- Email: [email protected]
School as a risk environment for children allergic to cats and a site for transfer of cat allergen to homes
School as a risk environment for children allergic to cats and a site for transfer of cat allergen to homes Catarina Almqvist, MD,a,b Per H. Larsson, MSc,c,d Ann-Charlotte Egmar, RN,a Marie Hedrén, BSc,c Per Malmberg, MD, PhD,c and Magnus Wickman, MD, PhD,a,e Stockholm, Sweden Background: Many children are allergic to furred pets and avoid direct pet contact. The school may be a site of indirect exposure to pet allergens, which may induce or maintain symptoms of allergic diseases. Objective: We sought to investigate airborne levels of cat allergen (Fel d 1) at schools and in homes with or without cats and to study clothes as a route for dissemination of allergens between homes and school. Methods: Airborne cat allergen was collected with personal samplers from (1) children attending classes with many (>25%) or few (<10%) cat owners and (2) homes with or without cats. A recently developed amplified ELISA assay, which detects low levels of airborne cat allergen in pet-free environments, was used. Dust samples were collected from clothes and mattresses. Results: There was a 5-fold difference in the median levels of airborne cat allergen between classes with many and few cat owners (2.94 vs 0.59 ng/m3; P < .001). The median airborne cat allergen concentration in classes with many cat owners was significantly higher than that found in the homes of non-cat owners (P < .001) but lower than that found in homes with cats (P < .001). Allergen levels in non-cat owners’ clothes increased after a school day (P < .001). Non-cat owners in classes with many cat owners had higher levels of mattress-bound cat allergen (P = .01). Conclusion: The results indicate significant exposure to cat allergen at school. Allergen is spread through clothing from homes with cats to classrooms. There the allergen is dispersed in air and contaminates the clothes of children without cats. The allergen levels in non-cat owners’ homes correlate with exposure to cat allergen at school. (J Allergy Clin Immunol 1999;103:1012-7.) Key words: Allergen, cat, Fel d 1, school, environment, air sampling, clothes, pets, transfer
The prevalence of allergic diseases and asthma has increased over the last few decades, especially among
From athe Department of Environmental Health, Karolinska Hospital; bAstrid Lindgren Children’s Hospital, Department of Woman and Child Health, Karolinska Institutet; cNational Institute for Working Life; dSachs Children’s Hospital, Karolinska Institutet, Stockholm; and ethe Department of Clinical Immunology and Transfusion Medicine, Karolinska Hospital. Supported by The Swedish Council for Work Life Research grant no 950150 and the Vårdal Foundation. Received for publication Aug 31, 1998; revised Jan 11, 1999; accepted for publication Jan 13, 1999. Reprint requests: Catarina Almqvist, MD, Department of Environmental Health, Norrbacka building, level 3, Karolinska Hospital, S-171 76 Stockholm, Sweden. Copyright © 1999 by Mosby, Inc. 0091-6749/99 $8.00 + 0 1/1/97672
1012
children and young people.1,2 In some areas of the world, 50% to 70% of children with asthma are sensitized to furred pets.3 Possession of furred pets has become more common over recent decades, and in Sweden it has been reported that 23% to 34% of schoolchildren own cats or dogs.1 Exposure to pet allergens has been shown to be a risk factor for sensitization and may induce or maintain symptoms of asthma and allergic diseases.4-7 In Sweden exposure to cockroaches is rare, and like in Los Alamos, NM,8 the most common sources of sensitization are allergens from furred pets and pollens. In areas with a high proportion of pet ownership, high levels of pet allergens have been found in dwellings with or without furred pets, as well as in dust from different public environments.9-12 Many allergic children who are sensitized to cat allergen have not had cats at home and avoid direct contact with pets. Indirect exposure to pet allergens might, however, be of sufficient magnitude to induce or maintain symptoms in sensitized children.13 Schools and day care centers where many children are together in a small area are possible sites of indirect allergen exposure.14,15 Cat owners’ clothes have been proposed to be the major source of allergen in the classroom.16 Exposure to airborne pet allergens in environments where pets have been kept has previously been studied, but because of the very low levels of airborne allergens, few studies have been performed in pet-free environments.17 The aim of this investigation was to study levels and routes of contamination of cat allergen at school and to assess further dissemination of allergens to homes without cats. A recently developed amplified ELISA has made it possible to detect very low levels of cat allergen and to measure airborne levels of allergen in pet-free environments by using personal samplers.
METHODS Questionnaires All secondary schools in 2 suburban districts of Stockholm, consisting of both rural and urban areas, were identified. Headmasters and teachers in grades 4 and 5 (age of students, 10 to 12 years) were informed about the study. A questionnaire regarding possession of domestic animals and furred pets was distributed to all 2042 pupils and their teachers. The answers were confirmed and signed by one of the pupil’s parents. The teachers compiled and mailed all questionnaires from their classes. If a class did not respond, the teacher received 2 reminders.
Almqvist et al 1013
J ALLERGY CLIN IMMUNOL VOLUME 103, NUMBER 6
Airborne cat allergen Exposure to airborne cat allergen Fel d 1 was examined in the breathing zone of 5 children in each of 12 classrooms on 2 different occasions. Six classes with many cat owners and 6 classes with few cat owners (Table I) were selected from 7 different schools for geographic and practical reasons. There was a similar number of pupils in the classes (average, 25; range, 18 to 31). In each class one cat owner, 2 children sitting next to a cat owner, and 2 children sitting far away (>1.5 m) were identified. Personal air samples were obtained by using IOM samplers (SKC Inc) equipped with 25-mm polytetrafluoroethylene filters (pore size 1 µm; Millipore AB, Sundbyberg, Sweden). An airflow rate of 2 L/min over the filter was obtained with Aircheck 50 personal sampling pumps (SKC Inc). Air was sampled for 3 hours during class. Effective sampling time and average airflow were used to calculate total volume of air sampled. Airborne levels of cat allergen Fel d 1 were also sampled in the homes of 36 children by one family member.
TABLE I. Distribution of classes with few or many cat owners Classes
Cat owners
Range
No. of classes
Few cat owners Intermediate cat owners Many cat owners
<10% 10%-25%
3%-9% —
12 classes 53 classes
>25%
26%-63%
17 classes*
*Two
classes had greater than 50% cat owners (ie, 57% and 63%).
