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137

104
1 _THS.
; AN ELECTROPHORETIC STUDY OF
ff SERUM AND PLASMA PROM NORMAL
; AND LELCOSlS-AFFECTED CHICKENS
i

Thesis fer the Degree of M S.
MICHKSAN STATE COLLEGE
Rail:- Evelyn Sanders

1943

 

 

 

 

 

AN ELECTROPHORETIC STUDY OF SERUM AND PLASMA FROM

NORMAL AND IEUCOSIS-AFFECTED CHICKENS

by
Ruth Evelyn Sanders

A THESIS
submitted to the Graduate School of Michigan
State College of Agriculture and Applied

Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE
Department of Chemistry

December , 1943

< x}!

_ u.

An Electrophoretic Study of Serum and Plasma from
Normal and Leucos Lie-Affected Chickens

Io IntrOduOtion eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeee 1
jIe Procedure eeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoee h

111'. Experimental and Results

A. Electrophoretic patterns of normal chicken
serum-and Plasma eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 6

B. Electrophoretic patterns of serum tram
leucosis-affected chickens eeeeeeeeeeeeeeeeeeeeeeeeoee 7

C. Electrophoretic patterns of serum obtained

at intervals after injection with active

transmissible tumor eeeeeeeeeeeeoeeeeeeeoeeeeeeeeeeeee 9
De Electrophoretic patterns of serum obtained

at intervals after injection with inactivated
tranflmdfisible tumDr eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeell

IV. Discussion eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee0913

V} Summary oeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee16

'VIe‘ Literature CitOd eeoeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeeeeela

H
w
(it?
C;
u

AN MTROPHOREI‘IC STUDY OF SERUM AND PLASMA FROM

NORMAL AND LEUCOSIS-AFFECTED CHICKENSu

It has been observed by poultry pathologists that when chickens
are affected with leucosis they may show one or more pathological
manifestations. It is a debated question whether the etiological
agent 1. the same in all tn. known manifestation of the disease.

In erythroblastic leucosis, according to Johnson (16), all stages

of red cells appear in the peripheral blood. in the capillaries of
the visceral orgns, and in the sinusoids of the narrow. In granulo-
blastic leucosis all stages of immature grannlocytes appear in the
peripheral blood as well as extravascular proliferation in many of
the visceral organs. In the other types of the disease there may

be an increase of lymphocytes and hemocytoblasts in the peripheral
blood. When these types of cells proliferate or infiltrate in the
nerves or central nervous system, the iris, or the visceral organs,
the disease is termed neuralymphcnatosis. ocular lynphomatosis, and

visceral lynphomatosis, respectively;

dhis study represents a cooperative project of
the Chemical Section and the Bacteriological
Section of the Michigan State Agricultural hperi-
ment Station and the Poultry Research Laboratory
of the United States Department of Agriculture.
The material used in the study was furnished by
the Poultry Research laboratory, and the work was
carried out in the Brucellosis Laboratory of the
Department of Bacteriology.

In neural lymphomatosis paralysis of the legs, wings, or neck may
be observed. The involved nerve is yellowish-gray or reddish-gray in
color and enlarged, due to the presence of many mononuclear cells.
Ocular lymphomatosis is characterised by a gray iris: small, irregular
pupil; asymmetrical papillary vacillations; and the carriage of the
head. the iris and ciliary body are thickened by the presence of my
mononculear cells. Subcutaneous, mscular, or visceral tumors are
present in visceral lymphomatosis. lalignant mononuclear cells are
aggregated in foci or are diffused throughout the organs or tissues
involved. In osteopetrotic lymphomatos is the bones are enlarged, dense,
and hard with a rough, irregular, porous surface. The enlargement of
the bones results from the formation of new abnormal bone. A chicken
affected with erythrogranuloblastosis is usually dull, weak, and anemic;
it. usually has an enlarged cherrybred liver, spleen, and kidney and
many immature cells of the erythrocytic and granulocytic series in the
blood.

