Total Spore Research Articles

Use of Microfiltration to Improve Fluid Milk Quality ,

The objectives of the research were to determine the growth characteristics of bacteria in commercially pasteurized skim milk as a function of storage temperature; to determine the efficacy of a microfiltration and pasteurization process in reducing the number of total bacteria, spores, and coliforms in skim milk; and to estimate the shelf life of pasteurized microfiltered skim milk as a function of storage temperature. For the first objective, commercially pasteurized skim milk was stored at 0.1, 2.0, 4.2, and 6.1°C. A total bacterial count >20,000 cfu/mL was considered the end of shelf life. Shelf life ranged from 16 d at 6.1°C to 66 d at 0.1°C. Decreasing storage temperature increased lag time and reduced logarithmic growth rate of a mixed microbial population. The increased lag time for the mixed microbial population at a lower storage temperature was the biggest contributor to longer shelf life. For the second objective, raw skim milk was microfiltered at 50°C using a Tetra Alcross M7 Pilot Plant equipped with a ceramic Membralox membrane (pore diameter of 1.4μm). The 50°C permeate was pasteurized at 72°C for 15s, and cooled to 6°C. Bacterial counts of raw skim milk were determined by standard plate count. Bacterial counts of microfiltered and pasteurized microfiltered skim milk were determined using a most probable number method. Across 3 trials, bacterial counts of the raw milk were reduced from 2,400, 3,600, and 1,475 cfu/mL to 0.240, 0.918, and 0.240 cfu/mL, respectively, by microfiltration. Bacterial counts in the pasteurized microfiltered skim milk for the 3 trials were 0.005, 0.008, and 0.005 cfu/mL, respectively, demonstrating an average 5.6 log reduction from the raw count due to the combination of microfiltration and pasteurization. For the third objective, pasteurized microfiltered skim milk was stored at each of 4 temperatures (0.1, 2.0, 4.2, and 6.1°C) and the total bacterial count was determined weekly over a 92-d period. At 6 time points in the study, samples were also analyzed for noncasein nitrogen and the decrease in casein as a percentage of true protein was calculated. After 92 d, 50% of samples stored at 6.1°C and 12% of samples stored at 4.2°C exceeded a total bacterial count of 20,000 cfu/mL. No samples stored at 0.1 or 2.0°C reached a detectable bacterial level during the study. When the bacterial count was <1,000 cfu/mL, shelf life was limited because sufficient proteolysis had occurred at 32 d at 6.1°C, 46 d at 4.2°C, 78 d at 2.0°C, and >92 d at 0.1°C to produce a detectable off-flavor in skim milk produced from a raw milk with a 240,000 somatic cell count.

Continuous Flow Nonthermal CO2 Processing: The Lethal Effects of Subcritical and Supercritical CO2 on Total Microbial Populations and Bacterial Spores in Raw Milk

The effect of pressurized (<50MPa) CO2 as a nonthermal process for bacterial reduction in raw skim milk was examined using a unique pressurized continuous flow system. The lethal effects of subcritical and super-critical CO2 applied at different temperatures and pressures toward total native psychrotrophic microbial populations, total inoculated Pseudomonas fluorescens, and total inoculated spore populations were studied and compared. Pressures between 10.3 and 48.3MPa; temperatures of 15, 30, 35, and 40°C; and CO2 concentrations of 0, 3, 66, and 132 g/kg of milk were studied. For both native populations and inoculated P. fluorescens, greater total microbial lethality was observed under supercritical CO2 conditions than under subcritical CO2 conditions. At 30°C, there was no effect on total microbial lethality of increasing pressure up to 20.7MPa with either 66 or 132 g/kg of CO2; at 35°C, there was a positive relationship between pressure and lethality at CO2 levels of 132 g/kg, but no relationship at 66 g/kg of CO2. For total microbial populations and P. fluorescens, CO2 applied at 132 g/kg at 30°C and pressures of 10.3 to 20.7MPa resulted in an average standard plate count reduction of 3.81 and 2.93 log, respectively; at 35°C and 20.7MPa, maximum reductions achieved were 5.36 and 5.02 log, respectively. For both total microbial populations and inoculated P. fluorescens, CO2 exhibited a greater overall lethal effect at 132 g/kg than at 66 g/kg and a greater effect at 35°C than at 30°C. At 24.1 and 48.3MPa and 40°C, microbial lethality in raw aged milk treated with 3 g/kg of CO2 was not significantly different than that observed for uncarbonated milk; lethality achieved in milk treated with 132 g/kg of CO2 was significantly higher than that achieved in these 2 low-level CO2 treatments. No treatment studied had any significant impact on spore populations. Our work shows that, using the studied system, pressurized CO2 results in greater microbial lethality in milk above critical temperatures than below and suggests that a critical concentration threshold level of CO2 is required for lethal effects. Our work also suggests that supercritical CO2 processing in a continuous flow system can achieve reductions in some microbial populations equal to or better than that typically achieved during high-temperature, short-time pasteurization.

