Genotyping characterization on clostridium perfringens affecting laboratory animals.
Laboratory animal diseases constitute one of the most important problems and do not occur as a single caused event, but are the end result of interactions of the microorganism's laboratory animals includes (Rat, Mice, Rabbits and Guinea pigs).Clostridia are large Gram positive rods, mostly motile, sporeforming fermentative and catalase negative. It is widely distributed in soil and presents as normal commensals in gastrointestinal tracts of many animal species and man . It causes dramatic infections and intoxications as protein exotoxins either singly or in combination are the major virulence factors that determine the clinical manifestion, morbidity and mortatity . The spores of some Clostridia species are very heat resistant and may survive to the heat treatment of canned foods. If the surviving spores germinate and the vegetative cells grow, spoilage will occur . The species of C.perfringens are divided into five types from A to E on the bases of production of four major toxins namely alpha (a), beta, (fi) epsilon (e) and iota (i), each of these types has been linked to specific diseases .Alpha toxin is the principle lethal toxin of C .perfringens, and a multifunctioinal phospholipase that prouced mainly by all types of the species . Epsilon toxin is protease activated protoxin that produced mainly by C. perfringens types B and D . Enteritis in laboratory animals mainly after weaning is the major cause of economic losses in the commercial laboratory animals the anaerobic bacteria including Clostridium spp. as etiological agents in necrotic enteritis in laboratory animals, many pathogenic anaerobic bacteria are normal inhabitants of the bacterial flora in animals  Diagnosis of enteritis that caused by C.perfhngens in laboratory animals mainly based on evaluation of clinical sings, gross and microscopical lesions, and bacrtriological culture of specimens and also detection of toxins in the specimens .Dermonecrotic test in albino guinea pig is a helpful method for typing of C.perfringens isolates . Polymease chain reaction (PCR) has been applied for detection of the genes encoding major toxins of C.perfringens. This method has been highlighted as a rapid and accurate method for detection of low copy numbers of genes. Also, the sensitivity and specificity of this method were confirmed by amplification of specific target DNA under a unique condition .This method is more accurate and faster than seroneutalization with mice or guinea pigs  .The aim of this work Detection and identification of C. perfringens toxins genes by conventional and multiplex PCR.
MATERIAL AND METHODS
A total of one hundred and ninety two (192) internal organs samples (liver, kidney, spleen, intestine) were used in this study. Samples were collected from apparently healthy rats 28, apparently healthy rabbits 28, diseased rats and rabbits 32 (each), while 24 water samples and 48 samples of pellets offered to theses rats and rabbits.
Culture media used:
Cooked meat medium and Nutrient agar medium (Oxoid) Neomycin sulphate sheep blood agar medium . Exotoxin production medium  Enriched egg yolk medium  Sugar fermentation medium  Aesculin hydrolysis medium  Gelatin medium  Indole medium. Antibiotic (oxoid): Neomycin sulphate (Up john)
Diagnostic C. perferingens antitoxin type A, B, C, D and E which was occurred in Animal Health Institute. (Burroguns, Welcome, Bechenham, London, England's) are used for typing of C. perfringens. Buffers and reagents used for conventional PCR [10;45;40].
Agarose gel electrophoresis buffers and reagents . DNA ladder (100pb, pharmacia, Catalogue number 27-4001-01, USA). (Spectroylyne Model TR-312 A).
