Molecular and Cellular Probes (1991) 5, 2 71 -275
Evaluation of three new techniques for the detection of STb-positive Escherichia coli strains Louis-Andre Lortie, Josee Harel, John M. Fairbrother and J . Daniel Dubreuil* Groupe de Recherche sur les Maladies Infectieuses du Porc (GREMIP), Faculte de Medecine Veterinaire, Universite de Montreal, 3200 rue Sicotte, C .P . 5000, St-Hyacinthe, Quebec, Canada J2S 7C6 (Received 17 September 1990, Accepted 21 January 1991)
A study was conducted to compare different techniques for the detection of heat-stable enterotoxin b (STb)-positive E . coli strains . Antisera against purified STb was used to develop an enzyme-linked immunosorbent assay (ELISA) . STb-positive strains identified by ELISA were tested for bioactivity in rat jejunal loops . Our ELISA was as sensitive as, but less specific than, the bioassay for detection of STbpositive strains . A non-radioactive DNA probe to detect the gene coding for STb was also developed by incorporating digoxigenin-11-dUTP into DNA by the random primed labelling technique . The nonradioactive digoxigenin-labelled DNA probe demonstrated a similar detectability to the radioactive probe and was more convenient to manipulate but was less sensitive and specific than the bioassay and the radioactive probe . In addition, the polymerase chain reaction (PCR) was used to amplify a specific portion of the gene coding for STb . The PCR was a highly specific and practical technique for the detection of STb-positive strains . All E . coli strains tested containing the STb gene produced the STb toxin .
KEYWORDS : Escherichia coli, enterotoxin, STb, DNA detection, digoxigenin, PCR, ELISA .
INTRODUCTION Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhoeal disease in swine and humans . 1,2 ETEC can produce a heat-labile toxin and a group of heat-stable toxins .' Heat-stable toxins are classified
Detection of STb has been done by the weaned pig intestinal loop assay which is laborious, expensive and not fully reproducible . Recently, it was demonstrated that STb can be detected by another biological model using adult rats ." , " Other workers have produced antibody against fusion proteins, 13,14 but
by their methanol solubility and biological activity .` STa (or ST-I) is methanol soluble and induces intestinal secretion in infant mice and neonatal pigs . STb (or ST-II) is methanol insoluble and induces intestinal secretion in neonatal pigs but does not affect infant mice .' The gene coding for STb has been cloned and sequenced .',' It codes for a 71-amino-acid peptide
antibodies directed against such fusion proteins may demonstrate cross-reactivity with non-STb-producing strains . Thus, use of antibodies directed against the purified active STb molecule should facilitate the development of a more specific in vitro immunoassay, such as an ELISA, for the quantitative determination of STb, thus obviating the need for the cumbersome pig or rat ligated loop assays ." Others have demonstrated that gene probes could be used to detect LT-positive and STa-positive ETEC as defined by bioassay . 1S Similarly, the gene coding
including a signal sequence and is totally different from that coding for STa . Even if the gene is commonly detected in porcine ETEC strains, the STb gene is uncommon in ETEC of human origin and STb does not appear to be a cause of human diarrhoeal disease .', " `Author to whom correspondence should be addressed
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© 1991 Academic Press Limited
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anaesthetized and, after a midline abdominal inci-
for STb can be detected by DNA hybridization with a 7,1,1' a technique not practical in radioactive probe the diagnostic laboratory . Non-isotopically labelled cloned probes for STb would constitute an alternative as they are easier to manipulate . In addition, a simple polymerase chain reaction (PCR)-based technique could provide a rapid and sensitive technique to determine the presence of the gene coding for STb in ETEC . The aim of this study was to develop practical techniques for the detection of STb-producing ETEC strains .
MATERIALS AND METHODS
sion, the small intestine was treated with a trypsin inhibitor (TI) (300 .tg ml -1 in 0 . 85% saline) . A series of eight ligated segments (loops), each 5 cm long, were made in the small intestine starting approximately 5 cm from the ileo-cecal junction . Loops were inoculated with 0 . 5 ml of test material containing 300 .tg ml - ' TI . All samples were dialysed against 20 mm TrisHCI (pH 6 . 8) and sterilized by passage through a 0 . 22 gm membrane (Acrodisc 13, Gelman) . Each sample was tested in at least two rats, in loops at different positions in the small intestine . A negative control consisting of 20 mm Tris-HCI (pH 6 . 8) was included in each animal . Abdominal incisions were closed and rats were allowed to regain conciousness . After 4 h, the rats were sacrificed and the volume of liquid in each loop
Bacterial strains Porcine Escherichia coli strains examined in this study are described in Table 1 . Escherichia coli strain K-12 (pRAS1) contained a multicopy recombinant plasmid carrying an STb toxin encoding gene .'•' 6 Strain HB101
was measured . Results were expressed as volume of liquid (ml) per length (cm) x diameter (cm) of intestine and were considered positive if greater than 0 . 05 .
was used as negative control .
