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Evaluation of the sensitization of poultry workers to Aspergillus fumigatus and Cladophialophora carrionii
Journal de Mycologie Médicale (2009) 19, 104—109
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Evaluation of the sensitization of poultry workers to Aspergillus fumigatus and Cladophialophora carrionii ´Evaluation de la sensibilisation `a Aspergillus fumigatus et `a Cladophialophora carrionii chez des e ´leveurs de volailles A.R. Khosravi a,*, J. Chabavizadeh b, H. Shokri a, H. Tadjbakhsh c a Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, Azadi Street, PO Box: 14155-6453, Tehran, Iran b Department of Mycology and Parasitology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran c Department of Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
Received 7 December 2008; received in revised form 2 February 2009; accepted 13 February 2009 Available online 8 April 2009
KEYWORDS Aspergillus fumigatus; Cl adophialophora carrionii; Poultry worker; Skin prick test; Specific IgE and IgG
MOTS CLÉS Aspergillus fumigatus ; Cladophialophora
Summary Objective. — To estimate the prevalence of allergic bronchopulmonary mycoses (ABPM) due to Aspergillus fumigatus and Cladophialophora carrionii in poultry workers with asthma. Materials and methods. — One hundred and five workers and 76 controls were enrolled with sequential trials. Sensitization to A. fumigatus and C. carrionii antigens has been evaluated in poultry workers by skin prick test (SPT) and by the presence of specific IgE and IgG antibodies in ELISA assay. Results. — Positive SPTs were obtained in 55.2 and 39% of the workers for A. fumigatus and C. carrionii, respectively (P < 0.05). Specific anti-A. fumigatus IgE and IgG were detected in 49 (46.7%) and 70 (66.7%) of the workers, while 39 (37.1%) and 75 (71.4%) of them had positive IgE and IgG responses to C. carrionii antigen (P < 0.05). Significant correlations were found among SPT, specific IgE and IgG results for two fungal species tested (P < 0.05). Conclusion. — Our data confirmed that both SPT and ELISA testing could be important in the identification of poultry workers with fungal sensitizations. # 2009 Elsevier Masson SAS. All rights reserved. Résumé Objectif. — Évaluer la prévalence d’allergies pulmonaires dues à Aspergillus fumigatus et à Cladophilophora carrionii chez des éleveurs de volailles. Mate´riels et me ´thodes. — Cent cinq patients atteints d’asthme et 76 témoins ont été inclus dans cette étude. La sensibilisation à des antigènes extraits d’A. fumigatus et de C. carrionii a été
* Corresponding author. E-mail address: [email protected] (A.R. Khosravi). 1156-5233/$ — see front matter # 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2009.02.004
Sensitization of poultry workers to A. fumigatus and C. carrionii carrionii ; Éleveurs de volaille ; Prick test ; IgE ; IgG
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évaluée par des tests cutanés ( prick test) et par la recherche d’IgE spécifiques et d’anticorps de type IgG en Elisa. Re ´sultats. — Des prick tests positifs ont été obtenus chez 55,2 et 39 % des éleveurs asthmatiques pour respectivement A. fumigatus et C. carrionii ( p < 0,05). Des IgE et des IgG anti-A. fumigatus ont été détectées respectivement chez 49 (46,7 %) et 70 (66,7 %) éleveurs asthmatiques, alors que 39 (37,1 %) et 75 (71,4 %) d’entre eux ont présenté respectivement des IgE et des IgG spécifiques de C. carrionii ( p < 0,05). Des corrélations significatives ont été trouvées pour les tests cutanés et pour les IgE et les IgG des deux espèces fongiques testées ( p < 0,05). Les patients témoins ont montré une faible sensibilisation. Conclusion. — Nos données ont confirmé que les tests cutanés et la recherche des IgE et des IgG par Elisa pourraient être importants pour la détection de la sensibilisation à des antigènes fongiques chez les éleveurs de volailles. # 2009 Elsevier Masson SAS. Tous droits réservés.
Introduction Animal rearing and processing on an industrial scale have introduced environments with a high contamination of organic dusts from various sources such as feed, litter, fecal material, animal matter and microbes [25]. Dust in poultry houses contains several inflammatory agents; among them are allergenic fungi, which have many biologically potent components in the cell wall and cytoplasm [4,10,16,17]. Over the last several years, as the general awareness of fungal exposure as a cause of illness has grown, more and more patients have been presenting themselves for diagnosis and treatment after exposure to different environments heavily contaminated by fungi. Results of the European Community Respiratory Health Survey (ECRHS) (including 1,132 patients in the European Union, United States and Australia) showed that fungal allergy is a strong risk factor for severe asthma in work places [31]. Among the fungi, Aspergillus fumigatus was reported to be the commonest allergenic fungus that play many a part in the occurrence of allergic bronchopulmonary mycoses (ABPM), but the role of Cladophialophora carrionii has not been completely explored [3,18]. In poultry houses C. carrionii was an abundant mold following A. fumigatus. Though allergic bronchopulmonary diseases are now common in Iran, a high index of suspicion is still required to establish the diagnosis. Early diagnosis and appropriate therapy could alter the natural course of the disease and prevent the development of endstage lung fibrosis [15]. Our study was carried out to document the prevalence of ABPM due to A. fumigatus and C. carrionii antigens and to test for immunologically mediated responses in a group of poultry workers with asthma.
