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Pollen and spore counts from Burkard traps for routine pollen and spore sampling placed at 15 m above ground and at ground level were compared. Daily counts of most pollen types were higher on the ground than at roof level, but the counts were significantly correlated. The ratios of pollen frequencies at high and low levels varied between 1.0 and 11.5. The most prominent differences were recorded for herbaceous pollen (e.g. Artemisia counts 11.5 and Poaccae counts 4.4 times higher at ground level) and in Botrytis and Ustilaginales spores. Tree pollen grains and basidiomycetous spores were more equally distributed. Wind speed did not affect the variation of pollen frequencies at either height. Large spores are not so unevenly distributed as previously supposed. Artemisia and grass pollen was detected 1 to 2 weeks earlier at ground level than on the roof. It is therefore concluded that especially the beginning of flowering should be monitored at a low level.

Spores of two microsporidia, Nosema pyrausta (from the European corn borer, Ostrinia nubilalis) and N. furnacalis (from the Asian corn borer, O. furnacalis) were harvested from laboratory-reared O. nubilalis caterpillars and purified by centrifugation through Percoll. Conditions permitting in vitro germination were defined for both species and found to be different. N. pyrausta spores were incubated in 0.1 N KOH for 30 min, recovered by centrifugation, and resuspended in 1 ml of an equal mixture of 1% low melting point (LMP) agarose and L-15B medium at 37 degrees C to induce germination. N. furnacalis spores were first washed in 10 mM Na2EDTA in 1 mM Tris base, pH 7.5, exposed to 0.01 N KOH in 0.17 M KCl for 30 min, centrifuged, and germinated in 1 ml of an equal mixture of 1% LMP agarose and 0.17 M KCl in 10 mM Na2EDTA (pH 8), at 37 degrees C. Eighty to 90% of the spores of each species germinated. Germinated spores were pipetted into a casting mold. Before electrophoresis, agarose blocks were incubated 48 hr at 50 degrees C in 10 mM Tris base/100 mM Na2EDTA, pH 7.8, with 1 mg/ml proteinase K and 1% N-laurylsarcosine to release the chromosomal DNA from sporoplasms. After pulsed-field electrophoresis, ethidium bromide staining revealed 13 chromosomal bands ranging in size from 1390- to 440-kb pairs and 1360- to 440-kb pairs in N. pyrausta and N. furnacalis, respectively. The difference in size estimates of corresponding chromosomes in the two species was not more than 60-kb pairs.

The physico-chemical properties of lipid components isolated from zoospores of the aquatic phycomycete, Blastocladiella emersonii, were investigated with electron spin resonance (ESR) spectroscopy using the spin label, 5-nitroxystearate. Lipid dispersions were made from zoospore phospholipids and glycolipids, both singly and in combination with each other and with isolated neutral lipid components. Plots of the hyperfine splitting parameter (2T parallel) vs. temperature indicate that it is the zoospore glycolipids rather than the phospholipids which are responsible for the phase transformations previously observed in aqueous dispersions of the total lipids extracted from zoospores and in zoospores in vivo. The discontinuities observed in the glycolipid dispersions seem to represent the onset and completion of a gel-to-liquid-crystalline phase transition. Over the temperature range tested, Ca2+ increased the rigidity of the glycolipid dispersions, the major component of which is probably a diglucosyldiglyceride, but had no effect on the phospholipid dispersions. The increase in 2T parallel was not affected by inclusion of neutral lipids into the glycolipid dispersion but was eliminated at high (5 : 1, w/w) phospholipid-to-glycolipid ratios. The Ca2+ effect was relatively independent of both the absolute rigidity of the dispersion and its phase (gel or liquid-crystalline), suggesting an interaction with the glycolipid head group rather than the hydrocarbon core. The Ca2+-induced increase in 2T was neither prevented nor reversed by the presence of K+. The presence of two spin label populations co-existing in a dynamic equilibrium was found in glycolipid/neutral lipid dispersions. Plots of the percentage ([HA/(HA + HB)] X 100 of the spin label population, as measured by the peak height of the low-field peaks, corresponding to the more immobilized component (HA) vs. temperature indicated two break points. The temperatures at which these break points occurred are similar to those obtained for the glycolipid dispersions, and match the break points (TL and TH) found in ESR experiments using zoospores in vivo. The importance of the glycolipids in the development of this organism is discussed.

