typing of clostridium perfringens from necrotic enteritis...
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Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
MS thesis submitted by
Amany El-Sayed
Under the supervision of
Prof. Dr. Heidy Shawky Prof. Dr. Mohamed Refai
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Introduction Material and Methods Results Conclusion and Recommendation
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Introduction
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Introduction
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Avian necrotic enteritis (NE) was first described in 1961 since then it has been reported to occur in almost all poultry-producing countries
Studies showed that the subclinical form of (NE) is a worldwide problem with an average of 80% of the flocks having had Clostridium diagnosed especially in broiler and turkey flocks
A follow-up study in 2005 indicated an increased incidence of Clostridial enteritis in all regions of the world.
Recent European surveys confirmed the severity and the widespread of the problem
(NE) is caused by toxins produced by Clostridium perfringens.
Introduction
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Isolation of C. perfringens and/or detection of its alpha toxin are of little value to confirm the disease because both are often found in the intestine of healthy birds
Clostridium perfringens is a Gram-positive, spore forming and anaerobic bacterium responsible for a wide range of diseases in humans and animals
Clostridium perfringens is commonly classified to Toxigenotypes: Type A, B, C, D and E. based on the types of toxins they produce. The main toxins produced by strains of Clostridium perfringens are α, β, ε and ι
Introduction
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
The conventional method of Clostridium perfringens typing is based on the detection and typing of the toxins with toxin neutralization test in mice. This procedure consumes a lot of antisera and experimental animals. Moreover, it is time consuming. In recent years, molecular techniques such as polymerase chain reaction (PCR) are increasingly used to type Clostridium perfringens
Toxin genotyping of Clostridium perfringens isolates utilizing PCR has usually required multiple reactions per isolate as the organism produces many different important toxins.
The large number of oligonucleotide primers needed to detect these genes in a single multiplex reaction.
Introduction
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
The aim of this research was to determine the prevalence of (NE) in turkeys and
identify the different types of Clostridium perfringens isolated from living and
slaughtered turkeys by Multiplex PCR molecular method.
Material and methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
A total number of 122 samples (39 intestinal samples and 83 fecal swabs
samples) was collected from apparently healthy and diseased turkey poults.
butchers' shops in Giza (27) and from Kafr Ghataty farm (95) The age of the turkey poults varied from 2 to 5 months. Media used for isolation and identification of C. perfringens:
1. Liquid media: Cooked meat medium enrichment. 1. Solid media: Neomycin sulphate sheep blood agar medium: isolation, purification observation of colonial morphology haemolytic activity of C. perfringens.
Materials
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Media used for preservation of C. perfringens:
Sheep blood agar for one week only.
Cooked meat medium for repeated subcultures.
Brain heart infusion with 16% glycerol for cryopreservation of C. perfringens
isolates till further use.
Stain: Gram's stain for microscopically identification.
Material used for DNA extraction, PCR, and agarose gel electrophoresis: Tris Edita buffer (TE). PCR master mix. Oligonucleotide primers.
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Primers of C. perfringens alpha toxin gene
Beta toxin gene
Epsilon toxin gene
iota toxin gene enterotoxin
(van Asten et al., 2009)
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Primer name and direction Nucleotide sequence ( 5 `to 3 `) Amplicon (bp)
cpa:
324 Forward GCTAATGTTACTGCCGTTGA
Reverse CCTCTGATACATCGTGTAAG`
cpb:
196 Forward GCGAATATGCTGAATCATCTA
Reverse GCAGGAACATTAGTATATCTTC`
etx:
655
Forward GCGGTGATATCCATCTATTC
Reverse CCACTTACTTGTCCTACTAAC
iA:
446 Forward ACTACTCTCAGACAAGACAG
Reverse CTTTCCTTCTATTACTATACG`
cpe:
233 Forward GGAGATGGTTGGATATTAGG
Reverse GGACCAGCAGTTGTAGATA
Table (2): Primers for the five toxins genes of C. perfringens used in multiplex PCR.
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Nuclease- free water.
Reagents used for agarose gel electrophoresis: Agarose gel (1.5%) Electrophoresis buffer Ethidium bromide solution
DNA loading dye.
DNA size markers: Gene ruler TM 100 bp DNA ladder. Gene ruler TM 50 bp DNA ladder.
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Isolation of C. perfringens: Inoculation of freshly prepared cooked meat medium Incubation anaerobically at 37°C for 24-48 hours. Subculture onto 10% sheep blood agar with neomycin sulphate (150-200 µg
/ml). The plate was incubated anaerobically at 37°C for 24-48 hours. Examination of suspected colonies for : morphological and cultural characters Gram stained films
Methods
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Colonial appearance Flat rounded with double zone of haemolysis Microscopically appearance: Gram positive bacilli with rounded ends rarely had central or oval non bulging endospores.
Methods
Materials and Methods
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
3. Identification of the isolates: Biochemical tests PCR for genotyping of C. perfringens: A total of 56 C. perfringens isolates subjected to genotyping by multiplex PCR.
