L. Renault, D. Mathieu, E. Le Bourhis. To cite this version: HAL Id: hal

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1 DETECTING ENTEROPATHOGENIC ESCHERICHIA COLI STRAINS OF PORCINE ORIGIN. 1. Correlations between 0 and K antigens and the enterotoxin production in strains isolated from the newborn piglet L. Renault, D. Mathieu, E. Le Bourhis To cite this version: L. Renault, D. Mathieu, E. Le Bourhis. DETECTING ENTEROPATHOGENIC ES CHERICHIA COLI STRAINS OF PORCINE ORIGIN. 1. Correlations between 0 and K antigens and the enterotoxin production in strains isolated from the newborn piglet. Annales de Recherches Vétérinaires, INRA Editions, 1977, 8 (3), pp <hal > HAL Id: hal Submitted on 1 Jan 1977

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3 _ DETECTING ENTEROPATHOGENIC ESCHERICHIA COLI STRAINS OF PORCINE ORIGIN 1. Correlations between 0 and K antigens and the enterotoxin production in strains isolated from the newborn piglet L. RENAULT D. MATHIEU E. LE BOURHIS* * Centre de Recherches et de Contrôle Biologiques, 19, quai de l Industrie, ATHIS MONS, France Centre National de la Recherche Scientifique, Service de Virologie, B.P. 8, VILLEJUIF, France Résumé DEPISTAGE DES SOUCHES D ESCHERICHIA COLI ENTEROPATHOGENES D ORIGINE PORGINE. 1. RECHERCHES SUR LA CORRELATION ENTRE LES SPECIFICITES ANTI GENIQUES 0 et K ET LA PRODUCTION D ENTEROTOXINE CHEZ LES SOUCHES ISOLEES DU PORCELET NOUVEAUNE. L étude de la production d entérotoxine par des tests biologiques (cellules Y1 et souriceau nouveauné) à partir de 67 souches d Escherichia coli responsables de diarrhée du porcelet nouveauné confirme l intérêt de ces méthodes et démontre une corrélation entre spécificité antigénique et production d entérotoxine. En particulier la présence de l antigène capsulaire K 88 est liée à celle de la fraction thermolabile ylt) de l entérotoxine chez 73,9 p. 100 des souches entéropathogènes étudiées. Introduction The discovery of an enterotoxin, along with its thermostable (S.T.) and thermolabile (L.T.) fractions, controlled by plasmides (Ent.) allowed H.W. Smith and Halls, in 1967, to individualize enteropathogenic Escherichia coli stock, in pork. The same group demonstrated at the same time, by studying the enteropathogenic potential in piglets, the correlation that exists between the thermolabile (L.T.) fraction and the presence of the capsular antigen K 88 (Smith and Gyles, 1970). Sojka in 1972, then proposed a simplified diagnostic outline based on research on capsular antigens in OK polyvalent serums. Thus, he distinguished a group of nonenteropathogenic strains, corresponding, in particular, to the 0139 : K 81 serogroup responsible for oedema disease and a group of enteropathogenic strains, the cause of diarrhea at birth and at weaning. The latter includes three subgroups : first, and the most important, with the K 88 capsular antigen and both the thermostable and thermolabile fractions of enterotoxin ; the other two, which are less frequent, and without the K 88 capsular antigen, possessed only the thermostable fraction of the enterotoxin. According to this author, since the 0 groups have in common only certain K antigens, research on the capsular agglutination allows

4 the somatic group with which this capsular antigen is most often associated to be deduced. Finally, physicians have more recently determined two new biological tests that are more simple, cheaper, and more sensitive for the two enterotoxin fractions of the strains responsible for infantile gastroenteritis. The first consists of determining the thermolabile fraction (LT) by the steroidogenesis effect of adrenal cells Y1 (Donta and Smith, 1974 ; Sack and Sack, 1975); and the second consists of recognizing the thermostable fractions (ST) presence by intragastric inoculation in suckling mice (Dean et al., 1972). Ellis and Kienholz, 1976, completely confirm these methods validity by showing the agreement with the inoculation method, in an intestinal loop of newborn piglets. Thus, it appeared interesting to us, in earlier research work, to determine whether there was a correlation between the capsular antigenic specificity, and the somatic antigenic specificity of Escherichia coli isolated in newborn piglets (Renault et al., 1975) and in weaned piglets (Renault et al., 1976). The present work seeks to determine if there was a correlation between the antigenic specificities and the production of enterotoxin, detected by these rapid biological tests. Material and methods. This study was carried out with 67 isolated Escherichia coli strains from the intestinal contents of newborn piglets, having the disease, from one to 6 days old, and with the stock taken from some feces of sows that had contaminated litters. The correlation between the 0 and K antigens in these strains had already been confirmed (Renault et al., 1975). Each strain was seeded in 5 ml trypticase soy liquid medium (Difco) in a 125 ml flask, incubated for 24 hours at 37 C, in an agitator of 200 movements per minute. The culture was then centrifuged at 12,000 g for 10 minutes. The supernatant was taken up and filtered on a millipore membrane of 2.5 mp. Determining the thermolabile (LT) fraction was effected according to the following methods,

