Molecular Detection of Echinococcus granulosis from Visceral Organs of Cattle Abattoirs-Kerbala Province

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Echino coccosis is a zoonotic disease of man and demoestic animals with world wide importance 1 caused by larved stage of Echinocossus granulosus , the common site for infections are liver , lung and other organs 2 .
The life cycle of this parasite is indirect , it has dogs and other canids as a final host and Omnivorus with harbivorus as intermediate host 3 .
The disease transmission , when food or water that contains the eggs of parasite is eaten or close contact with infected.
Animals 4 , in the human and intermediate host via infected handling soil or dirt animal hair with eggs 5 , but there are not biological or mechanical vectors for larval stage except birds and arthropods can act a mechanical vectors for eggs 6 .The disease occure without symptoms and signs but these appear deponton the cyst's size and location 7 , the disease has in both human and animals health considerable impact with economic loss in animal production 8 .
There are ten genotypes of E. granulosns (G1-G10), (G1) in sheep , (G2 -G3) in buffalo , (G4) in aquine , (G5) in cattle , (G6) in camel , (G7) in pigs and (G8 -G10) are cervid strains 9,10 , the strains have a biological and molecular aspects 11,12 with different in regions distribution and the pathological identification is difficult in the case of aberrant forms 13 also different in host specificity , pathogenicity life cycle pattern , transmission and rates of development finally response to the chemotherapeutic drugs 14 .
Polymerase chain reaction (PCR) based other methods has been vastly used for genotyping of E. granulous 15,16 , now the clinical sample are taken biopsy to PCR for amplified fragments of mitochondrial and nucleur DNA are subsequently sequenced 17 The aim of this study The aim of this study were to choose the optimal PCR technique with specific designing primer for cystic fluid to detect Echinococcus JAWAD et al.: MOLECULAR DETECTION OF Echinococcus granulosis granulosus hydatid cyst in internal visceral organs of cow abattoirs-kerbala governorate.

An internal organ of cow which includes
Liver, lung and liver with lung were sent to parasitological laboratory in the kerbala Veterinary medicine for parasite testing.For this, all cysts with organs were cut into one centimeter pieces and left in warm water for 20 minutes.Then, removed by compression, and the sediments were obtained 9 .DNA extraction were used for all cystic fluid and pieces by Tissue/Blood DNA Mini Kit (Geneaid, Korea), by following of manufacturer's instructions, The primer pair was used to amplify the partial mitochondrial NADH dehydrogenase subunit 1 (ND1) gene of Echinococcus granulosus , The sequence used in this study was from the National Center for Biotechnology ( http://www.18.nlm.nih.gov/)websites have accession numbers as (AY386216.1,KY766890.1,AF297617.1 and KX039956.1)the primer was designed from Primer3 plus ( http:// primer3.wi.19.edu/ ) website, The sequence of the primers are designed for the following.PCR amplification was completed using conventional PCR conditions in a final volume of twenty five microne : five microne of DNAtemplate (20 ng/ µl), 12¼lMilliQ water and 1.5µl for both primer forward and reverse primer (10 µM),all mixer were collected on each PCR tubes containing 5µlof AccuPower® ProFiTaq PCR PreMix (Bioneer, korea).The PCR reactivity was hold out in a Techne TC-512 thermocycler with the following cycling conditions: initial denaturation(95 °C, 15 min), succeed by 35 cycles of denaturation (95 °C, for thirty seconds), annealing (56 °C, 40 s) and sixty second for extension have (72 °C), and a last extension step (72 °C, 10 min).All the amplification of single fragment of the expected size was establish by gel electrophoresison a 1.5 % agarose gel (TBE, 1 %) stained with ethidium bromide.followed by 30 cycles using 1 min denaturation at 94°C, I min annealing at 55°C and 2 min extension at 72°C, PCR reactions were assayed on 1.5% agarose gels in the presence of 5 g/ml ethidium bromide.(B).

RESULTS AND DISCUSSION
Echinocossosis in Iraqi is major endom disease 20 and enzootic 21 , a total number of 35 slaughtered cattle were inspected in this study.The overall prevalence of hydatidosis in slaughtered livestock was 22 (62.8%).In total, 12 (63.6%),6 (27.2%) and 2(18.1%)Liver, lung and liver with lung, were condemned in slaughterhouses due to E. granulosus hydatid cysts in cattle, respectively.Additionally, PCR technique were used to detect these parasites in hydatid cysts in both organs.Figure 1 .The application of polymerase chain reaction (PCR) is based on studies involved large number of Echinococcus granulosus were limited due to cumbersome methods access to the genomes of these parasities (@) lytic Enzymes and protein Extraction to purified DNA parasities in particular Echinococcus granulosus involve weakening of the membrance of hydatied cyst by the prsesence of followed by enzymatic treatment and ionic detergents.

Table 1. Showed the primer instruments of this study
As detergents such as (sodium dodecyl sulfate )SDS have a deleterious effect on the protein denatyuration of membrance of hydtid cysts (@) it was necessary to modify a designing primer such procedures to use in PCR, the selection of appropriate primers for maximal specificity and efficiency.Primer specificity was affected by a number f factors ,including sequence , primer location and the PCR system used.General primar -designing rules for polymerase chain reaction were also applicable in PCR to avoid primaerdimer mispriming formation.

CONCLUSION
There are many studies reviewed importance in of echinoccosis but the program control against hydiat cyst cannot be successful unless the farmers education , charged people about this disease and controlled slaughters of livestock must be effective .