JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1981, p. 252-2570095-1 137/81/020252-06$02.00/0

Vol. 13, No. 2

K99 Antigen-Positive Enterotoxigenic Escherichia coli fromPiglets with Diarrhea in Sweden

CYRIL J. SMYTH,'* EVA OLSSON,' CRISTINA MONCALVO,lt OLOF SODERLIND,2FRITZ 0RSKOV,3 AND IDA 0RSKOV3

Department of Bacteriology and Epizootology, Swedish University ofAgricultural Sciences, College ofVeterinary Medicine, Biomedicum 583, S-751 23 Uppsala, Sweden'; National Veterinary Institute, S-750 07Uppsala, 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-enterotoxigenicstrains isolated from piglets with diarrhea were examined for K99 antigen byagglutination tests after growth on Minca-IsoVitaleX (BBL Microbiology Sys-tems, Cockeysville, Md.) agar medium. Of 64 K88-negative enterotoxigenic strainsfrom as many piglets, 17 were found to be K99 positive. Of these, 10 were Swedishand 7 were of Norwegian origin. AUl 17 produced heat-stable enterotoxin detect-able in the infant mouse assay, but only 2 gave positive ligated loop tests in 3- to7-week-old piglets. Ligated loop tests in 5- to 12-day-old piglets were positive foreach of the 15 K99-positive strains tested. Each of the Swedish K99-positiveisolates was from a piglet of c7 days of age. One piglet harboring a K99-positivestrain also harbored an 0141:K88 enterotoxigenic strain producing only heat-stable enterotoxin. Five of the Swedish piglets yielding K99-positive isolates werefrom dams vaccinated with a multicomponent bacterial vaccine containing K88antigen. The K99 strains were O:K:H serotyped. The O serogroups representedwere 08, 09, 064, 0101, and 0140. None of 101 non-enterotoxigenic porcineisolates, representing 42 serogroups and non-0-groupable and rough strains, wasfound to be K99 positive. The findings indicate that so-called class 2 or atypicalporcine enterotoxigenic E. coli should be routinely examined for the presence ofK99 antigen.

Certain specific ceil surface-associated adhe-sins on enterotoxigenic Escherichia coli (ETEC)have been shown to facilitate colonization of thesmall intestine of piglets, lambs, calves, and hu-mans (1, 3, 16, 17). Much epidemiological evi-dence (e.g., reference 37) and also experimentalstudies (e.g., references 2 and 39) suggest thatthe host species range of ETEC is determinedby the occurrence of these adhesins on the bac-terial surface. Many porcine ETEC possess theplasmid-mediated K88 antigen, a fibrillar pro-tein adhesin (7,8,27-29,34,39). However, ETEClacking K88 antigen, when fed orally to piglets,have been shown to cause diarrhea (12, 24) andto colonize the ileum (12, 15, 19, 24). Some suchstrains possess a fimbrial adhesin called 987P(15, 25).Another plasmid-mediated adhesin, desig-

nated K99 antigen (30), occurs on ETEC isolatedfrom cases of neonatal diarrhea in calves andlambs (8, 30, 32). The K99 antigen appeared atfirst to be associated only with such ETEC iso-lates. However, as a consequence of the studies

t Present address: Av. Rivera 3309 ap. 003, Montevideo,Uruguay.

on non-K88 ETEC enteropathogenic for piglets(15, 19, 24), K99 antigen was identified on por-cine ETEC (20). K99-positive porcine ETEChave also been described in reports from theNetherlands (6, 8). The prevalence of K99among K88-negative porcine ETEC in theUnited States has recently been recorded (18).The present investigation was initiated in 1978

(i) to determine whether K88-negative ETECisolates from piglet diarrhea in Sweden pos-sessed K99 antigen, (ii) to examine the entero-toxin types of such strains, and (iii) to fullyserotype any K99-positive porcine ETEC found.A small number of Norwegian K88-negative iso-lates were included for comparative purposes.

(Part of this study was reported at the 6thCongress of the International Pig VeterinarySociety, 30 June-3 July 1980, Copenhagen, Den-mark, abstract p. 143).

