it assesses the in vivo effect of a product on the adult parasite, given that the in vitro effects of bioactive substances do not always correspond to anthelmintic effectiveness in the target species. More recently, SEM has been used to demonstrate direct effects of products with potential anthelmintic effects (Martínez-Ortíz-de-Montelhano et al., 2013, Yoshihara et al., 2015, Andre et al., 2016). This technique assesses the interaction of compounds with the helminth cuticle (Martínez-Ortíz-de-Montellano et al., 2013). This is especially important in the evaluation of the direct effects of CTs because although the CT mechanism of action is not completely clear, there is a high correlation between phenolic content and the anthelmintic activity of the compounds (Akkari et al., 2016). In the present study, there was accentuated wrinkling of the cuticular ridges in the cephalic region and cuticle and structural changes in the H. contortus oral cavity region. The presence of transverse and longitudinal wrinkles on the cuticle of H. contortus upon contact with sources of CTs has been described previously (Martínez-Ortíz-de-Montellano et al., 2013, Yoshihara et al., 2015). Andre et al. (2016) showed loss of the normal appearance of the vulvar flap of female H. contortus and bubbles emerging from the worm cuticle after contact with carvacryl acetate. The description of skin wrinkles in H. contortus caused by substances with potential anthelmintic effects is unanimous in the literature, and it seems plausible because the worm cuticle is a protective barrier and is involved in both motility and metabolic exchanges that occur in the digestive tract of small ruminants (Martínez-Ortíz-de-Montellano et al., 2013). The toxicological safety of SaDec was evaluated in mice. Toxicity tests in rodents are important for assessing the safety of a drug; only then is the product is tested in the target species (Camurça-Vasconcelos et al., 2005). A dose of 5,000 mg/kg did not cause any mortality and/or change of animal behavior; therefore, the product was considered safe. Although the maximum recommended dose for acute toxicity testing is 2,000 mg/kg, we used the 5,000

13

mg/kg dose to ensure that SaDec was safe for target species. This decision was based on specific guidelines for the performance of acute toxicity (OECD, 2001). The low toxicity of SaDec may be associated with the molecular weight of the CTs present in the decoction if the CTs with higher molecular weight are not absorbed (McLeod, 1974). The effectiveness of SaDec and monepantel in the FECRT were 46.8% and 83% at a dose of 350 mg/kg and 2.5 mg/ kg, respectively. These effects are remarkably low for what is proposed for anthelmintics according to World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines (Wood et al., 1995). The poor efficacy of monepantel has also been reported in sheep farms in Uruguay and in goats from New Zealand (Scott et al., 2013, Mederos et al., 2014,). Thus, more detailed studies involving the description of the mode of action of the product and its effect on the controlled anthelmintic efficacy test in sheep should be conducted so that the efficacy of SaDec as a phytotherapic with potential effects on GINs can be reconsidered. In addition, tests with new pharmaceutical formulations of SaDec, multiple doses delivery or the usage of SaDec as a nutraceutical product can be considered in the validation of its anthelmintic effect. Although these are preliminary results, the low efficacy of SaDec does not invalidate further studies that may be developed to isolate and test the CTs and/or flavonoids present in SaDec, as it demonstrated an in vitro synergistic effect of these two classes of secondary metabolites against H. contortus (Klongsiriwet et al., 2015). Promising results were observed for 500 mg/kg S. anthelmia ethanolic extract, which decreased the epg by 76, 79.8, 75 and 12.3% for Strongyloides spp., Oesophagostomum spp., Haemonchus spp. and Trichostrongylus spp., respectively, after 12 days post-treatment (Ademola et al., 2007). In the present study, the effects were milder, but it is important to note that the ethanol extract dose was higher than that used for SaDec. Haemonchus spp. and Trichostrongylus spp. were the most prevalent genera in all of the coprocultures. In the present study, SaDec reduced of Haemonchus spp. prevalence as observed in Table 3. Consequently, a relative increase in Trichostrongylus spp. prevalence was observed. This effect might be due to the influence of pH in dissociation and reconstitution of tanninprotein complexes in the different infected organs (Hagerman et al. 1982, McNabb et al. 1998, Min et al. 2003, Minho et al. 2008). The present study demonstrated the effects of SaDec against small ruminant GINs. Additional studies are necessary to isolate and identify CT subgroups present in SaDec in an attempt to optimize the results obtained because studies related to the structural features of CTs and their anthelmintic activity are scarce (Quijada et al., 2015). In addition, the present study

14

contributes to the numerous reports of plants with anthelmintic effects. The use of herbal medicines as an alternative method of controlling hemonchosis in sheep should be pursued further, as the ineffectiveness of synthetic and semi-synthetic anthelmintics due to the resistance or multiresistance of GIN populations is a worldwide problem.

