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Background: Prototype nasal filters were developed to collect inhaled pollen. This study evaluated the efficacy of the filters for prevention of rhinitis symptoms during acute outdoor pollen exposure., Methods: A randomized double-blind design was used. Subjects (n=46) with a history of autumn exacerbation of rhinitis and positive skin test to ragweed, Bermuda and/or Bahia grass wore either active or placebo nasal filters for 2 h in autumn in a park containing these species. Major and Total Symptoms scores were recorded at 0, 30, 60, 90 and 120 min., Results: Subjects wearing active nasal filters had significantly reduced scores, at all time-points compared with placebo group (all P <0.05). Of 14 individual symptoms measured, seven were significantly reduced (number of sneezes, runny nose, itchy nose, sniffles, itchy throat; itchy eyes and watery eyes) and another three showed a trend towards lower severity. The nasal filters also enabled the resolution of existing symptoms. Maximal difference in symptoms was seen immediately after subjects had spent 20 min sitting beside a large patch of ragweed., Conclusion: This is the first clinical trial of a nasal filter. The results suggest it has potential for enhancing rhinitis management during acute allergen exposure.

Background: Assessment of personal exposure to dust mite allergen has relied on proxy measures. Only recently has a means to directly measure inhaled allergen particle number become available (the intra-nasal air sampler)., Objective: To quantify inspired dust mite group 1 and group 2 allergen-bearing particles in bed in undisturbed conditions prior to sleep by nasal air sampling and to investigate the relationship between inhaled particles and reservoir allergen levels., Methods: Twelve volunteers wore nasal samplers in bed for 6 evenings, nose-breathing in undisturbed conditions. Allergen-bearing particles ('halos') were detected by immunostaining for Der p 1, Der p 2, or Der p 1 and Der p 2 together, and counted by light microscopy. Count data were square root transformed for analysis of variance. Mattress dust samples were assayed for Der p 1 and Der p 2 concentrations., Results: Square root detransformed mean particle counts per 30-min sample were: Der p 1, 4.22; Der p 2, 5.9; Der p 1 + Der p 2, 4.87; and for all samples, 5.01, with no difference between the groups. With replicate samples, halo number correlated significantly with mattress allergen concentrations (Der p 1 r = 0.80, P < 0.01; Der p 2 r = 0.68, P < 0.02)., Conclusion: Nasal air sampling can be used to quantify nocturnal Der p exposure in undisturbed conditions in an area with moderate exposure to mite allergen and can provide a direct measure of inhaled mite allergen. The choice of either Der p 1 or Der p 2 is appropriate for this purpose.

Background: IgE-mediated responses to natural rubber latex allergens have become a major health problem among recognized risk groups., Objective: The purpose of this investigation was to measure the amounts of Hevea brasiliensis latex allergen (Hev b 1) inhaled and deposited on surfaces when latex or vinyl gloves were worn and compare the results with the conventional measures (breathing zone samplers) of occupational exposure., Methods: Hev b 1 exposure was measured by nasal sampling and breathing zone sampling. Latex allergen exposure was generated by having each subject don a pair of powdered latex examination gloves and continuing his or her normal daily activity for 30 minutes. By means of adhesive tape, surface dust samples were collected from the surfaces of gloves, the subject's hands, and work areas. Sampling was performed with subjects wearing no gloves, subjects wearing powdered vinyl gloves, subjects wearing powdered latex gloves, and nearby colleagues wearing latex gloves. All samples were assayed through use of the HALOgen assay (Inhalix, Sydney, Australia) with a Hev b 1-specific mAb. Particles transporting latex allergen were identified by a surrounding immunostain halo, and these were quantified and reported as total numbers of particles inhaled, airborne, or found on surface areas evaluated., Results: Study subjects inhaled 26 times more allergen when powdered latex gloves were worn than under the "no glove" and powdered vinyl glove conditions. During the same period, Hev b 1 particle levels measured in the ambient air through use of the breathing zone sampler increased by 24-fold. The median numbers of particles carrying Hev b 1 allergen per square centimeter on the surface of the hands after the wearing of latex and vinyl gloves were 1964 and 5, respectively. Latex allergen was physically associated both with cornstarch granules and with larger dust particles having a darker, more irregular appearance., Conclusion: In a laboratory where gloves are worn for protection, the use of latex gloves resulted in a 26-fold increase in inhaled latex allergen over background levels measured while vinyl gloves were worn as controls. Low levels of latex exposure also occurred when vinyl gloves or no gloves were worn; the reasons for this are under investigation.

