Campylobacter jejuni is the leading cause of bacterial gastroenteritis and is a major public heal... more Campylobacter jejuni is the leading cause of bacterial gastroenteritis and is a major public health concern worldwide. Despite its importance, our understanding of how C. jejuni causes diarrhoea and interacts with its hosts is limited due to the absence of appropriate infection models and established virulence factors found in other enteric pathogens. Additionally, despite its genetic diversity, non-pathogenic C. jejuni strains are unknown. Regardless of these limitations, significant progress has been made in understanding how C. jejuni uses a complex array of factors which aid the bacterium to survive and respond to host defences. This review provides an update on fitness and virulence determinants of this important pathogen and questions our knowledge on these determinants that are often based on inferred genomics knowledge and surrogate infection models.
Coccidiosis, caused by Eimeria parasites, significantly impacts poultry farm economics and animal... more Coccidiosis, caused by Eimeria parasites, significantly impacts poultry farm economics and animal welfare. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and caecal metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial BifidobacteriumBifidobacterium. Using a multi-omics factor analysis, data on caecal microbiota and metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offeringofferingofferingpotential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.
The article presents a processed dataset from amplicon se- quencing of the V4 region of the 16S r... more The article presents a processed dataset from amplicon se- quencing of the V4 region of the 16S rRNA gene to re- cover bacterial and archaeal taxa from the caeca of multi- ple chicken breeds of Pakistan. These include chicken breeds commonly raised at commercial level, Naked Neck, Black Australorp, Rhode Island Red, White Layer, and Broiler. All the breeds were challenged with Newcastle Disease Virus (NDV), with vaccination against the disease also explored. This resulted in samples belonging to four treatment groups as: Control; Vaccinated; Vaccinated and Challenged; and Non-vaccinated and Challenged. These were raised on an antibiotic free diet in a semi-controlled farming setup. 16S rRNA gene amplicon sequencing of caecal DNA from day old and mature chicken samples (22 weeks for Naked Neck, Black Australorp, Rhode Island Red and White Layer; 8 weeks for Broiler) of the four groups was performed. The paired-end reads from all the samples were quality trimmed, error cor- rected, and overlapped, on which unique Operational Taxo- nomic Units (OTUs) were obtained at 99 % similarity. Using predictive modelling, the MetaCyc functional pathways, as well as KEGG orthologs were also recovered. The generated data may be used to explore microbial interactions in gas- trointestinal tract with respect to NDV vaccination and infec- tion, together with increased understanding of chicken health and productivity.
Background Maintaining gut health is a persistent and unresolved challenge in the poultry industr... more Background Maintaining gut health is a persistent and unresolved challenge in the poultry industry. Given the critical role of gut health in chicken performance and welfare, there is a pressing need to identify effective gut health intervention (GHI) strategies to ensure optimal outcomes in poultry farming. In
This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 ... more This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 different groups of chicken raised in different farm setups in Pakistan. The groups comprise of three different breeds (Broilers, White Layers, and Black Australorp) of chicken raised in different farming setups that include antibiotic-free control, commercial (open and controlled shed), and backyard farms. We have recovered 569 Metagenomics-Assembled Genomes (MAGs) with a completeness of ≥50 % and contamination of ≤10 %. For each MAG, functional annotations were obtained that include KEGG modules, carbohydrate active enzymes (CAZymes), peptidases, geochemical cycles, antibiotic resistance genes, stress genes, and virulence genes. Furthermore, two different sets of Single Copy Genes (SCGs) were used to construct the phylogenetic trees. Based on the reconstructed phylogeny, phylogenetic gain of each MAG is calculated to give an account of novelty.
This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating f... more This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating from chicken caeca. The samples originate from broiler chickens, one group was infected with Newcastle Disease Virus (NDV) and one uninfected control group. There were four birds per group. Both groups were raised on commercially available antibiotic free feed under a semi-controlled setup. The binning step of the samples identified 130 MAGs with ≥50 % completion, and ≤10 % contamination. The data presented includes sequences in FASTA format, tables of functional annotation of genes, and data from two different approaches for phylogenetic tree construction using these MAGs. Major geochemical cycles at community level including carbon, sulfur, and nitrogen cycles are also presented.
Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the env... more Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the environment but also in the human body. The use of phages for the diagnosis of melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is emerging as a promising novel approach, but our understanding of conditions under which Burkholderia prophages can be induced remains limited. Here, we first demonstrated the isolation of Burkholderia phages from the hemocultures of melioidosis patients. The B.pseudomallei-positive hemoculture bottles were filtered to remove bacteria, and then phages were isolated and purified by spot and double agar overlay plaque assays. Forty blood samples (hemoculture-confirmed melioidosis) were tested, and phages were found in 30% of the samples. Transmission electron microscopy and genome analysis of the isolated phages, vB_HM387 and vB_HM795, showed that both phages are Myoviruses. These two phages were stable at a pH of 5–7 and temperatures of 25–37°C, suggesting their ability to survive in human blood. The genome sizes of vB_HM387 and vB_HM795 are 36.3 and 44.0 kb, respectively. A phylogenetic analysis indicated that vB_HM387 has homologs, but vB_HM795 is a novel Myovirus, suggesting the heterogeneity of Burkholderia phages in melioidosis patients. The key finding that Burkholderia phages could be isolated from the blood of melioidosis patients highlights the potential application of phage-based assays by detecting phages in blood as a pathogen-derived biomarker of infection.
