Clostridioides difficile Toxins: Host Cell Interactions and Their Role in Disease Pathogenesis.

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.

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.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

Dietary supplementation of prebiotic yeast Saccharomyces cerevisiae cell wall promotes growth performance and intestinal health in broiler chickens challenged with Clostridium perfringens.

1. This study evaluated the effectiveness of yeast (Saccharomyces cerevisiae) cell wall (YCW) supplementation on the growth performance, carcase characteristics, serum biomarkers, liver function, ileal histology and microbiota of broiler chickens challenged with Clostridium perfringens (C. perfringens).2. In a 35-d trial, 240 chicks aged 1-d-old were randomly assigned to one of four treatment groups, each with 10 replicates: control (CON) with no challenge or additives, challenged with C. perfringens (CHAL), CHAL and supplemented with YCW at either 0.25 g/kg (YCW0.25) or 0.5 g/kg (YCW0.5).3. In comparison to CON, the CHAL birds had reduced growth performance, survival rate, dressing percentage, breast meat yield, levels of total protein (TP), globulin (GLO), glucose (GLU), total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD), as well as a decreased Lactobacillus population (P < 0.01). Additionally, this group showed elevated levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and C. perfringens count (P < 0.01). Compared to CHAL, the YCW0.25 or YCW0.5 groups had improved growth performance, survival rate, dressing percentage, breast meat yield, levels of TP, GLO, GLU, and T-AOC, as well as the activities of T-SOD, GOT, and GPT, villus height, villus surface area, villus height to crypt depth ratio, and the populations of both Lactobacillus and C. perfringens; (P < 0.01).4. The data suggested that YCW supplementation at either 0.25 or 0.50 g/kg can restore the growth performance of broiler chickens during a C. perfringens challenge.

Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Cellular Immune Responses in Lymphoid Tissues of Broiler Chickens Experimentally Infected with Necrotic Enteritis-Producing Clostridium perfringens Strains.

Host cellular responses against Clostridium perfringens (CP), the causative agent of necrotic enteritis (NE) in chickens, are poorly understood. In the present study, we first tested the NE-producing ability of seven netB+ CP strains (CP5, CP18, CP26, CP64, CP67, CP68, and NCNE-1), using an experimental infection model of broiler chickens. Evaluation of intestinal gross lesions showed that all the strains, except CP5, were able to produce NE, while CP26 and CP64 strains produced relatively more severe lesions when compared with other groups. Next, cellular responses in the cecal tonsil (CT), bursa of Fabricius, and spleen were evaluated in chickens infected with strains representing variation in the level of virulence, namely, avirulent CP5, virulent CP18, and a relatively more virulent CP26 strain. Immunophenotyping analysis showed that CT or splenic macrophage frequencies were significantly higher in CP18- and CP26-infected chickens compared with uninfected controls, while the frequencies of γδ T-cells and B-cells in the CT of CP26-infected chickens were significantly higher than those in the uninfected, CP5- or CP18-infected groups. The T-cell analysis showed that chickens infected with CP18 and CP26 had a significantly higher number of splenic CD4+ and CD8+ T-cells expressing CD44 and CD28 activation molecules, while CP26-infected chickens also had significantly increased CT frequency of these activated CD4+ and CD8+ T-cells when compared with uninfected or CP5-infected groups. Collectively, our findings suggested that cellular responses, including activation of T-cells, are selectively induced against virulent CP strains and that the NE-producing characteristics of this pathogen may influence the outcome of immunity to NE.

