the molecular biology of pasteurella multocida
TRANSCRIPT
The molecular biology of Pasteurella multocida
Meredith L. Hunta,*, Ben Adlera, Kirsty M. Townsendb
aBacterial Pathogenesis Research Group, Department of Microbiology,
Monash University, Clayton Vic. 3168, AustraliabVeterinary Pathology and Anatomy, School of Veterinary Science & Animal Production,
The University of Queensland, St Lucia Qld 4072, Australia
Abstract
Pasteurella multocida is an important veterinary and opportunistic human pathogen. The species
is diverse and complex with respect to antigenic variation, host predeliction and pathogenesis.
Certain serological types are the aetiologic agents of severe pasteurellosis, such as fowl cholera in
domestic and wild birds, bovine haemorrhagic septicaemia and porcine atrophic rhinitis. The recent
application of molecular methods such as the polymerase chain reaction, restriction endonuclease
analysis, ribotyping, pulsed-®eld gel electrophoresis, gene cloning, characterisation and
recombinant protein expression, mutagenesis, plasmid and bacteriophage analysis and genomic
mapping, have greatly increased our understanding of P. multocida and has provided researchers
with a number of molecular tools to study pathogenesis and epidemiology at a molecular level.
# 2000 Elsevier Science B.V. All rights reserved.
Keywords: Pasteurella multocida; Molecular biology; Genetics
1. Introduction
Over a century has passed since the ®rst attempts by Louis Pasteur at immunisation
against infection with the Gram negative facultative bacterium, Pasteurella multocida, the
organism which bears his name. During this time, considerable research into the
mechanisms of immunity, host predilection, virulence and pathogenesis of P. multocida
has resulted in only very small increases in our understanding of the organism. Safe and
effective vaccines against pasteurellosis are still lacking, and until recently there had been
no extensive characterisation of this organism at the molecular level. The lack of genetic
tools for use in P. multocida hindered investigations at a time when great inroads were
Veterinary Microbiology 72 (2000) 3±25
* Corresponding author.
0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 1 8 3 - 2
being made into understanding the molecular basis of pathogenesis in many other
bacterial pathogens.
Substantial progress towards a better understanding has been recently made, with a
number of groups studying different aspects of the molecular biology of P. multocida.
This review brings together the current molecular knowledge of P. multocida, including
detection and identi®cation by polymerase chain reaction (PCR), molecular epidemiol-
ogy, cloning and characterisation of individual genes, genome mapping, extrachromo-
somal elements, tools for genetic manipulation and methods for mutagenesis.
2. Molecular identi®cation and differentiation of P. multocida
Since its ®rst isolation in 1881, detection, identi®cation and characterisation of
P. multocida has relied on the ability to cultivate or purify the organism in the laboratory.
The puri®ed organism is subsequently classi®ed according to phenotypic traits such as
morphology, carbohydrate fermentation patterns and serological properties. However,
culture conditions can in¯uence the expression of these attributes thus diminishing the
stability and reliability of phenotypic methods for strain identi®cation (Matsumoto and
Strain, 1993; Jacques et al., 1994). In recent years, identi®cation and characterisation has
favoured analyses that re¯ect one of the most fundamental properties of an organism, its
genetic information. Genotypic characterisation possesses versatility surpassing that of
traditional phenotypic methods, as nucleic acid analyses facilitate identi®cation and rapid
detection of an organism, determination of its taxonomic position, and investigation of
intra-species genetic relationships.
Molecular approaches such as DNA hybridisation and nucleic acid ampli®cation have
allowed bacterial detection directly from clinical samples, dramatically reducing the time
required for identi®cation. While such methods have had the greatest impact on the
detection of organisms that are generally dif®cult or slow to cultivate in the laboratory,
molecular technology has also signi®cantly in¯uenced the identi®cation and character-
isation of P. multocida.
2.1. Detection and identi®cation of P. multocida by speci®c PCR assays
Since the initial development of the PCR in 1985, the basic principle of in vitro nucleic
acid ampli®cation through repetitive cycling has had extensive applications in all aspects
of fundamental and applied clinical science (Rapley et al., 1992). The PCR method is no
longer used simply as a tool for the rapid production of large quantities of a de®ned target
sequence. The technique now plays a critical role in the clinical laboratory, as rapid and
speci®c detection of microorganisms has provided remarkable advances in the diagnosis
of infectious agents, particularly in cases where the presence of an organism has
signi®cance (Relman and Persing, 1996). Modi®cations to sample preparation have
allowed PCR analysis to be performed on clinical specimens, dramatically reducing the
time required for bacterial identi®cation. Furthermore, the incorporation of multiplex
PCRs can yield detailed information with regards to diagnosis, pathogenesis or antibiotic
resistance, if relevant oligonucleotide primers are available.
4 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
2.1.1. P. multocida-speci®c PCR assays
To date, only three areas of target speci®city have been addressed through the use of
PCR in P. multocida identi®cation. The most fundamental of these, namely a species-
speci®c PCR assay, was developed only recently for detection of P. multocida in mixed
cultures or clinical samples (Kasten et al., 1997a; Townsend et al., 1998a). Two distinct
approaches were used for the development of a P. multocida-speci®c PCR, one rational
and one fortuitous. Kasten et al. (1997a) described the use of oligonucleotide primers
constructed to amplify the psl gene encoding the P6-like protein (Psl) of P. multocida.
This gene demonstrates signi®cant similarity to the P6 protein of Haemophilus in¯uenzae
(Nelson et al., 1988) and H. parain¯uenzae (GenBank accession No. D28887). As
H. in¯uenzae is normally not isolated from poultry and negative results were obtained
with a wide range of avian pathogens, it was postulated that ampli®cation of this gene
could serve as a basis for P. multocida-speci®c detection (PCR-H). However, the
omission of reference strains from the genus Pasteurella sensu stricto is of some concern,
with unknown detection of all P. multocida subspecies and those species present in
the respiratory tract of healthy birds (P. langaa and P. volantium). With this in mind,
the speci®city of the PCR-H assay for P. multocida detection, in poultry or otherwise,
will remain questionable until ampli®cation in other Pasteurella species has been
examined.
