Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken

Fernanda de Oliveira Barbosa Oliveiro Caetano de Freitas Neto Diego Felipe Alves Batista Adriana Maria de Almeida Marcela da Silva Rubio Lucas Bocchini Rodrigues Alves Rosemeire de Oliveira Vasconcelos Paul Andrew Barrow Angelo Berchieri JuniorAbout the authors

ABSTRACT

Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE ΔmotB) or non-flagellated (SE ΔfliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE ΔmotB and SE ΔfliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5 dpi). The SE ΔmotB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE ΔfliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.

Keywords:
Mutation; Flagellum; Virulence factors; Poultry; Gut colonisation

Introduction

Salmonella enterica subsp. enterica serovar Enteritidis (SE) is a broad-host range micro-organism which poses a threat to both public and animal health.11 Mughini-Grass L, Enserink R, Friesema I, Heck M, Duynhoven Y, Pelt W. Risk factors for human salmonellosis originating from pigs, cattle, broiler chickens and egg laying hens: a combined case-control and source attribution analysis. PLoS ONE. 2014;9(2): http://dx.doi.org/10.1371/journal.pone.0087933.
http://dx.doi.org/10.1371/journal.pone.0...
It causes fowl paratyphoid, which is often associated with extensive gut colonisation and bacterial shedding in the faeces.22 Barrow PA, Lovell MA. Experimental infection of egg-laying hens with Salmonella Enteritidis phage type 4. Avian Pathol. 1991;20(2), http://dx.doi.org/10.1080/03079459108418769.
http://dx.doi.org/10.1080/03079459108418...
,33 Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE, eds. Diseases of Poultry. Ames: Iowa University Press; 2003. SE is one of the commonest serovars related to worldwide food-borne outbreaks.44 Threlfall EJ, Wain J, Peters T, et al. Egg-borne infectious of humans with Salmonella: not only an S. Enteritidis problem. Worlds Poult Sci J. 2014;70(1), http://dx.doi.org/10.1017/S0043933914000026.
http://dx.doi.org/10.1017/S0043933914000...

Following oral infection flagellated strains of Salmonella spp. colonise the intestines and flagellin, the main flagellar protein, is recognised through Toll-like receptor (TLR)-5 leading to activation of a pro-inflammatory response and release of the cytokines necessary to initiate the innate and adaptive immune responses.55 Paul MS, Brisbin JT, Abdul-Careem MF, Sharif S. Immunostimulatory properties of Toll-like receptor ligands in chickens. Vet Immunol Immunopathol. 2013;152(3-4), http://dx.doi.org/10.1016/j.vetimm.2012.10.013.
http://dx.doi.org/10.1016/j.vetimm.2012....
The intense local inflammation triggered during disease helps restrict the bacteria to the intestine and helps to control the systemic infection.66 Wigley P. Salmonella enterica in the chicken: how it has helped our understanding of immunology in a non-biomedical model species. Front Immunol. 2014;5(482), http://dx.doi.org/10.3389/fimmu.2014.00482.
http://dx.doi.org/10.3389/fimmu.2014.004...

Despite activating the innate immunity, possession of flagella is an important virulence trait which mediates bacterial attachment and invasion.77 Allen-vercoe E, Woodward MJ. The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant. J Med Microbiol. 1999;48(8), http://dx.doi.org/10.1099/00222615-48-8-771.
http://dx.doi.org/10.1099/00222615-48-8-...
,88 Parker CT, Guard Petter J. Contribution of flagella and invasion proteins to pathogenicity of Salmonella enterica serovar enteritidis in chicks. FEMS Microbiol Lett. 2001;204:287-291. In addition, flagella-mediated motility has also been considered as a virulence determinant for gut-associated Salmonella. Thus, a non-motile SE strain showed reduced ability to attach to cells in comparison to the parental strain.99 Van asten FJ, Hendriks HG, Koninkx JF, Van Dijk JE. Flagella-mediated bacterial motility accelerates but is not required for Salmonella serotype Enteritidis invasion of differentiated Caco-2 cells. Int J Med Microbiol. 2004;294(6), http://dx.doi.org/10.1016/j.ijmm.2004.07.012.
http://dx.doi.org/10.1016/j.ijmm.2004.07...

