MYCOSES 34, 261-266 (1991) ACCEPTED: JUNE 11, 1991

Ocular histoplasmosis due to Histoplasma farciminosum in Egyptian donkeys

Augen-Histoplasmose durch Histoplasma farciminosum an agyptischen Eseln

R. Soliman', M. Ebeid2, M. Essa3, M. A. Abd El-Hamid2, Y. Khamis2 and A. H. Said2

Key words. Histoplasma farciminosum, ocular mycosis, donkeys.

Schliisselworter. Histoplasma farciminosurn, Augenmykose, Esel

Summary. In this study 19 donkeys suffering from face excoriations below the medial canthas and le- sions in the lacrymal apparatus were subjected to clinical radiological and microbiological investiga- tions. Also histopathological examination of biopsy materials from ocular lesions were made. Eleven out of 19 donkeys (57.9%) showed specific lesions in the lacrymal apparatus. Mycological examination re- vealed the isolation of Histoplasma cells from 10 out of 19 examined cases. The isolated Histoplasma cul- ture, although not completely identical to H. far- ciminosum, was more related to this than to other Histoplasma species. Histopathological examina- tion revealed the presence of marked tissue infiltra- tion by lymphocytes, monocytes and macrophages. Also the fungal organism appeared as double-con- toured yeast cells lying within macrophages and giant cells.

Zusammenfassung. In dieser Studie wurden 19 Esel mit Hautabschurfungen am Vorderkopf unter den medialen Augenwinkeln und Lasionen im Tra- nenapparat klinisch, radiologisch und mikrobiolo- gisch untersucht. Auch histopathologische Unter- suchungen wurden durchgefuhrt. 11 der 19 unter- suchten Esel (57,9 yo) zeigten spezifische Lasionen im Tranenapparat. Die mykologische Untersu- chung fuhrte zur Isolierung von Histoplasma-Zellen bei 10 der 19 untersuchten Tiere. Die isolierte Hi-

' Dept. of Microbiology, Dept. of Pa- thology, Faculty of Veterinary Medicine, Cairo University, Giza,

Dept. of Surgery, and

Egypt.

Correspondence: Professor Dr R. Soliman, Dept. of Microbiol- ogy and Immunology, Fac. Veterinary Med., Cairo University, Giza, Egypt.

stoplasma-Kultur war zwar nicht vollstandig iden- tisch mit H. farciminosum, ahnelte dieser jedoch mehr als jeder anderen Histoplasma-Art. Die histo- pathologische Untersuchung zeigte eine deutliche Gewebsinfiltration mit Lymphozyten, Monozyten und Makrophagen. Die Pilzzellen erschienen auch als doppelt konturierte Hefezellen, intrazellular in Makrophagen und Riesenzellen gelegen.

Introduction

Occular mycosis involving the conjunctival and lac- rymal drainage system in horses and donkeys has been reported in tropical Africa [ I ] .

Dekester & Jeaume [2] found small granuloma- teous nodules at the entrance of the lacrymal sac and nasolacrymal duct in donkeys in Morocco. His- topathological section in these nodules revealed the presence of yeast cells within the giant cells. Cul- tures, however, were not successful.

Velu [3] and Jeaume & Dekester [4] showed that dacryocystitis in donkeys is caused by a fungus which is morphologically different from both His- toplasma farciminosum and H. capsulatum. They identified the isolated fungus as a new species under the name H. velu.

In Egypt, Fouad et al. [ 5 ] emphasized the role of H. farciminosum as the principle cause of lesions related to lacrymal apparatus of donkeys. However, El-Guindy et al. [6] identified the causative fungus as H. capsulatum.

The present investigation was undertaken to study the unclear situation of this disease in donkeys in Egypt, as well as to clear up the clinical and histopathological changes accompanied with this in- fection, moreover, to isolate, identify and describe the aetiological agent.

262 R. SOLIMAN ET AL.

Material and methods

Nineteen donkeys suffering from face excoriations below the medial canthus and lesions in the lacry- ma1 apparatus were subjected to clinical and ra- diological examination, microbiological and histo- pathological examinations.

The tissues were fixed in 10% neutral formalin for 48 h, dehydrated in gradual concentration of alcohol, cleared in xylol and embedded in parafin. Sections of 5 p thickness were made and stained with H & E and periodic acid Schiff (PAS) technique.

Results Clinical and radiological examination

Clinical and radiological examination Clinical and radiological examination involved: ( 1 ) Examination of the lesions for the presence of

any swelling, purulent materials, and tear ecze- ma or face excoriations below the nasal canthus.

(2) Nasolacrymal cannulation: The nasal punctum in each case was cannulated with a catheter and flushed with sterile physiological saline solution. The solution was recollected from the upper and lower puncta lacrimalia and centrifuged at 3000 r.p.m. for 10 minutes. The sediment was examined microscopically.

(3) Dacryocystorhinography was applied in order to ascertain the site of obstruction.

