Comparison of Ketamine-Xylazine, Butorphanol-Azaperone-Medetomidine, and Nalbuphine-Medetomidine-Azaperone for Raccoon (Procyon lotor) Immobilization.

Raccoons (Procyon lotor) are frequently handled using chemical immobilization in North America for management and research. In a controlled environment, we compared three drug combinations: ketamine-xylazine (KX), butorphanol-azaperone-medetomidine (BAM), and nalbuphine-medetomidine-azaperone (NalMed-A) for raccoon immobilization. In crossover comparisons, raccoons received a mean of the following: 8.66 mg/kg ketamine and 1.74 mg/kg xylazine (0.104 mL/kg KX); 0.464 mg/kg butorphanol, 0.155 mg/kg azaperone, and 0.185 mg/kg medetomidine (0.017 mL/kg BAM); and 0.800 mg/kg nalbuphine, 0.200 mg/kg azaperone, and 0.200 mg/kg medetomidine (0.020 mL/kg NalMed-A). Induction time was shortest with KX (mean±SE, 10.0±0.7 min) and longest with NalMed-A (13.0±1.3 min). A sampling procedure was completed on 89% (16/18), 72% (13/18), and 89% (16/18) of the raccoons administered KX, BAM, and NalMed-A, respectively. Reasons for incomplete sampling included inadequate immobilization (one KX and one NalMed-A), responsive behaviors (one each with KX, BAM, NalMed-A), or animal safety (four BAM). Mean recovery time for KX was 32.8±7.1 min without antagonizing and 28.6±5.2 min following delivery of an antagonist. Mean recovery time was 6.2±0.8 min for BAM and 5.1±0.5 min for NalMed-A after antagonizing. Only with KX were raccoons observed to recover without use of an antagonist. Supplemental oxygen was provided to 23% (3/13), 72% (13/18), and 71% (12/17) of raccoons immobilized with KX, BAM, and NalMed-A, respectively. Hypoxemia at <80% oxygen saturation occurred in 0% (0/17), 27% (4/15), and 6% (1/16) of the raccoons administered KX, BAM, and NalMed-A, respectively; all raccoons fully recovered from chemical immobilization. All combinations could be used for raccoon immobilization; however, the need for delivery of supplemental oxygen to a majority of raccoons immobilized with BAM and NalMed-A may limit broader use of these agents for certain field studies involving capture, sample, and release of free-ranging animals from a practical standpoint.

Demographic, environmental and physiological predictors of gastrointestinal parasites in urban raccoons.

Raccoons are host to diverse gastrointestinal parasites, but little is known about the ecology of these parasites in terms of their interactions with each other during coinfections, their interactions with host physiology and environmental factors, and their impact on raccoon health and survival. As a first step, we investigated the patterns of parasite infection and their demographic distribution in an urban-suburban population of raccoons trapped in the summers and autumns of 2018 and 2019. We collected faecal samples, demographic data, morphometric measurements, and blood smears, and used GPS data to classify trapping location by land cover type. Faecal floats were performed to detect and quantify gastrointestinal nematode eggs and coccidia oocysts, and white blood cell differentials were performed on blood smears to characterise white blood cell distributions. Data were analysed cross-sectionally and, where possible, longitudinally, using generalised linear models. Overall, 62.6% of sampled raccoons were infected with gastrointestinal nematodes, and 82.2% were infected with gastrointestinal coccidia. We analysed predictors of infection status and faecal egg count for three different morphotypes of nematode-Baylisascaris, strongyle, and capillariid nematodes-and found that infection status and egg count varied with Year, Month, Age class, Land cover, and coinfection status, though the significance of these predictors varied between nematode types. Gastrointestinal coccidia prevalence varied with Year, Month, Age class, strongyle infection status, and capillariid infection status. Coccidia oocyst counts were lower in adults and in October, but higher in females and in raccoons trapped in areas with natural land cover; furthermore, coccidia oocysts were positively associated with capillariid faecal egg counts. We found no evidence that gastrointestinal parasites influenced raccoon body condition or overwinter mortality, and so conclude that raccoons, though harbouring diverse and abundant gastrointestinal parasites, may be relatively tolerant of these parasites.

Serological Responses of Raccoons and Striped Skunks to Ontario Rabies Vaccine Bait in West Virginia during 2012-2016.

