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Veterinary Microbiology

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Short communication

Pasteurella multocida isolates associated with ovine pneumonia are toxigenic
D. Cida,⁎, A. García-Alvareza, L. Domíngueza,b, J.F. Fernández-Garayzábala,b, A.I. Velaa,b

a Animal Health Department, Veterinary School, Universidad Complutense de Madrid, Spain
b Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, Spain

A R T I C L E I N F O

Keywords:
Pasteurella multocida
PMT toxin
Sheep
Pneumonia

A B S T R A C T

The P. multocida toxin (PMT), a dermonecrotic protein encoded by the toxA gene, is the major virulence factor of
capsular type D P. multocida strains causing progressive atrophic rhinitis (PAR) in pigs. A high frequency of P.
multocida isolates harboring the toxA gene has been found among ovine pneumonic isolates, although the ability
of these isolates to express PMT has never been examined. In this study we have investigated the ability of ovine
toxA+ P. multocida isolates (n= 57) to express a functional toxin by detection of PMT toxin antigen using an
ELISA test and its cytopathic effect in a Vero cell assay. PMT antigen was expressed in the great majority (54/57;
94.7%) of toxA+ isolates. Moreover, the 100% toxA+ ovine isolates analyzed produced a cytopathic effect in
Vero cells within 24–48 h post-inoculation, identical to that described for porcine toxigenic P. multocida isolates.
These results show for the first time that, in addition to isolates associated with PAR, isolates of P. multocida
associated with pneumonia in sheep are also toxigenic. In addition, we found a total agreement (Kappa = 1; C.I.
0.75–1.25) between the detection of the toxA gene and the toxigenic capability of P. multocida isolates, in-
dicating the PCR detection of toxA would be a suitable predictive marker of the toxigenic fitness of P. multocida.

1. Introduction

Pasteurella multocida is associated with a number of diseases in do-
mestic and wild animals, including progressive atrophic rhinitis in pigs
(PAR) and pneumonia in pigs, cattle and sheep (Harper et al., 2006;
Wilson and Ho, 2013). The P. multocida toxin (PMT) is the major
virulence factor of P. multocida responsible for the turbinate bone de-
generation manifested during infection in PAR (Lax and Chanter,
1990). It is a dermonecrotic protein (146-kDa) encoded by the toxA
gene on a lysogenic bacteriophage residing only in the genome of
toxicogenic strains (Pullinger et al., 2004). Apart from its well-known
role in the pathogenesis of PAR, PMT has several other biological
functions. Inoculation of purified PMT can induce liver and kidney
damage in addition to atrophy of the nasal turbinate bones in pigs (Lax
and Chanter, 1990) and pneumonic lesions in rabbits (Chrisp and
Foged, 1991). PMT is also a potent mitogen (Rozengurt et al., 1990) and
interacts with several signal transduction pathways to disturb cell
growth and differentiation, favoring the evasion of the immune system
(Kubatzky et al., 2013). It has been suggested that PMT might dampen
the immune response by different toxin-related immune evasion stra-
tegies, facilitating the multiplication and survival of P. multocida in the
host (Kubatzky et al., 2013).

The synthesis of PMT is mainly associated with capsular type D-

toxA+ pig isolates causing PAR, although PMT has been occasionally
detected in porcine strains of capsular type A (Djordjevic et al., 1998;
Davies et al., 2003). Unexpectedly, a high frequency of toxA+ P. mul-
tocida isolates has been found among ovine pneumonic isolates of both
capsular types A and D in the last few years (Ewers et al., 2006; Sarangi
et al., 2015; Vougidou et al., 2015; Einarsdottir et al., 2016; Shirzad and
Tabatabaei, 2016; García-Alvarez et al., 2017), although the ability of
these isolates to express PMT has not been investigated. Isolates from
sheep and pigs belong to genetically different subpopulations of P.
multocida (García-Alvarez et al., 2017). This circumstance together with
the fact that not all genes are necessarily expressed (Bavananthasivam
et al., 2018) led us to investigate the ability of ovine toxA+ P. multocida
isolates to express a functional toxin by detection of PMT toxin antigen
using an ELISA test and its cytopathic effect in a Vero cell assay.

