Experimental Parasitology 157 (2015) 88e91
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Full length article
Evaluation of Neospora caninum truncated dense granule protein 2 for serodiagnosis by enzyme-linked immunosorbent assay in dogs Chunmei Jin 1, Longzheng Yu 1, Yinan Wang, Shiyue Hu, Shoufa Zhang* Department of Veterinary Medicine, Yanbian University, Yanji, Jilin Province 133002, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
NcGRA2t recombinant proteins do not exhibit cross-reactivity with Toxoplasma gondii. Established an ELISA based on NcGRA2t to detect Neospora caninum-infection in dogs. NcGRA2t could be a suitable source of antigen for the detection of early infection.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 August 2014 Received in revised form 20 May 2015 Accepted 8 July 2015 Available online 11 July 2015
Neosporosis is an infectious disease caused by Neospora caninum, and it primarily affects cattle and dogs. An infection by N. caninum causes fetal abortion and neonatal mortality. Previous proteomics and immunoscreening analyses revealed that N. caninum dense granule antigen 2 (NcGRA2) has potential for serodiagnosis of N. caninum. Consequently, we expressed the truncated NcGRA2 (NcGRA2t), which lacks a signal peptide. We compared the serodiagnostic performances of recombinant NcGRA2t with that of truncated surface antigen 1 of N. caninum (NcSAG1t). Specificity testing using sera from mice infected with Toxoplasma gondii indicated that the NcGRA2t recombinant protein does not cross-react with T. gondii. In addition, we detected anti-NcGRA2t antibody at the acute stage in experimentally infected dogs, while detecting anti-NcSAG1t antibody during both the acute and chronic stages. Our results suggest that the levels of anti-NcGRA2 antibody reflect parasite activation in dogs. In conclusion, antibodies against NcGRA2t and NcSAG1t are suitable indicators to distinguish the acute and chronic stages of N. caninum infection. © 2015 Elsevier Inc. All rights reserved.
Keywords: Neospora caninum NcGRA2t ELISA
1. Introduction Neospora caninum is an obligate intracellular apicomplexan parasite (Dubey et al., 1988). Domestic dogs, coyotes, dingoes and wolves are the known definitive host for N. caninum (McAllister * Corresponding author. E-mail address: [email protected] (S. Zhang). 1 The authors contributed equally to this study. http://dx.doi.org/10.1016/j.exppara.2015.07.003 0014-4894/© 2015 Elsevier Inc. All rights reserved.
et al., 1998; Gondim et al., 2004; King et al., 2010; Dubey et al., 2011), and many warm-blooded vertebrates, such as cattle, sheep, goats, deer, and horses are intermediate hosts (Dubey, 2003). Neosporosis now appears to be a major cause of bovine abortion or stillbirth and paralysis of rear limbs, often with contracture in dogs worldwide, and causes severe economic losses (Dubey et al., 2007). Hosts can acquire N. caninum by ingesting tissues of infected animals, or food or drinking water contaminated with oocysts shed by dogs, the definitive host (Dubey and Lindsay, 1996). Therefore,
C. Jin et al. / Experimental Parasitology 157 (2015) 88e91
specific and sensitive detection of this pathogen is very important to distinguish healthy animals from infected animals. Currently, a number of serological tests exist for detecting N. caninum specific antibodies, including the indirect fluorescence antibody test (IFAT), the N. caninum agglutination test (NAT), and immunoblotting tests, which are based on tachyzoite antigens (Dubey et al., 2006). Several enzyme-linked immunosorbent assays (ELISAs) using recombinant antigens have been widely evaluated to detect N. caninum-specific antibodies. A number of serodiagnostic assays for bovine or dog neosporosis use recombinant antigens, such as surface antigen 1 of N. caninum (NcSAG1), N. caninum dense granule antigen 7 (NcGRA7), and N. caninum subtilisin-like serine protease 1 (NcSUB1) (Chahan et al., 2003; Dubey and Schares, 2006; Huang et al., 2007; Ybanez et al., 2013). NcSAG1 is one of the antigens with the ability to detect anti-NcSAG1 antibody during both acute and chronic stages of infection (Hiasa et al., 2012). NcGRA7 is a suitable antigen for detection of N. caninum infection in the activation stage (Hiasa et al., 2012). Both tachyzoites and bradyzoites express the N. caninum dense granule antigen 2 gene (NcGRA2) (Strohbusch et al., 2008). NcGRA2 is a species-specific protein of N. caninum based on the comparison of two-dimensional gel electrophoresis (2-DE) profiles between N. caninum and Toxoplasma gondii (Lee et al., 2005). These findings suggest that NcGRA2 has potential use in the serodiagnosis of N. caninum, and requires further analyses to determine the utility of NcGRA2 in a diagnostic setting. In this study, we expressed truncated NcGRA2 (NcGRA2t) as a glutathione S-transferase (GST) fusion protein in Escherichia coli. Furthermore, we evaluated its diagnostic potential against N. caninum infection in dogs by comparing the results obtained from an established ELISA, based on truncated NcSAG1 (NcSAG1t).
