STUDIES ON HISTOPLASMOSIS I. C O M P A R A T I V E V I R U L E N C E OF V A R I A N T A N D PARENT STRAIN HISTOPLASMA CAPSULATUM IN H A M S T E R S 1) by ELIZABETH M. O ' H E R N , PH. n.

(Microbiology Department, George Washington University, School o/ Medicine, Washington 5, D.C.) (with 3 figs.)

(29,x.1963) Withir~ certain, endemic areas there is a high incidence of infection with Histoplasma capsulatum, yet histoplasmosis as an overt disease is infrequent (PALMER, 1945; GRAYSTON & FURCOLOW, 1953; SCHWARZ 6: BAUM, 1957). Both host factors, genetic and physiological (PROCKYOW, 1962), and factors pertaining to the organism, dosage (SAL¥IN, 1954) and strain virulence (LouRIA et al., 1959) contribute to the variation irt host response to infection. Among experimental animals tested the golden hamster has proved to be particularly susceptible to infection (MCLEOD et al., 1943; DROUtlET 6: $EGRETAIN, 1952) with Histoplasma capsulatum. Nevertheless, considerable variation m a y occur in response of individual animals to an equal, infecting dose of Histoplasma yeast cells. In a previous report (O'HERN, 1961) concerning such variation in hamsters with experimental histoplasmosis, the discussion of possible causal factors centered around tile development of resistance, i.e., the individual host response. In the above study it was noted that a soft-colony variant appeared as a secondary colony upon the Histoplasma isolated from the infected tissues of a few hamsters. When in subsequent tests it was found that a morphologically similar variant appeared in cultures from several animals, the organism was isolated and found to be stable in the yeast phase in subculture on blood agar at 37°C. That strain virulence of a specific organism m a y influence the course of disease in natural or experimental infection has been well documented in studies with a number of organisms (ToPLEY et al., 1) This work was initiated at the State University of New York, Downstate Medical Center, Brooklyn, N. Y., and supported in part by funds ((#12--1081) ~roln the National Fund for Medical Education, grant 7~ 12--1054.

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E.M. O'HERN

1931; WEBSTER & CLOUGH,1933; GEIMAN, 1943; FRIEDMAN et al., 1956; PAPPAGIANIS et al., 1956; HILL & MARCUS, 1959; HASENCLEVER & MITCHELL, 1960). In view of the variation ill response occurring among animals infected with the parent strain of Histoplasma and the repeated appearance of a morphologically similar variant in cultures of infected tissue from these animals, it seemed worthwhile to investigate whether a significant difference in virulence between the parent and the variant strains could be demonstrated. Young, prepubertal hamsters, and a moderately heavy infecting dose of organisms were used in the present study with a view to minimizing individual host differences. Test results based on survival time of 20 hamsters infected with each strain showed slight difference:between strains at this dose level, although, on statistical analysis, the parent strain was found to have a significantly greater virulence than the variant strain. The possible significance of these findings is discussed. MATERIALS AND METHODS Medium:

Brain heart infusion blood agar (BHIB) containing 20 units penicillin and 40 units streptomycin was used as the medium for isolating Histoplasma from animal tissue; antibiotics were omitted from the medium used for transfer and maintenance of the yeasts in culture. Yeast cultures were incubated at 37°C. Sabouraud's dextrose agar (SDA) was used for culture of the hyphal phase of Histoplasma; these cultures were incubated at ambient, room temperature. Organism:

T h e p a r e n t s t r a i n (I) of Histoplasma capsulatum (fig. 1) is a Department Stock Strain of unknown source showing white, cottony growth on SDA and producing micro- and macroconidia fairly abundantly in older cultures. As previously reported, cultural conversion of the hyphal form to the yeast form was not effected. on any medium tested (O'HERN, 1961). Cultures developing within the first or second week after inoculation of infected animal tissue onto B H I B at 37°C appear tan and somewhat granular but consist, in young cultures, solely of yeast cells. Colonies which appear in cultures of infected tissue after a delay of 3 to 4 weeks, as well as early cultures held for a similar period, consist of mixtures of yeasts and hyphae; on further holding or subculture to B H I B at 37°C, all become completely mycelial. Such colonies are firm and frequently wrinkled. T h e v a r i a n t s t r a i n (S) of Histoplasma capsulatum (fig. 2) was obtained in culture of tissue from hamsters infected with the parent strain. It appeared as a secondary colony upon a parent colony

VIRULENCE OF ttlSTOPLASMA VARIANT

Fig. 1.

169

Histoplasma capsulaEum p a r e n t

strain (I). Wrinkled, myceliaI colony on b r a i n h e a r t infusion blood agar; 14 d a y g r o w t h at 37 ° C. × 25.

which had reverted to hyphae and become firm and wrinkled. It differs from the parent yeast colony in exhibiting a more rapid growth rate and in retaining, grossly and microscopically, its yeastlike characteristics both in older cultures and in subcultures on B H I B at 37°C. On SDA, however, the growth of the variant is indistinguishable grossly and microscopically from that of the parent strain. A hyphal inoculum from the early growth, less than 1 month old, of the original mycelium obtained from a yeast inoculum, will convert to yeast on subculture to B H I B at 37°C, but this convertibility is lost in older cultures and in subcultures on SDA.

Preparation of inoculum: Since it was necessary to pass the parent strain (I) through an animal to obtain yeast ceils for inoculum, both organisms were passed through mice just prior to use. A suspension of mortarground mycelium from SDA was used as inoculum of the parent strain, while a saline suspension of yeasts from B H I B was used as the inoculum for the variant strain on the assumption that the

170

E.M. O'H~N

Fig. 2, Hisfoplasma capsulatum variant s t r a i n (S), Smooth, yeast-like colony on top of firm, mycelial parent colony; 14 day growth on brain heart infusion blood agar at 37 ° C.×25.

variant strain mycelium on SDA might well have undergone some change, as evidenced by inability of subcultures to convert to yeast on blood agar at 37°C. The infected mice were sacrified after 16 days of infection and the yeast cells of each strain obtained from cultures of spleen and liver were used as inoculum for virulence testing. Saline suspensions of twice-washed yeast cells were diluted to contain 100,000 cells per 0.5 ml used for intraperitoneat injection of hamsters. The animals: Forty male golden hamsters weighing 30 to 40 g were inoculated intraperitoneally with 0.5 ml of a saline suspension of Histoplasma yeast cells prepared from 3 day subcultures of organisms obtained from the tissues of infected mice. Twenty animals were inoculated with strain I (the parent) and 20 animals with strain S (the variant). Animals were isolated according to infecting strain, caged in groups of five in controlled temperature quarters, and allowed free access to food and water.

171

VIRULENCE OF ttlSTO.PLASMA VARIANT

RESULTS

Table I presents the variance ratio on survival time through the 38th day. Initial deaths with each strain occurred on the same day, the l l t h day, and within the short period of 19 days, all but 2 of the animals had died. Such results suggest that the infecting dose of organisms was not moderate for these animals but approached the overwhelming dose. However, an analysis of variance through the 18th day shows a significant difference in virulence of the parent strain, at the level of p -0.00 !, over the variant strain. TABLE I

Survival time o/hamsters in/ected with parent (I) a~d variant (S) Histoplasma capsulatum Animal n u m b e r 1 2 3 4 5 D a y s survived Strain I 10 l0 10 10 11 Strain S 10 I0 10 11 13 Difference: Sum, S m i n u s I S q u a r e d difference: Sum, S m i n u s Mean difference Variance t value t o.ooi value

6

7

8

9 10 11 11 13 14 15 i6 17 18 19 20

l I 11 l l l i l I 13 t3 I3 13 t3 13 13 16 17 20 13 13 13 13 13 15 15 15 16 I6 16 16 17 IZ* tZ

32 I

74 1.77 3.1 I 4.26 3.92

* tZ s u r v i v e d

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Slope = 13

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S = Variant

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IO DAYS

AFTER

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II

1 58

INOCULATION

Fig. 3. D e a t h rate of h a m s t e r s infected intraperitoneally with 100,000 y e a s t cells of Histoplasma capsulatum p a r e n t strain (I) and v a r i a n t s t r a i n (S).

172

B.M. o'nEla~

In fig. 3 the cumulative percentage of deaths is presented graphiically. The slope of the curve for the parent (I) strain between 20% and 85% deaths is 22; the slope for tile variant (S) strain within the same limits is 13, indicating the somewhat greater virulence of the parent strain. DISCUSSION Strain differences in Histoplasma have been reported for a number of cultural and morphological characters as well as for that of virulence (DRouHET & SCltWARZ, 1956; PINE & PEACOCK, 1958; LOURIA et al., 1959; PINE, 1962). No particular study appears to have beei1 made of variation within a single strain. The nature of the variation in the present case is not clear. The stability of the colonial and microscopic characters in subculture at 37°C suggests that the change m a y be a true mutation; however, the loss of convertibility after a certain, undetermined period on Sabouraud's medium at room temperature might be interpreted in several ways, e.g., the gradual loss of an induced enzyme or its end product presents irt the yeast and inducibIe in the animal but not in the medium tested; the selection under the influence of metabolic products in culture (GOODLOW et al., 1951) of a mutant with increased requirements for conversion; or even the integration of a cytoplasmic factor with consequent physiologic change known to occur in other organisms (CANTINO, 1956; FOLEY, 1958; CAMPBELL,1962). Tile possibility of the segregation of a heterocaryont shown in other fungi (Po~'TECORVO, 1946) to be involved in variation should probably not be wholly discounted despite the demonstration of the uninucleate condition in Histoplasma yeast cells (EDWARDS et al., 1959; EMMONS, 1959). What significance the lesser virulence of the smooth-colony variant m a y have in pathogenesis of the infection and apparent resistance of "individual animals is uncertain. The variant has not been observed from a sufficient number of animals for any pattern to become evident. Although significantly less virulent than the parent strain, the variant strain has obviously retained considerable virulence: the first fatalities occurred on the same day as those with the parent strain infections and 50% of the animals had died b y the 14th day. If the more rapid growth rate of the variant strain observed in vitro is maintained in the infected animal, it could prove quite disastrous, particularly in infections running a longer course, since it is probable that the variant differs antigenically from the parent strain. Such factors, differential growth rates and antigenic variation are well known determinants of pathogenesis and outcome in infections with other agents (SCHUHARDT & WILKERSON, 1951; HASENCLEVER & MITCHELL,1960). In addition, the insensitivity of the variant strain to the tissue inhibitory factor (O'HERN, 1963) further suggests that it m a y be an increased hazard for an animal to harbor such a variant strain.

VIRULENCE OF HISTOPLASMA VARIANT

173

Summary 1. A variant strain of Histoplasma capsulatum was isolated from a secondary colony in cultures of tissue from hamsters infected with the parent strain of Histoplasma. The variant strain was found to differ from the parent strain in colonial morphology, stability of the yeast phase in subculture, and in growth rate. 2. Results of virulence testing with these two strains showed the parent strain to be somewhat more virulent than the variant strain as measured b y survival time and slope of the death rate curve over a 19 day period. 3. Nevertheless, in view of the more rapid growth rate of the variant and its probable antigenic variation from the parent strain, the development of such a variant is viewed as an increased hazard to the host, particularly in a longer term infection. 4. The repeated isolation of a variant strain of Histoplasma suggests that strain variation m a y play a significant role in the outcome of naturally acquired infections.

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