DOCSLIB.ORG

Detection and Isolation of Plasmodium Liver Stages and Analysis of Circumsporozoite Protein Antigen Processing

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Detection and Isolation of Plasmodium Liver Stages and Analysis of Circumsporozoite Protein Antigen Processing STUDIES ON EXOERYTHROCYTIC DEVELOPMENT OF PLASMODIUM FALCIPARUM IN VITRO: DETECTION AND ISOLATION OF PLASMODIUM LIVER STAGES AND ANALYSIS OF CIRCUMSPOROZOITE PROTEIN ANTIGEN PROCESSING by Peter Dumoulin A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland May, 2015 Abstract Malaria, the disease caused by Plasmodium parasites, remains a major global health burden despite efforts to eradicate the parasite. Infection is initiated when a mosquito deposits sporozoites into the dermis that then actively invade hepatocytes. Sporozoites and the resulting liver stage of Plasmodium falciparum infection are leading targets for generation of a protective vaccine. The circumsporozoite protein is the major surface protein on sporozoites and is the target of vaccines aimed at preventing blood stage infection. During the process of invasion, sporozoites cleave the N-terminus of CSP and consequently two forms of CSP are potentially exposed to the immune system. We utilized ectopic expression of two natural form of CSP to quantify their abilities to serve as targets for MHC class I-restricted CD8+ T cells. We determined that presentation of both forms of CSP on MHC class I depends on proteasomal activity, however, presentation of full length CSP is more efficient and is conferred by the presence of N-terminal lysine residues absent in the cleaved form of CSP. To evaluate the presence of these two forms during in vitro infection, we developed methods to allow for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. We showed that exoerythrocytic forms can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Furthermore, we present an experimental evidence, that our ii methods can be applicable for the analysis of human host factors limiting development of P. falciparum EEFs. iii Main goals 1. Assess the ability of two forms of Plasmodium falciparum CSP, to serve as targets for MHC class I-restricted CD8+ T cell responses Specific Aims: A. To develop an in vitro system for monitoring the MHC class I processing and presentation of peptide epitopes derived from the P. falciparum CSP antigen B. To assess the ability of targets expressing one of the CSP forms to induce specific CD8+ T cell activation in a MHC class I-restricted and non-restricted manner C. To examine the role of major cellular proteolytic machineries in the processing of two forms of CSP D. To investigate the role of the ubiquitin system in the MHC class I-restricted processing of peptide epitopes from two forms of the CSP antigen 2. To develop an experimental system for detection and isolation of Plasmodium falciparum EEFs in vitro Specific Aims: A. To develop an in vitro hepatocellular model permissive for propagation of P. falciparum EEFs B. To develop flow cytometry-based detection and specific isolation of P. falciparum EEFs C. To compare dynamics of P. falciparum EEF development in vitro using different experimental hepatocellular systems D. To validate the applicability of our experimental model for the analysis of human host factors limiting development of P. falciparum EEFs. iv Table of Contents Abstract .............................................................................................................................. ii Main goals ......................................................................................................................... iv Table of Figures.............................................................................................................. viii Chapter 1 : General Introduction ................................................................................... 1 History of elucidating the Plasmodium life cycle ..................................................................... 5 From skin to merosome ............................................................................................................. 9 Targeting exoerythrocytic forms: whole organism vaccines ................................................ 13 Mechanisms of immune protection ........................................................................................ 17 Biology of CSP .......................................................................................................................... 23 Techniques to study the Plasmodium liver stage ................................................................... 26 Concluding remarks ................................................................................................................ 28 Chapter 2 : Processing and presentation of two forms of CSP on MHC class I ....... 29 Introduction .............................................................................................................................. 30 Presentation of peptides on MHC class I ........................................................................... 30 Targeting Plasmodium: role of the MHC class I antigen processing and presentation pathway ................................................................................................................................. 34 Two forms of CSP as targets of the CD8+ T cell immune response ................................. 