Fungal flora of normal eyes of healthy horses from the State of Rio de Janeiro, Brazil

Veterinary Ophthalmology (2003) 6, 1, 51 55 Blackwell Science, Ltd Fungal flora of normal eyes of healthy horses from the State of Rio de Janeiro, Brazil Maurílio Rosa,* Liane Maria Cardozo,* Jorge da Silva Pereira,* Dennis E. Brooks, Ana Lucia B. Martins,* Penha Sueli Silva Florido and Jussara Schwind Pedroso Stussi *CEPOV Centro de Estudo, Pesquisa e Oftalmologia Veterinária, R. Heitor de Moura, Estevão, 468, Teresópolis, RJ, Brazil, University of Florida, College of Veterinary Medicine, Gainesville, FL 32610-0126, USA, Mycology Laboratory of Universidade Federal Fluminense, Niterói, RJ, Brazil Address communications to: M. Rosa Tel.:/Fax: (5521) 2742 3900 e-mail: marcmax@openlink.com.br Abstract The conjunctival fungal flora of 32 adult horses with normal eyes (n = 64) from the State of Rio de Janeiro in Brazil was identified in the fall of 2000 using horses of different breeds, both genders and aged 5 19 years old. The culture samples were taken from the conjunctival sac of both eyes with a sterile cotton swab wetted with saline solution, seeded in Sabouraud s dextrose agar with chloramphenicol, and incubated for 5 days at an average temperature of 25 C. The number of fungal colonies per eye varied between 0 and 250 colony forming units (CFUs). There were often differences in colony types between eyes of the same animal. Filamentous fungi of genera were isolated and identified in the following proportion of the total genera of fungal colonies isolated: Aspergillus (32.2%), Penicillium (25.8%), Scopulariopsis (15.9%), Trichoderma (11.2%), Cladosporium (5.6%), Mucor (2.1%), Syncephalastrum (2.1%), Eurotium (1.7%), Geotrichum (0.9%), Rhizopus (0.9%), Gliomastix (0.4%), Fusarium (0.4%), Staphylotrichum (0.4%) and Verticillium (0.4%). Yeast genera represented 9% of the total isolates. Over half the horses had at least one normal eye with either Aspergillus, Penicillium, Trichoderma or Scopulariopsis isolated, which is a departure from other studies of the normal horse eye. Key Words: conjunctiva, fungi, horses INTRODUCTION Keratomycosis is an important disease of the equine eye. It may have an insidious onset with a prolonged healing period resulting in extensive fibrovascular corneal scarring. 1 Fungal organisms of ophthalmic importance are classified into filamentous fungi or molds that produce feathery, cottony or powdery aerial growth on culture media, and yeasts that produce opaque, creamy, pasty colonies on culture media. 2 Filamentous fungi produce tubular hyphae and are subdivided into those that produce hyphae with cross-walls (septate), and those that do not produce cross-walls (nonseptate). 2 The fungi involved in invasion and infection of the equine cornea are generally those of the normal equine conjunctival fungal flora, which varies with geographic region and season of the year. Aspergillus, Cladosporium, Alternaria, and Penicillium spp. predominate in most studies of the microbiology of the normal horse conjunctiva and cornea. 3 5 Fusarium and yeast spp. may also be commonly isolated in some areas. 3 5 The conjunctiva and cornea have unique and strong defensive systems against fungal infection. Immunologic, metabolic and antimicrobial mechanisms and physical tissue barriers protect the cornea from keratomycosis. The flushing action of the tears combined with eyelid movement tends to wash away fungi living on the surface of the conjunctiva and cornea. Tear film macrophages and tear film IgA, the qualitative and quantitative integrity of the tear film, the intact corneal epithelium, and the presence of conjunctival bacteria are all detrimental to fungal invasion and colonization of the cornea. The normal conjunctival bacterial flora protects against fungal infection by consumption of nutrients vital for fungal survival, as well as secreting metabolic substances that may have antifungal properties. 6,7 Initiation of fungal infection of the cornea requires the presence, inoculation, invasion and colonization of the cornea by viable fungi through an epithelial defect. 3,5,8 The ubiquitous presence of fungi on the surface of the equine conjunctiva and cornea, combined with suspected defects towards fungi in the equine corneal immune system, 1 make 2003 American College of Veterinary Ophthalmologists