It was also shown that neither mouse inoculation nor the PCR assay accomplished total
detection among infected ¯ocks. However, increased detection by PCR may be possible
with further optimisation of the sample preparation procedure. Another disadvantage of
this technique is that to achieve the maximum sensitivity of 10 organisms, additional
hybridisation with psl is required. While PCR technology is being increasingly
incorporated in laboratories around the world, hybridisation is still usually possible only
in specialised laboratories.
The PM-PCR developed by Townsend et al. (1998a) demonstrated a sensitivity of
less than 10 organisms (Lee et al., 1999) without the need for additional hybridisation.
This assay is based on the ampli®cation of a DNA sequence unique to P. multocida
(KMT1) that was isolated by subtractive hybridisation (Townsend et al., 1998a). While
the sensitivity of this assay is high, the speci®city is decreased slightly by the
ampli®cation of Pasteurella canis biovar 2, once recognised as an atypical P. multocida.
Indeed, 16S rRNA sequencing indicates that the two species are closely related (Dewhirst
et al., 1993). As species within Pasteurella sensu stricto other than P. multocida and
P. avium were not examined by Kasten et al. (1997a), it is not known whether P. canis
biovar 2 will remain positive with the PCR-H assay. DNA:DNA hybridisation
studies support the recognition of P. canis biovar 2 as a distinct species (Mutters et al.,
1985).
While false positives may occur with some isolates from pneumonic cattle and pigs
with the PM-PCR, P. multocida subspecies and P. canis biovar 2 can be distinguished on
the basis of indole and mannitol fermentation. Optimisation of the PM-PCR using regions
surrounding the KMT1 sequence may improve the speci®city of the assay, although this
requires further examination. Although some disadvantages in speci®city are evident in
both assays, the assay derived by (Townsend et al., 1998a) is more versatile as additional
hybridisation is not required for optimal sensitivity.
M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 5
2.1.2. PCR identi®cation of haemorrhagic septicaemia (HS)-causing serogroup B P.
multocida
Two independently isolated gene sequences unique to HS-causing seroproup B P.
multocida (Brickell et al., 1998; Townsend et al., 1998a) have been utilised in the
development of serogroup B-speci®c PCR assays. Comparative analysis with the H.
in¯uenzae Rd genome indicates that the DNA regions ampli®ed by Townsend et al.
(1998a) and Brickell et al. (1998) are potentially located within about 1 kb of each other.
However, this association has not been examined. Despite the implied close proximity of
these sequences, slight variation in speci®city is evident between the two assays.
To date, the HSB-PCR developed by Townsend et al. (1998a) remains speci®c for HS-
causing serogroup B P. multocida. Serogroup B cultures with the predominant somatic
antigen being either serotype 2 or 5 are speci®cally identi®ed by the ampli®cation of a
�620 bp fragment by the KTSP61 and KTT72 primers. These primers have recently been
employed for detection of HS-causing P. multocida from swine tonsil swabs with no
evidence of non-speci®c ampli®cation (Townsend et al., 1999). In contrast, while the PCR
assay developed by Brickell et al. (1998) demonstrated reasonably speci®c ampli®cation
of serogroup B isolates with HS signi®cance, ampli®cation was also observed with one of
the two serogroup E P. multocida isolates analysed.
Interestingly, both HS-causing serogroup B-speci®c PCR assays were developed by the
fortuitous isolation of DNA sequences unique to these serotypes. With the description of
the serotype A:1 (Chung et al., 1998) and serotype B:2 (Boyce et al., 1999) capsule
biosynthetic loci, rationally derived serogroup A- and B-speci®c PCR assays may be
developed in the near future. However, it should be noted that any PCR assay constructed
from the capsule biosynthetic locus will in theory, identify all organisms within that
capsular type. The speci®city will not extend to the somatic type until type-speci®c
regions have been identi®ed within the P. multocida homologue of the rfb (O-antigen
biosynthetic) gene cluster.
2.1.3. PCR detection of toxigenic P. multocida
The application of PCR technology for P. multocida identi®cation was ®rst reported in
1994 when primers constructed from the sequence of the toxA gene (encoding the
dermonecrotic toxin implicated in progressive atrophic rhinitis) were used to detect
toxigenic P. multocida strains (Nagai et al., 1994). Subsequent PCR assays have been
developed for the direct analysis of toxigenic P. multocida without additional
hybridisation for increased sensitivity (Kamp et al., 1996; Lichtensteiger et al., 1996;
Hotzel et al., 1997). The assay described by Kamp et al. (1996) appears to be the most
sensitive and effective method for large-scale analysis of nasal and tonsillar swabs.
However, the simplistic approach of Lichtensteiger et al. (1996) is more appealing for
small studies despite the potential for false positive ampli®cation (Townsend et al., 1999).
Other aberrant ®ndings when using the primers of Lichtensteiger et al. (1996) have been
reported (Amigot et al., 1998). Faint PCR products of the expected size were observed
from samples that were negative by both ELISA and cell culture. It was suggested that
these bands were a result of either low numbers of positive cells not detectable by
other methods, false ampli®cation or contamination with positive DNA. Additional
primer sets within the toxA gene for use in nested or multiplex PCRs may enhance
6 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
the sensitivity and speci®city of the assay, while eliminating the possibility of false
positive ampli®cation.
2.2. Detection of toxigenic P. multocida by colony hybridisation assays
A colony lift-hybridisation assay using a commercially available multicolour detection
kit was recently developed for rapid, highly sensitive and simultaneous detection of
toxigenic P. multocida and Bordetella bronchiseptica (Register et al., 1998). The major
advantage of this assay is the ability to screen the primary isolation plate for suspect
colonies, removing the need for pure cultures and allowing rapid analysis of large numbers
of samples. However, it was noted that while biotinylated probes are suggested for use
with the Genius Multicolor Detection Kit (Boehringer Mannheim), false positive reactions
have been shown to occur with biotinylated bacterial proteins in colony lift-hybridisation
assays (Register, 1998). It is, therefore, essential that biotin is not substituted for digoxigenin
and ¯uoroscein in the preparation of the labelled probe for this assay.
