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Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping
Veterinary Microbiology 103 (2004) 63–69 www.elsevier.com/locate/vetmic
Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping Zoran Jaglic*, Petra Svastova, Ivan Rychlik, Katerina Nedbalcova, Zdenka Kucerova, Ivo Pavlik, Milan Bartos Veterinary Research Institute Brno, Hudcova 70, 621 32 Brno, Czech Republic Received 24 February 2004; received in revised form 16 June 2004; accepted 5 July 2004
Abstract During the period of 2001–2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1–12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods. # 2004 Elsevier B.V. All rights reserved. Keywords: RFLP; Porcine pleuropneumonia; Epizootiology
1. Introduction Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumoniae which causes considerable economic losses throughout the world, including the Czech Republic (Sebunya and Saunders, * Corresponding author. Tel.: +420 533 331 216; fax: +420 541 211 229. E-mail address: [email protected] (Z. Jaglic).
1983; Satran and Nedbalcova, 2002). It can be categorised on the basis of nicotinamide adenine dinucleotide (NAD) requirement for growth into two biovars: NAD-dependent biovar 1 and NAD-independent biovar 2 (Pohl et al., 1983). Within these biovars, individual serotypes are differentiated on the basis of capsular polysaccharides and lipopolysaccharides (Nielsen, 1990). Until now 14 serotypes (1–12, 15 and K2:O7) of biovar 1 (Nicolet, 1992; Nielsen et al., 1996; Blackall et al., 2002) and two serotypes (13 and
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.07.010
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Z. Jaglic et al. / Veterinary Microbiology 103 (2004) 63–69
14) of biovar 2 (Fodor et al., 1989; Nielsen et al., 1996) have been described. However, the main complication of serotyping is cross-reacitivity. Multiple authors reported cross-reactions amongst strains of serotypes 1, 9, and 11 (Nakai et al., 1992; Mittal et al., 1993). Cross-reactivity has been also found in other strains of serotypes. For example, some authors reported crossreactions of serotype 7 with serotype 4 (Mittal and Bourdon, 1991; Barbosa et al., 1995) or with numerous other serotypes (Mittal and Bourdon, 1991; Tadjine and Mittal, 2001). Furthermore, Levonen et al. (1996) reported cross-reactions between serotypes 6/8, 1/9/11, and 5/6. Alternative methods for precise identification of individual serotypes were therefore tested. Individual serotypes could be distinguished into five groups on the basis of the Apx exotoxins production or on the gene-toxin profile (Frey et al., 1993; Beck et al., 1994). Such studies gradually evolved into a precise PCR identification system of A. pleuropneumoniae based on the presence or absence of apx genes in the genome of individual serotypes (Frey et al., 1995). The omlA gene is another suitable target for molecular typing by PCR or PCR-REA (Gram et al., 2000; Cho and Chae, 2003). Restriction fragment length polymorphism (RFLP) analysis was employed by Rychlik et al. (1994) and Fussing et al. (1998). The aim of our study was to serotype field isolates collected in the Czech Republic from 2001 to 2003. In the second part of this study, we attempted to differentiate selected A. pleuropneumoniae field isolates and reference strains of serotypes 1–12 of biovar 1 by ribotyping and PCR-REA because most of the field isolates showed cross-reactions among serotypes 9, 11, and/or 1 and could not be classified exactly.
