UNTANGLING PARELAPHOSTRONGYLUS TENUIS TRANSMISSION IN A DECLINING MOOSE POPULATION

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1014602
• Project Start Date: Oct 9, 2017
• Project End Date: Dec 31, 2021
• Program Code: [(N/A)]- (N/A)

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

Performing Department
Veterinary Population Medicine

Non Technical Summary
Minnesota's moose population is declining, due in large part to a fatal disease caused by a parasite, Parelaphostrongylus tenuis. The parasite has become known as "brainworm" based on a component of its life cycle in which it lives around the brain of white tailed deer and moose. It causes no ill effects to deer, but infection with the parasite often results in severe disease and can be fatal for moose. This disease is a well-recognized problem among moose anywhere their population overlaps with white tailed deer.Major gaps in our knowledge of the parasite'slife cycle challenge our ability to stop infection in moose. For example, scientists' methods for studying snails, an intermediate host required to bridge transmission between deer and moose,has involved attracting snails to wet cardboard left along the forest floor and sampling snails from there. The number of brainworm-infected snails collected with this method has been so low that scientists often have to screen tens of thousands of snails just to find a few with brainworm larvae. If this technique is not attracting the right species of snails (for example, the species that are primarily involved the brainworm life cycle)or if the behavior of snails changes after they are infected with brainworm tooso that they are no longer attracted to wet cardboard as they would if they were uninfected, this traditional technique for sampling snails may not be providing the information needed to fully understand transmission.Instead, our approach is to use a metagenomics approach to screen moose feces for DNA of snails that are eaten by moose. This approachwill help us identify the diversity and abundance ofsnail species consumed by moose in order to narrow the list of prospective snails that play a role brainworm transmission. We will simulatneously screen the feces for brainworm DNA to prioritize snail species as potentially important transmission hosts. Specifically, the simultaneous detection of certain snail species along with brainworm DNA suggests the detection of brainworm-infected snails. With a list of newly identified snail species known to be consumed by moose, we can refine field studies in our ongoing efforts to identify important habitats where transmission takes place.In addition, we will evaluate the genetic variability of brainworm larvae collected from infected deer in order to describe the structure of the parasite population in northeastern Minnesota. In evaluating how the genetics of the parasite population varies across space and habitats, we can attain a better understanding of transmission across the landscape. For example, in determining to what extent individual brainworm infections in deer are spatially related or not, we will be able to describe patterns of transmission and possiblyidentify landscape barriers to transmission that could facilitatecontrol of transmission to moose.

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
135	3130	1070	50%
135	3130	1080	50%

Knowledge Area
135 - Aquatic and Terrestrial Wildlife;

Subject Of Investigation
3130 - Nematodes;

Field Of Science
1070 - Ecology; 1080 - Genetics;

Keywords

moose

parasite transmission

metagenomics

parelaphostrongylus tenuis

white tailed deer

Goals / Objectives
Specific Aim 1: Utilize metabarcoding to identify potential gastropod intermediate hosts in moose feces. We hypothesize that, if moose are exposed to P. tenuis through the inadvertent consumption of the gastropod host, then we will be able to identify potential hosts through the detection of gastropod DNA in moose feces. The success of this Specific Aim will facilitate the prioritization of gastropod species for further study of their role in P. tenuis transmission.Specific Aim 2: Utilize metagenomics to characterize the genotype heterogeneity of P. tenuis in deer. We hypothesize that the genotypic population structure of P. tenuis is sufficiently heterogeneous to characterize transmission across the landscape. Characterization of the genotypic population structure of P. tenuis will allow us to explore P. tenuis genotypes across habitats and hosts to enhance our understanding of where and how P. tenuis transmission is occurring. Ultimately, by mapping the genotypic population structure of P. tenuis transmitted by deer to the landscape, we can progress to using the genotype of P. tenuis from moose to help identify where transmission would have occurred.

