Pioneering gut health improvements in piglets with phytogenic feed additives.

This research investigates the effects of phytogenic feed additives (PFAs) on the growth performance, gut microbial community, and microbial metabolic functions in weaned piglets via a combined 16S rRNA gene amplicon and shotgun metagenomics approach. A controlled trial was conducted using 200 pigs to highlight the significant influence of PFAs on gut microbiota dynamics. Notably, the treatment group revealed an increased gut microbiota diversity, as measured with the Shannon and Simpson indices. The increase in diversity is accompanied by an increase in beneficial bacterial taxa, such as Roseburia, Faecalibacterium, and Prevotella, and a decline in potential pathogens like Clostridium sensu stricto 1 and Campylobacter. Shotgun sequencing at the species level confirmed these findings. This modification in microbial profile was coupled with an altered profile of microbial metabolic pathways, suggesting a reconfiguration of microbial function under PFA influence. Significant shifts in overall microbial community structure by week 8 demonstrate PFA treatment's temporal impact. Histomorphological examination unveiled improved gut structure in PFA-treated piglets. The results of this study indicate that the use of PFAs as dietary supplements can be an effective strategy, augmenting gut microbiota diversity, reshaping microbial function, enhancing gut structure, and optimising intestinal health of weaned piglets providing valuable implications for swine production. KEY POINTS: • PFAs significantly diversify the gut microbiota in weaned piglets, aiding balance. • Changes in gut structure due to PFAs indicate improved resistance to weaning stress. • PFAs show potential to ease weaning stress, offering a substitute for antibiotics in piglet diets.

Campylobacter bilis sp. nov., isolated from chickens with spotty liver disease.

A novel species of Campylobacter was isolated from bile samples of chickens with spotty liver disease in Australia, making it the second novel species isolated from chickens with the disease, after Campylobacter hepaticus was isolated and described in 2016. Six independently derived isolates were obtained. They were Gram-stain-negative, microaerobic, catalase-positive, oxidase-positive and urease-negative. Unlike most other species of the genus Campylobacter, more than half of the tested strains of this novel species hydrolysed hippurate and most of them could not reduce nitrate. Distinct from C. hepaticus, many of the isolates were sensitive to 2,3,5-triphenyltetrazolium chloride (0.04%) and metronidazole (4 mg ml-1), and all strains were sensitive to nalidixic acid. Phylogenetic analysis using 16S rRNA and hsp60 gene sequences demonstrated that the strains formed a robust clade that was clearly distinct from recognized Campylobacter species. Whole genome sequence analysis of the strains showed that the average nucleotide identity and the genome blast distance phylogeny values compared to other Campylobacter species were less than 86 and 66%, respectively, which are below the cut-off values generally recognized for isolates of the same species. The genome of the novel species has a DNA G+C content of 30.6 mol%, while that of C. hepaticus is 27.9 mol%. Electron microscopy showed that the cells were spiral-shaped, with bipolar unsheathed flagella. The protein spectra generated from matrix-assisted laser desorption/ionization time of flight analysis demonstrated that they are different from the most closely related Campylobacter species. These data indicate that the isolates belong to a novel Campylobacter species, for which the name Campylobacter bilis sp. nov. is proposed. The type strain is VicNov18T (=ATCC TSD-231T=NCTC 14611T).

Stable Recombinant-Gene Expression from a Ligilactobacillus Live Bacterial Vector via Chromosomal Integration.

Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing. Alternative methods to control disease and maintain productivity need to be developed. Live vaccination is useful in preventing colonization of mucosa-dwelling pathogens by inducing a mucosal immune response. Native poultry isolate Ligilactobacillus agilis La3 (previously Lactobacillus agilis) has been identified as a candidate for use as a live vector to deliver therapeutic proteins such as bacteriocins, phage endolysins, or vaccine antigens to the gastrointestinal tract of chickens. In this study, the complete genome sequence of L. agilis La3 was determined and transcriptome analysis was undertaken to identify highly expressed genes. Predicted promoter regions and ribosomal binding sites from constitutively expressed genes were used to construct recombinant protein expression cassettes. A series of double-crossover shuttle plasmids were constructed to facilitate rapid selectable integration of expression cassettes into the Lagilis La3 chromosome via homologous recombination. Inserts showed 100% stable integration over 100 generations without selection. A positive relationship was found between protein expression levels and the predicted strength of the promoters. Using this system, stable chromosomal expression of a Clostridium perfringens antigen, rNetB, was demonstrated without selection. Finally, two recombinant strains, Lagilis La3::P eft -rnetB and Lagilis La3::P cwah -rnetB, were constructed and characterized, and they showed potential for future application as live vaccines in chickens.IMPORTANCE Therapeutic proteins such as antigens can be used to prevent infectious diseases in poultry. However, traditional vaccine delivery by intramuscular or subcutaneous injection generally has not proven effective for mucosa-dwelling microorganisms that live within the gastrointestinal tract. Utilizing live bacteria to deliver vaccine antigens directly to the gut immune system can overcome some of the limitations of conventional vaccination. In this work, Ligilactobacillus agilis La3, an especially effective gut colonizer, has been analyzed and engineered with modular and stable expression systems to produce recombinant proteins. To demonstrate the effectiveness of the system, expression of a vaccine antigen from poultry pathogen Clostridium perfringens was monitored over 100 generations without selection and found to be completely stable. This study demonstrates the development of genetic tools and novel constitutive expression systems and further development of L. agilis La3 as a live delivery vehicle for recombinant proteins.

