Frantisek Sisak - Academia.edu (original) (raw)

Papers by Frantisek Sisak

Infection and Immunity, 2011

In commercial poultry production, there is a lack of natural flora providers since chickens are h... more 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 ␤-defensins (i.e., gallinacins 1, 2, 4, and 6), expression of which dropped from day 4

Applied and Environmental Microbiology, 2013

Terminal restriction fragment length polymorphism and quantitative PCR showed that the cecal micr... more Terminal restriction fragment length polymorphism and quantitative PCR showed that the cecal microbiota of chicks up to the age of 21 days was dominated by representatives of the orders Enterobacteriales, Clostridiales, and Lactobacillales. Salmonella enterica serovar Enteritidis infection caused the greatest changes in the gut microbiota when 1-day-old chicks were infected, compared with the infection of 4-and 16-day-old chicks.

Poultry Science, 2015

After a ban on the use of antibiotics as growth promoters in farm animals in the European Union i... more After a ban on the use of antibiotics as growth promoters in farm animals in the European Union in 2006, an interest in alternative products with antibacterial or anti-inflammatory properties has increased. In this study, we therefore tested the effects of extracts from Curcuma longa and Scutellaria baicalensis used as feed additives against cecal inflammation induced by heat stress or Salmonella Enteritidis (S. Enteritidis) infection in chickens. Curcuma extract alone was not enough to decrease gut inflammation induced by heat stress. However, a mixture of Curcuma and Scutellaria extracts used as feed additives decreased gut inflammation induced by heat or S. Enteritidis, decreased S. Enteritidis counts in the cecum but was of no negative effect on BW or humoral immune response. Using next-generation sequencing of 16S rRNA we found out that supplementation of feed with the 2 plant extracts had no effect on microbiota diversity. However, if the plant extract supplementation was provided to the chickens infected with S. Enteritidis, Faecalibacterium, and Lactobacillus, both bacterial genera with known positive effects on gut health were positively selected. The supplementation of chicken feed with extracts from Curcuma and Scutelleria thus may be used in poultry production to effectively decrease gut inflammation and increase chicken performance.

Veterinární medicína, 1993

Infections with Salmonella enteritidis and S. typhimurium are frequent causes of food-borne disea... more Infections with Salmonella enteritidis and S. typhimurium are frequent causes of food-borne diseases in man and are responsible for considerable economic losses in the poultry industry (Fantasia et al., 1991; Pohl et al., 1991). Methods for the more careful differentiation and typing of these two serotypes are necessary for the investigation of the spread and transmission of the infection. Conventional methods of Salmonella differentiation (bioassays, phage-typing, resistance to antibiotics) often lack the necessary resolution potential (Wray et al., 1987). Plasmid profile analysis and restriction analysis of chromosomal DNA, based on restriction fragment length polymorphism (RFLP) have been used for the differentiation of Salmonellae (Wray et al., 1987; Nastasi et al., 1988; Franklin et al., 1990; Helmuth von et al., 1990; Martinetti and Altwegg, 1990). Rapid and unsophisticated analysis of the content of plasmid DNA tends to be preferred. However, some strains lack plasmids or may...

PLoS ONE, 2012

In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immun... more 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 nonvaccinated 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 cd Tlymphocytes 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)1b, IL6, IL8, IL18, LITAF, IFNc 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 IFNc expression in the CD4 T-lymphocytes in both the vaccinated and nonvaccinated chickens, and a variable IL17 expression oscillating around IFNc expression levels, the IL17:IFNc ratio in CD4 Tlymphocytes was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels than IFNc 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 IFNc as in the vaccinated chickens, the Th1 response with a higher resistance to S. Enteritidis infection dominated.

Veterinary Research, 2013

The characterization of the immune response of chickens to Salmonella infection is usually limite... more 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, IFNγ, iNOS, ES1, IL-1β, 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.

