Dr J. L. Anguita Unidad de Alergología Hospital Universitario Médico Quirúrgico Complejo Hospitalario de Jaén Avenida del Ejército Español 10 23007 Jaén Spain
Background: Sinapis alba (white mustard) is a entomophilic species included in the Brassicaceae family. To date it has not been related to allergic sensitization or clinical respiratory disease.
Methods: Twelve olive orchard workers had a history of rhinitis and/or bronchial asthma that occurred during control weed management and/or harvest, from January to March. They underwent skin prick tests (SPT) with S. alba pollen extract and a standard battery of aeroallergens. Sinapis alba pollen extract was prepared for performing quantitative skin tests, enzyme allergosorbent test and nasal challenge test (NCT). A portable monitoring station and an urban volumetric Hirst-type spore trap were used for the aerobiological study.
Results: Eleven patients suffered from rhinitis and bronchial asthma and one had only from rhinitis. All patients were sensitized to S. alba pollen extract, and they showed a positive NCT response. In the urban aerobiologic monitoring station the amount of S. alba pollen only exceptionally reached peaks of 21 grains/m3, whereas in the work environment peaks of 1801 grains/m3 were detected between 15 February and 7 April.
Conclusions: We demonstrate the existence of a new occupational allergen for olive farmers: S. alba pollen. We point out the importance of perform aerobiological sampling within the occupational environment for the detection and quantification of the allergenic source.
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Olive farming is a key element of agriculture in Andalucía, a Spanish region. There are a lot of different species of annual weeds which infest the olive orchard of Jaén, as it happens with some species of Brassicaceae family, like Sinapis alba (white mustard), a 20–100 cm weed with intensely yellow flowers (1). This species is very widespread in southern Europe and northern Africa, where it grows wild together with a great diversity of crops. It is also cultivated for obtaining white mustard. To date, it has not been related to allergic sensitization or clinical respiratory disease.
Weed control and harvesting are two critical activities in olive crop care. Both maneuvers, made by olive farmers, imply a narrow contact with olive orchard, at the time during which S. alba flowers (late winter to early spring). Like other Brassicaceae species, their pollen is prolate or subspheroidal, small to medium sized, trizonocolporate, isopolar, and radioisometric with a reticulate surface (2).
Patients and methods
Twelve patients fulfilled the following criteria: (i) they were olive orchard workers; and (ii) they had a history of rhinitis and/or bronchial asthma that occurred exclusively during control weed management and/or harvest during late winter (January–March). They showed sensitization without clinical relevance to another plants, which do not flower at the same time as S. alba.
The clinical data of the patients included age, sex, work-related symptoms, and atopic disease if any. The study was approved by the Medical Ethical Committee of our hospital and all participating subjects (or their parents if the subjects were younger than 18 years) gave their written informed consent.
Isolation of pollen
The isolation of pollen was carried out according to method described by Baer and Anderson (3). This method allows separating pollen grains with a purity >90% from waste remains of plant and flowers. The purity was determined by observing five random samples of isolated pollens under an optical microscope. Three hundred particles were counted from each sample, and the proportion between Sinapis pollen grains and other contaminated particles (e.g. flower remains) was calculated.
Preparation of the extract
Deffated S. alba pollen was extracted by magnetic stirring in phosphate buffer, clarified by centrifugation, filtered through 0.45 μm pore diameter membranes and dialyzed by ultrafiltration with 5 kDa cutoff. The dialyzed extract was sterilized through a 0.22-μm pore diameter membrane and freeze-dried (4).
Quantitative skin tests
Skin prick tests were performed in accordance with the recommendations of the European Academy of Allergology and Clinical Immunology (5). Skin tests were carried out with S. alba pollen extract and a routine battery of aeroallergens of our area (Lolium perenne, Olea europaea, Artemisia vulgaris, Chenopodium album, Salsola kali, Parietaria judaica, Plantago lanceolata, Dermatophagoides pteronyssinus, Alternaria alternata, dog and cat danders) (Lab Bial, Bilbao, Spain).
Sinapis alba lyophilized pollen extract was diluted in phenol glycerol saline solution at concentration of 0.002, 0.02, 0.2, 2 and 20 mg/ml and prick test with standardized lancets were performed. Histamine chlorohydrate at 10 mg/ml and phenol-glycerol saline solution were used as positive and negative controls, respectively. After 15 min, cello tape was applied over the wheals and the areas transferred to a blank record sheet (areas expressed in square millimeters).
The mean wheal area produced by each allergen concentration was plotted as a function of the allergen concentration in a log-log system and linear regression was performed. The allergen concentration eliciting a wheal equal to that produced by histamine (10 mg/ml) was defined as one histamine-equivalent prick (HEP) unit (6). One HEP unit of S. alba pollen extract was found to be equivalent to 1.4 mg/ml.
Nasal challenge tests
Nasal challenge tests (NCT) were performed in 12 patients with work-related respiratory symptoms and positive skin tests to S. alba pollen, and in eight subjects from the control group. This study was carried out in 2004, outside of the S. alba pollination period, at 9 am after adaptation to room temperature for 30 min, according to the method described by Gosepath et al. (7).