MATERIALS AND METHODSStrains. The Swedish strains (isolates with a B or

Bd prefix in Table 3) of K88-negative E. coli wererepresentative of routine isolates from piglet diarrheafrom 1975 through 1978. Of 153 strains from the same

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K99-POSITIVE ETEC IN SWEDEN 253

number of piglets, 56 were enterotoxigenic. Some ofthese strains had been used in previous studies (26, 33,35, 36, 38). In addition, 12 K88-negative isolates, ofwhich 8 were enterotoxigenic, originally received fromE. Liven, Royal Veterinary College of Norway, Oslo,were also included in this investigation (strains withN designation in Table 3). The Norwegian isolateswere derived from six herds.The laboratory strain K-12, carrying the K99 plas-

mid transferred from the reference strain E. coli B41,and its parent K-12 strain, both used for production ofanti-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 Trypti-

case soy broth (BBL Microbiology Systems, Cockeys-ville, Md.) at -70°C. Some had been stored at -20°Cin meat extract broth (Difco Laboratories, Detroit,Mich.) containing 5% (vol/vol) inactivated horse se-

rum. Ail strains had undergone minimal passage invitro before testing for K99 antigen.K99 antiserum. An anti-K99 antiserumn was pro-

duced using the immunization schedule of Evans et al.(5) for raising antibodies to colonization factor antigenI. The vaccine was strain K-12(K99+) grown on Minca-IsoVitaleX (BBL Microbiology Systems) agar medium(9) and suspended in 0.15 M NaCl containing 20 mMS0rensen phosphate buffer (pH 7.0) and 0.5% (vol/vol)formaldehyde. Antiserum. pools from two rabbits wereabsorbed extensively with heat-killed and live bacte-rial suspensions of the K-12(K99-) parent strain. Ceilsfrom Minca-IsoVitaleX agar medium (37°C, 18 h) wereused for absorption with live cells, whereas Trypticasesoy broth-grown cells (37°C, 18 h, 120 rpm) were

autoclaved for absorption with heat-killed cells. Im-munoglobulins were precipitated with ammonium sul-fate, and acetate dialysis was performed by the methodof Harboe and Ingild (11). The anti-K99 antiserumwas stored at 4°C in 0.1 M NaCI containing 15 mMNaN3. This antiserum was used routinely at a 1:10dilution for agglutination tests.The anti-K99 antisera used at Statens Seruminsti-

tut, Copenhagen were prepared against strain B41, thereference strain of antigen K99 (0101:K99), andagainst strain B85 (09:K99) (30).

Serological test for K99 antigen. Slide aggluti-nation tests using live bacterial suspensions were usedto demonstrate K99 antigen. Strains were grown on

5% horse blood agar medium and subcultured ontoMinca-IsoVitaleX agar medium. Confluent growthfrom half of a Minca-IsoVitaleX agar plate incubatedat 37°C for 18 h was suspended in 1 ml of 0.15 M NaCI(about 109 bacteria per ml). Reference K99-positiveand K99-negative strains were included as positiveand negative controls for agglutination tests. Pre-im-munization serum or saline was used for controls inslide agglutination. The procedure used at the Collab-orative Centre for Reference and Research on Esche-richia has been detailed (30).

Strains which were deemed K99 negative after di-rect testing in this manner were subcultured fromblood agar into tubes with 10 ml of Trypticase soybroth (14, 15). Tubes were incubated without shakingat 37°C for 48 to 72 h and passaged up to five times.

With the appearance of a distinct pellicle or floatingbacterial mass, bacteria were subcultured onto Minca-IsoVitaleX agar plates, and the test for K99 antigenwas repeated as above. When no pellicle was obtainedby passage 5, subculture and retesting were performedat this stage nonetheless. In addition, shake-flask cul-tures in Trypticase soy broth were performed withselected K99-negative (direct method) ETEC (13, 14).Subculture to Minca-IsoVitaleX agar medium wasdone after passage 4, based on the findings of Isaacsonet al. (14).