Personal Exposure to Airborne Dust and Microorganisms in Agricultural Environments

Airborne dust and microorganisms are associated with respiratory diseases and increased mortality and morbidity. Farmers are at high risk of exposure to both of these hazards. Very limited information, however, is available on the combined exposures to both hazards on different types of farms. Moreover, most of the previous studies have measured the mass concentration of particles ignoring the particle size. In this study, farmers' exposure to airborne dust and microorganisms was studied using our newly developed personal sampling system. Particle number concentration and size distribution were measured with an optical particle counter. Simultaneously, particles were collected on a filter and analyzed for microorganisms. The field measurements were conducted in animal confinements (swine, poultry, and dairy) and during grain harvesting (corn and soybean). The results show the following average concentrations on the workers' breathing zone: 1.7 × 10 6 to 2.9 × 10 7 particles/m 3 for total dust, 0.9 × 10 3 to 3.9 × 10 4 spores/m 3 for total fungal spores, 0.3 × 10 3 to 3.6 × 10 4 CFU/m 3 for culturable fungal spores, 0.3 × 10 4 to 3.3 × 10 8 CFU/m 3 for culturable bacteria, and limit of detection (LOD) to 2.8 × 10 3 CFU/m 3 for culturable actinomycetes in animal confinements. The respective concentrations were 4.4 × 10 6 to 5.8 × 10 7 particles/m 3 , 3.4 × 10 4 to 6.1 × 10 6 spores/m 3 , 8.2 × 10 4 to 7.4 × 10 6 CFU/m 3 , 0.4 × 10 5 to 1.4 × 10 6 CFU/m 3 , and LOD to 2.6 × 10 4 CFU/m 3 during grain harvesting. The highest contribution of large particles (3–10 μ m) in total particles was found during grain harvesting, whereas the size distribution was dominated by smaller particles (< 3 μ m) in animal confinements. High fraction (up to 37%) of particles between 2–10 μ m was found to be fungal spores. The results indicate that an increase in the concentration of large dust particles (2–10 μ m) during grain harvesting was partially attributed to the increase in the concentration of the fungal spores. Overall, the combined exposure to airborne dust and microorganisms was found to be more severe during harvesting than in animal confinements.

Culturability and concentration of indoor and outdoor airborne fungi in six single-family homes

In this study, the culturability of indoor and outdoor airborne fungi was determined through long-term sampling (24-h) using a Button Personal Inhalable Aerosol Sampler. The air samples were collected during three seasons in six Cincinnati area homes that were free from moisture damage or visible mold. Cultivation and total microscopic enumeration methods were employed for the sample analysis. The geometric means of indoor and outdoor culturable fungal concentrations were 88 and 102 colony-forming units (CFU) m −3, respectively, with a geometric mean of the I/ O ratio equal to 0.66. Overall, 26 genera of culturable fungi were recovered from the indoor and outdoor samples. For total fungal spores, the indoor and outdoor geometric means were 211 and 605 spores m −3, respectively, with a geometric mean of I/ O ratio equal to 0.32. The identification revealed 37 fungal genera from indoor and outdoor samples based on the total spore analysis. Indoor and outdoor concentrations of culturable and total fungal spores showed significant correlations ( r = 0.655 , p<0.0001 and r = 0.633 , p<0.0001, respectively). The indoor and outdoor median viabilities of fungi were 55% and 25%, respectively, which indicates that indoor environment provides more favorable survival conditions for the aerosolized fungi. Among the seasons, the highest indoor and outdoor culturability of fungi was observed in the fall. Cladosporium had a highest median value of culturability (38% and 33% for indoor and outdoor, respectively) followed by Aspergillus/Penicillium (9% and 2%) among predominant genera of fungi. Increased culturability of fungi inside the homes may have important implications because of the potential increase in the release of allergens from viable spores and pathogenicity of viable fungi on immunocompromised individuals.