89
proteins as previously described (Chahan et al., 2003). Briefly, incubate pGEX-NcSAG1t and pGEX-NcGRA2t transformants at 37 C at 250 rpm until the OD600 level reached 0.5. Synthesis of GST-NcSAG1t and GST-NcGRA2t were induced with 1 mM IPTG at 37 C for 4 h. Harvest cells by centrifugation and treated by TNE buffer (50 mM TriseHCl, pH7.5, 0.1 M NaCl, 2 mM EDTA) containing lysozyme (100 mg/ml) and 1% Triton X-100. After centrifugation, the supernatant was harvested, and the recombinant protein was purified with Glutathione-Sepharose 4B, according to the manufacturer's instructions (Amersham Pharmacia Biotech, Sweden). We measured the protein concentration using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA). 2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting We verified protein expression by SDS-PAGE after staining with Coomassie Brilliant Blue (MP Biomedicals, Inc., France). Thereafter, we used western blot analyses to determine the antigenicity of the NcGRA2t protein for application in serodiagnostic methods as previously described (Chahan et al., 2003). Briefly, After the proteins were transferred electrophoretically onto a polyvinylidene membrane (Millipore, USA), the membrane was blocked for overnight at 4 C with PBS containing 3% skim milk, and then the membrane was incubated with a diluted dog anti-N. caninum serum (1:100) for 1 h and washed three times with PBS. Following incubation with an HRP-conjugated goat anti-dog IgG (BETHYL, USA) (1:1000) for 1 h and wash three times, the membrane was placed into a substrate solution (0.5 mg/ml diaminobenzidine, 0.005% H2O2) to visualize the specific antigen bands. 2.4. Sera
2. Materials and methods 2.1. Parasite preparation We maintained N. caninum tachyzoites of the Nc-1 strain and T. gondii tachyzoites of the PLK strain in monkey kidney adherent fibroblast (Vero) cells cultured in Eagle's minimum essential medium (MEM; SigmaeAldrich, St. Louis, MO, USA) supplemented with 8% heat-inactivated fetal bovine serum (FBS) and 50 mg/ml kanamycin at 37 C in a 5% CO2 air environment. We purified tachyzoites by washing the parasites and host-cell debris in cold phosphate-buffered saline (PBS), and then we resuspended the final pellet in cold PBS, and passed it through a 27-gauge needle and a 5.0-mm pore filter (Millipore, Billerica, MA, USA). 2.2. Expression and purification of recombinant NcGRA2t in E. coli We prepared total RNA from Nc-1 tachyzoites using the RNeasy Plus Mini Kit (Qiagen, Germany), and used RNA as a template to amplify the coding region of NcGRA2t (GenBank accession no. NCLIV_045650) using the One Step RT PCR Kit (Takara, Japan). We performed all procedures according to the respective manufacturer's instructions. We designed a set of oligonucleotide primers, including EcoRI and XhoI restriction enzyme sites, to clone the truncated gene without a signal peptide (forward primer, 50 -AC GAATTC GCCGATTTTTCTGGCAGGG-30 ; reverse primer, 50 -CC CTCGAG TTAATTGACTTCAGCTTCTGG-30 ). We digested PCR products with EcoRI and XhoI before ligation into the glutathione Stransferase (GST) fusion protein in the E. coli expression vector, pGEX-4T1 (GE Healthcare, UK), which we digested with the same set of restriction enzymes. We prepared NcSAG1t as previously described (Chahan et al., 2003). We expressed the recombinant proteins of NcSAG1t and NcGRA2t in E. coli BL21 (DE3) as GST fusion
Sera used for the present study were as follows: 10 sera from BALB/c mice experimentally infected with 4 104 tachyzoites of the N. caninum Nc-1 strain, 10 sera from BALB/c mice experimentally infected with 4 102 tachyzoites of the T. gondii PLK strain, 10 sera from healthy BALB/c mice. We prepared experimentally infected dogs sera with N. caninum using two SPF (specific-pathogen-free) dogs as previously described (Hiasa et al., 2012). Before experimental infection, dogs were proved to be free of N. caninumand T. gondii-specific antibody by ELISA based on lysates of N. caninum and T. gondii. We obtained sera weekly from dogs via venipuncture of the radial vein and stored it at 20 C until use. Mice were given food and water ad libitum while the dogs were fed every day and given water ad libitum. All of the animals used in the present study were conducted in accordance with the Standards Relating to the Care and Management of Experimental Animals promulgated by the Yanbian University, China. 2.5. ELISA We performed the ELISA as previously described (Chahan et al., 2003). We diluted the GST-fused NcGRA2t and NcSAG1t recombinant proteins, and the GST control, in coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) to a final concentration of 0.1 mM. We coated each well of a 96-well microtiter plate (Nunc, Denmark) with 50 ml of the corresponding protein, and incubated the plate overnight at 4 C. Plates were washed once with PBST (PBS containing 0.05% Tween 20) and blocked with blocking solution (PBS containing 3% skim milk) at 37 C for 1 h. After washing once with PBST, 50 ml serum diluted (1:100) with blocking solution were added to duplicate wells and then incubated at 37 C for 1 h. After washing five times with PBST, the plates were incubated with 50 ml horseradish peroxidase (HRP)-conjugated goat anti-dog total IgG