Collection of samples:
One hundred and ninety two samples were collected for the isolation of clostridium perfringens (ICMSF, 1978)
Identification of the isolates Microscopical appearance:
Colonical appearance: Biochemical tests:
Catalase_test ; Gelatin liquefaction test; indole test;nitrate reduction test ;Sugar_fermentation_test; hydrogen Sulphide production test (Oxoid) and litmus milk_medium : Nagler's test by half antitoxin plate : Typing of Clostridium perfringens toxins dermonecrotic test in albino guinea pigs Toxin antitoxin neutralization test : Determination of the MLD of the prepared toxin : Antibiogram sensitivity test (Oxiod): Pathological, gross and histopathological examination
DNA extraction and purification : A) Extraction of C.perfringens DNA B) Purity assessment
PCR Amplification and cycling protocol: A) For conventional PCR [10;45;400] B) For multiplex PCR 
Detection of PCR products 
Incidence of C.perfringens from apparently healthy and diseased rats and rabbits and its pellets and water samples
One hundred and ninety two samples collected from apparently healthy rats (28), apparently healthy rabbits (28), diseased rats (32), diseased rabbits (32), water (24) and pellets of rats and rabbits (48). As shown in table (4), it was noticed that 57.1%, 53.5%, 81.2%, 84.3%, 50% and 58.3% of examined samples were positive for presence of C. perfringens microorganism, respectively
Antibiogram studies of the prevalent isolates of C.perfringens:
As shown in table (5) it was noticed that C. perfringens isolates are highly sensitive to Penicillin G (90%) and Clindamycine (90%), followed by Lincomycin (80%), finally Tylosin (75%) while, C. perfringens isolates weres resist to Cephaloridine, Ampicillin (35%), Chloramhemiol (30%), Erythromycine (25%) and finally Oxytetracycline (10%).
Histopatholgical studies of the internal organs in rat:.
1. Pathological feature in freshly sacrificed rats:-as shown in (photo7, 8).
2. Histopathological findings:-the portal vein and accompanied with nuclear pyknosis as shown in (photo, 10).
3. The liver showing marked haemolysis of RBCs within centeral vein & hepatic sinusoid as shown in (photo, 11)
4. Meanwhile, the liver showing different stages of necrosis of hepatic cells and pyknosis (first stage of necrosis) as shown in (photo, 12).
Results of polymerase chain reaction:
Conventional PCR results:
Conventional PCR technique was used for detection of alpha, epsilon of C.perfringens types "A" and "D" recovered from laboratory animal. PCR amplicons were electrophoresed in 1.5% agarose gel and stained with 0.5 [micro]g /ml ethidium bromide. Results of conventional PCR revealed that C. perfringens alpha toxin gave 1167 bp fragment as shown in (photo 18), C.perfringens epsilon toxin gave 960 bp fragment as shown in (photo 19).
Multiplex PCR results:
Multiplex PCR was used as a sensitive technique to identify C.perfringens isolates recovered from laboratory animals. PCR amplicons were electrophoresed in 1.5% agarose gel and stained with 0.5 pg/ml ethidium bromide. All the examined field identified as C.perfringens type "A" (alpha toxin) and gave 402 bp fragment and two examined field identified as C.perfringens type "D"(epsilon toxin) and gave 514 bp fragment as shown in (photo 20).
Anaerobic organisms are nation wide distribution. Although there is much that obligate anaerobic organisms are probably the principle sources of infection in human beings and domestic animals . Usually, C. perfringens has been classified into five toxigenic types (although E) on the basis of its ability to produce the major lethal toxins .
