Neosporosis is a very common disease worldwide, and losses to livestock farms that result from neosporosis-related abortions are a huge problem. Given that no therapy or effective vaccine is available currently, diagnosis and protection are very important to dairy farms. In this study, we developed a diagnostic method based on recombinant N. caninum proteins, MBP-NcGRA2, MBP-NcSRS2, and NcSAG1. An ELISA with co-immobilization of those three proteins gave ideal results in both the development and validation steps. We also tried to immobilize those proteins separately; however, the results were not as good as with co-immobilization. For instance, one more positive sample was misclassified when we immobilized only MBP-NcGRA2 for use in the ELISA. Individual immobilization of MBP-NcSRS2 or NcSAG1 resulted in lower PP values for positive serum samples than co-immobilization of three proteins.
Other proteins, such as NcMIC1 [22
], NcMIC3 [21
], and GRA7 [23
] have also been reported to have antigenicity. Therefore addition of those proteins to the diagnostic test might increase the sensitivity of the assay. In addition, some assays use inactivated total proteins of the parasite as a detector for the diagnosis of neosporosis. However, given that some proteins of N. caninum
have high similarity to those of T. gondii
], total proteins of parasites might result in misclassification. NcGRA2, NcSRS2 and NcSAG1 have identities of 33%, 43% and 51% to those of T. gondii
, respectively. According to the report of Nishikawa et al. [24
], no cross-reaction was observed between NcSRS2 and that of T. gondii
. Therefore, they could be used to distinguish these two infectious diseases.
Two proteins were expressed in an E. coli
protein expression system, which is a cost-effective method of producing large quantities of high-quality recombinant proteins [25
]. However, this approach is often insufficient for soluble expression of recombinant proteins. The Structural Genomics Center has estimated that up to 50% of all prokaryotic proteins are insoluble when expressed in E. coli
]. So far, many proteins of N. caninum
have been expressed in E. coli
, and most of them were expressed as inclusion bodies [27
]. In this case, refolding procedures are necessary in order to obtain biofunctional proteins. Although some proteins recover their activity after refolding treatments, it is still difficult to reproduce the complete properties of the native protein.
We expressed soluble NcGRA2 and NcSRS2 by fusing them with MBP, which is capable of functioning as a general molecular chaperone in the context of a fusion protein [28
]. Moreover, MBP did not hinder the immunoassay used in this study, which suggests that it may aid the production of protein on a large scale. We also tried to express NcSAG1, but unfortunately we could not obtain a soluble fraction. However, use of the BmNPV bacmid expression system, with both the cysteine-protease and chitinase genes deleted (BmNPV-CP−Chi−
), led to successful expression of NcSAG1 in silkworm larvae as a soluble fraction, with less protein degradation. The viral protease and chitinase activities in the hemolymph of the silkworm larvae were reduced by 95 and 50%, respectively [29
Given that the tachyzoite is a very infectious stage of N. caninum, many studies of vaccination and detection have been focused on this stage. NcGRA2 is usually contained in dense granules, and it is highly expressed in culture-derived tachyzoites, therefore it has been studied as a potential vaccine candidate. However, so far there has been no study on the diagnosis of neosporosis that has used this protein as a detector. In this study, this protein bound to N. caninum-specific antibodies in serum samples from cattle, which suggests that NcGRA2 is a good candidate protein for use in diagnostic assays. The combination of NcGRA2, NcSRS2, and NcSAG1 resulted in reliable detection of neosporosis.
The new method showed high values in the positive area in most positive samples than a commercial iscom ELISA kit, which suggests that this method may show a higher level of response to anti Neospora
antibodies than the commercial ELISA kit. Among the 104 samples tested, only one positive sample, no.7 serum sample, was misclassified as negative using our method ( Additional file 1
and Figure ). However, the PP value of this sample calculated with the commercial ELISA kit was 20.5, which was very close to the boundary value for interpretation; that obtained with our method was 16.8.
In addition to cattle, naturally occurring neonatal or fetal infections caused by Neospora
-like protozoa have been described in dogs, goats [30
], horses [31
], and sheep [3
]. Therefore, this diagnostic method may have wide applicability to other animal species.