Necrotic Enteritis In Broiler Chickens Research Articles

Evaluation of Immunization with Native NetB in Addition to Reactive Proteins Against Necrotic Enteritis in Broiler Chickens

Evaluation of Immunization with Native NetB in Addition to Reactive Proteins Against Necrotic Enteritis in Broiler Chickens

Clostridium perfringens chitinases, key enzymes during early stages of necrotic enteritis in broiler chickens.

The interaction between bacteria and the intestinal mucus is crucial during the early pathogenesis of many enteric diseases in mammals. A critical step in this process employed by both commensal and pathogenic bacteria focuses on the breakdown of the protective layer presented by the intestinal mucus by mucolytic enzymes. C. perfringens type G, the causative agent of necrotic enteritis in broilers, produces two glycosyl hydrolase family 18 chitinases, ChiA and ChiB, which display distinct substrate preferences. Whereas ChiB preferentially processes linear substrates such as chitin, ChiA prefers larger and more branched substrates, such as carbohydrates presented by the chicken intestinal mucus. Here, we show via crystal structures of ChiA and ChiB in the apo and ligand-bound forms that the two enzymes display structural features that explain their substrate preferences providing a structural blueprint for further interrogation of their function and inhibition. This research focusses on the roles of ChiA and ChiB in bacterial proliferation and mucosal attachment, two processes leading to colonization and invasion of the gut. ChiA and ChiB, either supplemented or produced by the bacteria, led to a significant increase in C. perfringens growth. In addition to nutrient acquisition, the importance of chitinases in bacterial attachment to the mucus layer was shown using an in vitro binding assay of C. perfringens to chicken intestinal mucus. Both an in vivo colonization trial and a necrotic enteritis trial were conducted, demonstrating that a ChiA chitinase mutant strain was less capable to colonize the intestine and was hampered in its disease-causing ability as compared to the wild-type strain. Our findings reveal that the pathogen-specific chitinases produced by C. perfringens type G strains play a fundamental role during colonization, suggesting their potential as vaccine targets.

Supplementation with organic yeast-derived selenium provides immune protection against experimental necrotic enteritis in broiler chickens

Necrotic enteritis (NE) is a potentially fatal poultry disease that causes enormous economic losses in the poultry industry worldwide. The study aimed to evaluate the effects of dietary organic yeast-derived selenium (Se) on immune protection against experimental necrotic enteritis (NE) in commercial broilers. Chickens were fed basal diets supplemented with different Se levels (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, Clostridium perfringens (C. perfringens) was orally administered at 14 days of age post hatch. The results showed that birds fed 0.25 Se mg/kg exhibited significantly increased body weight gain compared with the non-supplemented/infected birds. There were no significant differences in gut lesions between the Se-supplemented groups and the non-supplemented group. The antibody levels against α-toxin and NetB toxin increased with the increase between 0.25 Se mg/kg and 0.50 Se mg/kg. In the jejunal scrapings and spleen, the Se-supplementation groups up-regulated the transcripts for pro-inflammatory cytokines IL-1β, IL-6, IL-8, iNOS, and LITAF and avian β-defensin 6, 8, and 13 (AvBD6, 8 and 13). In conclusion, supplementation with organic yeast-derived Se alleviates the negative consequences and provides beneficial protection against experimental NE.

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 and/or in drinking water method twice, on d 8 and 13. Non-challenged 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 non-challenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Non-challenged 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 d19, birds received bromelain in drinking water had lower faecal 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.

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

BackgroundNecrotic 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.MethodsThis 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.ResultsOur 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.ConclusionOur 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.

Efficacy of administration of ampicillin and/or dimetridazole in the management of experimentally induced necrotic enteritis in broiler chickens.

Efficacy of administration of ampicillin and/or dimetridazole in the management of experimentally induced necrotic enteritis in broiler chickens.

Baicalin-aluminum alleviates necrotic enteritis in broiler chickens by inhibiting virulence factors expression of Clostridium perfringens.

