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Characterization of intestinal cnf1+ Escherichia coli from weaned pigs
IJMM
Int. J. Med. Microbial. 290, 539-542 (2000) © Urban & Fischer Verlag
http://www.urbanfischer.de/journals/ijmm
Characterization of intestinal cnf1+ Escherichia coli from weaned pigs Istvan T6th 1, Eric Oswald 2, Jacques G. Mainil 3, Mohamed Awad-Masalmeh 4, Bela Nagy1 Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary UMR 960, INRA-ENVT de Microbiologie Moleculaire, Ecole Nationale Veterinaire de Toulouse, Toulouse, France 3 Chaire de Bacteriologie et de Pathologie Bacterienne, Faculte de Medicine Veterinaire, Universite de Liege, Belgium 4 II. Medizinische Universitatsklinik fiir Klauentiere, Veterinarmedizinische Un ivers it at Wien, Austria 1
2
Received April 6, 2000 . Revision received July 11, 2000 . Accepted August 3, 2000
Abstract Escherichia coli isolated from 204 cases of porcine postweaning diarrhoea were tested by PCR for the genes of cytotoxic necrotic factors (CNF) and of cytolethal dystending toxin (CDT). Selected strains were also examined by PCR for the presence of papC-, sfa-, f17-, f18-, and afa-specific sequences encoding P, S, F17, F18 fimbriae and afimbrial adhesins. A 5.9 % (12/204) of the strains had enf1 gene, and two of them had edt gene as well. Further six edt+ strains were detected which were enf-negative. Most of the enfl + strains belonged to serogroups 02, 06, 08, 054 characteristic of necrotoxic E. coli (NTEC) of humans. All the cnfl + strains possessed the genes for P or S fimbriae or both, but were negative for F4, F17, or F18 or afimbrial adhesins. Results suggest that these enteric isolates may have entero- andlor uropathogenic significance in weaned pigs, and may have zoonotic potential for humans.
Key words: Escherichia coli - CNF - CDT - papC - sfa - pig
Introduction Diarrhoea of newly weaned pigs is often a multifactorial disease in which enterotoxigenic E. coli (ETEC) is thought to be the main factor in most cases. Verotoxigenic E. coli (VTEC) can also complicate postweaning diarrhoea or may cause oedema disease of weaned pigs (Nagy and Fekete, 1999). Both porcine postweaning ETEC and VTEC strains are known to carry fimbrial adhesins F18 (Rippinger et al., 1995). It is, however, possible that strains with no classical enterotoxic or verotoxic character are also involved in this multifactorial disease. One such logical possibility would be the involvement of necrotoxic E. coli (NTEC). NTEC producing cytotoxic necrotic factor (CNF) was first reported from an extract of an E. coli strain isolated from
enteritis of a child (Caprioli et al., 1983). Later on the existence of two distinct types of CNF was reported in isolates from calf diarrhoea: CNFI and CNF2 as antigenically similar toxins with distinct molecular weights (De Rycke et al., 1987, 1990). E. coli producing CNFI (NTECl) has been reported overwhelmingly from suckling pigs (Garabal et al., 1996; McLaren and Wray, 1986). NTECI strains were also reported to participate in an outbreak of necrotic enteritis and interstitial pneumonia (Penrith et al., 1995) but so far no specific studies have been conducted on the epidemiology and characteristics of NTEC in weaned pigs. Our aim was to investigate whether CNF could be an additional factor in porcine postweaning diarrhoea. Furthermore we also wanted to know whether a newly described toxin, known to be closely associated to
Corresponding author: Dr. Bela Nagy, Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Hungaria krt. 21, H-1143 Budapest, Hungary, Phone: +36-1-2522455, Fax: +36-1-2521069, E-mail: [email protected] 1438-4221100/29016-539 $ 15.00/0
540
1. T6th et al.
CNF and called cytolethal dystending toxin (CDT; Johnson and Lior, 1988), could also be produced by porcine NTEC and non-NTEC strains. Finally, the question was also asked whether enteric CNF+ strains from that age group of pigs could have adhesin genes coding for P, S, F17 fimbriae or afimbrial adhesins (AFA), characteristic of human uropathogenic E. coli (Hacker, 1990; Le Bouguenec et al., 1992; Le Bouguenec and Bertin, 1999; Lalioui et al., 1999; Mainil et al., 1999) or for F18 fimbriae characteristic of porcine ETEC (Rippinger et al., 1995), respectively.
