Roger Boivin

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.

Genetic parameters for resistance to the Salmonella abortusovis vaccinal strain Rv6 in sheep

An experimental population (1216 lambs from 30 sires) of the Inra401 sheep was created in an Inra flock to allow QTL detection for susceptibility to Salmonella infection, wool and carcass traits. The Inra401 is a sheep composite line developed from two breeds: Berrichon du Cher and Romanov. At 113 days of age on average, the lambs were inoculated intravenously with 108Salmonella abortusovis Rv6 (vaccinal strain). They were slaughtered 10 days after the inoculation. Several traits were measured at inoculation and/or slaughtering to estimate the genetic resistance of the lambs to Salmonella infection: specific IgM and IgG1 antibody titres, body weight loss, spleen and pre-scapular node weights and counts of viable Salmonella persisting in these organs. This paper presents a quantitative analysis of the genetic variability of the traits related to salmonellosis susceptibility. The heritabilities of the traits varied between 0.10 and 0.64 (significantly different from zero). Thus, in sheep as well as in other species, the determinism of resistance to Salmonella infection is under genetic control. Moreover, the correlations between the traits are in agreement with the known immune mechanisms. The genetic variability observed should help QTL detection.

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.

Quantitative trait loci for resistance to infection in sheep using a live Salmonella Abortusovis vaccine

Quantitative trait loci (QTL) mapping for susceptibility to a Salmonella Abortusovis vaccinal strain was performed using an experimental design involving 30 Romane sheep sire families (1216 progenies). Nine QTL corresponding to bacterial load, weight variations and antibody response criteria were mapped on eight chromosomes, including the major histocompatibility complex area on chromosome 20. Surprisingly, none was found to be significant in the SLC11A1 region (formerly NRAMP1) that has been shown to influence Salmonella susceptibility in other species.

Cytokine gene expression in lymph node and spleen of sheep in response to Salmonella infection by two serotypes displaying different host specificity

In order to investigate the determinism of the host specificity and to better understand the host resistance mechanisms, infections of sheep were performed with either S. abortusovis, serotype specific for ovine species, or with S. dublin, serotype adapted to cattle and accidentally transmissible to human. Following a subcutaneous challenge, S. dublin disseminated more rapidly towards lymphoid tissues than S. abortusovis. However, S. abortusovis tended to persist in spleen more efficiently than S. dublin. Using a quantitative RT-PCR method, the expression level of ovine cytokines genes was measured in the draining lymph node and in the spleen, in the course of infection. Inflammatory cytokine response was characterised by an early and strong increase of IL-1beta and TNFalpha mRNA in both lymphoid organs following S. dublin infection, while S. abortusovis challenge only induced IL-1beta mRNA increase in the spleen at day 3 post-inoculation. Likewise, S. dublin infection provoked a marked increase of IL-12 mRNA and a slight up-regulation of IFNgamma gene transcription in the local lymphoid site, in contrast to S. abortusovis infection. Elsewhere, both serotypes induced a strong and early IL-10 mRNA production and had no effect on IL-4 gene expression. Finally, taken together, these data suggest that the intensity of inflammatory and anti-infectious cytokine responses, but not the type 2 cytokine response, is serotype-dependent. They also suggest that the host-specific serotype, by limiting the host cytokine-mediated defence, could favour its persistence within lymphoid organs.