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PubMed2002

Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination.

The location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B. subtilis spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The ypeB gene is the second gene in a bicistronic operon with sleB. The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB - in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme's hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD(600) fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore's ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B. subtilis. Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination: the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

Amidohydrolases↗
PubMed2006

Significant passive protective effect against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids.

The protective-antigen (PA)-based cell-free vaccine is the only vaccine licensed for use against Bacillus anthracis infection in humans. Although the PA shows strong immunogenicity, the capsule or spore-associated somatic antigens may be important as additional vaccine targets for full protection against anthrax. In this study, the protective effect of spore-associated antigens against B. anthracis infection was determined. Rabbits were immunized with formalin-fixed spores of a non-toxigenic unencapsulated B. anthracis strain that lacked the two virulence plasmids pXO1 and pXO2, and the protective effects of the immune antibody were evaluated. Immunostaining and Western blot analysis revealed that the anti-B. anthracis (anti-BA)-spore IgG specifically bound to the surface of spores or endospores of B. anthracis, but not to vegetative cells, or closely related Bacillus species, such as Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis. Passively transferred anti-BA-spore IgG protected mice from intraperitoneal challenge with a lethal dose of fully virulent B. anthracis spores, and increased the survival rate in a dose-dependent manner. Pre-incubation of spores with antibody also reduced their infectivity in a dose-dependent manner. The number of bacteria (c.f.u.) in spleens and livers of infected mice was significantly lower in antibody-treated mice than in untreated mice. Treatment with anti-BA-spore IgG also inhibited the germination of spores in J774.1 macrophages, suggesting that opsonization of spores promotes phagocytosis and subsequent killing by macrophages. These results indicate the usefulness of spore surface antigens as vaccine targets. In combination with major virulence factors such as the PA, spore-associated antigens may offer a safer and more effective multicomponent vaccine for B. anthracis infection.

Animals↗
PubMed1990

Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae.

During sporulation of Saccharomyces cerevisiae, the four haploid nuclei generated by meiosis are encapsulated within multilayered spore walls. Taking advantage of the natural fluorescence imparted to yeast spores by the presence of a dityrosine-containing macromolecule in the spore wall, we identified and cloned two genes, termed DIT1 and DIT2, which are required for spore wall maturation. Mutation of these genes has no effect on the efficiency of spore formation or spore viability. The mutant spores, however, fail to accumulate the spore wall-specific dityrosine and lack the outermost layer of the spore wall. The absence of this cross-linked surface layer reduces the resistance of the spores to lytic enzymes, to ether, and to elevated temperature. Expression of the DIT and DIT2 genes is restricted to sporulating cells, with the DIT1 transcripts accumulating at the time of prospore enclosure and just prior to the time of dityrosine biosynthesis. Both genes act in a spore-autonomous manner implying that at least some of the activities responsible for forming the outermost layer of the spore wall reside within the developing spore rather than in the surrounding ascal cytoplasm. As the DIT2 gene product has significant homology with cytochrome P-450s, DIT2 may be responsible for catalyzing the oxidation of tyrosine residues in the formation of dityrosine.

Amino Acid Sequence↗
PubMed2006

How do spores germinate?

Spore germination, as defined as those events that result in the loss of the spore-specific properties, is an essentially biophysical process. It occurs without any need for new macromolecular synthesis, so the apparatus required is already present in the mature dormant spore. Germination in response to specific chemical nutrients requires specific receptor proteins, located at the inner membrane of the spore. After penetrating the outer layers of spore coat and cortex, germinant interacts with its receptor: one early consequence of this binding is the movement of monovalent cations from the spore core, followed by Ca2(+) and dipicolinic acid (DPA). In some species, an ion transport protein is also required for these early stages. Early events - including loss of heat resistance, ion movements and partial rehydration of the spore core - can occur without cortex hydrolysis, although the latter is required for complete core rehydration and colony formation from a spore. In Bacillus subtilis two crucial cortex lytic enzymes have been identified: one is CwlJ, which is DPA-responsive and is located at the cortex-coat junction. The second, SleB, is present both in outer layers and at the inner spore membrane, and is more resistant to wet heat than is CwlJ. Cortex hydrolysis leads to the complete rehydration of the spore core, and then enzyme activity within the spore protoplast resumes. We do not yet know what activates SleB activity in the spore, and neither do we have any information at all on how the spore coat is degraded.

