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Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)–PCR
Veterinary Microbiology 89 (2002) 323–328
Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)–PCR Wanderley Dias da Silveiraa,*, Alessandra Ferreiraa, Marcelo Lancellottia, Isildinha A.G.C.D. Barbosaa, Domingos S. Leitea, Antonio F.P. de Castrob, Marcelo Brocchic a
Department of Microbiology and Immunology, Biology Institute, Campinas State University, CP 6109, CEP 13083-970 Campinas, SP, Brazil b Department of Microbiology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil c Department of Cellular, Molecular Biology and Pathogenic Bio-Agents, University of Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil Received 12 February 2002; received in revised form 12 July 2002; accepted 16 August 2002
Abstract Forty-nine avian Escherichia coli isolates isolated from different outbreak cases of septicemia (24 isolates), swollen head syndrome (14 isolates) and omphalitis (11 isolates), and 30 commensal isolates isolated from poultry with no signs of illness were characterized by enterobacterial repetitive intergenic consensus (ERIC)–PCR technique and their serotypes were determined. The ERIC–PCR profile allowed the typing of the 79 isolates into 68 ERIC-types and grouped the isolates into four main clusters (A–D), with the omphalitis isolates being grouped with the commensals and separated from the septicaemia and swollen head syndrome. These results indicate that ERIC–PCR is a technique that could replace other molecular characterization techniques such as random amplification of polymorphic DNA (RAPD)–PCR and restriction fragment length polymorphism (RFLP), reinforce previous observations that omphalitis isolates are just opportunistic agents, and are consistent with many reports that specific genotypes are responsible for causing specific diseases. Most of the isolates were either nontypable or rough, supporting the need for alternative methods for typing these isolates. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Escherichia coli; Typing; ERIC–PCR
* Corresponding author. Tel.: þ55-193788-7945; fax: þ55-192378-87050. E-mail address: [email protected] (W.D. da Silveira).
0378-1135/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 2 5 6 - 0
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W.D. da Silveira et al. / Veterinary Microbiology 89 (2002) 323–328
1. Introduction Escherichia coli, besides being a normal species found in the gastrointestinal tract of most animals, can also cause a wide variety of diseases depending on the presence of virulence genes (Levine, 1987; Nataro and Kaper, 1998). In poultry, this bacterium causes diseases such as septicemia, swollen head syndrome, omphalitis, cellulitis, yolk-sack infection and respiratory tract infections (Sojka and Carnaghan, 1961; Morley and Thomson, 1984; Randall et al., 1984; Dho-Moulin and Fairbrother, 1999). The resulting morbidity and mortality have led to serious economic losses to the poultry industry (Gross, 1994). Avian pathogenic E. coli may possess genes or virulence factors that have not yet been described but could be involved in the disease processes (Dho-Moulin and Fairbrother, 1999). The discovery that prokaryotic genomes contain repeated sequences such as the enterobacterial repetitive intergenic consensus (ERIC) sequence (Hulton et al., 1991) has expanded the molecular biology tools that are available to assess the clonal variability of many bacterial isolates including E. coli (Versalovic et al., 1991; Dalla-Costa et al., 1998; Chansiripornchai et al., 2001). The purpose of this work was to evaluate by ERIC–PCR the clonal structure of APEC isolates isolated in Brazil and responsible for different diseases (septicemia, swollen head syndrome and omphalitis) in chickens. Escherichia coli isolates ‘‘comensals’’ isolated from the gastrointestinal tract of adult birds showing no clinical signs of these diseases were used as control for comparison. All isolates were recently investigated for the presence of pathogenic traits and for pathogenicity in the 1-day-old chick assay (Silveira et al., 2002).
2. Material and methods 2.1. Bacterial isolates Escherichia coli isolates were isolated from commercial adult chickens from different regions of Brazil presenting clinical with signs of septicemia (S: n ¼ 24) and swollen head syndrome (H: n ¼ 14), as well as from 1-day-old chicks with omphalitis (O: n ¼ 11); 30 commensal (N) were isolated from cloacal swabs from chickens showing no illness. MacConkey agar medium was used for primary isolation. Lacþ colonies were identified as E. coli with the Kit EPM Mille. For routine tests, LB or LA agar medium (Sambrook et al., 1989) were used. All isolates were stored at 80 8C in LB medium containing 15% glycerol. 2.2. ERIC–PCR conditions and primers Genomic bacterial DNA was extracted as described by Van Soolingen et al. (1991), and resuspended in TE buffer plus 10 mg/ml of RNAse. DNA integrity after extraction was determined using 0.7% agarose gels in TE buffer as described by Sambrook et al. (1989).