Serotyping. O antigen determination ofstrains wasperformed according to the method of Soderlind (34)to identify the 15 commonest 0 serogroups encoun-tered among isolates from piglets with neonatal diar-rhea in Sweden (O antigens 02, 06, 08, 09, 032, 045,064, 098, 0115, 0138, 0139, 0141, 0147, 0149, and0157). Strains not possessing these 0 antigens were Oserogrouped at Statens Seruminstitut, Copenhagen.0, K, and H antigen typing of K99-positive ETEC wasdone at this same Collaborative Reference Centre.The Norwegian isolates were serotyped on the abovebasis. Lack of K88 antigen was confirmed by slideagglutination tests (33, 34) before strains were includedin this investigation. All strains in Table 3 and manyin Table 4 were also tested for K99 antigen at StatensSeruminstitut.

Enterotoxigenicity tests. The procedures em-ployed have been detailed recently by Olsson andSoderlind (26). The following methods were used:ligated intestinal loop tests in 3- to 7-week-old pigletsand in 5- to 12-day-old piglets, using standardizedbacterial suspensions; the infant mouse assay in 2- to3-day-old mice with heat-treated culture supernatantfluids; and the Y1 adrenal cell test with culture super-natant fluids. A summary of the designations for en-terotoxigenicity types is given in Table 1.

Retrospective analyses ofisolates from pigletsyielding a K99-positive isolate. When 10 isolateswere available from individual piglets (36; 0. Soderlindand R. Mollby, Proc. 6th Congr. Int. Pig Vet. Soc.,Copenhagen, Denmark, p. 145, 1980) from which oneK99-positive isolate had been demonstrated, each wassubsequently screened for K99 antigen, O serogroupedor O:K:H serotyped, and tested for enterotoxigenicity.Data on the age of piglets was obtained from theoriginal records of case histories as submitted to the

TABLE 1. Designations of enterotoxigenicitypatterns ofporcine E. coli

Enterotoxigenic by test

Designation Piglet Infant Adrenlloop (3 te

os lcl7 weeks) moe Y1ci

ST+LT + + +ST pig + mouse + +ST pig + - -ST mouse - +LT + - +Ent- - - -Ent? - - +

a For details see Olsson and Soderlind (26).

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254 SMYTH ET AL.

National Veterinary Institute, Uppsala, by veterinari-ans.

RESULTSEnterotoxigenicity patterns and sero-

grouping of K88-negative strains. Table 2shows the O serogroups and enterotoxigenicitytype distributions of the E. coli isolates exam-ined. Of the 64 enterotoxigenic strains, 14 pro-duced heat-labile (LT) enterotoxin based on Y1adrenal cell tests (three ST+LT strains, sevenLT strains, and four Ent? strains, Table 2), and50 produced only heat-stable (ST) enterotoxinas guaged by intestinal loop tests in piglets andor the infant mouse assay. The ETEC isolatesbelonged to 20 O serogroups. In addition, threestrains, which were nongroupable with antiserato O antigens 1 through 16, and one rough strainwere enterotoxigenic. Of t4e 101 nonenterotoxi-genic isolates, 58 belonged to the same O sero-groups as the ETEC strains or were non-0-ser-ogroupable or rough. The other 43 nonentero-toxigenie strains were distributed among 28

TABLE 2. Enterotoxigenicity and O antigens of 165porcine K88-negative E. coli strains

No. of strains having enterotoxigenicity'o anti- S ' T Sgen ST + ST+pig ST ST LT Ent Ent?

LT pig mousemouse

6 1 68 1 3 1 3b lob9 1 4b 12b16 1 120 1 1 350 151 1 264 4 7b 375 176 178 198 2 1101 1 4 3115 2 1138 1 1 1 1140 1141 5 2 3147 1 1 1149 1 1 1157 1 3

