DETECTION OF ANTHELMINTIC RESISTANCE IN GASTROINTESTINAL NEMATODES OF SHEEP IN BALOCHISTAN THROUGH FAECAL EGG COUNT REDUCTION TEST AND EGG HATCH ASSAY

Sarhad J. Agric. Vol., No. 2, 14 DETECTION OF ANTHELMINTIC RESISTANCE IN GASTROINTESTINAL NEMATODES OF SHEEP IN BALOCHISTAN THROUGH FAECAL EGG COUNT REDUCTION TEST AND EGG HATCH ASSAY HAMDULLAH 1*, MOHAMMAD LATEEF 2, AZHAR MAQBOOL 2, MAKHDOOM ABDUL JABBAR 2, FARHAT ABBAS 3, SAADULLAH JAN 3, ABDUL RAZZAQ 4 and ESSA KAKAR 1 1. Livestock and Dairy Development Department, Berwary Road Quetta, Baluchistan - Pakistan. 2. University of Veterinary and Animal sciences, Outfall Road Lahore, Punjab - Pakistan. 3. Centre for Advanced Studies in Vaccinology and Biotechnology (CASVAB) University of Ba luchistan, Brewery Road Quetta - Pakistan. 4. Baluchistan Agricultural Research Centre (BARC), Pakistan Agriculture Research Council, West ern Bypass Quetta, Baluchistan - Pakistan. *Corresponding author: hamdullahkakar@gmail.com ABSTRACT The present study was conducted to investigate the efficacy of oxfendazole, levamisole and ivermectin against gastrointestinal nematodes of sheep. Three hundred and twenty sheep comprised four breeds (Balochi, Rakhshani, Kurakul and Balochi/Kurakulcross ) were selected for this experiment. The selected sheep were divided into four groups.first group was treated with oxfendazole, second with Levamisole, third with ivermectin and fourth kept as untreated to serve as control. Faecal samples were collected on day first prior to administration of treatment followed by day 14 post treatment. The efficacy of each anthelmintics was measured using the faecal egg count reduction test and egg hatch test. The arithmetic faecal egg count reduction with oxfendazole, levamisole and ivermectin were recorded %, % and % respectively, which indicated the susceptibility of these anthelmintics against gastrointestinal nematodes. The results of egg hatch test indicated that Lc0 were 0.031, 0.08, 0.092 and 0.086 ug/ml respectively for four sheep breeds, which are less than 0.1ug/ml of oxfendazole. Result indicates that no resistance was found in the eggs with oxfendazole. The egg hatch assay also confirmed the result of faecal egg count reduction test. The qualitative examination of faecal samples after Coproculture revealed four gastrointestinal nematode genera i.e., Haemonchus, Trichostrongylus, Nematodirus and Ostertagia. Key words: Sheep, anthelmintic resistance, Oxfendazole, Levamisole and Ivermectin, Balochistan. Citation: Hamdullah., M. Lateef., A. Maqbool., M. A. Jabbar., F. Abbas., S. Jan., A. Razzaq and E. Kakar. 14. Detection of anthelmintic resistance in gastrointestinal nematodes of sheep in Balochistan through faecal egg count reduction test and egg hatch assay. Sarhad J. Agric. (2): 179- INTRODUCTION The major gastrointestinal nematode species reported in Baluchistan were Haemonchus Strongyloides, Trichostrongylus, Nematodirus, and Trichuris (Razzaq et al., 13). These gastrointestinal nematodes are responsible for decreased productivity particularly in grazing conditions (Tembely et al., 17).The control of these nematodes for the past thirty years have mostly relied on the use of anthelmintics, but with the passage of time these have lost their efficacy due to the development of resistance (Waller, 17; Afaq, 03). Resistance to the major classes of anthelmintics has been recorded in Canada ( Falzon et al., 13), North America (Uhlinger et al., 12), Latin America (Eddi et al., 16; Nari et al., 16), Europ (Coles et al., 14, 06),Australia (Waller et al., 1), Asia (Gills, 13) and even in Pakistan (Afaq, 03).Resistance of benzimidazole group against Haemonchuscontortus was observed very early after their introduction (Drudge et al., 14). Similarly, levamisole/morantel resistance to H. contortus was detected in sheep (Le Jambre et al., 16). Macro cyclic lactones resistance was reported in South Africa (Carmichael et al., 17). In accordance with the prevailing climate, parasitic species and treatment regimes, the rate of emergence of anthelmintic resistance vary geographically adopted in the region (Prichard, ; Jackson, 13). Anthelmintic resistance is becoming a main constraint in small ruminant production throughout the world (Papadopoulos, 08) and has serious implications due to non availability of new drugs. Therefore the present study has been designed to evaluate the development of resistance against commonly used anthelmintics in sheep from Baluchistan, Pakistan.

