Transmission of aerosolized seasonal H1N1 influenza A to ferrets

Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID₅₀) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID₅₀ for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure.     

Total respiratory tract deposition of fine micrometer-sized particles in healthy adults: empirical equations for sex and breathing pattern.

Accurate dose estimation under various inhalation conditions is important for assessing both the potential health effects of pollutant particles and the therapeutic efficacy of medicinal aerosols. We measured total deposition fraction (TDF) of monodisperse micrometer-sized particles [particle diameter (Dp) = 1, 3, and 5 microm in diameter] in healthy adults (8 men and 7 women) in a wide range of breathing patterns; tidal volumes (Vt) of 350-1500 ml and respiratory flow rates (Q) of 175-1,000 ml/s. The subject inhaled test aerosols for 10-20 breaths with each of the prescribed breathing patterns, and TDF was obtained by monitoring inhaled and exhaled aerosols breath by breath by a laser aerosol photometer. Results show that TDF varied from 0.12-0.25, 0.26-0.68, and 0.45-0.83 for Dp = 1, 3, and 5 microm, respectively, depending on the breathing pattern used. TDF was comparable between men and women for Dp = 1 microm but was greater in women than men for Dp = 3 and 5 microm for all breathing patterns used (P < 0.05). TDF increased with an increase in Vt regardless of Dp and Q used. At a fixed Vt TDF decreased with an increase in Q for Dp = 1 and 3 microm but did not show any significant changes for Dp = 5 microm. The varying TDF values, however, could be consolidated by a single composite parameter (omega) consisting of Dp, Vt, and Q. The results indicate that unifying empirical formulas provide a convenient means of assessing deposition dose of particles under varying inhalation conditions.     

Production of early and late pulmonary responses with inhaled leukotriene D4 in allergic sheep

Some allergic sheep respond to inhalation of Ascaris suum antigen with both immediate and late increases in airflow resistance (late response). The mechanism of the late response is unknown but recent evidence suggests that the initial generation of slow-reacting substance of anaphylaxis (SRS-A) immediately after antigen challenge is a necessary pre-requisite for the physiologic expression of this late response. Based on this evidence we hypothesized that airway challenge with leukotriene D4 (LTD4), an active component of SRS-A would produce acute and late airway responses in allergic sheep similar to those observed with antigen. In five allergic sheep with documented early and late pulmonary responses to Ascaris suum antigen, inhalation of leukotriene D4 aerosol (delivered dose (mean +/- SE) 0.55 +/- 0.08 ug) resulted in significant early and late increases in specific lung resistance (SRL). In three allergic sheep which only demonstrated acute responses to antigen, LTD4 aerosol (delivered dose 0.59 +/- 0.09 ug) only produced an acute increase in SRL. In the late responders pretreatment with aerosol cromolyn sodium (1 mg/kg) did not affect the acute response but blunted the late increase in SRL. Pretreatment with aerosol FPL-57231 (1% w/v solution) completely blocked both the acute and late responses. These data support the hypothesis that initial release of LTD4 in the airways of sensitive animals is important for the physiologic expression of the late response.     

A comparison of the predicted risks of developing osteosarcoma for dogs exposed to 238PuO2 based on average bone dose or endosteal cell dose.

Dose-response relationships observed in laboratory animals can be used to identify possible human risk factors and may also be used in a quantitative manner when human data are not available. This paper presents an analysis of the dose dependency of osteosarcoma incidence in beagle dogs given a single inhalation exposure to a monodisperse aerosol of 238PuO2. We were particularly interested in comparing the predicted risks that were based on average bone dose with those based on endosteal cell dose and in evaluating the advantages of using a more biologically relevant cell-specific dose in risk estimation. The endosteal cell dose was calculated using the method of Marshall et al. (Health Phys. 35, 91-101, 1978), as extended to account for exposure by inhalation. The relationship between dose and time to tumor was analyzed by the proportional hazards regression model. The probability of developing osteosarcoma was strongly dependent on dose for dogs receiving low doses, but this was not true for dogs receiving high doses. The predicted risk based on endosteal cell dose was not consistently higher or lower than the risk based on average bone dose at various times after exposure, because the relationship between these two doses was not linear with respect to time. Also, as a result of the nonlinear relationship between these two doses, the risk estimated based on endosteal cell dose would not be a fixed factor of that based on the average dose. Random errors in the measured initial lung burden had a relatively large impact on the predicted risk based on endosteal cell dose, and the difference between the estimated risk of developing osteosarcoma based on endosteal cell dose and that based on average bone dose is likely to be within the error margins of the estimated risks.     