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ERIC–PCR was performed with the following primer sequences: ERIC1R, 50 -ATGTAAGCTCCTGGGGATTCAC-30 and ERIC2, 50 -AAGTAAGTGACTGGGGTG AGCG-30 (Versalovic et al., 1991). The PCR reaction conditions were as described by the same authors, in a final volume of 50 ml, with slight modifications as follows: an initial denaturation (94 8C, 7 min) followed by 30 cycles of denaturation (90 8C, 30 s), annealing (52 8C, 1 min), and extension (72 8C, 8 min) with a single final extension (72 8C, 15 min). The sizes of the amplified fragments were determined after electrophoresis in submersed agarose gel (1.2–1.5%) using 100 pb and 1 Kb DNA markers (Life Technologies) as standards. The PCR reaction for each strain was performed in three separated experiments to confirm the pattern of the amplified bands. Agarose gel electrophoresis was performed as described by Sambrook et al. (1989). 2.3. ERIC fingerprint analyses ERIC fingerprints of amplified DNA fragments obtained by agarose gel electrophoresis were recorded. The presence of a given band was coded as 1 and the absence of a given band was coded as 0 in a data matrix and analyzed using the POPGENE software (Version 1.31) (Yeh et al., 1999). A dendrogram of dissimilarity, having a smaller number of isolates but representative of all, was constructed. 2.4. O serogroup determination O serogroups were determined with antisera (O1–O175; antisera O47; O67: O70; O93; O94; O122 were not tested) purchased from the E. coli Reference Laboratory, School of Veterinary Medicine of Lugo, University of Santiago of Compostela, Spain, using the microtiter technique described by Guine´ e et al. (1972) and modified by Blanco et al. (1998).
3. Results The electrophoretic profiles of the DNA fragments obtained after PCR amplification using specific primers for ERIC sequences (Fig. 1) (Versalovic et al., 1991) were determined for the 79 avian E. coli isolates. The O serogroups were also determined for the isolates. The ERIC–PCR profiles allowed the differentiation of the 79 isolates into 68 ERIC-types which were grouped into four main clusters (A–D), with cluster A being sub-divided into two main sub-clusters (A1; A2) (Fig. 1). Cluster A1 comprised 91% of the omphalitis isolates and 90% of the isolates from healthy chickens but only 5% of the isolates considered to be pathogenic. On the other hand, 97% of septicemic and swollen head syndrome isolates were grouped into clusters A2 through D, and only two (7%) of the isolates from healthy chickens were located in these clusters. Particularly, clusters A2 through C grouped 93% of swollen head syndrome isolates, with just one isolate of the total located into cluster A1 and all but one of the septicemic isolates (96%) were grouped into cluster D.
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Fig. 1. Genetic distance of APEC and normal Escherichia coli strains based on ERIC–PCR analyses (S ¼ strain type, where N ¼ commensal, O ¼ omphalitis, S ¼ septicemia, H ¼ swollen head syndrome).
The O serogrouping of the isolates isolated from healthy chickens demonstrated that 43% of them were untypable with the antisera that were used, 13% were rough isolates and the other serogroups that were identified were: O5; O7; O8; O10; O15; O21; O49; O79; O139; O155; O159, each at a frequency of 3%; and O173 at a frequency of 7%. Omphalitis isolates were: untypable (82%), O6 (9%) and O2 (9%). Swollen head syndrome isolates were: untypable (13%), rough (7%), O1 (13%), O2 (20%), O8 (20%), O9 (7%) and O86 (13%). Septicemic isolates were: untypable (21%), rough (41%), O2 (8%), O8 (17%); O20 (4%); O111 (4%).
