Spore Release Research Articles

The on-line detection of biological particle emissions from selected agricultural materials using the WIBS-4 (Waveband Integrated Bioaerosol Sensor) technique

Agricultural activities have, for some time, been linked to adverse health effects such as Farmers' lung, hypersensitivity pneumonitis, aspergillosis and chronic obstructive pulmonary disease (COPD) This connection is known to be, at least in part, due to the numerous microbiological organisms that live and grow on materials found in occupational settings such as barns, animal shelters, stables and composting sites. Traditional techniques for determining biological release of fungal spores and bacteria require intensive, experienced human resources and considerable time to determine ambient concentrations. However more recently the fluorescence and light scattering signals obtained from primary biological aerosol particles (PBAP) have been utilised for their near real-time counting and characterisation abilities.In the current study, data collected for the bioaerosol types released from hay and silage were counted and identified using a combination of the WIBS-4 bioaerosol sensor approach and impaction/optical microscopy. Particle emissions were characterised according to particle numbers, their size distributions, particle asymmetry values and fluorescence characteristics. The variables obtained were shown to provide potential “fingerprint” signatures for PBAP emissions emanating from two important compost components, namely, silage and hay. Comparisons between the data acquired by the WIBS-4 bioaerosol sensor, optical microscopy findings and also previous literature suggest that the likely identification of Aspergillus/Penicillium type spores and bacterial species released from hay and silage was achieved on a relatively rapid time-scale.

Alternative Disease Assessment Method for Cercospora Spot (Pseudocercospora purpurea (Cooke) Deighton) of Avocado (Persea americana Mill.)

The most serious pre-harvest disease of avocado in South Africa is still Avocado Black spot (Cercospora spot). Two methods were compared to assess Cercospora infection. The conventional assessment method (CA) relies on a disease intensity rating system that classifies fruit as local, export or rejects market quality. The disease incidence and severity assessment method (ISA) combines spore release intensity (through a weather based prediction formula) with disease intensity on fruit (as measured for specific lesion sizes representing different infection periods). In the current study, Fuerte avocado fruit from the Limpopo Province in South Africa was subjected to both CA and ISA. Data obtained through CA proved to be very limited and only reflected total disease control at the end of the season. ISA allowed a more detailed assessment of the spray program and it was possible to evaluate the efficiency of individual spray applications. Both the effect of environmental influences on disease severity and the resulted disease intensity can also be evaluated through ISA visualization.

Portuguese marine fungi and the contribution of differential interference contrast (DIC) microscopy for their morphological identification

Marine fungi occur either in Open Ocean or in the intertidal zone of sandy beaches, salt marshes and mangroves, where their hosts and substrates are found. The development of morphological adaptations like appendages and sheaths of the spores are vital to the settlement and attachment to substrate surfaces, floatation and dispersion on seawater. The morphological features of these appendages and sheaths of spores also have an important role in the identification of marine fungi. Differential interference contrast (DIC) microscopy is an essential tool for the observation of these mucilaginous structures in marine fungi spores and was therefore applied to marine mycota from surveys along the Portuguese coast.Since 1991 Portuguese marine fungi have been studied and characterized based on morphological identifications. The studied environments included salt marshes, sandy beaches and marinas. On these environments different substrates were collected such as plants and baits of Spartina maritima, different categories of drift substrates and Fagus sylvatica and Pinus pinaster baits. These substrates, which had been exposed to different conditions of permanent and temporary submersion, were subjected to an initial examination under the stereoscope microscope in order to detect fruit bodies and spores of marine fungi. These structures were then observed in order to achieve the taxonomic identifications of these fungi, based on dichotomous keys for marine fungi.To observe and characterize the wide variety of appendages and mucilaginous sheaths of the ascospores, basidiospores and conidia often invisible in the bright field of the light microscope, the use of DIC microscopy was implemented, because three-dimensional images are produced, highlighting them. The identified fungi were microphotographed with a Leica Wild MPS 52 with Fujichrome RTP- 135, 64T Tungsten.The studies carried out with substrates subjected to conditions of permanent submersion highlighted the dominance of Ascomycota with unitunicate asci. The unitunicate asci are thin - walled, persistent or early deliquescing, favoring ascospores release on marine environmental conditions (fig 1a, 1b and 1c). On the other hand, Ascomycota with bitunicate asci, were mainly detected on temporary submersion conditions. These fungi presented asci with active spore discharge and mucilaginous sheaths that contributed to substrate attachment (fig. 2a and 2b). Ascospores and conidia presented morphological diversity on their appendages, which has particular importance on their success in marine environment (figs. 3, 4, 5, and 6).Marine Mycologists are in agreement that the best technique to observe the appendages and sheaths of spores, often invisible in the bright-field microscope is the use of differential interference or phase contrast microscopy. DIC microscopy was then applied to the observation of Portuguese marine fungi enabling to thoroughly characterize the structures essential to their morphological identifications.