Mechanical damage to rabbit tracheal epithelium from inhaling inert pyrite dust of needle-like structure. Part II.

The ultrastructure of rabbit tracheal epithelium was studied 2, 8 and 26 hours after termination of 8-hour inhalation of ground pyrite dust. Pyrite particles persist in the cytoplasm of ciliated cells throughout the interval followed up. Only through degeneration and elimination of these cells from the epithelium is the inhaled dust gradually removed from the mucous membrane. Goblet cells discharge pyrite-containing mucus into the ciliary border region for a period of 26 hours after cessation of inhalation thus inducing further damage to the ciliary border and the apical regions of the cells even after termination of inhalation. The morphological appearance of the ciliary border, especially in the first phase after termination of inhalation, suggests a disturbance of the self-cleaning function of the eipithelium of respiratory passages. The inhalation of aerosol of needle-like structure causes mechanical damage to tracheal epithelium. Substantial retardation of the onset of epithelial regeneration and slow rate of elimination of the inhaled aerosol from the tracheal mucosa was noted.     

Computational Analysis of Micron-particle Deposition in a Human Triple Bifurcation Airway Model

Steady laminar axisymmetric inhalation flow and wall deposition of micron-size particles in representative triple bifurcation airways have been simulated using a commercial finite-volume code with user-enhanced programs. Assuming spherical non-interacting particles (3 μm≤ d p ≤7 μm), various inlet Reynolds numbers (Re=500-2000) and Stokes numbers (St=0.02-0.23) were considered. The resulting particle deposition patterns were analyzed and then summarized in terms of deposition efficiencies, i.e. DE=DE(Re,St) Surprisingly high DE-values occur at relatively low Reynolds numbers (e.g., Re=500 ) in the third bifurcation. The quantitative results are of interest to researchers either conducting health risk assessment studies for inhaled particulate pollutants or analyzing drug aerosol inhalation and deposition at desired lung target sites.     

Production of early and late pulmonary responses with inhaled leukotriene D4 in allergic sheep

Some allergic sheep respond to inhalation of antigen with both immediate and late increases in airflow resistance (late response). The mechanism of the late response is unknown but recent evidence suggests that the initial generation of slow-reacting substance of anaphylaxis (SRS-A) immediately after antigen challenge is a necessary pre-requisite for the physiologic expression of this late response. Based on this evidence we hypothesized that airway challenge with leukotriene D₄ (LTD₄), an active component of SRS-A would produce acute and late airway responses in allergenic sheep similar to those observed with antigen. In five allergic sheep with documented early and late pulmonary responses to antigen, inhalation of leukotriene D₄ aerosol (delivered dose {mean ±SE} 0.55±0.08 ug) resulted in significant early and late increases in specific lung resistance (SR_L). In three allergic sheep which only demonstrated acute responses to antigen, LTD₄ aerosol (delivered dose 0.59±0.09ug) only produced an acute increase in SR_L. In the late responders pretreatment with aerosol cromolyn sodium (1 mg/kg) did not affect the acute response but blunted the late increase in SR_L. Pretreatment with aerosol FPL-57231 (1% w/v solution) completely blocked both the acute and late responses. These data support the hypothesis that initial release of LTD₄ in the airways of sensitive animals is important for the physiologic expression of the late response.     

Chromosomal abnormalities and sister-chromatid exchange in bone marrow cells of mice and Chinese hamsters after inhalation and intraperitoneal administration: I. Diepoxybutane

Diepoxybutane (DEB), a direct-acting animal carcinogen, was found to increase the frequency of structural chromosomal abnormalities (CA) and sister-chromatid exchange (SCE) in bone marrow cells of mice and Chinese hamsters, when inhaled from an aerosol during a 2-h head-only exposure or administered as a single intraperitoneal injection. For the purpose of comparing the genotoxicity in the 2 species, both after inhalation and intraperitoneal administration, the systemic DEB dose obtained by inhalation was determined on the basis of blood concentrations and inhalation duration after the investigation of the blood kinetics. The bone marrow cells of male and female NMRI mice were found to be more sensitive than those of Chinese hamsters to the genotoxic activity of DEB.     

