Fungal infections of the equine respiratory tract (Proceedings)

Fungi are eukaryotic organisms with a cell wall made up of chitins, glucans and mannans. The plasma membrane contains ergosterol, which is a sterol that is targeted by several antifungal drugs. Fungal infections in horses are relatively uncommon, although geographic prevalence is highly variable. In most cases of upper respiratory tract mycosis in horses, there are no obvious predisposing causes. Conidiobolus coronatus, Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum and Coccidioides immitis are considered primary pathogenic fungi, and usually infect immunologically normal horses. Opportunistic fungi including Pneumocystis carini, Aspergillus spp., Candida spp, Fusarium spp and Emmonsia crescens have caused fungal disease in horses that are immunocompromised or have concurrent severe disease.

Mycotic granulomas have been found in nasal passages, nasopharynx, paranasal sinuses, guttural pouch, trachea, bronchioles, lungs and mediastinum of infected horses. Upper respiratory disease caused by fungi is frequently acquired by the inhalation route.

Clinical signs

Horses with discrete or diffuse pulmonary fungal granulomas, or fungal pleuropneumonia can present with signs similar to bacterial pneumonia. There may be cough, nasal discharge, tachypnea, respiratory distress, hemoptysis and, if chronic, weight loss. Radiographic appearance of fungal pneumonia can be variable, with patchy bronchopneumonia or miliary patterns being the most common. Differentials include bacterial pneumonia, recurrent airway obstructive disease, silicosis, granulomatous disease complex or neoplasia.

Fungal organisms such as Conidiobolus coronatus, Cryptococcus neoformans and Coccidioides immitis can form granulomatous mass like lesions in the upper respiratory tract while Aspergillus spp. tend to form mycotic plaques. The most common clinical signs of upper respiratory fungal infection include unilateral or bilateral serosanguinous or mucopurulent nasal discharge, as well as inspiratory or expiratory noise. Other clinical signs include facial deformation, coughing and dyspnea caused by partial blockage of nasal passages by granulomatous masses. Differentials for mycotic granulomas of the respiratory tract include squamous cell carcinoma, ethmoidal hematoma, amyloidosis or exuberant granulation tissue. Horses with guttural pouch mycosis usually present with episodic serosanguinous nasal discharge that may progress to potentially fatal epistaxis as the fungal plaques are frequently located over an artery. Horses may also present with cranial nerve abnormalities. The duration of clinical signs can vary from days to many months.

Diagnosis

Fungal pneumonia may be diagnosed from samples obtained by tracheal wash or bronchoalveolar lavage fluid, or via a lung biopsy. Lung biopsy is associated with significant risk as large vessels are superficial and the lung is rich in plasminogen. The biopsy should be performed ideally after radiographic evaluation or with concurrent ultrasound guidance and should be obtained from the periphery of the lung using a spring-loaded biopsy needle.

Endoscopic examination can be used to directly observe lesions in the nasal passages, nasopharynx, guttural pouch, trachea and bronchioles, while masses in paranasal sinuses and lungs may be observed radiographically. Computed tomography or magnetic resonance imaging can be used to determine the extent of lesions and bony invasion in the skull. A sterile rigid arthroscope or flexible endoscope can be passed into the conchal or maxillary sinus via a hole drilled using an 8-20mm trephine to directly view lesions and obtain biopsy samples.

Large biopsy samples of the nasal passages or nasopharynx can be obtained for cytology, histopathology and culture by use of a uterine biopsy instrument passed nasally with visual guidance from a flexible endoscope. Excisional biopsy or surgical debulking may be performed through a sinus flap or via laryngotomy.

Diagnostic tests

Cytology- Fungal hyphae may be identified in airway fluid or in impression smears obtained from biopsied masses. Fungal hyphae, such as the non-pathogenic barn fungus Alternaria, are often present either free or in large mononuclear cells in tracheal aspirates from healthy horses. Clinicians must be careful in attributing significance to presence of fungal elements in tracheal aspirates, especially when cellular cytology is normal. With fungal pneumonia, aspirates may contain predominately neutrophils that often are degenerate and may contain intracellular fungal hyphae. Some fungi have characteristic morphological features that can give an early presumptive identification.

Histopathology

To identify fungal hyphae on histopathology samples slides are best stained with periodic acid Schiff (PAS), Gridley's fungus and Grocott-Gomori methenamine-silver nitrate (GMS). Immunohistochemistry, fluorescent in situ hybridization and DNA probes can be used to positively diagnose fungal organisms in histopathogy sections.

