Original article
Evaluation and comparison of serological methods for the detection of bovine neosporosis in Argentina
Evaluación y comparación de pruebas serológicas para la detección de la neosporosis bovina en Argentina
Lucía M. Camperoa,b,
, Lieselotte Minkec, Gastón Moréa,b, Magdalena Rambeauda,b, Diana Bacigalupea, Dadin P. Mooreb, Yanina Heckerb, Carlos M. Camperod, Gereon Scharesc, María C. Venturinia
Corresponding author
lcampero@fcv.unlp.edu.ar
Corresponding author: Universidad Nacional de La Plata, Facultad de Ciencias Veterinarias, Laboratorio de Inmunoparasitología. Calle 60 y 118 Zip code: 1900 La Plata, Argentina.
Corresponding author: Universidad Nacional de La Plata, Facultad de Ciencias Veterinarias, Laboratorio de Inmunoparasitología. Calle 60 y 118 Zip code: 1900 La Plata, Argentina.
a Laboratorio de Inmunoparasitología, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina
b Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
c Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Greifswald-Insel Riems, Germany
d Instituto Nacional de Tecnología Agropecuaria (INTA), Balcarce, Argentina
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The time to the most recent common ancestor (median and HPD95<span class="elsevierStyleHsp" style=""></span>%) and posterior probability values for relevant groups are shown at nodes.</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Héctor M. Marín, Carolina Torres, Gerardo D. Deluca, Viviana A. Mbayed" "autores" => array:4 [ 0 => array:2 [ "nombre" => "Héctor M." "apellidos" => "Marín" ] 1 => array:2 [ "nombre" => "Carolina" "apellidos" => "Torres" ] 2 => array:2 [ "nombre" => "Gerardo D." "apellidos" => "Deluca" ] 3 => array:2 [ "nombre" => "Viviana A." "apellidos" => "Mbayed" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115001194?idApp=UINPBA00004N" "url" => "/03257541/0000004700000004/v2_201602050024/S0325754115001194/v2_201602050024/en/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S0325754115000942" "issn" => "03257541" "doi" => "10.1016/j.ram.2015.06.007" "estado" => "S300" "fechaPublicacion" => "2015-10-01" "aid" => "53" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2015;47:282-94" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 1456 "formatos" => array:3 [ "EPUB" => 37 "HTML" => 889 "PDF" => 530 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>" "titulo" => "Genotyping and study of the <span class="elsevierStyleItalic">pauA</span> and <span class="elsevierStyleItalic">sua</span> genes of <span class="elsevierStyleItalic">Streptococcus uberis</span> isolates from bovine mastitis" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "282" "paginaFinal" => "294" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Genotipificación y estudio de los genes <span class="elsevierStyleItalic">pauA</span> y <span class="elsevierStyleItalic">sua</span> de aislamientos de <span class="elsevierStyleItalic">Streptococcus uberis</span> de mastitis bovina" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 2500 "Ancho" => 1487 "Tamanyo" => 434322 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Dendrogram of pulsed-field gel electrophoresis (PFGE) (A) and random amplified polymorphic DNA (RAPD) (B) profiles of 137 <span class="elsevierStyleItalic">Streptococcus uberis</span> subclinical mastitis isolates collected from 35 dairy herds. Isolate code and PFGE type (A) and RAPD type (B) of each strain are also represented in the dendrogram. The dendrogram was produced by using Dice coefficients and an unweighted pair group method using arithmetic averages (UPGMA).</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Melina S. Perrig, María B. Ambroggio, Fernanda R. Buzzola, Iván S. Marcipar, Luis F. Calvinho, Carolina M. Veaute, María Sol Barbagelata" "autores" => array:7 [ 0 => array:2 [ "nombre" => "Melina S." "apellidos" => "Perrig" ] 1 => array:2 [ "nombre" => "María B." "apellidos" => "Ambroggio" ] 2 => array:2 [ "nombre" => "Fernanda R." "apellidos" => "Buzzola" ] 3 => array:2 [ "nombre" => "Iván S." "apellidos" => "Marcipar" ] 4 => array:2 [ "nombre" => "Luis F." "apellidos" => "Calvinho" ] 5 => array:2 [ "nombre" => "Carolina M." "apellidos" => "Veaute" ] 6 => array:2 [ "nombre" => "María Sol" "apellidos" => "Barbagelata" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115000942?idApp=UINPBA00004N" "url" => "/03257541/0000004700000004/v2_201602050024/S0325754115000942/v2_201602050024/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>" "titulo" => "Evaluation and comparison of serological methods for the detection of bovine neosporosis in Argentina" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "295" "paginaFinal" => "301" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Lucía M. Campero, Lieselotte Minke, Gastón Moré, Magdalena Rambeaud, Diana Bacigalupe, Dadin P. Moore, Yanina Hecker, Carlos M. Campero, Gereon Schares, María C. Venturini" "autores" => array:10 [ 0 => array:4 [ "nombre" => "Lucía M." "apellidos" => "Campero" "email" => array:1 [ 0 => "lcampero@fcv.unlp.edu.ar" ] "referencia" => array:3 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] 2 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "Lieselotte" "apellidos" => "Minke" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">c</span>" "identificador" => "aff0015" ] ] ] 2 => array:3 [ "nombre" => "Gastón" "apellidos" => "Moré" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 3 => array:3 [ "nombre" => "Magdalena" "apellidos" => "Rambeaud" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 4 => array:3 [ "nombre" => "Diana" "apellidos" => "Bacigalupe" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 5 => array:3 [ "nombre" => "Dadin P." 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"apellidos" => "Venturini" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] ] "afiliaciones" => array:4 [ 0 => array:3 [ "entidad" => "Laboratorio de Inmunoparasitología, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Greifswald-Insel Riems, Germany" "etiqueta" => "c" "identificador" => "aff0015" ] 3 => array:3 [ "entidad" => "Instituto Nacional de Tecnología Agropecuaria (INTA), Balcarce, Argentina" "etiqueta" => "d" "identificador" => "aff0020" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author: Universidad Nacional de La Plata, Facultad de Ciencias Veterinarias, Laboratorio de Inmunoparasitología. Calle 60 y 118 Zip code: 1900 La Plata, Argentina." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Evaluación y comparación de pruebas serológicas para la detección de la neosporosis bovina en Argentina" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1254 "Ancho" => 1608 "Tamanyo" => 103743 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Time course of antibody response in EI heifers (mean) determined by IFAT and p38-ELISA. Each data point represents the mean value and error bars represent standard deviations of mean (SEM).</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Neospora caninum</span> is a protozoan parasite from the phylum Apicomplexa<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">7</span></a>, which is responsible for abortions in dairy and beef cattle worldwide<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">8</span></a>. Neosporosis causes important economic losses in the cattle industry, a situation that is of utmost importance in many South American countries. A recent study conducted in the Humid Pampa region of Argentina estimated annual economic losses of approximately US$ 44 million and US$ 13 million in the dairy and beef industry, respectively<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">14</span></a>, due to <span class="elsevierStyleItalic">N. caninum</span> abortions. The objective interpretation of data from different countries and laboratories depends on the reliability of serological diagnostic tests. Many serological techniques are available for <span class="elsevierStyleItalic">N. caninum</span> diagnosis in cattle. The indirect fluorescence antibody test (IFAT) is commonly used for this purpose, despite a possible subjective interpretation of results depending on the operator's experience. In addition, many commercial ELISA tests are available, which are more objective in terms of result interpretation<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">16</span></a>. However, in Argentina, commercial kits must be purchased abroad, implying high costs and long waiting periods. When considering the implementation of a new technique validated elsewhere, similar diagnostic characteristics should not be necessarily assumed, since local conditions, the target population and the epidemiological situation may vary from the original validation. Therefore, if the assay is to be applied in a different geographical region and/or population, revalidation of the assay under the new conditions is recommended, since sensitivity and specificity may vary within and among animal populations<a class="elsevierStyleCrossRefs" href="#bib0160"><span class="elsevierStyleSup">9,15,23</span></a>. Furthermore, cattle are presumably infected with diverse isolates of <span class="elsevierStyleItalic">N. caninum</span> and each region has a particular epidemiological situation.</p><p id="par0010" class="elsevierStylePara elsevierViewall">Schares <span class="elsevierStyleItalic">et al.</span><a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">17</span></a> evaluated an in-house ELISA based on the native p38 antigen of <span class="elsevierStyleItalic">N. caninum</span> having good diagnostic performance and different recommended cut-offs depending on the abortion pattern. In a multi-centered standardization study performed in Europe for several bovine neosporosis serological tests, a lower cut-off was recommended to obtain higher sensitivity values for p38-ELISA<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">21</span></a>. The p38-ELISA method is a low cost, practical and quick diagnostic assay; therefore its implementation for the diagnosis of bovine neosporosis in our country would be very beneficial. However, p38-ELISA has not been evaluated locally. In addition, to our knowledge, the diagnostic performance of available serological tests for bovine neosporosis in Argentina has not been thoroughly evaluated recently. Therefore, the aims of this study were: i) to evaluate the performance of p38-ELISA to diagnose bovine neosporosis in Argentina using a well-characterized local sera panel from experimentally infected (EI) and naturally exposed (NE) cattle and ii) to compare the diagnostic performance and agreement of three <span class="elsevierStyleItalic">N. caninum</span> serological tests: p38-ELISA, IFAT and immunoblotting (IB) using the same sera panel.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Serum samples</span><p id="par0015" class="elsevierStylePara elsevierViewall">All serum samples used in this work were kindly provided by the coauthors and belong to other research projects. Experimental sera (<span class="elsevierStyleItalic">n</span>= 36) provided by Dr. Hecker<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">11</span></a> came from 3 heifers intravenously inoculated with cell culture-derived tachyzoites from <span class="elsevierStyleItalic">N. caninum</span> NC-6 Argentina isolate<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">4</span></a>, and from 3 heifers subcutaneously inoculated with PBS. All 6 heifers were sampled at 0, 2, 3, 5, 9, 13 weeks post-inoculation (wpi), as described by Hecker <span class="elsevierStyleItalic">et al</span><a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">11</span></a><span class="elsevierStyleItalic">.</span> All animals used in the above study were handled in strict accordance with good animal practice and the conditions defined by the Ethical Committee of Animal Welfare (CICUAE) at INTA Balcarce.</p><p id="par0020" class="elsevierStylePara elsevierViewall">Serum samples from NE cattle, raised in dairy herds located in Santa Fe (<span class="elsevierStyleItalic">n</span>= 218) and beef herds located in Buenos Aires province (<span class="elsevierStyleItalic">n</span>= 44) were supplied by Dr. Campero CM. Additionally, sera from 37 mother-precolostrum calf pairs (<span class="elsevierStyleItalic">n</span>= 74), belonging to a dairy herd in Buenos Aires, were provided by Dr. Moré<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">12</span></a>.