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DIAGNOSTICS, TYPING AND IDENTIFICATION |
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK
Correspondence Robert L. Davies r.l.davies{at}bio.gla.ac.uk
Received5 July 2002 Accepted15 September 2002
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Abstract |
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 TOP Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Introduction |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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P. multocida is also frequently isolated from the lungs of pneumonic pigs and is thought to play a central role in porcine pneumonic pasteurellosis (Pijoan et al., 1983, 1984; Pijoan, 1992; Rubies et al., 2002; Zhao et al., 1992). Prior infection with other micro-organisms such as pseudorabies virus and Mycoplasma hyopneumoniae predisposes pigs to secondary infection with P. multocida (Ciprian et al., 1988; Fuentes & Pijoan, 1987). In contrast to isolates associated with PAR, P. multocida strains isolated from pneumonic lungs are usually non-toxigenic and of capsular type A (Pijoan et al., 1983, 1984; Rubies et al., 2002; Zhao et al., 1992). However, a small proportion of lung isolates are toxigenic and/or possess capsular type D (Pijoan et al., 1984; Rubies et al., 2002; Choi et al., 2001).
Despite the obvious differences between pneumonia and PAR and their association with P. multocida strains of different capsular types and toxin status, very little is known about the relationships of isolates responsible for each of these diseases (Djordjevic et al., 1998). A number of different methods such as bacteriophage typing, plasmid profiling, restriction endonuclease analysis, ribotyping and analysis of outer-membrane proteins (OMPs) have been used to examine diversity among P. multocida strains associated with either PAR (Gardner et al., 1994; Fussing et al., 1999; Harel et al., 1990; Donnio et al., 1999; Nielsen & Rosdahl, 1990; Lugtenberg et al., 1984; Vasfi Marandi & Mittal, 1995; Bowles et al., 2000) or pneumonia (Rubies et al., 2002; Zhao et al., 1992; Blackall et al., 2000). However, there have been very few comparative studies of P. multocida isolates derived from both PAR and pneumonia (Djordjevic et al., 1998).
The OMPs of Gram-negative bacteria play essential roles in host–pathogen interactions and in disease processes (Lin et al., 2002). These proteins are at the interface between pathogen and host and are subject to various selective pressures depending on their function. Consequently, OMPs exhibit varying degrees of inter-strain heterogeneity and this can be used to assess intra-species diversity and determine epidemiological relationships. The heat-modifiable and porin proteins are important classes of OMPs that are surface-exposed and exhibit molecular mass and antigenic variation (Sikkema & Murphy, 1992; Duim et al., 1997). P. multocida expresses heat-modifiable (OmpA) and porin (OmpH) proteins on the cell surface (Lugtenberg et al., 1984; Vasfi Marandi & Mittal, 1996, 1997; Luo et al., 1997, 1999; Vasfi Marandi et al., 1996), but very little is known about the precise roles of these proteins in pathogenesis. Heterogeneity of the OmpH protein in somatic serotype strains of P. multocida is due to the presence of hypervariable surface-exposed loop regions (Luo et al., 1999). In addition, the OmpH protein provides protection against P. multocida challenge in mice (Vasfi Marandi & Mittal, 1997) and chickens (Luo et al., 1999) and has potential as a vaccine candidate. However, very little is known about OmpA and OmpH heterogeneity among porcine P. multocida strains associated with PAR and pneumonia.
The aim of the present study was to characterize porcine strains of P. multocida recovered from cases of pneumonia and PAR by comparative analysis of their capsular types, toxA status and OMP profiles. In particular, molecular mass heterogeneity of the OmpA and OmpH proteins was examined and used as the basis of an OMP classification scheme to assess, in conjunction with toxA status and capsular type, inter-strain relatedness among porcine P. multocida isolates.
