W. L. HINDMARSH
257
fever, but its use was soon extended to other
diseases, though often with indifferent success
[Arkwright, 1931]. In addition to the testing
of sera for the presence of agglutinins against
bacteria suspected of having invaded the host,
agglutination was utilized in the identification
of unknown bacteria.
Specific agglutination is now regarded as
occurring in two stages. In the first place the
bacteria are acted upon by the agglutinin
present in the serum of the infected or immune
animals. This action confers on the bacteria
the property of clumping in the presence of neu-
tral salts in low concentration. In the second
place the bacteria are agglutinated into clumps
in the presence of electrolytes. The clumps
then fall to the bottom of the tube by gravity.
Wright and Smith [1897] used the agglutina-
tion test for the differentiation of Malta and
typhoid fever. Birt and Lamb [1899] describ-
ed the agglutination test for Malta fever, basing
their work on that done previously by Wright.
These authors recognized the necessity for a
standard method of preparing antigen, a know-
ledge of the agglutination titres of sera of normal
and infected individuals and a knowledge of the
period that elapsed between infection and the
presence of agglutinins in the serum.
Since this work was published the agglutina-
tion test has come into general use in the diag-
nosis of infection of cattle with Brucella abortus.
McFadyean and Stockman [1912] concluded
as a result of their tests that "one will be justi-
fied in regarding complete agglutination with a
serum dilution of 1 in 50 or 1 in 100 as strong
evidence of infection." They drew attention to
the fact that all animals whose sera give a posi-
tive reaction do not necessarily abort and that
certain infected animals which do abort may
not give a positive reaction at or shortly after
the event although later the serum will react
positively. The standardization of the bacterial
suspension used in these tests was 'when view-
ed in one of the small tubes employed for the
tests, it was faintly hazy in appearance." The
tubes were incubated at 37°C. to 38°C. for
24 hours before reading.
Seddon [1915] reported upon his investiga-
tions into use of the agglutination test. He
emphasized the necessity for a standardized
bacterial emulsion for use as antigen, using a
suspension of barium sulphate† as a standard
of capacity. In a later report Seddon [1919]
drew attention to the fact that, whilst aggluti-
nation due to non-specific agglutinins would take
place if sufficient serum was added to the bacte-
rial suspension, the concentration of these ag-
glutinins is never very high. With regard to
the agglutinins developed as a result of invasion
of the body by Br. abortus, he referred to the
definite relationship between the quantity of
specific agglutinins and of the bacterial sus-
pension which go together to produce the ag-
glutination reaction, pointing out the necessity
for using a definite unit of the suspension in the
test.
As a result of his investigations he concluded
that agglutination with 0.1 ml. of serum, using
the unit of antigen previously described, was
evidence of infection (N.B.: 0.5 ml. of suspen-
sion was used and the total volume of fluid in
the tubes was 2 ml.). Seddon compared the
agglutination responses with the clinical history
of the cattle. Like McFadyean and Stockman,
he noted that some infected animals failed to
give a positive reaction until after abortion and
that a small percentage of cattle, infected ap-
parently in the uterus only, may have a decreas-
ing agglutination titre and eventually give a
negative reaction.
The agglutination tests carried out at the
Glenfield Veterinary Research Station are based
upon Seddon's work, agglutination with 0.01
ml. of serum being regarded as a positive reac-
tion. For purpose of convenience, however, we
standardize the antigen by comparison with tube
2 of Burroughs Wellcome standard opacity
tubes. This gives a suspension slightly less
dense than Seddon's Barium Standard.
The wide use of the agglutination test for the
establishment of abortion-free herds has led to
many thousands of tests being carried out in
laboratories in almost all civilized countries.
From the accounts of tests as applied in various
centres, there is considerable variation in the
technique adopted, and unless the exact details
of the test are published it is difficult to make
comparisons between the results obtained by
different workers. Hence it is now generally
admitted that there is necessity for the determi-
nation of a standard for the technique and inter-
pretation of the test. In this connection
Stableforth [1936] has published some interest-
ing and pertinent data. He carried out tests
on the sera of 50 cows, with the bacterial sus-
pensions of different laboratories and using the
technique and methods of reading adopted in
†Three ml. of 1 per cent solution of barium chloride
in distilled water was added to 97 m 1. of 1 per cent.
solution of sulphuric acid in water.