Effect Aerosol Age Infectivity Airborne Pasteurella Macaca Man

JOURNAL OF BACTERIOLOGY, June, 1966 Copyright @ 1966 American Society for Microbiology

Vol. 91, No. 6

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Effect of Aerosol Age on the Infectivity of Airborne Pasteurella tularensis for Macaca mulatta and Man

WILLIAM D. SAWYER,1 JOSEPH V. JEMSKI, ARTHUR L. HOGGE, JR., HENRY T. EIGELSBACH, ELWOOD K. WOLFE, HARRY G. DANGERFIELD, WILLIAM S. GOCHENOUR, JR.,2 AND DAN CROZIER

U.S. Army Medical Unit and the U.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland Received for publication 12 February 1966

ABSTRACT SAWYER, WILLIAM D. (U.S. Army Medical Unit, Fort Detrick, Frederick, Md.), JOSEPH V. JEMSKI, ARTHUR L. HOGGE, JR., HENRY T. EIGELSBACH, ELWOOD K. WOLFE, HARRY G. DANGERFIELD, WILLIAM S. GOCHENOUR, JR., AND DAN CROZIER. Effect of aerosol age on the infectivity of airborne Pasteurella tularensis for Macaca

mulatta and man. J. Bacteriol. 91:2180-2184. 1966.-In aging aerosols of Pasteurella tularensis SCHU-S4, the respiratory infectivity for man and Macaca mulatta decreased more rapidly than the viability of the organisms. Infectivity was diminished after 120 min, and was reduced 10-fold after 180 min. These findings confirmed previous observations made in mice and guinea pigs, and also revealed that smaller losses of infectivity were detectable in the primate hosts.

The aerosol microenvironment is unfavorable

for most microorganisms, as evidenced by a decrease in viable organisms with time, i.e. "biological decay." (Viability is used here to mean the ability to produce a colony on suitable medium.) Phenotypic variations separable from death of the organism may also occur. One such phenotypic variation, diminished infectivity, is of considerable epidemiological significance. (The term infectivity is employed to indicate the capacity of an organism to invade a host and multiply detectably.) For example, drying of group A streptococci may diminish their human infectivity without destroying their viability (13). Because of this effect, aerosol spread of streptococcal upper respiratory infection is of minor importance in comparison with transmission by direct contact or by large droplets. Similar phenotypic variation has been demonstrated with other organisms, e.g. the gram-negative, intracellular parasites Pasteurella tularensis and P. pestis. The respiratory median lethal dose (LD5o) of P. tularensis for mice and for guinea pigs increases as aerosols are aged from 5.5 to 20 hr (7, 9, 17), and loss of infectivity from aerosol aging of only 40 min has been observed with P. pestis (7).

1 Present address: Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Md.

2 Present address: Walter Reed Army Institute of Research, Washington, D.C.

In the present studies, the effect of aerosol age upon the infectivity of P. tularensis for Macaca mulatta and man was determined by direct respiratory exposure. The results confirm the observations made in mice and guinea pigs that

infectivity declines more rapidly than viability.

MATERIALS AND METHODS

Animals. Healthy young M. mulatta (3 to 6 kg) and Hartley guinea pigs (300 to 400 g) were obtained from the Animal Farm, Fort Detrick, Md. Preexposure sera of the monkeys did not contain P. tularensis agglutinins. The principles of laboratory animal care proposed by the National Society for Medical Research were observed in this study.

Volunteers. Healthy young Seventh Day Adventist soldiers participated on a voluntary basis (2); they were informed of the nature of the studies prior to volunteering. These studies were supervised by the Commission on Epidemiological Survey of the Armed Forces Epidemiological Board. The cooperation of the War Service Commission of the Seventh Day Adventist Church is gratefully acknowledged. The men were observed closely in a hospital prior to and after exposure. Sera obtained prior to participation did not contain P. tularensis agglutinins.