38 Results ....................................................................................................................................... 40 Ectopic expression of CSP in hepatocytes .......................................................................... 40 Identification of CSP in traversed and infected hepatocytes during in vitro infection .. 41 Two forms of CSP differentially activate CD8+ T cells..................................................... 43 Targets expressing one of two forms of CSP are differentially lysed by CD8+ T cells... 49 Two forms of CSP do not alter immune phenotypes of targets or overall susceptibility to death stimuli ..................................................................................................................... 51 Half-life of two forms of CSP and dependence of proteasomal degradation on MHC class I presentation ............................................................................................................... 53 CSP-Full forms polyubiquitin chains more efficiently than CSP-Short ......................... 54 Individual lysine residues in the N-terminus of CSP are required for efficient MHC class I-restricted antigen presentation ............................................................................... 56 Discussion ................................................................................................................................. 59 Chapter 3 : Specific detection and isolation of P. falciparum EEFs in vitro .............. 67 v Introduction .............................................................................................................................. 68 Results ....................................................................................................................................... 72 Immortalized human hepatocyte cell lines are permissive for traversal by P. falciparum sporozoites ............................................................................................................................ 72 Human hepatocyte cell lines exhibit differential ability to support development of P. falciparum EEFs ................................................................................................................... 76 P. falciparum-infected HC-04 hepatocytes can be specifically isolated ........................... 81 Primary human hepatocytes support P. falciparum 3D7HT-GFP EEFs in vitro ........... 84 Discussion ................................................................................................................................. 93 Chapter 4 : Flow cytometry based assays of P. falciparum liver stages in vitro: from sporozoite infectivity to EEF development ................................................................. 100 Introduction ............................................................................................................................ 101 Results ..................................................................................................................................... 104 CD81 is required for P. falciparum infection of primary human hepatocytes, but is not essential for the infection of HC-04 .................................................................................. 104 Humanized CSP-specific antibody 2A10 inhibits traversal and reduces the number of P. falciparum EEFs in human hepatocytes in vitro ......................................................... 107 Discussion ............................................................................................................................... 109 Chapter 5 : Future Directions ...................................................................................... 113 Chapter 6 : Materials and Methods ............................................................................ 119 Human hepatocyte
Load more

Recommended publications
  • Hamilton County (Ohio) Naturalization Records – Surname M
    Hamilton County Naturalization Records – Surname M Applicant Age Country of Origin Departure Date Departure Port Arrive Date Entry Port Declaration Dec Date Vol Page Folder Naturalization Naturalization Date Maag, Frederick 46 Oldenburg Bremen New Orleans T 11/01/1852 5 132 F F Maag, Frederick 46 Oldenburg Bremen New Orleans T 11/01/1854 6 258 F F Maag, Sebastian 31 Baden Liverpool New York T 01/02/1858 16 296 F F Maahan, James 32 Ireland Toronto Buffalo T 01/20/1852 24 37 F F Maas, Anton 30 Prussia Bremen New York T 01/17/1853 7 34 F F Maas, Carl 18 Mannheim, Germany ? ? T 05/04/1885 T F Maas, Garrett William 25 Holland Rotterdam New York T 11/15/1852 5 287 F F Maas, Garrett William 25 Holland Rotterdam New York T 11/15/1852 6 411 F F Maas, Jacob 55 Germany Rotterdam New York F ? T T Maas, John 55 Germany Havre New York F ? T T Maas, John William 28 Holland Rotterdam New Orleans T 09/27/1848 22 56 F F Maas, Julius J. 48 Germany Bremen New York F ? T T Maas, Leonardus Aloysius 37 Holland Rotterdam New Orleans T 03/01/1852 24 6 F F Maass, F.W. 45 Hanover Bremen Baltimmore T 11/02/1860 18 3 F F Macalusa, Michael 23 Italy Palermo New York T 3/18/1903 T F MacAvoy, Henry 48 England Liverpool New York T 10/26/1891 T F MacDermott, Joseph England ? ? T 2/26/1900 T F Machenheimer, Christoph 29 Hesse Darmstadt Havre New York T 02/19/1850 2 100 F F Machnovitz, Moses 50 Russia Bremen Baltimore T 4/27/1900 T F Maciejensky, Martin 28 Prussia Hamburg New York T 04/28/1854 8 280 F F Maciejensky, Martin 28 Prussia Hamburg New York T 04/28/1854 9 151 F
