Rapid Immunochromatographic Test Using Recombinant SAG2 for Detection of Antibodies against Toxoplasma gondii in Cats (original) (raw)
2004
https://doi.org/10.1128/JCM.42.1.351-353.2004
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Abstract
An immunochromatographic test using recombinant truncated surface antigen 2 for detection of antibodies against Toxoplasma gondii was developed. Evaluation of detection of the antibody in mice and cats suggests that this test is rapid, simple, accurate, relatively inexpensive, and suitable for use under field conditions.
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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.
Infectious diseases (London, England), 2018
Toxoplasmosis is one of the most frequent parasitic infections in animals causing reproductive disorders and thus notable economic losses in productivity. Among food animals, pigs along with sheep and goats possess the highest incidence of Toxoplasma gondii cysts in meat, and play a role as a source of human infection. The commercial ELISA kit (PrioCHECK® Toxoplasma Ab SR, Prionics Schlieren-Zurich, Switzerland) for the detection of specific antibodies against T. gondii in swine serum was compared with a commercial IFAT (indirect fluorescent antibody test) (Toxo-Spot IF, bioMérieux, France), used as the reference test. The kappa value obtained comparing the results performed on sera by ELISA with the by IFAT was 1. By a receiver operating characteristics curve analysis, the commercial ELISA had a relative sensitivity of 100%, and a relative specificity of 100% respect to IFAT. The commercial ELISA showed a very good agreement with the commercial IFAT in the detection of serum antibo...
Pesquisa Veterinária Brasileira
The study determined the sensitivity and specificity of the indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT) for anti-Toxoplasma gondii antibody detection by analyzing sera from 46 experimentally infected pigs. Values for sensitivity were 95.7% (confidence interval 95%: 84.0-99.2%) and for specificity 97.8% (confidence interval 95%: 87.0-99.9%) in both tests. There was an optimum agreement of results between IFAT and MAT evidenced by a Kappa test of 0.86. These results validate these tests for the detection of T gondii infection in pigs. MAT and MAT despite methodologies with different characteristics and readings have similar accuracy in pig serum samples.
Comparison of Mat with Elisa and Lat Tests in Detecting Toxoplasma Gondii Antibodies in Human Sera
Science Journal of University of Zakho, 2017
The modified agglutination test (MAT) has been widely used for the detection of Toxoplasma gondii infection in numerous animal species. For the standard protocol of MAT test, T. gondii whole cell antigens were produced in the laboratory mice, which was a tedious process. The produced antigen in cell culture was used to assure its capability for MAT test. For detecting the antibodies of T. gondii in human sera, comparison was made between MAT, enzyme linked immunosorbent assay (ELISA) and latex agglutination test (LAT). A total of 96 human serum samples were tested. The anti-Toxoplasma IgG antibodies were found in 25.0% (24/96), 20.8% (20/96) and 13.5% (13/96) samples by MAT (cutoff 1:25), ELISA and LAT tests, respectively. The MAT and ELISA tests matched 95.8% in detection of IgG antibodies, with the positive percent agreement of 100% (20/20) and negative percent agreement of 94.7% (72/76). For the MAT versus LAT tests, the overall agreement was 88.5% (85/96), with the positive percent agreement of 100% (13/13) and negative percent agreement of 86.7% (72/83). These results suggest a strong correlation between the MAT and ELISA tests in detecting serum IgG to T. gondii in human sera. In conclusion, T. gondii prepared in cell culture provides an alternative solution to produce antigens for MAT test.
Seroprevalence of Toxoplasma gondii antibodies in clinically ill cats in the United States
American Journal of Veterinary Research, 2005
Objective—To determine regional seroprevalence estimates of Toxoplasma gondii-specific IgM and IgG in clinically ill cats throughout the United States. Sample Population—Sera from 12,628 clinically ill, client-owned cats. Procedure—Toxoplasma gondii-specific IgM and IgG antibodies were detected by use of ELISAs. Sera from clinically ill cats previously submitted for T gondii antibody testing were sequentially selected from our serum bank and the sample submission paperwork reviewed. The country was divided into 12 geographic regions. Overall prevalence as well as prevalence for each region, age group, season, sex (male vs female), and breed (domestic shorthair vs other) was calculated. Data were analyzed by logistic regression analysis. Results—Overall, 31.6% of the cats were seropositive for T gondii-specific IgM, IgG, or both. Percentage of cats seropositive for T gondii antibodies ranged from 16.1% (southwestern United States) to 43.5% (northeastern United States). As age increas...
