Allergy, 1990, 45, 293-297
Precipitating antibodies against pigeon bloom antigens as a specific and sensitive measure of pigeon exposure H. C. SiERSTED & N-C. G.
HANSEN
Department of Respiratory Diseases, Odense University Hospital, Denmark
Precipitating antibodies against pigeon bloom antigen and pigeon serum were evaluated as a measure of pigeon exposure in 62 male pigeon breeders compared with 40 unexposed control subjects. We found a combination of highly antigenic bloom extract and a sensitive counter current immunoelectrophoresis suitable for clinical estimation of pigeon exposure. The sensitivity of the test was 86% in never-smokers and 73% in smokers at the 98% specificity level. Smoking significantly reduced the humoral immune response to both antigens. Key words: bird fanciers lung; counterimmunoelectrophoresis; environmental exposure; extrinsic allergic alveolitis; pigeons; precipitins; smoking. Accepted for publication 21 December
1989
CLINICAL ASPECTS The diagnosis of pigeon breeders disease is to a wide extent dependent on the anamnesis, as the only diagnostic test available is provocation with the suspected antigen. Elevated amounts of precipitating antibodies against pigeon antigen can support an anamnesis of specific exposure, and can give rise to relevant suspicion in eases where exposure is less obvious. This paper describes a sensitive and specific test for pigeon exposure determined by precipitating antibodies against pigeon bloom extract in counter current immunoelectrophoresis.
Precipitating antibodies (PA) against pigeon antigens appear to indicate exposure rather than disease (5,6). The occurrence of such antibodies supports the exposure anamnesis in the often puzzling diagnosis of pigeon breeder's disease (PBD), especially in non-acute cases or when relevant exposure is not obvious. By double immune diffusion techniques, PA against pigeon serum (traditionally used) and against extract of pigeon bloom (a white, dusty material covering the feathers) has been found in 38 and 61 % of pigeon breeders respectively (2). This sensitivity is too low for clinical use. The aim of this study was to evaluate precipitating antibodies against pigeon bloom and
serum antigens as a measure of pigeon exposure, using a sensitive electrophoretic method (counter current immunoelectrophoresis). The correlation with smoking status was also investigated.
MATERIAL AND METHODS Sera were collected from 62 male pigeon breeders volunteering in a clinical survey as described elsewhere (8). These were the male responders among 200 breeders invited through the local breeders' association. The median age was 49 years (16-74 years). None of the breeders reported unequivocal symptoms of extrinsic
294
H. C. SIERSTED & N-C. G. HANSEN
f * '
6 G G Fig. 1. Gounter current immunoelectrophoresis. Two-lold titration of breeder's serum against (A) pigeon bloom extract and (B) pigeon serum.
allergic alveolitis, and most of them (61 %) reported no pigeon-related pulmonary symptoms at all. Sera from 15 breeders with high bloomtiters (median titer 128) were pooled and used for CIE. Forty healthy male blood donors who claimed that they had no contact with birds of any kind and that they had never had allergic disease were matched to the pigeon breeders with respect to age (5-year intervals) and grouped according to smoking habits (smokers, ex-smokers and never smokers). All samples were kept at -70°C. Saline bloom extracts were prepared from weekly washings of 20 clinically sound racing pigeons kept in a closed dovecote. The feathers of each bird were stroken with two sterile saline-wetted 25 cm' gauzeswabs, avoiding fecal material. Swabs were frozen at -20°C if not immediately washed out in three changes of sterile saline (pH 7.4), using a total of only 5ml/swab. The resulting white opaque mixture was centrifuged at 1700 g, 30', sterile filtered (poresize 45 |im, Millipore) and vacuum concentrated 10 to 15 times over 24 h in a multibag dialysis arrangement (10 3-ml bags made from Union Carbide dialysis tubing). The preparation was again sterile filtered and kept in small aliquots at -70°C until electrophoresis. The protein concentration of the final preparation was 0.2 mg/ml (estimated from OD
