The effects of a high-flavonoid corn cultivar on the gastrointestinal tract microbiota in chickens undergoing necrotic enteritis.

The search for alternative therapies to antimicrobial growth promoters (AGP) in poultry production has gained momentum in the past years because of consumer preference and government restrictions on the use of AGP in animal production. Flavonoids are plant-derived metabolites that have been studied for their health-promoting properties that could potentially be used as an alternative to AGP in poultry. In a previous study, we showed that the inclusion of a flavonoid-rich corn cultivar (PennHFD1) in the diet improved the health of broilers undergoing necrotic enteritis. However, the mechanisms of action by which the PennHFD1-based diet ameliorated necrotic enteritis are unknown. This study describes the microbial diversity and composition of the jejunum and ileum of chickens co-infected with Eimeria maxima and Clostridium perfringens and treated with a high-flavonoid corn-based diet. Luminal content and mucosal samples from the jejunum and ileum were collected for DNA extraction, 16S rRNA amplicon sequencing and data analyses. The infection model and the dietary treatments significantly changed the alfa diversity indices (Mucosal samples: ASVs, P = 0.04; Luminal content samples: ASVs, P = 0.03), and beta diversities (Mucosal samples: P < 0.01, Luminal content: P < 0.01) of the ileal samples but not those of the jejunal samples. The microbial composition revealed that birds fed the high-flavonoid corn diet had a lower relative abundance of C. perfringens compared to birds fed the commercial corn diet. The treatments also changed the relative abundance of other bacteria that are related to gut health, such as Lactobacillus. We concluded that both the infection model and the dietary high-flavonoid corn changed the broilers' gut microbial diversity and composition. In addition, the decrease in the relative abundance of C. perfringens corroborates with a decrease in mortality and intestinal lesions due to necrotic enteritis. Collecting different segments and sample types provided a broader understanding of the changes in the gut microbiota among treatments.

The Barrier Disruption and Pyroptosis of Intestinal Epithelial Cells Caused by Perfringolysin O (PFO) from Clostridium perfringens.

Clostridium perfringens (C. perfringens), a Gram-positive bacterium, produces a variety of toxins and extracellular enzymes that can lead to disease in both humans and animals. Common symptoms include abdominal swelling, diarrhea, and intestinal inflammation. Severe cases can result in complications like intestinal hemorrhage, edema, and even death. The primary toxins contributing to morbidity in C. perfringens-infected intestines are CPA, CPB, CPB2, CPE, and PFO. Amongst these, CPB, CPB2, and CPE are implicated in apoptosis development, while CPA is associated with cell death, increased intracellular ROS levels, and the release of the inflammatory factor IL-18. However, the exact mechanism by which PFO toxins exert their effects in the infected gut is still unidentified. This study demonstrates that a C. perfringens PFO toxin infection disrupts the intestinal epithelial barrier function through in vitro and in vivo models. This study emphasizes the notable influence of PFO toxins on intestinal barrier integrity in the context of C. perfringens infections. It reveals that PFO toxins increase ROS production by causing mitochondrial damage, triggering pyroptosis in IPEC-J2 cells, and consequently resulting in compromised intestinal barrier function. These results offer a scientific foundation for developing preventive and therapeutic approaches against C. perfringens infections.

Absent in Melanoma 2 Mediates Inflammasome Signaling Activation against Clostridium perfringens Infection.

Absent in melanoma 2 (AIM2), a key component of the IFI20X/IFI16 (PYHIN) protein family, is characterized as a DNA sensor to detect cytosolic bacteria and DNA viruses. However, little is known about its immunological role during pathogenic Clostridium perfringens (C. perfringens) infection, an extracellular bacterial pathogen. In a pathogenic C. perfringens gas gangrene model, Aim2-/- mice are more susceptible to pathogenic C. perfringens soft tissue infection, revealing the importance of AIM2 in host protection. Notably, Aim2 deficiency leads to a defect in bacterial killing and clearance. Our in vivo and in vitro findings further establish that inflammasome signaling is impaired in the absence of Aim2 in response to pathogenic C. perfringens. Mechanistically, inflammasome signaling downstream of active AIM2 promotes pathogen control. Importantly, pathogenic C. perfringens-derived genomic DNA triggers inflammasome signaling activation in an AIM2-dependent manner. Thus, these observations uncover a central role for AIM2 in host defense and triggering innate immunity to combat pathogenic C. perfringens infections.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors.

Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

Mechanisms of intestinal epithelial cell damage by Clostridiumperfringens.

Clostridium perfringens, a Gram-positive bacterium, causes intestinal diseases in humans and livestock through its toxins, related to alpha toxin (CPA), beta toxin (CPB), C. perfringens enterotoxin (CPE), epsilon toxin (ETX), Iota toxin (ITX), and necrotic enteritis B-like toxin (NetB). These toxins disrupt intestinal barrier, leading to various cell death mechanisms such as necrosis, apoptosis, and necroptosis. Additionally, non-toxin factors like adhesins and degradative enzymes contribute to virulence by enhancing colonization and survival of C. perfringens. A vicious cycle of intestinal barrier breach, misregulated cell death, and subsequent inflammation is at the heart of chronic inflammatory and infectious gastrointestinal diseases. Understanding these mechanisms is essential for developing targeted therapies against C. perfringens-associated intestinal diseases.

Toxigenic Clostridium perfringens Isolated from At-Risk Paediatric Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

[Study on biofilm formation and heterogeneity in Clostridium perfringens].

Many bacteria form biofilms and survive in the actual environment. Biofilms are not only a major form of bacteria but are also involved in tolerance to environmental stresses and antibiotics, suggesting the association with bacterial pathogenesis. Cells within biofilms display phenotypic heterogeneity; thus, even bacteria, unicellular organisms, can functionally differentiate and show multicellular behavior. Therefore, it is necessary to understand bacteria as a population to control their survival and pathogenesis in the actual environment. Previously, we found that Clostridium perfringens, an anaerobic pathogenic bacterium, form different structures in different temperatures and phenotypic heterogeneity on biofilm matrix gene expression within the biofilm. In this article, I summarize the results of our research on biofilms and their heterogeneity, the mechanisms of post-transcriptional gene expression regulation of virulence genes, and bacteria-host interactions mediated by extracellular membrane vesicles.

NanI sialidase contributes to toxin expression and host cell binding of Clostridium perfringens type G strain CP56 in vitro.

Necrotic enteritis, caused by NetB producing Clostridium perfringens type G strains, is a globally important poultry disease. An initial step in the pathogenesis of necrotic enteritis is the colonization and degradation of the intestinal mucus layer, a process in which C. perfringens sialidases - such as NanI sialidase - may play an important role. Sialidases cleave terminal sialic acid from complex carbohydrates on glycoconjugates, such as mucins. This study shows that NE-associated C. perfringens strain CP56 is able to use sialic acid (Neu5Ac) as a carbon source for bacterial growth. It is shown that supplementation of Neu5Ac in the growth medium does not only induce the production of extracellular sialidases of strain CP56, but also increases the production of both alpha toxin and NetB toxin. Moreover, it was found that pre-treating avian hepatocellular carcinoma cells (LMH cells) with the recombinant NanI sialidase increases the adherence of C. perfringens type G strain CP56 to these cells. As such, the data suggest an important role for sialidases in the pathogenesis of the disease.

Supplemental Bacillus subtilis PB6 Improves Growth Performance and Gut Health in Broilers Challenged with Clostridium perfringens.

