Clostridium perfringens gas gangrene caused by closed abdominal injury: A case report and review of the literature

BACKGROUND Abdominal Clostridium perfringens(C. perfringens) gas gangrene is a rare infection that has been described in the literature as most frequently occurring in postoperative patients with open trauma. Intra-abdominal gas gangrene caused by C.perfringens infection after closed abdominal injury is extremely rare, difficult to diagnose, and progresses rapidly with high mortality risk. Here, we report a case of C. perfringens infection caused by closed abdominal injury.CASE SUMMARY A 54-year-old male suffered multiple intestinal tears and necrosis after sustaining an injury caused by falling from a high height. These injuries and the subsequent necrosis resulted in intra-abdominal C. perfringens infection. In the first operation,we removed the necrotic intestinal segment, kept the abdomen open and covered the intestine with a Bogota bag. A vacuum sealing drainage system was used to cover the outer layer of the Bogota bag, and the drainage was flushed under negative pressure. The patient was transferred to the intensive care unit for supportive care and empirical antibiotic treatment. The antibiotics were not changed until the results of bacterial culture and drug susceptibility testing were obtained.Two consecutive operations were then performed due to secondary intestinal necrosis. After three definitive operations, the patient successfully survived the perioperative period. Unfortunately, he died of complications related to GuillainBarre syndrome 75 d after the first surgery. This paper presents this case of intraabdominal gas gangrene infection and analyzes the diagnosis and treatment based on a review of current literature.CONCLUSION When the intestines rupture leading to contamination of the abdominal cavity by intestinal contents, C. perfringens bacteria normally present in the intestinal tract may proliferate in large numbers and lead to intra-abdominal infection. Prompt surgical intervention, adequate drainage,appropriate antibiotic therapy, and intensive supportive care comprise the most effective treatment strategy. If the abdominal cavity is heavily contaminated, an open abdominal approach may be a beneficial treatment.

Cloning of α-β fusion gene from Clostridium perfringens and its expression

AIM： To study the cloning of α-β fusion gene from Closindium perfringens and the immunogenidty of 0-6 fusion expression. METHODS： Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of α-toxin and β-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of α-β fusion gene binding. In order to verify the exact location of the α-β fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed α-β fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. RESULTS： The protective co-toxin gene （cpa906） and the β-toxin gene （cpb930） were obtained. The recombinant plasmid pZCPAB carrying α-β fusion gene was constructed and transformed into BL21（DE3）. The recombinant strain BL21（DE3）（pZCPAB） was obtained. After the recombinant strain BL21（DE3）（pZCPAB） was induced by 42℃, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with α-βfusion protein could neutralize the toxicity of α-toxin and β-toxin. CONCLUSION： The obtained α-toxin and β-toxin genes are correct. The recombinant strain BL21（DE3）（pZCPAB） could produce α-β fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with α-β fusion protein could neutralize the toxicity of α-toxin and β-toxin.

Dietary ellagic acid ameliorated Clostridium perfringens-induced subclinical necrotic enteritis in broilers via regulating inflammation and cecal microbiota

