PART II. AIRBORNE ORGANISMSNorton Nelson, Chairman

VIABILITY AND INFECTIVITY OF MICROORGANISMS INEXPERIMENTAL AIRBORNE INFECTION

ROBERT J. GOODLOW AND FREDERIC A. LEONARD

U. S. Army Chemical Corps, Fort Detrick, Frederick, Maryland

Some appreciation of the implications andmeanings of the words airborne, viability, andinfectivity is prerequisite to any discussion ofairborne infection, although it seems wise toavoid speaking of "definitions." It is difficult toimprove on Langmuir's concept (9) of airborneinfection as involving the inhalation of dropletnuclei (resulting from the evaporation of aerosoldroplets) which remain suspended in air forrelatively long periods of time. This concepteliminates droplets per se, which are more prop-erly involved in contact infection.

Because the subject of this Conference impliesthat we are interested in the pathological andmedical aspects of airborne infection, our use ofthe word infectivity will connote pathogenicitysince infection per se is an example of parasitismthat may or may not include what we refer to asdisease. Still, the discussion of viability and in-fectivity may prove difficult because we are deal-ing with related but not mutually dependentproperties of a cell. Viability generally is con-sidered as the potentiality for multiplicationunder experimentally defined conditions. Such aprocess, of course, does seem essential for infec-tion to manifest itself, but certainly all cells thatare viable do not infect. For viruses, however,definitions become more complicated becausethe viruses' so-called viability is measured con-ventionally in terms of infectivity for the egg,tissue culture, or animal host.

In our laboratories and elsewhere, considerableeffort is currently devoted to studies of experi-mental airborne infection. MIany individuals areindependently investigating a number of ap-proaches to related problems in aerobiology; theresults are sometimes difficult to correlate andinterpret. WVe suspect that there will be times dur-ing this Conference when it will be difficult tointerpret reported data. The purpose of this paperis to point out, if possible, some of the many fac-tors which must be considered in evaluating andcorrelating data on this most complicated subject.

The viability and infectivity of airborne or-ganisms are relative processes and must be con-sidered only in relation to the experimental con-ditions employed. In view of the number ofvariables that affect the outcome of the host-parasite relationship and the fate of organismsin the aerosolized state, it seems an impossibletask to attempt to generalize. Some clarificationresults, however, if the processes of experimentalairborne infection are considered as follows: (i)effects of pre-aerosolization treatment on cells;(ii) effects of aerosolization per se; and (iii)effects of the postaerosolization environment.Even here, as Beveridge (2) points out, there isconsiderable discrepancy between the behaviorof parasites under natural versus laboratory con-ditions. For example, the agent of swine feverpersists in laboratory culture for long periods oftime but dies very rapidly under natural condi-tions in the pig sty. On the other hand, influenzaand foot-and-mouth viruses are quite fragile inthe laboratory but survive for some time innature.The two principal pre-aerosolization factors

that influence the viability and infectivity of cellsare strain selection and growth conditions, thelatter including such variables as choice of me-dium, aeration, length of incubation, pH, tem-perature, and conditions of harvest and storage.Manipulations of these factors usually influencethe growth and numbers of cells, the yield of anend product, or the selection of virulent oravirulent cells. For example, we know that bac-teria in the logarithmic stage of growth are moresusceptible than older cells to such stresses asheat, cold, antibiotics, desiccation, and radiation.Cells in the resting phase or lag phase of growthare more resistant to the stress of aerosolizationthan are cells in the stage of logarithmic growth(Fig. 1).

Genetic, nutritional, and physiological manipu-lation may play important roles in selecting cellsof increased aerosol stability and infectivity.

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CULTURE ACE 6 8 10 12 14 16 1i 20 22FIG. 1. Aerosol stability of Serratia marcescens as a function of culture age

Some obvious approaches such as culturing sur-viving aerosolized cells and re-aerosolizing themhave met with very limited success in selectingaerosol-resistant clones. Braun (3) used irradiatedcultures to select cells resistant to drying andantibiotics without success. Similarly, Koh, More-house, and Chandler (6) found that cells ofEscherichia coli that survived 10,000 to 70,000roentgens of ,3 radiation had all the known prop-erties of the parent cells. However, there is evi-dence in our laboratories that if we are able toisolate true thermophilic mutants of severalpathogens, these mutants will show increasedresistance to aerosolization. Furthermore, a salt-tolerant variant studied at Fort Detrick showsincreased resistance to aerosolization with reten-tion of infectivity. Delwiche et al. (4) showed thatthe usual reversion to avirulence when cells ofPasteurella pestis were grown in air at 37 C couldbe overcome by the addition of bicarbonate oraspartic acid to the medium or by growing thecells under nitrogen. Nutritional and physio-logical studies on some inherently aerosol-stablecells such as staphylococci, mycobacteria, andBacillus anthracis spores have not shown con-clusive relationships between properties of thesecells and aerosol stability.The role of storage conditions such as time, tem-