Clostridium perfringens type A is the main cause of necrotic enteritis (NE) in chickens. Since the use of antibiotics in feed is withdrawn, it is imperative to find out suitable alternatives to control NE. Baicalin-aluminum complex is synthesized from baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi. The present study investigated the effects of baicalin-aluminum on the virulence-associated traits and virulence genes expression of C. perfringens CVCC2030, it also evaluated the in vivo therapeutic effect on NE. The results showed that baicalin-aluminum inhibited bacterial hemolytic activity, diminished biofilm formation, attenuated cytotoxicity to Caco-2 cells, downregulated the expression of genes encoding for clostridial toxins and extracellular enzymes such as alpha toxin (CPA), perfringolysin O (PFO), collagenase (ColA), and sialidases (NanI, NanJ). Additionally, baicalin-aluminum was found to negatively regulate the expression of genes involved in quorum sensing (QS) communication, including genes of Agr QS system (agrB, agrD) and genes of VirS/R two-component regulatory system (virS, virR). In vivo experiments, baicalin-aluminum lightened the intestinal lesions and histological damage, it inhibited pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) expression in the jejunal and ileal tissues. Besides, baicalin-aluminum alleviated the upregulation of C. perfringens and Escherichia coli and raised the relative abundance of Lactobacillus in the ileal digesta. This study suggests that baicalin-aluminum may be a potential candidate against C. perfringens infection by inhibiting the virulence-associated traits and virulence genes expression.

Effects of Eimeria maxima infection doses on growth performance and gut health in dual-infection model of necrotic enteritis in broiler chickens.

The objective of this study was to investigate the effects of the different doses of Eimeria maxima (EM) oocysts on growth performance and intestinal health in broiler chickens challenged with a dual infection model of necrotic enteritis (NE) using EM and NetB+ Clostridium perfringens (CP). A total of 432 fourteen-d-old male Cobb 500 broiler chickens were divided into 6 groups with 6 replicates each. The six different groups were as follows: Control, non-challenged; T0+, challenged with CP at 1 × 109 colony forming unit; T5K+, T0+ + 5,000 EM oocysts; T10K+, T0+ + 10,000 EM oocysts; T20K+; T0+ + 20,000 EM oocysts; and T40K+; T0+ + 40,000 EM oocysts. The challenge groups were orally inoculated with EM strain 41A on d 14, followed by NetB+ CP strain Del-1 on 4days post inoculation (dpi). Increasing EM oocysts decreased d 21 body weight, body weight gain, feed intake (linear and quadratic, p < 0.001), and feed efficiency (linear, p < 0.001) from 0 to 7 dpi. Increasing EM oocysts increased jejunal NE lesion score and intestinal permeability on 5, 6, and 7 dpi (linear, p < 0.05). On 7 dpi, increasing the infection doses of EM oocysts increased jejunal CP colony counts (linear, p < 0.05) and increased fecal EM oocyst output (linear and quadratic, p < 0.001). Furthermore, increasing the infection doses of EM oocysts decreased the villus height to crypt depth ratios and the goblet cell counts (linear, p < 0.05) on 6 dpi. Increasing EM oocysts downregulated the expression of MUC2, B0AT, B0,+AT, PepT1, GLUT2, AvBD3 and 9, LEAP2, and TLR4, while upregulating CLDN1, CATHL3, IL-1β, IFN-γ, TNFSF15, TNF-α, IL-10, and Gam56 and 82 on 6dpi (linear, p < 0.05). Additionally, increasing EM oocysts decreased Pielou's evenness and Shannon's entropy (linear, p < 0.01). In conclusion, increasing the infection doses of EM significantly aggravated the severity of NE and exerted negative impact on intestinal health from 5 to 7 dpi.

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.

Subclinical doses of dietary fumonisins and deoxynivalenol cause cecal microbiota dysbiosis in broiler chickens challenged with Clostridium perfringens.

