MANAGEMENT OF ENTAMOEBA INVADENS IN THE HERPETOLOGICAL COLLECTION AT THE SINGAPORE ZOO.

Amebiasis caused by Entamoeba invadens is an important disease in reptile collections, causing severe morbidity and mortality. Surveillance of the parasite at the Singapore Zoo was carried out over a 4-yr period by PCR testing on reptiles that presented with lethargy and enteritis for disease investigation. Asymptomatic reptiles sharing the same enclosures as positive individuals were also tested as part of outbreak investigation. Animals in the collection that tested positive for the parasite were treated with metronidazole at various doses, with the addition of paromomycin for two cases, until a negative PCR test result was obtained at the end of the treatment course. A total of 97 samples from 49 individuals across 19 species of reptiles were obtained, of which 24 samples (24.7%) from 19 animals were positive for E. invadens. Of these positive samples, 11 samples were for disease investigation, eight samples for outbreak surveillance, and five samples for treatment monitoring. Treatment was initiated for 10 animals, four of which were showing clinical signs of disease. The parasite was cleared in nine of these 10 animals (90%), with eight animals receiving metronidazole as a sole therapeutic agent. A total of nine animals died of the disease, four of which (44.4%) presented dead or died within 24 h of presentation. Necrotizing enteritis was a consistent postmortem finding resulting in gastrointestinal perforation in two cases, and coelomic adhesions and hepatic trophozoites were each seen in five animals. The results suggest that the management of Entamoeba epizootics in the collection requires prompt outbreak investigation. Diagnosis of the disease with advanced diagnostic tools like PCR, endoscopy, and ultrasonography and treatment with metronidazole in both symptomatic and asymptomatic animals may reduce mortalities during an outbreak.

Infection with Nannizziopsis guarroi and Ophidiomyces ophiodiicola in reptiles in Taiwan.

Fungal infection is an emerging threat to reptiles. The main pathogens are fungi of the genera Nannizziopsis, Paranannizziopsis and Ophidiomyces. The clinical symptoms range from mild skin lesions to the dissemination of internal organs and even death. Most of the reported cases are from Europe, North America and Australia. In this study, we report the Nannizziopsis guarroi infection in one captive inland bearded dragon (Pogona vitticeps), one captive green iguana (Iguana iguana) and Ophidiomyces ophiodiicola infection in one wild red-banded snake (Dinodon rufozonatum) and one wild Chinese cobra (Naja atra) in Taiwan. The infections were confirmed by the presence of fungal elements in the tissue. The pathogens were identified based on their morphological and DNA sequence characteristics. The susceptibility profiles of the fungal strains to nine antifungal drugs were obtained using broth microdilution methods. The presence of both fungal species in Asia highlights the urgent need for surveillance and close monitoring of reptile infections to prevent them from spreading and to the possible collapse of reptile populations in the wild.

Disseminated Candidiasis and Candidemia Caused by Candida palmioleophila in a Green Sea Turtle (Chelonia mydas).

A sub-adult green sea turtle (Chelonia mydas) was rescued and treated for carapace and plastron shell fractures. The turtle was kept dry-docked for the first 2 months with a placement of a long-term jugular central venous catheter (CVC). Pain management, aggressive antibiotic and anthelmintic therapy, fluid therapy, force feeding, and wound debridement were provided to manage the shell fractures and control bacteremia. Human albumin was administered to treat severe hypoalbuminemia. On day 59, small budding yeasts were noted on the blood smears. Candidemia was confirmed by blood culture, as the yeasts were identified as Candida palmioleophila by the molecular multi-locus identification method. The CVC was removed, and the patient was treated with itraconazole. Although the carapace and plastron wounds had epithelized by 5.5 months after the rescue, the turtle died unexpectedly by 7.5 months. The postmortem examination revealed numerous necrogranulomas with intralesional yeasts, morphologically compatible with Candida spp., in joints, bones, brain, and lungs, suggestive of disseminated candidiasis. We describe a rare case of candidemia in the veterinary field. To our knowledge, this is the first report of candidiasis caused by C. palmioleophila in a reptile. The present results should improve veterinary medical care and, therefore, enhance the conservation of endangered sea turtle species.