Respuestas inmunes celulares en tejidos linfoides de pollos de engorde infectados experimentalmente con cepas de Clostridium perfringens productoras de enteritis necrótica. Las respuestas celulares del huésped contra Clostridium perfringens (CP), el agente causante de la enteritis necrótica (NE) en pollos, son poco conocidas. En el presente estudio, primero se analizó la capacidad de producción de enteritis necrótica de siete cepas de C. perfringens netB+ (CP5, CP18, CP26, CP64, CP67, CP68 y NCNE-1), utilizando un modelo de infección experimental de pollos de engorde. La evaluación de las lesiones macroscópicas intestinales mostró que todas las cepas, excepto CP5, podían producir enteritis necrótica, mientras que las cepas CP26 y CP64 produjeron lesiones relativamente más severas en comparación con los otros grupos. Posteriormente, se evaluaron las respuestas celulares en las tonsilas cecales (CT), la bolsa de Fabricio y en el bazo de pollos infectados con cepas que representan variaciones en el nivel de virulencia, por ejemplo las cepas CP5 avirulenta, CP18 virulenta y la cepa CP26 relativamente más virulenta. El análisis de inmunofenotipado mostró que las frecuencias de los macrófagos esplénicos o de las tonsilas cecales fueron significativamente más altas en los pollos infectados con las cepas CP18 y CP26 en comparación con los controles no infectados, mientras que las frecuencias de células T γd y células B en tonsilas cecales de los pollos infectados con la cepa CP26 fueron significativamente más altas que las de los pollos de los grupos no infectados, o infectados con las cepas CP5 o CP18. El análisis de células T mostró que los pollos infectados con las cepas CP18 y CP26 tenían un número significativamente mayor de células esplénicas T CD4+ y CD8+ que expresaban moléculas de activación CD44 y CD28, mientras que los pollos infectados con la cepa CP26 también tenían una frecuencia significativamente mayor en las tonsilas cecales de estas células T CD4+ y CD8+ activadas en comparación con grupos no infectados o infectados con la cepa CP5. En conjunto, estos hallazgos sugirieron que las respuestas celulares, incluida la activación de las células T, se inducen selectivamente contra las cepas virulentas de C. perfringens y que las características productoras de enteritis necrótica de este patógeno pueden influir en el resultado de la inmunidad contra la enteritis necrótica.

Molecular characterization and function of JAK/STAT pathway in IPEC-J2 cells during Clostridium perfringens beta2 toxin stimulation.

Intestinal infection with C. perfringens is responsible for outbreaks of diarrhea in piglets. Janus kinase / signal transducer and activator of transcription (JAK/STAT) is a vital signaling pathway that regulates cellular activity and inflammatory response, closely correlated with multiple diseases development and advances. Currently, the potential effect of JAK/STAT on C. perfringens beta2 (CPB2) treatment on porcine intestinal epithelial (IPEC-J2) cells has not been explored. The expression of JAK/STAT genes or proteins in IPEC-J2 cells induced by CPB2 were observed by qRT-PCR and Western blot, and further used WP1066 to explore the effect of JAK2/STAT3 on mechanism employed by CPB2 on apoptosis, cytotoxicity, oxidative stress and inflammatory cytokines of IPEC-J2 cells. JAK2, JAK3, STAT1, STAT3, STAT5A and STAT6 were highly expressed in CPB2-induced IPEC-J2 cells, among which STAT3 had the highest expression. Moreover, apoptosis, cytotoxicity and oxidative stress were attenuated via blocking the activation of JAK2/STAT3 by using WP1066 in CPB2-treated IPEC-J2 cells. Furthermore, WP1066 significantly suppressed the secretion of interleukin (IL)-6, IL-1ß and TNF-α induced by CPB2 in IPEC-J2 cells.Our findings provide some insights into the functional roles of JAK2/STAT3 in piglets against to C. perfringens infection.

Efficacy of Bacillus subtilis to replace in-feed antibiotics of broiler chickens under necrotic enteritis-challenged experiments: a systematic review and meta-analysis.

