Om Prakash et al,2018


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1361 Journal of Pharmacognosy and Phytochemistry 2018 75: 1361-1367 E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2018 75: 1361-1367 Received: 25-07-2018 Accepted: 26-08-2018 Om Prakash Veterinary Polytechnic Surajpur Chhattisgarh Kamdhenu Vishwavidyalaya Durg Chhattisgarh India S Gomathinayagam Department of Veterinary Parasitology Madras Veterinary College Tamil Nadu Veterinary and Animal Science University Chennai Tamil Nadu India Tirunelveli Jeyagopal Harikrishnan Tamil Nadu Veterinary and Animal Science University Chennai Tamil Nadu India Muthusamy Raman Transboundary Research Platform for Veterinary Biological Tamil Nadu Veterinary and Animal Science University Chennai Tamil Nadu India V Pandiyan Department of Veterinary Biochemistry Madras Veterinary College Chennai Tamil Nadu India P Azhahianambi Transboundary Research Platform for Veterinary Biological Tamil Nadu Veterinary and Animal Science University Chennai Tamil Nadu India Correspondence Om Prakash Veterinary Polytechnic Surajpur Chhattisgarh Kamdhenu Vishwavidyalaya Durg Chhattisgarh India Is L-buthionine sulfoximine reverse the benzimidazole resistance in Haemonchus contortus of small ruminants Om Prakash S Gomathinayagam Tirunelveli Jeyagopal Harikrishnan Muthusamy Raman V Pandiyan and P Azhahianambi Abstract Anthelmintic resistance AR in parasites of livestock is an emerging problem in many parts of the World. Hence finding new classes of anthelmintic drugs or anthelmintic resistance reversal agents is the need of the hour to sustain the global livestock production. The aim of present study was to study the Benzimidazole BZ resistance reversal property of L-buthionine sulfoximine L-BSO using in vitro egg hatch assay EHA. Thiabendazole TBZ alone and combination of TBZ and 200µM 500µM and700µML-BSO were used with proper control and the hatching percentage and ED50values were recorded. It was found that significant P0.001 reversal of BZ resistance occurred when H. contortus was treated with L-BSO 500µM and the effect was dose dependent. Hence L-BSO might potentially be used as a BZ resistance reversal agent to treat BZ resistant nematode parasites of livestock Keywords: Haemonchus benzimidazole buthionine sulfoximine small ruminants Introduction The Haemonchus contortus commonly known as the barber’s pole worm parasitic nematode a major cause of loss of production in sheep and goat industry. This parasite can inflict a considerable amount of damage to a flock or herd in a short span of time. Treatment and control of this gastrointestinal parasite have been successful using various anthelmintics such as benzimidazoles levamisole and ivermectin which exert selection pressure resulting in the development of anthelmintic resistance Torres-Acosta et al. 2012 56 . Unraveling the anthelmintic resistance mechanisms employed by H. contortus will help us to find solutions to overcome the anthelmintic resistance and use better treatment and control methods. The effect of glutathione GSH on the eggs of H. contortus susceptible/ resistance to anthelmintics was investigated using in vitro egg hatch assay EHA. The modulators or GSH analogue like Diethylemaleate D L-buthionine sulfoximine and patulin induce an unexpected decrease in the susceptibility of egg to thiabendazole Kerboeuf and Aycaedi 1999 31 . L-BSO increases the toxicity of nifurtimox benzidazole to the epimastigote trypomastigote and amastigote stages of Trypanosoma cruzi Faundez et al. 2005 19 . The enzyme activity of Burgia malayi the causative agent of lymphatic filariasis is irreversibly inhibited by the BSO Hussein and Walter 2005 29 . Buthionine sulfoximine is a potent and specific inhibitor of γ- glutamylcysteine synthetase and inhibits glutathione biosynthesis and causes depletion of cellular glutathione levels Griffith and Meister 1979 25 . Glutathione S- transferase GST which is part of parasite detoxification system associated with the establishment of parasitic nematode infections within the gastrointestinal environment of the mammalian host Rossum et al 2004 53 . On the above hypotheis the present study was conducted with the objective of is BSO potentiate the activity of TBZ in H. contortus. Material and Methods Collection of Parasites and Harvesting of Eggs Abomasums of sheep and goat were collected in normal saline from the slaughter house Perumbur Chennai India and transported to the laboratory. Abomasums were cut open along greater curvature and manual removal of adult H. contortus was carried out from the abomasums content and mucosal fold. The worms were washed two times with normal saline and female worms were separated and incubated in normal saline for an hour at 37 0 C for release of eggs. After incubation normal saline was collected in centrifuge tube and centrifuged Eppendorf 5810R Germany at 2000 rpm for 5 minutes to sediment the eggs. The supernatant was poured off and sediment was examined for the presence of eggs. The concentration of egg was adjusted to50 eggs/µl.

