no specific hemolysins were demonstrable in the blood serum. Again subcutaneous
injections of sheep blood cells in gradually increasing doses (six) were given, three to
four days apart, making a total of 250 c.c. in all. A test showed that the serum
reached its highest value (o.ooi c.c.) on the seventh day after the last injection, but
at the time of birth of kid it had greatly decreased (0.020-0.025 c.c). One normal
kid was bom 115 days after the last injection. Shortly after birth a blood sample
was taken from the kid; the seals were removed from the mother's nipples and a
sample of colostrum was drawn. Then the kid was allowed to remain with the mother
and suckle. On testing the samples the mother's senun showed a relatively low con-
tent (0.020-0.025 c.c.) of hemolysin, which remained quite constant for some days
after the birth of young. The colostrum while scant was especially high in its
hemolytic content (0.009 c-c.) on day of birth. On the following day the milk showed
a very low test (0.15 c.c.) and was entirely negative by the end of the week. A sample
of 0.30 c.c. of the kid's serum at birth did not show a trace of hemolysin by test, so
probably little if any passed the placenta from the mother. On the second day after
having suckled, only a trace of hemolysins could be demonstrated in the kid's serum;
this can be explained in part by the known fact that the mother had only a small
amount of colostrum, the volume of milk secreted was scant for several days, and the
kid showed signs of hunger and under-nourishment.
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352 L. W. Famulener
Experiment 7. Goat 6 (pregnant). — It may be of interest to record in this connec-
tion an observation made on another goat which might be classed with this series.
The mother showed signs of oncoming labor during the late afternoon, so both blood
and colostrum samples were taken. Next morning it was foimd that the animal had
given birth to two large (weight four lbs. each), apparently fully developed young which
were dead. Nothing in particular was shown on autopsy excepting some subcutaneous
edema in each; some bloody fluid was foimd, in the thoracic cavity, which was removed
and saved for test; the heart and large vessels were fairly well filled with pwutially
clotted blood which was removed and the serum collected; limgs, nondilated, on
section showed nothing unusual; the other organs appeared normal. Possibly a large
bleeding (between 300-400 c.c.) which was taken from the mother one week before,
might have had some influence upon the welfare of theyoimg. The weight of the
adult, on day following the birth of young, was 83 lbs. Upon centrifuging the fluid
from the thorax, and the blood from heart and great vessels, it was found that in one
kid the fluids came out quite clear, while those from the other were rather deeply
colored by the presence of hemoglobin. We shall therefore consider the tests made
upon the clearer fluids from the one kid. The following comparative values were
shown: Mother's seriun, 0.0025 c.c; Colostrum, 0.0015-0.0020 c.c; Kid — the
fluids from the blood and from the thorax failed to show a definite hemolysis in amounts
up to o. 20 c.c; a very slight tint was observed but it is questionable if this were due
to specific antibodies or traces of hemoglobin in the sample.
This adult was unique in the sense that she seemed to retain a very high antibody
content in her blood long after the last blood cell injection. The last blood injection
was given about six months (170 days) before birth of the yoimg. Most anunals lose
their antibodies much more quickly; few show them in appreciable quantities after
six months. This animal was immunized before conception had taken place, since
the period of gestation in goats is stated to be five months. If the hemolytic inmiime
bodies passed through the placenta to the fetus in this case, the amoimt was very
small.
From the experimental evidence given in this last series of
experiments it would appear that the colostrum plays a greater
rdle in the transmission of hemolytic inmiunity to the offspring than
the placenta — the latter probably transmits little if any appreciable
amount of hemolytic immune body, to the fetus in tUero.
b) Active immunization of the adult following immediately or
shortly after birth of young. — ^A number of workers have succeeded
in highly immunizing female animals, during the period of lacta-
tion, against toxins and getting the antibody in the milk in a
relatively high concentration. Since it has been proved that the
milk is an important factor in transmitting antibodies to the
suckling during the first days of life through gastro-intestinal
absorption, it was thought worth while to undertake similar experi-
ments at later periods in connection with our studies.
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Immunity Transmission from Mother to Offspring 353
Series 3. Animals Normal at Time op Parturition.
