Molecular Characterization of
Pasteurella multocida
Isolates of Clinical
significance
By
RABIA DURRANI
Department of Microbiology
QUAID-I-AZAM UNIVERSITY, ISLAMABAD.
Introduction
Objectives of Study
Materials and
Methods
Results

Focus of the study

Disease and it’s causative agents
INTRODUCTION
1.
Livestock and Poultry sectors are among the major
contributors in the economic growth of Pakistan
2. Both sectors are facing serious problems; among them
are
some
dangerous
diseases
like
Hemorrhagic
Septicemia (HS) which are posing a huge risk to the
economic development in these sectors
3. The focus of the research was to study the disease and
identify the agents responsible for.
Focus of the study


Pasteurella multocida is Gram negative, Non-spore forming,
cocobacillus bacterium
Commensals of upper respiratory tract of livestock and
Poultry

Causes severe Hemorrhagic septicemia (HS)

HS occurs as catastrophic epizootics in Asia and Africa
Disease and it’s causative agent(s)
Disease and it’s causative agent(s) (Continued….)

Several cases of HS reported in Wild animals also.

Common serotypes are Asian B:2 and African E:2

In wild animals B:2,5 strain is most common.

Vaccination is achieved by Whole cell preparations.

Common Vaccines are alum precipitated and Oil adjuvant.
Disease and it’s causative agents (Continued….)


Live Intranasal Vaccine B:3,4 serotype of deer origin found
successful in South East Asia. (Muneer and Afzal, 1989).
Common cause of HS disease is Vaccination Failure and that
is why death rate of animals is High in South East Asia.



Comparisons of isolates from clinically diseased animal (animal
suffering
from
Hemorrhagic
Septicemia)
for
their
microbiological profiles to explore any gross difference.
Study of molecular profiles to elucidate further proteomics.
To collate and analyze the data obtained and to examine
perception of the scientific workers that Pasteurella multocida
is an opportunistic pathogen.
Objectives of Study
 Sample collection & Identification of clinical isolates
 Antibiogram analysis of Pasteurella multocida
 Molecular Characterization of P. multocida
 P. multocida species specific PCR
 HS-associated type B serotype specific PCR
 Protein Profile Analysis of P. multocida by SDS-PAGE
 Whole Cell Profile of P. multocida
 Envelop profile of P. multocida
 Restriction enzyme analysis of P. multocida
MATERIALS AND METHODS
Sample Collection and Identification of clinical isolates

Samples were collected from National Veterinary Labs
Islamabad that were brought into the lab from different
cities

Nature of the samples were lungs and spleen tissues of
animals.

Gram staining of Organism

Subculturing and Purification




Biochemical characterization
Catalase test
Oxidase test
Growth in the absence of CO2
Identification of clinical isolates (Continued….)




Urease production test
H2S Production Test
Nitrate reduction test
Motility test

Injecting mice with clinical isolates of clinical Isolate

API 20 NE Test (Biomeurix )

Hyaluronidase Enzyme test for Pasteurella
(Smith et al., 1968)
multocida
Antibiogram analysis of Pasteurella multocida

Differentiation
of
clinical
microbiological staining dyes:
◦ Crystal violet
◦ Malachite Green
◦ Safranin
◦ Congo Red
isolates
by
Molecular
multocida
Characterization
of
Pasteurella

Crude method of DNA extraction

Amplification of Extracted DNA of Pasteurella
multocida by Polymerase Chain Reaction (PCR)

P. multocida species specific PCR assay
P. multocida species specific PCR
Primer sequences were:

KMT1SP6 5’ - GCTGTAAACGAACTCGTCGTCGCCAC3-3’

KMT1T7
5’- ATCCGCTATTTACCCAGTGG-3’
PCR Master Mix Preparation

PCR master mix about 20µl was prepared as follows:







H2O
MgCl2
dNTPs
Primer 1
Primer 2
PCR Buffer
Taq Polymerase
 Total Volume:

13µl
02µl
01µl
01µl
01µl
1.5µl
0.5µl
20µl
Then 5µl –extracted DNA was added, and makes the final
volume upto 25µl.
The thermal cycling parameters were as follows:
The initial denaturation at 94°C for 5minutes;

30cycles of:

Final extension at 72°C for 9minutes.
◦ 94°C for 1minute
◦ 53°C for 1minute (for primer annealing)
◦ 72°C for 1minute (extension)
Analysis of PCR product

About
8µl
of
PCR
product
were
separated
by
electrophoresis on 2% High melting Agarose gel in 1X TrisBoric acid running buffer (TBE) at 4V/cm for 1 hour .The
gel was stained with 1% ethidium bromide and DNA
fragments were viewed by Gel documentation system.
PCR assay for HS-associated type B serotype
Pasteurella multocida

PCR analysis for HS –associated type B serotype P. multocida
identification was performed.
◦ Primer pair used:
 KTSP61
 KTT72

These primers specifically amplify a product of approximately
560 base pair (bp) in all HS causing serotype of P. multocida.
HS causing Type B gene specific PCR test
Primer sequences were:

KTT72 5’- AGGCTCGTTTGGATTATGAAG-3’

KTSP61 5’ – ATCCGCTAACACACTCTC-3’
PCR master mix preparation
PCR master mix about 20ul was prepared as follows:
◦
◦
◦
◦
◦
◦
◦
◦

H2O
MgCl2
dNTPs
Primer 1
Primer 2
Buffer
Taq Polymerase
Total Volume:
13µl
02µl
01µl
01µl
01µl
1.5µl
0.5µl
20µl
Then 5ul of extracted DNA was added, and make the final
volume upto 25µl.
The thermal cycling parameters were as follows:

The initial denaturation at 94°C for 5minutes;

30cycles of:
◦ 94°C for 1minute
◦ 53°C for 1 minute
◦ 72°C for 1minute

Final extension at 72°C for 9minutes.
Protein Profile Analysis of P. multocida by SDSPAGE

Harvesting of Culture

Washing of Cells

Sonication (Disruption) of Cells for Whole Cell Preparation

Preparation of Whole-cell for SDS-PAGE

Preparation of Envelope

Staining and Destaining of Gel

Determination of Molecular Weight of Proteins
Whole Cell Profile of Pasteurella multocida

For the extraction of proteins, thick growth of pure P.
multocida were taken with the help of Pasteur pipette and
dipped in the ependorff tube containing 200µl-distilled
water.

Then they were vortexed with the help of (IKA USA mixer)
thoroughly to dissolve completely.

After
dissolving
the
samples
(dr.hielscher, type UP 400S).
were
sonicated
in

Sonicated for: ◦
◦
◦
◦


30 second stroke
30 second cooling
Amplitude 80 to 100
Cycle 0.5 to 1
After sonication:
◦ centrifugation at 12,000 rpm
◦ Time 5 min
The microcentrifuge was used to remove intact
cells. The supernatant was taken as whole cell and
boiled in boiling water along with 4X sample
loading buffer for 5minutes at 100°C before
loading on gel
High Energy Probe Sonicator
(DR. HEILSCHER Type 2005, Germany)
Envelop profile of P.multocida
Preparation of Envelope (Irfan et al.,2008)

Thick growth of P. multocida culture was:
◦ Suspended in 2ml of distilled water in eppendorff tube
◦ Centrifuged at 20,000rpm for 30minutes at 8˚C.
◦ The pellet was resuspended in 20mM Tris-HCl buffer and again
centrifuged at 20,000rpm for 30minutes.
◦ The final pellet rich in envelop was resuspended in 20mM Tris-HCl
buffer and stored at –20°C for further SDS-PAGE analysis.
Restriction enzyme analysis of P.multocida
Steps Involved







10 X restriction endonuclease buffer
DNA
Distilled water
Restriction enzyme and then gently mix the contents
Centrifuge for few seconds in a microfuge and the incubate
at 37°C for 1-4 hours
Heat the reaction at 65°C for 20 minutes
Run the digests on gel
Characterization of P.multocida

Cultural and microscopic

Biochemical

Analytical Profile Index (API) test

Antibiotic sensitivity

Hylouronidase production

Differentiation by microbiological staining dyes

PCR

SDS-PAGE
RESULTS
Cultural Characterization of P.multocida

On Brain Heart Infusion (BHI) agar and Nutrient agar:
◦
◦
◦
◦
Convex colonies
Entire edges
Mucoid and sticky nature
Approximate size of 2-3 mm diameter

On blood agar, all the pure culture showed:

On MacConkey agar:
◦ Luxuriant growth
◦ Translucent grayish or yellowish green colonies
◦ No haemolysis
◦ No growth
Staining of P.multocida

The Gram staining shows:
◦ Pleomorphic, gram negative, coccobacilli
◦ 0.2-0.4 mm in size
Colony morphology of P.multocida
Gram staining of P.multocida
Biochemical test results of P.multocida
Table 1. Common biochemical properties of Pasteurella multocida
API test results of P.multocida
API test results of P.multocida
Table 2. Analytical Profile Index (API) test results of Pasteurella multocida
Antibiotic sensitivity test for P.multocida
Antibiotic sensitivity test for P.multocida
Table 3. Number of clinical isolates sensitive to the antibiotics
Rapid plate screening test for Hyaluronidase production of P.multocida
S.No
Name of City
01
Number of
Isolates
Appearance of white
precipitate
Hyaluronidase (+)
Hyaluronidase
(-)
18
06
12
02
Kahutta
06
04
2
03
Badin
04
03
1
05 including
B:3,4
04
1
02
02
0
06
04
2
06
05
1
08
04
03
1
09
07
04
3
04
05
Taxila
06
07
Toba Tek Singh
10
Samundri
03
02
1
11
Faislabad
09
08
1
Table 4. Results of Hylouronidase production by P.multocida
Differentiation of Clinical strains of P.multocida by different staining
dyes
S.No
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
Name of City
Badin
Tando Jam
Taxila
Samundri
Khurrianwala
Toba Tek Singh
Jaranwala
Bakkar
Abbotabad
Khushab
Kahutta
Faislabad
Name of Dye (Conc. in µg)
Safranin
Malachite
Congo Red
Green
Observed
zone of
Inhibition
(mm)
No. of Crystal
Isolates Violet
0.1,1,5
37
0 0
04
0 0
07
0 0
05
0 0
12
0 0
04
0 0
03
0 0
03
0 0
0.1,1,5
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0.1,1,5
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0.1,1,5
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0
0
0
0
0
0
0
0
No zone
No zone
No Zone
No Zone
No Zone
No Zone
No Zone
No Zone
15
11
15
12
10
15
05
06
08
03
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
No Zone
No Zone
No Zone
No Zone
No Zone
No Zone
No Zone
No Zone
No Zone
No Zone
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Table 5. Differentiation of Clinical isolates by Microbiological
staining dyes
PCR results of P.multocida
Amplification of Extracted
multocida by PCR

DNA
of
Pasteurella
All samples when run on Agarose Gel Electrophoresis showed:
◦ Bands of base pairs 560 and 750
◦ The DNA ladder started from 100 base pairs.
Table 6. PCR results of P.multocida
Whole cell profiles of P.multocida HS and Non-HS (Badin)




Organism was genetically conserved and there is no demostratable
diversity with in the number of samples tested.
Isolates from wild life be included in such studies.
This organisms maintains harmlessly inhealthy animals and causes
disease as an opportunistic pathogen in extreme stress to animals.
By further studies we can devise better methods for control against
disease caused by this organism.
Conclusions & Future Prospect
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