Culture

Some fungi have fastidious growth requirements. They may be outcompeted by contaminant bacteria and may take up to several weeks to grow on culture media. For transport of tissue for microbiologic culture, the sample should be placed in a prepared culture media and transported at room temperature. Specific culture media such as Sabouraund's dextrose agar, inhibitory mold agar containing cycloheximide and chloramphenicol are useful. A Pan-fungal real-time PCR assay can be used to detect a variety of fungal organisms in body fluids and fungal isolates, followed by species specific real-time PCR to positively identify the organism.

Serological tests which utilize radioimmunoassays, immunodiffusion, complement fixation and enzyme-linked immunosorbent assays (ELISA) are available to detect circulating antibodies against several fungal organisms. These titers often decrease with resolution of disease, therefore repeated measurements can be used to help monitor response to treatment.

Immune function testing

Several fungal infections have been associated with host immune suppression caused by severe malnutrition, congenital immunodeficiency, or acquired immunodeficiency. Blood can be tested for immunoglobulin quantification by radial immunodiffusion and lymphocyte subpopulation phenotyping via flow cytometry.

Etiologic agents

Aspergillosis- Aspergillus spp. are common in the environment especially in moldy feed and bedding. They are opportunistic pathogens and often cause disease in horses that are immunosuppressed from debilitating disease, or that have been treated with immunosuppressive drugs. Infection is by inhalation of spores or by translocation of organisms across an inflamed gastrointestinal tract. Aspergillus spp. pneumonia is almost uniformly fatal, often with no or mild respiratory signs. Aspergillus spp. have broad septate hyphae with parallel sides and acute right angled branching, and may be observed or grown from a tracheal wash specimen. Care must be taken in such an interpretation as Aspergillus hyphae can be observed in tracheal aspirates of normal horses. Serologic diagnosis occasionally has been useful, but is often unreliable because many horses have titers to Aspergillus spp.

In human medicine, 50-90% of patients with invasive aspergillosis die despite treatment. Traditional treatment in humans has been amphotericin B, but reversible nephrotoxicity occurs in ~50 % of cases. Voriconazole is now considered the drug of choice against human aspergillosis. A foal with localized pulmonary aspergillosis was successfully treated with surgical resection of the lesion and oral voriconazole.

Treatment of aspergillus sinusitis and rhinitis in horses has been successful using oral itraconazole, topical natamycin (flushed via an endoscope or indwelling catheter placed into the sinus) and nystatin powder (insufflated up the nostril).

Candidiasis

Candidemia is a major problem in human ICU patients, with a mortality rate in patients with candidemia of 40-75%. Fluconazole is generally considered the drug of choice against Candida spp, although C. krusei is resistant to fluconazole. Itraconazole, amphotericin B, caspofungin or voraconazole are alternative antifungal agents.

Systemic candidiasis was diagnosed and successfully treated in 4 neonatal septic foals that had been aggressively treated with numerous antibiotics and parenteral nutrition. Three foals also had Candida glossitis and one had panophthalmitis and fungal keratitis. Two foals were treated with intravenous amphotericin B, while oral fluconazole was used on the other two foals.

Superficial Candida spp. infections of the mucous membranes (thrush) can occur in isolation or as part of a systemic infection. Candida spp. glossitis can be treated by rinsing the mouth either with potassium permanganate (0.025% q 24 hrs) or nystatin (0.3 g in 10 mls of water, q 8 hrs).

Coccidiomycosis

Coccidioides immitis is a soil saprophyte that grows in sandy, alkaline soils in semiarid areas. Inhaled arthroconidia enlarge to form non-budding spherules, which incite an inflammatory reaction in the lungs and lymph nodes. Horses have fever, weight loss, abdominal pain, and signs of respiratory disease. Diffuse infections, with granulomas in the lungs, liver, kidney or spleen have a grave prognosis. Localized, recurring nasal granulomas also have been reported. Przewalskii horses may be more susceptible.

Coccidioides immitis is difficult to culture, and spherules may not be observed histologically from antemortum lung biopsies. However, serology is very useful to diagnose infection, and decreasing titers are associated with clinical improvement. Serum antibodies are detected rarely in healthy horses. Antifungal agents successful in treatment of infected horses include fluconazole and itraconazole.