Necrotic enteritis (NE) and coccidiosis are among the most prevalent infectious diseases in broiler chickens, contributing to large profitability losses. Bacillus subtilis is a promising direct-fed probiotic to counter various pathogens infection in broiler chickens. Here, we performed a meta-analysis to investigate the effects of B. subtilis on broiler chickens performance. A total of 28 studies were selected according to a PRISMA checklist. Random-effect model and mixed-effect model of meta-analysis were fitted to estimate the overall effects of B. subtilis (BS) treatment compared to either the control group (CON) or NE-infected group (NEinf) as a baseline. Hedges' g effect size and its variance were used as estimators of standardized mean difference (SMD) calculation where the results were presented at a 95% confidence interval (95% CI) of the SMD. Overall, NEinf broiler chickens depressed (P < 0.01) body weight (BW), average daily gain (ADG), and feed intake, and elevated (P < 0.01) feed conversion ratio (FCR). Treatment with BS improved ADG and final BW of NEinf with no difference (P = 0.15) between BS and antibiotics (AB), indicating that they had comparable efficacy to treat NE in broiler chickens. BS supplemented to uninfected CON (BSS) improved (P < 0.01) final BW, ADG, and FCR. Compared to CON, BS, and AB failed to recover the FCR but these treatments decreased (P < 0.01) FCR when compared to the NEinf group with similar efficacy (P = 0.97). As expected, NEinf birds had a higher mortality rate (P < 0.01) and higher lesion score (P < 0.01) compared to CON, and treatment using AB and BS successfully decreased (P < 0.01) the mortality rate and lesion score. Compared to BS, AB was more effective to lower (P = 0.01) mortality rate, but comparable (P = 0.65) to minimize lesion score. To conclude, B. subtilis could be an effective natural additive to replace in-feed antibiotics in broiler chickens challenged with C. perfringens. However, the efficacy to reduce mortality rate was better with antibiotics treatment.

Nigella sativa as an antibiotic alternative to promote growth and enhance health of broilers challenged with Eimeria maxima and Clostridium perfringens.

The poultry industry has significant coccidiosis and necrotic enteritis (NE) challenges, leading to high mortality and unacceptable growth without antibiotic treatment. This research explored supplementing Nigella sativa (black cumin) seed oil in poultry feed to mitigate coccidiosis and prevent or lessen NE in broilers. In vivo studies consisted of 384 and 320 Cobb 500 male broiler chicks distributed in a randomized complete block experimental design for trials 1 and 2, respectively. The first trial compared 3 concentrations (1, 2, and 5 mL/kg) of black cumin seed oil (BCSO), and trial 2 compared 2 concentrations (2 and 5 mL/kg) BCSO, with birds challenged with Eimeria maxima and Clostridium perfringens (Cp) strains Cp#6 and Cp#4, respectively. Broiler live performance, NE disease outcomes, and Cp populations were measured for both trials. A commercially available BCSO oil product, determined in a preliminary in vitro study to have the highest anti-Cp activity, was selected for in vivo studies. Gas chromatography-mass spectrometry analysis indicated the major bioactive compounds p-cymene, thymoquinone, carvacrol, and thymol were present in the BCSO. In trial 1 with strain Cp#6, BCSO concentrations of 2 and 5 mL/kg reduced NE lesion score and mortality rate to 1.6% compared with 7.8% for positive control, with no adverse impact on live performance. In trial 2 with strain Cp#4, BCSO reduced NE lesion scores and mortality rate to 35.9% compared with 51.6% for positive control and also improved weight gain when there was a Cp infection in broiler chickens. The current study compared NE in broilers challenged with 2 different Cp strains producing different levels of NE. Following Cp infection, both the population of vegetative cells and spores of Cp in cecal contents decreased for all treatments in trial 2. In conclusion, BCSO at concentrations of 2 and 5 mL/kg enhanced broiler live performance and alleviated NE and has potential as a natural, non-medication antimicrobial nutritional supplement for use as a feed additive in chickens.

Experimental acute Clostridium perfringens type D enterotoxemia in sheep is not characterized by specific renal lesions.

