Isolation and Molecular Characterization of Foot and Mouth Disease SAT2 Virus during Outbreak 2012 in Egypt

Mohamed Shawky Abd El-Moety, Magdi Mahmoud Abd El-Aty, Hiam Mohamed Fakry, Hind Mohamed Daoud, Ehab El-Sayed Ibrahim, Wael Mossad Gamal El-Din, Sonia Ahmed Rizk, Hany abu El-Naga, Assem Abou Bakr Mohamed, Ahmed Salah Abd El-Krim, Ekbal Mohamed Farouk

Abstract
During April and May 2012, six outbreaks of FMD type SAT 2 were reported in Egyptian governorates: EL- Gharbiya (Kafr Qeretna, Al – Mahala Alkobra, Ebshaway Almalak, Qutoor), local cattle, buffaloes and dairy farms in Kafr El-Shaykh (Alsalmya, Fouah), AL - Minya (Village 3, Samalout, Mansafees, Abo Querkas), Dumyat (Ezbet 20, Kafr saad), ALexandria (Iraq Village, Al Aamerya), EL minufya (Monshaat Gerges, Ashmoun, Shatanoof, Ezbet Algalayla, Berket Alsabaa, Mahalet Sabak),Luxor (Al Gorna) and Al- Sharqiya (Al -Telleen, Menya Al kamh) Governorates. Suspected FMD virus samples (22 Tongue epithelium (TE), 12 vesicular fluid samples (VF)), in addition to, 10 cell culture grown virus of tongue epithelium origin, 6 cell culture grown virus of vesicular fluid origin. The assays used in this study involved CFT and Indirect ELISA typing kits for typing of FMDV virus samples; 3ABC-FMD for differentiation between vaccinated and infected animals in any revealed protective immune response in the examined sera from Egypt 2012 outbreak. PCR as an advanced confirmatory technique was done before submitting FMDV suspected clinical samples for Reference Laboratory of Foot and Mouth Disease in Pirbright, United Kingdom. Samples were isolated on primary bovine kidney and Baby Hamster kidney cell cultures, where all samples gave cytopathic effect. Also, all virus samples inoculated in swiss albino suckling baby mice showed pathognomonic paralysis effect of the virus in hind limbs of mice. Serum samples collected from suspected infected cattle were examined using FMD-3ABC. All positive samples to 3ABC antibodies indicated that the animals were infected by FMDV. FMD antibody detection revealed infection of 62%, 100%, 61%, 37.5%, 45.5% , 72.7% and 66.6% of sera collected from Al-Gharbiya, Kafr El-Shaykh, Dumyat, Alexandria, Al-Monufya, Luxor and Al-Sharqiya respectively. The obtained results of 3ABC antibodies were subsequently confirmed by Indirect ELISA Kit, which revealed that the causative agent of the outbreak was FMDV SAT2. The prefinal confirmatory technique was the Reverse transcription polymerase chain reaction (RT-PCR), which was done to detect clinically suspicious samples using target primers. Also, DNA sequencing of the current FMDV strain was performed and revealed that there was no significant divergence among the isolated strains as the divergence was 0.3 to 2.2% among them, and also there was no significant divergence between the isolated strains and FMDV SAT2/EGY/5/2012 and SAT2/EGY/15/2012 The closest percentage of identity of the isolated strains in comparison with reference strains was 89.4% with FMDV SAT2/LIB/2003. For official confirmation, the isolated viruses were sent to the Reference Laboratory for Foot and Mouth Disease in Pirbright, United Kingdom. Isolation of the causative agent is the first step for diagnosis of the disease. Finally, the PCR product was sequenced and analyzed using the gene bank data to confirm that the new outbreak of FMD in Egypt 2012 was due to FMD serotype SAT2.

Key words: FMDV, typing, isolation, CFT, cell culture, PCR, sequencing.