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METHODS |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The isolates were stored at -85 °C in 50 % (v/v) glycerol in brain heart infusion broth (BHIB). From -85 °C stock cultures, bacteria were streaked onto blood agar [brain heart infusion agar containing 5 % (v/v) defibrinated sheep blood] and incubated overnight at 37 °C. For preparation of DNA, a few colonies were inoculated into 10 ml volumes of BHIB and grown overnight at 37 °C at 120 r.p.m. For preparation of outer membranes, 0.4 ml of overnight growth in BHIB was inoculated into 400 ml volumes of BHIB in 2 l Erlenmeyer flasks and incubated for 7 h at 37 °C at 120 r.p.m.
Preparation of chromosomal DNA.
Cells from 1.0 ml overnight culture were harvested by centrifugation for 1 min at 13 000 g and washed once in sterile, distilled water. DNA was prepared with the InstaGene matrix (Bio-Rad) according to the manufacturer's instructions and stored at -20 °C.
Capsular PCR typing.
The capsular types were determined by multiplex capsular PCR typing (Townsend et al., 2001). Capsule-specific primers (CAPA, CAPB, CAPD, CAPE and CAPF) were synthesized by Sigma-GenoSys (Cambridge, UK) and the capsular gene fragments were amplified with a Taq DNA polymerase kit (Boehringer Mannheim) according to the manufacturer's instructions. PCRs were carried out in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) using the following amplification parameters: denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 1 min. Thirty cycles were performed and a final elongation step of 72 °C for 10 min was used. Production of PCR amplicons of the expected sizes was confirmed by electrophoresis and ethidium bromide staining in 2 % agarose gels. Pooled PCR amplicons of serotype A, B, D, E and F reference strains were used as internal size standards in each gel. Strains that were negative for all five capsular types were confirmed as being P. multocida in separate PCR assays with a P. multocida-specific primer set (Townsend et al., 2001) and classified as untypable.
PCR detection of the toxA gene.
Detection of the toxA gene was carried out by PCR as described previously (Lichtensteiger et al., 1996) with the exception that different oligonucleotide primers were used. The primers were designed after alignment and comparison of published toxA sequences (Lax et al., 1990; Buys et al., 1990; Petersen, 1990). The oligonucleotide primers were designed to amplify a 1854 bp fragment of toxA between nucleotides 2190 and 4043 (Petersen, 1990); the forward primer was 5'-CGTGAACTGCGTACTCAA-3' and the reverse primer was 5'-AAGAGGAGGCATGAAGAG-3'. PCR amplification of the toxA gene fragment was carried out as described above for capsular PCR typing except that an annealing temperature of 56 °C for 30 s was used. Production of PCR amplicons of the expected size was confirmed by electrophoresis and ethidium bromide staining in 1 % agarose gels. A 1 kb DNA ladder (Gibco Life Technologies) was used to size the fragments. The PCRs were carried out twice.
Preparation of OMPs.
Outer membranes were prepared by Sarkosyl extraction as described previously (Davies et al., 1992; Davies & Donachie, 1996). The protein concentrations were determined by the modified Lowry procedure (Markwell et al., 1978) and the OMPs were adjusted to 2.0 mg ml-1 in 20 mM Tris/HCl (pH 7.2) and stored at -85 °C.
SDS-PAGE.
The OMPs were separated by SDS-PAGE in 12 % (w/v) resolving gels (Hoefer SE600 electrophoresis apparatus) using the SDS discontinuous system of Laemmli (1970) as described previously (Davies et al., 1992; Davies & Donachie, 1996). Unless otherwise stated, all samples were heated at 100 °C for 5 min prior to electrophoresis. Twenty micrograms of protein were loaded per lane and the proteins were visualized by staining with Coomassie brilliant blue. Protein molecular mass standards (Pharmacia) consisted of phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and 
-lactalbumin (14.4 kDa). The molecular masses of individual proteins were calculated with the Labworks image acquisition and analysis computer software.
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RESULTS |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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PCR detection of the toxA gene
Successful amplification of the toxA gene resulted in a PCR fragment of the expected size (1854 bp) (Fig. 1). The toxA gene was detected in 14 (9 %) isolates. However, toxA was associated exclusively with four groups of strains that could be distinguished by their OMP and capsular types (Table 1).