Type D enterotoxemia, caused by Clostridium perfringens epsilon toxin (ETX), is one of the most economically important clostridial diseases of sheep. Acute type D enterotoxemia is characterized by well-documented lesions in the nervous, cardiocirculatory, and pulmonary systems. However, discrepancies and confusion exist as to whether renal lesions are part of the spectrum of lesions of this condition, which is controversial considering that for many decades it has been colloquially referred to as "pulpy kidney disease." Here, the authors assess renal changes in an experimental model of acute type D enterotoxemia in sheep and evaluate the possible role of ETX in their genesis. Four groups of 6 sheep each were intraduodenally inoculated with either a wild-type virulent C. perfringens type D strain, an etx knockout mutant unable to produce ETX, the etx mutant strain complemented with the wild-type etx gene that regains the ETX toxin production, or sterile culture medium (control group). All sheep were autopsied less than 24 hours after inoculation; none of them developed gross lesions in the kidneys. Ten predefined histologic renal changes were scored in each sheep. The proportion of sheep with microscopic changes and their severity scores did not differ significantly between groups. Mild intratubular medullary hemorrhage was observed in only 2 of the 12 sheep inoculated with the wild-type or etx-complemented bacterial strains, but not in the 12 sheep of the other 2 groups. The authors conclude that no specific gross or histologic renal lesions are observed in sheep with experimental acute type D enterotoxemia.

Intestinal bile acids provide a surmountable barrier against C. difficile TcdB-induced disease pathogenesis.

Intestinal bile acids play an essential role in the Clostridioides difficile lifecycle having been shown in vitro to modulate various aspects of pathogenesis, including spore germination, vegetative growth, and more recently the action of the primary virulence determinant, TcdB. Here, we investigated whether physiological levels of the total pool of intestinal bile acids in mice and humans protect against TcdB action. Small molecules extracted from the lumenal contents of the small intestine, cecum, colon, and feces were found to inhibit TcdB in accordance with the differential amounts of total bile acids in each compartment. Extracts from antibiotic-treated and germ-free mice, despite harboring dramatically altered bile acid profiles, unexpectedly also prevented TcdB-induced cell rounding to similar extents. We show that protection, however, is surmountable and can be overcome at higher doses of TcdB-typical to those seen during severe C. difficile infection-suggesting that the protective properties of intestinal bile acids are operant primarily under low to moderate toxin levels. Taken together, these findings demonstrate a role for intestinal bile acids in attenuating virulence, provide insights into asymptomatic carriage of toxigenic C. difficile, and inform strategies to manipulate bile acid levels for therapeutic benefit.

Clostridial diarrheas in piglets: A review.

Clostridium perfringens type C and Clostridioides difficile are the main enteric clostridial pathogens of swine and are both responsible for neonatal diarrhea in this species. The role of Clostridum perfringes type A is under discussion. History, clinical signs, gross lesions and histological findings are the basis for a presumptive diagnosis of C. perfringens type C or C. difficile infection. Confirmation is based upon detection of beta toxin of C. perfringens type C or toxin A/B of C. difficile, respectively, in intestinal contents or feces. Isolation of C. perfringens type C and/or C. difficile is highly suggestive of infection by these microorganisms but it is not enough to confirm a diagnosis as they may be found in the intestine of some healthy individuals. Diagnosis of C. perfringens type A-associated diarrhea is more challenging because the diagnostic criteria have not been well defined and the specific role of alpha toxin (encoded by all strains of this microorganism) and beta 2 toxin (produced by some type A strains) is not clear. The goal of this paper is to describe the main clostridial enteric diseases of piglets, including etiology, epidemiology, pathogenesis, clinical signs, pathology and diagnosis.

Clostridium perfringens strains proliferate to high counts in the broiler small intestinal tract, in accordance with necrotic lesion severity, and sporulate in the distal intestine.

