Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage.
OBJECTIVE
To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas.


METHODS
8 isolates of M. bovis.


METHODS
Filter-sterilized broth culture supernatants of M. bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M. bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay.


RESULTS
Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 X 10(7) d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxin-to-protein ratio. Frozen diafiltered retentate(-80 degrees C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd.


CONCLUSIONS
Diafiltered M. bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M. bovis isolates. Diafiltration could be useful for bulk semipurification of M. bovis cytolysin. Cytolysin-enriched vaccines of M. bovis could be contaminated by endotoxin.
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Lisle W
LW
George
John A
JA
Angelos
William W
WW
Ruehl
D000704:Analysis of Variance; D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification*; D002417:Cattle / Q000382:microbiology; D002840:Chromates; D002850:Chromatography, Gel; D002860:Chromium Radioisotopes; D003603:Cytotoxins / Q000276:immunology / Q000302:isolation & purification* / Q000633:toxicity*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects*; D005374:Filtration; D006461:Hemolysis / Q000187:drug effects; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007962:Leukocytes / Q000187:drug effects*; D016955:Moraxella bovis / Q000737:chemistry*; D017670:Sodium Compounds
D001427:Bacterial Toxins; D002840:Chromates; D002860:Chromium Radioisotopes; D003603:Cytotoxins; D007106:Immune Sera; D017670:Sodium Compounds; C028982:sodium chromate(VI)