Clostridium perfringens (CP) is the principal pathogenic bacterium of chicken necrotic enteritis (NE), which causes substantial economic losses in poultry worldwide. Although probiotics are known to provide multiple benefits, little is known about the potential effects of Bacillus subtilis (B. subtilis) application in preventing CP-induced necrotic enteritis. In this study, 450 male Arbor Acres broilers were divided into 5 experimental treatments: A: basal diet (control group); B: basal diet and CP challenge (model group); C: CP challenge+10 mg/kg enramycin (positive control group); D: CP challenge+4 × 107 CFU/kg of feed B. subtilis PB6 (PB6 low-dosage group); and E: CP challenge+6 × 107 CFU/kg of feed B. subtilis PB6 (PB6 high-dosage group). There were 6 replicate pens per treatment with 15 broilers per pen. The present research examined the effect of Bacillus subtilis PB6 (B. subtilis PB6) on growth performance, mRNA expression of intestinal cytokines and tight junctions, and gut flora composition in broilers challenged with CP. The entire experiment was divided into two phases: the non-CP challenge phase (d0-18) and the CP challenge phase (d18-26). PB6 did not increase the growth performance during the first stage, but the PB6 high-dosage group was found to have larger body weight gain and ADFI during the CP challenge stage. Feed supplementation with PB6 reduced the lesion score of challenged chicks, with increased tight junction-related gene expression (occludin and ZO-1) and decreased TNF-α expression compared with CP-infected birds. A decrease in the abundance of Clostridium XI, Streptococcus, and Staphylococcus was observed after CP infection (P < 0.05), while supplementation with PB6 restored the ileal microbial composition. In conclusion, administration of B. subtilis PB6 improved growth performance, enhanced intestinal barrier function, and mitigated intestinal inflammation/lesions, which might be due to its restoring effects on the ileal microbial composition in CP-challenged broilers.

Bacterial phospholipases C with dual activity: phosphatidylcholinesterase and sphingomyelinase.

Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.

A Novel PilR/PilS Two-Component System Regulates Necrotic Enteritis Pilus Production in Clostridium perfringens.

Clostridium perfringens causes necrotic enteritis (NE) in poultry. A chromosomal locus (VR-10B) was previously identified in NE-causing C. perfringens strains that encodes an adhesive pilus (NE pilus), along with a two-component system (TCS) designated here as PilRS. While the NE pilus is important in pathogenesis, the role of PilRS remains to be determined. The current study investigated the function of PilRS, as well as the Agr-like quorum-sensing (QS) system and VirSR TCS in the regulation of pilin production. Isogenic pilR, agrB, and virR null mutants were generated from the parent strain CP1 by insertional inactivation using the ClosTron system, along with the respective complemented strains. Immunoblotting analyses showed no detectable pilus production in the CP1pilR mutant, while production in its complement (CP1pilR+) was greater than wild-type levels. In contrast, pilus production in the agrB and virR mutants was comparable or higher than the wild type but reduced in their respective complemented strains. When examined for collagen-binding activity, the pilR mutant showed significantly lower binding to most collagen types (types I to V) than parental CP1 (P ≤ 0.05), whereas this activity was restored in the complemented strain (P > 0.05). In contrast, binding of agrB and virR mutants to collagen showed no significant differences in collagen-binding activity compared to CP1 (P > 0.05), whereas the complemented strains exhibited significantly reduced binding (P ≤ 0.05). These data suggest the PilRS TCS positively regulates pilus production in C. perfringens, while the Agr-like QS system may serve as a negative regulator of this operon. IMPORTANCE Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry, presenting a major challenge for poultry production in the postantibiotic era. Multiple factors in C. perfringens, including both virulent and nonvirulent, are involved in the development of the disease. We previously discovered a cluster of C. perfringens genes that encode a pilus involved in adherence and NE development, along with a predicted two-component regulatory system (TCS), designated PilRS. In the present study, we have demonstrated the role of PilRS in regulating pilus production and collagen binding of C. perfringens. In addition, the Agr-like quorum sensing signaling pathway was found to be involved in the regulation. These findings have identified additional targets for developing nonantibiotic strategies to control NE disease.

NanH Is Produced by Sporulating Cultures of Clostridium perfringens Type F Food Poisoning Strains and Enhances the Cytotoxicity of C. perfringens Enterotoxin.

