Use of ELISA based on NcSRS2 of Neospora caninumexpressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

Neospora caninum ; Pichia pastoris ; NcSRS2; ELISA

Authorship

Amanda Fernandes Pinheiro

Departamento de Microbiologia e Parasitologia, Instituto de Biologia, Universidade Federal de Pelotas – UFPel, Pelotas, RS, BrazilUniversidade Federal de PelotasBrazilPelotas, RS, BrazilDepartamento de Microbiologia e Parasitologia, Instituto de Biologia, Universidade Federal de Pelotas – UFPel, Pelotas, RS, Brazil

Sibele Borsuk

Núcleo de Tecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas – UFPel, Pelotas, RS, BrazilUniversidade Federal de PelotasBrazilPelotas, RS, BrazilNúcleo de Tecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas – UFPel, Pelotas, RS, Brazil

Maria Elisabeth Aires Berne

Luciano da Silva Pinto

Renato Andreotti

Centro Nacional Gado de Corte, Empresa Brasileira de Pesquisa Agropecuária – Embrapa, Campo Grande, MS, BrazilEmpresa Brasileira de Pesquisa AgropecuáriaBrazilCampo Grande, MS, BrazilCentro Nacional Gado de Corte, Empresa Brasileira de Pesquisa Agropecuária – Embrapa, Campo Grande, MS, Brazil

Talita Roos

Barbara Couto Roloff

Fábio Pereira Leivas Leite ^**Corresponding author: Fábio Pereira Leivas Leite, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Rua Gomes Carneiro, n. 1, Centro, CP 354, CEP 96010-610, Pelotas, RS, Brazil, e-mail: fabio_leite@ufpel.edu.br

*Corresponding author: Fábio Pereira Leivas Leite, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Rua Gomes Carneiro, n. 1, Centro, CP 354, CEP 96010-610, Pelotas, RS, Brazil, e-mail: fabio_leite@ufpel.edu.br

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (3)

Thumbnail

Figure 1
(a) Western Blot using recombinant NcSRS2 protein expressed in the P. pastoris system. The recombinant protein reacted with serum samples from animals (sheep and dogs) naturally infected with N. caninum. Full-range rainbow molecular weight marker (GE). (b) Samples 1 and 2 are from sheep serum that was IFAT-negative, but reacted above the ELISA threshold (i.e. they were positive), and samples 3, 4 and 5 were IFAT-positive and reacted under the ELISA threshold (i.e. they were negative). (c) Samples 1 to 4 are from dogs that were IFAT-negative, but reacted above the ELISA threshold (i.e. they were positive), and samples 5 to 9 were IFAT-positive and reacted under the ELISA threshold (i.e. they were negative).

Thumbnail

Figure 2
ROC analysis on the ELISA-NcSRS2 test using 37 confirmed positive and 73 confirmed negative sheep serum samples (as shown using the indirect fluorescence antibody test). (a) Frequency distribution of the confirmed positive (1) and confirmed negative (0) serum samples. The samples were considered positive when the cutoff values were greater than or equal to a mean ELISA absorbance value of 0.25. (b) ROC plot. Area under curve = 0.996 (0.008); 95% confidence interval from 0.958 to 0.997. (c) Negative predictive values (squares) and positive predictive values (diamonds) associated with the ELISA-NcSRS2 test for varying prevalence levels of neosporosis, determined using ROC analysis based on mean OD threshold values in ELISA.

Thumbnail

Figure 3
ROC analysis on the ELISA-NcSRS2 test using 21 confirmed positive and 44 confirmed negative canine serum samples (as shown using the indirect fluorescence antibody test). (a) Frequency distribution of the confirmed positive (1) and confirmed negative (0) serum samples. The samples were considered positive when the cutoff values were greater than or equal to a mean ELISA absorbance value of 0.21. (b) ROC plot. Area under curve = 0.986 (0.018); 95% confidence interval from 0.919 to 0.997. (c) Negative predictive values (squares) and positive predictive values (diamonds) associated with the ELISA-NcSRS2 test for varying prevalence levels of neosporosis, determined using ROC analysis based on mean OD threshold values in ELISA.

Figure 1 (a) Western Blot using recombinant NcSRS2 protein expressed in the P. pastoris system. The recombinant protein reacted with serum samples from animals (sheep and dogs) naturally infected with N. caninum. Full-range rainbow molecular weight marker (GE). (b) Samples 1 and 2 are from sheep serum that was IFAT-negative, but reacted above the ELISA threshold (i.e. they were positive), and samples 3, 4 and 5 were IFAT-positive and reacted under the ELISA threshold (i.e. they were negative). (c) Samples 1 to 4 are from dogs that were IFAT-negative, but reacted above the ELISA threshold (i.e. they were positive), and samples 5 to 9 were IFAT-positive and reacted under the ELISA threshold (i.e. they were negative).

Figure 2 ROC analysis on the ELISA-NcSRS2 test using 37 confirmed positive and 73 confirmed negative sheep serum samples (as shown using the indirect fluorescence antibody test). (a) Frequency distribution of the confirmed positive (1) and confirmed negative (0) serum samples. The samples were considered positive when the cutoff values were greater than or equal to a mean ELISA absorbance value of 0.25. (b) ROC plot. Area under curve = 0.996 (0.008); 95% confidence interval from 0.958 to 0.997. (c) Negative predictive values (squares) and positive predictive values (diamonds) associated with the ELISA-NcSRS2 test for varying prevalence levels of neosporosis, determined using ROC analysis based on mean OD threshold values in ELISA.

Figure 3 ROC analysis on the ELISA-NcSRS2 test using 21 confirmed positive and 44 confirmed negative canine serum samples (as shown using the indirect fluorescence antibody test). (a) Frequency distribution of the confirmed positive (1) and confirmed negative (0) serum samples. The samples were considered positive when the cutoff values were greater than or equal to a mean ELISA absorbance value of 0.21. (b) ROC plot. Area under curve = 0.986 (0.018); 95% confidence interval from 0.919 to 0.997. (c) Negative predictive values (squares) and positive predictive values (diamonds) associated with the ELISA-NcSRS2 test for varying prevalence levels of neosporosis, determined using ROC analysis based on mean OD threshold values in ELISA.

How to cite

copy

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

Acompanhe os números deste periódico no seu leitor de RSS

Versão para download de PDF

PDF

Inglês

Versões e tradução automática

Versão original do texto

English

Tradução automática

Google Translator
Microsoft Translator

Como citar

RIS

BIBTEX

Outros formatos de citação e exportação:

SciELO - Scientific Electronic Library Online
Rua Dr. Diogo de Faria, 1087 – 9º andar – Vila Clementino 04037-003 São Paulo/SP - Brasil
E-mail: scielo@scielo.org

Leia a Declaração de Acesso Aberto

Métricas

Use of ELISA based on NcSRS2 of Neospora caninumexpressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs

PlumX

Mensagem

[1] *Corresponding author: Fábio Pereira Leivas Leite, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Rua Gomes Carneiro, n. 1, Centro, CP 354, CEP 96010-610, Pelotas, RS, Brazil, e-mail: fabio_leite@ufpel.edu.br