[Show abstract][Hide abstract] ABSTRACT: Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.
PLoS ONE 10/2015; 10(10):e0141537. DOI:10.1371/journal.pone.0141537 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One of the recent trends in animal production is the revival of interest in organic farming. The increased consumer interest in organic animal farming is mainly due to concerns about animal welfare and the use of antibiotics in conventional farming. On the other hand, providing animals with a more natural lifestyle implies their increased exposure to environmental sources of different microorganisms including pathogens. To address these concerns, we determined the abundance of antibiotic resistance and diversity within fecal microbiota in pigs kept under conventional and organic farming systems in Sweden, Denmark, France and Italy. The abundance of sul1 , sul2 , strA , tet(A) , tet(B) and cat antibiotic resistance genes was determined in 468 samples by real-time PCR and the fecal microbiota diversity was characterized in 48 selected samples by pyrosequencing of V3/V4 regions of 16S rRNA. Contrary to our
PLoS ONE 07/2015; 10(7-7):e0132892. DOI:10.1371/journal.pone.0132892 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The response of chicken to non-typhoidal Salmonella infection is becoming well characterised
but the role of particular cell types in this response is still far from being understood.
Therefore, in this study we characterised the response of chicken embryo fibroblasts
(CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression
of chicken genes identified as significantly up- or down-regulated (�3-fold) by microarray
analysis was verified by real-time PCR followed by functional classification of the
genes and prediction of interactions between the proteins using Gene Ontology and
STRING Database. Finally the expression of the newly identified genes was tested in HD11
macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S.
Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen
in the caecum of orally infected chickens. The majority of these genes were assigned different
functions in the immune response, however five of them (LOC101750351, K123,
BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken
to Salmonella infection so far. K123 and G0S2 were the only ’non-immune’ genes inducible
by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection.
The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation
of fatty acids in mitochondria.
PLoS ONE 06/2015; 10(6). DOI:10.1371/journal.pone.0127708 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 12/2014; 9(12):e115142. DOI:10.1371/journal.pone.0115142 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chickens can be infected with Salmonella enterica at any time during their life. However, infections within the first hours and days of their life are epidemiologically the most important, as newly hatched chickens are highly sensitive to Salmonella infection. Salmonella is initially recognized in the chicken caecum by TLR receptors and this recognition is followed by induction of chemokines, cytokines and many effector genes. This results in infiltration of heterophils, macrophages, B- and T-lymphocytes and changes in total gene expression in the caecal lamina propria. The highest induction in expression is observed for matrix metalloproteinase 7 (MMP7). Expression of this gene is increased in the chicken caecum over 4000 fold during the first 10 days after the infection of newly hatched chickens. Additional highly inducible genes in the caecum following S. Enteritidis infection include immune responsive gene 1 (IRG1), serum amyloid A (SAA), extracellular fatty acid binding protein (ExFABP), serine protease inhibitor (SERPINB10), trappin 6-like (TRAP6), calprotectin (MRP126), mitochondrial ES1 protein homolog (ES1), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), avidin (AVD) and transglutaminase 4 (TGM4). The induction of expression of these proteins exceeds a factor of 50. Similar induction rates are also observed for chemokines and cytokines such as IL1β, IL6, IL8, IL17, IL18, IL22, IFNγ, AH221 or iNOS. Once the infection is under control, which happens approx. 2 weeks after infection, expression of IgY and IgA increases to facilitate Salmonella elimination from the gut lumen. This review outlines the function of individual proteins expressed in chickens after infection with non-typhoid Salmonella serovars.
Veterinary Research 12/2014; 45(1):119. DOI:10.1186/s13567-014-0119-2 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study we determined the complete nucleotide sequence of multidrug-resistance plasmid p9134, and its variants p9134dT and p9134dAT which spontaneously lost either tetracycline or both tetracycline and ampicillin resistance, respectively. The plasmids were 133,802 bp, 109,512 bp and 127,291 bp in size, respectively and their basic backbone was similar to that of IncI plasmids. Genes coding for ampicillin (blaTEM), chloramphenicol (catA1), streptomycin (strA, strB), tetracycline (tetA(A)) and gentamicin (aac(3)-IV) resistance were confirmed in wild-type p9134. Moreover, a gene for hygromycine resistance (hph) and a putative gene for apramycin resistance were newly determined. In p9134dAT, a continuous sequence coding for ampicillin and tetracycline resistances was lost. Genetic rearrangements in p9134dT were more complex and 2 recombination events must have occurred. During the first one, the tetracycline resistance locus was replaced with rck, srgB, srgA, orf7 and pefI originating from Salmonella virulence plasmid pSLT. During the second one, ydjA, pifA and repC genes from p9134 were replaced with repA2, PSLT025 and PSLT026 genes from pSLT. Our findings indicate that recombination event between unrelated plasmids might be quite common and may lead to the generation and selection of plasmids both transferring antibiotic resistance and increasing virulence of their host.
