Development of an Immunochromatographic Assay Based on Dense Granule Protein 7 for Serological Detection of Toxoplasma gondii Infection

ABSTRACTDense granule antigen proteins derived fromToxoplasma gondii(TgGRAs) are potential antigens for the development of diagnostic tools. TgGRA7 and TgGRA14 were detected in the peritoneal fluid ofT. gondii-infected mice, suggesting that TgGRAs may be highly antigenic proteins. Here, TgGRA7 and TgGRA14 were evaluated as candidates for the development of a marker for a rapid diagnostic test. The specificity and sensitivity of purified recombinant proteins of TgGRA7 and TgGRA14 were compared in an indirect enzyme-linked immunosorbent assay (iELISA) using a series of serum samples fromT. gondii-experimentally infected mice and using recombinantT. gondiimajor surface antigen 2 (TgSAG2) as a reference control. The iELISA with TgGRA7 showed the greatest diagnostic accuracy and could detect anti-TgGRA7 antibody in acute and chronic infections. A total of 59 field samples from pigs were also examined by the iELISAs, and the results compared with those of the latex agglutination test (LAT). Among the three recombinant antigens, TgGRA7 had the highest rates of positivity, with significant concordance (88.14) and kappa value (0.76) in comparison with the results using LAT. Furthermore, an immunochromatographic test (ICT) based on recombinant TgGRA7 was developed for rapid detection of antibodies to the infection. The ICT differentiated clearly between sera fromT. gondii-infected mice and uninfected orNeospora caninum-infected mice. Pig sera were examined with the ICT, and the results compared favorably with those of LAT and iELISA for TgGRA7, with kappa values of 0.66 and 0.70 to 0.79, respectively. These data suggest that the ICT based on TgGRA7 is a promising diagnostic tool for routine testing in the clinic and mass screening of samples in the field.

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Identification of a Highly Antigenic Region of Subtilisin-Like Serine Protease 1 for Serodiagnosis of Neospora caninum Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.00352-13 ◽

2013 ◽

Vol 20 (10) ◽

pp. 1617-1622 ◽

Cited By ~ 11

Author(s):

Rochelle Haidee D. Ybañez ◽

Mohamad Alaa Terkawi ◽

Kyohko Kameyama ◽

Xuenan Xuan ◽

Yoshifumi Nishikawa

Keyword(s):

Serine Protease ◽

Tandem Repeat ◽

Neospora Caninum ◽

Single Copy ◽

Enzyme Linked Immunosorbent Assay ◽

Infected Cattle ◽

Serum Samples ◽

Caninum Infection ◽

Content Type ◽

Field Samples

ABSTRACTNeospora caninumis an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that theN. caninumsubtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool forNeospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples fromN. caninumexperimentally infected cattle and mice and cattle from a farm with confirmed cases ofNeosporaabortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In theN. caninumexperimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera fromToxoplasma gondiiexperimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis ofN. caninum.

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Evaluation of Recombinant Dense Granule Antigen 7 (GRA7) of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies and Analysis of a Major Antigenic Domain

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.1.24-29.1999 ◽

1999 ◽

Vol 6 (1) ◽

pp. 24-29 ◽

Cited By ~ 56

Author(s):

Dirk Jacobs ◽

Martine Vercammen ◽

Eric Saman

Keyword(s):

Monoclonal Antibody ◽

Toxoplasma Gondii ◽

Immunoglobulin G ◽

Chronic Phase ◽

Dense Granule ◽

Leader Peptide ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Human Sera ◽

Antigenic Domain

ABSTRACT Dense granule protein 7 (GRA7) of Toxoplasma gondii was expressed in Escherichia coli as a fusion protein. The leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues. After purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG). For two sets of IgG-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was obtained. For chronic-phase sera, the sensitivity of detection was 79%, but chronic-phase sera with low titers were more difficult to detect (65% sensitivity for sera with immunofluorescence titer of 1/64). When GRA7 was combined with Tg34AR (rhoptry protein 2 C-terminal fragment), the sensitivity rose to 96%. For a set of acute-phase serum samples tested on GRA7, the sensitivity of detection was 94%, and high-titer IgM-positive sera were detected at an especially high rate. In contrast, when Tg34AR was used, the sensitivity was only 85% for this latter set of serum samples. Three truncated GRA7 fragments containing the same leader peptide as that of recombinant GRA7 were produced. The shortest fragment (97 N-terminal amino acids) was not reactive with human sera or with a specific anti-GRA7 monoclonal antibody, while the two larger fragments were reactive. The most important antigenic domain of GRA7 for human sera was localized between residues 97 and 146. The epitope for the specific monoclonal antibody could be further narrowed down by the use of synthetic peptides, but this epitope is not recognized by sera from T. gondii-infected humans. These results indicate that GRA7 may be considered as an additional tool for studying the immune response to T. gondii.