Lung tumor induction in Syrian hamsters with internally deposited particulate Pu: A synthesis based on microscopic dose distribution

Syrian hamsters inhaled a monodisperse aerosol of 238PuO2 and were serially sacrificed to study the microscopic distribution of particles, tissue at risk and dose as a function of time after exposure. The distribution of dose and tissue at risk around single particles in lung and the changes in distribution of particles with time have been reported previously. In the present paper, these measurements are applied to the computation of tissue-at-risk and radiation-dose-rate distributions within the lungs of Syrian hamsters. Based on these results, airway epithelium is irradiated at the same levels as other lung tissue and does not require separate consideration on the basis of dose to tissue. Incorporation of the measured microscopic radiation dose distribution into existing dose-effect models allowed data on lung tumor induction in Syrian hamsters from several laboratories to be adequately described by a model fit to data from a single laboratory.     

Theoretical study of nasal deposition

A theory is derived to calculate the regional and total deposition of aerosol particles in the nasal passages during inhalation. The particle size studied range from 0.2 to 10.0 μm diameter. The deposition is calculated in five regions; (I) the region filled with nasal hair, (II) the nasal valve, (III) the expansion region, (IV) the turbinate region and (V) the posterior bend. Equations are derived to determine the deposition caused by direct impaction on the nasal hairs and bends of the passages. The calculations show the deposition due to direct impaction does not account for the amount or location of deposited particles measured in experiments. Secondary flows have been speculated to exist in the expansion region after the nasal valve and an equation is derived to estimate the deposition caused by the secondary flows. The calculated deposition, due to direct impaction and secondary flows, shows general agreement with the experiment as to the predicted amount and location of deposited particles.     

Chromosomal abnormalities and sister-chromatid exchange in bone marrow cells of mice and Chinese hamsters after inhalation and intraperitoneal administration. II. Cyclophosphamide

The genotoxic effects of cyclophosphamide (CPP), a human and animal carcinogen requiring metabolic activation, were studied in bone marrow cells of mice and Chinese hamsters, analyzing chromosome abnormalities (CA) and sister-chromatid exchange (SCE) after a 2-h inhalation or a single intraperitoneal administration. In order to compare the genotoxicity after the different routes of administration in the dose range of 10-110 mg CPP/kg body weight, the systemic dose obtained by inhalation was calculated from blood concentrations and the inhalation duration after an analysis of the CPP blood kinetics. In NMRI mice the frequency of bone marrow cells with chromosome abnormalities was higher after aerosol exposure than after intraperitoneal administration of comparable CPP doses. In Chinese hamsters the CA frequency was similar with both exposure routes. Inhaled CPP was found to induce a higher frequency of CA and SCE in the bone marrow cells of mice compared to those of Chinese hamsters. The findings suggest that for genotoxins requiring metabolic activation species differences exist with respect to the influence of the route of entry and the sensitivity of bone marrow cells.     

Comparison of Streptococcus zooepidemicus and influenza virus pathogenicity in mice by three pulmonary exposure routes

Pulmonary infections may be induced in experimental animals by using several exposure routes. Inhalation of microbial aerosols is often viewed as the most relevant exposure route, although the comparability of either intranasal (i.n.) or intratracheal (i.t.) inoculation to aerosol inhalation is unclear. In these studies, the infection of mice with either Streptococcus zooepidemicus or influenza virus was compared following i.n., i.t., or aerosol challenge. The 50% lethal dose was determined by each exposure route for both microbes, and the organ clearance of a 50% lethal dose was determined. Mice were as or more sensitive to bacterial or viral infection following i.n. or i.t. instillation as compared with aerosol challenge. Organ clearance patterns of virus or bacteria varied slightly with exposure route.     

Comparison of Streptococcus zooepidemicus and influenza virus pathogenicity in mice by three pulmonary exposure routes.

Pulmonary infections may be induced in experimental animals by using several exposure routes. Inhalation of microbial aerosols is often viewed as the most relevant exposure route, although the comparability of either intranasal (i.n.) or intratracheal (i.t.) inoculation to aerosol inhalation is unclear. In these studies, the infection of mice with either Streptococcus zooepidemicus or influenza virus was compared following i.n., i.t., or aerosol challenge. The 50% lethal dose was determined by each exposure route for both microbes, and the organ clearance of a 50% lethal dose was determined. Mice were as or more sensitive to bacterial or viral infection following i.n. or i.t. instillation as compared with aerosol challenge. Organ clearance patterns of virus or bacteria varied slightly with exposure route.     

