PLEURO-PNEUMONIA 837 



Vaccination. Willens applying these observations vaccinated animals 

 against pleuro-pneumonia by inoculating a drop of the pulmonary exudate 

 into the cellular tissue of the tail. As the exudate quickly loses its virulence 

 a practical difficulty was at first experienced, for the exudate had to be 

 collected from a recently killed animal ; but after Pasteur had demonstrated 

 that the exudate when collected with aseptic precautions will retain its pro- 

 perties for several (up to 4) weeks vaccination became an easier matter. If 

 the exudate be inoculated 4-6 weeks after collection it produces no effect ; 

 hence the necessity, in countries where prophylactic vaccination is a regular 

 practice, of having vaccination centres where calves can be inoculated and 

 the fresh material collected 'at least once a month (but vaccination is now 

 effected with pure cultures (vide infra}). 



2. Methods of diagnosing- the disease Characteristics 

 of the organism. 



The microscope and the ordinary methods of cultivation being of no use 

 in searching for the micro-organism in pleuro-pneumonia, Nocard and Roux 

 cultivated the exudate in collodion sacs. 



Collodion sacs (p. 175) are filled with broth, sown with a drop of the 

 exudate from a case of pleuro-pneumonia and introduced into the peritoneal 

 cavities of rabbits. After remaining there for a fortnight or 3 weeks their 

 contents are cloudy, opalescent, and slightly albuminous. 1 



Microscopical examination of this cloudy fluid with a magnification of about 

 2000 diameters shows that it contains a large number of motile refractile 

 points but so small that their shape cannot be made out and they cannot 

 be stained. 



The virus is not without effect upon the rabbits used for the experiments : when 

 the sacs are removed after 15-20 days the animals are found to be very thin, and 

 they occasionally die before the twentieth day in a state of extreme emaciation 

 but without any appreciable lesion : their organs and body-fluids are sterile. The 

 control animals in which similar but sterile sacs are inserted remain healthy. The 

 symptoms are evidently of the nature of a toxaemia, which must be due to the diffusion 

 of products elaborated by the micro-organism : and the experiment shows that the 

 rabbit is susceptible to the toxin though it is immune to the organism itself. 



The contents of the collodion sacs removed from the rabbits cannot be 

 cultivated on ordinary culture media. The vitality of the virus can be 

 preserved by keeping it in collodion sacs in the peritoneal cavities of these 

 animals, but its virulence appears to diminish in the process. 



The organism cannot be grown in collodion sacs in the peritoneal cavities 

 of guinea-pigs. 



After a long series of experiments, Nocard and Roux have succeeded in 

 devising an artificial medium on which the micro-organism of pleuro-pneu- 

 monia can be grown in vitro. The medium is a mixture of twenty parts of 

 Martin's peptone solution (p. 32) and one part of rabbit or cow serum. 

 Tubes of this mixture sown aerobically with a drop of the exudate from a 

 case of pleuro-pneumonia or with some of the contents of a collodion-sac 

 culture, and incubated at 37 C. give a growth similar to that obtained in 

 collodion sacs : moreover the micro-organism retains its virulence on this 

 medium and a long series of sub-cultures can be made. 



By adding agar to the above solution a solid medium can be obtained on 

 which after incubating for 3 or 4 days the organism of pleuro-pneumonia 

 gives very minute colonies. When the colonies are very closely packed 



1 The contents of control sacs, prepared in the same manner but not sown with the 

 exudate remain clear under similar conditions. 