The term used in describing the types of the disease and the
pathological changes by which they are characterised were adopted by a
conference of investigators held at the U. 8. Regional Poultry Research
laboratory (3) (7)-

Invcstigators differ as to whether each of the various types of
leucosie is caused by the same or a different agent. It was thought,
therefore, that a study of the blood serum and plasma proteins of
affected chickens by the moving boundary method in the Tiselius
electrophoresis apparatus might furnish revealing informtion and aid
in clarifying the present state of knowledge pertaining to the different
types of the disease and also show whether there are changes that charac-

terize affected chickens from normal ones.

-3-

Electrophoret ic analysis by the moving boundary method finds its
widest application in the study of changes that occur in the blood
proteins accompanying diseases. Characteristic changes in the relative
composition of protein components of serum have been noted in many diseases

and pathological disturbances in man and animals by this method ()4) (18)
(29) (30).

-h-

PROCEDURE

Chickens of the single combed White Leghorn breed from 15 to 18
weeks of age were used. All the chickens wcrc‘killcd by electrocution
and eacsanquination and then examined for evidence of infection. Blood
was collected from two to nine chickens and pooled. They were usually
starved 211 hours previous to bleeding. For plasma samples the blood
was collected in 1/100 volume of a 25-per cent solution of sodium citrate.
The protein of the serum or plasma sample was determined by the refractive
differential method of Siebenmenn (a6), and the sample adjusted to a
protein concentration of 2 per cent with buffer solution. The diluted
sample was placed in cellophane tubing and dialyzed against three changes
of buffer at 3'0. The final dialysate was saved and used in filling the
electrode vessels and cell.

The buffer solution used on all samples consisted of diethyl barbituric
acid and sodium hydroxide adjusted to a pH 8.6 and ionic strength of 0.1'.
Longsworth (114), in a study of 12 buffers, found none superior to this one
for the electrophoretic study of human serum and plasma. In this study
phosphate hiffers of pH 7.7 and ionic strength of 0.1 or 0.2 were found
inferior to the barbiturate buffer.

Conductance measurements were made at 0'0. on the buffer and protein
after final dialysis. The Shedlovsky type conductivity cell was used (18)
(25) in a Dewar vessel containing cmshed ice and water.

Electrophoresis was carried out in the modified Tiselius apparatus
described by Longsworth and Maclnnes (15). The "schlicren scanning method"
of Longsworth (13) (18) was used in photographing the moving boundaries.
Electrophoresis was carried out at 0’0. and at a potential gradient of
6 to 7 volts per centimeter. The tine varied from 10,000 to 12.000 seconds.

.5-

The first objective was to obtain the clectrcphoretic patterns for
normal chicken serum and places. and to establish the mobilities and ratio
of normal protein components. After determination of the normals, a
comparison was made of serum from chickens showing different pathological
manifestations of the leucosis complex. Later, serum samples were obtained
from chickens at intervals following injection with a lymphoid humor and
also from chickens injected with this same hmor inactivated so that it

consistently failed to produce any manifestations of the disease.

.6.

EXPERIMENTAL AND REU‘LTS

A. Electrophoretic patterns of normal chicken serum and plasma.

Serum and plasn samples were obtained from apparently norml chickens
which showed no symptoms or lesions of any disease. Electrophoretic diagrams
of these samples are shown in Figs. 1, 2, 3, h, and 17. llobilities and
composition calculated from the patterns of the descending boundaries are
sent forth in Table 1.

San Clemente (22), in his report on patterns, mobilities, and composition
of normal, or more correctly, negative pullorum-agglutinating chicken serum,
used a phosphate buffer of pH 7.78 and 0.2 ionic strength. The protein
components showed lower mobilities and less separation than in barbital
mirror; Although good separation of ab. and or. was not attained in all
samples in barbital buffer, pH 8.6 and 0.1 ionic strength, no evidence of
separation of the two was found in the buffer of lower pH.