Project Methods
Specific Aim 1. Utilize metabarcoding to identify gastropod intermediate hosts in moose feces.We will use GPS data from collared moose on the Grand Portage Indian Reservation to locate bedding sites for the collection of fresh feces during spring and summer. Adult moose are collared with GPS Plus collars (Vectronic Aerospace GmbH), which are designed for real-time data transmission via satellite. With 30-40 collared moose monitored annually, we propose to collect samples from 20 individuals over three time periods (late spring, mid-summer, and late summer) and over a variety of habitat types used by moose (e.g. aspen/birch, upland conifer, swamp conifer, etc). It is during the snow-free period of the year when gastropods are active on the landscape and when moose would be exposed during foraging. This will give us a total of 60 samples with which to profile species of gastropods consumed across space and time.We will use a metabarcoding approach with the mitochondrial cytochrome c oxidase 1 (CO1) marker gene to assess gastropod diversity in moose fecal samples. Metabarcoding is a next generation sequencing tool that enables rapid and comprehensive biodiversity assessment from a variety of media, including feces. Metabarcoding of feces has revolutionized our understanding of wildlife diets, yet this technology has not been used, to our knowledge, to infer host-parasite dynamics. DNA will be extracted, PCR-amplified for COI, and sequenced by the University of Minnesota Genomics Center (UMGC). The UMGC has extensive experience with conducting amplicon-based marker gene profiling from fecal samples. This approach will enable identification of gastropod species ingested, as well as provide an estimate of abundance that will be critical in inferring the most important species for future study as intermediate hosts. In the event that amplification of host DNA is obscuring other signals, we will explore the use of a different amplicon-based marker gene or the use of peptide nucleic acid based strategies for blocking host DNA.Following amplicon sequencing, demultiplexing, and quality filtering of the data, an operational taxonomic unit (OTU) approach will be used in QIIME with 98% cutoff for the binning of amplicon sequences into species-level groups. These groups will be classified at the species level to identify and extract sequences with similarity to gastropods using the Barcode of Life database (University of Guelph, Ontario, Canada) to identify gastropod species based on 98% sequence similarity. We will analyze patterns of variation in gastropod species composition across moose samples using a cutting edge model-based Bayesian ordination and regression approach. In using these methods we will assess to what degree consumption of gastropods varies spatially, temporally or by habitat. Indicator species analysis will also be used to assess what species are the most important diet component in each habitat. Furthermore, using randomization tests we can analyze the extent to which gastropod species found in the moose diet are a unique or random component of the northern MN gastropod species pool (based on species distribution records).Simultaneously, we will also work with UMGC to develop a quantitative PCR assay to detect P. tenuis DNA in the fecal samples. While moose do not transmit P. tenuis by shedding larvae in feces, we hypothesize that we will detect P. tenuis DNA if infected gastropods have been ingested. Thus, the detection of P. tenuis by qPCR would provide another means to prioritize detected gastropod species for further study as potential intermediate hosts. Briefly, we will design three assays that target P. tenuis and interrogate them for efficiency using a five point one-to-five dilution series of DNA sample. Limit of detection will be determined by running this design against serially diluted P. tenuis DNA (isolated from deer feces; see Specific Aim 2) spiked in a pool of moose DNA at known concentrations. We will perform the pilot test on a small number of samples prior to processing the group of experimental samples using the developed assay. Overall, these analyses will provide new insights and inform future studies into the most important gastropod species and habitats for P. tenuis transmission to moose.Specific Aim 2. Utilize metagenomics to characterize the genotype heterogeneity of P. tenuis in deer.We will use GPS data from collared deer for establishing survey units for sampling fresh deer feces. As the shedding of larvae in deer feces peaks during winter and spring, we will collect fecal samples from January to May. As P. tenuis prevalence in deer feces has ranged between 46-69% among studies in MN, we will collect up to 400 fecal samples with the goal of obtaining larvae from approximately 200 samples. We will isolate P. tenuis larvae from feces using a modified Baermann floatation technique, as previously described, and submit larvae to UMGC for DNA extraction. Following DNA extraction, we will use restriction site associated DNA sequencing (RADseq) through UMGC to build a catalog of variants for P. tenuis using purified larvae DNA from deer feces. The benefit of this approach is that it incorporates both traditional restriction enzyme typing with SNP identification methods using DNA sequencing. Briefly, a restriction enzyme will be selected through experimental testing on two samples from candidate enzymes (BfuCI, BstYI, ApeKI, PstI, NsiI, BamHI, BglII, and SbfI) covering a broad range of specificities. P1 adapter will be ligated to the DNA fragments with a unique molecular identifier for each sample. DNA will then be pooled and randomly sheared, and P2 adapter will then be ligated to all fragments. Samples will then be amplified using P1-P2 adapters and sequenced at a depth of approximately 500,000 reads. This coverage will enable selection of an enzyme that yields >=1000 single nucleotide polymorphisms (SNPs) using the P1 adapter as a molecular identifier for SNP calling. Following identification of an appropriate restriction enzyme, we will then genotype 100 larvae DNA samples to build a catalog of variants. Using the SNP data, we will, for the first time, generate basic population level statistics, such as allele frequency for P. tenuis in deer. Bayesian cluster analysis will also be applied to estimate the number of populations and relative population size through time of P. tenuis that is likely in the GPIR area based on the SNP dataset. Population estimates are critical for future work as they provide baseline estimates with which to assess the effectiveness of future management strategies. We intend to perform this analysis using the Structure software packageand SNAPP. To model the spatial component of the population genetic structure, we will apply spatial principal component analysis (sPCA) using our geo-referenced SNP dataset. We intend to perform this analysis using the R package adegenet. This analysis will enable us to assess how finely structured the P. tenuis population is across our study area and will help untangle to what extent individual P. tenuis infections are spatially autocorrelated and where landscape barriers for gene flow might exist. Overall, these analyses will provide additional insights into where on the landscape P. tenuis transmission might be occurring, thus informing future studies on the transmission risk to moose.

