Stephanie D. Shaw

Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens

Microbial symbionts of vertebrate skin have an important function in defense of the host against pathogens. In particular, the emerging chytrid fungus Batrachochytrium dendrobatidis, causes widespread disease in amphibians but can be inhibited via secondary metabolites produced by many different skin-associated bacteria. Similarly, the fungal pathogens of terrestrial salamander eggs Mariannaea elegans and Rhizomucor variabilis are also inhibited by a variety of skin-associated bacteria. Indeed, probiotic therapy against fungal diseases is a recent approach in conservation medicine with growing experimental support. We present a comprehensive Antifungal Isolates Database of amphibian skin-associated bacteria that have been cultured, isolated, and tested for antifungal properties. At the start, this database includes nearly 2000 cultured bacterial isolates from 37 amphibian host species across 18 studies on five continents: Africa, Oceania, Europe, and North and South America. As the research community gathers information on additional isolates, the database will be updated periodically. The resulting database can serve as a conservation tool for amphibians and other organisms, and provides empirical data for comparative and bioinformatic studies. The database consists of a FASTA file containing 16S rRNA gene sequences of the bacterial isolates, and a metadata file containing information on the host species, life-stage, geographic region, and antifungal capacity and taxonomic identity of the isolate.

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis

The susceptibility of Archeys frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.

FLUOROSIS AS A PROBABLE FACTOR IN METABOLIC BONE DISEASE IN CAPTIVE NEW ZEALAND NATIVE FROGS (LEIOPELMA SPECIES)

This report describes the investigations into the cause and treatment of metabolic bone disease (MBD) in captive native New Zealand frogs (Leiopelma spp.) and the role of fluoride in the disease. MBD was diagnosed in Leiopelma archeyi and Leiopelma hochstetteri in 2008 at three institutions: Auckland Zoo, Hamilton Zoo, and the University of Otago. Most of these frogs had originally been held at the University of Canterbury for several years (2000-2004) but some were collected directly from the wild. Radiographs on archived and live frogs showed that MBD had been present at Canterbury, but at a lower rate (3%) than in the current institutions (38-67%). Microcomputed tomography showed that the femoral diaphyses of the captive frogs at Auckland Zoo had greater bone volume, bone surface, cross-sectional thickness, and mean total cross-sectional bone perimeter, which is consistent with osteofluorosis. On histology of the same femurs, there was hyperplasia, periosteal growth, and thickening of trabeculae, which are also consistent with skeletal fluorosis. An increase in fluoride levels in the water supply preceded the rise in the incidence of the above pathology, further supporting the diagnosis of osteofluorosis. Analysis of long-standing husbandry practices showed that ultraviolet B (UVB) exposure and the dietary calcium:phosphorus ratio were deficient when compared with wild conditions-likely causing chronic underlying MBD. To prevent multifactorial MBD in captive Leiopelma, the authors recommend increasing dietary calcium by incorporating into the captive diet inherently calcium-rich invertebrates; increasing exposure to natural or artificial (UVB) light; and using defluoridated water. Addressing these three factors at Auckland Zoo reduced morbidity, bone fractures, and mortality rates.

BASELINE CUTANEOUS BACTERIA OF FREE-LIVING NEW ZEALAND NATIVE FROGS (LEIOPELMA ARCHEYI AND LEIOPELMA HOCHSTETTERI) AND IMPLICATIONS FOR THEIR ROLE IN DEFENSE AGAINST THE AMPHIBIAN CHYTRID (BATRACHOCHYTRIUM DENDROBATIDIS)

