QUALITATIVE AND QUANTITATIVE STUDIES OF VIBRIO FETUS
ANTIGEN PRODUCED IN VARIOUS MEDIA, WITH EMPHASIS ON
THE EFFICACY OF A FLUID MEDIUM

 

By
MARVIN F. EEELD

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in.partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Bacteriology and Public Health

1950

TABLE OF CONTENTS

Introduction . . . . . . . . .
Historical.Review . . . . . .
Materials and.Methods . . . .

Procedure for Preparation
fetus Antigen . . . . .

Agglutination Tests . . .
Results and Discussion . . . .
511mm 0 O O O O O O O O O 0

Bibliography . . . . . . . . .

O O O O O

of Vibrio

The author wishes to express his sincere
appreciation to Dr. H. J. Stafseth for his
helpful suggestions and to Dr. J. J. Stockton
for his guidance and cooperation in this work.
He also wishes to thank Dr. C. H. Cunningham

for the use of the refrigerated centrifuge.

INTRODUCTION

The etiological role of'Vibrio fetus in abortions of cattle

 

and sheep has necessitated improved methods of diagnosis and control
of this organism. The use of semi-solid media has facilitated the
isolation and cultivation of the organism, but the fastidious growth
requirements of Z;_fgtus_have hindered the development of an efficient
method of antigen preparation. It is desirable, for antigen.prepar-
ation, that the organism of choice give a good yield of cells per
volume of medium and the antigen be easily'prepared. Semi-solid

media do not lend themselves well to the latter of these criteria.

Preparation of E:.£EEE§ antigen from a semi-solid medium.re-
quires tedious centrifugation and'washing to separate cells from agar.
The amount of work and time required make this method impractical for
antigen.preparation. It is, therefore, advantageous either to use a
medium which contains no agar or one which is sufficiently solid to

permit ready removal of the organisms from its surface.

Since 2: fetus requires an oxygen tension intermediate be-
tween aerobic and anaerobic conditions, a large inoculum, and from
three to four days incubation for perceptible growth, it is necessary
to employ a.medium which will permit maximum growth.under these con.

ditions.

The following experiments were carried out for the purpose of com-

paring solid, semi—solid and broth media for cultivation of'z; fetus for the

-1.

preparation of a satisfactory antigen. Special consideration was
given to the most efficient method of securing a high yield of cells.
An attempt was made to prove the usefulness of a broth as a satisfac-
tory medium for antigen production. The agglutination reaction, with
known negative and positive sera, was used to determine the aggluti-
nating titer of each antigen as influenced by the medium in which it

was cultivated.

HISTORICAL REVIEW

 

The first isolation of Y_._ f_e_t_1_1_s_ was reported by McFadyean and
Stockman (1913). Stockman in a later publication (1919) stated that
the disease was first discovered in sheep, and was commonest in this
species. Theobald Smith (1918) established L f_e_‘_t_u_s_ as the causative
agent in several bovine abortions in which Brucella abortus could not

be found. He named the organismVibrio fetus.

The occurrence of vibrionic abortions in sheep was reported
by Mattick (1925), Welch and Marsh (19214), Graham and Tharp (1930),
Lee and Scrivner (1910) and Ryff (19140). Bovine abortions were re—
ported by Schroeder (1920), Berger (1927), and more recently by
Plastridge and Williams (19143), Rhoades and Hardenbrook (19h?) and
Bell (1950). These authors reported the disease to be widespread,
having been found principally in New York, Illinois, Michigan, Cali—

fornia, Montana, Wyoming, and Connecticut.

-2-

In early cultural studies, Stockman (1919) obtained a good
initial growth in ordinary broth media, but was unable to success-
fully cultivate the organisms by subsequent transfer. However, he
'was able to maintain cultures by adding a small amount of potato to
the broth tubes. Growth was rarely observed on nutrient agar slants,
but if the agar was seeded while it was still liquid and then quickly
solidified, growth occurred just beneath the surface of the agar. He
stated that blood agar was very satisfactory for surface growth on
slants or plates, but for antigen preparation cultures in potato

broth were best.

