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.

Necrotic enteritis and antibiotic-free production of broiler chickens: Challenges in testing and using alternative products.

The global trend towards raising broiler chickens without the use of in-feed antibiotics (IFAs) means that there is an ongoing need to develop alternative treatments capable of delivering the benefits that IFAs previously provided. IFAs supported the productivity performance of chickens and played a key role in maintaining their health. Necrotic enteritis (NE) is an important disease of broilers that affects health, productivity, and welfare, and was previously well controlled by IFAs. However, with the reduction in IFA use, NE is resurgent in some countries. Vaccines and various feed additives, including pre-, pro-, and postbiotics, phytobiotics, fatty acids, and phage therapies have been introduced as alternative methods of NE control. While some of these feed additives have specific activity against the NE pathogen, Clostridium perfringens, most have the more general goal of reinforcing gut health. Extensive reviews of the effects of many of these feed additives on gut health have been published recently. Hence, rather than cover previously well reviewed areas of research this review focuses on the challenges and pitfalls in undertaking experimental assessment of alternative NE treatments and translating laboratory research to real world commercial production settings. The review is based on the author's particular experience, reading, thoughts, and analysis of the available information and inevitably presents a particular understanding that is likely to be at odds with others thinking on these issues. It is put forward to stimulate thinking and discussion on the issues covered.

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.

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.

Multi-omics analysis of hospital-acquired diarrhoeal patients reveals biomarkers of enterococcal proliferation and Clostridioides difficile infection.

Hospital-acquired diarrhoea (HAD) is common, and often associated with gut microbiota and metabolome dysbiosis following antibiotic administration. Clostridioides difficile is the most significant antibiotic-associated diarrhoeal (AAD) pathogen, but less is known about the microbiota and metabolome associated with AAD and C. difficile infection (CDI) with contrasting antibiotic treatment. We characterised faecal microbiota and metabolome for 169 HAD patients (33 with CDI and 133 non-CDI) to determine dysbiosis biomarkers and gain insights into metabolic strategies C. difficile might use for gut colonisation. The specimen microbial community was analysed using 16 S rRNA gene amplicon sequencing, coupled with untargeted metabolite profiling using gas chromatography-mass spectrometry (GC-MS), and short-chain fatty acid (SCFA) profiling using GC-MS. AAD and CDI patients were associated with a spectrum of dysbiosis reflecting non-antibiotic, short-term, and extended-antibiotic treatment. Notably, extended antibiotic treatment was associated with enterococcal proliferation (mostly vancomycin-resistant Enterococcus faecium) coupled with putative biomarkers of enterococcal tyrosine decarboxylation. We also uncovered unrecognised metabolome dynamics associated with concomitant enterococcal proliferation and CDI, including biomarkers of Stickland fermentation and amino acid competition that could distinguish CDI from non-CDI patients. Here we show, candidate metabolic biomarkers for diagnostic development with possible implications for CDI and vancomycin-resistant enterococci (VRE) treatment.

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.

Negative consequences of reduced protein diets supplemented with synthetic amino acids for performance, intestinal barrier function, and caecal microbiota composition of broiler chickens.

The consequences of feeding broiler chickens with reduced protein (RP) diets for gut health and barrier function are not well understood. This study was performed to elucidate the effect of reducing dietary protein and source of protein on gut health and performance parameters. Four experimental diets included 2 control diets with standard protein levels either containing meat and bone meal (CMBM) or an all-vegetable diet (CVEG), a medium RP diet (17.5% in growers and 16.5% in finisher), and a severe RP diet (15.6% in grower and 14.6% in finisher). Off-sex Ross 308 birds were assigned to each of the 4 diets and performance measurements were taken from d 7 to 42 post-hatch. Each diet was replicated 8 times (10 birds per replicate). A challenge study was conducted on additional 96 broilers (24 birds per diet) from d 13 to 21. Half of the birds in each dietary treatment were challenged by dexamethasone (DEX) to induce a leaky gut. Feeding birds with RP diets decreased weight gain (P < 0.0001) and increased feed conversion ratio (P < 0.0001) from d 7 to 42 compared with control diets. There was no difference between CVEG and CMBM control diets for any parameter. The diet containing 15.6% protein increased (P < 0.05) intestinal permeability independent of the DEX challenge. Gene expression of claudin-3 was downregulated (P < 0.05) in birds fed 15.6% protein. There was a significant interaction between diet and DEX (P < 0.05) and both RP diets (17.5% and 15.6%) downregulated claudin-2 expression in DEX-challenged birds. The overall composition of the caecal microbiota was affected in birds fed 15.6% protein having a significantly lower richness of microbiota in both sham and DEX-injected birds. Proteobacteria was the main phylum driving the differences in birds fed 15.6% protein. At the family level, Bifidobacteriaceae, Unclassified Bifidobacteriales, Enterococcaceae, Enterobacteriaceae, and Lachnospiraceae were the main taxa in birds fed 15.6% protein. Despite supplementation of synthetic amino acids, severe reduction of dietary protein compromised performance and intestinal health parameters in broilers, evidenced by differential mRNA expression of tight junction proteins, higher permeability, and changes in caecal microbiota composition.