Immunoassay (ELISA)
Biological assay The rat ligated loop assay was performed as described by Dubreuil et al ." Briefly 6- to 8-week-old white rats (Sprag Dawley C .D .) were fasted for 48 h,
The immunoassay was performed as described by Dubreuil et al." Crude culture supernatant (50 µl) diluted in 0 . 1 M sodium carbonate (pH 9 .6) was coated onto wells of polystyrene microtitre plates (Flow Laboratories, Inc., McLean, VA) overnight, Plates were washed three times with PBS-Tween 20,
Table 1 . Comparison of different techniques for the detection of porcine STb-positive E . coli strains Detection of STb Strains
79-5863 81-4035 82-4247 83-2240 84-442 84-3922 84-3948 88-261 88-5623 89-682 80-3972 88-3732 79-6755 80-3261 80-3531 81-7169 88-2467 pRAS 1 HB101
Serotypes
Rat loop assay
DNA probe "p
PCR'
ELISAt
0115 :KV165 0115:KV165 0115:KV165 0115 :KV165 0115 :KV165 0115-KV165 0115-KV165 0115 :KV165 0141 0141 0147 0147 0149 0149 0149 0149 0149
+ + +
+ + +
+ + +
+ + + + +
+ + + + + +
+
+
+
DNA probe Dig.
+
+1+ +
+1-
+ -
+1-
+
+ +
+ +
+ +
+ -
+ + +
+ + +
+ + +
+ + +
+
+
+
+
An amplified fragment at 377 pb corresponds to a positive result (Fig . 1) . t ELISA was considered positive if O .D . (414 nm) > 0. 20 .
Detection of STb-positive appropriate dilutions of rabbit anti-STb antibodies (50µl) were added to each well, and the plates incubated at 37° C for 1 h . Plates were washed three times with PBS-Tween 20, appropriate dilutions of goat anti-rabbit IgG (Daymar Laboratories Inc ., Toronto, Canada) in PBS-Tween 20 coupled to horseradish peroxidase (50 AD were added to each well and plates were incubated at 37 ° C for 15 min . Plates were washed and the chromogenic substrate 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS0. 4 mm) (Sigma), dissolved in citrate buffer (pH 4 . 0) with 2 mm H 2 02, was added . The optical density was measured with a Titertek Multiskan ® MC (Flow Laboratories, Mississauga, Canada) at 414 nm after incubation for 30 min at room temperature .
Probes The gene probe for STb enterotoxin was derived from a recombinant E . coli strain K-12 containing the plasmid pRAS1 as described previously . 7.16,11 Plasmid DNA was isolated by ultracentrifugation in a caesium chloride gradient." After digestion of the plasmid DNA by restriction endonucleases, generated fragments were separated by electrophoresis in agarose or acrylamide gels ." Probe fragments of 460 by were recovered by electro-elution and concentrated by ethanol precipitation . DNA fragments were labelled with 1a- 32 P1dCTP using a multiprimer DNA labelling kit (Amersham Corp., Arlington Heights, IL) . The non-radioactive probe was prepared by labelling the 460 by DNA fragment with digoxigenin-11dUTP by the random primed labelling technique according to the manufacturer's instructions (Boehringer Mannheim GmbH, Mannheim, Germany) .
Hybridization Colony hybridization with the radioactive probe was performed as previously described ." Briefly, strains were spot inoculated onto solid media, incubated and replicated onto a membrane . The membrane was consecutively placed, colony side up, onto filter papers saturated with lysing, denaturing and neutralizing solutions . The membrane was then prehybridized, hybridized and washed before being exposed to X-ray film . The film was developed according to the manufacturer's instructions . For the non-radioactive detection of the gene coding for STb, a plasmid DNA preparation of each sample was extracted by a rapid miniprep technique, 19 denatured and slot-blotted onto a nylon membrane (Zeta-Probe®, Bio-Rad Laboratories) with a
E. coli
2 73
Bio-Dot SF ® apparatus (Bio-Rad Laboratories) according to the manufacturer's instructions . The hybridization and the detection of the digoxigenin-labelled probe was performed as described by the manufacturer (Boehringer Mannheim GmbH, Mannheim, Germany). Sensitivity of DNA probes was evaluated by hybridization with 10-fold dilutions of target DNA .