Background: Subclinical necrotic enteritis(SNE), a common intestinal disease of broiler caused by Clostridium perfringens, could reduce production performance of broilers by chronic intestinal damage and poor absorption of nutrients. Ellagic acid(EA) has been reported to present antioxidant and anti-inflammatory properties on human and animals in many aspects. This study was conducted to evaluate the effect and mechanism of EA in relieving SNE in broilers induced by C. perfringens.Results: C. perfringens challenge decreased body weight(BW), average daily gain(ADG), jejunal villi height/crypt depth(V/C) ratio, the activity of catalase(CAT) and the mRNA expression of zonula occludens 1(ZO-1) in jejunal mucosa of broilers. While feed conversion ratios(FCR), jejunal crypt depth(CD), the activities of myeloperoxidase(MPO) and diamine oxidase(DAO), as well as the concentrations of interleukin 6(IL-6), C-reactive protein(CRP) and procalcitonin(PCT) in serum, the activities of inducible nitric oxide synthase(iNOS) and lysozyme(LZM), the concentration of malondialdehyde(MDA), and the mRNA expressions of claudin-2, TNF-α, IL-1β, TLR-4, TLR-2, NF-κB,JAK3, STAT6 and iNOS in jejunal mucosa of broilers were increased by C. perfringens challenge. Dietary EA supplement relieved these adverse effects, and heightened jejunal villi height(VH), the concentration of D-xylose in plasma, activity of superoxide dismutase(SOD), and the mRNA expression of occludin in jejunal mucosa of broilers.The alpha diversity of cecal microbiota indicated that dietary EA supplement increased observed species and Shannon index. C. perfringens challenge increased the relative abundance of Firmicutes and decreased the relative abundance of Desulfobacterota in cecal microbiota. EA increased the relative abundance of Firmicutes in cecal microbiota. LEfSe analysis showed that C. perfringens challenge triggered the imbalance of cecal microbiota in broilers, dietary EA supplementation led to a small beneficial effect on microbiota, while the simultaneous effect of them seemed to stimulate the immune function of broilers by improving the microbiota balance.Conclusions: Dietary EA ameliorated C. perfringens-induced SNE in broilers via regulating jejunal inflammation signaling pathways TLR/NF-κB and JAK3/STAT6, relieving jejunal oxidative stress and balancing cecal microbiota to inhibit intestinal barrier damage, prevent systemic inflammatory response and improve nutrient absorption capacity,finally protect and enhance growth performance of broilers.

Effect of Clostridium perfringens enterotoxin on gastric cancer cells SGC7901 which highly expressed claudin-4 protein

AIM To investigate the effects of Clostridium perfringens enterotoxin(CPE) on gastric cancer cells which highly expressed claudin-4(CL4) protein.METHODS In this study, we detected expression of CL4 protein in different gastric cancer cell lines. Then, we investigated the effects of CPE on SGC7901 cells which highly expressed CL4 protein and the effects of CPE on sub-cutaneous tumor in nude mice models.RESULTS CL4 are highly expressed in SGC7901 cells. CPE expressedsignificant cytotoxicity in SGC7901 cells. Suppression of CL4 expression significantly decreased CPE-mediated cytotoxicity. CPE also inhibited tumor growth in subcutaneous tumor xenograft models.CONCLUSION CPE showed CL4 mediated cytotoxicity on gastric cancer cells SGC7901 and inhib-ited tumor growth in nude mice models.

Clostridium perfringens bloodstream infection secondary to acute pancreatitis:A case report

BACKGROUND Clostridium perfringens(C.perfringens)is an opportunistic pathogen.It can cause infections after birth,after an abortion,and in patients with diabetes,malignancy,liver cirrhosis,or an immunosuppressive state.Here,we report a patient with C.perfringens infection secondary to acute pancreatitis,with no underlying diabetes,malignancy,or liver cirrhosis.CASE SUMMARY A 62-year-old Han Chinese woman presented to the Tianjin Hospital of ITCWM Nankai Hospital on January 8,2020 because of epigastric abdominal pain.Laboratory examination showed that urine amylase was 10403 U/L(reference:47-458),and blood amylase was 1006 U/L(reference:<100).Abdominal computed tomography showed pancreatic edema and peripancreatic exudation.She was diagnosed with mild acute pancreatitis and treated accordingly.She was readmitted the next day for similar symptoms.Two hours later,she went to the lavatory and urinated,and the urine color was like soy sauce.Oxygen saturation decreased to 77%,and she developed consciousness disturbance.She was admitted to the intensive care unit.After 8 h in the hospital,she had a high fever of 40℃,blood was drawn for culture,and 3 g of cefoperazone/sulbactam was administered.After 12 h,she had a cardiac arrest and died shortly.Blood culture confirmed a C.perfringens infection.CONCLUSION C.perfringens infection may be secondary to acute pancreatitis.Rapid recognition and aggressive early management are critical for the survival of patients with C.perfringens infection.

