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      Comparação entre diversos antígenos para o diagnóstico de Anaplasma marginale por ELISA Translated title: Comparison between several antigens for diagnosis of Anaplasma marginale by ELISA

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          Abstract

          Anaplasmose bovina é uma doença com grande importância nas regiões tropicais e subtropicais do mundo por determinar perdas econômicas devido à mortalidade e redução da produtividade. É causada por Anaplasma marginale, uma riquétsia intraeritrocítica obrigatória cujo controle requer, além de uma vacina eficiente, uma acurada identificação de bovinos cronicamente infectados. Apesar de existirem atualmente diversos métodos de diagnóstico dessa riquétsia, os métodos sorológicos, em particular o ensaio de imunoadsorção enzimática-ELISAs, são os mais utilizados devido à sua versatilidade e praticidade. No entanto, devido ao grande número de antígenos disponíveis, atualmente torna-se necessária uma avaliação para definir quais antígenos apresentam um melhor desempenho no diagnóstico da anaplasmose. Soros de bovinos positivos e negativos para A. marginale por PCR, e soros de animais provenientes do Brasil e Costa Rica, foram testados em ELISAs baseados em MSP1a, MSP2 e MSP5 recombinantes, um pool das três proteínas recombinantes, e antígeno de lisado de corpúsculos iniciais da riquétsia (CI). Utilizando soro de bovinos positivos para A. marginale por PCR, uma maior sensibilidade foi observada no ELISA CI. No entanto, uma maior especificidade, com soro de bovinos negativos a PCR, foi observada com os ELISAs recombinantes. O porcentual de bovinos positivos do Brasil e Costa Rica foi maior com ELISA CI. Razões para essas diferenças são discutidas.

          Translated abstract

          Bovine anaplasmosis is a major disease in tropical and subtropical regions of the world by determine economical loss due mortality and productive reduction. The disease is caused by Anaplasma marginale, an intraerythrocytic rickettsia whose control requires, besides an efficient vaccine, the accurate identification of chronically infected cattle. Although the existence of diverse methods of diagnosis of this rickettsia, the serological methods, in particular the enzyme immunosorbent assays (ELISAs), are the most used due to its versatility and practice. However, due to the high number of antigens currently available, an evaluation becomes necessary to define which antigens present the better performance in the diagnosis of anaplasmosis. Sera from cattle positive or negative to A. marginale by PCR, and sera from cattle proceeding from Brazil and Costa Rica, were tested by ELISAs based in recombinant MSP1a, MSP2, and MSP5, a pool of the three recombinant proteins, and initial body lisate antigen (CI). Using sera from A. marginale positive cattle by PCR, the highest sensitivity was shown by CI ELISA. Nevertheless, the highest specificity, with sera from negative cattle by PCR, was shown by recombinants ELISAs. The percentiles of positive cattle from Brazil and Costa Rica were higher with CI ELISA. Reasons for such differences were discussed.

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          Most cited references37

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          A statistically defined endpoint titer determination method for immunoassays.

          Results of immunoassays for which no positive standards are available are often expressed as endpoint titers. The endpoint titer is defined as the reciprocal of the highest analyte dilution that gives a reading above the cutoff. Unfortunately, there is no generally accepted rule for the determination of these cutoff values. In enzyme-linked immunosorbent assays (ELISA) a value two or three times the mean background or negative control reading is sometimes used. Other investigators set the cutoff arbitrarily at a certain absorbance value. These procedures do not provide statistically meaningful information about the risk of overtitration or false low titers. We have solved this problem by devising a practical method for establishing a statistically valid cutoff. The procedure involves calculating the upper prediction limit using the Student t-distribution. The mathematical formula which defines the upper prediction limit is expressed as the standard deviation multiplied by a factor which is based on the number of negative controls and the confidence level (1 - alpha). Appropriate factors are provided for 2 to 30 negative controls and for confidence levels ranging from 95% to 99.9%. Our new method is more reliable than other nonstatistical procedures yet does not require sophisticated computation. It can be applied to a variety of immunoassays provided that negative controls are available.
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            Antigens and alternatives for control of Anaplasma marginale infection in cattle.

            Anaplasmosis, a tick-borne cattle disease caused by the rickettsia Anaplasma marginale, is endemic in tropical and subtropical areas of the world. The disease causes considerable economic loss to both the dairy and beef industries worldwide. Analyses of 16S rRNA, groESL, and surface proteins have resulted in the recent reclassification of the order Rickettsiales. The genus Anaplasma, of which A. marginale is the type species, now also includes A. bovis, A. platys, and A. phagocytophilum, which were previously known as Ehrlichia bovis, E. platys, and the E. phagocytophila group (which causes human granulocytic ehrlichiosis), respectively. Live and killed vaccines have been used for control of anaplasmosis, and both types of vaccines have advantages and disadvantages. These vaccines have been effective in preventing clinical anaplasmosis in cattle but have not blocked A. marginale infection. Thus, persistently infected cattle serve as a reservoir of infective blood for both mechanical transmission and infection of ticks. Advances in biochemical, immunologic, and molecular technologies during the last decade have been applied to research of A. marginale and related organisms. The recent development of a cell culture system for A. marginale provides a potential source of antigen for the development of improved killed and live vaccines, and the availability of cell culture-derived antigen would eliminate the use of cattle in vaccine production. Increased knowledge of A. marginale antigen repertoires and an improved understanding of bovine cellular and humoral immune responses to A. marginale, combined with the new technologies, should contribute to the development of more effective vaccines for control and prevention of anaplasmosis.
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              Genetic diversity of anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development.

              The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes several pathogens of veterinary and human medical importance. An understanding of the diversity of Anaplasma major surface proteins (MSPs), including those MSPs that modulate infection, development of persistent infections, and transmission of pathogens by ticks, is derived in part, by characterization and phylogenetic analyses of geographic strains. Information concerning the genetic diversity of Anaplasma spp. MSPs will likely influence the development of serodiagnostic assays and vaccine strategies for the control of anaplasmosis.
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                Author and article information

                Journal
                pvb
                Pesquisa Veterinária Brasileira
                Pesq. Vet. Bras.
                Colégio Brasileiro de Patologia Animal - CBPA (Rio de Janeiro, RJ, Brazil )
                0100-736X
                1678-5150
                January 2010
                : 30
                : 1
                : 37-41
                Affiliations
                [02] Campo Grande MS orgnameEmbrapa Gado de Corte orgdiv1Área de Sanidade Animal Brasil
                [05] Heredia orgnameUniversidade Nacional Autônoma orgdiv1Escola de Medicina Veterinária Costa Rica
                [03] Seropédica RJ orgnameUniversidade Federal Rural do Rio de Janeiro Brasil
                [04] Campo Grande MS orgnameUniversidade Federal de Mato Grosso do Sul
                [01] Recife PE orgnameUniversidade Federal Rural de Pernambuco orgdiv1Departamento de Medicina Veterinária orgdiv2Laboratório de Doenças Parasitárias Brasil
                Article
                S0100-736X2010000100006 S0100-736X(10)03000106
                7751d899-a426-4298-995a-9190a1f95e46

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

                History
                : 19 August 2009
                : 03 June 2009
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 37, Pages: 5
                Product

                SciELO Brazil


                Anaplasma marginale,antígenos,proteínas recombinantes,ELISA,antigens,recombinant proteins

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