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Sgiﬁ'SLOGZCAL 75.133 {53“‘33 CF CATTLﬁ "5‘3

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“‘3' THE
CAM-39L EMENT P'iXATEC-N ’38? AM) EVE-E
AﬁGLUTWA'ﬁ‘iON ‘i’ES‘T

Thus}: for fhu Degrn of M. S.
MICHIGAN 37TAT‘E UNI‘V’ERSETY
Virginia. H. Ame”
E7955

SEROLOGICAL RESPONSES OF CATTLE TO_VIBRIO FETUS VACCINE
AS MEASURED BY THE COMPLEMENT FIXATION TEST

 

AND TUBE AGGLUTINATION TEST

By
Virginia H. Amell

A THESIS

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE
Department of Microbiology and Public Health
1955

(- I— (4’? l 2 ' (I '_')
J
A 1.} .? 1..

 

To
Zane Sullivan Amell

 

S:
Stockt:
pathic
istrat.
to Dr.
to the
the Ex

G
tunity
ment a
schola
from D
H- Cur
Dr, R1

many c

ACKNOWLEDGEMENTS

Sincere appreciation is expressed to Dr. J. J.
Stockton for the suggestion of the problem and em-
pathic guidance in this work, preparation and admin-
istration of vaccine, and collection of serum samples;
to Dr. W. A. Higgins for collection of serum samples;
to the Difco Laboratories Incorporated for furnishing
the Experimental Broth ;! 73 and a‘ 75.

Gratefully, and with much pleasure, this oppor—
tunity is taken to make recognition of the encourage-
ment and advice received throughout the author's
scholastic endeavors at Michigan State University,
from Dr. H. J. Stafseth, Dr. E. D. Devereux, Dr. C.
H. Cunningham, Dr. E. S° Beneke, Dr. J. J. Stockton,
Dr. Richard Schlegel, Mr. I. L. Dahljelm, and the

I“
I

many others.

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . .
REVIEWOFLITERATURE
MATERIALS AND METHODS . . . . . . . . . . . .
Preparation of Stock Antigen . . . . . .
Preparation of Vaccine and Inoculation of
Animals . . . . . . . . . . . . . . .
Procedure for Tube Agglutination Test . .
Procedure for Complement Fixation Test .
RESULTS AND DISCUSSION . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . .
LITERATURE CITED 0 . . . . . . . . . . . . .

Page
. l

19

LIST OF TABLES

TABLE

1.

m N] C3 C11 uh (JD N
o

Inoculation Data . . . . . . .
Tube Agglutination Test . .

Titration of Hemolysin . . .
Titration of Complement . . .
Titration of Antigen . . . . .
The Initial Complement Fixation
The Complement Fixation Test of

Test .
Diluted

Serum

Page
16
18
23
26
28
3O
31

Resultant Titers and Averages of Tube Agglutination

(AGG) and Complement Fixation (CF) Tests .

Figure I. Average Titers . . . .

34

35

INTRODUCTION

The term ”vibriosis" has been used to indicate the
presence of infeCtion with_Vibrio fetus which as yet appears
to occur naturally only in cattle and sheep (Plastridge_gt
.31., 1951). Vibrionic abortion was observed in Great Britain
as early as 1913 by McFadyean and Stockman (1913) and was
reported for the first time in the United States by Smith
(1918). It has subsequently been reported in Denmark,
Sweden, Germany, Japan, Australia, North Africa, Canada and
several areas of the United States (Prier, 1952).

Because the incidence of reported abortions due to in-

 

fection with Brugella‘gp., commonly referred to as brucel-
losis, has been far higher than that of abortions due to
vibriosis, comparatively little interest has been directed
toward the investigation of vibrionic abortion. .As the
diagnosis and prevention of brucellosis improved, more at-
tention was directed to the occurrence and significance of
vibriosis in cattle and sheep. In addition to abortions at-
tributed to vibrionic infections, the failure of cows to
conceive after repeated services during outbreaks of vibri—
osis in the herd has been observed. Therefore, the term
"vibriosis" includes those vibrionic infections causing

sterility as well as abortions.

Accurate figures are not available on the economic
importance of vibrionic abortion and "nonbreeding" but
it has been estimated that infertile cows as a result of the
infection, have cost the American cattle industry approxi—
mately $500,000,000 annually in lost calves and reduced
milk production (Osborne, 1952). In brucella free herds,
although brucellosis and vibriosis may be present simultan-
eously,;x..£g£g§ has caused an estimated annual abortion
rate of 4 to 20 percent and it has been suggested that many
abortions occur so early in gestation that they remain un—
detected (Plastridge, 1949; Plastridge 31.21,, 1951).
Tunnecliffe and Marsh (1954) of the Montana Veterinary Re—
search Laboratory reported that the number of abortions
due to vibriosis in the intermountain states during the 1952
lambing season was Iconservatively estimated to be 100,000.
This loss amounted to approximately $1,500,000 to $2,000,000.
These estimates serve as only a partial indication of the
losses attributable to vibriosis in cattle and sheep.

Attempts have been made to establish diagnostic pro-
cedure and to produce effective therapeutic agents for
vibriosis. However, the difficulties encountered in the
isolation and cultivation of the specific organisms_in
11359 have greatly hampered these efforts. Serological
tests are in use as supplementary aids but the sole method

of positive diagnosis is the isolation and identification

ofll..£glg§.from the fetus and/or the placental membranes
(Prier, 1952).

Antibiotics, streptomycin and penicillin, have been
reported as effective agents in the treatment of cattle
in which vibrionic abortion and nonbreeding have occurred
(Haubrich, 1950; Chambers, 1950; Easterbrooks and Plas-
tridge, 1952). The control of the disease by means of
vaccination has also been attempted but with relatively
little success (Plastridge.g£.§l., 1951; Osborne, 1952}.
However, these attempts do not preclude the possible control
of vibriosis by vaccination. In the development of a vac-
cine for such a purpose, serological tests may serve as
an indication of the immunological responses elicited in
the vaccinated animals by the vaccine.

The purpose of this thesis is to record the serologi-
cal responses, as determined by the complement fixation
test and tube agglutination test, of ten heifers to inocu—

lation with.X. fetus vaccine.

REVIEW OF LITERATURE

Apparently, vibrio-shaped organisms were first reported

found in aborted ovine fetuses by McFadyean and Stockman

£¢

(1913) in Great Britain. Vibrionic bovine abortion was re— ?E
ported by Smith (1918) in the United States and since that E
time, has been reported from many other regions of the f
world.(Prier, 1952). %

However, after the initial discoveries, little atten-
tion was devoted to vibrionic abortions during the following
decade. By virtue of the larger economic losses inflicted
by brucellosis, efforts were largely directed toward the
develOpment of effective controls of this disease. It has
been noted that, while brucellosis remains the most important
single cause of abortions and nonbreeding in the cattle popu-
lation as a whole despite the reduced incidence, the relative
importance of vibriosis tends to increase as the incidence
of brucellosis decreases (Plastridge.§t.§l., 1951).