Abstract Knowledge of baseline cutaneous bacterial microbiota may be useful in interpreting diagnostic cultures from captive sick frogs and as part of quarantine or pretranslocation disease screening. Bacteria may also be an important part of innate immunity against chytridiomycosis, a fungal skin disease caused by Batrachochytrium dendrobatidis (Bd). In February 2009, 92 distinct bacterial isolates from the ventral skin of 64 apparently healthy Leiopelma archeyi and Leiopelma hochstetteri native frogs from the Coromandel and Whareorino regions in New Zealand were identified using molecular techniques. The most-common isolates identified in L. archeyi were Pseudomonas spp. and the most common in L. hochstetteri were Flavobacterium spp. To investigate the possible role of bacteria in innate immunity, a New Zealand strain of Bd (Kaikorai Valley-Lewingii-2008-SDS1) was isolated and used in an in vitro challenge assay to test for inhibition by bacteria. One bacterial isolate, a Flavobacterium sp., inhibited growth of Bd. These results imply that diverse cutaneous bacteria are present and may play a role in the innate defense in Leiopelma against pathogens, including Bd, and are a starting point for further investigation.

Designing a diet for captive native frogs from the analysis of stomach contents from free-ranging Leiopelma

Abstract Diets for captive amphibians are often inadequate and lead to poor health. To determine the natural diet of two New Zealand frog species, we analysed the stomach contents of 16 Archeys frogs (Leiopelma archeyi) from the Moehau Range of the Coromandel Peninsula and nine Hochstetters frogs (Leiopelma hochstetteri) from the Moehau Range of the Coromandel Peninsula, the Hunua Ranges and Maungatautari. These specimens were obtained as by-catch from invertebrate pitfall traps from 2002 to 2008. Both species ate a wide range of invertebrates including springtails, mites, ants, parasitic wasps, amphipods and isopods. Leiopelma archeyi also ate snails. The mean ratio of maximum prey size ingested to snout–vent length in L. archeyi was 0.31 (range 0.16–0.5), and in L. hochstetteri was 0.42 (range 0.21–0.75). We suggest a reformulated captive diet based on the species and size of invertebrates ingested in the wild. This diet may assist in the prevention of metabolic bone disease.

Nematode and Ciliate Nasal Infection in Captive Archey's Frogs (Leiopelma archeyi)

Abstract:  Archeys frogs (Leiopelma archeyi) are first on the list of evolutionarily distinct and globally endangered (EDGE) amphibians. Captive breeding is an important strategy for protection of the species, but programs are hampered by a lack of information on diseases present in wild and captive populations. Two novel nematodes (Koerneria sp. and Rhabditis sp.) were found separately in four captive Archeys frogs showing clinical signs of hemorrhagic purulent nasal discharge and weight loss. One of these frogs also had a novel protozoal infection (Tetrahymena) in the nasal cavity. Koerneria, Rhabditis, and Tetrahymena have not previously been reported in amphibians in New Zealand. One frog was treated successfully with oral moxidectin at 0.4 mg/kg for the nematode infection and topical metronidazole at 10 mg/kg for the protozoal infection. The clinical signs abated only after both infections were cleared. The second frog died before treatment could be established. The third and fourth frogs were found dead.

The distribution and host range of Batrachochytrium dendrobatidis in New Zealand, 1930–2010

Chytridiomycosis caused by the fungal invasive pathogen Batrachochytrium dendrobatidis (Bd) was first detected in 1999 in Christchurch, New Zealand, in the Australian introduced frog species Litoria raniformis. It was detected in wild native frogs in the critically endangered Leiopelma archeyi in 2001 on the Coromandel Peninsula and has been suggested as responsible for a mass decline (88%) in that population between 1994 and 2002. We report the current distribution, host species and prevalence, where known, of Bd in New Zealand, which is essential for conservation management of New Zealand native frogs (Leiopelma spp.). The data set is structured so that it can be readily added to the Australian Bd database for further analyses. Our data included all regions in New Zealand and six offshore islands at 135 sites with 704 records from 23 contributors spanning collection dates 1930–2010. We report 54 positive sites from 132 positive individuals. We also detail negative findings, but declaring an area free from disease should consider the sensitivity of the test used and numbers of individuals tested. The data also included a comprehensive museum survey testing 152 individuals from five species (20 L. archeyi, 50 L. hochstetteri, 15 L. aurea, 40 L. ewingii, and 27 L. raniformis) from 1930–1999 using histology and Bd-specific immunohistochemistry. All museum specimens were negative, so the 1999 positive result is still the earliest record. In the L. archeyi Coromandel Ranges population, the period prevalence of Bd from 2006 to 2010 was relatively stable at 16%, but the number of animals tested remains low (up to N = 19) due to the now depleted population numbers. The period prevalence of Bd in the L. archeyi Whareorino population has remained both consistent and low (6%) between 2005 and 2010. In L. hochstetteri, L. hamiltoni, and L. pakeka all sampling for Bd has been negative. Positive Bd results have been found in all three Litoria spp., but Bd has not been found in the six offshore areas tested. Most data have been previously unpublished and represent the first confirmed reports of Bd in many regions and species in New Zealand.

