790 THE INTRA-CORPUSCULAR KLEMATOZOA 



towards the blunt end. When the parasite becomes rounded the chromatin 

 gradually fuses into one mass, which subsequently divides and forks in a 

 peculiar manner so as to give rise to a Y-shaped chromatin figure ; the two 

 processes are thin, and protrude into the two small buds and indicate coming 

 division. As the buds grow in size, the main mass of chromatin divides and 

 all the chromatin passes into the two pyriform bodies. 



[Moreover an amoeboid intra-corpuscular parasite may divide in the 

 .amoeboid stage into two parasites and the two daughter cells give rise inde- 

 pendently and perhaps simultaneously, to two pairs of pyriform parasites 

 in a similar manner to that described.] 



2. Appearance in films prepared from the spleen. Free parasites are much 

 more numerous in the spleen than in the blood of the peripheral circulation. 

 The small rounded or oval forms predominate and undergo multiplication 

 this organ being the main site of endogenous reproduction (schizogony). 

 These small forms being endowed with powers of amoeboid movement are 

 able to penetrate the red cells (Laveran and Mesnil). 



Appearance in ticks. The changes taking place in Piroplasma bigeminum 

 in the stomachs of ticks after the latter had fed on infected animals has been 

 followed by Koch. As has already been stated it is in the stomachs of certain 

 species of ticks that the piroplasma undergoes sexual (sporogonous) multi- 

 plication. About 12 or 20 hours after the infected meal it is not uncommon 

 to find the pyriform parasites, which have now left the red cells, changed 

 into spherical bodies each having twelve to twenty delicate straight pro- 

 longations arranged round it like rays round a star. These ray-like processes 

 subsequently disappear while the spherical bodies increase in size and later 

 large club-shaped structures (ookinetes) are seen, not only in the digestive 

 tube but also in the eggs. 



Methods of examination. 



Microscopical examination. The blood from an infected animal may be 

 examined either in the fresh condition or after drying. 

 Laveran and Nicolle give the following directions : 



1. Fix the films at 110 C. for a few minutes and then in a saturated aqueous 

 solution of perchloride of mercury for 1 minute. 



2. Stain for 1-2 hours in Laveran's eosin-methylene-blue solution, wash, 

 treat with tannin and proceed as described at p. 772. Before mounting in 

 balsam, make certain that the preparation is not stained too deeply : should 

 this be the case, decolourize in absolute alcohol. The nuclei of the parasites 

 should be stained violet-red. 



Cultures. A culture of P. bigeminum may be obtained if some blood rich 

 in parasites be sown in citrated blood or in serum rich in haemoglobin and 

 incubated at 37 C. (Lignieres). Growth appears in about a fortnight. The 

 piroplasma ta become rounded, leave the red cells and lose their nuclei : then 

 the nucleus is formed anew and divides into from two to five small spherical 

 bodies surrounded by protoplasm, constituting spores. These in their turn give 

 origin to new spherical bodies. Pyriform bodies are never seen in cultures. 



Experimental inoculation. Cattle are the only domestic animals susceptible 

 to infection. Infection of cattle can be produced experimentally by inocu- 

 lating infected blood beneath the skin, into the muscles or into the peripheral 

 circulation. The success of the experiment will depend upon the number of 

 parasites in the blood inoculated ; when it is rich in parasites a dose of 

 01-0-05 c.c. will be sufficient to produce infection, but it may be necessary 

 to use as much as 1, 2 or even 10 c.c. 



Cattle cannot be infected by the alimentary canal. 



