Effects of the Immunocontraceptive Gonacon on Pregnancy in Brucella-Seropositive American bison (Bison bison).

The purpose of this study was to determine if the number of pregnancies in naturally infected Brucella abortus-positive bison (Bison bison) cows would be reduced over a period of 5 yr after one treatment with 3000 µg gonadotropin-releasing hormone immunocontraceptive (GonaCon) compared to a similar group of naturally infected B. abortus-positive bison cows not treated with GonaCon. In each of the 5 yr, GonaCon-treated cows produced fewer offspring in relation to number of cows than the nontreated cows. Fisher's Exact test comparing offspring produced during the first reproductive season showed a significant difference between the two groups (P=0.0028). Differences in number of calves produced in GonaCon-treated and control groups were also noted in remaining years, but statistics were not applied because of data constraints. These data indicate that one treatment with GonaCon in brucellosis-seropositive female bison reduced pregnancies over five reproductive years. Thus, immunocontraception could potentially be used to manage brucellosis in affected herds.

Effects of Pregnancy Prevention on Brucella abortus Shedding in American bison (Bison bison).

Products of parturition are the predominant source of Brucella abortus for transmission in bison (Bison bison). Our objective was to assess whether preventing pregnancy in Brucella-seropositive bison reduced B. abortus shedding. Brucella-seropositive and -seronegative bison from Yellowstone National Park, Wyoming, USA were used in a replicated experiment. Each of two replicates (rep1, rep2) included a group of seropositive females treated with a single dose of gonadotropin-releasing hormone-based immunocontraceptive (Treatment rep1, n=15; Treatment rep2, n=20) and an untreated group (Control rep1, n=14; Control rep2, n=16) housed separately. Seronegative sentinel females were placed in each group to monitor horizontal transmission. Seronegative males were co-mingled for breeding each year. Pregnant females were removed from treatment groups in the first year, but not thereafter. Each January-June we monitored for B. abortus shedding events-any parturition associated with culture-positive fluids or tissues. We analyzed probability of shedding events using a negative binomial generalized linear mixed model fit by maximum likelihood using Laplace approximation. Over 5 yr, we observed zero shedding events in Treatment rep1 vs. 12 in Control rep1. All five Control rep1 sentinels but zero (0/5) Treatment rep1 sentinels seroconverted. In the second replicate, Treatment rep2 had two shedding events over 3 yr and Control rep2 had five events over 2 yr. Sentinels in both Control rep2 (3/6) and Treatment rep2 (5/6) seroconverted by trial endpoint. Treatment rep1 showed a reduced shedding probability relative to Control rep1, Treatment rep2, and Control rep2 (log odds value -25.36 vs. -1.71, -1.39, and -0.23, respectively). Fixed effect predictor covariates, year and age, had no explanatory value. These data suggest that successful contraception of brucellosis-seropositive female bison prevents shedding of B. abortus by individual animals. However, contraceptive treatment may or may not sufficiently reduce disease transmission to reduce brucellosis prevalence in an affected herd.

Immune Responses and Efficacy of Brucella Abortus Strain RB51 in Bison After Delivery in a Dry Dart Formulation or by Parenteral Inoculation.