measurements at 280 nm with human serum albumin as a reference). Pigeon serum was obtained by axillar venepuncture from clinically sound pigeons and kept at -70°C. Care was taken to avoid contamination with external material. Dilution of samples and electrophoresis was done in tris-barbital buffer: 70 mM tris(hydroxymethyl)aminomethane (Sigma 7-9R)/HCl, 24 mM 5,5'-diethyl barbituric acid and 0.01 % (w/v) NaNj at pH 8.6. Counter current immune electrophoresis (CCIE) was performed in 1.5 mm thick 1% agarose gel (0.5% Litex HSA lot 7091, 0.5% Litex LSA lot 4031, Litex, Denmark), 3 % polyethylene glycol MW 6000 (final weight/ volume concentrations) in tris-barbital buffer pH 8.6. Two-fold anodal titration rows of human sera were electrophoresed overnight at 2.5 V/cm against pigeon antigen in 6-^1 wells 10 mm apart, with 7.5 mm between each pair of wells. Both antigen preparations were used undiluted, as dilution ('As, Vio) resulted in weaker precipitation and poor spatial separation of the individual precipitation arcs. Antibody titers were defined as the reciprocal concentration in the most diluted sample causing visible precipitation. Crossed immunoelectrophoresis (CIE) was performed in 1.5 mm thick 1% HSA agarose, tris-barbital buffer, pH 8.6 essentially as described by Weeke (11). 17 \i\ of undiluted pigeon bloom extract was applied in an elongated well. The first dimensional electrophoresis (10 V/cm) was interrupted when the front of the bromphenol blue stained albumin marker had migrated approx. 35 mm. In the second dimensional run the upper gel contained 3 % (w/v) PEG-6000 and pigeon breeder sera pool diluted 1/90. All correlations were evaluated by Spearman rank correlation test at the 0.05 significance level. The use of other statistics are stated in the text. RESULTS The pigeon bloom extract was well suited for CCIE at the selected conditions, resulting in
295
EVALUATION OE PIGEON EXPOSURE sharp and easily read precipitation lines (Eig. lA). Up to five precipitation lines were detected. In CIE with pigeon breeder sera pool as antibody source the pigeon bloom extract gave rise to three distinct and four weak precipitation arcs (Fig. 2). The latter might not be recognized in the photograph. The double contouring of the anodal precipitates possibly reflects minor molecular heterogeneities. With the mobility of human serum albumin as reference (1.00) the relative mobilities of the bloom antigens were 0.84, 1.06 and 1.23 (distinct precipitation) and 0.37, 0.43, 1.19 and 1.43 (weak precipitation). The relative mobility of the albumin-bromphenol blue marker front was 1.08. Reactions of antigen non-identity were seen between the three distinct precipitates, whereas the two fast-moving weak precipitation arcs were continuous, indicating antigenic identity. All donors and pigeon breeders had precipitating antibodies against pigeon bloom antigen. Only 2.5% of the donors, but 77% of the pigeon breeders had titers > 8 (Eig. 3A). The bloom titer was, as the number of precipitation lines, significantly declining from never-smokers to ex-smokers and further to active smokers (Table 1, Fig. 3A). The period of smoking abstinence (median: 12 years, range 0.2-48 years) in ex-smoking breeders did not correlate to the titer value. Neither was correlation found between the tobacco consumption and the bloom titer in smoking or ex-smoking pigeon
breeders. In the donor group the median bloom titers were not significantly different for smokers, ex-smokers or never smokers (Table 1). In comparison with the bloom extract, pigeon serum gave fewer (Table 1) and more blurred (Fig. IB) precipitation lines in CCIE. With pigeon serum there was a considerable overlap between titers in unexposed and pigeon breeding individuals (Fig. 3B). Only 76% of the breeders had detectable antibodies against pigeon serum. Eight percent of the donors, but only 29% of the breeders had antibody titers exceeding titer 4. The effect of smoking was comparable with the two antigens (Table 1, Fig. 3). DISCUSSION Pigeon serum has traditionally been used as a convenient source of pigeon antigens suspected to be responsible for immunological sensitization in cases of allergic alveolitis in pigeon breeders (7). Attempts to develop a diagnostic test for this disease have been disappointing. Although some correlation between high titers of precipitating antibodies against pigeon antigens and pulmonary symptoms compatible with the diagnosis of PBD has been reported (10), this finding is not specific (3). The present study also failed to demonstrate any correlation between clinical symptoms and the level of precipitating antibodies against pigeon serum and bloom antigens in pigeon breeders (8). The antibody level more likely refiects a combination of normal variations in individual immune
CUM°
CUM =/ o 100-
100-
A
60604020-
'
// J/ i/ /
0-
/ /
60•
UNEXPOSED BLOOD DONORS
ff
A
80 -
40-
0
SMOKERS
A
EX-SMOKERS
o
NEVER-SMOKERS
- / / 20-1 :
/
// /
D ' /
PIGEON BREEDERS
/ /
/ /
0 r " 1 —1—1—1—I—r -]—\—I—1 0 2 8 32 128 512 6'. 256 U 16 1 PIGEON
Fig. 2. Grossed immunoelectrophoresis of pigeon bloom extract against a sera pool from pigeon breeders with high anti-bloom antibody titers in GGIE. Alb. denotes first dimension mobility of human albumin.
