Laboratorio de Referencia de E.coli (LREC) 
Departamento de Microbiología y Parasitología 
FACULTAD DE VETERINARIA 
Universidad de Santiago de Compostela, Campus de Lugo 27002, Lugo, España  
TEL-FAX  34-982-285936  
E-mail ecoli@lugo.usc.es

PRODUCTION OF TOXINS  (enterotoxins, verotoxins and necrotoxins)  AND COLICINS BY Escherichia coli STRAINS ISOLATED FROM SEPTICEMIC AND HEALTHY CHICKENS. RELATION WITH IN VIVO PATHOGENICITY

JESUS E. BLANCO, MIGUEL BLANCO, AZUCENA MORA, JORGE BLANCO*
Reference Laboratory of E. coli, Department of Microbiology and Parasitology,
Faculty of Veterinary, University of Santiago de Compostela, 27002 Lugo, Spain

 JOURNAL CLINICAL MICROBIOLOGY (1997) 35:2953-2957

INTRODUCTION. Toxigenic Escherichia coli that cause infections in human and domestic animals have been classified into three categories: enterotoxigenic E. coli (ETEC), verotoxigenic E. coli (VTEC), and necrotoxigenic E. coli (NTEC). The ETEC strains may synthesize thermolabile (LT) and/or thermostable (STa and STb) enterotoxins, and cause diarrhea in children, calves, lambs and piglets. The VTEC strains may produce three main types of verotoxins (VT1, VT2 and VT2v) that are similar to Shiga-toxin synthesized by Shigella dysenteriae type 1. VTEC (VT1+ and/or VT2+) are recognized as a cause of hemorrhagic colitis (HC) and the hemolytic-uremic syndrome (HUS), in humans who contract infection following the consumption of contaminated foodstuffs like meat and unpasteurized milk. Epidemiological investigation revelated that cattle frequently excrete VTEC (VT1+ and/or VT2+) in their feces and thus may represent a source of infection. Furthermore, VTEC producing VT2v are responsible for the edema disease in pigs. We have recently reported that NTEC strains can produce CNF1 and CNF2 cytotoxic necrotizing factors. CNF1 is produced by Hly+ E. coli that cause extraintestinal infections in humans and are isolated from stools of healthy cats, whereas CNF2 is synthesized by strains of bovine origin. A new type of hemolysin called enterohemolysin (EntHly), which is different from a -hemolysin (Hly) produced by ETEC strains of porcine origin and E. coli strains that cause extraintestinal infections in humans, was recently discovered in VTEC strains.

Colisepticemia is responsible for significant economic losses in aviculture in many parts of the world, although the mechanism of pathogenicity of E. coli strains that are potentially pathogenic to birds is not fully understood. Chicken lethal toxin obtained from avian E. coli O2, O45 and O109 was described by Truscott, but this toxin has not been observed in the majority of avian E. coli strains. However, recently studies indicate that E. coli strains isolated from chickens with septicemia produce other toxins that could be implicated with the in vivo pathogenicity. In the present study we have investigated the production of different types of toxins in avian E. coli strains. To the authors' knowledge, this is the first survey that describes the prevalence of ETEC, VTEC and NTEC strains among isolates from septicemic and healthy chickens. Furthermore, we have studied the in vivo pathogenicity of strains, a property that was correlated with the synthesis of colicins and the O serogroups of strains.

ABSTRACT. Since the mechanism of virulence of Escherichia coli strains pathogenic to birds is not fully understood, the prevalence of toxic factors produced by E. coli strains pathogenic to other animals was investigated. A total of 625 E. coli strains isolated from visceral organs of chickens with colisepticemia and from feces of healthy chickens in Spain were tested for production of enterotoxins (LT and STa), verotoxins (VT1, VT2 and VT2v), cytotoxic necrotizing factors (CNF1 and CNF2), a -hemolysin (Hly), enterohemolysin (EntHly), colicin V (Col V) and other types of colicins, and for necrotic and lethal activities. Only 45 (7%) of avian E. coli strains were toxigenic: 20 strains produced cytotoxic response in HeLa but not in Vero cells indicating the production of a cytotoxin not related with the verotoxins; 16 were EntHly-positive; five produced a new cytotonic product that causes the apparition of whitish vacuola in Vero and HeLa cells; three synthesized soluble factors that cause lethal activity in mice; and 1 elaborated heat-labile enterotoxin (LT). None of 625 avian E. coli strains was positive for production of verotoxins or cytotoxic necrotizing factors. In contrast, colicinogenicity occurred in 335 (73%) of the 458 septicemic strains and 97 (58%) of 167 fecal isolates (P < 0.01), and this property was correlated with in vivo pathogenicity of strains. Thus, 80% (P < 0.001) and 66% (P < 0.001) of strains producing Col V and other types of colicins were characterized as being of high pathogenicity, whereas only the 15% not-colicinogenic strains were classified as highly pathogenic. Our results clearly support the special pathogenicity theory, because the 60% of E. coli strains belonging to 18 serogroups (O1, O2, O5, O8, O12, O14, O15, O18, O20, O53, O78, O81, O83, O102, O103, O115, O116 and O132) most frequently identified among clinical septicemic strains were classified as highly pathogenic in vivo assays, whereas only the 24% of strains with O serogroups less prevalent among diseased chickens were considered highly pathogenic (P < 0.01).