Potential Use for Serosurveillance of Feral Swine to Map Risk for Anthrax Exposure, Texas, USA

Anthrax is a disease of concern in many mammals, including humans. Management primarily consists of prevention through vaccination and tracking clinical-level observations because environmental isolation is laborious and bacterial distribution across large geographic areas diffi cult to confi rm. Feral swine (Sus scrofa) are an invasive species with an extensive range in the southern United States that rarely succumbs to anthrax. We present evidence that feral swine might serve as biosentinels based on comparative seroprevalence in swine from historically defi ned anthrax-endemic and non–anthraxendemic regions of Texas. Overall seropositivity was 43.7% (n = 478), and logistic regression revealed county endemicity status, age-class, sex, latitude, and longitude were informative for predicting antibody status. However, of these covariates, only latitude was statistically signifi cant (β = –0.153, p = 0.047). These results suggests anthrax exposure in swine, when paired with continuous location data, could serve as a proxy for bacterial presence in specifi c areas

Rift Valley fever viral RNA detection by in situ hybridization in formalin-fixed, paraffin-embedded tissues

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.This work was done in partial completion of Izabela Ragan’s PhD dissertation.The USDA Agricultural Research Service and the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University’s startup funds for Dr. Davis. This work was also supported through the Department of Homeland Security (grant no. 2010-ST061-AG0001), Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD).http://online.liebertpub.com/VBZ2020-06-26hj2019Paraclinical Science

Bactrian camels shed large quantities of Middle East respiratory syndrome coronavirus (MERS-CoV) after experimental infection

ABSTRACTIn 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged. To date, more than 2300 cases have been reported, with an approximate case fatality rate of 35%. Epidemiological investigations identified dromedary camels as the source of MERS-CoV zoonotic transmission and evidence of MERS-CoV circulation has been observed throughout the original range of distribution. Other new-world camelids, alpacas and llamas, are also susceptible to MERS-CoV infection. Currently, it is unknown whether Bactrian camels are susceptible to infection. The distribution of Bactrian camels overlaps partly with that of the dromedary camel in west and central Asia. The receptor for MERS-CoV, DPP4, of the Bactrian camel was 98.3% identical to the dromedary camel DPP4, and 100% identical for the 14 residues which interact with the MERS-CoV spike receptor. Upon intranasal inoculation with 107 plaque-forming units of MERS-CoV, animals developed a transient, primarily upper respiratory tract infection. Clinical signs of the MERS-CoV infection were benign, but shedding of large quantities of MERS-CoV from the URT was observed. These data are similar to infections reported with dromedary camel infections and indicate that Bactrians are susceptible to MERS-CoV and given their overlapping range are at risk of introduction and establishment of MERS-CoV within the Bactrian camel populations

Hypoxic Regulation of <i>Hand1</i> Controls the Fetal-Neonatal Switch in Cardiac Metabolism

<div><p>Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia <i>in utero</i>. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several “fetal” genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.</p></div

<i>Hand1</i> levels fall in the heart immediately following birth, under control of hypoxia signaling.

<p>(A) RTPCR for <i>Hand1</i> RNA from whole hearts of perinatal mice at a range of stages around birth, showing a steep decline in expression from birth. Levels expressed as a multiple of average 6-wk-old adult levels (<i>n</i> = 4 each group). (B) Levels of cardiac <i>Hand2</i> RNA do not fall at birth. Levels of <i>Hand2</i> at p1.5 normalised to e18.5 levels (<i>n</i> = 6 each group). (C) Western blot of protein extract from e18 (prenatal) control, p0.5 <i>XMLC-Hand1</i>, and p0.5 control hearts showing reduction in Hand1 but not Hand2 protein levels after birth, and persistence of Hand1 expression in <i>XMLC2-Hand1</i> hearts. (D) RTPCR showing increased <i>Hand1</i> RNA levels in hearts of adult wild-type mice incubated at 12% oxygen for 2 wk (“hypoxia”) over controls at normoxia (20% O₂) (<i>n</i> = 4 each group) (<i>p</i> = 0.001, two-tailed <i>t</i> test). (E) RTPCR showing significantly increased <i>Hand1</i> RNA levels in the hearts of p0.5 neonatal <i>α-MHC-Cre::VHL^(fl/fl</i>⁾ mice compared with wild-type controls, <i>p</i> = 0.0002 two-tailed <i>t</i> test, <i>n</i> = 6 each group. (F) Western blot of protein extract from <i>VHL^(fl/fl)</i> and control hearts at p0.5, showing elevation of Hand1 and HIF1α in <i>VHL^(fl/fl)</i> hearts. (G) RTPCR of chromatin immunoprecipitation assay using anti-HIF1α antiserum and primers to the HIF motif-containing sequences in the <i>Hand1</i> promoter from e18 hearts, showing binding of HIF1α to two sites. Bars represent summation of three experiments, and results expressed as multiples of signal for nonamplified sequence. The <i>p</i> values are two-tailed <i>t</i> tests relative to nonamplified <i>γ-crystallin</i> primers.</p

