THE EFFICACY OF DIFFERENT ANTHELMINTIC BRANDS SOLD ON ETHIOPIAN MARKETS

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1 THE EFFICACY OF DIFFERENT ANTHELMINTIC BRANDS SOLD ON ETHIOPIAN MARKETS Yidnekachew Haramaya University, College of Veterinary Medicine, Haramaya, Ethiopia ABSTRACT A study was conducted to evaluate the efficacy of different anthelmintic brands sold on Ethiopian markets using the feacal egg count reduction test (FECRT) and controlled anthelmintic efficacy trial on Haramaya University goat and sheep farm in eastern Ethiopia. The 70 goats and 70 sheep were taken from 210 goats and 320 sheep systematic random sampling method and assigned to treat groups with three different ivermectins and three different albendazoles (albendazole, ivermectin and untreated control) from different manufacturers. Each treatment group included 10 goats and 10 sheep and treatments were administered according to weight of each goat and sheep with albendazole 7.5 mg/kg body weight, and ivermectin 0.2 mg/kg body weight as recommended by the manufacturers. Feacal samples were collected on day 0 before treatment, and again on day 14 post treatment. Efficacies of all the drugs were assessed on day 14 post-treatment by feacal egg count reduction test (FECRT). In goat farm after treatment the efficacy of ivermectin in all 3 groups (IU, ICHY, and ICHJ) was observed to higher than 99% (99-100%), while the effecacy of albendazole in all 3 groups (ALI, ACHT, ACHQ) was low, show by the low FECR, which was between 87-89% and In goat farm after treatment the efficacy of ivermectin in all 3 groups ( IU, ICHY, and ICHJ) was observed to higher than 97% (97-99%), while the effecacy of albendazole in all 3 groups (ALI, ACHT, ACHQ) was low, show by the low FECR, which was between 85-89%. Individual worm counts were done from feacal cultures very few Trichostrongylus spp. showed in samples taken from ivermectin treated animals and Trichostrongylus spp., Hemonchus spp., Oesophagostomum spp. Strongyoides spp. Bunostomum spp. Nematodirus spp. and Trichuris spp. were examined in samples taken from animals treated by albendazoles from different manufacturers. As we saw in these study ivermectins were more efficacious than albendazoles of different brands in both sheep and goat flock. Large scale studies, however, are needed to assess the current status of anthelmintic efficacy of the most commonly used anthelmintics which are sold on Ethiopian market in different species of animals and management systems in Ethiopia. Keywords : Anthelmintics, efficacy, Ethiopia, goat, nematode parasites, sheep. 1.INTRODUCTION Small ruminant livestock, such as sheep and goats, are extremely susceptible to internal parasites, especially gastrointestinal nematodes. Nematode parasites of small ruminants are known to be prevalent in various agro-ecological zones of Ethiopia causing significant losses. 59

2 About 80% of the national sheep population was reported to harbor varying degrees of infection with different species of nematode parasites (Asegede, 1990). Anthelmintics, drugs that remove the parasite from the intestines, are the most common method for managing helmintic parasites. Chemical anthelmintics are often used because they are cheap, simple and cost effective; but parasite resistance to them is growing (Stear et al., 2007; Yaday et al., 1995). Ivermectin as well as albendazole and fenbendezol (both benzimidazoles) have produced the highest levels of resistance, and resistance with levamisole and moxidectin is increasing (Burke, 2005; Schoenian, 2013). Among the main factors that are thought to contribute to the development of resistance are: frequent anthelmintic treatments; use of anthelmintics with a similar mode of action for several years; treatments when parasites have a small refugium; underdosing and other management practices such as frequent stock relocation after treatment, Under dosing was probably also common on some of the study farms (Chaka et al., 2008, Maingi et al., 1996). Recent attempts towards privatization of veterinary services in Ethiopia have resulted in a remarkable increase in veterinary pharmaceutical importers and distributors. This in turn contributed to the importation of different anthelmintic brands, mainly from the benzimidazole, imidazothiazole and macrocyclic lactone groups, under different trade names, from different manufacturers (Chaka et al., 2008). Despite the high use of benzimidazole, imidazothiazole and macrocyclic lactone groups in Ethiopia there are scarce of comprehensive reports on the efficacy of theses anthelmintics against economically important parasites. Periodical evaluation of commonly used anthelmintic is vital to know the efficacy and either resistance develop or not. With the above motive, the purpose of this study is therefore to determine and compare the efficacy of benzimidazole, imidazothiazole and macrocyclic lactone groups against economically important parasites of small ruminant in the study area. 2. MATERIAL AND METHODS 2.1. Study Animal and Study Design Randomized complete block design was employed for this field experimental study (Gomez and Gomez, 1984). The 70 goats and 70 sheep were taken from 210 goats and 320 sheep through systematic random sampling method and blocked in to seven treatment groups. Each groups included 10 goats and 10 sheep. On day 0, fecal samples were collected from each animal enrolled in the study, and assigned to treat with three different ivermectins, three different albendazoles from different manufacturers and untreated control. Treatments were administered according to weight of each goat and sheep with albendazole 7.5 mg/kg body 60