Bison (Bison bison) heifer calves (n = 32) were randomly assigned to control or vaccination with 1010 colony-forming units of Brucella abortus strain RB51 (RB51) vaccine by single or boostered parenteral delivery, or by surgical implantation of a dry dart formulation (n = 8/trt). Serum and/or peripheral blood mononuclear cells (PBMC) were obtained at 0, 4, 8, 13, 16, 21, and 24 wks after initial vaccination and at 0, 4, 8, 12, 15, 22, and 27 wks after booster vaccination to characterize humoral and cellular immune responses to RB51. Bison in both RB51 vaccination treatments demonstrated greater (P < 0.0001) serum humoral responses when compared to non-vaccinates, with parenteral vaccinates demonstrating greater (P < 0.01) responses when compared to mean responses of bison inoculated with the dry dart. Only the booster vaccinated treatment demonstrated greater (P < 0.0001) humoral responses than control bison in samples collected after re-inoculation. At 4, 8, 12, 16, and 24 wks after initial vaccination, PBMC from parenteral RB51 vaccinates demonstrated greater proliferative responses to RB51 when compared to responses of control animals. In comparison, bison inoculated with the RB51 dry dart did not demonstrate greater (P > 0.05) proliferative responses when compared to responses of non-vaccinates. Bison were pasture bred and pregnant animals experimentally challenged in mid-gestation with 107 CFU of B. abortus strain 2,308. Bison in parenteral vaccination treatments had reduced (P < 0.05) abortions and infection in uterine and fetal samples as compared to non-vaccinated bison, with booster vaccinates tending to have the lowest colonization (CFU/gm) in tissues. In comparison, the dry dart formulation did reduce abortion (P < 0.05) but not infection (P > 0.05) in most tissues when compared to non-vaccinated bison. The results of this study reaffirm the efficacy of boostered parenteral vaccination of bison with RB51 in preventing brucellosis. Our data also suggests that the novel dry dart RB51 formulation does not induce sufficient efficacy in bison after a single inoculation.

Non-Invasive Method to Detect Infection with Mycobacterium tuberculosis Complex in Wild Boar by Measurement of Volatile Organic Compounds Obtained from Feces with an Electronic Nose System.

More effective methods to detect bovine tuberculosis, caused by Mycobacterium bovis, in wildlife, is of paramount importance for preventing disease spread to other wild animals, livestock, and human beings. In this study, we analyzed the volatile organic compounds emitted by fecal samples collected from free-ranging wild boar captured in Doñana National Park, Spain, with an electronic nose system based on organically-functionalized gold nanoparticles. The animals were separated by the age group for performing the analysis. Adult (>24 months) and sub-adult (12-24 months) animals were anesthetized before sample collection, whereas the juvenile (<12 months) animals were manually restrained while collecting the sample. Good accuracy was obtained for the adult and sub-adult classification models: 100% during the training phase and 88.9% during the testing phase for the adult animals, and 100% during both the training and testing phase for the sub-adult animals, respectively. The results obtained could be important for the further development of a non-invasive and less expensive detection method of bovine tuberculosis in wildlife populations.

Production of embryos and a live offspring using post mortem reproductive material from bison (Bison bison bison) originating in Yellowstone National Park, USA.

Bison from Yellowstone National Park (YNP) have an important genetic history. As one of the few wild herds of bison with no evidence of cattle DNA introgression and a large enough population to maintain genetic diversity, they are considered a conservation priority for the species. Unfortunately, there is a high prevalence of the zoonotic disease brucellosis in the herd. Part of the management strategy for controlling the disease and herd size in YNP is to remove bison from the population during the winter migration out of the park. This interagency management cull provides an opportunity to collect a large number of oocytes from a wild bison population for genetic banking and research purposes. During the winters of 2014-2018, which is the nonbreeding season for bison, oocytes were collected post mortem and used to determine the effects of donor reproductive maturity and pregnancy status on oocyte quality and in vitro fertilization (IVF) outcomes, and to demonstrate the feasibility of producing healthy offspring. Cumulus oocyte complexes (COCs) were placed into an in vitro embryo production (IVP) system, and on days 7, 7.5, and 8 of in vitro culture (Day 0 = day of in vitro fertilization) embryos were assessed for developmental stage and quality prior to vitrification. Embryos were then stored in liquid nitrogen until the breeding season when a subset were warmed, cultured for 6 h, evaluated for survival, and transferred to healthy bison recipients. There were no significant differences in the ability of recovered COCs to support blastocyst development based on female reproductive maturity or pregnancy status (juvenile 79/959 (8.2%) vs sexually mature 547/6544 (8.4%); non-pregnant 188/2302 (8.2%) vs pregnant 556/6122 (9.1%)). Following the transfer of 15 embryos to 10 recipients, one healthy female calf was born. This work demonstrates that live offspring can be generated from COCs collected from YNP bison post mortem in the non-breeding season, and that gamete recovery can be a valuable tool for conservation of valuable genetics for this species while mitigating diseases like brucellosis.