Disseminated Fungal Infection and Fungemia Caused by Trichosporon asahii in a Captive Plumed Basilisk (Basiliscus plumifrons).

Trichosporon spp. are heavily arthroconidiating fungi and widely distributed in nature. Due to the similar fungal morphology, confusion among Trichosporon spp., Geotrichum spp., and Nannizziopsis spp. in reptiles is apparent and cannot be overlooked. Although few reptile Trichosporon isolates have been examined using the newer speciation criteria, the information on Trichosporon asahii in reptiles is still scarce. In the present study, we report the case of disseminated fungal infection and fungemia caused by T. asahii in a captive plumed basilisk (Basiliscus plumifrons). Multiple 0.2-0.5 cm, irregularly shaped, ulcerative nodules on the left hind foot were observed. The animal died due to the non-responsiveness to treatment. A microscopic evaluation revealed the fungal infection that primarily affected the left hind foot and right lung lobe with fungal embolisms in the lung and liver. The molecular identification of the fungal species by the DNA sequences of the ITS regions and D1/D2 gene from the fungal culture and ITS regions, from formalin-fixed paraffin-embedded (FFPE) lung tissues, were completely matched to those of T. asahii. The current report describes the first confirmed case of disseminated fungal infection and fungemia caused by T. asahii in a captive plumed basilisk.

Localization of goose haemorrhagic polyomavirus in naturally infected geese using in situ hybridization.

Goose haemorrhagic polyomavirus (GHPV) is the aetiological agent of haemorrhagic nephritis enteritis of geese (HNEG), a fatal disease that impacts geese and has been recorded only in Europe. The present study describes the first clinical cases of HNEG in Taiwan and the phylogenesis of Taiwanese GHPV, and it elucidates the pathogenesis of GHPV infection using in situ hybridization (ISH). The genomes of Taiwanese GHPV were highly similar to the previously reported strains. The diseased geese showed various degrees of vascular damage, especially in the digestive tract. The affected geese in the early stage showed transmural haemorrhagic enteritis in the intestine. In the middle to late stages, the most obvious lesion was hypoxic necrosis of renal tubules around intralobular central veins. Mineralization deposited in the kidney and systemic gout were also found. ISH revealed GHPV DNA in the vascular endothelial cells throughout the body, but not in the parenchymal cells of organs. Accordingly, the pathogenesis of GHPV infection was consistent with viral tropism in the endothelial cells. Specific attack of vascular endothelium by GHPV resulted in endothelial cell necrosis and subsequent increases of blood vessel permeability, as well as secondary circulation disorders, such as oedema, haemorrhage, and ischaemic necrosis in the adjacent parenchyma. RESEARCH HIGHLIGHTS Cell tropism of GHPV is determined by in situ hybridization. The tropism results in vascular dysfunction and subsequent pathobiology. Haemorrhagic nephritis and enteritis of geese described outside Europe for the first time.

Pulmonary hair embolism in a rescued free-ranging Eurasian otter Lutra lutra in Kinmen, Taiwan.

Eurasian otters Lutra lutra are listed as Near Threatened on the IUCN Red List and are imperiled by habitat loss, water pollution, and poaching. Harassment and attacks by stray animals are also recognized threats to the health of wild Eurasian otters. Pulmonary hair embolism is a possible complication in animals with deep traumatic injury, but to date no cases have been reported in wildlife. A free-ranging, adult male Eurasian otter was rescued due to severe emaciation and multiple bite wounds. The otter died 3 d after rescue and was necropsied. Grossly, a 1.5 × 1.5 × 1.5 cm firm nodule was observed in the left cranial lung lobe. Histologically, a fragment of hair shaft surrounded by multinucleated foreign body giant cells was observed in a medium-sized vein, and extensive eosinophilic infiltration was noted in the adjacent vascular wall and lung parenchyma. Based on the gross and histological findings, the pulmonary lesion was consistent with eosinophilic pneumonia and vasculitis induced by hair embolism. The presence of well-formed multinucleated foreign body giant cells and eosinophils may imply a late stage of foreign body reaction, and thus the presumptive source of hair embolism is an animal bite. This is the first report of pulmonary hair embolism associated with animal bite in a rescued free-ranging Eurasian otter.

Histopathology, Molecular Identification and Antifungal Susceptibility Testing of Nannizziopsis arthrosporioides from a Captive Cuban Rock Iguana (Cyclura nubila).

Chrysosporium-related fungi, the cause of superficial and deep mycoses, are an emerging infectious disease affecting not only reptiles but also immunocompromized humans. However, the information on Nannizziopsis arthrosporioides is extremely scarce. We herein characterized N. arthrosporioides isolated from a Cuban rock iguana (Cyclura nubila). Three skin ulcers were found in a Cuban rock iguana after captivity for 8 years. Microscopic examination revealed hyperplastic, hyperkeratotic, and ulcerative dermatitis coupled with numerous branched, septate fungal hyphae. The fungal culture yielded growth of zonate, felted cottony-powdery colonies with lobate margins on medium. Maximum-likelihood phylogeny tree based on the combined partial actin and partial ß-tubulin genes demonstrated that current isolates were mostly close to N. arthrosporioides. Furthermore, antifungal susceptibility test demonstrated that N. arthrosporioides had lowest minimal inhibitory concentration (MIC) values to isavuconazole, efinaconazole, and luliconazole, which may be the potential treatment of choice for N. arthrosporioides infection. The current study describes the first confirmed case of dermatomycosis caused by N. arthrosporioides of a captive reptile in Asia with detailed descriptions of the clinical, histopathological, and mycological features. The current findings provide new information on global distribution and host range of N. arthrosporioides and can raise the concern on the transboundary or emerging disease of N. arthrosporioides in Asian region.

Expression of HIF-1α and VEGF in feline mammary gland carcinomas: association with pathological characteristics and clinical outcomes.

BACKGROUND: The microenvironment within solid malignant tumors, including feline mammary gland carcinomas (FMGCs), is commonly hypoxic, possibly due to the lack of functional blood vessels in rapidly proliferating neoplastic tissue. Malignant cells can undergo genetic and adaptive changes that prevent them from dying due to oxygen deprivation through expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF). Therefore, HIF-1α and VEGF are ideal biomarkers for cancer therapy and prognostic evaluation. The aims of this study were to evaluate the expression of HIF-1α and VEGF in feline mammary carcinomas and analyze their correlations with clinical and pathological factors, such as clinical stage, histologic grading, regional metastasis, and overall survival rate. RESULTS: Paraffin-embedded tissue samples collected from 72 cats with FMGCs were retrospectively studied. Histologic pattern and histologic grading (Elston and Ellis grading system) of these FMGCs were determined. Our data indicated that grade II tubulopapillary carcinomas (43/72, 59.7%) prevailed in this study, and most FMCGs showed apparent necrosis, squamous metaplasia, and intratumoral stromal response. According to the results of immunohistochemical (IHC) stainings performed in tissue microarrays (TMAs), HIF-1α and VEGF overexpressions were respectively noted in 69.4% (50/72) and 77.8% (56/72) of FMGC cases. Chi-square test showed no correlation of HIF-1α overexpression with clinical and pathological factors. VEGF overexpression was significantly correlated with histologic pattern (p = 0.021), stromal response (p = 0.048), squamous metaplasia (p = 0.001), and lymphovascular invasion (p = 0.007). However, neither HIF-1α nor VEGF overexpression was correlated with histologic grading and metastasis. Of 38 cats with 1-year follow-up, IHC stainings of HIF-1α and VEGF were performed on whole tissue sections. The results showed that overexpression of HIF-1α was significantly correlated with the overall survival rate (p < 0.05) (log-rank test), whereas there was no significant correlation between VEGF overexpression and overall survival rate. CONCLUSIONS: This study suggests that the overexpression of HIF-1α may indicate poor prognosis/overall survival rate in cats with FMGCs. Developing compounds that inhibit HIF-1α may be a potential approach to FMGC treatment.