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Chimeric Protein Designed by Genome-Scale Immunoinformatics Enhances Serodiagnosis of Bovine Neosporosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01343-19 ◽

2020 ◽

Vol 58 (7) ◽

Author(s):

Higor Sette Pereira ◽

Ludmila Tavares e Almeida ◽

Vitória Fernandes ◽

Renato Lima Senra ◽

Patrícia Pereira Fontes ◽

...

Keyword(s):

Neospora Caninum ◽

Leukemia Virus ◽

Clinical Signs ◽

Chimeric Protein ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Noninvasive Test ◽

Content Type ◽

Bovine Neosporosis

ABSTRACT Neosporosis has become a concern since it is associated with abortion in cattle. Currently, in situ diagnosis is determined through anamnesis, evaluation of the history, and perception of the clinical signs of the herd. There is no practical and noninvasive test adapted to a large number of samples, which represents a gap for the use of new approaches that provide information about infections and the risks of herds. Here, we performed a search in the Neospora caninum genome by linear B-cell epitopes using immunoinformatic tools aiming to develop a chimeric protein with high potential to bind specifically to antibodies from infected cattle samples. An enzyme-linked immunosorbent assay with the new chimeric antigen was developed and tested with sera from natural field N. caninum-infected bovines. The cross-reactivity of the new antigen was also evaluated using sera from bovines infected by other abortive pathogens, including Trypanosoma vivax, Leptospira sp., Mycobacterium bovis, and Brucella abortus, and enzootic bovine leucosis caused by bovine leukemia virus, as well as with samples of animals infected with Toxoplasma gondii. The assay using the chimeric protein showed 96.6% ± 3.4% of sensitivity in comparison to healthy animal sera. Meanwhile, in relation to false-positive results provided by cross-reactivity with others pathogens, the specificity value was 97.0% ± 2.9%. In conclusion, immunoinformatic tools provide an efficient platform to build an accurate protein to diagnose bovine neosporosis based on serum samples.

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A New MIC1-MAG1 Recombinant Chimeric Antigen Can Be Used Instead of the Toxoplasma gondii Lysate Antigen in Serodiagnosis of Human Toxoplasmosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.05433-11 ◽

2011 ◽

Vol 19 (1) ◽

pp. 57-63 ◽

Cited By ~ 25

Author(s):

Lucyna Holec-Gąsior ◽

Bartłomiej Ferra ◽

Dorota Drapała ◽

Dariusz Lautenbach ◽

Józef Kur

Keyword(s):

Toxoplasma Gondii ◽

Expression System ◽

Chimeric Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Content Type ◽

Metal Affinity ◽

Positive Serum ◽

First Time ◽

Microneme Protein

ABSTRACTThis study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1)Toxoplasma gondiirecombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using anEscherichia coliexpression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondiiimmunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using aToxoplasmalysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis.

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Enzyme-Linked Immunosorbent Assays Based on Neospora caninum Dense Granule Protein 7 and Profilin for Estimating the Stage of Neosporosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.05669-11 ◽

2012 ◽

Vol 19 (3) ◽

pp. 411-417 ◽

Cited By ~ 12

Author(s):

Jun Hiasa ◽

Maki Nishimura ◽

Kazuhito Itamoto ◽

Xuenan Xuan ◽

Hisashi Inokuma ◽

...

