656 FILTERABLE VIRUSES— RICKETTSIA 
follows vaccination with cowpox virus have been amply confirmed in 
animals by the observations of Brinkerhoff and Tyzzer/ who showed 
that vaccination of monkeys protects them from subsequent infection 
with the smallpox virus. 
Originally vaccine virus was perpetuated by arm to arm inocula- 
tion, but the danger of transmitting syphilis or other disease as well 
as the uncertainty of the method have led to the use of calves as a source 
of vaccine virus. 
The source of the virus is three fold :- 
1.. Virus descended from spontaneous cowpox and continued through 
an indefinite series of animals— the true animal vaccine. 
2. Virus obtained from animals which have been inoculated with 
lymph from human vaccine pustules, either directly or indirectly, 
through a series of calves— this is knowTi as retro vaccine. 
3. Vaccine obtained by passing smallpox virus through the cow— 
the so-called variola vaccine. 
Preparation of Vaccine Virus.— Healthy female calves about three 
months of age are selected. After thorough cleansing the animal is 
fastened upon an operating table of special design and the abdomen 
and inner aspect of the thighs are shaved. If disinfectants have been 
used they are removed with sterile water. Shallow parallel incisions 
about ^ inch apart and just deep enough to become slightly reddened 
are made, and the vaccine is thoroughly rubbed into the scarified area. 
The quarters in which inoculated calves are kept are scrupulously 
clean; the animals are preferably fed an exclusive milk diet. Dust is 
reduced to a minimum and excreta are promptly removed by flushing 
with a stream of water. 
Four to six days after inoculation, depending upon the rate of 
development of the vaccine vesicles, the calf is again placed upon 
the table, the vaccinated area washed with sterile water and then 
rubbed gently with sterile absorbent cotton; any crusts or scabs are 
removed. The slightly elevated confluent eruption is curetted away 
and appears as a pulpy mass, which is thoroughly ground in a mill 
of special design with three or four times its volume of 60 per cent 
glycerin.^ The ground and comminuted glycerinized virus thus pre- 
pared contains variable numbers of bacteria ;■* as many as 700,000 
per cc. have been found. '^ Of the more common microorganisms, 
various molds, yeasts and members of the coccal group are usually 
present. Very rarely cases of tetanus have been reported following 
1 Jour. Med. Res., 1905, 14, 209. 
2 Theobald Smith: Med. Soc. Proc, June 10, 1903. 
^ Carbolic acid (0.1 per cent) is frequently added to the glycerin before mixing it with 
the pulp; experience indicates, however, that the carbolized vaccine virus loses its 
potency more rapidly than when glycerin alone is used. 
* See Rosenau (Am. Med., 1902, 3, 637) for bacteriology; also Bull. No. 12, 1902, 
U. S. P. H. and Marine Hospital Service). 
5 Theobald Smith: Loc. cit. 
