Gut Anaerobes Capable of Chicken Caecum Colonisation.

Chicks in commercial production are highly sensitive to enteric infections and their resistance can be increased by administration of complex adult microbiota. However, it is not known which adult microbiota members are capable of colonising the caecum of newly hatched chicks. In this study, we therefore orally inoculated chicks with pure cultures of 76 different bacterial isolates originating from chicken caecum on day 1 of life and determined their ability to colonise seven days later. The caecum of newly hatched chickens could be colonised by bacteria belonging to phyla Bacteroidetes, Proteobacteria, Synergistetes, or Verrucomicrobia, and isolates from class Negativicutes (phylum Firmicutes). On the other hand, we did not record colonisation with isolates from phyla Actinobacteria and Firmicutes (except for Negativicutes), including isolates from families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Lactobacillaceae. Representatives of genera commonly used in probiotics such as Lactobacillus, Enterococcus, or Bacillus therefore did not colonise the chicken intestinal tract after a single dose administration. Following challenge with Salmonella enterica serovar Enteritidis, the best protecting isolates increased the chicken's resistance to S. Enteritidis only tenfold, which, however, means that none of the tested individual bacterial isolates on their own efficiently protected chicks against S. Enteritidis.

Use of 16S rRNA gene sequencing for prediction of new opportunistic pathogens in chicken ileal and cecal microbiota.

In this study, we addressed differences in the development of gut microbiota in 4 successive batches of commercially hatched broiler parent chickens. When planning this study, we expected to find a batch with compromised performance which would allow identification of microbiota of suboptimal composition. Microbiota composition was determined only by sequencing the V3/V4 region of 16S rRNA genes in samples collected from chickens 5 to 18 wk of age. In a total, 100 and 160 samples originating from the ileum or cecum were processed, respectively. In one of the flocks with suboptimal performance we identified an increased abundance of Helicobacter brantae forming over 80% of ileal microbiota in individual chickens. Moreover, we also tested samples of 53-wk-old hens from the same genetic line in which egg production decreased. In this case, cecal microbiota was enriched for Fusobacterium mortiferum forming over 30% of total cecal microbiota. Although none of the identified unusual microbiota members have been well recognized as pathogenic, they may represent new opportunistic pathogens of chickens worth of further investigation. Analysis of gut microbiota composition by next generation sequencing thus proved as a useful and unbiased alternative to bacterial culture, especially in the cases of unspecific symptoms like decrease in flock performance.

Protein expression in the liver and blood serum in chickens in response to Salmonella Enteritidis infection.

Events occurring in the chicken caecum following Salmonella Enteritidis infection are relatively well-described. However, mechanisms of the immune response and defence beyond the intestinal tract are less well-described. In this study, we therefore determined changes in protein abundance in the liver and blood serum in response to S. Enteritidis infection using the unbiased approach of shotgun proteomics. Complement and coagulation cascades, TNF signalling, antigen processing and presentation was activated in the liver following infection with S. Enteritidis. Chicken proteins that decreased in the liver were involved in glycolysis, the citrate cycle, oxidative phosphorylation and fatty acid metabolism. No functional category was significantly activated or suppressed in the serum. Concerning individual proteins, VNN1, SAA, AVD, SERPINA3, SERPINB10, AGT, MRP126 or CP increased in abundance both in the liver and serum. MT4, MT3, PTGDS, GLRX and TGM4, though highly inducible in the liver, did not increase in the serum. PIGR, SERPINF2 and IGJ increased in the serum but not in the liver. SERPINA4, apoAIV, CLEC3B, SERPINF1, HRG, AHSG and ALB decreased both in the liver and serum. Avidin-like LOC431660, THRSP, GATM, GGACT, ACOX1, ALDOB or FABP7 decreased in the liver but not in the serum. Finally, CKM, CKB, PLTP, COMP, IGFALS, AMY1A or SERPIND1 decreased in the serum after S. Enteritidis infection but not in the liver. Differently abundant proteins characterise the chicken's response to infection and can be also used as markers of chicken health status.

Immune protection of chickens conferred by a vaccine consisting of attenuated strains of Salmonella Enteritidis, Typhimurium and Infantis.

