[show abstract][hide abstract] ABSTRACT: 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 theV3/V4 variable regions of genes coding for 16S rRNA.
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.
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.
BMC Veterinary Research 07/2013; 9(1):140. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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, IFNgamma, iNOS, ES1, IL-1beta, 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.
Veterinary Research 05/2013; 44(1):37. · 3.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study we were interested in the serovar cross-protection potential of Salmonella Pathogenicity Island 1 (SPI1) attenuated vaccine strains of Salmonella enterica serovars Enteritidis and Typhimurium and immune response of vaccinated and naive chickens to Salmonella infection. The immune response was characterized by real time PCR quantifying transcripts of interleukins IL1β, IL17, IL22, interferon gamma (IFNγ), inducible NO synthase (iNOS), immunoglobulins IgM, IgA, IgY and Ig light chain, and six genes of acute phase response including avidin, serum amyloid A, extracellular fatty acid-binding protein (Ex-FABP), immune responsive gene 1, chemokine AH221 and trappin-6. Vaccination with SPI1 mutants of both serovars protected chickens against Salmonella infection, independent of the serovar used for the challenge and the time post infection. However, expressions of all interleukins, iNOS and Ex-FABP showed that protection against homologous serovars was significantly higher than against heterologous serovars after intravenous challenge at 4 days post infection. The vaccination with a mixture of S. Enteritidis and S. Typhimurium SPI1 mutants induced an intermediate protection against challenge with both serovars, i.e. the mixed vaccine provided an additional protective effect when compared with the chickens vaccinated with a vaccine formed by only a single Salmonella serovar.
[show abstract][hide abstract] ABSTRACT: 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.
BMC Veterinary Research 02/2013; 9(1):30. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: The prevalence of Salmonella enterica serovar Enteritidis is gradually decreasing in poultry flocks in the EU, which may result in the demand for a vaccine that allows for the differentiation of vaccinated flocks from those infected by wild-type S. Enteritidis. In this study, we therefore constructed a (Salmonella Pathogenicity Island 1) SPI1-lon mutant with or without fliC encoding for S. Enteritidis flagellin. The combination of SPI1-lon mutations resulted in attenuated but immunogenic mutant suitable for oral vaccination of poultry. In addition, the vaccination of chickens with the SPI1-lon-fliC mutant enabled the serological differentiation of vaccinated and infected chickens. The absence of fliC therefore did not affect the immunogenicity of the vaccine strain and allowed for serological differentiation of the vaccinated chickens. The SPI1-lon-fliC mutant is therefore a suitable marker vaccine strain for oral vaccination of poultry.
PLoS ONE 01/2013; 8(6):e66172. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: T-RFLP and quantitative PCR showed that the cecal microbiota of chicks up to the age of 21 days was dominated by representatives of Enterobacteriales, Clostridiales and Lactobacillales. Salmonella Enteritidis infection caused the greatest changes in gut microbiota when one-day-old chicks were infected compared with infection of 4- and 16-day-old chicks.
Applied and environmental microbiology 11/2012; · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Helicobacter (H.) suis colonizes the stomach of pigs and is the most prevalent gastric non-H. pylori Helicobacter species in humans. Limited information is available on host immune responses after infection with this agent and it is unknown if variation in virulence exists between different H. suis strains. Therefore, BALB/c and C57BL/6 mice were used to compare colonization ability and gene expression of various inflammatory cytokines, as determined by real-time PCR, after experimental infection with 9 different H. suis strains. All strains were able to persist in the stomach of mice, but the number of colonizing bacteria at 59 days post inoculation was higher in stomachs of C57BL/6 mice compared to BALB/c mice. All H. suis strains caused an upregulation of interleukin (IL)-17, which was more pronounced in BALB/c mice. This upregulation was inversely correlated with the number of colonizing bacteria. Most strains also caused an upregulation of regulatory IL-10, positively correlating with colonization in BALB/c mice. Only in C57BL/6 mice, upregulation of IL-1beta was observed. Increased levels of IFN-gamma mRNA were never detected, whereas most H. suis strains caused an upregulation of the Th2 signature cytokine IL-4, mainly in BALB/c mice. In conclusion, the genetic background of the murine strain has a clear impact on the colonization ability of different H. suis strains and the immune response they evoke. A predominant Th17 response was observed, accompanied by a mild Th2 response, which is different from the Th17/Th1 response evoked by H. pylori infection.
