Experimental Tularemia in Macaca mulatta:Relationship of Aerosol Particle Size to theInfectivity of Airborne Pasteurella tularensis
WILLIAM C. DAY' AND RICHARD F. BERENDTAerobiology and Evaluation Laboratories, Fort Detrick, Frederick, Marylanld 21701
Received for publication 13 August 1971
Ninety-six Macaca mulatta were exposed to aerosol particles containing Pas-teurella tularensis. Four different aerosols were employed that contained particlesize distributions with median diameters of 2.1, 7.5, 12.5, or 24.0 Am. Size distribu-tions were calculated only for those particles observed by phase microscope tocontain organisms. Animals exposed to particles whose median diameters wereeither 2.1 or 7.5 ,4m were all infected and showed extensive infection of the lowerrespiratory tract, evidenced by large patches of consolidation with many necroticfoci on the surface. Death occurred in these animals 4 to 8 days after exposure.Monkeys exposed to 12.5- or 24.0-,um median diameter particles presented involve-ment of the cervical and mandibular lymph nodes, evidenced by swelling and ab-scess formation. Thirty-eight of the 45 animals in this group were infected. Thoseanimals succumbing to the disease died from 8 to 21 days after exposure. The res-piratory LDM values increased from 14 to 4,447 cells as the median diameter wasraised from 2.1 to 24.0 ,um.
When tularemia is acquired from an insectbite or contact with infected animal tissue, thedisease is usually manifested initially by theappearance of an ulcer or eschar at the portal ofentry, followed by enlargement of a regionallymph node (6, 8, 20). Approximately 36% ofthe patients who contract tularemia, however,display a typhoidal type of disease, often accom-panied by mild pleuropulmonary symptoms(27), in which the portal of entry is not dis-cernible. The clinical findings in the typhoidalvariety of the disease strongly suggest that theorganisms often gain access to the body throughthe respiratory tract (16, 18).To understand the conditions under which air-
borne Pasteurella tularensis cells are able toproduce infection, it is necessary to study thedisease after exposure of animals to artificiallyproduced aerosols. In this kind of study the sizeof the aerosol particles and the distribution ofcells within each particle size play extremelyimportant roles, since these factors control to alarge degree the size of the dose required to pro-duce infection (1-3, 7, 9).
Experiments have been reported in which both1 Present address: Biological Defense Research Laboratory,
animals and man have been infected by exposureto artificially produced aerosols containing P.tularensis (4, 13, 17, 21-24). In the majority ofthese studies, however, the particle size distribu-tions of the aerosols were relatively heterogene-ous, and it was difficult to ascertain the role ofparticle size in establishing infection.Only a few reports have been published in
which homogeneously sized P. tularensis aerosolswere used experimentally to infect Macacamulatta (rhesus monkeys) (9, 28). Data obtainedin our laboratories (9) have indicated that thenumber of inhaled P. tularensis cells required toproduce lethal infection in the rhesus monkeysincreased as the median diameter of the aerosolparticle increased.
This report presents experiments that weredesigned to determine the effect of aerosol par-ticle size on clinical findings in rhesus monkeysand to establish the relationship between the dis-tribution of cells within particles of various sizesand the dose required to produce infection.
MATERIALS AND METHODSCulture preparation. The SCHU S-4 strain of P.
tularensis was cultured at 37 C on a shaker in flaskscontaining culture media composed principally of amodified partial hydrolysate of casein (MCPH). Vi-
able cell counts after incubation averaged about4 X I'0/ml when assayed on glucose-cysteine-blood-agar (GCBA). Cultures were stored at 5 C betweenexperiments, and fresh cultures were obtained at3-week intervals.
Aerosol dissemination. Before dissemination, all cul-tures were diluted with MCPH to obtain the desiredviable airborne cell concentration. Aerosols were dis-seminated into a modified stainless-steel Reynierschamber (diameter, 42 inches; reference 21) at relativehumidities of 35 4 5%1(, and temperatures of 24 l 2C. Aerosol particles with diameters ranging from 1 to5 jim were disseminated with a University of ChicagoToxicity Laboratories (UCTL)-type atomizer (21)fittecd with an aerotec tube (12).
Dissemination of relatively homogeneous aerosolsof particles larger than 5 ,um was carried out with aspinning top (15). The particle size distribution pro-duced by this disseminator was controlled by regulat-ing the speed of the rotor with compressed air.
