A rapid, colourimetric assay for cytotoxin activity inCampylobacter jejuni

Abstract

Cell extracts and culture supernates of Campylobacter jejuni NCTC 11168 and three isolates from faecal samples from patients with enteritis were tested for cytotoxic activity on HeLa and Vero cells using a sensitive and rapid dye reduction assay which represents a simple assay for cytotoxin activity that can be assessed visually or spectrophotometrically in the wells of microplates. The assay was as sensitive as trypan blue exclusion and did not require the use of radioisotopes. A low level of cytotoxin activity, compared to that produced by a control verotoxin 2-producing Escherichia coli strain, was detected in cell extracts of all four strains, but no activity was detected in culture supernates. Production of an enterotoxin was evaluated by reverse passive latex agglutination with anti-cholera toxin antibody, a procedure which also represents a rapid and simple assay for this toxin. No enterotoxin activity was detected in cell extracts or culture supernates from any of the isolates.

Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

“…To detect and quantify cytotoxic activity during purification, we used an activity assay based on the lethal effects of the toxin on CHO cells. The TCID 50 of C31 strain for CHO cells was similar at 1–2 μg for a freshly prepared protein extract as well as a reconstituted form of the lyophilised extract as estimated by the visual method by direct microscopic observation of cytotoxic effect on cells [8] or by the indirect methyl thiazol tetrazolium (MTT) method by spectrophotometric measurement of formazin [9]. The cytotoxic effect of C31 toxin on CHO cells is shown in Figure 1.…”

“…The toxin titre was expressed as tissue culture infectivity 50 (TCID 50 ) dose, the concentration of the toxin that caused cytotoxicity in 50% of the monolayer. In some instances, we used the methyl thiazol tetrazolium (MTT) assay [9] to quantitate cytotoxin activity. Metabolically active CHO cells are able to reduce the formazin present in the MTT reagent resulting in a colour change, allowing spectrophotometric quantitation of the activity of the cytotoxin.…”

“…One hundred microliters of treated pool B fractions at a concentration of 2 μg/ml were added to a CHO cell monolayer in a microtitre plate. The MTT assay [9] for cytotoxicity was performed after a 24 h incubation period. Control samples consisted of PBS and trypsin inhibitor and were used in the calculation of percent cell death.…”

The Enterotoxin Production and Antimicrobial Resistance of Campylobacter Strains Originating from Slaughter Animals

“…A cytopathic effect correlating with cytotoxin production was confirmed by gene expression analysis in different cell cultures [ 13 , 38 , 45 ]; however, the production of enterotoxin by Campylobacter is still controversial [ 19 ]. The dominant perception is that Campylobacter enterotoxin is immunologically related to Vibrio cholere enterotoxin (CT) or Escherichia coli heat-labile enterotoxin (LT) [ 18 ]. The previous studies demonstrated that the capacity of Campylobacter spp.…”

“…Enterotoxins elevate the intracellular level of cyclic adenosine monophospate, and the subsequent ion flux changes cause the excess secretion of fluid, which results in watery diarrhea. In contrast, cytotoxic activity appears to be distinct from enterotoxin activity, as cytotoxins are associated with the destruction of target cells causing mucosal inflammation and hemorrhagic reactions [ 18 , 19 ]. However, although several Campylobacter cytotoxins have been identified, only cytolethal distending toxin (CDT) has been well characterized, and it was found that CDT activity is encoded by three closely linked or slightly overlapping genes termed cdtA , cdtB and cdtC [ 20 , 21 ].…”