Reduction in transplacental transmission of Neospora caninum in outbred mice by vaccination

Infection with the protozoan parasite Neospora caninum is an important cause of abortion in cattle. A major source of infection is transplacental transfer of the parasite from mother to offspring during pregnancy. This study describes investigations on the immunisation of outbred Qs mice before pregnancy with live or a crude lysate of N. caninum (NC-Nowra isolate) to prevent transplacental transfer of a challenge infection administered during pregnancy. Parasites present in the brains of pups from mice challenged with N. caninum (NC-Liverpool) were detected by PCR. Injection of live NC-Nowra tachyzoites before pregnancy dramatically reduced transplacental transfer from 75 to 0.8% in one experiment and from 76 to 8% in a second experiment. Injection of a crude lysate of NC-Nowra tachyzoites reduced transplacental transfer from 67 to 53% in one experiment and from 76 to 63% in a second experiment. Analysis of N. caninum-specific IgG1 and IgG2a antibody levels prior to pregnancy and challenge showed that NC-Nowra lysate induced a response skewed towards IgG1 whereas live parasites induced both IgG1 and IgG2a antibodies. After pregnancy and a challenge infection, a similar IgG1/IgG2a response was seen in all challenged groups. These results provide further positive support for the hypothesis that transplacental transmission of this parasite is preventable by vaccination.     

Prevention of lethal experimental infection of C57BL/6 mice by vaccination with Brucella abortus strain RB51 expressing Neospora caninum antigens

Bovine abortions caused by the intracellular protozoal parasite Neospora caninum are a major concern to cattle industries worldwide. A strong Th1 immune response is required for protection against N. caninum. Brucella abortus strain RB51 is currently used as a live, attenuated vaccine against bovine brucellosis. Strain RB51 can also be used as an expression vector for heterologous protein expression. In this study, putative protective antigens of N. caninum MIC1, MIC3, GRA2, GRA6 and SRS2, were expressed individually in B. abortus strain RB51. The ability of each of the recombinant RB51 strains to induce N. caninum-specific immunity was assessed in C57BL/6 mice. Mice were immunised by two i.p. inoculations, 4 weeks apart. Five weeks after the second immunisation, spleen cells from the vaccinated mice secreted high levels of IFN-gamma and IL-10 upon in vitro stimulation with N. caninum whole cell lysate antigens. N. caninum-specific antibodies of both IgG1 and IgG2a subtypes were detected in the serum of the vaccinated mice. Mice in the vaccinated and control groups were challenged with 2 x 10(7)N. caninum tachyzoites i.p. and observed for 28 days after vaccination. All unvaccinated control mice died within 7 days. Mice in the MIC1 and GRA6 vaccine groups were completely protected while the mice in the SRS2, GRA2 and MIC3 vaccinated groups were partially protected and experienced 10-50% mortality. The non-recombinant RB51 vector control group experienced an average protection of 69%. These results suggest that expression of protective antigens of N. caninum in B. abortus strain RB51 is a novel approach towards the development of a multivalent vaccine against brucellosis and neosporosis.     

Effects of re-infection with Neospora caninum in bulls on parasite detection in semen and blood and immunological responses

Three bulls with experimentally induced primary infection with Neospora caninum were re-infected intravenously with 10(8) live N. caninum tachyzoites of the NC-1 isolate at 300 days post-infection to investigate the presence of N. caninum in semen and blood, and the associated immune responses. In parallel, three bulls with experimentally induced primary infection with N. caninum and three non-infected bulls were also monitored. Re-infected and infected bulls showed an intermittent presence of N. caninum DNA in semen with a parasite load ranging from 0.1 to 15.6 (mean 4.4) and 0.1 to 11.1 (mean 4.1) parasites/ml, respectively. Re-infected bulls showed significant and persistent serum-specific IgM and IgG antibody responses. Specific IgG levels were detected in seminal plasma of all infected bulls, but the magnitude of the response was significantly higher in re-infected rather than in chronically infected animals. The mean specific IFN-gamma levels in re-infected bulls were significantly increased as early as 3 and 7 days after experimental infection when compared to bulls in other groups. This study showed that the intermittent presence and parasite load of N. caninum in the semen of re-infected bulls is very similar to that reported in chronically infected animals. The protozoa could not be isolated from BALB/c nu/nu mice inoculated with PCR-positive semen samples and inseminated heifers with pooled semen samples did not show seroconversion. Plasma IFN-gamma level seems to be a good indicator of a recent N. caninum infection.     

Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.     

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.     