A case report of reptile-associated nidovirus (serpentovirus) in a ball python (Python regius) in Taiwan.

Reptile-associated nidoviruses (serpentoviruses) have been reported to cause proliferative interstitial pneumonia in pythons and other reptile species. A captive, younger than 2 years old, intact female ball python (Python regius) showed increased oral mucus, wheezing, and audible breathing with weight loss. Gross and microscopic examination revealed large amounts of mucus in the esophagus and proliferative interstitial pneumonia. Serpentovirus genes were detected from the lung tissues by polymerase chain reaction. The current serpentoviruses was phylogenetically grouped with the serpentovirus previously identified in the US. No case of serpentovirus infection has been reported in Asia. The present report provides information of complete genome sequence and global distribution of serpentovirus.

Fatal canine parvovirus-2 (CPV-2) infection in a rescued free-ranging Taiwanese pangolin (Manis pentadactyla pentadactyla).

Carnivore protoparvovirus 1 includes feline parvovirus (FPV), variants of canine parvovirus-2 (CPV-2), mink enteritis virus, and raccoon parvovirus, important pathogens affecting both wild and domestic carnivores. In this report, we described a fatal CPV-2 infection in a rescued Taiwanese pangolin, which provides the first evidence of CPV-2 infection in a non-carnivore. Post-rescue, the Taiwanese pangolin died from complications resulting from a severe panleucocytopenia and bloody diarrhoea. A full autopsy was performed and microscopic examination of the tissues revealed ulcerative, necrotizing, and haemorrhagic glossitis, esophagitis and enteritis. The results of transmission electronic microscopy, polymerase chain reaction and in situ hybridization provided confirmatory evidence that the lesions in the tongue, oesophagus and intestine were associated with a protoparvovirus. Phylogenetic comparison of the whole VP2 gene from the current pangolin protoparvovirus strain showed close clustering with the CPV-2c strains from domestic dogs in Taiwan, China and Singapore. The amino acid sequence of the pangolin protoparvovirus showed 100% identity to the CPV-2c strains from domestic dogs in China, Italy, and Singapore. The current findings highlight that pangolins are susceptible to protoparvoviruses. The potential of cross-species transmission of protoparvoviruses between Carnivora and Pholidota should be considered when housing pangolins in close proximity to carnivores and adopting strict biosecurity measures to avoid cross-species transmission in rescue facilities and zoos.

Retroperitoneal extraosseous peripheral primitive neuroectodermal tumor in a Formosan serow: case report and literature review.

A 10-y-old female captive Formosan serow (Capricornis swinhoei) was inactive and was azotemic. An autopsy was performed following her death, and multiple irregularly shaped, white-to-gray masses of 0.5-2 cm diameter were noted on both ureters, the left adrenal gland, urinary bladder, and uterus. Microscopically, organs were effaced by a poorly demarcated, highly infiltrative neoplasm, composed of neoplastic round cells arranged in islands, sheets, or nests with occasional rosette formation. The neoplastic cells were small: ≤2 red blood cell (≤ 15 µm) diameter. The neoplastic cells were positive for CD56, CK, FLI-1, and NSE, but negative for desmin, GFAP, melan A, NF, PAX-8, S100, synaptophysin, and vimentin. Therefore, the diagnosis of retroperitoneal extraosseous peripheral primitive neuroectodermal tumor (pPNET) was made. pPNET with FLI-1 expression has not been reported previously in animals, to our knowledge.

Concurrent leukoencephalomyelitis and polyneuritis in a Maltese terrier: resembling combined central and peripheral demyelination in humans.