Amidohydrolases↗
PubMed2007

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants.

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.

Alloys↗
PubMed1989

Evaluating the penetration of Cladosporium spores into the human respiratory system on the basis of aerobiological sampling results.

The penetration of Cladosporium spores and spore aggregates into human airways was studied using three different spore sampling methods: 1) a Burkard spore trap for determining the aggregation degree of Cladosporium; two samplers, simulating the human respiratory system, 2) a 6-stage Andersen 2000 sampler, and 3) a new size-selective bioaerosol sampler (SSBAS), designed specifically for immunochemical and chemical analyses. The aggregation degree of Cladosporium spores varied between 1.0 and 1.3 spores per dispersal unit. Grouping seems to be of little if any importance to the penetration ability of Cladosporium spores into the respiratory tract. The distribution of spores in the Andersen and SSBAS differed significantly in the largest size class (spores greater than 7 microns in diameter); with the Andersen sampler only 10.8% of the spores were detected in stage 1, compared with 43% with the SSBAS. On the Andersen culture plates 95% of all colonies were detected in stages 1-4, where particles greater than 2.1 microns in diameter are trapped. In the SSBAS altogether 99.4% of spores were found in the first two filter stages (cutoff point approx. 2.5 microns in diameter). Conclusions regarding the penetration of spores to the lungs on the basis of aerobiological results should always be based on the use of properly calibrated spore traps.

Cladosporium↗
PubMed1990

Influence of transition metals added during sporulation on heat resistance of Clostridium botulinum 113B spores.

Sporulation of Clostridium botulinum 113B in a complex medium supplemented with certain transition metals (Fe, Mn, Cu, or Zn) at 0.01 to 1.0 mM gave spores that were increased two to sevenfold in their contents of the added metals. The contents of calcium, magnesium, and other metals in the purified spores were relatively unchanged. Inclusion of sodium citrate (3 g/liter) in the medium enhanced metal accumulation and gave consistency in the transition metal contents of independent spore crops. In citrate-supplemented media, C. botulinum formed spores with very high contents of Zn (approximately 1% of the dry weight). Spores containing an increased content of Fe (0.1 to 0.2%) were more susceptible to thermal killing than were native spores or spores containing increased Zn or Mn. The spores formed with added Fe or Cu also appeared less able to repair heat-induced injuries than the spores with added Mn or Zn. Fe-increased spores appeared to germinate and outgrow at a higher frequency than did native and Mn-increased spores. This study shows that C. botulinum spores can be sensitized to increased thermal destruction by incorporation of Fe in the spores.

Clostridium botulinum↗
PubMed1974

Ultrastructural analysis during germination and outgrowth of Bacillus subtilis spores.

Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.

Bacillus subtilis↗
PubMed1978

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.

Adenosine Triphosphate↗
PubMed1981

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).

Bacillus megaterium↗
PubMed1997

Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents.

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.

Acatalasia↗
PubMed1998

Analysis of outgrowth of Bacillus subtilis spores lacking penicillin-binding protein 2a.

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.

Bacillus subtilis↗
PubMed2000

Discrimination between viable and dead Encephalitozoon cuniculi (Microsporidian) spores by dual staining with sytox green and calcofluor white M2R.

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled) Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.

Analysis of Variance↗
PubMed1996

Binding of Aspergillus fumigatus spores to lung epithelial cells and basement membrane proteins: relevance to the asthmatic lung.

BACKGROUND: Aspergillus fumigatus is an opportunistic pathogen to which asthmatic subjects are particularly susceptible. The ability of spores of A fumigatus to bind to pulmonary cells and basement membrane proteins was investigated to determine the mechanisms involved in this susceptibility. METHODS: Cells of the A549 pulmonary epithelial cell line or purified basement membrane proteins were immobilised on the wells of microtitre plates. They were then exposed to spores of A fumigatus in suspension, with or without various pretreatments of the spores, cells, and proteins. Adherent spores were counted by light microscopy. RESULTS: Spores of A fumigatus bound in a concentration dependent manner to A549 epithelial cells and pretreatment of cells with interferon gamma (2500 units/ml) caused a significant doubling of spore binding. Binding of spores to A549 cells was inhibited by about a third by pre-incubation of the spores with fibrinogen (100 micrograms/ml). Spores bound specifically to extracellular matrix (ECM) components laid down by A549 cells, and pretreatment of the ECM components with hydrogen peroxide (25-80 microM) enhanced spore binding by approximately one third. They also bound specifically and in a saturable manner to purified fibrinogen, fibronectin, laminin, type I collagen, and type IV collagen. Pre-incubation of spores with Arg-Gly-Asp tripeptide (RGD; 50-200 micrograms/ ml) inhibited binding to fibronectin and type I collagen by 50%. CONCLUSIONS: This study suggests that the presence of activated epithelial cells and the exposure of basement membrane that occurs in asthma, together with oxidant stress, may facilitate the colonisation of the asthmatic lung by A fumigatus. The RGD sequence may be involved in spore binding to some ECM proteins. Free fibrinogen may protect against binding of A fumigatus spores to the pulmonary epithelium.