Ongc 3 5Rough 1 4Othersd 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 asbroad a spectrum of O antigens as possible.K99-positive porcine ETEC. Characteris-

tics of the 17 porcine strains identified as K99positive are given in Table 3. The majority ofthese strains were negative in the standard pigletligated loop test using 3- to 7-week-old pigletsand reading fluid accumulation after 18 to 20 h.However, each of 15 strains tested gave positivereactions in younger piglets (Table 3) when fluidaccumulation was read 10 to 12 h after inocula-tion of bacterial suspensions into ligated intes-tinal loops. No strain producing LT and none ofthe ST pig or Ent- strains (Table 2) was K99positive. Five O antigens were representedamong the K99-positive isolates, and all but twostrains were nonmotile. Of the eight NorwegianETEC, seven possessed K99 antigen. StrainsN52, N53, N60, and N61 and strains N58, N62,and N63 were isolates from individual piglets intwo herds, respectively. All 10 of the SwedishK99-positive isolates were from piglets whichwere c7 days old and from separate herds.In vitro detection ofK99 antigen. All K99-

positive isolates in Tables 3 and 4 were detectedby direct culture on Minca-IsoVitaleX agar me-dium. No strain which was deemed K99 negativeon the basis of direct culture was subsequentlyshown to produce K99 antigen after selection ofpellicle growth in tubes of Trypticase soy brothas described in Materials and Methods. Based

TABLE 3. Characteristics of 17 K99-positive porcineE. coli

Enterotoxi- Ligated pig-Straina 0:K:H sero- geict let loop test

type typem (5 to 12tye days old)'

Bd 2452/75 08:K85:H- ST mouse (+)Bd 2562/76 I 09:K35:H- ST mouse +B 1600/77 VI 064:H- ST mouse NTBd 3433/76 I 064:K-:H- ST pig + +

mouseBd 144/78 I 0101:K30:H- ST mouse +Bd 165/78 I 0101:K30:H- ST mouse (+)Bd 854/75 0101:K-:H- ST mouse +Bd 135/78 III 0101:K30:H- ST mouse NTBd 2068/75 0101:K30:H1 ST pig + +

mouseB 521/77 I 0140:K-:H18 ST mouse (+)

N58 09:K35:H- ST mouse +N62 09:K35:H- ST mouse +N63 09:K35:H- ST mouse (+)N52 064:K-:H- ST mouse +N53 064:K-:H- ST mouse (+)N60 064:K-:H- ST mouse +N61 064:K-:H- ST mouse +

aB and Bd strains are Swedish; N strains are Norwegianisolates 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.

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K99-POSITIVE ETEC IN SWEDEN 255

TABLE 4. Characteristics ofmultiple isolates ofE.coli from seven Swedish piglets harboring at least

one K99-positive isolate

No. of K99-negative isolateK99- No

Piglet no. positive Srotype or eioslts antigen genicity iof(n = 10) type lates

B 521/77 10Bd 2562/76 10Bd 165/78 10Bd 144/78 10Bd 3433/76 8 033:K-:H- Ent- 2Bd 135/78 5 015 Ent- 1

O? Ent- 4B 1600/77 5 0141:K88 ST pig + 1

mouse054 Ent- 1O? Ent- 3

K99-positive isolates had the same characteristics asshown for isolates from the respective piglets in Table 3.

b 0?, 0 antigen not determined but other than 02, 06, 08,09,032,045,064,098,0115,0138,0139,0141,0147,0149,and 0157. See Table 1 for explanation of enterotoxigenicitytypes. Ligated intestinal loop tests in 5- to 12-day-old pigletswere not performed for all K99-positive isolates from eachpiglet.

on the findings of Isaacson and co-workers (13,14), K99-negative isolates (direct method) ofSTmouse and ST pig + mouse enterotoxigenicitytypes with O antigens 08, 09, and 064 (sevenstrains) plus the three ST pig strains with Oantigen 08 (Table 2) were also grown using themodified passage procedure in shake flasks withTrypticase soy broth in an attempt to enhanceK99 detectability. ARl tests for K99 antigen pro-duction after passage 4 were negative.Examination ofmultiple isolates from in-

dividual piglets. For 7 ofthe 10 Swedish pigletsyielding a K99-positive isolate, 10 single-colonyisolates were available (Table 4). In four in-stances, all 10 isolates were K99 positive andhad the same O antigen or serotype and thesame enterotoxigenicity type as the first K99-positive isolates examined from the respectivepiglets. All but 1 of the 12 K99-negative isolatesin the other three piglets were nonenterotoxi-genic. The one exception, strain B 1600/77 II,was a K88-positive isolate with O antigen 0141and produced only ST enterotoxin(s).

