Porcine neutrophil function tests: Evaluation, optimization and validation of chemotaxis, phagocytosis and bacterial killing
Abstract
In vitro assays of chemotaxis under agarose, phagocytosis of opsonized zymosan particles and intracellular killing of bacteria were optimized and validated for porcine neutrophils. This study was done to evaluate the suitability of these tests for estimation of neutrophil function in experimental situations. Factors influencing chemotaxis under agarose were concentration of zymosan activated serum (ZAS), time of incubation and number of neutrophils inoculated. Recommended levels of these factors were: undiluted ZAS, 5 hours of incubation and 8 $\times$ 10$\sp5$ neutrophils. Pig-to-pig variation accounted for most of the total variation. Therefore, if adequate numbers of pigs are included in each sample group, the assay will permit valid estimation of chemotaxis in in vivo experiments. The assay is suitable for in vitro experiments because pig-to-pig variation can be eliminated. Optimization of the phagocytosis assay included evaluation of incubation time and consideration of the two response variables: average number of particles per neutrophil (INDEX) and percentage of neutrophils participating in phagocytosis (ACTIVITY). Incubation times between 0 and 40 min. were tested and 10, 20 and 30 min. were recommended. A close linear relationship (r$\sp2$ =.90) was evident between INDEX and ACTIVITY. Because determination of the INDEX was more tedious and subjective, ACTIVITY was the best response variable. Pig-to-pig variation contributed greatly to total variation at the times tested. Consequently, if adequate numbers of pigs are tested, this assay may permit valid evaluation of phagocytosis in in vivo experiments. The assay is useful for in vitro experiments using neutrophils from a single pig. Optimization of the killing assay included evaluation of 2 culture methods for estimation of surviving bacteria and time of incubation. Survival estimates by the colony counting method were less variable than by the microtiter plate method. Incubation times between 0 and 180 min. were tested and 0, 45 and 90 min. were recommended. Day-to-day variation contributed most to total variation and pig-to-pig contribution was small. Designs which eliminate day-to-day variation between groups will permit useful in vivo studies with this assay. The assay will also be useful with in vitro experiments.
Degree
Ph.D.
Advisors
Rebar, Purdue University.
Subject Area
Veterinary services
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