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">IFAT and immunoblot</span><p id="par0025" class="elsevierStylePara elsevierViewall">Detection of specific <span class="elsevierStyleItalic">N. caninum</span> antibodies was carried out by IFAT, as previously described<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">7</span></a>. A fluorescein isothiocyanate (FITC) labelled affinity-purified rabbit anti-bovine IgG antibody (Sigma-Aldrich, St. Louis, USA) conjugate was used. Reading was performed with an epifluorescence microscope (Leica). Unbroken fluorescence of the tachyzoite membrane and titer ≥1:25 was considered positive<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">13,17</span></a>.</p><p id="par0030" class="elsevierStylePara elsevierViewall">Detection of specific <span class="elsevierStyleItalic">N. caninum</span> antibodies was carried out by IB as described by Schares <span class="elsevierStyleItalic">et al</span><a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">18</span></a>. Briefly, 4<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">7</span> tachyzoites of NC-1 isolate were used for antigen preparation under non-reducing conditions, separated by electrophoresis in each SDS-polyacrylamide gel and electroblotted onto a PVDF membrane. Bovine serum samples were diluted 1:100. A conjugate anti-bovine IgG [H<span class="elsevierStyleHsp" style=""></span>+<span class="elsevierStyleHsp" style=""></span>L] peroxidase (1:1000) (Jackson Immunoresearch Lab. Inc., PA, USA) was used. The substrate solution consisted of 30<span class="elsevierStyleHsp" style=""></span>mg 4-chloro-1-naphthol (Sigma-Aldrich, St. Louis, USA), 10<span class="elsevierStyleHsp" style=""></span>ml methanol, 30<span class="elsevierStyleHsp" style=""></span>ml PBS and 40<span class="elsevierStyleHsp" style=""></span>μl 30<span class="elsevierStyleHsp" style=""></span>% H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2.</span> The reaction against five immunodominant antigens (IDAs) with relative molecular masses of 19, 29, 30, 33, 37<span class="elsevierStyleHsp" style=""></span>kDa was recorded. A sample was considered positive when two or more IDAs were recognized<a class="elsevierStyleCrossRefs" href="#bib0205"><span class="elsevierStyleSup">18,19</span></a>.</p><p id="par0035" class="elsevierStylePara elsevierViewall">Both techniques (IFAT and IB) used positive and negative control sera obtained from experimentally-infected and uninfected cows, respectively.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">p38-ELISA</span><p id="par0040" class="elsevierStylePara elsevierViewall">The 38<span class="elsevierStyleHsp" style=""></span>kDa antigen of <span class="elsevierStyleItalic">N. caninum</span> was obtained by affinity chromatography using monoclonal antibody 4.15.15 (IgG2a). Prior to affinity purification, 2<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">9</span> cell culture- derived tachyzoites from NC-1 isolate in 10<span class="elsevierStyleHsp" style=""></span>ml of PBS-0.5<span class="elsevierStyleHsp" style=""></span>% Triton X-100 were sonicated on ice for 90 s. The suspension was centrifuged at 13000<span class="elsevierStyleHsp" style=""></span>g for 30<span class="elsevierStyleHsp" style=""></span>min at 4<span class="elsevierStyleHsp" style=""></span>°C and the supernatant was used for p38 affinity purification. The indirect ELISA was performed as described by Schares <span class="elsevierStyleItalic">et al</span><a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">17</span></a>. Briefly, the antigen (1.5<span class="elsevierStyleHsp" style=""></span>ng/well) was diluted in coating buffer (0.1<span class="elsevierStyleHsp" style=""></span>M sodium bicarbonate, pH 8.3) and used to sensitize ELISA plates (Nunc-Immuno-Polysorb). A blocking solution with PBS-0.05<span class="elsevierStyleHsp" style=""></span>% Tween 20-20<span class="elsevierStyleHsp" style=""></span>% horse serum was applied; and the plate was incubated for 30<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>°C. A serum dilution of 1:200 was used and samples were analyzed in duplicate. A biotinylated monoclonal anti-bovine IgG (Sigma-Aldrich, St. Louis, USA) diluted 1:2000 and an extravidin-peroxidase conjugate (Sigma-Aldrich, St. Louis, USA) diluted 1:4000 were used. Antibodies were detected by incubation with substrate solution containing 100<span class="elsevierStyleHsp" style=""></span>μg/ml 3,3′,5,5′-tetramethylbenzidine and 0.004<span class="elsevierStyleHsp" style=""></span>% hydrogen peroxide in 0.2<span class="elsevierStyleHsp" style=""></span>M sodium acetate and 0.2<span class="elsevierStyleHsp" style=""></span>M citric acid for 15<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>°C. Optical density (OD) values were measured at 492<span class="elsevierStyleHsp" style=""></span>nm on a microplate reader (Labsystems MS Multiskan). Sample index values were recorded as the arithmetic mean of two index values, SI<span class="elsevierStyleInf">1</span> and SI<span class="elsevierStyleInf">2</span>. These index values were calculated by the formula SI<span class="elsevierStyleInf"><span class="elsevierStyleItalic">n</span></span>= (S<span class="elsevierStyleInf"><span class="elsevierStyleItalic">n</span></span>-<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">N</span>)/(<span class="elsevierStyleItalic">P</span>-<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">N</span>) where SI<span class="elsevierStyleInf"><span class="elsevierStyleItalic">n</span></span> is one of the two individual index values, and S<span class="elsevierStyleInf"><span class="elsevierStyleItalic">n</span></span> one of the two individual OD values obtained for a single sample. <span class="elsevierStyleItalic">N</span> is the arithmetic mean of two OD values obtained for the negative serum, and <span class="elsevierStyleItalic">P</span> represents the arithmetic mean of two OD values obtained for the positive serum. Monoclonal antibody 4.15.15 (IgG2a) and positive and negative control sera were kindly provided by Dr. Schares. The optimal cut-off index value for maximal sensitivity and specificity for p38-ELISA was determined by receiver operating characteristic (ROC) analysis considering a 95<span class="elsevierStyleHsp" style=""></span>% confidence interval relative to Relative Standards of Comparison (RSC).