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.

Effects of Bacillus coagulans supplementation on the growth performance and gut health of broiler chickens with Clostridium perfringens-induced necrotic enteritis

Background: The poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis(NE) due to Clostridium perfringens.Methods: This study was conducted to investigate the effects of feeding Bacil us coagulans on the growth performance and gut health of broiler chickens with C. perfringens-induced NE. Two hundred and forty 1-day-old broiler chicks were randomly assigned to a 2 × 2 factorial arrangement with two dietary B. coagulans levels(0 or 4 × 109 CFU/kg of diet) and two disease chal enge statuses(control or NE chal enged).Results: NE-induced reduction in body weight gain was relieved by the addition of B. coagulans into broiler diets compared with the NE-infected birds. NE infection damaged intestinal morphological structure, promoted intestinal C.perfringens growth and liver invasion, and enhanced anti-C. perfringens specific sI gA concentrations in the gut and specific IgG levels in serum compared with the uninfected birds. NE infection significantly(P < 0.05) decreased mucin-2(at 14 d post-infection(DPI), tol-like receptor 2(TLR2, at 7 and 14 DPI), TLR4(at 7 and 14 DPI), tumor necrosis factor super family15(TNFSF15, at 7 and 14 DPI), lysozyme(LYZ, at 14 DPI) and fowlicidin-2(at 7 and 14 DPI) mR NA levels, whereas it dramatical y(P = 0.001) increased IFN-γ mR NA levels at 7 DPI. However, chal enged birds fed diets supplemented with B.coagulans showed a significant(P < 0.01) decrease in gut lesion scores, decreased C. perfringens numbers in the cecum and liver, and an increase in fowlicidin-2 mR NA levels in compared with the uninfected birds. In addition, compared with the non-supplemented group, dietary inclusion of B. coagulans improved intestinal barrier structure, further increased specific sI gA levels and alkaline phosphatase(IAP) activity in the jejunum, enhanced the expression of jejunum lysozyme mR NA, and inhibited the growth, colonization, and invasion of C. perfringens; in contrast, it reduced serum-specific IgG concentrations and jejunum IFN-γ mR NA levels.Conclusion: These results indicated that dietary B. coagulans supplementation appeared to be effective in preventing the occurrence and reducing the severity of C. perfringens-induced NE in broiler chickens.

Effects of Lactobacillus acidophilus on the growth performance and intestinal health of broilers challenged with Clostridium perfringens

Background: Clostridium perfringens is the main etiological agent of necrotic enteritis. Lactobacil i show beneficial effects on intestinal health in infectious disease, but the protective functions of lactobacil i in C. perfringens-infected chickens are scarcely described. This study examined the effects of Lactobacil us acidophilus(L. acidophilus) on the growth performance and intestinal health of broiler chickens chal enged with Clostridium perfringens(C. perfringens) over a 28-day period. Using a 2 × 2 factorial arrangement of treatments, a total of 308 1-day-old male Arbor Acres broiler chicks were included to investigate the effects of Lactobacil us acidophilus(L. acidophilus) on the growth performance and intestinal health of broiler chickens chal enged with Clostridium perfringens(C. perfringens) during a 28-day trial.Results: During infection(d 14–21), C. perfringens challenge decreased the average daily gain(P < 0.05), and increased feed conversion ratio and the mortality rate(P < 0.05). However, dietary supplementation with L. acidophilus increased the body weight of C. perfringens-infected broilers on d 21(P < 0.05), and tended to decrease the mortality(P = 0.061).C. perfringens challenge decreased the villus height(P < 0.05), the ratio of villus height to crypt depth(P < 0.05) and OCLN(occludin) m RNA expression(P < 0.05), and increased the pro-inflammatory cytokine expression in the spleen and jejunum, the intestinal populations of C. perfringens and Escherichia(P < 0.05), and the serum content of endotoxin(P < 0.05), regardless of L. acidophilus supplementation. In contrast, dietary L. acidophilus reducedthe intestinal lesion score of challenged broilers(P < 0.05), the m RNA expression of pro-inflammatory cytokines, ileal populations of Escherichia and serum endotoxin content(P < 0.05), but increased the intestinal Lactobacillus populations(P < 0.05),irrespective of C. perfringens challenge.Conclusion: Dietary addition of L. acidophilus could improve the intestinal health and reduce the mortality of broilers suffering from necrotic enteritis.