Evidence of reproductive cost in the triphasic life history of the red alga Gracilaria chilensis (Gracilariales, Rhodophyta)

The extent of changes in basic physiological and demographic traits associated with reproduction was investigated in the highly cultivated haploid–diploid red alga, Gracilaria chilensis. Sixty individuals bearing vegetative and reproductive fronds collected in the natural population of Niebla (39°52′ S, 73°23′ W), in Chile, were cultivated under controlled culture conditions. Our results demonstrated that vegetative fronds have a higher survival rate and a better growth rate than reproductive ones irrespective of the type of individual analyzed (male gametophyte, female gametophyte, and tetrasporophyte). Moreover, the reproductive fronds clearly showed a decrease in photosynthetic activity compared to non-reproductive ones. In males and tetrasporophytes, the photosynthetic reduction in reproductive individuals could be explained by a physical effect of reproductive structure development as well as spores release, disrupting the continuity of the photosynthetic cortical tissues. Translocation of photoassimilates from nearby vegetative tissue or the previous accumulation of photosynthetic products seems to be a prerequisite for reproductive structure development in this species. Altogether, these results document for the first time in G. chilensis that reproduction has a strong physiological effect on male, female, and tetrasporophyte fronds. This trade-off between reproduction, growth, and survival suggest the existence of reproductive costs in the life history of G. chilensis.

납작돌잎(Lithophyllum yessoense)과 진분홍딱지(Hildenbrandia rubra)의 포자방출 및 생장에 미치는 수온의 영향

The effects of temperature on spore release, growth and photosynthetic efficiency of Lithophyllum yessoense and Hildenbrandia rubra were examined. L. yessoense was collected at Galnam and H. rubra was collected at Gyeokpo, Korea. The experimental temperatures were different for spore release (10, 15, <TEX>$20^{\circ}C$</TEX>), sporeling growth (10, 15, 20, 25, <TEX>$30^{\circ}C$</TEX>) and photosynthetic efficiency (10, 15, 20, <TEX>$25^{\circ}C$</TEX>). All other culture conditions were the same: 34 psu, 12:12 LD and <TEX>$50{\mu}mol$</TEX> photon <TEX>$m^{-2}s^{-1}$</TEX>. Spore liberation was maximal at <TEX>$10^{\circ}C$</TEX> for L. yessoense and at <TEX>$20^{\circ}C$</TEX> for H. rubra. After 14 days, the surface area of L. yessoense was 0.031 <TEX>$mm^2$</TEX> at <TEX>$25^{\circ}C$</TEX> and for H. rubra was 0.032 <TEX>$mm^2$</TEX> at <TEX>$20^{\circ}C$</TEX>. Sporelings of L. yessoense were a dark-red color and grew in a round shape. In contrast, H. rubra was bright pink and changed from a round shape in the early growth stage to later become flabelliform. Photosynthetic efficiency was highest between <TEX>$20-25^{\circ}C$</TEX> in both species. In conclusion, L. yessoense and H. rubra display different physiological features based on the optimal temperatures for spore release and sporling growth.

Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology

The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67–80bp reads with 94–100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores.

A Security Take on Safeguarding Children: Pediatric Medical Countermeasure Research

