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Effects of a transferring antibody against Neospora caninum infection in a murine model
Veterinary Parasitology 160 (2009) 60–65
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Effects of a transferring antibody against Neospora caninum infection in a murine model Yoshifumi Nishikawa *, Houshuang Zhang, Penglong Huang, Guohong Zhang, Xuenan Xuan National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 May 2008 Received in revised form 14 October 2008 Accepted 15 October 2008
We investigated the roles of a transferring antibody against Neospora caninum infection based on a murine model using recombinant vaccinia virus carrying NcSRS2 gene. Higher levels of anti-NcSRS2 antibody were detected in surviving offspring from vaccinated dams than controls while the transferring anti-NcSAG1 antibody was detected in the surviving offspring from unvaccinated dams. After mating, the female mice with transferring antibody against N. caninum were challenged with the parasites in mid-gestation. The transferring antibodies disappeared during pregnancy upon parasite infection. There was no significant difference on the parasite burden in dams and the survival rates of their offspring. Here, we have shown that N. caninum-specific transferring antibody does not control parasite infection in mice. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Neospora caninum Maternal immunity Transferring antibody
1. Introduction Infection with the coccidian parasite Neospora caninum causes paralysis and death in livestock and companion animals (Dubey and Lindsay, 1996). In cattle, N. caninum infection is associated with abortions, stillbirth, and neurological disease in calves, making neosporosis an important economic agricultural concern (Dubey, 1999). Transplacental transmission from a naturally infected dam to her fetus appears to be the major, and only confirmed intraspecific, natural route of transmission of the parasite (Pare´ et al., 1996; Anderson et al., 1997; Schares et al., 1998). Repeated in utero infections can occur in the same dam during subsequent pregnancies, resulting in abortion or congenital infection (Barr et al., 1993; Thurmond and Hietala, 1997). Congenitally infected calves remain persistently infected and can pass the infection onto their offspring (Pare´ et al., 1996; Anderson et al., 1997). Cows may also become infected by ingestion of oocysts that have
* Corresponding author. Tel.: +81 155 49 5886; fax: +81 155 49 5643. E-mail address: [email protected] (Y. Nishikawa). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.10.095
been shed by a definitive host, such as dogs (McAllister et al., 1998; De Marez et al., 1999). There is accumulating evidence that some N. caninuminfected cows can develop a degree of protective immunity against abortion and/or congenital transmission, indicating the advantage of vaccination. Although prevention of abortion might be a more realistic goal for a vaccine, the ultimate objective for control of this disease must be to prevent vertical transmission of the parasite. Several groups have investigated induction of protective immunity using recombinant vaccines, especially those based on a N. caninum surface antigen, NcSRS2. Our previous results showed that N. caninum infection was controlled in BALB/c mice that were immunized with a recombinant vaccinia virus carrying NcSRS2 (Nishikawa et al., 2001a,b). In addition, vaccination of mice with recombinant NcSRS2 produced by E. coli or the native protein, induced protective immunity against N. caninum infection (Cannas et al., 2003; Haldorson et al., 2005; Pinitkiatisakul et al., 2005, 2007). NcSRS2 peptides induce specific T cell activation, interferon-gamma secretion of peripheral blood mononuclear cells and antibody production in cattle (Staska et al., 2005; Baszler et al., 2008).
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Although prevention of vertical transmission of the parasite is important, the roles of maternal immunity are not well known. Transferring antibody is the antibody derived from the dam’s immune response to an antigen and transferred via placenta or colostrum to the offspring without need of the offspring’s active immune response to the same antigen. We have addressed the following question: Can the transferring antibodies derived from the dam infected with N. caninum or immunized with vaccine control the vertical transmission of the parasites? To determine the effects of the parasite-specific transferring antibodies, we used a murine model using a recombinant vaccinia virus carrying NcSRS2. This study would provide important insights for prevention of neosporosis. 2. Materials and methods 2.1. Preparation of N. caninum tachyzoites Neospora caninum tachyzoites of the Nc-1 isolate (Dubey et al., 1988) were maintained in monkey kidney adherent fibroblasts (Vero cells) cultured in Eagle’s minimum essential medium (EMEM, Sigma, St. Louis, MO) supplemented with 8% heat-inactivated fetal bovine serum (FBS). For the purification of tachyzoites, parasites and host-cell debris were washed in cold PBS, and the final pellet was resuspended in cold PBS and passed through a 27-gauge needle and a 5.0-mm-pore filter (Millipore, Bedford, MA). 2.2. Preparation of recombinant vaccinia viruses The recombinant vaccinia virus which expresses NcSRS2 was constructed as previously described (Nishikawa et al., 2000b). For preparation of viral inoculum, recombinant vaccinia viruses were propagated in rabbit kidney (RK13) cells in OPTI-MEM1I (OPTI-MEM, Gibco BRL, New York) without FBS.
