Alternative to antimicrobial growth promoters in the diets of broilers challenged with subclinical necrotic enteritis.

Necrotic enteritis (NE) is a disease of worldwide distribution, which affects young broilers and causes economic losses on a scale of 6 billion dollars per year. For decades, NE was controlled in poultry flocks by dietary administration of low doses of antimicrobial growth promoters (AGPs). However, an increase in NE incidence was noted after the AGP ban. This study aimed to compare the effect of an antibiotic (Enramycin) diet to a combination of sodium butyrate, hydrolyzed yeast, and zinc proteinate (ViligenTM) on broiler diets regarding performance, blood parameters, intestinal permeability, morphology and lesions, and carcass yield of broilers challenged with Eimeria spp. and Clostridium perfringens to simulate subclinical necrotic enteritis. A total of 1,150 one-day-old male broiler chickens with an initial average weight of 43.9 ± 0.65 g were allocated to 50 experimental pens. Animals were divided into 5 groups: Negative control (NC) without additives; Positive control (PC) with 0.12 g/ton of Enramycin (8%); V500, V1000, and V1500 with the addition of 500, 1.000, and 1.500 g/ton of Viligen, respectively. All animals were challenged by Eimeria spp. at 7 d of age and by C. perfringens at 17, 18, and 19 d for induction of subclinical NE. The broilers fed with all concentrations of Viligen showed similar performance, blood parameters, intestinal permeability, and carcass yield compared to PC broilers. However, NC broilers showed higher FCR compared to PC broilers from 1 to 33 d (1.42 vs. 1.39) (P = 0.048) and from 1 to 42 d (1.51 vs. 1.49) (P < 0.001). V1500 broilers had fewer intestinal lesions at 28 d when compared to the PC treatment (P < 0.05) and showed that higher Viligen inclusion resulted in lower intestinal damage. At 21 d, the V500 group showed higher intestinal morphology characteristics (VH:VD 4.9 vs. 3.5) compared to the PC treatment (P < 0.001). Thus, in this study, the dietary addition of Viligen to broilers challenged by an experimental model of subclinical NE resulted in lower intestinal damage and similar performance to that obtained by the addition of Enramycin.

Enterotoxemia produced by lambda toxin-positive Clostridium perfringens type D in 2 neonatal goat kids.

Enterotoxemia caused by Clostridium perfringens type D usually affects sheep and goats ≥ 2-wk-old. The main clinical signs and lesions of the disease are produced by the epsilon toxin (ETX) elaborated by this microorganism. However, ETX is produced in the form of a mostly inactive prototoxin that requires protease cleavage for activation. It has traditionally been believed that younger animals are not affected by type D enterotoxemia given the low trypsin activity in the intestinal content associated with the trypsin-inhibitory action of colostrum. Two Nigerian dwarf goat kids, 2- and 3-d-old, with a history of acute diarrhea followed by death, were submitted for postmortem examination and diagnostic workup. Autopsy and histopathology revealed mesocolonic edema, necrosuppurative colitis, and protein-rich pulmonary edema. Alpha toxin and ETX were detected in intestinal content, and C. perfringens type D was isolated from the colon of both animals. The isolates encoded the gene for lambda toxin, a protease that has been shown previously to activate ETX in vitro. Type D enterotoxemia has not been reported previously in neonatal kids, to our knowledge, and we suggest that lambda toxin activated the ETX.

Bacillus amyloliquefaciens CECT 5940 alone or in combination with antibiotic growth promoters improves performance in broilers under enteric pathogen challenge.

