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Int. Archs Allergy appl. Immun. 52: 3-14 (1976)
Immune Mechanisms Affecting Bovine Leucocytes during
Suppression of Tuberculin Skin Sensitivity in Cattle
L. A. Corner, P. M. Outteridge, Catherine W. Pearson and A. W. D. Lepper
CSIRO, Division of Animal Health, Animal Health Research Laboratory, Parkville, Vic.
Abstract. Complete desensitization to tuberculin skin-testing (anergy) was produced in
cattle by repeated intravenous injections of living BCG organisms into animals sensitized
by a prior subcutaneous dose of BCG. Two levels of desensitization were produced; com­
plete desensitization following 10 i. v. doses and partial desensitization following 5 i. v. doses
of 100 mg BCG. Intradermal tuberculin testing at the end of the experiment stimulated the
appearance of reactive blood lymphocytes in sensitized cattle as measured by in vitro 3Hthymidine uptake. The cattle which were partially desensitized showed this response but
the completely desensitized cattle did not. Reactivity of blood lymphocytes in vitro to PHA
and Brucella abortus antigen was not depressed in the anergic cattle. Serum antibody titres
to BCG polysaccharide, PPD, or whole BCG organisms showed remarkably little change
during the i. v. desensitizing injections of BCG. Differential blood leucocyte counts also
remained within normal limits during this period. The production of MIF by blood lympho­
cytes from the anergic cattle appeared to be unimpaired. Using 3H-thymidine uptake by
lymphocytes from sensitized cattle, it was found that serum from desensitized cattle did not
inhibit lymphocyte stimulation with tuberculin. It was concluded that the lack of tuberculinsensitized lymphocytes in the blood of anergic cattle may have been due to their removal
from the recirculating pool and their continued suppression in lymphoid tissue.
It has been found that some cattle that
are negative to the tuberculin skin test have
advanced lesions of tuberculosis. This loss
of skin responsiveness or ‘anergy’ is also a
well-recognized phenomenon in human
infections with Mycobacterium tuberculosis
Received: February 3, 1976.
and M. leprae [Citron and Scadding, 1957;
Bullock, 1968]. However, it is not yet clear
what causes anergy and whether it is due to
the absence of antigen-sensitive lympho­
cytes, to the presence of immunosuppressive
serum factors, or to both these influences.
In many of the experimental systems
used by other workers, a single intravenous
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injection of the sensitizing antigen has pro­
duced unresponsiveness to skin-testing with
the antigen. Schlossman et al. [1971], re­
ported that this treatment in guinea pigs was
followed by unresponsiveness to antigen in
peritoneal exudate cells but not lymph-node
cells. This was interpreted to mean that an­
tigen-reactive peripheral lymphocytes in in­
flammatory exudates or skin were depleted
but those in the lymph nodes were not af­
fected. On the other hand, Dwyer and Kantor [1973] reported that transfer of delayed
hypersensitivity by peritoneal exudate cells
from sensitized donor guinea pigs into de­
sensitized recipient guinea pigs was less suc­
cessful than a similar transfer into normal
recipients. They concluded that a humoral
suppressor was present in the desensitized
recipient guinea pig.
In cattle, there has been little work car­
ried out to clarify the immunological basis
for anergy to tuberculin testing. We have
previously reported that more than one
injection of tuberculin into the skin of tub­
erculin-sensitive cattle will suppress the ex­
pected delayed-type hypersensitivity re­
sponse at each skin-test site [Outteridge and
Lepper, 1973a]. Furthermore, the reactivity
of peripheral blood lymphocytes to tubercu­
lin in tissue culture is depressed 3 days after
intradermal injection of tuberculin in sensi­
tized cattle [Outteridge and Lepper, 1973b].