Background: Clothing has been proposed as an additional source of exposure to mite and cat allergens. Dispersal of allergen into public places has also been attributed to clothing., Objectives: We sought to study the contribution of various types of clothing on mite and cat exposure in a domestic environment. Also, we studied the ability of clothing to transfer allergen in a workplace., Methods: Personal exposure to mite and cat allergen from a range of clothing was measured by using intranasal air samplers in 11 homes. Five categories of clothing were tested. Wearing no upper clothing was the sixth category tested to distinguish the contribution of clothing over ambient background exposure. An adhesive tape was used to sample allergen from the surface of clothing, and reservoir dust samples were also collected. The above techniques were also used in the workplace to examine the amount of cat allergen transferred from cat owners to non-cat owners., Results: The amount of mite and cat allergen inhaled differed among the clothing types worn and whether they had been washed recently. Wearing a woolen sweater increased personal allergen exposure to cat and mite allergen by a mean of 11 and 10 times, respectively. Clothing items that were less frequently washed carried more allergen whether assessed by vacuuming or sampled with adhesive tape. This corresponded to the amount of allergen inhaled. We also found that cat levels on non-cat owners' clothing increased significantly at the end of a working day, which lead to the increase in their personal allergen exposure to cat., Conclusions: These studies strongly support the emerging model that personal clothing is an important source of both mite and cat allergen exposure. This article also demonstrates the importance of clothing as a means of distributing cat allergen into cat-free environments.

Background: Exposure to outdoor allergens has commonly been estimated by collecting airborne particles with a Hirst-type spore trap and then using morphologic criteria to identify the intact pollen grains and fungal spores that are recognized as allergen sources. Several antibody-based blotting or fixation methods have also been developed that enable the counting of amorphous airborne particles carrying allergen, but none of these methods allow the ready association of the released allergen with the morphologically identifiable particle of origin. A method has been developed that uses pressure-sensitive adhesive tape to sample the airborne particles and then allows the immunoidentification of the specific particles that are the allergen sources., Objective: Our purpose was to visualize and immunostain the particles carrying pollen allergen that are collected with a volumetric spore trap., Methods: A Burkard sampler was used to collect airborne particles onto pressure-sensitive adhesive tapes. The particles were permanently fixed between the tape and a protein-binding membrane when the tape was laminated with the membrane. Allergens that elute from the particles onto the membrane were detected with a range of antibodies. Both the particle and associated immunostained allergen were viewed through the transparent tape for final microscopic identification., Results: Polyclonal and monoclonal antibodies and IgE from allergic patients stained allergens in the periphery of particles collected on the tapes. Individual pollen grains and paucimicronic particles were seen with halos of immunostained allergen surrounding them. When IgE was used, the density of immunostaining in the halo surrounding Lolium perenne pollen grains was found to be proportional to the level of Lolium-specific IgE. The method is highly sensitive and can be used to detect different airborne particles that carry allergen. Both the particle and the immunostaining can be subjected to a range of simple measurement techniques., Conclusion: Individual particles carrying allergens and antigens were visualized. These particles included intact pollen grains, paucimicronic particles, and fungal spores.

