Veterinary Microbiology, 32 ( 1992 ) 163-175 Elsevier Science Publishers B.V., Amsterdam
163
Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs E. Salajka, Z. Salajkova, P. Alexa and M. Hornich Veterinary Research Institute, Brno, Czechoslovakia (Accepted 2 January 1992)
ABSTRACT Salajka, E., Salajkov~i,Z., Alexa, P. and Hornich, M., 1992. Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs. Vet. Microbiol., 32:163-175. A novel common colonization factor was detected in enterotoxigenic strains of Escherichia coli isolated from intestinal contents of piglets affected with postweaning diarrhoea. This factor was antigenically distinct from the previously described K88, K99, F41,987P, CFAI, CFAII and Att25 timbrial antigens. E. coli strains possessing this factor adhered to the pig intestinal brush borders and one strain, used in experimental infection in weanlings, colonized the intestinal epithelium and induced diarrhoea. Examination of 212 toxigenic strains of E. coli isolated from weanlings revealed the presence of the novel common colonization factor in 83 strains, belonging to serogroups 025, O108, O138, O141, O147 and O157. The antigen K88 was detected in 47 strains belonging to serogroups 08, O141, O147 and O149.
INTRODUCTION
Colonization factors (adhesins) are essential for virulence of enterotoxigenic Escherichia coli (ETEC) strains. Most ETEC strains isolated from newborn piglets possess one of the colonization factors K88, K99 and 987P. However, the situation is different in ETEC strains isolated from pigs affected with postweaning diarrhoea ( P W D ) . Svendsen et al. (1974) found haemolytic E. coli strains belonging to O serogroups 149, 8, 138 and 141 in such pigs, and noted that the K88 antigen was not present in any of them. Hoblet et al. (1986) failed to detect any of the common adhesins during the examination of 22 ETEC strains isolated from weanlings. Correspondence to: P. Alexa, Veterinary Research Institute, 621 32 Brno, Czechoslovakia.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
164
E. SALAJKA ET AL.
Nakazawa et al. ( 1987 ) examined 137 ETEC strains isolated from weanlings, using antisera specific for K88, K99, 987P and F41. K88 and K99 antigens were demonstrated in 4 and 32 strains, respectively, but the other antigens under study were not detected. Nagy et al. (1990) demonstrated the K88 antigen in 126 and the 987P antigen in one of the 205 E. coli strains isolated from PWD in pigs. None of the adhesins known so far was found in the remaining 78 strains. Of the 70 strains which lacked known adhesins, 19 produced neither enterotoxin, nor verocytotoxin. Frequent finding of ETEC, lacking any of the known adhesins, in PWDaffected piglets suggests that these strains possess other, so far unidentified adhesins. Aning et al. ( 1983 ) isolated 3 E. coli 0 6 5 : K?:H9 strains possessing an adhesin different from K88, K99, F41,987P, CFAI, or CFAII from normal weaned pigs, without commenting on their pathogenicity. Kennan and Monckton (1990) reported that the majority ofE. coli strains isolated from piglets with PWD in Australia belonged to O group 141 and did not possess any of the four characterized porcine fimbrial adhesins. They investigated 50 strains by transmission electron microscopy. Two strains that produced numerous fimbriae were chosen for further characterization. The isolated fimbriae were antigenically related and had a subunit molecular weight of 17 000. These fimbriae were not produced at 18 ° C. The authors proposed that these fimbriae were a new type of adhesive fimbriae associated with porcine ETEC of O group 141. Our experiments were based on the finding that the K88 antigen was present in only some of the ETEC strains, isolated from weanlings and examined in our laboratory. However, adhesion to piglet intestinal brush borders (IBB) was positive in many of the strains lacking any of the adhesins identified so far. Sera obtained from rabbits i m m u n i z e d with these ETEC strains belonged to various O serogroups, agglutinated ETEC strains not only of homologous, but also heterologous O serogroups. These findings, as well as the possibility of removing specific agglutinins from these sera by the culture of one of these strains, have demoristrated the presence of a novel c o m m o n surface antigen. This antigen with a provisional designation "8813" was found in a number of ETEC strains belonging to various O serogroups. MATERIALS AND METHODS
Escherichia coli strains E. coli reference strains for the antigens O1-O170 and K I - K 1 0 3 and for fimbrial antigens K88, K 9 9 : F 4 1 , 9 8 7 P , CFAI, CFAII and B pil 1 were obtained courtesy of the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark, through the Czechoslovak Col-
NOVEL COLONIZATION FACq'OR
16 5
lection of Microorganisms. The reference strain Att25-X 114/83 was kindly supplied by Dr. J.A. Morris, Central Veterinary Laboratory, Weybridge, UK. Field strains of E. coli were isolated in the period 1972-1987 from the intestines of approximately 550 dead pigs and from rectal swabs of approximately 750 pigs affected with PWD or oedema disease. The samples were inoculated onto 5% sheep blood agar plates and Endo agar plates. Two to four lactose-positive colonies, identified as E. coli by c o m m o n biochemical tests, were selected as strains for the identification of O antigens. Some of these strains with identified O antigens were maintained in our laboratory on Dorset's egg medium. Of this set, 393 strains, belonging to 10 dominant O serogroups, were examined for toxin production (heat-labile enterotoxin LT, heatstable enterotoxin - - ST, Verocytotoxin - - VT); 212 toxigenic strains were used for the examination of colonization factors. This subset included also E. coli strains isolated from pigs affected with PWD or oedema disease in 19881990. The field strains No. 7395 ( O 1 4 9 : K 8 8 : L T + S T a ÷) and No. 8810 (O 138 :"8813": STa ÷ ) were used for experimental infections of weaned pigs.
Diagnostic sera Reference E. coli strains were used for the preparation of anti-O and antiOK rabbit sera by the m e t h o d described by Sojka ( 1965 ). Thirty of the 103 reference strains for OK antigens were omitted during the preparation of the anti-OK sera for the identification of K antigens owing to the proposal of Orskov et al. (1977) to delete them. Anti-OK sera were prepared against six field isolates i.e. Nos. 8923-025, 8877-O108, 8804-O138, 8914-O141, 8872-O147 and 8 8 1 3 - / O 1 4 7 / . Strain 8813 possessed an antigen related to, but not identical with O 147. Antiserum against 8813 reacted with O antigens of the reference strains G1253O 147: K89 : K88ac and D 3 5 7 - O 147 : K89: H 19, but none of the sera to these reference strains reacted with the O antigen of the strain 8813. Therefore the O antigen designation of this strain is given in parentheses. E. coli cultures grown during 18 h at 37°C on No. 2 nutrient agar (Imuna, Sarissk6 Michalany, CSFR) on 10 cm diameter Petri dishes were used for absorption of sera. Cultures, rinsed from each of 5 dishes with 2 ml 0.85% N a C I + 0.15% phenol, were mixed, centrifuged at 1200 g for 15 min., washed with 0.85% NaCI + 0.15% phenol and added to 1 ml serum. The mixture was incubated in a water bath at 37 °C for 4 h, stored at 4 °C overnight, then centrifuged at 1500 g for 30 min. Antigen identification O antigens were identified in E. coli cultures grown in No. 2 nutrient broth (Imuna, Sarissk6 Michalany, CSFR). After 18 h incubation at 37°C the cultures were stained red by adding 0.05 ml 4% triphenyltetrazolium chloride
166
E. SALAJKA ET AL.
(TTC) to 5 ml culture and incubated at 37°C for 1 hour. Thereafter the culture was heated to 100°C for 1 h and cooled. Surface antigens were identified in cultures grown in parallel in No. 2 nutrient broth and Minca medium (Guin6e et al., 1977) at 37 °C overnight and at 18°C for 48 hours. The cultures were stained with TTC and left to stand for 4 hours to let the R forms settle to the bottom. The upper part of the culture was withdrawn and centrifuged at 800 g for 10 min. The sediment was resuspended in 0.85% NaCl+0.15% phenol to obtain a density of approximately 1 × 109 CFU per ml. The antigens were identified by agglutination in U-shaped microtitre plates. After pipetting 0.025 ml of serum diluted to its agglutination titre and bacterial suspension into each well the plates were covered and incubated at room temperature overnight. The results were read by the naked eye on the subsequent day. The agglutination titre was the reciprocal of the highest dilution which gave complete agglutination.
Preparation of intestinal brush borders (IBB) IBB were prepared from small intestines of piglets by the method described by Sellwood et al. ( 1975 ), which has been partly modified in our laboratory. Intestinal segments were washed with 0.85% NaC1 and, after ligating one end, filled with 0.01 M etylendiaminetetraacetate (EDTA) pH 6.8 (0.096 M NaC1, 0.008 M KH2PO4, 0.0056 M Na2HPO4, 0.0015 M KC1 and 0.01 M EDTA). After ligating the other end the segments were put into 0.85% NaC1. The EDTA solution was discarded after 20 min. and the intestine was half-filled with 0.3 M sucrose in the same buffer. Epithelial cells were detached into the fluid by gently rubbing of the intestine between the fingers. The fluid was collected and the loosening procedure was repeated twice to obtain as many cells as possible. The cell suspension was centrifuged at 1200 g for 15 min; the supernate was discarded and the sedimented cells were resuspended in a 10-fold volume of buffered 0.005 M EDTA pH 7.4 and homogenized in a mixer (ETA 012 ) at the lowest speed for 1 min. The homogenate was centrifuged at 300 g for 10 min. to separate IBB into the supernate. During a subsequent centrifugation at 1500 g for 15 min. the supernate was separated into 3 layers: EDTA solution, mucus, and IBB in the pellet. The pellet was washed twice with a 10fold volume of S/3rensen's buffer pH 7.4, diluted 1:4 (7.2 g Na2HPO4" 12 H20, 0.8 g NaH2PO4-2 H2, distilled water ad 1000 ml). After the last centrifugation, IBB was resuspended in a small volume of diluted S/Srensen's buffer pH 7.4 and kept in a refrigerator. In this way, IBB could be stored for at least 1 year. The capacity of each batch of IBB to bind with E. coli cultures possessing the adhesins K88, K99 or 987P was checked before use. Adhesion to IBB In the original method described by Sellwood et al. (1975) cultures of E. coli grown in nutrient broth, are washed twice with Krebs-Henseleit's buffer.
NOVEL COLONIZATION FACTOR
167
The washed culture (0.1 ml) is mixed with an equal amount of IBB and after 30 min of gentle mixing one drop is transferred onto a slide and examined by phase-contrast microscopy. This m e t h o d is rather time-consuming and hence not suitable for serial examination of large numbers of E. coli strains. The modification developed in our laboratory is based on the finding that a mixture of an E. coli culture and IBB, left to stand overnight, agglutinates if the E. coli strain possesses an adhesin. Thus the adhesion test can be done in the same way as the agglutination test by replacing the serum with the IBB suspension. The IBB suspension, washed in diluted S6rensen's buffer pH 7.4 is diluted in the same buffer to obtain an absorbance of 0.1 at 500 n m if 1cm-cuvettes are used in the Spekol photometer. Amounts of 0.025 ml of this suspension are dispensed into U-shaped wells of a microtitre plate and 0.025 ml of the TTC-stained cultures of E. coli grown in parallel in the nutrient broth and in the Minca medium, prepared as described above, are added. The plates are left to stand overnight and results are read in the same way as in the conventional agglutination test.
Haemagglutination Microhaemagglutination tests using guinea pig, cattle, sheep, and chicken erythrocytes with or without D-mannose were performed as described by Jones and Rutter (1974).
Demonstration of toxins Supernates ofE. coli cultures grown in Evans' m e d i u m (Evans et al., 1973 ) with agitation at 37°C for 18 h were used for demonstration o f V T , LT and STa. Penicillin G (1000 U per ml) and neomycin (1000/~g per ml) were added to the supernate after the centrifugation of the cultures at 2500 g for 30 min. Verocytotoxin was demonstrated in Vero cell cultures by a slight modification of the m e t h o d described by Konowalchuk et al. (1977), using 10 × 1.25 cm test tubes instead of microplates. One ml of the Vero cell suspension with a density of 2 X 105 cells per ml was distributed into each tube and 0.1 ml of the examined culture supernate was added on the subsequent day. The cytotoxic effect was examined on 3 subsequent days using an inversion microscope. The same procedure was used for the demonstration of LT on Y-1 cells and cytotonic effect was read one day after the addition of the supernates. STa was demonstrated by inoculation of newborn mice, as described by Stavric and Jeffrey ( 1977 ) and the ligated intestinal loop test in piglets (Smith and Halls, 1977) was used for the demonstration of STb. Only strains producing neither LT nor STa were examined for STb (Gyles, 1979).
168
E. SALAJKAET AL.
Intestinal colonization in vivo Four 28-day-old piglets were inoculated with ETEC cultures one day after weaning using the method of Sarmiento et al. (1988) Strain No. 7394 - O149:K88(LT+STa +) was used in two pigs and strain No. 8810 - O 138:"8813" (STa + ) in the remaining two. Profuse diarrhoea developed on post-infection day 2 in all of them and the pigs were sacrificed. Samples of various intestinal segments were collected and immediately frozen to - 60 ° C in cold heptane and stored on dry ice. Subsequently the presence of E. coli was demonstrated in histological slices by indirect immunofluorescence. Rabbit antiserum to the E. coli antigen was used in the first layer and fluorescein isothiocyanate-conjugated swine anti-rabbit IgG (SwaR/FITC), supplied by the Institute of Sera and Vaccines, Prague, CSFR, in the second one. RESULTS
All E. coli strains, isolated from the intestines and rectal swabs of 1300 pigs affected with PWD or oedema disease during the period 1972-1987, were haemolytic and most of them (72.4%) belonged to 10 O serogroups (Table i). Of the 393 E. coli strains stored in our laboratory, 322 (82%) produced toxins, namely LT, STa and/or VT. ETEC were distributed among 9 0 seroTABLE 1
Toxins produced by the predominant 0 groups of E. coli from postweaning diarrhoea and oedema disease in the period 1972-1987. Strains from 1300 piglets 0
Occurrence
No. of strains Tested
Producing toxins
group No.
%
08 045 0108 0119 0138 0139 0141 0147 0149 0157
142 45 107 19 132 85 61 145 154 53
10.9 3.5 8.2 1.5 10.1 6.5 4.7 11.1 11.8 4.1
Totals
943
72.4
LT
LT + ST
ST
52 26 37 14 61 54 36 31 55 27
25
13
2 5 14 11 31
393
98
12 2
22 37
2 5 18 7
17 3
45
95
VT
VT + ST
1
3 50 7
19 4
12
negative 12 20 11 3 6 4 6 1 8
61
LT=heat-labileenterotoxin;ST=heat-stableenterotoxin; VT=Verocytotoxin.
