Temporal Cell-mediated Immune Responses of Cattle Following Experimental and Natural Exposure to Living Brucella abortus J. M. B. Kaneene, R. D. Angus, D. W. Johnson, C. C. Muscoplat, R. K. Anderson and D. E. Pietz* ABSTRACT A study on cell-mediated immune responses in cattle with different exposure experiences to Brucella abortus was conducted by an in vitro lymphocyte stimulation assay. The purpose of this study was to determine how soon the cell-mediated immune responses would be detected following experimental exposure to B. abortus and to study the cell-mediated immune trend following experimental and natural exposure of cattle to B. abortus. The first positive cell-mediated immune responses occurred one to two weeks after experimental inoculation with living B. abortus strain 2308. The cell-mediated immune responses in these animals appeared at least one week before the appearance of B. abortus serum agglutinating antibodies. Animals which were naturally infected with B. abortus biotypes 1 and 2 demonstrated positive cell-mediated immune responses throughout the study.
ReSUMe Cette experience visait 'a utiliser une epreuve de stimulation des lymphocytes in vitro, pour etudier l'immunite cellulaire chez des bovins soumis a diverses infections experimentales avec Brucella abortus. On esperait ainsi determiner la rapidite avec laquelle on pouvait deceler les resultats de l'immunite cellulaire, a la suite d'une infection experimentale avec B. abortus, ainsi que le 'Department of Large Animal Clinical Sciences, College
of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 (Kaneene, Johnson and Muscoplat), National Animal Disease Center, Veterinary Services Laboratories, Ames, Iowa 50010 (Angus and Pietz) and Division of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, Minnesota 55455 (Anderson). Submitted July 17, 1978.
132
profil de cette immunite, a la suite d'une infection naturelle ou experimentale des bovins par ce microbe. On obtint les premiers resultats positifs de l'immunite cellulaire, environ une a deux semaines apres l'inoculation experimentale de la souche vivante 2308 de B. abortus, i.e. au moins une semaine avant l'apparition des anticorps agglutinants seriques. Les animaux atteints d'une infection naturelle attribuable aux biotypes 1 et 2 de B. abortus demontrerent une reaction positive d'immunite cellulaire, tout au long de 1'experience.
INTRODUCTION In our earlier studies, we reported that lymphocytes from Brucella abortus infected cattle (2-4) and Brucella suis infected pigs (5) exhibited positive in vitro lymphocyte stimulation responses when cultured with brucella antigens. Those initial studies were primarily directed at determination of a positive response and did not determine the duration of the response nor was it determined how soon after infecting cattle or pigs could the cell-mediated immune (CMI) response be detected by the lymphocyte stimulation test (LST). Thus, there was interest to follow infected animals and study the trend of CMI responses over a period of time. It was also desirable to determine how soon after infection with B. abortus, the CMI response could be detected by the in vitro LST and to study the trend of CMI thereafter. This experiment, therefore, was designed to: i) follow the trends of CMI responses in naturally infected animals, ii) experimentally inoculate cattle with living B. abortus to determine
Can. J. comp. Med.
TABLE I. Experimental Groups of Animals in the Study Arranged According to Type of Exposure to B. dbortus
Animal Group
Animal No.
1ba
Ib
II
26 43a 49 50 51
21 51 57 74
Age When Experimentally Infected With B. abortus
Brucella
Sex
15-16 months "
F M ssF " M " F " M
Not applicable " " "
F F F F
Exposure
Brucella Isolation Results
Inoculation
Neg.
Status Experimental "
Natural Infection " "t "
"
-Pos. " "
Not 891 F Not applicable Nonexposed applicable 892 " F " " 893 " F " " 894 F " " " III F " 895 " 900 M " F 901 902 " F 903 " M 904 F &Calfhood vaccinated bEach animal in this group was inoculated with 2.86 X 106 live B. abortus strain 2308 conjunctively
how soon the CMI responses would be detected and study the trend of CMI responses thereafter.
