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Development of a capture ELISA for the detection of antibodies to enteropathogenic Escherichia coli (EPEC) in rabbit flocks using intimin-specific monoclonal antibodies
Veterinary Microbiology 88 (2002) 351–366
Development of a capture ELISA for the detection of antibodies to enteropathogenic Escherichia coli (EPEC) in rabbit flocks using intimin-specific monoclonal antibodies D.G.F. Vandekerchovea,*, P.G. Kerrb, A.P. Callebautc, H.J. Ballb, T. Stakenborga, J. Marie¨na, J.E. Peetersa a
Department of Small Stock Pathology, Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium b Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT3 4SD, United Kingdom c Department of Quality and Security, Veterinary and Agrochemical Research Centre, Leuvensesteenweg 17, 3080 Tervuren, Belgium Received 13 December 2001; received in revised form 29 May 2002; accepted 29 May 2002
Abstract A capture enzyme-linked immunosorbent assay (cELISA) was developed using intimin-specific monoclonal antibodies to detect specific antibody in rabbits that have been in contact with enteropathogenic Escherichia coli (EPEC). Sera from 121 EPEC-negative, minimum-disease-level (MDL) rabbits were used for negative controls, and sera from 25 MDL rabbits, experimentally infected with EPEC of bio-/serotype 3/O15, for positive controls. These were used to determine a cut-off value for a positive cELISA result. The value selected gave the test a sensitivity of 80.0% and a specificity of 98.4% on an individual level. At this value, a flock level sensitivity and specificity of 79.2 and 85.2%, respectively were calculated for a flock with a prevalence of seven per cent, if 40 animals were tested, and a minimum of two reactors were obtained. The test characteristics improve with increasing prevalence. To evaluate the diagnostic potential of the cELISA, sera from 40 to 50 slaughter rabbits per flock from 25 rabbit flocks with bacteriologically determined EPEC status were tested. The results demonstrated that this test can be a useful tool to determine the EPEC status of a rabbitry, provided that it is used at regular intervals. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Enteropathogenic Escherichia coli; ELISA; Intimin; Rabbit; Serology
* Corresponding author. Tel.: þ32-2-3790438; fax: þ32-2-3790670. E-mail address: [email protected] (D.G.F. Vandekerchove).
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 1 2 5 - 6
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1. Introduction Rabbit enteropathogenic Escherichia coli (EPEC) are an important cause of economic losses through digestive disorders in commercial rabbitries, and can be divided into three groups, based on their bio/serotype (Peeters et al., 1988a). Strains with the bio/serotype or pathotype 1þ/O109 are highly pathogenic for young rabbits before weaning. The group comprising the pathotypes 2þ/O128 and 2þ/O132 shows variable pathogenicity for rabbits both before and after weaning. The highly pathogenic pathotypes 3/O15, 4þ/O26, and 8þ/O103 affect mostly weaned rabbits. In Belgium the pathotype 3/O15 is predominant (Peeters et al., 1988b). Colibacillosis causes losses through mortality, delayed growth, and poor feed conversion. Once EPEC are established in a rabbitry, they are extremely difficult to eradicate. There is no vaccine available that is both efficient and safe. Antibiotic resistance is increasing, and even when the antibiotic treatment is successful, three to seven per cent of the rabbits remain carriers of EPEC (Peeters, 1989), and may be the source of a renewed outbreak. Sooner or later the breeder is forced to empty the rabbitry and repopulate it with new animals. There is no guarantee, however, that the newly purchased animals are free of EPEC. Bacteriological culture has been used for diagnosis, but it is time consuming, expensive and could give false negative results in cases where antibiotics have been used. A serological test would not be influenced by the use of antibiotics, and would provide a relatively cheap method for the detection of contact with EPEC strains. We previously reported the development of an enzyme-linked immunosorbent assay (ELISA), based on 94-kDa proteins, partially purified from the outer membrane protein (OMP) fraction of EPEC cells (Vandekerchove and Peeters, 1998). This test had limited application since the protocol used to produce the target 94-kDa antigen had extremely low yields. However, it showed that systemic antibodies (IgG) could be used for the detection of EPEC infection per os. Detectable IgG levels were seen from 28 days after infection. This confirms the report of Levine et al. (1985), where after an infection per os with EPEC in two trials with human subjects, a seroconversion was seen in eight of ten and eight of nine subjects at 28 days after infection. EPEC are characterised by the presence of the locus for enterocyte effacement (LEE), which contains the eae gene, encoding intimin (Donnenberg et al., 1997). This implies that intimin is a suitable diagnostic antigen for the detection of contact with EPEC. Agin and Wolf (1997) reported a molecular weight for proteins that reacted with intiminantisera of approximately 97.5 kDa. Jerse and Kaper (1991) reported a molecular weight of 94 kDa for intimin. It was suspected that at least part of the 94-kDa protein band previously described (Vandekerchove and Peeters, 1998), consisted of intimin. The purposes of the present study were therefore: (1) confirmation of the presence of intimin in the 94-kDa protein fraction, (2) development of intimin-specific monoclonal antibodies (Mabs), and (3) development of a capture ELISA (cELISA) based on these Mabs. A cELISA, allowing the use of crude OMP rather than the purified 94-kDa proteins, would be easier to produce in large quantities and should have wider application potential.
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2. Materials and methods 2.1. Strains Strains 82/123 (pathotype 1þ/O109) and 82/90 (pathotype 2þ/O132) were cultured overnight in antibiotic medium 3 (Difco Laboratories, USA) as described by Peeters et al. (1988a), to be used for the production of the partially purified 94-kDa proteins, and in the case of 82/123, for the production of crude OMP. Strain 97/223.10 (pathotype 3/O15), which was used for the experimental infection of the rabbits, was also cultured overnight in antibiotic medium 3. 2.2. Production of the partially purified 94-kDa proteins This was done as described previously (Vandekerchove and Peeters, 1998). 2.3. Two-dimensional (2D) gel electrophoresis of the partially purified 94-kDa OMP fraction Two series of 2D gels were run. A first series was run with a non-linear (NL) IPG strip (Pharmacia, Brussels, Belgium) with pH range 3–10. Of the two gels run in parallel, one was used for silver staining, the other was used for Western blotting. Two microgram protein was used for the gel for the silver staining and 10 mg for the gel for Western blotting. For the second dimension a vertical sodium dodecyl sulphate (SDS) polyacryl amide 10% slab gel (180 mm 200 mm 1:5 mm) was used. The 2D polyacrylamide gel electrophoresis (PAGE) protocols and silver staining were performed as described by Hochstrasser et al. (1995). To identify the intimin, the nitrocellulose membrane was immunostained with an RDEC-1 intimin-specific antiserum (Agin and Wolf, 1997), kindly provided by T.S. Agin of the Walter Reed Army Institute of Research (Washington, DC, USA), at a dilution of 1:1400. The second series of gels was run as described previously, except for the use of an IPG strip with a pH range of 6–11. 2.4. In-gel protein digestion, mass spectrometry and database searching Two samples of a PrepCell-purified 94-kDa protein of strain 82/90 (2þ/O132) were further purified by 2D electrophoresis as described previously (IPG, NL, pH 3–10 and SDS-PAGE). On one gel the proteins were detected by the negative zinc-imidazole staining technique of Fernandez-Patron et al. (1995) using a commercial kit (Biorad, Nazareth, Belgium). The proteins on the second gel were stained with Coomassie Blue R-250 (Pharmacia, Brussels, Belgium). The immunogenic spots on the basic side of the gel were not clearly resolved as separate spots and therefore the negative stained spots (RS) were divided into four equal parts (RS1–RS4) and the Coomassie spots into five parts (CB1–CB5). The stained gel pieces were excised and in-gel digested as described by Williams et al. (1997) except for the omission of the alkylation step, as it was included in the 2D protocol. Negative stained spots were first washed with 1% citric acid followed by
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water washings and then treated as Coomassie stained spots. Spots were washed successively with 50% CH3CN, 50% C3HCN in 50 mM NH4HCO3 and 50% CH3CN in 10 mM NH4HCO3. Digestion was done with 0.5 mg modified trypsin (Promega, Leiden, The Netherlands) in 50 mM NH4HCO3 for 22 h at 37 8C. The peptides were extracted with 0.1% trifluoroacetic acid (TFA), 60% CH3CN, then dried in a Speedvac and kept at 20 8C until use. Peptide masses were determined by MALDI-TOF analysis on a Micromass TofSpec-2E and a Bruker Reflex TM III using standard procedures as described by Jensen et al. (1996). One digest spot (spot CB2) was desalted by capillary HPLC as described by de Jong (1998). The peptides were then eluted from the capillary column in a nanospray needle and the peptides were analysed by nano-ESI-MS and nano-ESI-MS-MS using an ion trap mass spectrometer (LCQ, Finnigan, Antwerp, Belgium). All peptide masses are given as protonated masses (M þ H)þ. Proteins were identified by searches in different databases (SWISS-PROT version 34, OWL version march 1998, EMBL) using the search programs Peptide Mass Search (http:// www.mdc-berlin.de/emu/peptide_mass.html) or ProteinLynx (Micromass, Vilvoorde, Belgium). Partial enzymatic cleavages leaving one or two cleavage sites and oxidation of methionine were considered in these searches. 2.5. Development of a hybridoma producing monoclonal antibodies (Mabs) against intimin A BALB/c mouse was immunised intraperitoneally with the partially purified 94-kDa solution. Three inoculations of 100, 50 and 50 ml of the antigen, each mixed with 50 ml adjuvant (125 mg Quil A per millilitre PBS, Superfos, DK-Vedbaek, Denmark) were given at approximately 3-week intervals. Ten days after the final inoculation, the mouse spleen cells were fused with NSO myeloma cells (Galfre and Milstein, 1981; Teh et al., 1984), and the hybridoma cells maintained in RPMI 1640 medium (Gibco, UK) supplemented with 20% gamma-globulin free horse serum. Microtitre wells coated with the immunising antigen or 1þ/O109 whole cells were used to screen the culture fluid of actively growing hybridomas by ELISA. Those giving high ELISA readings were further screened against strains from a variety of E. coli pathogroups and from related Enterobacteriaceae genera. Two hybridomas, considered to be specific to the immunising antigen, were cloned twice by limiting dilution. The tissue culture supernatants from the cloned hybridomas were also used to immunostain 2D Western blots of the partially purified 94-kDa proteins, as described in Section 2.3, as a final control of their intimin specificity. 2.6. Production of crude OMP A broth culture of reference strain 82/123 was harvested by centrifugation (17; 000 g), washed and resuspended in 1.25 M Tris buffer, pH 6.8. This was mixed with a 25% volume of SDS buffer (4% SDS, 1.25 M Tris (pH 6.8), 20% glycerol, 10% 2-mercapto-ethanol), heated to 100 8C for 5 min, cooled on ice, and centrifuged at 2000 g. The supernatant was recovered and diluted to 1.0 mg/ml for further use.