Typical Outdoor Levels of Airborne Non-Viable Fungal Spores Collected at the Ground Level During the Spring and Early Summer in Florida

RATIONALE: Ground-level air samples (GLAS) are collected during the course of building evaluations to determine types and levels of non-viable fungal spores (NVFS) indoors versus outdoors. However, outdoor reference levels of NVFS collected in GLAS are limited. This is a study to determine these levels during the spring and early summer in Florida.METHODS: 234 outdoor GLAS were collected for NVFS analysis from April to July 2005 in Florida, utilizing a Zefon Bio-PumpTM and Air-O-Cell cassettesTM. These samples were analyzed by optical microscopy. Non-parametric statistical analysis was performed. Percentile categories were established for continuous variables, according to the criteria proposed by the American Academy of Allergy Asthma and Immunology National Allergy Bureau, as low (<50th), moderate (50th-75th), high (75th-99th), and very high (>99th).RESULTS: Total spores as well as individual Ascospores, Basidiospores, Cladosporium, and Penicillium/Aspergillus follow continuous distributions. Low, moderate, high, and very high levels (in spores/m3) of total spores are <4,813; 4,813-8,164; 8,165-108,509; and >108,509, respectively. The corresponding individual Ascospore levels are <1,117; 1,117-2,206; 2,207-12,975; and >12,975, respectively. Basidiospore levels are <1,064; 1,064-2,506; 2,507-23,861; and >23,861, respectively. Cladosporium levels are <744; 744-1,635; 1,636-19,278; and >19,278, respectively. Penicillium/Aspergillus levels are <650; 650-1,076; 1,077-14,887; and >14,887, respectively.CONCLUSIONS: The levels of the most common types of outdoor NVFS collected in GLAS during the spring and early summer in Florida have scattered distributions. Year-round studies in Florida and other locations are needed to refine these reference levels during different seasons. This information would facilitate the interpretation of laboratory reports generated during the course of building evaluations. RATIONALE: Ground-level air samples (GLAS) are collected during the course of building evaluations to determine types and levels of non-viable fungal spores (NVFS) indoors versus outdoors. However, outdoor reference levels of NVFS collected in GLAS are limited. This is a study to determine these levels during the spring and early summer in Florida. METHODS: 234 outdoor GLAS were collected for NVFS analysis from April to July 2005 in Florida, utilizing a Zefon Bio-PumpTM and Air-O-Cell cassettesTM. These samples were analyzed by optical microscopy. Non-parametric statistical analysis was performed. Percentile categories were established for continuous variables, according to the criteria proposed by the American Academy of Allergy Asthma and Immunology National Allergy Bureau, as low (<50th), moderate (50th-75th), high (75th-99th), and very high (>99th). RESULTS: Total spores as well as individual Ascospores, Basidiospores, Cladosporium, and Penicillium/Aspergillus follow continuous distributions. Low, moderate, high, and very high levels (in spores/m3) of total spores are <4,813; 4,813-8,164; 8,165-108,509; and >108,509, respectively. The corresponding individual Ascospore levels are <1,117; 1,117-2,206; 2,207-12,975; and >12,975, respectively. Basidiospore levels are <1,064; 1,064-2,506; 2,507-23,861; and >23,861, respectively. Cladosporium levels are <744; 744-1,635; 1,636-19,278; and >19,278, respectively. Penicillium/Aspergillus levels are <650; 650-1,076; 1,077-14,887; and >14,887, respectively. CONCLUSIONS: The levels of the most common types of outdoor NVFS collected in GLAS during the spring and early summer in Florida have scattered distributions. Year-round studies in Florida and other locations are needed to refine these reference levels during different seasons. This information would facilitate the interpretation of laboratory reports generated during the course of building evaluations.

Relationship between indoor and outdoor bioaerosols collected with a button inhalable aerosol sampler in urban homes

This field study investigated the relationship between indoor and outdoor concentrations of airborne actinomycetes, fungal spores, and pollen. Air samples were collected for 24 h with a button inhalable aerosol sampler inside and outside of six single-family homes located in the Cincinnati area (overall, 15 pairs of samples were taken in each home). The measurements were conducted during three seasons - spring and fall 2004, and winter 2005. The concentration of culturable actinomycetes was mostly below the detection limit. The median indoor/outdoor ratio (I/O) for actinomycetes was the highest: 2.857. The indoor of fungal and pollen concentrations followed the outdoor concentrations while indoor levels were mostly lower than the outdoor ones. The I/O ratio of total fungal spores (median=0.345) in six homes was greater than that of pollen grains (median=0.025). The low I/O ratios obtained for pollen during the peak ambient pollination season (spring) suggest that only a small fraction penetrated from outdoor to indoor environment. This is attributed to the larger size of pollen grains. Higher indoor concentration levels and variability in the I/O ratio observed for airborne fungi may be associated with indoor sources and/or higher outdoor-to-indoor penetration of fungal spores compared to pollen grains. Practical Implication This study addresses the relationship between indoor and outdoor concentrations of three different types of bio-aerosols, namely actinomycetes, fungal spores, and pollen grains. The results show that actinomycetes are rare in indoor and outdoor air in Midwest, USA. Exposure to pollen occurs mainly in the outdoor air even during peak pollen season. Unexpectedly high fungal spore concentrations were measured outdoors during winter. The presented pilot database on the inhalable levels of indoor and outdoor bio-aerosols can help apportion and better characterize the inhalation exposure to these bio-aerosols. Furthermore, the data can be incorporated into existing models to quantify the penetration of biological particles into indoor environments from outdoors.

Mold Infestation of Wet Spray-Applied Cellulose Insulation

Mold investigations were conducted in four buildings that had been insulated with wet spray–applied cellulose insulation (WSACI). Bulk WSACI samples were collected and analyzed by quantitative polymerase chain reaction (QPCR) methods. Airborne mold was evaluated using both Burkard total mold spore and Andersen culturable/viable sampling methods. Although reportedly treated with biocidal borates, QPCR analyses indicated that elevated concentrations of mold cells (reported as spore equivalents per gram) may be present in WSACI. QPCR analyses showed the following: (1) very high concentrations of Penicillium chrysogenum in samples from two of four buildings; (2) very high concentrations of Stachybotrys chartarum in samples from one building and a more moderate presence in a second; (3) moderately high concentrations of Aspergillus versicolor in samples from one building and more moderate concentrations in a second; (4) the presence of the opportunistic pathogen, Aspergillus fumigatus, in samples from three of the four buildings, and (5) the presence of 22 of 23 target mold species. Elevated airborne total mold spore concentrations were observed in all four of the buildings investigated. Culturable/viable airborne mold concentrations were moderately elevated in three of the four buildings. Mold genera/types present were relatively consistent among airborne mold samples collected by both methods and bulk sample analyses. Results of this study suggest that WSACI has the potential to cause elevated airborne mold levels in buildings where it has been applied and pose significant mold exposure and public health risks.

Influence of pH Control Agents on Entomotoxicity Potency of Bacillus thuringiensis using Different Raw Materials

In this investigation, ammonium hydroxide and acetic acid were used as pH control agents during Bacillus thuringiensis (Bt) fermentation in a pilot scale fermentor (150-l) employing two secondary wastewater sludges from two different wastewater treatment plants (CUQS and JQS) and semi-synthetic soybean meal medium as raw materials. Regardless of the cultivation medium, a substantial increase in total cell count, spore count, protease activity and entomotoxicity was achieved when the pH of the culture was controlled using NH4OH/CH3COOH. At harvest, total cell count increased by almost 17%, 33% and 25%; protease activity was enhanced by 12%, 33% and 53% and maximal spore count augmented by almost 28%, 48% and 33% in CUQS, JQS and soybean medium, respectively. Entomotoxicity potency was improved by 22%, 21% and 14% in CUQS, JQS and soybean medium, respectively compared to results obtained with NaOH/H2SO4 as pH control agents. A higher entomotoxicity was also observed using sludge compared to the soybean medium. This improvement of the Bt process performance was a consequence of the addition of rapidly utilizable carbon and nitrogen source through pH control, which stimulated endotoxin production in the crystal and enhanced sporulation.

