Anticipating the species jump: surveillance for emerging viral threats.

Zoonotic disease surveillance is typically triggered after animal pathogens have already infected humans. Are there ways to identify high-risk viruses before they emerge in humans? If so, then how and where can identifications be made and by what methods? These were the fundamental questions driving a workshop to examine the future of predictive surveillance for viruses that might jump from animals to infect humans. Virologists, ecologists and computational biologists from academia, federal government and non-governmental organizations discussed opportunities as well as obstacles to the prediction of species jumps using genetic and ecological data from viruses and their hosts, vectors and reservoirs. This workshop marked an important first step towards envisioning both scientific and organizational frameworks for this future capability. Canine parvoviruses as well as seasonal H3N2 and pandemic H1N1 influenza viruses are discussed as exemplars that suggest what to look for in anticipating species jumps. To answer the question of where to look, prospects for discovering emerging viruses among wildlife, bats, rodents, arthropod vectors and occupationally exposed humans are discussed. Finally, opportunities and obstacles are identified and accompanied by suggestions for how to look for species jumps. Taken together, these findings constitute the beginnings of a conceptual framework for achieving a virus surveillance capability that could predict future species jumps.

NanoSIMS imaging of Bacillus spores sectioned by focused ion beam.

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top-sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift-out. The lift-out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top-cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is approximately 300 nm for top-cut sections versus approximately 100 nm for electron-transparent sections.

Comparative analysis of passive dosimetry and biomonitoring for assessing chlorpyrifos exposure in pesticide workers.

Under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the US Environmental Protection Agency (EPA) has the authority to regulate the use of pesticides to prevent unreasonable adverse human health effects associated with pesticide exposure. Accordingly, the EPA requires pesticide registrants to perform studies evaluating the potential for pesticide handler exposure. Data from five such studies that included exposure measurements based on both external measurements and biological monitoring were used to examine methods of assessment, routes and determinants of exposure and dose to the pesticide chlorpyrifos. Eighty workers across four job classes were included: mixer/loaders (M/L, n = 24), mixer/loader/applicators (M/L/A, n = 37), applicators (A, n = 9) and re-entry scouts (RS, n = 10). Results showed that doses were highly variable and differed by job class (P < 0.05) with median total (inhalation and dermal combined) exposure-derived absorbed doses (EDADtot) of 129, 88, 85 and 45 microg/application for A, M/L/A, M/L and RS, respectively. Doses derived from the measurement of 3,5,6-trichloro- 2-pyridinol (3,5,6-TCP) in urine were similar in magnitude but differed in rank with median values of 275, 189, 122 and 97 microg/application for A, M/L, RS, and M/L/A, respectively. The relative contribution of dermal to inhalation exposure was examined by their ratio. The median ratios of exposure-derived absorbed dermal dose (EDADderm) (assuming 3% absorption) to exposure-derived absorbed inhalation dose (EDADinh) (assuming 100% absorption) across job classes were 1.7, 1.5, 0.44 and 0.18 for RS, M/L, A and M/L/A, respectively, with an overall median of 0.6. For 34 of 77 workers (44%), this ratio exceeded 1.0, indicating the significance of the dermal exposure pathway. Different dermal absorption factor (DAF) assumptions were examined by comparing EDADtot to the biomarker-derived absorbed dose (BDAD) as a ratio where EDADtot was calculated assuming a DAF of 1, 3 and 10%. Median ratios of 0.45, 0.71 and 1.28, respectively, were determined suggesting the DAF is within the range of 3-10%. A simple linear regression of urinary 3,5,6-TCP against EDADtot indicates a positive association explaining 29% of the variability in the 3,5,6-TCP derived estimate of dose. A multiple linear regression model including the variables EDADderm, EDADinh and application type explained 46% of the variability (R2 = 0.46) in the urinary dose estimate. EDADderm was marginally significant (P = 0.066) while EDADinh was not (P = 0.57). The EDADderm regression coefficient (0.0007) exceeded the coefficient for EDADinh (0.00002) by a factor of 35. This study demonstrates the value of the pesticide registrant database for the purpose of evaluating pesticide worker exposure. It highlights the significance of the dermal exposure pathway, and identifies the need for methods and research to close the gap between external and internal exposure measures.

Physiological and genetic strategies for enhanced subtilisin production by Bacillus subtilis.

Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion. The beta-galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG), relative to co-genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.

Ultrastructural studies of sporulation in a conditionally temperature-sensitive ribonucleic acid polymerase mutant of Bacillus subtilis.

Morphological studies of a conditionally temperature-sensitive ribonucleic acid polymerase mutant of Bacillus subtilis have revealed that sporulation is inhibited at stage II when the cells are grown at 47.5 C. Growth and sporulation occur normally at 30 C with the mutant. The mutant grows normally at 47.5 C but is prevented from sporulating at the nonpermissive temperature by an abnormal septation during forespore membrane formation which prevents the subsequent engulfment process (stage III). The mutation affects the normal functioning of ribonucleic acid polymerase at the nonpermissive temperature resulting in abortive sporulation.

An RNA polymerase mutation causing temperature-sensitive sporulation in Bacillus subtilis.

