Research in Veterinary Science 1992, 52, 44-47
Immunoglobulin classes synthesised by the chicken Harderian gland after local immunisation M. GALLEGO, E. del CACHO, C. FELICES, J. A. BASCUAS, Departamento de Patologia Animal, Histologia y Anatomia Patologica, Facultad de Veterinaria, Miguel Server, 177, 50013Zaragoza, Spain
The immunoglobulin (Ig) levels in tears and sera were compared after antigen administration (salmonella O antigen) by eyedrop and injection into the nictitating membrane, to determine the lg classes synthesised by the plasma cells in the chicken Harderian gland. Samples of tears and sera were collected from immunised and control birds between 24 hours and 24 days after the antigen or sterile saline was administered. Samples were assayed for IgA, IgG and IgM concentrations using radial immunodiffusion. It is suggested that most of the IgG found in tears after local immunisation has an extraglandular origin.
THE Harderian gland of the chicken is the major contributor to locally produced antibodies which are active in protecting the upper portion of the respiratory tract (Davelaar et al 1982). Several groups have carried out studies on the immunoglobulin (Ig) classes produced by this gland. The presence of specific IgA and IgG antibodies against infectious bronchitis virus in tears of chickens vaccinated with this virus has been demonstrated (Davelaar et al 1982). Burns (1977) reported the presence of anti-BSA IgG and IgA antibodies after eyedrop stimulation with bovine serum albumin (BSA); specific IgM antibodies, on the other hand, were not detected. However, in naive chickens the type of antibodies produced by the Harderian gland plasma cells is not clear. According to immunofluorescence studies by Albini et al (1974) 48 per cent of lymphoid cells were positive for IgA, 22 per cent for IgG and 12 per cent for IgM. Bienenstock et al (1973) reported the prevalence oflgM and IgG, whereas IgA positive plasma cells were scarce. Mansikka et al (1989) demonstrated that unstimulated chickens have high levels of IgM, IgA and IgG
in tears and after ocular stimulation specific IgG and IgA antibodies appeared but IgM antibodies were barely detectable. The objective of this work was to compare the Ig levels in tears and serum after two different types of antigen administration: eyedrop and injection into the nictitating membrane, to determine the antibody classes synthesised by the plasma cells in the chicken Harderian gland. Materials and methods
Animals and immunisations Seven-week-old White Leghorn chickens were used. The birds were immunised with salmonella O antigen (Difco), 1.8 x 1010 organisms m1-1, diluted v/v in sterile saline, by injection into the nictitating membrane or by dropping it onto the eyeball. Fifteen chickens were injected twice, nine days apart, into the nictitating membrane (as close as possible to the formix) with 0.02 ml diluted commercial solution of salmonella O antigen. Fifteen control birds were injected at the same interval into the nictitating membrane with 0.02 ml sterile saline. Diluted commercial solution of salmonella O antigen was dripped twice, nine days apart, onto the eyeball (0.5 ml per eye) of 15 chickens. Sterile saline (0.5 ml) was dripped onto each eye of 15 control birds following the same pattern used with the salmonella O antigen.
Tear and blood collection Tears were collected using a 100 ml capillary tube held against the inner canthus of the eye. Manipulation of the third eyelid back and forth 44
Immunoglobulins synthesised by Harderian gland was sometimes necessary to facilitate the flow of tears. Chemical induction of lacrimation was not used. Chickens were bled by wing puncture. The volume of blood was 1 ml. Tears and blood were collected from every chicken, immunised and control, at 24 hours, two, four, five, six, seven, nine, 10, 11, 12, 14, 16, 18, 20, 22 and 24 days after the first administration of both the injection and eyedrop of salmonella O antigen and sterile saline.
Quantitation of Igs The Ig levels of blood and tears were determined by the radial immunodifusion method (RID) (Catty and Raykundalia 1989). The antisera used were goat anti-chicken IgM, rabbit antichicken IgG and goat anti-chicken IgA (ICN Biomedicals). They were characterised by immunoelectrophoresis and their specificity was checked by immunodiffusion. Optimal concentrations of antisera were 1 per cent for anti-IgM, 0.5 per cent for anti-IgA and 2.5 per cent for anti-]gG (Kowalski et al 1978). The upper and lower limits of sensitivity of RID were determined in relation to a previously calibrated standard chicken serum. Minimum detectable levels were 20 ~tg ml 1 for IgM, IgG and IgA. The diffusion diameters were measured and the results were plotted on a linear scale.