Two years ago I joined a group of experts across federal agencies to discuss the findings of an exercise dubbed “Dark Zephyr.” The Department of Health and Human Services (HHS) Office of the Assistant Secretary for Preparedness and Response (ASPR) developed this exercise to test the US government's capacity to deal with the aftermath of a large-scale attack involving a biological weapon. This particular simulation examined what would happen during the mass distribution of Bacillus anthracis spores, the causative agent of the disease anthrax. The projected outcomes, although alarming, did not surprise those who worked on issues pertaining to weapons of mass destruction (WMD). Nearly 8 million people could be exposed if a mass anthrax spore release took place in a city such as San Francisco, and almost a quarter of those exposed would be children. Many of those infected would die if left untreated. Despite a stressful situation rife with uncertainty, local, state, and federal agencies would have to provide an integrated response involving coordinated communication strategies, predictive plume modeling, forensics, intelligence collection, and distribution of medical products from the Strategic National Stockpile (SNS) through points of distribution. Dark Zephyr deliberately focused on the distribution of medical countermeasures (MCMs), including vaccine. Protocols developed by the US Centers for Disease Control and Prevention (CDC) to treat exposure to B. anthracis include the use of anthrax vaccine adsorbed (AVA) for both adults and children. Accordingly, much of the Dark Zephyr analysis focused on the amount of vaccine in the SNS, the speed with which vaccine could be delivered, and public acceptance of the MCM. One of the issues raised during the discussion was the absence of any definitive evidence regarding AVA use with children. Although vaccine administration is included in treatment protocols for children, did we know if it was effective? Did we know if the dosing was correct? Did we know whether it would even be accepted by parents and caregivers? These are difficult questions with no easy answers—answers that we need before we can be confident that preparedness and response plans for a mass anthrax distribution event would adequately provide for the care of children. Beyond the matter of MCMs for anthrax loomed the even larger question of how to obtain the best scientific evidence for pediatric treatment in the face of WMD threats more generally, particularly those cases where no therapy yet exists.

Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M1 (AFM1) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM1 sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM1 in milk. However, if there was no color change, presence of AFM1 at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM1 detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM1 in milk.

Carpospore release and sporeling development in Gracilaria gracilis (Gracilariales, Rhodophyta) from the southwestern Atlantic coast (Chubut, Argentina)

The agarophyte Gracilaria gracilis is an econom- ically valuable species exploited in Argentina and the car- pospore study is relevant since such spores are used as inoculum for commercial aquaculture. The aims of this work were to describe the carpospore release periodicity and carposporeling development and to evaluate the influ- ence of temperature, irradiance, and day length on carpo- spore release under laboratory conditions. Desiccation, osmotic shock, and spontaneous discharge were compared as sporulation induction methods and the last one was the best method for improved carpospore release (ANOVA: F= 6.4018, P 0.25). Sporulation time course analysis showed a peak during the first week with a total carpospore release at the end of the sixth week of about 2,000 carpospores cystocarp �1 day �1 .

Transcriptional Regulation and Characteristics of a Novel N -Acetylmuramoyl- l -Alanine Amidase Gene Involved in Bacillus thuringiensis Mother Cell Lysis

In Bacillus thuringiensis, a novel N-acetylmuramoyl-L-alanine amidase gene (named cwlB) was detected, and the CwlB protein was purified and characterized. Reverse transcription-PCR (RT-PCR) results indicated that cwlB and an upstream gene (named cwlA) formed one transcriptional unit. 5' rapid amplification of cDNA ends (5'-RACE)-PCR and transcriptional fusions with the lacZ gene indicated that transcription of the operon was directed by a promoter, P(cwlA), which is located upstream from the cwlA gene and that the transcription start site is a single 5'-end nucleotide residue T located 25 nucleotides (bp) upstream from the cwlA translational start codon. Moreover, the activity of P(cwlA) was controlled by σ(K). Morphological analysis suggested that the mutation of cwlB could delay spore release compared to the timing of spore release in the wild-type strain. Western blot assay demonstrated that purified CwlB bound to the B. thuringiensis cell wall. Observations with laser confocal microscopy and a green fluorescent protein-based reporter system demonstrated that the CwlB protein localizes to the cell envelope. All results suggest that the CwlB protein is involved in mother cell lysis in B. thuringiensis.

The photosynthetic performance of sterile and fertile sporophytes in a natural population of the fern Dryopteris affinis

We studied the photosynthetic performance of sterile and fertile sporophytes in a natural population of the fern Dryopteris affinis growing within a riparian forest (Central Italy) using chlorophyll (Chl) a fluorescence transients, the OJIP phase, where O is for the minimum fluorescence, P is for the peak (the maximum), and J and I are inflections. The "vitality" of the samples was assessed by the maximum quantum yield of primary photochemistry obtained indirectly from the fluorescence data (Fv/Fm); in the same way, the so-called performance index (PIABS) was obtained from fluorescence data. The photosynthetic performance (inferred from PIABS) of D. affinis changed significantly with the seasonal development of the fronds. The highest photosynthetic performance was recorded in the summer, corresponding to the period of spore release. The photosynthetic performance decreased in the winter, down to the minimal values of senescent fronds reached at the end of the seasonal cycle (May-June). On the whole, during the seasonal development, sterile and fertile fronds had a similar photosynthetic behaviour, as inferred from fluorescence data. At the end of spore maturation and dispersal (September-October), the fertile fronds showed somewhat lower photosynthetic performance than the sterile fronds, as revealed by PIABS. Being a long-lived fern, confined to humid and undisturbed sites in the Mediterranean, D. affinis deserves to be further investigated as a potential indicator of ecological continuity in Mediterranean riparian forests.