Therefore, type A exhibits several powerful toxicities, and infection with type A may result in myonecrosis, hemolysis, an increase in vascular permeability and platelet aggregation [25;14].The major lethal effects associated with this toxin are gas gangrene in human and necrotic enteritis and enterotoxaemia in animals .Epsilon toxin is produced by types B and D of C. perfringens and is responsible for a rapid fatal enterotoxaemia in economically important livestock *.This toxin is secreted as relatively inactive protoxin and is activated to a potent heat-laible toxin with the loss of an N-terminal peptide by a proteolytic enzyme produced by the organism . The activated protein is highly toxic and lethal, and have lethal, dermonecrotic activities . Neutralization with mice or guinea pigs has been performed to type C. perfringens. Neutralization depends mainly on having all the specific antitoxins against a, p, e and iota toxins. Therefore, the aim of the work is to assess the value of using biotechnology in rapid detection of C.perfringens toxins, to evaluate the role of C.perfringens toxins in laboratory animal diseases and to determine the incidence of C.perfringens in laboratory animals.In this study, one hundred and ninety two samples were collected from apparently healthy rats (28 apparently healthy rabbits (28), diseased rats (32), diseased rabbits (32), water (24) and pellets of rats and rabbits (48). Numbers of positive samples were 16, 15, 26, 27, 12 and 28, respectively. This result agrees with that recorded by Johnson and Gerding (1997).All the isolates were identified microscopically, colonial morphology, biochemical characteristics and analysis of fatty acids (Lecthinase test). All isolates were identified as C. perfringens were subjected to typing with mice serum neutralization test using commercial diagnostic antitoxin against alpha, epsilon and toxins (Burrough's Welcome, U.K.).All toxigenic isolates collected from apparently healthy rats classified into 2 types A and D types only .Result in table (11) show that 3 out of 10 isolates obtained from apparently healthy rats were type A and 2 type D and 5 non toxigenic isolates. Isolates recovered from apparently normal rabbits were classified as 4 type A, 2 type D and 6 non toxigenic C. perfringens. On the other side, isolates recovered fom diseased rats were 20 type A and 8 type D and 2 non toxigenic and diseased rabbits 22 type A and 7 type D and 3 non toxigenic. Finally in pellets of rats and rabbits 9 type A and 3 type D and 8 non toxigenic and in water 4 type A and 2 type D and 4 non toxigenic.Regarding to conventional methods for identification of C.perfringens recovered from laboratory animal, the obtained results revealed that C.perfringens is Gram positive short plumb rarely sporulated and non motile bacilli. It was apparent that sheep blood agar with neomycin sulphate (200 [micro]g/ml) is a perfect medium for isolation of C.perfringens rather than other Clostridium species and gave double zone of heamolysis. All the recovered strains in this work were fermentative to different sugars as glucose, maltose, lactose, sucrose and mannose with production of acid and gases, gelatin liquefiers, litmus milk positive, Catalase, Oxidase, and Indol test negative. Similar results were recorded by several authors as [19;2]. Nagler's test represented the action of C.perfringens alpha toxin (lecithinase) on lecithin of egg yolk onto enriched egg yolk agar medium which appeared as pearly opalescence zone surround the colonies while this reaction was inhibited by C.perfringens alpha toxin antiserum.These results go hand in hand with those recorded by Smith and Holdeman  who applied Nagler's test using half antitoxin plate to detect lecithinase activity of alpha toxin of different types of C.perfringens .Dermonecrotic test in albino guinea pigs. The isolated strains from laboratory animals were type A, D and. Action of C.perfringens type "A" isolates (producing only alpha toxin) appeared as an irregular area of yellowish necrosis tented to spread downward while that of type "D'' (produced alpha and epsilon toxin) appaered as a circular white necrosis with few small areas of purplish haemorrhagic necrosis as shown in (photo 6 and 7).These dermonecrotic criteria agree with those recorded by [29;37] for typing of C.perfringens.C.perfringens isolates recovered from laboratory animal were identified by dermonecrotic reactions in albino guinea pigs into toxigenic (67.3%) and non toxigenic (32.7%) strains. Typing of toxigenic C.perfringens strains recorded that C.perfringens type A was the most predominant one and represented by 47.3% while type D was 20%.
In the present study, toxin antitoxin neutralization test on the skin of albino guinea pigs and lethal toxicity test I/P in mice were applied using specific antitoxin to detect and identify the toxigenic strains of C.perfringens recovered from laboratory animal. The obtained results revealed that C.perfringens type A was the most predominant one among the total recovered isolates. These results are in accordance with . As shown in table (5) C. perfringens isolates were highly sensitive to Penicillin G (90%), Clindamycin (90%), Lincomycin (80%), Tylosin (75%) but the same strains were resistant to Cephaloridine (40%), Ampicillin (35%), Chloramphenicol (30%), Gentamicin (30%), followed by Doxyclline HCl (25%), Erythromycin (25%), Streptomycin (25%), Nalidexic acid (25%) and finally Oxytetracycline (10%).
