Allan P. Pessier

Cutaneous Chytridiomycosis in Poison Dart Frogs (Dendrobates spp.) and White's Tree Frogs (Litoria Caerulea)

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EXPERIMENTAL TRANSMISSION OF CUTANEOUS CHYTRIDIOMYCOSIS IN DENDROBATID FROGS

In a series of three experiments during March–October, 1998, two species of captive-bred poison dart frogs (Dendrobates tinctorius and D. auratus) were exposed to Batrachochytrium dendrobatidis, a recently-described chytridiomycete fungus (chytrid) that was originally isolated from a blue poison dart frog (D. azureus). All frogs exposed to the chytrids developed a fatal skin disease, whereas none of the control frogs developed skin lesions. The most consistent clinical sign in chytrid-exposed frogs was excessive shedding of skin. Gross lesions were subtle, usually affected the legs and ventrum, and consisted of mild skin thickening and discoloration. Microscopic examination of shed skin pieces and/or skin imprints demonstrated the presence of chytrids and was used for ante mortem and post mortem confirmation of chytrid infection. Histologically, there was epidermal hyperkeratosis, hyperplasia, and hypertrophy associated with low to moderate numbers of chytrids in the keratinized layers. These experiments demonstrated that Batrachochytrium dendrobatidis can be a fatal pathogen in poison dart frogs. The experimentally-induced disease in these frogs resembled cases of cutaneous chytridiomycosis that have recently been described in several other species of captive and wild amphibians.

Intersexuality and the cricket frog decline: historic and geographic trends.

Exposure to anthropogenic endocrine disruptors has been listed as one of several potential causes of amphibian declines in recent years. We examined gonads of 814 cricket frogs (Acris crepitans) collected in Illinois and deposited in museum collections to elucidate relationships between the decline of this species in Illinois and the spatial and temporal distribution of individuals with intersex gonads. Compared with the preorganochlorine era studied (1852–1929), the percentage of intersex cricket frogs increased during the period of industrial growth and initial uses of polychlorinated biphenyls (PCBs) (1930–1945), was highest during the greatest manufacture and use of p,p-dichlorodiphenyltrichloroethane (DDT) and PCBs (1946–1959), began declining with the increase in public concern and environmental regulations that reduced and then prevented sales of DDT in the United States (1960–1979), and continued to decline through the period of gradual reductions in environmental residues of organochlorine pesticides and PCBs in the midwestern United States (1980–2001). The proportion of intersex individuals among those frogs was highest in the heavily industrialized and urbanized northeastern portion of Illinois, intermediate in the intensively farmed central and northwestern areas, and lowest in the less intensively managed and ecologically more diverse southern part of the state. Records of deposits of cricket frog specimens into museum collections indicate a marked reduction in numbers from northeastern Illinois in recent decades. These findings are consistent with the hypothesis that endocrine disruption contributed to the decline of cricket frogs in Illinois.

Hepatic Hemorrhage, Hemocoelom, and Sudden Death due to Haemoproteus Infection in Passerine Birds: Eleven Cases

Haemoproteus spp. are ancient apicomplexan hemoparasites that have undergone extensive coevolution with their natural hosts and are typically species specific, with inapparent or minimal pathogenicity. A promiscuous genotype of Haemoproteus capable of undergoing host switching on a familial level was identified. This protozoan caused severe disease with high mortality in 6 species of exotic passerine birds housed in California at the San Diego Zoos Wild Animal Park: Surinam crested oropendola (Psarocolius decumanus decumanus), Guianan turquoise tanager (Tangara mexicana mexicana), blue-necked tanager (Tangara cyanicollis caeruleocephala, Guianan red-capped cardinal (Paroaria gularis gularis), magnificent bird of paradise (Diphyllodes magnificus hunsteini), and superb bird of paradise (Lophorina superba). The birds had few or no clinical signs. Necropsy findings consisted of hemocoelom and irregularly scattered areas of hemorrhage and hepatocellular necrosis. Affected areas of liver contained solitary protozoal megaloschizonts in varied states of degeneration and peripheral nonsuppurative inflammation. No other parasite life stages were found in parenchymal organs or blood smears. Polymerase chain reaction using consensus primers for an avian malarial mitochondrial cytochrome B gene segment was positive in all cases. Sequencing and BLAST analysis identified the protozoan as a Haemoproteus sp. related to Haemoproteus spp. found in asymptomatic passerine birds native to North America. In situ hybridization was performed in 3 animals with a mitochondrial cytochrome B probe and was positive only in megaloschizonts. These findings suggest the recognition of a genotype of Haemoproteus that exhibits high levels of host infidelity and causes severe disease in captive birds exotic to North America.

West Nile Virus Infection in Free-Ranging Squirrels in Illinois

West Nile virus (WNV) infection was diagnosed in 13 gray squirrels (Sciurus carolinensis) and 3 fox squirrels (Sciurus niger) that were observed with neurologic signs before death or found dead. All 16 had gliosis throughout all sections of the brain. Most had lymphoplasmacytic encephalitis or meningoencephalitis, many with admixed neutrophils. Neuronal necrosis and neuronophagia were also prominent features. West Nile virus antigen was demonstrated in the brain, spleen, heart or kidney in 10 of 13 gray squirrels and 3 of 3 fox squirrels by immunohistochemistry. Nucleic acid amplification tests (NAATs) confirmed the presence of WNV in the brain or spinal cord of 10/10 gray squirrels and 1/3 fox squirrels tested. Viral levels were quantified in various tissues of selected gray squirrels, and titers were highest in spleen and brain, with no virus detected in serum. This is the first description of lesions associated with WNV infection in gray and fox squirrels.