    [Show full text]
  • Human and Mouse CD Marker Handbook Human and Mouse CD Marker Key Markers - Human Key Markers - Mouse
    Welcome to More Choice CD Marker Handbook For more information, please visit: Human bdbiosciences.com/eu/go/humancdmarkers Mouse bdbiosciences.com/eu/go/mousecdmarkers Human and Mouse CD Marker Handbook Human and Mouse CD Marker Key Markers - Human Key Markers - Mouse CD3 CD3 CD (cluster of differentiation) molecules are cell surface markers T Cell CD4 CD4 useful for the identification and characterization of leukocytes. The CD CD8 CD8 nomenclature was developed and is maintained through the HLDA (Human Leukocyte Differentiation Antigens) workshop started in 1982. CD45R/B220 CD19 CD19 The goal is to provide standardization of monoclonal antibodies to B Cell CD20 CD22 (B cell activation marker) human antigens across laboratories. To characterize or “workshop” the antibodies, multiple laboratories carry out blind analyses of antibodies. These results independently validate antibody specificity. CD11c CD11c Dendritic Cell CD123 CD123 While the CD nomenclature has been developed for use with human antigens, it is applied to corresponding mouse antigens as well as antigens from other species. However, the mouse and other species NK Cell CD56 CD335 (NKp46) antibodies are not tested by HLDA. Human CD markers were reviewed by the HLDA. New CD markers Stem Cell/ CD34 CD34 were established at the HLDA9 meeting held in Barcelona in 2010. For Precursor hematopoetic stem cell only hematopoetic stem cell only additional information and CD markers please visit www.hcdm.org. Macrophage/ CD14 CD11b/ Mac-1 Monocyte CD33 Ly-71 (F4/80) CD66b Granulocyte CD66b Gr-1/Ly6G Ly6C CD41 CD41 CD61 (Integrin b3) CD61 Platelet CD9 CD62 CD62P (activated platelets) CD235a CD235a Erythrocyte Ter-119 CD146 MECA-32 CD106 CD146 Endothelial Cell CD31 CD62E (activated endothelial cells) Epithelial Cell CD236 CD326 (EPCAM1) For Research Use Only.
    [Show full text]
  • The Transcriptome of the Avian Malaria Parasite Plasmodium
    bioRxiv preprint doi: https://doi.org/10.1101/072454; this version posted August 31, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 The Transcriptome of the Avian Malaria Parasite 2 Plasmodium ashfordi Displays Host-Specific Gene 3 Expression 4 5 6 7 8 Running title 9 The Transcriptome of Plasmodium ashfordi 10 11 Authors 12 Elin Videvall1, Charlie K. Cornwallis1, Dag Ahrén1,3, Vaidas Palinauskas2, Gediminas Valkiūnas2, 13 Olof Hellgren1 14 15 Affiliation 16 1Department of Biology, Lund University, Lund, Sweden 17 2Institute of Ecology, Nature Research Centre, Vilnius, Lithuania 18 3National Bioinformatics Infrastructure Sweden (NBIS), Lund University, Lund, Sweden 19 20 Corresponding authors 21 Elin Videvall ([email protected]) 22 Olof Hellgren ([email protected]) 23 24 1 bioRxiv preprint doi: https://doi.org/10.1101/072454; this version posted August 31, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Abstract 26 27 Malaria parasites (Plasmodium spp.) include some of the world’s most widespread and virulent 28 pathogens, infecting a wide array of vertebrates. Our knowledge of the molecular mechanisms these 29 parasites use to invade and exploit hosts other than mice and primates is, however, extremely limited. 30 How do Plasmodium adapt to individual hosts and to the immune response of hosts throughout an 31 infection? To better understand parasite plasticity, and identify genes that are conserved across the 32 phylogeny, it is imperative that we characterize transcriptome-wide gene expression from non-model 33 malaria parasites in multiple host individuals.
    [Show full text]
  • And Toxoplasmosis in Jackass Penguins in South Africa
    IMMUNOLOGICAL SURVEY OF BABESIOSIS (BABESIA PEIRCEI) AND TOXOPLASMOSIS IN JACKASS PENGUINS IN SOUTH AFRICA GRACZYK T.K.', B1~OSSY J.].", SA DERS M.L. ', D UBEY J.P.···, PLOS A .. ••• & STOSKOPF M. K .. •••• Sununary : ReSlIlIle: E x-I1V\c n oN l~ lIrIUSATION D'Ar\'"TIGENE DE B ;IB£,'lA PH/Re El EN ELISA ET simoNi,cATIVlTli t'OUR 7 bxo l'l.ASMA GONIJfI DE SI'I-IENICUS was extracted from nucleated erythrocytes Babesia peircei of IJEMIiNSUS EN ArRIQUE D U SUD naturally infected Jackass penguin (Spheniscus demersus) from South Africo (SA). Babesia peircei glycoprotein·enriched fractions Babesia peircei a ele extra it d 'erythrocytes nue/fies p,ovenanl de Sphenicus demersus originoires d 'Afrique du Sud infectes were obto ined by conca navalin A-Sepharose affinity column natulellement. Des fractions de Babesia peircei enrichies en chromatogrophy and separated by sod ium dodecyl sulphate­ glycoproleines onl ele oblenues par chromatographie sur colonne polyacrylam ide gel electrophoresis (SDS-PAGE ). At least d 'alfinite concona valine A-Sephorose et separees par 14 protein bonds (9, 11, 13, 20, 22, 23, 24, 43, 62, 90, electrophorese en gel de polyacrylamide-dodecylsuJfale de sodium 120, 204, and 205 kDa) were observed, with the major protein (SOS'PAGE) Q uotorze bandes proleiques au minimum ont ete at 25 kDa. Blood samples of 191 adult S. demersus were tes ted observees (9, 1 I, 13, 20, 22, 23, 24, 43, 62, 90, 120, 204, by enzyme-linked immunosorbent assoy (ELISA) utilizing B. peircei et 205 Wa), 10 proleine ma;eure elant de 25 Wo.
