K99 Antigen-Positive Enterotoxigenic Escherichia coli from

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1981, p. 252-257 0095-1 137/81/020252-06$02.00/0

Vol. 13, No. 2

K99 Antigen-Positive Enterotoxigenic Escherichia coli from Piglets with Diarrhea in Sweden CYRIL J. SMYTH,'* EVA OLSSON,' CRISTINA MONCALVO,lt OLOF SODERLIND,2 FRITZ 0RSKOV,3 AND IDA 0RSKOV3 Department of Bacteriology and Epizootology, Swedish University of Agricultural Sciences, College of Veterinary Medicine, Biomedicum 583, S-751 23 Uppsala, Sweden'; National Veterinary Institute, S-750 07 Uppsala, Sweden2; and Collaborative Centre for Reference and Research on Escherichia (World Health Organization), Statens Seruminstitut, DK-2300 Copenhagen S, Denmark3

K88 antigen-negative enterotoxigenic Escherichia coli and non-enterotoxigenic strains isolated from piglets with diarrhea were examined for K99 antigen by agglutination tests after growth on Minca-IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.) agar medium. Of 64 K88-negative enterotoxigenic strains from as many piglets, 17 were found to be K99 positive. Of these, 10 were Swedish and 7 were of Norwegian origin. AUl 17 produced heat-stable enterotoxin detectable in the infant mouse assay, but only 2 gave positive ligated loop tests in 3- to 7-week-old piglets. Ligated loop tests in 5- to 12-day-old piglets were positive for each of the 15 K99-positive strains tested. Each of the Swedish K99-positive isolates was from a piglet of c7 days of age. One piglet harboring a K99-positive strain also harbored an 0141:K88 enterotoxigenic strain producing only heatstable enterotoxin. Five of the Swedish piglets yielding K99-positive isolates were from dams vaccinated with a multicomponent bacterial vaccine containing K88 antigen. The K99 strains were O:K:H serotyped. The O serogroups represented were 08, 09, 064, 0101, and 0140. None of 101 non-enterotoxigenic porcine isolates, representing 42 serogroups and non-0-groupable and rough strains, was found to be K99 positive. The findings indicate that so-called class 2 or atypical porcine enterotoxigenic E. coli should be routinely examined for the presence of K99 antigen. Certain specific ceil surface-associated adhesins on enterotoxigenic Escherichia coli (ETEC) have been shown to facilitate colonization of the small intestine of piglets, lambs, calves, and humans (1, 3, 16, 17). Much epidemiological evidence (e.g., reference 37) and also experimental studies (e.g., references 2 and 39) suggest that the host species range of ETEC is determined by the occurrence of these adhesins on the bacterial surface. Many porcine ETEC possess the plasmid-mediated K88 antigen, a fibrillar protein adhesin (7,8,27-29,34,39). However, ETEC lacking K88 antigen, when fed orally to piglets, have been shown to cause diarrhea (12, 24) and to colonize the ileum (12, 15, 19, 24). Some such strains possess a fimbrial adhesin called 987P (15, 25). Another plasmid-mediated adhesin, designated K99 antigen (30), occurs on ETEC isolated from cases of neonatal diarrhea in calves and lambs (8, 30, 32). The K99 antigen appeared at first to be associated only with such ETEC isolates. However, as a consequence of the studies t Present address: Av. Rivera 3309 ap. 003, Montevideo, Uruguay. 252

on non-K88

ETEC enteropathogenic for piglets (15, 19, 24), K99 antigen was identified on porcine ETEC (20). K99-positive porcine ETEC

have also been described in reports from the Netherlands (6, 8). The prevalence of K99 among K88-negative porcine ETEC in the United States has recently been recorded (18). The present investigation was initiated in 1978 (i) to determine whether K88-negative ETEC isolates from piglet diarrhea in Sweden possessed K99 antigen, (ii) to examine the enterotoxin types of such strains, and (iii) to fully serotype any K99-positive porcine ETEC found. A small number of Norwegian K88-negative isolates were included for comparative purposes. (Part of this study was reported at the 6th Congress of the International Pig Veterinary Society, 30 June-3 July 1980, Copenhagen, Denmark, abstract p. 143). MATERIALS AND METHODS Strains. The Swedish strains (isolates with a B or Bd prefix in Table 3) of K88-negative E. coli were representative of routine isolates from piglet diarrhea from 1975 through 1978. Of 153 strains from the same

