JOURNAL OF BACrERIOLOGY, Feb., 1966Copyright @ 1966 American Society for Microbiology

Host-Parasite Relationships in ExperimentalAirborne Tuberculosis

I. Preliminary Studies in BCG-Vaccinated and Nonvaccinated AnimalsD. W. SMITH, E. WIEGESHAUS, R. NAVALKAR,1 AND A. A. GROVER

Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin

Received for publication 18 September 1965

ABSTRACr

SMITH, D. W. (University of Wisconsin, Madison), E. WIEGESHAUS, R. NAVALKAR,AND A. A. GROVER. Host-parasite relationships in experimental airborne tubercu-losis. I. Preliminary studies in BCG-vaccinated and nonvaccinated animals. J. Bac-teriol. 91:718-724. 1966.-Previous studies from this laboratory on immunogenicityand allergenicity of defatted mycobacterial vaccines involved subcutaneous chal-lenge of guinea pigs and killing of the animals 6 weeks later to evaluate the amountof disease. This type of experiment has been discontinued in this laboratory in favorof an airborne challenge type of experiment, with the advantages that aniimals canbe challenged with small numbers of bacilli by a natural route, and the number ofprimary lesions, the rate of spread from those lesions, and the rate of bacillary mul-tiplication can be used to evaluate protection. Experiments to determine uniformityof infection showed that a fair degree of uniformity resulted when seven guinea pigswere exposed simultaneously, and were studied 3 weeks later to determine numbersof primary lesions and bacilli in the tissues. A less satisfactory degree of uniformitywas obtained when more animals were exposed at one time. BCG-vaccinated andnonvaccinated animals were studied to determine the earliest time and the optimaltime for killing the animals to detect the effects of vaccination. In guinea pigs, thedegree of protection assessed by lesion counts is time-dependent, but the degree ofprotection assessed by viable counts of bacilli in the tissues was relatively constant3 to 12 weeks after infection. Mice vaccinated subcutaneously with BCG were notprotected against infection at any interval between 2 and 19 weeks. Guinea pigsvaccinated subcutaneously with the same lot of vaccine were protected as judgedby counts of viable bacilli in the tissues 3 weeks after infection.

Progress in the elucidation of the factorsresponsible for acquired resistance in tuberculosishas not been commensurate with efforts made. Asurvey of the literature leads to the conclusionthat every product of the tubercle bacillus (cellwall, protoplasm, lipids, polysaccharides, andtuberculin) has been shown to be protective in thehands of at least one investigator. The reviews ofWeiss (14) and Crowle (1) serve as sources to theliterature. It is possible that protective substancesare found in all components of the tuberclebacillus, but experience with other infectiousagents suggests that this is very unlikely. If onlycertain fractions are protective, then these re-views fail to point the direction for future work.Some reasons for this failure include the following.

Present address: Bakteriologiska Institutionen,Goteborgs Universitet, G6teborg, Sweden.

(i) Few laboratories use the same criteria ordefinition of protection; in fact, the number ofevaluation methods almost equals the number oflaboratories studying the problem. (ii) Investi-gators often do not include control vaccines ofknown potency. (iii) Fractionation techniquesoften are inadequate to give clean separation ofimmunogenic materials. (iv) Insufficient attentionis given to statistical treatment of the data.

It is conceivable that little more progress willbe made in this field unless more attention isgiven to these problems, especially the first.Much thought must be given to the question:What is protection against tuberculosis and howcan it be measured? This is not to say that eachinvestigator should adopt a single staidrdmethod for evaluating protection, but carefulconsideration should be given to whether a

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method is intended to be relevant to tuberculosis.Protection against tuberculosis could mean anyof the following: prevention of infection, increasein survival time, increase in the 1D50 dose, re-duced rate of bacillary multiplication, preventionof spread from primary lesions, or failure todevelop clinically apparent infection. (In thiswork, "primary lesions" refers to the lesionswhich result from the multiplication of inhaledviable tubercle bacilli.)