23
7l
NOVEL COLONIZATION FACTOR
169
groups: 8, 45, 108, 119, 138, 141, 147, 149, and 157. The majority of strains producing VT belonged to the serogroup O 139 and the rest to the serogroups O 138 and O 141. The latter two groups also included strains which produced both VT and STa. In a preliminary attempt a majority of ETEC strains examined for the presence of adhesins by the agglutination test with IBB and sera to K88, K99: F41 and 987P showed positive agglutination with IBB. However, K88 was not demonstrable in some IBB positive strains and K99:F41 and 987P were absent from all of them. Six ETEC strains, reacting with IBB but not with any of the sera used, and belonging to the O serogroups 25, 108, 138, 141,147 o r / 1 4 7 / w e r e used for the immunization of rabbits to obtain OK sera. The diluted sera reacted with OK antigens not only of homologous strains, but also of all strains belonging to the set of six (Table 2). Antibodies responsible for these crossreactions could be absorbed by a single E. coli strain No. 8914-0141, belonging to the set (Table 3). It is therefore evident that the six strains, belonging to various O serogroups, share a c o m m o n surface antigen, which we have designated provisionally "8813". A collection of 212 toxigenic E. coli strains was examined for surface antigens and adhesion to IBB to ascertain the frequency of the "8813" antigen. A set of five sera to the antigens "8813" (two sera), K88, K99:F41 and 987P, and bacterial cultures grown at 37°C in both nutrient broth and Minca medium, were used. The bacterial suspensions were examined in parallel with TABLE 2 A d h e r e n c e o f e n t e r o t o x i g e n i c E. coli s t r a i n s to i n t e s t i n a l b r u s h b o r d e r s a n d a g g l u t i n a t i o n w i t h O K s e r a a g a i n s t t h r e e k n o w n f i m b r i a l a n t i g e n s a n d t h e n o v e l s u r f a c e a n t i g e n o f six f i e l d s t r a i n s Strains examined
Adherence
A g g l u t i n a t i o n w i t h O K sera: S t r a i n N o . / S e r o g r o u p
to IBB a Strain No.
7459 7564 8592 8923 8877 8804 8914 8872 8813
Serogroup
0149:K88 073:K99 064:987P 025 0108 0138 0141 0147 /0147/
+ + + + + + + + +
G205 08:
D1432 0101
987 09:
K88ac
K99: F41
K103: 987P
+ -
. + -
.
8923 025
. . + -
8877 0108
. .
. .
. + + + + + +
. .
.
8804 0138
. .
. + + + + + +
8914 0141
+ + + + + +
8813 /0147/
+ + + + + +
+ + + + + +
. .
.
8872 0147
. . + + + + + +
a T h e s a m e r e s u l t s w e r e o b t a i n e d in t h e b r u s h b o r d e r s f r o m t h e 3 pigs t h a t w e r e u s e d .
.
170
E. SALAJKA ET AL.
TABLE 3 Agglutin in titres of n o n - a b s o r b e d and a b s o r b e d sera to h o m o l o g o u s a n d heterologous E. coli strains possessing the novel c o m m o n surface a n t i g e n / s e r a a b s o r b e d by strain 8914, serogroup 0141 / OK serum Strain No.
Titres with O K a nt i ge ns of strains 0-group
8923
025
8877
0108
8804
0138
8914
0141
8872
0147
8813
/0147/
non-adsorbed n absorbed a
8923 025
8877 0108
8804 0138
8914 0141
n a n a n a n n a n a
1280 640 1280 1280 1280 1280 . 640 .
1280 . 1280 320 1280 1280 1280 . . 640 . .
1280 . 1280 1280 320 1280 1280 . 640 .
1280 . t 280 1280 2560 1280 640
8872 0147
8813 /0147/
1280
1280
1280 10 1280 1280 1280 40 1280 40
1280 10 1280 1280 1280
.
1280 40
both the sera and the IBB, prepared from 3 piglets. Results are summarized in Table 4, where the E. coli strains are arranged in terms of O antigens and each O group in terms of the production of toxins. The "8813" antigen was demonstrated in 83 strains belonging to eight O serogroups: 0 2 5 , 0 4 5 , O 108, O138, O141, O147, O157 a n d / O 1 4 7 / ; 47 strains belonging to 4 serogroups (08, O141, O147 and O149) reacted with the serum to K88 and 3 strains of serogroup O 149 reacted with the serum to K99:F41. None of the strains reacted with the serum to 987P and 79 strains did not react with any of the sera used. The adhesion test with IBB was positive in all strains reacting with the sera to "8813", K88, or K99:F41 and negative in all strains reacting with none of the sera used. Of the strains possessing the "8813" antigen, only cultures grown in nutrient broth were positive in the adhesion test. A brief summary of data given in Table 4 is presented in Table 5. Enterotoxins LT, LT + STa, STa or STb were produced by the strains possessing the "8813" antigen. The strains possessing the K88 antigen produced mostly LT or LT + STa and sporadically STb or VT. Thermostability, production at 37°C and 18°C and haemagglutinating properties of antigen "8813" were examined in 18 strains. Five control strains possessing K88 and two strains reacting with the serum to K99 : F41 were also examined in parallel. The antigen "8813" was inactivated at 60°C after 1 hour and its production was inhibited at 18 ° C. The control strains had identical properties. Strains possessing "8813" did not agglutinate any of the erythrocytes used, control strains agglutinated guinea pig erythrocytes and two strains, belonging to O149 serogroup and reacting with the serum to
171
NOVEL COLONIZATION FACTOR
TABLE4
Distribution of 212 toxigenic E. coli strains in terms of O-antigens, toxin production and surface antigens 0-group
No. o f
Toxin
strains
8813 IBB +
LT LT + STa
K88 IBB +
K99:F41 IBB +
8 6
987P
0
4 2
08
20
025
2
STa STb
045
4
STa VT
1
2 1
0108
16
El" STa STb
2 11 1
2
0119
6
STa
0138
22
LT STa STb VT V T + STa V T + STb
1 1
6
0139
27
VT V T + LT V T + STa
0141
20
LT+STa STa STb VT V T + STa
1 2
LT L T + STa STb
3
0147
23
25 1 1
7
22
LT L T + STa
0157
30
LT L T + STa STa STb
3 21
LT
20
Total (%)
20 212
1 6 2 4 3
1
0149
/0147/
2 2 1 1 8 1
6 1
7 4 2 10 9
3
2 2
2
83
47
3
79
39.2
22.2
1.4
37.2
IBB-intestinal b r u s h border; O = no agglutination with IBB a n d antisera u sed
172
E. SALAJKA ET AL.
TABLE 5 Summary from Table 4 indicating relations of surface antigens to toxic substances in 212 toxigenic E. coli strains Toxic substances
Surface antigens
Total
8813
K88
K99 : F41
negative
LT LT + STa STa STb VT VT+LT VT+STa VT + STb
28 22 21 12
25 19
3
8 3 18 5 31 1 12 1
64 44 39 19 32 1 12 1
Total
83
47
3
79
212
2 1
Fig. 1. J e j u n u m of a piglet infected with the strain No. 8 8 1 0 - O 138 : " 8 8 1 3 " / S T a ÷/. Fluorescein-labelled a n t i b o d y technique. C o n t i n u o u s a d h e r e n t coat o f bacteria on mucosal surface. Magnification X 250.
NOVEL COLONIZATION FACTOR
17 3
K99:F41, did not agglutinate sheep erythrocytes. Hence these strains possess the F41 but not the K99 antigen. Serological cross examinations using three ETEC strains possessing antigen "8813" with reference strains for the antigens K1-K103, CFAI, CFAII, pil 1 and Art25 confirmed that antigen "8813" is not related to any of them. Seven ETEC strains, possessing antigen "8813" and belonging to various O serogroups, were sent to the International Escherichia and Klebsiella Centre in Copenhagen for examination of their antigenic structure. Identification of O antigens was confirmed in all of them. The flagellar antigen H4 was demonstrated in the strain No. 8914-0141 and the remaining ones lacked H-antigens. Thus it has been confirmed that "8813" is not a flagellar antigen and has been called "preliminary F 18". Two weaned piglets were inoculated with strain 8810-O138 :"8813", STa ÷ to examine its adhesion to intestinal mucosa in vivo. Two separately housed piglets were infected with the strain 7395-0149: K88,LT+STa + for comparison. All four piglets developed profuse diarrhoea within 2 days. The piglets were sacrificed and several intestinal segments were examined using fluorescein-labelled antibody. Both ETEC strains produced marked coats on the epithelium of the small intestine (Fig. 1). That produced by strain 8810O 138 :"8813" was demonstrable not only by the homologous serum, but also by sera to the 5 ETEC strains listed in Table 2, belonging to various O serogroups, but sharing the c o m m o n surface antigen "8813". DISCUSSION
The surface antigen that has been detected is distinct from previously identified adhesins involved in colonization but behaves like them in a several respects. Thus, surface antigen "8813", a novel putative colonization factor, is produced in the intestine of experimentally infected pigs, is c o m m o n to a n u m b e r of different O serogroups, and is produced at 37 °C but not at 18 ° C. It is likely that this antigen is an adhesin which mediates colonization of the intestine, but proof of this requires further investigation. E. coli strains associated with PWD or oedema disease belong to a limited number of O serogroups (Sojka, 1965; Svedsen et al., 1974; Kennan and Monckton, 1990; Nagy et al., 1990). Our results were similar as indicated by data in Table 1. Our findings of E. coli strains belonging to the most frequently occurring serogroups and producing no known toxins correspond to those of Nagy et al. (1990). It is commonly accepted that enteropathogenicity of ETEC is associated with the presence of at least one of the known enterotoxins and a simultaneous presence of an adhesin binding bacteria to the enterocytes. This does not explain completely, however, the induction of diarrhoea caused by E. coli.