MATERIALS AND METHODS ANIMAL HISTORY
Dairy cattle were used in this study and information on each animal is given (Table I). Animals in group I were kept at the National Animal Disease Laboratories (NADL), Ames, Iowa, in separate units. Animals no. 10 and 43 were vaccinated with 5 ml of B. abortus strain 19 vaccine subcutaneously (approximately 5-15 x 109 cells/ml) at the age of three months. Animals in group I were purchased from the Tuberculosis School at Ames and had been exposed previously to Mycobacterium five months prior to inoculation of live B. abortus strain 2308. Animals 10, 26 and 43 were each inoculated with 0.1 mg of live M. avium strain D4 intratracheally. Animals 49, 50 and 51
Volume 43- April, 1979
were sensitized to M. bovis by inoculating (intradermally) 0.1 ml of Freund's incomplete adjuvant which had 1 mg/ml of heat killed M. bovis. All the six animals in group I were inoculated with 0.5 ml (2.86 x 106 cells) of live B. abortus strain 2308 conjunctively at the age of 16 months, five months after exposure to Mycobacterium. To prove that the animals in group I had developed delayed hypersensitivity, they were skin tested using M. bovis and M. arium antigens. Secondly, lymphocyte stimulation test was conducted on peripheral blood lymphocytes from these animals using M. bovis and M. avium purified protein derivatives. These animals developed typical delayed hypersensitivity skin reactions and positive lymphocyte stimulation responses. PREPARATION OF LYMPHOCYTE SUSPENSION
Following collection, heparinized blood was subjected to the following procedures: The lymphocytes were purified according
to the Ficoll-diatrizoate (FD) technique previously described (2). Briefly, a FD
133
solution' of specific gravity 1.077 was used. Heparinized blood was mixed with an equal volume of 0.15 M NaCl solution and this mixture was subsequently layered over the FD solution, using a sterile needle and syringe. Approximately 10 ml of diluted blood was layered over 5 to 6 ml of FD in 18 ml tubes fitted with plastic covers. All samples were centrifuged for 30 to 45 minutes with a force of 400 g delivered at the FD-blood interface. The separation procedure was done at ambient room temparture. Lymphocyte populations collected in this manner had greater than 95% pure mononuclear cells and a cell viability of 99% or more, as determined by the trypan blue exclusion method. The lymphocytes were washed twice in Hanks' balanced salt solution (HBSS) and resuspended in RPMI-16402 complete tissue culture medium. Cell counts were obtained, using a hemacytometer and cell concentrations were adjusted to 1.5 x 10 cells/ml. CULTURE MEDIUM, MITOGEN AND ANTIGEN Tissue culture medium was RPMI-16402 supplemented with 25 ml of Hepes buffer, penicillin (100 U/ml), streptomycin (100 ,ug/ml) and bovine fetal serum3 (BFS; 15% v/v). Concanavalin A (Con A)4 at a concentration of 2 ug/ culture was used as a positive control. A B. abortus-soluble antigen (BASA) prepared from cell of B. abortus strain 1119-3 was used at a concentration of 4.4 ,ig of protein/culture. The preparation of this antigen and some of the chemical analyses have been described (2). CELL CULTURES
After cell dilutions were made, suspensions of cells from each animal (200 ,ul) were added (in triplicate) to wells of a microtitration culture plate by using an automatic dispenser. Con A were added to appropriate wells of microtitration culture plates. As negative controls, triplicate wells for each animal were set up with lymphocyte suspension but neither
Con A nor BASA was added. All cultures were incubated for six days at 37°C in an incubator with a 5% C02-humidified air atmosphere. Approximately 16 to 18 hours before termination, 1 ,uCi of methyl-3Hthymidine5 (3HdR, 6.7 Ci/mmole) was added to each well. Cultures were terminated by cooling to 4°C and then the cells were harvested for liquid scintillation counting, as previously described (2). Animals in group II (Table I) were purchased from four different herds. B. abortus had been isolated from each of these herds prior to purchase of these animals. B. abortus was isolated repeatedly from the milk, and from tissues after slaughter, of each of the four animals in group II. Animals in group II were kept in separate isolation units of the College of Veterinary Medicine, University of Minnesota (U of M), while those in group III were kept in a brucellosis-free herd of the Department of Animal Science, U of M. All animals were bled once a week for a period of 18 to 24 weeks. Approximately 30 ml of blood were collected by jugular venipuncture from each animal. Twenty ml of each blood sample were put into sterile tubes containing heparin' (50 units/ml) and serum was recovered from the remaining 10 ml. SEROLOGICAL TESTS
All sera collected were subjected to the following tests: standard plate (SPT) and tube (STT) agglutination tests, buffered brucella antigen test (Card), Rivanol precipitation-plate agglutination test (Riv), 2-mercapto-ethanol tube test (2 ME) and acidified plate antigen test (APA) supplemental tests. These tests were conducted according to United States Department of Agriculture procedures (14). However, only the results of the STT are reported. Brucella abortus ISOLATION ATTEMPTS
At the end of the experiment the animals were slaughtered and the following tissues were collected for isolation of
lPharmacia Fine Chemicals, Piweataway, New Jersey.
