abstract

A 4 year old female captive born Atlantic Bottlenose Dolphin (Tursiops truncatus) presented with a history of anorexia and floating in the center of the pool. She had no previous episodes of illness. Bloodwork results demonstrated a leukocytosis with an absolute eosinophilia and an elevated erythrocyte sedimentation rate. Cytology of fecal and gastric samples was normal. Thoracic radiographs revealed an alveolar infiltrate in the caudal lung fields. Bronchoalveolar lavage was not performed due to the respiratory compromise seen in the acute disease. Porpoise and dolphin morbillivirus titers were negative. Serum samples taken on September 28, 2007, eighteen days after the onset of clinical signs were positive for Aspergillus and negative for Candida by immunodiffusion assay performed by Cerodex Laboratories, Inc. (2700 Technology Place, Norman, Oklahoma 73071). A fungal culture taken from the blowhole after the commencement of antifungal medication failed to grow fungus. Bacterial culture of the blowhole yielded Shewanella putrifaciens, Staphylococcus intermedius and a methacillin resistant Staphylococcus non-aureus, which was not demonstrated in additional bacterial cultures. The mainstay of treatment was trimethoprim sulfadiazine with supplemental folic acid, gastric protectants, tramadol, and itraconazole. Itraconazole therapy at 5 mg/kg BID yielded serum levels of 1.3 ug/ml, well within therapeutic range. Serum samples were then sent to Beacon Diagnostics Laboratory (124 Bernard E. Saint Jean Drive, East Falmouth, MA 02536-4445, (800) 568-0058) to run a Fungitell^TM assay to aid in interpreting clinical efficacy of our treatment.

This assay detects (13)-β-D-glucan, a cell wall component of most medically important fungi including Candida and Aspergillus. During the life cycle of the fungus minute quantities of (13)-β-D-glucan is sloughed and enters circulation of the host. This assay has been used in humans for the early detection of invasive fungal infections in immunocompromised patients². In humans this assay can detect picogram levels of (13)-β-D-glucan in serum. This assay does not detect certain fungal species that have zero to low levels of (13)-β-D-glucan in their cell wall including Cryptococcus and the Zygomycetes including Absidia, Mucor, and Rhizopus³. Time courses of serum (13)-β-D-glucan levels through the course of an infection have been shown to exhibit a rising level which falls in response to efficacious therapy¹. At present there are no established assay values specifically for cetaceans and therefore results were interpreted based on human guidelines. Normal human serum contains low levels of (13)- β-D-glucan, typically 10-40 pg/ml, presumably from commensal yeasts present in the gastrointestinal tract. Published human reference ranges are as follows: Negative (less than 60 pg/ml), Indeterminate (60-79 pg/ml), and Positive (greater than or equal to 80 pg/ml).

Fungitell^TM results from the Atlantic bottlenose dolphin on September 20, 2007 10 days after the beginning of clinical illness, was 277 pg/ml. Convalescent serum samples collected March 10, 2006 was 69 and September 11, 2007 <31 were negative, suggesting a current infection on September 20, 2007. Two other animals sharing the same pool had negative Fungitell values <31. Antifungal therapy was continued for approximately 3 months. During this time the erythrocyte sedimentation rate, absolute eosinophil count, and (13)- β-D-glucan levels decreased simultaneously. Antifungal therapy was discontinued 21 days post negative Fungitell assay, <31. Fungitell levels remained negative at least 2 months post discontinuation of Itraconazole and clinical signs have disappeared.

Further research to establish baseline values and clinical infection values for cetaceans is needed. These results suggest that this may be a useful tool for early diagnosis of invasive fungal disease and determining length of medical therapy as evaluated by clinical success. Because this is a non-specific fungal test further diagnostics should simultaneously be performed to identify the etiology of infection.

acknowledgements

The authors wish to thank Jim McBain for his assistance with providing diagnostic testing information, Malcolm Finkelman of Beacon Diagnostics for providing some diagnostic assays and special thanks to the staff at Florida's Gulfarium for providing excellent patient care and assistance with diagnostic tests.

References

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2.  Marty FM, Koo S, Bryar J, Baden LR. 2007. (13)-β-D-glucan assay positivity in patients with Pneumocystis (carinii) jiroveci pneumonia. Annals of Internal Medicine 147(1): 70-72.

3.  Odabasi Z, Paetznick VL, Rodriguez JR, Chen E, McGinnis MR, Ostrosky-Zeichner L. 2006. Differences in beta-glucan levels in culture supernatants of a variety of fungi. Medical Mycology 44: 267-272.