For verification of this paper, please visit on http://www.socialresearchfoundation.com/innovation.php#8

Fungi are significant as one of the causative agents of allergenic respiratory diseases in the tropics.The mycologists/plant pathologists, to know the diversity, long and short distance travel and dispersal mechanism in relation to the diseases appearing on economically important crop plants, have studied fungi of the outdoors. Similarly, from indoors fungi are known as the result of academic exercises undertaken by different researchers in our country (Rajan et al., 1952; Thirumalikolundusubramanian et al., 1980; Singh, 1981; Tilak et al., 1981; Jayaprakash and Ramalingam, 1981; D'silva and Freitas, 1982).

“Allergic-Type” reactions are known from the ancient’s times through persons who complained after eating certain foods or from coming into contact with certain substances. At that time no body knew the allergenic entities. The term 'Allergy' (Gr. 'allos"= other + 'ergon' = work) was proposed by Von Pirquet in 1906. He meant by allergy “any altered reactivity in humans or animals due to the introduction of a foreign substance”. Farr and Spector (1975) defined allergy as, "untoward physiologic events mediated by a variety of different immunological reactions." Organs of human body such as lung, skin and eyes come into the contact with various types of air pollutants. The air pollutants normally affect respiratory and skin systems of human body. The skin system is less affected than the respiratory system. The affect on respiratory system is on an increase.

Preparation of fungal extracts/antigen Techniques used for the antigen preparation were that of Sheldon et al. (1967) which are as follows: Mass-culture of the fungus The mass-culture of different fungi was done in Sabouraud broth (pH-8.0). The flasks containing 200 ml of the above broth were inoculated with the spore suspension of freshly prepared inoculum of the fungus and incubated at 28˚ C for fifteen to twenty days or until maximum growth and sporulation of different fungi was achieved. Harvesting and Drying After having sufficient growth and heavy sporulation of the fungus, the flasks were filtered with whatman no. 1 filter paper. The fungal mats so obtained were thoroughly washed four to five times with distilled water to remove the traces of adhering nutrient medium. The mats were dried under 0.2-mbar pressure at -45 to -55˚C using Lyophyliser (Model-Lyolab BII of LSL-Secfroid, Switzerland). Dried fungal pellicles were pulverized in pestle mortar to get fine powder so as to have maximum extraction of the antigenic material. Defatting The powdered fungal mass was defatted using solvent ether for 48 hours at 4˚C with periodical shaking and stirring. To ensure complete de-fatting frequent changes (6-10 times) of solvent ether were made till the supernatant became colourless. Finally, the ether was filtered out through Whatman no. 1 filter paper and the fungal mass was allowed to dry at room temperature. Extraction To obtain the active allergenic substances from the defatted fungal powder, extraction was done using cocas solution in weight/volume ratio of 1: 50 up to 24 hours on a magnetic stirrer at 4˚C. Clarification After the completion of the extraction process, the whole solution, containing allergenic ingredients was decanted and then centrifuged at 10,000 rpm in a refrigerated centrifuge for fifteen minutes in order to avoid clogging of the bacterial filter membranes during filtration. Sterilization In order to avoid the destruction of any thermo-labile component in the allergenic extract, the same was sterilized by filtration through bacterial filter of pore size 0.22 m. After filtration, the extract was transferred aseptically into the sterile vials and stored. Before using the extracts for the skin testing, they were tested for sterility. Storage All antigenic extracts were stored at –70˚C. Intra-dermal testing on patients After preliminary examinations/investigations/ case history recorded at allergy clinic, the patients having Bronchial Asthma, Rhinitis, Bronchial Asthma with Rhinitis or some other types of allergenic manifestations and where fungi could be suspected as a possible culprit, were subjected to intra-dermal tests for various fungal antigens to find out the probable offenders. The tests were performed over the upper and lower arms of the patients. The skin was surface sterilized with swabs (70% alcohol) and left till dryness. A sterile tuberculin syringe with disposable needle 26 gauges was used. The syringe was placed at an angle of 45 degrees to the arm with the level of needle down and facing towards the skin. The skin was stretched with one hand and the needle slightly inserted into it a little forward with lifting motion as if to pick up the skin with the tip of the needle. As the tip entered into the skin, 0.01 ml of the antigen (dilution 1: 500) was pushed into the intra-dermal layer. Similarly, control test of buffer saline was also performed on each patient before testing for any antigen. For different antigens, separate syringes were used. The results were recorded within 15-20 minutes after injection. The patients were also advised not to take any medication 48 hours prior to the testing, especially anti-histamines, broncho-dilators, symptomatic agents and cartico-steroids. The result of the skin tests was calculated using the formula of Voorhost. Index =½ [(L+W) flare + 3.7 + 11.0 log (L+W) wheal]