VOL. 13, 1981 number of piglets, 56 were enterotoxigenic. Some of these strains had been used in previous studies (26, 33, 35, 36, 38). In addition, 12 K88-negative isolates, of which 8 were enterotoxigenic, originally received from E. Liven, Royal Veterinary College of Norway, Oslo, were also included in this investigation (strains with N designation in Table 3). The Norwegian isolates were derived from six herds. The laboratory strain K-12, carrying the K99 plasmid transferred from the reference strain E. coli B41, and its parent K-12 strain, both used for production of anti-K99 antiserum, were kindly supplied by C. Gyles, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada. Most of the strains had been preserved in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) at -70°C. Some had been stored at -20°C in meat extract broth (Difco Laboratories, Detroit, Mich.) containing 5% (vol/vol) inactivated horse serum. Ail strains had undergone minimal passage in vitro before testing for K99 antigen. K99 antiserum. An anti-K99 antiserumn was produced using the immunization schedule of Evans et al. (5) for raising antibodies to colonization factor antigen I. The vaccine was strain K-12(K99+) grown on MincaIsoVitaleX (BBL Microbiology Systems) agar medium (9) and suspended in 0.15 M NaCl containing 20 mM S0rensen phosphate buffer (pH 7.0) and 0.5% (vol/vol) formaldehyde. Antiserum. pools from two rabbits were absorbed extensively with heat-killed and live bacterial suspensions of the K-12(K99-) parent strain. Ceils from Minca-IsoVitaleX agar medium (37°C, 18 h) were used for absorption with live cells, whereas Trypticase soy broth-grown cells (37°C, 18 h, 120 rpm) were autoclaved for absorption with heat-killed cells. Immunoglobulins were precipitated with ammonium sulfate, and acetate dialysis was performed by the method of Harboe and Ingild (11). The anti-K99 antiserum was stored at 4°C in 0.1 M NaCI containing 15 mM NaN3. This antiserum was used routinely at a 1:10 dilution for agglutination tests. The anti-K99 antisera used at Statens Seruminstitut, Copenhagen were prepared against strain B41, the reference strain of antigen K99 (0101:K99), and against strain B85 (09:K99) (30). Serological test for K99 antigen. Slide agglutination tests using live bacterial suspensions were used to demonstrate K99 antigen. Strains were grown on 5% horse blood agar medium and subcultured onto Minca-IsoVitaleX agar medium. Confluent growth from half of a Minca-IsoVitaleX agar plate incubated at 37°C for 18 h was suspended in 1 ml of 0.15 M NaCI (about 109 bacteria per ml). Reference K99-positive and K99-negative strains were included as positive and negative controls for agglutination tests. Pre-immunization serum or saline was used for controls in slide agglutination. The procedure used at the Collaborative Centre for Reference and Research on Escherichia has been detailed (30). Strains which were deemed K99 negative after direct testing in this manner were subcultured from blood agar into tubes with 10 ml of Trypticase soy broth (14, 15). Tubes were incubated without shaking at 37°C for 48 to 72 h and passaged up to five times.

253

K99-POSITIVE ETEC IN SWEDEN

With the appearance of a distinct pellicle or floating bacterial mass, bacteria were subcultured onto MincaIsoVitaleX agar plates, and the test for K99 antigen was repeated as above. When no pellicle was obtained by passage 5, subculture and retesting were performed at this stage nonetheless. In addition, shake-flask cultures in Trypticase soy broth were performed with selected K99-negative (direct method) ETEC (13, 14). Subculture to Minca-IsoVitaleX agar medium was done after passage 4, based on the findings of Isaacson et al. (14). Serotyping. O antigen determination of strains was performed according to the method of Soderlind (34) to identify the 15 commonest 0 serogroups encountered among isolates from piglets with neonatal diarrhea in Sweden (O antigens 02, 06, 08, 09, 032, 045, 064, 098, 0115, 0138, 0139, 0141, 0147, 0149, and 0157). Strains not possessing these 0 antigens were O serogrouped at Statens Seruminstitut, Copenhagen. 0, K, and H antigen typing of K99-positive ETEC was done at this same Collaborative Reference Centre. The Norwegian isolates were serotyped on the above basis. Lack of K88 antigen was confirmed by slide agglutination tests (33, 34) before strains were included in this investigation. All strains in Table 3 and many in Table 4 were also tested for K99 antigen at Statens Seruminstitut. Enterotoxigenicity tests. The procedures employed have been detailed recently by Olsson and Soderlind (26). The following methods were used: ligated intestinal loop tests in 3- to 7-week-old piglets and in 5- to 12-day-old piglets, using standardized bacterial suspensions; the infant mouse assay in 2- to 3-day-old mice with heat-treated culture supernatant fluids; and the Y1 adrenal cell test with culture supernatant fluids. A summary of the designations for enterotoxigenicity types is given in Table 1. Retrospective analyses of isolates from piglets yielding a K99-positive isolate. When 10 isolates were available from individual piglets (36; 0. Soderlind and R. Mollby, Proc. 6th Congr. Int. Pig Vet. Soc., Copenhagen, Denmark, p. 145, 1980) from which one K99-positive isolate had been demonstrated, each was subsequently screened for K99 antigen, O serogrouped or O:K:H serotyped, and tested for enterotoxigenicity. Data on the age of piglets was obtained from the original records of case histories as submitted to the TABLE 1. Designations of enterotoxigenicity patterns ofporcine E. coli Enterotoxigenic by test Designation