Enzymatic amplification The polymerase chain reaction (PCR) using a forward primer (5'-GCAATAAGGTTGAGGTGAT-3') covering the 5' end of the gene and a reverse primer (5'GCCTGCAGTGAGAAATGGAC-3') flanking the 3' extremity of the STb gene' was performed as previously described ." For each sample, three bacterial colonies were transferred with a sterile toothpick into 100 pl of sterile distilled water . Each sample was boiled for 1 min, then 67 . 5 pl was withdrawn and added to 10 pl of 10 x PCR buffer21 , 16 pl of 1 . 25 mm dNTPs, 1 pg of forward primer and 1 pg of reverse primer for a final volume of 99 . 5 pl . The samples were heated to 90 ° C for 5 min, 2 . 5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) was added, and the samples were subjected to PCR in a thermal cycler . The program used was 15 s at 94 ° C, 15 s at 40° C, 60 s at 72 ° C, for 30 cycles, followed by a final 7 min extension at 72 ° C . A volume of 5 pl of each reaction mixture was subjected to electrophoresis on a 1 . 2% agarose gel stained with ethidium bromide . 11-11,2' An amplified fragment of 377 by corresponds to a positive result, as determined in a previous study ."
RESULTS Ten of the 19 E. coli strains examined in this study were STb-positive by the rat intestinal loop assay . The same 10 strains were STb-positive using the radioactive DNA probe . No false-positive results were observed using this technique . The correlation between these two techniques was 100% (Table 1) . The 10 STb-positive E . coli strains identified by the bioassay were also positive by PCR . No false-positive results were obtained by this technique (Fig . 1) . The 10 strains positive in the bioassay were also positive by ELISA . However, of the nine strains that were negative in the bioassay, four were positive by ELISA . The correlation between the ELISA and the rat intestinal loop assay was 79% . The non-radioactive digoxigenin-labelled DNA probe demonstrated a detectability similar to that of the radioactive probe by detecting as little as 1 . 0 pg of target DNA (Fig . 2). This probe detected nine STb-
2 74
L.-A. Lortie et al.
Fig. 1 . Agarose gel analysis of PCR products of the 19 E . coli strains tested in this study . The order of the strains is the same as in Table 1 . Lane M shows a 750, 410 and 280 by DNA fragment as size markers . An amplified fragment of 377 by corresponds to an STb-positive strain .
500
100
10
1.0
0.1
0
pg
(a )
( b)
Fig . 2 . Evaluation of the detectability of the digoxigenin-labelled DNA probes (a) and of the 32 P-labelled DNA probe (b) for the detection of the gene coding for the heat-stable b (STb) enterotoxin of Escherichia coli . Both probes detected as little as 1 .0 pg of DNA .
positive strains and failed to detect one strain which
The other technique to detect the gene coding for
was positive by the bioassay . However, the digoxige-
STb using a non-radioactive probe was more con-
nin-labelled DNA probe weakly hybridized with three
venient to manipulate but was less sensitive and less
STb-negative strains, two of which were positive by
specific than the radioactive probe and the bioassay .
ELISA .
This may be intrinsic to the technique because other workers have observed 15% false-positive results using a digoxigenin-labelled DNA probe to
DISCUSSION AND CONCLUSIONS
detect
Campylobacter jejuni ." Regardless of the
sensitivity and specificity of these techniques used to In this study, we developed practical techniques for
detect the gene coding for STb, they do not
the detection of STb-producing ETEC strains . We
quantitatively evaluate the presence of the toxin in
evaluated the correlation between the presence of
a sample .
the gene coding for STb and the production of this
On the other hand, ELISA could constitute a way
enterotoxin . Identical results were obtained with the
to evaluate quantitatively STb production by ETEC
radioactively-labelled DNA probe and the rat intestinal loop assay . Although a small number of strains
strains . However, four false-positive results were obtained using this technique . Our antibodies raised
were examined, we conclude that an STb-positive
against purified STb gave positive ELISA with STb-
porcine strain by probe is also an STb producer as
negative strains of different serotypes . These strains
also observed in another study ."
cannot be considered STb-positive because the two
Two different techniques were then developed to
reference assays (the rat jejunal loop assay and the
detect the STb coding gene . The polymerase chain
DNA hybridization assay with a radioactive probe) were negative for them . We conclude, overall, that the ELISA and the digoxigenin-labelled DNA probe
reaction described in this study detected all STbpositive strains and no false-positive results were observed . This technique was easy to perform and was highly specific in our study .
were as sensitive as but less specific than the bioassay and the radioactive probe .