Coccidiosis, caused by Eimeria parasites, poses significant economic and welfare challenges in po... more Coccidiosis, caused by Eimeria parasites, poses significant economic and welfare challenges in poultry farming. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and host metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial Bifidobacterium. Using a multi-omics factor analysis (MOFA) machine learning model, data on caecal microbiota and host metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offering potential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.
This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating f... more This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating from chicken caeca. The samples originate from broiler chickens, one group was infected with Newcastle Disease Virus (NDV) and one uninfected control group. There were four birds per group. Both groups were raised on commercially available antibiotic free feed under a semi-controlled setup. The binning step of the samples identified 130 MAGs with ≥ 50% completion, and ≤ 10% contamination. The data presented includes sequences in FASTA format, tables of functional annotation of genes, and data from two different approaches for phylogenetic tree construction using these MAGs. Major geochemical cycles at community level including carbon, sulfur, and nitrogen cycles are also presented.
This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 ... more This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 different groups of chicken raised in different farm setups in Pakistan. The groups comprise of three different breeds (Broilers, White Layers, Black Australorp) of chicken raised in different farming setups that include antibiotic-free control, commercial (open and controlled shed), and backyard farms. We acquired 569 Metagenomics-Assembled Genomes (MAGs) with a completion rate of ≥50% and contamination of ≤10%. For each MAG, we have obtained functional annotations that include KEGG modules, carbohydrate active enzymes (CAZymes), peptidases, geochemical cycles, antibiotic resistance, stress, and virulence genes. Furthermore, we have used Single Copy Genes (SCGs) to construct phylogenetic tree utilizing several approaches, and then give an account of novelty of the MAGs using phylogenetic gain.
There was no effect of removing ZnO from piglet diets on the gut microbiome. These preliminary re... more There was no effect of removing ZnO from piglet diets on the gut microbiome. These preliminary results suggest that there is no benefit to extended lactation periods on the gut microbiome of weaning piglets and ZnO can indeed be successfully removed from piglet diets without impacting on the gut microbiome. However, the piglets in the experiments were raised in a challenge-free facility which is not representative of a commercial setting and so there may indeed be a need to find an alternative to ZnO in practice.
In recent years, there has been an unprecedented advancement in in situ analytical approaches tha... more In recent years, there has been an unprecedented advancement in in situ analytical approaches that contribute to the mechanistic understanding of microbial communities by explicitly incorporating ecology and studying their assembly. In this study, we have analyzed the temporal profiles of the healthy broiler cecal microbiome from day 3 to day 35 to recover the stable and varying components of microbial communities. During this period, the broilers were fed three different diets chronologically, and therefore, we have recovered signature microbial species that dominate during each dietary regime. Since broilers were raised in multiple pens, we have also parameterized these as an environmental condition to explore microbial niches and their overlap. All of these analyses were performed in view of different parameters such as body weight (BW-mean), feed intake (FI), feed conversion ratio (FCR), and age (days) to link them to a subset of microbes that these parameters have a bearing upon. We found that gut microbial communities exhibited strong and statistically significant specificity for several environmental variables. Through regression models, genera that positively/negatively correlate with the bird's age were identified. Some shortchain fatty acids (SCFAs)-producing bacteria, including Izemoplasmatales, Gastranaerophilales, and Roseburia, have a positive correlation with age. Certain pathogens, such as Escherichia-Shigella, Sporomusa, Campylobacter, and Enterococcus, negatively correlated with the bird's age, which indicated a high disease risk in the initial days. Moreover, the majority of pathways involved in amino acid biosynthesis were also positively correlated with the bird's age. Some probiotic genera associated with improved performance included Oscillospirales; UCG-010, Shuttleworthia, Bifidobacterium, and Butyricicoccaceae; UCG-009. In general, predicted antimicrobial resistance genes (piARGs) contributed at a stable level, but there was a slight increase in abundance when the diet was changed. To the best of the authors' knowledge, this is one of the first studies looking at the stability, complexity, and ecology of natural broiler microbiota development in a temporal setting.
