Perfringens Infection Research Articles

Necrotizing soft tissue infection: clinical characteristics, diagnosis, and management of 32 cases in Beijing.

ObjectiveNecrotizing soft tissue infection (NSTI) is a rare but life-threatening bacterial infection. This study was performed to analyze the clinical characteristics of patients with NSTI admitted to the intensive care unit of a trauma and burn center in Beijing and to summarize the treatment experience.MethodsThis retrospective study involved patients with NSTI admitted to the intensive care unit from January 2010 to January 2020. The clinical manifestations, pathogens, laboratory test results, and prognosis were compared between survivors and nonsurvivors.ResultsThirty-two patients were enrolled (28 men, 4 women), including 25 (78.1%) survivors and 7 (21.9%) nonsurvivors. The patients’ median age was 41.5 years (range, 30.0–52.5 years). Mortality was significantly higher in patients with a blood urea nitrogen concentration of ≥11.5 mg/dL, creatinine concentration of <3.4 mg/dL, prothrombin time of ≥15.9 s, and international normalized ratio of ≥1.3. Streptococcus pyogenes and Clostridium perfringens infections were associated with higher mortality.ConclusionsMore attention should be given to elderly patients and those with hyponatremia, coagulation disorder, and higher blood urea nitrogen or creatinine concentrations. The finding of large gram-positive rods or large numbers of gram-positive cocci in wound secretion smears is a helpful early warning sign of highly lethal NSTI.

Research Note: First report on the detection of necrotic enteritis (NE) B-like toxin in biological samples from NE-afflicted chickens using capture enzyme-linked immunosorbent assay

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type G. One of the pore-forming toxins, NE B-like (NetB) toxin, secreted by pathogenic C. perfringens type G, has been proposed to be the main virulent factor in NE pathogenesis. The present study aimed to detect the presence of NetB toxin in biological samples of NE-afflicted chickens using NetB-specific monoclonal-based enzyme-linked immunosorbent assay (ELISA). Biological samples, including serum, digesta, and fecal droppings, were obtained from three previous NE studies (designated as Trials 1 to 3). In Trials 1 and 2, broiler chicks were infected with Eimeria maxima strain 41A on day 1 and followed by the netB-positive C. perfringens on day 18. Serum samples were obtained at 20 d post-hatch (i.e., 2 d post C. perfringens infection). In addition, various samples, including serum, gut digesta, and fecal droppings, that had been collected 0, 6, 24, and 30 h post C. perfringens infection were obtained. In Trial 3, broiler chicks were indirectly infected with litter-contaminated E. maxima on d 14 and followed by netB-positive C. perfringens via drinking water on days 18, 19, and 20. Serum samples and fecal droppings were obtained 21 d post-hatch (i.e., 1 d post last C. perfringens infection). The results showed that NetB toxin was not detected in serum samples in Trials 1 and 3. No NetB toxin was detected in all samples obtained before C. perfringens infection in Trial 2. Low but detectable amounts of NetB toxin were found in the serum samples obtained 6 h post C. perfringens infection in Trial 2. While NetB toxin in digesta and fecal droppings was detected 6 h post C. perfringens infection, its level plateaued 24 and 30 h post C. perfringens infection. In Trial 3, NetB toxin was detected in fecal droppings from the NE group, and its concentration ranged from 2.9 to 3.1 ng/g of wet feces. In Trial 2, NE-specific lesions were not seen 0 and 6 h post C. perfringens infection but exhibited lesions were moderate to severe 24 h post infection, leading to a moderate association (r = +0.527) between NE lesions and NetB toxin in the gut digesta. This is the first study to use NetB-specific monoclonal-based capture ELISA to determine and report the presence of native NetB toxin in biological samples from NE-induced chickens.

Toll-Like Receptor 4 Protects Against Clostridium perfringens Infection in Mice.

