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Studies on Clostridium perfringens Types A and D Enterotoxins and their Sporulation
Keywords: Clostridium perfringens
enterotoxins
Studies on Clostridium perfringens Types A and D Enterotoxins and their Sporulation
Norhan, A. Khairy*; Ammar, A.M.*; Mona, A. Maghwery**; and Hanaa, A. Ahmed***
*Bacteriology, Mycology and Immunology Department, Fac. of Vet. Med., Zagazig University.
**Anaerobic Unit, Bacteriology Research Department, Animal Health Research Institute, Dokki, Giza.
***Mycoplasma Research Department, Animal Health Research Institute, Dokki, Giza.
ABSTRACT
In the present study, 10 C.perfringens strains (8 were type A and 2 were type D) were recovered from faecal samples of 10 enterotoxaemic calves and used for extraction of C.perfringens enterotoxin (CPE) using Duncan and Strong sporulation medium (DS), of the extracted toxins were purified by ammonium sulfate precipitation, then profiling of the enterotoxin proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Confirmative conventional PCR was done to C.perfringens isolates (one was type A and the other was type D) had the gene encoding CPE and gave a characteristic band at 233 bp. Electrophoretic profiles of the purified C.perfringens type A enterotoxins characterized by the presence of 2 characteristic protein bands with molecular masses of 40 and 66 kDa. Our study recorded that there is a direct relation between sporulation and the ability of C.perfringens to sporulate on DS sporulation medium as 8 out of 10 examined C.perfringens isolates formed subterminal and central oval non bulging endospores, 6 out of them produced enterotoxin.
INTRODUCTION
C.perfringens is Gram positive short plumb bacilli, about 3-7 µm in length and 0.4-1.2 µm in thickness rarely has central or subterminal oval non bulging endospores (Wilson and Miles, 1975). Certain strains of C.perfringens produce enterotoxin (CPE) which is an endotoxic component that formed exclusively in sporulating cells of C. perfringens and located in the vegetative part of the cell from which it is released when the vegetative remnants are lysed (Willis, 1977). The enterotoxin that produced by C. perfringens type A is a heat sensitive protein (320 amino acid), precipitable with ammonium sulfate, antigenic, non dialyzable and activated mainly by proteolytic activities (Hauschild, 1970 and Afaf and Basma 2003), it serves as an additional virulence factor in causing enteritis in domestic animals (Werdeling et al., 1991), it may be also produced by C. perfringens type D (Sayeed and McClane, 2007). The mode of action of C.perfringens enterotoxin occurredthrough multiple steps including binding to a specific receptor on cell membranes, insertion into the plasma membrane and complexing with membrane proteins (one toxin molecule of 35 kDa and membrane proteins of 70 and 50 kDa for a final complex of 160 kDa). The toxin doesn't enter cytoplasm but its presence in plasma membrane induces a rapid decrease in intracellular concentrations of ions and small molecules. Other effects include inhibition of synthesis of macromolecules, decreased energy metabolism, increased secretory flux, inhibition of glucose uptake and outpouring of water, sodium and chloride which manifested clinically by diarrhoea. (Songer, 1996). The enterotoxin production is directly related to the sporulation of the microorganism (Philippe et al., 2006) but the function of enterotoxin in sporulation is unknown (Stark and Duncan, 1972).
This work is aimed to extraction and purification of C. perfringens types A and D enterotoxins, detection of CPE gene using conventional polymerase chain reaction (PCR), electrophoretic profile of CPE to determine the antigenic relatedness between the isolates and to evaluate the correlation between CPE production and ability of the microorganism to sporulate.
MATERIAL AND METHODS
1- Samples: 10 C.perfringens strains (8 were type A and 2 were type D) were recovered from faecal samples of 10 enterotoxaemic calves (with haemorrhagic enteritis and severe diarrhoea) were used in this study.
2- Isolation and identification the microorganism: itwas performedby cultivation of each sample on an enrichment cooked meat medium (CMM) then detection of the haemolytic activity of C.perfringens toxins onto 10% sheep blood agar medium with neomycin sulphate (200 µg /ml) (Smith and Holdeman, 1968). Biochemical identification of the recovered isolates (fermentation of sugars, gelatin liquefaction, litmus milk, catalase and indole tests) (Koneman et al., 1992). Detection of lecithinase activity of C.perfringens alpha toxin on lecithin of an enriched egg yolk agar medium (Smith and Holdeman, 1968). Typing of the recovered isolates using dermonecrotic reactions in albino guinea pigs (Quinn et al., 2002).
3- Sporulation and enterotoxin production by C.perfringens isolates (Sakaguchi et al., 1973 and Enders and Duncan 1977): ten pure C.perfringens isolates were cultured in CMM, transferred to Duncan and Strong (DS) sporulation medium and incubated at 37°C for 8 hours under anaerobic condition then examined microscopically for the presence of central or subterminal oval non bulging endospores. The sporulated cells of four isolates were washed once in cold distilled water then suspended in 200 ml of cold saline. The cells were disrupted by sonic treatment [6 Hertz for 20 minutes using sonicator ultra-sonic (Farmingdale, New York, 11735, Model XL 2020, USA)] and debris was removed by centrifugation at 12000 xg for 20 minutes at 4°C in a cooling centrifuge (Nuaire, USA) to obtain a clear extract. The resultant cell extract was used for further purification of the enterotoxin.