Since the 1990s, oral rabies vaccination (ORV) has been used successfully to halt the westward spread of the raccoon rabies virus (RV) variant from the eastern continental USA. Elimination of raccoon RV from the eastern USA has proven challenging across targeted raccoon (Procyon lotor) and striped skunk (Mephitis mephitis) populations impacted by raccoon RV. Field trial evaluations of the Ontario Rabies Vaccine Bait (ONRAB) were initiated to expand ORV products available to meet the rabies management goal of raccoon RV elimination. This study describes the continuation of a 2011 trial in West Virginia. Our objective was to evaluate raccoon and skunk response to ORV occurring in West Virginia for an additional two years (2012-2013) at 75 baits/km2 followed by three years (2014-2016) of evaluation at 300 baits/km2. We measured the change in rabies virus-neutralizing antibody (RVNA) seroprevalence in targeted wildlife populations by comparing levels pre- and post-ORV during each year of study. The increase in bait density from 75/km2 to 300/km2 corresponded to an increase in average post-ORV seroprevalence for raccoon and skunk populations. Raccoon population RVNA levels increased from 53% (300/565, 95% CI: 50-57%) to 82.0% (596/727, 95% CI: 79-85%) during this study, and skunk population RVNA levels increased from 11% (8/72, 95% CI: 6-20%) to 39% (51/130, 95% CI: 31-48%). The RVNA seroprevalence pre-ORV demonstrated an increasing trend across study years for both bait densities and species, indicating that multiple years of ORV may be necessary to achieve and maintain RVNA seroprevalence in target wildlife populations for the control and elimination of raccoon RV in the eastern USA.

Variation in reversal learning by three generalist mesocarnivores.

Urbanization imposes novel challenges for wildlife, but also provides new opportunities for exploitation. Generalist species are commonly found in urban habitats, but the cognitive mechanisms facilitating their successful behavioral adaptations and exploitations are largely under-investigated. Cognitive flexibility is thought to enable generalists to be more plastic in their behavior, thereby increasing their adaptability to a variety of environments, including urban habitats. Yet direct measures of cognitive flexibility across urban wildlife are lacking. We used a classic reversal-learning paradigm to investigate the cognitive flexibility of three generalist mesocarnivores commonly found in urban habitats: striped skunks (Mephitis mephitis), raccoons (Procyon lotor), and coyotes (Canis latrans). We developed an automated device and testing protocol that allowed us to administer tests of reversal learning in captivity without extensive training or experimenter involvement. Although most subjects were able to rapidly form and reverse learned associations, we found moderate variation in performance and behavior during trials. Most notably, we observed heightened neophobia and a lack of habituation expressed by coyotes. We discuss the implications of such differences among generalists with regard to urban adaptation and we identify goals for future research. This study is an important step in investigating the relationships between cognition, generalism, and urban adaptation.

An oral bait vaccination approach for the Tasmanian devil facial tumor diseases.

Introduction: The Tasmanian devil (Sarcophilus harrisii) is the largest extant carnivorous marsupial. Since 1996, its population has declined by 77% primarily due to a clonal transmissible tumor, known as devil facial tumor (DFT1) disease. In 2014, a second transmissible devil facial tumor (DFT2) was discovered. DFT1 and DFT2 are nearly 100% fatal.Areas covered: We review DFT control approaches and propose a rabies-style oral bait vaccine (OBV) platform for DFTs. This approach has an extensive safety record and was a primary tool in large-scale rabies virus elimination from wild carnivores across diverse landscapes. Like rabies virus, DFTs are transmitted by oral contact, so immunizing the oral cavity and stimulating resident memory cells could be advantageous. Additionally, exposing infected devils that already have tumors to OBVs could serve as an oncolytic virus immunotherapy. The primary challenges may be identifying appropriate DFT-specific antigens and optimization of field delivery methods.Expert opinion: DFT2 is currently found on a peninsula in southern Tasmania, so an OBV that could eliminate DFT2 should be the priority for this vaccine approach. Translation of an OBV approach to control DFTs will be challenging, but the approach is feasible for combatting ongoing and future disease threats.

Immunogenicity of Ontario Rabies Vaccine for Small Indian Mongooses (Herpestes auropunctatus).