3 weight, and ivermectin 0.2 mg/kg body weight as recommended by the manufacturers. Fecal samples were collected again 10 to 14 days post-treatment from all animals included in the study, and the changes in the EPG were determined. All fecal samples were analyzed using a modified McMaster technique and culturing as described by Ministry of Agriculture, Fisheries and Food (1984) and Coles et al. (1992), with a minimum detection limit of 50 EPG (Cole et al., 2006) Data analysis The efficacy of the drugs was assessed by the percentage reduction of mean egg excretion on the 10 to 14 th day post-treatment, FECRT, following the methods described by the World Association for the Advancement of Veterinary Parasitology (WAAVP) or Coles et al., 1992: FECR% =100(1-Xt/Xc); where Xt and Xc are the arithmetic mean post-treatment FEC in the anthelmintic- treated (t) and control(c) groups, respectively. Efficacy of individual drug class was considered to be if the percentage reduction in egg count was less than 95% and also the 95% confidence level was less than 90%. The calculations are based on those of the Reso FECRT analysis program (Version 2.0 Revised ) by Leo Wursthorn and Paul Martin of CSIRO, Animal Health Research Laboratory, PARKVILLE, 3052 (based on those published in Anthelmintic Resistance Report of the Working Party for the Animal Health Committee of the SCA (CSIRO, 989). 3. RESULT 3.1. Feacal egg count reduction test (FECRT) The FECR (%) found by calculating results from FECRT by Coles et al., 1992 methods are shown in Table 1 and Table 2. The results of FECRT showed different degrees of reduction in egg count after treatment with different drugs. But the response of parasites to anthelmintics not significantly varied among the farms studied and the same drugs from different manifacturers. The response to albendazoles from different manifacturers were not effective with the eficacy of 85 89%. However, the treatment with ivermectins from different manifacturers were effective with 95 97% reduction in egg count Feacal egg count reduction test (FECRT) in goat farm The results of the mean FEC for the treated and control goats and the percentage FECR in the treated groups of goats for each anthelmintic drug tested are shown in Table 1. In this farms at the 14 th day after treatment the efficacy of ivermectin in all 3 groups (IU, ICHY, and ICHJ) 61

4 was observed to higher than 99% (99-100%), while the effecacy of albendazole in all 3 groups (ALI, ACHT, ACHQ) was low, show by the low FECR, which was between 87-89%. The average percentage reduction in FEC of albendazole groups was 88% on day 14 posttreatment. Some parasite eggs were recorded on the 14th day post-treatment in the ivermectin treatment groups, but several parasite eggs were recorded in the albendazole treatment groups. There was reduction in the FEC with all the groups of anthelmintics tested against helminthes parasites. In the feacal examination on the 14th day post-treatment, very high efficacies of the entire brand tested of ivermectin, (99-100%), but low efficacies of the entire brand tested of albendazole, (87-89%) were recorded. However, no stastically significant difference was found among the same treatment with three different manifucturers tested (p> 0.05%). Generally the over all effecacy against ivermectin in goat farm was high (97-99%) FECR with 5-30 egg per gram of feaces (EPG) in 14 days after treatment with between lower confidence limit (CL). However, insuffecient effecacies of albendazole (85-89%) of feacal egg count (FEC) reduction on goat flock among 30 animals which are treated, with EPG in 14 days after treatment, with between lower confidence limit (CL) Feacal egg count reduction test (FECRT) in sheep farm The results of the mean FEC for the treated and control sheep and the percentage FECR in the treated groups of sheep for each anthelmintic drug tested are shown in Table 2. In this farms at the 14 th day after treatment the efficacy of ivermectin in all 3 groups ( IU, ICHY, and ICHJ) was observed to higher than 97% (97-99%), while the effecacy of albendazole in all 3 groups (ALI, ACHT, ACHQ) was low, show by the low FECR, which was between 85-89%. The average percentage reduction in FEC of albendazole groups was 87% on day 14 post-treatment. Some parasite eggs were recorded on the 14th day post-treatment in the ivermectin treatment groups, but several parasite eggs were recorded in the albendazole treatment groups. While there was reduction in the FEC with all the groups of anthelmintics tested against helminthes parasite. In the feacal examination on the 14th day post-treatment, very high efficacies of the entire brand tested of ivermectin, (97-99%), but low efficacies of the entire brand tested of albendazol, (85-89%) were recorded using the methods of Coles et al. (1992). However, no stastically significant difference was found among the same treatment with three different manifucturers tested (p> 0.05%). Generally the over all effecacy against ivermectin in goat farm was high ( %) FEC reduction with 5-25 egg per gram of feaces (EPG) in 14 days after treatment with between lower confidence limit (CL). However, insuffecient effecacies of albendazole (85-89%) of feacal egg count (FEC) reduction on this goat flock among 30 animals which are treated, with EPG in 14 days after treatment, with between lower confidence limit (CL). 62