    [Show full text]
  • Systemic Induction of the Angiogenesis Switch by the Tetraspanin D6.1A/CO-029
    Research Article Systemic Induction of the Angiogenesis Switch by the Tetraspanin D6.1A/CO-029 Sabine Gesierich,1 Igor Berezovskiy,1 Eduard Ryschich,2 and Margot Zo¨ller1,3 1Department of Tumor Progression and Immune Defence, German Cancer Research Centre; 2Department of Surgery, University of Heidelberg, Heidelberg; and 3Department of Applied Genetics, Faculty of Chemistry and Bioscience, University of Karlsruhe, Karlsruhe, Germany Abstract transcription of angiogenic factors (6, 7). Recent studies in Expression of the tetraspanin CO-029 is associated with poor knockout and transgenic mouse models have provided further prognosis in patients with gastrointestinal cancer. In a evidence that tumor angiogenesis is not only guided by the pancreatic tumor line, overexpression of the rat homologue, tumor cell itself, but is also closely tied to the tumor microenvi- ronment (8). D6.1A, induces lethally disseminated intravascular coagula- tion, suggesting D6.1A engagement in angiogenesis. D6.1A- Tetraspanins are a large family of proteins grouped according to overexpressing tumor cells induce the greatest amount of structural relatedness. The key feature of tetraspanins is their angiogenesis in vivo, and tumor cells as well as exosomes potential to associate with each other and with a multitude of derived thereof strikingly increase endothelial cell branching molecules from other protein families (9–11), the most prominent in vitro. Tumor cell–derived D6.1A stimulates angiogenic partners being integrins (12). The tetraspanin, D6.1A (rat)/CO-029 a h a h factor transcription, which includes increased matrix metal- (human), associates with 3 1 and 6 1 and, after disassembly of a h loproteinase and urokinase-type plasminogen activator se- hemidesmosomes, with 6 4.
    [Show full text]
  • A Shared Pathway of Exosome Biogenesis Operates at Plasma And
    bioRxiv preprint doi: https://doi.org/10.1101/545228; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A shared pathway of exosome biogenesis operates at plasma and endosome membranes Francis K. Fordjour1, George G. Daaboul2, and Stephen J. Gould1* 1Department of Biological Chemistry Johns Hopkins University Baltimore, MD USA 2Nanoview Biosciences Boston, MA USA Corresponding author: Stephen J. Gould, Ph.D. Department of Biological Chemistry Johns Hopkins University Baltimore, MD USA Email: [email protected] Tel (01) 443 847 9918 1 bioRxiv preprint doi: https://doi.org/10.1101/545228; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Summary: This study of exosome cargo protein budding reveals that cells use a common pathway for budding exosomes from plasma and endosome membranes, providing a new mechanistic explanation for exosome heterogeneity and a rational roadmap for exosome engineering. Keywords: Protein budding, tetraspanin, endosome, plasma membrane, extracellular vesicle, CD9, CD63, CD81, SPIR, interferometry Abbreviations: EV, extracellular vesicles; IB, immunoblot; IFM, immunofluorescence microscopy; IPMC, intracellular plasma membrane-connected compartment; MVB, multivesicular body; SPIR, single-particle interferometric reflectance; SPIRI, single-particle interferometric reflectance imaging 2 bioRxiv preprint doi: https://doi.org/10.1101/545228; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved.