Toll-like receptor 4 (TLR4) has been reported to protect against Gram-negative bacteria by acting as a pathogen recognition receptor that senses mainly lipopolysaccharide (LPS) from Gram-negative bacteria. However, the role of TLR4 in Gram-positive bacterial infection is less well understood. Clostridium perfringens type A is a Gram-positive bacterium that causes gas gangrene characterized by severe myonecrosis. It was previously demonstrated that C. perfringens θ-toxin is a TLR4 agonist, but the role of TLR4 in C. perfringens infection is unclear. Here, TLR4-defective C3H/HeJ mice infected with C. perfringens showed a remarkable decrease in survival rate, an increase in viable bacterial counts, and accelerated destruction of myofibrils at the infection site compared with wild-type C3H/HeN mice. These results demonstrate that TLR4 plays an important role in the elimination of C. perfringens. Remarkable increases in levels of inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), were observed in C. perfringens-infected C3H/HeN mice, whereas the increases were limited in C3H/HeJ mice. Generally, increased G-CSF accelerates granulopoiesis in the bone marrow and the spleen to exacerbate neutrophil production, resulting in elimination of bacteria. The number of neutrophils in the spleen was increased in C. perfringens-infected C3H/HeN mice compared with non-infected mice, while the increase was lower in C. perfringens-infected C3H/HeJ mice. Furthermore, DNA microarray analysis revealed that the mutation in TLR4 partially affects host gene expression during C. perfringens infection. Together, our results illustrate that TLR4 is crucial for the innate ability to eliminate C. perfringens.

Phenotypic detection and genotyping of Clostridium perfringens associated with enterotoxemia in sheep in the Qassim Region of Saudi Arabia.

Background and Aim:Enterotoxemia caused by Clostridium perfringens toxinotypes is an often fatal disease of sheep of all ages, with a substantial economic loss to the sheep industry. This study was conducted to isolate C. perfringens from suspected cases of enterotoxemia in sheep in the central part of the Qassim Region, Saudi Arabia, and to determine the prevalent toxinotype by detecting alpha (cpA), beta (cpB), and epsilon (etX) toxin genes, which might help control this disease locally.Materials and Methods:A total of 93 rectal swabs and intestinal content samples were collected from diseased and animals suspected of having died of enterotoxemia in early 2020. Samples were subjected to bacteriological examination, biochemical analysis of isolates by VITEK 2, and molecular toxinotyping of isolates by LightCycler® real-time polymerase chain reaction (RT-PCR).Results:Our results revealed that only 14 isolates were confirmed by VITEK 2 as being C. perfringens, with excellent identification (probability of 95% and 97%). According to the toxinotyping of isolates by RT-PCR, all 14 isolates possessed both the cpA and etX toxin genes, while the cpB toxin gene was not detected in any of the isolates.Conclusion:Our findings demonstrated that C. perfringens type D was the only toxinotype found in the central part of the Qassim Region in 2020; moreover, according to the culture method, only 15% (14/93) of the suspected cases of enterotoxemia were confirmed to be caused by C. perfringens infection, which highlighted the importance of clinical and laboratory differential diagnosis of enterotoxemia in sheep.

A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm.

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

Protective Effects of Lactobacillus plantarum 16 and Paenibacillus polymyxa 10 Against Clostridium perfringens Infection in Broilers.

This study aimed to investigate the protective effects of Lactobacillus plantarum 16 (Lac16) and Paenibacillus polymyxa 10 (BSC10) against Clostridium perfringens (Cp) infection in broilers. A total of 720 one-day-old chicks were randomly divided into four groups. The control and Cp group were only fed a basal diet, while the two treatment groups received basal diets supplemented with Lac16 (1 × 108 cfu·kg−1) and BSC10 (1 × 108 cfu·kg−1) for 21 days, respectively. On day 1 and days 14 to 20, birds except those in the control group were challenged with 1 × 108 cfu C. perfringens type A strain once a day. The results showed that both Lac16 and BSC10 could ameliorate intestinal structure damage caused by C. perfringens infection. C. perfringens infection induced apoptosis by increasing the expression of Bax and p53 and decreasing Bcl-2 expression and inflammation evidence by higher levels of IFN-γ, IL-6, IL-1β, iNOS, and IL-10 in the ileum mucosa, and NO production in jejunal mucosa, which was reversed by Lac16 and BSC10 treatment except for IL-1β (P < 0.05). Besides, the two probiotics restored the intestinal microbiota imbalance induced by C. perfringens infection, characterized by the reduced Firmicutes and Proteobacteria and the increased Bacteroidetes at the phyla level and decreased Bacteroides fragilis and Gallibacterium anatis at the genus level. The two probiotics also reversed metabolic pathways of the microbiota in C. perfringens-infected broilers, including B-vitamin biosynthesis, peptidoglycan biosynthesis, and pyruvate fermentation to acetate and lactate II pathway. In conclusion, Lac16 and BSC10 can effectively protect broilers against C. perfringens infection through improved composition and metabolic pathways of the intestinal microbiota, intestinal structure, inflammation, and anti-apoptosis.