Evaluation of Volatile Organic Compounds Obtained from Breath and Feces to Detect Mycobacterium tuberculosis Complex in Wild Boar (Sus scrofa) in Doñana National Park, Spain.

The presence of Mycobacterium tuberculosis complex (MTBC) in wild swine, such as in wild boar (Sus scrofa) in Eurasia, is cause for serious concern. Development of accurate, efficient, and noninvasive methods to detect MTBC in wild swine would be highly beneficial to surveillance and disease management efforts in affected populations. Here, we describe the first report of identification of volatile organic compounds (VOC) obtained from the breath and feces of wild boar to distinguish between MTBC-positive and MTBC-negative boar. We analyzed breath and fecal VOC collected from 15 MTBC-positive and 18 MTBC-negative wild boar in Donaña National Park in Southeast Spain. Analyses were divided into three age classes, namely, adults (>2 years), sub-adults (12-24 months), and juveniles (<12 months). We identified significant compounds by applying the two-tailed statistical t-test for two samples assuming unequal variance, with an α value of 0.05. One statistically significant VOC was identified in breath samples from adult wild boar and 14 were identified in breath samples from juvenile wild boar. One statistically significant VOC was identified in fecal samples collected from sub-adult wild boar and three were identified in fecal samples from juvenile wild boar. In addition, discriminant function analysis (DFA) was used to build classification models for MTBC prediction in juvenile animals. Using DFA, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. Based on our results, further research is warranted and should be performed using larger sample sizes, as well as wild boar from various geographic locations, to verify these compounds as biomarkers for MTBC infection in this species. This new approach to detect MTBC infection in free-ranging wild boar potentially comprises a reliable and efficient screening tool for surveillance in animal populations.

Effects of Inactivated Mycobacterium bovis Vaccination on Molokai-Origin Wild Pigs Experimentally Infected with Virulent M. bovis.

The wild pig population on Molokai, Hawaii, USA is a possible reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and has been implicated in decades past as the source of disease for the island's domestic cattle. Heat-inactivated vaccines have been effective for reducing disease prevalence in wild boar in Spain and could prove useful for managing M. bovis in Molokai wild pigs. We designed an experiment to test this vaccine in wild pigs of Molokai genetics. Fifteen 3-4-month-old pigs were orally administered 106-107 colony forming units (cfu) of heat-inactivated M. bovis (Vaccinates; n = 8; 0.2 mL) or phosphate buffered saline (Controls; n = 7; 0.2 mL). Each dose was administered in a 0.5 mL tube embedded in a fruit candy/cracked corn mix. Boosters were given seven weeks post-prime in the same manner and dose. Nineteen weeks post-prime, pigs were orally challenged with 1 × 106 cfu of virulent M. bovis. Twelve weeks post-challenge, pigs were euthanized and necropsied, at which time 23 different tissues from the head, thorax, and abdomen were collected and examined. Each tissue was assigned a lesion score. Ordinal lesion score data were analyzed using non-parametric Wilcoxon Signed Rank test. Effect size was calculated using Cohen's d. Four of eight Vaccinates and four of seven Controls had gross and microscopic lesions, as well as culture-positive tissues. Vaccinates had statistically lower lesion scores than Controls in the following areas: gross thoracic lesion scores (p = 0.013 Cohen's d = 0.33) and microscopic thoracic lesion scores (p = 0.002, Cohen's d = 0.39). There were no differences in head lesion scores alone, both gross and microscopic, nor were there differences when comparing combined gross and microscopic head and thoracic lesion scores. These results are indicative that this vaccination protocol affords a modest degree of infection containment with this vaccine in Molokai wild pigs.

