typing of clostridium perfringens from necrotic enteritis...

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


Cairo University



Introduction


Cairo University



Introduction


Cairo University



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


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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


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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


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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


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Materials and Methods


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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



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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.



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Primers of C. perfringens alpha toxin gene

Beta toxin gene

Epsilon toxin gene

iota toxin gene enterotoxin

(van Asten et al., 2009)



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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.



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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.



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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



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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



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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


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Results


Cairo University



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


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

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Results


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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


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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


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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


95

27

Results


Cairo University



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


94

28

Results


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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


19

34

21

48

Results


Cairo University



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


38

84

Results


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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


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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


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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


Cairo University



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


Cairo University



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


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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


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Conclusion and Recommendations


Cairo University



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


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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

Thanks


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typing of clostridium perfringens from necrotic enteritis...

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