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A colourimetric, microplate assay for the leucotoxin of Pasteurella haemolytica
F F Craig1, R Dalgleish, A D Sutherland
1Dept. of Microbiology, University of Glasgow, Great Britain.
Abstract:
Culture supernates of Pasteurella haemolytica, which contain leucotoxin, inhibited the reduction of nitroblue tetrazolium (NBT) by bovine and ovine but not rabbit leucocytes in response to phorbol 12-myristate 13-acetate (PMA). Culture supernates of P. multocida, which contain no leucotoxin, had no inhibitory effect on the response of leucocytes from any species. The inhibition of NBT reduction was assessed visually or spectrophotometrically in the wells of microplates and used as a simple assay for leucotoxin. It was as sensitive as the trypan blue dye-exclusion method and did not require the use of radioisotopes. In addition, sera from P. haemolytica-infected calves inhibited leucotoxin activity in the microplate assay. Thus, inhibition of NBT reduction after stimulation of ruminant leucocytes with PMA can be used as a simple, specific assay for leucotoxin and for anti-leucotoxin antibodies.
Insights
Pasteurella haemolytica leucotoxin inhibits nitroblue tetrazolium reduction in ruminant leucocytes. This provides a simple, sensitive assay for detecting leucotoxin and antibodies against it.
Area of Science:
- Veterinary immunology
- Bacterial toxins
- Cellular immunology
Background:
- Pasteurella haemolytica produces leucotoxin, a key virulence factor in ovine and bovine respiratory disease.
- Assessing leucotoxin's effects on leucocyte function is crucial for understanding pathogenesis and developing diagnostics.
- Existing methods for leucotoxin detection can be complex or require specialized equipment.
Purpose of the Study:
- To develop a simple, sensitive, and specific assay for detecting Pasteurella haemolytica leucotoxin.
- To evaluate the utility of nitroblue tetrazolium (NBT) reduction inhibition as an indicator of leucotoxin activity.
- To explore the potential for detecting anti-leucotoxin antibodies using this assay.
Main Methods:
- Bovine, ovine, and rabbit leucocytes were stimulated with phorbol 12-myristate 13-acetate (PMA).
- The effect of Pasteurella haemolytica culture supernates (containing leucotoxin) and Pasteurella multocida supernates (control) on NBT reduction was measured spectrophotometrically.
- Inhibition of NBT reduction was assessed in microplates, and the effect of immune calf sera on leucotoxin activity was evaluated.
Main Results:
- Culture supernates of Pasteurella haemolytica significantly inhibited NBT reduction in bovine and ovine leucocytes but not rabbit leucocytes.
- Culture supernates of Pasteurella multocida had no inhibitory effect on leucocyte NBT reduction.
- The NBT reduction inhibition assay was as sensitive as the trypan blue dye-exclusion method and did not require radioisotopes.
- Sera from Pasteurella haemolytica-infected calves inhibited leucotoxin activity in the assay.
Conclusions:
- Inhibition of PMA-stimulated NBT reduction in ruminant leucocytes serves as a simple and specific assay for Pasteurella haemolytica leucotoxin.
- This assay is suitable for detecting both leucotoxin and antibodies against it.
- The method offers a practical alternative to existing, more complex diagnostic techniques.