2. Materials and methods 2.1. Bacterial strains and culture media A total of 591 field isolates have been included in this study. These strains originated from 136 farms located in the Czech Republic and were isolated from slaughtered or died animals in 2001–2003. Twenty five field isolates (2 of the serotype 9 and 11, 3 showing cross-reactivity 1/9/11, and 20 showing cross-reactivity 9/11), together with 12 reference
strains of biovar 1 (strains 4074, S1536, S1421, M62, K17, Femø, WF83, 405, CVJ13261, D13039, 56153, 8329, serotypes 1–12, respectively) were selected for molecular typing. The strains were grown on a Haemophilus influenzae medium (Haemophilus Test Medium Base complemented by Haemophilus Test Medium Supplement, Oxoid, England) with addition of Vitox (Oxoid, England). 2.2. Antisera and serotyping New Zealand white rabbits were immunised with the reference strains mentioned above. Antigen preparation and immunisation were carried out according to Mittal et al. (1982). Specificity of antisera with the reference strains was confirmed and serotyping of all 591 field isolates was carried out by coagglutination according to Mittal et al. (1983). 2.3. Ribotyping Chromosomal DNA was purified using QIAGEN Blood & Cell Culture DNA Kit (Qiagen, Germany) according to the manufacturer’s instruction. The DNA was digested by restriction endonucleases (REs) CfoI or HindIII and ribotyping was performed as described previously (Pavlik et al., 1999). 16S-rDNA hybridisation probe was amplified from DNA of A. pleuropneumoniae using the two specific primers according to Kirschner and Bottger (1998). Restriction endonuclease patterns (ribotypes) were analysed by the Gel Compar software (Applied Maths, Belgium). 2.4. PCR-REA analysis Two specific primers—APXIVAF (50 -GCC TCC GAC CTG AAT AAA CC-30 ) and APXIVAR (50 -CAA CCA TCT TCT CCA CC-30 )—were designed using GeneBase software (Applied Maths, Belgium) according to the published apxIVA sequence (Schaller et al., 1999). The expected size of resulting amplicon was 3529 bp. A single bacterial colony was resuspended in 50 ml distilled water and boiled for 20 min. After brief centrifugation, 2 ml of supernatant containing the genomic DNA was used for PCR amplification. PCR was carried out with Taq PCR Master Mix Kit (QIAGEN, Germany) in a final volume of 20 ml. For amplification the PCR mixtures were denatured by
Z. Jaglic et al. / Veterinary Microbiology 103 (2004) 63–69
incubation at 94 8C for 3 min followed by 35 cycles of (i) denaturation at 94 8C for 45 s, (ii) primer annealing at 60 8C for 45 s, and (iii) elongation at 72 8C for 3 min. The samples were then incubated at 72 8C for 7 min for completion of the elongation process of the final PCR products. Amplification products were separated by electrophoresis in 0.8% agarose gel in TBE buffer and visualised by ethidium bromide staining. The amplicons were digested with REs CfoI and HpaII for 4 h. The digests were checked by electrophoresis in 1.5% agarose gel, the restriction endonuclease patterns were compared by the Gel Compar software (Applied Maths, Belgium).
3. Results 3.1. Serotyping of field isolates by coagglutination From a total of 591 field isolates, only 175 isolates did not cross-react and could be classified to a single serotype. Cross-reactivities most frequently occurred amongst serotypes 9, 11 and/or 1, predominantly 9/11 (Table 1). This fact indicates that porcine pleuropneumoniae in the Czech Republic is frequently caused Table 1 Serotyping of Actinobacillus pleuropneumoniae field isolates from the Czech Republic from 2001 to 2003 Serotype
Isolate No.
1 2 3 4 5 6 7 8 9 10 11 12 1/9a 1/9/11a 2/8a 2/12a 9/11a
0 103 0 1 0 0 1 0 67 0 2 1 6 35 1 14 360
Total
591
a
Field isolates with the serotype cross-reactivity.
