SELECTED ARTICLES
IN VITRO STUDIES OF THE BASIS FOR SULFANILAMIDE THERAPY IN
BOVINE MASTITIS
By J. C. KAKAVAS, PH.D., Newark, Delaware
(With two charts)
THE subject of sulfanilamide therapy in bovine
mastitis has received considerable attention in recent
years. Although the early reports show contradic-
tory results, more recent controlled studies have
revealed that sulfanilamide administered orally to a
cow is of little or no curative value in permanently
eradicating the streptococci from the udders of cows
suffering from acute or chronic mastitis. A new
approach to the problem of sulfanilamide therapy in
bovine mastitis has been made in this laboratory,
and the results of the findings have been reported. 1
It was shown that where sulfanilamide is introduced
in large amounts directly into the udder via the teat
duct, mastitis streptococci are destroyed in most
cases. The purpose of this report is to present a
study of some of the fundamental principles which
govern the activity of sulfanilamide in respect to the
infecting organism.
Cultures and mediums
The strains of Streptococcus agalactiae employed
in this study, with the exception of three cultures,
had been isolated in this laboratory from chronic
cases of mastitis. Cultures 090R, V-8 and K-151A
were supplied by the Lederle Laboratories and
represent, respectively, types I, II, and III of Lance-
field group B. The identity of the cultures as Str-
agalactiae was established by means of physiologic
tests and serologic reactions, using the precipitin
method of Lancefield and the rapid agglutination
test. Some of the cultures had been carried on
stock culture medium for some time previous to this
study, while others were of recent isolation. The
test organisms were transferred every twenty-four
hours for two to three days to insure rapid growth.
Each culture was then diluted in sterile water blanks
sufficiently so that 1 c.c. contained approximately
500 to 2,000 living organisms. As will be shown
later in this paper, the number of organisms present
in the inoculum influences the degree and rapidity of
their destruction by the sulfanilamide.
The mediums employed in these studies were
tryptose broth and tryptose agar. Their composi-
tion was basically the same as described in a previous
publication. 1
Effect of temperature on bactericidal activity of
Sulfanilamide.—White and Parker 2 demonstrated
that sulfanilamide at 20 mg. per cent is germicidal
against group A streptococci at 40 C., but not at
37 C. Heishman and Miller 3 also reported that an
increase in incubation temperature enhances the
bactericidal action of sulfanilamide against group B
streptococci. In view of the above reports, the
following experiments were conducted to ascertain
by quantitative determinations the effect of sul-
fanilamide on Streptococcus agalactiae at different
temperatures. The tests were conducted as follows:
20 c.c. of tryptose broth containing 20 mg. per cent of
sulfanilamide were placed in 50 c.c. flasks. The
control flasks contained tryptose broth only. The
test organisms were grown in tryptose broth for
twenty-four hours and then diluted so that 1 c.c.
contained approximately 500 to 2,000 viable
organisms. One c.c. of the diluted culture was
placed in each flask. All tests were run in duplicate;
one set was incubated at 37 C. and the companion
set was incubated at 40.5 C. At intervals of from
five to twelve hours, a small portion was withdrawn
from each flask and placed in serial dilutions to
determine the number of living organisms at each
interval. With some of the cultures the-experiment
was repeated with a variation in the inoculum which
consisted of over one million organisms.
In chart 1, the results on four cultures are
presented. The ordinates represent the number of
living organisms at different intervals plotted i n
logarithmic scale, and the abscissas represent the
time intervals in hours. The results indicate that
the organisms in the flasks which were incubated at
37 C. grew at approximately the same rate in both
the control and the sulfanilamide flasks. At 40.5°
C. the rate of growth was first retarded in both
flasks, but soon a divergence occurred whereby the
organisms in the control flask continued to grow
until the maximum growth was reached, which was
approximately the same as that in the 37° C. flasks;
however, the organisms in the flasks with the 20 mg.
per cent sulfanilamide were rapidly decreasing in
number until all the organisms were killed.
There was some variation in the number of hours
it took to sterilize the various cultures. In some
cases, the organisms were killed in less than twenty
hours, whereas in others, it took slightly over thirty
hours. In chart 2, the results of the tests in which
the inoculum contained more than 1,000,000 bacteria
Presented before the Section on Sanitary Science and Food
Hygiene at the seventy-ninth annual meeting of the American
Veterinary Medical Association, Chicago, Aug. 24-27, 1942.
The author is associate bacteriologist, Haskell Research,
University of Delaware, Newark.
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