Dietary shifts have consequences for the repertoire of allergens produced by the European house dust mite.

Products manufactured from mass-cultured house dust mites, currently commercialized for the diagnosis and immunotherapy of allergy, are heterogeneous in terms of allergen composition and thus present concerns to regulatory authorities. The most abundant species, Dermatophagoides pteronyssinus (Trouessart) (Astigmata: Pyroglyphidae), produces 19 allergenic proteins. Many of these are putatively involved in mite digestive physiology and metabolism. This study aimed to evaluate the effects of mite-rearing media on allergen production. Mites were adapted to feed on culture media supplemented with proteins, lipids, carbohydrates or beard shavings, and collected to quantify major allergens (Der p 1 and 2) by immunodetection, transcription of allergen genes by real-time quantitative polymerase chain reaction, and allergen-related enzymatic activities. All culture media significantly affected the content of major allergens. Modification of macronutrients in the diet produced minor effects on the transcription of allergen genes, but significantly altered mite allergen-related activities. The most remarkable impacts were detected in mites feeding on beard shavings and were reflected in reductions in the content of major allergens, alterations in the transcription of nine allergen genes, and changes in eight allergen-related activities. These results demonstrate the importance of culture media to the quality and consistency of mite extracts used for pharmaceuticals, and highlight the need to further elucidate allergen production by mites in the laboratory and in domestic environments.

Effects of domestic chemical stressors on expression of allergen genes in the European house dust mite.

The expression of allergen genes in house dust mites is influenced by temperature and relative humidity, but little is known of the impacts of other environmental factors that may alter the repertoire of allergens released by mites in home microhabitats. Bioassays were conducted in concave microscope slides in combination with real-time quantitative polymerase chain reaction (RT-qPCR) to analyse gene expression of 17 allergens of Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) exposed to three chemical stressors that can be present in domestic environments. Short-term exposure (5-12 days) to diesel exhaust particles (DEPs) (1 µg/cm2 ), bacterial lipopolysaccharide endotoxin (0.1 µg/cm2 ) and benzyl benzoate (3.2 µg/cm2 ), at concentrations exceeding those expected in homes, had no significant effect on allergen transcription. A significant increase in the transcription of allergens Der p 3, Der p 8 and Der p 21 was observed only after exposing mites to a higher concentration of DEPs (10 µg/cm2 ) over a whole generation. In combination, the present results suggest that the analysed factors have low impact on allergen production. The methodology described here offers a sound and rapid approach to the broad-spectrum study of factors affecting allergen-related mite physiology, and allows the simultaneous screening of different factors in a relatively short period with consideration of the full spectrum of allergen genes.

Quality control of house dust mite extracts by broad-spectrum profiling of allergen-related enzymatic activities.

BACKGROUND: Diagnosis and immunotherapy of allergy against mites is based on complex extracts from large-scale cultures. However, the analysis of their composition using specific antibodies is limited. By taking advantage of the prevailing enzymatic nature of mite allergens, we have developed a broad-spectrum biochemical method for the standardization of native mite products. METHODS: Microplate-based assays have been implemented for thirteen Dermatophagoides pteronyssinus enzymatic activities, associated with Der p 1, 3, 4, 6, 8, 9, 15 and 20 allergens. The dynamics of these activities along culture growth, and their profile in purified fractions (bodies and faeces) and international reference standards (WHO/IUIS, two CBER/FDA), have been characterized. The stability of enzymatic activities and major allergens under stress conditions (40°C) has been assessed in the presence/absence of specific protease inhibitors. RESULTS: The analysis of enzymatic activities revealed distinct profiles along culture growth and between fractions (bodies and faeces). Remarkable differences were found when comparing international reference standards, being consistent with their source material (purified bodies or whole cultures). After 72 h at 40°C, only trypsin and alpha-amylase maintained high activity. Notably, the prominent role of trypsins in the hydrolytic degradation of major allergens is demonstrated by the use of inhibitors. CONCLUSIONS: Our method offers a robust approach to assess the complexity of mite extracts and highlights the critical importance of source materials for the composition and stability of finished products. The implementation of this approach in industry-based quality control procedures would contribute to the standardization of allergenic extracts used for diagnosis and immunotherapy.

Allergen expression in the European house dust mite Dermatophagoides pteronyssinus throughout development and response to environmental conditions.

House dust mites are a major source of allergy worldwide. While diagnosis and treatment based on mite extracts have remarkably advanced, little information exists on the expression of allergens in mites. We have studied gene expression of eight Dermatophagoides pteronyssinus (Trouessart) (Acari: Pyroglyphidae) allergens (Der p 1, 2, 3, 4, 5, 7, 10 and 21). All allergens showed higher transcription in nymphs compared with larvae or adults, with the only exception of Der p 10. The transcription of Der p 4 and Der p 10, together with the transcription and protein ratios Der p 1 to Der p 2, were higher in males than in females. One-week exposure of mite cultures to 16 or 35 °C (versus 24 °C) or low RH (44% versus 76%) significantly influenced the allergen gene transcription profile. Our results demonstrate that allergen expression is quantitatively and/or qualitatively influenced by mite development and sex, as well as by the environment. We suggest that monitoring allergen gene expression may be a useful tool to assist the optimization of mite cultures in the production of standardized allergenic extracts for clinical use.

Occurrence, characterization and insecticidal activity of Bacillus thuringiensis strains isolated from argan fields in Morocco.

Soils collected from five locations in the argan forest (an endemic plant) in Morocco were used to form the first collection of Bacillus thuringiensis (Bt) strains from this area (58 strains). Here we found that the argan forest is a major source of Bt, as 90.62% of the samples contained Bt strains. These strains produced mainly spherical or irregular crystals that in some cases remained adhered to the spore after cell lysis. There was no strain producing bipyramidal crystals, suggesting the absence of strains bearing crv1 genes. This was confirmed by PCR analysis using eight primer pairs that can potentially detect 13 different groups of cry and cyt genes. Strains containing cry7/8 were the most abundant (25.53%), followed by strains harbouring cry9A (14.89%), cry11 (8.51%) and cry4 (4.25%). The mixtures of spores and crystals as well as culture supernatants were assayed for toxicity towards Ceratitis capitata (Medfly), showing up to 30% mortality. Our findings suggest that the argan region is a suitable target for future and wider screening programmes looking for strains bearing toxins or combinations of them to develop more efficient Bt-based formulates.

Diversity of Bacillus thuringiensis strains isolated from citrus orchards in spain and evaluation of their insecticidal activity against Ceratitis capitata.

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDSPAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.