Previous studies from this laboratory (3, 12,13) on immunogenicity and allergenicity of de-fatted mycobacterial vaccines involved subcu-taneous challenge of guinea pigs followed bykilling of the animals after 6 weeks to evaluatethe extent of development of the disease. Al-though it is possible in such experiments to userelatively low levels of challenge, the route ofadministration is unnatural, the evaluationmethod usually employed (estimation of extentof gross and microscopic disease) is subjective,and normal statistical methods are not applicableto the data. For these reasons, subcutaneouschallenge experiments have been discontinued inthis laboratory in favor of an airborne challengetype of experiment. The advantages of thismethod are that animals can be challenged withsmall numbers of bacilli by a natural route andthe data can be evaluated by normal statisticalmethods. Determination of the number of pri-mary lesions and viable counts of tissues providcanswers to questions of whether infection hasbeen prevented, the rate of bacillary multiplica-tion, and whether there has been spread from theprimary lesion.The pioneering investigations in experimental

airbone infection have been those of Lurie (7)and Wells (15). O'Grady and Riley (10) reviewedthe work in this field.The purpose of this report is to present results

of preliminary experiments on airborne infectionof BCG-immunized and normal animals.

MATERIALS AND METHODSAnimals. Male and female guinea pigs, primarily

albino, weighing 500 to 800 g, obtained from a localsupplier, were fed standard guinea pig chow and watersupplemented daily with 160 ,ug/ml of vitamin C.They were randomly allocated to experimental groups,and they were housed either two or three per cage.CF-I female mice, 18 to 25 g, obtained from CarworthFarms (New City, N.Y.), were used in one experiment.They were caged in groups of five and were fed Purinachow and water.

Vaccine. Lyophillized BCG vaccine (supplied bythe Tice Laboratory of the Institution for Tuberculo-sis Research, affiliated with the University of Illinoisand Research Foundation, Chicago, Ill., now desig-nated as the source of Reference Standard Vaccine by

the American Trudeau Society Research DivisionStudy Group on Evaluation of Methods for Demon-strating Protective Activity of Mycobacterial Anti-gens) was resuspended in diluent to a final concen-tration of 1 mg/ml. Each vaccinated animal received0.1 mIl of the vaccine by either the subcutaneous or theintradermal route.

Tuberculin tests. Tuberculin tests after vaccinationwere made with either 0.1 or 5 ,g of purified proteinderivative (PPD) injected intradermally in 0.1 ml ofdiluent. Observations of the test sites were made usu-ally after 24 hr.

Preparation ofchallenge suspensioni. In some of theearly experiments, inocula were taken from culturesmaintained on Oleic Acid Albumin Agar (Difco) oron American Trudeau Society (ATS) medium. Main-tenance of challenge cultures on these media was dis-continued because of the possible progressive attenua-tion of the cultures and because components of ATSmedium interfered with nephelometric measurements.Mycobacterium tuberculosis strain H37Rv (obtainedfrom W. Steenken, Trudeau Laboratory), strainErdman (obtained from A. Crowle, Webb-WaringInstitute, Denver, Colo.), and strain 199-RB (ob-tained from the National Institutes of Health) weremaintained by semimonthly transfers of a thin surfacepellicle on Sauton medium. To prepare a challenge sus-pension, a loopful of the young pellicle was trans-ferred to 5 ml of Dubos broth (Difco) and was ho-mogenized with a Teflon-glass homogenizer; 0.2 mlwas inoculated into 10 ml of Dubos broth in 3-oz (ca.90 ml) prescription bottles. After 7 days of incubationat 37 C, the contents of several bottles were homoge-nized and filtered through a membrane of 5-,u poresize to remove clumps of bacilli. The resulting suspen-sion was adjusted to 20 nephelos units (Colemanmodel 9 Nephocolorimeter and Coleman NephelosStandards) by addition of Dubos broth, and was thenfurther diluted to 10-' with dilute Dubos broth (20%Cbroth in saline). A few drops of antifoam (Dow-Corning Antifoam AF Emulsion) were added to thefinal suspension. The number of viable tubercle bacilliin the suspension was determined from colony countsmade on Oleic Acid Albumin Agar.