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Analysis of OMP profiles
The stability of the OMP profiles was examined by comparing the profiles of two strains after repeated subculture and at different stages of the growth cycle. The profiles of these strains were identical after 5, 10, 15 and 20 rounds of subculture on blood agar and after 6, 8, 12 and 24 h of growth in BHIB (results not shown). The OMP profiles of the 158 strains were provisionally assigned to OMP-types based on profile similarity in SDS-PAGE gels (described below). Strains assigned to the same OMP-type were subsequently rerun on up to three or four occasions so that strains of the same OMP-type were compared directly on the same gel. An OMP classification scheme was devised based, firstly, on molecular mass variation of the two major proteins, OmpA and OmpH (OMP-type 1, 2, etc.), and, secondly, on variation of minor OMPs (OMP-type 1.1, 1.2, etc.).
The OmpA and OmpH proteins have overlapping molecular mass ranges (33–38 kDa) and were distinguished on the basis of their different behaviours in SDS-PAGE gels after heat treatment. The OmpH porin protein is tightly associated with peptidoglycan and does not migrate into the gel unless heated at a temperature of approximately 60 °C or higher prior to SDS-PAGE (Rosenbusch, 1974). Conversely, the OmpA protein is not associated with peptidoglycan and migrates freely into the gel after heat treatment at temperatures below 60 °C. However, the OmpA protein undergoes a characteristic conformational change when heated at 100 °C that results in an increase in its apparent molecular mass (Beher et al., 1980). Therefore, to identify OmpA and OmpH, one strain representing each OMP-type was subjected to heat treatment at 50, 60, 70, 80, 90 and 100 °C for 5 min prior to SDS-PAGE. The results for two strains of OMP-types 4.1 and 5.1 are shown in Fig. 2.
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The OMP profiles of the 158 isolates were typical of Gram-negative bacteria and consisted of two major proteins, OmpA and OmpH, and 20–30 minor proteins. The strains could be subdivided into six distinct groups that were classified as OMP-types 1–6 based on variation of OmpA and OmpH (described above). Strains of OMP-types 1, 3, 4 and 6 were further subdivided into OMP-types 1.1 and 1.2, 3.1 and 3.2, 4.1 and 4.2 and 6.1 and 6.2 based on variation of minor proteins. Profiles representing the most abundant OMP-types, namely 1.1, 2.1, 3.1, 4.1, 5.1 and 6.1, are shown in Fig. 3. The molecular mass of OmpA varied from 36.5 to 37.7 kDa, whereas that of OmpH varied from 33.5 to 38.1 kDa. The distribution of OMP-types among the porcine strains is shown in Table 1. The majority (82 %) of strains were represented by just four OMP-types, 1.1 (39 %), 2.1 (8 %), 3.1 (15 %) and 6.1 (20 %), whereas the remaining 18 % of strains were associated with six OMP-types.
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Relationship between OMP-types, capsular types, toxA status and clinical symptoms
Strains representing OMP-types 1.1, 2.1, 3.1 and 5.1 were associated exclusively with capsular type A and comprised 67 % of the total number of isolates. None of these isolates possessed the toxA gene. Eighty-nine per cent of the strains representing these four OMP-types were isolated from the lungs of pneumonic pigs. Two isolates were associated with PAR and the remainder with miscellaneous symptoms. Strains of OMP-types 1.2 and 3.2 accounted for only 5 % of the isolates but were respectively associated with capsular types A and D and A and F. None of these isolates possessed the toxA gene and they were also recovered mainly from cases of pneumonia.
Strains representing OMP-types 4.1 and 4.2 were associated with capsular types A and D, although two isolates of OMP-type 4.1 were classified as untypable. Isolates representing these two OMP-types accounted for only 6 % of the total number of strains. All four of the capsular type D strains of OMP-type 4.1 possessed the toxA gene and two of the isolates were associated with suspected PAR. The single capsular type D strain of OMP-type 4.2 was also associated with PAR, but it lacked the toxA gene.
