Modern Techniques for Detecting Salmonella enteritidis in Intestinal Epithelial Cells of 12 Hours Age Chicks

Abstract

Avian salmonellosis is frequently followed by mortality after transmission vertically or horizontally. The incubation period occurs at 12 hours, the per-acute form frequently leads to sudden death. Salmonella motivates epithelial cells to die and develop symptoms of diarrhea, then it leaves the intestinal epithelial cell through the basement membrane in the lamina propria before death induction. The present study was aimed to ensure the effects and relationship of Salmonella enteritidis on intestinal epithelial cells as well as to investigate the gradual effect of time interval on epithelial cells with the apoptosis phase induction. Prepared dilution of Salmonella cells from the Ten-Fold Dilution Technique using the spread plate method was used for counting Colony Form Unit (CFU). Four dilutions were used as infective dose in volume 0.5ml per bird. Ninety-five newly hatched chicks were inoculated with infective dose, the observation of clinical signs and deaths were recorded. LD50 was calculated from four groups of each serial dilution. The exponential phase was adjusted by culture broth at volume 2 ml and the applied protocol of Mel Lacey was followed as analysis in Excel Analysis Data, was then standardized in McFarland number this was selected as a point for labeling salmonella by fluorescence dye. The study modified the chang’s protocol by changing the label of Green Fluorescence Protein to Yellow Fluorescence Protein dye. The infective dose was determined as 2.5 ×10-7 and inoculated into forty-four newly hatched chicks and sacrificed at each 3hours of time intervals (3, 6, 9, and 12 hours), and naming groups according to intermittent time. Necropsy from characteristic lesions for histo-immuno-pathological analysis as well as studying relationships with salmonella cells located intracellular and extracellular. Five cm length of enteritis was prepared as Nik and Carlsson’s protocol. Extracellular salmonella was extracted from squeezed intestinal content which filtered by Celltrics before analysis by Flow Cytometer Technique. The singlet epithelium cells were prepared for permeabilization, using intracell’s protocol of immunostep manufacturer. Biomarker for detecting Apoptosis and death was applied by the technique of Annexin-V-FITC and Pi dye as described by Miltenyi Biotec Protocol was followed. Then analysis by flow cytometer was performed. It is worth mentioning that the current study was the first one in determining of LD50 for Salmonella enteritidis from White Nile State strain and the mortality rates were reported at diluted doses as 25% in group 243×10-5 (2.5×10-7), 15% in group 10-6, 15% group 10-7, and 6 % group 10-8. The exponential phase was determined at 30-90 minutes. This was the point of labeling V by Yellow Fluoresce Protein dye which successfully achieved as advised by Chang’s protocol. The finding of this study in histopathology was severe sluaphing of epithelial cells with infiltration in inflammatory cells such as heterophil and mononuclear cells during 3 hours post-inoculation. Salmonella cells that found extracellular were detected by Flow Cytometer and the viable cells were differentiated from dead one each 3 hours interval. Group 3 hours recorded 15-96.2% and 3.8-84.9% for live and dead cells respectively, group 6 hours at 4-96.4% and 3.6-95.8% for live and dead cells respectively, group 9 hours at 58.5-94.5% and 5.5-97.3% for living and dead respectively. The highest viable cell count for each group were about (57674 cells) in group 3, (37784 cells) in group 6, (49435 cells) in group 9, (93056 cells) in group 12. ANOVA test illustrated the normal pathway of cell cycle influenced by time intervals, the high effect of time that occurred on viable and apoptotic epithelial cells regarding the invasion process. Analysis of viable epithelial cells population that not invaded were not significant between group 3, 6, and 9 hours. Apoptotic epithelial cells free from salmonella were not significant between-group 6 hours with 9 and 12 hours and between 9 hours and 12 hours, While significant in a group of 3 hours. Dead epithelial cells that not carrier to salmonella was not significant between all groups under experiment during time intervals. Epithelial cells that suffering from salmonella had a complication, noticed increased strength of differences between groups of the time interval that stated in an apoptotic phase. New evidence was shown in the relationship between Salmonella enteritidis within four groups of time intervals. Salmonella cells that swam extracellularly were irreversibly correlated. Epithelial cells and salmonella in chicks who sacrificed at 3 hours were irreversibly correlated with those sacrificed for 6 and 9 hours. While the reversible link between groups 3 hours with 12 hours. Also, group 6 with 9 and 12 hours as well as group 9 with 12 hours. The conclusions were the natural cell cycle during the invasion was effected and the possibility of apoptosis induction may be due to extrinsic signals of salmonella clearly occurred. The descriptive study confirmed the ability of salmonella to traffic the function of epithelial cells according to effector proteins.