Publications by authors named "Mitchell V Palmer"

110 Publications

Use of blood matrices and alternative biological fluids for antibody detection in animal tuberculosis.

Vet Immunol Immunopathol 2021 Jul 22;239:110303. Epub 2021 Jul 22.

National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, 1920 Dayton Avenue, Ames, IA, 50010, USA.

Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97-0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB.
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http://dx.doi.org/10.1016/j.vetimm.2021.110303DOI Listing
July 2021

Genome Sequences of Mycobacterium tuberculosis Biovar bovis Strains Ravenel and 10-7428.

Microbiol Resour Announc 2021 Jun 17;10(24):e0041121. Epub 2021 Jun 17.

Pathobiology and Diagnostic Investigation Department, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA.

We report the draft genomes of two Mycobacterium tuberculosis biovar bovis strains. Strain Ravenel was isolated in the 1900s and has been shown to be attenuated in cattle. Strain 10-7428 is considered highly pathogenic in cattle and was isolated from a bovine tuberculosis outbreak.
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http://dx.doi.org/10.1128/MRA.00411-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210702PMC
June 2021

Distribution and persistence of atypical porcine pestivirus in experimentally inoculated pigs.

J Vet Diagn Invest 2021 Jun 2:10406387211022683. Epub 2021 Jun 2.

Veterinary Diagnostic and Production Animal Medicine, Iowa State University College of Veterinary Medicine, Ames, IA, USA.

Atypical porcine pestivirus (APPV) is a cause of congenital tremors (CTs) in piglets and has been found in swine populations around the globe. Although systemic distribution of the virus has been reported, there is limited information regarding viral localization at the cellular level and distribution at the tissue level. We collected multiple tissues from 2-d-old piglets ( = 36) born to sows inoculated at 45 or 62 d of gestation with APPV via 3 simultaneous routes: intravenous, intranasal, and directly in amniotic vesicles. In addition, 2 boars from APPV-inoculated sows with CT were raised and euthanized when 11 mo old. In situ hybridization performed on tissue samples from piglets demonstrated a broad and systemic distribution of viral RNA including endothelial cells, fibroblasts, and smooth muscle. Labeling in tissues was more pronounced in piglet tissues compared to boars, with the notable exception of diffuse labeling of the cerebellum in boars. Presence of APPV in boar tissues well after resolution of clinical signs suggests persistence of APPV similar to other pestiviruses.
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http://dx.doi.org/10.1177/10406387211022683DOI Listing
June 2021

Heterogeneity of Pulmonary Granulomas in Cattle Experimentally Infected With .

Front Vet Sci 2021 7;8:671460. Epub 2021 May 7.

Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States.

is the cause of tuberculosis in most animals, most notably cattle. The stereotypical lesion of bovine tuberculosis is the granuloma; a distinct morphological lesion where host and pathogen interact and disease outcome (i.e., dissemination, confinement, or resolution) is determined. Accordingly, it is critical to understand host-pathogen interactions at the granuloma level. Host-pathogen interactions within individual granulomas at different stages of disease have not been examined in cattle. We examined bacterial burden and cytokine expression in individual pulmonary granulomas from steers at 30, 90, 180, and 270 days after experimental aerosol infection with . Bacterial burdens within individual granulomas examined 30 days after infection were greater and more heterogenous (variable) than those examined 90 to 270 days after infection. Bacterial burdens did not correlate with expression of IFN-γ, TNF-α, TGF-β, granuloma stage, or lung lesion score, although there was a modest positive correlation with IL-10 expression. Granuloma stage did have modest positive and negative correlations with TNF-α and IL-10, respectively. Heterogeneity and mean expression of IFN-γ, IL-10 and TNF-α did not differ significantly over time, however, expression of TGF-β at 90 days was significantly greater than that seen at 30 days after infection.
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http://dx.doi.org/10.3389/fvets.2021.671460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138452PMC
May 2021

Mucosal IFNγ production and potential role in protection in Escherichia coli O157:H7 vaccinated and challenged cattle.

Sci Rep 2021 May 7;11(1):9769. Epub 2021 May 7.

USDA-ARS, National Animal Disease Center, 1920 Dayton Avenue, P.O. Box 70, Ames, IA, 50010, USA.

Shiga-toxin producing Escherichia coli O157:H7 (O157)-based vaccines can provide a potential intervention strategy to limit foodborne zoonotic transmission of O157. While the peripheral antibody response to O157 vaccination has been characterized, O157-specific cellular immunity at the rectoanal junction (RAJ), a preferred site for O157 colonization, remains poorly described. Vaccine induced mucosal O157-specific antibodies likely provide some protection, cellular immune responses at the RAJ may also play a role in protection. Distinct lymphoid follicles were increased in the RAJ of vaccinated/challenged animals. Additionally, increased numbers of interferon (IFN)γ-producing cells and γδ + T cells were detected in the follicular region of the RAJ of vaccinated/challenged animals. Likewise, adjuvanted-vaccine formulation is critical in immunogenicity of the O157 parenteral vaccine. Local T cell produced IFNγ may impact epithelial cells, subsequently limiting O157 adherence, which was demonstrated using in vitro attachment assays with bovine epithelial cells. Thus, distinct immune changes induced at the mucosa of vaccinated and challenged animals provide insight of mechanisms associated with limiting O157 fecal shedding. Enhancing mucosal immunity may be critical in the further development of efficacious vaccines for controlling O157 in ruminants and thus limiting O157 transmission to humans.
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http://dx.doi.org/10.1038/s41598-021-89113-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105325PMC
May 2021

Experimental Inoculation of Young Calves with SARS-CoV-2.