</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Statistical analysis</span><p id="par0045" class="elsevierStylePara elsevierViewall">Serum samples testing either positive or negative by both IFAT and IB were considered as RSC for p38-ELISA evaluation<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">2</span></a>. ROC analysis (Medcalc 13.0) was performed to determine the optimal cut-off value and relative sensitivity and specificity considering a 95<span class="elsevierStyleHsp" style=""></span>% confidence interval for the p38-ELISA relative to RSC. According to an arbitrary guideline by ROC analysis, the area under the curve (AUC) was evaluated as: non-informative (AUC= 0.5), less accurate (0.5< AUC≤ 0.7), moderately accurate (0.7< AUC≤ 0.9), highly accurate (0.9<span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>AUC≤1) and perfect tests (AUC= 1)<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">20</span></a>.</p><p id="par0050" class="elsevierStylePara elsevierViewall">The diagnostic characteristics of the tests were calculated according to the result of the “majority of tests” (Majority) since there is no gold standard test available for bovine neosporosis diagnosis<a class="elsevierStyleCrossRefs" href="#bib0120"><span class="elsevierStyleSup">1,21</span></a>.</p><p id="par0055" class="elsevierStylePara elsevierViewall">Agreement between serological tests was evaluated using Epidat 3.1 software (<span class="elsevierStyleItalic">Organización Panamericana de la Salud y Xunta de Galicia, Consellería de Sanidade</span>). Kappa values (<span class="elsevierStyleItalic">k</span>) were considered as follows: poor agreement (<span class="elsevierStyleItalic">k</span>= 0.00), slight agreement (<span class="elsevierStyleItalic">k</span>= 0.00–0.20), fair agreement (<span class="elsevierStyleItalic">k</span>= 0.21–0.40), moderate agreement (<span class="elsevierStyleItalic">k</span>= 0.41–0.60); substantial agreement (<span class="elsevierStyleItalic">k</span>= 0.61–0.80); almost perfect agreement (<span class="elsevierStyleItalic">k</span>> 0.81)<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">6</span></a>.</p></span></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Results</span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Experimentally-infected heifers</span><p id="par0060" class="elsevierStylePara elsevierViewall">The three EI heifers seroconverted at 2 wpi, showing an average titer of 1:400 determined by IFAT. An increase in the titer was observed in the third wpi that was maintained through 9 wpi and decreased at 13 wpi (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). The non-infected control group showed no positive IFAT reactions on all sampling dates.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0065" class="elsevierStylePara elsevierViewall">Testing of sera from EI heifers by p38-ELISA detected a peak at 3 wpi; however, 2 wpi index values were below the cut-off (= 0.0905, determined by the ROC analysis). Additionally, a slight decrease was observed from 3 to 5 wpi (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). The non-infected control group showed no positive ELISA reactions at all sampling dates.</p><p id="par0070" class="elsevierStylePara elsevierViewall">Immunoblot analysis of sera from the 3 EI heifers evidenced a positive reaction to the 5 IDAs at 2, 3, 5, 9, and 13 wpi (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>A). Sera collected at 0 wpi as well as sera from the non-infected control group showed no positive reactions by IB.</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065"><span class="elsevierStyleItalic">Neospora caninum</span> IDA recognition by NE cattle</span><p id="par0075" class="elsevierStylePara elsevierViewall">Based on the frequency and intensity of recognition by sera from NE cattle, the 37<span class="elsevierStyleHsp" style=""></span>kDa antigen and a 29<span class="elsevierStyleHsp" style=""></span>kDa antigen were detected by 100<span class="elsevierStyleHsp" style=""></span>% and 98<span class="elsevierStyleHsp" style=""></span>% of seropositive animals, respectively. The 30 and 33<span class="elsevierStyleHsp" style=""></span>kDa antigens were recognized with higher frequency and intensity in samples with IFAT titer ≥1:400 and were detected by 78<span class="elsevierStyleHsp" style=""></span>% and 68<span class="elsevierStyleHsp" style=""></span>% of seropositive animals, respectively. The 19<span class="elsevierStyleHsp" style=""></span>kDa protein was recognized in 70<span class="elsevierStyleHsp" style=""></span>% of sera from seropositive animals, mostly with IFAT titer ≥ 1:100. Finally, a protein ∼26<span class="elsevierStyleHsp" style=""></span>kDa was only detected by 13<span class="elsevierStyleHsp" style=""></span>% of samples, having IFAT titer ≥ 1:3200. When EI sera were analyzed by IB, similar immunoblotting patterns were observed compared to NE cattle (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>A). A clear relationship between an increasing IFAT titer and a more intense and diverse IDA recognition was observed (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>B) among NE cattle.</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Techniques performance comparison</span><p id="par0080" class="elsevierStylePara elsevierViewall">Antibodies to <span class="elsevierStyleItalic">N. caninum</span> were detected in 31<span class="elsevierStyleHsp" style=""></span>% (104/336), 28<span class="elsevierStyleHsp" style=""></span>% (95/336), and 27<span class="elsevierStyleHsp" style=""></span>% (93/336) of samples from NE cattle by IFAT, IB and p38-ELISA, respectively. RSC consisted of 94 sera that tested positive and 231 sera that tested negative by both IFAT and IB (<span class="elsevierStyleItalic">n</span>= 325) and were used for the evaluation of p38-ELISA. Ninety-two out of 94 positive RSC sera tested positive by p38-ELISA and 230 of 231 negative RSC sera tested negative by p38-ELISA (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). The negative sample by RSC that tested positive by p38-ELISA, did not show any reaction to the 38<span class="elsevierStyleHsp" style=""></span>kDa IDAs in IB. Agreement between RSC and p38-ELISA was almost perfect (<span class="elsevierStyleItalic">k</span>= 0.97). Relative sensitivity and specificity with a cut-off index value of 0.0905 were 97.8<span class="elsevierStyleHsp" style=""></span>% and 99.5<span class="elsevierStyleHsp" style=""></span>%, respectively. ROC analysis revealed that p38-ELISA was highly accurate (AUC= 0.982) relative to the RSC (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>).