Keyword(s):

Neospora Caninum ◽

Protozoan Parasite ◽

Dense Granule ◽

Acute Stage ◽

Neurological Symptoms ◽

Diagnostic Methods ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Antibody Response ◽

Content Type ◽

The Relationship

ABSTRACTNeospora caninumis an intracellular protozoan parasite that causes bovine and canine neosporosis, characterized by fetal abortion and neonatal mortality and by neuromuscular paralysis, respectively. Although many diagnostic methods to detect parasite-specific antibodies or parasite DNA have been reported, to date no effective serodiagnostic techniques for estimating pathological status have been described. Our study aimed to elucidate the relationship between the parasite-specific antibody response, parasite activation, and neurological symptoms caused byN. caninuminfection by using a recombinant antigen-based enzyme-linked immunosorbent assay. Among experimentally infected mice, anti-N. caninumprofilin (NcPF) antibody was only detected in neurologically symptomatic animals. Parasite numbers within the brains of the symptomatic mice were significantly higher than those in asymptomatic animals. In addition, anti-NcPF and anti-NcGRA7 antibodies were mainly detected at the acute stage in experimentally infected dogs, while anti-NcSAG1 antibody was produced during both acute and chronic stages. Furthermore, among anti-NcSAG1 antibody-positive clinical dogs, the positive rates of anti-NcGRA7 and anti-NcPF antibodies in the neurologically symptomatic dogs were significantly higher than those in the non-neurologically symptomatic animals. Our results suggested that the levels of anti-NcGRA7 and anti-NcPF antibodies reflect parasite activation and neurological symptoms in dogs. In conclusion, antibodies against NcGRA7 and NcPF may have potential as suitable indicators for estimating the pathological status of neosporosis.

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Toxoplasma gondii and Neospora caninum antibodies in goats in the Czech Republic

Veterinární Medicína ◽

10.17221/5850-vetmed ◽

2012 ◽

Vol 57 (No. 3) ◽

pp. 111-114 ◽

Cited By ~ 11

Author(s):

E. Bartova ◽

K. Sedlak

Keyword(s):

Czech Republic ◽

Toxoplasma Gondii ◽

Immunosorbent Assay ◽

Competitive Inhibition ◽

Neospora Caninum ◽

Fluorescent Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Antibodies ◽

Serum Samples ◽

The Czech Republic

Toxoplasma gondii is zoonotic protozoan parasite that causes infections in many vertebrate species. The present study determined the seroprevalence of T. gondii and N. caninum in goats from the Czech Republic. Serum samples were collected from 251 healthy adult goats in the Czech Republic during the years 2006 to 2009. Sera samples were tested for serum antibodies to Toxoplasma gondii by an enzyme-linked immunosorbent assay with cut off equal to or higher than 50% S/P. The same samples were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay with cut off equal to or higher than 30% inhibition; positive sera were confirmed by an indirect fluorescent antibody test with cut-off titre equal to or higher than 40. Sera positive in both tests were marked as positive. In total, 166 (66%) and 15 (6%) goat sera reacted positively for T. gondii and N. caninum antibodies, respectively. All sera positive for N. caninum antibodies were simultaneously positive for T. gondii antibodies. This is the first detection of N. caninum antibodies in goats in the Czech Republic. Our findings indicate that goats in the Czech Republic are frequently exposed to T. gondii, but less frequently to N. caninum.  

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Serological Survey and Associated Risk Factors on Toxoplasma gondii Infection in Goats in Mila District, Algeria

Folia Veterinaria ◽

10.2478/fv-2020-0007 ◽

2020 ◽

Vol 64 (1) ◽

pp. 48-59 ◽

Cited By ~ 1

Author(s):

A. Dahmane ◽

S. Boussena ◽

F. Hafsi ◽

F. Ghalmi

Keyword(s):