Clostridium perfringens type F food poisoning (FP) strains cause one of the most common foodborne illnesses. This FP develops when type F FP strains sporulate in the intestines and produce C. perfringens enterotoxin (CPE), which is responsible for the diarrhea and abdominal cramps of this disease. While C. perfringens can produce up to three different sialidases, the current study surveyed FP strains, which confirmed the results of a previous study that they consistently carry the nanH sialidase gene, often as their only sialidase gene. NanH production was found to be associated with sporulating cultures of the surveyed type F FP strains, including SM101 (a transformable derivative of a FP strain). The sporulation-associated regulation of NanH production by strain SM101 growing in modified Duncan-Strong medium (MDS) was shown to involve Spo0A, but it did not require the completion of sporulation. NanH production was not necessary for either the growth or sporulation of SM101 when cultured in MDS. In those MDS cultures, NanH accumulated in the sporulating mother cell until it was released coincidently with CPE. Since CPE becomes extracellular when mother cells lyse to release their mature spores, this indicates that mother cell lysis is also important for NanH release. The copresence of NanH and CPE in supernatants from lysed sporulating cultures was shown to enhance CPE cytotoxicity for Caco-2 cells. This enhancement was attributable to NanH increasing CPE binding and could be replicated with purified recombinant NanH. These in vitro findings suggest that NanH may be an accessory virulence factor during type F FP.IMPORTANCEClostridium perfringens type F strains cause the second most common bacterial foodborne illness in the United States. C. perfringens enterotoxin (CPE) is responsible for the diarrhea and cramping symptoms of this food poisoning (FP). Previous studies showed that NanI sialidase can enhance CPE activity in vitro While many type F FP strains do not produce NanI, they do consistently make NanH sialidase. This study shows that, like CPE, NanH is produced by sporulating type F FP strains and then released extracellularly when their sporulating cells lyse to release their mature spore. NanH was shown to enhance CPE cytotoxicity in vitro by increasing CPE binding to cultured Caco-2 cells. This enhancement could be important because many type F FP strains produce less CPE than necessary (in a purified form) to cause intestinal pathology in animal models. Therefore, NanH represents a potential accessory virulence factor for type F FP.

Comparative in silico genome analysis of Clostridium perfringens unravels stable phylogroups with different genome characteristics and pathogenic potential.

Clostridium perfringens causes a plethora of devastating infections, with toxin production being the underlying mechanism of pathogenicity in various hosts. Genomic analyses of 206 public-available C. perfringens strains´ sequence data identified a substantial degree of genomic variability in respect to episome content, chromosome size and mobile elements. However, the position and order of the local collinear blocks on the chromosome showed a considerable degree of preservation. The strains were divided into five stable phylogroups (I-V). Phylogroup I contained human food poisoning strains with chromosomal enterotoxin (cpe) and a Darmbrand strain characterized by a high frequency of mobile elements, a relatively small genome size and a marked loss of chromosomal genes, including loss of genes encoding virulence traits. These features might correspond to the adaptation of these strains to a particular habitat, causing human foodborne illnesses. This contrasts strains that belong to phylogroup II where the genome size points to the acquisition of genetic material. Most strains of phylogroup II have been isolated from enteric lesions in horses and dogs. Phylogroups III, IV and V are heterogeneous groups containing a variety of different strains, with phylogroup III being the most abundant (65.5%). In conclusion, C. perfringens displays five stable phylogroups reflecting different disease involvements, prompting further studies on the evolution of this highly important pathogen.

Transversal gene expression panel to evaluate intestinal health in broiler chickens in different challenging conditions.

There is a high interest on gut health in poultry with special focus on consequences of the intestinal diseases, such as coccidiosis and C. perfringens-induced necrotic enteritis (NE). We developed a custom gene expression panel, which could provide a snapshot of gene expression variation under challenging conditions. Ileum gene expression studies were performed through high throughput reverse transcription quantitative real-time polymerase chain reaction. A deep review on the bibliography was done and genes related to intestinal health were selected for barrier function, immune response, oxidation, digestive hormones, nutrient transport, and metabolism. The panel was firstly tested by using a nutritional/Clostridium perfringens model of intestinal barrier failure (induced using commercial reused litter and wheat-based diets without exogenous supplementation of enzymes) and the consistency of results was evaluated by another experiment under a coccidiosis challenge (orally gavaged with a commercial coccidiosis vaccine, 90× vaccine dose). Growth traits and intestinal morphological analysis were performed to check the gut barrier failure occurrence. Results of ileum gene expression showed a higher expression in genes involved in barrier function and nutrient transport in chickens raised in healthy conditions, while genes involved in immune response presented higher expression in C.perfringens-challenged birds. On the other hand, the Eimeria challenge also altered the expression of genes related to barrier function and metabolism, and increased the expression of genes related to immune response and oxidative stress. The panel developed in the current study gives us an overview of genes and pathways involved in broiler response to pathogen challenge. It also allows us to deep into the study of differences in gene expression pattern and magnitude of responses under either a coccidial vaccine or a NE.