With the increased recognition of vibrionic abortions,
it was also noted that impaired breeding efficiency by the
interference with conception occurred in herds of cattle
where active outbreaks of vibrionic abortions were in pro-
gress (Plastridge.gt.al., 1947; Stegenga and Terpstra, 1950;
Haubrich, 1950). This relation of breeding difficulties

to vibrionic infection has not been observed to occur in
sheep (Tunnecliffe and Marsh, 1954). It has been observed
that the organism is also one of the causes of retained
placentas in cattle (Moore, 1949, 1950). ward (1948)
reported finding the organism in minor skin infections of
man but its presence as a primary etiological agent of human
disease has not been demonstrated. Vibrionic abortions have
been induced in previously non-infected gravid guinea pigs
by various laboratory methods as well as by contact with
experimentally infected females and by transmission from
infected males during coitus (Ristic.gt‘§l., 1953, 1954).
Gravid guinea pigs are used for testing the pathogenicity

of cultures and as a medium for maintaining pathogenicity
(Osborne, 1952; Tunnecliffe and Marsh, 1954). However,
sheep and cattle appear to be the only natural hosts of

.E. fetus (Prier, 1952).

The source of infection is not definitely known but
cattle may become infected through the introduction of new
members into the herd, by a herd sire acting as a biologi-
cal or mechanical agent of transmission, or by other means
yet to be determined. Pregnant ewes have been experimentally
infected with'E.‘£gtg§ by ingestion and by vaginal, intra-
venous and intraperitoneal roums of inoculations (Tunne-
cliffe and Marsh, 1954). The same workers obtained evidence

which discredits ewes as carriers, or rams as agents of

transmission. It is generally accepted that viable organisms
are eliminated by infected animals before, during and after
abortion. The hay, soil and manure may thus be contaminated
and it has been shown that.E..£g£g§:may remain viable in such
materials for periods up to ten days (Lindenstruth and ward),
1948).

.. “a... 7'“

Following the introduction of infection into a herd of
cattle, all sexually mature females not previously exposed
appear to be equally susceptible and the greatest losses
occur during the first twelve months of the disease (Plas-
tridge.gt.gl., 1951). In older cattle, the disease appears
to be self limiting but reappears when new stock is introduced
into the herd. It has been suggested that a distribution
of positive blood tests among all age groups of a herd indi-
cates a recent infection, and that a low incidence of reactors,
limited primarily to animals 1 to 3 years of age, indicates
that the infection has been present in the herd for a year
or more (Plastridge.gilal., 1951).

Vibriosis in sheep appears to be self limiting also,
and generally appears and disappears within a year. The
relative susceptibility of previously infected ewes is as
yet a controversial question. Sheep which had been previously
infected were challenged by exposure to intravenous inocu-
lations of‘X..£gig§ and it was found that such sheep were as

susceptible as previously uninfected animals (Tunnecliffe

and Marsh, 1954). It was noted by these workers that the
challenge inoculations were so severe that exposure was in
no way comparable to that encountered under natural condi-
tions. Wiggins (1955) attempted to determine the lasting
effects of vibrionic abortion in sheep. No noticeable effect
was detected the year subsequent to the infection. This was
felt to be the result of two probable effects; the sustained
permanent injuries to genital tracts or general health, and
benefit from rest by not nursing lambs the previous year.
The agglutination test serves as an aid in establishing
the presence of infection. However, the transitory nature
of the positive reaction limits the use of this test despite
the improved methods of antigen production (Huddleson, 1949;
Plastridge.gtigl., 1949). McFadyean and Stockman (1913)
reported titers varying from 1:10 to 1:1000 by agglutination
tests on blood obtained from 4 ewes inoculated with infec-
tious material. Since that time the results of agglutination
tests performed by numerous workers, indicate the percentage
of positive reactors and the titers obtained varied from one
group of animals to another, and from animal to animal. A
rapid drop of titer between 14 to 60 days after abortion by
infected animals was noted by Blakemore and Gledhill (1946).
They also noted that titers were higher with homologous
antigens than heterologous antigens. Levi (1950) found that

a far higher titer was obtained with homologous antigen,

and approximately half of the infected ewes which reacted
significantly with homologous antigen, failed to do so with
a heterologous antigen. It was concluded by Marsh and Tunne-
cliffe (1955) that the agglutination test appeared to be of
limited diagnostic value due to the variable amounts of
agglutinins produced by infected animals, the transitory
nature of the titer, and recognition of the occurrence, fre—
quently within the same herd, of several serological types
of X} fetus. The agglutination test does have value as a
diagnostic aid, particularly when the antigen is prepared
from a homologous strain, and Osborne (1952) has suggested
that it be used as a basis for the diagnosis of a herd in-
fection. On the basis of agglutination tests made on cattle
with recent clinical evidence of vibriosis and cattle
slaughtered after failing to conceive,IPlastridge'gi.§l.
(1952a) have recommended agglutination tests of both blood
serum.and vaginal-cervical mucosa in diagnosing infertility.
There is little doubt that animals reacting to the test are
actual carriers and potential sources of infection (Prier,

1952).

Vibrio fetus antigen and specific anti-serum (rabbit-

 

origin) were reported capable of fixing complement (Batlin,
1949) but no published reports are available on the use of
complement fixation tests as a means of diagnosis. ‘Undoubt-

edly, many infected animals remain undetected and will

remain so as long as the isolation and identification of the
organism from aborted fetuses, placental membranes or
vaginal tracts remain the only accurate method of a positive
diagnosis of infection with'I..£g£g§.

The therapeutic value of some antibiotics has been in-

. ‘n.’

vestigated by a limited number of workers. Neomycin, chloro-
mycetin and terramycin have exhibited a marked.in‘gi1§g anti— i
vibrio activity while penicillin and streptomycin under the 5
same conditions produced less effects (Prier, 1952). Easter-
brooks and Plastridge (1951) found that streptomycin yielded
promising results.in;vit£g, and subsequent use of streptomycin
as an intrauterine infusion for cows which had been bred three
or more times without conceiving, indicated the value of such
treatment. This confirmed work conducted by Haubrich (1950)
in which streptomycin intrauterine infusions produced signi-
ficant results in combating sterility associated with vibrio-
sis. Penicillin was used effectively as intrauterine infusions
by Chambers (1950) in five nonbreeding cows. As a control
measure, it has been suggested that artificial insemination
in which antibiotics, streptomycin and/or penicillin, are
added to the semen, be employed routinely (Plastridge.g£.al,
1951; McEntee‘gt‘gl., 1954).

Vaccination is a potential control measure which offers
promise but is as yet little eXplored. There are two reports

available on the use of alX. fetus vaccine as a control

a; .. , all. ;. .,

10

measure against vibriosis. The first investigator employed

a formalinized-killed suspension of 25 fgjgs cells with a
density corresponding to a McFarland nephelometer reading of

5 (Plastridge gt $1., 1951). Serologically negative cows
heifers of breeding age were given three subcutaneous injec—
tions of 5 ml each of the vaccine at five day intervals.

The average number of services required was 2.5 for 63 animals

following the final inoculations; the average number of ser-

H.__.-... --—--l. mu ...—(.AhA—‘rj
I
,

vices required for a control group of 47 animals was 2.3.

Of the 63 inoculated animals, 4 aborted and 2 were sold be-
cause of sterility; of the 47 controls, 3 aborted and 4 were
sold because of sterility. From these observations, the
authors concluded that the bacterin was of no value in the
control of vibriosis since there were no significant differ-
ences in the abortion rates, number of animals sold due to
steriJJtyy and the conception rate between the treated and
untreated groups.