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secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) diluted at 1:4000 at 37 C for 1 h. After washing five times with PBST, the plates were incubated with 100 ml of the substrate solution (0.3 mg of 2,20 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) per ml, 0.1 M citric acid, 0.2 M sodium phosphate, 0.003% H2O2) each well at room temperature for 1 h. We calculated absorbance values as the difference in the mean optical densities at 415 nm (OD415) between the recombinant antigens (NcGRA2t, NcSAG1t) and the GST protein. We determined the cutoff value for dogs' sera samples as the mean OD415 for standard Neosporanegative sera (15 SPF dogs) plus three-fold standard deviations. 3. Results 3.1. Expression of NcGRA2t in E. coli We cloned the NcGRA2t gene lacking the N-terminal signal peptide sequence into the prokaryotic expression vector pGEX-4T1, and expressed it in E. coli as a soluble GST-fusion protein, with a molecular weight of approximately 48 kDa, including the 26 kDa GST tag (Fig. 1). Serum from a dog experimentally infected with N. caninum recognized the recombinant NcGRA2t protein by western blot, but there was no reactivity with the GST protein (Fig. 1).
Fig. 2. Cross-reactivity of NcGRA2t with closely related parasite-infected mice sera by ELISA. Lane 1, N. caninum infected-mice sera (n ¼ 10); Lane 2, T. gondii infected-mice sera (n ¼ 10); Lane 3, uninfected-mice sera (n ¼ 10).
antigen in the sera of two experimentally infected dogs. The results showed that all dogs infected with N. caninum produced IgG antibodies that were present in the samples 2 weeks after infection. Serum antibody levels against NcSAG1t peaked at 3 weeks after infection and remained at detectable levels until 28 weeks (Fig. 3B).
3.2. Evaluation of the diagnostic potential of NcGRA2t by ELISA In order to determine the specificity of NcGRA2t as a diagnostic antigen, we evaluated the cross-reactivity of NcGRA2t with the closely related T. gondii-infected mice sera by ELISA. We used the recombinant antigens to test the sera from 10 mice infected with N. caninum, 10 mice infected with T. gondii, and 10 uninfected mice. All of the sera samples from the N. caninum-infected mice showed reactivity against the NcGRA2t antigen, while the sera of T. gondiiinfected and uninfected mice showed no reactivity (Fig. 2). These results suggest it is possible to use an ELISA with antibodies specific to the NcGRA2t antigen to detect an N. caninum infection. In addition, we used an ELISA to evaluate the diagnostic potential of NcGRA2t for N. caninum serodiagnosis, and to compare NcGRA2t antigenicity against the established parasite antigen, NcSAG1t. The ELISA measured the IgG antibody levels against the
Fig. 1. SDS-PAGE and western blot analysis of recombinant NcGRA2t. Lane M, low molecular weight marker; Lanes 1e2, stained with Coomassie Brilliant Blue; Lanes 3e4, probed with N. caninum-infected dog serum; Lanes 1 and 3, rNcGRA2t with GST; Lane 2 and 4, GST.
Fig. 3. IgG antibody responses against NcGRA2t (A) and NcSAG1t (B) in dogs (n ¼ 2) at 0, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 28 weeks after experimental infection with N. caninum tachyzoites. Sera samples diluted 1:100 were tested by ELISA. The cutoff values were 0.22 for anti-NcGRA2t IgG and 0.12 for anti-NcSAG1t IgG.
C. Jin et al. / Experimental Parasitology 157 (2015) 88e91
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We detected antibody levels against NcGRA2t at 2 weeks after infection, with peak levels being detectable at 3 weeks after infection, and rapidly decreasing after 4 weeks (Fig. 3A). At 12 weeks after infection, we did not detect serum antibody against NcGRA2t, although we still observed reactivity with the antibody against NcSAG1t (Fig. 3B).
could be a valuable diagnostic tool for the detection of antibodies to N. caninum in dogs. Further serological surveys in dogs that rise under different living condition need to be accomplished, as well as, the association between seropositive status and neurological signs.