Preparation of Monoclonal Antibody Against Clostridium perfringensα-toxin and Screening and Identification of Phage Display Technology

Clostridium perfringens phospholipase C(plc),also calledα-toxin,is encoded by the plc gene Clostridium perfringers.The production ofα-toxin can lead to the occurrence of gas gangrene.Vaccination is considered as one of the best solutions against Clostridium infections.In this study,an anti-Cpα-toxin monoclonal antibody(mAbs)A10E5 was successfully prepared,which had better biological reactivity.Then,the phage random 12-peptide library was used to screen mAb A10E5 protein.After four rounds of screening,three peptides with high affinity to the anti-α-toxin mAbs were screened.Two 12-peptide peptide Q and peptide E with higher inhibition rate were obtained by indirect ELISA.Two polypeptides of 500μg·mL^(-1)synthesized in vitro were mixed with30μg·mL^(-1)α-toxin at a concentration to treat Hela cells.Cell viability was determined by MTT assay.The results showed that both of the peptides significantly increased the survival rate of Hela cells compared with theα-toxin group,and the effect of peptide Q was more obvious.The chickens were immunized with phages expressing two different affinity polypeptides and then challenged.The results of chicken weight change,intestinal lesion score,bacterial count,and antibody titer in peripheral blood showed that the two phages expressing the polypeptides had a certain protective effect on the chickens compared with the PBS group,and peptide Q had better protection effect.In conclusion,the high affinity peptide with mAb A10E5 was screened in this study,and the protective effect of the plc polypeptide vaccine was verified by in vivo and in vitro experiments,which was of great significance for the comprehensive prevention and treatment of the disease.

Analysis of the Presence of Clostridium perfringens in Feed and Raw Material Used in Poultry Production

Microbiological control of feeds used in industrial poultry production has been increasingly important due to the demands of the market for food safety, as well as the need to ensure better quality of the digestive system of the birds. Microbiological analysis carried out in raw material used in feed production, especially feather, meat, and organ meal, has shown contamination by Clostridium perfringens. In order to study the presence of Clostridium perfringens, a total of 354 samples of feed and raw material were analyzed from January 2011 to July 2013. Samples came from four companies located in the state of S&atilde;o Paulo, with a total of 166 samples of meat meal, 24 samples of feather meal, 43 samples of organ meal, and 121 samples of feed. The following results were obtained: 88 (53%), 15 (62.5%), 16 (37.21%), and 23 (19%) samples were positive for Clostridium perfringens in each group of samples, respectively, with counts ranging from 2.0 × 102 to 7.0 × 103 CFU/g.

Occurrence of Clostridium perfringens contamination in poultry feed ingredients:Isolation, identification and its antibiotic sensitivity pattern

This work has been undertaken to study the occurrence of Clostridium perfringens contamination in the poultry feed ingredients and find out its in-vitro antibiotic sensitivity pattern to various antimicrobial drugs. Two hundred and ninety-eight poultry feed ingredient samples received at Poultry Disease Diagnosis and Surveillance Laboratory, Namakkal, Tamil Nadu in South India were screened for the presence of C. perfringens. The organisms were isolated in Perfringens agar under anaerobic condition and subjected to standard biochemical tests for confirmation. In vitro antibiogram assay has been carried out to determine the sensitivity pattern of the isolates to various antimicrobial drugs. One hundred and one isolates of C. perfringens were obtained from a total of 298 poultry feed ingredient samples. Overall positivity of 33.89% could be made from the poultry feed ingredients. Highest level of C. perfringens contamination was detected in fish meal followed by bone meal, meat and bone meal and dry fish.Antibiogram assay indicated that the organisms are highly sensitive to gentamicin(100%), chlortetracycline(96.67%), gatifloxacin(93.33%), ciprofloxacin(86.67%), ofloxacin(86.67%) and lincomycin(86.67%). All the isolates were resistant to penicillin-G. Feed ingredients rich in animal proteins are the major source of C. perfringens contamination.