Mattress dust was stored at –20°C until extraction. Extraction (1/40 wt/vol) was made in PBS with 0.1% Tween 20 and 0.15% Kathon CG (Rohmand Haas, Hydrosupra Kemiservice, Helsingborg, Sweden) (PBSTK) for 2 hours at room temperature. Dust from clothes was immediately extracted in 20 mL of PBSTK and rotated for 2 hours at room temperature. Polytetrafluoroethylene filters containing airborne dust were left in the cassettes and kept at –20°C until extraction. The filters were extracted in 2 mL of PBSTK and rotated overnight at room temperature. All extracts were centrifuged twice (10 minutes × 1000g), divided into aliquots, and stored at –20°C until analysis.
were developed by adding 100 µL of KBlue (ELISA Technologies Inc). The blue color reaction was stopped after 20 minutes by the addition of 100 µL of 1 mol/L H2SO4. Endpoint yellow color was measured at 450 nm (minus 650 nm reference) on a ThermoMax reader by using SOFTmax version 2.3 for Windows software (Molecular Devices Inc). Clothes dust sample assay. Analysis of the amount of Fel d 1 in dust from children’s clothes was performed by essentially following the same ELISA protocol as for mattress dust. The following modifications were made. The concentration range of the standard curve was 0.039 to 2500 ng/mL. The in-house cat allergen control used had an assigned Fel d 1 concentration of 0.4 ng/mL. To achieve a higher sensitivity, an in-house prepared NeutrAvidin (ImmunoPure, product #31000; Pierce, Rockford, Ill) horseradish peroxidase conjugate (synthesized by Per H. Larsson) was used. Samples below 0.039 ng/mL were run in the amplified ELISA. Amplified assay. The analysis of air samples by an amplified ELISA was performed as above, with the following modifications. Coating with 6F9 was done by using 100 µL at 5 µg/mL in PBS overnight. Plates/strips were blocked for 1 hour by using PBS containing 1% BSA and then washed 4 times (throughout the assay). All dilutions were made in PBSTK containing 1% BSA, and 100-µL volumes were used in duplicates. Standards (2.5 to 160 pg Fel d 1/mL), samples, and an in-house control cat allergen extract (Fel d 1 at 40 pg/mL) were incubated for 90 minutes. After washing, plates were incubated with biotinylated 3EC4 at 1/1000 for 60 minutes, washed, and incubated for 30 minutes with an in-house streptavidin horseradish peroxidase conjugate (100 ng/mL). The amplifier NBiotinyl-4-Hydroxybenzohydrazone (13.5 µg/mL, molecular weight of 362 d; synthesized by Per H Larsson) in 0.1 mol/L borate/0.003% H2O2, pH 8.5, was added and allowed to react for 15 minutes according to the catalyzed reporter deposition concept.19 After washing, the enzyme conjugate was added and incubated as above. Readings were performed as for the dust sample assays. For all ELISAs, intraassay sample coefficients of variation less than 10% were accepted. Interassay coefficients of variation of controls less than 20% were considered acceptable. The results were expressed as micrograms of Fel d 1 per grams of dust for the mattresses, nanograms of Fel d 1 per garment for the clothes, and nanograms of Fel d 1 per cubic meter of air, respectively. Detection limits (as defined by the lowest standard point) of the assays were 25 ng Fel d 1/g mattress dust, 50 pg Fel d 1/g garment dust, and 42 pg Fel d 1/m3 of air at 60 minutes sampling time (amplified ELISA).
ELISA assays
Statistics
Mattress dust sample assay. Fel d 1–specific mouse mAbs 6F9 and biotinylated 3EC4 and cat allergen standard UVA 94/01 were purchased from Indoor Biotechnologies (University of Virginia, Charlottesville, Va),18 and ELISA was performed essentially as described in the accompanying instructions, with the following modifications. The coating antibody 6F9 was used at 10 µg/mL. The standard curve was 0.625 to 20 ng Fel d 1/mL and was run with an in-house control cat allergen extract (1.3 ng Fel d 1/mL). Plates
Levels of airborne cat allergen in each of the 12 classrooms were calculated from the mean values of the non-cat owners on 2 separate occasions. Values from cat owners were excluded. The median and 25-75 percentiles of the mean from classes with many or few cat owners were calculated. Comparisons were made by using the nonparametric unpaired Mann-Whitney U test. To compare the levels of airborne Fel d 1 in the breathing zone of cat owners with that of the children sitting next to or far from a cat owner, the nonpara-
Cat allergen in dust from clothes and mattresses Clothes were examined in 3 classes with many cat owners: 23%, 50%, and 56%, respectively. New T-shirts were purchased from a factory and delivered in sealed boxes. After school on day 1, all cat owners (n = 30) received a T-shirt to wear at home during the evening and all day at school on day 2. On the morning of day 2, 31 non-cat owners received a T-shirt to wear in class all day. All Tshirts were collected by the end of the school day. To compare the amount of cat allergen in T-shirts of cat owners before and after a school day, 15 of the cat owners received new T-shirts on day 2 to wear at home for an afternoon, and these were collected for vacuuming the following morning (day 3). Five unused T-shirts were vacuumed for reference. T-shirts were vacuumed in a standardized fashion16 with a vacuum cleaner equipped with a dust collector (Allergy Control Products Inc). The T-shirts were vacuumed on the front and back for 90 seconds each. Mattresses were vacuumed on the entire upper surface for 2.5 minutes with the dust collector. All airborne samples and dust samples from mattresses were obtained between March and June 1996, and the samples from the clothes were collected in May 1998.
Extraction of dust and air samples
1014 Almqvist et al
J ALLERGY CLIN IMMUNOL JUNE 1999
FIG 1. Levels of airborne cat allergen Fel d 1 in the breathing zones of non-cat owners in classes with many or few cat owners (P < .01). Box plots with medians marked are shown; box corresponds to 25-75 percentiles, and vertical lines correspond to 10% and 90% of the range, respectively.
metric paired Wilcoxon test was used. For comparisons of dust in mattresses and clothes, the nonparametric unpaired Mann-Whitney U test was used. Two-tailed tests were used, and P values less than .05 were regarded as statistically significant. The medians are shown in the results below, with the 25-75 percentiles in parenthesis. Permission for the study was obtained from the Ethics Committee of the Karolinska Institutet.
RESULTS The questionnaires were sent to 2042 pupils and teachers, and complete questionnaires were obtained from 1931 pupils (52% boys and 48% girls) and 83 teachers in the 85 school classes identified (response rate, 95%). One class had 2 teachers. Three classes did not respond despite reminders, and a few pupils in other classes declined participation or did not respond. Eighty-three percent of the children were between 10 and 11 years old, and 11% of the children were 12 years old. Fifty-eight percent of the children had domestic animals at home, and 43% owned furred pets (ie, cats, dogs, rabbits, or rodents). Eighteen percent of the children owned cats. The classes were divided into classes with many cat owners (>25%) or few cat owners (<10%) on the basis of less than 25 and greater than 75 percentiles of cat ownership (Table I).