Materials and patients Cultivation of A. fumigatus and C. carrionii A. fumigatus and C. carrionii, originally airborne isolates obtained from Fungal Collection of Mycology Research Center of Iran, were cultured into Sabouraud glucose broth containing 40 U/ml penicillin and 20 mg/l streptomycin (DaruPakhsh Co., Tehran, Iran) and incubated in a rotative incubator (150 rpm) at 32 8C for five days. At the end of incubation, fungal colonies containing both spores and mycelia were harvested by Whatman filter paper (No.1, Clifton,
USA) washed three times with 0.05 M PBS buffer, pH 7.4, and stored at —20 8C until used for the preparation of crude antigens. All chemicals used, unless otherwise stated, were obtained from Merck Company (Darmstadt, Germany).
Cell fractionation and crude antigen preparation A suspension of wet fungal colonies was prepared in lysing buffer (62.5 mM Tris, 1 mM dithiotritol, 0.2 mg/ml of phenyl sulfonyl fluoride and 15% glycerol, pH 6.8) and then subjected to cell disruption in two consecutive steps using glass beads (diameter: 1 mm) on a vortex mixer during 15 min with 5-minute intervals and then sonication method (Sonicator set, UP 200 s, dr. hielscher sonicator set, Germany). Cell disruption was carried out using 60% amplitude for 48 min (2:4 min pulse on:off basis) to achieve maximum disruption of cells. After cell disruption, the crude antigens were separated from other cell components by centrifugation at 3000 g for 20 min, subsequently ultracentrifuged at 35,000 g for 1 h. The antigenic extracts were sterilized using filter (0.2 m, Sartorious, USA), lyophilized by means of freezedryer (Labconco, USA) and stored at —20 oC until used.
Determination of protein Protein concentration of sample was determined after reconstituting in 50 and 100 ml of distilled water by Bradford method [6].
Study groups A total of 105 poultry workers with confirmed asthma by a physician were recruited in March and September 2008 from Mycology Research Center in Tehran, Iran. The whole patients had a history of bronchitis, defective breathing and limited use of the chest cages. All workers were men and their age range was from 23 to 57 years (mean age: 34.5 years). The controls, selected from the poultry houses, comprised 76 healthy volunteers (76 men) with no history of atopy (mean age: 36 years; age range: 23 to 59 years). None of them had any kind of medication. The information on respiratory and nonrespiratory allergic symptoms of all the subjects was recorded by a questionnaire. A thorough clinical examination with special attention to the respiratory system was performed. A history of the subject’s oral corticosteroid
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use and other therapies were recorded. All subjects gave informed consent to participate in the study.
Skin prick test (SPT) A. fumigatus and C. carrionii antigens were diluted with 0.05 M PBS buffer, pH 7.4, and containing 50% glycerin and then sterilized by means of filter (0.2 m, Sartorius, USA). Skin tests were performed in both workers and controls. It was ensured that neither the worker nor the control group had received antihistamines prior to undergoing the skin tests. Intradermal testing was performed with 3 10—6 ml of the standardized antigens (protein concentration: 20 mg/ml) along with negative control (0.05 M PBS buffer, pH 7.4, containing 50% glycerin) and positive control (0.1% histamine) on the volar aspect of the forearm and pricked with a 27 gauge needle. The tests were read after 15 min and any reaction with erythema and a mean wheal diameter at least 3 mm greater than negative control was taken as positive.
ELISA assay Total IgE level was determined by indirect-ELISA test using commercial kits (Immuno-Biological Laboratories, Hamburg, Germany) according to manufacture’s protocol. The specific serum IgE and IgG antibodies for A. fumigatus and C. carrionii in workers’ sera were detected as previously described [17]. Briefly, A. fumigatus and C. carrionii antigens were diluted in 0.05 M carbonate—bicarbonate buffer, pH 9.5, and a suitable titre was chosen using checker board titration. One hundred microliter of the antigen (30 mg/ml) was added to the wells and stored overnight at 4 8C. The wells were rinsed extensively with washing buffer (PBS-Tween 20 0.05%), and then 150 ml of blocking buffer was added to the wells and microplate was stored at 37 8C for 2.5 h. The wells were rinsed again. Serum samples were diluted 1:4 in PBS-Tween 20-BSA 1% and 100 ml were added to each well and incubated with shaking at 37 8C for 2 h, followed by washing with PBS-Tween 20 0.05%. Antihuman IgE conjugated with HRP (Sigma, St. Louis, USA) for IgE assay and antihuman IgG conjugated with HRP (Sigma, St. Louis, USA) for IgG assay were diluted 1/2000 and 1/4000, respectively, in PBS-Tween 20-BSA 1% and 100 ml was added to each well and incubated at 37 8C for 1.5 h and followed by washing with PBS-Tween 20-BSA 1%. Each well received 100 ml of a solution of 0.1 M diethanolamine (DEA), pH 9.8, 1 mM magnesium chloride and paranitrophenol phosphate (PNPD). After 30 min, the reaction was stopped with 50 ml of 3 M NaOH
and the OD was read at 405 nm. Each sample was tested in duplicate. Sera of control group were used as negative control. OD (405) values more than the OD value of the mean obtained with controls + 3 standard deviations (SDs) were considered positive.
Statistical analysis The incidences of positive SPT and ELISA tests for each individual antigen were computed using simple percentages. These proportions were compared using the x2 test. Correlations between SPT and specific serum IgE and/or IgG antibodies were calculated using a two-tailed Spearman’s rho with statistical significance determined using the t-test. Statistical significance was considered a P value less than 0.05.