Ninety patients were investigated with intradermal testing of developing stages of Pteris vittata L., whole spores, and their fractions. Allergenic principles were found to be present in both protoplasm and spore wall of the fertile tissue.

The antigenic properties of conidial preparations of Aspergillus fumigatus were compared with mycelial and culture filtrate extracts by use of sera of patients with aspergillosis and hyperimmune rabbit serum. Biologic activities were tested by intracutaneous tests. The immunologic parameters used were: precipitating properties in double-diffusion, IgG binding by ELISA, IgE binding by enzyme inhibition assay, and by enzyme allergosorbent test. It has been demonstrated that the components that are released after disintegration of spores have positive titers in all immunologic assays. The immunologic properties of these spore plasma extracts are comparable with mycelial and/or culture filtrate extracts that are in accordance with the corresponding skin reactivities. Components that are released spontaneously from spores are only weakly positive or negative in the immunologic assays and demonstrate a very low biologic activity.

A one-year study of potentially allergenic airborne biological particles was carried out in Bogotá, the capital city of Colombia. Bogotá is a populous city of approximately 6 million inhabitants. Located at 4 degrees Lat N and 2,600 m on the sea level, it has a mild tropical climate. The mean annual temperature of the city is 14 degrees C, the annual rainfall 1,013 mm and the relative humidity 72%. During the year June '86-May '87 the pollen count was 7,626 contributed by 72 grain types. Among these, 24 taxa have been identified. The majority of the grains came from imported trees; Cupressus spp was the main contributor. Weed pollen counts, which include the Compositae (ragweed), were notoriously low (4% of the total). The spore count was 13,264, almost twice as large as that of pollen. Fourty-four spore taxa were identified out of 72 types recorded. More than two-thirds (72%) of the spore count was contributed by the deuteromycetes. Cladosporium was the more frequently recorded genus, Penicillium/Aspergillus second next. Miscellaneous particles recorded were 609 Algae and 142 fern spores. Because of its location in the tropics, Bogotá has neither true seasons nor the large variations in atmospheric particle counting which are typical of temperate zones. However, distinct periodical increments in the counts of various species (peaks) may be the cause of seasonal allergic symptoms.

From the results of a number of studies it is concluded that: (1) Cattle of a large proportion (approximately 80 per cent) of the dairy farms in the Netherlands were infected with L3 larvae of Dictyocaulus viviparus during the grazing season in 1981. (2) Experiments showed a significant negative cubic effect on the level of infection with L3 larvae of D. viviparus on the growth of calves. (3) Sporangia of Pilobolus kleinii harbouring L3 larvae were shot significantly further away than sporangia not harbouring L3 larvae.

The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

The polysaccharides from the envelopes of heterocysts and spores of Anabaena cylindrica consist of repeating units containing 1 mannosyl and 3 glucosyl residues, all linked by beta(1 yields 3) glycosidic bonds, with glycosidic bonds, with glucose, xylose, galactose, and mannose present in side branches. Degradation of the polysaccharides with specific glycosidases has permitted identification of the linkages to almost all of the branches. When the polysaccharides, from which all but two types of side branches had been cleaved, were digested with a beta(1 yields 3) endoglucanase, glucose, a tri-, and a pentasaccharide were produced. The oligosaccharide products were identified as (see article of journal). The backbones of the polysaccharides were sequenced from the reducing terminus by a modified Smith degradation. Analysis with NaB3H4 at each stage of the degradation showed that the backbones terminate in the sequence Man-Glc-Glc-Glc and are therefore presumed to have the structure (Man-Glc-Glc-Glc)n, and that they contain an average of from 128 to 150 sugar residues. From the information obtained, the repeating sequences of the original polysaccharides from the two types of differentiated cells of A. cylindrica could be largely deduced and appeared to be identical.

The composition of amino acids of sporocysts, membranes of oocysts and oocysts of four species of Coccidia from hens is reported. A considerable figgerence was found to exist in the quantitative composition of amino acids in oocysts of different Coccidia species and in different structures of oocysts. It is necessary to carry out a search of preparations breaking the inclusions of some amino acids into the protein molecule of oocysts of Coccidia from hens.