Piglet

loop (3 te

Infant os

Adrenl lcl Y1ci

moe 7 weeks) + + ST+LT + + ST pig + mouse + ST pig + ST mouse + LT EntEnt? a For details see Olsson and Soderlind (26).

+

+

+

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National Veterinary Institute, Uppsala, by veterinarians.

RESULTS Enterotoxigenicity patterns and serogrouping of K88-negative strains. Table 2 shows the O serogroups and enterotoxigenicity type distributions of the E. coli isolates examined. Of the 64 enterotoxigenic strains, 14 produced heat-labile (LT) enterotoxin based on Y1 adrenal cell tests (three ST+LT strains, seven LT strains, and four Ent? strains, Table 2), and 50 produced only heat-stable (ST) enterotoxin as guaged by intestinal loop tests in piglets and or the infant mouse assay. The ETEC isolates belonged to 20 O serogroups. In addition, three strains, which were nongroupable with antisera to O antigens 1 through 16, and one rough strain were enterotoxigenic. Of t4e 101 nonenterotoxigenic isolates, 58 belonged to the same O serogroups as the ETEC strains or were non-0-serogroupable or rough. The other 43 nonenterotoxigenie strains were distributed among 28 TABLE 2. Enterotoxigenicity and O antigens of 165 porcine K88-negative E. coli strains No. of strains having enterotoxigenicity' o antigen 6 8 9 16 20 50 51 64 75 76 78 98 101 115 138 140 141 147 149 157

Ongc

' ST ST ST + SST+pig LT mouse pig mouse T

S

LT Ent

1 1 1 1

3

Ent?

6

1

3b

4b

lob 12b

1 1

1 3

1

2 3

TABLE 3. Characteristics of 17 K99-positive porcine E. coli 1

7b

4

Straina 1 1 1

2

1 3

4

1 2

1 1

1 1 1

5 1

2 1 1

3

1

1 1

3 1 1 3 5

1 4 Rough Othersd 43 a See Table 1 for designations of enterotoxigenicity types. bIncludes strains from E. Liven, Royal Veterinary College of Norway, Oslo. Non-0-serogroupable with antisera against O antigens 1 through 163. d Comprising the following O antigens (number of strains in parentheses): 01 (1), 02 (4), 05 (4), 011 (1), 015 (1), 019 (1), 038 (1), 040 (1), 045 (3), 049 (1), 054 (1), 069 (1), 071 (1), 079 (1), 088 (1), 089 (1), 091 (1), 093 (1), 0107 (1), 0108 (1), 0112 (1), 0118 (1), 0119 (1), 0126 (1), 0137 (1), 0139 (6), 0143 (1), OJF1 (1). '

