This paper delineates the significance of Aspergillus flavus as the possible cause of chronic rhinitis in a 4-year-male German shepherd dog. The affected dog exhibited the signs of dullness, intermittent anorexia, mild dyspnea, sneezing, and bilateral sanguineous nasal discharge. The fungus was repeatedly demonstrated in the nasal exudates both by direct microscopy in wet mount preparation of potassium hydroxide, and by the isolation on mycological media. There was no growth of Cryptococcus neoformans on Pal sunflower seed medium. The detailed microscopic examination of fungal isolates in ‘Narayan’ stain confirmed the identity as A. flavus. The epidemiological investigation revealed A. flavus in the immediate environment of the dog. Mycotic rhinitis should be suspected by a chronic serosanguineous bilateral nasal discharge. As A. flavus is an environmental contaminant, demonstration of fungus in repeated clinical samples is highly imperative to confirm an unequivocal diagnosis of disease. The housing of the dog in a well ventilated, dry pen, and periodical fumigation of the kennel can reduce the exposure to airborne fungi in the immediate environment. It is emphasized that immonocompromised dogs should not be exposed to dusty environment. As far as could be ascertained, this seems to be the first report on rhinomycosis in a dog due to A. flavus in India.
Rhinitis is the most common cause of upper respiratory tract malfunction, and involves multiple etiologies, such as viral, bacterial, fungal, and parasitic agents besides hypersensitivity reactions (Aiello and Mays, 1998). Rhinomycosis also known as fungal rhinitis, mycotic rhinitis, is a sporadic, acute mild, usually non-invasive ,opportunistic fungal disease of animals particularly of canines (Pal, 2007).The infection is chiefly caused by Aspergillus fumigatus, though other fungi such as Aspergillus flavus, A. niger, Cryptococcus neoformans, Penicillium, etc. are also implicated in the etiology of the disease (Goodall et al.,1984; Harvey,1984; Pal and Mehrotra,1984; Sharp et al,1991; Wolf,1992; Rozengurt and Sanchez,1993; Pal,2007). Mycological, serological, histopatholgical, and molecular techniques are employed for the diagnosis of Aspergillus infections (Pal and Mehrotra, 1985; Pal, 2007; Pal et al., 2014).Topical therapy with enilconazole, and clotimazole is proved useful. However, systemic treatment with ketoconazole, fluconazole, itraconazole, and thiabedazole has also been tried (Harvey, 1984; Sharp and Sullivan, 1989; Aiello and Mays, 1998; Pal, 2007).The paucity of information on rhinomycosis in pet animals prompted this investigation to report A. flavus as the cause of rhinitis in a young dog from Anand, Gujarat, India.
Materials and Methods
The nasal exudates collected on three consecutive days from the 4-year-old male German shepherd dog, were submitted by the Department of Clinic, Veterinary College, Anand, Gujarat, India constituted the materials for this study. The samples were processed immediately for the mycological examination by employing standard techniques (Pal, 2007). A part of the specimen was examined by potassium hydroxide technique for the presence of fungal elements (Pal, 2007).The remaining material was liberally inoculated on to the slants and plates of Sabouraud dextrose agar with chloramphenicol, and Pal sunflower seed medium (Pal, 1997). The former medium was incubated at 370 C, and the later was kept at 250 C. All the inoculated media were daily examined for the fungal growth. The detailed identification of the fungal isolates was done in a newly described Narayan stain (Pal, 2004). The new stain contained 0.5 ml of methylene blue (3% aqueous solution), 4.0 ml of glycerine, and 6.0 ml of dimethyl sulfoxide (DMSO). In order to establish the source of infection, the plates of Sabouraud agar and pal sunflower seed medium were exposed in the immediate environment of the sick dog (Pal, 2007). These plates were incubated at 370 C. There was no facility of molecular typing of fungal isolates in the VPH laboratory.
The clinical examination of the dog revealed sneezing, nasal pain, slight dyspnea, ulceration of nares, and bilateral serosanguineous nasal discharge. The rhinoscopic examination revealed fungal plaques. Hyaline, narrow, septate branching hyphae morphologically resembling to Aspergillus were demonstrated in the nasal exudate by potassium hydroxide technique. There was no growth of Cryptococcus neoformans on Pal sunflower seed medium .However, many colonies of Aspergillus grew on Sabouraud medium at 370C as well as Pal medium at 250C. The gross cultural characteristic on Sabouarud medium, and microscopic morphology of the isolates in Narayan stain confirmed the identity as A. flavus (Pal, 2007). Aero-mycological screening on Sabouraud agar and Pal sunflower seed medium indicated the heavy concentration of A. flavus in the environment where the dog was kept.
Fungal rhinitis principally caused by A. fumigatus, and rarely by other fungi has been recorded in camel, cat, horse, mule, and rat (Goodall et al., 1984; Pal and Mehrotra, 1984; Rozengurt and Sanchez, 1989; Pal, 2007). In the present investigation, the clinical and mycological observations conclusively established that the dog was suffering with fungal rhinitis due to A. flavus. The criteria of confirming the diagnosis of fungal infection was consistent with the earlier investigators that fungal agent must be detected, and isolated in pure and luxuriant form from the clinical materials on mycological medium (Pal and Mehrotra,1984; Aiello and Mays,1998; Pal,2007). Both the criteria was followed in the present case, as the causative pathogen A. flavuswas identified by direct microscopy in potassium hydroxide preparations as well as by cultural isolation on Sabouraud agar, and Pal sunflower seed medium. It is interesting to mention that repeated recovery of A. flavus from the nasal exudate of the patient unequivocally confirmed the diagnosis of rhinomycosis. As A. flavus is a soil borne fungus, which is ubiquitous in distribution, the repeated sampling is advised to rule out contamination. The cycloheximide should not be incorporated in Sabouraud medium as it inhibits the growth of A. flavus (Pal, 2007).There was no facility in the department to undertake molecular diagnosis of fungal infections. The epidemiological investigation helped to know the source of infection. The owner was advised to decontaminate the pen of the dog with 3 % formalin to kill the fungus in the environment. As the incidence of immunocompromised subjects is increasing, the role of opportunistic fungi should be prudently investigated in various clinical disorders of humans and animals. Further research on the development of safe, potent, cheap chemotherapeutic agents for the better management of mycotic infections should be conducted. The available literature scan revealed that this is perhaps the first record of A. flavus implicated in the etiology of rhinitis of a German shepherd dog from India. The successful isolation of A. flavus from clinical and environmental samples on Pal sunflower seed agar suggests that this medium, being a very cheap and easily available, can be routinely used by the microbiologists for the isolation of A. flavus and other opportunistic fungi from humans and animals.
The author is thankful to the Department of Clinics for the submission of clinical samples, Department of Veterinary Public Health for technical assistance, and the animal owner for his kind cooperation to permit in obtaining the clinical as well as environmental samples.