Anthracnose Caused by Colletotrichum horii on Sweet Persimmon in Korea: Dissemination of Conidia and Disease Development

AbstractAnthracnose disease caused by Colletotrichum horii (C. gloeosporioides), results in considerable economic damage to sweet persimmon in southern Korea yearly. This study deals with the life cycle of the pathogen in terms of seasonal fluctuations of spore dispersal and the development of disease based on field surveys, spore potential and fungal isolation. Anthracnose disease was observed first on twigs in the last week of May and reached an incidence of 1.2%. Subsequently, the disease increased rapidly and reached an incidence of 86% by the end of July. Infection of fruits started in mid‐June (2.8%) and increased gradually to 64.4% by the end of July. In severely infected orchards, 46.2% of diseased fruits were dropped. The pathogen began releasing conidia in the first week of May and continued until the end of September. The maximum release of spores was observed in mid‐July. To determine the optimal use of chemicals for control of anthracnose, the following spray programme was evaluated. Spraying two or three times resulted in 89.4 and 93% control, respectively, whereas spraying more than four times led to 100% control. In comparison, the disease rate of unsprayed trees was 89.8%. To control anthracnose effectively, it is recommended to take steps to eliminate inoculum sources in sweet persimmon orchards before spraying chemicals.

Incubation Temperature and Substrate Quality Modulate Sporulation by Aquatic Hyphomycetes

Frequency and amplitude of temperature oscillations can profoundly affect structure and function of ecosystems. Unless the rate of a biological process changes linearly within the range of these fluctuations, the cumulative effect of temperature differs from the effect measured at the average temperature (Jensen's inequality). Here, we measured numbers and types of spores released by aquatic hyphomycetes from oak and alder leaves that had been exposed in a Portuguese stream for between 7 and 94 days. Recovered leaves were incubated at four temperatures between 5 and 20 °C. Over this range, the sporulation response to temperature was decelerating, with an estimated optimum around 12.5 °C. Assuming a linear response, therefore, overestimates spore release from decaying leaves. The calculated discrepancy was more pronounced with recalcitrant oak leaves (greater toughness, phenolics concentration, lower N and P concentration than alder), and reached 26.6 % when temperature was assumed to oscillate between 1 and 9 °C, rather than remaining constant at 5 °C. The maximum fluctuation of water temperature over 48 h during the field experiment was approximately 3 °C, which would result in a discrepancy of up to 6 %. The composition of the fungal community (assessed by species identification of released spores) was significantly influenced by the state of decomposition, but not by leaf species or temperature. When quantifying the potential impact of global change on aquatic fungal communities, the average increase as well as fluctuations of the temperature have to be considered.

Spore Trapping and Pathogenicity of Fungi in the Botryosphaeriaceae and Diaporthaceae Associated with Avocado Branch Canker in California

Avocado branch canker in California is caused by a complex of fungal species in the families Botryosphaeriaceae and Diaporthaceae. As the popularity of avocado fruit increases, California growers are under pressure to increase their productivity in order to compete with imports. One way to increase production is through high-density planting, which entails intense canopy management, possibly leaving the trees vulnerable to infection through pruning wounds. A spore trap study was undertaken to determine the seasonal spore discharge of Botryosphaeriaceous and Diaporthaceous fungi. Based on colony counts, the highest population of Botryosphaeriaceous fungi (68%) occurred during or soon after precipitation events, which coincided with the winter months of December, January, and February. The spring and fall seasons had lower numbers of spores trapped (at 13 and 17%, respectively), with few spores trapped in the summer season. For members of the Diaporthaceae family, spores trapped were almost evenly split between winter and fall seasons (50 and 49%, respectively), with few to no spores trapped in the spring and summer seasons. A pathogenicity test of six fungi of Botryosphaeriaceae and one fungus of Diaporthaceae was conducted in the greenhouse. Internal plant vascular lesion lengths resulting from inoculation with any of the seven fungal species differed significantly from the noninoculated control. These studies suggest that pruning during the drier parts of the year would minimize infection by fungi in the Botryosphaeriaceae and Diaporthaceae families.

