Perfringens Culture Research Articles

Immunization with recombinant alpha toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens

Necrotic enteritis (NE) in poultry has re-emerged as a concern for poultry producers, due in part to banning, by many countries, of the use of antimicrobial growth promoters in feeds. This re-emergence has led to a search for alternative methods for control of the disease, particularly vaccination. The objective of this work was to determine if vaccination of broiler chicks with recombinant alpha toxin protected against experimental challenge. Broiler chicks were vaccinated subcutaneously at 5 and 15 days of age, followed 10 days later by challenge with Clostridium perfringens. Birds were challenged twice daily on 4 consecutive days by mixing C. perfringens cultures with feed (three parts culture: four parts feed). Non-vaccinated birds challenged with C. perfringens developed NE at the rate of 87.8%, while only 54.9% of vaccinated birds developed lesions. In addition, non-vaccinated birds had lesion scores averaging 2.37, while average scores in vaccinated birds were 1.35. Vaccination produced an antibody response, with post-vaccination anti-alpha toxin IgG (IgY) titers in vaccinated birds more than 5-fold greater than in non-vaccinated birds. After challenge, vaccinated birds had average IgG (IgY) titers >15-fold higher than those in non-vaccinated birds. These results suggest that alpha toxin may serve as an effective immunogen, and, as such, may play a role in pathogenesis.

Reproducible Infection Model for Clostridium perfringens in Broiler Chickens

Experiments were carried out to establish an infection and disease model for Clostridium perfringens in broiler chickens. Previous experiments had failed to induce disease and only a transient colonization with challenge strains had been obtained. In the present study, two series of experiments were conducted, each involving four groups of chickens with each group kept in separate isolators. A coccidial vaccine given at 10 times the prescribed dosage was used to promote the development of necrotic enteritis. In the first experiment, cultures of C. perfringens were mixed with the feed at day 9, 10, 11, and 12, and the coccidial vaccine was given at day 10, whereas in the second experiment, C. perfringens cultures were mixed with the feed at day 17, 18, 19, and 20, and the coccidial vaccine was given at day 18. Chickens were examined at day 9, 11, 12, and 15 (Experiment 1), and at day 17, 18, 20, and 24 (Experiment 2). There was no mortality in any of the groups; however, chickens in the groups receiving both coccidial vaccine and C. peifringens developed the subclinical form of necrotic enteritis, demonstrated by focal necroses in the small intestine, whereas chickens in control groups or groups receiving only coccidial vaccine or only C. perfringens cultures developed no necroses. The results underline the importance of predisposing factors in the development of necrotic enteritis.

Incidence and characterization of Clostridium perfringens isolated from antibiotic-associated diarrhoeal patients: a prospective study in an Indian hospital

Clostridium perfringens has been reported as causing between 2-15% of all cases of antibiotic-associated diarrhoea (AAD), and may be diagnosed by detection of enterotoxin in faeces. A prospective study comprising 150 diarrhoeal patients and 100 non-diarrhoeal controls was undertaken to assess the incidence of C. perfringens-associated diarrhoea in an Indian hospital. Methods used included C. perfringens culture, reverse passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) for detection of enterotoxin, and polymerase chain reaction (PCR) assay for the presence of enterotoxin gene. Attempts were made to type the isolates by multiplex PCR. Of the 150 diarrhoeal stool samples tested, 13 were culture positive. Of these, four were positive for C. perfringens enterotoxin by RPLA, two were positive by PCR and two were positive by RPLA and ELISA. Twenty-seven samples were positive for culture of C. perfringens in non-diarrhoeal controls but none were positive for enterotoxin either by RPLA or by PCR. The average incidence of C. perfringens AAD using these methods was 2.6%. Toxin typing showed that all the isolates belonged to type A. To conclude, the relatively low incidence of toxigenic C. perfringens suggests that enterotoxigenic C. perfringens is not a major cause of AAD in this population.

