The use of molecular techniques for detection and characterization of the Pasteurella multocida is very important for rapid and specific detection and characterization of the organism. During the period from 15th February, 2014 to 15th April, 2015, 425 nasopharyngeal swabs and 175 lung and spleen samples were collected and examined by conventional methods, 80 strains (18.82%) of P. multocida were isolated from the calves, sheep and goat with respiratory manifestation. Meanwhile, 77 strains (44%) were isolated from emergency slaughtered animals. All the recovered strains were positive for specific PCR for detection of P. multocida strains previously identified as P. multocida by standard microbiological techniques. Multiplex PCR for molecular typing of the capsular antigens of the recovered P. multocida revealed positive amplification of 1044 bp fragments specific to the capsular antigen type A with 105 strains (66.88%), and amplification 511 bp fragments of the capsular antigen type E with 52 strain (33.12%) and absence of B, D and F antigens. Multiplex PCR for molecular typing of the capsular antigens of P. multocida can be used as a simple, sensitive, rapid, reliable technique instead of the serological techniques for identification of the capsular antigens of P. multocid

Ashgan M. Hessain

Ayman S. Mubarak

Hussein M. Galal

Khalid S. Al-Maary

Moussa I. Mohamed

Saleh A. Kabli

Turki M. Dawoud

Saudi Journal of Biological Sciences

AbstractThe use of molecular techniques for detection and characterization of the Pasteurella multocida is very important for rapid and specific detection and characterization of the organism. During the period from 15th February, 2014 to 15th April, 2015, 425 nasopharyngeal swabs and 175 lung and spleen samples were collected and examined by conventional methods, 80 strains (18.82%) of P. multocida were isolated from the calves, sheep and goat with respiratory manifestation. Meanwhile, 77 strains (44%) were isolated from emergency slaughtered animals. All the recovered strains were positive for specific PCR for detection of P. multocida strains previously identified as P. multocida by standard microbiological techniques. Multiplex PCR for molecular typing of the capsular antigens of the recovered P. multocida revealed positive amplification of 1044bp fragments specific to the capsular antigen type A with 105 strains (66.88%), and amplification 511bp fragments of the capsular antigen type E with 52 strain (33.12%) and absence of B, D and F antigens. Multiplex PCR for molecular typing of the capsular antigens of P. multocida can be used as a simple, sensitive, rapid, reliable technique instead of the serological techniques for identification of the capsular antigens of P. multocid

Al-Maary, Khalid S.

Dawoud, Turki M.

Mubarak, Ayman S.

Hessain, Ashgan M.

Galal, Hussein M.

Kabli, Saleh A.

Mohamed, Moussa I.

Elsevier - Publisher Connector 

Molecular characterization of the capsular antigens of Pasteurella multocida isolates using multiplex PCR 

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Molecular characterization of the capsular antigens of Pasteurella multocida isolates using multiplex PCR

