Abstract
Bottlenose dolphins both in the wild and in managed-care facilities are known to suffer infections from numerous fungal agents, including Aspergillus, Blastomyces, Cryptococcus and Candida spp.1–3 The most reported symptoms in animals in human care are correlated to respiratory, skin and gastrointestinal infections.4–6
In the present case, a group of seven adult dolphins (5 females, 2 males) was monitored biweekly for blow, gastric and fecal cytology as part of the preventive medicine program. At the end of 2019, yeasts were observed in fecal cytologies of 3 animals. Fecal cultures confirmed the presence of different multi-resistant strains of Candida spp. Due to the lack of symptomatology and absence of susceptibility to antifungals, the animals were not treated with antimicrobials but started to receive daily probiotics to support their immune system.
All animals were examined monthly, and fecal culture was performed at least every 4 months to monitor the situation. In March 2021, one female presented with sudden anorexia. After hydration, it was possible to collect dark and very mucoid feces from this animal. Cytology showed abundant presence of pseudohyphae. Culture resulted positive for Candida albicans resistant to terbinafine, all the azoles and polyenes tested. Meanwhile, two other animals had respiratory symptoms with harsh cough, presence of peri-blowhole exfoliative dermatopathy, and behavior suggesting pruritus. Other 4 animals resulted positive to repeated cultures but never showed any symptoms. Out of 7 animals sharing the same pools, 4 animals tested positive to C. albicans, 2 females to C. glabrata and 1 male to C. tropicalis. All the strains were tested for susceptibility and showed multi-resistance patterns.
As none of the tested antifungals was eligible for the treatment on its own, combination therapy was considered. In vitro tests were performed to identify possible synergic activities. Four antifungals were chosen based on their described use in bottlenose dolphins: voriconazole, fluconazole, nystatin and terbinafine. Minimum inhibitory concentration (MIC) of each drug was tested and fractional inhibitory concentration (FIC) of 3 combinations of drugs was estimated with the checkerboard assay.7 The effect produced by each combination was categorized as synergism, additivity or indifference, and antagonism. Therapy was selected based on the lowest MIC value of the single drug or FIC value predicting synergic effect of the drug-drug combination.
Considering in vitro results, animals presenting C. albicans and C. tropicalis were treated with combination therapy of fluconazole (2 mg/kg PO, BID) and terbinafine (2 mg/kg PO, SID), while animals with C. glabrata were treated with nystatin (8000 IU/Kg PO, TID). The antifungal treatment plan started in March 2021 and lasted between 39 and 170 days. Treatments were discontinued after two bi-weekly negative fecal cultures. After treatments, all animals tested negative for Candida spp. Fluconazole and terbinafine showed a high MIC when tested alone, but a synergic FIC value. The combination therapy increased the inhibitory activity of the two drugs in vivo.
MIC breakpoints and pharmacokinetics of the drugs used are not currently available for bottlenose dolphins and further studies are ongoing to obtain these data in vivo.
Acknowledgments
The authors wish to thank the marine mammal staff from Oltremare.
Literature cited
1. Reidarson TH, McBain JF, Dalton LM, Rinaldi MG. 2001. Mycotic diseases. In: Dierauf, LA, Gulland FMD. (Eds.), CRC Handbook of Marine Mammal Medicine. Boca Raton (FL): CRC Press. p 337–353.
2. Reif JS, Fair PA, Adams J, Joseph B, Kilpatrick D, Sanchez R, Goldstein JD, McCulloch SD, Mazzoil M, Zolman ES, Hansen LJ, Bossart GD. 2008. Health status of bottlenose dolphins (Tursiops truncatus) from the Indian River Lagoon, FL and Charleston, SC. J. Am. Vet. Med. Assoc. 233: 299–307.
3. Morris PJ, Johnson WR, Pisani J, Bossart GD, Adams J, Reif JS, Fair PA. 2011. Isolation of culturable microorganisms from free-ranging bottlenose dolphins (Tursiops truncatus) from the southeastern United States. Veterinary Microbiology 148(2–4):24.
4. Marques GN, Silva NU, Leal MO, Flanagan CA. 2021. The use of posaconazole delayed-release tablets in the successful treatment of suspected mucormycosis in a bottlenose dolphin (Tursiops truncatus) calf. Medical Mycology Case Reports 32: 77–80.
5. Delaney MA, Terio KA, Colegrove KM, Briggs MB, and Kinsel MJ. 2012. Occlusive Fungal Tracheitis in 4 Captive Bottlenose Dolphins (Tursiops truncatus). Veterinary Pathology 50(1): 172–176.
6. Haulena M, Huff D, Ivančić M, Muhammad M, Hoang L, Zabek E, Raverty S. 2010. Intestinal Torsion Secondary to Chronic Candidiasis Caused by Candida krusei in a Pacific White-Sided Dolphin (Lagenorhynchus obliquidens). 41st Annual IAAAM conference proceedings, Vancouver, Canada.
7. Hall MJ, Middleton RF, Westmacott D. 1983. The fractional inhibitory concentration (FIC) index as a measure of synergy. J Antimicrob Chemother 11(5):427–33.