Airborne cat allergen Airborne Fel d 1 was investigated on 2 separate occasions in 6 classes with few cat owners and in 6 classes with many cat owners. There was a 5-fold difference in the median level of airborne cat allergen between classes
with many cat owners compared with those with few cat owners (median [25-75 percentiles], 2.94 ng/m3 [2.374.77] vs 0.59 ng/m3 [0.45-0.81]; P < .01; Fig 1). In classes with few cat owners, the median level of sampled airborne Fel d 1 in the breathing zones of cat owners was 2.1 ng/m3 (1.53-12.0), that for children next to cat owners was 0.66 ng/m3 (0.40-1.14), and that for children far from cat owners was 0.52 ng/m3 (0.35-0.75). The cat owners were exposed to significantly higher concentrations of cat allergen compared with children sitting next to (P = .05) or far from cat owners (P = .05). Comparing non-cat owners sitting next to cat owners with those sitting far from cat owners, no significant difference in exposure to airborne levels of Fel d 1 was found (P = .17). In classes with many cat owners, the median level of sampled airborne Fel d 1 in the breathing zones of cat owners was 3.79 ng/m3 (1.80-13.9). Children sitting next to cat owners were found to have cat allergen levels of 4.09 ng/m3 (2.44-5.27) in their breathing zone, whereas children far from cat owners had levels of 2.77 ng/m3 (1.70-3.87). No significant differences were found between cat owners and non-cat owners nor between the different groups of non-cat owners (Fig 2). The levels of airborne cat allergen were significantly higher in classrooms (1.54 ng/m3 [0.59-2.94], n = 12) than in homes without a cat (0.15 ng/m3 [0.04-0.27], n = 26; P < .001) and significantly lower than in homes with a cat (19.8 ng/m3 [7.54-45.4], n = 10; P < .001; Fig 3). There was thus a 7-fold difference in the median values of airborne cat allergen in homes with cats compared
Almqvist et al 1015
J ALLERGY CLIN IMMUNOL VOLUME 103, NUMBER 6
FIG 2. Levels of airborne Fel d 1 in breathing zones of cat owners, children sitting next to cat owners, and children sitting far from cat owners in classes with many or few cat owners, respectively. Classes with few cat owners: Cat owner/near cat owner, P = .05; cat owner/far from cat owner, P = .05; next to cat owner/far from cat owner, P = .17. Classes with many cat owners, not significant.
with school classes with many cat owners. Whether the child attended a class with many or few cat owners did not affect the airborne levels of Fel d 1 in homes without cats.
Cat allergen in dust from clothes and mattresses The levels of Fel d 1 found in dust from vacuumed Tshirts are shown in Table II. The levels of Fel d 1 in the classes with 50% and 56% cat owners were higher in the evening than in the morning, whereas the opposite was found in the class with 23% cat owners, although this was not significant. After a day at school, the levels of Fel d 1 in the T-shirts of non-cat owners were significantly higher than those found in the unused T-shirts (P < .001). In dust from the children’s mattresses, the median levels of Fel d 1 for cat owners (n = 45) was 87.4 µg Fel d 1/g dust (29.4-199.4), and that for non-cat owners (n = 181) was 0.5 µg Fel d 1/g dust (0.2-1.4) (P < .001). The median levels of Fel d 1 in mattresses from homes without cats where the child attended a class with many cat owners (0.85 µg/g dust [0.32-1.64], n = 75) were significantly higher than if the child attended a class with few cat owners (0.38 µg/g dust [0.19-1.21], n = 106; P = .01).
DISCUSSION This study provides new data partly based on methods not previously available. There are 2 main findings. There is an association between the levels of airborne cat allergen and the number of cat owners in classes, with higher levels of Fel d 1 in classes with greater than 25% cat owners and lower levels in classes with less than 10% cat owners. The levels could be sufficient to influence the allergic inflammation in children with cat allergy.13,20 Airborne cat allergen levels were not strongly influenced by location in relation to a cat owner within the classroom; however, it was clearly shown that the clothes of cat owners were a potential source of cat allergen contamination in the classroom. The second main finding is that cat allergen is transferred from cat owners’ clothes to non-cat owners’ clothes. The transferral of allergens may either be by direct contact or by deposition of allergen elsewhere in the classroom, which is then picked up by non-cat owner’s clothes. This may contribute to the observation that children in classes with many cat owners have higher allergen concentrations in their mattresses at home than children in classes with few cat owners, thus providing new insights into the mechanisms of allergen dispersion in society.
1016 Almqvist et al
J ALLERGY CLIN IMMUNOL JUNE 1999
FIG 3. Levels of airborne Fel d 1 in breathing zones of non-cat owners in all classrooms compared with children in homes without cats (P < .001) and children in homes with cats (P < .001).
TABLE II. Median levels of Fel d 1 found in dust from clothes (median and 25-75 percentiles) Parameters
Cat owner morning, n = 15 Cat owner evening, n = 15 Cat owner total, n = 30 Non-cat owner, n = 31 Unused T-shirt, n = 5
Fel d 1/garment (ng)
2720 (700-12,950) 3970 (950-18,430) 3030 (900-15,620) 146 (86-234) 0.3 (0.2-2.2)
Cat owner total versus non-cat owner, P < .001; non-cat owner versus unused T-shirts, P < .001.
Airborne allergen levels The amplified ELISA method for airborne cat allergen Fel d 1 used in this study makes it possible to measure allergen levels even in pet-free environments and to compare allergen levels in the breathing zones of cat owners and non-cat owners despite the low levels. The levels of airborne Fel d 1 in homes with cats correspond well with what has been found in earlier studies.17,21,22 However, very few studies on the levels of airborne Fel d 1 in cat-free environments have been carried out. Woodcock et al23 concluded that Fel d 1 was undetectable in the absence of disturbance in homes without a cat. Bollinger et al17,24 found levels of 1 ng/m3 in cat-free environments, even with an amplified ELISA. The levels of Fel d 1 in homes without cats in our study were found to be as low as 0.01 ng/m3. Within each classroom, sitting next to or far from a cat owner was nonsignificant for the measured levels of cat allergen. In classes with few cat owners, the cat owners had significantly higher levels of Fel d 1 in ambient air
than did the non-cat owners, which could not be shown in classes with many cat owners. In classes with many cat owners, most pupils are likely to be sitting in the vicinity of a cat owner. In this study we chose an arbitrary distance of more than 1.5 m from a cat owner, but this may be too close to detect a gradient in concentration in the classroom, depending on distance from the cat owner. On the other hand, during the sampling of airborne cat allergen, teachers and pupils kept going in and out of the classroom, and the pupils rarely remained in their seats for a whole 40 minutes. It is also very likely that there are reservoirs of cat allergen in the classroom that contribute to the exposure. Therefore location within the classroom may have little influence on exposure levels. We have not found any previous study where this kind of comparison in exposure has been done.