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at 1 week after boost and were inspected twice daily for the presence of vaginal plugs. The first day a plug was noted was designated as day 0 of pregnancy for each individual. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week post-vaccination). The surviving offspring (F1) were used for investigation of maternal immunity (Fig. 1). The surviving F1 female mice or naı¨ve female mice, 10 weeks of age, were mated with naı¨ve males, 10 weeks of age, for 3 days (1 female with 1 male per cage) at 1 week. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation. The surviving offspring were named F2 (Fig. 1). Numbers and survival rates of F2 offspring were measured at 30 days after birth. 2.5. DNA isolation and PCR analysis For DNA preparation, each whole brain of BALB/c mice was thawed in 10 time-volumes of extraction buffer (0.1 M Tris–HCl pH 9.0, 1% SDS, 0.1 M NaCl, 1 mM EDTA) and 1 mg/ml of Proteinase K at 55 8C. The DNA was purified by phenol-chloroform extraction and ethanol precipitation. The DNA concentration was adjusted to 100 mg/ml for the brain as a template DNA. The DNA amplified by PCR was suspended in 10 ml reaction mixture containing 2.5 ml of template DNA, 1 ml of 10 PCR buffer which contained 15 mM MgCl2 (PerkinElmer, Boston, MA), 1 ml of 10 mM dNTP mix, 0.1 ml of 5 U/ml Ampli GoldTMTaq DNA polymerase (PerkinElmer) and 2 ml of 10 pmol/ml N. caninum specific primers, Np6 and Np21 (Liddell et al., 1999). Amplification was done in a thermal cycler, GeneAmp PCR System 2400 (PerkinElmer) employing 40 cycles for denaturation (94 8C, 1 min), annealing (63 8C, 1 min) and primer extension (74 8C, 3.5 min). At the end of cycle reaction, a primer extension was continued for 10 min at 74 8C and then kept at 4 8C. The PCR products were visualized by electrophoresis in agarose gels. 2.6. Measurement of N. caninum specific antibodies
2.3. Mice BALB/c male and female mice, 6–7 weeks of age, were obtained from Clea Japan (Tokyo, Japan). Until their use at 7–8 weeks of age, mice were housed under specific pathogen-free conditions in the animal facility of the National Research Center for Protozoan Diseases at the Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. Mice were cared for and used under the Guiding Principles for the Care and Use of Research Animals promulgated by the Obihiro University of Agriculture and Veterinary Medicine. 2.4. Vaccination, mating and challenge infection Female mice, 6–7 weeks of age, were injected intraperitoneally (i.p.) with OPTI-MEM or 5 106 plaque forming units of recombinant vaccinia virus and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males, 10 weeks of age, for 3 days (1 female with 1 male per cage)
Serum (20 ml) was obtained from F1 mice at 30 days after birth and female F1 mice or naı¨ve female mice at 0, 17 and 30 days after mating via tail vein for measuring N. caninum-specific antibodies by ELISA. To confirm the lack of an antibody response in unvaccinated and uninfected mice, control serum was taken from all the animals. The recombinant proteins of NcSAG1, NcSRS2, and NcGRA7 were expressed in E. coli as glutathione S-transferase (GST) fusion proteins and then purified using Glutathione Sepharose 4B (Amersham Pharmacia Biotech, Sweden) as described previously (Chahan et al., 2003; Gaturaga et al., 2005; Hara et al., 2006). The lysate of N. caninum (NLA) was prepared as previously reported (Liao et al., 2005). Fifty microliters of purified NcSAG1, NcSRS2, NcGRA7, and their control GST, as well as NLA at a final concentration of 5 mg/ml was coated on ELISA plates (Nunc, Denmark) overnight at 4 8C with a carbonatebicarbonate buffer (pH 9.6). After blocking with PBS containing 3% skim milk (PBS-SM) for 1 h at 37 8C, the plates were washed twice with PBS containing 0.05%
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Fig. 1. Procedure for vaccination, mating and challenge infection. Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week post-vaccination). The surviving F1 female mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve males for 3 days at 1 week. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation. The surviving offspring were named F2.
Fig. 2. Transferring antibody in F1 offspring. Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week postvaccination). Sera of the surviving F1 offspring from vaccinated and control groups at 30 days after birth were collected and examined by ELISA using the parasite lysates (NLA), NcSRS2, NcSAG1 and NcGRA7 as antigens. Vaccinated (N = 21); control (N = 19). The statistical difference was determined by twosided Mann–Whitney’s U-test. Differences of P < 0.001 were considered significant. Data are representative of repeated experiments.
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Tween20 (PBS-T), and 100 ml of serum samples diluted at 1:250 with PBS-SM was added to duplicate wells. Plates were incubated at 37 8C for 1 h. After washing five times with PBS-T, plates were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Bethyl Laboratories, USA) diluted at 1:4000 with PBS-SM at 37 8C for 1 h. The plates were washed five times, and then substrate solution (0.1 M citric acid, 0.2 M sodium phosphate, 0.003% H2O2, and 0.3 mg/ml 2,20 -azide-bis[3ethylbenzthiazoline-6-sulfonic acid]; Sigma, St. Louis, MO) was added to each well in 100-ml aliquots. The absorbance at 415 nm was read after 1 h of incubation at room temperature using an ELISA reader (Corona Microplate Reader MTP-120; Corona, Tokyo, Japan). The ELISA result was determined by the difference in the mean optical density at a value of 415 nm (OD415nm) between the recombinant antigen (NcSGA1, NcSRS2, or NcGRA7) and the GST protein. For ELISA using NLA, the result was determined by simply taking the OD415nm value. 3. Results 3.1. Transferring antibody in surviving offspring Previous studies have shown that vaccination of dams with recombinant vaccinia virus expressing NcSRS2 (vvNcSRS2) appeared to confer effective protection against
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vertical transmission to F1 offspring (Nishikawa et al., 2001a). The survival rate of F1 offspring in the group vaccinated with vvNcSRS2 was 83.3%, while it was 27.3% in the group vaccinated with recombinant vaccinia virus expressing green fluorescence protein (control virus) and 28.9% in the group inoculated with medium. Therefore, we focused on maternal immunity induced by vaccination with vvNcSRS2 against N. caninum infection. Fig. 2 represents the transferring antibody in F1 offspring. The offspring from vvNcSRS2-vaccinated dams had higher levels of anti-NLA and NcSRS2 antibodies than controls. In F1 offspring from control mice, higher levels of antiNcSAG1 antibody were detected than in experimentally vaccinated animals. There was no significant difference in the production of anti-NcGRA7 antibody. 3.2. Antibody production in F1 mice during pregnancy following N. caninum infection Next, we examined parasite-specific antibody production in the F1 mice during pregnancy following N. caninum infection (Fig. 3). After mating, female F1 mice or naı¨ve female mice were challenged with N. caninum. The lack of an antibody response against NLA, NcSRS2, NcSAG1 and NcGRA7 in unvaccinated and uninfected mice (Day 0 of naı¨ve group) was confirmed. Although high levels of antiNcSRS2 antibody were detected at Day 0 in the F1 mice from
Fig. 3. Antibody production of F1 female mice following N. caninum infection. Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6– 9 days of gestation (4-week post-vaccination). Female F1 mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve male mice and were then challenged with N. caninum at 6–9 days gestation. Sera of dams (N = 6) were collected at 0, 17 and 30 days after mating and examined by ELISA using the parasite lysates (NLA), NcSRS2, NcSAG1 and NcGRA7 as antigens. Data are representative of repeated experiments.