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1362 Journal of Pharmacognosy and Phytochemistry Assessment of benzimidazole resistance by EHA Assessment of benzimidazole resistance by egg hatch assay EHA was performed as using harvested eggs from the slaughter house Perumbur Chennai India using pure thiabendazole -99 percent Sigma Aldrich- T8904 USA in five concentration of 0.05 0.1 0.3 0.5 and 1.0 µg/ml as per the procedure of World Association for Advancement of Veterinary Parasitology WAAVP proposed by Coles et al. 1992 12 with slight modification Lourde Raj et al. 2006 Laksmipriya 2012 41 36 . Assessment of reversal effect of buthionine sulfoximine by EHA The effect of BSO was performed as similar to egg hatch assay in this test different concentrations 200µM 500µM and 700µM of BSO B2515 Sigma Aldrich USA added along with thiabendazole. Estimation of Intracellular glutathione concentration The total intracellular glutathione GSH was determined by a calorimetric method described by Greech et al. 1999 24 . TBZ resistant eggs and larvae of H. contortus were used in this assay. One gram of eggs and larvae were exposed to 1.0 µg/ml of TBZ and 200µM 500µM and 700µM of BSO at 2 4 and 6 hours intervals. The observance was read at 413nm against a blank and the amount of glutathione is expressed as nano mole per gram of larvae by UV-VIS mini-1240 spectrophotometer A109035i Shimadzu Asia Pacific Pte Ltd Singapore. Statistical Analysis Data analysis was carried out using SPSS 20 version software SPSS 20.0 Inc. Chicago Illinois USA. To analysis the mean hatching percentage and intracellular glutathione level between resistant populations before and after treatment with BSO data were subjected to a multivariate analysis ANOVA with Tukey’s post-host test. The differences were considered statistically significant when P0.001. The ED 50 were calculated using probit analysis. Result In the present study a total of 1003 abomasal samples of sheep and goat were examined at the slaughter house Perambur Chennai India. Out of which 534 samples were found to contain male and female worms of Haemonchus contortus on dissection. Prevalence of H. contortus in small ruminants slaughtered in Perambur slaughter house Chennai Tamil Nadu India was 53.2 recorded. Assessment of resistance by Egg hatch assay EHA The assessment of resistance and susceptible worms were based on the ability of eggs to hatch at concentration greater than 0.1µg/ml of thiabendazole TBZ. In present study samples were found to be resistant to TBZ by EHA where larvae could be seen even at concentrations higher than 1.0 µg/ml of TBZ. The mean hatching percentage at different concentrations i.e. control 0.05 0.1 0.3 0.5 and 1.0 µg/ml was recorded and analysed Table 1. Using probit analysis ED 50 value for benzimidazole resistance worm was 0.193µg/ml. Table 1: Comparative egg hatching percentage Mean ±SEM after in vitro egg hatch assay EHA to see the anthelmintic resistance and reversal of anthelmintic resistant in Haemonchus contortus in sheep and goat treatment with thiabendazole Alone and a combination of thiabendazole and 200 500 and 700µM L- buthionine sulfoximine L-BSO compared with control Treatment Comparative egg hatching percentage Mean ±SEM No L-BSO Alone TBZ L-BSO 200µM L-BSO 500µM L-BSO 700µM Control No TBZ 82.44 ±1.59 78.02 ±1.31 77.31±1.29 79.75 ±1.14 TBZ 0.05 µg/ml 74.78 ±1.58 62.53 ±1.64 52.73±1.57 36.70 ±1.77 TBZ 0.1 µg/ml 62.04 ±1.90 54.34±1.96 40.04±1.88 26.29 ±1.18 TBZ 0.3 µg/ml 48.80 ±2.82 40.09±2.53 34.24 ±2.25 22.48±1.68 TBZ 0.5 µg/ml 37.56 ±2.16 24.80 ±1.93 16.39 ±1.39 4.53 ±0.86 TBZ 1.0 µg/ml 4.02±0. 