Saudi Journal of Biological Sciences (2017) 24, 367–370King Saud University
Saudi Journal of Biological Sciences
www.ksu.edu.sa
www.sciencedirect.comORIGINAL ARTICLEMolecular characterization of the capsular antigens
of Pasteurella multocida isolates using multiplex
PCR* Corresponding authors at: Department of Health Science, College of Applied Studies and Community Service, King Saud Un
P.O. Box 22459, Riyadh 11495, Saudi Arabia (A.M. Hessain). Department of Botany and Microbiology, College of Science, King Saud Un
P.O. Box 2455, Riyadh, Saudi Arabia (M.I. Mohamed).
E-mail address: ahessan@ksu.edu.sa (A.M. Hessain).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
http://dx.doi.org/10.1016/j.sjbs.2016.06.006
1319-562X  2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Khalid S. Al-Maary a, Turki M. Dawoud a, Ayman S. Mubarak a,
Ashgan M. Hessain b,c,*, Hussein M. Galal c, Saleh A. Kabli d,
Moussa I. Mohamed a,c,*aDepartment of Botany and Microbiology, College of Science, King Saud University, Saudi Arabia
bDepartment of Health Science, College of Applied Studies and Community Service, King Saud University, P.O. Box 22459,
Riyadh 11495, Saudi Arabia
cDepartment of Microbiology, Faculty of Veterinary Medicine, Cairo University, P.O. 2446, Cairo, 14242 Giza, Egypt
dDepartment of Biology, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah, Saudi ArabiaReceived 29 February 2016; revised 23 May 2016; accepted 10 June 2016
Available online 17 June 2016KEYWORDS
Molecular typing;
P. multocida;
Multiplex PCR;
Capsular antigens;
Microbiological techniquesAbstract The use of molecular techniques for detection and characterization of the Pasteurella
multocida is very important for rapid and specific detection and characterization of the organism.
During the period from 15th February, 2014 to 15th April, 2015, 425 nasopharyngeal swabs and
175 lung and spleen samples were collected and examined by conventional methods, 80 strains
(18.82%) of P. multocida were isolated from the calves, sheep and goat with respiratory manifesta-
tion. Meanwhile, 77 strains (44%) were isolated from emergency slaughtered animals. All the recov-
ered strains were positive for specific PCR for detection of P. multocida strains previously identified
as P. multocida by standard microbiological techniques. Multiplex PCR for molecular typing of the
capsular antigens of the recovered P. multocida revealed positive amplification of 1044 bp fragments
specific to the capsular antigen type A with 105 strains (66.88%), and amplification 511 bp frag-
ments of the capsular antigen type E with 52 strain (33.12%) and absence of B, D and F antigens.iversity,
iversity,
368 K.S. Al-Maary et al.Multiplex PCR for molecular typing of the capsular antigens of P. multocida can be used as a sim-
ple, sensitive, rapid, reliable technique instead of the serological techniques for identification of the
capsular antigens of P. multocida
 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is
an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).1. Introduction
Pasteurella multocida is a Gram-negative bacterium responsi-
ble for Pasteurellosis (acute septicaemic disease characterized
by high morbidity and mortality rate as well as severe econom-
ical losses) in cattle, sheep, goat and poultry (Prabhakar et al.,
2012; Jakeen et al., 2016). It has a zoonotic impact in human; it
can cause soft tissue infection after animal bites or via inhala-
tion causing respiratory tract infection.
Based on its capsular antigens P. multocida had been clas-
sified into five groups (capA, B, D, E and F) (Espinosa
et al., 2012; Prabhakar et al., 2012). P. multocida is incrimi-
nated in many other disease conditions such as mastitis,
meningitis, peritonitis, atrophic rhinitis and ear infections
due to the effect of P. multocida toxins (Atashpaz et al.,
2009; Prabhakar et al., 2010).
The outbreak of P. multocida showed acute respiratory dis-
ease manifested with high fever, nasal discharge, respiratory
distress, polypnoea and death within few days. The post-
mortem findings showed severe congestion in the lung, trachea,
liver and small intestine (Azizi et al., 2011). The diagnosis of
Pasteurellosis in farm animals is still under taken by the clini-
cal manifestation, post mortem finding and by conventional
bacteriological methods for identification of the causative
agents, such methods are not reliable and time consuming
and at the same time it is very difficult to differentiate between
P. multocida and the other Pasteurella species specially the
Pasteurella haemolytica (Mannheimia haemolytica) (Berge
et al., 2006; Bell, 2008; Rajeev-Gautam et al., 2006).
Therefore, the use of molecular techniques, specially the
polymerase chain reaction (PCR) for molecular detection
and characterization of the capsular antigens of the P. multo-
cida is very important for rapid and specific detection and
characterization of the organism which play an important role
in the control of the disease among the farm animals and
reducing the economical losses (Rajeev-Gautam et al., 2006;
Jakeen et al., 2016). Therefore, the current study is aimed to
detect and characterize the recovered strains of P. multocida
from samples collected from different abattoirs located in
Riyadh, Kingdom of Saudi Arabia.
2. Materials and methods
2.1. Samples
During the period from 15th February, 2014 to 15th April,
2015, 425 nasopharyngeal swabs were collected under aseptic
conditions from calves (n= 200) from 6 to 12 months age,
sheep (n= 125) 6 to 12 months and goat (n= 100) from 1
to 2 years age suffering from respiratory manifestations. Also
175 lung samples were collected from emergency slaughtered
calves (n= 75), sheep (n= 50) and goat (n= 50). The
samples collected from different abattoirs located in Riyadh,Kingdom of Saudi Arabia and transferred under complete
aseptic condition to the College of science laboratory at King
Saud University for standard bacteriological techniques and
for molecular detection and characterization of P. multocida.
2.2. Standard bacteriological examination
All the collected samples were cultured primary into casein
sucrose yeast broth (Oxoid) for 6–8 h and a loopful from each
broth were sub-cultured on to blood agar and MacConkey
agar media and incubated for 48 h at 37 C. The recovered
strains of P. multocida were completely identified with API
20 E tests (BioMe´rieu) according to Jakeen et al. (2016).