The second investigation, made by Osborne (1952), held
more promise of vaccination as a potential control of vibri-
onix: infections. An avianized vaccine was prepared by
diJthing cells, grown in embryonating eggs, with physiological
saljJue to yield 76 percent transmission on a photoelectric
colorimeter. Nine virgin heifers were selected for vaccina-
All had been found to be free of vibriosis as indicated

tion.
by eight negative agglutination tests over a nine month period,

11

and free of brucellosis as determined by three negative
agglutination tests, prior to the initial inoculation.

Three inoculations were made subcutaneously in the lateral
cervical region at seven—day intervals. The amount of inocu-
lum varied: 1 or 2 ml at the first inoculation, 3 or 5 ml
at the second inoculation, and 5 ml for all animals at the
final inoculation. Ten heifers, also found to be vibriosis

and brucellosis free, served as controls. On the 2lst day-

-_._ .. “...—.-‘n—‘n..- .
..4
o

1

following the final inoculation, the nineteen heifers were

I!"

challenged by intrauterine (or intracervical) infusion.

The challenge dose was 1 ml of a freshly isolated strain of
.E.‘£§13§‘which was standardized to 76 percent transmission on
the photoelectric colorimeter.

After the challenge dose, all heifers were observed.
daily, and bred by artificial insemination on all observed
estrous periods during the following six months. Sixty-six
,percent of the vaccinated heifers conceived by the end of
the six months period as compared with thirty percent of
the unvaccinated group. Agglutination tests indicated that
all vaccinated heifers became reactors by two weeks after
the initial inoculation, and maximum titers were recorded
twentybone days after the final inoculation. It was con-
cluded by Osborne that, although the titer proved highly

transitory and not as high in all instances as should be

12

elicited by a potent vaccine, the increased conception
rate of the vaccinated heifers appeared to justify further

investigation of a vaccine to control vibrionic infections

in cattle and sheep.

n v“! RM'QAvn‘y
‘ 1

13

MATERIALS AND METHODS
Preparation of Stock Antigen

The antigen employed for the inoculation of the test
animals and for use in the serological tests was provided
by Dr. J. J. Stockton of the Department of Microbiology and
Public Health, Michigan State University, East Lansing,
Michigan. The strain of E} fgjgs used was designated as
Strain 1236. It was isolated from a swab from the cervical
mucosa of heifer N00 1236 from the Experimental Dairy Herd
at the Larro Research Farm, Detroit, Michigan, in May, 1950.

Until ready for use in the preparation of antigen, the
culture was lyophilized following the method described by
Stockton and Newman (1950). The strain was grown in thiol
medium (Difco), and the upper layer containing the growth
was removed from five tubes of four-day-old cultures and
suspended in sterile skim milk. After thorough mixing,

0.25 ml of this suspension was dispensed to vials made from
Pyrex tubing (7 mm outside diameter). The vials were placed
in a dry-ice—alcohol bath (-70 C) and after freezing, the
contents were dried for 48 hours as a pressure of approxi-
mately 100l( . After drying, the cultures were sealed in

vacuo and stored at 4 C.

...-e . . ...-....-- -*-_—__-.__
_ J'Il’

14

Seed cultures for the preparation of stock antigen were
made by reconstituting lyophilized specimens in 10 ml quan-
tities of sterile Difco Experimental Broth #73 or #75. These
were incubated 48 hours at approximately 35 C and under approx-
imately 10 percent C02 tension. Organisms were then sub-
cultured in sterile broth. Following incubation, microsc0pic
examinations were made employing a phase contrast microsc0pe.
These specimens were examined for purity and motility. Only
pure, actively motile cultures were used for inoculation of
stock antigen medium.

Twenty ml of seed culture was placed into 1 liter of
sterile Difco Experimental Broth #73 or #75 contained in a
3 liter compressed Erlenmeyer flask. These were incubated
in the same manner as the seed cultures. Following incubation,
the organisms were concentrated by centrifugation employing
an IEC Model PR-l refrigerated centrifuge. Specimens were
subjected to a relative centrifugal force of approximately
3000 x.G for 30 minutes. The sedimented cells were washed
thrice with 0.3 percent formalin neutral 0.85 percent NaCl
solution. The washed cells, which constituted the stock
antigen, were suspended in a suitable volume of 0.3 percent
formalin neutral 0.85 percent NaCl solution and stored at 4 C.

This stock antigen was used for preparation of the vaccine,

and also for the tube agglutination and complement fixation

tests.

15

Preparation of Vaccine and Inoculation of Test Animals

For preparation of the vaccine, cell counts were made
of the stock antigen using a Petroff Hauser Bacterial Counting
Chamber and the phase contrast microscope. As determined from

this count, the suspension was concentrated or diluted to

9

yield a cell count of approximately 50 x 10 cells per ml.

Ten heifers, ranging in age from 8 1/2 to 16 3/4 months,
were selected from the dairy herd at the Larro Research Farm,
Detroit, Michigan. These animals were brucella—negative as
determined by the plate agglutination test. Eight of the
heifers had been vaccinated with Brucella M vaccine. The
vibrio vaccine was administered subcutaneously in either the
right or left cervical region. The vaccine was administered
to the heifers at the intervals and in the quantities indi-
cated in Table 1.

Blood samples were collected from all animals at the
time of the initial inoculation and periodically thereafter
for approximately nine months. The samples were obtained to
determine the serological responses of the vaccinated heifers
by the complement fixation and tube agglutination tests. The
samples were taken on the dates indicated in the tabulated
results of the serological tests (Table 8).

The serum of each blood sample collected was separated

from the clot by centrifugation. The sera were decanted into

INOCULATION DATA

TABLE 1

16

 

 

Date V. fet. Vacc.

 

 

 

 

 

 

 

 

knimal Date of Age at Date

umber Birth lst Inoc. Bruc. M. (amounts in m1)
(mo.) Vacc. Oct. 8 NOv 5 Dec 21
1954 1954 1954
1655 5/15/53 16 3/4 12/29/53 1.0 2.0 2.0
1656 5/21/53 16 1/2 12/29/53 1.0 2.0 2.0
1663 6/26/53 15 1/2 12/29/53 1.0 2.0 2.0
1666 7/22/53 14 1/2 12/29/53- 1.0 2.0 2.0
1663 7/31/53 14 1/4 12/30/53 1.0 2.0 2.0
1682 10/5/53 12 7/16/54 1.0 2.0 2.0
1635 10/15/53 12 1/4 7/16/54’ 1.0 2.0 2.0
1688 11/3/53 11 7/16/54 1.0 2.0 2.0
1702 1/3/54 9 1/4 1.0 2.0 2.0
1707 1/24/54 3 1/2 1.0 2.0 2.0

 

 

 

'1'!

 

'ﬁ --.1 .

17

individual sterile tubes, plugged and centrifuged to insure
complete separation from red blood cells. After the second
centrifugation, the sera were transferred to individual

sterile milk vials, stoppered and stored at -40 C.

Procedure for the Tube Agglutination Test

. .. .l'
_ .3.