strains by multiplex PCR using primers designed for the consensus region of the edtB genes (E. Oswald, personal communication): 5' -GAAAATAAATGGAATATAAATGTCCG-3' (CDT-sense1), 5' -GAAAATAAATGGAACACACATGTCCG3' (CDT-sense2), 5'-AAATCACCAAGAATCATCCAGTTA3' (CDT-antisensel), 5'-AAATCTCCTGCAATCATCCAGTTA-3' (CDT antisense2). The amplification reaction consisted of 30 cycles (94 DC for 1 min, 55 DC for 1 min, and 72 DC for 1 min), with an initial hot start at 94 DC for 5 min, and with a final extension at 72 DC for 10 min. Results of the above-mentioned PCR methods on the en[+ strains were controlled by DNA hybridisation studies for en!, pap, sfa, afa fimbrial genes and extended to the analysis for presence of the f17 gene family as described (Mainil et al., 1997).
Materials and methods A total of 204 E. coli strains were obtained from the small intestine of different weaned pigs that died as a result of post weaning diarrhoea. Phenotypic tests of the strains included O-typing and haemolysin production of selected strains as described (Mainil et al., 1999), as well as agglutination tests for the presence of F4 (K88; Fairbrother, 1992) and F18 fimbrial antigens (Rippinger et al., 1995). The presence of mannose resistant haem agglutinins (MRHA) was tested using human A, and 0, as well as ovine and bovine red blood cells in repeated experiments as described (Faris et al., 1981). E. coli J96 (04:H5, papG+) was used as MRHA + control (Johnson et al., 1997). Toxin production of E. coli bacteria was examined using HeLa cell cultures (De Rycke et al., 1996). CNF production was also tested by rabbit skin test as described (Caprioli et al., 1983). PCR methods for detection of the different sequences were used as follows: enfl and enf2 (Blanco et al., 1996), pap, sfa, as well as afa (Le Bougenec et al., 1992.), afa8 (Lalioui et al., 1999). All the intestinal en(+ strains were also phenotypically tested for the presence of F4 (K88) and F18 antigens (as above). Furthermore, enf+ strains were PCR tested for f18 (Imberects et al., 1994) and for the genes of enterotoxins (It, sta, sth) and verotoxins (sltI, sltII) as described (Alexa et al., 1997; Brian et al., 1992), respectively. The presence of the edt gene was examined in all
Table 1. Phenotypic (0 and haemolytic) and genotypic (cnf, edt, papC and sfa) characters of necrotoxic E. coli strains isolated from the small intestine of diarrheal weaned pigs. Strains
o group
Hly
cnfl
edt
papC
sfa
AII-40 AI-9 AI-85 AI-154 AI-210 AI-222 AII-37 AI-136 AI-137 AI-164 AI-219 AI-220
06 0164 02 08 08 08 054 054 054
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ +
+
+ + +
OX OX OX
+ + + + + + + + + +
+ +
+ + + +
Results CNF-specific sequences were detected in 5.9 % (12/204) of the porcine intestinal strains. All the 12 cnr strains proved to be cnfl +and all produced haemolysin. Two strains also possessed a edt-specific sequence in addition to cnfl (Table 1), and further six cdt+ strains were detected which were cnf-negative. The cytotoxic effect of the cnfl + strains and the cytolethal distending activity of strains AI-9 and AII-40 were demonstrated in tissue cultures. In addition, the necrotoxic effect of two cnfl + strains (AII-37 and AII-40) was proven in rabbit skin test. All the 12 NTEC and further 10 haemolytic, cnf-negative intestinal porcine E. coli strains of this collection were examined for the papC, sfa, and afa sequences by multiplex PCR, and for f18 genes by single PCR. All the 12 enteric cnfl + strains contained papC and/or sfa genes (Table 1). Results of DNA hybridisation agreed with those of PCR (not shown). None of the strains harboured genes of afa and afa8, or f17 and f18, and none of them expressed MRHA or F4 or F18 fimbriae either. Furthermore, the 10 haemolytic, non-necrotoxic, non-enterotoxic enteric isolates of this collection were also negative for such adhesin or edt genes, although all of them were haemolytic.