Acknowledgments

We thank Dr. J. Cabaret from INRA for providing the ISE isolate of H. contortus. The authors also thank Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) (CI3-0093-001020100/14) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (458011/2014-2) for their financial support. Mr. Ribeiro received a doctoral research scholarship from Coordenação de Pessoal de Nível Superior (CAPES). Dr. Bevilaqua was supported by a fellowship from CNPq (303018/2013-5).

Conflicts of interest The authors declare that they have no conflicts of interest.

References Achenbach, H., Hübner, H., Vierling, W., Brandt, W., Reiter, M., 1995. Spiganthine, the cardioactive principle of Spigelia anthelmia. J. Nat. Prod.58, 1092–1096.

Ademola, O., Fagbemi, B.O., Idowu, S.O., 2007. Anthelmintic activity of Spigelia anthelmia extract against gastrointestinal nematodes of sheep. Parasitol Res. 101, 63–69. Akkari, H., Rtibi, K., B’chir, F., Rekik, M., Darghouth, M.A., Gharbi, M., 2014. In vitro evidence that the pastoral Artemisia campestris species exerts an anthelmintic effect on Haemonchus contortus from sheep. Vet. Res. Commun. 38, 249–255. Akkari, H., Hajaji, S., B’chir, F., Rekik, M., Gharbi, M., 2016. Correlation of polyphenolic content with radical-scavenging capacity and anthelmintic effects of Rubus ulmifolius (Rosaceae) against Haemonchus contortus. Vet. Parasitol. 221, 46–53.

15

Andre, W.P.P., Ribeiro, W.L.C., Cavalcante, G.S., dos Santos, J.M., Macedo, I.T.F., Paula, H.C.B., Freitas, R.M., Morais, S.M., Melo, J.V., Bevilaqua, C.M.L., 2016. Comparative efficacy and toxic effects of carvacryl acetate and carvacrol on sheep gastrointestinal nematodes and mice. Vet. Parasitol. 218, 52–58.

Assis, L.M., Bevilaqua, C.M.L., Morais, S.M., Vieira, L.S., Costa, C.T.C., Souza, J.A.L., 2003. Ovicidal and larvicidal activity in vitro of Spigelia anthelmia Linn. Extracts on Haemonchus contortus. Vet. Parasitol. 117, 43–49.

Athanasiadou, S., Kyriazakis, I., Jackson, F., Coop, R.L., 2001. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies. Vet. Parasitol. 99, 205–219.

Braga, R. (Ed.), 2001. Plantas do Nordeste: Especialmente do Ceará. Fundação Guimarães Duque, Fortaleza, p. 496 pp.

Cabaret, J., 2014. Reliable Phenotypic Evaluations of Anthelmintic Resistance in Herbivores: How and When Should They Be Done? In: Quick, W. (Ed.), Anthelmintics – Clinical Pharmacology uses in Veterinary Medicine and efficacy. Nova Science Publisher, New York, pp. 1–26.

Camurça-Vasconcelos, A.L.F., Melo, L.M., Nascimento, N.R.F., Teixeira, P.G.M., Menezes, D.B., Silva, R.A., Souza, I.P., Queiroz, M.G.R., Morais, S.M., Bevilaqua, C.M.L., 2005. Avaliação toxicológica do extrato acetato de etila de Spigelia anthelmia Linn, em ratos e camundongos. Rev. Bras. Ciênc. Vet. 12, 46–52.

Camurça-Vasconcelos, A.L.F., Nascimento, N.R.F., Sousa, C.M., Melo, L.M., Morais, S.M., Bevilaqua, C.M.L., M.F.G. Rocha, M.F.G., 2004. Neuromuscular effects and acute toxicity of an ethyl acetate extract of Spigelia anthelmia Linn. J. Ethnopharmacol. 92, 257–261.

