Microbiological Research 174 (2015) 1–8

Contents lists available at ScienceDirect

Microbiological Research journal homepage: www.elsevier.com/locate/micres

Distribution of the ompA-types among ruminant and swine pneumonic strains of Pasteurella multocida exhibiting various cap-locus and toxA patterns C. Vougidou a , V. Sandalakis b,c , A. Psaroulaki b , V. Siarkou d , E. Petridou d , L. Ekateriniadou e,∗ a

Institute of Infectious and Parasitic Diseases of Thessaloniki, Ministry of Rural Development and Food, 54627 Thessaloniki, Greece Laboratory of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical Medicine, University of Crete, 71110 Crete, Greece c Regional Laboratory of Public Health (Crete), 71110 Crete, Greece d Laboratory of Microbiology and Infectious Diseases, Veterinary School, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece e Veterinary Research Institute of Thessaloniki, Hellenic Agricultural Organisation-Demeter (NAGREF), Campus of Thermi, 57001 Thessaloniki, Greece b

a r t i c l e

i n f o

Article history: Received 7 November 2014 Received in revised form 4 February 2015 Accepted 15 February 2015 Available online 27 February 2015 Keywords: Pasteurella multocida ompA toxA PCR-RFLP Sequence analysis

a b s t r a c t Pasteurella multocida is an important pathogen in food-producing animals and numerous virulence genes have been identified in an attempt to elucidate the pathogenesis of pasteurellosis. Currently, some of these genes including the capsule biosynthesis genes, the toxA and the OMPs-encoding genes have been suggested as epidemiological markers. However, the number of studies concerning ruminant isolates is limited, while, no attempt has ever been made to investigate the existence of ompA sequence diversity among P. multocida isolates. The aim of the present study was the comparative analysis of 144 P. multocida pneumonic isolates obtained from sheep, goats, cattle and pigs by determining the distribution of the ompA-types in conjunction with the cap-locus and toxA patterns. The ompA genotypes of the isolates were determined using both a PCR-RFLP method and DNA sequence analysis. The most prevalent capsule biosynthesis gene among the isolates was capA (86.1%); a noticeable, however, rate of capD-positive isolates (38.6%) was found among the ovine isolates that had been associated primarily with the capsule type A in the past. Moreover, an unexpectedly high percentage of toxA-positive pneumonic isolates was noticed among small ruminants (93.2% and 85.7% in sheep and goats, respectively), indicating an important epidemiological role of toxigenic P. multocida for these species. Despite their great heterogeneity, certain ompA-genotypes were associated with specific host species, showing evidence of a host preference. The OmpA-based PCR-RFLP method developed proved to be a valuable tool in typing P. multocida strains. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction Pasteurella multocida is one of the most important pathogens in food-producing animals involved in the emergence and final progression of lower respiratory tract (LRT) infections, such as the Bovine Respiratory Disease Complex (BRDC), enzootic pneumonia of swine and pneumonia in sheep, while it plays the primary role in the pathogenesis of progressive atrophic rhinitis (PAR) in pigs, avian cholera, haemorrhagic septicaemia in both domesticated and wild animals and snuffles in rabbits (Wilkie et al., 2012).

∗ Corresponding author. Tel.: +30 2310365392; fax: +30 2310365371. E-mail address: [email protected] (L. Ekateriniadou). http://dx.doi.org/10.1016/j.micres.2015.02.003 0944-5013/© 2015 Elsevier GmbH. All rights reserved.

Numerous P. multocida genes critical for virulence have been identified in an attempt to elucidate the pathogenesis of pneumonic pasteurellosis which remains enigmatic in all host-species (Fuller et al., 2000; Harper et al., 2006; Hunt et al., 2001; May et al., 2001; Wilkie et al., 2012). The importance of capsule as a virulence determinant in the pathogenesis of P. multocida has been established with the construction of genetically defined mutants unable either to synthesize or to export capsule to their surface, hence, being attenuated for virulence (Boyce and Adler, 2000; Chung et al., 2001). Identification of the capsule biosynthesis genes (capA, capB, capD, capE and capF) led to the development of a DNA-based typing system (Boyce et al., 2000; Chung et al., 1998; Townsend et al., 2001), alternative to the traditionally used serological methods (serogroups A, B, D, E and F based on the antigenicity of their polysaccharide capsule) (Carter, 1967; Rimler and Rhoades, 1987).

