ECO-EPIDEMIOLOGY OF AMPHIBIAN HEALTH AND DISEASE IN MINNESOTA

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: TERMINATED
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1007228
• Project No.: MIN-62-084
• Project Start Date: Jul 24, 2015
• Project End Date: Jun 30, 2017

Project Director
Travis, D.

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Veterinary Population Medicine

Non Technical Summary
Amphibians are an important part of global biodiversity and provide important ecosystem services such as new peptide/drug discovery and as environmental indicators for land use change and agricultural and urban/rural chemical use. However, they are facing a serious extinction crisis worldwide and are among the most threatened vertebrates on the planet. According to the IUCN's 2008 Global Amphibian Assessment "nearly one-third (32 %) of the world's 6,000 amphibian species are known to be threatened or extinct." Drivers of this decline include habitat loss and fragmentation, invasive species, climate change, and environmental contaminants, and most importantly infectious diseases such as Batrachochytrium dendrobatidis (Bd) and Ranavirus spp. The ultimate goal of this proposal is to pilot an approach to understanding the eco-epidemiology of these two diseases in Minnesota. It will be conducted through a unique new collaboration between UMN CVM, the Amphibian Disease Laboratory of the San Diego Zoological Society, and the Minnesota Zoo. The objectives of this work are to 1) validate protocols to establish baseline health of amphibians in and around the MN Zoo, 2) evaluate spatial and temporal dynamics of Bd and Ranavirus prevalence in association with environmental risk factors in order to generate hypotheses for a larger study within MN, and 3) conduct a risk assessment for MN Zoo to devise best practices for collecting wild amphibians for educational purposes.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
136	0830	1170	100%

Knowledge Area
136 - Conservation of Biological Diversity;

Subject Of Investigation
0830 - Wild animals;

Field Of Science
1170 - Epidemiology;

Keywords

amphibian disease;

ranavirus;

chytrid fungus

disease surveillance,

disease risk analysis

Goals / Objectives
The ultimate goal is to pilot an approach to understanding the eco-epidemiology oftwo amphibian pathogens in Minnesota. The pilot will be conducted in collaboration with the Amphibian Disease Laboratory of the San Diego Zoological Society (SDZ), and the Minnesota Zoo (MN Zoo). The Wildlife Disease Laboratories at the San Diego Zoo Institute for Conservation Research offer low-cost testing for the amphibian Bd and Ranaviruses in zoo collections and for wildlife conservation projects. The larger goal is to encourage widespread surveillance of amphibians for these potentially population-limiting infectious diseases in conjunction with the goals of the MNDNRObjective 1: Validate protocols to establish baseline health of amphibians in and around the MN Zoo landscape in Dakota County.Objective 2: Evaluate spatial and temporal dynamics of Bd and Ranavirus prevalence in association with environmental risk factors in the study site in order to generate hypotheses for a larger study within MN.Objective 3: Conduct a risk assessment for MN Zoo to devise best practices for this aspect of their business.