Production and purification of Clostridium perfringens type D epsilon toxin and IgY antitoxin.

The aim of this study was to purify Clostridium perfringens type D epsilon toxin and produce and purify anti-epsilon chicken immunoglobulin Y (IgY). A single-step ion exchange chromatography resulted in a high-yield and high-purity toxin, while ion exchange chromatography followed by gel filtration resulted in the highest purity of the toxin, but at a lower yield. Purified and inactivated epsilon toxin were then administered in chickens via four inoculations and IgY was obtained at a high purity and yield, with an antibody titer of 50 IU/mL and high levels of avidity (73.2%). In summary, C. perfringens type D epsilon toxin and chicken anti-epsilon IgY were successfully produced and purified, and may be used for the diagnosis of enterotoxemia caused by the epsilon toxin, as well as in potency tests of existing and future vaccines against enterotoxemia.

Differential expression of intestinal genes in necrotic enteritis challenged broiler chickens with 2 different Clostridium perfringens strains.

The primary cause of necrotic enteritis (NE) disease in chickens is the NetB-positive Clostridium perfringens bacterium. Many factors are known to affect the severity of NE in the challenge models of broiler chickens, and one of these factors is the virulence of C. perfringens strain. This study was conducted to evaluate the effect of 2 pathogenic C. perfringens strains in a NE challenge model on gut health and mRNA expression of genes encoding apoptosis, tight junction, immunity, and nutrient transporters in broilers. Day-old Ross-308 male broilers (n = 468) were allocated in a 2 × 3 factorial arrangement of treatments with in-feed antibiotics (no or yes) and challenge (Non, C. perfringens strain NE18, and C. perfringens strain NE36) as the factors. The birds in the challenged groups were inoculated with Eimeria species on day 9 and with a fresh suspension of C. perfringens NE18 or NE36 on day 14 and 15. Sample collection was performed on 2 birds of each pen on day 16. Necrotic enteritis challenge, impaired feed conversion ratio during day 0 to 16 compared with the control group where the effect of the NE36 challenge was more severe than that with NE18 (P < 0.001). The mRNA expression of mucin-2, immunoglobulin-G, occludin (P < 0.001), and tight junction protein-1 (P < 0.05) genes were downregulated in both challenged groups compared with the nonchallenged counterparts. Antibiotic supplementation, on the other hand, increased weight gain, and feed intake in all challenged birds (P < 0.01), but upregulated mucin-5ac and alanine, serine, cysteine, and threonine transporter-1 (P < 0.05) only in the NE18 challenged birds. The challenge with NE36 significantly upregulated caspase-8 and claudin-1 (P < 0.001), but downregulated glucose transporter-2 (P < 0.001) compared with the NE18 challenge. These results suggest that NE challenge is detrimental to the performance of broilers through compromised intestinal health, and different C. perfringens strains can affect the severity of the disease through modulating the expression of intestinal genes encoding proteins responsible for apoptosis, gut integrity, immunity, mucus production, and nutrient transporters.

Clostridium perfringens Produces an Adhesive Pilus Required for the Pathogenesis of Necrotic Enteritis in Poultry.