% 0 17.4 0 0.2 0 0 0.2 0 11.3 0 0.3 0.2 1.0 5.9 0.2 2.4 60.8 100
65
by A. pleuropneumoniae isolates which are usually difficult to classify by serotyping. 3.2. Ribotyping Ribotyping of reference strains of serotypes 1–12 generated with RE HindIII resulted in nine different ribotypes described as ribotypes HindIII/1–9 (Fig. 1). All of the reference strains, except for the strains of serotypes 1, 3, and 6, and serotypes 9 and 11, could be differentiated. Ribotyping generated with RE CfoI resulted in only four different ribotypes described as ribotypes CfoI/1–4. However, the reference strain of serotype 1, which was grouped together with strains of serotypes 3 and 6 when digesting with RE HindIII, could be easily differentiated from these strains after the digestion with RE CfoI (Fig. 2). All of 25 field isolates were of profiles identical to that of reference strains of serotypes 9 and 11 (data not shown). 3.3. PCR-REA analysis After amplification of the sequence of the apxIVA gene, amplicons of two different sizes were observed. An amplification product of estimated size 3529 bp was observed in most reference strains of serotypes 1– 12; except for the strains of serotypes 4, 6, 9, and 11, in which a slightly smaller amplicon of 3000 bp was recorded (not shown). Digestion of the amplicons with RE CfoI resulted in 10 different restriction profiles amongst reference strains of serotypes 1–12. All of the reference strains, except for the strains of serotypes 8 and 10, and serotypes 9 and 11, could be differentiated. Use of an additional RE HpaII allowed differentiation of the reference strains of serotypes 8 and 10 but not serotypes 9 and 11 (Fig. 3). All of 25 field isolates had identical profiles as reference strains of serotypes 9 and 11 (not shown).
4. Discussion In the Czech Republic the most frequent crossreactivities were observed amongst serotypes 9, 11, and/or 1. Cross-reactivities amongst these serotypes have also been described by several authors (Mittal, 1990; Inzana et al., 1992; Nakai et al., 1992; Mittal et al., 1993). Besides the most frequent cross-reacting
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Fig. 1. RFLP analysis of A. pleuropneumoniae reference strains of serotypes 1–12 after digestion with RE HindIII and hybridisation with the 16S-rDNA probe (left side of the image), schematic presentation of HindIII RFLP patterns of all 12 serotype reference strains. M: molecular marker l DNA/HindIII (right side of the image).
Fig. 2. RFLP analysis of A. pleuropneumoniae reference strains of serotypes 1–12 after digestion with RE CfoI and hybridisation with the probe 16S-rDNA (left side of the image), schematic presentation of CfoI RFLP patterns of the 12 reference strains. M: molecular marker l DNA/ HindIII (right side of the image).
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Fig. 3. apxIVA amplification products from A. pleuropneumoniae reference strains of a) serotypes 1–12 digested with RE CfoI, and b) serotypes 8, 9, 10, and 11 digested with RE HpaII. M1 = l DNA/HindIII, M2 = 100 bp ladder.
isolates 1/9/11 so far, unpublished cross-reactions 2/12 were observed. By ribotyping we observed different profiles in reference strains from those described by Fussing et al. (1998), although the same REs were used in both studies. Unlike Fussing et al. (1998), we were unable to differentiate reference strains of serotype 3 and 6 using HindIII. After digestion with CfoI, we observed a much lower number of different ribotypes when compared with the study of Fussing et al. (1998). However, as far as the most frequently cross-reacting strains of serotypes 9/11 are concerned, none of studies could differentiate between them. Serotype-specificity of the apxI–III genes was described earlier (Beck et al., 1994). Due to this fact several authors differentiated A. pleuropneumoniae serotypes by PCR-based detection of apxI–III genes (Frey et al., 1995; Gram et al., 2000; Sthitmatee et al., 2003). Unlike the apxI–III genes, the apxIVA gene is present in all A. pleuropneumoniae serotypes (Schaller et al., 1999). Moreover, the recently reported modular structure of apxIVA, with variable numbers of modules of glycine-rich nonapeptides in different strains, increases diversity of apxIVA (Schaller et al., 1999) and makes it a suitable target for DNA typing methods. Consistent with this, immediately after the amplification of apxIVA, variation in the size of amplification products in strains of different serotypes
