Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease. .

Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

Background & objectives: Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Methods: Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. Results: For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Interpretation & conclusions: Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

Development of Multiple ELISAs for the Detection of Antibodies against Classical Swine Fever Virus in Pig Sera / 中国病毒学·英文版

The major immunogenic proteins (Ems,E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E.coli and purified by affinity chromatography.The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies in pig sera.Optimum cut-off values were determined by receiver operating characteristic (ROC) analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets.The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results.The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination.

Seroprevalencia de Leishmaniasis canina en las localidades TOPO I, TOPO II y las Cumbres de Zamora de la parroquia capital de San Mateo municipio Bolívar, estado Aragua / Seroprevalence of Canine Leishmaniasis in the Localities Topo I, Topo II, and Las Cumbres de Zamora of The Parish Capital of San Mateo Bolivar Municipality, Aragua State

La leishmaniasis visceral (LV) o Kalazar es una enfermedad parasitaria zoonótica conocida desde la antigüedad, ampliamente distribuida en el mundo. Dada la variabilidad, gravedad y complejidad de las manifestaciones clínicas se considera un problema de salud pública a nivel mundial, estimándose una incidencia anual de 500.000 casos, con una prevalencia de 14 millones de personas enfermas, siendo los más afectados los niños menores de cinco años. En vista del papel que juega el perro como reservorio, es prioritario identificar los casos de LV canina. Para contribuir con el control de la leishmaniasis, se determinó la prevalencia en tres localidades de San Mateo Municipio Bolívar del estado Aragua, tomando en cuenta factores de riesgo asociados a la vivienda, al control sanitario del canino, así como la edad, raza y procedencia. Para el diagnóstico, se empleó la técnica de ELISA usando el antígeno recombinante rk39. Se ensayaron muestras séricas de 74 caninos, resultando 13 seropositivos con una prevalencia de 17,56%. Las variables tipo de vivienda, raza, edad, sexo y procedencia del animal, no mostraron asociación con la transmisión de la enfermedad. Además, si se obtuvo asociación con el tipo de tenencia de los caninos utilizados para la protección de animales con 38,46% de prevalencia. Esta investigación aporta elementos importantes para el conocimiento epidemiológico y para el control de esta zoonosis en el Municipio Bolívar del estado Aragua.

Visceral leishmaniasis (VL) or Kalazar is a zooonotic parasitic disease known since ancient times and is widely distributed throughout the world. Given the variability, severity, and complexity of clinical manifestations, VL is considered a public health problem worldwide, with an estimated annual incidence of 500,000 cases, with a prevalence of 14 million sick people, being children under five years old the most affected. In view of the role dogs play as reservoirs, it is of top priority to identify cases of VL. To contribute to the leishmaniasis control, the prevalence of VL in three localities of San Mateo, Bolivar Municipality, in the State of Aragua, Venezuela, was determined, taking into consideration risk factors associated with housing, sanitary control of the dog, age, breed, and origin. For diagnostic purposes, the ELISA technique was employed, using the recombinant antigen rK39. Serum samples of 74 canines were tested. Of all samples tested, only 13 resulted positives, with a prevalence of 17.56%. The variables type of housing, breed, age, sex, and origin, did not show any association with the disease transmission. Nonetheless, there was an association with the type of canine tenure used for the protection of animals; with a prevalence of.38.46%. This investigation provides important elements for both the epidemiological knowledge and control of this zoonosis in the Bolívar Municipality of the State of Aragua.

Profile of Anti-Tp47 antibodies in patients with positive serology for syphilis analized by western blot

In Brazil, syphilis is still a great problem of public health. Serological test is essential for syphilis diagnosis and the current trend is the use of recombinant antigen in the treponemal tests, due to its confirmed higher sensibility and specificity. The purpose of the present study was to analyze the profile of anti-Tp47 antibodies in patients with positive serology for syphilis. One hundred positive sera samples were analyzed by Western Blot (WB) technique, using the recombinant antigen (rTp47). Ten of them did not present antibodies against the fraction rTp47, the results were confirmed by WB using native T. pallidum antigen. All ten samples had antibodies against the fractions Tp17 and Tp15 and presented low reactivity in VDRL, negative results or title below than 1:4. Considering that VDRL is used for therapeutic monitoring due to seroreversion of nontreponemal antibodies in response to the treatment, and that some studies reported loss of treponemal antibodies after treatment, we could speculate if these ten samples are cases of serological memory from patients previously treated for syphilis. In addition, although several features state the Tp47 fraction as one of the major antigenic components, based on our results we point out to the importance of including other antigenic proteins such as Tp17 and Tp15 in addition to Tp47 in tests for serological screening of syphilis.

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.

INDUCTION OF PROTECTIVE IMMUNITY OF SCHISTOSOMA JAPONICUM RECOMBINANT ANTIGENS IN MICE / 中国血吸虫病防治杂志

Schistosoma japonicum recombinant antigens were used to immunize mice in order to ob-serve their protective immunity against challenge. It was assumed that the recombinant anti-gens could reduce worm burden (29. 2%- 51. 0%) and inibit the fecundity of female schisto-some (52. 1% - 74. 3%). The antibody titers increased significant 3 weeks after the first im-munization and remained high lever. The results indicated that the recombinant antigens ex-pressed in the E. coli could induce protective immunity against challenge. It might be pro-duced in large scale as an important candidate component of a multivalent vaccine against schistosomiasis japonicum.