Analysis of Variance↗
PubMed1997

Effects of intranasal exposure to spores of Stachybotrys atra in mice.

The effects of highly toxic and nontoxic spores of Stachybotrys atra were investigated in mice after six intranasal administrations of 1 x 10(5) and 1 x 10(3) spores in phosphate-buffered saline during a 3-week period. Toxic spores contained the trichothecene mycotoxins, satratoxins G and H, as well as the immunosuppressant stachybotrylactones and -lactams. No trichothecenes were detected in the nontoxic spores, and they contained only minor amounts of stachybotrylactones and -lactams. In mice injected with toxic and nontoxic spores, the platelet count was decreased and leucocyte and erythrocyte counts, hemoglobin concentration, and hematocrit were increased. No IgG antibodies to S. atra were detected in sera of mice exposed intranasally to spores. No histological changes were detected in spleen, thymus, or intestines of mice. The mice receiving 1 x 10(5) toxic spores intranasally developed severe inflammatory changes within both bronchioles and alveoli. Hemorrhage was detected in alveoli. The mice receiving 1 x 10(5) nontoxic spores also developed inflammatory changes in the lungs, but these changes were significantly milder than those in mice receiving toxic spores. The mice receiving 1 x 10(3) toxic spores developed inflammatory changes in the lungs that were less severe than those in the mice receiving 1 x 10(5) toxic spores. No inflammatory changes were detected in the mice receiving 1 x 10(3) of nontoxic spores. The present findings indicate that exposure to S. atra spores containing toxins (satratoxins) can be a significant health risk.

Administration, Intranasal↗
PubMed1964

EFFECT OF SPORULATION MEDIUM ON HEAT RESISTANCE, CHEMICAL COMPOSITION, AND GERMINATION OF BACILLUS MEGATERIUM SPORES.

Levinson, Hillel S. (U.S. Army Natick Laboratories, Natick, Mass.), and Mildred T. Hyatt. Effect of sporulation medium on heat resistance, chemical composition, and germination of Bacillus megaterium spores. J. Bacteriol. 87:876-886. 1964.-Bacillus megaterium spores, grown on variously supplemented media, had varying concentrations of P, Ca, Mn, or dipicolinic acid. Supplementation with CaCl(2) yielded spores with increased heat resistance; addition of l-glutamate, l-proline, or increase of the phosphate concentration yielded spores with reduced heat resistance. Germination characteristics depended on both the sporulation medium and the germinant (glucose, l-alanine, l-leucine, or KNO(3)); pronounced differences were demonstrable with glucose and l-alanine, which trigger germination via different metabolic pathways. An increase in CaCl(2) during sporulation yielded spores with increased germination in glucose but not in l-alanine. Germination in l-alanine was optimal with spores produced on media containing 0.1 mm MnCl(2), but germination of such spores was minimal in glucose. An increase in the sporulation medium phosphate decreased the initial germination rate in glucose, but not in l-alanine. Spores produced in CaCl(2)-supplemented media had increased heat-activation requirements (increased dormancy) for germination induced by l-alanine, and decreased heat-shock requirements for glucose-induced germination. An increase of sporulation phosphate yielded spores with reduced dormancy for germination induced by l-alanine, but with unchanged dormancy on the other germinants. Spores produced with added l-glutamate had reduced dormancy for glucose-induced germination, and increased dormancy for germination induced by l-alanine. Addition of CaCl(2) or l-glutamate to the sporulation medium yielded spores with increased sensitivity to "ionic germination" (with KI). Spores from synthetic medium were incapacitated for full postgerminative development, as shown by repression of the changes in oxygen-uptake rate which accompany normal cell division.