174
E. SALAJKAETAL.
Diarrhoea was induced, for example, by an E. coli strain K88+Ent - (Smith and Linggood, 1971 ) and increased fluid secretion into the intestinal lumen resulted from the impaired sucrase activity due to intestinal colonization with a non-toxigenic strain of E. coli (Schlager et al., 1990). It has also been reported that adhesion of enteropathogens to the enterocytes is not necessary if adherence to the mucus coat of the intestine takes place (K6tyi, 1990; Yamamoto and Yokota, 1988 ). Frequent isolation of ETEC strains lacking any of the known adhesion factors from PWD-affected piglets suggested the existence of other, yet unidentified factors (Hoblet et al., 1986; Nakazawa et al., 1987). This was supported by our finding of the surface antigen "8813", which was demonstrated in many strains belonging to various O serogroups. This antigen has properties similar to colonization factor K88: it is heat-labile, its production is inhibited at 18 °C and it induces intestinal colonization in a similar way as K88. Our ETEC strains possessing antigen "8813" showed adhesion to IBB analogous to that reported by Aning et al. (1983) for 3 strains ofE. coli isolated from normal animals and adhering to freshly prepared IBB only. The authors did not comment their pathogenicity, however. Our strains were isolated from piglets affected with PWD and agglutinated with both freshly prepared IBB and with IBB stored ad 4 °C for one year. All our strains produced LT, STa or STb. Strain 8810-O 138 :"8813", STa + induced diarrhoea and colonized the small intestine in experimentally infected piglets. Presently, we are not able to determine whether the colonization factor "8813" is identical to that described by Aning et al. ( 1983 ). Kennan and Monckton (1990) proposed that fimbriae associated with porcine ETEC of the O 141 serogroup isolated in Australia were a new type of adhesive fimbriae, although the adhesive property was not investigated. Some of our ETEC strains with "8813" antigen belonged to O 141 group. Likewise in this case we cannot determine whether the common colonizing factor "8813" is related to fimbriae described by these authors. ACKNOWLEDGEMENTS
We express our sincere thanks to Dr. C.L. Gyles, Ontario Veterinary College, Guelph, Canada, for the critical review of the manuscript and to Drs. I. and F. Orskov, WHO Escherichia and Klebsiella Centre Copenhagen, Denmark, for their kind serotyping ofE. coli strains.
REFERENCES Aning, K.G., Thomlinson, J.R., Wray, C., Sojka, W.J. and Coulter, J., 1983. Adhesion factor distinct from K88, K99, F41,987P, CFAI and CFAII in porcine Escherichia coll. Vet. Rec., 112:251.
NOVEL COLONIZATION FACTOR
175
Burrows, M.R., Sellwood, R. and Gibbons, R.A., 1976. Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Escherichia coil J. Gen. Microbiol., 96: 269-275. Evans, D.G., Evans, D.J., Jr. and Pierce, N.F., 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coil Infect. Immun., 7: 873-880. Guin6e, P.A.M., Jansen, W.H. and Agterberg, C.M., 1976. Detection of the K99 antigen by means of agglutination and immunoelectrophoresis in Escherichia coli isolates from calves and its correlation with enterotoxigenicity. Infect. Immun., 13:1369-1377. Gyles, C.L., 1979. Limitation of the infant mouse test for Escherichia coil heat stable enterotoxin. Can. J. Comp. Med., 43: 371-379. Hoblet, K.H., Kohler, E.M., Saif, L.J., Theil, K.W. and Ingalls, W.L., 1986. Study of porcine postweaning diarrhea involving K88 ( - ) hemolytic Escherichia colL Am. J. Vet. Res., 47: 1910-1912. Jones, G.W. and Rutter, J.M., 1974. The association of K88 antigen with haemagglutinating activity in porcine strains of Escherichia coil J. Gen. Microbiol., 84:135-144. Kennan, R.M. and Monckton, R.P., 1990. Adhesive fimbriae associated with porcine enterotoxigenic Escherichia coli of the O141 serotype. J. Clin. Microbiol., 28:2006-2011. K6tyi, I., 1990. Binding of enteric bacteria to hog gastric mucin. Acta Microbiol. Hung., 37: 4553. Konowalchuk, J., Speirs, J.l. and Stavric, S., 1977 Vero response to a cytotoxin of Escherichia coli. Infect. Immun., 18: 775-779. Nagy, B., Casey, T.A. and Moon, H.W., 1990. Phenotype and genotype ofEscherichia coli isolated from pigs with postweaning diarrhea in Hungary. J. Clin. Microbiol., 28:651-653. Nakazawa, M., Sugimoto, Ch., Isayama, Y. and Kashiwazaki, M., 1987. Virulence factors in Escherichia coli isolated from piglets with neonatal and post-weaning diarrhea in Japan. Vet. Microbiol., 13:291-300. Orskov, I., Orskov, F., Jann, B. and Jann, K., 1977. Serology, chemistry, and genetics of O and K antigens ofEscherichia colL Bact. Rev., 41:667-710. Sarmiento, J.I., Casey, T.A. and Moon, H.W., 1988. Postweaning diarrhea in swine: Experimental model of enterotoxigenic Escherichia coli infection. Am. J. Vet. Res., 49:1154-1159. Sellwood, R., Gibbons, R.A., Jones, G.W. and Rutter, J.M., 1975. Adhesion of enteropathogenic Escherichia coli to pig intestinal brush borders: The existence of two pig phenotypes. J. Med. Microbiol., 8:405-411. Schlager, T.A., Wanke, Ch.A. and Guerrant, R.L., 1990. Net fluid secretion and impaired villous function induced by colonization of the small intestine by nontoxigenic colonizing Escherichia colL Infect. Immun., 58:1337-1343. Smith, H.W. and Halls, S., 1967. Observations by the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Path. Bact., 93: 499-529. Smith, H.W. and Linggood, M.A., 1971. Observations on the pathogenic properties of the K88, Hly and Ent plasmids of Escherichia coli with particular reference to porcine diarrhoea. J. Med. Microbiol., 4: 467-485. Sojka, W.J., 1965. Escherichia coli in domestic animals and poultry. Review series No. 7 of the Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England: 205-206. Stavric, S. and Jeffrey, D., 1977. A modified bioassay for heat-stable Escherichia coli enterotoxin. Can. J. Microbiol., 23:331-336. Svendsen, J., Larsen, J.L. and Bille, N., 1974. Outbreaks of post weaning Escherichia coli diarrhoea in pigs. Nord. Vet. Med., 26:314-322. Yamamoto, T. and Yokota, T., 1988. Electron microscopic study of Vibrio cholerae 01 adherence to the mucus. Infect. Immun., 56: 2753-2759.