Experiment 8, Goal 8 {norma]). Preliminary test. — ^This animal gave birth to
one normal kid which was allowed to remain with and suckle mother. Blood samples
were taken from the mother and kid, then the mother received a subcutaneous injec-
tion of 75 c.c. of sheep corpuscles. Following this, loo c.c. corpuscles were given daily
for three consecutive days — a total of 375 c.c. Blood samples were drawn from the
mother and kid on alternate days for two weeks, after which samples were taken every
third day for some time longer. In this experiment no milk samples were taken during
the first weeks as the supply was scant; the senmi samples were tested at the same
time using o. 20 c.c. rabbit serum per tube for complement. It was foimd that anti-
bodies did not appear in definite amoimts in the mother's senmi until five days after
the last blood injection, then they rapidly increased imtil the nth day, when the
highest concentration (0.0035 c-c.) was found; the antibodies slowly declined until on
the 46th day after last injection the value had fallen to 0.037 c.c. Serum samples
from the kid when tested in amounts as high as 0.5 c.c. failed to show positive results.
It is questionable if any appreciable amoimt of hemolysin was excreted in the milk
when the mother's serum was at its greatest concentration. If so, the kid at that age
failed to absorb (unchanged) a sufficient quantity to give a definite hemoljrtic reaction
in the test.
Experiments g and 10, — ^Similar experiments were performed with goats 4 and 5,
with the exception that milk samples were taken throughout and tested at the same
time as the blood serum samples of the respective mother and kid. In neither case
did we succeed in getting very high inununity in the mother animals, and it was foimd
that an appreciable amoimt of antibody failed to pass over in the milk. Naturally the
serum of the kids did not show antibodies.
These experiments proved quite unsatisfactory. It appears
that an exceedingly high immunization of the female is necessary
before a fairly high antibody content occurs in the milk. In
neither of the two goats last mentioned was the degree of immuni-
zation sufficient to cause a demonstrable overflow of antibodies in
their milk.
An obvious objection to the active immunization of normal
females immediately after birth of young is the necessary delay
before the production of antibodies in high concentration in the
mother's body, and their appearance in the milk. In the mean-
while the young animal becomes more mature; its digestive powers
are more pronounced, and probably anatomical changes occur in
the gastro-intestinal mucosa which retard or prevent the absorption
of the unchanged antibodies.
Series 4. Animals Partly Immunized at Time of Parturition.
In order partly to overcome certain objections to which atten-
tion has just been directed, animals which had been previously
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354
L. W. Famulener
immunized and still showed a low antibody content in their blood
at the time of birth of the young, were reimmunized by more blood
injections following parturition. In this way it was hoped that a
high immunity could be induced in the mother, and more quickly
than was possible in the strictiy normal animals, and that the milk
would either remain high or increase in immune body value.
Possibly kids receiving such milk in quantity during the first weeks
of life would be able to absorb the inmiune body unchanged, and
as a result their sera would remain about constant or even show
some decided rise in the hemolysins. In opposition, other factors
enter, such as (a) animal growth (increase in body fluid), (6) loss
through blood samples withdrawn, and the {c) metabolic destruc-
tion of antibodies. Therefore, such experiments should be well
controlled.
Experimtni ii. Goat 6 {previously immunized), — ^Found in the forenoon with
normal twins, and as they had suckled, only a small sample of milk could be obtained
from the mother. Both remained with mother imtil late in the afternoon. The
mother was given a large injection of washed sheep corpuscles the same evening.*
The blood injections were repeated every second day imtil four in all were given — a
total of 425 c.c. Blood samples (adult and kid) and milk were taken for several weeks.
All were tested simultaneously. The results of the test may be tabulated as follows:
Time
Mother's Serum
Milk
Kid "A" Serum
(Mother's Milk)
Kid "B" Scrum
(Cow's Milk)
Dav of birth of young
acl day after birth o
0.0080 c.c.
0.05-0.06 c.c.
0.002 CO.
0.0035 C.C.
f young..
0.0077 "
0.27 C.C.
0.002 "
0.0030 "
4th •• " " '
2th " •• " •
' * . .
0.0040
0.16 "
0.003 ;;
0.003s "
* " . .
0.0020 "
0.08 "
0.003 "
0.0040
10th " " " •
* " . .