perature, and concentration have been studiedextensively in our laboratories. Although general-izations can be misleading, it appears thatprolonged storage of cells is accompanied bydecreases in viability and infectivity even atrelatively low temperatures. Aerosol stability mayor may not be affected by storage. The additionof chemical stabilizers will be discussed later.Suffice it to say at this point that the pre-aero-solization treatment of organisms used in studieson airborne infection is of great importance andrequires close attention to strain selection, con-ditions of growth and harvest, and preparationsfor the act of aerosolization. Studies in our labora-tories concerning the effects of aerosolization perse will be discussed by other speakers.The third phase of our studies, the effect of

the postaerosolization environment, is mostcritical inasmuch as we now are dealing with thehost-parasite relationship. We will discuss only afew factors which affect this relationship. Suchsubjects as host susceptibility and the effects ofstress on the experimental host cannot be dis-cussed even though a fascinating literature isaccumulating on the role of body irradiation,nutritional deprivations, and the effect of chemi-cals such as 6-mercaptopurine and endotoxinpretreatment.

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GOODLOW AND LEONARD

Certainly one of the most important param-eters involving airborne microorganisms is thesize of the particles or droplets comprising theaerosol (7, 8). Many workers have demonstratedthat LD5o or ID5o values of certain airbornepathogens decrease as the aerosol particle sizedecreases. This has been clearly demonstratedwith B. anthracis, Pasteurella tularensis, Coxiellaburnetti, Brucella suis, and the virus of Venezuelanequine encephalomyelitis. Critical sizes rangefrom approximately 1 to 5 A. Such particles areretained maximally in the lung and, indeed, mustbe in this size range to reach the terminal bron-chioles and alveoli.The effects of particle size in experimental

airborne tularemia in the guinea pig and themonkey are shown in Table 1. Note the quantita-tive difference in LD5o values between guineapigs and monkeys exposed to the same aerosol.Obviously this does not reflect a difference insusceptibility to P. tularensis because bothspecies demonstrate equal susceptibility to thel-/. cloud. Reasons postulated for the lowerLD50 values in monkeys include greater filtrationefficiency of the guinea pig lung for larger particu-late aerosols and mouth breathing in the monkey.Besides illustrating the importance of the particlesize of the aerosol, the data do emphasize theobvious fact that the choice of laboratory animalin experiments of this kind is important.A major problem in obtaining quantitative data

on the response to different particle sizes involvesthe difficulty in producing homogeneously sizedparticle clouds. As various disseminating devicesproduce aerosols of large particles, invariablysatellite, small 1-, particles are produced. We areaware of no atomizing device that will producea truly homogeneous cloud. Obviously, if enoughpopulated satellites are produced to initiate infec-tion, the number of larger particles inhaled and

TABLE 1. LD5o dose in animals exposed to aerosolsof Pasteurella tularensis

LDso dose forParticle size

diameterGuinea pigs Rhesus monkeys

no. of cells no. of cells1 3 177 6,500 240

12 20,000 54022 170,000 3,000

retained is relatively immaterial. It is importantto note also that organisms within particles of aheterogeneous aerosol do not distribute them-selves evenly throughout the droplets. The dis-tribution of organisms throughout the availableparticles of the aerosol is influenced by the con-centration of organisms in the material aeroso-lized. The smaller particles of the aerosol remainunpopulated at low organism concentrations,whereas at higher concentrations the small par-ticles of the aerosol contain organisms. This is animportant observation and illustrates the neces-sity for microscopic examination of collectedsamples of the aerosol. Use of the proper stainand phase-contrast microscopy makes it possibleto visualize bacteria and some rickettsiae withincollected aerosol particles, thus enabling the in-vestigator to determine the organism distribu-tion within particles, an important and some-times critical piece of information.

Figure 2 shows P. pestis within an aerosolparticle. The slide was prestained with basicfuchsin, the aerosol was allowed to settle on theslide and was examined with the phase-contrastmicroscope. As mentioned, quantitative studieson the role of particle size in relation to the in-fectivity of aerosols were difficult because devicesthat would disseminate homogeneously sizedparticles were unavailable. Some time ago weemployed a vibrating reed device which is capableof producing particles 5 ,u and above with a re-markable degree of homogeneity and uniformity,thus eliminating the confusing satellite particles.Particles produced by this device vary in diam-eter no more than 0.2 to 0.3,. Table 2 showssome of the particle size and dose response dataobtained using the vibrating reed and exposingrestrained, unanesthetized rhesus monkeys. Wewere surprised by the obviously low LD5o valuefor the 8-Au cloud. If calculated, that value wouldbe expected to be below 60 inhaled cells.

