Recombinant gp19 as a potential antigen for detecting anti-<i>Ehrlichia canis</i> antibodies in dog sera

The canine monocytic ehrlichiosis, caused by Ehrlichia canis, is endemic in several regions of Brazil. Some serological diagnostic techniques using immunodominant proteins of E. canis as antigens are available, but their specificities and sensitivities are questionable. Based on this, the objective of this study was to test the antigenic potential of the recombinant gp19 protein (rGP19) for subsequent use in diagnostic tests. The rGP19 expressed in the Escherichia coli strain BL21 (DE3) C41 was recognized in the sera from experimentally infected dogs using ELISA and Western blotting. Thus, it was possible to obtain a promising antigen with the ability to differentiate between E. canis-positive and -negative animals, even 1 week after infection.

Diagnosis; serology; rickettsiae; ehrlichiosis

Authorship

Rômulo Silva de Oliveira

Departamento de Microbiologia e Parasitologia, Universidade Federal de Pelotas – UFPel, Pelotas, RS, BrasilUniversidade Federal de PelotasBrasilPelotas, RS, BrasilDepartamento de Microbiologia e Parasitologia, Universidade Federal de Pelotas – UFPel, Pelotas, RS, Brasil

Rodrigo Casquero Cunha

Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas – UFPel, Pelotas, RS, BrasilUniversidade Federal de PelotasBrasilPelotas, RS, BrasilCentro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas – UFPel, Pelotas, RS, Brasil

Jonas Moraes-Filho

Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, São Paulo, SP, BrasilUniversidade de São PauloBrasilSão Paulo, SP, BrasilFaculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo – USP, São Paulo, SP, Brasil

Relber Aguiar Gonçales

Pós-graduação em Biologia Celular e Molecular, Universidade de São Paulo – USP, São Paulo, SP, BrasilUniversidade de São PauloBrasilSão Paulo, SP, BrasilPós-graduação em Biologia Celular e Molecular, Universidade de São Paulo – USP, São Paulo, SP, Brasil

Ana Paula de Souza Stori de Lara

Luciana Farias da Costa de Avila

Marcelo Bahia Labruna

Fábio Pereira Leivas Leite ^**Corresponding author: Fábio Pereira Leivas Leite. Centro de Desenvolvimento Tecnológico – CDTEC, Biotecnologia, Campus Capão do Leão, Universidade Federal de Pelotas – UFPel, prédio 19, Corredor da Embrapa, s/n, Capão do Leão, CEP 96160-000, Pelotas, RS, Brasil. e-mail: fabio@leivasleite.com.br

*Corresponding author: Fábio Pereira Leivas Leite. Centro de Desenvolvimento Tecnológico – CDTEC, Biotecnologia, Campus Capão do Leão, Universidade Federal de Pelotas – UFPel, prédio 19, Corredor da Embrapa, s/n, Capão do Leão, CEP 96160-000, Pelotas, RS, Brasil. e-mail: fabio@leivasleite.com.br

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Figures

Figures (9)

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Figure 1
The plasmid pAE-gp19 with the gp19 gene inserted between the KpnI and EcoRI restriction enzyme sites.

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Figure 2
Solubility test for GP19. 12% SDS-PAGE stained with Coomassie Blue. M: Molecular marker (Bio-Rad); 1: total extract sonicated; 2: pellet of the sonication with Akta Wash buffer; 3: supernatant of the sonication with Akta Wash buffer; 4: pellet of the solubilization with Akta Wash buffer with N-lauroylsarcosine; 5: supernatant of the solubilization with Akta Wash buffer with N-lauroylsarcosine; 6: pellet of the solubilization with Akta Wash buffer with urea; 7: supernatant of the solubilization with Akta Wash buffer with urea. Arrows indicate gp19.

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Figure 3
12% SDS-PAGE stained with Coomassie Blue. M: Molecular marker (Bio-Rad); 1:1 µg of polyhistidine-tag protein with 73 kDa; 2:10 µg of bovine serum albumin (BSA); 4:3 µg of purified rGP19.