Discussion Our studies confirm earlier findings of Dozois et al. (1997), and de Brito et al. (1999) describing the occurrence of cnfl+, paV and/or sfa+ E. coli among enteric and extraintestinal isolates from suckling pigs, and provide additional evidence for the presence of NTEC with similar properties in diarrhoeal weaned pigs. Furthermore, we provided evidence for the presence of CDT among porcine NTEC and non-NTEC strains. To our knowledge this is the first report about CDT in porcine E. coli. These studies have also revealed that
Urovirulence genotypes of Escherichia coli in pigs
the majority of porcine postweaning NTEC strains from the intestine of weaned pigs possess adhesins, which confirm uropathogenic significance for man, although in vitro expression of these adhesins could not be proven. However, the potential uropathogenic significance of these porcine cnf+ strains is indicated by the fact that the serogroups 02, 06, 08, 054 of the NTEC1 strains in our study show an overlap with those of NTEC1 strains in man (Garabal et aI., 1996; De Rycke et aI., 1999) raising the question of potential zoonotic transmission of cn[+ strains. Acknowledgements. Supports from OMFB-EU-98-DI0-115, FAIR3-CT96-1335, OTKA-T026150, -A312, Ministry of Health T-I0 186/96 and from OECD (Paris, France), furthermore personal contributions of Marta Toth-Szekrenyi and Peter-Zsolt Fekete, are acknowledged. We also thank Colin Bell (Belfast), and Dr. Lothar Beutin (Berlin) for 0 typing the strains, and Dr. Levente Emody (Pees, Hungary) providing advise and pap+ strains for haem agglutination studies.
References Alexa, P., Rychlik, I., Nejezchleb, A., Hamrik, J.: Identification of enterotoxin-producing Escherichia coli by PCR and biological methods. Vet. Med. Czech. 42, 97-100 (1997). Blanco, M., Blanco, J. E., Blanco, J., Alonso M. P., Balsobre, c., Mourino M., Madrid, c., Juarez, A.: Polymerase chain reaction for detection of Escherichia coli strains producing cytotoxic necrotizing factor type 1 and type 2 (CNFI and CNF2). J.Microbiol.Methods 26, 95-101 (1996). Brian, M.J., Frosolono, M., Murray, B. E., Miranda, A., Lopez, E. L., Gomez, H. E, Cleary, T. G.: Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and haemolytic-uremic syndrome. J. Clin. Microbiol. 30, 1801-1806 (1992). Caprioli, A., Falbo, V., Roda, L. G., Ruggeri, EM., Zona, c.: Partial purification and characterization of an Escherichia coli toxic factor that induces morphological cell alterations. Infect. Immun. 39, 1300-1306 (1983). De Brito, B. G., Leite, D. S., Linhares, R. E., Viditto, M. c.: Virulence-associated factors of uropathogenic Escherichia coli strains isolated from pigs. Vet. Microbial. 65, 123132 (1999). De Rycke, J., Gonzalez, E. A., Blanco, J., Oswald, E., Bianco, M., Boivin, R.: Evidence for two types of cytotoxic necrotizing factor in human and animal clinical isolates of Escherichia coli. J. Clin. Microbial. 28, 694-699 (1990). De Rycke, J., Guillot, J. E, Boivin, R.: Cytotoxins in nonenterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves. Vet. Microbiol. 15, 137-150 (1987). De Rycke, J., Mazars, P., Nougayrede, J. P., Tasca, c., Boury, M., Herault E, Valette, A., Oswald E.: Mitotic block and delayed lethality in HeLa epithelial cells exposed to
541
