- RIKILT - Business Unit Safety & Health (2)
- Animal Nutrition (1)
- Aquaculture (1)
- ID - Dier en Omgeving (1)
- IMARES Aquaculture (1)
- M. Geens (1)
- B.M. Goddeeris (3)
- W. Haasnoot (2)
- M. Loos (1)
- J. Meulen van der (1)
- T.A. Niewold (1)
- B. Schacht (1)
- E. Schacht (1)
- S. Schauvliege (1)
Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli
Loos, M. ; Geens, M. ; Schauvliege, S. ; Gasthuys, F. ; Meulen, J. van der; Dubreuil, J.D. ; Goddeeris, B.M. ; Niewold, T.A. ; Cox, E. - \ 2012
PLoS ONE 7 (2012)7. - ISSN 1932-6203
toll-like-receptor - pancreatitis-associated protein - intestinal epithelial-cells - postweaning diarrhea - labile enterotoxin - gene-expression - dendritic cells - cytokine expression - human monocytes - net absorption
Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.
|Innovatie en duurzaamheid hoog in het vaandel op Food Professional Day (interview met Wout Abbink)
Cox, E. ; Abbink, W. - \ 2011
Generation of group-specific antibodies against sulfonamides
Cliquet, P. ; Cox, E. ; Haasnoot, W. ; Schacht, E. ; Goddeeris, B.M. - \ 2003
Journal of Agricultural and Food Chemistry 51 (2003)20. - ISSN 0021-8561 - p. 5835 - 5842.
enzyme-immunoassay - monoclonal-antibodies - sulfamethazine residues - sulfathiazole - elisa - tissue - recognition - antibiotics - milk - meat
To develop a sulfonamide-specific ELISA, different attempts were made to obtain monoclonal antibodies specific for the common structure of sulfonamides. In a first approach, sulfanilamide was linked to albumins using glutaraldehyde or a succinimide ester as cross-linker. A weak immune response or none at all was induced after immunization of mice with those conjugates. High antibody titers were obtained with conjugates where sulfanilamide was linked to albumins or casein (azocasein) with a diazotation reaction. However, the antibodies were only highly specific for the bound sulfanilamide molecule. In a second approach, sulfonamide-protein conjugates were used in which the sulfonamide molecule is linked at its side chain, leaving the common structure of sulfonamides unchanged. Three sulfonamide derivatives (S, TS, and PS, previously described in the literature) containing a carboxyl group in their side chain were linked to proteins using a carbodiimide mediated reaction. Immunization with the S-conjugates led to high antibody titers, but the antibodies were only highly specific for the bound S-molecule. Group-specific antibodies were obtained after immunization with the PS- and TS-conjugates. It was described that immunization with PS-conjugates lead to the recognition of other sulfonamides (sulfamethazine, -merazine, -diazine, and -dimethoxine) that are not well recognized by antibodies induced after immunization with TS-conjugates. Therefore, we tried to guide the immune response in the direction of recognition of the common structure of sulfonamides by immunizing the animals alternately with PS- and TS-conjugates. The polyclonal antibodies of the mice indeed had a broader specificity, but the specificity of the monoclonals obtained after fusion experiments was not influenced. Immunization with TS-conjugates seemed sufficient to obtain sulfonamide-specific monoclonal antibodies. With the best monoclonal (mAb 3B5B10E3) two competitive inhibition (ci) ELISA's were developed: one coated with antigen and the other coated with the monoclonal antibody. Sulfadiazine, -dimethoxine, -thiazole, -pyridine, and -methoxazole were detected in both ELISA's at their MRL-value (100 ppb) in buffer solution. Sulfadiazine, sulfathiazole, and sulfamethoxazole could even be detected at 10 ppb.
Extraction procedure for sulfachloropyridazine in porcine tissues and detection in a sulfonamide-specific enzyme-linked immunosorbent assay (ELISA)
Cliquet, P. ; Cox, E. ; Haasnoot, W. ; Schacht, B. ; Goddeeris, B.M. - \ 2003
Analytica Chimica Acta 494 (2003)1-2. - ISSN 0003-2670 - p. 21 - 28.
sulfamethazine residues - monoclonal-antibodies - animal-tissues - swine urine - immunoassay - sulfathiazole - sulfadimidine - antibiotics - meat - recognition
Sulfonamide-specific polyclonal rabbit antibodies were obtained after immunization with a sulfathiazole derivative (N1-[4-(carboxymethyl)-2-thiazolyl]sulfanilamide = TS) coupled to keyhole lympet hemocyanin. Using these antibodies, two sulfonamide-specific enzyme-linked immunosorbent assays (ELISAs) were developed differing in coating antigen: TS-ovalbumin (TS-ova) and PS-ovalbumin (PS-ova, PS = N1-[4-methyl-5-[2-4-carboxyethyl-1-hydroxyphenyl]-azo-2-pyridyl]sulfanilamide). The detection of sulfamethazine, sulfamerazine, sulfadimethoxine, sulfadiazine, sulfathiazole, sulfapyridine, sulfachloropyridazine and sulfisoxazole in buffer was studied. Higher antibody titers were obtained in the ELISA coated with TS-ova (TS-ciELISA) as compared to the ELISA coated with PS-ova (PS-ciELISA), but the detection of sulfonamides was more sensitive in the PS-ciELISA, allowing the detection of all tested sulfonamides at the maximum residue level (MRL) value (100 ng ml¿1). In a subsequent step, an extraction procedure was developed for the detection of sulfonamides in muscles, kidney, liver and fat by both ELISAs using sulfachloropyridazine as a model. As extraction buffer a carbonate/hydrogen carbonate buffer (pH 10) was chosen in which sulfonamides are highly soluble. Differences in homogenizing techniques (high-speed mixer (Ultraturax) versus vortex) and the effect of kaolin (hydrated aluminum silicate) treatment, to diminish the background signal in the ELISA, were evaluated. The best extraction procedure was the one using a vortex mixer as homogenizer and no kaolin treatment. Sulfachloropyridazine was easily detected at the MRL in all tissues.