163
Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs E. Salajka, Z. Salajkova, P. Alexa and M. Hornich Veterinary Research Institute, Brno, Czechoslovakia (Accepted 2 January 1992)
ABSTRACT Salajka, E., Salajkov~i,Z., Alexa, P. and Hornich, M., 1992. Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs. Vet. Microbiol., 32:163-175. A novel common colonization factor was detected in enterotoxigenic strains of Escherichia coli isolated from intestinal contents of piglets affected with postweaning diarrhoea. This factor was antigenically distinct from the previously described K88, K99, F41,987P, CFAI, CFAII and Att25 timbrial antigens. E. coli strains possessing this factor adhered to the pig intestinal brush borders and one strain, used in experimental infection in weanlings, colonized the intestinal epithelium and induced diarrhoea. Examination of 212 toxigenic strains of E. coli isolated from weanlings revealed the presence of the novel common colonization factor in 83 strains, belonging to serogroups 025, O108, O138, O141, O147 and O157. The antigen K88 was detected in 47 strains belonging to serogroups 08, O141, O147 and O149.
INTRODUCTION
Colonization factors (adhesins) are essential for virulence of enterotoxigenic Escherichia coli (ETEC) strains. Most ETEC strains isolated from newborn piglets possess one of the colonization factors K88, K99 and 987P. However, the situation is different in ETEC strains isolated from pigs affected with postweaning diarrhoea ( P W D ) . Svendsen et al. (1974) found haemolytic E. coli strains belonging to O serogroups 149, 8, 138 and 141 in such pigs, and noted that the K88 antigen was not present in any of them. Hoblet et al. (1986) failed to detect any of the common adhesins during the examination of 22 ETEC strains isolated from weanlings. Correspondence to: P. Alexa, Veterinary Research Institute, 621 32 Brno, Czechoslovakia.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
164
E. SALAJKA ET AL.
Nakazawa et al. ( 1987 ) examined 137 ETEC strains isolated from weanlings, using antisera specific for K88, K99, 987P and F41. K88 and K99 antigens were demonstrated in 4 and 32 strains, respectively, but the other antigens under study were not detected. Nagy et al. (1990) demonstrated the K88 antigen in 126 and the 987P antigen in one of the 205 E. coli strains isolated from PWD in pigs. None of the adhesins known so far was found in the remaining 78 strains. Of the 70 strains which lacked known adhesins, 19 produced neither enterotoxin, nor verocytotoxin. Frequent finding of ETEC, lacking any of the known adhesins, in PWDaffected piglets suggests that these strains possess other, so far unidentified adhesins. Aning et al. ( 1983 ) isolated 3 E. coli 0 6 5 : K?:H9 strains possessing an adhesin different from K88, K99, F41,987P, CFAI, or CFAII from normal weaned pigs, without commenting on their pathogenicity. Kennan and Monckton (1990) reported that the majority ofE. coli strains isolated from piglets with PWD in Australia belonged to O group 141 and did not possess any of the four characterized porcine fimbrial adhesins. They investigated 50 strains by transmission electron microscopy. Two strains that produced numerous fimbriae were chosen for further characterization. The isolated fimbriae were antigenically related and had a subunit molecular weight of 17 000. These fimbriae were not produced at 18 ° C. The authors proposed that these fimbriae were a new type of adhesive fimbriae associated with porcine ETEC of O group 141. Our experiments were based on the finding that the K88 antigen was present in only some of the ETEC strains, isolated from weanlings and examined in our laboratory. However, adhesion to piglet intestinal brush borders (IBB) was positive in many of the strains lacking any of the adhesins identified so far. Sera obtained from rabbits i m m u n i z e d with these ETEC strains belonged to various O serogroups, agglutinated ETEC strains not only of homologous, but also heterologous O serogroups. These findings, as well as the possibility of removing specific agglutinins from these sera by the culture of one of these strains, have demoristrated the presence of a novel c o m m o n surface antigen. This antigen with a provisional designation "8813" was found in a number of ETEC strains belonging to various O serogroups. MATERIALS AND METHODS
Escherichia coli strains E. coli reference strains for the antigens O1-O170 and K I - K 1 0 3 and for fimbrial antigens K88, K 9 9 : F 4 1 , 9 8 7 P , CFAI, CFAII and B pil 1 were obtained courtesy of the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark, through the Czechoslovak Col-
NOVEL COLONIZATION FACq'OR
16 5
lection of Microorganisms. The reference strain Att25-X 114/83 was kindly supplied by Dr. J.A. Morris, Central Veterinary Laboratory, Weybridge, UK. Field strains of E. coli were isolated in the period 1972-1987 from the intestines of approximately 550 dead pigs and from rectal swabs of approximately 750 pigs affected with PWD or oedema disease. The samples were inoculated onto 5% sheep blood agar plates and Endo agar plates. Two to four lactose-positive colonies, identified as E. coli by c o m m o n biochemical tests, were selected as strains for the identification of O antigens. Some of these strains with identified O antigens were maintained in our laboratory on Dorset's egg medium. Of this set, 393 strains, belonging to 10 dominant O serogroups, were examined for toxin production (heat-labile enterotoxin LT, heatstable enterotoxin - - ST, Verocytotoxin - - VT); 212 toxigenic strains were used for the examination of colonization factors. This subset included also E. coli strains isolated from pigs affected with PWD or oedema disease in 19881990. The field strains No. 7395 ( O 1 4 9 : K 8 8 : L T + S T a ÷) and No. 8810 (O 138 :"8813": STa ÷ ) were used for experimental infections of weaned pigs.
Diagnostic sera Reference E. coli strains were used for the preparation of anti-O and antiOK rabbit sera by the m e t h o d described by Sojka ( 1965 ). Thirty of the 103 reference strains for OK antigens were omitted during the preparation of the anti-OK sera for the identification of K antigens owing to the proposal of Orskov et al. (1977) to delete them. Anti-OK sera were prepared against six field isolates i.e. Nos. 8923-025, 8877-O108, 8804-O138, 8914-O141, 8872-O147 and 8 8 1 3 - / O 1 4 7 / . Strain 8813 possessed an antigen related to, but not identical with O 147. Antiserum against 8813 reacted with O antigens of the reference strains G1253O 147: K89 : K88ac and D 3 5 7 - O 147 : K89: H 19, but none of the sera to these reference strains reacted with the O antigen of the strain 8813. Therefore the O antigen designation of this strain is given in parentheses. E. coli cultures grown during 18 h at 37°C on No. 2 nutrient agar (Imuna, Sarissk6 Michalany, CSFR) on 10 cm diameter Petri dishes were used for absorption of sera. Cultures, rinsed from each of 5 dishes with 2 ml 0.85% N a C I + 0.15% phenol, were mixed, centrifuged at 1200 g for 15 min., washed with 0.85% NaCI + 0.15% phenol and added to 1 ml serum. The mixture was incubated in a water bath at 37 °C for 4 h, stored at 4 °C overnight, then centrifuged at 1500 g for 30 min. Antigen identification O antigens were identified in E. coli cultures grown in No. 2 nutrient broth (Imuna, Sarissk6 Michalany, CSFR). After 18 h incubation at 37°C the cultures were stained red by adding 0.05 ml 4% triphenyltetrazolium chloride
166
E. SALAJKA ET AL.
(TTC) to 5 ml culture and incubated at 37°C for 1 hour. Thereafter the culture was heated to 100°C for 1 h and cooled. Surface antigens were identified in cultures grown in parallel in No. 2 nutrient broth and Minca medium (Guin6e et al., 1977) at 37 °C overnight and at 18°C for 48 hours. The cultures were stained with TTC and left to stand for 4 hours to let the R forms settle to the bottom. The upper part of the culture was withdrawn and centrifuged at 800 g for 10 min. The sediment was resuspended in 0.85% NaCl+0.15% phenol to obtain a density of approximately 1 × 109 CFU per ml. The antigens were identified by agglutination in U-shaped microtitre plates. After pipetting 0.025 ml of serum diluted to its agglutination titre and bacterial suspension into each well the plates were covered and incubated at room temperature overnight. The results were read by the naked eye on the subsequent day. The agglutination titre was the reciprocal of the highest dilution which gave complete agglutination.