Estimating the Number of Droplets and Drug Particles Emitted from MDIs

The objective of this paper is to assess the number of drug particles or droplets contained in metered dose inhaler (MDI) aerosols. Equations were developed to estimate this. The number of drug particles was estimated to be as high as about 300 million for QVAR solution MDIs and as low as 670,000 for Beclovent MDIs. The number of particles in MDI aerosols was shown to be highly dependent on the mass median aerodynamic diameter (MMAD) and geometric standard deviation, and to a lesser extent the total mass of the aerosol. It was demonstrated that when the number of particles are calculated assuming that the aerosol is monodisperse and using the MMAD as the particle size, the number of particles are significantly underestimated. The number of droplets atomized from HFA-134a MDIs was estimated to range from about 220 million to about 1.1 billion droplets per actuation. For solution MDIs, each of the atomized droplets contains drug and thus the number of drug particles is the same as the number of atomized droplets. However, for suspension MDI formulations many of the droplets do not contain any micronized drug particles and the number of drug particles is much lower than the number of atomized droplets.     

The principles of aerosol samplers and sampling

The principles of aerosol sampling involve both a) the scientific factors which govern the way in which particles are transported from the air outside the sampler to a filter or sensing region inside it, and b) recognition of particle size-selective criteria which relate the act of sampling to the reason it is being carried out (e.g., assessment of health risk associated with inhalation). This paper reviews the state-of-the-art in both these areas, and goes on to mention some recent technical developments concerning practical sampling instruments.     

Measurement of resuspended aerosol in the Chernobyl area

Results of measurements of the resuspended radioactive aerosols in the Chernobyl area are presented which were obtained soon after the Chernobyl reactor accident and in a European project in 1992–1993. The measurements were carried out with the intention of obtaining a data base for dose assessment of resuspended radioactive particles. Potential significant dose contributions may result from inhalation and secondary contamination due to resuspended radionuclides. In this first article of a series of three papers, the instrumentation and the measurement uncertainties are discussed. An effort was made to sample quantitatively giant aerosol particles (particles larger than 10 μm aerodynamic diameter) as well. The comparison of the samplers shows, in general, an agreement of concentration measurements of ¹³⁷Cs and ⁷Be within a factor of two. One sampler was identified with larger discrepancies and needs additional investigation of its sampling characteristics; for another device, the recalibration of the analysing system is recommended. Ordinary integrating samplers have a loss of about 30% in ¹³⁷Cs activity compared to an isokinetic sampler collecting giant particles as well. The mean ratio of ¹³⁷Cs activity concentration between an instrument sampling only particles larger than 10 μm and an ordinary integrating sampler is 0.39 ± 0.15 during anthropogenic-enhanced resuspension. These findings demonstrate the significant contribution of giant particles to resuspended airborne radioactivity. The results of this study concerning integral measurements during wind-driven resuspension proved to be in good agreement with previously published data on resuspension. Received: 15 May 1997 / Accepted: 4 August 1997     

Estimating Total Effective Dose Equivalents from Terrorist Use of Radiological Dispersion Devices

Radiological dispersion devices (RDDs), commonly called “dirty bombs,” utilize a conventional explosive to deliberately disperse non-fissile material as an aerosol. This analysis models total effective dose equivalent (Sv) at various locations down-wind from the detonation site subsequent to terrorists detonating a ²⁴¹Am, ¹³⁷Cs, ⁶⁰Co, ¹⁹²Ir, or⁹⁰Sr RDD. A source term for each isotope equaling 3.7 × 10¹³ Bq with an instantaneous release by either high explosives or low explosives at street level is assumed in order to evaluate total effective dose equivalent (TEDE) under various meteorological scenarios for intentional releases of non-fissile materials by terrorists. The inhalation pathway on average contributes most to TEDE. The inhalation pathway accounts for 96% (0.22 Sv) of the mean exposure estimate of 0.2321 Sv and occurs over an extremely short time frame (i.e., a few minutes). Ground shine, on average, contributes the second most to TEDE estimates accounting for approximately 4% (0.009 Sv) of the estimate. A cautionary note with regard to ground shine is warranted, however, because Hotspot estimates for this pathway are based on the assumption that a person is exposed for 4 days (96 hours). The TEDE for submersion (i.e., passing through the plume without inhaling particles) is negligible for the scenarios evaluated contributing less than 1% (5.2 × 10^?6 Sv) to the TEDE estimate averaged across all 140 model runs (5 nuclides × 2 rainfall scenarios × 2 explosive scenarios × 7 wind and atmospheric stability scenarios). The TEDE value for ²⁴¹Am from inhalation is much greater, on average, than the inhalation TEDE value for ⁶⁰Co, ¹³⁷Cs, ¹⁹²Ir, or ⁹⁰Sr. This underscores the potentially high risk to human health posed by exposure to ²⁴¹Am. Ground shine is the primary exposure pathway for ⁶⁰Co and ¹³⁷Cs due to the energetic and penetrating gamma rays those radionuclides emit. ¹⁹²Ir and ⁹⁰Sr have relatively low mean TEDE values for all of the pathways examined.     