5 described by Donta and Smith (1974) and by Sack and Sack (1975). The Y1 adrenal cells were cultivated in monocellular layers in an F 10 HAM medium, with 12.5 % horse serum and 8.5 % calf foetus serum added ; Falcon boxes (35/10 mm) were incubated in a humid atmosphere at 37 C, in the presence of 5 % C02. Each Falcon box received 0.2 ml culture filtrate in 1.8 ml of medium. After 18 to 24 hours at 37 C, the enterotoxin s stimulation of steroidogenesis was evaluated as a function of the percentage of balloonshaped cells. Effectively the Yl cell, at rest, appeared to be a starlike cell with fine elongations that stuck onto surrounding cells to form little islets, then an almost continous layer (photo n 1). On the other hand, the cells stimulated by the toxin s action assumed a completely different appearance : these cells were rounded off, become refracting, and separated themselves one from another (photo n 2). The reaction was considered to be positive only if the percentage of rounded cells was above 70 %. The activity s specificity was confirmed by performing the test with heated supernatant (30 min. at 100 C) : the test should become negative (control carried out for all the strains), and by neutralizing the supernatant with an antitoxic serum for one hour at 37 C (test carried out on a few strains). Determining the thermostable fraction (ST) was effected according to the Dean et al., method (1972). The filtrate (0.1 ml) coloured by a methylene blue solution (2 %) was injected through an intradermic needle in the stomach of 4 suckling mice, aged 1 to 4 days. After 4 hours, at 28 C, the suckling mice were killed and the intestinal mass was weighed. The presence of the thermostable fraction was evaluated as a function of the small intestine s dilatation (photo n 3) and especially, as a function of the intestinal weight vs body weight. If this ratio was less than 0.070, the test was considered negative ; if greater than positive. The two tests specificity was verified at each titration with reference strains that come from the C.D.C. of Atlanta, kindly supplied by Dr. Le Minor.

6 The Escherichia coli strains were the following : N 11 : Negative for toxins. N 13 : ST positive. N 14 : LT and ST positive. Strain n 14 proved to be clearly positive as far as the test on Y1 adrenal cells was concerned. Strain 13 and 14 proved to be clearly positive in the test on suckling mice ( ) and strain 11 negative ( ). Results 1 ) Determining the THERMOLABILE FRAC TION : Of the 67 strains studied in newborn piglets, a total of 34 (or 50.7!) had the thermolabile fraction of enterotoxin. The 34 positive strains all belonged to the serological group of Sojka, which, theoretically, possesses both fractions, ST and LT, of enterotoxin (34 out of 46, or 73.9 %) [Tabl. 1]. The enteropathogenic strains belonging to the Sojka serological group, producing only the ST fraction, nor any of the strains with different somatic groupings, nor any strains that had 0139 : K 82, without enterotoxin, reacted positively in the adrenal cell test. 2) Determining the THERMOSTABLE FRAC TION : Of the 67 strains studied, in newborn piglets, a total of 43 (or 64.1 percent) possessed the thermostable fraction of the enterotoxin (Tabl. 2). They correspond to the following breakdown : strains out of 46 (or 73.9 %) belonged to the serological group of Sojka, with theoretically the 2 fractions LT and LT of the enterotoxin ; strains out of 6, belonged to the Sojka serological groups which, in theory, had only the ST fraction ; strains out of 12, were from somatic groups different from those of Sojka.