The correlation between flagella and flagella-mediated motility and whether or not they contribute independently to Salmonella pathogenesis is unknown. To investigate this, non-motile but fully flagellated (SE ΔmotB) and non-motile and non-flagellated (SE ΔfliC) mutant strains were constructed and the roles of flagella and flagella-mediated motility on intestinal colonisation and systemic invasion of chickens were assessed.

Materials and methods

Bacteria

This study used the spontaneous nalidixic acid resistant strain P125109 (SE). The parent strain was isolated from a case of food-poisoning in humans and is virulent for young chickens and capable of contaminating eggs when inoculated in laying hens.1010 Barrow PA, Lovell MA. Experimental infection of egg-laying hens with Salmonella enteritidis phage type 4. Avian Pathol. 1991;20(2), http://dx.doi.org/10.1080/03079459108418769.
http://dx.doi.org/10.1080/03079459108418...
All bacteria were cultured in lysogeny broth (LB - Becton Dickinson, Sparks, Maryland, USA) at 37 °C for 24 h at 150 revolutions per min (rpm).1111 Berchieri A Jr, Wigley P, Page K, Murphy CK, Barrow PA. Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens. Avian Pathol. 2001;30(4), http://dx.doi.org/10.1080/03079450120066304.
http://dx.doi.org/10.1080/03079450120066...

Mutant construction

Two mutant strains, SE ΔfliC and SE ΔmotB, were constructed using the Lambda-red method1212 Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97(12), http://dx.doi.org/10.1073/pnas.120163297.
http://dx.doi.org/10.1073/pnas.120163297...
and transduction with the phage P22 was used to transfer the mutation to a clean genetic background. Putative mutants were selected in Lysogeny agar (LA - DifcoTM, Detroit, Michigan, US) containing 20 µg/mL chloramphenicol and confirmed by polymerase chain reaction (PCR). After selection, the chloramphenicol-resistance gene was eliminated by using a helper plasmid expressing the FLP recombinase (pCP20), which acts on the directly repeated FRT (FLP recognition target) sites flanking the resistance gene. Specific primers were designed through PrimerBlast tool1313 Ye J, Colouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012;13(134), http://dx.doi.org/10.1186/1471-2105-13-134.
http://dx.doi.org/10.1186/1471-2105-13-1...
and are available in Table 1.

Table 1
Primer sequences used to construct the SE ΔfliC and SE ΔmotB mutant strains.

Flagella and flagella-mediated motility detection

SE, SE ΔmotB and SE ΔfliC swimming motility was detected by propagation on semi-solid agar (SSA), after inoculation onto the surface of semi-solid plates consisting of 0.9% heart infusion broth (Oxoid, Basingstoke, Hampshire, UK) and 0.25% LA (Difco, Detroit, Michigan, US), after 24 h incubation at 28 °C assessed by bacterial spread through the soft agar. Flagella expression was additionally confirmed through serum-agglutination using specific anti-H:g,m antibodies (Remel, Dartford, Kent, UK).

Chickens

One hundred and seventy one one-day-old male chickens from a commercial line of egg layer were used in the two experiments. Birds were housed in acclimatised rooms and received water and feed ad libitum. On arrival, samples of faeces in the transport cardboard boxes were collected and processed to exclude infection with Salmonella spp.1111 Berchieri A Jr, Wigley P, Page K, Murphy CK, Barrow PA. Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens. Avian Pathol. 2001;30(4), http://dx.doi.org/10.1080/03079450120066304.
http://dx.doi.org/10.1080/03079450120066...
In each experiment birds in infected groups received 1 × 109 colony forming units (CFU) of SE, SE ΔmotB or SE ΔfliC, respectively, into the crop using oral gavage needles. Experiments were approved by the institutional ethical committee (Process 1.353/15; approved on 03 March 2015).

Experiment 1 - mortality, clinical signs and faecal shedding

Forty-five chickens were distributed randomly into three groups of 15 animals and then infected. Birds of infected groups received 1 × 109 CFU of SE, SE ΔmotB or SE ΔfliC, respectively, into the crop as above. Birds were observed for four weeks. Mortality and other clinical signs were recorded daily and bacterial shedding in faeces was monitored by cloacal swabs twice a week.1111 Berchieri A Jr, Wigley P, Page K, Murphy CK, Barrow PA. Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens. Avian Pathol. 2001;30(4), http://dx.doi.org/10.1080/03079450120066304.
http://dx.doi.org/10.1080/03079450120066...