Molecular screening of Bacillus thuringiensis strains from wastewater sludge for biopesticide production

Ten strains of Bacillus thuringiensis (Bt) were isolated from sludges collected from wastewater treatment plants and two strains were isolated from dead Tortricidae larvae sample collected from forest (Quebec, Canada). The bacterial isolates were characterized by PCR method using specific primers for amplification of the CryI gene (Cry IA and Cry IC genes codes active toxin against Lepidoptera species), Cry III gene (active against some Coleoptera species) and Cry IV gene (toxic against Diptera species). This study showed that all the 12 bacterial strains yielded a PCR product for the Cry IA gene and five of them also possessed the Cry IC gene. The insecticidal activity or entomotoxicity of these strains against Lepidoptera species was confirmed through bioassays using third and fourth instar Choristoneura fumiferana larvae. Growth of new (INRS) strains in wastewater sludge gave higher values of viable total cell count and spore counts in comparison to standard strains. The entomotoxicity potential of the INRS isolated strains varied between 10650 and 14750 SBU/μl. These results of insecticidal activity were substantially higher than the reference strain of Bt var. kurstaki with an entomotoxicity value of 10789 SBU/μl. Thus the isolated Bt strains could be used to develop a more efficient process for biopesticide production.

Exploitative and Hierarchical Antagonism in a Cooperative Bacterium

Social organisms that cooperate with some members of their own species, such as close relatives, may fail to cooperate with other genotypes of the same species. Such noncooperation may take the form of outright antagonism or social exploitation. Myxococcus xanthus is a highly social prokaryote that cooperatively develops into spore-bearing, multicellular fruiting bodies in response to starvation. Here we have characterized the nature of social interactions among nine developmentally proficient strains of M. xanthus isolated from spatially distant locations. Strains were competed against one another in all possible pairwise combinations during starvation-induced development. In most pairings, at least one competitor exhibited strong antagonism toward its partner and a majority of mixes showed bidirectional antagonism that decreased total spore production, even to the point of driving whole populations to extinction. Differential response to mixing was the primary determinant of competitive superiority rather than the sporulation efficiencies of unmixed populations. In some competitive pairings, the dominant partner sporulated more efficiently in mixed populations than in clonal isolation. This finding represents a novel form of exploitation in bacteria carried out by socially competent genotypes and is the first documentation of social exploitation among natural bacterial isolates. Patterns of antagonistic superiority among these strains form a highly linear dominance hierarchy. At least some competition pairs construct chimeric, rather than segregated, fruiting bodies. The cooperative prokaryote M. xanthus has diverged into a large number of distinct social types that cooperate with clone-mates but exhibit intense antagonism toward distinct social types of the same species. Most lengthy migration events in nature may thus result in strong antagonism between migratory and resident populations, and this antagonism may have large effects on local population sizes and dynamics. Intense mutual antagonism appears to be more prevalent in this prokaryotic social species than has been observed in the eukaryotic social slime mold Dictyostelium discoideum, which also exhibits multicellular development. The finding of several cases of facultative social exploitation among these natural isolates suggests that such exploitation may occur frequently in nature in many prokaryotes with cooperative traits.

Wastewater Sludge Pre-treatment for Enhancing Entomotoxicity Produced by Bacillus thuringiensis var. kurstaki

Wastewater sludge is a complex raw material for fermentation and requires pre-treatment in order to transform less biodegradable compounds into more easily degradable ones. In this study, sludge was treated by thermo-alkaline and oxidative pre-treatment methods and subjected to Bacillus thuringiensis fermentation in bench scale fermentors under controlled conditions. Thermo-alkaline treatment was found to be an effective process in order to enhance the entomotoxicity tested against spruce budworm. The total cell and spore counts were improved by 40 and 46%, respectively as compared to that using the untreated sludge. The final entomotoxicity potency increased from 12.3 × 109 SBU/l of the raw sludge to 16.6 × 109 SBU/l of the thermo-alkaline pre-treated sludge. The improvement of the process performance was attributed to a better oxygen transfer due to decrease in media viscosity and an improvement of nutrient availability due to the sludge solubilization and biodegradability.

Influence of Environment on Atmospheric Concentrations of Peronospora antirrhini Sporangia in Field-Grown Snapdragon.