52 ROSA ET AL. infection of the horse cornea more likely than in other domestic animal species. 5,8,9 Equine keratomycosis is not recognized, however, as frequently in temperate regions of the world where bacterial keratitis is the major problem. 4,5,7,10 Fungal keratitis or keratomycosis of horses is caused by infection of the cornea by saprophytic filamentous fungi or molds, and yeasts. 1,7,8,10 12 More than 30 genera of fungi are reported to cause keratitis in the horse. The use of topical antibiotics and the improper utilization of topical corticosteroids in corneal ulcer therapy may lead to or intensify the clinical signs of fungal keratitis in horses. 1,4,5,7,9,10,14 Differences in geographic prevalence of fungi in horse eyes, the knowledge of risk factors for the development of keratomycosis, the clinical signs of fungal keratitis, and the types of effective antifungal drugs are important to equine clinicians and veterinary ophthalmologists. The objective of this research was to identify the most common ocular fungi of horses from the geographic area of the State of Rio de Janeiro, Brazil, in order to better understand and more effectively treat keratomycosis in horses in this South American region. 3,7,11,13 MATERIALS AND METHODS This research was conducted from April to December 2000, in the mycology laboratory of the Universidade Federal Fluminense, Niterói, Brazil. The eyes of 32 healthy horses (n = 64 eyes) of different breeds, both genders, and ages varying from 5 to 19 years old, were studied after ophthalmic examination failed to show any evidence of ocular inflammation and infection. Ophthalmic examinations consisted of slit lamp biomicroscopy and direct ophthalmoscopy. Culture material was collected from the conjunctival sac using a sterile cotton swab wetted with saline solution, taking care not to contaminate the swab by contact with the skin. This procedure was carried out in both eyes using a different swab per eye. After the collection, each swab was placed in a sterilized assay tube, identified, and sent to the laboratory to be processed. 13,15 17 These collections were done in the morning from April to July, 2000 (fall season) in animals that were used in jumping, training, and/or other outdoor activities (18 horses), and in animals being stabled in their stalls (14 horses). In the laboratory each swab was streaked onto a Petri dish containing 2% Sabouraud s dextrose agar with 1% chloramphenicol, and incubated in an inverted position at between 24 C and 26 C for 5 days, after which fungi in CFUS were counted. Next, the isolated colonies were transferred to an assay tube containing 2% Sabouraud s dextrose agar. All media were left for 21 days to identify slow growing organisms. 16,17 Slides were mounted with Amann s lactophenol or cotton blue, and when necessary microcultures were created for identification of fungi to genus. In some cases cultures on potato dextrose agar were made to stimulate fungal reproduction and sporulation. 15 17 RESULTS The majority of fungal isolates appeared on culture media within 3 5 days of inoculation. Table 1 shows the total number of fungal CFUs comprising the colonies (molds and yeast) found in each eye of each horse. None were negative in both eyes. The least number of isolates was obtained in animal number 20 with one colony forming one CFU isolated in the right eye, and the greatest number were found in animal number 5, with 250 CFUs of two fungal genera in the left eye. The greatest number of fungi isolated in one horse was animal 17 with an average of 141.5 CFUs. There was no observed positive correlation between the fungal number isolated, the breed, or the animal s age, but there was an increase in the incidence of fungi in animals that were being stabled compared to those that were outside. An average of 90 CFUs was found in stalled animals and 70 CFUs in active animals (Tables 1 3). The temperature and wind conditions on April 13, May 18, June 29 and July 6, 2000 in Rio de Janeiro were 81.7 F and 3.7 mph, 75.4 F and 7.4 mph, 75.1 F and 5.1 mph, and 70.5 F and 6.7 mph, respectively. There was no rain during this time. The CFU average for all eyes in all the horses sampled on those dates was 62, 11.2, 59.5, and 33.4 CFUs, respectively (Table 1). There were 256 total colonies (often composed of multiple CFUs) of mold and yeast genera isolated and identified (Tables 2 and 3). The prevalence of filamentous fungi or molds was 91% (n = 233 colonies), and of yeasts was 9% (n = 23 colonies). Filamentous fungi of the following genera were identified in the following proportion of the total fungal colonies isolated: Aspergillus (32.2%), Penicillium (25.8%), Scopulariopsis (15.9%), Trichoderma (11.2%), Cladosporium (5.6%), Mucor (2.1%), Syncephalastrum (2.1%), Eurotium (1.7%), Geotrichum (0.9%), Rhizopus (0.9%), Gliomastix (0.4%), Fusarium (0.4%), Staphylotrichum (0.4%), and Verticillium (0.4%) (Tables 2 and 3). Aspergillus, Penicillium, Trichoderma and Scopulariopsis were the most frequent isolates in at least one eye of 23 (71.9%), 21 (65.6%), 19 (59.4%) and 16 (50%) horses, respectively. DISCUSSION The most commonly reported fungal genera isolated in keratomycosis of horses are Aspergillus, Penicillium and Fusarium. 1,7,8,11 13 The fungi most often identified in normal eyes in horses in Brazil were Aspergillus, Penicillium, Trichoderma and Scopulariopsis. Although Alternaria and Cladosporium have also been described in normal horse eyes with a high incidence in other geographical areas, 3 5,13,17 they were not found in large numbers in the present study. Fusarium in particular was only isolated once in Brazil (0.4% of total), and not at all in other areas. 3,5,7,14 The isolation of Staphylotrichum, Verticillium and Gliomastix in