Treatment of chytridiomycosis with reduced-dose itraconazole

Effective treatment methods to eliminate infection with Batrachochytrium dendrobatidis (Bd) are required for development of sustainable captive survival assurance populations of amphibians and to reduce the risk of introducing Bd to new locations as part of amphibian trade or reintroduction programs. Treatment with itraconazole baths at 100 mg l-1 is commonly used in captive amphibians, but side effects are observed in some amphibian species and life stages. Naturally occurring outbreaks of chytridiomycosis in Wyoming toads Anaxyrus baxteri and Whites tree frogs Litoria caerulea were treated with lower-dose itraconazole baths (e.g. 50 mg l-1 for Whites tree frogs) and followed post-treatment with serial Taqman PCR testing to confirm elimination of Bd infection. Post-treatment PCR tests were consistently negative for the presence of Bd and treatment was deemed successful. Although this was not a controlled clinical trial, results suggest that lower doses of itraconazole may be effective for treatment of chytridiomycosis with resulting cost savings to amphibian conservation programs and a potential for a reduction in dose-related side effects from itraconazole treatment. Prospective clinical trials of alternative itraconazole treatment protocols are encouraged.

Isolation of a Bohle-like iridovirus from boreal toads housed within a cosmopolitan aquarium collection

A captive survival assurance population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy. Preliminary DNA sequence analysis of the MCP, DNA polymerase, and neurofilament protein (NFP) genes demonstrated highest identity with Bohle iridovirus (BIV). A virus, tentatively designated zoo ranavirus (ZRV), was subsequently isolated, and viral protein profiles, restriction fragment length polymorphism analysis, and next generation DNA sequencing were performed. Comparison of a concatenated set of 4 ZRV genes, for which BIV sequence data are available, with sequence data from representative ranaviruses confirmed that ZRV was most similar to BIV. This is the first report of a BIV-like agent outside of Australia. However, it is not clear whether ZRV is a novel North American variant of BIV or whether it was acquired by exposure to amphibians co-inhabiting the same facility and originating from different geographic locations. Lastly, several surviving toads remained PCR-positive 10 wk after the conclusion of the outbreak. This finding has implications for the management of amphibians destined for use in reintroduction programs, as their release may inadvertently lead to viral dissemination.

Neospora caninum Infection in a Free-Ranging Raccoon (Procyon lotor) with Concurrent Canine Distemper Virus Infection

During a canine distemper virus (CDV) outbreak in raccoons (Procyon lotor) from Cook County, Illinois, a juvenile female suffering from seizures was killed and necropsied. Gross and histologic findings of necrotizing encephalitis and proliferative bronchopneumonia were attributed to CDV infection and considered the cause of clinical signs. A section of cerebellum stained immunohistochemically for Neospora caninum revealed an approximately 40 μm diameter, round to oval cyst with a 2- to 3-μm-thick wall and filled with 1–2 μm diameter, round to oval bradyzoites. Polymerase chain reaction (PCR) results were positive for N. caninum using DNA extracted from the brain. Specific PCR for the closely related organisms Toxoplasma gondii and Hammondia heydorni yielded negative results. This case report provides histologic, immunohistochemical, and molecular evidence that raccoons are a naturally occurring intermediate host of N. caninum.

Rhabditid nematode-associated ophthalmitis and meningoencephalomyelitis in captive Asian horned frogs (Megophrys montana).

Between 2006 and 2008, 4 captive Asian horned frogs (Megophrys montana) were diagnosed with ocular and neurologic disease associated with rhabditid nematodiasis. Mortality, either spontaneous or by humane euthanasia, was high (3/4, 75%). Gross and histologic findings included varying degrees of ulcerative keratitis, histiocytic uveitis and retinitis, meningoencephalomyelitis, and epidermal chromatophore (iridophore) hyperplasia with intralesional nematodes. Entry into the host was presumed to be by direct invasion of the skin and the cornea with migration through the optic nerve to the brain and spinal cord. One frog was diagnosed with rhabditid nematodiasis antemortem, and clinical signs and lesions in the frog did not progress after unilateral enucleation and anthelminthic treatment were completed. Gross and tissue morphology of the nematodes were consistent with the order Rhabditida. DNA was extracted separately from 2 individual nematodes that were isolated from frozen and ethanol-preserved eye and brain tissue. These DNA templates were used for polymerase chain reaction amplification and sequencing of nuclear 28S large subunit (LSU) and internal transcribed spacer (ITS) ribosomal DNA regions. Comparison of the LSU and ITS sequences to those deposited in GenBank revealed an exact match for Caenorhabditis elegans.

Survey for the Pathogenic Chytrid Fungus Batrachochytrium dendrobatidis in Southwestern North Carolina Salamander Populations

Batrachochytrium dendrobatidis is a fungal pathogen responsible for a potentially fatal disease of amphibians. We conducted a survey for B. dendrobatidis in the Appalachian Mountains of southwestern North Carolina, USA, from 10 June to 23 July 23 2009. Ventral skin swabs were collected from plethodontid salamanders (n=278) and real-time PCR was performed to test for the presence of B. dendrobatidis. We found no evidence of B. dendrobatidis, suggesting that B. dendrobatidis is absent or present in such low levels that it was undetected. If B. dendrobatidis was present at the time of our sampling, this survey supports evidence of low prevalence of B. dendrobatidis in North American headwater stream salamander populations.