Protection of chickens against fowl typhoid using field vaccine programs formulated with the live attenuated strain Salmonella Gallinarum ΔcobSΔcbiA

<div><p>ABSTRACT: Salmonella enterica serovar Gallinarum biovar Gallinarum (SG) is a host-specific bacteria that causes the fowl typhoid (FT). This disease is highly pathogenic to commercial chickens, specially brown layers and breeders, causing acute septicemia followed by high morbidity and mortality. Vaccination is extensively adopted in the fields as a biosafety tool for prevention of isolated infections and outbreaks in commercial poultry flocks. The present study evaluated the use of an attenuated SG with deletions on genes cobS and cbiA (SGΔcobSΔcbiA) as a live vaccine, using vaccination schemes adjusted for field conditions. To this end, brown layers were used in two different experiments, to evaluate the long-term protection, necessary in the fields. The vaccination scheme on the first experiment consisted of two doses, the first at 4 th week-of-age and the booster dose at 8 th week-of-age with challenge at 16 th week-of-age with wild SG strain. On the second experiment, the vaccination was carried out by different routes using three doses of the live vaccine, at 4 th , 8 th and 12 th weeks-of-age, and the challenge was done at 20 th weeks-of-age. After the challenge, the mortality was recorded during 28 days, and the egg production (experiment 2) was evaluated and compared with the group of unvaccinated layers. In both experiments, the mortality was significantly reduced, and the egg production was not affected in vaccinated layer-hens. In summary, this study shows the efficacy and the protection of different vaccination schemes against FT that can be applied under field conditions in commercial poultry farms.</p></div

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

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

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

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