Extensive genome analysis identifies novel plasmid families in Clostridium perfringens.

Globally, the anaerobic bacterium Clostridium perfringens causes severe disease in a wide array of hosts; however, C. perfringens strains are also carried asymptomatically. Accessory genes are responsible for much of the observed phenotypic variation and virulence within this species, with toxins frequently encoded on conjugative plasmids and many isolates carrying up to 10 plasmids. Despite this unusual biology, current genomic analyses have largely excluded isolates from healthy hosts or environmental sources. Accessory genomes, including plasmids, also have often been excluded from broader scale phylogenetic investigations. Here we interrogate a comprehensive collection of 464 C. perfringens genomes and identify the first putative non-conjugative enterotoxin (CPE)-encoding plasmids and a putative novel conjugative locus (Bcp) with sequence similarity to a locus reported from Clostridium botulinum. We sequenced and archived 102 new C. perfringens genomes, including those from rarely sequenced toxinotype B, C, D and E isolates. Long-read sequencing of 11 C. perfringens strains representing all toxinotypes (A-G) identified 55 plasmids from nine distinct plasmid groups. Interrogation of the 464 genomes in this collection identified 1045 plasmid-like contigs from the nine plasmid families, with a wide distribution across the C. perfringens isolates. Plasmids and plasmid diversity play an essential role in C. perfringens pathogenicity and broader biology. We have expanded the C. perfringens genome collection to include temporal, spatial and phenotypically diverse isolates including those carried asymptomatically in the gastrointestinal microbiome. This analysis has resulted in the identification of novel C. perfringens plasmids whilst providing a comprehensive understanding of species diversity.

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.

Prevalence of Campylobacter hepaticus specific antibodies among commercial free-range layers in Australia.

Spotty liver disease (SLD) caused by Campylobacter hepaticus affects the health and productivity of layer hens and is a disease of concern in poultry. In this study, blood and cloacal swab samples were collected from 709 birds across 11 free-range layer farms from different regions of Australia. The prevalence of C. hepaticus specific antibodies and DNA was assessed using a C. hepaticus specific ELISA and PCR and its correlation with mortalities and changes in egg production was analyzed to better understand the seroprevalence of C. hepaticus in Australian free-range layer farms. C. hepaticus specific antibodies were detected from birds in four of the five farms that had no history of SLD with seroprevalence as high as 41% in one of the farms. Seroprevalence of anti-C. hepaticus antibodies among flocks that had an active or previous SLD outbreak varied between 2 and 64%. C. hepaticus DNA was detected from birds in three farms with no known SLD history and five farms with confirmed SLD outbreaks. A good correlation was observed between the ELISA and PCR results with a Pearson correlation coefficient value of 0.85 (p-value = 0.001). No correlation was observed between the flock size or flock age and ELISA or PCR outcomes, and no significant difference between the seroprevalence of anti-C. hepaticus antibodies among flocks with or without a known history of SLD was established (p = 0.143). This study demonstrates the usefulness of C. hepaticus specific ELISA and PCR in identifying the occurrence of mild or sub-clinical SLD and provides a broader and more complete understanding of SLD epidemiology that will inform future research aimed at the development of methods to control SLD, such as appropriate biosecurity measures, vaccines, and feed additives.

Design and Evaluation Challenges of Conversational Agents in Health Care and Well-being: Selective Review Study.

BACKGROUND: Health care and well-being are 2 main interconnected application areas of conversational agents (CAs). There is a significant increase in research, development, and commercial implementations in this area. In parallel to the increasing interest, new challenges in designing and evaluating CAs have emerged. OBJECTIVE: This study aims to identify key design, development, and evaluation challenges of CAs in health care and well-being research. The focus is on the very recent projects with their emerging challenges. METHODS: A review study was conducted with 17 invited studies, most of which were presented at the ACM (Association for Computing Machinery) CHI 2020 conference workshop on CAs for health and well-being. Eligibility criteria required the studies to involve a CA applied to a health or well-being project (ongoing or recently finished). The participating studies were asked to report on their projects' design and evaluation challenges. We used thematic analysis to review the studies. RESULTS: The findings include a range of topics from primary care to caring for older adults to health coaching. We identified 4 major themes: (1) Domain Information and Integration, (2) User-System Interaction and Partnership, (3) Evaluation, and (4) Conversational Competence. CONCLUSIONS: CAs proved their worth during the pandemic as health screening tools, and are expected to stay to further support various health care domains, especially personal health care. Growth in investment in CAs also shows the value as a personal assistant. Our study shows that while some challenges are shared with other CA application areas, safety and privacy remain the major challenges in the health care and well-being domains. An increased level of collaboration across different institutions and entities may be a promising direction to address some of the major challenges that otherwise would be too complex to be addressed by the projects with their limited scope and budget.