2Biolabs, Chicago, Illinois. 3Gibco, Grand Island, New York. 4Miles-Yeda, Rehovot, Israel.
134
5Schwartz/Mann, Orangesburg, New York. 6Upjohn Company, Kalamazoo, Michigan.
Can. J. comp. Med.
Brucella abortus: supramammary, mandibular, retropharyngeal, gastrohepatic, internal and external iliacs and lumbar lymph nodes; spleen, udder tissue, part of the uterus, fetal membranes, testicles, liver, heart and bone marrow. The laboratory procedures used were those recommended by United States Department of Agriculture (15).
t200
No. 10
Stimulation Index * _ Titer
t,oo
t5 0o a
_
'C 0
._
I
tc
S~10
1 Ng3-
1:25
0
RESULTS
_t
in.5 1-25
EXPRESSION OF LYMPHOCYTE STIMULATION RESULTS .
These are expressed in two ways: i)
difference in counts per minute (ACPM) = mean cpm of triplicate cultures without either Con A or BASA subtracted from mean cpm of triplicate cultures with Con A or BASA, ii) stimulation index (SI) = mean cpm of triplicate culture with Con A or BASA divided by mean cpm of triplicate culture without either Con A or BASA.
-
Volume 43- April, 1979
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4 6 8 10 12 14 16 20 22 Weeks from Challenge Fig. 1. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 10, group I, B. abortus strain 19 vaccinated and later experimentally inoculated with living B. abortus strain 2308 - pregnant heifer. A Stimulation Index s 3.0 (-3) was considered a positive response. A titer : 1:200 (4--) in B. abortus strain 19 vaccinated cattle was considered a reactor (16).
40 35
No.26
30 25
LYMPHOCYTE STIMULATION
Either Figs. 1 through 6 or Tables II through IV show the results of lymphocyte stimulation tests for group I animals which were experimentally inoculated with living B. abortus. In our laboratory, a stimulation index equal or greater than 3.0 was used as indicative of positive responses. This criterion was based on accumulated data in the last two years. This criterion should be taken as purely for this laboratory and does not represent an official classification criterion. Using the criterion that a stimulation index equal to or greater than 3.0 is a positive response, it can be observed that BASA first induced responses of 3.0 at postinoculation week 1 in SI lymphocytes from cattle no. 26, 43, 49 and 51 and at postinoculation week 2 in samples from cattle no. 10 and 50. Animals no. 10, 26, 43 and 51 maintained positive lymphocyte stimulation responses (LSR) from the time they first became positive until week 16 when the experiment was terminated. Animal no. 49 had a positive LSR at week 1 through 4 and then 12 through 14 while animal no. 50 had a positive LSR at weeks 2, 3 and 5 only.
.
-2 0 2
A
20 x
I\"
PI
15
II
~10
i
.O II
%..
'
i
I~~~~~~
U)
II n -.
.
V-2
StInuItlon
-
I
~
a'
I' h---s
I
5,
t2s a
us
mat
--- Titer .
.
.
.
.
.
.
.
.
.
.
.
.
......