Adenomatous hyperplasia of the mucous glands in captive Archey’s frogs (Leiopelma archeyi)

Abstract AIMS: To describe the gross and light microscopic characteristics of skin lesions observed on the ventral skin of captive Archey’s frogs (Leiopelma archeyi) between 2000 and 2012, and to investigate their occurrence, possible aetiology and association with survival. METHODS: Postmortem skin samples were obtained for histological evaluation from 37 frogs, with and without skin lesions, that died while in captivity at Auckland Zoo between 2000 and 2012. Four frogs with skin lesions were biopsied under general anaesthesia and samples used for both light and transmission electron microscopy. The records of 94 frogs held at the University of Otago and Auckland Zoo between 2000–2012 were reviewed, which included some frogs recently collected from the wild. Information about the occurrence of skin lesions, and mortality associated with skin lesions was collated. RESULTS: Grossly the skin lesions varied in appearance; most were circular, pale grey papules, which measured from <0.5–1.5 mm in diameter with no umbilication. The overlying epidermis was not fragile and there was no associated inflammation. Contents often appeared clear or semi-transparent. Lesions were located predominantly on ventral surfaces including trunk, thighs, lower legs and forearms, and gular region, but not on digits. The number ranged from single to multiple, often confluent lesions covering the entire ventral surface of the frog. Histologically the lesions consisted of enlarged proliferating mucous glands that expanded the dermis and elevated the epidermis. They were semi-organised, solid or occasionally cavitated acinar structures with central lumina which sometimes contained mucus. Nuclei showed moderate anisokaryosis and mitotic figures were uncommon. Transmission electron microscopy did not show any infectious agents. Between 2000 and 2012, skin lesions were recorded in 35/94 (37%) frogs. The size and location of skin lesions varied over time, with some resolving and sometimes reappearing. Skin lesions were not associated with an increased risk of death. CONCLUSIONS: The skin lesions had the gross and microscopic characteristics of adenomatous hyperplasia of the dermal mucous glands. CLINICAL RELEVANCE: The aetiology of this adenomatous hyperplasia is unknown, but factors associated with the captive environment are the most likely cause. This is the first description of adenomatous hyperplasia of the cutaneous mucous glands in amphibians.

Historical trends in frog populations in New Zealand based on public perceptions

Surveys were distributed to New Zealand land users in 1998 and 2008 to acquire information about New Zealand frogs with the aim of compiling and mapping their distribution and inferred population trends without costly and time-consuming field surveys. The overall frog population trend was reported as declining, with possible causes reported as an increase in agriculture, an increase in the distribution of predatory fish and disease. The resultant maps could be used for four main purposes: 1) to identify regions where Litoria populations are known to occur, which can be eliminated when considering suitable regions for translocation of Leiopelma; 2) to identify growing or stable populations of Litoria species, which may assist future disease surveys, population monitoring and to identify sources of genetic material that may serve as an Ark for declining Australian populations; 3) to highlight populations that are in decline to enable effective targeting of detailed disease studies; and 4) to approximate the stability of amphibian populations in the absence of more accurate, but costly, scientific monitoring.