Viruses 2021 03 9;13(3). Epub 2021 Mar 9.

Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, 240 Farrier Rd, Ithaca, NY 14853, USA.

The host range of SARS-CoV-2 and the susceptibility of animal species to the virus are topics of great interest to the international scientific community. The angiotensin I converting enzyme 2 (ACE2) protein is the major receptor for the virus, and sequence and structural analysis of the protein has been performed to determine its cross-species conservation. Based on these analyses, cattle have been implicated as a potential susceptible species to SARS-CoV-2 and have been reported to have increased ACE2 receptor distribution in the liver and kidney, and lower levels in the lungs. The goal of the current study was to determine the susceptibility of cattle to SARS-CoV-2 utilizing inoculation routes that facilitated exposure to tissues with increased ACE2 receptor distribution. For this, colostrum-deprived calves approximately 6 weeks of age were inoculated via the intratracheal or intravenous routes. Nasal and rectal swab samples, as well as blood and urine samples, were collected over the course of the study to evaluate viral shedding, viremia, and seroconversion. Pyrexia was used as the primary criteria for euthanasia and tissue samples were collected during necropsy. Importantly, SARS-CoV-2 RNA was detected in only two nasal swab samples collected on days 3 and 10 post-inoculation (pi) in two calves; one calf in the intratracheal group and the other calf in the intravenous group, respectively. Additionally, the calf in the intratracheal group that was positive on the nasal swab on day 3 pi also had a positive tracheobronchial lymph node on day 9 pi. Viral nucleic acid load on these samples, based on PCR cycle threshold values, were low and infectious virus was not recovered from the samples. These results suggest that there was no productive replication of SARS-CoV-2 in calves following intratracheal and intravenous inoculation.
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http://dx.doi.org/10.3390/v13030441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000368PMC
March 2021

Susceptibility of white-tailed deer () to SARS-CoV-2.

J Virol 2021 Mar 10. Epub 2021 Mar 10.

Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.
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http://dx.doi.org/10.1128/JVI.00083-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139686PMC
March 2021

Severity of bovine tuberculosis is associated with innate immune-biased transcriptional signatures of whole blood in early weeks after experimental Mycobacterium bovis infection.

PLoS One 2020 9;15(11):e0239938. Epub 2020 Nov 9.

Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States of America.

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a pathogen that impacts both animal and human health. Consequently, there is a need to improve understanding of disease dynamics, identification of infected animals, and characterization of the basis of immune protection. This study assessed the transcriptional changes occurring in cattle during the early weeks following a M. bovis infection. RNA-seq analysis of whole blood-cell transcriptomes revealed two distinct transcriptional clusters of infected cattle at both 4- and 10-weeks post-infection that correlated with disease severity. Cattle exhibiting more severe disease were transcriptionally divergent from uninfected animals. At 4-weeks post-infection, 25 genes had commonly increased expression in infected cattle compared to uninfected cattle regardless of disease severity. Ten weeks post-infection, differential gene expression was only observed when severely-affected cattle were compared to uninfected cattle. This indicates a transcriptional divergence based on clinical status following infection. In cattle with more severe disease, biological processes and cell type enrichment analyses revealed overrepresentation of innate immune-related processes and cell types in infected animals. Collectively, our findings demonstrate two distinct transcriptional profiles occur in cattle following M. bovis infection, which correlate to clinical status.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239938PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652326PMC
January 2021

Mycobacterium bovis and you: A comprehensive look at the bacteria, its similarities to Mycobacterium tuberculosis, and its relationship with human disease.

Tuberculosis (Edinb) 2020 12 2;125:102006. Epub 2020 Oct 2.

Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA.

While Mycobacterium tuberculosis is the primary cause of tuberculosis in people, multiple other mycobacteria are capable of doing so. With the World Health Organization's goal of a 90% reduction in tuberculosis by 2035, all tuberculous mycobacteria need to be addressed. Understanding not only the similarities, but importantly the differences between the different species is crucial if eradication is ever to be achieved. Mycobacterium bovis, while typically thought of as a disease of cattle, remains a possible source of human infection worldwide. Although this species' genome differs from Mycobacterium tuberculosis by only 0.05%, significant differences are present, creating unique challenges to address. This review focuses on features which distinguish this bacterium from Mycobacterium tuberculosis, including differences in origin, structure, environmental persistence, host preferences, infection and disease, host immune response, diagnostics and treatment.
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http://dx.doi.org/10.1016/j.tube.2020.102006DOI Listing
December 2020

Changes in circulating lymphocytes and lymphoid tissue associated with vaccination of colostrum deprived calves.