other serogroups and were chosen to cover as broad a spectrum of O antigens as possible. K99-positive porcine ETEC. Characteristics of the 17 porcine strains identified as K99 positive are given in Table 3. The majority of these strains were negative in the standard piglet ligated loop test using 3- to 7-week-old piglets and reading fluid accumulation after 18 to 20 h. However, each of 15 strains tested gave positive reactions in younger piglets (Table 3) when fluid accumulation was read 10 to 12 h after inoculation of bacterial suspensions into ligated intestinal loops. No strain producing LT and none of the ST pig or Ent- strains (Table 2) was K99 positive. Five O antigens were represented among the K99-positive isolates, and all but two strains were nonmotile. Of the eight Norwegian ETEC, seven possessed K99 antigen. Strains N52, N53, N60, and N61 and strains N58, N62, and N63 were isolates from individual piglets in two herds, respectively. All 10 of the Swedish K99-positive isolates were from piglets which were c7 days old and from separate herds. In vitro detection of K99 antigen. All K99positive isolates in Tables 3 and 4 were detected by direct culture on Minca-IsoVitaleX agar medium. No strain which was deemed K99 negative on the basis of direct culture was subsequently shown to produce K99 antigen after selection of pellicle growth in tubes of Trypticase soy broth as described in Materials and Methods. Based

sero0:K:H type

Bd 2452/75 Bd 2562/76 I B 1600/77 VI Bd 3433/76 I

08:K85:H09:K35:H064:H064:K-:H-

Bd 144/78 I Bd 165/78 I Bd 854/75 Bd 135/78 III Bd 2068/75

0101:K30:H0101:K30:H0101:K-:H0101:K30:H0101:K30:H1

B 521/77 I

0140:K-:H18

Enterotoxigeict

typem tye

ST mouse ST mouse ST mouse ST pig + mouse ST mouse ST mouse ST mouse

Ligated piglet loop test (5 to 12

days old)' (+) +

NT + +

(+) +

ST mouse ST pig + mouse

NT

ST mouse

(+)

+

+ ST mouse 09:K35:H+ ST mouse 09:K35:HST mouse 09:K35:H(+) + ST mouse 064:K-:HST mouse 064:K-:H(+) + ST mouse 064:K-:H+ ST mouse 064:K-:HaB and Bd strains are Swedish; N strains are Norwegian isolates from two herds. bSee Table 1 for explanation. Volume-to-length ratios (milliiters/centimeter of gut): +, -1.0; (+), 0.5-1.0; NT, not tested.

N58 N62 N63 N52 N53 N60 N61


VOL. 13, 1981

TABLE 4. Characteristics of multiple isolates of E. coli from seven Swedish piglets harboring at least one K99-positive isolate

255

prevalence of K99 antigen on porcine ETEC (6, 8, 18, 20) and on its presence on strains with O antigens 09,064, and 0101. The serotype 0101:

K30:H- appears to be the commonest in the studies to date including the present. The seroNo type 0101:K28:NM has only been reported from Piglet no. the Netherlands (6, 8). No direct comparisons of iof (n 10) type lates frequencies of K99 antigen among porcine isolates can be made, however, as the present study B 521/77 10 10 Bd 2562/76 and those mentioned above all have used "seBd 165/78 10 lected" or "representative" collections of strains Bd 144/78 10 rather than being studies of a longitudinal char8 033:K-:HBd 3433/76 Ent2 acter. The serotypes 08:K85:H- and 0140:K-: 5 Bd 135/78 015 Ent1 O? H18 have not been previously described among Ent4 5 B 1600/77 0141:K88 ST pig + 1 porcine K99-positive ETEC, although the formouse mer is characteristic of bovine K99-positive 054 Ent1 strains (23, 30). O? Ent3 Anionic and cationic components of the K99 K99-positive isolates had the same characteristics as surface-associated adhesin have been described shown for isolates from the respective piglets in Table 3. b 0?, 0 antigen not determined but other than 02, 06, 08, (22). However, Morris et al. (23) have presented 09,032,045,064,098,0115,0138,0139,0141,0147,0149, evidence that these are two distinct adhesive and 0157. See Table 1 for explanation of enterotoxigenicity antigens. The cationic antigen is considered to types. Ligated intestinal loop tests in 5- to 12-day-old piglets be K99 antigen. The reference K99 strain E. coli were not performed for all K99-positive isolates from each B41 produces in addition the anionic adhesin piglet. which is antigenically distinct from K99 antigen. on the findings of Isaacson and co-workers (13, These authors also found that the anionic ad14), K99-negative isolates (direct method) of ST hesin is produced by K99-positive bacteria posmouse and ST pig + mouse enterotoxigenicity sessing 0 antigens 09 and 0101, whereas K99types with O antigens 08, 09, and 064 (seven positive strains within O serogroups 08, 020, strains) plus the three ST pig strains with O and 064 and the laboratory strain K-12(K99+) antigen 08 (Table 2) were also grown using the only produce the cationic K99 antigen (23). modified passage procedure in shake flasks with Thus, the antiserum raised in the present study Trypticase soy broth in an attempt to enhance in Sweden and that produced at the National K99 detectability. ARl tests for K99 antigen pro- Animal Diseases Center, Ames, Iowa (20) by duction after passage 4 were negative. using a K-12(K99+) vaccine both contain only Examination of multiple isolates from in- agglutinins specific for K99 antigen. Accorddividual piglets. For 7 of the 10 Swedish piglets ingly, demonstration of the presence of the anyielding a K99-positive isolate, 10 single-colony ionic adhesin would not have been possible with isolates were available (Table 4). In four in- the anti-K99 antiserum raised in Sweden. Ail of the K99 antigen-positive porcine isolates stances, all 10 isolates were K99 positive and had the same O antigen or serotype and the in the present investigation produced only ST same enterotoxigenicity type as the first K99- enterotoxin detectable in the infant mouse assay positive isolates examined from the respective and in ligated loop tests in 5- to 12-day-old piglets. All but 1 of the 12 K99-negative isolates piglets. None of the porcine K99-positive E. coli in the other three piglets were nonenterotoxi- studied by Guinée and Jansen (8) or by Moon et genic. The one exception, strain B 1600/77 II, al. (18) produced LT enterotoxin detectable by was a K88-positive isolate with O antigen 0141 the adrenal cell test or by stimulation of cyclic and produced only ST enterotoxin(s). adenosine 3',5'-monophosphate synthesis in baby hamster kidney cells. Moon et al. (18) DISCUSSION described one K99-positive porcine isolate that Porcine enteropathogenic E. coli with O an- would be described as ST pig by our criteria, tigens 09, 064, and 0101 have been described whereas the remaining seven in their study and as class 2 (21) or as atypical (32). Such porcine the seven K99-positive isolates described by Guiisolates were first shown to produce the plasmid- née and Jansen (8) were all reactive in the infant mediated K99 antigen in 1977 (6, 20). At least 15 mouse assay. Thus, all K99-positive porcine E. of the K99 isolates in Table 3 fit the class 2 coli described to date have been only ST enterdefinition (21). otoxigenic. Indeed, of 117 bovine E. coli isolates Previous investigators have reported on the tested by Guinée and Jansen (8), the 74 that K99-negative isolate No. of K99positive Srotype or ei oslts antigen genicity =

