258
Agglutination Test for Brucella Infection in Cattle
these laboratories. The results were far from
uniform.
19 cows reacted negative or doubtful.
24 cows reacted negative or doubtful or
positive.
2 cows reacted doubtful or positive.
1 cow reacted negative or positive.
4 cows only gave a positive reaction to all
the methods used.
Thus 46. of the 50 cattle would have been
judged positive, negative or doubtful accord-
ing to the technique adopted.
Similarly in America sera have been examin-
ed simultaneously in a number of laboratories
with reactions that varied to a considerable
degree. When one considers the large number
of institutions throughout the world in which
the test for Bang's disease is carried out, the
almost innumerable variations in technique
and interpretation, and the fact that, in spite
of these variations, herds are being rid of the
infection, we can only marvel at the results
being attained. However, it is undoubtedly
true that the less exact methods lead to eco-
nomic loss in causing the disposal of non-
infected animals and in lengthening the period
of eradication by the failure to select some
infected cattle.
It is obvious that in deciding the signi-
ficance of the results of the test we are not
concerned merely with the highest or lowest
dilution which gives agglutination; we have to
take into consideration:
(a) The suspension of bacilli.
(b) The blood serum.
(c) The diluting fluid.
(d) The treatment of the combination of
serum and antigen.
Frei [1937] discussed these matters very
fully, and his opinions are freely quoted in the
following summary.
Scrum. Coagulation can exercise an action
on the colloids of the serum and notably on
the agglutinins. Coagulation at low tempera-
tures or shaking during coagulation must be
avoided, otherwise the agglutination titre will
be lowered. Tests should be put up as soon
as possible after collection of the serum, since
the titre may fall if the serum is held for
lengthy periods, although delay of three or
four days is not of much significance. How
far the holding of uncontaminated sera is of
importance is difficult to state, but the sera
used as known positive controls at Glenfield
have been for some months carbolized without
any appreciable loss of titre in the diagnostic
range. These sera are, however, strongly
positive, and it can be understood that a slight
loss of agglutinin may be of significance with
sera on the border line; of course contamina-
tion of the sera, which frequently occurs in
field collection and at high temperatures, exer-
cises an adverse influence and renders some
sera untestable. Similarly haemolysis and
consequent staining of the serum is undesir-
able, and according to Frei may lessen the
titre. In our experience haemolysis frequent-
ly leads to a deposit in the control tube not
containing antigen and thus renders the inter-
pretatior. of the test very difficult. Frei,
quoting the report of the U. S. A. Commission
on Bang's Disease, states that, whilst varia-
tions of pH within fairly wide limits (4.7 to
8.9) do not have any noticeable effect on the
titre, unclean bottles, especially those which
have held disinfectants such as cresol or soaps,
tend to lower the titre.
Bacterial suspension. It is generally known
that the agglutinability of the antigen is in-
fluenced by the method of culture and type of
culture media employed. Stableforth showed
that when using the same bacterial strain there
was a difference in the agglutinability of sus-
pensions prepared in the different media, pre-
pared in similar media but using organs from
different species of animals, and even in
different flasks using the same medium.
Hence it is well-nigh impossible to guarantee
that supplies of media prepared under similar
conditions will be identical. Such difficulties
could be overcome if each batch of antigen
could be titrated against a standard serum;
the recent work in the preparation of standard
dry serum offers hope in this direction.
Whilst many laboratories prepare their anti-
gens from cultures when they have attained
their apparent maximum growth, e.g., after
two or three days. Frei prefers to use 24-
hour cultures, since, in his opinion, at this
age of culture there is maximum agglutin-
ability. Culture is sometimes raked off the
medium with a glass rod or a platinum needle.
In the U. S. A., however, the usual practice
is to flood the flask or tube with carbol saline,
and allow the fluid to remain in contact with
the culture two or three hours before washing
off. In any case the suspension must be
filtered to remove any particles of agar, which,
if left in the antigen, may increase the degree
of agglutination.
The use of living bacteria in the suspension
is favoured by some, but the consensus of