Background: Cockroach allergen is recognized as a causal factor for asthma. However, airborne cockroach allergen has not been detected in undisturbed conditions, and therefore the behavior and properties of airborne cockroach allergen have been poorly characterized. A new aeroallergen sampling method and sensitive system of immunoassay have been used to examine cockroach allergen exposure., Objective: Our purpose was to measure and characterize airborne cockroach allergens during normal domestic exposure in the homes of Sydney, Australia., Methods: Air sampling with Institute of Occupational Medicine, Edinburgh (IOM) samplers was performed in the living rooms of 10 houses during low- and no-disturbance environments. In addition, inhaled particles were collected by each home occupant during low domestic exposure with use of intra-nasal samplers that impact particles onto an adhesive surface. The particles collected on the IOMs and the intra-nasal samplers were immunostained with Bla g 1 monoclonal antibodies. Particle size, morphologic characteristics, and the relative Bla g 1 content of particles were estimated. Reservoir dust samples from the kitchen, living room, and bedroom were assayed by an ELISA. Two forms of repeatability of IOM air sampling were examined. The first measure tested the repeatability of 2 IOM samples collected simultaneously in the same room during low- and no-disturbance activities. The second measure examined the repeatability of IOM sampling over time on 10 consecutive days., Results: Bla g 1 was detected in reservoir dust samples taken from all homes (geometric mean 1.5 U/g, range 0.2-9.4 U/g). Inhaled particles containing Bla g 1 were detected during 1 hour of intra-nasal sampling in 8 of 10 homes during low disturbance. Cockroach particles were detected on all of the IOM samples collected for both 4-hour low-disturbance and overnight no-disturbance sampling environments. Particles containing Bla g 1 collected with the IOM samplers during low disturbance ranged in size from 3 to 350 microm. These particles are amorphous and irregular in shape, and a majority of the large particles were described as flakes (flat, transparent particles) and fibers (threadlike). A relationship was demonstrated between the allergen content of cockroach particles and their particle size. The larger particles elute more Bla g 1. The coefficient of repeatability for measurements made during low and no disturbance was 3.62 and 2.09, respectively. For measurements repeated over time at the same site, the coefficient of repeatability was 2.61. This represents the fold range within which 95% of pairs of measurements made at an interval of 1 day would be expected to lie., Conclusions: Airborne cockroach allergen is present in both undisturbed and low-disturbance environments in homes with relatively low reservoir levels of Bla g 1. In agreement with previous reports, airborne particles containing cockroach allergen (Bla g 1) are mainly associated with particles >10 microm. These particles are amorphous and irregular in shape and can be described as flakes and fibers.

Background: Measurement of personal exposure to Der p 1 aeroallergen has previously been limited by the low quantity of material collected by sampling systems and the assay sensitivity. This has meant that exposure could only be detected if long sampling periods were used or reservoir dust was artificially disturbed. We have developed a sampling method to sample true personal exposure and combined it with a novel method which is sensitive enough to measure allergen exposure over shorter time frames., Objective: To describe normal domestic exposure to dust mite allergen during a range of activities in houses in Sydney, Australia., Methods: Inhaled particles containing mite allergen Der p 1 were collected using a nasal air sampler which impacts particles (> approximately 5 microm) onto a protein-binding membrane coated with a thin, porous, adhesive film. The allergen is bound to the membrane in the immediate vicinity of the particle and detected by immunostaining with monoclonal antibodies specific for Der p 1. In addition, samples were collected using a standard Institute of Occupational Medicine (IOM) personal air sampler and the amount of eluted Der p 1 was assayed by ELISA., Results: The median number (range) of inhaled particles containing Der p 1 collected in each 10-min sampling period was: dust raising 5 (2-10); lying in bed, 0 (0-2); sitting on the bed, 1 (0-2); walking around the bedroom, 0 (0-2). This represented 0-5.1% of all particles captured. The Der p 1 concentration of floor and bed dust was 19.4 and 55.1 microg/g, respectively. The standard IOM personal sampler and ELISA were unable to detect Der p 1 for any of the activities performed., Conclusions: We were able to count individual allergen-carrying particles inhaled over short time periods, during different domestic exposure situations. This will offer new insight into several aspects of personal allergen exposure.

