# Virulence Genotyping ofPasteurella multocida Isolated from Multiple Hosts from India

Authors: Laxmi Narayan Sarangi; Adyasha Priyadarshini; Santosh Kumar; Prasad Thomas; Santosh Kumar Gupta; Viswas Konasagara Nagaleekar; Vijendra Pal Singh
Journal: The Scientific World Journal
 (2014)

---

## Abstract

In this study, 108P. multocida isolates recovered from various host animals such as cattle, buffalo, swine,
poultry (chicken, duck, and emu) and rabbits were screened for carriage of 8 virulence associated genes.
The results revealed some unique information on the prevalence of virulence associated genes among Indian isolates.
With the exception of toxA gene, all other virulence associated genes were found to be regularly
distributed among host species. Association study between capsule type and virulence genes suggested that
pfhA, nanB, and nanH genes were regularly distributed among all serotypes with the exception of CapD,
whereas toxA gene was found to be positively associated with CapD and CapA. The frequency
of hgbA and nanH genes among swine isolates of Indian origin was found to be less in comparison
to its equivalents around the globe. Interestingly, very high prevalence of tbpA gene was observed among poultry, swine,
and rabbit isolates. Likewise, very high prevalence of pfhA gene (95.3%) was observed among Indian isolates, irrespective
of host species origin.


---

## Body

## 1. Introduction

Pasteurella multocida belonging to family Pasteurellaceae is a ubiquitous organism affecting multihost species, thus causing several diseases like haemorrhagic septicaemia in cattle and buffalo, enzootic bronchopneumonia in cattle, sheep, and goats, atrophic rhinitis in swine, fowl cholera in poultry and snuffles in rabbits [1, 2]. These diseases are known to cause severe financial loss to livestock industry, especially in tropical countries. Conventional vaccines have been used for several decades as a control strategy, but major limitation of these vaccines is their ineffectiveness in inducing long acting cross protective immunity [3, 4]. Therefore, several outer membrane proteins (OMPs) have been proposed as candidate antigen for subunit vaccine [3, 5].The OMPs of Gram negative bacteria play an essential role in the disease process. They are involved in the process of nutrient uptake, transport of molecules in and out of the cell, colonization and invasion of the host, evasion of host immune response, injury to host tissue, and so forth, required for productive infection [6]. These proteins are subjected to different selection pressures, thereby exhibiting varying degree of interstrain heterogeneity. Therefore, these virulence associated genes can be used to assess intraspecies diversity and also to obtain epidemiological relationships [7]. In addition, these OMPs are good immunogens and can be used as vaccine components to provide protection [8–10]. Hence, virulence profiling can be used as a typing method for characterization of bacterial pathogens [11] and also for development of subunit vaccine in vaccine strain selection. For the first time, virulence profiling ofP. multocida isolates was carried out by Ewers et al. [12], and subsequently it has been used by many workers to understand the diversity of the pathogen recovered from different host origin [13–19].The previous study carried out in our laboratory on carriage of 19 virulence genes amongP. multocida isolates, recovered from small ruminants, revealed some novel information on the frequency of virulence genes like very high prevalence ofpfhA gene, 48.9% prevalence oftoxA gene with the highest prevalence among serotype A followed by serotype D, and in one isolate each of capsular types B and F (Sarangi et al., submitted for publication). These findings from small ruminant isolates encouraged sampling of more isolates of Indian origin from various hosts to have a clear understanding on the heterogeneity of the bacteria. Therefore, this study was extended toP. multocida isolates recovered from multiple host species for 8 important virulence associated genes, encoding proteins involved in bacterial survival and pathogenesis. It included genes encoding transferrin binding protein (TbpA) and haemoglobin binding protein (HgbA, HgbB) associated with iron acquisition, filamentous haemagglutinin (PfhA), subunit of type IV fimbriae (PtfA), sialidases (NanB, NanH) involved in initial colonization and adhesion, and dermonecrotoxin (ToxA).