BIOCHEMICAL CONFIRMATORY TEST ON SUSPECTED ISOLATES OF CLOSTRIDIUM PERFRINGENS USING INDIRECT ACID PHOSPHATASE

The effectiveness of indirect acid phosphatase of confirmatory identification of Clostridium perfringens (Bacteria) isolates from faecal samples of goats and sheep is described C. perfringens infections has not the isolates belonged to the desired species been much reported in the Nigerian animal husbandry systems, thus this method could help for easy identification of this disease. The results proved acid phosphatase, a quick, reliable, simple and accurate method for the biochemical confirmatory test for C.perfringens. This test proved same to the reference strains obtained for comparison.

Phytobiotic role of essential oil-loaded microcapsules in improving the health parameters in Clostridium perfringens-infected broiler chickens

Nowadays, the development of phytobiotics, an antibiotic-replacing agent, has attracted a considerable attention as poultry feed additive. The present study aims to develop the microcapsules loaded with a blend of essential oils (thyme, peppermint, savoury, and black pepper), and evaluate its health-promoting activity among the Clostridium perfringens-challenged broiler chickens. Based on the results, the spherical microcapsules containing a blend of thyme, summer savoury, peppermint, and black pepper essential oils had the particle size of 158.6 µm and encapsulation efficiency of 94.2%. The major bioactive compounds were thymol, carvacrol, p-cymene, and γ-terpinene. The microencapsulation of essential oil effectively prevented the release of bioactive phenolic compounds in the upper parts of the GIT. Additionally, it increased the delivery of the phenolic compounds to the lower segments of the GIT in the broiler chickens. The administration of the essential oil-loaded microcapsules (0.5, 1, and 2 kg/ton) in the C. perfringens-infected broiler chickens enhanced growth parameters, total feed intake, FCR, body antioxidant status, ileum morphostructure, and intestinal microbial population. Further, this treatment resulted in regulating the antioxidant and inflammation genes in ileum tissue. Consequently, the essential oil-loaded microcapsules can be considered as a promising phytobiotic against C. perfringens infection in broiler chickens. HIGHLIGHTS The spherical microcapsules containing a blend of EO had the particle size of 158.6 µm and encapsulation efficiency of 94.2%. The microencapsulation of essential oil effectively prevented the release of bioactive phenolic compounds in the upper parts of the GIT. The EO-loaded microcapsules led to an improvement in growth parameters, body antioxidant status, ileum morphostructure, and intestinal microbial population. The microcapsules regulated the antioxidant and inflammation genes in ileum tissue.

Primary Emphysematous Pancreatitis Caused by Clostridium Perfringens Infection

A 66-year-old male was admitted to a close hospital for acute pancreatitis. 48h hours after developed clinical worsening. Therefore, a CT scan was performed showing signs of Emphysematous Severe Acute Pancreatitis. A transfer to our hospital was granted, being admitted to the ICU. A follow-up CT scan was performed, showing an increase in the size of abdominal collections and pneumoretroperitoneum. For this reason, an open pancreatic necrosectomy was performed. However, the patient showed an overall excellent response allowing for weaning and extubation. 15 days after the surgery, bleeding from the abdominal wall is detected. A CT scan was performed showing epigastric artery bleeding. After infructuous endovascular embolization, a surgery was required. During the stay in the ICU the patient suffered through diverse nosocomial infections by multiresistant bacteria. The patient’s condition worsened. Due to an absence in response to therapy, the patient died 58 days after being admitted to the hospital. Discussion: Clostridium’s perfringens necrotizing acute pancreatitis is a rare condition that can usually be suspected by the presence of gas around the pancreas and characteristically pneumoretroperitoneum. Its origin is usually linked to invasive procedures of the bile duct. It can also appear on collections or necrotic tissue related to previous acute pancreatitis or chronic pancreatitis. Conclusion: The Clostridium perfringens confers a raise in morbidity and mortality in spite of optimal treatment decisions. Therefore, an early diagnosis and admission to ICUs, as well as an early appliance of surgical measures, might lower the chances of adverse outcomes.