Suppressive effect of Clostridium perfringens-produced heat-stable substance(s) on proliferation of human colon adenocarcinoma HT29 cells in culture

Clostridium perfringens has been regarded as one of the intestinal bacteria increasing colon cancer risk. In previous studies, we have shown that the oral administration of C. perfringens culture medium can inhibit the mutagen-induced formation of pre-neoplastic lesions in rat colon, thus proposing the existence of factor(s) preventing colon tumorigenesis in this culture medium. However, the properties of effective factor(s) and the mechanism of this inhibitory action still remain to be investigated. Then, the effect of C. perfringens culture medium on human colon adenocarcinoma HT29 cells was examined. The exposure of HT29 cells to C. perfringens culture medium was found to suppress the proliferation of these cells probably through the reduction of immediate early gene egr-1 expression. These observations suggest that C. perfringens culture medium has a cytostatic action on colon tumor cells, which may be responsible for the prevention of pre-neoplastic formation in rat colon.

Topical mafenide hydrochloride aqueous spray in initial management of massive contaminated wounds with devitalized tissue.

Since at least WWII, some open, contaminated wounds involving massive soft tissue injury and vascular damage have resulted in "irreversible shock," despite prompt rescue, hemorrhage control, and blood and fluid replacement, without signs of clinical infection. In animal studies, survival time was related statistically to the dosage of Clostridium perfringens in multicontaminated explosive wounds. Survival time was lengthened by the application of some topical antibacterial agents, but actual recovery was achieved only with topical mafenide hydrochloride solution aqueous spray, which resulted in negative clostridium. perfringens cultures. Although not related statistically to survival time, the mafenide hydrochloride spray also controlled the Pseudomonas aeruginosa in these wounds. Mafenide hydrochloride had the American trade name of Sulfamylon from about 1942 until 1998, when another pharmaceutical company patented Sulfamylon as the trade name for mafenide acetate, a weaker antibacterial agent. However, mafenide hydrochloride still is available from chemical companies. Mafenide hydrochloride solution spray has been used successfully in treatment of patients with severe by contaminated wounds and deep burns, and its use in initial care should be revisited.

Experimental Clostridium perfringens type D enterotoxemia in goats.

The effects of intraduodenal administration of Clostridium perfringens cultures and culture products in goats were evaluated to develop a reliable experimental model of enterotoxemia in this species. Five conventionally reared, 11-16-week-old Angora goat kids were dosed intraduodenally with whole cultures of C. perfringens type D; five similar animals were dosed with C. perfringens type D filtered culture supernatant; and a third group of five kids was dosed with C. perfringens type D washed cells. Two kids were used as controls and received sterile, nontoxic culture medium intraduodenally. All animals received starch solution into the abomasum. All five kids inoculated with whole culture and three of five dosed with culture supernatant and with washed cells developed central nervous system signs. Diarrhea was observed in two of five kids inoculated with whole culture, in all five of those dosed with culture supernatant, and in three of five of those that received washed cells. The most striking postmortem findings consisted of lung edema, necrotizing pseudomembranous colitis, and cerebral vasogenic edema. The protocol thus provided a reasonable model of naturally occurring enterotoxemia in goats, producing a range of clinical signs and postmortem changes similar to those observed in the natural disease.

Sporulation-promoting ability of Clostridium perfringens culture fluids.

The culture supernatant fluids (CSFs) of 12 strains of Clostridium perfringens types A, B, C, and D stimulated sporulation of test strains NCTC 8238 and NCTC 8449 of this organism. The sporulation-promoting ability was present in vegetative and sporulating CSFs of both enterotoxin-positive (Ent+) and Ent- strains. The sporulation factor possessed a molecular weight between 1,000 and 5,000 and was heat and acid stable. This study suggests a potential role for Ent- strains in food-borne disease outbreaks caused by Ent+ strains of C. perfringens type A.

Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens

Clostridium perfringens enterotoxin (CPE) is an important sporulation-associated virulence factor in several illnesses of humans and domestic animals, including C. perfringens type A food poisoning. Therefore, the ability to determine the enterotoxigenicity of food or fecal C. perfringens isolates with simple, rapid assays should be helpful for epidemiologic investigations. In this study, Western immunoblotting (to detect CPE production in vitro) was compared with PCR assays and digoxigenin-labeled probe assays (to detect all or part of the cpe gene) as a method for determining the enterotoxigenicity of C. perfringens isolates. The cpe detection assays yielded reliable results with DNA purified from vegetative C. perfringens cultures, while Western immunoblots required in vitro sporulation of C. perfringens isolates to detect CPE production. Several cpe-positive C. perfringens isolates from diarrheic animals did not sporulate in vitro under commonly used sporulation-inducing conditions and consequently tested CPE negative. This result indicates that cpe gene detection and serologic CPE assays do not necessarily yield similar conclusions about the enterotoxigenicity of a C. perfringens isolate. Until further studies resolve whether these cpe-positive isolates which do not sporulate in vitro can or cannot sporulate and produce CPE in vivo, it may be preferable to use cpe detection assays for evaluating C. perfringens isolate enterotoxigenicity and thereby avoid potential false-negative conclusions which may occur with serologic assays.

Clostridial enteritis in free‐living lorikeets(Trichoglossusspp.)

Numerous free-living lorikeets (Trichoglossus spp.) in 19 flocks in eastern Australia either developed diarrhoea, depression and died or were found dead. Grossly, the duodenum and jejunum of affected birds were enlarged and haemorrhagic with yellow, necrotic mucusa. Histological examination of affected intestines revealed acute necrosis and oedema of villous structures, with vascular congestion, haemorrhage and a moderate infiltration of heterophils and lymphocytes. Gram-stained sections of affected intestines revealed numerous Gram-positive bacilli at the brush border. Anaerobic bacteriological culture of selected intestines revealed Clostridium species in profuse growths. Gas-liquid chromatography and biochemical tests revealed a complex of C. perfringens. Beta toxin was detected in intestinal contents and from half the C. perfringens cultures from affected birds.

DETECTION of ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN GROUND BEEF

Clostridium perfringens is widely distributed in foods. This experiment was performed to assess occurrence of C. perfringens cultures and toxigenic strains isolated from ground beef Samples (118) were collected from 20 locations in Northeast Kansas and the number of C. perfringens was enumerated in these samples by Fung's Double-tube method with tryptose sulfite cycloserine agar medium. Out of 118 samples, 54 (46%) were found positive for C. perfringens. Pure isolates of C. perfringens were further grown in cooked meat medium for 24 h at 42C then heat shocked at 75C for 20 min and inoculated into modified Duncan and Strong medium for production of C. perfringens enterotoxin. Presence of enterotoxin was tested by the reverse passive latex agglutination test (Oxoid), which can detect enterotoxin up to a minimum level of 2 ng/mL. the data indicate that 46% of the beef samples harbored C. perfringens, but only 32 (6%) of 525 isolates were found to produce enterotoxin. This study emphasized the importance of continued surveillance of C. perfringens in meats and meat products and assessment of the toxigenesis of isolates.

A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of Clostridium perfringens enterotoxin (CPE) by a sandwich method with polystyrene beads was elaborated. The ELISA was very sensitive with a detection limit of 1 pg/ml of CPE. Clostridium perfringens culture fluid did not interfere with the assay. This ELISA may be useful for the mass screening for Cl. perfringens producing small amounts of CPE.

Influence of Bacteria on Clostridium perfringens Infections in Young Chickens

When monoflora chickens with Lactobacillus acidophilus or Streptococcus faecalis were inoculated with Clostridium perfringens either in broth culture or resuspended in Gifu anaerobic medium broth or supernatant fluid, few or no chickens died. Approximately 50% of germ-free chickens died after inoculation of C. perfringens culture, whereas no conventional birds died after inoculation of broth culture. C. perfringens in the contents of duodenum from germ-free chickens numbered about 10(4) colony-forming units (CFU)/g after inoculation 10(8) CFU broth culture per bird, but in gnotobiotic and conventional chickens this organism decreased or was not detected. When C. perfringens was cultured in intestinal contents collected from germ-free chickens, C. perfringens proliferated but alpha toxin was not detected. These findings indicate that the pathogenicity of C. perfringens was suppressed by L. acidophilus or S. faecalis administered previously or inhibited by normal intestinal flora.