## 2. Materials and Methods

### 2.1. Bacterial Strains

In the present study, 108P. multocida isolates recovered from large ruminants (buffalo, n
=
23, cattle, n
=
18), avians (chicken, n
=
18, duck, n
=
8, and emu, n
=
4), swine (n
=
34), and rabbit (n
=
3), maintained at Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, were used. Selections of isolates were carried out on the basis of host origin, year and place of isolation in order to incorporate isolates from all over India. The details of the isolates (isolate number, host origin, capsular type, year and place of isolation, and disease symptom (if available)) are given in Table 1.Table 1
Details of the isolates (host origin, serotype detected, place of isolation, year of isolation, symptom, presence/absence of individual virulence genes).
Sample id
Species
Serotype
Place
Year
Disease/symptom
tbpA
hgbA
hgbB
pfhA
ptfA
toxA
nanB
nanH
10
Cattle
F
Pune
1992
N.A.
P
P
P
P
P
A
P
P
11
Cattle
F
Pune
1992
N.A.
P
P
P
P
P
A
P
P
51
Pig
A
UP
1995
N.A.
P
P
P
P
P
P
P
A
53
Buffalo
B
Pune
1996
N.A.
A
P
P
P
P
A
P
A
98
Duck
A
Tripura
2001
N.A.
P
P
P
P
P
A
P
P
117
Cattle
B
Bhubaneswar
2001
N.A.
P
P
P
P
P
A
P
P
118
Cattle
B
Bhubaneswar
2001
N.A.
P
P
P
P
P
A
P
P
120
Cattle
B
Bhubaneswar
2001
N.A.
P
P
P
P
P
A
P
P
128
Cattle
B
Bangalore
2001
N.A.
P
P
P
P
P
A
P
P
132
Buffalo
A
Palampur
2001
N.A.
P
P
P
P
P
A
P
P
133
Buffalo
A
Palampur
2001
N.A.
A
A
P
P
P
A
P
A
134
Buffalo
A
Palampur
2001
N.A.
P
P
P
P
P
A
P
P
141
Chicken
A
Chennai
2001
N.A.
P
P
P
P
P
A
P
P
202
Chicken
A
Chennai
2002
N.A.
A
P
P
P
P
A
P
A
206
Chicken
B
Chennai
2002
N.A.
P
P
P
P
P
A
P
A
222
Buffalo
A
Mathura
2002
N.A.
P
P
A
P
P
A
P
P
258
Chicken
A
Nasik
2002
N.A.
P
P
P
P
P
A
P
P
330
Chicken
B
Anand
2002
N.A.
P
P
P
P
P
A
P
P
288
Buffalo
B
Bhubaneswar
2003
N.A.
P
P
P
P
P
A
P
P
291
Pig
B
Guwahati
2003
N.A.
P
P
A
P
P
A
P
P
292
Pig
B
Guwahati
2003
N.A.
P
P
P
P
P
A
P
A
366
Cattle
B
Palampur
2004
N.A.
P
A
A
P
P
A
P
P
390
Buffalo
B
Palampur
2005
N.A.
P
P
P
P
P
A
P
P
400
Buffalo
B
Ludhiana
2005
N.A.
P
P
P
P
P
A
P
P
407
Chicken
B
Ludhiana
2005
N.A.
P
P
P
P
P
A
P
P
409
Buffalo
B
Jammu
2005
N.A.
P
P
P
P
P
A
P
P
410
Buffalo
B
Jammu
2005
N.A.
P
P
P
P
P
A
P
P
425
Duck
B
Chennai
2005
N.A.
P
P
P
P
P
P
P
P
448
Rabbit
B
Palampur
2006
N.A.
P
P
A
P
P
A
P
P
456
Chicken
A
Chennai
2006
N.A.
P
P
P
P
P
A
P
P
460
Chicken
A
Chennai
2006
N.A.
A
A
A
P
P
A
P
A
464
Chicken
A
Chennai
2006
N.A.
A
P
P
P
P
A
P
P
569
Chicken
A
Chennai
2007
N.A.
P
A
A
P
P
A
P
A
537
Pig
A
Guwahati
2007
N.A.
A
A
A
P
P
A
A
P
540
Pig
A
Guwahati
2007
N.A.
P
P
P
P
P
P
P
P
543
Pig
D
Guwahati
2007
N.A.
P
P
P
P
P
P
P
A
550
Duck
A
Guwahati
2007
N.A.
P
A
P
P
P
P
P
P
555
Buffalo
B
Anand
2007
N.A.
P
P
A
P
P
A
P
A
559
Rabbit
B
Palampur
2007
Nasal discharge
P
P
A
P
P
A
P
P
563
Cattle
B
Anand
2007
N.A.
P
P
A
P
P
A
P
P
585
Pig
A
Guwahati
2008
N.A.
P
P
A
P
P
P
P
P
587
Pig
A
Guwahati
2008
N.A.
P
P
P
P
P
P
P
P
602
Buffalo
B
Palampur
2008
N.A.
P
P
P
P
P
A
P
P
608
Rabbit
A
Palampur
2008
Nasal discharge
P
P
P
P
P
A
P
P
610
Buffalo
B
Ludhiana
2008
N.A.
A
A
A
P
P
A
P
A
618
Chicken
B
Palampur
2008
N.A.
P
P
P
P
P
A
P
P
632
Buffalo
B
Anand
2008
N.A.
A
A
A
P
P
A
P
P
633
Chicken
B
Bangalore
2008
N.A.
P
A
P
P
P
A
P
P
655
Buffalo
D
Guwahati
2008
N.A.
P
P
P
P
P
A
P
P
701
Pig
A
Guwahati
2009
N.A.
P
P
A
P
P
P
P
P
702
Pig
A
Guwahati
2009
N.A.
P
P
P
P
P
A
P
P
703
Pig
D
Guwahati
2009
N.A.
P
P
P
P
P
P
P
A
704
Cattle
B
Guwahati
2009
N.A.
P
P
A
A
P
A
P
P
720
Pig
B
UP
2009
N.A.
P
P
P
P
P
A
P
P
721
Pig
B
UP
2009
N.A.
P
P
P
P
P
A
P
P
722
Pig
B
UP
2009
N.A.
P
P
A
P
P
P
P
P
725
Buffalo
A
Ludhiana
2009
N.A.
P
P
P
P
P
P
P
A
733
Pig
D
Guwahati
2009
N.A.
P
A
P
P
P
P
P
A
736
Pig
D
Guwahati
2009
N.A.
P
P
P
A
P
A
P
A
737
Pig
D
Guwahati
2009
N.A.
P
P
P
A
P
A
P
A
749
Cattle
A
Palampur
2009
N.A.
P
P
P
P
P
A
P
P
746
Cattle
A
Palampur
2009
N.A.
P
P
P
P
P
A
P
P
747
Cattle
A
Palampur
2009
N.A.
P
P
P
P
A
A
P
P
754
Cattle
A
Palampur
2009
N.A.
P
P
P
P
P
A
P
P
782
Chicken
A
Anand
2009
N.A.
A
P
A
P
P
A
P
A
794
Chicken
A
Thrissur
2009
Necrotic foci in liver and haemorrhage in heart
P
P
P
P
P
A
P
A
652
Buffalo
B
Guwahati
2010
N.A.
P
P
P
P
P
A
P
P
653
Buffalo
B
Guwahati
2010
N.A.
P
P
P
P
P
A
P
P
784
Chicken
A
Anand
2010
N.A.
P
P
P
P
P
A
P
P
803
Chicken
A
Chennai
2010
N.A.
P
P
P
P
P
A
P
P
804
Chicken
A
Anand
2010
N.A.
P
P
P
P
P
A
P
P
811
Cattle
A
Palampur
2010
N.A.
P
P
P
P
P
A
P
P
852
Pig
A
Guwahati
2011
Diseased
P
P
P
P
P
P
P
P
860
Pig
B
Guwahati
2011
Diseased
P
P
P
P
P
A
P
P
876
Pig
A
Thrissur
2011
Fever
A
A
A
P
P
A
A
P
877
Pig
A
Thrissur
2011
Fever
A
A
P
P
P
A
P
P
879
Pig
A
Thrissur
2011
Fever
A
P
A
P
P
A
P
A
890
Emu
A
Chennai
2011
N.A.
P
P
P
P
P
A
P
P
2751
Cattle
B
Palampur
2011
Nasal discharge
P
P
A
P
P
A
P
P
2766
Cattle
B
Palampur
2011
Nasal discharge
P
P
P
P
P
A
P
P
3324
Cattle
B
Palampur
2011
Nasal discharge
P
P
A
P
P
A
P
P
4312
Cattle
B
Palampur
2011
Nasal discharge
P
P
A
P
P
A
P
P
BP23
Pig
B
Guwahati
2011
N.A.
P
A
A
P
P
A
P
P
BP28
Pig
A
Guwahati
2011
N.A.
P
A
P
P
P
P
P
P
BP37
Pig
A
Guwahati
2011
N.A.
P
P
A
P
P
P
P
P
EMU 2
Emu
A
Chennai
2011
N.A.
P
P
P
P
P
A
P
P
JP18
Pig
A
Guwahati
2011
N.A.
P
P
P
P
P
P
P
P
NP23
Pig
B
Guwahati
2011
N.A.
P
P
P
P
P
A
P
P
NP37
Pig
B
Guwahati
2011
N.A.
P
P
P
P
P
A
P
P
PP1A
Pig
A
Thrissur
2011
N.A.
P
P
P
P
P
A
P
P
PP2A
Pig
A
Thrissur
2011
N.A.
P
P
P
P
P
P
P
P
PP4A
Pig
A
Thrissur
2011
N.A.
P
P
P
P
P
A
P
P
914
Duck
A
Thrissur
2012
N.A.
P
P
P
P
P
A
P
P
920
Emu
A
Anand
2012
N.A.
P
P
P
P
P
A
P
P
922
Emu
A
Anand
2012
N.A.
P
P
P
P
P
A
P
P
DP53
Duck
A
Thrissur
2013
N.A.
P
P
P
P
P
A
P
A
DP54
Duck
A
Thrissur
2013
N.A.
P
P
P
P
P
P
P
A
DP55
Duck
A
Thrissur
2013
N.A.
A
P
P
P
P
A
P
A
DP56
Duck
A
Thrissur
2013
N.A.
P
P
P
P
P
A
P
A
P14
Pig
D
Guwahati
2013
N.A.
A
A
A
A
P
P
A
A
P15
Pig
A
Guwahati
2013
N.A.
A
A
A
A
P
A
P
A
P16
Pig
D
Guwahati
2013
N.A.
A
A
P
A
P
A
A
A
PAB 78
Buffalo
B
Anand
2013
N.A.
P
P
A
P
P
A
P
P
PAB 80
Buffalo
B
Anand
2013
N.A.
P
P
A
P
P
A
P
P
PAB 86
Buffalo
B
Anand
2013
N.A.
A
A
A
A
A
A
P
P
PAP 88
Chicken
A
Anand
2013
N.A.
P
P
P
P
P
A
P
A
LDHB 106
Buffalo
B
Ludhiana
2014
N.A.
A
P
A
P
P
A
P
P
MSRB 108
Buffalo
B
Ludhiana
2014
N.A.
A
P
P
P
P
A
P
P
(N.A. = not available; A = absence of virulence gene; P = presence of virulence gene as detected in PCR reaction).