Cannabis-derived cannabidiol and nanoselenium improve gut barrier function and affect bacterial enzyme activity in chickens subjected to C. perfringens challenge

Revealing the multifocal mechanisms affecting cross-talk between Clostridium perfringens pathogenesis and the host response is an urgent need in the poultry industry. Herein, the activity of Cannabis sativa-derived cannabidiol (CBD) and selenium nanoparticles (Nano-Se) in modulating the host response to Clostridium perfringens challenge was investigated in broiler chickens subjected to a mild infection model. The infected chickens exhibited no clinical manifestations, confirming the potential hazard of pathogen transmission to the food chain in the commercial sector. However, both CBD and Nano-Se affected the responses of chickens to C. perfringens challenge. The beneficial actions of both agents were manifested in the upregulated expression of genes determining gut barrier function. Both CBD and Nano-Se promoted shifts in gut bacterial enzyme activity to increased energy uptake in challenged chickens and upregulated potential collagenase activity. There was no opposite effect of CBD and Nano-Se in mediating the host response to challenge, whereas an additive effect was evidenced on the upregulation of gene determining gut integrity. Collectively, these findings indicate that understanding the action mechanisms of CBD and Nano-Se is of great interest for developing a preventive strategy for C. perfringens infection in broilers.

Inflammatory responses, energy metabolism enzymes, oxidative status in Clostridium perfringens infection in broilers

The aim of this study was to determine whether infection by Clostridium perfringens negatively interferes with the oxidant/antioxidant status and activity of energy metabolism enzymes (creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK)), as well as the zootechnical performance of broilers. A completely randomized design with three treatments, with five replications by treatment, and 10 birds by repetitions was used: T1: non-infected group; T2: group infected with C. perfringens; T3: group infected with C. perfringens, and basal diet with performance enhancers (antibiotic and coccidiostatic). At 21 days of age, the birds were experimentally infected orally with 4.0 x 108 CFU/mL C. perfringens. At strategic moments during the experimental period, zootechnical data and blood samples were collected. There were no significant differences among treatments in terms of weight gain, feed intake, or feed conversion. At 20 days, heterophils counts were significantly lower in the T3 group birds than in the others. A significant reduction in the number of total leukocytes, because of the lower number of lymphocytes, was observed in groups T2 and T3 on day 34. Neutrophil and monocyte counts were significantly lower in group T3 than in group T1 on day 34. On day 42, there were significantly lower levels of total protein and globulin in birds in T3 than in T1. On day 27, PK activity was significantly higher in groups T2 and T3; similar behavior observed on day 34 when CK and PK activities in T2 and T3 were significantly higher than those of T1. AK activity was significantly higher in the serum of T3 birds than in those of the other groups at 34 days. At 41 days, CK and PK activities were significantly higher only in birds in group T3 compared to other groups. The levels of reactive oxygen species and lipoperoxidation, as well as the activity of glutathione S-transferase, were significantly lower in birds from group T3 compared to other groups at age 20 days. After a challenge with C. perfringens, there were fluctuations in the behavior of oxidative stress biomarkers; in particular, at 41 days of age, birds in T2 had elevated levels of lipoperoxidation, different from what was seen in birds in T3 that consumed feed with a performance enhancer. Preliminary results suggest that clostridiosis affects serum activity of enzymes in the phosphotransfer network, requiring compensatory enzymatic changes in an attempt to maintain energy homeostasis. In addition, the infection reduces the number of inflammatory cells and causes oxidant/antioxidant imbalance that may contribute to the pathophysiology of the disease. Además, la infección reduce el número de células inflamatorias y provoca un desequilibrio en el estado oxidante / antioxidante que puede contribuir a la fisiopatología de la enfermedad.