Hamdullah, et al. Detection of anthelmintic resistance in gastrointestinal nematodes of sheep 180 MATERIALS AND METHODS Experimental Design Selection of Farm and Anthelmintics Karakul sheep research farm at Maslakh District Pishin was selected to evaluate the anthelmintic resistance against gastrointestinal nematodes in sheep. The anthelmintic resistance in gastrointestinal nematodes of sheep was detected through faecal egg count reduction test (FECR) against oxfendazole, levamizole and ivermectin and egg hatch assay (EH) for oxfendazole (Coles et al., 12; Lyndal-Murphy, 12). Selection of Animals Four homogeneous groups of 80 sheep representing Balochi, Rakhshani, Karakul and cross-bred each equally were constituted for the trial. Animals were selected randomly aging below one year. Animals, previously parasitized with gastro-intestinal nematodes were selected. Group A and B were comprised of animals dosed with Oxfendazole (M/S ICI, Pakistan Ltd.) @ 4.3 mg/kg body weight and Levamisole (M/S ICI, Pakistan Ltd.) @ 7. mg/kg body weight orally with a calibrated drenching gun. Ivermectin (M/S P.D H. Lab. Pvt. Ltd.) @ 0ug/kg body weight was administered subcutaneously to group C. Group D was kept as control. Medicine was administered according to the manufacturer recommendations. Faecal Egg Count Reduction Test Five grams faecal sample were collected directly from the rectum of individual animal and put in ice box before treatment and day 14 post treatment. Faecal examination was conducted using standard procedure described by (Coles et al., 12). The identification of species of nematodes were conducted through Coproculture and by microscopic studies of their eggs and counted Eggs Per Gram () were assessed by a modified McMaster technique (Coles, 16). The brief procedure of coproculture was comprised that the faecal samples were placed in petri-dish and covered with moisten cotton layer. These dishes were placed at room temperature for seven days and examined under the microscope for identification of nematodes larvae. The briefly procedure of McMaster technique consists, 3 grams of faecal material was thoroughly mixed/homogenized in 42 ml of water in a plastic container using a homogenizer. The homogenate was poured through a fine mesh sieve (aperture um or to 1 inch) and filtrate was agitated and filled in a 1 ml test tube. The filtrate was than centrifuged at00 rpm for two minutes and discarded the supernatant. The sediment was agitated and filled in glass tube to previous level with floatation solution (saturated solution of sodium chloride with 1. to 1. specific gravity). Each glass tube was inverted six times and fluid was aspirated with pipette and filled both chambers of McMaster slide. Examined one chamber and multiplied the number of eggs/larvae under one etched area by, for eggs per gram of faeces. Statistical Analysis The percentage reduction was calculated by the formula R%= (1-Xt/Xc) where Xt was treated group and Xc was control group (Coles et al., 12, 06). A reduction in eggs per gram of faeces less than 9% with confidence interval between % to 9% were taken as an indication (Coles et al., 12) for the presence of anthelmintic resistant nematodes in treated animals. Calculations were made according to (Coles et al., 12) using a spreadsheet that was created by Angus Cameron, Aus. Vet. Animal Health Services for the University of Sydney. Its calculation are based on those of the Reso FECR test analysis program (Version 2.0) Egg Hatch Test Egg hatch test were conducted using a standard procedure described by (Le jamber, 16) and (Coles et al., 12). Egg hatch test was carried out following the standard procedure described by (Le jamber, 16; Coles et al., 12; Taylor et al., 02). Statistical Analysis Logarithmic concentration (LC) value was calculated for the eggs by log probit analysis (Finney, 11). Eggs having LC value in excess of 0.1ug anthelmintic/ml was indicative of anthelmintic resistance against oxfendazole (Le Jambre, 16; Coles et al., 12). RESULT AND DISCUSSION Faecal egg count reduction test of naturally nematodes infected Balochi, Rakhshani, Karakul and Cross breed sheep The present study results revealed that, reduction in eggs per gram () of faeces was %, %, %, 9% respectively in four breeds of sheep (Balochi, Rakhshani, Karakul and Cross breed sheep) treated with oxfendazole. The lower confidence interval was %, %, %, % and upper confidence interval was %,