### 2.2. Confirmation ofP. multocida Isolates

The isolates were revived in brain heart infusion broth by 18–24 h incubation at 37°C and plated subsequently onto blood agar to study cultural characteristics. The cultures were then tested for purity by biochemical tests as per standard techniques [20]. The genomic DNA of the isolates was extracted by CTAB method [21], and the isolates were reconfirmed asP. multocida by PM-PCR followed by determination of capsular type by multiplex PCR [22, 23].


### 2.3. Detection of Virulence Associated Genes by PCR

The isolates were then subjected to screening of 8 virulence genes encoding iron binding proteins (TbpA, HgbA, HgbB), colonization and adhesion related protein (PfhA, PtfA), sialidases (NanB, NanH), and dermonecrotoxin (ToxA) by individual PCR reactions, utilizing oligonucleotide primers described previously. The details of the virulence genes, sequences of the oligonucleotide primers, and citations used are listed in Table2.Table 2
Details of primers and citations used for the detection of capsular type and virulence associated genes in strains ofPasteurella multocida.
Gene
Primer
Primer sequence (5′-3′)
Reference
PM-PCR and Capsular serotypes
KMT1
PMPCR-FPMPCR-R
ATCCGCTATTTACCCAGTGGGCTGTAAACGAACTCGCCAC
[22]
hyaD-hyaC
capA FcapA R
GATGCCAAAATCGCAGTCAGTGTTGCCATCATTGTCAGTG
[23]
bcbD
capB FcapB R
CATTTATCCAAGCTCCACCGCCCGAGAGTTTCAATCC
[23]
dcbF
capD FcapD R
TTACAAAAGAAAGACTAGGAGCCCCATCTACCCACTCAACCATATCAG
[23]
ecbJ
capE FcapE R
TCCGCAGAAAATTATTGACTCGCTTGCTGCTTGATTTTGTC
[23]
fcbD
capF FcapF R
AATCGGAGAACGCAGAAATCAGTTCCGCCGTCAATTACTCTG
[23]
Iron acquisition genes
tbpA
tbpA FtbpA R
GGACAGTGCATATAACTTGTTGGACAGTGCATATAACTTGTTTACTA
[32]
hgbA
hgbA FhgbA R
CATATCGGATCCTTGAAACCAGAGGAAGCAAAAAGAATCGGAGCTCACGACCTGGTGAGTAAAAACGAT
In-house [33]
hgbB
HgbB FHgbB R
ACCGCGTTGGAATTATGATTGCATTGAGTACGGCTTGACAT
[12]
Adhesins
ptfA
ptfA FptfA R
AGGATCCATGAAAAAAGCCATTTGGAGCTCTTATGCGCAAAATCCTG
In-house
pfhA
pfhA FpfhA R
TAAGCCTATCGGTTCAAGTCGGATAAATCTACCCCGTCCTCT
In-house
Sialidases
NanB
NanB FNanB R
GTCCTATAAAGTGACGCCGAACAGCAAAGGAAGACTGTCC
[12]
nanH
nanH FnanH R
CACTGCCTTATAGCCGTATTCAGCACTGTTACCCGAACCC
[12]
Dermonecrotoxin
ToxA
ToxA FToxA R
TCTTAGATGAGCGACAAGGGAATGCCACACCTCTATAG
[34]


### 2.4. Statistical Analysis

Statistical analysis of the data generated from the study was performed with SPSS 16.0 (SPSS Inc., Chicago).P values of <0.05 were considered as statistically significant.


