Evaluation of pathogenicity of <i>Salmonella</i> Gallinarum strains harbouring deletions in genes whose orthologues are conserved pseudogenes in <i>S</i>. Pullorum

<div><p>The diseases caused by <i>Salmonella</i> Gallinarum and <i>S</i>. Pullorum in chickens known as fowl typhoid and pullorum disease, respectively, pose a great threat to the poultry industry mainly in developing countries, since they have already been controlled in the developed ones. These bacteria are very similar at the genomic level but develop distinct host-pathogen relationships with chickens. Therefore, a deep understanding of the molecular mechanisms whereby <i>S</i>. Gallinarum and <i>S</i>. Pullorum interact with the host could lead to the development of new approaches to control and, perhaps, eradicate both diseases from the chicken flocks worldwide. Based on our previous study, it was hypothesised that metabolism-related pseudogenes, fixed in <i>S</i>. Pullorum genomes, could play a role in the distinct host-pathogen interaction with susceptible chickens. To test this idea, three genes (<i>idnT</i>, <i>idnO</i> and <i>ccmH</i>) of <i>S</i>. Gallinarum str. 287/91, which are pseudogenes on the <i>S</i>. Pullorum chromosomes, were inactivated by mutations. These genetically engineered strains grew well on the solid media without any colony morphology difference. In addition, similar growth curves were obtained by cultivation in M9 minimal medium containing D-gluconate as the sole carbon source. Infection of chickens with <i>idnTO</i> mutants led to increased numbers of bacteria in the livers and spleens at 5 days post-infection, but with slightly decreased heterophil infiltration in the spleens when compared to the wild-type strain. On the other hand, no significant phenotypic change was caused by mutation to <i>ccmH</i> genes. Apart from the above-mentioned alterations, all <i>S</i>. Gallinarum strains provoked similar infections, since mortality, clinical signs, macroscopic alterations and immune response were similar to the infected chickens. Therefore, according to the model applied to this study, mutation to the <i>idnTO</i> and <i>ccmH</i> genes showed minor impact on the fowl typhoid pathogenesis and so they may be relics from the ancestor genome. Our data hints at a more complex mechanism driving the distinct host-pathogen interaction of <i>S</i>. Gallinarum/Pullorum with chickens than differential inactivation of a few genes.</p></div

Transcription of the cytokine-related genes evaluated in this study by RT-qPCR.

<p>Chickens were orally infected with the strains here tested on the 15 day of age. Samples of caecal tonsils and spleen tissues were collected at 1, 3 and 5 dpi. (*) This symbol shows groups whose mRNA transcription statistically differs by one-way ANOVA followed by Tukey’s test (* <i>P</i> < 0.05 / ** <i>P</i> < 0.01). CXCLi2: CXC-like chemokine previously named as interleukin 8; IL6: Interleukin 6; IFN: Interferon gamma; (CT): Caecal tonsil; (SP): Spleen; Control: Non-infected chickens; SP449/87: Chickens infected with <i>S</i>. Pullorum str. 449/87 (pullorum disease positive control); SG287/91: Chickens infected with <i>S</i>. Gallinarum str. 287/91 (fowl typhoid positive control); SGΔ<i>ccmH</i>: Chickens infected with SGΔ<i>ccmH</i>; SGΔ<i>idnTO</i>: Chickens infected with SGΔ<i>idnTO</i>; SGΔ<i>ccmHidnTO</i>: Chickens infected with SGΔ<i>ccmHidnTO</i>.</p

Number of viable colony-forming units (CFU) per gram (g) of liver or spleen tissue collected from chickens infected on the 15 day of age.

<p>Organs were collected at 1, 3 and 5 days post-infection (dpi). Graph interpretation: Groups compared by day-post infection for each analysed tissue. Different letters stand for statistically distinct CFU / g through one-way ANOVA followed by Bonferroni correction. Results are expressed as mean ± standard deviation. (*) A single liver sample which was positive for SGΔ<i>idnTO</i> after enrichment; SP449/87: <i>S</i>. Pullorum str. 449/87 (pullorum disease positive control); SG287/91: <i>S</i>. Gallinarum str. 287/91 (fowl typhoid positive control); SGΔ<i>ccmH</i>: <i>S</i>. Gallinarum str. 287/91 harbouring mutation on the genes <i>ccmH</i> alone; SGΔ<i>idnTO</i>: <i>S</i>. Gallinarum str. 287/91 harbouring mutation on the genes <i>idnTO</i> alone; SGΔ<i>ccmHidnTO</i>: <i>S</i>. Gallinarum str. 287/91 harbouring both mutations.</p

Growth curves of SG287/91 and its derivative mutants cultured in M9 minimal media containing D-gluconate as the sole carbon source.

<p>(*) On the comparison by time point, the growth of at least two strains statistically differed by one-way ANOVA followed by Bonferroni correction for multiple comparisons. Symbols on the curve express mean ± standard deviation. SG287/91: <i>S</i>. Gallinarum str. 287/91 (parental strain); SGΔ<i>ccmH</i>: <i>S</i>. Gallinarum str. 287/91 harbouring mutation in the genes <i>ccmH</i>; SGΔ<i>ccmHidnTO</i>: <i>S</i>. Gallinarum str. 287/91 harbouring mutations in the genes <i>idnTO</i> and <i>ccmH</i>; SGΔ<i>idnTO</i>: <i>S</i>. Gallinarum str. 287/91 harbouring mutation in the genes <i>idnTO</i>.</p

Effects of <i>in ovo</i> injection of bacterial peptides and CpG-ODN on <i>Salmonella enterica</i> serovar Heidelberg infection in specific pathogen-free (SPF) chicks

The increasing prevalence of pathogenic and antimicrobial-resistant isolates of Salmonella Heidelberg (SH) represents a global concern. Consequently, novel strategies for preventing and controlling infections caused by this bacterium are needed. This study aimed to investigate the protective effects of in ovo injection with a formulation based on bacterial peptides, either alone or in combination with oligodeoxynucleotides containing cytosine-phosphodiester-guanine (CpG-ODN), against SH infection in chicks. Genomic data of SH available in public databases were analyzed to select amino acid sequences of structural proteins or those with greater relevance for intestinal colonization. These sequences were subjected to linear epitope prediction tools to identify highly immunogenic peptides. SPF eggs (n = 180) were incubated and injected via the allantoic cavity at day 18 of incubation. Results showed that in ovo injection of peptides + CpG-ODN reduced SH colonization in the caecal content during the first week post-infection, although it did not reduce overall faecal excretion throughout the study. Furthermore, CpG-ODN injection may positively affect intestinal health, as evidenced by reduced crypt depth at 21 dpi and increased villus height at 28 dpi. Levels of secretory IgA did not differ between chicks in any groups, and no detectable SH counts were found in the livers of chicks in any of the infected groups throughout the study. In conclusion, in ovo injection with peptides + CpG-ODN may help reduce caecal colonization by SH in the early stages of infection, but it does not impact total faecal excretion. Additionally, CpG-ODN injection may improve intestinal health parameters in chicks. Peptides + CpG-ODN reduced SH in caeca at the first week post-infection.Administered formulations did not reduce SH-faecal excretion.Levels of intestinal IgA were similar between all groups.CpG-ODN improved some parameters associated with chick intestinal health. Peptides + CpG-ODN reduced SH in caeca at the first week post-infection. Administered formulations did not reduce SH-faecal excretion. Levels of intestinal IgA were similar between all groups. CpG-ODN improved some parameters associated with chick intestinal health.</p