Sarhad J. Agric. Vol., No. 2, 14 181 %, %, % respectively. It seems no resistance as higher percentage than the criteria i.e., percent reduction 9%, CI (-9%). The levamizole treated sheep showed % reduction in (-% CI) the lower and upper confidence levels were %, %, %, 9% and %, %, %, % respectively. Similarly the percent reduction in was,,, % in ivermectin treated sheep. The lower and upper confidence level was %, %, %, % and %, %, %, % respectively. The result indicated that no resistance of gastrointestinal nematodes was found against oxfendazole, levamisole and ivermectin (Table 1).The Coproculture examination revealed four nematodes species, following the comparison of pre and post treatment of oxfendazole treated groups. Table 1. Mean faecal egg count in sheep naturally infected with mixed species of Nematodes before and after treatment with different anthelmintics in Balochi (B), Rukhshani (R), Karakul (K) and Cross (C) breed Anthelmintics (Mean 0- day; n=) (Mean 14- day; n=) Reduction % Lower confidence level Upper confidence level Remarks Oxfendazole B 219 R 232 K 246 C 2 Levamisole B 23 R 2400 K 238 C 21 Ivermectin B 19 R 240 K 2240 C 24 Untreated B 2 R 22 K 240 C 260 70 12 70 9 9 2 3 40 0 9 9 _ The total counts 219, 232, 246 and 2 (Table 1) of Balochi, Rakhshani, Karakul and Cross sheep breed included approximately (6/219), 27.79%, 28.60%, 26.77%, 27.22% for Haemonchus, (72/219) 33%, 32%, %, 29.1% for Trichostrongylus, (440/219) %, %, 21.70%, 21.23% for Nematodirus and (4/219) 19%, 19%, 21.0% and 22.39% for Ostertagia (Table 2). The data in table 2 revealed that all nematode species were susceptible to oxfendazole based on the criteria considered. Table 2 Mean faecal egg counts of different species of nematodes in sheep before and after treatment with oxfendazol in Balochi (B), Rukhshani (R), Karakul (K) and Cross (C) breed Nematodes species (Mean 0- (Mean14- Reduction % Lower confidence level Upper confidence level Remarks day; n=) Haemonchus B 6 R 66 K 660 C 70 Trichostongylus B 72 R 70 K 740 C 7 Nematodirus B 440 R 46 K 3 C 0 Ostertagia B 4 R 44 K C 80 day; n=) 2 3 1 2 3 40 2 9 9 9 9 92 The total counts of Balochi, Rukhshani, Karakul and Cross-bred in levamisole treated groups were 23, 2400, 238, 21 (Table 1), which were (660/23) 28.44%, 29.37%, 28.1%, 27.63% for Haemonchuscontortus, (7/23) 31.46%, 31.87%, 29.76%, 29.42% for Trichostrongylus, (4/23) 19.61%, 19.16%,.7%, 21% for Nematodirus and (47/23).47%, 19.8%, 21.38%, 21.86% for Ostertagia (Table 3). The data in table 3 shows that all nematode species were susceptible to levamisole.

Hamdullah, et al. Detection of anthelmintic resistance in gastrointestinal nematodes of sheep 182 Table 3. Mean faecal egg counts of different species of nematodes in sheep before and after treatment with Levamizol in Balochi (B), Rukhshani (R), Karakul (K) and Cross (C) breed. Nematodes species (Mean 0-day; n=) (Mean 14-day; n=) Reduction % Lower confidence Upper confidence Remarks Haemonchus B 660 R 70 K 680 C 69 Trichostongylus B 7 R 76 K 7 C 740 Nematodirus B 4 R 460 K 49 C Ostertagia B 47 R 470 K C 0 1 3 1 1 1 1 9 9 level 92 level The total counts of Balochi, Rukhshani, Karakul and Cross-bred in ivermectin treated groups were 19, 240, 2240 and 24 (Table 1) in which Haemonchuscontortus was (4/19) 2.2%, 29.%, 29%, 27.68%, Trichostrongylus(/19) 28.%, 32.22%, %, 29.33%, Nematodirus (4/19) 23.69%, 18.71%,.7%, 21.28% and Ostertagia (4/19) 21.87%, 19.12%,.3%, 21.69% (Table 4). The data in Ta ble 4 revealed that all nematode species were susceptible to ivermectin. Table 4. Mean faecal egg counts of different species of nematodes in sheep before and after treatment with Ivermictin in Balochi (B), Rukhshani (R), Karakul (K) and Cross (C) breed. Nematodes species (Mean 0-day; n=) (Mean 14-day; n=) Reduction % Lower confidence level Upper confidence level Remarks Haemonchus B 4 R 7 K 60 C 670 Trichostongylus B R 77 K 67 C 7 Nematodirus B 4 R 40 K 46 C 1 Ostertagia B 4 R 460 K 460 C 2 2 1 1 1 92 92 Egg hatch assay for evaluation of oxfendazoleresistance in Balochi, Rakhshani, Karakul and Cross sheep at Muslukh Karakul sheep Farm Egg hatch test was performed for confirmation of oxfendazole susceptibility through faecal egg count reduction (FECR) test. The results of egg hatch test indicated (Table to 8) that Lc 0 were 0.031, 0.08, 0.092 and 0.086 ug/ml, which are less than 0.1ug/ml of oxfendazole. This indicates that no resistance was found against nematodes with oxfendazole. The qualitative examination of faecal samples after Coproculture revealed four gastrointestinal nematode genera, Haemonchus, Trichostrongylus, Nematodirus and Ostertagia.

