Enteritis In Chickens Research Articles

Clinicopathology and Ultrastructural Alterations of Experimentally Induced Necrotic Enteritis in Chickens

Background: This study was designed to elucidate the clinicopathological and ultrastructural alterations in experimentally induced necrotic enteritis (NE) in chickens and the present study acknowledged pronounced clinical signs, gross, histopathological and ultrastructural lesions in the chickens infected by both Clostridium perfringens type A, type C and coccidia infection. Methods: The day-old chickens were divided in to five groups including control. NE was induced by Clostridium perfringens, type-A and C with and without coccidia infection to the designated experimental groups in this study. Result: The clinical signs, hematology and biochemical parameters were evaluated in this study. Clinical indicators included diarrhea, ruffled feathers, dehydration, depression and a drop in TEC, Hb% and PCV% were noticeable. There was also a large increase in TLC and DC, a significant increase in ALT, AST and a decrease in total protein. In the intestine and other organs, there were noticeable inflammatory gross lesions and histological lesions. Transmission electron microscopic (TEC) observation of intestine revealed disruption of intercellular junction complexes, delimitation of enterocytes, disintegration of nucleus, dilatation of endoplasmic reticulum and cristeolysis of mitochondria. The present work will be an admiring contribution to inclusive study of the NE in chicken in terms of clinicopathology and ultrastructural lesions.

Natural intraepithelial lymphocytes are critical intestinal defense against necrotic enteritis in chickens

Abstract In chickens, the subsets of intraepithelial lymphocytes (IEL) and their responses to enteric diseases remain unclear. Therefore, our study aimed to elucidate the roles of IEL populations in necrotic enteritis (NE) in chickens. At 14 days of age, 63 SPF chicks were randomly divided into 3 groups: Control (sham challenge), Eimeria maxima (EM) challenge, and Clostridium Perfringens (CP) challenge. Birds in the EM and CP groups were orally infected with EM at day 14 of age, and while CP birds received additional oral infection with CP bacteria at days 18 and 19 of age. At 20 days of age (1 day post CP infection; dpi), and 27 days of age (7 dpi), 7 birds per group were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. Birds exhibited subclinical NE based on lesion scores, and CP-infected birds showed lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The CP group showed substantial increases in the total number of natural IEL subsets, including TCRαβ+CD4-CD8-, TCRαβ+CD8αα+, TCRγδ+, TCRneg and iCD8α IEL. By 7 dpi, only the number of TCRαβ+CD4-CD8- and TCRαβ+CD8αα+ IEL maintain their increase in the CP group. The CP group had significant higher expression of IL-1β and IFN-γ and osteopontin genes at 1 dpi. These findings suggest that natural IEL play a critical role in the host response during subclinical NE, potentially conferring protection against CP infection.

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.

Efficacy of both antibiotic and probiotic for control of necrotic enteritis in chickens experimentally

Efficacy of both antibiotic and probiotic for control of necrotic enteritis in chickens experimentally

Molecular and cellular characterization of immunity conferred by lactobacilli against necrotic enteritis in chickens.