</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0085" class="elsevierStylePara elsevierViewall">Sensitivity and specificity values were calculated for each test in relation to the Majority criterion (<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>). The Agreement of each test with respect to the Majority criterion, as well as agreement between tests, was almost perfect (<a class="elsevierStyleCrossRefs" href="#tbl0010">Tables 2 and 3</a>).</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><elsevierMultimedia ident="tbl0015"></elsevierMultimedia></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Discussion</span><p id="par0090" class="elsevierStylePara elsevierViewall">Serological tests are the most suitable tool to evaluate <span class="elsevierStyleItalic">N. caninum</span> infection in animals, and contribute important information to control measures. Despite their importance, there is scarce information regarding the evaluation and comparison of serological diagnostic tests using a well-characterized sera panel. Such studies are highly recommended prior to considering a certain diagnostic technique as a routine diagnostic tool<a class="elsevierStyleCrossRefs" href="#bib0120"><span class="elsevierStyleSup">1,10</span></a>. Even though IFAT is considered subjective, time-consuming and limited for large-scale investigations, it is still the most frequently used test for neosporosis detection in many South American countries. In addition, different cut-off values are commonly used, making comparisons more difficult<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">22</span></a>. The present study aimed to evaluate an in-house ELISA using local sera and to compare the diagnostic performance of serological tests available in Argentina for the detection of antibodies to <span class="elsevierStyleItalic">N. caninum</span>. Since a true gold standard is not available for bovine neosporosis, RSC are necessary when evaluating new techniques and ideally include results from more than one technique<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">2</span></a>.</p><p id="par0095" class="elsevierStylePara elsevierViewall">Evaluation of EI sera with p38-ELISA detected a peak of antibodies at 3 wpi, however, at 2 wpi, index values were below the cut-off. Nonetheless, IFAT titers at 2 wpi were 1:400. Potentially the cut-off value applied in the ELISA could have underestimated low avidity IgG produced in early or recent infections<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">5</span></a>. A lower cut-off value was evaluated to detect seropositive animals at 2 wpi; however, under such conditions, the specificity of the test was considerably lower in the ROC analysis (data not shown). A slight decrease in the antibody response was detected by IFAT between 9 and 13 wpi, but not by the p38-ELISA.</p><p id="par0100" class="elsevierStylePara elsevierViewall">We also determined the immunoreactivity of sera by IB. Cattle are presumably infected with a diverse population of <span class="elsevierStyleItalic">N. caninum</span> isolates, with potential immunogenic differences. In our study, though, the IB banding pattern found for EI and NE sera was similar. This fact is highly relevant for the selection of target antigens for diagnostic purposes, especially for the development and validation of serological tests in different geographical regions where cattle could be exposed to different <span class="elsevierStyleItalic">N. caninum</span> isolates. The reaction to the 38<span class="elsevierStyleHsp" style=""></span>kDa IDA in 100<span class="elsevierStyleHsp" style=""></span>% of positive serum samples is in agreement with the high relative sensitivity and specificity obtained in this work for the p38-ELISA method. Additionally, a ∼26<span class="elsevierStyleHsp" style=""></span>kDa antigen was only found in samples having IFAT titers ≥1:3200. Immunoreaction to this antigen could be indicative of high serological titers developed in different situations: cows with recrudescence of infection, cows that aborted as a result of <span class="elsevierStyleItalic">N. caninum</span> infection, and cows or calves evidencing vertical transmission<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">3</span></a>. Further studies should be performed in order to clarify the role of this antigen in the outcome of the disease.</p><p id="par0105" class="elsevierStylePara elsevierViewall">Ideally, laboratories should perform internal evaluation and validation studies to analyze the diagnostic performance of serological tests with local sera and everyday routine conditions. Using the decision of the “majority of tests” as gold standard, high sensitivity, specificity and agreement values were demonstrated for IFAT, IB and p38-ELISA. Based on our results, IFAT was very sensitive and was able to detect early stages of infection in EI heifers. However, a greater number of samples should be analyzed in order to determine the capacity of IFAT to detect recent infections, since only 3 EI animals were analyzed in our study. In addition, IFAT can be a useful tool to study serological profiles in infected cattle. Moreover, p38-ELISA displayed accurate diagnostic performance and is a practical and quick diagnostic tool for large-scale studies. In our country, commercial ELISAs are purchased abroad, implying high costs and delay. Therefore, a local in-house ELISA would be a low-cost and time-saving alternative.</p><p id="par0110" class="elsevierStylePara elsevierViewall">The IB method can be used as a confirmatory technique for samples yielding inconclusive serological results from other tests<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">8</span></a>. It allows an unequivocal serological diagnosis with the highest sensitivity and specificity values among the tests analyzed, as shown in this work. However, only a few reference laboratories are performing this technique, since it is laborious, time-consuming and requires specialized equipment and training.