Vaccine 2020 10 26;38(46):7268-7277. Epub 2020 Sep 26.

Ridpath Consulting, LLC, Gilbert, IA 50105, USA.

The objective of this study was to compare immunological responses and lymphoid depletion in young, colostrum deprived calves following administration of vaccines containing modified-live bovine viral diarrhea virus (BVDV). A group of calves exposed to a typical virulence non-cytopathic (ncp) BVDV-2 field strain (ncp exposed) was included to compare responses of calves receiving vaccine to responses generated against a field strain (mimicking a natural infection). A negative control group administered a placebo was used in all comparisons. All vaccines used in the study were administered per manufacturer recommendations while ncp BVDV exposed calves received 5 ml intranasally (2.5 ml/nare; 4.2 × 10 TCID/ml) of the BVDV-2 field strain. Samples collected at each time point included nasal swabs for virus detection, blood samples for complete blood counts and detection of viremia, PBMCs for flow cytometric analysis, serum for virus neutralization titers, and thymus tissue at necropsy for evaluation of lymphoid depletion. A measurable neutralizing BVDV titer was observed for all treatment groups excluding the control animals, which remained negative during the study period. Virus shedding was only detected from the ncp vaccinated and ncp exposed calves. A decline from baseline was observed for peripheral lymphocyte and CD4 cells for the groups receiving the adjuvanted cytopathic (cp) vaccine, the double deleted genetically modified (ddGM) vaccine, the ncp vaccine and ncp exposed calves, but not for the control group or groups receiving cp vaccines. Thymus depletion was observed for the ncp vaccine and ncp exposed calves and to a lesser extent for the ddGM vaccine calves. Collectively, these data suggest that the virus biotype, method of attenuation, presentation, and use of adjuvant will influence vaccine impacts on lymphoid tissues and the immune response. As such, multiple variables should be considered when determining costs and benefits of vaccination.
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http://dx.doi.org/10.1016/j.vaccine.2020.09.046DOI Listing
October 2020

Case Report: Fading Elk Syndrome in a Herd of Captive Elk () in the North American Midwest.

Front Vet Sci 2020 21;7:497. Epub 2020 Aug 21.

Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States.

Fading elk syndrome, or chronic ill-thrift of elk, is a disease associated with abomasal parasitism with species, of which elk appear to be particularly susceptible. While this syndrome has been extensively reported to affect wapiti-type red deer hybrids farmed in New Zealand since the mid 1980's, there is only a single report of this disease in North America. Here, we report a case of fading elk syndrome in a herd of 34 elk () in Ames, Iowa, at the National Animal Disease Center. Analysis of complete blood counts were unremarkable, but blood chemistry demonstrated a severe hypoalbuminemia. Fecal floatations were also unremarkable, and non-diagnostic. Histological examination of tissues collected at necropsy revealed proliferative abomasitis and nematodes consistent with spp. Anthelmintic treatment consisting of a combination of pour-on Cydectin and injectable Noromectin Plus, at double the recommended dose for cattle, showed positive results, as all remaining animals in the herd recovered. The work presented here is the first report of naturally-acquired disease in a herd of captive elk used for research and sheds light on this seldomly-reported disease in North America.
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http://dx.doi.org/10.3389/fvets.2020.00497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472827PMC
August 2020

Case report: characterization of a persistent, treatment-resistant, novel Staphylococcus aureus infection causing chronic mastitis in a Holstein dairy cow.

BMC Vet Res 2020 Sep 15;16(1):336. Epub 2020 Sep 15.

USDA-ARS-National Animal Disease Center, Ruminant Disease and Immunology Research Unit, Ames, IA, USA.

Background: Mastitis is the most common health concern plaguing the modern dairy cow and costs dairy producers estimates of two billion dollars annually. Staphylococcus aureus infections are prevalent, displaying varied disease presentation and markedly low cure rates. Neutrophils are considered the first line of defense against mastitis causing bacteria and are frequently targeted in the development of treatment and prevention technologies. We describe a case of naturally occurring, chronic mastitis in a Holstein cow (1428), caused by a novel strain of S. aureus that was not able to be cleared by antibiotic treatment.