Detection of STb-positive E. coli In conclusion, the technique with the greatest specificity and sensitivity for a rapid and non-radioactive diagnostic test of STb-positive strains was the detection of the gene coding for STb by the polymerase chain reaction . Moreover, this technique was easy to perform, rapid, specific, may be automated and is low cost-effective .
ACKNOWLEDGEMENTS This study was supported in part by a grant to J . D . D . from the National Sciences and Engineering Council of Canada (OGP0046569) and Fonds pour la formation de Chercheurs et I'Aide a la Recherche (15728540) and to J . H . from Conseil des Recherches en Peche et Agro-alimentaire du Quebec (2243) and to L.A .L . from Ministere de I'Enseignement Superieur et de la Science . We thank Stephane Bolduc for his useful assistance in the bioassay, Bernadette Foiry for her technical assistance and Charles M . Dozois for reviewing the manuscript .
REFERENCES 1 . Gyles, C . L., Stevens, J . B . & Craven, J . A . (1971) . A study of Escherichia coli strains isolated from pigs with gastrointestinal disease . Canadian Journal of Comparative Medicine 35, 258-66. 2 . Sack, R . B . (1975) . Human diarrhea[ disease caused by enterotoxic Escherichia coli . Annual Review of Microbiology 29, 333-53 . 3 . Guerrant, R. L ., Holmes, R . K., Robertson, D. C . & Greenberg, R . N . (1985) . Roles of enterotoxins in the pathogenesis of Escherichia coli diarrhea . In Microbiology-1985 . (Leive, L., Bonventre, P . F ., Morello, J . A ., Schlesinger, S ., Silver, S. D . & Wu, H . C ., eds) pp . 68-73 . ASM Publication . 4 . Burgess, M . N ., Bywater, R . J ., Cowley, C . M., Mullan, N . A . & Newsome, P . M . (1978). Biological evaluation of a methanol-soluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves . Infection & Immunity 21, 526-31 . 5. Robichaud, N ., Lallier, R . & Lariviere, S . (1978). Comparison of the various test systems for the detection of Escherichia coli enterotoxin . In Proceeding of the 2nd International symposium on neonatal diarrhea (S . D . Acres ed .) Saskatoon, Saskatchewan, Canada . 6 . Weikel, C . S ., Nellans, H . N . & Guerrant, R . L. (1986) . M vivo and in vitro effects of a novel enterotoxin, STb, produced by Escherichia coli . Journal of Infectious Diseases 153, 893-901 . 7 . Lee . C. H ., Moseley, S . L ., Moon, H . W ., Whipp, S . C ., tyles, C . L. & So, M . (1983) . Characterization of the gene encoding heat-stable toxin II and preliminary molecular epidemiological studies of enterotoxigenic Escherichia coli heat-stable toxin II producers. Infection & Immunity 42, 264-8. 8 . Picken, R. N ., Mazaitis, A . J ., Maas, W . K ., Rey, M. & Heyneker, H . (1983) . Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli . Infection & Immunity 42, 269-75 . 9 . Echeverria, P., Seriwatana, J ., Patamaroj, U ., Moseley,
275
S . L., McFarland, A ., Chityothin, 0 . & Chaicumpa, W . (1984). Prevalence of heat-stable II enterotoxigenic Escherichia coli in pigs, water, and people at farms in Thailand as determined by DNA hybridization . Journal of Clinical Microbiology 19, 489-91 . 10 . Weikel, C . S ., Tiemens, K . M., Moseley, S . L ., Huq, I . M . & Guerrant, R . L. (1986) . Species specificity and lack of production of STb enterotoxin by Escherichia coli isolated from humans with diarrheal disease. Infection & Immunity 52, 323-5 . 11 . Dubreuil, J . D ., Fairbrother, J . M ., Lallier, R . & Lariviere, S . (1991) . Production and purification of heat-stable enterotoxin b (STb) from a porcine Escherichia coli strain . Infection & Immunity 59, 198-203 . 12 . Whipp, S . C . (1990) . Assay of enterotoxigenic Escherichia coli heat-stable toxin b in rats and mice . Infection & Immunity 58, 930-4 . 13 . Lawrence, R . M ., Huang, P .-T ., Click, J ., Oppenheim, J . D . & Maas, W. K . (1990). Expression of the cloned gene for enterotoxin STb of Escherichia coli . Infection & Immunity 58, 970-7 . 