was observed. Even more subtle differentiation was reached when the PCR products were digested with REs CfoI and HpaII. It has been shown that the discriminatory power of the apxIVA based PCR-REA was higher than that of apxI–III PCR (Frey et al., 1995; Gram et al., 2000; Sthitmatee et al., 2003). Unfortunately, with both methods, we failed to differentiate reference strains of serotypes 9 and 11 and field isolates showing these cross-reactions. Many previous authors have also been unable to differentiate these serotypes, irrespective of whether PCR (Gram et al., 2000; Cho and Chae, 2003; Sthitmatee et al., 2003) or RFLP techniques (Rychlik et al., 1994; Fussing et al., 1998) were used. Two studies reported on successful differentiation of these two serotypes. Hennessy et al. (1993) used arbitrarily primed PCR to distinguish these two serotypes and de la PuenteRedondo et al. (2000) reported on variability of tbpA gene in serotypes 9 and 11. However, arbitrarily primed PCR is known to be quite difficult to standardise (Black, 1993) and concerning the tbpA gene variability, we amplified the gene sequence and digested the PCR product with restriction endonuclease AsnI exactly as described (de la PuenteRedondo et al., 2000) but no variability among reference strains of serotypes 9 and 11, and crossreacting isolates 9/11 were observed (unpublished observations). This further confirms that a certain genetic relationship exists between these serotypes.
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Acknowledgement This work was supported by the grant no. QC0195 of the National Agency for Agricultural Research, the Ministry of Agriculture (The Czech Republic).
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Hennessy, K.J., Iandolo, J.J., Fenwick, B.W., 1993. Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain-reaction. J. Clin. Microbiol. 31, 1155–1159. Inzana, T.J., Todd, J., Koch, C., Nicolet, J., 1992. Serotype specificity of immunological assays for the capsular polymer of Actinobacillus pleuropneumoniae serotype 1 and serotype 9. Vet. Microbiol. 31, 351–362. Kirschner, P., Bottger, E.C., 1998. Species identification of mycobacteria using rDNA sequencing. Methods Mol. Biol. 101, 349– 361. Levonen, K., Seppanen, J., Veijalainen, P., 1996. Antibodies against 12 serotypes of Actinobacillus pleuropneumoniae in Finnish slaughter sows. J. Vet. Med. B 43, 489–495. Mittal, K.R., 1990. Cross-reactions between Actinobacillus (Haemophilus) pleuropneumoniae strains of serotype 1 and serotype 9. J. Clin. Microbiol. 28, 535–539. Mittal, K.R., Bourdon, S., 1991. Cross-reactivity and antigenic heterogeneity among Actinobacillus pleuropneumoniae strains of serotype 4 and serotype 7. J. Clin. Microbiol. 29, 1344–1347. Mittal, K.R., Higgins, R., Lariviere, S., 1982. Evaluation of slide agglutination and ring precipitation tests for capsular serotyping of Haemophilus pleuropneumoniae. J. Clin. Microbiol. 15, 1019–1023. Mittal, K.R., Higgins, R., Lariviere, S., 1983. Detection of typespecific antigens in the lungs of Haemophilus-Pleuropneumoniae-infected pigs by coagglutination test. J. Clin. Microbiol. 18, 1355–1357. Mittal, K.R., Kamp, E.M., Kobisch, M., 1993. Serological characterization of Actinobacillus pleuropneumoniae strains of serotype 1, serotype 9 and serotype 11. Res. Vet. Sci. 55, 179–184. Nakai, T., Kawahara, K., Danbara, H., Kume, K., 1992. Identification of the cross-reacting antigen among Actinobacillus pleuropneumoniae strains of serotype 1, serotype 9 and serotype 11 by use of monoclonal antibodies. J. Vet. Med. Sci. 54, 707–710. Nicolet, J., 1992. Actinobacillus pleuropneumoniae. In: Leman, A.D., Straw, B.E., Mengeling, W.L., D’Allaire, S., Taylor, D.J. (Eds.), Diseases of Swine,Iowa State University Press, Ames, IO, pp. 401–408. Nielsen, R., 1990. New diagnostic techniques: a review of the HAP group of bacteria. Can. J. Vet. Res. 54, 68–72. Nielsen, R., Andresen, L.O., Plambeck, T., 1996. Serological characterization of Actinobacillus pleuropneumoniae biotype 1 strains antigenically related to both serotypes 2 and 7. Acta Vet. Scand. 37, 327–336. Pavlik, I., Horvathova, A., Dvorska, L., Bartl, J., Svastova, P., du Maine, R., Rychlik, I., 1999. Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis. J. Microbiol. Meth. 38, 155–167. Pohl, S., Bertschinger, H.U., Frederiksen, W., Mannheim, W., 1983. Transfer of Hemophilus pleuropneumoniae and the Pasteurella haemolytica-like organism causing porcine necrotic pleuropneumonia to the genus Actinobacillus (Actinobacillus pleuropneumoniae comb. nov.) on the basis of phenotypic and deoxyribonucleic-acid relatedness. Int. J. Syst. Bacteriol. 33, 510–514.