A FATAL CASE OF C. PERFRINGENS NECROTIZING INFECTION WITH MYONECROSIS FOLLOWING AN ELECTIVE OUTPATIENT LIPOSUCTION

SESSION TITLE: Medical Student/Resident Critical Care Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: C. Perfringens is an uncommon cause of Necrotizing Fasciitis in immunocompetent patients. It typically leads to infections in unsterile conditions and in traumatic injuries or following natural disasters such as earthquakes. We present a case of an immunocompetent patient who developed a fatal case of Necrotizing Fasciitis secondary to C. Perfringens after an elective outpatient procedure. CASE PRESENTATION: 47 year-old previously healthy female who underwent outpatient bilateral liposuction for posterior upper arm lipodystrophy presented to her surgeon’s office later that day with increasing redness, pain, and swelling. Physical examination was unrevealing other than erythema in right arm and a temperature of 101F. Patient was sent home with Amoxicillin-Clavulanic Acid. On day two, her wound continued to worsen and was referred to the hospital. Examination showed an ill-appearing woman with markedly swollen right upper extremity including the hand with discoloration and blisters. Due to the high suspicion of Necrotizing Fasciitis and the need for immediate intervention, she was transferred to our facility for management. A puncture incision was immediately performed to drain fluid and reduce tension. Cultures grew gram positive rods and wound debridement was performed pending culture results. She underwent multiple wound debridements and was started on Vancomycin, Piperacillin/Tazobactam and Clindamycin. Patient developed septic shock and respiratory failure. She was intubated and resuscitated with fluids and vasopressors. She was also placed on CRRT for renal failure. Cultures grew Clostridium Perfringens and the regimen shifted to Penicillin and Clindamycin. Her condition continued to worsen eventually developing ARDS. Given her deterioration, family decided to pursue DNR with patient expiring on Day 6. DISCUSSION: Clostridium Perfringens is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium that is found in nature as part of decaying vegetation, marine sediment and soil. Infection leads to fast developing tissue necrosis, bacteremia, and gas gangrene. The infection is most commonly seen in contaminated injuries, unsterile procedures, or in immune-compromised patients. However, C. Perfringens infection following a sterile procedure is extremely rare. Literature review revealed five cases of C. perfringens infection following elective procedures in immune-competent patients. Cases reviewed involved in-patient procedures and non-elective surgical interventions whereas our case involved an outpatient elective liposuction procedure in a previously healthy individual. CONCLUSIONS: Given the rapidly progressive nature of tissue destruction and clinical deterioration of this condition, we suggest that clinicians consider this infection as part of their differential when they are presented with signs of C. Perfringens infection following surgery to prevent a fatal outcome. Reference #1: Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections. N Engl J Med. 2018;378(10):971. doi:10.1056/NEJMc1800049 Reference #2: Bryant AE, Stevens DL. Clostridial myonecrosis: new insights in pathogenesis and management. Curr Infect Dis Rep. 2010;12(5):383-391. doi:10.1007/s11908-010-0127-y DISCLOSURES: No relevant relationships by Sarah Al-Obaydi, source=Web Response No relevant relationships by Firas Alzaiem, source=Web Response No relevant relationships by James DeMaio, source=Web Response No relevant relationships by Tharun Paruchuri, source=Web Response

Clostridium perfringens α and β recombinant toxoids in equine immunization

ABSTRACT: Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the β toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400μg) of C. perfringens α and β recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.

Research Note: Effects of organic zinc on broiler intestinal permeability and integrity in Clostridium perfringens–challenged condition

This study evaluates the effects of a zinc–amino acid complex on broiler's intestinal permeability and integrity challenged with Clostridium perfringens. A total of 180 Arbor Acres 1-day-old male broilers were assigned to 6 treatments in a completely randomized 2 × 3 factorial design. The experiment investigated the comparative effects of inorganic and organic zinc supplements, that is (ZnSO4 treatment: 80 mg zinc/kg from ZnSO4; iso-dose replacement group. (ISO) treatment:40 mg zinc/kg from ZnSO4 plus 40 mg zinc/kg from a zinc–amino acid complex; and organic treatment: 40 mg zinc/kg from a zinc–amino acid complex) on C. perfringens–challenged broilers. C. perfringens, on the one hand, compromised intestinal mucosal barrier function by increasing the intestinal permeability of fluorescein isothiocyanate–labeled dextran (P < 0.05) and plasma endotoxin level, on the other hand, decreased both the transepithelial electrical resistance and the relative expression of occludin levels in the ileum at day 21 (P < 0.05). However, zinc supplement alleviates C. perfringens-induced pathologic changes in the intestinal permeability. ISO treatment, in particular, enhanced the villus height–to–crypt depth ratio and transepithelial electrical resistance and also reduced plasma endotoxin levels. In addition, zinc supplement relatively enhanced the expression of occludin levels in the ileum compared with the C. perfringens–challenged group. ISO treatment had the highest relative expression of occludin levels in the ileum. Thereby, the results indicated that partial replacement of ZnSO4 with a zinc–amino acid complex in the broiler diet could promote intestinal mucosal barrier function during C. perfringens infection via increased expression of occludin in the ileum.