Y. Nishikawa et al. / Veterinary Parasitology 160 (2009) 60–65
64 Table 1 PCR detection of N. caninum in dams. Treatment
Trial
Total number of dams positive/total number of dams
Vaccinated
Trial 1 Trial 2 Trials 1 and 2
2/7 (28.6%) 5/6 (83.3%) 7/13 (53.8%)
Control
Trial 1 Trial 2 Trials 1 and 2
4/6 (66.7%) 3/5 (60.0%) 7/11 (63.6%)
Naı¨ve
Trial 1 Trial 2 Trials 1 and 2
2/4 (50.0%) 3/5 (60.0%) 6/10 (60.0%)
Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve male mice for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week postvaccination). Female F1 mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve male mice and were then challenged with N. caninum at 6–9 days gestation. DNA was extracted from brain of the dams at 40 days after the infection, and were then analyzed by PCR. The statistical difference was determined by x2-test.
dams vaccinated with vvNcSRS2, the blood levels of the antibody decreased upon parasite infection. All antibodies tested here were detected at Day 30 in the F1 mice from dams vaccinated with vvNcSRS2 or control dams. 3.3. Effects of transferring antibody against N. caninum infection The PCR detection assay allows examination of tissue localization of N. caninum. Although N. caninum DNA was detected brain, lungs, liver and spleen of BALB/c mice until 6 days after the infection, parasite DNA were found almost exclusively in the brain at 10 and 26 days after the infection (Nishikawa et al., 2001b). To confirm the effects of transferring antibody against N. caninum infection, PCR analyses were carried out using brain tissue from F1 mice challenged with the parasites. As shown in Table 1, there
were no statistically significant differences among the tested group (P > 0.05). In addition, the numbers and survival rates of F2 offspring were examined (Table 2). There was no statistically significant difference in the numbers of F2 offspring and the survival rates among the groups (P > 0.05). 4. Discussion Since N. caninum infection has emerged as an important reproductive disease in cattle throughout the world (Anderson et al., 2000), the development of a vaccine against this parasite is thought to be advantageous. Previous studies have demonstrated that recombinant vaccines based on NcSRS2 afforded effective protection against N. caninum infection (Nishikawa et al., 2001a,b; Cannas et al., 2003; Pinitkiatisakul et al., 2005, 2007; Haldorson et al., 2006). However, the role maternal antibody may play in the next generation after N. caninum infection has not yet been evaluated. Our study indicates that anti-NcSRS2 IgG antibodies induced by vaccination of dams with recombinant vaccinia virus expressing NcSRS2 were detected in the offspring (Fig. 2). Interestingly, levels of anti-NcSAG1 antibody in offspring from control mice were higher that those from the vaccinated group (Fig. 2). These results indicated that anti-NcSRS2 and anti-NcSAG1 antibodies were transferred from vaccinated and nonvaccinated dams to the offspring, respectively. However, serum levels of the transferred antibodies decreased upon parasite infection (Fig. 3). This may be due to the binding of the transferred antibodies to the parasite. Although previous studies have shown that anti-NcSAG1 antibody or anti-NcSRS2 antibody inhibited N. caninum invasion into the host cells in an in vitro model (Nishikawa et al., 2000a; Haldorson et al., 2006), the reactions of the parasitespecific antibody do not affect the inhibition of the parasite burden in an in vivo model. These results showed that the transferring antibody does not contribute to protective immunity against N. caninum infection in mice. Our observation suggests the repeated in utero infection in the same dam during bovine pregnancy.
Table 2 Numbers and survival rates of F2 offspring. Treatment
Trial
Mean number pups/litter (S.D.)
Number of surviving pups/number of pups in litter
Total number of surviving pups/total number of pups
Vaccinated
Trial 1 Trial 2 Trials 1 and 2
6.0 (2.10) 6.0 (1.00) 6.0 (1.61)
4/4, 1/9, 1/4, 5/6, 3/8, 2/5 4/7, 0/5, 1/6, 4/7, 0/5
16/36(44.4%) 9/30 (30.0%) 25/66 (37.9%)
Control
Trial 1 Trial 2 Trials 1 and 2
5.6 (2.70) 7.6 (0.55) 6.6 (2.12)
4/4, 1/3, 8/9, 0/4, 4/8 1/8, 3/7, 2/7, 0/8, 1/8
17/28(60.7%) 7/38(18.4%) 24/66 (36.4%)
Naı¨ve
Trial 1 Trial 2 Trials 1 and 2
5.3 (1.53) 4.7 (0.58) 5.0 (1.10)
3/5, 0/4, 3/7 0/5, 3/5, 1/4
6/16(37.5%) 4/14(28.6%) 10/30(33.3%)
Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve male mice for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week post-vaccination). Female F1 mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve male mice and were then challenged with N. caninum at 6–9 days gestation. Numbers of surviving F2 offspring at 30 days after birth were analyzed by ANOVA, and the differences were then analyzed using Turkey–Kramer multiple comparison tests. For the survival rates of offspring, the statistical difference was determined by x2-test.
