Diseases of New Zealand native frogs

Shaw, Stephanie D. (2012) Diseases of New Zealand native frogs. PhD thesis, James Cook University.

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The aims of this project were to evaluate the health status of New Zealand native frogs (Leiopelma spp.) and to investigate what diseases, if any, were limiting their survival both in captivity and in the wild. Issues for captive frogs included nutritional and infectious diseases. For wild frogs I investigated the occurrence of declines and conducted mapping and experimental studies on chytridiomycosis.

Mortality rates and causes of death were analysed for 252 wild-caught Leiopelma spp. that were held in captivity in a research program at the University of Canterbury and later transferred to other institutions between 2000 and 2006. Leiopelma archeyi and Leiopelma hochstetteri had similar overall average mortality by year (12.4% and 14.9% respectively) but different yearly mortality patterns, whereas Leiopelma pakeka had much lower overall mortality (3.5%).

On further investigation, metabolic bone disease (MBD) was diagnosed in L. archeyi and L. hochstetteri in 2008 at three institutions: Auckland Zoo, Hamilton Zoo, and the University of Otago. 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%). Micro-computed 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 was consistent with osteofluorosis. On histology of the same femurs there was hyperplasia, periosteal growth, and thickening of trabeculae which was 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. To determine the natural diet of Leiopelma spp., stomach contents of sixteen L. archeyi from the Coromandel and nine L. hochstetteri from the Coromandel, the Hunua Ranges and Maungatautari were analysed. Both species ate a wide range of invertebrates including springtails, mites, ants, parasitic wasps, amphipods and isopods, while L. 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). Analysis of long-standing husbandry practices showed that ultraviolet-B exposure and the dietary calcium:phosphorus ratio was deficient when compared with wild conditions – likely attributing to chronic underlying MBD.

Two novel nematodes (Koerneria sp. and Rhabditis sp.) were found separately in four captive Archey's frogs showing clinical signs of haemorrhagic purulent nasal discharge and weight loss. One of these frogs also had a novel protozoal infection (Tetrahymena) in the nasal cavity. 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.

Multifocal small domed lesions occurred extensively on the ventral skin of captive Leiopelma archeyi at two institutions between 2000 and 2012. Incidence was 41% (34/83) of frogs at Auckland Zoo and 9% (1/11) at the University of Otago and were not linked with an increased risk of death. The lesions had the gross and microscopic characteristics of adenomatous hyperplasia (AH) of the dermal mucous glands which are widely distributed over the skin of normal Archey's frogs. In affected frogs the size and location of lesions varied over time, even resolved completely in some animals, and sometimes reappeared. Histologically the lesions were composed of enlarged mucous glands that expanded the dermis and elevated the epidermis. They were semi-organized, with occasional acinar structures with central lumina sometimes containing mucus. Nuclei had moderate anisokaryosis and mitotic figures were uncommon. The aetiology of this adenomatous hyperplasia is unknown, but factors associated with the captive environment are most likely.

Surveys were distributed to New Zealand land users in 1998 and 2008 to acquire information about the distribution and population levels of both native (Leiopelma spp.) and non-native (Litoria spp.) frogs. Overall frog populations in New Zealand were reported as declining, but were stable or increasing in a few regions. Possible causes reported for declines were disease (chytridiomycosis), increase in agriculture and an increase in the distribution of predatory fish.

The current distribution, host species and prevalence where known of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) is reported in New Zealand. I conducted histology and PCR on new and archived specimen and also collated previous test records. The data included all regions in New Zealand and six off shore islands at 135 sites with 704 records from over eleven contributors spanning collection dates 1930-2010. The earliest case was from 1999 and we report 132 positive individuals from 54 widespread sites. Bd was detected in all three non-native Litoria spp. in five out of sixteen regions but not in the six off-shore areas tested. Bd was not detected in native L. hochstetteri, L. hamiltoni and L. pakeka. Included in the data is a museum survey of 152 individuals from five species from 1930-1999 using histology and Bd specific immunohistochemistry. All museum specimens were negative. In L. archeyi at a study site in the Coromandel Ranges, the prevalence of Bd from 2006-2010 was relatively stable at 14-18%. The prevalence of Bd in Whareorino has remained both consistent and low (<50% for the 95% confidence interval upper limit) between 2005-2010.

An experiment infection trial revealed L. archeyi may be innately resistant to chytridiomycosis. Six wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. All six L. archeyi became reinfected at low intensities, but rapidly self-cured, most by two weeks. In contrast another species, Litoria ewingii, developed severe chytridiomycosis when exposed to the same inoculum.

As inhibition by skin bacteria has been suggested as a factor in resistance to Bd, I investigated baseline cutaneous bacterial flora in native NZ frogs. Ninety-two unique bacterial isolates were identified from the ventral skin of 62 apparently healthy L. archeyi and L. hochstetteri frogs from the Coromandel and Whareorino regions in New Zealand were identified using DNA extraction and polymerase chain reaction techniques. A New Zealand strain of Bd (KVLe08SDS1) was also isolated for the first time from a Litoria ewingii from the Dunedin area. This Bd strain was used against 21 bacterial isolates in an in vitro challenge assay to test for Bd inhibition. One bacterial isolate, a Flavobacterium sp., inhibited the growth of Bd. This positive result may indicate that cutaneous bacteria are part of the innate immunity of L. archeyi against chytridiomycosis and is the first report of its kind in Leiopelma spp.