Cat allergen in dust from clothes and mattresses The allergen levels found in dust from clothes indicate that cat allergen is carried to school in cat owners’ clothes and is there transferred to the clothes of children without cats. This has also been suggested in former studies.16,25 The schoolchildren without cats at home may bring the allergen home in their clothes and deposit it in their homes and beds. This hypothesis is supported by the levels of Fel d 1 found in dust from mattresses of children without cats, which were significantly higher among children attending a class with many cat owners than those attending classes with few cat owners. Our data, however, do not allow a distinction between the possibility that the allergen in the mattresses could be derived from the child’s clothes contaminated at school or from
Almqvist et al 1017
J ALLERGY CLIN IMMUNOL VOLUME 103, NUMBER 6
visiting cat owners’ clothes. It is likely that children who attend a class with many cat owners also meet more cat owners outside school than do children attending a class with few cat owners.
Clinical effects The levels of cat allergen exposure in classrooms found in this study may be high enough to worsen or maintain an inflammation in the mucous membranes of the airways and to elicit or aggravate symptoms of asthma.13,20 Previous studies have shown that Fel d 1 levels of 8 µg/g dust may cause sensitization and elicit asthma.26 The corresponding airborne levels are not known, but worsening of symptoms caused by inhaled cat allergen cannot be excluded at the levels of cat allergen found in classrooms in our study. The difference in exposure to cat allergen between classes with many or few cat owners is significant, but it is evident that children are exposed to allergens even when they are in a classroom with very few cat owners. This is important to families who try to avoid pets because of allergic disposition. Bollinger et al17 studied Fel d 1 in cat-free environments in relation to symptoms found in a cat challenge room for 1 hour, in which levels less than 500 ng/m3 were found to give rise to respiratory symptoms in individuals allergic to cats. To reduce cat allergen levels in schools, it would be of considerable interest to look into possible intervening measures, preferably to take steps to minimize the allergen source from the clothes of cat owners. Whether a child allergic to cats clinically improves if placed in a class with few cat owners compared with a class with many cat owners should also be investigated. The ethical and moral aspects should be considered when taking intervening steps, but our data imply a necessity to investigate health effects and to reduce exposure to pet allergens, which is also of interest from a health economics point of view. We thank André Lauber for expert computer assistance and Inger Kull and Siw Siljerud for sample collection. We also thank the children and teachers in the schools, without whose assistance this study would not have been possible.
REFERENCES 1. Åberg N, Hesselmar B, Åberg B, Eriksson B. Increase of asthma, allergic rhinitis and eczema in Swedish schoolchildren between 1979 and 1991. Clin Exp Allergy 1995;25:815-9. 2. Burr ML, Butland BK, King S, Vaughan-Williams E. Changes in asthma prevalence: two surveys 15 years apart. Arch Dis Child 1989;64:1452-6. 3. Croner S, Kjellman N-I M. Natural history of bronchial asthma in childhood—a prospective study from birth to 14 years of age. Allergy 1992;47:150-7. 4. Wickman M, Nordvall L, Pershagen G. Risk factors in early childhood for sensitization to airborne allergens. Pediatr Allergy Immunol 1992;3:128-33. 5. Wahn U, Lau S, Bergmann R, Kulig M, Forster J, Bergmann K, et al. Indoor allergen exposure is a risk factor for sensitization during the first three years of life. J Allergy Clin Immunol 1997;99:763-9. 6. Warner JA, Little SA, Pollock I, Longbottom JL, Warner JO. The influ-
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
25.
26.
ence of exposure to house dust mite, cat, pollen and fungal allergens in the home on primary sensitization in asthma. Pediatr Allergy Immunol 1990;1:79-86. Platts-Mills TAE, Vervloet D, Thomas WR, Aalberse RC, Chapman MD. Indoor allergens and asthma: report of the third international workshop. J Allergy Clin Immunol 1997;100(Suppl):S1-24. Ingram JM, Sporik R, Rose G, Honsinger R, Chapman MD, Platts-Mills TAE. Quantitative assessment of exposure to dog (Can f 1) and cat (Fel d 1) allergens: relation to sensitization and asthma among children living in Los Alamos, New Mexico. J Allergy Clin Immunol 1995;96:449-56. Wood RA, Eggleston PA, Lind P, Ingemann L, Schwartz B, Graveson S, et al. Antigenic analysis of household dust samples. Am Rev Respir Dis 1988;137:358-63. Egmar AC, Emenius G, Almqvist C, Wickman M. Cat and dog allergen in mattresses and textile-covered floors of homes which do or do not have pets, either in the past or currently. Pediatr Allergy Immunol 1998;9:315. Egmar AC, Almqvist C, Emenius G, Lilja G, Wickman M. Deposition of cat (Fel d 1), dog (Can f 1) and horse allergen over time in public environments—a model of dispersion. Allergy 1998;53:957-61. Custovic A, Fletcher A, Pickering CAC, Francis HC, Green R, Smith A, et al. Domestic allergens in public places III: house dust mite, cat, dog and cockroach allergens in British hospitals. Clin Exp Allergy 1998;28:53-9. Ihre E, Zetterström O. Increase in non-specific bronchial responsiveness after repeated inhalation of low doses of allergen. Clin Exp Allergy 1993;23:298-305. Munir AKM, Einarsson R, Dreborg SKG. Mite (Der p 1, Der f 1), cat (Fel d 1) and dog (Can f 1) allergens in dust from Swedish day-care centres. Clin Exp Allergy 1995;25:119-26. Smedje G, Norbäck D, Edling C. Asthma among secondary schoolchildren in relation to the school environment. Clin Exp Allergy 1997;27:1270-8. Enberg RN, Shamie SM, McCullough J, Ownby DR. Ubiquitous presence of cat allergen in cat-free buildings: probable dispersal from human clothing. Ann Allergy 1993;70:471-4. Bollinger ME, Eggleston PA, Flanagan E, Wood RA. Cat antigen in homes with and without cats may induce allergic symptoms. J Allergy Clin Immunol 1996;97:907-14. Chapman MD, Aalberse RC, Brown MJ, Platts-Mills TAE. Monoclonal antibodies to the major feline allergen Fel d 1. II. Single step affinity purification of Fel d 1, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d 1 exposure. J Immunol 1988;140:812-8. Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. J Immunol Methods 1989;125:279-85. Roquet A, Lagging E, Ihre E, van Hage-Hamsten M, Halldén G, Härfast B, et al. Induction of increased bronchial reactivity by repeated low-dose allergen exposure. APMIS 1998;106:293-9. Sakaguchi M, Inouye S, Irie T, Miyazawa H, Watanabe M, Yasueda H, et al. Airborne cat (Fel d 1), dog (Can f 1), and mite (Der 1 and Der 2) allergen levels in the homes of Japan. J Allergy Clin Immunol 1993;92:797802. Luczynska CM, Li Y, Chapman MD, Platts-Mills TAE. Airborne concentrations and particle size distribution of allergen derived from domestic cats (Felis domesticus). Am Rev Respir Dis 1990;141:361-7. Woodcock A, Taggart SCO, Custovic A. Airborne cat allergen [abstract]. J Allergy Clin Immunol 1995;95:326. Bollinger ME, Wood RA, Chen P, Eggleston PA. Measurement of cat allergen levels in the home by use of an amplified ELISA. J Allergy Clin Immunol 1998;101:124-5. Patchett K, Lewis S, Crane J, Fitzharris P. Cat allergen (Fel d 1) levels on school children’s clothing and in primary school classrooms in Wellington, New Zealand. J Allergy Clin Immunol 1997;100:755-9. Gelber LE, Seltzer LH, Bouzoukis JK, Pollart SM, Chapman MD, PlattsMills TAE. Sensitization and exposure to indoor allergens as risk factors for asthma among patients presenting to hospital. Am Rev Respir Dis 1993;147:573-8.