Cavalcante, G.S., Morais, S.M., Andre, W.P.P., Ribeiro, W.L.C., Rodrigues, A.L.M., Lira, F.C.M.L., Viana, J.M., Bevilaqua, C.M.L., 2016. Chemical composition and in vitro activity of Calotropis procera (Ait.) latex on Haemonchus contortus. Vet. Parasitol. 226, 22–25.

16

Chan-Pérez, I., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., CastañedaRamírez, G.S., Vilarem, G., Mathieu, C., 2016. In vitro susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of two tannin rich plants. Vet. Parasitol. 217, 53–60.

Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor M.A., Waller P.J., 1992. World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44.

CONCEA, 2013. Diretrizes da prática de Eutanásia. Conselho Nacional de Controle de Experimentação Animal, Brasília, 54 pp.

Dash, K., Hall, K., Barger, I.A., 1988. The role of arithmetic and geometric worm egg counts in faecal egg count reduction test and in monitoring strategic drenching programs in sheep. Aust Vet. J. 65, 66–68.

Dos Santos, J.M.L., Monteiro, J.P., Ribeiro, W.L.C., Macedo, I.T.F., Araújo-Filho, J.V., Andre, W.P.P., Araújo, P.R.M., Vasconcelos, J.F., Freitas, E.P., Camurça-Vasconcelos, A.L.F., Vieira, L.S., Bevilaqua, C.M.L., 2017. High levels of benzimidazole resistance and β-tubulin isotype 1 SNP F167Y in Haemonchus contortus populations from Ceará State, Brazil. Small Ruminant Res. 146, 48-52.

Engström, M.T., Karonen, M., Ahern, J.R., Baert, N., Payré, B., Hoste, H., Salminen, J.P., 2016. Chemical structures of plant hydrolyzable tannins reveal their in vitro activity against egg hatching and motility of Haemonchus contortus nematodes. J. Agric. Food Chem. 64,840–851. Gaudin, E., Simon, M., Quijada, J., Schelcher, F., Sutra, F.J., Lespine, A., Hoste, H., 2016. Efficacy of sainfoin (Onobrychis viciifolia) pellets against multi resistant Haemonchus contortus and interaction with oral ivermectin: Implications for on-farm control. Vet. Parasitol. 227, 122–129.

Hagerman, A.E., Robbins, C.T., Weerasuriya, Y., Wilson, T.C., McArthur, C., 1992. Tannin chemistry in relation to digestion. J. Range Manage. 45, 57–62.

17

Hoste, H., Martinez-Ortiz-de-Montellano, C., Manolaraki, F., Brunet, S., Ojeda-Robertos, N., Fourquaux, I., Torres-Acosta, J.F.J., Sandoval-Castro. C.A., 2012. Direct and indirect effects of bioactive tannin-rich tropical and temperate legumes against nematode infection. Vet. Parasitol. 186, 18–27. Hoste, H., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Mueller-Harvey, I., Sotiraki, S., Louvandini, H., Thamsborg, S.M., Terrill, T.H., 2015. Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock. Vet.Parasitol. 212, 5–17. Hoste, H., Jackson, F., Athanasiadou, S., Thamsborg, S.M., Hoskin, S.O., 2006. The effects of tannin-rich plants on parasitic nematodes in ruminants. Trends Parasitol. 22, 253–261.

Hoste, H., Torres-Acosta, J.F.J., 2011. Non chemical control of helminths in ruminants: Adapting solutions for changing worms in a changing world. Vet. Parasitol. 180, 144–154. Hoste, H., Torres-Acosta, J.F.J., Quijada, J., Chan-Perez, I., Dakheel, M.M., Kommuru, D.S. Mueller-Harvey, I., Terrill, T.H., 2016. Interactions between nutrition and infections with Haemonchus contortus and related gastrointestinal nematodes in small ruminants. Adv. Parasitol. 93, 239–351.

Hounzangbe-Adote, M.S., Paolini, V., Fouraste, I., Moutairou, K., Hoste, H. 2005. In vitro effects of four tropical plants on three life-cycle stages of the parasitic nematode, Haemonchus contortus. Res. Vet. Sci. 78, 155–160.

Hubert, J., Kerboeuf, D., 1992. A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Vet. Rec.130, 442–446.

Hübner, H., Vierling, W., Brandt, W., Reiter, M., Achenbach, H., 2001. Minor constituents of Spigelia anthelmia and their cardiac activities. Phytochemistry. 57, 285–296.

