238 
amounts as large as 3 ¢.c. were easily tolerated 
and gave similar results. 
3. In protectively inoculated rabbits a slight re- 
sistance was manifested within 3 to 4 days, but 
complete immunity did not appear until the 
seventh day after the protective inoculation. 
4. That the resistance in question was not a 
‘¢zonal’’ or local immunity was shown by inocula- 
tion of previously protected rabbits in the ear, 
flank and back; also by intravenous and intra- 
peritoneal inoculations, none of which were fatal. 
5. The resistance produced by inoculation with 
2 ec. of Culture 52 was sufficient to protect 
against at least 3 cc. of the virulent culture 
when the M.L.D. of the latter was one hundred- 
quintillionth (0.000,000,000,000,000,000,01 c.c.) of 
one cubic centimeter. 
6. Both fowls and pigeons have been rendered 
immune by inoculation with Culture 52. But, 
whereas a single inoculation is invariably sufficient 
for rabbits, fowls and pigeons may require two. 
7. Attempts to produce resistance by inocula- 
tion with killed and attenuated cultures have 
failed; likewise attempts to produce resistance by 
successive inoculation with gradually increasing 
numbers of living organisms; this last is probably 
due to the fact that inoculation with even 4 organ- 
isms is fatal. 
8. In rabbits, immunity to the virulent culture 
has been found to endure for at least 8 months. 
9. The immunity obtained through inoculation 
with Culture 52 is inherited; female rabbits, six 
and one half months after the protective inocula- 
tion, are able to give birth to young which are 
perfectly immune. 
10. The blood serum from resistant rabbits is 
protective. 
Serum Diagnosis of Glanders: JOHN R. MOHLER. 
In 1909 Schiitz and Schubert published the 
results of their important work on the application 
of the method of complement fixation for the diag- 
nosis of glanders. Their experiments were fol- 
lowed by splendid results, exceeding by far those 
obtained by either the mallein or agglutination 
test. Consequently, they recommended that this 
method of diagnosis in combination with the ag- 
glutination test be taken as the official test in 
Germany. ‘This method, overcoming as it does 
the disadvantages of the mallein and agglutina- 
tion tests, constitutes the most reliable method for 
the diagnosis of glanders which we have at our 
command at the present time. It has recently 
been thoroughly studied by the Bureau of Animal 
SCIENCE 
[N.S. Vou. XXXV. No. 893 
Industry and has proved to be highly satisfactory. 
The principle of this test is presented in the phe- 
nomenon of hemolysis, which was first discovered 
and studied by Bordet and Gengou. This phe- 
nomenon consists of the well-known fact that if 
red blood cells of one animal are introduced into 
another of a different species, the blood of the 
latter acquires the power to dissolve the blood 
cells of the former when mixed with them in a test 
tube. The substances necessary for hemolysis are, 
(1) the hemolytic amboceptor, which is the serum 
of a rabbit that has been injected with washed 
sheep corpuscles; (2) the complement in the form 
of normal guinea-pig serum, and (3) washed 
blood corpuscles of the sheep. In the complement 
fixation test there are also used, besides the hem- 
olytic system, the serum of the horse to be exam- 
ined and an extract of glanders bacilli, termed 
antigen. 
The complement fixation test is so called on 
account of the fact that the complement is fixed 
by the combination of glanders bacilli extract 
with antibodies in the serum of a glandered horse, 
and is thus prevented from participating in the 
hemolytic process in which it is essential in order 
for hemolysis to take place. By this method, even 
small quantities of glanders antibodies (ambo- 
ceptors) can be demonstrated in a serum. 
The complement fixation accordingly represents 
a specific test, as only in the presence of glanders 
antibodies and glanders antigen will a reaction 
take place. The results of the test should be in- 
terpreted as follows: 
1. Horses in which the serum produces a com- 
plete fixation of the complement in quantities of 
0.1 ec. and 0.2 ¢.c. should be considered as glan- 
dered. 
2. Horses in which the serum gives a complete 
fixation in the quantity of 0.2 ¢.c., and an incom- 
plete fixation in the quantity of 0.1 ¢.., should 
likewise be considered as glandered. 
3. Horses in which the serum produces incom- 
plete fixation in quantities of 0.1 ¢.c. and 0.2 ce. 
should also be considered as glandered. 
4. Horses in which the serum shows no fixation 
of the complement should be considered free from 
glanders. 
In order to reduce the possibility of error to 
a mimimum, the agglutination test may be ap- 
plied to the negative cases, and if this shows a 
value of 1:1,000 or over the animal should be 
considered as glandered. However, such cases are 
extremely rare. 
