Iraqi J Pharm Sci, Vol.18(2) .2009 Genetic variations of Echinococcus granulosus 61 Genetic Variations of Echinococcus granulosus Isolated from Sheep and Cows by Using Fingerprint DNA Method in Iraq Zaman A.A. Ibrahim * ,1 , Salwa S.Muhsin ** and Farhan A. Risan *** * Department of Basic Medical Sciences, College of Nursing, University of Baghdad, Baghdad, Iraq, ** Department of X RAY, Institute of Medical technology, Foundation of Technical Education, Baghdad, Iraq, *** Department of Medical Pathological Technology , College of Health and Medical Technology ,Baghdad , Iraq. Abstract The fingerprinting DNA method which depends on the unique pattern in this study was employed to detect the hydatid cyst of Echinococcus granulosus and to determine the genetic variation among their strains in different intermediate hosts (cows and sheep). The unique pattern represents the number of amplified bands and their molecular weights with specialized sequences to one sample which different from the other samples. Five hydatitd cysts samples from cows and sheep were collected, genetic analysis for isolated DNA was done using PCR technique and Random Amplified Polymorphic DNA reaction(RAPD) depending on (4) random primers, and the results showed: 1-Ability of OPF-13 primer to find fingerprinting for DNA of hydatid cysts collected from cows with (3) unique patterns and from sheep with (5) unique patterns. 2- Ability of OPF-06 primer to found (4) unique patterns for cows and (2) unique patterns for sheep. 3-Ability of OPF-19 primer to find fingerprinting for DNA of hydatid cysts collected from cows (5) unique patterns and (4) unique patterns for sheep. 4-OPF-16 was unable to find the finger prints of DNA isolated from cows and sheep Keywords : Hydatid cyst. OPF Primer .PCR. RAPD. صة الخال لذ اسخخذهج في ُزٍ Unique patternالخي حعخوذ على الٌسك الوخويض Fingerprinting DNAاى طشيمت البظوت الْساثيت الذساست لخشخيض طفيلي األكيبط ألعذسيَ ّهعشفت االخخالفبث الْساثيت في هضبئفَ الْسطيت الوخخلفت "األغٌبم ّاألبمبس" . أر يوثل عذد الحضم الٌبحجت هع أّصاًِب الجضيئيت الخبطت ّألخخببع ألخبص ببلعيٌت ّالخي حويضُب عي بميت العيٌبث ليذ الذساست ّالخي الٌسك الوخويض كبًج حشخول على خوست عيٌبث لكل هي األكيبط العذسيَ ألوعضّلَ هي األغٌبم ّاألبمبس . ّبعذ ححليل العيٌبث ببسخخذام حمبًت الـ RAPD ببدئبث عشْائيت. أظِشث الٌخبئج هب يلي 4ببالعخوبد على– ّبخٌسيك OPF-6حن إيجبد بظوت ّساثيت للعيٌبث الخوست لذًب األكيبط العذسيَ الوعضّلت هي األغٌبم ّاألبمبس ببسخخذام الببدئ -١ للعيٌبث جويعب. unique patternهخويض ظوت ّساثيت لذًب عيٌبث األكيبط العذسيَ الوعضّلت هي األغٌبم ّبخٌسيك هخويض على الخْطل إلى إيجبد ب OPF -13لذسة الببدئ -٢ ( أًسبق لألبمبس.۳بعذد خوست اًسبق ّ) على إيجبد بظوت ّساثيت لذًب عيٌبث األكيبط العذسيَ الوعضّلت هي الوضبئف الْسطيت األبمبس ّبٌسك OPF -19لذسة الببدئ -٣ ( أًسبق لألغٌبم.4) ( ٥ّهخويض بعذد ) على إيجبد بظوت ّساثيت لذًب عيٌبث األكيبط العذسيَ الوعضّلت هي الوضبئف الْسطيت OPF -16عذم لذسة الببدئ -٤ ّاألبمبس( الوسخخذهت في الذساست. األغٌبم ) Introduction Hydatidosis is a most serious zoonosis disease caused by larval stage hydatid cysts Of tapeworms Echinococcus granulosus which effect human and wide rang of livestock (1,2) and the ultimate growth of the cyst depends on location inside the host, so in some areas of the body they are unable to expand freely whereas in other most growth results in serious impairments to the function of vital structures or even in death (3) . These parasites live for long times in the hostile medium of the host and their struggle for life gave them various strategies that allow them to feed, reproduce and evade the immune system and immunosurveillance and promote chronic infection (4) .Many methods were used for treatment of this disease by (surgically, chemotherapy, physically…etc), surgical remain the primary choice and method, others still ineffective or complementary) for controlling and inhibition of the disease distribution (5) , and many advanced laboratory methods were used for the diagnosis of this parasite in human and animals (6) . 