However, these phenomena are temporary
and may differ from the prolonged desensi­
tization found in anergic cattle with tuber­
In an attempt to produce anergy which
could be compared with that found in tuber­
culous cattle, a long-term experiment was
set up in BCG-sensitized cattle. These sensi­
tized animals were given repeated intrave­
nous injections of living Mycobacterium
bovis var. BCG organisms for a period of
18 weeks. Levels of sensitization were test­
ed before, during and after this period by
tuberculin skin testing and in vitro tritiated
[3H]-thymidine uptake by tuberculin-sti­
mulated blood lymphocytes. Leucocyte mi­
gration inhibition factor (MIF) was also
measured in supernates of tuberculin-sti­
mulated blood lymphocytes. The serum an­
tibody responses to several mycobacterial
antigens were measured during the experi­
ment. Finally, serums from anergic cattle
were tested in vitro for immunosuppressive
activity against stimulated blood lympho­
Materials and Methods
Twelve Hereford heifers were kept in an isola­
tion unit and fed a mixture of concentrates, lu­
cerne and oaten chaff. They were randomly as­
signed to 4 equal groups. All the heifers were vac­
cinated at 4 months of age with Brucella abortus,
strain 19. At about 1 year of age, 9 of the cattle
were injected subcutaneously in the brisket with
50 mg of BCG organisms from Commonwealth
Serum Laboratories, Parkville, Vic. (CSL) in 2.5 ml
of sterile Hornybrooks medium.
Preparation of BCG for Injection
The BCG organisms were suspended in Homybrooks medium at a concentration of 20 mg/ml by
stirring for 3 days at 4 °C.
5 ml of Hornybrooks medium containing
100 mg of BCG were injected intravenously into 2
groups of 3 cattle (table I). Rectal temperatures
were measured in these cattle hourly for the first
8 h after intravenous injection of BCG.
Tuberculin purified protein derivative (PPD),
prepared from human strains of M. tuberculosis
was obtained from CSL. This contained 100,000
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Blood Leucocytes in Anergic Cattle
Skin Testing
Approximately 10,000 TU of tuberculin SM
(CSL) or 1 mg B. abortus antigen were injected intradermally into the skin of the neck of each heif­
er tested. Measurements of the increase in skin
thickness to the nearest millimeter were made on
a fold of skin over the injection site using vernier
Blood Collection and Leucocyte Cultures
Blood samples from each animal were collect­
ed with aseptic precautions from the jugular vein.
They consisted of:
(1) 5 ml, collected each week and immediately
mixed with 2 mg of the dipotassium salt of ethylenediaminetetraacetic acid (EDTA) for total and
differential leucocyte counts. Leucocyte counts
were carried out on a haemocytometer and differ­
ential counts were carried out on blood smears
stained with May-Griinwald-Giemsa.
(2) 20 ml, collected each week and allowed to
clot. Serum was removed and stored at 4 °C with
1/5000 merthiolate. This was used for all antibody
(3) 40 ml, collected every 2 weeks and mixed
with disodium EDTA at a concentration of
2 mg/ml. Leucocytes were prepared from this
blood by centrifugation and washing in EDTA sa­
line. They were transferred into tubes of Eagle’s
minimal essential medium (MEM) containing 20%
fetal calf serum (FCS) and cultured in triplicate
for 5 days as previously described [Outteridge and
Lepper, 1973b], Three concentrations of either
tuberculin, B. abortus antigen or PHA (5, 50 and
100 /tg/ml) were used in the medium. Measure­
ment of lymphocyte transformation was carried
out by ’H-thymidine uptake as previously de­
scribed [Outteridge and Lepper, 1973b].
Dilutions of serum from all the cattle were
tested for inhibition of PPD stimulation of blood
lymphocytes from 2 sensitized donor cattle. One
of these was from group A and the other from
outside the experimental groups.
Leucocyte Migration Inhibition Test
Leucocyte migration inhibition factor (MIF)
was assayed under agarose according to the meth­
od of Tautz et al. [1974]. The agarose was made
up as a 1.2% solution in Dulbecco modified Ea­
gle’s medium (CSL) with 10% FCS. A volume of
4.2 ml of this mixture was poured into 5 cm plas­
tic Petri dishes (Camelec Medical Products, South
Australia) and allowed to set. Eight holes, each
accommodating 6 /A, were punched in the agar.
