JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p. 1743–1746 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.4.1743–1746.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 41, No. 4
Use of Single-Enzyme Amplified Fragment Length Polymorphism for Typing Pasteurella multocida subsp. multocida Isolates from Pigs A. M. Moreno,1* M. R. Baccaro,1 A. J. P. Ferreira,1 and A. F. Pestana de Castro2 Faculdade de Medicina Veterina ´ria e Zootecnia, Universidade de Sa ˜o Paulo,1 and Instituto de Cieˆncias Biome´dicas, Universidade de Sa ˜o Paulo,2 05508-000 Sa ˜o Paulo, Sao Paulo, Brazil Received 4 September 2002/Returned for modification 31 October 2002/Accepted 7 January 2003
Single-enzyme amplified fragment length polymorphism (SE-AFLP) analyses were used to differentiate 97 isolates of porcine Pasteurella multocida subsp. multocida. The strains, isolated from animals with pneumonia, rhinitis, and septicemia, were classified as capsular types A, D, and F. SE-AFLP showed a discriminatory index of 0.87 and identified 18 different profiles. methods of characterizing strains of P. multocida have been described by several investigators (4, 6, 10). In this study, we report the use of a rapid PCR-based technique, single-enzyme amplified fragment length polymorphism (SE-AFLP), for typing of Pasteurella multocida subsp. multocida isolates from pigs with pneumonia, atrophic rhinitis, and septicemia. Bacteriology. Ninety-seven strains of P. multocida subsp. multocida were identified through biochemical characteriza-
Pasteurella multocida is an important veterinary and opportunistic human pathogen. Certain serological types are the etiologic agents of severe pasteurellosis, such as fowl cholera in domestic and wild birds, bovine hemorrhagic septicemia, porcine progressive atrophic rhinitis, and pneumonia (10). Pig herds commonly present pneumonia or porcine progressive atrophic rhinitis caused by P. multocida, and therefore there is a serious need in Brazil for further investigation of the epidemiology of this pathogen (5, 16). Various molecular
FIG. 1. SE-AFLP profiles of P. multocida subsp. multocida produced by primer HIG. Lanes 1 and 20, 100-bp molecular weight standard. The remaining lanes show AFLP profiles (A to R). * Corresponding author. Mailing address: Laborato ´rio de Patologia Suína, Faculdade de Medicina Veterina´ria e Zootecnia-Universidade de Sa˜o Paulo, Av. Prof. Dr. Orlando Marques de Paiva, n. 87, Cidade Universita´ria, 05508-000 Sa˜o Paulo, SP, Brazil. Phone: 55 11 30911294. Fax: 55 11 3091-7829. E-mail:
[email protected]. 1743
FIG. 2. Dendrogram showing the relationship between P. multocida subsp. multocida isolates on the basis of AFLP patterns. Percentages of similarity between patterns were calculated by use of Jaccard’s coefficient. The dendrogram was constructed by use of UPGMA (unweighted pair group method using arithmetic average). 1744
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tion, including production of catalase, oxidase, and indol, urease activity, production of ornithine decarboxylase, and carbohydrate fermentation (1, 7). The strains were obtained between 1999 and 2002 from 67 herds from nine states in Brazil: Sa˜o Paulo, Santa Catarina, Parana´, Espírito Santo, Minas Gerais, Mato Grosso do Sul, Goia´s, Rio de Janeiro, and Rio Grande do Sul. The animals presented pneumonia, atrophic rhinitis, and septicemia. For PCR and SE-AFLP, cultures were grown in brain heart infusion broth (Difco) at 37°C for 18 to 24 h, and DNA was extracted using the guanidine thiocyanate method (2). Capsular typing. Capsular serotyping was conducted employing the indirect hemagglutination test as previously described (17). Results were confirmed by the multiplex capsular PCR assay recently described and which generates fragments of 1,044, 760, 657, 511, and 851 bp by P. multocida types A, B, D, E and F, respectively (18). Toxin gene detection. All strains were tested for the presence of the gene that codifies toxin production using the PCR previously described (13). SE-AFLP subtyping. Ten microliters of DNA was digested overnight at 37°C with 24 U of HindIII in the buffer of enzyme and water in a volume of 20 l. Five microliters of digested DNA was used in a ligation reaction containing 0.2 g of each adapter oligonucleotide (15), 1 U of T4 DNA ligase, ligase buffer, and water, in a final volume of 20 l incubated at room temperature for 3 h. Ligated DNA was heated to 80°C for 10 min and diluted 1/5 in sterile distilled water, and 5 l was used for each PCR. PCRs consisted of 5 l of ligated DNA, 2.5 mM MgCl2, 300 ng of primer (15), and 1.25 U of Taq DNA polymerase, PCR buffer in a final volume of 50 l. Cycles consisted of 94°C for 4 min followed by 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2.5 min. The amplicons were analyzed on a 2.0% agarose gel and registered by an image capturing system (ImageMaster VDS, Amersham Pharmacia Biotech). The 100-bp DNA ladder (Invitrogen) was included twice on each gel. Banding patterns were assessed visually and under a code, with no knowledge about serotyping results or epidemiological data. The discriminatory power of typing methods was calculated following the description provided by Hunter and Gaston (11). Infection with P. multocida subsp. multocida is widespread in Brazilian swine herds. Capsular typing of the 97 strains showed 72 strains to be type A, 22 strains to be type D, and three strains to be type F. Eleven strains were toxigenic, where three were type A and eight were type D strains. The distribution of type A and type D strains in the lungs and nasal cavity was similar to the data provided previously (12, 14). There had been no reports of type F infection in pigs before. Using SE-AFLP, 7 to 12 DNA fragments ranging between approximately 400 and 1,400 bp were studied. Eighteen SEAFLP profiles, designated A to R, were observed (Fig. 1). To test the reproducibility, three separate preparations of 22 P. multocida isolates were subjected to SE-AFLP, and no band variation was seen. However, some variations in the intensities of the bands were observed in different PCR runs. The discriminatory index of the SE-AFLP was 0.87. A comparison between plasmid profile, phage typing, and HindIII ribotyping showed discriminatory indexes of 0.16, 0.77, and
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0.78, respectively (9). Discriminatory indexes of 0.74 and 0.84 were registered using RFLP of the ompH gene with HinfI and RsaI restriction enzymes (3). The relationship that exists among SE-AFLP profiles and the phenotypic data are illustrated in Fig. 2. Strains from the same herd showed one to three different SE-AFLP profiles; however, they were clustered together. The majority of type D strains isolated from the nasal cavity and toxin-producing strains were allocated in the same cluster, i.e., AIIa. Strains isolated from the nasal cavity were present in other clusters, too. Some authors suggest that this occurs because P. multocida from pneumonic lung lesions can colonize the nasal cavity and vice versa (9). Type F strains showed two different profiles, which were not observed in type A and D strains but which were allocated in the same cluster of three type A strains (AIIId) with 80% similarity. Among type A strains, a high level of genetic heterogeneity was observed. Previous reports had also described the heterogeneous nature of serogroup capsular A isolated from different animal species (8). With respect to the states of origin of pigs, strains with common profiles were observed in different regions of Brazil. This can be attributed to the distribution of breeders from different genetic sources across the country. SE-AFLP was simple and easy to use or standardize and proved to have great potential for subtyping strains of P. multocida subsp. multocida isolated from pigs. REFERENCES 1. Blackall, P. J., J. L. Pahoff, D. Maks, N. Fegan, C. J. Morrow. 1995. Characterization of Pasteurella multocida isolates from fowl cholera outbreaks on turkey farms. Aust. Vet. J. 72:135–138. 2. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. WertheinVan Dillen, and J. Van Der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495–503. 3. Borowski, S. M. 2001. Tese de Doutorado. Universidade Federal do Rio Grande do Sul. Porto Alegre, Brazil. 4. Bowles, R., J. L. Pahoff, B. N. Smith, and P. J. Blackal. 2000. Ribotype diversity of porcine Pasteurella multocida from Australia. Aust. Vet. J. 78: 630–635. 5. Brito, J. R. F., I. A. Piffer, I. Wentz, and M. A. V. P. Brito. 1993. Capsular types and toxin production by strains of Pasteurella multocida isolated from pigs in southern Brazil. Rev. Microbiol. 24:94–97. 6. Chaslus-Dancla, E., M. C. Lesage-Descauses, S. Leroy-Se´trin, J. L. Martel, P. Coudert, and J. P. Lafont. 1996. Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals. Vet. Microbiol. 52:91–102. 7. Cowan, S. T., K. J. Steel, G. I. Barrow, and R. K. A. Feltham. 1993. Cowan and Steel’s manual for the identification of medical bacteria, 3rd ed. Cambridge University Press, Cambridge, United Kingdom. 8. Dziva, F., H. Christensen, J. E. Olsen, and K. Mohan. 2001. Random amplification of polymorphic DNA and phenotypic typing of Zimbabwean isolates of Pasteurella multocida. Vet. Microbiol. 82:361–372. 9. Fussing, V., J. P. Nielsen, M. Bisgaard, and A. Meyling. 1999. Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark. Vet. Microbiol. 65:61–74. 10. Hunt, M. L., B. Adler, and K. M. Townsend. 2000. The molecular biology of Pasteurella multocida. Vet. Microbiol. 72:3–25. 11. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J. Clin. Microbiol. 26:2465–2466. 12. Iwamatsu, S., and T. Sawada. 1988. Relationship between serotypes, dermonecrotic toxin production of Pasteurella multocida isolates and pneumonic lesions of porcine lung. Jpn. J. Vet. Sci. 50:1200–1206. 13. Kamp, E. M., G. C. A. M. Bokken, T. M. M. Vermeulen, M. F. de Jong, H. E. C. M. Buys, F. H. Reek, and M. A. Smits. 1996. A specific and sensitive PCR assay for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs. J. Vet. Diagn. Investig. 8:304–309. 14. Larivie`re, S., L. Leblanc, K. R. Mittal, and G. P. Martineau. 1992. Characterization of Pasteurella multocida from nasal cavities of piglets from farms with or without atrophic rhinitis. J. Clin. Microbiol. 30:1398–1401. 15. McLauchlin, J., G. Ripabelli, M. M. Brett, and, E. J. Threlfall. 2000. Am-
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