Progress 10/09/17 to 12/31/21

Outputs
Target Audience:We have four main target audiences for this project 1) tribal communities that rely on moose as a subsistence species, 2) natural resource managers whose management decisions will be informed by this project, 3) the general population of Minnesota, particularly the agricultural communities that raise domestic ruminant species, and 4) the scientific community at large. Over the past project period our efforts among these groups have included: 1. Tribal communities: Our goal is to provide tribal communities, and the Grand Portage Indian Reservation in particular, as a project partner, with regular updates of our research activities and findings. To accomplish this, our team meets biweekly with Grand Portage partners in the Department of Biology and Environment to share research updates. 2. Natural Resource Managers: We continue to work and communicate directly with MN Department of Natural Resources. In the last reporting period we shared results from this project at an Annual Moose Research and Management meeting held by the MN Department of Natural Resources on July 28-29, 2021. 3. Larger Minnesota Community: We have worked alongside the Grand Portage Department of Biology and Environment to develop short informational videos that can be shared freely with collaborators and the general public about the ongoing projects. 4. Scientific Community: Our team's efforts to reach the larger scientific community are through student education and training and scientific meetings. In this reporting period, we presented efforts on this project at the International Wildlife Disease Association Conference and the North American Moose Conference. We also presented the work in several forums of University of Minnesota. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has served as dissertation research for a PhD student who is obtaining skills in field sampling techniques, molecular and bioinformatics. The student has recently progressed to PhD candidate. It has also provided a unique research experience for a DVM student. How have the results been disseminated to communities of interest?The project has been shared through four presentations in the past reporting period; at the International Wildlife Disease Association Conference, at the North American Moose Conference, at the Three Minute Thesis Competition at the University of Minnesota College of Veterinary Medicine, and as an Oral Examination Seminar at the University of Minnesota College of Veterinary Medicine. What do you plan to do during the next reporting period to accomplish the goals?We are nearing completion of this project. At this stage, we have generated more data than we originally proposed, and we anticipate that data generation will be complete by the final report. We intend to utilize these data in upcoming landscape genetics studies to enhance our understand of transmission across the northeaster Minnesota landscape.

Impacts
What was accomplished under these goals? Specific Aim 1: Since last report, we extracted DNA from 226 moose pellet samples, successfully amplified COI from 159 of those samples, and sequenced those amplicons with the Illumina MiSeq platform. When added to previously sequenced samples, this gives us a total of 250 moose samples collected and 179 sequenced. The sequenced samples were subjected to a highly sensitive and efficient bioinformatic pipeline that allowed us to compare our sequences to all Gastropoda COI sequences available in the Barcode of Life Database. Though this process, we detected gastropods in 5 samples. More specifically, we detected two species of upland, ground-dwelling snails in Autumn (Punctum minutissimum on September 14, 2018; Zonitoides arboreus on September 17, 2019) and 1 species of aquatic snail twice during the summer (Helisoma trivolvis on June 20, 2019 and June 24, 2020). We additionally detected Punctum minutissimum in a pilot sample that did not have associated metadata. This is the first time any researcher has empirically documented moose consumption of gastropods. Although we do not have enough gastropod "hits" to do a formal analysis of gastropod consumption patterns by moose, these results point us in exciting new research directions. Specific Aim 2: Using the cleaning and whole genome amplification protocal described in the previous report, we worked with the University of Minnesota Genomics Center (UMGC) on an eight-sample pilot project to ensure that the updated protocal produced results that would be useable for a landscape genetics analysis. Five of the eight samples contained mostly P. tenuis DNA. Using those five samples, we identified 20,694-54,574 informative single nucleotide polymorphisms (depending on sequencing depth), which is more than enough for a high-resolution landscape genetics analysis of P. tenuis. Since the pilot sample was successful, we cleaned larvae, extracted DNA, and amplified DNA for 143 P. tenuis larval samples. These are now in the UMGC pipeline for RAD-seq analysis. We anticipate having the resulting sequence data within a month, at which point we will commence with landscape genetics analyses.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Garwood, T., Moore, S., Fountain-Jones, N., Larsen, P., Wolf, T. 2021. Presentation: Using metabarcoding to study effective contact for brainworm transmission between moose and gastropods. 54th North American Moose Conference and Workshop, November 30- December 2, 2021, Virtual conference.
• Type: Other Status: Other Year Published: 2021 Citation: Garwood, T., 2021. Presentation: Investigating brainworm ecology to reduce aberrant transmission to Minnesota Moose. Three Minute Thesis Competition, College of Veterinary Medicine, University of Minnesota
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Garwood, T., Moore, S., Fountain-Jones, N., Larsen, P., Wolf, T. 2021. Presentation: Using metabarcoding to study effective contact for brainworm transmission between moose and gastropods. Annual Wildlife Disease Association/Biennial European Wildlife Disease Association Joint Virtual Conference, Cuenca, Spain.
• Type: Other Status: Other Year Published: 2021 Citation: Garwood, T., 2021. Presentation: Investigating brainworm ecology to reduce aberrant transmission to Minnesota Moose. Comparative and Molecular Bioscience Preliminary Oral Examination Seminar, April 19, 2021, College of Veterinary Medicine, University of Minnesota