Temporal dynamics of gut microbiota in caged laying hens: a field observation from hatching to end of lay.

Gut health has major implications for the general health of food-producing animals such as the layer birds used in the egg industry. In order to modulate gut microbiota for the benefit of gut health, an understanding of the dynamics and details of the development of gut microbiota is critical. The present study investigated the phylogenetic composition of the gut microbiota of a commercial layer flock raised in cages from hatch to the end of the production cycle. This study also aimed to understand the establishment and development of gut microbiota in layer chickens. Results showed that the faecal microbiota was dominated by phyla Firmicutes and Proteobacteria in the rearing phase, but Bacteroidetes in mid lay and late lay phase. The gut microbiota composition changed significantly during the transfer of the flock from the rearing to the production shed. The richness and diversity of gut microbiota increased after week 6 of the flocks age and stabilized in the mid and late lay phase. The overall dynamics of gut microbiota development was similar to that reported in earlier studies, but the phylogenetic composition at the phylum and family level was different. The production stage of the birds is one of the important factors in the development of gut microbiota. This study has contributed to a better understanding of baseline gut microbiota development over the complete life cycles in layer chickens and will help to develop strategies to improve the gut health. KEY POINTS: • Faecal microbiota of caged hens was dominated by phyla Firmicutes and Proteobacteria in the rearing phase. • The gut microbiota composition changed significantly during the transfer of the flock from the rearing to the production shed. • The richness and diversity of gut microbiota increased after week 6 of the flocks age and stabilized in the mid and late lay phase.

Development of an enzyme-linked immunosorbent assay for detecting Campylobacter hepaticus specific antibodies in chicken sera - a key tool in Spotty Liver Disease screening and vaccine development.

Spotty Liver Disease (SLD) is an emerging disease of serious concern in the egg production industry, as it causes significant egg loss and mortality in layer hens. The causative agent is a newly identified Gram-negative bacterium, Campylobacter hepaticus, and knowledge about C. hepaticus pathogenesis and the potential for vaccine development is still in its infancy. Current detection methods for SLD, such as PCR and culturing, only detect an active infection and will not give any indication of a past infection from which the bacteria have been cleared. An immunological assay, on the other hand, can provide information on previous infections and therefore is crucial in vaccine development against SLD. In the present study, we have developed the first immunoassay capable of detecting C. hepaticus-specific antibodies present in the sera of infected birds. The assay uses C. hepaticus total protein extract (TPE) as the antigen coating on enzyme-linked immunosorbent assay (ELISA) plates. The cross reactivity of C. hepaticus antibodies with closely related C. jejuni and C. coli antigens was successfully overcome by pre-absorbing the sera using C. jejuni cell extracts. The assay was validated using sera samples from both naturally- and experimentally-infected birds, birds vaccinated with formalin-killed bacteria, and serum samples from SLD-negative birds (control group). The optimized ELISA assay had 95.5% specificity and 97.6% sensitivity. The immunoassay provides a useful tool for monitoring the exposure of poultry flocks to C. hepaticus infection and can be used to direct and support vaccine development. HIGHLIGHTS The first immunoassay developed for Spotty Liver Disease (SLD). A useful method for detecting C. hepaticus-specific antibodies in birds. Highly specific (95.5%) and sensitive (97.6%) assay. A key tool for use in epidemiological studies and vaccine development.

An intermittent hypercaloric diet alters gut microbiota, prefrontal cortical gene expression and social behaviours in rats.