0 2 4 8 8 10 12 14 1 1 20 22 Weeks trom Challeg Fig. 2. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 26 of group I, young bull experimentally inoculated with living B. abortus strain 2308, no history of strain 19 vaccination. A Stimulation Index -- 3.0 (-e) was considered a positive response. A titer :- 1 :100 (4-) in nonvaccinated cattle was considered a reactor (16).
Either Fig. 7 or Table V shows the results of samples from group II, naturally infected with B. abortus. Brucella abortussoluble antigen induced a positive LSR in samples from all the animals in this group from the first testing through week 22. These animals were purchased from the field as already infected with B. abortus so no preinfection samples could be tested.
135
1:200
No. 50 ---Stimulation Index --- Titer
4.-
0
-1:100
I-c
.° t50
a ._
x
0
0
c
1:25 3
0 L
° 10
a U,
D
Neg 'F noo at 1:25
l~~~~~ /1' OC Weeks from Challenge Fig. 3. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 43, group I, B. abortus strain 19 vaccinated as a calf and later experimentally inoculated with living B. abortus strain 2308 - nonpregnant heifer. A Stimulation Index !. 3.0 (-e) was considered a positive response. A titer =- 1:200 (-v) in B. abortus strain 19 vaccinated cattle was considered a reactor (16).
No. 49 ---
41100
50. No. 51
45
L
40 .
0
35
-
o
30
.
c
25 .
C
1:50 x
2 4 6 8 10 12 14 16 Weeks from Challenge
Fig. 5. Lymphocyte stimulation and tube seroagglutination responses of animal no. 50, group I, experimentally inoculated with living B. abortus strain 2308 - nonpregnant heifer with no history of strain 19 vaccination. A Stimulation Index s 3.0 (-*) was considered a positive response. A titer : 1:200 (+-) in B. abortus strain 19 vaccinated cattle was considered a reactor (16).
11:200
Stimulation Index Titer
-2 0
t200
4.i *t100kX I.c
0
CD
.2
4
150 g
a
1:25 L
10
E
0 n
(.)
r
K.-.
4
t251
in10.
Neg at 1:25
at
°°°A IE°° a~~~~~t
15
..
-
_Neg at
t25
.
_"E~~~~~~~--
-2
0
2
6
4
8
----
10 12 14 16
Weeks from Challenge
-
a
A
Stimulation
positive
response.
Index A
titer
strain 19 vaccinated cattle (16).
Results III, (Fig. 7 or BASA did
group
136
3.0
(-e)
1:200
was
ol.' -2 0 .
Fig. 4. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 49, group I, young bull experimentally inoculated with living B. no history of strain 19 vaccinaabortus strain 2308 tion.
was
(
ReSUMe Cette experience visait 'a utiliser une epreuve de stimulation des lymphocytes in vitro, pour etudier l'immunite cellulaire chez des bovins soumis a diverses infections experimentales avec Brucella abortus. On esperait ainsi determiner la rapidite avec laquelle on pouvait deceler les resultats de l'immunite cellulaire, a la suite d'une infection experimentale avec B. abortus, ainsi que le 'Department of Large Animal Clinical Sciences, College
of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 (Kaneene, Johnson and Muscoplat), National Animal Disease Center, Veterinary Services Laboratories, Ames, Iowa 50010 (Angus and Pietz) and Division of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, Minnesota 55455 (Anderson). Submitted July 17, 1978.
132
profil de cette immunite, a la suite d'une infection naturelle ou experimentale des bovins par ce microbe. On obtint les premiers resultats positifs de l'immunite cellulaire, environ une a deux semaines apres l'inoculation experimentale de la souche vivante 2308 de B. abortus, i.e. au moins une semaine avant l'apparition des anticorps agglutinants seriques. Les animaux atteints d'une infection naturelle attribuable aux biotypes 1 et 2 de B. abortus demontrerent une reaction positive d'immunite cellulaire, tout au long de 1'experience.