Case Presentation: The infection was identified in a single quarter, 2 months into the cow's first lactation. The infection persisted for the following 20 months, including through dry off, and a second calving and lactation. This case of mastitis was associated with a consistently high somatic cell count, however presented with no other clinical signs. This cow was unsuccessfully treated with antibiotics commonly used to treat mastitis, consisting of two rounds of treatment during lactation and an additional round at the beginning of dry off. The chronic infection was also unchanged through an experimental mid-lactation treatment with pegylated granulocyte-colony stimulating factor (PEG-gCSF) and an additional periparturient treatment with PEG-gCSF. We isolated milk neutrophils from 1428 and compared them to two cows challenged with experimental S. aureus, strain Newbould 305. Neutrophils from 1428's milk had higher surface expression of myeloperoxidase compared to experimental Newbould challenged animals, as well as increased presence of Neutrophil Extracellular Traps. This suggests a heightened activation state of neutrophils sourced from 1428's naturally occurring infection. Upon postmortem examination, the affected quarter revealed multifocal abscesses separated by fibrous connective tissues. Abscesses were most common in the gland cistern and collecting duct region. Microscopically, the inflammatory reaction was pyogranulomatous to granulomatous and consistent with botryomycosis. Colonies of Gram-positive cocci were found within the eosinophilic matrix of the Splendore-Hoeppli reaction within granulomas and intracellularly within the acinar epithelium.

Conclusions: Collectively, we describe a unique case of chronic mastitis, the characterization of which provides valuable insight into the mechanics of S. aureus treatment resistance and immune escape.
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http://dx.doi.org/10.1186/s12917-020-02528-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491080PMC
September 2020

Evaluation of A Baculovirus-Expressed VP2 Subunit Vaccine for the Protection of White-Tailed Deer () from Epizootic Hemorrhagic Disease.

Vaccines (Basel) 2020 Jan 31;8(1). Epub 2020 Jan 31.

Center of Excellence for Emerging and Zoonotic Animal Diseases, and the Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.

Epizootic hemorrhagic disease virus (EHDV) is an arthropod-transmitted RNA virus and the causative agent of epizootic hemorrhagic disease (EHD) in wild and domestic ruminants. In North America, white-tailed deer (WTD) experience the highest EHD-related morbidity and mortality, although clinical disease is reported in cattle during severe epizootics. No commercially licensed EHDV vaccine is available in North America. The objective of this study was to develop and evaluate a subunit vaccine candidate to control EHD in WTD. Recombinant VP2 (rVP2) outer capsid proteins of EHDV serotypes 2 (EHDV-2) and 6 (EHDV-6) were produced in a baculovirus-expression system. Mice and cattle vaccinated with EHDV-2 or EHDV-6 rVP2 produced homologous virus-neutralizing antibodies. In an immunogenicity/efficacy study, captive-bred WTD received 2 doses of EHDV-2 rVP2 or sham vaccine, then were challenged with wild-type EHDV-2 at 30 d post vaccination. None of the rVP2-vaccinated deer developed clinical disease, no viral RNA was detected in their blood or tissues (liver, lung, spleen, kidney), and no EHDV-induced lesions were observed. Sham-vaccinated deer developed clinical disease with viremia and typical EHD vascular lesions. Here, we demonstrate a rVP2 subunit vaccine that can provide protective immunity from EHDV infection and which may serve as an effective tool in preventing clinical EHD and reducing virus transmission.
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http://dx.doi.org/10.3390/vaccines8010059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157196PMC
January 2020

Vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis bacille Calmette-Guérin (BCG) results in positive tuberculin skin test results in a dose-dependent fashion.

Res Vet Sci 2020 Apr 11;129:70-73. Epub 2020 Jan 11.

National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, United States Department of Agriculture, 1920 Dayton Avenue, Ames, IA, 50010, USA.

Mycobacterium bovis is the cause of tuberculosis in most mammalian species, most notably cattle and other members of the family Bovidae; however, many species of the family Cervidae are also susceptible. In North America, tuberculosis has been identified in both captive and free-ranging cervids. Captive cervids are tested for tuberculosis following many of the same guidelines applied to cattle, including intradermal tuberculin testing using M. bovis purified protein derivative (PPD). Both captive and free-ranging deer and elk have been implicated as the source of infection for many cattle herds. Vaccination with the human vaccine M. bovis BCG has been considered as one possible tool to aid in eradication of tuberculosis from cattle and both captive and free-ranging cervids. Studies in cattle have demonstrated that BCG vaccination can induce false positive intradermal tuberculin test reactions in some cattle. Similar findings have been reported for red deer. We orally vaccinated white-tailed deer with BCG and showed that vaccination can induce false positive skin test reactions in some deer and that the rate of false positive reactions is greater with a higher vaccine dose.
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http://dx.doi.org/10.1016/j.rvsc.2020.01.010DOI Listing
April 2020

Biomarkers of cell-mediated immunity to bovine tuberculosis.

Vet Immunol Immunopathol 2020 Feb 30;220:109988. Epub 2019 Nov 30.

National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture (USDA), Ames, Iowa, USA.