Pneumocystosis

Pneumocystis carinii has been reclassified from a protozoan to a saprophytic fungus, but some researches even consider it to be a plant because it lacks ergosterol. Pneumocystis carinii cannot be cultured, and diagnosis is based on the cytological identification of bronchoalveolar lavage fluid. A fluorescent in situ hybridization method has been developed recently to detect P. carinii in histologic sections. Immunohistochemistry also can be used.

Pneumocystis carinii is common in human AIDs patients, and people undergoing immunosuppressive therapy after organ transplantation. P. carinii causes diffuse interstitial pneumonia, especially in immunocompromised horses such as Arabian foals with severe combined immunodeficiency. Trimethoprim- sulfamethozazole (TMS) (25-30 mg/kg PO q 12 hrs) is the treatment of choice. Dapsone (3 mg/kg PO daily for 2 months) was used to successfully treat a foal that developed Salmonella enterocolitis after treatment with TMS.

Histoplasmosis

Histoplasmosis is caused by the saprophytic fungus Histoplasma capsulatum, and is most prevalent in moist soil containing bird or bat waste. Yeast organisms are 2-4 μm in diameter, with a thin clear halo surrounding a round or crescent- shaped basophilic cytoplasm. Histoplasmosis has been reported in less than 10 equine cases, and thus horses are considered to be relatively resistant to disease. H. capsulatum may occur in an enteric, pulmonary or disseminated form. Successful treatment with amphotericin B was reported in a filly with pulmonary histoplasmosis diagnosed by cytological identification of the organism on a tracheal wash smear and from a lung aspirate. Treatment with amphotericin B or itraconazole is recommended.

Blastomycosis

Blastomycosis is caused by inhalation of Blastomyces dermatitidis conidiae. Blastomyces yeasts can be identified on cytological examination, often within multinucleated giant cells. They are spherical, with basophilic protoplasm and unstained refractile walls. Unilateral, broad-based budding is characteristic. Systemic blastomycosis has been reported in two horses, both of which were euthanized. Serology is useful for diagnosis and treatment with amphotericin B, itraconazole or fluconazole is recommended.

Cryptococcosis

Cryptococcosis is caused by C. neoformans var grubii (serotype A), C. neoformans var neoformans (serotype D) and C. gattii (serotypes B and C), which are ubiquitous, saprophytic, round, yeast-like fungi. There is an epidemiologic relationship between C. gattii and the Australian river redgum tree, and C. neoformans var neoformans and bird (particularly pigeon) excreta. Serologic testing is useful, with resolution of lesions correlated with declining titers.

Cryptococcosis in horses primarily causes pneumonia, rhinitis, meningitis and abortion. Surgical resection of a jejunal lesion was successful in 1 horse. C. gattii pneumonia diagnosed by TTW and lung mass aspirates, was treated successfully with IV amphotericin B. Fluconazole was successful in the treatment of cryptococcus meningitis and sinonasal granulomas.

Conidiobolomycosis

Conidiobolus coronatus is a saprophytic fungus that causes single to multiple granulomatous lesions in the nasal passages, soft palate or trachea that can be observed endoscopically. Hyphae of C. coronatus are thin-walled, highly septate, and irregularly branched. The lesions typically have large numbers of eosinophils surrounding hyphae. Definitive diagnosis is based on microbiological culture, immunodiffusion or PCR.

Conidiobolomycosis lesions can be treated with surgical excision, laser or cryotherapy or long-term administration of iodides or antifungals. Amphotericin B has been administered intralesionally or topically in combination with DMSO to treat C. coronatus. It is important to remember that long-term therapy and reevaluation is essential, as recurrence can occur. Oral fluconazole was successful in treating two pregnant mares with nasal conidiobolomycosis.

Pseudallescheriosis

Pseudallescheria boydii is a saprophytic ascomycete. Infection most commonly involves the extremities and in human patients it is known as Madura foot. Pseudallescheria boydii cultured from the nasal cavity and sinus of a horse with chronic, malodorous nasal discharge, resolved after debriding the plaque, followed by twice daily topical application of miconazole cream that was infused for 4 weeks through lavage tubing placed through the frontal bone and into the sinuses. Adjunctive iodide therapy was also administered.

Other rare causes of fungal pneumonia that have been reported to affect horses include Scopulariopsis which was successfully treated a combination of ketaconazole and aerosolization of enilconazole; Acremonium strictum and Emmonsia crescens.

Treatment and prognosis

Treatment of upper respiratory tract fungal granulomas may involve surgical (debulking, laser or cryotherapy) and/or medical therapy (systemic, topical or intralesional).

Table 1: antifungal drugs used in horses