Fusarium toxins are one of the most common contaminants in poultry diets. The co-occurrence of fumonisins (FUM) and deoxynivalenol (DON), even at a subclinical dose, negatively affects the growth performance, intestinal integrity and induce subclinical necrotic enteritis in broiler chickens. Loss of gut integrity can be expected to alter the intestinal microbiota's composition. The objective of this study was to identify the effects of combined FUM and DON on the cecal microbiome profile and predicted metabolic functions and a short chain fatty acid profile in broilers challenged with Clostridium perfringens. A total of 240 1 day-old chicks were randomly assigned to two treatments: a control diet and the control diet with 3 mg/kg FUM + 4 mg/kg DON each with eight replications. All the birds were received cocci vaccine at d0. All birds in both treatment groups were challenged with C. perfringens 1 × 108 CFU via feed on d 19 and 20 to achieve 5% mortality. On d 35, the FUM and DON contaminated diet numerically (P = 0.06) decreased the body weight gain (BWG) by 84 g compared to the control group. The bacterial compositions of the cecal contents were analyzed by sequencing the V3-V4 region of the 16S rRNA gene. Overall, microbial richness and diversity increased (P < 0.02) during the studied period (d 21-35). Cecal contents of birds in the FUM + DON group had greater (P < 0.05) microbial evenness and diversity (Shannon index) compared to the control group. FUM + DON exposure decreased (P = 0.001) the relative abundance of Proteobacteria in the cecal content, compared to the control group. The combined FUM + DON significantly increased the relative abundance of the Defluviitaleaceae and Lachnospiraceae families (P < 0.05) but decreased the abundances of the Moraxellaceae and Streptococcaceae (P < 0.05) compared to the control group birds. At the genus level, FUM + DON exposure decreased (P < 0.05) Acinetobacter and Pseudomonas abundance and had a tendency (P = 0.08) to decrease Thermincola abundance compared to the control group. In the ileum, no NE-specific microscopic abnormalities were found; however, the tip of the ileal villi were compromised. The present findings showed that dietary FUM and DON contamination, even at subclinical levels, altered cecal microbial composition, dysregulated intestinal functions, and impaired the gut immune response, potentially predisposing the birds to necrotic enteritis.

No change in avilamycin (Surmax® Premix) minimum inhibitory concentration for Clostridium perfringens isolates recovered from poultry up to 7 years post-approval in Canada.

Clostridium perfringens, the causative agent of necrotic enteritis (NE) in chickens, has an enormous economic impact on global broiler production. The non-medically important antibiotic avilamycin was approved in Canada in 2014 to prevent and control NE in broiler chickens. To compare avilamycin susceptibility in C. perfringens isolates collected pre- and 7 years post-avilamycin approval in Canada and determine the avilamycin resistance mutation frequency rate in C. perfringens. The MICs of avilamycin were determined for 89 strains of C. perfringens recovered from clinically relevant NE field cases pre-avilamycin approval between 2003 and 2013 (n = 50) and post-avilamycin approval between 2014 and 2021 (n = 39) across Canada. For determining the mutant prevention concentration (MPC) of avilamycin for C. perfringens strains, a strain with avilamycin MIC of 1 mg/L was randomly selected. MIC studies showed no difference in avilamycin susceptibility in pre-avilamycin and post-avilamycin isolates (MIC50/90: pre-avilamycin approval 2/2 mg/L and post-avilamycin approval 1/2 mg/L). The MPC was 8 × MIC (8 mg/L) for the selected strain. These findings suggest that the susceptibility of C. perfringens strains to avilamycin was not impacted by its continued use in the 7 years following its approval in Canada. Avilamycin, a non-medically important antibiotic, poses no threat to human health regarding cross-resistance or co-selection of other medically important antibiotics. These factors make avilamycin an appropriate choice for continued use in broiler chickens to prevent and control NE without increased antimicrobial resistance concerns.

Clostridium perfringens in Broiler Chickens: Isolation, Identification, Typing, and Antimicrobial Susceptibility

Necrotic enteritis (NE) is a common worldwide poultry disease caused by the bacterium of Clostridium perfringens (C. perfringens) which has significant economic losses in the poultry industry as well as the cost of treatment and preventive measures. The current study was conducted to evaluate the incidence of NetB toxin positive in C. perfringens on different farms in Egypt. In the years 2020 and 2021, on industrial broiler farms (15-45 days- old), 100 intestinal samples were collected consisting of 30 healthy Ross broiler chickens and 70 unhealthy Ross broiler chickens. Culture and biochemical characterization (Catalase, urease, sugar fermentation, gelatin liquefaction, nitrate reduction, and lecithinase reaction tests) confirmed that C. perfringens was isolated at a rate of 10% (3/30) from apparently healthy broiler chickens and 40% from unhealthy broiler chickens. Thirty-one isolates were tested for toxigenicity and typing by ELISA kits and the results showed that 80% of the isolates from unhealthy broiler chickens were C perfringens type A alpha-toxin (toxigenic), 20% were non-toxigenic, and 66.7% isolates from apparently normal broiler chickens were toxigenic. The same thirty-one (44%) C. perfringens isolates were detected by PCR to investigate the presence of the NetB toxin gene in apparently healthy and unhealthy broilers and subsequently detect the role of NetB toxin in inducing NE. Of the samples, 82% of the isolates from unhealthy chicks were found to incode NetB gene, while none of the isolates from healthy broiler chickens had NetB. Clostridium perfringens showed sensitivity to amoxicillin, amoxicillin with clavulanic acid and ampiclox, intermediate for ofloxacin, and high resistance to cephalexin, streptomycin, colistin sulfate, erythromycin, sulfa trimethoprim, gentamycin, and oxytetracycline. The present study revealed the importance of NetB gen in the appearance of clinical signs of NE in broiler chickens. Keywords: Alpha toxin, Antibiotic sensitivity test, Clostridium perfringens, Necrotic enteritis, NetB