Results Patient characteristics and SPT reactions A total of 181 subjects were tested with two antigens for a total of 362 skin tests. All poultry workers were male with an average age of 34.5 (range: 23—57). Of the 181 subjects analyzed, 62 (59%) workers and 40 (52.6%) controls had a smoking history. Smokers showed significantly more positive reactions in SPT to A. fumigatus than nonsmokers (P < 0.05), while this difference was not significant to C. carrionii. As illustrated in Table 1, 58 (55.2%) of the workers responded with positive SPT reaction to A. fumigatus antigens and 41 (39%) of them also responded with a positive SPT reaction to C. carrionii antigens (P < 0.05). None of the controls reacted with a positive SPT to both fungal antigens. Significant differences were observed between the SPT results of the workers and controls to both A. fumigatus and C. carrionii antigens (P < 0.05). The results did not show any significant differences among different ages in poultry workers.
ELISA test A positive total IgE result was defined as a value of at least 100 IU/ml. Positive sera were recorded in 48 (45.7%) of the workers and six (7.9%) of the controls (P < 0.05). Concerning the specific IgE and IgG antibodies assays, the mean OD (405) + 3 SDs control group was statistically calculated 0.75 for A. fumigatus-specific serum IgE, 0.90 for A. fumigatusspecific serum IgG, 0.52 for C. carrionii-specific serum IgE and
Table 1 Frequency of positive skin prick, total IgE and specific IgE and IgG-ELISA tests to Aspergillus fumigatus and Cladophialophora carrionii antigens in poultry workers and controls (n, %). Re ´sultats des tests cutane´s et de la recherche des IgE et des IgG contre des antige `nes extraits d’Aspergillus fumigatus et de Cladophialophora carrionii chez des e´leveurs de volailles (n, %). Group
Worker Control
Total IgE
48 (45.5) 6 (7.9)
Sensitization to A. fumigatus
Sensitization to C. carrionii
SPT
Specific IgE
Specific IgG
SPT
Specific IgE
Specific IgG
58 (55.2) 0 (0)
49 (46.7) 2 (2.6)
70 (66.7) 2 (2.6)
41 (39) 0 (0)
39 (37.1) 2 (2.6)
75 (71.4) 2 (2.6)
SPT: skin prick test. Positive skin prick test: any reaction with erythema and a mean wheal diameter at least 3 mm greater than negative control was taken as positive; positive serologic tests: OD(405) values more than the OD value of the mean obtained with controls + 3 standard deviations (SDs) were considered positive.
Sensitization of poultry workers to A. fumigatus and C. carrionii
Figure 1 Evaluation of the presence of specific serum IgE and IgG antbodies to A. fumigatus and C. carrionii antigens in poultry workers and controls. Évaluation des IgE et des IgG anti-A. fumigatus et anti-C. carrionii chez les éleveurs de volailles asthmatiques et chez les patients témoins.
0.50 for C. carrionii-specific serum IgG antibodies. Positive sera were defined with ODs more than 0.75, 0.90, 0.52 and 0.50. According to Table 1, of 105 sera obtained from workers, 49 (46.7%) and 39 (37.1%) were found to have specific IgE antibodies to A. fumigatus and C. carrionii, while 70 (66.7%) and 75 (71.4%) showed IgG responses against A. fumigatus and C. carrionii, respectively. Only two controls (2.6%) had positive responses in specific IgE and IgG antibodies against both antigens tested. Significant correlations were observed between A. fumigatus-specific serum IgE and C. carrionii-specific serum IgE, A. fumigatus-specific serum IgG and C. carrioniispecific serum IgG, and also between specific IgE and IgG antibodies in both fungi (P < 0.05) (Fig. 1). Significant positive correlations were found between the SPT reactions and A. fumigatus- and C. carrionii-specific serum IgE (rs = 0.75 and rs = 0.88, P < 0.05), but not for SPT reactions with A. fumigatus- and C. carrionii-specific serum IgG. Concordance between the tests was 64.5%, leaving 35.5% of patients with a positive test using either SPT or ELISA. There was also no significant correlation between the total IgE level and the levels of specific IgE and IgG antibodies to both A. fumigatus and C. carrionii.