Whole blood based assays, particularly interferon gamma (IFN-γ) release assays (IGRAs), are used for the diagnosis of both bovine and human tuberculosis (TB). The aim of the current study was to evaluate a panel of cytokines and chemokines for potential use as diagnostic readouts indicative of Mycobacterium bovis (M. bovis) infection in cattle. A gene expression assay was used to determine the kinetics of the response to M. bovis purified protein derivative and a fusion protein consisting of ESAT-6, CFP10, and Rv3615c upon aerosol infection with ∼10 cfu of M. bovis. The panel of biomarkers included: IFN-γ, CXCL9, CXCL10, CCL2, CCL3, TNF-α, IL-1α, IL-1β, IL-1Ra, IL-22, IL-21 and IL-13. Protein levels of IFN-γ, CXCL9, and CXCL10 were determined by ELISA. Findings suggest that CXCL9, CXCL10, IL-21, IL-13, and several acute phase cytokines may be worth pursuing as diagnostic biomarkers of M. bovis infection in cattle.
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http://dx.doi.org/10.1016/j.vetimm.2019.109988DOI Listing
February 2020

Characteristics of subclinical ssp. infection in a captive white-tailed deer herd.

J Vet Diagn Invest 2019 Nov 11;31(6):844-851. Epub 2019 Sep 11.

Bacterial Diseases of Livestock Research Unit (Palmer, Kanipe), Animal Resources Unit (Cox), National Animal Disease Center, Agricultural Research Service.

Paratuberculosis (Johne's disease) is caused by ssp. (MAP), and affects both domestic and wild ruminants, including cattle, goats, sheep, and deer. In cattle, most infections occur during calfhood followed by a prolonged incubation period of 1-2 y or more before cows shed culturable numbers of MAP bacilli in their feces. As disease progresses, infected animals develop protein-losing enteropathy, intractable diarrhea, and weight loss. In a cohort of 32 clinically normal deer from a herd with a history of periodic clinical paratuberculosis, we found that subclinical infection was characterized by high rates of infection, common involvement of mesenteric lymph nodes, minimal lesion formation, few intralesional acid-fast bacilli, and low-level fecal shedding of MAP. The characteristics of subclinical paratuberculosis in white-tailed deer resemble those of cattle and red deer, although microscopic lesions were less common in subclinical deer than reported for subclinical cattle, and we did not see necrotizing granulomas as described in subclinical red deer and elk.
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http://dx.doi.org/10.1177/1040638719873028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900728PMC
November 2019

Characterization of γδ T Cell Effector/Memory Subsets Based on CD27 and CD45R Expression in Response to Infection.

Immunohorizons 2019 06 12;3(6):208-218. Epub 2019 Jun 12.

Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50010; and

Tuberculosis (TB) remains a leading cause of death from infectious diseases worldwide. is the causative agent of bovine TB and zoonotic TB infection. γδ T cells are known to participate in the immune control of mycobacterial infections. Data in human and nonhuman primates suggest that mycobacterial infection regulates memory/effector phenotype and adaptive immune functions of γδ T cells. To date, the impact of infection on bovine γδ T cells and their effector and memory differentiation remains unknown. In this study, we show that circulating γδ T cells from -infected cattle can be differentiated based on the expression of CD27, which is indicative of their capacity to respond to virulent infection: CD27 γδ T cells proliferated in response to Ag and, thus, may comprise the adaptive γδ T cell compartment in cattle. We further show that bovine -specific γδ T cells express surface markers characteristic of central memory T cells (CD45RCD27CD62L) and that -specific CD4 and γδ T cells both upregulate the expression of the tissue-homing receptors CXCR3 and CCR5 during infection. Our studies contribute significantly to our understanding of γδ T cell differentiation during TB infection and provide important insights into the link between phenotypic and functional subsets in the bovine. Accurate characterization of γδ T cell effector and memory-like responses induced during mycobacterial infection will contribute to improved strategies for harnessing the γδ T cell response in protection against TB for humans and animals.
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http://dx.doi.org/10.4049/immunohorizons.1900032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6875775PMC
June 2019

Research with Agricultural Animals and Wildlife.

ILAR J 2019 12;60(1):66-73

Infectious Bacterial Disease of Livestock Research Unit, National Animal Disease Center, National Centers for Animal Health, Agricultural Research Service, United States Department, Ames, Iowa.

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.
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http://dx.doi.org/10.1093/ilar/ilz006DOI Listing
December 2019

Early Pulmonary Lesions in Cattle Infected via Aerosolized .

Vet Pathol 2019 07 21;56(4):544-554. Epub 2019 Mar 21.

3 National Veterinary Services Laboratories, Ames, IA, USA.

is a serious zoonotic pathogen and the cause of tuberculosis in many mammalian species, most notably, cattle. The hallmark lesion of tuberculosis is the granuloma. It is within the developing granuloma where host and pathogen interact; therefore, it is critical to understand host-pathogen interactions at the granuloma level. Cytokines and chemokines drive cell recruitment, activity, and function and ultimately determine the success or failure of the host to control infection. In calves, early lesions (ie, 15 and 30 days) after experimental aerosol infection were examined microscopically using in situ hybridization and immunohistochemistry to demonstrate early infiltrates of CD68+ macrophages within alveoli and alveolar interstitium, as well as the presence of CD4, CD8, and γδ T cells. Unlike lesions at 15 days, lesions at 30 days after infection contained small foci of necrosis among infiltrates of macrophages, lymphocytes, neutrophils, and multinucleated giant cells and extracellular acid-fast bacilli within necrotic areas. At both time points, there was abundant expression of the chemokines CXCL9, MCP-1/CCL2, and the cytokine transforming growth factor (TGF)-β. The proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as the anti-inflammatory cytokine IL-10, were expressed at moderate levels at both time points, while expression of IFN-γ was limited. These findings document the early pulmonary lesions after infection in calves and are in general agreement with the proposed pathogenesis of tuberculosis described in laboratory animal and nonhuman primate models of tuberculosis.
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http://dx.doi.org/10.1177/0300985819833454DOI Listing
July 2019