5 3.2. Anthelmintic efficacy in goat flok Results of the FECRT of the goat flock shown that ivermectin groups of anthelmintics were found to be effective, while the different albendazoles were not effective (Table 1). Table 1. Feacal egg count reduction tests of the Haramaya University goat flock FECRT Control Ivermectin Albendazole IU ICHY ICHJ ALI ACHT ACHQ Numbers Mean epg (pretreatment) Mean epg (posttreatment) % Reduction Upper 95% CL(%) Lower 95% CL(%) Interpretation - Effective Effective Effective Not Effective Not Effective Not Effective IU=Ivermectin from Uraguay (1%, 1ml/50 kg body wt). ICHY=Ivermectin from China Hebei Yuanzheng Pharmaceutical Co., Ltd. (10mg/ml, 0.5ml/25kg body wt). ICHJ=Ivermectin from China Shandong jinyang Biological Pharmaceutical Co., Ltd. (1%, 0.5ml/25kg body wt). ALI= Albendazole from India (300mg, 7.5mg/kg body wt). ACHT=Albendazole China Shanghai Tongren Pharmaceutical Co., Ltd. (300mg USP24, 10-15mg/kg body wt). ACHQ=Albendazole from China Chengdu Qiankun Veterinary Pharmaceutical Co., Ltd. (300mg, 7.5mg/kg body wt) Anthelmintic efficacy in sheep flock Results of the FECRT of the HU sheep flock shown that ivermectin groups of anthelmintics were found to be effective, while the different albendazoles were not effective (Table 2). Table 2. Feacal egg count reduction tests of the Haramaya University sheep flock FECRT Control Ivermectin Albendazole IU* ICHY ICHJ ALI ACHT ACHQ 63

6 Numbers Mean epg (pretreatment) Mean epg (post treatment) % Reduction Upper 95% CL(%) Lower 95% CL(%) Interpretation - Effective Effective Effectiv e Not Effective Not Effective Not Effective *For abbreviations see table 3.4. Feacal culture The results of the post-treatment feacal cultures of all the albendazole treatment groups during FECRT in both sheep and goat flocks revealed that, Trichostrongylus spp. and Haemonchus contortus were the dominated nematode spp. Others also including Oesophagostomum spp. Strongyoides spp. Bunostomum spp. Nematodirus spp. and Trichuris spp. were found in varying numbers. This is used to know which species of parasite is more survival against albendazole drugs from different manifactureres. However, some Trichostrongylus spp. were found in samples taken from ivermectin drugs from different manufacturers treated animals. 4. DISCUSSION The results revealed that albendazoles from different manifacturers at the recommended doses could only reduce 85 89% of eggs of GI nematodes of sheep and goat farms showing low efficacy of albendazole in sheep and goat farms of Haramaya Univeristy. On the other hand, ivermectin has shown % reduction in FEC which has been described as highly effective by computer programme RESO. The results, thus indicates ivermectin was more effective drug than albendazole against GI nematodes of sheep and goat in Haramaya Univeristy. In conturary to this (Hussien et al., 1995; Asmare et al., 2005, Sissay et al., 2006) in field studies from different parts of Ethiopia under communal grazing system have shown the effectiveness of albendazole in small ruminants against GINs were reported. This difference is due to the continuous stream of new classes (modes-of-action) of anthelmintics had for several decades compensated for parallel development of resistance (von SamsonHimmelstjerna and Blackhall, 2005). It has also been shown that the problem of anthelmintic resistance is clearly linked to the drenching frequency, the presence of GINs and the prevailing type of grazing management (Waller et al., 1995). Biological fitness of 64