2. Material and methods

2.1. P. multocida strains

This study included 57 pneumonic ovine P. multocida isolates har-
boring the toxA gene (toxA+): 43 capsular type A, 13 capsular type D
and one nontypeable (NT) as determined by multiplex PCR (García-
Alvarez et al., 2017). Detailed information about the 57 toxA+ ovine

https://doi.org/10.1016/j.vetmic.2019.04.006
Received 15 February 2019; Received in revised form 1 April 2019; Accepted 5 April 2019

⁎ Corresponding author.
E-mail address: lcid@ucm.es (D. Cid).

Veterinary Microbiology 232 (2019) 70–73

0378-1135/ © 2019 Elsevier B.V. All rights reserved.

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isolates is shown in Table 1. In addition, three pneumonic toxA− iso-
lates of capsular types A (M172), D (P69) and NT (M478), were also
included as negative controls in both the ELISA and Vero cell assays. A
toxigenic capsular type D P. multocida strain (M91996) from porcine
atrophic rhinitis kindly supplied by Exopol (Pol. Río Gállego D/8,
Zaragoza, Spain) was used as positive control in the Vero cell assays.

2.2. Detection of PMT toxin

PMT toxin was determined in the 57 toxA+ P. multocida isolates
using a commercial enzyme immunoassay kit (PMT ELISA Kit, Dako
Corporation, Denmark) according to the manufacturer’s instructions.
Assays were performed from overnight pure cultures of each isolate in
Columbia blood agar plates (bioMérieux). According to manufacturer
instructions, agar plate growth was harvested with two ml of deionized
water and 200 μL of this bacterial suspension were inoculated into two
uncoated microwells, covered with sealing tape and incubate overnight
at 37 °C for extraction. Bacterial extracts were transferred to microwell
coated with monoclonal antibody to PMT in volumes of 50 μL each.
After incubation 60 min. at room temperature and washing, conjugate
was added. After incubation for 2 h at room temperature and washing,
chromogenic substrate was added. The reaction was stopped by the
addition of 100 μL of 0.46 mol/L sulfuric acid. Presence of PMT in the
extracts of the bacterial cultures was indicated by a yellow color in the
microwells. The optical density (O.D) was measured at 490 nm. The
final OD-value for each bacterial specimen was the mean of the OD of
the two microwells. ELISA assay for each isolate was performed by
duplicate in two independent experiments.

2.3. Cytopathic effect of PMT

The in vitro biological effect of PMT of 16 isolates (13 toxA+ ran-
domly selected isolates and three toxA+ isolates that were negative on
ELISA; Table 2) was assayed in a Vero cell line (Pennings and Storm,
1984). Bacterial extracts were prepared as described previously
(Amigot et al., 1998) with a few modifications. Briefly, bacteria were
grown on Columbia agar (BioMerieux) and harvested directly from the
agar using 4 ml of phosphate-buffered saline, pH 7.4. The suspensions
were treated by sonication (three cycles of 50 s each, alternating 3 s on
and 2 s off), centrifuged (10 min, 5000 rpm), and filtered through a
sterile 0.22-mm pore membrane filter (Millipore). Vero cell monolayers
were grown in 24-well tissue culture plates with flat bottoms at 37 °C,
5% CO2 in a humidified atmosphere, using Dulbecco's Modified Eagle
Medium (DMEM) containing 10% fetal calf serum (FCS). Before in-
oculation, the medium was removed and 200 μl of crude extracts was
added to cells in duplicate (Amigot et al., 1998). After 20 min of in-
cubation, 1 ml of DMEN without FCS was added to each well and the
plates were incubated at 37 °C, in a humidified atmosphere with 5%
CO2 for a maximum of 5 days. Cell morphology and cytopathic effects
were observed daily with an inverted microscope.

2.4. Sequencing of the toxA gene

The toxA gene of two isolates representative of the most frequent
capsular types and genotypes of ovine P. multocida isolates (M297,
capsular type A, genotype ST50 and M135, capsular type D and ST19;
García-Alvarez et al., 2017) were PCR-amplified sequenced using the
primers and conditions described by García (2009). Both isolates gave
positive results in both PMT ELISA and Vero cell assays. The amplified
products were purified by using a QIAquick PCR purification kit
(Qiagen) and sequenced using an automatic DNA sequencer (ABI
PRISM 3730; Applied Biosystem). Sequences of both isolates were as-
sembled and compared each other and with the sequence of toxA gene
of the reference porcine strain NCTC 12178 (accession number X51512)
using the FASTA sequence comparison tool (https://fasta.bioch.
virginia.edu/fasta_www2/fasta_list2.shtml). Moreover, the toxA se-
quences of isolates M297 and M135 were compared with toxA se-
quences available in the public ENA-database (formely EMBL-database)
using the FASTA server (https://www.ebi.ac.uk/Tools/sss/fasta/
nucleotide.html).