Clostridium (C.) perfringens is the causative agent of necrotic enteritis (NE), an important enteric disease in poultry. Although a variety of virulence factors have been identified and as such the pathogenesis is well studied, data on colonization and sporulation during passage in the intestinal tract are scarce. This study, therefore, evaluated the behaviour of C. perfringens in the different intestinal compartments of broiler chickens during a NE trial. Necrotic enteritis-associated lesions were mostly found in the jejunum, where they were significantly more severe compared to the duodenum and ileum. Furthermore, a positive correlation between the total number of vegetative C. perfringens cells in the duodenum, jejunum, ileum, or distal colon and disease severity was observed. Additionally, in the caecum and distal colon, C. perfringens was mainly present as a spore. This observation has important consequences for NE treatment and prevention, as both the vegetative cells and C. perfringens spores should be targeted to avoid uptake of spores from the litter and reinfection of the birds after antibiotic treatment.

Spray-dried porcine plasma enhances feed efficiency, intestinal integrity, and immune response of broilers challenged with necrotic enteritis.

Re-emergence of enteric diseases in the postantibiotic era has imposed severe loss to the poultry industry leading to the urgent need for appropriate additives to maintain gut health. Recently, more attention has been paid to animal plasma due to its high concentrations of active components such as albumins and globulins. The objective of this study was to evaluate the effects of spray-dried porcine plasma (SDP) supplementation during the starter phase (d 0-10) on growth performance, intestine health, and immune response of broilers under necrotic enteritis (NE) challenge. A total of 720 day-old male broiler parental line chicks (Ross 308) were randomly assigned to a 2 (NE challenge: no, yes) × 2 (SDP: 0, 2%) factorial arrangement with 12 replications of 15 chicks each. To induce NE, birds were inoculated with live Eimeria vaccine on d 9 and Clostridium perfringens on d 14. The body weight of birds and feed consumption were measured per pen on d 8, 10, 24, and 29 to calculate performance parameters. On d 16, three birds per pen were sampled to analyse the intestinal lesion score, gut permeability, villi morphology, relative weight of organs, and immune response. Results showed that SDP improved (P < 0.001) FCR in the pre-challenge phase (d 0-8). The results indicated that supplementing SDP lowered (P < 0.01) FCR at the end of the experiment (d 29). Dietary SDP decreased (P < 0.05) the concentration of FITC-d in serum samples of challenged broilers, although it did not affect the intestinal morphology and lesion score. Birds fed with SDP had a higher (P < 0.05) relative weight of bursa (g/kg live body weight) compared to non-supplemented birds. Supplementing SDP reduced the concentration of interleukin-6 (P < 0.05) and α-1 acid glycoprotein (P = 0.051) in serum samples of broilers. In conclusion, supplementation of SDP in the starter phase enhanced feed efficiency and gut integrity in NE challenged broilers, possibly through manipulating the immune response, while further studies targeting intestinal microflora and key genes are required to explore the mode of action.

Discovery of a novel natural product inhibitor of Clostridioides difficile with potent activity in vitro and in vivo.

Clostridioides difficile infection is a global health threat and remains the primary cause of hospital-acquired infections worldwide. The burgeoning incidence and severity of infections coupled with high rates of recurrence have created an urgent need for novel therapeutics. Here, we report a novel natural product scaffold as a potential anticlostridial lead with antivirulence properties and potent activity both in vitro and in vivo. A whole cell phenotypic screening of 1,000 purified natural products identified 6 compounds with potent activity against C. difficile (minimum inhibitory concentration (MIC) range from 0.03 to 2 µg/ml). All these 6 compounds were non-toxic to human colorectal cells. The natural product compounds also inhibited the production of key toxins, TcdA and TcdB, the key virulence determinants of C. difficile infection pathology. Additionally, the compounds exhibited rapid bactericidal activity and were superior to the standard-of-care antibiotic vancomycin, in reducing a high inoculum of C. difficile in vitro. Furthermore, a murine model of C. difficile infection revealed that compound NP-003875 conferred 100% protection to the infected mice from clinical manifestations of the disease. Collectively, the current study lays the foundation for further investigation of the natural product NP-003875 as a potential therapeutic choice for C. difficile infection.

Recombinant Limosilactobacillus (Lactobacillus) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis.