INTRODUCTION In our earlier studies, we reported that lymphocytes from Brucella abortus infected cattle (2-4) and Brucella suis infected pigs (5) exhibited positive in vitro lymphocyte stimulation responses when cultured with brucella antigens. Those initial studies were primarily directed at determination of a positive response and did not determine the duration of the response nor was it determined how soon after infecting cattle or pigs could the cell-mediated immune (CMI) response be detected by the lymphocyte stimulation test (LST). Thus, there was interest to follow infected animals and study the trend of CMI responses over a period of time. It was also desirable to determine how soon after infection with B. abortus, the CMI response could be detected by the in vitro LST and to study the trend of CMI thereafter. This experiment, therefore, was designed to: i) follow the trends of CMI responses in naturally infected animals, ii) experimentally inoculate cattle with living B. abortus to determine
Can. J. comp. Med.
TABLE I. Experimental Groups of Animals in the Study Arranged According to Type of Exposure to B. dbortus
Animal Group
Animal No.
1ba
Ib
II
26 43a 49 50 51
21 51 57 74
Age When Experimentally Infected With B. abortus
Brucella
Sex
15-16 months "
F M ssF " M " F " M
Not applicable " " "
F F F F
Exposure
Brucella Isolation Results
Inoculation
Neg.
Status Experimental "
Natural Infection " "t "
"
-Pos. " "
Not 891 F Not applicable Nonexposed applicable 892 " F " " 893 " F " " 894 F " " " III F " 895 " 900 M " F 901 902 " F 903 " M 904 F &Calfhood vaccinated bEach animal in this group was inoculated with 2.86 X 106 live B. abortus strain 2308 conjunctively
how soon the CMI responses would be detected and study the trend of CMI responses thereafter.
MATERIALS AND METHODS ANIMAL HISTORY
Dairy cattle were used in this study and information on each animal is given (Table I). Animals in group I were kept at the National Animal Disease Laboratories (NADL), Ames, Iowa, in separate units. Animals no. 10 and 43 were vaccinated with 5 ml of B. abortus strain 19 vaccine subcutaneously (approximately 5-15 x 109 cells/ml) at the age of three months. Animals in group I were purchased from the Tuberculosis School at Ames and had been exposed previously to Mycobacterium five months prior to inoculation of live B. abortus strain 2308. Animals 10, 26 and 43 were each inoculated with 0.1 mg of live M. avium strain D4 intratracheally. Animals 49, 50 and 51
Volume 43- April, 1979
were sensitized to M. bovis by inoculating (intradermally) 0.1 ml of Freund's incomplete adjuvant which had 1 mg/ml of heat killed M. bovis. All the six animals in group I were inoculated with 0.5 ml (2.86 x 106 cells) of live B. abortus strain 2308 conjunctively at the age of 16 months, five months after exposure to Mycobacterium. To prove that the animals in group I had developed delayed hypersensitivity, they were skin tested using M. bovis and M. arium antigens. Secondly, lymphocyte stimulation test was conducted on peripheral blood lymphocytes from these animals using M. bovis and M. avium purified protein derivatives. These animals developed typical delayed hypersensitivity skin reactions and positive lymphocyte stimulation responses. PREPARATION OF LYMPHOCYTE SUSPENSION
Following collection, heparinized blood was subjected to the following procedures: The lymphocytes were purified according
to the Ficoll-diatrizoate (FD) technique previously described (2). Briefly, a FD
133
solution' of specific gravity 1.077 was used. Heparinized blood was mixed with an equal volume of 0.15 M NaCl solution and this mixture was subsequently layered over the FD solution, using a sterile needle and syringe. Approximately 10 ml of diluted blood was layered over 5 to 6 ml of FD in 18 ml tubes fitted with plastic covers. All samples were centrifuged for 30 to 45 minutes with a force of 400 g delivered at the FD-blood interface. The separation procedure was done at ambient room temparture. Lymphocyte populations collected in this manner had greater than 95% pure mononuclear cells and a cell viability of 99% or more, as determined by the trypan blue exclusion method. The lymphocytes were washed twice in Hanks' balanced salt solution (HBSS) and resuspended in RPMI-16402 complete tissue culture medium. Cell counts were obtained, using a hemacytometer and cell concentrations were adjusted to 1.5 x 10 cells/ml. CULTURE MEDIUM, MITOGEN AND ANTIGEN Tissue culture medium was RPMI-16402 supplemented with 25 ml of Hepes buffer, penicillin (100 U/ml), streptomycin (100 ,ug/ml) and bovine fetal serum3 (BFS; 15% v/v). Concanavalin A (Con A)4 at a concentration of 2 ug/ culture was used as a positive control. A B. abortus-soluble antigen (BASA) prepared from cell of B. abortus strain 1119-3 was used at a concentration of 4.4 ,ig of protein/culture. The preparation of this antigen and some of the chemical analyses have been described (2). CELL CULTURES