2

C. Vougidou et al. / Microbiological Research 174 (2015) 1–8

The dermonecrotic P. multocida toxin (PMT), a 146 kDa protein encoded by the toxA gene, is responsible for the clinical and pathological signs of atrophic rhinitis in swine and it is mainly associated with serogroup D strains (Ewers et al., 2006; Harper et al., 2006; Lax and Chanter, 1990). In general, P. multocida strains that cause pneumonia are considered to be non-toxigenic (Harper et al., 2006). However, the toxA gene has been detected at low rates in type A and type D porcine strains (Bethe et al., 2009; Davies et al., 2003a) and at various rates among ruminant strains, the role of which in the pathogenesis of pneumonia remains unclear (Ewers et al., 2006). Outer membrane proteins (OMPs) are surface-exposed and, therefore, subject to diversifying selection within the host resulting in inter-strain heterogeneity; the different OMP profiles are, therefore, useful markers of clonality (Davies et al., 2004). One of the major OMPs of P. multocida is the heat-modifiable outer membrane protein A (OmpA). The protein, a structural homologue of the Escherichia coli OmpA (Carpenter et al., 2007; Khalid et al., 2008), is immunogenic and involved in adhesion, thus playing an important role in host-pathogen interaction (Dabo et al., 2003; Hatfaludi et al., 2010). The ompA gene may experience high selection pressure and undergo more frequent genetic variation than do housekeeping genes. Therefore, ompA typing might prove valuable in epidemiologic investigations, especially for short term tracing of clones (Tang et al., 2010). Currently, some of the virulence genes including the capsule biosynthesis genes, the toxA and the OMPs-encoding genes have been suggested as epidemiological markers, and their distribution among isolates recovered from different sources and/or disease status has become the subject of recent studies (Ewers et al., 2006; Garcia et al., 2011; Tang et al., 2009; Verma et al., 2013). However, the number of studies concerning the virulence gene patterns of ruminant isolates focuses primarily on cattle (Ewers et al., 2006; Verma et al., 2013). Furthermore, to the best of our knowledge, no attempt has been made up to date to investigate the existence of ompA sequence diversity among P. multocida isolates originated from various food-producing animals. The aim of the present study was the comparative analysis of P. multocida pneumonic isolates obtained from sheep, goats and cattle as well as from pigs by determining the distribution of the ompA-types in conjunction with the cap-locus and toxA patterns. The ompA genotypes of the isolates were determined using both a PCR-RFLP method and DNA sequence analysis. The study was undertaken to establish whether specific virulence patterns were associated mainly with a distinct species, potentially revealing some kind of host adaptation.

2. Materials and methods 2.1. Bacterial strains Pneumonic lungs from randomly selected sheep (n = 247), goats (n = 47), cattle (n = 66) and pigs (n = 131) were obtained in 21 slaughterhouses throughout Greece from March 2006 until February 2008. One hundred and forty-four P. multocida isolates (ovine = 44, caprine = 7, bovine = 19 and porcine = 74) were recovered. Moreover, one ovine, two caprine and two bovine isolates recovered from animals that died of pneumonia over the same period (kindly provided by the Regional State Laboratories) were included in the study. Pneumonic lung tissue samples were inoculated onto nonselective 5% sheep blood agar plates and incubated aerobically for 18–24 h at 37 ◦ C. Following subculture onto fresh blood agar, isolates were phenotypically determined to be P. multocida by cultural characteristics and biochemical testing (Gram-negative coccobacilli, non-haemolytic, catalase positive, cytochrome-oxidase positive, indole positive, showing no growth on MacConkey agar)