Poultry is frequently associated with campylobacteriosis in humans, with Campylobacter jejuni bei... more Poultry is frequently associated with campylobacteriosis in humans, with Campylobacter jejuni being the most usual Campylobacter associated with disease in humans. Far-reaching research on Campylobacter was undertaken over the past two decades. This has resulted in interventions being put in place on farms and in processing plants. Despite these interventions, coupled with increased media coverage to educate the consumer on Campylobacter prevalence and campylobacteriosis, human health incidents are still high. Recent research is now shifting toward further understanding of the microorganisms to challenge interventions in place and to look at further and more relevant interventions for the reduction in human incidents. Farm practices play a key role in the control of colonization within poultry houses and among flocks. Prevalence at the farm level can be up to 100% and time of colonization may vary widely between flocks. Considerable research has been performed to understand how farm management and animal health practices can affect colonization on farms. This review will focus on farm practices to date as a baseline for future interventions as the microorganism becomes better understood. Further research is required to understand the chicken microbiome and factors influencing vertical transmission. The persistence of Campylobacter in animal and environmental reservoirs within and around farms requires further investigation to tailor farm practices toward preventing such reservoirs.
Chickens are a key food source for humans yet their microbiome contains bacteria that can be path... more Chickens are a key food source for humans yet their microbiome contains bacteria that can be pathogenic to humans, and indeed potentially to chickens themselves. Campylobacter is present within the chicken gut and is the leading cause of bacterial foodborne gastroenteritis within humans worldwide. Infection can lead to secondary sequelae such as Guillain-Barré syndrome and stunted growth in children from low-resource areas. Despite the global health impact and economic burden of Campylobacter, how and when Campylobacter appears within chickens remains unclear. As such, there has been a motivation to decrease the number of Campylobacter within chickens and thus reduce the risk of infection to humans. The lack of day-to-day microbiome data with replicates, relevant metadata, and a lack of natural infection studies have delayed our understanding of the chicken gut microbiome and Campylobacter. Here, we performed a comprehensive day-to-day microbiome analysis of the chicken cecum from day 3 to 35 (12 replicates each day; n=396) combining metadata such as chicken weight and feed conversion rates to investigate what the driving forces are for the microbial changes within the chicken gut over time, and how this relates to Campylobacter appearance within a natural habitat setting. We found a rapidly increasing microbial diversity up to day 12 with variation observed both in terms of genera and abundance, before a stabilisation of the microbial diversity after day 20. In particular, we identified a shift from competitive to environmental drivers of microbial community from days 12 to 20 creating a window of opportunity whereby Campylobacter appears. Campylobacter was identified at day 16 which was one day after the most substantial changes in metabolic profiles observed. In addition, microbial variation over time is most likely influenced by the diet of the chickens whereby significant shifts in OTU abundances and beta dispersion of samples often corresponded with changes in feed. This study is unique in comparison to the most recent studies as neither sampling was sporadic nor Campylobacter was artificially introduced, thus the experiments were performed in a natural setting. We believe that our findings can be useful for future intervention strategies and can help elucidate the mechanism through which Campylobacter within chickens can be reduced.
Campylobacter jejuni is the leading cause of food-borne bacterial enteritis in humans, with conta... more Campylobacter jejuni is the leading cause of food-borne bacterial enteritis in humans, with contaminated poultry products considered the main source of infection. To survive the food chain, C. jejuni utilizes multiple defense mechanisms that counter oxidative and aerobic stresses. In this study, we phenotypically characterised 63 C. jejuni strains with oxidative stress survival and antimicrobial susceptibility testing to investigate correlations between these two phenotypes against the source of the strains and the presence of the MarR regulators RrpA and RrpB which have a role in regulating the response to oxidative and aerobic stress. C. jejuni strains isolated from meat and neck skin displayed the highest resistance to oxidative stress. In addition, C. jejuni strains that have an rrpA+rrpB− profile exhibit increased resistance to oxidative stress and to antimicrobials. Here we establish a preliminary link between the distribution of RrpA and RrpB and the increased resistance to antimicrobials. This study provides insight into how the genotypic make up of C. jejuni can influence the ability of the bacterium to survive within areas of high oxygen stress, such as the food chain, and subsequently can have a potential negative impact on human health.
Listeriosis is a foodborne disease, with a high mortality rate, that predominantly affects the el... more Listeriosis is a foodborne disease, with a high mortality rate, that predominantly affects the elderly. Under European Union legislation, EC 2073/2005, food business operators are encouraged to undertake sampling to ensure that the food processing environment, and required to ensure that food products, are free of Listeria monocytogenes. To determine the prevalence of L. monocytogenes in smaller food processing facilities in Northern Ireland, 24 companies submitted six processing environment swabs and two food samples every two months for eighteen months (July 2015 to November 2016) for L. monocytogenes examination. The prevalence of L. monocytogenes was 4.6% in food samples, and 6.3% in processing environment swabs. Over the duration of the study, 96 isolates of L. monocytogenes were obtained, one from each positive sample, except for two meat samples that had >100 cfu/g, where two isolates were obtained from each sample. No seasonality in occurrence of L. monocytogenes was seen for food isolates but significantly higher numbers of positive processing environment swabs were found in the warmer months of May, July and September (p = .007). Pulsed Field Gel Electrophoresis (PFGE) analysis revealed the presence of 27 pulsotypes; 9 pulsotypes were shared between different facilities and 9 were persistent. Based on a Combase predictive growth model, 77.5% (n = 130) of the foods tested were predicted to support the growth of L. monocytogenes. All of the isolates carried the pathogenicity genes inlA and actA and 71.4% carried qacH, which confers resistance to quaternary ammonium compounds which are frequently used in sanitizers. Whole genome sequencing of the isolates allowed multi-locus sequence typing to be undertaken. The data indicated that the sequence types identified included those with disease-causing ability, highlighting the disease-causing potential of the isolates.
Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an... more Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence‐associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E‐cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence‐associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST‐OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST‐OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs‐associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs‐induced cleavage of E‐cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence‐associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.
The current trend in reducing the antibiotic usage in animal production imposes urgency in the id... more The current trend in reducing the antibiotic usage in animal production imposes urgency in the identification of novel biocides. The essential oil carvacrol, for example, changes the morphology of the cell and acts against a variety of targets within the bacterial membranes and cytoplasm, and our in vitro results show that it reduces adhesion and invasion of chicken intestinal primary cells and also biofilm formation. A trial was conducted to evaluate the effects of dietary supplementation of carvacrol at four concentrations (0, 120, 200, and 300 mg/kg of diet) on the performance of Lactobacillus spp., Escherichia coli, Campylobacter spp., and broilers. Each of the four diets was fed to three replicates/trial of 50 chicks each from day 0 to 35. Our results show that carvacrol linearly decreased feed intake, feed conversion rates and increased body weight at all levels of supplementation. Plate count analysis showed that Campylobacter spp. was only detected at 35 days in the treatment groups compared with the control group where the colonization occurred at 21 days. The absence of Campylobacter spp. at 21 days in the treatment groups was associated with a significant increase in the relative abundance of Lactobacillus spp. Also, carvacrol was demonstrated to have a significant effect on E. coli numbers in the cecum of the treatment groups, at all supplementation levels. In conclusion, this study shows for the first time that at different concentrations, carvacrol can delay Campylobacter spp., colonization of chicken broilers, by inducing changes in gut microflora, and it demonstrates promise as an alternative to the use of antibiotics.
Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed w... more Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world. The reference and original sequenced strain C. jejuni NCTC11168 has low levels of motility compared to clinical isolates. Here, we describe the draft genome of the laboratory derived hypermotile variant named 11168H.
Campylobacter jejuni is the leading cause of bacterial gastroenteritis and is a major public heal... more Campylobacter jejuni is the leading cause of bacterial gastroenteritis and is a major public health concern worldwide. Despite its importance, our understanding of how C. jejuni causes diarrhoea and interacts with its hosts is limited due to the absence of appropriate infection models and established virulence factors found in other enteric pathogens. Additionally, despite its genetic diversity, non-pathogenic C. jejuni strains are unknown. Regardless of these limitations, significant progress has been made in understanding how C. jejuni uses a complex array of factors which aid the bacterium to survive and respond to host defences. This review provides an update on fitness and virulence determinants of this important pathogen and questions our knowledge on these determinants that are often based on inferred genomics knowledge and surrogate infection models.
Coccidiosis, caused by Eimeria parasites, significantly impacts poultry farm economics and animal... more Coccidiosis, caused by Eimeria parasites, significantly impacts poultry farm economics and animal welfare. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and caecal metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial BifidobacteriumBifidobacterium. Using a multi-omics factor analysis, data on caecal microbiota and metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offeringofferingofferingpotential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.
The article presents a processed dataset from amplicon se- quencing of the V4 region of the 16S r... more The article presents a processed dataset from amplicon se- quencing of the V4 region of the 16S rRNA gene to re- cover bacterial and archaeal taxa from the caeca of multi- ple chicken breeds of Pakistan. These include chicken breeds commonly raised at commercial level, Naked Neck, Black Australorp, Rhode Island Red, White Layer, and Broiler. All the breeds were challenged with Newcastle Disease Virus (NDV), with vaccination against the disease also explored. This resulted in samples belonging to four treatment groups as: Control; Vaccinated; Vaccinated and Challenged; and Non-vaccinated and Challenged. These were raised on an antibiotic free diet in a semi-controlled farming setup. 16S rRNA gene amplicon sequencing of caecal DNA from day old and mature chicken samples (22 weeks for Naked Neck, Black Australorp, Rhode Island Red and White Layer; 8 weeks for Broiler) of the four groups was performed. The paired-end reads from all the samples were quality trimmed, error cor- rected, and overlapped, on which unique Operational Taxo- nomic Units (OTUs) were obtained at 99 % similarity. Using predictive modelling, the MetaCyc functional pathways, as well as KEGG orthologs were also recovered. The generated data may be used to explore microbial interactions in gas- trointestinal tract with respect to NDV vaccination and infec- tion, together with increased understanding of chicken health and productivity.