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:We have four main target audiences for this project 1) tribal communities that rely on moose as a subsistence species, 2) natural resource managers who's management decisions will be informed by this project, 3) the general population of Minnesota, particularly the agricultural communities that raise domestic ruminant species, and 4) the scientific community at large. Over the past project period our efforts among these groups have included: 1. Tribal communities: Our goal is to provide tribal communities, Grand Portage Indian Reservation in particular, as a project partner, with regular updates of our research activities and findings. To accomplish this, our team meets regularly with Grand Portage partners in the Department of Biology and Environmentto share research updates. 2. Natural Resource Managers: We continue to work and communicate directly with MN Department of Natural Resources. 3. Larger Minnesota Community: We have worked alongside the Grand Portage Department of Biology and Environment to develop short informational videos that can be shared freely with collaborators and the general public about the ongoing projects. Our team also shared project updates through the popular media, which included interviews with the MN Star Tribune and Minnesota Daily. A presentation of the project was also delivered remotely to the Minnesota Native Plant Society. 4. Scientific Community: Our team's efforts to reach the larger scientific community are through student education and training and scientific meetings. In the previous reporting period, we presented efforts on this project at the National meeting of the American Veterinary Medical Association, and the project was also featured as a news artcle in the Journal of the American Veterinary Medical Association. Changes/Problems:The laboratory methods used in this project are novel and have required substantial time and effort in development and trouble-shooting. During the current reporting period we experienced a laboratory shutdown of three months that delayed laboratory research efforts. Despite that, we continue to move forward and make progress toward achieving our project goals. What opportunities for training and professional development has the project provided?The graduate student, Tyler Garwood, on this project has been going through substantial training in molecular methods and bioinformatics in partnership with our collaborator, Peter Larsen. This supplements a Genomics for Wildlife Diseases workshop hosted by Colorado State University that TG attended in the previous reporting period to obtain a broad introduction to bioinformatic methods. How have the results been disseminated to communities of interest?Results are quite preliminary at this stage, but the project has been described through two presentations in the past reporting period, one to the national conference of the American Veterinary Medical Association and another to the Minnesota Native Plant Society. We have also shared project related news to the general public through local media outlets, including the MN Star Tribune and Minnesota Daily newspapers. What do you plan to do during the next reporting period to accomplish the goals?Planned activities for the upcoming reporting period Specific Aim 1: Over the next reporting periodour activities are as follows: 1. Extract DNA from 2019 and 2020 moose fecal samples, perform PCR, and submit for metabarcoding. 2. Run new sequences through metabarcoding pipeline 3. Summarize data and perform preliminary statistical analyses Specific Aim 2: Planned activities over the next reporting period are as follows: 1. Finalize validation of cleaning protocal for P. tenuis larvae 2. Extract DNA and perform whole genome amplification on larval samples. 3. Submit larval samples to UMGC for RADseq. 4. Perform PCR to confirm morphological identification ofP. tenuisin samples