The very pronounced '/-g1cbulin disturbance is present in the patterns
of all chicken serum and plasm. It appears as a tall spike on all descending
patterns and as a call peak on ascending patterns. According to Longs-
worth, _e_t_ :_1_'. (17) (18) a similar disturbance in the floglcbulin is due
to convection, resulting from reaction in the neighborhood of the boundary
following electrophoretic separation of the constituents. He has shown
that at least one of the )6 -g1obulins of normal serum has an affinity for
lipid material which migrates with the ,6" -globulin. Abramson (1) has
discussed the physics of the )5 -anomaly and other possible explanations.

The position of the disturbance with respect to the mobilities of
the ,5 -and f-glohulin is not always the same on the ascending and descending
patterns (Figs. 6, 11, and 114).

It may be noted that the total protein in the normal serum samples

varied from 2.19 to 3.71; grams per 100 ml. The fibrinogen concentration,

 

 

r—a———r <———a-1

Figure 1. Electrophoretic patterns of normal chicken serum.
The patterns were obtained after electrophoresis for 10.000
seconds at 6.0} volts per cm.

     

I—d————> ‘——————0

Figure 2. Electrophoretic patterns of normal chicken serum. The
tterns were obtained after electrophoresis for 12.000 seconds at
.h9 volts per cm.

 

nL

Figure 3. Electrophorctic patterns of normal chicker serum. The
patterns were obtained after electrophoresis for 11,?00 seconds
at 6.75 volts per cm.

.14 in

Figure L. Electrophoretic patterns of normal chicken plasma. The
patterns were obtained after electro;horosis for 12.150 seconds at
7.16 volts per cm.

 

       

 

 

 

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.7-

calculated from percentage of total area in the two normal plasma samples,
was 10.6 and 16.1; per sent.

Pig. 2 shows the patterns of serum from apparently normal chickens.
However, since the Y-globulin peak shows a slight bulge in the region
where a new component was found in serum from leucosis-affected chickens,
there is a possibility that the sample was from affected chickens, er at

least a portion of the composite sample was from an affected chicken.

B. Electrophoreticiatterns of serum from leucosis-affected

chickens.

Studies of the various manifestations of the avian-leucosis complex
have resulted in differences of opinion as to whether all are manifestations
of the same fundamental disease and due to a semen causative agent.

Olsen (19) found that none of three experimntal strains produced
only one type of leucosis but that “the transmissible agent of fowl
leukosis is not responsible for conditions known as lymphocytoma or
neurolymphomtosis gallinarum.” In experiments by Stubbs (27) no tumors
were produced, and no paralysis occurred in chickens injected with either
blood or tissues r... birds showing crythroleucosis and myeloid leucosis.
Brandly and associates (2) observed that in six serial passages with "strain
59, lymphomatosis-osteopetrosis, 110.8 per cent of the chickens injected
developed various manifestations of lymphomtosis and of osteopetrosis.

Of the affected chickens, 21.9 Per cent showed evidence of osteopetrosis.
In the contact and non-contact controls 21.2 per cent showed lesions of
lymphomtosis, but only one control chicken showed osteopetrosis.

The results of Lee, lilckc, _e‘_t_ =1. (11) (12) indicate a unity of
etiology of erythroleucosis, mloid leucosis, lymphocytoma, irites, and
neurolymphomatosis gallinarum. They reported that the disease in all its

-5-

manifestations may be transmitted to healthy chicks by injection of tissue
suspensions of affected organs, by injection of cell-free filtrates, by
diract pen contact, or by rearing chicks on contaminated litter. In
another experiment 80 per cent of the chicks hatched from eggs coming from
matings of males and females affected with the iritis form of leucosis
succumbed to some form of leucosis complex, that is, iritis, lymphocytoma,
myeloid leucosis, or osteopetrosis. In similar experiments (6) (21) others
have reached the same conclusions.

In working with a naturally occurring strain of neural lymphomatosis
with leucemic tendencies Hall, _e_t_ _a_l_. (5) reported that after 17 serial
passages through young chicks the strain became an apparently pure leucosis
type. There was a mrked reduction in the incubation period and an increase
in the percentage of takes. Thus, the agent itself my undergo changes with
regard to its pathological manifestation and its qualities.