Veterinary Research 10/2012; 43(1):75. · 3.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: The significance of maternal immunity against non-typhoid Salmonella spp. acquired by piglets via colostrum and milk was evaluated in a Salmonella enterica serovar Typhimurium challenge experiment. Piglets from sows vaccinated with an experimental inactivated vaccine exhibited high levels of serum immunoglobulins G and A against S. Typhimurium 4days after birth, just prior to experimental oral challenge. The S. Typhimurium load in the ileal and caecal wall of piglets 3days after experimental inoculation was lower by a 2-log magnitude compared to unvaccinated controls. Such a vaccine, delivering colostral/lactogenic immunity to piglets thus has the potential to reduce the prevalence non-typhoid Salmonella spp. infection.
The Veterinary Journal 09/2012; · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Within the last decade, macrophages have been shown to be capable of differentiating toward a classically activated phenotype (M1) with a high antimicrobial potential or an alternatively activated phenotype (M2). Some pathogens are capable of interfering with differentiation in order to down-regulate the anti-microbial activity and enhance their survival in the host.
To test this ability in Salmonella enterica serovar Typhimurium, we infected porcine alveolar macrophages with wild-type Salmonella Typhimurium and its isogenic mutants devoid of two major pathogenicity islands, SPI-1 and SPI-2. The induction of genes linked with M1 or M2 polarization was determined by quantification of gene expression by RT-qPCR. The ΔSPI-1 mutant induced a high, dose-dependent M1 response but a low M2 response in infected macrophages. On the other hand, wild-type Salmonella Typhimurium induced a low M1 response but a high, dose-dependent M2 response in infected macrophages. The response to ΔSPI-2 mutant infection was virtually the same as the wild-type strain.
We therefore propose that Salmonella Typhimurium DT104 studied here can polarize macrophages towards the less bactericidal M2 phenotype and that this polarization is dependent on the type III secretion system encoded by SPI-1.
BMC Veterinary Research 07/2012; 8:115. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.
[show abstract][hide abstract] ABSTRACT: In this study we were interested in the vaccine potential of two attenuated mutants of Salmonella enterica serovar Enteritidis for poultry. The first mutant was attenuated by the removal of the whole Salmonella Pathogenicity Island 1 (SPI1) and the second mutant was devoid of the whole SPI2. These 2 mutants were used for oral vaccination of 2 chicken lines; Lohmann Brown and ISA Brown. Chickens were vaccinated orally on day 1 of life, revaccinated on day 21 and challenged on day 42. The challenge was performed either orally or intravenously. Despite a slightly different response between the two chicken lines, both the mutants gave protection to poultry against S. Enteritidis challenge as documented by findings such as the bacterial counts in tissues, spleen weight, antibody production and cytokine response (namely IL-17 and IL-22). When the 2 mutants were compared, vaccination with the SPI1 mutant proved to be more effective in the protection of poultry against S. Enteritidis challenge than the vaccination with the SPI2 mutant. On the other hand, vaccination with the SPI2 mutant stimulated a slightly higher antibody production and such a mutant might therefore be a better choice if Salmonella is used as a vector for the delivery of heterologous antigens with a desired stimulation of the humoral part of the immune system.