Y. Nishikawa et al. / Veterinary Parasitology 160 (2009) 60–65
The present study suggests that each generation should have a vaccination to prevent N. caninum infection because of no protective efficiency of the transferring antibody. However, to control the neosporosis, we have to consider the following points: vaccines which can not be neutralized by the transferring antibody and can induce cellular immunity against N. caninum: diagnostic systems which distinguish between naturally infected and vaccinated animals. Previous studies have suggested that nonpregnant animals might develop sufficient immunity to protect against vertical transmission during a subsequent pregnancy (Williams et al., 2000; Innes et al., 2001; Nishikawa et al., 2001a). Therefore, after the above-mentioned problems are overcome, vaccine therapy for nonpregnant animals could help to control vertical transmission of N. caninum. Acknowledgments We thank Dr. Dubey (United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, and Parasite Biology and Epidemiology Laboratory) for the gift of Neospora caninum, NC-1 isolate. This work was supported by Grant-in-Aid for Young Scientists (Start-up) from Japan Society for the Promotion of Science (18880003). References Anderson, M.L., Andrianarivo, A.G., Conrad, P.A., 2000. Neosporosis in cattle. Anim. Reprod. Sci. 60–61, 417–431. Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.W., Packham, A.E., Barr, B.C., Conrad, P.A., 1997. Evidence of vertical transmission of Neospora sp. infection in dairy cattle. J. Am. Vet. Med. Assoc. 210, 1169–1172. Barr, B.C., Conrad, P.A., Breitmeyer, R., Sverlow, K., Anderson, M.L., Reynolds, J., Chauvet, A.E., Dubey, J.P., Ardans, A.A., 1993. Congenital Neospora infection in calves born from cows that had previously aborted Neospora-infected fetuses: four cases (1990–1992). J. Am. Vet. Med. Assoc. 202, 113–117. Baszler, T.V., Shkap, V., Mwangi, W., Davies, C.J., Mathison, B.A., Mazuz, M., Resnikov, D., Fish, L., Leibovitch, B., Staska, L.M., Savitsky, I., 2008. Bovine immune response to inoculation with Neospora caninum surface antigen SRS2 lipopeptides mimics immune response to infection with live parasites. Clin. Vaccine Immunol. 15, 659–667. Cannas, A., Naguleswaran, A., Mu¨ller, N., Eperon, S., Gottstein, B., Hemphill, A., 2003. Vaccination of mice against experimental Neospora caninum infection using NcSAG1- and NcSRS2-based recombinant antigens and DNA vaccines. Parasitology 126, 303–312. Chahan, B., Gaturaga, I., Huang, X., Liao, M., Fukumoto, S., Hirata, H., Nishikawa, Y., Suzuki, H., Sugimoto, C., Nagasawa, H., Fujisaki, K., Igarashi, I., Mikami, T., Xuan, X., 2003. Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1. Vet. Parasitol. 118, 177–185. De Marez, T., Liddell, S., Dubey, J.P., Jenkins, M.C., Gasbarre, L., 1999. Oral infection of calves with Neospora caninum oocysts from dogs: Humoral and cellular immune responses. Int. J. Parasitol. 29, 1647–1657. Dubey, J.P., 1999. Recent advances in Neospora and neosporosis. Vet. Parasitol. 84, 349–367. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Carpenter, J.L., Speer, C.A., Topper, M.J., Uggla, A., 1988. Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192, 1269–1285.
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Gaturaga, I., Chahan, B., Xuan, X., Huang, X., Liao, M., Fukumoto, S., Hirata, H., Nishikawa, Y., Takashima, Y., Suzuki, H., Fujisaki, K., Sugimoto, C., 2005. Detection of antibodies to Neospora caninum in cattle by enzyme-linked immunosorbent assay with truncated NcSRS2 expressed in Escherichia coli. J. Parasitol. 91, 191–192. Haldorson, G.J., Stanton, J.B., Mathison, B.A., Suarez, C.E., Baszler, T.V., 2006. Neospora caninum: antibodies directed against tachyzoite surface protein NcSRS2 inhibit parasite attachment and invasion of placental trophoblasts in vitro. Exp. Parasitol. 112, 172–178. Haldorson, G.J., Mathison, B.A., Wenberg, K., Conrad, P.A., Dubey, J.P., Trees, A.J., Yamane, I., Baszler, T.V., 2005. Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice. Int. J. Parasitol. 35, 1407–1415. Hara, A.O., Liao, M., Baticados, W., Bannai, H., Zhang, G., Zhang, S., Lee, E.G., Nishikawa, Y., Claveria, F., Igarashi, M., Nagasawa, H., Xuan, X., 2006. Expression of recombinant dense granule protein 7 of Neospora caninum and evaluation of its diagnostic potential for canine neosporosis. J. Protozool. Res. 16, 34–41. Innes, E.A., Wright, S.E., Maley, S., Rae, A., Schock, A., Kirvar, E., Bartley, P., Hamilton, C., Carey, I.M., Buxton, D., 2001. Protection against vertical transmission in bovine neosporosis. Int. J. Parasitol. 31, 1523–1534. Liao, M., Xuan, X., Huang, X., Shirafuji, H., Fukumoto, S., Hirata, H., Suzuki, H., Fujisaki, K., 2005. Identification and characterization of crossreactive antigens from Neospora caninum and Toxoplasma gondii. Parasitology 130, 481–488. Liddell, S., Jenkins, M.C., Collica, C.M., Dubey, J.P., 1999. Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination. J. Parasitol. 85, 1072–1075. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., MuGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–1478. Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2000a. Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells. Int. J. Parasitol. 30, 51–58. Nishikawa, Y., Kousaka, Y., Fukumoto, S., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2000b. Delivery of Neospora caninum surface protein, NcSRS2 (Nc-p43), to mouse using recombinant vaccinia virus. Parasitol. Res. 86, 934–939. Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2001a. Prevention of vertical transmission of Neospora caninum in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene. Vaccine 19, 1710–1716. Nishikawa, Y., Inoue, N., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2001b. Protective efficacy of vaccination by recombinant vaccinia virus against Neospora caninum infection. Vaccine 19, 1381–1390. Pare´, J., Thurmond, M.C., Hietala, S.K., 1996. Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. Res. 60, 133–139. Pinitkiatisakul, S., Mattsson, J.G., Wikman, M., Friedman, M., Bengtsson, K.L., Sta˚hl, S., Lunde´n, A., 2005. Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms. Vet. Parasitol. 