/
I
f1
B
BLOOM TITER
0
8
2
1
1.
i
1
32 16
61.
PIGEON SERUM TITER
Fig. 3. Gumulated frequencies of pigeon bloom titers (A) and pigeon serum titers (B) in all unexposed blood donors and in pigeon breeders grouped according to smoking status.
296
H. C. SIERSTED & N-C. G. HANSEN Table 1
Median antibody titers and median number of precipitation lines (p-lines) in GGIE with the two pigeon antigens in donors and pigeon breeders in relation to smoking status. The P-values refer to Kruskal-Wallis test. P< 0.05 implicates rejection of the null-hypothesis, that the 3 samples are from the same population Group
Unexposed donors {n = 40)
Smoking status {n) Bloom Bloom Serum Serum
titer p-lines titer p-lines
Never(20)
Ex(5)
Active (15)
4 2 2
2 2 2
4 1 2
1
1
1
reactivity and the degree of antigen exposure (3, 6). As a measure of pigeon exposure, however, the conventionally used immunodiffusion test for PA is much too insensitive to be a reliable support to the clinical anamnesis, especially in doubtful cases with no obvious bird contact, as about 40% of heavily exposed individuals do not develop detectable antibodies (2). In an inter-laboratory study of precipitins to inhaled avian antigens using bird sera or dropping extracts in different electrophoretic methods, CCIE was found to be the most sensitive (4). The present report is the first to evaluate CCIE in estimating pigeon antigen exposure. All breeders had PA against the bloom extract, but only 76% had PA against pigeon serum. The technical quality of the precipitation was superior with the bloom antigen, as pigeon serum tended to give blurred precipitation, possibly as a result of the high concentration of irrelevant protein. The specificity and sensitivity of the exposure-test was most favorable with pigeon bloom antigen. By choosing a suitable cut-off point, the performance of the test could be adjusted (Fig. 3). For clinical use, bloom titers exceeding 8 indicate relevant exposure with high specificity (98%) and sensitivity (86% in never-smokers, 73% in smokers). The suppressive effect of tobacco smoking on the humored immune response to inhaled pigeon antigens has been described earlier (1, 9). In our material the median bloom-titers in nonsmoking pigeon breeders were 4-fold higher than in smoking breeders.
Pigeon breeders (n = 62) P
0.14 0.09 0.82 0.23
Never(21) 64
3 8 3
Ex-
(18) 32 3 1.5 2
Active (23) 16 2 1 1
P
0.0031 0.0011 0.0001 0.0001
The occurrence of low titers of PA against pigeon bloom as well as serum antigens in the majority of individuals claiming to be unexposed to birds was not further investigated, but might be the result of continuous inhalation of small amounts of bird antigens by daily breathing. In CCIE several precipitation lines were demonstrated with both antigen preparations. The number of precipitation lines were positively correlated to the titers, but offered no advantages in evaluating exposure. The antigen heterogeneity of the bloom extract was confirmed in CIE. Seven different precipitation arcs could be recognized, showing a wide spectrum of electrophoretic mobilities (Fig. 3). The number of precipitation lines and their appearance in CCIE varied considerably, suggesting individual differences in the response to bloom antigens. Thus the antigen heterogeneity of the preparation seems to be advantageous in testing for pigeon exposure. In conclusion, CCIE is suitable for the detection of PA against pigeon bloom extract. The test is recommended as a reliable indicator of pigeon exposure. ACKNOWLEDGEMENTS We thank The National Association against Lung Diseases and The Hojbjerg Foundation for financial support.
REFERENCES 1. Andersen, P. & Ghristensen, K. M.: Serum antibodies to pigeon antigens in smokers and nonsmokers. Acta Med. Scand. 213, 191-193, 1983.