Hourly concentrations of airborne sporangia of Peronospora antirrhini at a commercial snapdragon farm were investigated over three growing seasons to determine the influence of the environment on their occurrence and concentration. Hourly concentrations of sporangia of P. antirrhini were estimated using a Burkard volumetric spore sampler. Atmospheric sporangial concentrations followed a diurnal pattern and were greatest during 0500 to 1200 h. Minimum daily temperatures <10.0°C appeared to have a moderate limiting effect on atmospheric sporangial concentrations, whereas temperatures <6.0°C had more severe limiting effects. Maximum daily temperatures ≥30.0°C limited concentrations of atmospheric sporangia. Long dew periods (≥6 h) were associated with relatively large sporangia releases. On 69 days (1999 to 2001), the total number of sporangia trapped was >100/m3/day, and the average length of leaf wetness duration prior to these releases was 11 h. Consecutive days with short leaf wetness periods were associated with low atmospheric sporangial concentrations. Significant positive correlations (P = 0.0502 and P = 0.0174) were found between total rainfall and total spore count for both research plots in 2000. Information gained from this study will contribute to the development of a disease management program that utilizes environmental cues to prompt fungicide applications, thus increasing the efficiency of fungicide programs and delaying the development of pathogen resistance.

Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus

The development of a rapid method for the selective detection and enumeration of the total and viable vegetative cell and spore content of thermophilic bacilli in milk powder by PCR. Quantitative PCR and microscopy indicate the presence of up to 2.9 log units more cells in milk powder than accounted for by plate counting due to the majority of cells being killed during milk processing. Two approaches for viable and dead cell differentiation of thermophilic bacilli by quantitative PCR were evaluated, these being the nucleic binding dye ethidium monoazide (EMA) and DNase I digestion. The former agent exposed to a viable culture of Anoxybacillus flavithermus caused considerable cell inactivation. In contrast, DNase I treatment had no effect on cell viability and was utilized to develop DNA extraction methods for the differential enumeration of total, viable vegetative cells and spores in milk powder. Moreover, the methods were further applied and evaluated to 41 factory powder samples taken throughout eight process runs to assess changes in numbers of vegetative cells and spores with time. DNase I treatment reduced vegetative cell numbers enumerated with PCR by up to 2.6 log units. The quantification of spores in the factory milk powders investigated indicates on average the presence of 1.2 log units more spores than determined by plate counting. The method presented in this study provides the ability to selectively enumerate the total and viable cell and spore content of reconstituted milk. The current study provides a tool to monitor the extent of thermophilic contamination during milk powder manufacturing 60-90 min after sampling.

Patterns of airborne conidia of Stemphylium vesicarium, the causal agent of brown spot disease of pears, in relation to weather conditions

Seven-day volumetric spore samplers were installed in pear orchards of northern Italy, in the years between 1993 and 2002, and operated continuously during the development of brown spot epidemics (mid-April–mid-August), caused by Stemphylium vesicarium. Aerial concentration of conidia was recorded at 2 h intervals to study their diurnal and seasonal patterns and the influence of weather conditions. The diurnal periodicity of aerial conidia showed a peak around midday and low counts in the dark. The increase in spore concentration was significantly correlated with the reduction of relative humidity and wetness in early morning, and the increase of wind in late morning and afternoon. Conidia of S. vesicarium became easily airborne to form a regular component of the air-spora in pear orchards, while ascospores were caught only sporadically. Differences between years concerned total spore counts and numbers of peaks (defined as days with more than 30 conidia/m3 air per day). Periods with highest spore counts occurred in late-May to early-June (in 2 years), mid to end of June (5 years), or after mid-July (3 years). There was a significant correlation between spore peaks and days with favourable weather conditions, defined as days with air temperature between 15 and 25°C and high humidity, particularly a wet period longer than 10 h. Occurrence of one or more consecutive days with favourable weather conditions determined an increase in the airborne concentration of conidia, which usually lasted some days and then decreased.