## 2.1. Bacterial Strains

In the present study, 108P. multocida isolates recovered from large ruminants (buffalo, n
=
23, cattle, n
=
18), avians (chicken, n
=
18, duck, n
=
8, and emu, n
=
4), swine (n
=
34), and rabbit (n
=
3), maintained at Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, were used. Selections of isolates were carried out on the basis of host origin, year and place of isolation in order to incorporate isolates from all over India. The details of the isolates (isolate number, host origin, capsular type, year and place of isolation, and disease symptom (if available)) are given in Table 1.Table 1
Details of the isolates (host origin, serotype detected, place of isolation, year of isolation, symptom, presence/absence of individual virulence genes).
Sample id
Species
Serotype
Place
Year
Disease/symptom
tbpA
hgbA
hgbB
pfhA
ptfA
toxA
nanB
nanH
10
Cattle
F
Pune
1992
N.A.
P
P
P
P
P
A
P
P
11
Cattle
F
Pune
1992
N.A.
P
P
P
P
P
A
P
P
51
Pig
A
UP
1995
N.A.
P
P
P
P
P
P
P
A
53
Buffalo
B
Pune
1996
N.A.
A
P
P
P
P
A
P
A
98
Duck
A
Tripura
2001
N.A.
P
P
P
P
P
A
P
P
117
Cattle
B
Bhubaneswar
2001
N.A.
P
P
P
P
P
A
P
P
118
Cattle
B
Bhubaneswar
2001
N.A.
P
P
P
P
P
A
P
P
120
Cattle
B
Bhubaneswar
2001
N.A.
P
P
P
P
P
A
P
P
128
Cattle
B
Bangalore
2001
N.A.
P
P
P
P
P
A
P
P
132
Buffalo
A
Palampur
2001
N.A.
P
P
P
P
P
A
P
P
133
Buffalo
A
Palampur
2001
N.A.
A
A
P
P
P
A
P
A
134
Buffalo
A
Palampur
2001
N.A.
P
P
P
P
P
A
P
P
141
Chicken
A
Chennai
2001
N.A.
P
P
P
P
P
A
P
P
202
Chicken
A
Chennai
2002
N.A.
A
P
P
P
P
A
P
A
206
Chicken
B
Chennai
2002
N.A.
P
P
P
P
P
A
P
A
222
Buffalo
A
Mathura
2002
N.A.
P
P
A
P
P
A
P
P
258
Chicken
A
Nasik
2002
N.A.
P
P
P
P
P
A
P
P
330
Chicken
B
Anand
2002
N.A.
P
P
P
P
P
A
P
P
288
Buffalo
B
Bhubaneswar
2003
N.A.
P
P
P
P
P
A
P
P
291
Pig
B
Guwahati
2003
N.A.
P
P
A
P
P
A
P
P
292
Pig
B
Guwahati
2003
N.A.
P
P
P
P
P
A
P
A
366
Cattle
B
Palampur
2004
N.A.
P
A
A
P
P
A
P
P
390
Buffalo
B
Palampur
2005
N.A.
P
P
P
P
P
A
P
P
400
Buffalo
B
Ludhiana
2005
N.A.
P
P
P
P
P
A
P
P
407
Chicken
B
Ludhiana
2005
N.A.
P
P
P
P
P
A
P
P
409
Buffalo
B
Jammu
2005
N.A.
P
P
P
P
P
A
P
P
410
Buffalo
B
Jammu
2005
N.A.
P
P
P
P
P
A
P
P
425
Duck
B
Chennai
2005
N.A.
P
P
P
P
P
P
P
P
448
Rabbit
B
Palampur
2006
N.A.
P
P
A
P
P
A
P
P
456
Chicken
A
Chennai
2006
N.A.
P
P
P
P
P
A
P
P
460
Chicken
A
Chennai
2006
N.A.
A
A
A
P
P
A
P
A
464
Chicken
A
Chennai
2006
N.A.
A
P
P
P
P
A
P
P
569
Chicken
A
Chennai
2007
N.A.
P
A
A
P
P
A
P
A
537
Pig
A
Guwahati
2007
N.A.
A
A
A
P
P
A
A
P
540
Pig
A
Guwahati
2007
N.A.
P
P
P
P
P
P
P
P
543
Pig
D
Guwahati
2007
N.A.
P
P
P
P
P
P
P
A
550
Duck
A
Guwahati
2007
N.A.
P
A
P
P
P
P
P
P
555
Buffalo
B
Anand
2007
N.A.
P
P
A
P
P
A
P
A
559
Rabbit
B
Palampur
2007
Nasal discharge
P
P
A
P
P
A
P
P
563
Cattle
B
Anand
2007
N.A.
P
P
A
P
P
A
P
P
585
Pig
A
Guwahati
2008
N.A.
P
P
A
P
P
P
P
P
587
Pig
A
Guwahati
2008
N.A.
P
P
P
P
P
P
P
P
602
Buffalo
B
Palampur
2008
N.A.
P
P
P
P
P
A
P
P
608
Rabbit
A
Palampur
2008
Nasal discharge
P
P
P
P
P
A
P
P
610
Buffalo
B
Ludhiana
2008
N.A.
A
A
A
P
P
A
P
A
618
Chicken
B
Palampur
2008
N.A.
P
P
P
P
P
A
P
P
632
Buffalo
B
Anand
2008
N.A.
A
A
A
P
P
A
P
P
633
Chicken
B
Bangalore
2008
N.A.
P
A
P
P
P
A
P
P
655
Buffalo
D
Guwahati
2008
N.A.
P
P
P
P
P
A
P
P
701
Pig
A
Guwahati
2009
N.A.
P
P
A
P
P
P
P
P
702
Pig
A
Guwahati
2009
N.A.
P
P
P
P
P
A
P
P
703
Pig
D
Guwahati
2009
N.A.
P
P
P
P
P
P
P
A
704
Cattle
B
Guwahati
2009
N.A.
P
P
A
A
P
A
P
P
720
Pig
B
UP
2009
N.A.
P
P
P
P
P
A
P
P
721
Pig
B
UP
2009
N.A.
P
P
P
P
P
A
P
P
722
Pig
B
UP
2009
N.A.
P
P
A
P
P
P
P
P
725
Buffalo
A
Ludhiana
2009
N.A.
P
P
P
P
P
P
P
A
733
Pig
D
Guwahati
2009
N.A.
P
A
P
P
P
P
P
A
736
Pig
D
Guwahati
2009
N.A.
P
P
P
A
P
A
P
A
737
Pig
D
Guwahati
2009
N.A.
P
P
P
A
P
A
P
A
749
Cattle
A
Palampur
2009
N.A.
P
P
P
P
P
A
P
P
746
Cattle
A
Palampur
2009
N.A.
P
P
P
P
P
A
P
P
747
Cattle
A
Palampur
2009
N.A.
P
P
P
P
A
A
P
P
754
Cattle
A
Palampur
2009
N.A.
P
P
P
P
P
A
P
P
782
Chicken
A
Anand
2009
N.A.
A
P
A
P
P
A
P
A
794
Chicken
A
Thrissur
2009
Necrotic foci in liver and haemorrhage in heart
P
P
P
P
P
A
P
A
652
Buffalo
B
Guwahati
2010
N.A.
P
P
P
P
P
A
P
P
653
Buffalo
B
Guwahati
2010
N.A.
P
P
P
P
P
A
P
P
784
Chicken
A
Anand
2010
N.A.
P
P
P
P
P
A
P
P
803
Chicken
A
Chennai
2010
N.A.
P
P
P
P
P
A
P
P
804
Chicken
A
Anand
2010
N.A.
P
P
P
P
P
A
P
P
811
Cattle
A
Palampur
2010
N.A.
P
P
P
P
P
A
P
P
852
Pig
A
Guwahati
2011
Diseased
P
P
P
P
P
P
P
P
860
Pig
B
Guwahati
2011
Diseased
P
P
P
P
P
A
P
P
876
Pig
A
Thrissur
2011
Fever
A
A
A
P
P
A
A
P
877
Pig
A
Thrissur
2011
Fever
A
A
P
P
P
A
P
P
879
Pig
A
Thrissur
2011
Fever
A
P
A
P
P
A
P
A
890
Emu
A
Chennai
2011
N.A.
P
P
P
P
P
A
P
P
2751
Cattle
B
Palampur
2011
Nasal discharge
P
P
A
P
P
A
P
P
2766
Cattle
B
Palampur
2011
Nasal discharge
P
P
P
P
P
A
P
P
3324
Cattle
B
Palampur
2011
Nasal discharge
P
P
A
P
P
A
P
P
4312
Cattle
B
Palampur
2011
Nasal discharge
P
P
A
P
P
A
P
P
BP23
Pig
B
Guwahati
2011
N.A.
P
A
A
P
P
A
P
P
BP28
Pig
A
Guwahati
2011
N.A.
P
A
P
P
P
P
P
P
BP37
Pig
A
Guwahati
2011
N.A.
P
P
A
P
P
P
P
P
EMU 2
Emu
A
Chennai
2011
N.A.
P
P
P
P
P
A
P
P
JP18
Pig
A
Guwahati
2011
N.A.
P
P
P
P
P
P
P
P
NP23
Pig
B
Guwahati
2011
N.A.
P
P
P
P
P
A
P
P
NP37
Pig
B
Guwahati
2011
N.A.
P
P
P
P
P
A
P
P
PP1A
Pig
A
Thrissur
2011
N.A.
P
P
P
P
P
A
P
P
PP2A
Pig
A
Thrissur
2011
N.A.
P
P
P
P
P
P
P
P
PP4A
Pig
A
Thrissur
2011
N.A.
P
P
P
P
P
A
P
P
914
Duck
A
Thrissur
2012
N.A.
P
P
P
P
P
A
P
P
920
Emu
A
Anand
2012
N.A.
P
P
P
P
P
A
P
P
922
Emu
A
Anand
2012
N.A.
P
P
P
P
P
A
P
P
DP53
Duck
A
Thrissur
2013
N.A.
P
P
P
P
P
A
P
A
DP54
Duck
A
Thrissur
2013
N.A.
P
P
P
P
P
P
P
A
DP55
Duck
A
Thrissur
2013
N.A.
A
P
P
P
P
A
P
A
DP56
Duck
A
Thrissur
2013
N.A.
P
P
P
P
P
A
P
A
P14
Pig
D
Guwahati
2013
N.A.
A
A
A
A
P
P
A
A
P15
Pig
A
Guwahati
2013
N.A.
A
A
A
A
P
A
P
A
P16
Pig
D
Guwahati
2013
N.A.
A
A
P
A
P
A
A
A
PAB 78
Buffalo
B
Anand
2013
N.A.
P
P
A
P
P
A
P
P
PAB 80
Buffalo
B
Anand
2013
N.A.
P
P
A
P
P
A
P
P
PAB 86
Buffalo
B
Anand
2013
N.A.
A
A
A
A
A
A
P
P
PAP 88
Chicken
A
Anand
2013
N.A.
P
P
P
P
P
A
P
A
LDHB 106
Buffalo
B
Ludhiana
2014
N.A.
A
P
A
P
P
A
P
P
MSRB 108
Buffalo
B
Ludhiana
2014
N.A.
A
P
P
P
P
A
P
P
(N.A. = not available; A = absence of virulence gene; P = presence of virulence gene as detected in PCR reaction).


