Jpn. J. Med. Sci. Biol., 48, 103-115, 1995. SEASONAL FLUCTUATIONS OF DERMATOPHAGOIDES MITE POPULATION IN HOUSE DUST Hiroyuki MATSUOKA, Noriko MAEDA, Yutaka ATSUTA1, Katsuhiko ANDO and Yasuo CHINZEI Department of Medical Zoology, Mie University School of Medicine, 2-174 Edobashi, Tsu-shi, Mie 514 and latsuta Pediatrics Clinic, 13-1 Okura, Tsu-shi, Mie 514 (Received January 30, 1995. Accepted April 12, 1995) SUMMARY: To evaluate seasonal fluctuations of Dermatophagoides species (sp.) in residential houses in Mie Prefecture, Japan, we employed the ELISA inhibition method with rabbit polyclonal antibody to eggs and adult mites. House dust accumulated for seven days in vacuum cleaners was collected from 14 houses of atopic patients every month for one year. The one-week-dust samples weighed from 2.5 to 117 g (mean 35.9 g). The density of Dermatophagoides spp. in the dust samples were 5 to 755 mites per gram (mean 109). During one week, 100 to 20,400 (mean 4,200) mites of Dermatophagoides sp. were collected. Among 142 samples in one year, 50 (35.2%) contained more than 100 mites per gram, which is a critical number for atopic symptoms. From December to February, 12 (34.5%) out of 35 samples contained more than 100 mites per gram. These results suggest that Dermatophagoides sp. have become a year-round allergen in Japan. INTRODUCTION Dermatophagoides farinae (D. farinae) and D. pteronyssinus are major species of house dust mites (1-3). Bodies and feces of these species are well known as major indoor allergens (4,5). According to previous reports (6-8), the number of
house dust mite reaches a peak in summer or early autumn and decreases in winter. In this decade in Japan, however, atopic symptoms of the patients who have a high titers of anti-dermatophagoides IgE continue throughout the year. We suspect that enough Dermatophagoides allergen exists in houses all year round to act as a trigger of atopic attack. It has been proposed that a level of 100 mites per gram dust should be regarded as a risk factor for sensitization and the development of asthma. Such a higher level as 500 mites per gram dust has been proposed as a major risk factor for development of acute asthma in mite-allergic individuals (9). Evaluation of seasonal fluctuations of the populations of house dust mites is presently of value. House dust mites can be counted directly under a microscope (6). This requires much experience and effort. We have developed an enzyme-linked immunosorbent assay (ELISA) inhibition method to detect mite antigens in environmental samples (10). Rabbit serum was used as the antibody after several injections with an extract of D. farinae. A positive correlation between the ELISA value and the direct counts of mites has been confirmed (11). In the present experiments, we measured Dermatophagoides antigen in the dust collected from residential houses of atopic patients during a one-year period. MATERIALS AND METHODS House dust samples: House dust accumulated for 7 days in vacuum cleaners was collected from 14 houses of atopic patients every month from August, 1989 to July, 1990 in Mie Prefecture. The houses were constructed during 1970 to 1986. After weighing, each dust sample was mixed vigorously; one gram of dust was extracted in 20 ml of phosphate buffered saline (PBS) by stirringfor 10 min on a magnetic stirrer. The mixture was centrifuged at 2,000 ~g for 10 min. The supernatant served as a sample for ELISA inhibition assay. Atopic sera: Sera were collected from 12 patients, who consulted periodically a pediatric clinic for treatment of asthmatic bronchitis. These sera showed a radioallergosorbent test (BAST) score of 4 or 5 to Dermatophagoides sp. The sera were mixed and used as a pooled positive serum. Culture and extraction of D. farinae: D, farinae was cultured accoarding to the method of Sasa et al (12). The cultured medium containing about 20,000 104