PLoS ONE, 2014

In this study we characterised the development of caecal microbiota in egg laying hens over their... more In this study we characterised the development of caecal microbiota in egg laying hens over their commercial production lifespan, from the day of hatching until 60 weeks of age. Using pyrosequencing of V3/V4 variable regions of 16S rRNA genes for microbiota characterisation, we were able to define 4 different stages of caecal microbiota development. The first stage lasted for the first week of life and was characterised by a high prevalence of Enterobacteriaceae (phylum Proteobacteria). The second stage lasted from week 2 to week 4 and was characterised by nearly an absolute dominance of Lachnospiraceae and Ruminococcaceae (both phylum Firmicutes). The third stage lasted from month 2 to month 6 and was characterised by the succession of Firmicutes at the expense of Bacteroidetes. The fourth stage was typical for adult hens in full egg production aged 7 months or more and was characterised by a constant ratio of Bacteroidetes and Firmicutes formed by equal numbers of the representatives of both phyla.

Salmonella enterica subsp. enterica originating from poultry and poultry products is responsible ... more Salmonella enterica subsp. enterica originating from poultry and poultry products is responsible for the vast majority of human gastrointestinal disorders in Europe. For this reason different measures that seek to decrease its incidence in poultry including vaccination with inactivated vaccine continue to be tested. In this study we compared four different inactivated vaccines of S. Typhimurium in chickens which were

Veterinary Microbiology, 2014

Interaction between pigs and Salmonella enterica serovar Derby (Salmonella Derby) is much less un... more Interaction between pigs and Salmonella enterica serovar Derby (Salmonella Derby) is much less understood in comparison with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). To study interactions of weaned piglets with Salmonella Derby, we compared the course of infections with Salmonella Derby De1 and Salmonella Typhimurium DT104 strains, both isolated from pig herds with a long history of asymptomatic infection. Salmonella Derby strain used was shed during the 28-day experiment period, while Salmonella Typhimurium strain was not found in faeces after day 17 post-infection. When the piglets were co-infected with both strains, Salmonella Derby was present in faeces until the end of the experiment, whilst Salmonella Typhimurium disappeared after day 21 post-infection. At the end of the experiment, Salmonella Derby was present in more tissues when compared with Salmonella Typhimurium. Piglets infected with Salmonella Typhimurium responded earlier with synthesis of anti-lipopolysaccharide IgM and IgG antibodies and with higher antibody levels compared to piglets infected with Salmonella Derby. Cellular immune response to both strains was very low and was detected later than was the onset of IgG antibody production.

Vaccine, 2009

If any new live Salmonella vaccine is introduced in the future, it is quite probable that detaile... more If any new live Salmonella vaccine is introduced in the future, it is quite probable that detailed characterisation of its attenuation will be required. In this study we therefore compared 34 isogenic mutants of S. Enteritidis in aroA, aroD, galE, ssrA, sseA, phoP, rpoS, ompR, htrA, clpP, lon, rfaL, rfaG, rfaC, hfq, sodCI, hilA, sipA, avrA, sopB, sopA, sopE, sifA, shdA, fliC, fur, relA, spoT, rel-spoT, misL, rmbA, STM4258, STM4259 and spvBC genes for their resistance to stresses likely to be expected in the host and for their virulence and immunogenicity in Balb/C mice. We found that the cold and bile resistances essentially did not correlate with the resistances to other stress factors. Resistance to acid pH, heat, polymyxin and serum correlated with each other and also with the attenuation. When the residual virulence and immunogenicity were both considered, mutants in htrA, ompR, aroA, aroD and lon performed the best in mice. Furthermore, when a detailed comparison of polymyxin and serum sensitive mutants was performed, the serum sensitive mutants were more immunogenic.

PLoS ONE, 2012

In this study we were interested in identification of new markers of chicken response to Salmonel... more 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-6like protein (TRAP6) and serum amyloid A (SAA). Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and cd 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, 2014

Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonell... more Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.