129, 25–34. Pinitkiatisakul, S., Friedman, M., Wikman, M., Mattsson, J.G., Lo¨vgrenBengtsson, K., Sta˚hl, S., Lunde´n, A., 2007. Immunogenicity and protective effect against murine cerebral neosporosis of recombinant NcSRS2 in different iscom formulations. Vaccine 25, 3658–3668. Schares, G., Peters, M., Wurm, R., Ba¨rwald, A., Conraths, F.J., 1998. The efficiency of vertical transmission of Neospora caninum in dairy cattle analysed by serological techniques. Vet. Parasitol. 80, 87–98. Staska, L.M., Davies, C.J., Brown, W.C., McGuire, T.C., Suarez, C.E., Park, J.Y., Mathison, B.A., Abbott, J.R., Baszler, T.V., 2005. Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferon-secreting T lymphocytes of infected Holstein cattle. Infect. Immun. 73, 1321–1329. Thurmond, M.C., Hietala, S.K., 1997. Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle. Am. J. Vet. Res. 58, 1381–1385. Williams, D.J., Guy, C.S., McGarry, J.W., Guy, F., Tasker, L., Smith, R.F., MacEachern, K., Cripps, P.J., Kelly, D.F., Trees, A.J., 2000. Neospora caninum-associated abortion in cattle: the time of experimentallyinduced parasitaemia during gestation determines foetal survival. Parasitology 121, 347–358.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Effects of a transferring antibody against Neospora caninum infection in a murine model Yoshifumi Nishikawa *, Houshuang Zhang, Penglong Huang, Guohong Zhang, Xuenan Xuan National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 May 2008 Received in revised form 14 October 2008 Accepted 15 October 2008
We investigated the roles of a transferring antibody against Neospora caninum infection based on a murine model using recombinant vaccinia virus carrying NcSRS2 gene. Higher levels of anti-NcSRS2 antibody were detected in surviving offspring from vaccinated dams than controls while the transferring anti-NcSAG1 antibody was detected in the surviving offspring from unvaccinated dams. After mating, the female mice with transferring antibody against N. caninum were challenged with the parasites in mid-gestation. The transferring antibodies disappeared during pregnancy upon parasite infection. There was no significant difference on the parasite burden in dams and the survival rates of their offspring. Here, we have shown that N. caninum-specific transferring antibody does not control parasite infection in mice. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Neospora caninum Maternal immunity Transferring antibody
1. Introduction Infection with the coccidian parasite Neospora caninum causes paralysis and death in livestock and companion animals (Dubey and Lindsay, 1996). In cattle, N. caninum infection is associated with abortions, stillbirth, and neurological disease in calves, making neosporosis an important economic agricultural concern (Dubey, 1999). Transplacental transmission from a naturally infected dam to her fetus appears to be the major, and only confirmed intraspecific, natural route of transmission of the parasite (Pare´ et al., 1996; Anderson et al., 1997; Schares et al., 1998). Repeated in utero infections can occur in the same dam during subsequent pregnancies, resulting in abortion or congenital infection (Barr et al., 1993; Thurmond and Hietala, 1997). Congenitally infected calves remain persistently infected and can pass the infection onto their offspring (Pare´ et al., 1996; Anderson et al., 1997). Cows may also become infected by ingestion of oocysts that have
* Corresponding author. Tel.: +81 155 49 5886; fax: +81 155 49 5643. E-mail address: [email protected] (Y. Nishikawa). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.10.095
been shed by a definitive host, such as dogs (McAllister et al., 1998; De Marez et al., 1999). There is accumulating evidence that some N. caninuminfected cows can develop a degree of protective immunity against abortion and/or congenital transmission, indicating the advantage of vaccination. Although prevention of abortion might be a more realistic goal for a vaccine, the ultimate objective for control of this disease must be to prevent vertical transmission of the parasite. Several groups have investigated induction of protective immunity using recombinant vaccines, especially those based on a N. caninum surface antigen, NcSRS2. Our previous results showed that N. caninum infection was controlled in BALB/c mice that were immunized with a recombinant vaccinia virus carrying NcSRS2 (Nishikawa et al., 2001a,b). In addition, vaccination of mice with recombinant NcSRS2 produced by E. coli or the native protein, induced protective immunity against N. caninum infection (Cannas et al., 2003; Haldorson et al., 2005; Pinitkiatisakul et al., 2005, 2007). NcSRS2 peptides induce specific T cell activation, interferon-gamma secretion of peripheral blood mononuclear cells and antibody production in cattle (Staska et al., 2005; Baszler et al., 2008).
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Although prevention of vertical transmission of the parasite is important, the roles of maternal immunity are not well known. Transferring antibody is the antibody derived from the dam’s immune response to an antigen and transferred via placenta or colostrum to the offspring without need of the offspring’s active immune response to the same antigen. We have addressed the following question: Can the transferring antibodies derived from the dam infected with N. caninum or immunized with vaccine control the vertical transmission of the parasites? To determine the effects of the parasite-specific transferring antibodies, we used a murine model using a recombinant vaccinia virus carrying NcSRS2. This study would provide important insights for prevention of neosporosis. 2. Materials and methods 2.1. Preparation of N. caninum tachyzoites Neospora caninum tachyzoites of the Nc-1 isolate (Dubey et al., 1988) were maintained in monkey kidney adherent fibroblasts (Vero cells) cultured in Eagle’s minimum essential medium (EMEM, Sigma, St. Louis, MO) supplemented with 8% heat-inactivated fetal bovine serum (FBS). For the purification of tachyzoites, parasites and host-cell debris were washed in cold PBS, and the final pellet was resuspended in cold PBS and passed through a 27-gauge needle and a 5.0-mm-pore filter (Millipore, Bedford, MA). 2.2. Preparation of recombinant vaccinia viruses The recombinant vaccinia virus which expresses NcSRS2 was constructed as previously described (Nishikawa et al., 2000b). For preparation of viral inoculum, recombinant vaccinia viruses were propagated in rabbit kidney (RK13) cells in OPTI-MEM1I (OPTI-MEM, Gibco BRL, New York) without FBS.