(Quinn et al., 1994) prior to PCR identification based on the amplification of the P. multocida-specific clone KMT1 (Townsend et al., 2001). PCR amplifications were performed on whole genomic DNA (NucleoSpin® Tissue/Macherey-Nagel). 2.2. Detection and analysis of genes 2.2.1. Capsule biosynthesis genes (capA, B, D, E and F) P. multocida isolates were subjected to multiplex PCR analysis to investigate the presence of cap genes. Besides the capsule-specific primers (CAPA, CAPB, CAPD, CAPE and CAPF), the P. multocidaspecific primer set (KMT1T7/KMT1SP6) was included in the assay to confirm the identification of the isolates at the species level (Townsend et al., 2001). 2.2.2. toxA gene Detection of the toxA gene was carried out based on two previously described PCR protocols. The protocol described by Davies et al. (2003a) was used to amplify a fragment between nucleotides 2190 and 4043 (Petersen, 1990), whereas a fragment of the gene between nucleotides 2096 and 2942 was amplified by the second protocol (Lichtensteiger et al. 1996). Reactions, for both protocols, were carried out in a 25 ␮l volume containing (final concentrations): 1× PCR Buffer (Invitrogen), 2 mM MgCl2 , 1 U Taq DNA polymerase (Invitrogen), each dNTP at a concentration of 200 ␮M and 40 ng of sample DNA. Primers were added at a final concentration of 0.8 ␮M. 2.2.3. ompA gene 2.2.3.1. PCR-RFLP analysis of the ompA gene. Primers PmompaF 5 -CAAGCTGCACCACAACCTAA-3 and PmompaR 5 CGATCGTCAGCTAAACATGC-3 were designed to amplify an ompA gene segment between nucleotides 58 and 1023 (GenBank accession numbers: AY643794, AY643795, AY643796, AY643797, AY643798, AY035341, AY903603 – NCBI, 2009 (http://www.ncbi. nlm.nih.gov/), using the Primer3Plus tool (Untergasser et al., 2007) (http://www.bioinformatics.nl/primer3plus). Reactions were carried out in a 25 ␮l volume containing (final concentrations): 1× PCR Buffer (Invitrogen), 2 mM MgCl2 , 1.25 U Taq DNA polymerase (Invitrogen), 200 ␮M of each deoxynucleoside triphosphate and 40 ng of sample DNA. Primers were added at a final concentration of 0.8 ␮M. DNA amplification was performed in a PTC-200 Peltier Thermal Cycler (MJ Research) using the following parameters: initial denaturation at 95 ◦ C for 10 min and, then, 35 cycles of denaturation at 95 ◦ C for 30 sec, annealing at 58.5 ◦ C for 30 sec and elongation at 72 ◦ C for 1 min, followed by a final elongation at 72 ◦ C for 7 min. The restriction endonucleases EcoRI, EcoRV and DraI (New England Biolabs) were used to analyze the ompA gene. The three endonucleases were selected after analysis of the published ompA sequences with the NEBcutter tool (http://tools.neb.com/ NEBcutter2/index.php) in order to find no, one and two cutters of the target sequence. PCR amplicons were digested with the selected enzymes by standard procedures and according to the manufacturer’s instructions. Restriction fragments were separated by electrophoresis in 2% agarose gels stained with ethidium bromide and in 8% polyacrylamide gels and photographed under UV light by a Kodak gel Doc XR system. 2.2.3.2. Sequence analysis of the ompA gene. Nine hundred sixtyfive (965) bp amplicons of the ompA gene from 94 isolates were obtained using the PCR primers (Lark Technologies Inc.). The retrieved nucleotide and protein sequence data were analyzed using the Sequence Scanner version 1.0 (Applied Biosystems) and the EditSeq and MegAlign modules of the Lasergene Ver.7.1 software (DNASTAR Inc., Madison, WI, USA).

C. Vougidou et al. / Microbiological Research 174 (2015) 1–8

3

Table 1 ompA-types of the P. multocida isolates and information on the polymorphic sites in relation to strains AE004439, AY643795 and CP003022 (NCBI). ompA-type (Allele)

GenBank accession no

No of isolates

No of polymorphic codon sites

Insertions/deletions

Synonymous substitutions

Non-synonymous substitutions

Reference strain (NCBI)a

ompA1 ompA2.1 ompA2.2 ompA3 ompA4 ompA5.1 ompA5.2 ompA5.3 ompA5.4 ompA5.5 ompA5.6 ompA6.1 ompA6.2 ompA6.3 ompA6.4 ompA6.5 ompA7.1 ompA7.2 ompA7.3 ompA7.4 ompA8.1 ompA8.2 ompA9 ompA10.1 ompA10.2 ompA10.3 ompA10.4 ompA11.1 ompA11.2

JQ621915 JQ621930 JQ621926 JQ621910 JQ621923 JQ621905 JQ621914 JQ621932 JQ621908 JQ621929 JQ621909 JQ621911 JQ621925 JQ621913 JQ621931 JQ621918 JQ621924 JQ621922 JQ621919 JQ621928 JQ621917 JQ621916 JQ621920 JQ621921 JQ621927 JQ621906 JQ621912 JQ621904 JQ621907

1 10 1 18 1 4 1 1 1 1 1 3 1 1 1 1 1 1 1 1 3 2 17 5 2 1 1 10 2

35 18 19 1 54 6 7 8 7 7 5 3 2 2 4 6 – 1 1 1 3 2 4 6 5 7 5 4 5

– 1 – triplet ins. 1 – triplet ins. – 3 – triplets ins. – – – – – – – – – – 3 – triplets ins. – – – – – – 2 – triplets del. – – – – – –

23 8 9 – 28 4 4 4 4 4 4 – – 1 1 1 – – – 1 1 – – 1 1 1 1 – –

12 9 9 1 23 2 3 4 3 3 1 3 2 1 3 2 – 1 1 – 3 2 2 5 4 6 4 4 5

AE004439 AY643795 AY643795 AY643795 AY643795 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022 CP003022

a

The best BLAST result.

Additional alignments were performed using the ClustalW2 (Thompson et al., 1994) multiple sequence alignment software (http://www.ebi.ac.uk/Tools/msa/clustalw2). The MEGA software package v6.06 was used to conduct the evolutionary analysis (Tamura et al., 2013). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The dataset was subjected to bootstrap analysis of 1000 replicates (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980) and they are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. The topology of the transmembrane strands of the ␤-barrel OmpA was predicted using the PRED-TMBB web server (http://biophysics.biol.uoa.gr/PRED-TMBB/) (Bagos et al., 2004a, 2004b).

