336 VETERINARY BACTERIOLOGY 



only by experimentation, as there are many unknown or uncon- 

 trolled factors involved. Chamberland and Roux found that atten- 

 uation could likewise be accomplished by the use of small amounts 

 of certain antiseptics in the culture-medium. One part in 600 : 

 800 of phenol, and one part in 2000 : 5000 of potassium bichro- 

 mate were found to reduce the virulence so that ten days' exposure 

 rendered the organism non- virulent for the sheep. Numerous other 

 methods of attenuation have been proposed, such as heating to a 

 temperature just below the thermal death-point, and injection into 

 resistant animals, such as the frog and white rat. These latter have 

 no practical significance. Before use the virulence of cultures grown 

 from sixteen to eighteen hours at 37 must be determined. Rab- 

 bits, guinea-pigs, and mice are inoculated with varying amounts 

 usually from y 1 ^ to unnr f a standard loopful of the culture is in- 

 jected subcutaneously. An exposure to a temperature of 42.5 for 

 eleven days renders the organism non- virulent for the rabbit, but 

 an exposure of twenty days is required before it shows diminished 

 virulence for the guinea-pig, and of ten weeks before it becomes 

 non-virulent for the mouse. Experiments have shown that the 

 organisms once attenuated do not regain at once their virulence 

 when cultivated at 37. Pasteur believed that the attenuated 

 forms lost their power of spore production, but avirulent types 

 have since been shown to be sporogenous and not to be differen- 

 tiated morphologically from the fully virulent form. Slight 

 cultural differences have been described. Pasteur prepared vac- 

 cine of two grades of virulence. The " Premier vaccine " (vaccine 

 I.) killed white mice with certainty, was irregular in pathogenicity 

 for guinea-pigs and a 1 virulent for rabbits. The " Deuxieme vac- 

 cine " (vaccine II.) killed guinea-pigs and was somewhat virulent 

 for the rabbit. About 0.25 c.c. of vaccine I. is used for cattle, half 

 the amount for sheep. Twelve days later a similar injection of vac- 

 cine II. is made. The use of the Pasteur vaccination method has 

 been attended with highly satisfactory results in European coun- 

 tries, although not so good in the United States. Variations in the 

 virulence of vaccine II. and also differences in susceptibility in 

 the vaccinated animals lead to a loss among such animals which 

 averages about 1 per cent, in cattle and somewhat more in sheep us 

 a direct result of the vaccination. Many modifications of the 