In vitro antibacterial activity of thymol and carvacrol and their effects on broiler chickens challenged with Clostridium perfringens

Background： In the post-antibiotic era, essential oils （EO） are promising alternatives to growth-promoting antibiotics. The aim of the present study was to investigate the antibacterial activities of an EO product and its components thymol and carvacrol in vitro, and the efficacy of EO to control Clostridium perffingens challenge in broiler chickens. Results： The in vitro minimum inhibitory concentration assay showed strong antibacterial activity of the EO product, thymol, and carvacrol against pathogenic Escherichia col~, C. perffingens, and Salmonella strains, and weak activity towards beneficial Lactobacillus strains. Besides, an additive effect was observed between thymol and carvacrol. The in vivo study was carried out with 448 male broiler chicks following a 4 x 2 factorial arrangement to test the effects of EO supplementation （0, 60, 120, or 240 mg/kg EO in wheat-based diet）, pathogen challenge （with or without oral gavage of C. perfr/ngens from day 14 to day 20） and their interactions. Each treatment consisted of eight replicate pens （seven birds/pen）. The challenge led to macroscopic gut lesions, and resulted in a significant increase in ileal populations of C. perffingens and Eschefichia subgroup （P ≤ 0.05） on day 21. Dietary EO supplementation did not influence C. perfringens numbers, but linearly alleviated intestinal lesions on day 21 and 28 （P= 0.010 and 0.036, respectively）, and decreased Eschefichia populations in ileum with increased EO dosages （P = 0.027 and 0.071 for day 21 and 28, respectively）. For caecum, EO quadratically influenced Lactobacillus populations on day 21 （P = 0.002）, and linearly decreased the numbers of total bacteria and Eschefichio on day 28 （P = 0.026 and 0.060, respectively）. Mean thymol and carvacrol concentrations in the small intestine were 0.21 and 0.20 μg/g in intestinal digesta （wet weight）, respectively, for birds fed 60 mg/kg EO, and 0.80 and 0.71 μg/g, respectively, for birds fed 240 mg/kg EO. Conclusions： These results indicated that dietary EO supplementation could affect intestinal microbiota and alleviate intestinal lesions in broilers, which may contribute in controlling C. perffingens infection in broiler chickens.

A middle-aged lady with a pyogenic liver abscess caused by Clostridium perfringens

The pyogenic liver abscess caused by Clostridium perfringens (C. perfringens ) is a rare, but rapidly fatal infection. It is usually associated with malignancy and immunosuppression. We report the case of 50-year-old lady with the secondary liver metastases from rectal cancer presented with fever and epigastric pain. The identification of Grampositive bacilli septicaemia, the presence of gas-forming liver abscess and massive intravascular hemolysis should lead to the suspicion of C. perfringens infection. Here we review twenty cases published since 1990 and their clinical features are discussed. The importance of "an aggressive treatment policy" with multidisciplinary team approach is emphasized.

In vivo and in vitro protective effect of arginine against intestinal inflammatory response induced by Clostridium perfringens in broiler chickens