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at 1 week after boost and were inspected twice daily for the presence of vaginal plugs. The first day a plug was noted was designated as day 0 of pregnancy for each individual. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week post-vaccination). The surviving offspring (F1) were used for investigation of maternal immunity (Fig. 1). The surviving F1 female mice or naı¨ve female mice, 10 weeks of age, were mated with naı¨ve males, 10 weeks of age, for 3 days (1 female with 1 male per cage) at 1 week. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation. The surviving offspring were named F2 (Fig. 1). Numbers and survival rates of F2 offspring were measured at 30 days after birth. 2.5. DNA isolation and PCR analysis For DNA preparation, each whole brain of BALB/c mice was thawed in 10 time-volumes of extraction buffer (0.1 M Tris–HCl pH 9.0, 1% SDS, 0.1 M NaCl, 1 mM EDTA) and 1 mg/ml of Proteinase K at 55 8C. The DNA was purified by phenol-chloroform extraction and ethanol precipitation. The DNA concentration was adjusted to 100 mg/ml for the brain as a template DNA. The DNA amplified by PCR was suspended in 10 ml reaction mixture containing 2.5 ml of template DNA, 1 ml of 10 PCR buffer which contained 15 mM MgCl2 (PerkinElmer, Boston, MA), 1 ml of 10 mM dNTP mix, 0.1 ml of 5 U/ml Ampli GoldTMTaq DNA polymerase (PerkinElmer) and 2 ml of 10 pmol/ml N. caninum specific primers, Np6 and Np21 (Liddell et al., 1999). Amplification was done in a thermal cycler, GeneAmp PCR System 2400 (PerkinElmer) employing 40 cycles for denaturation (94 8C, 1 min), annealing (63 8C, 1 min) and primer extension (74 8C, 3.5 min). At the end of cycle reaction, a primer extension was continued for 10 min at 74 8C and then kept at 4 8C. The PCR products were visualized by electrophoresis in agarose gels. 2.6. Measurement of N. caninum specific antibodies
2.3. Mice BALB/c male and female mice, 6–7 weeks of age, were obtained from Clea Japan (Tokyo, Japan). Until their use at 7–8 weeks of age, mice were housed under specific pathogen-free conditions in the animal facility of the National Research Center for Protozoan Diseases at the Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. Mice were cared for and used under the Guiding Principles for the Care and Use of Research Animals promulgated by the Obihiro University of Agriculture and Veterinary Medicine. 2.4. Vaccination, mating and challenge infection Female mice, 6–7 weeks of age, were injected intraperitoneally (i.p.) with OPTI-MEM or 5 106 plaque forming units of recombinant vaccinia virus and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males, 10 weeks of age, for 3 days (1 female with 1 male per cage)
Serum (20 ml) was obtained from F1 mice at 30 days after birth and female F1 mice or naı¨ve female mice at 0, 17 and 30 days after mating via tail vein for measuring N. caninum-specific antibodies by ELISA. To confirm the lack of an antibody response in unvaccinated and uninfected mice, control serum was taken from all the animals. The recombinant proteins of NcSAG1, NcSRS2, and NcGRA7 were expressed in E. coli as glutathione S-transferase (GST) fusion proteins and then purified using Glutathione Sepharose 4B (Amersham Pharmacia Biotech, Sweden) as described previously (Chahan et al., 2003; Gaturaga et al., 2005; Hara et al., 2006). The lysate of N. caninum (NLA) was prepared as previously reported (Liao et al., 2005). Fifty microliters of purified NcSAG1, NcSRS2, NcGRA7, and their control GST, as well as NLA at a final concentration of 5 mg/ml was coated on ELISA plates (Nunc, Denmark) overnight at 4 8C with a carbonatebicarbonate buffer (pH 9.6). After blocking with PBS containing 3% skim milk (PBS-SM) for 1 h at 37 8C, the plates were washed twice with PBS containing 0.05%
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Fig. 1. Procedure for vaccination, mating and challenge infection. Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week post-vaccination). The surviving F1 female mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve males for 3 days at 1 week. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation. The surviving offspring were named F2.
Fig. 2. Transferring antibody in F1 offspring. Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week postvaccination). Sera of the surviving F1 offspring from vaccinated and control groups at 30 days after birth were collected and examined by ELISA using the parasite lysates (NLA), NcSRS2, NcSAG1 and NcGRA7 as antigens. Vaccinated (N = 21); control (N = 19). The statistical difference was determined by twosided Mann–Whitney’s U-test. Differences of P < 0.001 were considered significant. Data are representative of repeated experiments.
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Tween20 (PBS-T), and 100 ml of serum samples diluted at 1:250 with PBS-SM was added to duplicate wells. Plates were incubated at 37 8C for 1 h. After washing five times with PBS-T, plates were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Bethyl Laboratories, USA) diluted at 1:4000 with PBS-SM at 37 8C for 1 h. The plates were washed five times, and then substrate solution (0.1 M citric acid, 0.2 M sodium phosphate, 0.003% H2O2, and 0.3 mg/ml 2,20 -azide-bis[3ethylbenzthiazoline-6-sulfonic acid]; Sigma, St. Louis, MO) was added to each well in 100-ml aliquots. The absorbance at 415 nm was read after 1 h of incubation at room temperature using an ELISA reader (Corona Microplate Reader MTP-120; Corona, Tokyo, Japan). The ELISA result was determined by the difference in the mean optical density at a value of 415 nm (OD415nm) between the recombinant antigen (NcSGA1, NcSRS2, or NcGRA7) and the GST protein. For ELISA using NLA, the result was determined by simply taking the OD415nm value. 3. Results 3.1. Transferring antibody in surviving offspring Previous studies have shown that vaccination of dams with recombinant vaccinia virus expressing NcSRS2 (vvNcSRS2) appeared to confer effective protection against
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vertical transmission to F1 offspring (Nishikawa et al., 2001a). The survival rate of F1 offspring in the group vaccinated with vvNcSRS2 was 83.3%, while it was 27.3% in the group vaccinated with recombinant vaccinia virus expressing green fluorescence protein (control virus) and 28.9% in the group inoculated with medium. Therefore, we focused on maternal immunity induced by vaccination with vvNcSRS2 against N. caninum infection. Fig. 2 represents the transferring antibody in F1 offspring. The offspring from vvNcSRS2-vaccinated dams had higher levels of anti-NLA and NcSRS2 antibodies than controls. In F1 offspring from control mice, higher levels of antiNcSAG1 antibody were detected than in experimentally vaccinated animals. There was no significant difference in the production of anti-NcGRA7 antibody. 3.2. Antibody production in F1 mice during pregnancy following N. caninum infection Next, we examined parasite-specific antibody production in the F1 mice during pregnancy following N. caninum infection (Fig. 3). After mating, female F1 mice or naı¨ve female mice were challenged with N. caninum. The lack of an antibody response against NLA, NcSRS2, NcSAG1 and NcGRA7 in unvaccinated and uninfected mice (Day 0 of naı¨ve group) was confirmed. Although high levels of antiNcSRS2 antibody were detected at Day 0 in the F1 mice from
Fig. 3. Antibody production of F1 female mice following N. caninum infection. Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve males for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6– 9 days of gestation (4-week post-vaccination). Female F1 mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve male mice and were then challenged with N. caninum at 6–9 days gestation. Sera of dams (N = 6) were collected at 0, 17 and 30 days after mating and examined by ELISA using the parasite lysates (NLA), NcSRS2, NcSAG1 and NcGRA7 as antigens. Data are representative of repeated experiments.
