J. Parasitol., 101(1), 2015, pp. 37–40 Ó American Society of Parasitologists 2015
EVALUATION OF AN INDIRECT ELISA USING RECOMBINANT GRANULE ANTIGEN GRA7 FOR SERODIAGNOSIS OF TOXOPLASMA GONDII INFECTION IN CATS Yufeng Cai, Zedong Wang*, Jiping Li*, Nan Li*, Feng Wei*, and Quan Liu* College of Life Science, Jilin Agricultural University, 2888 Xincheng Street, Changchun, Jilin Province 130118, PR China. Correspondence should be sent to: [email protected] ABSTRACT:
The precise detection of Toxoplasma gondii infection in cats has important public health significance. In the present study, recombinant granule antigen protein GRA7 was evaluated as a potential diagnostic marker for T. gondii infection in cats by an indirect enzyme-linked immunosorbent assay (ELISA), using the classified serum samples from cats, by immunofluorescence assay (IFA) and by modified agglutination test (MAT). There was a perfect agreement (97.2%) between GRA7-ELISA and MAT/IFA (Kappa ¼ 0.92; 95% confidence interval [CI], 0.85 to 0.99), and GRA7-ELISA had a sensitivity of 94.9% and a specificity of 97.9%. No significant difference (P . 0.05) was observed between the detection results by GRA7- and Toxoplasma lysate antigen-based ELISA. Receiver operating characteristic analysis showed a relative sensitivity and specificity of 89.7 and 92.5% at the cut-off value of 0.1 for GRA7-ELISA. These data demonstrate that GRA7 is a promising serodiagnostic marker for T. gondii infection in cats.
2008). GRA7-ELISA shows the highest positive rate in pregnant women compared with the rhoptry (ROP1), the major surface antigen (SAG1), matrix antigens (MAG1), and GRA8 (Pfrepper et al., 2005). However, there are few reports on evaluation of GRAs as potential diagnostic markers for T. gondii infection in cats. In the present study, recombinant protein GRA7 was expressed and evaluated for serodiagnosis of T. gondii infection in cats by an indirect ELISA. The results demonstrate that GRA7 is a promising serodiagnostic marker for T. gondii infection in cats that may provide the foundation for the development of reliable and standard diagnostic methods of feline T. gondii infection and eventually contribute to the control of transmission of toxoplasmosis from animals to humans.
Toxoplasma gondii is an obligate intracellular parasite that can infect nearly all warm-blooded animals, including humans. Humans become infected post-natally by ingesting undercooked or raw meat containing tissue cysts or by consuming food or drink contaminated with T. gondii sporulated oocysts (Zhou et al., 2011). The life cycle of the parasite includes a sexual reproduction in definitive hosts and asexual reproduction in intermediate hosts. Cats serve as both definitive and intermediate hosts for T. gondii, thus playing a key role in transmission of the infection and making it necessary to develop sensitive and specific diagnostic tests for the detection of T. gondii infection (Dubey, 2009). Diagnosis of T. gondii in cats can be conducted by parasite isolation, fecal examination of oocysts, and serologic tests. Isolation of the parasite by living animal or cell culture is costly and time consuming. Microscopic detection of fecal oocysts is low sensitive and requires an experienced technician. In addition, it is easy to reach a wrong conclusion due to the short period during which cats shed oocysts (Dubey, 2010). Therefore, serologic tests may be the most suitable and practical method for detecting T. gondii infection in cats. Many serologic tests, including the Sabin-Feldman dye test (DT), indirect hemagglutination (IHA), latex agglutination test (LAT), modified agglutination test (MAT), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA), have been developed to detect T. gondii infection in humans and animals (Robert-Gangneux and Darde, 2012). In spite of the satisfactory results of serological tests, it is necessary to develop reliable and standard reagents for serodiagnosis of T. gondii infection. Most conventional tests using tachyzoites as detection antigens are difficult to standardize and the test results are difficult to evaluate. Toxoplasma gondii dense granule antigen proteins (GRAs) are secretory proteins, expressed by tachyzoite and bradyzoite, in which GRA7 is secreted into the cytoplasm of bradyzoite-infected cells and within the parasitophorous vacuole (PV) and the PV membrane in tachyzoite-infected cells. GRA7-based ELISA has presented overall specificity of 98 to 100% and sensitivity of 81 to 88% in humans and goats (Jacobs et al., 1999; Velmurugan et al.,