7

Density determinations of the antigens used in the

zl— ... ‘ era; but" I.

serological tests were made with a Cenco Industrial Phote-

lometer using a red filter (650 mu). The instrument was

7".\ '-
~..I
—-w.

adjusted to read 100 percent transmission through a tube 3
containing 5 ml of 0.3 percent formalinized saline. Stock
antigen was added in 0.01 ml increments to 5 m1 formalinized
saline in a tube corresponding in light transmission to the
tube used to standardize the instrument. After each addi-
tion, the suspension was thoroughly agitated and the density
determined photometrically. The process was repeated until
the transmission through the saline suspension was reduced
to 76 percent. The dilution of antigen thus determined was
used to test each serum sample as indicated in Table 2. All
quantities are given in ml, and a separate tube of 1 ml of
antigen only served as control.

Serum-antigen mixtures were incubated 48 hours at 37 C and
were read over a concave mirror using artificial, reflected
light. The serum titer was recorded as the highest dilution
of sermm in which there was perceptible agglutination which

persisted during slight manual agitation.

 

TABLE 2

TUBE AGGLUTINATION TEST

18

 

 

 

 

 

 

 

ube 1 2_‘ 3 4 5 6 7 Controﬂ
tigen 2.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Lerum .08 0 0 0 0 0 0 0
Transfer .
to right 1.0 1.0 1.0 1.0 1.0 1.0 1.0 O
inal
ilutiona 1:25 1:50 1:100 1:200 lg400 1:800 1:1600 -

 

 

 

 

 

 

 

 

 

 

*Serial dilutions continued for higher titers.

19

Procedure for the Complement Fixation Test

Preparation of the Diluent

Eighty-five hundredths percent buffered saline solution
was used in all complement fixation tests and for the dilution
and titration of all reagents used in this test. The concen-
tration of the sodium chloride was maintained above 0.80 per-
cent and below 0.90 percent to avoid laking of red blood
cells employed in the indicator system.

A stock solution of buffered saline (pH 7.3) was pre—

pared according to the following formula:

NaCl .. ................... 170.00 gm
m2PO4 coco... oooooooooo 0 2.78m
NaZHPO4 ....... ......... . 11.30 gm
H20 qDSOadO c o c oooooooo 0 1000.00 m1

For use, dilute 1:20 in distilled water (Kent 31 21., 1946).

Preparation of Sheep Red Blood Cell Suspension
Modified Alsever's solution was used to preserve sheep
red blood cells (Kent.gt.al., 1946) to be used in the com—

plement fixation test. This solution contained the following:

Dextrose ............. 2.05 gm
Na citrate ............. 0.80 gm
NaCl ....... ...... 0.42 gm
Citric acid ............ 0.055 gm

H20 q.s.ad. .......... 100 m1

._“s~A&-—‘F ’
‘ .m ~'
I" '
1 L

‘wm1~“'

20

After thorough mixing, the solution was dispensed in
25 ml quantities into 100 ml Florence flasks. Sterilization
was accomplished by autoclaving at 121 C for 10 min. The
solution had a final pH of 6.1 and was stored at 4 C.

Whole blood was drawn aseptically from sheep and added
immediately to an equal volume of modified Alsever's solution.
The contents were briskly rotated 15 minutes to insure thor—
ough mixing and to avoid clotting. The Alsever's—whole-blood
mixture (hereafter refered to as Alsever—WBM) was stored at
4 C and allowed to stand 3 to 4 days to permit the suscepti-
bility of the cells to lysis by complement and hemolysin to
become constant (Kabat and Mayer, 1948). The level oﬁ sus-
ceptibility remained unchanged 8 to 12 weeks.

A two percent suspension of red blood cells was used in
the complement fixation tests and in the titrations of the re-
agents. When the total amount of the two percent suSpension
required for one day's use was estimated, the amount of Alsever—
WBM needed to yield that quantity was calculated to insure
sufficient suspension and avoid waste. An approximate
relation was found between the desired volume of two percent
suspension of red blood cells and the required amount of
Alsever-WBM. If the final volume of the two percent sus—
pension was referred to as x, then x times 0.02 indicated
the amount of packed cells to be diluted 1:50. This amount

of packed cells was present in that quantity of Alsever-WBM

§ 1""

21

found by dividing the amount of packed cells required by
the percentage of red blood cells present in the Alsever-
WBM. Since the whole blood was added to an equal volume
of Alsever's solution, the concentration of the red cells

was reduced approximately from 45 percent to 22.5 percent.

:1 jlir

Therefore, (x) (.02) / (.225) equalled the amount of Alsever— é
WBM required. Since (.02) / (.225) was constant and equalled
0.089, the amount of Alsever-WBM required for a day's use

was calculated simply as 0.089 times the amount of two per-

.r. Ana-urn v ..‘L‘ d. ‘

cent suspension of the red blood cells needed. For example,
if 80 ml of two percent suspension were wanted, 0.089 times
80 ml indicated that 7.12 ml of Alsever-WBM would yield the
needed quantity.

The quantity of Alsever-WBM thus determined was placed
in 15 m1 graduated conical centrifuge tubes. The red blood
cells contained in the centrifuge tubes were washed thrice
by the addition of buffered saline, centrifugation and removal
of the supernatant. Each centrifugation lasted for a period
of ten minutes in an International Clinical centrifuge, Model
CI. In the event the supernatant was red, the cells were
discarded as too fragile.

The packed volume of red blood cells obtained in the
graduated centrifuge tubes was diluted 1:50 with buffered
saline after the removal of the supernatant, to yield a

two percent suspension. This suspension was prepared daily

22

to avoid any increase in the susceptibility of the red cells

to hemolysis.

Titration of Hemolysin

The minimal hemolytic dose, hereafter referred to as

M.H.D., of hemolysin is the smallest quantity of a given T7
dilution of hemolysin which will yield complete hemolysis it:
of a given amount of red blood cells in the presence of an A
excess of complement at a given temperature in a specified 1
length of time. This is also referred to as one unit of a;

hemolysin and is used interchangeably with M.H.D.

To determine the M.H.D. of hemolysin or complement, one
must be titered against the other. The titer of a particular
lot of hemolysin is relatively stable and once titrated, need
not be determined daily. This is the standard against which
the complement, much less stable than hemolysin, can be
titrated daily immediately prior to use.

Commercially prepared hemolysin obtained from Sharpe
and Dohme (Antisheep Hemolysin Glycerinized Rabbit) was used
for the complement fixation tests and titration of the other
reagents. To determine the M.H.D. of hemolysin, the commercial
preparation was diluted 1:100 and titrated against complement
from a pool of fresh guinea pig sera which was diluted 1:10.
The titration was accomplished as shown in Table 3. All quan—

tities are given in m1.

23

TABLE 3
TITRATION OF HEMOLYSIN

 

 

 

Tube 1 2 3 4 5 6 7 8
Saline 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 e-
komp. 0.1 0.1 0.1 0.1 0.1 0.1‘ 0 0.1 E

 

 

 

 

 

 

 

 

Incubated at 37 C for 30 min. in water bath

 

 

lJ-A An .15. 0‘ L a II.