Oral rabies vaccination is the principal strategy used to control rabies in wildlife. No oral rabies vaccine is licensed for small Indian mongooses (Herpestes auropunctatus). The Ontario Rabies Vaccine Bait (ONRAB) is a human adenovirus type-5 rabies glycoprotein recombinant vaccine licensed for rabies control in striped skunks (Mephitis mephitis) in Canada and is under experimental evaluation in the US. We evaluated varying doses of ONRAB vaccine by direct instillation into the oral cavity with three groups of 10 mongooses: Group 1 received 109.5 TCID50, group 2 received 108.8 TCID50, and group 3 received 108.5 TCID50 of vaccine. Six control mongooses were sham-vaccinated with culture media. We collected a serum sample prior to vaccination and on days 14 and 30 postvaccination (PV). We quantified the level of rabies virus neutralizing antibodies (RVNA) from mongoose sera and compared titers among vaccinated groups and time points PV, where values greater than or equal to 0.1 IU/mL were considered positive. On day 14 PV, 87% (26 of 30, 95% confidence interval 70-95%) of vaccinates had seroconverted, whereas all vaccinates demonstrated RVNA by day 30 PV. There was a marginal effect of vaccine dose on group means of log-transformed RVNA titers at day 14 PV (F=2.5, P=0.099), but not day 30 PV. Sham-vaccinated animals were seronegative during all time points.

Volatile metabolomic signatures of rabies immunization in two mesocarnivore species.

Rabies is a zoonotic disease caused by infection with rabies virus, which circulates naturally in several wild carnivore and bat reservoirs in the United States (US). The most important reservoir in the US from an animal and public health perspective is the raccoon (Procyon lotor). To prevent the westward expansion of a significant raccoon rabies epizootic along the eastern seaboard, an operational control program implementing oral rabies vaccination (ORV) has existed in the US since the 1990s. Recently, two vaccine efficacy studies conducted with raccoons and striped skunks (Mephitis mephitis) provided the opportunity to determine if volatile fecal metabolites might be used to non-invasively monitor ORV programs and/or predict virus protection for these species. The volatile metabolome is a rich source of information that may significantly contribute to our understanding of disease and infection. Fecal samples were collected at multiple time points from raccoons and striped skunks subjected to oral treatment with rabies vaccine (or sham). Intramuscular challenge with a lethal dose of rabies virus was used to determine protection status at six (raccoons) and 11 (skunks) months post-vaccination. In addition to fecal samples, blood was collected at various time points to permit quantitative assessment of rabies antibody responses arising from immunization. Feces were analyzed by headspace gas chromatography with mass spectrometric detection and the chromatographic responses were grouped according to cluster analysis. Cluster scores were subjected to multivariate analyses of variance (MANOVA) to determine if fecal volatiles may hold a signal of immunization status. Multiple regression was then used to build models of the measured immune responses based on the metabolomic data. MANOVA results identified one cluster associated with protective status of skunks and one cluster associated with protective status of raccoons. Regression models demonstrated considerably greater success in predicting rabies antibody responses in both species. This is the first study to link volatile compounds with measures of adaptive immunity and provides further evidence that the volatile metabolome holds great promise for contributing to our understanding of disease and infections. The volatile metabolome may be an important resource for monitoring rabies immunization in raccoons and striped skunks.

EFFECT OF HIGH-DENSITY ORAL RABIES VACCINE BAITING ON RABIES VIRUS NEUTRALIZING ANTIBODY RESPONSE IN RACCOONS ( PROCYON LOTOR).

From 2014 to 2016, we examined the effect of distributing oral rabies vaccine baits at high density (150 baits/km2) in an area of Virginia, US that was naïve to oral rabies vaccination prior to the study. We also compared the effect of baiting at high density in a naïve area to baiting at standard density (75 baits/km2) in an area that had been baited annually for 12 yr. Our results suggested that rabies virus seroconversion in raccoons ( Procyon lotor) gradually increased each year under the highdensity bait treatment. However, we did not detect a difference in seroconversion between bait density treatments. Virginia opossums ( Didelphis virginiana) were abundant in the study area and were a potentially important nontarget species that competed for oral rabies vaccine baits, but the ratio of opossums to raccoons in this study did not affect rabies virus neutralizing antibody response of the raccoon populations.

FIELD TRIALS OF ONTARIO RABIES VACCINE BAIT IN THE NORTHEASTERN USA, 2012-14.

