dematiaceous fungal pathogens isolated from nature

Mycopathologia vol. 70, 3: 153-161, 1980

DEMATIACEOUS FUNGAL PATHOGENS ISOLATED FROM NATURE

D.M. DIXON 1, H.J. SHADOMY 2 & S. SHADOMY 2

1 Biology Department, Loyola College, Baltimore, MD 21210, USA 2 Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

23298, USA

Abstract

This study was conducted to demonstrate the presence of pathogenic dematiaceous fungi in nature. Using hamster and mouse inoculation techniques, 43 isolates ofdematiace- ous fungi were recovered from 39 samples of woody plant material and soil from the Virginia environment. Thirteen species were identified and included 4 Phialophora spp., 3 Cladosporium spp., 2 Exophiala spp., Sporothrix sp., Wangiella dermatitidis, Bispora betulina, and Scytalidium lignicola. Evidence is presented for the first isolations of C. trichoides from nature in the United States; these isolates proved to be pathogenic for mice in which they produced disease and death in a course similar to that seen in man. Natural isolates of Phialophora verrueosa, Phialophora repens, Exophiala jeanselmei, and Wangiella dermat#idis were identical to those species isolated from man using the following criteria: morphology, 12 % gelatin reaction, and survival in laboratory animals.

Introduction

Knowledge of the distribution of zoopathogenic dematiaceous fungi in nature is incomplete. Further, few reports exist regarding the isolation of these etiological agents from nature. Most reports have dealt with the natural isolation of Phialophora verrucosa which is known to occur on wood pulp (28). This fungus has been isolated from wood of a barn (10), a Finnish sauna (26), from plant debris (16), and from soil (19). Isolation of P. pedrosoi from nature has been reported on five occasions. The fungus was recovered from plant debris, soil, and air (5). Only one

Reprint Requests: Dr. D. M. Dixon, Biology Department, Loyola College, Baltimore, MD 21210.

reported isolation of Wangiella dermatitidis from natural sources can be found in the medical literature (5). Clado- sporium carrionii was isolated from wooden fence posts in Australia by Ridley (24) while C. trichoides has been reported to occur in Panamanian soil (19). We have recently reported the isolation of C. trichoides from the bark of Juniperus virginiana in Suffolk, Virginia (7).

Knowledge of the ecology and classification of the pathogenic dematiaceous fungi is essential to the under- standing of their role in medical mycology. Discovery of the sources of human pathogenic fungi in nature provides information useful in diagnosing fungal infections. De- monstrating the presence of pathogenic fungi in a given geographical area can alert the physician to consider these fungi in his differential diagnoses. Pathogenic dematiaceous fungi are generally believed to be limited to tropical and subtropical areas (10). To date, no systematic search has been conducted for these organisms from natural source in the United States. Therefore, the purpose of this study was to demonstrate the presence of these fungi in nature in this country.

Materials and methods

Collection and treatment of samples; Suffolk, Virginia

Twenty samples of soil and woody material (Table 1) were collected from rural Suffolk, Virginia on April 15, 1976. Utilizing a hunting knife which was rinsed with sterile water after each use, approximately 25 ml sample volumes were placed in 100 ml glass bottles containing a small crystal of paradichlorobenzene (for arthropod control), and returned tO the laboratory for processing on the same day. The procedure used was a modification of the mineral oil flotation technique of Smith and Furcolow (27). The samples were incubated for 1 hour at 25 ~ in 50 ml

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Table 1. Field Collection Data and Sample Material from Suffolk, Va.

Site No. Site Sample

H-1 Stump of Juniperus virginiana Bark H-2 Crayfish burrow Soil F- 1 Tool shed wall Wood F-2 Tool shed foundation Wood F-3 Chicken coop roof Wood F-5 Ground adjacent to F-3 Soil F-6 Dog pen Soil F-7 Ground adjacent to F-3 Soil F-9 Woodpile Wood F- 10 Lawn furniture Wood F- 11 Tree trunk Bark F- 12 Lawn furniture Wood F- 13 House wall Wood F-14 Ground beneath oil tank Soil F- 15 Freezer support Wood F- 17 Trunk of Juniper~s virginiana Bark F-18 Base of F-17 Bark