E:mol. .01. .02 .03 .05 .08 0.1 0.1 (3

 

 

 

 

 

 

 

C 2 percent 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

 

Incubated at 37 C for 30 min. in water bath

 

 

 

The complement fixation tests relies on an indicator
system comprised of hemolysin and sheep red blood cells.
Under the proper conditions, if there is no interference
by other factors, complement lyses red blood cells which
have been sensitized by hemolysin. Some specific antigen—
antibody combinations are capable of 'fixing' complement so
the complement is no longer free to lyse the sensitized red
blood cells. Therefore, in the titration of hemolysin
(Table 3), if hemolysis occurs in either tubes 7 or 8,

hemolysin or complement respectively, is capable of lysing

24

the red blood cells. Any tests employing such hemolysin or
complement are of no value.

In reference to Table 3, the smallest amount of hemo-
lysin, contained in the 1:100 dilution, which caused complete
lysis of the RBC, was designated as the M.H.D.

For the titration of other reagents and for complement E
fixation tests, commercially prepared hemolysin was diluted 2
with buffered saline to yield three M.H.D. per 0.1 ml. §

§
Preparation and Titration of Complement E

Complement was obtained by aseptically drawing five to
eight m1 of blood from each of five to eight unanaesthetized
guinea pigs by cardiac puncture. After the blood was drawn,
it was placed in sterile test tubes, the tubes plugged and
the blood allowed to clot in a slanted position. After
clotting, the tubes were placed upright in a rack and allowed
to stand at room temperature for 1 hour. They were then
refrigerated at 8 C for 2 hours. After reaming the clots,
the tubes were centrifuged for 20 minutes. The serum was
decanted into sterile tubes, the tubes plugged and again
centrifuged for 20 minutes. The supernatant of all tubes
in which there was not more than slight hemolysis of the red
cells, was poured into sterile flasks and stoppered. Care

was exercised to avoid the transfer of any remaining cells.

25

The sera were pooled in one flask to assure uniformity of
the hemolytic activity of the complement obtained.

The pooled sera were dispensed into sterile pyrex tubes
in the approximate amounts required for daily use. The tubes
were hermetically sealed with a cross-fire oxygen torch,
exercising caution to avoid any heat deterioration of the

complement. The tubes were rotated in a dry-ice—alcohol bath

_ i _-_ 1-1. ____...._,‘1

and when the contents were frozen, were removed to a C02

chest (—40 C) for storage.

q”*""

The hemolytic activity of the complement remained
relatively stable throughout a three-to-four-week period
when prepared and stored in the above manner. However, it
is perhaps the most critical reagent employed in the com—
plement fixation test and was therefore titrated daily. An
occasional tube of complement was titrated which had under-
gone sufficient deterioration to necessitate its disposal.
This was believed to have resulted from faulty sealing of
the tube and the subsequent deleterious effect of CO2 on the
complement.

The M.H.D. of complement is the smallest quantity of a
given dilution of complement which causes complete hemolysis
of a given amount of red blood cells in the presence of an
excess of hemolysin (3 units) at a specified temperature
in a definite period of time. Immediately prior to titration

of the complement to determine the M.H.D., the complement was

allowed to thaw and the tube was opened.

removed and diluted 1:10.

to avoid any loss of hemolytic activity. The diluted com-
plement was titered as indicated in Table 4.

are given in m1.

TABLE 4

The remainder was refrigerated

TITRATION OF COMPLEMENT

26

One tenth ml was

All quantities

 

 

* ¥
IF:;e

 

 

 

 

 

 

 

 

 

 

1 2 3 4 5 6 7
#aline 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Comp. .01 .03 .05 .07 0.1 0.1 0
Incubated at 37 C for 30 min. in water bath
emol. 0.11P0.l 0.1 0.1 0.1 0 0.1
C 0.5 0.5 0.5 0.5 0.5 0.5 0.5

 

 

 

 

 

 

 

 

 

Incubated at 37 C for 30 min.

in water bath

 

Tubes 6 and 7 (Table 4) should show no hemolysis as

explained previously in describing the titration of hemoly—

sin.

If no hemolysis occurred in the other tubes, or if

hemolysis occurred only in tube 5, the complement, from

which the 0.1 ml was taken for titration, was discarded as

having too little hemolytic activity.

In addition, it was

 

27

found to be advisable to include 2 units (Table 5) of antigen
in the daily titrations of complement. A few lots of antigen
which exhibited no anticomplementary action when titrated
with one pool of complement, were found to be slightly anti-
complementary with another pool of complement.

The M.H.D. of complement was that quantity in the tube
(Table 4) containing the smallest amount of complement in
which there was complete hemolysis of the red blood cells.
The remaining complement was diluted with buffered saline
to yield 2 M.H.D. per 0.1 ml for the titration of antigen

and complement fixation tests.

Titration of Antigen

Stock antigen was diluted with buffered saline to cor-
respond to a light transmission equal to that of tube 3
of a McFarland Nephelometer. The adjustment was made using
a Cenco photelometer with a red filter (650 mu). It should
be noted that formalinized saline which was used as a
diluent for the antigen in the agglutination tests, was
not a satisfactory diluent for the antigen used in the com-
plement fixation tests. The dilution required for the dif—
ferent lots of antigen varied and it was believed that the
formalinized saline was directly anticomplementary or acted
on the antigen in such a manner that the anticomplementary

activity of the antigen was increased.

28

An undiluted, known-positive antiserum of bovine origin
was inactivated by heating in a water bath at 56 C for 30
minutes and used in the titration of the antigen. Only
those lots of antigen which yielded the specified results
in Table 5 were used in complement fixation tests. All quan-

tities are given in ml.

TABLE 5
TITRATION OF ANTIGEN

A

 

 

 

 

 

 

 

 

 

 

The. 1 2 3 4 5 6 7 3
Saline 1.0 21.0 .1.0 ..1.0“.1.0..1.0 1.0 1.0
Antigen .01 ' .03 .05 0.1 0.1 0 0 0
Comp. 0.1 0.1 0.1 0.1 0 0.1 0 0.1
Antiser. .02 .02 .02 0 0 0.1 0.1 0

 

Incubated at 37 C for 30 min. in water bath

 

Hemol. 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

 

 

 

 

 

 

RBC 2 percent 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

 

 

Incubated at 37 C for 30 min. in water bath

 

Required Results* H PH NH H NH H NH H

 

 

 

 

 

 

 

 

 

 

 

* H 4 hemolysis PH - partial hemolysis NH - no hemolysis

29

The minimal fixing dose, or unit, of the antigen was
contained in .05 ml (Table 5). This yielded two units per
0.1 m1 of diluted antigen and this was the amount used in
the complement fixation tests. Tubes 4 through 8 were con-
trols as follows: 4-antigen not anticomplementary; 5-antigen
not hemolytic; 6-antiserum not anticomplementary; 7—antiserum
inactivated; and 8-hemolytic system properly adjusted.

The procedure outlined in Table 5 was repeated using a
known negative serum. The same results were obtained with
the exception of tubes 2 and 3. Complete hemolysis was
necessary in both of these tubes to indicate the inability

of negative serum-antigen mixture to fix complement.