Mucosal and systemic lymphoid immune responses against Clostridium perfringens strains with variable virulence in the production of necrotic enteritis in broiler chickens

ABSTRACT Necrotic enteritis (NE), caused by Clostridium perfringens, is an economically important disease of chickens. Although NE pathogenesis is moderately well studied, the host immune responses against C. perfringens are poorly understood. The present study used an experimental NE model to characterize lymphoid immune responses in the caecal tonsils (CT), bursa of Fabricius, Harderian gland (HG) and spleen tissues of broiler chickens infected with four netB+ C. perfringens strains (CP1, CP5, CP18, and CP26), of which CP18 and CP26 strains also carried the tpeL gene. The gross and histopathological lesions in chickens revealed CP5 to be avirulent, while CP1, CP18, and CP26 strains were virulent with CP26 being “very virulent”. Gene expression analysis showed that, while the virulent strains induced a significantly upregulated expression of pro-inflammatory IL-1β gene in CT, the CP26-infected birds had significantly higher CT transcription of IFNγ and IL-6 pro-inflammatory genes compared to CP5-infected or uninfected chickens. Furthermore, CP26 infection also led to significantly increased bursal and HG expression of the anti-inflammatory/regulatory genes, IL-10 or TGFβ, compared to control, CP5 and CP1 groups. Additionally, the splenic pro- and anti-inflammatory transcriptional changes were observed only in the CP26-infected chickens. An antibody-mediated response, as characterized by increased IL-4 and/or IL-13 transcription and elevated IgM levels in birds infected with virulent strains, particularly in the CP26-infected group compared to uninfected controls, was also evident. Collectively, our findings suggest that lymphoid immune responses during NE in chickens are spatially regulated such that the inflammatory responses against C. perfringens depend on the virulence of the strain.

Essential Oils as an Alternative to Antibiotics to Reduce the Incidence and Severity of Necrotic Enteritis in Broiler Chickens: A Short Review

Due to the removal of antibiotic growth promoters (AGPs) and consumer pressure for antibiotic-free (ABF) or no antibiotics ever (NAE) poultry production, there is a need for sustainable alternatives to prevent disease in commercial poultry operations. Without AGPs, there has been a rise in diseases that were traditionally controlled by subtherapeutic levels of antibiotics in the diet. This has impacted the health of commercial poultry and has been a significant cost to poultry producers. To mitigate this, the industry has started to investigate alternatives to antibiotics to treat these forthcoming health issues, such as necrotic enteritis (NE). NE is an enteric disease caused by an over proliferation of toxigenic Clostridium perfringens (CP) in the gastrointestinal tract. Although CP is a commensal in the avian intestinal tract, dysbiosis caused by inflammation and impaired intestinal integrity facilitates uncontrolled replication of CP. Infectious agents, such as Eimeria maxima, appear to be a predominant predisposing factor that promotes NE. However, non-infectious stressors, including dietary changes, have also been associated with NE to some degree. As a result of increased pressure to restrict the use of antibiotics, there is a need for research evaluating the efficacy of alternatives, such as plant-derived essential oils, as potential tools to mitigate NE in commercial poultry flocks. The aim of this study is to review the effects of essential oils as an alternative to antibiotics to reduce the incidence and severity of necrotic enteritis in broiler chickens.

Necrotic Enteritis in Broiler Chickens: A Review on the Pathogen, Pathogenesis, and Prevention.