Utility of the Neonatal Calf Model for Testing Vaccines and Intervention Strategies for Use against Human RSV Infection.

Vaccines (Basel) 2019 Jan 8;7(1). Epub 2019 Jan 8.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA 50010, USA.

Respiratory syncytial virus (RSV) is a significant cause of pediatric respiratory tract infections. It is estimated that two-thirds of infants are infected with RSV during the first year of life and it is one of the leading causes of death in this age group worldwide. Similarly, bovine RSV is a primary viral pathogen in cases of pneumonia in young calves and plays a significant role in bovine respiratory disease complex. Importantly, naturally occurring infection of calves with bovine RSV shares many features in common with human RSV infection. Herein, we update our current understanding of RSV infection in cattle, with particular focus on similarities between the calf and human infection, and the recent reports in which the neonatal calf has been employed for the development and testing of vaccines and therapeutics which may be applied to hRSV infection in humans.
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http://dx.doi.org/10.3390/vaccines7010007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466205PMC
January 2019

Milk biosynthesis requires the Golgi cation exchanger TMEM165.

J Biol Chem 2019 03 8;294(9):3181-3191. Epub 2019 Jan 8.

From the Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218 and

Milk is a hallmark of mammals that is critical for normal growth and development of offspring. During biosynthesis of lactose in the Golgi complex, H is produced as a by-product, and there is no known mechanism for maintaining luminal pH within the physiological range. Here, using conditional, tissue-specific knockout mice, immunostaining, and biochemical assays, we test whether the putative H/Ca/Mn exchanger known as TMEM165 (transmembrane protein 165) participates in normal milk production. We find TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and for normal growth rates of nursing pups. The milk of TMEM165-deficient mice contained elevated concentrations of fat, protein, iron, and zinc, which are likely caused by decreased osmosis-mediated dilution of the milk caused by the decreased biosynthesis of lactose. When normalized to total protein levels, only calcium and manganese levels were significantly lower in the milk from TMEM165-deficient dams than control dams. These findings suggest that TMEM165 supplies Ca and Mn to the Golgi complex in exchange for H to sustain the functions of lactose synthase and potentially other glycosyl-transferases. Our findings highlight the importance of cation and pH homeostasis in the Golgi complex of professional secretory cells and the critical role of TMEM165 in this process.
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http://dx.doi.org/10.1074/jbc.RA118.006270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398142PMC
March 2019

Use of the Human Vaccine, Bacillus Calmette Guérin in Deer.

Front Vet Sci 2018 8;5:244. Epub 2018 Oct 8.

Infectious Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States.

The only vaccine ever approved for human tuberculosis was developed a century ago from an isolate of derived from a tuberculous cow. Initial safety and efficacy studies of an attenuated version of this isolate were conducted in cattle and other animals. In 1921 the first human, an infant, was orally dosed with this attenuated strain that came to be known as bacillus Calmette-Guérin (BCG); named for Albert Calmette and Camille Guérin, the two French scientists that developed the strain. Since 1921, billions of people have been vaccinated with BCG making it the oldest, most widely used, and safest vaccine in use today. It is also the tuberculosis vaccine most studied for use in wildlife, including deer. While BCG vaccination of deer may not reliably prevent infection, it consistently decreases lesion severity, minimizing large, necrotic lesions, which often contain large numbers of bacilli. It is believed that decreased lesion severity correlates with decreased disease transmission; however, this hypothesis remains to be proven. Safety studies in white-tailed deer show BCG may persist in lymphoid tissues for up to 12 months; a factor to be considered in deer used for food. Beyond efficacy and safety, methods of vaccine delivery to free-ranging deer are also under investigation, both in the laboratory and in the field. The ideal delivery method is effective, efficient and safe for non-target species, including livestock. Ingestion of BCG by cattle is of special concern as such cattle may present as "false positives" using currently approved diagnostic methods, thus interfering with efforts by animal health agencies to monitor cattle for tuberculosis. An effective BCG vaccine for deer would be of value in regions where free-ranging deer represent a potential source of for livestock. Such a vaccine would also be beneficial to farmed deer where represents a serious threat to trade and productivity.
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http://dx.doi.org/10.3389/fvets.2018.00244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186790PMC
October 2018

Inbred Rats as a Model to Study Persistent Renal Leptospirosis and Associated Cellular Immune Responsiveness.