Antibody titration
45
Results
Higher levels of specific antibody were found in both sera and tears after eyedrop application (Figs 1 and 2). Following the eyedrop application 8
6t I(.5 4 --
2 0 1
r AI
I
Tlo
5
15
'
J
I
20
25
30
Days FIG 1 :Agglutinating antibody levels in serum. Antigen administration by eyedrop (1~) and by injection (¢) twice at a nine day interval. Antigen administration { .~-). Geometric mean titres (GMT). Control Birds (El)
8 -
~_ 4
The antibodies in serum and tears were titrated following the microtitration technique proposed by Williams and Whittemore (1972). A commercial solution of salmonella O antigen was used as antigen.
2
0
t =#' 0
Anaesthesia Chickens were anaesthetised with sodium thiopental (30 mg k g -1 bodyweight) before the injection into the nictitating membrane.
Statistical analysis The Kolgomorov-Smirnov test was used to determine the significance of differences between the mean values of IgA and IgG concentrations which were detected daily in immunised and control animals.
t
'~'~==' k# ' ~ = ' = = 5 "PIO
/
I, = '= "~ =' '~ I 15 20 25
I 30
Days
FIG 2: Agglutinating antibody levels in tears. Antigen administered by eyedrep (rq) and by injection ( , ) twice at a nine day interval. Antigen administration ( ~- ). Geometric mean titres (GMT). Control birds (r#l)
of the antigen, IgA and IgG levels increased both in sera and tears (Figs 3 and 4). However, after the injection o f the antigen into the nictitating m e m b r a n e the IgA level increased both in tears and serum but the IgG level did not increase either in serum or
46
M. Gallego, E. del Cacho, C. Felices, J. A. Bascuas 6
10 ~-" I
8
-
I
g g 4
g =o B
c
°° 2 e3
0
'
l
'
"]:&lI
5
0
'~
1
I
I
15
20
25
'
l
I
30
0
'tl
5
I
10
Days
15
'
1
I
20
25
'
I
30
Days
FIG 3: Immunoglobulin levels in serum after eyedrop administration. Serum IgA (+) and serum IgG ([:3). Antigen administration ( ~-)
FIG 4: Immunoglobulin levels in tears after eyedrop administration. IgA (*) and IgG (El). Antigen administration ( ~) 8 _
6 -
~i__ " 6 E
I
o~ 4 E
5_o
c
E
E
o
o~ 2 g
=
8 g
o o
4
2
0 ~
0
T
5
' AI
T10
I
I
I
I
15
20
25
30
0
I 5
Days
'j~l
I 10
15
20
25
30
Days
FIG 5: Immunoglobulin levels in tears after two antigen injections into the nictitating membrane. IgA ( , ) and IgG (El). Antigen administration ( >)
FIG 6: Immunoglobulin levels in serum following two antigen injections into the nictitating membrane. IgA (,,) and IgG ([B). Antigen administration ( ~)
tears (Figs 5 and 6). IgG levels in serum started to increase four days after the first administration of the antigen by eyedrop. It reached the maximum level between days 7 and 9, and then declined. The IgG level increased again 48 hours after the second antigen administration reaching the maximum level on day 16 (Fig 3). The IgG level in tears started to increase at the same time as serum did (Figs 3 and 4). After the injection of the antigen, the IgA level in tears was higher than that found after eyedrop administration (Figs 4 and 5). The IgM concentration was constant in all cases (1 mg m1-1 in serum and 0.04 mg m1-1 in tears).