Z. Jaglic et al. / Veterinary Microbiology 103 (2004) 63–69 Rychlik, I., Bartos, M., Sestak, K., 1994. Use of DNA-fingerprinting for accurate typing of Actinobacillus pleuropneumoniae. Vet. Med. - Czech. 39, 167–174. Satran, P., Nedbalcova, K., 2002. Prevalence of serotypes, production of Apx toxins, and antibiotic resistance in strains of Actinobacillus pleuropneumoniae isolated in the Czech Republic. Vet. Med. - Czech. 47, 92–98. Schaller, A., Kuhn, R., Kuhnert, P., Nicolet, J., Anderson, T.J., MacInnes, J.I., Seger, R..P.A.M., Frey, J., 1999. Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae. Microbiol. UK 145, 2105–2116.
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Sebunya, T.N.K., Saunders, J.R., 1983. Haemophilus pleuropneumoniae infection in swine: a review. J. Amer. Vet. Med. Assoc. 182, 1331–1337. Sthitmatee, N., Sirinarumitr, T., Makonkewkeyoon, L., Sakpuaram, T., Tesaprateep, T., 2003. Identification of the Actinobacillus pleuropneumoniae serotype using PCR based apx genes. Mol. Cell. Probes 17, 301–305. Tadjine, M., Mittal, K.R., 2001. Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains. Vet. Microbiol. 78, 49–60.
Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping Zoran Jaglic*, Petra Svastova, Ivan Rychlik, Katerina Nedbalcova, Zdenka Kucerova, Ivo Pavlik, Milan Bartos Veterinary Research Institute Brno, Hudcova 70, 621 32 Brno, Czech Republic Received 24 February 2004; received in revised form 16 June 2004; accepted 5 July 2004
Abstract During the period of 2001–2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1–12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods. # 2004 Elsevier B.V. All rights reserved. Keywords: RFLP; Porcine pleuropneumonia; Epizootiology
1. Introduction Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumoniae which causes considerable economic losses throughout the world, including the Czech Republic (Sebunya and Saunders, * Corresponding author. Tel.: +420 533 331 216; fax: +420 541 211 229. E-mail address: [email protected] (Z. Jaglic).
