Introduction
Sheep production is an attractive source of income for farmers in the Kashmir Valley, due to low capital input and the ability of sheep to thrive on native pasture grasses. These factors and a high demand for sheep meat has led to a tremendous growth of the sheep industry in the Kashmir Valley. However, productivity is constrained by parasitic infections (Dhar et al., Reference Dhar, Sharma and Bansal1982; Tariq et al., Reference Tariq, Chishti, Fayaz and Shawl2006). Infections with gastrointestinal nematodes in sheep are responsible for specific clinical signs and can cause severe economic losses and endanger animal welfare. Reduced feed intake is a well-known feature of trichostrongyle infections in ruminants (Coop & Holmes, Reference Coop and Holmes1996). Clinical parasitic gastroenteritis is characterized by anaemia, abortion, anorexia, dull hair coat, loss of blood and plasma proteins, alterations in protein, energy and mineral metabolism, changes in water balance, diarrhoea, reduced body weight gain, wool growth and reproduction, and death due to secondary infections; the consequences of this are changes in body composition and carcass quality (Fox, Reference Fox1993), resulting in great economic losses to the farmers and livestock industry.
A number of control measures to combat the helminth infections of ruminants are available nowadays. The main methods for control of nematodes are prophylactic treatment with synthetic anthelmintics. The perceivable drug residues in animal products, the increasing prevalence of anthelmintic-resistant strains of helminths and high cost of conventional anthelmintics is alarming. For the sheep industry, this situation has now reached a crisis point in some countries (Waller, Reference Waller1997). There is an urgent need for the development of sustainable alternatives to anthelmintics in ruminant production systems (Athanasiadou et al., Reference Athanasiadou, Tzamaloukas, Kyriazakis, Jackson and Coop2005). Considerable research has shown that some plants not only affect the nutrition of animals, but also have antiparasitic effects (Waghorn & McNabb, Reference Waghorn and McNabb2003). The vast majority of studies investigating the effects of plant extracts on helminth parasites, either in experimental or in grazing conditions, have been conducted using sheep (Sharma et al., Reference Sharma, Bahga and Srivastava1971; Niezen et al., Reference Niezen, Waghorn and Charleston1998; Molan et al., Reference Molan, Waghorn, Min and McNabb2000; Athanasiadou et al., Reference Athanasiadou, Kyriazakis, Jackson and Coop2001; Githiori et al., Reference Githiori, Hoglund, Waller and Baker2002, Reference Githiori, Hoglund, Waller and Baker2003, Reference Githiori, Hoglund, Waller and Baker2004, Reference Githiori, Athanasiadou and Thamsborg2006; Iqbal et al., Reference Iqbal, Lateef, Ashraf and Jabbar2004, Reference Iqbal, Lateef, Jabbar, Mohammad and Khan2005, Reference Iqbal, Lateef, Akhter, Gayur and Gilani2006a, Reference Iqbal, Lateef, Jabbar, Ghayur and Gilanib, Reference Iqbal, Lateef, Khan, Jabbar and Akhterc; Eguale et al., Reference Eguale, Tilahun, Debella, Feleke and Makonnen2007). Various plant species have been screened for anthelmintic efficacy against different helminth parasites of ruminants. Phytochemicals such as isoflavones (irisflorentin, irigenin, junipigenin-A, iridin) and stilbene glycoside (piceid) have been isolated from the rhizomes of Iris hookeriana (Shawl et al., Reference Shawl and Dar1985). On the basis of its constituents, the present study was carried out with the objective of evaluating the anthelmintic efficacy of I. hookeriana rhizome against gastrointestinal nematodes of sheep (Ovis aries Linn.) under in vitro and in vivo conditions, in comparison with the synthetic anthelmintic, albendazole. The present study is the first scientific study of this nature in this part of the world.
Materials and methods
Iris hookeriana Foster (Iridaceae), locally called mazarmund, is a small herb with long stem, slender rhizome, short perianth tube and flowers blue-purple with darker blotches, found on open grassy slopes at 2400–3300 feet (730–1000 m) (Polunin & Stainton, Reference Polunin and Stainton1985). It differs from other Iris species in having one or several blotched flowers. The plant material (rhizome) was collected from subalpine meadows of the Gulmarg area of the Kashmir Valley in late July 2005, identified and authenticated by a botanist in Department of Botany, University of Kashmir, Srinagar, India.