64 2.80 ±0.39 1.21 ± 0.27 1.02 ±.23 ED50 0.193µg/ml 0.137 µg/ml 0.077 µg/ml 0.033µg/ml Result Resistant Resistant Susceptible Reversed Susceptible Reversed Level of significance: P0.001 Assessment of reversal effect of buthionine sulfoximine The mean percentage of egg hatch at different concentration of TBZ i.e. 0.05 01 0.3 0.5 and 1.0µg/ml with combination of 200µM 500µ and 700µM were recorded and It was significantly P0.001 reduced in all concentrations. Using probit analysis the ED 50 values after the combination of TBZ and 200 µM 500 µM and 700 µM concentrations of reversal agent BSO as 0.137 0.077 and 0.033µg/ml TBZ were recorded respectively. Fig 1: Thiabendazole Resistance and effect of BSO in Thiabendazole resistance

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1363 Journal of Pharmacognosy and Phytochemistry Estimation of intracellular glutathione Intracellular glutathione has been estimated in eggs and larvae of TBZ resistant H. contortus before and after exposure to different concentration of 200 500 and 700µµM of BSO and OD values were taken at 412nm in2 4 6 hour intervals. It was found that the glutathione level was significantly P0.001 reduced in all concentrations Table. 2. Regression coefficient R square between intracellular reduced glutathione GSH and egg hatching percentage at 1.0µg/ml of TBZ in 2 4 and 6 hours intervals were 0.372 0.615 and 0.354 calculated respectively. Table 2: Reduced intracellular glutathione Mean±SEM in Haemonchus contortus when treatment with thiabendazole 1.0 µg/ml alone and combination of thiabendazole and L-BSO 200 µM L-BSO 500 µM and L-BSO 700 µM at two four and six hours intervals Treatment Intracellular glutathione nmol/g Mean± SEM Two hours Four hours Six hours TBZ 1.0 µg/ml 39.09±1.08 24.65 ±0.61 14.04 ±0.82 TBZ+ L-BSO 200 µM 28.94±1.23 15.30 ±1.05 7.52 ±0.61 TBZ+ BSO 500 µM 18.02±0.69 8.08 ±0.19 2.14 ±0.30 TBZ+ BSO 700 µM 8.03±0.39 4.01±0.40 1.17 ±0.97 Level of significance: P 0.001 Fig 2: Estimated intracellular reduced glutathione nmol/gin H. contortus of sheep/goat after treatment with thiabendazole alone and combination of thiabendazole 200µM L-BSO 500µM L-BSO and 700 µM L-BSO at two four and six hour interval Discussion The previous studied about prevalence of H. contortus reported by scientist time to time. Fakae 1990 18 conducted survey in the Nigerian derived savanna from August 1987 to July 1988 on the seasonal fluctuations in the composition of Haemonchus contortus burden of naturally infected West African Dwarf sheep and goats and found that the incidence of H. contortus infection was high 77.8–100 with no definite seasonal distinction. H. contortus infection lowers in the summer season as compared to winter and rainy season Garg et al. 2003 20 . Yadav et al. 2006 60 recorded that the seasonal variation throughout the year and was highest during the rainy season 88.54 followed by summer 83.15 and winter 76.01. Shugufta et al. 2005 54 studied the incidence of gastrointestinal nematodes in sheep of Kashmir valley. Five types of nematodes viz. strongyles Trichostrongylus spp. Haemonchus spp. Nematodirus spp. and Marshallagia spp. were identified. The seasonal prevalence of infections indicated that the nematode infection overall was highest in summer 67.14 and lowest in winter 44.31. Al- shaibani et al. 2008 1 observed for 12 months and H. contortus 24.6 were found to be predominantly of gastrointestinal nematode parasites. Qamar et al. 2008 50 found overall the highest 43.69 seasonal prevalence in sheep and Goats was recorded during summer followed by autumn 38.46 spring 37.12 while the lowest 28.79 was recorded during winter. Lashari et al. 2015 37 reported the prevalence of Fasciolia hepatica Avitellina centripunctata Haemonchus contortus