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2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia) Comparative Estimation of Total Protein Content and Enzymatic Activities of Hydatid Cyst of Ecchinococcus granulosus Isolated from Sheep and Goats in Duhok Province, Kurdistan Region of Iraq Wijdan M.S. Mero and Arshad Mohammad Abdullah become infected accidentally by ingesting food, drink or other materials contaminated with the egg of this parasite[2]. There are 4 known species of Echinococcus, namely: Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli and Echinococcus oligarthrus. [9]. Three of these species are of medical importance, Echinococcus granulosus, causing cystic echinococcosis (CE) which is the most prevalent species in all continents, causing a considerable public health problems in many regions of the world [2]. Furthermore, it is also common in Iraq [1]. E. multilocularis, causing alveolar echinococcosis (AE) is more widely distributed in the northern hemisphere, and represents a considerable public health burden as it’s the most virulent species and infection with it is lethal in most untreated patients. However, biochemical studies are also useful in differentiating strain variations of E.granulosus in different countries [11]. The present study was designed to evaluate the Some biochemical studies on the parasite and its intermediate hosts, which included the activity of some key enzymes and total protein contents. These findings will give us some information about the strain identification and treatment of hydatid disease. Abstract—These This study included comparative Enzymatic and total protein contents of hydatid cyst of Ecchinococcus granulosus isolated from liver and lungs of infected sheep and goats in Duhok abattoirs. With respect to enzymes the activity of the following enzymes were measured namely acid phosphatases (ACP), alcaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamate oxaloacetate transminase (GOT) and glutamate Pyruvate transminase (GPT) in cysts isolated from both liver and lungs of infected sheep and goats. Regarding protein content of protoscolices was higher than hydatid cyst fluid, but it was lower than infected host tissues.The activities of all these enzymes were higher in prtoscolices as compared with their activities in hydatid fluid, but in general infected host tissue showed the highest enzymatic activities as compared with hydatid cyst. II. MATERIALS AND METHODS For this study a total of 36 hydatid cysts (Fluid and tissue of infected organs) of sheep and goats in addition to 8 samples of organs (liver and lung) from non infected animals (sheep and goat) were collected to be used as a control. In this study we used total protein kit and some enzyme kits acid phosphatases (ACP), alcaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamate oxaloacetate transminase (GOT) and glutamate Pyruvate transminase (GPT) and some Buffer systems (Citrate buffer, Carbonate-Bicarbonate buffer and Tris-HCl buffer) for homogenization of tissue. Hydatid cyst fluids were taken in a special test tubes (5-8 mL), separated by centrifugation at 4000 rpm for 30 minutes and the supernatants was separated and stored in deep freezer at 40˚C until used. The protoscolices separated by centrifugation of hydatid cyst fluid then homogenization of protoscolices and centrifugation of extract at 4000 rpm for 30 Fig. 1 Hydatid cysts and infected organs Keywords— ACP, ALP, Ecchinococcus granulosus, GOT, GPT. I. INTRODUCTION YDATID disease (hydatidosis or echinococcosis) is a silent cyclozoonotic infection of man and domestic animals, caused by the larval stage of Echinococcus