1983; Satran and Nedbalcova, 2002). It can be categorised on the basis of nicotinamide adenine dinucleotide (NAD) requirement for growth into two biovars: NAD-dependent biovar 1 and NAD-independent biovar 2 (Pohl et al., 1983). Within these biovars, individual serotypes are differentiated on the basis of capsular polysaccharides and lipopolysaccharides (Nielsen, 1990). Until now 14 serotypes (1–12, 15 and K2:O7) of biovar 1 (Nicolet, 1992; Nielsen et al., 1996; Blackall et al., 2002) and two serotypes (13 and
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.07.010
64
Z. Jaglic et al. / Veterinary Microbiology 103 (2004) 63–69
14) of biovar 2 (Fodor et al., 1989; Nielsen et al., 1996) have been described. However, the main complication of serotyping is cross-reacitivity. Multiple authors reported cross-reactions amongst strains of serotypes 1, 9, and 11 (Nakai et al., 1992; Mittal et al., 1993). Cross-reactivity has been also found in other strains of serotypes. For example, some authors reported crossreactions of serotype 7 with serotype 4 (Mittal and Bourdon, 1991; Barbosa et al., 1995) or with numerous other serotypes (Mittal and Bourdon, 1991; Tadjine and Mittal, 2001). Furthermore, Levonen et al. (1996) reported cross-reactions between serotypes 6/8, 1/9/11, and 5/6. Alternative methods for precise identification of individual serotypes were therefore tested. Individual serotypes could be distinguished into five groups on the basis of the Apx exotoxins production or on the gene-toxin profile (Frey et al., 1993; Beck et al., 1994). Such studies gradually evolved into a precise PCR identification system of A. pleuropneumoniae based on the presence or absence of apx genes in the genome of individual serotypes (Frey et al., 1995). The omlA gene is another suitable target for molecular typing by PCR or PCR-REA (Gram et al., 2000; Cho and Chae, 2003). Restriction fragment length polymorphism (RFLP) analysis was employed by Rychlik et al. (1994) and Fussing et al. (1998). The aim of our study was to serotype field isolates collected in the Czech Republic from 2001 to 2003. In the second part of this study, we attempted to differentiate selected A. pleuropneumoniae field isolates and reference strains of serotypes 1–12 of biovar 1 by ribotyping and PCR-REA because most of the field isolates showed cross-reactions among serotypes 9, 11, and/or 1 and could not be classified exactly.
2. Materials and methods 2.1. Bacterial strains and culture media A total of 591 field isolates have been included in this study. These strains originated from 136 farms located in the Czech Republic and were isolated from slaughtered or died animals in 2001–2003. Twenty five field isolates (2 of the serotype 9 and 11, 3 showing cross-reactivity 1/9/11, and 20 showing cross-reactivity 9/11), together with 12 reference
strains of biovar 1 (strains 4074, S1536, S1421, M62, K17, Femø, WF83, 405, CVJ13261, D13039, 56153, 8329, serotypes 1–12, respectively) were selected for molecular typing. The strains were grown on a Haemophilus influenzae medium (Haemophilus Test Medium Base complemented by Haemophilus Test Medium Supplement, Oxoid, England) with addition of Vitox (Oxoid, England). 2.2. Antisera and serotyping New Zealand white rabbits were immunised with the reference strains mentioned above. Antigen preparation and immunisation were carried out according to Mittal et al. (1982). Specificity of antisera with the reference strains was confirmed and serotyping of all 591 field isolates was carried out by coagglutination according to Mittal et al. (1983). 2.3. Ribotyping Chromosomal DNA was purified using QIAGEN Blood & Cell Culture DNA Kit (Qiagen, Germany) according to the manufacturer’s instruction. The DNA was digested by restriction endonucleases (REs) CfoI or HindIII and ribotyping was performed as described previously (Pavlik et al., 1999). 16S-rDNA hybridisation probe was amplified from DNA of A. pleuropneumoniae using the two specific primers according to Kirschner and Bottger (1998). Restriction endonuclease patterns (ribotypes) were analysed by the Gel Compar software (Applied Maths, Belgium). 2.4. PCR-REA analysis Two specific primers—APXIVAF (50 -GCC TCC GAC CTG AAT AAA CC-30 ) and APXIVAR (50 -CAA CCA TCT TCT CCA CC-30 )—were designed using GeneBase software (Applied Maths, Belgium) according to the published apxIVA sequence (Schaller et al., 1999). The expected size of resulting amplicon was 3529 bp. A single bacterial colony was resuspended in 50 ml distilled water and boiled for 20 min. After brief centrifugation, 2 ml of supernatant containing the genomic DNA was used for PCR amplification. PCR was carried out with Taq PCR Master Mix Kit (QIAGEN, Germany) in a final volume of 20 ml. For amplification the PCR mixtures were denatured by
Z. Jaglic et al. / Veterinary Microbiology 103 (2004) 63–69
incubation at 94 8C for 3 min followed by 35 cycles of (i) denaturation at 94 8C for 45 s, (ii) primer annealing at 60 8C for 45 s, and (iii) elongation at 72 8C for 3 min. The samples were then incubated at 72 8C for 7 min for completion of the elongation process of the final PCR products. Amplification products were separated by electrophoresis in 0.8% agarose gel in TBE buffer and visualised by ethidium bromide staining. The amplicons were digested with REs CfoI and HpaII for 4 h. The digests were checked by electrophoresis in 1.5% agarose gel, the restriction endonuclease patterns were compared by the Gel Compar software (Applied Maths, Belgium).
