National Animal Disease Center

Ames, IA, United States

National Animal Disease Center

Ames, IA, United States
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Zeng X.,Stanford University | Wei Y.-L.,Stanford University | Huang J.,Stanford University | Newell E.,Stanford University | And 8 more authors.
Immunity | Year: 2012

γδ T cells contribute uniquely to immune competence. Nevertheless, how they function remains an enigma. It is unclear what most γδ T cells recognize, what is required for them to mount an immune response, and how the γδ T cell response is integrated into host immune defense. Here, we report that a noted B cell antigen, the algae protein phycoerythrin (PE), is a murine and human γδ T cell antigen. Employing this specificity, we demonstrated that antigen recognition activated naive γδ T cells to make interleukin-17 and respond to cytokine signals that perpetuate the response. High frequencies of antigen-specific γδ T cells in naive animals and their ability to mount effector response without extensive clonal expansion allow γδ T cells to initiate a swift, substantial response. These results underscore the adaptability of lymphocyte antigen receptors and suggest an antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. © 2012 Elsevier Inc.


Cross B.M.,Johns Hopkins University | Breitwieser G.E.,Weis Center for Research | Reinhardt T.A.,National Animal Disease Center | Rao R.,Johns Hopkins University
American Journal of Physiology - Cell Physiology | Year: 2014

Breast cancer is the second leading cause of cancer mortality in women, estimated at nearly 40,000 deaths and more than 230,000 new cases diagnosed in the U.S. this year alone. One of the defining characteristics of breast cancer is the radiographic presence of microcalcifications. These palpable mineral precipitates are commonly found in the breast after formation of a tumor. Since free Ca2+ plays a crucial role as a second messenger inside cells, we hypothesize that these chelated precipitates may be a result of dysregulated Ca2+ secretion associated with tumorigenesis. Transient and sustained elevations of intracellular Ca2+ regulate cell proliferation, apoptosis and cell migration, and offer numerous therapeutic possibilities in controlling tumor growth and metastasis. During lactation, a developmentally determined program of gene expression controls the massive transcellular mobilization of Ca2+from the blood into milk by the coordinated action of calcium transporters, including pumps, channels, sensors and buffers, in a functional module that we term CALTRANS. Here we assess the evidence implicating genes that regulate free and buffered Ca2+in normal breast epithelium and cancer cells and discuss mechanisms that are likely to contribute to the pathological characteristics of breast cancer. © 2014 the American Physiological Society.


Hester S.E.,Pennsylvania State University | Lui M.,University of California at Los Angeles | Nicholson T.,National Animal Disease Center | Nowacki D.,Pennsylvania State University | Harvill E.T.,Pennsylvania State University
PLoS ONE | Year: 2012

Sensing the environment allows pathogenic bacteria to coordinately regulate gene expression to maximize survival within or outside of a host. Here we show that Bordetella species regulate virulence factor expression in response to carbon dioxide levels that mimic in vivo conditions within the respiratory tract. We found strains of Bordetella bronchiseptica that did not produce adenylate cyclase toxin (ACT) when grown in liquid or solid media with ambient air aeration, but produced ACT and additional antigens when grown in air supplemented to 5% CO2. Transcriptome analysis and quantitative real time-PCR analysis revealed that strain 761, as well as strain RB50, increased transcription of genes encoding ACT, filamentous hemagglutinin (FHA), pertactin, fimbriae and the type III secretion system in 5% CO2 conditions, relative to ambient air. Furthermore, transcription of cyaA and fhaB in response to 5% CO2 was increased even in the absence of BvgS. In vitro analysis also revealed increases in cytotoxicity and adherence when strains were grown in 5% CO2. The human pathogens B. pertussis and B. parapertussis also increased transcription of several virulence factors when grown in 5% CO2, indicating that this response is conserved among the classical bordetellae. Together, our data indicate that Bordetella species can sense and respond to physiologically relevant changes in CO2 concentrations by regulating virulence factors important for colonization, persistence and evasion of the host immune response.