After cell dilutions were made, suspensions of cells from each animal (200 ,ul) were added (in triplicate) to wells of a microtitration culture plate by using an automatic dispenser. Con A were added to appropriate wells of microtitration culture plates. As negative controls, triplicate wells for each animal were set up with lymphocyte suspension but neither
Con A nor BASA was added. All cultures were incubated for six days at 37°C in an incubator with a 5% C02-humidified air atmosphere. Approximately 16 to 18 hours before termination, 1 ,uCi of methyl-3Hthymidine5 (3HdR, 6.7 Ci/mmole) was added to each well. Cultures were terminated by cooling to 4°C and then the cells were harvested for liquid scintillation counting, as previously described (2). Animals in group II (Table I) were purchased from four different herds. B. abortus had been isolated from each of these herds prior to purchase of these animals. B. abortus was isolated repeatedly from the milk, and from tissues after slaughter, of each of the four animals in group II. Animals in group II were kept in separate isolation units of the College of Veterinary Medicine, University of Minnesota (U of M), while those in group III were kept in a brucellosis-free herd of the Department of Animal Science, U of M. All animals were bled once a week for a period of 18 to 24 weeks. Approximately 30 ml of blood were collected by jugular venipuncture from each animal. Twenty ml of each blood sample were put into sterile tubes containing heparin' (50 units/ml) and serum was recovered from the remaining 10 ml. SEROLOGICAL TESTS
All sera collected were subjected to the following tests: standard plate (SPT) and tube (STT) agglutination tests, buffered brucella antigen test (Card), Rivanol precipitation-plate agglutination test (Riv), 2-mercapto-ethanol tube test (2 ME) and acidified plate antigen test (APA) supplemental tests. These tests were conducted according to United States Department of Agriculture procedures (14). However, only the results of the STT are reported. Brucella abortus ISOLATION ATTEMPTS
At the end of the experiment the animals were slaughtered and the following tissues were collected for isolation of
lPharmacia Fine Chemicals, Piweataway, New Jersey.
2Biolabs, Chicago, Illinois. 3Gibco, Grand Island, New York. 4Miles-Yeda, Rehovot, Israel.
134
5Schwartz/Mann, Orangesburg, New York. 6Upjohn Company, Kalamazoo, Michigan.
Can. J. comp. Med.
Brucella abortus: supramammary, mandibular, retropharyngeal, gastrohepatic, internal and external iliacs and lumbar lymph nodes; spleen, udder tissue, part of the uterus, fetal membranes, testicles, liver, heart and bone marrow. The laboratory procedures used were those recommended by United States Department of Agriculture (15).
t200
No. 10
Stimulation Index * _ Titer
t,oo
t5 0o a
_
'C 0
._
I
tc
S~10
1 Ng3-
1:25
0
RESULTS
_t
in.5 1-25
EXPRESSION OF LYMPHOCYTE STIMULATION RESULTS .
These are expressed in two ways: i)
difference in counts per minute (ACPM) = mean cpm of triplicate cultures without either Con A or BASA subtracted from mean cpm of triplicate cultures with Con A or BASA, ii) stimulation index (SI) = mean cpm of triplicate culture with Con A or BASA divided by mean cpm of triplicate culture without either Con A or BASA.
-
Volume 43- April, 1979
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4 6 8 10 12 14 16 20 22 Weeks from Challenge Fig. 1. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 10, group I, B. abortus strain 19 vaccinated and later experimentally inoculated with living B. abortus strain 2308 - pregnant heifer. A Stimulation Index s 3.0 (-3) was considered a positive response. A titer : 1:200 (4--) in B. abortus strain 19 vaccinated cattle was considered a reactor (16).