</p><p id="par0115" class="elsevierStylePara elsevierViewall">In conclusion, this study describes the accurate performance of p38-ELISA and the almost perfect diagnostic performance of the studied tests for the detection of anti-<span class="elsevierStyleItalic">N. caninum</span> antibodies, evaluated with a well-characterized sera panel of cattle from Argentina. The selection of the technique and cut-off applied will depend on the purpose of diagnosis as well as on feasibility in each laboratory. Results from the present work emphasize the importance of comparative studies and provide useful information for future serological diagnostic standardization programs among different laboratories in South America.</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Funding</span><p id="par0120" class="elsevierStylePara elsevierViewall">El presente trabajo fue financiado con fondos del PIP 2012-2014 N° 112-20110100488 otorgado por CONICET y recursos del Laboratorio de Inmunoparasitología, FCV, UNLP.</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Ethical disclosures</span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Protection of human and animal subjects</span><p id="par0125" class="elsevierStylePara elsevierViewall">The authors declare that the procedures followed were in accordance with the conditions defined by the Code of Ethics of the World Medical Association (Declaration of Helsinki), and approved by the Ethical Committee of Animal Welfare (CICUAE) of INTA Balcarce, Argentina.</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Confidentiality of data</span><p id="par0130" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article.</p></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Right to privacy and informed consent</span><p id="par0135" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article.</p></span></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Conflict of interest</span><p id="par0140" class="elsevierStylePara elsevierViewall">The authors state that they have no conflicts of interest to declare.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:13 [ 0 => array:3 [ "identificador" => "xres602665" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec616715" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres602664" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec616716" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Serum samples" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "IFAT and immunoblot" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "p38-ELISA" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Statistical analysis" ] ] ] 6 => array:3 [ "identificador" => "sec0035" "titulo" => "Results" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0040" "titulo" => "Experimentally-infected heifers" ] 1 => array:2 [ "identificador" => "sec0045" "titulo" => "Neospora caninum IDA recognition by NE cattle" ] 2 => array:2 [ "identificador" => "sec0050" "titulo" => "Techniques performance comparison" ] ] ] 7 => array:2 [ "identificador" => "sec0055" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0060" "titulo" => "Funding" ] 9 => array:3 [ "identificador" => "sec0065" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0070" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0075" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0080" "titulo" => "Right to privacy and informed consent" ] ] ] 10 => array:2 [ "identificador" => "sec0085" "titulo" => "Conflict of interest" ] 11 => array:2 [ "identificador" => "xack202825" "titulo" => "Acknowledgments" ] 12 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2015-04-04" "fechaAceptado" => "2015-07-21" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec616715" "palabras" => array:6 [ 0 => "Bovine neosporosis" 1 => "Argentina" 2 => "p38-ELISA" 3 => "Indirect fluorescence antibody test" 4 => "Immunoblotting" 5 => "Diagnostic performance" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec616716" "palabras" => array:6 [ 0 => "Neosporosis bovina" 1 => "Argentina" 2 => "ELISA-p38" 3 => "Inmunofluorescencia indirecta" 4 => "Inmunoblot" 5 => "<span class="elsevierStyleItalic">Performance</span> diagnóstica" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Neospora caninum</span> is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. The accurate diagnosis of neosporosis is essential for management and control measures. The aims of this study were: i) to evaluate the performance of an in-house enzyme-linked immunosorbent assay based on the 38<span class="elsevierStyleHsp" style=""></span>kDa native antigen (p38-ELISA) to diagnose bovine neosporosis in Argentina using a well- characterized local sera panel from experimentally infected and naturally exposed cattle and ii) to compare the diagnostic performance and agreement of three <span class="elsevierStyleItalic">N. caninum</span> serological tests: p38-ELISA, indirect fluorescence antibody test (IFAT) and immunoblotting (IB) using the same sera panel. Serum samples testing either positive or negative by IFAT and IB were considered “Relative Standards of Comparison” (RSC) and used for p38-ELISA evaluation. Receiver operating characteristics analysis revealed that p38-ELISA was highly accurate (area under the curve= 0.982) according to RSC with a cut-off index of 0.0905. Relative sensitivity and specificity of p38-ELISA were 97.8<span class="elsevierStyleHsp" style=""></span>% and 99.5<span class="elsevierStyleHsp" style=""></span>%, respectively and agreement between RSC and p38-ELISA was almost perfect (k= 0.97). The evaluation and performance comparison of serological tests were performed according to the definition of gold standard based on the decision of the “majority of tests”. All tests displayed high sensitivity and specificity values (greater than 95<span class="elsevierStyleHsp" style=""></span>%); and excellent agreement. This study describes the accurate performance of p38-ELISA evaluated locally and the highly accurate diagnostic performance of the studied tests for the detection of anti-<span class="elsevierStyleItalic">N. caninum</span> antibodies in cattle from Argentina.