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Health effects of predatory beneficial mites and wasps in greenhouses

8 Exposure estimation and individual sensitivity

• 8.1 Background
• 8.2 Material and methods
  • 8.2.1 Sampling strategy
  • 8.2.2 Sampling methods
  • 8.2.3 Analytical methods
• 8.3 Results
  • 8.3.1 Individual sensitivity
• 8.4 Discussion and Conclusions

8.1 Background

In the test of HR (see chapter 6), the predator which showed the strongest exposure response relationship was Amblyseius cucumeris. An exposure study was therefore set up for this.

Several methods can be used to determine the allergenic content in sample material (foods & dust). Usually the determination is based on RAST (Radio Allergo Sorbent Test) or EAST (Enzyme Allergo Sorbent Test) – inhibition. These methods are based on the fact that IgE-allergen binding in a solid phase system is inhibited by addition of free allergen. Based on the standard inhibition curve using known amount of allergen the inhibitory capacity in an unknown sample can be detected. In some cases it is time-consuming and laborious to develop a reliable inhibition assay due to a less reliable IgE      determination for the allergen in question or interfering compounds in the sample material. Another problem may be lack of sufficient sample material or high tittered IgE sera.

An alternative to the above mentioned assays is to perform a biological determination of the allergen in a sample. This can be done in vitro by measuring allergen induced histamine release from human basophiles sensitized with a high tittered IgE serum. These passively sensitized basophiles will release histamine by incubation with different concentrations of the allergen and a dose-response curve can be established. A sample containing unknown amount of the allergen in question will induce histamine release and the biological concentration can be established from the standard curve. This method is very sensitive, provided the access to high tittered sera, and it is relatively unaffected by contaminating material.

In the present study we used allergen induced histamine release from passively sensitized basophiles.

The finding of several high tittered sera specifically directed against A. cucumeris in the previous histamine release screening (see chapter 6) made the investigation possible.

8.2 Material and methods

In a large greenhouse firm A. cucumeris has been used routinely for several years and is actually applied in their R&D department and in their production of mother plants of Campanula.

The mites, product name Amblyline cu CRS were supplied from GARTA A/S, produced by Syngenta Bioline, Little Clacton, Essex, UK and delivered the day before appliance.

A. cucumeris was applied in small letters (sachets) with an average of 1.000 mites in each including some fodder. The letters are then placed with interval among the plants.

In the R&D department the mite letters are put with a high density, about one letter per 50 cm on normal growth tables. They are renewed about every thirty days. The R&D department is situated in a series of small older greenhouses. Three to four persons are continuously working in the area with a high plant contact, handling and controlling the plants. Two persons handled the sachets. On the same day the bug Orius was applied.

Figure 8.1. The sachets with Amblyseius cucumeris were placed in dishes and placed on each table. (Photo J.Bælum)

The production of mother plants is situated in two glasshouses of about 10,000 m² on mobile tables. Tasks are sorting, irrigating, and controlling.

The production of sales plants where no mites are applied takes place in a large greenhouse. Plants on mobile tables are automatically moved around in the house. A number of persons work here packing and controlling the plants.

8.2.1 Sampling strategy

Three areas have been taken out:

1.       the R&D glasshouses,

2.       the production of mother plants,

3.       a control area, the packing area for the normal production of Campanula, where A. cucumeris is not used.

The study was carried out from May 31 to June 2. In the production of mother plants and in the R&D department A. cucumeris was applied on June1 thereby giving possibility for a pre day and a post day.

From the logger of the climate computer the average outdoor temperature on the three days was 12.5° 11.5°, and 11.7°C, respectively while the temperature during the workday (07:00-16:00) was 15.5° 13.2°, and 14.6°C. 5 % of the time it was sunny and there was no rain.

In the greenhouses temperatures ranged between 19 and 21°C. The relative humidity was 57-67 % in the packing area and 62-78 % in the other areas.

Table 8-1. The program for the sampling of antigen against Amblyseius cucumeris.

8.2.2 Sampling methods

Sampling of the inhalable dust fraction was done with IOM Personal samplers and SKC portable pumps type 224-5 (SKC, Dorset, UK, www.skcinc.com) equipped with 25 mm Teflon-filters with an average pore size of 1,0 μm (MFS, Frisinette, Ebeltoft). The flow was 2,0 l/min and sample time 3-4 hours excluding pauses or other stays outside the production area.

8.2.3 Analytical methods

In the study we determined allergenic activity in 25 filters, three different batches of A. cucumeris (in the original bags), labelled: 22 14 6; 17 106 17 & 17 106 (1-6-06) and one sample from a dish. Unexposed filters were included as blanks.

Standard A. cucumeris-extract was a mixture of crude A. cucumeris (21 μg/ml) and pure A. colemani extract (5 μg/ml). Filters and cassettes were weighed after acclimatization, before and after sampling at Department of Environmental and Occupational Medicine, Aarhus University. Filters were sent to RefLab, Copenhagen. Samples were then extracted in pipes buffer and tested against sera from: 1) two persons with a clear reaction to A. cucumeris and 2) two persons with no reaction to A. cucumeris but reaction to P. persimilis, which has not been in use at the actual greenhouse.

Five high titter sera (HR-Test = class 5) against A. colemani from five different persons were included.

8.2.3.1 Extraction of material from filters

Before and after extraction each filter was examined by eye and visible colour of the filter was noted as 0; (+); + and ++. There was no change in the colour after 3 hours extraction and the extraction was therefore continued for another 69 hours, but no change in colour was observed. This crude inspection indicates that the extraction is far from complete.

Each of the 25 filters and one unexposed were cut into four pieces and extracted 3 hours in 3 ml Pipes buffer at room temperature. After the three hour extraction period a sample of the extract (500 μl) was removed. The extraction was then continued for 72 hours at 4º C.

8.2.3.2 Extraction of A. cucumeris from original bags

Samples from each of the three different bag-batches were extracted in pipes buffer (100 mg/mL) for one hour at 4º C. Then the samples were centrifuged and the supernatants used in the experiments.

8.2.3.3 Histamine release method

Blood bank buffy coat basophiles were used in the study. The basophiles were screened for activity against 10 inhalation and 10 food allergens and Anti-IgE. Only cells showing no histamine release response to the 20 allergens and a good release (> 30 %) to Anti-IgE were included in the study. Before sensitizing the cells with the above mentioned sera, IgE from the basophile surface was removed by a short exposure to low pH.

The IgE deprived cells were passively sensitized with the above mentioned sera for 60 min. at 37º C. The passively sensitized cells were incubated with extracts from the 25 filters, one unexposed filter, and the three samples as well as the standard A. cucumeris sample. All extracts and samples were tested in 6 concentrations by 3.5 fold dilutions (the standard A. cucumeris sample was used in concentrations from 26 to 0.26 μg/mL). Control experiments using cells sensitized to healthy non allergic sera were included.

8.3 Results

The amount of dust on filters varied considerably in a non-predictable manner.

No significant reactions were found when sensitized basophiles were incubated with the 3 hour extracts. The standard A. cucumeris sample showed a class 6 reaction (release already at 0.26 μg/mL) corresponding to and hereby confirming common allergenicity with the samples from the large screening study.

Some of the 72 hour filter extracts showed however reactions. As can be seen from table 8-1 there is some concordance to positive reactions in the histamine release and the amount of dust determined by weighing the filters.

The three crude samples were potent inducers of histamine release from the passively sensitized cells. By extrapolation from the release curve induced by the standard extract the allergenic material constituted about 5-10 % of the samples. In the three original batch bags the content was 8-10 % allergen content (w/w) while extraction from the dish indicated about 15 % allergen content (w/w).

Table 8-2. The results of the measurements of antigen against Amblyseius cucumeris in air.

8.3.1 Individual sensitivity

There were only 4 filters with quantifiable antigen concentrations so there was no possibility of testing individual sensitivity to these.

8.4 Discussion and Conclusions

Our results indicate that it is possible to determine allergenic material from A. cucumeris in the filters. The extraction efficacy from the filters seem however to be low. This is indicated by the visual inspection and the relatively low histamine release responses observed in the filter extracts where the allergenic material constitutes varying amounts from 0.1 to about 10 % of the actual material deposited on the filters.

On the other hand, the results show that high concentrations of antigen were found in the samples of material, in both fresh and old sachets as well as dust from the dishes. This implies that an airborne exposure is relevant.

Further studies are needed to establish a more efficient extraction method to enhance the sensitivity. It is also possible that the use of less hydrophobic filters will allow a more efficient extraction. It should be noted that allergens eliciting an IgE mediated reaction are almost exclusively water soluble. This is indeed demonstrated in this study where crude material extracted with buffer releases up to 15 % allergenic material on a weight to weight basis.

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Version 1.0 August 2007, © Danish Environmental Protection Agency