[Show abstract][Hide abstract] ABSTRACT: Poultry meat is the most common protein source of animal origin for humans. However, intensive breeding of animals in confined spaces has led to poultry colonisation by microbiota with a zoonotic potential or encoding antibiotic resistances. In this study we were therefore interested in the prevalence of selected antibiotic resistance genes and microbiota composition in feces of egg laying hens and broilers originating from 4 different Central European countries determined by real-time PCR and 16S rRNA gene pyrosequencing, respectively. strA gene was present in 1 out of 10,000 bacteria. The prevalence of sul1, sul2 and tet(B) in poultry microbiota was approx. 6 times lower than that of the strA gene. tet(A) and cat were the least prevalent being present in around 3 out of 10,000,000 bacteria forming fecal microbiome. The core chicken fecal microbiota was formed by 26 different families. Rather unexpectedly, representatives of Desulfovibrionaceae and Campylobacteraceae, both capable of hydrogen utilisation in complex microbial communities, belonged among core microbiota families. Understanding the roles of individual population members in the total metabolism of the complex community may allow for interventions which might result in the replacement of Campylobacteraceae with Desulfovibrionaceae and a reduction of Campylobacter colonisation in broilers, carcasses, and consequently poultry meat products.
PLoS ONE 10/2014; 9(10). DOI:10.1371/journal.pone.0110076 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Following infection and initial multiplication in the gut lumen, Salmonella Typhimurium crosses the intestinal epithelial barrier and comes into contact with cells of the host immune system. Mononuclear phagocytes which comprise macrophages and dendritic cells (DC) are of key importance for the outcome of Salmonella infection. Although macrophages and DC may differentiate from a common precursor, their capacities to process and present antigen differ significantly. In this study, we therefore compared the response of porcine macrophages and DC differentiated from peripheral blood monocytes to S. Typhimurium and one of the most potent bacterial pathogen associated molecular patterns, bacterial lipopolysaccharide. To avoid any bias, the expression was determined by protein LC-MS/MS and verified at the level of transcription by quantitative RT-PCR.ResultsWithin 4 days of culture, peripheral blood monocytes differentiated into two populations with distinct morphology and expression of MHC II. Mass spectrometry identified 446 proteins in macrophages and 672 in DC. Out of these, 433 proteins were inducible in macrophages either after infection with S. Typhimurium or LPS exposure and 144 proteins were inducible in DC. The expression of the 46 most inducible proteins was verified at the level of transcription and the differential expression was confirmed in 22 of them. Out of these, 16 genes were induced in both cell types, 3 genes (VCAM1, HMOX1 and Serglycin) were significantly induced in macrophages only and OLDLR1 and CDC42 were induced exclusively in DC. Thirteen out of 22 up-regulated genes contained the NF-kappaB binding site in their promoters and could be considered as either part of the NF-kappaB feedback loop (IkappaBalpha and ISG15) or as NF-kappaB targets (IL1beta, IL1alpha, AMCF2, IL8, SOD2, CD14, CD48, OPN, OLDLR1, HMOX1 and VCAM1).Conclusions
The difference in the response of monocyte derived macrophages and DC was quantitative rather than qualitative. Despite the similarity of the responses, compared to DC, the macrophages responded in a more pro-inflammatory fashion.
BMC Veterinary Research 10/2014; 10(1):244. DOI:10.1186/s12917-014-0244-1 · 1.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background Salmonellae are food-borne pathogens of great health and economic importance. To pose a threat to humans, Salmonellae normally have to cope with a series of stressful conditions in the food chain, including low temperature. In the current study, we evaluated the importance of the Clp proteolytic complex and the carbon starvation protein, CsrA, for the ability of Salmonella Typhimurium to grow at low temperature.ResultsA clpP mutant was severely affected in growth and formed pin point colonies at 10°C. Contrary to this, rpoS and clpP/rpoS mutants were only slightly affected. The clpP mutant formed cold resistant suppressor mutants at a frequency of 2.5¿×¿10¿3 and these were found not to express RpoS. Together these results indicated that the impaired growth of the clpP mutant was caused by high level of RpoS. Evaluation by microscopy of the clpP mutant revealed that it formed filamentous cells when grown at 10°C, and this phenotype too, disappered when rpoS was mutated in parallel indicating a RpoS-dependency. A csrA (sup) mutant was also growth attenuated a low temperature. An rpoS/csrA (sup) double mutant was also growth attenuated, indicating that the phenotype of the csrA mutant was independent from RpoS.Conclusions
The cold sensitivity of clpP mutant was associated with increased levels of RpoS and probably caused by toxic levels of RpoS. Although a csrA mutant also accumulated high level of RpoS, growth impairment caused by lack of csrA was not related to RpoS levels in a similar way.