Background:Necrotic enteritis is a widespread disease in poultry caused by Clostridium perfringens.We previously reported that dietary arginine supplementation protected the intestinal mucosa of broiler chickens with necrotic enteritis,but the related protective mechanisms remain unclear.The in vivo trial was designed as a 2×2 factorial arrangement to evaluated the effects of arginine supplementation on inflammatory responses,arginine transporters,arginine catabolism and JAK-STAT signalling pathway in broiler chickens challenged with C.perfringens or without C.perfringens.Furthermore,we validated the in vivo results using intestinal epithelial cells of chicken embryos.Results:C.perfringens infection markedly increased gut gross pathological and histopathological lesion scores,promoted liver C.perfringens invasion,reduced serum arginine levels,and elevated jejunal mucosal lysozyme activities(P<0.05),but these effects were significantly reversed by arginine supplementation in vivo(P<0.05).The challenge significantly increased serum procalcitonin levels,jejunal mucosal iNOS activities and jejunal IL-6,TGF-β3,cationic amino acid transporter(CAT)-1,and CAT-3 mRNA expression(P<0.05),whereas arginine supplementation significantly reduced jejunal IFN-γ,IL-1β,IL-6,IL-10,TGF-β3,and CAT-3 mRNA expression(P<0.05).Arginine supplementation significantly attenuated the C.perfringens challenge-induced increases in jejunal i NOS,arginase 2,arginine decarboxylase,arginine:glycine amidinotransferase,JAK1,JAK3,STAT1,and STAT6 mRNA expression(P<0.05).The in vitro experiment showed that C.perfringens challenge markedly increased cellular cytotoxicity and the mRNA expression of IL-1β,IL-8,IL-10,CAT-1 and CAT-3(P<0.05),which were significantly reversed by 50μmol/L and/or400μmol/L arginine pre-treatment(P<0.05).Conclusions:Arginine prevented C.perfringens challenge-induced circulated arginine deficiency,normalized intestinal arginine transport and catabolism,down-regulated JAK-STAT signalling pathway and attenuated the inflammatory response,which exerted protective effects on the intestine of broiler chickens.

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.

The Challenges of Diagnosis and Control of Enterotoxaemia Caused by <i>Clostridium perfringens</i>in Small Ruminants

Enterotoxaemia is one of the important pathologies caused by Clostridium perfringens, which produces intestinal and systemic disease in goats, sheep and other animals. These Gram-positive anaerobic bacteria are normally resident in the intestinal tract of ruminants but during favourable conditions, proliferate uncontrollably and release toxins which produce disease in the host. Different strains of C. perfringens are responsible for several clinical syndromes, including lamb dysentery, pulpy kidney disease and struck. However, the pathology and pathogenesis of caprine enterotoxaemia is not well understood, with limited studies available in goats. Caprine enterotoxaemia can be controlled with the better understanding of its risk factors and pathogenesis. The diagnosis of enterotoxaemia in animals is complex and often requires group of tests than one single test for better specificity and sensitivity. Tentative diagnosis of enterotoxaemia in sheep and goats is based on the history, clinical signs and gross lesions during post-mortem examination of animals;however, confirmatory diagnosis of enterotoxaemia requires different laboratory diagnostic tools. Toxin detection of C. perfringens in case of enterotoxaemia is furthermost accepted benchmark in establishing a definitive diagnosis of enterotoxaemia in intestinal contents. Measuring urine glucose or observing Gram-stained smears of intestinal mucosa can be used as supplementary tests. However, it is also imperative that enterotoxaemia cannot be ruled out in the event of negativity of aforementioned diagnostic tests. Hence, definitive diagnosis of enterotoxaemia in goats can be achieved with the use of molecular techniques (PCR, ELISA and immune-fluorescence) coupled with toxin detection in intestine or biological assays including mouse inoculation test (MIT). In case of goats, vaccine efficacy is poor which may be due to need of high to moderate level of serum antibodies to protect against both systemic and enteric effects because intestinal form of disease is partially independent of the circulating anti-toxin antibodies. Thus, for the prevention and control of enterotoxaemia in goats and sheep, these aspects must be considered to develop more holistic control measures.

Efficient and Soluble Expression of α Protein of Clostridium perfringens and Preparation of Genetic Engineering Subunit Vaccine

[Objective] The paper was to develop genetic engineering vaccine that can express α exotoxin antigen protein efficiently without destroying its immunogenicity for preventing and controlling the diseases caused by Clostridium perfringens. [Method] Efficiently expressed soluble recombinant α protein was obtained from Escherichia coli expression system by optimizing codon,removing signal peptide,selecting sequences with better hydrophilicity and antigenicity,and optimizing expression conditions. [Result] Mice obtained higher serum antibody level when immunized by α protein,and the immune protection rates against type A,type B,type C and type D C. perfringens were 100%,90%,85% and 90%,respectively. The antibody titer of mice within 7-14 d after the third immunization reached the peak. [Conclusion]The α protein has good immunogenicity,and can be further used to develop genetic engineering subunit vaccines for preventing C. perfringens.