Serum samples for electrophoretic study were obtained from typical
cases of neurolymphomatosis, visceral lymphomtosis, osteopetrotic lymphomtosis,
and erythrogranuloblastosis. Electrophoretic patterns of these samples (Figs.
5, 6, 7, and 9) show, in addition to the five normal proteins components, a
peak which is designated as the "L” component. The new component is closely
associated with y-globulin and moved with that component in electrophoresis
of the neurolymphomatosis serum. Since the ”L" component was apparently
masked on the descending patterns by the boundary disturbances, calculations
of mobility and relative composition were made from the ascending pattern.
According to Longsworth (16), close agreement of mobility on ascending and
descending patterns may be obtained by using an equation which includes the
ratio of area of the ascending peak to the area of the descending peak and

correction for the e-boundary. This equation could be applied only to those

 

   

r-d———-> <—s-1

Figure 5. Electrophoretic patterns of composite serum from chickens
showing visceral lymphomatosis. The patterns were obtained after
electrophoresis for 12,000 seconds at 6.95 volts per cm.

I—d———> +——a—1

Figure 6. ElectrOphoretic patterns of composite serum from chickens
showing neurolymphomatosis. The tterns were obtained after electro-
phoresis for 12,000 seconds at 6. volts per cm.

ML

‘Fd———_.

 

 

Figure 7. Electrophoretic patterns of composite serum from chickens
showing osteopetrotic lymphomstosis. The patterns were obtained
after electrophoresis for 12,000 seconds at 6. 69 volts per cm.

uh

I—4———-> s————44
Figure 8. Electrophoretic patterns of composite serum from chickens
showing erythrogranulohlastosis. The patterns were obtainol after
electrophoresis for 12,100 seconds at 6.5} volts per cm.

 

 

 

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.9-

patterns in which the area of the ”L" component could be measured on both
ascending and descending patterns.“

In order to sake comparisons of mobility of the “L” component calculations
were made on all ascending patterns and are included in Table 2. The average
value is 2.9 x 10.5. In a later experiment the "L" component was visible
on the descending patterns, and more accurate mobility data were obtained
from these.

Included in Table 3 are relative compositions of components of all
normal and pathological serum studied. A comparison of normal serum with
visceral, neuro-, and osteopetrotic lymphomatosis, and erythrogranuloblastosis
shows significant changes in composition. There was an average decrease in
albumin content from a normal 146.3 to 32.2 per cent; the Oc-globulins
increased from 13.1 to 20.0 per cent, and ’f-globulin decreased from 26.7
to 19.1 per cent. In one sample, neurclymphonatosis, the "L” component
moved with the Y-globulin which accounted for 37.2 per cent of the total
protein. The "L" component represented an average of 10.5 per cent of the
total protein.-

Changes in concentrations of the components resulted in the alteration

of the albumin/globulin ratio from 0.87 to 0.06.

C. Electrophoretic patterns of serum obtained at intervals after

injection with active transmissible tumor.

 

Following the observation of the presence of the '1." component in
serum from chickens showing all the different manifestations of the avian
leucosis complex, the next logical step was to correlate the appearance
and development of symptoms of leucosis with the appearance of the ”L"

component in the blood. This study was performed on the blood of chickens

affected with a transmissible lymphoid tumor. The strain used was the

one recovered by Olsen (20) and termed "R.P.L. 12" by the Regional Poultry
Research Laboratory. Tumors are produced in the. mscle tissues of chickens
within five days after injection and in the visceral organs after a longer
period of time.

Pectoral tumors obtained from three chickens were minced and an equal
volume of 0.85-per cent NaCl added. One ml. of this suspension was in-
jected intramuscularly in susceptible chickens. Composite serum samples
for electrophoresis were obtained from three chickens at one day inter-
vals after injection. The last was collected on the nineteenth day.

The chickens killed on the third day after injection showed no
symptoms or internal lesions. However, the electr0phoretic patterns of
the serum (Fig. 9) show the ”L" component present. The composition data
shown in Table 3 indicate a change in the relative compositions of protein
components. The- albumin content decreased from a normal 146.3 to h3.l per
cent, and V-globulin from a norml 26.5 to 17.1 per cent.

