1st. A culture of the bacillus made in the thermostat at 42 C. (107.6° F.) so as to prevent the formation of spores is then sterilized by heating to 100° C. for one hour and then injected subcutem in a dose of 2 drams, to be repeated on the second day. This, like all the other methods named should be done by some one accustomed to bacteriological manipulation and the sterilization completed by superheating the neck of the vessel containing the mixture. Any germs escaping on the hands, instruments or other objects used will prove fatal in spite of all the appearance of precautions.
2d. Roux sterilized his cultures by filtering them through a porcelain (Pasteur) filter and using only the filtrate for injection. This requires even greater precaution in manipulation as what is left in the filter is most virulent, and must be thoroughly sterilized to obviate dangers from its dissemination.
3d. Intravenous injection of a small quantity of virus, containing but a few bacilli produces no local swelling, but only a slight temporary hyperthermia and permanent immunity. The greatest care is necessary in the manipulation, to prevent any contact of the bacillus with the subcutaneous tissues or the walls of the vein. The virulent exudate swarming with bacilli is taken and a drop or two added to a normal salt solution, which is diluted and shaken in a stoppered bottle, until each drop contains but one, or at most two bacilli. Then the hands having been thoroughly washed with soap and warm water and rinsed in a 5 per cent. solution of carbolic acid, and the instruments having been boiled, the vein is raised as for bleeding, and penetrated by a short cannula and trochar, which after boiling has been dipped in the carbolic acid solution, the trochar is withdrawn, and the nozzle of the syringe containing the virulent solution is inserted through the cannula, so that its point is free in the centre of the blood stream, into which a few drops of the virulent solution are discharged. The nozzle is left in place for a few seconds to ensure the washing of any infecting matter from its point, when it is withdrawn, followed immediately after by the cannula. Great care should be taken to avoid any scratching of the inner coat of the vein with the cannula, trochar or nozzle.
4th. Another method of immunizing is by the injection of the virulent liquid into the trachea and bronchia. This appears to bring it so directly in contact with the blood, that the microbes are destroyed as rapidly as if it were introduced into the blood stream direct. The injection is made between two tracheal rings, the manipulation being essentially the same as in the cases of the vein, the tissues being first perforated by a sterilized cannula and trochar, and the sterilized nozzle subsequently inserted through the cannula.
5th. Inoculation into the tip of the tail can be successfully employed, the coldness of the region and the scantiness of the connective tissue preventing any dangerous increase of the bacilli in the cooler season. In the heat of summer, however, this is to be avoided as dangerous. The tail is first washed with soap and water followed by a 3 per cent. solution of phenol. It is then punctured with a fine trochar or needle, (sterilized), within two inches of the tip and in a downward direction and the instrument is moved slightly from side to side so as to form a small sac, and is then withdrawn. The sterilized nozzle of the hypodermic syringe is now inserted in the opening and a few drops of the virus injected into the sac. When the nozzle has been withdrawn the thumb may be placed on the external orifice and the end of the tail manipulated to diffuse the virus in the connective tissue. This is usually followed by an insignificant swelling, and a slight rise of temperature. Should the swelling exceed the size of a duck’s egg or if others appear higher up on the tail, they may be freely scarified and covered with a carbolic acid bandage. Or the tail may be amputated above the highest swelling and the stump treated with antiseptics.
6th. The virus prepared by the Pasteur institutes, that of Arloing, Cornevin and Thomas, is the most extensively employed. Forty grammes of the diseased muscle are dried rapidly at 32° C (90° F.) and triturated in 80 grammes of water. This is divided in 12 equal parts and put on plates in two thermostats, six at 100° C. (212° F.) and six at 85° C. (185° F.) where they are kept for six hours, when it forms a dry, brownish powder. One tenth of a gramme (1½ gr.) of this powder is dissolved in five grammes of distilled or boiled water and will furnish ten doses. The animal to be protected is first injected in the tip of the tail or elsewhere with the virus prepared at 100° C., and ten days later with that prepared at 85° C.
By the use of this method in hundreds of thousands of animals on infected lands the mortality has been reduced to less than one tenth of its former amount. It is attended by the one danger which is not always duly appreciated, that unless its use is restricted to herds on ground that is already infected, it endangers the infection of new districts. The spores are not absolutely sterilized at 85° C. Arloing and Cornevin and later, Nocard and Roux have shown that the addition of lactic acid to the liquid which has been weakened for inoculation, restores it to its former virulence, making it a most deadly agent. Galtier says that the virus weakened by heating to 100° C. for seven hours until it will no longer kill a mature Guinea pig, will still kill a new born Guinea pig and acquire all its original virulence in the act. Also that the injection of large enough doses will not only kill the full grown Guinea pig, but at the same time restore the microbes to their former virulence. While recognizing the great economy of the judicious use of such weakened virus, we cannot but condemn the reckless sale by the Pasteur institutes of their products, to be used on animals on all kinds of lands, the uninfected as well as the infected. A great and valuable prophylactic measure should not be used in such a way as to increase the area of prevalence of the disease which is to be prevented, and also the yearly demands for more of the preventive agent. This may appeal to the business instinct, but this should ever be held subordinate to sanitary considerations. The danger might be avoided by making the state the sole distributor of such prophylactic agents, but in any case their use should be forbidden, and as far as possible prevented, upon dense and wet soils that are not yet contaminated by the bacillus.
7th. Kitt secured immunization by inoculating once only, with dried virus which had been subjected for six hours to steam at 100° C.
8th. In different outbreaks, I have taken the blood from the sick animal, or one that has just died, and heated it for over one hour in a water bath, at 100° C., then broken up the coagulated mass in well boiled water, filtered the liquid and used the filtrate for inoculation in doses of 2 drams, repeated the second day. Great care is taken in keeping the whole mass at 100° C. for the requisite length of time; then in heating the upper part of the vessel, which was above the contents and the water so as to char anything adhering to it; to see that hands, instruments, and all articles used have been thoroughly sterilized; and to dip the hypodermic nozzle in carbolic acid before each injection.
It is not claimed that this method is perfect, since severe, advanced cases may have bacilli and even spores in the blood, and the latter would not be sterilized but only weakened. It has, however, several manifest advantages that may be held to more than counterbalance this danger.