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

Abstract

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.

Serological evaluation of Neospora caninum in pregnant women treated at referral center for prenatal screening in Mato Grosso do Sul, Brazil

“…Regional conditions must be taken into account so that there is a better understanding of the interaction between antigen and antibody for N. caninum (Lima et al, 2007). Although the rNcSRS2 protein (Nc-p43) did not cross-react with T. gondii in the serum of cattle, dogs and sheep (Borsuk et al, 2011;Pinheiro et al, 2015), further studies are needed to assess the interaction of this antigen with antibodies, from human samples positive for T. gondii.…”

High seroprevalance of Neospora caninum in dogs in Victoria, Australia, compared to 20 years ago

“…Therefore, genetic analysis of oocysts by PCR is required for confirmation [ 5 , 25 ]. Exposure of both intermediate and definitive hosts to N. caninum is commonly evaluated using IFAT [ 16 , 26 ] or ELISA [ 5 , 32 ] to detect anti- N. caninum antibodies in serum. Despite its subjectivity, which is dependent on user observation of fluorescence, IFAT is currently considered the ‘gold standard’ for canine N. caninum serology, because it shows virtually no cross-reactivity with related protozoan parasites [ 2 , 33 , 34 ].…”

“…Competitive ELISA N. caninum positive ( n = 39) and 40 negative ( n = 41) dog serum samples were re-tested for the presence of anti- N. caninum antibodies using an indirect fluorescence test (IFAT). As in previous studies [ 11 , 25 , 26 ], IFAT tachyzoite N. caninum 12-well slides, FITC anti-canine serum, N. caninum canine positive serum and N. caninum canine negative serum (all from VMRD) were used according to the manufacturer’s instructions. Slides were examined with an Olympus BX60 microscope equipped to detect fluorescein isothiocyanate fluorescence (FITC, maximum excitation wavelength 490 nm; mean emission wavelength 530 nm).…”

Detecção De Anticorpos Anti-Neospora Caninum Por Ensaio Imunoenzimático Em Ovinos Do Município De São Roque No Estado De São Paulo

“…Pouco são os relatos de neosporose clínica, entretanto sabe-se que alguns surtos de aborto e desordens reprodutivas em ovinos e bovinos devem estar relacionados aos testes positivos frente a Neospora caninum (CABRAL et al, 2009;ROSA et al, 2011;MACEDO et al, 2013;WARLETA et al, 2014;MACEDO et al, 2017;GUERRA et al, 2019;SANTOS et al, 2020) Testes sorológicos indiretos como o ELISA e testes diretos como o PCR, são prérequisitos para confirmação do diagnóstico do agente infeccioso (ASADPOUR et al, 2012;PINHEIRO et al, 2015). A associação dos testes direto e indireto pode ser necessária tendo em vista que animais, mesmo livres de infecção, podem permanecer soropositivos por aproximadamente 6 meses (CARDOSO et al, 2012).…”

“…Poucos são os estudos sobre a neosporose na região sudeste apesar do crescente interesse na produção de ovinos, portanto este trabalho objetivou apresentar a ocorrência desta enfermidade em propriedades da região de São Roque no Estado de São Paulo. PINHEIRO et al (2015) no teste ELISA observa-se especificidade de 94% e sensibilidade de 100% para detecção de N. caninum em ovinos, sendo utilizada em comparação com outras técnicas sorológicas (ÁLVAREZ-GARCÍA, 2003).…”