Vol. 33, No. 2INFECTION AND IMMUNITY, Aug. 1981, p. 395-4000019-9567/81/080395-06$02.00/0

Comparative Virulence of Absidia corymbifera Strains inMice

DENNIS J. KITZ,It* ROBERT W. EMBREE,1 AND JOHN CAZIN, JR.2

Departments ofBotany' and Microbiology,2 University ofIowa, Iowa City, Iowa 52242

Received 26 January 1981/Accepted 28 April 1981

The comparative virulence of six different strains of Absidia corymbifera forcortisone-treated and untreated Swiss mice was determined. Spores of the sixstrains were inoculated into mice by the intravenous, intraperitoneal, and intra-nasal routes. All six strains were found to be virulent in cortisone-treated anduntreated mice by the intravenous route. Up to a 16-fold difference in strainvirulence was observed for cortisone-treated mice and up to a 10-fold differencefor untreated mice. When spores were administered by the intraperitoneal route,50% lethal dose values could be calculated only for the cortisone-treated mice,although a few deaths were seen in untreated mice challenged with 107 spores.Each of the six isolates of A. corymbifera, when administered in an intranasaldosage of 106 spores, produced death in some cortisone-treated mice. Studiesmade to determine the viability of spores produced by each strain revealed thatgermination was 90% or greater on Littman and YpSs agars at an incubationtemperature of 40°C in less than 12 h.

Absidia corymbifera (Cohn) Saccardo andTrotter is one of a number of Mucorales speciesthat have been identified as etiological agents ofmucorymcosis (1). This species has been subjectto considerable taxonomic study; 44 previouslydescribed species have been reduced by Ellisand Hesseltine (7) to synonomy with two spe-cies, A. corymbifera and Absidia ramosa. Not-tebrock et al. (14) later reduced A. ramosa tosynonomy with A. corymbifera.

In many of the experimental models employ-ing A. corymbifera for the study of mucormy-cosis, challenge dosages for animals appear tohave been chosen arbitrarily, with little or noattempt being made to deal with the infectiousdosage in a quantitative manner. Corbel andEades (3) commented only briefly on the effectthat the source of an A. ramosa isolate mighthave on the pathogenicity of the isolate for mice.They felt that there was little difference in thevirulence of A. ramosa isolates which weretested intravenously. Corbel and Eades also in-vestigated the acquired immunity developed inmice in response to experimental mucormycosis(4), in addition to determining the susceptibilityof mice to experimental infections with A. ra-mosa (5). In neither of these two studies, how-ever, was an initial 50% lethal dose (LD50) valuedetermined. Rather than use a predeterminedoptimal challenge dosage, they elected to useextremely large challenge dosages of up to 5 x

t Present address: Division of Infectious Diseases, Depart-ment of Intemal Medicine, Washington University School ofMedicine, St. Louis, MO 63110.

107 spores in test groups as small as six animalsper group. It is questionable that such an ap-proach would allow for a meaningful statisticalanalysis or that an individual experiment wouldbe sensitive enough to show a cause-effect rela-tionship.Few reports ofLD50 values for mice challenged

with spores of Mucorales species can be foundin the literature. Smith and Jones (17) reportedan LD50 value for one isolate ofA. ramosa of 104spores for normal, 5-week-old Swiss mice chal-lenged intravenously. Corbel and Eades (3)found that doses between 5 x 105 and 5 x 106spores of A. ramosa isolate administered intra-venously caused the death of approximately 50%of 19- to 22-day-old C3H mice. More recently,Kitz et al. (10) have found that three differentisolates ofa thermotolerant species, Radiomycesembreei, are able to kill Swiss mice. The LD5ovalues for 5-week-old mice challenged intrave-nously with these three isolates of R. embreeirange from 4.5 x 102 to 2.0 x 103 spores foruntreated mice and from 0.8 x 102 to 1.5 x 102spores for cortisone-treated mice.The present study was designed to determine

whether an isolate-related virulence exists forsix strains ofA. corymbifera inoculated into miceby the intravenous, intraperitoneal, and intra-nasal routes and to quantitate the LD5o valuesfor the intravenous and intraperitoneal routes.

MATERIALS AND METHODSExperimental design. Ten groups of mice, each

group consisting of five males and five females, were395

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396 KITZ, EMBREE, AND CAZIN

used for each intravenous experiment. Five of the tengroups received cortisone treatment to increase sus-ceptibility to infection. Four cortisone-treated and un-treated groups were inoculated intravenously via thelateral tail vein with 102, 103, 104, or 105 spores in 0.1ml of saline. One group of cortisone-treated and onegroup of untreated animals served as controls andwere inoculated intravenously with 0.1 ml of saline.The intraperitoneal experiments were similar to the

intravenous experiments in design, except that groupsof six cortisone-treated and six untreated mice, eachgroup composed of three males and three females,received graded dosages of 105, 106, or 107 spores in 0.1ml of saline. In the intranasal experiments, one groupof 10 male cortisone-treated mice and one group of 10male untreated mice received challenge doses of ap-proximately 106 spores in 0.03 ml of saline. These micewere lightly anesthesized with ether before sporeswere administered to the nares. The intraperitonealand intranasal experiments also included groups ofcortisone-treated and untreated mice which served ascontrols and received sham inocula of saline.

Observations were carried out over a period of 30days, during which the mice were allowed free accessto food and water. Animals were checked at least twicea day, and deaths were recorded on a daily basis.Necropsies were performed on all animals that died,as well as on challenged survivors that were sacrificedafter the 30-day observation period. Evidence of infec-tion by the challenge organism was determined bymicroscopic observation of tissues for the presence ofhyphae and by incubation of tissue specimens onpotato-dextrose agar at 37°C. Tissues examined atnecropsy were taken from the brain, heart, liver,spleen, kidneys, and lungs. Slides were prepared byplacing a piece of each organ in a drop of 10% KOHcombined with a drop of blue-black ink (Parker) andcrushing the piece under a cover slip.Fungus strains. The six strains of A. corymbifera

tested were provided by J. J. Ellis and came from theculture collection maintained by the Northern Re-gional Research Laboratories at Peoria, Ill. Strain 2981was isolated from soil and has been designated theneotype of the species by Ellis and Hesseltine (7).Strain 6249 was isolated from a calf with stomatitis,and strains 6248, 6250, 6251, and 6252 were clinicalisolates from human beings.

Mice. Animals used for this study were Swiss mice(Webster strain) maintained by the Department ofMicrobiology at the University of Iowa. At the begin-ning of each experiment, the mice were 5 to 6 weeksold and weighed 18 to 20 g. The animals were caged ingroups at least 1 week before the beginning of eachexperiment to allow them to acclimate to their newenvironment.Spore suspensions. Spore suspensions ofthe fungi

were obtained from colonies grown on Emerson YpSsagar (8) in crystalizing dishes (90 by 50 mm) at 250Cfor 14 days. Spores were harvested in sterile 0.15 MNaCl containing 0.1% Tween 80 by scraping the sur-face of each colony with a bent glass rod and filteringthe scrapings through a fine-mesh screen and cottongauze to remove hyphal fragments. The resulting sus-pension consisted primarily of sporangiospores and afew short pieces of hyphae which lacked protoplasm.

The spore suspension was centrifuged, and the sporepellet was washed three times in sterile 0.15 M NaClto remove residual medium.

Counts to determine the number of viable spores insuspensions prepared for injection were obtained asfollows. Three separate serial dilutions of the sporesuspensions were prepared, and each of the threedilution series-was plated in triplicate. Each samplewas pipetted into a sterile petri dish, to which meltedLittman agar (12) was added and allowed to solidify.Littman agar was employed to slow the radial spreadof fungus hyphae, allowing for the formation of dis-crete colonies. The agar plates were incubated at 400C.The stock spore suspensions were checked for bacte-rial contamination by plating a sample onto bloodagar. The remaining spore suspension was stored at40C until viability counts were available. The effect ofstorage at 40C for 24 h on spore germination was alsodetermined.Spore germination. Each strain ofA. corymbifera

was examined individually for spore germination bymicroscopic observation of the spores on the surfaceof Littman agar. Since it was not known whetherLittman agar had an effect on spore germination,identical studies were carried out on YpSs agar tocompare the percentages of spore germination on eachmedium. Spore suspensions were prepared as de-scribed above, and 0.1-ml volumes of saline containing104 spores (based on hemacytometer counts) werespread with a glass rod over the surface of petri dishes(60 by 15 mm) containing 9 ml of Littman or YpSsagar. A number of plates were prepared and incubatedat 400C. Initially, a pair of plates was removed at 2-hintervals, and growth was stopped by inverting thedishes over formaldehyde-soaked filter paper. Oncespore germination had begun, pairs of plates werechecked at 1-h intervals. A minimum of 250 randomspores were examined on each of two plates, and theresults were expressed as the percentage of sporesshowing the formation of a germ tube.

Steroid treatment. Cortisone acetate (Merck,Sharp & Dohme) was administered to the animals bysubcutaneous injection. A solution containing 10 mgof cortisone acetate per ml was prepared, and five 0.1-ml doses were administered to each animal. The sched-ule for cortisone treatment (13) involved administra-tion of the first dose 24 h before challenge, followedby four additional cortisone injections at 48-h inter-vals.

Virulence determination. Virulence of individualfungus strains in mice infected intravenously and in-traperitoneally was determined on the basis of LD50values over a 30-day observation period. LD5o valueswere calculated by the method of Spearman and Kar-ber (9). ET50 values (the time in days required for 50%of the animals to die from a given infectious dose)were used to compare the virulence of individualstrains of A. corymbifera administered intravenouslyin cortisone-treated and untreated mice. The ET50values were calculated by the method of Litchfield(11).

RESULTSThe LD50 values for intravenous and intraper-

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VIRULENCE OF A. CORYMBIFERA 397

itoneal experiments with A. corymbifera areshown in Tables 1 and 2. A range in virulencewas seen for the six- strains of A. corymbiferawith both the intravenous and intraperitonealroutes of challenge. In the intravenous series ofexperiments (Table 1), the LD50 values for un-treated mice ranged from 1.4 x 104 to 13.8 x 104spores, and for the cortisone-treated mice, theLD5o values ranged from 0.4 x 103 to 6.6 x 103spores. The differences in LD5o values betweencortisone-treated and untreated mice for eachstrain tested intravenously was significant at P=0.001.For the intraperitoneal series of experiments

with A. corymbifera (Table 2), LD6o values couldbe calculated only for the cortisone-treated mice,although a few deaths did occur among theuntreated animals after a challenge dose of 107spores. The LD5o values for cortisone-treated

TABLE 1. LD50 values for mice challengedintravenously with spores ofA. corymbiferaa

LD50 Sig-Fungus nifi-strain Untreated mice Cortisone-treated cance

mice (P)2981 1.4 x 104 2.8 x 103 0.001

(0.7 x 104-2.9 x 104)b (1.1 x 103-4.4 x 103)6248 10.7 x 104 4.3 x 103 0.001

(5.1 x 104-22.3 x 104) (2.0 x 103-9.1 x 103)6249 1.4 x 104 0.4 x 10' 0.001

(0.5 x 104-4.0 x 104) (0.2 x 103-1.0 x 103)6250 8.5 x 104 1.7 x 103 0.001

(4.0 x 104-18.2 x 104) (0.9 x 103-3.1 x 103)6251 13.8 x 104 4.4 x 103 0.001

(6.5 x 104-29.5 x 104) (2.3 x 103-8.3 x 103)6252 5.2 x 104 6.6 x 103 0.001

(2.6 x 105-10.5 x 104) (2.9 x 103-15.1 x 1(3)a Observation period, 30 days.b Ranges in parentheses represent 95% confidence intervals.

mice infected intraperitoneally ranged from 2.7X 105 to 4.9 x 10c spores.The difference in susceptibility between cor-

tisone-treated and untreated mice is shown notonly by a reduction in LD5o values, but also bya reduction in the survival time after a givenspore dosage, as determined by ET5o values.ET50 values for the intravenous series of experi-ments with A. corymbifera are shown in Table3. With five of the six strains tested at thechallenge dosage of 105 spores, the ET5o valuesfor the cortisone-treated animals were lowerthan those for untreated animals. At a challengedosage of 104 spores, only cortisone-treated miceshowed a 50% lethal response.

Results of the intranasal experiments areshown in Table 4. Lethal infections could beestablished with all six strains ofA. corymbifera

TABLE 2. LD5o values for mice challengedintraperitoneally with spores ofA. corymbiferaa

Fungus LDsostrain Untreated mice Cortisone-treated mice

2981 >8.3 x 106 2.7 x 105(0/6)6 (0.6 x 105-11.2 x 105)

6248 >10.9 x 10 8.5 x 105(1/6) (2.3 x 105-36.2 x 105)

6249 >9.0 x 10 2.9 x 105(2/6) (0.8 x 105-11.0 x 105)

6250 >10.3 x 106 10.2 x 105(0/6) (3.0 x 105-34.7 x 105)

6251 >10.3 x 106 >103.0 x 105(0/6) (2/6)

6252 >10.6 x 106 49.0 x 105(0/6) (14.5 x 105-166.0 x 105)

Observation period, 30 days.bFractions in parentheses represent number dead per total

number, at challenge dose of 10' spores.' Ranges in parentheses represent 95% confidence intervals.

TABLE 3. ET50 values for mice challenged intravenously with A. corymbiferaaET50 values at indicated spore dosages (days)

Fungus Cortisone-treated animals Untreated animalsstraIn

10( 104 1(9 1(2 105 104 1(9 1(2

2981 2.9 4.0 >30 >30 2.6 >30 >30 >30(2.7-3.1)b (3.1-5.2) (1/10)C (0/10) (1.9-3.6) (4/10) (0/10) (0/10)

6248 4.8 7.4 >30 >30 18.0 >30 >30 >30(4.7-4.9) (6.1-9.0) (1/10) (0/10) (10.6-30.6) (0/10) (0/10) (0/10)

6249 3.5 4.4 8.0 >30 6.7 >30 >30 >30(3.3-3.7) (3.8-5.1) (6.0-10.6) (1/10) (3.4-13.4) (4/10) (1/10) (0/10)

6250 4.7 6.2 >30 >30 13.5 >30 >30 >30(3.9-5.7) (5.1-7.6) (2/10) (0/10) (8.2-22.3) (0/10) (0/10) (0/10)

6251 4.4 7.5 >30 >30 11.0 >30 >30 >30(3.7-5.2) (6.2-9.1) (1/10) (0/10) (6.1-19.8) (0/10) (0/10) (0/10)

6252 5.1 9.3 >30 >30 7.6 >30 >30 >30(4.4-6.0) (6.8-12.7) (0/10) (0/10) (4.5-12.9) (0/10) (0/10) (0/10)

a Observation period, 30 days.bRanges in parentheses represent 95% confidence intervals.'Fractions in parentheses represent number dead per total number.

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administered by the intranasal route at a dosageof 106 spores, but only when the mice weretreated with cortisone. LD50 values were notdetermined for the intranasal route of challenge,since the uptake of spores by the animals couldnot be quantified. The spore suspension wasplaced in the nares of each mouse and, althougha number of the spores were inhaled, some couldhave been swallowed or forcibly discharged bythe animal at the time of inoculation.The use of blue-black ink-10% KOH crush

preparations of tissues provided a fast and sim-ple method for determining which organs con-tained invading hyphae. Figure 1 shows hyphae

TABLE 4. Intranasal challenge of mice with A.corymbifera sporesa

No. dead/total no.Fungus Spore dosagestrain (x 106) Untreated Cortisone

niice treatedmice

2981 0.98 x 106 0/10 3/106248 0.98 X 106 0/10 4/106249 0.95 X 106 0/10 1/106250 1.00 X 106 0/10 1/106251 1.02 X 106 0/10 4/106252 0.89 x 106 0/10 1/10

a Observation period, 30 days.

of A. corymbifera in kidney tissue. In the intra-venous series of experiments, hyphae usuallywere observed in the brains of untreated animalsthat died and sometimes also in the kidneys. Inthe cortisone-treated animals, hyphae were seenmost often in the brains and kidneys and occa-sionally in the hearts and lungs when the largerdosages of 104 and 105 spores were used forchallenge. Hyphae usually were found in thekidneys of animals that died from the 102- and103-spore dosages. In the intraperitoneal seriesof experiments, hyphae were seen in the kidneysof all the animals that died and sometimes inthe spleens of cortisone-treated animals. In thecortisone-treated animals that died from intra-nasal infections, hyphae were seen in the lungsand occasionally in the brains or kidneys. A.corymbifera could be subcultured from organsof dead animals in which hyphae were observed,as well as from organs in which hyphae were notseen.

After the 30-day observation period for eachexperiment, all surviving animals were sacri-ficed, and cultures were made of the organs. Inthe mice challenged intravenously or intraperi-toneally, A. corymbifera often could be re-covered from some organs, usually from thespleens and livers. Occasionally, unilateral kid-ney infections were seen in some of the surviving

FIG. 1. Hyphae of A. corymbifera in a 10% KOH-blue-black ink crush preparation of kidney tissue.Magnification, x500.

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mice, and the presence of A. corymbifera wasconfirmed by microscopic observation and byculture. In the intranasal series of experiments,46 cortisone-treated animals were necropsiedand 8 had positive lung cultures, 10 had positivebrain cultures, and 7 had positive brain and lungcultures both, whereas A. corymbifera was notisolated from 21 animals. Of the 60 untreatedmice necropsied, 23 had positive lung cultures,6 had positive brain cultures, and 5 had positivebrain and lung culture both, whereas A. corym-bifera was not isolated from 26 -animals. Allcontrol animals survived the 30-day observationperiod, and cultures made from their organswere negative for A. corymbifera.

In Table 5, the maximum spore germinationof each A. corymbifera isolate is shown, alongwith the number of hours of incubation at 400Cnecessary to achieve maximum germination onLittman and YpSs agars. Germination of sporesfrom the six isolates ofA. corymbifera was foundto be greater than 90% under all of the cultureconditions tested, and for most strains, germi-nation approached 100%. Therefore, the viablespore counts on spore suspensions prepared foranimal injection corresponded very closely tothe total number of spores injected into theanimals. Storage of the spore suspensions at 40Cfor 24 h did not appear to affect spore viability.

DISCUSSIONOur study has determined that a range ofLD50

values exists for the six isolates ofA. corymbiferatested and that several of the values obtainedfor untreated mice challenged intravenously aresimilar to the value reported by Smith and Jones(17) for mice of the same strain and age. Smith(16) also studied the in vivo development of A.ramosa spores administered intravenously tocortisone-treated and untreated mice. He ex-amined the livers, lungs, and kidneys of mice atvarious intervals after challenge with 106 spores

TABLE 5. Spore germination ofA. corymbiferaisolatesa

Fungus % Germination on agar:strain YpSs Littman Littman2981 95/7b 96/llb 91/llC6248 97/7 97/10 97/106249 98/6 97/8 97/96250 97/7 98/9 98/96251 97/6 98/8 97/86252 97/7 97/9 97/10

a Incubation temperature, 40°C.b Percent germination per number of hours.C Percent germination per number of hours after

storage for 24 h at 4°C.

VIRULENCE OF A. CORYMBIFERA 399

and found that only the kidneys of untreatedanimals were infected. In addition, he found thathyphae were present in the livers, lungs, andkidneys of cortisone-treated animals and thatspore germination was much slower in the liverthan in the lungs or the kidneys.The results of our study also demonstrate that

cortisone-treated animals experience hyphal in-vasion more widespread than that experiencedby untreated animals. At the larger dosages of10' and 105 spores administered intravenously,hyphae were observed in the brains and kidneysof untreated mice and the brains, kidneys, andoccasionally the hearts and lungs of cortisone-treated mice. Perhaps even more interesting wasthe high percentage of recovery of A. corymbi-fera from the organs of dead animals subcul-tured onto potato-dextrose agar that was incu-bated at 370C. Baker (2) has stated that theetiological agent is isolated from only about 16%of those patients suspected of having mucor-mycosis. One could speculate that many of theseunsuccessful attempts to isolate the fungus weremade with biopsy specimens taken from livingpatients. In our study, major organs were notremoved for subculturing until the animals haddied; yet, A. corymbifera was recovered fromnearly 100% of the animals, including those ani-mals in which tissue autolysis had begun tooccur. One possible explanation for our successin isolating the etiological agent may be that thecultures were incubated at 370C. Since growthof A. corymbifera occurs at a much faster rateat 370C than at room temperature, the funguswas able to grow out of the organs and colonizethe plate even when bacterial contamination waspresent.

It was of interest also that A. corymbiferacould be isolated, in some instances, from organssuch as the spleen and liver, in which hyphaewere not observed. This was true regardless ofwhether the animals died from infection or weresacrificed and necropsied after the 30-day obser-vation period. Smith and Jones (17) reportedthat although A. ramosa spores are eliminatedfrom the lungs of the host by 4 days after intra-venous challenge, no such elimination appearsto occur in the spleen or liver. Corbel and Eades(6) found that the liver and spleen contain sub-stances which appear to inhibit germination ofA. corymbifera spores in vitro. It may be thatthese inhibitory substances prevent germinationbut allow viable spores to persist up to a monthor more in these two organs.The ET5o values for the intravenous series of

experiments reveal that a critical dose size isnecessary to produce a 50% lethal response in agroup of mice. The spore dosage must be large

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enough to establish an acute infection in theanimals and produce death in a relatively shortperiod of time, or death usually will not occur.Chronic infections were sometimes seen in thesurviving challenged animals, and these usuallywere characterized by a unilateral kidney infec-tion. At necropsy after the 30-day observationperiod, A. corymbifera hyphae could be ob-served, and the fungus was subcultured from theunilaterally infected kidneys.

Intranasal infections were successfully estab-lished with all six isolates of A. corymbifera, afinding which reflects what is thought to be thenatural route of infection. Among the six isolatestested in this study, isolate 2981, the same isolatewith which Reinhardt et al. (15) failed to estab-lish intranasal infections in alioxan-diabetic rab-bits, produced three deaths in a group of 10cortisone-treated mice. Reinhardt et al. basedtheir findings on a challenge of only three rab-bits. Because of the natural variation in resist-ance among animals, Reinhardt et al. might havebeen able to establish infections if a larger testgroup had been utilized.

Results of this study demonstrate that a rangeof virulence exists for the six strains of A. cor-ymbifera tested and that virulence can be accu-rately quantitated in terms of LD50 values. Re-alization that such quantitation can be accom-plished should allow for more accurate design offuture experiments to study mucormycosis.

LITERATURE CITED1. Ajello, L., D. F. Dean, and R. S. Irwin. 1976. The

zygomycete Saksenaea vasiformis as a pathogen ofhumans with a critical review of the etiology of zygo-mycosis. Mycologia 68:52-62.

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2. Baker, R. D. 1975. Opportunistic fungal infections, p.204-214. Charles C Thomas, Publishers, Springfield, Ill.

3. Corbel, M. J., and S. M. Eades. 1975. Factors determin-ing the susceptibility of mice to experimental phyco-mycosis. J. Med. Microbiol. 8:551-564.

4. Corbel, M. J., and S. M. Eades. 1976. Experimentalphycomycosis in mice; examination of acquired immu-nity in resistance to Absidia ramosa. J. Hyg. 77:221-233.

5. Corbel, M. J., and S. M. Eades. 1976. The relativesusceptibility of New Zealand Black and CBA mice toinfection with opportunistic fungal pathogens. Sabour-audia 14:17-32.

6. Corbel, M. J., and S. M. Eades. 1978. Observations onthe localization of Absidia corymbifera in vivo. Sa-bouraudia 16:125-132.

7. Ellis, J. J., and C. W. Hesseltine. 1966. Species ofAbsidia with ovoid sporangiospores. II. Sabouraudia 5:59-77.

8. Emerson, R. 1941. An experimental study of the lifecycles and taxonomy of Allomyces. Lloydia 4:77-144.

9. Finney, D. J. 1964. Statistical methods in biological assay,p. 524-553, 2nd ed. Hafner Publishing Co., New York.

10. Kitz, D. J., R. W. Embree, and J. Cazin, Jr. 1980.Radiomyces, a genus in the Mucorales pathogenic formice. Sabouraudia 18:115-121.

11. Litchfield, J. T. 1949. A method for rapid graphic solutionof time-percent effect curves. J. Pharmacol. Exp. Ther.97:399-408.

12. Littman, M. L. 1947. A culture medium for the primaryisolation of fungi. Science 106:109-111.

13. Lupan, D. M., and J. Cazin, Jr. 1973. Pathogenicity ofAllescheria boydii for mice. Infect. Immun. 8:743-751.

14. Nottebrock, H., H. J. Scholer, and M. Wall. 1974.Taxonomy and identification of mucormycosis-causingfungi. I. Synonymity of Absidia ramosa with A. cor-ymbifera. Sabouraudia 12:64-74.

15. Reinhardt, D. J., W. Kaplan, and L. Ajello. 1970.Experimental cerebral zygomycosis in alloxan-diabeticrabbits. Infect. Immun. 2:404-413.

16. Smith, J. M. B. 1976. In vivo development of spores ofAbsidia ramosa. Sabouraudia 14:11-15.

17. Smith, J. M. B., and R. H. Jones. 1973. Localization andfate of Absidia ramosa spores after intravenous inoc-ulation of mice. J. Comp. Pathol. 83:49-55.

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