Necrotic enteritis is an important enteric disease of poultry that can be controlled with in-feed antibiotics. However, with the concerns over antimicrobial resistance, there is an increased interest in the use of alternatives. Probiotics are one of the alternatives that have gained considerable attention due to their antimicrobial and immunomodulatory activities. Therefore, in the present study, we evaluated the effects of two different Lactobacillus species alone or as a cocktail on prevention of necrotic enteritis. Day-old male broiler chickens were divided into five groups and on days 1, 8, 15, and 22, birds in groups 2 and 3 received 1×108 colony forming units (CFU) of L. johnsonii and L. reuteri, respectively. Group 4 received probiotic cocktails containing both bacteria (108 CFU/bird) and the negative and positive control groups did not receive any lactobacilli. Starting on day 23 post-hatch, birds in all groups (except the negative control group) were orally challenged twice per day with 3×108 CFU of a pathogenic C. perfringens strain for 3 days. Tissue and cecal samples were collected before and after challenge to assess gene expression, lymphocyte subsets determination, and microbiome analysis. On day 26 of age, lesion scoring was performed. The results demonstrated that the group that received the lactobacilli cocktail had significantly reduced lesion scores compared to the positive control group. In addition, the expression of interleukin (IL)-12 in the jejunum and CXC motif chemokine ligand 8 (CXCL8), IL-13, and IL-17 in the ileum were downregulated in the group that received the lactobacilli cocktail when compared to the positive control. Treating chickens with the lactobacilli cocktail prior to challenge enhanced the percentage of CD3-CD8+ cells and Bu-1+IgY+ B cells in the ileum and increased the frequency of monocyte/macrophages, CD3-CD8+ cells, Bu-1+IgM+, and Bu-1+IgY+ B cells in the jejunum. Treatment with the lactobacilli cocktail reduced the relative expression of Gamma-Protobacteria and Firmicutes compared to the positive control group. In conclusion, the results presented here suggest that treatment with the lactobacilli cocktail containing L. johnsonii and L. reuteri reduced necrotic enteritis lesions in the small intestine of chickens, possibly through the modulation of immune responses.

Gastrointestinal Impaction and Necrotic Enteritis in a Backyard Chicken.

The carcass of a 4-mo-old, female, mixed-breed backyard chicken was submitted for postmortem evaluation and diagnostic workup. The bird was previously presented to a veterinary clinic because of chronic weight loss and loose stool, and was euthanized before submission to the California Animal Health and Food Safety, Turlock lab. On gross examination, the proventriculus, gizzard, and duodenum were markedly distended and impacted with a mixture of fibrous plant material, cereal grain, and litter material. The koilin layer of the gizzard was eroded. There were multifocal to coalescing, 0.2-1-cm diameter white nodules on the serosal surface of the duodenal loop and lesions extended into the distal jejunum. The duodenum had multifocal, transmural, umbilicated, and ulcerated mucosal lesions, which were covered with a white pseudomembrane. Microscopically, there was segmental, transmural necrosis of the intestinal wall with diffuse sloughing of villi epithelium and accumulation of fibrino-hemorrhagic exudate with numerous bacterial colonies in the lumen. The gross and microscopic findings were indicative of gastrointestinal impaction and necrotic enteritis. Proliferation of Clostridium perfringens within the intestine was demonstrated by anaerobic bacterial culture, intestinal gram stains, and immunohistochemistry. The C. perfringens isolate was type F (encoding the gene for alpha toxin -cpa- and for enterotoxin -cpe) by PCR toxinotyping. Overgrowth of C. perfringens was likely exacerbated by the rough fibrous forage and highly fermentable grain diet. To our knowledge, gastrointestinal impaction concurrent with necrotic enteritis has not been described in backyard chickens. In addition, to our knowledge, C. perfringens type F has not been associated with necrotic enteritis in chickens.

Probiotics as Alternatives to Antibiotics for the Prevention and Control of Necrotic Enteritis in Chickens.

Necrotic enteritis (NE) in poultry is an economically important disease caused by Clostridium perfringens type A bacteria. A global trend on restricting the use of antibiotics as feed supplements in food animal production has caused a spike in the NE incidences in chickens, particularly in broiler populations. Amongst several non-antibiotic strategies for NE control tried so far, probiotics seem to offer promising avenues. The current review focuses on studies that have evaluated probiotic effects on C. perfringens growth and NE development. Several probiotic species, including Lactobacillus, Enterococcus, Bacillus, and Bacteroides bacteria as well as some yeast species have been tested in chickens against C. perfringens and NE development. These findings have shown to improve bird performance, reduce C. perfringens colonization and NE-associated pathology. The underlying probiotic mechanisms of NE control suggest that probiotics can help maintain a healthy gut microbial balance by modifying its composition, improve mucosal integrity by upregulating expression of tight-junction proteins, and modulate immune responses by downregulating expression of inflammatory cytokines. Collectively, these studies indicate that probiotics can offer a promising platform for NE control and that more investigations are needed to study whether these experimental probiotics can effectively prevent NE in commercial poultry operational settings.

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.

Avian Macrophage Responses to Virulent and Avirulent Clostridium perfringens.