Impacts
What was accomplished under these goals? Specific Aim 1: Acquisition of additional funding (as a result of the pilot work supported by this grant) supports the collection and analysis of additional samples above what was originally proposed in this grant application.Since the last report, we collected 112 moose pellet samples over the period of June-October 2020. After collection, we prepared each sample by randomly selecting 3 pellets, removing the outer layer of the pellet to minimize DNA contamination, and homogenizing them together to minimize the likelihood of missing gastropod DNA between pellets. All samples from 2020 have been processed for molecular analysis.We have collected and prepped 246 moose pellets total between 2018-2020, and consider this sample set complete. Since last report, we improved our bioinformatic pipeline to be more efficient and accurate. We changed mapping parameters to reduce the likelihood of obtaining false positives and created a custom upper Midwest slugs and snails database that allows for more efficient sequence mapping. Using this new pipeline and database, we identified a single gastropod (Punctum minutissimum) in one of the 20 early sequenced moose samples from our dataset. Specific Aim 2: Additional funding acquired has also been used to support more sampling in association with this aim. We continued to collect deer pellets from January-June 2020. Similarly to 2019, the Grand Portage Indian Reservation (GPIR) was spatially broken up into 1 km2survey cells from which we targeted the collection of 2 fecal samples from each of 50 randomly selected survey cells that fell within deer summer and winter range and 25 additional samples collected opportunistically from cells that fall outside of primary deer range and within primary moose range in GPIR. We employed a scat detection dog to find samples in cells of low deer density, which greatly improved our efficiency and overall success rate. We collected and processed a total of 455 samples, 60% (271) of which areP. tenuispositive, with counts rangingfrom 0.28-842.1 larvae per gram of feces. Larvae were recovered and frozen from all positive samples. We consider sample collection complete for this aim and have extracted larvae via modified Baermann's protocal from all samples. We generated a whole genome sequence of P. tenuis since last report with the help of the University of Minnesota Genomics Center (UMGC). Upon aligning our restriction site associated DNA sequencing (RADseq) reads obtained from previous sequencing runs, we found that in most submitted samples, contaminate (ie non- P. tenuis) DNA made up the majority of DNA in those extractions. With this knowledge, we have incorporated cleaning of larvae extracted from fecal samples 4 times in nuclease free water to remove contaminates. We tested the efficacy of this cleaning using an in-lab Oxford Nanopore Flongle sequencing run, and preliminary results show that the new protocol stepswere successful in increasingthe amount of P. tenuis DNA in our extract. We are also now using whole genome amplification to ensure sufficient quantities ofDNA for the RADseq protocal and other downstream applications.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Wolf TM, Moore SA, Swader T, Travis DA. Moose Population Decline in Minnesota: Evaluating Ecosystem Health Through Tribal-Academic Partnerships. AVMA Virtual Convention 2020, August 19-22, 2020.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:We have four main target audiences for this project 1) tribal communities that rely on moose as a subsistence species, 2) natural resource managers who's management decisions will be informed by this project, 3) the general population of Minnesota, particularly the agricultural communities that raise domestic ruminant species, and 4) the scientific community at large. Over the past project period our efforts among these groups have included: 1. Tribal communities: Our goal is to provide tribal communities, Grand Portage Indian Reservation in particular, as a project partner, with regular updates of our research activities and findings. To accomplish this, our team has met with the Grand Portage Tribal Council to share research updates, participated in an interview about the project on the Grand Marias public radio station WTIP, and participated in community activities in Grand Portage for two weeks in May 2019to informally share information about the project directly with community members. 2. Natural Resource Managers: We continue to work and communicate directly with MN Department of Natural Resources. 3. Larger Minnesota Community: We have worked alongside the Grand Portage Department of Biology and Environment to develop short informational videos that can be shared freely with collaborators and the general public about the ongoing projects. Our team also shared project updates with MN Zoo, which is in process of developing information for the public on MN moose research.The combined results from this project, along with a parallel project examining P. tenuis risk to goats, will be shared with the MN agricultural community, where P. tenuis infection poses a risk to domestic ruminants, particularly small ruminants (i.e. goats) and camelidae (i.e. llamas and alpaca). https://www.youtube.com/playlist?list=PLnC4yTjRBxQeDnfIW1Oxd2BlHnBKc4_bY 4. Scientific Community: Our team's efforts to reach the larger scientific community are through student education and training and scientific meetings. This project has been in early stages of development over the past year and not yet ready for dissemination of results through scientific meetings; however, this project has provided a platform for educating students on scientific research, animals as biosentinels, and ecosystem health in three University of Minnesota classes over the 2019-2020academic year, reaching undergraduates, graduate students, and professional students in primarily in the College of Veterinary Medicine, School of Public Health, College of Biological Sciences, College of Food, Agriculture, and Natural Resource Sciences, and Humphrey School of Public Affairs. Preliminary results were also presented at the UMN COllege of Veterinary Medicine's annual Points of Pride Research day. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has served in the training and development of a veterinary summer scholars student over the period of MayAugust 2018. In this regard, a second-year veterinary student optimized laboratory protocols for the extraction and recovery of larvae through the modified Baermann technique from deer pellets. Accordingly, she became familiar with standard techniques in parasitology for the quantification of lungworm larvae, participated in protocol optimization and troubleshooting, and epidemiological analysis of P. tenuis data. Her efforts on this project were reported in August at a national meeting for veterinary summer scholars students as a scientific poster, and will again be showcased at the UMN College of Veterinary Medicine's (CVM) annual Points of Pride research day. This project will also serve as a component of the thesis research of a Ph.D. student, Tyler Garwood, who as recently begun (August 2018) the graduate program in the CVM's Molecular and Biosciences program. How have the results been disseminated to communities of interest?We are still in the stages of our projectwhere results are limited; however, preliminary results were presented as scientific posters: Mayer T, Moore S, Fountain-Jones N, Johnson D, Anderson S, Marchetto K, Wolf T. Characterizing transmission epidemiology of the meningeal worm (Parelaphostrongylus tenuis). National Veterinary Summer Scholars Symposium, Texas A&M University, College Station, TX, August 4, 2018. Garwood T., Moore S., Fountain-Jones N., Larsen P., Wolf T. 2019. Poster: Understanding Brainworm Landscape Genomics on Grand Portage Indian Reservation to Prevent Minnesota Moose Declines. Points of Pride Research Day Poster Competition at UMN College of Veterinary Medicine, St. Paul, MN, October 2, 2019. We are also working with MNZoo in their preparation of an exhibit in 2021 to display MN moose research to the general public. What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1: Over the next reporting periodour activities are as follows: 1. Revise bioinformaticpipeline to remove primer/library preparations sequences and BLAST to a custom database of Upper Midwest slugs and snails. 2. Extract DNA from 2019 moose fecal samples, perform PCR, and submit for metabarcoding. 3. Process all sequencing reads through revised bioinformatic pipeline, summarize and report findings. Specific Aim 2: Planned activities over the next reporting period are as follows: 1. Build adequate reference genome of P. tenuis to remove potential contamination from RADseq reads. 2. RecoverP. tenuislarvae from remaining deer pellet samples collected through the 2019 field season. 3. Submit larval samples to UMGC for RADseq. 4. Summarize and report results.

