Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways.

Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology for deposition.

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions.

AIMS: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. METHODS AND RESULTS: We evaluated the kinetics of uptake, germination and proliferation of Bacillus anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3 and A549 cell lines. We also analysed the influence of various cell culture medium formulations related to spore germination. CONCLUSIONS: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the serum-free extracellular environment was evident. Spore germination was appreciably higher in immortalized cell cultures than in primary epithelial cells. Additionally, spores still germinated apically at a mucus-secreting air-liquid interface lung barrier that was devoid of cell culture medium much earlier than medium-only controls. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of lung epithelial cells in B. anthracis spore dissemination after inhalation remains poorly defined and rather controversial. These results are novel as they show spore germination is appreciably enhanced in the presence of lung cells in vitro, however, the cell line and cell state (air-liquid interface vs submerged in medium) dictates the extent of germination and in some cases proliferation.

Defining cell culture conditions to improve human norovirus infectivity assays.

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

Hazards from pathogenic microorganisms in land-disposed sewage sludge.

Sewage sludge is a complex mixture of organic and inorganic compounds of biological and mineral origin that are precipitated from wastewater and sewage during primary, secondary, and tertiary sewage treatment. Present in these sludges are significant numbers of microorganisms that include viral, bacterial, protozoan, fungal, and helminth pathogens. The treatment of sludge to reduce biochemical oxygen demand, solids content, and odor is not always effective in reducing numbers of pathogens. This becomes a public health concern because the infectious dose for some of these pathogens may be as low as 1 particle (virus) to 50 organisms (Giardia). When sludge is applied to land for agricultural use and landfill compost, these pathogens can survive from days (bacteria) to months (viruses) to years (helminth eggs), depending on environmental conditions. Shallow aquifers can become contaminated with pathogens from sludge and, depending on groundwater flow, these organisms may travel significant distances from the disposal site. Communities that rely on groundwater for domestic use can become exposed to these pathogens, leading to a potential disease outbreak. Currently, methods to determine the risk of disease from pathogens in land-disposed sludge are inadequate because the sensitivity of pathogen detection is poor. The application of recombinant DNA technology (gene probes and polymerase chain reaction) to environmental samples may provide increased sensitivity for detecting specific pathogens in land-disposed sludge and greatly improved risk assessment models for our exposure to these sources of pathogens.

Detection of naturally occurring enteroviruses and hepatitis A virus in undigested and anaerobically digested sludge using the polymerase chain reaction.

Four undigested and four anaerobically digested sewage sludge samples were analyzed for enteroviruses and hepatitis A virus using seminested and double polymerase chain reaction (PCR), respectively. For enteroviruses, all eight samples were positive when detection was by seminested PCR. Using cell culture all samples except two digested sludge samples were positive. For hepatitis A virus, seven out of eight samples were positive by PCR detection. In all samples, PCR inhibitory substances were removed by passage through Sephadex G-50 and Chelex 100 columns. Overall the PCR methodology was highly successful in identifying the presence of both viruses; however, with this methodology, there was no indication as to whether enteroviruses or hepatitis A viruses not confirmed in cell culture were infectious.

Comparison of PCR and cell culture for detection of enteroviruses in sludge-amended field soils and determination of their transport.

PCR and cell culture assays for enteroviruses were conducted on soil samples collected from an experimental farm that had received mesophilic anaerobically digested sludge for the past 7 years. Of 24 samples assayed, 21 samples were positive by PCR, implying that at least some viral nucleic acid sequences remained intact. However, these viral particles were unable to infect the Buffalo Green Monkey cell line used in subsequent cell culture assays. It is significant that positive PCR detection of nucleic acid sequences occurred even though the most recent sludge application was 3 months prior to soil sampling. Viral nucleic acid sequences were detected by PCR at points vertically and laterally displaced from sludge injections, illustrating significant transport of viruses. Rainfall and irrigation events may have contributed to viral transport.

Persistence of viruses in desert soils amended with anaerobically digested sewage sludge.

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log(10) reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 x 10 Pa for both soils) and temperatures of 15, 27, and 40 degrees C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40 degrees C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40 degrees C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15 degrees C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log(10) reduction per day in clay loam and sandy loam soils, respectively. At 27 degrees C, this rate increased to 0.629 log(10) reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15 degrees C (k = 0.064 log(10) reduction per day, clay loam; k = 0.095 log(10) reduction per day, sandy loam) and 27 degrees C (k = 0.133 log(10) reduction per day, clay loam; k = 0.154 log(10) reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40 degrees C. No reduction of PRD-1 was observed after 28 days at 15 degrees C and after 16 days at 27 degrees C. At 40 degrees C, the inactivation rates were 0.208 log(10) reduction per day in amended clay loam soil and 0.282 log(10) reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15 degrees C, within 3 days at 27 degrees C, and within 2 days at 40 degrees C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.

Inactivation of coliphage MS-2 and poliovirus by copper, silver, and chlorine.

The efficacy of electrolytically generated copper and silver ions (400 and 40 micrograms/L, respectively) was evaluated separately and in combination with free chlorine (0.2 and 0.3 mg/L) for the inactivation of coliphage MS-2 and poliovirus type 1 in water at pH 7.3. The inactivation rate was calculated as log10 reduction/min: k = -(log10 Ct/C0)/t. The inactivation of both viruses was at least 100 times slower in water containing 400 and 40 micrograms/L copper and silver, respectively (k = 0.023 and 0.0006 for MS-2 and poliovirus, respectively), compared with water containing 0.3 mg/L free chlorine (k = 4.88 and 0.036). Significant increases in the inactivation rates of both viruses were observed in test systems containing 400 and 40 micrograms/L copper and silver, respectively, with 0.3 mg/L free chlorine when compared with the water systems containing either metals or free chlorine alone. Poliovirus was approximately 10 times more resistant to the disinfectants than coliphage MS-2. This observation suggests either a synergistic or an additive effect between the metals and chlorine for inactivation of enteric viruses. Use of copper and silver ions in water systems currently used in swimming pools and spas may provide an alternative to high levels of chlorination.

Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.

Cell culture and PCR determination of poliovirus inactivation by disinfectants.

Inactivation of poliovirus type 1 by 1 N HCl, 1 N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR). A minimum contact time of 45 min with HCl, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 10(2) PFU of poliovirus type 1 per ml undetectable by seminested PCR. In cell culture, a minimum contact time of 5 min to HCl, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method. No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light. These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.

A method to detect enteroviruses in sewage sludge-amended soil using the PCR.

PCR detection of seeded poliovirus type 1 in sludge-amended soil was made possible by utilizing Sephadex G-50 and Chelex-100 resins to remove compounds present in sludge-amended soil that may inhibit PCR. With this method, enteroviruses indigenous to an anerobically digested sludge were detected by PCR in 10 different soils amended with this sludge.