The present study evaluated the avian macrophage responses against Clostridium perfringens that varied in their ability to cause necrotic enteritis in chickens. Strains CP5 (avirulent-netB+), CP1 (virulent-netB+), and CP26 (highly virulent-netB+tpeL+) were used to evaluate their effect on macrophages (MQ-NCSU cells) and primary splenic and cecal tonsil mononuclear cells. The bacilli (whole cells) or their secretory products from all three strains induced a significant increase in the macrophage transcription of Toll-like receptor (TLR)21, TLR2, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and CD80 genes as well as their nitric oxide (NO) production and major histocompatibility complex (MHC)-II surface expression compared to an unstimulated control. The CP1 and CP26-induced expression of interferon (IFN)γ, IL-6, CD40 genes, MHC-II upregulation, and NO production was significantly higher than that of CP5 and control groups. Furthermore, splenocytes and cecal tonsillocytes stimulated with bacilli or secretory products from all the strains showed a significant increase in the frequency of macrophages, their surface expression of MHC-II and NO production, while CP26-induced responses were significantly higher for the rest of the groups. In summary, macrophage interaction with C. perfringens can lead to cellular activation and, the ability of this pathogen to induce macrophage responses may depend on its level of virulence.

Necrotic enteritis in chickens: a review of pathogenesis, immune responses and prevention, focusing on probiotics and vaccination.

Necrotic enteritis (NE), caused by Clostridium perfringens (CP), is one of the most common of poultry diseases, causing huge economic losses to the poultry industry. This review provides an overview of the pathogenesis of NE in chickens and of the interaction of CP with the host immune system. The roles of management, nutrition, probiotics, and vaccination in reducing the incidence and severity of NE in poultry flocks are also discussed.

Assessment of Antibactrial Effect of Different Extracts of Thymus Vulgaris against Clostridium Perfringens in Chicken

Necrotic enteritis in chicken is caused by the Clostridium perfringens, which have been found on almost chickensand for decades antimicrobial therapy was the only strategy to control it. However the misuse of antimicrobials linked to many impacts like reduced susceptibility of C. perfringens strains and cause cross-resistance to antimicrobial agents. Thisworkdemonstrates that, the uses of herbal extracts with antibacterial effectsconsider as an effective way to prevent and reducethe Necrotic enteritis.The present study determines antimicrobial activities of methanolic, ethanolic and watery extracts of thymus vulguarisagainst Clostridium Perfringenstype Ain chicken.The antibacterial activity was assessed through measuring sensitivity of isolated strains to different extracts by agar diffusiontechniqueand minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)by using the broth macrodilution method. Thymus vulgaris different extracts consider as effective anticlostridial herbal agent. The result of agar well diffusion assayand broth macrodilution methodshowed that, the tested strains were more sensitive to theethanolic and methanolic extracts which give the lowest (MIC) and (MBC) (32, 16 respectively) than the watery extracts (512, 256 respectively) .

Immunization with oral and parenteral subunit chimeric vaccine candidate confers protection against Necrotic Enteritis in chickens

Following the ban on the use of in-feed antimicrobials, necrotic enteritis (NE) NE is the most important clostridial disease. Vaccination has been considered as a possible approach to prevent NE. Our previous study showed that a chimeric protein product consisting of antigenic epitopes of NetB, Alpha-toxin and Zinc metallopeptidase (Zmp) triggered immune response against C. perfringens. In the current study we optimized the chimeric gene and constructed a fusion protein containing NetB, Alpha-toxin and Metallopeptidase (NAM) for expressing in tobacco plant to use as an edible vaccine for immunizing the chicken against NE. Simultaneously, we expressed and purified a His-tagged recombinant version of the NAM (rNAM) expressed in E. coli BL21 for subcutaneous immunization of chickens. Immunized birds produced strong humoral immune responses against both edible plant-based and parenteral purified rNAM.The responses were determined by the mean titer of antibody in blood samples to be around 9000 and 32,000, for edible and injected rNAM, respectively. Birds immunized subcutaneously showed the most striking responses. However the edible vaccine provided a more long lasting IgY response 14 days after the third vaccination compared to the injected birds. Chickens immunized with either lyophilized leaves expressing rNAM or purified rNAM, subsequently were subjected to the challenge with a virulent C. perfringens strain using an NE disease model. Our results showed that birds immunized both parenterally and orally with recombinant chimeric vaccine were significantly protected against the severity of lesion in the intestinal tract, but the protection provided with the injectable form of the antigen was greater than that of the oral form. Further analysis is needed to check whether these strategies can be used as the potential platform for developing an efficient vaccine against NE.

