A novel modified-indirect ELISA based on spherical body protein 4 for detecting antibody during acute and long-term infections with diverse Babesia bovis strains.
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ABSTRACT: Cattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission. Since B. bovis is considered a persistent infection, developing a reliable, high-throughput assay that detects antibody during all stages of the infection could be pivotal for establishing better control protocols.A modified indirect enzyme-linked immunosorbent assay (MI-ELISA) was developed using the spherical body protein-4 (SBP4) of B. bovis to detect antibody against diverse strains through all infection stages in cattle. This SBP4 MI-ELISA was evaluated for sensitivity and specificity against field sera from regions with endemic and non-endemic B. bovis. Sera were also evaluated from cattle infected experimentally with various doses and strains during acute and persistent infection with parasitemia defined by nested PCR.The format variables for SBP4 MI-ELISA were optimized and the cutoff for positive and negative interpretation was determined based on receiver operating characteristic curve analysis using B. bovis positive and negative sera tested in the reference immunofluorescence assay (IFA). The diagnostic specificity of the SBP4 MI-ELISA using IFA-negative sera collected from Texas was 100%, significantly higher than the cELISA (90.4%) based on an epitope in the rhoptry-associated protein-1 (RAP-1 cELISA). The diagnostic sensitivity of the SBP4 MI-ELISA was 98.7% using the IFA-positive sera collected from several areas of Mexico, in contrast to that of the RAP-1 cELISA at 60% using these same sera. In cattle infected with low and high doses of three B. bovis strains, the SBP4 MI-ELISA remained antibody positive for 11 months or more after initial detection at 10 to 13 days post-inoculation. However, the RAP-1 cELISA did not reliably detect antibody after eight months post-inoculation despite the fact that parasitemia was occasionally detectable by PCR. Furthermore, initial antibody detection by RAP-1 cELISA in low-dose infected animals was delayed approximately nine and a half days compared to the SBP4 MI-ELISA.These results demonstrate excellent diagnostic sensitivity and specificity of the novel SBP4 MI-ELISA for cattle with acute and long-term carrier infections. It is posited that use of this assay in countries that have B. bovis-endemic herds may be pivotal in preventing the spread of this disease to non-endemic herds.
A novel modified-indirect ELISA based on spherical body protein 4 for detecting antibody during acute and long-term infections with diverse Babesia bovis strains.
<h4>Background</h4>Cattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission. Since B. bovis is considered a persistent infection, developing a reliable, high-throughput assay that detects antibody during all stages of the infection could be pivotal for establishing better control protocols.<h4>Methods</h4>A modified indirect enzyme-linked immunosorbent assay (MI-ELISA) was developed using the spherical body protein-4 (SBP4) of B. bovis to detect antib ...[more]
Project description:The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.
Project description:Babesia bovis, a tick-borne intraerythrocytic protozoan parasite that belongs to the phylum Apicomplexa, is one of the etiological agents of bovine babesiosis, a highly prevalent disease in tropical and subtropical countries that causes significant morbidity and deaths in cattle. This report presents the draft genome sequences of attenuated and virulent B. bovis strains of Mexican origin.
Project description:Porcine deltacoronavirus (PDCoV) is a novel coronavirus that can cause vomiting and watery diarrhea in pigs and death in piglets. Since PDCoV was first detected in 2009 in Hong Kong, the prevalence of PDCoV has increased in recent years, resulting in serious economic losses to the swine industry. The coronavirus spike (S) protein is an antigen that has been demonstrated to contain epitopes that induce neutralizing antibodies. The presence of serum and milk IgA antibodies against pathogens that replicate primarily on mucosal surfaces is important for mucosal immunity. Here, an indirect anti-PDCoV IgA antibody enzyme-linked immunosorbent assay (PDCoV S1 IgA ELISA) using the purified S1 portion of S protein as the coating antigen was developed to detect PDCoV IgA antibodies in serum and sow's milk. A receiver operating characteristic (ROC) curve analysis showed high specificity and sensitivity of the PDCoV-S1-IgA-ELISA based on samples confirmed by IFA. Anti-PDCoV IgA antibodies in 152 serum samples and 65 milk samples collected from six farms that had experienced diarrhea outbreaks within previous last two years were detected by this assay, and 62.5% of the serum samples and 100% of the milk samples were positive for PDCoV. The indirect ELISA method established in this study will provide a convenient tool for measurement of serum and milk IgA levels against PDCoV in pig herds, rapid detection of PDCoV infection in pigs, and evaluation of the immunogenicity of vaccines.
Project description:BackgroundSalmonella enterica serovar Pullorum is a host-restricted serotype causing infection in poultry. The pathogen can not only cause acute infection in young chicks with high mortality and morbidity, but also persist in adult chickens without evident clinical symptoms and lead to vertical transmission. To eradicate S. Pullorum in poultry farms, it is necessary to establish an efficient method to monitor the prevalence of the pathogen in adult chickens. The protein IpaJ is a specific immunogen in S. Pullorum and is not detected in closely related serotypes, such as S. Gallinarum and S. Enteritidis.ResultsIn the present study, IpaJ was expressed as a recombinant fusion protein MBP-IpaJ in E. coli. The purified MBP-IpaJ was used as a coating antigen to develop an indirect ELISA assay, which was applied to the detection of S. Pullorum infection in chickens. The indirect ELISA assay demonstrated that antibodies produced against IpaJ were detectable in antisera of chickens infected with S. Pullorum in the second week, stably increased until the tenth week, and persisted at a high level in the following two weeks. Furthermore, the ELISA method detected four positive samples out of 200 clinical antiserum samples collected from a poultry farm, and the positive samples were confirmed to be reacted with S. Pullorum using the standard plate agglutination test.ConclusionsThe established indirect ELISA using the IpaJ protein is a novel method for specific detection of S. Pullorum infection, and contribute to eradication of pullorum disease in the poultry industry.
Project description:Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.
Project description:African swine fever (ASF) is a disease that causes severe economic losses to the global porcine industry. As no vaccine or drug has been discovered for the prevention and control of ASF virus (ASFV), accurate diagnosis and timely eradication of infected animals are the primary measures, which necessitate accurate and effective detection methods. In this study, the truncated ASFV I329L (amino acids 70-237), was induced using IPTG and expressed in Escherichia coli cells. The highly antigenic viral protein I329L was used to develop an indirect enzyme-linked immunosorbent assay (iELISA), named I329L-ELISA, which cut-off value was 0.384. I329L-ELISA was used to detect 186 clinical pig serum samples, and the coincidence rate between the indirect ELISA developed here and the commercial kit was 96.77%. No cross-reactivity was observed with CSFV, PRRSV, PCV2, or PRV antibody-positive pig sera, indicating good specificity. Both intra- assay and inter-assay coefficients were below 10%, and the detection sensitivity of the iELISA reached 1:3200. In this study, an iELISA for ASFV antibody detection was developed based on the truncated ASFV I329L protein. Overall, the I329L-ELISA is a user-friendly detection tool that is suitable for ASFV antibody detection and epidemiological surveillance.