Small tumors were found present in the pectoral miscle in two of
the three chickens examined on the fifth day. in increase in y-globulin
to 31.8 per cent of the total protein indicates that the ”L" component
moved as a fraction of V—globulin (Fig. 10). A mrther‘decrease in
albumin to 38.5 per cent occurred.

From the seventh to the nineteenth day tumors in the pectoral muscles
increased in size in all chickens, and intradermal tumors also were present.
The ”I.” component is present in all ascending patterns, as a separate
fraction in the seventh, thirteenth, and seventeenth day samples (Figs. 11,
13, and 114), and as a component in y-globulin in the nineteenth day

sample (Fig. 15). The albumin content dropped to a minunnm of 32.9 per

n L

t a .___> 2———— a 4|
Figure 9. Electrophoretic patterns of composite serum
collected from chickens three days after injection with
transmissible tumor. The patterns were obtained after
electroyhoresis for 10,220 seconds at 6.19 volts per cm.

10L
{+1.

[— d_————-> <————— a -{
Figure 10. Electrophoretic patterns of composite serum collected
from chickens five days after injection with transmissible tumor.

The patterns were obtained after electrOphoresis for 12,000 seconds
at 5.99 volts per cm.

in L.

Fd——> 2__————a-1
Figure 11. Electrophoretic patterns of composite serum collected
from chickens seven days after injection with transmissible tumor.
The patterns were obtained after electrophoresis for 11,500 seconds
at 6.L2 volts per on.

1.1 L1.

*—
H———> a-1
Figure 12. Electroyhoretic patterns of composite serum collected
fron chickens nine days after injection with transmissible tumor.
The patterns were obtained after electrophoresis for 12,000 seconds
at 5.79 volts per cm.

 

 

 

n L

<4—————————————-—— 0-4
Figure 13. Electrophoretic patterns of composite serum collected
from chickens thirteen days after injection with transmissible
tumor. The patterns were obtained after electrophoresis for
12,260 seconds at 6.33 volts per cm.

.JL

4———a-q
Figure dlh. Electrophoretic patterns of composite serum collected
from chickens seventeen days after injection with transmissible
tumor. The patterns were obtained after electrophoresis for
12,000 seconds at 6. 57 volts per cnu

 

 

1+1.
AWLJ L 0

Figure 15. Electrophoretic patterns of composite serum collected
from chickens nineteen days after injection with transmissible
tumor. The patterns were obtained after electrOphoresis for
12,000 seconds at 6.39 volts per cm.

1
L A

[.4_. 2—0—1
Figure 16. Electrophorctic patterns of a protein
fraction removed from composite serum by precipi-
tation with 15 per cent NaBSC,. Samples were
collected from chickens nineteen days after in-
jection with transmissible tumor. The patterns

were cbtaincd after electrophoresis for 12,000
Succnds at 6.38 volts per cm.

 

 

.151 LA
[-4 :7 4 a

Figure 17. Electrophoretic patterns of normal chicken plasma. The
patterns were obtained after electrophoresis for 12,000 seconds at
6.3 volts per cm.

L L

I—d—_———>

Figure 18. Electrophoretic patterns of composite plasma collected
from chickens injected with transmissible tumor. The patterns were
obtained after electrophoresis for 12,000 seconds at 6.61 volts per cm.

-11..

cent on the seventh day. The albumin/globulin ratio showed a gradual
decrease from 0.76 on the third day to a minumum OJi9 on the seventh.

Calculations on the ascending patterns (Table 2) show the average
mobility of the "L" component to be -3.0 x 10.5 as compared with the
average -2.9 x 10'5 in the earlier studies of serum from chickens showing
different types of the avian leucosis complex. The ”L" component occurs
as a separate peak in both ascending and descending patterns shown in
Figs. 11 and 153 a more accurate mobility value for the "L" component was
calculated from these patterns , giving an average of -2.5 x 10.5 cn’?’ sect'l
volt'l.