Pathological features in freshly sacrificed rats showed mainly congestion in liver, while in few friable livers with grayish red pin point foci were observed. In kidney and urinary bladder appeared severely increased in size and congested, the intestine and spleen showed congestion .
Histopathological findings in liver mostly showed vascular degeneration associated with nuclear pyknosis; also it is found in liver marked depletion and congestion of the portal vein accompanied with nuclear pyknosis. Finally, liver also showing marked haemolysis of RBCs within the central vein and liver showed different stages of hepatic cell necrosis.
The kidney mostly showed marked interstitial haemorrahges within the renal cortex, thrombosis of renal vasculature, accumulation of eosinophilic proteinaious material within the glomerular space and showed in the kidney metastatic calcification with the glomerular tuft, severe congestion in renal blood vessels.
The spleen showed numerous numbers of haemosidrin laden macrophages.
Polymerase chain reaction (PCR) allows the specific and exponential synthesis of a predetermined DNA region via the use of two small and specifically designed fragments of DNA (primers or oligonucleotides) which form the two termini of the nucleic acid molecule to be amplified. PCR amplification reactions in general are highly specific and the specificity being determined by the correct hybridisation of primer specific sequences to be complementary sequences present on the target DNA molecule to be amplified . PCR is used for diagnostic purposes as it is much faster than conventional microbiological techniques for isolation and identification of causative agents. It has been proved to be a very sensitive and specific technique for detection of the genes encoding alpha and epsilon exotoxins of C.perfringens for rabid typing and evaluation of the virulence of the microorganism [14;10;45;26;1].
In the present work, conventional PCR was applied to detect the genes encoding alpha and epsilon of C.perfringens strains recovered from laboratory animals as a recent confirmatory technique for the traditional methods of identification. The recorded results revealed that C. perfringens type A contained alpha toxin gene which gave a characteristic band at 1167bp when visualized under UV light. Similar results were recorded by several authors as Fach and Guillou  Augustynowicz et al.,  and Eman et al., . Different results were obtained by Piatti et al.,  who developed a PCR assay for detection of C.perfringens alpha toxin gene using different oligonucleotide primers than those used in this study and it gave a characteristic band at 324 bp.C.perfringens type D isolates were proved to contain alpha and epsilon exotoxins which gave characteristic bands at 1167 and 960 bp respectively. These results coincide with several authors as Warren et al.,  Gkioutzidis et al.,  and Eman et al., . In the present study, multiplex PCR was proved to be a reliable and sensitive protocol for genotyping of the untypable C.perfingens isolates recovered form laboratory animals. PCR primers were selected for the genes encoding alpha and epsilon exotoxins of C.perfringens .The recorded results revealed that all tested strains were identified as C.perfringens type A and gave a characteristic band at 402 bp and tested isolates number (3 & 5) were identified as C.perfringens type D and gave a characteristic band at 514bp.
These results go hand in hand with those obtained by Augustynowicz et al.,  who developed a multiplex PCR assay for determination of the toxin genes of C.perfringens and found that C.perfringens alpha toxin gene amplicon was recorded in all testes isolates and gave a characteristic band at 402 bp. And C.perfringens epsilon toxin gene amplicon was recorded in isolates number (3 &5s) and gave a characteristic band at 514 bp.
Received 23 January 2014
Received in revised form 19 April 2014
Accepted 6 April 2014
Available online 15 May 2014
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(1) Nashwa A. Ezzeldeen, (2) Mamdouh Mahmoud El Shorbagy, (3) Heba Mohammad Diaa Abd El Hafez, (4) Eman M.S.Halwani, (5) Mohamed A. Abdelmonem
(1) Professor of Microbiology, Faculty of Veterinary Medicine,Cairo University Professor of Microbiology, Faculty of science, Taif University.
(2) Chief Researcher and Head of Anaerobic Unit, Microbiology Dept., Animal Health Research Institute, Dokki, Giza.
(3) (B.V.Sc.) Faculty of Veterinary Medicine, Cairo University.
