- Email: [email protected]
Effect of bursectomy on development of a spontaneous postnatal amelanosis
C L INI<
41
IMLlL’NOI.OGY
AND
21, 407-411
IMMUNOPAl’HDLOGY
(1981)
BRIEF COMMUNICATION Effect
The vitiligo. terized
of Bursectomy on Development Postnatal Amelanosis
delayed The
amelanotic DAM line
by the absence
(DAM) displays of normal
day of hatching significantly examined 12 to 20 weeks phocytes play an important
chicken line a spontaneous
has
pigmentation
in emerging
decreased after hatching. role in the
been postnatal
the
incidence The results development
of a Spontaneous
developed as a model amelanosis which feathers.
Bursectomy
of human is characon the
and severity of amelanosis in birds suggest that bursa-dependent lymof delayed amelanosis in the DAM
chicken.
INTRODUCTION
The delayed amelanotic (DAM) chicken line displays a high incidence of spontaneous feather amelanosis associated with a high incidence of ocular amelanosis and occasional integumentary defects (I). Hypothyroidism is also present in a greater than normal frequency in the DAM line. These characteristics resemble the clinical picture of human patients with vitiligo, a disease characterized by a spontaneous loss of cutaneous melanin (2-6). The chicken represents an excellent model for investigation of the immune system because of an anatomical and functional dissociation into bursa-dependent and thymus-dependent compartments (7, 8). The thymus-dependent system is basic to immunological recognition which is manifested in graft vs host reactions, delayed hypersensitivity, and homograft rejection (9, 10). The thymic system also plays a role in the response by B cells to some antigens, and is represented morphologically by generally small lymphoid cells. In contrast, the bursadependent system is responsible for the production of antibody and is represented by plasma cells and larger lymphoid cells such as those found in germinal centers. The purpose of this investigation was to determine the role of the bursadependent compartment of the immune system on the development of delayed amelanosis in the DAM chicken line. MATERIALS
AND METHODS
Animuls. All birds used were of the delayed amelanotic (DAM) chicken line, developed at the University of Massachusetts Poultry Research Center (1). Bursectomized and unoperated hatchmate control birds were all produced by the same pen mating. Animals were housed together in battery-type cages and allowed free access to food and water. 407 009@1229/81/120407-05$01.0010 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved
40X
BRIEI-
C‘OMMUNIC
A I ION
Bursectom~. Chicks were surgically bursectomized on the day of hatch, under sodium barbitol anesthesia ( II). The completeness of the bursectomy was evaluation by challenging birds at I2 weeks of age with 0.5 ml of 509: SRBC. Seven days later, birds were bled and the level of circulating anti-SRBC antibody was determined by a direct microhemagglutination assay ( 12). All unoperated birds showed titers of 5 or greater. Only those operated birds having anti-SRBC titers of 3 or less (background levels) were included in evaluation of the bursectomized group. E\*rrlucrtion (!f’ ott~c~lutto.~i,s. Bursectomized and unoperated hatchmate control birds were inspected biweekly for onset and extent of amelanosis. Birds were classified as ameianotic when any emerging feathers were white, rather than normally pigmented. The severity of amelanosis was classified according to the percentage of total plumage which was amelanotic. Birds were assigned to one of three groups, consisting of birds displaying less than lOc7( amelanosis, lo-90% amelanosis. or greater than 9OV amelanosis.
RESULTS A comparison of the percentages of birds exhibiting amelanosis in the bursectomized and hatchmate control groups was made (Table I). The difference in incidence of amelanosis between the bursectomized and control groups was significant at 12 weeks V’ < 0.005), 16 weeks (P < O.Ol), and 20 weeks (P < 0.005). Thus, it is evident that surgical bursectomy on the day of hatching significantly reduced the incidence of amelanosis in DAM line chickens. The mean onset time of amelanosis for affected control birds was 11.6 weeks, while it was slightly later for affected bursectomized birds, 12.4 weeks. The observation period was terminated at 20 weeks because the bursectomized birds exhibited high mortality after that age. There was, however, no increase in the incidence of amelanosis in the control group after 20 weeks, suggesting that the onset of amelanosis in this population had reached its full expression by this time. Sc~l~rrity
of Attlrlat1osi.r
A comparison of the severity with which birds were affected by amelanosis was made (Table 2). Grouping birds according to the percentage of their plumage that
Percentage 8 Weeks
of birds
exhibiting
amelanosih
12 Weeks
16 Week5
20 Week\
Bursectomized
13.9
77.8
50.0
55.6
II = 36 Control
77 7 --.-
51.9
70.3
77.x
P c 0.005
P <. 0.01
P _ 0.00s
I, = 27 n.b.