40 35
No.26
30 25
LYMPHOCYTE STIMULATION
Either Figs. 1 through 6 or Tables II through IV show the results of lymphocyte stimulation tests for group I animals which were experimentally inoculated with living B. abortus. In our laboratory, a stimulation index equal or greater than 3.0 was used as indicative of positive responses. This criterion was based on accumulated data in the last two years. This criterion should be taken as purely for this laboratory and does not represent an official classification criterion. Using the criterion that a stimulation index equal to or greater than 3.0 is a positive response, it can be observed that BASA first induced responses of 3.0 at postinoculation week 1 in SI lymphocytes from cattle no. 26, 43, 49 and 51 and at postinoculation week 2 in samples from cattle no. 10 and 50. Animals no. 10, 26, 43 and 51 maintained positive lymphocyte stimulation responses (LSR) from the time they first became positive until week 16 when the experiment was terminated. Animal no. 49 had a positive LSR at week 1 through 4 and then 12 through 14 while animal no. 50 had a positive LSR at weeks 2, 3 and 5 only.
.
-2 0 2
A
20 x
I\"
PI
15
II
~10
i
.O II
%..
'
i
I~~~~~~
U)
II n -.
.
V-2
StInuItlon
-
I
~
a'
I' h---s
I
5,
t2s a
us
mat
--- Titer .
.
.
.
.
.
.
.
.
.
.
.
.
......
0 2 4 8 8 10 12 14 1 1 20 22 Weeks trom Challeg Fig. 2. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 26 of group I, young bull experimentally inoculated with living B. abortus strain 2308, no history of strain 19 vaccination. A Stimulation Index -- 3.0 (-e) was considered a positive response. A titer :- 1 :100 (4-) in nonvaccinated cattle was considered a reactor (16).
Either Fig. 7 or Table V shows the results of samples from group II, naturally infected with B. abortus. Brucella abortussoluble antigen induced a positive LSR in samples from all the animals in this group from the first testing through week 22. These animals were purchased from the field as already infected with B. abortus so no preinfection samples could be tested.
135
1:200
No. 50 ---Stimulation Index --- Titer
4.-
0
-1:100
I-c
.° t50
a ._
x
0
0
c
1:25 3
0 L
° 10
a U,
D
Neg 'F noo at 1:25
l~~~~~ /1' OC Weeks from Challenge Fig. 3. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 43, group I, B. abortus strain 19 vaccinated as a calf and later experimentally inoculated with living B. abortus strain 2308 - nonpregnant heifer. A Stimulation Index !. 3.0 (-e) was considered a positive response. A titer =- 1:200 (-v) in B. abortus strain 19 vaccinated cattle was considered a reactor (16).
No. 49 ---
41100
50. No. 51
45
L
40 .
0
35
-
o
30
.
c
25 .
C
1:50 x
2 4 6 8 10 12 14 16 Weeks from Challenge
Fig. 5. Lymphocyte stimulation and tube seroagglutination responses of animal no. 50, group I, experimentally inoculated with living B. abortus strain 2308 - nonpregnant heifer with no history of strain 19 vaccination. A Stimulation Index s 3.0 (-*) was considered a positive response. A titer : 1:200 (+-) in B. abortus strain 19 vaccinated cattle was considered a reactor (16).
11:200
Stimulation Index Titer
-2 0
t200
4.i *t100kX I.c
0
CD
.2
4
150 g
a
1:25 L
10
E
0 n
(.)
r
K.-.
4
t251
in10.
Neg at 1:25
at
°°°A IE°° a~~~~~t
15
..
-
_Neg at
t25
.
_"E~~~~~~~--
-2
0
2
6
4
8
----
10 12 14 16
Weeks from Challenge
-
a
A
Stimulation
positive
response.
Index A
titer
strain 19 vaccinated cattle (16).
Results III, (Fig. 7 or BASA did
group
136
3.0
(-e)
1:200
was
ol.' -2 0 .
Fig. 4. Lymphocyte stimulation and tube seroagglutination antibody responses of animal no. 49, group I, young bull experimentally inoculated with living B. no history of strain 19 vaccinaabortus strain 2308 tion.
was
(