Discussion
Injecting the antigen into the nictitating membrane seems to be more efficient in producing an immune response in the Harderian gland than administering the antigen by eyedrop onto the eyeball. Both the greater cellular activation observed in the gland (Gallego et al 1992) and a remarkable increase of antibodies (IgA) noticed in tears after the antigen administration by injection support this suggestion. These results show that the eyedrop administration of the antigen produces an antibody concentration in serum higher than that achieved by injection into the third eyelid. Furthermore, most of the antibodies
Immunoglobulins synthesised by Harderian gland are IgG. These results suggest that the antigen administered by eyedrop produces an Ig systemic immune response. The antigen administered b y eyedrop could stimulate other lymphoid organs, such as the Peyer's patches, as an important amount of the dropped antigen is drained into the oral cavity and swallowed. Consequently, the salmonella O antigen swallowed may produce an IgG systemic immune response similar to that described by Lascelles et al (1988) in mammals following the oral administration of Salmonella typhimurium. Local IgG produced in the gut associated lymphoid tissue (Burns 1982) contributes to the serum Ig pool and eventually reaches the Harderian gland. In the gland, the IgG could be actively transported from the serum to the tears by the epithelial cells lining the glandular ducts. This selective transport of IgG by the epithelial cells could explain the results obtained by Survashe and Aitken (1977) in birds whose Harderian glands had been removed. In these animals the serum antibody response to the antigen dropped onto the eyeball was higher than it was in the intact birds. These results indicate that most of the IgG level in tears after the eyedrop administration of the antigen has an extraglandular origin as Davelaar et al (1982) and Baba et al (1990) have suggested. Gallego et al (1992) have reported that the number of plaque forming cells in the Harderian glands to sheep red blood cells increased 10-fold after third eyelid injection compared to eyedrop. In the present study, however, the results indicated that the antibody levels detected in tears to salmonella O antigen after third eyelid injection do not increase at the same rate. It could be due to the difference in the sensitivity of the techniques which were used (plaque forming assay and microagglutination) as well as to the data analysis and presentation. Furthermore, the haemolytic plaque assay is a very sensitive technique. As Gilbert and Dresser (1987) have reported 50 plaque forming cells in a lymph node draining a site of injection could be a highly significant result, although the antibody secreted by these cells into the circulation is probably undetectable.
47
Acknowledgement This work was supported by DGICYT (grant PB-86 0532).
References ALBINI, B., WICK, G., ROSE, E. & ORLANS, E. (1974) Immunoglobulin production in chicken Harderian gland. International Archives of Allergy 47, 23-34 BABA, T., KAWATA, K., MASUMOTO, K. & KAJIKAWA, T. (1990) Role of the Harderian gland in immunoglobulin A production in chicken lacrimal fluid. Research in Veterinary Science 49, 20-24 BIENENSTOCK, J., GALDIE, J. & PEREY, D. (1973) Synthesis of IgG, IgA, IgM by chicken tissues: Immunofluorescent and 14C amino acid incorporation studies. Journal oflmmunology 111, 112118 BURNS, R. B. (1977) Possible route of antigen uptake by the Harderian gland of domestic fowl. British Poultry Science 118, 407-409 BURNS, R. B. (1982) Histology and immunology of Peyer's patches in the domestic fowl (Gallus domesticus). Research in Veterinary Science 32, 359-367 CATTY, D. & RAYKUNDALIA, C. (1989) Gel immunodiffusion, immunoelectrophoresis, and immunostaining methods. In Antibodies. Vol 1. Ed D. Catty. Oxford, IRL Press. pp 137-167 DAVELAAR, F. G., NOORDZIJ, A. & VAN DER DONK, J. A. (1982) A study on the synthesis and secretion of immunoglobulins by the Harderian gland of the fowl after eyedrop vaccination against infections bronchitis at 1-day-old. Avian Pathology 11, 63-79 GALLEGO, M., DEL CACHO, E., ARNAL, C. & BASCUAS, J. A. (1992) Local immune response in the chicken Harderian gland to antigen given by different ocular routes. Research in Veterinary Science 52, 38-43 GILBERT, K. & DRESSER, D. W. (1987) The induction and enumeration of antibody-forming cells in vitro. In 'Lymphocytes a practical approach'. Ed G. G. B. Klaus. Oxford, IRL Press. pp 109-129 KOWALSKI, W. J., MALKINSON, M., LESLIE, G. A. & SMALL, P. A. (1988) The secretory immunological system of the fowl. Immunology 34, 663-667 LASCELLES, A. K., BEH, K. J., MUKKUR, T. K. & WILLIS, G. (1988) Immune response of sheep to oral and subcutaneous administration of live aromatic dependent mutant of S typhimurium. Veterinary Immunology and Immunopathology 18, 259-2267 MANSIKKA, A., SANDBERG, N., VEROMAA, T., VAINIO, O., GRANFORS, K. & TOIVANEN, P. (1989) B cell maturation in the chicken Harderian gland. Journal of Immunology 142, 18261834 SURVASHE, B. D. & AITKEN, I. D. (1977) Further observations on functional deletion of paraocular glands in the fowl (Gallus domesticus). Research in Veterinary Science 23, 217-223 WILLIAMS, J. E. & W H I T T E M O R E , A. D. (1972) Microantiglobulin test for detecting S typhimurium agglutinins. Applied Microbiology 23, 931-937
Received March 20, 1991 Accepted July 30, 1991
Immunoglobulin classes synthesised by the chicken Harderian gland after local immunisation M. GALLEGO, E. del CACHO, C. FELICES, J. A. BASCUAS, Departamento de Patologia Animal, Histologia y Anatomia Patologica, Facultad de Veterinaria, Miguel Server, 177, 50013Zaragoza, Spain
The immunoglobulin (Ig) levels in tears and sera were compared after antigen administration (salmonella O antigen) by eyedrop and injection into the nictitating membrane, to determine the lg classes synthesised by the plasma cells in the chicken Harderian gland. Samples of tears and sera were collected from immunised and control birds between 24 hours and 24 days after the antigen or sterile saline was administered. Samples were assayed for IgA, IgG and IgM concentrations using radial immunodiffusion. It is suggested that most of the IgG found in tears after local immunisation has an extraglandular origin.