& Soil F- 19 Tree trunk Bark F-20 Dog pen Wood

sterile water containing.50/~g polymyxin B, 5,000 units penicillin, and 1,000 #g streptomycin per ml. Ten ml volumes of sterile mineral oil (E.R. Squibb) were added to each bottle which were then mixed by vigorous shaking for 5 minutes. Materials which gathered at the oil-water

interphase were then removed with 1 ml pipettes and

stored at 4 ~ overnight. Male golden Syrian hamsters were inoculated with the

material from the oil-water interphase (1 animal per sample; 0.5 ml intraperitoneally and 0.2 ml intratesticular- ly) using tuberculin syringes With 18 gauge needles. Ani- mals were killed 7 weeks post inoculation, and lungs, livers, spleens, and testes removed and homogenized separately in sterile plastic bags (Whirlpak, A.H. Thomas) containing 5 ml sterile 0.85 ~o saline. One-half ml portions of homogenate were cultured in duplicate on Mycosel (BBL) and Sabhi(Difco) agar, each supplemented with 250/zg per ml chloramphenicol. Plates were incubated at

room temperature for 4 weeks. Fungal growth was observed and subcultured (at monthly intervals) onto the same type of medium for further study. When multiple colonies displaying identical morphology were isolated from a single animal, only representative colonies were maintained.

Collection and Treatmentof Samples ; Williamsburg, Virginia

Nineteen samples of woody material (Table 2) were

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collected from Williamsburg, Virginia on September 15, 1977. Samples were collected and processed as described above except that the volume of mineral oil added was reduced from 10 to 5 ml. Animals (1 mouse and 1 golden Syrian hamster per sample) were inoculated and cultured

as before except that the incubation period was 4-5 weeks and mouse organ homogenates were prepared using 1 ml saline, with 0.2 ml portions per plate cultured using Mycobiotic (Difco) agar and Sabhi agar without the addition of chloramphenicol. The resulting fungal growth was treated as before.

Identification of Fungi

Identifications were based primarily upon colonial mor-

phologies exhibited on isolation media and upon microscopic study ofconidial ontogeny as described by Hughes (17).

Additional criteria were considered in the taxonomy of

Cladosporium and other dematiaceous species. Tem- perature tolerance was assessed by presence or absence of growth on Sabouraud (Difco) agar for 4 weeks with incuba-

tion at 43 ~ The ability to liquify 12 ~o gelatin (Difco) was determined with the method described by Ajello et aI. (1).

In Vivo and in Vitro experiments with Cladosporium spp.

The Fungus Collection, Division of Infectious Diseases,

Table 2. Field Collection Data and Sample Material from Williamsburg, Va.

Site No. Site Sample

WMBG-I Mulched border beneath Pinus sp. Sawdust WMBG-2 Mulched border beneath Pinus sp. Sawdust &

pine cone WMBG-3 Mulched border beneath Pinus sp. Sawdust WMBG-4 Mulched border beneath Pinus sp. Sawdust WMBG-5 Trunk of Lagerstromia indica Bark WMBG-6 Border beneath WMBG-5 Sawdust WMBG-8 Sawdust storage pile Sawdust WMBG-9 Lumber in WMBG-7 Wood WMBG-10 Lumber in WMBG-7 Rotten wood WMBG-11 Lumber in WMBG-7 Rotten wood WMBG- 12 Base of Pinus sp. in WMBG-7 Bark WMBG- 13 Base of Pinus sp. in WMBG-7 Bark WMBG-14 Trunk ofJuniperus virginiana Bark WMBG-15 Trunk of Pinussp. Bark WMBG-16 Tree stump Wood WMBG-17 Tree stump Wood WMBG-18 Trunk of Cornusflorida Bark WMBG-19 Trunk of Quercus sp. Bark WMBG-20 Beneath Pinus sp. Pine cone

Medical College of Virginia, Virginia Commonwealth

University (MCV Fungus Collection) was the source of the clinical isolates and the control organisms. Inocula were prepared by growing colonies on Mycobiotic agar plates for 6-12 days at room temperature and then flooding the colonies with 4 ml sterile 0.85 % saline while gently scraping them with a pipete tip. The resulting suspensions were allowed to stand until the large pieces settled out (2-3 minutes). The supernatants were used as inocula. This method was found to be reproducible. Hemacyto- meter counts of 1 • 106 units per ml were consistently obtained with 6 isolates of Cladosporium spp. These corresponded to viable counts of 1 • 10 s to 1 x 109 colony forming units per ml as determined with 4 isolates of

Cladosporium spp. following serial dilutions and culturing on Mycobiofic agar.