The prevalence of allergic diseases and asthma has increased over the last few decades, especially among
From athe Department of Environmental Health, Karolinska Hospital; bAstrid Lindgren Children’s Hospital, Department of Woman and Child Health, Karolinska Institutet; cNational Institute for Working Life; dSachs Children’s Hospital, Karolinska Institutet, Stockholm; and ethe Department of Clinical Immunology and Transfusion Medicine, Karolinska Hospital. Supported by The Swedish Council for Work Life Research grant no 950150 and the Vårdal Foundation. Received for publication Aug 31, 1998; revised Jan 11, 1999; accepted for publication Jan 13, 1999. Reprint requests: Catarina Almqvist, MD, Department of Environmental Health, Norrbacka building, level 3, Karolinska Hospital, S-171 76 Stockholm, Sweden. Copyright © 1999 by Mosby, Inc. 0091-6749/99 $8.00 + 0 1/1/97672
1012
children and young people.1,2 In some areas of the world, 50% to 70% of children with asthma are sensitized to furred pets.3 Possession of furred pets has become more common over recent decades, and in Sweden it has been reported that 23% to 34% of schoolchildren own cats or dogs.1 Exposure to pet allergens has been shown to be a risk factor for sensitization and may induce or maintain symptoms of asthma and allergic diseases.4-7 In Sweden exposure to cockroaches is rare, and like in Los Alamos, NM,8 the most common sources of sensitization are allergens from furred pets and pollens. In areas with a high proportion of pet ownership, high levels of pet allergens have been found in dwellings with or without furred pets, as well as in dust from different public environments.9-12 Many allergic children who are sensitized to cat allergen have not had cats at home and avoid direct contact with pets. Indirect exposure to pet allergens might, however, be of sufficient magnitude to induce or maintain symptoms in sensitized children.13 Schools and day care centers where many children are together in a small area are possible sites of indirect allergen exposure.14,15 Cat owners’ clothes have been proposed to be the major source of allergen in the classroom.16 Exposure to airborne pet allergens in environments where pets have been kept has previously been studied, but because of the very low levels of airborne allergens, few studies have been performed in pet-free environments.17 The aim of this investigation was to study levels and routes of contamination of cat allergen at school and to assess further dissemination of allergens to homes without cats. A recently developed amplified ELISA has made it possible to detect very low levels of cat allergen and to measure airborne levels of allergen in pet-free environments by using personal samplers.
METHODS Questionnaires All secondary schools in 2 suburban districts of Stockholm, consisting of both rural and urban areas, were identified. Headmasters and teachers in grades 4 and 5 (age of students, 10 to 12 years) were informed about the study. A questionnaire regarding possession of domestic animals and furred pets was distributed to all 2042 pupils and their teachers. The answers were confirmed and signed by one of the pupil’s parents. The teachers compiled and mailed all questionnaires from their classes. If a class did not respond, the teacher received 2 reminders.
Almqvist et al 1013
J ALLERGY CLIN IMMUNOL VOLUME 103, NUMBER 6
Airborne cat allergen Exposure to airborne cat allergen Fel d 1 was examined in the breathing zone of 5 children in each of 12 classrooms on 2 different occasions. Six classes with many cat owners and 6 classes with few cat owners (Table I) were selected from 7 different schools for geographic and practical reasons. There was a similar number of pupils in the classes (average, 25; range, 18 to 31). In each class one cat owner, 2 children sitting next to a cat owner, and 2 children sitting far away (>1.5 m) were identified. Personal air samples were obtained by using IOM samplers (SKC Inc) equipped with 25-mm polytetrafluoroethylene filters (pore size 1 µm; Millipore AB, Sundbyberg, Sweden). An airflow rate of 2 L/min over the filter was obtained with Aircheck 50 personal sampling pumps (SKC Inc). Air was sampled for 3 hours during class. Effective sampling time and average airflow were used to calculate total volume of air sampled. Airborne levels of cat allergen Fel d 1 were also sampled in the homes of 36 children by one family member.
TABLE I. Distribution of classes with few or many cat owners Classes
Cat owners
Range
No. of classes
Few cat owners Intermediate cat owners Many cat owners
<10% 10%-25%
3%-9% —
12 classes 53 classes
>25%
26%-63%
17 classes*
*Two
classes had greater than 50% cat owners (ie, 57% and 63%).
Mattress dust was stored at –20°C until extraction. Extraction (1/40 wt/vol) was made in PBS with 0.1% Tween 20 and 0.15% Kathon CG (Rohmand Haas, Hydrosupra Kemiservice, Helsingborg, Sweden) (PBSTK) for 2 hours at room temperature. Dust from clothes was immediately extracted in 20 mL of PBSTK and rotated for 2 hours at room temperature. Polytetrafluoroethylene filters containing airborne dust were left in the cassettes and kept at –20°C until extraction. The filters were extracted in 2 mL of PBSTK and rotated overnight at room temperature. All extracts were centrifuged twice (10 minutes × 1000g), divided into aliquots, and stored at –20°C until analysis.
