﻿CULTIVATION OF A BOVINE PIROPLASMA. 87 



of much interest and one which needs further investigation and therefore 

 our observations on the parasites were extended further. The results of 

 this study are here presented in as brief a manner as possible. 



Observation 3. — Twenty-one native cattle were carefully examined 

 both by microscopic and cultural methods at the same time. Of this 

 number, nine were shown by microscopic examination to be infected with 

 Piroplasma parvumj in only seven of these cases were the flagellate 

 organisms found in the cultures ; in the remaining two, the cultures 

 gave no growth of flagellates. Microscopic tests failed to detect any 

 parasite in the other twelve cases and, similarly, the cultural method 

 gave a negative result. Therefore, the almost constant occurrence of 

 piroplasma in the blood and of flagellates in the culture renders the 

 existence of certain relations between them very probable and we can 

 hardly regard this occurrence as a merely accidental phenomenon. 



I have also ascertained the minimum doses of infected blood which 

 would give a growth of the flagellates in cultures. For this purpose a 

 freshly prepared culture medium was used, which consisted of the blood 

 of an uninfected calf or rabbit and common bouillon. Each tube 

 of the blood-bouillon was inoculated with a different amount of the 

 infected blood which harbored a fair number of intraglobular parasites. 

 I have obtained some interesting results from a series of experiments 

 along this line, namely, that the quantity of the incubated material is 

 a matter of indifference in respect to the development of trypanosomata 

 in the cultures, since one platinum -loop full of the blood gave exactly 

 the same result as if one cubic centimeter of the same material was used. 

 In other words, the germ which gives rise to trypanosomata in the 

 culture exists even in so small an amount of the blood as one platinum 

 loop-full, or one drop. These facts suggested a possible means of 

 detecting the flagellates by direct blood examination, if they exist origi- 

 nally in the blood of an infected calf. However, a large number of blood 

 preparations, both fresh and stained, were thoroughly investigated, but 

 no flagellates were observed. This mode of observation was repeated 

 several times, but every series gave negative results. Furthermore, we 

 have centrifugated a mixture of the infected blood and salt-solution at 

 a low temperature, to prevent a possible development of the parasites. 

 The sediment was than removed drop by drop from the upper layer of 

 the centrifugated material and subjected to careful microscopic examina- 

 tion. It was expected by this procedure to obtain direct evidence of the 

 existence of trypanosomata in the blood, but our trials failed to detect 

 any motile or flagellated organism. 



Observation Jf. — The most effective evidence in favor of the view that 

 the flagellates originate in the culture may be secured by a morphologic 

 study of each developmental stage of the parasites, but many difficulties 

 are encountered in such investigations. On the one hand the scanty 



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