A one-year-old male Maltese terrier presented with mild ataxia and disorientation for 4 months. Over time, clinical signs progressed from paraparesis to non-ambulatory tetraparesis, voice change and dysphagia. Histological examination revealed concurrent leukoencephalomyelitis and polyneuritis. Infectious etiologies, including dengue, Japanese encephalitis, Zika, canine distemper, pseudorabies, rabies, toxoplasmosis, neosporosis, leishmaniasis, and encephalitozoonosis, were ruled out by PCR and/or immunohistochemical (IHC) staining. IHC tested on neurological tissues highlighted a heterogeneous population of infiltrating T and B lymphocytes admixed macrophages. Therefore, this case was diagnosed with current leukoencephalomyelitis and polyneuritis, resembling combined central and peripheral demyelination (CCPD), an autoimmune inflammatory demyelinating disease affecting both the CNS and PNS in humans.

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus).

BACKGROUND: Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect animals at high trophic levels, such as cetaceans and humans, via the food chain. Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in the modulation of immune system and can be classified into two types: Th1 and Th2. Th1/Th2 balance can be evaluated by the ratios of their polarizing cytokines (i.e., interferon [IFN]-γ/Interleukin [IL]-4), and animals with imbalanced Th1/Th2 response may become more susceptible to certain kinds of infection. Therefore, the present study evaluated the in vitro cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP20) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). METHODS: Blood samples were collected from six captive common bottlenose dolphins (Tursiops truncatus). The cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses. The cytokines evaluated included IL-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. The geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ß2-microglobulin (B2M), of each sample were determined and used to normalize the mRNA expression levels of target genes. RESULTS: The ratio of late apoptotic/necrotic cells of cPBMCs significantly increased with or without concanavalin A (ConA) stimulation after 24 h of 10 µg/ml C-AgNP20 treatment. At 4 h of culture, the mRNA expression level of IL-10 was significantly decreased with 1 µg/ml C-AgNP20 treatment. At 24 h of culture with 1 µg/ml C-AgNP20, the mRNA expression levels of all cytokines were significantly decreased, with the exceptions of IL-4 and IL-10. The IFN-γ/IL-4 ratio was significantly decreased at 24 h of culture with 1 µg/ml C-AgNP20 treatment, and the IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 µg/ml C-AgNP20 treatment, respectively. Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 µg/ml C-AgNP20 after 24 h of culture. DISCUSSION: The present study demonstrated that the sublethal dose of C-AgNP20 (≤1 µg/ml) had an inhibitory effect on the cytokine mRNA expression levels of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased the mRNA expression level of TNF-α. Th2 cytokine bias is associated with enhanced immunity against parasites but decreased immunity to intracellular microorganisms. TNF-α is a contributing factor for the inflammatory response against the infection of intracellular pathogens. In summary, our data indicate that C-AgNP20 suppresses the cellular immune response and thereby increases the susceptibility of cetaceans to infection by intracellular microorganisms.

Immunotoxicity of Silver Nanoparticles (AgNPs) on the Leukocytes of Common Bottlenose Dolphins (Tursiops truncatus).

Silver nanoparticles (AgNPs) have been extensively used and are considered as an emerging contaminant in the ocean. The environmental contamination of AgNPs is expected to increase greatly over time, and cetaceans, as the top ocean predators, will suffer the negative impacts of AgNPs. In the present study, we investigate the immunotoxicity of AgNPs on the leukocytes of cetaceans using several methods, including cytomorphology, cytotoxicity, and functional activity assays. The results reveal that 20 nm Citrate-AgNPs (C-AgNP20) induce different cytomorphological alterations and intracellular distributions in cetacean polymorphonuclear cells (cPMNs) and peripheral blood mononuclear cells (cPBMCs). At high concentrations of C-AgNP20 (10 and 50 µg/ml), the time- and dose-dependent cytotoxicity in cPMNs and cPBMCs involving apoptosis is demonstrated. C-AgNP20 at sub-lethal doses (0.1 and 1 µg/ml) negatively affect the functional activities of cPMNs (phagocytosis and respiratory burst) and cPBMCs (proliferative activity). The current study presents the first evidence of the cytotoxicity and immunotoxicity of AgNPs on the leukocytes of cetaceans and improves our understanding of environmental safety concerning AgNPs. The dose-response data of AgNPs on the leukocytes of cetaceans are invaluable for evaluating the adverse health effects in cetaceans and for proposing a conservation plan for marine mammals.