Saccharomyces boulardii attenuates inflammatory response induced by Clostridium perfringens via TLR4/TLR15-MyD8 pathway in HD11 avian macrophages

Macrophages are professional phagocytic cells that play a critical role in initiating immune responses by presenting antigen and phagocytic clearance. The macrophages can be targeted for immunomodulation by beneficial microbes, such as probiotics. The aim of this study is to investigate the protective effect of Saccharomyces boulardii against Clostridium perfringens infection in avian macrophage cell line HD11. In this study, HD11 macrophages were prestimulated with S. boulardii for 6 h and then infected with C. perfringens for 3 h. Results showed that S. boulardii enhanced phagocytosis and bactericidal capacity against C. perfringens by HD11 cells. The S. boulardii effectively promoted the mRNA expression of CD80, CD83, and CD197 cell-surface molecules in C. perfringens-infected HD11 cells. Moreover, we found that prestimulation with S. boulardii reduced the mRNA expression of CD40, toll-like receptor [TLR] 4, and TLR15 induced by C. perfringens and thereby downregulated the mRNA expression of myeloid differentiation primary response 88, TNF receptor associated factor 6, nuclear factor kappa-B p65 subunit, and c-Jun N-terminal kinase genes in HD11 cells. The upregulation of cytokines (interleukin [IL]-6, tumor necrosis factor alpha, and IL-10) and inducible nitric oxide synthase mRNA expression in C. perfringens-infected HD11 cells were noticeably inhibited by S. boulardii pretreatment. Conclusively, these results might provide a new insight into the role of S. boulardii in regulating avian immune defense against C. perfringens invasion and immune escape.

C. perfringens challenge reduces matrix metalloproteinase activity in the jejunal mucosa of Eimeria-infected broiler chickens

Matrix metalloproteinases (MMPs) play an important role in intestinal extracellular matrix homeostasis. An overexpression of MMPs results in tissue destruction and local inflammation and has been associated with multiple inflammatory diseases. These host proteases might also be important in tissue damage caused by infectious agents, such as in intestinal damage in Clostridium perfringens-induced avian necrotic enteritis (NE). The aim of the present study was to elucidate the effect of a C. perfringens infection on the MMP activity in the small intestine of birds with a pre-disposing coccidial infection to obtain a more thorough understanding of the pathogenesis of NE. For this purpose, the gelatinolytic activity present in jejunal tissue of Eimeria infected birds which were challenged with either a pathogenic C. perfringens type G strain or a commensal C. perfringens type A strain was analyzed using substrate zymography. The results show that infection of broilers with Eimeria and different C. perfringens strains, independent of their pathogenicity, decreases the expression of a 40–45 kDa host collagenase in the jejunum, as compared to the expression in Eimeria-infected control birds. It was also shown that the expression of 2 MMPs with molecular weights of approximately 50–60 and 60–70 kDa was significantly lower in necrotic tissue as compared to the activity in macroscopically healthy tissue adjacent to the lesion. These results indicate that host collagenases are not elicited by the C. perfringens infection for permeabilizing the host mucosa to allow penetration of the NetB toxin in Eimeria infected broilers.

Bioinformatics analysis of NetF proteins for designing a multi-epitope vaccine against Clostridium perfringens infection

Clostridium perfringens is an important human and animal pathogen that is the primary causative agent of necrotizing enteritis and enterotoxemia in many types of animals. C. perfringens produces a variety of toxins, including NetF which may plays a crucial role in the pathogenesis of foal and canine necrotizing enteritis. In this study, we used several bioinformatics methods to analyze various aspects of the NetF proteins, including the physicochemical properties, secondary and tertiary structures, and the dominant B-cell and T-cell epitopes. The results showed that NetF protein was a stable and hydrophilic protein. The secondary structure of the NetF protein consisted of 2.62% alpha helixes, 6.56% beta turns, 38.69% extended strands and 52.13% random coils. Moreover, several potential B and T-cell epitopes were identified for NetF. In addition, the obtained findings from antigenicity and allergenicity evaluation remarked that this protein is immunogenic and non-allergen.Based on the results of Ramachandran plot, 94.22%, 5. 42%, and 0.36% of amino acid residues were incorporated in the favored, allowed, and outlier regions, respectively. This study provides a foundation for further investigations, and laid a theoretical basis for the development of an appropriate vaccine against C. perfringens infection.