Smith and Taylor (1919) found that growth was readily db—
tained from sealed nutrient agar slants provided that a few drops of
defibrinated horse blood had been added to the water of condensation.
Growth appeared only on the circumference of the agar near the water
of condensation and gradually spread in a fine white film between the
agar and the tube after 2 to 3 days incubation at 37° 0. Growth did
not occur in nutrient gelatin or beef peptone bouillion, but did occur

in both media when a few drops of defibrinated bloodwere added.

Welch and Marsh (1921;) obtained results similar to those of
Smith and Taylor by using serum agar slants in sealed tubes. They
also used a brain medium in liver broth which had been used in their
laboratory for routine anaerobic cultivation. Good growth was ob-
tained in 21; hours. Rhoades and Hardenbrook (19h?) used blood agar

and tryptose crystal—violet agar plates in an atmosphere of 10 per

cent 002 for initial cultures, but found subcultures to die out after
one or more transfers. Bell (1950) isolated Z;_f§tu§ by inoculating
blood agar plates and semi-solid beef heart infusion broth (0.3 per
cent agar) with the stomach contents of an aborted fetus. Growth on
both media was observed after 3 days incubation at 37° C. in an at-
mosphere of 10 per cent 002. Subcultures were established in the
semi-solid heart infusion agar but not on the blood agar. Lee and
Scrivner (l9hl) successfully used oystine veal-infusion broth in

sealed tubes for cultivation of E; fetus.

Ryff and Lee (1916) in studies on various cultural media,
stated that Difco veal infusion with added salt and agar was superb
ior to nutrient agar, tryptose agar, Difco tryptose glucose agar and
Difco brain veal agar. However, the addition of blood to these media
stimulated growth equal to that in the veal infusion. Their studies
on hydrogen ion concentration with Difco semi—solid veal infusion
agar revealed optimum growth at about pH 7.0 with lesser growth at

both pH 6.0 and pH 8.0.

After several unsuccessful attempts to grow cells for anti-
gen on solid media and in agar-free broth, Plastridge and Williams
(l9h3) found it necessary to use a semi-solid liver-infusion agar
(0.3 per cent agar). The antigen was prepared by differential cenp
trifugation to remove the agar. The cells were washed and then
suspended in a small amount of 0.3 per cent formalinized saline.

For use in the tube agglutination test, the cells were diluted with

formalinized saline to a density of 1.5 on the McFarland nephelometer

scale.

Huddleson (19h8) stated that Difco "Thiol medium" was satis-
factory for the isolation and.maintenance of’E:_£§tu§. Plastridge,
Williams, and Roman (l9h9) reported that isolation and cultivation
of E:_f§tu§ has been facilitated by the use of a semi—solid medium,
but it is difficult and expensive to prepare satisfactory agglutinp
ation antigens from a semi~solid.medium. They suggested the use of
Difco Thiol medium (0.1 per cent agar) with the addition of 2.5 per
cent agar. The antigen was prepared by washing the culture from
the agar surface with a small volume of 0.3 per cent formalinized

saline.

MATERIALS AND METHODS

 

For cultural purposes a strain of E: fgtgg'was obtained from
Dr. W. N. Plastridge, of Storrs Agricultural Experiment Station,
University of Connecticut, Storrs. Stock cultures were maintained
in Thiol medium, with subcultures being made every two or three

weeks.

The following media used for antigen production are listed

beloW:

l.

3.

“Thiol medium", Difco

 

Bacto proteose peptone #3 . . . . . 10 gm.
Bacto yeast extract . . . . . . . . 5 gm.
Bacto dextrose . . . . . . . . . . 1 gm.
Sodium.Chloride . . . . . . . . . . 5 gm.

8 gm.

Thiol complex .
Bacto agar . . . . . . . . . . . . 1 gm.

p-amino benzoic acid . . . . . . 0.05 gm.

Thirty grams of dehydrated.powder was dissolved
in 1000 m1. of distilled water, adjusted to pH
6.8 and tubed in 10 m1. portions. The medium was
sterilized in the autoclave for 20 minutes at

121° C. (15 lb. pressure).