Long term trends in outdoor Aspergillus/Penicillium spore concentrations in Derby, UK from 1970 to 2003 and a comparative study in 1994 and 1996 with the indoor air of two local houses

Aspergillus/Penicillium spore concentrations have been monitored in Derby since 1970 using a volumetric spore trap, with full year data from 1991. In addition a short comparative study with the indoor air was undertaken at two local houses in 1994 and 1996. Aspergillus/Penicillium spores were present in the Derby air throughout the year and often reached maximum monthly cumulative concentrations in the autumn, although they were occasionally the dominant spores in the winter when total spore concentrations were low. Very high daily concentrations could occur at any time of year with a count of over 5000 recorded. Peak days in the autumn and winter of 2002–2003 were examined on a two hourly basis showing higher concentrations in the middle of the day. There was a positive correlation of cumulative monthly Aspergillus/Penicillium totals with maximum temperature. Indoor data from the two houses was examined on a daily basis and compared with simultaneously sampled outdoor daily spore concentrations. The elevated Aspergillus/Penicillium spore levels found in the older of the two houses occurred on all of the days sampled. Compared to the modern house, the Aspergillus/Penicillium spore concentrations in the old house represented a much higher percentage of the total spore count than in the modern one. The correlation between outdoor Aspergillus/Penicilliumspore concentrations and the indoor air of the old house was 0.62, whereas in the modern house it was 0.31. Peak hourly samples of Aspergillus/Penicillium spore counts occurred at times of greatest activity.

Effects of long-term NP-fertilization on abundance and diversity of arbuscular mycorrhizal fungi under a maize cropping system

Diversity of arbuscular mycorrhizal fungi (AMF) in 27-year long-term NP-fertilization plots under a maize cropping system in Thailand was studied through spore morphological characterization. The plots received 0-0, 60-60, 120-120 and 180-180 kg N-P2O5 ha-1 year -1 as ammonium sulfate and triple superphosphate. The plots were sampled monthly for one year, the AMF spores were counted and morphotyped, and taxa were identified after morphotyping and monospecific pot culture. Spore number g-1 soil, relative spore abundance and Shannon-Wiener indexes were calculated. Sixteen putative taxa were recorded from the field of which nine sporulated on maize roots in pot culture. The long-term fertilization caused decreases in AMF total spore numbers and variation in species diversity depended on sampling time. Effects of fertilization on spore number and also relative spore abundance varied with species and sampling time. Among the nine species sporulating under maize, only Acaulospora sp.1 showed no change (P > 0.003 after Bonferroni correction) in spore number with fertilization in the field; and was therefore classified as an AMF species insensitive to fertilization. Spores of Entrophospora schenckii, Glomus mosseae, Glomus sp.1, Glomus geosporum-like and Scutellospora fulgida, though they decreased in absolute numbers in response to fertilization, showed no change (P > 0.003 after Bonferroni correction) in relative abundance; these species were classified as AMF species slightly sensitive to fertilization. Three unidentified species of Glomus, though they decreased in absolute numbers in response to fertilization, showed decreases (P < 0.003 after Bonferroni correction) in relative abundance; these species were classified as AMF species highly sensitive to fertilization.

Presence of Bacillus cereus in Street Foods in Gaborone, Botswana

The purpose of this study was to evaluate the microbiological safety and quality of street foods sold in Gaborone, Botswana. A total of 148 point-of-sale composite street food samples were bought and analyzed between June 2001 and May 2002. The analysis focused on the level of contamination of various street foods with Bacillus cereus. The B. cereus (vegetative and spores), total spore, and total viable counts were determined on all the samples. Also B. cereus isolates from 444 individual point-of-sale food samples were characterized with respect to their biochemical profiles and enterotoxigenic properties. The B. cereus contamination rate for point-of-sale foods was 65%. The B. cereus counts ranged from not detectable to levels as high as 9.1 log CFU/g. Despite the high rate of contamination of some samples, generally, most samples had B. cereus counts of less than 4 log CFU/g; hence, they were of acceptable microbiological quality. Bacillus diarrheal enterotoxin was detected from 52 isolates from individual portions of meals using the B. cereus enterotoxin reversed passive latex agglutination kit. Results of the assay revealed that 59.6% of the B. cereus isolates were enterotoxigenic. Most of the enterotoxigenic isolates were obtained from vegetable samples.