Impacts
What was accomplished under these goals? Specific Aim 1: Since the last report, we collected 113 moose pellet samples over the period of June-October 2019. Note: the NIFA grant supports the analysis of 60 samples for this aim, and we have obtained new funding to support the additional samples collected during the same field season. After collection, we prepared each sample by randomly selecting 3 pellets, removing the outer layer of the pellet to minimize DNA contamination, and homogenizing them together to minimize the likelihood of missing gastropod DNA between pellets. All samples from 2019 have been prepped, and we are currently extracting DNA from these samples.To date, we have collected and prepped 134 moose pellets total. Although we ran all sequenced pilot samples through our bioinformatic pipeline, we noticed several errors in the pipeline that make those results unreliable. We are working with experts to improve both the quality and speed of the pipeline, which will produce results more reliably and quickly. Specific Aim 2: Last year, we validated a restriction site associated DNA sequencing (RADseq) protocol developed by University of Minnesota Genomics Center (UMGC), which we will use to achieve Aim 2. However, we need a reference genome to be sure we are analyzing P. tenuis DNA and not contaminant DNA that might be in the sample. Reliable reference genomes do not currently exist for P. tenuis; hence, we are using outside funds and working with UMGC to develop a high-quality whole genome sequence with Pacific Biosciences SMRT sequencing technology. We have submitted adult female worms for high molecular weight DNA extraction and are collaborating with the Minnesota Department of Natural Resources to acquire more adult worms for extraction and sequencing. After acquiring this sequence, we can analyze the sequence data collected from P. tenuis larvae. We continued to collect deer pellets from January-December 2019. Similarly to 2018, the Grand Portage Indian Reservation (GPIR) was spatially broken up into 1 km2survey cells from which we targeted the collection of 2 fecal samples from each of 50 randomly selected survey cells that fell within deer summer and winter range and 25 additional samples collected opportunistically from cells that fall outside of primary deer range and within primary moose range in GPIR. We have collected and processed a total of 313 samples, 50% (158) of which are P. tenuis positive, with counts rangingfrom 0.3-840 larvae per gram of feces. Larvae were recovered and frozen from all positive samples. We performed RADseq on 139 samples and will submit larvae from the remaining 2019 samples once all samples have been extracted. Note: the NIFA grant supports the analysis of 60 samples for this aim, and we have obtained new funding to support the additional samples collected during the same field season. ?

