Molecular identification of house dust mites and storage mites

Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR–RFLP of ITS2 region. With the established PCR–RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR–RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.     

Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification

The identification of allergy‐causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full‐length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite‐specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species‐specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi‐nested re‐amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR–RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR–RFLP system and the rDNA sequences obtained can be of use in the identification of allergy‐causing mite species.     

Structural biology of mite allergens

Mite allergens contribute to a significant proportion of human allergic symptoms, including asthma and rhinitis. The development of therapies to treat and prevent these symptoms depends largely on our understanding of the properties of these allergens. Much effort has been devoted to determining the structure and organization of mite allergens, particularly of the house dust mites, toward understanding their activities and how they elicit immunological responses in humans. Here, we review the structural biology of the major allergens from two species of house dust mites, Dermatophagoides farinae and D. pteronyssinus, as well as allergens from a storage mite, Blomia tropicalis. The knowledge gained from the structural biology of these allergens will enable progress in producing novel, more effective treatments for mite allergies based on specific immunotherapy approaches.     

Clinical significance of dust mite allergens

Dust mites are an important cause of allergic diseases worldwide. The traces of Dermatophagoides farinae and Dermatophagoides pteronyssinus can be found all over the world, while Blomia tropicalis are more common allergenic mites in tropical areas. A variety of allergenic components in dust mites have been found, apart from the different positive rates of IgE reactions in dust mite allergic patients, their biological characteristics, effects on the innate immune system, and especially their distribution characteristics in patients are different. Studying the relationship between dust mite allergens and clinical significance will help for diagnosis of patients and formulation of corresponding Allergen Specific Immunotherapy.

     

Seasonal prevalence of allergenic mites in house dust of Kolkata Metropolis,India

During the past few decades, house dust mites have attracted worldwide interest among medical entomologists and acarologists because of their importance in causing nasobronchial allergic disorders in human beings. House dust mites are present throughout the year; however, their relative densities differ in different seasons and habitats. Because the prevalence of house dust mite allergen is important epidemiologically and clinically, detailed knowledge on the seasonal abundance of important allergenic mites is of great importance for better understanding of the pathogenesis of the disease. In view of this, a systematic survey was carried out on the prevalence of total mites and four common allergenic mites in the city of Kolkata for two consecutive years. Both bed and bedroom floor dust were collected separately from homes inhabited by asthmatic patients situated in different corners of the city on monthly basis from January 2004 to December 2005. The population levels of total mites and four common allergenic mites, namely Dermatophagoides pteronyssinus, D. farinae, Austroglycyphagus geniculatus, and Blomia tropicalis separately, were highest during the pre-monsoon period (March–May), irrespective of habitat, whereas densities were low in all cases during winter (December–February). The study indicates that season had the most significant effect on the relative abundance of house dust mites except Dermatophagoides farinae, irrespective of habitat.     

Detection of serine proteases in extracts of the domestic mite Blomia tropicalis

Previous studies have shown that the domestic mites Dermatophagoides pteronyssinus and D. farinae contain allergens with serine protease activity. These proteolytic allergens include trypsin, chymotrypsin, elastase, kallikrein, and C3/C5 convertase. However, it is not known whether the domestic mite Blomia tropicalis shares with other mite species the serine protease activities. The enzymatic activity present in extracts obtained from food-free B. tropicalis was investigated using specific substrates and inhibitors. Based upon the concentration response and inhibition profiles, and the digestion of specific substrates our data demonstrate that extracts from B. tropicalis exhibit several serine-protease-like activities. The enzyme activities detected in the B. tropicalis extracts are trypsin, elastase, chymotrypsin, kallikrein, C3/C5 convertase, and mast cell protease. Our results also demonstrate that kallikrein and C3/C5 convertase-like activities were not significantly affected by the α₁-antiprotease, a naturally occurring serine protease inhibitor which protects lung mucosa from the enzymatic action. These data strongly suggest that the Echymyopodidae mite B. tropicalis shares at least five serine proteases with members of other mite families, the Glycyphagidae and Pyroglyphidae. In addition, our data demonstrate the potential use of biochemical methods to detect serine proteases for evaluation of mite growth in vitro, or to detect environmental exposures to these enzymes. This revised version was published online in July 2006 with corrections to the Cover Date.     