and Trichuris globulosa was 21.41 12.23 6.50 and 5.73 respectively and suggest that the age sex body weight and breed are important factors which influence the prevalence of gastrointestinal parasites. Tasawar et al. 2010 55 reported the overall prevalence of Haemonchus contortus was 77.7 in sheep at Government Research Centre for Conservation of Sahiwal Cattle RCCSC Jehangirabad District Khanewal from February 2007 to June 2007. Mesele et al. 2014 46 perfomeda cross sectional was conducted to estimate the prevalence of haemonchosis in small ruminants through examination of 613 Abomasum of small ruminants 355 sheep and258 goats. The overall prevalence in this study was 38.6 with a prevalence of 22.8 and 15.8 were recorded for sheep and goats respectively. similarly number of studied about prevalence of haemonchosis in sheep and goat conducted time to time with 57.8 9.18 Degheidy et al. 2014 13 40.9 Gebresilassie and Tadele 2015insheep and goats 67.2 and 56.6respectivelyBulbul et al. 2015 12.1 Boukhari et al. 2016 7 and 38.0 Castle 2017 11 . Coles et al. 1992 12 given the procedure of egg hatch assay and eggs hatching with an ED 50 value in excess of 0.1 g TBZ per ml were indicative of benzimidazole resistance. Varady and Corba 1999 59 found egg hatch assay to be sensitive and accurate to determine benzimidazole resistance in H. contortus. They demonstrated that the ED 50 in resistant strain was different from the susceptible strain by resistant factors. Calvete et al. 2012 10 also found 11 per cent of

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1364 Journal of Pharmacognosy and Phytochemistry TBZ resistant nematodes in sheep farms in Northeast Spain using egg hatch assay. The mean percentage of egg hatch in 42 resistant samples was above the discriminating dose of 0.1 µg per ml of TBZ and larvae hatched even at higher concentration of TBZ. This is similar to the report of Le Jambre 1976 38 who showed that TBZ resistant strains hatched in higher concentration of TBZ than non-resistant strains. Taylor et al. 2002 57 reported eggs from susceptible individuals rarely hatch at concentrations greater than 0.1µg / ml of thiabendazole. In the previous studied many scientist and researchers reported the anthelmintic resistance by egg hatch assay with range of ED 50 Value as 0.586 Arunachalam et al. 2005 2 0.627 0.678 and 0.388 µg / ml Easwaran et al. 2009 16 17 and 0.8 and 0.6 µg/ml of benzimidazole Lourde raj et al. 2006 41 17.9 Lakshmipriya 2012 36 ED50 for egg hatch was 0.196 with lower and upper limit ofED 50 of 0.051 and 0.329 Dinesh 2013 14 0.059 µg/ml Rialch et al. 2013 51 0.299µg/ml of albendazole Minakshisundram et al. 2014 45 The ED 50 for egg hatch was 0.196 indicating suspected resistance to benzimidazole anthelmintics Kumbhakar et al. 2015 35 Egg hachability 19.66 Goncalves et al. 2016 23 . Buthionine sulfoximine BSO was used as reversal agent in the present study and found significant reversal effect which was dose depended. The reversal of resistance occurred when TBZ resistance eggs were treated with BSO and resulted in inhibition of egg hatching. Beugnet et al. 1997 5 performed egg hatch assay EHA and demonstrated the relationship between P-glycoproteins p-gp and benzimidazole resistance through the use of the p-gp inhibitor verapamil a calcium channel blocker. They had shown that in the presence of verapamil the toxicity of the drug increased and that benzimidazole resistance could be partially reversed. Kerboeuf et al. 1999 31 examined membrane drug transport mechanism of resistance to anthelmintics by flow cytometry using rhodamine 123 a p-gp transport probe and found a higher level of green fluorescence in eggs of