Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

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).

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.

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

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

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

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

The Gut Microbiota of Laying Hens and Its Manipulation with Prebiotics and Probiotics To Enhance Gut Health and Food Safety.

The microbiota plays a vital role in maintaining gut health and influences the overall performance of chickens. Most gut microbiota-related studies have been performed in broilers, which have different microbial communities compared to those of layers. The normal gut microbiota of laying chickens is dominated by Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria at the phylum level. The composition of the gut microbiota changes with chicken age, genotype, and production system. The metabolites of gut microbiota, such as short-chain fatty acids, indole, tryptamine, vitamins, and bacteriocins, are involved in host-microbiota cross talk, maintenance of barrier function, and immune homeostasis. Resident gut microbiota members also limit and control the colonization of foodborne pathogens. In-feed supplementations of prebiotics and probiotics strengthen the gut microbiota for improved host performance and colonization resistance to gut pathogens, such as Salmonella and Campylobacter The mechanisms of action of prebiotics and probiotics come through the production of organic acids, activation of the host immune system, and production of antimicrobial agents. Probiotic candidates, including Lactobacillus, Bifidobacterium, Bacillus, Saccharomyces, and Faecalibacterium isolates, have shown promising results toward enhancing food safety and gut health. Additionally, a range of complex carbohydrates, including mannose oligosaccharides, fructo-oligosaccharides, and galacto-oligosaccharides, and inulin are promising candidates for improving gut health. Here, we review the potential roles of prebiotics and probiotics in the reshaping of the gut microbiota of layer chickens to enhance gut health and food safety.

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

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

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

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

Phytogenic products, used as alternatives to antibiotic growth promoters, modify the intestinal microbiota derived from a range of production systems: an in vitro model.

The removal of antibiotics from the feeds used in the livestock industry has resulted in the use of a wide range of alternative antimicrobial products that aim to deliver the productivity and health benefits that have traditionally been associated with antibiotics. Amongst the most popular alternatives are phytogenic product-based extracts from herbs and spices with known antimicrobial properties. Despite embracing such alternatives, the industry is still largely unaware of modes of action, their overall effects on animal health, and interactions with other feed additives such as probiotics. To address some of these issues, three phytogenic products were selected and their interactions with caecal microbiota of layers, grown under six different production systems, were investigated in vitro. Caecal microbiotas were grown with and without phytogenic products, and the changes in microbiota composition were monitored by sequencing of 16S rRNA gene amplicons. Phytogenic products and production system both significantly influenced microbiota composition. The three phytogenic products all altered the relative abundance of species within the Lactobacillus genus, by promoting the growth of some and inhibiting other Lactobacillus species. There were also significant alterations in the Bacillus genus. This was further investigated by comparing the effects of the phytogenic products on the growth of a commercially used Bacillus-based probiotic. The phytogens affected the probiotic mix differently, with some promoting the growth of Bacillus sp. at lower phytogenic concentrations, and fully suppressing growth at higher concentrations, indicating the importance of finding an optimal concentration that can control pathogens while promoting beneficial bacteria. KEY POINTS: • After removal of antibiotics from animal feed, urgent solutions for pathogen control were needed. • Alternative products entered the market without much knowledge on their effects on animal health. • Probiotic products are used in combination with phytogens despite the possible incompatibility.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Spotlight on avian pathology: Campylobacter hepaticus, the cause of Spotty Liver Disease in layers.

Campylobacter hepaticus was recently identified as the aetiological agent of Spotty Liver Disease (SLD). SLD causes significant health and productivity losses in the Australian egg industry and the disease is present in other countries. Following the isolation and characterization of C. hepaticus, molecular tools and refined culturing methods have been developed to identify the pathogen. It is suspected that the application of these tools will lead to identification of the pathogen in many poultry production systems throughout the world. As C. hepaticus has only recently been identified, little is known about the mechanisms of pathogenesis and, hence, new research needs to be directed towards understanding SLD epidemiology and C. hepaticus virulence mechanisms to inform efforts to develop intervention strategies.

Characterisation of the intestinal microbiota of commercially farmed saltwater crocodiles, Crocodylus porosus.

The Australian saltwater crocodile (Crocodylus porosus) industry began commercially in the 1980s, producing skins for export and crocodile meat as a by-product. Industry research has thus far focused on strategies to improve production efficiency. In the current study, we utilised 16S rRNA sequencing to characterise the intestinal microbiome of Australian saltwater crocodiles. Samples were collected from 13 commercially farmed crocodiles from six sample sites along the length of the intestinal tract. The results indicate a similar microbiome composition to that found in the freshwater alligator, with the dominate phyla represented by Firmicutes, primarily Clostridia, and Fusobacteria, which appears to be distinct from mammalian, fish, and other reptile phyla which are generally dominated by Firmicutes and Bacteroidetes. The high abundance of 'pathogenic' bacteria, with no apparent consequence to the host's health, is of great interest and warrants further additional investigation. This will enable expansion of the current understanding of host immune function and how it is modified by host and intestinal microbiome interactions.

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

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

Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus (TMEV) is one of the most common viral pathogens that circulate widely in captive mouse colonies. A molecular biology detection method would be a useful tool to use in an integrated program to monitor and prevent TMEV infection and transmission. Thus, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect TMEV infection. The sensitivity of the RT-RPA assay approached 8 copies per reaction, which is equivalent to the sensitivity of RT-qPCR reactions. This assay did not detect RNA extracts from other murine pathogens included in this study or TMEV negative samples. Brain tissues and contaminated biological materials were used to assess the clinical performance of the RT-RPA. The detection results of RT-RPA and RT-qPCR were very similar, except that a contaminated biological material sample which was positive by RT-qPCR, with a CT value of 38, was negative by RT-RPA. In summary, the developed RT-RPA assay offers a rapid, sensitive and specific alternative method for monitoring of TMEV, especially in resource-limited conditions.

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

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

Correlations between intestinal innate immune genes and cecal microbiota highlight potential for probiotic development for immune modulation in poultry.

Immune function is influenced by the diversity and stability of the intestinal microbiota. A likely trade-off of immune function for growth has been demonstrated in heavier breeds of poultry that have been genetically selected for growth and feed efficiency traits. We investigated the expression of selected innate immune genes and genes encoding products involved in intestinal barrier function to determine whether function changes could be consistently linked to the phenotypic expression of feed conversion ratio (FCR), a common measure of performance within poultry broiler flocks. In addition, we compared individual cecal microbial composition with innate immune gene expression. Samples were utilised from two replicate trials termed P1E1 and P1E2. High (n = 12) and low (n = 12) performing birds were selected based on their individual FCR data from each replicate and combined for microbiota phylogenetic composition and immune gene expression analysis. Toll-like receptor 1 (TLR1La) and zonula occludens 1 (ZO1) were differentially expressed between high- and low-performing broilers. Several taxa were correlated with FCR; of these, unclassified YS2 and ZO1 were also positively correlated with each other. Interactions between taxa and differentially expressed innate immune genes between P1E1 and P1E2 were much greater compared to relationships between high- and low-performing birds. At the level of phylum, reciprocal correlations between tight junction proteins and Toll-like receptors with Bacteroidetes and Firmicutes were evident, as were correlations at the genus level.

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

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

Expansion of the Clostridium perfringens toxin-based typing scheme.

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, ß-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not ß-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.