2.2.3.3. Nucleotide sequence accession numbers. The GenBank accession numbers for the ompA sequences obtained in this study are JQ621904–JQ621932.

3. Results and discussion The prevalence of P. multocida varied significantly among the different species studied in this study. While a high prevalence (56.5%) of the isolates was found in pigs, the prevalence was lower in ruminants. The prevalence among the bovine isolates was 28.8%, similar to that of previous studies conducted in pneumonic lungs collected either after slaughter (Vogel et al., 2001) or at necropsy (Fulton et al., 2009). In small ruminants, for which there is no available data up to date, the prevalence was 17.8% and 14.9% for sheep and goats respectively.

3.1. Distribution of capsule biosynthesis genes The most prevalent capsule biosynthesis gene among the 144 P. multocida isolates examined was capA (86.1%) followed by capD (13.2%). Interestingly, a capF-positive isolate was recovered from a goat that died from pneumonia (Table S1). Capsule type F isolates were originally recovered from turkeys (Rimler, 1987) but, since then, they have been obtained from diseased pigs, cattle and sheep (Davies et al., 2004, 2003a) and currently from goat, revealing a potentially important role as pathogenic strains for various host species other than poultry. Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micres. 2015.02.003. In small ruminants, the prevalence of capsular type A isolates was high (61.4% and 71.4% for ovine and caprine isolates, respectively). Remarkably, a noticeable rate of capD-positive isolates (38.6%) was found among the ovine isolates that had been associated primarily with the capsule type A in the past (Blanco-Viera et al., 1995; Davies et al., 2003b; Soriano-Vargas et al., 2012). In agreement with previous studies that have predominantly linked pneumonic strains from swine to the capsule antigen A (Davies et al., 2003a; Fussing et al., 1999; Pors et al., 2011; Prescott et al., 1984; Soriano-Vargas et al., 2012), all the porcine isolates harboured the capA gene (Table S1 in Supplementary material). The vast majority of the bovine isolates (94.7%) also harboured the capA gene (Table S1), consistently with previous studies (Davies et al., 2004; Ewers et al., 2006; Purdy et al., 1997). 3.2. Prevalence of the toxA gene Among the 114 strains examined for the presence of the toxA gene, 49 strains (42.9%) were found positive using both protocols (Table S1). We found a low prevalence of porcine pneumonic isolates harbouring the toxA (2.3%). In general, the action of

4

C. Vougidou et al. / Microbiological Research 174 (2015) 1–8

Fig. 1. Evolutionary analysis showing the relationships among 94 P. multocida isolates based on the genetic diversity of their ompA gene sequences. The phylogenetic tree was constructed using the MEGA v6.06 software package (Tamura et al., 2013).

PMT encoded by the toxA gene has been mainly associated with atrophic rhinitis (Wilkie et al., 2012) and the low rates (0–10%) of toxA-positive isolates that have also been detected among pigs with pneumonia by other studies (Bethe et al., 2009; Davies et al., 2003a; Djordjevic et al., 1998; Garcia et al., 2011; Hariharan et al., 2000; Jamaludin et al., 2005; Tang et al., 2009) seem to confirm this. A low prevalence rate (5.3%) of toxA-positive bovine pneumonic isolates was also found being in consistency with previous studies (Ewers et al., 2006). In contrast, an unexpectedly high percentage of toxA-positive pneumonic isolates was noticed among small ruminants (93.2% and 85.7% in sheep and goats, respectively). These results strengthen the notion that toxigenic strains of P. multocida

may have an important epidemiological role for these species, as Ewers et al. (2006) have suggested for sheep; the authors detected the toxA at a 66.7% prevalence rate among 13 isolates recovered from sheep of unknown, however, disease status. Moreover, given the fact that the strains investigated in the present study originated from pneumonic lungs, the significance of PMT for the induction of pneumonic lesions and the pathogenesis of pneumonia should be further investigated in the future. In relation to the capsular type, the distribution of the toxA gene revealed a clear association to type D strains (85% of which were toxA-positive) in contrast to 39% of toxA-positive type A strains. However, the percentage of type A isolates harbouring the

C. Vougidou et al. / Microbiological Research 174 (2015) 1–8

5

Fig. 2. (A–C) Positions of the substitutions and insertions/deletions shown in the 2D OmpA structure representations. The 2D representations were calculated using the CP003022, AY643795, and AE004439 OmpA amino acid sequences available in the NCBI database. All underlined amino acid positions refer to non-synonymous substitutions. In A there is an insertion between positions 98–99 and a deletion between positions 90–92. Substitutions after the last transmembrane section are not depicted.

toxA gene was considerably higher from what has been previously reported [e.g. 6.3% by Ewers et al. (2006) and approximately 10% by Garcia et al. (2011)], a discrepancy that was due to the large number of toxA-positive isolates from small ruminants; interestingly enough, in the latter, the prevalence of the toxA gene among type A strains was even higher than in type D strains (96.77% and 89.47%, respectively). Finally, the capsule type F caprine isolate did not harbour the toxA gene. In recent publications it has been demonstrated or at least hypothesized that different subpopulations of P. multocida may have been evolved by horizontal transfer of cap genes and/or of toxA or by a conversion of toxA-encoding phages, which could account for the low proportion of toxA detected in this study among bovine and porcine isolates due to simple loss of this gene (Bethe et al., 2009; Davies et al., 2003a). On the other hand, the high prevalence rate of toxA-positive strains among small ruminants may result in a reintroduction of the toxA locus at any time.