The Complement Fixation Test

To test the individual serum samples obtained from
the inoculated heifers, complement was diluted to yield 2
units per 0.1 ml after daily titration (Table 4), hemolysin
was diluted to yield 3 units per 0.1 ml (Table 3), stock
antigen was diluted to yield 2 units per 0.1 ml (Table 5),
and a 2 percent suspension of red blood cells was prepared.
It is to be noted that all reagents were refrigerated when
not in use and all glassware used was meticulously clean.
Serum samples were allowed to thaw, inactivated at 56 C for
30 minutes in a water bath and tested as shown in Table 6.

All quantities given are in ml.

TABLE 6

THE INITIAL COMPLEMENT FIXATION TEST

30

 

 

 

Tube 1 2 3 4 5 6

._

Saline 1.0 1.0 1.0 1.0 .1.0 1.0
Antigen 0.1 0.1 0.1 0.1 o 0

Wcomp. 0.1 0.1 0.1 0.1 0.1 0

Serum .01 .02{ .04 o 0.1 0.1

 

 

 

 

 

 

 

Incubated at 37 C for 30 min. in water bath

 

 

Emu. 0.1 0.1 0.1 0.1 0.1 0.1
D 0.5 0.5 0.5 0.5 0.5 0.5

Incubated at 37 C for 30 min. in water bath

 

 

 

 

 

 

 

 

 

Tubes 4, 5, and 6 served as controls: 4-antigen not
anticomplementary; 5-serum not anticomplementary; 6-serum
inactivated.

The initial serum samples, and subsequent samples, were
tested as shown in Table 6, until complement fixation was
obtained. As the response of the heifers developed, as
indicated by a positive fixation test, i.e., a lack of
hemolysis in tubes 2 and 3 (Table 6), it was necessary to

make serial dilutions with buffered saline of the serum

 

31

samples. Serial two-fold dilutions were made from a dilution
of 1:5 through a dilution of 1:1280. Thereafter, the follow-
ing dilutions were made: 1:1920, 1:2560, 1:3200, 1:3840,
1:4480, 1:5120, 1:5760, and 1:6400. The range of dilution
was anticipated on the basis of the reSults obtained from

the previous samples and were tested as shown in Table 7,

if, for example, a previous titer had been 1:40. Quantities

are given in m1.

TABLE 7
THE COMPLEMENT FIXATION TEST OF DILUTED SERUM

 

 

 

 

 

 

 

 

 

 

Tube 1 2 3 4 5 6 7
Saline 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Antigen 0.1 0.1 0.1 0.1 0.1 0 0
temp. 0.1 0.1 0.1 0.1 0.1 0.1 0
523;"‘3111; 1:20 1:40 1:80 1:160 1:32) 1:1 1:1

 

 

 

 

 

 

 

 

 

Incubated at 37 C for 30 min. in water bath
Hemo. 0.1 0.1 0.1 0.1 0.1 0.1 0.1
C 0.5 0.5 0.5 0.5 0.5 0.5 0.5

 

 

Incubated at 37 C for 30

min. in water bath

 

 

IRA.“- In: F!

“-15 A

32

The highest dilution of the serum in which there was
complete fixation of the complement employing 0.02 ml of
that dilution, was recorded as the titer of the serum.
If the serum proved to be improperly inactivated as indicated
by hemolysis in tube 7, or if the serum appeared anti-
complementary, as indicated by no hemolysis in tube 6, the

results were not recorded.

1"
r

a. -..:nuﬂ-‘4—1h‘ -..-l. t M‘s-.110

T.

33

RESULTS AND DISCUSSION

The complement fixation and tube agglutination titers
obtained from the test animals are recorded in Table 8.
The complement fixation (hereafter referred to as CF) 5%
titer was the highest dilution of serum which completely t
fixed complement (Tables 6 and 7). The tube agglutination

. .-' 'rT"‘-T\—'- -..- .. ~

titer was the highest dilution of serum which produced a

1- :- . ‘1‘
“VJ—

perceptible agglutination (Table 2).

Average CF and agglutination titers were determined
for all serum samples obtained on the same day. ,These
averages are recorded in Table 8, and are graphically illus-
trated in Figure I.

In general, both tests indicated a definite response
by all test animals following the initial injection of
.X..£gtg§'vaccine, the highest individual agglutination
titer being 1:800, and the highest individual CF titer
being 1:640 on thesame date. While the CF titers con-
tinued to increase slightly, the agglutination titers ex-
hibited a gradual decline until the time of the second
inoculation. Following the second inoculation, administered
four weeks after the initial injection, a rapid increase
of titer was found by both tests at the end of the subse~

quent week; the highest individual agglutination titer was

TABLE 8

34

RESULTANT TITERS AND AVERAGES OF TUBE AGGLUTINATION

<£AGG)(ANDJCOMPLEMENT FIXATION (CF) TESTS

 

 

 

 

Eats Test Number of Heifer . Ave —
1655 1656 1663 1666 1668 1682 1685 1688 1702 1707 age
10/8 .AGG' 0 0 0 0 200 100 0 25 0 0 32.
54 CF 0 0 0 0 0 0 0 0 0 0 0
10/1st AGG 400 200 400 200 800 400 800 200 400 400 42
54 CF 10 5 5 1 10 10 10 1 20 40 11.
10/22 .AGG 800 800 800 800 400 800 800 200 400 400 62.
54 ‘. CF 160 40 80 160 80 160 160 80 320 640 18:
10/29. .AGG 400 400 400 800 400 800 400 200 400 200 44.
L54 CF 320 80 160 320 160 640 320 160 320 1280 37.
11/5 AGG 200 400 200 400 400 400 200 200 400 200 30.
64 CF 320 160 640 1280 320 1280 640 160 320 1280 _640
11/12 AGG 3200 1600 800 1600 800 800 800 800 1600 3200 152.
54 CF 1920 1280 1920 2560 1280 3200 1920 1920 2560 3840 224.
11/19 AGG' 3200 800 1600 800 1600 1600 800 .400 800 800 124.
54 CF 2560 1280 1280 1920 1280 3200 1920 1280 2560 3840 211.
11/26. AGG 1600 " 400 800 800 400 200 4 400 800 67~
54 CF 2560 1280 1920 640 2560 1920 2560 3200 208.
12/31 AGG 800 400 400 400 200 400 400 400 200 800 44.
54 CF 2560 640 1280 1280 640 2560 1920 640 1920 3200 1664
12/101 AGG 800 200 400 400 200 800 200 200 50 400 36-
54 CF 2560 640 1280 640 640 2560 1280 640 1920 2560 147.
12/21 AGG 400 400 400 400 400 800 400 400 100 400 41.
54 CF 3200 640 640 1280 640 1920 1280 640 1920 2560 147.
12/31 AGG ~3200 3200 1600 3200 1600 800 800 800 3200 6400 243.
54 CF 5120 3840 3200 3840 3840 5760 3200 2560 5120 3840 4014
1/14 AGG 200 800 400 1600 1600 400 400 800 1600 1600 94.
55 CF 3200 1920 1920 2560 1280 3840 1280 640 3200 1280 211/
d/28 AGG 200 400 400 1600 800 200 400 1600 1600 800 32.
55 CF 640 160 320 640 160 1280 160 80 1280 320 50.
2/10 AGG 100 400 400 400 200 200 200 400 400 800 35.
55 CF 160 80 80 160 80‘ 320 40 40 160 80 12.
2/28 AGG 200 200 200 200 400 100 100 200 400 400 24.
55 CF 80 20 4o 80 40 80 20 40 40 20 4.
3/11 AGG 100 M 200 100 200 100 100 200 200 100 144
55 CF 40 .10 20 10 20 5 10 20 5 1
3/25 AGG 100 200 200 200 100 200 200 200 200 17
55 CF 10 5 1 1 5 1 1 1 5 3
4/22 AGG 100 200 9 200 100 100 100 100 200 12
55 ‘ CF 5 1 4 4* 1 1 43 1 5 .
6/24 AGG 25 M 50 100 25 100 50 50 3 7
55 CF + ‘ + + + + + + + +

 

 

 

 

 

*Test not performed

**Sample anti-complementary
+Comp1ement fixed only with undiluted serum

35

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36

1:3200, the highest individual CF titer was 1:3840. During
the next five weeks, a slight decline in CF titers occurred
and the agglutination titers fell to approximately the

same level as that found prior to the second inoculation.