Lindberg's combined gas chromatography-mass spectrometry techniques for the analysis of partially methylated alditol acetate sugar derivatives were used to study the structures of polysaccharides from the envelopes of heterocysts and spores of Anabaena cylindrica. Polysaccharides from both envelopes are highly branched. Glucose, mannose, galactose, and xylose are at terminal positions, whereas glucose and mannose are at internal positions in these polymers. The molar percentages of the 11 partially methylated alditol acetate derivatives observed were approximately the same for both envelopes, suggesting that the envelope polysaccharides may be identical or almost identical. Smith degradation (periodate oxidation followed by reduction with sodium borohydride and mild acid hydrolysis) of the polysaccharides from the two kinds of envelopes removes the side branches without measurable fragmentation of the backbones. Gas chromatographic analysis of partially methylated alditol acetate derivatives of the sugars showed that the backbones of both envelope polysaccharides consist of glucose (Glc) and mannose (Man) linked by 1 leads to 3 glycosidic bonds. Disaccharides, trisaccharides, and tetrasaccharides obtained from the backbone polysaccharides by partial acid hydrolysis were fractionated by column chromatography and separated by high voltage paper electrophoresis. Analysis of these oligosaccharides established that the backbone polysaccharides from both heterocysts and spores consist of repetitions of the structural unit Glc leads to Glc leads to Glc leads to Glc leads to Man, and that all linkages in the backbones are in the beta configuration.

Microsporida are potentially useful as biological control agents for insects of economic and medical importance. Prior to their responsible use, however, an accurate and reliable means of identification to the species and subspecies level is required. Current methods used for identification are not adequate, due to variability of identifiable characters and to the occurrence of dimorphism. Recently, progress has been made in the use of biochemical characteristics to support the more traditional methods of distinguishing between morphologically similar species. We report on an improved method of characterization of microsporidan spore proteins, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). This method increased the number of spore polypeptides resolved from Nosema locustae spore protein extracts 2-3-fold over 1-dimensional PAGE. Also, each of the 2D-PAGE spore protein fingerprints of the species examined, namely Nosema locustae, Nosema bombycis, and Vairimorpha necatrix, were unique and differences in their spore protein composition were easily determined. The major structural proteins of Nosema locustae spores co-electrophoresed with alpha and beta tubulin from calf brain and had similar pI and molecular weight values as reported for tubulin in other species. Each species' 2D-PAGE fingerprint contained a few polypeptides that were present in relatively high concentration and these polypeptides may represent the major proteins of the structural components of the spore.

The levels of fatty acids and their distribution were determined in cultures of Bacillus megaterium during growth, sporulation, and germination. Branched-chain pentadecanoates (br-C15) were the principal fatty acids of log-phase cells. Synthesis of branched-chain tetradecanoates (br-C14) during sporulation increased the relative proportion of these branched fatty acids in sporulating cells and in mature spores. The log-phase distribution was reestablished during outgrowth of the spore. The ratio of br-C15 to br-C14 could be radically altered by addition of their respective amino acid precursors, isoleucine and valine, without seriously affecting the sporulation process. The fatty acid composition of each of the purified phospholipids from log-phase cells was the same, indicating that each phospholipid receives a portion of the fatty acid pool present in the cell at the time of its synthesis. Similarly, the fatty acids of each of the spore phospholipids resembled those of the spore extract. Phospholipids accounted for two-thirds of the fatty acids of the log-phase but only one-third of those of the spore.

Spheroplasts of Clostridium botulinum 62A were prepared with the use of lysozyme. These spheroplasts were then exposed to ferritin-labeled type A antitoxin. Ultrathin sections of these specimens revealed the ferritin-labeled antibody symmetrically arranged around the outer spore coats but not within the spore cortex. The ferritin-labeled antibody was also observed in the bacterial cytoplasm. Here it was arranged in aggregates and strands, although it was not associated with any identifiable cell structure. Controls included sections of C. botulinum spheroplasts treated with a 1.5% solution of ferritin as well as spheroplasts of C. roseum and Bacillus subtilis treated with conjugated type A antitoxin or a 1.5% solution of ferritin. No intracellular or extracellular ferritin was demonstrable in these specimens.