The response was evaluated by measuring nasal resistance with active anterior rhinomanometry and by scoring the clinical symptoms. Total symptom scores represented the sum of the scores for: sneezing (0–2 sneezes: 0 points; 3–5: 1 point; >5: 2 points); rhinorrhea (moderate: 1 point; severe: 2 points); tearing, itching (eyes, throat): 1 point; conjunctivitis, cough, urticaria and/or dyspnea: 2 points. Positive clinical challenge was defined as >3 points (7).
Active anterior rhinomanometry was performed according to the criteria of the Committee Report on Standardization of Rhinomanometry (8). A rhinospir 164 rhinomanometer (Sibelmed, Barcelona, Spain) programmed to perform nasal provocation test was used. Airflow and resistances were recorded in an X-Y mirror image.
After spraying 0.2 ml of the diluent, increasing concentrations of allergen (0.002, 0.02, 0.2, 2 and 20 mg/ml) were sprayed into the same nostril every 15 min until symptoms appeared and resistances doubled those induced by the diluent.
Specific IgE measurement
Specific IgE was measured by means of the enzyme allergosorbent test (EAST) against S. alba pollen. The solid phase was obtained by coupling the extract solution (10 mg/ml) to the 6-mm diameter cyanogen bromide-activated paper disks as described by Ceska and Lundqvist (9). EAST was performed in accordance with the manufacturer's instructions (Specific IgE EIA Kit; HYTEC.HYCOR Biomedical Ltd, Penicuik, UK).
A volumetric Hirst-type spore trap was used (10) for aerobiological sampling in the urban environment. This trap was placed 15–25 m above the ground level, ensuring free air circulation. We used a portable monitoring station (VPPS 1000 Lanzoni; Lanzoni s.r.l., Bologna, Italy) for the camp sampling, into an olive crop which contains high densities of S. alba. Sampling was carried out in a continuous way during the whole flowering period of the plant population during 2003.
Daily pollen counts were made using the methodology proposed by the Spanish Aerobiology Network (11) and the data are expressed as number of pollen grains per cubic meter of air (grains/m3).
The clinical features of the 12 patients are summarized in Table 1. They showed symptoms between January and March, exclusively in the work environment: five of them during weed control management and seven during harvesting.
Table 1. Clinical characteristics and specific anti-Sinapis alba IgE class of 12 patients sensitized to S. alba pollen
They had positive results in skin prick tests (SPT) and NCT to S. alba pollen extract. The symptom scores and functional alterations during NCT are summarized in Table 2. Specific IgE to S. alba pollen was positive (>0.35 kU/ml) in all patient sera (Table 1).
Table 2. Nasal challenge test results in 12 patients with Sinapis alba sensitization
Negative control resistance*
S. alba pollen extract concentration- positive NCT†
The seasonal variation of the daily production of S. alba pollen during the study period is shown in Fig. 1. The total grain count of S. alba pollen during its main pollen season (31 days: between 15 February and 15 April) was 29.994 (with a mean value of 968 grains/m3) in the portable monitoring station placed in S. alba community, and 81 (with a mean value of 3 grains/m3) in the urban monitoring station.
The highest amounts of pollen are released early in the morning (09:00–13:00), when floral anthesis occurs, whereas during night hours (23:00–07:00) the pollen emission to the atmosphere is suppressed (Fig. 2).
We have demonstrated the existence of a new occupational allergen: S. alba pollen in olive farmers.
We demonstrate that exposure when working directly in olive orchard can be 300-fold higher than that observed in the general population. This broad difference probably reflects the aerobiological behavior of an entomophilic plant, with a limited pollen liberation rate into the atmosphere and in which the daily pattern of pollen emission may vary throughout the day (12). Other authors have also suggested the need of this aerobiological sampling of the patient's environment, with portable devices, for the detection and quantification of proximity aeroallergens, in some species of the Brassicaceae family (13).
After suspecting an occupational disease and precisely measuring the nature and timing of exposure in olive orchard workers, the diagnosis must be confirmed in each worker through inhalation challenges. Reports about the allergenicity of Brassicaceae pollen are limited, but they have applied this same diagnostic stepwise approach. Diplotaxis erucoides pollen has been identified as a relevant source of aeroallergens with capacity to induce IgE-mediated sensitization in agricultural workers, directly involved in viniculture, with clinical respiratory symptoms and occupational sensitization. These patients showed sensitization (SPT and specific IgE) to D. erucoides pollen and rhinoconjunctival symptoms after nasal inhalation challenges (14).
Thus, we have demonstrated the IgE-mediated nature of these reactions by means of the positive SPT and the positive specific anti-S. alba IgE. Specific inhalation challenge with active anterior rhinomanometry confirmed that respiratory allergy was caused by S. alba pollen, eliciting an immediate clinical and physiologic response.
We conclude that S. alba pollen is a potential cause of IgE-mediated occupational respiratory disease. Epidemiologic studies are necessary to assess the importance of this aeroallergen among exposed olive orchard workers and among the general population living nearby S. alba-growing areas.