THE Harderian gland of the chicken is the major contributor to locally produced antibodies which are active in protecting the upper portion of the respiratory tract (Davelaar et al 1982). Several groups have carried out studies on the immunoglobulin (Ig) classes produced by this gland. The presence of specific IgA and IgG antibodies against infectious bronchitis virus in tears of chickens vaccinated with this virus has been demonstrated (Davelaar et al 1982). Burns (1977) reported the presence of anti-BSA IgG and IgA antibodies after eyedrop stimulation with bovine serum albumin (BSA); specific IgM antibodies, on the other hand, were not detected. However, in naive chickens the type of antibodies produced by the Harderian gland plasma cells is not clear. According to immunofluorescence studies by Albini et al (1974) 48 per cent of lymphoid cells were positive for IgA, 22 per cent for IgG and 12 per cent for IgM. Bienenstock et al (1973) reported the prevalence oflgM and IgG, whereas IgA positive plasma cells were scarce. Mansikka et al (1989) demonstrated that unstimulated chickens have high levels of IgM, IgA and IgG
in tears and after ocular stimulation specific IgG and IgA antibodies appeared but IgM antibodies were barely detectable. The objective of this work was to compare the Ig levels in tears and serum after two different types of antigen administration: eyedrop and injection into the nictitating membrane, to determine the antibody classes synthesised by the plasma cells in the chicken Harderian gland. Materials and methods
Animals and immunisations Seven-week-old White Leghorn chickens were used. The birds were immunised with salmonella O antigen (Difco), 1.8 x 1010 organisms m1-1, diluted v/v in sterile saline, by injection into the nictitating membrane or by dropping it onto the eyeball. Fifteen chickens were injected twice, nine days apart, into the nictitating membrane (as close as possible to the formix) with 0.02 ml diluted commercial solution of salmonella O antigen. Fifteen control birds were injected at the same interval into the nictitating membrane with 0.02 ml sterile saline. Diluted commercial solution of salmonella O antigen was dripped twice, nine days apart, onto the eyeball (0.5 ml per eye) of 15 chickens. Sterile saline (0.5 ml) was dripped onto each eye of 15 control birds following the same pattern used with the salmonella O antigen.
Tear and blood collection Tears were collected using a 100 ml capillary tube held against the inner canthus of the eye. Manipulation of the third eyelid back and forth 44
Immunoglobulins synthesised by Harderian gland was sometimes necessary to facilitate the flow of tears. Chemical induction of lacrimation was not used. Chickens were bled by wing puncture. The volume of blood was 1 ml. Tears and blood were collected from every chicken, immunised and control, at 24 hours, two, four, five, six, seven, nine, 10, 11, 12, 14, 16, 18, 20, 22 and 24 days after the first administration of both the injection and eyedrop of salmonella O antigen and sterile saline.
Quantitation of Igs The Ig levels of blood and tears were determined by the radial immunodifusion method (RID) (Catty and Raykundalia 1989). The antisera used were goat anti-chicken IgM, rabbit antichicken IgG and goat anti-chicken IgA (ICN Biomedicals). They were characterised by immunoelectrophoresis and their specificity was checked by immunodiffusion. Optimal concentrations of antisera were 1 per cent for anti-IgM, 0.5 per cent for anti-IgA and 2.5 per cent for anti-]gG (Kowalski et al 1978). The upper and lower limits of sensitivity of RID were determined in relation to a previously calibrated standard chicken serum. Minimum detectable levels were 20 ~tg ml 1 for IgM, IgG and IgA. The diffusion diameters were measured and the results were plotted on a linear scale.
