Clinical Allergy, 1979, Volume 9, pages 43-52
Microbiological and serological studies of farmers' lung in Finland E. O. TERHO and J. LACEY* Department of Diseases of the Chest, University of Turku, Finland, and * Department of Plant Pathology, Rothamsted Experimental Station, Harpenden, Hertfordshire (Received 24 July 1978; accepted for publication 8 August 1978)
Summary
Sera from few Finnish patients with clinical farmers' lung react in precipitin tests with extracts of the thermophilic actinomycetes that commonly cause the disease elsewhere. Hays associated with the disease in Finland showed less evidence of spontaneous heating and contained fewer actinomycete spores than British hays. Only Thermoactinomyces vulgaris was sometimes abundant. Some species of mesophilic fungi were more abundant than in Britain and one, Aspergillus umbrosus, reacted with most sera from farmers' lung patients in precipitin tests. A panel of antigens, including thermophilic actinomycetes, A. umbrosus and other species of the Aspergillus glaucus group, is recommended for screening farmers' lung sera. Introduction
Mouldy hays associated with farmers' lung (FL) in England are characterized by a heavy growth of actinomycetes (Gregory & Lacey, 1963). The main causative agent of FL has been considered to be Micropolyspora faeni (Pepys et al., 1963; Parratt & Boyd, 1976), a thermophilic actinomycete that grows in hay that has been stored damp and heats spontaneously. Precipitating antibodies to M. faeni are present in a large proportion of the sera of English FL patients, and evidence of exposure to the spores is given by some unaffected farm workers (Pepys & Jenkins, 1965). Since 1972, precipitin tests have been used routinely at the Department of Diseases of the Chest, University of Turku, Finland, for the investigation of FL. Although antigens relevant to FL in England were used in the same agar gel double diffusion technique employed by Pepys et al. (1963), few sera from subjects with a typical clinical history of FL gave positive reactions to M. faeni. It was therefore thought that micro-organisms other than M. faeni might be implicated in the disease in Finland. Correspondence: Dr E. O. Terho, Department of Pulmonary Diseases, University Central Hospital, Kuopio, Finland. 0009-9090/79/0100-0043 $02.00 © 1979 Blackwell Scientific Publications 43
44
E. O. Terho and J. Lacey
The present study was designed to identify the types of micro-organisms to which Finnish FL patients were exposed. Hay samples associated with FL were analysed microbiologically and the sera of clinically proven cases of FL were tested by an agar gel double diffusion technique against a panel of antigens selected on the basis of their occurrence in hay samples. Materials and methods Sources of hay samples Patients suffering from FL were asked to supply samples of hay, straw or grain which they were handling at the onset of their illness. Additionally, one sample of good Finnish hay was examined. Samples were sent to Rothamsted Experimental Station, Harpenden, England, for microbiological examination. Microbiological examination of hay samples All hay samples were examined by the wind tunnel method of Gregory & Lacey (1963). Spores blow off the hay were trapped on microscope slides in a cascade impactor, for microscopic classification and enumeration, or on agar media in petri dishes in an Andersen sampler, for growth in culture. Fungi were isolated on 2% malt extract agar containing 20 units penicillin and 40 units streptomycin per ml medium and incubated at either 25°C or 40°C. Actinomycetes and other bacteria were grown on either half-strength Oxoid nutrient agar incubated at 25°C or 40°C or half-strength Table 1. Antigen panel used to test Finnish FL sera Actinomycetes Mieropolyspora faeni 9535 Mieropolyspora faeni A 1311 Saccharomonospora viridis A 66 Streptomyces griseoflavus A. 159 Thermoactinomyces vulgaris A 64 Thermoactinomyces vulgaris Hollister-Stier Fungi Acremonium strictum C 11 Aspergillus clavatus C 572 Aspergillus flavus C 1217 Aspergillus fumigatus Bencard* Aspergillus glaucus Bromptonf Aspergillus terreus 3/11 A Aspergillus umbrosus C 2234+ Aspergillus umbrosus C 22351 Aspergillus versicolor 1/11 Penicillium notatum Brompton t Penicillium sp. C2163t Penicillium sp. C 2233$ * Two separate antigen solutions. t Crude antigen preparation received from the Department of Clinical Immunology, Cardiothoracic Institute, Brompton, London t Strain isolated from a Finnish hay sample.
Farmers^ lung in Finland
45
Table 2. Occurrence of different spore types in Finnish hays Spore type Actinomycetes plus bacteria Fungi Acremoniella atra Acremonium Alternaria Aspergillus glaucus group Other Aspergillus/Penicillium Chrysosporium Cladosporium Doratomyces/Microascus Fusarium Helminthosporium Humicola lanuginosa Mucoraceae Paecilomyces Scopulariopsis Tilletia Trichothecium roseum Waltemia sebi Yeasts
Mean spore content (x 10"** spores/g dry weight) 110-5 21 1
Percentage samples* containing spores 100 100 12
29 (6) 12
71 (24) 82 (47) 6 (6) 77 (12) 12 6 6
41 (12) 18 (6) 18 (12) 12 6
12 (6) 6 6
* Figures in parentheses indicate samples containing more than 3 x 10* spores per g dry weight.
Oxoid Tryptone soya agar incubated at 55°C, both with 50 jug actidione per ml added to inhibit fungi (Lacey & Dutkiewicz, 1976). Sources of FL sera Sera were collected from patients examined at four chest hospitals in Finland between 1973 and 1975, and tested for precipitins at Turku. The sera came from patients with the clinical features of FL (Ministry of Social Security, 1967) and miliary mottling on chest radiographs. They were all taken in the acute or subacute phase of the disease. The total number of sera examined was fifty-four. The mean age of the patients was 45-2 (range: 25 to 71) years; 22% ofthe patients were men and 78% women. Control sera Sera from fifty-four hospital personnel without known exposure to mouldy materials were used as controls in serological testing. The mean age ofthe donors of control sera was 40-2 (22-62) years, and their sex distribution was the same as that of patients. Antigens To supplement commercially produced antigens from Bencard Ltd and HollisterStier Laboratories, antigens were produced from micro-organisms cultured on either solid or liquid media incubated at temperatures appropriate to the species. The antiD
46
E. O. Terho and J. Lacey
Table 3. Frequency of isolation of micro-organisms from Finnish hays Colony type Fungi Absidia spp. Acremonium strietum Aspergillus fumigatus A. glaueus group A. versieolor Cladosporium spp. Doratomyces spp. Humicola lanuginosa Mueor pusillus Mycelia sterilia Penicillium spp. Scopulariopsis brevicaulis Talaromyces thermophilus Triehotheeiutn roseutn Wallemia sebi Actinomycetes Mieropolyspora faeni Saecharomotwspora viridis Streptotnyces albus S. griseus Grey Streptomyces spp. Thermoactinomyces vulgaris Bacteria Bacillus licheniformis Other bacteria
Percentage samples yielding colonies*
59 24 (12) 41 (6) 82 (18) 41 (6) 59 (18) 12 (6) 35 (12) 18 (6) 53 94 (47) 47 (6) 35 (18) 24 (6) 12 (12) 59 43 (28) 36 79 (50) 71 (14) 82 (43) 79 (28) 100 (64)
Other species isolated from fewer than 20% samples: Aeremoniella atra, Alternaria spp., Aspergillus eandidus, A. nidulans, A. ochraeeus, Aureobasidium pullulans, Botrytis cinerea, Chaetomium sp., Chrysosporium luteum, C. panttorum, C. pruinosum, Fusarium sp., Malbranehea pulchella var sulfurea, Microascus sp., Mucor spp. (mesophilic), Neetria inventa (stat. conid. Verticillium laterttlum), Oidiodendron sp., Paecilomycesvarioti, Phoma sp., Sporobolomyces spp., Thermoascus crustaceus, Thielavia thermophila, yeasts, Thermomonospora sp. Figures in parentheses indicate samples yielding more than 10' colonies per g dry weight. gens were extracted from the mycelium and culture medium together using carbolsaline, and were freeze-dried (Pepys et al, 1963). These crude antigens were reconstituted at 30 mg/ml, 10 mg/ml and sometimes also at 5-0 and 2-0 mg/ml for double diffusion tests. Commercial antigen solutions were used only at the concentrations supplied (15 mg/ml for 'Bencard' Aspergillus fumigatus and 50 mg/ml for HollisterStier Thermoactinomyces vulgaris). The panel of antigens used is listed in Table 1. Serological tests Precipitating antibodies to antigens were demonstrated by double diffusion in agar gel. A semi-micro method, modified from the method described by Longbottom &
Farmers' lung in Finland
47
Pepys (1964) was used. Ionagar No. 2 (Oxoid) was made at 1-5% in a solution containing equal volumes of isotonie saline and Mellvaine's eitrie aeid phosphate buffer (pH 7-0). The pattern consisted of a central well, 6-0 mm diameter, for the serum surrounded at a distance of 3-4 mm by six peripheral wells, 3-0 mm diameter, for the antigens. The plates were incubated at 28°C for 4 days. Precipitin lines were stained with naphthalene black. Results Microbiology of hay samples The frequency of occurrences of different spore types in the seventeen hay samples examined is shown in Table 2 and the frequency of isolation of different colony types in Table 3. Numbers of 'actinomycetes plus bacteria' varied between O-l and 580 x 10^ per g dry weight. Only A. glaucus group, other Aspergillus/Penicillium and Cladosporium type spores were found in more than half the samples and both Aspergillus categories most often occurred in large numbers. The range of species isolated was similar to that found in British hays (Gregory & Lacey, 1963; Lacey, 1974). However, the frequency of isolation of thermophilous species such as Absidia spp., A. fumigatus, Humicola lanuginosa, Mucor pusillus, M.
Table 4. Combinations of precipitin reactions obtained with group II sera Positive sera Combination Micropolyspora faeni only Thermoactinomyces vulgaris only M. faeni plus T. vulgaris M. faeni plus Saccharomonospora viridis T. vulgaris plus S. viridis M. faeni plus 5. viridis plus T. vulgaris Total
Number 2 8 3 1 1
2 17
0/
/o
12 47 18 6 6 12
Table 5. Combinations of precipitin reactions obtained with group III sera Positive sera Combination Aspergillus spp. only Aspergillus spp. plus Penicillium spp. Acremonium strictum plus Aspergillus spp. plus Penicillium spp. Total
Number 2 9 1 12
0/
/o
17 75 8 —
48
E.O. Terho andJ. Lacey
Table 6. Combinations of precipitin reactions to the main groups of antigens obtained with group IV sera Positive sera Number
Combination Actinomycetes Actinomycetes Actinomycetes Actinomycetes Actinomycetes Total
plus Acremoniutn strietum plus Aspergillus spp. plus Acremonium strietum plus Aspergillus spp. plus Aspergillus spp. plus Penicillium spp. plus Acremonium strietum plus Aspergillus spp. plus Penicillium spp.
20
Table 7. Precipitin reactions between FL sera and individual antigens Positive sera Antigen Actinomycetes Micropolyspora faeni 9535 Mieropolyspora faeni A 1311 Saccharomonospora viridis A 66 Streptomyees griseofiavus A 159 Thermoactinomyces vulgaris A 64 Thermoactinomyces vulgaris Hollister-Stier Fungi Acremonium strietum O 17 Aspergillus elavatus C 572 Aspergillus flavus C 1217 Aspergillus fumigatus Bencsird 1 Aspergillus fumigatus Bencard 2 Aspergillus glaueus Brompton Aspergillus terreus 3/72 A Aspergillus umbrosus C 2234 Aspergillus umbrosus C 2235 Aspergillus versieolor 2/72 Penieillium notatum Brompton Penicillium sp. C 2163 Penicillium sp. C 2233 Total positive sera Total negative sera Total sera * Percentage of the total number of FL sera.
1 8 5 3 3
Number
0/
/o
17 18 11 4 7 29
32 33 20 7 13 54
10 9 16 11 15 23 17 28 27 9 12 12 11
19 17 30 20 28 43 32 52 50 17 22 22 20
49 5 54
91 9
/o
5 40 25 15 15
Farmers'' lung in Finland
49
faeni and T. vulgaris tended to be lower. This suggests that spontaneous heating was less common in this small sample of hays than in comparable British hays associated with FL. Also, Acremonium strictum, Doratomyces spp. and Penicillium spp. occurred more frequently and in larger numbers than in Britain, while the composition of the A. glaucus group also differed in some hays, with abundant A. umbrosus, a species rarely found in British hays, replacing the more usual A. amstelodami and A. repens. The actinomycetes usually associated with FL, M. faeni and T. vulgaris were present, respectively, in 59% and 82% of the hay samples. However, M. faeni was never abundant while T. vulgaris occurred in numbers usually associated with farmer's lung type hays with a similar frequency to England (Lacey, 1974). Immunology of FL patients Precipitins were detected in forty-nine (91%) out of fifty-four FL sera, but there were no positive reactions with the fifty-four control sera. FL sera could be divided into four distinct groups on the basis of their precipitin reactions: (I) precipitin-negative sera (five out of fifty-four, 9%); (II) precipitins to actinomycetes only (seventeen out of fifty-four, 32%); (III) precipitins to fungi only (twelve out of fifty-four, 22%); and (IV) precipitins to both actinomycetes and fungi (twenty out of fifty-four, 37%). Table 4 shows the various combinations of precipitin reactions to actinomycete antigens obtained with group II sera. Reactions to pairs of isolates of M. faeni (9535 and A 1311), and of T. vulgaris (A 64 and Hollister-Stier) are combined. Precipitin reactions with group III and group IV sera are shown in Tables 5 and 6.
Table 8. Number of positive reactions obtained to different groups of antigens
Antigen group
Micropolyspora faeni Thermoactinomyces vulgaris Thermophilic actinomycetest Mesophilic actinomycetes* Actinomycetes§ Aspergillus fumigatus Aspergillus umbrosus Aspergillus glaucus groupif Aspergillus spp. Penicillium isolates** Pencillium spp.
Fungi t t
Number of reacting sera*
Percentage of total positive sera
Percentage of FL sera
20 32 37 11 37 16 30 30 31 13 16 32
41 65 76 22 76 33 61
37 59 69 20 69 30 56 56 57 24 30 59
6t 63 27 33 65
* Precipitins present to at least one micro-organism within the group. t M. faeni, T. vulgaris. X S. viridis, Streptomyces griseoflavus. § M. faeni, T. vulgaris, S. viridis, S. griseoflavus. % A. glaucus, A. umbrosus. ** Pencillium spp. C 2163 and C 2233. t t Acremonium strictum, Aspergillus spp., Penicillium spp.
50
E. O. Terho and J. Lacey
Table 9. Reactions of FL sera to antigen preparations of two T. vulgaris isolates Reaction with T. vulgaris A 64 (r. thalpophilus) Positive Negative Total
Reaction with vulgaris Hollister-Stier r. Positive 4 25 29
Negative 3 22 25
Total 7 47 54
Precipitins to A. glaucus and to A. umbrosus were present in all sera within group III and A. umbrosus was the most common fungus giving positive reactions with group IV sera. Numbers of FL sera reacting to individual antigen preparations are shown in Table 7, and to particular groups in Table 8. Using McNemar's tests there were no statistically significant differences ( P > 0 05) between reactions to M. faeni 9535 and M. faeni A 1311, to A. umbrosus C 2234 and A. umbrosus C2235, to Penicillium sp. C2163 and Penicillium sp. C2234 or to the two solutions oi A. fumigatus in Bencard's sero-diagnostic set. However, there was a highly significant difference (/'
Microbiological and serological studies of farmers' lung in Finland E. O. TERHO and J. LACEY* Department of Diseases of the Chest, University of Turku, Finland, and * Department of Plant Pathology, Rothamsted Experimental Station, Harpenden, Hertfordshire (Received 24 July 1978; accepted for publication 8 August 1978)
Summary
Sera from few Finnish patients with clinical farmers' lung react in precipitin tests with extracts of the thermophilic actinomycetes that commonly cause the disease elsewhere. Hays associated with the disease in Finland showed less evidence of spontaneous heating and contained fewer actinomycete spores than British hays. Only Thermoactinomyces vulgaris was sometimes abundant. Some species of mesophilic fungi were more abundant than in Britain and one, Aspergillus umbrosus, reacted with most sera from farmers' lung patients in precipitin tests. A panel of antigens, including thermophilic actinomycetes, A. umbrosus and other species of the Aspergillus glaucus group, is recommended for screening farmers' lung sera. Introduction
Mouldy hays associated with farmers' lung (FL) in England are characterized by a heavy growth of actinomycetes (Gregory & Lacey, 1963). The main causative agent of FL has been considered to be Micropolyspora faeni (Pepys et al., 1963; Parratt & Boyd, 1976), a thermophilic actinomycete that grows in hay that has been stored damp and heats spontaneously. Precipitating antibodies to M. faeni are present in a large proportion of the sera of English FL patients, and evidence of exposure to the spores is given by some unaffected farm workers (Pepys & Jenkins, 1965). Since 1972, precipitin tests have been used routinely at the Department of Diseases of the Chest, University of Turku, Finland, for the investigation of FL. Although antigens relevant to FL in England were used in the same agar gel double diffusion technique employed by Pepys et al. (1963), few sera from subjects with a typical clinical history of FL gave positive reactions to M. faeni. It was therefore thought that micro-organisms other than M. faeni might be implicated in the disease in Finland. Correspondence: Dr E. O. Terho, Department of Pulmonary Diseases, University Central Hospital, Kuopio, Finland. 0009-9090/79/0100-0043 $02.00 © 1979 Blackwell Scientific Publications 43
44
E. O. Terho and J. Lacey
The present study was designed to identify the types of micro-organisms to which Finnish FL patients were exposed. Hay samples associated with FL were analysed microbiologically and the sera of clinically proven cases of FL were tested by an agar gel double diffusion technique against a panel of antigens selected on the basis of their occurrence in hay samples. Materials and methods Sources of hay samples Patients suffering from FL were asked to supply samples of hay, straw or grain which they were handling at the onset of their illness. Additionally, one sample of good Finnish hay was examined. Samples were sent to Rothamsted Experimental Station, Harpenden, England, for microbiological examination. Microbiological examination of hay samples All hay samples were examined by the wind tunnel method of Gregory & Lacey (1963). Spores blow off the hay were trapped on microscope slides in a cascade impactor, for microscopic classification and enumeration, or on agar media in petri dishes in an Andersen sampler, for growth in culture. Fungi were isolated on 2% malt extract agar containing 20 units penicillin and 40 units streptomycin per ml medium and incubated at either 25°C or 40°C. Actinomycetes and other bacteria were grown on either half-strength Oxoid nutrient agar incubated at 25°C or 40°C or half-strength Table 1. Antigen panel used to test Finnish FL sera Actinomycetes Mieropolyspora faeni 9535 Mieropolyspora faeni A 1311 Saccharomonospora viridis A 66 Streptomyces griseoflavus A. 159 Thermoactinomyces vulgaris A 64 Thermoactinomyces vulgaris Hollister-Stier Fungi Acremonium strictum C 11 Aspergillus clavatus C 572 Aspergillus flavus C 1217 Aspergillus fumigatus Bencard* Aspergillus glaucus Bromptonf Aspergillus terreus 3/11 A Aspergillus umbrosus C 2234+ Aspergillus umbrosus C 22351 Aspergillus versicolor 1/11 Penicillium notatum Brompton t Penicillium sp. C2163t Penicillium sp. C 2233$ * Two separate antigen solutions. t Crude antigen preparation received from the Department of Clinical Immunology, Cardiothoracic Institute, Brompton, London t Strain isolated from a Finnish hay sample.
Farmers^ lung in Finland
45
Table 2. Occurrence of different spore types in Finnish hays Spore type Actinomycetes plus bacteria Fungi Acremoniella atra Acremonium Alternaria Aspergillus glaucus group Other Aspergillus/Penicillium Chrysosporium Cladosporium Doratomyces/Microascus Fusarium Helminthosporium Humicola lanuginosa Mucoraceae Paecilomyces Scopulariopsis Tilletia Trichothecium roseum Waltemia sebi Yeasts
Mean spore content (x 10"** spores/g dry weight) 110-5 21 1
Percentage samples* containing spores 100 100 12
29 (6) 12
71 (24) 82 (47) 6 (6) 77 (12) 12 6 6
41 (12) 18 (6) 18 (12) 12 6
12 (6) 6 6
* Figures in parentheses indicate samples containing more than 3 x 10* spores per g dry weight.
Oxoid Tryptone soya agar incubated at 55°C, both with 50 jug actidione per ml added to inhibit fungi (Lacey & Dutkiewicz, 1976). Sources of FL sera Sera were collected from patients examined at four chest hospitals in Finland between 1973 and 1975, and tested for precipitins at Turku. The sera came from patients with the clinical features of FL (Ministry of Social Security, 1967) and miliary mottling on chest radiographs. They were all taken in the acute or subacute phase of the disease. The total number of sera examined was fifty-four. The mean age of the patients was 45-2 (range: 25 to 71) years; 22% ofthe patients were men and 78% women. Control sera Sera from fifty-four hospital personnel without known exposure to mouldy materials were used as controls in serological testing. The mean age ofthe donors of control sera was 40-2 (22-62) years, and their sex distribution was the same as that of patients. Antigens To supplement commercially produced antigens from Bencard Ltd and HollisterStier Laboratories, antigens were produced from micro-organisms cultured on either solid or liquid media incubated at temperatures appropriate to the species. The antiD
46
E. O. Terho and J. Lacey
Table 3. Frequency of isolation of micro-organisms from Finnish hays Colony type Fungi Absidia spp. Acremonium strietum Aspergillus fumigatus A. glaueus group A. versieolor Cladosporium spp. Doratomyces spp. Humicola lanuginosa Mueor pusillus Mycelia sterilia Penicillium spp. Scopulariopsis brevicaulis Talaromyces thermophilus Triehotheeiutn roseutn Wallemia sebi Actinomycetes Mieropolyspora faeni Saecharomotwspora viridis Streptotnyces albus S. griseus Grey Streptomyces spp. Thermoactinomyces vulgaris Bacteria Bacillus licheniformis Other bacteria
Percentage samples yielding colonies*
59 24 (12) 41 (6) 82 (18) 41 (6) 59 (18) 12 (6) 35 (12) 18 (6) 53 94 (47) 47 (6) 35 (18) 24 (6) 12 (12) 59 43 (28) 36 79 (50) 71 (14) 82 (43) 79 (28) 100 (64)
Other species isolated from fewer than 20% samples: Aeremoniella atra, Alternaria spp., Aspergillus eandidus, A. nidulans, A. ochraeeus, Aureobasidium pullulans, Botrytis cinerea, Chaetomium sp., Chrysosporium luteum, C. panttorum, C. pruinosum, Fusarium sp., Malbranehea pulchella var sulfurea, Microascus sp., Mucor spp. (mesophilic), Neetria inventa (stat. conid. Verticillium laterttlum), Oidiodendron sp., Paecilomycesvarioti, Phoma sp., Sporobolomyces spp., Thermoascus crustaceus, Thielavia thermophila, yeasts, Thermomonospora sp. Figures in parentheses indicate samples yielding more than 10' colonies per g dry weight. gens were extracted from the mycelium and culture medium together using carbolsaline, and were freeze-dried (Pepys et al, 1963). These crude antigens were reconstituted at 30 mg/ml, 10 mg/ml and sometimes also at 5-0 and 2-0 mg/ml for double diffusion tests. Commercial antigen solutions were used only at the concentrations supplied (15 mg/ml for 'Bencard' Aspergillus fumigatus and 50 mg/ml for HollisterStier Thermoactinomyces vulgaris). The panel of antigens used is listed in Table 1. Serological tests Precipitating antibodies to antigens were demonstrated by double diffusion in agar gel. A semi-micro method, modified from the method described by Longbottom &
Farmers' lung in Finland
47
Pepys (1964) was used. Ionagar No. 2 (Oxoid) was made at 1-5% in a solution containing equal volumes of isotonie saline and Mellvaine's eitrie aeid phosphate buffer (pH 7-0). The pattern consisted of a central well, 6-0 mm diameter, for the serum surrounded at a distance of 3-4 mm by six peripheral wells, 3-0 mm diameter, for the antigens. The plates were incubated at 28°C for 4 days. Precipitin lines were stained with naphthalene black. Results Microbiology of hay samples The frequency of occurrences of different spore types in the seventeen hay samples examined is shown in Table 2 and the frequency of isolation of different colony types in Table 3. Numbers of 'actinomycetes plus bacteria' varied between O-l and 580 x 10^ per g dry weight. Only A. glaucus group, other Aspergillus/Penicillium and Cladosporium type spores were found in more than half the samples and both Aspergillus categories most often occurred in large numbers. The range of species isolated was similar to that found in British hays (Gregory & Lacey, 1963; Lacey, 1974). However, the frequency of isolation of thermophilous species such as Absidia spp., A. fumigatus, Humicola lanuginosa, Mucor pusillus, M.
Table 4. Combinations of precipitin reactions obtained with group II sera Positive sera Combination Micropolyspora faeni only Thermoactinomyces vulgaris only M. faeni plus T. vulgaris M. faeni plus Saccharomonospora viridis T. vulgaris plus S. viridis M. faeni plus 5. viridis plus T. vulgaris Total
Number 2 8 3 1 1
2 17
0/
/o
12 47 18 6 6 12
Table 5. Combinations of precipitin reactions obtained with group III sera Positive sera Combination Aspergillus spp. only Aspergillus spp. plus Penicillium spp. Acremonium strictum plus Aspergillus spp. plus Penicillium spp. Total
Number 2 9 1 12
0/
/o
17 75 8 —
48
E.O. Terho andJ. Lacey
Table 6. Combinations of precipitin reactions to the main groups of antigens obtained with group IV sera Positive sera Number
Combination Actinomycetes Actinomycetes Actinomycetes Actinomycetes Actinomycetes Total
plus Acremoniutn strietum plus Aspergillus spp. plus Acremonium strietum plus Aspergillus spp. plus Aspergillus spp. plus Penicillium spp. plus Acremonium strietum plus Aspergillus spp. plus Penicillium spp.
20
Table 7. Precipitin reactions between FL sera and individual antigens Positive sera Antigen Actinomycetes Micropolyspora faeni 9535 Mieropolyspora faeni A 1311 Saccharomonospora viridis A 66 Streptomyees griseofiavus A 159 Thermoactinomyces vulgaris A 64 Thermoactinomyces vulgaris Hollister-Stier Fungi Acremonium strietum O 17 Aspergillus elavatus C 572 Aspergillus flavus C 1217 Aspergillus fumigatus Bencsird 1 Aspergillus fumigatus Bencard 2 Aspergillus glaueus Brompton Aspergillus terreus 3/72 A Aspergillus umbrosus C 2234 Aspergillus umbrosus C 2235 Aspergillus versieolor 2/72 Penieillium notatum Brompton Penicillium sp. C 2163 Penicillium sp. C 2233 Total positive sera Total negative sera Total sera * Percentage of the total number of FL sera.
1 8 5 3 3
Number
0/
/o
17 18 11 4 7 29
32 33 20 7 13 54
10 9 16 11 15 23 17 28 27 9 12 12 11
19 17 30 20 28 43 32 52 50 17 22 22 20
49 5 54
91 9
/o
5 40 25 15 15
Farmers'' lung in Finland
49
faeni and T. vulgaris tended to be lower. This suggests that spontaneous heating was less common in this small sample of hays than in comparable British hays associated with FL. Also, Acremonium strictum, Doratomyces spp. and Penicillium spp. occurred more frequently and in larger numbers than in Britain, while the composition of the A. glaucus group also differed in some hays, with abundant A. umbrosus, a species rarely found in British hays, replacing the more usual A. amstelodami and A. repens. The actinomycetes usually associated with FL, M. faeni and T. vulgaris were present, respectively, in 59% and 82% of the hay samples. However, M. faeni was never abundant while T. vulgaris occurred in numbers usually associated with farmer's lung type hays with a similar frequency to England (Lacey, 1974). Immunology of FL patients Precipitins were detected in forty-nine (91%) out of fifty-four FL sera, but there were no positive reactions with the fifty-four control sera. FL sera could be divided into four distinct groups on the basis of their precipitin reactions: (I) precipitin-negative sera (five out of fifty-four, 9%); (II) precipitins to actinomycetes only (seventeen out of fifty-four, 32%); (III) precipitins to fungi only (twelve out of fifty-four, 22%); and (IV) precipitins to both actinomycetes and fungi (twenty out of fifty-four, 37%). Table 4 shows the various combinations of precipitin reactions to actinomycete antigens obtained with group II sera. Reactions to pairs of isolates of M. faeni (9535 and A 1311), and of T. vulgaris (A 64 and Hollister-Stier) are combined. Precipitin reactions with group III and group IV sera are shown in Tables 5 and 6.
Table 8. Number of positive reactions obtained to different groups of antigens
Antigen group
Micropolyspora faeni Thermoactinomyces vulgaris Thermophilic actinomycetest Mesophilic actinomycetes* Actinomycetes§ Aspergillus fumigatus Aspergillus umbrosus Aspergillus glaucus groupif Aspergillus spp. Penicillium isolates** Pencillium spp.
Fungi t t
Number of reacting sera*
Percentage of total positive sera
Percentage of FL sera
20 32 37 11 37 16 30 30 31 13 16 32
41 65 76 22 76 33 61
37 59 69 20 69 30 56 56 57 24 30 59
6t 63 27 33 65
* Precipitins present to at least one micro-organism within the group. t M. faeni, T. vulgaris. X S. viridis, Streptomyces griseoflavus. § M. faeni, T. vulgaris, S. viridis, S. griseoflavus. % A. glaucus, A. umbrosus. ** Pencillium spp. C 2163 and C 2233. t t Acremonium strictum, Aspergillus spp., Penicillium spp.
50
E. O. Terho and J. Lacey
Table 9. Reactions of FL sera to antigen preparations of two T. vulgaris isolates Reaction with T. vulgaris A 64 (r. thalpophilus) Positive Negative Total
Reaction with vulgaris Hollister-Stier r. Positive 4 25 29
Negative 3 22 25
Total 7 47 54
Precipitins to A. glaucus and to A. umbrosus were present in all sera within group III and A. umbrosus was the most common fungus giving positive reactions with group IV sera. Numbers of FL sera reacting to individual antigen preparations are shown in Table 7, and to particular groups in Table 8. Using McNemar's tests there were no statistically significant differences ( P > 0 05) between reactions to M. faeni 9535 and M. faeni A 1311, to A. umbrosus C 2234 and A. umbrosus C2235, to Penicillium sp. C2163 and Penicillium sp. C2234 or to the two solutions oi A. fumigatus in Bencard's sero-diagnostic set. However, there was a highly significant difference (/'