EVALUATION OE PIGEON EXPOSURE 2. Banham, S. W., McKenzie, H., McSharry, G., Lynch, P. P. & Boyd, G.: Antibody against a pigeon bloomextract: a further antigen in pigeon fanciers' lung. Glin. Allergy 12, 173-178, 1982. 3. Barboriak, J. J., Fink, J. N., Sosman, A. J. & Dhaliwal, K. S.: Precipitating antibody against pigeon antigens in sera of asymptomatic pigeon breeders. J. Lab. Glm. Med. 82, 372-376, 1973. 4. Bergmann, K-G., Aiache, J. M., Bartmann, K., et al.: Precipitins to inhaled avian antigens: Results of an inter-laboratory study. Glin. Allergy 13, 451-457, 1983. 5. Burrell, R. & Rylander, R.: A critical review of the role of precipitins in hypersensitivity pneumonitis. Eur. J. Respir. Dis. 62, 332-343, 1981. 6. Fink, J. N.: Epidemiologic aspects of hypersensitivity pneumonitis. Monogr. Allergy 21, 59-69, 1987. 7. Fink, J. N., Barboriak, J. J., Sosman, A. J., Bukosky, R. J. & Arkins, J. A.: Antibodies against pigeon serum proteins in pigeon breeders. J. Lab. Glin. Med. 71, 20-25, 1968.
297
8. Hansen, N-G. G. & Siersted, H. G.: Allergic alveolitis among pigeon breeders in the county of Funen (summary in English). Ugeskr. Laeger, 152, 25-28, 1990 . 9. McSharry, G., Banham. S. W. & Boyd, G.: Effect of cigarette smoking on the antibody response to inhaled antigens and the prevalence of extrinsic allergic alveolitis among pigeon breeders. Glin. Allergy 15, 487-494. 1985. 10. McSharry, G., Banham, S. W., Lynch, P. P. & Boyd, G: Antibody measurement in extrinsic allergic alveolitis. Eur. J. Respir. Dis. 65, 259-265, 1984. 11. Weeke, B.: Grossed immunoelectrophoresis. In Axelsen, N. H., Kroll, J. & Weeke, B. (eds.): A manual of quantitative immunoelectrophoresis, pp. 47-56. Universitetsforlaget, Oslo, 1973. Address: Hans Chr. Siersted
Department of Respiratory Diseases Odense University Hospital DK-5000 Odense G Denmark
Precipitating antibodies against pigeon bloom antigens as a specific and sensitive measure of pigeon exposure H. C. SiERSTED & N-C. G.
HANSEN
Department of Respiratory Diseases, Odense University Hospital, Denmark
Precipitating antibodies against pigeon bloom antigen and pigeon serum were evaluated as a measure of pigeon exposure in 62 male pigeon breeders compared with 40 unexposed control subjects. We found a combination of highly antigenic bloom extract and a sensitive counter current immunoelectrophoresis suitable for clinical estimation of pigeon exposure. The sensitivity of the test was 86% in never-smokers and 73% in smokers at the 98% specificity level. Smoking significantly reduced the humoral immune response to both antigens. Key words: bird fanciers lung; counterimmunoelectrophoresis; environmental exposure; extrinsic allergic alveolitis; pigeons; precipitins; smoking. Accepted for publication 21 December
1989
CLINICAL ASPECTS The diagnosis of pigeon breeders disease is to a wide extent dependent on the anamnesis, as the only diagnostic test available is provocation with the suspected antigen. Elevated amounts of precipitating antibodies against pigeon antigen can support an anamnesis of specific exposure, and can give rise to relevant suspicion in eases where exposure is less obvious. This paper describes a sensitive and specific test for pigeon exposure determined by precipitating antibodies against pigeon bloom extract in counter current immunoelectrophoresis.
Precipitating antibodies (PA) against pigeon antigens appear to indicate exposure rather than disease (5,6). The occurrence of such antibodies supports the exposure anamnesis in the often puzzling diagnosis of pigeon breeder's disease (PBD), especially in non-acute cases or when relevant exposure is not obvious. By double immune diffusion techniques, PA against pigeon serum (traditionally used) and against extract of pigeon bloom (a white, dusty material covering the feathers) has been found in 38 and 61 % of pigeon breeders respectively (2). This sensitivity is too low for clinical use. The aim of this study was to evaluate precipitating antibodies against pigeon bloom and
serum antigens as a measure of pigeon exposure, using a sensitive electrophoretic method (counter current immunoelectrophoresis). The correlation with smoking status was also investigated.