Jackson, F., Varady, M., Bartley, D., 2012. Managing anthelmintic resistancein goats—can we learn lessons from sheep? Small Ruminant Res. 103, 3–9. Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol. 20, 477–81.

18

Karnovsky, M. J., 1965. A formaldehyde-glutaraldehyde fixate of hight osmolality for use in electron microscopy. J. Cell Biol. 27, 137–138.

Klongsiriwet, C., Quijada, J., Williams, A.R., Mueller-Harvey, I., Williamson, E.M., Hoste, H., 2015. Synergistic inhibition of Haemonchus contortus exsheathment by flavonoid monomers and condensed tannins. Int. J. Parasitol. Drugs Drug Resist.5, 127–134. Kommuru, D.S., Whitley, N.C., Miller, J.E., Mosjidis, J.A., Burke, J.M., Gujja, S., Mechineni, A., Terrill, T.H., 2015.Effect of Sericea lespedeza leaf meal pellets on adult female Haemonchus contortus in goats. Vet. Parasitol. 207, 170–175. Lifschitz, A., Ballent, M., Virkel, G., Sallovitz, J., Lanusse, C., 2014. Accumulation of monepantel and its sulphone derivative in tissues of nematode location in sheep: Pharmacokinetic support to its excellent nematodicidal activity. Vet. Parasitol. 203, 120–126.

Macedo, I.T.F., Bevilaqua, C.M.L., Oliveira, L.M.B., Camurça-Vasconcelos, A.L.F., Morais, S.M., Machado, L.K.A., Ribeiro, W.L.C. 2012. In vitro activity of Lantana camara, Alpinia zerumbet, Mentha villosa and Tagetes minuta decoctions on Haemonchus contortus eggs and larvae. Vet. Parasitol. 190, 504–509.

Macedo, I.T.F., Oliveira, L.M.B., Ribeiro, W.L.C., Santos, J. M. L., Silva, K.C., Araújo-Filho, J.V., Camurça-Vasconcelos, A.L.F., Bevilaqua, C.M.L., 2015. Anthelmintic activity of Cymbopogon citratus against Haemonchus contortus. Rev. Bras. Parasitol. Vet. 24, 268–275.

Makkar, H.P.S. (Ed.), 2003.Quantification of Tannins in Tree and Shrub Foliages. A Laboratory Manual. Springer Netherlands, Amsterdam, 56 pp. Mangan, J.L., 1988. Nutritional effects of tannins in animal feeds. Nutr. Res. Rev. 1, 209–231. Martínez-Ortíz-de-Montellano, C., Arroyo-López, C., Fourquaux, I., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., 2013. Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions. Exp. Parasitol. 133, 281–286.

19

Martínez-Valladares, M., Geurden, T., Bartram, D.J., Martínez-Pérez, J.M., Robles-Pérez, D., Bohórquez, A., Florez, E., Meana, A., Rojo-Vázquez, F.A., 2015. Resistance of gastrointestinal nematodes to the most commonly used anthelmintics in sheep, cattle and horses in Spain. Vet. Parasitol. 211, 228–233. Matos, F.J.A. (Ed.), 2009. Introdução à fitoquímica experimental. Edições UFC, Fortaleza, 150 pp.

McLeod, M.N., 1974. Plant tannins - Their role in forage quality. Nutr. Abst. Rev. 44, 803– 812.

McNabb, W.C., Peters, J.S., Foo, L.Y., Waghorn, G.C., Jackson, S.J., 1998. Effect of condensed tannins prepared from several forages on the in vitro precipitation of ribulose-1,5bisphospathe carboxilase (rubisco) protein and its digestion by trypsin (EC 2.4.21.4) and chymotrypsin (EC 2.4.21.1). J. Sci. Food Agric. 77, 201–212.

Mederos, A.E., Ramos, Z., Banchero, G.E., 2014. First report of monepantel Haemonchus contortus resistance on sheep farms in Uruguay. Parasit. Vectors. 7, 598-601. Min, B.R.; Barry, T.N.; Attwood, G.T.; McNabb, W.C., 2003. The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages: a review. Anim. Feed Sci. Tech. 106, 3–19. Minho, A.P., Gennari, S.M., Amarante, A.F.T., Abdalla, A.L., 2010. Anthelmintic effects of condensed tannins on Trichostrongylus colubriformis in experimentally infected sheep. Semin: Ciênc. Agrár. 31, 1009–1016

Monteiro, M.V.B., Bevilaqua, C.M.L., Morais, S.M., Andrade, M.L.K., Camurça-Vasconcelos, A.L.F., Campello, C.C., Ribeiro, W.L.C., Mesquita, M.A., 2011. Anthelmintic activity of Jatropha curcas L. seeds on Haemonchus contortus. Vet. Parasitol. 182, 259–263. Morais, S.M., Bevilaqua, C.M.L., Souza, J.A.L., Assis, L.M., 2002. Chemical investigation of Spigelia anthelmia Linn. used in Brazilian folk medicine as anthelmintic Rev. Bras. Farmacogn. 12, 81–82.