Strains of OMP-type 6.1 represented 20 % of the total number of isolates. Twenty-eight OMP-type 6.1 isolates possessed capsular type D and four strains were of capsular type A. The four capsular type A isolates and five of the capsular type D isolates possessed the toxA gene. The five capsular type D, toxA+ strains and two of the capsular type A, toxA+ strains were associated with PAR. In contrast, the majority (70 %) of the capsular type D, toxA- strains were associated with pneumonia. There were only two isolates representing OMP-type 6.2, one each of capsular types A and D. The capsular type A strain possessed the toxA gene, whereas the capsular type D isolate did not.
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DISCUSSION |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Genetic diversity of porcine strains of P. multocida has been examined in previous studies by restriction endonuclease analysis and ribotyping (Gardner et al., 1994; Fussing et al., 1999; Rubies et al., 2002; Zhao et al., 1992; Djordjevic et al., 1998; Harel et al., 1990; Donnio et al., 1999; Blackall et al., 2000). A comparative analysis of OMP variation in a large population of P. multocida strains associated with pneumonia and PAR has not been undertaken. The overall diversity of the OMP profiles was relatively low, since 82 % of the isolates were represented by only four OMP-types, 1.1, 2.1, 3.1 and 6.1. The remaining 18 % of the strains were associated with just six OMP-types. The relatively low degree of diversity of the OMP profiles was not unexpected, because porcine P. multocida strains have previously been shown to have limited genetic diversity (Gardner et al., 1994; Fussing et al., 1999; Rubies et al., 2002; Zhao et al., 1992; Harel et al., 1990; Bowles et al., 2000; Blackall et al., 2000). The association of a small number of OMP-types with the majority of the strains suggests that a relatively small number of virulent groups (or clones) are responsible for most cases of infection (discussed in further detail below). These findings are in contrast to the high degree of diversity observed in the OMP profiles of avian isolates of P. multocida (Davies et al., 2003).
The OmpA and OmpH proteins both exhibited molecular mass heterogeneity (Fig. 3), although the molecular mass range of OmpH (33.5–38.1 kDa) was greater than that of OmpA (36.5–37.7 kDa). The shift in molecular mass of OmpH was most pronounced in OMP-types 6.1 and 6.2 (33.5 kDa). Comparative analysis of the deduced amino acid sequences of the OmpH protein in somatic serotype strains of P. multocida has indicated that molecular mass heterogeneity is due to variation in the number of amino acids in two discrete hypervariable regions that are thought to correspond to external surface-exposed loops (Luo et al., 1999). Molecular mass heterogeneity also occurs in the corresponding P2 (OmpH) and P5 (OmpA) proteins of Haemophilus influenzae and is also due to differences in the size of hypervariable surface-exposed loop regions (Sikkema & Murphy, 1992; Duim et al., 1997). These surface-exposed loops are thought to interact with the host immune system and, by undergoing antigenic variation, provide the bacterium with an important defence mechanism (Yi & Murphy, 1997; Neary et al., 2001). The observed heterogeneity of the OmpA and OmpH proteins of porcine P. multocida strains might also be due to variation in the size of hypervariable surface-exposed loop regions and might play important roles in the pathogenesis of pneumonic pasteurellosis and PAR.