Micro biological examination

Purulent discharge from medial canthal region was collected from the diseased animals using sterile dis- posable syringes. The samples were collected by cre- ation of a mild pressure on the medial canthus of the diseased eyes. The collected 19 samples were subjected to the following investigations: (1) Direct microscopic examination of wet prepa-

rations and smears stained with Gram's stain. (2) Bacteriological examination: All samples were

inoculated on nutrient agar, blood agar and McConkey's agar and were incubated at 37 "C for 48 hours. The isolated bacteria were iden- tified according to Breed et al. [7] and Scott & Baiely [8].

(3) Mycological examination: The collected sam- ples were inoculated on the following media: Brain heart infusion agar with 10% horse blood, Sabouraud dextrose agar with 2.5% glycerol, and PPLO nutrient agar (Difco) with 2% dextrose and 2.5% glycerol-pH 7.8 [9]. Each sample was inoculated on 20 slants from each medium, 10 of which were incubated a t 25 "C, and the remaining at 37 "C. The isolated fungi were identified according to Emmons et al. [lo].

His to pa thological examination

The collected overgrowths together with biopsy specimens taken from ulcerated areas of the skin of the face were subjected to histopathological exam- ination.

Eleven out of the examined nineteen donkeys (57.8%) showed specific lesions at the lacrymal drainage system. The lesions were characterized by swelling of the lids in the area of the medial canthus with mucopurulent to purulent discharges, compli- cated with tear eczema and swelling of the maxillae. Unhealthy granulomatous overgrowth of a size ranged from 2 to 10 cm, originating from the ca- runcula lacrimalis and the conjunctiva of the third eyelid, was observed (Figs 1 A and B).

The nasolacrymal duct in most of the examined cases proved to be obstructed since retrograde flush- ing was unsuccessful. The intraosseous part of the duct together with the osseous lacrymal canal were the main site of obstruction.

The post mortem examination and dacryocysto- rhinography (Figs 1 C and D) revealed that the osseous lacrymal canal was completely destroyed with perforation of the lacrymal bone, creating a fistula between the orbit and maxillary sinus. In one case the obstruction was observed near the orbit as a granuloma involving the osseous lacrymal canal.

Micro biological findings

Ten out of the nineteen samples were positive for the presence of Histoplasma-like organisms both in the wet preparations (Fig. 2 A) and in Gram's stained smears (Fig. 2 B). The organisms appeared as a double-contoured ovoid yeast-like cells meas- uring 4-5 x 2-3 pm in diameter. Many of these cells were pointed at one end taking a lemon-shaped appearance. The cell wall was highly refractile and the cellular contents were clear and homogeneous. Intracytoplasmic refractile granules, were also seen.

Isolation of the fungus was only successful in 5 cases. In some cases primary cultures were obtained on SDA and on PPLO agar after 6-8 weeks incu- bation at 25 "C, as a small, convoluted, whitish, yellow pasty colonies (Fig. 3 A). After 4 months incubation a t the same conditions, the colonies be- came brown, raised convoluted and reached a di- ameter of 20 mm (Fig. 3 B). The isolated Histo- plasma cultures were markedly pleomorphic and exhibited different macroscopical colonial forms fol- lowing repeated subculture on SDA and PPLO agar. The isolated Histoplasma cultures showed

rnycoses 34, 261-266 (1991)

HISTOPLASMA FARCIMINOSUM IN EGYPTIAN DONKEYS 263

Figure 1. Dacryocystitis due to Histoplasma infection. (A) Dacryocystitis with an overgrowth in the third eyelid. (B) Tear eczema. (C) Granulating mass involving osseous lacrymal canal (arrow). (D) Dacryocystorhinography: the nasolacrymal duct was discontinued near the orbit.

Figure 2. nisms. (A) Unstained, (B) Gram's stain.

Double-contoured yeast cells of Histoplasma orga-

strong urease activity on Christensen's urea medium at 25 "C.

The microscopical morphology of the isolated cultures revealed the presence of double-contoured yeast-like cells identical to those demonstrated in ocular discharges from the diseased animals. Most of these cells became enlarged and transformed to a sac-like structure containing one or more highly refractile granules. Budding yeast-like cells with narrow neck were also found. In cultures incubated

Figure 3. Culture of Histoplasma organisms on PPLO agar after 6 weeks incubation at 25 "C (A) and after 16 weeks a t the same conditions (B).

at 25 "C, germ tubes, short hyphae, in addition to long hyphae were observed (Fig. 4 A). Incubation for extended period of times up to 8 months revealed no noticeable macroscopical growth, however, the microscopical examination of the inoculum showed the transformation of most yeast cells into a rela- tively large double-walled sac containing refractile granules (Fig. 4 B).

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264 R. SOLIMAN ET AL.

Bacteriological findings: No specific bacterial Histopathological findings pathogens could be isolated from the examined cases. The examined overgrowths showed diffuse granu-

lomatous reaction in the form of aggregation of large number of macrophages, mature lymphocytes and plasma cells as well as a considerable number of giant cells, mostly of the Langerhans type (Fig. 5 A).

Blood capillaries could be seen between the pro- liferated inflammatory cells. Also, young capillaries and fibroblasts were commonly seen in the periph- ery of the preceding cellular reaction.