Sarhad J. Agric. Vol., No. 2, 14 183 Table. Eggs hatched (%) at different concentrations of oxfendazole in Balochi sheep. S# Oxfendazoleug/ml Oxfendazoleng/ml Log(oxf) Hatching Probit Regression (%) hatching 1 22.6 2260 4.7 2 2.6 2.9243 2 11.33 113 4.0423 4.66 3. 3.18 3.67 670 3.738 9.33 3.67 3.437 4 2.84 2840 3.4332 12.6 3.8 3.7181 1.42 14 3.1229 16.66 4.026 3.31 6 0.71 7 2.8126 22.66 4.24 4.2481 7 0.36 360 2.63 29.33 4.447 4.077 8 0.18 180 2.227 36.66 4.6 4.7727 9 0.09 1.24 41.33 4.773.0377 0.04 4 1.6321 0.27 11 0.023 23 1.36173 64.38. 12 0.012 12 1.078 72.83.808 13 0.006 6 0.7781 82.66.4 6.073 14 0.003 3 0.47712.66 6. 6.338 1 0.001 1 0.33 7.326 6.78 Log Lc0=1.7 Lc0=.31ng/ml= 0.031ug/ml The present study results revealed that, percent reduction in of faeces was susceptible with oxfendazole, levamisole and ivermectin, in all four breeds of sheep against Haemonchus,Trichostrongylus, Nematodirus and Ostertagia infection which are in agreements with (Swarnkar et al., 19), who reported no anthelmintic resistance in Karakuk sheep in India. They observed that, levamizole and fenbendazole were % effective through faecal egg count reduction, while Lc 0 value on egg hatch assay was 0.074± 0.01ug thiabendazole/ml. Similarly Menkir(et al., 06) also observed no resistance to tetramisole, albendazole, combination of these two drugs and also ivermectin in sheep. Bersisa Kumsa and AjebuNurfeta (08) and Asmare et al. (0) also evaluated the efficacy of anthelmintic against sheep nematodes in southern Ethiopia. They also observed that albendazole, tetramisole and ivermectin were.34%,.77%, and.% effective through faecal egg count reduction. It might be due to the parasite populations of small ruminants in the region within and surrounding would have common ancestry. Han-Bo et al., (17) and Sheferaw and Asha.,() found that ivermectin was % and.3% effective against gastrointestinal nematodes of sheep. Mirhadi et al., (11) also presented.1% efficacy of ivermectin against Nematodirusspathiger. Yadav et al., (1) reported ivermectin and closantel % effective against Haemonchuscontortus. But benzimidazole, levamizole and morantel 6% and 81% effective against Haemonchuscontortus, which in contrast to the present study. However Borgsteede et al., (16) reported no resistance for Levamisole and ivermectin against Haemonchuscontortus, Trichostrongylu scolubriformis, Teladorsagia circumcincta and Cooperiacurticei which is similar in the present study, but in contrast Haemonchuscontortus, Trichostrongylu scolubriformis, Cooperiacurticei and Teladorsagia circumcincta were resistant to benzimidazole. Gill (16) observed resistance to albendazole and levamisole against sheep nematodes, which in contrast to the present study. However no resistance was found against ivermectin which is similar to the present study. Table 6. Eggs hatched (%) at different concentration of oxfendazole in Rukhshani sheep. S# Oxfendazoleug/ml Oxfendazoleng/ml Log(oxf) Hatching Probit Regression (%) hatching 1 22.6 2260 4.7 0.66 2.676 2.8 2 11.33 113 4.0423 4 3.249 3.123 3.67 670 3.738 8 3.9 3.392 4 2.84 2840 3.4332 14.66 3.2 3.66 1.42 14 3.1229 19.33 4.112 3.928 6 0.71 7 2.8126 22.66 4.228 4.1 7 0.36 360 2.63 29.33 4.447 4.46 8 0.18 180 2.227 36 4.642 4.729 9 0.09 1.24 43.33 4.824 4.8 0.04 4 1.6321 46.66 4.2.266 11 0.023 23 1.36173 0.27 12 0.012 12 1.078 66.66.426.779 13 0.006 6 0.7781 84.4 6.047 14 0.003 3 0.47712 9.33 6.64 6.316 1 0.001 1 0.33 7.326 6.742 LogLc0=1.917 Lc0=89.4473ng/ml=0.08ug/ml.