Objectives: Excessive consumption of high fat and high sugar (HFHS) diets alters reward processing, behaviour, and changes gut microbiota profiles. Previous studies in gnotobiotic mice also provide evidence that these gut microorganisms may influence social behaviour. To further investigate these interactions, we examined the impact of the intermittent access to a HFHS diet on social behaviour, gene expression and microbiota composition in adolescent rats. Methods: Male rats were permitted intermittent daily access (2 h / day) to a palatable HFHS chow diet for 28 days across adolescence. Social interaction, social memory and novel object recognition were assessed during this period. Following testing, RT-PCR was conducted on hippocampal and prefrontal cortex (PFC) samples. 16S ribosomal RNA amplicon sequencing was used for identification and relative quantification of bacterial taxa in faecal samples. Results: We observed reduced social interaction behaviours, impaired social memory and novel object recognition in HFHS diet rats compared to chow controls. RT-PCR revealed reduced levels of monoamine oxidase A (Maoa), catechol-O-methyltransferase (Comt) and brain derived neurotrophic factor (Bdnf) mRNA in the PFC of HFHS diet rats. Faecal microbiota analysis demonstrated that the relative abundance of a number of specific bacterial taxa differed significantly between the two diet groups, in particular, Lachnospiraceae and Ruminoccoceae bacteria. Discussion: Intermittent HFHS diet consumption evoked physiological changes to the brain, particularly expression of mRNA associated with reward and neuroplasticity, and gut microbiome. These changes may underpin the observed alterations to social behaviours.

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens.

BACKGROUND: Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome. RESULTS: Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis. CONCLUSIONS: The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

Selenium nanoparticles in poultry feed modify gut microbiota and increase abundance of Faecalibacterium prausnitzii.

The poultry industry aims to improve productivity while maintaining the health and welfare of flocks. Pathogen control has been achieved through biosecurity, vaccinations and the use of antibiotics. However, the emergence of antibiotic resistance, in animal and human pathogens, has prompted researchers and chicken growers alike to seek alternative approaches. The use of new and emerging approaches to combat pathogen activity including nanotechnology, in particular, silver nanoparticles (NPs), has been found to not only eradicate pathogenic bacteria but also include issues of toxicity and bioaccumulation effects. Other novel metal nanoparticles could provide this pathogen reducing property with a more tailored and biocompatible nanomaterial for the model used, something our study represents. This study investigated the benefits of nanomaterial delivery mechanisms coupled with important health constituents using selenium as a biocompatible metal to minimise toxicity properties. Selenium NPs were compared to two common forms of bulk selenium macronutrients already used in the poultry industry. An intermediate concentration of selenium nanoparticles (0.9 mg/kg) demonstrated the best performance, improving the gut health by increasing the abundance of beneficial bacteria, such as Lactobacillus and Faecalibacterium, and short-chain fatty acids (SCFAs), in particular butyric acid. SCFAs are metabolites produced by the intestinal tract and are used as an energy source for colonic cells and other important bodily functions. Selenium nanoparticles had no significant effect on live weight gain or abundance of potentially pathogenic bacteria.

Rapid and Specific Methods to Differentiate Foodborne Pathogens, Campylobacter jejuni, Campylobacter coli, and the New Species Causing Spotty Liver Disease in Chickens, Campylobacter hepaticus.

Campylobacter jejuni and Campylobacter coli play a major role in bacteria-related foodborne illness in humans. Recently, a newly identified species, Campylobacter hepaticus, was shown to be the causative agent of spotty liver disease in chickens. The pathogenic potential of C. hepaticus in humans is unknown. This new species contains genes usually used to detect C. jejuni and C. coli in DNA-based detection methods, such as the hippuricase (hipO) gene and the glyA (serine hydroxymethyltransferase) gene, with a high degree of similarity. Therefore, polymerase chain reaction (PCR) primers used to detect these species need to be evaluated carefully to prevent misidentification of these important Campylobacter species. A multiplex PCR was developed and optimized to simultaneously and specifically identify the presence of C. jejuni, C. coli, and C. hepaticus in chicken samples containing high-complexity microbiota. The assay represents a new diagnostic tool for investigating the epidemiology of Campylobacter colonization in poultry and environmental samples. It may also be applicable to the investigation of Campylobacter contamination in food and in outbreaks of campylobacteriosis.

Campylobacter hepaticus sp. nov., isolated from chickens with spotty liver disease.