Escherichia coli BM2-1 producing cytotoxic necrotizing factor type 1. Infect. Immun. 64,1694-1705 (1996). De Rycke, J., Milan, A., Oswald, E.: Necrotoxic Escherichia coli (NTEC): two emerging categories of human and animal pathogens. Vet. Res. 30,221-233 (1999). Dozois, C. M., Clement, S., Desautels, C., Oswald, E., Fairbrother, J. M.: Expression of P, S, and Fl C adhesins by cytotoxic necrotizing factor I-producing Escherichia coli from septicemic and diarrheic pigs. FEMS Microbiol. Lett. 152, 307-312 (1997). Fairbrother, J. M.: Enteric colibacillosis. In: Diseases of Swine, 7 th edition (Leman, A. D., Straw, B. E. et aI., eds.), pp. 489-497. Iowa State University Press, Ames, Iowa 1992. Faris, A., Wadstrom, T., Freer, J.: Hydrophobic and hemagglutinating properties of Escherichia coli possessing colonization factor antigens (CFA/I or CFNII), Type 1 pili or other pili. Current Microbiol. 5,67-72 (1981). Garabal, J. I., Gonzalez, E. A., Vazquez, E, Blanco, J., Bianco, J. E.: Serogroups of Escherichia coli isolated from piglets in Spain. Vet. Microbiol. 48, 113-123 (1996). Hacker, J.: Genetic determinants coding for fimbriae and adhesins of extra intestinal Escherichia coli. Curro Topics Microbiol.Immunol.151, 1-27 (1990). Imberects, H., Van Pelt, N., De Greeve, H., Lintermans, P.: Sequences related to the major subunit gene FedA of Fl 07 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae. FEMS Microbiol. Lett. 119, 309-314 (1994). Johnson, J. R., Stapleton, A. E., Russom T. A., Scheutz, E, Brown, J. J., Maslow, J. N.: Characteristics and prevalence within serogroup 04 of a J96-like clonal group of uropathogenic Escherichia coli 04:H5 containing the class I and class III alleles of papG. Infect. Immun. 65, 2153-2159 (1997). Johnson, W. M., Lior, H.: A new heat labile cytolethal distending toxin (CLDT) produced by Escherichia coli isolates from clinical material. Microb. Pathog. 4, 103-113 (1988). Lalioui, L., Jouve, J., Gounon, 0., Le Bouguenec, c.: Molecular cloning and characterization of the afa-7 and afa-8 gene clusters encoding afimbrial adhesins in Escherichia coli strains associated with diarrhea or septicemia in calves. Infect. Immun. 7,5048-5059 (1999). Le Bouguenec, c., Archaumband, M., Labigne, A.: Rapid and specific detection of pap, afa, and sfa adhesinencoding operons in uropathogenic strains by polymerase chain reaction. J. Clin. Microbiol. 30, 1189-1193 (1992). Le Bouguenec, C., Bertin, Y.: AFA and F17 adhesins produced by pathogenic strains in domestic animals. Vet. Res. 30, 317-342 (1999). Mainil, J. G., Jacquemin, E., Herault, E, Oswald, E.: Presence of pap-, sfa-, and afa-related sequences in necrotoxigenic Escherichia coli isolates from cattle: evidence for new variants of the AFA family. Can.J. Vet. Res. 61, 193-199 (1997). Mainil, J. G., Jacquemin, E., Pohl, P., Fairbrother, J. M., Ansuini, A., Le Bouguenec, c., Ball H.J., De Rycke, J., Oswald, E.: Comparison of necrotoxigenic Escherichia coli
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I. T6th et al.
isolates from farm animals and from humans. Vet. MicrobioI. 70, 123-135 (1999). McLaren, I., Wray, C.: Another animal Escherichia coli cytopathic factor. Vet. Rec. 119, 576-577 (1986). Nagy, B., Fekete, P. Zs.: Enterotoxic Escherichia coli in farm animals. Vet. Res. 30, 259-284 (1999). Penrith, M. L., Henton, M. M., Clay, C. G.: CNF1 toxinproducing strains of Escherichia coli isolated from wean-
er pigs with necrotic enteritis in South Africa. Vet. Rec. 136, 493-494 (1995). Rippinger, P., Bertschinger, H. u., Imberechts, H., Nagy, B., Sorg, I., Stamm, M., Wild, P., Wittig, W.: Designation F18ab and F18ac for the related fimbrial types F107, 2134P and 8813 of E. coli isolated from porcine postweaning diarrhoea from oedema disease. Vet. Microbiol. 45,281-295 (1995).