2.3. Molecular characterisation of P. multocida
Repeated efforts have been made to classify the P. multocida species according to
phenotypic attributes such as serological antigen presentation (capsular and somatic) and
biochemical fermentation pro®les. However, these techniques provide limited character-
isation and insuf®cient information for epidemiologic studies of P. multocida (Wilson
et al., 1992). The ability to differentiate phenotypically similar isolates is critically important
in epidemiology, particularly when establishing the identity of bacterial vaccine strains
(Stull et al., 1988). The development of DNA-based techniques has provided alternative
methods of characterisation that overcome the limitations of phenotyping, while identifying
precisely individual strains of closely related bacteria (Owen, 1989). During the last decade,
genomic characterisation techniques have supplemented or replaced traditional typing
methods for the discrimination of isolates from a wide range of bacterial pathogens (Tenover
et al., 1995). Initially, the equipment and reagents required for molecular characterisation
were expensive, and few laboratories were capable of performing these `complicated'
procedures (Wachsmuth, 1986). In recent years, technique optimisation and the increased
availability of equipment have allowed such methods to be incorporated on a routine basis
in most laboratories throughout the world.
Molecular characterisation, or DNA ®ngerprinting as we know it today, encompasses a
large range of methods with variable speci®city and discriminatory power, most of which
have been used to differentiate phenotypically similar P. multocida isolates. The
application of these techniques to P. multocida epidemiology has generated a large
number of publications, many of which could not be reviewed in detail. However,
selected references are reviewed that illustrate areas in which the application has had a
major impact.
2.3.1. Restriction endonuclease analysis
Restriction endonuclease analysis (REA) has proved to be a valuable component of
bacterial epidemiologic studies, particularly in investigations of outbreaks of pasteur-
M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 7
ellosis. This method is a highly reproducible technique that is not in¯uenced by the
inconsistent expression of phenotypic traits that limit the sensitivity and speci®city of
conventional typing methods (Snipes et al., 1989). The use of REA, either solely or in
conjunction with ribotyping (see below), can provide sensitive, distinctive banding
pro®les capable of differentiating isolates of similar serotype. Several restriction enzymes
have been used for DNA ®ngerprinting of P. multocida isolates by REA, with HhaI and
HpaII yielding the most informative and easily distinguished pro®les from a wide range
of serotypes (Wilson et al., 1992, 1993; Blackall et al., 1995, 1996; Diallo et al., 1995).
Wilson et al. (1992) demonstrated a high level of differentiation among the
P. multocida somatic reference serotype strains, with each of the 16 serotypes producing
a unique HhaI ®ngerprint pro®le. Such discrimination was also seen within 71 capsular
serogroup B isolates, with 20 HhaI pro®les evident among eight somatic serotypes.
Thirteen of these were recognised among 54 isolates possessing classic HS-causing
P. multocida serotypes (B:2, B:5 or B:2,5), with several instances where isolates of
similar serotype exhibited distinct DNA ®ngerprints.
However, the discriminatory power of REA with HhaI observed among avian and
serogroup B P. multocida did not extend to serogroup E isolates, in which a single unique
HhaI pro®le was shown among 13 E:2 strains (Wilson et al., 1992). Minor pro®le
differences were demonstrated following HpaII digestion, illustrating the potential
requirement for multiple REA typing to characterise fully some P. multocida isolates.
Isolates from outbreaks of septicaemic pasteurellosis in elks have been characterised
by HhaI and HpaII DNA ®ngerprinting, and compared with pro®les previously
established in HS and septicaemic pasteurellosis strains (Wilson et al., 1995). All 70
isolates from the 1987 and 1993 outbreaks were identi®ed as serotype B:3,4 and shown to
exhibit a single common DNA pro®le, regardless of whether HhaI or HpaII endonuclease
was used. These ®ndings indicated that P. multocida isolated from these outbreaks was
endemic to the National Elk Refuge, and clearly distinct from other HS and septicaemic
pasteurellosis isolates of similar serotype.
Considerable genetic heterogeneity has been observed among swine isolates, allowing
detailed epidemiological studies to be performed (Buttenschùn and Rosendal, 1990; Zhao
et al., 1992; Gardner et al., 1994). DNA pro®ling using BamHI digests of paired isolates
of P. multocida from swine lungs and kidneys indicated that despite the high genetic
diversity among serogroup A isolates associated with bronchopneumonia, genotypic
identity was observed among strains isolated from different sites of the same animal
(Buttenschùn and Rosendal, 1990). It was concluded that P. multocida isolated from
kidney lesions represented blood borne dissemination from primary bronchopneumonic
lesions. SmaI ®ngerprinting results of P. multocida associated with progressive atrophic
rhinitis (PAR) supported the hypothesis that a common infectious source exists in
Australian swine herds (Gardner et al., 1994) and that PAR in Australian herds was
associated with the importation of breeder pigs.
The high level of discrimination exhibited by REA has allowed investigations into the
vaccinal safety and ef®cacy of the two principal live attenuated fowl cholera vaccines
(M9 and CU) currently in use in turkeys in the US (Snipes et al., 1990; Wilson et al.,
1993). REA of somatic serotype 3,4 P. multocida isolates from M9-vaccinated and
unvaccinated turkey ¯ocks using the enzyme SmaI revealed eight distinct pro®les that
8 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
were con®rmed by ribotyping (Snipes et al., 1990). It was shown that the pro®le observed
for the M9 vaccine strain was uncommon among unvaccinated turkeys, yet the majority
of isolates from M9-vaccinated ¯ocks possessed the M9 ®ngerprinting pro®le.
Kim and Nagaraja (1990) were able to demonstrate further subtle differences between
the CU (Clemson University) and M9 (a slow-growing variant of CU) strains following
REA of BglII-digested DNA. Several ®eld isolates were shown to possess DNA pro®les
similar to those of either CU or M9 (Kim and Nagaraja, 1990; Wilson et al., 1993),
although the vaccination history of the birds from which the isolates were obtained was
unknown. Once it is ®rmly established that the DNA pro®le of either M9 or CU is
uncommon among unvaccinated birds, REA using HhaI or BglII could become a valuable
tool to assess the level of mortality in fowl cholera outbreaks due to these vaccine strains.
2.3.2. Ribotyping
The banding patterns produced by REA are often complex, making visual
interpretation of results dif®cult. Ribotyping, like REA, utilises restriction enzyme
digestion of genomic DNA and agarose gel electrophoresis for DNA fragment separation.