P. tularensis SCHU-S4. Cultures were grown in modified casein hydrolysate medium (Mills et al., Bacteriol. Proc., p. 37, 1949) for 16 hr with continuous shaking at 37 C, and were stored for 14 to 21 days at 4 C until used for exposures of men and monkeys. In the interval, samples were used for preliminary trials (see below). On one occasion, aerosols were created after 35 days of storage; the results of

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this exposure were consistent with similar trials with

the fresher cultures. No attempt was made to evalu-

ate the effect of culture age upon the rate at which infectivity for M. mulatta and man declines during

aerosol aging. From studies of the guinea pig infectivity of 20-hr aerosol-aged P. tularensis, Hood (9)

concluded that older cultures lose infectivity more rapidly than fresh cultures. The cultures contained

approximately 30 X 109 viable cells per milliliter, and subcutaneous dose-response titration demonstrated that one to five organisms routinely killed guinea pigs and rabbits. The cultures were similar in viscosity, surface tension, specific gravity, pH, and content of total solids.

Tularemia immunization. Live tularemia vaccine (3)

was administered by multiple cutaneous puncture.

All men developed P. tularensis agglutinins after vac-

cination.

Aerosolization. Aerosols were generated within 10

sec by use of a 2-fluid nozzle (11) and undiluted culture. Clouds were created and held in a 1,000,000liter spherical static chamber in the absence of sunlight; the temperature was 24 C, and the relative humidity was 85%. Biological decay was similar in all tests, 1.5 to 2% per minute.

With the use of the experience gained fiom preliminary trials, desired aerosol concentrations were achieved by adjusting the amount of culture disseminated, and by diluting the aerosol with clean air equilibrated with chamber conditions. At the time of exposure, at least 65% of the viable organisms were contained in particles 5 A or less in diameter.

Aerosol sampling. Samples of aerosols were col-

lected in impingers, AG13o, operated at 12.5 liters

per min (11) and containing gelatin-peptone-cysteine with 0.25% Antifoam A (Dow-Corning Corp., Midland, Mich.). The gelatin-peptone-cysteine had the following composition: 0.1% gelatin, 3% Difco peptone, 0.1% L-cysteine, 0.85% NaCl, 0.4% K2HP04, 0.002% spermadine phosphate; pH 7.1. Impingers were equipped routinely with a preimpinger (11) which excluded particles greater than 5 ,u in diameter. To estimate the distribution of organisms in large and small particles (see above), the GP20 sampler (11), a non-discriminatory total collector, was employed in addition to the AGI30 with preimpinger.

Enumeration of P. tularensis. Serial dilutions of

each sample were plated on glucose-cysteine-bloodagar, and colonies were counted after 72 hr of incubation at 37 C.

Aerosol exposure. Caged monkeys were placed in the test chamber at the equator for 3 or 10 min (10). A tightly fitting face mask connected to the chamber at the equator was used for human exposure. Subjects inhaled through the nose and exhaled through a mouthpiece connected to filters and an air flow meter. Prescribed volunteer exposure was 10 breaths of 1 liter in 60 sec; only minor deviations occurred in the entire series.

The product of (i) the duration of exposure, (ii) the respiratory minute volume, and (iii) concentration of viable organisms in particles 5 ,u or less in diameter was taken as the inhaled dose. Respiratory volumes

of monkeys were estimated from Guyton's (8) formula. To simplify presentation, doses inhaled by groups of men and animals have been recorded as the mid-point of the range. This procedure and the division into dosage groups, although arbitrary, were consistent with the accuracy of dose estimation, and reflected the pattern of responses when analyzed subject by subject.

Criteria of infection. Only monkeys showing a serological response or evidence of tularemia at autopsy were considered to be infected; 72% of autopsies included histological or cultural studies, or both.

The criterion for infection of nonvaccinated volunteers was a rise in P. tularenisis agglutinins within 6 weeks of exposure. Because postinfection changes in agglutinin titer. are variable in vaccinated subjects (15), only clinical criteria of infection were used in this- group: symptoms, fever, elevated erythrocyte sedimentation rate, and the demonstration of C-reactive protein.