Sheep bred for resistance (R) or susceptibility (S) to fleece rot and myiasis (blowfly strike) have been shown to differ in inflammatory response to intradermal administration of blowfly (Lucilia cuprina) antigens and artificial challenge. The current paper describes analysis of antibody responses to L. cuprina antigens in the R and S animals. Serum antibody titres and specificities to larval antigens were examined and the specificity of wound exudate antibodies was also investigated in animals artificially challenged with L. cuprina. Titres of L. cuprina specific serum IgA, IgM, IgG2 and IgG1 were measured by ELISA, while specificities were examined on two-dimensional immunoblots of larval homogenates. Exposure to L. cuprina stimulated the production of specific antibody in both R and S animals, however antibody titres did not differ between the R and S animals. There was large variation in antibody specificity between individual animals and some L. cuprina proteins appear to be more frequently recognized by sera from either resistant or susceptible animals, however the recognition of a specific protein could not be solely attributed to the resistance status of the animal. It appears that resistance in these animals may be independent of serum antibody and is likely to be an innate response. Despite high levels of IgG in wound exudates, this antibody recognized few antigens in comparison with serum from the same animal, suggesting that exudate contains little functional antibody in comparison to serum.

Sheep bred for resistance (R) or susceptibility (S) to fleece rot and myiasis (blowfly strike) were experimentally infected with L. cuprina larvae. Exudates released from the wound site were collected during the infection at 6, 12, 18 and 24 h. The exudates were separated using two-dimensional gel electrophoresis, and proteins were silver stained and identified by immunoblotting with specific antibody and by their isoelectric points and molecular weights. Comparisons of exudate composition were made over time and between R and S sheep. Between 6 and 12 h post larval implantation the exudate was rich in IgG and fibrinogen, which is before extensive tissue damage and suggests that the exudate is not simply tissue haemorrhage but the result of an inflammatory response by the sheep to L. cuprina. The exudate grew in complexity between 12 and 18 h and contained a maximum of 74 distinct peptide spots by 24 h. Exudate from wounds on resistant sheep contained many more peptides in the first 12 h of infection, suggesting a more rapid inflammatory response. The source of proteins from the exudate remains speculative; it appears to be composed of many acute-phase proteins, large amounts of immunoglobulin G and proportionally low levels of serum albumin. Exudate composition is likely to be influenced by the local synthesis of acute-phase proteins and perhaps immunoglobulins, selective transport to the infection site and also enzymic degradation by L. cuprina larval enzymes. The more rapid exudation of acute-phase and serum proteins at infection sites on R sheep may allow the inhibition of the establishment of fleece rot bacteria or L. cuprina larvae under natural challenge.

The relationship between K-agglutination antibody titres and protection against experimental challenge with Dichelobacter nodosus, the effect of increasing the number of D. nodosus fimbrial antigens, and the importance of the nature of additional antigens in multivalent vaccines on antibody response and protection against experimental challenge with D. nodosus were examined in Merino sheep. A total of 204 Merino sheep were allocated to one of 12 groups, and vaccinated with preparations containing a variable number of rDNA D. nodosus fimbrial antigens. The most complex vaccine contained ten fimbrial antigens from all major D. nodosus serogroups, while the least complex contained a single fimbrial antigen. In addition to D. nodosus fimbrial antigens, other bacterial rDNA fimbrial antigens (Moraxella bovis Da12d and Escherichia coli K99), and bovine serum albumin (BSA) were used in some vaccines. Antibody titres to fimbrial antigens and BSA were measured by agglutination and ELISA tests, respectively. Antibody titres were determined on five occasions (Weeks 0, 3, 6, 8, and 11 after primary vaccination). All sheep were exposed to an experimental challenge with virulent isolates of D. nodosus from either serogroup A or B, 8 weeks after primary vaccination. For D. nodosus K-agglutinating antibody titres, a strong negative correlation between antibody titre and footrot lesion score was observed. This relationship was influenced by the virulence of the challenge strain. Increasing the number of fimbrial antigens in experimental rDNA D. nodosus fimbrial vaccines resulted in a linear decrease in K-agglutinating antibody titres to individual D. nodosus serogroups. Similarly, a linear decrease in protection to challenge with homologous serogroups was observed as the number of D. nodosus fimbrial antigens represented in the vaccine increased. The reduction in antibody titres in multicomponent vaccines is thought to be due to antigenic competition. The level of competition between individual antigens is not constant and appears to be related to the immunodominance (nature) of the competing antigens. Both BSA ELISA, and M. bovis K-agglutinating antibody titres were adversely affected by the presence of two D. nodosus fimbrial preparations, whereas the antigenicity of E. coli K99 was unchanged by the presence of two additional D. nodosus antigens. Further studies are required to determine the step(s) in the immune response which are influenced by antigenic competition. Our results suggest that antigen presentation, particularly following primary vaccination, is the step most strongly influenced by antigenic competition.