"Thiol Broth", Difco

 

This medium, supplied by Difco for experimental
purposes, contains the same ingredients as "Thiol
medium? except agar. The medium was prepared in a
manner similar to that employed for Thiol.medium
using 29 grams of dehydrated powder for each 1000

ml. of distilled water.

Solid Thiol Agar (used as slants)

 

This medium was recommended by Plastridge for
the preparation of agglutination antigen. The

medium was prepared by dissolving 25 grams of agar

h.

and 0.05 gm. glutathione in 1000 ml. of “Thiol.mediumfi.
After sterilization in the autoclave, the agar was

allowed to harden in a slanting position.

Chicken Infusion Broth

 

The chicken broth was prepared by adding one
liter of distilled water to each two pounds of
lean chicken.meat. It was placed in the steamer
for three to four hours, and the resulting broth
decanted from the cooked meat. The broth was
allowed to cool so the fat present would solidify,
and was filtered through filter paper. This gave
a double strength infusion.broth which was diluted
'with equal parts of distilled water before use.

‘ Ten grams of Bacto peptone was dissolved in the
chicken broth, and the medium adjusted to pH 6.8,
tubed in 10 ml. portions and sterilized in the

autoclave for 20 minutes at 121° 0.

Chicken Infusion semi-solid medium

 

This medium.was prepared from chicken infusion
broth, with the addition of 3 grams of agar for
each 1000 ml. of medium. Plastridge and'Williams
described the successful use of a similar medium
containing beef liver broth instead of chicken

brOth o

6. Chicken Infusion Broth with added_glutathione

 

To 1000 ml. of chicken infusion broth 0.05
gram of glutathione was added before steriliza-
tion. Glutathione, a reducing agent, was added
to remove part of the dissolved oxygen in the
liquid for the purpose of providing a more satis-
factory oulture medium.for'!:.f§tgs. The medium
must be used shortly after sterilization before

the glutathione is oxidized.

In making subcultures of X;_£§tug from stock cultures of
semi-solid.medium.it was necessary to suspend the organisms in broth.
For this purpose brain heart infusion broth, Difco, was prepared.

The medium was tubed in 5 ml. portions and sterilized in the autoclave
for 15 minutes at 121° C. Suspensions of‘z:_f§tg§fwere prepared by
placing h.ml. of brain heart infusion over the agar surface of each
stock culture and the sub—surface growth suspended by agitating the

broth with a pipette.

PROCEDURE FOR THE PREPARATION OF VIBRIO FETUS ANTIGEN

l. "Thiol Medium”

 

Seed cultures were prepared by transferring 0.25 ml. of

brain heart infusion suspension from stock cultures to a

-8-

series of 10 tubes containing 10 ml. each of Thiol medium.

The tubes were incubated aerobically at 37° C. for 3 days,

and sub-cultures from this series were made and incubated in

the same manner. The third transfer was made into 100 tubes

of medium. One seed tube was used for each 10 tubes inocu- 1
lated. After incubating at 37° C. for 3 days, growth was

found within the first 0.5 cm. from the surface of the medium.

The organisms were removed by a'water aspirator into a large

Erlenmeyer flask, and treated with 0.3 per cent formalinized

saline.

The suspension was centrifuged at 1600 r.p.m. for 30 mine
utes to remove the agar by sedimentation and the cell-containing
supernatant decanted. The agar was resuspended in formalinized
saline and again centrifuged. The supernatant fluids were com-
bined and centrifuged at h500 to 5000 r.p.m. for 60 minutes in
an International Equipment Company PR1 refrigerated centrifuge.
The cells were washed with formalinized saline and re-centrifuged.
The sediment of cells was then diluted'with formalinized saline

to 100 ml. and stored at h° c.

2. Thiol Broth

 

The procedure for the preparation of antigen from Thiol
broth was similar to that described under Thiol medium. Pre-
liminary tests showed the minimum inoculum required to sustain
growth to be 0.5 ml. for each transfer. The initial inoculap

tion was with a brainpheart-infusion suspension from stock

kin

 

 

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cultures and subsequent cultures could be made directly from
the broth.medium. Elimination of agar also permitted the
direct removal of cells from the broth by centrifugation. A
black precipitate which was present in the medium after incu-

bation, was removed by filtering the antigen through absorbent

cotton.

