This paper describes the in vivo anthelmintic activity of Curcuma longa hydro alcoholic rhizome extract (CHE 50:50) in naturally infected benzimidazole resistant gastrointestinal nematodes of goats maintained in a farm. Thirty non-descript goats, infected with gastrointestinal nematodes were divided into three groups of ten animals each consisting of Group A (untreated controls), Group B (ABZ at 7.5mgkg-1 body weight) and Group C (CHE at 500mgkg-1 body weight). Prior to conducting faecal egg count reduction test (FECRT), egg hatch assay (EHA) was performed by pooling samples from each group showing ED50 value 0.594 indicating high resistance to benzimidazole. Faecal egg count reduction of 64% and 66% on day 10 post-infection was observed in albendazole treated and CHE treated goats, respectively.
Helminth infections are a major cause for reducing the productivity in livestock, particularly in poor world (Githiori et al., 2006). The control of parasitic helminths in domestic animals relies largely on the use of anthelmintic drugs (Taylor et al., 2002). However, increasing reports of parasitic populations that have developed anthelmintic resistance have become increasingly common, and severely threatens the beneficial exploitations of this control strategy (Waller, 1997). The resistance menace particularly poses a great threat in the viability of organic small ruminant production system (Thamsborg and Roepstorff, 2014). In India Thiabendazole resistance was first reported in sheep from Rishikesh (Uttarakhand) (Varshney and Singh, 1976). Other reports are from Madhya Pradesh (Dixit et al., 2017), Assam (Phukan et al., 2012) Rajasthan (Maharshi et al., 2011) and Southern India (Easwaran et al., 2009, Manikkavasagan et al., 2015). Anthelmintic resistance is widespread in several states of India (Singh and Swarnkar, 2008) resulting in decreasing efficacy of the valuable control option. This has set up a stage for using alternative control options. World Health Organization has now realized that the traditional system of medicine may play an important role in the development of livestock of the third world countries (WHO, 1993). Juice of turmeric (Curcumma longa) traditionally has long been traditionally used to cure worm infections in south and Southeast Asia (Nadkarni, 1976). It has been documented that curcumin inhibit thioredoxinreductases (TrxRs) of nematehelminthes, which are essential for cell growth and survival and thereby may play a vital role in alternative medicine (Hudson et al., 2010). Therefore, the present study was undertaken to know the efficacy of hydro-alcoholic extract of Curcuma longa against benzimidazole resistant nematode parasites of goats.
Materials and Methods
Raw turmeric tubers were purchased from the local market and were identified and authenticated at the Department of Botany, Government V.Y.T. Post Graduate Autonomous College, Durg, Chhattisgarh. These rhizomes were cut into small pieces and sun dried till they became brittle or easily breakable. These were powdered and extraction was done by using Soxhlet apparatus with 50% ethanol and 50% distilled water (hydro-alcoholic) as solvent. The extract was yellowish-brown, while its consistency was semiliquid to solid and stored at 40C in air tight containers for further studies. Thirty non-descript goats of either sex, naturally infected with gastrointestinal nematodes were selected from Goat Farm of College of Veterinary Science & A.H., Anjora, Durg, Chhattigarh, India. These animals were divided in three groups, of 10 animals each. Group A animals served as untreated controls while Group B animals were subjected to oral administration of albendazole (ABZ) at 7.5mgkg-1 body weight and Group C animals with oral administration of Curcuma longa hydro ethanolic (CHE) extract (50:50) at 500mgkg-1 body weight. The particular dose and concentration of hydro-ethanol for preparation of CHE extract is as per Pande, (2011). From each group of animal fresh faecal sample were collected per rectum, pooled and subjected to egg hatch assay (EHA). The pooled sample was mixed very well and around 60-70 gm faecal sample was put in a 500 ml beaker and water added in the beaker. It was kept for 20-30 minutes so that faecal matter gets soaked and loosened and then mixed thoroughly with stirrer. After mixing faecal slurry was strained 2-3 times using tea strainer to remove the faecal debris. Strained faecal slurry was put in centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. After centrifugation supernatant was thrown from all centrifuge tubes. Sediment was well agitated and saturated sodium chloride solution was poured up to top in each centrifuge tube for floatation of parasitic eggs. Microscopic cover glasses were put on each centrifuge tube for adhering of eggs. After 10 min cover glass were held with forcep and dipped in a beaker containing distilled water. In vitro egg hatch assay (EHA) was performed on ﬁve replicates of eggs separated from pooled faeces collected on day 0, using pure thiabendazole (TBZ; Sigma, St. Louis, Missouri, USA) in a two-fold dilution series of 0.0625, 0.125, 0.25, 0.50 and 1.0 µg TBZ ml-1 (Coles et al., 1992, 2006). Eggs (n=100) per well, along with varying concentrations of TBZ, were incubated in 24-multiwell plates for 24h at 280C in a BOD incubator, followed by addition of a drop of Lugol’s iodine to each well. Unhatched eggs and larvae (L1) were counted to determine the proportion of eggs hatched. Data were analysed by Probit Analysis to obtain ED50 values for egg hatch. An ED50 value in excess of 0.1µg TBZ ml-1 was considered to indicate the emergence of benzimidazole resistance.