## 2.2. Confirmation ofP. multocida Isolates

The isolates were revived in brain heart infusion broth by 18–24 h incubation at 37°C and plated subsequently onto blood agar to study cultural characteristics. The cultures were then tested for purity by biochemical tests as per standard techniques [20]. The genomic DNA of the isolates was extracted by CTAB method [21], and the isolates were reconfirmed asP. multocida by PM-PCR followed by determination of capsular type by multiplex PCR [22, 23].


## 2.3. Detection of Virulence Associated Genes by PCR

The isolates were then subjected to screening of 8 virulence genes encoding iron binding proteins (TbpA, HgbA, HgbB), colonization and adhesion related protein (PfhA, PtfA), sialidases (NanB, NanH), and dermonecrotoxin (ToxA) by individual PCR reactions, utilizing oligonucleotide primers described previously. The details of the virulence genes, sequences of the oligonucleotide primers, and citations used are listed in Table2.Table 2
Details of primers and citations used for the detection of capsular type and virulence associated genes in strains ofPasteurella multocida.
Gene
Primer
Primer sequence (5′-3′)
Reference
PM-PCR and Capsular serotypes
KMT1
PMPCR-FPMPCR-R
ATCCGCTATTTACCCAGTGGGCTGTAAACGAACTCGCCAC
[22]
hyaD-hyaC
capA FcapA R
GATGCCAAAATCGCAGTCAGTGTTGCCATCATTGTCAGTG
[23]
bcbD
capB FcapB R
CATTTATCCAAGCTCCACCGCCCGAGAGTTTCAATCC
[23]
dcbF
capD FcapD R
TTACAAAAGAAAGACTAGGAGCCCCATCTACCCACTCAACCATATCAG
[23]
ecbJ
capE FcapE R
TCCGCAGAAAATTATTGACTCGCTTGCTGCTTGATTTTGTC
[23]
fcbD
capF FcapF R
AATCGGAGAACGCAGAAATCAGTTCCGCCGTCAATTACTCTG
[23]
Iron acquisition genes
tbpA
tbpA FtbpA R
GGACAGTGCATATAACTTGTTGGACAGTGCATATAACTTGTTTACTA
[32]
hgbA
hgbA FhgbA R
CATATCGGATCCTTGAAACCAGAGGAAGCAAAAAGAATCGGAGCTCACGACCTGGTGAGTAAAAACGAT
In-house [33]
hgbB
HgbB FHgbB R
ACCGCGTTGGAATTATGATTGCATTGAGTACGGCTTGACAT
[12]
Adhesins
ptfA
ptfA FptfA R
AGGATCCATGAAAAAAGCCATTTGGAGCTCTTATGCGCAAAATCCTG
In-house
pfhA
pfhA FpfhA R
TAAGCCTATCGGTTCAAGTCGGATAAATCTACCCCGTCCTCT
In-house
Sialidases
NanB
NanB FNanB R
GTCCTATAAAGTGACGCCGAACAGCAAAGGAAGACTGTCC
[12]
nanH
nanH FnanH R
CACTGCCTTATAGCCGTATTCAGCACTGTTACCCGAACCC
[12]
Dermonecrotoxin
ToxA
ToxA FToxA R
TCTTAGATGAGCGACAAGGGAATGCCACACCTCTATAG
[34]