[show abstract][hide abstract] ABSTRACT: In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immune chickens interact differently when exposed to S. enterica. In this study we therefore determined the immune response of vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enterica serovar Enteritidis (S. Enteritidis). Using flow cytometry we showed that 4 days post infection (DPI), counts of CD4 and B-lymphocytes did not change, CD8 and γδ T-lymphocytes decreased and macrophages and heterophils increased in the spleen. When vaccinated and non-vaccinated chickens were compared, only macrophages and heterophils were found in significantly higher counts in the spleens of the non-vaccinated chickens. The non-vaccinated chickens also expressed higher anti-LPS antibodies than the vaccinated chickens. The expression of interleukin (IL)1β, IL6, IL8, IL18, LITAF, IFNγ and iNOS did not exhibit any clear pattern in the cells sorted from the spleens of vaccinated or non-vaccinated chickens. Only IL17 and IL22 showed a differential expression in the CD4 T-lymphocytes of the vaccinated and non-vaccinated chickens at 4 DPI, both being expressed at a higher level in the non-vaccinated chickens. Due to a similar IFNγ expression in the CD4 T-lymphocytes in both the vaccinated and non-vaccinated chickens, and a variable IL17 expression oscillating around IFNγ expression levels, the IL17∶IFNγ ratio in CD4 T-lymphocytes was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels than IFNγ in the non-vaccinated chickens, the Th17 immune response with a higher macrophage and heterophil infiltration in the spleen dominated. However, when the expression of IL17 was lower than that of IFNγ as in the vaccinated chickens, the Th1 response with a higher resistance to S. Enteritidis infection dominated.
PLoS ONE 01/2012; 7(2):e32346. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study we were interested in identification of new markers of chicken response to Salmonella Enteritidis infection. To reach this aim, gene expression in the spleens of naive chickens and those intravenously infected with S. Enteritidis with or without previous oral vaccination was determined by 454 pyrosequencing of splenic mRNA/cDNA. Forty genes with increased expression at the level of transcription were identified. The most inducible genes encoded avidin (AVD), extracellular fatty acid binding protein (EXFABP), immune responsive gene 1 (IRG1), chemokine ah221 (AH221), trappin-6-like protein (TRAP6) and serum amyloid A (SAA). Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and γδ T-lymphocytes, we found that the above mentioned genes were preferentially expressed in macrophages. AVD, EXFABP, IRG1, AH221, TRAP6 and SAA were induced also in the cecum of chickens orally infected with S. Enteritidis on day 1 of life or day 42 of life. Unusual results were obtained for the immunoglobulin encoding transcripts. Prior to the infection, transcripts coding for the constant parts of IgM, IgY, IgA and Ig light chain were detected in B-lymphocytes. However, after the infection, immunoglobulin encoding transcripts were expressed also by T-lymphocytes and macrophages. Expression of AVD, EXFABP, IRG1, AH221, TRAP6, SAA and all immunoglobulin genes can be therefore used for the characterization of the course of S. Enteritidis infection in chickens.
PLoS ONE 01/2012; 7(10):e48101. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: There is increasing evidence that activation of innate immunity, in animals and man, by live vaccines, sub-unit vaccines or synthetic or non-synthetic stimulants can induce a profound and rapidly induced resistance to pathogens, including infectious agents that are unrelated to the stimulating antigen or agent. We review the evidence for this phenomenon and present the proposition that this approach might be used to stimulate immunity during the life of the animal when susceptibility to infection is high and when normal vaccination procedures may be inappropriate.
Research in Veterinary Science 10/2011; 93(1):7-12. · 1.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: In commercial poultry production, there is a lack of natural flora providers since chickens are hatched in the clean environment of a hatchery. Events occurring soon after hatching are therefore of particular importance, and that is why we were interested in the development of the gut microbial community, the immune response to natural microbial colonization, and the response to Salmonella enterica serovar Enteritidis infection as a function of chicken age. The complexity of chicken gut microbiota gradually increased from day 1 to day 19 of life and consisted of Proteobacteria and Firmicutes. For the first 3 days of life, chicken cecum was protected by increased expression of chicken β-defensins (i.e., gallinacins 1, 2, 4, and 6), expression of which dropped from day 4 of life. On the other hand, a transient increase in interleukin-8 (IL-8) and IL-17 expression could be observed in chicken cecum on day 4 of life, indicating physiological inflammation and maturation of the gut immune system. In agreement, the response of chickens infected with S. Enteritidis on days 1, 4, and 16 of life shifted from Th1 (characterized mainly by induction of gamma interferon [IFN-γ] and inducible nitric oxide synthase [iNOS]), observed in younger chickens, to Th17, observed in 16-day-old chickens (characterized mainly by IL-17 induction). Active modification of chicken gut microbiota in the future may accelerate or potentiate the maturation of the gut immune system and increase its resistance to infection with different pathogens.