3.3. Heterogeneity of the ompA gene Sequence analysis of the ompA gene revealed great heterogeneity among the 94 isolates tested. Twenty nine unique sequences, representing distinct alleles, were identified. However, they were assigned to one of 11 genotypic clusters of allelic variants designated ompA1 to ompA11 types (Table 1; Fig. 1). The variation degree between alleles representing each cluster was very low (less than 2%). ompA3-type was the most prevalent one, consisting of 18 (19.1%) isolates, followed by types ompA9 (17 isolates – 18.1%), ompA11 (12 isolates – 12.8%) and ompA2 (11 isolates – 11.7%). The remaining ompA-types were less common (Table 1; Fig. 1). Based on the BLAST analysis the sequences were separated into three groups as shown in Table 1; the best BLAST matches (GenBank accession numbers: AE004439, AY643795 and CP003022) were chosen as reference ompA sequences. Most of the polymorphic nucleotide sites were detected in the four hypervariable

6

C. Vougidou et al. / Microbiological Research 174 (2015) 1–8

Fig. 3. Partial alignments of the ompA amplicons of the eleven genotypes compared to strain AY643795 (Genbank accession number) with highlighted the cleavage sites for the restriction endonucleases EcoRV (A) and DraI (B). RFLP patterns of the amplicons after digestion with EcoRV (C) and DraI (D). The different profiles (E0, E1, E2 and D1, D2, D3) are noted above the pictures and the restriction fragments (in bp) on the right and left. The numbers below the pictures correspond to the samples that were digested. (C) 1: 100 bp DNA ladder, 2: PPM907C, 2: PPM106P, 4: PPM507Ga, 5: PPM 2307Sa, 6: PPM507P, 6: PPM1607P and 7: M71. (D) 1: 10 bp DNA ladder, 2: PPM107C, 3: PPM206P, 4: M44, 5: PPM5107Sc, 6: 100 bp DNA ladder, 7: PPM207C, 8: PPM207 Pa, 9: PPM4307Sb, 10: 100 bp DNA ladder, 11: PPM307Pe and 12: PPM 307Pst. (E) Physical map that shows the EcoRV (numerals above the line) and DraI (numerals below the line) restriction fragments and cleavage sites (in bp).

domains situated within the surface-exposed loop regions of the eight stranded ␤-barrel structure (Fig. 2). The rest of the protein amino acid sequence revealed a high degree of amino acid sequence conservation. Interestingly, most of the substitutions detected outside the transmembrane domains were non-synonymous, while synonymous substitutions were mostly observed within the transmembrane areas. The 63 sequences presenting higher similarity to the CP003022 reference sequence showed all of their non-synonymous substitutions and insertions/deletions to be located outside the transmembrane domains (Fig. 2A). A greater number of variations were observed in the group of the 30 sequences showing high similarity to the AY643795 reference strain. A closer examination of the sites of interest showed that, as with the previous group, most of the non-synonymous substitutions and insertions/deletions were located outside the transmembrane domains; however, whole loop regions (positions 46 till 55 and 144 till 152) as well as a few amino acids at the

beginning of the transmembrane domain (positions 119, 173, 185 and 197) showed increased variability (Fig. 2B). The only sequence showing similarity to AE004439 presented two non-synonymous substitutions within the transmembrane domain (positions 196, 197) and another two very close to the beginning of the transmembrane chains (positions 175, 187) (Fig. 2C). Two dimensional analyses (2D) of the acquired amino acid sequences showed that in all cases the overall characteristic structure of the OmpA protein was retained (data not shown). Notably, while examining the amino acid substitutions, we observed that, although non-synonymous, the replacing amino acids were also non polar and neutral in charge, like the substituted ones, with the exception of position 197 (Fig. 2C) were Leucine, a non polar neutral amino acid was replaced by Lysine, a polar positively charged amino acid with almost equal but opposite hydropathy indexes (3.8 for L and -3.9 for K). It is not possible to know if these substitutions affect the actual functionality of the protein. Undoubtedly, further research is needed