A Regional Comparison of Mold Spore Concentrations Outdoors and Inside “Clean” and “Mold Contaminated” Southern California Buildings

A total of 625 buildings and outdoor locations in the San Diego, California, area were monitored using the Allergenco Sampl-Air MK-3 impaction sampler or the Zefon Air-O-Cell slit bioaerosol cassette. Locations were classified by rigid criteria as clean commercial, commercial with mold growth, clean residential, residential with water staining, and residential with mold growth. In addition, coastal and inland outdoor locations were measured. Seven categories (total spores, Ascospores/Basidiospores, Cladosporium, Smut/Myxomyceteslike, Aspergillus/Penicillium (AS/PE), Alternaria, and Unidentified/Other) were detected frequently enough that maximum likelihood estimate techniques could be used to determine distribution parameters and, thus, treat these as continuous variables. For total counts (no nondetectables) an analysis of variance was used to examine differences in location means. For the other categories Land's confidence limits were generated and visually compared for differences among locations. For 12 other categories (Curvularia, Dreschlera, Epicoccum, Fusa-rium, Mildew-like, Pithomyces, Rusts, Stachybotrys, Stemphyllium, Torula, Ulocladium, and Zygomycetes-like), detection generally occurred in less than 10% of samples. These genera were treated as dichotomous (detect/nondetect) data, and Chi-square analyses differentiated between locations. For total counts, values were significantly different on the order of clean < outdoor < moldy. There was a large difference between the moldy and other location classes. For AS/PE, moldy location means were clearly higher than those for clean buildings and outdoors, although the clean and outdoor means could not be differentiated. For all other genera the results tend to indicate little or no ability to discriminate location. For example, there were no differences in the probabilities of detecting Stachybotrys among the various locations. In our study only total counts, usually driven by AS/PE concentrations, had value in determining whether a building is mold contaminated employing our set of rigorous location classification criteria.

Mixed inoculations of a microsporidian parasite with horizontal and vertical infections

Mixed infections, where more than one parasite genotype is present in a single host, have been suggested to be an important factor in host-parasite interactions. As the host represents a limited resource, co-infecting parasite genotypes are expected to be under resource competition. Competition will not only modify the survival of the co-infecting genotypes, but is also likely to affect total within-host parasite growth as well as host survival and reproduction. We measured parasite infectivity and spore production of seven isolates of Octosporea bayeri (Microsporidia) and their effect on the reproduction and longevity of its host Daphnia magna (Cladocera), after single- or double-isolate inoculations through vertical and horizontal transmission. Double-isolate inoculations increased parasite infectivity and total spore production in horizontal infections, but had no significant effect on host reproduction or longevity. The increase in spore production in double-isolate inoculations was not found when infections occurred vertically. Our results suggest that, depending on the way the infection was acquired, within-host reproduction can increase as a result of parasite genetic diversity, without necessarily affecting the host's fitness. Whether this challenges the current views of virulence evolution depends on the definitions used.

Association of early‐onset otitis media in infants and exposure to household mould

Otitis media is one of the most common infections of early childhood. Children who first experience acute otitis media at an early age (before 6 months) are at increased risk for recurrent otitis media. This prospective study investigated exposure to measured levels of airborne household mould and the risk of early otitis media in the first 6 months of life among a cohort of infants at high risk for asthma. Between September 1996 and December 1998, women were invited to participate if they had at least one other child with physician-diagnosed asthma. Mothers were given a standardised questionnaire within 4 months of their infant's birth. Airborne mould samples were also taken at this time, and culturable fungi were categorised into four levels according to the report of the Commission of European Communities: 0 (undetectable), 1-499 colony forming units (CFU)/m(3) (low), 500-999 CFU/m(3) (medium), > or =1000 CFU/m(3) (high). Infant respiratory symptoms were collected during quarterly telephone interviews at 6, 9 and 12 months of age. Of the 806 children in the study, 27.8% experienced otitis media before six months of age. Household levels of Penicillium and Cladosporium were modestly associated with the number of otitis media episodes (P = 0.056 and 0.081 respectively). After controlling for potential confounders, Penicillium and Cladosporium were not associated with early otitis media. High levels of 'other' mould (defined as total spore count minus counts for Penicillium, Cladosporium, and yeast) were associated with early otitis media (OR 3.49; 95% CI [1.38, 8.79]). We also found associations between day-care outside of the home and birth during the summer or fall season with early otitis media. This study is suggestive of a relationship between otitis media and mould that warrants further study.