Clostridium perfringens epsilon toxin: Toxic effects and mechanisms of action

Clostridium perfringensε-toxin(ETX)is the main toxin resulting in enterotoxemia of sheep and goats.ETX can cause lesions of multiple organs,such as hydropericardium,pulmonary edema and focal symmetrical encephalomalcia(FSE).Significant advancements have been made in studies of the cytotoxicity and neurotoxicity of ETX;however,candidates of ETX receptor still remain unconfirmed.Evidence has indicated several candidate molecules to be involved in the recognition and binding of ETX to different target cells.Myelin and lymphocyte(MAL)protein might be one of the candidate receptors for ETX,although this action needs further confirmation.Multiple signaling pathways of cell death are involved in the cytotoxicity of ETX.In Madin-Darby Canine Kidney(MDCK)cells cytotoxicity of ETX was thought to be associated with pore formation,which induces destruction of cellular ion balance.Hemolysis of human erythrocytes induced by ETX required activation of P2 receptors.Therefore,host-related factors are involved in ETX-induced cytotoxicity,which provides potential targets to block the toxic effect of ETX.

Stimbiotic supplementation modulated intestinal inflammatory response and improved broilers performance in an experimentally-induced necrotic enteritis infection model

Background:Two experiments were conducted to establish an optimal NE challenge model and evaluate the efficacy of stimbiotic(STB)supplementation in necrotic enteritis(NE)challenged broilers.In Exp.1,a total of 120 Arbor Acres(AA)broilers(45.0±0.21 g)were randomly assigned to 6 treatments in a 3×2 factorial arrangement.Vaccine treatments included non-challenge(0),×10 the recommended dose(×10)or×20 the recommended dose(×20)by the manufacturer.Clostridium perfringens(CP)treatments were non-challenge(No)or 3 mL of 2.2×10^(7)CFU CP challenge(Yes).In Exp.2,a total of 72 AA broilers(40.17±0.27 g)were randomly assigned to 6 treatments in a 3×2 factorial arrangement.Dietary treatments included non-additive(CON),100 mg/kg STB(STB)and 100 mg/kg STB on top of a typical commercial blend including an essential oil,probiotics,and enzyme(CB).Challenge treatments included non-NE challenge(No)and NE challenge(Yes)as established in Exp.1.Results:In Exp.1,CP and vaccine challenge decreased(P<0.05)body weight(BW),body weight gain(BWG)and feed intake(FI),and increased(P<0.05)the number of broilers with diarrhea and intestinal lesions.The oral administration of×20 recommended dose of vaccines coupled with 3 mL of 2.2×10^(7)CFU CP resulted in(P<0.01)a significantly increased incidence of wet litter and intestinal lesions.Thus,this treatment was chosen as the challenge model for the successful inducement of NE in Exp.2.In Exp.2,the NE challenge negatively affected(P<0.01)growth performance,ileal morphology,immunoglobulin contents in blood,caecal microbiota in the caecum,footpad dermatitis,intestinal lesion scores,tumour necrosis factor(TNF-α)and endotoxin in the serum compared with the non-NE challenged birds.The supplementation of STB and CB in diets enhanced(P<0.05)growth performance,intestinal microbiota,and blood profiles by stimulating ileal morphology(VH and VH:CD)and propionate production in the cecum,and there were no differences in measured variables between STB and CB supplemented birds.Conclusion:Overall,these results indicate that STB supplementation was able to reduce the inflammatory response and improve the performance of NE challenged birds,and the supplementation of STB alone was as effective as a typical commercial blend containing a number of other additives.