Epidemiology and Infection, 1996

A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELIS... more A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELISAs used for the serological detection of Salmonella enteritica serotype Enteritidis in chickens. The aim was to ascertain how far agreement could be reached on the interpretation of optical density readings for high titre, intermediate titre and low titre sera. Two sets of sera were sent to 14 participants. The first set compared high, medium and low titre sera raised in specified-pathogen-free and commercial broiler breeder chickens. The second set comprised 20 sera of different antibody titres raised in commercial birds reared under laboratory conditions and sent blind. Both indirect and double-antibody sandwich blocking ELISAs were used with a number of different detecting antigens. With a few exceptions good agreement was reached on the interpretation of results obtained from high and low titre sera from the optical density obtained with a single serum dilution. Differences were observed in the interpretation of medium titre sera. The results suggested that most ELISAs produce reasonably comparable results and that practical problems may arise from interpretation of the results mainly as a result of the choice of the criteria used for differentiating sera obtained from infected and uninfected chickens. These problems are discussed.

BMC Microbiology, 2009

Background: Salmonella is a highly successful parasite of reptiles, birds and mammals. Its abilit... more Background: Salmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in oneday-old chickens, together with the innate immune response of this host.

BMC Microbiology, 2010

Background: In this study we were interested in the colonisation and early immune response of Bal... more Background: In this study we were interested in the colonisation and early immune response of Balb/C mice to infection with Salmonella Enteritidis and isogenic pathogenicity island free mutants.

Applied and Environmental Microbiology, 2003

Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was... more Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The S. enterica serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for S. enterica serovar Typhimurium and Escherichia coli (indigenous in manure). After the 1 March manure application, S. enterica serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application, S. enterica serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil, S. enterica serovar Typhimurium died at a similar rate (P >or= 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10 degrees C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with S. enterica serovar Typhimurium. Manure application under warmer (daily average maximum temperature >20 degrees C) summer conditions is not recommended when vegetable planting is done between the time of manure application and late summer. A late fall manure application will not increase the risk of contaminating vegetables planted the next spring, since further experiments showed that repeated freeze-thaw cycles were detrimental to the survival of S. enterica serovar Typhimurium and E. coli in manure-fertilized soil. The number of indigenous E. coli in soil was never significantly lower (P < 0.05) than that of S. enterica serovar Typhimurium, suggesting its usefulness as an indicator organism for evaluating the risk of vegetable contamination with manure-borne S. enterica serovar Typhimurium.

Antimicrobial Agents and Chemotherapy, 2003

In a collection of 66 Salmonella enterica serovar Typhimurium strains isolated between 1984 and 2... more In a collection of 66 Salmonella enterica serovar Typhimurium strains isolated between 1984 and 2002 in the Czech Republic, genes coding for antibiotic resistance were determined by using specific PCRs. We found that the pentadrug-resistant ACSSuT clone first appeared in the Czech Republic in 1990. A new variant of the aadA gene designated aadA21 is described, the 5 end of which was identical to aadA2 and the 3 end of which was identical to aadA1.

Archives of Microbiology, 2002

sdiA in Salmonella enterica serovar Typhimurium encodes a protein belonging to the LuxR family of... more sdiA in Salmonella enterica serovar Typhimurium encodes a protein belonging to the LuxR family of transcriptional regulators. Initial computer analysis revealed the presence of a fur box 19 bp upstream of the start codon of the sdiA gene and a helix-turn-helix motif in the carboxy-terminal part of the SdiA protein typical for transcriptional regulators. Deletion of the fur box resulted in twofold increase of sdiA transcription. Furthermore, addition of dipyridyl, an iron chelator, to culture media increased sdiA transcription to the level observed in the fur box mutant, confirming that sdiA is suppressed in the presence of iron. When S. enterica sv. Typhimurium was grown in conditioned medium, sdiA transcription was repressed to 30% of that in cells grown in fresh LB broth; this repression was independent of the fur box. Oral infection of mice with the strain lacking the helix-turn-helix domain of SdiA indicated increased virulence of this S. enterica sv. Typhimurium mutant. sdiA, dually controlled by iron concentration and culture-density-derived signals, may therefore play an important role in S. enterica sv. Typhimurium virulence regulation.

