A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1)

Abstract

S U M M A R YNeospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue †.

Comparison of an ELISA assay for the detection of adhesive/invasive Neospora caninum tachyzoites

“…Thus, the first two methods are suitable for an initial evaluation of parasite invasion and real-time PCR participates as an unequivocal quantitative assay. The incubation of N. caninum or T. gondii at 37°C is a current way to determine the in vitro effects of drugs or sera on the invasion phenomenon (NAGULESWARAN et al, 2003;PEREIRA et al, 2011;SAWMYNADEN et al, 2008). Our results indicate that all these methods show losses of invasive/adhesive tachyzoites incubated at 37°C; therefore, this is an important effect to take into account when designing assays that require long incubation times.…”

“…caninum direct or indirect detection in cell cultures and tissues is well established by traditional methods, such as IFAT (YAKHCHALI et al, 2010), and highly specific methods such as real-time PCR (DEBACHE et al, 2010;GHALMI et al, 2008;NAGULESWARAN et al, 2003;PEREIRA et al, 2011). The three methods tested in this study for the detection of N. caninum invasion and/or adherence in cell culture showed a similar pattern, indicating that different methods may be complementary, since Figure 3.…”

“…After washing with PBS, antiserum against N. caninum (anti-Nc tachyzoite protein extract, 1:1000 (PEREIRA et al, 2011)) was incubated for 1 h, followed by three washes in PBS. The secondary antibody goat anti-rabbit-peroxidase conjugate (1:10000, ZyMed-Invitrogen) was incubated for 1 h and then washed three times.…”

“…caninum DNA was extracted and purified from each well according to the manufacturer's instructions (Wizard SV Genomic DNA Purification System, Promega). Parasites were quantified by real-time PCR, using Nc 5 primers with the forward primer linked to FAM fluorophore Lux (Invitrogen) (PEREIRA et al, 2011). The experiment was carried out in a Mastercycler ep RealPlex (Eppendorf ) and the results analyzed using RealPlex software (Eppendorf ).…”

“…X84238) is widely applied as a quantitative method due to its fidelity and sensitivity (COLLANTES-FERNANDEZ et al, 2002;GHALMI et al, 2008;NAGULESWARAN et al, 2003). N. caninum expressing β-galactosidase is a useful tool to detect tachyzoite integrity in secretion assays (LOVETT et al, 2000;PEREIRA et al, 2011), especially when stored as a stable cytoplasmic enzyme (HOWE et al, 1997). The reproducible and stable reaction with CPRG is well established and widely applied in several microorganisms and cell lineages (D'ANGELO et al, 2009;ESPERANDIM et al, 2010;VAVROVA et al, 2010).…”