Preparation of intestinal brush borders (IBB) IBB were prepared from small intestines of piglets by the method described by Sellwood et al. ( 1975 ), which has been partly modified in our laboratory. Intestinal segments were washed with 0.85% NaC1 and, after ligating one end, filled with 0.01 M etylendiaminetetraacetate (EDTA) pH 6.8 (0.096 M NaC1, 0.008 M KH2PO4, 0.0056 M Na2HPO4, 0.0015 M KC1 and 0.01 M EDTA). After ligating the other end the segments were put into 0.85% NaC1. The EDTA solution was discarded after 20 min. and the intestine was half-filled with 0.3 M sucrose in the same buffer. Epithelial cells were detached into the fluid by gently rubbing of the intestine between the fingers. The fluid was collected and the loosening procedure was repeated twice to obtain as many cells as possible. The cell suspension was centrifuged at 1200 g for 15 min; the supernate was discarded and the sedimented cells were resuspended in a 10-fold volume of buffered 0.005 M EDTA pH 7.4 and homogenized in a mixer (ETA 012 ) at the lowest speed for 1 min. The homogenate was centrifuged at 300 g for 10 min. to separate IBB into the supernate. During a subsequent centrifugation at 1500 g for 15 min. the supernate was separated into 3 layers: EDTA solution, mucus, and IBB in the pellet. The pellet was washed twice with a 10fold volume of S/3rensen's buffer pH 7.4, diluted 1:4 (7.2 g Na2HPO4" 12 H20, 0.8 g NaH2PO4-2 H2, distilled water ad 1000 ml). After the last centrifugation, IBB was resuspended in a small volume of diluted S/Srensen's buffer pH 7.4 and kept in a refrigerator. In this way, IBB could be stored for at least 1 year. The capacity of each batch of IBB to bind with E. coli cultures possessing the adhesins K88, K99 or 987P was checked before use. Adhesion to IBB In the original method described by Sellwood et al. (1975) cultures of E. coli grown in nutrient broth, are washed twice with Krebs-Henseleit's buffer.
NOVEL COLONIZATION FACTOR
167
The washed culture (0.1 ml) is mixed with an equal amount of IBB and after 30 min of gentle mixing one drop is transferred onto a slide and examined by phase-contrast microscopy. This m e t h o d is rather time-consuming and hence not suitable for serial examination of large numbers of E. coli strains. The modification developed in our laboratory is based on the finding that a mixture of an E. coli culture and IBB, left to stand overnight, agglutinates if the E. coli strain possesses an adhesin. Thus the adhesion test can be done in the same way as the agglutination test by replacing the serum with the IBB suspension. The IBB suspension, washed in diluted S6rensen's buffer pH 7.4 is diluted in the same buffer to obtain an absorbance of 0.1 at 500 n m if 1cm-cuvettes are used in the Spekol photometer. Amounts of 0.025 ml of this suspension are dispensed into U-shaped wells of a microtitre plate and 0.025 ml of the TTC-stained cultures of E. coli grown in parallel in the nutrient broth and in the Minca medium, prepared as described above, are added. The plates are left to stand overnight and results are read in the same way as in the conventional agglutination test.
Haemagglutination Microhaemagglutination tests using guinea pig, cattle, sheep, and chicken erythrocytes with or without D-mannose were performed as described by Jones and Rutter (1974).
Demonstration of toxins Supernates ofE. coli cultures grown in Evans' m e d i u m (Evans et al., 1973 ) with agitation at 37°C for 18 h were used for demonstration o f V T , LT and STa. Penicillin G (1000 U per ml) and neomycin (1000/~g per ml) were added to the supernate after the centrifugation of the cultures at 2500 g for 30 min. Verocytotoxin was demonstrated in Vero cell cultures by a slight modification of the m e t h o d described by Konowalchuk et al. (1977), using 10 × 1.25 cm test tubes instead of microplates. One ml of the Vero cell suspension with a density of 2 X 105 cells per ml was distributed into each tube and 0.1 ml of the examined culture supernate was added on the subsequent day. The cytotoxic effect was examined on 3 subsequent days using an inversion microscope. The same procedure was used for the demonstration of LT on Y-1 cells and cytotonic effect was read one day after the addition of the supernates. STa was demonstrated by inoculation of newborn mice, as described by Stavric and Jeffrey ( 1977 ) and the ligated intestinal loop test in piglets (Smith and Halls, 1977) was used for the demonstration of STb. Only strains producing neither LT nor STa were examined for STb (Gyles, 1979).
168
E. SALAJKAET AL.
Intestinal colonization in vivo Four 28-day-old piglets were inoculated with ETEC cultures one day after weaning using the method of Sarmiento et al. (1988) Strain No. 7394 - O149:K88(LT+STa +) was used in two pigs and strain No. 8810 - O 138:"8813" (STa + ) in the remaining two. Profuse diarrhoea developed on post-infection day 2 in all of them and the pigs were sacrificed. Samples of various intestinal segments were collected and immediately frozen to - 60 ° C in cold heptane and stored on dry ice. Subsequently the presence of E. coli was demonstrated in histological slices by indirect immunofluorescence. Rabbit antiserum to the E. coli antigen was used in the first layer and fluorescein isothiocyanate-conjugated swine anti-rabbit IgG (SwaR/FITC), supplied by the Institute of Sera and Vaccines, Prague, CSFR, in the second one. RESULTS
All E. coli strains, isolated from the intestines and rectal swabs of 1300 pigs affected with PWD or oedema disease during the period 1972-1987, were haemolytic and most of them (72.4%) belonged to 10 O serogroups (Table i). Of the 393 E. coli strains stored in our laboratory, 322 (82%) produced toxins, namely LT, STa and/or VT. ETEC were distributed among 9 0 seroTABLE 1
Toxins produced by the predominant 0 groups of E. coli from postweaning diarrhoea and oedema disease in the period 1972-1987. Strains from 1300 piglets 0
Occurrence
No. of strains Tested
Producing toxins
group No.
%
08 045 0108 0119 0138 0139 0141 0147 0149 0157
142 45 107 19 132 85 61 145 154 53
10.9 3.5 8.2 1.5 10.1 6.5 4.7 11.1 11.8 4.1
Totals
943
72.4
LT
LT + ST
ST
52 26 37 14 61 54 36 31 55 27
25
13
2 5 14 11 31
393
98
12 2
22 37
2 5 18 7
17 3
45
95
VT
VT + ST
1
3 50 7
19 4
12
negative 12 20 11 3 6 4 6 1 8
61
LT=heat-labileenterotoxin;ST=heat-stableenterotoxin; VT=Verocytotoxin.