Operation of infection chamber. Five to thirty ani-mals were exposed at one time in three verticallyspaced compartmented baskets in a model A3 air-borne infection chamber (Tri-R Instruments Co.,Jamaica, N.Y.) (8). A nebulizer-venturi unit con-structed from a Vaponephrine nebulizer (Vapone-phrine Co., Upper Darby, Pa.) was used to nebulizethe suspension. The unit (which should be capable ofnebulizing at least 1.3 g of water per 5 min under thestated conditions) was operated with 30 psi of airpressure (primary air) at a flow rate of 5 liters/min.The aerosol was drawn through the chamber at 24liters per min (secondary airflow) by means of avacuum pump. To improve mixing, a fan [4-inch (10cm) blade, 3,000 rev/min] was mounted on the baffleplate at the bottom of the chamber. An electric in-cinerator (4) placed in the line between the chamberand pump rendered the air noninfectious before it wasdischarged. At the end of the nebulization period

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(cloud buildup), the primary air was stopped, and air-flow through the chamber was continued for 45 nin.

Autopsy. At various periods after infection, groupsof animals were killed by the intraperitoneal injectionof 2.0 ml of pentobarbital sodium (60 mg/ml). Bodyweight, spleen weight, and when possible the numberof primary lesions on the surface of the lungs were re-corded. Specimens of lung, liver, spleen, and mediasti-nal lymph nodes were fixed in formalin for histo-pathology.

Enumeration of viable tubercle bacilli in the tissues.At autopsy, the right lower lobe of the lung and a por-tion of spleen were removed aseptically and cultured.Each tissue was ground for 2 to 3 min in a Teflon-glasshomogenizer with 5 ml of 2% bovine albumin frac-tion V solution. The homogenates were diluted ingelatin-saline (0.1% gelatin), and appropriate dilu-tions were plated on Oleic Acid Albumin Agar by thedrop technique recommended by Fenner (2), or byspreading 0.2 ml over the surface of the medium. After12 to 14 days of incubation at 37 C, colonies werecounted at appropriate dilutions, and the results wereused to calculate the number of viable bacilli in thetissue.

RESULTS

The purpose of the first three experiments wasto gain some insight into the degree of uniformityof infection that could be expected betweenanimals and between experiments, and to gainexperience in the quantitative enumeration ofviable tubercle bacilli in the tissues.Ten guinea pigs were infected by the respira-

tory route with H37Rv. In an attempt to assessthe number of viable bacilli inhaled, three animalswere killed 1 day after infection, and homogenatesof the right lower lobe of the lung were plated onOleic Acid Albumin Agar. No colonies developedeven from the undiluted homogenates. The re-maining seven animals were killed 3 weeks after

TABLE 1. Number ofprimary lesions andviable tubercle bacilli in tissues ofguinea pigs killed 3 weeks afterairborne infection with H37Rv

Primar lung No. of bacilliGuinea lesions (logarithms)

pig -_ _ _

RLL Total Lung Spleen

A-1 5 24 5.33 4.12A-2 5 30 5.19 4.27A-3 6 24 5.20 3.39B-1 5 22 4.90 4.29B-2 5 22 5.16 3.38C-2 6 31 5.43 3.15C-3 6 24 5.02 2.78

Mean and 5.4 d:0.5 25 d 4 5.18 41:0.18 3.62 i 0.60SD

*Right lower lobe.

infection. Primary lung lesions were counted, andthe number of viable bacilli in the lung and spleenwas determined. The results of this experiment(Table 1) indicated a fair degree of uniformityboth in the number of lesions that developed andin the number of bacilli that were cultured.To determine whether similar results could be

obtained with another strain of mycobacteria,animals in the next experiment were exposed to anaerosol prepared from M. tuberculosts stain199-RB, and were killed 3 weeks after infection.The results (Table 2) indicated that the number ofprimary lesions and the number of bacilli culturedfrom the tissues were comparable to those seen inTable 1, where H37Rv was the challenge strain,and where the concentration of organisms in thechallenge suspension was similar.Although a fairly satisfactory degree of uni-