1 Corresponding author E – mail - : zamanbrahim@yahoo.com Received : 17 / 3 / 2009 Accepted : 27/ 10 /2009 Iraqi J Pharm Sci, Vol.18(2) .2009 Genetic variations of Echinococcus granulosus 62 Aim of the study The experimental studies at molecular level till now absent or hardly very rare about this parasite(adult and larval stage) in Iraq , so this study was carried out using DNA technique depending on Polymerase Chain Reaction(PCR) method and strictly indicator Random Amplified Polymorphic DNA(RAPD) to find out the fingerprinting which used to specify the sequences characterize the DNA which isolated from cows and sheep because first of all ,the high sensitivity of this technique to determine the genetic variations between all animals hydatid cysts isolates, and secondly easy and perfect method to be used (7) . Materials and Methods. Samples Collection. Genetic materials DNA had been isolated from hydatid cysts specimens of cows and sheep which collected from different slaughterhouses in north, middle, and south of Iraq during the period from June 2005 - June 2006 Design of experiments. The DNA isolation was done as follow. - One cm³ specimens from tissues germinal layer of hydatid cyst were isolate from cows and sheep & preserved into 70% ethanol with700µL of proteinase buffer was added {Method was cited by (8) and minor modified by ( 9) .} - The tissues had been cut by small and sharp scissor into small pieces, and then 35L of proteinase –K enzyme solution has been added and incubated for 24 hours at 55C in the incubator, - Ten µL RNAase enzyme solution has been added and incubated at 37C for 1hour, then the same volume of (Chloroform, phenol, isomil alcohol) solution was added and mixed by vortex For 1 minute and centrifuged at 12000 rpm for 15 minutes. - The upper layer pulled out and transferred to a clean, dry, tube, this process had been repeated many times till the middle white layer disappeared, then o.6 ml volume of Isopropanol was added and then left at room temperature for 1hour and centrifuged at 1400 rpm for 10 minutes. - The supernatants then removed and the precipitants washed with (70%, 95%) ethanol respectively, the tube left open to complete drying from ethanol, then the DNA Dissolved by 150 µL Tris-EDTA (TE) with pH 8 and incubated for 2 hours at 65 C to complete dissolving. - Then whole solution was kept at deep freeze (20C). - After the DNA characterization by measuring DNA concentration and estimation of its Purity ( 10, 11) using Spectrophotometer with wave length 260nm, RAPD was done according to (12) method which includes the following steps: 1- The master mixture solution was prepared by mixing the following constituents In sterile plain tubes with following concentrations for each constituent: Cows (5) samples Sheep (5) samples Final concentration Volume of each sample constituents 424.8 354 - 17.7 Distilled water 60 50 1 X 2.5 Buffered solution x 10 12 10 200 µMole 0.5 dNTPs 08 40 Pmol/R 2 Primer 7.2 6 1.5µL 0.3 µL Polymerase enzyme 2- The constituents mixed well by vortex for 30 seconds and centrifuged for 30 seconds to precipitate all solution drops that suspended on tube wall. 3- Each tube which contained cows and sheep samples had been distributed in very sterile 0.5 ml Eppendorf tube labeled with names of 5 hydatid cysts isolates of the study. 4- Two µL from each DNA sample of both sheep and cows after performing several dilutions for the concentrated samples by D.W to reach the final required concentration starting the RAPD reaction Of DNA chain which ranged between 10-50ngm / each 1 µL of DNA sample and the final volume became 25µL (13) . 5- The constituents were mixed and left for few minutes in centrifugation to collect the contents of Reaction in the bottom of the tube, then 20-25µL of mineral oil to prevent the evaporation during the duplication process when the temperature reaches up to 94 C (14,15) . 6- The tubes were transferred to thermocycler to start the duplication reaction according to the the following program:  One round for 2 minutes at 94 C for primary denaturation of DNA strands .  Forty duplicated rounds, each round for 2 minutes at 92 C to denatures the matrix ,1 minute at 36 C to bind the primers with DNA matrix, and 2 minutes at 72 C for elongation of the bonded primers ,with last round for final elongation for 10 minutes at 72 C. Iraqi J Pharm Sci, Vol.18(2) .2009 Genetic variations of Echinococcus granulosus 63 7- The tubes were removed from thermocycler, and volume of (23-24) µL from the contents was pulled from under the oil and mixed with 3 µL loaded solution. 8-Agarose gel with concentration 1.2% was prepared and electrophoresis by well method was done On the Samples with volumetric indicator formed from DNA treated by (ECO1) enzyme for 4-5 hours. 9- The gel was examined after staining with ethidium bromide (1mg/ml) under Ultra - Violet and black and white Polaroid 667 photos were taken to the gel. 10- The molecular weights of the duplicating segments were calculated depending on the bands positions with known molecular weights which produced from DNA cutting enzyme (ECO1) only that considered standard volumetric agents. Four primers were tested. 1- OPF – 13 3' GGCTGCAGAA 5' 2-OPF – 06 3' GGGAATTCGG 5' 3- OPF – 19 3' CCTCTAGACC 5 4- OPF– 16 3' CGAGTACTGG 5' These primers were produced by (Operon Technologies Alameda–A) to be used in final analysis Of RAPD experiments, and the results which appeared in the gel were converted into Binary characters tables by putting number (1) if the band was found and number (0) if the band was not found and the molecular weights were calculated for the duplicated products in comparison with volumetric agent (ECO1) only. In order to characterize each sample for the studied sample, we investigate the finger- prints or the duplicated bands and we determined the organized sequences characterization (14) . Results and Discussion: In Iraq the studies about genetic variation between animals infected with hydatid cysts at molecular level almost very rare specially using fingerprint for identification. By using the four primers (OPF 6, 13, 16, 19) the results showed their reactions in RAPD technique that they were different in duplicated bands production and their molecular weights with each hydatid cyst DNA samples taken from cows and sheep. - The primer OPF-13. This primer was able to find (3) types for unique patterns of DNA which isolated from cows (Figure 1A, Table 1A) and ( 5 ) types for unique patterns of DNA isolated from sheep (Figure 1B, Table 1B), and these results agree with the results conducted by (16) who they said that there are differences in sequences characterizes of duplicated bands and this primer gave a description for genetic material and genetic pattern of DNA isolated from cows and sheep (17) . Cows A NO. of sample Sheep B NO. of sample Figure 1: show the duplication production on agarose gel (1.2%) for hydatid cysts samples isolated from cows (A) and sheep (B) using OPF-13 primer 3.5 hours and with 65 volts . M 1 2 3 4 5 M 1 2 3 4 5 Iraqi J Pharm Sci, Vol.18(2) .2009 Genetic variations of Echinococcus granulosus 64 Table1: show the calculation method of sequences characterizes using primer OPF- 13. A Numbers of unique patterns (3) Isolated from cows. (0) = duplicated bands not exist (1)=Duplicated bands exit Different colors means numbers Of unique patterns. B Number of unique patterns (5) For DNA isolated from sheep. (0) = duplicated bands not exist (1) =Duplicated bands exit Different colors means numbers Of unique patterns. The primer OPF-6. This primer was able to found the fingerprint with (4) types of unique patterns for DNA hydatid cysts which isolated from cows (Figure 2A , Table 2A) and ( 2) types for unique patterns of DNA isolated