Rhabditid Nematode-Associated Ophthalmitis and Meningoencephalomyelitis in Captive Asian Horned Frogs (\u3ci\u3eMegophrys montana\u3c/i\u3e) [Case reports]

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 (LSD) and internal transcribed spacer (ITS) ribosomal DNA regions. Comparison of the LSD and ITS sequences to those deposited in GenBank revealed an exact match for Caenorhabditis elegans

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

Emerging Pathogen in Wild Amphibians and Frogs (Rana catesbeiana) Farmed for International Trade

Chytridiomycosis is an emerging disease responsible for global decline and extinction of amphibians. We report the causative agent, Batrachochytrium dendrobatidis, in North American bullfrogs (Rana catesbeiana) farmed for the international restaurant trade. Our findings suggest that international trade may play a key role in the global dissemination of this and other emerging infectious diseases in wildlife

Cutaneous Bacteria of the Redback Salamander Prevent Morbidity Associated with a Lethal Disease

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an infectious disease that causes population declines of many amphibians. Cutaneous bacteria isolated from redback salamanders, Plethodon cinereus, and mountain yellow-legged frogs, Rana muscosa, inhibit the growth of Bd in vitro. In this study, the bacterial community present on the skin of P. cinereus individuals was investigated to determine if it provides protection to salamanders from the lethal and sub-lethal effects of chytridiomycosis. When the cutaneous bacterial community was reduced prior to Bd exposure, salamanders experienced a significantly greater decrease in body mass, which is a symptom of the disease, when compared to infected individuals with a normal bacterial community. In addition, a greater proportion of infected individuals with a reduced bacterial community experienced limb-lifting, a behavior seen only in infected individuals. Overall, these results demonstrate that the cutaneous bacterial community of P. cinereus provides protection to the salamander from Bd and that alteration of this community can change disease resistance. Therefore, symbiotic microbes associated with this species appear to be an important component of its innate skin defenses

Do Frogs Get Their Kicks on Route 66? Continental U.S. Transect Reveals Spatial and Temporal Patterns of Batrachochytrium dendrobatidis Infection

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has been devastating amphibians globally. Two general scenarios have been proposed for the nature and spread of this pathogen: Bd is an epidemic, spreading as a wave and wiping out individuals, populations, and species in its path; and Bd is endemic, widespread throughout many geographic regions on every continent except Antarctica. To explore these hypotheses, we conducted a transcontinental transect of United States Department of Defense (DoD) installations along U.S. Highway 66 from California to central Illinois, and continuing eastward to the Atlantic Seaboard along U.S. Interstate 64 (in sum from Marine Corps Base Camp Pendleton in California to Naval Air Station Oceana in Virginia). We addressed the following questions: 1) Does Bd occur in amphibian populations on protected DoD environments? 2) Is there a temporal pattern to the presence of Bd? 3) Is there a spatial pattern to the presence of Bd? and 4) In these limited human-traffic areas, is Bd acting as an epidemic (i.e., with evidence of recent introduction and/or die-offs due to chytridiomycosis), or as an endemic (present without clinical signs of disease)? Bd was detected on 13 of the 15 bases sampled. Samples from 30 amphibian species were collected (10% of known United States' species); half (15) tested Bd positive. There was a strong temporal (seasonal) component; in total, 78.5% of all positive samples came in the first (spring/early-summer) sampling period. There was also a strong spatial component—the eleven temperate DoD installations had higher prevalences of Bd infection (20.8%) than the four arid (<60 mm annual precipitation) bases (8.5%). These data support the conclusion that Bd is now widespread, and promote the idea that Bd can today be considered endemic across much of North America, extending from coast-to-coast, with the exception of remote pockets of naïve populations

Seasonal Pattern of Batrachochytrium dendrobatidis Infection and Mortality in Lithobates areolatus: Affirmation of Vredenburg's “10,000 Zoospore Rule”

To fully comprehend chytridiomycosis, the amphibian disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), it is essential to understand how Bd affects amphibians throughout their remarkable range of life histories. Crawfish Frogs (Lithobates areolatus) are a typical North American pond-breeding species that forms explosive spring breeding aggregations in seasonal and semipermanent wetlands. But unlike most species, when not breeding Crawfish Frogs usually live singly—in nearly total isolation from conspecifics—and obligately in burrows dug by crayfish. Crayfish burrows penetrate the water table, and therefore offer Crawfish Frogs a second, permanent aquatic habitat when not breeding. Over the course of two years we sampled for the presence of Bd in Crawfish Frog adults. Sampling was conducted seasonally, as animals moved from post-winter emergence through breeding migrations, then back into upland burrow habitats. During our study, 53% of Crawfish Frog breeding adults tested positive for Bd in at least one sample; 27% entered breeding wetlands Bd positive; 46% exited wetlands Bd positive. Five emigrating Crawfish Frogs (12%) developed chytridiomycosis and died. In contrast, all 25 adult frogs sampled while occupying upland crayfish burrows during the summer tested Bd negative. One percent of postmetamorphic juveniles sampled were Bd positive. Zoospore equivalents/swab ranged from 0.8 to 24,436; five out of eight frogs with zoospore equivalents near or >10,000 are known to have died. In summary, Bd infection rates in Crawfish Frog populations ratchet up from near zero during the summer to over 25% following overwintering; rates then nearly double again during and just after breeding—when mortality occurs—before the infection wanes during the summer. Bd-negative postmetamorphic juveniles may not be exposed again to this pathogen until they take up residence in crayfish burrows, or until their first breeding, some years later