20

OECD, 2001. Guidelines for the testing of chemicals. Acute oral toxicity – Acute toxic class Method. 423. Organisation for Economic Cooperation and Development, Paris, 14 pp. Oliveira, L.M.B., Macedo, I.T.F., Vieira, L.S., Camurça-Vasconcelos, A.L.F., Tomé, A.R., Sampaio, R.A., Louvandini, H., Bevilaqua, C.M.L., 2013. Effects of Mimosa tenuiflora on larval establishment of Haemonchus contortus in sheep. Vet. Parasitol. 196, 341–346.

Paulino, R.C., Henriques, G.P.S.A., Moura, O.N.S., Coelho, M.F. B., Azevedo, R.A.B., 2012. Medicinal plants at the Sítio do Gois, Apodi, Rio Grande do Norte State, Brazil. Rev. Bras. Farmacogn. 22, 29-39.

Pavarini, D.P., Pavarini, S.P., Niehues, M.,Lopes, N.P., 2012. Exogenous influences on plant secondary metabolite levels. Anim. Feed Sci. Tech. 176, 5–16.

Qi, H., Wang, W.X., Dai, J.L., Zhu, L., 2015. In vitro anthelmintic activity of Zanthoxylum simulans essential oil against Haemonchus contortus. Vet. Parasitol. 2011, 223–227.

Quijada, J., Fryganas, C., Ropiak, H.M., Ramsay, A., Mueller-Harvey, I., Hoste, H., 2015. Anthelmintic Activities against Haemonchus contortus or Trichostrongylus colubriformis from Small Ruminants are influenced by structural features of condensed tannins. J. Agric. Food Chem. 63. 6346–6354.

Ribeiro J.C., Ribeiro W.L.C., Camurça-Vasconcelos A.L.F., Macedo I.T.F., Dos Santos J.M.L., Paula H.C.B., Araújo-Filho J.V., Magalhães R.D., Bevilaqua C.M.L., 2014. Efficacy of free and nanoencapsulated Eucalyptus citriodora essential oils on sheep gastrointestinal nematodes and toxicity for mice. Vet. Parasitol. 204, 243–248.

Ribeiro, W.L.C., Camurça-Vasconcelos, A.L.F., Macedo, I.T.F., dos Santos, J.M.L., Ribeiro, J.C., Pereira, V.A., Viana, D.A., Paula, H.C.B., Bevilaqua, C.M.L., 2015. In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents. Vet. Parasitol. 135, 124–129. Salminen, J.P., Karonen, M., 2011. Chemical ecology of tannins and other phenolics: we need a change in approach. Funct. Ecol. 25, 325-338.

21

Sandoval-Castro, C.A., Torres-Acosta, J.F.J., Hoste, H., Salem, A.Z.M., Chan-Pérez, J.I., 2012. Using plant bioactive materials to control gastrointestinal tract helminths in livestock. Anim. Feed Sci. Tech. 176, 192–201. Scott, I., Pomroy, B., Paul, K., Greg, S., Barbara, A., Moss, A., 2013. Lack of efficacy of monepantel against Teladorsagia circumcincta and Trichostrongylus colubriformis. Vet Parasitol. 198, 166–171. Ueno, H., Gonçalves, P.C. (Eds.), 1998. Manual para o diagnóstico das helmintoses de ruminantes. Japan International Cooperation Agency, Tokyo, 143 pp.

Vermerris, W., Nicholson, R., 2006. Isolation and Identification of Phenolic Compounds. In: Vermerris, W., Nicholson, R. (Eds.), Phenolic Compound Biochemistry. Springer Netherlands, Amsterdam, pp. 151–196.