Allergenic Diseases

On the basis of affected target organs, allergenic diseases can be classified into the following 3 major groups.

1.     Allergy of Respiratory Tract

a.     Allergic Rhinitis

b.     Allergic Bronchial Asthma

2.     Allergy of Skin

3.     Allergy of Gastro-Intestinal Tract

In the present study only respiratory allergy has been considered.

Allergy of Respiratory Tract

In a large   number   of individuals, hostility to the environment is manifested through exposure of host to noxious agents, which assault the respiratory tract.  The majority of these assaults occur by inhalation of inorganic and organic substances including dusts, moulds, animal-dander, silica and other living organisms.  In addition to inhalants, the allergy of respiratory tract is also triggered by injestants  (Foods and Drugs) and uninjectants  (Drugs and Vaccines). Triggering of the respiratory tract allergy may be accompanied by bronchospasm, sneezing or secretion of nasal fluid.

Classification of Allergic Reactions

Following four groups of allergenic reactions are known (Cooms and Gell, 1963).

Type   I   - Hypersensitivity   reactions   -   Immediate-Hypersensitivity or Anaphylactic type

The term immediate hypersensitivity denotes an immunological sensitivity to antigens that manifest it by tissue reactions occurring within minutes after the antigen combines with the appropriate antibody.  The response is mainly induced by IgE antibody (Ishizaka et al., 1966).

Antibodies commonly called reagins cause this common and clinically important type of allergy. Cytophilicity is a characteristic of the reagins, that is, a strong tendency to attach to the mast cells and basophilic granulocytes. This property distinguishes them from other antibodies. It is localized to the Fc-fragment of the IgE molecule, which is composed of heavy chains in the molecule.  In humans, mast cells are   found especially in the lungs, in the membranes of the upper respiratory tract, in the skin and in the intestinal tract. Mast cells are rich in granules or small intracellular organelles, which contain histamine and several other biologically active substances.  Reagins are found circulating free in the serum and in certain secretions. It varies from individual to individual.

Type II  - Hypersensitivity reactions - Ccomplement mediated cytotoxic type

In these immune reactions, the antibodies are free in the serum, while the antigen is bound to the surface of certain cells or is a component of the cell membrane. When the antibodies react with the antigen, complement is activated, the wall is damaged and lysis may ensure. These reactions are classically considered to by cytotoxic and involve the combination of IgG or IgM antibodies.

Type III - Hypersensitivity reaction - Late, Arthus type

In this type, extracellular antigens interact with free circulating precipitating (IgG) antibodies within tissue space to form antigen-antibody complexes, usually involving complement. Damage of the surrounding tissues occurs by activated complement induced granulocyte accumulation and release of histamine.  The diseases like Serum sickness, Farmer’s lung and Extrinsic Allergic Alveolotis can exemplify type III-respiratory diseases. If the allergic condition is allowed to persist the tissue damage may be irreversible.

Type    IV  - Hypersensitivity    reaction - Delayed Hypersensitivity

It differs from all the aforesaid reactions. It is not caused by antibodies but by immuno-component cells (lymphocytes).

These reactions, also called delayed hypersensitivity reactions, occur as a rule within 12-48 hours after exposure to the allergen. These reactions lead to inflammatory tissue damage and infiltration   of cells, which are principally mononuclear (lymphocytes and macrophages). The inflammatory reaction leads to irreversible damage with deterioration of the tissue. The examples are positive Tuberculin reaction, contact dermatitis and rejection of tissues transplanted from other individuals.