Int. J. Med. Microbial. 290, 539-542 (2000) © Urban & Fischer Verlag
http://www.urbanfischer.de/journals/ijmm
Characterization of intestinal cnf1+ Escherichia coli from weaned pigs Istvan T6th 1, Eric Oswald 2, Jacques G. Mainil 3, Mohamed Awad-Masalmeh 4, Bela Nagy1 Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary UMR 960, INRA-ENVT de Microbiologie Moleculaire, Ecole Nationale Veterinaire de Toulouse, Toulouse, France 3 Chaire de Bacteriologie et de Pathologie Bacterienne, Faculte de Medicine Veterinaire, Universite de Liege, Belgium 4 II. Medizinische Universitatsklinik fiir Klauentiere, Veterinarmedizinische Un ivers it at Wien, Austria 1
2
Received April 6, 2000 . Revision received July 11, 2000 . Accepted August 3, 2000
Abstract Escherichia coli isolated from 204 cases of porcine postweaning diarrhoea were tested by PCR for the genes of cytotoxic necrotic factors (CNF) and of cytolethal dystending toxin (CDT). Selected strains were also examined by PCR for the presence of papC-, sfa-, f17-, f18-, and afa-specific sequences encoding P, S, F17, F18 fimbriae and afimbrial adhesins. A 5.9 % (12/204) of the strains had enf1 gene, and two of them had edt gene as well. Further six edt+ strains were detected which were enf-negative. Most of the enfl + strains belonged to serogroups 02, 06, 08, 054 characteristic of necrotoxic E. coli (NTEC) of humans. All the cnfl + strains possessed the genes for P or S fimbriae or both, but were negative for F4, F17, or F18 or afimbrial adhesins. Results suggest that these enteric isolates may have entero- andlor uropathogenic significance in weaned pigs, and may have zoonotic potential for humans.
Key words: Escherichia coli - CNF - CDT - papC - sfa - pig
Introduction Diarrhoea of newly weaned pigs is often a multifactorial disease in which enterotoxigenic E. coli (ETEC) is thought to be the main factor in most cases. Verotoxigenic E. coli (VTEC) can also complicate postweaning diarrhoea or may cause oedema disease of weaned pigs (Nagy and Fekete, 1999). Both porcine postweaning ETEC and VTEC strains are known to carry fimbrial adhesins F18 (Rippinger et al., 1995). It is, however, possible that strains with no classical enterotoxic or verotoxic character are also involved in this multifactorial disease. One such logical possibility would be the involvement of necrotoxic E. coli (NTEC). NTEC producing cytotoxic necrotic factor (CNF) was first reported from an extract of an E. coli strain isolated from
enteritis of a child (Caprioli et al., 1983). Later on the existence of two distinct types of CNF was reported in isolates from calf diarrhoea: CNFI and CNF2 as antigenically similar toxins with distinct molecular weights (De Rycke et al., 1987, 1990). E. coli producing CNFI (NTECl) has been reported overwhelmingly from suckling pigs (Garabal et al., 1996; McLaren and Wray, 1986). NTECI strains were also reported to participate in an outbreak of necrotic enteritis and interstitial pneumonia (Penrith et al., 1995) but so far no specific studies have been conducted on the epidemiology and characteristics of NTEC in weaned pigs. Our aim was to investigate whether CNF could be an additional factor in porcine postweaning diarrhoea. Furthermore we also wanted to know whether a newly described toxin, known to be closely associated to
Corresponding author: Dr. Bela Nagy, Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Hungaria krt. 21, H-1143 Budapest, Hungary, Phone: +36-1-2522455, Fax: +36-1-2521069, E-mail: [email protected] 1438-4221100/29016-539 $ 15.00/0
540
1. T6th et al.
CNF and called cytolethal dystending toxin (CDT; Johnson and Lior, 1988), could also be produced by porcine NTEC and non-NTEC strains. Finally, the question was also asked whether enteric CNF+ strains from that age group of pigs could have adhesin genes coding for P, S, F17 fimbriae or afimbrial adhesins (AFA), characteristic of human uropathogenic E. coli (Hacker, 1990; Le Bouguenec et al., 1992; Le Bouguenec and Bertin, 1999; Lalioui et al., 1999; Mainil et al., 1999) or for F18 fimbriae characteristic of porcine ETEC (Rippinger et al., 1995), respectively.