1. Extraction of C. perfringens DNA 2. PCR amplification and cycling protocol 3. Visualization of PCR products
Methods
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
1. Symptoms observed on the examined diseased turkey poults.
2. P.M. lesions.
3. Classification of the collected samples:
I. Classification of the collected samples according to location
II. Classification of the collected samples according to sex
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Results Classification of the collected samples according to location
Kafr Ghatati's Farm
slaughter house and butcher’s shops
0
10
20
30
40
50
60
70
80
Apparently heathy birds
Diseased Poults
20
75
27
Kafr Ghatati's Farm slaughter house and butcher’s shops
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Classification of the collected samples according to sex
Apparently heathy birds
Diseased Poults
0
5
10
15
20
25
30
35
40
45
50
Male
Female
27
11.5
50
11.5
Apparently heathy birds Diseased Poults
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results of isolation of C. perfringens from turkey with regard to health states
0
10
20
30
40
50
60
70
80
Diseased
Apparently healthy
30
36
45
11
No. of Negative samples No. of positive samples
75
47
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results of isolation of C. perfringens from turkey with regard to location
0
10
20
30
40
50
60
70
80
90
100
Kafr Ghatati farm
Slaughter houses
45
21
50
6
No. of Negative samples No. of positive samples
95
27
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results of isolation of C. perfringens from turkey with regard to sex
0
10
20
30
40
50
60
70
80
90
100
Male
Female
48
18
46
10
No. of Negative samples No. of positive samples
94
28
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results of isolation of C. perfringens from turkey with regard to age
0
5
10
15
20
25
30
35
40
45
50
2 M
3 M
4 M
5 M
12 22
16
16
7
12
5
32
No. of Negative samples No. of positive samples
19
34
21
48
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results of isolation of C. perfringens from turkey with regard to season
0
10
20
30
40
50
60
70
80
90
Winter
Summer
24 42
14
42
No. of Negative samples No. of positive samples
38
84
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
655pb 446pb 324bp 300 233pb 196pb 150 100
50
50 bp
DNA
Marker
Control
+ve
1 2 3 4 5 6 7 8 9 10 11 12
Control
-ve
13
PCR Result Photo (1): Multiplex PCR gel amplification product of Alpha toxin (324 bp), beta toxin (196 bp), epsilon toxin (655 bp), iota toxin (446 bp) and enterotoxin (233 bp) Lane 1: DNA Marker (50 bp), Lane 2: positive control, Lane15: negative control and Lane 3-14 and 16: C. perfringens field isolates.
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
PCR Result Photo (2): Multiplex PCR gel amplification product of Alpha toxin (324 bp), beta toxin (196 bp), epsilon toxin (655 bp), iota toxin (446 bp) and enterotoxin (233 bp) Lane 1: DNA Marker (100bp), Lane 2: negative control, Lane 3: positive control and Lane 4-12: C. perfringens field isolates.
655pb 446pb
324bp 300pb 233pb 196pb 100pb
100bp
DNA
Marker
Control
-ve
Control
+ve 14 15 16 17 18 19 20 21 22
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
PCR Result Photo (3): Multiplex PCR gel amplification product of Alpha toxin (324 bp), beta toxin (196 bp), epsilon toxin (655 bp), iota toxin (446 bp) and enterotoxin (233 bp) Lane 1: DNA Marker (100bp), Lane 2: positive control, Lane16: negative control and Lane 3-15: C. perfringens field isolates.
655pb 446pb 400pb 324bp 233bp 200pb 196pb 100pb
100bp
DNA
Marker
Control
+ve 23 24 25 26 27 28 29 30 31 32 33 34 35
Control
-ve
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
PCR Result Photo (4): Multiplex PCR gel amplification product of Alpha toxin (324 bp), beta toxin (196 bp), epsilon toxin (655 bp), iota toxin (446 bp) and enterotoxin (233 bp) Lane 1: DNA Marker (100bp), Lane 2-5: C. perfringens field isolates.
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results of molecular typing of Clostridium perfringens isolates
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
Type A
Type A withenterotoxins Type C
Type E
Negative
43.50%
0.80% 0.80%
0.80%
54.10%
Clostridium perfringens Percentage
Results
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Results of molecular typing of Clostridium perfringens isolates type A
according to status of the bird
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
Diseased living poults
Diseased Dead poults
Apparently healthy livingpoults Apparently healthy
slaughtered poults
29.50%
7.40%
4.10%
2.50%
Conclusion and recommendations
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Conclusion and Recommendations
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
C. perfringens plays a serious role in turkey enteritis through proliferation in the
intestinal tract with stormy fermentation gas and acid formation causing
obstructions of intestinal tract and production of several exotoxins as Alpha,
Beta, Epsilon and Iota as well as endotoxin (enterotoxin).
(NE) and hepatitis caused by C. perfringens and other associated micro-
organisms have great economic value as following:
Multiplex PCR is highly sensitive, rapid and accurate method for differentiation
between different C. perfringens types.
The most found type of C. perfringens isolated from enteritis in turkey is type A
producing Alpha toxin. Enterotoxins also detected.
Conclusion and Recommendations
Typing of Clostridium Perfringens from necrotic enteritis in turkeys by multiplex PCR
Cairo University
Faculty of Veterinary Medicine
Microbiology Department
Type C and Type E were also detected
Type B and Type D were not detected.
We recommended to take care of the following:
Improve hygienic condition in turkey flocks.
Provide good quality ration.
Carry out further studies on pathogenic genes of C. perfringens and produce
suitable vaccines to control the infection without misuse of antibiotics or
growth promotors which is forbidden
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