Dual Type I and Type III reactions

Evidence of dual response on the skin and bronchial challenge is found in three major groups of respiratory tract allergy, viz., Asthma, Pulmonary Eosinophilia and Extrinsic Allergic Alveolitis.

Steps/Events Involved in Allergenic Attacks

1.     Entering of fungal spores in the nose, eyes and glands on mucous membrane of the upper or lower respiratory tracts.

2.     Solubilization of the fungal allergens, which gets into the mucous tissues having mast cells and high concentration of antibodies (IgE).

3.     The antigens quickly complex with antibodies (IgE).

4.     Activation of enzymes, which cause the release of 'chemical mediators' from these cells.

5.     The chemical mediators (Histamines) induce the allergic symptoms like dilation of blood capillaries, contraction of mucous membrane, hypersecretion of watery nasal fluids and contraction of nasal or bronchial passage. These depend on the concentration of various factors like, the concentration of the spores, their types, method of contact, the person’s prior history of exposure, and weather conditions, etc.

A total of 199 patients belonging to either sex were tested for the allergenic responses to 8 fungal antigens, which were suspected to be etiologically significant in causing various allergenic maladies in them.  Out of them, 129 were males while 70 females.  The patients were classified into four groups on the basis of the diseases manifested among them. The groups were: Rhinitis, Bronchial Asthma, Rhinitis with Bronchial Asthma and ‘Others’ having allergenic manifestations other than the aforesaid ones (Urinary and Dermatitis). As is evident from table-2, the maximum number of patients belonged to Rhinitis group (67 patients) followed by Bronchial Asthma and the minimum being to 'others' (24 patients). The patients belonging to Rhinitis with Bronchial Asthma group were 51 in number. Data on the basis of sex, was also quite significant among different groups of patients.  Out of the 67 Rhinitis patients, 53 were males, while females were 14 in number. In Bronchial Asthma group, both male and females were equal in number (29 males and 28 females).  Patients showing symptoms of both Rhinitis and Bronchial Asthma together were represented by 15% male  (29 in number) and 11.0% females (22 in number).  Patients exhibiting other allergenic diseases, grouped as 'others', were 18 males and 6 females.

The relationship between the food-habit and incidence of various allergenic diseases among the males has been shown in table-4. It is evident from the table that non-vegetarians (91 in number) outnumbered the vegetarians (38 in number). Data presented indicated that Rhinitis with Bronchial Asthma was sufficiently higher among the vegetarian males whereas, Bronchial Asthma cases (20 patients) were more among the non-vegetarian females  (Table-5).

As regards the family-history of the patients for the allergenic diseases is concerned, it could be noted that the number of male and female patients having positive family-history for the allergenic diseases was much higher than those showing negative family-history for such diseases (Table-6).

Habitat-wise distribution of the patients is presented in table-7, and as was evident from the table, the incidence of allergenic diseases was much higher in the urban patients than the rural ones. This was the case for both the sexes.

The results of the tests conducted for various antigens over the patients using intra-dermal technique are presented in tables-4.8 to 4.15. The antigens of fungi, viz.,  Alternaria alternata (32),  Aspergillus flavus (32), A. fumigatus (25),  A. niger  (24), A. terreus (27), Chaetomium globosum (14),  Pestalotia pezizoides (20), Trichoderma lignorum (25) were tested. The figures given in parentheses against the name of each fungus indicated the number of patients tested for that particular antigen.

Alternaria alternata antigen had been tested over 32 patients. There were 22 males and 10 females.  Twenty-eight cases were above 20 years of age. Almost all were from urban areas (22 in number).  Non-vegetarians were more in number than vegetarians and 65% cases had positive family history for the respiratory diseases. Voorhost Index was found almost five times more than the control in 9 cases, while in the rest of the cases the reaction was positive, but not very significant (Table-8).

Aspergillus flavus for which 32 patients were tested, 22 cases were males and 10 females. Except four, all were above 20 years of age. Four patients were vegetarians and 28 non-vegetarians including both sexes. Twenty-two belonged to urban areas while 10 to rural areas. Out of these, 11 were Bronchial Asthamatics. Out of the 32 patients, 21 had positive family-history while 11 had negative. Although most of the patients showed increased Voorhost Index than the control, 10 cases had Voorhost Index significantly greater than control (Table-9).

Chaetomium globosum antigen was tested on 14 cases. There were 8 males and 6 females; 7 belonged to urban while the rest to rural areas. There were ten vegetarians. Three patients   were with positive family history.  Voorhost Index was significant in 5 cases (table-13).

Pestalotia pezizoides antigen was tested for 20 patients.  Of which 12 and 8 were males and females, respectively.  Thirteen belonged to the rural while the rest to the urban areas.  Non-vegetarian were 15 and 5 were vegetarians.  As regards the family history of cases for the allergenic diseases, they were positive and negative in equal numbers.  Although, except for one, all cases showed positive reactions, none was found significant (Table-14).

A total of 25 patients were tested for Trichoderma  lignorum, out of which 16 were males, while 09 females. Three patients were below the age group of 20 and twelve were drawn from the urban areas. The vegetarians and non-vegetarians were 5 and 20, respectively.  Nine cases had positive family history. The patients of Rhinitis were higher in number (10) followed by those with Rhintis with Bronchial Asthma (7). None of the cases showed higher Voorhost Index over control, although most of them were positive (Table- 15). 

The survey data collected during the present investigation indicated that the rural and economically weak and those unaware of their health used to come to cities to earn their livelihood.  Even the so-called urban workers also belonged to rural areas because they had their roots in the rural areas.  Other members of the worker’s family also get involved in the profession.  The males dominated the occupation—ginning. The females worked as fillers and stitchers.  The age of the workers ranged from 17-65 years indicating the entire family's involvement in the occupation.  Data also indicated that the family history for allergenic diseases among the workers played important role, although most of the interviewed ones had negative family history.

The results of the skin tests for different fungal antigens are presented in tables 8 to 15.

Although almost all the fungal antigens tested gave positive reactions on the patients tested, the number of significantly positive cases were not encouraging. This points out that fungi may be allergenic to a greater population but restricted to a few as a potent allergenic agentSince the allergenicity of different fungal forms varies with their spore size, density, surface topography and their wall chemistry and also the mycelium, varied allergenic behaviour of the forms tested have been reported by the others as well (Gregory, 1961; Hearn and Mackenzie, 1979, 1981; Hearn et al., 1980; Lacey, 1981; Barreto-Berter et al., 1981; Bartnicki-Garcia and Lippman, 1981; Gomez-Miranda and Leal, 1981; Cole et al., 1982;  San-Blas, 1982).

The aforesaid biologic features of various fungal forms, that seem to have association with allergenic diseases, are bound to vary with different fungi and hence variation in the allergenic behaviour of the tested forms for varying population, as also presently observed, appears logical.

Table- 1. Classification of the patients on the basis of allergenic diseases (Total patients= 199).

Table-  2. Distribution of allergenic diseases in different age groups (considering both sexes together)

 

Table- 3.  Food habit and the incidence of allergenic diseases among males

Table-4.  Food habit and the incidence of allergenic diseases among females

Table-  5.  Family history of the patients (Total= 199).

Table-6. Habitat-wise distribution of the patients (Total=199).

Table-7. Case history of the patients and their allergenic response tested for Alternaria alternata antigen

Table-8. Case history of the patients and their allergenic response tested for Aspergillus flavus antigen

Table-9. Case history of the patients and their allergenic response tested for Aspergillus fumigatus antigen

Table-10. Case history of the patients and their allergenic response tested for Aspergillus niger antigen

Table-11. Case history of the patients and their allergenic response tested for Aspergillus terreus antigen

Table-12. Case history of the patients and their allergenic response tested for Chaetomium globosum antigen

Table-13. Case history of the patients and their allergenic response tested for Pestalotia pezizoides antigen.

Table-14. Case history of the patients and their allergenic response tested for Trichoderma lignorum antigen.

1. Aas, K., Leegaard, J., Aukrust, L. and Grimmer, O. (1980). Immediate type hyper-sensitivity to common molds; comparison of different diagnostic materials. Allergy (Copenh), 35:443-452. 2. Beaumont, F., Haufman, H. F., Sluiter H. J. and De Vries K. (1985). Sequential sampling of fungal air spores inside and outside the homes of mold sensitive, asthamatic patients: A search for a relationship of obstructive reactions. Ann. Allergy, 55:740-746. 3. Chakraverty, R. (1974). Fungi in air and human allergy. J. Sci. Club, 28: 83-106. 4. D'silva, A. M. and Freitas, Y. M. (1982). A survey of the aerial mycoflora of Bombay. Ind. J. Micro, 22 : 27-33. 5. Ellis, D. (1933). Incidence of asthma in Port Sudan with special reference to influence of Molds. Guy’s Hosp. Rep. 83: 102. 6. Fink, J. N. (1985). Allergic Bronchopulmonary aspergillus. Chest., 87: 815-845. 7. Farr, R. S. and Spector, S. L. (1975). What is asthma ? In: The Asthmatic patients in trouble. T. Greenwich, Petty (Eds.) C.P.C. Communications. 8. Gravesen, S. (1978). Identification and prevalence of culturable mesophilic microfungi in house dust from 100 Danish homes: Comparison between airborne and dust bound fungi. Allergy (Copenh), 33: 268-272. 9. Gregory, P. H. (1961). The microbiology of the atmosphere. Leonard Hill (Books) Ltd. London. 251 pp. 10. Gomez-Miranda, B. and Leal, J. A. (1981). Extracettular and cell wall polysaccharides of Aspergillus alliaceus. Trans. Br. Mycol. Soc., 76: 249. 11. Hopkins, J. G., Benham, R. W. and Keston, B. M. (1930). Asthma due to a fungus Alternaria. J. Amer. Med. Ass., 94: 6. 12. Jayaprakash, K. B and Ramalingam, A. (1981). Aspergillus fumigatus in the air of environments at Mysore. Indian J. Bot., 4: 17-23. 13. Lawande, R. V. and Onyemelukwe, G. C. (1984). Air borne fungi during Hartmattan in Zaria, Nigeria. Ann. Allergy, 52:47-49. 14. Lacey, J. (1981). The aerobiological of conidial fungi. In Biology of conidial fungi. Vol. I. G.T. Cole and B. Kendrick (Eds.). Academic Press, New York. 15. Lawler, G. L., Fischer, and Thomas, J. (Eds.). Manual of Allergy and Immunology: Diagnosis and therapy (1981). In: Little Brown and Company, Boston. 504 pp. 16. Longbottom, J. L. and Pepys, J. (1964). Pulmonary Aspergillosis: Diagnostic and Immunological significance of antigens and ‘C’ substances in A. fumigatus. J. Path. Bact., 88: 141. 17. Wadhwani, K. (1979). Air borne fungal flora of Lucknow (University area) J. Palynology, 15:105-110. 18. Maunsel, K. (1954). Respiratory allergy to fungus spores. Prog. Allergy, 4: 457-520. 19. Schumacher, M. J. and Jeffery, S. E. (1976). Variability of Alternaria alternata: Biochemical and immunological characteristics of culture filtrates from seven isolates. J. Allergy Clin. Immunol., 58: 263-277. 20. Sward-Nordmo, M., Almeland, T. L. and Aukrust, L. (1984a). Variability in different strains of cladosporium herbarum with special attention to carbonhyderates and contents of two important allergens (Ag-31 and Ag-54). Allergy, 39: 387-394. 21. Sreeramulu, T. (1961). Concentration of fungus spores in the air inside cattle shed. Acta Allergol. Kbh., 16:337-346. 22. Sandhu, R. S., Khan. Z. U. and Randhawa, H. S. (1977). Natural occurrence of A. fumigatus in cane-sugar mills.Sabouraudia, 15: 263-272. 23. Sarna, N. J. and Govil, C. M. (1979). Survey of air spora of Jaipur city. J. Indian Bot. Soc., 58: 215-225. 24. Tilak, S. T. and Babu, M. (1981). Aerobiological approach to leaf spot disease of Bajra. Indian J. Bot., 4:87-90. 25. Tom, B., and Wolf, B. F. (2012). Food for Thought: Plant Research Remains Grossly Underfunded, Despite the Demand for Increased Crop Production to Support a Growing Population. The Scientist Magazine, LabX Media Group, (Ontario, ON).