Allergic inflammation induced by a Penicillium chrysogenum conidia-associated allergen extract in a murine model.

BACKGROUND: Recent evidence has shown that viable conidia from the fungus Penicillium chrysogenum induce allergic effects in mice. The present study was conducted to determine the specific allergic dose response of C57BL/6 mice to the protease extract, Pen ch, isolated from viable P. chrysogenum conidia. METHODS: Mice were treated with primary intraperitoneal (IP) injections of 10 or 100 microg of Pen ch adsorbed to alum, followed by weekly IP injections of 0.1, 1.0, or 10.0 microg Pen ch with alum for 4 weeks, and with 10.0 microg of Pen ch by intranasal (IN) inoculations the final 2 weeks before killing. RESULTS: Intraperitoneal injections of 10 and 100 microg of Pen ch for 5 weeks followed by 2 weeks of IN instillation of 10 microg induced significant increases of total serum immunoglobulin (Ig)E and IgG(1). Bronchoalveolar lavage cell counts revealed increased numbers of eosinophils and neutrophils. Histopathological examination of lungs detected perivascular inflammation by eosinophils and neutrophils and increased mucous production. CONCLUSIONS: The data presented in this study indicate that sensitization to protease allergens released by viable P. chrysogenum conidia in vivo induce a strong allergic inflammatory response in a murine model, which could have implications for people exposed to high levels of conidia of this organism.

Airborne microbial flora in a cattle feedlot.

A total of 1,408 cattle held in eight commercial feedlot pens were used to examine the quantity and diversity of microorganisms in cattle feedlot air. The effect of two feeding patterns on the generation of airborne dust and the total numbers of microorganisms was also examined (four feedlot pens/treatment). Microbial samples were collected, and dust particles that were 2.5 microm or less in diameter were measured with a Dustrak monitor during the evening dust peak for 4 days at sites both upwind and downwind of the feedlot pens. An Andersen biological cascade sampler was employed with different medium and incubation combinations for the capture and identification of bacteria and fungi. The results showed that when bacteria were considered, only nonpathogenic gram-positive organisms were recovered. However, gram-negative bacteria may have been present in a viable but nonculturable state. Fungi were recovered in smaller numbers than bacteria, and none of the fungi were pathogenic. The Dustrak results showed that one feeding pattern resulted in cattle behavior that generated levels of downwind dust lower (P = 0.04) than the levels generated by the behavior resulting from the other feeding pattern. However, the Andersen sampler results showed that there were no differences between feeding patterns with regard to the total number or diversity of microorganisms. The disparity may have been due to the different operating principles of the two systems. The overall numbers of microorganisms recovered were lower than those reported in studies of intensively housed farm animals in which similar recovery techniques were used.

Evaluation of fungal growth on cellulose-containing and inorganic ceiling tile.

Buildings with poor indoor air quality (IAQ) frequently have many areas with surface fungal contamination. Studies have demonstrated that certain fungal genera (e.g., Cladosporium, Penicillium, and Stachybotrys) are able to grow on building materials such as wallpaper, drywall, and ceiling tiles, particularly after water damage has occurred. Due to the increasing awareness of sick building syndrome (SBS), it has become essential to identify building materials that prevent the interior growth of fungi. The objective of this study was to identify building materials that would not support the growth of certain fungal genera, regardless of whether an external food source was made available. The growth of three fungal genera (Cladosporium, Penicillium, and Stachybotrys) was evaluated on cellulose-containing ceiling tile (CCT) and inorganic ceiling tile (ICT). Both types of ceiling tile were exposed to environmental conditions which can occur inside a building. Our results show that ICT did not support the growth of these three fungal genera while CCT did. Our data demonstrate that ICT could serve as an ideal replacement for CCT.

An animal model for allergic penicilliosis induced by the intranasal instillation of viable Penicillium chrysogenum conidia.