C. Vougidou et al. / Microbiological Research 174 (2015) 1–8

to enlighten the contribution of the detected OmpA substitutions, especially of those within the transmembrane sections and the ones in the closest proximity. It is worth noting that the polymorphic sites previously described for the OmpA of M. haemolytica were also located in the four hypervariable domains within the surface-exposed loop regions (Davies and Lee, 2004). Furthermore, research on the OmpA of M. haemolytica has indicated that the protein is under strong selective pressure from the host and plays an important role in host-pathogen relationships; in that research it was suggested that there is a host-specific ligand-like function of the surface exposed loops of OmpA which, therefore, need to be different in bovine and ovine strains (Davies and Lee, 2004; Hounsome et al., 2011). 3.4. Relationship between ompA-types, cap-locus and toxA patterns, and host specificity Despite their great heterogeneity, certain ompA-genotypes were associated with specific host species (Fig. 1). Genotypes ompA4, ompA5 and ompA7 comprised the bovine strains, genotypes ompA8-ompA11 the ovine and caprine strains and clusters ompA1-ompA3 the porcine strains. Among the 94 isolates that were analyzed, only one bovine (PMM107C) and two ovine (PMM4307Sa and PMM4307Sb) strains clustered within clusters ompA2 and ompA3, i.e. with the porcine isolates. Finally, three ovine strains (allele ompA6.1) clustered with the bovine strains (Fig. 1). Although a strict host adaptation could not be established, the majority of the genotypic clusters showed evidence of a host preference. It is not possible to know whether this association is due to the existence of host specific epitopes as in M. haemolytica (Davies and Lee, 2004; Hounsome et al., 2011) but, in such a case, ompA typing might prove valuable in epidemiologic investigations, especially for short term tracing of clones. Furthermore, since Omps are highly immunogenic and have a great potential of being used in future vaccines (Dabo et al., 2008), the correlation of certain ompA genotypes with specific hosts could bring one step closer to the development of highly efficacious host specific vaccines. No correlation was observed among the ompA-types, the capsule types and the presence or absence of the toxA gene. In contrast, strains representing cluster ompA9 were associated exclusively with capsular type D and comprised 89.5% of the total number of capsular type D strains (Fig. 1). 3.5. RFLP ompA-profiling Treatment with the restriction endonuclease EcoRV either did not cut (E0) or cut at one (E1) or two (E2) sites (Fig. 3A) producing three different patterns (Fig. 3C), whereas DraI cut at one (D1), two (D2) or three (D3) sites (Fig. 3B) producing three different patterns (Fig. 3D). Treatment with EcoRI did not cut any of the sequences. The observed restriction sites were the same with the predicted ones based on sequence analysis. The combination of the different patterns produced after the treatment with the restriction endonucleases revealed five profiles [I (E0D1), II (E1D2), III (E1D3), IV (E1D1) and V (E2D2)] among the 114 P. multocida isolates examined with this method (Table S1). Profile I (Fig. 3, Table S1) was the predominant one among the bovine (94.7%), the caprine (85.7%) and the ovine (95.5%) isolates, whereas, in swine, profiles II (54.6%) and IV (42.4%) prevailed. A distinct correlation was observed between the RFLP profiles and the ompA genotypes: Profile II was associated with genotype ompA3, profile IV with genotype ompA2 and profile V with ompA1, whereas the predominant in ruminants profile I included the remaining genotypes (ompA4-ompA11) (Table S1). Only two ruminant isolates (bovine strain PMM107C and ovine strain PMM4307Sb) showed profile II and only one (ovine strain