7 unselected worms (Coles, 2005). The most important factor in anthelmintic resistance is the proportional contribution of genetic material that worms surviving therapy transfer to the next generation (Jabar et al., 2006). Small ruminants are kept in large number and frequent anthelmintic treatment is practiced (Sissay et al., 2006). Small ruminant production in Ethiopia is mainly carried out by small-scale subsistence farmers with limited resources and financial competence to use anthelmintics regularly. This together with communal grazing practice and the absence of coordinated approach to anthelmintic treatment is believed to delay the development of anthelmintic resistance. Selective treatment of only seriously parasitized animals based on the level of anemia (FAMACHA chart) in areas where Haemonchosis is the predominant problem could help maintain the efficacy of currently available anthelmintics (Ejlertsen et al., 2006). FAMACHA chart is a tool used to identify animals that are critically affected by H. contortus and that should get treatment by looking at the degree of paleness of the conjunctival mucus membrane. This approach reduces selection pressure for anthelmintic resistance leaving many untreated animals in which unselected, susceptible worms survive and reproduce thus ensuring predominance of susceptible worms in the refugia (van Wyk 2001; Biffa et al., 2006) 4.1. Anthelmintic efficacy in goat flock A total of 70 goats were selected by using random sampling method to evaluate the efficacy of ivermectins and albendazole drugs from different manufacturers which are the major groups of broad-spectrum anthelmintics sold on Ethiopian markets against natural infection of helminthes parasite. In the present finding ivermectin from different manfacturers were found to be effective with FECRT and lower 95% confidence limits. However, all the albendazole goups were not effective with FECRT and lower 95% confidence limits. This is in agreement with Waruiru et al., 1997 who reportd 100%and 29% of effecacy for ivermectin and albendazole respectively. In the current study ivermectin groups of different manfacturers were very effective with FECRT and lower 95% confidence limit in goat flock. The result agreed with the reports of Borgsteede et al., 1996, Chandrawathani et al., 1999, Sissay et al., 2005 in goat flock of the same farms as that of the present study, Sissay et al., 2006 in goat flock of eastern Ethiopia owned by smallholder farmers with the efficacy of ( %, >95%, 96-98%, 96-99%) respectively. Contrary to our study, Zajac and Gipson, 2000, Terrill et al., 2001, in goat herds of USA was reported there is resistance of ivermectin with the efficacy of 54-90% and 78% respectively. In the present study albendazoles from different manfacturers were found to be not effective with FECRT and lower 95% confidence limit in goat flock. The result in agreement with the reports of Ancheta and Dumilon, 1999, Chandrawathani et al.,1999, 65