P value” ” x2 test
with
control
birds
representing
the
expected
frequencies
of amelanosis.
per group
21
31
2
2 “.S.
3 4
” No visible amelanosis. ‘, Less than IO? of total plumage is amelanotic. ’ IO-9OV of total plumage is amelanotic. ‘I Greater than 9OQ of total plumage is amelanotic. ’ y2 test with control birds representing the expected
Control P value’
Number of birds Bursectomized
0”
0
0
~ 3” ~-
degree
P < 0.025
4
5 10
0
0
3
P c 0.005
3
8
I
of amelanosis.
8
I8
~ . 0
16 Weeks
OF AMELANOSIS
of amelanosis
of severity
5
12 Weeks .~~ ~~ I 2
of each
II
26
0
-~~
Severity
TABLE 2 BLVISEC.TOMY ON THE SFVERITY
frequencies
OF NEOIU~TZI
8 Weeks .___ I lJ 2”
EFFECT
I6
9
2
0
I
3
6
I6
0
I2
2
s I3 P c 0.01
7
I
20 Weeks
3
I
3
s 5 z
:: zi
z
::
!z F; tl
410
BRIEF
COMMUNIC
A r‘lON
was amelanotic demonstrated significant differences between the bursectomized and control groups at 12 weeks (P < 0.0259, 16 weeks (P c 0.005). and 20 weeks (P < 0.01). Removal of the bursa reduced the severity of amelanosis. Thus, bursectomy influenced not only the incidence of amelanosis, but also the severity of expression of the disease in DAM line chickens. DISCUSSION
Neonatal bursectomies were performed to investigate the role of the bursadependent compartment of the lymphoid system on the development of delayed amelanosis in the DAM chicken. The present study demonstrated that neonatal bursectomy significantly decreased the incidence and severity of amelanosis. This suggests that bursa-dependent cells may play a major role in the development of the disease. The observation that bursectomy reduced, but did not eliminate, the development of amelanosis may be attributable to different causes. For example, at the time of hatching, the migration of some bursal cells to the peripheral lymphoid organs has already occurred (13). This may allow a slight expression of bursal-dependent functions in neonatally bursectomized birds. Alternatively, bursal-dependent cells may be only one of several undetermined factors involved in the pathogenesis of delayed amelanosis. The influence of neonatal bursectomy on delayed amelanosis is similar to the effect of this operation on one established spontaneous autoimmune disease. The Obese strain (OS) of chickens is well documented as a model of hereditary spontaneous autoimmune chronic thyroiditis (review (14)). In OS birds, neonatal bursectomy decreased the incidence and severity of the disease (15), a situation similar to that occurring in DAM line birds. Thymectomy of OS birds increased the incidence and severity of thyroiditis (16). The effect of thymectomy on DAM line birds is currently under investigation. An autoimmune reaction has been hypothesized as one possible cause of amelanosis in the DAM line (1). Numerous immune cells have been observed to be associated with melanocyte destruction and elimination in the choroid of amelanotic birds ( 17). While further experimentation is necessary to elicit the exact mechanisms by which bursal-dependent cells are involved in the pathogenesis of delayed amelanosis in DAM chickens, the present study establishes a role for this cell compartment in the disease. Neonatal bursectomy significantly reduced the OCcurrence and severity of delayed amelanosis. indicating that bursal-derived cells play an important role in the development of this spontaneous postnatal amelanosis. ACKNOWLEDGMENTS This work Station.
was supported
by NIH
Grant
AM25252
and the Massachusetts
Agricultural
Experiment
REFERENCES I. 2. 3. 4. 5.
Smyth, Albert, BO~SSY, Lerner, Lerner,
J. R., Jr., Boissy, R. E., and Fite. K. V., J. Hrwd. 72, 150, D. M., Nordlund, J. J.. and Lerner, A., Op/ha/,no/~,~;c,rr 86, R. E.. Fite. K. V., and Smyth. J. R.. Jr., Ytr/~ .f. Bio/. Met/. A. B.,A~zrr. J. Med. 51, 141. lY71. A. B.. Nordlund. J. J., and Albert, D. M., N. &g/. J. ,$fe‘/.