4. Discussion
Acknowledgments
Neosporosis is a common disease worldwide, and intermediate host infection may occur by ingestion of excreted Neospora oocysts by dogs, after infection (Dubey and Lindsay, 1996). Diagnosis and protection are important to intermediate host and definitive host. Total proteins of N. caninum as a detector for the serodiagnosis of neosporosis have cross-reaction with T. gondii. Other proteins, such as NcSAG1, NcSRS2 and NcGRA7, no cross-reaction was observed between N. caninum and T. gondii (Nishikawa et al., 2002, Hiasa et al., 2012). Therefore, they could be used to distinguish these two infectious parasites. Even though N. caninum has a similar morphology to T. gondii, early research indicated the protein sequence of NcGRA2 exhibited only 50% sequence identity to TgGRA2 (Ellis et al., 2000). Previous reports suggested NcGRA2 as a potential species-specific protein of N. caninum, based on the 2-DE maps comparing the proteomes between N. caninum and T. gondii (Lee et al., 2005). In this study, we developed an ELISA diagnostic method based on the recombinant N. caninum protein, rNcGRA2t for the detection of the dogs. rNcGRA2t was expressed in E. coli as a soluble GST-fusion protein, and western blot analysis showed that serum from a dog experimentally infected with N. caninum reacted with rNcGRA2t, indicating that rNcGRA2t was antigenic. In addition, a specificity test using sera from mice experimentally infected with T. gondii indicated that the NcGRA2t recombinant proteins do not exhibit cross-reactivity with T. gondii. The development of diagnostic tools identifying active and chronic stages of infection is important in order to control neosporosis. The NcGRA2 gene is highly expressed during N. caninum asexual development (Ellis et al., 2000), therefore, secreted antigens could be a suitable source of antigen for the detection of early infection, and it may reflect a more accurate infection status of the host. Previous research showed that anti-NcGRA7 IgG was detected during acute infection of N. caninum dogs (Hiasa et al., 2012). Similarly, our study demonstrates that in experimentally infected dogs, the levels of IgG antibody against NcGRA2t are detectable during the acute stage of infection. In addition, antibody levels of anti-NcGRA2t were higher than those levels of anti-NcSAG1t at the acute stage, suggesting NcGRA2t has a higher antigenicity. Using NcSAG1t as a potential candidate antigen for identification of infection has previously proven useful for both cattle and dogs. Our study provides additional data showing maintenance of high levels of IgG against NcSAG1t over an extended period. These results suggest that the anti-NcSAG1t antibody detected the N. caninum infection at both the acute and chronic stages, and that the production of anti-NcSAG1t and anti-NcGRA2t antibodies identify NcSAG1t and NcGRA2t as potential candidate antigens to discriminate between the active and chronic stages of infection. Further study. In conclusion, we demonstrated the dynamics of anti-NcSAG1t and anti-NcGRA2t antibodies in experimentally N. caninum-infected dogs. This suggests that ELISA with NcGRA2t and NcSAG1t
We thank Dr. Dubey (United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, and Parasite Biology and Epidemiology Laboratory) for the gift of Neospora caninum, Nc-1 isolate. This research was supported by Jilin Provincial Agriculture Research System, China (201327).
References Chahan, B., Gaturaga, I., Huang, X., Liao, M., Fukumoto, S., Hirata, H., Nishikawa, Y., Suzuki, H., Sugimoto, C., Nagasawa, H., Fujisaki, K., Igarashi, I., Mikami, T., Xuan, X., 2003. Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1. Vet. Parasitol. 118, 177e185. Dubey, J.P., 2003. Review of Neospora caninum and neosporosis in animals. Korean J. Parasitol. 41, 1e16. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1e59. Dubey, J.P., Schares, G., 2006. Diagnosis of bovine neosporosis. Vet. Parasitol. 140, 1e34. Dubey, J.P., Hattel, A.L., Lindsay, D.S., Topper, M.J., 1988. Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. J. Am. Vet. Med. Assoc. 193, 1259e1263. Dubey, J.P., Buxton, D., Wouda, W., 2006. Pathogenesis of bovine neosporosis. J. Comp. Pathol. 134, 267e289. Dubey, J.P., Vianna, M.C., Kwok, O.C., Hill, D.E., Miska, K.B., Tuo, W., Velmurugan, G.V., Conors, M., Jenkins, M.C., 2007. Neosporosis in Beagle dogs: clinical signs, diagnosis, treatment, isolation and genetic characterization of Neospora caninum. Vet. Parasitol. 