Specific Carbohydrate from Asterococcus mycoides for Serologic Tests of Bovine Pleuropneumonia

It is known that a substance active in the specific precipitative reaction is present in the serum of infected animals and can also be extracted from the affected lungs in cases of bovine pleuropneumonia. These observations suggest the presence of a haptenic carbohydrate. An attempt, therefore, was made in the present experiment to isolate a specific carbohydrate from pure cultures of Asterococcus mycoides, the causal agent of bovine pleuropneumonia, and to determine its serologic activity with immune rabbit's serum.

The strain of the organism used here was isolated from the pleural exudate of a fatal case of pleuropneumonia in a cow. Forty flasks, each containing 200 cc. of liver-digest broth enriched by addition of 5% unheated horse serum, were inoculated with the organism and incubated for 5 days at 37°C. The growth was centrifuged for 2 hours at 3,000 r.p.m. and the sediment was collected, washed in saline, centrifuged again and finally resuspended in distilled water. For the sediment obtained from one liter of culture 20 cc. of water was employed. This suspension was left in the icechest overnight. At the end of this period, 0.5% of potassium hydroxide was added following which the suspension became transparent and markedly slimy. This was centrifuged and to the clear fluid glacial acetic acid was added. A floccular precipitate, designated here as nucleo-protein, developed. The precipitate was collected, dried in the incubator and preserved for serologic tests. The remaining clear fluid was passed through a Seitz filter and further precipitation accomplished by addition of 5 volumes of 95% ethyl alcohol. The mixture was left in the icechest for 18 hours. During this time a small amount of white precipitate was formed. This was collected, dried in the incubator and used in the serologic tests.