Verbascoside Protects Mice From Clostridial Gas Gangrene by Inhibiting the Activity of Alpha Toxin and Perfringolysin O.

Gas gangrene, caused mainly by the anaerobic bacterium Clostridium perfringens (C. perfringens), causes death within 48 h of onset. Limited therapeutic strategies are available, and it is associated with extremely high mortality. Both C. perfringens alpha toxin (CPA) and perfringolysin O (PFO) are important virulence factors in the development of gas gangrene, suggesting that they are therapeutic targets. Here, we found that verbascoside, a phenylpropanoid glycoside widely distributed in Chinese herbal medicines, could effectively inhibit the biological activity of both CPA and PFO in hemolytic assays. The oligomerization of PFO was remarkably inhibited by verbascoside. Although no antibacterial activity was observed, verbascoside treatment protected Caco-2 cells from the damage caused by CPA and PFO. Additionally, infected mice treated with verbascoside showed significantly alleviated damage, reduced bacterial burden, and decreased mortality. In summary, verbascoside has an effective therapeutic effect against C. perfringens virulence both in vitro and in vivo by simultaneously targeting CPA and PFO. Our results provide a promising strategy and a potential lead compound for C. perfringens infections, especially gas gangrene.

Effects of Clostridium perfringens beta2 toxin on apoptosis, inflammation, and barrier function of intestinal porcine epithelial cells

Clostridium perfringens beta2 (CPB2) toxin is an important virulence factor that causes enteric diseases in both humans and animals. To investigate the underlying mechanism in CPB2-induced inflammation and damage in the small intestinal epithelium, intestinal porcine epithelial cells (IPEC-J2) were treated with recombinant CPB2 (rCPB2) toxin. The results showed that IPEC-J2 cell viability was decreased by rCPB2 toxin treatment in a dose- and time-dependent manner. Analysis of cell morphology and Annexin V-FTIC/PI staining revealed that rCPB2 toxin induces cell apoptosis. Indeed, the expression of caspase-3, caspase-8, and caspase-9 was significantly increased at both the mRNA and protein levels in IPEC-J2 cells treated with rCPB2 toxin. The caspase-3 inhibitor Ac-DEVD-CHO reduced rCPB2 toxin-induced cell apoptosis. Moreover, exposure to the toxin increased the expression of interleukin (IL)-6, IL-7, IL-12, and IL-1β, while decreasing that of transforming growth factor beta 1 (TGFβ1). Additionally, rCPB2 toxin treatment also induced intestinal barrier dysfunction, as evidenced by the degradation of zonula occludens (ZO)-1, claudin-1, and E-cadherin, as well as an increase in paracellular permeability. Overall, the results indicated that rCPB2 toxin induces apoptosis and inflammation, in addition to impairing intestinal barrier function in IPEC-J2 cells. Our findings provide a foundation to better understand the pathogenesis of C. perfringens infection and inform strategies to effectively prevent and treat C. perfringens-induced enteric diseases.

Prevalence and characterization of Clostridium perfringens isolated from feces of captive cynomolgus monkeys (Macaca fascicularis)

Clostridium perfringens is ubiquitous in the environment and the gastrointestinal tract of warm-blooded animals. While part of the gut microbiome, abnormal growth of C. perfringens causes histotoxic, neurologic, and enteric diseases in a variety of animal species, including humans, due to the production of toxins. There is extremely limited information on C. perfringens infection in non-human primates. Presently, 10 strains were successfully isolated from 126 monkeys and confirmed by molecular and biochemical analyses. All isolates were genotype A based on molecular analysis. Alpha toxin was identified in all isolates. Beta 2 toxin was detected in only three isolates. No other toxins, including enterotoxin, beta, iota, epsilon, and net B toxin, were identified in any isolate. All isolates were highly susceptible to β-lactam antibiotics. Double hemolysis and lecithinase activity were commonly observed in all strains. Biofilm formation, which can increase antibiotic resistance, was identified in 90% of the isolates. The data are the first report the prevalence and characteristics of C. perfringens isolated from captive cynomolgus monkeys.