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.

Quantitative Assessment of the Risk Linked to the Consumption of Braised Beef Meat “<i>Choukouya</i>” Contaminated with Pathogenic <i>Clostridium perfringens</i>in Côte D’Ivoire

Inadequate handling, processing and sales of meat products have resulted in microbial contamination responsible for most infectious human food-borne illness. The study conducted a stochastic assessment of the risk of infection linked to the consumption of braised beef meat “Choukouya” contaminated by Clostridium perfringens in C?te d’Ivoire. We conducted “Choukouya” consumers (n = 900) and vendors (n = 300) survey to characterize the actors behavior. 189 samples of “Choukouya” were collected and microbiologically analyzed according to French standard protocols NF V 08-061 and XPV 08-061. A risk model was developed and the risk of infection linked to the consumption of “Choukouya” was estimated by Monte Carlo simulation procedure. The consumer’s surveys showed that the percentage of population consuming “Choukouya” was 74.4%, with an average consumption of 114.3 ± 0.5 g/person/intake. The microbiological analysis revealed the isolation of 70% of Clostridium perfringens in the vegetative form and 60.3% in sporulated form with an average loading of 3.7 ± 2.6 log10·cfu/g and 1.1 ± 1.0 log10·cfu/g respectively. The probability of ingesting a dose greater than 109 bacteria of Clostridium prefringens varied between 7.36% and 7.93%. The braised beef meat “Choukouya” sold in the streets of cities in C?te d’Ivoire represents a real risk of infection, and Clostridium perfringens is one of the causes. This risk could be mitigated by the establishment of good hygiene practices and adequate handling processes in this informal sector.

Fatal liver abscess caused by <i>Clostridium perfringens</i>complicated with transarterial chemoembolization

A 77-year-old man who received transarterial chemoembolization for large hepatocellular carcinomas complained of acute pain in the upper abdomen and suddenly developed severe jaundice, anemia, and massive hematuria. Abdominal computed tomography demonstrated gas gangrene at the tumor site. Clostridium perfringens was identified from blood samples and drainage cultures from the liver abscess. Despite intensive treatment, the patient died 55 hours after chemoembolization due to multiple organ failure. When treating patients with large tumors, such as in our case, this rare but fatal complication that causes sepsis and hemolysis with lightning-like rapidity should be considered.

Molecular Evaluation of the Enterotoxigenicity of <i>Clostridium difficile</i>and <i>Clostridium perfringens</i>Swine Isolates by PCR Assays

Clostridium difficile and C. perfringens are enteric pathogens affecting a variety of mammals. This study evaluated the molecular enterotoxigenicity of Clostridium swine isolates by PCRs. One hundred and ten swine faeces were analyzed by culture assay. The faecal samples were from sixty-seven healthy animals and 43 with gastrointestinal tract disease. C. difficile strains were PCR-screened for the presence of tcdA/tcdB and cdtA/cdtB genes. All C. perfringens isolates were tested for the characterization of the toxinotype. Overall, sixty-five swine resulted positive: 38 for C. difficile and 17 for C. perfringens. One sample tested C. perfringens and C. difficile-positive, at the same time: on the whole, 39 C. difficile strains were isolated. Thirty-eight C. difficile isolates (all from healthy animals) resulted tcdA/tcdB and cdtA/cdtB-negative by PCRs and toxins A/B-negative by immunological tests. All C. perfringens strains were type A;eight were also cpb2-positive. In the sample (diarrhoeic), with double infection, C. difficile tested tcdA/tcdB and cdtA/cdtB-positive by PCRs and toxins A/B-positive by immunoassays;C. perfringens resulted cpb2-positive. The molecular genotypeing/toxinotyping should be applied to establish a final diagnosis and to assess properly the full implications and the epidemiological impact of these findings in particular in samples of healthy animals and aid in the development of effective intervention methods for controlling clostridial disease outbreaks.

