Introduction
In developing countries such as India, small ruminants make an important contribution to human livelihood. Thirty-seven percent of the world's sheep population (1.2 billion) and 56% of the world's goat population (1 billion) are bred and reared in Asia (FAO, Reference Scherf and Pilling2015). Parasitic gastroenteritis accounts for heavy production losses in the small ruminant industry (Dhar et al., Reference Dhar, Sharma and Bansal1982). Amongst helminthic infections of small ruminants, infection with Haemonchus contortus has been a major threat (Swarnkar et al., Reference Swarnkar2008; Santos et al., Reference Santos, Silva and Amarante2012). The ubiquitous prevalence of H. contortus makes it a predominant helminth in both tropical and temperate climates. In India alone, the annual cost of anthelmintic treatment against H. contortus has been estimated to be USD103 million (McLeod, Reference McLeod, Sani, Gray and Baker2004). For decades, the application of broad spectrum anthelmintics has been the primary strategy for the control of Haemonchus infection. However, resistance to these anthelmintics continues to be documented in Haemonchus populations around the world, including in India (Singh et al., Reference Singh, Swarnkar and Khan2002). In earlier studies at the University goat farm in Jabalpur, levamisole and fenbendazole resistance was detected in the strongyles (Das et al., Reference Das2015). Further, the resistant nematodes were identified as H. contortus by coproculture and molecular methods, and the mechanism of fenbendazole resistance was linked to a single nucleotide polymorphism at position 200 of the β-tubulin isotype I gene (Dixit et al., Reference Dixit2017).
Rotational use of anthelmintics with different pharmacokinetics and/or herbal medicine is a possible alternative treatment to counter the heavy losses that are associated with gastroenteritis caused by nematodes resistant to the benzimidazole group of anthelmintics. Herbal products offer the advantage of sustainable supply and are ecologically acceptable. Evaluation of the anti-parasitic effect of plants with respect to H. contortus has been previously reported (Ferreira et al., Reference Ferreira2013). However, studies on the efficacy of botanicals against known drug-resistant gastrointestinal nematodes are scarce. The present study was therefore planned to determine the efficacy of a conventional dewormer (closantel) and some unexploited herbal extracts possessing anti-parasitic activity in goats infected with fenbendazole-resistant H. contortus.
Materials and methods
The herbal leaves
The plant leaves were identified and authenticated by the botanist in the Department of Botany, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, India.
Annona squamosa (sitaphal)
This is commonly known as sugar apple, custard apple or sitaphal, and is cultivated as an edible fruit throughout India.
Azadirachta indica (neem)
The tree is synonymously known as Melia azadirachta L and is commonly referred to as the Neem tree. It is a hardy tree growing to 15–20 m in height and is usually found throughout the tropics and subtropics.
Nicotiana tabacum (tobacco)
Nicotiana tabacum leaves are well-known to contain nicotine as a main alkaloid, which is probably responsible for the anti-nematicidal activity of the plant extract.
The animals
Fifty goats with faecal egg counts of >600 eggs, from whom anthelmintic medication was withheld for 8–12 weeks prior to the trial, were randomly selected and assigned to five groups of 10 animals for the experiment, irrespective of age, sex and breed. Each animal was weighed, and anthelmintics were administered on the basis of individual body weight.
Aqueous herbal extract
Leaves of A. indica, A. squamosa and N. tabacum were harvested directly and/or purchased from the local market. The leaves were dried under shade, powdered using a commercial stainless steel electrical blender and weighed. Powdered leaves of A. indica (1025 g), A. squamosa (950 g) and N. tabacum (550 g) were soaked in 6.8 l, 6.75 l and 3 l, respectively, of distilled water at room temperature (30°C). The suspension was shaken vigorously every 24 hours for 10 days. The aqueous suspension was filtered using muslin cloth. The filtrate was concentrated using a rotary vacuum evaporator until it acquired a pasty consistency, and was poured into Petri dishes. The paste was further dried at 45°C using a hot air oven, and was then stored at 4°C until use. The percentage yield of extract was calculated as follows:
$$\eqalign{& \% {\rm yield\,of\,extract} = \cr & \,\,\,\left[ {{\rm Weight\,of\,extract} \left( {\rm g} \right)/{\rm Weight\,of\,dried\,leaves} \left( {\rm g} \right)} \right] \times 100} $$The yield of aqueous leaf extract from A. indica, A. squamosa and N. tabacum was 21.96%, 34.21% and 68.19%, respectively. The extracts were administered to the animals by adding 10–15 g jaggery for easy and complete administration.
Medication schedule
Group I animals were given aqueous leaf extract of A. indica at 1 g/kg body weight (Chandrawathani et al., Reference Chandrawathani2013a), Group II animals received a single oral dose of aqueous leaf extract of A. squamosa at 1.5 g/kg body weight (Githiori et al., Reference Githiori2004) and Group III animals were given aqueous leaf extract of N. tabacum at 1 g/kg body weight (Hamad et al., Reference Hamad2013). Group IV animals were treated as a control group. Group V animals were given closantel at 10 mg/kg body weight orally.