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Garwood T., Moore S., Fountain-Jones N., Larsen P., Wolf T. 2019. Poster: Understanding Brainworm Landscape Genomics on Grand Portage Indian Reservation to Prevent Minnesota Moose Declines. Points of Pride Research Day Poster Competition at UMN College of Veterinary Medicine, St. Paul, MN, October 2, 2019.

Progress 10/09/17 to 09/30/18

Outputs
Target Audience:We have four main target audiences for this project 1) tribal communities that rely on moose as a subsistence species, 2) natural resource managers who's management decisions will be informed by this project,3) the general population of Minnesota, particularly the agricultural communities that raise domestic ruminant species, and 4) thescientific community at large. Over the past project period our efforts among these groups have included: 1. Tribal communities: Our goal is to provide tribal communities, Grand Portage Indian Reservation in particular, as a project partner, with regular updates of our research activities and findings. To accomplish this, our team has met with the Grand Portage Tribal Council to share research updates,participated in an interview about the project on the Grand Marias public radio station WTIP (see link below), and participated in community activities in Grand Portage for two weeks in May 2018 and 2 days in August 2018 to informally share information about the project directly with community members. http://www.wtip.org/researchers-share-update-moose-brainworm-study 2. Natural Resource Managers:In December 2017, our team hosted a 2-day workshop that brought together natural resource managers working with deer and moose and infectious disease experts to identify gaps in research and ensure project activities continue to meet the needs of resource managers. 3. Larger Minnesota Community: We have worked alongside the Grand Portage Department of Biology and Environment to develop short informational videos that can be shared freely with collaborators and the general public about the ongoing projects. In addition to this, Dr. Wolf provides semi-annual project updates directly to donors who demonstrated a committed interest in this research througha crowd-funding campaign in 2017. The combined results from this project, along with a parallel project examining P. tenuis risk to goats, will be shared with the MN agricultural community, where P. tenuis infection poses a risk to domestic ruminants, particularly small ruminants (i.e. goats) and camelidae (i.e. llamas and alpaca). https://www.youtube.com/playlist?list=PLnC4yTjRBxQeDnfIW1Oxd2BlHnBKc4_bY 4. Scientific Community: Our team's efforts to reach the larger scientific community are through student education and training and scientific meetings. This project has been in early stages of development over the past year and not yet readyfor dissemination of results through scientific meetings; however, this project has provided a platform for educating students on scientific research, animals as biosentinels, andecosystem health in three University of Minnesota classes over the 2017-2018 academic year, reaching undergraduates, graduate students, and professional students in primarily in the College of Veterinary Medicine, School of Public Health, College of Biological Sciences, College of Food, Agriculture, and Natural Resource Sciences, and Humphrey School of Public Affairs. Changes/Problems:The only challenge faced during this project period has been in the recruitment/start of a grad student to conduct much of the field sampling, where the funding cycle and academic recruitment cycle was mismatched. We have successfully recruited a grad student to this project, but the start date followed much of the field season. Thus, we will use a second field season to obtain remaining samples. What opportunities for training and professional development has the project provided?This project has served in the training and development of a veterinary summer scholars student over the period of May-August 2018. In this regard, a second-year veterinary student optimized laboratory protocols for the extraction and recovery of larvae through the modified Baermann technique from deer pellets. Accordingly, she became familiar with standard techniques in parasitology for the quantification of lungworm larvae, participated in protocol optimization and troubleshooting, and epidemiological analysis of P. tenuis data. Her efforts on this project were reported in August at a national meeting for veterinary summer scholars students as a scientific poster, and will again be showcased at the UMN College of Veterinary Medicine's (CVM) annual Points of Pride research day. This project will also serve as a component of the thesis research of a Ph.D. student, Tyler Garwood, who as recently begun (August 2018) the graduate program inthe CVM's Molecular and Biosciences program. How have the results been disseminated to communities of interest?We are still in the very early stages of our project, where results are limited; however, preliminary resultswere presented by the veterinary summer scholars student on her efforts on the project this summer: Mayer T, Moore S, Fountain-Jones N, Johnson D, Anderson S, Marchetto K, Wolf T. Characterizing transmission epidemiology of the meningeal worm (Parelaphostrongylus tenuis). National Veterinary Summer Scholars Symposium,Texas A&M University, College Station, TX, August 4, 2018. What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1: Over the next reporting period our activities are as follows: 1. Finalize bioinformaticanalyses of pilot samples to determine the optimal primer set for the metabarcoding analyses of accrued moose pellet samples. 2. Process 2018 samples at end of the season (November 2018) and submit to UMGC for DNA extraction and metabarcoding. 3. Process resulting sequencing reads through the recently developed bioinformatics protocols and summarize data for reporting. Specific Aim 2: Planned activities over the next reporting period are as follows: 1. Continue to work with UMGC in the enhancement of the DNA extraction protocol for recovered P. tenuis larvae. 2. Recover P. tenuis larvae from remaining deer pellet samples collected through the 2018 field season. 3. Submit larval samples to UMGC for RADseq. 4. Analyze and report on the resulting genotyping data.

