Confirmed Cases of Ophidiomycosis in Museum Specimens from as Early as 1945, United States

Ophidiomycosis represents a conservation threat to wild snake populations. The disease was reported in North America early in the 21st century, but the history of ophidiomycosis has not been investigated. We examined museum specimens and confirmed cases of ophidiomycosis >50 years before the disease’s reported emergence.

ognized as major threats to global biodiversity, causing population declines and extinction events in a variety of host species (1). Ophidiomyces ophidiicola, the causative agent of ophidiomycosis, is one such pathogen recognized as a conservation threat to wild snakes (2). The disease first gained attention in 2008 when fatal infections emerged in eastern massasauga rattlesnakes (Sistrurus catenatus) in Illinois, USA (3), and has since been documented throughout North America and Europe (2,4). The earliest retrospective detection of O. ophidiicola in snakes was from 2000 (5). We report the earliest known confirmed cases of ophidiomycosis in free-living snakes in the United States, dating back to 1945.
We investigated the historical occurrence of ophidiomycosis in snakes in the United States by examining specimens preserved in formalin or ethanol at the University of Wisconsin Zoological Museum (UWZM; Madison, WI, USA) and Morehead State University Museum Collection (Morehead, KY, USA). We visually examined 524 specimens representing 30 snake species from 19 states in the eastern United States collected during 1900-2012 (Appendix 1, https://wwwnc.cdc.gov/EID/article/27/7/20-4864-App1.xlsx). To reduce risk for cross-contamination, we first examined snakes for clinical signs of ophidiomycosis within the glass jars in which they were stored. When specimens were removed from the jars for sampling, new gloves were worn to handle Ophidiomycosis represents a conservation threat to wild snake populations. The disease was reported in North America early in the 21st century, but the history of ophidiomycosis has not been investigated. We examined museum specimens and confirmed cases of ophidiomycosis >50 years before the disease's reported emergence.
Clinical signs of ophidiomycosis are not pathognomonic, and a confirmed diagnosis requires compatible histopathologic lesions and the detection of O. ophidiicola (6). Because these confirmatory steps involve destructive sampling of museum material, we selected a subset of snakes (n = 12) for these analyses. We targeted specimens with large (>0.5 cm 2 ) or multiple skin lesions from distant geographic areas and collected >25 years before the reported 2008 emergence of ophidiomycosis (3) (Table). From selected snakes, we excised and formalin-fixed portions of lesioned skin, routinely processed them for light microscopy, and stained with periodic acid-Schiff and Grocott methenamine silver methods. We also collected small pieces of lesioned skin (≈4 mm 2 ) for PCR-based detection of O. ophidiicola. We extracted DNA from dehydrated tissue by using the Gentra Puregene Tissue Kit (QIAGEN, https://www.qiagen.com); we used 10 µL of the kit-provided proteinase K per sample.  Negative controls consisted of blank extractions. For PCR, we used existing primers that specifically target the internal transcribed spacer region (ITS) of O. ophidiicola (6) and a newly designed PCR assay that targets mitochondrial NADH dehydrogenase subunit 1 (nad1) (Appendix 2, https://wwwnc.cdc.gov/EID/ article/27/7/20-4864-App2.pdf). We targeted these 2 loci, which exist at high copy numbers in the genome, because amplifiable DNA was expected to be at low abundance in the preserved specimens. We cloned and sequenced PCR amplicons of the appropriate size to confirm the presence of O. ophidiicola. We conducted tissue collection, DNA extraction, and PCR under strict protocols (e.g., unidirectional workflow and regular decontamination of work surfaces and equipment) to prevent contamination of samples.
Of the 12 snakes subjected to histopathological analyses, 7 (58.3%) had microscopic lesions with intralesional fungi consistent with ophidiomycosis (6) (Table; Figure). We detected DNA from O. ophidiicola in 3 (50%) of the 6 specimens from UWZM that had been stored in 70% ethanol (Table). We did not detect DNA of O. ophidiicola in snakes from the Morehead State University Museum Collection (n = 6), likely because these specimens were stored long-term in formalin, which is known to affect the recovery of amplifiable nucleic acid. These results highlight the importance of targeting specimens stored in ethanol rather than formalin for molecular-based detection of pathogens in archival material.
We amplified the ITS target from 2 of the 3 specimens and nad1 target from all 3 specimens; these sequences were 100% identical to existing O. ophidiicola sequences in GenBank. The 3 additional specimens from UWZM were strongly suspected to represent cases of ophidiomycosis on the basis of the presence of arthroconidia in histologic sections of lesioned skin (6); however, fungal DNA from these specimens may not have been suitable for PCR amplification. Negative controls performed as expected. The 3 PCR-positive specimens met the diagnostic criteria for confirmed cases of ophidiomycosis (6); they were collected in Florida in 1945, Wisconsin in 1958, and Tennessee in 1973 (Table). These cases predate the earliest previously known detection of O. ophidiicola in free-living snakes in North America by as much as 55 years (5).
Museum specimens can provide crucial insights into the history of emerging infectious diseases. Preserved animal specimens have been used to trace the origin and spread of other fungal pathogens, such as Pseudogymnoascus destructans (white-nose syndrome in bats) and Batrachochytrium spp. (chytridiomycosis in amphibians) (8-10). By using a similar approach, we demonstrate that ophidiomycosis was circulating in the eastern United States for decades before its recognition as an emerging disease. Future work focusing on how such factors as climate change, environmental disturbance, and underlying health of snake populations influence ophidiomycosis dynamics might reveal the mechanism by which ophidiomycosis is emerging (2).  (1)(2)(3) and also in humans (3). We describe an outbreak affecting 120 lactating buffaloes and 40 lactating cows in Kannivadi, Navapatti, Alathuranpatti, Maniakaranpatti, Muthukumaranpatti, S.Pudur, and E.Chittor, Dindigul district; and in Krishnarayapuram, Karur district, in Tamil Nadu, India in 2004. Pock lesions (0.5-1 cm diameter) were seen over the bodies of lactating buffaloes but restricted to only the udder and teats of lactating cows (Figure, panel A). Buffalopox did not cause death in the animals we reviewed; it affected more buffaloes (30%-50%) than cows (20%-30%). Suckling calves developed pock lesions on the forehead, lips, and mouth. Three milkers who worked with the affected animals experienced multiple pock lesions (1 cm diameter) on the fingers, interdigital webs, wrist and forearm (Figure, panel B) and generalized effects including fever (100°F) and enlargement of axillary lymph nodes.
To investigate the causative agent, we used existing clinical samples. Scab samples were collected randomly from 20 affected animals (both buffaloes and cows). We examined scab suspensions under transmission electron microscope (EM) at 80 KV and inoculated the suspension in BHK21 cell line for virus isolation. We examined scab homogenates and cell culture fluid by PCR for differential diagnosis of cowpox virus and VACV infection (4). EM revealed typical brick-shaped pox virus particles of ≈290 × 270 nm with irregularly arranged superficial filaments formed by tubules (Figure, panel C). After 2 blind passages, we noticed in BHK21 cell lines cytopathogenic effects such as cellular rounding, cellular fusion, and intracytoplasmic inclusion bodies ( Figure, panel D) after 48-60 hours of infection; PCR analysis revealed the causative agent to be VACV.
During the global eradication of smallpox, strains of VACV were used as vaccine. VACV infection sometimes transmitted from the vasicular lesion of vaccinae to domestic animals, usually cattle; in turn, infected animals transmitted VACV to susceptible humans (5). Several outbreaks in cattle and humans that were thought to be cowpox were in fact caused by VACV (1,6,7). The infected animals were treated with parenteral injection of antimicrobial drugs for 1 week to control secondary bacterial infection and an antiinflammatory drug for 3 days to reduce pain and inflammation. Animal workers were also advised to clean the animals' lesions with 1% potassium permanganate solution followed by tropical application over the pock lesions with indigenous product of neem leaf extract and turmeric powder suspended in glycerin. Individual animals recovered in ≈1 month. Similarly affected humans were diagnosed at primary healthcare centers and treated with oral antimicrobial drugs and analgesics for 1 week, which reduced pain and pustules. Healing was complete in 3 weeks' time.