A blastema-predominant canine renal nephroblastoma with gingival metastasis: case report and literature review.

Nephroblastomas are uncommon embryonal tumors in dogs. We report herein a blastema-predominant nephroblastoma with gingival metastasis in an 8-y-old Miniature Pinscher dog. Histologically, the mass was composed mainly of blastemal elements with minor epithelial and mesenchymal differentiation. Metastatic masses in the gingiva had histologic and immunohistochemical features similar to those of the primary renal nephroblastoma. Neoplastic cells were extensively positive for both vimentin and PAX8, and scattered positive for cytokeratin. Using the clinical staging of human Wilms tumor, we staged our case as stage IV with <4 mo of survival time. We summarized previous studies of canine renal and spinal nephroblastomas, and analyzed the correlations among clinical staging, histologic classification, and mean survival time of dogs with renal nephroblastomas. Clinical staging was significantly correlated with survival time, as shown in humans. In dogs, however, additional factors can potentially influence the outcome of treatment and disease development.

Investigation of silver (Ag) deposition in tissues from stranded cetaceans by autometallography (AMG).

Silver, such as silver nanoparticles (AgNPs), has been widely used in commercial products and may be released into the environment. The interaction between Ag deposition and biological systems is raising serious concerns because of one health consideration. Cetaceans, as the top predators of the oceans, may be exposed to Ag/Ag compounds and suffer negative health impacts from the deposition of these compounds in their bodies. In the present study, we utilized autometallography (AMG) to localize the Ag in the liver and kidney tissues of cetaceans and developed a model called the cetacean histological Ag assay (CHAA) to estimate the Ag concentrations in the liver and kidney tissues of cetaceans. Our results revealed that Ag was mainly located in hepatocytes, Kupffer cells and the epithelial cells of some proximal renal tubules. The tissue pattern of Ag/Ag compounds deposition in cetaceans was different from those in previous studies conducted on laboratory rats. This difference may suggest that cetaceans have a different metabolic profile of Ag, so a presumptive metabolic pathway of Ag in cetaceans is advanced. Furthermore, our results suggest that the Ag contamination in cetaceans living in the North-western Pacific Ocean is more severe than that in cetaceans living in other marine regions of the world. The level of Ag deposition in cetaceans living in the former area may have caused negative impacts on their health condition. Further investigations are warranted to study the systemic Ag distribution, the cause of death/stranding, and the infectious diseases in stranded cetaceans with different Ag concentrations for comprehensively evaluating the negative health effects caused by Ag in cetaceans.

Locally extensive meningoencephalitis caused by Miamiensis avidus (syn. Philasterides dicentrarchi) in a zebra shark.

Scuticociliatosis, caused by ciliated protozoa in the subclass Scuticociliatia of the phylum Ciliophora, can cause fatal disease in teleost fish species. However, information on scuticociliatosis in elasmobranchs is still scarce. In this report, we describe a case of locally extensive meningoencephalitis caused by Miamiensis avidus (syn. Philasterides dicentrarchi) in a 2 yr old captive zebra shark Stegostoma fasciatum. Granulocytic meningoencephalitis was observed through histological assessment. Inflammation was confined to the ventral aspect of the brain with a large number of ciliated protozoa, transforming into non-suppurative meningitis in the lateral aspect, and gradually vanished in the dorsal aspect. No histopathological and polymerase chain reaction (PCR) evidence of systemic dissemination of M. avidus was found. PCR targeting the gene coding the small-subunit ribosomal RNA (SSUrRNA) of M. avidus was performed on the brain, liver, and gill tissues, and only brain tissue yielded a positive result. The DNA sequences from amplicons of the protozoal SSUrRNA gene were completely matched to that of M. avidus. The distribution of protozoa in the current case was mainly located in the brain and suggests the possibility of a direct neural invasive pathway of M. avidus through the nasal cavity/ampullary system and/or a unique tissue tropism of M. avidus specific to the brain in zebra sharks. Further investigations on the pathogenesis of M. avidus in elasmobranchs, especially zebra sharks, are needed.