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4. Discussion Pulsed field gel electrophoresis (PFGE) is considered to be the most reliable molecular finger-printing technique to differentiate organisms but restriction fragment length polymorphism (RFLP) is the one that is used most frequently. However both techniques require large quantities of DNA, are time consuming, and require expensive equipment. Other techniques such as ERIC–PCR and REP–PCR (Gilson et al., 1984; Hulton et al., 1991) and random amplification of polymorphic DNA (RAPD)–PCR (Welsh and McClelland, 1990) have been proposed as alternatives and used to characterize Escherichia coli isolates of avian origin (Chansiripornchai et al., 2001; de Moura et al., 2001). The 79 pathogenic and nonpathogenic avian Escherichia coli isolates analyzed here by the ERIC–PCR technique posses a high degree of polymorphism in their DNA sequences, but the isolates were grouped into four main clusters (Fig. 1). Most omphalitis isolates and isolates from healthy chickens were grouped together, reinforcing our previous investigation where we proposed that omphalitis isolates are just opportunistic and normally nonpathogenic agents since they do not behave as pathogenic agents in the 1-day-old chick assay (Silveira et al., 2002). In addition, ERIC–PCR grouped septicaemic and swollen head syndrome isolates in closer clusters but separated them from nonpathogenic and omphalitis ones, and suggested that APEC isolates, independently of the type of syndrome caused (septicemia or swollen head syndrome), belong to specific clones with genetic backgrounds different from those of the other groups. These data also suggest a possible correlation between pathogenicity and clonal origin. All septicemic isolates except one were grouped into cluster D. This cluster diverged considerably from the others suggesting a common origin for these isolates. These data are different from those previously published by de Moura et al. (2001) who used ERIC–PCR technique and suggested that no specific genotype would be responsible for colibacillosis. Our results grouped most of the pathogenic and nonpathogenic isolates into different clusters, suggesting that specific genotypes are responsible for each kind of disease. Comparing our results with those described by Chansiripornchai et al. (2001), who used RAPD–PCR and did not find differences between avian pathogenic and nonpathogenic E. coli isolates, lead us to suggest that ERIC–PCR has a better discriminating capacity and could substitute either RAPD–PCR or RFLP, which are claimed to show a lower level of genetic variability (Maurer et al., 1998). Because most of the isolates were either nontypable or rough for both nonpathogenic and pathogenic isolates, molecular typing methods such as ERIC–PCR are particularly useful in discriminating between APEC and commensal E. coli isolates.
Acknowledgements This work was supported by Grants no. 96/03683-0 and no. 99/04097-0 from The Foundation for the Support of Research of the State of Sa˜ o Paulo (FAPESP) and no. 300121/90-3 from The National Council for Scientific and Technological Development (CNPq).
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References Blanco, J.E., Blanco, M., Mora, A., Jansen, W.H., Garcı´a, V., Va´ zquez, M.L., Blanco, J., 1998. Serotypes of Escherichia coli isolated from septicaemic chickens in Galicia (northwest Spain). Vet. Microbiol. 61, 229–235. Chansiripornchai, N., Ramasoota, P., Sasipreyajan, J., Svenson, S.B., 2001. Differentiation of avian Escherichia coli (APEC) isolates by random amplified polymorphic DNA (RAPD) analysis. Vet. Microbiol. 80, 77–83. de Moura, A.C., Irino, K., Vidotto, M.C., 2001. Genetic variability of avian Escherichia coli isolates evaluated by enterobacterial reptitive intergenic consensus and repetitive extragenic palindromic polymerase chain reation. Avian Dis. 45, 173–181. Dalla-Costa, L.M., Irino, K., Rodrigues, J., Rivera, I.N.G., Trabulsi, L.R., 1998. Characterisation of diarrhoeagenic Escherichia coli clones by ribotyping and ERIC–PCR. J. Med. Microbiol. 