Experiment 2 - local and systemic infection and pathological changes

One hundred and five chickens were distributed randomly into three groups of 35 animals and then infected. Birds in infected groups were infected orally inoculated with 1 × 109 CFU of SE, SE ΔmotB or SE ΔfliC, respectively. A fourth group of 21 chicks was kept as the uninfected control for histopathology. Birds of uninfected control group were also mock-infected with 0.2 mL of sterile lysogeny broth (LB - Becton Dickinson, Sparks, Maryland, USA). At 2, 3, 5, 7, 14, 21 and 28 days post-infection (dpi), five birds from each infected group were euthanased by cervical dislocation and samples of spleen, liver and caecal content collected for bacterial enumeration.1111 Berchieri A Jr, Wigley P, Page K, Murphy CK, Barrow PA. Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens. Avian Pathol. 2001;30(4), http://dx.doi.org/10.1080/03079450120066304.
http://dx.doi.org/10.1080/03079450120066...
Gross pathologies were also recorded.

At the time points above samples of liver, caecum and ileum were collected from the same infected chicks and also from three non-infected animals for histopathology.1515 Bancroft JD, Stevens A, eds. Theory and Practice of Histological Techniques. Philadelphia: Churchill Livingstone; 2008. Samples were formalin-fixed and paraffin-embedded. Tissues were sectioned at 4-µm thickness, stained with haematoxylin and eosin and observed by light microscopy. Lesions were classified as mild, moderate and severe as described previously.1616 Lopez PD, Freitas Neto OC, Batista DFA, et al. Experimental infection of chickens by a flagellated motile Salmonella Gallinarum mutant strain gives insights into the immunopathology of fowl typhoid. Vet J. 2016;214, http://dx.doi.org/10.1016/j.tvjl.2016.05.006.
http://dx.doi.org/10.1016/j.tvjl.2016.05...

Statistical analysis

Data on mortality and faecal shedding were compared by chi-square test.1717 Greenwood PE, Nikulin MS, eds. A Guide To Chi-Squared Testing. New York: Wiley and Sons; 1996. Statistical differences amongst viable bacteria numbers recovered from caecal contents, livers and spleens were determined using Tukey's test.66 Wigley P. Salmonella enterica in the chicken: how it has helped our understanding of immunology in a non-biomedical model species. Front Immunol. 2014;5(482), http://dx.doi.org/10.3389/fimmu.2014.00482.
http://dx.doi.org/10.3389/fimmu.2014.004...
Statistical tests were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, California, USA).

Results

Mutagenesis - flagella and flagella-mediated motility assessment

Deletion of fliC and motB genes from the SE chromosome was first confirmed by PCR. The mutant strains showed impairment in their ability to spread throughout the SSA after 24 h incubation and only a small halo, nearly 6-mm diameter, was noticeable in the centre of the agar. By contrast, SE was able to cover the whole semi-solid surface after 24 h of incubation. The serum agglutination test targeting the flagellar antigens (H: g,m) was positive for SE and SE ΔmotB strains and negative for SE ΔfliC.

Experiment 1 - mortality and faecal shedding

Clinical manifestations began at 4 dpi in all infected chickens in which somnolence, closed eyes and persistent diarrhoea (up to 13 dpi) containing smears of blood were observed. SE and SE ΔfliC infections produced 13% mortality (n = 2/15 infected) whereas no mortality occurred amongst SE ΔmotB-infected chickens. Despite this, no statistical significance was found between the mortality rates (p > 0.05). Additionally, the number of positive cloacal swabs from which the inoculated strain was recovered was very similar amongst the animals infected with SE (92.5%), SE ΔmotB (87.5%) and SE ΔfliC (93.3%), and it was not statistically significant (p > 0.05).