Palmer M.V.,National Animal Disease Center | Thacker T.C.,National Animal Disease Center | Waters W.R.,National Animal Disease Center | Robbe-Austerman S.,National Veterinary Services Laboratories
PLoS ONE | Year: 2014

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis from livestock, particularly cattle. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to other deer and cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer, thus interfering with the intraspecies and interspecies transmission cycles. Thirty-three white-tailed deer were assigned to one of two groups; oral vaccination with 1x108 colony-forming units of M. bovis BCG Danish (n = 17); and non-vaccinated (n = 16). One hundred eleven days after vaccination deer were infected intratonsilarly with 300 colony-forming units of virulent M. bovis. At examination, 150 days after challenge, BCG vaccinated deer had fewer gross and microscopic lesions, fewer tissues from which M. bovis could be isolated, and fewer late stage granulomas with extensive liquefactive necrosis. Fewer lesions, especially those of a highly necrotic nature should decrease the potential for dissemination of M. bovis within the host and transmission to other susceptible hosts.


Cross B.M.,Johns Hopkins University | Hack A.,Johns Hopkins University | Reinhardt T.A.,National Animal Disease Center | Rao R.,Johns Hopkins University
PLoS ONE | Year: 2013

An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca2+-ATPase, and the Ca2+ influx channel Orai1, has previously been shown to contribute to elevated Ca2+ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca2+ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca2+ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca2+ entry (SICE), which mediates the massive basolateral Ca2+ influx into mammary epithelia to support the large calcium transport requirements for milk secretion. © 2013 Cross et al.


Allen H.K.,National Animal Disease Center | An R.,University of Kentucky | Handelsman J.,Yale University | Moe L.A.,University of Kentucky
PLoS ONE | Year: 2015

Functional metagenomic analysis of soil metagenomes is a method for uncovering as-yet unidentified mechanisms for antibiotic resistance. Here we report an unconventional mode by which a response regulator derived from a soil metagenome confers resistance to the β-lactam antibiotic carbenicillin in Escherichia coli. A recombinant clone (βlr16) harboring a 5,169 bp DNA insert was selected from a metagenomic library previously constructed from a remote Alaskan soil. The βlr16 clone conferred specific resistance to carbenicillin, with limited increases in resistance to other tested antibiotics, including other β-lactams (penicillins and cephalosporins), rifampin, ciprofloxacin, erythromycin, chloramphenicol, nalidixic acid, fusidic acid, and gentamicin. Resistance was more pronounced at 24°C than at 37°C. Zone-of-inhibition assays suggested that the mechanism of carbenicillin resistance was not due to antibiotic inactivation. The DNA insert did not encode any genes known to confer antibiotic resistance, but did have two putative open reading frames (ORFs) that were annotated as a metallopeptidase and a two-component response regulator. Transposon mutagenesis and subcloning of the two ORFs followed by phenotypic assays showed that the response regulator gene was necessary and sufficient to confer the resistance phenotype. Quantitative reverse transcriptase PCR showed that the response regulator suppressed expression of the ompF porin gene, independently of the small RNA regulator micF, and enhanced expression of the acrD, mdtA, and mdtB efflux pump genes. This work demonstrates that antibiotic resistance can be achieved by the modulation of gene regulation by heterologous DNA. Functional analyses such as these can be important for making discoveries in antibiotic resistance gene biology and ecology.


Cheville N.F.,Iowa State University | Stasko J.,National Animal Disease Center
Veterinary Pathology | Year: 2014

Technical improvements in electron microscopy, both instrumental and preparative, permit increasingly accurate analyses. Digital images for transmission electron microscopy (TEM) can be processed by software programs that automate tasks and create custom tools that allow for image enhancement for brightness, contrast and coloration; for creation of rectangular, ellipsoidal or irregular area selections; and for measurement of mean area and standard deviation. Sample preparation remains a source of error since organelles and spatial arrangements of macromolecules rapidly change after anoxia. Guidelines for maintaining consistency in preparation, examination and interpretation are presented for different electron microscopy (EM) modalities. © The Author(s) 2013.