The influence of household pets on the composition and quantity of allergenic mite fauna within Irish homes: a preliminary investigation

Allergenic mites are responsible for inducing hypersensitive reactions in genetically predisposed people worldwide. Mites in dust from 30 Irish homes with pets (dogs, n = 23; cats, n = 7) were compared with those in 30 homes without pets. House dust mites constituted 78% of all mites recorded, with Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) representing 57–72% of mites in furniture and mattresses in both home types compared with only 22% of mites in pet beds. Although storage mites accounted for just 13% of all mites recorded, they represented 46% of mites recorded in pet beds. Median levels of the dust mite allergen Der p 1 (µg/g) in dust samples from mattresses in homes without pets were significantly greater than in mattresses from homes with pets, reflecting the greater densities of D. pteronyssinus found in the former home category. Mite species richness was greater in homes with pets (17 species) than in homes without pets (13 species). This suggests that although the presence of pets can result in a wider variety of epidemiologically important mite species within households, increased competition among mite species may result in a more balanced mite fauna in the home, inhibiting the dominance of any one species and hence lowering allergen‐associated risks.     

Enzymatic analysis of Blomia tropicalis and Blomia kulagini (Acari: Echimyopodidae) allergenic extracts obtained from different phases of culture growth

The majority of important allergenic extracts from arthropods present enzymatic activity. This activity has been studied particularly in Dermatophagoides house dust mites because of its implication in the stability and immunogenicity of extracts used as tools for the diagnosis and specific treatment of allergic diseases. Extracts from cultures of Blomia tropicalis [van Bronswijk (1973a, b). Acarologia 15:477–489, 490–505] and Blomia kulagini (Zakhvatkin 1936) were used to study enzymatic profiles during three growth periods of the mite population: latency phase, maximum mite concentration during exponential growth, and drop stage. The activities of 19 enzymes were analyzed using the Api Zym system. The results show a large variety of enzymes. Some enzymatic activity was found to be (almost) exclusively attributable to mites. The activity levels of proteases, glycosidases and lipases overlapped with the growth curve. Only phosphatase activity showed no significant change during mite growth when compared with the culture medium. We suggest that the glycosidases (β-galactosidase, β-glucuronidase, β-N-acetylglucosaminidase, α-mannosidase and α-fucosidase) and proteases (leucine aminopeptidase and trypsin) may constitute suitable parameters for inclusion in the quality control process for the production of allergenic mite extracts, and may help define a new index for conducting environmental controls.     

NMR Structure and IgE Epitopes of Blo t 21, a Major Dust Mite Allergen from Blomia tropicalis

Blo t 21 is a paralogue of the group 5 allergen, Blo t 5, a major allergen from the dust mite Blomia tropicalis. Blo t 21 has moderate sequence identity (40.7%) to Blo t 5 and low to moderate cross-reactivity to Blo t 5. In B. tropicalis, the most prevalent and allergenic allergens are in the order of Blo t 21, Blo t 5, and Blo t 7. Here, we determined the NMR solution structure of Blo t 21, which represents the first structure of the group 21 dust mite allergen. The structure of Blo t 21 closely resembles the structures of Blo t 5 and Der p 5, comprising three anti-parallel α-helices arranged in a helical bundle. Using site-directed mutagenesis and specific IgE binding ELISA, Blo t 21 was found to contain both conserved and unique charged IgE epitope residues at the L2 loop region and on helix α3. Cross-inhibition assays confirmed that Blo t 21 has a low to moderate cross-reactivity with Blo t 5 and Der p 5 and represents a novel group of major allergen in B. tropicalis. In addition to group 5 allergens, Blo t 21 has also a low to moderate cross-reactivity with group 21 allergens from Dermatophagoides mites, confirming that B. tropicalis is a major and distinct source of dust mite allergens.     

