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

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

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

Pathological findings associated with toxoplasmosis in free-ranging marmosets (<i>Callithrix</i> spp.).

(A) Random lytic necrotizing hepatitis with adjacent mild lipidosis, Liver, HE, Scale bar = 100 μm. (B) Immuno staining for T. gondii zoites, Liver, DAB, Scale bar = 60 μm. (C) Splenitis, histiocytic, with fibrin deposition in red pulp, Spleen, HE, Scale bar = 60 μm. (D) Immuno staining for T. gondii zoites, Spleen, DAB, Scale bar = 60 μm. (E) Interstitial pneumonia, characterized by alveolar walls expanded by histiocytes, lymphocytes and plasma cells, with moderate number of foamy macrophages, mild alveolar hemorrhage, diffuse congestion, and intracytoplasmic Toxoplasma gondii zoites (inset), Lung, HE, Scale bar = 60 μm. (F) Immuno staining for T. gondii zoites, Lung, DAB, Scale bar = 60 μm.</p

Distribution of toxoplasmosis cases in free-ranging marmosets from Rio de Janeiro state from January 2017 to July 2019.

Map from Rio de Janeiro mesoregions (IBGE 2021) with total number of animals with by each mesoregion (grey box). SRC: EPCG 4674 –SIRGAS 2000 (QGIS Version 3.24.3).</p

General data and pathological findings of free-ranging marmosets (<i>Callithrix</i> spp.) from the State of Rio de Janeiro (Brazil) with toxoplasmosis from January 2017 to July 2019.

General data and pathological findings of free-ranging marmosets (Callithrix spp.) from the State of Rio de Janeiro (Brazil) with toxoplasmosis from January 2017 to July 2019.</p