The additional use of Southern blotting and hybridisation with a labelled DNA probe
reduces the complexity of the restriction patterns, and highlights restriction fragment
length polymorphisms (RFLPs) within the bacterial genome without the computer
analysis that is sometimes required.
Ribosomal RNA (rRNA) molecules are highly conserved, ubiquitous molecules that
constitute the major proportion of RNA in the bacterial cell (Grimont and Grimont,
1986). As rRNA operons vary in copy number and genomic location between strains and
species, DNA probes speci®c for rRNA gene sequences can be used to identify RFLPs
within and/or around the ribosomal operon, thus providing the basis for bacterial strain
differentiation.
Ribotyping, in conjunction with REA, has been used to characterise successfully and
differentiate avian (Snipes et al., 1989; Blackall et al., 1995) and porcine (Zhao et al.,
1992; Gardner et al., 1994) isolates of P. multocida. Ribotyping of avian P. multocida
isolated from fowl cholera outbreaks in turkeys in Australia (Blackall et al., 1995) and the
US (Carpenter et al., 1991) has demonstrated considerable genomic heterogeneity,
providing suf®cient evidence to discount the relatedness of outbreaks previously
indistinguishable by serotyping and biotyping.
Ribotyping of HS-causing isolates of P. multocida demonstrated increased discrimina-
tion among strains of similar serotype than ®eld alternation gel electrophoresis (FAGE),
but more inconsistencies within classi®cations were observed (Townsend et al., 1997a). It
appeared that the limited view of RFLPs within or around the ribosomal operon
occasionally compounded the confusion of the phenotypic classi®cations. Ribotyping in
combination with other genotypic methods could provide greater discrimination among
HS-causing isolates, although a distorted view of genetic relatedness could occur if
ribotyping alone is used.
2.3.3. Pulsed-®eld gel electrophoresis (PFGE)
Field alternation electrophoretic methods, more commonly known as pulsed-®eld gel
electrophoresis (PFGE), remain the `gold standard' ®ngerprinting method for molecular
M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 9
epidemiology (Goering, 1993), as polymorphisms throughout the chromosome are
examined without the complexity of REA patterns and the restricted view of genetic
variation produced by ribotyping. Detailed reviews of the development and application of
all PFGE methods have been published previously (Dawkins, 1989; Townsend and
Dawkins, 1993).
PFGE analysis has consistently shown greater discrimination in identi®cation and
differentiation of bacterial species than ribotyping (Prevost et al., 1992; Kristjansson
et al., 1994; Townsend et al., 1997a). REA provides comparable discrimination among
isolates; however, the complex banding patterns sometimes require computer software
analysis to achieve de®nitive interpretation and accurate typing (Wilson et al., 1993;
Kristjansson et al., 1994).
While PFGE analysis has become an integral component of bacterial genetics and
molecular epidemiology during the last decade, it has had limited application in the
comparative typing of P. multocida isolates (Donnio et al., 1994, 1999; Blackwood et al.,
1996; Townsend et al., 1997a). The initial study by Donnio et al. (1994) demonstrated
heterogeneity among P. multocida serotype D:2 isolated from the oropharynx of pig
breeders whose livestock had suffered from pasteurellosis, although there was no
indication of the genetic relatedness between P. multocida isolated from the breeders and
their pigs. Recently, characterisation by PFGE of dermonecrotic toxin (DNT)-producing
strains of P. multocida isolated from humans and swine revealed signi®cant DNA
polymorphisms, although no correlation with host species was evident (Donnio et al.,
1999). It was suggested that the lack of discrimination between toxigenic isolates of
porcine and human origin might indicate colonisation of people from a porcine reservoir.
Discrimination between isolates of similar serotype was observed following PFGE
analysis of HS-causing isolates of P. multocida (Townsend et al., 1997a), with some
correlation to geographic location. HS-causing serogroup B isolates from North America
were clearly differentiated from Asian serogroup B strains, with the latter exhibiting a
high degree of homogeneity. The ability of PFGE to demonstrate genetic relatedness, yet
maintain discrimination, was particularly evident after analysis of the North American
isolates. Cultures representing the original Yellowstone Park Buffalo `B' strain
(Gochenour, 1924) and isolations made from horse blood during subsequent passages
(Stein et al., 1949) were indistinguishable by serotyping, protein and ribotyping studies
(Johnson et al., 1991; Townsend et al., 1997a). PFGE analysis demonstrated polymorphic
restriction pro®les clearly identifying each isolate, illustrating the value of this technique
in bacterial molecular epidemiology.
Recently, an extensive study by Gunarwardana et al. (1999) con®rmed the standing of
PFGE as the `gold standard' technique for molecular epidemiology, as it was shown that
PFGE was more discriminatory than repetitive extragenic palindromic PCR (REP-PCR)
for the characterisation of avian P. multocida. However, REP-PCR compared favourably
with PFGE classi®cations, and appears to be an extremely useful technique for
laboratories without the specialised PFGE equipment.
2.3.4. PCR ®ngerprinting
Several reports have detailed the use of PCR ®ngerprinting for the differentiation of P.
multocida isolates (Chaslus-Dancla et al., 1996; Zucker et al., 1996; Schuur et al., 1997;
10 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
Townsend et al., 1997b, 1998b, 1999; Hopkins et al., 1998; Gunarwardana et al., 1999).
Arbitrarily primed PCR (AP-PCR) was effective in discriminating P. multocida isolates
from the respiratory tract of pigs (Zucker et al., 1996) and also post-vaccination isolates
from turkeys (Hopkins et al., 1998). However, the use of a radioactive label by Hopkins
et al. (1998) to increase sensitivity may limit the feasibility of these primers in some
laboratories. The 32P-dCTP procedure will enhance detection of ampli®ed fragments in
organisms with a high percentage of cytosine in their genome. This bias will also limit the
sensitivity of A�T-rich amplimers, but this did not seem to affect the level of
discrimination among avian P. multocida.
