34                      Pyosepticaemia of Calves in India

Fermentation of Carbohydrates.—The following are fermented with the
production of acid and gas :—Dextrose, maltose mannitol, xylose, dulsitol,
dextrin, sorbitol, rhamnose, glucose, galactose, laevulose, mannose and
trehalose. Acid is produced in starch. Lactose, sucrose, salicin, inocitol,
glycerol, inulin, rhaffinose, adonite and arabinose are not attacked.

Other biochemical reactions.—Litmus-milk is rendered slightly acid in
24 hours, but later becomes alkaline. Tested for Indol in 1 per cent peptone
water after 5 days' growth, using Bohme's reagents, a faint colour appears
which disappears with time. It is negative for the Voges-Proskauer test,
but positive for the methyl-red test. It reduces nitrates, produces hydro-
gen-sulphide and is positive for catalase and methyline blue reductase.

The morphological, cultural and biochemical characters are typical
of the salmonella group and resemble those of S. enteritidis, S. newport, S.
dar-es-salaam, S. stanley and certain other members of the salmonella group.
A preliminary agglutination test was undertaken to ascertain which of the
various heat-stabile somatic antigens occurring in the salmonella group was
concerned in the organism under study.

The serum for this agglutination test was obtained as follows : the
Shirlaw's organism was grown in plain agar in a Petri-dish for 24 hours. It
was then washed with normal saline solution containing 0.1 per cent of for-
malin. It was then adjusted to an opacity of 300 million bacteria per c.c.
with saline containing 0.1 per cent of formalin. This emulsion was injected
intravenously, after a storage of 3 days at room temperature, into a rabbit
five times at intervals of five to six days. The doses for the successive in-
jections were 0.1 c.c., 0.5 c.c. for the first and second injections respectively,
and 1 c.c. for the subsequent three injections. The rabbit was bled for serum
on the ninth day after the last injection. Its titre then was over 12,000,
the end titre not having been determined.

The heat-stabile antigens were made according to the technique
of Bruce-White [1926]. Roux flasks containing agar with a 1 : 800 con-
centration of phenol, were sown with the selected cultures. After an
incubation of 48 hours, the cultures were washed in 0.1 per cent saline and
heated in a water-bath at the boiling point for about thirty to forty minutes.
The opacity of the emulsions was then made up to 200 million bacteria per
c.c. and formalin was added to give a concentration of 0.1 per cent.

The serum was diluted in series in such a manner, that, when an equal
quantity of the bacterial emulsion was added, the final dilution of the serum
in the successive tubes became 25, 50, 100, 200, 400, and 800 respectively.

The strains were selected for this test so that all the somatic factors
then known to occur in the salmonella group were represented.

The test was conducted in the water-bath at 55°C. and readings were
taken at the end of four hours and twenty-four hours. The results are tabu-
lated in Table No. I.