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The primary objective of the current study was to identify two of the highly pathogenic avian influenza virus (HPAIV) isolates of subtype H5N1 genotypically using one step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), followed by sequence and phylogenetic analyses. A further objective was to determine in vitro the virucidal efficacy of four types of chemical disinfectants, namely Formalin, Glutaraldehyde, TH4® and Virkon®S at different concentrations and contact times on the two HPAI isolates. A/chicken/Egypt/0626/2006 (EGY06) and A/chicken/Egypt/1094/2010 (EGY10) were isolated from cloacal and tracheal swabs from broiler during HPAI H5N1 outbreaks in Egypt in 2006 and 2010. The first strain, EGY06, was isolated from a non-vaccinated flock in February 2006 in the Alexandria governorate. The second strain, EGY10, was isolated from a vaccinated flock in November 2010 in the Marsa Matrouh governorate. Classical identification of the two isolates was carried out in the Department of Poultry and Hygiene, Faculty of Veterinary Medicine, Alexandria University, Egypt. Molecular identification and genetic analyses were conducted in the Gene Analysis Unit of the National Laboratory for Veterinary Quality Control on Poultry Production (NLQP), Egypt.
Using RT-PCR with specific sets of primers for H5 and N1 genes of AIV it was confirmed that the two isolates belonged to AI subtype H5N1. After molecular characterization and phylogenetic analysis of the HA and NA genes, the strain EGY06 was closely related to the 2006 predecessor Egyptian viruses of 2.2.1 clade, whereas EGY10 clustered within the classic 2.2.1/c group that commonly isolated from small-scale commercial farms and human since 2009.
The efficacy of four chemical disinfectants to inactivate both isolates was carried out in accordance to the guidelines of the German Veterinary Medical Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) for testing of disinfection procedures and chemical disinfectants. The experiments were performed using suspension tests without and with protein load (40% Bovine Calf Serum "BCS") as well as wood and gauze as a carriers (also loaded with BCS), at room temperature and incubation times of 15 to 120 min.
The obtained results showed that the use of Glutaraldehyde, Formalin or TH4® 0.5% without protein load led to complete inactivation of the virus after 15, 30, 60 or 120 min contact time. Use of Virkon®S 0.5% with and without protein load led to survival of the virus even after 60 min. In contrast, using Formalin and TH4® (1% and 2%) with and without protein load led to complete inactivation of the virus even at the shortest contact time of 15 min. Similar results were obtained after using Glutaraldehyde 1%, while treatment of H5N1 with Glutaraldehyde 2% led to gel formation.
After treatment of contaminated carriers (poplar wood and gauze) with Formalin, Glutaraldehyde and TH4® 0.5%, the virus was inactivated after 30 min. Concentration of 1% of the three disinfectants was sufficient to inactivate the two isolates within 15 min contact time, except in case of Virkon®S which required higher concentrations to give similar results.
The study indicated that the four chemical disinfectants could efficiently inactivate the two tested H5N1 viruses when used at higher concentration than the manufacturers recommended. The results of the present thesis highlight the sensitivity of HPAIV H5N1 to different disinfectants, which may improve biosecurity measures on the farms and reduce the economic losses caused by HPAIV H5N1.