Necrotic enteritis (NE), caused by Clostridium perfringens, is an intestinal disease with devastating economic losses to the poultry industry. NE is a complex disease and predisposing factors that compromise gut integrity are required to facilitate C. perfringens proliferation and toxin production. NE is also characterized by drastic shifts in gut microbiota; C. perfringens is negatively correlated with Lactobacilli. Vaccines are only partially effective against NE and antibiotics suffer from the concern of resistance development. These strategies address only some aspects of NE pathogenesis. Thus, there is an urgent need for alternative strategies that address multiple aspects of NE biology. Here, we developed Limosilactobacillus (Lactobacillus) reuteri vectors for in situ delivery of nanobodies against NetB and α toxin, two key toxins associated with NE pathophysiology. We generated nanobodies and showed that these nanobodies neutralize NetB and α toxin. We selected L. reuteri vector strains with intrinsic benefits and demonstrated that these strains inhibit C. perfringens and secrete over 130 metabolites, some of which play a key role in maintaining gut health. Recombinant L. reuteri strains efficiently secreted nanobodies and these nanobodies neutralized NetB. The recombinant strains were genetically and phenotypically stable over 480 generations and showed persistent colonization in chickens. A two-dose in ovo and drinking water administration of recombinant L. reuteri strains protected chickens from NE-associated mortality. These results provide proof-of-concept data for using L. reuteri as a live vector for delivery of nanobodies with broad applicability to other targets and highlight the potential synergistic effects of vector strains and nanobodies for addressing complex diseases such as NE.

The effects of feed supplementing Akkemansia muciniphila on incidence, severity, and gut microbiota of necrotic enteritis in chickens.

Akkermansia muciniphila (AM) is a mucin-degrading anaerobe, exerting beneficial effects on gut integrity improvement, inflammatory alleviation, and metabolic regulations in humans. Excess amounts of mucin and mucogenesis in the gut facilitate the development of necrotic enteritis (NE) in chickens. The study aimed to evaluate the effects of oral inoculation of AM on NE prevention and gut modulation in a NE-reproduced model coinfecting with Clostridium perfringens (CP) and Eimeria parasites. A total of 105 commercial 1-day-old broilers were randomly allocated into 5 groups, respectively challenged with Eimeria (Eimeria group), Eimeria and CP (Eimeria+CP group), Eimeria and CP with AM (Eimeria+CP+AM group), Eimeria and AM (Eimeria+AM group), and a placebo (Noninfected group). The treatment of AM exhibited a low degree of amelioration on NE severity. The application neither protected broilers from NE by decreasing NE-positive numbers nor reached a significant reduction in lesion scores in the small intestines. The development of NE reduced species diversity in jejunal microbiota; the pretreatments of AM exacerbated the consequence by losing species richness and promoted the similarity of the jejunal microbial community presented in the Eimeria+CP group. The participation of AM enhanced the increments of genera Clostridium sensu stricto 1 and Escherichia_Shigella and decreased the number of Lactobacillus. The significant variations of genera Clostridium sensu stricto 1 and Lactobacillus in jejunal microbiota were associated with NE development and promotion. In conclusion, oral inoculation of AM promoted the development of NE and modulated the jejunal microbiota favorable for CP overgrowth in broilers. The application of AM as a probiotic in broilers should be cautious on account of the effects to predispose NE.

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection.

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.

Antimicrobial activity of sophorolipids against Eimeria maxima and Clostridium perfringens, and their effect on growth performance and gut health in necrotic enteritis.