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2.7. Experimental sera One group of eight (group A) and one group of 18 (group B) 8-week-old Cunistar minimum-disease-level rabbits (N.V. Verlabreed, Nevele, Belgium) were screened for the presence of EPEC, Clostridium spiroforme and Eimeria as described by Peeters et al. (1986). In addition, they were screened for rotavirus according to the method of Vanopdenbosch (1980). Both groups were infected per os with strain 97/223.10, of pathotype 3/O15, and became excretors of the strain. Blood samples were taken on days 27–34 post-infection (dpi 27–34) from group A animals and on dpi 7 (15 animals), dpi 14 (17 animals) and dpi 21 (17 animals) for group B animals. All 25 samples taken at dpi 21 or later (dpi 21) were used for positive controls. In addition, the group B sera taken at dpi 7, dpi 14 and dpi 21 were examined to determine the test results as a function of time after infection. A collection of 121 blood samples taken at dpi 0, originating from three different groups of rabbits, all from the same origin and screened as described previously, were used for negative controls. Thirty-nine of these samples were taken from 4-week-old rabbits and 82 from 8-week-old rabbits. 2.8. Sampling of slaughter rabbits A total of 25 rabbit flocks, originating from 23 different farms, were sampled. Sera were collected at slaughter from 40 to 50 rabbits per rabbitry. The EPEC status of a farm was determined by the culture of rectal swabs collected from live animals, or the caecal content after slaughter, following the procedure as described by Peeters et al. (1986). For practical reasons the number of animals culture sampled was limited to 20 per flock, allowing the detection of a prevalence of 13.6%. The respective breeders provided information on the use of antibiotics. This was confirmed by assessing the proportion of Escherichia coli positive cultures, as described by Padilha et al. (1996) who reported that E. coli can be isolated from 70% healthy untreated rabbits after weaning. 2.9. cELISA The microtiter plates (Nunc, Maxisorp, Denmark) were coated overnight at 4 8C with 0.5 mg/ml of the intimin-specific Mabs in 0.05 M carbonate buffer (pH 9.5) containing 0.5% of bovine serum albumin (BSA), at a rate of 100 ml per well. The plates were then washed six times with phosphate buffered saline (PBS). One hundred microliter of crude OMP was added per well, and the plate was incubated at 37 8C for 1 h. The wells were washed six times with PBS–Tween (PBS, 0.25% Tween 20 diluted at 20% (v/v)). The rabbit sera were diluted to 1:625 using PTN buffer (PBS, with 2% NaCl (w/v), 0.005% Tween 80 (v/v), 0.5% BSA (w/v)) and added at a rate of 100 ml per well. The plates were incubated at 37 8C for 1 h. The plates were washed six times with PBS–Tween, and 100 ml per well of goat-anti-rabbit IgG peroxidase conjugate (Sigma, Belgium), diluted to 1:2000 using PTN buffer, was added. After incubation at 37 8C for 1 h, the plates were washed six times with PBS–Tween. TMB (3,30 ,5,50 -tetramethylbenzidine, Kierkegaard & Perry Laboratories Inc., Maryland, USA) mixed with an equal volume of H2O2 was used as
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substrate, 100 ml per well. The plates were then incubated in the dark for 10 min at 25 8C. One hundred microliter of 1 M o-phosphoric acid per well was used to stop the reaction. The optical density (OD) values were read at 450 nm (counter filter 690 nm). A positive control serum of an animal infected with and excretor of the reference strain 1þ/O109, and a negative control serum of a 4-week-old non-infected MDL-rabbit were also tested on each cELISA microtitre plate. The control sera were tested in four wells each, the test sera in duplicate. A serum was considered to react positively if its average OD was higher than the cut-off value.
3. Results Fig. 1 shows the partially purified 94-kDa fraction of strain 82/123 (1þ/O109) after 2D electrophoresis and silver staining, using an IPG pH 3–10 strip. Fig. 2a and b show the nitrocellulose membrane after immunoblotting with the RDEC 1-intimin-specific antibody, using an IPG pH 6–11 strip. This high-resolution method clearly revealed that the 94kDa band was composed of several different proteins. Proteins recognised by the intiminspecific antiserum were found in the pI-region of 8–10 of the gel, as deduced from the position of standard proteins and the manufacturer’s information. Figs. 1 and 2 not only show differences in the pI of the intimin spots, but also in their molecular weight. Table 1 shows some peptide ions found in the tryptic digest of the 94-kDa protein after 2D electrophoresis. Only the peptides corresponding with the calculated tryptic peptides from the E. coli RDEC-1 intimin (hits) are shown. This sequence is the translated E. coli RDEC-1 eae gene sequence (Agin and Wolf, 1997). MALDI-TOF mass spectrometry gave between 18 and 27 hits per spot (corresponding with a coverage ranging from 30 to 47%) identifying the RDEC-1 gene or, with similar probabilities, the intimin sequence from REPEC strain 84/110/1, which has bio/serotype 8þ/O103. The data from all spots together covered 60% of the sequence.
Fig. 1. Pattern found for strain 82/123 (pathotype 1þ/O109) after two-dimensional SDS-PAGE and silverstaining of the partially purified 94-kDa fraction, using a non-linear IPG strip (pH 3–10) for the first dimension. The intimin spots are outlined.
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Fig. 2. (a) Pattern found for strain 82/123 (pathotype 1þ/O109) after two-dimensional SDS-PAGE and immunostaining with the RDEC 1-intimin-specific antibodies of the partially purified 94-kDa fraction, using a non-linear IPG strip (pH 6–11). The intimin spots are outlined and shown in greater detail in part (b); (b) detail from part (a) showing the intimin spots not only having a different pI, but also a different molecular weight.
The identity of the spots was further confirmed by sequence information from the nanospray ESI-MS-MS data (Table 2). From seven peptides at least three and up to seven consecutive amino acid (aa) sequences were obtained. This permitted a very specific search, using PeptideSearch based on a sequence tag and information on the peptide mass. For four of the seven peptides, PeptideSearch only matched one protein sequence: intimin from the RDEC-1 or REPEC 84/110/1 strains as previously. The peptide with mass 930.2 (Table 2) was not recognised as intimin, but the FindMod tool (http://www.hcuge.ch) suggested that deamidation of the theoretical peptide with mass 929.4 and sequence SSVNGYFR (position 302–309), whereby asparagine (N) would be transformed to aspartic acid (D), could have interfered with the PeptideSearch recognition. The MS–MS fragmentation spectrum of peptide 930.2 clearly confirmed this suggestion. In CB1 a peptide was found with mass 1639.6. FindMod suggests a phosphorylation of the sequence 802–816. This peptide contains four potential residues which can be phosphorylated: aa 804, 806, 808 and 812.
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Table 1 Peptide masses measured by MALDI-TOF (M þ H)þ observeda RS1b
(M þ H)þ RS3c
CB1d
3809.66 1532 2855 1235 1363 2664 1977 1034 2839 1050 1568 1498 2170 2847 1882 2568 2489 1074 3451 2603
2664.41 1977.04 2825.36 1034.53 2839.31 1050.57 2914.5 1567.7 1497.75 2169.93 2847.48 1881.88 2568.25 1120.55 1074.54 3450.67
2792.11 2663.99 1976.75
2838.88 1050.4 2914.03 1567.46 1498.5 2847.09 1881.54 2567.87
1074.39 3450.13
2123.02 1499 2283
2283.74 2230.18
1711 1135
1135.55 1940.88
3050 2665 1651 964.8 a
1135.36 1962.8 1940.58
964.29
Calculatede
Residuesf
3809.85 1531.85 2854.4 1234.75 1362.85 2792.09 2664.44 1977.01 2825.41 1034.54 2839.3 1050.54 2915.34 1567.71 1498.7 2169.98 2847.47 1881.85 2568.25 1120.6 2488.28 1074.56 3450.67 2602.36 2123.07 1498.7 2283.1 2229.24 1710.86 1135.59 1962.01 1940.98 3049.57 2665.35 1650.85 964.44
19–52 79–92 79–103 104–115 104–116 116–144 117–144 162–180 162–188 255–264 265–288 289–297 310–333 321–333 334–347 348–365 366–392 393–408 409–428 441–449 450–471 485–493 494–526 587–613 638–657 708–721 708–729 722–744 745–760 775–783 775–792 784–801 817–847 862–886 872–886 887–894
Protonated mass of the peptide; peptide masses are monoisotopic. Spot 1, reverse-stained. c Spot 3, reverse-stained. d Spot 1, Coomassie-stained. e Theoretical protonated peptide masses from a tryptic digest of RDEC-1 (SwissProt accession number P77067). f Corresponding start and end of amino acid sequence. b
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Table 2 Peptide masses and sequence information measured by nano-ESI-MS-MS (M þ H)þ observeda
Corresponding peptide
Sequenceb
1212.8 1499.4 1268.6 1143.6 1129.6 1019.4 930.2
921–931 708–721 672–683 626–637 53–62 69–78 302–309
NQLINVGVNNK TEATTDQNGYATVK ADGSDAITYTVR SATPGQVVVSAK LLTQNAAQDR TGETVANISK SSVNGYFR
a b
Protonated mass of the peptide. Underlined sequence was experimentally detected.