DISCUSSIONPorcine enteropathogenic E. coli with O an-

tigens 09, 064, and 0101 have been describedas class 2 (21) or as atypical (32). Such porcineisolates were first shown to produce the plasmid-mediated K99 antigen in 1977 (6, 20). At least 15of the K99 isolates in Table 3 fit the class 2definition (21).

Previous investigators have reported on the

prevalence of K99 antigen on porcine ETEC (6,8, 18, 20) and on its presence on strains with Oantigens 09,064, and 0101. The serotype 0101:K30:H- appears to be the commonest in thestudies to date including the present. The sero-type 0101:K28:NM has only been reported fromthe Netherlands (6, 8). No direct comparisons offrequencies of K99 antigen among porcine iso-lates can be made, however, as the present studyand those mentioned above all have used "se-lected" or "representative" collections of strainsrather than being studies of a longitudinal char-acter. The serotypes 08:K85:H- and 0140:K-:H18 have not been previously described amongporcine K99-positive ETEC, although the for-mer is characteristic of bovine K99-positivestrains (23, 30).

Anionic and cationic components of the K99surface-associated adhesin have been described(22). However, Morris et al. (23) have presentedevidence that these are two distinct adhesiveantigens. The cationic antigen is considered tobe K99 antigen. The reference K99 strain E. coliB41 produces in addition the anionic adhesinwhich is antigenically distinct from K99 antigen.These authors also found that the anionic ad-hesin is produced by K99-positive bacteria pos-sessing 0 antigens 09 and 0101, whereas K99-positive strains within O serogroups 08, 020,and 064 and the laboratory strain K-12(K99+)only produce the cationic K99 antigen (23).Thus, the antiserum raised in the present studyin Sweden and that produced at the NationalAnimal Diseases Center, Ames, Iowa (20) byusing a K-12(K99+) vaccine both contain onlyagglutinins specific for K99 antigen. Accord-ingly, demonstration of the presence of the an-ionic adhesin would not have been possible withthe anti-K99 antiserum raised in Sweden.

Ail ofthe K99 antigen-positive porcine isolatesin the present investigation produced only STenterotoxin detectable in the infant mouse assayand in ligated loop tests in 5- to 12-day-oldpiglets. None of the porcine K99-positive E. colistudied by Guinée and Jansen (8) or by Moon etal. (18) produced LT enterotoxin detectable bythe adrenal cell test or by stimulation of cyclicadenosine 3',5'-monophosphate synthesis inbaby hamster kidney cells. Moon et al. (18)described one K99-positive porcine isolate thatwould be described as ST pig by our criteria,whereas the remaining seven in their study andthe seven K99-positive isolates described by Gui-née and Jansen (8) were all reactive in the infantmouse assay. Thus, all K99-positive porcine E.coli described to date have been only ST enter-otoxigenic. Indeed, of 117 bovine E. coli isolatestested by Guinée and Jansen (8), the 74 that

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256 SMYTH ET AL.

were K99 positive gave positive infant mouseassays and positive calf intestinal loop tests,whereas the remaining 43 strains, which be-longed to the same 0-antigen groups, were neg-ative in all three respects. Again, the K99-posi-tive bovine strains were negative in Y1 adrenalcell tests for LT. Thus, there appears to be ageneral correlation between possession of K99antigen and ST-only enterotoxigenicity amonganimal strains of E. coli.

In agreement with Moon et al. (18), no K99-positive strain in this investigation originatedfrom piglets over 2 weeks old. The study ofMoon and Whipp (21) showed a development ofresistance with age by the piglet intestine toclass 2 porcine enteropathogenic E. coli. Morerecent observations (31) have indicated that ep-ithelial cells from piglet intestine become resist-ant to K99 antigen-mediated adhesion with in-creasing age of piglets, possibly due to a reduc-tion in the numbers of receptors for K99 antigen,i.e., the piglet intestine becomes refractory tocolonization by K99-positive ETEC that havebeen described as atypical or class 2 enteropath-ogenic E. coli. Our case history data supportthese observations.

