The microbiology of actinomycetes organisms known to cause farmer's lung disease and their occurrence from coal mines of Telangana

Introduction

FARMER'S LUNG is an occupational disease which was registered in 1965 under the National Insurance Act (Industrial Injuries Act) of 1946. For the purpose of the Act, it is defined as "pulmonary disease due to the inhalation of the dust of mouldy hay or of other mouldy vegetable produce, and characterized by symptoms and signs attributable to a reaction in the peripheral part of the bronco pulmonary system and giving rise to a defect in gas exchange". The disease is allergic in character, allergy now being taken to imply a greatly enhanced ability of an individual to react immunologically to a specific antigen following the development of hypersensitivity. Particles which carry one or more antigens implicated as causative of an allergy are said to be allergens. The inhalation of particulate allergens in the form of spores of actinomycetes, fungi and the proteins of animals is responsible for a number of diseases akin to and including farmers lung, of which those listed in Table 1 are well known. The organisms concerned, although clearly eliciting an allergic pulmonary response, do not cause infection by colonization of the respiratory airways or lung parenchyma (Blyth, 1970) [7] . Aspergillums fumigates, known to effect such colonization, can also cause pulmonary allergy when inhaled in large quantities. Allergic disorders such as farmer's lung disease have been variously classified according to the emphasis placed on details of their pathology. In the U.S.A. they are commonly categorized as hypersensitivity pneumonitis, in this country they are commonly described as examples of allergic alveolitis, or extrinsic allergic alveolitis to emphasize the external source of the allergens.Cattle are also susceptible to Extrinsic allergic alveolitis. The first confirmed case of farmer's lung in a Cow was described by Pirie et al. (1971a) [31] in spite of worldwide recognition of Farmer's Lung, there is limited microbiological data from Indian coal mine environments. This study aims to isolate and identify actinomycetes and fungal species from coal mine dust in Telangana to understand their potential role in occupational extrinsic allergic alveolitis.

Some examples of extrinsic allergic alveolitis due to actinomycetes and fungi** Disease

Source of allergens Precipitating antibodies identified against Farmer's lung (Pepys & Jenkins, 1965) Mouldy hay, grain and straw Micropolyspora faeni, Thermoactinomyces vulgaris Mushroom worker's lung (Sakula, 1967) Mushroom compost M. faeni and T. vulgaris Bagassosis (Salvaggio et al., 1967; Lacey, 1971) Mouldy sugar cane T. vulgaris, T. sacchari Maple bark disease (Emanuel et al., 1966) [19] Mouldy maple bark Cryptostroma corticale Sequoiosis (Cohen et al., 1967) Mouldy redwood sawdust Species of Graphium and Aureobasidium Malt worker's lung (Riddle et al., 1968) Mouldy malt Aspergillus clavatus

Material and Methodology Sample collection

Ecological sample were collect from Telangana coal mines operated by The Singareni Collieries Company Limited (SCCL). Sampling targeted both core zones (mining lease areas) and buffer zones (within a 10 km radius) to assess occupational exposure and environmental distribution of thermophilic actinomycetes and fungi. Samples included coal dust, soil, and air particulates, collected aseptically in sterile containers and transported to the laboratory under controlled conditions.

Sputum culture

Sputum consists of mucus and enmeshed material expectorated following -coughing. When sputum is cultured directly or after digestion by pancreatin, the =viable population of micro-organisms can be selectively grown over a wide range of temperatures, and it yields a wide variety of filamentous and yeastlike fungi (Kahanpaa, 1971) [22] . Asp. [25] . Trichloracetic acid extracts from mouldy hay were progressively precipitated with 50-60%, 60-70% and 70-90% of ethanol. Immunoelectrophoresis of the original extracts showed the presence of A, B and C precipitation areas when reacted against positive sera. Antigen a was fractionated by ethanol into 3 components, a ^ 1 a ^ 2 and a ^ 3 and antigen b into 2, b ^ 1 b ^ 2 Cultures of M. faeni recovered from the mouldy hay sample gave identical TCAsoluble extracts. Extracts obtained in phosphate buffer after ultrasonic treatment of the organism, followed by TCA in the cold, yielded 5 antigens, 3 of which were acid soluble. In addition, the medium in which the organism had been grown gave a TCA-soluble antigen not isolated from mouldy hay or M. faeni. Ethanol precipitated antigens, chromatographed after purifica-tion by passing through a column of DEAE (diethylaminoethyl) cellulose, gave evidence of a glycopeptide constitution (LaBerge & Stahmann, 1966b) [25] . The polysaccharide moeity was found to be composed largely of arabinose, galactose and glucosamine, all known to occur widely as cell wall components of Gram positive organisms.

Much essentially basic work has been done on the development of generally acceptable techniques for the conduct of serological tests to diagnose and prognose farmer's lung disease. A working party of the Public Health Laboratory Service (Murray, 1967) [29] reported that gel diffusion was marginally superior to complement fixation in the diagnosis of mild forms of the disease, but that the 2 techniques gave identical results with moderate and severe cases. Murray, Pepys & Crighton (1967) [30] showed that latex particles could readily be coated with antigens from M. faeni and used for agglutination (flocculation) tests with sera. The titres recorded gave some indication of severity of the disease and the test compared favourably with gel diffusion, but was more rapidly and easily conducted. In both latex agglutination and complement fixation tests, antigens used were prepared from 45° broth cultures of M. faeni by extraction with glycine after homogenization and ultrasonic treatment. Doubtfully positive reactions may be enhanced by increasing the relative concentration of antibody, either by concentrating serum by diffusion against polyethylene glycol (Carbo wax) or by reconstituting it to a predetermined volume in saline after lyophilization. Jameson (1968) has, however, described a rapid and very sensitive precipitin test using immuno-osmophoresis to eliminate losses due to dilution and undirected migration of reactants in gel diffusion. In a group of 12 cases negative in gel diffusion, 11 became positive after concentration of sera and immuno-osmophoresis. Gray, Wenzel & Emanuel (1969) [19] have described an immunofluorescence test for the detection of M. faení: Parratt (in Grant et al., 1972) has used a similar method to identify cases in Scotland.

(iv)The antigenic potency of extra-and intra-cellular antigens of M. faeni in test systems

A detailed serological analysis of the extracellular antigens of M. faeni recovered by concentrating supernatant culture fluids by dialysis against 40% polyethylene glycol was made by Fletcher et al. (1970). Two batches of broth tested against highly reactive sera from 2 farmer's lung patients showed the presence of 29 antigenic components: antigens 1-5 were identified as com-ponents of antigen c of Pepys & Jenkins (1965). In a series of cases of farmer's lung disease investigated, 75% of sera were positive; 20% of cases suffering from other respiratory disorders, but exposed to the same environment, were also positive. Severity of disease did not influence the distribution of the zones of precipitation, although their numbers were greater in severe or moderate clinically established cases. Gibson (1971) has shown that most antigens occur during exponential growth in broth cultures, and Edwards (1971) [18] has used a double diffusion method for separating antigens from culture fluids as a means of purifying antigenic componentsof purifying antigenic components. Jameson (1968, 1969) reported that not only broths but also the concen-trated dialysates from broth cultures were instrumental in reacting with positive sera by immunoosmophoresis. From the work described in 1969, it was evident that the use of broths containing extracellular exudates resulted in more reactions with test sera than were obtainable using mycelial extracts. Despite this, concentrated extracts of M. faeni which were run through a column of Sephadex 150 gave 3 fractions rich in antigen a of Pepys & Jenkins (1965) when subjected to conventional electrophoresis. Among other tests, the Sephadex fraction in immuno-osmophoresis was selective in distinguishing between 2 groups in a series of farmer's lung cases, those possessing antibody to antigen a and those deficient in this antibody. This finding may be aetiologically significant and indicate that sources of sensitization other than M. faeni need to be sought, especially as in this series of sera deficient in A antibody, only one of six reacted to T. vulgaris, the other accredited allergen.

(b) Fungal antigens

Although the evidence that the thermophilic actinomycetes are the organisms carrying the antigens essential for provoking farmer's lung disease is convincing, sera from patients may react to a number of extracts from other sources. Pepys et al. (1962) showed that 60 of 71 patients with the disease and who had precipitins to mouldy hay also possessed them to Asp. fumigatus in 25% of Cases, Cladosporium herbarum in 16%, P. notatum in 6%, Mucor spp. in 52% and Humicola lanuginosa in 11%. Inhibition tests showed, however, that only extracts of mouldy hay when added to positive sera caused loss of reactivity in gel diffusion. Pepys et al. (1963) stated that extracts from hays inoculated individually with M. pusillus, Asp. fumigatus, H. lanuginosa, H. stellata or Paecilomyces spp. reacted with more farmer's lung sera than did untreated hay. Hapke et al. (1968) have forwarded the suggestion that another antigen or antigens besides those carried by M. faeni may be implicated in the disease. This followed from their observations that if chronic patients were tested against 12 hay extracts additional to the individual hay samples implicated in their own environments, a sharp rise in positivity was observed. It may also be significant that Williams (1963) reported an attack of farmer's lung disease when testing the coarse crepitations, but not the fine, fraction of mouldy hay dust where elements of the actinomycetes would be most likely to occur. Grant et al. (1972) commenting on the seronegative, clinically established cases, stated that the courses of their sensitization remained obscure. In cattle, Pepys & Jenkins (1965) reported that precipitins to M. faeni were identifiable whether clinical respiratory disease was present or absent, and tended to correlate positive results with 'fog fever'. By contrasting groups of cattle showing fog fever with those suffering from indoor respiratory disease, Pirie et al. (1971b) [31] found that reactivity to M. faeni and Asp. fumigatus occurred solely in the latter

Result

The microbiological study of coal mine samples collected from Telangana exposed the presence of diverse fungal species and thermophilic actinomycetes. These microbes were isolated from sputum samples, soil, air, and coal dust, representative extensive environmental distribution and potential occupational exposure. Sputum consists of mucus and enmeshed material expectorated following -coughing. When sputum is cultured directly or after digestion by pancreatin, the =viable population of micro-organisms can be selectively grown over a wide range of temperatures, and it yields a wide variety of filamentous and yeastlike fungi (Kahanpaa, 1971) [22] . Asp. fumigatus, Penicillium spp., Geotrichum candidum, Candida albicans and Torulopsis spp. [5] . Fink, Hensley & Barboriak (1970) have created an experimental allergic alveolitis in the rat which may act as a model for further investigations.

Freeze-thaw and ultrasonic treatment are procedures commonly regarded as releasing large amounts of intracellular components from micro-organisms. Trichloracetic acid extraction is likely to produce a wider overall spectrum of substances by adding some cell wall components to those leached from the cytoplasm. Much of the serological evidence has suggested that the antigens derived by these methods are more restricted in type and number than those of extracellular origin extracted from growth media and their dialysates. These observations might be construed to indicate that sensitization to antigens may follow retention of actinomycete spores in lung parenchyma sufficiently long to allow diffusion into the alveolar environment. Spore germination may also occur (Gibson, 1971). Points in the respiratory airways where branching takes place are areas provoking an interruption in air flow conducive to an accumulation of particles (Casarett, 1958) [9] , and retention may equally occur on isolated patches of squamous epithelium (Casarett, 1960) [10] . Druett (1967) [17] has suggested that 2/3 of a deposited insoluble dust passes rapidly to the gastrointestinal tract, 1/6 more reaching the same location during the first day. The remaining 1/6 may show prolonged retention with a half life of c. 120 days. Channell et al. (1969) [11] reported that spores of Asp. clavatus, the cause of maltworkers lung, could be recovered up to a month from volunteers who had been exposed only once. Particle clearance by alveolar phagocytes adds innumerable potential sites of sensitization to those already possible in the respiratory and gastrointestinal tracts.Based on these result, serological study was performed to assess the immune response against microbial antigens present in showing individuals. [27] . The environmental distribution of microbial load was assessed across different zones of the coal mining area, including core and buffer zones. It has always been difficult for the microbiologist to give an objective opinion as to the potential safety or otherwise of a particular mouldy hay or grain crop, either with a view to mycotoxin content or respiratory hazard. An account of the occurrence of precipitins to M. faeni in cattle herds of south Scotland and northwest England (Pirie et al., 1972) [31] has shown that in a farm where a farmer complained of a recurrence of disease, the incidence of precipitins in his cows had increased from 9 to 76%. None of 75 animals on a farm feeding good hayand only 1 of 58 animals fed silage were seropositive. A high incidence of precipitins in a herd would seem to indicate a hazardous situation for the farmer and his stock. It is interesting to speculate on the utilization of this evidence as a means of preventing disease.

Discussion

Organisms known to cause Farmer's Lung and their Occurrence

Two thermophilic actinomycetes are known to cause the disease, the more important being originally known as Thermopolyspora polyspora. The species was given a detailed examination by Cross, Maciver & Lacey (1968) [13] , who allocated it to the genus Micropolyspora and described it as a new species, M. faeni. [12,14,15,16] . Both actinomycetes are of wide distribution in natural habitats and occur as strains varying in substrate and temperature ranges. The strains implicated in farmer's lung disease are primarily encountered in overheated hay and grain. Overheating may result in temperatures of 50-70° when produce is stored with moisture contents>35% (Gregory, Lacey, Festenstein & Skinner, 1963; Gregory & Lacey, 1963a, b). A range of fungal species including Asp. fumigatus, Absidia ramosa and Mucor pusillus is also encountered.

It is generally assumed that sensitization of a susceptible subject follows the inhalation of the high concentrations of spores likely to be found in the enclosed, ill ventilated conditions of farm stock buildings when feeding stuffs are being worked. Lacey & Lacey (1964) estimated the concentrations of airborne spores before, during and after the shaking of mouldy hay in cowsheds. Using a cascade impactor (May, 1945) and an Andersen sampler (Andersen, 1958) [3] , they showed that concentrations of up to 1-6 x 10 spores/m³ occurred in the air whilst the material was being shaken and that 98% of the spores were of actinomycetes. Of this peak concentration 90% was deposited from the air within 20 min. The comparatively modern practice of moist grain storage in silos is most adequate and economic when efficient sealing restricts air supplies, and under these conditions mould growth is arrested. If, however, the seal is broken, moulding is rapid and during subsequent unloading procedures, aerial sporeconcentrations as high as 3 x 109/m³ have been recorded (Lacey, 1969). Fifty % of the cases recorded by Grant et al. (1972) were due to mouldy hay and 27% were due to mouldy grain; mouldy straw was also a source of allergens.

Inhalation, Retention and Elimination of Respiratory Allergens

The organic dusts inhaled in enclosed buildings consist of a wide range of particles, amongst the insoluble representatives of which are pollens, propagules of algae, fragments of higher plant tissues, spores and mycelia of fungi and actinomycetes, bacteria and parts of the exoskeletal system of insects, along with an inorganic fraction rich in silica. Austwick (1966) [4] , in a review of respiratory fungal disease, illustrated diagrammatically the limits of penetration of different spore types in relation to air inhaled at an initial velocity of 150 cm/sec declining finally to 1 cm/sec in the alveolar ducts and sacs. The largest spore forms tend to become deposited in the nasopharynx and trachea, the smallest may penetrate to the alveolar spaces.

As the spores of the allergenic actinomycetes measure 0-5-1-3 µm diam, penetration deep into the pulmonary spaces is assured. The results of the examination of traces from volumetric spore traps has shown that the lung normally copes with an intake of 12-15,000 fungal spores/m³ of air inspired during June-September in Britain (Gregory, 1961). Estimated figures of this sort contrast with those which may be compiled from the data of Lacey & Lacey (1964). Assuming a spore concentration of 16 x 10°/m³, in enclosed conditions it may be computed that an exposure of 30 min would result in the inhalation of 480 x 10 spores and retention of perhaps 200 x 106 in the lower respiratory tract. The fate of inhaled particles depends largely on the area of deposition. With the exception of the alveoli, their sacs and ducts, the respiratory airways are shrouded in mucus. Mucus is composed of 95-97% of water, 1-2% of salt and 2-5-3-5% of mucin, a mucopolysaccharide complex containing sialic acid and mucoprotein constituents. It is produced by glands and goblet cells of the mucosa (Kilburn, 1967) [23] , and is regarded as a blanket of two layers, the outer nearest the lumen of the airway being a stiff gel transport layer of high tensile strength and the inner a more fluid (sol) layer in contact with underlying mucosal cells. The outer gel layer is kept in motion by the beating of cilia from mucosal cells which are bathed in the sol layer. Particulate material adhering to the gel layer is passed upwards towards the hypopharynx and enters the gastrointestinal tract after being swallowed. Speed of movement of the gel layer mucus has been estimated at 0-15-0-30 cm/min in the bronchioles, 0-25-1-0 cm/min in the bronchi and 3-0-4-0 cm/min in the trachea (Hilding, 1957). Apart from its protective influence against the penetration of mucosal cells by particles, mucus necessarily prevents the transudation of fluid and preserves equilibrium in water relations.

As both mucus and cilia are absent from alveoli many of the inhaled spores of actinomycetes will impinge on an area in which alveolar cells and their processes are covered solely by a surfactant layer (Pattle, 1967). This layer is composed of an outer insoluble lipoprotein area c. 5 nm thick, adjoining the alveolar lumen and overlying an inner slime or jelly of similar constitution. The surfactant, by lowering surface tension in the alveoli (James, 1965), acts as a stabilizer in which inhaled particles become coated. The coating renders them hydrophobic and so they remain unaggregated and suitable for phagocytosis. Phagocytic alveolar macrophages are assumed to swim in or on the surfactant. One of the primary responses of the lung to inhaled particles is to increase the number of macrophages proportionally to the total dust exposure (Morrow, 1967) [28] . Macrophages attempt to digest ingested material and it is thought that those containing indigestible debris enter the mucus blanket and are eliminated by the gastrointestinal tract. If, however, the ingested particles are of pathogenic significance, i.e. capable of eliciting an inflammatory response, the macrophages involved may migrate to the lymphatics. It is estimated by Davis (1963) that 10% of alveolar dusts pass to the lymphatics and the rest are moved in the mucus blanket.

The efficiency with which particulate matter is eliminated from the lung depends largely on ciliary action and phagocytosis. These activities may be impaired by a number of factors including pre-existing respiratory disease, tobacco smoking, alcoholic intoxication and anoxia. Some factors affect ciliary function; others, secretion of mucus. Although most of the particles are eliminated during the first few hours after deposition, some may take weeks or years to be eliminated (Rylander, 1968; Channel et al., 1969) [11] .

Common types of respiratory hypersensitivity

In a review of the immunological basis of allergy, Lessof (1972) summarized the principal allergic diseases of the respiratory system. Inhaled allergens such as pollens, house dust, feathers, moulds and animal danders sensitize some 10% of the population which has a heritable predisposition to allergy, and is known as a topic. In atopic individuals, allergic rhinitis (including hay fever) and allergic asthma predominate. The reaction (type 1) is mediated by reaginic antibodies which are cell bound and belong to immunoglobulin antibodies of the E class (Stanworth, 1971). The reaction may be summarized as follows: Antigen + cell-bound reaginin mast cells release of histamine, Slow Reacting Substance (SRS), etc. If the vasoactive substances released act on the upper respiratory tract, dilation of capillaries together with an increase in their permeability give increased secretions and tissue fluid, and the symptoms of hay fever are felt. If, however, the reaction occurs lower in the respiratory tract, it may result in bronchospasm, increased secretions and the development of an asthmatic attack. Identification of the antigen responsible may be made by intradermal skin testing. A weal and flare are seen at the test site after a short time if the result is positive.

In contrast to the above reaction, which is essentially of immediate anaphylactic type, allergic alveolitis is usually, but not exclusively, found in nonatopic subjects who show a type III Arthus reaction mediated by circulating, cell free antibodies of the classes IgA, IgG or IgM, which can be made to precipitate with antigen in serological investigations. The pathological features of the reaction are thought to be due to the formation of immune complexes between antigen and antibody in the lung capillaries. In farmer's lung disease, these complexes, which also combine with some of the enzymes of serum complement, may disappear slowly from circulation. The very solubility of the complexes may be responsible for provoking symptomatology and be controlled by antigen excess. Patients with more antibodies may have milder symptoms because of the removal of insoluble complexes by the reticuloendothelial system.

Clinical features

The syndrome is described by Grant et al. (1972) and Stretton (1972) as one having an acute or insidious onset, possibly determined by the degree of exposure to antigen. Acute illness results several hours after exposure and is characterized by malaise, sweating, chills, anorexia, breathlessness and a sensation of tightness in the chest. Cough is usually distressing and often nonproductive. With more gradual onset, progressively increasing breathlessness may be accompanied by weight loss. On X-ray examination there is a micronodular pulmonary shadowing. There is also restrictive ventilatory defect due to reduced distensibility and reduced diffusing capacity. In consequence Forced Vital Capacity (FVC), carbon mon oxide transfer factor (TCO), and partial pressure of oxygen in the blood (Pa.02) are reduced. Resolution of all the abnormalities follows withdrawal from and subsequent avoidance of antigens. Irreversible chronic disease is marked by dyspnoea and cough.

Histopathological features

Lung biopsies from patients suffering acute attacks of the disease show a number of characteristic changes (Emanuel, Wenzel, Bowerman & Lawton, 1964, Hapke et al, 1968, Seal et al., 1968) [19] . Interalveolar septa are usually infiltrated by large histiocytic and small round mononuclear cells, and a diffuse interstitial inflammatory infiltrate results. Noncaseating sarcoid-like granulomata with epithelioid and giant cell elements are common. They appear within 3 weeks and resolve over some 12 months. Neither bacterial nor fungal elements are visualized in these lesions, although Wenzel & Emanuel (1965) recovered M. faeni from biopsy material incubated at 60°. Granulomata together with intraalveolar exudate account for X-ray changes. In chronic disease, a diffuse, fine interstitial fibrosis is common, although focal areas of peribronchial distribution may be frequent. Cystic change, resulting in cavities several cm in diam can arise in heavily fibrosed areas (honeycomb lung). In the lung parenchyma from both acute and chronic stages of the disease, foreign body giant cells, some of which contain birefringent inclusions are not uncommon. Their significance has not been fully assessed, although considerable diagnostic value has been attached to them by Emanuel er al. (1964) [19] . Except in the fatal, early, acute case reported by Barrowcliff & Arblaster (1968) [6] , surprisingly little severe vasculitis, a dominant feature of Arthus reactions, has been reported for farmer's lung disease. Some workers have therefore questioned the resemblance between the disease and the classic Arthus reaction in which inflammatory change and necrosis of the vascular system result in haemorrhage. Considerable intraalveolar haemorrhage was reported by Pirie et al. (1971a) [31] in bovine farmer's lung and also vascular infiltration by polymorphonuclear leukocytes in intradermal skin tests. The mechanism is said to involve the liberation of lysozomal proteolytic enzymes (cathepsins) which effect subsequent vascular changes (Kabat, 1967) [21] . That immune complexes, using Bovine Serum Albumin (BSA) and anti-BSA serum at the region of antigen-antibody equivalence may cause lesions in rat skin similar to those of farmer's lung has recently been demonstrated by Spector & Heesom (1969) in their studies of granulomata. Cases of the disease in which radiological evidence indicates an increase in fibrosis despite avoidance of exposure to antigen, pose greater problems for diagnosis than acute episodes. Decrease in antibody renders such cases unreactive serologically and symptoms resemble those met in diffuse idiopathic pulmonary fibrosis, pulmonary tuberculosis and other pneumoconioses (Fletcher, Rondle & Murray, 1970).

Conclusion

Treatment of severely ill patients in the acute stage of farmer's lung disease is by the administration of corticosteroids which probably stabilize the lysozymal membranes of polymorphonuclear leukocytes thus obviating the effects of tissue damage from proteolytic enzymes. The hypoxic patient may require 28-35% of oxygen. Rest and supportive treatment are adjuncts (Stretton, 1972). Grant et al. (1972) suggested a number of measures regarded as necessary to reduce the prevalence of the disease. These included (1) an educational programme to familiarize workers with information on the disease and its prevention; (2) financial assistance to enable farmers of smaller properties to buy equipment for drying hay and cereal crops; (3) official propaganda to encourage silage production; (4) encouragements to aid in the construction of well ventilated and mechanically equipped stock buildings;

(5) personal protection by the use of efficient respirators (Gourley & Braidwood, 1971). Nash & Easson (1972) commented on the proposals and suggested that a new attitude towards moulding of stored crops was necessary especially in view of the popularity of hay as opposed to silage production. They advocated a reappraisal of current standards of field management even where artifical drying is practised. The use of organic acid additives, particularly those containing propionic acid has had considerable success in inhibiting mould growth in stored cereal grain, but technical difficulties of evenly applying the additives to hay while baling are considerable.

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