A secondary bile acid from microbiota metabolism attenuates ileitis and bile acid reduction in subclinical necrotic enteritis in chickens

BackgroundClostridium perfringens-induced chicken necrotic enteritis (NE) is responsible for substantial economic losses worldwide annually. Recently, as a result of antibiotic growth promoter prohibition, the prevalence of NE in chickens has reemerged. This study was aimed to reduce NE through titrating dietary deoxycholic acid (DCA) as an effective antimicrobial alternative.Materials and methodsDay-old broiler chicks were assigned to six groups and fed diets supplemented with 0 (basal diet), 0.8, 1.0 and 1.5 g/kg (on top of basal diet) DCA. The birds were challenged with Eimeria maxima (20,000 oocysts/bird) at d 18 and C. perfringens (109 CFU/bird per day) at d 23, 24, and 25 to induce NE. The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology, mRNA accumulation and C. perfringens colonization by real-time PCR, targeted metabolomics of bile acids, fluorescence in situ hybridization (FISH), or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.ResultsAt the cellular level, birds infected with E. maxima and C. perfringens developed subclinical NE and showed shortening villi, crypt hyperplasia and immune cell infiltration in ileum. Dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds. Consistent with the increased histopathological scores, subclinical NE birds suffered body weight gain reduction compared to the uninfected birds, an effect attenuated with increased doses of dietary DCA. At the molecular level, the highest dose of DCA at 1.5 g/kg reduced C. perfringens luminal colonization compared to NE birds using PCR and FISH. Furthermore, the dietary DCA reduced subclinical NE-induced intestinal inflammatory gene expression and cell apoptosis using PCR and TUNEL assays. Upon further examining ileal bile acid pool through targeted metabolomics, subclinical NE reduced the total bile acid level in ileal digesta compared to uninfected birds. Notably, dietary DCA increased total bile acid and DCA levels in a dose-dependent manner compared to NE birds.ConclusionThese results indicate that DCA attenuates NE-induced intestinal inflammation and bile acid reduction and could be an effective antimicrobial alternative against the intestinal disease.

Developing an experimental necrotic enteritis model in turkeys - the impact of Clostridium perfringens, Eimeria meleagrimitis and host age on frequency of severe intestinal lesions

BackgroundNecrotic enteritis is a significant problem to the poultry industry globally and, in Norway up to 30% of Norwegian turkey grow-outs can be affected. However, despite an awareness that differences exist between necrotic enteritis in chickens and turkeys, little information exists concerning the pathogenesis, immunity, microbiota or experimental reproduction of necrotic enteritis in turkeys. In particular, it is important to determine the appearance of the gross lesions, the age dependency of the disease and the role of netB toxin of Clostridium perfringens. To this end, we report our findings in developing an in vivo experimental model of necrotic enteritis in turkeys.ResultsA four tier (0–3) scoring system with clearly defined degrees of severity of macroscopic intestinal lesions was developed, based on 2312 photographic images of opened intestines from 810 B.U.T. 10 or B.U.T. Premium turkeys examined in nine experiments. Loss of macroscopically recognizable villi in the anterior small intestine was established as the defining lesion qualifying for a score 3 (severe intestinal lesions). The developed scoring system was used to identify important factors in promoting high frequencies of turkeys with severe lesions: a combined Eimeria meleagrimitis and Clostridium perfringens challenge, challenge at five rather than 3 weeks of age, the use of an Eimeria meleagrimitis dose level of at least 5000 oocysts per bird and finally, examination of the intestines of 5-week-old turkeys at 125 to 145 h after Eimeria meleagrimitis inoculation. Numbers of oocysts excreted were not influenced by Clostridium perfringens inoculation or turkey age. Among three different lesion score outcomes tested, frequency of severe lesions proved superior in discriminating between impact of four combinations of Clostridium perfringens inoculation and turkey age at challenge.ConclusionsThis study provides details for the successful establishment of an in vivo model of necrotic enteritis in turkeys.

