The Salmonella virulence island genes SPI-1 and SPI-2 encode T3SS-1 and T3SS-2 in the type Ⅲ secretion system(T3SS),respectively,and which can transport effector proteins secreted by Salmonella into host cells.In order to analyze the effects of the deletion of virulence island genes SPI-1 and SPI-2 on the biological characteristics,pathogenicity and immunogenicity of Salmonella enterica,the laboratory constructed a double deletion strains of the virulence island genes SPI-1 and SPI-2 in the Salmonella enterica strain SM6(SM6△T3SS)in the previous period,and plotted the growth curves of the parental strain and the deletion strain.The morphology of the two cells was observed by scanning electron microscopy,and the adhesion and invasion of the parental strain SM6 and the deletion strain SM6△T3SS to human colorectal adenocarcinoma cells(Caco-2 cells)were determined.The growth curves showed that the growth rate of the SM6△T3SS strain was significantly faster than that of parental strain SM6 at 5 hours(P＜0.05),and the growth rate of the SM6△T3SS strain was highly significant faster than that of the SM6 strain from 6 hours onwards(P＜0.01).Scanning electron microscopic observation showed that the parental strains were mostly present singly,while the deletion strains had obvious bacterial aggregation,and their surfaces were coated by a gel-like substance.The results of adhesion and invasion showed that the deletion strain SM6△T3SS adhered to and invaded Caco-2 cells in a highly significant decrease compared with the parental strain SM6(P＜0.001,P＜0.001).These results suggest that the deletion of virulence island genes SPI-1 and SPI-2 may have altered the growth,metabolism,secretion and/or structure of bacteria,resulting in the alteration of the biological characteristics of Salmonella and the reduction of its adherence and invasion ability to intestinal epithelial cells.The 7-day-old SPF chickens were infected by intraperitoneal injection at the dose of 1×107cfu/100 μL,and the bacteria-carrying capacity of the deletion strain in the cecum,liver and spleen of the SPF chickens was detected at different times after infection;the chickens were immunized by intraperitoneal injection at a dose of 1 × 107cfu/100 μL with the deletion strain at the age of 7,21 and 35 days,respectively.Blood samples was collected at different times after the first immunization;and the blood of each group of chickens was analyzed by indirect ELISA,and the blood of each group of chickens was analyzed by indirect ELISA.Blood was collected at different times after the first immunization and indirect ELISA was used to detect the IgG antibody levels in the serum of chickens in each group.The serum levels of IL-4 and IFN-y secretion in each group were detected by indirect ELISA.The clinical symptoms of the chickens in each group were observed within 14 days after the first immunization,and the mortality rate of the chickens in each group was counted.At 7 days post-toxicity,some chickens in each group were dissected and their cecum and liver tissues were collected to observe the histopathological changes of the tissues and to evaluate the immunological efficacy of the deletion strain.The results of bacterial load showed that 2 days after infection,the bacterial load of the deletion strain in spleen and liver was extremely significantly lower than that of the parental strain(P＜0.001),but the bacterial load in cecum was extremely significantly higher than that of the parent strain(P＜0.01).On the 4 days after infection,the bacterial load of the deletion strain in liver and cecum was extremely significantly and significantly lower than that of the parent strain(P＜0.01,P＜0.05).On the 8 days after infection,the bacterial load of the deletion strain in spleen was extremely and significantly lower than that of the parent strain(P＜0.01),and the bacterial load in the above organs of chickens in both groups dropped to a lower level.The ELISA results of antibodies and cytokines showed that the antibody level of chickens in the immunized group was significantly higher than that of the control group at 14 days after the first immunization(P＜0.05).From 21 days after immunization,the antibody levels of all chickens in the immunized group were extremely significantly higher than those of the control group(P＜0.001).There was no significant change in serum IFN-γ secretion level during the immunization period,but the content of IL-4 was significantly higher than that of the control group at 42 days after immunization(P＜0.01).The content of IFN-γ and IL-4 in the serum of SPF chickens was significantly higher than that of the control group after the tapping of the virus(P＜0.05).The chickens in the SM6△T3SS-immunized group survived throughout the experimental period with no obvious clinical symptoms,no deaths,and only slight pathological damage to the liver and intestinal mucosa,while the chickens in the control group had a mortality rate of 80％within 14 days after the attack,with obvious lesions on the liver and intestinal mucosa.These results indicate that the SM6△T3SS strain can stimulate the production of high levels of humoral and cellular immune responses in SPF chickens,reduce the pathogenicity of SPF chickens,and have better immunoprotective effects on chickens.The construction of the double-deficiency strain laid a good foundation for elucidating the pathogenic mechanism of Salmonella and developing safe and effective live attenuated Salmonella vaccines,and also provides a new ideas for the prevention and control of Salmonellosis.
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