47, 227–234. Dho-Moulin, M., Fairbrother, J.M., 1999. Avian pathogenic Escherichia coli (APEC). Vet. Res. 30, 299–316. Gilson, E., Cle´ ment, J.M., Brutlag, D., Hofnung, M., 1984. A family of dispersed repetitive extragenic palindromic DNA sequences in Escherichia coli. EMBO J. 3, 1417–1421. Gross, W.B., 1994. Diseases due to Esherichia coli in poultry. In: Gyles, C.L. (Ed.), Escherichia coli in Domestic Animals and Man. CAB International, Wallingford, UK. pp. 237–259. Guine´ e, P.A.M., Agterberg, C.M., Jansen, W.H., 1972. Escherichia coli O antigen typing by means of a mechanized microtechnique. Appl. Microbiol. 24, 127–131. Hulton, C.S., Higgins, C.F., Sharp, P.M., 1991. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimuirum and other enterobacteria. Mol. Microbiol. 5, 825–834. Levine, M.M., 1987. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorragic, and enteroadherent. J. Infect. Dis. 155, 377–389. Maurer, J.J., Lee, M.D., Lobsinger, C., Brown, T., Maier, M., Thayer, S.G., 1998. Molecular typing of avian Escherichia coli strains isolated by random amplification of polymorphic DNA. Avian Dis. 42, 431–451. Morley, A.J., Thomson, D.K., 1984. Swollen head syndrome in broiler chickens. Avian Dis. 28, 238–243. Nataro, J.P., Kaper, J.B., 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11, 142–201. Randall, C.J., Meakins, P.A., Harris, M.P., Watt, D.J., 1984. A new skin disease in broilers? Vet. Rec. 114, 246. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, NY, pp. 1584. Silveira, W.D., Ferreira, A., Brocchi, M., Hollanda, L.M., de Castro, A.F.P., Yamada, A.T., Lancellotti, M., 2002. Biological characteristics and pathogenicity of avian Escherichia coli strains. Vet. Microbiol. 85 (1), 47–53. Sojka, W.J., Carnaghan, R.B.A., 1961. Escherichia coli infection in poultry. Res. Vet. Sci. 2, 340–352. Van Soolingen, D., Hermans, P.W.M., Hass, P.E.W., Sool, D.R., Van Embden, J.D.A., 1991. The occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains evaluation of IS-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29, 2578–2586. Versalovic, J., Koeuth, T., Lupski, J.R., 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acid Res. 19, 6823–6831. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218. Yeh, F.C., Yang, R.-C., Boyle, T., 1999. Popgene version 1.31. Microsoft Windows-based freeware for Population Genetic Analysis (website: http://www.ualberta. ca/fyeh/).
Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)–PCR Wanderley Dias da Silveiraa,*, Alessandra Ferreiraa, Marcelo Lancellottia, Isildinha A.G.C.D. Barbosaa, Domingos S. Leitea, Antonio F.P. de Castrob, Marcelo Brocchic a
Department of Microbiology and Immunology, Biology Institute, Campinas State University, CP 6109, CEP 13083-970 Campinas, SP, Brazil b Department of Microbiology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil c Department of Cellular, Molecular Biology and Pathogenic Bio-Agents, University of Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil Received 12 February 2002; received in revised form 12 July 2002; accepted 16 August 2002
Abstract Forty-nine avian Escherichia coli isolates isolated from different outbreak cases of septicemia (24 isolates), swollen head syndrome (14 isolates) and omphalitis (11 isolates), and 30 commensal isolates isolated from poultry with no signs of illness were characterized by enterobacterial repetitive intergenic consensus (ERIC)–PCR technique and their serotypes were determined. The ERIC–PCR profile allowed the typing of the 79 isolates into 68 ERIC-types and grouped the isolates into four main clusters (A–D), with the omphalitis isolates being grouped with the commensals and separated from the septicaemia and swollen head syndrome. These results indicate that ERIC–PCR is a technique that could replace other molecular characterization techniques such as random amplification of polymorphic DNA (RAPD)–PCR and restriction fragment length polymorphism (RFLP), reinforce previous observations that omphalitis isolates are just opportunistic agents, and are consistent with many reports that specific genotypes are responsible for causing specific diseases. Most of the isolates were either nontypable or rough, supporting the need for alternative methods for typing these isolates. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Escherichia coli; Typing; ERIC–PCR
* Corresponding author. Tel.: þ55-193788-7945; fax: þ55-192378-87050. E-mail address: [email protected] (W.D. da Silveira).