Some Characters of <i>Cl. perfringens</i>Isolated from Fresh and Marketed Processed Meat

This study was carried out a fresh meat immediately after slaughter and a marketed processed cattlemeat (sausage and minced meat). A total of 530 meat samples were examined for the presence of Cl. perfringens, 423 were from fresh meat obtained immediately after slaughtering (108 cattle meat, 101 sheep meat, 100 camel meat and 114 buffaloe meat) and 107 processed meat (57 from sausage and 50 from minced meat). Cl. perfringens was isolated from 204 (48.2%) of fresh meat samples, 61 (56.5%) from cattle, 53 (52.5%) from sheep meat, 45 (45%) from camel meat and 45 (39.5%) from buffaloe meat. The isolation rate of Cl. perfringens was higher in processed meat, it was isolated from 68 (63.6%) of which 45 (78.9%) from sausage and 23 (46%) in minced meat. The processed meat was found to harbour higher viable count ranging between 4 × 102 - 7 × 106 Cl. perfringens cells/gm meat than that Fresh meat in which the number ranged from 102:5 × 106 cells/gm meat. Typing of isolated strains revealed that the majority of it was of Cl. perfringens type A, 2 of type B, 3 of type C and one type D. Sixty strains of Cl. perfringens type A were randomized and tested for heat resistance at 100℃ and the results were recorded. Production of enterotoxin by 10 strains of Cl. perfringens was performed by ligated ileal loop test in rabbits. It was done by injection of whole culture in skimmed milk, cell extracts and concentrated culture filtrates of the organism in the ileal ligated loop of rabbits and the results were recorded.

A Preliminary Molecular Typing by PCR Assays of <i>Clostridium perfringens</i>and <i>Clostridium difficile</i>Isolates from Dogs

Clostridium perfringens and C. difficile have been associated with acute and chronic large and small bowel diarrhoea, and acute haemorrhagic diarrhoeal syndrome in dogs. The objective of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. perfringens and 10 C. difficile isolates from 95 canine faeces (n = 36, diarrhoeic and n = 59, non-diarrhoeic). Concerning C. perfringens, 13 strains (92.9%) were type A, of which 3 (23.1%) also possessed the beta 2 toxin (CPB2)-encoding gene. One isolate (7.1%) was type D and possessed CPB2 gene. On the whole, 4 of the 14 strains (28.6%) tested cpb2-positive. Six C. difficile isolates (60.0%) demonstrated tcdA+/tcdB+ and cdtA+/vcdtB+ genotype and tested positive for, in vitro, toxin production by EIA. Eight distinct ribotypes were observed. In conclusion, the PCR assays may provide useful and reliable tools for C. perfringens and C. difficile molecular typing in routine veterinary diagnostics.

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.

Osthole ameliorates myonecrosis caused by Clostridium perfringens type A infection in mice

This study aimed to investigate the protective effect of the nature product osthole(OST)against Clostridium perfrin-gens type A infection-caused myonecrosis in a mouse model.Male mice were divided into(1)control,(2)infected,(3)OST50 and(4)OST100 treatment groups.In the infected groups,mice were intramuscularly injected with 1×10^(8) CFU of C.perfringens per day for 6 days.Mice in the OST50 and OST100 groups were administrated intraperitoneally with OST at the doses of 50 or 100 mg/kg per day post C.perfringens infection.Our results showed that C.perfringens infection caused marked necrosis and inflammatory cell infiltration in the muscle tissues of mice.Mice in the OST50 and OST100 treatment groups displayed significantly attenuated C.perfringens infection-induced lipid peroxida-tion,oxidative stress,and apoptosis in their muscle tissue.Furthermore,OST treatment significantly downregulated the expressions of NF-κB,IL-1β,and TNF-αmRNA and protein levels,while concomitantly upregulating the expressions of Nrf2 and HO-1 mRNA and protein.OST treatments also inhibited the expression of phosphorylation(p)-p38,p-mTOR,and p-Erk1/2 proteins,and upregulated LC3II and Beclin1 proteins.In summary,our results reveal that OST therapy confers a protective effect against C.perfringens infection-induced oxidative stress and inflammation in muscle tissue,via activation of Nrf2/HO-1 and autophagy pathways and inhibition of p38,Erk1/2 and NF-κB pathways.