23
7l
NOVEL COLONIZATION FACTOR
169
groups: 8, 45, 108, 119, 138, 141, 147, 149, and 157. The majority of strains producing VT belonged to the serogroup O 139 and the rest to the serogroups O 138 and O 141. The latter two groups also included strains which produced both VT and STa. In a preliminary attempt a majority of ETEC strains examined for the presence of adhesins by the agglutination test with IBB and sera to K88, K99: F41 and 987P showed positive agglutination with IBB. However, K88 was not demonstrable in some IBB positive strains and K99:F41 and 987P were absent from all of them. Six ETEC strains, reacting with IBB but not with any of the sera used, and belonging to the O serogroups 25, 108, 138, 141,147 o r / 1 4 7 / w e r e used for the immunization of rabbits to obtain OK sera. The diluted sera reacted with OK antigens not only of homologous strains, but also of all strains belonging to the set of six (Table 2). Antibodies responsible for these crossreactions could be absorbed by a single E. coli strain No. 8914-0141, belonging to the set (Table 3). It is therefore evident that the six strains, belonging to various O serogroups, share a c o m m o n surface antigen, which we have designated provisionally "8813". A collection of 212 toxigenic E. coli strains was examined for surface antigens and adhesion to IBB to ascertain the frequency of the "8813" antigen. A set of five sera to the antigens "8813" (two sera), K88, K99:F41 and 987P, and bacterial cultures grown at 37°C in both nutrient broth and Minca medium, were used. The bacterial suspensions were examined in parallel with TABLE 2 A d h e r e n c e o f e n t e r o t o x i g e n i c E. coli s t r a i n s to i n t e s t i n a l b r u s h b o r d e r s a n d a g g l u t i n a t i o n w i t h O K s e r a a g a i n s t t h r e e k n o w n f i m b r i a l a n t i g e n s a n d t h e n o v e l s u r f a c e a n t i g e n o f six f i e l d s t r a i n s Strains examined
Adherence
A g g l u t i n a t i o n w i t h O K sera: S t r a i n N o . / S e r o g r o u p
to IBB a Strain No.
7459 7564 8592 8923 8877 8804 8914 8872 8813
Serogroup
0149:K88 073:K99 064:987P 025 0108 0138 0141 0147 /0147/
+ + + + + + + + +
G205 08:
D1432 0101
987 09:
K88ac
K99: F41
K103: 987P
+ -
. + -
.
8923 025
. . + -
8877 0108
. .
. .
. + + + + + +
. .
.
8804 0138
. .
. + + + + + +
8914 0141
+ + + + + +
8813 /0147/
+ + + + + +
+ + + + + +
. .
.
8872 0147
. . + + + + + +
a T h e s a m e r e s u l t s w e r e o b t a i n e d in t h e b r u s h b o r d e r s f r o m t h e 3 pigs t h a t w e r e u s e d .
.
170
E. SALAJKA ET AL.
TABLE 3 Agglutin in titres of n o n - a b s o r b e d and a b s o r b e d sera to h o m o l o g o u s a n d heterologous E. coli strains possessing the novel c o m m o n surface a n t i g e n / s e r a a b s o r b e d by strain 8914, serogroup 0141 / OK serum Strain No.
Titres with O K a nt i ge ns of strains 0-group
8923
025
8877
0108
8804
0138
8914
0141
8872
0147
8813
/0147/
non-adsorbed n absorbed a
8923 025
8877 0108
8804 0138
8914 0141
n a n a n a n n a n a
1280 640 1280 1280 1280 1280 . 640 .
1280 . 1280 320 1280 1280 1280 . . 640 . .
1280 . 1280 1280 320 1280 1280 . 640 .
1280 . t 280 1280 2560 1280 640
8872 0147
8813 /0147/
1280
1280
1280 10 1280 1280 1280 40 1280 40
1280 10 1280 1280 1280
.
1280 40
both the sera and the IBB, prepared from 3 piglets. Results are summarized in Table 4, where the E. coli strains are arranged in terms of O antigens and each O group in terms of the production of toxins. The "8813" antigen was demonstrated in 83 strains belonging to eight O serogroups: 0 2 5 , 0 4 5 , O 108, O138, O141, O147, O157 a n d / O 1 4 7 / ; 47 strains belonging to 4 serogroups (08, O141, O147 and O149) reacted with the serum to K88 and 3 strains of serogroup O 149 reacted with the serum to K99:F41. None of the strains reacted with the serum to 987P and 79 strains did not react with any of the sera used. The adhesion test with IBB was positive in all strains reacting with the sera to "8813", K88, or K99:F41 and negative in all strains reacting with none of the sera used. Of the strains possessing the "8813" antigen, only cultures grown in nutrient broth were positive in the adhesion test. A brief summary of data given in Table 4 is presented in Table 5. Enterotoxins LT, LT + STa, STa or STb were produced by the strains possessing the "8813" antigen. The strains possessing the K88 antigen produced mostly LT or LT + STa and sporadically STb or VT. Thermostability, production at 37°C and 18°C and haemagglutinating properties of antigen "8813" were examined in 18 strains. Five control strains possessing K88 and two strains reacting with the serum to K99 : F41 were also examined in parallel. The antigen "8813" was inactivated at 60°C after 1 hour and its production was inhibited at 18 ° C. The control strains had identical properties. Strains possessing "8813" did not agglutinate any of the erythrocytes used, control strains agglutinated guinea pig erythrocytes and two strains, belonging to O149 serogroup and reacting with the serum to
171
NOVEL COLONIZATION FACTOR
TABLE4
Distribution of 212 toxigenic E. coli strains in terms of O-antigens, toxin production and surface antigens 0-group
No. o f
Toxin
strains
8813 IBB +
LT LT + STa
K88 IBB +
K99:F41 IBB +
8 6
987P
0
4 2
08
20
025
2
STa STb
045
4
STa VT
1
2 1
0108
16
El" STa STb
2 11 1
2
0119
6
STa
0138
22
LT STa STb VT V T + STa V T + STb
1 1
6
0139
27
VT V T + LT V T + STa
0141
20
LT+STa STa STb VT V T + STa
1 2
LT L T + STa STb
3
0147
23
25 1 1
7
22
LT L T + STa
0157
30
LT L T + STa STa STb
3 21
LT
20
Total (%)
20 212
1 6 2 4 3
1
0149
/0147/
2 2 1 1 8 1
6 1
7 4 2 10 9
3
2 2
2
83
47
3
79
39.2
22.2
1.4
37.2
IBB-intestinal b r u s h border; O = no agglutination with IBB a n d antisera u sed
172
E. SALAJKA ET AL.
TABLE 5 Summary from Table 4 indicating relations of surface antigens to toxic substances in 212 toxigenic E. coli strains Toxic substances
Surface antigens
Total
8813
K88
K99 : F41
negative
LT LT + STa STa STb VT VT+LT VT+STa VT + STb
28 22 21 12
25 19
3
8 3 18 5 31 1 12 1
64 44 39 19 32 1 12 1
Total
83
47
3
79
212
2 1
Fig. 1. J e j u n u m of a piglet infected with the strain No. 8 8 1 0 - O 138 : " 8 8 1 3 " / S T a ÷/. Fluorescein-labelled a n t i b o d y technique. C o n t i n u o u s a d h e r e n t coat o f bacteria on mucosal surface. Magnification X 250.
NOVEL COLONIZATION FACTOR
17 3
K99:F41, did not agglutinate sheep erythrocytes. Hence these strains possess the F41 but not the K99 antigen. Serological cross examinations using three ETEC strains possessing antigen "8813" with reference strains for the antigens K1-K103, CFAI, CFAII, pil 1 and Art25 confirmed that antigen "8813" is not related to any of them. Seven ETEC strains, possessing antigen "8813" and belonging to various O serogroups, were sent to the International Escherichia and Klebsiella Centre in Copenhagen for examination of their antigenic structure. Identification of O antigens was confirmed in all of them. The flagellar antigen H4 was demonstrated in the strain No. 8914-0141 and the remaining ones lacked H-antigens. Thus it has been confirmed that "8813" is not a flagellar antigen and has been called "preliminary F 18". Two weaned piglets were inoculated with strain 8810-O138 :"8813", STa ÷ to examine its adhesion to intestinal mucosa in vivo. Two separately housed piglets were infected with the strain 7395-0149: K88,LT+STa + for comparison. All four piglets developed profuse diarrhoea within 2 days. The piglets were sacrificed and several intestinal segments were examined using fluorescein-labelled antibody. Both ETEC strains produced marked coats on the epithelium of the small intestine (Fig. 1). That produced by strain 8810O 138 :"8813" was demonstrable not only by the homologous serum, but also by sera to the 5 ETEC strains listed in Table 2, belonging to various O serogroups, but sharing the c o m m o n surface antigen "8813". DISCUSSION
The surface antigen that has been detected is distinct from previously identified adhesins involved in colonization but behaves like them in a several respects. Thus, surface antigen "8813", a novel putative colonization factor, is produced in the intestine of experimentally infected pigs, is c o m m o n to a n u m b e r of different O serogroups, and is produced at 37 °C but not at 18 ° C. It is likely that this antigen is an adhesin which mediates colonization of the intestine, but proof of this requires further investigation. E. coli strains associated with PWD or oedema disease belong to a limited number of O serogroups (Sojka, 1965; Svedsen et al., 1974; Kennan and Monckton, 1990; Nagy et al., 1990). Our results were similar as indicated by data in Table 1. Our findings of E. coli strains belonging to the most frequently occurring serogroups and producing no known toxins correspond to those of Nagy et al. (1990). It is commonly accepted that enteropathogenicity of ETEC is associated with the presence of at least one of the known enterotoxins and a simultaneous presence of an adhesin binding bacteria to the enterocytes. This does not explain completely, however, the induction of diarrhoea caused by E. coli.