All MDL rabbits were EPEC negative at the time of purchase. C. spiroforme and rotavirus were only sporadically present. A number of animals were infected with Eimeria. These were treated with toltrazuril (Baycox, Bayer, Germany) for 1 week. This treatment did not influence the experiment, except for its coccidiostatic effect. Using eight different cut-off values in the OD range of 0.200–0.550, with intervals of 50 units, the sensitivity and specificity of the test was calculated with the 25 positive control sera, and the 121 negative control sera. A cut-off value of 0.350 gave the test a specificity of 98.4% and a sensitivity of 80.0% (Table 3). Table 4 shows the performance of the assay at different time points after infection. One week after infection only 53.3% of the animals reacted. Two weeks after infection this percentage had increased to 88.2%, with a slight decrease 1 week later, to 82.4%. Because the negative control sera originated from rabbits at two different ages, a supplementary calculation of the test specificity for these two age groups is presented in Table 5. Upper and lower limits of the 95% confidence interval (CI) were calculated with the continuity correction except when the obtained proportions exceeded 80% (Agresti, 1996). In such cases the binomial distribution was used (Clopper and Pearson, 1934). With a test sensitivity of 80.0% and a specificity of 98.4% on the individual level, the calculations presented in Table 6 can be made for the flock level (Martin et al., 1992). For example, if 40 animals are tested in a flock with a prevalence of seven per cent, a flock level sensitivity and specificity of 79.2 and 85.2%, respectively, are reached. In a negative flock, Table 3 Characteristics of the cELISA when used to analyse negative and positive control sera to calculate test sensitivity and specificity on an individual level Test characteristic
Sera taken at
N
Number of sera negative
Number of sera positive
%
Lower limit–upper limit (95% CIa)
Specificity Sensitivity
dpi 0d dpi 21d
121 25
119 5
2 20
98.4 80.0
94.2–99.8b 64.3–95.7c
a
Confidence interval. Calculated using the binomial distribution. c Calculated using the normal distribution. d Day post-infection. b
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Table 4 Sensitivity of the cELISA on an individual level when used to analyse sera obtained from a group of rabbits experimentally infected with and excreting a 3/O15 strain, at different time points Sera taken at
N
Number of positive sera
Sensitivity (%)
Lower limit–upper limit (95% CIa)
dpi 7d dpi 14d dpi 21d
15 17 17
8 15 14
53.3 88.2 82.4
28.1–78.6c 63.6–98.5b 56.6–96.2b
a
Confidence interval. Calculated using the binomial distribution. c Calculated using the normal distribution. d Day post-infection. b
Table 5 Specificity of the cELISA on the individual level when used to analyse the negative control sera, split by age of the rabbits Age of rabbits (day post-infection)
N
Number of negative sera
Specificity (%)
Lower limit–upper limit (95% CIa)
4 weeks (dpi 0) 8 weeks (dpi 0) 4 þ 8 weeks (dpi 0)
39 82 121
39 80 119
100.0 97.6 98.4
91.0–100.0b 91.5–99.7b 94.2–99.8b
a b
Confidence interval. Calculated using the binomial distribution.
a test with these characteristics will give up to one false positive result. Therefore, a flock is considered positive only if more than one reactor is seen. Table 7 summarises the survey results obtained with the intimin-based cELISA on the rabbit flocks. The assay gave positive results, irrespective of the pathotype or combination of pathotypes recorded in the rabbit flock. The use of antibiotics did not appear to have an influence on the serological results. Flocks number 6a and 6b, and number 18a and 18b originated from the same farms. In the 15 flocks bacteriologically positive for one EPEC type, 2/48 (4.2%) to 17/48 (35.4%) of the sera tested positively. In the four flocks Table 6 Predicted characteristics of the cELISA on flock level with a sensitivity and specificity on the individual level of 80.0 and 98.4%, respectively Flock prevalence (%) 3 N sampled per flock Minimum expected number of reactors if positive flock Expected number of reactors if negative flock Flock level sensitivity if cutpoint > 1 reactor (%) Flock level specificity if cutpoint > 1 reactor (%)
7
10
40 45 50 40 45 50 40 45 50 2 2 2 3 3 4 4 4 5 1 1 1 1 1 1 1 1 1 48.5 54.9 60.6 79.2 84.3 88.3 90.5 93.7 95.8 85.2 82.2 79.1 85.2 82.2 79.1 85.2 82.2 79.1
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Table 7 Results obtained with sera taken at the slaughter plant Flock number
Type of EPEC
Use of antibiotics
Number of sera tested
Number (%) of sera positive
1 2 3 4 5 6a 7 8 9 10 11 12 13 14 15 16 17 18b
1þ/O109 1þ/O109 1þ/O109 2þ/O132 2þ/O132 2þ/O132 2þ/O132 3/O15 3/O15 3/O15 3/O15 3/O15 3/O15 3/O15 8þ/O103 2þ/O132, 2þ/O132, 1þ/O109, 3/O15 1þ/O109, 2þ/O132, None None None None None None
Yes No No Yes No No No Yes Yes Yes No No No No No No No No
47 50 48 48 48 48 40 50 48 48 48 47 48 48 48 50 48 50
6 17 12 10 2 5 4 8 5 4 7 7 12 17 6 9 9 2
No
48
Yes Yes Yes No No No
50 48 48 48 40 48
6b 19 20 21 18a 22 23
3/O15 3/O15 2þ/O132, 2þ/O128, 3/O15
(12.8%) (34.0%) (25.0%) (20.8%) (4.2%) (10.4%) (10.0%) (16.0%) (10.4%) (8.3%) (14.6%) (14.9%) (25.0%) (35.4%) (12.5%) (18.0%) (18.8%) (4.0%)
6 (12.5%) 10 1 1 5 4 0
(20.0%) (2.1%) (2.1%) (10.4%) (10.0%) (0.0%)
The flocks are not chronologically ordered. a First sampling of the rabbitry. b Second sampling of the rabbitry.
bacteriologically positive for two or more EPEC types, 2/50 (4.0%) to 9/48 (18.8%) sera were found positive. Pathotype 3/O15 was found in 11 of the 25 flocks analysed. Pathotypes 2þ/O132 and 1þ/O109 were found in eight and five flocks, respectively. Pathotypes 2þ/O128 and 8þ/O103 were each found in only one flock. Six rabbit flocks were bacteriologically negative for EPEC. Of these six, three had been using antibiotics, and the sera collected from these flocks tested positive in 1/48 (2.1%) to 10/50 (20.0%) of the cases. Flocks bacteriologically negative and not treated with antibiotics, gave 0/48 (0.0%) to 5/48 (10.4%) positive sera.
4. Discussion The antigen used to create a hybridoma, producing the monoclonal antibodies for use in the cELISA, was found to be intimin by immunostaining. Moreover, the identity of the
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corresponding spots of another EPEC strain, 82/90 (pathotype 2þ/O132), was confirmed to be intimin by in-gel-digest and peptide fingerprinting with MALDI-TOF, and by peptide sequencing with nano-ESI-MS-MS. The peptide with mass 3809.66 (Table 1) corresponds with the aa sequence 19–52. Agin and Wolf (1997) showed by N-terminal sequencing that the Coomassie detected protein started on residue 40. Our data support the possibility that not all the intimin of cultured E. coli is processed by cleaving off the first 39 amino acids. This can explain the difference in molecular mass of the immunogenic spots, expressed as the doublet band detected both after silverstaining (Fig. 1) and immunoblotting (Fig. 2a and b). Proteolytic degradation (Agin and Wolf, 1997) can also provide an explanation for the different molecular weights of the intimin spots. Using the ProtParam tool in the SWISS-PROT database a pI of 8.55 was obtained for E. coli intimin (strain RDEC-1, sequence 40–939). For the total sequence (1–939) of RDEC-1, ProtParam gives a pI of 8.95. Different processing by cleaving off different sequences can explain the heterogeneity seen in pI. On the other hand, chemical modifications like deamidation or phosphorylation can also explain the differences in pI seen on the 2D gel. As the digested spots were not fully separated and as the detected peptides did not cover the total sequence it was impossible to characterise the chemical differences between the different spots. The intimin-specific monoclonal antibodies captured intimin from the crude OMP preparation that was more readily produced in larger quantities than the partially purified 94-kDa proteins described by Vandekerchove and Peeters (1998). The results produced with the cELISA demonstrate its ready application to the routine diagnostic testing of field samples. EPEC are characterised by the presence of the Locus for Enterocyte Effacement (LEE), which contains the eae gene, encoding intimin (Donnenberg et al., 1997). Intimin is therefore a suitable diagnostic antigen for the detection of contact with EPEC. Agin and Wolf (1997) reported the existence of different intimin families. Several different intimin subtypes have been identified since then (Adu-Bobie et al., 1998; Oswald et al., 2000). Only b-type intimin has been detected in EPEC strains thus far (Oswald et al., 2000). When testing strains 82/123 and 82/90 by PCR, they too were found to produce b-intimin (results not shown). If different intimin subtypes are present in rabbit EPEC, it did not affect the test results in this study. The results obtained with the slaughter sera demonstrated a good detection potential for all EPEC types found. At the time of the development of the 94-kDa-based ELISA, six reference strains of different pathotypes were available for the production of the 94-kDa antigen. Initial purification experiments with strains 82/123 (1þ/O109) and 85.91.2 (8þ/O103) yielded better results with strain 82/123, so the 1þ/ O109 strain was chosen for antigen production. In later experiments, rabbits were experimentally infected with strain 97/223.10 (3/O15). Earlier work had shown that serum from rabbits infected with reference strains of different EPEC-pathotypes (1þ/O109, 2þ/O128, 2þ/O132, 3/O15, 4þ/O26 and 8þ/O103) reacted with the 94-kDa-antigen produced by these reference strains (Vandekerchove and Peeters, 1998). Furthermore, all EPEC strains have the eae gene and the 94-kDa antigen had been proved to contain intimin. Therefore the sera from the 3/O15-infected rabbits could be used as positive control sera in combination with a 1þ/O109-antigen.