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64 Table 1 PCR detection of N. caninum in dams. Treatment
Trial
Total number of dams positive/total number of dams
Vaccinated
Trial 1 Trial 2 Trials 1 and 2
2/7 (28.6%) 5/6 (83.3%) 7/13 (53.8%)
Control
Trial 1 Trial 2 Trials 1 and 2
4/6 (66.7%) 3/5 (60.0%) 7/11 (63.6%)
Naı¨ve
Trial 1 Trial 2 Trials 1 and 2
2/4 (50.0%) 3/5 (60.0%) 6/10 (60.0%)
Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve male mice for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week postvaccination). Female F1 mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve male mice and were then challenged with N. caninum at 6–9 days gestation. DNA was extracted from brain of the dams at 40 days after the infection, and were then analyzed by PCR. The statistical difference was determined by x2-test.
dams vaccinated with vvNcSRS2, the blood levels of the antibody decreased upon parasite infection. All antibodies tested here were detected at Day 30 in the F1 mice from dams vaccinated with vvNcSRS2 or control dams. 3.3. Effects of transferring antibody against N. caninum infection The PCR detection assay allows examination of tissue localization of N. caninum. Although N. caninum DNA was detected brain, lungs, liver and spleen of BALB/c mice until 6 days after the infection, parasite DNA were found almost exclusively in the brain at 10 and 26 days after the infection (Nishikawa et al., 2001b). To confirm the effects of transferring antibody against N. caninum infection, PCR analyses were carried out using brain tissue from F1 mice challenged with the parasites. As shown in Table 1, there
were no statistically significant differences among the tested group (P > 0.05). In addition, the numbers and survival rates of F2 offspring were examined (Table 2). There was no statistically significant difference in the numbers of F2 offspring and the survival rates among the groups (P > 0.05). 4. Discussion Since N. caninum infection has emerged as an important reproductive disease in cattle throughout the world (Anderson et al., 2000), the development of a vaccine against this parasite is thought to be advantageous. Previous studies have demonstrated that recombinant vaccines based on NcSRS2 afforded effective protection against N. caninum infection (Nishikawa et al., 2001a,b; Cannas et al., 2003; Pinitkiatisakul et al., 2005, 2007; Haldorson et al., 2006). However, the role maternal antibody may play in the next generation after N. caninum infection has not yet been evaluated. Our study indicates that anti-NcSRS2 IgG antibodies induced by vaccination of dams with recombinant vaccinia virus expressing NcSRS2 were detected in the offspring (Fig. 2). Interestingly, levels of anti-NcSAG1 antibody in offspring from control mice were higher that those from the vaccinated group (Fig. 2). These results indicated that anti-NcSRS2 and anti-NcSAG1 antibodies were transferred from vaccinated and nonvaccinated dams to the offspring, respectively. However, serum levels of the transferred antibodies decreased upon parasite infection (Fig. 3). This may be due to the binding of the transferred antibodies to the parasite. Although previous studies have shown that anti-NcSAG1 antibody or anti-NcSRS2 antibody inhibited N. caninum invasion into the host cells in an in vitro model (Nishikawa et al., 2000a; Haldorson et al., 2006), the reactions of the parasitespecific antibody do not affect the inhibition of the parasite burden in an in vivo model. These results showed that the transferring antibody does not contribute to protective immunity against N. caninum infection in mice. Our observation suggests the repeated in utero infection in the same dam during bovine pregnancy.
Table 2 Numbers and survival rates of F2 offspring. Treatment
Trial
Mean number pups/litter (S.D.)
Number of surviving pups/number of pups in litter
Total number of surviving pups/total number of pups
Vaccinated
Trial 1 Trial 2 Trials 1 and 2
6.0 (2.10) 6.0 (1.00) 6.0 (1.61)
4/4, 1/9, 1/4, 5/6, 3/8, 2/5 4/7, 0/5, 1/6, 4/7, 0/5
16/36(44.4%) 9/30 (30.0%) 25/66 (37.9%)
Control
Trial 1 Trial 2 Trials 1 and 2
5.6 (2.70) 7.6 (0.55) 6.6 (2.12)
4/4, 1/3, 8/9, 0/4, 4/8 1/8, 3/7, 2/7, 0/8, 1/8
17/28(60.7%) 7/38(18.4%) 24/66 (36.4%)
Naı¨ve
Trial 1 Trial 2 Trials 1 and 2
5.3 (1.53) 4.7 (0.58) 5.0 (1.10)
3/5, 0/4, 3/7 0/5, 3/5, 1/4
6/16(37.5%) 4/14(28.6%) 10/30(33.3%)
Female mice were injected intraperitoneally (i.p.) with OPTI-MEM (control group) or 5 106 plaque forming units of vvNcSRS2 (vaccinated group) and boosted with the same inoculum at 2 weeks after the first inoculation. Female mice were housed with naı¨ve male mice for 3 days at 1 week after boost. All pregnant dams were challenged on the same day with 1 105 Nc-1 tachyzoites at 6–9 days of gestation (4-week post-vaccination). Female F1 mice from vaccinated and control groups or naı¨ve female mice were mated with naı¨ve male mice and were then challenged with N. caninum at 6–9 days gestation. Numbers of surviving F2 offspring at 30 days after birth were analyzed by ANOVA, and the differences were then analyzed using Turkey–Kramer multiple comparison tests. For the survival rates of offspring, the statistical difference was determined by x2-test.