Clostridium perfringens type A and C are the primary etiological agents associated with necrotic enteritis (NE) in poultry. The predisposing factors implicated in the incidence of NE changes the physical properties of the gut, immunological status of birds, and disrupt the gut microbial homeostasis, causing an over-proliferation of C. perfringens. The principal virulence factors contributing to the pathogenesis of NE are the α-toxin, β-toxin, and NetB toxin. The immune response to NE in poultry is mediated by the Th1 pathway or cytotoxic T-lymphocytes. C. perfringens type A and C are also pathogenic in humans, and hence are of public health significance. C. perfringens intoxications are the third most common bacterial foodborne disease after Salmonella and Campylobacter. The restrictions on the use of antibiotics led to an increased incidence of NE in poultry. Hence, it is essential to develop alternative strategies to keep the prevalence of NE under check. The control strategies rely principally on the positive modulation of host immune response, nutritional manipulation, and pathogen reduction. Current knowledge on the etiology, pathogenesis, predisposing factors, immune response, effect on the gut microbial homeostasis, and preventative strategies of NE in this post-antibiotic era is addressed in this review.

Immunization of broiler chickens with recombinant alpha-toxin protein for protection against necrotic enteritis

Necrotic enteritis ( NE ), caused by Clostridium perfringens ( CP ), has been reported in a wide variety of avian species including chickens. Necrotic enteritis has been reported in most parts of the world with acute outbreaks occurring sporadically and most often in broiler chickens after maternal immunity declines with bird age. This study was conducted to develop computer-aided design of a recombinant CP protein vaccine in an experimental model of necrotic enteritis. A synthetic rCPA which has been designed using predictive tools expressed and obtained through cloning, used to immunize and assess intestinal lesion scores, antibody levels, growth performance, and CP reduction in vivo. Broilers were immunized on days 7, 14, and 21 (V groups) with the purified recombinant clostridium alpha-toxin. On days 24, 25, and 26, the birds were orally challenged with CP (1.0 × 10 9 CFU/bird). Increased body weight (BW), clearance of CP, increased antibodies, and intestinal lesion scores were measured as protection parameters after oral co-infection with CP. Birds immunized and challenged with recombinant protein (CHV) experienced significantly less body weight loss and less intestinal lesions compared to the positive control group (PC). Higher anti-recombinant anti-rCPA antibody titers were observed in the challenged vaccinated (CHV) and only vaccinated (V) groups with an increased level CP clearance of intestinal contents in immunized birds. Based on these results, vaccination with computer-aided clostridial recombinant proteins, particularly rCPA toxin, enhances protective immunity against experimental necrotic enteritis in broiler chickens.

Lactobacillus casei displaying Clostridium perfringens NetB antigen protects chickens against necrotic enteritis.

Necrotic enteritis is a serious economical disease of poultry caused by Clostridium perfringens. NetB toxin of Clostridium perfringens is considered the causative agent of necrotic enteritis. Following the withdrawal of in-feed antibiotic growth promoters, there has been an urgent need to develop alternative approaches such as vaccination. Currently, there are no commercially available vaccines to control necrotic enteritis especially in broiler chickens as the target population. In the present study, we constructed a recombinant Lactobacillus casei strain expressing NetB protein of C. perfringens on the cell surface and used this probiotic-based vaccine strain to immunize broiler chickens orally against experimental induction of necrotic enteritis. The birds immunized with the oral vaccine strain were significantly protected against necrotic enteritis challenge and developed strong serum anti-NetB antibody responses to NetB protein. Furthermore, the immunized birds showed higher body weight gains during the challenge experiment compared with control birds. This study showed, for the first time, that a probiotic-based vector vaccine could be a promising vaccine candidate to provide protection against necrotic enteritis in broiler chickens. KEYPOINTS: • The probiotic L. casei carrying pT1NX-netB plasmid displayed NetB antigen on the cell surface. • The LC-NetB vaccine strain induced high anti-toxin antibody response in broiler chickens. • The LC-NetB vector vaccine provided significant protection against experimental NE challenge.

Immunization with Pooled Antigens for Clostridium perfringens Conferred Partial Protection against Experimental Necrotic Enteritis in Broiler Chickens.