Table 1
Details of the 57 toxA+ pneumonic ovine P. multocida isolates used in this study.

Capsular type Geographical origin Year of isolation No. of isolates Isolates

A (n = 43) Badajoz 2008 19 M66, M77, M78, M84, M96, M113, M114, M117, M128, M135, M160, M166, M174, M176, M187,
M193, M199, M206, M208

Cáceres 2008 2 M321, M326
Cáceres 2009 4 P71, P73, P82, P85
Madrid 2009 18 P61, P63, P67, P68, P70, P72, P74, P75, P76, P78, P79, P80, P81, P83, P84, P86, P87, P88

D (n = 13) Badajoz 2008 3 M67, M93, M134
Cáceres 2008 7 M279, M285, M293, M297, M337, M343, M344
Madrid 2009 3 P64, P60, P65

NT (n = 1) Madrid 2009 1 P77

NT, nontypeable as determined by the multiplex PCR described by Townsend et al. (2001).

Table 2
Pasteurella multocida toxin (PMT) antigen detection by enzyme immunoassay
(PMT ELISA) and cytopathic effect detection by Vero cell assay in pneumonic
ovine P. multocida isolates harboring the toxA gene (toxA+).

Capsular type Sequence
type (ST)a

No. of
isolates

PMT detection
by ELISA (no.
of isolates)

No. of isolates with
cytopathic effect on
Vero cells/ No. of
isolates analyzedb

A (n = 43) ST19 2 1 + (2/2)
ST48 1 1
ST49 1 1 + (1/1)
ST50 10 10 + (5/5)
ST54 1 1
ST56 4 4
ST57 1 1
ST58 1 1
ST59 1 1
ST60 1 1
nd 20 19 + (5/5)

D (n = 13) ST19 4 4 + (2/2)
ST20 1 0 + (1/1)
ST50 1 1
ST53 1 1
ST55 1 1
nd 5 5

NT (n = 1) ST52 1 1

NT, nontypeable as determined by the multiplex PCR described by Townsend
et al. (2001).
nd, not determined.

a Sequence types (STs) were previously determined by García-Alvarez et al.
(2017) based on the Multi-host MLST Database (http://pubmlst.org/
pmultocida_multihost/).

b 16 isolates (13 randomly toxA+ and PMT producing isolates and three
toxA+ isolates but PMT negative on ELISA test.

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=X51512
https://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml
https://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml
https://www.ebi.ac.uk/Tools/sss/fasta/nucleotide.html
https://www.ebi.ac.uk/Tools/sss/fasta/nucleotide.html
http://pubmlst.org/pmultocida
http://pubmlst.org/pmultocida


2.5. Statistical analysis

Analysis of the association between PMT antigen expression de-
termined by ELISA test and capsular type of isolates was done calcu-
lating the Odds ratio (OR) and the Fisher exact test with a significance
level set at P < 0.05. Frequency and association measures and their
confidence intervals (C.I.) were determined using the Epi InfoTM 7
program of the Centers for Disease Control and Prevention (CDC)
(http://wwwn.cdc.gov/). The agreement between toxA gene detection
by PCR and Cytopathic effect detection on Vero cell assay was esti-
mated by calculating the Kappa coefficient on the WinEpi website
(http://www.winepi.net/uk/index.htm). The confidence level was set
at 95%. A value k equal to 0 indicates that there is no agreement once
the chance factor is discounted while a value of kappa equal to 1 in-
dicates a total agreement.

