Studies on phagocytosis; determination of blood opsonin for Brucella.

The bactericidal properties of dog blood for Brucella suis have been described previously (Victor, Wells et al., 1952). Because leucocytes were essential for bactericidal activity toward this strain, the relation between bactericidal and phagocytic activity of dog blood was investigated. In order to measure phagocytosis satisfactorily a method must be employed yielding reproducible results. The opsonocytophagic test of Huddleson, Johnson, and Hamaan (1933) was inadequate for our purposes because it employs 0.8 per cent sodium citrate which inhibits the bactericidal action of dog blood (Victor, Wells et al., 1952). Many investigators have found that sodium citrate also inhibited phagocytosis of brucellae by normal blood, presumably without interfering with the phagocytic activity of leucocytes of individuals with brucellosis. Others have suggested that heparin, which did not interfere with phagocytosis, be employed as an anticoagulant. The test devised by Huddleson et al. (1933) is done in tubes only intermittently agitated even though the influence of continuous agitation on phagocytosis had been demonstrated (Fenn, 1923). Furthermore, evaluation of the intensity of phagocytosis by counting the number of brucellae within individual leucocytes was a highly subjective one. Finally, the opsonocytophagic test of Huddleson et al. (1933) did not distinguish between opsonin titer and the phagocytic activity of leucocytes. Experiments were performed to define factors which influence phagocytosis of Brucella suis by dog leucocytes; namely, phagocytic power of various types of blood leucocytes, activity in heparinized and citrated blood, effect of continuous and of intermittent agitation, effect of temperature, effect of time of agitation, comparison of phagocytosis of living and of formalin killed bacteria, variability of a standardized test procedure, and effect on phagocytes of repeated washing in an isotonic solution of electrolytes and gelatin. From these experiments a method for determination of opsonin titer in plasma was developed.