0378-1135/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 2 5 6 - 0
324
W.D. da Silveira et al. / Veterinary Microbiology 89 (2002) 323–328
1. Introduction Escherichia coli, besides being a normal species found in the gastrointestinal tract of most animals, can also cause a wide variety of diseases depending on the presence of virulence genes (Levine, 1987; Nataro and Kaper, 1998). In poultry, this bacterium causes diseases such as septicemia, swollen head syndrome, omphalitis, cellulitis, yolk-sack infection and respiratory tract infections (Sojka and Carnaghan, 1961; Morley and Thomson, 1984; Randall et al., 1984; Dho-Moulin and Fairbrother, 1999). The resulting morbidity and mortality have led to serious economic losses to the poultry industry (Gross, 1994). Avian pathogenic E. coli may possess genes or virulence factors that have not yet been described but could be involved in the disease processes (Dho-Moulin and Fairbrother, 1999). The discovery that prokaryotic genomes contain repeated sequences such as the enterobacterial repetitive intergenic consensus (ERIC) sequence (Hulton et al., 1991) has expanded the molecular biology tools that are available to assess the clonal variability of many bacterial isolates including E. coli (Versalovic et al., 1991; Dalla-Costa et al., 1998; Chansiripornchai et al., 2001). The purpose of this work was to evaluate by ERIC–PCR the clonal structure of APEC isolates isolated in Brazil and responsible for different diseases (septicemia, swollen head syndrome and omphalitis) in chickens. Escherichia coli isolates ‘‘comensals’’ isolated from the gastrointestinal tract of adult birds showing no clinical signs of these diseases were used as control for comparison. All isolates were recently investigated for the presence of pathogenic traits and for pathogenicity in the 1-day-old chick assay (Silveira et al., 2002).
2. Material and methods 2.1. Bacterial isolates Escherichia coli isolates were isolated from commercial adult chickens from different regions of Brazil presenting clinical with signs of septicemia (S: n ¼ 24) and swollen head syndrome (H: n ¼ 14), as well as from 1-day-old chicks with omphalitis (O: n ¼ 11); 30 commensal (N) were isolated from cloacal swabs from chickens showing no illness. MacConkey agar medium was used for primary isolation. Lacþ colonies were identified as E. coli with the Kit EPM Mille. For routine tests, LB or LA agar medium (Sambrook et al., 1989) were used. All isolates were stored at 80 8C in LB medium containing 15% glycerol. 2.2. ERIC–PCR conditions and primers Genomic bacterial DNA was extracted as described by Van Soolingen et al. (1991), and resuspended in TE buffer plus 10 mg/ml of RNAse. DNA integrity after extraction was determined using 0.7% agarose gels in TE buffer as described by Sambrook et al. (1989).
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ERIC–PCR was performed with the following primer sequences: ERIC1R, 50 -ATGTAAGCTCCTGGGGATTCAC-30 and ERIC2, 50 -AAGTAAGTGACTGGGGTG AGCG-30 (Versalovic et al., 1991). The PCR reaction conditions were as described by the same authors, in a final volume of 50 ml, with slight modifications as follows: an initial denaturation (94 8C, 7 min) followed by 30 cycles of denaturation (90 8C, 30 s), annealing (52 8C, 1 min), and extension (72 8C, 8 min) with a single final extension (72 8C, 15 min). The sizes of the amplified fragments were determined after electrophoresis in submersed agarose gel (1.2–1.5%) using 100 pb and 1 Kb DNA markers (Life Technologies) as standards. The PCR reaction for each strain was performed in three separated experiments to confirm the pattern of the amplified bands. Agarose gel electrophoresis was performed as described by Sambrook et al. (1989). 2.3. ERIC fingerprint analyses ERIC fingerprints of amplified DNA fragments obtained by agarose gel electrophoresis were recorded. The presence of a given band was coded as 1 and the absence of a given band was coded as 0 in a data matrix and analyzed using the POPGENE software (Version 1.31) (Yeh et al., 1999). A dendrogram of dissimilarity, having a smaller number of isolates but representative of all, was constructed. 2.4. O serogroup determination O serogroups were determined with antisera (O1–O175; antisera O47; O67: O70; O93; O94; O122 were not tested) purchased from the E. coli Reference Laboratory, School of Veterinary Medicine of Lugo, University of Santiago of Compostela, Spain, using the microtiter technique described by Guine´ e et al. (1972) and modified by Blanco et al. (1998).