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The cut-off value defined for detection of positive sera, allowed a reasonable sensitivity and specificity both on an individual and flock level. Some discrepancy was seen in the results shown in Table 4, for test sensitivity at different ages of the positive control group B rabbits, where 15 reacted positive at dpi 14, but only 14 at dpi 21. This result was due to differences in the individuals tested. Eighteen animals were present in the group, but sometimes only low volumes of serum were obtained. The sera were also used in other tests. In some cases there was not enough serum remaining for use in the cELISA, whereby the available number of sera was reduced to 15 or 17. This test was developed for use on flock level. When analysing a flock with a test that does not have 100% sensitivity and specificity, false positive and false negative results are bound to occur. The flock level sensitivity and specificity of the test was calculated to minimise the possibility of falsely declaring a flock as infected with or free of EPEC (Martin et al., 1992). Here the number of animals tested and the critical number of reactors used to decide the health status of the herd are very important. Peeters (1989) reported a prevalence of three to seven per cent of carriers in a contaminated rabbitry after successful antibiotic treatment. The prevalence of carriers is the proportion of animals that still have the causative strain in their digestive system. The proportion of animals that have been in contact with the strain, and will therefore have produced antibodies, will be substantially higher in recently infected flocks. As shown in Table 6, with an increasing prevalence within the flock, the performance of the test on flock level improves (Martin et al., 1992). If for example 40 animals are tested in a flock where seven per cent of the animals have been infected with EPEC, and the same number is tested in a flock with a prevalence of 10%, the flock level sensitivity increases from 79.2 to 90.5%. The flock level specificity remains 85.2%. Table 6 shows the results obtained for 40, 45 and 50 animals sampled. Up to one reactor was allowed for negative flocks, and the flock was considered serologically positive only if a minimum of two animals reacted. For practical reasons, the number of samples bacteriologically analysed was limited to 20 per flock. This allowed a detection of a prevalence of no less than 13.6% in a flock of 500 animals. After infection, an animal excretes the pathogenic strain continuously only during the first couple of weeks, before the excretion becomes intermittent. If antibiotics are used, excretion is lowered further, and the chance of falsely declaring a flock bacteriologically negative is increased. The sera of the EPEC-positive commercial rabbitries generally reacted strongly in the cELISA, even if antibiotics had been used, or if only a few animals had been found bacteriologically positive for EPEC. Ten sera from flock number 19 were cELISA positive, but EPEC were not detected bacteriologically. This flock had close contacts with another flock that was positive for pathotype 3/O15, so there had probably been a contamination with this pathotype. The negative bacteriological results were most likely due to the use of antibiotics. Retesting fecal material from this flock would have helped to clarify its bacteriological status, if it could have been done shortly after taking the blood samples. The cELISA was developed 4 years later. In view of the elapsed time after the blood sampling, resampling of the fecal material was no longer significant. Two of the three rabbitries that were bacteriologically negative and had been using antibiotics, produced only one positive serum. Flock 20 had problems caused by EPEC that had started only a couple of weeks before the blood sampling. Here the sampling has probably been performed too soon after
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the onset of the infection. The experimental infections (Tables 3 and 4) show that the production of a detectable level of antibodies after EPEC infection can take up to 4 weeks or more. Flock 21 had been treated with antibiotics as a preventive measure. This rabbitry had no history of infections with EPEC. Flock 18a was found to be bacteriologically negative with no antibiotics used, but repeating the analyses 4 months later, several pathotypes were detected (see results for flock 18b). The bacteriological result for 18a may have been falsely negative, but positive results were obtained with the cELISA at both samplings. The positive cELISA results with flock 22 were also not confirmed by the culture results. This rabbitry had possibly been infected with EPEC some months before, but this had not been confirmed by laboratory analysis. Flock 23 was considered to be ‘‘genuinely’’ negative. For more than 2 years, bacteriological tests on samples received regularly from this rabbitry, resulted only in the isolation of non-pathogenic E. coli strains. A direct comparison of the cELISA results with the results obtained using the 94-kDabased ELISA was not possible because a different number of sera were tested, and the sera were tested blindly, without individual numbering. The positive and negative control sera used to calculate the sensitivity and specificity of the 94-kDa-based ELISA, were no longer available at the time of the cELISA’s development, so there was no comparison possible for these sera, either. However, the sensitivity and specificity of the 94-kDa-based ELISA were recalculated using the same method as for the cELISA. In this way, a specificity of 95.8% was found, and a sensitivity of 96.2%. The cELISA had a higher specificity (98.4%), and a lower sensitivity (80.0%). The higher specificity means that fewer false positive results will be obtained. On the other hand, the lower sensitivity means that more false negatives are likely to be found. Indeed the cELISA generally yielded a lower number of positive sera for the rabbitries than the 94-kDa-based ELISA. However, when used on flock level, the performance of the cELISA is quite satisfactory. Sanitation of livestock from enzootic diseases has been proved possible before, like for Salmonella enteritidis in poultry (McIlroy et al., 1989; Edel, 1994) or Mycoplasma hyopneumoniae in swine (Heinonen et al., 1999). Serological tests proved to be a valuable tool in this process. However, a number of measures must accompany the serological testing to minimise the risk of reintroducing the pathogen in the rabbitry: thorough cleaning and desinfection before restocking, use of a hygiene lock, and screening of all animals that are newly introduced into the flock. In view of the asymptomatic carriers that can be expected after antibiotic treatment (Peeters, 1989) it is not advisable to try and sanitise a rabbitry using antibiotics, even though this method has been used successfully in other species, like poultry (Edel, 1994). In this case, the flocks became serologically negative 2–3 months after eradication of S. enteritidis. To avoid rabbitries being declared serologically negative because they are sampled too soon after an EPEC infection, the cELISA should be repeated regularly. A rabbit is weaned at the age of 4–5 weeks, and slaughtered at 11–12 weeks. Most rabbits will be infected during the first 2 weeks after weaning, and a seroconversion can be detected for the majority of the animals at 4 weeks after infection. On the other hand, reproduction stock may remain in the rabbitry for up to a year, and, if they are carriers, play a key role in the infection of their offspring. A good rule, therefore, would be to test a rabbitry at every flock ‘‘turnover’’, which would imply testing the rabbitry every 6 weeks. Both slaughter rabbits and does should be tested. If a farm has negative results at each repeated sampling, it could
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take on a quality label, which assures the prospective buyers of rabbits that these animals are free of EPEC. If the serological results leave room for doubt, even after repeating the test, the status of the animals should be confirmed by extensive bacteriological sampling.
Acknowledgements Part of this work was supported by a grant of the Section of Contractual Research, Directorate General Research and Development of the Belgian Federal Ministry of Agriculture. The authors wish to thank the breeders and slaughterhouses that have collaborated with this study. Also many thanks to T. Agin and M. Wolf for sending us the intimin-specific antiserum, and to M. de Haan, I. Heymans, D. Vandergheynst, M. Van Hessche and L. Van Muylem for their excellent technical support.
References Adu-Bobie, J., Frankel, G., Bain, C., Goncalves, A.G., Trabulsi, L.R., Douce, G., Knutton, S., Dougan, G., 1998. Detection of intimins a, b, g, and d, four intimin derivatives expressed by attaching and effacing microbial pathogens. J. Clin. Microbiol. 36, 662–668. Agin, T.S., Wolf, M.K., 1997. Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine. Infect. Immun. 65, 320–326. Agresti, A., 1996. An Introduction to Categorical Data Analysis. Wiley/Interscience, New York. Clopper, C.J., Pearson, E.S., 1934. The use of confidence or fiducial limits in the case of the binomial. Biometrika 26, 404–413. de Jong, A., 1998. Contribution of mass spectrometry to contemporary immunology. Mass Spectrom. Rev. 17, 311–315. Donnenberg, M.S., Kaper, J.B., Finlay, B.B., 1997. Interactions between enteropathogenic Escherichia coli and host epithelial cells. Trends Microbiol. 5, 109–114. Edel, W., 1994. Salmonella enteritidis eradication programme in poultry breeder flocks in The Netherlands. Int. J. Food Microbiol. 21, 171–178. Fernandez-Patron, C., Calero, M., Collazo, P., Garcia, J., Madrazo, J., Musacchio, A., Soriano, F., Estrada, R., Frank, R., Castellanos-Serra, L., 1995. Protein reverse staining: high-efficiency microanalysis of unmodified proteins detected on electrophoresis gels. Anal. Biochem. 224, 203–211. Galfre, G., Milstein, C., 1981. Preparation of monoclonal antibodies: strategies and procedures. Meth. Enzymol. 73B, 3–46. Heinonen, M., Autio, T., Saloniemi, H., Tuovinen, V., 1999. Eradication of Mycoplasma hyopneumoniae from infected swine herds joining the LSO 2000 health class. Acta Vet. Scand. 40, 241–252. Hochstrasser, D.F., Sanchez, J.-Ch., Pasquali, Ch., Ravier, F., Golaz, O., Appel, R., 1995. Two-Dimensional Polyacrylamide Gel Electrophoresis Training Course Manual. Geneva University Hospital, Switzerland. Jensen, O.N., Podtelejnikov, A., Mann, M., 1996. Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps. Rap. Comm. Mass Spectrom. 10, 1371– 1378. Jerse, A.E., Kaper, J.B., 1991. The eae gene of enteropathogenic Escherichia coli encodes a 94-kDa membrane protein, the expression of which is influenced by the EAF plasmid. Infect. Immun. 59, 4302–4309. Levine, M.M., Nataro, J.P., Karch, H., Baldini, M.M., Kaper, J.B., Black, R.E., Clements, M.L., O’Brien, A.D., 1985. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J. Infect. Dis. 152, 550–559. Martin, S.W., Shoukri, M., Thorburn, M.A., 1992. Evaluating the health status of herds based on tests applied to individuals. Prev. Vet. Med. 14, 33–43.