In H & E stained sections the Histoplasma cells appeared with bizarre-shaped dark basophilic nu- clear elements embedded in eosinophilic cytoplasm and surrounded by an unstained halo. The fungal

the cytoplasm of macrophages and giant cells (Fig. Figure 4. Microscopical morphology of Histoplasma culture. cells were found scattered, single or in groups within (A) Mold form (young culture), (B) yeast form.

Figure 5. Granulomatous reaction caused by Histoplasma infection showing (A) aggregation of macrophages, giant cells and a considerable number of lymphocytes (H & E staining), (B) macrophages and giant cells containing Histoplasma organisms (arrows; H & E staining), ( C ) double-contoured Histoplasma cells within giant cells ( x 1000; H & E staining), and (D) Histoplasma organisms within macrophages and giant cells (PAS staining).

mycoses 34, 261-266 (1991)

HISTOPLASMA FARCIMINOSUM IN EGYPTIAN DONKEYS 265

5 B). Some of the fungal cells escaped from the orig- inally intracellular location. In the central area of the lesion giant cells appeared heavily infected with Histoplasma cells, about 20-25 yeast cells/host cell (Fig. 5 C), while towards the periphery of the lesion the number of fungal cells diminished markedly. Sections stained with periodic acid Schiff (PAS) re- action showed that the nuclear elements of the or- ganisms appeared strongly positive, refractile, cir- cular or triangular in shape. They were surrounded by a thin film of cytoplasm which stained less in- tensely with PAS and appeared surrounded by an unstained halo (Fig. 5 D).

Discussion

Histoplasma infection of the eye and its lacrymal drainage system is a frequent syndrome affecting donkeys in Egypt [5, 61. Two clinical types of the disease were recognized, those with canalicular signs predominating in the form of dacryocystitis which in most of the cases was accompanied by obstruction of the nasolacrymal duct; and those with marked conjunctival granulomatous reaction with no ap- parent canalicular cases, changes in the facial bones were observed particularly in the lacrymal bone. Those changes might be attributed to the rarefac- tion of the bone (rarefying ostitis) accompanying the inflammatory processes in the area as described by Runnells et al. [ 1 11 rather than the actual effect of the fungus on the bones.

The direct microscopical examination of the ocu- lar discharge revealed the presence of double-con- toured yeast-like cells identical to that characteristic of the genus Histoplasma [lo, 121. The detected yeast-like cells were morphologically more similar to H. farciminosum, especially by exhibiting a pro- jection at one pole taking a lemon-shaped appear- ance and also by the narrow cell junction of the budding yeast cells [9, 131.

The macroscopical and microscopical morphol- ogy of the isolated fungi differed completely from those of H. capsulatum [ 10, 121. On the other hand the macroscopical morphology of the cultures dif- fered also from those of H. farciminosum as de- scribed by Selim et al. [9]. Moreover the urease activity of the isolated fungal cultures was similar to that of H. farciminosum [9]. Microscopically the isolated fungal cultures showed the presence of many double-contoured yeast-like cells similar to those found in the discharge. In addition, short hy- phae and germ tube formation were seen. The mi- cro- and macroscopical morphology of the isolated fungal cultures were identical to those described for Histoplasma velu, (Cryptococcus mirandi) Velu (1924) and Jeaume and Dekester (1925). In con- clusion the isolated fungal cultures were more re-

lated to H. farciminosum and may represent a var- iant of the fungus. More investigations, however, are necessary to obtain a clear result.

The tissue reaction induced by the fungal cells in the eyes of donkeys was a typical granulomatous reaction characteristical for mycotic infection.

The ocular reaction differed from the cutaneous reaction caused by H. farciminosum mentioned by Khater et al. [14] because the cutaneous reaction appeared in the form of four zones; the central nec- rotic zone, a vascular zone, a thick cellular zone and a thick fibreous tissue capsule. This difference may be attributed to the softness of the tissue of the eye.

Regarding the location of the fungal organism it was found largely intracellularly, particularly in macrophages and giant cells which is in agreement with the observations of Khater et al. [14], Junger- man & Schwartzman [ 151 and Emmons et al. [lo].

The isolated Histoplasma strains showed bizarre- shaped dark basophilic nuclear elements with eosi- nophilic cytoplasm which in turn was encircled by an unstained halo (H & E stain). These observations contradict those reported by Khater et al. [ 141 and Jungerman & Schwartzman [ 151: in their studies the fungus occurred as a central spherical basophilic body surrounded by an unstained halo which in turn was encircled by a thin cell wall. On the other hand, our findings agreed with those of Emmons et al. [ 101 in which a single tiny nucleus was observed.

Sections stained with PAS showed that the cen- tral nuclear element stained intensely red in colour surrounded by less intensely stained cytoplasm. The unstained halo observed in H & E staining was also PAS negative. These findings were not in agreement with those of Khater et al. [14], Jungerman & Schwartzman [15] and Fouad et al. [5] in which the PAS-stained fungi appeared as signet rings.

The persistence of the unstained halo even after PAS staining as reported, supported the idea that this halo has to be regarded as an artifact due to the contraction of the cytoplasm during the tissue processing as mentioned by Emmons et al. [ 101.

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mycoses 34, 261-266 (1991)