Temporal dynamics of spore release of the crayfish plague pathogen from its natural host, American spiny-cheek crayfish (Orconectes limosus), evaluated by transmission experiments

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to indigenous European crayfish species. The North American invasive spiny-cheek crayfish, Orconectes limosus, is an important source of this pathogen in central and western Europe. We evaluated potential changes in A. astaci spore release rate from infected individuals of this species by experiments investigating the pathogen transmission to susceptible noble crayfish, Astacus astacus. We filtered defined volumes of water regularly to quantify spore concentration, and sampled crayfish tissues at the end of the experiment. The filters and tissues were then tested for the presence of A. astaci DNA by species-specific quantitative PCR. Additionally, we tested the efficiency of horizontal transmission to apparently uninfected O. limosus. The experiments confirmed that A. astaci can be transmitted to susceptible crayfish during intermoult periods, and that the pathogen was more frequently detected in noble crayfish recipients than in American ones. The pathogen spore concentrations substantially varied in time, and significantly increased during moulting of infected hosts. Our study strengthens the evidence that although the likelihood of crayfish plague transmission by water transfer from localities with infected American crayfish might increase when these are moulting or dying, no time-periods can be proclaimed safe.

An orthogonal design for optimization of growth conditions for all life history stages of Gracilariopsis lemaneiformis (Rhodophyta)

Gracilariopsis lemaneiformis is an important economic macroalgae worldwide. At present it is mainly cultured by vegetative propagation, which is labor intensive, bulky, and requires large amounts of propagating material. To identify the optimal conditions for spore release, spore development, and fertilization success of Gp. lemaneiformis, four levels of each of four factors (light, temperature, salinity, and photoperiod) were tested using an orthogonal experimental design. Variations in temperature and salinity had significant effects on carpospore yield, and the optimal conditions for carpospore release were 20°C, 35‰, 15μmolm−2s−1, and 8/16–14/10hL/D. The main factors that influenced tetraspore release were temperature, light, and salinity, with the optimal combination being 25°C, 15μmolm−2s−1, 35‰, and 8/16–14/10hL/D. Temperature variation also had an effect on spore development, whereas the effects of the other factors were not significant. The optimal conditions for tetraspore development were 25°C, 45μmolm−2s−1, 12/12hL/D, and 25‰, and for carpospore development they were 25°C, 45μmolm−2s−1, 30‰, and 12/12hL/D. Temperature significantly affected fertilization success of male and female gametophytes, whereas salinity, photoperiod, and light intensity had no significant effect (P>0.05). The optimal combination of factors for fertilization success was 25°C, 30‰, 12/12hL/D, and 15–45μmolm−2s−1. The culture time of sporeling growth was reduced by about 3–4months in the study, the results of which can also provide valuable information for genetic breeding, large-scale culture of spores, and algal cultivation.

Environmental predictors and incubation period of AIDS-associated penicillium marneffei infection in Ho Chi Minh City, Vietnam.

Penicillium marneffei is an emerging dimorphic mycosis endemic in Southeast Asia, and a leading cause of mortality among human immunodeficiency virus (HIV)-infected people in the region. Factors governing the seasonal incidence of P. marneffei infection are unknown, and may yield critical insights into possible reservoirs or modes of acquisition. This study included HIV-infected patients presenting with P. marneffei (n = 719) and Cryptococcus neoformans (n = 1598) infection to the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam, from 2004 to 2010, and temperature, humidity, wind, precipitation, and HIV-related admissions data for the corresponding period. We used multivariate regression modeling to identify factors associated with P. marneffei and C. neoformans admissions. We estimated the P. marneffei incubation period by considering profile likelihoods for different exposure-to-admission delays. We found that P. marneffei admissions were strongly associated with humidity (P < .001), and that precipitation, temperature, and wind did not add explanatory power. Cryptococcus neoformans admissions were not seasonal, and P. marneffei admissions were more common relative to C. neoformans admissions during months of high (≥85%) humidity (odds ratio, 1.49; 95% confidence interval [CI], 1.10-2.01). Maximum likelihood estimation suggested a P. marneffei incubation period of 1 week (95% CI, 0-3 weeks). Our findings suggest that humidity is the most important environmental predictor of P. marneffei admissions, and may drive exposure by facilitating fungal growth or spore release in the environment. In addition, it appears that a high proportion of penicilliosis patients present to the hospital with primary disseminated infection within 3 weeks of exposure.