BACKGROUND: A study was undertaken to determine the consequences of long term intranasal instillation of Penicillium chrysogenum propagules in a mouse model. METHODS: C57 Black/6 mice were inoculated intranasally each week for six weeks with 10(4) viable and non-viable P chrysogenum conidia. Cytokine levels and cellular responses in these animals were then measured. RESULTS: Compared with controls, mice inoculated intranasally each week for six weeks with 10(4) P chrysogenum conidia (average viability 25%) produced significantly more total serum IgE (mean difference 1823.11, lower and upper 95% confidence intervals (CI) 539.09 to 3107.13), peripheral eosinophils (mean difference 5.11, 95% CI 2.24 to 7.99), and airway eosinophilia (rank difference 11.33, 95% CI 9.0 to 20.0). With the exception of airway neutrophilia (mean difference 20.89, 95% CI 3.72 to 38.06), mice inoculated intranasally with 10(4) non-viable conidia did not show significant changes in total serum IgE, peripheral or airway eosinophils. However, when compared with controls, this group (10(4) non-viable) had a significant increase in total serum IgG(2a) (mean difference 1990.56, 95% CI 790.48 to 3190.63) and bronchoalveolar lavage (BAL) fluid levels of interferon (IFN)-gamma (mean difference 274.72, 95% CI 245.26 to 304.19). In addition, lung lavages from mice inoculated intranasally with 10(4) viable P chrysogenum conidia had significantly increased levels of interleukin (IL)-4 (mean difference 285.28, 95% CI 108.73 to 461.82) and IL-5 (mean difference 16.61, 95% CI 11.23 to 21.99). The IgG(2a)/IgE ratio and the IFN-gamma/IL-4 ratio was lower in the group of mice inoculated intranasally with 10(4) viable conidia than in the 10(4) non-viable conidia group and the controls. When proteins were extracted from P chrysogenum conidia, attached to microtitre plates and incubated with serum from the 10(4) viable group, significant increases in conidia-specific IgE and IgG(1) were observed compared with controls, while serum from the 10(4) non-viable group was similar to controls. CONCLUSIONS: These data suggest that long term inhalation of viable P chrysogenum propagules induces type 2 T helper cell mediated (Th2) inflammatory responses such as increases in total and conidia-specific serum IgE and IgG(1), together with BAL fluid levels of IL-4 and IL-5 and peripheral and airway eosinophilia, which are mediators of allergic reactions.

Lipase activity from strains of Pasteurella multocida.

Thirteen clinical isolates of Pasteurella multocida from a variety of different animals and humans were examined for their ability to produce lipase. Lipase substrates used included Tween 20, Tween 40, Tween 80, and Tween 85. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in Roswell Park Memorial Institute-1640 defined media (RPMI-1640), but activity increased in the filtrates when the cultures were allowed to proceed to the stationary phase. All strains examined (except for serotype 2) showed lipase activity against at least one of the Tweens. Tween 40 was the best substrate to demonstrate lipase activity. Pasteurella multocida serotype 8 produced the most active lipase against Tween 40 (3,561.7 units of activity/microgram of protein). This activity continued to increase after P. multocida entered a stationary growth phase. P. multocida lipase activity was optimal at pH 8.0. Lipase activity of P. multocida serotype 8 was eluted from a Sepharose 2B column at several points, indicating that several lipases may be produced in vitro by this organism. These data demonstrate that clinical isolates of P. multocida produce lipase; therefore, this enzyme should be considered a potential virulence factors for this organism.

Performance of the Affymetrix GeneChip HIV PRT 440 platform for antiretroviral drug resistance genotyping of human immunodeficiency virus type 1 clades and viral isolates with length polymorphisms.

The performance of a silica chip-based resequencing method, the Affymetrix HIV PRT 440 assay (hereafter referred to as the Affymetrix assay), was evaluated on a panel of well-characterized nonclade B viral isolates and on isolates exhibiting length polymorphisms. Sequencing of human immunodeficiency virus type 1 (HIV-1) pol cDNAs from clades A, C, D, E, and F resulted in clade-specific regions of base-calling ambiguities in regions not known to be associated with resistance polymorphisms, as well as a small number of spurious resistance polymorphisms. The Affymetrix assay failed to detect the presence of additional serine codons distal to reverse transcriptase (RT) codon 68 that are associated with multinucleoside RT inhibitor resistance. The increasing prevalence of non-clade B HIV-1 strains in the United States and Europe and the identification of clinically relevant pol gene length polymorphisms will impact the generalizability of the Affymetrix assay, emphasizing the need to accommodate this expanding pool of pol genotypes in future assay versions.

Continually measured fungal profiles in sick building syndrome.

Buildings with indoor air quality (IAQ) complaints frequently have high airborne concentrations of Penicillium species, while buildings with few IAQ complaints have an indoor air (IDA) fungal ecology similar to outdoor air (ODA), where Cladosporium species is usually the dominant microorganism. These studies compared fungal air profiles, measured continually over 6 h in a documented sick building, in IDA in a room experiencing IAQ problems with fungal profiles measured concurrently in ODA. The dominant species collected at both sites were Penicillium species, Cladosporium species, and Alternaria species. In the IDA, Penicillium species were always the dominant organisms, ranging from 150 to 567 cfu/m3 (89.8-100% of the total fungi). In the ODA, Cladosporium species were dominant in four samples (40.0-70.6%), while Penicillium species were dominant (52.7-79.6%) in two. These data demonstrate that, even though ODA fungal profiles are changing continuously, IDA fungal profiles in "sick" buildings tend to remain unchanged.

Correlation between the prevalence of certain fungi and sick building syndrome.

OBJECTIVE: To examine the role of fungi in the production of sick building syndrome. METHODS: A 22 month study in the United States of 48 schools (in which there had been concerns about health and indoor air quality (IAQ). Building indoor air and surface samples, as well as outdoor air samples were taken at all sites to look for the presence of fungi or their viable propagules. RESULTS: Five fungal genera were consistently found in the outdoor air and comprised over 95% of the outdoor fungi. These genera were Cladosporium (81.5%), Penicillium (5.2%), Chrysosporium (4.9%), Alternaria (2.8%), and Aspergillus (1.1%). At 20 schools, there were significantly more colony forming units per cubic metre (CFU/m3) (p < 0.0001) of propagules of Penicillium species in the air samples from complaint areas when compared with the outdoor air samples and the indoor air samples from noncomplaint areas. At five schools, there were more, although not significant (p = 0.10), Penicillium propagules in the air samples from complaint areas when compared with the outdoor air samples and the indoor air samples from noncomplaint areas. In 11 schools, the indoor air (complaint areas) fungal ratios were similar to that in the outdoor air. In these 11 schools Stachybotrys atra was isolated from swab samples of visible growth under wetted carpets, on wetted walls, or behind vinyl wall coverings. In the remaining 11 schools, the fungal ratios and CFU/m3 of air were not significantly different in different areas. Many of the schools took remedial action that resulted in an indoor air fungal profile that was similar to that outdoors. CONCLUSIONS: Propagules of Penicillium and Stachybotrys species may be associated with sick building syndrome.

Pasteurella haemolytica ultraviolet-irradiated vaccine by parenteral and aerosol routes compared in the goat model.

Positive control 1 (PC1) (n = 9) goats were injected transthoracically into the left lung with live Pasteurella haemolytica biovar A, serovar 1 (PhA1) in polyacrylate (PA) beads on days 0 and 21. Positive control 2 (PC2) (n = 6) goats were nebulized with live PhA1 and PA beads on days 0 and 21. Negative control (NC) goats (n = 6) were each injected transthoracically into the left lung with PA beads alone on days 0 and 21. Four groups (n = 6) were administered PA beads mixed with ultraviolet (UV) killed PhA1 on days 0 and 21. The treatment doses of bacteria for these groups were principal group 1 (PR1) injected into the left lung (7.7 x 10(10) cfu); PR2, 7.7 x 10(10) UV-killed PhA1 injected subcutaneously (SC); PR3, 7.7 x 10(10) UV-killed PhA1 injected SC only on day 21; PR4, nebulized with PA beads mixed with 5.6 x 10(10) cfu of UV-killed PhA1; and PR5, nebulized with PA beads mixed with 5 x 10(8) cfu of UV-killed PhA1. All goats were challenged transthoracically in the right lung with 1 x 10(8) cfu of live PhA1 on day 42 and necropsied on day 46. The sizes of consolidated lung lesions at the challenge site were used as a measure of immunity. The data show that the introduction of live PhA1 into the lungs of goats, either by injection or aerosolization, offers excellent protection against a subsequent homologous challenge. The data also demonstrate that two transthoracic injections (21 days apart) of UV-killed PhA1 (PR1), and subcutaneous injection of UV-killed PhA1(PR2) also offer excellent protection against a subsequent homologous live PhA1 challenge. One SC injection of UV-killed PhA1 (PR3) appears to offer only partial protection against a subsequent homologous live PhA1 challenge. Inhalation of UV-killed PhA1 mixed with PA beads (PR4 and PR5) induced no protection in goats against a subsequent live PhA1 transthoracic challenge.