Necrotic enteritis (NE) is a multifactorial and important enteric infectious disease etiologically caused by pathogenic C. perfringens infection, accounting for the estimated loss of around USD 6 billion in the global poultry industry. The increasing incidence of NE was found to be associated with the voluntary reduction or withdrawal of antibiotic growth promoters from animal feed during recent years. Therefore, the development of effective vaccines specific to NE assumes a priority for the poultry industry. This study aimed to identify the potential C. perfringens proteins as vaccine targets for NE. Three recombinant C. perfringens proteins targeting five antigens were prepared: two chimeric proteins (alpha-toxin and NetB, fructose-1,6-bisphosphate aldolase (FBA) and a zinc metalloprotease (Zm)), and one single collagen adhesion protein (Cna). Their protection efficacies were evaluated with a potent challenge model of Eimeria maxima/C. perfringens dual infections using a netB+tpeL+ C. perfringens strain. Young chicks were immunized twice subcutaneously with adjuvanted C. perfringens proteins on Days 4 and 15. At six days after the second immunization, the chickens immunized with Cna, FBA, and Zm antigens, and alpha-toxin had much higher serum antibody titers than unvaccinated controls prior to the challenge. Following the challenge, the pooled antigen-immunized group demonstrated no mortality and the least lesion scores against virulent challenge. The results indicate that the immunization with multicomponent antigens, including C. perfringens housekeeping protein Cna, may confer partial protection.

Comparative efficacy of postbiotic, probiotic, and antibiotic against necrotic enteritis in broiler chickens

Prevention of necrotic enteritis (NE), caused by Clostridium perfringens (C. perfringens), is one of the most important goals to improve the profitability of broiler chickens. This work aimed to compare the efficacy of 2 antibiotic alternatives including a postbiotic (dry feed additive and aqueous nonviable Lactobacillus (L.) species fermentation) and a probiotic (dry feed additive and aqueous Bacillus (B.) subtilis and B. lischeniformis mixture) with an antibiotic (amoxicillin in water) against NE. Four hundred, day-old broiler chicks were divided into 8 equal groups (Gs), n = 50 each (5 replicates; 10 each). Chickens of G1 (postbiotic dry-feed additive), G2 (postbiotic and antibiotic in drinking water), G3 (postbiotic dry and aqueous), G4 (probiotic dry-feed additive), G5 (probiotic and antibiotic in drinking water), G6 (probiotic dry and aqueous), and G7 (nontreated) were orally inoculated with a toxigenic C. perfringens type A on the d 19 to 21 of age and predisposed with 3X coccidial vaccine for induction of NE. However, chickens of G8 were kept nontreated or challenged. The severity of NE signs was markedly decreased in G3 in comparison with other challenged treatment groups, and the mortality rates were 22%, 10%, 16%, 22%, 12%, 20%, and 36% in Gs 1, 2, 3, 4, 5, 6, and 7, respectively. The best significant (P ≤ 0.05) feed conversion ratio was detected in G3 (1.51), G6 (1.54), and G2 and G8 (1.61). In addition, the European production efficiency factor was significantly (P ≤ 0.05) improved in G3 (279.33) and G2 (266.67), but it was decreased in G7 (177.33) when compared with G8 (339.33). An improvement in intestinal and hepatic pathology and liver function tests, as well as a significant (P ≤ 0.05) decrease in bacterial counts were observed in Gs 2, 5, 3, 6, 1, and 4, respectively in comparison with G7. Immunologically, the highest significant (P ≤ 0.05) hemagglutination inhibition antibody titers for Newcastle disease virus vaccine were in Gs 1 and 3 (6.4 log2). In conclusion, the combined feed and water postbiotic treatment demonstrated promising results in ameliorating the severity of NE and improving the hepatic and the immune status of broiler chickens when compared with the commonly used probiotic and antibiotic.

Inclusion of high-flavonoid corn in the diet of broiler chickens as a potential approach for the control of necrotic enteritis

Avian necrotic enteritis (NE) is an infectious disease that impacts poultry worldwide causing economic losses. Discontinued use of antimicrobial growth promoters has been associated with high incidence of the disease, which has led to a necessity for finding new therapeutic alternatives. Flavonoids are polyphenolic compounds that have been studied for their health-promoting properties in animals and humans. This study presents a flavonoid-rich corn (PennHFD), as a potential alternative for ameliorating NE in broiler chickens. The effect of a diet formulated with PennHFD was compared to a diet based on commercially available corn in chickens subjected to a controlled challenge of NE based on a co-infection of Eimeria maxima and Clostridium perfringens. Birds fed on the PennHFD-based diet had lower incidence of intestinal lesions (P = 0.048), higher body weight gain (P < 0.01), lower feed conversion ratio (P < 0.01), and lower mortality rates (P = 0.023) compared to the control diet. Therefore, we concluded that the inclusion of the high-flavonoid PennHFD reduces the severity of an experimental challenge of NE in broiler chickens.