3. Results 3.1. Serotyping of field isolates by coagglutination From a total of 591 field isolates, only 175 isolates did not cross-react and could be classified to a single serotype. Cross-reactivities most frequently occurred amongst serotypes 9, 11 and/or 1, predominantly 9/11 (Table 1). This fact indicates that porcine pleuropneumoniae in the Czech Republic is frequently caused Table 1 Serotyping of Actinobacillus pleuropneumoniae field isolates from the Czech Republic from 2001 to 2003 Serotype
Isolate No.
1 2 3 4 5 6 7 8 9 10 11 12 1/9a 1/9/11a 2/8a 2/12a 9/11a
0 103 0 1 0 0 1 0 67 0 2 1 6 35 1 14 360
Total
591
a
Field isolates with the serotype cross-reactivity.
% 0 17.4 0 0.2 0 0 0.2 0 11.3 0 0.3 0.2 1.0 5.9 0.2 2.4 60.8 100
65
by A. pleuropneumoniae isolates which are usually difficult to classify by serotyping. 3.2. Ribotyping Ribotyping of reference strains of serotypes 1–12 generated with RE HindIII resulted in nine different ribotypes described as ribotypes HindIII/1–9 (Fig. 1). All of the reference strains, except for the strains of serotypes 1, 3, and 6, and serotypes 9 and 11, could be differentiated. Ribotyping generated with RE CfoI resulted in only four different ribotypes described as ribotypes CfoI/1–4. However, the reference strain of serotype 1, which was grouped together with strains of serotypes 3 and 6 when digesting with RE HindIII, could be easily differentiated from these strains after the digestion with RE CfoI (Fig. 2). All of 25 field isolates were of profiles identical to that of reference strains of serotypes 9 and 11 (data not shown). 3.3. PCR-REA analysis After amplification of the sequence of the apxIVA gene, amplicons of two different sizes were observed. An amplification product of estimated size 3529 bp was observed in most reference strains of serotypes 1– 12; except for the strains of serotypes 4, 6, 9, and 11, in which a slightly smaller amplicon of 3000 bp was recorded (not shown). Digestion of the amplicons with RE CfoI resulted in 10 different restriction profiles amongst reference strains of serotypes 1–12. All of the reference strains, except for the strains of serotypes 8 and 10, and serotypes 9 and 11, could be differentiated. Use of an additional RE HpaII allowed differentiation of the reference strains of serotypes 8 and 10 but not serotypes 9 and 11 (Fig. 3). All of 25 field isolates had identical profiles as reference strains of serotypes 9 and 11 (not shown).