The collected plants were processed for shade drying in a well-ventilated room (drying room, Indian Institute of Integrative Medicine, Sanat Nagar, Srinagar). The dried plant parts were milled to a fine powder using an electric blender. The powdered plant material was stored in an airtight container/cellophane bags at 4°C until extraction.
Preparation of ethanolic extracts
The ethanolic extracts were prepared by dissolving 200 g of powdered plant material in a conical glass percolator to which 1000 ml of 95% ethanol (EtOH) was added. Plant material was allowed to macerate for 16 h at room temperature and the percolate was collected by filtering through non-absorbent cotton wool. The process of maceration/percolation was repeated three times ( × 1000 ml). The combined filtrate was evaporated in a vacuum rotary evaporator under reduced pressure at 50°C to give a crude EtOH extract. The extract was scrapped off and transferred to a container and kept air tight for storage at 4°C until further use.
Preparation of aqueous extracts
Aqueous extracts were prepared by dissolving 100 g of powdered plant material in 500 ml of distilled water in a glass percolator. Plant material was allowed to macerate for 24 h at room temperature and the brew was filtered using Whatman No. 1 filter paper. The process of percolation was repeated three times ( × 500 ml). The combined filtrate was then concentrated in a water bath until the solvent had evaporated. The final crude aqueous extract was transferred to a vial and kept air tight for storage at 4°C until further use.
In vitro anthelmintic activity
For the evaluation of anthelmintic activity of the crude aqueous and ethanolic extracts under in vitro conditions, the method adopted was a worm motility inhibition assay as described by Iqbal et al. (Reference Iqbal, Lateef, Ashraf and Jabbar2004). In vitro anthelmintic activity was assessed on adult Trichuris ovis worms, to determine the inhibitory effect of extracts against adult worms. Mature T. ovis worms were collected from intestines of freshly slaughtered sheep in the local abattoir of Hazratbal, Srinagar. The worms were washed and finally suspended in phosphate-buffered saline (PBS: 0.1 m phosphate, 0.05 m NaCl, pH = 7.2). Twenty worms were exposed to each of the following treatments in separate Petri dishes at room temperature:
(1) albendazole at 0.55 mg ml− 1 (positive control);
(2) crude aqueous extract (CAE) at 25 mg ml− 1;
(3) crude ethanolic extract (CEE) at 25 mg ml− 1;
(4) PBS (negative control).
Crude aqueous extracts (CAE) were diluted in distilled water and crude ethanolic extracts (CEE) in dimethyl sulphoxide (DMSO). There were five replicates for each treatment.
The inhibition of motility of the worms was used as the criterion for anthelmintic activity. The motility was observed at 0, 1, 2, 4 and 8 h intervals. Percentage worm motility inhibition (%WMI) was determined according to Rabel et al. (Reference Rabel, McGregor and Douch1994) using the following formula: [(number of mobile worms in negative control Petri dish − number of mobile worms in treatment Petri dish)/number of mobile worms in negative control Petri dish] × 100.
The mortality index (MI) was calculated by the following formula: total number of immobile worms/total number of worms per Petri dish.
The 50% lethal concentration (LC50) of the individual plant extracts tested for anthelmintic activity was calculated by the following formula, as given by Assis et al. (Reference Assis, Bevilqua, Morais, Vieira, Costa and Souza2003): [number of living (mobile) worms/total number of worms in Petri dish (plant extract)]/[number of living (mobile) worms/total number of worms in control Petri dish (PBS)].
In vivo anthelmintic activity
The in vivo trials were performed by adopting the faecal egg count reduction (FECR) assay. Sheep of both sexes (female and male young stock ≤ 1 year), were used for in vivo trials. Before the start of the experiment the animals were confirmed to be naturally infected with mixed species of gastrointestinal nematodes by qualitative and quantitative faecal examination using standard parasitological procedures (Soulsby, Reference Soulsby1982). Only those animals whose egg counts exceeded 750 eggs per gram of faeces (EPG) were used (Ademola et al., Reference Ademola, Fagbemi and Idowu2005).
The sheep used for experiment were randomly divided into four groups of five animals each on the basis of faecal egg counts and assigned to different treatments. Group 1 received a single oral dose of 1.0 g kg− 1 body weight of extract. Group 2 received single oral dose of 2.0 g kg− 1 body weight of extract. Group 3 was a positive control which was given a single dose of albendazole orally at 5 mg kg− 1 body weight (Ethicare Animal Health, New Delhi, India; trade mark aldazole-2.5). Group 4 served as negative control and received no treatment. Faecal samples of each group were collected in the morning, starting from day 0 post-treatment (PT) and at days 5, 10 and 15 PT. The eggs were counted by Stoll's dilution method. Egg count per cent reduction (ECR%) was calculated using the following formula: [(pre-treatment egg count per gram − post-treatment egg count per gram)/pre-treatment egg count per gram] × 100.