Clostridium perfringens type G strains cause necrotic enteritis (NE) in poultry, an economically important disease that is a major target of in-feed antibiotics. NE is a multifactorial disease, involving not only the critically important NetB toxin but also additional virulence and virulence-associated factors. We previously identified a C. perfringens chromosomal locus (VR-10B) associated with disease-causing strains that is predicted to encode a sortase-dependent pilus. In the current study, we sought to provide direct evidence for the production of a pilus by C. perfringens and establish its role in NE pathogenesis. Pilus structures in virulent C. perfringens strain CP1 were visualized by transmission electron microscopy (TEM) of immunogold-labeled cells. Filamentous structures were observed extending from the cell surface in wild-type CP1 but not from isogenic pilin-null mutant strains. In addition, immunoblotting of cell surface proteins demonstrated that CP1, but not the null mutant strains, produced a high molecular weight ladder-like pattern characteristic of a pilus polymer. Binding to collagen types I, II, and IV was significantly reduced (Tukey's test, P < 0.01) in all three pilin mutants compared to CP1 and could be specifically blocked by CnaA and FimA antisera, indicating that these pilins participate in adherence. Furthermore, fimA and fimB null mutants were both severely attenuated in their ability to cause disease in an in vivo chicken NE challenge model. Together, these results provide the first direct evidence for the production of a sortase-dependent pilus by C. perfringens and confirm its critical role in NE pathogenesis and collagen binding.IMPORTANCE In necrotic enteritis (NE), an intestinal disease of chickens, Clostridium perfringens cells adhere tightly to damaged intestinal tissue, but the factors involved are not known. We previously discovered a cluster of C. perfringens genes predicted to encode a pilus, a hair-like bacterial surface structure commonly involved in adherence. In the current study, we have directly imaged this pilus using transmission electron microscopy (TEM). We also show that inactivation of the pilus genes stops pilus production, significantly reducing the bacterium's ability to bind collagen and cause disease. Importantly, this is the first direct evidence for the production of a sortase-dependent pilus by C. perfringens, revealing a promising new target for developing therapeutics to combat this economically important disease.

A case of Crimean-Congo hemorrhagic fever with the bacteremia of Clostridium perfringens.

Crimean-Congo hemorrhagic fever (CCHF) is a worldwide tick-borne viral infection in humans. The aim of the study is to report a case of a female patient with severe CCHF with the bacteremia of Clostridium perfringens. An 18-year-old woman admitted to the emergency department with sudden onset of fever, nausea and vomiting, myalgia, headache, generalized abdominal pain. It was learned that the patient was living in a rural area and had a history of tick bite 3 days before the admission. At laboratory examination, bicytopenia, abnormal liver function tests, and abnormal coagulation parameters were observed. The diagnosis of the case was confirmed with a positive real-time polymerase chain reaction. On the third day of hospitalization, she had an increase in abdominal pain, confusion, and respiratory distress. She was transferred to the intensive care unit for close monitoring. On the fifth day of hospitalization, she developed fever again. Catheter and peripheral anaerobic blood cultures grew C. perfringens. No evidence of perforation was observed on abdominal tomography. It has been successfully treated with a multidisciplinary approach. CCHF demonstrates different types of clinical presentations, except for common symptoms of fever and hemorrhage. A case of CCHF with C. perfringens bacteremia has not been previously reported before.

The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin.

The necrotic enteritis toxin B-like (NetB) toxin secreted by Clostridium perfringens is a key virulence agent in the pathogenesis of avian necrotic enteritis, a disease that causes significant economic loss to the poultry industry worldwide. NetB was purified from Clostridium perfringens type G (CNEOP004) that was isolated from chickens with necrotic enteritis in Japan. EC50 of this purified NetB toward chicken liver-derived LMH cells was 0.63 µg/ml. In vivo pathogenicity of NetB to chicks produced characteristic lesions of necrotic enteritis. Analysis of the localization of the NetB monomer and oligomer molecules on LMH cells showed that both molecules of the toxin were localized in non-lipid raft regions. Moreover, removal of cholesterol with the cholesterol depletion assay carried out in LMH cells detected both oligomers and monomers of the NetB molecule. These data suggest that the NetB toxin may recognize membrane molecules different from cholesterol in non-raft region. Furthermore, NetB-binding molecules on LMH cell membranes using the toxin overlay assay with immunoblotting showed that protein molecules of different molecular sizes were bound to NetB on non-lipid raft fractions. Further studies are necessary to characterize these protein molecules to examine their specific association with NetB binding and oligomerization.