Comparison of indirect ELISA based on recombinant protein NcSRS2 and IFAT for detection of Neospora caninum antibodies in sheep

Abstract

Neospora caninum, an Apicomplexan parasite that can causes abortion, is responsible for considerable economic and reproductive losses in livestock. The purpose of the present study was to determine whether recombinant NcSRS2 is a suitable indirect ELISA antigen for determining specific immune response to N. caninum in sheep. A total of 441 serum samples were subjected to IFAT and rNcSRS2 based-ELISA, with both tests performing similarly. The sensitivity and specificity of indirect ELISA were 98.6 and 98.3%, respectively. The kappa index shows 0.98 concordance between the two tests, which is considered excellent. Seroprevalences of 30.8 and 32.0% were detected by IFAT and indirect ELISA, respectively, showing these tests did not differ significantly on this measure (p > 0.05). Serological analysis showed that HisG tag was detected by Western Blotting recognizing rNcSRS2 protein. The potential value of rNcSRS2-based ELISA as a highly specific and sensitive tool for serological diagnosis is also supported by the strong agreement found between IFAT and ELISA. The results support the potential use of recombinant protein NcSRS2 as an antigen in indirect ELISA in sheep.Keywords: NcSRS2, ELISA, IgG, diagnosis, ovine. ResumoNeospora caninum é um parasito Apicomplexa que pode causar abortos e é reconhecido como agente importante responsável por perdas econômicas e reprodutivas. Este estudo avaliou a proteína recombinante NcSRS2 como antígeno para ELISA indireto na determinação de resposta imune para N. caninum em ovinos. 441 amostras de soro foram analisadas por IFAT e ELISA indireto com rNcSRS2 e ambos os testes revelaram comportamento similar. A sensibilidade e especificidade de ELISA indireto foram 98,6 e 98,3%, respectivamente. O índice kappa mostrou uma concordância entre os dois testes com valor de 0,98, que é considerado excelente. Prevalências de 30,8 e 32,0% detectadas por IFAT e ELISA indireto, respectivamente, mostraram que os testes não diferiram significativamente nesse aspecto (P > 0.05). A análise sorológica revelou que os anticorpos específicos da cauda de histidina reconheceu por Western Blotting a proteína recombinante NcSRS2. O valor potencial do ELISA indireto baseado no antígeno rNcSRS2 como ferramenta altamente específica e sensível para diagnóstico sorológico é também reforçado pela alta concordância dos valores obtidos com IFAT e com ELISA indireto. Esses resultados respaldam o uso potencial da proteína rNcSRS2 como antígeno em ELISA indireto em ovinos.Palavras-chave: NcSRS2, ELISA, IgG, diagnóstico, ovinos.

Evaluation of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlândia, Minas Gerais State, Brazil, by different serological methods

“…Regarding N. caninum, the occurrence of 23% seropositivity in sheep from this region was also higher than the majority of data reported in different regions of Brazil, ranging from 3.2% in the southern region (Vogel et al, 2006) to 15% in the northeast region (Uzeda et al, 2007), except for a higher seroprevalence of 29% in Rondônia, in the Amazon region and 31-32% in Campo Grande, in the central region of the country (Andreotti et al, 2009). Although positive association was found between seropositivity for T. gondii and the age of the animals, such association was not found for N. caninum only, as previously reported by other studies (Figliuolo et al, 2004;Romanelli et al, 2007;Spilovská et al, 2009), suggesting that vertical transmission could occur in sheep farms, similar to bovine infections.…”

Factors associated with the seroprevalence of Neospora caninum (Apicomplexa: Toxoplasmatinae) in sheep from the State of Sergipe, Brazil

“…Among the studies conducted in Brazil with identical techniques and cut-off points, similar results were found by Faria et al (2010) in Alagoas (9.6%) and Munhóz et al (2010) in São Paulo (13.9%). However, the prevalence verified in this study was higher than the rates identified by Salaberry et al (2010) in Minas Gerais (8.1%) and Figliuolo et al (2004) in São Paulo (9.2%), and lower than the rates found by Andreotti et al (2009) in farms in the city of Campo Grande, central-western Brazil (30.8%), by Rossi et al (2011) in Minas Gerais (47.1%) and by Tembue et al (2011) in Pernambuco (64.2%). Variations by location might be related to differences in the region, climate, animal age, sample size (Dubey et al, 2011), production systems (Melo et al, 2001), or even pathogenic synergism (Melo et al, 2004).…”

Seroprevalence of Toxoplasma gondii and Neospora caninum infections in goats in Poland

“…These values were used to calculate the individual-level true seroprevalence according to Noordhuizen et al (2001). SE and SP of N. caninum ELISA were 98.6% and 98.3%, respectively (Andreotti et al, 2009). For calculating the herd-level true seroprevalence, the test SE and SP were adjusted from individual-to herdlevel using the Herdacc program (Jordan and McEwen, 1998), for a median herd size of 73 animals, herd cut-off value of one animal positive indicating a positive herd and 12-28 animals sampled per herd.…”