Ten strains of an unknown Campylobacter species were isolated from the livers of chickens with spotty liver disease in Australia. The strains were Gram-stain-negative, microaerobic, catalase- and oxidase-positive and urease-negative. Unlike most other species of the genus Campylobacter, most of the tested strains of this novel species hydrolysed hippurate and half of them could not reduce nitrate. All strains showed resistance, or intermediate resistance, to nalidixic acid and most of them were resistant to cephalothin. Examination of negatively stained cells under transmission electron microscopy revealed that they were S-shaped, with bipolar unsheathed flagella. Phylogenetic analyses based on the 16S rRNA gene and the heat shock protein 60 (hsp60) gene sequences indicated that the strains formed a robust clade that was clearly distinct from recognized Campylobacter species. Unusually, they had a DNA G+C content of 27.9 mol%, lower than any previously described Campylobacter species, and they showed less than 84 % average nucleotide identity to the nearest sequenced species. Taken together, these data indicate that the strains belong to a novel Campylobacter species, for which the name Campylobacter hepaticus sp. nov. is proposed. The type strain is HV10T (=NCTC 13823T=CIP 111092T).

Layer chicken microbiota: a comprehensive analysis of spatial and temporal dynamics across all major gut sections.

BACKGROUND: The gut microbiota influences chicken health, welfare, and productivity. A diverse and balanced microbiota has been associated with improved growth, efficient feed utilisation, a well-developed immune system, disease resistance, and stress tolerance in chickens. Previous studies on chicken gut microbiota have predominantly focused on broiler chickens and have usually been limited to one or two sections of the digestive system, under controlled research environments, and often sampled at a single time point. To extend these studies, this investigation examined the microbiota of commercially raised layer chickens across all major gut sections of the digestive system and with regular sampling from rearing to the end of production at 80 weeks. The aim was to build a detailed picture of microbiota development across the entire digestive system of layer chickens and study spatial and temporal dynamics. RESULTS: The taxonomic composition of gut microbiota differed significantly between birds in the rearing and production stages, indicating a shift after laying onset. Similar microbiota compositions were observed between proventriculus and gizzard, as well as between jejunum and ileum, likely due to their anatomical proximity. Lactobacillus dominated the upper gut in pullets and the lower gut in older birds. The oesophagus had a high proportion of Proteobacteria, including opportunistic pathogens such as Gallibacterium. Relative abundance of Gallibacterium increased after peak production in multiple gut sections. Aeriscardovia was enriched in the late-lay phase compared to younger birds in multiple gut sections. Age influenced microbial richness and diversity in different organs. The upper gut showed decreased diversity over time, possibly influenced by dietary changes, while the lower gut, specifically cecum and colon, displayed increased richness as birds matured. However, age-related changes were inconsistent across all organs, suggesting the influence of organ-specific factors in microbiota maturation. CONCLUSION: Addressing a gap in previous research, this study explored the microbiota across all major gut sections and tracked their dynamics from rearing to the end of the production cycle in commercially raised layer chickens. This study provides a comprehensive understanding of microbiota structure and development which help to develop targeted strategies to optimise gut health and overall productivity in poultry production.

Bacillus amyloliquefaciens Probiotics Mix Supplementation in a Broiler Leaky Gut Model.

The supplementation of antimicrobial growth promoters (AGPs) has been banned in many countries because of the emergence of antimicrobial-resistant pathogens in poultry products and the environment. Probiotics have been broadly studied and demonstrated as a promising AGP substitute. Our study is centred on the effects of a multi-strain Bacillus-based probiotic product on broiler production performance and gut microbial profile in a dexamethasone-induced leaky gut challenge. Two hundred and fifty-six broiler chicks were hatched and randomly assigned into four groups (wheat-soybean meal basal diet (BD) = non-supplemented control (C), BD supplemented with dexamethasone in week 4 (CD), BD containing a probiotic from day one (P), and BD containing a probiotic from day one and supplemented with dexamethasone during challenge week 4 (PD)). The production performance and caecal, gizzard, jejunal lumen and jejunal mucosa swab microbiota were studied by 16S rRNA gene sequencing. The Bacillus probiotic product significantly improved production performance and altered caecal gut microbiota (p ≤ 0.05), but no significant impact on microbiota was observed in other gut sections.

Dust sprinkling as an effective method for infecting layer chickens with wild-type Salmonella Typhimurium and changes in host gut microbiota.