Antibody titration
45
Results
Higher levels of specific antibody were found in both sera and tears after eyedrop application (Figs 1 and 2). Following the eyedrop application 8
6t I(.5 4 --
2 0 1
r AI
I
Tlo
5
15
'
J
I
20
25
30
Days FIG 1 :Agglutinating antibody levels in serum. Antigen administration by eyedrop (1~) and by injection (¢) twice at a nine day interval. Antigen administration { .~-). Geometric mean titres (GMT). Control Birds (El)
8 -
~_ 4
The antibodies in serum and tears were titrated following the microtitration technique proposed by Williams and Whittemore (1972). A commercial solution of salmonella O antigen was used as antigen.
2
0
t =#' 0
Anaesthesia Chickens were anaesthetised with sodium thiopental (30 mg k g -1 bodyweight) before the injection into the nictitating membrane.
Statistical analysis The Kolgomorov-Smirnov test was used to determine the significance of differences between the mean values of IgA and IgG concentrations which were detected daily in immunised and control animals.
t
'~'~==' k# ' ~ = ' = = 5 "PIO
/
I, = '= "~ =' '~ I 15 20 25
I 30
Days
FIG 2: Agglutinating antibody levels in tears. Antigen administered by eyedrep (rq) and by injection ( , ) twice at a nine day interval. Antigen administration ( ~- ). Geometric mean titres (GMT). Control birds (r#l)
of the antigen, IgA and IgG levels increased both in sera and tears (Figs 3 and 4). However, after the injection o f the antigen into the nictitating m e m b r a n e the IgA level increased both in tears and serum but the IgG level did not increase either in serum or
46
M. Gallego, E. del Cacho, C. Felices, J. A. Bascuas 6
10 ~-" I
8
-
I
g g 4
g =o B
c
°° 2 e3
0
'
l
'
"]:&lI
5
0
'~
1
I
I
15
20
25
'
l
I
30
0
'tl
5
I
10
Days
15
'
1
I
20
25
'
I
30
Days
FIG 3: Immunoglobulin levels in serum after eyedrop administration. Serum IgA (+) and serum IgG ([:3). Antigen administration ( ~-)
FIG 4: Immunoglobulin levels in tears after eyedrop administration. IgA (*) and IgG (El). Antigen administration ( ~) 8 _
6 -
~i__ " 6 E
I
o~ 4 E
5_o
c
E
E
o
o~ 2 g
=
8 g
o o
4
2
0 ~
0
T
5
' AI
T10
I
I
I
I
15
20
25
30
0
I 5
Days
'j~l
I 10
15
20
25
30
Days
FIG 5: Immunoglobulin levels in tears after two antigen injections into the nictitating membrane. IgA ( , ) and IgG (El). Antigen administration ( >)
FIG 6: Immunoglobulin levels in serum following two antigen injections into the nictitating membrane. IgA (,,) and IgG ([B). Antigen administration ( ~)
tears (Figs 5 and 6). IgG levels in serum started to increase four days after the first administration of the antigen by eyedrop. It reached the maximum level between days 7 and 9, and then declined. The IgG level increased again 48 hours after the second antigen administration reaching the maximum level on day 16 (Fig 3). The IgG level in tears started to increase at the same time as serum did (Figs 3 and 4). After the injection of the antigen, the IgA level in tears was higher than that found after eyedrop administration (Figs 4 and 5). The IgM concentration was constant in all cases (1 mg m1-1 in serum and 0.04 mg m1-1 in tears).