Evaluation of Toxoplasma Gondii IgG Antibodies in Stray and Household Dogs by Elisa
Toxoplasma gondii is one of the most prevalent multisystemic protozoal organisms affecting warm-blooded vertebrates. The aim of study was to determine Toxoplasma sero-epidemiology and also to investigate chronic form of the Toxoplasmosis. Blood samples were taken from 90 dogs including 45 household (pet) and 45 stray dogs in order to detect anti-Toxoplasma IgG antibodies. ELISA were used for diagnosing IgG antibodies. Based on our results 40% of household dogs and 77.77% of stray dogs had anti-Toxoplasma IgG antibodies. It was also seen that antibody titer in stray dogs was statistically much higher than that of household dogs (P<0.05). Antibodies titer in pets which were kept outdoor was significantly higher than that of those which were kept indoor dogs (P<0.05). Regarding the kind of food (raw, cooked or both) and the amount of antibody, it was seen that the antibody titer in dogs which were fed with raw food was significantly higher (P<0.05). Considering the sero-positi...
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Brazilian Journal of Veterinary Research and Animal Science, 2003
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Background: Toxoplasma gondii infects all warm-blooded animals, including humans. Early diagnosis and determining the infective stage are critical for effectively treating immunosuppressed individuals and pregnant women with toxoplasmosis. Among the rhoptry proteins of the parasite, Rhoptry protein 8 (ROP8), is known to be expressed during the early stages of T. gondii infection and is involved in parasitophorous vacuole formation. In this study, we have investigated the diagnostic efficacy of recombinant ROP8 (rROP8). Methods: The ROP8 gene was cloned into pCOLD I DNA vector and expressed as a soluble recombinant antigen in Escherichia coli. Expressed ROP8 protein was evaluated using western blot method.
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Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).
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Toxoplasma gondii is a ubiquitous zoonotic parasite, with felines being the only definitive hosts. Cats shed oocysts with their faeces, and seroprevalence studies can be used to indirectly assess the environmental contamination. The current study aimed to evaluate T. gondii seroprevalence in Greek cats and identify possible risk factors. In total, 1554 blood samples were analyzed from different cats across all nine geographic regions of Greece, and a short questionnaire was completed for each cat. A rapid immunochromatographic test was used to detect anti-T. gondii antibodies, IgG type, and 21.8% of cats were seropositive. Regarding risk factors, when chi-square tests were applied, seropositivity was significantly higher (p < 0.05) in rural cats, cats with outdoor access, and hunting cats. Gender, age, ownership, and raw feeding were not significant risk factors, although female, adult, stray, and raw-feeding cats had a higher seroprevalence than their counterparts. Binary logist...
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Frontiers in Cellular and Infection Microbiology, 2020
Toxoplasmosis is a widely distributed zoonotic infection caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii. It is mainly transmitted through the ingestion of oocysts shed by an infected cat acting as its definitive host. The key to effective control and treatment of toxoplasmosis is prompt and accurate detection of T. gondii infection. Several laboratory diagnostic methods have been established, including the most commonly used serological assays such as the dye test (DT), direct or modified agglutination test (DAT/MAT), indirect hemagglutination test (IHA), latex agglutination test (LAT), indirect immunofluorescent test (IFAT), enzyme-linked immunosorbent assays (ELISA), immunochromatographic tests (ICT), and the western blot. Nonetheless, creating specific and reliable approaches for serodiagnosis of T. gondii infection, and differentiating between acute and chronic phases of infection remains a challenge. This review provides information on the current trends in the serodiagnosis of human toxoplasmosis. It highlights the advantages of the use of recombinant proteins for serological testing and provides insight into the possible future direction of these methods.