Solid Thiol Agar Slants

Seed tubes were prepared by transferring four hemm loop-
fuls of stock culture to solid thiol agar slants and spreading
each loopful over an area of 1-2 sq. cm. After incubating at
37° C. for h days in an atmosphere of 10 per cent 002, the
colonies were spread over a larger area and re-incubated.
Sub—cultures were made from these seed tubes to 100 slants
for antigen production. The cells were washed from the
surface with a small volume of formalinized saline, and the
suspension filtered through absorbent cotton. The antigen was
then washed by centrifugation as described under Thiol medium

and diluted to a.volume of 100 ml.

Chicken Infusion Broth

The method of preparing antigen from this medium was the
same as used for Thiol broth. Preliminary tests showed the
minimum required inoculum to be 0.5 ml. per 10 ml. of broth

when cultured in an atmosphere of 10 per cent C02 at 37° C.

-10-

 

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Cultures could be initiated aerobically with a 1.0 ml. ino-

culum, but died out suddenly in the following transfer.

5. Chicken Infusion Semi-solid Agar Medium
The preparation of antigen from this semi-solid.medium
'was described under Thiol medium, with the exception that
all cultures were incubated in an atmosphere of 10 per cent

C02 at 37° C.

6. Chicken Infusion Broth with added glutathione'

The method of preparation was the same as for chicken
infusion broth. It was found that a heavy black precipitate
formed in the broth tubes similar to that in Thiol broth.
Since this sediment did not form in ordinary chicken infu-
sion broth, it was concluded that its presence was due to
glutathione. The precipitate was easily removed by filterb

ing the cell suspension through absorbent cotton.

Standardization of Antigen

 

The six antigen suspensions from each medium were prepared in
100 tubes containing 10 ml. per tube. As mentioned before, the
volume of cells for each preparation was diluted to 100 ml. This
'was an arbitrary volume, but it did give a basis for determining
the dilution factor required to give a density equal to that of the
standard antigen. This standard antigen had a density corresponding
to 1.5 on the McFarland Nephelometer scale and was supplied by Dr.

'W. N. Plastridge.

All density determinations of the antigen were made with the
Beckman MOdel B Spectrophotometer using a light wave of 650 mu (red)
and a slit opening of 0.82 mm. The instrument was adjusted to 100
per cent transmission of light with formalinized saline as a blank.
After the instrument was adjusted, slight variations in current were
observed. Consequently, it was necessary to reset the blank reading

after determination of the density of each antigen.

Before density readings of the antigens could be taken, all
suspensions had to be shaken vigorously with a few glass beads for
15 minutes to break up clumps of cells. The standard antigen was
found to give 76 per cent transmitted light or an optical density
of 0.118. Each concentrated antigen was diluted to give the same
per cent transmission of light as the standard antigen. Dilutions
were made by adding 0.01 ml. portions of concentrated antigen from
a serological pipette to 1.8 ml. of formalinized saline. The sus-
pension was thoroughly mixed after each addition of antigen and

readings were taken until the desired density was reached.

Agglutination tests

 

For determination of the agglutination titers of each antigen,
h serum.samp1es were supplied by Dr. J. J. Stockton, Department of
Bacteriology and Public Health, Michigan State College. In previous
tests, Sera Nos. 1332 and 1298 were positive while sera Nos. 1250
and 1326 were negative in the tube agglutination test for Z;_fgtu§

infection.

The sera were diluted from 1-50 through 1-3200 by placing
0.08 ml. of serum.in a tube containing h ml. of the respective
standardized antigen and making subsequent serial two—fold dilutions
in tubes containing 2 ml. of the antigen. The control tube contained

2 ml. of antigen.

All tubes were incubated at 37° C. and readings taken at 2h
and NB hours. The readings were the same at both intervals. Com-
plete agglutination was designated as h, no agglutination as 0, and
partial agglutination as 3, 2, or 1, depending on the degree of

agglutination.