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.

C型产气荚膜梭菌生产用菌种种子批的建立与鉴定

为建立C型产气荚膜梭菌(Clostridium perfringens)生产用菌种种子批,以C型产气荚膜梭菌C59-2株第2代基础种子作为原始种子,对原始种子通过接种改良厌气肉肝汤和肉肝胃酶消化汤连续传代至第13代,分别对基础种子1代(F1)、第5代(F5)、第10代(F10)和第13代(F13)基础种子进行纯粹检验、培养特性鉴定、生化特性鉴定、产毒素能力的测定、血清型鉴定、免疫原性鉴定。结果表明,各项指标均合格,可作为仔猪大肠杆菌病、C型产气荚膜梭菌病二联灭活疫苗的生产用菌种用于疫苗的生产,基础种子批定为F1~F10代。

Modulations of genes related to gut integrity,apoptosis,and immunity underlie the beneficial effects of Bacillus amyloliquefaciens CECT 5940 in broilers fed diets with different protein levels in a necrotic enteritis challenge model

Background:The ban of in-feed antimicrobial additives has negatively affected the poultry industry by causing necrotic enteritis(NE)to emerge in the flocks.Alternatives such as Bacillus probiotics have shown to be effective on eliminating the negative effects of this disease.Two experiments were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940(BA)in broiler chickens under NE challenge and/or fed diets with different protein levels.Methods:In both experiments,480 day-old mix-sexed Ross-308 broilers were arranged in a 2×2 factorial arrangement of treatments.In experiment 1,the factors were NE challenge(yes or no)and probiotic(yes or no).In experiment 2,the factors were dietary crude protein levels(standard or reduced)and probiotic(yes or no)and were used under NE challenge condition.Oral administration of Eimeria oocysts(day 9)followed by inoculation with Clostridium perfringens(day 14 and 15)was used to induce NE challenge.On day 16,two birds from each treatment were gavaged with fluorescein isothiocyanate-dextran(FITC-d)and blood samples were collected for gut integrity evaluation,and jejunal samples were collected for gene expression assay.Results:In experiment 1,BA supplementation decreased caspase-3(CASP3)(P<0.001)and caspase-8(CASP8)(P<0.05)and increased occludin(OCLD)(P<0.05)expression regardless of the challenge.Additionally,BA supplementation downregulated interfron-γ(IFN-γ)expression(P<0.01)and upregulated immunoglobulin-G(IgG)(P<0.01)and immunoglobulin-M(IgM)(P<0.05)only in challenged birds.In experiment 2,the expression of genes encoding mucin-2(MUC2)(P<0.001),tight junction protein-1(TJP1)(P<0.05)and OCLD(P<0.05)were upregulated by the addition of BA in the diet,regardless of the crude protein level.Further,BA supplementation downregulated INF-γ(P<0.01)and upregulated immunoglobulin-A(IgA)(P<0.05),IgM(P<0.05)and IgG(P<0.01)regardless of the crude protein level.Conclusion:These findings suggest that supplementation of BA in broiler diets can improve gut health by modulation of genes related to the mucosal barrier,tight junction,and immunity in broilers challenged by unfavourable conditions such as NE challenge.

Dietary encapsulated essential oils and organic acids mixture improves gut health in broiler chickens challenged with necrotic enteritis