Eighteen groups of strains were identified based on specific combinations of OMP-type, capsular type and toxA status (Table 1). Strains representing each of these groups have most likely been derived from a common ancestral cell and are considered here to belong to the same clonal group. Preliminary sequence data from three housekeeping genes (adk, g6pd and mdh) confirm the clonal nature of porcine P. multocida strains (unpublished data). Avian P. multocida strains have been shown to have a clonal population structure by multilocus enzyme electrophoresis (Blackall et al., 1998) and the association of specific OMP and lipopolysaccharide types was considered to mark clones of porcine strains of P. multocida (Lugtenberg et al., 1984). Our data provide strong evidence to suggest that different subpopulations (clones) of P. multocida are responsible for pneumonia and PAR in pigs. The majority (75 %) of cases of pneumonia were associated exclusively with capsular type A strains of OMP-types 1.1, 2.1, 3.1 and 5.1. Notably, the toxA gene was absent from all of these strains. In addition, a small proportion (13 %) of pneumonia cases were caused by non-toxigenic capsular type D strains of OMP-type 6.1. In contrast, the majority (76 %) of cases of PAR were associated with toxA-containing capsular type D strains of OMP-type 4.1 and capsular type A and D strains of OMP-type 6.1. The association of capsular type D strains of OMP-type 6.1 with PAR or pneumonia was strongly correlated with the presence or absence, respectively, of the toxA gene. A strong correlation between PAR and possession of the toxA gene has been well documented in previous studies (Eamens et al., 1988; Gardner et al., 1994; Lariviere et al., 1992; Fussing et al., 1999; Sakano et al., 1992).
There have been very few studies of the molecular epidemiology of porcine pasteurellosis, which is poorly understood (Zhao et al., 1992; Blackall et al., 2000). Predominant restriction endonuclease analysis patterns or ribotypes have been described among P. multocida strains associated with pneumonia (Rubies et al., 2002; Zhao et al., 1992; Bowles et al., 2000; Blackall et al., 2000) and PAR (Gardner et al., 1994; Fussing et al., 1999) but, in some cases, these may be associated with the nature of pig production (Rubies et al., 2002; Bowles et al., 2000; Blackall et al., 2000). A characteristic feature of most pathogenic bacteria is that the majority of cases of infectious disease are normally caused by a small number of clones (Selander & Musser, 1990). Conversely, opportunistic infections are often associated with non-pathogenic strains that have a high level of diversity (Whittam, 1995; White et al., 1990). Pneumonic pasteurellosis is often considered to be a secondary infection, which follows initial infection with micro-organisms such as pseudorabies virus and M. hyopneumoniae (Ciprian et al., 1988; Fuentes & Pijoan, 1987). Djordjevic et al. (1998) suggested that pneumonic P. multocida isolates are opportunistic pathogens of low virulence, although these authors based their conclusion on a study of only 10 strains isolated from lung lesions. In the present study, the majority (88 %) of cases of porcine pneumonia were associated with non-toxigenic capsular type A strains of OMP-types 1.1, 2.1, 3.1 and 5.1 and non-toxigenic capsular type D strains of OMP-type 6.1. In particular, 57 (46 %) strains recovered from cases of pneumonia were of OMP-type 1.1. Furthermore, the strains studied in this investigation were isolated from widespread geographical locations in England and Wales over a 12-year period and represented mostly single sporadic cases.
We suggest two hypotheses to account for the limited diversity among P. multocida strains associated with porcine pneumonia in England and Wales. Firstly, the diversity of commensal strains occupying the nasopharynx of healthy pigs is relatively low. Strains isolated from diseased animals are opportunistic pathogens that have low diversity because they represent the normal background flora of the porcine nasopharynx. Secondly, the diversity of commensal strains occupying the nasopharynx of healthy pigs is relatively high. In this case, strains isolated from infected animals have low diversity because they represent a small number of virulent clones that are capable of causing disease under certain circumstances. The second scenario implies that P. multocida is not a secondary or opportunistic pathogen, as is generally considered, but instead has a primary role in the pathogenesis of porcine pneumonia. Examination of these hypotheses will require further characterization of P. multocida isolates representing the normal flora of the porcine nasopharynx.
The toxA gene was not distributed randomly among P. multocida strains, but was associated exclusively with capsular type A strains of OMP-types 6.1 and 6.2 and capsular type D strains of OMP-types 4.1 and 6.1 (Table 1). These findings support the view that distinct combinations of capsular types, toxA status and OMP-types represent clones of P. multocida. Toxigenic strains of capsular type A are relatively uncommon, although they are sometimes associated with outbreaks of PAR (Sakano et al., 1992) and pneumonia (Fussing et al., 1999; Choi et al., 2001). Our data show that toxigenic capsular type A strains represent two closely related but distinct clonal groups of P. multocida that are characterized by OMP-types 6.1 and 6.2. Clearly, toxigenic capsular type A strains of OMP-types 6.1 and 6.2 represent a subpopulation of P. multocida that is distinct from the non-toxigenic capsular type A strains of OMP-types 1.1, 2.1, 3.1 and 5.1 associated mainly with pneumonia (Table 1).
Strains of OMP-types 1.1, 2.1, 3.1 and 5.1 were associated only with capsular type A, whereas isolates of OMP-types 1.2, 4.1, 4.2, 6.1 and 6.2 were associated with capsular types A and D (Table 1). The most likely explanation for these observations is that capsular switching, possibly involving horizontal transfer of capsular biosynthesis genes, has occurred among strains of the latter, but not the former, OMP-types (Musser et al., 1988). Toxigenic capsular type D strains of OMP-type 6.1 may also have been derived from non-toxigenic capsular type D strains of OMP-type 6.1 by horizontal transfer of the toxA gene. The association of toxA with capsular type D in the majority of the toxA-containing strains also suggests that the toxA gene is in linkage disequilibrium with the type D capsular gene cluster. In particular, the existence of toxA-containing capsular type D strains of OMP-type 4.1 suggests that both the toxA and type D capsular genes may have been acquired by horizontal gene transfer in these isolates. The absence of both toxA and the type D capsule in pneumonic strains of OMP-types 1.1, 2.1, 3.1 and 5.1 (which comprise 82 % of the isolates) suggests that these isolates are not susceptible to the horizontal transfer of these genes. Therefore, our data suggest that horizontal transfer of the toxA and capsular biosynthesis genes is restricted mainly to specific subpopulations of P. multocida represented by OMP-types 4.1 and 4.2 and 6.1 and 6.2. It is interesting to speculate that strains representing these OMP-types have fundamental differences in their biology, with respect to horizontal transfer of the toxA and capsular genes, in comparison with isolates of OMP-types 1.1, 2.1, 3.1 and 5.1. One explanation that may account for these observations is that these two groups of strains occupy different ecological niches within the porcine respiratory tract and are effectively isolated from each other.
Gardner et al. (1994) identified toxigenic and non-toxigenic strains among isolates of the same genotype and suggested that lysogenic phage might account for their observations. These authors also identified a single toxigenic, capsular type A strain and suggested that the toxA gene may have been derived from a capsular type D strain. A variety of toxin genes expressed by pathogenic bacteria are carried and transferred by bacteriophages (Cheetham & Katz, 1995) and various lines of evidence suggest that phage-mediated transduction might also represent the mechanism of horizontal transfer of toxA in P. multocida. Nielsen & Rosdahl (1990) isolated 24 different bacteriophages from porcine strains of P. multocida for typing toxigenic and non-toxigenic strains. Flanking sequences of toxA were shown to be homologous with bacteriophages isolated from P. multocida, and induction of toxigenic strains yielded DNA fragments that hybridized with a toxA probe (Andresen et al., 1990). It has also been suggested that phage conversion is responsible for the generation of a particular ribotype that is itself associated with toxin production (Donnio et al., 1999).
In summary, this study has demonstrated that different subpopulations of P. multocida are responsible for pneumonia and PAR in pigs. The association of a small number of clones with porcine pneumonia in England and Wales suggests that these strains are not opportunistic pathogens, but are virulent isolates that have a primary role in the pathogenesis of this disease. The association of both capsular types A and D, and the toxA gene, with a small number of specific OMP-types in strains associated primarily with PAR suggests that horizontal transfer of capsular biosynthesis and toxA genes has occurred among these isolates.
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Acknowledgments |
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Footnotes |
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REFERENCES |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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