Clarke J M, Morton R J
Department of Infectious Diseases and Physiology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078, USA.
Am J Vet Res. 2000 Feb;61(2):129-32. doi: 10.2460/ajvr.2000.61.129.
To develop an in vitro fluorometric assay to assess Pasteurella haemolytica adherence to bovine respiratory and epithelial cells and compare adherence of single strains of P. haemolytica serovars A1 and A2 (PhA1 and PhA2, respectively).
Monolayers of bovine turbinate and Madin Darby bovine kidney (MDBK) cells.
To determine optimal inoculum concentration and incubation time, various concentrations of P. haemolytica were labeled with fluorescein isothiocyanate and incubated with monolayers of bovine cells for various times. Bovine cells were washed to remove nonadherent bacteria, and percentage of bacteria adhered (percentage of adherence) was estimated fluorometrically. Percentage of adherence of PhA1 was compared with that of PhA2.
The optimal inoculum concentration that resulted in measurable fluorescence of adherent bacteria was 1 x 10(8) colony-forming units/ml, and the optimal incubation time was 45 minutes. Percentage of adherence of PhA1 to MDBK and turbinate cells was significantly greater than that determined for PhA2.
The in vitro fluorometric assay is a time-efficient, inexpensive, and labor-saving method for evaluation of P. haemolytica adherence to bovine cells. The concentration of bacteria used to inoculate bovine cells in this assay is similar to that typically used in other types of in vitro adherence assays. The predominance of PhA1 over PhA2 during the early stages of bovine respiratory disease may be attributable to the ability of PhA1 to adhere more avidly to nasopharyngeal tissue.
开发一种体外荧光测定法，以评估溶血巴斯德氏菌对牛呼吸道和上皮细胞的黏附情况，并比较溶血巴斯德氏菌A1和A2血清型（分别为PhA1和PhA2）单菌株的黏附情况。
牛鼻甲和马-达二氏牛肾（MDBK）细胞单层。
为确定最佳接种物浓度和孵育时间，将不同浓度的溶血巴斯德氏菌用异硫氰酸荧光素标记，并与牛细胞单层孵育不同时间。洗涤牛细胞以去除未黏附的细菌，并通过荧光测定法估算黏附细菌的百分比（黏附百分比）。比较PhA1和PhA2的黏附百分比。
导致黏附细菌产生可测量荧光的最佳接种物浓度为1×10⁸ 菌落形成单位/毫升，最佳孵育时间为45分钟。PhA1对MDBK和鼻甲细胞的黏附百分比显著高于PhA2。
体外荧光测定法是一种评估溶血巴斯德氏菌对牛细胞黏附情况的省时、廉价且省力的方法。本测定法中用于接种牛细胞的细菌浓度与其他类型体外黏附测定法中通常使用的浓度相似。在牛呼吸道疾病早期阶段，PhA1比PhA2占优势可能归因于PhA1更 avidly黏附于鼻咽组织的能力。 （avidly这个词在医学语境中可能不太常见，此处直接保留英文未准确翻译出对应的中文词汇，因为不确定具体合适的中文表述，仅按要求保留英文原文。）