Background Maintaining gut health is a persistent and unresolved challenge in the poultry industr... more Background Maintaining gut health is a persistent and unresolved challenge in the poultry industry. Given the critical role of gut health in chicken performance and welfare, there is a pressing need to identify effective gut health intervention (GHI) strategies to ensure optimal outcomes in poultry farming. In
This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 ... more This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 different groups of chicken raised in different farm setups in Pakistan. The groups comprise of three different breeds (Broilers, White Layers, and Black Australorp) of chicken raised in different farming setups that include antibiotic-free control, commercial (open and controlled shed), and backyard farms. We have recovered 569 Metagenomics-Assembled Genomes (MAGs) with a completeness of ≥50 % and contamination of ≤10 %. For each MAG, functional annotations were obtained that include KEGG modules, carbohydrate active enzymes (CAZymes), peptidases, geochemical cycles, antibiotic resistance genes, stress genes, and virulence genes. Furthermore, two different sets of Single Copy Genes (SCGs) were used to construct the phylogenetic trees. Based on the reconstructed phylogeny, phylogenetic gain of each MAG is calculated to give an account of novelty.
This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating f... more This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating from chicken caeca. The samples originate from broiler chickens, one group was infected with Newcastle Disease Virus (NDV) and one uninfected control group. There were four birds per group. Both groups were raised on commercially available antibiotic free feed under a semi-controlled setup. The binning step of the samples identified 130 MAGs with ≥50 % completion, and ≤10 % contamination. The data presented includes sequences in FASTA format, tables of functional annotation of genes, and data from two different approaches for phylogenetic tree construction using these MAGs. Major geochemical cycles at community level including carbon, sulfur, and nitrogen cycles are also presented.
Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the env... more Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the environment but also in the human body. The use of phages for the diagnosis of melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is emerging as a promising novel approach, but our understanding of conditions under which Burkholderia prophages can be induced remains limited. Here, we first demonstrated the isolation of Burkholderia phages from the hemocultures of melioidosis patients. The B.pseudomallei-positive hemoculture bottles were filtered to remove bacteria, and then phages were isolated and purified by spot and double agar overlay plaque assays. Forty blood samples (hemoculture-confirmed melioidosis) were tested, and phages were found in 30% of the samples. Transmission electron microscopy and genome analysis of the isolated phages, vB_HM387 and vB_HM795, showed that both phages are Myoviruses. These two phages were stable at a pH of 5–7 and temperatures of 25–37°C, suggesting their ability to survive in human blood. The genome sizes of vB_HM387 and vB_HM795 are 36.3 and 44.0 kb, respectively. A phylogenetic analysis indicated that vB_HM387 has homologs, but vB_HM795 is a novel Myovirus, suggesting the heterogeneity of Burkholderia phages in melioidosis patients. The key finding that Burkholderia phages could be isolated from the blood of melioidosis patients highlights the potential application of phage-based assays by detecting phages in blood as a pathogen-derived biomarker of infection.
Coccidiosis, caused by Eimeria parasites, poses significant economic and welfare challenges in po... more Coccidiosis, caused by Eimeria parasites, poses significant economic and welfare challenges in poultry farming. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and host metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial Bifidobacterium. Using a multi-omics factor analysis (MOFA) machine learning model, data on caecal microbiota and host metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offering potential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.
This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating f... more This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating from chicken caeca. The samples originate from broiler chickens, one group was infected with Newcastle Disease Virus (NDV) and one uninfected control group. There were four birds per group. Both groups were raised on commercially available antibiotic free feed under a semi-controlled setup. The binning step of the samples identified 130 MAGs with ≥ 50% completion, and ≤ 10% contamination. The data presented includes sequences in FASTA format, tables of functional annotation of genes, and data from two different approaches for phylogenetic tree construction using these MAGs. Major geochemical cycles at community level including carbon, sulfur, and nitrogen cycles are also presented.
This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 ... more This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 different groups of chicken raised in different farm setups in Pakistan. The groups comprise of three different breeds (Broilers, White Layers, Black Australorp) of chicken raised in different farming setups that include antibiotic-free control, commercial (open and controlled shed), and backyard farms. We acquired 569 Metagenomics-Assembled Genomes (MAGs) with a completion rate of ≥50% and contamination of ≤10%. For each MAG, we have obtained functional annotations that include KEGG modules, carbohydrate active enzymes (CAZymes), peptidases, geochemical cycles, antibiotic resistance, stress, and virulence genes. Furthermore, we have used Single Copy Genes (SCGs) to construct phylogenetic tree utilizing several approaches, and then give an account of novelty of the MAGs using phylogenetic gain.