Nothing a Hot Bath Won't Cure: Infection Rates of Amphibian Chytrid Fungus Correlate Negatively with Water Temperature under Natural Field Settings

Dramatic declines and extinctions of amphibian populations throughout the world have been associated with chytridiomycosis, an infectious disease caused by the pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd). Previous studies indicated that Bd prevalence correlates with cooler temperatures in the field, and laboratory experiments have demonstrated that Bd ceases growth at temperatures above 28°C. Here we investigate how small-scale variations in water temperature correlate with Bd prevalence in the wild. We sampled 221 amphibians, including 201 lowland leopard frogs (Rana [Lithobates] yavapaiensis), from 12 sites in Arizona, USA, and tested them for Bd. Amphibians were encountered in microhabitats that exhibited a wide range of water temperatures (10–50°C), including several geothermal water sources. There was a strong inverse correlation between the water temperature in which lowland leopard frogs were captured and Bd prevalence, even after taking into account the influence of year, season, and host size. In locations where Bd was known to be present, the prevalence of Bd infections dropped from 75–100% in water <15°C, to less than 10% in water >30°C. A strong inverse correlation between Bd infection status and water temperature was also observed within sites. Our findings suggest that microhabitats where water temperatures exceed 30°C provide lowland leopard frogs with significant protection from Bd, which could have important implications for disease dynamics, as well as management applications

Ex situ diet influences the bacterial community associated with the skin of red-eyed tree frogs (Agalychnis callidryas)

Amphibians support symbiotic bacterial communities on their skin that protect against a range of infectious pathogens, including the amphibian chytrid fungus. The conditions under which amphibians are maintained in captivity (e.g. diet, substrate, enrichment) in ex situ conservation programmes may affect the composition of the bacterial community. In addition, ex situ amphibian populations may support different bacterial communities in comparison to in situ populations of the same species. This could have implications for the suitability of populations intended for reintroduction, as well as the success of probiotic bacterial inoculations intended to provide amphibians with a bacterial community that resists invasion by the chytrid fungus. We aimed to investigate the effect of a carotenoid-enriched diet on the culturable bacterial community associated with captive red-eyed tree frogs (Agalychnis callidryas) and make comparisons to bacteria isolated from a wild population from the Chiquibul Rainforest in Belize. We successfully showed carotenoid availability influences the overall community composition, species richness and abundance of the bacterial community associated with the skin of captive frogs, with A. callidryas fed a carotenoid-enriched diet supporting a greater species richness and abundance of bacteria than those fed a carotenoid-free diet. Our results suggest that availability of carotenoids in the diet of captive frogs is likely to be beneficial for the bacterial community associated with the skin. We also found wild A. callidryas hosted more than double the number of different bacterial species than captive frogs with very little commonality between species. This suggests frogs in captivity may support a reduced and diverged bacterial community in comparison to wild populations of the same species, which could have particular relevance for ex situ conservation projects

A novel copro-diagnostic molecular method for qualitative detection and identification of parasitic nematodes in amphibians and reptiles

© 2017 Huggins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Anthropogenic disturbance via resource acquisition, habitat fragmentation and climate change, amongst other factors, has led to catastrophic global biodiversity losses and species extinctions at an accelerating rate. Amphibians are currently one of the worst affected classes with at least a third of species categorised as being threatened with extinction. At the same time, they are also critically important for many habitats and provide man with a powerful proxy for ecosystem health by acting as a bioindicator group. Whilst the causes of synchronised amphibian losses are varied recent research has begun to highlight a growing role that macroparasites are playing in amphibian declines. However, diagnosing parasite infection in the field can be problematic, principally relying on collection and euthanasia of hosts, followed by necropsy and morphological identification of parasites in situ. The current study developed a non-invasive PCR-based methodology for sensitive detection and identification of parasitic nematode DNA released in the faeces of infected amphibians as egg or tissue fragments (environmental DNA). A DNA extraction protocol optimised for liberation of DNA from resilient parasite eggs was developed alongside the design of a novel, nematode universal, degenerate primer pair, thus avoiding the difficulties of using species specific primers in situations where common parasite species are unknown. Used in conjunction this protocol and primer pair was tested on a wide range of faecal samples from captive and wild amphibians. The primers and protocol were validated and detected infections, including a Railletnema nematode infection in poison dart frogs from ZSL London Zoo and Mantella cowani frogs in the wild. Furthermore, we demonstrate the efficacy of our PCR-based protocol for detecting nematode infection in other hosts, such as the presence of pinworm (Aspiculuris) in two tortoise species and whipworm (Trichuris muris) in mice. Our environmental DNA approach mitigates problems associated with microscopic identification and can be applied to detect nematode parasitoses in wild and captive hosts for infection surveillance and maintenance of healthy populations