RESULTS AND DISCUSSION

 

It was found in the case of Thiol medium and chicken infusion
semi-solid agar that a 0.25 ml. inoculum.of broth suspension was suf-
ficient to sustain growth in 10 ml. of the media. For the three broth
media, however, a minimum inoculum of 0.5 ml. was required. It was
noted that growth in'a semi-solid medium was concentrated near the
surface while in broth the growth was dispersed throughout the liquid.
These observations suggest two factors responsible for growth oij;
fetus in artificial media: '

l. The inoculum should be of sufficient volume
to initiate satisfactory growth in the respec-

tive media.

-13-

2. Conditions for growth in a broth medium are
favorable only at a depth in the liquid where
the oxygen tension is intermediate between

aerobic and anaerobic conditions.

It was observed that colonies on solid Thiol agar slants
would develop only where the inoculum contained large numbers of
organisms. ‘When each loopful of organisms was spread over the
surface, care had to be taken not to streak them over too large

an area.

The black precipitate formed during the incubation of Thiol
broth and glutathione infusion broth cultures was believed to be
due to the presence of sulfur in the Thiol broth and in the gluta—

thione infusion broth.

Table 1 shows the dilution required for each antigen to give
an optical density equal to that of the standard antigen, and the
total volume of standardized antigen obtained from each 100 ml. of
concentrated cell suspension. The standard antigen was found to
give 76 per cent transmitted light or an optical density of 0.118.
'When each of the 100 m1. volumes of concentrated antigen had been
diluted to give the same density as the standard antigen, it was
found that solid Thiol slants and chicken infusion semi-solid medium
gave the largest volumes, 20h0 ml. and 2000 ml. respectively; Thiol

medium yielded 1900 ml. while Thiol broth, chicken infusion broth

.4111...

Standardization of’y: fetus Antigen and a

Table 1

Comparison of the Total Volume from Various Media*

 

 

 

Dilution of Total Volume
Concentrated Antigen (ml.) of
to Equal Density of Standardized
Culture medium. Standard Antigen** Antigen***
Thiol medium. 1.00-18 1900
Thiol Broth 1.35-18 1670
Solid Thiol 0.95-18 ZONO
Slants
Chicken Inf. 0.93-18 lth
Broth
Chicken Inf. 1.15-18 2000
Semi-Solid
Glutathione 1.h2-18 1370
Chicken Inf.
Broth

 

 

 

* Determinations made with Beckman Model B Spectrophotometer
“with'wave length 650 mu and slit opening of 0.82 mm,

-** Standard antigen gave 76 per cent transmitted light or
optical density of 0.118.

*** This volume represents the total volume obtained from each

100 cc. of concentrated antigen suspension.

 

and glutathione chicken infusion broth yielded 1670, 1hh0 and 1370

ml. of antigen respectively.

These results would indicate that solid and semi-solid media
'were superior to broth media in supporting growth of E; fetus, but
the amount of work and time required in preparing antigens from the
semi-solid media must also be considered. 'When a semi-solid medium
was used, two or more additional periods of centrifugation'were
required to separate the agar from the cells. When solid Thiol
slants were used it was found that loop transfers took consider-

ably more time than pipette transfers.

Agglutination Tests

 

Table 2 shows agglutination titers for each antigen with pos-
itive serum No. 1332. Antigen from Thiol broth, chicken infusion
broth, and glutathione chicken infusion broth gave a titer of 1-h00
while antigen from Thiol.medium, chicken infusion semi-solid, and
solid Thiol slants gave titers of 1-200, 1-200 and 1-100 respectively.
Table 3 supports the results of Table 2 by showing that consistently
high titers were obtained with all antigens from broth media while
antigens from agar media gave titers about 1 or 2 two-fold dilutions

IOWBI‘O

Tables h and 5 show agglutination titers with two negative sera
Nos. 1250 and 1326. Antigen from Thiol broth and glutathione chicken

infusion broth gave a non-specific titer of 1-100 while antigen from

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the other media gave a 1-50 nonpspecific titer. The only exception
to this was encountered with the antigen from Thiol medium which did

not give a nonpspecific titer with serum No. 1326.

Indications are that the three broth media were superior to the
agar media with respect to agglutination titers. Even though broth
media were not as suitable as agar’media for producing large quantities
of antigen the time and'work saved by using a broth.medium compensated

for the differences in antigen volume obtained.