There was no effect of removing ZnO from piglet diets on the gut microbiome. These preliminary re... more There was no effect of removing ZnO from piglet diets on the gut microbiome. These preliminary results suggest that there is no benefit to extended lactation periods on the gut microbiome of weaning piglets and ZnO can indeed be successfully removed from piglet diets without impacting on the gut microbiome. However, the piglets in the experiments were raised in a challenge-free facility which is not representative of a commercial setting and so there may indeed be a need to find an alternative to ZnO in practice.
In recent years, there has been an unprecedented advancement in in situ analytical approaches tha... more In recent years, there has been an unprecedented advancement in in situ analytical approaches that contribute to the mechanistic understanding of microbial communities by explicitly incorporating ecology and studying their assembly. In this study, we have analyzed the temporal profiles of the healthy broiler cecal microbiome from day 3 to day 35 to recover the stable and varying components of microbial communities. During this period, the broilers were fed three different diets chronologically, and therefore, we have recovered signature microbial species that dominate during each dietary regime. Since broilers were raised in multiple pens, we have also parameterized these as an environmental condition to explore microbial niches and their overlap. All of these analyses were performed in view of different parameters such as body weight (BW-mean), feed intake (FI), feed conversion ratio (FCR), and age (days) to link them to a subset of microbes that these parameters have a bearing upon. We found that gut microbial communities exhibited strong and statistically significant specificity for several environmental variables. Through regression models, genera that positively/negatively correlate with the bird's age were identified. Some shortchain fatty acids (SCFAs)-producing bacteria, including Izemoplasmatales, Gastranaerophilales, and Roseburia, have a positive correlation with age. Certain pathogens, such as Escherichia-Shigella, Sporomusa, Campylobacter, and Enterococcus, negatively correlated with the bird's age, which indicated a high disease risk in the initial days. Moreover, the majority of pathways involved in amino acid biosynthesis were also positively correlated with the bird's age. Some probiotic genera associated with improved performance included Oscillospirales; UCG-010, Shuttleworthia, Bifidobacterium, and Butyricicoccaceae; UCG-009. In general, predicted antimicrobial resistance genes (piARGs) contributed at a stable level, but there was a slight increase in abundance when the diet was changed. To the best of the authors' knowledge, this is one of the first studies looking at the stability, complexity, and ecology of natural broiler microbiota development in a temporal setting.
Poultry is frequently associated with campylobacteriosis in humans, with Campylobacter jejuni bei... more Poultry is frequently associated with campylobacteriosis in humans, with Campylobacter jejuni being the most usual Campylobacter associated with disease in humans. Far-reaching research on Campylobacter was undertaken over the past two decades. This has resulted in interventions being put in place on farms and in processing plants. Despite these interventions, coupled with increased media coverage to educate the consumer on Campylobacter prevalence and campylobacteriosis, human health incidents are still high. Recent research is now shifting toward further understanding of the microorganisms to challenge interventions in place and to look at further and more relevant interventions for the reduction in human incidents. Farm practices play a key role in the control of colonization within poultry houses and among flocks. Prevalence at the farm level can be up to 100% and time of colonization may vary widely between flocks. Considerable research has been performed to understand how farm management and animal health practices can affect colonization on farms. This review will focus on farm practices to date as a baseline for future interventions as the microorganism becomes better understood. Further research is required to understand the chicken microbiome and factors influencing vertical transmission. The persistence of Campylobacter in animal and environmental reservoirs within and around farms requires further investigation to tailor farm practices toward preventing such reservoirs.
Chickens are a key food source for humans yet their microbiome contains bacteria that can be path... more Chickens are a key food source for humans yet their microbiome contains bacteria that can be pathogenic to humans, and indeed potentially to chickens themselves. Campylobacter is present within the chicken gut and is the leading cause of bacterial foodborne gastroenteritis within humans worldwide. Infection can lead to secondary sequelae such as Guillain-Barré syndrome and stunted growth in children from low-resource areas. Despite the global health impact and economic burden of Campylobacter, how and when Campylobacter appears within chickens remains unclear. As such, there has been a motivation to decrease the number of Campylobacter within chickens and thus reduce the risk of infection to humans. The lack of day-to-day microbiome data with replicates, relevant metadata, and a lack of natural infection studies have delayed our understanding of the chicken gut microbiome and Campylobacter. Here, we performed a comprehensive day-to-day microbiome analysis of the chicken cecum from day 3 to 35 (12 replicates each day; n=396) combining metadata such as chicken weight and feed conversion rates to investigate what the driving forces are for the microbial changes within the chicken gut over time, and how this relates to Campylobacter appearance within a natural habitat setting. We found a rapidly increasing microbial diversity up to day 12 with variation observed both in terms of genera and abundance, before a stabilisation of the microbial diversity after day 20. In particular, we identified a shift from competitive to environmental drivers of microbial community from days 12 to 20 creating a window of opportunity whereby Campylobacter appears. Campylobacter was identified at day 16 which was one day after the most substantial changes in metabolic profiles observed. In addition, microbial variation over time is most likely influenced by the diet of the chickens whereby significant shifts in OTU abundances and beta dispersion of samples often corresponded with changes in feed. This study is unique in comparison to the most recent studies as neither sampling was sporadic nor Campylobacter was artificially introduced, thus the experiments were performed in a natural setting. We believe that our findings can be useful for future intervention strategies and can help elucidate the mechanism through which Campylobacter within chickens can be reduced.