RESULTS

Aging of aerosols of the SCHU-S4 strain of P. tularensis at 24 C and 85% relative humidity reduced infectivity for M. mulatta (Table 1 and Fig. 1). The decline in infectivity began only after aging of the aerosols for 60 min, but after

TABLE 1. Infectivity of aged aerosols of Pasteurella tularensis for Macaca mulatta

Inhaled dose (viable cells)

No. infected/no. exposed to aerosol aged for

60 min

120 min

180 min

50 150 350 750 2,000 6,500 25,000

7/8

1/8

1/19

7/8

10/12

2/20

12/12 12/12

1/16

12/12 7/8

3/8

4/4

8/8

24/24

8/8

28/28

4/4

8/8

20/20

z U

tL

J 4

2

cj 4

a

z

50

150 350 750 2p00 6,500 25,000

VIABLE ORGANISMS INHALED

FIG. 1. Infectivity of aged aerosols of Pasteurella

tularensis for Macaca mulatta. Inhaled dose plotted on logarithmic scale. Aerosol ages were: 60 min (0), 120 min (A), and 180 min (O).

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180 min was approximately 10-fold. The results of exposure to aerosols aged 60 min corresponded to those obtained with dynamic aerosols only a few seconds old (4, 5, 7).

The course of disease was similar regardless

of the age of aerosol. Although incubation periods

among animals exposed to clouds of the same age varied inversely with dose, the incubation periods, irrespective of dose, were roughly the same for aerosols of different ages (Fig. 2). Most deaths occurred between 6 and 10 days after exposure, and the 30-day mortality was similar in all groups (Fig. 3). The small number of animals infected by low doses of the 180-min aerosol probably accounts for the wide fluctuation of

z

60-

4

u

40-

z

a

20-

z

AEROSOL AGE

* 60 Minutes

C] 120 Minutes

I

C3 180 Minutes

oofl

7: oFfl 00.r,i

2

3

4

5

6

7

8

II

INCUBATION PERIOD (days)

FIG. 2. Incubation period of monkeys infected by aerosol-aged Pasteurella tularensis.

AEROSOL AGE * 60 Minutes O1 120 Minutes D 180 Minutes

Coo)

80-

H60-

-j

H40-

20-

0

50 150 350 750 2,000 6,500 25.000 VIABLE ORGANISMS INHALED

FIG. 3. Mortality of monkeys infected by aerosolaged Pasteurella tularensis. Only a few animals were infected by low doses of 180-min aerosol, i.e., one, two, and one at doses of50, 150, and 350 organisms, respectively. (Note that mortality percentages refer only to animals that were infected. and therefore do not include animals that were exposed but failed to contract the disease.)

mortality in these groups (see legend to Fig. 3). Postmortem examination in all groups revealed widespread acute lesions, usually of bronchopneumonia, lymphadenitis, and splenitis; meningitis or encephalitis was also frequently observed (1).

The effect of aerosol age on infectivity of airborne P. tularensis for man was similar (Table 2). Infectivity of aerosols aged 30 and 60 min was comparable to that of dynamic aerosols (12, 16), but was reduced when the clouds were aged 120 or 180 min. Neither the incubation period (Table 3) nor the severity of the clinical illness, however, varied with the age of the aerosol. Onset of illness was usually abrupt, and the predominant manifestations were fever, headache, myalgia, anorexia, and retrosternal discomfort. Early therapy with streptomycin (1 g every 12 hr for 7 days) rapidly terminated the illness without complications or relapses. Agglutinin responses were regularly demonstrated after overt illness,

but were not detected in patients who remained

free from symptoms.

TABLE 2. Human infectivity of aged aerosols oj Pasteurella tularensis

Inhaled dose (viable cells)

No. infected/no. exposed to aerosol aged for

30 min 60 min 120 min 180 min

80 150 350 750 7,500 12,500 50,000

0/4

2/4 3/4

0/8

2/4

1/4 0/4

4/4

0/4

8/8 8/8

3/4

7/8 10/12

TABLE 3. Incubation periods in volunteers infected with aged aerosols of Pasteurella tularensis

Incubation period 30-min aerosol

No. of men 60-min 120-min aerosol aerosol

180-min aerosol

days

2

1

2

3

3

14

4

1

4

5

3

5

4

1

6

2

1

7

11

81

1

101

121

131

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INFECTIVITY OF P. TULARENSIS IN AGING AEROSOLS

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Because men and monkeys were exposed simultaneously to certain aerosols, a direct comparison of susceptibilities could be made (Table 4). Because of their lower respiratory volumes, monkeys exposed for 10 min inhaled approximately the same number of organisms as men exposed for 1 min; monkeys exposed 3 min therefore inhaled approximately 30% of the 1-min human dose. With allowances made for this difference, the data suggest that M. mulatta is about three times as susceptible to airborne P. tularensis as man.

