Amy Turmelle Gilbert

Biomarkers contribute to measuring uptake in wildlife baiting applications, such as oral rabies vaccination (ORV), when marking capabilities are adequately validated. One biomarker, iophenoxic acid (IPA), can exist in multiple analogues and has the potential of being a quantitative marker for ORV uptake, but the marking capabilities of the different analogues have not been adequately validated for target species, such as raccoons ( Procyon lotor ). We experimentally fed captive raccoons 4 different encapsulated IPA analogues, methyl, ethyl, butyl, and pentyl, at 3 different doses, 2.5 mg, 5 mg, and 10 mg, to evaluate marking and decay relative to dose. We then evaluated marking of 2 IPA analogues, ethyl and propyl, incorporated within the matrix of placebo ORV baits. We evaluated the concentration of each IPA analogue in raccoon serum using liquid chromatography tandem mass spectrometry. For marking and decay evaluation, all 4 IPAs at all doses marked raccoons beyond 454 days, and we observed dose‐dependent relationships with serum IPA concentration. Testing with ethyl or propyl in an ORV bait matrix at 1 mg IPA per bait marked raccoon sera for at least 67 days post‐consumption. All IPAs evaluated have use for estimating bait uptake by raccoons. The IPA analogue, sampling window, and dose are all factors to consider when selecting and using an IPA for estimating bait uptake in wildlife.

The common vampire bat ( Desmodus rotundus ) is expanding in geographic range, increasing concern about the spread of this species to the United States (U.S.) and the associated risks of rabies virus (RABV) transmission. In 2020, we used a structured approach to convene a panel of experts to assess the risks of common vampire bat and vampire bat RABV incursion into the U.S. and to identify strategies for surveillance, management, research, and outreach/education. Expert opinion estimated a relatively low probability (25%) of common vampire bat establishment in the U.S. within 5 years and an even lower probability (12%) regarding the establishment of vampire bat RABV. However, both probabilities increased (60% for the common vampire bat and 48% for vampire bat RABV) within a 20‐year time horizon. Experts attributed increasing risks of common vampire bat range expansion to increasing temperatures, land cover use changes, and vampire bat population growth. If established, the highest perceived risk of vampire bat RABV transmission was to livestock and domestic animals, followed by other wildlife and humans. Vaccination of livestock and enhanced rabies surveillance were identified as key strategies for mitigating and detecting potential incursions of vampire bat RABV, yet culling or population control of common vampire bats was not widely supported by experts to reduce risks of range expansion nor viral transmission. Education and outreach were identified as critical components of a vampire bat RABV prevention and response strategy, including public awareness about identifying vampire bats and their bites, the health risks of RABV infection, and the importance of case reporting and preventive vaccination. The process also underscored the need for proactive, interagency communication and coordination to ensure readiness to detect common vampire bats within U.S. borders. This work demonstrates how expert elicitation can provide both quantitative risk estimates and qualitative insights to support evidence‐based management in the face of ecological uncertainty.

Ethyl-iophenoxic acid (Et-IPA) is a widely used biomarker to confirm oral bait consumption in eutherian species. Although previously believed to have limited use for marsupial species, a 1 mg oral dose was detectable in a marsupial; Tasmanian devils (Sarcophilus harrisii) were marked for ≥56 days, supporting the biomarker’s use in a devil bait vaccine program. Using liquid chromatography with tandem mass spectrometry, we measured serum Et-IPA in four off-target marsupial species administered 1 mg oral doses of Et-IPA. By Day 14, Et-IPA was undetectable in all four species, suggesting it is not a useful biomarker for these species.

Ethyl-iophenoxic acid (Et-IPA) is widely recognized as a useful biomarker to confirm oral bait consumption in eutherian species. In historical studies on marsupials, Et-IPA was rapidly eliminated from brushtail possums ( Trichosurus vulpecula ) and swamp wallabies ( Wallabia bicolor ) suggesting limited use for marsupial species. However, a 1 mg oral dose of Et-IPA was detectable in the marsupial Tasmanian devils ( Sarcophilus harrisii ) for ≥ 56 days suggesting the biomarker can be used in a devil bait vaccine program. To assess Et-IPA marking in off-target marsupials that may consume baits, we administered 1 mg oral doses of Et-IPA to brushtail possums, forester kangaroos ( Macropus giganteus tasmaniensis ), spotted-tailed quolls ( Dasyurus maculatus ) and eastern quolls ( Dasyurus viverrinus ). Liquid chromatography with tandem mass spectrometry was used to detect and quantify serum Et-IPA. Et-IPA was detected in the serum on day 2 but was not detected by day 14 in any of the species tested, including the two quoll species which are in the same carnivorous Dasyuridae family as the devils. The rapid elimination of Et-IPA in the marsupials included in this study suggests it is not useful as a biomarker for these species. Furthermore, rapid elimination in the kangaroos and possums suggests that Et-IPA is unlikely to accumulate in the food chain following distribution of Et-IPA-marked oral bait vaccines for Tasmanian devils. Short summary for non-experts: A recent study in Tasmanian devils ( Sarcophilus harrisii ) challenged the concept that ethyl iophenoxic acid (Et-IPA) is not a useful serum biomarker for marsupials. Using the same sensitive liquid chromatography-tandem mass spectrometry method we detected serum Et-IPA in four marsupial species on day two post-ingestion but by day 14, serum Et-IPA was undetectable. These findings indicate that Et-IPA is an unsuitable biomarker for these species and suggest that Et-IPA from devil bait vaccines is unlikely to bioaccumulate in the Tasmanian environment.