were developed by adding 100 µL of KBlue (ELISA Technologies Inc). The blue color reaction was stopped after 20 minutes by the addition of 100 µL of 1 mol/L H2SO4. Endpoint yellow color was measured at 450 nm (minus 650 nm reference) on a ThermoMax reader by using SOFTmax version 2.3 for Windows software (Molecular Devices Inc). Clothes dust sample assay. Analysis of the amount of Fel d 1 in dust from children’s clothes was performed by essentially following the same ELISA protocol as for mattress dust. The following modifications were made. The concentration range of the standard curve was 0.039 to 2500 ng/mL. The in-house cat allergen control used had an assigned Fel d 1 concentration of 0.4 ng/mL. To achieve a higher sensitivity, an in-house prepared NeutrAvidin (ImmunoPure, product #31000; Pierce, Rockford, Ill) horseradish peroxidase conjugate (synthesized by Per H. Larsson) was used. Samples below 0.039 ng/mL were run in the amplified ELISA. Amplified assay. The analysis of air samples by an amplified ELISA was performed as above, with the following modifications. Coating with 6F9 was done by using 100 µL at 5 µg/mL in PBS overnight. Plates/strips were blocked for 1 hour by using PBS containing 1% BSA and then washed 4 times (throughout the assay). All dilutions were made in PBSTK containing 1% BSA, and 100-µL volumes were used in duplicates. Standards (2.5 to 160 pg Fel d 1/mL), samples, and an in-house control cat allergen extract (Fel d 1 at 40 pg/mL) were incubated for 90 minutes. After washing, plates were incubated with biotinylated 3EC4 at 1/1000 for 60 minutes, washed, and incubated for 30 minutes with an in-house streptavidin horseradish peroxidase conjugate (100 ng/mL). The amplifier NBiotinyl-4-Hydroxybenzohydrazone (13.5 µg/mL, molecular weight of 362 d; synthesized by Per H Larsson) in 0.1 mol/L borate/0.003% H2O2, pH 8.5, was added and allowed to react for 15 minutes according to the catalyzed reporter deposition concept.19 After washing, the enzyme conjugate was added and incubated as above. Readings were performed as for the dust sample assays. For all ELISAs, intraassay sample coefficients of variation less than 10% were accepted. Interassay coefficients of variation of controls less than 20% were considered acceptable. The results were expressed as micrograms of Fel d 1 per grams of dust for the mattresses, nanograms of Fel d 1 per garment for the clothes, and nanograms of Fel d 1 per cubic meter of air, respectively. Detection limits (as defined by the lowest standard point) of the assays were 25 ng Fel d 1/g mattress dust, 50 pg Fel d 1/g garment dust, and 42 pg Fel d 1/m3 of air at 60 minutes sampling time (amplified ELISA).
ELISA assays
Statistics
Mattress dust sample assay. Fel d 1–specific mouse mAbs 6F9 and biotinylated 3EC4 and cat allergen standard UVA 94/01 were purchased from Indoor Biotechnologies (University of Virginia, Charlottesville, Va),18 and ELISA was performed essentially as described in the accompanying instructions, with the following modifications. The coating antibody 6F9 was used at 10 µg/mL. The standard curve was 0.625 to 20 ng Fel d 1/mL and was run with an in-house control cat allergen extract (1.3 ng Fel d 1/mL). Plates
Levels of airborne cat allergen in each of the 12 classrooms were calculated from the mean values of the non-cat owners on 2 separate occasions. Values from cat owners were excluded. The median and 25-75 percentiles of the mean from classes with many or few cat owners were calculated. Comparisons were made by using the nonparametric unpaired Mann-Whitney U test. To compare the levels of airborne Fel d 1 in the breathing zone of cat owners with that of the children sitting next to or far from a cat owner, the nonpara-
Cat allergen in dust from clothes and mattresses Clothes were examined in 3 classes with many cat owners: 23%, 50%, and 56%, respectively. New T-shirts were purchased from a factory and delivered in sealed boxes. After school on day 1, all cat owners (n = 30) received a T-shirt to wear at home during the evening and all day at school on day 2. On the morning of day 2, 31 non-cat owners received a T-shirt to wear in class all day. All Tshirts were collected by the end of the school day. To compare the amount of cat allergen in T-shirts of cat owners before and after a school day, 15 of the cat owners received new T-shirts on day 2 to wear at home for an afternoon, and these were collected for vacuuming the following morning (day 3). Five unused T-shirts were vacuumed for reference. T-shirts were vacuumed in a standardized fashion16 with a vacuum cleaner equipped with a dust collector (Allergy Control Products Inc). The T-shirts were vacuumed on the front and back for 90 seconds each. Mattresses were vacuumed on the entire upper surface for 2.5 minutes with the dust collector. All airborne samples and dust samples from mattresses were obtained between March and June 1996, and the samples from the clothes were collected in May 1998.
Extraction of dust and air samples
1014 Almqvist et al
J ALLERGY CLIN IMMUNOL JUNE 1999
FIG 1. Levels of airborne cat allergen Fel d 1 in the breathing zones of non-cat owners in classes with many or few cat owners (P < .01). Box plots with medians marked are shown; box corresponds to 25-75 percentiles, and vertical lines correspond to 10% and 90% of the range, respectively.
metric paired Wilcoxon test was used. For comparisons of dust in mattresses and clothes, the nonparametric unpaired Mann-Whitney U test was used. Two-tailed tests were used, and P values less than .05 were regarded as statistically significant. The medians are shown in the results below, with the 25-75 percentiles in parenthesis. Permission for the study was obtained from the Ethics Committee of the Karolinska Institutet.
RESULTS The questionnaires were sent to 2042 pupils and teachers, and complete questionnaires were obtained from 1931 pupils (52% boys and 48% girls) and 83 teachers in the 85 school classes identified (response rate, 95%). One class had 2 teachers. Three classes did not respond despite reminders, and a few pupils in other classes declined participation or did not respond. Eighty-three percent of the children were between 10 and 11 years old, and 11% of the children were 12 years old. Fifty-eight percent of the children had domestic animals at home, and 43% owned furred pets (ie, cats, dogs, rabbits, or rodents). Eighteen percent of the children owned cats. The classes were divided into classes with many cat owners (>25%) or few cat owners (<10%) on the basis of less than 25 and greater than 75 percentiles of cat ownership (Table I).
Airborne cat allergen Airborne Fel d 1 was investigated on 2 separate occasions in 6 classes with few cat owners and in 6 classes with many cat owners. There was a 5-fold difference in the median level of airborne cat allergen between classes
with many cat owners compared with those with few cat owners (median [25-75 percentiles], 2.94 ng/m3 [2.374.77] vs 0.59 ng/m3 [0.45-0.81]; P < .01; Fig 1). In classes with few cat owners, the median level of sampled airborne Fel d 1 in the breathing zones of cat owners was 2.1 ng/m3 (1.53-12.0), that for children next to cat owners was 0.66 ng/m3 (0.40-1.14), and that for children far from cat owners was 0.52 ng/m3 (0.35-0.75). The cat owners were exposed to significantly higher concentrations of cat allergen compared with children sitting next to (P = .05) or far from cat owners (P = .05). Comparing non-cat owners sitting next to cat owners with those sitting far from cat owners, no significant difference in exposure to airborne levels of Fel d 1 was found (P = .17). In classes with many cat owners, the median level of sampled airborne Fel d 1 in the breathing zones of cat owners was 3.79 ng/m3 (1.80-13.9). Children sitting next to cat owners were found to have cat allergen levels of 4.09 ng/m3 (2.44-5.27) in their breathing zone, whereas children far from cat owners had levels of 2.77 ng/m3 (1.70-3.87). No significant differences were found between cat owners and non-cat owners nor between the different groups of non-cat owners (Fig 2). The levels of airborne cat allergen were significantly higher in classrooms (1.54 ng/m3 [0.59-2.94], n = 12) than in homes without a cat (0.15 ng/m3 [0.04-0.27], n = 26; P < .001) and significantly lower than in homes with a cat (19.8 ng/m3 [7.54-45.4], n = 10; P < .001; Fig 3). There was thus a 7-fold difference in the median values of airborne cat allergen in homes with cats compared
Almqvist et al 1015
J ALLERGY CLIN IMMUNOL VOLUME 103, NUMBER 6
FIG 2. Levels of airborne Fel d 1 in breathing zones of cat owners, children sitting next to cat owners, and children sitting far from cat owners in classes with many or few cat owners, respectively. Classes with few cat owners: Cat owner/near cat owner, P = .05; cat owner/far from cat owner, P = .05; next to cat owner/far from cat owner, P = .17. Classes with many cat owners, not significant.
