Perfringens Infection Research Articles

Innovative unified impact of magnetite iron nanoparticles and quercetin on broiler chickens: performance, antioxidant and immune defense and controlling of Clostridium perfringens infection

Innovative unified impact of magnetite iron nanoparticles and quercetin on broiler chickens: performance, antioxidant and immune defense and controlling of Clostridium perfringens infection

An outbreak of Clostridium perfringens infection on a training ship anchored in Busan, Korea.

In September 2023, an outbreak of food-borne disease occurred among students on a training ship docked in Busan. This was an epidemiological investigation with the aim of improving infection prevention activities and group meal service practices on board ships. In this study, a case was defined as an individual who experienced diarrhea more than twice a day during their training period aboard the training ship. A total of 171 exposed individuals including 6 food handlers was well-defined; therefore, a retrospective cohort study was conducted. We administered a questionnaire and conducted laboratory tests including 38 rectal swab samples. Relative risk (95% confidence interval) for each food item was calculated. Of the 165 students and school staff members, 41 met the case definition, resulting in an attack rate of 24.8%; all cases were students. The shape of the epidemic curve was unimodal, with the peak from 0:00 to 06:00 on September 7, 2023. Clostridium perfringens was detected in 9 cases, and no other pathogens were found. Significant relative risk was shown in 11 different food items. Clostridium perfringens was the causative pathogen of this outbreak on the training ship. Due to the lack of preserved food samples, the exact source of infection could not be confirmed. Ships are not classified as collective dining facilities, leaving them in a management blind spot. Therefore, specialized guidelines, voluntary inspections by the operating entities, and continuous education for managers and staff are necessary.

Elevated levels of butyric acid in the jejunum of an animal model of broiler chickens: from early onset of Clostridium perfringens infection to clinical disease of necrotic enteritis

BackgroundNecrotic enteritis (NE) is an economically important disease of broiler chickens caused by Clostridium perfringens (CP). The pathogenesis, or disease process, of NE is still not clear. This study aimed to identify the alterations of metabolites and metabolic pathways associated with subclinical or clinical NE in CP infected birds and to investigate the possible variations in the metabolic profile of birds infected with different isolates of CP.MethodologyUsing a well-established NE model, the protein content of feed was changed abruptly before exposing birds to CP isolates with different toxin genes combinations (cpa, cpb2, netB, tpeL; cpa, cpb2, netB; or cpa, cpb2). Metabolomics analysis of jejunal contents was performed by a targeted, fully quantitative LC-MS/MS based assay.ResultsThis study detected statistically significant differential expression of 34 metabolites including organic acids, amino acids, fatty acids, and biogenic amines, including elevation of butyric acid at onset of NE in broiler chickens. Subsequent analysis of broilers infected with CP isolates with different toxin gene combinations confirmed an elevation of butyric acid consistently among 21 differentially expressed metabolites including organic acids, amino acids, and biogenic amines, underscoring its potential role during the development of NE. Furthermore, protein-metabolite network analysis revealed significant alterations in butyric acid and arginine-proline metabolisms.ConclusionThis study indicates a significant metabolic difference between CP-infected and non-infected broiler chickens. Among all the metabolites, butyric acid increased significantly in CP-infected birds compared to non-infected healthy broilers. Logistic regression analysis revealed a positive association between butyric acid (coefficient: 1.23, P < 0.01) and CP infection, while showing a negative association with amino acid metabolism. These findings suggest that butyric acid could be a crucial metabolite linked to the occurrence of NE in broiler chickens and may serve as an early indicator of the disease at the farm level. Further metabolomic experiments using different NE animal models and field studies are needed to determine the specificity and to validate metabolites associated with NE, regardless of predisposing factors.

Replacing Hydrolyzed Soybean Meal with Recombinant β-Glucosidase Enhances Resistance to Clostridium perfringens in Broilers Through Immune Modulation.