Experiment 2

Caecal colonisation and systemic invasion

The results of bacterial enumeration in livers, spleens and caecal contents are shown in Fig. 1. There was no statistically significant difference between the bacterial numbers in caecal content (p = 0.7225), liver (p = 0.5618) and spleen (p = 0.5294) at 2 dpi. SE colonised the caecal contents in higher numbers early (3, 5 and 7 dpi) in infection (p < 0.05). However, from 14 dpi onward the bacterial counts of all strains in caecal contents decreased to similar numbers (p = 0.6257). Bacterial recovery from livers and spleens was very similar for all three strains. SE reached the spleens in higher numbers at 3 dpi (p < 0.05) but at 5 dpi onward all strains showed a similar behaviour (p = 0.1880). The bacterial numbers in the livers were low (103 CFU/g) throughout the experiment for all three strains and no statistical significance was found (p = 0.3513).

Fig. 1
Bacterial counts (log10 CFU/g) in livers, spleens and caecal contents collected from one-day-old chicks infected with SE, SE ΔfliC and SE ΔmotB. Different letters on the plots mean there was statistical significance by Tukey's test (p < 0.05) between distinct treatments by day.

Pathological changes

No gross pathology was observed in any infected animal at 2 dpi. From 3 dpi, mild hepatosplenomegaly and mild haemorrhagic enteritis were observed in SE-infected chickens, whereas no noticeable changes occurred in the intestines of SE ΔfliC- and SE ΔmotB-infected chickens. The greatest changes, however, were noticed at 7 dpi when congestive hepatosplenomegaly and thickened intestinal mucosa were noticeable in all necropsied animals. From 14 to 28 dpi gross pathologies became mild but present in all infected animals.

The most severe histopathological changes were observed in the liver, ileum and caeca of SE-infected chickens. SE induced hepatocyte degeneration and lymphoid reactivity from 2 dpi, but at 7 dpi, the former became severe and diffused and the latter moderate and mostly surrounding the portal triads and perivascular areas. During this same span of time (2-7 dpi) SE ΔmotB induced milder hepatocyte degeneration and moderate lymphoid reactivity surrounding the portal triads and perivascular areas whereas SE ΔfliC provoked mild foci of necrosis with mild adjacent infiltration of mononuclear cells in the hepatic parenchyma. At 14, 21 and 28 dpi mild hepatocyte degeneration with lymphoid reactivity at parenchyma was seen in livers in all infected animals.

In the gut SE elicited moderate multifocal lymphocyte infiltration in the ileal lamina propria mucosa from 2 to 7 dpi. At 5dpi SE ΔmotB elicited mild multifocal lymphocyte infiltration in the ileal lamina propria (Fig. 2). Meanwhile in SE ΔfliC-infected chickens this alteration was observed later, at 7 dpi. From 14 dpi onwards, lymphocyte infiltration in the ileal lamina propria became moderate in birds infected with both mutant strains. By contrast, SE caused diffuse and moderate lymphocyte infiltration in caecal lamina propria during all experiments. The SE ΔfliC and SE ΔmotB strains caused the same lesions, but at 3 and 5 dpi mild lymphocyte infiltration in the caecal lamina propria was observed. Mononuclear cell infiltration in lamina propria in addition to villus fusion and submucosal oedema became mild and similar in all infected birds after 21 dpi.

Fig. 2
Transverse sections of ilea collected at 5 dpi from chicks infected at 1 day of life with SE, SE ΔmotB or SE ΔfliC. (A) Healthy ileum collected from an uninfected bird showing no mononuclear infiltration (arrow); (B) presence of mild mononuclear inflammatory infiltrate in the mucosa (arrow) of a SE ΔmotB-infected chick; (C) no detectable changes in ileal mucosa (arrow) of a SE ΔfliC-infected chick; (D) presence of severe mononuclear inflammatory infiltrate (arrow) in fused and shortened villi in the ileal mucosa of a SE-infected chick. Haematoxylin and eosin staining. Scale bars: 100 µM (small images) and 40 µM (large images).

