The characterization of the immune response of chickens to Salmonella infection is usually limited to the quantification of expression of genes coding for cytokines, chemokines or antimicrobial peptides. However, processes occurring in the cecum of infected chickens are likely to be much more diverse. In this study we have therefore characterized the transcriptome and proteome in the chicken cecum after infection with Salmonella Enteritidis. Using a combination of 454 pyrosequencing, protein mass spectrometry and quantitative real-time PCR, we identified 48 down- and 56 up-regulated chicken genes after Salmonella Enteritidis infection. The most inducible gene was that coding for MMP7, exhibiting a 5952 fold induction 9 days post-infection. An induction of greater than 100 fold was observed for IgG, IRG1, SAA, ExFABP, IL-22, TRAP6, MRP126, IFNγ, iNOS, ES1, IL-1β, LYG2, IFIT5, IL-17, AVD, AH221 and SERPIN B. Since prostaglandin D2 synthase was upregulated and degrading hydroxyprostaglandin dehydrogenase was downregulated after the infection, prostaglandin must accumulate in the cecum of chickens infected with Salmonella Enteritidis. Finally, above mentioned signaling was dependent on the presence of a SPI1-encoded type III secretion system in Salmonella Enteritidis. The inflammation lasted for 2 weeks after which time the expression of the “inflammatory” genes returned back to basal levels and, instead, the expression of IgA and IgG increased. This points to an important role for immunoglobulins in the restoration of homeostasis in the cecum after infection.

Salmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host.

Background: In this study, we characterised the microbiota present in the faeces of 15- and 46-week-old egg laying hens before and after tetracycline or streptomycin therapy. In the first experiment, the layers were subjected to 7 days of therapy. In the second experiment, the hens were subjected to two days of therapy, which was repeated for an additional two days after 12 days of antibiotic withdrawal. This enabled us to characterise dynamics of the changes after antibiotic administration and withdrawal, and to identify genera repeatedly resistant to tetracycline and streptomycin. Results: Real-time PCRs specific for Enterobacteriales, Lactobacillales, Clostridiales and Bifidobacteriales showed that changes in the microbiota in response to antibiotic therapy and antibiotic withdrawal were quite rapid and could be observed within 24 hours after the change in therapy status. Pyrosequencing of PCR amplified V3/V4 variable regions of 16S rRNA genes showed that representatives of the orders Clostridiales, Lactobacillales, Bacteroidales, Bifidobacteriales, Enterobacteriales, Erysipelotrichales, Coriobacteriales, Desulfovibrionales, Burkholderiales, Campylobacterales and Actinomycetales were detected in the faeces of hens prior to the antibiotic therapy. Tetracycline and streptomycin therapies decreased the prevalence of Bifidobacteriales, Bacteroidales, Clostridiales, Desulfovibrionales, Burkholderiales and Campylobacterales in faecal samples in both experiments. On the other hand, Enterobacteriales and Lactobacillales always increased in prevalence in response to both therapies. Within the latter two orders, Escherichia and Enterococcus were the genera prevalence of which increased after all the antibiotic treatments. Conclusions: The changes in microbiota composition induced by the antibiotic therapy were rapid and quite dramatic and only representatives of the genera Enterococcus and Escherichia increased in response to the therapy with both antibiotics in both experiments.

Background: Infection of newly hatched chicks with Salmonella enterica serovar Enteritidis (S. Enteritidis) results in an inflammatory response in the intestinal tract which may influence the composition of gut microbiota. In this study we were therefore interested whether S. Enteritidis induced inflammation results in changes in the cecal microbiota. To reach this aim, we compared the cecal microbiota of non-infected chickens and those infected by S. Enteritidis by pyrosequencing the V3/V4 variable regions of genes coding for 16S rRNA. Results: Cecal microbiota of chickens up to 19 days of life was dominated by representatives of Enterobacteriaceae, Lachnospiraceae and Ruminococcaceae, followed by Lactobacillaceae. The presence of Lachnospiraceae did not change after S. Enteritidis infection. Enterobacteriaceae increased and Ruminococcaceae decreased after S. Enteritidis infection in two independent experiments although these results were not significant. A significant increase in both experiments was observed only for the representatives of Lactobacillaceae which may correlate with their microaerophilic growth characteristic compared to the obligate anaerobes from the families Lachnospiraceae and Ruminococcaceae. Conclusions: We conclude that S. Enteritidis infection influences the composition of the cecal microbiota in chickens but these changes are minor in nature and should be understood more as an indirect consequence of infection and inflammation rather than a positively selected evolutionary trait.

The colonization of poultry with different Salmonella enterica serovars poses an issue throughout the world. In this study we therefore tested the efficacy of a vaccine consisting of attenuated strains of Salmonella enterica serovars Enteritidis, Typhimurium and Infantis against challenge with the same serovars and with S. Agona, Dublin and Hadar. We tested oral and aerosol administration of the vaccine, with or without co-administration of cecal microbiota from adult hens. The protective effect was determined by bacterial counts of the challenge strains up to week 18 of life and by characterizing the immune response using real-time PCR specific for 16 different genes. We have shown that a vaccine consisting of attenuated S. Enteritidis, S. Typhimurium and S. Infantis protected chickens against challenge with the wild type strains of the same serovars and partially protected chickens also against challenge with isolates belonging to serovars Dublin or Hadar. Aerosol vaccination was more effective at inducing systemic immunity whilst oral vaccination stimulated a local immune response in the gut. Co-administration of cecal microbiota increased the protectiveness in the intestinal tract but slightly decreased the systemic immune response. Adjusting the vaccine composition and changing the administration route therefore affects vaccine efficacy.

Lactobacilli are commonly used as probiotics in poultry to improve production parameters and to increase chicken resistance to enteric infections. However, lactobacilli do not efficiently colonise the chicken intestinal tract, and also, their anti-infection effect in vivo is sometimes questionable. In this study, we therefore evaluated the potential of a mixture of four Lactobacillus species (L. salivarius, L. reuteri, L. ingluviei and L. alvi) for the protection of chickens against Salmonella Enteritidis infection. Whenever the chickens were inoculated by lactobacilli and S. Enteritidis separately, there was no protective effect of lactobacilli. This means that when lactobacilli and S. Enteritidis are exposed to each other as late as in the crop of chickens, lactobacilli did not influence chicken resistance to S. Enteritidis at all. The only positive effect was recorded when the mixture of lactobacilli and S. Enteritidis was used for the inoculation of feed and the feed was anaerobically fermented for 1 to 5 days. In this case, chickens fed such a diet remained S. Enteritidis negative. In vitro experiments showed that the protective effect was caused by acidification of feed down to pH 4.6 due to lactobacilli fermentation and was associated with S. Enteritidis inactivation. The probiotic effect of lactobacilli was thus expressed in the feed, outside the chicken host.