A single-site mutant of Bacillus subtilis with a rifampin-resistant RNA polymerase has been isolated; this mutation causes temperature-sensitive sporulation. The temperature-sensitive mutation was only expressed during a limited time period, covering the middle third of the sporulation sequence. Mutant cells grown at the nonpermissive temperature exhibited the normal change in RNA polymerase template specificity, accumulated extracellular proteolytic activity and antibiotic activity, but failed to accumulate alkaline phosphatase and, hence, were blocked at or near stage III in the sporulation sequence. Pulse-labeled RNA synthesis was seriously deranged during postexponential growth phase in mutant cells incubated at the nonpermissive temperature.

Physiological and biochemical changes associated with macroconidial germination in Microsporum gypseum.

A study was made of the metabolic processes associated with macroconidial germination in Microsporum gypseum. The optimum conditions for stimulation of endogenous respiration, changes in chemical composition as germination proceeds, and the uptake and synthetic fates of amino acids, glucose, and uracil were investigated. The assimilation and conversion of (14)C-glucose, (14)C-amino acids, and (14)C-uracil into the cell pool and into trichloroacetic acid-precipitable material were studied during the early stages of germination (i.e., prior to germ-tube emergence). The macroconidia were not metabolically inert for any significant period of time after exposure to germination conditions. Rather, the spores rapidly assimilated all metabolites and slowly converted them into macromolecules. Investigations of the effect of inhibitors of nucleic acid and protein synthesis prior to germ-tube emergence and during early germ-tube elongation suggested significant changes in metabolism and cell permeability may be correlated with the emergence of germ tubes. Radioactivity of incorporated glucose was found to be associated largely with the lipid fractions of the macroconidia early in germination.

Ultrastructural analysis of sporulation in a conditional serine protease mutant of Bacillus subtilis.

The morphological characteristics of wild-type Bacillus subtilis and a temperature-sensitive serine protease derivative have been observed during vegetative and sporulation time periods. At 30 C wild-type and mutant cells grow and sporulate identically. At 47.5 C wild-type and mutant cells grow identically, but the mutant cells are blocked at stage 0 or I in the sporulation sequence. Wild-type cells sporulate normally at 47.5 C.

Absence of histones from the chromosomal proteins of fungi.

Interphase chromosomes were isolated in good yield from four species of fungi. In no case does the chromatin contain histones such as are characteristic of the chromosomes of other eukaryotic organisms.

Isolation and preliminary characterization of developmental mutants from Microsporum gypseum.

Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed.

Relationship of nuclear segregation and macroconidial germination in Microsporum gypseum.

Microsporum gypseum macroconidia germinated at 37 C possessed from one to eight nuclei per germinated spore compartment. The distribution of nuclei per spore compartment was the result of a random packaging of nuclei from the available nuclear population. Partial inhibition of germination by incubation at 25 C or at 37 C in the presence of 10(-4)m phenyl methyl sulfonyl-fluoride resulted in an enrichment of germinated spores containing high numbers of nuclei per compartment. The selection for higher nuclear numbers was statistically significant. Compartments possessing high numbers of nuclei appeared to be precommitted to spore germination since they were not sensitive to germination inhibition. The effect of incubation temperature variation on spore germination is discussed with respect to the organism's natural environment.

Macroconidial germination in Microsporum gypseum.

Biochemical events which occur during macroconidial germination have been studied in the dermatophyte Microsporum gypseum. The specific activity levels of various metabolic enzymes have been assayed during germination time periods. The accumulated levels of several of these enzymes, as a function of exogenous carbohydrate source, have been investigated. M. gypseum was found to possess a constitutive glyoxalate shunt, a constitutive glucokinase, a fructose phosphoenolpyruvate transferase, and a mannitol phosphoenolpyruvate transferase. The integration of endogenous reserve utilization during germination is discussed. The purification and properties of an alkaline phosphatase and its possible relationship to sporulation and spore germination also are described.

Biochemical changes during fungal sporulation and spore germination. I. Phenyl methyl sulfonyl fluoride inhibition of macroconidial germination in Microsporum gypseum.

Macroconidia of Microsporum gypseum release free amino acids into the medium during germination. A single alkaline protease is also found in the germination supernatant fraction. The purified protease is capable of hydrolyzing isolated spore coats in vitro. Phenyl methyl sulfonyl fluoride (PMSF) is an effective inhibitor of the protease. Incorporation of PMSF at 10(-4)m into the germination system inhibits spore germination and the release of free amino nitrogen. Addition of PMSF after germ tube emergence is completed has no effect on subsequent outgrowth. The addition of exogenous purified protease to quiescent spores results in more than a 2.5-fold increase in germinated spores. It is concluded that spore coat proteolysis is an essential event in the germination of dermatophyte macroconidia. A model system to explain macroconidial germination response to inhibition, temperature shift, and addition of protease is presented.

Heat-induced macroconidia germination in Microsporum gypseum.

A method for obtaining purified ungerminated macroconidia is described, and a technique for obtaining 85 to 90% germination of macroconidia under normal nutritional conditions is presented.