[Show abstract][Hide abstract] ABSTRACT: International trade with ornamental fish is gradually recognized as an important source of a wide range of different antibiotic resistant bacteria. In this study we therefore characterized the prevalence of selected antibiotic resistance genes in the microbiota found in the carriage water of ornamental fish originating from 3 different continents. Real-time PCR quantification showed that the sul1 gene was present in 11 out of 100 bacteria. tet(A) was present in 6 out of 100 bacteria and strA, tet(G), sul2 and aadA were present in 1-2 copies per 100 bacteria. Class I integrons were quite common in carriage water microbiota, however, pyrosequencing showed that only 12 different antibiotic gene cassettes were present in class I integrons. The microbiota characterized by pyrosequencing of the V3/V4 variable region of 16S rRNA genes consisted of Proteobacteria (48%), Bacteroidetes (29.5%), Firmicutes (17.8%), Actinobacteria (2.1%) and Fusobacteria (1.6%). Correlation analysis between antibiotic resistance gene prevalence and microbiota composition verified by bacterial culture showed that major reservoirs of sul1 sul2, tet(A), tet(B) tet(G), cat, cml, bla, strA, aacA, aph and aadA could be found among Alpha-, Beta- and Gammaproteobacteria with representatives of Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae and Comamonadaceae being those most positively associated with the tested antibiotic resistance genes.
PLoS ONE 08/2014; 9(8):e103865. DOI:10.1371/journal.pone.0103865 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human salmonellosis is one of the most common food-borne diseases worldwide with poultry being the most frequent reservoir of non-typhoidal Salmonella enterica. Understanding the interactions between chickens and S. enterica serovars is therefore important in order to design vaccines and subsequently decrease the incidence of human salmonellosis. In this study we characterized the immune response-related gene expression in the cecum of chickens inoculated with 4 deletion mutants of Salmonella Enteritidis (phoP, aroA, SPI1 and SPI2). Inoculation of newly hatched chickens with the phoP mutant did not induce expression of any of the 36 tested genes, in other words, did not cause inflammation. Challenge of these chickens consequently led to similar immune response as in non-inoculated, challenged control chickens. Inoculation of chickens with the SPI1 mutant significantly induced 17 genes (EX-FABP, iNOS, STAT3, MPEG1, SAA and MMP7 in particular) and such chickens were well-protected as none of the genes was significantly induced after the challenge. Inoculation of chickens with the aroA or SPI2 mutant specifically induced the expression of C3 complement, HCLS1, IgM and immunoglobulin light chain (IgL). Challenge of chickens inoculated with SPI2 or aroA mutant resulted in upregulation of 11 and 8 genes, respectively. While challenge of chickens inoculated with the SPI2 mutant induced higher IgA expression, the aroA mutant stimulated higher IgY expression. phoP, aroA, SPI1 and SPI2 mutants were recognized differently by the chicken immune system and these differences can be used for both a more detailed understanding of the chicken immune system and a targeted vaccine design.
XXIII. Tomáškovy days young microbiologists, Brno, Czech Republic; 07/2014
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 04/2014; 9(4):e96116. DOI:10.1371/journal.pone.0096116 · 3.23 Impact Factor
[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. DOI:10.1186/1746-6148-9-140 · 1.78 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 06/2013; 8(6):e66172. DOI:10.1371/journal.pone.0066172 · 3.23 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. DOI:10.1186/1297-9716-44-37 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Salmonella enterica serovar Typhimurium requires the Type III Secretion System, encoded by Salmonella pathogenicity island 1 (SPI1) and controlled by the master regulator, HilA, to penetrate the intestinal epithelium. Numerous regulators affect virulence through influence on this system, including the proteolytic component ClpP, the stationary phase regulator RpoS and the carbon storage regulator CsrA. However, the mechanism behind the ClpP regulation is not fully understood. To elucidate this we examined differentially expressed genes in a ΔclpP mutant compared with wild-type using global transcriptomic analysis. SPI1 and SPI4 virulence genes were significantly down-regulated in the ΔclpP mutant whereas several RpoS-dependent genes and the fliC gene encoding flagellin were up-regulated. While the ΔclpP mutant was attenuated in cell invasion, this attenuation was not present in a ΔclpP/rpoS::amp double mutant suggesting the repression of invasion was directed through RpoS. The expression of the csrA virulence regulator was increased in the ΔclpP mutant and decreased in the rpoS::amp and ΔclpP/rpoS::amp mutants indicating that ClpP affects the csrA expression level as well. Thus, this study suggests that ClpP affects SPI1 expression and thereby virulence indirectly through its regulation of both RpoS and CsrA.
[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.