The early appearance of the "L" component in the serum and the early
shift in composition indicate that there is a rapid change in the blood
serum proteins of chickens affected with the transmissible tumor agent”

Two electrophoretic patterns of plasma samples from apparently
normal chickens (Figs. h, and 17) were compared with the patterns of a
plasma sample from chickens 19 days after injection with a tumor producing
agent (Fig. 18). The comparison revealed the same decrease in the albumin/
globulin ratio that was observed in serum (Table 3). The normal plasma
contained 114.1 per cent fibrinogen; the increase to 27.1.. per cent of this
component in the leucosis plasma indicates tint the ”L" component moved

at approximately the same rate as fibrinogen.

D. Electrophoretic patterns of serum obtained at intervals after
ingeetien with inactivated transmissible tumor.
The inactivated mterial was prepared from the tumor-produc ing agent

described in Section c, by rapidly freezing to 46%: and thawing twice.

-12-

As in the previous experiment, a composite serum sample for electrophoretic
study was obtained from three chickens at one day intervals after injection.
Tissues taken at necropsy showed no evidence of tumor formation in any of
the chickens injected with the inactivated material.

The electrophoretic pattern of the serum sample collected on the
seventh day after injection (Fig. 19) is apparently normal. Calculations
on the ascending pattern (Table 3) show a decrease in the albumin below
that observed in the normal. The fifteenth day sample (Fig. 21) shows
the presence of the '1." component, accouxflsing for 11.9 per cent of the
total protein in the sample. There is close agreement between the
mobility value -2.6 x 10"5 of the "L” component in this serum with
that found in serum samples from affected chickens which was -2.5 x 10-5.
The relative compositions of the serum with respect to albumin remained
virtually the same as that found on the seventh day, but the Yoglobulin
decreased to 18.6 per cent.

The electrophoretic patterns of the sample collected on the nineteenth
day after injection (Fig. 22) show the ”L” component present and moving
with the i-globulin as evidenced by an increase of that component to
30.3 per cent of total protein.

In Table )4 are sent forth the comparative mobility. composition,
and ratio values of the proteins in a composite serum sample from normal
chickens, one from chickens injected with active tumor agent, and one
from chickens injected with inactivated transmissible tumor agent. These
values, as calculated from the descending patterns, are more accurate than

those calculated for the ascmding patterns and shown in Table 3.

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Figure I”l9. Electrophoretic patterns of ccmp.site serum collected

from chickens seven days after injection with inactivated transmissible
tumor. The patterns were obtained after electrophoresis for 12.620
seconds at 5.88 volts per cm.

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Figure 20. Electrophoretic patterns of composite serum collect d from
chickens fifteen days after injection with inactivated transmissible‘
tumor. The patterns were obtained after electrophoresis for 12, 350
seconds at 6.h7 VOlts per cm.

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Figure 21. Electrophoretic patterns of composite serum collected from
chickens nineteen days after injection with inactivated transmissible
tumor. The patterns were obtained after electrophoresis for 12.000
seconds at 6.614 volts per cm.

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-13-

DISCUSSION

The electrophoretic analysis of the proteins in serum from chickens
affected with leucosis revealed the presence of a protein component which
is not found in normal chicken serum. The new component is closely
associated with Y-globulin and has been desigmted by the letter ”L".

The ‘1." component in blood serum samples taken from chickens that
showed gross pathological conditions of leucosis represented approximately
10 per cent of the total serum proteins. It does not seem likely that the
protein of the leucosis agent would be present in blood serum in such a
high concentration. By crude electrophoretic methods, Lee and Wilcke
(10) studied the filtrate of a tumor-producing material and reported that
the causative agent of fowl leucosis migrated to the cathode at pH 14.01
to 6.01 and to the anode at pH 7.01 to 9.01. They found that the causative
agent was inactivated at a pH of h and 9.

