J.I. Garabal

Serogroups of Escherichia coli isolated from piglets in Spain.

Serogroups of 1334 E. coli colonies isolated in Spain between 1986 and 1991 from piglets with diarrhoea, oedema disease and from healthy piglets, were determined. The serogroups determined in E. coli from diarrhoea were: O1, O2, O4, O5, O7, O8, O9, O12, O20, O21, O23, O25, O26, O32, O39, O45, O54, O75, O78, O80, O83, O91, O92, O101, O103, O113, O115, O116, O118, O125, O127, O138, O139, O141, O149, O153 and O157; from pigs with oedema disease: O8, O101, O138, O149 and O157; and from healthy piglets: O1, O2, O5, O7, O8, O9, O20, O21, O26, O29, O45, O64, O71, O80, O81, O91, O101, O105, O113, O115, O116, O126, O128, O132, O138, O139, O142, O146, O149, O152, O153, and O168. Serogroups O138, O141 and O149 were found to be statistically associated with enteric porcine colibacillosis (diarrhoea and oedema disease) (0.025 > P < 0.01). In addition, enterotoxigenic (ETEC) strains belonged to serogroups: O8, O9, O20, O101, O138, O141 and O149; verotoxigenic (VTEC) strains (VTEC) strains to serogroups 091 and 0138; and necrotizing (NTEC) strains to serogroups O2, O4, O8, O54, O78 and O83. Furthermore, 91.2% (249 out 273) of the ETEC (LT and/or STa) and/or VTEC strains belonged to only seven different serogroups. These major serogroups to which the ETEC and VTEC strains belonged, were determined in a lower percentage (21.2%) among non-toxigenic E. coli colonies isolated from sick and healthy piglets.

Colonization antigens of enterotoxigenic Escherichia coli strains isolated from piglets in Spain

Eighty-eight enterotoxigenic E.coli strains isolated from 69 pigs with enteric infections (diarrhoea or oedema disease) were investigated for the presence of F4 (K88), F5 (K99), F6 (987P) and F41 colonization antigens. The commonest colonization antigen was F6 (987P), which was detected in ETEC strains from 31.9% pigs, followed by F5 (K99) 11.6%, F4 (K88) 10.1% and F41 8.7%. Presence of F6 (987P) and F5 (K99) fimbriae was statistically associated (0.025 > p < 0.005) with diarrhoeic piglets younger than 15 days. F4 (K88) colonization antigen was only expressed by ETEC isolated from piglets older than 15 days. 90.5% of ETEC isolated from 90.0% of piglets younger than 15 days expressed F5 (K99), F6 (987P) or F41 antigens, whereas only 31.3% ETEC isolated from piglets older than 15 days were positive for F4 (K88), F5 (K99), F6 (987P) or F41 antigens (p < 0.001). None of the ETEC pigs with oedema disease produced any of the four colonization antigens. ETEC bearing colonization antigens were associated with particular serogroups and toxic phenotypes, whereas 4P- ETEC strains showed diverse phenotypic characteristics.

Isolation of K88 Antigen Variants (ab, ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

Fimbriae isolation by means of thermal shock was applied to fifteen K88‐positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88‐positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris‐HCl for isolation of K88 antigen. The molecular weights by SDS‐PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.

Toxigenic Escherichia coli in Spanish piggeries from 1986 to 1991

Four-hundred and fourteen faecal samples from pigs with diarrhoea, oedema disease or healthy pigs, were collected from 65 piggeries located in different areas of Spain from 1986 to 1991. A total of 1334 Escherichia coli cultures were isolated from the pigs and studied for production of heat-labile (LT) and heat-stable (STa) enterotoxins, verotoxin (VT) and for type 1 (CNF1) and type 2 (CNF2) cytotoxic necrotizing factors. Strains producing enterotoxins (P < 0.001) or verotoxin (P < 0.05) were associated with enteric diseases of pigs. In the majority (82.3%) of piglets with strains, producing verotoxin the strains were also positive for production of STa enterotoxin. The most frequent toxin detected was STa. Although we isolated strains producing CNF1 from 1.5% of sick pigs, they were not statistically associated (P < 0.7) with enteric disease. Pigs may constitute a natural reservoir of CNF1 producing E. coli strains in Spain; their presence in the porcine intestine may be of significance in public health because such strains have been associated with human extraintestinal infections.

Fimbriae extracts from enterotoxigenic Escherichia coli strains of bovine and porcine origin with K99 and/or F41 antigens.

Fimbriae extracts obtained using the thermal shock method, from bovine and porcine enterotoxigenic Escherichia coli strains with K99 and/ or F41 antigens, were analyzed by SDS-PAGE, immunoblotting and haemagglutinating activity. Three major protein bands with molecular weights 17 kDa, 29.3 kDa and 30.9 kDa were detected depending on the strain assayed. A 17 kDa band was identified as the fimbrial subunit for K99 fimbriae and was detected in strains of bovine and porcine origin. The 30.9 kDa band was identified as the fimbriae subunit for F41 fimbriae and was detected in all porcine strains with F41 antigen and only in the bovine strains of serogroups O9 and 0101 that proved positive for F41 antigen. The 29.3 kDa band was shown to be antigenically related to F41 and K88, and was only detected in bovine strains of serogroups O8 (5 strains) and O20 (a single strain). We speculate that the 29.3 kDa band may be related to the CS31A antigen.

Prevalence of enterotoxigenic Escherichia coli strains in outbreaks and sporadic cases of diarrhoea in Spain.

Escherichia coli strains isolated 1985–1988 in Spain from patients with diarrhoea were examined; 1170 strains were isolated from 582 sporadic cases of diarrhoea in children, and seven strains were associated with seven outbreaks of diarrhoea. Strains positive for STa enterotoxin production in the infant mouse test were also assayed for production of LT enterotoxin on Vero cells and by a coagglutination test. Thirty-one strains were STa positive: 28 were isolated from 16 (2.7 %) sporadic cases of diarrhoea and three were responsible for outbreaks. The majority of STa+LT− strains from both outbreaks and sporadic cases were serotype O153.H45 and expressed the CFA/I colonization factor antigen. Enterotoxigenic STa+LT− strains of serotype O27:H7 and STa+LT+ CFA/II+ strains of serotype O6:H15: H16 were also isolated frequently from sporadic cases.

Enterotoxic, Cytotoxic, Necrotic and Lethal Activities in Cell-Free Extracts of Salmonella Strains Isolated from Humans

Unconcentrated cell-free sonic extracts from thirty Salmonella strains isolated from the faeces and blood of humans were investigated for the production of enterotoxins in various tests (Vero cell, infant mouse, rabbit skin permeability and rabbit ileal loop), as well as for lethal activity in adult mice. Sonic extracts from 23 (76.7%) strains were lethal for mice, 21 (70%) increased skin permeability and 3 (10%) showed necrotizing activity for the rabbit skin. No Salmonella strain producing typical Escherichia coli toxins, such as thermolabile (LT) or thermostable (STa) enterotoxins, Verotoxin (VT) or cytotoxic necrotizing factor (CNF) cytotoxins, were detected. Non-repetitive fluid accumulation in rabbit loops was obtained when unconcentrated sonic extracts from 10 selected strains were assayed in seven rabbits. Growth of Salmonella in casamino acid yeast extract medium, followed by treatment of bacterial cells with polymyxin B, was demonstrated to be a rapid and sensitive method for releasing the delayed permeability factor.