A nose-only apparatus for airborne delivery of Mycobacterium tuberculosis to mice: calibration of biological parameters

Mycobacterium tuberculosis is the main cause of tuberculosis and is still a public health concern worldwide. This mycobacterium is transmitted through aerosols from human beings suffering from pulmonary tuberculosis to susceptible persons. To study this natural route of infection, we designed a new nose-only aerosol apparatus--system of aerosolisation of microorganisms (SAM)--in a carefully designed biohazard facility. For safety reasons, Mycobacterium smegmatis was first used to calibrate several parameters, such as inoculum density, atmospheric conditions (i.e. hygrometry) and particle size distribution. We present evidence that our apparatus is totally adapted to airborne delivery; the particle size of generated aerosol ranges from 1 to 7 microm, which is ideal for an infection by inhalation. We found that 99% of generated particles (<7 microm) could be retained by the respiratory tract, and among these particles, 62-79% (<3.3 microm) were able to reach pulmonary compartments. The next step was to simultaneously challenge 48 mice with M. tuberculosis in a highly reproducible way. We showed that a moderate dose (4 log10 colony-forming units (CFU) per mice) of M. tuberculosis was capable of causing progressive lung pathology and death in mice 30 days post-aerosolisation. Therefore, our apparatus, once calibrated, is easy to handle, safe, and can be used with any pathogen, which is spread by aerosol.     

A model for early death caused by radiation pneumonitis and pulmonary fibrosis after inhaling insoluble radioactive particles

Large radiation doses to the lung can cause early death from cardiopulmonary insufficiency resulting from radiation pneumonitis and pulmonary fibrosis. A model for early death following inhalation of insoluble radioactive particles is propose. The model is based on three assumptions: (1) early death results from damage to a cluster of cells from a large number of cell clusters at risk, (2) the dose that causes early death depends on how the radiation is delivered in time and (3) the cell clusters at risk to damage are equally sensitive ro radiation. Results from asymptotic theory of extreme values, along with biophysical considerations, suggest that the cumultive distribution function for the absorbed radiation dose to the production of pulmonary injury sufficient to cause early death is best estimated by the third asymptotic distribution without a threshold. This distribution function is identical to the Weibull cumulative distribution function. Data for Beagle dogs after inhaling relatively insoluble forms of alpha- or beta-gamma-emitting particles are shown to support the Weibull model.     

Distribution and biological effects of inhaled 239Pu(NO3)4 in cynomolgus monkeys.

Twenty male cynomolgus monkeys were exposed by inhalation either to an aerosol of 239Pu(NO3)4 to produce projected initial lung burdens of either 40, 10, or 4 kBq or to a carrier aerosol as a control. Animals died or were sacrificed at 0.01, 1, 3, 6, 12, 24, 40, and 99 months after inhalation, and the distribution and biological effects of the 239Pu were determined. The 239Pu cleared efficiently from the lungs so that less than 0.05 kBq remained at 99 months after exposure to 40 kBq. Total skeletal 239Pu activity was nearly constant after the first year, but the fraction of the body burden in skeleton at sacrifice increased with time up to 99 months because of clearance from other organs. Plutonium in the liver increased to a peak at 1 year and then decreased to about 10% of the peak value at 99 months. Plutonium in the testes was localized in the interstitial tissue with only 0.01 to 0.002% of the projected lung burden remaining in testes at 99 months after inhalation. Three animals exposed to 40 kBq of 239Pu died of radiation-related pulmonary pneumonitis and fibrosis. A primary papillary adenocarcinoma of the lung was identified in one animal exposed to 40 kBq initial lung burden and sacrificed 99 months after inhalation. The frequency of chromosome aberrations in blood lymphocytes was significantly elevated only in monkeys with projected deposits of 40 kBq of 239Pu. There was no change in aberration frequency in other exposure groups as a function of inhaled activity, time after exposure, or calculated total dose to the lungs. Only in monkeys that had marked radiation-induced pathological changes in the lung did the frequency of chromosome-type aberrations increase significantly, to a value about twice the control level. In cynomolgus monkeys, chromosome aberration frequency in blood lymphocytes is not a good indicator of radiation dose or damage from inhaled soluble plutonium.