MATERIALS AND METHODS Preparation of Toxoplasma lysate antigen (TLA) Soluble Toxoplasma gondii lysate antigens were prepared by sonicating the purified T. gondii tachyzoites of GT1 strain (kindly provided by Professor J. P. Dubey, Animal Parasitic Disease Laboratory, Animal Natural and Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland) and diluted to a final concentration of 1 mg/ml in phosphate-buffered saline (PBS) as described elsewhere (Zhu et al., 2008). Expression and purification of recombinant GRA7 Based on the nucleotide sequence of GRA7 (JX045574), the PCR primers for amplification of the gene products of 675-bp GRA7 (forward: 5 0 -ATTCCATGGCGGCCACCGCGTCAGAT-3 0 ; reverse: 5 0 GCGAATTCCTCTTCTGTGTCTGTCTGCCTCTC-3 0 ) were designed. The underlined letters in the sequence of the primers represented the EcoRI and NcoI linker sites in the expression vector pET-28a (Novagen, Darmstadt, Germany). The PCR was performed at an annealing temperature of 58 C for 45 sec. The resulting gene product was cloned into the EcoR1/NcoI site of pET-28a to generate a recombinant plasmid pET28-GRA7 and processed for the expression of recombinant gene products in Escherichia coli BL21 (DE3) according to the standard techniques. The recombinant GRA7 was analyzed by SDS-PAGE, and the reactivity with T. gondii-positive sera was tested by immunoblot using T. gondii-positive mice sera. The recombinant protein was purified using an Ni-NTA purification system (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
Received 20 May 2014; revised 6 September 2014; accepted 9 September 2014. * Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Institute, Academy of Military Medical Sciences, Cangchun, Jilin Province 130122, PR China. DOI: 10.1645/14-575.1
Serum samples A total of 190 blood samples were collected from cats between November and December, 2010 in Lanzhou, China. These animals were all clinically healthy and included 154 household cats (83 male, 71 female) and 36 stray cats (14 male, 22 female). The age distributions of the 154 cats 37
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TABLE I. Detection of T. gondii infection in cats by Toxoplasma lysate antigen (TLA)- and GRA7-ELISA in comparison to the techniques of modified agglutination test/ immunofluorescence assay (MAT/IFAT). MAT/IFAT
Detection method
False-positive False-negative rate/specificity rate/sensitivity Positive Negative Total (%) (%)
TLA-ELISA Positive Negative Total
36 3 39
3 143 146
GRA7-ELISA Positive Negative Total
37 2 39
3 143 146
2.1/97.9
7.7/92.3
2.1/97.9
5.1/94.9
39 146 185 40 145 185
were as follows: 45 were ,1 yr old, 80 were 1–2 yr, 33 were 2–3 yr, and 32 were .3 yr. Sera were separated by centrifugation at 1,500 g for 10 min and stored at 20 C until use. The serum samples were classified into positive and negative for T. gondii antibodies by IFA and MAT. IFA was carried out as described elsewhere (Marca et al., 1996; Macri et al., 2009). Briefly, slides with formalized tachyzoites attached were incubated with cat sera diluted 1:16 and with fluorescein isothiocyanatelabeled goat anti-cat IgG (Abcam, Cambridge, Massachusetts) diluted 1:32 in 0.01 M PBS with 1% of bovine serum albumin. The positive and negative control sera were included. MAT was conducted using cat serum samples diluted at 1:25 as previously described (Dubey and Desmonts, 1987). ELISA ELISA was performed to test T. gondii infection in cats as described by De Craeye et al. (2008) and Huang et al. (2002). Briefly, microplates were coated with TLA or GRA7, respectively. After washing, 50 ll of cat serum diluted 1:64 was added to each well for 1 hr at 37 C, then 50 ll of horseradish peroxidase-conjugated goat anti-cat IgG antibodies (Abcam) diluted 1:20,000 were added. After incubation for 1 hr at 37 C and washing, color was developed by the addition of a substrate 3,3 0 ,5,5 0 tetramethylbenzidine (Beyotime Institute of Biotechnology, Haimen City, Jiangsu Province, China) and stopped by 2 M H2SO4. The optical densities (OD) were measured at 450 nm in a microplate reader (model ELx800, Biotek, Winooski, Vermont). ELISA results were determined for each serum in duplicate. The cut-off point of OD values of a positive sample was set to be at least 2 times higher than that of the negative sample at any dilution point (Zhu et al., 2008). Data analysis The significance of association between the different tests was analyzed using the McNemar chi-square test, and the degree of agreement was quantified using Kappa statistics. The expected performance of the ELISAs at different cut-off points was examined using the receiver operating characteristic (ROC) curves.
