Feb 14, 1972 - We thank Joseph V. Jemski and Ellis J. Tonik for supervising the aerosol ... for their assistance with the necropsies; and Richard H. Pettit,. Charles H. ... 280:1310. 11. Jellison, W. L., C. R. Owen, J. F. Bell, and G. M. Kohls. 1961 ...
Vol. 5, No. 5
INFECT-ION AND IMMtjNITY, May 1972, p. 734-744 Copyright li; 1972 American Socicty for Micr-obiology
Prinzted in U.S.A'.
Pathogenesis of Tularemia in Monkeys Aerogenically Exposed to Francisella tularensis 425 ROBERT L. SCHRICKER,l HENRY T. EIGELSBACH, JOHN Q. MITTEN,2 AND WILLIAM C. HALL' U.S. Army Biological Defense Research Laboratory, Fort Detrick, Frederick, Marylanfd 21701 Received for publication 14 February 1972
The pathogenesis of tularemia was studied in groups of rhesus monkeys (Macaca mulatta) that inhaled graded 10-fold doses ranging from 10 through 106 organisms of Francisella tularensis 425, a strain highly virulent for the white mouse but of reduced virulence for the domestic rabbit. Mean incubation periods ranged from 3 to 6 days followed by acute illness lasting 5 to 11 days with subsequent recovery of most animals. The higher inhaled doses resulted in shorter incubation periods, longer and more severe acute illnesses, and 18% mortality at the highest dose. Strain 425 multiplied in the lungs, disseminated to the regional lymph nodes, and became systemic. Maximal bacterial populations in tissues were reached by the 7th day after exposure of the animals regardless of the number of organisms inhaled. F. tularensis was no longer recoverable from any of six tissues examined 2 months after exposure. The most significant tissue changes occurred in the lungs; these consisted of foci of liquefaction necrosis, lobular consolidation, and pleural effusion and adhesions. The data indicate that the inhaled dose of strain 425 determined the maximal growth of the organism in the lungs which in turn influenced the severity of the usually selflimiting pneumonia and systemic infection. Although the monkey is less resistant to tularemia than is man, this laboratory animal when infected with F. tularensis 425 provides a useful model for the self-limiting type of human pulmonary tularemia usually observed in Europe and Asia but to a lesser extent in North America. It is not generally recognized that two major rope and Asia where it is sometimes called paletypes of tularemia occur in North America al- arctica tularemia (15, 16). The pathogenesis of fatal tularemia resulting though this fact was reported years ago (1, 11). Each of the two types is characterized by an epi- from type A fully virulent F. tularensis strain demiologic pattern and virulence of the infecting SCHU S4 (4) and the immuLnogenicity against Francisella tularensis strain. Strains of high viru- it produced by live vaccine prophylaxis with lence for man, designated type A by Jellison et al. strain LVS (5) have been well documented for (11), are usually associated with tick-borne tula- laboratory animals including the monkey (7, 13, remia of rabbi:s (Langomorpha). Nearly 90% of 23, 24, 25); controlled vaccine evaluation and human cases reported in North America are of this therapy studies have also been reported for man type. Strains of lowered virulence for man (type (9, 19, 20, 21). The purpose of the present study B) are usually associated with the water-borne was to characterize the pathogenesis of type-B disease of rodents (Rodentia). They are respon- tularemia that is usually nonfatal for man. The sible for only 5 to 10%, of reported human cases rhesus monkey (Macaca mulatta) was chosen as in North America (8) although many cases are the test animal because of similarities in tularemic probably undiagnosed (1). However, a major out- infection in man and monkey (6). F. tularensis break of relatively mild tularemia was recently 425 (2) was selected for this study because our reported among muskrat trappers in Vermont (3, previous experience, as well as that of others 10, 26). The morbidity of the disease was com- (18), had shown it to have the virulence and culparable to that most frequently observed in Eu- tural characteristics of most Eurasian strains as well as of strains involved in the Vermont 1 Prescent address: Veterinarly Biologics-V/S APHS, Hyattsoutbreak in contrast to those of strain SCHU ville, Md. 20782. S4. Several F. tularensis strains were studied with 2 Present address: Dept. of Pathology, School of Medicine, to fermentative characteristics and viruregard Johns Hopkins Univ., Baltimore, Md. 21205. 3 Present address: 140 Pinecrest, Saln Antonio, Tex. 78209. lence for mice and rabbits. The infection was 734
VOL.
51
1 972
PATHOGENESIS OF TULAREMIA IN MONKEYS
evaluated by determining and correlating (i) the clinical course of the illness and (ii) bacterial dissemination, multiplication, and persistence as well as (iii) the character and course of the anatomic pathologic process. MATERIALS AND METHODS F. tularensis strains. The origin of strains 425 (2), 503 (16), and SCHU S4 (4) have been previously reported. We isolated strain Vt 68 from a sample of muskrat spleen from an animal trapped during the 1968 Vermont outbreak (26); the sample was provided by Lowell S. Young of the National Center for Disease Control, U.S. Department of Health, Education, and Welfare, Atlanta, Ga. All organisms were cultivated in a modified casein partial hydrolysate liquid medium (5). Cells harvested after 18 hr of incubation with shaking at 37 C contained approximately 3 X 1010 viable organisms per ml determined by a colony count on glucose-cystein-blood-agar (GCBA) (5). Median subcutaneous lethal doses for white mice, 20 mice per group, and domestic rabbits (Oryctolagus), 6 animals per group, as well as glycerol fermentation were used to distinguish type A from type B (1, 15, 16). Type A strains of F. tularensis ferment glycerol and require less than 10 organisms to kill rabbits inoculated subcutaneously, whereas strains of type B do not ferment glycerol and require more than 106 organisms administered subcutaneously to kill rabbits. Monkeys. Rhesus monkeys were caged individually and conditioned for 6 months before use. Animals weighed between 2 and 3.5 kg and were randomly distributed by weight and sex. Aerogenic exposures. Strain 425 aerosols were generated in a large sphere from liquid suspensions with a nebulizer producing particles primarily in the range of 1 to 5 ,um in diameter. The aerosols were allowed to equilibrate at 21 C and 85% relative humidity before the animals were exposed. Equipment and methods for generating static aerosols, determining inhaled doses, and maintaining whole-body exposed animals have been described by Jemski and Phillips (12). Clinical study. One group of 10 monkeys inhaled approximately 10 organisms, and other groups of 20 to 30 monkeys inhaled 102, 103, l04, 105, or 106 cells. Monkeys were examined daily for signs of disease (exemplified by acute illness) and 2 to 3 times a week thereafter until the studies were terminated 120 days after exposure. Body weights were determined at approximately weekly intervals throughout each study, and data were calculated as per cent change from preexposure weight. Standard clinical laboratory determinations made on heparinized venous blood samples of all monkeys included plasma C-reactive protein (CRP), corrected erythrocyte sedimentation rate (ESR), and erythrocyte packed-cell volume (PCV). Tests were made on monkeys at 2- to 4-day intervals through the acute illness and at 35, 60, 90, and 120 days after challenge. Base line values were established over a 3-week period before exposure. Blood-urea-nitrogen (BUN) and creatinine (Auto Analyzer, Technicon Co.), as well as serum glutamic-pyruvic transaminase (SGP-T, Sigma
735
Chemical Co.), values were determined on serum samples of 10 of the monkeys inhaling 105 organisms. Serum F. tularelnsis agglutinin titers (5) were determined on all animals before exposure and on survivors 14, 35, 60, 90, and 120 days after inhaling strain 425. Chest roentgenograms were made of approximately one-half of the monkeys from each group (5 to 15 animals) before and at 2- to 4-day intervals through the 2nd week after exposure and again at 3 and 4 weeks or until there was no longer radiographic evidence of pulmonary disease. Roentgenographic classification of pulmonary lesions and other clinical criteria for classifying the severity of the illness are summarized in Table 1. Bacteriological and pathological studies. Groups of 36 monkeys each were administered 104 or 106 cells of strain 425 via the respiratory route in the manner previously described. Two to four previously assigned monkeys from each group were to be killed (Nembutal, Abbot Co.) and necropsies to be performed 1, 3, 6, 10, 15, 21, 35, 60, and 120 days after challenge. Moribund animals were killed and examined; animals found dead were necropsied immediately. Samples of tissues were removed aseptically from the lungs, tracheobronchial lymph nodes, spleen, liver, femoral bone marrow, and blood. Tissues, except blood, were weighed and then ground in ten Broeck tissue grinders containing 2 ml of sterile 0.1% gelatin-saline solution. Blood and dilutions of tissue suspensions were plated on GCBA and incubated at 37 C; colonies were counted after 96 hr. Weights of the lungs, spleen, and liver were recorded. Viable strain 425 populations were reported on the basis of an entire organ, per gram of bone marrow or lymph nodes, and per milliliter of blood. At the necropsy periods designated above, two to four monkeys from each group were examined for macroscopic and histologic lesions. Lungs were inflated with 10% buffered Formalin instilled through the trachea which was then ligated, and the specimen was submerged in Formalin. Samples of tracheobronchial lymph nodes, liver, spleen, and any other organs and tissues that appeared abnormal macroscopically were also preserved in Formalin. Fixed tissues were embedded in paraffin, cut into 5- to 7-,um sections, and stained by hematoxylin-eosin and Geimsa methods (13). Chest roentgenograms were made of one or two of the monkeys from each group at each time period for comparison with necropsy observations.
RESULTS Strain comparison. Virulence and glycerol fermentation data of four strains of F. tularensis are shown in Table 2. Strains 425, Vermont strain VT 68, and Eurasian strain 503 produced comparable data. These three strains differed markedly in rabbit virulence and glycerol fermentation from North American strain SCHU S4. Clinical. Clinical data on monkeys inhaling 10-fold doses ranging irom 10 to 106 cells of strain 425 are shown in Table 3. All animals became infected. Group mean incubation periods
736
SCHRICKER ET AL. TABLE
1.
INFECT. IMMUNITN'
Criteria for classifying severity of tularemia ini monzkeys Severity of illness'
Parameter
Temperature, rectal.
Mild
Moderate
Febrile to 3 days, maximum > 103.5 F (ca. 39.7 C)
Febrile to a week, maximum > 104.0 F (ca. 40.0 C)
None
Anorexia
present I
or
to
3
Present to
a
Severe
Febrile longer than
a
week, maximum > 105.0 F (ca. 40.6 C)
week
Present
days
longer
than
a
longer
than
a
week
Weakness, depression, or
both
.............
None
Present to
a
week
Present
week Body weight ......No change X ray, chest
..........
No lesions
or
gain
or+
No
for
to
6 days only C-reactive
protein..
Negative
to
+1
for
3
+1
week or longer >+I for about
15 for
>
week,
a
1
to 3 days
>15 for week
or
10%"
loss
+3 to +5, lesions 2 to
5
weeks >+3 for
a
longer
than
week
maximum +3
days
Erythrocyte sedimentation rate (mm/hrl)
i oss change or to +3, lesions for
>25 for longer than week
longer
a
a Roentgenographic classification of pulmonary lesions: +1, minimal infiltrate, diffuse or localized; +2, definite infiltrate, diffuse or localized (if diffuse-miliary or 1-cm bronchopneumonic patches; if localized-an ill defined 1- to 2-cm bronchopneumonic patch); +3, diffuse 0.5- to 1-cm or well defined 2- to 3-cm bronchopneumonic patches; +4, multiple 1-cm infiltrates or early lobar consolidation (animals exhibiting these lesions classified severely ill regardless of other data); +5, coalescence of the above multiple infiltrate with or without extensive lobar consolidation, obscuring both mediastinum and heart shadow. b Normal:
