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Chickens in commercial production are hatched in a clean hatchery environment in the absence of any contact with adult hens. However, Gallus gallus evolved to be hatched in a nest in contact with an adult hen which may act as a donor of gut microbiota. In this study, we therefore addressed the issue of microbiota development in newly hatched chickens with or without contact with an adult hen. We found that a mere 24-hour-long contact between a hen and newly hatched chickens was long enough for transfer of hen gut microbiota to chickens. Hens were efficient donors of Bacteroidetes and Actinobacteria. However, except for genus Faecalibacterium and bacterial species belonging to class Negativicutes, hens did not act as an important source of Gram-positive Firmicutes. Though common to the chicken intestinal tract, Lactobacilli and isolates from families Erysipelotrichaceae, Lachnospiraceae and Ruminococcaceae therefore originated from environmental sources instead of from the hens. These observation may have considerable consequences for the evidence-based design of the new generation of probiotics for poultry.

Based on previous knowledge on changes in the gut microbiota of weaned piglets, a mixture of five different Prevotella species, Anaerovibrio lipolyticus, and Mitsuokella multacida (a Prevotella mixture) was tested as potentially novel type of probiotics for weaned Large White piglets of mixed sexes. The mixture was provided orally on the day of weaning to piglets in the experimental group, and the microbiota composition at weaning and one week later was determined by 16S rRNA sequencing in rectal swabs of 14 control and 27 experimental piglets. Bacteroides and Escherichia significantly decreased, and Prevotella, Blautia, or Faecalibacterium increased in the microbiota of both control and experimental piglets one week after weaning. Bacteria from the Prevotella mixture were detected in the gut microbiota of experimental piglets; however, the same bacteria of environmental origin were also recorded in control piglets. Despite this, early and uniform administration of the Prevotella mixture affected the composition of the gut microbiota of experimental piglets one week after weaning. The families Lactobacillaceae and Lachnospiraceae were more abundant in the gut microbiota of experimental piglets, while Pasteurellaceae, Coriobacteriaceae, Bacteroidales RF16 group, and Methanobacteriaceae were more abundant in control piglets. The Prevotella-based bacterial mixture thus may represent a novel approach to modify gut microbiota and consequently gut health in weaned piglets.

Due to the low populations of Bacteroides sp. in the gut microbiota of sows compared to nursed piglets, sows may not represent an ideal source of Bacteroides sp. for newborn piglets. In this study, we therefore tested the effect of oral administration of a mixture of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides fragilis and Bacteroides xylanisolvens on the microbiota development of newborn piglets. Oral administration of such a mixture to piglets within 12 h after parturition did not result in any adverse effects. Sequencing of 16S rRNA showed that 4 days after administration, these species formed approx. 20% of total faecal microbiota and affected the development of gut microbiota in treated piglets. The treatment resulted in an increased abundance of Veillonella caviae, Fusobacterium gastrosuis, Dialister sp., Clostridium jeddahitimonense, C. cadaveris, Butyricicoccus pullicaecorum, Actinobacillus indolicus, A. minor, Streptococcus pasteurianus, S. parasuis, S. equinus, S. pluranimalium, S. thoraltensis and S. suis. On the other hand, administration of the Bacteroides mixture suppressed piglet colonisation by C. disporicum and multiple species from family Prevotellaceae. Bacteroides-treated piglets exhibited significantly higher body weight than untreated controls at 3 months of age. Administration of a mixture of Bacteroides shaped the development of gut microbiota in nursed piglets, which resulted in improved parameters at the end of the pre-fattening period.

There are extensive differences in the caecal microbiota of chicks from hatcheries and those inoculated with faecal material from adult hens. Besides differences in microbial composition, the latter chickens are highly resistant to Salmonella Enteritidis challenges, while the former are susceptible. In this study, we tested whether strains from genera Bacteroides, Megamonas, or Megasphaera can increase chicken resistance to Salmonella and Campylobacter jejuni when defined microbial mixtures consisting of these bacterial genera are administered. Mixtures consisting of different species and strains from the above-mentioned genera efficiently colonised the chicken caecum and increased chicken resistance to Salmonella by a factor of 50. The tested mixtures were even more effective in protecting chickens from Salmonella in a seeder model of infection (3–5 log reduction). The tested mixtures partially protected chickens from C. jejuni infection, though the effect was lower than that against Salmonella. The obtained data represent a first step for the development of a new type of probiotics for poultry.

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.

Lactobacilli are commonly used as probiotics in poultry to improve production parameters and to increase chicken resistance to enteric infections. However, lactobacilli do not efficiently colonise the chicken intestinal tract, and also, their anti-infection effect in vivo is sometimes questionable. In this study, we therefore evaluated the potential of a mixture of four Lactobacillus species (L. salivarius, L. reuteri, L. ingluviei and L. alvi) for the protection of chickens against Salmonella Enteritidis infection. Whenever the chickens were inoculated by lactobacilli and S. Enteritidis separately, there was no protective effect of lactobacilli. This means that when lactobacilli and S. Enteritidis are exposed to each other as late as in the crop of chickens, lactobacilli did not influence chicken resistance to S. Enteritidis at all. The only positive effect was recorded when the mixture of lactobacilli and S. Enteritidis was used for the inoculation of feed and the feed was anaerobically fermented for 1 to 5 days. In this case, chickens fed such a diet remained S. Enteritidis negative. In vitro experiments showed that the protective effect was caused by acidification of feed down to pH 4.6 due to lactobacilli fermentation and was associated with S. Enteritidis inactivation. The probiotic effect of lactobacilli was thus expressed in the feed, outside the chicken host.

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 one-day-old chickens, together with the innate immune response of this host. Results The mutant lacking all 5 major SPIs was still capable of colonising the caecum while colonisation of the liver and spleen was dependent on the presence of both SPI-1 and SPI-2. In contrast, the absence of SPI-3, SPI-4 or SPI-5 individually did not influence virulence of S . Enteritidis for chickens, but collectively they contributed to the colonisation of the spleen. Proinflammatory signalling and heterophil infiltration was dependent on intact SPI-1 only and not on other SPIs. Conclusions SPI-1 and SPI-2 are the two most important pathogenicity islands of Salmonella Enteritidis required for the colonisation of systemic sites in chickens.

In this study, we addressed the origin of chicken gut microbiota in commercial production by a comparison of eggshell and feed microbiota with caecal microbiota of 7-day-old chickens, using microbiota analysis by 16S rRNA sequencing. In addition, we tested at which timepoint during prenatal or neonatal development it is possible to successfully administer probiotics. We found that eggshell microbiota was a combination of environmental and adult hen gut microbiota but was completely different from caecal microbiota of 7-day-old chicks. Similarly, we observed that the composition of feed microbiota was different from caecal microbiota. Neither eggshell nor feed acted as an important source of gut microbiota for the chickens in commercial production. Following the experimental administration of potential probiotics, we found that chickens can be colonised only when already hatched and active. Spraying of eggs with gut anaerobes during egg incubation or hatching itself did not result in effective chicken colonisation. Such conclusions should be considered when selecting and administering probiotics to chickens in hatcheries. Eggshells, feed or drinking water do not act as major sources of gut microbiota. Newly hatched chickens must be colonised from additional sources, such as air dust with spores of Clostridiales. The natural colonisation starts only when chickens are already hatched, as spraying of eggs or even chickens at the very beginning of the hatching process did not result in efficient colonisation.

The gut microbiota of warm-blooded vertebrates consists of bacterial species belonging to two main phyla; Firmicutes and Bacteroidetes. However, does it mean that the same bacterial species are found in humans and chickens? Here we show that the ability to survive in an aerobic environment is central for host species adaptation. Known bacterial species commonly found in humans, pigs, chickens and Antarctic gentoo penguins are those capable of extended survival under aerobic conditions, i.e., either spore-forming, aerotolerant or facultatively anaerobic bacteria. Such bacteria are ubiquitously distributed in the environment, which acts as the source of infection with similar probability in humans, pigs, chickens, penguins and likely any other warm-blooded omnivorous hosts. On the other hand, gut anaerobes with no specific adaptation for survival in an aerobic environment exhibit host adaptation. This is associated with their vertical transmission from mothers to offspring and long-term colonisation after administration of a single dose. This knowledge influences the design of next-generation probiotics. The origin of aerotolerant or spore-forming probiotic strains may not be that important. On the other hand, if Bacteroidetes and other host-adapted species are used as future probiotics, host preference should be considered.

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.