1981. 1145, 1979. 50, (Suppl.issue5). 296,
232. 1977.
197.
BRIEF
COMMUNICATION
411
6. Smyth, J. R., Jr., Boissy, R. E., Fite. K. V.. and Albert, D. M.. IIII.~Y~. Op/rtlru[/&. Vjs~~l/ SC.;. 20, 799, 1981. 7. Szenberg, A., and Warner, N. L.. Nature (London) 194, 146, 1962. 8. Warner. N. L., Szenberg, A., and Bumet, F. M., Aust. J. E~J. Bio/. ,uet/. .‘j~~c. 40, 373. 1962. 9. Cooper, M. D., Peterson, R. D. A.. and South, M. A., Nrrturc NPW B;o/. 2135, 143, 1965. JO. Cooper. M. D., Peterson, R. D. A.. South, M. A., and Good, R. A.. ./. E.ITJJ. Med. 123,75. 1966. 11. Peterson, R. D. A., Burmeister, B. R.. Frederickson, T. N.. Purchase, H. G., and Good. R. A.../. Nut. C‘uncer Inst. 32, 1343, 1964. 12. Wegman. T., and Smithies, 0.. Trumfitsion 6, 67, 1966. 13. Cooper, M. D., Cain, W. A., Van Alten, P. J., and Good, R. A.. Irlr. Arc./r A//crgy 35, 242, 1969. 14. Wick, G., Sundick. R., and Albini, B., C/in. I/n/,rltn~/. IIIIItINfIfPUthO/. 3, 272, 1974. 15. Wick, G., Kite, J. H., Cole, R. K., and Witebsky, E.. .I. I,t~rtntr~~,/. 104, 45. 1970. 16. Wick. G., Kite, J. H., Jr., and Witebsky, E.. .I. Ifmnu&. 104, 54, 1970. 17. Boissy. R. E., Master’s thesis, University of Massachusetts, Amherst, 1979. Received
May
6, 1981; accepted
with
revisions
July 20. 1981
41
IMLlL’NOI.OGY
AND
21, 407-411
IMMUNOPAl’HDLOGY
(1981)
BRIEF COMMUNICATION Effect
The vitiligo. terized
of Bursectomy on Development Postnatal Amelanosis
delayed The
amelanotic DAM line
by the absence
(DAM) displays of normal
day of hatching significantly examined 12 to 20 weeks phocytes play an important
chicken line a spontaneous
has
pigmentation
in emerging
decreased after hatching. role in the
been postnatal
the
incidence The results development
of a Spontaneous
developed as a model amelanosis which feathers.
Bursectomy
of human is characon the
and severity of amelanosis in birds suggest that bursa-dependent lymof delayed amelanosis in the DAM
chicken.
INTRODUCTION
The delayed amelanotic (DAM) chicken line displays a high incidence of spontaneous feather amelanosis associated with a high incidence of ocular amelanosis and occasional integumentary defects (I). Hypothyroidism is also present in a greater than normal frequency in the DAM line. These characteristics resemble the clinical picture of human patients with vitiligo, a disease characterized by a spontaneous loss of cutaneous melanin (2-6). The chicken represents an excellent model for investigation of the immune system because of an anatomical and functional dissociation into bursa-dependent and thymus-dependent compartments (7, 8). The thymus-dependent system is basic to immunological recognition which is manifested in graft vs host reactions, delayed hypersensitivity, and homograft rejection (9, 10). The thymic system also plays a role in the response by B cells to some antigens, and is represented morphologically by generally small lymphoid cells. In contrast, the bursadependent system is responsible for the production of antibody and is represented by plasma cells and larger lymphoid cells such as those found in germinal centers. The purpose of this investigation was to determine the role of the bursadependent compartment of the immune system on the development of delayed amelanosis in the DAM chicken line. MATERIALS
AND METHODS
Animuls. All birds used were of the delayed amelanotic (DAM) chicken line, developed at the University of Massachusetts Poultry Research Center (1). Bursectomized and unoperated hatchmate control birds were all produced by the same pen mating. Animals were housed together in battery-type cages and allowed free access to food and water. 407 009@1229/81/120407-05$01.0010 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved
40X
BRIEI-
C‘OMMUNIC
A I ION
Bursectom~. Chicks were surgically bursectomized on the day of hatch, under sodium barbitol anesthesia ( II). The completeness of the bursectomy was evaluation by challenging birds at I2 weeks of age with 0.5 ml of 509: SRBC. Seven days later, birds were bled and the level of circulating anti-SRBC antibody was determined by a direct microhemagglutination assay ( 12). All unoperated birds showed titers of 5 or greater. Only those operated birds having anti-SRBC titers of 3 or less (background levels) were included in evaluation of the bursectomized group. E\*rrlucrtion (!f’ ott~c~lutto.~i,s. Bursectomized and unoperated hatchmate control birds were inspected biweekly for onset and extent of amelanosis. Birds were classified as ameianotic when any emerging feathers were white, rather than normally pigmented. The severity of amelanosis was classified according to the percentage of total plumage which was amelanotic. Birds were assigned to one of three groups, consisting of birds displaying less than lOc7( amelanosis, lo-90% amelanosis. or greater than 9OV amelanosis.