Prevention of neonatal <i>Hand1</i> down-regulation in transgenic mouse hearts leads to cardiomyopathy and death.

<p>(A) Cardiac RNA levels of <i>Hand1</i> in e18.5 and p0.5 wild-type, and e18.5 and p0.5 transgenic (TG 18.5 and TG0.5, respectively) showing RNA levels in the transgenic heart around 2.5 times that of wild-type p0.5 heart. (B) Cardiac <i>Hand1</i> elevating pups appear grossly normal, but are cyanosed (c, control; oe, Hand1 overexpressing). (C, D) H and E stain of cryostat section through thorax of control and <i>Hand1</i> overexpressing hearts, showing thin ventricular wall of the Hand1 overexpressing heart, and ventricular rupture (arrowed) with blood in the pericardial space (rv, right ventricle; lv, left ventricle). (E, F) EFIC sectioning and reconstruction of control and Hand1 overexpressing hearts from 4-h-old fostered pups, showing small size but no gross structural defect. (G, H) Periodic acid-Schiff stain of control and Hand1 overexpressing heart, showing decreased glycogen levels in <i>Hand1</i> overexpressing heart (purple). Glycogen stain in intercostal muscle of transgenic pup arrowed in (H). (I) Quantification of glucose enzymatically released from glycogen in hearts of neonates 2 h after caesarian section. Levels of glycogen in XMLC-Hand1 hearts are 17.5% of XMLC controls (<i>p</i> value, two-tailed <i>t</i> test, <i>n</i> = 6 hearts each group).</p

Prolongation of neonatal cardiac <i>Hand1</i> expression prevents transcriptional up-regulation of lipid metabolizing genes.