resistant H. contortus indicating high levels of expression of pgp in resistant eggs. Riou et al. 2003 52 confirmed that cholesterol could modulate p-gp activity on nematode eggs and change the level of resistance to anthelmintics. They suggested changes in membrane lipid contents could provide another way to improve the reversion of resistance and to increase the efficacy of anthelmintics. Kerboeuf et al. 2008 32 suggested that the combination of anthelmintic targeting nematodes with an inhibitor of p-gp efflux pumps had a significant effect on both egg excretion and the number of worms coming from anthelmintics resistant nematodes. They opined that this discovery would open up new perspectives in nematode control by maintaining a good efficiency of the treatment while reducing the doses of active compound. They analyzed the functional consequences of the localization for xenobiotic transport and drug resistance in nematodes and compared with results obtained in vertebrates. They suggested that understanding of such mechanisms was crucial in overcoming the failure of drug treatments due to the development of resistance. In this study addition of BSO to TBZ has inhibited hatching of resistant eggs by increasing toxicity of TBZ. Blackhall et al. 2008 32 studied an association between a specific allele of p-gp and survival of benzimidazole treatment. They suggested that by reducing the amount of drug to reach its target p-gp could act as a general defensive mechanism against xenobiotics. Bartley et al. 2009 a 3 showed the influence of various p-gp interfering compounds on the efficacy of ivermectin sensitive and resistant nematode isolates. They also demonstrated that in the presence of p-gpinterfering agents the in vitro susceptibility to ivermectin of both sensitive and resistant isolates of T. circumcincta and H. contortus was increased. Bartley et al. 2009 b 4 described the increased sensitivity of resistant larvae to ivermectin after the co-incubation with pluronic 85 but in vivo co-administration of ivermectin with this p-gp modulator to sheep did not show an improved efficacy against resistant H. contortus. Lifschitz et al. 2010 a 39 showed by field trial done in Argentina that the efficacy of both ivermectin and moxidectin against resistant Cooperia spp. in cattle tended to increase after their co-administration with loperamide LPM as a p-gp modulator. Lifschitz et al. 2010 b 40 reported the effects of loperamide LPM a p-gp modulating agent on both ivermectin kinetic behaviour and anthelmintic activity in infected lambs. Described that the raft-like structures RLSs in egg shell colocalized with a large proportion of the p-gp. They analyzed the functional consequences of the colocalization for xenobiotic transport and drug resistance in nematodes and compared with results obtained in vertebrates. They suggested that understanding of such mechanisms was crucial in overcoming the failure of drug treatments due to the development of resistance. Godoy 2010 22 studied that the expression of HcPgp-A in transfected LLC-PK1 cells and to see the effect of ivermectin and moxidectin on inhibition of rhodamine 123 transport by the transfected cells. Rhodamine 123 was actively transported by HcPgp-A. Ivermectin was four fold more potent at inhibiting rhodamine 123 transport by HcPgp-A than was moxidectin. The result provided the first information that MLs can inhibit the transport of Pgp substrates by a parasitic nematode ABC transporter and may indicate an active role for H. contortus Ppgs in ML resistance. Heckler et al. 2014 26 evaluated the in vitro effect of eight P-gp modulating drugs to potentiate IVM efficacy against an IVM-resistant field isolate