Repetitive extragenic palindromic PCR (REP-PCR) ®ngerprinting was recently shown
to compare very favourably with PFGE in discrimination of avian and swine P. multocida
isolates, exhibiting a high level of differentiation (Townsend et al., 1997b, 1998b, 1999;
Gunarwardana et al., 1999). Interestingly, P. multocida isolated from outbreaks of fowl
cholera (FC) and HS in Vietnam demonstrated minimal variation, with a single REP
pro®le observed among serotype A:1 and HS-causing serogroup B isolates, respectively
(Townsend et al., 1998b; Gunarwardana et al., 1999). Analysis of P. multocida isolates
from the tonsils of healthy pigs at slaughter (Townsend et al., 1999) exhibited genetic
heterogeneity, with some indication that healthy pigs may act as a reservoir for
FC-associated serotypes.
3. Genetics of P. multocida
3.1. Metabolic genes
There have been surprisingly few metabolic genes cloned or characterised in P.
multocida. Mock et al. (1991) described the cloning and sequencing of a gene encoding
the P. multocida adenylate cyclase. Not surprisingly, it was similar to its E. coli
homologue with the deduced protein sequence indicating N-terminal catalytic and C-
terminal regulatory domains. The gene could express adenylate cyclase activity in E. coli
and was subject to similar regulatory mechanisms.
The beta-subunit of the P. multocida tryptophan synthase was cloned, with sequence
analysis indicating high levels of similarity to homologues from other Gram negative
bacteria (Jablonski et al., 1996). The trpB gene encodes a protein of 43.6 kDa that
contains the GGGSNA motif involved in binding to pyridoxal phosphate. The P.
multocida trpB was able to complement a de®ned E. coli mutant, thus con®rming
functional expression of the gene in E. coli. The gene encoding the alpha-subunit, trpA,
was located adjacent to trpB.
The galE gene of P. multocida could complement a galE mutant of Salmonella
(Fernandez de Henestrosa et al., 1997), while the GalE protein was most closely related
to its H. in¯uenzae homologue, with which it shared 85% identity. Of particular
importance was the ®nding that a galE mutant of P. multocida constructed by allelic
exchange was attenuated in virulence for mice. A similar mutant complementation
strategy was used to clone the aroA gene of P. multocida and construct an aroA mutant,
which was also attenuated for virulence in mice (Homchampa et al., 1992). Moreover,
M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 11
the mutant could induce immunity against subsequent lethal challenge. A marker-free
aroA mutant was shown to cross protect against heterologous serogroup A strains in
mice (Homchampa et al., 1997) and showed vaccine potential in chickens (Scott et al.,
1999).
Cloning of the ®rA and skp genes of P. multocida was reported by Delamarche et al.
(1995), but no further characterisation was carried out. ®rA encodes a glucosamine
transferase (Dicker and Seetharam, 1991) which appears to be involved in the
biosynthesis of Lipid A, while the function of the skp gene remains uncertain. However,
the skp homologue from H. in¯uenzae was recently shown to elicit protective immunity
against infection in an infant rat model (El-Adhami et al., 1999), suggesting that further
investigation into the role of skp in immunity to pasteurellosis is warranted.
Although no restriction endonucleases have been isolated from P. multocida, a
restriction/modi®cation system has been reported (Hoskins and Lax, 1997) in which
unmodi®ed DNA was cleaved at or near PstI sites. It was not characterised further.
3.2. Outer membrane protein (OMP) genes
The P6 OMP of H. in¯uenzae has been shown to elicit protective immunity in animal
models of infection. Kasten et al. (1995) cloned a gene encoding the P. multocida
homologue of P6 and showed it to be present in all 16 somatic serotypes. However,
immunisation of turkeys with recombinant P6 failed to protect them against subsequent
challenge (Kasten et al., 1997b).
The ompH porin of P. multocida was puri®ed and its N-terminus sequenced
(Chevalier et al., 1993). OmpH is a homologue of the P2 porin of H. in¯uenzae and
a monoclonal antibody against OmpH could passively protect mice against infection
(Vas® Marandi and Mittal, 1997). Luo et al. (1997) cloned the ompH gene and showed
experimentally that ompH had porin activity. Immunisation of chickens with the
recombinant mature length ompH elicited immunity against homologous challenge,
although heterologous protection was not investigated. Subsequent analysis of ompH
from different serotypes showed a high degree of conservation and predicted the presence
of two large external loops. A cyclic synthetic peptide which mimicked the predicted
structure of loop 2 was able to induce partial homologous protection in chickens (Luo
et al., 1999).
The gene encoding the Oma87 OMP was cloned and characterised (Ruffolo and Adler,
1996). Oma87 showed high similarity to the D15 protective outer membrane protein of H.
in¯uenzae (Loosmore et al., 1997) and rabbit antiserum against Oma87 was able to
passively protect mice against infection. Oma87/D15 homologues have been subse-
quently identi®ed in a number of Gram negative species including Neisseria (Manning et
al., 1998) and Shigella (GenBank accession No. AAD23568). The protective epitope(s) of
D15 were localised to the N-terminus of the protein (Yang et al., 1998). Immunisation of
chickens with a serogroup D GST-Oma87 fusion protein containing the N-terminal 25%
of Oma87, failed to protect chickens against challenge with a serogroup A:1 strain
(Harper et al., 1999) despite the >95% sequence identity between Oma87 from the two
strains. Further work is required to determine unequivocally the role of Oma87 in
immunity to pasteurellosis.
12 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
3.3. Heat shock proteins
P. multocida produced increased amounts of several proteins at 428C (Love and
Hirsh, 1994). A 60 kDa protein was subsequently shown to be the GroEL homologue
encoded by the groESL operon (Love et al., 1995). Although the regulation of GroES
and GroEL was not investigated, analysis of the upstream sequences suggests that
P. multocida utilises a s32-based system (Bukau, 1993) to upregulate its heat shock genes.
The 70 kDa protein upregulated at 428C was almost certainly DnaK, but it was not
identi®ed.