The infectivity of aged P. tularensis aerosols for immunized subjects was also diminished. Whereas none of 12 volunteers immunized 9 weeks earlier with live vaccine became ill after inhaling approximately 50,000 viable organisms in a 120-min aerosol, 7 of 8 nonvaccinated volunteers developed acute tularemia. In contrast, previous experiments have shown that a comparable dose from a dynamic aerosol produces illness in most vaccinated subjects (4, 12, 16).

Unlike the respiratory results in primates, the subcutaneous infectivity of P. tularensis for guinea pigs was not decreased by aging the aerosol for 180 min. Other experiments have shown that more prolonged aerosol aging of P. tularensis results in diminished intraperitoneal infectivity for guinea pigs (9).

DISCUSSION

The hazard of exposure to airborne bacterial pathogens is not determined by the number of inhaled organisms that are viable, but rather by

the number that are infectious. That infectivity and viability may become dissociated during aerosol aging is demonstrated here and in earlier studies (7, 9, 17). Failure to recognize this fact may lead to misdirected efforts to control disease transmission by eliminating airborne microorganisms, as illustrated by the failure of dust control to prevent the spread of group A streptococcal infections (13). Unless structural or biochemical determinants of infectivity can be measured, the infectious potential of a microbial aerosol must be determined by direct exposure of the host concerned. Injection of air samples into susceptible animals by nonaerosol routes may be misleading, as illustrated in the present studies. Although susceptible laboratory animals may be used to measure the infectiousness of airborne bacteria, it must be borne in mind that their respiratory volumes are substantially less than that of man. Only if they have a relatively high degree of susceptibility, therefore, are they useful in studies of human airborne infection (14, 19).

The way in which aerosol aging affects infectivity of P. tularensis is unknown. Whatever the mechanism may be, the state of the bacteria at the time of aerosolization has an important bearing on the reduction of infectivity. The presence of chloride ions, yeast extract, or casein hydrolysate in either the growth medium or the

spray suspension, for example, accelerates loss of

infectivity in the aerosol (9). The age of the cul-

ture also influences the rate at which infectivity is lost (9); i.e., aerosols of stored cultures lose

TABLE 4. Infectivity of aged aerosols of Pasteurella tularensis for man and Macaca mulatta exposed simultaneously

Aerosol concn (viable organisms per liter)

Speci.es

Exposure time

Inhaled dose (viable cells)

No. infected/no. exposed to aerosol aged for

60 min

120 min

180 min

8-18

Man

1

Monkey

3

Monkey

10

20-40

Man

1

Monkey

3

Monkey

10

70-100

Man

1

Monkey

3

Monkey

10

500-1,500

Man

1

Monkey

3

Monkey

10

4,500-6,500

Man

1

Monkey

3

Monkey

10

80-180

3/4

25-50

80-180

7/8

230-360

80-110

280-360

700-1,000

4/4

300-350

700-1,000

8/8

5,000-15,000

1,700-4,100

6,000-15,000

45,000-65,000

13 ,500-20,000

45,000-50,000

0/4

0/8

0/4

0/8

1/4

0/8

0/4

0/4

0/4

1/4

0/4

4/4

0/4

4/4

0/4

8/8

11/12

8/8

12/12

12/12

10/12

12/12

4/4

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infectivity more rapidly than those of fresh cultures (9). Factors operating during and after aerosolization, e.g., temperature, relative humidity, and particle size, may be important as well. Until all such variables have been defined, quantitative results must be interpreted with caution.