of H.placei. The association of IVM with cyclosporin-A ceftriaxone dexametha-sone diminazene aceturate quercetin trifluoperazine verapamil or vinblastine resulted in increased IVM 10−4M efficacy of 5.1 49.06 76.42 3.31 28.85 13.74 45.64 and 43.61 respectively. Hussein et al. 1996 28 reported that effect of DL- buthionine- S R sulfoximine BSO a selective glutathione GSH depleting agent on the GSH synthesis of Ascaris suum. The GSH concentration of reproductive and muscle tissues of A. suum was 8.5 ± 0.3 and 14.3 ± 1.3 nmol/mg protein respectively and after treatment of parasites with BSO for 24 h the GSH content of reproductive tissue was totally depleted as compare to untreated control. Faundez et al. 2005 19 found that L - buthionine-S R sulfoximine BSO increased the toxicity of nifurtimox and benzimidazole towards the epmastigaote tryapomastigote and mastigote form of Trypanosoma cruzi. BSO at 500µm decreased the total glutathione-derived thiol by 70 to 80 in 48 hours. Hussein et al. 2005 29 the Treatment of the Brugia malayi with different concentrations of 20 100 200 and 500 μM BSO lowered the GSH content by 70 47 35 and 23 respectively. The effect of BSO is dose dependent. Buthionine sulfoximine S-n-buty1 homocysteine sulfoximine is a potent and specific inhibitor of y-glutamylcysteinesynthetase administered to animals orincorporated into tissue culture media it inhibits glutathione biosynthesis and causes depletion of cellular glutathione levels. Glutathione the most abundant intracellular nonproteinsulihydryl compound has various cellular functions mainly related to the thiolgroupof the cysteine

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1365 Journal of Pharmacognosy and Phytochemistry residue. The reduced glutathione GSH as a cosubstrate of glutathione peroxidase plays an essential protective role against oxygen-reactive species that may be generated under various conditions. This protective mechanism results in increased formation of oxidized glutathione GSSG which is actively transported across the cell membrane so that its intracellular concentrations are kept low. Drug resistance has been correlated to increased GST and GSH level in certain nematodes like Haemonchus contortus Kwalek et al. 1984 30 . Ullah et al. 2017 58 estimated reduced glutathione as total acid soluble sulfhydryl concentrations colorimetrically using Ellmans reagent 5 5-dithiobis-2-nitrobenzoic acid or DTNB according to thymoquinone and curcumin effect on Fasciola gigantica. The level of reduced glutathione was significantly inhibited by both curcumin as well as thymoquinone at the highest concentration 60 μM used. The present study showed the increase GSH level in resistance H. contortus and after treatment with GSH inhibitors BSO level of GSH was decreased. The our studied showed the relationship between egg hatching percentage and reduced glutathione with regression of coefficient at 24and 6 hours interval after treating with thinbendazole and L-BSO. When reduced intracellular glutathione is responsible for is 61.5 hatching of egg when treated with 500µM L-BSO. Conclusion The present experiment was conducted to see the potentiation of thiabendazole efficacy by buthionine sulfoximine BSO. BSO can be used as AR reversal agent along with BZ drugs. Enhances the TBZ toxicity by reducing the GSH concentration. Suppressing the antioxidant/redox potential of the worms. Acknowledgement The Dean Madras Veterinary College Tamil Nadu Veterinary and animal Science University Chennai India 600 007 for providing facilities to conduct research work. References 1. AL-Shaibani IRM Phulan MS Arijo A Qureshi TA. 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