Genetic Variations of Echinococcus granulosus Isolated from Sheep and Cows by Using Fingerprint DNA Method in Iraq

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 fro m 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. 2Ability 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


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 me thods 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) .

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: .

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 ma trix ,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.

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 w as 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 fingerprints 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 w ith 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) .The primer OPF-6.

Cows
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) .

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 1: show the duplication production on agarose gel (1.2%) for hydatid cysts sample s isolated from cows (A) and sheep (B) using OPF-13 primer 3.5 hours and with 65 volts .

Figure 2
Figure 2 :show the duplication production on agarose gel (1.2%) for hydatid cysts sample s Isolate d from cows (A) and shee p (B) using OPF-6 primer for 3.5 hours and with 65volts

Figure 3
Figure 3: show the duplication production on agarose gel (1.2%) for hydatid cysts sample s Isolated from cows (A) and sheep (B) using OPF-19 primer for 3.5 hours and with 65 volts .