Pathology and molecular diagnosis of necrotic enteritis in chicken

The present research work was carried out with an aim to study the pathology of necrotic enteritis in chickens followed by isolation and molecular detection of C. perfringens. Total 320 numbers of samples were collected from 15 districts in Assam. Isolation and identification of C. perfringens by cultural, morphological characteristics and confirmation was done by detection of cpa gene of C. perfringens by PCR with virulence toxin genes of C. perfringens such as TpeL and NetB. The clinical signs shown by the birds were diarrhoea, dehydration, depression, reluctance to move, loss of appetite, ruffled feathers, drooping of wings and head and huddling. The gross lesions of intestine revealed haemorrhagic, eroded, detached dead mucosal tissues, diphtheritic membrane, and distention. Liver, kidneys and lungs showed congestion, haemorrhage and focal areas of necrosis. Histopathology of NE in chickens revealed congestion of blood vessels in the lamina propria and submucosa with vacuolation of epithelial cells of intestinal villi with necrosis, making the villi broader and shorter. Different developmental stages of coccidia were also seen in the mucosal epithelial cells in few birds during this study. Liver, kidneys, heart, lungs, spleen, bursa of Fabricius and brain showed variable nature of histopathological lesions like congestion, haemorrhage and focal areas of coagulative necrosis.

Clostridium perfringens type A Causing Necrotic Enteritis Outbreaks among Chickens in Egypt

Undoubtedly, necrotic enteritis is identified as one of the most threatening diseases which face poultry industry in Egypt and need radical solution to avoid huge economic losses. The present work was designed for typing Clostridium perfringens isolated from diseased chickens suspected to have necrotic enteritis from different outbreaks in six Governorates in Egypt (Gharbia, Dakhalia, Sharkia, Ismailia, North Sinai and Kafr El Sheikh) during the period from August to December 2018. Intestine and liver samples were obtained from eighty six diseased broiler chickens representing 60 flocks. C. perfringens was isolated and toxigenicity of the recovered isolates was determined by Nagler's and dermonecrotic reactions. Furthermore, multiplex polymerase chain reaction (PCR) targeting alpha, beta, epsilon and iota toxins' genes was performed for result confirmation and typing of the toxigenic isolates. Sixty-six C.perfringens isolates (38.37%) were recovered from 172 intestine and liver samples and twenty isolates (30.3%) were toxigenic and typed as C. perfringens type A producing alpha toxin only. These findings established the fact that alpha toxin is the only main toxin of C. perfringens type A which is basically responsible for its pathogenicity and virulence. In addition, most of the positively toxigenic isolates were isolated from hepatic lesions (15 isolates) rather than intestinal lesion (5 isolates). In conclusion, alpha toxin is a major toxin for NE development in chickens. Genotyping of Clostridium perfringens by multiplex PCR is a useful adjunct to diagnosis of necrotic enteritis in chickens.

The Effect of Clostridium butyricum on Gut Microbiota, Immune Response and Intestinal Barrier Function During the Development of Necrotic Enteritis in Chickens.