For the past 20 years footrot vaccines have evolved from simple bacterins to highly specific recombinant DNA (rDNA) fimbrial vaccines. The development of these vaccines has left a trail of discoveries, challenges and solutions; these processes continue as we move closer to understanding the requirements of a footrot vaccine. The initial whole cell vaccines were unsuccessful due to the short duration of immunity and incorporation of limited serotypes. A multistrain vaccine eliminated the problem of serotype inclusion, although the duration of immunity in many cases is still inadequate. The proteases of Dichelobacter nodosus appear to be cross protective; however, little is known of their ability to protect sheep against footrot. The major protective immunogen is the bacterial fimbriae, which also forms the basis for the K-agglutination serotyping system. K-agglutinin titre correlates directly with resistance to challenge. The protective fimbrial epitope is conformationally dependent, suggesting little advantage in the development of synthetic peptide vaccines. To enhance the efficiency of vaccine production D. nodosus fimbrial genes were eventually cloned and successfully expressed in Ps. aeruginosa. Monovalent vaccines based on recombinant fimbriae are omnipotent, inducing high levels of agglutinins and long lasting immunity. In multivalent vaccines, on the other hand, incorporation of each additional serogroup into the vaccine results in reduced efficacy both in terms of reduced K-agglutinin titres and reduced protection following challenge. The least effective are multivalent formulations representing all major serogroups. In addition, considerable genetic variation has been observed in the ability of sheep to respond optimally to each serogroup in a multivalent vaccine. Results show that the limitation of the sheep to mount an effective immune response, rather than the quality or quantity of the immunogen, limits the efficacy of current footrot vaccines. Studies are being undertaken to examine in detail the immune response of sheep to potentially highly effective footrot vaccines.

Sheep repeatedly infected with L. cuprina at 2- but not 4-week intervals developed partial resistance to infection after five infections, as measured by larval recovery. However, resistance did not persist for more than three infections. Skin weal responses were measured after injection of larval products simultaneously with each infection. The only correlation between weal size and larval recoveries occurred at infection 1 and indicated a relationship between skin sensitivity and innate rather than acquired resistance. The results suggest that resistance to L. cuprina can develop after repeated infections but that it is short lived and requires frequent larval exposure. A role for hypersensitivity responses was not confirmed by the weal responses but was suggested by the size of wound developed per larva recovered.

The specific serum antibody responses of sheep exposed to 10 consecutive infections of L. cuprina have been analysed by enzyme-linked immuno-sorbent assay and immunoblotting using monoclonal antibodies specific for sheep immunoglobulin isotypes. Recognition of a number of larval excretory-secretory products by IgM antibodies appeared to be non-specific. IgG1 was the major antibody class stimulated by the infection protocol and marked increases in antibody to specific excretory-secretory antigens were observed. Three molecules of 35, 30 and 25 kDa were particularly recognized although the extent of recognition of these molecules varied considerably between individual sheep serum. A pooled serum composed of sera collected after five to seven infections significantly inhibited larval growth in in vitro cultures when compared to a sera pool consisting of sera collected both prior to infection and after infections 1 and 2. The degree of inhibition was greater when serum with high specific antibody titre was used.

Sheep which have been bred for resistance (R) or susceptibility (S) to fleece rot and blowfly strike, were tested for intradermal inflammatory responses to excretory and secretory products of Lucilia cuprina larvae. R rams and lambs gave significantly larger skin weals than S animals. In addition, R and S rams were infected with L cuprina first instar larvae and wound exudates were collected. In the first 12 hours of infection R rams released significantly more exudate protein at the wound site than S rams. Correlations suggested that exudate production was stimulated by both larval burden and inflammatory responses, however, in the R group the inflammatory correlation was positive while in the S group it was negative. The results imply that inflammatory responses may play a role in innate resistance to L cuprina. The difference in inflammatory responses suggests genetic differences between the flocks and therefore could show some potential as a trait for indirect selection for resistance to fleece rot and body strike.