Infection and Immunity, 2011

In commercial poultry production, there is a lack of natural flora providers since chickens are h... more 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 ␤-defensins (i.e., gallinacins 1, 2, 4, and 6), expression of which dropped from day 4

Applied and Environmental Microbiology, 2013

Terminal restriction fragment length polymorphism and quantitative PCR showed that the cecal micr... more Terminal restriction fragment length polymorphism and quantitative PCR showed that the cecal microbiota of chicks up to the age of 21 days was dominated by representatives of the orders Enterobacteriales, Clostridiales, and Lactobacillales. Salmonella enterica serovar Enteritidis infection caused the greatest changes in the gut microbiota when 1-day-old chicks were infected, compared with the infection of 4-and 16-day-old chicks.

Poultry Science, 2015

After a ban on the use of antibiotics as growth promoters in farm animals in the European Union i... more After a ban on the use of antibiotics as growth promoters in farm animals in the European Union in 2006, an interest in alternative products with antibacterial or anti-inflammatory properties has increased. In this study, we therefore tested the effects of extracts from Curcuma longa and Scutellaria baicalensis used as feed additives against cecal inflammation induced by heat stress or Salmonella Enteritidis (S. Enteritidis) infection in chickens. Curcuma extract alone was not enough to decrease gut inflammation induced by heat stress. However, a mixture of Curcuma and Scutellaria extracts used as feed additives decreased gut inflammation induced by heat or S. Enteritidis, decreased S. Enteritidis counts in the cecum but was of no negative effect on BW or humoral immune response. Using next-generation sequencing of 16S rRNA we found out that supplementation of feed with the 2 plant extracts had no effect on microbiota diversity. However, if the plant extract supplementation was provided to the chickens infected with S. Enteritidis, Faecalibacterium, and Lactobacillus, both bacterial genera with known positive effects on gut health were positively selected. The supplementation of chicken feed with extracts from Curcuma and Scutelleria thus may be used in poultry production to effectively decrease gut inflammation and increase chicken performance.

Veterinární medicína, 1993

Infections with Salmonella enteritidis and S. typhimurium are frequent causes of food-borne disea... more Infections with Salmonella enteritidis and S. typhimurium are frequent causes of food-borne diseases in man and are responsible for considerable economic losses in the poultry industry (Fantasia et al., 1991; Pohl et al., 1991). Methods for the more careful differentiation and typing of these two serotypes are necessary for the investigation of the spread and transmission of the infection. Conventional methods of Salmonella differentiation (bioassays, phage-typing, resistance to antibiotics) often lack the necessary resolution potential (Wray et al., 1987). Plasmid profile analysis and restriction analysis of chromosomal DNA, based on restriction fragment length polymorphism (RFLP) have been used for the differentiation of Salmonellae (Wray et al., 1987; Nastasi et al., 1988; Franklin et al., 1990; Helmuth von et al., 1990; Martinetti and Altwegg, 1990). Rapid and unsophisticated analysis of the content of plasmid DNA tends to be preferred. However, some strains lack plasmids or may...

PLoS ONE, 2012

In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immun... more 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 nonvaccinated 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 cd Tlymphocytes 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)1b, IL6, IL8, IL18, LITAF, IFNc 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 IFNc expression in the CD4 T-lymphocytes in both the vaccinated and nonvaccinated chickens, and a variable IL17 expression oscillating around IFNc expression levels, the IL17:IFNc ratio in CD4 Tlymphocytes was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels than IFNc 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 IFNc as in the vaccinated chickens, the Th1 response with a higher resistance to S. Enteritidis infection dominated.

Veterinary Research, 2013

The characterization of the immune response of chickens to Salmonella infection is usually limite... more 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, IFNγ, iNOS, ES1, IL-1β, 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.