3.4. Capsule biosynthetic genes
The entire capsule biosynthetic locus of P. multocida A:1 was cloned and sequenced
(Chung et al., 1998). Analysis revealed a typical Gram negative type of organisation
(Boulnois and Roberts, 1990) in which a central Region 2 encoding sugar biosynthetic
genes and glycosyl transferases is bounded by two regions (1 and 3) which encode
transport functions. The four Region 1 genes were homologues of the H. in¯uenzae
bexABCD genes which encode proteins involved in exporting the polysaccharide capsule
to the bacterial cell surface, including the principle ABC transporter BexA (designated
HexA in P. multocida A:1). The two Region 3 genes (phyAB) appeared to be involved in
phospholipid substitution to anchor the capsule in the outer membrane. The ®ve genes
comprising Region 2 encode enzymes for the synthesis and assembly of the serogroup A
hyaluronic acid capsule. One of the genes, hyaD, was identi®ed as the hyaluronic acid
synthase gene reported by DeAngelis et al. (1998).
In contrast, the organisation of the serogroup B capsule locus differed signi®cantly
(Boyce et al., 1999). The four Region 1 genes (cexABCD) together with phyA constitute
Region 1, while the single phyB gene makes up Region 3. Of the nine genes in Region 2,
three were similar to sugar biosynthesis or glycosyl transferase genes. However, the
deduced protein products of the remaining six showed no similarity to any sequences in
the databases. Nevertheless, based on their genetic and transcriptional organisation, it was
suggested that they were involved in capsule biosynthesis. The structure of the serogroup
B capsule remains to be determined. Interestingly, there is a relatively low level of
sequence similarity between the serogroup A and serogroup B homologues, but the genes
¯anking the loci exhibit almost 100% nucleotide sequence identity (Boyce et al., 1999),
suggesting a similar chromosomal location. The cap locus has been mapped in serogroup
A (see below) but not in serogroup B.
3.5. Toxin genes
Strains of P. multocida serogroup D which cause atrophic rhinitis produce a
dermonecrotic toxin (PMT, for P. multocida toxin), which is the principle virulence
factor in atrophic rhinitis. PMT induces localised osteolysis in the nasal turbinates,
primarily through increased osteoclastic bone resorption. Recombinant toxin derivatives
have been used as vaccine candidates (Nielsen et al., 1991; Petersen et al., 1991).
Although toxin related sequences have occasionally been found in other serotypes, the
M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 13
synthesis of PMT is usually restricted to serogroup D. The toxA gene was cloned and
shown to express functional PMT in E. coli (Petersen and Foged, 1989). Sequence
analysis of toxA revealed a gene of 4381 bp encoding a protein of 146.5 kDa as well as
the presence of an upstream negative regulator gene, txaR (Petersen, 1990). Deletion of
txaR resulted in a 10-fold increase in toxin expression. However, a subsequent study
found no effect on toxin production following deletion of txaR (Hoskins and Lax, 1996).
Growth at 308C or under iron replete conditions caused less than a four-fold decrease in
the expression of toxA and these authors suggested that PMT is essentially expressed
constitutively.
4. Bacteriophages in P. multocida
Various researchers have demonstrated the presence of bacteriophages in P. multocida.
11 temperate bacteriophages were isolated from a variety of avian P. multocida strains
(Kirchner and Eisenstark, 1956). Bacteriophages were also isolated from bovine strains of
P. multocida, three of which were demonstrated to be genus and species speci®c (Rifkind
and Pickett, 1954; Gadberry and Miller, 1977). Studies on 21 P. multocida bacteriophages
showed a number with morphological similarity to the P2 and T7 coliphages (Ackermann
and Karaivanov, 1984). A set of 24 bacteriophages was recovered after mitomycin
treatment of mainly atrophic rhinitis isolates. These were subsequently used in a proposed
typing system to discriminate between the toxigenic and non-toxigenic strains of P.
multocida (Nielsen and Rosdahl, 1990), but the scheme has not gained widespread
acceptance.
More recently, some sequence similarity to bacteriophage Mu has been found. Regions
with identity to the Mu sequence appear to be present only in the HS causing serogroup B
strains, and have been used to develop a B:2 speci®c PCR (Brickell et al., 1998). None of
the bacteriophages recovered to date have been studied in suf®cient detail for their use as
tools for the genetic manipulation of P. multocida.
5. Native plasmids of P. multocida
Several groups have undertaken studies to determine the presence of plasmids in
numerous strains of P. multocida and to investigate the correlation between antibiotic
resistance pro®les, virulence attributes and the presence of plasmids. The rate of plasmid
carriage has been shown to vary considerably between different P. multocida collections.
Plasmid carriage in avian isolates from two studies varied from 24 (Price et al., 1993) to
70.7% (Hirsh et al., 1985) of isolates tested. Studies with bovine and porcine strains
by Schwarz et al. (1989) found that 47% of strains carried plasmids. A similar ®gure of
51% of porcine serogroup D isolates carrying plasmids was found by Cote et al. (1991).
P. multocida isolated from rabbits demonstrated the highest plasmid carriage rate of
92% of the 28 isolates tested (Gunther et al., 1991).
P. multocida has been shown to harbour plasmids from 1.3 kb (Diallo et al., 1995) to
ca. 100 kb (Hirsh et al., 1989) in size. However the majority of plasmids identi®ed have
14 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
been between 2 and 6 kb in size. Many phenotypically cryptic plasmids have been
found in P. multocida isolated from avian (Hirsh et al., 1989; Price et al., 1993) and
mammalian hosts (Haghour et al., 1987; Cote et al., 1991; Gunther et al., 1991). A
number of R-plasmids have been identi®ed, which confer various antibiotic resistances.
Most R-plasmids were non-conjugative, but could be readily transferred to other
P. multocida strains and E. coli by transformation. Conjugal transfer of a small non-
conjugative R-plasmid in P. multocida has been reported previously, but this required
the presence of a helper fertility plasmid (Hirsh et al., 1981). Subsequently, Hirsh et al.
(1989) identi®ed a large conjugative R-plasmid that was capable of transferring multiple
antibiotic resistance to E. coli and P. multocida.
Many of the R-plasmids carried resistance to streptomycin (Stm) and sulphonamides
(Sul). Cote et al. (1991) found an R-plasmid carrying the Stm and Sul determinants to be
highly similar to the salmonella plasmid RSF1010. Tetracycline (Tet) resistance was
sometimes present with stm and sul genes on a single R-plasmid or carried on a separate
R-plasmid (Silver et al., 1979; Hirsh et al., 1985). Similar R-plasmids from human and
bovine isolates of P. multocida were found to carry the ROB-1 û-lactamase gene and
confer signi®cant resistance to penicillins (Livrelli et al., 1988; Rosenau et al., 1991).