Identification of house-dust mites

The term house-dust mite usually refers to those species of the mite family Pyroglyphidae, that are known to commonly occur, although sometimes regionally, in the dust of human dwellings. These species belong to five genera:Dermatophagoides, Euroglyphus, Hirstia, andMalayoglyphus. Related species ofDermatophagoides have the most world-wide occurrence, the commonest being:D. farinae, D. microceras, andD. pteronyssinus. A correct taxonomic identification of house-dust mites is very important, not only from a biological stand point, but also regarding the consequences of their respective allergenical properties. Several immuno-chemical studies revealed differences between the products of two hard to distinguish sibling species. A preliminary practical taxonomic key for the most common and important house-dust mites is presented.     

Do the microorganisms from laboratory culture spent growth medium affect house dust mite fitness and microbiome composition?

The interaction of house dust mites(HDM)and microorganisms is the key factor in the survival of these mites in human-made environments.Spent growth medium(SPGM)provides the rest of the dict,along with dead mite bodies and microorganisms.SPGM represents a source of microorganisms for the recolonization of mite food and the mite digestive tract.An experiment was performed to observe how adding SPGM to the HDM diet affects HDM population growth,the microbiome composition and the microbial respiration in microcosms.We analyzed American house dust mite(Dermatophagoides farinae)and European house dust mite(Dermatophagoides pleronyssinus)originating from control diets and diets treated with an extract of SPGM from 1-and 3-month-old mite cultures.The microbiome was described using 16S and 18S barcode sequencing.The composition of the bacterial and fiungal microbiomes differed between the HDM species,but the SPGM treatment influenced only the bacterial profile of D.farinae.In the D.farinae microbiome of specimens on SPGM-treated dicts compared to those of the control situation,the Lactobacillus profile decreased,while the Candinium,Staphylococ-cus,Acinetobacter,and Sphingomonas profiles increased.The addition of SPGM extract decreased the microbial respiration in the microcosms with and without mites in almost all cascs.Adding SPGM did not influence the population growth of D.farinae,but it had a variable effect on D.pteronyssimus.The results indicated that the HDM are marginally influenced by the microorganisms in their feces.     

How relevant are house dust mite-fungal interactions in laboratory culture to the natural dust system?

Both house dust and house dust mitesDermatophagoides pteronyssinus contained a wider range of fungi than laboratory mite cultures. In total, nine species of fungi were isolated fromD. pteronyssinus in house dust, and these included three xerophilic species (Eurotium amstelodami, Aspergillus penicillioides andWallemia sebi) commonly found in laboratory cultures ofD. pteronyssinus. It is concluded that mites do interact with a similar range of fungi in natural dust and in laboratory culture, but that the diversity of fungal species in the laboratory is reduced and the density of individual fungal species in culture exceeds that of house dust. In a second experiment, dust samples were incubated at room temperature with 75% relative humidity. The diversity of fungi invariably declined from up to 13 genera to the few species recorded in laboratory culture. This suggests that the dominance of xerophilic fungi in laboratory mite rearings is mediated primarily by low relative humidity, and the exclusion of air-borne spores.     

热带无爪螨体内特异性变应原定位

【目的】探讨热带无爪螨Blomia tropicalis特异性变应原在体内的定位。【方法】制作热带无爪螨石蜡切片, HE染色后光镜观察其内部形态结构。选用对尘螨过敏患者的阳性血清特异性IgE 抗体作探针,免疫组织化学染色分析其特异性抗原存在位置。【结果与结论】免疫组织化学染色可见热带无爪螨中肠、盲囊、结肠的肠壁和内容物及生殖腺等均有阳性反应。其中肠壁组织和肠腔内容物呈强阳性反应,提示肠为特异性抗原的主要存在部位。     

Population dynamics of mites of the family pyroglyphidae and micromycetes in laboratory cultures