Density estimation for unmarked animals is particularly challenging, yet density estimates are often necessary for effective wildlife management. Raccoons (Procyon lotor) are the primary terrestrial wildlife reservoir for Lyssavirus rabies within the United States. The raccoon rabies variant (RRVV) is actively managed at landscape scales using oral rabies vaccination (ORV) within the eastern United States. To effectively manage RRVV, it is important to know the density of raccoons to appropriately scale the density of ORV baits distributed on the landscape. We compared methods to estimate raccoon densities from camera‐trap data versus more intensive capture‐mark‐recapture (CMR) estimates across 2 land cover types (upland pine and bottomland hardwood) in the southeastern United States during 2019 and 2020. We evaluated the effect of alternative camera configurations and durations of camera trapping on density estimates and used an N‐mixture model to estimate raccoon densities, including covariates on abundance and detection. We further compared different methods of scaling camera‐based counts, with the maximum number of raccoons seen on any given image within a day best explaining density. Camera‐trap density estimates were moderately correlated with CMR estimates (r = 0.56). However, densities from camera‐trap data were more reliable when classifying category of density as an index used to inform management (83% correct when compared to CMR estimates), although the densities in our study fell into the 2 lowest density classes only. Using more cameras reduced bias and uncertainty around density estimates; however, if ≤6 camera traps were used at a site, a line transect approach proved less biased than a grid design. Camera trapping should be conducted for at least 3 weeks for more accurate estimates of raccoon population density in our study area (<5% bias). We show that camera‐trap data can be used to assign raccoon densities to management‐relevant density index bins, but more studies are needed to ensure reliability across a greater range of environmental conditions and raccoon densities.

Oral baits containing vaccines, medicants or toxicants have been delivered to a broad range of wildlife species to protect against or treat disease, or suppress populations. Ethyl-Iophenoxic acid (Et-IPA) is used as a biomarker to determine oral bait consumption by wildlife species to assess and refine baiting strategies. Et-IPA is a persistent biomarker in many eutherian mammal species but not in the two marsupial species, swamp wallaby (Wallabia bicolor) and brushtail possum (Trichosurus vulpecula), tested to date. Et-IPA has not previously been evaluated in carnivorous marsupials. The Tasmanian devil (Sarcophilus harrisii), the largest extant carnivorous marsupial, is threatened by devil facial tumour disease (DFTD). Development of an oral DFTD vaccine bait is underway. In this study, eight devils were fed between 1 mg and 50 mg Et-IPA via oral baits to assess the effectiveness of Et-IPA as a serum biomarker for this species. Using liquid chromatography with tandem mass spectrometry, serum Et-IPA was detected in the devils up to 206 days after ingestion. This study demonstrates the utility of Et-IPA for estimating oral bait consumption by devils, the first carnivorous marsupial species to which this applies.

The reservoir for zoonotic o’nyong-nyong virus (ONNV) has remained unknown since this virus was first recognized in Uganda in 1959. Building on existing evidence for mosquito blood-feeding on various frugivorous bat species in Uganda, and seroprevalence for arboviruses among bats in Uganda, we sought to assess if serum samples collected from bats in Uganda demonstrated evidence of exposure to ONNV or the closely related zoonotic chikungunya virus (CHIKV). In total, 652 serum samples collected from six bat species were tested by plaque reduction neutralization test (PRNT) for neutralizing antibodies against ONNV and CHIKV. Forty out of 303 (13.2%) Egyptian rousettes from Maramagambo Forest and 1/13 (8%) little free-tailed bats from Banga Nakiwogo, Entebbe contained neutralizing antibodies against ONNV. In addition, 2/303 (0.7%) of these Egyptian rousettes contained neutralizing antibodies to CHIKV, and 8/303 (2.6%) contained neutralizing antibodies that were nonspecifically reactive to alphaviruses. These data support the interepidemic circulation of ONNV and CHIKV in Uganda, although Egyptian rousette bats are unlikely to serve as reservoirs for these viruses given the inconsistent occurrence of antibody-positive bats.

The Virginia opossum (Didelphis virginiana), North America's only marsupial, has a range extending from southern Ontario, Canada, to the Yucatan Peninsula, Mexico, and from the Atlantic seaboard to the Pacific. Despite the Virginia opossum's taxonomic uniqueness in relation to other mammals in North America and rapidly expanding distribution, its ecology remains relatively understudied. Our poor understanding of the ecology of this important mesopredator is especially pronounced in the rural southeastern United States. Our goal was to estimate effects of habitat on opossum density within an extensive multi‐year spatial capture‐recapture study. Additionally, we compared the results of this spatial capture‐recapture analysis with a simple relative abundance index. Opossum densities in the relatively underdeveloped regions of the southeastern United States were lower compared to the more human‐dominated landscapes of the Northeast and Midwest. In the southeastern United States, Virginia opossums occurred at a higher density in bottomland swamp and riparian hardwood forest compared to upland pine (Pinus spp.) plantations and isolated wetlands. These results reinforce the notion that the Virginia opossum is commonly associated with land cover types adjacent to permanent water (bottomland swamps, riparian hardwood). The relatively low density of opossums at isolated wetland sites suggests that the large spatial scale of selection demonstrated by opossums gives the species access to preferable cover types within the same landscape.

Rabies is an ancient lethal scourge that has plagued humankind for centuries