FOOT-AND-MOUTH DISEASE IN EXPERIMENTALLY INFECTED MULE DEER (ODOCOILEUS HEMIONUS).

The only known outbreak of foot-and-mouth disease (FMD) in wildlife in the US occurred in mule deer (Odocoileus hemionus) in California in 1924-25. There is little recorded information on the pathogenesis and epidemiology of the disease in deer in that outbreak. In this experimental study, we compared the susceptibility of mule deer to FMD virus (FMDV) serotype O to that of cattle (Bos taurus). We also determined the potential for intra- and interspecies transmission of FMDV serotype O in mule deer and cattle, and assessed conventional laboratory tests in their ability to detect FMDV in mule deer. Two mule deer and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. The inoculated steer and deer were kept in the same room with contact animals of both species. Exposed contact animals were moved to rooms with unexposed animals after becoming febrile. All mule deer (n=14) and cattle (n=6) developed clinical signs and lesions consistent with FMDV infection. Deer had a high prevalence of myocarditis and high mortality. Virus was transmitted between mule deer, from cattle to mule deer, and from mule deer to cattle. Virus and antibodies against nonstructural FMDV proteins in mule deer and cattle were detected by conventional laboratory tests. Virus shedding was detected by PCR and virus isolation up to 9 d postexposure in deer.

PARTIAL PROTECTION IN BALB/C HOUSE MICE (MUS MUSCULUS) AND ROCKY MOUNTAIN ELK (CERVUS CANADENSIS) AFTER VACCINATION WITH A KILLED, MUCOSALLY DELIVERED BRUCELLA ABORTUS VACCINE.

Brucellosis, caused by Brucella abortus, has been eliminated from livestock in the US. Remaining wildlife reservoirs are the bison (Bison bison) and elk (Cervus canadensis) populations in Yellowstone National Park and the surrounding area, from which there is periodic exposure and transmission to surrounding livestock herds. Elk account for nearly all of the livestock exposure, and the infection appears to be expanding in the elk population. Currently, there are no known effective vaccines for brucellosis in elk. We conducted three experiments to evaluate the efficacy and practicality of delivering a killed B. abortus vaccine compounded with montmorillonite clay as a carrying agent to oral, nasal, and conjunctival mucosa. The first study, conducted in laboratory mice (Mus musculus), demonstrated protection against infection equal to that produced by the currently approved cattle (Bos taurus) vaccine RB51. The second experiment, conducted as a pilot study in a small sample of elk, demonstrated partial protection against B. abortus infection. Results of the third experiment showed that elk consumed the majority of a surrogate vaccine compounded with montmorillonite mixed in hay with oral, nasal, conjunctival, and gastrointestinal exposure to the vaccine. These results suggest that multiple exposures to a mucosally delivered vaccine may provide an effective method of vaccinating wildlife.

Brucella pinnipedialis in lungworms Parafilaroides sp. and Pacific harbor seals Phoca vitulina richardsi: proposed pathogenesis.

Brucella spp. were first isolated from marine mammals in 1994 and since have been described in numerous pinniped and cetacean species with nearly global distribution. Microscopic, electron microscopic, or culture results have shown lungworms in harbor seals to be infected with brucellae, suggesting that the lungworms may serve a role in this infection. In this study, we reviewed archived and more recent case material from 5 Pacific harbor seals from Washington State (USA) with evidence of B. pinnipedialis infection in the lungworm Parafilaroides sp. Twenty-two sections of lung containing approximately 220 Parafilaroides sp., stained with an immunohistochemical technique using antibody to B. abortus, showed approximately 80 (36%) infected nematodes. A few brucellae were also present in lung parenchyma in proximity to nematodes. Infection was present in the first- and fourth-stage larvae in the seal lung and intestines, as well as in the male and female reproductive organs of adult nematodes. Infected sperm deposits in the nematode uterus were suggestive of venereal transmission between lungworms. Massive infection of some degenerate adult lungworms and evidence of degeneration of some developing larvae in utero were observed. Based on these observations, we suggest that Parafilaroides sp., rather than the Pacific harbor seal Phoca vitulina richardsi, is the preferred host of B. pinnipedialis infection.