    [Show full text]
  • Tetraspanin CD151 Plays a Key Role in Skin Squamous Cell Carcinoma
    Oncogene (2013) 32, 1772–1783 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Tetraspanin CD151 plays a key role in skin squamous cell carcinoma QLi1, XH Yang2,FXu1, C Sharma1, H-X Wang1, K Knoblich1, I Rabinovitz3, SR Granter4 and ME Hemler1 Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared with other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3 (signal transducers and activators of transcription), a regulator of cell proliferation and apoptosis. CD151 also supports protein kinase C (PKC)a–a6b4 integrin association and PKC-dependent b4 S1424 phosphorylation, while regulating a6b4 distribution. CD151–PKCa effects on integrin b4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including 7,12-dimethylbenz[a]anthracene (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting epidermal growth factor receptor, PKC, Jak2/Tyk2 and STAT3. Hence, CD151 ‘co-targeting’ may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151/laminin-binding integrin complexes.
    [Show full text]
  • Extracellular Vesicle Human CD9/CD63/CD81 Antibody Panel
    Extracellular Vesicle Human CD9/CD63/CD81 Antibody Panel Antibody panel for the detection of extracellular vesicles using CD9, CD63, and CD81 markers Catalog #100-0211 1 Kit Product Description The Extracellular Vesicle Human CD9/CD63/CD81 Antibody Panel is suitable for the detection of extracellular vesicles (EVs) derived from human cells. It comprises three primary antibodies that are immunoreactive toward human CD9, CD63, and CD81; these are proteins that are typically expressed on EVs and widely used as markers to analyze and isolate these cell-derived particles. CD9, CD63, and CD81 belong to the tetraspanin family of membrane proteins, which possess four transmembrane domains and interact with diverse proteins on the cell surface to form multimolecular networks termed tetraspanin-enriched microdomains. CD9, CD63, and CD81 proteins are expressed on the surface of many cells, including B cells, T cells, NK cells, monocytes, dendritic cells, thymocytes, endothelial cells, and fibroblasts, and are involved in modulating a variety of cellular processes including cell activation, adhesion, differentiation, and tumor invasion. The antibodies provided in this panel have been reported for use in analyzing primary cells, cell lines, and EVs by ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and Western blotting. They have been reported to cross-react with their cognate antigens in non-human primates, including baboons and rhesus and cynomolgus macaques. Product Information The following products comprise the Extracellular
    [Show full text]
  • Aberrant Expression of Tetraspanin Molecules in B-Cell Chronic Lymphoproliferative Disorders and Its Correlation with Normal B-Cell Maturation
    Leukemia (2005) 19, 1376–1383 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu Aberrant expression of tetraspanin molecules in B-cell chronic lymphoproliferative disorders and its correlation with normal B-cell maturation S Barrena1,2, J Almeida1,2, M Yunta1,ALo´pez1,2, N Ferna´ndez-Mosteirı´n3, M Giralt3, M Romero4, L Perdiguer5, M Delgado1, A Orfao1,2 and PA Lazo1 1Instituto de Biologı´a Molecular y Celular del Ca´ncer, Centro de Investigacio´n del Ca´ncer, Consejo Superior de Investigaciones Cientı´ficas-Universidad de Salamanca, Salamanca, Spain; 2Servicio de Citometrı´a, Universidad de Salamanca and Hospital Universitario de Salamanca, Salamanca, Spain; 3Servicio de Hematologı´a, Hospital Universitario Miguel Servet, Zaragoza, Spain; 4Hematologı´a-hemoterapia, Hospital Universitario Rı´o Hortega, Valladolid, Spain; and 5Servicio de Hematologı´a, Hospital de Alcan˜iz, Teruel, Spain Tetraspanin proteins form signaling complexes between them On the cell surface, tetraspanin antigens are present either as and with other membrane proteins and modulate cell adhesion free molecules or through interaction with other proteins.25,26 and migration properties. The surface expression of several tetraspanin antigens (CD9, CD37, CD53, CD63, and CD81), and These interacting proteins include other tetraspanins, integri- F 22,27–30F their interacting proteins (CD19, CD21, and HLA-DR) were ns particularly those with the b1 subunit HLA class II 31–33 34,35 analyzed during normal B-cell maturation and compared to a moleculesFeg HLA DR -, CD19, the T-cell recep- group of 67 B-cell neoplasias. Three patterns of tetraspanin tor36,37 and several other members of the immunoglobulin expression were identified in normal B cells.