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K99 positive gave positive infant mouse tion). The present findings indicate that atypical and positive calf intestinal loop tests, or class 2 porcine ETEC should be routinely whereas the remaining 43 strains, which be- checked for K99 antigen. longed to the same 0-antigen groups, were negACKNOWLEDGMENTS ative in all three respects. Again, the K99-posiWe express our thanks to R. Mollby for help, interest, and tive bovine strains were negative in Y1 adrenal and to Gunnel Sigstam, Kristina Zimmerman, Ulla cell tests for LT. Thus, there appears to be a criticismand R. Mattson for excellent technical assistance. general correlation between possession of K99 Nestor, This study was supported by the Swedish Council for antigen and ST-only enterotoxigenicity among Forestry and Agricultural Research. animal strains of E. coli. LITERATURE CITED In agreement with Moon et al. (18), no K991. Arbuthnott, J. P., and C. J. Smyth. 1979. Bacterial positive strain in this investigation originated adhesion in host/pathogen interactions in animals, p. from piglets over 2 weeks old. The study of 165-198. In D. C. Ellwood, J. Melling, and P. Rutter Moon and Whipp (21) showed a development of (ed.), Adhesion of microorganisms to surfaces. Acaresistance with age by the piglet intestine to demic Press, London. class 2 porcine enteropathogenic E. coli. More 2. Bertschinger, H. U., H. W. Moon, and S. C. Whipp. 1972. Association of Escherichia coli with the small recent observations (31) have indicated that epintestinal epithelium. I. Comparison of enteropathoithelial cells from piglet intestine become resistgenic and nonenteropathogenic porcine strains in pigs. ant to K99 antigen-mediated adhesion with inInfect. Immun. 5:595-605. creasing age of piglets, possibly due to a reduc- 3. Duguid, J. P., and D. C. Old. 1980. Adhesive properties of Enterobacteriaceae, p. 185-217. In E. H. Beachey tion in the numbers of receptors for K99 antigen, (ed.), Bacterial adherence: receptors and recognition, i.e., the piglet intestine becomes refractory to series B, vol. 6. Chapman and Hall, London. colonization by K99-positive ETEC that have 4. Ellis, R. P. 1979. Serologic and epidemiologic investigations of colibacillosis in pigs, p. 161-166. In S. Acres been described as atypical or class 2 enteropath(ed.), Proceedings of the 2nd International Symposium ogenic E. coli. Our case history data support on Neonatal Diarrhea. Veterinary Infectious Diseases these observations. Organization, Saskatoon, Canada. In piglets from which K99-positive ETEC 5. Evans, D. G., R. P. Silver, D. J. Evans, Jr., D. G. Chase, and S. L. Gorbach. 1975. Plasmid-controlled were isolated, these enterotoxigenic bacteria colonization factor associated with virulence in Eschedominated. Interestingly, in one piglet one 0141: richia coli enterotoxigenic for humans. Infect. Immun. K88, ST pig + mouse isolate was detected in 12:656-667. addition to five K99-positive isolates, indicating 6. Guinée, P. A. M., C. M. Agterberg, W. H. Jansen, and J. F. Frik. 1977. Serological identification of pig enterthat ETEC infections may in some instances otoxigenic Escherichia coli strains not belonging to the involve strains with different adhesins, simultaclassical serotypes. Infect. Immun. neously. The dam of this piglet had been vacci- 7. Guinée, P. A. M., and W. H. Jansen.15:549-555. 1979. Behavior of nated with a commercial, multicomponent, Escherichia coli K antigens K88ab, K88ac, and K88ad in immunoelectrophoresis, double diffusion, and hemagwhole bacterial vaccine containing K88 antigen. glutination. Infect. Immun. 23:700-705. Four of the other piglets yielding K99-positive 8. Guinée, P. A. M., and W. H. Jansen. 1979. Detection of ETEC were also from similarly vaccinated dams enterotoxigenicity and attachment factors in Escherichia coli strains of human, porcine and bovine origin; (0. Soderlind and R. Mollby, Proc. 6th. Congr. a comparative study. Zentralbl. Bakteriol. Parasitenkd. Int. Pig Vet. Soc., Copenhagen, Denmark, p. 145, Infektionskr. Hyg. Abt. 1 Orig. Reihe A 243:245-257. 1980; 0. Soderlind, E. Olsson, C. J. Smyth, and 9. Guinée, P. A. M., J. Veldkamp, and W. H. Jansen. R. Mollby, manuscript in preparation). 1977. Improved Minca medium for the detection of K99 In conclusion, K99-positive porcine ETEC antigen in calf enterotoxigenic strains of Escherichia Immun. 15:676-678. producing only ST enterotoxin(s) have been 10. coli. Infect. Gyles, C. L. 1979. Limitations of the infant mouse test for identified among Swedish and Norwegian K88Escherichia coli heat stable enterotoxin. Can. J. Comp. negative ETEC isolates. Most of these belong to Med. 43:371-379. serotypes, and have enterotoxigenicity charac- 11. Harboe, N., and A. Ingild. 1973. Immunization, isolation of immunoglobulin, estimation of antibody titre, p. teristics, of ETEC previously described as so161-164. In N. H. Axelsen, J. Kroll, and B. Weeke (ed.), cailed atypical or class 2 isolates. Atypical or A manual of quantitative immunoelectrophoresis. Univclass 2 isolates, many of which may probably ersitetsforlaget, Oslo. bear K99 antigen, have increased in frequency 12. Hohmann, A., and M. R. Wilson. 1975. Adherence of enteropathogenic Escherichia coli to intestinal epitheamong ETEC from neonatal diarrhea in both hum in vivo. Infect. Immun. 12:866-880. the United States (4) and Canada (M. Wilson 13. Isaacson, R. E. 1980. Factors affecting expression of the and C. Gyles, unpublished data cited in reference Escherichia coli pilus K99. Infect. Immun. 28:190-194. 10). Some epidemiological factors influencing 14. Isaacson, R. E., H. W. Moon, and R. A. Schneider. 1978. Distribution and virulence of Escherichia coli in the occurrence of K99-positive strains in piglet the small intestines of calves with and without diarrhea. diarrhea will form the subject of a separate Am. J. Vet. Res. 39:1750-1755. report (Soderlind et al., manuscript in prepara- 15. Isaacson, R. E., B. Nagy, and H. W. Moon. 1977. were

assays

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16.

17.

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26.

27.

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