In the US, rabies virus (RV) has been enzootic in raccoons ( Procyon lotor) since the late 1940s. Oral rabies vaccination (ORV) was implemented in the 1990s to halt the spread of raccoon RV and continues to be used as a wildlife management tool. Our objective was to evaluate a recombinant human adenovirus-rabies virus glycoprotein vaccine in northern New York, Vermont, and New Hampshire over a 3-yr period, using changes in RV neutralizing antibody (RVNA) seroprevalence in raccoon populations as an immunologic index of ORV impact. Vaccine baits were distributed at 75 baits/km2 and 750-m flight-line spacing in the study area. Animal sampling occurred during 10-d intervals pre- and post-ORV during 2012-14 within eight study cells: four northern cells had a history of ORV with a different vaccine for 3 or more years prior and four southern cells were ORV naive. Baseline raccoon RVNA seroprevalence was 27.3% ( n=1,079, 95% confidence interval [CI]: 24.8-30.1) before ORV in 2012. Raccoon RVNA seroprevalence averaged 68.5% ( n=1,551, 95% CI: 66.2-70.8) post-ORV during the 3-yr study. The RVNA seroprevalence levels in this study were considered to be adequate for stopping raccoon RV transmission and supported and expanded the results from a West Virginia field trial, as well as earlier evaluations along the Canada-US border.

BAIT DEVELOPMENT FOR ORAL DELIVERY OF PHARMACEUTICALS TO RACCOONS (PROCYON LOTOR) AND STRIPED SKUNKS (MEPHITIS MEPHITIS).

Oral vaccination is one tool used to control wildlife diseases. A challenge to oral vaccination is identifying baits specific to target species. The US has been conducting oral vaccination against rabies since the 1990s. Improvements in bait development will hasten disease elimination. In Colorado, we examined a novel bait for oral vaccination and offered two different flavors, sweet and fish, to captive raccoons ( Procyon lotor ) and striped skunks ( Mephitis mephitis ) to assess consumption and flavor preference and observed bait removal by target and nontarget species in the field. During captive trials, raccoons and skunks consumed 98% and 87% of offered baits, respectively. Baits contained a sachet to simulate a vaccine package. Raccoons and skunks consumed 98% and 94% of the sachets, respectively. All unconsumed sachets were punctured, suggesting that animals had oral exposure to the contents. Raccoons preferred fish-flavored bait, but skunks did not have a preference. In the field, raccoons consumed the most baits, followed by fox squirrels ( Sciurus niger ). Other rabies host species (striped skunks, red foxes [ Vulpes vulpes ], coyotes [ Canis latrans ]) had very low visitation and were never observed consuming baits. High consumption rates by raccoons and skunks in captivity and observance of raccoons consuming baits in the field suggest that these baits may be useful for oral delivery of pharmaceuticals. Further field research is warranted to determine how to best optimize bait delivery.

Exposure to Rabies in Small Indian Mongooses (Herpestes auropunctatus) from Two Regions in Puerto Rico.

The small Indian mongoose (Herpestes auropunctatus) was introduced to several Caribbean Islands to control rat (Rattus spp.) damage to sugarcane plantations. Mongooses failed at suppressing rat populations and are now considered pests throughout most of their introduced range. Importantly, mongooses are rabies reservoirs on several Caribbean Islands. In Puerto Rico, mongooses have been implicated in up to 70% of reported animal rabies cases. There is no rabies vaccination program for wildlife in Puerto Rico, and data on rabies in mongooses are limited. We conducted a serosurvey of mongooses in two different ecologic environments in Puerto Rico: El Yunque National Forest and Cabo Rojo National Wildlife Refuge. We collected 119 serum samples from 112 mongooses, 44 (39.3%) of which were positive for rabies virus-neutralizing antibodies. We also collected oral swabs from 147 mongooses, including 88 from which we also collected serum. No oral swabs were positive for rabies virus RNA. Our data support previous research suggesting rabies virus is circulating within the mongoose population on Puerto Rico.

EVALUATION OF ANTHELMINTIC FISHMEAL POLYMER BAITS FOR THE CONTROL OF BAYLISASCARIS PROCYONIS IN FREE-RANGING RACCOONS (PROCYON LOTOR).