Role of dust in Salmonella transmission on chicken farms is not well characterised. Salmonella Typhimurium (ST) infection of commercial layer chickens was investigated using a novel sprinkling method of chicken dust spiked with ST and the uptake compared to a conventional oral infection. While both inoculation methods resulted in colonisation of the intestines, the Salmonella load in liver samples was significantly higher at 7 dpi after exposing chicks to sprinkled dust compared to the oral infection group. Infection of chickens using the sprinkling method at a range of doses showed a threshold for colonisation of the gut and organs as low as 1000 CFU/g of dust. Caecal content microbiota analysis post-challenge showed that the profiles of chickens infected by the sprinkling and oral routes were not significantly different; however, both challenges induced differences when compared to the uninfected negative controls. Overall, the study showed that dust sprinkling was an effective way to experimentally colonise chickens with Salmonella and alter the gut microbiota than oral gavage at levels as low as 1000 CFU/g dust. This infection model mimics the field scenario of Salmonella infection in poultry sheds. The model can be used for future challenge studies for effective Salmonella control.

A live attenuated Salmonella Typhimurium vaccine dose and diluent have minimal effects on the caecal microbiota of layer chickens.

Among the Salmonella reduction strategies in poultry production, one option is to use a Salmonella vaccine. The aim of vaccinating layer flocks is to reduce the shedding of wild-type Salmonella in the poultry environment, thereby reducing the contamination of poultry products (eggs and meat). Nutritive diluent and a higher dose of vaccine may enhance its colonization potential in the gut of chickens. In this study, a commercially available live attenuated vaccine (Vaxsafe® ST) was reconstituted in different media and delivered orally to day-old chicks at three different doses (107, 108, and 109 CFU/chick). Gut colonization of the vaccine strain and the effects of vaccination on gut microbiota were assessed in commercial-layer chickens. The vaccine diluent and dosage minimally affected microbiota alpha diversity. Microbiota beta diversity was significantly different (P < 0.05) based on the vaccine diluent and dose, which indicated that the vaccinated and unvaccinated chickens had different gut microbial communities. Differences were noted in the abundance of several genera, including Blautia, Colidextribacter, Dickeya, Enterococcus, Lactobacillus, Pediococcus, and Sellimonas. The abundance of Colidextribacter was significantly lower in chickens that received vaccine reconstituted in Marek's and water diluents, while Lactobacillus abundance was significantly lower in the water group. The highest vaccine dose (109 CFU/chick) did not significantly alter (P > 0.05) the abundance of microbial genera. Chicken age affected the microbiota composition more significantly than the vaccine dose and diluent. The abundance of Lactobacillus, Blautia, Caproiciproducens, Pediococcus, and Colidextribacter was significantly higher on day 14 compared with day 7 post-vaccination. The Salmonella Typhimurium vaccine load in the caeca was not significantly affected by diluent and vaccine dose; however, it was significantly lower (P < 0.0001) on day 14 compared with day 7 post-vaccination. Overall, the S. Typhimurium vaccine minimally affected the gut microbiota structure of layer chicks, whereas changes in microbiota were more significant with chicken age.

Nanocapsules Comprised of Purified Protein: Construction and Applications in Vaccine Research.

Nanoparticles show great promise as a platform for developing vaccines for the prevention of infectious disease. We have been investigating a method whereby nanocapsules can be formulated from protein, such that the final capsules contain only the cross-linked protein itself. Such nanocapsules are made using a silica templating system and can be customised in terms of size and porosity. Here we compare the construction and characteristics of nanocapsules from four different proteins: one a model protein (ovalbumin) and three from infectious disease pathogens, namely the influenza virus, Helicobacter pylori and HIV. Two of the nanocapsules were assessed further. We confirm that nanocapsules constructed from the urease A subunit of H. pylori can reduce subsequent infection in a vaccinated mouse model. Further, we show that capsules constructed from the HIV gp120 protein can be taken up by dendritic cells in tissue culture and can be recognised by antibodies raised against the virus. These results point to the utility of this method in constructing protein-only nanocapsules from proteins of varying sizes and isoelectric points.

Pre-existing immunity against vaccine vectors--friend or foe?

Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.

The temporal fluctuations and development of faecal microbiota in commercial layer flocks.