The data were analysed using Statistical packages MINITAB software version 13.2 (Minitab Inc., Pennsylvania, USA). In all the analyses, the confidence level was held at 95% and P < 0.05 was set for significance.
Results and discussion
The results are presented separately for in vitro and in vivo evaluation of anthelmintic activity of I. hookeriana rhizome against gastrointestinal nematodes of sheep. The ethanol solvent was chosen in the present study because it extracts compounds with a wide range of polarities, is volatile and has relatively low toxicity (Farnsworth, Reference Farnsworth1990).
In vitro anthelmintic activity
The results of in vitro anthelmintic activity of CAE and CEE of I. hookeriana compared with albendazole against T. ovis were close to significance (P = 0.06). Crude aqueous extract compared with PBS was statistically non-significant (P>0.05) in its activity against T. ovis. Crude ethanolic extract compared with PBS was statistically significant (P = 0.05). The CAE and CEE of I. hookeriana exhibited time-dependent anthelmintic activity against T. ovis of sheep, as revealed from the inhibition of motility and/or death of the worms (table 1). The CAE of I. hookeriana resulted in mean %WMI of 54.0%, while CEE resulted in mean %WMI of 84.6%, as observed after the worms were put in PBS for 30 min after exposure to different treatments. The mean MI of CAE was 0.55 while for CEE it was 0.85. The LC50 for aqueous extracts was 0.45 mg ml− 1 and for ethanolic extracts was 0.15 mg ml− 1. Thus CEE exhibited greater anthelmintic activity, which could be due to the presence of alcohol-soluble active principle(s) in I. hookeriana. The worms that were exposed to albendazole were found paralysed and/or dead at 8 h (100% mortality or paralysis); whereas the worms that were kept as untreated negative controls in PBS did not show any paralysis or mortality.
In vitro anthelmintic efficacy of crude aqueous and crude ethanolic extracts of Iris hookeriana on Trichuris ovis of sheep.
CAE, Crude aqueous extracts; CEE, crude ethanolic extracts; h, hour; SEM, standard error of mean.
aWorms were exposed to phosphate-buffered saline for 30 min after exposure to the different treatments to confirm their mortality.
bPBS (phosphate-buffered saline) indicates negative control.
Our observations on in vitro anthelmintic activity of I. hookeriana against T. ovis of sheep are supported by various studies where various plant species have been screened for anthelmintic efficacy using the same methods under in vitro conditions against different gastrointestinal nematodes of sheep. Iqbal et al. (Reference Iqbal, Lateef, Jabbar, Mohammad and Khan2005) reported higher in vitro anthelmintic activity in crude aqueous extracts as compared to crude methanol extracts and crude powder against Haemonchus contortus, which they considered as an indication of presence of water-soluble active ingredients in Calotropis procera latex. Iqbal et al. (Reference Iqbal, Lateef, Khan, Jabbar and Akhter2006c) also revealed significant in vitro anthelmintic activity (P ≤ 0.05) of crude aqueous extracts and crude methanol extracts of Swertia chirata against H. contortus. Garg & Atal (Reference Garg and Atal1963) also reported in vitro paralysis of Oesophagostomum columbianum and Bunostomum trigonocephalum 4 and 6 h, respectively, after treatment with 1% calotropin isolated from the latex of C. procera. Al-Qarawi et al. (Reference Al-Qarawi, Mahmoud, Sobaih, Haroun and Adam2001) observed a concentration-dependent larvicidal activity against H. contortus in vitro within 20 min of application of C. procera latex. Khobragade et al. (Reference Khobragade, Jangede and Maske1994) found that crude aqueous extracts of Allium sativum caused complete cessation of motility (death) of B. trigonocephalum after 6, 12, 16 and 20 h of exposure in concentrations of 200, 100, 50, and 25 mg ml− 1, respectively. Results of the present study suggest that crude aqueous and ethanolic extracts of I. hookeriana exhibit significant in vitro anthelmintic activity against T. ovis of sheep, and have the potential to contribute to the control of gastrointestinal nematode parasites of sheep.