## 2.4. Statistical Analysis

Statistical analysis of the data generated from the study was performed with SPSS 16.0 (SPSS Inc., Chicago).P values of <0.05 were considered as statistically significant.


## 3. Results and Discussion

P. multocida is an economically important veterinary pathogen, causing wide range of diseases in livestock and poultry. The bacteria have been classified into five capsular types (A, B, D, E, and F) based on capsular typing, with each capsule type being predominantly associated with a particular disease in a host species. But isolation of other capsular types from such hosts by cross species infection is not uncommon [2, 7]. Ability of the bacteria to infect and survive in several hosts as commensal exposes it to various selection pressures, resulting in emergence of divergent strains in field scenario. Molecular epidemiological study by employing REP-PCR, ERIC-PCR, MLST analysis, and so forth has confirmed the diversity ofP. multocida circulating in India and also the possibility of transboundary spread of strains across evolutionary time [24, 25]. Therefore, a detailed study on the presence of virulence associated genes recovered from different host species in Indian subcontinent will be helpful to understand the disease process and to develop disease control measures in future.In this study,P. multocida isolates recovered from various host species were screened for presence of 8 important virulence associated genes (tbpA, hgbA, hgbB, pfhA, ptfA, nanB, nanH, andtoxA) involved in bacterial pathogenesis. The results confirmed that, with the exception oftoxA gene, all other virulence associated genes are regularly distributed among the isolates of different host origin. The result of individual PCR reaction for each isolate is presented in Table 1. Among the genes encoding iron binding proteins,tbpA gene was present in 82.4% of isolates which range from 69.6% in buffalo to 100% in cattle, emu, and rabbits (Table 3). Similarly,hgbA gene was found to be regularly distributed among all isolates affecting different hosts with the lowest prevalence among swine isolates (73.5%). GenehgbB has the lowest prevalence among the three iron binding proteins screened in this study and was found in 72.2% of the isolates. The percentage prevalence of this gene was found to be more among avian isolates (90%) in comparison to large ruminants (65.9%) and swine (67.6%) isolates. Of the two sialidases present inP. multocida isolates, the percentage prevalence ofnanB gene was found to be more thannanH gene. Overall very high prevalence ofpfhA gene (93.5%) was observed in this study with 100% prevalence among avian isolates. Dermonecrotoxin gene (toxA) was found only in 17.6% of strains with majority of the isolates belonging to porcine origin. One buffalo and 3 duck isolates were also found to carrytoxA gene (Table 3).Table 3
Prevalence of virulence associated genes amongPasteurella multocida isolates recovered from various host species of India.
Host origin/capsular type
No. of strains
tbpA(%)
hgbA(%)
hgbB (%)
pfhA (%)
ptfA (%)
nanB (%)
nanH (%)
toxA (%)
Buffalo
23
69.6
82.6
65.2
95.7
95.7
100
78.3
4.3
Cap type A
5
80.0
80.0
80.0
100
100
100
60.0
20.0
Cap type B
17
64.7
82.3
58.8
94.1
94.1
100
82.3
0.0
Cap type D
1
100
100
100
100
100
100
100
0.0
Cap type F
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cattle
18
100
94.4
66.7
94.4
94.4
100
100
0.0
Cap type A
5
100
100
100
100
80.0
100
100
0.0
Cap type B
11
100
90.9
45.4
90.9
100
100
100
0.0
Cap type D
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cap type F
2
100
100
100
100
100
100
100
0.0
Large ruminants (cattle + buffalo)
41
82.9
87.8
65.9
95.1
95.1
100
87.8
2.4
Cap type A
10
90.0
90.0
90.0
100
90.0
100
80.0
10.0
Cap type B
28
78.5
85.7
53.5
92.8
96.4
100
89.2
0.0
Cap type D
1
100
100
100
100
100
100
100
0.0
Cap type F
2
100
100
100
100
100
100
100
0.0
Chicken
18
77.8
83.3
83.3
100
100
100
61.1
0.0
Cap type A
13
69.2
84.6
76.9
100
100
100
53.8
0.0
Cap type B
5
100
80.0
100
100
100
100
80.0
0.0
Cap type D
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cap type F
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Duck
8
87.5
87.8
100
100
100
100
50.0
37.5
Cap type A
7
85.7
85.7
100
100
100
100
42.8
28.5
Cap type B
1
100
100
100
100
100
100
100
100
Cap type D
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cap type F
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Emu
4
100
100
100
100
100
100
100
0.0
Cap type A
4
100
100
100
100
100
100
100
0.0
Cap type B
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cap type D
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cap type F
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Avian (chicken + duck + emu)
30
83.3
86.7
90.0
100
100
100
63.3
10.0
Cap type A
24
79.1
87.5
87.5
100
100
100
58.3
8.3
Cap type B
6
100
83.3
100
100
100
100
83.3
16.6
Cap type D
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cap type F
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Rabbit
3
100
100
33.3
100
100
100
100
0.0
Cap type A
1
100
100
100
100
100
100
100
0.0
Cap type B
2
100
100
0.0
100
100
100
100
0.0
Cap type D
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Cap type F
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Pig
34
79.4
73.5
67.6
85.3
100
88.2
67.6
44.1
Cap type A
18
72.2
72.2
61.1
94.4
100
88.8
83.3
44.2
Cap type B
9
100
88.8
66.6
100
100
100
88.8
11.1
Cap type D
7
71.4
57.1
85.7
42.8
100
71.4
0.0
57.1
Cap type F
0
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Total (all isolates)
108
82.4
83.3
72.2
93.5
98.1
96.3
75.0
17.6
Cap type A
53
79.2
83.0
79.2
98.0
98.0
96.2
71.7
24.5
Cap type B
45
86.7
86.7
60.0
95.6
97.8
100
88.9
4.4
Cap type D
8
75.0
62.5
87.5
50.0
100
75.0
12.5
50.0
Cap type F
2
100
100
100
100
100
100
100