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Figure 4
Western blotting. M: Molecular marker Dual Color (Bio-Rad); 1 and 5) 1 µg of polyhistidine-tag protein; 2) 5 µg of bovine serum albumin (BSA); 3 and 4) 3 µg of purified rGP19; 1, 2, and 3) incubated with anti-His; 4 and 5) incubated with dog anti-E. canis. Arrows: rGP19 recognized by anti-His and anti-E. canis; asterisk: dimers and trimers of rGP19 recognized by anti-His and anti-E. canis.

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Figure 5
ELISA standardization. P50, P100, P150, and P200 are positive sera, incubated with 50, 100, 150, and 200 ng of rGP19 per well, respectively. N50, N100, N150, and N200 are negative sera, incubated with 50, 100, 150, and 200 ng of rGP19 per well, respectively.

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Figure 6
Comparison between the titration by IIFA of the sera collected from dog 1 and its OD_492nm from ELISA.

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Figure 7
Comparison between the titration by IIFA of the sera collected from dog 2 and its OD_492nm from ELISA.

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Figure 8
Comparison between the titration by IIFA of the sera collected from dog 3 and its OD_492nm from ELISA.

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Figure 9
Comparison between the titration by IIFA of the sera collected from the three dogs and their OD_492nm from ELISA.

Figure 1 The plasmid pAE-gp19 with the gp19 gene inserted between the KpnI and EcoRI restriction enzyme sites.

Figure 2 Solubility test for GP19. 12% SDS-PAGE stained with Coomassie Blue. M: Molecular marker (Bio-Rad); 1: total extract sonicated; 2: pellet of the sonication with Akta Wash buffer; 3: supernatant of the sonication with Akta Wash buffer; 4: pellet of the solubilization with Akta Wash buffer with N-lauroylsarcosine; 5: supernatant of the solubilization with Akta Wash buffer with N-lauroylsarcosine; 6: pellet of the solubilization with Akta Wash buffer with urea; 7: supernatant of the solubilization with Akta Wash buffer with urea. Arrows indicate gp19.

Figure 3 12% SDS-PAGE stained with Coomassie Blue. M: Molecular marker (Bio-Rad); 1:1 µg of polyhistidine-tag protein with 73 kDa; 2:10 µg of bovine serum albumin (BSA); 4:3 µg of purified rGP19.

Figure 4 Western blotting. M: Molecular marker Dual Color (Bio-Rad); 1 and 5) 1 µg of polyhistidine-tag protein; 2) 5 µg of bovine serum albumin (BSA); 3 and 4) 3 µg of purified rGP19; 1, 2, and 3) incubated with anti-His; 4 and 5) incubated with dog anti-E. canis. Arrows: rGP19 recognized by anti-His and anti-E. canis; asterisk: dimers and trimers of rGP19 recognized by anti-His and anti-E. canis.

Figure 5 ELISA standardization. P50, P100, P150, and P200 are positive sera, incubated with 50, 100, 150, and 200 ng of rGP19 per well, respectively. N50, N100, N150, and N200 are negative sera, incubated with 50, 100, 150, and 200 ng of rGP19 per well, respectively.

Figure 6 Comparison between the titration by IIFA of the sera collected from dog 1 and its OD_492nm from ELISA.

Figure 7 Comparison between the titration by IIFA of the sera collected from dog 2 and its OD_492nm from ELISA.

Figure 8 Comparison between the titration by IIFA of the sera collected from dog 3 and its OD_492nm from ELISA.

Figure 9 Comparison between the titration by IIFA of the sera collected from the three dogs and their OD_492nm from ELISA.

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Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br

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Recombinant gp19 as a potential antigen for detecting anti-<i>Ehrlichia canis</i> antibodies in dog sera

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[1] *Corresponding author: Fábio Pereira Leivas Leite. Centro de Desenvolvimento Tecnológico – CDTEC, Biotecnologia, Campus Capão do Leão, Universidade Federal de Pelotas – UFPel, prédio 19, Corredor da Embrapa, s/n, Capão do Leão, CEP 96160-000, Pelotas, RS, Brasil. e-mail: fabio@leivasleite.com.br