Risk Factors ◽

Toxoplasma Gondii ◽

Protozoan Parasite ◽

Latex Agglutination ◽

Enzyme Linked Immunosorbent Assay ◽

Seroprevalence Rate ◽

Serum Samples ◽

Serological Tests ◽

North Eastern ◽

Eastern Algeria

AbstractToxoplasma gondii is a protozoan parasite prevalent in humans and other animals worldwide having medical and veterinary importance on account of reproductive failure causing significant socioeconomic losses. The aims of this study were to estimate the seroprevalence of T. gondii infection in goats, determined the possible risk factors associated, and evaluate the performances of the latex agglutination test (LAT) to anti-T. gondii antibodies screening using the indirect Enzyme-linked immunosorbent assay as a reference test (iELISA). A total of 184 serum samples from goats reared on 25 farms in Mila district from North-Eastern Algeria were collected and tested for anti-T. gondii IgG antibodies using two commercial serological tests (ELISA and LAT). A seroprevalence rate of 71.73 % and 63.58 % was obtained by both ELISA and LAT tests, respectively. The analysis of some factors thought to be related to the onset of this infection such as age, sex, management system, locality and presence of cats showed no significant relationship (P > 0.05); these factors did not seem to affect the frequency of the infection. The seropositivity level of T. gondii was significantly higher in aborted goats (P = 0.007), which suggested that they may play a significant role in pregnancy failure. In the concordance evaluation between the two serological tests (ELISA and LAT), the Cohen’s Kappa value was calculated and the results showed a K of 0.519 (p = 0.000) belonging to the range of 0.41—0.60 indicating just average agreement. The results of the Mc Nemar test showed that both tests gave significantly different results and seropositivity values (P < 0.05). The high prevalence observed in this study indicated a widespread exposure to T. gondii from goats and the potential risk of T. gondii infection for humans in North-Eastern Algeria. These results elucidate the challenges of applying serology to estimate goat exposure to T. gondii. The choice between the two serological tests will depend on their performances, as well as the availability of the equipment, laboratory conditions and the number of samples to be tested.

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Enzyme-Linked Immunosorbent Assay Using Recombinant SAG1 Antigen To Detect Toxoplasma gondii-Specific Immunoglobulin G Antibodies in Human Sera and Saliva

Clinical and Vaccine Immunology ◽

10.1128/cvi.00512-12 ◽

2013 ◽

Vol 20 (4) ◽

pp. 468-473 ◽

Cited By ~ 16

Author(s):

Nouha Chahed Bel-Ochi ◽

Aïda Bouratbine ◽

Mohamed Mousli

Keyword(s):

Toxoplasma Gondii ◽

Sensitivity And Specificity ◽

Specific Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Native Form ◽

Content Type ◽

Percent Agreement ◽

Human Sera ◽

Commercial Kits

ABSTRACTSerologic detection ofToxoplasma gondiiIgG antibodies is widely accepted as a means to determine immune status and susceptibility toToxoplasmainfection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinantT. gondiiSAG1 antigen (rSAG1) to assess its diagnostic performance inToxoplasmaIgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

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Limited Value of Assays Using Detection of Immunoglobulin G Antibodies to the Two Recombinant Dense Granule Antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for Distinguishing between Acute and Chronic Infections in Pregnant Women

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.6.1016-1021.2004 ◽

2004 ◽

Vol 11 (6) ◽

pp. 1016-1021 ◽

Cited By ~ 28

Author(s):

Josette Ferrandiz ◽

Corinne Mercier ◽

Martine Wallon ◽

Stéphane Picot ◽

Marie-France Cesbron-Delauw ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Pregnant Women ◽

Immunoglobulin G ◽

Dense Granule ◽

Enzyme Linked Immunosorbent Assay ◽

Chronic Infections ◽

Serum Samples ◽

Positive Serology ◽

Group 2 ◽

Group 1

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection. Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T. gondii (group 3) were examined for immunoglobulin G (IgG) reactivity. For both antigens, the specificity reached 98%. In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt. In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively. Combination of the readings obtained with both antigens yielded a sensitivity of 91%. A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results. Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections.

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Dense-Granule Protein NcGRA7, a New Marker for the Serodiagnosis of Neospora caninum Infection in Aborting Cows

Clinical and Vaccine Immunology ◽

10.1128/cvi.00251-07 ◽

2007 ◽

Vol 14 (12) ◽

pp. 1640-1643 ◽

Cited By ~ 27

Author(s):

Penglong Huang ◽

Min Liao ◽

Houshuang Zhang ◽

Eung-goo Lee ◽

Yoshifumi Nishikawa ◽

...

Keyword(s):

Immunosorbent Assay ◽

Specific Antibody ◽

Neospora Caninum ◽

Dense Granule ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Caninum Infection ◽

Recombinant Antigens ◽

Granule Protein ◽

Bovine Abortion

ABSTRACT To investigate whether the production of an antigen-specific antibody is associated with Neospora caninum-induced bovine abortion, 62 serum samples were tested with an enzyme-linked immunosorbent assay using the recombinant antigens NcSAG1, NcSRS2, and NcGRA7. Our study suggested that NcGRA7 would be a new marker for the serodiagnosis of N. caninum infection resulting in abortion.

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