Development of an Indirect ELISA Using Different Fragments of Recombinant Ncgra7 for Detection of Neospora caninum Infection in Cattle and Water Buffalo



Background: Dense granules are immunodominant proteins for the standardi-zation of immunodiagnostic procedures to detect neosporosis. In the presented study different fragment of a dense-granule protein was evaluated for serodiagno-sis of Neospora caninum in cattle and water buffalo.

Methods:NcGRA7, from N. caninum tachyzoites was amplified. PCR product and pMAL-c2X plasmid were digested with EcoR1 restriction enzyme and ex-pressed in Escherichia coli to evaluate its competence for detection of anti- N. cani-num antibodies with ELISA in comparison with commercial IDEXX ELISA. Fur-thermore, 230 sera of presumably healthy cattle and water buffaloes (108 cattle and 122 water buffaloes) were analyzed by both tests to determine the agreement of these two procedures.

Results:Sensitivities and specificities of NcGRA7-based ELISA were 94.64% and 90.38% respectively using sera of cattle, but were 98.57% and 86.54% in the case of buffaloes respectively. A good correlation between the results of IDEXX ELISA and ELISA based on recombinant NcGRA7 for detecting N. caninum an-tibodies was appeared. Analyzing by Mc Nemar´s showed that NcGRA7-based ELISA has acceptable capability to differentiate the positive results in comparison with IDEXX ELISA.

Conclusion:NcGRA7-based ELISA considering utilized new fragment of ge-nomic DNA is a good tool for serodiagnosis of anti- N. caninum antibodies for screening and epidemiological purposes on cattle herd and water buffaloes as well.

Dubey JP, Schares G. Diagnosis of bovine neosporosis. Vet Parasitol. 2006; 140: 1–34.

Anderson M, Barr B, Rowe J, Conrad P. Ne-osporosis in dairy cattle. Jpn J Vet Res. 2012; 60: 51-4.

Bjerkas I, Mohn SF, Presthus J. Unidentified cyst-forming sporozoon causing encephalomy-elitis and myositis in dogs. Z Parasitenkunde 1984; 70: 271–274.

Dubey JP, Lindsay DS. A review of Neospora caninum and neosporosis. Vet Parasitol. 1996; 67: 1–59.

McAllister MM, Dubey JP, Lindsay DS et al. Dogs are definitive hosts of Neospora caninum. Int J Parasitol. 1998; 28: 1473–1478.

Gondim LFP, McAllister MM, Pitt WC, Zem-licka DE. Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int J Parasitol. 2004; 34: 159–161.

Klevar S. Tissue cyst forming coccidian; Toxo-plasma gondii and Neospora caninum as a cause of disease in farm animals. Acta Vet Scand. 2007. 49, (Suppl1): S1.

Sharifdini M, Mohebali M, Keshavarz H, Hos-seininejad M, Hajjaran H, Akhoundi B, Rahimi Foroushani A, Zarei Z, Charehdar S. Neospora caninum and Leishmania infantum co-infection in domestic dogs (Canis familiaris) in Meshkin-Shahr district, Northwestern Iran. Iran J Ar-thropod-Borne Dis. 2011; 1(3): 60–68.

Dubey JP. Recent advances in Neospora and neosporosis. Vet Parasitol. 1999; 84: 349-367.

Hall CA, Reichel MP, Ellis JT. Neospora abor-tions in dairy cattle: diagnosis, mode of trans-mission and control. Vet Parasitol. 2005; 128: 231–241.

Osawa T, Wastling J, Maley S et al. A multiple antigen ELISA to detect Neospora-specific anti-bodies in bovine sera, bovine foetal fluids, ovine and caprine sera. Vet Parasitol. 1998; 79: 19–34.

Packham AE, Sverlow KW, Conrad PA et al. A modified agglutination test for Neospora cani-num: Development, optimization, and compari-son to the indirect fluorescent-antibody test, and comparison to the indirect fluorescent-antibody test and enzyme-linked immuno-sorbent assay. Clin Diagn Lab Immunol. 1998; 5: 467–473.

Atkinson R, Harper PAW, Reichel MP, Ellis JT. Progress in the serodiagnosis of Neospora caninum infections of cattle. Parasitol Today. 2000; 3: 110–114.

Silva DAO, Lobato J, Mineo TWP, Mineo JR. Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimiza-tion of cut-off titers and inhibition studies of cross reactivity with Toxoplasma gondii. Vet Para-sitol. 2007; 143:234–244.

Howe DK, Crawford AC, Lindsay D, Sibley LD. The p29 and p35 immunodominant anti-gens of Neospora caninum tachyzoites are ho-mologous to the family of surface antigens of Toxoplasma gondii. Infect Immun. 1998; 66:5322–5328.

Aguado-Martı´nez A, lvarez-Garcı´a A´, Fer-na´ndez-Garcı´a G et al. Usefulness of rNcGRA7- and rNcSAG4-based ELISA tests for distinguishing primo-infection, recrudes-cence, and chronic bovine neosporosis. Vet Parasitol. 2008; 157: 182–195.

Romano MI. An applied printing immunoassay with recombinant Nc-SAG1 for detection of antibodies to Neospora caninum in cattle. J Vet Diagn Inves. 2011; 23: 971–976.

Hiasa J, Nishimura M, Itamoto K et al. En-zyme-linked immunosorbent assays based on Neospora caninum dense granule protein 7 and profilin for estimating the stage of neosporosis. Clin Vaccine Immunol. 2012; 19:411-417.

Jiménez-Ruiz E, Bech-Sàbat G, Alvarez-García G et al. Specific antibody responses against Ne-ospora caninum recombinant rNcGRA7, rNcSAG4, rNcBSR4 and rNcSRS9 proteins are correlated with virulence in mice. Parasitol-ogy. 2013; 140:569-79.

Terkawi MA, Kameyama K, Rasul NH et al. Development of an immunochromatographic assay based on dense granule protein 7 for se-

rological detection of Toxoplasma gondii infec-tion. Clin Vaccine Immunol. 2013; 20:596-601.

Dubey JP, Desmonts G. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Vet J. 1987; 19: 337-9.

Nigro M, Gutierrez A, Hoffer AM et al. Evalu-ation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired tox-oplasmosis by an immunoglobulin G analysis. Diagn Micr Infect Dis. 2003; 47: 609–613.

Pietkiewicz H, Hiszczyn´ ska-Sawicka E, Kur J et al. Usefulness of Toxoplasma gondii-specific re-combinant antigens in serodiagnosis of human toxoplasmosis. J Clin Microbiol. 2004; 42: 1779–1781.

Chung CT, Niemela SL, Miller RH. One-step preparation of competent Escherichia coli: trans-formation and storage of bacterial cells in the same solution. Biochemistry. 1989; 86: 2172–2175.

Dohoo IR, Martin SW, Stryhn H. In: Dohoo IR. (Ed.). Vet Epidemiol Res. AVC Inc., Char-lottetown, 2003; p: 706.

Dubey JP, Schares G, Ortega-Mora LM. Epi-demiology and control of neosporosis and Ne-ospora caninum. Clin Microbiol Rev. 2007; 20: 323–367.

Chahan B, Gaturaga I, Huang X et al. Serodi-agnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1. Vet Parasitol. 2003; 118: 177–185.

Dubey JP, Hattel AL, Lindsay DS, Topper MJ. Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experi-mental transmission. J Am Vet Med Assoc. 1988; 193: 1259–1263.

Paré J, Hietala SK, Thurmond MC. An en-zyme-linked immunosorbent assay (ELISA) for serological diagnosis of Neospora sp. infec-tion in cattle. J Vet Diagn Invest. 1995; 7:352-599.

Bjorkman C, Alvarez-Garcia G, Conraths FJ et al. Neospora caninum IgG avidity tests: an interla-boratory comparison. Vet Parasitol. 2006; 140: 273–280.

Hara AO, Liao M, Baticados W, Bannai H et al. Expression of recombinant dense granule protein 7 of Neospora caninum and evaluation of its diagnostic potential for canine neosporosis. J Protozool Res. 2006; 16: 34-41.

Sonda S, Fuchs N, Gottstein B, Hemphill A. Molecular characterization of a novel micro-neme antigen in Neospora caninum. Mol Bio-chem Parasitol. 2000. 108:39–51.

Alvarez-Garcia G, Pitarch A, Zaballos A et al. The NcGRA7 gene encodes the immunodom-inant 17 kDa antigen of Neospora caninum. Para-sitology. 2007; 134: 41–50.

Huang P, Liao M, Zhang H, Lee G et al. Dense-granule protein NcGRA7, a new mark-er for the serodiagnosis of Neospora caninum in-fection in aborting cows DI196. Clin Vaccine Immunol. 2007; 14: 1640–1643.

Alvarez-Garcia G, Lopez-Perez I, Innes E Col-lantes-Fernandez et al. Use of an immu-

nodominant P17 antigenic fraction of Neospora caninum in detection of antibody response in cattle. Mem Inst Oswaldo Cruz. 2006; 101: 529–534.

Liddell S, Parker C, Vinyard B et al. Immuniza-tion of mice with plasmid DNA coding for NcGRA7 or NcsHSP33 confers partial protec-tion against vertical transmission of Neospora caninum. J Parasitol. 2003; 89: 496–500.

Wilkowsky SE, Bareiro GG, Mon ML et al. An applied printing immunoassay with recombi-nant Nc-SAG1 for detection of antibodies to Neospora caninum in cattle. J Vet Diagn Invest. 2011; 23:971-6.

IssueVol 10 No 1 (2015) QRcode
Cattle ELISA Iran NcGRA7 Neospora caninum Recombinant antigen Water buffalo

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Hamidinejat H, Seifi Abad Shapouri MR, Namavari MM, Shayan P, Kefayat M. Development of an Indirect ELISA Using Different Fragments of Recombinant Ncgra7 for Detection of Neospora caninum Infection in Cattle and Water Buffalo. Iran J Parasitol. 1;10(1):69-77.