149 (3e4), 158e166. Dubey, J.P., Jenkins, M.C., Rajendran, C., Miska, K., Ferreira, L.R., Martins, J., Kwok, O.C., Choudhary, S., 2011. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Vet. Parasitol. 181 (2e4), 382e387. Ellis, J.T., Ryce, C., Atkinson, R., Balu, S., Jones, P., Harper, P.A., 2000. Isolation, characterization and expression of a GRA2 homologue from Neospora caninum. Parasitology 120 (Pt 4), 383e390. Gondim, L.F.P., McAllister, M.M., Pitt, W.C., Zemlicka, D.E., 2004. Coyotes (Canis latrans) are denitive hosts of Neospora caninum. Int. J. Parasitol. 34, 159e161. Hiasa, J., Nishimura, M., Itamoto, K., Xuan, X., Inokuma, H., Nishikawa, Y., 2012. Enzyme-linked immunosorbent assays based on Neospora caninum dense granule protein 7 and profilin for estimating the stage of neosporosis. Clin. Vaccine Immunol. 19, 411e417. Huang, P., Liao, M., Zhang, H., Lee, E.G., Nishikawa, Y., Xuan, X., 2007. Dense-granule protein NcGRA7, a new marker for the serodiagnosis of Neospora caninum infection in aborting cows. Clin. Vaccine Immunol. 14, 1640e1643. King, J.S., Slapeta, J., Jenkins, D.J., Al-Qassab, S.E., Ellis, J.T., Windsor, P.A., 2010. Australian dingoes are definitive hosts of Neospora caninum. Int. J. Parasitol. 40 (8), 945e950. Lee, E.G., Kim, J.H., Shin, Y.S., Shin, G.W., Kim, Y.R., Palaksha, K.J., Kim, D.Y., Yamane, I., Kim, Y.H., Kim, G.S., Suh, M.D., Jung, T.S., 2005. Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 815, 305e314. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473e1478. Nishikawa, Y., Claveria, F.G., Fujisaki, K., Nagasawa, H., 2002. Studies on serologicalcross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. J. Vet. Med. Sci. 64 (2), 161e164. Strohbusch, M., Muller, N., Hemphill, A., Greif, G., Gottstein, B., 2008. NcGRA2 as a molecular target to assess the parasiticidal activity of toltrazuril against Neospora caninum. Parasitology 135, 1065e1073. Ybanez, R.H., Terkawi, M.A., Kameyama, K., Xuan, X., Nishikawa, Y., 2013. Identification of a highly antigenic region of subtilisin-like serine protease 1 for serodiagnosis of Neospora caninum infection. Clin. Vaccine Immunol. 20, 1617e1622.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Full length article
Evaluation of Neospora caninum truncated dense granule protein 2 for serodiagnosis by enzyme-linked immunosorbent assay in dogs Chunmei Jin 1, Longzheng Yu 1, Yinan Wang, Shiyue Hu, Shoufa Zhang* Department of Veterinary Medicine, Yanbian University, Yanji, Jilin Province 133002, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
NcGRA2t recombinant proteins do not exhibit cross-reactivity with Toxoplasma gondii. Established an ELISA based on NcGRA2t to detect Neospora caninum-infection in dogs. NcGRA2t could be a suitable source of antigen for the detection of early infection.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 August 2014 Received in revised form 20 May 2015 Accepted 8 July 2015 Available online 11 July 2015
Neosporosis is an infectious disease caused by Neospora caninum, and it primarily affects cattle and dogs. An infection by N. caninum causes fetal abortion and neonatal mortality. Previous proteomics and immunoscreening analyses revealed that N. caninum dense granule antigen 2 (NcGRA2) has potential for serodiagnosis of N. caninum. Consequently, we expressed the truncated NcGRA2 (NcGRA2t), which lacks a signal peptide. We compared the serodiagnostic performances of recombinant NcGRA2t with that of truncated surface antigen 1 of N. caninum (NcSAG1t). Specificity testing using sera from mice infected with Toxoplasma gondii indicated that the NcGRA2t recombinant protein does not cross-react with T. gondii. In addition, we detected anti-NcGRA2t antibody at the acute stage in experimentally infected dogs, while detecting anti-NcSAG1t antibody during both the acute and chronic stages. Our results suggest that the levels of anti-NcGRA2 antibody reflect parasite activation in dogs. In conclusion, antibodies against NcGRA2t and NcSAG1t are suitable indicators to distinguish the acute and chronic stages of N. caninum infection. © 2015 Elsevier Inc. All rights reserved.
Keywords: Neospora caninum NcGRA2t ELISA
1. Introduction Neospora caninum is an obligate intracellular apicomplexan parasite (Dubey et al., 1988). Domestic dogs, coyotes, dingoes and wolves are the known definitive host for N. caninum (McAllister * Corresponding author. E-mail address: [email protected] (S. Zhang). 1 The authors contributed equally to this study. http://dx.doi.org/10.1016/j.exppara.2015.07.003 0014-4894/© 2015 Elsevier Inc. All rights reserved.