Spotty liver disease adversely affect the gut microbiota of layers hen.

Spotty Liver Disease (SLD) is a serious infectious disease which occurs mainly in laying chickens in free range production systems. SLD outbreaks can increase mortality and decrease egg production of chickens, adversely impact welfare and cause economic hardship for poultry producers. The bacterium Campylobacter hepaticus is the primary cause of the disease. This study aimed to identify the effects of C. hepaticus on chicken gut microbiota and gut structure. Three C. hepaticus strains (HV10T, NSW44L and QLD19L), isolated from different states of Australia, were used in the study. Chickens at 26-weeks post-hatch were orally dosed with one of the C. hepaticus strains (challenged groups) or Brucella broth (unchallenged or control group). Six days after the challenge, birds were necropsied to assess liver damage, and caecal content and tissue samples were collected for histology, microbiology, and 16S rRNA gene amplicon sequencing to characterize the composition of the bacterial microbiota. Strain C. hepaticus NSW44L produced significantly more disease compared to the other C. hepaticus strains and this coincided with more adverse changes observed in the caecal microbiota of the birds challenged with this strain compared to the control group. Microbial diversity determined by Shannon and Simpson alpha diversity indices was lower in the NSW44L challenged groups compared to the control group (p = 0.009 and 0.0233 respectively, at genus level). Short-chain fatty acids (SCFAs) producing bacteria Faecalibacterium, Bifidobacterium and Megamonas were significantly reduced in the challenged groups compared to the unchallenged control group. Although SLD-induction affected the gut microbiota of chickens, their small intestine morphology was not noticeably affected as there were no significant differences in the villus height or ratio of villus height and crypt depth. As gut health plays a pivotal role in the overall health and productivity of chickens, approaches to improve the gut health of the birds during SLD outbreaks such as through diet and keeping the causes of stress to a minimum, may represent significant ways to alleviate the impact of SLD.

Enhancement of live vaccines by co-delivery of immune modulating proteins.

Vaccines are very effective in providing protection against many infectious diseases. However, it has proven difficult to develop highly efficacious vaccines against some pathogens and so there is a continuing need to improve vaccine technologies. The first successful and widely used vaccines were based on attenuated pathogens (e.g., laboratory passaged Pasteurella multocida to vaccinate against fowl cholera) or closely related non-pathogenic organisms (e.g., cowpox to vaccinate against smallpox). Subsequently, live vaccines, either attenuated pathogens or non-pathogenic microorganisms modified to deliver heterologous antigens, have been successfully used to induce protective immune responses against many pathogens. Unlike conventional killed and subunit vaccines, live vaccines can deliver antigens to mucosal surfaces in a similar manner and context as the natural infection and hence can often produce a more appropriate and protective immune response. Despite these advantages, there is still a need to improve the immunogenicity of some live vaccines. The efficacy of injectable killed and subunit vaccines is usually enhanced using adjuvants such mineral salts, oils, and saponin, but such adjuvants cannot be used with live vaccines. Instead, live vaccines can be engineered to produce immunomodulatory molecules that can stimulate the immune system to induce more robust and long-lasting adaptive immune responses. This review focuses on research that has been undertaken to engineer live vaccines to produce immunomodulatory molecules that act as adjuvants to increase immunogenicity. Adjuvant strategies with varying mechanisms of action (inflammatory, antibody-mediated, cell-mediated) and delivery modes (oral, intramuscular, intranasal) have been investigated, with varying degrees of success. The goal of such research is to define adjuvant strategies that can be adapted to enhance live vaccine efficacy by triggering strong innate and adaptive immune responses and produce vaccines against a wider range of pathogens.

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).

Campylobacter hepaticus, the cause of Spotty Liver Disease in chickens, can enter a viable but nonculturable state.

