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.

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.

Screening of Microbial Volatile Organic Compounds for Detection of Disease in Cattle: Development of Lab-scale Method.

The primary hurdle for diagnosis of some diseases is the long incubation required to culture and confirm the presence of bacteria. The concept of using microbial VOCs as "signature markers" could provide a faster and noninvasive diagnosis. Finding biomarkers is challenging due to the specificity required in complex matrices. The objectives of this study were to (1) build/test a lab-scale platform for screening of microbial VOCs and (2) apply it to Mycobacterium avium paratuberculosis; the vaccine strain of M. bovis Bacillus Calmette-Guérin; and M. kansasii to demonstrate detection times greater those typically required for culture. SPME-GC-MS was used for sampling, sample preparation, and analyses. For objective (1), a testing platform was built for headspace sampling of bacterial cultures grown in standard culture flasks via a biosecure closed-loop circulating airflow system. For (2), results show that the suites of VOCs produced by Mycobacteria ssp. change over time and that individual strains produce different VOCs. The developed method was successful in discriminating between strains using a pooled multi-group analysis, and in timepoint-specific multi- and pair-wise comparisons. The developed testing platform can be useful for minimally invasive and biosecure collection of biomarkers associated with human, wildlife and livestock diseases for development of diagnostic point-of-care and field surveillance.

Research with Agricultural Animals and Wildlife.

In fiscal year 2016, agricultural animals such as swine, sheep, goats, and cattle represented 10% of the 820 812 animals used in USDA-regulated research. In addition to traditional agricultural animals, research studies using captive wildlife are becoming increasingly important as human and livestock populations encroach upon, and thus expand interactions with, wildlife populations on the landscape. Optimum healthcare of both livestock and captive wildlife in a research setting requires proper husbandry, management, and veterinary care. Regardless of animal species, proper care and management are essential for animal well-being, valid research data, and the health and safety of animal care personnel. Using wildlife in research presents unique challenges as there is generally limited peer-reviewed research on wildlife welfare, husbandry, and nutrition. Animals often become excited during handling or transport, and care must be taken to avoid injury. When severe injuries do occur, differences may exist in methods of euthanasia. Many wildlife species are evolutionarily programmed to conceal signs of illness, making assessment of their condition difficult; moreover, attending veterinarians are often not as experienced in the care of wildlife as they are in the care of traditional laboratory animals or livestock. These differences are further magnified in the context of wildlife field research. The concepts of replace, reduce, and refine are as valid in livestock and wildlife research as in biomedical research, and investigators should work closely with their Institutional Animal Care and Use Committees to ensure humane animal care. The Institutional Animal Care and Use Committee is centrally important in providing guidelines relative to ethical use of animal subjects for research and can serve as a valuable resource for research accountability.

Use of faecal volatile organic compound analysis for ante-mortem discrimination between CWD-positive, -negative exposed, and -known negative white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is a naturally occurring infectious, fatal, transmissible spongiform encephalopathy of cervids. Currently, disease confirmation relies on post-mortem detection of infectious prions in the medial retropharyngeal lymph nodes or obex in the brain via immunohistochemistry (IHC). Detection of CWD in living animals using this method is impractical, and IHC and other experimental assays are not reliable in detecting low concentrations of prion present in biofluids or faeces. Here, we evaluate the capability of faecal volatile organic compound analysis to discriminate between CWD-positive and -exposed white-tailed deer located at two positive cervid farms, and two groups of CWD-negative deer from two separate disease-free farms.

Use of fecal volatile organic compound analysis to discriminate between non-vaccinated and BCG-Vaccinated cattle prior to and after Mycobacterium bovis challenge.

Bovine tuberculosis is a zoonotic disease of global public health concern. Development of diagnostic tools to improve test accuracy and efficiency in domestic livestock and enable surveillance of wildlife reservoirs would improve disease management and eradication efforts. Use of volatile organic compound analysis in breath and fecal samples is being developed and optimized as a means to detect disease in humans and animals. In this study we demonstrate that VOCs present in fecal samples can be used to discriminate between non-vaccinated and BCG-vaccinated cattle prior to and after Mycobacterium bovis challenge.

Fecal Volatile Organic Ccompound Profiles from White-Tailed Deer (Odocoileus virginianus) as Indicators of Mycobacterium bovis Exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) Vaccination.

