Key fungal diseases of Australian reptiles Dr Joshua Llinas The Unusual Pet Vets Jindalee Shop 1/62 Looranah Street, Jindalee QLD, 4074 Introduction With an ever-expanding differential list for dermal lesions in reptiles, it is important for the clinician to be across emerging conditions. This presentation will discuss important fungal diseases found in Australian reptiles, their clinical presentation, pathogenesis, and the diagnostic approach and treatment. The focus will be on the group previously referred to as Chrysosporium anamorph of Nannizziopsis vriesii now reassigned to the Order Onygenaceae 23, the fungal pathogen that is responsible for “yellow fungus disease” and the recently described microsporidia, Encephalitozoon pogonae 32. Finally, there will a brief discussion on lesser diagnosed fungi, Aspergillus spp, Basidiobolus spp. Geotrichium spp., Paecilomyces spp., and Trichophyton spp. Onygenaceae- Yellow Fungus Disease and the CANV complex Previously Chrysosporium anamorph of Nannizziopsis vriesii, this pathogen has undergone a taxonomy reassignment to the Order Onygenaceae30. Three groups, Nannizziopsis and Paranannizziopsis in lizards along with Ophidiomyces in snakes, they contain at least 16 species of pathogenic fungi to reptiles, all causing deep dermal lesions12,2,4,27. Of these, the most frequently isolated in Australia are two of the nine currently known species of Nannizziopsis, Nannizziopsis barbata, and less commonly detected in Australia, Nannizziopsis guarroi . The list of lizard species with confirmed infection of N. barbata has recently been expanded to include, free living and captive Australian Eastern Water dragon (Intellagama lesueurii), central bearded dragon (Pogona vitticeps), Coastal bearded dragon (Pogona barbata), Tommy Round head lizard (Diporiphora australis), a captive Eastern Blue tongue skink (Tiliqua scincoides), Centralian blue tongue skink (Tiliqua multifasciata ), and a Kimberly rock monitor (Varanus glauerti)27,20,5. The recently reported outbreaks of fungal dermatomycoses in multiple colonies of Eastern water dragons in South East Queensland with Nannizziopsis most similar to N. barbata highlights the need for the veterinary community to be aware of this condition (N.Peterson and C. Frere Personal Communication Dec 2018). Pathogenesis Nannizziopsis spp have been found to be a primary pathogen reptiles that results in a dermatomycosis of the skin and mucocutaneous junctions27. Reptile skin has an outer layer of keratin which is the preferred substrate for these fungi. In experimentally infected veiled Chameleons (Chamaeleo calyptratus), although damaged skin increased the risk of infection, this was not required for infection to take hold fulfilling Koch’s postulate and indicating it is a primary pathogen 21. Increased environmental or social stressors such as overcrowding, suboptimal temperatures or humidity, poor hygiene which all lead to suppressed immune system, likely contribute to the pathogenicity in both captive and free- living reptiles 28. Very little is known about the epidemiology of these pathogens, but reports suggest it is not a common part of the microbiota of the skin of reptiles 22 but, is found naturally in the environment and substrates 4. Proceedings of VetFest 2020 Llinas, J – Key fungal diseases of Australian reptiles 224 Gross appearance Typical lesions range from hyperkeratotic and granulomatous to moist, vesicular, liquified necrosis due to the keratinolytic activity of this genus of fungi4,12,21-23,30. The lesions range in colour from yellow to tan-brown giving rise to the colloquial description, “Yellow Fungus Disease”. Initial lesions may appear as a small, superficial discolouration when the keratin layer of the stratum corneum is infected. Lesions can appear anywhere on the dermis or mucocutaneous junctions with a predilection for the head, ventral abdomen, limbs and tail27,20,12. If left to progress , penetration into the muscle and bone can occur5. The author has observed the eastern water dragons to have more hyperkeratotic lesions while captive bearded dragons display more, liquified necrosis when in advanced stages of the disease process. Systemic dissemination is rare but does occur, with one report demonstrating it had spread to the liver4. Diagnosis Definitive diagnosis is made through a combination of histopathology, cultures and PCR testing30,20. Histopathological findings of intralesional fungal hyphae and granulomas would suggest further investigation is warranted. Cultures can be performed and if Nannizziopsis is suspected, it is important to communicate to the lab and pathologist as the need for lower incubation temperatures (25-35C) as higher temperatures can restrict growth resulting in false negatives20. Increased