HPLC Comparison of Tear Film Protein Pattern in Normal and Infectious Bovine Keratoconjunctivitis Affected Cattle

Abstract

Purpose: This project was undertaken to establish the tear protein pattern in normal cattle and changes in the pattern at different stages of IBK, and disease of great economic importance.

Methods: Non-stimulated tears were atraumatically collected with a 50μ1 capillary tube. The study included normal and infected animals in three stages of naturally occurring IBK. Analysis was performed with a TSK 3000 SWXL size exclusion HPLC column at a flow rate of 0.3ml/min with 0.5M NaCl, 0.1M NaH₃PO₄, pH 5. Quantitation was completed with computerized integration of peaks.

Results: In all animals 7 major peaks were found at retention times of 17, 19, 21, 27, 29, 33, and 39 minutes. During the active course of the disease, peaks 1, 2, 3, 5, 6, and 7 showed various changes. Separation for different bacterial culture result within the group had statistically significant differences.

Conclusions: The tear protein pattern in IBK affected cattle versus normal shows distinct changes. In addition to the stage of the disease changes are markedly influenced by the type of recovered organism. The first three peaks that changed in size during the course of the disease are likely to contain immunoglobulins.