4. Discussion In the Czech Republic the most frequent crossreactivities were observed amongst serotypes 9, 11, and/or 1. Cross-reactivities amongst these serotypes have also been described by several authors (Mittal, 1990; Inzana et al., 1992; Nakai et al., 1992; Mittal et al., 1993). Besides the most frequent cross-reacting
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Fig. 1. RFLP analysis of A. pleuropneumoniae reference strains of serotypes 1–12 after digestion with RE HindIII and hybridisation with the 16S-rDNA probe (left side of the image), schematic presentation of HindIII RFLP patterns of all 12 serotype reference strains. M: molecular marker l DNA/HindIII (right side of the image).
Fig. 2. RFLP analysis of A. pleuropneumoniae reference strains of serotypes 1–12 after digestion with RE CfoI and hybridisation with the probe 16S-rDNA (left side of the image), schematic presentation of CfoI RFLP patterns of the 12 reference strains. M: molecular marker l DNA/ HindIII (right side of the image).
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Fig. 3. apxIVA amplification products from A. pleuropneumoniae reference strains of a) serotypes 1–12 digested with RE CfoI, and b) serotypes 8, 9, 10, and 11 digested with RE HpaII. M1 = l DNA/HindIII, M2 = 100 bp ladder.
isolates 1/9/11 so far, unpublished cross-reactions 2/12 were observed. By ribotyping we observed different profiles in reference strains from those described by Fussing et al. (1998), although the same REs were used in both studies. Unlike Fussing et al. (1998), we were unable to differentiate reference strains of serotype 3 and 6 using HindIII. After digestion with CfoI, we observed a much lower number of different ribotypes when compared with the study of Fussing et al. (1998). However, as far as the most frequently cross-reacting strains of serotypes 9/11 are concerned, none of studies could differentiate between them. Serotype-specificity of the apxI–III genes was described earlier (Beck et al., 1994). Due to this fact several authors differentiated A. pleuropneumoniae serotypes by PCR-based detection of apxI–III genes (Frey et al., 1995; Gram et al., 2000; Sthitmatee et al., 2003). Unlike the apxI–III genes, the apxIVA gene is present in all A. pleuropneumoniae serotypes (Schaller et al., 1999). Moreover, the recently reported modular structure of apxIVA, with variable numbers of modules of glycine-rich nonapeptides in different strains, increases diversity of apxIVA (Schaller et al., 1999) and makes it a suitable target for DNA typing methods. Consistent with this, immediately after the amplification of apxIVA, variation in the size of amplification products in strains of different serotypes
was observed. Even more subtle differentiation was reached when the PCR products were digested with REs CfoI and HpaII. It has been shown that the discriminatory power of the apxIVA based PCR-REA was higher than that of apxI–III PCR (Frey et al., 1995; Gram et al., 2000; Sthitmatee et al., 2003). Unfortunately, with both methods, we failed to differentiate reference strains of serotypes 9 and 11 and field isolates showing these cross-reactions. Many previous authors have also been unable to differentiate these serotypes, irrespective of whether PCR (Gram et al., 2000; Cho and Chae, 2003; Sthitmatee et al., 2003) or RFLP techniques (Rychlik et al., 1994; Fussing et al., 1998) were used. Two studies reported on successful differentiation of these two serotypes. Hennessy et al. (1993) used arbitrarily primed PCR to distinguish these two serotypes and de la PuenteRedondo et al. (2000) reported on variability of tbpA gene in serotypes 9 and 11. However, arbitrarily primed PCR is known to be quite difficult to standardise (Black, 1993) and concerning the tbpA gene variability, we amplified the gene sequence and digested the PCR product with restriction endonuclease AsnI exactly as described (de la PuenteRedondo et al., 2000) but no variability among reference strains of serotypes 9 and 11, and crossreacting isolates 9/11 were observed (unpublished observations). This further confirms that a certain genetic relationship exists between these serotypes.
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Acknowledgement This work was supported by the grant no. QC0195 of the National Agency for Agricultural Research, the Ministry of Agriculture (The Czech Republic).
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