0.0009 "
0.0s "
0.004 "
0.00s "
1 2th " " " •
0.00098 "
0.045 ;;
0.0042 "
0.00s "
i6th " " " •
* " . .
o.ooia "
0.0s
0.006 "
0.007 "
a2d " •• " '
( «
0.0017 "
0.1S+ ;
0.0078 "
"
28th " " " •
' " . .
0.0022 "
O.IC+ "
0.18 "
0.009 "
0.019 "
34th " " " •
( It
0.0027 "
o.ois "
0.03 "
40th " " " •
0.003 "
0.27
0.022 "
0.04s "
(When 45 dajrs old, Kid "A" weighed 14J lbs,, and Kid "B," 17 lbs.)
As evident from the above data, the kid which was with the
mother and getting a bountiful supply of milk containing large
amounts of immune body, showed in its blood serum an antibody
content practically running parallel with that of a control (brother)
which was fed upon cow's milk after the first day following birth.
* At the same time this animal received injections of killed cultures of the colon bacillus in small but
increasing doses on days alternating with the blood injections. These injections were continued over two
weeks without any harmful efifects in so far as noted.
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Immunity Transbiission from Mother to Offspring 355
The ratio of weight between the two kids probably ran roughly
parallel throughout the test. So it would appear, at least in this
instance, that not many of the antibodies pass over unchanged to
the circulation from the gastro-intestinal tract of a suckling animal
after the first days following birth.
This experiment would suggest that even when we can get an
animal highly immunized and the milk shows the presence of con-
siderable antibodies, hardly an appreciable amount is absorbed
unchanged from the digestive tract by the suckling after the first
days of life. These results were supported by other observations,
but in experiments where a control twin was not available.
c) Active immunization of newly bom or older kids against
foreign blood cells.
Semes 5. Blood Injections into Kms.
In connection with our studies where mothers received very
large injections of blood during gestation, especially in the very
last weeks, we must consider the possibility of soluble antigens
passing the placenta to the fetus. Bang and Forssman^ state that
ox corpuscles gave an ether soluble fraction which possessed anti-
genic properties when injected into suitable animals. It is possible,
therefore, that in the body also similar soluble products are liberated
in quantity and eventually reach the blood stream from the
large subcutaneous blood injections. Hektoen and Carlson*^ have
shown by transfusion experiments with dogs, which had been
previously injected (intravenous) with small amounts of goat's
blood cells, that after several hours the antigen is fixed in the body,
of the injected animal, and not transmitted in sufficient quantity
to the normal recipient to cause specific antibody production in the
latter. But when large blood injections were given to a donor,
then later a direct transfusion was made to a normal animal, the
recipient as well as donor produced antibodies in due course of time.
This showed that the body's ability to fix the formed antigen was
limited. Therefore the possibility exists that, from large sub-
cutaneous blood injections into gravid goats, soluble antigen might
eventually reach the fetus. The question arises, If the fetus
receives antigen shortiy before birth, has it the power to react and
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3s6 L. W. Famulener
form antibodies, or may it retain the antigen and form antibodies
shortiy after birth ? Any attempt directiy to inject the fetus* in
uiero is necessarily surrounded by numerous technical difficulties.
Moreover, the results thus derived would be jusUy subject to
criticism. A few experiments were carried out on kids at different
periods after their birth to test their reactive ability against sheep
erythrocytes. The results may throw some light upon this side
of the immimity problem.
ExperimetU 12, Kid g6 B {newly born). — ^This kid was a twin bom from a mother
which had been immunized against sheep corpuscles, over five months before parturi-
tion. The mother's blood taken six days before birth of the young showed a trace of
hemoljrtic immune body in o. 10 c.c. senmi, while a sample taken on the day of birth
failed to show any hemolysin in like amount of serum. The colostrum on day of birth
of young showed a small amount of immime body in o.io c.c, but the milk on the
following day was negative. A blood sample was taken from the kid before it was
allowed to suckle, then it was placed with the mother. The same evening 12.5 c.c*
of sheep corpuscles were injected into the kid subcutaneously and the next afternoon
another injection of the same amount of corpuscles was given. Blood samples (eight)
were taken at intervals during the 15 days following the last injection. On testing it
was found that o. 10 c.c. serum from blood samples taken the second and third days
after birth showed a small amount of hemolysin, but this practically disappeared and
soon showed only as a trace. Since the kid's blood sample taken before suckling
showed no hemolytic action in ihe same amount, one must look to the colostrum as a
source of its antibody on second and third days. The tests of samples, covering two
weeks following last blood injection, failed to show more than a trace of hemolysis at
any time, but perhaps this trace was slightly greater at the end of the second week.