14. Handl, C ., Ronnberg, B ., Nilsson, B., Olsson, E ., Jonsson, H . & Flock, J .-I . (1988) . Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II . Journal of Clinical Microbiology 26, 1555-60. 15 . Bopp, C. A ., Threatt, V. L ., Moseley, S . L ., Wells, J . G . & Wachsmuth, I . K . (1990) . A comparison of alkaline phosphatase and radiolabelled gene probes with bioassays for enterotoxigenic Escherichia coli . Molecular and Cellular Probes 4, 193-203. 16 . Mainil, J . G ., Moseley, S . L ., Schneider, R . A ., Sutch, K ., Casey, T . A . & Moon, H . W. (1986) . Hybridization of bovine Escherichia coli isolates with gene probes for four enterotoxins (STaP, STaH, STb, LT) and one adhesion factor (K99). American Journal of Veterinary Research 47, 1145-8 . 17 . Broes, A ., Fairbrother, J . M., Mainil, J ., Harel, J . & Lariviere, S . (1988) . Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli serotype 08 : K'2829' strains isolated from piglets with diarrhea . Journal of Clinical Microbiology 26, 2402-9 18. Maniatis, T ., Fritsch, E. F . & Sambrook, J . (1982) . Molecular cloning: A Laboratory Manual. New York : Cold Spring Harbor . 19 . Ausubel, F . M ., Brent, R., Kingston, R . E., Moore, D . D ., Smith, J . A ., Seidman, J . G . & Struhl, K . (1987) . Preparation and analysis of DNA . In Current Protocols in Molecular B iology . pp . 2 .4.1-2 .4 . 2 New York : Greene Publishing Associates and Wiley-Interscience . 20 . Lortie, L .-A., Dubreuil, J . D . & Harel, J . (1991). Characterization of Escherichia coli strains producing heat-stable enterotoxin b (STb) isolated from humans with diarrhea . Journal of Clinical Microbiology 29, 656-9. 21 . Zon, L . I ., Dorfman, D . M . & Orkin, S . H . (1989) . The polymerase chain reaction colony miniprep. Biotechniques 7, 696-8 . 22 . Harel, J ., Lapointe, H ., Fallara, A ., Lortie, L .-A ., BigrasPoulin, M ., Lariviere, S . & Fairbrother, J . M . (1991) . Detection of genes for fimbrial antigens and enterotoxins associated with Escherichia coli serogroups isolated from pigs with diarrhea . Journal of Clinical Microbiology 29, 745-52 . 23. Taylor, D . E . & Hiratsuka, K . (1990). Use of non-radioactive DNA probes for detection of Campylobacter jejuni and Campylobacter coli in stool specimens . Molecular and Cellular Probes 4, 261-71 .
Evaluation of three new techniques for the detection of STb-positive Escherichia coli strains Louis-Andre Lortie, Josee Harel, John M. Fairbrother and J . Daniel Dubreuil* Groupe de Recherche sur les Maladies Infectieuses du Porc (GREMIP), Faculte de Medecine Veterinaire, Universite de Montreal, 3200 rue Sicotte, C .P . 5000, St-Hyacinthe, Quebec, Canada J2S 7C6 (Received 17 September 1990, Accepted 21 January 1991)
A study was conducted to compare different techniques for the detection of heat-stable enterotoxin b (STb)-positive E . coli strains . Antisera against purified STb was used to develop an enzyme-linked immunosorbent assay (ELISA) . STb-positive strains identified by ELISA were tested for bioactivity in rat jejunal loops . Our ELISA was as sensitive as, but less specific than, the bioassay for detection of STbpositive strains . A non-radioactive DNA probe to detect the gene coding for STb was also developed by incorporating digoxigenin-11-dUTP into DNA by the random primed labelling technique . The nonradioactive digoxigenin-labelled DNA probe demonstrated a similar detectability to the radioactive probe and was more convenient to manipulate but was less sensitive and specific than the bioassay and the radioactive probe . In addition, the polymerase chain reaction (PCR) was used to amplify a specific portion of the gene coding for STb . The PCR was a highly specific and practical technique for the detection of STb-positive strains . All E . coli strains tested containing the STb gene produced the STb toxin .