Front Cell Infect Microbiol 2018 14;8:66. Epub 2018 Mar 14.

Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States.

Pathogenic species of cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Rats are regarded as one of the most significant reservoir hosts of infection for human disease, and in the absence of clinical signs of infection, excrete large numbers of organisms in their urine. A unique biological equilibrium exists between pathogenic leptospires and reservoir hosts of infection, but surprisingly, little is known concerning the host's cellular immune response that facilitates persistent renal colonization. To address this deficiency, we established and applied an immunocompetent inbred rat model of persistent renal colonization; leptospires were detected in urine of experimentally infected rats by 3 weeks post-infection and remained positive until 8 weeks post-infection. However, there was little, if any, evidence of inflammation in colonized renal tubules. At 8 weeks post-infection, a robust antibody response was detected against lipopolysaccharide and protein outer membrane (OM) components. Purified B and T cells derived from the spleen of infected and non-infected rats proliferated in response to stimulation with 0.5 μg of OM fractions of , including CD4+ T cells, which comprised 40% of proliferating cells, compared to 25% in non-infected controls. However, analysis of gene expression did not determine which immunoregulatory pathways were activated. Lymphocytes purified from the lymph node draining the site of colonization, the renal lymph node, also showed an increase in percentage of proliferating B and T cells. However, in contrast to a phenotype of 40% CD4+ T cells in the spleen, the phenotype of proliferating T cells in the renal lymph node comprised 65% CD4+ T cells. These results confirm that the renal lymph node, the local lymphoid organ, is a dominant site containing reactive CD4+ T cells and highlight the need to consider the local, vs. systemic, immune responses during renal colonization infection. The use of inbred immunocompetent rats provides a novel tool to further elucidate those pathophysiological pathways that facilitate the unique biological equilibrium observed in reservoir hosts of leptospirosis.
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http://dx.doi.org/10.3389/fcimb.2018.00066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861151PMC
March 2019

Pathology of the Emerging Mycobacterium tuberculosis Complex Pathogen, Mycobacterium mungi, in the Banded Mongoose ( Mungos mungo).

Vet Pathol 2018 03 19;55(2):303-309. Epub 2017 Dec 19.

5 Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Ames, IA, USA.

Wild banded mongooses ( Mungos mungo) in northeastern Botswana and northwest Zimbabwe are infected with a novel Mycobacterium tuberculosis complex (MTC) pathogen, Mycobacterium mungi. We evaluated gross and histologic lesions in 62 infected mongooses (1999-2017). Many tissues contained multifocal irregular, lymphohistiocytic to granulomatous infiltrates and/or multifocal or coalescing noncaseating to caseating granulomas with variable numbers of intralesional acid-fast bacilli. Over one-third of nasal turbinates examined had submucosal lymphohistiocytic to granulomatous infiltrates, erosion and ulceration of the nasal mucosa, bony remodeling, and nasal distortion. Similar inflammatory cell infiltrates expanded the dermis of the nasal planum with frequent ulceration. However, even in cases with intact epidermis, acid-fast bacilli were present in variable numbers among dermal infiltrates and on the epidermal surface among desquamated cells and debris, most commonly in small crevices or folds. In general, tissue involvement varied among cases but was highest in lymph nodes (50/54, 93%), liver (39/53, 74%), spleen (37/51, 73%), and anal glands/sacs (6/8, 75%). Pulmonary lesions were present in 67% of sampled mongooses (35/52) but only in advanced disseminated disease. The pathological presentation of M. mungi in the banded mongoose is consistent with pathogen shedding occurring through scent-marking behaviors (urine and anal gland secretions) with new infections arising from contact with these contaminated olfactory secretions and percutaneous movement of the pathogen through breaks in the skin, nasal planum, and/or skin of the snout. Given the character and distribution of lesions and the presence of intracellular acid-fast bacilli, we hypothesize that pathogen spread occurs within the body through a hematogenous and/or lymphatic route. Features of prototypical granulomas such as multinucleated giant cells and peripheral fibrosis were rarely present in affected mongooses. Acid-fast bacilli were consistently found intracellularly, even in regions of necrosis. The mongoose genome has a unique deletion (RD1) that includes part of the encoding region for PPE68 (Rv3873), a gene co-operonic with PE35. These proteins can influence the host's cellular immune response to mycobacterial infections, and it remains uncertain how this deletion might contribute to observed patterns of pathology. M. mungi infection in banded mongooses is characterized by both a unique transmission and exposure route, as well as accompanying pathological features, providing an opportunity to increase our understanding of MTC pathogenesis across host-pathogen systems.
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http://dx.doi.org/10.1177/0300985817741730DOI Listing
March 2018

Emerging Understanding of Tuberculosis and the Granuloma by Comparative Analysis in Humans, Cattle, Zebrafish, and Nonhuman Primates.