RESULTS A comparison of the percentages of birds exhibiting amelanosis in the bursectomized and hatchmate control groups was made (Table I). The difference in incidence of amelanosis between the bursectomized and control groups was significant at 12 weeks V’ < 0.005), 16 weeks (P < O.Ol), and 20 weeks (P < 0.005). Thus, it is evident that surgical bursectomy on the day of hatching significantly reduced the incidence of amelanosis in DAM line chickens. The mean onset time of amelanosis for affected control birds was 11.6 weeks, while it was slightly later for affected bursectomized birds, 12.4 weeks. The observation period was terminated at 20 weeks because the bursectomized birds exhibited high mortality after that age. There was, however, no increase in the incidence of amelanosis in the control group after 20 weeks, suggesting that the onset of amelanosis in this population had reached its full expression by this time. Sc~l~rrity
of Attlrlat1osi.r
A comparison of the severity with which birds were affected by amelanosis was made (Table 2). Grouping birds according to the percentage of their plumage that
Percentage 8 Weeks
of birds
exhibiting
amelanosih
12 Weeks
16 Week5
20 Week\
Bursectomized
13.9
77.8
50.0
55.6
II = 36 Control
77 7 --.-
51.9
70.3
77.x
P c 0.005
P <. 0.01
P _ 0.00s
I, = 27 n.b.
P value” ” x2 test
with
control
birds
representing
the
expected
frequencies
of amelanosis.
per group
21
31
2
2 “.S.
3 4
” No visible amelanosis. ‘, Less than IO? of total plumage is amelanotic. ’ IO-9OV of total plumage is amelanotic. ‘I Greater than 9OQ of total plumage is amelanotic. ’ y2 test with control birds representing the expected
Control P value’
Number of birds Bursectomized
0”
0
0
~ 3” ~-
degree
P < 0.025
4
5 10
0
0
3
P c 0.005
3
8
I
of amelanosis.