Bovine blood was obtained from a single donor
outside the experimental groups and leucocyte
suspensions were prepared containing approxi­
mately 20% monocytes and 80% lymphocytes. The
cells were preincubated for 1 h at 37 °C with supernates from 5-day cultures of blood lympho­
cytes with PPD. The cells were dispensed into the
wells using a Ziptrol microliter pipette (Drum­
mond Scientific Co., Pennsylvania) each test being
prepared in quadruplicate. After 18 h in the COa
incubator, the Petri dishes were filled with metha­
nol and formalin each for 10 min, the agar re­
moved and the cells stained with May-GriinwaldGiemsa for 90 min.
Measurement of Leucocyte Migration
The radius of the stained migration area was
measured using a dissecting microscope with an
ocular micrometer. The migration index was cal­
culated as a ratio according to Curtis and Hersh
[1973] and Rocklin et al. [1970]. This ratio was:
percentage migration = —x 100, where
area of migration in supernate with antigen
area of migration in medium with antigen
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tuberculin units (TU)/ml with 0.5°/o added phenol.
For in vitro lymphocyte stimulation, portions of
the tuberculin were dialyzed against normal saline
to remove the added phenol and then sterilized by
filtration through a 0.3 um Millipore filter.
Antigen was prepared from B. abortus strain
19. The bacteria were grown on potato agar and
washed off in 1.5% phenol saline. The organisms
were disrupted in a Ribi cell fractionator (Sorvall
Instruments) and a cell-free supernate was pre­
pared by centrifugation at 80,000 g. The supernate
was dialyzed against 4 changes of distilled water
and then freeze-dried. This material was dissolved
in normal saline and sterilized by Millipore filter
for use in skin-test injections and in the lympho­
cyte culture experiments.
Phytohaemagglutinin-P (PHA; Difco Corpora­
tion) was used in the cell culture experiments.
Table I
Sensitizing BCG
injection (50 mg)
Desensitizing i.v.
injections of BCG
(100 mg)
10 at 2-weekly intervals
5 at 4-weekly intervals
Blood samples for lymphocyte culture with
PHA were obtained at 25 and 26 weeks after sen­
sitization. Similar samples for culture with Brucel­
la antigen were obtained at 26 and 27 weeks after
sensitization with BCG. Serum samples for anti­
body assays were obtained at weekly intervals dur­
ing the experiment.
Inhibition of migration was considered to have
taken place when the ratio was <0.8.
Antibody Assays
Antibodies to tuberculopolysaccharide were
measured in serum as previously described [Lepper et al., 1973]. Antibodies to tuberculin PPD
were measured by a passive haemagglutination
test [Boyden, 1951]. Antibodies to whole organ­
isms (BCG) were classified as IgG or IgM using
an indirect immunofluorescence test [Lepper and
Pearson, 1975].
Experimental Design
Groups of heifers were treated as shown in ta­
ble I.
Five weeks after BCG sensitization, desensiti­
zation of groups B and C was commenced and
then maintained for a period of 18 weeks. All
heifers were skin-tested with PPD at 1 week be­
fore sensitization and at 4 weeks after sensitiza­
tion. They were then not tested again with tuber­
culin until 20 weeks after sensitization. This
avoided skin-testing th e a n im als du rin g th e p eriod
of desensitization. Subsequent tuberculin skin tests
were at 28, 36, 45 and 50 weeks after sensitization
with BCG. The Brucella skin test was carried out
27 weeks after BCG sensitization.
Blood samples for lymphocyte culture with
tuberculin PPD were obtained at 4 and 5 weeks
after sensitization and then at 2-weekly intervals.
These collection times alternated with the weeks
that the heifers were given i.v. desensitizing injec­
tions of BCG. Ten samples were taken for lym­
phocyte culture from each heifer during the de­
sensitization procedure.