In conclusion, captive leiopelmatids had high mortality rates due to inadequate husbandry. To prevent multi-factorial MBD in captive Leiopelma spp., dietary calcium should be increased, exposure to ultraviolet-B light increased and de-fluoridated water used as a minimum standard. Attempting to recreate natural diets and conditions will improve the chances of establishing a healthy breeding collection. Chytridiomycosis was not identified as a cause of death in any captive cases. Amphibian chytrid is geographically widespread in New Zealand and has been found in all Litoria spp. and L. archeyi. Populations of L. archeyi infected with Bd appear to be stable at present and as individuals self-cured when reinfected in captivity, this species appears to have some natural resistance to chytridiomycosis. In contrast, populations of non-native Litoria spp. have generally declined. Cutaneous bacteria of L. archeyi may play a role in their innate immunity. Bd has not been found in any other leiopelmatids despite widespread testing. Hence chytridiomycosis does not appear to be a current threat to L. archeyi or L. hochstetteri, although further surveys are needed to understand population impacts on L. archeyi. The continued use of field hygiene protocols to reduce the risk of introducing Bd (or new strains in the case of populations where it is already present) or other pathogens to threatened frog populations are recommended.

This project has exemplified the importance of integrating the baseline data obtained from healthy wild-caught frogs to aid disease investigation of captive frogs. It also demonstrates the value of both clinical disease experience and an ecological viewpoint when investigating and managing disease in wildlife.

Item ID: 24600
Item Type: Thesis (PhD)
Keywords: amphibians; Archey's frog; diseases; frog populations; frogs; Hochstetter's frog; infections; Leiopelma archeyi; Leiopelma hochstetteri; Leiopelmatidae; metabolic bone disease (MBD); native frogs; New Zealand;
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Additional Information:

This is a thesis by publication. A published article included as chapter 5 has been redacted from the thesis due to copyright restrictions. The full thesis may either be requested via document delivery at your local library or viewed in the Eddie Koiki Mabo Library at JCU, Townsville.

Eight publications arising from this thesis are stored in ResearchOnline@JCU, at the time of processing. Please see the Related URLs. The publications are:

Chapter 2: Shaw, Stephanie, and Holzapfel, Avi (2008) Mortality of New Zealand native frogs in captivity. DOC Research and Development Series, 295. pp. 1-30.

Chapter 3: Shaw, S.D., Skerratt, L.F., Kleinpaste, R., Daglish, L., and Bishop, P. J. (2012) Designing a diet for captive native frogs from the analysis of stomach contents from free-ranging Leiopelma. New Zealand Journal of Zoology, 39 (1). pp. 47-56.

Chapter 4: Shaw, Stephanie D., Bishop, Phillip J., Harvey, Catherine, Berger, Lee, Skerratt, Lee F., Callon, Karen, Watson, Maureen, Potter, John, Jakob-Hoff, Richard, Goold, Mike, Kunzmann, Nicole, West, Peter, and Speare, Rick (2012) Fluorosis as a probable factor in metabolic bone disease in captive New Zealand native frogs (Leiopelma species). Journal of Zoo and Wildlife Medicine , 43 (3). pp. 549-565.

Chapter 5: Shaw, Stephanie, Speare, Richard, Lynn, Denis H, Yeates, Gregor, Zhao, Zeng, Berger, Lee, and Jakob-Hoff, Richard (2011) Nematode and ciliate nasal infection in captive Archey's frogs (Leiopelma archeyi). Journal of Zoo and Wildlife Medicine, 42 (3). pp. 473-479.

Chapter 7: Shaw, Stephanie, Berger, Lee, Bell, Sara, Dodd, Sarah, James, Tim Y., Skerratt, Lee F., Bishop, Phillip J., and Speare, Rick (2014) 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). Journal of Wildlife Diseases, 50 (4). p. 732.

Chapter 8: Shaw, Stephanie D., Skerratt, Lee F., Haigh, Amanda, Bell, Ben D., Daglish, Lisa, Bishop, Phillip J., Summers, Rachel, Moreno, Virginia, Melzer, Sabine, Ohmer, Michel, Herbert, Sarah, Gleeson, Dianne, Rowe, Lucy, and Speare, Richard (2013) The distribution and host range of Batrachochytrium dendrobatidis in New Zealand, 1930–2010. Ecology, 94 (9). p. 2108.

Chapter 9: Shaw, Stephanie D., Bishop, Phillip J., Berger, Lee, Skerratt, Lee Francis, Garland, Stephen, Gleeson, Dianne M., Haigh , Amanda, Herbert, Sarah, and Speare, Rick (2010) Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms, 92 (2-3). pp. 159-163.

Chapter 10: Shaw, Stephanie, Berger, Lee, Bell, Sara, Dodd, Sarah, James, Tim Y., Skerratt, Lee F., Bishop, Phillip J., and Speare, Rick (2014) 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). Journal of Wildlife Diseases, 50 (4). p. 732.

Date Deposited: 18 May 2016 04:33
FoR Codes: 05 ENVIRONMENTAL SCIENCES > 0502 Environmental Science and Management > 050202 Conservation and Biodiversity @ 50%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0702 Animal Production > 070204 Animal Nutrition @ 50%
SEO Codes: 96 ENVIRONMENT > 9604 Control of Pests, Diseases and Exotic Species > 960499 Control of Pests, Diseases and Exotic Species not elsewhere classified @ 50%
96 ENVIRONMENT > 9608 Flora, Fauna and Biodiversity > 960805 Flora, Fauna and Biodiversity at Regional or Larger Scales @ 50%
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