Abstract
Mucormycotic infections caused by Apophysomyces spp. have been reported in multiple genera of cetaceans.1,2 These infections are aggressive and rapidly fatal making early detection and treatment paramount for survival.1,3,4 Mucormycosis infections can be diagnosed histologically, with definitive diagnosis of Apophysomyces spp. requiring culture and species identification.3,4 The aim of our research is to develop an accurate and sensitive enzyme-linked immunosorbent assay (ELISA) for the early detection of Apophysomyces spp. infections in cetaceans. In this study, archived sera from bottlenose dolphins, Tursiops truncatus, and a Pantropical spotted dolphin, Stenella attenuata, were evaluated to determine sensitivity and specificity of a newly developed ELISA.
Archived serum samples from bottlenose dolphins and a Pantropical spotted dolphin were obtained from two repositories in Florida. A total of 73 serum samples from 60 individual dolphins were analyzed. Serum samples representing 8 Apophysomyces positive dolphins, 7 dolphins with other fungal diseases, 15 dolphins with non-fungal diseases, and 30 healthy dolphins, were evaluated. All positive samples were confirmed positive for Apophysomyces spp. by culture and/or by histology. Serum samples from each culture positive dolphin had been collected at various time points following the appearance of clinical signs, which generally included anorexia and an elevated erythrocyte sedimentation rate. Apophysomyces infections were classified as either acute or subacute. Two dolphins with acute infections survived less than 5 days after the onset of clinical symptoms, while six dolphins with prolonged illness survived 5 days or more after the onset of clinical symptoms.
Results from this initial feasibility study found the test to be quite sensitive, with 100% of culture positive serum samples from 6 dolphins with prolonged disease testing "positive" and the 2 dolphins with acute illness testing "suspect". In 4 of the 8 dolphins, serum samples were obtained in the earliest stages of illness, some as soon as day 1. Early diagnosis was possible in the majority of these cases, with 3 of 4 dolphins classified as "positive" or "suspect" within four days of the appearance of clinical signs. Serum samples from the 4 remaining dolphins had been collected 7 days or more after the development of clinical symptoms, and all samples resulted in "strong positive" titers. The test specificity was 95%, due to cross-reactivity from two dolphins with a related mucormycoticfungal infection which classified these dolphins as "suspect". However, these animals showed little increase in titer over a period of several weeks to several months, as opposed to rapid titer increases observed in all true positives. No false positives occurred from serum samples of healthy dolphins or those with non-fungal illnesses.
With this work, antibodies to Apophysomyces spp. were detected in the sera of dolphins for the first time. While additional studies are necessary to validate sensitivity and specificity of the ELISA, current results are promising that the assay may offer valuable information for the early detection and long-term monitoring of treatment effectiveness for mucormycosis caused by Apophysomyces spp. in dolphins.
Acknowledgements
The authors wish to thank Deanna Sutton and the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio for providing fungal cultures used in the development of this assay.
References
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