Project Methods
A. Sampling Frame and Data CollectionFieldwork will start in mid-May 2015 and go through the end of September 2015. All sampling will take place within the MN Zoo site (Apple Valley, MN) with expansion around the zoo in Lebanon Hills Regional Park in order to adequately replicate habitat types since all of this land was surveyed ecologically by the same team at the same time. Data will be collected three times (spring, summer, early fall) per pond over the 5-month period. Sampling sites will include two habitat types: 4 altered wetland areas and 4 ponds with high quality marsh (Chapman, 2012) (Figure 1). Individuals encountered through sight or sound will be captured by hand, by investigators wearing sterile gloves. After data collection, each frog will be placed in a separate sterile, ventilated plastic holding container; this will prevent individual frogs from being sampled twice during the same sampling session. After the appropriate number of individuals are captured during a sampling session, all animals will be released. Before moving from one pond to the next to collect samples, equipment will be disinfected, gloves changed (they will be changed between individual frogs), and proper PPE will be used to avoid cross-contamination between bodies of water as outlined in The Manual for Control of Infectious Diseases in Amphibian Survival Assurance Colonies and Reintroduction Programs (Pessier et al.). In addition, sick or dead animals will be collected and submitted to SDZ for necropsy; these could be valuable for disease surveillance and could lead to the detection of other amphibian pathogens such as the alveolate protozoan responsible for amphibian larval mortality events throughout the US.Figure 1. Example of proposed habitat sampling sites: Quality marsh/wetland: Vegetation communities of ponds have been given above average environmental quality rankings (B/C) through an environmental survey conducted by Applied Ecological Services, Inc in 2012. Altered marsh/wetland: These habitats are adjacent to areas of high anthropogenic impact (e.g. high traffic, adjacent to roads, or runoff from developed areas).B. Environmental Data Collection:The following environmental data will be collected coinciding with amphibian disease sampling:HabitatWater QualityPond/lake location and sizeDissolved oxygen (DO; ppm)Vegetation type (description)Ammonia, Nitrate, Nitrite (ppm)Animal species seenPhosphorous (ppm)Water temperatureTurbidity (secchi depth)ColiformsTotal Organic CarbonBaseline salinity (ppt)C. Amphibian Data Collection:Amphibians will be captured every other month from each proposed habitat location from May-September 2014. Species selection for sampling is based on most common species encountered in 2014 surveys, including:Wood frog (Lithobates sylvatica): 10 frogs per location per sampling sessionBoreal chorus frog (Pseudacris triseriata): 10 frogs per location and per sampling sessionGrey tree frogs (Hyla sp.): 10 frogs per location per sampling sessionBased on disease prevalence levels observed in 2014 study, sample size calculations indicate that the proposed number of frogs and habitat replicates will be sufficient to detect differences in prevalence by habitat type at alpha=0.05 with a power estimate of 97%.A basic health exam will be conducted on every frog collected and will include morphometric measurements, documentation of behavior, presence of skin lesions, bloating, abnormal body condition, and any other morphological abnormalities. Any physical abnormalities will be documented with photography when possible. Diagnostic sampling will follow standard protocols for Bd and Ranavirus recommended by the Institute for Conservation Research (San Diego Zoo) (http://www.sandiego zooglobal.org/News/Amphibian_Disease_Laboratory/). Briefly, animals will be manually restrained for swabbing of skin on ventral surfaces and the oral cavity for Bd and Ranavirus testing, respectively. Swabs will be placed in sterile cryovials and stored on ice until they can be returned to MN Zoo Animal Health for longer term storage in a -80°C freezer. Sick or dead animals will be collected and submitted for necropsy at the Pessier lab as part of Objective 1. In addition to seasonal sampling, survey techniques will be piloted to estimate frog populations at each site and to improve our ability to detect mortality events as a result of disease.D. Analysis of samples (Diagnostic)All amphibian samples will be sent to the Institute for Conservation Research (San Diego Zoo) where they will be analyzed by Real-Time (Taqman) PCR according to diagnostic standards established by Pessier et al. (Boyle et al, 2004; Pallister et al., 2007). Genotyping of Ranavirus positive samples will be conducted to get an idea of the major Ranavirus group present (Frog Virus 3 vs Ambystoma tigrinum-like viruses vs. exotic Ranaviruses like Bohle iridovirus). This involves conventional PCR for the major capsid protein and neurofilament-like protein (Holopainen et al, 2009).E. Data analysisDescriptive statistics will be generated (measures of central tendency and variance/uncertainty/confidence limits etc.) for all health and habitat data by spp., collection location and season (Objective 1). Disease and health prevalence data will be described spatially and temporally by spp. and habitat type and compared to 2014 data. Dependent variables (health outcomes) will be compared to independent variables (spp., season, habitat and environmental variables) via mixed regression modeling to generate epidemiological hypotheses for spp predilection and associated risk factors such as seasonality and habitat/environmental variables. This data will be used to conduct a feasibility analysis for a State-wide program. Finally, data will be used to estimate the risk of importing disease into the captive collection at MN Zoo over a ten-year period using methods established in OIE-IUCN joint guidelines.