The recombinant protein GRA7 was purified using an Ni-NTA purification system, showing the purity to be more than 95%. Classification of cat serum samples by IFA and MAT A total of 190 serum samples from cats were tested for anti-T. gondii antibodies by using IFA and MAT, respectively, showing 39 positive samples and 146 negative samples. The other 5 samples had different results by the 2 tests and were removed from the present study. Evaluation of the diagnostic performance of TLA and GRA7 To evaluate the potential of recombinant protein for serodiagnosis of T. gondii infection in cats, 2 separate ELISAs were developed using TLA and GRA7 as coating antigens. The optimal working dilution, determined by checkerboard assays using serial dilutions of antigens and sera, was shown to be 10 lg/ ml for TLA and 5 lg/ml for GRA7. The classified cat serum samples were tested for anti-T. gondii IgG antibodies by TLA- and GRA7-ELISA, respectively. There were 39 positive and 146 negative samples by TLA-ELISA, while there were 40 positive and 145 negative samples tested by GRA7ELISA (Table I), demonstrating that the seroprevalence was 21.1% by TLA-ELISA and 21.6% by GRA7-ELISA. There was no significant difference between positive and negative results when comparing TLA-ELISA or GRA7-ELISA with MAT/IFA using McNemar chi-square (P . 0.05). A substantial agreement (96.8%) was found between TLA-ELISA and MAT/IFA results (Kappa ¼ 0.90; 95% CI, 0.83 to 0.98), and a perfect agreement (97.2%) was observed between GRA7-ELISA and MAT/IFA results (Kappa ¼ 0.92; 95% CI, 0.85 to 0.99). Compared with the true results tested by MAT/IFA, 3 falsepositive and 3 false-negative samples were found in TLA-ELISA and 3 false-positive and 2 false-negative samples were found in GRA7-ELISA, resulting in the low false-positive rate (2.1%) and false-negative rate (5.1%) for GRA7-ELISA between the tests (Table I). ROC analysis ROC analysis revealed an area under curve (AUC) of 0.954 (95% CI, 0.915 to 0.994) for TLA-ELISA and 0.969 (95% CI, 0.944 to 0.994) for GRA7-ELISA, respectively (Fig. 1). The estimated sensitivities and specificities for different ELISA index were obtained from ROC analysis (Fig. 2), indicating that the cutoff value at 0.26 for TLA-ELISA shows a sensitivity of 84.6% and a specificity of 99.3%, and the 0.10 for GRA7-ELISA shows a sensitivity of 89.7% and a specificity of 92.5%.
RESULTS
DISCUSSION
Expression and purification of recombinant T. gondii GRA7
Many serological tests (DT, IHA, IFA, and ELISA) have been used to detect T. gondii infection in cats. Despite it high specificity and sensitivity in humans, DT requires live parasites and a suitable human serum as a source of accessory factor, a fact which severely limits the application to other species (Balfour et al., 1982). IFA has been regarded as a standard test detecting T. gondii infection in humans and animals, but fluorescence microscopy is necessary for the test; thus it is difficult to use in the field. IHA is species independent and does not need specific
The GRA7 encoding genes were amplified by RT-PCR from total RNA of T. gondii GT1 strain and cloned into expression vector pET-28a to construct recombinant plasmids pET28GRA7, which were transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The immunoreactivity of the expressed protein was confirmed by western blot assay using the mice serum samples positive for anti-T. gondii IgG antibodies.
CAI ET AL.—FELINE T. GONDII INFECTION DETECTED BY RECOMBINANT GRA7
39
FIGURE 1. Receiver operating characteristics (ROC) analysis of the ELISAs. ROC analysis shows an area under the curve of 0.954 (95% CI, 0.915 to 0.994) for Toxoplasma lysate antigen-ELISA and 0.969 (95% CI, 0.944 to 0.994) for GRA7-ELISA, respectively.
conjugates, but it is less sensitive (Dubey, 2010). MAT is a sensitive and specific test to distinguish acute and chronic infection, but it requires large amounts of purified parasites (Dubey, 2010). ELISA has been shown to be both sensitive and specific to T. gondii infection and is also suitable to screen large numbers of clinical samples (Dabritz et al., 2007). However, the present ELISAs using native tachyzoite antigens may vary significantly between laboratories or between batches. An alternative approach is to develop serological tests using recombinant proteins due to the precise antigen composition and easy standardization (Holec-Gasior, 2013). A lot of recombinant antigens have been produced and used for development of ELISA for serodiagnosis of T. gondii infection in humans and animals including surface antigens (SAG1 and SAG2), rhoptry proteins (ROP2 and ROP4), microneme proteins (MIC1 and MIC3), and dense granule antigens (GRA1, GRA2, GRA5, GRA6, GRA7, and GRA8) (Pietkiewicz et al., 2004; Holec-Gasior 2013). Among these antigens, GRA7 induces a very strong antibody response in the acute infection (Pietkiewicz et al., 2004). We evaluated recombinant GRA7 for serodiagnosis of T. gondii infection in cats by indirect ELISA, showing a high sensitivity and specificity, suggesting that GRA7 is a good serological marker for the detection of T. gondii infection in cats. GRA7 is expressed by both T. gondii tachyzoites and bradyzoites and is abundant on the surface and cytoplasmic matrix of host cells, the PV membrane, and within the PV lumen (Pfrepper et al., 2005). In humans, GRA7 appears significantly earlier than other antigens such as SAG1 and MAG1 (Fischer et al., 1998; Neudeck et al., 2002). When GRA7 is released from tachyzoites and bradyzoites, it has direct contact with the host immune system and induces strong antibody responses in both early and late stages of infection (Jacobs et al., 1998, 1999). These antigenic properties of GRA7 make it a good serological maker for the detection of anti-T. gondii antibodies in chronic and acute infection (Nigro et al., 2003; Kotresha et al., 2012). ROC analysis
FIGURE 2. Relative sensitivity and specificities of Toxoplasma lysate antigen (TLA)- and GRA7-ELISA at different cut-offs. Vertical line shows the selected cut-offs of 0.26 for (A) TLA-ELISA and 0.10 for (B) GRA7-ELISA.