Aerosol sampling techniques. Aerosol particles weresampled with Shipe impingers (25) for the determina-tion of the viable cell concentration. The sampler wasoperated at a flow rate of 9.5 liters/min and contained25 ml of gelatine-saline solution and three drops ofDow-Corning antifoam (Dow-Corning Company,Midland, Mich.). Each sample was collected for 1min at selected times during animal exposure. Theconcentration of viable cells was determined by rou-tine dilution and plating of impinger fluid on thesurface of GCBA plates. The resulting colonies werecounted after 48 hr of incubation, and the number ofviable cells per liter of aerosol was calculated.
Samples for the determination of particle size andthe distribution of organisms within particles werecollected from each aerosol by permitting the par-ticles to sediment over a 3-hr period onto the surfaceof glass microscope slides that were coated with 50%Permount (Fisher Scientific Co., Silver Spring, Md.).This material preserved the particles, reduced flat-tening, and enhanced adherence to the slide. Samplescollected in this manner were then disinfected by ex-posure to ultraviolet light before microscopic examina-tion. Repeated sampling studies conducted on quies-cent chamber air with a Cascade impactor (14)immediately after the 3-hr settling period failed todemonstrate the presence of airborne particles.
Animal exposure. Ninety-six male monkeys thatweighed 4 to 5 kg were employed for respiratoryvirulence titrations. Before exposure, all animals wereanesthetized by intravenous inoculation of a mixtureof sodium pentothal and sodium pentobarbital (Com-butal, Abbott Laboratories, Berlin, Md.). The numberof cells inhaled by each animal was controlled byvarying exposure time. The dose of airborne bacteriaadministered to each animal was calculated by meansof Guyton's respiratory factor (10). The respiratoryLD5o for each particle size level was calculated fromthe dose-response data by the probit method (5).
Clinical studies. After exposure, all monkeys werehoused individually in closed cages in which the airwas continuously filtered to prevent cross-infection.The majority of clinical laboratory tests and observa-tions were carried out over a 21-day period. The blood
of surviving monkeys was titrated for P. tutlarensisagglutinins at 3 weeks postexposure.
Daily clinical observations included the recordingof appetite, sensitivity to light, body temperature,auscultation of the chest, palpation of the abdomenfor hepatosplenic sensitivity and enlargement, radio-graphic changes in the lung, white blood cell counts,hematocrits, erythrocyte sedimentation rates, andblood cultures.
Detailed necropsies were performed promptly onmonkeys that died and also on sacrificed survivorsafter 21 days. At autopsy, portions of liver and spleenwere aseptically removed from each monkey, groundin a Waring Blendor containing 10 ml of MCPH, andcultured on GCBA plates to determine the presence ofP. tularensis.
Particle size analysis. Microscopic sizing and count-ing of particles, as well as the counting of cells withinparticles, was carried out with a phase microscopefitted with an eyepiece graticule (14). Approximately2,000 particles were sized and classified into selectedsize ranges for each aerosol. The number of cells withinparticles was determined at the same time as theparticle size distribution to avoid any changes in sizedue to drying. No attempt was made to distinguishbetween viable and nonviable cells. Particles werecounted but not examined or sized unless they con-tained cells.The parameter of particle size employed in this
study was entitled the "organism number mediandiameter" (ONMD). This term is an original one de-veloped by the investigator and is somewhat similarto the "bacterial median diameter" developed bySonkin (26). The ONMD is defined as the diameter(in micrometers) of that particle representative of themedian number of organisms. The method used tocalculate the ONMD was similar to that employed forthe determination of the particle number mediandiameters described by Wolf et al. (19), with theexception that the cumulative percentage of cells wassubstituted for the per cent of particles occurring ineach size range.
This type of particle size and organism distributionanalysis was used because it best expressed the rela-tionship between particle size and the infective dose,especially in the case of aerosols that were not ho-mogeneous.
RESULTSParticle size and organism distribution. The
size distribution of the aerosol particles and thenumber of cells contained within each size rangeto which monkeys were exposed are recorded inTables 1 and 2.The data presented in Tables 1 and 2 indicate
that aerosols disseminated by the spinning top(Table 2) were more homogeneous than thosedisseminated by the UCTL atomizer-aerotectube combination (Table 1). The average numberof cells per particle and the percentage of cellsobserved for each size range differed widely(Tables 1 and 2) for each experimental condition.
a Spray concentration was 4 X 107 cells/ml.bONMD, organism number median diameter.
See text for a full explanation.c SD, geometrical standard deviation.
TABLE 2. Size distribution of aerosol particles andPasteurella tularensis cells disseminated by
the spinning topa
Particle sizerange (,m)
Aerosol 13.8-5.45.4-7.67.6-10.8
Aerosol 27.6-10.810.8-12.512.5-17.6
Aerosol 312.5-17.617.6-24.924.9-35.0
Particlefre-
quency(%)
4.885.49.8
7.283.89.0
3.683.612.8
ismgfr- Avg no.
quencye of cells/quny particle
0.279.420.4
3.182.114.8
1.676.921.5
5.112.226.7
35.678.4151.3
241.5490.0895.0"
ONMDb(Am)
7.5
12.5
24.0
a Spray concentrations were 4 X 108 (aerosol 1),4 X 109 (aerosol 2), and 4 X 10lo (aerosol 3) cellsper ml.bONMD, organism number median diameter.