Discussion
Injecting the antigen into the nictitating membrane seems to be more efficient in producing an immune response in the Harderian gland than administering the antigen by eyedrop onto the eyeball. Both the greater cellular activation observed in the gland (Gallego et al 1992) and a remarkable increase of antibodies (IgA) noticed in tears after the antigen administration by injection support this suggestion. These results show that the eyedrop administration of the antigen produces an antibody concentration in serum higher than that achieved by injection into the third eyelid. Furthermore, most of the antibodies
Immunoglobulins synthesised by Harderian gland are IgG. These results suggest that the antigen administered by eyedrop produces an Ig systemic immune response. The antigen administered b y eyedrop could stimulate other lymphoid organs, such as the Peyer's patches, as an important amount of the dropped antigen is drained into the oral cavity and swallowed. Consequently, the salmonella O antigen swallowed may produce an IgG systemic immune response similar to that described by Lascelles et al (1988) in mammals following the oral administration of Salmonella typhimurium. Local IgG produced in the gut associated lymphoid tissue (Burns 1982) contributes to the serum Ig pool and eventually reaches the Harderian gland. In the gland, the IgG could be actively transported from the serum to the tears by the epithelial cells lining the glandular ducts. This selective transport of IgG by the epithelial cells could explain the results obtained by Survashe and Aitken (1977) in birds whose Harderian glands had been removed. In these animals the serum antibody response to the antigen dropped onto the eyeball was higher than it was in the intact birds. These results indicate that most of the IgG level in tears after the eyedrop administration of the antigen has an extraglandular origin as Davelaar et al (1982) and Baba et al (1990) have suggested. Gallego et al (1992) have reported that the number of plaque forming cells in the Harderian glands to sheep red blood cells increased 10-fold after third eyelid injection compared to eyedrop. In the present study, however, the results indicated that the antibody levels detected in tears to salmonella O antigen after third eyelid injection do not increase at the same rate. It could be due to the difference in the sensitivity of the techniques which were used (plaque forming assay and microagglutination) as well as to the data analysis and presentation. Furthermore, the haemolytic plaque assay is a very sensitive technique. As Gilbert and Dresser (1987) have reported 50 plaque forming cells in a lymph node draining a site of injection could be a highly significant result, although the antibody secreted by these cells into the circulation is probably undetectable.
47
Acknowledgement This work was supported by DGICYT (grant PB-86 0532).
References ALBINI, B., WICK, G., ROSE, E. & ORLANS, E. (1974) Immunoglobulin production in chicken Harderian gland. International Archives of Allergy 47, 23-34 BABA, T., KAWATA, K., MASUMOTO, K. & KAJIKAWA, T. (1990) Role of the Harderian gland in immunoglobulin A production in chicken lacrimal fluid. Research in Veterinary Science 49, 20-24 BIENENSTOCK, J., GALDIE, J. & PEREY, D. (1973) Synthesis of IgG, IgA, IgM by chicken tissues: Immunofluorescent and 14C amino acid incorporation studies. Journal oflmmunology 111, 112118 BURNS, R. B. (1977) Possible route of antigen uptake by the Harderian gland of domestic fowl. British Poultry Science 118, 407-409 BURNS, R. B. (1982) Histology and immunology of Peyer's patches in the domestic fowl (Gallus domesticus). Research in Veterinary Science 32, 359-367 CATTY, D. & RAYKUNDALIA, C. (1989) Gel immunodiffusion, immunoelectrophoresis, and immunostaining methods. In Antibodies. Vol 1. Ed D. Catty. Oxford, IRL Press. pp 137-167 DAVELAAR, F. G., NOORDZIJ, A. & VAN DER DONK, J. A. (1982) A study on the synthesis and secretion of immunoglobulins by the Harderian gland of the fowl after eyedrop vaccination against infections bronchitis at 1-day-old. Avian Pathology 11, 63-79 GALLEGO, M., DEL CACHO, E., ARNAL, C. & BASCUAS, J. A. (1992) Local immune response in the chicken Harderian gland to antigen given by different ocular routes. Research in Veterinary Science 52, 38-43 GILBERT, K. & DRESSER, D. W. (1987) The induction and enumeration of antibody-forming cells in vitro. In 'Lymphocytes a practical approach'. Ed G. G. B. Klaus. Oxford, IRL Press. pp 109-129 KOWALSKI, W. J., MALKINSON, M., LESLIE, G. A. & SMALL, P. A. (1988) The secretory immunological system of the fowl. Immunology 34, 663-667 LASCELLES, A. K., BEH, K. J., MUKKUR, T. K. & WILLIS, G. (1988) Immune response of sheep to oral and subcutaneous administration of live aromatic dependent mutant of S typhimurium. Veterinary Immunology and Immunopathology 18, 259-2267 MANSIKKA, A., SANDBERG, N., VEROMAA, T., VAINIO, O., GRANFORS, K. & TOIVANEN, P. (1989) B cell maturation in the chicken Harderian gland. Journal of Immunology 142, 18261834 SURVASHE, B. D. & AITKEN, I. D. (1977) Further observations on functional deletion of paraocular glands in the fowl (Gallus domesticus). Research in Veterinary Science 23, 217-223 WILLIAMS, J. E. & W H I T T E M O R E , A. D. (1972) Microantiglobulin test for detecting S typhimurium agglutinins. Applied Microbiology 23, 931-937
Received March 20, 1991 Accepted July 30, 1991