8 Terrill et al., 2001, Eguale et al., 2009 with the efficacy of (30-65%, 50-90%, 62%, <84%) respectively. However, this is different which reported by different authors. Sissay et al., 2005 in goat farm on the same farms of this study, reported there was no anthelmintic resistance of albendazole with FECR% 95% this difference is expected due to either the development of resistance of parasite to the drug from 2003 until now or during anthelmintic treatments, a small number of worms survive, these being the most resistant proportion of the population. These worms contaminate the pasture with a majority of resistant larvae for subsequent generations, leading gradually to the selection pressure of AR). Sissay et al., 2006 in Eastern Ethiopia goat flocks owned by small holder farmers with FECR% 95-97% this difference is due to different in the frequency of the drug taken Anthelmintic efficacy in sheep flock A total of 70 goats were selected by using random sampling method to evaluate the efficacy of ivermectin and albendazole drugs from different manufacturers which are the major groups of broad-spectrum anthelmintics sold on Ethiopian markets against natural infection of helminthes parasite. In the present finding ivermectin from different manfacturers were found to be effective with FECRT and lower 95% confidence limits. However, all the albendazole goups were not effective with FECRT and lower 95% confidence limits. This is in agreement with George et al., 2011 with 95-97%, 46-62% of effecacy ivermectin and albendazole respectively. The present study also showed ivermectins from different manfacturers were very effective with FECRT and lower 95% confidence limits in sheep flock. This result agrees with the reports of Chartier et al., 1997, Chandrawathani et al., 1999, Arece et al., 2003, Sissay et al., 2005 in sheep flocks of the same farms of the present study, Sissay et al., 2006 in in sheep flock of eastern Ethiopia owned by smallholder farmers. With the efficacy of (98-100%, -, 95-98, 95-99). This disagreed to Cernanska et al., 2006 with FECR%=52-94% and lower 95% CL= Albendazole from different manfacturers in the current study were not effective with 85-89% FECRT and lower 95% confidence limits in the sheep flock. This result is in agreement with the results of Ancheta and Dumilon, 1999, Chandrawathani et al.,1999, Cernanska et al., 2006, George et al., 2011 with the efficacy of (<50-90%, 30-91%,69.8%, 46-62% ) respectively. In contorary different results were reported by authors: Arece et al., 2003 in sheep of Matanzas, Cuba, with the effecacy of %, Sissay et al., 2005 in sheep farm on the same farms of this study, reported there was no anthelmintec resistance of albendazole with FECR% this difference is due to either the efficacy of the drugs were low or in the parasite of animals develops resistance from 2003 until now. Sissay et al., 2006 in Eastern Ethiopia sheep and goat flocks owned by small holder farmers with FECR% 95-97%, this is 66

9 due to the frequency of the in smallholder is less than the farms. Chaka. et al., 2008 with the efficacy of in experimental infected sheep. 5. CONCLUSION AND RECOMMENDATIONS The study objectively assessed and evaluated ivermectin broad-spectrum anthelmintics from different manufacturers which are sold in Ethiopian markets was more effective than that of albendazole drugs from different manufacturers which less effective to most gastrointestinal parasites such as, Trichostrongylus spp., Haemonchus contortus, Oesophagostomum spp. Strongyoides spp. Bunostomum spp. Nematodirus spp. and Trichuris spp. Without any significant variation either with in different manufacturers of the same drug or with different species of animals. To know the resistance of anthelmintics on FECR only are often liable to uncertainty, it will often be necessary to use complementary investigations (Egg hatch assays, Larval paralysis, migration and motility tests, Larval development tests, Adult development test, Biochemical tests, Molecular techniques). Based on the above conclusion, the following recommendations are forwarded: Give advice for the farmers and persons who are contact to animals to use anthelmintic drugs which are more effective. Fulfill good infrastructure for a reasonable set of strategic sanitation, and adequate nutrition. Done research on AR should be a priority area, enabling constant monitoring, reevaluation and adjustment of control strategies To adapt biological control or use of parasite resistant breeds. Farmers should be educated with proper veterinary extension about the importance of use of efficacious anthelmintics. Further study is suggested to identify the species of GI nematodes involved in the anthelmintic resistance. Different study should be done to verify the development of resistance of nematode parasites against commonly using anthelmintics in Haramaya Univeristy by using In vitro methods (Egg hatch assays, Larval paralysis, migration and motility tests, Larval development tests, Adult development test, Biochemical tests, Molecular techniques) to support these findings. 6. REFERENCES Ancheta, B.P. and Dumilo, A.R. (1999). Anthelmintic resistance of small ruminants in the Philippines. Proceeding of the PSAS 3 th Annual Convention, October 1999, Metro Manila, Philippines. 67

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13 Zajac, M.A. and Gipson, A.T. (1999). Multiple anthelmintic resistance in goat herds. Vet. Parasitol. 87 (2000) ANNEXES Annex-I. Material, Preparation and procedures for saline flotation technique. Materials Pestle and mortar Feacal samples Tea strainer Measuring cylinders Test tube and racks Pipette and flasks Screw capped bottles Cover slip and slides Microscope Balance and water Ncl/solid/ and spatula Stirring rod Tea spoon Plastic containers Preparation of saturated saline floatation fluid 400 grams of Ncl was measured using balance 100 ml of water was made warm (heated) The salt was added into the water and stirred until the salt is left undissolved to produce saturated saline solution. Procedure 1. 3 grams of feaces was measured by tea spoon and placed in the mortar 2. The feacal mass was ground by pestle 3. The grounded feaces was placed into the container-1 4. The floatation fluid (45ml) was added into container-1 5. The mixture was stirred (mixed) thoroughly 6. The feacal suspension was poured through tea strainer into container-2 for filtration 7. The filtrate was transferred to test tubes making positive meniscus 8. Cover slip was applied on the top and 9. The cover slip was taken up vertically after 10 min and placed on slide 10. Examined under microscope (10x) Annex-II. Material, Preparation and procedures for Bearmann technique Materials 71