Progress 07/24/15 to 06/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems:We are currently planning to complete Objective 3 (disease risk analysis) in the Summer of 2017. This is beyond our proposed timeline due to a delay in the receipt of test results; however, this offers an opportunity to train an additional veterinary student in epidemiological research. In addition, Wolf was on maternity leave in Fall 2016, which delayed the publication of Obj. 1-2. What opportunities for training and professional development has the project provided?Several second year veterinarystudents (summer scholars) have led field seasons, and numerous others helped with sample collection. Thus resarch and field work training have occured. How have the results been disseminated to communities of interest?Presentations to local reptile/amphibian interest groups, as well as one scientific conference (notes in refs), as well as the MN staff and community via presentation. What do you plan to do during the next reporting period to accomplish the goals?We will publish results of obj. 1-2, and conduct obj #3. We will report #3 to zoo stakeholders and discuss publication.

Impacts
What was accomplished under these goals? To meet Objectives 1 and 2, three species of frogs, Tree frogs (Hyla spp.), Boreal Chorus frogs (Pseudacris maculate), and Wood frogs (Lithobates sylvaticus), were sampled over three seasons in 2015, spring, summer, and fall. The number of frogs sampled are included in Table 1. Table 1. Total number of frogs sampled of each species over each season in 2015. Species Spring Summer Fall Total Tree frogs 50 42 35 127 Boreal chorus frogs 58 21 11 90 Wood frogs 4 34 7 45 Tree frogs- 50 in spring, 42 in summer, and 35 in fall for a total of 127 sampled this year; Boreal Chorus frogs- 58 in spring, 21 in summer, and 11 in fall for a total of 90 sampled this year; Wood frogs- 4 in spring, 34 in summer, and 7 in fall for a total of 45 sampled this year. Spring field activities resulted in the sampling of 112 frogs, summer activities sampled 97 frogs, and fall activities sampled 53 frogs for a total of 262 frogs. Frogs were captured from seven ponds of varying environmental conditions in Dakota County - a golf course pond, three ponds at the Minnesota Zoo (Dragonfly, Galaxy Little, and Vernal Pond), and three ponds at Lebanon Park (Equestrian Trailhead, Frog Pond, and Tamarack Bog). The average number of frogs caught at each pond over the course of the field season was 37 (s.d. ± 13). At this time, disease screening results have been completed for two of the three sampling seasons (Spring and Summer). As compared to disease screening of the same species in 2014, results from spring and summer of 2015 show a change in prevalence of Bd by species and a decreasing trend in the prevalence of ranavirus. In 2014, prevalences of Bd and ranavirus were highest in Wood frogs (Bd: 66.7%; ranavirus: 74.1%) followed by Boreal Chorus frogs (Bd: 40.3%; ranavirus: 49.3%) and Tree frogs (Bd: 10.4%; ranavirus: 32.8%). In 2015, the prevalence of Bd was highest in Boreal Chorus frogs (82.3%) followed by Wood frogs (21.1%), and Tree frogs (14.1%). The prevalence of ranavirus in spring and summer of 2015 were too low (0.5%) to assess species trends. This project is ongoing. We expect to receive disease screening results from our final sampling season (Fall) in the next month. These results will be analyzed along with additional pilot data collected in 2014. We are working with collaborators to generate a final publication of all results in early to mid-2017. Additionally, results from Objectives 1 and 2 will be used this summer (2017) to complete a formal disease risk analysis for reduction of disease risks through the collection of wild amphibians to supplement captive collections (Objective 3).