</p></span>" ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Neospora caninum</span> es un parásito protozoo responsable de abortos y pérdidas económicas en bovinos. La realización de un diagnóstico serológico preciso y con resultados comparables obtenidos por diferentes pruebas contribuye al manejo de este problema y a encarar medidas de control. Los objetivos del presente trabajo fueron los siguientes: 1) evaluar en Argentina una prueba de enzimoinmunoensayo <span class="elsevierStyleItalic">in-house</span> con el antígeno nativo de 38<span class="elsevierStyleHsp" style=""></span>kDa de <span class="elsevierStyleItalic">N. caninum</span> (ELISA-p38) para el diagnóstico de la neosporosis bovina, utilizando un panel de sueros locales bien caracterizados, procedentes de bovinos infectados de modo experimental o naturalmente expuestos; 2) comparar el desempeño y establecer el nivel de concordancia de tres pruebas serológicas para la detección de <span class="elsevierStyleItalic">N. caninum</span>, ELISA-p38, inmunofluorescencia indirecta (IFI) e inmunoblot (IB), con el mismo panel de sueros. Los sueros que resultaron positivos o negativos a IFI e IB fueron considerados como estándares relativos de comparación (ERC) para evaluar la prueba de ELISA-p38. El análisis de característica operativa del receptor determinó que la prueba de ELISA-p38 fue altamente precisa (área bajo la curva= 0,982) usando el punto de corte 0,0905. La sensibilidad y especificidad relativa del ELISA-p38 fue 97,8<span class="elsevierStyleHsp" style=""></span>% y 99,5<span class="elsevierStyleHsp" style=""></span>%, respectivamente, con una concordancia casi perfecta (k= 0,97) respecto del ERC. La comparación del desempeño de las pruebas se realizó usando como <span class="elsevierStyleItalic">gold standard</span> el criterio de la decisión de la “mayoría de las pruebas”. Las pruebas exhibieron altos valores de sensibilidad y especificidad (mayores del 95<span class="elsevierStyleHsp" style=""></span>%) y excelente concordancia. Este trabajo describe un buen desempeño de la prueba de ELISA-p38 evaluada localmente y adecuada <span class="elsevierStyleItalic">performance</span> diagnóstica de las pruebas serológicas analizadas para la detección de anticuerpos anti <span class="elsevierStyleItalic">N. caninum</span> en bovinos de Argentina.</p></span>" ] ] "multimedia" => array:6 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1254 "Ancho" => 1608 "Tamanyo" => 103743 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Time course of antibody response in EI heifers (mean) determined by IFAT and p38-ELISA. Each data point represents the mean value and error bars represent standard deviations of mean (SEM).</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1547 "Ancho" => 2303 "Tamanyo" => 237238 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Representative pattern of <span class="elsevierStyleItalic">N. caninum</span> immunodominant antigen recognition in <span class="elsevierStyleBold">A)</span> naturally and experimentally infected cattle and <span class="elsevierStyleBold">B)</span> naturally exposed cattle at different IFAT titers. MW= molecular weight pattern; a= sera from a naturally infected cow; b= sera from an experimentally infected heifer; (–)= seronegative cow; IFAT titers are expressed as the reciprocal of the highest serum dilution showing positive fluorescence.</p>" ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1547 "Ancho" => 1484 "Tamanyo" => 89932 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Receiver operating characteristic (ROC) PLOT of p38- ELISA using relative standard of comparison sera. Optimal cut-off index value= 0.0905. Area under the curve= 0.982; 95<span class="elsevierStyleHsp" style=""></span>% confidence interval between 0.96 and 0.99.</p>" ] ] 3 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="3" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">p38-ELISA</th></tr><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Positive \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Negative \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Total \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry " colspan="4" align="left" valign="top"><span class="elsevierStyleItalic">RSC</span></td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>Positive \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">92 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">94 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>Negative \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">230 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">231 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleHsp" style=""></span>Total \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">93 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">232 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">325 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab986744.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">p38-ELISA results in relation to the relative standards of comparison (RSC)</p>" ] ] 4 => array:7 [ "identificador" => "tbl0010" "etiqueta" => "Table 2" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td-with-role" title="table-head ; entry_with_role_rowhead " align="left" valign="top" scope="col">Tests \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="3" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Majority of tests</th></tr><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Sensitivity, 95<span class="elsevierStyleHsp" style=""></span>% (CI) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Specificity, 95<span class="elsevierStyleHsp" style=""></span>%(CI) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">k</span>-values, 95<span class="elsevierStyleHsp" style=""></span>%(CI) \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">IFAT \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">100 (99.47–100) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">95.87 (93.15–98.58) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.93 (0.88–0.97) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">IB \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">100 (99.47–100) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">99.59 (98.57–100) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.99 (0.98–1) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">p38-ELISA \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">97.87 (94.42–100) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">99.59 (98.57–100) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.98 (0.95–1) \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab986745.