3. Results The electrophoretic profiles of the DNA fragments obtained after PCR amplification using specific primers for ERIC sequences (Fig. 1) (Versalovic et al., 1991) were determined for the 79 avian E. coli isolates. The O serogroups were also determined for the isolates. The ERIC–PCR profiles allowed the differentiation of the 79 isolates into 68 ERIC-types which were grouped into four main clusters (A–D), with cluster A being sub-divided into two main sub-clusters (A1; A2) (Fig. 1). Cluster A1 comprised 91% of the omphalitis isolates and 90% of the isolates from healthy chickens but only 5% of the isolates considered to be pathogenic. On the other hand, 97% of septicemic and swollen head syndrome isolates were grouped into clusters A2 through D, and only two (7%) of the isolates from healthy chickens were located in these clusters. Particularly, clusters A2 through C grouped 93% of swollen head syndrome isolates, with just one isolate of the total located into cluster A1 and all but one of the septicemic isolates (96%) were grouped into cluster D.
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Fig. 1. Genetic distance of APEC and normal Escherichia coli strains based on ERIC–PCR analyses (S ¼ strain type, where N ¼ commensal, O ¼ omphalitis, S ¼ septicemia, H ¼ swollen head syndrome).
The O serogrouping of the isolates isolated from healthy chickens demonstrated that 43% of them were untypable with the antisera that were used, 13% were rough isolates and the other serogroups that were identified were: O5; O7; O8; O10; O15; O21; O49; O79; O139; O155; O159, each at a frequency of 3%; and O173 at a frequency of 7%. Omphalitis isolates were: untypable (82%), O6 (9%) and O2 (9%). Swollen head syndrome isolates were: untypable (13%), rough (7%), O1 (13%), O2 (20%), O8 (20%), O9 (7%) and O86 (13%). Septicemic isolates were: untypable (21%), rough (41%), O2 (8%), O8 (17%); O20 (4%); O111 (4%).
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4. Discussion Pulsed field gel electrophoresis (PFGE) is considered to be the most reliable molecular finger-printing technique to differentiate organisms but restriction fragment length polymorphism (RFLP) is the one that is used most frequently. However both techniques require large quantities of DNA, are time consuming, and require expensive equipment. Other techniques such as ERIC–PCR and REP–PCR (Gilson et al., 1984; Hulton et al., 1991) and random amplification of polymorphic DNA (RAPD)–PCR (Welsh and McClelland, 1990) have been proposed as alternatives and used to characterize Escherichia coli isolates of avian origin (Chansiripornchai et al., 2001; de Moura et al., 2001). The 79 pathogenic and nonpathogenic avian Escherichia coli isolates analyzed here by the ERIC–PCR technique posses a high degree of polymorphism in their DNA sequences, but the isolates were grouped into four main clusters (Fig. 1). Most omphalitis isolates and isolates from healthy chickens were grouped together, reinforcing our previous investigation where we proposed that omphalitis isolates are just opportunistic and normally nonpathogenic agents since they do not behave as pathogenic agents in the 1-day-old chick assay (Silveira et al., 2002). In addition, ERIC–PCR grouped septicaemic and swollen head syndrome isolates in closer clusters but separated them from nonpathogenic and omphalitis ones, and suggested that APEC isolates, independently of the type of syndrome caused (septicemia or swollen head syndrome), belong to specific clones with genetic backgrounds different from those of the other groups. These data also suggest a possible correlation between pathogenicity and clonal origin. All septicemic isolates except one were grouped into cluster D. This cluster diverged considerably from the others suggesting a common origin for these isolates. These data are different from those previously published by de Moura et al. (2001) who used ERIC–PCR technique and suggested that no specific genotype would be responsible for colibacillosis. Our results grouped most of the pathogenic and nonpathogenic isolates into different clusters, suggesting that specific genotypes are responsible for each kind of disease. Comparing our results with those described by Chansiripornchai et al. (2001), who used RAPD–PCR and did not find differences between avian pathogenic and nonpathogenic E. coli isolates, lead us to suggest that ERIC–PCR has a better discriminating capacity and could substitute either RAPD–PCR or RFLP, which are claimed to show a lower level of genetic variability (Maurer et al., 1998). Because most of the isolates were either nontypable or rough for both nonpathogenic and pathogenic isolates, molecular typing methods such as ERIC–PCR are particularly useful in discriminating between APEC and commensal E. coli isolates.
Acknowledgements This work was supported by Grants no. 96/03683-0 and no. 99/04097-0 from The Foundation for the Support of Research of the State of Sa˜ o Paulo (FAPESP) and no. 300121/90-3 from The National Council for Scientific and Technological Development (CNPq).
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