PLoS ONE, 2014

In this study we characterised the development of caecal microbiota in egg laying hens over their... more In this study we characterised the development of caecal microbiota in egg laying hens over their commercial production lifespan, from the day of hatching until 60 weeks of age. Using pyrosequencing of V3/V4 variable regions of 16S rRNA genes for microbiota characterisation, we were able to define 4 different stages of caecal microbiota development. The first stage lasted for the first week of life and was characterised by a high prevalence of Enterobacteriaceae (phylum Proteobacteria). The second stage lasted from week 2 to week 4 and was characterised by nearly an absolute dominance of Lachnospiraceae and Ruminococcaceae (both phylum Firmicutes). The third stage lasted from month 2 to month 6 and was characterised by the succession of Firmicutes at the expense of Bacteroidetes. The fourth stage was typical for adult hens in full egg production aged 7 months or more and was characterised by a constant ratio of Bacteroidetes and Firmicutes formed by equal numbers of the representatives of both phyla.

Salmonella enterica subsp. enterica originating from poultry and poultry products is responsible ... more Salmonella enterica subsp. enterica originating from poultry and poultry products is responsible for the vast majority of human gastrointestinal disorders in Europe. For this reason different measures that seek to decrease its incidence in poultry including vaccination with inactivated vaccine continue to be tested. In this study we compared four different inactivated vaccines of S. Typhimurium in chickens which were

Veterinary Microbiology, 2014

Interaction between pigs and Salmonella enterica serovar Derby (Salmonella Derby) is much less un... more Interaction between pigs and Salmonella enterica serovar Derby (Salmonella Derby) is much less understood in comparison with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). To study interactions of weaned piglets with Salmonella Derby, we compared the course of infections with Salmonella Derby De1 and Salmonella Typhimurium DT104 strains, both isolated from pig herds with a long history of asymptomatic infection. Salmonella Derby strain used was shed during the 28-day experiment period, while Salmonella Typhimurium strain was not found in faeces after day 17 post-infection. When the piglets were co-infected with both strains, Salmonella Derby was present in faeces until the end of the experiment, whilst Salmonella Typhimurium disappeared after day 21 post-infection. At the end of the experiment, Salmonella Derby was present in more tissues when compared with Salmonella Typhimurium. Piglets infected with Salmonella Typhimurium responded earlier with synthesis of anti-lipopolysaccharide IgM and IgG antibodies and with higher antibody levels compared to piglets infected with Salmonella Derby. Cellular immune response to both strains was very low and was detected later than was the onset of IgG antibody production.

Vaccine, 2009

If any new live Salmonella vaccine is introduced in the future, it is quite probable that detaile... more If any new live Salmonella vaccine is introduced in the future, it is quite probable that detailed characterisation of its attenuation will be required. In this study we therefore compared 34 isogenic mutants of S. Enteritidis in aroA, aroD, galE, ssrA, sseA, phoP, rpoS, ompR, htrA, clpP, lon, rfaL, rfaG, rfaC, hfq, sodCI, hilA, sipA, avrA, sopB, sopA, sopE, sifA, shdA, fliC, fur, relA, spoT, rel-spoT, misL, rmbA, STM4258, STM4259 and spvBC genes for their resistance to stresses likely to be expected in the host and for their virulence and immunogenicity in Balb/C mice. We found that the cold and bile resistances essentially did not correlate with the resistances to other stress factors. Resistance to acid pH, heat, polymyxin and serum correlated with each other and also with the attenuation. When the residual virulence and immunogenicity were both considered, mutants in htrA, ompR, aroA, aroD and lon performed the best in mice. Furthermore, when a detailed comparison of polymyxin and serum sensitive mutants was performed, the serum sensitive mutants were more immunogenic.

PLoS ONE, 2012

In this study we were interested in identification of new markers of chicken response to Salmonel... more 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-6like protein (TRAP6) and serum amyloid A (SAA). Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and cd 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, 2014

Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonell... more Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.