8
I8
~ . 0
16 Weeks
OF AMELANOSIS
of amelanosis
of severity
5
12 Weeks .~~ ~~ I 2
of each
II
26
0
-~~
Severity
TABLE 2 BLVISEC.TOMY ON THE SFVERITY
frequencies
OF NEOIU~TZI
8 Weeks .___ I lJ 2”
EFFECT
I6
9
2
0
I
3
6
I6
0
I2
2
s I3 P c 0.01
7
I
20 Weeks
3
I
3
s 5 z
:: zi
z
::
!z F; tl
410
BRIEF
COMMUNIC
A r‘lON
was amelanotic demonstrated significant differences between the bursectomized and control groups at 12 weeks (P < 0.0259, 16 weeks (P c 0.005). and 20 weeks (P < 0.01). Removal of the bursa reduced the severity of amelanosis. Thus, bursectomy influenced not only the incidence of amelanosis, but also the severity of expression of the disease in DAM line chickens. DISCUSSION
Neonatal bursectomies were performed to investigate the role of the bursadependent compartment of the lymphoid system on the development of delayed amelanosis in the DAM chicken. The present study demonstrated that neonatal bursectomy significantly decreased the incidence and severity of amelanosis. This suggests that bursa-dependent cells may play a major role in the development of the disease. The observation that bursectomy reduced, but did not eliminate, the development of amelanosis may be attributable to different causes. For example, at the time of hatching, the migration of some bursal cells to the peripheral lymphoid organs has already occurred (13). This may allow a slight expression of bursal-dependent functions in neonatally bursectomized birds. Alternatively, bursal-dependent cells may be only one of several undetermined factors involved in the pathogenesis of delayed amelanosis. The influence of neonatal bursectomy on delayed amelanosis is similar to the effect of this operation on one established spontaneous autoimmune disease. The Obese strain (OS) of chickens is well documented as a model of hereditary spontaneous autoimmune chronic thyroiditis (review (14)). In OS birds, neonatal bursectomy decreased the incidence and severity of the disease (15), a situation similar to that occurring in DAM line birds. Thymectomy of OS birds increased the incidence and severity of thyroiditis (16). The effect of thymectomy on DAM line birds is currently under investigation. An autoimmune reaction has been hypothesized as one possible cause of amelanosis in the DAM line (1). Numerous immune cells have been observed to be associated with melanocyte destruction and elimination in the choroid of amelanotic birds ( 17). While further experimentation is necessary to elicit the exact mechanisms by which bursal-dependent cells are involved in the pathogenesis of delayed amelanosis in DAM chickens, the present study establishes a role for this cell compartment in the disease. Neonatal bursectomy significantly reduced the OCcurrence and severity of delayed amelanosis. indicating that bursal-derived cells play an important role in the development of this spontaneous postnatal amelanosis. ACKNOWLEDGMENTS This work Station.
was supported
by NIH
Grant
AM25252
and the Massachusetts
Agricultural
Experiment
REFERENCES I. 2. 3. 4. 5.
Smyth, Albert, BO~SSY, Lerner, Lerner,
J. R., Jr., Boissy, R. E., and Fite. K. V., J. Hrwd. 72, 150, D. M., Nordlund, J. J.. and Lerner, A., Op/ha/,no/~,~;c,rr 86, R. E.. Fite. K. V., and Smyth. J. R.. Jr., Ytr/~ .f. Bio/. Met/. A. B.,A~zrr. J. Med. 51, 141. lY71. A. B.. Nordlund. J. J., and Albert, D. M., N. &g/. J. ,$fe‘/.
1981. 1145, 1979. 50, (Suppl.issue5). 296,
232. 1977.
197.
BRIEF
COMMUNICATION
411
6. Smyth, J. R., Jr., Boissy, R. E., Fite. K. V.. and Albert, D. M.. IIII.~Y~. Op/rtlru[/&. Vjs~~l/ SC.;. 20, 799, 1981. 7. Szenberg, A., and Warner, N. L.. Nature (London) 194, 146, 1962. 8. Warner. N. L., Szenberg, A., and Bumet, F. M., Aust. J. E~J. Bio/. ,uet/. .‘j~~c. 40, 373. 1962. 9. Cooper, M. D., Peterson, R. D. A.. and South, M. A., Nrrturc NPW B;o/. 2135, 143, 1965. JO. Cooper. M. D., Peterson, R. D. A.. South, M. A., and Good, R. A.. ./. E.ITJJ. Med. 123,75. 1966. 11. Peterson, R. D. A., Burmeister, B. R.. Frederickson, T. N.. Purchase, H. G., and Good. R. A.../. Nut. C‘uncer Inst. 32, 1343, 1964. 12. Wegman. T., and Smithies, 0.. Trumfitsion 6, 67, 1966. 13. Cooper, M. D., Cain, W. A., Van Alten, P. J., and Good, R. A.. Irlr. Arc./r A//crgy 35, 242, 1969. 14. Wick, G., Sundick. R., and Albini, B., C/in. I/n/,rltn~/. IIIIItINfIfPUthO/. 3, 272, 1974. 15. Wick, G., Kite, J. H., Cole, R. K., and Witebsky, E.. .I. I,t~rtntr~~,/. 104, 45. 1970. 16. Wick. G., Kite, J. H., Jr., and Witebsky, E.. .I. Ifmnu&. 104, 54, 1970. 17. Boissy. R. E., Master’s thesis, University of Massachusetts, Amherst, 1979. Received
May
6, 1981; accepted
with
revisions
July 20. 1981