Aglycone soy isoflavones have notable immune-regulatory bioactivity, while glycosidic forms in soybean meal pose challenges for absorption. β-Glucosidase (EC 3.2.1.21) catalyzes the non-reducing terminal β-d-glucosidic bonds, releasing β-d-glucan and aglycones. This study evaluated the impact of enzymatically hydrolyzed soybean meal (ESM) using recombinant β-glucosidase from Aspergillus niger on the growth performance and intestinal immune function of broilers under Clostridium perfringens infection. Prior to the feeding trial, soybean meal was enzymatically digested with recombinant β-glucosidase, ensuring almost complete conversion of glycosides to aglycones. After a week of pre-feeding, a total 180 healthy AA broilers were randomly assigned to three groups-control, semi-replacement of ESM (50% ESM), and full-replacement of ESM (100% ESM)-with 6 replicates of 10 chickens, and the trial lasted 28 days. On the 36th day, broilers were challenged with 1 mL of 1 × 1010 CFU/mL Clostridium perfringens (Cp) via gavage for 3 days. The results showed that the substitution of ESM had no effect on the body weight gain of broilers but significantly reduced the feed consumption and feed-to-gain ratio (p < 0.01). The study revealed that Cp significantly disrupted jejunal morphology, while ESM significantly mitigated this damage (p < 0.05). Real-time PCR results demonstrated that compared to the Cp group, ESM restored Cp-induced intestinal barrier impairments (e.g., Occludin, Claudin-1, Muc2), normalized aberrant cellular proliferation (PCNA) and apoptosis (Caspase-1 and Caspase-3), and upregulated the expression of anti-inflammatory factor Il-10 while suppressing pro-inflammatory cytokines (Il-1β, Il-6, and Il-8) (p < 0.05). Moreover, flow cytometry analyses demonstrated that ESM promoted Treg cell-derived Il-10, which alleviated macrophage-derived inflammation. Substituting conventional soybean meal with β-glucosidase, enzymatically treated, significantly reduced feed consumption and alleviated the intestinal damage and immune dysfunctions induced by Clostridium perfringens infection in broilers.

Identification of immunogenic antigens and evaluation of vaccine candidates against Clostridium perfringens

Necrotic enteritis (NE) caused by Clostridium perfringens (C. perfringens) has resulted in significant losses for the poultry industry worldwide. Currently, there is no widely promoted vaccine for NE. In this study, immunoprecipitation (IP) was employed to isolate immunogenic proteins of C. perfringens, and 118 potential candidate antigens were identified through liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS). From these, three candidate antigen proteins were selected based on their predicted antigenicity, hydrophilicity, stability, and transmembrane signalling properties: ArcB (an ornithine aminotransferase), TmpC (a probable membrane lipoprotein), and EntB (a possible enterotoxin). These three proteins were successfully produced in large quantities using Escherichia coli (E. coli), with confirmed good solubility. Both in vitro and in vivo research demonstrated that these antigens possess strong immunogenicity, eliciting robust antigen-specific humoral and cellular immune responses in chickens and mitigating NE symptoms caused by C. perfringens. The candidate antigens identified through immunoproteomics hold potential as subunit vaccines against C. perfringens infection.

Probiotic Bacillus subtilis QST713 improved growth performance and enhanced the intestinal health of yellow-feather broilers challenged with coccidia and Clostridium perfringens