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8 It It Either The 0139 : K 82 strain, at the root of o3dema disease, and which does not carry any enterotoxin, did not prove to be positive. 3) COMPARED RESULTS OF SIMULTA NEOUS DETERMINATION OF TWO ENTERO TOXIN FRACTIONS : Of the 46 strains, supposed to produce these two enterotoxin fractions, as a function of their antigenic formula, 27 (or 58.6 %) actually revealed a biological activity on adrenal cells (Y1) and, at the same time, on suckling mice, particularly strains from group 0149 : K 91, K 88 a (23 out of 32, or 71.8 %). enterotoxin at the same time, were far more numerous at birth than at weaning. As for the absence of Biological reaction, on adrenal cells and suckling mice, of many strains isolated from diarrhea and which theoretically, should have carried both thermolabile and thermostable fractions according to their antigenic formula, this absence may have several explanations : might be a question of biologically inactive strains, in spite of their antigenic formula corresponding to that of pathogenic strains. Moon et al. (1966), observed such negative variations in performing the test using the intestinal loop of the piglet. might be a question, also, of strains that had lost after a certain period of conservation, their coding plasmides for both enterotoxin fractions and the capsular antigen K 88 (Smith and Lingood, 1972). Dlscussion These results show that a correlation exists between the antigenic specificities 0 and K, researched by the capsular agglutination test established by Sojka, and the production of enterotoxins researched by the biological activity tests on the suprarenals, and a newborn baby mice. In particular, the association is confirmed for the majority of enteropathogenic strains, between the presence of the capsular antigen K 88 and the presence of the thermolabile fraction of enterotoxin. This association is further confirmed between the existence of antigenic formulae allowing the presence of the thermostable fraction to be suspected, and the demonstration of the real presence of this fraction. Yet, it still should be noted that the test on suckling mice allowed the thermostable fraction of the enterotoxin to be brought to evidence, in certain cases, in strains which, by considering their antigenic formula, should not be enteropathogenic, or in strains known to be septicemic, as with the serogroup 078 : K 80. The close relationship between the presence of the capsular antigen K 88 and the production of the thermolabile fraction is probably due to the fact that in our preliminary serological investigation, the enteropathogenic strains having both the thermostable and thermolabile fractions of the Conclusion A rapid detection of enteropathogenic Escherichia coli strains in newborn piglets may use the following methods : serological determination by capsular agglutination simplified by the Sojka model, or by biological determination of the two enterotoxin fractions 1) by the cellular culture test of Y1 adrenal, for the (LT) thermolabile fractions; 2) by the intragastric inoculation test on suckling mice for thermostable fraction (ST). The excellent correlation between the presence of the capsular antigene K 88 and that of the thermolabile fraction (LT) allows to limit the process theoretically to rapid capsular detection. On the other hand, the presence of the thermostable fraction (ST) of the enterotoxin in strains which were considered to be nonenteropathogenic on the basis of their antigenic formula, justifies resorting to the biological test in suckling mice. (Accepted for publication, July 1977.J

9 Summary A study of enterotoxin production by means of biological tests (Y1 adrenal cells and suckling mice) using 67 Escheriehia coli strains responsible for diarrhea in newborn piglets, confirmed the advantages of these methods and demonstrated the correlation between antigenic specificity and enterotoxin production. In particular the presence of capsular antigen K 88 is related to the thermolabile (LT) fraction of the enterotoxin in 73.9 % of the enteropathogenic strains studied. References. biol., DEAN A.G&dquo; CHING Y.C., WILLIAMS R.G., HARDENL.B., Test for Escherichia coli enterotoxin using infant mice : application in a study of diarrhea in children in Honolulu. J. Inf. Dis., 125, DONTA S.T., SMITH D.M., Stimulation of steroidogenesis in tissue culture by enterotoxigenic Escherichia coli and its neutralization by specific antiserum. Inf. Immun., 9, ELLIS R.P., KIENHOLZ J.C., Detection of enteropathogenic Eseherichia coli : comparison ot porcine gut ligation, suckling mouse inoculation, and Y1 adrenal cell assays. Congrbs Int. Pathol. porcine, Ames 1976, J. 11. MOON H.W., SORENSEN D.K.. SAUTTER J.H., Eseheriehia coli infection of the ligated intestinal loop of the newborn pig. Am, J. Vet. Res., 27, RENAULT L Colibacillose du porc. Ann. Rech. V6t., 6, RENAULT L., LE BOURHIS E., MAIRE C., VAISSAIRE J., Enqudte sur la colibacillose des porcelets nouveaun6s. Bull. Acad. V6t. Fr., 48, RENAULT L., LE BOURHIS E., MAIRE C., VAISSAIRE J., Enqu8te sur la colibacillose du por celet au moment du sevrage. Bull. Acad. V6t. Fr., 49, SACK D.A., SACK R.B., Test for enterotoxigenic Escherichia coli using Y1 adrenal cells in miniculture. lnf. Immun., 11, SMITH H.W., HALLS S., Observations by the ligated intestinal segment and oral inoculatior methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Pathol. Bacteriol., 93, SMITH H.W., GYLES C.L., The relationship between two apparently different enterotoxins produced by enteropathogenic strains of Eseherichia coli of porcine origin., J. Med. Micro. 3, SMITH H.W., LINGOOD M.A., Further observation on Escherichia coli enterotoxins with partb cular regard to those produced by atypical piglet strains and by calf and lamb strains : the transmissible nature of these enterotoxins and of a K antigen possessed by calf and lamb strains J. Med. Microbiol., 5, SOJKA W.J., Colibacillose intestinale des porcelets. Ann. M6d. V6t., 116,