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Sensitivity, specificity and agreement (<span class="elsevierStyleItalic">k</span>-values) relative to the “majority of tests” decision</p>" ] ] 5 => array:7 [ "identificador" => "tbl0015" "etiqueta" => "Table 3" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td-with-role" title="table-head ; entry_with_role_rowhead " align="left" valign="top" scope="col">Tests \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="3" align="center" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">k</span>-values (95<span class="elsevierStyleHsp" style=""></span>% CI)</th></tr><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">IFAT(≥1:25<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">IB(≥ 2 IDAs<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>) \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">p38-ELISA (0.0905<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>) \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">IFAT \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.92 (0.88–0.97) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.91 (0.86–0.96) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">IB \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.97 (0.94–1) \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab986746.png" ] ] ] "notaPie" => array:1 [ 0 => array:3 [ "identificador" => "tblfn0005" "etiqueta" => "a" "nota" => "<p class="elsevierStyleNotepara" id="npar0005">Cut-offs used.</p>" ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Agreement (<span class="elsevierStyleItalic">k</span>-values) among different serological tests.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:23 [ 0 => array:3 [ "identificador" => "bib0120" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Serological diagnosis of bovine neosporosis: A comparative study of commercially available ELISA tests" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "G. 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Ercoli and S. Peñaloza for their excellent technical assistance. Financial support for this study was provided by CONICET, through PIP 2012-2014 N° 112-20110100488 and by resources from Laboratorio de Inmunoparasitología, FCV, UNLP.</p>" "vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/03257541/0000004700000004/v2_201602050024/S0325754115000929/v2_201602050024/en/main.assets" "Apartado" => array:4 [ "identificador" => "37861" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Microbiología clínica y enfermedades infecciosas" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/03257541/0000004700000004/v2_201602050024/S0325754115000929/v2_201602050024/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115000929?idApp=UINPBA00004N" ]
| Year/Month | Html | Total | |
|---|---|---|---|
| 2024 November | 4 | 0 | 4 |
| 2024 October | 22 | 6 | 28 |
| 2024 September | 58 | 2 | 60 |
| 2024 August | 36 | 11 | 47 |
| 2024 July | 28 | 4 | 32 |
| 2024 June | 24 | 5 | 29 |
| 2024 May | 22 | 2 | 24 |
| 2024 April | 15 | 4 | 19 |
| 2024 March | 37 | 5 | 42 |
| 2024 February | 29 | 8 | 37 |
| 2024 January | 28 | 2 | 30 |
| 2023 December | 27 | 11 | 38 |
| 2023 November | 27 | 6 | 33 |
| 2023 October | 43 | 17 | 60 |
| 2023 September | 12 | 0 | 12 |
| 2023 August | 22 | 5 | 27 |
| 2023 July | 13 | 5 | 18 |
| 2023 June | 21 | 4 | 25 |
| 2023 May | 43 | 12 | 55 |
| 2023 April | 34 | 4 | 38 |
| 2023 March | 25 | 3 | 28 |
| 2023 February | 15 | 0 | 15 |
| 2023 January | 12 | 1 | 13 |
| 2022 December | 31 | 9 | 40 |
| 2022 November | 31 | 5 | 36 |
| 2022 October | 21 | 13 | 34 |
| 2022 September | 15 | 15 | 30 |
| 2022 August | 22 | 10 | 32 |
| 2022 July | 20 | 7 | 27 |
| 2022 June | 10 | 6 | 16 |
| 2022 May | 20 | 9 | 29 |
| 2022 April | 28 | 12 | 40 |
| 2022 March | 36 | 11 | 47 |
| 2022 February | 59 | 6 | 65 |
| 2022 January | 59 | 6 | 65 |
| 2021 December | 41 | 10 | 51 |
| 2021 November | 59 | 13 | 72 |
| 2021 October | 79 | 9 | 88 |
| 2021 September | 54 | 11 | 65 |
| 2021 August | 71 | 13 | 84 |
| 2021 July | 41 | 9 | 50 |
| 2021 June | 51 | 8 | 59 |
| 2021 May | 31 | 11 | 42 |
| 2021 April | 39 | 12 | 51 |
| 2021 March | 19 | 5 | 24 |
| 2021 February | 19 | 7 | 26 |
| 2021 January | 16 | 12 | 28 |
| 2020 December | 22 | 7 | 29 |
| 2020 November | 27 | 12 | 39 |
| 2020 October | 18 | 9 | 27 |
| 2020 September | 18 | 12 | 30 |
| 2020 August | 46 | 15 | 61 |
| 2020 July | 17 | 11 | 28 |
| 2020 June | 32 | 10 | 42 |
| 2020 May | 22 | 8 | 30 |
| 2020 April | 28 | 6 | 34 |
| 2020 March | 32 | 7 | 39 |
| 2020 February | 32 | 6 | 38 |
| 2020 January | 29 | 7 | 36 |
| 2019 December | 33 | 8 | 41 |
| 2019 November | 39 | 6 | 45 |
| 2019 October | 44 | 9 | 53 |
| 2019 September | 42 | 6 | 48 |
| 2019 August | 18 | 5 | 23 |
| 2019 July | 20 | 10 | 30 |
| 2019 June | 37 | 17 | 54 |
| 2019 May | 74 | 9 | 83 |
| 2019 April | 46 | 25 | 71 |
| 2019 March | 15 | 4 | 19 |
| 2019 February | 27 | 8 | 35 |
| 2019 January | 9 | 10 | 19 |
| 2018 December | 17 | 19 | 36 |
| 2018 November | 15 | 7 | 22 |
| 2018 October | 17 | 10 | 27 |
| 2018 September | 27 | 7 | 34 |
| 2018 August | 10 | 2 | 12 |
| 2018 July | 9 | 8 | 17 |
| 2018 June | 7 | 1 | 8 |
| 2018 May | 5 | 7 | 12 |
| 2018 April | 6 | 1 | 7 |
| 2018 March | 2 | 1 | 3 |
| 2018 February | 4 | 3 | 7 |
| 2018 January | 8 | 0 | 8 |
| 2017 December | 4 | 3 | 7 |
| 2017 November | 5 | 0 | 5 |
| 2017 October | 8 | 5 | 13 |
| 2017 September | 8 | 17 | 25 |
| 2017 August | 16 | 9 | 25 |
| 2017 July | 10 | 5 | 15 |
| 2017 June | 19 | 2 | 21 |
| 2017 May | 21 | 10 | 31 |
| 2017 April | 15 | 16 | 31 |
| 2017 March | 20 | 53 | 73 |
| 2017 February | 14 | 1 | 15 |
| 2017 January | 16 | 2 | 18 |
| 2016 December | 25 | 9 | 34 |
| 2016 November | 17 | 7 | 24 |
| 2016 October | 34 | 13 | 47 |
| 2016 September | 15 | 9 | 24 |
| 2016 August | 24 | 9 | 33 |
| 2016 July | 37 | 15 | 52 |
| 2016 June | 32 | 19 | 51 |
| 2016 May | 23 | 18 | 41 |
| 2016 April | 29 | 38 | 67 |
| 2016 March | 41 | 28 | 69 |
| 2016 February | 33 | 29 | 62 |
| 2016 January | 29 | 28 | 57 |
| 2015 December | 8 | 5 | 13 |
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