<p>(A) Schematic showing myocardial lipid metabolism (adapted from Kodde et al. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666-Kodde1" target="_blank">[55]</a>). (B) RTPCR showing RNA expression in e16 control, p0.5 control, and p0.5 hand1 up-regulating hearts. Levels of <i>ACC</i>, <i>MCD</i>, <i>FABP4</i>, <i>ACSL</i>, <i>CPT1A CPT1B</i>, and <i>HSL</i> are significantly up-regulated around birth (<i>p</i><0.05 two-tailed <i>t</i> test, <i>n</i> = 4 each group). No postnatal rise in <i>ACC</i>, <i>MCD</i>, <i>FABP4</i>, <i>CPT1A</i>, and <i>HSL</i> is seen in Hand1 up-regulating hearts. Significantly increased RNA expression of <i>ACBP</i> and <i>ATGL</i> is seen in Hand1 up-regulating hearts. Genes whose expression is reduced in Hand1 overexpressing hearts are in red in (A). <i>ACC</i>, acetyl coA carboxylase; <i>MCD</i>, malonyl coA decarboxylase; <i>FABP</i>, fatty acid binding protein; <i>FATP</i>, fatty acid transport protein; <i>ACSL</i>, acyl coA synthase long chain 1; <i>HSL</i>, hormone sensitive lipase; <i>ATGL</i>, adipose triglyceride lipase; <i>ACBP</i>, acylcoA binding protein; <i>CPT</i>, Carnitine Palmitoyl Transferase. (C) RTPCR of mRNA from 2-mo-old adult XMLC2-Hand1 mice following doxycycline induction for 2 wk, showing changes in expression of RNA encoding fatty acid metabolising proteins relative to control non-up-regulating mice (*<i>p</i><0.05, **<i>p</i><0.005, two-tailed <i>t</i> test, <i>n</i> = 4 each group). (D) RTPCR of mRNA from e14.5 embryo hearts from αMHC-Cre::Hand1^(fl/fl) and control pups, showing up-regulation of genes encoding fatty acid metabolising enzymes(*<i>p</i><0.05, two-tailed <i>t</i> test, <i>n</i> = 4 each group). (E) RTPCR showing significant <i>PGC1-α</i> elevation in the heart around birth (<i>n</i> = 4 each group, <i>p</i> = 0.008, two-tailed <i>t</i> test), with no significant drop in <i>Hand1</i> up-regulating hearts (<i>p</i> = 0.26, <i>n</i> = 6 each group). (F) Western blot of protein extract from cultured HL1 cardiomyocytes nontransfected (“control”) and transfected with <i>PGC1-α</i> and <i>HIF1</i>, showing no elevation of Hand1 in <i>PGC1-α</i> elevated PGC1<i>α</i> and Hand1 but not Hand2 protein expression in HIF1 expressing cells. (G) PCR of nuclear genomic (<i>globin</i>) and mitochondrial (<i>COX2</i>) DNA showing unchanged ratio in <i>Hand1</i> elevating neonatal hearts and Hand1-transfected HL1 cells compared with controls, implying no change in mitochondrial number. Control HL1 cells are transfected with an empty vector. (H) Map of 5′ promoters of several putative <i>Hand1</i> transcriptional targets in the e18.5 heart. Numbers refer to fold enrichment over <i>γ crystallin</i> in chromatin immunoprecipitation assay using anti-Hand1 serum. For more detailed chromatin immunoprecipitation data, please see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666.s009" target="_blank">Text S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666.s002" target="_blank">Figure S2</a>. (I) Site-directed mutagenesis of the Hand1-binding canonical CANNTG e-box in the 5′ HSL luciferase promoter de-represses expression of luciferase in HL1 cells, both in untransfected cells and cells stably expressing Hand1 (transfections in triplicate, measurement in quadruplicate, <i>p</i> values, two-tailed <i>t</i> test).</p

Lipid metabolism is inhibited in neonatal <i>Hand1</i> overexpressing hearts.

<p>(A) LC-MS trace showing typical output for intact lipid extracted from control hearts (green trace) and Hand1 up-regulating hearts (red), showing significantly lower levels of triacylglycerides (TAG) compared to phospholipid (PL) in Hand1 up-regulating hearts. (B) Quantitative analysis of cardiac triacylglyceride levels showing significant reduction in Hand1 up-regulating hearts, expressed as the ratio of TAG to phospholipid (<i>n</i> = 6 each group, <i>p</i> = 0.006, two-tailed <i>t</i> test). (C) Quantitative analysis of cardiac malonyl coA levels showing significant reduction in Hand1 up-regulating hearts (<i>n</i> = 6 each group, <i>p</i> = 0.04, two-tailed <i>t</i> test). (D) Reduced levels of C6, C14, and C18 containing acylcarnitine species in Hand1 prolonging neonatal hearts compared with controls. For full dataset, please refer to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666.s007" target="_blank">Table S3</a>. (E) Multivariate partial least squares discriminant analysis (PLS-DA) scores of acylcarnitine profiles showing a significant decrease in global levels of acylcarnitines in Hand1 up-regulated hearts relative to controls (R²X = 33%, R²Y = 62%, and Q² = 48%). (F) BODIPY-500/510C₁, C₁₂ uptake is significantly reduced in HL1 cells by transfection with Hand1. Graph shows quantification of fluorescence in Hand1 transfected and nontransfected controls, 10 high power fields each. (i) and (ii) show representative fluorescence micrographs of control and Hand1 transfected cells following labeled lipid incubation (<i>p</i> = 0.037, two-tailed <i>t</i> test).</p