Alanine↗
PubMed2007

Incubation period, spore egestion and horizontal transmission of Nosema fumiferanae (Microsporidia: Nosematidae) in spruce budworm (Choristoneura sp., Lepidoptera: Tortricidae): the role of temperature and dose.

Various instars of Choristoneura occidentalis were fed with a range of doses of Nosema fumiferanae and reared at 20, 24 and 28 degrees C to determine the influence of temperature and dose on the time to spore egestion and the number of spores egested in the frass. When larvae were fed in the third stadium, as few as 10(2) spores per larva initiated infection, and both onset of spore egestion and the number of spores egested were affected by a complex relationship between temperature and inoculation dose. Onset of spore egestion varied from 11 to 15 days postinoculation. At 20 degrees C, the onset was delayed and spore production decreased with increasing inoculation dose whereas at higher temperatures spores were first egested at the lowest dose and spore production increased with dose. When larvae were fed spores in the fifth and sixth stadium, no spores were egested because pupation occurred before completion of the incubation period. To assess the effect of temperature on horizontal transmission, Choristoneura fumiferana larvae fed with 10(4) N. fumiferanae spores per larva were reared with uninfected larvae at 15, 20 and 25 degrees C. At 15 degrees C, we observed the highest degree of horizontal transmission, defined by the largest change in N. fumiferanae prevalence, even though the density of spores available for horizontal transmission was the lowest. Infected adults eclosed later than uninfected adults and the time to eclosion was also dependent on sex and temperature. We relate our experimental findings to consequences for horizontal and vertical transmission of N. fumiferanae in spruce budworm populations.

Animals↗
PubMed2003

Histological, immunohistochemical and morphometric changes in lung tissue in juvenile mice experimentally exposed to Stachybotrys chartarum spores.

Stachybotrys chartarum is an important toxigenic fungus often associated with chronically wet cellulose-based building materials. The purpose of this study was to evaluate some histological, immunohistochemical and morphometric changes in mouse lung tissues exposed intratracheally to either 50 microl of 1.4 x 10(6) S. chartarum spores (< or = 35 ng toxin/kg BW), isosatratoxin-F (35 ng/kg BW), 50 microl of 1.4 x 10(6) Cladosporium cladosporioides spores, or 50 microl saline. Exposure of lung tissues to S. chartarum or C. cladosporioides spores resulted in granuloma formation at the sites of spore impaction. Some of the lung tissues impacted by S. chartarum spores also showed erythrocyte accumulation in the alveolar air space, dilated capillaries engorged with erythrocytes, and hemosiderin accumulation at spore impaction sites, which were features not noted in the C. cladosporioides-spore treated animals. Immunohistochemistry revealed reduced collagen IV distribution in lung granulomas in S. chartarum-treated animals especially at 48 and 72 hr post-exposure compared to that in lungs of mice with C. cladosporioides-spore induced granulomas. Quantitative analysis of pooled S. chartarum and C. cladosporioides spore impacted lungs revealed significant depression (P < 0.05) of alveolar air space from 71.4 +/- 6.1% in untreated animals to 56.04 +/- 6.1% in the S. chartarum- and 60.24 +/- 5.5% in the C. cladosporioides-spore treated animals. It also revealed that alveolus air space in S. chartarum treated animals declined significantly from 63.74 +/- 3.1% at 12 hr post-exposure to 42.94 +/- 7.9% at 72 hr post-exposure and was increased to 54.84 +/- 5.2% at 96 hr post-exposure. Alveolus air space in C. cladosporioides treated animals also decreased significantly from 64.84 +/- 7.1% at 12 hr exposure to 54.94 +/- 5.4% at 48 hr post-exposure and was increased to 64.64 +/- 10.1% at 96 hr post-exposure. It also revealed significant (P < 0.05) alveolar accumulation of erythrocytes from 1.24 +/- 1.4% in the untreated animals to 3.44 +/- 1.5% in the pooled S. chartarum spore treated animals. Erythrocyte abundance in S. chartarum treated animals increased significantly (P < 0.001) from 2.14 +/- 1.7% at 12 hr post-exposure to 5.54 +/- 1.5% at 72 hr and 4.94 +/- 1.4% at 96 hr post-exposure. These results further reveal that exposure to S. chartarum spores elicit tissue responses in vivo significantly different from those associated with exposure to pure trichothecene toxin and to spores of a non-toxigenic fungus.

Animals↗