Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis

Identification by PCR signature-tagged mutagenesis of attenuated Salmonella Pullorum mutants and corresponding genes in a chicken embryo model

A key feature of the fowl-specific pathogen Salmonella Pullorum is its vertical transmission to progeny via the egg. In this study, PCR signature-tagged mutagenesis identified nine genes of a strain of S. Pullorum that contributed to survival in the chicken embryo during incubation. The genes were involved in invasion, cell division, metabolism and bacterial defence. The competition index in vivo and in vitro together with a virulence evaluation for chicken embryos of all nine mutant strains confirmed their attenuation

Revisiting Persistent Salmonella Infection and the Carrier State: What Do We Know?

One characteristic of the few Salmonella enterica serovars that produce typhoid-like infections is that disease-free persistent infection can occur for months or years in a small number of individuals post-convalescence. The bacteria continue to be shed intermittently which is a key component of the epidemiology of these infections. Persistent chronic infection occurs despite high levels of circulating specific IgG. We have reviewed the information on the basis for persistence in S. Typhi, S. Dublin, S. Gallinarum, S. Pullorum, S. Abortusovis and also S. Typhimurium in mice as a model of persistence. Persistence appears to occur in macrophages in the spleen and liver with shedding either from the gall bladder and gut or the reproductive tract. The involvement of host genetic background in defining persistence is clear from studies with the mouse but less so with human and poultry infections. There is increasing evidence that the organisms (i) modulate the host response away from the typical Th1-type response normally associated with immune clearance of an acute infection to Th2-type or an anti-inflammatory response, and that (ii) the bacteria modulate transformation of macrophage from M1 to M2 type. The bacterial factors involved in this are not yet fully understood. There are early indications that it might be possible to remodulate the response back towards a Th1 response by using cytokine therapy

Identification and Immune Functional Characterization of Pigeon TLR7

Toll-like receptor 7 (TLR7) is activated by single-stranded RNA and synthetic imidazoquinoline components, and induces interferon production. In this study, we cloned the TLR7 gene from King pigeon (Columba livia). The TLR7 open reading frame is 3144 bp and encodes a 1047-amino acid protein, consisting of a canonical TLR composition with 15 leucine-rich repeats (LRRs). Amino acid-inserting modifications were found at position 15 of LRR2, LRR11, LRR13, and LRR14 and position 10 of LRR10. The tissue distribution of pigeon TLR7 suggests that immune-associated tissues, especially the spleen and liver, have high TLR7 expression. HEK293T cells transfected with pigeon TLR7 plasmid responded to the agonist R848, indicating a functional TLR7 homolog. Following R848 stimulation of pigeon peripheral blood mononuclear cells, the levels of IFN-γ, IL-6, IL-8, CCL5, and IL-10 mRNA, assessed using quantitative real-time PCR, were significantly up-regulated. After Newcastle disease virus vaccine strain LaSota inoculation and agonist R848 injection, the level of TLR7 mRNA in the spleen of pigeons increased significantly in the R848-injected group, but decreased in the LaSota-inoculated group at three day post-infection (d.p.i.). The mRNA levels of inflammatory cytokines and chemokines were significantly upregulated in both LaSota-inoculated and R848-injected groups. Triggering pigeon TLR7 leads to robust up-regulation of inflammatory cytokines and chemokines, suggesting an important role in the innate immune response

Evaluation of the Safety and Protection Efficacy of spiC and nmpC or rfaL Deletion Mutants of Salmonella Enteritidis as Live Vaccine Candidates for Poultry Non-Typhoidal Salmonellosis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a host-ranged pathogen that can infect both animals and humans. Poultry and poultry products are the main carriers of S. Enteritidis, which can be transmitted to humans through the food chain. To eradicate the prevalence of S. Enteritidis in poultry farms, it is necessary to develop novel vaccines against the pathogen. In this study, we constructed two vaccine candidates, CZ14-1∆spiC∆nmpC and CZ14-1∆spiC∆rfaL, and evaluated their protective efficacy. Both mutant strains were much less virulent than the parental strain, as determined by the 50% lethal dose (LD50) for three-day-old specific-pathogen free (SPF) White Leghorns and Hyline White chickens. Immunization with the mutant candidates induced highly specific humoral immune responses and expression of cytokines IFN-γ, IL-1β, and IL-6. In addition, the mutant strains were found to be persistent for almost three weeks post-infection. The survival percentages of chickens immunized with CZ14-1∆spiC∆nmpC and CZ14-1∆spiC∆rfaL reached 80% and 75%, respectively, after challenge with the parental strain. Overall, these results demonstrate that the two mutant strains can be developed as live attenuated vaccines

Prime-Boost Immunization Using a DNA Vaccine Delivered by Attenuated Salmonella enterica Serovar Typhimurium and a Killed Vaccine Completely Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus▿

H5N1 highly pathogenic avian influenza virus (HPAIV) has posed a great threat not only for the poultry industry but also for human health. However, an effective vaccine to provide a full spectrum of protection is lacking in the poultry field. In the current study, a novel prime-boost vaccination strategy against H5N1 HPAIV was developed: chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella enterica serovar Typhimurium, and boosting with a killed vaccine followed. Chickens in the combined vaccination group but not in single vaccination and control groups were completely protected against disease following H5N1 HPAIV intranasal challenge, with no clinical signs and virus shedding. Chickens in the prime-boost group also generated significantly higher serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA titers against avian influenza virus (AIV) and higher host immune cellular responses than those from other groups before challenge. These results demonstrated that the prime-boost vaccination strategy provides an effective way to prevent and control H5N1 highly pathogenic avian influenza virus