Canis ISSN: 2398-2942

Bactericidal assay

Contributor(s): Michael Day, Helen Milner

Overview

  • Phagocytic cells (neutrophils and macrophages) take up particulate antigen, eg micro-organisms, and destruction of the particle occurs within the enzymatic environment of the phagolysosome.
  • The bactericidal assay tests the ability of a phagocyte (usually a neutrophil) to kill a bacterium after it has been taken in to the cell cytoplasm.

Sampling

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Tests

Methodologies

  • The test is performed with a pure population of viable neutrophils isolated from whole blood.
  • A variety of methods are available to produce such a population.
  • Neutrophils are then incubated with a suspension of bacteria, at a predetermined optimum ratio of neutrophils:bacteria.
  • The bacteria are usually opsonized by prior incubation with a standard pool of fresh canine serum.
  • A series of replicate cultures of neutrophils + bacteria will be established.
  • The neutrophils will be permitted to phagocytose the opsonized bacteria over a defined time period, eg 10 minutes, following which extracellular (non-phagocytosed) bacteria will be removed from the system.
  • In the case of Staphylococcus as a target bacterium, this is readily achieved by the addition of the enzyme lysostaphin to the culture system. Lysostaphin selectively destroys extracellular organisms.
  • After phagocytosis and removal of extracellular organisms (to prevent ongoing phagocytosis), the replicate cultures will be stopped at set time points, eg 0, 30, 60 and 90 minutes, to establish the kinetics of neutrophil killing of the internalized bacteria.
  • At each time point, the neutrophils will be lysed by the addition of water to the cultures. Any bacteria which are present within the cytoplasm (dead and alive) will be released from the cell.
  • Samples of the lysed cultures will be collected, and serial dilutions made and plated out on nutrient agar.
  • Following a period of culture, the number of colonies can be counted.
  • This information can be used to determine the number of intracellular bacteria at time 0, ie following the period of phagocytosis, and the numbers of bacteria at times 30, 60 and 90 minutes. The latter numbers may be expressed as a percentage of the original number of organisms at time 0. The number of viable bacteria should decrease in linear fashion over the period of culture.
  • An alternative method for enumerating dead and alive bacteria following phagocytosis, involves the addition of the fluorochrome acridine orange.
  • Viable bacteria (with intact DNA) will fluoresce green, whereas dead bacteria (with denatured DNA) will fluoresce red or yellow-red.

Control

  • The test should include appropriate controls, particularly the concomitant testing of samples from clinically normal control animals.

Availability

  • These tests will rarely be available outside research institutions.

 

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references from VetMed Resource and PubMed.
  • Chamas P P C & Hagiwara M K (1998) Evaluation of neutrophilic function (chemotaxis, phagocytosis and microbicidal activity) in healthy dogs and in dogs suffering from recurrent deep pyoderma. Vet Immunol Immunopathol 64, 123-131.
  • Shearer D H & Day M J (1997) An investigation of phagocytosis and intracellular killing of Staphylococcus intermedius by canine neutrophils in vitro. Vet Immunol Immunopathol 58, 219-230.

Other sources of information

  • Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, London.


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