Wood, I.B., Amaral, N.K., Bairden, K., Duncan, J.L., Kassai, T., Malone Jr., J.B. , Pankavich J.A., Reinecke, R.K., Slocombe, O., Taylor, S.M., Vercruysse, J., 1995. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine, caprine). Vet. Parasitol. 58 181–213. Yoshihara, E., Minho, A.P., Tabacow, V.B.D., Cardim, S.T., Yamamura, M.H., 2015. Ultrastructural changes in the Haemonchus contortus cuticle exposed to Acacia mearnsii extract. Semin: Ciênc. Agrár. 36, 3763–3768.

22

100 Negative control

Live worms (%)

80

IVM (100 µg/ml) 1.6 mg/ml

60

0.8 mg/ml 0.4 mg/ml

40

0.2 mg/ml 0.1 mg/ml

20

0.05 mg/ml

0 0

6

12 Time (h)

18

24

Fig. 1. The effect of a Spigelia anthelmia decoction (SaDec) on the inhibition of adult Haemonchus contortus motility. Phosphate buffered saline (1 ml) containing 4% penicillin/streptomycin was used as the negative control. The positive control was 100 µg/ml ivermectin. The error bars indicate standard deviation of the mean (P<0.05).

23

Fig. 2. A scanning electron microscopy image showing ultrastructural changes in the cephalic region and cuticle of adult female Haemonchus contortus after incubation in 1 ml phosphate buffered saline (PBS) containing 4% penicillin/streptomycin (A1 and B1) (negative control) or 0.8 mg/ml Spigelia anthelmia decoction (A2 and B2).

Table 1: Mean effects (± standard deviation) of Spigelia anthelmia decoction on Haemonchus contortus in the egg hatching test (EHT) and larval development test (LDT). Concentration (mg/ml)

EHT

LDT

10

99.3±1.1A

-

5

90.0±3.3B

97.8±2.4A

2.5

71.1±7.8C

83.3±3.8B

1.25

42.1±4.9D

50.0±4.9C

0.62

23.9±5.9E

20.4±6.1D

0.31

2.8±2.3F

2.8±2.2E

24

Negative Control

2.9±2.1F

2.2±2.5E

Positive Control

98.0±1.8A

96.4±2.2A

Capital letters denote comparisons of the means in the columns. Different letters indicate significantly different values (P<0.05). Distilled water was the negative control in both tests. The positive controls for EHT and LDT was 0.025 mg/ml thiabendazole and 0.008 mg/ml ivermectin, respectively. The EHT and LDT were performed in three repetitions with five replicates for each treatment and for each control.

Table 2. The egg counts per gram of feces (epg) and percentage efficacy ± 95% confidence interval (95% CI) of 350 mg/ml Spigelia anthelmia decoction (SaDec) in the fecal egg count reduction test (FECRT). Treated groups

Day 0

Day 7

Day 14

Mean epg

6,095Aa

5,008Aa

3,240Ba

Efficacy (%) (95% CI)

--

9 (-200 to 79)

47 (-69 to 88)

Mean epg

3,250Ab

955Bb

530Bb

Efficacy (%) (95% CI)

--

67 (-41 to 96)

83 (36 to 99)

Mean epg

2,800Ab

2,825Aa

2,980Aa

Efficacy (%) (95% CI)

--

NA

NA

SaDec

Positive control

Negative Control

Capital letters compare the means in the lines, and the lowercase letters denote comparisons of the means in the rows. Different letters indicate significantly different values (P<0.05). The positive control group (G2) was treated with 2.5 mg/kg monepantel (Zolvix®), and the negative control group (G3) received distilled water. NA = not applicable.

Table 3: Prevalence (%) of nematode genera in the fecal egg count reduction test (FECRT) on days 0, 7 and 14 post-treatment. Treated groups

Day 0

Day 7

Day 14

61

57

49

SaDec Haemonchus spp.

25

Trichostrongylus spp.

37

42

47

Oesophagostumum spp.

2

1

1

Haemonchus spp.

68

65

69

Trichostrongylus spp.

27

31

26

Oesophagostumum spp.

5

4

5

Haemonchus spp.

66

69

68

Trichostrongylus spp.

31

2

28

Oesophagostumum spp.

3

29

4

Positive control

Negative control

The SaDec group was treated with 350 mg/ml Spigelia anthelmia decoction. The positive control group (G2) was treated with 2.5mg/kg monepantel (Zolvix®), and the negative control group (G3) received distilled water.