Original data on the study of the population dynamics in allergenic house dust mites of the family Pyroglyphidae (Dermatophagoides pteronyssinus (Trouessart 1897) and D. farinae Hughes 1961) during long (37 weeks) joint cultivation with the micromycetes Aspergillus penicillioides Speg. in simple periodical cultures without addition of food (SPC) with different initial population density of mites are given. The micromycete A. penicillioides Speg., dominating in laboratory cultures of pyroglyphids, was cultivated in parallel without mites. It was found that during joint cultivation of A. penicillioides and mites, population dynamics of D. pteronyssinus and D. farinae in SPC depended on the initial population density of mites, which affected the duration of mite developmental stages and the degree of the maximal population density. Cultures of D. farinae developed more rapidly than cultures of D. pteronyssinus, independently of the initial population density (50 or 200 specimens per gram of the substrate). A high degree of the initial population density in both mite species resulted in the shortening of the lag-phase, in more rapid reaching of the maximal population density, and in the higher degree of the maximal population density. Population density of the fungus A. penicillioides did not depend on the presence of both mite species. On the basis of our own data and literary analysis, we assume that A. penicillioides can affect the ability of mites to explore the trophic substrate, the rate of the population development, and the degree of their maximal population density. Mites, in their turn, did not significantly affect the development of A. penicillioides in our experiments.     

A review of recent immunochemical studies ofBlomia tropicalis andEuroglyphus maynei allergens

Exposure to mites other thanDermatophagoides spp., particularlyBlomia tropicalis andEuroglyphus maynei, has been increasingly recognized as a cause of asthma. Positive skin tests and serum IgE antibodies toB. tropicalis have been reported in asthmatic patients from several areas of the world, including São Paulo (Brazil), Hong Kong and Tampa (Florida, USA). Analysis ofB. tropicalis extracts showed undetectable levels of the major Group I and Group IIDermatophagoides spp. allergens. Immunoabsorption experiments showed that most of the IgE antibodies toB. tropicalis ( 70%) reacted with species-specific allergens. Murine monoclonal antibodies toB. tropicalis could present antigens that were recognized by human IgG antibodies. Sensitization toE. maynei has been reported in Europe, North and South America and Australia. Analysis of four differentE. maynei extracts by ELISA and RIA showed thatE. maynei produces an allergen that is antigenically related toDermatophagoides Group I allergens. The amino acid sequence of this allergen (Eur m I) has recently been reported. Further identification and purification ofB. tropicalis andE. maynei allergens is required to develop specific assays for measuring these allergens in dust samples. This will make it possible to investigate the relationship between exposure toB. tropicalis orE. maynei and the development of sensitization and allergic disease.     

Interference in foraging behaviour of European and American house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) by catmint, Nepeta cataria (Lamiaceae)

The European and American house dust mites, Dermatophagoides pteronyssinus and D. farinae, have a huge impact upon human health worldwide due to being the most important indoor trigger of atopic diseases such as asthma, rhinitis and atopic dermatitis. Preceding studies have shown that the behavioural response of house dust mites towards volatile chemicals from food sources can be assessed using a Y-tube olfactometer assay. In the current study, we used this assay to investigate, for the first time, the ability of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), known to repel other ectoparasites affecting human and animal health, to interfere with the attraction of D. pteronyssinus and D. farinae towards a standard food source (fish flakes). Two distinct chemotypes (A and B), enriched in the iridoid compounds (4aS,7S,7aR)-nepetalactone and (4aS,7S,7aS)-nepetalactone, and the sesquiterpene (E)-(1R,9S)-caryophyllene, were used. Initial assays with a hexane extract of fish flakes (FF extract) confirmed attraction of mites to this positive control (P < 0.001 and P < 0.05 for D. pteronyssinus and D. farinae respectively), but when presented in combination with either N. cataria chemotype, tested across a range of doses (10, 1, 0.1 and 0.01 μg), decreasing attraction of mites to their food source was observed as the dose augmented. Our study shows that N. cataria, enriched in iridoid nepetalactones and (E)-(1R,9S)-caryophyllene, exhibits potent repellent activity for house dust mites, and has the potential for deployment in control programmes based on interference with normal house dust mite behaviour.     