Greenlee J.J.,National Animal Disease Center | Smith J.D.,National Animal Disease Center | Greenlee M.H.,Iowa State University | Nicholson E.M.,National Animal Disease Center
PLoS ONE | Year: 2012

The majority of bovine spongiform encephalopathy (BSE) cases have been ascribed to the classical form of the disease. H-type and L-type BSE cases have atypical molecular profiles compared to classical BSE and are thought to arise spontaneously. However, one case of H-type BSE was associated with a heritable E211K mutation in the prion protein gene. The purpose of this study was to describe transmission of this unique isolate of H-type BSE when inoculated into a calf of the same genotype by the intracranial route. Electroretinograms were used to demonstrate preclinical deficits in retinal function, and optical coherence tomography was used to demonstrate an antemortem decrease in retinal thickness. The calf rapidly progressed to clinical disease (9.4 months) and was necropsied. Widespread distribution of abnormal prion protein was demonstrated within neural tissues by western blot and immunohistochemistry. While this isolate is categorized as BSE-H due to a higher molecular mass of the unglycosylated PrP Sc isoform, a strong labeling of all 3 PrP Sc bands with monoclonal antibodies 6H4 and P4, and a second unglycosylated band at approximately 14 kDa when developed with antibodies that bind in the C-terminal region, it is unique from other described cases of BSE-H because of an additional band 23 kDa demonstrated on western blots of the cerebellum. This work demonstrates that this isolate is transmissible, has a BSE-H phenotype when transmitted to cattle with the K211 polymorphism, and has molecular features that distinguish it from other cases of BSE-H described in the literature.


Allen H.K.,National Animal Disease Center | Stanton T.B.,National Animal Disease Center
Annual Review of Microbiology | Year: 2014

The human food chain begins with upwards of 1,000 species of bacteria that inhabit the intestinal tracts of poultry and livestock. These intestinal denizens are responsible for the health and safety of a major protein source for humans. The use of antibiotics to treat animal diseases was followed by the surprising discovery that antibiotics enhanced food animal growth, and both led to six decades of antibiotic use that has shaped food animal management practices. Perhaps the greatest impact of antibiotic feeding in food animals has been as a selective force in the evolution of their intestinal bacteria, particularly by increasing the prevalence and diversity of antibiotic resistance genes. Future antibiotic use will likely be limited to prudent applications in both human and veterinary medicine. Improved knowledge of antibiotic effects, particularly of growth-promoting antibiotics, will help overcome the challenges of managing animal health and food safety. Copyright © 2014 by Annual Reviews. All rights reserved.


Looft T.,National Animal Disease Center | Allen H.K.,National Animal Disease Center | Casey T.A.,National Animal Disease Center | Alt D.P.,National Animal Disease Center | Stanton T.B.,National Animal Disease Center
Frontiers in Microbiology | Year: 2014

Antibiotics are used in livestock and poultry production to treat and prevent disease as well as to promote animal growth. Carbadox is an in-feed antibiotic that is widely used in swine production to prevent dysentery and to improve feed efficiency. The goal of this study was to characterize the effects of carbadox and its withdrawal on the swine gut microbiota. Six pigs (initially 3-weeks old) received feed containing carbadox and six received unamended feed. After 3-weeks of continuous carbadox administration, all pigs were switched to a maintenance diet without carbadox. DNA was extracted from feces (n = 142) taken before, during, and following (6-week withdrawal) carbadox treatment. Phylotype analysis using 16S rRNA sequences showed the gradual development of the non-medicated swine gut microbiota over the 8-week study, and that the carbadox-treated pigs had significant differences in bacterial membership relative to non-medicated pigs. Enumeration of fecal Escherichia coli showed that a diet change concurrent with carbadox withdrawal was associated with an increase in the E. coli in the non-medicated pigs, suggesting that carbadox pre-treatment prevented an increase of E. coli populations. In-feed carbadox caused striking effects within 4 days of administration, with significant alterations in both community structure and bacterial membership, notably a large relative increase in Prevotella populations in medicated pigs. Digital PCR was used to show that the absolute abundance of Prevotella was unchanged between the medicated and non-medicated pigs despite the relative increase shown in the phylotype analysis. Carbadox therefore caused a decrease in the abundance of other gut bacteria but did not affect the absolute abundance of Prevotella. The pending regulation on antibiotics used in animal production underscores the importance of understanding how they modulate the microbiota and impact animal health, which will inform the search for antibiotic alternatives. © 2014 Looft, Allen, Casey, Alt and Stanton.

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