The in vitro antimicrobial activity of sophorolipids (SLs) against Eimeria maxima and Clostridium perfringens, and the in vivo effects of SLs on growth performance and gut health in necrotic enteritis (NE)-afflicted broiler chickens were studied. To test the direct killing effects of SLs on enteric pathogens, 2.5 × 105 freshly prepared sporozoites of each Eimeria acervulina, E. maxima, and E. tenella were placed in each well of a 96-well plate, and the vegetative stage of Clostridium perfringens was prepared at 1 × 109 cfu/well. Four different SLs (C18:1 lactonic diacetyled SL [SL1], C18:1 deacetyled SL [SL2], C18:1 monoacetyled SL [SL3], and C18:1 diacetyled SL [SL4]), and 2 anticoccidial chemical controls, decoquinate and monensin, were evaluated at 3 dose levels (125 µg/mL, 250 µg/mL, and 500 µg/mL). Samples were incubated at 41°C for 3 h, and microbial survival ratios were measured by using a cell counter to quantify the number of live microbes stained by fluorescent dye. A total of 336 (0-day-old) male commercial broiler chickens were used to assess the effects of SLs in vivo. Chickens were randomly allocated to 6 treatment groups (7 chickens per cage, 8 cages per treatment) as follows: a control group which received a basal diet (CON), a negative control group (NC) which received a basal diet and NE challenge, and 4 SL treatment groups with NE (NC+SL1, NC+SL2, NC+SL3, and NC+SL4). The inclusion rates of SLs in each group were 200 mg/kg of feed. NE-induced chickens were orally infected with E. maxima (10,000 oocysts/chicken) on d 14, followed by C. perfringens (1 × 109 cfu/chicken) on d 19. Disease parameters measured included gut lesion scores, intestinal cytokine production, and level of tight junction protein expression. Data were analyzed using a Mixed Model (PROC MIXED) in SAS. In vitro (Experiment 1), all SLs dose-dependently decreased (P < 0.001) the viability of the three species of Eimeria sporozoites and C. perfringens. In vivo (Experiment 2), dietary SLs increased (P < 0.001) body weight and average daily gain of broiler chickens infected with NE. Dietary SL1 and SL4s increased (P < 0.05) feed conversion ratio compared to NC. Furthermore, SL1 and SL4 decreased (P < 0.05) gut lesion scores in combination with increased expression of IL1ß, IL8, TNFSF15, and IL10 genes (P < 0.05) in NE-afflicted chickens. Overall, dietary SLs promoted growth performance, intestinal immune responses, and intestinal barrier integrity of NE-afflicted, young broiler chickens.

Intestinal changes and immune responses during Clostridium perfringens-induced necrotic enteritis in broiler chickens.

Clostridium perfringens-induced necrotic enteritis (NE) is an economically important disease of broiler chickens. The present study evaluated the effect of C. perfringens on the intestinal histomorphometry, enteric microbial colonization, and host immune responses using 3 experimental NE reproduction methods. The experimental groups consisted of 1) unchallenged Control diet (corn-soybean meal), 2) Control diet + Eimera inoculation at d 11 followed by C. perfringens challenge at d 15 (ECp), 3) Wheat-based diet + C. perfringens challenge (WCp), and 4) Wheat-based diet + Eimeria inoculation followed by C. perfringens challenge (WECp). The results showed that chickens receiving ECp and WECp had reduced (P < 0.05) bird performance coupled with enteric gross lesions and epithelial damage at d 17 and 24 of age compared to unchallenged control birds. These ECp and WECp administered birds also had increased (P < 0.05) ileal colonization by clostridia and E. coli at d 17 and 24, while the resident Lactobacillus counts were reduced (P < 0.05) at d 24 of age. Furthermore, at d 24, jejunal transcription of IL-6, IL-10, annexin-A1 and IL-2 genes was upregulated (P < 0.05) in the ECp group, whereas the transcription of TNF receptor associated factor (TRAF)-3 gene was increased (P < 0.05) in WECp treated birds when compared to unchallenged control group. Additionally, stimulation of chicken splenocytes and cecal tonsilocytes with virulent C. perfringens bacilli or their secretory proteins resulted in a higher (P < 0.05) frequency of T cells and their upregulation of MHC-II molecule, as determined by flow cytometry. These findings suggest that C. perfringens, while inducing epithelial damage and changes in microbiota, can also trigger host immune responses. Furthermore, NE reproduction methods using coccidia with or without the wheat-based dietary predisposition seem to facilitate an optimal NE reproduction in broiler chickens and thus, may provide better avenues for future C. perfringens research.