strains by multiplex PCR using primers designed for the consensus region of the edtB genes (E. Oswald, personal communication): 5' -GAAAATAAATGGAATATAAATGTCCG-3' (CDT-sense1), 5' -GAAAATAAATGGAACACACATGTCCG3' (CDT-sense2), 5'-AAATCACCAAGAATCATCCAGTTA3' (CDT-antisensel), 5'-AAATCTCCTGCAATCATCCAGTTA-3' (CDT antisense2). The amplification reaction consisted of 30 cycles (94 DC for 1 min, 55 DC for 1 min, and 72 DC for 1 min), with an initial hot start at 94 DC for 5 min, and with a final extension at 72 DC for 10 min. Results of the above-mentioned PCR methods on the en[+ strains were controlled by DNA hybridisation studies for en!, pap, sfa, afa fimbrial genes and extended to the analysis for presence of the f17 gene family as described (Mainil et al., 1997).
Materials and methods A total of 204 E. coli strains were obtained from the small intestine of different weaned pigs that died as a result of post weaning diarrhoea. Phenotypic tests of the strains included O-typing and haemolysin production of selected strains as described (Mainil et al., 1999), as well as agglutination tests for the presence of F4 (K88; Fairbrother, 1992) and F18 fimbrial antigens (Rippinger et al., 1995). The presence of mannose resistant haem agglutinins (MRHA) was tested using human A, and 0, as well as ovine and bovine red blood cells in repeated experiments as described (Faris et al., 1981). E. coli J96 (04:H5, papG+) was used as MRHA + control (Johnson et al., 1997). Toxin production of E. coli bacteria was examined using HeLa cell cultures (De Rycke et al., 1996). CNF production was also tested by rabbit skin test as described (Caprioli et al., 1983). PCR methods for detection of the different sequences were used as follows: enfl and enf2 (Blanco et al., 1996), pap, sfa, as well as afa (Le Bougenec et al., 1992.), afa8 (Lalioui et al., 1999). All the intestinal en(+ strains were also phenotypically tested for the presence of F4 (K88) and F18 antigens (as above). Furthermore, enf+ strains were PCR tested for f18 (Imberects et al., 1994) and for the genes of enterotoxins (It, sta, sth) and verotoxins (sltI, sltII) as described (Alexa et al., 1997; Brian et al., 1992), respectively. The presence of the edt gene was examined in all
Table 1. Phenotypic (0 and haemolytic) and genotypic (cnf, edt, papC and sfa) characters of necrotoxic E. coli strains isolated from the small intestine of diarrheal weaned pigs. Strains
o group
Hly
cnfl
edt
papC
sfa
AII-40 AI-9 AI-85 AI-154 AI-210 AI-222 AII-37 AI-136 AI-137 AI-164 AI-219 AI-220
06 0164 02 08 08 08 054 054 054
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ +
+
+ + +
OX OX OX
+ + + + + + + + + +
+ +
+ + + +
Results CNF-specific sequences were detected in 5.9 % (12/204) of the porcine intestinal strains. All the 12 cnr strains proved to be cnfl +and all produced haemolysin. Two strains also possessed a edt-specific sequence in addition to cnfl (Table 1), and further six cdt+ strains were detected which were cnf-negative. The cytotoxic effect of the cnfl + strains and the cytolethal distending activity of strains AI-9 and AII-40 were demonstrated in tissue cultures. In addition, the necrotoxic effect of two cnfl + strains (AII-37 and AII-40) was proven in rabbit skin test. All the 12 NTEC and further 10 haemolytic, cnf-negative intestinal porcine E. coli strains of this collection were examined for the papC, sfa, and afa sequences by multiplex PCR, and for f18 genes by single PCR. All the 12 enteric cnfl + strains contained papC and/or sfa genes (Table 1). Results of DNA hybridisation agreed with those of PCR (not shown). None of the strains harboured genes of afa and afa8, or f17 and f18, and none of them expressed MRHA or F4 or F18 fimbriae either. Furthermore, the 10 haemolytic, non-necrotoxic, non-enterotoxic enteric isolates of this collection were also negative for such adhesin or edt genes, although all of them were haemolytic.