Faecal egg counts
Faecal samples were collected from each goat on day zero (pre-treatment) and again on days 10 and 14 post treatment. Samples were collected rectally and placed in plastic bags bearing the animal's identification number, and parasitological analysis was undertaken in the laboratory. One gramme of faeces from each goat was mixed in 14 ml of saturated sodium chloride solution and the eggs were counted in one chamber of a McMaster's egg counting slide. The total egg count was multiplied by 100 (Zajac and Conboy, Reference Zajac and Conboy2012).
Faecal egg count reduction test (FECRT)
The percentage reduction in faecal egg count was calculated as per Coles et al. (Reference Coles1992) using the following formula:
$${\rm R} = 100\left( {1 - {\rm Xt}/{\rm Xc}} \right)$$where R is the percentage reduction in faecal egg count, Xt is the mean FEC of the treatment group and Xc is the mean FEC of the control group.
Faecal culture
Thirty grammes of faeces from the infected animals were mixed in a Petri dish, which was placed in an incubator at 27°C for 10 days to obtain third-stage larvae. One hundred larvae were obtained from faecal cultures and identified to genus level based on morphological characteristics such as the shape of the anterior portion and tail, as well as caudal and sheath length (Van Wyk et al., Reference Van Wyk, Cabaret and Michael2004).
Results
The mean FEC on day 10 in Groups I, II and III was 2020, 3800 and 2380, respectively, and 2570 in the untreated control group (Group IV). Comparison of FEC revealed no reductions on day 10 post infection in animals treated with herbal extracts. The mean FEC of the control group (Group IV) and closantel-treated individuals (Group V) on day 14 post infection was 2750 and 120, respectively. FEC reduction and lower 95% confidence interval for closantel were 95.64 and 87, respectively (Table 1). Haemonchus contortus (81%), Strongyloides spp. (8%), Oesophagostomum spp. (6%), Trichostrongylus spp. (4%), and other larvae (1%) were identified in the pre-treatment faecal cultures. Post-treatment coproculture in the closantel-treated group revealed Strongyloides spp. (54%), Oesophagostomum spp. (38%), Trichostrongylus spp. (6%) and H. contortus (2%). Evidently, the H. contortus strain of goats in Central India was found to be susceptible to closantel.
Table 1. Mean faecal egg count (FEC) and FEC reduction (FECR, %) in goats treated with three botanical aqueous extracts and closantel.
Discussion
Resistance in H. contortus in Central India to fenbendazole was documented recently using allele-specific polymerase chain reaction (PCR). The frequency of resistant allele (r) was quite high (74%) incidental to widespread occurrence of resistance to benzimidazoles (Dixit et al., Reference Dixit2017). Consequently, closantel is currently used widely to control the parasite in small ruminants. Clearly, closantel has been a drug of choice, being an anthelmintic in the salicylanilide drug class. Salicylanilides become highly bound to plasma proteins, and therefore they specifically target parasites that ingest blood, such as H. contortus. Salicylanilides uncouple oxidative phosphorylation, decrease the availability of adenosine triphosphate and nicotinamide adenine dinucleotide in the mitochondria and thus decrease the energy available to the parasites. Closantel also disrupts mechanisms that maintain pH homeostasis in the parasite (Lanusse et al., Reference Lanusse, Guillermo, Alvarez, Riviere and Papich2009). However, periodically, reports have suggested developing resistance to closantel as well (Chandrawathani et al., Reference Chandrawathani2013b; Premaalatha et al., Reference Premaalatha2014). According to Coles et al. (Reference Coles1992), if FEC reduction is <95% and the lower confidence limit <90%, only then is it considered to be resistant. If only one of these two criteria is fulfilled, then resistance is suspected. In our study the FEC reduction was above 95% and the lower confidence limit was below 90%. Therefore, as per the guidelines of the World Association for the Advancement of Veterinary Parasitology, it should be considered to be a case of suspected resistance. Closantel is an anthelmintic with persistent effect only against haematophagous parasites such as H. contortus. After treatment with closantel, Haemonchus larvae were reduced from 81% to 2%. Therefore, when the percentage FEC reduction was calculated only for H. contortus using RESO software, it was found to be susceptible to closantel, which is similar to observations reported by Westers et al. (Reference Westers2016).