Kanamycin resistance was found in conjunction with Stm, Sul and Tet resistance on the
large conjugative plasmid of an avian strain (Hirsh et al., 1989) and on an R-plasmid from
serogroup D which also carried Stm, Sul and chloramphenicol resistance genes
(Yamamoto et al., 1990). The production of chloramphenicol acetyltransferases
conferring chloramphenicol resistance in P. multocida has also been shown to be
mediated by R-plasmids of 5.1, 5.5 and 17 kb (Vassort-Bruneau et al., 1996).
Plasmid analysis from complement resistant P. multocida (Lee and Wooley, 1995)
demonstrated a correlation between plasmid carriage in the P. multocida CU strain and an
increase in complement resistance, invasiveness and virulence. However, other reports
have found that plasmids were unrelated to virulence (Gunther et al., 1991; Price et al.,
1993; Diallo et al., 1995).
In most cases the antibiotic resistance genes identi®ed on plasmids were not
characterised further; their presence was deduced on the basis of antibiotic resistance
phenotypes. However, the Tet(B) and Tet(M) classes of tetracycline resistance
determinants were detected in two strains (Chaslus-Dancla et al., 1995) with the authors
suggesting that the genes were chromosomally borne. Hansen et al. (1993) identi®ed a
novel, plasmid-borne tetracycline resistance determinant, designated Tet(H), in an avian
strain of P. multocida. Tet(H) belongs to the ef¯ux-mediated class of tetracycline
resistance determinants. In a subsequent study (Hansen et al., 1996), the tet(H) gene
was detected in the majority of a set of North American isolates of P. multocida and
P. haemolytica. Based on the fact that tet(H) was found located both on the chromosome
as well as on plasmids, it was hypothesised that it might be borne on a transposable
element. This was con®rmed by the discovery of the transposon Tn5706 (Kehrenberg
et al., 1998) which was shown to carry the tet(H) gene with its regulator tetR.
Interestingly, its 228 amino acid transposase enzyme was more closely related to
transposases found in Gram positive bacteria. Other transposons have been used for
insertional mutagenesis described below, but Tn5706 remains the only P. multocida
transposable element identi®ed to date.
M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 15
6. Vectors for genetic manipulation
Some native P. multocida plasmids have been modi®ed for use as cloning vehicles.
Vectors such as pBAC64 (Bills et al., 1993), pAKA16 (Azad et al., 1994) and their
derivatives and pIG112 (Wright et al., 1997) have been engineered largely for use as
shuttle vectors between E. coli, P. multocida and other members of the Haemophilus,
Actinobacillus and Pasteurella (HAP) group. Other shuttle vectors based on the broad
host range plasmid RSF1010, have also been used in P. multocida (Lee and Henk, 1997).
Tools such as these provide a useful resource for research into the molecular aspects of
pathogenesis.
The conjugative plasmids, pJM703.1 and pGP704 are unable to replicate in host strains
lacking the pir gene product, and as such have been used as suicide vectors in other
bacteria (Miller and Mekalanos, 1988; Herrero et al., 1990). However, when introduced
into serotype A:1 (PBA100) or D:12 (PM25) P. multocida strains, respectively, these
vectors failed to act as suicide replicons (Homchampa et al., 1992; Fernandez de
Henestrosa et al., 1997). These ®ndings suggest that a lambda pir homologue may be
present on the chromosome of P. multocida. Both groups found that these plasmids could
be lost from P. multocida by culturing for 60 to 100 generations without selection.
Alternative suicide vectors that may be useful in P. multocida are those carrying ColE1
replication origins (Nnalue and Stocker, 1989; Azad et al., 1994) or temperature sensitive
derivatives of RSF1010.
7. Mutagenesis of P. multocida
7.1. Transposon mutagenesis
Transposon mutagenesis has the advantage of generating large numbers of random
mutants that can then be tested for any alteration of their virulence phenotype.
Several transposons have been examined in P. multocida with varying degrees of
success. Nnalue and Stocker (1989) found that while Tn5 did not transpose in
P. multocida, transposition of Tn7 was successful. However, Tn7 was found to
insert into a single site in the P. multocida genome, and as such was not practical
for use in the generation of random mutants (Nnalue, 1990). Two transposons of
potential use in P. multocida as tools for mutagenesis are Tn10 and Tn916. Prior to
their use in avian strains of P. multocida, these transposons were shown to be useful
in Actinobacillus and Haemophilus, respectively (Kauc and Goodgal, 1989; Tascon
et al., 1993).
Using a mini-Tn10 vector and delivery by conjugation, insertion of Tn10 into the
P. multocida genome was shown to be random and stable by Lee and Henk (1996).
More recently, Tn916 has been shown to insert in a quasi-random fashion throughout
the genome of P-1059, a serotype A:3 strain (DeAngelis, 1998). Mutants in a region
related to capsule production were thus identi®ed. By sequencing the P. multocida
DNA ¯anking the transposon in one such mutant, DeAngelis (1998) was able to identify
the P. multocida hyaluronan synthase gene. Both authors have foreshadowed the
16 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
application of these transposons for the identi®cation of genes involved in virulence and
pathogenesis.
7.2. Chemical mutagenesis
Agents such as N-methyl-N-nitro-N-nitrosoguanidine and acridinium salts have been
employed by a number of groups attempting to generate P. multocida mutants that are
suf®ciently attenuated for use as live vaccines (Wei and Carter, 1978; Kucera et al.,
1981). Attenuated strains, some displaying temperature sensitivity or streptomycin
dependence, have been isolated and have shown varying degrees of success as live
vaccines (Chengappa et al., 1979; Hofacre et al., 1989). The genetic basis of attenuation
in these mutants is not de®ned and some of these empirically derived vaccine strains have
been implicated in outbreaks of fowl cholera, suggesting a reversion to virulence and
indicating the instability of the mutant phenotype (Hofacre and Glisson, 1986).