4- Enterotoxin purification (Waters et al., 2003): the supernatant protein was precipitated by addition of ammonium sulfate (4.76g of (NH4)2SO4 /10 ml supernatant) and incubation overnight at 4°C. The precipitated protein was then collected by centrifugation at 12000 rpm for 30 minutes at 4°C and resuspended in 25µl of sterile distilled water.
5- PCR for detection of C.perfringens enterotoxin gene:
A- Extraction of C.perfringens DNA (Sheedy et al., 2004):
1 ml of culture from each of the six sporulated C.perfringens isolates was centrifuged at 5000 xg for 15 minutes; the cell pellet was washed twice with 1 ml phosphate buffer saline (PH 7.2) then resuspended in 50µl PBS. The mixture was vortexed, heated directly at 100°C for 10 minutes in a heat block for cell lysis then cooled on refrigerator for 5 minutes. Finally the suspension was centrifuged at 13,000 xg for 2 minutes. 10 µl of supernatant fluid was used as template DNA.
B- Primer selection for CPE gene (Tong and Labbe, 2003):
Forward 5' GGA GAT GGT TGG ATA TTA GG 3'
Reverse 5' GGA CCA GCA GTT GTA GAT A 3'
C- PCR amplification and cycling protocol (Tong and Labbe, 2003):
DNA samples were amplified in a total of 25 μl of the following reaction mixture: 5µl 10X buffer, 1µl MgCl2, 1µl dNTPs, 0.5µl Taq polymerase enzyme, 0.25µl of each primers, 12µl DNase-RNase- free deionized water and 5µl of template DNA. PCR cycling program was performed in Ptc-100 Peltier thermal cycler MJ Research as following: initial denaturation at 94°C for 5 minutes then 30 cycles consisting of (denaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes and extension at 72°C for 3 minutes) thus followed by final extension at 72°C for 5 minutes.
D- Detection of PCR products: as reported by (Augustynowicz et al., 2000) and the image analysis was done by Image QuantTL-V2003.03 (Amersham Biosciences).
E- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of C.perfringens enterotoxins (Oconnor, 2006):
An equal volume of lysis buffer (50mM Tris-HCl [pH 6.8], 10% SDS, 50% glycerol, 2% β-mercaptoethanol, 0.1% bromophenol blue) was added to the precipitated protein then boiling at 100°C for 5 minutes in a water bath and centrifugation in a microcentrifuge at 11.600 rcf for 10 minutes to remove cell debris. Supernatant containing the purified CPE was stored at – 4°C until used. Estimation of the protein concentration was determined (Bradford, 1976). The proteins were separated on 10% acrylamide gels by adjusting the current to provide 1.5 mA per lane for 4-5 hours in a Hoefer SE 400 electrophoresis unit (Hoefer Scientific Instrument, San Francisco, California, USA). Gels were stained with Coomassie brilliant blue. SDS-PAGE molecular weight standard was used as a marker (Pharmacia). Image analysis was done by Image QuantTL-V2003.03 (Amersham Biosciences).
Results
1- Isolation and identification C.perfringens:
All the examined samples gave double zones of haemolysis on neomycin sulphate sheep blood agar medium, fermentative to different sugars (glucose, maltose, lactose, sucrose and mannose) with production of acid and gases, gelatin liquefiers, litmus milk positive, catalase, oxidase and indole tests negative, positive Nagler`s reaction and by dermonecrotic reaction in albino guinea pigs, 8 isolates were identified as C.perfringens type A and 2 were type D.
2- PCR results:
Two out of six C.perfringens isolates had had the gene encoding CPE (one was type A and the other was type D) and gave a characteristic band at 233 bp (photo 1).
Photo (1): Agarose gel electrophoresis of C. perfringens enterotoxin genes.
| 1 2 3 4 5 6 7 7 |
| 233 |
| bp 500 200 100 |
Lane 1: 100 bp DNA ladder (Pharmacia).
Lane 2, 3, 5 and7: Negative for C. perfringens enterotoxin field isolate
Lane 4 and 6: C.perfringens type D enterotoxin field isolate
3- SDS-PAGE of C.perfringens type A enterotoxin results:
Electrophoretic profile of the four purified C.perfringens type A enterotoxins gave 3–9 protein bands with molecular weights ranged from 12.17 -128.7 kDa. All isolates shared a common characteristic protein bands at 40 and 66 kDa. (table 1) and (photo2).
Table (1): Electrophoretic profile of the four purified C.perfringens type A enterotoxins
| | C. perfringens enterotoxin field isolate (1) | C. perfringens enterotoxin field isolate (2) | C. perfringens enterotoxin field isolate (3) | C. perfringens enterotoxin field isolate (4) |
| | MW (kd) | MW (kd) | MW (kd) | MW (kd) |
| 1 | | 128.39 | | 128.7 |
| 2 | | 113.78 | | 114.13 |
| 3 | | 90.13 | | |
| 4 | 77.71 | | | |
| 5 | 65.31 | 66.96 | 65.18 | 65.18 |
| 6 | 60.4 | 59.01 | | 59.01 |
| 7 | 55.41 | | | |
| 8 | 48.75 | | 49.2 | 48.9 |
| 9 | 39.77 | 38
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