In piglets from which K99-positive ETECwere isolated, these enterotoxigenic bacteriadominated. Interestingly, in one piglet one 0141:K88, ST pig + mouse isolate was detected inaddition to five K99-positive isolates, indicatingthat ETEC infections may in some instancesinvolve strains with different adhesins, simulta-neously. The dam of this piglet had been vacci-nated with a commercial, multicomponent,whole bacterial vaccine containing K88 antigen.Four of the other piglets yielding K99-positiveETEC were also from similarly vaccinated dams(0. Soderlind and R. Mollby, Proc. 6th. Congr.Int. Pig Vet. Soc., Copenhagen, Denmark, p. 145,1980; 0. Soderlind, E. Olsson, C. J. Smyth, andR. Mollby, manuscript in preparation).

In conclusion, K99-positive porcine ETECproducing only ST enterotoxin(s) have beenidentified among Swedish and Norwegian K88-negative ETEC isolates. Most of these belong toserotypes, and have enterotoxigenicity charac-teristics, of ETEC previously described as so-

cailed atypical or class 2 isolates. Atypical orclass 2 isolates, many of which may probablybear K99 antigen, have increased in frequencyamong ETEC from neonatal diarrhea in boththe United States (4) and Canada (M. Wilsonand C. Gyles, unpublished data cited in reference10). Some epidemiological factors influencingthe occurrence of K99-positive strains in pigletdiarrhea will form the subject of a separatereport (Soderlind et al., manuscript in prepara-

J. CLIN. MICROBIOL.

tion). The present findings indicate that atypicalor class 2 porcine ETEC should be routinelychecked for K99 antigen.

ACKNOWLEDGMENTSWe express our thanks to R. Mollby for help, interest, and

criticism and to Gunnel Sigstam, Kristina Zimmerman, UllaNestor, and R. Mattson for excellent technical assistance.

This study was supported by the Swedish Council forForestry and Agricultural Research.

LITERATURE CITED1. Arbuthnott, J. P., and C. J. Smyth. 1979. Bacterial

adhesion in host/pathogen interactions in animals, p.165-198. In D. C. Ellwood, J. Melling, and P. Rutter(ed.), Adhesion of microorganisms to surfaces. Aca-demic Press, London.

2. Bertschinger, H. U., H. W. Moon, and S. C. Whipp.1972. Association of Escherichia coli with the smallintestinal epithelium. I. Comparison of enteropatho-genic and nonenteropathogenic porcine strains in pigs.Infect. Immun. 5:595-605.

3. Duguid, J. P., and D. C. Old. 1980. Adhesive propertiesof Enterobacteriaceae, p. 185-217. In E. H. Beachey(ed.), Bacterial adherence: receptors and recognition,series B, vol. 6. Chapman and Hall, London.

4. Ellis, R. P. 1979. Serologic and epidemiologic investiga-tions of colibacillosis in pigs, p. 161-166. In S. Acres(ed.), Proceedings of the 2nd International Symposiumon Neonatal Diarrhea. Veterinary Infectious DiseasesOrganization, Saskatoon, Canada.

5. Evans, D. G., R. P. Silver, D. J. Evans, Jr., D. G.Chase, and S. L. Gorbach. 1975. Plasmid-controlledcolonization factor associated with virulence in Esche-richia coli enterotoxigenic for humans. Infect. Immun.12:656-667.

6. Guinée, P. A. M., C. M. Agterberg, W. H. Jansen, andJ. F. Frik. 1977. Serological identification of pig enter-otoxigenic Escherichia coli strains not belonging to theclassical serotypes. Infect. Immun. 15:549-555.

7. Guinée, P. A. M., and W. H. Jansen. 1979. Behavior ofEscherichia coli K antigens K88ab, K88ac, and K88adin immunoelectrophoresis, double diffusion, and hemag-glutination. Infect. Immun. 23:700-705.