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in layer hens, resulting in mortality and productivity losses. Like other Campylobacter species, C. hepaticus is a fastidious organism that requires microaerobic conditions to grow and efficiently replicate. Despite its apparent vulnerability to environmental conditions, it is suspected that there are environmental sources of C. hepaticus that cause infections in chickens newly placed in production houses. Although C. hepaticus DNA has been detected in insects, rodent and wild bird droppings, and in environmental samples such as water and soil, it has not been possible to culture C. hepaticus from these sources. Therefore, it is unclear whether these environments harbor viable bacteria or the remnants of dead bacteria. Determining the viability of C. hepaticus in challenging conditions has implications for understanding the potential relevance of environmental reservoirs and routes of transmission. Other Campylobacters are known to enter viable but nonculturable (VBNC) states that result in prolonged survival in hostile environmental conditions. This study has demonstrated that C. hepaticus can also enter a VBNC state when stored in water or a simple salt solution (Ringer's solution). Cells in the VBNC state could not be recovered on media normally used for primary isolation, but media modifications were tested, and a 'resuscitation' media was developed. VBNC cells could be recovered from Ringer's solution stored at 4 °C for up to 112 days. VBNC cells are postulated to play an important role in the epidemiology of SLD.

A lytic bacteriophage isolate reduced Clostridium perfringens induced lesions in necrotic enteritis challenged broilers.

Background: Bacteriophages are viral predators of bacteria and are common in nature. Their host-specific infections against specific bacteria make them an attractive natural agent to control bacterial pathogens. Interest in the potential of bacteriophages as antibacterial agents in the production animal industries has increased. Methods: A total of 18 bacteriophages were isolated from Australian commercial poultry environments, from which three highly active phages were chosen for enrichment. Sequencing libraries were prepared using a Nextera XT kit (Illumina) and sequenced on an Illumina MiSeq instrument using 2 × 300 bp paired-end chemistry. The sequence data were then assembled and aligned with a2 bacteriophage as the reference. An animal trial was performed by oral gavaging Clostridium perfringens netB containing strain EHE-NE18 to the Ross 308 broiler chickens prior inoculation with Eimeria species. The chickens were raised following the management guide for Ross 308 from d 0 to d 21 and fed with starter and grower diets met the specific breed nutrient requirements. Body weight gain and feed intake were measured on d 9 and d 21 and FCR adjusted with mortality was calculated. Results: The isolated bacteriophages only had only 96.7% similarity to the most closely related, previously characterized, Clostridium bacteriophage indicated that they might represent a novel strain of bacteriophage. A "cocktail" containing the three bacteriophages was capable of lysing four known disease-inducing C. perfringens strains in vitro. Oral administration of the bacteriophages cocktail to broilers challenged with necrotic enteritis markedly alleviated intestinal necrotic lesions in the duodenum and jejunum on day 16 post-hatch. The phage treatment significantly reduced the lesion scores of birds challenged with NE (P < 0.01), and the lesion scores between birds treated with the bacteriophages and the unchallenged birds were not statistically different (P > 0.05). However, no effect on the growth performance was observed during the recorded period of days 9-21. Conclusion: These findings suggest that bacteriophage treatment is a promising approach to protect intestinal health from C. perfringens induced necrotic enteritis. Further research will be required on the dosing, route of administration, and large scale validation studies to further advance this approach to pathogen control.

The use of filamentous hemagglutinin adhesin to detect immune responses to Campylobacter hepaticus infections in layer hens.

Campylobacter hepaticus is the aetiological agent of Spotty Liver Disease (SLD). SLD can cause significant production loss and mortalities among layer hens at and around peak of lay. We previously developed an enzyme linked immunosorbent assay (ELISA), SLD-ELISA1, to detect C. hepaticus specific antibodies from bird sera using C. hepaticus total proteins and sera pre-absorbed with Campylobacter jejuni proteins. The high specificity achieved with SLD-ELISA1 indicated the presence of C. hepaticus specific antibodies in sera of infected birds. However, some of the reagents used in SLD-ELISA1 are time consuming to prepare and difficult to quality control. This understanding led to the search for C. hepaticus specific immunogenic proteins that could be used in recombinant forms as antibody capture antigens in immunoassay design. In this study, an immunoproteomic approach that combined bioinformatics analysis, western blotting, and LC MS/MS protein profiling was used, and a fragment of filamentous hemagglutinin adhesin (FHA), FHA1,628-1,899 with C. hepaticus specific antigenicity was identified. Recombinant FHA1,628-1,899 was used as antigen coating on ELISA plates to capture FHA1,628-1,899 specific antibodies in sera of infected birds. SLD-ELISA2, based on the purified recombinant FHA fragment, is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study is the first report of the use of FHA from a Campylobacter species in immunoassays, and it also opens future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate.