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Characterization and Biochemical Behavior

Assessment of Vaccinal Strain of Pasteurella
multocida type 1 B6
Article December 2015
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Dildar Hussain Kalhoro

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Asian Institute of Technology

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Kalhoro et al., IJAVMS, Vol. 9, Issue 1, 2015: 29-35

CHARACTERIZATION AND BIOCHEMICAL BEHAVIOR

ASSESSMENT OF VACCINAL STRAIN OF PASTEURELLA
MULTOCIDA TYPE 1 B6
Dildar Hussain Kalhoro1, Rahmatullah Rind1, Muhammad Saleem Kalhoro1, Alam
Zeb2
Ali Zaman2, Shahnawaz Kumbhar 3, Mazhar Hussain Mangi 1 and Rahimdad
Birohi1
1
Department of Veterinary Microbiology, Sindh Agriculture University Tandojam,
Pakistan
3
Department of Veterinary Pathology, Sindh Agriculture University Tandojam, Pakistan
2
Gomal Collage of Veterinary Sciences, Gomal University D. I. Khan KPK, Pakistan
ABSTRACT
This study was carried out to record the morphological and biochemical properties of Pasteurella
vaccinal strain. Pasteurella multocida Type 1 B-6 was observed as gram-negative, short rods,
arranged singly, bipolar. Coccobacillary in shape and recorded as non-motile and non-spore
forming. In old culture, the organism showed pleomorphic and long rod characteristics. During
the investigation, various types of the colonies of the organism were also observed on blood agar.
The organism appeared as non-haemolytic and exhibited moist, mucoid and shiny growth. It
failed to grow on MacConkeys agar. It was observed that vaccinal strain of Pasteurella
multocida fermented glucose, Sucrose ,Maltose, Galactose , Mannose and Fructose but did not
ferment lactose and salacin. It did not liquefy gelatin.
Key Words: Pasteurella multocida, Vaccinal strain, Morphology, Biochemical
characteristics.
INTRODUCTION
Pasteurella multocida is gram-negative, non-motile, rod shaped, facultative
anaerobic bacterium and has been isolated from a wide range of host throughout the world. This
bacterium is the etiologic agent of a variety of economically significant diseases in both wild and
domestic animals, including fowl cholera in poultry, haemorrhagic septicemia in cattle and
buffalo, atrophic rhinitis in swine, and snuffles in rabbits 1.
Pasteurellosis is among the important contagious diseases in the world. As this disease
has many similarities with respiratory diseases of cattle and sheep, scant attention is being paid to
it. Organism is present in the natural flora of the buccal-pharyngeal region, therefore in the
animals which are under stress, like the ones that are being transferred, have respiratory
infections, have bad nutrition and ventilation, and are being kept in overcrowded places, bacterial
growth and proliferation occur in the region and later gets extended to the lower respiratory tract
and causes pasteurellosis 8.9.5. Haemorrhagic septicaemia locally known as Gal Ghoto is one of
the economically important disease of buffaloes, cattle and sheep. The young animals, specially
growing heifers, calves and kids/lambs and the most common victims of this deadly disease. In

CHARACTERIZATION AND BIOCHEMICAL BEHAVIOR ASSESSMENT OF VACCINAL STRAIN OF PASTEURELLA MULTOCIDA TYPE 1 B6

the absence of proper treatment and preventive measures, it gives classical losses to farmers as it
has high morbidity and mortality in bovines. Most cases are acute resulting in death within 8 to
24 hours after onset 4-5. Disease can be prevented by use of antibiotics such as enrofloxacin,
oxytetracycline chloramphenicol and ampicillin 6-7.1.
The purpose of the study was to determine the morphological, cultural and biochemical
properties of Pasteurella vaccinal strain.
MATERIALS AND METHODS
Study was carried out at the Department of Veterinary Microbiology, Faculty of Animal
Husbandry and Veterinary Sciences, Sindh Agriculture University, Tando Jam and Vaccine
Production Unit, Tando Jam. Pasteurella multocida seed type I (B6) was obtained from
Veterinary Research Institute, Lahore. The morphological, cultural and biochemical tests were
carried out on different media and chemicals. To conduct the study different preliminary steps
were taken like sterilization of glassware, media preparation and stains preparation. New glass
wares were immersed in HCl solution overnight to remove the surface alkali. Autoclave was used
to sterilize the used glassware, petri dishes, conical flasks, bijoux bottles, universal bottles. All
other glassware containing media were sterilized at temperature of 121oC with 15lb pressure for
15 minutes and washed in basin containing low concentration of detergent and antiseptic (2%
lysol). The vaccinal strain of Pasteurella multocida was inoculated on MacConkeys agar, Muller
Hinton agar, Brain Heart Infusion Agar, Blood agar and peptone agar and colonies were observed
after 24 hours. When the colonies were found morphological, cultural and staining characteristics
of Pasteurella multocida were studied. All the isolates were passed through the biochemical tests
i-e Oxidase, Catalase, Urease, Voges Proskauer, Tripe sugar iron agar ,Indole, Gelatin
liquefaction, Nitrate reduction and Sugar fermentation test for further verification of bacteria.

RESULTS AND DISCUSSION

Morphologically Pasteurella multocida was observed as Gram negative. Cells of
the species were short rods, bipolar and some were coccobacillary in shape. Other morphological,
cultural and staining characteristics of the Pasteurella multocida on different culture media
observed during present study are present in Table 1, and Plates 1, 2. Holt et al. (1991) reported
that Pasteurella multocida was Gram negative and non-motile. Cells were spherical, ovoidal or
rod shaped, occurred singly or less frequently in pairs or short chains and having bipolar
characteristics. Finegold and Baron (1986) reported similar characteristics of Pasteurella species
as reported by above authors. It was observed as small coccoid rod shaped bacillus that often
exhibited bipolar staining. The species produced small, translucent and smooth or rough colonies.
Therefore present findings are in the complete agreement to those of the above workers.
Biochemical values of Pasteurella multocida were determined and recorded during study (Table
2 and Plate 3). Biochemically, Pasteurella multocida showed positive interaction in catalase,
oxidase, indole, and nitrate reduction, but was found to be negative for geletin liquefaction,
urease and Voges-Proskauer. However, triple sugar iron medium was consumed and produced
change in the medium and was observed as acid slant and acid butt. During this experimental
work, sugar fermentation tests were also used to confirm biochemical properties. Eight different

Kalhoro et al., IJAVMS, Vol. 9, Issue 1, 2015: 29-35

CHARACTERIZATION AND BIOCHEMICAL BEHAVIOR ASSESSMENT OF VACCINAL STRAIN OF PASTEURELLA MULTOCIDA TYPE 1 B6

sugars were used and their interactions such as acid and gas production were noted. Pasteurella
multocida fermented various sugars like glucose, fructose, galactose, sucrose, maltose and
mannose but failed to ferment lactose and salicin.
Sugar fermentation and biochemical values of Pasteurella multocida were determined
and recorded during the study are present in Table 2, 3 and Plate 3. Karaivanov (1984) stated that
Pasteurella multocida was indole positive, reduced nitrate to the nitrates, produced of hydrogen
sulfide, lysine-decarboxylase-, and phosphatase activity, and fermented substrates such as manite,
glucose, galactose, saccharose, manose, levulose, dulcite, lactose, maltose, rafinose, trechalose,
salicin, melobiose, icelobiose, arabinose, xylose, and sorbite. Unchitti et al., (1992) reported that
Pasteurella multocida produced catalase, oxydase, indol, nitrate reduction and ornithine
decarboxylase but failed to produce urease, gelatinase, methyl red, acetoin and could not grow on
MacConkey agar, SS-agar, in nutrient broth with 0% or 6% NaCl. Holt et al. (1994) investigated
that Pasteurella multocida was urease negative. Glucose and other carbohydrates were
catabolised with the production of acid but no gas, catalase-positive and oxidase-positive; nitrates
were reduced to nitrites. The findings of present study regarding the biochemical and sugar
fermentation properties are in agreement to the properties demonstrated by the above workers in
their studies.
Table 1. Morphological, cultural and staining chacteristics of Pasteurella multocida .

S.No

Bacterial
species

Microscopic chacteristics
Arrangement

1.

Pasteurella
multocida

Single

Colony chacteristics

Shape

Staining
reaction

Motility

Small rods,
Coccobacillary,
bipolar.

Gram-ve

-ve

Solid

Broth

On blood agar the

organism appeared as nonhaemolytic and exhibited
moist, mucoid and shiny
growth. It failed to grow
on MacConkeys agar

In broth
medium,
granular
deposits were
formed in the
bottom of the
tube.

Table 2. The bio-chemical properties of Pasteurella multocida type 1(B6).

S.NO

Specie

Bio-chemical tests
1

A/A

Pasteurella multocida type 1(B6).

1. Triple sugar iron 2. Indole 3. Oxidase

4. Catalase
6. Nitrate reduction
7. Urease
8. Voges praskauer

5. Gelatin liquefaction

Kalhoro et al., IJAVMS, Vol. 9, Issue 1, 2015: 29-35

CHARACTERIZATION AND BIOCHEMICAL BEHAVIOR ASSESSMENT OF VACCINAL STRAIN OF PASTEURELLA MULTOCIDA TYPE 1 B6

Table 3. Sugar fermentation properties of Pasteurella multocida type 1(B6)

S.NO

1.

Specie

Sugars

Pasteurella multocida type

1(B6)

Glucose

Sucrose

Maltose

Galactose

Mannose

Fructose

Lactose

Salicin

+ =
- =

Positive result
Negative result
a

Plate 1. Morphological characteristics Pasteurella multocida type 1(B6).

Kalhoro et al., IJAVMS, Vol. 9, Issue 1, 2015: 29-35

CHARACTERIZATION AND BIOCHEMICAL BEHAVIOR ASSESSMENT OF VACCINAL STRAIN OF PASTEURELLA MULTOCIDA TYPE 1 B6

a.

Pasteurella multocida type 1(B6)

Gv.r

b.

Plate 2.

d.

= Gram-negative rods s =

Pasteurella multocida type 1(B6)

BHI = Brain heart infusion broth gd

single cb =

coccobacillary

= granular deposits nor = normal

Cultural characteristics of Pasteurella multocida type 1(B6) on different

media.
a.
Blood agar
B/A = blood agar
nhc = non-hemolytic colonies
b.
MacConkeys agar
M/A =
MacConkeys agar ng
= no growth
c.
Muller Hinton agar
MHA = Muller Hinton agar wc = light white colonies
Brain heart infusion agar
BHI
= Brain heart infusion agar lwc
= white colonies

Kalhoro et al., IJAVMS, Vol. 9, Issue 1, 2015: 29-35

CHARACTERIZATION AND BIOCHEMICAL BEHAVIOR ASSESSMENT OF VACCINAL STRAIN OF PASTEURELLA MULTOCIDA TYPE 1 B6

Plate 3.

Bio-chemical and sugar fermentation properties of Pasteurella

multocida type 1(B6).
a. Triple sugar iron test
A/A =
acid slant and acid butt n
=
normal
b. Indole test
Ind+ve
=
indole positive nor
=
normal
c. Sugar fermentation test
sf +ve
= sugar fermentation positive
npw-sf
= normal peptone water for sugar fermentation
d. Oxidase test
Oxi + ve =
positive
e. Catalase test Cat +ve = positive
CONCLUSION
It is concluded that Vaccinal strain Pasteurella multocida Type 1 B-6 was
observed as Gram-negative. Cells were short rods, arranged singly, bipolar and some were
coccobacillary in shape and recorded as non-motile and non-spore forming. Organism failed to
grow on MacConkeys agar. Pasteurella multocida showed positive interaction in catalase,
oxidase, indole, and nitrate reduction, but was found to be negative for geletin liquefacation,
urease and Voges-Proskauer, fermented various sugars like glucose, Sucrose, Maltose, Galactose,
Mannose and Fructose but failed to ferment lactose and salicin. It is further concluded that
present study would help in better understanding of the identification procedures for the
organisms of Pasteurella species specially Pasteurella multocida during laboratory identification.
REFERENCES:
1. Aiello, S.E,. The Merck Veterinary Manual. Merck and Co. Inc., N.J., USA.1998th ed.,pp
537 539.

Kalhoro et al., IJAVMS, Vol. 9, Issue 1, 2015: 29-35

CHARACTERIZATION AND BIOCHEMICAL BEHAVIOR ASSESSMENT OF VACCINAL STRAIN OF PASTEURELLA MULTOCIDA TYPE 1 B6

2. Araghy Soure, A. Etiological study of calf pneumonia by analysis of bronchoalveolar

fluid. Ph.D.Thesis Veterinary Faculty, Uni of Tehran. 2007
3. Finegold, S.W, Sydney.M. and Baron.EJ , Diagnostic Microbiology.The C.V.Mosby
Company St. Louis. 1986. .7th ed
4. Holt, J.G, Krieg.N.R, Sneath.P.H.A., Staley. J.T. and S.T.Williams.S.T,. Bergeys
Manual of Determinative Bacteriology, Williams & Wilkins Baltimore, USA. 1994, 9th
ed.p.196
5. Kalhoro. D.H.,Rind.R , Kalhoro.S and Baloch.H,. In-Vito antimicrobial susceptibility of
Pasteurella multocida. Pak. J. A.A.E.V..S. 2010, 26(2):80-86.
6. Karaivanov.K. Biochemical tests for identifying Pasteurella multocida. Vet. Med. Nauki.
1984,21 (9): 38-44.
7. Morishita, T.Y., Lowenstine. L.J, Hirsh.D.C and Brooks.D.L Pasteurella multocida in
raptors: prevalence and characterization. Avian Diseases, Snipes, K.P., Hirsh.1996.
40:908918.
8. Muneer , A, Mumtaz.M.D and Ahmed.J ,. Haemorrhagic septicemia in bovine respiratory
diseases. Agro Vety. News. 1998Vol: 9.p.6.
9. Unchitti, K, Wongsawang.S, Saitanu.K and Thoongsuwan.S, Characteristics of Pasteurella
multocida isolated from humans, swine and poultry in Thailand J.S.A..Trop.Med
public Health, 1992,23(3): 520-525.

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