Previous electrophoretic studies of blood serum have shown that the
antibody proteins are closely associated with V-glcbulin and migrate as
a component of that globulin or as a separate fraction of a mobility
slightly greater than V-glcbulin (22) (23) (28) (31). Tiselius (29)
found that the pneumoccous carbohydrate-precipitating serum-antibody
produced in the horse migrated as a new component, and a similar antibody
produced in the rabbit appeared as an addition to the normal Y-globulin.
The new component was no longer present in horse serum after the antibody
had been removed by homologous type specific polysaccharide or by a heavy
suspension of specific pneumococci. In the rabbit antiserum the amount of

V-globulin increased to 56 per cent of the total protein as compared with
17 per cent in normal serum. Pneumococcus antibody was shown by Van der

Scheer and associates (30) to migrate with the 7-globulin component of

-m.

serum from 11. out of 15 hyperinnnunised horses. In the one exception,
shortly after hyperimmunisation, the antibody protein migrated as a
separate component between ,6 -and V-glcbulins.‘ The mobility was

-2.1 x 10'”5 cm‘3 sec."1 volt":l

in a .02! phosphate buffer solution of
pH 7.6. This mobility is very close to that calculated for the ”I."
component in serum from leucosis affected chickens. The mobility of
the 'T" component demonstrated in diphtheria and in tetanus antitcxin
from the horse was between that of )3 -and Y-globulins (32).

Shapiro and associates (24) separated a viscous protein from the
blood plasma of a patient with multiple myelcma; the pattern of the
extra protein appeared between I -and V-globulin and moved with
fibrinogen.

It is postulated thatthe new ”L" component described herein is
of the nature of an antibody protein and occurs in all types of the
avian leucosis complex. Also. it my be produced by the injection of
an inactivated transmissible tuner.

Due to the position of the Y-glcbclin disturbance noted in all
chicken serum, it is not improbable that one would find evidence of
the 'L" component in serum from normal chickens or from chickens that
have been exposed to infective material.

The occurrence of the "L"

component in serum after the injection
of inactivated tumor material suggests further investigations into its
inmmnological and diagnostic possibilities. The presence of the new
component suggests that it might be possible to develop an active
immunity against the disease or that the antibody—like substance may
be used in the development of a diagnostic test for the detection of

the disease.

-15-

Production of neutralizing antibodies in serum against leucosis
agents has been demonstrated by the protective value of such a serum
against the disease. According to Rabat and Forth (8), the injection
of materials from fowl tumors into rabbits will produce virus-
neutralizing antibodies in rabbits. Lee (9) produced protective
antiserum by the immunization of ducks and turkeys with repeated
injections of leucosis material. The antiserum obtained protected
susceptible chickens against disease-producing doses of leucosis
agent. The results of his experiments lend support to the hypothesis
that the ”L” component found in the serum of chickens affected with
different terns of leucosis is antibody protein. lee states,
"Neutralization of the agent of one type or form of the disease by
duck or turkey serum produced by a series of injections of infectious
mterial from lesions of a different form or type of the complex more
closely indicates the presence of a single etiology.”

Since the electrophoretic patterns of chicken serum show that the
"L“ component is closely associated with V-glcbulin, it was thought
possible that the salting out of the 7/-globulin might also remove
the antibody protein. This was attempted by adding 30-per cent Hagso‘
to an equal volume of serum from chickens showing leucosis tumors.
The precipitated globulin was centrifuged, washed, dissolved, and
dialysed against 0.85-per cent sodium chloride solution. The protein
solution was diluted to a protein concentration of 2 per cent. The
electrOphoretio pattern of this fraction as shown in Fig. 16 indicates
apparently pure V-globulin. There was no separation of the "L"

component.

-16-

SUMMARY

A cowarative electrophoretic analysis by the moving boundary
method has been made of the proteins in serum and plasma from normal
chickens and from chickens affected with avian leucosis. The average
mobilities of the constituents in normal serum and plasma, as found
under the conditions employed in this study, are as follows: albumin,
-5.9 x 10'53 09, t-globulin, 5.0 x 10-53 02:44.14 1 10.53%:‘35 x 10.53
7,-2.0 1 10.5; fibrinogen, -2.5 x 10'"5 cm’3 sec.”1 volt-1. The
average relative concentrations of the components in norml serum with
respect to total protein are: albumin, 143.8 per cent; a‘v’rl' act-globulin,
19.3 per cent; )3 ,13.0 per cent; 7’, 23.9 per cent.