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McIlroy, S.G., McCracken, R.M., Neill, S.D., O’Brien, J.J., 1989. Control, prevention and eradication of Salmonella enteritidis infection in broiler and broiler breeder flocks. Vet. Rec. 125, 545–548. Oswald, E., Schmidt, H., Morabito, S., Karch, H., Marches, O., Caprioli, A., 2000. Typing of intimin genes in human and enteropathogenic Escherichia coli: characterization of a new intimin variant. Infect. Immun. 68, 64–71. Padilha, M.T.S., Licois, D., Coudert, P., 1996. Frequency of the carriage and enumeration of Escherichia coli in caecal content of 15- to 49-day-old rabbits. In: Proceedings of the VI World Rabbit Congress, Toulouse, France, July 1996. Peeters, J.E., Geeroms, R., Carman, R., Wilkins, T., 1986. Significance of Clostridium spiroforme in the enteritis-complex of commercial rabbits. Vet. Microbiol. 12, 25–31. Peeters, J.E., Geeroms, R., Ørskov, F., 1988a. Biotype, serotype, and pathogenicity of attaching and effacing enteropathogenic Escherichia coli strains isolated from diarrhoeic commercial rabbits. Infect. Immun. 56, 1442–1448. Peeters, J.E., Geeroms, R., Vroonen, C., Pohl, P., 1988b. A selective citrate-sorbose medium for screening certain enteropathogenic attaching and effacing Escherichia coli in weaned rabbits. Vlaams Diergeneeskd. Tijdschr. 57, 264–270. Peeters, J.E., 1989. Ademhalings—en spijsverteringsstoornissen in de industrie¨ le slachtkonijnenhouderij. Diergeneesk. Memo. 36, 126. Teh, C.-Z., Wong, E., Lee, C.-Y.G., 1984. Generation of monoclonal antibodies to human gonadotrophin by a facile cloning procedure. J. Appl. Biochem. 6, 48–55. Vandekerchove, D.G.F., Peeters, J.E., 1998. Detection of specific antibodies against strains of enteropathogenic Escherichia coli (EPEC) in the rabbit by an ELISA using 94-kDa proteins. World Rabbit Sci. 6, 303–310. Vanopdenbosch, E., 1980. Immunodiffusion test for the detection of rotavirus antigen in faecal material and gut homogenates. Curr. Topics Vet. Med. Anim. Sci. 13 (6899), 118–120. Williams, K., LoPresti, M., Stone, K., 1997. Techniques in Protein Chemistry VIII. Academic Press, San Diego, CA, USA, pp. 79–90.
Development of a capture ELISA for the detection of antibodies to enteropathogenic Escherichia coli (EPEC) in rabbit flocks using intimin-specific monoclonal antibodies D.G.F. Vandekerchovea,*, P.G. Kerrb, A.P. Callebautc, H.J. Ballb, T. Stakenborga, J. Marie¨na, J.E. Peetersa a
Department of Small Stock Pathology, Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium b Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT3 4SD, United Kingdom c Department of Quality and Security, Veterinary and Agrochemical Research Centre, Leuvensesteenweg 17, 3080 Tervuren, Belgium Received 13 December 2001; received in revised form 29 May 2002; accepted 29 May 2002
Abstract A capture enzyme-linked immunosorbent assay (cELISA) was developed using intimin-specific monoclonal antibodies to detect specific antibody in rabbits that have been in contact with enteropathogenic Escherichia coli (EPEC). Sera from 121 EPEC-negative, minimum-disease-level (MDL) rabbits were used for negative controls, and sera from 25 MDL rabbits, experimentally infected with EPEC of bio-/serotype 3/O15, for positive controls. These were used to determine a cut-off value for a positive cELISA result. The value selected gave the test a sensitivity of 80.0% and a specificity of 98.4% on an individual level. At this value, a flock level sensitivity and specificity of 79.2 and 85.2%, respectively were calculated for a flock with a prevalence of seven per cent, if 40 animals were tested, and a minimum of two reactors were obtained. The test characteristics improve with increasing prevalence. To evaluate the diagnostic potential of the cELISA, sera from 40 to 50 slaughter rabbits per flock from 25 rabbit flocks with bacteriologically determined EPEC status were tested. The results demonstrated that this test can be a useful tool to determine the EPEC status of a rabbitry, provided that it is used at regular intervals. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Enteropathogenic Escherichia coli; ELISA; Intimin; Rabbit; Serology
* Corresponding author. Tel.: þ32-2-3790438; fax: þ32-2-3790670. E-mail address: [email protected] (D.G.F. Vandekerchove).
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 1 2 5 - 6
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1. Introduction Rabbit enteropathogenic Escherichia coli (EPEC) are an important cause of economic losses through digestive disorders in commercial rabbitries, and can be divided into three groups, based on their bio/serotype (Peeters et al., 1988a). Strains with the bio/serotype or pathotype 1þ/O109 are highly pathogenic for young rabbits before weaning. The group comprising the pathotypes 2þ/O128 and 2þ/O132 shows variable pathogenicity for rabbits both before and after weaning. The highly pathogenic pathotypes 3/O15, 4þ/O26, and 8þ/O103 affect mostly weaned rabbits. In Belgium the pathotype 3/O15 is predominant (Peeters et al., 1988b). Colibacillosis causes losses through mortality, delayed growth, and poor feed conversion. Once EPEC are established in a rabbitry, they are extremely difficult to eradicate. There is no vaccine available that is both efficient and safe. Antibiotic resistance is increasing, and even when the antibiotic treatment is successful, three to seven per cent of the rabbits remain carriers of EPEC (Peeters, 1989), and may be the source of a renewed outbreak. Sooner or later the breeder is forced to empty the rabbitry and repopulate it with new animals. There is no guarantee, however, that the newly purchased animals are free of EPEC. Bacteriological culture has been used for diagnosis, but it is time consuming, expensive and could give false negative results in cases where antibiotics have been used. A serological test would not be influenced by the use of antibiotics, and would provide a relatively cheap method for the detection of contact with EPEC strains. We previously reported the development of an enzyme-linked immunosorbent assay (ELISA), based on 94-kDa proteins, partially purified from the outer membrane protein (OMP) fraction of EPEC cells (Vandekerchove and Peeters, 1998). This test had limited application since the protocol used to produce the target 94-kDa antigen had extremely low yields. However, it showed that systemic antibodies (IgG) could be used for the detection of EPEC infection per os. Detectable IgG levels were seen from 28 days after infection. This confirms the report of Levine et al. (1985), where after an infection per os with EPEC in two trials with human subjects, a seroconversion was seen in eight of ten and eight of nine subjects at 28 days after infection. EPEC are characterised by the presence of the locus for enterocyte effacement (LEE), which contains the eae gene, encoding intimin (Donnenberg et al., 1997). This implies that intimin is a suitable diagnostic antigen for the detection of contact with EPEC. Agin and Wolf (1997) reported a molecular weight for proteins that reacted with intiminantisera of approximately 97.5 kDa. Jerse and Kaper (1991) reported a molecular weight of 94 kDa for intimin. It was suspected that at least part of the 94-kDa protein band previously described (Vandekerchove and Peeters, 1998), consisted of intimin. The purposes of the present study were therefore: (1) confirmation of the presence of intimin in the 94-kDa protein fraction, (2) development of intimin-specific monoclonal antibodies (Mabs), and (3) development of a capture ELISA (cELISA) based on these Mabs. A cELISA, allowing the use of crude OMP rather than the purified 94-kDa proteins, would be easier to produce in large quantities and should have wider application potential.