Histopathological observations of monkeyssacrificed at 24 and 48 hr after exposure indicatedthat all primary tularemia lesions in the lungwere located in the terminal respiratory bronchi-oles regardless of 1- or 8-,u particle size. In noease were primary lesions observed at the depthof the alveolus. Animals in both groups showedX-ray evidence of pneumonic lesions.

Consequently, in a third series, animals wereexposed to P. tularensis aerosols containinghomogeneous particles 18 ,u in diameter. These

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FIG. 2. Pasteurella pestis within an aerosol particle

monkeys became ill and died. None showed X-rayevidence of pneumonic disease. Pathological evi-dence indicated that the distribution of lesionssupported a nasopharyngeal portal of entry withspread to the regional lymph nodes, sepsis, dis-semination, and death.

In experimental airborne tularemia in themonkey, it is clear that the particle size of theaerosol is of great importance in determining theportal of entry and the nature of the consequentdisease. There is evidence that this is likewisetrue in the case of experimental pneumonicplague.

Problems associated with measuring aerosoldecay, both physical and biological, will be dis-cussed by subsequent speakers. With severalexceptions, aerosols of microorganisms decay orlose viability very rapidly. The mechanisms ofdeath after aerosolization are very obscure. Webb(11-13) showed that decay of an aerosol occursin at least two stages, a very rapid death in thefirst several seconds of cloud age and a slower

TABLE 2. Dose response in rhesus monkeys exposedto aerosols of Pasteurella tularensis

Particle size No. of cells No. ill/no. No. dead/no.diameter inhaled exposed exposed

;A

1 35 8/9 7/91 170 10/10 9/101 1,360 10/10 10/108 60 5/6 5/68 230-320 19/20 15/20

death rate thereafter, which may result in longperiods of time for cloud extinction. These decayrates can be correlated with a mathematicalfunction of both temperature and humidity. Lowrelative humidity (ca. 20 to 30%7) hastens thedecay rate for most cells as does very high hu-midity (ca. 95%); optimal humidity ranges from40 to 80%. We have done considerable work onthe effect of solar radiation on aerosols (1). Aspecial aerosol chamber has been constructed to

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GOODLOW AND LEONARD

TABLE 3. Effect of aerosol age on respiratory virulence of Pasteurellatularensis for guinea pigs

Average aerosol age

Experimental parameters UCTL atomizer Collision atomizer

1.37 min 330 min 1.37 min 330 min 24 hr

Respiratory LD50.................... 18 cells 199 cells 11 cells 156 cells 540 cells95% confidence limit* ............... (16-23) (158-250) (9-17) (108-307) (426-704)

* The majority of these results are an average of 11 tests.

TABLE 4. Effect of aerosol age on respiratoryvirulence of Pasteurella pestis for guinea pigs

Expt no. Aerosol No. animals Inhaled dose Per centage inoculated mortality

min

1 5 10 9.2 X 104 10045 10 1.7 X 105 20

2 5 10 6.8 X 104 10045 10 7.0 X 104 10

3 5 10 1.2 X 105 9045 10 1.2 X 105 30

permit exposure of aerosols to natural sunlightunder controlled conditions of temperature andhumidity. Preliminary studies indicate that (i)large-particulate clouds are more resistant thansmall-particulate clouds to the lethal affect ofsolar radiation, (ii) dry disseminated aerosols(dust) were more resistant to solar radiation thanwet disseminated aerosols, and (iii) in wet aero-

sols atmospheric moisture (relative humidityabove 70%) afforded significant protectionagainst the lethal affect of solar radiation.Data supporting the various hypotheses re-

lating to viability and infectivity of airborne cellsare painfully meager and probably reflect our

inadequate techniques for recognizing and meas-

uring the various phenomena in operation. Scott(10), Ferry, Brown, and Damon (5), and Webb(11) all confirm the thesis that the one common

factor affecting airborne viability is the rela-tionship between cellular proteins and water.Webb suggests that death results from the move-

ment of water molecules in and out of the cellin an equilibrium system, resulting in the col-lapse of the natural structure of cell protein.Treatment of aerosol-stable cells with ribonu-

clease or lysozyme rendered them aerosol sensi-tive. Cells of E. coli which are freely permeableto ions, small molecules, sugars, and peptidesare air sensitive, whereas cells of Staphylococcuscitreus and Staphylococcus albus are not so per-meable and are more aerosol stable.Webb (12) and others have shown that aerosol

stability can be improved by adding a variety ofcompounds to the spray material. These com-pounds include amino acids, long chain proteinderivatives, some sugars, and polyhydroxycyclo-hexanes. In our laboratories, the roles of non-metabolized carbohydrates, metabolic inhibitors,and many other classes of compounds are beingstudied. If we are able to determine the causesof death of aerosolized organisms, we are confi-dent that the geneticist, the nutritionist, andphysiologist will be better able to develop strainsor mutants which meet our requirements.