Infection and immunity 07/2011; 79(7):2755-63. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Natural variation in the presence or the absence of STM0517-0529 genes allowing allantoin utilisation has been described in field isolates of the multidrug resistant Salmonella enterica serovar Typhimurium belonging to the phage type DT104. Interestingly, S. enterica subspecies enterica serovar Typhimurium DT104 is quite frequent in pigs and cattle, but rarely present in egg-laying hens. Taking into account the different mode of allantoin metabolism in birds and mammals, we were interested in whether the absence of STM0517-0529 genes may disable this clone in poultry colonisation. We have therefore constructed the allB (also designated as STM0523) mutants in S. enterica subspecies enterica serovar Typhimurium and S. enterica subspecies enterica serovar Enteritidis, and with these, we infected mice, newly hatched chickens and adult egg-laying hens to show that the defect in allantoin utilisation does not influence S. enterica virulence for mice or adult hens, but slightly decreases virulence of S. enterica for chickens. The decrease in virulence of the allB mutant was relatively minor as it could be observed only after a mixed infection model, consistent with a lower prevalence, but not a total absence of such clones in poultry flocks.
[show abstract][hide abstract] ABSTRACT: In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.
[show abstract][hide abstract] ABSTRACT: In this study we have compared protein secretion in the wild type of S. Typhimurium and the rfaC mutant. We found out that the rfaC mutant was defective in protein secretion. In addition, the rfaC mutant was defective in its invasion into an IPEC-J2 porcine epithelial cell line and also in motility in semisolid agar. Consistent with this, reduced flagella numbers were observed in the rfaC mutant. In the rfaC mutant, there were no defects in flagellin expression as detected by western blot and immune electron microscopy which demonstrated equal amounts of flagellin in the cytoplasm of both the rfaC mutant and the wild-type S. Typhimurium. However, in the wild-type strain only, the flagellin was assembled to spatially restricted areas on the inner side of cytoplasmic membrane. The oligosaccharide core of LPS is therefore required for the assembly of flagella and T3SS secretion machinery followed by protein secretion.
[show abstract][hide abstract] ABSTRACT: Unlike all of the other retrons, the bacterial retron reverse transcriptase RrtE is capable of synthesizing small double-stranded DNA (sdsDNA) from template RNA. In this study, we analyzed the biosynthesis of the sdsDNA by RrtE in detail. We found out that the initiation of reverse transcription was dependent on a novel self-priming mechanism utilizing a free 3'OH of RNA that is reverse-transcribed into sdsDNA. The priming of the sdsDNA synthesis was not dependent on any particular nucleotide being used as a donor of 3'OH (unlike all of the other retrons, which prime from 2'OH of a particular guanosine) or any particular nucleotide being introduced into the sdsDNA first. Due to the relaxed demands for the initiation of reverse transcription, RrtE has the potential to generate dsDNA from different RNA transcripts in vivo.
Cellular and Molecular Life Sciences CMLS 03/2011; 68(21):3607-17. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Salmonella Enteritidis mutants with deletions in phoP, fliC or phoPfliC were tested for their virulence and their ability to induce parameters of the innate and adaptive immunity in addition to their potential for serological differentiation between vaccinated, non-vaccinated and infected chickens. The double phoPfliC deletion mutant was sufficiently attenuated but not diminished in its capability to inhibit the caecal colonisation and systemic invasion of homologous Salmonella Enteritidis shortly after administration of the vaccine strain to very young chicks. Immunisation with the attenuated ΔphoPfliC mutant resulted in protective effects which were only slightly and insignificantly lower than after "immunisation" with a Salmonella wild-type strain, indicating the capability to induce an intense adaptive immune response and protection against Salmonella exposure in older chickens. The deletion in fliC enabled the effective the differentiation between immunised and infected chickens using a commercially available ELISA kit. The double phoPfliC deletion mutant of Salmonella Enteritidis might be a potential and promising live Salmonella vaccine candidate with novel characteristics for use in poultry.