In this study, we investigated the effects of dietary supplementation with Bacillus subtilis (QST713) on the performance and intestinal health of yellow feather broilers under Coccidia and Clostridium perfringens (CP) challenge or CP alone. One-day-old yellow-feathered broiler roosters (n = 600) were randomly assigned to 5 groups (6 replicates with 20 roosters per replicate): the Con blank group, the CIC.p group (d24 Coccidia+d28-30 of CP challenge), the CIC.p + BS group (CIC.p +100 mg/kg B. subtilis), the C.p group (d 28-34 of CP challenge), and the C.p +BS group (C.p +100 mg/kg B. subtilis). The experiment lasted 80 d. The birds were evaluated for parameters such as average daily gain (ADG), average daily feed intake (ADFI), feed efficiency (F/G), intestinal lesion score, villus histomorphometry, intestinal tight junctions, inflammatory factors, and cecal microorganisms. The results revealed that 1) C.p. increased the F/G of broilers from 22 to 42 d (P < 0.05), whereas CIC.p. significantly decreased the 42 d and 80 d body weights (BW) and 22-42 d and 1-80 d ADG (P < 0.05) and significantly increased the 22 to 42 d and 1 to 80 d F/G (P < 0.05). The number of intestinal lesions significantly increased at 35 d and 42 d (P < 0.05). CIC.p significantly decreased the jejunum and ileum villus height (VH) and the ileum villus height/crypt depth (P < 0.05) at 35 d. The challenge significantly upregulated the expression of Claudin-1 and IL-4 mRNAs in the jejunum at 35 d and significantly downregulated the expression of IL-10 mRNA in the ileum at 35 d (P < 0.05); the number of unique OTUs in the challenge group decreased significantly after challenge treatment, and the relative abundances of Romboutsia at 35 d and Cladomyces and Lactobacillus at 42 d decreased significantly (P < 0.05). 2) Compared with the challenge groups, the addition of BS decreased the F/G of broilers from 22 to 42 d. Compared with the CIC group, the addition of BS significantly increased the F/G of broilers from 22 to 42 d. Compared with that in the CIC.p group, the addition of BS significantly increased the VH in the jejunum and ileum at 35 d (P < 0.05). Compared with the challenge groups, the BS groups presented significantly lower mRNA expression levels of Claudin-1 (P < 0.05) in the jejunum at 35 d. The Shannon and Chao indices suggested that BS increased the alpha diversity of cecum microorganisms in broilers. Dietary supplementation with B. subtilis can alleviate the damage to intestinal morphology and intestinal barrier function, as well as the altered cecal flora structure in broilers caused by Coccidia and C. perfringens infections.

In vivo overexpression of the avian interleukin-17 in a necrotic enteritis disease model modulates the expression of antimicrobial peptides in the small intestine of broilers

In humans and mice, the induction of interleukin (IL)-17 expression enhances epithelial barrier integrity through the secretion of antimicrobial peptides (AMP), thereby improving antibacterial defense. However, it is unclear whether IL-17 has similar antibacterial effects in chickens by modulating the expression of AMPs, such as avian beta-defensins (also known as gallinacins) and cathelicidins. This study evaluated the in vivo effects of inoculating 20-day-old broiler chickens with two doses of a plasmid encoding chicken IL-17 (pCDNA3.1/rchIL-17-V5-HIS TOPO plasmid [pCDNA3.1-IL-17]; 5 or 10 μg/bird). On day 23 of age, all broilers, except those in the negative control group, were orally challenged with a virulent Clostridium perfringens strain for three days. To investigate IL-17-mediated effects against C. perfringens infection, the expression of avian beta-defensin 1 (avBD1), avBD2, avBD4, avBD6, cathelicidins, and inducible nitric oxide synthase (iNOS) genes were quantified, and gross necrotic enteritis (NE) lesion scores were assessed in the small intestine. The results showed that broilers receiving the higher dose of pCDNA3.1-IL-17 (10 μg) had significantly lower NE lesion scores compared to those receiving the lower dose (5 μg), the vector control, and the positive control groups. Furthermore, the expression of all avian beta-defensins and cathelicidin genes was detectable across all groups, regardless of treatment and time points. IL-17 treatment led to significantly higher expression of avBD1, avBD2, avBD4, avBD6, cathelicidin, and iNOS in the duodenum, jejunum, and ileum compared to control chickens. In C. perfringens-infected chickens, the expression of avBD1, avBD2, avBD4, cathelicidin, and iNOS in the ileum was significantly higher than in control chickens. Pre-treatment with the higher dose of pCDNA3.1-IL-17 (10 μg) in infected chickens was associated with reduced NE lesion severity and increased expression of avBD1, avBD2, cathelicidin, and iNOS in the ileum, but not avBD4 and avBD6. These findings provide new insights into the potential effect of IL-17 and reduction in NE lesion severity by modulating AMP expression which may be involved in mediating protective immunity against intestinal infection with C. perfringens.

Gut Lactococcus garvieae promotes protective immunity to foodborne Clostridium perfringens infection.

