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A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis

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A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis
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  Accepted Manuscript Title: A bacterial engineered glycoprotein as a novel antigenfor diagnosis of bovine brucellosisAuthor: Andr´es E. Ciocchini Diego A. Rey Serantes LucianoJ. Melli Leticia S. Guidolin Jeremy A. Iwashkiw Sebasti´anElena Cristina Franco Ana M. Nicola Mario F. Feldman DiegoJ. Comerci Juan E. UgaldePII: S0378-1135(14)00214-4DOI: http://dx.doi.org/doi:10.1016/j.vetmic.2014.04.014Reference: VETMIC 6598To appear in:  VETMIC  Received date: 14-1-2014Revised date: 14-3-2014Accepted date: 19-4-2014Please cite this article as: Ciocchini, A.E., Serantes, D.A.R., Melli, L.J.,Guidolin, L.S., Iwashkiw, J.A., Elena, S., Franco, C., Nicola, A.M., Feldman,M.F., Comerci, D.J., Ugalde, J.E.,A bacterial engineered glycoprotein as a novelantigen for diagnosis of bovine brucellosis,  Veterinary Microbiology  (2014),http://dx.doi.org/10.1016/j.vetmic.2014.04.014This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.Themanuscriptwillundergocopyediting,typesetting,andreviewoftheresultingproof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.  Page 1 of 34    A  c  c  e   p   t  e  d    M  a  n   u  s  c  r   i   p   t 1   A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis 1   2   Andrés E. Ciocchini 1 , Diego A. Rey Serantes 1 , Luciano J. Melli 1 , Leticia S. Guidolin 1 , Jeremy A. 3   Iwashkiw 3 , Sebastián Elena 2 , Cristina Franco 2 , Ana M. Nicola 2 , Mario F. Feldman 3 , Diego J. 4   Comerci 1,4*  and Juan E. Ugalde 1* . 5   6   1- Instituto de Investigaciones Biotecnológicas “Dr. Rodolfo A. Ugalde”, Instituto Tecnológico de 7   Chascomús (IIB-INTECH), Universidad Nacional de San Martín, CONICET, San Martín, 1650, 8   Buenos Aires, Argentina; 9   2- Laboratorio de Referencia de la OIE para Brucelosis, Dirección General de Laboratorio y 10   Control Técnico (DiLab), Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), 11   Martínez, 1640, Buenos Aires, Argentina; 12   3- Alberta Glycomics Centre, Department of Biological Sciences, University of Alberta, Edmonton, 13   AB, TG6 2E9, Canada; 14   4- Comisión Nacional de Energía Atómica, Grupo Pecuario, Centro Atómico Ezeiza, Buenos Aires, 15   Argentina. 16   17   * Corresponding authors:   18   Tel.: +54 11 4006 1500 (2129); Fax: +54 11 4006 1559 19   E-mail addresses: jugalde@iibintech.com.ar (J. E. Ugalde); dcomerci@iibintech.com.ar (D. J. 20   Comerci) 21   Complete correspondence address to which the proofs should be sent: Instituto de 22   Investigaciones Biotecnológicas “Dr. Rodolfo A. Ugalde”, Instituto Tecnológico de Chascomús 23   (IIB-INTECH), Universidad Nacional de San Martín. Campus Miguelete, Av. 25 de Mayo y 24   Francia, San Martín, 1650, Buenos Aires, Argentina. E-mail addresses: jugalde@iibintech.com.ar 25   (J. E. Ugalde). 26   27    Page 2 of 34    A  c  c  e   p   t  e  d    M  a  n   u  s  c  r   i   p   t 2   Abstract 27   28   Brucellosis is a highly contagious zoonosis that affects livestock and human beings. Laboratory 29   diagnosis of bovine brucellosis mainly relies on serological diagnosis using serum and/or milk 30   samples. Although there are several serological tests with different diagnostic performance and 31   capacity to differentiate vaccinated from infected animals, there is still no standardized reference 32   antigen for the disease. Here we validate the first recombinant glycoprotein antigen, an  N  - 33   formylperosamine O -polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of bovine 34    brucellosis. This antigen can be produced in homogeneous batches without the need of culturing 35    pathogenic brucellae; all characteristics that make it appropriate for standardization. An indirect 36   immunoassay based on the detection of anti O -polysaccharide IgG antibodies in bovine samples 37   was developed coupling OAg-AcrA to magnetic beads or ELISA plates. As a proof of concept and 38   to validate the antigen, we analyzed serum, whole blood and milk samples obtained from non- 39   infected, experimentally infected and vaccinated animals included in a vaccination/infection trial 40    performed in our laboratory as well as more than 1000 serum and milk samples obtained from 41   naturally infected and S19-vaccinated animals from Argentina. Our results demonstrate that OAg- 42   AcrA-based assays are highly accurate for diagnosis of bovine brucellosis, even in vaccinated 43   herds, using different types of samples and in different platforms. We propose this novel 44   recombinant glycoprotein as an antigen suitable for the development of new standard 45   immunological tests for screening and confirmatory diagnosis of bovine brucellosis in regions or 46   countries with brucellosis-control programs. 47   48   Keywords: Bovine brucellosis; diagnosis; serological tests ; recombinant glycoprotein antigen.   