A study was conducted to investigate the effects of Bacillus amyloliquefaciens CECT 5940 as a direct-fed microbial (DFM) alone or in association with bacitracin methylene disalicylate (BMD) in broilers under enteric pathogen challenge. A total of 1,530-day-old male Cobb500 chicks were randomly assigned to 5 treatments, with 9 replicate pens with 34 birds each. Treatments included positive control (PC, basal diet without additives or challenge); negative control (NC, basal diet without additive and challenged birds); NC + 0.05 g/kg BMD; NC + 1 g/kg DFM (106 CFU B. amyloliquefaciens CECT 5940/g of feed); and NC + 0.05 g/kg BMD + 1 g/kg DFM. The challenge consisted of oral gavage with Eimeria maxima and Clostridium perfringens inoculum. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were evaluated on days 21, 35, and 42. Ileal and cecal content were collected on days 21 and 28 for C. perfringens enumeration by real-time PCR assay and the intestinal health was evaluated by scores. Uniformity (UN), carcass (CY), and breast meat yields (BMY) were evaluated on day 42. After 14 and 21 d post-inoculation, birds in the challenged groups had significant lower FI and BWG compared to the PC group (P < 0.05). However, the groups receiving DFM, BMD, or its combination presented better FCR, CY, BMY, UN, and lower incidence of footpad lesion and litter quality visual scores, compared to the NC group without feed additives (P < 0.05). Mortality was not affected by treatments (P > 0.05). Broilers fed DFM, BMD, or its combination presented lower C. perfringens in ileal content at 21 and 28 d compared to NC group without additives (P < 0.05) and also maintained gut health by keeping the frequency of ballooning, abnormal content, and swollen mucosa comparable to the PC group (P > 0.05). The study indicates that Bacillus amyloliquefaciens CECT 5940 is effective as BMD to provide similar performance and gut health in challenged broilers.

Optimization of solid-state fermentation conditions of Bacillus licheniformis and its effects on Clostridium perfringens-induced necrotic enteritis in broilers

In the present study, we examined the growth parameters of Bacillus licheniformis in solid-state fermentation (SSF) and evaluated the effects of Bacillus licheniformis-fermented products on Clostridium perfringens-challenged broilers. During four and six days of SSF, the highest viable biomass was observed at 5% glucose, 10% soybean meal, 3% yeast, and 50% initial moisture content. The Bacillus licheniformis SSF products were heat- and acid-resistant. Furthermore, the fermented products were able to inhibit the growth of Clostridium perfringens and Staphylococcus aureus in vitro. In feeding experiments, in a similar manner to the antibiotic treatment group, dietary supplementation of Bacillus licheniformis-fermented products significantly improved intestinal morphology and necrotic lesions under Clostridium perfringens challenge, accompanied by increased IFN-γ mRNA expression in the spleen and bursa of Fabricius. These results together suggest that Bacillus licheniformis-fermented products have potential for development as feed additives and use as possible substitutes for antibiotics to treat Clostridium perfringens in the poultry industry.(AU)

Adhesion and invasion of Clostridium perfringens type A into epithelial cells

ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells.

Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses.

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens . Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens. Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.

Interactions between Clostridium perfringens spores and Raw 264.7 macrophages.

Clostridium perfringens is the causative agent of a variety of histotoxic infections in humans and animals. Studies on the early events of C. perfringens infections have been largely focused on the interactions between their vegetative cells and macrophages. Consequently, in the current study we have examined the interactions between C. perfringens spores and Raw 264.7 macrophages. Raw 264.7 cells were able to interact and phagocytose Clostridium perfringens spores of a food poisoning isolate, strain SM101, and a non-food borne isolate, strain F4969, albeit to different extents. Phagocytosis and to a lesser extent, association, of C. perfringens spores by Raw 2647 macrophages was completely inhibited in presence of cytochalasin D. Complement increased association and phagocytosis of C. perfringens spores by Raw 264.7 macrophages. Survival of C. perfringens spores during macrophage infection seems to depend on the ability of spore germination during infection as: (i) F4969 spores germinated during infection with Raw 264.7 macrophages and subsequently killed by macrophages; and (ii) SM101 spores remained dormant inside Raw 264.7 macrophages and thus survived up to 24 h of infection. The in vitro spore-resistance factors, α/ß-type SASP, SpmA/B proteins and spore's core water content, seems to play no role in mediating SM101 spore-resistance to macrophages. Collectively, these results might well have implications in understanding the initial stages of infections by C. perfringens spores.

Carbon catabolite repression of type IV pilus-dependent gliding motility in the anaerobic pathogen Clostridium perfringens.