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2. Materials and methods 2.1. Strains Strains 82/123 (pathotype 1þ/O109) and 82/90 (pathotype 2þ/O132) were cultured overnight in antibiotic medium 3 (Difco Laboratories, USA) as described by Peeters et al. (1988a), to be used for the production of the partially purified 94-kDa proteins, and in the case of 82/123, for the production of crude OMP. Strain 97/223.10 (pathotype 3/O15), which was used for the experimental infection of the rabbits, was also cultured overnight in antibiotic medium 3. 2.2. Production of the partially purified 94-kDa proteins This was done as described previously (Vandekerchove and Peeters, 1998). 2.3. Two-dimensional (2D) gel electrophoresis of the partially purified 94-kDa OMP fraction Two series of 2D gels were run. A first series was run with a non-linear (NL) IPG strip (Pharmacia, Brussels, Belgium) with pH range 3–10. Of the two gels run in parallel, one was used for silver staining, the other was used for Western blotting. Two microgram protein was used for the gel for the silver staining and 10 mg for the gel for Western blotting. For the second dimension a vertical sodium dodecyl sulphate (SDS) polyacryl amide 10% slab gel (180 mm 200 mm 1:5 mm) was used. The 2D polyacrylamide gel electrophoresis (PAGE) protocols and silver staining were performed as described by Hochstrasser et al. (1995). To identify the intimin, the nitrocellulose membrane was immunostained with an RDEC-1 intimin-specific antiserum (Agin and Wolf, 1997), kindly provided by T.S. Agin of the Walter Reed Army Institute of Research (Washington, DC, USA), at a dilution of 1:1400. The second series of gels was run as described previously, except for the use of an IPG strip with a pH range of 6–11. 2.4. In-gel protein digestion, mass spectrometry and database searching Two samples of a PrepCell-purified 94-kDa protein of strain 82/90 (2þ/O132) were further purified by 2D electrophoresis as described previously (IPG, NL, pH 3–10 and SDS-PAGE). On one gel the proteins were detected by the negative zinc-imidazole staining technique of Fernandez-Patron et al. (1995) using a commercial kit (Biorad, Nazareth, Belgium). The proteins on the second gel were stained with Coomassie Blue R-250 (Pharmacia, Brussels, Belgium). The immunogenic spots on the basic side of the gel were not clearly resolved as separate spots and therefore the negative stained spots (RS) were divided into four equal parts (RS1–RS4) and the Coomassie spots into five parts (CB1–CB5). The stained gel pieces were excised and in-gel digested as described by Williams et al. (1997) except for the omission of the alkylation step, as it was included in the 2D protocol. Negative stained spots were first washed with 1% citric acid followed by
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water washings and then treated as Coomassie stained spots. Spots were washed successively with 50% CH3CN, 50% C3HCN in 50 mM NH4HCO3 and 50% CH3CN in 10 mM NH4HCO3. Digestion was done with 0.5 mg modified trypsin (Promega, Leiden, The Netherlands) in 50 mM NH4HCO3 for 22 h at 37 8C. The peptides were extracted with 0.1% trifluoroacetic acid (TFA), 60% CH3CN, then dried in a Speedvac and kept at 20 8C until use. Peptide masses were determined by MALDI-TOF analysis on a Micromass TofSpec-2E and a Bruker Reflex TM III using standard procedures as described by Jensen et al. (1996). One digest spot (spot CB2) was desalted by capillary HPLC as described by de Jong (1998). The peptides were then eluted from the capillary column in a nanospray needle and the peptides were analysed by nano-ESI-MS and nano-ESI-MS-MS using an ion trap mass spectrometer (LCQ, Finnigan, Antwerp, Belgium). All peptide masses are given as protonated masses (M þ H)þ. Proteins were identified by searches in different databases (SWISS-PROT version 34, OWL version march 1998, EMBL) using the search programs Peptide Mass Search (http:// www.mdc-berlin.de/emu/peptide_mass.html) or ProteinLynx (Micromass, Vilvoorde, Belgium). Partial enzymatic cleavages leaving one or two cleavage sites and oxidation of methionine were considered in these searches. 2.5. Development of a hybridoma producing monoclonal antibodies (Mabs) against intimin A BALB/c mouse was immunised intraperitoneally with the partially purified 94-kDa solution. Three inoculations of 100, 50 and 50 ml of the antigen, each mixed with 50 ml adjuvant (125 mg Quil A per millilitre PBS, Superfos, DK-Vedbaek, Denmark) were given at approximately 3-week intervals. Ten days after the final inoculation, the mouse spleen cells were fused with NSO myeloma cells (Galfre and Milstein, 1981; Teh et al., 1984), and the hybridoma cells maintained in RPMI 1640 medium (Gibco, UK) supplemented with 20% gamma-globulin free horse serum. Microtitre wells coated with the immunising antigen or 1þ/O109 whole cells were used to screen the culture fluid of actively growing hybridomas by ELISA. Those giving high ELISA readings were further screened against strains from a variety of E. coli pathogroups and from related Enterobacteriaceae genera. Two hybridomas, considered to be specific to the immunising antigen, were cloned twice by limiting dilution. The tissue culture supernatants from the cloned hybridomas were also used to immunostain 2D Western blots of the partially purified 94-kDa proteins, as described in Section 2.3, as a final control of their intimin specificity. 2.6. Production of crude OMP A broth culture of reference strain 82/123 was harvested by centrifugation (17; 000 g), washed and resuspended in 1.25 M Tris buffer, pH 6.8. This was mixed with a 25% volume of SDS buffer (4% SDS, 1.25 M Tris (pH 6.8), 20% glycerol, 10% 2-mercapto-ethanol), heated to 100 8C for 5 min, cooled on ice, and centrifuged at 2000 g. The supernatant was recovered and diluted to 1.0 mg/ml for further use.
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2.7. Experimental sera One group of eight (group A) and one group of 18 (group B) 8-week-old Cunistar minimum-disease-level rabbits (N.V. Verlabreed, Nevele, Belgium) were screened for the presence of EPEC, Clostridium spiroforme and Eimeria as described by Peeters et al. (1986). In addition, they were screened for rotavirus according to the method of Vanopdenbosch (1980). Both groups were infected per os with strain 97/223.10, of pathotype 3/O15, and became excretors of the strain. Blood samples were taken on days 27–34 post-infection (dpi 27–34) from group A animals and on dpi 7 (15 animals), dpi 14 (17 animals) and dpi 21 (17 animals) for group B animals. All 25 samples taken at dpi 21 or later (dpi 21) were used for positive controls. In addition, the group B sera taken at dpi 7, dpi 14 and dpi 21 were examined to determine the test results as a function of time after infection. A collection of 121 blood samples taken at dpi 0, originating from three different groups of rabbits, all from the same origin and screened as described previously, were used for negative controls. Thirty-nine of these samples were taken from 4-week-old rabbits and 82 from 8-week-old rabbits. 2.8. Sampling of slaughter rabbits A total of 25 rabbit flocks, originating from 23 different farms, were sampled. Sera were collected at slaughter from 40 to 50 rabbits per rabbitry. The EPEC status of a farm was determined by the culture of rectal swabs collected from live animals, or the caecal content after slaughter, following the procedure as described by Peeters et al. (1986). For practical reasons the number of animals culture sampled was limited to 20 per flock, allowing the detection of a prevalence of 13.6%. The respective breeders provided information on the use of antibiotics. This was confirmed by assessing the proportion of Escherichia coli positive cultures, as described by Padilha et al. (1996) who reported that E. coli can be isolated from 70% healthy untreated rabbits after weaning. 2.9. cELISA The microtiter plates (Nunc, Maxisorp, Denmark) were coated overnight at 4 8C with 0.5 mg/ml of the intimin-specific Mabs in 0.05 M carbonate buffer (pH 9.5) containing 0.5% of bovine serum albumin (BSA), at a rate of 100 ml per well. The plates were then washed six times with phosphate buffered saline (PBS). One hundred microliter of crude OMP was added per well, and the plate was incubated at 37 8C for 1 h. The wells were washed six times with PBS–Tween (PBS, 0.25% Tween 20 diluted at 20% (v/v)). The rabbit sera were diluted to 1:625 using PTN buffer (PBS, with 2% NaCl (w/v), 0.005% Tween 80 (v/v), 0.5% BSA (w/v)) and added at a rate of 100 ml per well. The plates were incubated at 37 8C for 1 h. The plates were washed six times with PBS–Tween, and 100 ml per well of goat-anti-rabbit IgG peroxidase conjugate (Sigma, Belgium), diluted to 1:2000 using PTN buffer, was added. After incubation at 37 8C for 1 h, the plates were washed six times with PBS–Tween. TMB (3,30 ,5,50 -tetramethylbenzidine, Kierkegaard & Perry Laboratories Inc., Maryland, USA) mixed with an equal volume of H2O2 was used as
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substrate, 100 ml per well. The plates were then incubated in the dark for 10 min at 25 8C. One hundred microliter of 1 M o-phosphoric acid per well was used to stop the reaction. The optical density (OD) values were read at 450 nm (counter filter 690 nm). A positive control serum of an animal infected with and excretor of the reference strain 1þ/O109, and a negative control serum of a 4-week-old non-infected MDL-rabbit were also tested on each cELISA microtitre plate. The control sera were tested in four wells each, the test sera in duplicate. A serum was considered to react positively if its average OD was higher than the cut-off value.
3. Results Fig. 1 shows the partially purified 94-kDa fraction of strain 82/123 (1þ/O109) after 2D electrophoresis and silver staining, using an IPG pH 3–10 strip. Fig. 2a and b show the nitrocellulose membrane after immunoblotting with the RDEC 1-intimin-specific antibody, using an IPG pH 6–11 strip. This high-resolution method clearly revealed that the 94kDa band was composed of several different proteins. Proteins recognised by the intiminspecific antiserum were found in the pI-region of 8–10 of the gel, as deduced from the position of standard proteins and the manufacturer’s information. Figs. 1 and 2 not only show differences in the pI of the intimin spots, but also in their molecular weight. Table 1 shows some peptide ions found in the tryptic digest of the 94-kDa protein after 2D electrophoresis. Only the peptides corresponding with the calculated tryptic peptides from the E. coli RDEC-1 intimin (hits) are shown. This sequence is the translated E. coli RDEC-1 eae gene sequence (Agin and Wolf, 1997). MALDI-TOF mass spectrometry gave between 18 and 27 hits per spot (corresponding with a coverage ranging from 30 to 47%) identifying the RDEC-1 gene or, with similar probabilities, the intimin sequence from REPEC strain 84/110/1, which has bio/serotype 8þ/O103. The data from all spots together covered 60% of the sequence.
Fig. 1. Pattern found for strain 82/123 (pathotype 1þ/O109) after two-dimensional SDS-PAGE and silverstaining of the partially purified 94-kDa fraction, using a non-linear IPG strip (pH 3–10) for the first dimension. The intimin spots are outlined.
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Fig. 2. (a) Pattern found for strain 82/123 (pathotype 1þ/O109) after two-dimensional SDS-PAGE and immunostaining with the RDEC 1-intimin-specific antibodies of the partially purified 94-kDa fraction, using a non-linear IPG strip (pH 6–11). The intimin spots are outlined and shown in greater detail in part (b); (b) detail from part (a) showing the intimin spots not only having a different pI, but also a different molecular weight.
The identity of the spots was further confirmed by sequence information from the nanospray ESI-MS-MS data (Table 2). From seven peptides at least three and up to seven consecutive amino acid (aa) sequences were obtained. This permitted a very specific search, using PeptideSearch based on a sequence tag and information on the peptide mass. For four of the seven peptides, PeptideSearch only matched one protein sequence: intimin from the RDEC-1 or REPEC 84/110/1 strains as previously. The peptide with mass 930.2 (Table 2) was not recognised as intimin, but the FindMod tool (http://www.hcuge.ch) suggested that deamidation of the theoretical peptide with mass 929.4 and sequence SSVNGYFR (position 302–309), whereby asparagine (N) would be transformed to aspartic acid (D), could have interfered with the PeptideSearch recognition. The MS–MS fragmentation spectrum of peptide 930.2 clearly confirmed this suggestion. In CB1 a peptide was found with mass 1639.6. FindMod suggests a phosphorylation of the sequence 802–816. This peptide contains four potential residues which can be phosphorylated: aa 804, 806, 808 and 812.