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The present study suggests that each generation should have a vaccination to prevent N. caninum infection because of no protective efficiency of the transferring antibody. However, to control the neosporosis, we have to consider the following points: vaccines which can not be neutralized by the transferring antibody and can induce cellular immunity against N. caninum: diagnostic systems which distinguish between naturally infected and vaccinated animals. Previous studies have suggested that nonpregnant animals might develop sufficient immunity to protect against vertical transmission during a subsequent pregnancy (Williams et al., 2000; Innes et al., 2001; Nishikawa et al., 2001a). Therefore, after the above-mentioned problems are overcome, vaccine therapy for nonpregnant animals could help to control vertical transmission of N. caninum. Acknowledgments We thank Dr. Dubey (United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, and Parasite Biology and Epidemiology Laboratory) for the gift of Neospora caninum, NC-1 isolate. This work was supported by Grant-in-Aid for Young Scientists (Start-up) from Japan Society for the Promotion of Science (18880003). References Anderson, M.L., Andrianarivo, A.G., Conrad, P.A., 2000. Neosporosis in cattle. Anim. Reprod. Sci. 60–61, 417–431. Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.W., Packham, A.E., Barr, B.C., Conrad, P.A., 1997. Evidence of vertical transmission of Neospora sp. infection in dairy cattle. J. Am. Vet. Med. Assoc. 210, 1169–1172. Barr, B.C., Conrad, P.A., Breitmeyer, R., Sverlow, K., Anderson, M.L., Reynolds, J., Chauvet, A.E., Dubey, J.P., Ardans, A.A., 1993. Congenital Neospora infection in calves born from cows that had previously aborted Neospora-infected fetuses: four cases (1990–1992). J. Am. Vet. Med. Assoc. 202, 113–117. Baszler, T.V., Shkap, V., Mwangi, W., Davies, C.J., Mathison, B.A., Mazuz, M., Resnikov, D., Fish, L., Leibovitch, B., Staska, L.M., Savitsky, I., 2008. Bovine immune response to inoculation with Neospora caninum surface antigen SRS2 lipopeptides mimics immune response to infection with live parasites. Clin. Vaccine Immunol. 15, 659–667. Cannas, A., Naguleswaran, A., Mu¨ller, N., Eperon, S., Gottstein, B., Hemphill, A., 2003. Vaccination of mice against experimental Neospora caninum infection using NcSAG1- and NcSRS2-based recombinant antigens and DNA vaccines. Parasitology 126, 303–312. Chahan, B., Gaturaga, I., Huang, X., Liao, M., Fukumoto, S., Hirata, H., Nishikawa, Y., Suzuki, H., Sugimoto, C., Nagasawa, H., Fujisaki, K., Igarashi, I., Mikami, T., Xuan, X., 2003. Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1. Vet. Parasitol. 118, 177–185. De Marez, T., Liddell, S., Dubey, J.P., Jenkins, M.C., Gasbarre, L., 1999. Oral infection of calves with Neospora caninum oocysts from dogs: Humoral and cellular immune responses. Int. J. Parasitol. 29, 1647–1657. Dubey, J.P., 1999. Recent advances in Neospora and neosporosis. Vet. Parasitol. 84, 349–367. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Carpenter, J.L., Speer, C.A., Topper, M.J., Uggla, A., 1988. Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192, 1269–1285.
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Gaturaga, I., Chahan, B., Xuan, X., Huang, X., Liao, M., Fukumoto, S., Hirata, H., Nishikawa, Y., Takashima, Y., Suzuki, H., Fujisaki, K., Sugimoto, C., 2005. Detection of antibodies to Neospora caninum in cattle by enzyme-linked immunosorbent assay with truncated NcSRS2 expressed in Escherichia coli. J. Parasitol. 91, 191–192. Haldorson, G.J., Stanton, J.B., Mathison, B.A., Suarez, C.E., Baszler, T.V., 2006. Neospora caninum: antibodies directed against tachyzoite surface protein NcSRS2 inhibit parasite attachment and invasion of placental trophoblasts in vitro. Exp. Parasitol. 112, 172–178. Haldorson, G.J., Mathison, B.A., Wenberg, K., Conrad, P.A., Dubey, J.P., Trees, A.J., Yamane, I., Baszler, T.V., 2005. Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice. Int. J. Parasitol. 35, 1407–1415. Hara, A.O., Liao, M., Baticados, W., Bannai, H., Zhang, G., Zhang, S., Lee, E.G., Nishikawa, Y., Claveria, F., Igarashi, M., Nagasawa, H., Xuan, X., 2006. Expression of recombinant dense granule protein 7 of Neospora caninum and evaluation of its diagnostic potential for canine neosporosis. J. Protozool. Res. 16, 34–41. Innes, E.A., Wright, S.E., Maley, S., Rae, A., Schock, A., Kirvar, E., Bartley, P., Hamilton, C., Carey, I.M., Buxton, D., 2001. Protection against vertical transmission in bovine neosporosis. Int. J. Parasitol. 31, 1523–1534. Liao, M., Xuan, X., Huang, X., Shirafuji, H., Fukumoto, S., Hirata, H., Suzuki, H., Fujisaki, K., 2005. Identification and characterization of crossreactive antigens from Neospora caninum and Toxoplasma gondii. Parasitology 130, 481–488. Liddell, S., Jenkins, M.C., Collica, C.M., Dubey, J.P., 1999. Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination. J. Parasitol. 85, 1072–1075. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., MuGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–1478. Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2000a. Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells. Int. J. Parasitol. 30, 51–58. Nishikawa, Y., Kousaka, Y., Fukumoto, S., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2000b. Delivery of Neospora caninum surface protein, NcSRS2 (Nc-p43), to mouse using recombinant vaccinia virus. Parasitol. Res. 86, 934–939. Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2001a. Prevention of vertical transmission of Neospora caninum in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene. Vaccine 19, 1710–1716. Nishikawa, Y., Inoue, N., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H., Mikami, T., 2001b. Protective efficacy of vaccination by recombinant vaccinia virus against Neospora caninum infection. Vaccine 19, 1381–1390. Pare´, J., Thurmond, M.C., Hietala, S.K., 1996. Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. Res. 60, 133–139. Pinitkiatisakul, S., Mattsson, J.G., Wikman, M., Friedman, M., Bengtsson, K.L., Sta˚hl, S., Lunde´n, A., 2005. Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms. Vet. Parasitol. 129, 25–34. Pinitkiatisakul, S., Friedman, M., Wikman, M., Mattsson, J.G., Lo¨vgrenBengtsson, K., Sta˚hl, S., Lunde´n, A., 2007. Immunogenicity and protective effect against murine cerebral neosporosis of recombinant NcSRS2 in different iscom formulations. Vaccine 25, 3658–3668. Schares, G., Peters, M., Wurm, R., Ba¨rwald, A., Conraths, F.J., 1998. The efficiency of vertical transmission of Neospora caninum in dairy cattle analysed by serological techniques. Vet. Parasitol. 80, 87–98. Staska, L.M., Davies, C.J., Brown, W.C., McGuire, T.C., Suarez, C.E., Park, J.Y., Mathison, B.A., Abbott, J.R., Baszler, T.V., 2005. Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferon-secreting T lymphocytes of infected Holstein cattle. Infect. Immun. 73, 1321–1329. Thurmond, M.C., Hietala, S.K., 1997. Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle. Am. J. Vet. Res. 58, 1381–1385. Williams, D.J., Guy, C.S., McGarry, J.W., Guy, F., Tasker, L., Smith, R.F., MacEachern, K., Cripps, P.J., Kelly, D.F., Trees, A.J., 2000. Neospora caninum-associated abortion in cattle: the time of experimentallyinduced parasitaemia during gestation determines foetal survival. Parasitology 121, 347–358.