with school classes with many cat owners. Whether the child attended a class with many or few cat owners did not affect the airborne levels of Fel d 1 in homes without cats.
Cat allergen in dust from clothes and mattresses The levels of Fel d 1 found in dust from vacuumed Tshirts are shown in Table II. The levels of Fel d 1 in the classes with 50% and 56% cat owners were higher in the evening than in the morning, whereas the opposite was found in the class with 23% cat owners, although this was not significant. After a day at school, the levels of Fel d 1 in the T-shirts of non-cat owners were significantly higher than those found in the unused T-shirts (P < .001). In dust from the children’s mattresses, the median levels of Fel d 1 for cat owners (n = 45) was 87.4 µg Fel d 1/g dust (29.4-199.4), and that for non-cat owners (n = 181) was 0.5 µg Fel d 1/g dust (0.2-1.4) (P < .001). The median levels of Fel d 1 in mattresses from homes without cats where the child attended a class with many cat owners (0.85 µg/g dust [0.32-1.64], n = 75) were significantly higher than if the child attended a class with few cat owners (0.38 µg/g dust [0.19-1.21], n = 106; P = .01).
DISCUSSION This study provides new data partly based on methods not previously available. There are 2 main findings. There is an association between the levels of airborne cat allergen and the number of cat owners in classes, with higher levels of Fel d 1 in classes with greater than 25% cat owners and lower levels in classes with less than 10% cat owners. The levels could be sufficient to influence the allergic inflammation in children with cat allergy.13,20 Airborne cat allergen levels were not strongly influenced by location in relation to a cat owner within the classroom; however, it was clearly shown that the clothes of cat owners were a potential source of cat allergen contamination in the classroom. The second main finding is that cat allergen is transferred from cat owners’ clothes to non-cat owners’ clothes. The transferral of allergens may either be by direct contact or by deposition of allergen elsewhere in the classroom, which is then picked up by non-cat owner’s clothes. This may contribute to the observation that children in classes with many cat owners have higher allergen concentrations in their mattresses at home than children in classes with few cat owners, thus providing new insights into the mechanisms of allergen dispersion in society.
1016 Almqvist et al
J ALLERGY CLIN IMMUNOL JUNE 1999
FIG 3. Levels of airborne Fel d 1 in breathing zones of non-cat owners in all classrooms compared with children in homes without cats (P < .001) and children in homes with cats (P < .001).
TABLE II. Median levels of Fel d 1 found in dust from clothes (median and 25-75 percentiles) Parameters
Cat owner morning, n = 15 Cat owner evening, n = 15 Cat owner total, n = 30 Non-cat owner, n = 31 Unused T-shirt, n = 5
Fel d 1/garment (ng)
2720 (700-12,950) 3970 (950-18,430) 3030 (900-15,620) 146 (86-234) 0.3 (0.2-2.2)
Cat owner total versus non-cat owner, P < .001; non-cat owner versus unused T-shirts, P < .001.
Airborne allergen levels The amplified ELISA method for airborne cat allergen Fel d 1 used in this study makes it possible to measure allergen levels even in pet-free environments and to compare allergen levels in the breathing zones of cat owners and non-cat owners despite the low levels. The levels of airborne Fel d 1 in homes with cats correspond well with what has been found in earlier studies.17,21,22 However, very few studies on the levels of airborne Fel d 1 in cat-free environments have been carried out. Woodcock et al23 concluded that Fel d 1 was undetectable in the absence of disturbance in homes without a cat. Bollinger et al17,24 found levels of 1 ng/m3 in cat-free environments, even with an amplified ELISA. The levels of Fel d 1 in homes without cats in our study were found to be as low as 0.01 ng/m3. Within each classroom, sitting next to or far from a cat owner was nonsignificant for the measured levels of cat allergen. In classes with few cat owners, the cat owners had significantly higher levels of Fel d 1 in ambient air
than did the non-cat owners, which could not be shown in classes with many cat owners. In classes with many cat owners, most pupils are likely to be sitting in the vicinity of a cat owner. In this study we chose an arbitrary distance of more than 1.5 m from a cat owner, but this may be too close to detect a gradient in concentration in the classroom, depending on distance from the cat owner. On the other hand, during the sampling of airborne cat allergen, teachers and pupils kept going in and out of the classroom, and the pupils rarely remained in their seats for a whole 40 minutes. It is also very likely that there are reservoirs of cat allergen in the classroom that contribute to the exposure. Therefore location within the classroom may have little influence on exposure levels. We have not found any previous study where this kind of comparison in exposure has been done.
Cat allergen in dust from clothes and mattresses The allergen levels found in dust from clothes indicate that cat allergen is carried to school in cat owners’ clothes and is there transferred to the clothes of children without cats. This has also been suggested in former studies.16,25 The schoolchildren without cats at home may bring the allergen home in their clothes and deposit it in their homes and beds. This hypothesis is supported by the levels of Fel d 1 found in dust from mattresses of children without cats, which were significantly higher among children attending a class with many cat owners than those attending classes with few cat owners. Our data, however, do not allow a distinction between the possibility that the allergen in the mattresses could be derived from the child’s clothes contaminated at school or from
Almqvist et al 1017
J ALLERGY CLIN IMMUNOL VOLUME 103, NUMBER 6
visiting cat owners’ clothes. It is likely that children who attend a class with many cat owners also meet more cat owners outside school than do children attending a class with few cat owners.