et al., 1998; Gondim et al., 2004; King et al., 2010; Dubey et al., 2011), and many warm-blooded vertebrates, such as cattle, sheep, goats, deer, and horses are intermediate hosts (Dubey, 2003). Neosporosis now appears to be a major cause of bovine abortion or stillbirth and paralysis of rear limbs, often with contracture in dogs worldwide, and causes severe economic losses (Dubey et al., 2007). Hosts can acquire N. caninum by ingesting tissues of infected animals, or food or drinking water contaminated with oocysts shed by dogs, the definitive host (Dubey and Lindsay, 1996). Therefore,
C. Jin et al. / Experimental Parasitology 157 (2015) 88e91
specific and sensitive detection of this pathogen is very important to distinguish healthy animals from infected animals. Currently, a number of serological tests exist for detecting N. caninum specific antibodies, including the indirect fluorescence antibody test (IFAT), the N. caninum agglutination test (NAT), and immunoblotting tests, which are based on tachyzoite antigens (Dubey et al., 2006). Several enzyme-linked immunosorbent assays (ELISAs) using recombinant antigens have been widely evaluated to detect N. caninum-specific antibodies. A number of serodiagnostic assays for bovine or dog neosporosis use recombinant antigens, such as surface antigen 1 of N. caninum (NcSAG1), N. caninum dense granule antigen 7 (NcGRA7), and N. caninum subtilisin-like serine protease 1 (NcSUB1) (Chahan et al., 2003; Dubey and Schares, 2006; Huang et al., 2007; Ybanez et al., 2013). NcSAG1 is one of the antigens with the ability to detect anti-NcSAG1 antibody during both acute and chronic stages of infection (Hiasa et al., 2012). NcGRA7 is a suitable antigen for detection of N. caninum infection in the activation stage (Hiasa et al., 2012). Both tachyzoites and bradyzoites express the N. caninum dense granule antigen 2 gene (NcGRA2) (Strohbusch et al., 2008). NcGRA2 is a species-specific protein of N. caninum based on the comparison of two-dimensional gel electrophoresis (2-DE) profiles between N. caninum and Toxoplasma gondii (Lee et al., 2005). These findings suggest that NcGRA2 has potential use in the serodiagnosis of N. caninum, and requires further analyses to determine the utility of NcGRA2 in a diagnostic setting. In this study, we expressed truncated NcGRA2 (NcGRA2t) as a glutathione S-transferase (GST) fusion protein in Escherichia coli. Furthermore, we evaluated its diagnostic potential against N. caninum infection in dogs by comparing the results obtained from an established ELISA, based on truncated NcSAG1 (NcSAG1t).
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proteins as previously described (Chahan et al., 2003). Briefly, incubate pGEX-NcSAG1t and pGEX-NcGRA2t transformants at 37 C at 250 rpm until the OD600 level reached 0.5. Synthesis of GST-NcSAG1t and GST-NcGRA2t were induced with 1 mM IPTG at 37 C for 4 h. Harvest cells by centrifugation and treated by TNE buffer (50 mM TriseHCl, pH7.5, 0.1 M NaCl, 2 mM EDTA) containing lysozyme (100 mg/ml) and 1% Triton X-100. After centrifugation, the supernatant was harvested, and the recombinant protein was purified with Glutathione-Sepharose 4B, according to the manufacturer's instructions (Amersham Pharmacia Biotech, Sweden). We measured the protein concentration using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA). 2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting We verified protein expression by SDS-PAGE after staining with Coomassie Brilliant Blue (MP Biomedicals, Inc., France). Thereafter, we used western blot analyses to determine the antigenicity of the NcGRA2t protein for application in serodiagnostic methods as previously described (Chahan et al., 2003). Briefly, After the proteins were transferred electrophoretically onto a polyvinylidene membrane (Millipore, USA), the membrane was blocked for overnight at 4 C with PBS containing 3% skim milk, and then the membrane was incubated with a diluted dog anti-N. caninum serum (1:100) for 1 h and washed three times with PBS. Following incubation with an HRP-conjugated goat anti-dog IgG (BETHYL, USA) (1:1000) for 1 h and wash three times, the membrane was placed into a substrate solution (0.5 mg/ml diaminobenzidine, 0.005% H2O2) to visualize the specific antigen bands. 2.4. Sera