0.0Iron acquisition and uptake are essential for bacterial survival, and many bacteria have developed different iron sequestering system for uptake of iron. The expression of iron acquisition proteins increases under iron limiting condition, as well asin vivo condition (reviewed in [5]).P. multocida utilizes various receptors for adapting to variations in supply of different haem iron sources [5]. Among these, TbpA protein is necessary for extraction of iron from transferrin and has been reported to be an important virulence factor and epidemiological marker in cattle [12, 19, 26]. Previous studies reported thattbpA gene is either absent or rarely present in poultry, swine, and rabbit isolates (Table 4) [12–16]. In contrast to these findings, we observed a very high occurrence oftbpA gene among poultry (83.3%), swine (79.4%), and rabbit (100%) isolates (Tables 3 and 4). Among the host species, the prevalence of this gene was found to be lowest (69.6%) in buffalo (Table 3). The difference in prevalence of this gene among isolates of cattle and buffalo origin was found to be statistically significant (P
<
0.05), which is quite unexpected. Therefore more number of isolates from both host origins should be carried out before reaching any conclusion. In this study,tbpA gene was found to be frequently distributed among four capsule types, including CapF (Table 3). This is in contrast to Ewers et al. [12], who observedtbpA gene in 70% of CapB strains, followed by 37% of CapA, 9.5% of CapD strains and nil in CapF strains. Similarly, Katsuda et al. [18] reported positive association of CapA strain withtbpA gene.Table 4
Comparison of the distribution of virulence associated genes amongPasteurella multocida isolates recovered from various host species across the globe.
Gene/host
Cattle
Buffalo
Poultry
Pig
Rabbit
Country (reference)
No. of strains tested
Prevalence (%)
Country (reference)
No. of strains tested
Prevalence (%)
Country (reference)
No. of strains tested
Prevalence (%)
Country (reference)
No. of strains tested
Prevalence (%)
Country (reference)
No. of strains tested
Prevalence (%)
tbpA
India (this study)
18
100
India (this study)
23
69.6
India (this study)
30
83.3
India (this study)
34
79.4
India (this study)
03
100
Germany [12]
104
70.2
Germany [12]
07
57.1
Germany [12]
20
0.0
Germany [12]
52
0.0
Germany [12]
20
0.0
Japan [18]
378
76.2
Spain [15]
205
0.0
Brazil [16]
46
8.6
India [19]
23
100
Germany [13]
382
0.0
hgbA
India (this study)
18
94.4
India (this study)
23
82.6
India (this study)
30
86.7
India (this study)
34
73.5
India (this study)
03
100
Germany [12]
104
95.2
Germany [12]
07
100
Germany [12]
20
90.0
Germany [12]
52
98.1
Germany [12]
20
100
Japan [18]
378
95.5
Brazil [17]
25
100
Spain [15]
205
100
Brazil [16]
46
73.9
India [19]
23
100
Germany [13]
382
100
China [14]
233
96.6
hgbB
India (this study)
18
66.7
India (this study)
23
65.2
India (this study)
30
90.0
India (this study)
34
67.6
India (this study)
03
33.3
Germany [12]
104
57.7
Germany [12]
07
85.7
Germany [12]
20
85.0
Germany [12]
52
86.5
Germany [12]
20
100
Japan [18]
378
61.4
Brazil [17]
25
100
Spain [15]
205
60.5
Brazil [16]
46
30.4
India [19]
23
26.1
Germany [13]
382
84.3
ptfA
India (this study)
18
94.4
India (this study)
23
95.7
India (this study)
30
100
India (this study)
34
100
India (this study)
03
100
Germany [12]
104
99.0
Germany [12]
07
100
Germany [12]
20
100
Germany [12]
52
100
Germany [12]
20
100
Japan [18]
378
94.7
Brazil [17]
25
92.0
Spain [15]
205
100
Brazil [16]
46
93.4
India [19]
23
86.9
Germany [13]
382
100
China [14]
233
93.6
pfhA
India (this study)
18
94.4
India (this study)
23
95.7
India (this study)
30
100
India (this study)
34
85.3
India (this study)
03
100
Germany [12]
104
46.2
Germany [12]
07
100
Germany [12]
20
45.0
Germany [12]
52
21.2
Germany [12]
20
75.0
Japan [18]
378
52.4
Brazil [17]
25
60.0
Spain [15]
205
40.5
Brazil [16]
46
0.0
India [19]
23
100
Germany [13]
382
20.9
China [14]
233
15.0
nanB
India (this study)
18
100
India (this study)
23
100
India (this study)
30
100
India (this study)
34
88.2
India (this study)
03
100
Germany [12]
104
100
Germany [12]
07
100
Germany [12]
20
100
Germany [12]
52
100
Germany [12]
20
100
Japan [18]
378
100
Brazil [17]
25
100
Spain [15]
205
100
Brazil [16]
46
95.6
India [19]
23
0.0
Germany [13]
382
100
China [14]
233
81.5
nanH
India (this study)
18
100
India (this study)
23
78.3
India (this study)
30
63.3
India (this study)
34
67.6
India (this study)
03
100
Germany [12]
104
88.5
Germany [12]
07
100
Germany [12]
20
65.0
Germany [12]
52
98.1
Germany [12]
20
100
Japan [18]
378
88.4
Brazil [17]
25
96.0
Spain [15]
205
100
Brazil [16]
46
67.3
India [19]
23
100
Germany [13]
382
100
China [14]
233
97.0
toxA
India (this study)
18
0.0
India (this study)
23
4.3
India (this study)
30
10.0
India (this study)
34
44.1
India (this study)
03
0.0
Germany [12]
104
5.8
Germany [12]
07
0.0
Germany [12]
20
5.0
Germany [12]
52
36.5
Germany [12]
20
0.0
India [19]
23
0.0
Brazil [17]
25
0.0
Spain [15]
205
7.8
Brazil [16]
46
0.0
Germany [13]
382
3.4
China [14]
233
4.7P. multocida utilizes two proteins (HgbA and HgbB) for acquiring iron directly from haem component. Morton et al. [27] reported that the presence of both proteins might provide increased uptake of iron and protection against negative effects of mutation in one of the encoding genes. Between these two proteins,hgbA gene was found to be regularly distributed (>95% prevalence) among isolates [12–15, 17, 18]. In the present study, 73.5% of porcine isolates were found to carry this gene, which is lower in comparison to previous findings, that is, nearly 100% prevalence (Table 4) [12–15]. The frequency ofhgbB gene varies among strains of different host origin and also with disease status of the animal [12, 15, 16, 18, 19]. In this study, 72.2% of the isolates were found to carry this gene with highest frequency observed among avian strains (90%), which is in agreement with previous reports (Table 4) [12, 17].Among the genes encoding proteins involved in bacterial colonization and adhesion,ptfA gene has the highest (98.1%) prevalence (Table 3). This gene encodes type 4 fimbria subunit and has been associated with bovine diseases [19]. Worldwide, this gene is regularly distributed with more than 85% prevalence amongP. multocida isolates, irrespective of host origin and capsule type (Table 4).pfhAgene encoding filamentous haemagglutinin is an important epidemiological marker and the