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          Abstract

          Summary Effective control and monitoring of foot‐and‐mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype‐specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD‐endemic countries have motivated the development of simple diagnostic platforms to support local decision‐making. Using a portable thermocycler, the T‐COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan‐serotype‐specific real‐time RT‐PCR (rRT‐PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa‐specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan‐serotype‐specific lyophilized assay were comparable to that of an OIE‐recommended laboratory‐based rRT‐PCR (determined using a panel of 57 FMDV‐positive samples and six non‐FMDV vesicular disease samples for differential diagnosis). The FMDV‐typing assay was able to correctly identify the serotype of 33/36 FMDV‐positive samples (no cross‐reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre‐clinical, clinical and clinically recovered cattle. These data support the use of field‐ready rRT‐PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

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          Review of the status and control of foot and mouth disease in sub-Saharan Africa.

          Six of the seven serotypes of foot and mouth disease (FMD) virus (i.e. all but Asia 1) are prevalent in Africa although there are marked regional differences in distribution. Three of these serotypes are unique to Africa, namely the three South African Territories (SAT) serotypes. Serotype C may also now be confined to Africa because it has not been reported elsewhere recently. In southern Africa at least, the SAT serotypes have an intimate and probably ancient association with African buffalo (Syncerus caffer) that is instrumental in their maintenance. Within each of the six prevalent serotypes, with the possible exception of C, there are a number of different lineages with more or less defined distributions (i.e. topotypes) that in some cases are sufficiently immunologically different from one another to require specific vaccines to ensure efficient control. This immunological diversity in prevalent serotypes and topotypes, in addition to uncontrolled animal movement in most parts of the continent, render FMD difficult to control in present circumstances. This fact, together with poorly developed intercontinental trade in animals and animal products has resulted in the control of FMD being afforded a low priority in most parts of the continent, although the northern and southern regions of the continent are an exception. As a consequence, eradication of FMD from Africa as a whole is not a prospect within the foreseeable future. In southern Africa, the use of fencing and other means to strictly control the movement of wildlife and livestock as well as judicious application of vaccine has resulted in countries of the region being able to access beef and other livestock markets in Europe and elsewhere in the developed world. Significant marketing of livestock and livestock products from Africa outside the continent is unlikely to be achieved unless similar approaches can be developed for other regions of Africa. This will result in continuing under-exploitation of a valuable resource in the arid and semi-arid regions of Africa, with increasing marginalisation of human populations living there.
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            Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay.

            A fluorogenic RT-PCR (5'-nuclease probe-based) assay using a primer/probe set designed from the internal ribosomal entry site region of the virus genome was developed for the specific detection of all seven serotypes of foot-and-mouth disease (FMD) virus in epithelial suspensions and cell culture virus preparations. The reverse transcription polymerase chain reaction (RT-PCR) specifically detected FMD virus in sample submissions from the UK 2001 FMD outbreak with greater sensitivity than our conventional RT-PCR procedure and our routine diagnostic procedures of ELISA and virus isolation in cell culture. The fluorogenic RT-PCR provides relatively fast results, enables a quantitative assessment to be made of virus amounts and can handle more samples and/or replicates of samples in a single assay than the conventional RT-PCR procedure. Therefore it is seen as a valuable tool to complement the routine diagnostic procedures for FMD virus diagnosis.
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              Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-and-mouth disease.

              An automated one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) protocol was optimised and evaluated for the routine diagnosis of foot-and-mouth disease (FMD). Parallel testing of RNA samples (n=257) indicated that this assay has a diagnostic sensitivity at least equivalent to the automated two-step rRT-PCR protocol previously used for the laboratory detection of FMD virus (FMDV). This more rapid and economical one-step protocol will play a key role in contingency planning for any future outbreaks of FMD in the United Kingdom (UK).
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                Author and article information

                Contributors
                veronica.fowler@pirbright.ac.uk
                Journal
                Transbound Emerg Dis
                Transbound Emerg Dis
                10.1111/(ISSN)1865-1682
                TBED
                Transboundary and Emerging Diseases
                John Wiley and Sons Inc. (Hoboken )
                1865-1674
                1865-1682
                30 July 2017
                February 2018
                : 65
                : 1 ( doiID: 10.1111/tbed.2018.65.issue-1 )
                : 221-231
                Affiliations
                [ 1 ] The Pirbright Institute Pirbright Surrey UK
                [ 2 ] Institute of Biodiversity, Animal Health and Comparative Medicine College of Medical Veterinary & Life Sciences University of Glasgow Glasgow UK
                [ 3 ] European Commission for the Control of Foot‐and‐Mouth Disease (EuFMD) Animal Production and Health Division FAO Rome Italy
                [ 4 ] Foot‐and‐Mouth Disease Laboratory, Embakasi Ministry of Agriculture, Livestock, Fisheries and Blue Economy Nairobi Kenya
                [ 5 ] Department of Biochemistry, Molecular Biology and Biotechnology College of Veterinary and Medical Sciences Sokoine University of Agriculture, Chuo Kikuu Morogoro Tanzania
                [ 6 ] Department of Veterinary Science Faculty of Agriculture University of Miyazaki Miyazaki Japan
                [ 7 ] National Animal Health Diagnostic & Investigation Centre Sebeta Oromia Ethiopia
                [ 8 ] Tetracore Inc Rockville MD USA
                Author notes
                [*] [* ] Correspondence

                V. Fowler, The Pirbright Institute, Pirbright, Surrey, UK.

                Email: veronica.fowler@ 123456pirbright.ac.uk

                Author information
                http://orcid.org/0000-0002-4993-1901
                http://orcid.org/0000-0002-7874-8014
                Article
                TBED12684
                10.1111/tbed.12684
                5811823
                28758346
                9b1850fd-634f-492e-9f4e-4ff4ca6a0c9b
                © 2017 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 31 March 2017
                Page count
                Figures: 7, Tables: 1, Pages: 11, Words: 6695
                Funding
                Funded by: The Pirbright Institute Business Development Fund
                Award ID: 289364
                Funded by: Biotechnology and Biological Sciences Research Council (BBSRC)
                Award ID: 289364
                Funded by: European Union
                Award ID: 289364
                Categories
                Original Article
                Original Articles
                Custom metadata
                2.0
                tbed12684
                February 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.3.2.2 mode:remove_FC converted:14.02.2018

                Infectious disease & Microbiology
                diagnostics,disease control,foot‐and‐mouth disease,foot‐and‐mouth disease virus,lyophilized,rrt‐pcr

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