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems:We are currently planning to complete Objective 3 (disease risk analysis) in the Summer of 2017. This is beyond our proposed timeline due to a delay in the receipt of test results; however, this offers an opportunity to train an additional veterinary student in epidemiological research. In addition, Dr. Wolf was on leave in Fall 2016, which delayed the publication of Objectives1-2. What opportunities for training and professional development has the project provided?Several second year veterinarystudents (summer scholars) have led field seasons, and numerous others helped with sample collection. Thus resarch and field work training have occured. How have the results been disseminated to communities of interest?Presentations to local reptile/amphibian interest groups, as well as one scientific conference (notes in refs), as well as the MN staff and community via presentation. What do you plan to do during the next reporting period to accomplish the goals?We will publish results of objectives1-2, and conduct objectives #3. We will report #3 to zoo stakeholders and discuss publication.

Impacts
What was accomplished under these goals? To meet Objectives 1 and 2, three species of frogs, Tree frogs (Hyla spp.), Boreal Chorus frogs (Pseudacris maculate), and Wood frogs (Lithobates sylvaticus), were sampled over three seasons in 2015, spring, summer, and fall. The number of frogs sampled are included in Table 1. Table 1. Total number of frogs sampled of each species over each season in 2015. Species Spring Summer Fall Total Tree frogs 50 42 35 127 Boreal chorus frogs 58 21 11 90 Wood frogs 4 34 7 45 Tree frogs- 50 in spring, 42 in summer, and 35 in fall for a total of 127 sampled this year; Boreal Chorus frogs- 58 in spring, 21 in summer, and 11 in fall for a total of 90 sampled this year; Wood frogs- 4 in spring, 34 in summer, and 7 in fall for a total of 45 sampled this year. Spring field activities resulted in the sampling of 112 frogs, summer activities sampled 97 frogs, and fall activities sampled 53 frogs for a total of 262 frogs. Frogs were captured from seven ponds of varying environmental conditions in Dakota County - a golf course pond, three ponds at the Minnesota Zoo (Dragonfly, Galaxy Little, and Vernal Pond), and three ponds at Lebanon Park (Equestrian Trailhead, Frog Pond, and Tamarack Bog). The average number of frogs caught at each pond over the course of the field season was 37 (s.d. ± 13). At this time, disease screening results have been completed for two of the three sampling seasons (Spring and Summer). As compared to disease screening of the same species in 2014, results from spring and summer of 2015 show a change in prevalence of Bd by species and a decreasing trend in the prevalence of ranavirus. In 2014, prevalences of Bd and ranavirus were highest in Wood frogs (Bd: 66.7%; ranavirus: 74.1%) followed by Boreal Chorus frogs (Bd: 40.3%; ranavirus: 49.3%) and Tree frogs (Bd: 10.4%; ranavirus: 32.8%). In 2015, the prevalence of Bd was highest in Boreal Chorus frogs (82.3%) followed by Wood frogs (21.1%), and Tree frogs (14.1%). The prevalence of ranavirus in spring and summer of 2015 were too low (0.5%) to assess species trends. This project is ongoing. We expect to receive disease screening results from our final sampling season (Fall) in the next month. These results will be analyzed along with additional pilot data collected in 2014. We are working with collaborators to generate a final publication of all results in early to mid-2017. Additionally, results from Objectives 1 and 2 will be used this summer (2017) to complete a formal disease risk analysis for reduction of disease risks through the collection of wild amphibians to supplement captive collections (Objective 3).