The Multilayered Interlaced Network (MIN) in the sporoplasm of the Microsporidium Anncaliia algerae is derived from Golgi

This study provides evidence for the Golgi-like activity of the multilayered interlaced network (MIN) and new ultrastructural observations of the MIN in the sporoplasm of Anncaliia algerae, a microsporidium that infects both insects and humans. The MIN is attached to the end of the polar tubule upon extrusion from the germinating spore. It surrounds the sporoplasm, immediately below its plasma membrane, and most likely maintains the integrity of the sporoplasm, as it is pulled through the everting polar tube. Furthermore, the MIN appears to deposit its dense contents on the surface of the sporoplasm within minutes of spore discharge thickening the plasma membrane. This thickening is characteristic of the developmental stages of the genus Anncaliia. The current study utilizes transmission electron microscopy (TEM), enzyme histochemistry, and high voltage TEM (HVEM) with 3D tomographic reconstruction to both visualize the structure of the MIN and demonstrate that the MIN is a Golgi-related structure. The presence of developmentally regulated Golgi in the Microsporidia has been previously documented. The current study extends our understanding of the microsporidial Golgi and is consistent with the MIN being involved in the extracellular secretion in Anncaliia algerae. This report further illustrates the unique morphology of the MIN as illustrated by HVEM using 3D tomography.

Comparative development and tissue tropism of Nosema apis and Nosema ceranae

The two etiological agents of nosema disease in honey bees, Nosema apis and Nosema ceranae (Microsporidia: Nosematidae), reproduce in the midgut tissues of the host. N. apis is tissue specific but the development and tissue tropism of N. ceranae is not well understood. Our investigations compared development of the two phylogenetically related pathogens in all major host tissues. Using microscopy, PCR and qPCR quantification to evaluate tissue tropism of infected bees in communal cages and of individually restrained infected bees, we found no detectable spores in cephalic or other body tissues except midgut tissues. Nosema DNA was detected in Malpighian tubules but the tubules could not be separated from the alimentary tract without release of spores from the midgut. Nosema DNA was not detected in hemolymph sampled from the head capsule or the abdomen of infected bees. We confirmed that N. ceranae only develops in midgut tissues. Spores of both species released from host midgut cells accumulated in the hindgut lumen, and we noted differences in numbers and ratios of spore types and in growth curves between the two pathogens. N. apis reached a consistent level of spore production after 12days post inoculation (dpi); N. ceranae spore production increased linearly from 12 to 20 dpi and the number of mature N. ceranae spores was consistently higher.

Operational Evaluation of the Rapid Viability PCR Method for Post-Decontamination Clearance Sampling

The Rapid Viability Polymerase Chain Reaction (RV-PCR) method was evaluated during the Bio-Response Operational Testing and Evaluation (BOTE), an interagency project to evaluate field-level facility biological remediation, using leading decontamination technologies. The tests were performed using an intentional release (aerosolization) of spores of Bacillus atrophaeus subspecies globigii (BG), as a surrogate for Bacillus anthracis, the etiologic agent for anthrax. Three decontamination methods were assessed including fumigation with vaporized hydrogen peroxide (VHP), fumigation with chlorine dioxide (CD), and a surface treatment process using pH-adjusted bleach.The RV-PCR method was developed to rapidly detect live B. anthracis spores during a bioterrorism event. The method uses a change in realtime PCR response before and after a nine hour incubation step, to determine the presence of viable bacterial spores in the sample; the method was recently verified for air filter, wipe and water samples at the 10-spore level for B. anthracis Ames spores, and was also developed for swab, sponge-stick, and vacuum sock/filter samples. In the method, high throughput sample processing is combined with PCR-based analysis before and after a rapid culture step to speed viability determination, especially for complex surface and environmental samples that present challenges to current culture-based methods. In the BOTE project, a total of 159 surface wipe samples from post-decontamination events were analyzed by splitting the suspension after spore recovery into two equal parts, with one part analyzed by RV-PCR and the other part by culture after concentrating to the same volume. In the BOTE project, the RV-PCR method provided rapid results for post-decontamination samples that were 98% (156/159 samples) consistent with results from culture analysis. The percentage agreement was noteworthy, given the large number of samples containing low spore levels. For the Post-VHP, Post-Bleach, and Post-CD event samples, the percentage agreement was 93% (41/44 samples), 100% (47/47 samples), and 100% (68/68 samples), respectively. The RV-PCR method performed well for the surrogate BG spores exposed to decontaminants at real-world application levels, and with wipe samples containing background debris and indigenous microbial populations.