Epidemiology and Infection, 1996

A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELIS... more A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELISAs used for the serological detection of Salmonella enteritica serotype Enteritidis in chickens. The aim was to ascertain how far agreement could be reached on the interpretation of optical density readings for high titre, intermediate titre and low titre sera. Two sets of sera were sent to 14 participants. The first set compared high, medium and low titre sera raised in specified-pathogen-free and commercial broiler breeder chickens. The second set comprised 20 sera of different antibody titres raised in commercial birds reared under laboratory conditions and sent blind. Both indirect and double-antibody sandwich blocking ELISAs were used with a number of different detecting antigens. With a few exceptions good agreement was reached on the interpretation of results obtained from high and low titre sera from the optical density obtained with a single serum dilution. Differences were observed in the interpretation of medium titre sera. The results suggested that most ELISAs produce reasonably comparable results and that practical problems may arise from interpretation of the results mainly as a result of the choice of the criteria used for differentiating sera obtained from infected and uninfected chickens. These problems are discussed.

BMC Microbiology, 2009

Background: Salmonella is a highly successful parasite of reptiles, birds and mammals. Its abilit... more Background: Salmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in oneday-old chickens, together with the innate immune response of this host.

BMC Microbiology, 2010

Background: In this study we were interested in the colonisation and early immune response of Bal... more Background: In this study we were interested in the colonisation and early immune response of Balb/C mice to infection with Salmonella Enteritidis and isogenic pathogenicity island free mutants.

Applied and Environmental Microbiology, 2003

Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was... more Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The S. enterica serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for S. enterica serovar Typhimurium and Escherichia coli (indigenous in manure). After the 1 March manure application, S. enterica serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application, S. enterica serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil, S. enterica serovar Typhimurium died at a similar rate (P >or= 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10 degrees C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with S. enterica serovar Typhimurium. Manure application under warmer (daily average maximum temperature >20 degrees C) summer conditions is not recommended when vegetable planting is done between the time of manure application and late summer. A late fall manure application will not increase the risk of contaminating vegetables planted the next spring, since further experiments showed that repeated freeze-thaw cycles were detrimental to the survival of S. enterica serovar Typhimurium and E. coli in manure-fertilized soil. The number of indigenous E. coli in soil was never significantly lower (P < 0.05) than that of S. enterica serovar Typhimurium, suggesting its usefulness as an indicator organism for evaluating the risk of vegetable contamination with manure-borne S. enterica serovar Typhimurium.

Antimicrobial Agents and Chemotherapy, 2003

In a collection of 66 Salmonella enterica serovar Typhimurium strains isolated between 1984 and 2... more In a collection of 66 Salmonella enterica serovar Typhimurium strains isolated between 1984 and 2002 in the Czech Republic, genes coding for antibiotic resistance were determined by using specific PCRs. We found that the pentadrug-resistant ACSSuT clone first appeared in the Czech Republic in 1990. A new variant of the aadA gene designated aadA21 is described, the 5 end of which was identical to aadA2 and the 3 end of which was identical to aadA1.

Archives of Microbiology, 2002

sdiA in Salmonella enterica serovar Typhimurium encodes a protein belonging to the LuxR family of... more sdiA in Salmonella enterica serovar Typhimurium encodes a protein belonging to the LuxR family of transcriptional regulators. Initial computer analysis revealed the presence of a fur box 19 bp upstream of the start codon of the sdiA gene and a helix-turn-helix motif in the carboxy-terminal part of the SdiA protein typical for transcriptional regulators. Deletion of the fur box resulted in twofold increase of sdiA transcription. Furthermore, addition of dipyridyl, an iron chelator, to culture media increased sdiA transcription to the level observed in the fur box mutant, confirming that sdiA is suppressed in the presence of iron. When S. enterica sv. Typhimurium was grown in conditioned medium, sdiA transcription was repressed to 30% of that in cells grown in fresh LB broth; this repression was independent of the fur box. Oral infection of mice with the strain lacking the helix-turn-helix domain of SdiA indicated increased virulence of this S. enterica sv. Typhimurium mutant. sdiA, dually controlled by iron concentration and culture-density-derived signals, may therefore play an important role in S. enterica sv. Typhimurium virulence regulation.