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Serological detection of IgG antibodies to antigens one soil isolate 0f thermophilic actinomycetes belonged to the species Thermoactinomyces vulgaris in (70) sera of patients with farmer’s lung disease and hay fever patients compared with control normal peoples in the same environmental condition were carried out using ELISA method. The results indicated that (19) sera from symptomatic patients having IgG level between (1.007-1.626 ) I.U/ml, four of them having farmer’s lung disease and the remaining have hay fever as diagnosed by clinical . The IgG level between (0.293-0.944) I.U/ml indicated negative and which was found in(16) patients two of them having farmer’s lung disease and the remaning have hay fever. The IgG level of normal people were devided in two groups: first account positive control was between (1.089-2.147) I.U/ml and the second account negative was between (0.8190.959) I.U/ml. The present study has demonstrated the usefulness of estimation of specific IgG antibodies activity to thermophilic actinomyces by ELISA for the diagnosis of farmer’s lung disease as a screening test of large number of samples with sufficient high sensitivity. Keyword: Thermoactinomyces vulagris, Antigen,lung disease, ELISA INTRODUCTION Hypersensitivity pneumonitis (Hp) is not a uniform disease entity, but rather a complex dynamic clinical syndrome that varies in its initial presentation and clinical course, resulting in the emergence of different patterns of disease over time. Hp has been described as occurring in acute, sub acute and chronic forms. The acute form manifests as recurrent episodes of dyspnoea and cough with fever, chills and malaise occurring about 4-8h after exposure to antigens, and usually resolving within about 24-48h, lung function tests typically show a restrictive defect with reduced gas diffusion and hypoxaemia, and a chest radiography may show alveolar shadowing(Calvert et al., 1999). The chronic form was characterized by the insidious development of dyspnoea and pulmonary fibrosis in patients that have not experienced acute symptoms. The subacute form is similar to the chronic form in that dyspnoea develops insidiously, but these patients also have discrete episodes of acute symptoms after antigens exposure. Classification of HP into acute and chronic forms has tended to cause confusion as it is often assumed that there is an inveitable progression from acute to chronic disease if antigen exposure continues (Bourke et al., 2001). Farmer’s lung disease (FLD), the most common form of extrinsic allergic alveolitis is a pulmonary disease with symptoms of dyspnea and cough resulting from repeated exposure to high concentration or prolonged exposure to low concentrations of inhaled antigens from moldy hay or straw, both of which lead to Bassima A. Abdulla et al. 60 sensitization and development of this disease. Its diagnosis has most often relied on an array of nonspecific clinical symptoms and signs developed in an appropriate setting, with the demonstration of interstitial marking on chest x-ray, serum precipitins against offending antigens, lymphocytic alveolitis on bronchoalveolar lavage, and or agranulomatous reaction on lung biopsies (Reboux et al., 2001).The thermophilic actinomycetes are responsible for hypersensitivity pneumonitis, an allergic reaction to these agents. This occupational disease occurs in farmers, factory workers, and others who are repeatedly exposed to these agents, there are acute and chronic forms of this disease. Acute hypersensitivity pneumonitis typically symptoms resolve within a day and a continued exposure to the organisms form a chronic disease in which symptoms progressively worsen with subsequent development of irreversible lung fibrosis (Forbes et al., 1998). The genus Thermoactinomyces spp. was one of the earliest known actinomycete taxa that was first proposed with Thermoactinomyces vulgaris, the type species of the genus, there was no doubt in recognizing Thermoactinomyces species as actinomycetes because of their morphological characteristics of forming aerial and substrate mycelia. Thermoactinomyces was thermophilic grows between (45 and 60C) and forms single spores on both its aerial and substrate mycelia, it is commonly found in damp hay stacks, compost piles and other high-temperature habitate, unlike other actinomycete spores. Spores of Thermoactinomyces spp. are true endospores and very heat-resistant, they can survive at 90C for more than 30 minutes, they are formed within the hyphae and appear to have typical endospore, gram positive, aerobic and saprophytic chemorganotrophic utilize a range of sugars and are able to degrade various polymeric substrates (Holt et al., 1994). However, some studies provided evidence that the genus Thermoactinomyces spp. should no longer be classified within the order Actinomycetales (Yoon and Park, 2000). Thermoactinomyces species produce endosproes as shown in bacilli and have lower G+C contents than those of actinomycets. 16sr RNA oligonucleotide sequencing revealed that the genus Thermoactinomyces is more closely related to Bacillus species than to actinomycetes and should be placed within the family Bacillaceae (Prescott et al., 2008). Identifying the etiological agents is of primary importance for the immunological diagnosis of this disease.It is also a necessary step in the development of preventive methods. Saccharopolyspora rectivigula and Thermoactinomyces vulgaris remain the main recognized etiologies of FLD, other bacterial and fungal species have been demonstrated or suspected to play a role in FLD (Reboux et al., 2001). The aim of this work to estimate the specific IgG antibody using ELISA in sera of patients with famer’s lung disease in farmer’s workers compared with hay fever patient’s and control people. MATERIALS AND METHODS Cahort study was carried out from April 2007 to April 2008 in Iraq (Mosul).Thirty five sera from males and females patients, five of them having farmer’s lung disease and thirty with hay fever attending the private clinic in Mosul. Protocol for diagnosis of farmer’s lung disease by serology was cared out using IgG ELISA Euroimmun. Antibody of Thermophilic bacterial antigen belongs to one soil isolate of the species Thermoactinomyces vulagris was carried out as the flowing: Serological Detection of the T. vulagris Antigen in Farmer’s lung disease 61 Preparation of Thermoactinomyces vulgaris antigen: Bacteria was cultured in trypticase soy agar with 2%-Glycerol in pH 7.0 for 3-5 days in 45C. Bacterial growth was harvested by centrifugation at 3000 cycle for 10 min and washed 2 times with sterile phosphate buffer saline (PBS in pH7.3). Two milliliters of the bacterial pellet was resuspended in 10 ml of PBS. Sonicated (in 24,000 Ampudance/sec) 5 sec. to each cycle between each cycle 1⁄2 min. modified from (Kohler et al., 2003). 1-1ooL of antigen was dispensed in to desirved number of coated wells in to the holder and incubated in 45C to 24 hours. 2-1:40 diluted of serum negative and positive control was dispensed into the appropriate wells. 3-Incubate at 37C for 30 minutes. 4The liquid from all wells was removed at the end of incubation. Wells was rinsed and flicked the microtiter wells 4 times with diluted wash buffer (1x). 5Liquid from all wells was disposed by trapping it in absorbent paper with the openings facing downwards to remove all residual wash buffer. 6100L of enzyme conjugate (peroxidaselabelld anti-human IgG)was dispensed into each wells and incubated for 30 minutes of room temperature. 7Wells was washed as described above in

Serological Detection of the Thermoactinomyces vulagris Antigen in Farmer’s lung disease Patients using ELISA method