7

PMM4307Sa) showed profile IV. These three ruminant isolates that showed the same PCR-RFLP profiles as porcine isolates (Table S1), also clustered with the porcine strains following sequence analysis (Fig. 1). Finally, the only type F strain as well as the only porcine toxA-positive strain showed unique profiles (III and V, respectively) (Fig. 3, Table S1). These satisfactory results of the PCR-RFLP combined with its easy and rapid application render the method a valuable tool in typing P. multocida strains. 4. Conclusions The unexpectedly high percentage of toxA-positive isolates that was noticed among small ruminants (93.2% and 85.7% in sheep and goats, respectively) strengthens the notion that toxigenic strains of P. multocida may have an important epidemiological for these species. Given the fact that the strains investigated in the present study originated from pneumonic lungs, the role of PMT in the induction of pneumonic lesions and the pathogenesis of pneumonia in small ruminants seems to be of great significance and should be further clarified in the future. Great heterogeneity of the ompA gene was found among the isolates, certain ompA-genotypes, however, were associated with specific host species showing evidence of host preference. Most of the polymorphisms detected outside the transmembrane domains of the OmpA protein were non-synonymous, while synonymous mutations were mostly observed within the transmembrane areas. Since OmpA is involved in adhesion and undergoes strong selective pressure, further investigation is needed to clarify whether these polymorphisms affect the functionality of the protein and if they account for the observed host preference. The PCR-RFLP method developed for the ompA-profiling of the isolates proved to be a valuable tool in typing P. multocida strains. Funding This study was supported by grants from the project “Pythagoras II” [MIS: 97436(5A)], with the title “Phenotypic and genotypic characterization of Pasteurella multocida and Mannheimia haemolytica strains isolated from sheep and goats in Greece”. References Bagos PG, Liakopoulos TD, Spyropoulos IC, Hamodrakas SJ. PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins. Nucleic Acids Res 2004a;32:400–4. Bagos PG, Liakopoulos TD, Spyropoulos IC, Hamodrakas SJ. A Hidden Markov Model method, capable of predicting and discriminating beta-barrel outer membrane proteins. BMC Bioinform 2004b;5:29. Bethe A, Wieler LH, Selbitz HJ, Ewers C. Genetic diversity of porcine Pasteurella multocida strains from the respiratory tract of healthy and diseased swine. Vet Microbiol 2009;139:97–105. Blanco-Viera FJ, Trigo FJ, Jaramillo-Meza L, Aguilar-Romero F. Serotypes of Pasteurella multocida and Pasteurella haemolytica isolated from pneumonic lesions in cattle and sheep from Mexico. Rev Latinoam Microbiol 1995;37:121–6. Boyce JD, Adler B. The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B:2). Infect Immun 2000;68:3463–8. Boyce JD, Chung JY, Adler B. Genetic organisation of the capsule biosynthetic locus of Pasteurella multocida M1404 (B:2). Vet Microbiol 2000;72:121–34. Carpenter T, Khalid S, Sansom MS. A multidomain outer membrane protein from Pasteurella multocida: modelling and simulation studies of PmOmpA. Biochim Biophys Acta 2007;1768:2831–40. Carter GR. Pasteurellosis Pasteurella multocida and Pasteurella haemolytica. Adv Vet Sci 1967;11:321–79. Chung JY, Wilkie I, Boyce JD, Townsend KM, Frost AJ, Ghoddusi M, et al. Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A. Infect Immun 2001;69:2487–92. Chung JY, Zhang Y, Adler B. The capsule biosynthetic locus of Pasteurella multocida A:1. FEMS Microbiol Lett 1998;166:289–96. Dabo SM, Confer A, Montelongo M, York P, Wyckoff JH 3rd. Vaccination with Pasteurella multocida recombinant OmpA induces strong but non-protective and deleterious Th2-type immune response in mice. Vaccine 2008;26:4345–51. Dabo SM, Confer AW, Quijano-Blas RA. Molecular and immunological characterization of Pasteurella multocida serotype A:3 OmpA: evidence of its role in

8

C. Vougidou et al. / Microbiological Research 174 (2015) 1–8

P. multocida interaction with extracellular matrix molecules. Microb Pathog 2003;35:147–57. Davies RL, Lee I. Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompA) of Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi. J Bacteriol 2004;186:5741–52. Davies RL, MacCorquodale R, Reilly S. Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin. Vet Microbiol 2004;99:145–58. Davies RL, MacCorquodale R, Baillie S, Caffrey B. Characterization and comparison of Pasteurella multocida strains associated with porcine pneumonia and atrophic rhinitis. J Med Microbiol 2003a;52:59–67. Davies RL, Watson PJ, Caffrey B. Comparative analyses of Pasteurella multocida strains associated with the ovine respiratory and vaginal tracts. Vet Rec 2003b;152:7–10. Djordjevic SP, Eamens GJ, Ha H, Walker MJ, Chin JC. Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis. J Med Microbiol 1998;47:679–88. Ewers C, Lubke-Becker A, Bethe A, Kiebling S, Filter M, Wieler LH. Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Vet Microbiol 2006;114:304–17. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783–91. Fuller TE, Kennedy MJ, Lowery DE. Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis. Microb Pathog 2000;29:25–38. Fulton RW, Blood KS, Panciera RJ, Payton ME, Ridpath JF, Confer AW, et al. Lung pathology and infectious agents in fatal feedlot pneumonias and relationship with mortality, disease onset, and treatments. J Vet Diagn Invest 2009;21:464–77. Fussing V, Nielsen JP, Bisgaard M, Meyling A. Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp, multocida in Denmark. Vet Microbiol 1999;65:61–74. Garcia N, Fernandez-Garayzabal JF, Goyache J, Dominguez L, Vela AI. Associations between biovar and virulence factor genes in Pasteurella multocida isolates from pigs in Spain. Vet Rec 2011;169:362. Hariharan H, Cepica A, Qian B, Heaney S, Hurnik D. Toxigenic and drug resistance properties of porcine Pasteurella multocida isolates from Prince Edward Island. Can Vet J 2000;41:798. Harper M, Boyce JD, Adler B. Pasteurella multocida pathogenesis: 125 years after Pasteur. FEMS Microbiol Lett 2006;265:1–10. Hatfaludi T, Al-Hasani K, Boyce JD, Adler B. Outer membrane proteins of Pasteurella multocida. Vet Microbiol 2010;144:1–17. Hounsome JD, Baillie S, Noofeli M, Riboldi-Tunnicliffe A, Burchmore RJ, Isaacs NW, et al. Outer membrane protein A of bovine and ovine isolates of Mannheimia haemolytica is surface exposed and contains host species-specific epitopes. Infect Immun 2011;79:4332–41. Hunt ML, Boucher DJ, Boyce JD, Adler B. In vivo-expressed genes of Pasteurella multocida. Infect Immun 2001;69:3004–12. Jamaludin R, Blackall PJ, Hansen MF, Humphrey S, Styles M. Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand. N Z Vet J 2005;53:203–7. Khalid S, Bond PJ, Carpente T, Sansom MS. OmpA: gating and dynamics via molecular dynamics simulations. Biochim Biophys Acta 2008;1778:1871–80. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111–20.