CLINICOPATHOLOGIC FEATURES OF INFECTION WITH NOVEL BRUCELLA ORGANISMS IN CAPTIVE WAXY TREE FROGS ( PHYLLOMEDUSA SAUVAGII) AND COLORADO RIVER TOADS ( INCILIUS ALVARIUS).

Two novel and distinct Brucella strains were recovered from 5 of 10 adult, sex undetermined, captive waxy tree frogs ( Phyllomedusa sauvagii) and two of five adult, sex undetermined, captive Colorado river toads ( Incilius alvarius) held in a zoologic collection with clinical and pathologic findings of bacterial disease. These amphibians originated from three separate private breeding facilities over several years and exhibited disease 9-49 mo following release from quarantine. Common presenting signs were vague but included focal abscessation, weight loss, change in coloration, anorexia, and decreased perching. Two waxy tree frogs and one Colorado river toad recovered with supportive care and antimicrobial treatment based on susceptibility testing. Microgranulomatosis, subcutaneous and renal abscessation, femoral osteomyelitis, and multicentric infection were the most common histologic findings. The organisms were identified antemortem in samples from subcutaneous abscesses, cloaca, and skin and from a variety of organ systems postmortem, and demonstrated a consistent susceptibility pattern. Initial isolates were misidentified as Ochrobactrum anthropi. Polymerase chain reaction and sequencing of the 16S rRNA gene identified the two organisms as novel Brucella strains similar to Brucella inopinata-like sp. and other novel organisms within the emerging "BO clade." Brucella strain oaks (isolated from waxy tree frogs) and Brucella strain leathers (isolated from Colorado river toads) differed from each other by 16 of 571 base pairs in a region of chromosome 2, and did not closely match any previous GenBank entries. This report describes the clinicopathologic features of infection by these bacteria in two amphibian species and expands the range of novel Brucella organisms from amphibian reservoirs.

Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish.

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals ( Phoca vitulina richardii) and North Atlantic hooded seals ( Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS) 711 gene and sequence type (ST)27 primer-probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS 711 and ST27 primer-probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS 711 primer-probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS 711 primer-probe was used to test Atlantic cod ( Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS 711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

Environmental and behavioral changes may influence the exposure of an Arctic apex predator to pathogens and contaminants.

Recent decline of sea ice habitat has coincided with increased use of land by polar bears (Ursus maritimus) from the southern Beaufort Sea (SB), which may alter the risks of exposure to pathogens and contaminants. We assayed blood samples from SB polar bears to assess prior exposure to the pathogens Brucella spp., Toxoplasma gondii, Coxiella burnetii, Francisella tularensis, and Neospora caninum, estimate concentrations of persistent organic pollutants (POPs), and evaluate risk factors associated with exposure to pathogens and POPs. We found that seroprevalence of Brucella spp. and T. gondii antibodies likely increased through time, and provide the first evidence of exposure of polar bears to C. burnetii, N. caninum, and F. tularensis. Additionally, the odds of exposure to T. gondii were greater for bears that used land than for bears that remained on the sea ice during summer and fall, while mean concentrations of the POP chlordane (ΣCHL) were lower for land-based bears. Changes in polar bear behavior brought about by climate-induced modifications to the Arctic marine ecosystem may increase exposure risk to certain pathogens and alter contaminant exposure pathways.

Evaluation of antibody response to an adjuvanted hapten-protein vaccine as a potential inhibitor of sexual maturation for farmed Atlantic salmon.