Obstetric Haemorrhage: Causes and Management

A total of 405 mixed sex broilers with a BW of 42±1 g (1 d of age) were used in a 35-d trial to investigate the effects of phytogenic feed additive (PFA) containing essential oils of thyme and star anise as main active components on growth performance, energy and nutrient apparent total tract digestibility (ATTD), blood metabolites, intestinal microflora, meat color, and relative organ weight after oral challenge with Clostridium perfringens. Broilers were randomly assigned to 1 of 3 treatments (9 replicate pens per treatment with 15 broilers per pen). Dietary treatments were: control (CON); antibiotics (ANT), CON+10 mg/kg of avilamycin); and PFA, CON+250 mg/kg of PFA. Broilers were oral challenged with 5 mL C. perfringens (107 cfu/mL) culture suspensions on d 19, 20, and 21. Broilers fed PFA diets had greater (P<0.01) feed conversion ratio than those fed CON and ANT diets during the finisher phase (d 21–35). Broilers fed PFA diet got lower (P<0.01) FCR than those fed CON diet during d 0–35. The chicks fed PFA had greater (P<0.01) ATTD of dry matter, and gross energy than that fed CON and ANT diets at d 21. The ATTD of crude protein and crude ash were increased (P<0.05) by dietary supplementation of avilamycin or PFA at d 21. Dietary supplementation of avilamycin or PFA improved (P<0.05) the ATTD of gross energy at d 35. At d 35, supplementation of PFA reduced (P<0.01) the serum total cholesterol (TC) content and increased (P<0.01) high density lipoprotein-cholesterol (HDL-C) than CON and ANT treatments. The HDL-C concentration was increased (P<0.01) by avilamycin inclusion in the diet compared with that of broilers in CON. The PFA inclusion reduced (P<0.01) C. perfringens and Escherichia coli counts in small and large intestine. The lesion score of small intestine was also reduced (P<0.05) by supplementation of the diet with PFA compared with ANT and CON diets. In conclusion, our results indicated that supplementation of PFA improved growth performance, reduced blood TC, and also inhibited C. perfringens and E. coli proliferation in small and large intestines in broiler chicks under oral C. perfringens challenge.

Metabolism of primary bile acids by Clostridium perfringens

C. perfringens degraded chenodeoxycholic acid (CDC) into two major products: 7α-hydroxy-3-oxo-5β-cholanoic acid and 3β,7α-dihydroxy-5β-cholanoic acid, the latter appearing very quickly and the former very slowly in whole cell cultures. Yields of about 60 and 16% were obtained for the respective products. Both products, as substrates in C. perfringens cultures, were quickly transformed back to CDC (about 84%) and 3β,7α-dihydroxy-5β-cholanoic acid (about 16%), whereas 7α-hydroxy-3-oxo-5β-cholanoic acid was formed slowly (4–5 days). In contrast to CDC, cholic acid was not epimerized at 3α-OH position; however, 7α, 12α-dihydroxy-3-oxo-cholanoic acid was formed slowly. When 7α,12α-dihydroxy-3-oxo cholanoic acid was substrate, rapid quantitative reduction, back to cholic acid then slow oxidation back to the 3-oxo product was observed. Thirty-six strains of C. perfringens (18 from a culture collection and 18 isolated from human feces) were screened for 3α-OH epimerization and oxidation of the primary bile acids. All but three strains epimerized CDC (yields 14–20%), while none epimerized cholic acid. Oxidation at the 3 position varied from 0–30% for CDC and 0–90% for cholic acid. Cell-free preparations of C. perfringens in the presence of NADP produced both 3-oxo and 3β-hydroxyl products with CDC, but only the 3-oxo product with cholic acid; these activities were non-inducible. The authors propose that 3α-OH isomerization takes place early in log phase via a 3α- and 3β-hydroxysteroid dehydrogenase (HSDH) system. Accumulation of 3-oxo product occurs late in stationary phase as the Redox (Eh) value rises above —100 mV.