(4) Departement of Biology, Faculty of science, Taif University.
(5) Department of Microbiology, Central Laboratory of Residue Analysis of Pesticides & Heavy Metals in Foods (QCAP), Dokki, Giza, Egypt
Corresponding Author: Nashwa A. Ezzeldeen, Professor of Microbiology, Faculty of Veterinary Medicine,Cairo University Professor of Microbiology, Faculty of science, Taif University.
Table 1: Distribution of total examined samples recovered from apparently healthy and diseased rats, rabbits, pellets and dinking water. Samples (Sources) Number of examined samples % 1- apparently healthy rats 28 14.6 2-apparently healthy rabbits 28 14.65 3-diseased rats 32 16.65 4-diseased rabbits 32 16.6 5-water 24 12.5 6-pellets of rats and rabbits 48 25 Total 192 100 Table 2: Specific oligonucleotide primers sequence used for the toxins genes (Cpa) alpha-toxin and (Etx) epsilon-toxin of C. perfringens. Primer (direction) Nucleotide sequence Amplicon(bp) Cpa (alpha toxin) 5' AAG ATT TGT AAG GCG CTT-3' 1167 Forward 5'- ATT TCC TGA AAT CCA CTC-3' Reverse Etx (epsilon toxin) 961 Forward 5' AAG TTT AGC AAT CGC ATC-3' Reverse 5'- TAT TCC TGG TGC CTT AAT-3' Buffers and reagents used for multiplex PCR . Primers for the two toxin genes (alpha, epsilon,) of C. perfringens were selected from published paper  shown in table (3). Table 3: Primers for the two toxins genes of C. perfringens used in multiplex PCR. Primer name and Nucleotide sequence Amplified direction Product size (bp) Cpa Forward 5' GTT GAT AGC GCA GGA CAT GTT AAG 3' 402 Reverse 5 'CAT GTA GTC ATC TGT TCC AGC ATC 3' Cpe Forward 5ACT GCA ACT ACT ACT CAT ACT GTG 3'' 541 Reverse 5'CTG GTG CCT TAA TAG AAA GAC TCC 3' Table 4: Incidence of C.perfringens in apparently healthy and diseased rats, rabbits and its pellets and water samples: Type of samples No. of examined samples positive samples No. Apparently healthy rats 28 16 Apparently healthy 28 15 rabbits Diseased rats 32 26 Diseased rabbits 32 27 Water 24 12 Pellets of rats and rabbits 48 28 Total 192 124 Type of samples Negative samples % No. % Apparently healthy rats 57.1 12 42.9 Apparently healthy 53.5 13 46.5 rabbits Diseased rats 81.2 6 18.1 Diseased rabbits 84.3 5 15.7 Water 50 12 50 Pellets of rats and rabbits 58.3 20 41.7 Total 64.6 68 33.4 Table 5: Antibiogram studies of the prevalent isolates of C. perfringens. Antimicrobial discs Concentration C. perfringens (20) No. % A.A. Ampicillin 10 pg 7 35 R Cephaloridine 30 pg 8 40 R Chloramphenicol 30 pg 6 30 R Clindomycin 2 pg 18 90 S Doxycillin HCl 30 pg 5 25 R Erythromycin 15 pg 5 25 R Gentamicin 10 Pg 6 30 R Lincomycin 15 pg 16 80 S Nalidix acid 30 pg 5 25 R Oxytetracycline 30 pg 2 10 R Pencillin G 10 i.u 18 90 S Streptomycin 10 pg 5 25 R Tylosin 16 pg 15 75 S No. = number of sensitive isolates % = percentage of sensitive isolates in relation to total isolate species.A.A. = antibiogram activity.
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|Author:||Ezzeldeen, Nashwa A.; El Shorbagy, Mamdouh Mahmoud; El Hafez, Heba Mohammad Diaa Abd; Halwani, M.S.|
|Publication:||Advances in Environmental Biology|
|Date:||Apr 1, 2014|
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