MATERIAL AND METHODS Sera were collected from 62 male pigeon breeders volunteering in a clinical survey as described elsewhere (8). These were the male responders among 200 breeders invited through the local breeders' association. The median age was 49 years (16-74 years). None of the breeders reported unequivocal symptoms of extrinsic
294
H. C. SIERSTED & N-C. G. HANSEN
f * '
6 G G Fig. 1. Gounter current immunoelectrophoresis. Two-lold titration of breeder's serum against (A) pigeon bloom extract and (B) pigeon serum.
allergic alveolitis, and most of them (61 %) reported no pigeon-related pulmonary symptoms at all. Sera from 15 breeders with high bloomtiters (median titer 128) were pooled and used for CIE. Forty healthy male blood donors who claimed that they had no contact with birds of any kind and that they had never had allergic disease were matched to the pigeon breeders with respect to age (5-year intervals) and grouped according to smoking habits (smokers, ex-smokers and never smokers). All samples were kept at -70°C. Saline bloom extracts were prepared from weekly washings of 20 clinically sound racing pigeons kept in a closed dovecote. The feathers of each bird were stroken with two sterile saline-wetted 25 cm' gauzeswabs, avoiding fecal material. Swabs were frozen at -20°C if not immediately washed out in three changes of sterile saline (pH 7.4), using a total of only 5ml/swab. The resulting white opaque mixture was centrifuged at 1700 g, 30', sterile filtered (poresize 45 |im, Millipore) and vacuum concentrated 10 to 15 times over 24 h in a multibag dialysis arrangement (10 3-ml bags made from Union Carbide dialysis tubing). The preparation was again sterile filtered and kept in small aliquots at -70°C until electrophoresis. The protein concentration of the final preparation was 0.2 mg/ml (estimated from OD
measurements at 280 nm with human serum albumin as a reference). Pigeon serum was obtained by axillar venepuncture from clinically sound pigeons and kept at -70°C. Care was taken to avoid contamination with external material. Dilution of samples and electrophoresis was done in tris-barbital buffer: 70 mM tris(hydroxymethyl)aminomethane (Sigma 7-9R)/HCl, 24 mM 5,5'-diethyl barbituric acid and 0.01 % (w/v) NaNj at pH 8.6. Counter current immune electrophoresis (CCIE) was performed in 1.5 mm thick 1% agarose gel (0.5% Litex HSA lot 7091, 0.5% Litex LSA lot 4031, Litex, Denmark), 3 % polyethylene glycol MW 6000 (final weight/ volume concentrations) in tris-barbital buffer pH 8.6. Two-fold anodal titration rows of human sera were electrophoresed overnight at 2.5 V/cm against pigeon antigen in 6-^1 wells 10 mm apart, with 7.5 mm between each pair of wells. Both antigen preparations were used undiluted, as dilution ('As, Vio) resulted in weaker precipitation and poor spatial separation of the individual precipitation arcs. Antibody titers were defined as the reciprocal concentration in the most diluted sample causing visible precipitation. Crossed immunoelectrophoresis (CIE) was performed in 1.5 mm thick 1% HSA agarose, tris-barbital buffer, pH 8.6 essentially as described by Weeke (11). 17 \i\ of undiluted pigeon bloom extract was applied in an elongated well. The first dimensional electrophoresis (10 V/cm) was interrupted when the front of the bromphenol blue stained albumin marker had migrated approx. 35 mm. In the second dimensional run the upper gel contained 3 % (w/v) PEG-6000 and pigeon breeder sera pool diluted 1/90. All correlations were evaluated by Spearman rank correlation test at the 0.05 significance level. The use of other statistics are stated in the text. RESULTS The pigeon bloom extract was well suited for CCIE at the selected conditions, resulting in
295
EVALUATION OE PIGEON EXPOSURE sharp and easily read precipitation lines (Eig. lA). Up to five precipitation lines were detected. In CIE with pigeon breeder sera pool as antibody source the pigeon bloom extract gave rise to three distinct and four weak precipitation arcs (Fig. 2). The latter might not be recognized in the photograph. The double contouring of the anodal precipitates possibly reflects minor molecular heterogeneities. With the mobility of human serum albumin as reference (1.00) the relative mobilities of the bloom antigens were 0.84, 1.06 and 1.23 (distinct precipitation) and 0.37, 0.43, 1.19 and 1.43 (weak precipitation). The relative mobility of the albumin-bromphenol blue marker front was 1.08. Reactions of antigen non-identity were seen between the three distinct precipitates, whereas the two fast-moving weak precipitation arcs were continuous, indicating antigenic identity. All donors and pigeon breeders had precipitating antibodies against pigeon bloom antigen. Only 2.5% of the donors, but 77% of the pigeon breeders had titers > 8 (Eig. 3A). The bloom titer was, as the number of precipitation lines, significantly declining from never-smokers to ex-smokers and further to active smokers (Table 1, Fig. 3A). The period of smoking abstinence (median: 12 years, range 0.2-48 years) in ex-smoking breeders did not correlate to the titer value. Neither was correlation found between the tobacco consumption and the bloom titer in smoking or ex-smoking pigeon