Discussion ABPM are devastating pulmonary diseases that result from an aggressive allergic response to fungal colonizations in the airways. The environmental conditions of poultry houses favored the contamination by fungi [5,18]. Therefore, it is important to look for major fungal agents and their relevant immunologic profiles to assess the real etiology of respiratory allergic disorders in poultry workers. Early fungal exposure itself can trigger innate immune system and after a few days or weeks the adaptive system is likely to become involved as antibodies and T-cells specifically reactive to fungal antigens. Several investigators showed specific and nonspecific mechanisms of defense in poultry workers and the following characters were found: an increase in the blood leucocyte phagocytic activity, nonspecific neutrophilic damage values, specific neutrophilic damage and specific lymphocyte damage values in blood serum [24] and the shifts in immunoregulation expressed in terms of the decrease of the number of T-lymphocyte suppressors, precursors of
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effectory and immunoregulatory T-lymphocytes, reduction of functional activity of T- and B-cells along with an increase in the content of B-lymphocytes with antibody-dependent killers’ sensitization and suppression of nonspecific protective factors [8]. In poultry workers different respiratory allergic disorders have been reported by investigators [25,30,32]. Zuskin et al. [32] demonstrated that poultry male smokers had significantly higher prevalence of respiratory allergic disorders than poultry male nonsmokers (P < 0.05), which is consistent with our finding. In our study from Iran, we screened 105 poultry workers for A. fumigatus and C. carrionii sensitizations, and found that 58 (55.2%) of our 105 workers had positive skin reactivity to A. fumigatus. The reported frequency of skin test reactivity to Aspergillus antigens in patients with ABPA has varied from 16 to 38% in different parts of the world [29]. It shows that the prevalence of positive SPT is different from study to study. In this study, the prevalence of sensitization to Aspergillus antigen is higher than other studies and is likely explained by higher fungal exposure in our population, different contents of the allergens in various strains tested and the lack of a single diagnostic criterion with a standardized test [28]. Separate batches of extract produced by the same strains of a fungal species grown under same cultural conditions and duration may show batch to batch variability in potency. This variability in potency has been well documented by Salvaggio and Aukrust [26]. As expected, we observed that the group of workers had significantly a higher mean total IgE (45.5%), specific IgE (46.7%) and IgG (66.7%) values against A. fumigatus antigen than those of controls. Among the workers, between 1.9 and 8.5% of IgE negative sera were identified to have positive SPT towards A. fumigatus. Some of the patients presenting symptoms of fungal illness with positive SPT do not show IgE antibody to the fungus [9]. This may be the result of isotype switching from IgE to IgG production as stimulation of the immune system increases as well as cross-reactivity among different Aspergillus antigens. These illnesses were originally described in association with various occupational exposures in unprotected workers [9]. Anderson and Schonheyder [1] showed the levels of IgG and IgE antibodies to Aspergillus antigen were significantly higher in poultry workers than in blood donors (P < 0.05). In another study of humoral immunity, it was shown the presence of IgG antibody (76% positive reactions in the farms, 63% in the slaughter house) in sera of workers [30], which is consistent with our study. In contrast, Maurya et al. [20] demonstrated that 28.5% of their asthmatic patients had a positive skin test reaction to Aspergillus antigens and that 10.4% of the patients had test results that were positive for specific IgG. In a comparative study of the prevalence of sensitization to Aspergillus antigens among asthmatic subjects in Cleveland and London, it was observed that 28% of asthmatic subjects from Cleveland and 23% of those from London had immediate skin reactivity to Aspergillus antigens. Furthermore, 7.5% of the patients from Cleveland and 10.5% from London had anti-Aspergillus IgG antibody in their serum [27]. A Canadian study found that 21.5% of 200 asthmatic subjects reacted to Aspergillus antigens [19], while another report from South Africa observed a skin test positivity of 22%, while 5% were positive for serum precipitins [2]. The investigators found that, in an appropriate clinical setting,
108 the high serum levels of IgE-A. fumigatus or IgG-A. fumigatus can be diagnostic of allergic respiratory disorders [11,21]. Although our findings are consistent with previous reports that fungal sensitization is common in patients with respiratory allergic disorders, differences observed between our results and other investigations are related to a lack of standardized antigenic extracts, the large number of possible antigenic epitopes, and the poor sensitivity of the skin immediate hypersensitivity response, the stability of crude antigens, length of the cultured period, the variations in the fungal strains, the allergenic extracts used and the different assay systems employed. In addition, as allergic disorders are dependent on environmental and genetic factors, immune responses to the allergens may vary in dependence of populations and races [11]. Therefore, standardization of the fungal extracts is imperative to improve the diagnosis of fungal sensitization. Cladophialophora species are the most important allergenic species. Information on the prevalence of Cladophialophora in different indoor environments is limited and occupational health risk associated with exposure to Cladophialophora species has not been thoroughly assessed. A study on poultry houses revealed a total fungal spore concentration of 2 107 m—3 [22]. Various Cladophialophora species are often associated with wood products. Timber, wood chips and firewood found in different indoors such as poultry houses can be profoundly colonized by Cladophialophora species carrying the potential risk of increased exposure to this fungus [7]. Based on a study in Iran, C. carrionii was the predominant fungus in poultry houses. It may be resulted from wet wood chips on the litter, high humidity and weak air-conditioning [23]. In this study, 39% of 105 asthmatic workers had a positive skin test result for C. carrionii antigens, while 37.1 and 71.4% had positive test results for the presence of specific serum IgE and IgG antibodies, respectively. In our knowledge, there is little information on clinically Cladophialophora sensitization in allergic subjects [3,13,14]. Hasnain et al. [12] investigated 605 individuals, representing positive SPTs to Cladophialophora species in 19.6% individuals. In another study by Jaakkola et al. [13], the relations between fungi-specific IgE antibodies to six fungi were analyzed. IgE antibodies to Cl. herbarium were associated with an increased risk of adult-onset asthma. Explanation for the variation in the results obtained in various studies is associated with some factors such as various extraction methods and different criteria to select the patients and also the protocol of SPT can affect the SPT results. Regarding to the existence of evidence in the occupational health literature documenting that poultry workers are at risk for developing respiratory disease, there is a need to protect the health of poultry workers. Tests of allergy and hypersensitivity are associated with exposure, but not with disease. The possibility of useful tests of sensitization has not been excluded; a prospective study design is likely to be more rewarding than cross-sectional approaches such as in this study.
Acknowledgements This work was supported by the Research Council of University of Tehran. The authors would like to thank Dr. Kimiaei
A.R. Khosravi et al. and his colleagues at the Tehran Allergy Clinic for cooperation in skin test of patients. Also, the authors thank all the subjects who volunteered.
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Evaluation of the sensitization of poultry workers to Aspergillus fumigatus and Cladophialophora carrionii ´Evaluation de la sensibilisation `a Aspergillus fumigatus et `a Cladophialophora carrionii chez des e ´leveurs de volailles A.R. Khosravi a,*, J. Chabavizadeh b, H. Shokri a, H. Tadjbakhsh c a Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, Azadi Street, PO Box: 14155-6453, Tehran, Iran b Department of Mycology and Parasitology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran c Department of Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
Received 7 December 2008; received in revised form 2 February 2009; accepted 13 February 2009 Available online 8 April 2009
KEYWORDS Aspergillus fumigatus; Cl adophialophora carrionii; Poultry worker; Skin prick test; Specific IgE and IgG
MOTS CLÉS Aspergillus fumigatus ; Cladophialophora
Summary Objective. — To estimate the prevalence of allergic bronchopulmonary mycoses (ABPM) due to Aspergillus fumigatus and Cladophialophora carrionii in poultry workers with asthma. Materials and methods. — One hundred and five workers and 76 controls were enrolled with sequential trials. Sensitization to A. fumigatus and C. carrionii antigens has been evaluated in poultry workers by skin prick test (SPT) and by the presence of specific IgE and IgG antibodies in ELISA assay. Results. — Positive SPTs were obtained in 55.2 and 39% of the workers for A. fumigatus and C. carrionii, respectively (P < 0.05). Specific anti-A. fumigatus IgE and IgG were detected in 49 (46.7%) and 70 (66.7%) of the workers, while 39 (37.1%) and 75 (71.4%) of them had positive IgE and IgG responses to C. carrionii antigen (P < 0.05). Significant correlations were found among SPT, specific IgE and IgG results for two fungal species tested (P < 0.05). Conclusion. — Our data confirmed that both SPT and ELISA testing could be important in the identification of poultry workers with fungal sensitizations. # 2009 Elsevier Masson SAS. All rights reserved. Résumé Objectif. — Évaluer la prévalence d’allergies pulmonaires dues à Aspergillus fumigatus et à Cladophilophora carrionii chez des éleveurs de volailles. Mate´riels et me ´thodes. — Cent cinq patients atteints d’asthme et 76 témoins ont été inclus dans cette étude. La sensibilisation à des antigènes extraits d’A. fumigatus et de C. carrionii a été
* Corresponding author. E-mail address: [email protected] (A.R. Khosravi). 1156-5233/$ — see front matter # 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2009.02.004
Sensitization of poultry workers to A. fumigatus and C. carrionii carrionii ; Éleveurs de volaille ; Prick test ; IgE ; IgG
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évaluée par des tests cutanés ( prick test) et par la recherche d’IgE spécifiques et d’anticorps de type IgG en Elisa. Re ´sultats. — Des prick tests positifs ont été obtenus chez 55,2 et 39 % des éleveurs asthmatiques pour respectivement A. fumigatus et C. carrionii ( p < 0,05). Des IgE et des IgG anti-A. fumigatus ont été détectées respectivement chez 49 (46,7 %) et 70 (66,7 %) éleveurs asthmatiques, alors que 39 (37,1 %) et 75 (71,4 %) d’entre eux ont présenté respectivement des IgE et des IgG spécifiques de C. carrionii ( p < 0,05). Des corrélations significatives ont été trouvées pour les tests cutanés et pour les IgE et les IgG des deux espèces fongiques testées ( p < 0,05). Les patients témoins ont montré une faible sensibilisation. Conclusion. — Nos données ont confirmé que les tests cutanés et la recherche des IgE et des IgG par Elisa pourraient être importants pour la détection de la sensibilisation à des antigènes fongiques chez les éleveurs de volailles. # 2009 Elsevier Masson SAS. Tous droits réservés.
Introduction Animal rearing and processing on an industrial scale have introduced environments with a high contamination of organic dusts from various sources such as feed, litter, fecal material, animal matter and microbes [25]. Dust in poultry houses contains several inflammatory agents; among them are allergenic fungi, which have many biologically potent components in the cell wall and cytoplasm [4,10,16,17]. Over the last several years, as the general awareness of fungal exposure as a cause of illness has grown, more and more patients have been presenting themselves for diagnosis and treatment after exposure to different environments heavily contaminated by fungi. Results of the European Community Respiratory Health Survey (ECRHS) (including 1,132 patients in the European Union, United States and Australia) showed that fungal allergy is a strong risk factor for severe asthma in work places [31]. Among the fungi, Aspergillus fumigatus was reported to be the commonest allergenic fungus that play many a part in the occurrence of allergic bronchopulmonary mycoses (ABPM), but the role of Cladophialophora carrionii has not been completely explored [3,18]. In poultry houses C. carrionii was an abundant mold following A. fumigatus. Though allergic bronchopulmonary diseases are now common in Iran, a high index of suspicion is still required to establish the diagnosis. Early diagnosis and appropriate therapy could alter the natural course of the disease and prevent the development of endstage lung fibrosis [15]. Our study was carried out to document the prevalence of ABPM due to A. fumigatus and C. carrionii antigens and to test for immunologically mediated responses in a group of poultry workers with asthma.
Materials and patients Cultivation of A. fumigatus and C. carrionii A. fumigatus and C. carrionii, originally airborne isolates obtained from Fungal Collection of Mycology Research Center of Iran, were cultured into Sabouraud glucose broth containing 40 U/ml penicillin and 20 mg/l streptomycin (DaruPakhsh Co., Tehran, Iran) and incubated in a rotative incubator (150 rpm) at 32 8C for five days. At the end of incubation, fungal colonies containing both spores and mycelia were harvested by Whatman filter paper (No.1, Clifton,
USA) washed three times with 0.05 M PBS buffer, pH 7.4, and stored at —20 8C until used for the preparation of crude antigens. All chemicals used, unless otherwise stated, were obtained from Merck Company (Darmstadt, Germany).
Cell fractionation and crude antigen preparation A suspension of wet fungal colonies was prepared in lysing buffer (62.5 mM Tris, 1 mM dithiotritol, 0.2 mg/ml of phenyl sulfonyl fluoride and 15% glycerol, pH 6.8) and then subjected to cell disruption in two consecutive steps using glass beads (diameter: 1 mm) on a vortex mixer during 15 min with 5-minute intervals and then sonication method (Sonicator set, UP 200 s, dr. hielscher sonicator set, Germany). Cell disruption was carried out using 60% amplitude for 48 min (2:4 min pulse on:off basis) to achieve maximum disruption of cells. After cell disruption, the crude antigens were separated from other cell components by centrifugation at 3000 g for 20 min, subsequently ultracentrifuged at 35,000 g for 1 h. The antigenic extracts were sterilized using filter (0.2 m, Sartorious, USA), lyophilized by means of freezedryer (Labconco, USA) and stored at —20 oC until used.
Determination of protein Protein concentration of sample was determined after reconstituting in 50 and 100 ml of distilled water by Bradford method [6].
Study groups A total of 105 poultry workers with confirmed asthma by a physician were recruited in March and September 2008 from Mycology Research Center in Tehran, Iran. The whole patients had a history of bronchitis, defective breathing and limited use of the chest cages. All workers were men and their age range was from 23 to 57 years (mean age: 34.5 years). The controls, selected from the poultry houses, comprised 76 healthy volunteers (76 men) with no history of atopy (mean age: 36 years; age range: 23 to 59 years). None of them had any kind of medication. The information on respiratory and nonrespiratory allergic symptoms of all the subjects was recorded by a questionnaire. A thorough clinical examination with special attention to the respiratory system was performed. A history of the subject’s oral corticosteroid
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use and other therapies were recorded. All subjects gave informed consent to participate in the study.
Skin prick test (SPT) A. fumigatus and C. carrionii antigens were diluted with 0.05 M PBS buffer, pH 7.4, and containing 50% glycerin and then sterilized by means of filter (0.2 m, Sartorius, USA). Skin tests were performed in both workers and controls. It was ensured that neither the worker nor the control group had received antihistamines prior to undergoing the skin tests. Intradermal testing was performed with 3 10—6 ml of the standardized antigens (protein concentration: 20 mg/ml) along with negative control (0.05 M PBS buffer, pH 7.4, containing 50% glycerin) and positive control (0.1% histamine) on the volar aspect of the forearm and pricked with a 27 gauge needle. The tests were read after 15 min and any reaction with erythema and a mean wheal diameter at least 3 mm greater than negative control was taken as positive.
ELISA assay Total IgE level was determined by indirect-ELISA test using commercial kits (Immuno-Biological Laboratories, Hamburg, Germany) according to manufacture’s protocol. The specific serum IgE and IgG antibodies for A. fumigatus and C. carrionii in workers’ sera were detected as previously described [17]. Briefly, A. fumigatus and C. carrionii antigens were diluted in 0.05 M carbonate—bicarbonate buffer, pH 9.5, and a suitable titre was chosen using checker board titration. One hundred microliter of the antigen (30 mg/ml) was added to the wells and stored overnight at 4 8C. The wells were rinsed extensively with washing buffer (PBS-Tween 20 0.05%), and then 150 ml of blocking buffer was added to the wells and microplate was stored at 37 8C for 2.5 h. The wells were rinsed again. Serum samples were diluted 1:4 in PBS-Tween 20-BSA 1% and 100 ml were added to each well and incubated with shaking at 37 8C for 2 h, followed by washing with PBS-Tween 20 0.05%. Antihuman IgE conjugated with HRP (Sigma, St. Louis, USA) for IgE assay and antihuman IgG conjugated with HRP (Sigma, St. Louis, USA) for IgG assay were diluted 1/2000 and 1/4000, respectively, in PBS-Tween 20-BSA 1% and 100 ml was added to each well and incubated at 37 8C for 1.5 h and followed by washing with PBS-Tween 20-BSA 1%. Each well received 100 ml of a solution of 0.1 M diethanolamine (DEA), pH 9.8, 1 mM magnesium chloride and paranitrophenol phosphate (PNPD). After 30 min, the reaction was stopped with 50 ml of 3 M NaOH
and the OD was read at 405 nm. Each sample was tested in duplicate. Sera of control group were used as negative control. OD (405) values more than the OD value of the mean obtained with controls + 3 standard deviations (SDs) were considered positive.
Statistical analysis The incidences of positive SPT and ELISA tests for each individual antigen were computed using simple percentages. These proportions were compared using the x2 test. Correlations between SPT and specific serum IgE and/or IgG antibodies were calculated using a two-tailed Spearman’s rho with statistical significance determined using the t-test. Statistical significance was considered a P value less than 0.05.
Results Patient characteristics and SPT reactions A total of 181 subjects were tested with two antigens for a total of 362 skin tests. All poultry workers were male with an average age of 34.5 (range: 23—57). Of the 181 subjects analyzed, 62 (59%) workers and 40 (52.6%) controls had a smoking history. Smokers showed significantly more positive reactions in SPT to A. fumigatus than nonsmokers (P < 0.05), while this difference was not significant to C. carrionii. As illustrated in Table 1, 58 (55.2%) of the workers responded with positive SPT reaction to A. fumigatus antigens and 41 (39%) of them also responded with a positive SPT reaction to C. carrionii antigens (P < 0.05). None of the controls reacted with a positive SPT to both fungal antigens. Significant differences were observed between the SPT results of the workers and controls to both A. fumigatus and C. carrionii antigens (P < 0.05). The results did not show any significant differences among different ages in poultry workers.
ELISA test A positive total IgE result was defined as a value of at least 100 IU/ml. Positive sera were recorded in 48 (45.7%) of the workers and six (7.9%) of the controls (P < 0.05). Concerning the specific IgE and IgG antibodies assays, the mean OD (405) + 3 SDs control group was statistically calculated 0.75 for A. fumigatus-specific serum IgE, 0.90 for A. fumigatusspecific serum IgG, 0.52 for C. carrionii-specific serum IgE and
Table 1 Frequency of positive skin prick, total IgE and specific IgE and IgG-ELISA tests to Aspergillus fumigatus and Cladophialophora carrionii antigens in poultry workers and controls (n, %). Re ´sultats des tests cutane´s et de la recherche des IgE et des IgG contre des antige `nes extraits d’Aspergillus fumigatus et de Cladophialophora carrionii chez des e´leveurs de volailles (n, %). Group
Worker Control
Total IgE
48 (45.5) 6 (7.9)
Sensitization to A. fumigatus
Sensitization to C. carrionii
SPT
Specific IgE
Specific IgG
SPT
Specific IgE
Specific IgG
58 (55.2) 0 (0)
49 (46.7) 2 (2.6)
70 (66.7) 2 (2.6)
41 (39) 0 (0)
39 (37.1) 2 (2.6)
75 (71.4) 2 (2.6)
SPT: skin prick test. Positive skin prick test: any reaction with erythema and a mean wheal diameter at least 3 mm greater than negative control was taken as positive; positive serologic tests: OD(405) values more than the OD value of the mean obtained with controls + 3 standard deviations (SDs) were considered positive.
Sensitization of poultry workers to A. fumigatus and C. carrionii
Figure 1 Evaluation of the presence of specific serum IgE and IgG antbodies to A. fumigatus and C. carrionii antigens in poultry workers and controls. Évaluation des IgE et des IgG anti-A. fumigatus et anti-C. carrionii chez les éleveurs de volailles asthmatiques et chez les patients témoins.
0.50 for C. carrionii-specific serum IgG antibodies. Positive sera were defined with ODs more than 0.75, 0.90, 0.52 and 0.50. According to Table 1, of 105 sera obtained from workers, 49 (46.7%) and 39 (37.1%) were found to have specific IgE antibodies to A. fumigatus and C. carrionii, while 70 (66.7%) and 75 (71.4%) showed IgG responses against A. fumigatus and C. carrionii, respectively. Only two controls (2.6%) had positive responses in specific IgE and IgG antibodies against both antigens tested. Significant correlations were observed between A. fumigatus-specific serum IgE and C. carrionii-specific serum IgE, A. fumigatus-specific serum IgG and C. carrioniispecific serum IgG, and also between specific IgE and IgG antibodies in both fungi (P < 0.05) (Fig. 1). Significant positive correlations were found between the SPT reactions and A. fumigatus- and C. carrionii-specific serum IgE (rs = 0.75 and rs = 0.88, P < 0.05), but not for SPT reactions with A. fumigatus- and C. carrionii-specific serum IgG. Concordance between the tests was 64.5%, leaving 35.5% of patients with a positive test using either SPT or ELISA. There was also no significant correlation between the total IgE level and the levels of specific IgE and IgG antibodies to both A. fumigatus and C. carrionii.
Discussion ABPM are devastating pulmonary diseases that result from an aggressive allergic response to fungal colonizations in the airways. The environmental conditions of poultry houses favored the contamination by fungi [5,18]. Therefore, it is important to look for major fungal agents and their relevant immunologic profiles to assess the real etiology of respiratory allergic disorders in poultry workers. Early fungal exposure itself can trigger innate immune system and after a few days or weeks the adaptive system is likely to become involved as antibodies and T-cells specifically reactive to fungal antigens. Several investigators showed specific and nonspecific mechanisms of defense in poultry workers and the following characters were found: an increase in the blood leucocyte phagocytic activity, nonspecific neutrophilic damage values, specific neutrophilic damage and specific lymphocyte damage values in blood serum [24] and the shifts in immunoregulation expressed in terms of the decrease of the number of T-lymphocyte suppressors, precursors of
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effectory and immunoregulatory T-lymphocytes, reduction of functional activity of T- and B-cells along with an increase in the content of B-lymphocytes with antibody-dependent killers’ sensitization and suppression of nonspecific protective factors [8]. In poultry workers different respiratory allergic disorders have been reported by investigators [25,30,32]. Zuskin et al. [32] demonstrated that poultry male smokers had significantly higher prevalence of respiratory allergic disorders than poultry male nonsmokers (P < 0.05), which is consistent with our finding. In our study from Iran, we screened 105 poultry workers for A. fumigatus and C. carrionii sensitizations, and found that 58 (55.2%) of our 105 workers had positive skin reactivity to A. fumigatus. The reported frequency of skin test reactivity to Aspergillus antigens in patients with ABPA has varied from 16 to 38% in different parts of the world [29]. It shows that the prevalence of positive SPT is different from study to study. In this study, the prevalence of sensitization to Aspergillus antigen is higher than other studies and is likely explained by higher fungal exposure in our population, different contents of the allergens in various strains tested and the lack of a single diagnostic criterion with a standardized test [28]. Separate batches of extract produced by the same strains of a fungal species grown under same cultural conditions and duration may show batch to batch variability in potency. This variability in potency has been well documented by Salvaggio and Aukrust [26]. As expected, we observed that the group of workers had significantly a higher mean total IgE (45.5%), specific IgE (46.7%) and IgG (66.7%) values against A. fumigatus antigen than those of controls. Among the workers, between 1.9 and 8.5% of IgE negative sera were identified to have positive SPT towards A. fumigatus. Some of the patients presenting symptoms of fungal illness with positive SPT do not show IgE antibody to the fungus [9]. This may be the result of isotype switching from IgE to IgG production as stimulation of the immune system increases as well as cross-reactivity among different Aspergillus antigens. These illnesses were originally described in association with various occupational exposures in unprotected workers [9]. Anderson and Schonheyder [1] showed the levels of IgG and IgE antibodies to Aspergillus antigen were significantly higher in poultry workers than in blood donors (P < 0.05). In another study of humoral immunity, it was shown the presence of IgG antibody (76% positive reactions in the farms, 63% in the slaughter house) in sera of workers [30], which is consistent with our study. In contrast, Maurya et al. [20] demonstrated that 28.5% of their asthmatic patients had a positive skin test reaction to Aspergillus antigens and that 10.4% of the patients had test results that were positive for specific IgG. In a comparative study of the prevalence of sensitization to Aspergillus antigens among asthmatic subjects in Cleveland and London, it was observed that 28% of asthmatic subjects from Cleveland and 23% of those from London had immediate skin reactivity to Aspergillus antigens. Furthermore, 7.5% of the patients from Cleveland and 10.5% from London had anti-Aspergillus IgG antibody in their serum [27]. A Canadian study found that 21.5% of 200 asthmatic subjects reacted to Aspergillus antigens [19], while another report from South Africa observed a skin test positivity of 22%, while 5% were positive for serum precipitins [2]. The investigators found that, in an appropriate clinical setting,
108 the high serum levels of IgE-A. fumigatus or IgG-A. fumigatus can be diagnostic of allergic respiratory disorders [11,21]. Although our findings are consistent with previous reports that fungal sensitization is common in patients with respiratory allergic disorders, differences observed between our results and other investigations are related to a lack of standardized antigenic extracts, the large number of possible antigenic epitopes, and the poor sensitivity of the skin immediate hypersensitivity response, the stability of crude antigens, length of the cultured period, the variations in the fungal strains, the allergenic extracts used and the different assay systems employed. In addition, as allergic disorders are dependent on environmental and genetic factors, immune responses to the allergens may vary in dependence of populations and races [11]. Therefore, standardization of the fungal extracts is imperative to improve the diagnosis of fungal sensitization. Cladophialophora species are the most important allergenic species. Information on the prevalence of Cladophialophora in different indoor environments is limited and occupational health risk associated with exposure to Cladophialophora species has not been thoroughly assessed. A study on poultry houses revealed a total fungal spore concentration of 2 107 m—3 [22]. Various Cladophialophora species are often associated with wood products. Timber, wood chips and firewood found in different indoors such as poultry houses can be profoundly colonized by Cladophialophora species carrying the potential risk of increased exposure to this fungus [7]. Based on a study in Iran, C. carrionii was the predominant fungus in poultry houses. It may be resulted from wet wood chips on the litter, high humidity and weak air-conditioning [23]. In this study, 39% of 105 asthmatic workers had a positive skin test result for C. carrionii antigens, while 37.1 and 71.4% had positive test results for the presence of specific serum IgE and IgG antibodies, respectively. In our knowledge, there is little information on clinically Cladophialophora sensitization in allergic subjects [3,13,14]. Hasnain et al. [12] investigated 605 individuals, representing positive SPTs to Cladophialophora species in 19.6% individuals. In another study by Jaakkola et al. [13], the relations between fungi-specific IgE antibodies to six fungi were analyzed. IgE antibodies to Cl. herbarium were associated with an increased risk of adult-onset asthma. Explanation for the variation in the results obtained in various studies is associated with some factors such as various extraction methods and different criteria to select the patients and also the protocol of SPT can affect the SPT results. Regarding to the existence of evidence in the occupational health literature documenting that poultry workers are at risk for developing respiratory disease, there is a need to protect the health of poultry workers. Tests of allergy and hypersensitivity are associated with exposure, but not with disease. The possibility of useful tests of sensitization has not been excluded; a prospective study design is likely to be more rewarding than cross-sectional approaches such as in this study.
Acknowledgements This work was supported by the Research Council of University of Tehran. The authors would like to thank Dr. Kimiaei
A.R. Khosravi et al. and his colleagues at the Tehran Allergy Clinic for cooperation in skin test of patients. Also, the authors thank all the subjects who volunteered.
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