The third and final inoculation, given six weeks

after the second injection, was again followed by a rapid
increase in titers; the highest individual agglutination !
titer was 1:6400, the highest individual CF titer was 3
1:5760. This peak was reached ten days after the final ino-

Ti

culation after which the titers again decreased. It should
be noted that while a peak occurred seven days after the
second inoculation and the highest ten days after the third
inoculation, the difference in days was a result of col-
lection dates of samples rather than a difference in days
established by titers. Had titers been determined at the
same interval after the second and third inoculation, the
height of the peaks or the relation of the two peaks to
each other would have not, in all probability, differed
significantly.

Initially, CF antibodies developed more slowly than
agglutinins but higher average peaks were reached by CF
antibodies than by agglutinins after the second and third
inoculations. The highest average CF titer, observed ten
days after the third inoculation, was 1:4012; the highest

average agglutination titer, also observed ten days after

37

the third inoculation, was 1:2480. The individual titers
at that time varied from 1:2560 to 1:5760 by the CF test,
and from 1:800 to 116400 by the agglutination test.

The transitory nature of titers following the adminis-
tration of a vaccine of‘l..fgtg§ has been noted by Osborne
(1952) as well as natural infections (Plastridge.gt.§;.,
1951; Blakemore and Gledhill, 1946). Approximately 17
weeks after the third inoculation and approximately seven
months after the initial injection, the average agglutination:
titer had decreased to 1:120 and complement was fixed by a
serum dilution of 1:2. Approximately nine months after the
initial inoculation and 26 weeks after the final, the
average agglutination titer was 1:71 and complement was
fixed only with undiluted serum.

Three heifers, 1668, 1682, and 1688, yielded serum
agglutination titers of 1:200, 1:100 and 1:25 respectively
immediately prior to the original inoculatibn. These ani-
mals did not differ significantly in their responses to
E} ffgyg vaccine although following the second and third
inoculations when the highest responses were obtained from
all test animals, the agglutination titers of the three ani—
mals did not reach the average titers on these peakdates.
The presence of agglutinins at the time of the initial
inoculation might be attributed to previous natural ex—

perience witth. fetus. There have been reports of cross

38

agglutination reactions with some strains of‘!.'fg£g§ and
Brucella abortus anti serum of rabbit origin (Kiggins gt
31,, 1955). Agglutinin-adsorption testing indicated the
antigens to be related but not identical (Kiggins §t_al.,
1955). However, all test animals had been found to be
brucella negative to the plate agglutination tests prior :
to inoculations with‘E,.£gtg§ vaccine. In addition, a
complement fixation tests in which‘g. abortus antigen was .
substituted for 1. m antigen (Table 6 and/or 7) at the
time of the initial inoculations and four weeks thereafter, 5
indicated no fixation.
No relation between the observations made following
the.E..£gtg§ vaccine as outlined in this thesis and that
of the bacterin administered by Plastridge $3.31.,(1951)
can be made since no serological testing was reported by
the latter. Osborne (1952) however, recorded serum agglu-
tination titers obtained from nine heifers following the
administration of an avianized‘z..£gtg§ vaccine. As pre—
viously mentioned, three inoculations were made which
varied from 2 to 5 ml per inoculation at seven day inter-
vals. Maximum titers of 1:640 for two heifers were re-
corded on the twenty-first day after the final inoculation,
incomplete agglutination at 1:640 for five heifers, and no

agglutination for two heifers at 1:640.

39

On the basis of the titers obtained, the formalinized
vaccine as administered (Table 1) appears to have elicited
more agglutinins than the avianized vaccine. Osborne did
'not report titers at intervals prior to twenty one days
after the final inoculation at which time maximum titers
were obtained. Had tests been performed seven or ten days
following the final inoculation, a higher production of
agglutinins might have been indicated. However, twenty
four days after the final inoculation of the formalinized
vaccine, the average titer was 1:940, 1:200 for one heifer,
1:400 for three heifers, 1:800 for two heifers and 1:1600
for four heifers. These would seem to indicate a greater
response by the test animals to the formalinized vaccine as
administered and as the responses were tested.

The significant reaction of all test animals by the

development of agglutinins and complement fixing antibodies

might indicate the value of further investigation of seros ~

‘logical responses of animals. ’It is recognized that

results of serological tests of vaccinated animals do not
necessarily measure the degree of immunity produced by a
vaccine. However, a relation may be established and the
serological responses elicited by the vaccine implies the
need of further investigation of vaccination as a future

control of vibriosis.

34d“?!

2 -' ”....

4'.

40

SUMMARY

Ten heifers were selected at the Larro Research Farm,
Detroit, Michigan, to receive a Vibrio fetus vaccine. The
test animals, ranging in age from eight and one-half to
sixteen and three—fourths months, were negative to the
brucella plate agglutination test. .

The formalinized suspension of‘!,.£§£g§ (50 x 109 ,{:L
cells per ml) was administered subcutaneously into the-
right or left cervical region of the test animals. Three
injections were made, the primary injection of 1.0 ml was
followed by two injections of 2.0 ml each at four and ten
'week intervals.

Blood samples were collected from the test animals
periodically for approximately nine months after the ini—
tial inoculation. Serum titers for antibodies vs.;!.
'fgtgg were determined by means of complement fixation and
tube agglutination tests. A significant acceleration of
antibody production was indicated by a rapid rise in serum
titers following vaccine inoculations.’

The serological responses of the test animals to the

vaccine suggest the merit of attempting to determine any

possible standardization of serological reSponses as an

41

aid, perhaps, in detecting the presence of infection, and
that further investigation of vaccination as a potential

measure against vibriosis may be highly worthwhile.

42

LITERATURE CITED

Batlin, Alex. 1949. Serological Relationship of Vibrio
fetus. Master of Science Thesis, U. of Wis.

Blakemore, F., and Gledhill, A. W. 1946. Studies on
Vibrionic Abortion of Sheep. I. The Agglutination
Test as a Means of Diagnosis. J. Comp. Path. and f
Therap.,_§§, 69-74. ‘ 3

Chambers, E. E. 1948. Penicillin in the Treatment of 2
Nonbreeding Cows. N. Am. Vet., _2__9_, 640-641. ’1.

Easterbrooks, H. L. and Plastridge, W. N. 1950. Intra- i
uterine Antibiotic Treatment of Vibrionic Sterility 5*
in Dairy Cows. J;A.V.M.AI, 117, 388.

Easterbrooks, H. L. and Plastridge, W. N. 1952. Experi-
mental Transmission of Vibrio fetus in Diluted Semen
and by Contact. J.A.V.MJA., 120, 199-201.

Haubrich, W. H. 1950. Vibrionic Abortion in Cattle. Vet.
Med.,.4§, 389—391.

Huddleson, I. F. 1949. A Satisfactory Medium for the
Isolation, Cultivation and Maintenance of Viability
of Vibrio fetus (bovine)., J. Bact., 56, 508.

Kabat, J. F. and Mayer, M. M. 1948. Experimental Immuno-
chemistry. Charles C. Thomas Publisher, Springfield,
Illinois, lst edition. 107.

Kent, J. F., Bukantz, S. C. and Rein, C. R. 1946. Studies
in Complement Fixation. J. Imm.,.§§, 37-49.

Kiggins, E. M., Plastridge, W. N. and Easterbrooks, H. L.
1955. Cross Agglutination Between Brucella abortus
and Vibrio fetus. Am. J. Vet. Res.,‘l§, 291-294.

Levi, M. L. 1950. The Agglutination Test in Vibrionic
Abortion of Sheep. J. Comp. Path. and Therap.,
.69, 65—70. ’

43

Lindenstruth, R. W., and Ward, B. Q. 1948. Viability of
Vibrio fetus in Hay, Soil and Manure. J.A.V.M.A., 113,
63.

Marsh, H., and Tunnecliffe, E. A. 1955. The Diagnostic
Significance of the Agglutination Reaction for Vibriosis
in Sheep. J.A.V.M.A., 126, 100-103.

McEntee, K., Hughes, D. E., and Gilman, H. L. 1954. Pre--‘
vention of Vibriosis in Inseminated Heifers by Treating
the Semen from Vibrio-infected Bulls with Penicillin, 1
Streptomycin and Sulfanilamide. Cornell Vet., Vol.
XLIV, #3, 395-462.

- 96” «(Q-s ‘n--_. -..

McFadyean, J., and Stockman, S. 1913. Part III. Abortion
in Sheep. Rept. Dept. Comm., Apptd.by the Bd. of Agr.
and Fish. to Inquire into Epizootic Abortion. London,
1909, London,Wyman and Sons Ltd. (cited from Tunne- '
cliffe and Marsh, 1954). g

Moore, G. R. 1949. Retained Afterbirth. N. Am. Vet.,
ﬂ, 365-370, 389.

Moore, G. R. 1950. Clinical Aspects of Vibrio fetus
Infection in Cattle. J.A.V.M.A., 102, 89-95}

Osborne, J. C. 1952. Avianized Vibrio fetus Vaccine and
Some Preliminary Observations of its USe. Pro. Bk.
Am. V. M.A., 89th Ann. Mtg., June 23-26, 112-115.

Plastridge, W. N. 1949. Abst. of Paper Presented at NE
States Conf. on Brucellosis in Domestic Animals, N. Y.
N. Y., Jan. 24-25 (cited from Prier, 1952).

Plastridge, W. N., Easterbrooks, H. L. 1952a. Diagnosis,
Diagnostic Tests and Their Reliability. Pro. Bk. A.V.
M.A., 89th Ann. Mtg., 331-334.

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Plastridge, W. N., Williams, L. F., Chernak, C. and Easter-
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44

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Vibrionic Abortion in Cattle. Am. J. Vet. Res.,.g,
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|

Smith, Theo. 1918. Spirilla Associated with Disease of

 

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J. Exptl. Med., 28, 701-719. (cited from Prier, 1952). 23

Ristic, M., Morse, E. V., Wipf, L., and.McNutt, S. H.
1954. Transmission of Experimental Vibrio fetus
Infection Among Guinea Pigs and HamSters. Am. J.
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Vibrio fetus by Lyophilization. Cornell Vet., XL, N00

2 - °

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'Ward, B. Q. 1948. The Apparent Involvement of Vibrio
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v01. XVI, ﬁg, 9 214-216.

SEROLOGICAL RESPONSES OF CATTLE TO VIBRIO FETUS VACCINE
AS MEASURED BY THE COMPLEMENT FIXATION TEST
AND TUBE AGGLUTINATION TEST

By
Virginia H. Amell

AN ABSTRACT

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

955
Approved by / . /. W
- /, ,

Virginia H. Amell
1

Infection with Vibrio fetus, clinically manifested in
sexually mature female cattle as infertility or abortion of
fetuses, has been estimated to have cost the American cattle
industry approximately 500 billion dollars annually. Ovine

vibrionic abortion, during one lambing season in the mid-

I'N": .‘7

western intermountain states, inflicted a loss of approximately'
two billion dollars in lost lambs. The greater incidence of
brucellosis, now declining as a result of reliable diagnostic .
procedures and effective immunization, has apparently delayed.
extensive investigation necessary to determine adequate means
for the prevention of vibriosis and a rapid and accurate di-
agnosis of the infection. Determining the presence of the
infection by means of serological tests has thus far been
delayed or hampered by the difficulty encountered originally
in cultivation, the existence of heterologous strains of the
organism, and lack of standardization of antigen production.
.As yet, the only reliable diagnosis is the isolation of V.
iEEIEE from aborted fetuses, placental membranes and/or
vaginal mucosa.

To obtain an indication of the possible value of a
Vnaccine, as determined by serological resoonses, complement
fVixation and tube agglutination,tests were performed on the
srxra of ten heifers inoculated with X, fetus vaccine. Three

iiioculations per animal were made; 1 ml was given in the

Virginia H. Amell
2

primary injection, and 2 ml in each of two subsequent in-
jections at four and ten week intervals.

' The stock antigen, from which the vaccine was prepared,
was produced from cultures maintained in lyophile. The organ-
isms had originally been iSolated from the cervical mucosa of
a Vibrio-infected animal. The lyophilized cultures were re-
constituted and grown in Difco Experimental Broth #73 and/or
#75 under approximately 10 percent CO2 tension. Subcultures
of actively motile organisms were harvested and suspended in
formalinized saline. This suspension constituted the stock
antigen from which the vaccine and the antigen for complement

fixation and agglutination tests were prepared. The vaccine

9

was standardized to contain approximately 50 x 10 cells per

m1o The titer of the serum obtained from inoculated test
animals, as determined by the tube agglutination test, was
the highest dilution with which there was perceptible agglu-
tination after 48 hour incubation. The titer determined by
complement fixation, was the highest dilution of serum with
hﬂiich .02 ml of the serum dilution completely fixed 2 units
of complement .

Significant responses elicited from the test animals
'bjr the X. fgtus vaccine were clearly indicated by titers
otrtained by both tests employed. It was not assumed that

truese responses measured the degree of immunity conferred by

Virginia H. Amell
3

the vaccine. From the observed production of agglutinins and
complement fixing antibodies, it seems that significant serolog-
ical responses might be expected from the administration

of a suitable vaccine. The protection, if any, afforded by 3

this serological response, has yet to be evaluated.

10:. use 0117

 

 

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