An experimental contraceptive vaccine was evaluated in Atlantic salmon (Salmo salar). A peptide derived from the beta subunit of luteinizing hormone (LH) was conjugated to two different carrier proteins, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), and formulated with one of four immunostimulants in a water-in-oil emulsion. Specific antibody responses to the peptide and each carrier protein were evaluated. While the antibody response to KLH was stronger than the response to BSA, both carrier proteins stimulated comparable antibody responses to the LH peptide. The immunostimulant proved to be more important for enhancing the LH peptide antibody response than the carrier protein selection; vaccines containing a combination of Aeromonas salmonicida and Vibrio anguillarum stimulated significantly greater LH peptide antibody production than any of the other three immunostimulants evaluated at 12 weeks post-vaccination. This study provides proof-of-concept for specific antibody production against a hapten-carrier protein antigen in Atlantic salmon and reinforces the importance of vaccine immunostimulant selection.

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.

ISOLATION AND CHARACTERIZATION OF A NOVEL MARINE BRUCELLA FROM A SOUTHERN SEA OTTER (ENHYDRA LUTRIS NEREIS), CALIFORNIA, USA.

We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.

FOOT-AND-MOUTH DISEASE IN A SMALL SAMPLE OF EXPERIMENTALLY INFECTED PRONGHORN (ANTILOCAPRA AMERICANA).

There is limited information on the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American wildlife and none concerning pronghorn ( Antilocapra americana ). In an experimental study of 13 pronghorn and six steers ( Bos taurus ), we compared the susceptibility of pronghorn to FMD virus (FMDV) strain O, with that of cattle ( Bos taurus ). We also determined the potential for intra- and interspecies transmission of FMDV strain O in pronghorn and cattle, assessed the application of conventional laboratory tests in their suitability to detect FMDV infection in pronghorn, and evaluated the potential role of pronghorn as efficient long-term carriers of FMDV. After acclimation to containment at Plum Island Animal Disease Center, two pronghorn and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were housed with contact animals. When contact-exposed animals developed fever they were placed in rooms with previously unexposed animals. All inoculated and exposed cattle and pronghorn developed clinical disease typical of FMD. Pronghorn developed severe foot lesions and mild to moderate oral lesions, primarily on the tongue. Duration of clinical signs in both species was 2-3 wk with foot abnormalities evident to the end of the study (51 d postexposure). Other lesions included pancreatitis, myositis of the tongue, and secondary lesions including pleuritis, pneumonia, decubital ulcers, and tenosynovitis. Virus transmission occurred between pronghorn, from cattle to pronghorn, and from pronghorn to cattle. Conventional laboratory tests detected virus and antibodies against nonstructural and structural FMDV proteins in pronghorn and cattle. Virus was present in some animals for 1 wk but was not detectable by virus isolation or PCR at 3 wk postinfection or afterward.

Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock.

Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations.

Vaccination of Elk (Cervus canadensis) with Brucella abortus Strain RB51 Overexpressing Superoxide Dismutase and Glycosyltransferase Genes Does Not Induce Adequate Protection against Experimental Brucella abortus Challenge.

In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosyltransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further, work is needed for development of an effective brucellosis vaccine for use in elk.

Determining the persistence of Mycobacterium bovis bacille Calmette-Guerin Danish in select tissues of orally vaccinated feral swine (Sus scrofa ssp.).

Mycobacterium bovis bacille Calmette-Guerin (BCG) is being considered for vaccination of feral swine (Sus scrofa ssp.). Since BCG is a live bacterium, evaluation of its safety and persistence in tissues is important. Fifteen feral swine received approximately 4.5 × 10(6) colony forming units of BCG Danish via oral bait. Four animals received bait without BCG. At 1, 3, 6, and 9 months post-vaccination, four vaccinates were euthanized. Non-vaccinates were euthanized at 9 months. Clinical signs were not noted in vaccinated pigs at any time. Tissues from all 20 pigs were culture-negative for mycobacteria. Based on our data, BCG is safe and appears not to persist in feral swine tissues after one month post-oral vaccination. However, further work must be performed at higher doses, and on a larger number of animals representing the target population, and further evaluation of persistence in tissues within the first month post-vaccination is needed.