Mice received 0.5 ml inocula of freshly harvested

suspensions intravenously (lateral tail vein) and were kept for up to 4 weeks. Brains of the first 4 mice infected with C. trichoides (MCV 53.20, Table 7) were removed for both cultural and histopathological studies. One-half of each brain was fixed in 10 % neutral buffered formalin and sent

to the Department of Academic Pathology, Medical College of Virginia, Virginia Commonwealth University for the following histopathological preparations: hema- toxylin and eosin (H & E), periodic acid-Schiff (PAS),

and methenamine silver. The remaining portions of the

brains of these animals and the lungs, livers, spleens, and

testes of the other animals (Table 7) were cultured as described above. The organ homogenates (0.05 ml in duplicate) were digested with 10 % KOH and examined

microscopically for fungi.

Animals

Adult male mice (Mus musculis, white Swiss, DUB/ICR) were obtained from Flow Laboratories, Dublin, Virginia. Adult male hamsters (Mesocricetus auratus, golden Syrian) were obtained from Charles River Breeding Laboratories, Wilmington, Massachusetts. All animals were housed separately and provided Purina Laboratory Chow and water ad libitum.

Results

Fungi recovered from Suffolk, Virginia

Results of cultures isolated from hamsters inoculated with processed samples from Suffolk, Virginia are shown in Table 3. Twelve of the 20 samples were positive for fungi by this technique and 4 samples were positive for 2 dif-

ferent organisms. There was little correlation noted

Table 3. Fungi Recovered from Suffolk, Va.

Organism S i tes Cultures Positive b Medium Positiv&

Testes Spleen Liver Mycosel Sabhi

Exophialajeanselmei F-I 1 4 2 2 F-12 3 2 1 F-14 4 2 2 F-15 4 2 2

Phialophora verrucosa F-2 1 1 F-8 4 4 4 4 F-12 2 2

Phialophora repens F-7 4 2 2 Phialophora hoffrnannii F- 14 1 1 Sporothrix sp. F-8 1 1

F-9 1 1 Cladosporium trichoides H- 1 2 1 2 1

N V c F-5 1 1 Porosporae F- 11 2 2 Lost to Aspergillus sp. overgrowth F-19 1 1 N V F-20 1 1

a Suffolk, Virginia site numbers. See Table 1 for description of site and collection material. b Number of isolates from plating in duplicate on Mycosel and Sabhi agar.

Cultures became nonviable and could not be identified.

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between the type of material sampled, the source of the

sample (natural wood from living or dead trees, or struc-

tural wood from dwelling or lawn furniture) and the fungi

recovered. Sixteen fungal isolates were recovered from 20

samples. Six species were identified; these included 4

isolates of Exophiala jeanselmei, 3 of Phialophora verrucosa, 2 of Sporothrix sp., and 1 each of P. repens, P. hoffmannii, and Cladosporium trichoides. Five isolates

remained unidentified; these included 1 member of the

Porosporae, 1 isolate lost to contamination, and 2 or-

ganisms which died before being identified.

Homogenates of hamster testes were culturally positive

for fungi more often than homogenates of either spleen or

liver. No fungi were recovered from lungs while only

twice did testes fail to yield positive cultures of organisms

which were grown from liver or spleen. This occurred

when a Sporothrix species was grown from liver and spleen

of the animals injected with samples from F-8 and F-9.

Mycosel agar yielded positive cultures more often than

did Sabhi agar. Two isolates grew on Sabhi agar that

failed to grow on Mycosel agar; conversely, 5 isolates grew

on Mycosel agar which failed to grow on Sabhi agar. More

than 1 organism was isolated per plate on two occasions.

In general, multiple colonies of a given organism were

obtained per plate.

Fungi recovered from Williamsburg, Viryinia

Fungi were recovered from 9 to 18 sites from Williams-

burg, Virginia using the hamster inoculation technique

(Table 4). Ten different organisms were found, all but 2 of

which were identified. Six genera were represented; these

included Cladosporium, Exophiala, Phialophora, Rhino- cladieIla, Scytalidium, and Wangiella. Cladosporium spe-

cies A (see Table 6) was a commonly encountered isolate

from 8 sites. The isolate of C. triehoides was subjected to

the same identification criteria as the isolate from Suffolk,

Virginia (7). It was found to be gelatin negative, to grow

well at 43 ~ and was recovered from the brains of experi-

mentally infected mice.

Table 4. Fungi Recovered from Williamsburg, Va. Using a Hamster Inoculation Technique.