14 Feacal culture and water Test tube and racks Petri dish and iodine solution Scissors and plastic tube/ rubber band Centrifuge and centrifuge tubes Gauze and Pasteur pipette Funnel and funnel stand Suspensor sticks Microscope, slide, and cover slip Spoon/spatula Preparation or installation The funnel was fitted to the funnel stand The rubber tube was installed at lower end of the funnel extended through the stand The end portion of the rubber tube was crushed/ closed by clamp Water was made warm Procedure 1. About 5 grams of cultured feacal mass was taken by spatula and placed on the prepared gauze 2. Making an pouch, the feaces was closed and tied near the feacal mass 3. The stick was passed just through the tie 4. Warmed water was poured into the funnel (filled) 5. The end of the stick which was passed through the tie was placed on the edges of the funnel in such a way that the feacal mass could be fully immersed into the water and allowed to settle for 24 hours 6. After 24 hours, some fluid withdrawn into test tubes from the bottom portion of the funnel 7. The two test tubes were closed and centrifuge at 1500 rpm for 5 min 8. The supernatant was discarded carefully and the sediment was poured into Petri dishes 9. Iodine solution was added into the sediment to kill larvae ( for larvae immobilization) 10. Pasteur pipette was used to take the larval drop and place on the slide 11. Cover sip applied and examined under microscope (10X, 40X) Annex-III. Materials, Preparation and procedures for Feacal egg count / McMaser egg counting technique/ Materials Pestle and mortar Beakers /2/ and plastic containers Balance and Tea strainer Measuring cylinders Stirring rod and spatula 72

15 McMaster egg counting chamber Compound microscope, pipette Flotation fluid and feacal mass Tea spoon Preparation of saturated saline solution (Annex-I) Procedure 1. 3 grams of feaces were measured by tea spoon and placed in mortar 2. The feaces was ground by pestle and transferred into container-1 3. About 42 ml of saturated saline solution was added into container-1 4. Using stirring rod, the mixture was dissolved 5. The suspension was then poured though tea strainer into container-2 6. The filtrate was taken by pasture pipette and the McMaster egg counting chambers was filled 7. The chamber was allowed to float the eggs for 3 min 8. The counting chamber was then examined under the microscope /10X/ 9. The eggs touching bottom and right lines were excluded while counting 10. The total counts from both chambers were added together and the result obtained multiplied by 50 to get eggs per gram/epg Annex-III. Materials, procedures for Feacal culture Materials Feaces Bottles /plastics with cover/ Spatula Water Mortar and pestle Procedure 1. 5 grams of feaces was measured by spoon and placed in mortar 2. The feaces then ground by pestle 3. Some water was added to moisten the feaces 4. The moistened feaces then transferred to the bottles and loosely covered 5. The bottle then kept at room temperature for 14 days under suitable moisture 6. The feaces were then moistened every 2 days 7. The larvae were then recovered by bearmann apparatus Annex-V. Material, Preparation and procedures for sedimentation technique. Materials 1%methyle blue and pipette Pestle and mortar Spatula and tea strainer Test tube and test tube racks Measuring cylinders and plastic containers 73

16 Microscope, microscopic slide and cover slip Water and glass beakers Stirrer / stirring rod/ Preparation of 1% methylene blue 1 grams of solid methylene blue crystal was weighed using balance 100 ml of water was poured into beaker The methylene crystal was added into the water producing 1% methylene blue Procedure 1. 3 grams of feaces was measured by tea spoon and placed in the mortar 2. The feacal mass was ground by pestle 3. The grounded feaces was placed into the container ml of water was added into container-1 5. The mixture was stirred (mixed) thoroughly with stirring rod 6. The feacal suspension was poured through tea strainer into container-2 and the residue removed and allowed to sediment for 15 min. 7. The supernatant was discarded and the sediment re-suspended in 5 ml of water and allowed to sediment for 5 min. 8. The sediment was stained with methylene blue (2-3) drops 9. The mixture was taken by pipette and placed on the microscopic slide 10. Cover slip was applied and examined under microscope (10X) 74