Background Beauveria bassiana  is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of  Beauveria  species has not been widely studied.
Background
Beauveria bassiana  is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of  Beauveria  species has not been widely studied.Beauveria bassianaBeauveriaMethods IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to  B. bassiana  extracts was measured using intradermal skin testing.
Methods
IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to  B. bassiana  extracts was measured using intradermal skin testing.B. bassianaResults Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in  B. bassiana  crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including;  Alternaria alternata ,  Aspergillus fumigatus ,  Cladosporium herbarum ,  Candida albicans ,  Epicoccum purpurascens , and  Penicillium notatum . Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent  B. bassiana  specific allergens. Intradermal skin testing confirmed the  in vitro  results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to  B. bassiana .
Results
Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in  B. bassiana  crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including;  Alternaria alternata ,  Aspergillus fumigatus ,  Cladosporium herbarum ,  Candida albicans ,  Epicoccum purpurascens , and  Penicillium notatum . Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent  B. bassiana  specific allergens. Intradermal skin testing confirmed the  in vitro  results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to  B. bassiana .B. bassianaAlternaria alternataAspergillus fumigatusCladosporium herbarumCandida albicansEpicoccum purpurascensPenicillium notatumB. bassianain vitroB. bassianaConclusions Beauveria bassiana  possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential  B. bassiana  specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of  B. bassiana .
Conclusions
Beauveria bassiana  possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential  B. bassiana  specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of  B. bassiana .Beauveria bassianaB. bassianaB. bassiana

Background Microorganisms are currently under intensive study for use as biopesticides [ 1 - 3 ]. Several fungal species including  Metarhizium anisopliae ,  Verticillium lecanii , and  Beauveria bassiana  are being used as biocontrol agents for a number of crop, livestock, and human nuisance pests [ 4 - 7 ]. Strains of  B. bassiana  have been licensed for commercial use against whiteflies, aphids, thrips, and numerous other insect and arthropod pests.  B. bassiana  fungal formulations are being spread onto a range of vegetables, melons, tree fruits and nuts, as well as organic crops. As alternatives to chemical pesticides these agents are natural occurring and are considered to be non-pathogenic to humans, although a few cases of  B. bassiana  mediated tissue infections have been reported [ 8 , 9 ]. Airborne mold spores are widespread, and many have been identified as inhalant allergens eliciting type I hypersensitive reactions in atopic individuals [ 10 - 14 ]. Common allergenic moulds include the anamorphs of ascomycetes and constitute many species within the  Alternaria ,  Aspergillus , and  Cladosporium  genera [ 15 - 19 ]. The genes encoding for numerous fungal allergens have been isolated, and their protein products expressed and characterized. Purified fungal allergens have been shown to be bound by human IgEs and to elicit allergic reactions in atopic individuals using skin prick tests. Patients with mould allergies often display IgE-mediated responses to multiple fungi, a phenomenon typically thought to result from the presence of common cross-reactive allergen(s) [ 15 , 20 - 22 ], although parallel independent sensitization to multiple fungal allergens can also occur. In this regards, identification of genus and/or species specific allergens would provide useful tools in differentiating allergic reactions due to primary sensitization and those mediated by cross-reactive epitopes. In the present study, we demonstrate  Beauveria bassiana  crude extracts contain numerous allergens capable of being recognized by human serum IgEs. The allergens were proteinaceous in nature, and immunoblot inhibition experiments revealed the presence of shared epitopes between  Beauveria  and several other common fungal moulds. Potential  Beauveria-specific  allergens were also identified, including a strongly reactive ~35-kDa protein band. Intradermal skin testing using  B. bassiana  extracts resulted in allergenic reactions in several individuals, including some who have had occupational exposure to the fungus.
Background
Microorganisms are currently under intensive study for use as biopesticides [ 1 - 3 ]. Several fungal species including  Metarhizium anisopliae ,  Verticillium lecanii , and  Beauveria bassiana  are being used as biocontrol agents for a number of crop, livestock, and human nuisance pests [ 4 - 7 ]. Strains of  B. bassiana  have been licensed for commercial use against whiteflies, aphids, thrips, and numerous other insect and arthropod pests.  B. bassiana  fungal formulations are being spread onto a range of vegetables, melons, tree fruits and nuts, as well as organic crops. As alternatives to chemical pesticides these agents are natural occurring and are considered to be non-pathogenic to humans, although a few cases of  B. bassiana  mediated tissue infections have been reported [ 8 , 9 ].Metarhizium anisopliaeVerticillium lecaniiBeauveria bassianaB. bassianaB. bassianaB. bassianaAirborne mold spores are widespread, and many have been identified as inhalant allergens eliciting type I hypersensitive reactions in atopic individuals [ 10 - 14 ]. Common allergenic moulds include the anamorphs of ascomycetes and constitute many species within the  Alternaria ,  Aspergillus , and  Cladosporium  genera [ 15 - 19 ]. The genes encoding for numerous fungal allergens have been isolated, and their protein products expressed and characterized. Purified fungal allergens have been shown to be bound by human IgEs and to elicit allergic reactions in atopic individuals using skin prick tests. Patients with mould allergies often display IgE-mediated responses to multiple fungi, a phenomenon typically thought to result from the presence of common cross-reactive allergen(s) [ 15 , 20 - 22 ], although parallel independent sensitization to multiple fungal allergens can also occur. In this regards, identification of genus and/or species specific allergens would provide useful tools in differentiating allergic reactions due to primary sensitization and those mediated by cross-reactive epitopes.AlternariaAspergillusCladosporiumIn the present study, we demonstrate  Beauveria bassiana  crude extracts contain numerous allergens capable of being recognized by human serum IgEs. The allergens were proteinaceous in nature, and immunoblot inhibition experiments revealed the presence of shared epitopes between  Beauveria  and several other common fungal moulds. Potential  Beauveria-specific  allergens were also identified, including a strongly reactive ~35-kDa protein band. Intradermal skin testing using  B. bassiana  extracts resulted in allergenic reactions in several individuals, including some who have had occupational exposure to the fungus.Beauveria bassianaBeauveriaBeauveria-specificB. bassianaMethods Strains and cultures Beauveria bassiana  (ATCC 90517) was grown on Sabouraud dextrose + 0.5–1% yeast extract or Potato dextrose (PD) media on either agar plates or in liquid broth. Plates were incubated at 26°C for 10–12 days and conidia were harvested by flooding the plate with sterile dH 2 O containing 0.01% Tween-20. Liquid cultures were inoculated with conidia harvested from plates at 0.5–1 × 10 5  conidia/ml. Extract preparation Alternaria alternata ,  Aspergillus fumigatus ,  Candida albicans ,  Cladosporium herbarum ,  Epicoccum purpurascens , and  Penicillium notatum  were acquired from Greer Laboratories inc., (Lenoir, NC). Extracts were resuspended in TE (40 mM Tris-HCl, pH 8.0, 1 mM EDTA) to a final concentration of 2 mg/ml.  Beauveria bassiana  was grown in Sabouraud's broth containing 1% yeast extract with aeration at 25°C for 3–5 d. Cellular mass was harvested by centrifugation (10,000 × g, 10 min) and freeze-dried. Cells were resuspended in TE containing 0.1% phenylmethylsulfonyl fluoride (PMSF) and homogenized using a bead-beater apparatus. Precipitations Crude extracts of  B. bassiana  were subjected to three successive precipitations before use in Western blots. Acetone precipitation Homogenized  B. bassiana  extracts (50 ml) were mixed with 8 × volume (400 ml) of acetone (kept at -20°C), with rapid stirring, and incubated overnight at -20°C. The precipitate was collected by centrifugation (30 min, 4000 × g), and the pellet was air dried (10 min) before being resuspended in TE containing 0.1% PMSF. Streptomycin precipitation (removal of DNA) Streptomycin sulfate (5 ml of 10% solution) was added dropwise to resuspended acetone precipitated extracts (40 ml) at 4°C with rapid stirring. Samples were incubated for an additional 30 min on ice before being centrifuged (15 min, 10,000 × g) in order to remove the precipitate. Proteins in the resultant supernatant were precipitated using ammonium sulfate. Ammonium sulfate The proteins present in the streptomycin sulfate treated supernatant were precipitated using ammonium sulfate (75%, final concentration). Saturated ammonium sulfate (120 ml) was added dropwise to the  Beauveria  extract (40 ml) at 4°C with rapid stirring. The solution was allowed to stir overnight at 4°C and precipitated proteins were harvested by centrifugation (30 min, 100,000 × g). The protein pellet was resuspended in TE containing 0.1% PMSF (40 ml) and extensively dialyzed against the same buffer before use. SDS-Polyacrylamide gel electrophoresis (PAGE) Protein samples (30–40 μg) were analyzed by sodium-dodecyl-sulphate-polyacrylaminde gel electrophoresis (SDS-PAGE, 10% Bis-tris gel, Invitrogen, Carlsbad, CA) using standard protocols. Gels were stained with Gelcode blue stain reagent (Pierce, Rockford, IL) and subsequently de-stained with dH20. Western blotting Protein samples were separated under reducing conditions using 10% Bis-tris polyacrylamide gels (Invitrogen Mops system) and transferred to polyvinylidene-fluoride (PVDF) membranes (Invitrogen) as described. Immunoblot experiments were performed using individual and pooled human sera as the primary antibody solution as indicated. Typically, sera were diluted 1:5 with Tris-HCl buffered saline (TBS) containing 5% dry milk + 0.1% Tween-20. IgE-specific reactivity was visualized using a horseradish peroxidase (HRP) conjugated goat anti-human IgE (polyclonal) secondary antibody (BioSource International, Los Angeles, CA). Membranes were washed with TBS containing 0.1% Tween-20 and bands were visualized using the Immuno-Star HRP detection system (Biorad, Hercules, CA). Enzyme Treatments The ammonium sulfate fraction of  B. bassiana  crude extracts was treated with Proteinase K (ICN-Biomed, Aurora, Oh) following standard protocols. Typically, samples (36 μl) were incubated with 4 μl Proteinase K solution (10 mg/ml in 50 mM Tris-HCl, pH 7.5) for 2 hr at 37°C before analysis. Samples were also treated with endoglycosidase-H (EndoH, New England Biolabs, Beverly, MA) and peptide: N-Glycosidase F (PNGaseF, New England Biolabs) according to the manufacturer's recommendations. For EndoH and PNGaseF treatments, samples (36 μl) were denatured in 4 μl 10 × denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) at 100°C for 10 min prior to the addition of the EndoH (5 μl of 10 × G5 Reaction Buffer, 50 mM sodium citrate, pH 5.5) and PNGaseF reaction buffers (50 mM sodium phosphate pH 7.5) and enzymes (5 μl), respectively. Reactions were incubated at 37°C for 2 h before being analyzed by SDS-PAGE and Western blotting. Immunoblot inhibition IgE binding to  B. bassiana  proteins were competed with proteins of other fungal extracts. SDS-PAGE resolved  B. bassiana  proteins were electroblotted to PVDF membranes as described above. Membranes were blocked with TBS containing 5% dry milk + 0.1% Tween-20 and strips were incubated with pooled human sera (1:5 v/v in same buffer) containing 100–500 μg of the indicated fungal crude protein extract. Skin sensitivity profiles to fungal extracts Patients were tested with 9 common fungal extracts for allergy diagnosis using a skin prick assay. The following extracts were obtained from ALA-Abello (Round Rock, TX);  Alternaria tenius ,  Aspergillus fumigatus ,  Cephalosporium  ( Acremonium strictum ),  Curvularia  spp.  Bipolaris ,  Epicoccum nigram ,  Fusarium  spp.,  Helminthosporium sativum ,  Hormodendrum horde ,  Penicillium  (mixed,  P. chrysogenum  and  P. notatum ). Extracts were tested using a 1:10 dilution of the 20,000 PNU/ml stock solution, and skin sensitivity was recorded on a relative scale from 0–4 reflecting the size of induration or weal (4 representing the highest reactivity) and using histamine (0.1 mg/ml) reaction scored as a 3 if no interference was present. Intradermal skin testing B. bassiana  crude extracts were prepared as described above but were extensively dialyzed against 0.15 N NaCl and filtered through a 0.22 μm filter before use. Subjects were given intradermal injections of 0.1 ml crude extract ranging in concentration from 0.01–1 mg/ml. Control injections included saline and histamine (0.1 mg/ml). Allergenic reactions were allowed to develop for 15–30 min before the height and width of the reactions were recorded.
Methods
Strains and cultures Beauveria bassiana  (ATCC 90517) was grown on Sabouraud dextrose + 0.5–1% yeast extract or Potato dextrose (PD) media on either agar plates or in liquid broth. Plates were incubated at 26°C for 10–12 days and conidia were harvested by flooding the plate with sterile dH 2 O containing 0.01% Tween-20. Liquid cultures were inoculated with conidia harvested from plates at 0.5–1 × 10 5  conidia/ml.
Strains and cultures
Beauveria bassiana  (ATCC 90517) was grown on Sabouraud dextrose + 0.5–1% yeast extract or Potato dextrose (PD) media on either agar plates or in liquid broth. Plates were incubated at 26°C for 10–12 days and conidia were harvested by flooding the plate with sterile dH 2 O containing 0.01% Tween-20. Liquid cultures were inoculated with conidia harvested from plates at 0.5–1 × 10 5  conidia/ml.Beauveria bassianaExtract preparation Alternaria alternata ,  Aspergillus fumigatus ,  Candida albicans ,  Cladosporium herbarum ,  Epicoccum purpurascens , and  Penicillium notatum  were acquired from Greer Laboratories inc., (Lenoir, NC). Extracts were resuspended in TE (40 mM Tris-HCl, pH 8.0, 1 mM EDTA) to a final concentration of 2 mg/ml.  Beauveria bassiana  was grown in Sabouraud's broth containing 1% yeast extract with aeration at 25°C for 3–5 d. Cellular mass was harvested by centrifugation (10,000 × g, 10 min) and freeze-dried. Cells were resuspended in TE containing 0.1% phenylmethylsulfonyl fluoride (PMSF) and homogenized using a bead-beater apparatus.
Extract preparation
Alternaria alternata ,  Aspergillus fumigatus ,  Candida albicans ,  Cladosporium herbarum ,  Epicoccum purpurascens , and  Penicillium notatum  were acquired from Greer Laboratories inc., (Lenoir, NC). Extracts were resuspended in TE (40 mM Tris-HCl, pH 8.0, 1 mM EDTA) to a final concentration of 2 mg/ml.  Beauveria bassiana  was grown in Sabouraud's broth containing 1% yeast extract with aeration at 25°C for 3–5 d. Cellular mass was harvested by centrifugation (10,000 × g, 10 min) and freeze-dried. Cells were resuspended in TE containing 0.1% phenylmethylsulfonyl fluoride (PMSF) and homogenized using a bead-beater apparatus.Alternaria alternataAspergillus fumigatusCandida albicansCladosporium herbarumEpicoccum purpurascensPenicillium notatumBeauveria bassianaPrecipitations Crude extracts of  B. bassiana  were subjected to three successive precipitations before use in Western blots. Acetone precipitation Homogenized  B. bassiana  extracts (50 ml) were mixed with 8 × volume (400 ml) of acetone (kept at -20°C), with rapid stirring, and incubated overnight at -20°C. The precipitate was collected by centrifugation (30 min, 4000 × g), and the pellet was air dried (10 min) before being resuspended in TE containing 0.1% PMSF. Streptomycin precipitation (removal of DNA) Streptomycin sulfate (5 ml of 10% solution) was added dropwise to resuspended acetone precipitated extracts (40 ml) at 4°C with rapid stirring. Samples were incubated for an additional 30 min on ice before being centrifuged (15 min, 10,000 × g) in order to remove the precipitate. Proteins in the resultant supernatant were precipitated using ammonium sulfate. Ammonium sulfate The proteins present in the streptomycin sulfate treated supernatant were precipitated using ammonium sulfate (75%, final concentration). Saturated ammonium sulfate (120 ml) was added dropwise to the  Beauveria  extract (40 ml) at 4°C with rapid stirring. The solution was allowed to stir overnight at 4°C and precipitated proteins were harvested by centrifugation (30 min, 100,000 × g). The protein pellet was resuspended in TE containing 0.1% PMSF (40 ml) and extensively dialyzed against the same buffer before use. SDS-Polyacrylamide gel electrophoresis (PAGE) Protein samples (30–40 μg) were analyzed by sodium-dodecyl-sulphate-polyacrylaminde gel electrophoresis (SDS-PAGE, 10% Bis-tris gel, Invitrogen, Carlsbad, CA) using standard protocols. Gels were stained with Gelcode blue stain reagent (Pierce, Rockford, IL) and subsequently de-stained with dH20.
Precipitations
Crude extracts of  B. bassiana  were subjected to three successive precipitations before use in Western blots.B. bassianaAcetone precipitation Homogenized  B. bassiana  extracts (50 ml) were mixed with 8 × volume (400 ml) of acetone (kept at -20°C), with rapid stirring, and incubated overnight at -20°C. The precipitate was collected by centrifugation (30 min, 4000 × g), and the pellet was air dried (10 min) before being resuspended in TE containing 0.1% PMSF.
Acetone precipitation
Homogenized  B. bassiana  extracts (50 ml) were mixed with 8 × volume (400 ml) of acetone (kept at -20°C), with rapid stirring, and incubated overnight at -20°C. The precipitate was collected by centrifugation (30 min, 4000 × g), and the pellet was air dried (10 min) before being resuspended in TE containing 0.1% PMSF.B. bassianaStreptomycin precipitation (removal of DNA) Streptomycin sulfate (5 ml of 10% solution) was added dropwise to resuspended acetone precipitated extracts (40 ml) at 4°C with rapid stirring. Samples were incubated for an additional 30 min on ice before being centrifuged (15 min, 10,000 × g) in order to remove the precipitate. Proteins in the resultant supernatant were precipitated using ammonium sulfate.
Streptomycin precipitation (removal of DNA)
Streptomycin sulfate (5 ml of 10% solution) was added dropwise to resuspended acetone precipitated extracts (40 ml) at 4°C with rapid stirring. Samples were incubated for an additional 30 min on ice before being centrifuged (15 min, 10,000 × g) in order to remove the precipitate. Proteins in the resultant supernatant were precipitated using ammonium sulfate.Ammonium sulfate The proteins present in the streptomycin sulfate treated supernatant were precipitated using ammonium sulfate (75%, final concentration). Saturated ammonium sulfate (120 ml) was added dropwise to the  Beauveria  extract (40 ml) at 4°C with rapid stirring. The solution was allowed to stir overnight at 4°C and precipitated proteins were harvested by centrifugation (30 min, 100,000 × g). The protein pellet was resuspended in TE containing 0.1% PMSF (40 ml) and extensively dialyzed against the same buffer before use.
Ammonium sulfate
The proteins present in the streptomycin sulfate treated supernatant were precipitated using ammonium sulfate (75%, final concentration). Saturated ammonium sulfate (120 ml) was added dropwise to the  Beauveria  extract (40 ml) at 4°C with rapid stirring. The solution was allowed to stir overnight at 4°C and precipitated proteins were harvested by centrifugation (30 min, 100,000 × g). The protein pellet was resuspended in TE containing 0.1% PMSF (40 ml) and extensively dialyzed against the same buffer before use.BeauveriaSDS-Polyacrylamide gel electrophoresis (PAGE) Protein samples (30–40 μg) were analyzed by sodium-dodecyl-sulphate-polyacrylaminde gel electrophoresis (SDS-PAGE, 10% Bis-tris gel, Invitrogen, Carlsbad, CA) using standard protocols. Gels were stained with Gelcode blue stain reagent (Pierce, Rockford, IL) and subsequently de-stained with dH20.
SDS-Polyacrylamide gel electrophoresis (PAGE)
Protein samples (30–40 μg) were analyzed by sodium-dodecyl-sulphate-polyacrylaminde gel electrophoresis (SDS-PAGE, 10% Bis-tris gel, Invitrogen, Carlsbad, CA) using standard protocols. Gels were stained with Gelcode blue stain reagent (Pierce, Rockford, IL) and subsequently de-stained with dH20.Western blotting Protein samples were separated under reducing conditions using 10% Bis-tris polyacrylamide gels (Invitrogen Mops system) and transferred to polyvinylidene-fluoride (PVDF) membranes (Invitrogen) as described. Immunoblot experiments were performed using individual and pooled human sera as the primary antibody solution as indicated. Typically, sera were diluted 1:5 with Tris-HCl buffered saline (TBS) containing 5% dry milk + 0.1% Tween-20. IgE-specific reactivity was visualized using a horseradish peroxidase (HRP) conjugated goat anti-human IgE (polyclonal) secondary antibody (BioSource International, Los Angeles, CA). Membranes were washed with TBS containing 0.1% Tween-20 and bands were visualized using the Immuno-Star HRP detection system (Biorad, Hercules, CA).
