Identification of a Highly Antigenic Region of Subtilisin-Like Serine Protease 1 for Serodiagnosis of Neospora caninum Infection

Abstract

Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples from N. caninum experimentally infected cattle and mice and cattle from a farm with confirmed cases of Neospora abortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In the N. caninum experimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera from Toxoplasma gondii experimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis of N. caninum.

Cloning and Sequencing of Truncated Toxoplasma gondii Subtilisin-Like 1 Antigen

“…They demonstrated that NcSUB1 is highly conserved in N. caninum and have only 63% sequence identity to the T. gondii orthologue. Therefore, they demonstrated no cross-react with T. gondii in their ELISA method [35]. Hruzik et al introduced Cterminal domain of TgSUB1 as a potential marker for serodiagnosis of acute toxoplasmosis.…”

Markers related to the diagnosis and to the risk of abortion in bovine neosporosis

“…Potentially useful markers for serodiagnosis are continuously being analyzed, and some recent studies have focused on the highly antigenic region of subtilisinlike serine protease 1 (Ybañez et al, 2013), NcSRS2 surface protein (Pinheiro et al, 2013), Neospora-surface antigen (p40) (He et al, 2013), monoclonal antibodies against NcSAG1 (Dong et al, 2013), and anti-rNcp-43 polyclonal antibodies (Sá et al, 2014), among others.…”

Importance of serological cross-reactivity amongToxoplasma gondii, Hammondiaspp.,Neosporaspp.,Sarcocystisspp. andBesnoitia besnoiti

“…A protease with 42 kDa was localized in the rhoptry of T. gondii by means of a MAb; this MAb also reacted to a 42 kDa protein in N. caninum, which was also localized in the rhoptry of this parasite (Ahn et al 2001). (Howe et al 1998; T. gondii (Howe et al 2002;Chahan et al 2003;Liao et al 2005b;Huang et al 2007;Ybanez et al 2013) Sarcocystis spp. (Howe et al 2002) rNcSRS2 (Ba/SRS2p44, NcSRS2t, tNcSRS2) Surface (35-43 kDa) (Hemphill and Gottstein, 1996;Hemphill et al 1997;Howe et al 1998) T. gondii (Nishikawa et al 2001(Nishikawa et al , 2002Gaturaga et al 2005;Huang et al 2007;Liu et al 2007) H. heydorni (Nishikawa et al 2001 Surface (18 kDa Dense granules (68 kDa, 37 kDa) (Liddell et al 1998) T. gondii (Lally et al 1996;Jenkins et al 1997) Sarcocystis spp.…”