In vivo anthelmintic activity
The in vivo anthelmintic activity of crude aqueous and ethanolic extracts of I. hookeriana in sheep naturally infected with mixed species of gastrointestinal (GI) nematodes demonstrated moderate anthelmintic activity at both of the doses and extracts tested. The animals were suffering from mixed GI nematode species, including H. contortus, T. ovis, Ostertagia circumcincta, B. trigonocephalum and O. columbianum. The CAE resulted in maximum of 31.53% reduction in faecal egg counts on day 10 post-treatment (PT) with 1 g kg− 1 (table 2). The maximum (45.62%) ECR was recorded in sheep treated with CEE at 2 g kg− 1 on day 10 PT, closely followed by CEE at 1 g kg− 1 (43.54%) on day 10 PT (table 3). From day 10 onwards the egg counts again showed a tendency to increase in both CAE- and CEE-treated animals.
Mean±SEM of in vivo anthelmintic activity of crude aqueous extracts (CAE) of Iris hookeriana in sheep naturally infected with mixed species of gastrointestinal nematodes.
Figures in parentheses indicate egg count per cent reduction (ECR%) after treatment. SEM, standard error of the mean.
Mean±SEM of in vivo anthelmintic activity of crude ethanolic extracts (CEE) of Iris hookeriana in sheep naturally infected with mixed species of gastrointestinal nematodes.
Figures in parenthesis indicate egg count percent reduction (ECR%) after treatment. SEM, standard error of mean.
Our results on in vivo anthelmintic efficacy of I. hookeriana against gastrointestinal nematodes of sheep were again in agreement with the findings of other authors who have worked on the anthelmintic activities of some other plant extracts in other parts of the world. In Pakistan, Iqbal et al. (Reference Iqbal, Lateef, Akhter, Gayur and Gilani2006a) showed that crude powder and aqueous extract of ginger exhibited a dose- and a time-dependent anthelmintic activity against GI nematodes of sheep, with a maximum reduction in eggs per gram of faeces of 25.6% and 66.6%, respectively, on day 10 post-treatment. Seeds of Butea monosperma administered as crude powder at doses of 1, 2 and 3 g kg− 1 body weight to sheep naturally infected with mixed species of GI nematodes also exhibited significant anthelmintic effects (Iqbal et al., Reference Iqbal, Lateef, Jabbar, Ghayur and Gilani2006b). Iqbal et al. (Reference Iqbal, Lateef, Khan, Jabbar and Akhter2006c) also revealed significant in vivo anthelmintic activity (P ≤ 0.05) of ethanolic and methanolic extracts of Swertia chirata against mixed species of GI nematodes of sheep. Iqbal et al. (Reference Iqbal, Lateef, Jabbar, Mohammad and Khan2005) recorded ECR% of 88.4 and 77.8% in sheep (naturally suffering from mixed species of H. contortus, Trichostrongylus colubriformis, Trichostrongylus axei, Strongyloides papillosus and T. ovis) treated with crude aqueous extracts and crude powder of C. procera at 3 g kg− 1 body weight on days 7 and 10 post-treatment, respectively. Ademola et al. (Reference Ademola, Fagbemi and Idowu2005) revealed anthelmintic action of Spondias mombin extracts on Haemonchus spp., Trichostrongylus spp., Oesophagostomum spp., Strongyloides spp. and Trichuris spp. of sheep, and a reported maximum reduction (78.4%) in eggs per gram of faeces on day 10 post-treatment with 3 g kg− 1 body weight. Infected sheep drenched with Quebracho extract from Schinopsis had lower faecal egg count post treatment (P < 0.05) as compared to control (Athanasiadou et al., Reference Athanasiadou, Kyriazakis, Jackson and Coop2000). Githiori et al. (Reference Githiori, Hoglund, Waller and Baker2003) observed a significant reduction in faecal egg counts of H. contortus of sheep after treatment with water extracts of Albizia anthelmintica, Olea europaea, Annona squamosa and Ananas cosmosus.