House-dust mite sensitivity among rural and urban asthmatics of West Bengal, India: a comparison

House-dust mite allergy is a fairly common problem in West Bengal among individuals sensitive to dust inhalation. House-dust mites belonging to the genusDermatophagoides are abundant in the homes of asthmatic patients residing in urban as well as rural areas of West Bengal. The frequency of positive skin reaction to different dust-related allergens tested was higher (χ²=5.4777, df = 1;P < 0.05) among patients of urban areas compared with that among the patients of rural areas. Urban patients showed more frequent skin reaction towards cockroach allergen, while rural patients are more sensitive to hay-dust allergen and these are very much related to their local environmental conditions. Analysis of radioallergosorbent test (RAST) results against house dust (HD) and mites reveal that 73 and 90% patients of both urban and rural areas responded positively towardsDermatophagoides pteronyssinus (DP) andDermatophagoides farinae (DF) antigens, respectively. The present study indicates no significant difference in house-dust mite sensitivity and mite levels in homes among the rural and urban asthmatics of West Bengal, India as evidenced from the results of analysis of dust samples, allergy skin test and detection of mite-specific IgE antibodies by RAST.     

Modelling and Bioinformatics Analysis of the Dimeric Structure of House Dust Mite Allergens from Families 5 and 21: Der f 5 Could Dimerize as Der p 5

Abstract

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.     

Distribution and abundance of house-dust mites (Acarina: Pyroglycphidae) in Rome,Italy

For the first time in Rome, house-dust mite infestation was studied in 90 randomly selected houses. In each house, mite infestation was assessed in three sites: mattress, bedroom and living room. In total, 87.8% of the sampled houses were positive for dust mites. In the houses infested, 11.4% showed densities of >100 mites/g of dust, 15.2% registered densities between 50 and 99, and in the remaining houses (73.4%), the densities were between 1 and 49 mites/g dust. The percentages of infested houses were positively correlated with the relative humidity (RH) values (r=0.89,P=0.02). At the lowest range of RH (between 46 and 50), the infestation was 50% and at the highest range of RH (between 73 and 78) it was 100%. The mattress was significantly the most infested (71.1%) of the tested sites. Only wool and spring mattresses were infested, and they did not show any significant differences in mite concentrations.Dermatophagoides farinae was the most abundant species (53.1%), followed byGlycyphagus domesticus (34.5%),D. pteronyssinus (5.2%), andEuroglyphus maynei (0.2%);D. farinae was also the most frequent species (56.9%). The remaining specimens (7.0%) were predator species commonly found in houses. The prevalence ofD. farinae in Rome is discussed.     

Age structure and dynamics of house dust mite populations

Age structure—the relative numbers of eggs, immatures and adults—in populations of the house dust mitesDermatophagoides pteronyssinus andEuroglyphus maynei was investigated in four sequential monthly samples taken from mattresses in each of eight homes in Glasgow, Scotland. Additionally, age structure ofD. pteronyssinus was determined in samples taken bimonthly for 6 months from nine quadrats of a double mattress. It was found that although age structure varied considerably with time, forD. pteronyssinus in different homes the most common structure was one in which immatures were dominant, then eggs and then adults (31% of samples). Immatures or eggs were dominant in 75% of samples. ForE. maynei the age structure was quite different: the most common structure was one in which adults were dominant, then immatures and then eggs (69% of samples). In different quadrats of a double mattress, mean age structure ofD. pteronyssinus underwent a shift towards higher proportions of immatures and then eggs during the sampling period, which reflected the increase in population density detected during this period.Life and fecundity tables were constructed forD. pteronyssinus andE. maynei using previously-available in vitro data on fecundity and survivorship rates and hypothetical values based on means derived from a number of studies. From the tables the stable age distributions were calculated and compared with the age structures of the natural populations. It was found that mean age structure of natural populations ofD. pteronyssinus was fairly close to the predicted stable age distribution, but those ofE. maynei indicated the populations were in decline during the sampling period, a fact confirmed by abundance data. The concept that the rate of increase of house dust mite populations can be estimated by determining age structure of mites isolated from dust samples was explored using the hypothetical population parameters ofD. pteronyssinus. It was predicted that quite large differences in fecundity and mortality would not drastically alter the proportions of eggs, immatures and adults in stable populations.Eggs as components of the house dust mite population are considered seriously for the first time. Those ofD. pteronyssinus andE. maynei were identified and differentiated by allometry. It is stressed that forD. pteronyssinus, during the sampling period, half or more of the mites in a dust sample may be represented as eggs, and to ignore them is to deliberately make a less accurate estimate of population density than could be otherwise achieved.