incubation times of 2-3 weeks have been suggested due to slower growth at these lower temperatures20. Recent development of highly sensitive and specific PCR assays has made definitive diagnosis possible by this method 30,5,27. Treatment options A combination of surgical debridement, topical and systemic treatments may be required to effectively treat these pathogens27. Surgery to debulk larger lesions may allow topical antifungal preparations to work more effectively but this has not been thoroughly investigated. Topical preparations commonly suggested including 0.125% chlorhexidine, 2% chlorhexidine, terbinafine and miconazole and povidone iodine1,34. Systemic treatment with a triazole is the mainstay of treatment, with ketoconazole, itraconazole and voriconazole most frequently used27,10,34. Voriconazole appears to be more affective and carries fewer side effects than the other triazoles although side effects are still possible34. Response to treatments have been varied with combination of surgery and voriconazole showing the most promise at this stage Collection management is focused around optimal husbandry and diet, cleanliness and disinfection of the environment. Proper quarantine procedures should be in place to minimse the risk of transmissible. Using easily replaceable substrate is required to ensure frequent changing is possible will also help with reduced infection rates. Stress reduction in both captive and free-living populations is paramount to minimising susceptibility. Microsporidia in Pogona vitticeps Microsporidia are obligate, intracellular pathogenic fungi with over 1300 species described and have been distributed into approximately 200 genera 8,26,35,6. Microsporidia have a small, fast evolving genome that lack many of the genes that would allow them to live independently 17. Although initially categorized as a protozoan 37, publication of a microsporidium genome 13 and investigations into the phylogeny of their alpha and beta tubulins 16,15, suggest a fungal origin. Proceedings of VetFest 2020 Llinas, J – Key fungal diseases of Australian reptiles 225 Pathogenesis Affected individuals are usually immunocompromised 7,8,31,36 and a number of clinical signs have been reported including lethargy, anorexia, diarrhoea, weight loss, and sudden death 36,11,19,14. Gross appearance of lesions The lesions are seen as multiple small white to pale yellow nodules or larger granulomatous lesions with visible necrosis which may be widely disseminated throughout the organs. Diagnosis Due to the non-specific clinical signs seen with microsporidiosis 32, a diagnosis is generally made on post mortem examination. Microsporidium spores have been detected by light microscopy 3, electron microscopy 9, immunohistochemistry 11, and PCR 25,24. Multiple cases of ovarian granulomas have been reported with the author recently finding two individuals from two separate collections after large masses were palpated on routine physical exams. These masses were confirmed by histopathology and PCR to be the microsporidia E. pogonae. It is difficult to recommend screening tests as the validity in asymptomatic animals has not been clarified and further studies are required to determine the normal prevalence in reptiles. However, if granulomatous lesions are present, a combination of histopathology, blood and PCR can be used to confirm a diagnosis of microsporidiosis. Molecular techniques have been used to identify microsporidia to the genus level in only a few reptile cases with three genera being reported: Encephalitozoon, Heterosporis and Pleistophora of which only Encephalitozoon has so far been described in P. vitticeps 32. Histological lesions are typically granulomatous with necrotic foci. Lesions have been reported in in a wide variety of tissues including muscle, testes, ovaries, colon, kidneys, the oral cavity, liver, spleen, lungs, adrenals and fat bodies 33,14,25. The yolk of hatchlings born to parents with confirmed microsporidia was found to contain the organism, indicating the potential for vertical transmission 19. In a report by Sokolova et al. 32, sequence analysis of a microsporidium found in a P.vitticeps suggested it was a separate species, unique to reptiles, and it was named Encephalitozoon pogonae 29. The author has confirmed E. pogonae in two Australian captive collections by histopathology, PCR testing and DNA sequencing. Antemortem detection of E. pogonae was also possible in blood and oral- cloacal swabs. Treatment Localised lesions such as those found on the nictitans have been treated with a combination of surgery and fenbendazole18. To date, the two unpublished cases of granulomatous disease of the ovaries were treated with a combination of surgery and fenbendazole with one patient dying shortly after