2. Materials and methods 2.1. Parasite preparation We maintained N. caninum tachyzoites of the Nc-1 strain and T. gondii tachyzoites of the PLK strain in monkey kidney adherent fibroblast (Vero) cells cultured in Eagle's minimum essential medium (MEM; SigmaeAldrich, St. Louis, MO, USA) supplemented with 8% heat-inactivated fetal bovine serum (FBS) and 50 mg/ml kanamycin at 37 C in a 5% CO2 air environment. We purified tachyzoites by washing the parasites and host-cell debris in cold phosphate-buffered saline (PBS), and then we resuspended the final pellet in cold PBS, and passed it through a 27-gauge needle and a 5.0-mm pore filter (Millipore, Billerica, MA, USA). 2.2. Expression and purification of recombinant NcGRA2t in E. coli We prepared total RNA from Nc-1 tachyzoites using the RNeasy Plus Mini Kit (Qiagen, Germany), and used RNA as a template to amplify the coding region of NcGRA2t (GenBank accession no. NCLIV_045650) using the One Step RT PCR Kit (Takara, Japan). We performed all procedures according to the respective manufacturer's instructions. We designed a set of oligonucleotide primers, including EcoRI and XhoI restriction enzyme sites, to clone the truncated gene without a signal peptide (forward primer, 50 -AC GAATTC GCCGATTTTTCTGGCAGGG-30 ; reverse primer, 50 -CC CTCGAG TTAATTGACTTCAGCTTCTGG-30 ). We digested PCR products with EcoRI and XhoI before ligation into the glutathione Stransferase (GST) fusion protein in the E. coli expression vector, pGEX-4T1 (GE Healthcare, UK), which we digested with the same set of restriction enzymes. We prepared NcSAG1t as previously described (Chahan et al., 2003). We expressed the recombinant proteins of NcSAG1t and NcGRA2t in E. coli BL21 (DE3) as GST fusion
Sera used for the present study were as follows: 10 sera from BALB/c mice experimentally infected with 4 104 tachyzoites of the N. caninum Nc-1 strain, 10 sera from BALB/c mice experimentally infected with 4 102 tachyzoites of the T. gondii PLK strain, 10 sera from healthy BALB/c mice. We prepared experimentally infected dogs sera with N. caninum using two SPF (specific-pathogen-free) dogs as previously described (Hiasa et al., 2012). Before experimental infection, dogs were proved to be free of N. caninumand T. gondii-specific antibody by ELISA based on lysates of N. caninum and T. gondii. We obtained sera weekly from dogs via venipuncture of the radial vein and stored it at 20 C until use. Mice were given food and water ad libitum while the dogs were fed every day and given water ad libitum. All of the animals used in the present study were conducted in accordance with the Standards Relating to the Care and Management of Experimental Animals promulgated by the Yanbian University, China. 2.5. ELISA We performed the ELISA as previously described (Chahan et al., 2003). We diluted the GST-fused NcGRA2t and NcSAG1t recombinant proteins, and the GST control, in coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) to a final concentration of 0.1 mM. We coated each well of a 96-well microtiter plate (Nunc, Denmark) with 50 ml of the corresponding protein, and incubated the plate overnight at 4 C. Plates were washed once with PBST (PBS containing 0.05% Tween 20) and blocked with blocking solution (PBS containing 3% skim milk) at 37 C for 1 h. After washing once with PBST, 50 ml serum diluted (1:100) with blocking solution were added to duplicate wells and then incubated at 37 C for 1 h. After washing five times with PBST, the plates were incubated with 50 ml horseradish peroxidase (HRP)-conjugated goat anti-dog total IgG
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secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) diluted at 1:4000 at 37 C for 1 h. After washing five times with PBST, the plates were incubated with 100 ml of the substrate solution (0.3 mg of 2,20 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) per ml, 0.1 M citric acid, 0.2 M sodium phosphate, 0.003% H2O2) each well at room temperature for 1 h. We calculated absorbance values as the difference in the mean optical densities at 415 nm (OD415) between the recombinant antigens (NcGRA2t, NcSAG1t) and the GST protein. We determined the cutoff value for dogs' sera samples as the mean OD415 for standard Neosporanegative sera (15 SPF dogs) plus three-fold standard deviations. 3. Results 3.1. Expression of NcGRA2t in E. coli We cloned the NcGRA2t gene lacking the N-terminal signal peptide sequence into the prokaryotic expression vector pGEX-4T1, and expressed it in E. coli as a soluble GST-fusion protein, with a molecular weight of approximately 48 kDa, including the 26 kDa GST tag (Fig. 1). Serum from a dog experimentally infected with N. caninum recognized the recombinant NcGRA2t protein by western blot, but there was no reactivity with the GST protein (Fig. 1).
Fig. 2. Cross-reactivity of NcGRA2t with closely related parasite-infected mice sera by ELISA. Lane 1, N. caninum infected-mice sera (n ¼ 10); Lane 2, T. gondii infected-mice sera (n ¼ 10); Lane 3, uninfected-mice sera (n ¼ 10).
antigen in the sera of two experimentally infected dogs. The results showed that all dogs infected with N. caninum produced IgG antibodies that were present in the samples 2 weeks after infection. Serum antibody levels against NcSAG1t peaked at 3 weeks after infection and remained at detectable levels until 28 weeks (Fig. 3B).
3.2. Evaluation of the diagnostic potential of NcGRA2t by ELISA In order to determine the specificity of NcGRA2t as a diagnostic antigen, we evaluated the cross-reactivity of NcGRA2t with the closely related T. gondii-infected mice sera by ELISA. We used the recombinant antigens to test the sera from 10 mice infected with N. caninum, 10 mice infected with T. gondii, and 10 uninfected mice. All of the sera samples from the N. caninum-infected mice showed reactivity against the NcGRA2t antigen, while the sera of T. gondiiinfected and uninfected mice showed no reactivity (Fig. 2). These results suggest it is possible to use an ELISA with antibodies specific to the NcGRA2t antigen to detect an N. caninum infection. In addition, we used an ELISA to evaluate the diagnostic potential of NcGRA2t for N. caninum serodiagnosis, and to compare NcGRA2t antigenicity against the established parasite antigen, NcSAG1t. The ELISA measured the IgG antibody levels against the
Fig. 1. SDS-PAGE and western blot analysis of recombinant NcGRA2t. Lane M, low molecular weight marker; Lanes 1e2, stained with Coomassie Brilliant Blue; Lanes 3e4, probed with N. caninum-infected dog serum; Lanes 1 and 3, rNcGRA2t with GST; Lane 2 and 4, GST.
Fig. 3. IgG antibody responses against NcGRA2t (A) and NcSAG1t (B) in dogs (n ¼ 2) at 0, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 28 weeks after experimental infection with N. caninum tachyzoites. Sera samples diluted 1:100 were tested by ELISA. The cutoff values were 0.22 for anti-NcGRA2t IgG and 0.12 for anti-NcSAG1t IgG.
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We detected antibody levels against NcGRA2t at 2 weeks after infection, with peak levels being detectable at 3 weeks after infection, and rapidly decreasing after 4 weeks (Fig. 3A). At 12 weeks after infection, we did not detect serum antibody against NcGRA2t, although we still observed reactivity with the antibody against NcSAG1t (Fig. 3B).
could be a valuable diagnostic tool for the detection of antibodies to N. caninum in dogs. Further serological surveys in dogs that rise under different living condition need to be accomplished, as well as, the association between seropositive status and neurological signs.
4. Discussion
Acknowledgments
Neosporosis is a common disease worldwide, and intermediate host infection may occur by ingestion of excreted Neospora oocysts by dogs, after infection (Dubey and Lindsay, 1996). Diagnosis and protection are important to intermediate host and definitive host. Total proteins of N. caninum as a detector for the serodiagnosis of neosporosis have cross-reaction with T. gondii. Other proteins, such as NcSAG1, NcSRS2 and NcGRA7, no cross-reaction was observed between N. caninum and T. gondii (Nishikawa et al., 2002, Hiasa et al., 2012). Therefore, they could be used to distinguish these two infectious parasites. Even though N. caninum has a similar morphology to T. gondii, early research indicated the protein sequence of NcGRA2 exhibited only 50% sequence identity to TgGRA2 (Ellis et al., 2000). Previous reports suggested NcGRA2 as a potential species-specific protein of N. caninum, based on the 2-DE maps comparing the proteomes between N. caninum and T. gondii (Lee et al., 2005). In this study, we developed an ELISA diagnostic method based on the recombinant N. caninum protein, rNcGRA2t for the detection of the dogs. rNcGRA2t was expressed in E. coli as a soluble GST-fusion protein, and western blot analysis showed that serum from a dog experimentally infected with N. caninum reacted with rNcGRA2t, indicating that rNcGRA2t was antigenic. In addition, a specificity test using sera from mice experimentally infected with T. gondii indicated that the NcGRA2t recombinant proteins do not exhibit cross-reactivity with T. gondii. The development of diagnostic tools identifying active and chronic stages of infection is important in order to control neosporosis. The NcGRA2 gene is highly expressed during N. caninum asexual development (Ellis et al., 2000), therefore, secreted antigens could be a suitable source of antigen for the detection of early infection, and it may reflect a more accurate infection status of the host. Previous research showed that anti-NcGRA7 IgG was detected during acute infection of N. caninum dogs (Hiasa et al., 2012). Similarly, our study demonstrates that in experimentally infected dogs, the levels of IgG antibody against NcGRA2t are detectable during the acute stage of infection. In addition, antibody levels of anti-NcGRA2t were higher than those levels of anti-NcSAG1t at the acute stage, suggesting NcGRA2t has a higher antigenicity. Using NcSAG1t as a potential candidate antigen for identification of infection has previously proven useful for both cattle and dogs. Our study provides additional data showing maintenance of high levels of IgG against NcSAG1t over an extended period. These results suggest that the anti-NcSAG1t antibody detected the N. caninum infection at both the acute and chronic stages, and that the production of anti-NcSAG1t and anti-NcGRA2t antibodies identify NcSAG1t and NcGRA2t as potential candidate antigens to discriminate between the active and chronic stages of infection. Further study. In conclusion, we demonstrated the dynamics of anti-NcSAG1t and anti-NcGRA2t antibodies in experimentally N. caninum-infected dogs. This suggests that ELISA with NcGRA2t and NcSAG1t
We thank Dr. Dubey (United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, and Parasite Biology and Epidemiology Laboratory) for the gift of Neospora caninum, Nc-1 isolate. This research was supported by Jilin Provincial Agriculture Research System, China (201327).
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