breeders. In the donor group the median bloom titers were not significantly different for smokers, ex-smokers or never smokers (Table 1). In comparison with the bloom extract, pigeon serum gave fewer (Table 1) and more blurred (Fig. IB) precipitation lines in CCIE. With pigeon serum there was a considerable overlap between titers in unexposed and pigeon breeding individuals (Fig. 3B). Only 76% of the breeders had detectable antibodies against pigeon serum. Eight percent of the donors, but only 29% of the breeders had antibody titers exceeding titer 4. The effect of smoking was comparable with the two antigens (Table 1, Fig. 3). DISCUSSION Pigeon serum has traditionally been used as a convenient source of pigeon antigens suspected to be responsible for immunological sensitization in cases of allergic alveolitis in pigeon breeders (7). Attempts to develop a diagnostic test for this disease have been disappointing. Although some correlation between high titers of precipitating antibodies against pigeon antigens and pulmonary symptoms compatible with the diagnosis of PBD has been reported (10), this finding is not specific (3). The present study also failed to demonstrate any correlation between clinical symptoms and the level of precipitating antibodies against pigeon serum and bloom antigens in pigeon breeders (8). The antibody level more likely refiects a combination of normal variations in individual immune
CUM°
CUM =/ o 100-
100-
A
60604020-
'
// J/ i/ /
0-
/ /
60•
UNEXPOSED BLOOD DONORS
ff
A
80 -
40-
0
SMOKERS
A
EX-SMOKERS
o
NEVER-SMOKERS
- / / 20-1 :
/
// /
D ' /
PIGEON BREEDERS
/ /
/ /
0 r " 1 —1—1—1—I—r -]—\—I—1 0 2 8 32 128 512 6'. 256 U 16 1 PIGEON
Fig. 2. Grossed immunoelectrophoresis of pigeon bloom extract against a sera pool from pigeon breeders with high anti-bloom antibody titers in GGIE. Alb. denotes first dimension mobility of human albumin.
/
I
f1
B
BLOOM TITER
0
8
2
1
1.
i
1
32 16
61.
PIGEON SERUM TITER
Fig. 3. Gumulated frequencies of pigeon bloom titers (A) and pigeon serum titers (B) in all unexposed blood donors and in pigeon breeders grouped according to smoking status.
296
H. C. SIERSTED & N-C. G. HANSEN Table 1
Median antibody titers and median number of precipitation lines (p-lines) in GGIE with the two pigeon antigens in donors and pigeon breeders in relation to smoking status. The P-values refer to Kruskal-Wallis test. P< 0.05 implicates rejection of the null-hypothesis, that the 3 samples are from the same population Group
Unexposed donors {n = 40)
Smoking status {n) Bloom Bloom Serum Serum
titer p-lines titer p-lines
Never(20)
Ex(5)
Active (15)
4 2 2
2 2 2
4 1 2
1
1
1
reactivity and the degree of antigen exposure (3, 6). As a measure of pigeon exposure, however, the conventionally used immunodiffusion test for PA is much too insensitive to be a reliable support to the clinical anamnesis, especially in doubtful cases with no obvious bird contact, as about 40% of heavily exposed individuals do not develop detectable antibodies (2). In an inter-laboratory study of precipitins to inhaled avian antigens using bird sera or dropping extracts in different electrophoretic methods, CCIE was found to be the most sensitive (4). The present report is the first to evaluate CCIE in estimating pigeon antigen exposure. All breeders had PA against the bloom extract, but only 76% had PA against pigeon serum. The technical quality of the precipitation was superior with the bloom antigen, as pigeon serum tended to give blurred precipitation, possibly as a result of the high concentration of irrelevant protein. The specificity and sensitivity of the exposure-test was most favorable with pigeon bloom antigen. By choosing a suitable cut-off point, the performance of the test could be adjusted (Fig. 3). For clinical use, bloom titers exceeding 8 indicate relevant exposure with high specificity (98%) and sensitivity (86% in never-smokers, 73% in smokers). The suppressive effect of tobacco smoking on the humored immune response to inhaled pigeon antigens has been described earlier (1, 9). In our material the median bloom-titers in nonsmoking pigeon breeders were 4-fold higher than in smoking breeders.