Western blotting
Protein samples were separated under reducing conditions using 10% Bis-tris polyacrylamide gels (Invitrogen Mops system) and transferred to polyvinylidene-fluoride (PVDF) membranes (Invitrogen) as described. Immunoblot experiments were performed using individual and pooled human sera as the primary antibody solution as indicated. Typically, sera were diluted 1:5 with Tris-HCl buffered saline (TBS) containing 5% dry milk + 0.1% Tween-20. IgE-specific reactivity was visualized using a horseradish peroxidase (HRP) conjugated goat anti-human IgE (polyclonal) secondary antibody (BioSource International, Los Angeles, CA). Membranes were washed with TBS containing 0.1% Tween-20 and bands were visualized using the Immuno-Star HRP detection system (Biorad, Hercules, CA).Enzyme Treatments The ammonium sulfate fraction of  B. bassiana  crude extracts was treated with Proteinase K (ICN-Biomed, Aurora, Oh) following standard protocols. Typically, samples (36 μl) were incubated with 4 μl Proteinase K solution (10 mg/ml in 50 mM Tris-HCl, pH 7.5) for 2 hr at 37°C before analysis. Samples were also treated with endoglycosidase-H (EndoH, New England Biolabs, Beverly, MA) and peptide: N-Glycosidase F (PNGaseF, New England Biolabs) according to the manufacturer's recommendations. For EndoH and PNGaseF treatments, samples (36 μl) were denatured in 4 μl 10 × denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) at 100°C for 10 min prior to the addition of the EndoH (5 μl of 10 × G5 Reaction Buffer, 50 mM sodium citrate, pH 5.5) and PNGaseF reaction buffers (50 mM sodium phosphate pH 7.5) and enzymes (5 μl), respectively. Reactions were incubated at 37°C for 2 h before being analyzed by SDS-PAGE and Western blotting.
Enzyme Treatments
The ammonium sulfate fraction of  B. bassiana  crude extracts was treated with Proteinase K (ICN-Biomed, Aurora, Oh) following standard protocols. Typically, samples (36 μl) were incubated with 4 μl Proteinase K solution (10 mg/ml in 50 mM Tris-HCl, pH 7.5) for 2 hr at 37°C before analysis. Samples were also treated with endoglycosidase-H (EndoH, New England Biolabs, Beverly, MA) and peptide: N-Glycosidase F (PNGaseF, New England Biolabs) according to the manufacturer's recommendations. For EndoH and PNGaseF treatments, samples (36 μl) were denatured in 4 μl 10 × denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) at 100°C for 10 min prior to the addition of the EndoH (5 μl of 10 × G5 Reaction Buffer, 50 mM sodium citrate, pH 5.5) and PNGaseF reaction buffers (50 mM sodium phosphate pH 7.5) and enzymes (5 μl), respectively. Reactions were incubated at 37°C for 2 h before being analyzed by SDS-PAGE and Western blotting.B. bassianaImmunoblot inhibition IgE binding to  B. bassiana  proteins were competed with proteins of other fungal extracts. SDS-PAGE resolved  B. bassiana  proteins were electroblotted to PVDF membranes as described above. Membranes were blocked with TBS containing 5% dry milk + 0.1% Tween-20 and strips were incubated with pooled human sera (1:5 v/v in same buffer) containing 100–500 μg of the indicated fungal crude protein extract.
Immunoblot inhibition
IgE binding to  B. bassiana  proteins were competed with proteins of other fungal extracts. SDS-PAGE resolved  B. bassiana  proteins were electroblotted to PVDF membranes as described above. Membranes were blocked with TBS containing 5% dry milk + 0.1% Tween-20 and strips were incubated with pooled human sera (1:5 v/v in same buffer) containing 100–500 μg of the indicated fungal crude protein extract.B. bassianaB. bassianaSkin sensitivity profiles to fungal extracts Patients were tested with 9 common fungal extracts for allergy diagnosis using a skin prick assay. The following extracts were obtained from ALA-Abello (Round Rock, TX);  Alternaria tenius ,  Aspergillus fumigatus ,  Cephalosporium  ( Acremonium strictum ),  Curvularia  spp.  Bipolaris ,  Epicoccum nigram ,  Fusarium  spp.,  Helminthosporium sativum ,  Hormodendrum horde ,  Penicillium  (mixed,  P. chrysogenum  and  P. notatum ). Extracts were tested using a 1:10 dilution of the 20,000 PNU/ml stock solution, and skin sensitivity was recorded on a relative scale from 0–4 reflecting the size of induration or weal (4 representing the highest reactivity) and using histamine (0.1 mg/ml) reaction scored as a 3 if no interference was present.
Skin sensitivity profiles to fungal extracts
Patients were tested with 9 common fungal extracts for allergy diagnosis using a skin prick assay. The following extracts were obtained from ALA-Abello (Round Rock, TX);  Alternaria tenius ,  Aspergillus fumigatus ,  Cephalosporium  ( Acremonium strictum ),  Curvularia  spp.  Bipolaris ,  Epicoccum nigram ,  Fusarium  spp.,  Helminthosporium sativum ,  Hormodendrum horde ,  Penicillium  (mixed,  P. chrysogenum  and  P. notatum ). Extracts were tested using a 1:10 dilution of the 20,000 PNU/ml stock solution, and skin sensitivity was recorded on a relative scale from 0–4 reflecting the size of induration or weal (4 representing the highest reactivity) and using histamine (0.1 mg/ml) reaction scored as a 3 if no interference was present.Alternaria teniusAspergillus fumigatusCephalosporiumAcremonium strictumCurvulariaBipolarisEpicoccum nigramFusariumHelminthosporium sativumHormodendrum hordePenicilliumP. chrysogenumP. notatumIntradermal skin testing B. bassiana  crude extracts were prepared as described above but were extensively dialyzed against 0.15 N NaCl and filtered through a 0.22 μm filter before use. Subjects were given intradermal injections of 0.1 ml crude extract ranging in concentration from 0.01–1 mg/ml. Control injections included saline and histamine (0.1 mg/ml). Allergenic reactions were allowed to develop for 15–30 min before the height and width of the reactions were recorded.
Intradermal skin testing
B. bassiana  crude extracts were prepared as described above but were extensively dialyzed against 0.15 N NaCl and filtered through a 0.22 μm filter before use. Subjects were given intradermal injections of 0.1 ml crude extract ranging in concentration from 0.01–1 mg/ml. Control injections included saline and histamine (0.1 mg/ml). Allergenic reactions were allowed to develop for 15–30 min before the height and width of the reactions were recorded.B. bassianaResults Identification of IgE reactive bands An ammonium sulfate fraction of  B. bassiana  proteins was resolved on SDS-PAGE (Fig.  1 , lane B) and transferred to PVDF membranes as described in the Materials and Methods. Membranes were probed with sera from individual patients who were reactive to various moulds (Table  1 ), which was pooled and used to demonstrate IgE reactivity against antigens present in  B. bassiana  extracts (Fig.  1 ). Serum mix-I represents pooled sera derived from patients E, J, K, L, and M, as well as three additional patients that were only tested (skin prick) against  Aspergillus  and  Penicillium , displaying test scores of 3–4 for each. These data demonstrate human IgE binding of allergens present in  B. bassiana  extracts. Initial blots showed 12–15 distinct reactive protein bands, ranging in molecular mass from 12 kDa to >95 kDa (under denaturing conditions); with the most prominent bands located around 64, 45, and 35 kDa. Control experiments omitting either the primary or secondary antibody incubation steps resulted in complete loss of signal. Proteinase K digestion of samples also resulted in loss of all signal (Fig.  1 , lane 4), indicating the proteinaceous nature of the IgE reactive bands. Since the carbohydrate moieties of several protein allergens are known to play a role in their allergenicity and even cross-reactivity [ 20 - 22 ], samples were treated with the deglyocosylating enzymes EndoH and PNGaseF. Control experiments incubating samples in the EndoH denaturing buffer without any enzyme altered the IgE-reactive signals (Fig.  1 , lane 5), however, samples treated with EndoH did not appear any different than control reactions (Fig.  1 , lane 6). Similar results were obtained in PNGaseF digests (data not shown). These data appear to indicate that the  B. bassiana  IgE-antigen profiles observed on Western blots are proteins with minimal contributions due to glycosylation. Figure 1 SDS-PAGE and immunoblot analysis of  Beauveria bassiana  crude extracts. SDS-PAGE, Gelcode blue stained, lanes A) 5 μg protein standards, and B) 40 μg  B. bassiana  crude extract. Immunoblots probed with pooled serum mix-I (patients displaying mould allergies), lanes 1), 5 μg protein standards, 2) 20 μg  B. bassiana  crude extract, 3) 40 μg crude extract, 4) 40 μg crude extract, Proteinase K treated, 5) 40 μg crude extract, denaturing buffer control (no EndoH), 6) 40 μg crude extract, EndoH treated Table 1 Allergic profile of patients A–G, obtained by skin prick testing. Patient ID Individual Skin Reactivity to Fungal Extracts* Alt † Asp Cep Cur Epi Fusa Helmin Hormo Pen A 3 2 0 3 2 2 0 0 3 B 3 2 0 2 3 2 2 2 0 C 4 0 0 3 0 2 0 0 0 D 0 0 2 2 0 2 0 2 2 E 3 2 0 3 2 3 3 0 0 F 4 1 1 2 4 0 2 0 0 G 3 0 0 4 3 0 2 0 0 *Skin test scores were registered using a relative scale from 0–4 with 4 representing the highest reactivity as described in the Materials and Methods.  † Abbreviations used;  Alt ,  Alternaria tenius ;  Asp ,  Aspergillus fumigatus ;  Cep ,  Cephalosporium  ( Acremonium strictum );  Cur ,  Curvularia  spp.  Bipolaris ;  Epi ,  Epicoccum nigram ;  Fusa ,  Fusarium  spp.;  Helmin ,  Helminthosporium sativum ;  Hormo ,  Hormodendrum horde ;  Pen ,  Penicillium  (mixed,  P. chrysogenum  and  P. notatum ). Immunoprint Analysis of  B. bassiana : Reactivity with Individual Sera In order to determine the variation and distribution of serum IgEs reactive to  B. bassiana  extracts, individual sera from patients displaying mould allergies (Fig.  2 , lanes A–G) as well as random sera from the general population (Fig.  2 , lanes H–M) were used as probes for Western blots (Fig.  2 ). The reactivity of pooled sera from patients A–G (termed serum mix-II) is also shown (Fig.  2 , lane 2). Skin prick test results for patients A–G are shown for comparative purposes (Table  1 ) and represent the clinically determined reactivity of each patient to extracts of the tested fungal species. Patients (A–G) were selected based skin prick reactivity to at least 4 different fungi with scores of 2 or greater. Identical concentrations of  B. bassiana  extract (40 μg) were resolved by SDS-PAGE, blotted to PVDF membranes, and the lanes were cut into separate strips. Each strip was treated with a 1:5 dilution of each respective serum as described in the Materials and Methods (Fig.  3 , lane 2 is the sera pool). A total of 16 individual sera were tested, with the sera from three patients displaying no IgEs reactive to proteins present in the  B. bassiana  extracts. The results for the remaining 13 sera are shown in Fig.  2 . The data show a large individual variation in serum IgEs capable of binding epitopes present in  B. bassiana  extracts, both in terms of banding distribution and the intensity of the reaction. No correlation was observed between measurements of total IgE and the observed binding to  B. bassiana  allergens. Some patients displayed strong reactions to multiple bands, whereas others to a more limited set of epitopes. Distinct strongly reactive bands ranging from 40 kDa to approximately 200 kDa could be seen in sera A, E, and to a lesser extent L. A strongly reactive 35 kDa band was visible in sera C, G, E, and L. Several sera displayed IgEs that bound to only a limited set of 2–3 allergens (C, F, G, weak bands in B, I, J, K, and M). Blots probed with one serum (H) resulted in a large smear ranging from ~30 kDa to 55 kDa. The reason for the observed smear is unknown and efforts to distinguish separate bands by manipulating the concentrations of either sera or extract were unsuccessful. A number of bands (based upon molecular mass) appeared to be common to several sera including proteins of approximately 35, 42–48, and 60 kDa. A number of allergens of high molecular weight (~100–200 kDa) were also visible; however the resolution in this range on the Western blots is poor. Figure 2 Immunoprint analysis of  B. bassiana  extracts (40 μg crude extract/strip) probed with individual sera. Lane 1) 5 μg protein standards, 2) pooled serum mix-II (patients displaying mould allergies). Lanes A)–G) membrane strips treated with individual sera, Lanes H)–M) membrane strips probed with individuals having had occupational exposure to  B. bassiana  and other fungi (see intra-dermal skin test results for individuals J–M, Table 2). Figure 3 IgE immunoblot inhibition with fungi.  B. bassiana  protein strips (40 μg crude extract) were blocked and incubated with mix containing (500 μl) pooled sera (mix-II) and 2) no additions, 3) 40 μg  Alternaria alternata  crude extract, 4) 400 μg  Alternaria alternata , 5) 40 μg  Aspergillus fumigatus , 6) 400 μg  Aspergillus fumigatus , 7) 400 μg  Cladosporium herbarum , 8) 400 μg  Candida albicans , 9) 400 μg  Epicoccum purpurascens , and 10) 400 μg  Penicillium notatum  protein. Intradermal Skin Testing A total of ten individuals were tested for allergenic reactivity to  B. bassiana  crude extracts using an intradermal delivery procedure. Data using 1 mg/ml  B. bassiana  crude extract and histamine controls are presented in Table  2 . Seven out of the ten individuals (ID #s, J–O, and Q) displayed skin reactivity reactions to the  B. bassiana  extracts (Table  2 , also see corresponding Western blot results for individuals J, K, L, and M; Fig.  2 ). It is interesting to note that 4 (J–M) of 5 individuals (plus S) that have had occupational exposure to  B. bassiana  displayed skin reactivity as well as bands on Western blots. A preliminary correlation was observed between the  B. bassiana /histamine ration and the  in vitro  reactivity of individual sera in Western blots. Individuals J, K, and M, displayed  B. bassiana /histamine control ratios <1, also showed weak bands in Western blots (Fig.  2 ), whereas individual L who had a  B. bassiana /histamine ratio = 1.65, reacted against numerous epitopes in the extract and with a higher intensity. Table 2 Intradermal skin test results using  B. bassiana  extract Patient ID Histamine control 1  (0.1 mg/ml) B. bassiana  Extract (1 mg/ml) B. bassiana /Histamine Induration 2 Erythema 2 Induration 2 Erythema 2 Induration ratio 3 J 4,5 7 × 6 12 × 16 8 × 8 12 × 13 0.65 K 4,5 20 × 15 55 × 50 13 × 12 14 × 13 0.52 L 4,5 11 × 10 16 × 33 13 × 14 26 × 28 1.65 M 4,5 15 × 16 36 × 44 10 × 12 10 × 12 0.30 N 16 × 14 38 × 58 10 × 11 21 × 17 0.49 O 21 × 16 39 × 59 9 × 8 18 × 21 0.21 P 15 × 17 44 × 45 5 × 4 5 × 4 0.08 Q 15 × 14 36 × 38 9 × 12 10 × 13 0.51 R 15 × 15 55 × 38 4 × 4 11 × 13 0.07 S 4 20 × 19 38 × 43 4 × 4 4 × 4 0.04 1 In all instances saline control injections produced indurations of 3–4 mm × 3–4 mm. 2 Values recorded in mm × mm. 3 Induration ratio expressed as  B. bassiana  reaction area (mm 2 )/histamine reaction area (mm 2 ). 4 Individual with occupational exposure to  Beauveria bassiana . 5 See Western Blot results for individual, Fig. 2. Cross-reactivity among different fungi In order to determine the extent of cross-reactivity of  B. bassiana  allergens with other fungi, immunoblot inhibition experiments were performed. Identical concentrations of  B. bassiana  crude extract (40 μg) were resolved by SDS-PAGE, blotted to PVDF membranes, and lanes were cut into separate strips. Each strip was treated with a 1:5 dilution pooled sera (serum mix-II) as the primary antibody supplemented with concentrations of fungal crude extracts as described in the Materials and Methods. Fig.  3  shows Western blots in which the binding of human IgEs to allergens present in  B. bassiana  extracts were competed with: excess crude extracts from  Alternaria alternata  (Fig  3 , lanes 3, 4),  Aspergillus fumigatus  (lanes 5, 6),  Cladosporium herbarum  (lanes 7) , Epicoccum purpurascens  (Lane 8) , Penicillium notatum  (lane 9) ,  and  Candida albicans  (lane 10). There was complete loss of all signals using 2-fold excess  B. bassiana  extract as the competitor (data not shown). These data indicate that while  Beauveria  possess many epitopes in common with several other fungi, notably  Alternaria  and  Penicillium , a 35-kDa major reactive band was not inhibited by any extract tested.
Results
Identification of IgE reactive bands An ammonium sulfate fraction of  B. bassiana  proteins was resolved on SDS-PAGE (Fig.  1 , lane B) and transferred to PVDF membranes as described in the Materials and Methods. Membranes were probed with sera from individual patients who were reactive to various moulds (Table  1 ), which was pooled and used to demonstrate IgE reactivity against antigens present in  B. bassiana  extracts (Fig.  1 ). Serum mix-I represents pooled sera derived from patients E, J, K, L, and M, as well as three additional patients that were only tested (skin prick) against  Aspergillus  and  Penicillium , displaying test scores of 3–4 for each. These data demonstrate human IgE binding of allergens present in  B. bassiana  extracts. Initial blots showed 12–15 distinct reactive protein bands, ranging in molecular mass from 12 kDa to >95 kDa (under denaturing conditions); with the most prominent bands located around 64, 45, and 35 kDa. Control experiments omitting either the primary or secondary antibody incubation steps resulted in complete loss of signal. Proteinase K digestion of samples also resulted in loss of all signal (Fig.  1 , lane 4), indicating the proteinaceous nature of the IgE reactive bands. Since the carbohydrate moieties of several protein allergens are known to play a role in their allergenicity and even cross-reactivity [ 20 - 22 ], samples were treated with the deglyocosylating enzymes EndoH and PNGaseF. Control experiments incubating samples in the EndoH denaturing buffer without any enzyme altered the IgE-reactive signals (Fig.  1 , lane 5), however, samples treated with EndoH did not appear any different than control reactions (Fig.  1 , lane 6). Similar results were obtained in PNGaseF digests (data not shown). These data appear to indicate that the  B. bassiana  IgE-antigen profiles observed on Western blots are proteins with minimal contributions due to glycosylation. Figure 1 SDS-PAGE and immunoblot analysis of  Beauveria bassiana  crude extracts. SDS-PAGE, Gelcode blue stained, lanes A) 5 μg protein standards, and B) 40 μg  B. bassiana  crude extract. Immunoblots probed with pooled serum mix-I (patients displaying mould allergies), lanes 1), 5 μg protein standards, 2) 20 μg  B. bassiana  crude extract, 3) 40 μg crude extract, 4) 40 μg crude extract, Proteinase K treated, 5) 40 μg crude extract, denaturing buffer control (no EndoH), 6) 40 μg crude extract, EndoH treated Table 1 Allergic profile of patients A–G, obtained by skin prick testing. Patient ID Individual Skin Reactivity to Fungal Extracts* Alt † Asp Cep Cur Epi Fusa Helmin Hormo Pen A 3 2 0 3 2 2 0 0 3 B 3 2 0 2 3 2 2 2 0 C 4 0 0 3 0 2 0 0 0 D 0 0 2 2 0 2 0 2 2 E 3 2 0 3 2 3 3 0 0 F 4 1 1 2 4 0 2 0 0 G 3 0 0 4 3 0 2 0 0 *Skin test scores were registered using a relative scale from 0–4 with 4 representing the highest reactivity as described in the Materials and Methods.  † Abbreviations used;  Alt ,  Alternaria tenius ;  Asp ,  Aspergillus fumigatus ;  Cep ,  Cephalosporium  ( Acremonium strictum );  Cur ,  Curvularia  spp.  Bipolaris ;  Epi ,  Epicoccum nigram ;  Fusa ,  Fusarium  spp.;  Helmin ,  Helminthosporium sativum ;  Hormo ,  Hormodendrum horde ;  Pen ,  Penicillium  (mixed,  P. chrysogenum  and  P. notatum ).
Identification of IgE reactive bands
An ammonium sulfate fraction of  B. bassiana  proteins was resolved on SDS-PAGE (Fig.  1 , lane B) and transferred to PVDF membranes as described in the Materials and Methods. Membranes were probed with sera from individual patients who were reactive to various moulds (Table  1 ), which was pooled and used to demonstrate IgE reactivity against antigens present in  B. bassiana  extracts (Fig.  1 ). Serum mix-I represents pooled sera derived from patients E, J, K, L, and M, as well as three additional patients that were only tested (skin prick) against  Aspergillus  and  Penicillium , displaying test scores of 3–4 for each. These data demonstrate human IgE binding of allergens present in  B. bassiana  extracts. Initial blots showed 12–15 distinct reactive protein bands, ranging in molecular mass from 12 kDa to >95 kDa (under denaturing conditions); with the most prominent bands located around 64, 45, and 35 kDa. Control experiments omitting either the primary or secondary antibody incubation steps resulted in complete loss of signal. Proteinase K digestion of samples also resulted in loss of all signal (Fig.  1 , lane 4), indicating the proteinaceous nature of the IgE reactive bands. Since the carbohydrate moieties of several protein allergens are known to play a role in their allergenicity and even cross-reactivity [ 20 - 22 ], samples were treated with the deglyocosylating enzymes EndoH and PNGaseF. Control experiments incubating samples in the EndoH denaturing buffer without any enzyme altered the IgE-reactive signals (Fig.  1 , lane 5), however, samples treated with EndoH did not appear any different than control reactions (Fig.  1 , lane 6). Similar results were obtained in PNGaseF digests (data not shown). These data appear to indicate that the  B. bassiana  IgE-antigen profiles observed on Western blots are proteins with minimal contributions due to glycosylation.B. bassianaB. bassianaAspergillusPenicilliumB. bassianaB. bassianaAllergic profile of patients A–G, obtained by skin prick testing.AltAspCepCurEpiFusaHelminHormoPen*Skin test scores were registered using a relative scale from 0–4 with 4 representing the highest reactivity as described in the Materials and Methods.  † Abbreviations used;  Alt ,  Alternaria tenius ;  Asp ,  Aspergillus fumigatus ;  Cep ,  Cephalosporium  ( Acremonium strictum );  Cur ,  Curvularia  spp.  Bipolaris ;  Epi ,  Epicoccum nigram ;  Fusa ,  Fusarium  spp.;  Helmin ,  Helminthosporium sativum ;  Hormo ,  Hormodendrum horde ;  Pen ,  Penicillium  (mixed,  P. chrysogenum  and  P. notatum ).AltAlternaria teniusAspAspergillus fumigatusCepCephalosporiumAcremonium strictumCurCurvulariaBipolarisEpiEpicoccum nigramFusaFusariumHelminHelminthosporium sativumHormoHormodendrum hordePenPenicilliumP. chrysogenumP. notatumImmunoprint Analysis of  B. bassiana : Reactivity with Individual Sera In order to determine the variation and distribution of serum IgEs reactive to  B. bassiana  extracts, individual sera from patients displaying mould allergies (Fig.  2 , lanes A–G) as well as random sera from the general population (Fig.  2 , lanes H–M) were used as probes for Western blots (Fig.  2 ). The reactivity of pooled sera from patients A–G (termed serum mix-II) is also shown (Fig.  2 , lane 2). Skin prick test results for patients A–G are shown for comparative purposes (Table  1 ) and represent the clinically determined reactivity of each patient to extracts of the tested fungal species. Patients (A–G) were selected based skin prick reactivity to at least 4 different fungi with scores of 2 or greater. Identical concentrations of  B. bassiana  extract (40 μg) were resolved by SDS-PAGE, blotted to PVDF membranes, and the lanes were cut into separate strips. Each strip was treated with a 1:5 dilution of each respective serum as described in the Materials and Methods (Fig.  3 , lane 2 is the sera pool). A total of 16 individual sera were tested, with the sera from three patients displaying no IgEs reactive to proteins present in the  B. bassiana  extracts. The results for the remaining 13 sera are shown in Fig.  2 . The data show a large individual variation in serum IgEs capable of binding epitopes present in  B. bassiana  extracts, both in terms of banding distribution and the intensity of the reaction. No correlation was observed between measurements of total IgE and the observed binding to  B. bassiana  allergens. Some patients displayed strong reactions to multiple bands, whereas others to a more limited set of epitopes. Distinct strongly reactive bands ranging from 40 kDa to approximately 200 kDa could be seen in sera A, E, and to a lesser extent L. A strongly reactive 35 kDa band was visible in sera C, G, E, and L. Several sera displayed IgEs that bound to only a limited set of 2–3 allergens (C, F, G, weak bands in B, I, J, K, and M). Blots probed with one serum (H) resulted in a large smear ranging from ~30 kDa to 55 kDa. The reason for the observed smear is unknown and efforts to distinguish separate bands by manipulating the concentrations of either sera or extract were unsuccessful. A number of bands (based upon molecular mass) appeared to be common to several sera including proteins of approximately 35, 42–48, and 60 kDa. A number of allergens of high molecular weight (~100–200 kDa) were also visible; however the resolution in this range on the Western blots is poor. Figure 2 Immunoprint analysis of  B. bassiana  extracts (40 μg crude extract/strip) probed with individual sera. Lane 1) 5 μg protein standards, 2) pooled serum mix-II (patients displaying mould allergies). Lanes A)–G) membrane strips treated with individual sera, Lanes H)–M) membrane strips probed with individuals having had occupational exposure to  B. bassiana  and other fungi (see intra-dermal skin test results for individuals J–M, Table 2). Figure 3 IgE immunoblot inhibition with fungi.  B. bassiana  protein strips (40 μg crude extract) were blocked and incubated with mix containing (500 μl) pooled sera (mix-II) and 2) no additions, 3) 40 μg  Alternaria alternata  crude extract, 4) 400 μg  Alternaria alternata , 5) 40 μg  Aspergillus fumigatus , 6) 400 μg  Aspergillus fumigatus , 7) 400 μg  Cladosporium herbarum , 8) 400 μg  Candida albicans , 9) 400 μg  Epicoccum purpurascens , and 10) 400 μg  Penicillium notatum  protein.
Immunoprint Analysis of  B. bassiana : Reactivity with Individual Sera
B. bassianaIn order to determine the variation and distribution of serum IgEs reactive to  B. bassiana  extracts, individual sera from patients displaying mould allergies (Fig.  2 , lanes A–G) as well as random sera from the general population (Fig.  2 , lanes H–M) were used as probes for Western blots (Fig.  2 ). The reactivity of pooled sera from patients A–G (termed serum mix-II) is also shown (Fig.  2 , lane 2). Skin prick test results for patients A–G are shown for comparative purposes (Table  1 ) and represent the clinically determined reactivity of each patient to extracts of the tested fungal species. Patients (A–G) were selected based skin prick reactivity to at least 4 different fungi with scores of 2 or greater. Identical concentrations of  B. bassiana  extract (40 μg) were resolved by SDS-PAGE, blotted to PVDF membranes, and the lanes were cut into separate strips. Each strip was treated with a 1:5 dilution of each respective serum as described in the Materials and Methods (Fig.  3 , lane 2 is the sera pool). A total of 16 individual sera were tested, with the sera from three patients displaying no IgEs reactive to proteins present in the  B. bassiana  extracts. The results for the remaining 13 sera are shown in Fig.  2 . The data show a large individual variation in serum IgEs capable of binding epitopes present in  B. bassiana  extracts, both in terms of banding distribution and the intensity of the reaction. No correlation was observed between measurements of total IgE and the observed binding to  B. bassiana  allergens. Some patients displayed strong reactions to multiple bands, whereas others to a more limited set of epitopes. Distinct strongly reactive bands ranging from 40 kDa to approximately 200 kDa could be seen in sera A, E, and to a lesser extent L. A strongly reactive 35 kDa band was visible in sera C, G, E, and L. Several sera displayed IgEs that bound to only a limited set of 2–3 allergens (C, F, G, weak bands in B, I, J, K, and M). Blots probed with one serum (H) resulted in a large smear ranging from ~30 kDa to 55 kDa. The reason for the observed smear is unknown and efforts to distinguish separate bands by manipulating the concentrations of either sera or extract were unsuccessful. A number of bands (based upon molecular mass) appeared to be common to several sera including proteins of approximately 35, 42–48, and 60 kDa. A number of allergens of high molecular weight (~100–200 kDa) were also visible; however the resolution in this range on the Western blots is poor.B. bassianaB. bassianaB. bassianaB. bassianaB. bassianaIntradermal Skin Testing A total of ten individuals were tested for allergenic reactivity to  B. bassiana  crude extracts using an intradermal delivery procedure. Data using 1 mg/ml  B. bassiana  crude extract and histamine controls are presented in Table  2 . Seven out of the ten individuals (ID #s, J–O, and Q) displayed skin reactivity reactions to the  B. bassiana  extracts (Table  2 , also see corresponding Western blot results for individuals J, K, L, and M; Fig.  2 ). It is interesting to note that 4 (J–M) of 5 individuals (plus S) that have had occupational exposure to  B. bassiana  displayed skin reactivity as well as bands on Western blots. A preliminary correlation was observed between the  B. bassiana /histamine ration and the  in vitro  reactivity of individual sera in Western blots. Individuals J, K, and M, displayed  B. bassiana /histamine control ratios <1, also showed weak bands in Western blots (Fig.  2 ), whereas individual L who had a  B. bassiana /histamine ratio = 1.65, reacted against numerous epitopes in the extract and with a higher intensity. Table 2 Intradermal skin test results using  B. bassiana  extract Patient ID Histamine control 1  (0.1 mg/ml) B. bassiana  Extract (1 mg/ml) B. bassiana /Histamine Induration 2 Erythema 2 Induration 2 Erythema 2 Induration ratio 3 J 4,5 7 × 6 12 × 16 8 × 8 12 × 13 0.65 K 4,5 20 × 15 55 × 50 13 × 12 14 × 13 0.52 L 4,5 11 × 10 16 × 33 13 × 14 26 × 28 1.65 M 4,5 15 × 16 36 × 44 10 × 12 10 × 12 0.30 N 16 × 14 38 × 58 10 × 11 21 × 17 0.49 O 21 × 16 39 × 59 9 × 8 18 × 21 0.21 P 15 × 17 44 × 45 5 × 4 5 × 4 0.08 Q 15 × 14 36 × 38 9 × 12 10 × 13 0.51 R 15 × 15 55 × 38 4 × 4 11 × 13 0.07 S 4 20 × 19 38 × 43 4 × 4 4 × 4 0.04 1 In all instances saline control injections produced indurations of 3–4 mm × 3–4 mm. 2 Values recorded in mm × mm. 3 Induration ratio expressed as  B. bassiana  reaction area (mm 2 )/histamine reaction area (mm 2 ). 4 Individual with occupational exposure to  Beauveria bassiana . 5 See Western Blot results for individual, Fig. 2.
Intradermal Skin Testing
A total of ten individuals were tested for allergenic reactivity to  B. bassiana  crude extracts using an intradermal delivery procedure. Data using 1 mg/ml  B. bassiana  crude extract and histamine controls are presented in Table  2 . Seven out of the ten individuals (ID #s, J–O, and Q) displayed skin reactivity reactions to the  B. bassiana  extracts (Table  2 , also see corresponding Western blot results for individuals J, K, L, and M; Fig.  2 ). It is interesting to note that 4 (J–M) of 5 individuals (plus S) that have had occupational exposure to  B. bassiana  displayed skin reactivity as well as bands on Western blots. A preliminary correlation was observed between the  B. bassiana /histamine ration and the  in vitro  reactivity of individual sera in Western blots. Individuals J, K, and M, displayed  B. bassiana /histamine control ratios <1, also showed weak bands in Western blots (Fig.  2 ), whereas individual L who had a  B. bassiana /histamine ratio = 1.65, reacted against numerous epitopes in the extract and with a higher intensity.B. bassianaB. bassianaB. bassianaB. bassianaB. bassianain vitroB. bassianaB. bassianaIntradermal skin test results using  B. bassiana  extractB. bassianaB. bassianaB. bassiana1 In all instances saline control injections produced indurations of 3–4 mm × 3–4 mm.2 Values recorded in mm × mm.3 Induration ratio expressed as  B. bassiana  reaction area (mm 2 )/histamine reaction area (mm 2 ).B. bassiana4 Individual with occupational exposure to  Beauveria bassiana .Beauveria bassiana5 See Western Blot results for individual, Fig. 2.Cross-reactivity among different fungi In order to determine the extent of cross-reactivity of  B. bassiana  allergens with other fungi, immunoblot inhibition experiments were performed. Identical concentrations of  B. bassiana  crude extract (40 μg) were resolved by SDS-PAGE, blotted to PVDF membranes, and lanes were cut into separate strips. Each strip was treated with a 1:5 dilution pooled sera (serum mix-II) as the primary antibody supplemented with concentrations of fungal crude extracts as described in the Materials and Methods. Fig.  3  shows Western blots in which the binding of human IgEs to allergens present in  B. bassiana  extracts were competed with: excess crude extracts from  Alternaria alternata  (Fig  3 , lanes 3, 4),  Aspergillus fumigatus  (lanes 5, 6),  Cladosporium herbarum  (lanes 7) , Epicoccum purpurascens  (Lane 8) , Penicillium notatum  (lane 9) ,  and  Candida albicans  (lane 10). There was complete loss of all signals using 2-fold excess  B. bassiana  extract as the competitor (data not shown). These data indicate that while  Beauveria  possess many epitopes in common with several other fungi, notably  Alternaria  and  Penicillium , a 35-kDa major reactive band was not inhibited by any extract tested.
Cross-reactivity among different fungi
In order to determine the extent of cross-reactivity of  B. bassiana  allergens with other fungi, immunoblot inhibition experiments were performed. Identical concentrations of  B. bassiana  crude extract (40 μg) were resolved by SDS-PAGE, blotted to PVDF membranes, and lanes were cut into separate strips. Each strip was treated with a 1:5 dilution pooled sera (serum mix-II) as the primary antibody supplemented with concentrations of fungal crude extracts as described in the Materials and Methods. Fig.  3  shows Western blots in which the binding of human IgEs to allergens present in  B. bassiana  extracts were competed with: excess crude extracts from  Alternaria alternata  (Fig  3 , lanes 3, 4),  Aspergillus fumigatus  (lanes 5, 6),  Cladosporium herbarum  (lanes 7) , Epicoccum purpurascens  (Lane 8) , Penicillium notatum  (lane 9) ,  and  Candida albicans  (lane 10). There was complete loss of all signals using 2-fold excess  B. bassiana  extract as the competitor (data not shown). These data indicate that while  Beauveria  possess many epitopes in common with several other fungi, notably  Alternaria  and  Penicillium , a 35-kDa major reactive band was not inhibited by any extract tested.B. bassianaB. bassianaB. bassianaAlternaria alternataAspergillus fumigatusCladosporium herbarum, Epicoccum purpurascens, Penicillium notatum,Candida albicansB. bassianaBeauveriaAlternariaPenicilliumDiscussion Although it is well known that fungi are important triggers of respiratory allergies, the potential allergenicity of entomopathogenic fungi used in biocontrol has largely been untested. Aerobiological surveys of conidial fungi and skin sensitivity tests to fungal extracts performed in the 1980s in the Netherlands revealed that although  Beauveria  could barely be detected in airborne samples, and represented less than 0.1% of the airborne fungal "flora", the incidence of allergic skin test reaction to  Beauveria  was the highest of all fungal species tested [ 10 , 23 , 24 ]. In rural areas, the use of fungi in agricultural pest management practices can greatly increase the potential for human exposure to these agents. Likewise, in urban settings, the commercialization of fungal products for household use may potentiate a much wider problem since indoor air concentrations of the moulds can greatly increase. For these reasons, an examination of the allergenic potential of  Beauveria bassiana  is imperative. The present study demonstrated the allergenic potential of  B. bassiana  directly by intradermal skin testing of individuals and  in vitro  by revealing the presence of serum IgEs capable of binding allergens present in fungal crude extracts. Over 20 different IgE binding proteins were observed using Western blots probed with sera from patients displaying mould allergies. Results using individual sera revealed a wide variation in IgE-binding proteins between sera, although several common bands, including a protein with an apparent molecular mass of 35 kDa were visible among the sera of several patients. Our  in vitro  observations were confirmed by intradermal skin testing on individuals using  B. bassiana  extracts. While the testing sample population was small, these results indicated that our extracts were able to elicit allergic reactions in individuals, including some that have had occupational exposure to the fungus. Concentrations of ~1 mg/ml of  B. bassiana  extracts were required to elicit indurations equivalent to 0.1 mg/ml histamine in most individuals, indicating the possibility of potent allergens in the  Beauveria  extract. Interestingly, not all individuals specifically exposed to  B. bassiana  displayed allergic reactions and individuals J, K, and M (who did display mild allergic reactions, Table  2 ) did not react to the 35 KDa protein based upon Western blotting results (Fig.  2 ). We do not, however, have any quantifiable index of exposure for the individuals in our sample and any interpretations should be made with some caution. Numerous studies have revealed the presence of cross-reactive proteins among fungal species between genera [ 15 , 20 - 22 , 25 - 27 ]. In our experiments, (excess) crude extract from a test organism was added during the primary antibody (human sera) incubation. Common or shared epitopes between  B. bassiana  and the test fungus would result in a loss of signal due to competition for reactive IgEs. However, IgEs reactive to  Beauveria-specific  allergens would not be affected, resulting in no change in the corresponding reactive bands on a Western blot. Loss of a signal would indicate that a homolog or shared epitope (IgE-reactive) exists between the two fungal species, implying that primary sensitization by one organism can result in an allergic reaction when exposed to the homologous allergen of another organism. Competitive immunoblot inhibition experiments revealed significant epitope homology between  B. bassiana  and several clinically important fungi responsible for IgE-mediated allergic reactions in atopic individuals. Thus, an allergic reaction to  Beauveria  exposure may arise in patients sensitized to other fungi. Extracts from  A. alternata  and  E. purpurascens  almost completely competed with allergens present in the  B. bassiana  extract with the notable exception of the ~35 kDa allergen. Competition experiments using  A. fumigatus ,  C. herbarum ,  C. albicans , and  P. notatum  extracts also indicated the presence of many shared epitopes, although distinct (non-competed) IgE-binding  B. bassiana  proteins of 35 kDa, 64 kDa, and >200 kDa molecular mass were detectable. These proteins, particularly the 35 kDa allergens may represent  B. bassiana  specific allergens. Experiments are underway to characterize the 35 kDa allergen, which may lead to a diagnostic assay for  B. bassiana  sensitization. Finally, our analysis of potential  B. bassiana  allergens was limited to cell extracts grown under specific conditions and did not include the culture filtrate. Extracellular proteases, an important class of fungal proteins that can elicit allergenic reactions, have been characterized from a number of fungal species [ 28 - 31 ], and are likely to be present in  B. bassiana . A careful examination of culture growth conditions is also warranted in order to provide a standardized reagent for testing purposes.
Discussion
Although it is well known that fungi are important triggers of respiratory allergies, the potential allergenicity of entomopathogenic fungi used in biocontrol has largely been untested. Aerobiological surveys of conidial fungi and skin sensitivity tests to fungal extracts performed in the 1980s in the Netherlands revealed that although  Beauveria  could barely be detected in airborne samples, and represented less than 0.1% of the airborne fungal "flora", the incidence of allergic skin test reaction to  Beauveria  was the highest of all fungal species tested [ 10 , 23 , 24 ]. In rural areas, the use of fungi in agricultural pest management practices can greatly increase the potential for human exposure to these agents. Likewise, in urban settings, the commercialization of fungal products for household use may potentiate a much wider problem since indoor air concentrations of the moulds can greatly increase. For these reasons, an examination of the allergenic potential of  Beauveria bassiana  is imperative.BeauveriaBeauveriaBeauveria bassianaThe present study demonstrated the allergenic potential of  B. bassiana  directly by intradermal skin testing of individuals and  in vitro  by revealing the presence of serum IgEs capable of binding allergens present in fungal crude extracts. Over 20 different IgE binding proteins were observed using Western blots probed with sera from patients displaying mould allergies. Results using individual sera revealed a wide variation in IgE-binding proteins between sera, although several common bands, including a protein with an apparent molecular mass of 35 kDa were visible among the sera of several patients.B. bassianain vitroOur  in vitro  observations were confirmed by intradermal skin testing on individuals using  B. bassiana  extracts. While the testing sample population was small, these results indicated that our extracts were able to elicit allergic reactions in individuals, including some that have had occupational exposure to the fungus. Concentrations of ~1 mg/ml of  B. bassiana  extracts were required to elicit indurations equivalent to 0.1 mg/ml histamine in most individuals, indicating the possibility of potent allergens in the  Beauveria  extract. Interestingly, not all individuals specifically exposed to  B. bassiana  displayed allergic reactions and individuals J, K, and M (who did display mild allergic reactions, Table  2 ) did not react to the 35 KDa protein based upon Western blotting results (Fig.  2 ). We do not, however, have any quantifiable index of exposure for the individuals in our sample and any interpretations should be made with some caution.in vitroB. bassianaB. bassianaBeauveriaB. bassianaNumerous studies have revealed the presence of cross-reactive proteins among fungal species between genera [ 15 , 20 - 22 , 25 - 27 ]. In our experiments, (excess) crude extract from a test organism was added during the primary antibody (human sera) incubation. Common or shared epitopes between  B. bassiana  and the test fungus would result in a loss of signal due to competition for reactive IgEs. However, IgEs reactive to  Beauveria-specific  allergens would not be affected, resulting in no change in the corresponding reactive bands on a Western blot. Loss of a signal would indicate that a homolog or shared epitope (IgE-reactive) exists between the two fungal species, implying that primary sensitization by one organism can result in an allergic reaction when exposed to the homologous allergen of another organism. Competitive immunoblot inhibition experiments revealed significant epitope homology between  B. bassiana  and several clinically important fungi responsible for IgE-mediated allergic reactions in atopic individuals. Thus, an allergic reaction to  Beauveria  exposure may arise in patients sensitized to other fungi. Extracts from  A. alternata  and  E. purpurascens  almost completely competed with allergens present in the  B. bassiana  extract with the notable exception of the ~35 kDa allergen. Competition experiments using  A. fumigatus ,  C. herbarum ,  C. albicans , and  P. notatum  extracts also indicated the presence of many shared epitopes, although distinct (non-competed) IgE-binding  B. bassiana  proteins of 35 kDa, 64 kDa, and >200 kDa molecular mass were detectable. These proteins, particularly the 35 kDa allergens may represent  B. bassiana  specific allergens. Experiments are underway to characterize the 35 kDa allergen, which may lead to a diagnostic assay for  B. bassiana  sensitization. Finally, our analysis of potential  B. bassiana  allergens was limited to cell extracts grown under specific conditions and did not include the culture filtrate. Extracellular proteases, an important class of fungal proteins that can elicit allergenic reactions, have been characterized from a number of fungal species [ 28 - 31 ], and are likely to be present in  B. bassiana . A careful examination of culture growth conditions is also warranted in order to provide a standardized reagent for testing purposes.B. bassianaBeauveria-specificB. bassianaBeauveriaA. alternataE. purpurascensB. bassianaA. fumigatusC. herbarumC. albicansP. notatumB. bassianaB. bassianaB. bassianaB. bassianaB. bassianaConclusions Although  Beauveria  holds promise as an arthropod biological control agent, there have been few reports on the allergenic potential of these organisms. Identification of  B. bassiana  specific allergens can lead diagnostic methods for determining sensitization to this organism and may provide a rational basis for allergen attenuation in order to yield safer biocontrol products. The observed cross-reactivity among proteins of  B. bassiana  and the fungi tested, highlight the importance of considering the possibility that multiple fungal sensitivity can occur due to exposure to a single fungus. Further testing should be performed to determine the scope, severity, and range of allergenic reactions to  B. bassiana .
Conclusions
Although  Beauveria  holds promise as an arthropod biological control agent, there have been few reports on the allergenic potential of these organisms. Identification of  B. bassiana  specific allergens can lead diagnostic methods for determining sensitization to this organism and may provide a rational basis for allergen attenuation in order to yield safer biocontrol products. The observed cross-reactivity among proteins of  B. bassiana  and the fungi tested, highlight the importance of considering the possibility that multiple fungal sensitivity can occur due to exposure to a single fungus. Further testing should be performed to determine the scope, severity, and range of allergenic reactions to  B. bassiana .BeauveriaB. bassianaB. bassianaB. bassianaCompeting Interests The author(s) declare that they have no competing interests.
Competing Interests
The author(s) declare that they have no competing interests.Authors' contributions GSW carried out the immunoassays and other  in vitro  experiments. SWH performed the clinical experiments and participated in the design of the study. NOK conceived of the study, and participated in its design and coordination, and drafted the manuscript.
Authors' contributions
GSW carried out the immunoassays and other  in vitro  experiments. SWH performed the clinical experiments and participated in the design of the study. NOK conceived of the study, and participated in its design and coordination, and drafted the manuscript.in vitro


Background Early identification of permanent hearing impairment in children enables appropriate intervention which reduces adverse developmental outcomes. The UK Government has introduced a universal hearing screening programme for neonates. All involved health professionals, including those in Primary Care, need to be aware of the service to enable them to offer appropriate support to their patients. A programme of information dissemination within Primary Care was therefore undertaken. The aim of the current study was to determine the extent to which the information had reached General Practitioners (GPs), the GPs' preferred mode of dissemination and the sources from which GPs accessed information
Background
Early identification of permanent hearing impairment in children enables appropriate intervention which reduces adverse developmental outcomes. The UK Government has introduced a universal hearing screening programme for neonates. All involved health professionals, including those in Primary Care, need to be aware of the service to enable them to offer appropriate support to their patients. A programme of information dissemination within Primary Care was therefore undertaken. The aim of the current study was to determine the extent to which the information had reached General Practitioners (GPs), the GPs' preferred mode of dissemination and the sources from which GPs accessed informationMethods Postal questionnaire survey of a randomised sample of 1000 GPs in the Phase I pilot sites of the Neonatal Hearing Screening Programme (NHSP).
Methods
Postal questionnaire survey of a randomised sample of 1000 GPs in the Phase I pilot sites of the Neonatal Hearing Screening Programme (NHSP).Results Responses were received from 54.2% of the sample. Just under 50% of those responding had received information, 62.2% of respondents said they would like to receive more information and the preferred methods of dissemination were the written word and web-sites to allow access when needed. Few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject. Many are keen to delegate detail to a third party, usually the health visitor, who has traditionally had responsibility for hearing screening.
Results
Responses were received from 54.2% of the sample. Just under 50% of those responding had received information, 62.2% of respondents said they would like to receive more information and the preferred methods of dissemination were the written word and web-sites to allow access when needed. Few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject. Many are keen to delegate detail to a third party, usually the health visitor, who has traditionally had responsibility for hearing screening.Conclusions Dissemination efforts for service developments of relevance to GPs should concentrate on advertising a website address via brief but memorable posted literature and/or articles in relevant journals and magazines. The website should be GP-friendly, and have a dedicated area for GPs including information of specific relevance and downloadable information sheets.
Conclusions
Dissemination efforts for service developments of relevance to GPs should concentrate on advertising a website address via brief but memorable posted literature and/or articles in relevant journals and magazines. The website should be GP-friendly, and have a dedicated area for GPs including information of specific relevance and downloadable information sheets.

Background Approximately 1.65 per 1000 babies born in the UK have a significant permanent hearing impairment (>40 dB HL) detectable at birth [ 1 ]. Early recognition and appropriate intervention can reduce the proven negative impact on the development of communication skills [ 2 ]. Thus, based on robust evidence [ 3 ], in June 2000 the UK Government announced an initiative to introduce effective and appropriate screening for women and children by 2004 [ 4 ] which included the implementation of a novel screen for hearing impairment for all newborns in England. Although targeted hearing screening has been in place in some areas for several years, the introduction of a universal service at birth and the proposal to abandon the health visitor distraction screen at the age of 8 months, has implications for the knowledge General Practitioners (GPs) are now expected to have. Thus a crucial part of the implementation of the new service was to provide information to GPs and other primary care staff. Previous studies addressing the issue of the most appropriate method of dissemination of information to GPs are inconclusive [ 5 ]. Rapidly developing technology is adding further methods [ 6 ], the ease of which may adversely contribute to the overload. This study uses the introduction of the Newborn Hearing Screening Programme (NHSP) to illustrate the issues around dissemination to GPs [ 6 , 7 ]. Introduction of a novel screening service is a good example because a screen is only the beginning of a long process that integrates testing, diagnosis, intervention and monitoring [ 8 ], and in the case of permanent hearing impairment, long-term follow up. The National Screening Committee recognises information dissemination to be key to effective delivery of screening programmes stating that, "There is a need for all those involved in service delivery to understand the objectives and principles of the relevant screening programme(s) and be able to adequately interpret and explain the results and implications of the screening tests to patients as appropriate." [ 4 ] Primary care professionals are often the most accessible people with whom patients, in this case parents, can raise their questions. The phased implementation of NHSP in the UK began in 2001 in 23 pilot sites and was accompanied by a campaign of information dissemination including distribution of leaflets and posters to all general practices, the development of a dedicated website and appointment of local co-ordinators. The aim of the current study was to determine the extent to which the dissemination information had reached the GPs, the GPs' preferred mode of dissemination and the sources from which GPs accessed information.
Background
Approximately 1.65 per 1000 babies born in the UK have a significant permanent hearing impairment (>40 dB HL) detectable at birth [ 1 ]. Early recognition and appropriate intervention can reduce the proven negative impact on the development of communication skills [ 2 ]. Thus, based on robust evidence [ 3 ], in June 2000 the UK Government announced an initiative to introduce effective and appropriate screening for women and children by 2004 [ 4 ] which included the implementation of a novel screen for hearing impairment for all newborns in England. Although targeted hearing screening has been in place in some areas for several years, the introduction of a universal service at birth and the proposal to abandon the health visitor distraction screen at the age of 8 months, has implications for the knowledge General Practitioners (GPs) are now expected to have. Thus a crucial part of the implementation of the new service was to provide information to GPs and other primary care staff. Previous studies addressing the issue of the most appropriate method of dissemination of information to GPs are inconclusive [ 5 ]. Rapidly developing technology is adding further methods [ 6 ], the ease of which may adversely contribute to the overload.This study uses the introduction of the Newborn Hearing Screening Programme (NHSP) to illustrate the issues around dissemination to GPs [ 6 , 7 ]. Introduction of a novel screening service is a good example because a screen is only the beginning of a long process that integrates testing, diagnosis, intervention and monitoring [ 8 ], and in the case of permanent hearing impairment, long-term follow up. The National Screening Committee recognises information dissemination to be key to effective delivery of screening programmes stating that, "There is a need for all those involved in service delivery to understand the objectives and principles of the relevant screening programme(s) and be able to adequately interpret and explain the results and implications of the screening tests to patients as appropriate." [ 4 ] Primary care professionals are often the most accessible people with whom patients, in this case parents, can raise their questions.The phased implementation of NHSP in the UK began in 2001 in 23 pilot sites and was accompanied by a campaign of information dissemination including distribution of leaflets and posters to all general practices, the development of a dedicated website and appointment of local co-ordinators. The aim of the current study was to determine the extent to which the dissemination information had reached the GPs, the GPs' preferred mode of dissemination and the sources from which GPs accessed information.Methods We designed and piloted a one-page questionnaire [see  Additional file 1 ] and used it to conduct a postal questionnaire survey early in 2003 of 1000 general practitioners in ten purposively sampled primary care trusts implementing the NHSP in the pilot sites. Five of the sites were those implementing the screen as a community based service and the remaining five were implementing the screen as a hospital based service sampled to match the community sites for annual birth rate. In each site 100 GPs were randomly selected to receive the questionnaire incorporating the restriction of only one questionnaire per practice. Identification of non-respondents was not possible as all information on returned questionnaires was anonymous. A reminder would have involved a total mailing. This option was rejected for reasons of inappropriate mailings to those GPs who had responded and cost.
Methods
We designed and piloted a one-page questionnaire [see  Additional file 1 ] and used it to conduct a postal questionnaire survey early in 2003 of 1000 general practitioners in ten purposively sampled primary care trusts implementing the NHSP in the pilot sites. Five of the sites were those implementing the screen as a community based service and the remaining five were implementing the screen as a hospital based service sampled to match the community sites for annual birth rate. In each site 100 GPs were randomly selected to receive the questionnaire incorporating the restriction of only one questionnaire per practice. Identification of non-respondents was not possible as all information on returned questionnaires was anonymous. A reminder would have involved a total mailing. This option was rejected for reasons of inappropriate mailings to those GPs who had responded and cost.Results Response rate Of the 960 questionnaires not returned by the Post Office and therefore assumed to have reached their destination, 520 (54.2%) were returned at least partially completed. The answers from the questionnaire are summarised in Table  1 . Table 1 Positive response to questionnaire items Total number of responses Positive response N % Information received: Received any information 506 252 49.8 Seen leaflets 508 125 24.6 Visited website 505 12 2.4 Got poster 495 64 12.9 Knows contact details of local co-ordinator 503 78 15.5 Information requested: 429 267 62.2 Via written information 104 39.0 Via website 82 30.7 Via seminars 39 14.6 Via open day 16 6.0 Via meetings 15 5.6 Via e-mail 4 1.5 Information sources: Meetings: Existence of local GP forum 436 268 61.5 Attend local GP forum 39 14.6 Attend local child health seminar 437 84 19.2 Journals read: British Medical Journal 457 363 79.4 British Journal of General Practice 457 142 31.1 The Lancet 457 4 0.9 Update 457 194 42.5 BMA News 457 165 36.1 PCT Newsletter 457 144 31.5 Practitioner 457 130 28.4 Pulse 457 32 7.0 GP 457 13 2.8 Doctor 457 6 1.3 Information received The responses indicate that just less than half of our sample had received any information about NHSP. Of those, half had seen (but not necessarily read) the leaflets but only 2.4% of responders (4.8% of those receiving information) had visited the website. Information requested When asked if they would like more information about NHSP 62.2% said yes. Of those, the route most commonly cited was via the written word (39.0%) presumably leaflets and letters but 82 (30.7%) requested information via a website. Meetings and e-mail notification were not popular preferences. Information sources To ensure that information reaches the target audience we should consider from which sources they get their information. Here it was clear that attending meetings is not common, less than 20% of respondents attended the local GP forum or child health seminar but GPs do read journals and magazines, notably the British Medical Journal (79.4%), Update (42.5%) and the British Journal of General Practice (31.1%). Open comments In open comments (N = 84 respondents, 16.6%) it appeared that many of those that had received information had actually heard about the programme from the media or their own patients. A key theme in the comments was the delegation of responsibility for the knowledge to a colleague. While few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject, many are keen to delegate detail to a colleague, usually the health visitor, who has traditionally had responsibility for hearing screening.
Results
Response rate Of the 960 questionnaires not returned by the Post Office and therefore assumed to have reached their destination, 520 (54.2%) were returned at least partially completed. The answers from the questionnaire are summarised in Table  1 . Table 1 Positive response to questionnaire items Total number of responses Positive response N % Information received: Received any information 506 252 49.8 Seen leaflets 508 125 24.6 Visited website 505 12 2.4 Got poster 495 64 12.9 Knows contact details of local co-ordinator 503 78 15.5 Information requested: 429 267 62.2 Via written information 104 39.0 Via website 82 30.7 Via seminars 39 14.6 Via open day 16 6.0 Via meetings 15 5.6 Via e-mail 4 1.5 Information sources: Meetings: Existence of local GP forum 436 268 61.5 Attend local GP forum 39 14.6 Attend local child health seminar 437 84 19.2 Journals read: British Medical Journal 457 363 79.4 British Journal of General Practice 457 142 31.1 The Lancet 457 4 0.9 Update 457 194 42.5 BMA News 457 165 36.1 PCT Newsletter 457 144 31.5 Practitioner 457 130 28.4 Pulse 457 32 7.0 GP 457 13 2.8 Doctor 457 6 1.3
Response rate
Of the 960 questionnaires not returned by the Post Office and therefore assumed to have reached their destination, 520 (54.2%) were returned at least partially completed. The answers from the questionnaire are summarised in Table  1 .Positive response to questionnaire itemsInformation received:Received any informationInformation requested:Via written informationVia written information10439.0Via websiteVia website8230.7Via seminarsVia seminars3914.6Via open dayVia open day166.0Via meetingsVia meetings155.6Via e-mailVia e-mail41.5Meetings:Existence of local GP forumAttend local GP forumAttend local GP forum3914.6Attend local child health seminarJournals read:British Medical JournalBritish Journal of General PracticeThe LancetUpdateBMA NewsPCT NewsletterPractitionerPulseGPDoctorInformation received The responses indicate that just less than half of our sample had received any information about NHSP. Of those, half had seen (but not necessarily read) the leaflets but only 2.4% of responders (4.8% of those receiving information) had visited the website.
Information received
The responses indicate that just less than half of our sample had received any information about NHSP. Of those, half had seen (but not necessarily read) the leaflets but only 2.4% of responders (4.8% of those receiving information) had visited the website.Information requested When asked if they would like more information about NHSP 62.2% said yes. Of those, the route most commonly cited was via the written word (39.0%) presumably leaflets and letters but 82 (30.7%) requested information via a website. Meetings and e-mail notification were not popular preferences.
Information requested
When asked if they would like more information about NHSP 62.2% said yes. Of those, the route most commonly cited was via the written word (39.0%) presumably leaflets and letters but 82 (30.7%) requested information via a website. Meetings and e-mail notification were not popular preferences.Information sources To ensure that information reaches the target audience we should consider from which sources they get their information. Here it was clear that attending meetings is not common, less than 20% of respondents attended the local GP forum or child health seminar but GPs do read journals and magazines, notably the British Medical Journal (79.4%), Update (42.5%) and the British Journal of General Practice (31.1%).
Information sources
To ensure that information reaches the target audience we should consider from which sources they get their information. Here it was clear that attending meetings is not common, less than 20% of respondents attended the local GP forum or child health seminar but GPs do read journals and magazines, notably the British Medical Journal (79.4%), Update (42.5%) and the British Journal of General Practice (31.1%).Open comments In open comments (N = 84 respondents, 16.6%) it appeared that many of those that had received information had actually heard about the programme from the media or their own patients. A key theme in the comments was the delegation of responsibility for the knowledge to a colleague. While few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject, many are keen to delegate detail to a colleague, usually the health visitor, who has traditionally had responsibility for hearing screening.
Open comments
In open comments (N = 84 respondents, 16.6%) it appeared that many of those that had received information had actually heard about the programme from the media or their own patients. A key theme in the comments was the delegation of responsibility for the knowledge to a colleague. While few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject, many are keen to delegate detail to a colleague, usually the health visitor, who has traditionally had responsibility for hearing screening.Discussion The response rate of more than 50% is acceptable for a survey of GPs. A second mailing might have improved this but its advantage was weighed against the attraction of anonymity. To minimise the number of questions and encourage response, no demographic details were requested. Analysis of any response bias by age of practitioner, size of practice etc was therefore not possible. A response might be more likely from people who knew something of the subject and our estimates for the level of awareness might therefore be high but it is likely that the views expressed concerning resources accessed and preferred channels of dissemination are representative. Although considerable effort had been expended in distributing letters, leaflets and posters to every general practice, less than half the respondents said they had seen it. It is possible that mail had been screened and intercepted by administrative staff in the practice or rejected as "junk mail" by the GP. It is important therefore to ensure such mailed information is identified as relevant. Some health service developments will not be high on the average GP's agenda. In terms of neonatal hearing screening s/he will see only 20 newborns per year on average and approximately one permanently hearing-impaired child in a working lifetime. Nearly two-thirds of respondents said they would like to receive more information even though many complained about "information overload". This is a weakness inherent in surveys of this nature where stated attitudes may reflect socially desirable responses rather than respondents' true attitudes. Further exploration of this issue using qualitative techniques of personal interview would contribute to the general issue of information dissemination to GPs. GPs cannot know everything about everything and do not necessarily need to but they do need to know where they can find the relevant information and to be able to access it easily [ 9 ]. Our survey indicates that brief written information combined with the internet would be useful routes to use.
Discussion
The response rate of more than 50% is acceptable for a survey of GPs. A second mailing might have improved this but its advantage was weighed against the attraction of anonymity. To minimise the number of questions and encourage response, no demographic details were requested. Analysis of any response bias by age of practitioner, size of practice etc was therefore not possible. A response might be more likely from people who knew something of the subject and our estimates for the level of awareness might therefore be high but it is likely that the views expressed concerning resources accessed and preferred channels of dissemination are representative.Although considerable effort had been expended in distributing letters, leaflets and posters to every general practice, less than half the respondents said they had seen it. It is possible that mail had been screened and intercepted by administrative staff in the practice or rejected as "junk mail" by the GP. It is important therefore to ensure such mailed information is identified as relevant. Some health service developments will not be high on the average GP's agenda. In terms of neonatal hearing screening s/he will see only 20 newborns per year on average and approximately one permanently hearing-impaired child in a working lifetime.Nearly two-thirds of respondents said they would like to receive more information even though many complained about "information overload". This is a weakness inherent in surveys of this nature where stated attitudes may reflect socially desirable responses rather than respondents' true attitudes. Further exploration of this issue using qualitative techniques of personal interview would contribute to the general issue of information dissemination to GPs.GPs cannot know everything about everything and do not necessarily need to but they do need to know where they can find the relevant information and to be able to access it easily [ 9 ]. Our survey indicates that brief written information combined with the internet would be useful routes to use.Conclusions We suggest that any dissemination effort for service developments of relevance to GPs should concentrate on advertising the website address via brief but memorable posted literature and/or articles in relevant journals and magazines. It is also crucial that any website be GP-friendly by having a dedicated area for GPs including information of specific relevance and downloadable information sheets.
Conclusions
We suggest that any dissemination effort for service developments of relevance to GPs should concentrate on advertising the website address via brief but memorable posted literature and/or articles in relevant journals and magazines. It is also crucial that any website be GP-friendly by having a dedicated area for GPs including information of specific relevance and downloadable information sheets.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions PM designed and carried out the survey, performed the analyses and wrote the original report. HF supervised the design and implementation of the study and drafted the manuscript. Both authors read and approved the final manuscript.
Authors' contributions
PM designed and carried out the survey, performed the analyses and wrote the original report. HF supervised the design and implementation of the study and drafted the manuscript. Both authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:Supplementary Material Additional File 1 A survey of awareness of the NHS Newborn Hearing Screening Programme (NHSP) amongst General Practitioners (GPs) Questionnaire Click here for file
Supplementary Material
Additional File 1
A survey of awareness of the NHS Newborn Hearing Screening Programme (NHSP) amongst General Practitioners (GPs) QuestionnaireClick here for file