-21-

 

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SUMMARY

Six media were studied as to suitability for'!:_fgtg§ antigen
production. The total volume of standardized antigen obtained.per
volume of medium and the agglutination titers of each antigen were
determined. Consideration was given to the time and work required

to prepare each antigen.

Solid Thiol agar slants, Thiol medium and chicken infusion
semi-solid agar were superior to broth media in supporting growth
of E: fetus, but antigen from broth cultures was more easily prepared.
Agglutination titers were higher in the antigens made from.2:_£gtu§

grown in broth media.

It is suggested that E:_fetus antigen for agglutination tests
be prepared from cultures in Thiol broth or chicken infusion broth

according to the method described in this paper.

-22-

BIBLIOGRAPHY

 

Barger, E. H. Report of a case of abortion induced by Vibrio fetus.
J. Am. Vet. Med. Assoc. 1.2.: 1468—1471:, 1927.

 

Bell, W. B. Vibrio fetus infection in Virginia. J. Am. Vet. Med.
Assoc. lg? 35, 1950.

 

Carpenter, C. M. Researches upon a spirillum associated with abor-
tion in ewes. Ann. Report to N. Y. State Vet. College, 1920.

Graham, R. and Thorp, F. Jr. Vibrionic abortions in sheep. J. Am.
Vet. Med. Assoc. 16: 568-573, 1930.

Huddleson, I. Forest. A satisfactory medium for the isolation, cul-
tivation and maintenance of viability of Vibrio fetus (bovine).
J. Bact. _5_6_: 508, 19h8.

 

Lee, A. M. and Scrivner, L. H. Experimental Work upon recent outbreaks
of abortions in ewes. Am. J. Res. _2_: 50-5h, 19hl.

Mattick, A. T. R. Abortion in cattle caused by Vibrio fetus. J. Comp.
Path. and Ther. 38: 221-223, 1925.

 

McFadyean, Sir John and Stockman, Sir 8. Report to the Board of Agri-
culture and Fisheries, Great Britain, 1913. (cited by Stockman,
Sir S. 1919).

Plastridge, W. N. and Williams, L. F. Observations on Vibrio fetus
infection in cattle. J. Am. Vet. Med. Assoc. :_L_g_g_: 89-99, 191.3.

 

Plastridge, W. N., Williams, L. F. and Petrie, P. Vibrionic abortion
in cattle. Am. J. Vet. Res. _8_: 178-183, 191.7.

Plastridge, W. N. and Williams, L. F. Vibrionic abortion in cattle:
second report. Cornell Vet. 3_8_: 165-180, 19h8.

Plastridge, W. N. and Williams, L. F. and Roman, J. An improved method
for the preparation of Vibrio fetus agglutination antigen.
Jo Of BaCto 22: 657-658, 19390

 

Rhoades, H. E. and Hardenbrook, H. Jr. The occurrence of vibrionic
abfirtion in an Illinois dairy herd. Cornell Vet. 31: 8-13
19 7.

Ryff, J. F. Vibrionic abortion in Michigan sheep. J. Am. Vet. Med.
Assoc. 21: 52, 19110.

-23-

Ryff, J. F. and Lee, A. H. Vibrio fetus in sheep. Am. J. Vet. Res.
9: 1149-158, 1915. — —“

Schroeder, E. C. A review of several publications on infectious
abortion disease. J. Am. Vet. Med. Assoc. 19: 270—281, 1920.

Smith, T. Spirilla associated with disease of the fetal membranes in
cattle (infectious abortion). J. Expt. Med. gg: 701-719, 1918.

Smith, T. and Taylor, M. 5. Some morphological and biological charac-
ters of the spirilla.(Vibrio fetus, m. Sp.) associated with
disease of the fetal membranes in cattle. J. Exp. Med. 39:
299-311, 1919.

 

Stockman, Sir S. Vibrionic abortion. J. Am. Vet. Med. Assoc. 8:
149 9’50h, 1919 o

Welch, H. and Marsh, H. ‘Vibrionic abortion in sheep. J. Am. Vet Med.
Assoc. 65: 203-210, 192k.

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