الفهرس 
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Abstract
Poultry and poultry products are considered as important source of Salmonella that cause human illness and even death. Salmonella Typhimurium is also the most frequently isolated serovar from global food-borne outbreaks.
Salmonella enterica is mainly transmitted to humans following consumption of contaminated eggs and poultry products. Hence, a rapid detection and identification method of this serovar is necessary in the food industry and public health. So, that prevention of Salmonella infection is important for poultry health and for food processing industries.
Salmonella Typhimurium has long served as a model organism for genetic studies, and a wide variety of classical and molecular genetic tools exist for the identification and characterization of potential Salmonella virulence genes. In addition, the availability of in vitro tissue culture and small-animal models of infection facilitates study of complex interactions between host and pathogen during various stages of infection.
The molecular approach to microbial pathogenesis has resulted in an impressive amount of data on bacterial virulence genes. Bacterial genome sequences rapidly add candidate virulence genes to electronic databases.
The interpretation of this great information is unclear because every gene involved in pathogenicity is called a virulence gene, regardless of its function in the complex process of virulence. A refined definition of virulence genes is proposed in which the function of the gene in the virulence process is incorporated. With the development of molecular biological techniques, it became possible to identify the genes encoding those factors responsible for virulence. This resulted in molecular microbiology, in which the role and function of specific genes in (bacterial) virulence was the subject of investigation. The search for virulence genes evolved together with the technical development of molecular biology and genetic modification of microorganisms. In the beginning of molecular microbiology, genes were identified that encoded virulence factors of known reputation and these were used as probes to find analogs in other organisms.
Many diverse bacterial pathogens share common mechanisms of their abilities to adhere, invade and cause cytotoxicity of host cells. Many of these characters of infection appear is related to large blocks of virulence genes from a common microbial ancestor, which can be transmited to other bacteria via horizontal transfer.
When study microbial pathogenicity it is essential to understanding the molecular mechanisms of microbial virulence and to the development of novel vaccines and other therapeutic agents for the treatment and prevention of infectious diseases.
The aim of this study is to investigate the pathogenicity of different isolates of Salmonella Typhimurium on cell culture to study the different virulence factors such as: invasion, adherence and cytotoxicity.
Twelve strains of Salmonella Typhimurium (10 chicken: eggs, layers, broilers and 2 ducks) isolated from poultry flocks in different geographical areas (Giza, Kalubia, Damietta and Kafr EL-Sheikh) and these isolates were serotype Typhimurium. All strains in this study were characterized by phenotypic and genotypic methods to compare the usefulness of the methods in epidemiological studies.
The obtained results of the 12 isolates of S.Typhimurium were sensitive to ciprofloxacin and sulfamethaxazone-trimethoprim, Norfloxacin and Ofloxacin, gentamycin, ampicillin, chloramphenicol, tetracyclin, amoxicillin and finally flucloxacillin by percentage of 91.7%, 83.3%, 75%, 66.7%, 50%, 33.3%, 16.7% and 0% respectively.While 100%, 83.3%, 66.7%, 50%, 33.3%, 25%, 16.7% and 8.3% of the isolates were resistant to flucloxacillin, amoxicillin , tetracyclin, chloramphenicol, ampicillin, gentamycin, Norfloxacin and Ofloxacin, ciprofloxacin and sulfamethaxazone-trimethoprim respectively.
All isolates could be classified as either invasive according to gentamycin survival assay or cytotoxic according to crystal violet dye exclusion assays. The results showed that isolates of S.Typhimurium (all of them were able to invade the Vero cells in the tissue culture plates by different percent, 33.3% have high invasion capability while 41.7% of them have moderate invasion capability but only 25% have low invasion capability) with high invasiveness capability exhibited high epithelial cell cytotoxicity (the cytotoxicity occurred ranged from 49% to 80%) i.e. there is a direct correlation existed between invasion and cytotoxicity.
All S.Typhimurium local strains were able to adhere to Vero cells by different degrees (the 75% of Salmonella strains have high adherence capability while 25% of them have moderate capability) but have inverse correlation with the invasion and cytotoxicity capability.
The nucleotide sequence of Local Egyptian isolate were
compared with invA of S.Typhimurium published sequences on Genbank. The multiple alignment of nucleotide sequence of Egyptian isolate with other published S.T. strains on Genbank showed important difference at position (470) from (G to A) nucleotides.
The multiple alignment of deduced amino acid of Egyptian isolate with other published S.T. strains on Genbank showed only one amino acid difference at (F 530 S) and this significant difference led to high identity percentage between S.Typhimurium strains.
The homology percentage of nucleotide sequence results showed high homology (99.4%) between ST/Egypt/Poultry/2014 strain and ST.LT2, ST.ATCC13311, ST.VNP20009, ST.DT2, ST.DT104, ST.UK1, ST.SL1344, ST.14028S and ST.4/74 but it gives (99.2%) with STYINVA strain.
The homology percentage of deduced amino acid results showed high homology (99.9%) between ST/Egypt/Poultry/2014 strain and ST.LT2, ST.ATCC13311, ST.VNP20009, ST.DT2, ST.DT104, ST.UK1, ST.SL1344, ST.14028S and ST.4/74 but it gives (99.6%) with STYINVA strain. These results agree with Shi Q. et al., 2012 which found the high InvA homology between Salmonella strains (72.9-97.2%).
For more clarification of the genetic relatedness of Egyptian isolate with other published S.Typhimurium strains, phylogenetic analysis of nucleotide sequence showed two major clusters or branches also, one representing the Egyptian isolate ST/Egypt/Poultry/2014 and the second divided in two sub-branches, the first for STYINVA strain and the second divided in two small subgroups, the first contains ST.4/74 and ST.SL1344 strains and the second contains ST.LT2, ST.ATCC13311, ST.VNP20009, ST.DT2, ST.DT104, ST.UK1 and ST.14028S strains.
Phylogenetic analysis of deduced amino acid showed two major clusters or branches, one representing the STYINVA strain and the second one divided in two sub-branches, the first for ST/Egypt/Poultry/2014 Egyptian local strain (KR185982) and the second for remaining strains: ST.LT2, ST.ATCC13311, ST.VNP20009, ST.DT2, ST.DT104, ST.UK1, ST.SL1344, ST.14028S and ST.4/74.
Both phylogenetic trees demonstrated that the Egyptian isolate (ST/Egypt/Poultry/2014) is closely related to the other S.Typhimurium isolates but in a separate cluster because the presence of single, significant amino acid substitution at position (F 530 S), due to the change of nucleotide at position (A 470 G), and non-significant changes in another nucleotides.