KEYWORDS : Escherichia coli, enterotoxin, STb, DNA detection, digoxigenin, PCR, ELISA .
INTRODUCTION Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhoeal disease in swine and humans . 1,2 ETEC can produce a heat-labile toxin and a group of heat-stable toxins .' Heat-stable toxins are classified
Detection of STb has been done by the weaned pig intestinal loop assay which is laborious, expensive and not fully reproducible . Recently, it was demonstrated that STb can be detected by another biological model using adult rats ." , " Other workers have produced antibody against fusion proteins, 13,14 but
by their methanol solubility and biological activity .` STa (or ST-I) is methanol soluble and induces intestinal secretion in infant mice and neonatal pigs . STb (or ST-II) is methanol insoluble and induces intestinal secretion in neonatal pigs but does not affect infant mice .' The gene coding for STb has been cloned and sequenced .',' It codes for a 71-amino-acid peptide
antibodies directed against such fusion proteins may demonstrate cross-reactivity with non-STb-producing strains . Thus, use of antibodies directed against the purified active STb molecule should facilitate the development of a more specific in vitro immunoassay, such as an ELISA, for the quantitative determination of STb, thus obviating the need for the cumbersome pig or rat ligated loop assays ." Others have demonstrated that gene probes could be used to detect LT-positive and STa-positive ETEC as defined by bioassay . 1S Similarly, the gene coding
including a signal sequence and is totally different from that coding for STa . Even if the gene is commonly detected in porcine ETEC strains, the STb gene is uncommon in ETEC of human origin and STb does not appear to be a cause of human diarrhoeal disease .', " `Author to whom correspondence should be addressed
0890-8508/91/040271 + 05 $03 .00/0
271
© 1991 Academic Press Limited
L .-A . Lortie et al.
272
anaesthetized and, after a midline abdominal inci-
for STb can be detected by DNA hybridization with a 7,1,1' a technique not practical in radioactive probe the diagnostic laboratory . Non-isotopically labelled cloned probes for STb would constitute an alternative as they are easier to manipulate . In addition, a simple polymerase chain reaction (PCR)-based technique could provide a rapid and sensitive technique to determine the presence of the gene coding for STb in ETEC . The aim of this study was to develop practical techniques for the detection of STb-producing ETEC strains .
MATERIALS AND METHODS
sion, the small intestine was treated with a trypsin inhibitor (TI) (300 .tg ml -1 in 0 . 85% saline) . A series of eight ligated segments (loops), each 5 cm long, were made in the small intestine starting approximately 5 cm from the ileo-cecal junction . Loops were inoculated with 0 . 5 ml of test material containing 300 .tg ml - ' TI . All samples were dialysed against 20 mm TrisHCI (pH 6 . 8) and sterilized by passage through a 0 . 22 gm membrane (Acrodisc 13, Gelman) . Each sample was tested in at least two rats, in loops at different positions in the small intestine . A negative control consisting of 20 mm Tris-HCI (pH 6 . 8) was included in each animal . Abdominal incisions were closed and rats were allowed to regain conciousness . After 4 h, the rats were sacrificed and the volume of liquid in each loop
Bacterial strains Porcine Escherichia coli strains examined in this study are described in Table 1 . Escherichia coli strain K-12 (pRAS1) contained a multicopy recombinant plasmid carrying an STb toxin encoding gene .'•' 6 Strain HB101
was measured . Results were expressed as volume of liquid (ml) per length (cm) x diameter (cm) of intestine and were considered positive if greater than 0 . 05 .
was used as negative control .
Immunoassay (ELISA)
Biological assay The rat ligated loop assay was performed as described by Dubreuil et al ." Briefly 6- to 8-week-old white rats (Sprag Dawley C .D .) were fasted for 48 h,
The immunoassay was performed as described by Dubreuil et al." Crude culture supernatant (50 µl) diluted in 0 . 1 M sodium carbonate (pH 9 .6) was coated onto wells of polystyrene microtitre plates (Flow Laboratories, Inc., McLean, VA) overnight, Plates were washed three times with PBS-Tween 20,
Table 1 . Comparison of different techniques for the detection of porcine STb-positive E . coli strains Detection of STb Strains
79-5863 81-4035 82-4247 83-2240 84-442 84-3922 84-3948 88-261 88-5623 89-682 80-3972 88-3732 79-6755 80-3261 80-3531 81-7169 88-2467 pRAS 1 HB101
Serotypes
Rat loop assay
DNA probe "p
PCR'
ELISAt
0115 :KV165 0115:KV165 0115:KV165 0115 :KV165 0115 :KV165 0115-KV165 0115-KV165 0115 :KV165 0141 0141 0147 0147 0149 0149 0149 0149 0149
+ + +
+ + +
+ + +
+ + + + +
+ + + + + +
+
+
+
DNA probe Dig.