Baylisascaris procyonis is a common gastrointestinal parasite of raccoons (Procyon lotor) and is a zoonotic helminth with the potential to cause severe or fatal infection. Raccoons thrive in human-dominated landscapes, and the fecal-oral transmission pathway and lack of effective treatment make B. procyonis a serious threat to public health. The distribution of medicinal baits has emerged as a socially acceptable and cost-effective method for managing disease in free-ranging wildlife. We assessed the suitability of a mass-producible anthelmintic bait for B. procyonis mitigation by evaluating the willingness of free-ranging raccoons to consume anthelmintic baits and determining whether bait consumption successfully cleared B. procyonis infections from raccoons. Anthelmintic baits were modified from standard fishmeal polymer baits, the food attractant commonly used in oral rabies vaccine baits, with the introduction of 220 mg of pyrantel pamoate into the fishmeal mixture. We captured 16 naturally infected raccoons, presented one anthelmintic bait, and monitored B. procyonis infection over 90 d by screening feces for eggs. Treatment cleared B. procyonis infections for nine of 12 raccoons that consumed >10 g of the 15 g bait. We used remote cameras to monitor in situ patterns of bait consumption for anthelmintic baits relative to standard baits. Both anthelmintic and standard baits were rapidly consumed, with no differences in the rate of consumption between bait types. However, after bait contact, raccoons demonstrated a greater willingness to consume standard baits while ignoring anthelmintic baits more frequently (P = 0.06). Initial trials of anthelmintic baits show promise, although refinement in both dose and palatability is needed. At mass production scales, the addition of pyrantel pamoate to fishmeal polymer baits would be inexpensive, potentially making anthelmintic baits a viable management option when coupled with an oral rabies vaccine or used independently for B. procyonis mitigation.

A behaviorally-explicit approach for delivering vaccine baits to mesopredators to control epizootics in fragmented landscapes.

Despite the widespread use of aerial baiting to manage epizootics among free-ranging populations, particularly in rabies management, bait acceptance and seroconversion rates often are lower than required to eliminate spread of disease. Our objectives in this study, therefore, were to evaluate the performance of stratified bait distribution models derived from resource selection functions (RSF) on uptake of placebo rabies baits by raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana), as well as the probability of bait uptake as a function of proximity to bait distribution areas in fragmented agricultural ecosystems. Among 478 raccoons and 108 opossums evaluated for presence of Rhodamine B (RB) across 8 sites, only 26% of raccoons and 20% of opossums exhibited marking consistent with bait consumption 14-24 days post-baiting. The effective area treated, based on 90% kernel density estimators of marked individuals, ranged from 99-240 ha larger than bait distribution zones, with RB marked individuals captured up to 753 m beyond the bait zone. Despite incorporation of RSF data into bait distribution models, no differences in uptake rates were observed between treatment and control sites. These data likely reflect the underlying constraints imposed by the loss and fragmentation of habitat on animal movement in heterogeneous landscapes, forcing individuals to optimize movements at coarse (i.e., patch-level) rather than fine spatial scales in highly fragmented environments. Our data also confirm that the probability of bait acceptance decreases with increasing distance from bait zone interiors, even within the zone itself. Thus, although bait acceptance was confirmed beyond bait zone boundaries, the proportion of vaccinated individuals may comprise a small minority of the population at increasing distances from baiting interiors. These data suggest focal baiting creates a buffered area of treated individuals around bait zones or bait stations, but repeated treatments may be needed to achieve sufficient uptake to eradicate disease.

Use of infrared thermography to detect thermographic changes in mule deer (Odocoileus hemionus) experimentally infected with foot-and-mouth disease.

Infrared thermography (IRT) measures the heat emitted from a surface, displays that information as a pictorial representation called a thermogram, and is capable of being a remote, noninvasive technology that provides information on the health of an animal. Foot-and-mouth disease (FMD) caused by FMD virus (FMDV) is a severe, highly communicable viral disease of cloven-hoofed animals, including both domestic and wild ruminants. Early detection of the disease may reduce economic loss and loss of susceptible wildlife. The objective of this study was to evaluate the use of IRT to detect possible heat changes associated with sites of infection with FMDV in experimentally infected mule deer (Odocoileus hemionus). Infection occurred through either inoculation with FMDV or exposure to inoculated animals. Early vesicular lesions were observed on the mouth, feet, or both within 24 hrs postinoculation and 48-96 hrs post-exposure. From internal temperature sensors, the exposed animals' body temperatures elevated significantly from the pre-infection temperature (38.8 degrees C, P < or = 0.002) starting the day before any lesions were observed. Body temperature was also found not to be significantly different from eye temperatures of well-focused thermograms. For feet thermograms, the mean of the daily maximum (MMAX) foot temperature rose significantly (P = 0.017) from two days before (27.3 degrees C +/- 1.9 degrees C SE) to the maximum MMAX observed (33.0 degrees C +/- 2.0 degrees C SE) at two days after the first foot lesion occurrence. These observed changes indicate that IRT may be a rapid, remote, and noninvasive method to screen for suspect animals in order to test further for FMDV infection during an FMD outbreak.