Necrotic enteritis (NE) causes huge economic losses to the poultry industry. Probiotics are used as potential alternatives to antibiotics to prevent NE. It is known that Clostridium butyricum can act as a probiotic that can prevent infection. However, whether or not it exerts a beneficial effect on NE in chickens remains elusive. Therefore, we investigated the impact of C. butyricum on immune response and intestinal microbiota during the development of NE in chickens, including experimental stages with basal diets, high-fishmeal-supplementation diets, and Clostridium perfringens challenge. Chickens were divided into two groups from day 1 to day 20: one group had its diet supplemented with C. butyricum supplementation and one did not. At day 20, the chickens were divided into four groups: C. perfringens challenged and unchallenged chickens with and without C. butyricum supplementation. All groups were fed a basal diet for 13 days and thereafter a basal diet with 50% fishmeal from day 14 to 24. Chickens were infected with C. perfringens from day 21 to 23. At days 13, 20 and 24, samples were collected for analysis of the relative expression of immune response and intestinal mucosa barrier-related genes and intestinal microbes. The results show that C. butyricum can inhibit the increase in IL-17A gene expression and the reduction in Claudin-1 gene induced-expression caused by C. perfringens challenge. Moreover, C. butyricum was found to increase the expression of anti-inflammatory IL-10 in infected chickens. Although C. butyricum was found to have a significant beneficial effect on the structure of intestinal bacteria in the basal diet groups and decrease the abundance of C. perfringens in the gut, it did not significantly affect the occurrence of intestinal lesions and did not significantly correct the shift in gut bacterial composition post C. perfringens infection. In conclusion, although C. butyricum promotes the expression of anti-inflammatory and tight junction protein genes and inhibits pro-inflammatory genes in C. perfringens-challenged chickens, it is not adequate to improve the structure of intestinal microbiota in NE chickens. Therefore, more effective schemes of C. butyricum supplementation to prevent and treat NE in chickens need to be identified.

Immunization with subunits of a novel pilus produced by virulent Clostridium perfringens strains confers partial protection against necrotic enteritis in chickens

Necrotic enteritis (NE) is an economically important disease of broiler chickens that is caused primarily by Clostridium perfringens strains that produce the NetB toxin. It is controlled in North America principally through the application of in-feed antimicrobials, but alternative control methods, such as vaccination, are urgently needed. We previously identified a cluster of C. perfringens genes prevalent in disease-causing strains, denominated VR-10B, that is predicted to encode a pilus. The current study evaluated the ability of three predicted pilin structural subunits (CnaA, FimA, FimB) to protect against NE in two immunization studies. In the first study, young broiler chickens were immunized twice intramuscularly (i.m.) with CnaA or FimA, which resulted in only a weak serum antibody response, and no reduction in the severity of intestinal lesions following experimental challenge with C. perfringens strain CP1. In the second study, chickens were injected subcutaneously (s.c.) with CnaA, FimB, or a combination of all three proteins, on days 7, 14 and 19, which resulted in a marked antibody response specific to each antigen. Chickens immunized with either CnaA or FimB had significantly reduced NE lesion severity, whereas immunization with all three proteins in combination did not provide protection. Western blot experiments using serum from immunized birds were also performed, providing the first experimental evidence to suggest that this locus may in fact encode a functional pilus structure.

Clostridium perfringens-mediated necrotic enteritis is not influenced by the pre-existing microbiota but is promoted by large changes in the post-challenge microbiota

Problem addressed: Clostridium perfringens is the etiological agent of necrotic enteritis in chickens. As necrotic enteritis is a gastrointestinal disease, the interactions of pathogenic C. perfringens strains with the complex microbiota of the gastrointestinal tract may influence disease development and severity of disease.Objective: In this study the interactions of a pathogenic strain of C. perfringens, WER-NE36, with the microbiota of broilers was investigated to determine whether the pre-existing microbiota could influence disease outcomes in the necrotic enteritis challenge model.Methods and approach: Faecal microbiota compositions were measured before and after C. perfringens challenge and caecal microbiota was also characterised at necropsy. The microbiota profiles from individual birds were related back to the degree of necrotic enteritis that each bird developed.Results: Under the experimental conditions used the pre-existing microbiota did not have an effect on disease outcomes. However, C. perfringens challenge was shown to have a significant effect on the microbiota of broilers, regardless of disease status, by displacement of commensal clostridia.Conclusions: The microbiota signature after challenge resembled that of lower productivity birds, supporting the finding that physically obvious disease (necrotic lesions), as well as dysbiosis, are associated with shifts in gut microbiota and affect broiler performance, increasing costs to the poultry industry.

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens

BackgroundClostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome.ResultsDistinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis.ConclusionsThe high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.