from sheep ( Figure 2B, Table 2B)( 18) . Figure 2 :show the duplication production on agarose gel (1.2%) for hydatid cysts samples Isolated from cows (A) and sheep (B) using OPF-6 primer for 3.5 hours and with 65volts ١ ٢ ٣ ٤ ٥ Mw bp 8 8 1 1 1 ١011 8 8 1 1 1 ١٦11 8 1 1 1 1 ١١11 1 1 1 1 1 011 8 1 1 1 1 001 1 1 8 8 8 001 1 8 1 1 1 0٥1 1 1 8 8 8 0٣1 1 1 1 1 1 ٦01 1 1 1 1 1 ٦٣1 8 1 8 8 8 ٥٤1 1 8 8 8 8 ٤٢1 1 8 8 8 8 ٣01 1 1 1 1 1 ٣٦1 ١ ٢ ٣ ٤ ٥ Mw bp 1 1 8 1 1 ١011 8 8 8 1 1 ١٦11 8 1 8 1 1 ١١11 1 1 1 1 1 011 1 8 1 8 1 001 1 1 8 8 8 001 1 1 1 8 8 0٥1 1 1 8 8 8 0٣1 8 1 1 1 1 ٦01 1 1 8 1 1 ٦٣1 8 1 1 8 8 ٥٤1 8 1 1 8 8 ٤٢1 8 1 1 8 8 ٣01 1 1 1 1 1 ٣٦1 Cows A M 1 2 3 4 5 Sheep B M 1 2 3 4 5 Iraqi J Pharm Sci, Vol.18(2) .2009 Genetic variations of Echinococcus granulosus 65 Table2 : show the calculation method of sequences characterize using primer OPF-6 A Mw Bp 1 2 3 4 5 1100 0 1 1 1 0 1000 1 0 0 1 0 990 1 1 1 1 1 800 1 0 0 0 0 770 1 1 1 1 1 650 1 1 1 1 1 450 1 1 1 1 1 Numbers of unique patterns (4) For DNA Isolated from cows. (0) =Duplicated bands not exist (1) =Duplicated bands exist Different colors means number of Unique patterns. B Mw Bp 1 2 3 4 5 1100 0 0 1 1 0 1000 0 0 1 1 0 990 0 0 1 1 0 800 0 0 1 1 0 770 1 1 1 1 1 650 1 1 1 1 1 450 0 0 1 1 0 Numbers of unique patterns (2) For DNA isolated from sheep. (0)=Duplicated bands not exist (1)= Duplicated bands exist Different colors means number of Unique patterns. The Primer OPF -19 This primer showed (5) unique patterns for DNA hydatid cysts samples of cows (Figure 3A, Table 3A), and (4) Unique patterns for sheep DNA hydatid Cysts samples (Figure 3B, Table3B). It is clear from unique patterns number differences between the samples that the complementary loci for this primer which used in RAPD indicators not evenly distributed between the samples (19) , and the appearance of unique pattern specific for cow's hydatid cysts DNA represent fingerprint for these samples differentiated them from other samples by using this primer (20) . From this study, we could diagnosis the DNA of sheep and cows hydatid cysts using RAPD method with banding patterns and their molecular weights, so, by fingerprints the only three primers were able to diagnosis the genetic variations and differences between all hydatid cysts samples (21). NO. of sample NO. of sample Figure 3: show the duplication production on agarose gel (1.2%) for hydatid cysts samples Isolated from cows (A) and sheep (B) using OPF-19 primer for 3.5 hours and with 65 volts . Cows A Sheep B M 1 2 3 4 5 M 1 2 3 4 5 Iraqi J Pharm Sci, Vol.18(2) .2009 Genetic variations of Echinococcus granulosus 66 Table-3 show the calculation method of sequences characterizes using primer OPF-19. A Mw bp 1 2 3 4 5 1050 0 1 1 1 0 1000 0 1 1 1 1 980 8 1 1 8 8 860 8 8 1 1 8 750 1 1 1 1 1 630 0 1 1 8 1 Numbers of unique patterns (5) For DNA Isolated from cows. (0) =Duplicated bands not exist (1)= Duplicated bands exist Different colors means numbers of Unique patterns. B Mw bp 1 2 3 4 5 1050 0 1 0 0 0 1000 1 1 1 1 1 980 1 1 1 1 1 860 8 1 1 1 8 750 8 1 1 1 1 630 0 1 1 1 1 Numbers of unique patterns (4) For DNA isolated from sheep. (0) =Duplicated bands not exist (1)= Duplicated bands exist Different colors means numbers of Unique patterns. The Primer OPF-16 This primer had no ability to found hydatid cyst DNA and gave them unique patterns by finger prints for cows and sheep, this primer failed to recognize the genetic variations by using RAPD reaction and fingerprints method in the presence of hydatid cysts sample which had no broad genetic background and its high recognition ability failed to show a unique pattern of the studied samples (18) . Acknowledgements Sincerely, we must gift our deep thanks to all staff members in all Baghdad–city slaughter- houses for their great help to obtain intact hydatid cyst with our best heart wishes for them. 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