C. perfringens necrotic enteritis leads to losses of about US $2 billion to the poultry industry worldwide every year. Worse, US Centers for Disease Control and Prevention (CDC) has estimated that C. perfringens causes nearly 1 million foodborne illnesses in the United States annually. Nowadays, the treatment recommendation is a combination of a broad-spectrum synergistic penicillin with clindamycin or a carbapenem, despite growing scientific concern over antibiotic resistance. The global understanding of the gut microbiome for C. perfringens infection may provide important insights into the intervention. L. garvieae originated from Mongolian sheep intestine, exhibited potentially probiotic properties, and was able to limit C. perfringens enterocolitis and pathogenic colonization. Importantly, we found that L. garvieae limits C. perfringens invasion via improving intestinal mucosal barrier function. Also, L. garvieae alleviates C. perfringens-induced gut microbiota dysbiosis. It allowed us to convince that utilization of probiotics to promote protective immunity against pathogens infection is of pivotal importance.

Clostridium perfringens in the Intestine: Innocent Bystander or Serious Threat?

The Clostridium perfringens epidemic threatens biosecurity and causes significant economic losses. C. perfringens infections are linked to more than one hundred million cases of food poisoning annually, and 8-60% of susceptible animals are vulnerable to infection, resulting in an economic loss of more than 6 hundred million USD. The enzymes and toxins (>20 species) produced by C. perfringens play a role in intestinal colonization, immunological evasion, intestinal micro-ecosystem imbalance, and intestinal mucosal disruption, all influencing host health. In recent decades, there has been an increase in drug resistance in C. perfringens due to antibiotic misuse and bacterial evolution. At the same time, traditional control interventions have proven ineffective, highlighting the urgent need to develop and implement new strategies and approaches to improve intervention targeting. Therefore, an in-depth understanding of the spatial and temporal evolutionary characteristics, transmission routes, colonization dynamics, and pathogenic mechanisms of C. perfringens will aid in the development of optimal therapeutic strategies and vaccines for C. perfringens management. Here, we review the global epidemiology of C. perfringens, as well as the molecular features and roles of various virulence factors in C. perfringens pathogenicity. In addition, we emphasize measures to prevent and control this zoonotic disease to reduce the transmission and infection of C. perfringens.

Combined Effect of Nigella sativa and Kefir on the Live Performance and Health of Broiler Chickens Affected by Necrotic Enteritis.

Coccidiosis and necrotic enteritis (NE) are prevalent poultry ailments worldwide, leading to decreased live performance and elevated mortality rates without antibiotic usage. This study evaluated Nigella sativa (black cumin) seeds (BCS) and kefir as alternatives to antibiotics for broilers. An in vivo study over a 28-day period, using 384 Cobb 500 male broilers organized into six treatment groups as part of a completely randomized block experimental design was conducted. Each treatment group included eight replicates, with each replicate containing eight birds. The treatments included positive control, negative control, antibiotic control, 5% BCS in feed, 20% kefir in drinking water, and a combination of 5% BCS and 20% kefir. NE was induced in broilers by administering ~5000 oocysts of Eimeria maxima orally on day 14, followed by inoculation with about 108 CFU/mL of Clostridium perfringens (Cp) (strain Cp#4) on days 19, 20, and 21. Live performance metrics including feed intake, body weight gain, and feed conversion were assessed in broilers. Additionally, NE disease outcomes such as lesion scores, mortality rates, and Cp populations in cecum were determined during the study. The BCS, kefir, and the combination had no detrimental effect on broiler live performance. BCS-treated and combination groups had lower NE scores (p > 0.05) in comparison to the positive control and exhibited no significant difference (p > 0.05) from antibiotic control. Additionally, treatment groups and antibiotic control were not significantly different (p > 0.05) in mortality, whereas the BCS and kefir combination significantly reduced (p < 0.05) mortality to 14.1% compared to 31.3% for the positive control. C. perfringens vegetative cells significantly decreased (p < 0.05) in treatments with BCS, kefir, and their combination on days 22 and 28 compared to the positive control. On day 22, Cp sores were significantly lower (p < 0.05) for the kefir and combination treatments compared to the positive control. In conclusion, BCS and kefir successfully reduced C. perfringens infection and mortality without any detrimental impact on broiler live performance with the combined treatment being the most effective. These results suggest that BCS and kefir could serve as potential alternatives to antibiotics in managing NE.

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.

Cell-Free Supernatant of Bacillus subtilis G2B9-Q Improves Intestinal Health and Modulates Immune Response to Promote Mouse Recovery in Clostridium perfringens Infection.