Response to Tuberculin Skin Testing
The increase in skin thickness (mm) for
all the cattle after tuberculin skin testing is
shown in figure 1. This figure presents diagrammatically the effect of either 10 desen­
sitizing doses (group B) or 5 desensitizing
doses (group C) of BCG on the tuberculin
skin response. Also shown are the skin reac­
tions in the sensitized control group A and
the untreated group D tested at different
times during the experiment.
It can be seen that, following subcuta­
neous injection of BCG (vertical arrows) in
groups A, B and C, the response to tubercu­
lin skin-testing increased but no change was
found in the uninjected group D. During the
period of desensitizing injections (inverted
arrows) the increase in skin thickness, elicit­
ed by the skin test, dropped in groups B and
C. At the same time, the skin reactions in
the sensitized control group A were main­
tained. Tuberculin skin tests carried out
subsequent to the last desensitizing doses,
showed that the skin reactivity in groups B
and C remained low.
Systemic Temperatures after Intrave­
nous Injections of BCG
The times taken to reach peak rectal
temperatures recorded after each intrave­
nous injection of BCG are shown for groups
B and C in figure 2a. It was found that the
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y _ area of migration in supernate without antigen
area of migration in medium without antigen
Blood Leucocytes in Anergic Cattle
Fig. 1. The graph depicts the increase in skin
thickness (mm) on the neck 72 h after i.d. injec­
tion of PPD for each group at different times af­
ter BCG sensitization. Groups B and C received
10 and 5 i.v. BCG injections, respectively, and
these are indicated by inverted arrows. The verti­
cal arrows indicate the time that the sensitizing
doses of BCG were injected into the brisket of 9
of the heifers.
mean time taken to reach peak temperature
was shortened from 8.3 h at the first dose,
to 6 h at the fifth dose in groups B and C.
In group B, at the subsequent doses (6-10),
the mean time taken to reach peak tempera­
tures was further shortened from 3.8 h at
the sixth dose, to 3.5 h at the tenth dose.
The peak temperatures recorded after
each intravenous injection are shown in fig­
ure 2b. On average, the greatest increase in
temperature (3 °C) was found to occur at
the third and fourth dose of BCG in groups
B and C. In the cattle given 5 doses (group
C), the fifth dose produced a temperature
rise which was close to the maximum re­
corded. In group B, subsequent doses
(6-10) were found to produce less tempera­
ture increase than the first 5 doses. Howev­
er, a mean increase of 1.5 °C was still re­
corded at the tenth and last dose of BCG. A
control series of injections of Homybrooks
medium alone were made in the 3 cattle of
group A, but systemic temperatures were
not significantly raised during the 8 h after
injection in these cattle.
In summary, the results showed that the
greater the number of desensitizing doses of
BCG, the shorter was the time taken to
reach the peak and the lower the peak tem­
perature recorded.
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Fig. 2. Systemic temperatures after intrave­
nous injections of BCG in groups B and C. a
The mean time (h) taken to reach peak tempera­
ture. b The mean temperature increase (°C) after
each desensitizing dose.
Antibody to Mycobacterial Antigens
The sensitizing injection of BCG stimu­
lated anti-tuberculopolysaccharide antibod­
ies but these did not persist in high titre be­
yond 8 weeks. Only low titres of antibodies
to PPD were stimulated by the BCG injec­
tions. The classes of antibody to whole BCG
organisms were IgG and IgM with IgM pre­
dominating. The intravenous injections of
BCG produced remarkably little effect on
all these antibodies in groups B and C.
Blood Leucocyte Counts
Absolute and differential leucocyte
counts were carried out every week on
blood from each animal. No significant
changes in these counts, attributable to the
intravenous injections of BCG, were found
in groups B and C.
Peripheral Blood Lymphocyte Response
to Tuberculin during Desensitization
Samples of blood were taken every 2
weeks and the progressive changes in reac­
tivity of the lymphocyte cultures were mea­
sured. The results of the lymphocyte culture
work are shown in figure 3. In this figure
the mean disintegrations per minute per
culture for each group are shown for each
concentration of tuberculin.