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Table 1 Peptide masses measured by MALDI-TOF (M þ H)þ observeda RS1b
(M þ H)þ RS3c
CB1d
3809.66 1532 2855 1235 1363 2664 1977 1034 2839 1050 1568 1498 2170 2847 1882 2568 2489 1074 3451 2603
2664.41 1977.04 2825.36 1034.53 2839.31 1050.57 2914.5 1567.7 1497.75 2169.93 2847.48 1881.88 2568.25 1120.55 1074.54 3450.67
2792.11 2663.99 1976.75
2838.88 1050.4 2914.03 1567.46 1498.5 2847.09 1881.54 2567.87
1074.39 3450.13
2123.02 1499 2283
2283.74 2230.18
1711 1135
1135.55 1940.88
3050 2665 1651 964.8 a
1135.36 1962.8 1940.58
964.29
Calculatede
Residuesf
3809.85 1531.85 2854.4 1234.75 1362.85 2792.09 2664.44 1977.01 2825.41 1034.54 2839.3 1050.54 2915.34 1567.71 1498.7 2169.98 2847.47 1881.85 2568.25 1120.6 2488.28 1074.56 3450.67 2602.36 2123.07 1498.7 2283.1 2229.24 1710.86 1135.59 1962.01 1940.98 3049.57 2665.35 1650.85 964.44
19–52 79–92 79–103 104–115 104–116 116–144 117–144 162–180 162–188 255–264 265–288 289–297 310–333 321–333 334–347 348–365 366–392 393–408 409–428 441–449 450–471 485–493 494–526 587–613 638–657 708–721 708–729 722–744 745–760 775–783 775–792 784–801 817–847 862–886 872–886 887–894
Protonated mass of the peptide; peptide masses are monoisotopic. Spot 1, reverse-stained. c Spot 3, reverse-stained. d Spot 1, Coomassie-stained. e Theoretical protonated peptide masses from a tryptic digest of RDEC-1 (SwissProt accession number P77067). f Corresponding start and end of amino acid sequence. b
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Table 2 Peptide masses and sequence information measured by nano-ESI-MS-MS (M þ H)þ observeda
Corresponding peptide
Sequenceb
1212.8 1499.4 1268.6 1143.6 1129.6 1019.4 930.2
921–931 708–721 672–683 626–637 53–62 69–78 302–309
NQLINVGVNNK TEATTDQNGYATVK ADGSDAITYTVR SATPGQVVVSAK LLTQNAAQDR TGETVANISK SSVNGYFR
a b
Protonated mass of the peptide. Underlined sequence was experimentally detected.
All MDL rabbits were EPEC negative at the time of purchase. C. spiroforme and rotavirus were only sporadically present. A number of animals were infected with Eimeria. These were treated with toltrazuril (Baycox, Bayer, Germany) for 1 week. This treatment did not influence the experiment, except for its coccidiostatic effect. Using eight different cut-off values in the OD range of 0.200–0.550, with intervals of 50 units, the sensitivity and specificity of the test was calculated with the 25 positive control sera, and the 121 negative control sera. A cut-off value of 0.350 gave the test a specificity of 98.4% and a sensitivity of 80.0% (Table 3). Table 4 shows the performance of the assay at different time points after infection. One week after infection only 53.3% of the animals reacted. Two weeks after infection this percentage had increased to 88.2%, with a slight decrease 1 week later, to 82.4%. Because the negative control sera originated from rabbits at two different ages, a supplementary calculation of the test specificity for these two age groups is presented in Table 5. Upper and lower limits of the 95% confidence interval (CI) were calculated with the continuity correction except when the obtained proportions exceeded 80% (Agresti, 1996). In such cases the binomial distribution was used (Clopper and Pearson, 1934). With a test sensitivity of 80.0% and a specificity of 98.4% on the individual level, the calculations presented in Table 6 can be made for the flock level (Martin et al., 1992). For example, if 40 animals are tested in a flock with a prevalence of seven per cent, a flock level sensitivity and specificity of 79.2 and 85.2%, respectively, are reached. In a negative flock, Table 3 Characteristics of the cELISA when used to analyse negative and positive control sera to calculate test sensitivity and specificity on an individual level Test characteristic
Sera taken at
N
Number of sera negative
Number of sera positive
%
Lower limit–upper limit (95% CIa)
Specificity Sensitivity
dpi 0d dpi 21d
121 25
119 5
2 20
98.4 80.0
94.2–99.8b 64.3–95.7c
a
Confidence interval. Calculated using the binomial distribution. c Calculated using the normal distribution. d Day post-infection. b
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Table 4 Sensitivity of the cELISA on an individual level when used to analyse sera obtained from a group of rabbits experimentally infected with and excreting a 3/O15 strain, at different time points Sera taken at
N
Number of positive sera
Sensitivity (%)
Lower limit–upper limit (95% CIa)
dpi 7d dpi 14d dpi 21d
15 17 17
8 15 14
53.3 88.2 82.4
28.1–78.6c 63.6–98.5b 56.6–96.2b
a
Confidence interval. Calculated using the binomial distribution. c Calculated using the normal distribution. d Day post-infection. b
Table 5 Specificity of the cELISA on the individual level when used to analyse the negative control sera, split by age of the rabbits Age of rabbits (day post-infection)
N
Number of negative sera
Specificity (%)
Lower limit–upper limit (95% CIa)
4 weeks (dpi 0) 8 weeks (dpi 0) 4 þ 8 weeks (dpi 0)
39 82 121
39 80 119
100.0 97.6 98.4
91.0–100.0b 91.5–99.7b 94.2–99.8b
a b
Confidence interval. Calculated using the binomial distribution.
a test with these characteristics will give up to one false positive result. Therefore, a flock is considered positive only if more than one reactor is seen. Table 7 summarises the survey results obtained with the intimin-based cELISA on the rabbit flocks. The assay gave positive results, irrespective of the pathotype or combination of pathotypes recorded in the rabbit flock. The use of antibiotics did not appear to have an influence on the serological results. Flocks number 6a and 6b, and number 18a and 18b originated from the same farms. In the 15 flocks bacteriologically positive for one EPEC type, 2/48 (4.2%) to 17/48 (35.4%) of the sera tested positively. In the four flocks Table 6 Predicted characteristics of the cELISA on flock level with a sensitivity and specificity on the individual level of 80.0 and 98.4%, respectively Flock prevalence (%) 3 N sampled per flock Minimum expected number of reactors if positive flock Expected number of reactors if negative flock Flock level sensitivity if cutpoint > 1 reactor (%) Flock level specificity if cutpoint > 1 reactor (%)
7
10
40 45 50 40 45 50 40 45 50 2 2 2 3 3 4 4 4 5 1 1 1 1 1 1 1 1 1 48.5 54.9 60.6 79.2 84.3 88.3 90.5 93.7 95.8 85.2 82.2 79.1 85.2 82.2 79.1 85.2 82.2 79.1
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Table 7 Results obtained with sera taken at the slaughter plant Flock number
Type of EPEC
Use of antibiotics
Number of sera tested
Number (%) of sera positive
1 2 3 4 5 6a 7 8 9 10 11 12 13 14 15 16 17 18b
1þ/O109 1þ/O109 1þ/O109 2þ/O132 2þ/O132 2þ/O132 2þ/O132 3/O15 3/O15 3/O15 3/O15 3/O15 3/O15 3/O15 8þ/O103 2þ/O132, 2þ/O132, 1þ/O109, 3/O15 1þ/O109, 2þ/O132, None None None None None None
Yes No No Yes No No No Yes Yes Yes No No No No No No No No
47 50 48 48 48 48 40 50 48 48 48 47 48 48 48 50 48 50
6 17 12 10 2 5 4 8 5 4 7 7 12 17 6 9 9 2
No
48
Yes Yes Yes No No No
50 48 48 48 40 48
6b 19 20 21 18a 22 23
3/O15 3/O15 2þ/O132, 2þ/O128, 3/O15
(12.8%) (34.0%) (25.0%) (20.8%) (4.2%) (10.4%) (10.0%) (16.0%) (10.4%) (8.3%) (14.6%) (14.9%) (25.0%) (35.4%) (12.5%) (18.0%) (18.8%) (4.0%)
6 (12.5%) 10 1 1 5 4 0
(20.0%) (2.1%) (2.1%) (10.4%) (10.0%) (0.0%)
The flocks are not chronologically ordered. a First sampling of the rabbitry. b Second sampling of the rabbitry.
bacteriologically positive for two or more EPEC types, 2/50 (4.0%) to 9/48 (18.8%) sera were found positive. Pathotype 3/O15 was found in 11 of the 25 flocks analysed. Pathotypes 2þ/O132 and 1þ/O109 were found in eight and five flocks, respectively. Pathotypes 2þ/O128 and 8þ/O103 were each found in only one flock. Six rabbit flocks were bacteriologically negative for EPEC. Of these six, three had been using antibiotics, and the sera collected from these flocks tested positive in 1/48 (2.1%) to 10/50 (20.0%) of the cases. Flocks bacteriologically negative and not treated with antibiotics, gave 0/48 (0.0%) to 5/48 (10.4%) positive sera.