Pigeon breeders (n = 62) P
0.14 0.09 0.82 0.23
Never(21) 64
3 8 3
Ex-
(18) 32 3 1.5 2
Active (23) 16 2 1 1
P
0.0031 0.0011 0.0001 0.0001
The occurrence of low titers of PA against pigeon bloom as well as serum antigens in the majority of individuals claiming to be unexposed to birds was not further investigated, but might be the result of continuous inhalation of small amounts of bird antigens by daily breathing. In CCIE several precipitation lines were demonstrated with both antigen preparations. The number of precipitation lines were positively correlated to the titers, but offered no advantages in evaluating exposure. The antigen heterogeneity of the bloom extract was confirmed in CIE. Seven different precipitation arcs could be recognized, showing a wide spectrum of electrophoretic mobilities (Fig. 3). The number of precipitation lines and their appearance in CCIE varied considerably, suggesting individual differences in the response to bloom antigens. Thus the antigen heterogeneity of the preparation seems to be advantageous in testing for pigeon exposure. In conclusion, CCIE is suitable for the detection of PA against pigeon bloom extract. The test is recommended as a reliable indicator of pigeon exposure. ACKNOWLEDGEMENTS We thank The National Association against Lung Diseases and The Hojbjerg Foundation for financial support.
REFERENCES 1. Andersen, P. & Ghristensen, K. M.: Serum antibodies to pigeon antigens in smokers and nonsmokers. Acta Med. Scand. 213, 191-193, 1983.
EVALUATION OE PIGEON EXPOSURE 2. Banham, S. W., McKenzie, H., McSharry, G., Lynch, P. P. & Boyd, G.: Antibody against a pigeon bloomextract: a further antigen in pigeon fanciers' lung. Glin. Allergy 12, 173-178, 1982. 3. Barboriak, J. J., Fink, J. N., Sosman, A. J. & Dhaliwal, K. S.: Precipitating antibody against pigeon antigens in sera of asymptomatic pigeon breeders. J. Lab. Glm. Med. 82, 372-376, 1973. 4. Bergmann, K-G., Aiache, J. M., Bartmann, K., et al.: Precipitins to inhaled avian antigens: Results of an inter-laboratory study. Glin. Allergy 13, 451-457, 1983. 5. Burrell, R. & Rylander, R.: A critical review of the role of precipitins in hypersensitivity pneumonitis. Eur. J. Respir. Dis. 62, 332-343, 1981. 6. Fink, J. N.: Epidemiologic aspects of hypersensitivity pneumonitis. Monogr. Allergy 21, 59-69, 1987. 7. Fink, J. N., Barboriak, J. J., Sosman, A. J., Bukosky, R. J. & Arkins, J. A.: Antibodies against pigeon serum proteins in pigeon breeders. J. Lab. Glin. Med. 71, 20-25, 1968.
297
8. Hansen, N-G. G. & Siersted, H. G.: Allergic alveolitis among pigeon breeders in the county of Funen (summary in English). Ugeskr. Laeger, 152, 25-28, 1990 . 9. McSharry, G., Banham. S. W. & Boyd, G.: Effect of cigarette smoking on the antibody response to inhaled antigens and the prevalence of extrinsic allergic alveolitis among pigeon breeders. Glin. Allergy 15, 487-494. 1985. 10. McSharry, G., Banham, S. W., Lynch, P. P. & Boyd, G: Antibody measurement in extrinsic allergic alveolitis. Eur. J. Respir. Dis. 65, 259-265, 1984. 11. Weeke, B.: Grossed immunoelectrophoresis. In Axelsen, N. H., Kroll, J. & Weeke, B. (eds.): A manual of quantitative immunoelectrophoresis, pp. 47-56. Universitetsforlaget, Oslo, 1973. Address: Hans Chr. Siersted
Department of Respiratory Diseases Odense University Hospital DK-5000 Odense G Denmark