Campylobacter jejuni is the leading cause of food-borne bacterial enteritis in humans, with conta... more Campylobacter jejuni is the leading cause of food-borne bacterial enteritis in humans, with contaminated poultry products considered the main source of infection. To survive the food chain, C. jejuni utilizes multiple defense mechanisms that counter oxidative and aerobic stresses. In this study, we phenotypically characterised 63 C. jejuni strains with oxidative stress survival and antimicrobial susceptibility testing to investigate correlations between these two phenotypes against the source of the strains and the presence of the MarR regulators RrpA and RrpB which have a role in regulating the response to oxidative and aerobic stress. C. jejuni strains isolated from meat and neck skin displayed the highest resistance to oxidative stress. In addition, C. jejuni strains that have an rrpA+rrpB− profile exhibit increased resistance to oxidative stress and to antimicrobials. Here we establish a preliminary link between the distribution of RrpA and RrpB and the increased resistance to antimicrobials. This study provides insight into how the genotypic make up of C. jejuni can influence the ability of the bacterium to survive within areas of high oxygen stress, such as the food chain, and subsequently can have a potential negative impact on human health.
Listeriosis is a foodborne disease, with a high mortality rate, that predominantly affects the el... more Listeriosis is a foodborne disease, with a high mortality rate, that predominantly affects the elderly. Under European Union legislation, EC 2073/2005, food business operators are encouraged to undertake sampling to ensure that the food processing environment, and required to ensure that food products, are free of Listeria monocytogenes. To determine the prevalence of L. monocytogenes in smaller food processing facilities in Northern Ireland, 24 companies submitted six processing environment swabs and two food samples every two months for eighteen months (July 2015 to November 2016) for L. monocytogenes examination. The prevalence of L. monocytogenes was 4.6% in food samples, and 6.3% in processing environment swabs. Over the duration of the study, 96 isolates of L. monocytogenes were obtained, one from each positive sample, except for two meat samples that had >100 cfu/g, where two isolates were obtained from each sample. No seasonality in occurrence of L. monocytogenes was seen for food isolates but significantly higher numbers of positive processing environment swabs were found in the warmer months of May, July and September (p = .007). Pulsed Field Gel Electrophoresis (PFGE) analysis revealed the presence of 27 pulsotypes; 9 pulsotypes were shared between different facilities and 9 were persistent. Based on a Combase predictive growth model, 77.5% (n = 130) of the foods tested were predicted to support the growth of L. monocytogenes. All of the isolates carried the pathogenicity genes inlA and actA and 71.4% carried qacH, which confers resistance to quaternary ammonium compounds which are frequently used in sanitizers. Whole genome sequencing of the isolates allowed multi-locus sequence typing to be undertaken. The data indicated that the sequence types identified included those with disease-causing ability, highlighting the disease-causing potential of the isolates.
Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an... more Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence‐associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E‐cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence‐associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST‐OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST‐OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs‐associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs‐induced cleavage of E‐cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence‐associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.
The current trend in reducing the antibiotic usage in animal production imposes urgency in the id... more The current trend in reducing the antibiotic usage in animal production imposes urgency in the identification of novel biocides. The essential oil carvacrol, for example, changes the morphology of the cell and acts against a variety of targets within the bacterial membranes and cytoplasm, and our in vitro results show that it reduces adhesion and invasion of chicken intestinal primary cells and also biofilm formation. A trial was conducted to evaluate the effects of dietary supplementation of carvacrol at four concentrations (0, 120, 200, and 300 mg/kg of diet) on the performance of Lactobacillus spp., Escherichia coli, Campylobacter spp., and broilers. Each of the four diets was fed to three replicates/trial of 50 chicks each from day 0 to 35. Our results show that carvacrol linearly decreased feed intake, feed conversion rates and increased body weight at all levels of supplementation. Plate count analysis showed that Campylobacter spp. was only detected at 35 days in the treatment groups compared with the control group where the colonization occurred at 21 days. The absence of Campylobacter spp. at 21 days in the treatment groups was associated with a significant increase in the relative abundance of Lactobacillus spp. Also, carvacrol was demonstrated to have a significant effect on E. coli numbers in the cecum of the treatment groups, at all supplementation levels. In conclusion, this study shows for the first time that at different concentrations, carvacrol can delay Campylobacter spp., colonization of chicken broilers, by inducing changes in gut microflora, and it demonstrates promise as an alternative to the use of antibiotics.
Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed w... more Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world. The reference and original sequenced strain C. jejuni NCTC11168 has low levels of motility compared to clinical isolates. Here, we describe the draft genome of the laboratory derived hypermotile variant named 11168H.