presence of this gene has been correlated with occurrence of disease in cattle, swine and sheep [12, 13, 18, 19, 28]. Almost all previous studies reported low prevalence of this gene with varying frequencies in between 46–52%, 45–60%, and 15–40.5% among isolates of cattle, poultry, and pig origin, respectively (Table 4) [12–15]. But interestingly, very high prevalence, 85.3% (pig) to 100% (avian), of this gene was observed among Indian isolates (Table 3). This suggestspfhA gene might be providing survival advantage to the bacterium in the host and the occurrence of horizontal gene transfer has led to such high prevalence among Indian strains/clones.Sialidases play an important role in colonization to epithelial surface. They enhance bacterial virulence by unmasking key host receptor and by reducing the effectiveness of mucin [5, 29]. Of the two sialidases (NanB and NanH) present inP. multocida isolates, thenanB gene was found in almost all isolates, whereas the prevalence ofnanH varied according to host origin and geographical location. In this study, the frequency ofnanH gene among poultry isolates was found to be low (63.3%), which is in contrast to the report of Furian et al. [17] (Table 4). Similarly, the carriage ofnanH gene among isolates of pig origin from India was also found to be lower (67.6%) in comparison to isolates from other parts of the globe, which reported higher (>97%) frequency (Table 4) [12–15].Dermonecrotoxin (sometimes calledP. multocida toxin) is encoded bytoxA gene. This gene was initially detected in serotype D isolates and was found to be associated with atrophic rhinitis in pigs. Later on, it was detected in strains of serotype A from pigs and other hosts [30]. In this study,toxA gene was detected in 44.1% of pig isolates. Further, one buffalo and three duck isolates were also found positive fortoxA gene (Table 3). Two serotype B isolates were found to carry this gene which is in agreement with our previous findings (Sarangi et al., submitted for publication). A lysogenic bacteriophage infection ofP. multocida resulting in horizontal gene transfer could be the reason [31].The association of virulence associated genes with particular capsular type and host origin was assessed by the Chi-square and Fisher’s exact test. Out of the 8 virulence associated genes studiedtoxA, pfhA, nanB, andnanH were found to be associated (positive or negative) with capsular type.pfhA, nanB, andnanH genes were found to be regularly distributed among all serotypes with the exception of serotype D. Negative association ofpfhA gene with CapD strains has been reported previously [12, 14, 18]. Dermonecrotoxin encoded bytoxAgene was found to be positively associated with capD and CapA. Ewers et al. [12] observed clear association oftoxA gene with CapD strains which was later supported by similar reports from other workers [13, 14]. Among cattle isolates, a significant difference (P
=
0.021) was observed in the distribution ofhgbB gene among serotypes (Table 3). Similarly, for pig isolates the frequency ofpfhA andnanH gene among serotypes was found to be statistically significant (Table 3). However, as the number of strains tested under each serotype was less, more number of samples should be tested before reaching any definite conclusion. In order to ascertain any trend in the distribution of virulence genes over time period, the strains used in the study were divided into two groups, contemporary (2009–2014) and archived (1992–2008), and statistical analysis was carried out. But no statistically significant difference was observed between the two groups with respect to virulence gene distribution (Table 1).The prevalence of virulence associated genes was found to vary amongP. multocida isolates recovered from various host species. Significant association betweentoxA andnanH genes with host origin was also observed. Dermonecrotoxin gene was found to be positively associated with porcine isolates, whereasnanH gene was found to be positively associated with large ruminant isolates, more specifically with cattle isolates, which agrees well with the findings of Ewers et al. [12].The combination of genes amongP. multocida isolates was assessed by the Chi-square and Fisher’s exact test. Significant association was observed betweentbpA-hgbA, tbpA-hgbB, tbpA-pfhA, tbpA-nanB, tbpA-nanH, hgbA-hgbB, hgbA-pfhA, hgbA-nanB, hgbA-nanH, pfhA-nanB, andpfhA-nanH. Similar association among iron acquisition genes, as well as between various virulence associated genes, has been reported previously by Ewers et al. [12].To sum up, the present study revealed some unique epidemiological information on the prevalence of virulence associated genes among Indian strains in comparison to its equivalents in other parts of the globe. The result shows that with the exception oftoxA gene the virulence associated genes are regularly distributed amongP. multocida isolates. The occurrence ofptfA, hgbA, andnanH genes among swine isolates of Indian origin was found to be less in comparison to other countries. Gene encoding dermonecrotoxin was observed in 17.6% of the total isolates studied. This gene is present mostly among swine isolates, with few occurrences in buffalo and duck isolates. Interestingly, very high prevalence oftbpA gene was observed among poultry, swine, and rabbit isolates. Likewise, very high prevalence ofpfhA gene was observed among Indian isolates, irrespective of host species origin. As proper history of majority of the isolates with respect to its disease status was not available, it was not possible to perform association study between virulence gene and disease status of the animal, which could have enhanced the significance of this study. Therefore, more number of isolates with proper history on disease status of the host should be carried out in future, which will be helpful to make a more definite conclusion, to provide insight into mechanism of pathogenesis, association of genes with outcome of the disease, and in future vaccine strategies.



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*Source: 814109-2014-11-18.xml*