Virginiamycin Effects on Controlling Necrotic Enteritis Infection in Chickens

Duplicate trials were conducted with male broiler chickens to evaluate virginiamycin as treatment against experimentally induced necrotic enteritis infection. Each trial consisted of seven treatments, each replicated four times, with 10 birds per replicate. Two treatments were fed control ration (noninfected control and infected control) and the five remaining treatments were fed virginiamycin at 5, 10, 15, 20, or 40 g/ton. Birds were orally dosed with 10 ml of Clostridium perfringens culture at 14 days of age. At 5 weeks of age, surviving birds were killed and necropsied to obtain lesion scores. Birds fed virginiamycin had significantly less mortality and lower intestinal lesion scores than nonmedicated birds when experimentally infected with necrotic enteritis.

New presumptive identification test for Clostridium perfringens: reverse CAMP test.

The reverse CAMP test proved to be a highly sensitive test since 97.0% of all Clostridium perfringens cultures tested gave an easily discernible positive reaction.

The prevention of experimental Clostridium novyi and Cl. perfringens gas gangrene in high-velocity missile wounds by active immunisation.

SUMMARY Sheep were immunised with a mixed vaccine containing the toxoids of Clostridium perfringens, Cl. septicum and Cl. novyi and challenged at different intervals after immunisation. The sheep were wounded and challenged in the thigh muscle by a high-velocity bullet that passed through a piece of battledress impregnated with a suspension of either Cl. novyi spores or Cl. perfringens culture. Immunisation was almost completely effective in preventing the onset of gas gangrene due to Cl. novyi even 1 yrafter immunisation, the survival rate being 82.5-94.5 percent, within 90 per cent, confidence limits (rate for unvaccinated controls: 2·5-8·5 per cent.). Vaccinated sheep that survived challenge with Cl. novyi werelater challenged with Cl. perfringens and survived this, the rate of survival being 83-94.5 per cent, (rate for unvaccinated controls : 12-50 per cent.). The role that such a vaccine might play in the prevention of gas gangrene in man is discussed.

A Technique for Producing Large Yields of Vegetative Cell-Free Refractile Clostridium perfringens Spores of Unaltered Heat Resistance1

Sonic treatment of sporulated Clostridium perfringens cultures produced suspensions free of vegetative cells without altering viable spore counts or thermal resistance.

The effectiveness of sorbed trianatoxin in combating gas gangrene in monkeys

Monkeys infected with 0.25 to 2 ml of an 18-hour culture ofCl. perfringens developed the general and local symptoms characteristic of gas gangrene. A dose of 0.5 ml of an 18-hourCl. perfringens culture was absolutely lethal for monkeys. Immunization by sorbed trianatoxin, and subsequent reinoculation led to the accumulation of 1 to 4 units ofCl. perfringens antitoxin in the blood. Monkeys which were twice immunized with sorbed trianatoxin and subsequently reimmunized six months later were then able to survive infection with 3 to 6 times the lethal dose of the culture.

Characterization of the bacterial enzyme “Thromboplastinase”

1. 1. The action of the enzyme “thromboplastinase” has been compared with that of a Clostridium perfringens culture filtrate and purified α-toxin, and of a Bacillus cereus phospholipase. 2. 2. All enzymes released acid-soluble phosphorus from purified lecithin. In the conentrations used, the clostridial enzymes were most active. In addition, all enzymes liberated acid-soluble phosphorus from human brain thromboplastin and reduced the clot-accelerating activity of thromboplastin. In these cases, however, thromboplastinase was most active. 3. 3. The acid-soluble compounds released by thromboplastinase, by the B. cereus phospholipase, and by C. perfringens lecithinase were identified as phosphocholine, phosphoethanolamine,sb and phosphoserine and were measured quantitatively. Thromboplastinase liberated phosphocholine most actively, and the other compounds approximately equally. The clostridial enzymes liberated as much phosphocholine as did thromboplastinase, but less phosphoethanolamine, and little or no phosphoserine. The B. cereus phospholipase released small amounts of all three compounds. 4. 4. The destruction of thromboplastic activity does not appear primarily due to the lecithinase activity of thromboplastinase, but rather to the breakdown of phosphatidylethanolamine and possibly phosphatidylserine by this enzyme. The relation between thromboplastinase action and that of the other enzymes studied, especially that of the B. cereus phospholipase, is discussed.