A new component, designated the "L” component, was found in serum
from chickens affected with the several forms of leucosis. The new

1 .volt'1

protein component has a mobility -2.55 x 10'5 on” see." and
represents approximately 10 per cent of the total protein in the serum.
The relative composition of the components in leucosis serum
- (Table 3) as compared with the normal showed a decrease in albumin
and V-globulin and an increase in “-globulins. The electrophoretic
patterns in which the new component failed to migrate as a separate
peak show a very high concentration of V-globulin. This fact would
indicate a close relationship between the "L“ component and V-globulin.
A series of blood samples drawn from chickens at intervals after
injection with an active transmissible tumor agent share the presence
of the ”L" component as early as the third day. Chickens injected will:
the inactivated tumor agent also show the presence of the ”L" component
but not until the fifteenth day after injection.
Attempts to separate the new protein component from 7/-globulin by

salting out methods were unsuccessful.

-17..

The presence of the "L" component in the serum from leucosis-affected

chickens has immnologioal and diagnostic significance.

ACKNOWLEDGMENT
I wish to thank Dr. I. F. Huddleson, Dr. P. J. Schaible, and
members of the staff of the Poultry Research Laboratory for suggestions

and assistance in this project.

l.

2.

3.

h.

5.

7.

8.

9.

10'.

LITERATURE CITED

Abramson, H. A., L. S. Moyer and M. H. Gorin. Electrophoresis of
Proteins. Reinhold Publishing Corp., New York (1912).

Brandly, C. A., N. 31. Nelson and G. E. Cottral. Serial passage of
lymphomatosis-osteopetrosis in chickens. Am. J. Vet. Res.

2,. 289 (19M).

Cottral, c. E. Typical cases illustrating the main clinical,
necropsy, and histopathological findings for the various
mnifestations of the avian-leukosis complex. Collaborators
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Gray, 8. J. and E. S. G. Barron. The electrophoretic analyses of
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Hall, W. J., C. W. Bean, and Morris Pollard. Transmission of fowl
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Johnson, E. P. Fowl leukosis. Ve. Agri. Exp. Sta. Bul. 76 (min).

Jungherr, E. Tentative pathologic nomenclature, etc. Am. J. Vet.
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Kabat, E. A. and J. F‘irth. Neutralization of the agent causing
leukosis and sarcoma of fowls by rabbit antisera. J. Exp.
see. 11,. 257 (19M). ,

Lee, C. D. Studies on the production of specific antibodies against
the agent of fowl leucosis. Amer. J. Vet. Res. _3_, 336 (19h2).

Lee, C. D. and H. I... Wilcke. The migration of the etiologic agent
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-19-

Lee, C. D. and H. L. Wilcke. Transmission experiments with iritis
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Lee, 0. 1)., H. L. Wilcke, c. mil-ray and s. 11'. Henderson. Fowl
leukosis. J. Infect. Dis. _6_Z_l_, l (1937).

Longsworth, L. G. The observation of electrophoretic boundaries.
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Longsworth, L. G. Recent advances in the study of proteins by
electrophoresis. Chem. Rev. .32, 323 (19142).

Longsworth, L. G. and D. A. Maclnnes. Electrophoresis of proteins
by the Tiselius method. Chem. Rev. 3)"? 271 (1939).

Longsworth, L. G. and D. A. Machines. The interpretation of simple
electrophoretic patterns. J. An. Chem. Soc. fig, 705 (191.10).

Longsworth, L. G., and D. A. Ethnnes. An electrophoretic study
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Longsworth, L. G., T. Shedlovsky and D. A. Eaclnnes. Electrophoretic
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Olson, Carl. A study of transmissible fowl leukosis. J..Am. V.t..
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Patterson, F. D., H. L. Wilcke, C. Murray and E. F. Henderson.
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.20-

Shapiro, 8., V. Ross and D. H. Moore. A viscous protein obtained
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electrophoretic examination of digested sera. J. Immunol.

9.3,. 31.9 (1911).

 

 

 

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