Clinical effects The levels of cat allergen exposure in classrooms found in this study may be high enough to worsen or maintain an inflammation in the mucous membranes of the airways and to elicit or aggravate symptoms of asthma.13,20 Previous studies have shown that Fel d 1 levels of 8 µg/g dust may cause sensitization and elicit asthma.26 The corresponding airborne levels are not known, but worsening of symptoms caused by inhaled cat allergen cannot be excluded at the levels of cat allergen found in classrooms in our study. The difference in exposure to cat allergen between classes with many or few cat owners is significant, but it is evident that children are exposed to allergens even when they are in a classroom with very few cat owners. This is important to families who try to avoid pets because of allergic disposition. Bollinger et al17 studied Fel d 1 in cat-free environments in relation to symptoms found in a cat challenge room for 1 hour, in which levels less than 500 ng/m3 were found to give rise to respiratory symptoms in individuals allergic to cats. To reduce cat allergen levels in schools, it would be of considerable interest to look into possible intervening measures, preferably to take steps to minimize the allergen source from the clothes of cat owners. Whether a child allergic to cats clinically improves if placed in a class with few cat owners compared with a class with many cat owners should also be investigated. The ethical and moral aspects should be considered when taking intervening steps, but our data imply a necessity to investigate health effects and to reduce exposure to pet allergens, which is also of interest from a health economics point of view. We thank André Lauber for expert computer assistance and Inger Kull and Siw Siljerud for sample collection. We also thank the children and teachers in the schools, without whose assistance this study would not have been possible.
REFERENCES 1. Åberg N, Hesselmar B, Åberg B, Eriksson B. Increase of asthma, allergic rhinitis and eczema in Swedish schoolchildren between 1979 and 1991. Clin Exp Allergy 1995;25:815-9. 2. Burr ML, Butland BK, King S, Vaughan-Williams E. Changes in asthma prevalence: two surveys 15 years apart. Arch Dis Child 1989;64:1452-6. 3. Croner S, Kjellman N-I M. Natural history of bronchial asthma in childhood—a prospective study from birth to 14 years of age. Allergy 1992;47:150-7. 4. Wickman M, Nordvall L, Pershagen G. Risk factors in early childhood for sensitization to airborne allergens. Pediatr Allergy Immunol 1992;3:128-33. 5. Wahn U, Lau S, Bergmann R, Kulig M, Forster J, Bergmann K, et al. Indoor allergen exposure is a risk factor for sensitization during the first three years of life. J Allergy Clin Immunol 1997;99:763-9. 6. Warner JA, Little SA, Pollock I, Longbottom JL, Warner JO. The influ-
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
25.
26.
ence of exposure to house dust mite, cat, pollen and fungal allergens in the home on primary sensitization in asthma. Pediatr Allergy Immunol 1990;1:79-86. Platts-Mills TAE, Vervloet D, Thomas WR, Aalberse RC, Chapman MD. Indoor allergens and asthma: report of the third international workshop. J Allergy Clin Immunol 1997;100(Suppl):S1-24. Ingram JM, Sporik R, Rose G, Honsinger R, Chapman MD, Platts-Mills TAE. Quantitative assessment of exposure to dog (Can f 1) and cat (Fel d 1) allergens: relation to sensitization and asthma among children living in Los Alamos, New Mexico. J Allergy Clin Immunol 1995;96:449-56. Wood RA, Eggleston PA, Lind P, Ingemann L, Schwartz B, Graveson S, et al. Antigenic analysis of household dust samples. Am Rev Respir Dis 1988;137:358-63. Egmar AC, Emenius G, Almqvist C, Wickman M. Cat and dog allergen in mattresses and textile-covered floors of homes which do or do not have pets, either in the past or currently. Pediatr Allergy Immunol 1998;9:315. Egmar AC, Almqvist C, Emenius G, Lilja G, Wickman M. Deposition of cat (Fel d 1), dog (Can f 1) and horse allergen over time in public environments—a model of dispersion. Allergy 1998;53:957-61. Custovic A, Fletcher A, Pickering CAC, Francis HC, Green R, Smith A, et al. Domestic allergens in public places III: house dust mite, cat, dog and cockroach allergens in British hospitals. Clin Exp Allergy 1998;28:53-9. Ihre E, Zetterström O. Increase in non-specific bronchial responsiveness after repeated inhalation of low doses of allergen. Clin Exp Allergy 1993;23:298-305. Munir AKM, Einarsson R, Dreborg SKG. Mite (Der p 1, Der f 1), cat (Fel d 1) and dog (Can f 1) allergens in dust from Swedish day-care centres. Clin Exp Allergy 1995;25:119-26. Smedje G, Norbäck D, Edling C. Asthma among secondary schoolchildren in relation to the school environment. Clin Exp Allergy 1997;27:1270-8. Enberg RN, Shamie SM, McCullough J, Ownby DR. Ubiquitous presence of cat allergen in cat-free buildings: probable dispersal from human clothing. Ann Allergy 1993;70:471-4. Bollinger ME, Eggleston PA, Flanagan E, Wood RA. Cat antigen in homes with and without cats may induce allergic symptoms. J Allergy Clin Immunol 1996;97:907-14. Chapman MD, Aalberse RC, Brown MJ, Platts-Mills TAE. Monoclonal antibodies to the major feline allergen Fel d 1. II. Single step affinity purification of Fel d 1, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d 1 exposure. J Immunol 1988;140:812-8. Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. J Immunol Methods 1989;125:279-85. Roquet A, Lagging E, Ihre E, van Hage-Hamsten M, Halldén G, Härfast B, et al. Induction of increased bronchial reactivity by repeated low-dose allergen exposure. APMIS 1998;106:293-9. Sakaguchi M, Inouye S, Irie T, Miyazawa H, Watanabe M, Yasueda H, et al. Airborne cat (Fel d 1), dog (Can f 1), and mite (Der 1 and Der 2) allergen levels in the homes of Japan. J Allergy Clin Immunol 1993;92:797802. Luczynska CM, Li Y, Chapman MD, Platts-Mills TAE. Airborne concentrations and particle size distribution of allergen derived from domestic cats (Felis domesticus). Am Rev Respir Dis 1990;141:361-7. Woodcock A, Taggart SCO, Custovic A. Airborne cat allergen [abstract]. J Allergy Clin Immunol 1995;95:326. Bollinger ME, Wood RA, Chen P, Eggleston PA. Measurement of cat allergen levels in the home by use of an amplified ELISA. J Allergy Clin Immunol 1998;101:124-5. Patchett K, Lewis S, Crane J, Fitzharris P. Cat allergen (Fel d 1) levels on school children’s clothing and in primary school classrooms in Wellington, New Zealand. J Allergy Clin Immunol 1997;100:755-9. Gelber LE, Seltzer LH, Bouzoukis JK, Pollart SM, Chapman MD, PlattsMills TAE. Sensitization and exposure to indoor allergens as risk factors for asthma among patients presenting to hospital. Am Rev Respir Dis 1993;147:573-8.