Background:The poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis(NE)due to Clostridium perfringens.In the present study,we investigated the effects of dietary supplementation with a blend of encapsulated essential oils and organic acids(BLJ)on growth performance and gut health using a coinfection model of NE in broiler chickens.Methods:Two hundred and eighty-eight one-day-old male Arbor Acres broiler chicks were randomly assigned using a 2×2 factorial design into two groups fed either 0 or 500 mg/kg dietary BLJ and co-challenged(or not challenged for the control)with Eimeria spp./C.perfringens.Results:Infected birds fed the BLJ-supplemented diet exhibited an improved feed conversion ratio throughout the trial(P<0.01),a higher villus height and villus height/crypt depth ratio,and reduced intestinal C.perfringens counts,liver C.perfringens carriage,gut lesion scores and serum fluorescein isothiocyanate dextran(FITC-D)concentrations at 7 d postinfection compared with those of birds without BLJ supplementation(P<0.05).NE-infected birds fed BLJ exhibited significantly upregulated claudin-1 and IGF-2 mRNA levels(P<0.05),increased A20 mRNA expression and significantly downregulated TRAF-6,TNFSF15 and TOLLIP mRNA levels in the jejunum at 7 d post-infection compared with those in birds without BLJ supplementation(P<0.05).Compared with the uninfected and untreated birds,the uninfected birds fed BLJ displayed increased relative abundances of Lactobacillus and Coprococcus but reduced Rikenellaceae levels.Compared with the unsupplemented NE-challenged birds,infected birds fed BLJ showed an increased relative abundance of Unclassified_Lachnospiraceae and a significantly decreased relative abundance of Erysipelotrichaceae.Conclusion:BLJ supplementation improved growth performance and gut health in NE-infected broiler chickens by strengthening the intestinal barrier function,positively modulating the gut microbiota community and differentially regulating intestinal immune responses.Our results also suggested that adding BLJ effectively controlled NE infections after experimental Eimeria and Clostridium perfringens coinfection.

Necrotic enteritis and antibiotic-free production of broiler chickens:Challenges in testing and using alternative products

The global trend towards raising broiler chickens without the use of in-feed antibiotics(IFAs)means that there is an ongoing need to develop alternative treatments capable of delivering the benefits that IFAs previously provided.IFAs supported the productivity performance of chickens and played a key role in maintaining their health.Necrotic enteritis(NE)is an important disease of broilers that affects health,productivity,and welfare,and was previously well controlled by IFAs.However,with the reduction in IFA use,NE is resurgent in some countries.Vaccines and various feed additives,including pre-,pro-,and postbiotics,phytobiotics,fatty acids,and phage therapies have been introduced as alternative methods of NE control.While some of these feed additives have specific activity against the NE pathogen,Clostridium perfringens,most have the more general goal of reinforcing gut health.Extensive reviews of the effects of many of these feed additives on gut health have been published recently.Hence,rather than cover previously well reviewed areas of research this review focuses on the challenges and pitfalls in undertaking experimental assessment of alternative NE treatments and translating laboratory research to real world commercial production settings.The review is based on the author's particular experience,reading,thoughts,and analysis of the available information and inevitably presents a particular understanding that is likely to be at odds with others thinking on these issues.It is put forward to stimulate thinking and discussion on the issues covered.

Fatal hemolysis due to clostridium perfrigens blood stream infection

Here we reported a 59-year-old woman with diabetes who was found to have massive hemolysis associatedwith clostridium （C.） perfringens blood stream infection. She died 13 hours after admission in spite of aggressive treatment. The lady referred to our hospital with high fever, chills, body ache and right hypochondriac pain for 1 day. Physical examination revealed a temperature of 39, heart rate of 110 beats/min, blood pressure of 101/69 mmHg, and a respiratory rate of 25 breaths/min. Pulse oximetry was 94% on room air. Chest auscultation revealed good air movement without rales. Tenderness of right upper abdomen was found. Abnormal laboratory results included white blood cell （WBC） count of 20x109/L with 90.9% neutrophils,

Influence of feeding crimped kernel maize silage on the course of subclinical necrotic enteritis in a broiler disease model

