Identification Of Salmonella Pullorum Genes Expressed During The Infection Of Macrophages By Selective Capture Of Transcribed Sequences

Salmonella pullorum is distributed in worldwide. Nowadays, the pullorum disease has been eliminated or partially eliminated in the developed countries, however, there it is an important avain disease causing serious losses in the poultry industry in China. Salmonella pullorum mainly causes the death of 2-3 weeks old chickens, and the adult chickens appear as bacteria carrier without clinical symptoms. Most of the survival chickens show the reproductive disorders, including decreased egg production, production of contaminated eggs, which cause severe economic losses. The disease can spread vertically, as well as transmit horizontally, which is the main reason for the reproductive disorders. It is important to explore the novel potential virulence-related gene and develop new strategies for the control of the pullorum disease.With the development of various molecular biological and immunological research methods, the gene expression of pathogens in infection process can be evaluated. It is notable that identification of transcribed sequences of pathogens in vivo is very important for exploring the pathogenetic mechanism. SCOTS is applicable to many kinds of bacteria from which transcribed sequences can be isolated. In this study, it was carried out an invasion test both in chicken model and macrophage cell line to compare the growth property of Salmonella pullorum in vivo and in vitro. In avian macrophage cell line HD-11, the bacterial transcribed sequences of S. pullorum was captured using SCOTS and the expression develop captured sequence 7-6 was analyzed by real-time quantitative PCR.1 Comparison of invasion process in vivo and in vitro of Salmonella pullorumIn order to investigate the specific infection of Salmonella pullorum in chicken model and in the avian macrophages cell line HD-11 in vitro, 9-days-old chickens were inoculated orally or intramuscularly with S. pullorum S06004. The bacteria within macrophages of spleen and liver can reach the highest point at the third day after inocularion, and would be less than 500 cfu/g at the ninth day, which can be persist more than 5 days. The result of invasion in vivo indicatied that bacteria in macrophages were proliferated at first, then reduced to a lower level which can last some time. Meanwhile bacteria infected the macrophage in vitro, intracellular bacteria reached the highest point in 3h at proliferation stage, then reduced to a lower level which also lasted a few hours. So the in vitro invasion of HD-11 cells by S. pullorum can simulate the invasion in vivo in some extent.2 Identification of Salmonella pullorum genes expressed within macrophages by selective capture of transcribed sequencesThe selective capture of transcribed sequences(SCOTS) was used to identify Salmonella pullorum S06004 genes expressed during the process of infection of avian macrophage cell line HD-11. According to bacterial invasion in vivo and in vitro, the invasion of Salmonella pullorum S06004 in avian macrophage cell line HD-11 can simulate in vivo. The transcribed sequences were captured, and identified the virulence genes and related regulation genes expressed within macrophages. 16 sequences were obtained. Except an unknown sequence, these sequences included the coding genes of virulence-related secreted proteins in type III secretion system, outer membrane protein coding genes of typeâ… secretion system, sensitive kinase related to metabolism and nucleoside hydrolase coding genes, transport genes, and some plasmid genes, environmental adaptability functional gene.3 Kinetic detection of the expression level of a captured sequence 7-6 during the infection process by real-time quantitative PCRIt was designed a pair of primers of the captured sequence 7-6 of S. pullorum for the specific detection and the primers of 16sRNA gene were used as internal control. The total RNA of infected cells at different time was extracted, and reverse transcribed into cDNAs, and then the gene expression levels of 7-6 was monitored using real-time PCR. The results showed: the captured sequence 7-6 was not expressed in vitro, and then there are the highest expression in early invasion, at the intracellular bacteria proliferation phase, the expression level reached to the highest at 3h proliferation, and then decreased to a very low level after 5h proliferation. The changes in transcriptional level of 7-6 were related to the infection process of macrophages.