Monoclonal Antibodies Preventing Invasion of Neospora caninum Tachyzoites into Host Cells

Abstract

ABSTRACT. Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells. KEY WORDS: invasion process, monoclonal antibody, Neospora caninum, surface antigen.J. Vet. Med. Sci. 66(11): 1355-1358, 2004 Neospora caninum is a coccidian protozoon first isolated from the brain of a puppy by Dubey in 1988, which developed paralysis in the hind limbs [5]. Dogs infected with this parasite mainly show neurological symptoms and infected cows are attached with abortion and stillborn, so serious economical damages are produced [4]. In 1998, McAllister et al. reported that definitive host of the protozoon is dogs [14]. However, the life cycle of N. caninum is not completely clarified yet, and the procedures for prevention and treatment of neosporosis also are not established. The pathogenesis of N. caninum is known to be caused by multiplication of parasites resulting destruction of many kinds of host cells by invasion [2,3,20]. To develop the prevention and treatment of neosporosis, analyses of antigens related with the invasion process into host cells are required. Antigens of some protozoan parasites belonging to phylum Apicomplexa, such as Toxoplasma gondii participate in invading the host cells and were thought to be surface antigens, secretory proteins and those in the structures of apical complex such as microneme, rhoptry and dense granule [6,11,15,18]. Also in N. caninum, some antigens, possibly affecting the invasion, were studied, and the antigens on the body surface [9,10] In this study, we prepared monoclonal antibodies (MAbs) against N. caninum tachyzoites which obstruct the invasion of tachyzoites into host cells, and further, we tried to specify the antigen of N. caninum concerning with attachment and invasion into the host cells. MATERIALS AND METHODSParasites: Neospora caninum JPA-1 strain used was isolated and passaged by Yamane et al. in the National Institute of Animal Health, Japan [23]. The isolate was inoculated into monolayer Vero cells maintained in Eagle's minimum essential medium (MEM; NISSUI PHARMACEUTICAL Co., Japan) supplemented with 10% fetal calf serum (FCS; HARLAN SERA-LAB, England), 2 mM L-glutamine (Wako Pure Chemical Industries, Japan), 1% MEM Vitamin solution (GIBCO BRL, U.S.A.), 13 mM sodium hydrogen carbonate (NaHCO 3 ) (Wako Pure Chemical Industries), and 4 µl/ml Fungizone (GIBCO BRL). The tachyzoites cultured were used for examination. Purification of tachyzoites:The tachyzoites cultured were purified according to an improved metho...

Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract

“…These results are in agreement with previous reports demonstrat-ing that antibodies specific for different membrane proteins of N. caninum are capable of interfering with, and may even block, the parasite entry into host cells. [44][45][46] However, such a protective role needs to be confirmed in further experiments, namely as the effect of IgG opsonization on leucocyte killing of tachyzoites of the closely related protozoan T. gondii remains controversial with previous studies showing either enhanced killing 47 or no effect in this regard. 48 Only a limited number of reports specifically studied the mucosal immune response to this parasite 26,27 and to the best of our knowledge no study had previously addressed the immune response in the intestinal mucosa and associated lymphoid tissue of mice immunized with N. caninum antigens.…”

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

“…MAbs raised against T. gondii reacted with N. caninum tachyzoites by IFAT, but only at low titres (10–40) (Latif and Jakubek, 2008). A number of reports demonstrated the usefulness of MAbs against T. gondii , which do not cross-react with N. caninum (Baszler et al 2001; Uchida et al 2004; Srinivasan et al 2006; Cunha-Junior et al 2010). …”

Gene Cloning and Characterization of the Protein Encoded by theNeospora caninumBradyzoite-Specific Antigen GeneBag1

“…As a result, 3 Mabs were established; Mab 20-2-57 recognized only rNcBAG1, which was selected for use in this study, because the other 2 Mabs recognized both NcBAG1 and TgBAG1. Mabs against TgBAG1 (GenBank X82213) and NcSAG4 (AY763105) were previously obtained using the same protocol (Uchida et al, 2004) and are hereafter designated 6-4 and 4-D-1, respectively. The Mab against NcSAG1 7-2-73 has been previously described (Ishigaki et al, 2012).…”