Vet Pathol 2018 01;55(1):8-10

1 Infectious Bacterial Diseases of Livestock, National Animal Disease Center, United States Department of Agriculture, Ames, IA, USA.

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http://dx.doi.org/10.1177/0300985817712795DOI Listing
January 2018

Experimental Transmission of Bovine Digital Dermatitis to Sheep: Development of an Infection Model.

Vet Pathol 2018 03 16;55(2):245-257. Epub 2017 Nov 16.

2 Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.

Digital dermatitis is an infectious cause of lameness primarily affecting cattle but also described in sheep, goats, and wild elk. Digital dermatitis is a polymicrobial infection, involving several Treponema species and other anaerobic bacteria. Although the exact etiology has not been demonstrated, a number of bacterial, host, and environmental factors are thought to contribute to disease development. To study host-bacterial interactions, a reproducible laboratory model of infection is required. The objective of this study was to demonstrate key aspects of bovine digital dermatitis lesions in an easy-to-handle sheep model. Crossbred sheep were obtained from a flock free of hoof disease. Skin between the heel bulb and dewclaw was abraded before wrapping to emulate a moist, anaerobic environment. After 3 days, abraded areas were inoculated with macerated lesion material from active bovine digital dermatitis and remained wrapped. By 2 weeks postinoculation, experimentally inoculated feet developed erosive, erythematous lesions. At 4 weeks postinoculation, microscopic changes in the dermis and epidermis were consistent with those described for bovine digital dermatitis, including erosion, ulceration, hyperkeratosis, ballooning degeneration of keratinocytes, and the presence of neutrophilic infiltrates. Silver staining of lesion biopsy sections confirmed that spirochetes had penetrated the host epidermis. The model was then perpetuated by passaging lesion material from experimentally infected sheep into naïve sheep. This model of bovine digital dermatitis will allow for future novel insights into pathogenic mechanisms of infection, as well as the development of improved diagnostic methods and therapeutics for all affected ruminants.
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http://dx.doi.org/10.1177/0300985817736572DOI Listing
March 2018

Measuring bovine γδ T cell function at the site of Mycobacterium bovis infection.

Vet Immunol Immunopathol 2017 Dec 27;193-194:38-49. Epub 2017 Oct 27.

Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS, USA. Electronic address:

Bovine γδ T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/γδ T cell co-culture system to elucidate the role of γδ T cells in local immunity to M. bovis infection. Transcriptomics analysis revealed that γδ T cells upregulated expression of several novel, immune-associated genes in response to stimulation with M. bovis antigen. BCG-infected macrophage/γδ T cell co-cultures confirmed the results of our RNAseq analysis, and revealed that γδ T cells from M. bovis-infected animals had a significant impact on bacterial viability. Analysis of γδ T cells within late-stage M. bovis granulomas revealed significant expression of IFN-γ and CCL2, but not IL-10, IL-22, or IL-17. Our results suggest γδ T cells influence local immunity to M. bovis through cytokine secretion and direct effects on bacterial burden.
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http://dx.doi.org/10.1016/j.vetimm.2017.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703227PMC
December 2017

Identification of Novel Antigens Recognized by Serum Antibodies in Bovine Tuberculosis.

Clin Vaccine Immunol 2017 Dec 5;24(12). Epub 2017 Dec 5.

National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, USA.

Bovine tuberculosis (TB), caused by , remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (∼95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.
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http://dx.doi.org/10.1128/CVI.00259-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717178PMC
December 2017

Using White-tailed Deer () in Infectious Disease Research.

J Am Assoc Lab Anim Sci 2017 Jul;56(4):350-360

Deputy Director Office, National Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Ames, Iowa.

Between 1940 and 2004, more than 335 emerging infectious disease events were reported in the scientific literature. The majority (60%) of these events involved zoonoses, most of which (72%) were of wildlife origin or had an epidemiologically important wildlife host. Because this trend of increasing emerging diseases likely will continue, understanding the pathogenesis, transmission, and diagnosis of these diseases in the relevant wildlife host is paramount. Achieving this goal often requires using wild animals as research subjects, which are vastly different from the traditional livestock or laboratory animals used by most universities and institutions. Using wildlife in infectious disease research presents many challenges but also provides opportunities to answer questions impossible to address by using traditional models. Cervid species, especially white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and red deer (Cervus elaphus), are hosts or sentinels for several important pathogens, some of which are zoonotic. The long history of infectious disease research using white-tailed deer, conducted at ever-increasing levels of sophisticated biosecurity, demonstrates that this type of research can be conducted safely and that valuable insights can be gained. The greatest challenges to using wildlife in infectious disease research include animal source, facility design, nutrition, animal handling, and enrichment and other practices that both facilitate animal care and enhance animal wellbeing. The study of Mycobacterium bovis infection in white-tailed deer at the USDA's National Animal Disease Center serves to illustrate one approach to address these challenges.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517323PMC
July 2017

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

PLoS One 2017 7;12(7):e0179914. Epub 2017 Jul 7.

Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, Iowa, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179914PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501492PMC
October 2017
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