10 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 
always necessary to establish the causal relationship between the 
germ and the disease. 
Because of the absence of any of the higher animal parasites and 
of fungi in bees suffering from Nosema-disease these groups of para- 
sites naturally can be eliminated as possible causal factors. Maiden 
(1912, 1913) studied the bacteriology of Nosema-infected bees. He 
found that the number of bacteria in the diseased bees was much 
greater than in normal ones, the proportion being as 12 to 1. He 
found, however, no evidence of a direct etiological relation existing 
between these bacteria and the disease. Whether they play a 
secondary role is a question which admits of much discussion but 
one which is somewhat foreign to the present paper. 
Some preliminary experiments were made by the writer in regard 
to the possibility of the presence of a filtrable virus in Nosema- 
disease. The results obtained indicate that no such virus is present. 
By thus eliminating, at least tentatively, the higher animal para- 
sites, the fungi, the bacteria, and the filtrable viruses — groups of 
parasites which cause diseases in animals — there remains another 
group, the protozoa. Of this group there is only one species, Nosema 
apis, that is constantly present in Nosema-disease. Other protozoa 
are occasionally encountered in adult bees, but when found are 
present usually in small numbers only. The conclusion is naturally 
reached, therefore, that Nosema apis is the cause of Nosema-disease. 
Such a conclusion is in harmony with views generally accepted at the 
present time in regard to proof necessary to establish the causal re- 
lation of such a germ to the disease. 
PREDISPOSING CAUSES. 
AGE. 
Experimental inoculations have shown that in general adult bees 
of all ages are susceptible to Nosema infection. In nature it is found 
that the youngest bees are always free from infection and that the 
old shiny bees usually are. The absence of Nosema apis in the younger 
ones may be attributed simply to the fact that they have not yet been 
infected through the taking of food containing the germ. In the case 
of the shiny bees it seems probable that they have escaped infection, 
although it is possible that some of them might have been infected 
at one time and later recovered. 
The brood does not seem to be at all susceptible to infection with 
Nosema apis. In heavily infected colonies the larvae and pupae appar- 
ently remain healthy. In these studies larvae were inoculated more 
or less directly by means of a pipette and examinations 1 were made 
daily following the inoculation. The spores were found mixed with 
1 The examinations were made through fixing and sectioning inoculated iarvae. 