49   50    Page 3 of 34    A  c  c  e   p   t  e  d    M  a  n   u  s  c  r   i   p   t 3   Introduction   50   Brucellosis is a highly contagious zoonosis caused by Gram-negative bacteria of the genus 51    Brucella  that affects livestock, wild animal species and humans. The main pathogenic species 52   worldwide are  B. abortus , responsible for bovine brucellosis;  B. melitensis , the major etiologic 53   agent of ovine and caprine brucellosis; and  B. suis , responsible for swine brucellosis (Corbel, 1997). 54   These three  Brucella  species remain an important cause of veterinary morbidity and mortality. 55   Brucellosis causes important economic losses not only because it affects animal production 56   (reduced milk, abortion, delayed conception and impaired fertility) but also because detection of the 57   disease in a region or country imposes, due to international veterinary regulations, restrictions on 58   animal movements and trade (Seleem et al., 2010). Brucellosis in humans can be severely 59   debilitating and disabling and remains an important public health concern (Young, 1995). In the 60   absence of a human brucellosis vaccine, prevention of the disease depends predominantly on 61   control of brucellosis in animals that constitute the natural disease’s reservoirs (Godfroid et al., 62   2010). For these reasons, many programs have been implemented worldwide to control and/or 63   eradicate brucellosis mainly in cattle, small ruminants, and pigs. 64   Control of bovine brucellosis depends on vaccination and/or detection of infected animals 65   and slaughter. Due to the lack of pathognomonic signs for the clinical diagnosis of brucellosis in 66   animals, laboratory confirmation of bovine brucellosis by isolation of the pathogen or 67   demonstration of specific antibodies is essential. Bacteriological isolation of the microorganism 68   confirms the diagnosis; however, slow growth of brucellae primary cultures (up to 7 days), the risk 69   involved in their handling and poor sensitivity makes diagnosis based exclusively on isolation of 70    brucellae not always feasible and effective. Therefore, laboratory diagnosis mainly relies on 71   serological diagnosis using serum and/or milk samples (Godfroid et al., 2010; Saegerman C., 2010). 72   The most commonly used serological tests for diagnosis of bovine brucellosis are the 73   agglutination tests such as the buffered plate agglutination test (BPAT), rose Bengal plate 74   agglutination test (RBPT) and serum agglutination test (SAT), and the complement fixation test 75    Page 4 of 34    A  c  c  e   p   t  e  d    M  a  n   u  s  c  r   i   p   t 4   (CFT) (Aznar et al., 2012; DILAB-SENASA, 2009; Godfroid et al., 2010; OIE, 2012). Indirect 76   enzyme-linked immunosorbent assays (iELISA) have also been developed for serum and m. For 77   analyzing individual and bulk tank milk samples the milk ring test (MRT) is the most widely used 78   test (Gall and Nielsen, 2004). All these assays use as antigen the whole bacteria, bacterial extracts 79   containing high concentrations of smooth lipopolysaccharide (sLPS) or purified sLPS. These tests 80   suffer from false positive reactions and, because S19 vaccine is antigenically similar to virulent 81   strains, does not allow a precise differentiation of vaccinated from infected animals. Consequently, 82   other tests have been developed including the competitive ELISA (CELISA) and the fluorescence 83    polarization assay (FPA), which have eliminated most reactions due to cross-reacting antibodies 84   and residual antibodies produced in response to S19 vaccination (McGiven et al., 2003; Nielsen, 85   1990; Nielsen et al., 1996; Nielsen et al., 1995). Both assays measure specific antibodies against the 86   immunodominant O -polysaccharide section of the LPS (Caroff et al., 1984b). Additionally, a 87   common problem to all serological tests currently used for diagnosis of brucellosis is the lack of a 88   standardized reference antigen, and one of the main reasons for this is the source of the antigens as 89   well as the preparation methods used (Al Dahouk et al., 2003). Therefore, for harmonization of the 90   serological diagnosis of brucellosis there is a need to develop a new reference antigen easy to 91   standardize. 92   Previously, we have produced and characterized a recombinant glycoprotein consisting of a 93   homopolymer of  N  -formylperosamine, the O -polysaccharide of  B. abortus  (OAg) covalently linked 94   to the carrier protein AcrA (hereinafter OAg-AcrA) (Iwashkiw et al., 2012). OAg-AcrA was 95    produced using an in vivo  engineered bacterial glycosylation system based on the combination of 96   the LPS biosynthesis pathway of Yersinia enterocolitica   O:9, whose O-polysaccharide is identical 97   to the  B. abortus  OAg, and the  N  -glycosylation pathway of Campylobacter jejuni  (Feldman et al., 98   2005). OAg-AcrA was applied to the development and validation of a new indirect immunoassay 99   for diagnosis of human brucellosis using magnetic beads (Ciocchini et al., 2013). We have 100  
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