Potential application of rLc36 protein for diagnosis of canine visceral leishmaniasis

Visceral leishmaniasis (VL) is fatal if left untreated. Infected dogs are important reservoirs of the disease, and thus specific identification of infected animals is very important. Several diagnostic tests have been developed for canine VL (CVL); however, these tests show varied specificity and sensitivity. The present study describes the recombinant protein rLc36, expressed by Leishmania infantum, as potential antigen for more sensitive and specific diagnosis of CVL based on an immunoenzymatic assay. The concentration of 1.0 μg/mL of rLc36 enabled differentiation of positive and negative sera and showed a sensitivity of 85% and specificity of 71% (with 95% confidence), with an accuracy of 76%.

Key words:
canine visceral leishmaniasis; immunodiagnosis; recombinant protein

Authorship

Camila Tita Nogueira

Universidade Estadual Paulista, Instituto de Química, Campus de Araraquara, Araraquara, SP, BrasilUniversidade Estadual PaulistaBrasilAraraquara, SP, BrasilUniversidade Estadual Paulista, Instituto de Química, Campus de Araraquara, Araraquara, SP, Brasil

Mayara Lúcia Del Cistia

Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas, Campus de Araraquara, Araraquara, SP, BrasilUniversidade Estadual PaulistaBrasilAraraquara, SP, BrasilUniversidade Estadual Paulista, Faculdade de Ciências Farmacêuticas, Campus de Araraquara, Araraquara, SP, Brasil

Ana Carolina Urbaczek

Universidade de São Paulo, Instituto de Química, Campus de São Carlos, São Carlos, SP, BrasilUniversidade de São PauloBrasilSão Carlos, SP, BrasilUniversidade de São Paulo, Instituto de Química, Campus de São Carlos, São Carlos, SP, Brasil

Márcia MG Jusi

Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Campus de Jaboticabal, Jaboticabal, SP, BrasilUniversidade Estadual PaulistaBrasilJaboticabal, SP, BrasilUniversidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Campus de Jaboticabal, Jaboticabal, SP, Brasil

Angela Maria Arenas Velásquez

Rosângela Zacarias Machado

Henrique Ferreira

Universidade Estadual Paulista, Instituto de Biociências, Campus de Rio Claro, Rio Claro, SP, BrasilUniversidade Estadual PaulistaBrasilRio Claro, SP, BrasilUniversidade Estadual Paulista, Instituto de Biociências, Campus de Rio Claro, Rio Claro, SP, Brasil

Flávio Henrique-Silva

Universidade Federal de São Carlos, Departamento de Genética e Evolução, São Carlos, SP, BrasilUniversidade Federal de São CarlosBrasilSão Carlos, SP, BrasilUniversidade Federal de São Carlos, Departamento de Genética e Evolução, São Carlos, SP, Brasil

Hélio Langoni

Universidade Estadual Paulista, Faculdade de Medicina Veterinária e de Zootecnia, Campus de Botucatu, Botucatu, SP, BrasilUniversidade Estadual PaulistaBrasilBotucatu, SP, BrasilUniversidade Estadual Paulista, Faculdade de Medicina Veterinária e de Zootecnia, Campus de Botucatu, Botucatu, SP, Brasil

Paulo Inácio da Costa

Márcia AS Graminha ⁺+ Corresponding author: graminha@fcfar.unesp.br

+ Corresponding author: graminha@fcfar.unesp.br

AUTHORS' CONTRIBUTION

CTN and MLDC provided the experimental data, wrote and reviewed the full manuscript; MASG and PIC designed and reviewed the full manuscript; ACU, MMGJ, AMAV, RZM, HF, FHS and HL provided assistance with experimental data.

CTN and MLDC contributed equally to the study.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (3)

Thumbnail

Fig. 1
quantitative analysis of Lc36 gene expression in different life-cycle stage forms of Leishmania infantum.

Thumbnail

Fig. 2
purification and reactivity of protein rLc36. (A) Western blot analysis of purified His6-tagged rLc36. rLc36 was expressed in Escherichia coli BL21(DE3) after isopropyl β-d-thiogalactopyranoside induction for 4 h and purified by nickel-affinity chromatography using a gradient of imidazole (20, 40, 80, and 200 mM). The purified recombinant protein was assessed by western blotting using anti-histidine mouse antibody. Non-soluble fraction (IF) obtained after cell lysis and before column purification step. Protein molecular weight determined by Kaleidoscope. (B) Reactivity of antigens rLc36. The cut-off used was 0.191, for a confidence of 95%, using 20 negative sera for visceral leishmaniasis.

Thumbnail

Fig. 3
reactivity of 39 positive sera for canine visceral leishmaniasis (CVL) versus rLc36. The dotted line refers to a cut-off of 0.181, calculated based on a confidence of 99%, using 20 negative sera for VL.

Fig. 1 quantitative analysis of Lc36 gene expression in different life-cycle stage forms of Leishmania infantum.

Source: authors.

Fig. 2 purification and reactivity of protein rLc36. (A) Western blot analysis of purified His6-tagged rLc36. rLc36 was expressed in Escherichia coli BL21(DE3) after isopropyl β-d-thiogalactopyranoside induction for 4 h and purified by nickel-affinity chromatography using a gradient of imidazole (20, 40, 80, and 200 mM). The purified recombinant protein was assessed by western blotting using anti-histidine mouse antibody. Non-soluble fraction (IF) obtained after cell lysis and before column purification step. Protein molecular weight determined by Kaleidoscope. (B) Reactivity of antigens rLc36. The cut-off used was 0.191, for a confidence of 95%, using 20 negative sera for visceral leishmaniasis.

Source: authors.

Fig. 3 reactivity of 39 positive sera for canine visceral leishmaniasis (CVL) versus rLc36. The dotted line refers to a cut-off of 0.181, calculated based on a confidence of 99%, using 20 negative sera for VL.

Source: authors.

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Potential application of rLc36 protein for diagnosis of canine visceral leishmaniasis

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[1] + Corresponding author: graminha@fcfar.unesp.br