An aqueous two-phase system made with polyethylene glycol (PEG) 1000 and potassium phosphate gives much higher recovery of Bacillus macerans spores in the upper phase (PEG rich) than does a similar system utilizing PEG 4000. The upper phase completely rejects vegetative cells, which collect at the interface. The system may be useful in purifying spores of other species. Scanning electron micrographs of B. macerans spores cleaned in this system show no obvious attached sporangial fragments. The micrographs show that the ridged coat may form polygonal structures at the poles, as previously observed in Bacillus polymyxa spores.

The spore appendages of Clostridium taeniosporum NI were removed from the spores by sonic treatment and were isolated by using discontinuous sucrose gradients. The amino acid composition of the appendages, which are elaborations of the spore coat, was similar to but not identical with the amino acid composition of the coats. Approximately 80% of the appendage dry weight was composed of 17 common amino acids, whereas 68% of the spore coat dry weight was amino acids. Mole ratios of the amino acids differed between the appendages and spore coats. The appendages contained neither diaminopimelic acid nor hydroxyproline. Glucosamine was an abundant constituent but muramic acid was absent. Approximately 10% of appendage dry weight consisted of three sugars, one of which was glucose. Phosphorus content was high and dipicolinic acid was absent. Appendage fine structure was not affected by common buffers, dilute acids and bases, hydrogen bond-breaking agents, certain proteolytic enzymes, or lysozyme.

The principal phospholipids of Bacillus megaterium throughout the cycle of growth and sporulation were found to be phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and a hitherto unidentified isomer of glycosaminyl-phosphatidylglycerol. Phosphatidylglycerol predominated during vegetative cell growth and then declined as spores developed, whereas diphosphatidylglycerol became more prominent during spore maturation. The latter phosphatide was relatively inaccessible in the vegetative cell and was more accessible in the spore, as judged by solvent extraction under various conditions.

Coccidian oocysts resembling those of Isospora bigemina were excreted by cats fed Toxoplasma. In order to identify these oocysts with Toxoplasma infectivity a number of critical comparisons were made. The appearance of oocysts and Toxoplasma infectivity was simultaneous in the feces of 23 of 24 adult cats, 3-5 days after feeding of Toxoplasma cysts; in the feces of 4 out of 9 cats, 7-10 days after feeding of trophozoites; and in 8 out of 17 cats, 20-24 days after feeding of cat feces containing oocysts. Oocysts and infectivity were present in similar numbers, and they disappeared simultaneously from the feces of cats. Oocysts and infectivity were also observed simultaneously in the feces of 9 kittens, 1-2 days old, fed Toxoplasma cysts. Oocysts could not be separated from infectivity by filtration, by continuous particle electrophoresis, or by density gradient centrifugation. Excystation of oocysts was followed by an increase in titer of Toxoplasma infectivity. Unsporulated oocysts in fresh cat feces were noninfectious to mice, but oocyst sporulation was associated quantitatively with the development of infectivity at different temperatures and conditions of oxygenation. Maximum oocyst sporulation at 48 hr correlated with the development of maximum Toxoplasma infectivity. 1 and 2% sulfuric acid, and 2.5% potassium dichromate were found to be the best preservatives for sporulation of oocysts and for the development of Toxoplasma infectivity. Low sporulation rates in 0.1% formalin, 20% ethanol, and in water were associated with low infectivity in these reagents. Neither Toxoplasma infectivity nor oocysts developed in 0.3% formalin, 1% ammonium hydroxide, or 1% iodine in 20% ethanol. Oocysts, sporocysts, and sporozoites were stained specifically with Toxoplasma antibody in the indirect fluorescent antibody test. Typical coccidian stages, schizonts, and male and female gametocytes were found in the epithelium of the small intestine of kittens fed Toxoplasma cysts. The classification of T. gondii is discussed in relation to that of other isosporan coccidia of cats and dogs. The term "Toxoplasma oocyst" is introduced and Toxoplasma is classified in the family Toxoplasmidae of the suborder Eimeriina. The species Isospora bigemina is restricted to dogs, and I. cati to cats. I. felis and so-called I. rivolta from cats were noninfectious to dogs, and did not confer immunity to subsequent infection with I. canis and I. rivolta from dogs.