Differentiation Between Brucella melitensis and Brucella abortus by Precipitin Reaction

Abstract

The precipitin reaction has been used in an attempt to develop a simple and rapid method by which strains of Brucella melitensis can be differentiated from those of Brucella abortus. Strains utilized for the immunization of rabbits were B. melitensis 428 and B. abortus 456 obtained from the National Institute of Health at Washington, and B. suis 1529, isolated by Huddleson in 1931 from the supramammary gland of a hog.

The organisms were grown for 48 hours on freshly prepared tryptose agar slant and the growth suspended in 0.9% saline, the opacity adjusted to match barium sulphate standard No. 3. One-tenth of a cc of this living suspension was then injected into the ear vein of rabbits every other day until 4 injections had been given. On successive days following the last injection, trial bleedings were made from the ear vein and precipitin reactions carried out with formamide extracts of homologous organisms. If after 10 days precipitins were not demonstrable, another series of injections was started, followed by daily bleedings. Such a procedure was continued until precipitins appeared in the blood, their presence being indicated by a definite ring at the interphase between the serum and the extract and appearing in about 10 to 15 minutes. Immediately thereafter, the animal was bled and the blood collected into sterile paraffined test tubes; the serum was then used for the test. Emphasis must be laid on the fact that the animal has to be bled as soon as the presence of precipitin in the blood is detected.

The preparation of the extract followed Fuller's¹ method for the extraction of polysaccharides from hemolytic streptococci. In this case, however, the organisms were grown for 48 hours on tryptose phosphate agar slants and the amount of growth, that could be scraped off at one time with a loop 1.5 mm in diameter, used for the extraction.