Original Article

Designing and Evaluation of a Recombinant Multiepitope Pro-tein by Using ELISA for Diagnosis of Leishmania infantum In-fected in Dogs

Abstract

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Correct identification of infected patients and reservoirs is vital to control the spread of VL. One important step in the control of Zoonotic Visceral leishmaniasis (ZVL) is the identification of infected dogs, which are the main domestic reservoir hosts of Leishmania infantum. We aimed to prepare and evaluate a new recombinant antigen using Bioinformatics tools for diagnosis of ZVL in domestic dogs.

Methods: The present study was carried out in Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran  during 2015- 2018. Three L. infantum (JPCM5 strain) proteins were analyzed as follows: Nucleotide sequences of the surface proteins, putative amastin-like surface protein (P1), surface antigen protein 2 precursor (P2) and surface antigen-like protein (P3). The epitopes were predicted by several different bioinformatics servers using different methods. The predicted epitopes were selected with the highest immunogenic potential (P1P2P3) linked to each other with linkers (Gly, Se) and synthesized. Then the expression and protein purification were performed. In total, 114 serum samples were collected at 7 months. Positive and negative sera were confirmed using direct agglutination test (DAT). These recombinant antigens from L. infantum were used by indirect ELISA.

Results: Considering the cut-off point of 0.23, the test showed a sensitivity of 98% (95%CI=89.50%-99.90%) and a specificity of 95.31% (95%CI=87.10%-98.72%). Kappa analysis indicated very good agreement (kappa=0.831) between ELISA and DAT (P<0.05).

Conclusion: ELISA using the recombinant protein P1P2P3 has great potential for the diagnosis of canine visceral leishmaniasis (CVL).