mites per gram was mixed with saturated sodium chloride and 0.1% Tween 20. Most mites and eggs floated on the surface of the mixture. The part floating on the mixture was put in to 100 volumes of 0.02 M Tris-HCl buffer, ph 7.6, which was mixed and centrifuged at 2,000 ~g for 10 min. Mites and eggs were concentrated in the sediment, which was homogenized in 10 volumes of 0.02 M Tris-HCl buffer, ph 7.6, with a Teflon-glass homogenizer, extracted by stirring at 4 C overnight, and centrifuged at 10,000 ~g for 30 min. The supernatant was used as a crude mite extract. Protein concentration was determined with a protein assay kit (Protein Assay Kit I, Bio-Rad, Hercules, CA) with bovine serum globulin as a standard. Antiserum to D, farinae: A Japanese white rabbit was injected subcutaneously in several places with D, farinae crude extract containing 15 mg of protein. Three boosters each of 10 mg protein were injected at two-week intervals. Freund complete adjuvant was used for the primary injection and Freund incomplete adjuvant for the boosters. Serum was taken 7 days after the last booster and stored at -20 C until used. Characterization of the anti-d, farinae antibody by Western blotting D. farinae mites and eggs were collected under a microscope from the surface of the mixture of mite culture and a saturated sodium chloride solution. Mites and eggs were respectively homogenized and electrophorested on sodium dodecyl sulfate (SDS)-polyacrylamide gel (7.5%) under nonreducing conditions. Separated proteins were stained with silver (Silver Stain Plus Kit, Bio-Rad) or transferred to nitrocellulose sheet for Western blotting. The nitrocellulose sheets were allowed to react with rabbit serum [1:1,000 in PBS containing 5% skim milk and 0.05% Tween 20 (SM/PBS/Tw)] followed by incubation with the secondary antibody of anti-rabbit IgG conjugated to horseradish peroxidase (1:1,000 in SM/PBS/Tw). Positive antigen bands of mites were developed with a substrate kit (Konica Immuno Stain HRP-1000, Konica, Co., Tokyo). One gram of mite culture containing 20,000 adult and immature mites and their eggs and feces of mites was extracted with 10 ml of PBS by stirring for 10 min on a magnetic sterer. The mixture was centrifuged at 2,000 ~g for 10 min. Twenty microliters of the supernatant per lane, which was equivalent to soluble extract of 2 mg of culture (40 mites), was electrophoresed on 5-15% gradient SDSpolyacrylamide gel under nonreducing conditions. Separated proteins were stained with silver or transferred to nitrocellulose sheet for Western blotting. The nitrocellulose sheets were treated with pooled atopic serum (undiluted) or rabbit immune serum (1:1,000 in SM/PBM/Tw) as the first antibody followed by incubation with the second antibody of anti-human IgE or anti-rabbit IgG conjugated to horseradish peroxidase (1:1,000 in SM/PBS/Tw). Positive antigen bands were developed with the substrate kit as above. ELISA inhibition test with common indoor antigens: A 96-microwell flatbottom plate was coated overnight with 100ƒÊl of D, farinae crude antigen (1ƒÊg 105
protein/ml) in 0.05 M carbonate buffer, ph 9.6. Next day, the antigen solution was removed, and each well was washed with PBS and filled with 200ƒÊl of PBS containing 1% bovine serum albumin (BSA/PBS) to block uncoated areas. During the process of blocking, 120ƒÊl of each of diluted rabbit anti-d, farinae serum as the first antibody [1:10,000 in PBS containing 1% BSA and 0.05% Tw (BSA/PBS/Tw)] and 120ƒÊl of a series of diluted inhibitors (mite, house dust, cotton, silk, feather and wool antigens despatched by Torii & Co., Tokyo) were mixed and incubated at 25 C for 60 min. After removal of the blocking solution, 100ƒÊl of the mixture was placed in each well and incubated for 60 min. The plate was washed three times with PBS/Tw, and after addition of the secondary antibody (100ƒÊl of goat anti-rabbit IgG conjugated with horseradish peroxidase, 1:2,000 in BSA/PBS/Tw), incubated for 60 min. The plate was then washed three times with PBS/Tw, and after addition of 100ƒÊl of substrate solution [0.04% 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (Sigma, St. Louis, MO), 0.02% of H2O2 in 0.05 M citrate-0.1 M phosphate buffer, ph 4.5] to each well, incubated for 15 to 30 min. Absorbance was measured at a wavelength of 410 nm. Two wells were used for each sample, and the mathematical mean was obtained as the absorbance of each sample. Absorbance of the wells with the first antibody without inhibitors was considered as 0% inhibition, and that without the first antibody as 100% inhibition. Determination of Dermatophagoides antigen concentrations in dust extract: The ELISA inhibition test was done as above. A 120-ƒÊl portion of dust extract was pre-incubated with diluted first antibody (1:10,000 in BSA/PBS/Tw). The mixture was added to the mite-extract-coated plate. Standard solutions of D. farinae antigen (400, 100, 25, 6.4, 1.6, 0.4, 0.1 and 0ƒÊg protein/ml) were prepared, pre-incubated with diluted first antibody and allowed to react in each plate for a standard curve, with which the concentration of Dermatophagoides antigen in each extracted dust sample was determined. Estimation ofdermatophagoides mite popullation in house dust: According to the correlation graph between the mite antigen content obtained by the ELISA inhibition method and the mite density obtained by direct counting of mites in the dust (11), 1ƒÊg of D, fariane antigen is roughly equivalent to one adult mite. The number of Dermatophagoides mite per gram of house dust was therefore estimated from the dust extract. With the number of Dermatophagoides mite per gram of house dust and the weight of house dust, the number of Derma tophagoides mites in house dust for one week was calculated.106
RESULTS Reactivity Separation of Rabbit Immune patterns other when compared ern blotting of proteins in adult mites and in eggs were similar in the silver stained showed that rabbit mites and eggs. Serum to Adult Mites and Eggs The molecular immune weights each SDS-polyacrylamide serum reacted gel (Fig. 1). Westto similar bands in adult of the bands that reacted with rabbit se - rum were 400, 190, 140 and 110 kda, respectively. The rabbit serum did not react to lower molecular weight bands such as Der f I (24 kda) or Der f II (15 kda) whereas human atopic serum reacted to these bands (Fig. 2). Specificity The reaction microplates farinae of rabbit was strongly concentration occurred of Rabbit Immune inhibited Serum to Common Indoor Antigens anti-d. farinae inhibited slightly antibody to D. farinae antigen-coated (Fig. 3). House dust at a high but not significantly. No inhibition by mite extract the reaction with any other antigen antibody er antigen specifically contained, extract. This indicates that the rabbit anti-d. reacts to mite antigen and does not react to any oth - in house dust. Fig. 1. Reactivity of rabbit immune serum to adult mites and eggs of D. farinae. Egg extract (lanes 1, 3 and 5; 20 eggs per lane) and adult ex tract (lanes 2, 4 and 6; 4 mites per lane) were separated. Proteins in lanes 1 and 2 were stained with silver. Proteins in the other lanes were analyzed by Western blotting with rabbit serum before (lanes 5, 6) and after (lanes 3, 4) immunization. 107
Fig. 2. Reactivity of atopic human sera and rabbit immune serum to the extract of mite culture. Lane 1, silver stain of extract separated. Lane 2, Western blot analysis probed with atopic sera sensitized with Dermatophagoides allergens. Lane 3, Western blot analysis probed with rabbit serum against D. farinae adult mites. Fig. 3. Specificity of the ELISA inhibition method to common indoor antigen. Extract of D. farinae adults was used for coating. Rabbit immune serum was used as the first antibody, which was preincubated with soluble antigens of mite ( œ), house dust ( ), cotton ( ), silk ( ), feather ( ) and wool ( ) as ELISA inhibitors. 108
Fig. 4. Standard curve for estimation of Dermatophgoides antigen concentration. Each curve was prepared from each ELISA plate. Fig. 5. Monthly changes in house-dust weight. House dust was accumulated for 7 days in vacuum cleaners. Bars on the columns indicate standard error. 109
Sensitivity of ELISA Inhibition Method The ELISA inhibition curve was obtained with standard solutions of D. farinae antigen and diluted rabbit anti-d, farinae serum (Fig. 4). This shows that the concentration of the mite antigen can be measured within a range from 0.1 to 100ƒÊg protein/ml, corresponding to 2 to 2,000 mites/g dust. Amount of House Dust in Vacuum Cleaners During one year, 142 samples of one-week-accumulated dust were collected from 14 houses. The weights of the dust samples were distributed from 2.5 to 117 g (mean 35.9 g). No particular monthly changes in house-dust weight were observed (Fig.5). Monthly Changes of Dermatophagoides Antigen As shown in Table I, the number of Dermatophagoides mite in dust samples ranged from 5 to 755 mites per gram (mean 109). In each house and each month, Fig. 6. Monthly changes in Dermatophagoides mite population. The number of Dermatophagoides mite in 7-day-accumulated house dust was estimated from the product of the actual dust weight and the number of Dermatophagoides mite per gram dust obtained in Table I. Bars on the columns indicate standard error. 110
111
the number of mites varied. Among 142 samples collected in one year, 50 (35.2%) contained more than 100 mites per gram, which is a critical number for atopic symptoms (9). The average number of the mites per gram dust was high in August, 1989 and July, 1990, but did not decrease in the winter. From December, 1989 to February, 1990, 12 (34.5%) out of 35 dust samples contained more than 100 mites per gram. As shown in Fig. 6, the average number of Derma tophagoides mite in house dust collected during one week showed similar tendencies to the number of Dermatophagoides mites per gram dust. During one week, 100 to 20,400 (mean 4,200) the mites were removed from each house, respectively. These results suggest that Dermatophagoides species have become a year-round allergen. DISCUSSION To estimate the amount of mites in house dust, several methods have been developed, such as [1] counting mites directly in the dust under a microscope (6), [2] measuring the concentration of guanine (13), a component of mite excret, and [3] measuring mite-specific protein by radioimmunoassay (14,15) or ELISA (16-18) with monoclonal or polyclonal antibodies. We used an ELISA inhibition method to measure the amount of mites in house dust. We used the serum of a rabbit immunized with crude extract of cultured D, farinae adults for this test. A positive correlation was observed between the mite density obtained by direct counting of mites in the dust and the mite antigen content obtained by the ELISA-inhibition method (11). It is not clear, however, which protein reacted to the immune serum. In the present study, we confirmed that the rabbit serum reacted to some of egg components with relatively high molecular weights, and did not react to either Der f I or Der f II. We prepared the immunogen from the floating part of a mixture of mite culture in a saturated sodium chloride solution. The floating part contained adults, nymphs and eggs but lacked most soluble antigens such as Der f I and Der f II. The number of eggs can be estimated at 2,000 per gram of culture according to a previous report (19). Egg proteins of D, farinae would include most adult mite proteins and many immunogenic proteins. might have reacted to egg proteins when mite extract was injected. The rabbit immune system On the other hand, mite extract should contain some other proteins, e.g., nymph- and adult male-specific proteins. Although we did not examine the reac- 112
tivity of the immune serum to nymph or adult male, the serum will possibly react to them. We recognize that further effort will be needed to assess accurately the mite population in house dust with such polyclonal antibodies. The rabbit immune serum did not react to any other house-dust content tested, but reacted to either D, farinae or D, pteronyssinus (data not shown) antigen. We did not test the reactivity of the serum to other genera of mites. The ELISA inhibition test system can be used to estimate the amount of mites in house dust, since the majority of house dust mites are Dermatophagoides species (2,6,11). In this study we found little seasonal fluctuation in the amount of Dermatophagoides antigen. This indicates that adult mites and mite eggs have become a year-round antigen. In past reports (6-8), the number of mites seasonally fluctuations during the year. The number of mites was high in summer to autumn and low in winter. Our data show, however, that Derma tophagoides antigen is high in summer and remains similarly high throughout the other seasons. Recently, the quality of building materials in Japanese houses with aluminium framed windows and tightened floors, walls and ceilings has made it possible to keep the inside of the house warm in winter. Even when the outdoor temperature is below 0 C, the floor temperature in newly constructed houses does not decrease below 10 C, whereas that in houses constructed 30 years ago drops to 5 C (unpublished data). Since the houses in which we collected dust for the present study were constructed during 1970 to 1986, the floor temperature may not have decreased below 10 C even in winter. Humidity is another important factor for survival of mites. Optimum relative humidity (RH) for D, farinae is 55-75%, and the mites can not survive under RH conditions below 40% or over 85% (20,21). Japanese resident houses may presently have such areas that maintain the optimum RH to allow mites to survive in winter. House conditions have become more comfortable in Japan, and house dust mites appear to maintain their populations even in winter. ACKNOWLEDGEMENTS This work was financially supported by a grant in aid to H. M. from the Ministry of Education, Science and Culture, Japan (02857066). We thank Dr. DeMar Taylor, Tsukuba University, for critical reading of the manuscript. 113
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