FUNGAL FLORA OF NORMAL EYES OF HEALTHY HORSES 53 Table 1. Gender, age, breed and total number of colony forming units (CFUs) of all molds and yeasts of 32 healthy horses (n = 64 eyes) in the State of Rio de Janeiro, Brazil Date of Animal CFU* sample collection No. Activity Gender Age Breed Right eye Left eye Mean** Average 13/04/2000 1 + MC 16 PSI 36 46 41 2 + MC 12 PSI 25 40 32.5 3 F 14 SRD 13 61 37 4 + F 10 BH 75 45 60 5 + MC 9 HANV 29 250 139.5 62 18/05/2000 6 + MC 8 PSI 8 28 18 7 + MC 5 BH 0 5 2.5 8 MC 8 PSI 6 16 11 9 F 8 BH 25 12 18.5 10 MC 19 SRD 4 2 3 11 MC 12 SRD 12 31 21.5 12 + MC 15 BH 1 7 4 11.2 29/06/2000 13 F 7 PSI 47 45 46 14 F 8 PSI 30 70 50 15 + F 8 SB 65 70 67.5 16 + MC 5 PSI 19 12 15.5 17 MC 11 SRD 153 130 141.5 18 + MI 5 BH 32 17 24.5 19 + MC 10 SRD 140 121 130.5 20 + F 6 BH 1 0 0.5 59.5 06/07/2000 21 F 12 SRD 106 21 63.5 22 MC 10 BH 36 15 25.5 23 + MI 7 PSI 31 2 16.5 24 + MI 10 BH 10 27 18.5 25 F 10 SRD 42 21 31.5 26 F 8 PSI 13 51 32 27 MC 13 SRD 177 95 136 28 + MC 9 BH 17 25 21 29 + F 10 SRD 5 4 4.5 30 + F 8 SF 20 19 19.5 31 MI 15 PSI 7 26 16.5 32 + F 8 SRD 18 13 15.5 33.4 *Total colony forming units (CFUs) of all genera of molds and yeasts isolated; (+) animal in racing activity; ( ) animal stabled; (F) female; (MC) gelded male; (MI) male; Age (in years); (PSI) English pure blood; (BH) Brazilian Racing Horse; (SRD) no breed defined; (HANV) Hanoverian; (SF) French Saddle; (SB) Belgium Saddle; **Mean CFU of all fungal types for both eyes of each horse; Mean CFU of all eyes of all horses by collection date. Brazilian horses appears to be the first time that they have been identified in the horse. Gliomastix has previously been isolated from a dog s eye, and Staphylotrichum from a cow s eye. 4 More fungi were isolated from the eyes of stabled horses than horses living outside in Brazil, similar to findings in another study in the USA. 3,9 This could reflect increased humidity, dust and other hygienic problems in the stables, and might represent a higher risk for development of keratomycosis in horses confined to stables. 3 Whether a horse lives in a temperate or tropical environment can cause differences in the number and types of microbial fungal flora found in horse eyes, and also in the intensity of equine keratomycosis. 1,6,11 The weather during the fall collection period in Rio de Janeiro was warm and windy with no precipitation recorded. Seasonal changes were not evaluated in the present research, but some fluctuations in the CFU average for the four dates of specimen collection might be related to climatic variation in temperature, pollen levels, rain and/or wind in the days prior to and including the day of the culturing. There did appear to be a slight trend of more fungi collected on days with lower temperatures and higher wind. CONCLUSION The greatest number of fungi were obtained from animals that lived in stalls in Rio de Janeiro, Brazil. Molds were more prevalent (91%) than yeasts, and the genera Aspergillus, Penicillium, Trichoderma and Scopulariopsis were the most frequent fungal genera cultured from the eyes of normal horses in Brazil.

54 ROSA ET AL. Table 2. Number of genera of fungi isolated and identified per eye from CFUs found in 16 healthy horses (n = 32 eyes; horse #1 16) in the State of Rio de Janeiro, Brazil 13/04/2000 (81.7 F, 3.7 mph) 18/05/2000 (75.4 F, 7.4 mph) 29/06/2000 (75.1 F, 5.1 mph) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Sample OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS Aspergillus 1 2 5 6 4 2 4 4 1 2 2 1 3 3 1 5 1 1 2 4 2 1 Cladosporium 2 1 1 1 2 Eurotium 3 1 Fusarium 1 Geotrichum Gliomastix 1 Mucor 1-1 Penicillium 3 4 2 2 1 1 1 2 2 4 1 3 1 3 2 Rhizopus Scopulariopsis 1 1 2 2 5 1 2 3 1 1 2 1 Staphylotrichum 1 Syncephalastrum 2 2 1 - Trichoderma 1 1 1 1-1 19 3 Verticillium 1 - Yeast 7 1 3 1 1 2 Table 3. Number of genera of fungi isolated and identified per eye from 16 healthy horses (n = 32 eyes; horse #17 32) in the State of Rio de Janeiro, Brazil 29/06/2000 (77.9 F, 5.1 mph) 06/07/2000 (70.5 F, 6.7 mph) 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Sample OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS Aspergillus 1 2 1 2 1 1 1 3 1 2 3 Cladosporium 1 1 1 1 2 Eurotium Fusarium Geotrichum 2 Gliomastix Mucor 1 1 1 Penicillium 4 3 1 1 2 1 3 1 2 2 4 1 3 Rhizopus 1 1 Scopulariopsis 1 3 1 1 2 4 1 1 1 Staphylotrichum Syncephalastrum Trichoderma 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 2 Verticillium Yeast - - 1 1 3 1 1 1

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