The colonization of poultry with different Salmonella enterica serovars poses an issue throughout the world. In this study we therefore tested the efficacy of a vaccine consisting of attenuated strains of Salmonella enterica serovars Enteritidis, Typhimurium and Infantis against challenge with the same serovars and with S. Agona, Dublin and Hadar. We tested oral and aerosol administration of the vaccine, with or without co-administration of cecal microbiota from adult hens. The protective effect was determined by bacterial counts of the challenge strains up to week 18 of life and by characterizing the immune response using real-time PCR specific for 16 different genes. We have shown that a vaccine consisting of attenuated S. Enteritidis, S. Typhimurium and S. Infantis protected chickens against challenge with the wild type strains of the same serovars and partially protected chickens also against challenge with isolates belonging to serovars Dublin or Hadar. Aerosol vaccination was more effective at inducing systemic immunity whilst oral vaccination stimulated a local immune response in the gut. Co-administration of cecal microbiota increased the protectiveness in the intestinal tract but slightly decreased the systemic immune response. Adjusting the vaccine composition and changing the administration route therefore affects vaccine efficacy.

Composition of Gut Microbiota Influences Resistance of Newly Hatched Chickens to Salmonella Enteritidis Infection.

Since poultry is a very common source of non-typhoid Salmonella for humans, different interventions aimed at decreasing the prevalence of Salmonella in chickens are understood as an effective measure for decreasing the incidence of human salmonellosis. One such intervention is the use of probiotic or competitive exclusion products. In this study we tested whether microbiota from donor hens of different age will equally protect chickens against Salmonella Enteritidis infection. Newly hatched chickens were therefore orally inoculated with cecal extracts from 1-, 3-, 16-, 28-, and 42-week-old donors and 7 days later, the chickens were infected with S. Enteritidis. The experiment was terminated 4 days later. In the second experiment, groups of newly hatched chickens were inoculated with cecal extracts of 35-week-old hens either on day 1 of life followed by S. Enteritidis infection on day 2 or were infected with S. Enteritidis infection on day 1 followed by therapeutic administration of the cecal extract on day 2 or were inoculated on day 1 of life with a mixture of the cecal extract and S. Enteritidis. This experiment was terminated when the chickens were 5 days old. Both Salmonella culture and chicken gene expression confirmed that inoculation of newly hatched chickens with microbiota from 3-week-old or older chickens protected them against S. Enteritidis challenge. On the other hand, microbiota from 1-week-old donors failed to protect chickens against S. Enteritidis challenge. Microbiota from 35-week-old hens protected chickens even 24 h after administration. However, simultaneous or therapeutic microbiota administration failed to protect chickens against S. Enteritidis infection. Gut microbiota can be used as a preventive measure against S. Enteritidis infection but its composition and early administration is critical for its efficacy.

Important Metabolic Pathways and Biological Processes Expressed by Chicken Cecal Microbiota.

The gut microbiota plays important roles in its host. However, how each microbiota member contributes to the behavior of the whole population is not known. In this study, we therefore determined protein expression in the cecal microbiota in chickens of selected ages and in 7-day-old chickens inoculated with different cecal extracts on the day of hatching. Campylobacter, Helicobacter, Mucispirillum, and Megamonas overgrew in the ceca of 7-day-old chickens inoculated with cecal extracts from donor hens. Firmicutes were characterized by ABC and phosphotransferase system (PTS) transporters, extensive acyl coenzyme A (acyl-CoA) metabolism, and expression of l-fucose isomerase. Anaerostipes, Anaerotruncus, Pseudoflavonifractor, Dorea, Blautia, and Subdoligranulum expressed spore proteins. Firmicutes (Faecalibacterium, Butyrivibrio, Megasphaera, Subdoligranulum, Oscillibacter, Anaerostipes, and Anaerotruncus) expressed enzymes required for butyrate production. Megamonas, Phascolarctobacterium, and Blautia (exceptions from the phylum Firmicutes) and all Bacteroidetes expressed enzymes for propionate production pathways. Representatives of Bacteroidetes also expressed xylose isomerase, enzymes required for polysaccharide degradation, and ExbBD, TonB, and outer membrane receptors likely to be involved in oligosaccharide transport. Based on our data, Anaerostipes, Anaerotruncus, and Subdoligranulum might be optimal probiotic strains, since these represent spore-forming butyrate producers. However, certain care should be taken during microbiota transplantation because the microbiota may behave differently in the intestinal tract of a recipient depending on how well the existing communities are established.

phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens.

Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the interactions between chickens and S. enterica is therefore important for vaccine design and subsequent decrease in the incidence of human salmonellosis. In this study we therefore characterized the interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested the response of HD11 chicken macrophage-like cell line to S. Enteritidis infection monitoring the transcription of 36 genes related to immune response. All the mutants and the wild type strain induced inflammatory signaling in the HD11 cell line though the response to SPI1 mutant infection was different from the rest of the mutants. When newly hatched chickens were inoculated, the phoP as well as the SPI1 mutant did not induce an expression of any of the tested genes in the cecum. Despite this, such chickens were protected against challenge with wild-type S. Enteritidis. On the other hand, inoculation of chickens with the aroA or SPI2 mutant induced expression of 27 and 18 genes, respectively, including genes encoding immunoglobulins. Challenge of chickens inoculated with these two mutants resulted in repeated induction of 11 and 13 tested genes, respectively, including the genes encoding immunoglobulins. In conclusion, SPI1 and phoP mutants induced protective immunity without inducing an inflammatory response and antibody production. Inoculation of chickens with the SPI2 and aroA mutants also led to protective immunity but was associated with inflammation and antibody production. The differences in interaction between the mutants and chicken host can be used for a more detailed understanding of the chicken immune system.

Curcuma and Scutellaria plant extracts protect chickens against inflammation and Salmonella Enteritidis infection.

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.

Succession and replacement of bacterial populations in the caecum of egg laying hens over their whole life.

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.

The early innate response of chickens to Salmonella enterica is dependent on the presence of O-antigen but not on serovar classification.

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.

Immune response of pigs to Salmonella enterica serovar Derby and Typhimurium infections.

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.

Influence of Salmonella enterica serovar Enteritidis infection on the composition of chicken cecal microbiota.

BACKGROUND: 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 the V3/V4 variable regions of genes coding for 16S rRNA. RESULTS: 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. CONCLUSIONS: 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.

Vaccination of chickens with SPI1-lon and SPI1-lon-fliC mutant of Salmonella enterica Serovar Enteritidis.

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.

Chicken innate immune response to oral infection with Salmonella enterica serovar Enteritidis.

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-1ß, 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.

SPI1 defective mutants of Salmonella enterica induce cross-protective immunity in chickens against challenge with serovars Typhimurium and Enteritidis.

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 IL1ß, 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.

Chicken faecal microbiota and disturbances induced by single or repeated therapy with tetracycline and streptomycin.

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.

Characterization of chicken spleen transcriptome after infection with Salmonella enterica serovar Enteritidis.

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.

Cytokine signaling in splenic leukocytes from vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enteritidis.

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)1ß, 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.

Mycobacterium avium Subsp. avium and Salmonella enterica serotype Typhimurium var. Copenhagen phage type DT2 in pigeons.

We report on a coinfection of Mycobacterium avium subsp. avium and Salmonella enterica serotype Typhimurium var. Copenhagen phage type DT2 in pigeons from one flock, from which squabs were occasionally consumed by humans. Triplex quantitative real-time PCR and culture methods were used for M. a. avium detection in livers and culture method was used for the detection of Salmonella sp. in samples of liver and caecum of 33 examined birds. M. a. avium was detected in a total of 31 (93.9%) and Salmonella Typhimurium in a total of 11 (33.3%) pigeons. Coinfection with both pathogens was found in 10 (30.3%), infection with Salmonella Typhimurium alone in 1 (3.0%), and infection with M. a. avium alone in 21 (63.7%) pigeons. Neither pathogen was detected in one pigeon. There was no difference in clinical symptoms exhibited by pigeons infected by M. a. avium and/or Salmonella Typhimurium. All Salmonella Typhimurium isolates were sensitive to all 15 antimicrobials tested. According to these results we emphasize good heat treatment of consumed squabs.

allB, allantoin utilisation and Salmonella enterica serovar Enteritidis and Typhimurium colonisation of poultry and mice.

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.