granulosus (small dog tapeworm). Man and domestic animals H Arshad Mohammad Abdullah, M.Sc. in Biology/ Parasitology, University of Duhok, phone: 009647504572331, e-mail: [email protected]. Prof. Dr.Wijdan M.S. Mero, Professor of Parasitology, Head of Biology School, Faculty of Science,University of Zakho, 14 2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia) minutes and the supernatants were collected and stored in a deep freezer at -40˚C till used. The liver and lung tissues of infected and non infected animals homogenized with a desired buffer, the supernatant was collected and stored in deep freezer at -40˚C till used [8]. Hydatid cyst fluid and extracts of protoscolices and host tissues were used for determination of total proteins and enzyme activities. enzyme in high activity in both hydatid fluids and protoscolices of E. granulosus as well as infected host tissue. Similar high activities of ACP enzyme was observed by McManus and Bryant (1995) [6] in sheep hydatid cysts of E. granulosus, Sherif et al., (1984) [17], they reported it in hydatid fluid. TABLE2 ACTIVITY OF ACP ENZYME III. RESULTS AND DISCUSSION A. Total protein content of hydatid cyst isolated from sheep and goats liver and lung: Table (1). Similarly Smyth (1979) [18], reported lower protein content in infected host tissue, and this low protein content was due to its use by the parasite, since the protoscolices of E. granulosus can digest host proteins. Meduri et al., (1990) [7], also reported low protein content in hepatic and pulmonary hydatid cysts of infected cattle which were 0.2 mg/100ml, 0.09mg/100ml respectively, these values were much lower than the values reported in the present study. On the other hand, the results of the present study disagree with those reported by Frayha and Haddad (1980) [3]; Ul-Haq (1994) [21] and Radfar and Iranyar (2004) [11], who stated that hydatid fluid and protoscolices of sheep liver hydatid cysts contain high concentration of protein. TABLE 1 TOTAL PROTEIN Specimens Hydatid Fluids ( g/dL ) Protoscolices (IU/100g) Infected Organ (IU/100g) Control Organ (IU/100g) Sheep Liver M ± SEM Sheep Lung M ± SEM Goats Liver M ± SEM Goats Lung M ± SEM 1.600 ± 0.084 N=7 2.086 ± 0.265 N=7 1.840 ± 0.13 N=5 2.386 ± 0.143 N=7 2.186 ± 0.135 N=7 2.229±0.18 7 N=7 1.925 ± 0.2810 N=4 ± 2.557 ± 0.146 N=7 2.420 ± 0.1463 N=5 2.050 ± 0.1708 N=4 2.904 ± 0.029 N=2 2.705 ± 0.033 N=2 2.503 ± 0.43 N=2 2.206 ± 0.128 N=2 1.557 ± 0.036 N=7 2.757 0.187 N=7 TABLE 3 ACTIVITY OF ALP ENZYME B. ACP activity in hydatid cyst isolated from sheep and goats liver and lung: Table (2). ACP is an important enzyme and is found in Liver and other organs [19]. McManus and Barrett (1985) [5], reported the presence of ACP enzyme in the sheep liver protoscolices. The results of the present study revealed the presence of this 15 2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia) TABLE 4 ACTIVITY OF LDH ENZYME TABLE 5 ACTIVITY OF GPT ENZYME Specimens Sheep Liver M ± SEM Sheep Lung M ± SEM Goats Liver M ± SEM Goats Lung M ± SEM Specimens Sheep Liver M ± SEM Sheep Lung M ± SEM Goats Liver M ± SEM Goats Lung M ± SEM Hydatid Fluids (IU/L) 583.7 ± 21.70 N=6 705.6 ±20.25 N=7 722.8 ± 4.176 N=5 749.3 ± 41.29 N=6 Hydatid Fluids (IU/L) 93.17 ± 2.393 N=5 108.0 ± 5.007 N=5 103.5 ± 3.08 N=7 107.0 ± 6.484 N=6 Protoscolices (IU/100g) 645.5 ± 33.70 N=4 707.3 ±30.06 N=7 740.5 ± 34.38 N=6 751.1 ±20.61 N=7 Protoscolices (IU/100g) 100.5 ± 4.786 N=6 112.2 ± 8.801 N=5 107.7 ± 6.53 N=7 109.9 ± 5.870 N=6 Infected Organ (IU/100g) 735.5 ± 16.61 N=6 732.1± 10.67 N=7 889.6 ± 37.82 N=5 896.8 ± 48.13 N=4 Infected Organ (IU/100g) 106.9 ± 7.951 N=7 114.0 ± 12.73 N=7 109.8 ± 8.68 