Baluyut C S, Simonson R R, Bemrick W J, Maheswaran S K
Am J Vet Res. 1981 Nov;42(11):1920-6.
Timed cultures of Pasteurella haemolytica 12296 strain in RPMI 1640 medium (with L-glutamine, pH 7.4) were used to determine the correlation between cytotoxin production and the age of the culture. Cytotoxic activity was measured by a 51Cr-release assay and trypan blue exclusion test with bovine neutrophils as target cells. Results demonstrated that optimal cytotoxin production occurred during the logarithmic phase (peaked at 6 hours) and decreased during the stationary phase of bacterial growth. The cytotoxin was concentrated by sequential ultrafiltration on Diaflo XM 50, XM 100, and XM 300 membranes. The cytotoxin was retained on an XM 300 membrane. These studies indicated that the molecular weight of cytotoxic substance was 300,000 or more. The cytotoxin was heat labile, oxygen stable, and susceptible to extremes of pH and killed bovine neutrophils and mononuclear leukocytes. It was not hemolytic to bovine or ovine RBC. The cytotoxic activity was inactivated by trypsin and did not contain any detectable endotoxin. Bovine fetal serum and serum collected before immunization from neonatal calves did not neutralize the cytotoxic effects of toxin on neutrophils. However, adult bovine serum from 6 cows and an antiserum (against the cytotoxin) neutralized the cytotoxin, as revealed by both the 51Cr-release assay and the trypan blue exclusion test. This was confirmed by transmission electron microscopy. These results indicated that the cytotoxin may be antigenic in cattle. The significance and implications of these findings to bovine pasteurellosis are discussed.
采用溶血巴斯德菌12296菌株在RPMI 1640培养基（含L - 谷氨酰胺，pH 7.4）中进行定时培养，以确定细胞毒素产生与培养时间之间的相关性。细胞毒性活性通过51Cr释放试验和以牛中性粒细胞为靶细胞的台盼蓝排斥试验进行测定。结果表明，最佳细胞毒素产生发生在对数生长期（6小时达到峰值），并在细菌生长的稳定期下降。通过在Diaflo XM 50、XM 100和XM 300膜上进行连续超滤浓缩细胞毒素。细胞毒素保留在XM 300膜上。这些研究表明，细胞毒性物质的分子量为300,000或更大。该细胞毒素热不稳定、对氧稳定，对极端pH敏感，可杀死牛中性粒细胞和单核白细胞。它对牛或羊红细胞不具有溶血作用。细胞毒性活性可被胰蛋白酶灭活，且不含有任何可检测到的内毒素。牛胎血清和新生犊牛免疫前采集的血清不能中和毒素对中性粒细胞的细胞毒性作用。然而，来自6头奶牛的成年牛血清和一种抗血清（针对细胞毒素）可中和细胞毒素，这通过51Cr释放试验和台盼蓝排斥试验均得到证实。透射电子显微镜也证实了这一点。这些结果表明，该细胞毒素在牛体内可能具有抗原性。讨论了这些发现对牛巴氏杆菌病的意义和影响。