Adults usually show specific hemolysin production by the end of the first week after
injection, and often this is at a maximiun on the ninth to 12th day. We must con-
clude that the young in this case produced hemolysins only to a very slight extent
(minute quantity) in response to the injection of sheep blood corpuscles in relatively
large amounts.
Experiment 13. Kid is B {10 days old). — ^This kid was also bom from an immu-
nized mother, but failed to show any appreciable amount of specific hemolysins in its
blood serum at the end of the first week after birth. On the loth day after its birth
the kid was given a subcutaneous injection of 10 c.c. of sheep blood corpuscles and an
equal dose was given in the same way on the 12th day. Samples were collected at
intervals for three weeks after the last injection. By the test.f all were negative (o. 10
c.c.) imtil the ninth day, when a slight trace of hemolysin was shown; on the 12th day
this was somewhat increased, a definite but slight hemolysis; i6th day, questionable
trace; 20th day, o. 10 c.c. gave negative results. This would indicate that a slight but
transient immimity reaction took place against the sheep blood cells.
* Kreidl and Mandl {loc. ciL) attempted injection of the fetus (goats) in utero, but were unsuccessful
in the outcome in practically every case.
t Used the regular technic excepting 3.0 c.c. of a 5 per cent instead of a 7.5 per cent suspension
of sheep erythrocytes were added per tube.
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Immunity Transmission from Mother to Offspring 357
Experiment 14. 68 B {18 days old). — ^The mother of this kid had been immunized
against sheep blood cells, some time previously. It was removed from the mother on
day of birth and fed during the first days upon goat's milk and then later upon cow's
milk. On the i8th day after its birth, 20 c.c. sheep blood cells were injected into
it subcutaneously and two days later an equal volume was given in the same way.
Blood samples were taken during three weeks following the last injection. Testing
by the regular technic, it was found that o. 10 c.c. of serum showed a trace of antibody,
on the 1 8th day after birth (first injection of blood). Probably it got colostrum, which
contained inunune body, on day of birth. These antibodies quickly disappeared and
further samples were negative in amount tested, until sbc days after last blood injec-
tion, when a slight trace again appeared and gradually increased so that on the 12th
day after the last injection of blood, a slight but definite hemolysis was produced by
o.io c.c. of serum; this had again practically disappeared within a week. This
experiment indicates that only a very slight immunity reaction occurs in a kid after
it has reached the age of 18 to 20 days.
If we are permitted to draw any conclusions from these few
experiments we should say .that the newly bom animal has only
slight ability to form hemolysins during the first days following
birth. Consequently we cannot assume that the fetus in utero has
greater power, even if antigen should reach it through the placenta.
But, on the other hand, it is more probable that the fetus has even
lesser power to form immunity reaction product than the newly
bom. This seems to be a property which is only gradually acquired
by the growing young, as they adjust themselves to their environ-
ment in the external world. The work of several investigators
(Schkarin,7'> MoU^') would seem to substantiate the view that the
organism of very young infants or animals have only a slight
ability for antibody production.
d) Gastro-intestinal absorption of antibodies.
Series 6. Feeding of Hemolytic Serum to Newly Born Kids.
As apparent from the data submitted, the newly bom kid
readily absorbs in an unchanged condition the specific hemolytic
immune body from colostrum through the gastro-intestinal tract.