Impacts
What was accomplished under these goals? Specific Aim 1: Since the start of the grant period we were able to obtain samples from nine moose, collected over the period of May-July 2017, which were utilized in the research and development of the metabarcoding platform with which to screen for gastropod DNA, the development of which has been a major accomplishment since the beginning of this grant period. We worked directly with the University of Minnesota Genomics Center in the development and optimization of a metagenomic assay involving the PCR amplification of the mitochondrial cytochrome c oxidase 1 (CO1) marker geneof DNA extracted from the sampled moose pellets. In the design of this assay, we utilized two different PCR primer sets for the amplification of the marker gene to determine the optimal primer set to assess gastropod diversity in moose fecal samples.We also included DNA extracted from zebra mussels as a positive control to confirm assay success in sequencing the CO1 gene of mollusk species. The reads that have been produced from these sequencing activities are currently being processed through a bioinformatics pipeline to finalize the metagenomic protocol. Simultaneously, we have collaborated with biology staff of the Grand Portage Indian Reservation (GPIR) to build the sample set we intend to analyze for this project. Our goal for this project was to collect moose pellet samples from collared animals (using known location data) throughout the time period when gastropods are on the landscape (June-October). Despite the limitation of lacking a dedicated graduate student on the project during the majority of the sampling season (due to mismatch in the funding period of the grant and recruitment of a graduate student into our program), GPIR staff were able to collect an additional twelve samples over the period June-August 2018 for a total of 21 samples across three months of the year. Sampling is still underway and will continue through the 2019 sampling season. The results of this aim will enhance our understanding of what gastropod species are consumed by moose, as a pathway for P. tenuis infection. These analyses will inform further research into habitat preferences and P. tenuis prevalence among the identified gastropods, enhancing our understanding of P. tenuis risk for both moose and susceptible domestic ruminant species. Specific Aim 2: A major accomplishment since the start of this grant period has been in our work withUniversity of Minnesota Genomics Center (UMGC) on the research and development of a restriction site associated DNA sequencing (RADseq) protocol to build a catalog of variants for P. tenuis using purified larvae DNA from deer feces. To achieve this, we utilized 8 duplicate deer pellet samples collected in July 2017 from GPIR. We first subjected the fecal samples to a modified Baerman procedure for the extraction of P. tenuis larvae from the pellets and established a protocol for larval recovery. DNA was extracted directly from the recovered larvae and submitted to UMGC for RADseq development and optimization through the selection of restriction enzymes and amplification and sequencing conditions.In this pilot, the data were analyzed at four read depths: 0.5, 1, 2, and 4 million reads per sample and a depth of 2M reads were considered sufficient to obtain a level of genotype diversity needed to evaluate transmission patterns. At this sequencing depth, we should obtain >1,000 single nucleotide polymorphisms for genotyping. We are currently working with UMGC to enhance our DNA extraction methods to maximize DNA quantity when pellet larval counts are low. Simultaneously, we have worked with biologists from GPIR to obtain deer pellets from January-July 2018. To do this, the GPIR was spatially broken up into 1 km2survey cells from which we targeted the collection of 2 fecal samples from each of 50 randomly selected survey cells that fell within deer summer and winter range and 25 additional samples collected opportunistically from cells that fall outside of primary deer range and within primary moose range in GPIR. Over this time period, 93 total pellet samples have been collected across 62 survey cells. Up to the writing ofthis report 78 of the pellet samples had been subjected to the modified Baermann. Sixty-two percent (n=48)of the samplescontained larvae, with counts rangingfrom 0.3-480 larvae per gram of feces. Larvae were recovered from all positive samples and are currently stored frozen until RADseq will be performed. Field sampling is ongoing and intended through the 2019 summer season. The development of this methodology and ecological application of results will facilitate ongoing endeavors to understand transmission pathways on a landscape scale, informing both risk to moose as well as domestic ruminant species also susceptible to P. tenuis infection.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Mayer T, Moore S, Fountain-Jones N, Johnson D, Anderson S, Marchetto K, Wolf T. Characterizing transmission epidemiology of the meningeal worm (Parelaphostrongylus tenuis). National Veterinary Summer Scholars Symposium, Texas A&M University, College Station, TX, August 4, 2018.