ACTINOBACILLUS-PLEUROPNEUMONIAE CULTURE SUPERNATANTS INTERFERE WITH KILLING OF PASTEURELLA-MULTOCIDA BY SWINE PULMONARY ALVEOLAR MACROPHAGES

The effect of Actinobacillus pleuropneumoniae culture supernatant on s wine pulmonary alveolar macrophage (PAM) functions was studied. The A. pleuropneumoniae culture supernatant was toxic to PAMs when tested by MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and l actate dehydrogenase (LDH) release assays. Biological activity of the supernatant was ascribed to cytotoxins. Both the LDH and MTT assays we re used for measurement of crude A. pleuropneumoniae cytotoxin concent ration with good reproducibility. A preparation containing 6,800 toxic units/mL (determined by MTT assay) was used for subsequent experiment s. The objective was to study the effect of crude cytotoxin on the abi lity of swine PAMs to kill Pasteurella multocida. Phagocytosis of opso nized P. multocida type A by PAMs was not efficient. Only 8% of incuba ted organisms were ingested by noncytotoxin-treated PAMs after 30 min phagocytosis. The bactericidal effect of noncytotoxin-treated PAMs onl y last for 60 min, after which, the rate of growth of surviving P. mul tocida exceeded the rate of bacterial killing by PAMs. Complete elimin ation of P. multocida by PAMs was not observed in this study. A total loss of ability to kill P. multocida by PAMs was seen when the PAMs we re pretreated with a high concentration (340 toxic units/mL) of A. ple uropneumoniae cytotoxin. If the PAMs were pretreated with a low concen tration (3.4 toxic units/mL) of cytotoxin, a significant reduction in the killing of P. multocida was still observed. The reductions in phag ocytosis, phagosome-lysosome fusion (demonstrated using yeast particle s of Candida albicans), and oxidative burst (demonstrated by nitro blu e tetrazolium reduction (NBT) assay) may have contributed to the impai red killing of P. multocida by PAMs. These results suggest that A. ple uropneumoniae cytotoxin induces damage and impaired function of PAMs. These changes may favor the survival of A. pleuropneumoniae in pig lun gs and also render the infected animal more susceptible to secondary b acterial infection, by organisms such as P. multocida.