RESUMEN
Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.
Asunto(s)
Antígenos de Protozoos/sangre , Enfermedades de los Perros/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Leishmania infantum/inmunología , Leishmaniasis Visceral/veterinaria , Proteínas Protozoarias/sangre , Animales , Anticuerpos Antiprotozoarios/sangre , Enfermedades de los Perros/parasitología , Perros , Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Proteínas Recombinantes/sangre , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Feline heartworm disease, caused by the filarial nematode Dirofilaria immitis, has been diagnosed with increased frequency in areas endemic for canine heartworm infection. The routine methods for determining the infection status of dogs, such as identification of circulating microfilariae in blood or identification of circulating antigen in serum, plasma or blood, have proven inadequate for screening cats. The inadequacies are due to the likelihood of single-sex infections and clinical disease during prepatent infections. Current antibody detection methodologies rely on crude or partially purified worm antigen preparations that may result in poor specificity. This report describes the cloning, expression, and diagnostic utility of the D. immitis homologue (PDi33) of the Onchocerca volvulus aspartyl protease inhibitor (Ov33). PDi33 is present in all stages that occur in the mammalian host (microfilariae, L3, L4, adult males, and females) and is released by adults cultured in vitro. An indirect enzyme-linked immunosorbent assay (ELISA) using antibody to recombinant PDi33 as a diagnostic marker for infection in cats was very sensitive and was useful for identifying prepatent infections. Testing of sera from cats infected with common gastrointestinal parasites also indicated excellent specificity. The same ELISA in dogs, although demonstrating reasonable sensitivity and specificity, appeared to be of less value as compared with the currently accepted antigen detection methodologies.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Enfermedades de los Gatos/diagnóstico , Dirofilaria immitis/inmunología , Dirofilariasis/diagnóstico , Inhibidores de Proteasas/inmunología , Animales , Secuencia de Bases , Enfermedades de los Gatos/inmunología , Gatos , Clonación Molecular , ADN Complementario/química , ADN de Helmintos/química , Dirofilaria immitis/genética , Dirofilariasis/inmunología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/inmunología , Perros , Electroforesis en Gel de Poliacrilamida/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Regulación de la Expresión Génica , Immunoblotting/veterinaria , Masculino , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Polyclonal and monoclonal antibodies were used to study the immunogenic and antigenic characteristics of chlamydiae. We focused on the most predominant proteins in the outer membrane complex, the major outer membrane protein (MOMP) and the doublet consisting of proteins of 57 and 62 kilodaltons (57-62 kDa doublet). Immunoblot analyses were performed with chlamydial elementary bodies by using (i) immune sera from sheep which had undergone a recent episode of abortion due to the ovine abortion (OA) strain of C. psittaci, (ii) rabbit hyperimmune anti-C. psittaci (OA) and -C. trachomatis sera, and (iii) monoclonal antibodies to the MOMP of C. trachomatis. The typical pattern of response with polyclonal antisera against heterologous elementary bodies was reactivity with the 57-62 kDa doublet and lipopolysaccharide with weak and sometimes no anti-MOMP activity. Three distinct genus-specific anti-C. trachomatis MOMP monoclonal antibodies showed different patterns of reactivity with the MOMPs of the two immunotypes of C. psittaci and C. trachomatis serovars. Our data confirm the predominance of a genus-specific 57-62 kDa doublet response despite the presence of genus-specific epitopes on the MOMP.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/análisis , Chlamydia trachomatis/inmunología , Chlamydophila psittaci/inmunología , Epítopos/análisis , Especificidad de la Especie , Animales , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/inmunología , Pollos , Epítopos/inmunología , Femenino , Cabras , Immunoblotting , Serotipificación , OvinosRESUMEN
An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Toxoplasma gondii infections on single serum dilutions was developed. This test system is a standardized kit designed to detect circulating specific antibodies to Toxoplasma gondii in human sera. It consists of Toxoplasma gondii soluble antigen-coated microtitration multiwell plates, specific immunoglobulin-enzyme conjugate and other required reagents. In a clinical trial performed on sera from 1,035 clinically suspected toxoplasmosis cases, the Sabin Feldman Dye Test (SFDT) and this ELISA system agreed closely. Relative to the SFDT, the sensitivity and specificity of the latter was 98.0% and 97.6% respectively with a correlation coefficient of 0.97. In a further study of 121 sera, the Indirect Fluorescent Antibody Test (IFAT), the Indirect Haemagglutination Test (IHAT) and this ELISA procedure showed over 90% agreement, with correlation coefficient of 0.98 and 0.95 respectively. Within the working concentration of specific antibody to T. gondii in human serum, there was a linear relationship between the ELISA values and the WHO international standard for human anti-Toxoplasma serum.