has been used to evaluate the relative sensitivity and specificity of the developed ELISA in which the AUC shows a statistical summary of the overall diagnostic performance of the detection method (Swets, 1988; Greiner et al., 2000). In this study, the AUC of 0.969 for GRA7-ELISA suggests that it represents a highly accurate test with good discrimination of positive from negative samples, and the cut-off at 0.10 showed the most appropriate sensitivity and specificity (89.7 and 92.5%, respectively). This study develops an ELISA test for the serological diagnosis of T. gondii infection in cats and can be used with relatively high sensitivity and specificity. ACKNOWLEDGMENTS This study was supported by the National Basic Research Program of China (‘‘973’’ Program) (2012CB722501), the Special Fund for Agroscientific Research in the Public Interest (Grant 201303042), and the National Natural Science Foundation of China (Grant 31372430).
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Toxoplasma gondii in human sera. Journal of Clinical Pathology 35: 228–232. DABRITZ, H. A., I. A. GARDNER, M. A. MILLER, M. R. LAPPIN, E. R. ATWILL, A. E. PACKHAM, A. C. MELLI, AND P. A. CONRAD. 2007. Evaluation of two Toxoplasma gondii serologic tests used in a serosurvey of domestic cats in California. Journal of Parasitology 93: 806–816. DE CRAEYE, S., A. FRANCART, J. CHABAUTY, V. DE VRIENDT, S. VAN GUCHT, I. LEROUX, AND E. JONGERT. 2008. Prevalence of Toxoplasma gondii infection in Belgian house cats. Veterinary Parasitology 157: 128–132. DUBEY, J. P. 2009. History of the discovery of the life cycle of Toxoplasma gondii. International Journal for Parasitology 39: 877–882. ———. 2010. Toxoplasmosis of animals and humans, 2nd ed. CRC Press, Boca Raton, Florida, 313 p. ———, AND G. DESMONTS. 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337–339. FISCHER, H. G., S. STACHELHAUS, M. SAHM, H. E. MEYER, AND G. REICHMANN. 1998. GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by infected host cells. Molecular and Biochemical Parasitology 91: 251–262. GREINER, M., D. PFEIFFER, AND R. D. SMITH. 2000. Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Preventive Veterinary Medicine 45: 23–41. HOLEC-GASIOR, L. 2013. Toxoplasma gondii recombinant antigens as tools for serodiagnosis of human toxoplasmosis: Current status of studies. Clinical and Vaccine Immunology 20: 1343–1351. HUANG, X., X. XUAN, E. N. KIMBITA, B. BATTUR, T. MIYAZAWA, S. FUKUMOTO, M. MISHIMA, L. H. MAKALA, H. SUZUKI, C. SUGIMOTO, ET AL . 2002. Development and evaluation of an enzyme-linked immunosorbent assay with recombinant SAG2 for diagnosis of Toxoplasma gondii infection in cats. Journal of Parasitology 88: 804– 807. JACOBS, D., J. F. DUBREMETZ, A. LOYENS, F. BOSMAN, AND E. SAMAN. 1998. Identification and heterologous expression of a new dense granule protein (GRA7) from Toxoplasma gondii. Molecular and Biochemical Parasitology 91: 237–249. ———, M. VERCAMMEN, AND E. SAMAN. 1999. 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 6: 24–29. KOTRESHA, D., D. POONAM, Y. HAFIZNUR MUHAMMAD, G. SAADATNIA, O. NURULHASANAH, O. SABARIAH, S. Y. TAN, A. K. ZAHIDAH IZZATI, AND N. RAHMAH. 2012. Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis. Tropical Biomedicine 29: 129–137.
MACRI, G., M. SALA, A. M. LINDER, N. PETTIROSSI, AND M. SCARPULLA. 2009. Comparison of indirect fluorescent antibody test and modified agglutination test for detecting Toxoplasma gondii immunoglobulin G antibodies in dog and cat. Parasitology Research 105: 35–40. MARCA, M. C., J. J. RAMOS, A. LOSTE, T. SAEZ, AND M. C. SANZ. 1996. Comparison of indirect immunofluorescent antibody test and modified direct agglutination test methods for detection of Toxoplasma gondii antibodies in adult sheep in Spain. Veterinary Parasitology 67: 99–103. NEUDECK, A., S. STACHELHAUS, N. NISCHIK, B. STRIEPEN, G. REICHMANN, AND H. G. FISCHER. 2002. Expression variance, biochemical and immunological properties of Toxoplasma gondii dense granule protein GRA7. Microbes and Infection 4: 581–590. NIGRO, M., A. GUTIERREZ, A. M. HOFFER, M. CLEMENTE, F. KAUFER, L. CARRAL, V. MARTIN, E. A. GUARNERA, AND S. O. ANGEL. 2003. Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis. Diagnostic Microbiology and Infectious Disease 47: 609– 613. PFREPPER, K. I., G. ENDERS, M. GOHL, D. KRCZAL, H. HLOBIL, D. WASSENBERG, AND E. SOUTSCHEK. 2005. Seroreactivity to and avidity for recombinant antigens in toxoplasmosis. Clinical and Diagnostic Laboratory Immunology 12: 977–982. ´ PIETKIEWICZ, H., E. HISZCZYNSKA -SAWICKA, J. KUR, E. PETERSEN, H. V. NIELSEN, M. STANKIEWICZ, I. ANDRZEJEWSKA, AND P. MYJAK. 2004. Usefulness of Toxoplasma gondii-specific recombinant antigens in serodiagnosis of human toxoplasmosis. Journal of Clinical Microbiology 42: 1779–1781. ROBERT-GANGNEUX, F., AND M. L. DARDE. 2012. Epidemiology of and diagnostic strategies for toxoplasmosis. Clinical Microbiology Reviews 25: 264–296. SWETS, J. A. 1988. Measuring the accuracy of diagnostic systems. Science 240: 1285–1293. VELMURUGAN, G. V., A. K. TEWARI, J. R. RAO, S. BAIDYA, M. U. KUMAR, AND A. K. MISHRA. 2008. High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis. Veterinary Parasitology 154: 185–192. ZHOU, P., Z. CHEN, H. L. LI, H. ZHENG, S. HE, R. Q. LIN, AND X. Q. ZHU. 2011. Toxoplasma gondii infection in humans in China. Parasites & Vectors 4: 165. ZHU, J., J. YIN, Y. XIAO, N. JIANG, J. ANKARLEV, J. LINDH, AND Q. CHEN. 2008. A sero-epidemiological survey of Toxoplasma gondii infection in free-range and caged chickens in northeast China. Veterinary Parasitology 158: 360–363.
EVALUATION OF AN INDIRECT ELISA USING RECOMBINANT GRANULE ANTIGEN GRA7 FOR SERODIAGNOSIS OF TOXOPLASMA GONDII INFECTION IN CATS Yufeng Cai, Zedong Wang*, Jiping Li*, Nan Li*, Feng Wei*, and Quan Liu* College of Life Science, Jilin Agricultural University, 2888 Xincheng Street, Changchun, Jilin Province 130118, PR China. Correspondence should be sent to: [email protected] ABSTRACT:
The precise detection of Toxoplasma gondii infection in cats has important public health significance. In the present study, recombinant granule antigen protein GRA7 was evaluated as a potential diagnostic marker for T. gondii infection in cats by an indirect enzyme-linked immunosorbent assay (ELISA), using the classified serum samples from cats, by immunofluorescence assay (IFA) and by modified agglutination test (MAT). There was a perfect agreement (97.2%) between GRA7-ELISA and MAT/IFA (Kappa ¼ 0.92; 95% confidence interval [CI], 0.85 to 0.99), and GRA7-ELISA had a sensitivity of 94.9% and a specificity of 97.9%. No significant difference (P . 0.05) was observed between the detection results by GRA7- and Toxoplasma lysate antigen-based ELISA. Receiver operating characteristic analysis showed a relative sensitivity and specificity of 89.7 and 92.5% at the cut-off value of 0.1 for GRA7-ELISA. These data demonstrate that GRA7 is a promising serodiagnostic marker for T. gondii infection in cats.
2008). GRA7-ELISA shows the highest positive rate in pregnant women compared with the rhoptry (ROP1), the major surface antigen (SAG1), matrix antigens (MAG1), and GRA8 (Pfrepper et al., 2005). However, there are few reports on evaluation of GRAs as potential diagnostic markers for T. gondii infection in cats. In the present study, recombinant protein GRA7 was expressed and evaluated for serodiagnosis of T. gondii infection in cats by an indirect ELISA. The results demonstrate that GRA7 is a promising serodiagnostic marker for T. gondii infection in cats that may provide the foundation for the development of reliable and standard diagnostic methods of feline T. gondii infection and eventually contribute to the control of transmission of toxoplasmosis from animals to humans.
Toxoplasma gondii is an obligate intracellular parasite that can infect nearly all warm-blooded animals, including humans. Humans become infected post-natally by ingesting undercooked or raw meat containing tissue cysts or by consuming food or drink contaminated with T. gondii sporulated oocysts (Zhou et al., 2011). The life cycle of the parasite includes a sexual reproduction in definitive hosts and asexual reproduction in intermediate hosts. Cats serve as both definitive and intermediate hosts for T. gondii, thus playing a key role in transmission of the infection and making it necessary to develop sensitive and specific diagnostic tests for the detection of T. gondii infection (Dubey, 2009). Diagnosis of T. gondii in cats can be conducted by parasite isolation, fecal examination of oocysts, and serologic tests. Isolation of the parasite by living animal or cell culture is costly and time consuming. Microscopic detection of fecal oocysts is low sensitive and requires an experienced technician. In addition, it is easy to reach a wrong conclusion due to the short period during which cats shed oocysts (Dubey, 2010). Therefore, serologic tests may be the most suitable and practical method for detecting T. gondii infection in cats. Many serologic tests, including the Sabin-Feldman dye test (DT), indirect hemagglutination (IHA), latex agglutination test (LAT), modified agglutination test (MAT), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA), have been developed to detect T. gondii infection in humans and animals (Robert-Gangneux and Darde, 2012). In spite of the satisfactory results of serological tests, it is necessary to develop reliable and standard reagents for serodiagnosis of T. gondii infection. Most conventional tests using tachyzoites as detection antigens are difficult to standardize and the test results are difficult to evaluate. Toxoplasma gondii dense granule antigen proteins (GRAs) are secretory proteins, expressed by tachyzoite and bradyzoite, in which GRA7 is secreted into the cytoplasm of bradyzoite-infected cells and within the parasitophorous vacuole (PV) and the PV membrane in tachyzoite-infected cells. GRA7-based ELISA has presented overall specificity of 98 to 100% and sensitivity of 81 to 88% in humans and goats (Jacobs et al., 1999; Velmurugan et al.,
MATERIALS AND METHODS Preparation of Toxoplasma lysate antigen (TLA) Soluble Toxoplasma gondii lysate antigens were prepared by sonicating the purified T. gondii tachyzoites of GT1 strain (kindly provided by Professor J. P. Dubey, Animal Parasitic Disease Laboratory, Animal Natural and Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland) and diluted to a final concentration of 1 mg/ml in phosphate-buffered saline (PBS) as described elsewhere (Zhu et al., 2008). Expression and purification of recombinant GRA7 Based on the nucleotide sequence of GRA7 (JX045574), the PCR primers for amplification of the gene products of 675-bp GRA7 (forward: 5 0 -ATTCCATGGCGGCCACCGCGTCAGAT-3 0 ; reverse: 5 0 GCGAATTCCTCTTCTGTGTCTGTCTGCCTCTC-3 0 ) were designed. The underlined letters in the sequence of the primers represented the EcoRI and NcoI linker sites in the expression vector pET-28a (Novagen, Darmstadt, Germany). The PCR was performed at an annealing temperature of 58 C for 45 sec. The resulting gene product was cloned into the EcoR1/NcoI site of pET-28a to generate a recombinant plasmid pET28-GRA7 and processed for the expression of recombinant gene products in Escherichia coli BL21 (DE3) according to the standard techniques. The recombinant GRA7 was analyzed by SDS-PAGE, and the reactivity with T. gondii-positive sera was tested by immunoblot using T. gondii-positive mice sera. The recombinant protein was purified using an Ni-NTA purification system (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
Received 20 May 2014; revised 6 September 2014; accepted 9 September 2014. * Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Institute, Academy of Military Medical Sciences, Cangchun, Jilin Province 130122, PR China. DOI: 10.1645/14-575.1
Serum samples A total of 190 blood samples were collected from cats between November and December, 2010 in Lanzhou, China. These animals were all clinically healthy and included 154 household cats (83 male, 71 female) and 36 stray cats (14 male, 22 female). The age distributions of the 154 cats 37
38
THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 1, FEBRUARY 2015
TABLE I. Detection of T. gondii infection in cats by Toxoplasma lysate antigen (TLA)- and GRA7-ELISA in comparison to the techniques of modified agglutination test/ immunofluorescence assay (MAT/IFAT). MAT/IFAT
Detection method
False-positive False-negative rate/specificity rate/sensitivity Positive Negative Total (%) (%)
TLA-ELISA Positive Negative Total
36 3 39
3 143 146
GRA7-ELISA Positive Negative Total
37 2 39
3 143 146
2.1/97.9
7.7/92.3
2.1/97.9
5.1/94.9
39 146 185 40 145 185
were as follows: 45 were ,1 yr old, 80 were 1–2 yr, 33 were 2–3 yr, and 32 were .3 yr. Sera were separated by centrifugation at 1,500 g for 10 min and stored at 20 C until use. The serum samples were classified into positive and negative for T. gondii antibodies by IFA and MAT. IFA was carried out as described elsewhere (Marca et al., 1996; Macri et al., 2009). Briefly, slides with formalized tachyzoites attached were incubated with cat sera diluted 1:16 and with fluorescein isothiocyanatelabeled goat anti-cat IgG (Abcam, Cambridge, Massachusetts) diluted 1:32 in 0.01 M PBS with 1% of bovine serum albumin. The positive and negative control sera were included. MAT was conducted using cat serum samples diluted at 1:25 as previously described (Dubey and Desmonts, 1987). ELISA ELISA was performed to test T. gondii infection in cats as described by De Craeye et al. (2008) and Huang et al. (2002). Briefly, microplates were coated with TLA or GRA7, respectively. After washing, 50 ll of cat serum diluted 1:64 was added to each well for 1 hr at 37 C, then 50 ll of horseradish peroxidase-conjugated goat anti-cat IgG antibodies (Abcam) diluted 1:20,000 were added. After incubation for 1 hr at 37 C and washing, color was developed by the addition of a substrate 3,3 0 ,5,5 0 tetramethylbenzidine (Beyotime Institute of Biotechnology, Haimen City, Jiangsu Province, China) and stopped by 2 M H2SO4. The optical densities (OD) were measured at 450 nm in a microplate reader (model ELx800, Biotek, Winooski, Vermont). ELISA results were determined for each serum in duplicate. The cut-off point of OD values of a positive sample was set to be at least 2 times higher than that of the negative sample at any dilution point (Zhu et al., 2008). Data analysis The significance of association between the different tests was analyzed using the McNemar chi-square test, and the degree of agreement was quantified using Kappa statistics. The expected performance of the ELISAs at different cut-off points was examined using the receiver operating characteristic (ROC) curves.
The recombinant protein GRA7 was purified using an Ni-NTA purification system, showing the purity to be more than 95%. Classification of cat serum samples by IFA and MAT A total of 190 serum samples from cats were tested for anti-T. gondii antibodies by using IFA and MAT, respectively, showing 39 positive samples and 146 negative samples. The other 5 samples had different results by the 2 tests and were removed from the present study. Evaluation of the diagnostic performance of TLA and GRA7 To evaluate the potential of recombinant protein for serodiagnosis of T. gondii infection in cats, 2 separate ELISAs were developed using TLA and GRA7 as coating antigens. The optimal working dilution, determined by checkerboard assays using serial dilutions of antigens and sera, was shown to be 10 lg/ ml for TLA and 5 lg/ml for GRA7. The classified cat serum samples were tested for anti-T. gondii IgG antibodies by TLA- and GRA7-ELISA, respectively. There were 39 positive and 146 negative samples by TLA-ELISA, while there were 40 positive and 145 negative samples tested by GRA7ELISA (Table I), demonstrating that the seroprevalence was 21.1% by TLA-ELISA and 21.6% by GRA7-ELISA. There was no significant difference between positive and negative results when comparing TLA-ELISA or GRA7-ELISA with MAT/IFA using McNemar chi-square (P . 0.05). A substantial agreement (96.8%) was found between TLA-ELISA and MAT/IFA results (Kappa ¼ 0.90; 95% CI, 0.83 to 0.98), and a perfect agreement (97.2%) was observed between GRA7-ELISA and MAT/IFA results (Kappa ¼ 0.92; 95% CI, 0.85 to 0.99). Compared with the true results tested by MAT/IFA, 3 falsepositive and 3 false-negative samples were found in TLA-ELISA and 3 false-positive and 2 false-negative samples were found in GRA7-ELISA, resulting in the low false-positive rate (2.1%) and false-negative rate (5.1%) for GRA7-ELISA between the tests (Table I). ROC analysis ROC analysis revealed an area under curve (AUC) of 0.954 (95% CI, 0.915 to 0.994) for TLA-ELISA and 0.969 (95% CI, 0.944 to 0.994) for GRA7-ELISA, respectively (Fig. 1). The estimated sensitivities and specificities for different ELISA index were obtained from ROC analysis (Fig. 2), indicating that the cutoff value at 0.26 for TLA-ELISA shows a sensitivity of 84.6% and a specificity of 99.3%, and the 0.10 for GRA7-ELISA shows a sensitivity of 89.7% and a specificity of 92.5%.
RESULTS
DISCUSSION
Expression and purification of recombinant T. gondii GRA7
Many serological tests (DT, IHA, IFA, and ELISA) have been used to detect T. gondii infection in cats. Despite it high specificity and sensitivity in humans, DT requires live parasites and a suitable human serum as a source of accessory factor, a fact which severely limits the application to other species (Balfour et al., 1982). IFA has been regarded as a standard test detecting T. gondii infection in humans and animals, but fluorescence microscopy is necessary for the test; thus it is difficult to use in the field. IHA is species independent and does not need specific
The GRA7 encoding genes were amplified by RT-PCR from total RNA of T. gondii GT1 strain and cloned into expression vector pET-28a to construct recombinant plasmids pET28GRA7, which were transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The immunoreactivity of the expressed protein was confirmed by western blot assay using the mice serum samples positive for anti-T. gondii IgG antibodies.
CAI ET AL.—FELINE T. GONDII INFECTION DETECTED BY RECOMBINANT GRA7
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FIGURE 1. Receiver operating characteristics (ROC) analysis of the ELISAs. ROC analysis shows an area under the curve of 0.954 (95% CI, 0.915 to 0.994) for Toxoplasma lysate antigen-ELISA and 0.969 (95% CI, 0.944 to 0.994) for GRA7-ELISA, respectively.
conjugates, but it is less sensitive (Dubey, 2010). MAT is a sensitive and specific test to distinguish acute and chronic infection, but it requires large amounts of purified parasites (Dubey, 2010). ELISA has been shown to be both sensitive and specific to T. gondii infection and is also suitable to screen large numbers of clinical samples (Dabritz et al., 2007). However, the present ELISAs using native tachyzoite antigens may vary significantly between laboratories or between batches. An alternative approach is to develop serological tests using recombinant proteins due to the precise antigen composition and easy standardization (Holec-Gasior, 2013). A lot of recombinant antigens have been produced and used for development of ELISA for serodiagnosis of T. gondii infection in humans and animals including surface antigens (SAG1 and SAG2), rhoptry proteins (ROP2 and ROP4), microneme proteins (MIC1 and MIC3), and dense granule antigens (GRA1, GRA2, GRA5, GRA6, GRA7, and GRA8) (Pietkiewicz et al., 2004; Holec-Gasior 2013). Among these antigens, GRA7 induces a very strong antibody response in the acute infection (Pietkiewicz et al., 2004). We evaluated recombinant GRA7 for serodiagnosis of T. gondii infection in cats by indirect ELISA, showing a high sensitivity and specificity, suggesting that GRA7 is a good serological marker for the detection of T. gondii infection in cats. GRA7 is expressed by both T. gondii tachyzoites and bradyzoites and is abundant on the surface and cytoplasmic matrix of host cells, the PV membrane, and within the PV lumen (Pfrepper et al., 2005). In humans, GRA7 appears significantly earlier than other antigens such as SAG1 and MAG1 (Fischer et al., 1998; Neudeck et al., 2002). When GRA7 is released from tachyzoites and bradyzoites, it has direct contact with the host immune system and induces strong antibody responses in both early and late stages of infection (Jacobs et al., 1998, 1999). These antigenic properties of GRA7 make it a good serological maker for the detection of anti-T. gondii antibodies in chronic and acute infection (Nigro et al., 2003; Kotresha et al., 2012). ROC analysis
FIGURE 2. Relative sensitivity and specificities of Toxoplasma lysate antigen (TLA)- and GRA7-ELISA at different cut-offs. Vertical line shows the selected cut-offs of 0.26 for (A) TLA-ELISA and 0.10 for (B) GRA7-ELISA.
has been used to evaluate the relative sensitivity and specificity of the developed ELISA in which the AUC shows a statistical summary of the overall diagnostic performance of the detection method (Swets, 1988; Greiner et al., 2000). In this study, the AUC of 0.969 for GRA7-ELISA suggests that it represents a highly accurate test with good discrimination of positive from negative samples, and the cut-off at 0.10 showed the most appropriate sensitivity and specificity (89.7 and 92.5%, respectively). This study develops an ELISA test for the serological diagnosis of T. gondii infection in cats and can be used with relatively high sensitivity and specificity. ACKNOWLEDGMENTS This study was supported by the National Basic Research Program of China (‘‘973’’ Program) (2012CB722501), the Special Fund for Agroscientific Research in the Public Interest (Grant 201303042), and the National Natural Science Foundation of China (Grant 31372430).
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