7.3. Targeted mutagenesis
A targeted or rational approach to mutagenesis has also been applied for the study of
pathogenesis in P. multocida. A speci®c gene is identi®ed and cloned and then inactivated
either by a deletion of a portion of the gene or by the insertion of a cassette often
encoding antibiotic resistance. This inactivated gene construct is then delivered to the
wildtype organism and reintroduced into the genome via allelic replacement. Isolates
carrying the mutated construct are then selected by virtue of an altered phenotype such as
the resistance to an antibiotic and subsequent genetic analysis to con®rm the mutant
genotypic pro®le.
The aroA and galE metabolic genes have been targets for mutation in P. multocida
using this approach. The aroA mutants PMP1 and PMP3 were derived from the strains
X-73 and P-1059, respectively. They have been shown to be signi®cantly attenuated and
studies illustrate their potential for use as stable, non-reverting, live vaccines against
pasteurellosis (Homchampa et al., 1997). The safety and ef®cacy of PMP1 and PMP3 as
vaccines for use in chickens was recently demonstrated (Scott et al., 1999). High doses of
the live attenuated mutants were administered and provided solid cross-protective
immunity against a serotype 4 strain (PM206). No vaccine associated clinical signs or
lesions were detected and neither mutant strain could be reisolated �24 h after
vaccination. The authors have indicated further work is currently being undertaken to
assess the safety of these live vaccine strains in immunosuppressed birds and to determine
the longevity and extent of heterologous protection provided (Scott et al., 1999).
The galE mutant generated by Fernandez de Henestrosa et al. (1997) was constructed
using a system similar to that of the aroA allelic exchange. An inactivated construct was
introduced using a conjugative plasmid which failed to suicide in the P. multocida D:12
recipient strain. The plasmid was then cured by passaging without antibiotics. The galE
mutant showed reduced virulence when tested in the mouse model. However, before this
strain could be considered as a live attenuated vaccine candidate the authors suggest that
additional mutations in other loci are required to ensure its safety (Fernandez de
Henestrosa et al., 1997).
M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 17
8. The genome of P. multocida
8.1. Genome mapping and organisation
A physical and genetic map of an Australian serotype A:1 fowl cholera isolate
(PBA100) was generated by Hunt et al. (1998) using the restriction enzymes ApaI, NotI
and CeuI. This study found the chromosome to be a 2.35 Mb single circular molecule,
with no extrachromosomal elements. Similar sized genomes within the Pasteurellaceae
family have been observed in H. in¯uenzae, 1.8 Mb, (Fleischmann et al., 1995) and
Actinobacillus pleuropneumoniae with sizes ranging between 2.3 and 2.4 Mb (Chevallier
et al., 1998; Oswald et al., 1999). The genetic organisation of P. multocida A:1 strain
PBA100 appeared to have many features common to other bacterial species. A putative
location for the origin of replication was suggested and various genes involved in
transcription were also located in the surrounding regions. Several genes involved in
virulence and immunity in P. multocida and other bacterial pathogens were localised onto
the physical map, such as the type IV ®mbrial subunit gene ptfA, capsule biosynthesis
genes, genes involved in iron acquisition, the skp gene and those encoding outer
membrane porin ompH and the oma87 OMP.
8.2. Organisation of rrn genes
The intron encoded endonuclease CeuI which recognises a 26 bp sequence found
exclusively in the 23S rRNA gene (rrl) (Liu et al., 1993; Toda and Itaya, 1995) was used
to determine the number of rrl genes present in P. multocida. Hunt et al. (1998) found the
rRNA genes of P. multocida to exist as ®ve operons with the gene order rrs-rrl-rrf similar
to that of many other eubacteria. The rrn operons were arranged in two groups being
transcribed divergently away from the oriC region. Single ApaI and CeuI sites were found
in all the rrs and rrl genes, respectively. These restriction enzymes may prove useful for
tracking the number and arrangement of rRNA genes in other P. multocida isolates.
8.3. Genomic diversity
Comparison of rare restriction sites and marker genes between genomic maps of
different species or strains provides one measure of genetic variation at the molecular
level. As no other P. multocida genomic maps are currently available, the genetic map of
P. multocida was compared to that of H. in¯uenzae Rd (Fleischmann et al., 1995). Short
regions of gene order conservation, but no long range co-linearity of gene order, were
found. The genome of A. pleuropneumoniae has recently been mapped (Oswald et al.,
1999). However a comparison to the P. multocida map was not possible due to the low
resolution of these genetic maps.
Genomic heterogeneity within P. multocida has long been recognised from the many
methods used for molecular epidemiology discussed earlier. Avian strains in particular
have been found to exhibit considerable diversity (Blackall et al., 1998). PFGE restriction
pro®le differences have been observed between both ApaI, NotI and CeuI genomic
digests of avian isolates (Hunt et al., 1998; Gunarwardana et al., 1999) As CeuI cleaves
18 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25
only in the rrl gene of eubacteria, differences in CeuI restriction pro®les indicate
alterations in the rrn backbone of the chromosome between these strains. Genomic
diversity may have arisen through recombination events such as insertions, deletions,
inversions and duplications or may be due to the large number of separate serotypes being
grouped together in the species. Alternatively the restriction polymorphism seen may be
due merely to point mutations in the rare restriction sites indicating an alteration in the
physical map but no signi®cant alteration of the gene order.
The generation of genetic maps of other P. multocida strains and serotypes to compare
the organisation may help to determine if these restriction polymorphisms relate to a
distinct association between the genome architecture and properties such as virulence,
host speci®city and other metabolic or pathogenic phenotypic aspects.
8.4. Genome sequencing
Sequencing of the P. multocida genome has been undertaken by the Advanced Genetic
Analysis Centre at the University of Minnesota, (http://www.cbc.umn.edu/ResearchPro-
ject/AGAC/Pm/index.html). A valuable database is being constructed from the random
sequencing of an A:3,4 turkey strain. Knowing the sequence of a bacterial pathogen's
genome provides much information about the unique biology of the organism. However,
the genome sequence per se will provide only limited information about the role of
individual genes in pathogenesis. The challenge will be to determine which of the 2000 or
so genes being uncovered by the sequencing project are distinctly involved in virulence,
pathogenesis, host predilection or in the induction of a protective immune response.
Indeed, a number of molecular tools such as transposon mutagenesis discussed above and
microarray (gene chip) technology will be critical in the next wave of P. multocida
research and will elucidate the role of many genes.
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