Discussion

Flagella and flagella-mediated motility are considered important factors for salmonellosis.1818 Josenhans C, Suerbaum S. The role of motility as a virulence factor in bacteria. Int J Med Microbiol. 2002;291(8), http://dx.doi.org/10.1078/1438-4221-00173.
http://dx.doi.org/10.1078/1438-4221-0017...
Their contribution to S. Enteritidis (SE) pathogenicity in poultry has been evaluated in separate studies,88 Parker CT, Guard Petter J. Contribution of flagella and invasion proteins to pathogenicity of Salmonella enterica serovar enteritidis in chicks. FEMS Microbiol Lett. 2001;204:287-291.,1919 Dibb-Fuller MP, Woodward MJ. Contribution of fimbriae and flagella of Salmonella Enteritidis to colonization and invasion of chicks. Avian Pathol. 2000;29(4), http://dx.doi.org/10.1080/03079450050118412.
http://dx.doi.org/10.1080/03079450050118...
,2020 Robertson JMC, McKenzie NH, Duncan M, et al. Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat. J Med Microbiol. 2003;52(1), http://dx.doi.org/10.1099/jmm.0.04901-0.
http://dx.doi.org/10.1099/jmm.0.04901-0...
but the role of flagella as opposed to motility still requires further investigation. To shed light on this subject, the present study compared the infection biology of the motile and fully flagellated SE strain P125109 and its derivative mutant strains, one non-motile and non-flagellated (SE ΔfliC) and other non-motile but flagellated (SE ΔmotB) using one-day-old male chickens as the model.

Over the 4-week experiment 1 bacterial recovery from faeces was similar for all strains independent on the phenotype. In agreement with this result, a previous report showed that the infection of chicks by a wild-type SE and a non-motile flagellated resulted in a similar degree of faecal excretion.2020 Robertson JMC, McKenzie NH, Duncan M, et al. Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat. J Med Microbiol. 2003;52(1), http://dx.doi.org/10.1099/jmm.0.04901-0.
http://dx.doi.org/10.1099/jmm.0.04901-0...
These findings, combined with the absence of significant mortality in the present study, show that neither the absence of flagella nor its related motility alter the faecal excretion ability of SE in chickens.

Although the mutations introduced into the SE chromosome did not impair bacterial shedding by faeces, the ability to colonise the caeca early was altered since the counts of SE ΔfliC and SE ΔmotB in caecal contents at 3 and 5 dpi were significantly lower. Previous studies using chicken infection, chicken gut explants or cultured epithelial cells infected with non-motile strains of Salmonella, also reported the reduced ability of these mutant strains to colonise/adhere to the cells when comparing to the wild type flagellated strains, in the early stages of colonisation.77 Allen-vercoe E, Woodward MJ. The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant. J Med Microbiol. 1999;48(8), http://dx.doi.org/10.1099/00222615-48-8-771.
http://dx.doi.org/10.1099/00222615-48-8-...
,1919 Dibb-Fuller MP, Woodward MJ. Contribution of fimbriae and flagella of Salmonella Enteritidis to colonization and invasion of chicks. Avian Pathol. 2000;29(4), http://dx.doi.org/10.1080/03079450050118412.
http://dx.doi.org/10.1080/03079450050118...
,2121 Schmitt CK, Ikeda JS, Darnell SC, et al. Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis. Infect Immun. 2001;69(9), http://dx.doi.org/10.1128/IAI.69.9.5619-5625.200.
http://dx.doi.org/10.1128/IAI.69.9.5619-...
Taken together these results suggest that flagella and flagella-mediated motility would play important roles early (up to 5 dpi), but not later, during SE infection in chickens. This conclusion is also supported by the fact that at 7 dpi both mutant strains started to cause intestinal histopathological changes similar to those induced by the wild-type strain.

It has been hypothesised that recognition of flagellated Salmonella strains through intestinal TLR5 leads to activation of pro-inflammatory response which in turn helps to restrict the bacteria to the intestine and to prevent systemic infection.55 Paul MS, Brisbin JT, Abdul-Careem MF, Sharif S. Immunostimulatory properties of Toll-like receptor ligands in chickens. Vet Immunol Immunopathol. 2013;152(3-4), http://dx.doi.org/10.1016/j.vetimm.2012.10.013.
http://dx.doi.org/10.1016/j.vetimm.2012....
,77 Allen-vercoe E, Woodward MJ. The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant. J Med Microbiol. 1999;48(8), http://dx.doi.org/10.1099/00222615-48-8-771.
http://dx.doi.org/10.1099/00222615-48-8-...
However, in the present study, at 3 dpi the wild type SE strain was recovered from spleen in higher counts than the non-flagellated SE ΔfliC. Very similar results were reported in rats infected with flagellated and non-flagellated-SE strains.2020 Robertson JMC, McKenzie NH, Duncan M, et al. Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat. J Med Microbiol. 2003;52(1), http://dx.doi.org/10.1099/jmm.0.04901-0.
http://dx.doi.org/10.1099/jmm.0.04901-0...
It seems that the absence of flagella in SE was in fact disadvantageous in establishing systemic infection. Further studies must be carried out in order to better characterise the immunological bases of the infection by these strains and to assess whether or not the lower systemic colonisation of SE ΔfliC is a consequence of its reduced invasion ability due to the absence of motility.99 Van asten FJ, Hendriks HG, Koninkx JF, Van Dijk JE. Flagella-mediated bacterial motility accelerates but is not required for Salmonella serotype Enteritidis invasion of differentiated Caco-2 cells. Int J Med Microbiol. 2004;294(6), http://dx.doi.org/10.1016/j.ijmm.2004.07.012.
http://dx.doi.org/10.1016/j.ijmm.2004.07...