The first blood sample was taken 1
month after subcutaneous injection of BCG
and just before a tuberculin skin test. It can
be seen that blood from groups A and B
contained strongly reactive lymphocytes but
that from group C contained only weakly
reactive cells. The tuberculin skin test stim­
ulated the appearance of reactive lympho­
cytes 1 week later in the unsensitized group
In the absence of further skin testing, the
in vitro reactivity of the lymphocytes in all
groups declined and at 17 weeks none of the
groups reacted. This decline was most rapid
in group B which had received 3 desensitiz­
ing BCG injections by the time the lympho­
cytes became unreactive. Conversely, group
C, which had received only 2 injections of
BCG by this time, showed some persistent
lymphocyte reactivity. In group A, which
was not desensitized, a decline in reactivity
was also noted.
At the time the heifers were again skintested, 20 weeks after BCG sensitization, all
the groups had unreactive blood lympho­
cytes. Tuberculin skin testing stimulated the
reappearance of reactive blood lymphocytes
in groups A and C but not in groups B and
D. This was taken as evidence that group B
had been completely desensitized by 8 i.v.
injections of BCG. On the other hand,
group C was partially desensitized by 4 i.v.
injections of BCG and blood lymphocytes
from heifers in this group still reacted to
Stimulation of Blood Lymphocytes with
PHA or B. abortus Antigen
Samples of blood were obtained from all
heifers 2 and 3 weeks after the last desensi­
tizing injection in group B. The results of
lymphocyte cultures with 3 concentrations
of PHA are shown in figure 4. The response
of blood lymphocytes for each heifer at both
sampling times is shown according to the
group in which it was placed. All groups re­
sponded strongly to PHA and although
there was some variation between heifers,
groups and sampling times, the ratios of
stimulated to control lymphocytes were sim­
ilar for each group.
Samples of blood lymphocytes were cul­
tured with B. abortus antigen 3 and 4 weeks
after the last desensitizing dose of BCG. In
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Blood Leucocytes in Anergic Cattle
Fig. 3. The mean uptake of
3H-thymidine by blood lympho­
cytes from cattle in each group is
shown for the period from 4 to
22 weeks after BCG sensitization.
The graphs show mean incorpo­
ration of the label by lymphocytes
for dilutions of 5, 50 and 100 ,«g
PPD/ml medium, as well as for
control unstimulated lymphocyte
cultures. The vertical arrows indi­
cate the times at which the cat­
tle were skin-tested with tuber­
These results, together with those of the
tuberculin lymphocyte culture experiments,
indicate a tuberculin-specific suppression of
reactivity in the desensitized heifers both in
the in vitro test and the skin test.
Effect of Serum on the in vitro Response
of Blood Lymphocytes to Tuberculin
Serum samples were obtained from all
the cattle 27 weeks after sensitization. At
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the period between the two blood samples, a
Brucella skin test was carried out on all the
heifers. 6 of the 12 heifers responded to the
in vitro test and 3 of these heifers were in
the groups desensitized with BCG. All the
heifers reacted strongly to the intradermal
injection of the Brucella antigen but there
was no evidence of increased in vitro lym­
phocyte reactivity 1 week after the skin test
was initiated.
Fig. 4. This shows the effect of PHA stimula­
tion on 3H-thymidine uptake by blood lympho­
cytes from each animal in the four groups of heif­
ers. The results are expressed as a ratio of stimu­
lated to control cultures for dilutions of 5, 50 and
100 «g PHA/ml of medium. The responses are
shown for samples taken at 2 weeks (— ) and 3
weeks (— ) after the last desensitizing injection in
group B.
this stage both desensitized groups B and C
had failed to respond in a tuberculin skin
test 1 week before. These serum samples
were diluted in culture medium containing
25 pg PPD/ml and homologous tuberculinsensitive lymphocytes were added. This test
was carried out twice but no inhibition of
lymphocyte stimulation by tuberculin was
found with serum from any of the cattle.
In experiments on delayed hypersensitiv­
ity, Uhr and Pappenheimer [1958] reported
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p g PHA/ml o f culture medium
Comparison of Leucocyte Migration In­
hibition with Lymphocyte Transformation
Tests during Desensitization of Cattle
Leucocyte MIF was measured with supemates from tuberculin-stimulated lym­
phocytes obtained during desensitization
with BCG. The supernates were pooled ac­
cording to their experimental group and
tested with blood leucocytes obtained from
an unrelated cow. The results, expressed as
a migration index for each pooled sample
containing 50 /<g PPD/ml medium are
shown in table II. Also shown are the re­
sults of 3H-thymidine uptake on samples of
blood lymphocytes obtained from each
group during the period of desensitization
and until the tuberculin skin test at 20 weeks.
These results are expressed as the mean
stimulation index for each group of samples
stimulated with 50 pg PPD/ml medium.
The main finding was that in groups B
and C which had received the desensitizing
doses of BCG, leucocyte migration indices
below 0.8 were found at times which were
not always associated with high stimulation
indices. In these groups there was therefore
a poor correlation between lymphocyte
transformation and leucocyte MIF release.
The control nonsensitized group D had mi­
gration indices above 0.8 throughout the ex­
periment using the MIF test. In this group
there was an association between high mi­
gration indices and low stimulation indices.
The highest transformation index of 1.6 was
obtained 1 week after a tuberculin skin test
in these cattle.
Blood Leucocytes in Anergic Cattle
Table II. In vitro blood leucocyte cultures in the presence of tuberculin during desensitization with BCG.
Lymphocyte transformation (DNA synthesis) compared with production of leucocyte migration inhibition
factor in pooled culture supernates
Weeks after
Tuberculin test (20)
Group A
Group B
10 x desensitized
Group C
5 x desensitized
Group D
migration1 trans.2
migration trans.
that a single intravenous injection of soluble
protein antigen would desensitize guinea
pigs. This desensitization lasted 6-10 days,
using a large dose, and was specific for the
antigen injected. The cattle in our experi­
ments received either 5 or 10 doses of BCG
intravenously, which produced partial and
complete desensitization, respectively. This
desensitization was specific for tuberculin
and did not affect the skin reaction to the
unrelated B. abortus antigen. The desensiti­
zation lasted longer in the cattle given 5-10
doses of BCG than in the guinea pigs given
one dose of soluble antigen by Uhr and
Pappenheimer. However, the progressive
nature of the desensitization process in the
cattle was reflected by the systemic temper­
atures recorded after BCG injection. It
seemed that the greater the number of intra­
venous, desensitizing doses of BCG, the
shorter the time taken to attain the tempera­
ture peak and the lower the peak tempera­
ture reached. Atkins and Francis [1973]
have suggested that activated lymphocytes
can cause blood leucocytes to release an en­
dogenous pyrogen. If this was the cause of
the pyrexia in the cattle, as desensitization
progressed, the pyrogen was released more
rapidly from the leucocytes but in smaller
quantity. This suggests a progressive in­
crease in the lymphocyte receptor affinity
for antigens of BCG had occurred but, at
the same time, there had been a diminution
in the number of cells reacting to antigen.
The results of the lymphocyte culture
work support the idea that tuberculin-sen­
sitive lymphocytes were either removed
from the blood or were inhibited from
transforming in the desensitized cattle. The
anamnestic response of the blood lympho­
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1 Mean migration index for pooled samples containing 50 fig PPD/ml medium. Figures in italics below 0.8
are regarded as positive.
2 Mean stimulation index for lymphocyte sample with 50 fig PPD/ml medium.
cytes, after the tuberculin skin test at 20
weeks, showed that the sensitized control
cattle (group A) could be restimulated to re­
lease reactive lymphocytes into the blood.
The anamnestic response was lower in the
cattle which had received the 4th desensitiz­
ing dose (group C) than in group A but it
was absent in the cattle which had received
the 8th desensitizing dose (group B). This
again supports the idea that there were two
degrees of desensitization in the two groups
of cattle.