Cross-protection studies with three serotypes of Pasteurella haemolytica in the goat model.

Cross-protection studies employing three serotypes of Pasteurella haemolytica (Ph) were performed in goats, with challenge exposure by transthoracic injection. Indirect hemagglutination (IHA) serum titers showed that the herd had been naturally infected with Ph biovar A, serovar 2 (PhA2) prior to the study. Sixty-four weanling male Spanish goats were randomly allotted to 16 groups. Fifteen goats were given two transthoracic injections into the lungs 21 days apart with live Pasteurella haemolytica biovar A, serovar 1 (PhA1) in agar beads. Fifteen goats were given two transthoracic injections into the lungs 21 days apart with live PhA2 in agar beads. Sixteen goats were given two transthoracic injections into the lungs 21 days apart with live P. haemolytica biovar A, serovar 6 (PhA6) in agar beads. Eighteen control (CON) goats were given two transthoracic injections into the lungs 21 days apart with agar beads alone. Fourteen days after the second injection, goats were challenge-exposed to either live PhA1, PhA2, or PhA6 by transthoracic injection into the lung, and 4 days later, all goats were euthanatized and necropsied. Serum antibody to P. haemolytica antigens was measured throughout the experiment. Mean volumes of consolidated lung tissue for the CON goats challenged with PhA1, PhA2, and PhA6 were 28.29 cm3, 8.36 cm3, and 16.29 cm3, respectively. Mean volumes of consolidated lung tissue for the PhA1-immunized goats challenged with PhA1, PhA2, and PhA6 were 4.38 cm3, 0.25 cm3, and 1.90 cm3, respectively. Mean volumes of consolidated lung tissue for the PhA2-immunized goats challenged with PhA1, PhA2, and PhA6 were 9.68 cm3, 0.05 cm3, and 3.39 cm3, respectively. Mean volumes of consolidated lung tissue for the PhA6-immunized goats challenged with PhA1, PhA2, and PhA6 were 14.05 cm3, 1.27 cm3, and 4.53 cm3, respectively. These data demonstrate protection in immunized goats challenged with the homologous serotype of P. haemolytica. PhA1-immunized animals were protected against serotype 2 challenge as well as against serotype 6 challenge. PhA2-immunized animals were not protected against serotype 1 challenge, but were protected against transthoracic PhA6 challenge. PhA6-immunized animals were not protected against serotype 1 challenge, but were protected against transthoracic PhA2 challenge. There appears to be some cross-protection among the P. haemolytica serotypes, and this fact should be taken into consideration when developing vaccines against this organism.

In vivo production of neuraminidase by Pasteurella multocida A:3 in goats after transthoracic challenge.

Six goats were injected transthoracically with live Pasteurella multocida A:3 to examine if an extracellular enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 6) each received 1 ml of live 7.5 x 10(4) cfu of P. multocida mixed with polyacrylate beads transthoracically in the left lung on day 0 and 1 ml of live P. multocida (2.2 x 10(8) cfu) mixed with polyacrylate beads transthoracically in the left lung on day 22. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 22. Serum was obtained from all animals on days 0, 7, 14, 22, 29, and 36. Preimmune sera from all animals showed no detectable antibody to P. multocida A:3 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals demonstrated a significant antibody titer against the P. multocida A:3 neuraminidase. On day 36, serum samples from the six infected animals possessed complete enzyme-neutralizing activity. Anti-neuraminidase antibody could be detected as early as day 14 in the infected animals. These data show that neuraminidase is produced in vivo during an active P. multocida A:3 lobar infection.