In the present study, following administration of a single dose of crude aqueous extract of N. tabacum, a 7% reduction in FEC was found on day 10 post treatment, whereas Hamad et al. (Reference Hamad2013) reported 87.5% and 88.6% reductions in sheep on day 14 post treatment using crude aqueous methanolic extract of N. tabacum at 2 and 4 g/kg body weight, respectively, against benzimidazole-resistant H. contortus. In our study, the comparatively smaller reduction in FEC may be due to the difference in solvents used (water vs 70% methanol), host species (goat vs sheep), dosage (1 g vs 2 g) and assessment days post treatment (10 vs 14). Nicotiana tabacum leaves contain nicotine, which may cause spastic paralysis of worms via action on nicotinic receptors. Levamisole is also known to stimulate nicotinic receptors for exhibiting anthelmintic activity. In our study, the nematodes were both levamisole- and fenbendazole-resistant, whereas Hamad et al. (Reference Hamad2013) studied benzimidazole-resistant H. contortus only. This may also explain the non-effectiveness of N. tabacum extract as the gastrointestinal nematodes were also levamisole-resistant.
Iqbal et al. (Reference Iqbal2006) studied the effect of N. tabacum aqueous and methanolic extract on H. contortus in sheep and found that the aqueous extract was comparatively less effective. In addition, the efficacy of aqueous extract was dose dependent and increased efficacy was evident when 3 g/kg body weight was used instead of 1 or 2 g/kg body weight. Interestingly, the anthelmintic effect was rapid. It was observed at 5 days post treatment but was not evident at 10 days post treatment after a single dose of N. tabacum extract.
The efficacy of A. indica administered to small ruminants as fresh leaves (Chandrawathani et al., Reference Chandrawathani2006; Chagas and Vieira, Reference Chagas and Vieira2007) and crude leaf powder (Akhtar and Riffat, Reference Akhtar and Riffat1984; Dongre et al., Reference Dongre2015) was variable. In an experiment, a significant reduction in the number of worms was recorded among sheep that received 3 g/kg of fresh neem leaves for 6 weeks, on necropsy, compared to the control group, although this was not reflected in a reduction in the FEC (Chandrawathani et al., Reference Chandrawathani2006). Chagas and Vieira (Reference Chagas and Vieira2007) reported another experiment that revealed no anthelmintic effect of neem at a dosage of 30 g of dried leaves per goat/day given for 5 days. In the present study, compared to the control group, only a 21% reduction in FEC was observed at day 10 post treatment in the group treated with A. indica. Similar to our findings, Worku et al. (Reference Worku, Franco and Miller2009) reported that aqueous leaf extract containing water-soluble proteins from neem was not an effective anthelmintic in goats. Nawaz et al. (Reference Nawaz2014) reported 89% efficacy of aqueous extract of A. indica leaves against H. contortus in sheep 12 days post treatment. This is the only study whose findings are dissimilar to our own.
Investigations carried out with different species of the genus Annona have shown that aqueous leaf extracts of Annona senegalensis (Ndjonka et al., Reference Ndjonka2011) and A. muricata (Ferreira et al., Reference Ferreira2013), and methanol and ethyl acetate leaf extracts of A. squamosa (Kamaraj and Rahuman, Reference Kamaraj and Rahuman2011) demonstrate in vitro anthelmintic activity against different nematodes, including H. contortus. The anthelmintic activities of Annona extracts have been attributed to the annonaceous acetogenins, a class of natural compounds extracted from leaves and seeds. Ferreira et al. (Reference Ferreira2013) indicated the presence of phenolic compounds in the aqueous leaf extract of A. muricata. In our study, aqueous leaf extract of A. squamosa has shown no effect on fenbendazole-resistant H. contortus when used in vivo in goats. However, compounds or substances that are effective in vitro do not necessarily work equally well in vivo. In addition, acetogenins may not be extracted effectively from a plant using water as a solvent. Vieira et al. (Reference Vieira1999) administered A. squamosa at 1 g/kg for four consecutive days and found a 51.90% reduction in the adult Oesophagostomum columbianum population, although it was ineffective in eliminating H. contortus, Trichostrongylus colubriformis and Strongyloides papillosus in goats. Githiori et al. (Reference Githiori2004) evaluated in vivo anthelmintic efficacy of fresh leaves of seven plants, including A. squamosa and A. indica, and found no significant difference in FEC 2–3 weeks post treatment in lambs. These findings are consistent with the observations of the anti-parasitic efficacy of herbal extracts reported here.
In conclusion, the rotational use of closantel is the preferred choice over the benzimidazole group of anthelmintics and/or herbal extracts to combat the problem of in situ development of drug-resistant gastrointestinal nematodes, especially H. contortus, and to ensure increased productivity in small ruminants in Central India.
Acknowledgements
The authors are grateful to the Dean of the College of Veterinary Science and Animal Husbandry NDVSU, Jabalpur, for providing the facilities to enable this study to be undertaken.
Financial support
The authors thank ICAR, New Delhi, for financial assistance under the All India Network Programme on Gastrointestinal Parasitism.
Conflict of interest
None.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guidelines on the care and use of laboratory animals.