+
+1+ +
+1-
+ -
+1-
+
+ +
+ +
+ +
+ -
+ + +
+ + +
+ + +
+ + +
+
+
+
+
An amplified fragment at 377 pb corresponds to a positive result (Fig . 1) . t ELISA was considered positive if O .D . (414 nm) > 0. 20 .
Detection of STb-positive appropriate dilutions of rabbit anti-STb antibodies (50µl) were added to each well, and the plates incubated at 37° C for 1 h . Plates were washed three times with PBS-Tween 20, appropriate dilutions of goat anti-rabbit IgG (Daymar Laboratories Inc ., Toronto, Canada) in PBS-Tween 20 coupled to horseradish peroxidase (50 AD were added to each well and plates were incubated at 37 ° C for 15 min . Plates were washed and the chromogenic substrate 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS0. 4 mm) (Sigma), dissolved in citrate buffer (pH 4 . 0) with 2 mm H 2 02, was added . The optical density was measured with a Titertek Multiskan ® MC (Flow Laboratories, Mississauga, Canada) at 414 nm after incubation for 30 min at room temperature .
Probes The gene probe for STb enterotoxin was derived from a recombinant E . coli strain K-12 containing the plasmid pRAS1 as described previously . 7.16,11 Plasmid DNA was isolated by ultracentrifugation in a caesium chloride gradient." After digestion of the plasmid DNA by restriction endonucleases, generated fragments were separated by electrophoresis in agarose or acrylamide gels ." Probe fragments of 460 by were recovered by electro-elution and concentrated by ethanol precipitation . DNA fragments were labelled with 1a- 32 P1dCTP using a multiprimer DNA labelling kit (Amersham Corp., Arlington Heights, IL) . The non-radioactive probe was prepared by labelling the 460 by DNA fragment with digoxigenin-11dUTP by the random primed labelling technique according to the manufacturer's instructions (Boehringer Mannheim GmbH, Mannheim, Germany) .
Hybridization Colony hybridization with the radioactive probe was performed as previously described ." Briefly, strains were spot inoculated onto solid media, incubated and replicated onto a membrane . The membrane was consecutively placed, colony side up, onto filter papers saturated with lysing, denaturing and neutralizing solutions . The membrane was then prehybridized, hybridized and washed before being exposed to X-ray film . The film was developed according to the manufacturer's instructions . For the non-radioactive detection of the gene coding for STb, a plasmid DNA preparation of each sample was extracted by a rapid miniprep technique, 19 denatured and slot-blotted onto a nylon membrane (Zeta-Probe®, Bio-Rad Laboratories) with a
E. coli
2 73
Bio-Dot SF ® apparatus (Bio-Rad Laboratories) according to the manufacturer's instructions . The hybridization and the detection of the digoxigenin-labelled probe was performed as described by the manufacturer (Boehringer Mannheim GmbH, Mannheim, Germany). Sensitivity of DNA probes was evaluated by hybridization with 10-fold dilutions of target DNA .
Enzymatic amplification The polymerase chain reaction (PCR) using a forward primer (5'-GCAATAAGGTTGAGGTGAT-3') covering the 5' end of the gene and a reverse primer (5'GCCTGCAGTGAGAAATGGAC-3') flanking the 3' extremity of the STb gene' was performed as previously described ." For each sample, three bacterial colonies were transferred with a sterile toothpick into 100 pl of sterile distilled water . Each sample was boiled for 1 min, then 67 . 5 pl was withdrawn and added to 10 pl of 10 x PCR buffer21 , 16 pl of 1 . 25 mm dNTPs, 1 pg of forward primer and 1 pg of reverse primer for a final volume of 99 . 5 pl . The samples were heated to 90 ° C for 5 min, 2 . 5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) was added, and the samples were subjected to PCR in a thermal cycler . The program used was 15 s at 94 ° C, 15 s at 40° C, 60 s at 72 ° C, for 30 cycles, followed by a final 7 min extension at 72 ° C . A volume of 5 pl of each reaction mixture was subjected to electrophoresis on a 1 . 2% agarose gel stained with ethidium bromide . 11-11,2' An amplified fragment of 377 by corresponds to a positive result, as determined in a previous study ."
RESULTS Ten of the 19 E. coli strains examined in this study were STb-positive by the rat intestinal loop assay . The same 10 strains were STb-positive using the radioactive DNA probe . No false-positive results were observed using this technique . The correlation between these two techniques was 100% (Table 1) . The 10 STb-positive E . coli strains identified by the bioassay were also positive by PCR . No false-positive results were obtained by this technique (Fig . 1) . The 10 strains positive in the bioassay were also positive by ELISA . However, of the nine strains that were negative in the bioassay, four were positive by ELISA . The correlation between the ELISA and the rat intestinal loop assay was 79% . The non-radioactive digoxigenin-labelled DNA probe demonstrated a detectability similar to that of the radioactive probe by detecting as little as 1 . 0 pg of target DNA (Fig . 2). This probe detected nine STb-
2 74
L.-A. Lortie et al.
Fig. 1 . Agarose gel analysis of PCR products of the 19 E . coli strains tested in this study . The order of the strains is the same as in Table 1 . Lane M shows a 750, 410 and 280 by DNA fragment as size markers . An amplified fragment of 377 by corresponds to an STb-positive strain .
500
100
10
1.0
0.1
0
pg
(a )
( b)
Fig . 2 . Evaluation of the detectability of the digoxigenin-labelled DNA probes (a) and of the 32 P-labelled DNA probe (b) for the detection of the gene coding for the heat-stable b (STb) enterotoxin of Escherichia coli . Both probes detected as little as 1 .0 pg of DNA .
positive strains and failed to detect one strain which
The other technique to detect the gene coding for
was positive by the bioassay . However, the digoxige-
STb using a non-radioactive probe was more con-
nin-labelled DNA probe weakly hybridized with three
venient to manipulate but was less sensitive and less
STb-negative strains, two of which were positive by
specific than the radioactive probe and the bioassay .
ELISA .
This may be intrinsic to the technique because other workers have observed 15% false-positive results using a digoxigenin-labelled DNA probe to
DISCUSSION AND CONCLUSIONS
detect
Campylobacter jejuni ." Regardless of the
sensitivity and specificity of these techniques used to In this study, we developed practical techniques for
detect the gene coding for STb, they do not
the detection of STb-producing ETEC strains . We
quantitatively evaluate the presence of the toxin in
evaluated the correlation between the presence of
a sample .
the gene coding for STb and the production of this
On the other hand, ELISA could constitute a way
enterotoxin . Identical results were obtained with the
to evaluate quantitatively STb production by ETEC
radioactively-labelled DNA probe and the rat intestinal loop assay . Although a small number of strains
strains . However, four false-positive results were obtained using this technique . Our antibodies raised
were examined, we conclude that an STb-positive
against purified STb gave positive ELISA with STb-
porcine strain by probe is also an STb producer as
negative strains of different serotypes . These strains
also observed in another study ."
cannot be considered STb-positive because the two
Two different techniques were then developed to
reference assays (the rat jejunal loop assay and the
detect the STb coding gene . The polymerase chain
DNA hybridization assay with a radioactive probe) were negative for them . We conclude, overall, that the ELISA and the digoxigenin-labelled DNA probe
reaction described in this study detected all STbpositive strains and no false-positive results were observed . This technique was easy to perform and was highly specific in our study .
were as sensitive as but less specific than the bioassay and the radioactive probe .
Detection of STb-positive E. coli In conclusion, the technique with the greatest specificity and sensitivity for a rapid and non-radioactive diagnostic test of STb-positive strains was the detection of the gene coding for STb by the polymerase chain reaction . Moreover, this technique was easy to perform, rapid, specific, may be automated and is low cost-effective .
ACKNOWLEDGEMENTS This study was supported in part by a grant to J . D . D . from the National Sciences and Engineering Council of Canada (OGP0046569) and Fonds pour la formation de Chercheurs et I'Aide a la Recherche (15728540) and to J . H . from Conseil des Recherches en Peche et Agro-alimentaire du Quebec (2243) and to L.A .L . from Ministere de I'Enseignement Superieur et de la Science . We thank Stephane Bolduc for his useful assistance in the bioassay, Bernadette Foiry for her technical assistance and Charles M . Dozois for reviewing the manuscript .
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