8. Guinée, P. A. M., and W. H. Jansen. 1979. Detection ofenterotoxigenicity and attachment factors in Esche-richia coli strains of human, porcine and bovine origin;a comparative study. Zentralbl. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt. 1 Orig. Reihe A 243:245-257.

9. Guinée, P. A. M., J. Veldkamp, and W. H. Jansen.1977. Improved Minca medium for the detection of K99antigen in calf enterotoxigenic strains of Escherichiacoli. Infect. Immun. 15:676-678.

10. Gyles, C. L. 1979. Limitations of the infant mouse test forEscherichia coli heat stable enterotoxin. Can. J. Comp.Med. 43:371-379.

11. Harboe, N., and A. Ingild. 1973. Immunization, isolationof immunoglobulin, estimation of antibody titre, p.161-164. In N. H. Axelsen, J. Kroll, and B. Weeke (ed.),A manual of quantitative immunoelectrophoresis. Univ-ersitetsforlaget, Oslo.

12. Hohmann, A., and M. R. Wilson. 1975. Adherence ofenteropathogenic Escherichia coli to intestinal epithe-hum in vivo. Infect. Immun. 12:866-880.

13. Isaacson, R. E. 1980. Factors affecting expression of theEscherichia coli pilus K99. Infect. Immun. 28:190-194.

14. Isaacson, R. E., H. W. Moon, and R. A. Schneider.1978. Distribution and virulence of Escherichia coli inthe small intestines of calves with and without diarrhea.Am. J. Vet. Res. 39:1750-1755.

15. Isaacson, R. E., B. Nagy, and H. W. Moon. 1977.

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Colonization of porcine intestine by Escherichia coli:colonization and adhesion factors of pig enteropatho-gens that lack K88. J. Infect. Dis. 135:531-539.

16. Jones, G. W. 1977. The attachment of bacteria to thesurfaces of animal cells, p. 139-176. In L. Reissig (ed.),Microbial interactions: receptors and recognition, seriesB, vol. 3. Chapman and Hall, London.

17. Moon, H. W., R. E. Isaacson, and J. Pohlenz. 1979.Mechanisms of association of enteropathogenic Esche-richia coli with intestinal epithelium. Am. J. Clin. Nu-trit. 32:119-127.

18. Moon, H. W., E. M. Kohler, R. A. Schneider, and S.C. Whipp. 1980. Prevalence of pilus antigens, entero-toxin types, and enteropathogenicity among K88-nega-tive enterotoxigenic Escherichia coli from neonatalpigs. Infect. Immun. 27:222-230.

19. Moon, H. W., B. Nagy, and R. E. Isaacson. 1977.Intestinal colonization and adhesion by enterotosigenicEscherichia coli: ultrastructural observations on adher-ence to ileal epithelium of the pig. J. Infect. Dis.36(Suppl.):S124-S129.

20. Moon, H. W., B. Nagy, R. E. Isaacson, and I. 0rskov.1977. Occurrence of K99 antigen on Escherichia coliisolated from pigs and colonization of pig ileum by K99+enterotosigenic E. coli from calves and pigs. Infect.Immun. 15:614-620.

21. Moon, H. W., and S. C. Whipp. 1970. Development ofresistance with age by swine intestine to effects ofenteropathogenic Escherichia coli. J. Infect. Dis. 122:220-223.

22. Morris, J. A., A. E. Stevens, and W. J. Sojka. 1978.Anionic and cationic components of the K99 surfaceantigen from Escherichia coli B41. J. Gen. Microbiol.107:173-175.

23. Morris, J. A., C. J. Thorns, and W. J. Sojka. 1980.Evidence for two adhesive antigens on the K99 refer-ence strain Escherichia coli B41. J. Gen. Microbiol.118:107-113.

24. Nagy, B., H. W. Moon, and R. E. Isaacson. 1976.Colonization of porcine small intestine by Escherichiacoli: ileal colonization and adhesion by pig enteropath-ogens that lack K88 antigen and by some acapsularmutants. Infect. Immun. 13:1214-1220.