This experiment was carried out with 375 male broilers(Ross 308) from days 1 to 28 to evaluate the influence of crimped kernel maize silage(CKMS) on the manifestation of subclinical necrotic enteritis,microbiota counts, organic acid production and relative weights of gastrointestinal segments. A necrotic enteritis disease model was applied. Birds were allocated into 3 different dietary treatments: a maizebased feed(MBF, control diet), and 2 diets supplemented with 15%(CKMS15) or 30%(CKMS30) of crimped ensiled kernel maize. The disease model involved a 10-time overdose of an attenuated live vaccine against coccidiosis given orally on day 17, followed by oral inoculation of Clostridium perfringens Type A(S48,10~8 to 10~9 bacteria/bird) twice daily on days 18,19, 20 and 21. Scoring of intestinal lesions was performed on days 22, 23, 25 and 28. Ileal and caecal digesta samples were collected for the quantification of selected bacterial groups and organic acids. The results showed that there was no effect of dietary treatments on small intestinal lesion scores(P> 0.05). Lesions scores peaked on days 23 and 25 and decreased again on day 28(P = 0.001). No effect of age on microbiota counts was observed, but feeding of CKMS30 reduced the number of coliforms in ileal contents(P = 0.01). Dietary treatments did not affect organic acid concentrations in ileum and caeca, but there was an effect of age; butyric acid was higher on days 22, 23 and 25 than on day 28(P = 0.04). Acetic acid and propionic acid concentrations in caeca were the highest on days 22 and 28 but the lowest on days 23 and 25. Relative gizzard and caeca weights were increased, and relative ileum weights were decreased when birds were fed CKMS30(P < 0.05). In conclusion, the inclusion of CKMS in broiler diets had no effects on the course of necrotic enteritis but had potential benefits in terms of inhibition of potentially harmful microorganisms.

Necrotic enteritis challenge regulates peroxisome proliferator-1 activated receptors signaling and β-oxidation pathways in broiler chickens

Necrotic enteritis(NE)is an important enteric disease in poultry and has become a major concern in poultry production in the post-antibiotic era.The infection with NE can damage the intestinal mucosa of the birds leading to impaired health and,thus,productivity.To gain a better understanding of how NE impacts the gut function of infected broilers,global mRNA sequencing(RNA-seq)was performed in the jejunum tissue of NE challenged and non-challenged broilers to identify the pathways and genes affected by this disease.Briefly,to induce NE,birds in the challenge group were inoculated with 1 mL of Eimeria species on day 9 followed by 1 mL of approximately 108 CFU/mL of a NetB producing Clostridium per-fringens on days 14 and 15.On day 16,2 birds in each treatment were randomly selected and euthanized and the whole intestinal tract was evaluated for lesion scores.Duodenum tissue samples from one of the euthanized birds of each replicate(n=4)was used for histology,and the jejunum tissue for RNA extraction.RNA-seq analysis was performed with an Illumina RNA HiSeq 2000 sequencer.The differ-entially expressed genes(DEG)were identified and functional analysis was performed in DAVID to find protein-protein interactions(PPI).At a false discovery rate threshold<0.05,a total of 377 DEG(207 upregulated and 170 downregulated)DEG were identified.Pathway enrichment analysis revealed that DEG were considerably enriched in peroxisome proliferator-activated receptors(PPAR)signaling(P<0.01)andβ-oxidation pathways(P<0.05).The DEG were mostly related to fatty acid metabolism and degradation(cluster of differentiation 36[CD36],acyl-CoA synthetase bubblegum family member-1[ACSBG1],fatty acid-binding protein-1 and-2[FABP1]and[FABP2];and acyl-coenzyme A synthetase-1[ACSL1]),bile acid production and transportation(acyl-CoA oxidase-2[ACOX2],apical sodium-bile acid transporter[ASBT])and essential genes in the immune system(interferon-,[IFN-γ],LCK proto-oncogene,Src family tyrosine kinase[LCK],zeta chain of T cell receptor associated protein kinase 70 kDa[ZAP70],and aconitate decarboxylase 1[ACOD1]).Our data revealed that pathways related to fatty acid digestion were significantly compromised which thereby could have affected metabolic and immune responses in NE infected birds.