2.3. Pathogenicity test
For evaluation of the pathogenicity of the recovered P. multo-
cida isolates each strain was cultured in brain heart infusion
broth and after 24 h incubation, the turbidity of each tube was
adjusted to McFarland’s tube no 4. From each test tube 0.1 ml
of 24 h broth culture were inoculated into 3 mice intramuscu-
larly and the mice were kept under observation for 72 h. Dead
micewere subjected to postmortem examination andblood films
were prepared and stained with Leishman’s stain and examined
for the presence of the characteristic bipolarity of P. multocida.
2.4. Serotyping of the somatic and capsular antigens using
ELISA
ELISA had been carried out according to Jakeen et al. (2016)
using somatic antigens and capsular antigens as coating
antigens and standard positive control hyperimmune serum
prepared in rabbit according to the previously reported tech-
niques by OIE (2008).
2.5. Molecular detection of P. multocida by PCR
Molecular detection of P. multocidawas carried out according to
the previously reported method by Jakeen et al. (2016) using
Qiagen kits, species primers for detection of P. multocida;
forward primer KMT1T7 (50-ATC CGC TAT TTA CCC
AGT GG-30), reverse primer KMT1SP6 (50-GCT GTA AAC
GAA CTC GCC AC-30). The molecular characterization of the
capsular antigens of P. multocida was carried out by multiplex
PCR using specific primer pairs for different serogroups (A, B,
D, E and F), previously reported by Townsend et al. (1998).
3. Results
The collected samples from animals with respiratory manifes-
tation revealed positive isolation of 80 strains (18.82%) of
P. multocida, while, 77 strains (44%) were isolated from lung
Table 1 Rate of isolation of P. multocida from different species of animals suffering from respiratory manifestation.
Types of samples Samples from animals with
respiratory manifestation
Number of
samples
Percent of isolation of P. multocida
Number Percentage (%)
Sterile nasopharyngeal swabs Calves 200 35 17.5
Sheep 125 26 20.8
Goat 100 19 19
Total number 425 80 18.82
Lung and spleen Calves 75 30 40
Sheep 50 22 44
Goat 50 25 50
Total number 175 77 44
Figure 1 Agarose gel electrophoresis showing amplification of 460 bp fragments specific for P. multocida field isolates.
Figure 2 Agarose gel electrophoresis showing amplification of 1044 bp fragments specific to the capsular antigen type A and 511 bp
fragments specific to the capsular antigen type E.
Capsular antigens of Pasteurella multocida 369and spleen of emergency slaughtered animals as shown in
Table 1. The characteristic colonial morphology of P. multo-
cida had been observed with all the recovered strains. More-
over, all the recovered strains showed severe pathogenicity
and killed all the inoculated mice within 24–72 h and the char-
acteristic bipolarity could be observed in the stained blood and
spleen smear prepared from the dead mice with Leishman’s
stain. The results of ELISA test using somatic and capsular
antigens as coating antigens to test the serum samples collected
from all animals with respiratory manifestation and emergency
slaughtered animals were 64% and 68% respectively.Molecular identification of the recovered strains with the
specific primers KMT1T7 and KMT1SP6 revealed positive
amplification of 460 bp fragments with all the recovered strains
previously identified as P. multocida by standard microbiolog-
ical techniques as shown in Fig. 1. Multiplex PCR for
molecular typing of the capsular antigens of the recovered
P. multocida revealed positive amplification of 1044 bp frag-
ments specific to the capsular antigen type A with 105 strains
(66.88%), and amplification 511 bp fragments of the capsular
antigen type E with 52 strain (33.12%) as shown in Fig. 2
and absence of B, D and F antigens.
370 K.S. Al-Maary et al.4. Discussion
The infections with P. multocida most commonly occur as sec-
ondary infections to the viral infection and other diseases con-
ditions (Worarach et al., 2014). The interaction between
bacterial infection and viral affection in cases of pneumonia
of sheep and goats is usually occurring. A primary viral pneu-
monia may be an insignificant disease until the intervention of a
secondary pasteurellosis which converts it into an outbreak of
pneumonia of major economic importance (Rad et al., 2009).
The present study is aimed to detect and characterize the
recovered strains of P. multocida from samples collected from
different abattoirs located in Riyadh, Kingdom of Saudi
Arabia on molecular basis. 157 strains of P. multocida were
isolated from the examined samples all of them showed posi-
tive results with, catalase, oxidase, ornithine decarboxylase,
indole and negative for urea hydrolyze, gelatin liquefaction,
and cannot ferment lactose and mannitol sugar (Jakeen
et al., 2016). To overcome the disadvantage of the conven-
tional methods used for diagnosis of P. multocida PCR assay
reported by Townsend et al. (1998) using KMT1T7 and
KMT1SP6 primers has been used to rapidly confirm the results
of the standard bacteriological methods. All the recovered
strains showed positive amplification of 460 bp fragments
specific for P. multocida within few hours which indicate the
higher sensitivity and specificity of the molecular techniques
(Kumar et al., 2009; Jakeen et al., 2016). Multiplex PCR has
been carried out in the current study for molecular character-
ization of the capsular antigens to overcome the disadvantage
of the serological methods used for the typing of P. multocida.
Positive amplification of 1044 bp fragments specific to the
capsular antigen type A with 105 strains (66.88%), and
amplification 511 bp fragments of the capsular antigen type
E with 52.
5. Conclusion
Molecular techniques based on multiplex PCR for molecular
typing of the capsular antigens of P. multocida can be used
as a simple, sensitive, rapid, reliable technique instead of the
serological techniques used for identification of the capsular
antigens of P. multocida.
Acknowledgment
The authors extend their appreciation to the Deanship of
Scientific Research at King Saud University for funding of
the work through the research group project No.: RGP-176.References
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Molecular characterization of the capsular antigens of Pasteurella multocida isolates using multiplex PCR

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