4. Discussion The antigen used to create a hybridoma, producing the monoclonal antibodies for use in the cELISA, was found to be intimin by immunostaining. Moreover, the identity of the
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corresponding spots of another EPEC strain, 82/90 (pathotype 2þ/O132), was confirmed to be intimin by in-gel-digest and peptide fingerprinting with MALDI-TOF, and by peptide sequencing with nano-ESI-MS-MS. The peptide with mass 3809.66 (Table 1) corresponds with the aa sequence 19–52. Agin and Wolf (1997) showed by N-terminal sequencing that the Coomassie detected protein started on residue 40. Our data support the possibility that not all the intimin of cultured E. coli is processed by cleaving off the first 39 amino acids. This can explain the difference in molecular mass of the immunogenic spots, expressed as the doublet band detected both after silverstaining (Fig. 1) and immunoblotting (Fig. 2a and b). Proteolytic degradation (Agin and Wolf, 1997) can also provide an explanation for the different molecular weights of the intimin spots. Using the ProtParam tool in the SWISS-PROT database a pI of 8.55 was obtained for E. coli intimin (strain RDEC-1, sequence 40–939). For the total sequence (1–939) of RDEC-1, ProtParam gives a pI of 8.95. Different processing by cleaving off different sequences can explain the heterogeneity seen in pI. On the other hand, chemical modifications like deamidation or phosphorylation can also explain the differences in pI seen on the 2D gel. As the digested spots were not fully separated and as the detected peptides did not cover the total sequence it was impossible to characterise the chemical differences between the different spots. The intimin-specific monoclonal antibodies captured intimin from the crude OMP preparation that was more readily produced in larger quantities than the partially purified 94-kDa proteins described by Vandekerchove and Peeters (1998). The results produced with the cELISA demonstrate its ready application to the routine diagnostic testing of field samples. EPEC are characterised by the presence of the Locus for Enterocyte Effacement (LEE), which contains the eae gene, encoding intimin (Donnenberg et al., 1997). Intimin is therefore a suitable diagnostic antigen for the detection of contact with EPEC. Agin and Wolf (1997) reported the existence of different intimin families. Several different intimin subtypes have been identified since then (Adu-Bobie et al., 1998; Oswald et al., 2000). Only b-type intimin has been detected in EPEC strains thus far (Oswald et al., 2000). When testing strains 82/123 and 82/90 by PCR, they too were found to produce b-intimin (results not shown). If different intimin subtypes are present in rabbit EPEC, it did not affect the test results in this study. The results obtained with the slaughter sera demonstrated a good detection potential for all EPEC types found. At the time of the development of the 94-kDa-based ELISA, six reference strains of different pathotypes were available for the production of the 94-kDa antigen. Initial purification experiments with strains 82/123 (1þ/O109) and 85.91.2 (8þ/O103) yielded better results with strain 82/123, so the 1þ/ O109 strain was chosen for antigen production. In later experiments, rabbits were experimentally infected with strain 97/223.10 (3/O15). Earlier work had shown that serum from rabbits infected with reference strains of different EPEC-pathotypes (1þ/O109, 2þ/O128, 2þ/O132, 3/O15, 4þ/O26 and 8þ/O103) reacted with the 94-kDa-antigen produced by these reference strains (Vandekerchove and Peeters, 1998). Furthermore, all EPEC strains have the eae gene and the 94-kDa antigen had been proved to contain intimin. Therefore the sera from the 3/O15-infected rabbits could be used as positive control sera in combination with a 1þ/O109-antigen.
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The cut-off value defined for detection of positive sera, allowed a reasonable sensitivity and specificity both on an individual and flock level. Some discrepancy was seen in the results shown in Table 4, for test sensitivity at different ages of the positive control group B rabbits, where 15 reacted positive at dpi 14, but only 14 at dpi 21. This result was due to differences in the individuals tested. Eighteen animals were present in the group, but sometimes only low volumes of serum were obtained. The sera were also used in other tests. In some cases there was not enough serum remaining for use in the cELISA, whereby the available number of sera was reduced to 15 or 17. This test was developed for use on flock level. When analysing a flock with a test that does not have 100% sensitivity and specificity, false positive and false negative results are bound to occur. The flock level sensitivity and specificity of the test was calculated to minimise the possibility of falsely declaring a flock as infected with or free of EPEC (Martin et al., 1992). Here the number of animals tested and the critical number of reactors used to decide the health status of the herd are very important. Peeters (1989) reported a prevalence of three to seven per cent of carriers in a contaminated rabbitry after successful antibiotic treatment. The prevalence of carriers is the proportion of animals that still have the causative strain in their digestive system. The proportion of animals that have been in contact with the strain, and will therefore have produced antibodies, will be substantially higher in recently infected flocks. As shown in Table 6, with an increasing prevalence within the flock, the performance of the test on flock level improves (Martin et al., 1992). If for example 40 animals are tested in a flock where seven per cent of the animals have been infected with EPEC, and the same number is tested in a flock with a prevalence of 10%, the flock level sensitivity increases from 79.2 to 90.5%. The flock level specificity remains 85.2%. Table 6 shows the results obtained for 40, 45 and 50 animals sampled. Up to one reactor was allowed for negative flocks, and the flock was considered serologically positive only if a minimum of two animals reacted. For practical reasons, the number of samples bacteriologically analysed was limited to 20 per flock. This allowed a detection of a prevalence of no less than 13.6% in a flock of 500 animals. After infection, an animal excretes the pathogenic strain continuously only during the first couple of weeks, before the excretion becomes intermittent. If antibiotics are used, excretion is lowered further, and the chance of falsely declaring a flock bacteriologically negative is increased. The sera of the EPEC-positive commercial rabbitries generally reacted strongly in the cELISA, even if antibiotics had been used, or if only a few animals had been found bacteriologically positive for EPEC. Ten sera from flock number 19 were cELISA positive, but EPEC were not detected bacteriologically. This flock had close contacts with another flock that was positive for pathotype 3/O15, so there had probably been a contamination with this pathotype. The negative bacteriological results were most likely due to the use of antibiotics. Retesting fecal material from this flock would have helped to clarify its bacteriological status, if it could have been done shortly after taking the blood samples. The cELISA was developed 4 years later. In view of the elapsed time after the blood sampling, resampling of the fecal material was no longer significant. Two of the three rabbitries that were bacteriologically negative and had been using antibiotics, produced only one positive serum. Flock 20 had problems caused by EPEC that had started only a couple of weeks before the blood sampling. Here the sampling has probably been performed too soon after
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the onset of the infection. The experimental infections (Tables 3 and 4) show that the production of a detectable level of antibodies after EPEC infection can take up to 4 weeks or more. Flock 21 had been treated with antibiotics as a preventive measure. This rabbitry had no history of infections with EPEC. Flock 18a was found to be bacteriologically negative with no antibiotics used, but repeating the analyses 4 months later, several pathotypes were detected (see results for flock 18b). The bacteriological result for 18a may have been falsely negative, but positive results were obtained with the cELISA at both samplings. The positive cELISA results with flock 22 were also not confirmed by the culture results. This rabbitry had possibly been infected with EPEC some months before, but this had not been confirmed by laboratory analysis. Flock 23 was considered to be ‘‘genuinely’’ negative. For more than 2 years, bacteriological tests on samples received regularly from this rabbitry, resulted only in the isolation of non-pathogenic E. coli strains. A direct comparison of the cELISA results with the results obtained using the 94-kDabased ELISA was not possible because a different number of sera were tested, and the sera were tested blindly, without individual numbering. The positive and negative control sera used to calculate the sensitivity and specificity of the 94-kDa-based ELISA, were no longer available at the time of the cELISA’s development, so there was no comparison possible for these sera, either. However, the sensitivity and specificity of the 94-kDa-based ELISA were recalculated using the same method as for the cELISA. In this way, a specificity of 95.8% was found, and a sensitivity of 96.2%. The cELISA had a higher specificity (98.4%), and a lower sensitivity (80.0%). The higher specificity means that fewer false positive results will be obtained. On the other hand, the lower sensitivity means that more false negatives are likely to be found. Indeed the cELISA generally yielded a lower number of positive sera for the rabbitries than the 94-kDa-based ELISA. However, when used on flock level, the performance of the cELISA is quite satisfactory. Sanitation of livestock from enzootic diseases has been proved possible before, like for Salmonella enteritidis in poultry (McIlroy et al., 1989; Edel, 1994) or Mycoplasma hyopneumoniae in swine (Heinonen et al., 1999). Serological tests proved to be a valuable tool in this process. However, a number of measures must accompany the serological testing to minimise the risk of reintroducing the pathogen in the rabbitry: thorough cleaning and desinfection before restocking, use of a hygiene lock, and screening of all animals that are newly introduced into the flock. In view of the asymptomatic carriers that can be expected after antibiotic treatment (Peeters, 1989) it is not advisable to try and sanitise a rabbitry using antibiotics, even though this method has been used successfully in other species, like poultry (Edel, 1994). In this case, the flocks became serologically negative 2–3 months after eradication of S. enteritidis. To avoid rabbitries being declared serologically negative because they are sampled too soon after an EPEC infection, the cELISA should be repeated regularly. A rabbit is weaned at the age of 4–5 weeks, and slaughtered at 11–12 weeks. Most rabbits will be infected during the first 2 weeks after weaning, and a seroconversion can be detected for the majority of the animals at 4 weeks after infection. On the other hand, reproduction stock may remain in the rabbitry for up to a year, and, if they are carriers, play a key role in the infection of their offspring. A good rule, therefore, would be to test a rabbitry at every flock ‘‘turnover’’, which would imply testing the rabbitry every 6 weeks. Both slaughter rabbits and does should be tested. If a farm has negative results at each repeated sampling, it could
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take on a quality label, which assures the prospective buyers of rabbits that these animals are free of EPEC. If the serological results leave room for doubt, even after repeating the test, the status of the animals should be confirmed by extensive bacteriological sampling.
Acknowledgements Part of this work was supported by a grant of the Section of Contractual Research, Directorate General Research and Development of the Belgian Federal Ministry of Agriculture. The authors wish to thank the breeders and slaughterhouses that have collaborated with this study. Also many thanks to T. Agin and M. Wolf for sending us the intimin-specific antiserum, and to M. de Haan, I. Heymans, D. Vandergheynst, M. Van Hessche and L. Van Muylem for their excellent technical support.
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