Lax AJ, Chanter N. Cloning of the toxin gene from Pasteurella multocida and its role in atrophic rhinitis. J Gen Microbiol 1990;136:81–7. May BJ, Zhang Q, Li LL, Paustian ML, Whittam TS, Kapur V. Complete genomic sequence of Pasteurella multocida, Pm70. Proc Natl Acad Sci U S A 2001;98:3460–5. Petersen SK. The complete nucleotide sequence of the Pasteurella multocida toxin gene and evidence for a transcriptional repressor, TxaR. Mol Microbiol 1990;4:821–30. Pors SE, Hansen MS, Christensen H, Jensen HE, Petersen A, Bisgaard M. Genetic diversity and associated pathology of Pasteurella multocida isolated from porcine pneumonia. Vet Microbiol 2011;150:354–61. Prescott JF, Bhasin JL, Sanford SE, Binnington BD, Kierstead ME, Percy DH, et al. Serotypes and antimicrobial susceptibility of Pasteurella multocida isolated from cattle and pigs in Ontario. Can Vet J 1984;25:117–8. Purdy CW, Raleigh RH, Collins JK, Watts JL, Straus DC. Serotyping and enzyme characterization of Pasteurella haemolytica and Pasteurella multocida isolates recovered from pneumonic lungs of stressed feeder calves. Curr Microbiol 1997;34: 244–9. Quinn PJ, Carter ME, Markey B, Carter GR. Pasteurella species. In: Quinn PJ, Carter ME, Markey B, Carter GR, editors. Clinical veterinary microbiology. London: Mosby; 1994. p. 254–8. Rimler RB. Cross-protection factor(s) of Pasteurella multocida: passive immunization of turkeys against fowl cholera caused by different serotypes. Avian Dis 1987;31:884–7. Rimler RB, Rhoades KR. Serogroup F, a new capsule serogroup of Pasteurella multocida. J Clin Microbiol 1987;25:615–8. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406–25. Soriano-Vargas E, Vega-Sanchez V, Zamora-Espinosa JL, Acosta-Dibarrat J, Aguilar-Romero F, Negrete-Abascal E. Identification of Pasteurella multocida capsular types isolated from rabbits and other domestic animals in Mexico with respiratory diseases. Trop Anim Health Prod 2012;44:935–7. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 2013;30:2725–9. Tang C, Zhang B, Yue H, Yang F, Shao G, Hai Q, et al. Characteristics of the molecular diversity of the outer membrane protein A gene of Haemophilus parasuis. Can J Vet Res 2010;74:233–6. Tang X, Zhao Z, Hu J, Wu B, Cai X, He Q, et al. Isolation, antimicrobial resistance, and virulence genes of Pasteurella multocida strains from swine in China. J Clin Microbiol 2009;47:951–8. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80. Townsend KM, Boyce JD, Chung JY, Frost AJ, Adler B. Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system. J Clin Microbiol 2001;39:924–9. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007;35:71–4. Verma S, Sharma M, Katoch S, Verma L, Kumar S, Dogra V, et al. Profiling of virulence associated genes of Pasteurella multocida isolated from cattle. Vet Res Commun 2013;37:83–9. Vogel G, Nicolet J, Martig J, Tschudi P, Meylan M. Pneumonia in calves: characterization of the bacterial spectrum and the resistance patterns to antimicrobial drugs. Schweiz Arch Tierheilkd 2001;143:341–50. Wilkie IW, Harper M, Boyce JD, Adler B. Pasteurella multocida: diseases and pathogenesis. Curr Top Microbiol Immunol 2012;361:1–22.

Distribution of the ompA-types among ruminant and swine pneumonic strains of Pasteurella multocida exhibiting various cap-locus and toxA patterns.

Pasteurella multocida is an important pathogen in food-producing animals and numerous virulence genes have been identified in an attempt to elucidate ...
2MB Sizes 0 Downloads 6 Views