Immunization with live vaccine is quite effective in preventing tularemia in populations at high risk, such as residents of endemic areas (18), laboratory workers (4), and other occupationally exposed groups (6). Whereas immunized volunteers exposed to dynamic aerosols of P. tularensis are fully protected only against relatively small doses (4, 12, 16), the present studies have shown that those exposed to aged aerosols are resistant to relatively large doses, e.g., 50,000 viable organisms. These results suggest that the aged aerosols may simulate more closely the conditions of natural airborne transmission than do the dynamic aerosols.

LITERATURE CITED

1. ARBITER, D. 1963. Brain lesions in monkeys infected with Pasteurella tularensis. J. Infect. Diseases 112:237-242.

2. ARMY REGULATION 70-25. 1962. Use of volunteers as subjects of research. Department of the Army, Washington, D.C.

3. EIGELSBACH, H. T., AND C. M. DOWNS. 1961. Prophylactic effectiveness of live and killed tularemia vaccines. I. Production of vaccine and evaluation in the white mouse and guinea pig. J. Immunol. 87:415-425.

4. EIGELSBACH, H. T., W. D. TIGERTT, S. SASLAW, AND F. R. MCCRUMB. 1962. Live and killed tularemia vaccines: evaluation in animals and

man. Proc. Army Sci. Conf., U.S. Military Acad., West Point 1:235-246. 5. EIGELSBACH, H. T., J. J. TULIS, E. L. OVERHOLT, AND W. R. GRIFFITH. 1961. Aerogenic immun-

ization of the monkey and guinea pig with live tularemia vaccine. Proc. Soc. Exptl. Biol. Med. 108:732-734.

6. EVANS, L. R. 1965. Experiences with tularemia vaccine. Am. J. Med. Sci. 249:548-550.

7. GOODLOW, R. J., AND F. A. LEONARD. 1961. Viability and infectivity of microorganisms in experimental airborne infection. Bacteriol. Rev. 25:182-187.

8. GUYTON, A. C. 1947. Measurement of the respiratory volume of laboratory animals. Am. J. Physiol. 150:70-77.

9. HOOD, A. M. 1961. Infectivity of Pasteurella tularensis clouds. J. Hyg. 59:497-504.

10. JEMSKI, J. V. 1962. Maintenance of monkeys experimentally infected with organisms pathogenic for man. Proc. Animal Care Panel 12:8998.

11. JEMSKI, J. V., AND G. B. PHILIPS. 1965. Aerosol challenge of animals, p. 287-341. In W. I. Gay [ed. ], Methods of animal experimentation. Academic Press, Inc., New York.

12. MCCRUMB, F. R., JR. 1961. Aerosol infection of man with Pasteurella tularensis. Bacteriol. Rev. 25:262-267.

13. PERRY, W. D., A. C. SIEGEL, AND C. H. RAMMELKAMP, JR. 1957. Transmission of group A streptococci. II. The role of contaminated dust. Am. J. Hyg. 66:96-101.

14. RILEY, R. L. 1961. Airborne pulmonary tuberculosis. Bacteriol. Rev. 25:243-248.

15. SASLAW, S., AND S. CARHART. 1961. Studies with tularemia vaccines in volunteers. III. Serologic aspects following intracutaneous or respiratory challenge in both vaccinated and nonvaccinated volunteers. Am. J. Med. Sci. 241: 689-699.

16. SASLAW, S., H. T. EIGELSBACH, J. A. PRIOR, H. E. WILSON, AND S. CARHART. 1961. Tularemia vaccine study. II. Respiratory challenge. Arch. Internal Med. 107:702-714.

17. SCHLAMM, N. A. 1960. Detection of viability in aged or injured Pasteurella tularensis. J. Bacteriol. 80:818-822.

18. TIGERTT, W. D. 1962. Soviet viable Pasteurella tularensis vaccines. Bacteriol. Rev. 26:354-373.

19. TIGERTT, W. D., A. S. BENENSON, AND W. S. GOCHENOUR. 1961. Airborne Q fever. Bac-

teriol. Rev. 25:285-293.

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