N=7 113.2 ± 10.94 N=7 442.4 ± 2.568 N=2 453.2 ± 1.843 N=2 430.7 ± 0.391 N=2 427.1 ± 0.948 N=2 84.59 ± 1.359 N=2 79.74 ± 0.172 N=2 76.92 ± 0.931 N=2 67.65 ± 1.508 N=2 Control Organ (IU/100g) Control Organ (IU/100g) increases dramatically in acute liver damage and in infected host tissue with echinococcusis [24, 10]. In this study high GPT activity was observed in hydatid cyst and infected tissue. results in were reported by Sanchez and Sanchez, (1971) [13]; Frayha and Haddad (1980) [3]; McManus and Bryant (1995) [6] ; Zeheer (1997) [24]. Furthermore Zeheer (1997) [24], reported high GPT activity in hydatid cysts wall also. C. ALP activity in hydatid cyst isolated from sheep and goats liver and lung: Table (3). ALP is an enzyme which plays an important role in the metabolism, physiology, immunology and nutrient absorption of the cystic echinococcosis [4, 20, 15]. Frayha and Haddad (1980) [3], reported a higher activity of ALP enzyme in protoscolices as compared with hydatid cyst fluid. The higher activity of ALP in hydatid cysts (both protoscolices and hydatid fluid) than that of uninfected host tissue, is in agreement with the finding of Sarciron et al., (1991) [14], who also reported higher activity of this enzyme in hydatid cyst as compared to control host liver. F. GOT activity in hydatid cyst isolated from sheep and goats liver and lung: Table (6). The enzyme glutamate oxaloacetate transminase (GOT) is distributed in all body tissue with a high activity in liver parenchymal cells, and its level increases during liver damage [24, 19, 22]. In the present study, high GOT activity was observed in hydatid cysts(hydatid cyst fluid and protoscolices) isolated from sheep and goats liver and lung as well as infected sheep and goats liver and lung tissue. These results are similar to what have been reported by Sanchez and Sanchez (1971) [13]; Frayha and Haddad (1980) [3]; McManus and Bryant (1995) [6] ; Zeheer (1997) [24], who also detected high GOT activity in sheep hydatid cyst fluids, protoscolices, liver and hydatid cysts wall. D. LDH activity in hydatid cyst isolated from sheep and goats liver and lung: Table (4). LDH is an enzyme that catalyzes the conversion of lactate to pyruvate, it is present in large amounts in heart, kidney, liver, and other body organs [16]. The higher activity in sheep protoscolices as compared with the hydatid fluid LDH in this study is in agreement with the finding of Frayha and Haddad (1980) [3] and Zeheer (1997) [24], while McManus and Bryant, (1995) [6], Rouhani and Vatankhah (2008) [12], reported high LDH in hydatid fluid of sheep. E. GPT activity in hydatid cyst isolated from sheep and goats liver and lung: Table (5). Glutamate Pyruvate transminase (GPT) is an enzyme present in hepatocytes (liver cells), kidney and other body organs, it is important for metabolism of amino acid in krebs cycle and 16 2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia) TABLE 6 ACTIVITY OF GOT ENZYME Specimens Sheep Liver M ± SEM Sheep Lung M ± SEM Goats Liver M ± SEM Goats Lung M ± SEM Hydatid Fluids (IU/L) 90.03 ± 2.78 N=7 98.69 ± 3.91 N=7 73.93 ± 5.316 N=4 100.9 ± 4.04 N=5 Protoscolices (IU/100g) 94.05 ± 7.16 N=7 114.5 ± 12.87 N=7 81.78 ± 4.566 N=6 103.3 ± 4.98 N=6 Infected Organ (IU/100g) 105.2 ± 6.12 N=7 125.4 ± 12.71 N=7 94.55 ± 4.203 N=6 115.5 ± 4.796 N=7 63.46 ± 1.151 N=2 71.41 ± 1.04 N=2 54.47 ± 0.359 N=2 57.48 ± 0.982 N=2 Control Organ (IU/100g) [10] H. Nyblom,; U. Berggren,; J. Balldin, and R. Olsson,.High AST/ALT ratio may indicate advanced alcoholic liver disease rather than heavy drinking". Alcohol Alcohol. 2004, pp.336[11] M. H. 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