Organisms Recovered Sites Cultures Positive b Medium Positive"

Testes Spleen Lung Myco- Sabhi biotic

Cladosporium sp. A. ~ WMBG-1 1 1 WMBG-4 1 1 WMBG-6 1 1 1 1 WMBG-9 1 1 WMBG-10 1 1 WMBG-11 1 1 WMBG-13 1 d 1

Cladosporium sp. B WMBG-13 1 1 C. triehoides WMBG-10 1 4 2 3 Exophilia spinifera WMBG-13 1 d 1 Phialophora verrucosa WMBG- 11 3 1 2

WMBG-13 1 d 1 Rhinoeladiella ? WMBG-17 2 1 1 Seytalidium lionicola WMBG-6 1 1 Wanoiella dermatitidis WMBG-1 1 1

WMBG-4 1 1 W-41 e WMBG-10 4 2 2 W-50 WMBG-19 1 1

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a Williamsburg, Virginia site numbers. See Table 2 for description of site and collection material.

Number of isolates from plating in duplicate on Mycobiotic and Sabhi agar. c See Table 6 for explanation of Cladosporium sp. A and sp. B. d Fungi recovered from enlarged, necrotic testicle. e Williamsburg, Virginia. Fungus Collection accession numbers; unidentified isolate.

Hamster organs most frequently postive for fungi were ;estes and spleen. No fungi were recovered from liver. Mycobiotic agar and Sabhi agar produced 18 and l l ~ositive cultures, respectively. Only in 3 instances did Lsolates grow on Sabhi agar that did not grow on Mycobio- fic agar; 12 isolates grew exclusively on Mycobiotic agar.

A mass developed in the scrotum of the hamster injected with material from site WMBG-13 which was approximately twice the size of the normal appearing testicles. The pathologist who viewed the slides identified the material as a third necrotic testicle (H.I. Lurie, personal communication). No fungi were seen in H & E stained and unstained sections of the mass. Cladosporium species B (Table 6), E. spinifera, and P. verrucosa were cultured from the mass. The remaining hamsters appeared normal at autopsy, and uninoculated control hamsters were culturally negative for fungi.

Fungi were recovered from 9 of 19 samples from Wi- liamsburg, Virginia using the mouse inoculation technique (Table 5). These included Bispora betulina (3 isolates); Cladosporium species B (2 isolates); Phialophora sp. (1 isolate ); W. dermatitidis (1 isolate); and 2 unidentified isolates, W-19 and W-27. Fungi were recovered from testes, spleen, and liver, but not lungs. More isolates were obtained on Sabhi agar than Mycobiotic agar. Mice appeared normal at autopsy and uninoculated control mice were culturally negative for fungi.

Identification of Cladsporium spp. in Vitro and in Vivo

Cladosporium species isolated from Williamsburg, Virginia

are listed by animal inoculation in Table 6. Three species of Cladosporium were found including C. trichoides (1 isolate) and 2 unidentified species: species A (10 isolates) and species B (1 isolate).

In order to investigate the pathogenicity and neurotropism of C. trichoMes MCV 53.20 (Suffolk, Virginia field isolate), 4 mice were injected with saline suspensions as described in Materials and Methods. Results of this first in vivo experiment are shown in Table 7. From days 11-14 post injection, all 4 mice developed ruffled coats, shallow breathing and muscular paralysis. Two mice died on day 15. Two additional mice were found to be moribund on days 11 and 16, respectively and were killed at that time.

Small masses of dematiaceous hyphae could be seen upon gross inspection of the brains of the above mice. All brains showed abundant, dematiaceous, septate, branching hyphae upon digestion with 10 ~ KOH and microscopic examination. Histopathological sections of portions of all 4 brains revealed the presence of large amounts of hyphae. Hyphae were most easily located when stained with methenamine silver. The hyphae were PAS positive, olivaceou~ green on H & E, and appeared as septate, branching filaments distributed throughout the brain. The pathologist who viewed the slides reported meningitis with isolated microabcess formation (H.I. Lurie, personal communication). The details of the in vivo and in vitro morphology of this isolate have been published previously (7).

In a second in vivo experiment, 2 mice each were inoculated as described above with C. trichoides MCV 53.12

Table 5. Fungi Recovered from Williamsburg, Va. Using a Mouse Inoculation Technique.