Hamdullah, et al. Detection of anthelmintic resistance in gastrointestinal nematodes of sheep 184 Similar study was carried out by Farias et al., (17) and reported resistance to benzimidazole and Levamisole against Trichostrongylus, Ostertagia and Haemonchus, but no resistance was found to ivermectin against Trichostrongylus, Ostertagia and Haemonchus. Present findings disagree with Chandrawathani et al., (19) who observed resistance to benzimidazole, levamizole, the combination of benzimidazole/ levamizole, ivermectin and closantel against Haemonchuscontortus in sheep. (Falzon et al., 13) also recorded resistance of ivermectin and benzimidazoles in sheep flock in Canada.Sargison (12) reported multiple anthelmintic resistances in sheep and goat. Marian et al., (06) used egg hatch test for benzimidazole resistance and compared it with FECR test and declared resistance with two tests. The LD 0 was higher than 0.1mg/ml thiabendazole indicating resistance, which in contrast to the present study. Table 7. Eggs hatched (%) at different concentration of oxfendazole in Karakul sheep S# Oxfendazoleug/ml Oxfendazoleng/ml Log(oxf) Hatching Probit Regression (%) hatching 1 22.6 2260 4.68 1.3333 2.676 2.87246 2 11.33 113 4.04229.3333 3.3 3.1406 3.67 670 3.7383 8.6666 3.628 3.40868 4 2.84 2840 3.43318 13.3333 3.874 3.67637 1.42 14 3.12288 18.6666 4.4 3.473 6 0.71 7 2.8128 21.3333 4.1 4.2131 7 0.36 360 2.62 4.476 4.47606 8 0.18 180 2.2272 34.6666 4.601 4.74442 9 0.09 1.242 42.6666 4.811.01279 0.04 4 1.63212 49.3333 4..28116 11 0.023 23 1.361727 3.3333.07.42 12 0.012 12 1.0781 70.6666.24.792 13 0.006 6 0.77811 8.3333 6.036 6.06128 14 0.003 3 0.477121 6.71 6.32 1 0.001 1 0.6666 7.17 6.7 Log Lc0=1.8 Lc0=.0234ng/ml=0.092ug/ml Table 8. Eggs hatched (%) at different concentration of oxfendazole in Cross-bred sheep. S# Oxfendazoleug/ml Oxfendazoleng/ml Log(oxf) Hatching Probit Regression (%) hatching 1 22.6 2260 4.7 0.66 2.676 2.813 2 11.33 113 4.0423 3.33 3.119 3.0884 3.67 670 3.738 7.33 3.24 3.376 4 2.84 2840 3.4332 14 3.92 3.628 1.42 14 3.1229 18.66 4.122 3.1 6 0.71 7 2.8126 22 4.228 4.178 7 0.36 360 2.63 28.66 4.447 4.436 8 0.18 180 2.227 3.33 4.61 4.71173 9 0.09 1.24 44 4.849 4.381 0.04 4 1.6321 47.33 4.92.288 11 0.023 23 1.36173 0.66.02.12 12 0.012 12 1.078 6.33.38.77469 13 0.006 6 0.7781 82.66. 6.04676 14 0.003 3 0.47712.66 6.64 6.31883 1 0.001 1 0.33 7.326 6.700 LogLc0= 1.632 Lc 0= 86.3614 ng/ ml = 0.086 ug/ ml CONCLUSION AND RECOMMENDATIONS The present study indicates that no anthelmintics resistance was found against nematodes infection in native sheep of Baluchistan. However it is recommended that the proper anthelmintic dose with 3-4 months interval and rotation of anthelmintics for minimized the nematode infection and enhance sheep productivity. REFERENCES Afaq, M. 03. Parasitic control practices and anthelmintic resistance against GIT nematodes of Sheep. PhD thesis, Deptt. of Vet. Parasitol. Univ. Agri., Faisalabad. Borgsteede, F.H., M.J.J. Pekelder, D.P. Dercksen. 16. Anthelmintic resistant nematodes in goats in the Netherlands. Vet. Parasitol., 6: 83-87.

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