174
E. SALAJKAETAL.
Diarrhoea was induced, for example, by an E. coli strain K88+Ent - (Smith and Linggood, 1971 ) and increased fluid secretion into the intestinal lumen resulted from the impaired sucrase activity due to intestinal colonization with a non-toxigenic strain of E. coli (Schlager et al., 1990). It has also been reported that adhesion of enteropathogens to the enterocytes is not necessary if adherence to the mucus coat of the intestine takes place (K6tyi, 1990; Yamamoto and Yokota, 1988 ). Frequent isolation of ETEC strains lacking any of the known adhesion factors from PWD-affected piglets suggested the existence of other, yet unidentified factors (Hoblet et al., 1986; Nakazawa et al., 1987). This was supported by our finding of the surface antigen "8813", which was demonstrated in many strains belonging to various O serogroups. This antigen has properties similar to colonization factor K88: it is heat-labile, its production is inhibited at 18 °C and it induces intestinal colonization in a similar way as K88. Our ETEC strains possessing antigen "8813" showed adhesion to IBB analogous to that reported by Aning et al. (1983) for 3 strains ofE. coli isolated from normal animals and adhering to freshly prepared IBB only. The authors did not comment their pathogenicity, however. Our strains were isolated from piglets affected with PWD and agglutinated with both freshly prepared IBB and with IBB stored ad 4 °C for one year. All our strains produced LT, STa or STb. Strain 8810-O 138 :"8813", STa + induced diarrhoea and colonized the small intestine in experimentally infected piglets. Presently, we are not able to determine whether the colonization factor "8813" is identical to that described by Aning et al. ( 1983 ). Kennan and Monckton (1990) proposed that fimbriae associated with porcine ETEC of the O 141 serogroup isolated in Australia were a new type of adhesive fimbriae, although the adhesive property was not investigated. Some of our ETEC strains with "8813" antigen belonged to O 141 group. Likewise in this case we cannot determine whether the common colonizing factor "8813" is related to fimbriae described by these authors. ACKNOWLEDGEMENTS
We express our sincere thanks to Dr. C.L. Gyles, Ontario Veterinary College, Guelph, Canada, for the critical review of the manuscript and to Drs. I. and F. Orskov, WHO Escherichia and Klebsiella Centre Copenhagen, Denmark, for their kind serotyping ofE. coli strains.
REFERENCES Aning, K.G., Thomlinson, J.R., Wray, C., Sojka, W.J. and Coulter, J., 1983. Adhesion factor distinct from K88, K99, F41,987P, CFAI and CFAII in porcine Escherichia coll. Vet. Rec., 112:251.
NOVEL COLONIZATION FACTOR
175
Burrows, M.R., Sellwood, R. and Gibbons, R.A., 1976. Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Escherichia coil J. Gen. Microbiol., 96: 269-275. Evans, D.G., Evans, D.J., Jr. and Pierce, N.F., 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coil Infect. Immun., 7: 873-880. Guin6e, P.A.M., Jansen, W.H. and Agterberg, C.M., 1976. Detection of the K99 antigen by means of agglutination and immunoelectrophoresis in Escherichia coli isolates from calves and its correlation with enterotoxigenicity. Infect. Immun., 13:1369-1377. Gyles, C.L., 1979. Limitation of the infant mouse test for Escherichia coil heat stable enterotoxin. Can. J. Comp. Med., 43: 371-379. Hoblet, K.H., Kohler, E.M., Saif, L.J., Theil, K.W. and Ingalls, W.L., 1986. Study of porcine postweaning diarrhea involving K88 ( - ) hemolytic Escherichia colL Am. J. Vet. Res., 47: 1910-1912. Jones, G.W. and Rutter, J.M., 1974. The association of K88 antigen with haemagglutinating activity in porcine strains of Escherichia coil J. Gen. Microbiol., 84:135-144. Kennan, R.M. and Monckton, R.P., 1990. Adhesive fimbriae associated with porcine enterotoxigenic Escherichia coli of the O141 serotype. J. Clin. Microbiol., 28:2006-2011. K6tyi, I., 1990. Binding of enteric bacteria to hog gastric mucin. Acta Microbiol. Hung., 37: 4553. Konowalchuk, J., Speirs, J.l. and Stavric, S., 1977 Vero response to a cytotoxin of Escherichia coli. Infect. Immun., 18: 775-779. Nagy, B., Casey, T.A. and Moon, H.W., 1990. Phenotype and genotype ofEscherichia coli isolated from pigs with postweaning diarrhea in Hungary. J. Clin. Microbiol., 28:651-653. Nakazawa, M., Sugimoto, Ch., Isayama, Y. and Kashiwazaki, M., 1987. Virulence factors in Escherichia coli isolated from piglets with neonatal and post-weaning diarrhea in Japan. Vet. Microbiol., 13:291-300. Orskov, I., Orskov, F., Jann, B. and Jann, K., 1977. Serology, chemistry, and genetics of O and K antigens ofEscherichia colL Bact. Rev., 41:667-710. Sarmiento, J.I., Casey, T.A. and Moon, H.W., 1988. Postweaning diarrhea in swine: Experimental model of enterotoxigenic Escherichia coli infection. Am. J. Vet. Res., 49:1154-1159. Sellwood, R., Gibbons, R.A., Jones, G.W. and Rutter, J.M., 1975. Adhesion of enteropathogenic Escherichia coli to pig intestinal brush borders: The existence of two pig phenotypes. J. Med. Microbiol., 8:405-411. Schlager, T.A., Wanke, Ch.A. and Guerrant, R.L., 1990. Net fluid secretion and impaired villous function induced by colonization of the small intestine by nontoxigenic colonizing Escherichia colL Infect. Immun., 58:1337-1343. Smith, H.W. and Halls, S., 1967. Observations by the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Path. Bact., 93: 499-529. Smith, H.W. and Linggood, M.A., 1971. Observations on the pathogenic properties of the K88, Hly and Ent plasmids of Escherichia coli with particular reference to porcine diarrhoea. J. Med. Microbiol., 4: 467-485. Sojka, W.J., 1965. Escherichia coli in domestic animals and poultry. Review series No. 7 of the Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England: 205-206. Stavric, S. and Jeffrey, D., 1977. A modified bioassay for heat-stable Escherichia coli enterotoxin. Can. J. Microbiol., 23:331-336. Svendsen, J., Larsen, J.L. and Bille, N., 1974. Outbreaks of post weaning Escherichia coli diarrhoea in pigs. Nord. Vet. Med., 26:314-322. Yamamoto, T. and Yokota, T., 1988. Electron microscopic study of Vibrio cholerae 01 adherence to the mucus. Infect. Immun., 56: 2753-2759.