Organism Sites Cultures Positive b Medium Positive a

Testes Spleen Liver Myco- Sabhi biotic

Bispora betulina WMBG-3 1 1 WMBG-5 1 1 WMBG-6 1 1

Cladosporium sp. A c WMBG-4 1 1 WMBG- 11 1 1

Phialophora sp. WMBG-26 2 2 Wangiella dermatitidis WMBG-9 1 1 W-19 d lost to Asperoillus WMBG-I 1 1 W-27 WMBG-17 1 1

Williamsburg, Virginia site numbers. See Table 2 for description of site and collection material. b Number of isolates from plating in duplicate on Mycobiotic and Sabhi agar.

See Table 6 for explanation of Cladosporium sp. A. d Williamsburg, Virginia. Fungus Collection accession numbers; unidentified isolate.

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Table 6. Cladosporium Species Isolated from Williamsburg, Va.

Fungus (No. isolates) Sites Positive a Isolation Technique

Cladosporium trichoides b (1) WMBG-10 Hamster inoculation Cladosporium species A c (14) WMBG-8; WMBG-10; WMBG-11; Direct plating

WMBG-14 WMBG-4; WMBG-11 Mouse inoculation WMBG- 1 ; WMBG-4; WMBG-6; Hamster inoculation WMBG-9; WMBG-10; WMBG-11 WMBG-13

Cladosporium species B d (1) WMBG-13 Hamster inoculation

" Williamsburg, Virginia site numbers. See Table 2 for description of site and collection material. b Cladosporium triehoides. Colonies on malt extract agar (after 19 days, 30 ~ 34- cm in diam, rounded, flat, powdery, velvety. Colonies grey-green with a black advancing border (0.2 cm) formed by tightly appressed, immersed hyphae. Colony reverse dark black, advancing border light green. Conidia smooth, 0-septate, 4.4-8.8(6.6) • 2.2-2.7(3.3) # and borne in long, flexuous chains. c Cladosporium species A. Colonies on malt extract agar (after 19 days, 30 ~ 3-4 cm in diam, velvety. Advancing border (0.5 cm) formed by radiating strands of immersed hyphae. Colonies dark olive green with greenish-black reverse. Conidia smooth, 0-septate, 1.64.4(3.0) x 2.8-7.2(4.8) ~ and borne in short chains of 4-5 conidia. d Cladosporium species B. Colonies on malt extract agar (after 19 days, 30 ~ ovoid (2 x 4 cm in diam), flat smooth, and dull black. Colony reverse black. Conidia echinulate, 0-septate, 7.8-15.6(10.4) • 7.8-23.4(13.8) it, solitary or in chains of 2-3 conidia.

Table 7. In Vivo Studies with Cladosporium Species.

Cultural Recovery of Fungus Symptoms, Day of Organs Organism; (MCV Fungus Mouse No. Positive/No. Cultured

Collection No. Day of Death (D), Positive Number)" Onset Kill (K) Brain Lungs Liver Spleen Direct KOH

Cladosporium trichoides (MCV 53-20) 1 11 11 (K) 2/2 b b b Brain

2 13 15 (D) 2/2 b b b Brain 3 14 15 (D) 2/2 b b b Brain

1st Experiment 4 14 15 (K) 2/2 b b b Brain 1 14 16 (D) 4/4 b b b Brain

2nd Experiment 2 25 26 (K) 4/4 b b b Brain 3rd Experiment 1 28 28 (K) 3/3 0/4 4/4 4/4 Brain

Cladosporium trichoides (MCV 53-12) 1 ,15 25 (D) 2/2 0/2 2/2 1/2 Brain 2nd Experiment 12 13 15 (K) 2/2 2/2 2/2 2/2 Brain

Cladosporium sp. (MCV 53.13) 1 - 25 (K) b 1/2 0/2 0/2 - 2nd Experiment 2 15 (K) 2/2 2/2 2/2 2/2 -

1 - 2 6 ( K ) 0/4 1/4 4/4 4/4 -

3 r d Experiment 2 - 26 (K) 0/4 0/4 1/4 4/4 -

a MCV 53-20 Suffolk, Virginia field isolate. MCV 53-12 Human brain abscess isolate. MCV 53-13 National Center for Disease Control - gelatin positive saprophyte.

b Not done.

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(isolated from a human brain abcess) and Cladosporium

sp. MCV 53.13 (Center for Disease Control's gelatin positive saprophyte). Results of the experiment are shown in Table 7. Both groups of mice inoculated with the isolates of C. trichoides reacted similarly. One mouse from each of the 2 groups died. Brains from all 4 mice were positive for the fungus by microscopic examination and by culture. Positive cultures from homogenized lung, liver, and spleen tissues were obtained in both groups. No fungi were seen in these tissues upon microscopic examination. Neither animal injected with Cladosporium sp. (MCV 53.13) showed any of the symptoms of cerebral cladosporiosis. Cladosporium sp. was recovered from cultures of the brain, lungs, liver, and spleen of mouse # 2 and the lungs of mouse # 1 (Table 7). It is important to note that fungi were not seen in the brain tissue upon microscopic examination.

Evidence of the neurotropism of C. trichoides (MCV 53.20) was demonstrated in a third experiment by microscopic examination and cultural recovery of the fungus from the brain of a mouse which had been injected 28 days previously (Table 7). Brains of 2 control mice injected with Cladosporium sp. (MCV 53.13) were found to be negative for fungi upon culture and microscopic examination although the fungus was cultured from lungs, liver, and spleen in both animals.

Isolates of Cladosporium sp. A (W-29, W-30), were selected for in vivo study. Mice were inoculated with saline suspensions of the isolates (1 mouse per isolate) as described in Materials and Methods. Animals appeared normal 58 days post-injection when they were killed, and lungs, livers, spleens, and brains cultured as described for the previous in vivo experiments with Cladosporium. The fungus was recovered only from the lungs of the 2 animals. All organs were negative for fungi by microscopic examination.

Discussion

Results of the Suffolk and Williamsburg, Virginia field trips indicate that pathogenic dematiaceous fungi are common in the environment in this region. It is puzzling that similar reports are so infrequent in the literature. Many of the epidemiological studies of the systemic mycoses have involved sampling the environment of Virginia and surrounding area for pathogenic fungi, yet no dematiaceous pathogens have been reported (8, 9, 11, 12). Factors involved may be the isolation techniques used, the nature of the sample material, and difficulty in recognizing

and identifying dematiaceous fungi. Comparison of ma- thods for isolation and identification of dematiaceous fungi from nature has been reported separately. (Compari- son of methods for isolation of pathogenic dematiaceous fungi from nature, Dixon, D.M. and H.J. Shadomy, companion submission to Mycopathologia. ).

Wood rarely has been considered as a source for zoopathogenic fungi in nature. Dixon et al. (6) demonstrated the ability of Blastomyces dermatitidis, a systemic fungal pathogen of man, to grow and sporulate in vitro on various types of woody plant material; as a result, it was suggested that such materials be considered as sources for the isolation of other zoopathogenic fungi from nature. Accordingly, woody plant material proved to be a good sample source for pathogenic dematiaceous fungi in this study. Most of the dematiaceous fungal pathogens reported from nature have been recovered from similar materials (5, 24, 26). Gezuele et al. (16) found wood to be a better sample source than soil for isolation of P. verrucosa and P. pedrosoi in Uruguay. The 2 isolations of C. trichoides

from the Virginia environment were from woody samples. The only other reported isolation of this fungus from nature is that of Klite et al. (19) where the sample material was poorly described.

Most of the cultures recovered from hamsters were grown from the testes. The 18 gauge needle used for intratesticular inoculation may have caused enough tissue trauma to result in more favorable growth conditions for fungi. The lower temperature of the testicles in relation to the body temperature (39 ~ may also be important. Gezuele et al. (16) found that natural isolates of P. verru-

cosa and P. pedrosoi could grow at 38 ~ but not at 39 ~ This temperature difference would not be expected to be as important in mice inoculated with these same fungi by various routes, because the mouse body temperature is 37.4 ~

Both isolates of C. trichoides from nature displayed animal pathogenicity with neurotropism comparable to the isolate from a human brain abcess. The times of death for mice inoculated with these isolates corresponded to those reported by other workers (3, 4). Although the most common form of cerebral cladosporiosis in humans is a localized brain abcess, diffuse forms of the disease may occur where the pathology is that of an inflammatory response involving the meninges (3). Mice in the present study and in the study of Binford et al. (4) demonstrated the diffuse meningeal form of the disease when injected intravenously with C. trichoides. Several naturally acquired infections reported in cats have also presented as meningi-

159

tis (18). The paralysis noted in the mice inoculated with C. trichoides in this study has been noted by others in mice (4) and in humans (21) infected with this fungus. It should be noted that both hamsters from which the 2 isolates of C. trichoides were recovered appeared grossly

normal: at autopsy. None of the mice inoculated with saprophytic Clados-

porium sp. (MCV 53.13) exhibited any of the symptoms of cerebrabcladosporiosis, and no fungi could be seen upon microscopic examination of brain tissue. The fungus was, however, cultured from homogenates of brain, lung, and liver!tissue on several occasions. Emmons and Carrion (14) produced lesions in the brains of rats following inoculation with saprophytic Hormodendrum sp. and took this as evidence of similarity of the saprophytic to the parasitic species. Recently, Cladosporium cladosporiodes has been reported as the etiological agent of a fungus ball in a human (20). These and other Cladosporium species are ubiquitous in nature where they are saprophytes on vegetation. It is likely that more such cases of human infection will be ascribed to these 'saprophytic' species in

the future. Fourteen isolates of Cladosporium sp. isolated from

Williamsburg, Virginia were designated as species A. This species microscopically resembled the organisms recovered from nature by Gezuele et al. (16) and identified as Phialophora pedrosoi var. cladosporiodes. However, no phialides were seen in any of the 14 isolates, so they cannot yet be placed in the genus Phialophora.

Absence of proteolytic activity in vitro has been corre- lated with pathogenicity of the dematiaceous fungi (1, 15, 22, 25). Fuentes and Bosch (15) found that 12 ~ gelatin, inspissated serum, and milk could be used to determine in vitro proteolytic activity and that all the saprophytic species tested exhibited this activity while none of the pathogens did so. Pathogenic species of P. verrucosa recovered from nature gave the same results as those isolated from disease in man (15). Only one isolate in the present study was found to be gelatin positive (Phialophora sp. Table 5).

Many of the dematiaceous fungi recovered from nature in this study have been referred to as pathogenic. In the truest sense of the word pathogenicity means the ability to cause disease. When the species of dematiaceous fungi described here are isolated from disease processes, there is less difficulty with use of the term. However, with isolation of these species from nature, additional care must be taken before classifying the organisms as pathogens. The most conclusive test of human pathogeni-

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city is to demonstrate the ability of these organisms to cause disease in man. Although this has been done with natural isolates of C. carrioii, it is neither the most desirable nor the safest means of confirming pathogenicity (24). For the systemic fungal pathogens recovered from nature, laboratory animals are used to fulfill Koch's postulates (13). This is not always possible when dealing with the pathogenic dematiaceous fungi (with the notable exception of C. trichoides) because these organisms do not ordina- rily produce overt disease in laboratory animals (23).

With these points in mind, other factors must be considered when classifying natural isolates of the dematiaceous fungi as pathogens. The primary criteria of pathogenic potential used in this study were morphological identity of the natural isolates with those of the same species known to cause disease in man, inability to liquefy 12 ~ gelatin, and the ability of the organism to survive passage in laboratory animals. Recovery of viable fungi from homogenized tissue of hamsters and mice 6-7 weeks post inoculation is considered especially significant because it demonstrates the latent pathogenic potential of these fungi as defined by Ajello et al. (2). For this reason, the isolates of S. lignicola and B. betulina recovered from animals are also of interest. Because of these results, clinical isolates of these species or of any dematiaceous fungi must be carefully considered as possible etiological agents rather than as simple laboratory contaminants.

Acknowledgements

The senior author was supported by NIH Training Grant A1 00382. Thanks are due to Dr. H.I. Lurie for examining histopathological specimens, to Drs. T. Kerkering and M. Tipple for assistance in collecting and processing the samples of wood and soil and to Margaret Daley for typing the manuscript. Special thanks are extei~ded to Dr. M.R. McGinnis for his many helpful discussions and for his identifications of selected isolates of E. jeanselmei, P. hoffmannii and P. repens.

References

1. Ajello, L., L.K. Georg, W. Kaplan, & L. Kaufman. 1966. Laboratory manual for medical mycology. U.S. Govern- ment Printing Office, Washington, D.C., p. F-21.

2. Ajello, L., LK. Georg, R.T. Steigbigel, & C.J.K. Wang.

1974. A case of phaeohyphomycosis caused by a new species of Phialophora. Mycologia 66: 490-498.

3. Bennett, J.E., H. Bonner, A.E. Jennings, & R.I. Lopez. 1973. Chronic meningitis caused by Cladosporium trichoides. Amer. J. Clin. Pathol. 59: 398-407.

4. Binford, C.H., R.K. Thompson, M.E. Gorham, & C.W.E. Emmons. 1952. Mycotic brain abcess due to Cladosporium trichoides, a new species. Amer. J. Clin. Pathol. 22: 535-542.

5. Brygoo, E.R. & P. Destombes. 1976. Epidemiologie de la chromoblastomycose humaine. Bull. Inst. Pasteur 74: 219: 243.

6. Dixon, D.M., H.J. Shadomy, & S. Shadomy. 1977. In vitro growth and sporulation of Blastomyces dermatitidis on woody plant material. Mycologia 69:1193-1195.

7. Dixon, D.M., H.J. Shadomy, & S. Shadomy. 1977. Isolation of Cladosporium trichoides from nature. Mycopathologia 62: 125-127.

8. Emmons, C.W. 1949. Isolation of Histoplasma capsulatum from soil. Pub. Hlth. Rep. 64: 892-896.

9. Emmons, C.W. 1951. Isolation of Cryptococcus neoformans from soil. J. Bact. 62: 685-690.

10. Emmons, C.W. 1954. The significance of saprophytism in the epidemiology of the mycoses. Trans. N.Y. Aead. Sci. II. 17: 157-166.

11. Emmons, C.W. 1955. Saprophytic sources of Cryptococcus neoformans associated with the pigeon (Columba !ivia). Am. J. Hyg. 62: 227-232.

12. Emmons, C.W. 1958. Association of bats with histoplasmo- sis. Pub. Hlth. Rep. 73: 590-595.

13. Emmons, C.W., C.H. Binford, J.P. Utz, & K.J. Kwon- Chung. 1977. Medical mycology. Lea & Febiger, Phila- delphia, pp. 386-482.

14. Emmons, C.W., & A.L. Carrion. 1936. The phialophora type of sporulation in Hormodendrum pedrosoi and Hormodendrum compactum. Puerto Rico J. Pub, Hlth. Trop. Med. 11 : 703-710.

15. Fuentes, C.A., & Z.E. Bosch. 1960. Biochemical differen- tiation of the etiological agents of chromoblastomycosis from non-pathogenic Cladosporium species. J. Invest. Dermatol. 34: 419-421.

16. Gezuele, E., J.E. Mackinnon, & I.A. Conti-Diaz. 1972. The frequent isolation of Phialophora verrucosa and Phialophora pedrosoi from natural sources. Sabourandia 10: 266-273.

17. Hughes, S.J. 1953. Conidiophores, conidia and classification. Canad. J. Bot. 31 : 577 659.

18. Jang, S.S., E.L. Biberstein, M.G. Rinaldi, A.M. Henness, G.A. Boorman, & R.F. Taylor. 1977. Feline brain abcess due to Cladosporium trichoides. Sabourandia 15 : 115-I 23.

19. Klite, P.B., H.B. Kelley, & F.N. Dierks. 1965. A new soil sampling technique for pathogenic fungi. Amer. J. Epi- demiol. 31 : 124-130.

20. Kwon-Chung, K.J., I.S. Schwarz, & B.J. Rybak. 1975. A pulmonary fungus ball produced by Cladosporium cladosporioides. Amer. J. Clin. Pathol. 64: 565-568.

21. Middleton, F.G., P.F. Jurgenson, J.P. Utz, S. Shadomy, & H.J. Shadomy. 1976. Brain abcess caused by Cladosporium trichoides. Arch. Intern. Med. 136:444 448.

22. Nielson, H.S., Jr. 1974. Dematiaceous fungi. In: E.H. Lennette, E.H. Spaulding, & J.P. Truant (eds.), Manual of Clinical Microbiology, 2nd ed. American Society for Micro- biology, Washington, D.C. pp. 528-540.

23. Rao, V.K., & C.R. Prasad. 1973. Experimental maduro- mycosis. I. Infection of mice with Phialophora jeanselmei. Mycopathol. Mycol. Appl. 51:119 126.

24. Ridley, M.F. 1957. The natural habitat of Cladosporium carrionii, a cause of chromoblastomycosis in man. Austra- lian J. Derm. 4: 23-27.

25. Rippon, J.W. 1974. Medical mycology. The pathogenic fungi and the pathogenic actinomycetes. W.B. Saunders Company, Philadelphia, London, Toronto, p. 241.

26. Salonen, A., & A.L. Roukola. 1969. Mycoflora of the Finnish 'sauna.' Mycopathol. Mycol. Appl. 38: 327-328.

27. Smith, C.D., & M.L. Fureolow. 1964. Efficiency of three techniques for isolating Histoplasma capsulatum from soil, including a new flotation method. J. Lab. Clin. Med. 64: 343-348.

28. Wang, C.J.K. 1965. Fungi of pulp and paper of New York. State Univ. Coll. Forrestry Tech. Pub. 87, p. 76.

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