Neospora caninum: high susceptibility to the parasite in C57BL/10ScCr mice

C57BL/10ScCr mice, lack Toll-like receptor 4 and a functional Interleukin-12 receptor. Taking this into account, susceptibility of these mice to Neospora caninum infection was assessed comparatively to that of immunocompetent C57BL/10ScSn mice. C57BL/10ScCr mice inoculated intraperitoneally with 5x10(5)N. caninum tachyzoites showed a high susceptibility to this parasite. All infected C57BL/10ScCr mice were dead by day 8 post-infection whereas all control C57BL/10ScSn mice survived this parasitic challenge. Immunohistochemical analysis of infected C57BL/10ScCr mice showed N. caninum tachyzoites spread in the pancreas, liver, lung, intestine, heart and brain whereas no parasites were detected in similarly infected C57BL/10ScSn controls. The higher susceptibility of C57BL/10ScCr mice to neosporosis correlates with reduced interferon-gamma mRNA expression and increased IL-4 mRNA expression, comparatively to C57BL/10ScSn controls, detected in the spleen after the parasitic challenge. C57BL/10ScCr mice could thus be used as a new experimental model where to study immunobiological mechanisms associated with host susceptibility to neosporosis.     

Natural killer cells act as early responders in an experimental infection with Neospora caninum in calves

The intracellular protozoan parasite Neospora caninum is a cause of abortion and congenital disease in cattle worldwide. We have previously shown that natural killer (NK) cells produce IFN-gamma in response to N. caninum tachyzoites in vitro. This study aimed to investigate the role of NK cells and other cellular immune responses in an experimental N. caninum infection model in calves. Phenotyping of peripheral blood lymphocytes showed a drop in the percentage of NK cells at days 4-6 after i.v. inoculation, followed by an increase in the percentage of both NK cells and CD8+ T cells which peaked at days 11-15. A whole blood flow cytometric assay showed that CD4+ T cells were the major IFN-gamma producing cells, but in the early stages of the infection both NK cells and CD8+ T cells contributed to IFN-gamma production. We also compared the ability of two different N. caninum antigen preparations--sonicated soluble antigens and intact heat-inactivated parasites--to induce proliferation and IFN-gamma production in various cell types. Heat-inactivated tachyzoites induced a 3.7 times greater increase in the number of IFN-gamma producing NK cells compared with sonicated soluble antigens. This indicated the presence of some NK cell-stimulating antigens in the intact tachyzoite that were absent from the sonicated soluble antigens. The heat-inactivated whole tachyzoites also inhibited gammadelta T cell proliferation while the soluble antigens from N. caninum did not. We believe this is the first time NK cells have been demonstrated to be early responders in N. caninum infection in calves.     

Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens

Bovine abortions caused by the apicomplexan parasite Neospora caninum have been responsible for severe economic losses to the cattle industry. Infected cows either experience abortion or transmit the parasite transplacentally at a rate of up to 95%. Neospora caninum vaccines that can prevent vertical transmission and ensure disruption in the life cycle of the parasite greatly aid in the management of neosporosis in the cattle industry. Brucella abortus strain RB51, a commercially available vaccine for bovine brucellosis, can also be used as a vector to express plasmid-encoded proteins from other pathogens. Neospora caninum protective antigens MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in strain RB51. Female C57BL/6 mice were vaccinated with a recombinant strain RB51 expressing N. caninum antigen or irradiated tachyzoites, boosted 4 weeks later and then bred. Antigen-specific IgG, IFN-gamma and IL-10 were detected in vaccinated pregnant mice. Vaccinated mice were challenged with 5 x 10(6)N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups 3 weeks after birth and examined for the presence of N. caninum by real-time PCR. The RB51-MIC3, RB51-GRA6, irradiated tachyzoite vaccine, pooled strain RB51-Neospora vaccine, RB51-MIC1 and RB51-SRS2 vaccines elicited approximately 6-38% protection against vertical transmission. However, the differences in parasite burden in brain tissue of pups from the control and vaccinated groups were highly significant for all groups. Thus, B. abortus strain RB51 expressing the specific N. caninum antigens induced substantial protection against vertical transmission of N. caninum in mice.     

Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice

NcSRS2, a tachyzoite surface protein of Neospora caninum, is an immunodominant protein with respect to induction of antibody production and has a role in attachment and invasion of host cells. Native NcSRS2 was isolated from whole tachyzoite lysate antigen by affinity chromatography using NcSRS2 specific monoclonal antibody and used to immunize BALB/c mice in a congenital transmission study. NcSRS2 was a highly conserved protein as indicated by comparison of deduced amino acid sequence obtained from NcSRS2 gene sequences of 10 geographically distinct N. caninum isolates. Mice immunized with purified native NcSRS2 produced antigen-specific antibody, primarily of IgG 1 subtype. Following challenge during gestation with 10(7) tachyzoites, immunized mice had a statistically significant decreased frequency of congenital transmission compared to non-immunized mice (P相似文献   

Quantitative analysis of parasite DNA in the blood of immunized and naïve mice after infection with Neospora caninum

Real-time PCR was used to study the duration and level of parasitaemia in mice immunized with immune-stimulating complexes (iscoms) containing recombinant NcSRS2, one of the immunodominant surface antigens of Neospora caninum. After challenge infection, blood was collected daily for 9 days. During this period the amounts of parasite DNA detected in immunized mice were significantly lower (P<0.001), and the duration of parasitaemia appeared to be shorter, than in non-immunized controls. Furthermore, the degree of parasitaemia seemed to correlate well with the amount of N. caninum DNA in the brain 3 weeks post-inoculation and with disease severity measured as changes in body weight. These results indicate that the protective immunity induced by the NcSRS2-iscoms was sufficient to reduce the level of parasitaemia, which probably reduced the number of parasites reaching the brain, and could be the reason for the reduction in brain parasite load and clinical symptoms. Furthermore, real-time PCR was found to be a sensitive means for rapid assessment of N. caninum in blood.     