25. Nagy, B., H. W. Moon, and R. E. Isaacson. 1977.Colonization of porcine intestine by enterotoxigenicEscherichia coli: selection of piliated forms in vivo,adhesion of piliated forms to epithelial cells in vitro,and incidence of a pilus antigen among porcine entero-pathogenic E. coli. Infect. Immun. 16:344-352.

26. Olsson, E., and O. Sôderlind. 1980. Comparison of dif-ferent assays for definition of heat-stable enterotoeigen-icity of Escherichia coli porcine strains. J. Clin. Micro-biol. 11:6-15.

27. 0rskov, I., and F. 0rskov. 1964. Episome-carried sur-face antigen K88 of Escherichia coli: I. Transmissionof the determinant of the K88 antigen and influence onthe transfer of chromosomal markers. J. Bacteriol. 91:

K99-POSITIVE ETEC IN SWEDEN 257

69-75.28. 0rskov, I., F. 0rskov, B. Jann, and K. Jann. 1977.

Serology, chemistry, and genetics of O and K antigensof Escherichia coli. Bacteriol. Rev. 41:667-710.

29. 0rskov, I., F. 0rskov, W. J. Sojka, and W. Wittig.1964. K antigens K88ab(L) and K88ac(L) in E. coli. Anew O antigen: 0147 and a new K antigen: K89(B).Acta Pathol. Microbiol. Scand. 62:439-447.

30. 0rskov, I., F. 0rskov, H. W. Smith, and W. J. Sojka.1975. The establishment of K99, a thermolabile, trans-missible Escherichia coli K antigen, previously called"Kco", possessed by calf and lamb enteropathogenicstrains. Acta Pathol. Microbiol. Scand. Sect. B 83:31-36.

31. Runnels, P. L, H. W. Moon, and R. A. Schneider.1980. Development of resistance with host age to adhe-sion of K99' Escherichia coli to isolated intestinalepithelial cells. Infect. Immun. 28:298-300.

32. Smith, H. W., and M. A. Linggood. 1972. Further ob-servations on Escherichia coli enterotoeins with partic-ular regard to those produced by atypical piglet strainsand by calf and lamb strains: the transmissible natureof these enterotoxins and of a K antigen possessed bycalf and lamb strains. J. Med. Microbiol. 5:243-250.

33. Smyth, C. J., P. Jonsson, E. Olsson, O. Scderlind, J.Rosengren, S. Hjertén, and T. Wadstrom. 1978.Differences in hydrophobic surface characteristics ofporcine enteropathogenic Escherichia coli with andwithout K88 antigen as revealed by hydrophobic inter-action chromatography. Infect. Immun. 22:462-472.

34. Sôderlind, O. 1971. Studies on Escherichia coli in pigs.II. Serological investigations. Zentralbl. Veterinaermed.Reihe B 18:569-590.

35. Sôderlind, O., and R. Mollby. 1978. Studies on Esche-richia coli in pigs. V. Determination of enterotoxicityand frequency of O groups and K88 antigen in strainsfrom 200 piglets with neonatal diarrhoea. Zentralbl.Veterinaermed. Reihe B 25:719-728.

36. Soderlind, O., and R. MolIby. 1979. Enterotoxins, Ogroups, and K88 antigen in Escherichia coli from neo-natal piglets with and without diarrhea. Infect. Immun.24:611-616.

37. Sojka, W. J. 1971. Enteric diseases in new-born piglets,calves and lambs due to Escherichia coli infection. Vet.Bull. 41:509-522.

38. Wadstrom, T., C. J. Smyth, A. Faris, P. Jonsson, andJ. H. Freer. 1979. Hydrophobic adsorptive and hem-agglutinating properties of enterotoxigenic Escherichiacoli with different colonizing factors: K88, K99, andcolonization factor antigens and adherence factor, p.29-55. In S. Acres (ed.), Proceedings of the 2nd Inter-national Symposium on Neonatal Diarrhea. VeterinaryInfectious Diseases Organization, Saskatoon, Canada.

39. Wilson, M. R., and A. W. Hohmann. 1974. Immunity toEscherichia coli in pigs: adhesion of enteropathogenicEscherichia coli to isolated intestinal epithelial cells.Infect. Immun. 10:776-782.

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