1. Abass E, Kang C, Martinkovic F, et al et al. Heterogeneity of Leishmania donovani para-sites complicates diagnosis of visceral leishmaniasis: comparison of different se-rological tests in three endemic regions. PLoS One. 2015;10(3):e0116408.
2. Mohebali M. Visceral leishmaniasis in Iran: Review of the Epidemiological and Clinical Features. Iran J Parasitol. 2013;8(3):348-358.
3. Mohebali M, Moradi-Asl E, Rassi Y. Geo-graphic distribution and spatial analysis of Leishmania infantum infection in domestic and wild animal reservoir hosts of zoono-tic visceral leishmaniasis in Iran: A system-atic review. J Vector Borne Dis. 2018;55(3):173-183.
4. Bern C, Maguire JH, Alvar J. Complexities of assessing the disease burden attributa-ble to leishmaniasis. PLoS Negl Trop Dis. 2008;2(10): e313.
5. Maia C, Campino L. Cytokine and Pheno-typic Cell Profiles of Leishmania infantum In-fection in the Dog. J Trop Med. 2012; 541-571.
6. Mohebali M, Edrissian GH, Shirzadi MR, et al. An observational study on the cur-rent distribution of visceral leishmaniasis in different geographical zones of Iran and implication to health policy. Travel Med Infect Dis. 2011;9(2):67-74.
7. Reiner SL, Locksley RM. The regulation of immunity to Leishmania major. Annu Rev Immunol. 1995;13:151-177.
8. Dantas-Torres F, de Brito ME, Brandao-Filho SP. Seroepidemiological survey on canine leishmaniasis among dogs from an urban area of Brazil. Vet Parasitol. 2006;140(1-2):54-60.
9. Moshfe A, Mohebali M, Edrissian G, et al. Canine visceral leishmaniasis: asymptomat-ic infected dogs as a source of L. infantum infection. Acta Trop. 2009;112(2):101-105
10. Souza AP, Soto M, Costa JM, et al. To-wards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins. PLoS One. 2013;8(6):e66110.
11. Caballero ZC, Sousa OE, Marques WP, Saez-Alquezar A, Umezawa ES. Evaluation of serological tests to identify Trypanosoma cruzi infection in humans and determine cross-reactivity with Trypanosoma rangeli and spp. Clin Vaccine Immunol. 2007;14(8):1045-1049.
12. Denise H, Poot J, Jimenez M, et al. Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5. BMC Mol Biol. 2006;7:42.
13. Harith A, Kolk A, Kager P, et al. A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leish-maniasis. Trans R Soc Trop Med Hyg. 1986;80(4):583-536.
14. Hosseininejad M, Mohebali M, Hosseini F, Karimi S, Sharifzad S, Akhoundi B. Sero-prevalence of canine visceral leishmaniasis in asymptomatic dogs in Iran. Iranian Journal of Veterinary Research, Shiraz University. 2012; 13(1):1-4.
15. Rafati S, Nakhaee A, Taheri T, et al. Pro-tective vaccination against experimental ca-nine visceral leishmaniasis using a combi-nation of DNA and protein immunization with cysteine proteinases type I and II of L. infantum. Vaccine. 2005; 23(28): 3716–3725.
16. Greiner M, Pfeiffer D, Smith RD. Princi-ples and practical application of the receiv-er-operating characteristic analysis for di-agnostic tests. Prev Vet Med. 2000;45(1-2):23-41.
17. Molina R, Amela C, Nieto J, et al. Infectivi-ty of dogs naturally infected with Leishma-nia infantum to colonized Phlebotomus pernicio-sus. Trans R Soc Trop Med Hyg. 1994;88(4):491-493.
18. Mohebali M, Hajjaran H, Hamzavi Y, et al. Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Vet Parasitol. 2005;129(3-4):243-251.
19. Barati M, Mohebali M, Alimohammadian MH, Khamesipour A, Akhoundi B, Zarei Z. Canine visceral leishmaniasis: sero-prevalence survey of asymptomatic dogs in an endemic area of northwestern Iran. J Parasit Dis. 2015;39(2):221-224.
20. Faria AR, Costa MM, Giusta MS, et al .High-throughput analysis of synthetic peptides for the immunodiagnosis of ca-nine visceral leishmaniasis. PLoS Negl Trop Dis. 2011;5(9):e1310.
21. Zanette MF, Lima VM, Laurenti MD, et al. Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Ne-ospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs. Rev Soc Bras Med Trop. 2014;47(1):105-107.
22. Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol. 2002;9(5):951-958.
23. EL‐Manzalawy Y, Dobbs D, Honavar V. Predicting linear B‐cell epitopes using string kernels. J Mol Recognit. 2008;21(4):243-255.
24. Skwarczynski M, Toth I. Peptide-based synthetic vaccines. Chem Sci. 2016;7(2):842-854.
25. Larsen JEP, Lund O, Nielsen M. Im-proved method for predicting linear B-cell epitopes. Immunome Res. 2006;2:2.
26. Chavez-Fumagalli MA, Martins VT, Tes-tasicca MC, et al. Sensitive and specific se-rodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an im-munoproteomic approach. Clin Vaccine Immunol. 2013;20(6):835-841.
27. Taran M, Mohebali M, Modaresi M, Mamishi S, Mahmoudi M, Mojarad M. Diagnosis of canine visceral leishmaniasis by ELISA using K39sub recombinant an-tigen. Iran. J Public Health. 2007;36(2):1-6.
28. Porrozzi R, Santos da Costa MV, Teva A, et al. Comparative evaluation of enzyme-linked immunosorbent assays based on crude and recombinant leishmanial anti-gens for serodiagnosis of symptomatic and asymptomatic Leishmania infantum vis-ceral infections in dogs. Clin Vaccine Im-munol. 2007;14(5):544-548.
29. Costa DN, Codeco CT, Silva MA, Wer-neck GL. Culling dogs in scenarios of im-perfect control: realistic impact on the prevalence of canine visceral leishmaniasis. PLoS Negl Trop Dis. 2013;7(8): e2355.
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IssueVol 16 No 3 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v16i3.7090
Keywords
Visceral leishmaniasis Leishmania infantum; Multi-epitope Domestic dog
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How to Cite
1.
Yaghoubi P, Bandehpour M, Mohebali M, Akhoundi B, Kazemi B. Designing and Evaluation of a Recombinant Multiepitope Pro-tein by Using ELISA for Diagnosis of Leishmania infantum In-fected in Dogs. Iran J Parasitol. 2021;16(3):377-385.