Rapid Immunochromatographic Test Using Recombinant SAG2 for Detection of Antibodies against Toxoplasma gondii in Cats (original) (raw)
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Veterinary Parasitology, 2001
The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8 M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.
Memórias do Instituto Oswaldo Cruz, 1997
We evaluated the titers of anti-T. gondii antibodies by various serological tests in 40 serum samples from dogs exhibiting clinical signs of infectious diseases. Indirect immunofluorescence (IgG-IFI), indirect haemagglutination (IHA and M-Toxo) and immunoenzymatic (ELISA and PA-ELISA) tests were carried out. Titers ≥ 64 were considered as positive. Anti-Toxoplasma antibodies were found in 9 (22.5%), 14 (35%), 14 (35%) and 12 (30%) samples, respectively for IHA, IgG-IFI, ELISA and PA-ELISA. The results showed that 57% were negative in all tests and 43% of the dogs presented antibodies to T. gondii; from these, 20% were positive in all three tests with high titers of antibodies and 23% were positive in only one or two tests with low titers of antibodies and mainly related to the IFI and ELISA tests. We observed 5 (12.5%) and 1 (2.5%) reactive samples, respectively, by M-Toxo and IHA with or without 2mercapthoethanol, in the attempt to detect specific IgM. We can conclude that serodiagnosis of toxoplasmosis in dog have to be based on the combination of serological tests (IFI and ELISA) and with emphasis at the determination of the titers and the classes of the specific antibodies.
Pathogens
Toxoplasma gondii is the zoonotic parasite responsible for toxoplasmosis in warm-blooded vertebrates. This systematic review compares and evaluates the available knowledge on enzyme-linked immunosorbent assays (ELISAs), their components, and performance in detecting T. gondii antibodies in animals. Four databases were searched for published scientific studies on T. gondii and ELISA, and 57 articles were included. Overall, indirect (95%) and in-house (67%) ELISAs were the most used types of test among the studies examined, but the ‘ID Screen® Toxoplasmosis Indirect Multi-species’ was common among commercially available tests. Varying diagnostic performance (sensitivity and specificity) and Kappa agreements were observed depending on the type of sample (serum, meat juice, milk), antigen (native, recombinant, chimeric) and antibody-binding reagents used. Combinations of recombinant and chimeric antigens resulted in better performance than native or single recombinant antigens. Protein ...
Tropical Animal Health and Production, 2014
An antibody detection recombinant enzyme-linked immunosorbent assay (ELISA) specific for Toxoplasma gondii was laboratory standardized using recombinant truncated surface antigen 2 (SAG2) protein of T. gondii. A 483-bp sequence coding for truncated tachyzoite stagespecific SAG2 protein was amplified and ligated in pPROExHT-b expression vector to transform Escherichia coli DH5α cells. A high-level expression of the histidine-tagged fusion protein was obtained after 8 h of incubation. The recombinant protein was affinity purified using Ni-NTA agarose column and characterized by SDS-PAGE and Western blot analysis. Subsequently, the diagnostic potential of the recombinant protein was assessed with 168 field sera samples from sheep, goats and cattle. Among the small ruminants, 50 % (n=60) sheep sera samples and 41.26 % (n=63) goat samples were detected positive for T. gondii-specific antibodies. As far as seroprevalence of toxoplasmosis in cattle is concerned, 64.44 % (n=45) of sera samples assayed were found to be positive. When compared to indirect fluorescent antibody test (IFAT), the sensitivity of the recombinant truncated SAG2 antigen-based ELISA (rec-SAG2-ELISA) ranged from 81.25 to 87.10 % while the specificity was 85.71 to 91.43 % with substantial agreement between the tests.
Animals
Toxoplasmosis is a zoonotic disease, caused by the protozoan Toxoplasma gondii, affecting most warm-blooded animals. Assessing the seroprevalence of T. gondii in different animal species gives a good estimate of the global circulation of the parasite and the risk for human infections. However, the seroprevalence of T. gondii in dogs is not studied as much as other species, despite their close contact with wildlife and humans in rural or urban environments and evidence that dogs can also be a potential source for human contaminations. A commercial enzyme-inked immunosorbent assay (ELISA) kit to detect anti-T. gondii antibodies in sera of hunting dogs potentially naturally infected, was compared to the modified agglutination test (MAT), used as the reference method. The ELISA presented a sensitivity of 76.5% (CI 95%: 60.0–87.6) and a specificity of 87.7% (CI 95%: 76.7–93.9) and a substantial agreement with the MAT for the detection of canine anti-T. gondii antibodies. Both tests can t...
Aim: The aims of the study are to detect the presence of Toxoplasma gondii antigen and to determine its distribution location in several organs of domestic cat using immunohistochemistry (IHC) method with Labeled-[Strept] Avidin-Biotin (LAB-SA). Material and Methods: Four domestic cats aged 1-2 years were used as sample in this research. The sample divided into two groups with two cats each. Cats in Group I were positive Toxoplasma based on serologically screening test, while cats in Group II were orally infected with 1×10 6 Toxoplasma oocyst. All samples then necropsied, and the organs including brain, liver, kidney, duodenum, jejunum, ileum, lungs, and spleen were collected for IHC method with LAB-SA. Result: The result showed that Toxoplasma antigens were detected in ileum of both serologically positive domestic cat and the experimentally infected cats. Toxoplasma was also observed in kidney of serologically positive domestic cat. In the serologically positive domestic cat, necrotic lesions were found on ileum, kidney, and liver, whereas in experimentally infected cat, the lesion was only found on ileum. Conclusion: The presence of Toxoplasma antigen is successfully detected in several organs of domestic cat using IHC method with the LAB-SA.
Experimental Parasitology, 2006
Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 · 10 7 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days À6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as ''gold standard'' and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA · IFAT (j = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs.