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Abstract
Salmonella infection is one of the most serious problems that affect poultry industry causing high economical losses not only due to high mortality in young birds but also for the debilitating effect which predisposes for many other diseases. Investigations into the epidemiology of animal and human salmonellosis require the use of several typing methods for differentiation of the strains into groups below the level of serovars especially when traditional typing (phenotyping) indicates close relationships between isolates.
In the present investigation, the bacteriological isolation and identification of Salmonella showed varied recovery rates between isolates. The colonial characteristics revealed that 9 out of 14 apparently healthy samples, 17 out of 25 diseased samples and 9 out of 11 freshly dead samples were showed typical characteristics of Salmonella. On MacConkey agar showed colorless non lactose fermenting colonies while on Salmonella Shigella agar media the colonies of non lactose fermenting salmonella appeared transparent with black centers. In the same time, On Xylose Lysine Desoxycholate (XLD) agar, colonies of non-lactose fermenting Salmonella appeared pink with black pigment indicating H2S production.
The morphological characteristics revealed that 9 suspected isolates out of 14 from the apparently healthy samples, 17 out of 25 from the diseased samples and 9 suspected isolates out of 11 from the freshly dead samples were Gram negative non-spore forming medium size straight rods, and usually motile.
Depending on the results of API 20E identification system, 6 suspected Salmonella isolates were recovered out of 9 from the apparently healthy samples, 13 suspected isolates out of 17 diseased samples and 7 isolates out of 9 freshly dead samples representing recovery rates 67%, 76% and78% respectively.
from the results of API 20E system, the obtained isolates subgrouped into three groups, the first group suspected to be S. Pullorum and the second group suspected to be either S. Enteritidis or S. Montevideo while the third group suspected to be either S. Typhimurium or S. Gallinarum.
By using polyvalent and monovalent antisera, 5 serotypes of salmonella represented as S. Pullorum, S. Typhimurium, S. Enteritidis, S. Gallinarum, and S. Montevideo were identified serologically where 2 isolates out of 6 suspected samples from the apparently healthy cases, 4 isolates out of 13 diseased cases and 2 isolates out of 7 obtained from the freshly dead cases were identified as S. Pullorum representing a recovery rate of 31%. Also one isolate out of 6 recovered from the apparently healthy cases and one isolate out of 13 diseased cases were identified as S. Montevideo representing a recovery rate of 8 %. At the same time 3 isolates out of 6 were obtained from the apparently healthy, 2 out of 13 were obtained from the diseased cases and 2 out of 7 from the freshly dead cases were identified as S. Enteritidis representing a recovery rate of 27%. On the other hand 2 isolates out of 13 diseased cases and 1 out of 7 freshly dead cases were identified as S. Gallinarum representing a recovery rate of 11.5%. While 4 isolates out of 13 diseased cases and 2 out of 7 freshly dead cases were identified as S. Typhimurium representing a recovery rate of 23%.
The results of bacteriological isolation and identification of Salmonellae indicate that Salmonella were detected in 26 isolates out of 50 samples with recovery rate 52%.
The genomic DNA of different isolated salmonella strains was extracted and the estimation size of the genomic DNA of the different strains was more than 23Kb which used later in the PCR assay.
Most of the isolates showed a degree of variation in plasmid number (from 1 plasmid up to 6 plasmids) and molecular weight (from 583 bp to more than 23,130 bp). All salmonella serotypes harbored plasmid of 11425bp.
Concerning PCR assays, 4 different specific primers were used, each of them was specific for spvC gene, rfbS gene, IE gene and fliC gene.
Regarding to the PCR amplification of plasmid spvC gene using its specific primer, the PCR product of molecular weight of 570 bp was obtained in all 5 serotypes.
As regards to rfbS gene amplification using its specific primer, the PCR product of molecular weight of 720bp was obtained only in Salmonella Pullorum and Salmonella Gallinarum.
Concerning PCR amplification of IE gene using its specific primer, the PCR product with molecular weight of 559 bp was obtained only in Salmonella Enteritidis.
As the result of PCR amplification of fliC gene using its specific primer, the PCR product with a molecular weight of 183 bp was obtained only in Salmonella Typhimurium.
SDS-PAGE of Salmonella serotypes revealed common electrophoretic patterns of cell protein especially among the molecular weight of 11 KDa and 45KDa shared in all serotypes which constituting highest percent of total protein patterns of serotype pullorum. Also there are other protein patterns shared in the most of the isolated serotypes like that obtained at 16KDa, 18 KDa, 29KDa, 33KDa, 36KDa, 38KDa and 40 KDa. On the other hand, there were additional proteins bands related to each serotype like that obtained at 13KDa, 20 KDa, 24 KDa, 25 KDa, 53 KDa, 80 KDa, 84 KDa, 93 KDa, 105 KDa, 112 KDa, 139 KDa, and 164 KDa which represent a low percent of total protein patterns of each serotype.
Overall, this study demonstrated the importance and advantages of different modern techniques like PCR and SDS-PAGE over the traditional isolation and identification procedures in the rapid identification and differentiation of the different Salmonella serovars. It is more clearly that the use of PCR assay able to differentiate between the different serotypes of Salmonella in the suspected samples saving time, money and effort.