FIGURE 73.—Comparative growth of tubercle bacilli obtained from 1,000 cubic centimeters of culture medium: A, Growth from broth medium; B, growth from synthetic medium.
TUBERCULIN of Greater Purity and Efficiency Developed by Department
The constant search for improved methods of producing biological products for livestock disease prevention and control, by scientists of the Bureau of Animal Industry, has led to the production, from cultures on a new synthetic medium, of a tuberculin that is more efficient as a diagnostic agent than tuberculin produced by other methods. Prior to the development of the new tuberculin, the testing of cattle and other susceptible animals for tuberculosis was conducted with tuberculin made essentially in the same manner as the original product developed by Robert Koch more than 40 years ago.
Although tuberculin made according to the Koch method has given excellent results, it is not a perfect product. In some cases about 2 in 1,000 animals tested, it has been impossible to find lesions of tuberculosis in reacting cattle. In other cases, tuberculous cattle have failed to react to the first test. It was with the hope that facts might be developed that would lead to a reduction of this small percentage of error that the investigations here discussed were undertaken.
The medium used for the Koch tuberculin consists of a clear broth, made from lean beef or veal, to which 1 percent of peptone, 4 to 7 percent of glycerin, and 0.5 percent of salt are added.
This mixture is then inoculated with pure cultures of tuberele bacilli. The bacteria grow on the surface of the broth, forming a film or pellicle which gradually extends until it covers the entire surface. This growth takes place over a period of about 2 months.
At the end of the growing period the broth cultures are sterilized, the dead bacteria are removed by filtration, and the clear, sterile filtrate is concentrated to the desired degree. A suitable preservative is then added. The final product, which is used in testing cattle, contains not only the soluble substances derived from the growth of the tubercle bacilli on broth but also any portions of the culture medium which have not been used up during the growth of bacilli. It is generally recognized that the Koch or broth tuberculin is extremely complex. It always contains considerable quantities of unused glycerin. In addition there are unused nitrogenous substances derived from the beef as well as similar nitrogenous protein materials derived from the peptone which is added to the broth.
The new synthetic medium developed by the Bureau for bovine tuberculosis contains no protein whatever. The nitrogen required by the bacteria for their growth is supplied by the pure, crystalline amino acid, asparagin, while the carbohydrate and mineral needs of the bacteria are furnished by pure glycerin, dextrose, magnesium sulphate, potassium phosphate, and derivatives of sodium and iron.
Since the active substance in any tuberculin is derived from the growth of the bacilli, the strength must depend primarily on the amount of growth per 100 cubic centimeters of culture fluid, provided the bacteria are the same. Careful investigations and weighings of tubercle bacilli have shown that, under favorable conditions, on the average 0.5 gram of bacilli, dry weight, may be obtained from 100 cubic centimeters of the ordinary glycerinated broth. The synthetic medium affords a vastly greater amount of growth. From each 100 cubic centimeters of culture fluid there is obtained, at the height of the development, an average of 2 grams of tubercle bacilli, or approximately four times as much as is obtained from the same quantity of glycerinated broth medium (fig. 73). So far as known, no other synthetic culture medium for tubercle bacilli has afforded such abundant growth. As was to be expected from the greater growth of bacteria, tuberculin prepared from the synthetic-medium cultures was found, when tested on tuberculous guinea pigs, to be very much more potent than that derived from cultures on the broth medium.
Another advantage afforded by the use of the synthetic medium lies in the purity of the final product, that is, the tuberculin. As previously stated, the Koch, or broth tuberculin, always contains, as impurities, considerable unused residues of the culture medium. The newer tuberculin, on the other hand, is essentially a pure solution of the products of the tuberculosis bacillus. This result was attained by adjusting the constituents of the synthetic culture medium so that the bacteria use practically all of them. The final tuberculin contains only products which are derived from the tubercle bacillus itself. Since the reaction of tuberculous cattle to tuberculin is caused only by the products of the tubercle bacillus, it is evident that the new tuberculin is much purer than the older product.
The real test of a biological product, however, is in the actual potency when applied to animals. More than 40,000 comparative tests of the old and new tuberculins have been made on cattle. In one series 13,288 cattle were tested simultaneously with both tuberculins by the intradermic method. Of these, 1,127 gave reactions (swellings at site of injection) to the broth tuberculin and 1,268 reacted to the new tuberculin. All these reactors were slaughtered and lesions of tuberculosis were found in 1,205. Every one of these reacted to the new tuberculin but 135 did not react to the old tuberculin. No tuberculous animal in the series reacted to the broth tuberculin without at the same time reacting to the new tuberculin. In the case of the cattle which reacted to both tuberculins, the great majority showed more pronounced and more clear-cut reactions to the new tuberculin.
Since the new tuberculin was thus proved to be more effective when used for diagnosis under practical field conditions, the Bureau discontinued the production of the old tuberculin in April 1934. In its place, the tuberculin produced from cultures on: the new synthetic medium is now being used exclusively by the Bureau of Animal Industry in official tuberculosis-eradication work. This new tuberculin is produced in amount sufficient to test more than 18,000,000 cattle annually.