These immune bodies do not appear in the kid's blood serum unless
present in considerable amounts in the ingested colostrum. How-
ever, the amount in colostrum-milk may be comparatively low
and still cause a decided increase in hemolytic value of the suckling's
blood, if the total volimie taken is large. The question suggested
itself. Was this free absorption of immune body taken through the
milk-colostnmi pecxiliar to the combination ? Or, are the immune
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3S8 L. W. Famulener
bodies readily absorbed by the newly bom kid, when fed hemolytic
serum, irrespective of the nature of the medium in which they are
present ? As we had an opportimity, this question was put to test
in two cases by feeding newly bom kids with homologous serum
(goat) which contained specific hemolysins. The kids were taken
away from the mothers immediately after birth and were never
allowed to suckle. Owing to the lack of animals at the time this
investigation was in progress, exact quantitative experiments upon
this question, imfortunately, could not be continued. The follow-
ing results may prove to be of some interest in that direction:
Experiment 15, Kid 112 B (newly horn, weight 1,350 gms.). — ^A twin, which was
not allowed to suckle mother, was removed inmiediately after birth and a blood
sample was drawn. An inmiimized goat (15) was bled at once, the blood defibrinated
and centrifuged to remove all corpuscles. About 30 c.c. of this supernatant serum
was fed to the kid and no other feeding was made until the next day, when it was given
cow*s milk, which was continued from that time on. Unfortunately on testing this
serum it was found to be of much lower value than suspected; test showed that 0.02
c.c. produced, however, a well marked hemolysis when tested by the regular technic,
with the exception that 2.0 c.c. of 5 per cent instead of 7.5 per cent sheep blood
suspension was used.
Samples of blood were taken upon four consecutive days after the feeding of the
immune serimi. When these samples (including the sample taken from the kid before
feeding) were tested, in the manner just mentioned, none showed hemolysis in amounts
of o . 15-0 . 20 c.c. If one- third of the antibodies were absorbed (unchanged) one woidd
expect the test to give at least a slightly positive reaction in the volume of kid's senun
which was tested. It follows that the absorption of imchanged antibodies from the
digestive tract in this animal was not pronounced.
Experiment 16. Kid gy B (newly born, weight 2,53s gf^ts.). — Immediately after
birth this kid was removed without being permitted to suckle. A blood sample was
taken, then it was fed by pipette 15 c.c. of specific immime senmi (goat 15) which had
been kept under good condition, in freezing ice-box, for almost four months. This
hemolytic serum, tested shortly after the feeding experiment, was of such potency that
0.002 c.c. almost completely hemolysed 3 c.c. of 5 per cent sheep blood suspension
(o. 10 c.c. guinea-pig serum for complement), while o. 001 c.c. gave a definite hemolysis.
About one hour after this feeding the kid was given 150 c.c. of cow's milk. No further
feeding was made until the next morning, when it was put upon cow's milk regularly.
Blood samples were drawn daily at first, then at longer intervals, covering in all 15
days' observation. The tests were carried out upon these samples by the same technic
(amount of blood samples, complemelit, and volume fluid) employed in the preceding
experiment (Kid 112 B). It was found that the sample taken before the feeding of
hemolytic senmi Was negative in o.io c.c; the day following the immune serum
feeding, o. 10 c.c. serum showed a well marked action; the second day after, the same
amoimt of serum showed less hemolytic action; the hemolytic power gradually dimin-
ishing until, on the 13th and 15th days after the serum feeding, no action was shown
by o.io C.C. This experiment showed that considerable absorption (approximately
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Immunity TRANsinssioN from Mother to Offspring 359
10 per cent) of the unchanged hemolytic serum from the digestive tract took place,
and appeared in the general circulation of the young animal. Recognizing the fact
that no definite quantitative conclusions can be drawn from as few as two experiments,
no more can be said than that these experiments suggest that antibody absorption
while taking place is relatively meager when hemolytic sera are fed to newly bom
kids.
5. General discussion of results, — Before attempting to draw any
definite conclusions from the above experimental data, it may be
well to consider more fully some of the findings, and the related
questions. In this brief discussion, not only our own results, but
the correlated results of other workers, will be considered. * The
experiments clearly demonstrate that goats, when highly immu-
nized against sheep erythrocytes during the latter part of gestation,
transmit the specific antibodies to their sucklings. Apparently
the placenta plays a very small part in this transmission, but the
principal r61e is played by the antibodies which are present in the
colostrum-milk and are readily absorbed from the digestive tract
when taken by the newly bom offspring. In those cases where the
young bom from immime mothers were prevented from taking the
colostrum-milk until after a blood sample had been drawn,' tests
failed to show the presence of hemolysins in the kid's blood serum
in suflScient amoimt to be appreciable by the method used. So we