The animals treated with albendazole showed decreased faecal egg counts from the first day of treatment, to 97.76% ECR at day 15 post-treatment, depicting significant anthelmintic activity of albendazole. Thus although I. hookeriana exhibited anthelmintic activity under in vivo conditions against mixed species of gastrointestinal nematodes of sheep, the results were not comparable to the effects of albendazole, which resulted in 97.76% reduction in faecal egg counts. Thus synthetic anthelmintics possessed greater anthelmintic activity as compared to the plant extracts tested in the present study. Lateef et al. (Reference Lateef, Iqbal, Khan, Akhtar, Akhtar and Jabbar2003) found that although aqueous and methanolic extracts of roots of Adhatoda vesica possessed anthelmintic activity against H. contortus under in vivo conditions, this was not comparable with the effects of levamisole. Iqbal et al. (Reference Iqbal, Lateef, Ashraf and Jabbar2004) also observed that although whole plants of Artemisia brevifolia possessed anthelmintic activity against GI nematodes of sheep, the activity was not comparable with that of levamisole, which resulted in 99.2% reduction in faecal egg count. In vivo controlled studies have showed that plant remedies have, in most instances, caused less pronounced reductions in the level of parasitism in sheep than those due to the use of anthelmintic drugs, and only rarely have reductions similar to those caused by synthetic anthelmintics been achieved (Githiori et al., Reference Githiori, Athanasiadou and Thamsborg2006). Therefore, there is the need for identification of active principles from plant extracts, so that standardization of dose and toxicity studies for drug development can be carried out.
According to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines, a faecal egg count reduction (FECR) of 99% or higher is considered to be highly efficacious, whereas a reduction of 80% or around is just adequate (Githiori et al., Reference Githiori, Athanasiadou and Thamsborg2006). In the present study crude aqueous and ethanolic extracts of the rhizome of I. hookeriana could not produce more than 45.62% reduction in faecal egg counts at either of the doses tested. The dosage had no significant effect on the anthelmintic efficacy of I. hookeriana against gastrointestinal nematodes in sheep. When considering the WAAVP guidelines, it can be concluded that I. hookeriana does possess anthelmintic efficacy, but that it does not fall into the two above-mentioned categories.
The physiology and resistance status of a parasite could play a significant role in evaluating the anthelmintic efficacy of the plant extracts. Therefore, the present study would obviously have had different results if the in vitro work had used H. contortus or some parasite other than T. ovis. Certainly, previous research has shown that plant extracts affect some nematode parasites and have no anthelmintic effect on other nematodes. For example, Felix et al. (Reference Felix, Dieter, Veronika, Markus and Hubertus2007) showed that administration of chicory (Cichorium intybus), birdsfoot trefoil (Lotus corniculatus), sainfoin (Onobrychis viciifolia) or a ryegrass/lucerne mixture (control) for 17 days was associated with significant reductions of total daily faecal egg output specific to H. contortus (chicory: 89%; birdsfoot trefoil: 63%; sainfoin: 63%; all tests P < 0.05). However, no anthelmintic effects were found against another nematode parasite, Cooperia curticei.
The animals used in the present study were suffering from naturally mixed species of H. contortus, T. ovis, O. circumcincta, B. trigonocephalum and O. columbianum. The FECR was calculated collectively for all the parasites. There was the possibility that I. hookeriana may be affecting some parasites and not others in a mixed infection. Previous research has shown that some plant extracts were quite active against infection by one parasite species, but could not pose any effect against mixed infections of gastrointestinal nematodes in sheep. For example, Muraleedharan et al. (Reference Muraleedharan, Sahadev, Nagasampagi and Joshi2003) observed a reduction in faecal egg count in H. contortus of goats on day 14 after treatment with Neemrich 180-EC, an oil-based product extracted from neem (Azadirachta indica) seeds. However, Costa et al. (Reference Costa, Bevilaqua, Maciel, Camurca-Vasconcelos, Morais, Monteiro, Farias, da Silva and Souza2006) reported that leaves of A. indica had no anthelmintic effect against mixed gastrointestinal nematodes of sheep. Similarly, Marley et al. (Reference Marley, Fraser, Fychan, Theobald and Jones2005) have shown that lambs grazed on red clover (Trifolium pratense) or white clover (Trifolium repens), but not lucerne (Medicago sativa), had lower pooled mean faecal egg count as compared to lambs grazing ryegrass (Lolium perenne), and concluded that legume forages have the potential to contribute to the control of abomasal but not small intestine nematode parasites in finishing lamb systems.
In conclusion, the results from the present study on I. hookeriana against gastrointestinal nematodes of sheep suggest an alternative to the use of commercially available anthelmintics for the treatment of gastrointestinal nematodiasis in sheep. There is a need to undertake detailed phytochemical and pharmacological studies of the anthelmintic activity of I. hookeriana. Research is recommended for anthelmintic studies on experimentally induced infections of particular species of gastrointestinal nematodes.
Acknowledgements
We are grateful to the Council of Scientific and Industrial Research, New Delhi, India for financial assistance. We are also highly thankful to Mr Rafiq Ahmad for his valuable contribution to plant collection and allocation of animals.