Clostridium perfringens is one of the critical causative agents causing diarrhea in piglets, with significant economic losses to the pig industry. Under normal gut microbiota homeostasis and well-managed barns, diarrhea caused by C. perfringens could be controlled. Some reports show that probiotics, such as Bacillus subtilis, are beneficial in preventing necrotic enteritis (NE) in chickens, but few reports on piglets. Clostridium perfringens was found in the piglets' diarrhea with intestinal microbiota dysbiosis in our survey. Bacillus subtilis G2B9-Q, which was isolated from the feces of healthy pigs, was found to have anti-Clostridium activity after screening. Clostridium perfringens was used to challenge mice by intraperitoneal injection for modeling to evaluate the anti-infective activity of cell-free supernatant (CFS) of B. subtilis G2B9-Q and different concentrations of B. subtilis G2B9-Q by oral administration. The results showed that G2B9-Q can mitigate intestinal lesions caused by C. perfringens infection, reduce inflammatory reactions, and modulate intestinal microbiota. The CFS of G2B9-Q can alleviate the pathological damage of intestinal tissues caused by C. perfringens infection, reduce the concentration of TNF-α and IL-10 in the sera of mice, as well as the relative expression levels of alpha toxin (CPA), perfringolysin O (PFO) toxin, IL-10, IL-22, andTNF-α in the jejunum and colon tissues, and alleviate the changes in gut microbiota structure caused by C. perfringens infection, which showed better therapeutic effects and indicated that the metabolites of G2B9-Q are essential mediators for their beneficial effects. Therefore, the CFS of G2B9-Q could potentially replace antibiotics in treating C. perfringens infection.

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.

Ex vivo pharmacokinetic/pharmacodynamic of hexahydrocolupulone against Clostridium perfringens in broiler chickens.

The economic impact of necrotizing enteritis (NE) resulting from Clostridium perfringens infection has been significant within the broiler industry. This study primarily investigated the antibacterial efficacy of hexahydrocolupulone against C. perfringens, and its pharmacokinetics within the ileal contents of broiler chickens. Additionally, a dosing regimen was developed based on the pharmacokinetic/pharmacodynamic (PK/PD) model specific to broiler chickens. Results of the study indicated that the minimum inhibitory concentration (MIC) of hexahydrocolupulone against C. perfringens ranged from 2 mg/L to 16 mg/L in MH broth. However, in ileal content, the MIC ranged from 8 mg/L to 64 mg/L. The mutation prevention concentration (MPC) in the culture medium was found to be 128 mg/L. After oral administration of hexahydrocolupulone at a single dosage of 10-40 mg/kg bodyweight, the peak concentration (Cmax), maximum concentration time (Tmax), and area under the concentration-time curve (AUC) in ileal content of broiler chickens were 291.42-3519.50 μg/g, 1-1.5 h, and 478.99-3121.41 μg h/g, respectively. By integrating the in vivo PK and ex vivo PD data, the AUC0-24h/MIC values required for achieving bacteriostatic, bactericidal, and bacterial eradication effects were determined to be 36.79, 52.67, and 62.71 h, respectively. A dosage regimen of 32.9 mg/kg at 24 h intervals for a duration of 3 days would yield therapeutic efficacy in broiler chickens against C. perfringens, provided that the MIC below 4 mg/L.

STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING‑deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

Extract of Scutellaria baicalensis and Lonicerae flos improves growth performance, antioxidant capacity, and intestinal barrier of yellow-feather broiler chickens against Clostridium perfringens