White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis Bacille Calmette Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no method exists to non-invasively monitor the presence of bovine tuberculosis in deer. In this study, volatile organic compound profiles of BCG-vaccinated and non-vaccinated deer, before and after experimental challenge with M. bovis strain 95-1315, were generated using solid phase microextraction fiber head-space sampling over suspended fecal pellets with analysis by gas chromatography/mass spectrometry. Chromatograms were processed using XCMS Online to characterize ion variation among treatment groups. The principal component scores resulting from significant (α = 0.05) ion responses were used to build linear discriminant analysis models. The sensitivity and specificity of these models were used to evaluate the feasibility of using this analytical approach to distinguish within group comparisons between pre- and post-M. bovis challenge: non-vaccinated male or female deer, BCG-vaccinated male deer, and the mixed gender non-vaccinated deer data. Seventeen compounds were identified in this analysis. The peak areas for these compounds were used to build a linear discriminant classification model based on principal component analysis scores to evaluate the feasibility of discriminating between fecal samples from M. bovis challenged deer, irrespective of vaccination status. The model best representing the data had a sensitivity of 78.6% and a specificity of 91.4%. The fecal head-space sampling approach presented in this pilot study provides a non-invasive method to discriminate between M. bovis challenged deer and BCG-vaccinated deer. Additionally, the technique may prove invaluable for BCG efficacy studies with free-ranging deer as well as for use as a non-invasive monitoring system for the detection of tuberculosis in captive deer and other livestock.

A pilot study exploring the use of breath analysis to differentiate healthy cattle from cattle experimentally infected with Mycobacterium bovis.

Bovine tuberculosis, caused by Mycobacterium bovis, is a zoonotic disease of international public health importance. Ante-mortem surveillance is essential for control; however, current surveillance tests are hampered by limitations affecting ease of use or quality of results. There is an emerging interest in human and veterinary medicine in diagnosing disease via identification of volatile organic compounds produced by pathogens and host-pathogen interactions. The objective of this pilot study was to explore application of existing human breath collection and analysis methodologies to cattle as a means to identify M. bovis infection through detection of unique volatile organic compounds or changes in the volatile organic compound profiles present in breath. Breath samples from 23 male Holstein calves (7 non-infected and 16 M. bovis-infected) were collected onto commercially available sorbent cartridges using a mask system at 90 days post-inoculation with M. bovis. Samples were analyzed using gas chromatography-mass spectrometry, and chromatographic data were analyzed using standard analytical chemical and metabolomic analyses, principle components analysis, and a linear discriminant algorithm. The findings provide proof of concept that breath-derived volatile organic compound analysis can be used to differentiate between healthy and M. bovis-infected cattle.

Detection of volatile organic compounds in Brucella abortus-seropositive bison.

Brucellosis is of great public health and economic importance worldwide. Detection of brucellosis currently relies on serologic testing of an antibody response to Brucella infection, which suffers from cross-sensitivities to other antibody responses. Here we present a new method for identifying Brucella exposure that is based on profiling volatile organic compounds (VOCs) in exhaled breath. Breath samples from Brucella-seropositive bison and controls were chemically analyzed and demonstrated statistically significant differences in the concentration profiles of five VOCs. A point-of-care device incorporating an array of nanomaterial-based sensors could identify VOC patterns indicative of Brucella exposure with excellent discriminative power, using a statistical algorithm. We show that the patterns were not affected by the animals' environment and that the discriminative power of the approach was stable over time. The Brucella-indicative VOCs and collective patterns that were identified in this pilot study could lead to the development of a novel diagnostic screening test for quickly detecting infected animals chute-side, pen-side, or even remotely in populations of free-ranging ungulates. The promising preliminary results presented encourage subsequent larger scale trials in order to further evaluate the proposed method.

Ecology and geography of human monkeypox case occurrences across Africa.

As ecologic niche modeling (ENM) evolves as a tool in spatial epidemiology and public health, selection of the most appropriate and informative environmental data sets becomes increasingly important. Here, we build on a previous ENM analysis of the potential distribution of human monkeypox in Africa by refining georeferencing criteria and using more-diverse environmental data to identify environmental parameters contributing to monkeypox distributional ecology. Significant environmental variables include annual precipitation, several temperature-related variables, primary productivity, evapotranspiration, soil moisture, and pH. The potential distribution identified with this set of variables was broader than that identified in previous analyses but does not include areas recently found to hold monkeypox in southern Sudan. Our results emphasize the importance of selecting the most appropriate and informative environmental data sets for ENM analyses in pathogen transmission mapping.