formity was obtained with a limited number ofanimals exposed in a given basket in the chamber,questions were raised about possible differencesof exposure of animals placed at differt levelsin the chamber. Therefore, seven guinea pigs in

the top basket and seven in the bottom basketwere exposed to an aerosol of H37Rv and werekilled 3 weeks later. Table 3 shows that the totalnumber of lesions was about one-third that seenin the first experiment. It should also be notedthat the number of bacilli in the nebulizer sus-pension was about one-third less than in the firstexperiment. No difference was seen between themean number of lesions on the lungs of animalsin the top and bottom baskets. The coefficient ofvariation of the number of lesions was greaterthan when there were only seven animals in thechamber.The purpose of the next two experiments was to

TABLE 2. Number ofprimary lesions andviable tubercle bacilli in tissues ofguinea pigs killed 3 weeks afterairborne infection with Myco-bacterium tuberculosis 199-RB

Primary lung No. of bacilliGuinea lesions (logarithms)pig ..

RLL Total Lung S1leim

S-332 6 27 4.78 3.40S-333 8 40 5.10 4.30S-334 7 32 4.88 4.20S-335 8 38 5.10 5.44S-336 7 32 5.18 3.85S-337 8 39 4.78 5.77S-338 7 25 5.00 5.74

Mean and 7.3 4 0.8 33 1 6 4.97 4 0.16 4.67 1 0.96SD

* Right lower lobe.

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TABLE 3. Number ofprimary lesiotns and viable tubercle bacilli in tissues ofguinea pigs exposed in top andbottom baskets of airborne infection chamber to aerosol of H37Rv and killed 3 weeks later

Primary lung lesions No. of bacilli (logarithms)Position Guinea pig _-

RLL* Total Lung Spleen

Top S-304 3 7 5.00 2.70S-305 3 10 4.65 2.48S-306 2 12 4.54 4.54S-307 2 8 1.70S-308 1 6 4.40 3.88S-309 3 9 3.40 2.40S-310 1 7 4.18

Mean and SD 2.1 4- 0.9 8.4 1- 2.1 4.36 :t1 0.54 2.95 -L 1.05

Bottom S-311 2 10 4.65 2.60S-312 0 5 4.30 4.78S-313 1 4 4.88 2.40S-314 4 9 5.13 4.54S-315 4 9 4.48 2.54S-316 3 6 2.70S-317 0 6 4.10 3.30

Mean and SD 2.0 -- 1.7 7.0 1 2.3 4.31 IE 0.79 3.36 4- 1.05

* Right lower lobe.

FIG. 1. Photograph offixed specimens oflungs ofguinea pigs killed 5 weeks after airborne infection (498 and499 BCG-vaccinated, 641 and 642 nonvaccinated).

gain experience in the study of vaccinated andnonvaccinated animals challenged by the respira-tory route, specifically to determine the earliesttime and the optimal time for killing the animalsto detect the effects of vaccination. In the firstexperiment, one group of BCG vaccinated guineapigs and a comparable nonvaccinated group wereexposed to infection 8 weeks later with H37Rv.Twenty-one animals from each group were killed3 weeks after infection to determine the uni-formity of infectionona larger sample of animals.Thereafter, seven animals from each group werekilled at each interval. At 3 weeks, the meannumber of primary lesions in the vaccinated

animals was 5 i 3, whereas in the nonvaccinatedanimals it was 9 i 4. Comparison of numbers ofprimary lesions in nonvaccinated animals at dif-ferent intervals indicates an increase from 9 i 3lesions at 4 weeks to 15 7 lesions at 5 weeks,with no further increase at 6 weeks. Figures 1 and2 show the gross appearance of lungs of vac-cinated and nonvaccinated animals 5 and 8 weeksafter infection. The results of this experiment arepresented in Table 4. Little change was seen in thenumbers of bacilli cultured from the lungs andspleens of nonvaccinated animals. Bacillarypopulations in the lungs of vaccinated animalswere relatively constant at a level 100-fold lower

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at most intervals for the first 12 weeks, followedby a period of progressive decline. Viable bacilliin the spleens of vaccinated animals remained ata 1,000-fold lower level at most intervals, with a

suggestion of increase in the last three periods.In the next experiment, CF-1 mice vaccinated

with BCG cells by the subcutaneous route and a

nonvaccinated group were infected 5 weeks laterwith M. tuberculosis (Erdman strain) and were

FIG. 2. Photograph offixed specimens of lungs ofguinea pigs killed 8 weeks after airborne infection (631and 677 nonvaccinated, 543 and 545 BCG-vaccinated).

killed at various intervals. The results presentedin Table 5 indicate little difference between vac-

cinated and nonvaccinated mice in the number ofbacilli in lungs and spleens at most intervals.The last experiment to be reported compared

intradermal and subcutaneous routes of BCGvaccination. Eight guinea pigs were vaccinatedby each route. The animals were infected withH37Rv 7 weeks after vaccination and were killed3 weeks later. The results presented in Table 6indicate that the number of primary lesionsamong the three groups was not markedly dif-ferent. The numbers of viable bacilli culturedfrom lung and spleen were strikingly differentbetween vaccinated and nonvaccinated animals,but not between the groups vaccinated by dif-ferent routes.

Table 7 shows the relationship between thenumber of primary lesions and the concentrationof the suspension nebulized for the experimentswith guinea pigs.

DIscussIoN

The results of preliminary experiments suggesta fair degree of uniformity of infection whenseven animals are exposed and studied 3 weekslater for numbers of primary lesions and bacillarypopulations in the tissues. As the number ofanimals exposed at one time is increased, however,experience shows that the uniformity of infectionis less satisfactory. To conduct long-term experi-ments on the natural course of the disease invaccinated and nonvaccinated animals,- largenumbers of animals will have to be infected undervery uniform conditions. Further work andfurther modifications in equipment will berequired before a satisfactory degree of uniformityis achieved.

TABLE 4. Logarithm of the number of viable tubercle bacilli in lung and spleen of vaccinated andnonvaccinated guinea pigs* at several intervals after infection with H37Rv

Lungt Spleent

post-infectionNonvaccinated Vaccinated Nonvaccinated Vaccinated

3 5.34 ± 0.44t 3.29 4 0.72 3.96 1.08 0.47 :1: 1.034 5.42 :i4 1.21 3.53 4 0.61 5.34 4 0.93 0.005 4.14 i 1.93 3.42 i 0.66 4.54 ± 1.00 0.47 A 0.126 4.16 i 0.44 3.41 :1= 0.72 5.27 ± 0.67 1.70 i 2.218 3.79 ± 0.47 3.26 ± 0.90 3.95 4 0.72 1.04 4 1.3610 5.32 i 0.81 3.05 : 0.85 4.97 i 1.43 1.37 ± 1.8212 4.85 ± 0.52 2.96 ± 0.60 3.68 : 1.74 0.34 i 0.9014 5.28 ± 0.57 2.81 4 0.76 4.49 i 0.46 1.40 ± 1.7917 4.84 ± 0.68 0.88 ± 1.60 4.37 ± 1.01 1.74 ± 2.1720 4.97 4 0.55 0.90 4 1.28 4.58 ± 1.03 2.48 ± 1.14

* Seven animals per group except at 3 weeks when there were 21 per group.t Calculated as the total number of bacilli per spleen or right lower lobe of the lung.I Standard deviation.

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TABLE 5. Logarithm of the number of viable tubercle bacilli in lung and spleen of vaccinated andnonvaccinated mice at several intervals after infection with Mycobacterium tuberculosis

(Erdman strain)

Lung SpleenNo. of weekspost-infection

Nonvaccinated Vaccinated Nonvaccinated Vaccinated

2 5.34 i 0.27 5.25 i 0.29 0.00 0.39 4 1.023 4.84 i 2.37 5.25 i 0.55 3.15 i 2.48 4.70 ± 0.684 6.19 ± 0.52 5.14 i 0.41 4.00 i 0.48 2.93 4 0.826 5.74 ± 0.19 4.99 ± 0.57 4.04 + 0.35 3.32 ± 0.8710 5.14 ± 0.32 3.84 i 0.66 3.53 i 0.42 2.92 i 1.3819 4.16 i 0.58 4.04 i 0.61 2.90 i 0.10 2.85 i 0.79

TABLE 6. Number ofprimary lesions andviable tubercle bacilli in the tissues ofBCG-vaccinated and nonvaccinated

guinea pigs killed 3 weeks afterinfection with H37Rv

No. of bacilli

Group Total lung (logarithms)lesions*

Lung Spleen

BCG, intradermal. 8 3.28 0.00BCG, subcutaneous ....... 5 3.24 1.54Nonvaccinated ............ 13 5.65 4.72

* Secondary airflow double that of previous ex-periments.

TABLE 7. Relationship between the numberof primary lesions and the concentration

of the nebulizer suspension

Nebulizer fluid Total lung lesionsExpt in Table (viable units/ml 3 weeks after

X 103) infection

1 25 252 35 333 9.5 84 10 86* 30 13

* Secondaryperiment.

airflow was doubled for this ex-

Evidence was obtained in one experiment (seeTable 4) that at several intervals after infectiontubercle bacilli were present in the tissues of non-vaccinated guinea pigs at a level at least 100-foldhigher than in vaccinated animals. This is similarto the results reported by Middlebrook (9) inairborne-infected guinea pigs examined 3 weeksafter infection.

In these experiments, vaccinated guinea pigsexamined 3 weeks after infection did not exhibitthe 10-fold reduction in the number of primarylesions reported by Middlebrook (9). Ribi et al.

(11) reported that 4 weeks after airborne infectionpulmonary lesions are absent in the lungs of 80%of mice vaccinated intravenously with BCG.Larson and Wicht (6) reported that, althoughnone of the vaccinated mice examined at 4 weeksexhibited pulmonary tubercles, all vaccinatedmice studied 10 weeks after infection had lesions.Lesion counts are difficult to assess in guineapigs because the number of primary lesions invaccinated animals is probably maximal at 2 to3 weeks, after which they begin to diminish innumber and size with macroscopic evidence ofresolution. The number of primary lesions innonvaccinated animals is low at 3 weeks andincreases up to 6 weeks. Further consideration ofthis question will probably require histopatho-logical observations of sections of the lung sur-faces. This information would be important be-cause it would permit an interpretation of whetheror not vaccination prevents the development of aproportion of inhaled viable particles, and hencewhether vaccination could prevent infection.The data in Table 4 suggest that populations

of bacilli in the lungs of vaccinated animals beganto decrease at 17 and 20 weeks, whereas in thesame period populations of bacilli in the spleensof the same animals began to increase. Assess-ment of the significance of these observations willrequire experiments conducted over longerperiods of time and with larger groups of animals.Guinea pigs have continued to be the animal of

choice in these experiments because acquiredresistance and delayed hypersensitivity can bestudied in the same animal, and because theyrespond to vaccines administered by routesusually employed in the immunization of humansubjects. It has been shown in these experimentsthat mice were not protected against respiratorychallenge when they were vaccinated by the sub-cutaneous route with the same lot of vaccinewhich protected guinea pigs (see Tables 5 and 6).Results reported by Larson and Wicht (6) in-dicate that H37Ra and BCG produced protection

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against airborne infection when given by theintravenous or respiratory route, but not whengiven by subcutaneous, intraperitoneal, or oralroute.One disadvantage of experiments involving

airborne infection is the associated health hazardfor laboratory personnel. Precautions must betaken during the exposure and the subsequenthandling of animals. In these experiments, theairborne infection chamber was operated in thesame adequately ventilated room where theinfected animals were to be housed. Precautionsroutinely taken in this work include: vaccinationof all skin test-negative persons, use of protectiveclothing (gown, trousers, surgical gloves, boots),effective face mask (see 5), and germicidal lampsmounted so that the upper portion of eachanimal room is exposed to ultraviolet irradiation.The effectiveness of these procedures to date isindicated by the fact that none of the personnelinvolved has shown any evidence of diseaseon routine chest X ray taken every 4 months.The underlying philosophy of this work is that

the use of the respiratory route of challenge to-gether with a low level of infection (5 to 10 pri-mary lesions) in animals will lead to observationsmore likely to be relevant to the natural course ofpulmonary tuberculosis in man. Evaluation ofhost response to experimental airborne tuberculo-sis will include a determination of the number ofprimary lung lesions that develop and thenumber of viable bacilli in the tissues at variousintervals after infection. These data would per-mit the following assessments of protection: (i)prevention of infection, (ii) rate of spread fromthe primary lesion, (iii) rate of bacillary multipli-cation in vivo.

Future studies will be concerned with thedevelopment of techniques leading to a re-producible predictable infection in the range of 5to 10 primary lesions in large numbers of animals.This infection system will be used to characterizethe natural course of the disease in nonvaccinatedanimals. Detailed knowledge of the naturalcourse of the disease is essential for recognitionof the true influence of vaccination and fordetermining the stage of the disease likely topresent the best point of departure for studies ofmechanisms of acquired resistance in tuberculo-sis.

AcKNowLEDrmErnThis investigation was supported by Public Health

Service research grant AI-00646 from the NationalInstitute of Allergy and Infectious Diseases.We acknowledge the participation of H. K. Kim

and K. Kanai (presently at the Department of Tuber-culosis, National Institute of Health, Tokyo, Japan)

in some of these experiments, and w6 thank KatrinaJones, Philip Rose, and Curlee Seals for their abletechnical assistance.

LIrEAruRE CIrED

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3. FREGNAN, G. B., AND D. W. SMrrH. 1963. Im-munogenicity and allergenicity in guinea pigsof a defatted mycobacterial vaccine and itsfractions. Am. Rev. Respirat. Diseases 87:877-888.

4. GREMILLION, G. G., L. F. MILLER, AND G. A.BODMER. 1958. An electric incinerator for sterili-zation of small volumes of air. Appl. icrobiol.6:274-276.

5. GUYTON, H. G., AND H. M. Dsciu., 1963.Respiratory protection provided by five newcontagion masks. Appl. Microbiol. 11:66-68.

6. LARSON, C. L., AND W. C. WICHT. 1962. Studies ofresistance to experimental tuberculosis in micevaccinated with living attenuated tuberclebacilli and challenged with virulent organisms.Am. Rev. Respirat. Diseases 85:833446.

7. LURIE, M. B. 1964. Resistance to tuberculosis:experimental studies in native and acquired de-fensive mechanisms. Harvard Univ. Press,Cambridge, Mass.

8. MIDDLEBROOK, G. 1952. An apparatus for air-borne infection of mice. Proc. Soc. ExptL Biol.Med. 80:105-110. T'

9. MIDDLEBROOK, G. 1961. Immunological aspectsof airborne infection: reactions to inhaled- anti-gens. Bacteriol. Rev. 25:331-346.

10. O'GRADY, F., AMD R. L. RILEY. 1963. Expri-mental airborne tuberculosis. Advan. Tuberc.Res. 12:150-190.

11. RIBI, E., C. L. LARSON, W. WICHf, R. Lwr, ANDG. GOODE. 1965. Resistance to expermentaltuberculosis stimulated by fractions from at-tenuated tubercle bacilli. Proc. Soc. Exptl. Biol.Med. 118.926-933.

12. SMrIrH, D. W., AND G. P. KuBIcA. 1955. Compari-son of extracts of tuberclel bacilli and BCG asimmunizing agents in guinea pigs. Proc. Soc.Exptl. Biol. Med. 901629-635.

13. SMITH, D. W., G. B. FRtNAN, L. DL -RIcHARDsON, AND E. VALDIVIA. 1964.of acquired resistance in guinea PIgw'fatted mycobacterium tuberculosis vanos4tBacteriol. 88.87-92.

14. WEIss, D. W. 1959. Vaccination a.ins,4Ue ^ulosis with nonliving vaccines. I. 1 hand its historical background. AM. gev. 3e-spirat. Diseases 80:340-358, 495- `, 8

15. WmEs, W. F. 1955. Airboome co nandirhygiene. Harvard Univ. Press, Cadhbride,Mass.

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