Interestingly, SE ΔfliC, but not SE ΔmotB, showed improved ability to colonise the caeca at 7 dpi. Similar results were previously reported for Salmonella Typhimurium (STM) in a murine ligated-loop invasion assay. It was postulated that the extracellular electrostatic repulsion produced around the paralysed flagella prevented the contact between bacteria and intestinal cells, thus affecting the gut colonisation.2222 Jones BD, Lee CA, Falkow L. Invasion by Salmonella Typhimurium is affected by the direction of flagellar rotation. Infect Immunol. 1992;60:2475-2480. This phenomenon, designated as steric hindrance, could also be the possible explanation for the longer lower level caecal recovery of SE ΔmotB compared to SE ΔfliC observed in the present study.

The wild-type strain induced, at the early stages of infection, more severe hepatic lesions. According to Xiao et al.,2323 Xiao Y, Liu F, Yang J, et al. Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice. Cell Mol Immunol. 2014;12(6), http://dx.doi.org/10.1038/cmi.2014.110.
http://dx.doi.org/10.1038/cmi.2014.110...
flagella expression is inhibited in the liver although a minimal amount of flagellin released by flagellated strains is sufficient to stimulate the immune system via TLR5 recognition and induce subsequent function abnormality and damage to the liver. SE ΔfliC does not produce flagellin which is thought to be the reason by which only mild hepatic lesions were produced by this strain early in infection. In this study SE ΔmotB induced inflammatory infiltration in ileal and caeca mucosae. This result agrees with that shown by Xiao et al.2323 Xiao Y, Liu F, Yang J, et al. Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice. Cell Mol Immunol. 2014;12(6), http://dx.doi.org/10.1038/cmi.2014.110.
http://dx.doi.org/10.1038/cmi.2014.110...
in which paralysed flagella were associated with a significant reduction in in vitro invasiveness although presumably still able to signal through TLR5.

Data generated in this study showed that the lack of either flagella or flagella-mediated motility impairs SE pathogenicity in young chickens, chiefly in the intestine and early during infection. The paralysed flagella also appeared to be more detrimental than the complete absence of flagella, a fact demonstrated previously in epithelial cells.99 Van asten FJ, Hendriks HG, Koninkx JF, Van Dijk JE. Flagella-mediated bacterial motility accelerates but is not required for Salmonella serotype Enteritidis invasion of differentiated Caco-2 cells. Int J Med Microbiol. 2004;294(6), http://dx.doi.org/10.1016/j.ijmm.2004.07.012.
http://dx.doi.org/10.1016/j.ijmm.2004.07...
These results imply that motility in Salmonella contributes to the early stages of intestinal colonisation.

Acknowledgments

This work was supported by São Paulo Research Foundation (FAPESP) [grant numbers 2014/02014-1 (F.O. Barbosa) and 2013/26127-7 (A. Berchieri Junior)] and National Council of Technological and Scientific Development (CNPq).

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    » http://dx.doi.org/10.1016/j.vetimm.2012.10.013
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    » http://dx.doi.org/10.3389/fimmu.2014.00482
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    Allen-vercoe E, Woodward MJ. The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant. J Med Microbiol. 1999;48(8), http://dx.doi.org/10.1099/00222615-48-8-771
    » http://dx.doi.org/10.1099/00222615-48-8-771
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Publication Dates

  • Publication in this collection
    Oct-Dec 2017

History

  • Received
    16 Aug 2016
  • Accepted
    20 Jan 2017
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br