    [Show full text]
  • Adirondack Recreational Trail Advocates (ARTA)
    Adirondack Recreational Trail Advocates (ARTA) Proposal for the Adirondack Rail Trail Photo: Lake Colby Causeway, Lee Keet, 2013 Submitted by the Board of Directors of ARTA Tupper Lake: Hope Frenette, Chris Keniston; Maureen Peroza Saranac Lake: Dick Beamish, Lee Keet, Joe Mercurio; Lake Clear: David Banks; Keene: Tony Goodwin; Lake Placid: Jim McCulley; Beaver River: Scott Thompson New York State Snowmobile Association: Jim Rolf WWW.TheARTA.org Adirondack Recreational Trail Advocates P.O. Box 1081 Saranac Lake, N.Y. 12983 Page 2 This presentation has been prepared by Adirondack Recreational Trail Advocates (ARTA), a not-for- profit 501(c)(3) corporation formed in 2011 and dedicated to creating a recreational trail on the largely abandoned and woefully underutilized rail corridor . © 2013, Adirondack Recreational Trail Advocates, Inc. Page 3 Contents Executive Summary ...................................................................................................................................... 6 Original UMP Criteria Favor the Rail Trail .................................................................................................. 7 Changing the Status of the Corridor ........................................................................................................... 10 Classification as a Travel Corridor ......................................................................................................... 10 Historic Status ........................................................................................................................................
    [Show full text]
  • Host-Parasite Interactions Between Plasmodium Species and New Zealand Birds: Prevalence, Parasite Load and Pathology
    Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Host-parasite interactions between Plasmodium species and New Zealand birds: prevalence, parasite load and pathology A thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science in Wildlife Health At Massey University, Palmerston North New Zealand © Danielle Charlotte Sijbranda 2015 ABSTRACT Avian malaria, caused by Plasmodium spp., is an emerging disease in New Zealand and has been reported as a cause of morbidity and mortality in New Zealand bird populations. This research was initiated after P. (Haemamoeba) relictum lineage GRW4, a suspected highly pathogenic lineage of Plasmodium spp. was detected in a North Island robin of the Waimarino Forest in 2011. Using nested PCR (nPCR), the prevalence of Plasmodium lineages in the Waimarino Forest was evaluated by testing 222 birds of 14 bird species. Plasmodium sp. lineage LINN1, P. (Huffia) elongatum lineage GRW06 and P. (Novyella) sp. lineage SYATO5 were detected; Plasmodium relictum lineage GRW4 was not found. A real- time PCR (qPCR) protocol to quantify the level of parasitaemia of Plasmodium spp. in different bird species was trialled. The qPCR had a sensitivity and specificity of 96.7% and 98% respectively when compared to nPCR, and proved more sensitive in detecting low parasitaemias compared to the nPCR. The mean parasite load was significantly higher in introduced bird species compared to native and endemic species.
    [Show full text]
  • A New One-Step Multiplex PCR Assay for Simultaneous Detection and Identification of Avian Haemosporidian Parasites
    Parasitology Research (2019) 118:191–201 https://doi.org/10.1007/s00436-018-6153-7 GENETICS, EVOLUTION, AND PHYLOGENY - ORIGINAL PAPER A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites Arif Ciloglu1,2,3 & Vincenzo A. Ellis2 & Rasa Bernotienė4 & Gediminas Valkiūnas4 & Staffan Bensch2 Received: 25 May 2018 /Accepted: 12 November 2018 /Published online: 7 December 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Accurate detection and identification are essential components for epidemiological, ecological, and evolutionary surveys of avian haemosporidian parasites. Microscopy has been used for more than 100 years to detect and identify these parasites; however, this technique requires considerable training and high-level expertise. Several PCR methods with highly sensitive and specific detection capabilities have now been developed in addition to microscopic examination. However, recent studies have shown that these molecular protocols are insufficient at detecting mixed infections of different haemosporidian parasite species and genetic lineages. In this study, we developed a simple, sensitive, and specific multiplex PCR assay for simultaneous detection and discrimination of parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon in single and mixed infections. Relative quantification of parasite DNA using qPCR showed that the multiplex PCR can amplify parasite DNA ranging in concentration over several orders of magnitude. The detection specificity and sensitivity of this new multiplex PCR assay were also tested in two different laboratories using previously screened natural single and mixed infections. These findings show that the multiplex PCR designed here is highly effective at identifying both single and mixed infections from all three genera of avian haemosporidian parasites.
    [Show full text]