See text for a full explanation.c SD, geometrical standard deviation.d Cell count was obtained by extrapolation.
These differences were caused by the variousdissemination procedures and by the differencesbetween the initial spray concentrations selectedto provide the desired dose of cells.
Microscopic examination of the aerosols pro-duced by the UCTL-aerotec tube indicated thatapproximately 50%X- of the particles smaller than1 ,um and 10% of the particles in the 1 to 4 ,imrange contained no cells. However, all particles5 Am or larger in diameter contained bacterialcells regardless of the disseminator employed.
Selected photomicrographs of P. tularensis cellsobserved in aerosol particles produced by eachdisseminator are presented in Fig. 1.
Clinical and pathological findings. The infec-tivity and lethality of four particle size rangeswere determined. The dose responses (Table 3)indicated that the total number of animals ex-posed to aerosol particles whose median diame-ters were 2.1 and 7.5 ,um showed a greater infec-tion rate and died earlier in greater numbersthan those exposed to 12.5 and 24.0 ,um aerosols.(Although 21 of 22 animals exposed to 12.5-j,mparticles ultimately became infected, consider-ably more cells were required than for the smallerparticles.) Approximately 16%o of the monkeysexposed to large particles (12.5 and 24.0 Mm)survived with no signs of infection, whereas allanimals exposed to 2.1- and 7.5-jpm aerosolsbecame infected.The clinical laboratory findings for animals
exposed to particles of 2.1 and 7.5 j,m were simi-lar. The time of onset of clinical disease was 2 to3 days after exposure and was first evidenced byelevation of rectal temperature to a range of 40to 41.1 C. After the initial rise, which usuallylasted from 2 to 4 days, the rectal temperaturedropped to a range of 35 to 36.7 C. Monkeysdisplaying this reaction usually succumbed to theinfection. Animals also displayed such symptomsat the onset of illness as anorexia, sensitivity tolight, and sensitivity to abdominal palpation. Agradual rise in the erythrocyte sedimentation ratewas seen throughout the course of the disease.Little or no change in hematocrit readings wasobserved. A twofold increase in leukocytes wasseen, usually accompanied by a bacteremia.Animals also developed pneumonitis. Radiologi-cal examinations showed severe lobar infiltrationin most animals, in which the density of thelungs increased with time, suggesting necrosisand abscess formation. The blood of all infectedsurvivors had antibody agglutinin titers of 1:320or greater 3 weeks after exposure. Base line titerswere uniformly negative.The incubation time of the disease for monkeys
exposed to 12.5- and 24.0-Mm aerosols waslonger (6 to 10 days) than for those exposed tosmaller particles (2 to 3 days), and milder clinicalsymptoms were seen at onset than were observedfor small particles. Clinical illness was firstmanifested by slight rise in rectal temperature, aslightly elevated white blood cell count, and thefinding of 2 to 3 bacterial cells per ml of blood.
L Anorexia, accompanied by chronic diarrhea, wasi observed as the disease progressed. These symp-
toms were usually followed by prostration anddeath, the latter usually occurring 7 to 10 daysafter exposure. In addition, a total of seven ani-
mals exposed to 12.5- and 24.0-,um particlesdeveloped severe conjunctivitis accompanied bya purulent discharge from the nose and eyes, arise in rectal temperature, and an increase in thenumber of bacteria in the blood. The monkeysthat developed conjunctivitis died between 10 to21 days after exposure, in contrast to those de-scribed below.At necropsy, the lungs of animals exposed to
2.1- or 7.5-,um aerosol particles were congestedand covered with large patches of consolidationwith many necrotic foci on the surface. Thebronchial lymph nodes were yellow and firm, aswere the spleen and liver, which also showedmany small foci surrounded by irregular areas ofnecrosis. On the other hand, autopsy of monkeysexposed to 12.5- and 24.0-,um particles showedthat lesions were confined chiefly to the upperrespiratory tract with mucosal congestion in thenasopharyngeal area, and enlargement of thecervical lymph nodes. An average of three to fivesmall lesions were found in the lower respiratorytract of those animals showing infection. Theselesions, however, were not as large as those ob-served in animals exposed to small particles. Thisfinding may have been due to hematogenousspread, although no lesions were found elsewherein the reticuloendothelial system.
Respiratory LD50 and ID50 values obtained formonkeys at each particle size level are shown inTable 4. The data indicate that a 317-fold greaternumber of organisms was required for mortalitywith aerosols whose median diameter was 24.0Am than was needed with aerosols whose mediandiameter was 2.1 ,um. However, the effect ofparticle size on infectivity (Table 4) in the ab-sence of lethality was more pronounced between2.1 and 7.5 Am than between 7.5 and 24.0 ,um,as evidenced by the decrease in the ratio betweendoses as the particle size was increased.
DISCUSSIONThe most pertinent finding in this study was
the significant increase in dose required to pro-duce lethal infection as the particle size increased.The data, however, indicated that in each aerosolthe particles that contained cells were not ofuniform size. The important question raised bythese findings is that of the role that each particlesize plays in the initiation of lethal infection. It ispossible that all particles in the inhaled dosewere not equally infective. In the case of thoseaerosols having median particle diameters of 2.1
FIG. 1. Photomicrographs of aero'sol particles con-
taining Pasteurella tularensis cells (magnification2,000 X). A, Spinning top, 12.5- to 6.5-pum particles
a ONMD, organism number median diameter. See text for a full explantation.I D/T, dead/total exposed; 1/T, infected/total exposed.c One animal died at each dose due to the effects of anesthesia.
TABLE 4. Respiratory LD5c and ID50 values formonzkeys, obtaired with aerosols of Pasteurella
tularenisis conitainiing different particle sizedistributionzs
a ONMD, organism number median diameter.See text for a full explanation.
and 7.5 ,m, the LD50 values differed significantly,but clinical symptoms were similar. This findingsuggests that the effective particle size may havebeen approximately the same in both cases. Inthis instance it can be speculated that all inhaledparticles contained in the 2.1-Am median aerosolwere infective, whereas, in the 7.5-,Am medianaerosol, only those cells contained in the smallestparticles were responsible for lethality. Therefore,many of the organisms in the 7.5-Am medianaerosol were contained in particles that werecapable of initiating infection, but were either toolarge in size or too few in number to play a sig-nificant role in establishing a lethal infection, yetthey were counted as though they were part ofthe lethal dose.The smallest particles in aerosols having a
median of 12.5 ,m were 7.6 ,um in diameter, andthe smallest in 24.0 Am aerosols were 12.5 Am.The same situation existed, therefore, as withsmaller particles, viz. the calculated LD50 valuesmay be overestimated because some of thelarger particles contained organisms that playedno active role in producing a discernible infec-tion. An important difference between the largerand smaller size ranges, however, is that enoughbacteria were present in the former to produceupper respiratory infection with a concomitantchange in clinical symptoms. These assumptionsare emphasized by the findings of Harper andMorton (11), who reported that a greater numberof small particles (1 to 2 Mm) impacted in thelower respiratory tract (tracheobronchial andpulmonary areas) of the monkey than did largeparticles (10 Mm). The majority of the large par-ticles impacted in the upper tract (nasopharyn-geal area). In this instance it is possible that cellscontained in large particles, impacted in theupper tract, were less able to establish infectionthan small particles in the lower tract. In relationto this hypothesis, it was significant to note theability of 12.5- and 24.0-,m particles to initiateconjunctivitis and subsequent fatal infection.However, the initiation of infection in the eyewith large particles did require a large dose, and,because of these size and dose requirements,tularemia infection in the eye is not frequentlyobserved.
factor conferred a higher level of protection inthe tissues of the upper respiratory tract thanthose of the lower, thus making tissues of thelower tract more susceptible to invasion. Theimportance of the site of particle deposition asrelated to tissue susceptibility has been empha-sized by Druett et al. (3) to explain the loss ofinfectivity of airborne Brucella suis for guineapigs as the particle size was increased. It followsthat the factors of site of deposition, clearance,and tissue susceptibility adversely affect theprobability of infection with large particles, and,therefore, a smaller number of cells are requiredto produce a lethal infection with small particlesthan with large.The observation that an increase in particle
size requires an increase in dose to initiate alethal infection is not unique for monkeys.Studies conducted in our laboratories (7) indi-cated that relatively homogeneous aerosols of P.tularensis became less infectious for guinea pigsas the particle diameter was increased from 1 to13 Am.
In conclusion, a change in the particle sizedistribution not only produced a significantchange in the LD50, but also produced differentclinical symptoms. The findings indicated thatparticle size plays a very important role in theinitiation of infection in the respiratory tract ofthe rhesus monkey.
ACKNOWLEDGMENTS
We thank Charles M. Beard for carrying out the clinicalevaluations reported in this paper, and we also acknowledge thetechnical assistance of Gerda Pirsch and Ruth R. Bailey.
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