3. Results and discussion

PMT antigen was detected in 54 of the 57 toxA+ isolates (94.7%,
C.I. 85.4–98.9%; Table 2). No significant differences (P > 0.05; OR
1.7, CI 0.14–20.5) were observed between isolates of capsular type A
(95.3%; 41/43) and D (92.3%; 12/13), indicating that the toxA gene is
transcribed and expressed in the great majority of the ovine toxA+ P.
multocida isolates investigated, regardless of their capsular type. Ana-
lysis of the DNA sequences of toxA gene of strains M297 and M135
(capsular types A and D, respectively) showed similarity values of
99.9% between them (in 3711 nucleotide overlap) and between 99.6
and 100% with other toxA gene sequences available in the public ENA-
database. These results indicate that the toxA gene is highly conserved
in P. multocida regardless of their serotype or host origin (Frandsen
et al., 1991; Donnio et al., 1999; Einarsdottir et al., 2016). The nearly
complete sequences of the toxA gene of the ovine strains M135 and
M297 have been deposited in the Gene Bank under the accession
numbers LS974118 and LR215670, respectively. Comparative analyses
of the deduced amino acid sequences of the toxA gene of strains M135
and M297 with that from the porcine reference strain NCTC 12,178
revealed a similarity of 99.6% and 99.8%, respectively, suggesting that
PMT toxin of the ovine isolates would be biologically functional. This
assumption was supported by the fact that culture extracts of the 16
toxA+ ovine isolates analyzed produced a cytopathic effect in the Vero
cells within 24–48 h post-inoculation (Table 2) identical to that de-
scribed for porcine toxigenic P. multocida isolates in Vero cells
(Pennings and Storm, 1984) and characterized by the development of
knots throughout the monolayer (Fig. 1A). On the other hand, culture
extracts of the three toxA- isolates did not produce damage to the Vero
cells (Fig. 1B). PMT was not detected by the ELISA assay in three of the
toxA+ isolates even though they exhibited an evident cytopathic effect
in Vero cells (Table 2). Nevertheless, we found a total agreement
(k = 1; C.I. 0.75–1.25) between the detection of the toxA gene and the

toxigenic capability of P. multocida isolates determined through cell
culture assay. These results indicate that although the ELISA technique
has been widely used to detect toxigenic P. multocida isolates (Amigot
et al., 1998; Hariharan et al., 2000; MacInnes et al., 2008), the PCR
detection of the toxA gene would be a suitable predictive marker of the
toxigenic fitness of P. multocida.

Epidemiological data have suggested a relevant role for toxA+
isolates in ovine pneumonia (Ewers et al., 2006; Sarangi et al., 2015;
Vougidou et al., 2015; Einarsdottir et al., 2016; Shirzad and Tabatabaei,
2016; García-Alvarez et al., 2017), but the production of PMT has been
mainly associated with capsular type D, and also sporadically with
capsular type A, porcine isolates causing PAR (Djordjevic et al., 1998;
Davies et al., 2003). The results of this study show for the first time that
isolates of P. multocida associated with pneumonia in sheep are also
toxigenic. PMT toxin is able to induce a persistent inflammatory re-
sponse (Kubatzky et al., 2013). This persistent inflammatory response
could be responsible for the consolidated areas observed in lungs of
sheep with subclinical pneumonia caused by P. multocida. More than
half of the 57 toxA+ isolates of this study were previously character-
ized by MLST typing (García-Alvarez et al., 2017) belonging to a rela-
tively large number of STs (Table 2). These data suggest therefore that
the toxigenic capacity of sheep P. multocida isolates from pneumonia
would not be related to particular genetic lineages. These results should
be confirmed by the analysis of a large collection of ovine isolates from
other geographical origins and genetic backgrounds.

PMT is considered a suitable and effective antigen for vaccination
that is able to induce a protective immune response (Foged, 1992;
Harper et al., 2006) and is commonly included as a toxoid in many
commercial vaccines to control PAR (Zhang et al., 2018). While de-
termining the precise role of PMT in the development of pneumonia in
sheep, this study lends some support for the inclusion of this toxin in
vaccines for prophylaxis against ovine pneumonic pasteurellosis.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

The authors thank Paula Marino Rey for her excellent technical
assistance. This study was supported by AGL2009-10136 (Ministerio de
Ciencia e Innovación, Spain).

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	Pasteurella multocida isolates associated with ovine pneumonia are toxigenic
	Introduction
	Material and methods
	P. multocida strains
	Detection of PMT toxin
	Cytopathic effect of PMT
	Sequencing of the toxA gene
	Statistical analysis

	Results and discussion
	Conflict of interest
	Acknowledgements
	References