Detection of Escherichia coli strains producing cytotoxic necrotizing factor type two (CNF2) by enzyme-linked immunosorbent assay

Sheep and rabbit antisera were produced against lysates of E. coli strain 711 (pVir). This K-12 strain carries the Vir plasmid which codes for Cytotoxic Necrotizing Factor type 2 (CNF2). Immunoglobulin G (IgG) fractions of both immune sera were subsequently purified by a two-step precipitation method. To increase the specificity for CNF2, the sheep IgG preparation was extensively adsorbed against both a sonicated extract of isogenic K-12 strain 711 and intact phenol-treated cells of vaccine strain 711 (pVir). An enzyme-linked immunosorbent assay (ELISA) was developed to detect clinical isolates of E. coli producing CNF2, using the final preparations of rabbit and sheep IgG in a double sandwich technique. The results obtained with this CNF2-ELISA were compared to those obtained with the conventional HeLa cell cytotoxicity assay. The testing of 133 E. coli strains (49 CNF2 positive strains and 84 negative strains) resulted in no false-negative and no false-positive. Therefore, the CNF2-ELISA offers a good alternative to the HeLa cell culture assay for the detection of CNF2-producing strains where facilities for and experience with cell cultures is lacking.     

Cytotoxins in non-enterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves

We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E. coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves. Three types of cytotoxic responses were observed. Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E. coli from infant and piglet enteritis. Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated. Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains. Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures. Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts. Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically. In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.     

Necrotoxic Escherichia coli (NTEC): two emerging categories of human and animal pathogens.

Necrotoxic Escherichia coli (NTEC) were originally defined as strains of E. coli producing a toxin called cytotoxic necrotising factor (CNF). Two types of CNF have been identified, each of them being genetically linked to several other specific virulence markers, a situation that allows the definition of two distinct homogeneous categories of NTEC called NTEC-1 and NTEC-2. CNF1 and CNF2 are highly homologous holoproteins containing 1,014 amino acids that exert both lethal and necrotic activities in vivo and induce multinucleation and actin stress fibres in cell cultures. The activity of CNFs on mammal cells is due to their ability to constitutively activate by deamidation the Rho proteins, a family of small GTPases that regulate the physiology of the cell cytoskeleton. In NTEC-1, the gene encoding CNF1 belongs to a pathogenicity island which also comprises the genes encoding for alpha-haemolysin and P-fimbriae. In NTEC-2 strains, CNF2 is encoded by a plasmid that also encodes, in 100% of the isolates, a new member of the cytolethal distending toxin family (CDT-III) and in about 50% of the isolates, the F17b-fimbrial adhesin that confers the ability to adhere to calf intestinal villi. The presence of CDT is also suspected in a large majority of NTEC-1 strains. NTEC-1 strains can be found in humans and in all species of domestic mammals, whereas NTEC-2 strains have only been reported in ruminants. The implication of NTEC strains has been clearly established in extra-intestinal infections of humans and animals, for instance in urinary tract infections for NTEC-1 strains. Their role in severe dysenteric syndromes, both in humans and animals, is substantiated by several clinical reports, but there is little published information on this pathogenicity in animal models of infection. The combined production of several powerful toxins (haemolysin, CNF, CDT) by NTEC strains makes them, however, potentially aggressive pathogens which deserve to be searched for on a larger scale. Moreover, NTEC-1 from man and animals appear to be highly related according to available molecular markers, which indicates that domestic animals could constitute reservoirs of NTEC strains which are pathogenic for humans.     

ULTRASOUND-GUIDED TISSUE-CORE BIOPSY OF LIVER, SPLEEN AND KIDNEY IN NORMAL DOGS

Six normal dogs were subjected to ultrasound-guided biopsy of the liver, spleen and kidney to examine the accuracy of the technique (i.e. the presence of targeted tissue) and the histologic quality of the biopsies. Five consecutive tissue-core biopsies of each organ were taken on one or more occasions. The accuracy of the technique was 77% for hepatic, 90% for splenic, 53.5% for left kidney and 40% for right kidney biopsies. The histologic quality of the liver and kidney samples was sufficient, although for some samples the diagnostic value was limited by their size and in renal samples either cortical or medullary tissue was sometimes lacking. In contrast, the quality of the splenic sections was not good. The effect of reused and resterilized needles on the quality of the specimens was evaluated by histologic inspection of the samples and by the amount of biopsies lacking tissue. All tissue samples, including those taken with reused or resterilized needles had sharp-cut edges. Twenty-two of the total number of 120 biopsies (18%) contained no tissue. Absence of tissue in the samples was observed in biopsies taken with all needle types. The animals were observed for possible complications of the repeated needle biopsy. Apart from one case of hematuria, no complications were encountered.     

Toxic effects for lambs of cytotoxic necrotising factor from Escherichia coli

The lethal effect, clinical signs and lesions caused by the intravenous inoculation into six lambs (seven to 10 days old) of a partly purified preparation of cytotoxic necrotising factor (CNF) from Escherichia coli, strain BM2-1, were investigated. Two control preparations were also tested in two lambs each: (i) the same as above but heated for one hour at 60 degrees C, a treatment which inactivates CNF and preserves residual endotoxic activity; and (ii) purified material from a CNF-defective mutant of BM2-1. Whereas none of the lambs in either of the control groups died or showed significant clinical signs or lesions, all the lambs inoculated with partly purified CNF developed severe clinical signs starting six hours after inoculation which consisted mainly of neurological signs and mucoid diarrhoea. The most striking lesions were oedema and haemorrhages in the central nervous system, and foci of coagulation necrosis in the myocardium. Mucus hypersecretion in the gastrointestinal tract was not associated with cellular inflammation.     

Comparison of necrotoxigenic Escherichia coli isolates from farm animals and from humans

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.