Campylobacter jejuni is the major cause of human bacterial gastroenteritis. As a microaerophilic ... more Campylobacter jejuni is the major cause of human bacterial gastroenteritis. As a microaerophilic bacterium, C. jejuni will be exposed to reactive oxygen species (ROS) during the course of normal bacterial metabolism as well as during colonisation or infection, from the host immune system. C. jejuni contains a number of different mechanisms for countering the effects of oxidative stress and the control of the C. jejuni oxidative stress response is complex involving multiple inter-linked levels of regulation, with two new regulators of the oxidative stress response recently identified. In this chapter, we cover both the mechanisms of C. jejuni oxidative stress defence and the current understanding of the increasingly complex regulation of this oxidative stress response.
The human pathogen Campylobacter jejuni is part of the genus Campylobacter that lies within the e... more The human pathogen Campylobacter jejuni is part of the genus Campylobacter that lies within the epsilon proteobacteria subclass of bacteria. The nearest family in phylogenetic terms is the Helicobacteraceae which includes the Helicobacter and Wolinella genuses. Campylobacter species are Gram-negative, curved rod shaped or spiral and are motile (via polar flagella).
Cryptosporidium is an enteric protozoan parasite that causes gastrointestinal disorders in humans... more Cryptosporidium is an enteric protozoan parasite that causes gastrointestinal disorders in humans and in a wide range of animals, mainly in calves. As there is no available efficient treatment for cryptosporidiosis, in this study we evaluated the effect of Auranta 3001, a natural feed additive on animal growth, number of days with liquid diarrhoea and oocyst excretion, mean oocysts/gram faeces and on biochemical and physical parameters. The study showed that calves fed with Auranta 3001 as a feed additive administered prior to infection with C. parvum, significantly reduced (P < 0.05) the number of days with liquid diarrhoea, the number of days with oocyst excretion, the number of days of antibiotic administration and mean oocysts/gram faeces. Moreover, the prophylactic administration of Auranta 3001, significantly (P < 0.05) reduced the percentage of calves with fever and increased the body weight at day 56. However, significant differences were not seen between IgG, total protein intake and haematocrit percentage. This study showed the efficacy of Auranta 3001 in reducing cryptosporidiosis manifestations in calves.
Antimicrobial agents have been in use for therapeutic purposes for over a century, with most of t... more Antimicrobial agents have been in use for therapeutic purposes for over a century, with most of the development occurring in the latter half of the twentieth century. Penicillin was the first of the naturally occurring antimicrobials to be used in medicine and its structure includes a beta-lactam ring. Further compounds, such as the cephalosporins, were discovered and these also included beta-lactam structures. Subsequently bacteria which were resistant to these compounds were found, and their resistance was due to their production of enzymes, beta-lactamases, which hydrolysed the beta-lactam ring. Synthetic derivatives of the beta-lactam antimicrobials were developed to render them recalcitrant to beta-lactamases but enzymes with a broader substrate range evolved, and were categorised as extended substrate beta-lactamases (ESBL). Since the antimicrobials had a significant role to play in human medicine the emergence of ESBL caused significant concerns. Further, similar antimicrobials were used by veterinarians, raising the prospect that bacteria in the commensal flora of livestock could acquire ESBL resistance properties and exchange them via genetic exchange. Thus, pathogenic bacteria present in livestock could become resistant to antimicrobials with adverse consequences should zoonotic infections occur. In this review we consider the emergence of ESBL, the problems involved in detecting and reporting such properties, and consider the consequences for consumers of potentially contaminated food products.
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Papers by Ozan Gundogdu
concern worldwide. Despite its importance, our understanding of how C. jejuni causes diarrhoea
and interacts with its hosts is limited due to the absence of appropriate infection models and
established virulence factors found in other enteric pathogens. Additionally, despite its genetic
diversity, non-pathogenic C. jejuni strains are unknown. Regardless of these limitations, significant
progress has been made in understanding how C. jejuni uses a complex array of factors which aid
the bacterium to survive and respond to host defences. This review provides an update on fitness
and virulence determinants of this important pathogen and questions our knowledge on these
determinants that are often based on inferred genomics knowledge and surrogate infection
models.
concern worldwide. Despite its importance, our understanding of how C. jejuni causes diarrhoea
and interacts with its hosts is limited due to the absence of appropriate infection models and
established virulence factors found in other enteric pathogens. Additionally, despite its genetic
diversity, non-pathogenic C. jejuni strains are unknown. Regardless of these limitations, significant
progress has been made in understanding how C. jejuni uses a complex array of factors which aid
the bacterium to survive and respond to host defences. This review provides an update on fitness
and virulence determinants of this important pathogen and questions our knowledge on these
determinants that are often based on inferred genomics knowledge and surrogate infection
models.
https://www.cabidigitallibrary.org/doi/abs/10.1079/9781800622746.0007