Publications

Progress 07/24/15 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems:We are currently planning to complete Objective 3 (disease risk analysis) in the Summer of 2016. This is beyond our proposed timeline due to a delay in the receipt of test results; however, this offers an opportunity to train an additional veterinary student in epidemiological research. What opportunities for training and professional development has the project provided?This project provided an opportunity to train a veterinary Summer Scholar student (Meagan Abraham) in field data collection and epidemiological research. We also anticipate an additional opportunity to train a second veterinary Summer Scholar student in risk analysis with the completion of Objective 3. How have the results been disseminated to communities of interest?Preliminary results have been reported through scientific meetings and a popular magazine published by MN Zoo (see Publications section of this report). We also intend to develop a final manuscript from Objective 1 and 2 for publication in a peer-reviewed journal, such as the Journal of Wildlife Disease. We anticipate an additional manuscript of Objective 3, for publication in Zoo Biology or the Journal of Zoo and Wildlife Medicine. What do you plan to do during the next reporting period to accomplish the goals?We will collect final diagnostic data, analyze and prepare submission ofresults for peer review. We willhire a veterinary student to lead objective three.

Impacts
What was accomplished under these goals? Three species of frogs,Tree frogs (Hyla spp.), Boreal Chorus frogs (Pseudacris maculate), and Wood frogs (Lithobates sylvaticus), were sampled over three seasons in 2015, spring, summer, and fall. The total number of frogs sampled of each species over each season in 2015 arelisted below. A total of 50 Tree frogs were sampled in the spring, 42 in the summer, and 35 in the fall, for a total of 127 sampled this year. A total of 58 Boreal chorus frogs were sampled in the spring, 21 in the summer ,and 11 in the fall, for a total of 90 sampled this year. A total of 4 wood frogs were sampled in the spring, 34 in the summer, and 7 in the fall,for a total of 45 sampled this year. Spring field activities resulted in the sampling of 112 frogs, summer activities sampled 97 frogs, and fall activities sampled 53 frogs for a total of 262 frogs. Frogs were captured from seven ponds of varying environmental conditions in Dakota County - a golf course pond, three ponds at the Minnesota Zoo (Dragonfly, Galaxy Little, and Vernal Pond), and three ponds at Lebanon Park (Equestrian Trailhead, Frog Pond, and Tamarack Bog). The average number of frogs caught at each pond over the course of the field season was 37 (s.d. ± 13). At this time, disease screening results have been completed for two of the three sampling seasons (Spring and Summer). As compared to disease screening of the same species in 2014, results from spring and summer of 2015 show a change in prevalence of Bd by species and a decreasing trend in the prevalence of ranavirus. In 2014, prevalences of Bd and ranavirus were highest in Wood frogs (Bd: 66.7%; ranavirus: 74.1%) followed by Boreal Chorus frogs (Bd: 40.3%; ranavirus: 49.3%) and Tree frogs (Bd: 10.4%; ranavirus: 32.8%). In 2015, the prevalence of Bd was highest in Boreal Chorus frogs (82.3%) followed by Wood frogs (21.1%), and Tree frogs (14.1%). The prevalence of ranavirus in spring and summer of 2015 were too low (0.5%) to assess species trends. This project is ongoing. We expect to receive disease screening results from our final sampling season (Fall) in the next month. These results will be analyzed along with additional pilot data collected in 2014. We are working with collaborators to generate a final publication of all results in early to mid-2016. Additionally, results from Objectives 1 and 2 will be used this summer to complete a formal disease risk analysis for reduction of disease risks through the collection of wild amphibians to supplement captive collections (Objective 3).

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Abraham et al. Investigating population health and prevalence of Bd and ranavirus in three amphibian species in Dakota Co. MN. In: Merial-NIH National Veterinary Scholar Symposium; July 30-August 2, 2015; UC Davis CVM; Davis, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Abraham et al. Investigating population health and prevalence of Bd and ranavirus in three amphibian species in Dakota Co. MN. In: University of Minnesota Points of Pride; October 6-7, 2015; UMN CVM; Saint Paul, MN.
• Type: Other Status: Published Year Published: 2015 Citation: Wolf TM. The importance of amphibians: Collecting data at the zoo. Zoo Tracks, Minnesota Zoo June 2015: 22-23.