The microbiota of the gastrointestinal tract influences gut health, which in turn strongly impacts the general health and productivity of laying hens. It is essential to characterise the composition and temporal development of the gut microbiota in healthy layers raised under different management systems, to understand the variations in typical healthy microbiota structure, so that deviations from this might be recognised and correlated with production and health issues when they arise. The present investigation aimed to study the temporal development and phylogenetic composition of the gut microbiota of four commercially raised layer flocks from hatch to end of the production cycle. Non-intrusive faecal sampling was undertaken as a proxy to represent the gut microbiota. Sequencing of 16S rRNA gene amplicons was used to characterise the microbiota. Beta diversity analysis indicated that each faecal microbiota was different across the four flocks and had subtly different temporal development patterns. Despite these inter-flock differences, common patterns of microbiota development were identified. Firmicutes and Proteobacteria were dominant at an early age in all flocks. The microbiota developed gradually during the rearing phase; richness and diversity increased after 42 d of age and then underwent significant changes in composition after the shift to the production farms, with Bacteroidota becoming more dominant in older birds. By developing a more profound knowledge of normal microbiota development in layers, opportunities to harness the microbiota to aid in the management of layer gut health and productivity may be more clearly seen and realised.

Heterologously expressed SacP23, a novel bacteriocin from Paenibacillus polymyxa #23, is active against methicillin resistant Staphylococcus aureus.

Antimicrobial peptides have the potential to be used in a range of applications, including as an alternative to conventional antibiotics for the treatment of bacterial infections of humans and animals. Therefore, there is interest in identifying novel bacteriocins which have desirable physico-chemical properties or antimicrobial activities. Paenibacillus polymyxa #23, isolated from a marine sponge, has wide spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. To explore the basis of this antimicrobial activity, the complete genome sequence of the strain was examined. Multiple genes predicted to encode antimicrobial peptides were identified. One gene was predicted to encode a novel sactipeptide bacteriocin, named SacP23. To confirm that SacP23 does have antimicrobial activity and to explore the antimicrobial spectrum of the peptide it was heterologously expressed in Bacillus subtilis. A cluster of eight genes, encoding a full complement of accessory genes as well as the structural gene expressed from the native promoter, was cloned into B. subtilis BS54A. The recombinant strain displayed antimicrobial activity against several Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus. Heterologous expression of a whole gene cluster offers a powerful way to interrogate and resolve the various antimicrobial activities expressed by native strains that encode multiple compounds of interest.

Characterisation of N-linked protein glycosylation in the bacterial pathogen Campylobacter hepaticus.

Campylobacter hepaticus is an important pathogen which causes Spotty Liver Disease (SLD) in layer chickens. SLD results in an increase in mortality and a significant decrease in egg production and therefore is an important economic concern of the global poultry industry. The human pathogen Campylobacter jejuni encodes an N-linked glycosylation system that plays fundamental roles in host colonization and pathogenicity. While N-linked glycosylation has been extensively studied in C. jejuni and is now known to occur in a range of Campylobacter species, little is known about C. hepaticus glycosylation. In this study glycoproteomic analysis was used to confirm the functionality of the C. hepaticus N-glycosylation system. It was shown that C. hepaticus HV10T modifies > 35 proteins with an N-linked heptasaccharide glycan. C. hepaticus shares highly conserved glycoproteins with C. jejuni that are involved in host colonisation and also possesses unique glycoproteins which may contribute to its ability to survive in challenging host environments. C. hepaticus N-glycosylation may function as an important virulence factor, providing an opportunity to investigate and develop a better understanding the system's role in poultry infection.

Campylobacter bilis, the second novel Campylobacter species isolated from chickens with Spotty Liver Disease, can cause the disease.

Spotty Liver Disease (SLD) is a significant disease of commercial layer hens. It can cause up to 10 % flock mortalities and reduce egg production by 25 %. Campylobacter hepaticus has been identified as the main cause of the disease, although it also appears that predisposing factors, such as some form of stress, may increase the likelihood of clinical disease occurring. Recently, a newly identified species, Campylobacter bilis, was isolated from bile samples of clinical SLD affected chickens. To investigate the pathogenic potential of C. bilis two independent isolates were used in infection trials of layer hens. Within 6 days of oral challenge birds developed typical SLD liver lesions, demonstrating that both strains induced SLD. C. bilis could be recovered from all the challenged birds that developed SLD. Thus, each of the steps in Koch's postulates have been fulfilled, confirming that C. bilis is an additional cause of SLD. A PCR method was developed which can specifically detect C. bilis from samples with complex microbiota. The identification of this newly discovered Campylobacter species as a second cause of SLD and the provision of a rapid method to detect the SLD causing bacterium will help with SLD vaccine development and epidemiology, thus assisting in the control of this important disease of poultry.