In this study, we aimed to investigate the effect of Scutellaria baicalensis and Lonicerae Flos (SL) extract on the growth performance and intestinal health of yellow-feather broilers following a Clostridium perfringens challenge. In total, 600 one-day-old yellow-feather broilers were divided into five treatments (6 replicate pens of 20 birds per treatment), including a control (Con) group fed a basal diet and the infected group (iCon) fed a basal diet and infected with Clostridium perfringens, the other 3 groups receiving different doses of SL (150, 300, and 450 mg/kg) and infected with Clostridium perfringens. The total experimental period was 80 d. When the birds were 24-days-old, a subclinical necrotizing enteritis model was induced by orally inoculating the birds with 11,000 oocysts of mixed Eimeria species on d 24, followed by C. perfringens (108 CFU/mL) from d 28 to 30. The birds were evaluated for parameters such as average weight gain (AWG), average daily feed intake (ADFI), mortality, feed conversion ration (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. Results indicated that C. perfringens infection led to reduced AWG and the levels of tight junction proteins, increased the FCR, ileum E. coli load, and intestinal permeability, causing damage to the intestinal mucosal barrier (P < 0.05). Compared with the infected group, supplementing 300 mg/kg of SL significantly increased AWG at 43 to 80 d, the ratio of villus height to crypt depth in the jejunum and ileum at 35 d, and the activity of superoxide dismutase (SOD) in serum. It also significantly reduced the FCR at 22 to 42 d, intestinal lesion score, and the amount of C. perfringens in the ileum (P < 0.05). Additionally, compared with the infected group, the addition of 300 mg/kg SL significantly increased mRNA levels of claudin-2, claudin-3, mucin-2, and toll-like receptor 2 (TLR-2) in the ileum of infected birds at 35 d of age. In conclusion, supplementation with SL extract could effectively mitigate the negative effects of C. perfringens challenge by improving intestinal barrier function and histomorphology, positively influencing the growth performance of challenged birds.

Lactobacilli-derived adjuvants combined with immunoinformatics-driven multi-epitope antigens based approach protects against Clostridium perfringens in a mouse model

Clostridium perfringens is ubiquitously distributed and capable of secreting toxins, posing a significant threat to animal health. Infections caused by Clostridium perfringens, such as Necrotic Enteritis (NE), result in substantial economic losses to the livestock industry annually. However, there is no effective commercial vaccine available. Hence, we set out to propose an effective approach for multi-epitope subunit vaccine construction utilizing biomolecules. We utilized immunoinformatics to design a novel multi-epitope antigen against C. perfringens (CPMEA). Furthermore, we innovated novel bacterium-like particles (BLPs) through thermal acid treatment of various Lactobacillus strains and selected BLP23017 among them. Then, we detailed the structure of CPMEA and BLPs and utilized them to prepare a multi-epitope vaccine. Here, we showed that our vaccine provided full protection against C. perfringens infection after a single dose in a mouse model. Additionally, BLP23017 notably augmented the secretion of secretory immunoglobulin A (sIgA) and enhanced antibody production. We conclude that our vaccine possess safety and high efficacy, making it an excellent candidate for preventing C. perfringens infection. Moreover, we demonstrate our approach to vaccine construction and the preparation of BLP23017 with distinct advantages may contribute to the prevention of a wider array of diseases and the novel vaccine development.

Broad host range phages target global Clostridium perfringens bacterial strains and clear infection in five-strain model systems.

Clostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αβ T cells) and natural IEL (TCRαβCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαβ+CD4-CD8-, TCRαβ+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαβ+CD4-CD8- and TCRαβ+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1β and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

Association of Bacillus subtilis and Bacillus amyloliquefaciens: minimizes the adverse effects of necrotic enteritis in the gastrointestinal tract and improves zootechnical performance in broiler chickens

This study aimed to evaluate the efficiency and capacity of the probiotic composed of Bacillus subtilis and Bacillus amyloliquefaciens, in improving the zootechnical performance of broiler chickens challenged with Eimeria spp. and Clostridium perfringens. The broilers were distributed in a completely randomized design in poultry isolators (12 birds each), resulting in 3 treatments: T1 (control, no challenge and no Bacillus in diet), T2 (challenged with Eimeria spp., followed by Clostridium perfringens infection and no Bacillus in the diet), and T3 (challenged with Eimeria spp., Clostridium perfringens and treated with Bacillus subtilis and Bacillus amyloliquefaciens). They were evaluated for a period of 29 d, divided into preinitial (1-7 d of age), initial (8-21 d), and growth (22-29 d) phases. Assessments of body weight, weight gain, feed consumption, and feed conversion were conducted, along with the classification of the scores and optical microscopy of the tract gastrointestinal. The animals challenged and treated with the probiotic containing Bacillus spp. showed improved indicators of zootechnical performance. Additionally, the animals challenged and treated (T3) had a better score for intestinal lesions compared to the other treatment groups. Therefore, the probiotic consisting of Bacillus subtilis and Bacillus amyloliquefaciens could be considered an effective option for disease prevention and improving the zootechnical performance of broiler chickens.