In addition to loss of viability of cells suspendedin the aerosolized state for prolonged periods oftime, it has become apparent that in the cases ofP. tularensis and P. pestis, at least, there aredemonstrable losses of virulence or infectivity(Tables 3 and 4). The mechanisms involved arequite obscure and apparently are somewhat in-dependent of losses in viability. This is illus-trated by the difference in mortality caused byessentially equal doses of P. pestis. It is notknown whether the "less" virulent cells in the45-min-old cloud would regain their originalvirulence if regrown in a growth medium and re-aerosolized for 5 min.

In summary, investigators concerned withexperimental airborne infection should pay closeattention to:

1) Specific strain of organism chosen for study.2) Conditions of growth and harvest of the

organism.3) Age of culture at time of aerosolization.

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4) Suspending medium of the organism to beaerosolized.

5) Technique of aerosolization, including dis-seminating device, temperature, and relativehumidity.

6) Techniques of aerosol sampling.7) Particle size distribution of the aerosol.8) Distribution of organisms (if possible)

within the aerosol particles.9) The age of the aerosol at time of animal

exposure.10) The decay rate, both physical and bio-

logical, of the aerosol.11) Choice of experimental animal

LITERATURE CITED

1. BEEBE, J. M. 1959. Stability of disseminatedaerosols of Pasteurella tularensis subjectedto simulated solar radiations at varioushumidities. J. Bacteriol. 78:18-24.

2. BEVERIDGE, W. I. B. 1957. Properties of someinfective agents in relation to their modesof transmission, p. 129-133. In C. Horton-Smith led.], Biological aspects of the trans-mission of disease. Oliver and Boyd, Edin-burgh.

3. BRAUN, W. 1958. Quoted, p. 59-60. In Reportof conference on factors that influence via-bility and function in microorganisms. Na-tional Academy of Sciences-National Re-search Council, Washington, D. C.

4. DELWICHE, E. A., G. M. FUKUI, A. W. AN-DREWS, AND M. J. SURGALLA. 1959. Environ-mental conditions affecting the populationdynamics and the retention of virulence ofPasteurella pestis: the role of carbon dioxide.J. Bacteriol. 77:355-360.

5. FERRY, R. M., W. F. BROWN, AND E. B.DAMON. 1958. Studies on the loss of viabilityof stored bacterial aerosols. II. Death ratesof several non-pathogenic organisms in re-lation to biological and structural charac-teristics. J. Hyg. 66:125-150.

6. KOH, W. Y., C. T. MOREHOUSE, AND V. L.CHANDLER. 1956. Incidence and character-istics of beta radiation survivors (Escker-ichia coli). Appl. Microbiol. 4:153-155.

7. LANDAHL, H. D. 1950. On the removal of air-borne droplets by the human respiratorytract. I. The lung. Bull. Math. Biophys.12:43-56.

8. LANDAHL, H. D. 1950. On the removal of air-borne droplets by the human respiratorytract. II. The nasal passages. Bull. Math.Biophys. 12:161-169.

9. LANGMUIR, A. D. 1956. Air-borne infection, p.152-167. In K. F. Maxcy [ed.], Rosenaupreventive medicine and public health. 8thed. Appleton-Century-Crofts, New York.

10. SCOTT, W. J. 1958. The effect of residual wateron the survival of dried bacteria duringstorage. J. Gen. Microbiol. 19:624-633.

11. WEBB, S. J. 1959. Factors affecting the viabil-ity of air-borne bacteria. I. Bacteria aeroso-lized from distilled water. Can. J. Microbiol.6:649-669.

12. WEBB, S. J. 1960. Factors affecting the viabil-ity of air-borne bacteria. II. The effect ofchemical additives on the behavior of air-borne cells. Can. J. Microbiol. 6:71-87.

13. WEBB, S. J. 1960. Factors affecting the via-bility of air-borne bacteria. III. The role ofbonded water and protein structure in thedeath of air-borne cells. Can. J. Microbiol.6:89-105.

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