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium responsible for the production of severe histotoxic and gastrointestinal diseases in humans and animals. In silico analysis of the three available genome-sequenced C. perfringens strains (13, SM101, and ATCC13124) revealed that genes that encode flagellar proteins and genes involved in chemotaxis are absent. However, those strains exhibit type IV pilus (TFP)-dependent gliding motility. Since carbon catabolite regulation has been implicated in the control of different bacterial behaviors, we investigated the effects of glucose and other readily metabolized carbohydrates on C. perfringens gliding motility. Our results demonstrate that carbon catabolite regulation constitutes an important physiological regulatory mechanism that reduces the proficiencies of the gliding motilities of a large number of unrelated human- and animal-derived pathogenic C. perfringens strains. Glucose produces a strong dose-dependent inhibition of gliding development without affecting vegetative growth. Maximum gliding inhibition was observed at a glucose concentration (1%) previously reported to also inhibit other important behaviors in C. perfringens, such as spore development. The inhibition of gliding development in the presence of glucose was due, at least in part, to the repression of the genes pilT and pilD, whose products are essential for TFP-dependent gliding proficiency. The inhibitory effects of glucose on pilT and pilD expression were under the control of the key regulatory protein CcpA (catabolite control protein A). The deficiency in CcpA activity of a ccpA knockout C. perfringens mutant strain restored the expressions of pilT and pilD and gliding proficiency in the presence of 1% glucose. The carbon catabolite repression of the gliding motility of the ccpA mutant strain was restored after the introduction of a complementing plasmid harboring a wild-type copy of ccpA. These results point to a central role for CcpA in orchestrating the negative effect of carbon catabolite regulation on C. perfringens gliding motility. Furthermore, we discovered a novel positive role for CcpA in pilT and pilD expression and gliding proficiency in the absence of catabolite regulation. Carbon catabolite repression of gliding motility and the dual role of CcpA, either as repressor or as activator of gliding, are analyzed in the context of the different social behaviors and diseases produced by C. perfringens.

Inorganic phosphate induces spore morphogenesis and enterotoxin production in the intestinal pathogen Clostridium perfringens.

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for food poisoning and non-food borne gastrointestinal (GI) diseases. Although CPE production is strongly regulated by sporulation, the nature of the signal(s) triggering sporulation remains unknown. Here, we demonstrated that inorganic phosphate (Pi), and not pH, constitutes an environmental signal inducing sporulation and CPE synthesis. In the absence of Pi-supplementation, C. perfringens displayed a spo0A phenotype, i.e., absence of polar septation and DNA partitioning in cells that reached the stationary phase of growth. These results received support from our Northern blot analyses which demonstrated that Pi was able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0A expression, indicating that Pi acts as a key signal triggering spore morphogenesis. In addition to being the first study reporting the nature of a physiological signal triggering sporulation in clostridia, these findings have relevance for the development of antisporulation drugs to prevent or treat CPE-mediated GI diseases in humans.

Adhesive properties of an outer structure of Clostridium perfrigens type A isolated from piglets with catarrhal enteritis

One strain (S32) of Clostridium perfringens type A was isolated from a case of catarrhal enteritis of piglets. This strain was able to adhere to HeLa cells showing an adherence index (AI) of 25.15ñ1.26(mean ñ 1 standard error of the mean). Treatment of the bacterial cells with trypsin (025mg/ml) decreased in 70 (per cent)-80 (per cent) the AI and metaperiodate (10mg/ml) abolished completely the adherence, suggesting that the structure responsible for this phenomenon was probably a glycoprotein. Heating of bacterial suspensions (100§C/5 min) before carrying out the adhesion test decreased the AI rendering it equal to the negative controls. Rabbit homologous S32 antiserum inhibited the adherence up to dilutions of 1:640, at least. The piglet ileal loop assay carried out with strains S32 and Jab-1 (negative control) demonstrated that the strain S32 was able to adhere to the intestinal epithelial cells when examined after Gram staining. Transmission electron microscopy (TEM) demonstrated that S32 strain displayed a loose fibrillar material not seen with Jab-1. Stabilization of the bacterial cells with homologous antiserum of strain S32, followed by staining with rhutenium red, revealed loose long fibrillar material on the outer surface of the cells, that sometimes could be seen spreading out from the cells and linking bacterial cells. The question whether this structure might be an adhesin for this strain of Cl. perfringes type A, perhaps playing a role in the pathogenesis of the catarrhal enteritis of piglets, is dependent on further studies.

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).

Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.

Growth, sporulation and enterotoxin production by Clostridium perfringens type A in the presence of human bile salts.

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.