Discussion Our studies confirm earlier findings of Dozois et al. (1997), and de Brito et al. (1999) describing the occurrence of cnfl+, paV and/or sfa+ E. coli among enteric and extraintestinal isolates from suckling pigs, and provide additional evidence for the presence of NTEC with similar properties in diarrhoeal weaned pigs. Furthermore, we provided evidence for the presence of CDT among porcine NTEC and non-NTEC strains. To our knowledge this is the first report about CDT in porcine E. coli. These studies have also revealed that
Urovirulence genotypes of Escherichia coli in pigs
the majority of porcine postweaning NTEC strains from the intestine of weaned pigs possess adhesins, which confirm uropathogenic significance for man, although in vitro expression of these adhesins could not be proven. However, the potential uropathogenic significance of these porcine cnf+ strains is indicated by the fact that the serogroups 02, 06, 08, 054 of the NTEC1 strains in our study show an overlap with those of NTEC1 strains in man (Garabal et aI., 1996; De Rycke et aI., 1999) raising the question of potential zoonotic transmission of cn[+ strains. Acknowledgements. Supports from OMFB-EU-98-DI0-115, FAIR3-CT96-1335, OTKA-T026150, -A312, Ministry of Health T-I0 186/96 and from OECD (Paris, France), furthermore personal contributions of Marta Toth-Szekrenyi and Peter-Zsolt Fekete, are acknowledged. We also thank Colin Bell (Belfast), and Dr. Lothar Beutin (Berlin) for 0 typing the strains, and Dr. Levente Emody (Pees, Hungary) providing advise and pap+ strains for haem agglutination studies.
References Alexa, P., Rychlik, I., Nejezchleb, A., Hamrik, J.: Identification of enterotoxin-producing Escherichia coli by PCR and biological methods. Vet. Med. Czech. 42, 97-100 (1997). Blanco, M., Blanco, J. E., Blanco, J., Alonso M. P., Balsobre, c., Mourino M., Madrid, c., Juarez, A.: Polymerase chain reaction for detection of Escherichia coli strains producing cytotoxic necrotizing factor type 1 and type 2 (CNFI and CNF2). J.Microbiol.Methods 26, 95-101 (1996). Brian, M.J., Frosolono, M., Murray, B. E., Miranda, A., Lopez, E. L., Gomez, H. E, Cleary, T. G.: Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and haemolytic-uremic syndrome. J. Clin. Microbiol. 30, 1801-1806 (1992). Caprioli, A., Falbo, V., Roda, L. G., Ruggeri, EM., Zona, c.: Partial purification and characterization of an Escherichia coli toxic factor that induces morphological cell alterations. Infect. Immun. 39, 1300-1306 (1983). De Brito, B. G., Leite, D. S., Linhares, R. E., Viditto, M. c.: Virulence-associated factors of uropathogenic Escherichia coli strains isolated from pigs. Vet. Microbial. 65, 123132 (1999). De Rycke, J., Gonzalez, E. A., Blanco, J., Oswald, E., Bianco, M., Boivin, R.: Evidence for two types of cytotoxic necrotizing factor in human and animal clinical isolates of Escherichia coli. J. Clin. Microbial. 28, 694-699 (1990). De Rycke, J., Guillot, J. E, Boivin, R.: Cytotoxins in nonenterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves. Vet. Microbiol. 15, 137-150 (1987). De Rycke, J., Mazars, P., Nougayrede, J. P., Tasca, c., Boury, M., Herault E, Valette, A., Oswald E.: Mitotic block and delayed lethality in HeLa epithelial cells exposed to
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