The disappearance of reactivity to tuber­
culin in blood lymphocytes from the com­
pletely desensitized cattle was not accompa­
nied by loss of reactivity to PHA or B.
abortus antigen. Nor was it accompanied by
the appearance of specific immunosuppres­
sive factors in the serum of the desensitized
cattle. Surprisingly, there was little change
in serum antibodies to tuberculopolysaccharide, PPD or whole BCG organisms
following the intravenous injections of
BCG. Differential counts of blood leuco­
cytes remained stable during the desensitiz­
ing period and in general, the results gave
no indication of specific humoral inhibition
of lymphocyte reactivity in the desensitized
cattle. The possibility of a defect in the pro­
duction of leucocyte MIF, accompanying
desensitization, has been suggested by Jokipii and Kosunen [1974]. However, our re­
sults indicated MIF production by lympho­
cytes from these cattle occurred at intervals
during desensitization.
These results also did not support the
hypothesis that desensitization was caused
by immunosuppressive factors from trans­
forming lymphocytes, as reported in a pre­
vious paper by Outteridge and Lepper
[1973a]. In this earlier work we found that
a temporary desensitization to the tubercu­
lin skin test followed the subcutaneous
injection of tuberculin, tuberculin-stim­
ulated lymphocytes or supernates from
lymphocyte cultures stimulated with tuber­
culin. An attempt was made in those experi­
ments to demonstrate specific immunosup­
pressive factors in the serum of tuberculous
cattle, but was without success. Another at­
tempt, made with serum of anergic cattle in
the present experiments, was also unsuc­
cessful. If there were specific immunosup­
pressive factors such as antigen-antibody
complexes in these serums, they were not
detected by 3H-thymidine uptake, the meth­
od used in these experiments. The results of
these and earlier experiments favour the idea
that specifically sensitized lymphocytes were
removed from the blood of desensitized cattle.
Evidence from laboratory animal experi­
ments suggests that a single intravenous
injection of antigen can remove recirculat­
ing antigen-reactive lymphocytes [Sc/1/ 055 man et al., 1971; Sprent et al., 1971; Rowley et al., 1972]. However, it is not certain
where the lymphocytes go, although it
seems possible that they accumulate in the
lymph nodes and spleen. It is noteworthy
that raised specific activities of antigenreactive lymphocytes per gram of lymphoid
tissue are not commonly reported. In the
desensitized cattle, no gross abnormalities
were found in the lymph nodes and spleen
at postmortem examination. However, a
single granuloma containing acid-fast bacilli
was found in the liver of two cattle in group
B. More granulomata might have been ex­
pected in lymphoid tissue in view of the
large numbers of living BCG organisms in­
jected. If sensitized lymphocytes were
sequestered in the lymphoid tissue, as sug­
gested by Schlossman et al. [1971], it would
be expected that they would reappear in the
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blood soon after the desensitizing injections
ceased. However, this did not happen in the
completely desensitized cattle within the
time limit of our experiment. It could be
concluded that the cells were removed from
the circulation and either continued to be
suppressed in the lymphoid tissue [Rook,
1975], or were destroyed.
The idea of a suppression mechanism
has been further developed by other work­
ers to involve bone marrow derived (B)
cells or their products in suppression of
T-cell functions such as delayed hypersensi­
tivity [Katz et al., 1974; Neta and Salvin,
1974]. According to this hypothesis, the de­
sensitized cattle in our experiments could
have possessed suppressor B cells in their
lymphoid tissue which prevented the expres­
sion of delayed hypersensitivity. However,
using cells or serum from desensitized
guinea pigs, Dwyer and Kantor [1975] failed
to demonstrate a suppression effect when
these preparations were transferred into
sensitized recipient animals. It therefore re­
mains an open question as to whether sup­
pressor cells exist. As yet there is no general
agreement on the cell type or the mechanism
of action of these cells.
We would like to thank Dr. G. G. Alton for
growing the B. abortus organisms and Dr. P.
Plackett for help in preparation of the skin-test
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Correspondence to: Dr. L. A. Corner, CSIRO,
Division of Animal Health, Animal Health Re­
search Laboratory, Private Bag No. 1, PO, Parkville, Vic. 3052 (Australia)
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