In vivo faecal egg count reduction test (FECRT) was performed using faecal samples collected per rectum from each animal on day 0 (pre-treatment) and day 10 (post-treatment) for Group A, B and C (Coles et al., 1992, 2006). The intensity of infection was determined by modified McMaster technique (Skerman and Hillard, 1966). The left over faecal samples were pooled on both pre-treatment and post-treatment and subjected to larval culture at room temperature for 7 days. Infective larvae were harvested and identified (Hansen and Perry, 1990). The data were analysed for faecal egg count reduction (%FECR) using RESO calculator (Martin and Wursthorn, 1991).
Result and Discussion
Observations on egg hatch assay (EHA) to know the status of benzimidazole resistance in gastrointestinal nematodes of goats revealed ED50 for egg hatch was 0.594 (Table 1), which is indicative of high degree of resistance towards benzimidazole in goats prevailed in the farm.
Table 1: Status of benzimidazole resistance in gastrointestinal nematodes of goats maintained in farm by egg hatch assay (EHA)
|In vitro EHA (For Benzimidazole only)|
|Variance of Slope||5.30E-02|
|Lower Limit of ED50||0.406|
|Upper Limit of ED50||0.909|
Earlier, Sanyal et al. (2014) observed ED50 value of 0.150 in the same farm. The results of the faecal egg count reduction test revealed 64%and 66% reduction in faecal egg counts for ABZ and CHE, respectively (Table 2). There is significant reduction in faecal egg counts in group B and C in comparison to group A on tenth day whereas there was non-significant difference between animals of Group B and C (Table 2). Various workers from India and abroad had shown variable efficacy for ABZ showing resistance for the drug which is dependent on many factors like frequency of use, abuse or extensive use, genetic changes in the worm etc. Efficacy of CHE extract is not upto the mark as per the recommendation of World Association for Advancement of Veterinary Parasitology (WAAVP) against benzimidazole resistant nematode parasites of goats but certainly this work provide vital information regarding alternative medicine for sustainable control of internal parasites.
Table 2: Faecal egg count reduction following treatment with albendazole 7.5mgkg-1 body weight and Curcuma longa hydro ethanolic (CHE) extract (50:50) at 500mgkg-1 body weight
|In vivo Faecal Egg Count Reduction Test|
|Parameter||Group A(Control)||Group B(ABZ)||Group C (CHE)|
|No. of Animals||10||10||10|
|Mean EPG (Day 0)||900||730||1683|
|Mean EPG (Day 10)||900||200||275|
Afrin et al. (2016) obtained lowered faecal egg counts and ant-inflammatory effect in Black Bengal goats using 0.2% and 0.3% curcumin for 60 days in the ration. Ullah et al. (2013) found anthelmintic effect of C. longa due to cucurbitacin as secondary metabolite. Nasai et al. (2016) observed an increase in the number of dead larvae of Haemonchus contortus over time as turmeric concentrations exhibited the highest antihelmintic activity in a dose-dependent way with the maximum effect observed at the highest dose of turmeric extract (200 mg/ml), where 78% of the worms died 24 h post-exposure. Bazh and El-Bahy (2013) screened the in vitro and in vivo activity of ginger and curcumin on Ascaridia galli. Contrary to the available published results CHE at 500mgkg-1 body weight had non-appreciable anthelmintic effect in benzimidazole resistant nematode parasites of goats and needs further investigations on its dose-dependent efficacy.
In these days where an increased resistance to conventional drugs is one of major causes of concern to livestock industry and also is the major drivers of drug discovery (Anthony et al., 2005). Recent advance and an increased understanding of phytotherapeutics together with the availability of the structural and functional information of the parasite molecules and the plant products further crusade the search for a novel anti-parasitic agent which may be affordable and sustainable.
Parasitic diseases are a global problem and considered as a major obstacle in the health and product performance of livestock. Chemotherapeutics agents are the first choice for effective control but due to drug resistance problem there is urgent need for alternative therapy. At present time many medicinal plants have been studied in traditional medicine including Ayurveda and Chinese medicine. Curcumin extracted from turmeric have anti-parasitic effects against many endo-parasites. However, the efficacy is not up to the mark but there is always scope for future research work to achieve the ultimate outcome of getting product with minimal problem of drug resistance, economical and sustainable. This present study not only validates our ancient knowledge about using herbal medicines as anthelmintic but also open up new areas for sustainable control of gastrointestinal nematodes.