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.     

First isolation of Neospora caninum from an aborted bovine fetus in Spain

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.     

Experimental neosporosis in bulls: parasite detection in semen and blood and specific antibody and interferon-gamma responses

AIM: To investigate the presence of Neospora caninum in semen and blood, and the development of specific antibody and interferon-gamma (IFN-gamma) responses in experimentally infected bulls. METHODS: Eight bulls were intravenously infected with 10(8) live N. caninum tachyzoites of NC-1 isolate. The presence of N. caninum in semen and blood was assessed using a nested-PCR procedure. PCR-positive semen samples were bioassayed using a BALB/c nu/nu mouse model. Specific anti-N. caninum antibody and IFN-gamma responses were also examined. In parallel, eight seronegative bulls were studied as non-infected controls. All bulls were monitored for 26 weeks. RESULTS: All eight experimentally infected bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. Parasite load in semen ranged from 0.1 to 14.5 parasites/ml (mean 6.0). N. caninum could not be detected in BALB/c nu/nu mice inoculated with PCR-positive semen samples. A significant increase in mean serum specific IgM antibody response to N. caninum was detected between 10 and 28 days post-infection (p.i.). Serum specific IgG, IgG1, and IgG2 antibody levels in experimentally infected bulls were significantly different after 21, 10, and 14 days p.i. as compared to controls, respectively. Specific anti-N. caninum IgG were detected in seminal plasma from infected bulls and values obtained were different from controls after 25 days p.i. Mean specific IFN-gamma responses in experimentally infected bulls were significantly higher than controls 3 days p.i. CONCLUSIONS: This is the first study to report the presence of N. caninum DNA in the semen and blood of experimentally infected bulls. Our observations indicate an intermittent presence of N. caninum in low numbers in semen and associated with chronic stage of the infection. This study is also the first to report the detection of anti-N. caninum IgG in seminal plasma of experimentally infected bulls.     

Infected dendritic cells facilitate systemic dissemination and transplacental passage of the obligate intracellular parasite Neospora caninum in mice

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites.     

Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination

Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.     

Toxoplasma gondii Protein Disulfide Isomerase (TgPDI) Is a Novel Vaccine Candidate against Toxoplasmosis

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect all warm-blooded animals, including both mammals and birds. Protein disulfide isomerase (PDI) localises to the surface of T. gondii tachyzoites and modulates the interactions between parasite and host cells. In this study, the protective efficacy of recombinant T. gondii PDI (rTgPDI) as a vaccine candidate against T. gondii infection in BALB/c mice was evaluated. rTgPDI was expressed and purified from Escherichia coli. Five groups of animals (10 animals/group) were immunised with 10, 20, 30, 40 μg of rTgPDI per mouse or with PBS as a control group. All immunisations were performed via the nasal route at 1, 14 and 21 days. Two weeks after the last immunisation, the immune responses were evaluated by lymphoproliferative assays and by cytokine and antibody measurements. The immunised mice were challenged with tachyzoites of the virulent T. gondii RH strain on the 14th day after the last immunisation. Following the challenge, the tachyzoite loads in tissues were assessed, and animal survival time was recorded. Our results showed that the group immunised with 30 μg rTgPDI showed significantly higher levels of specific antibodies against the recombinant protein, a strong lymphoproliferative response and significantly higher levels of IgG2a, IFN-gamma (IFN-γ), IL-2 and IL-4 production compared with other doses and control groups. While no changes in IL-10 levels were detected. After being challenged with T. gondii tachyzoites, the numbers of tachyzoites in brain and liver tissues from the rTgPDI group were significantly reduced compared with those of the control group, and the survival time of the mice in the rTgPDI group was longer than that of mice in the control group. Our results showed that immunisation with rTgPDI elicited a protective immune reaction and suggested that rTgPDI might represent a promising vaccine candidate for combating toxoplasmosis.     

Fatal Neospora caninum infection in kittens

Three 3-day-old kittens were inoculated subcutaneously and orally with Neospora caninum tachyzoites. A littermate and the queen were not inoculated with N. caninum and served as controls. Kitten 1 died between 14 and 16 days postinoculation (DPI) and was eaten by the mother. Kitten 2 died 17 DPI and kitten 3 was euthanized 29 DPI in a moribund condition. The control littermate and the dam remained healthy. Granulomatous skeletal myositis and nonsuppurative encephalomyelitis were the main lesions and were associated with numerous N. caninum tachyzoites in kittens 2 and 3. Cysts were found in kitten 3. Oocysts were not found in any cats. Neither lesions nor parasites were found in control cats.     

Neospora caninum (Protozoa: apicomplexa) infections in mice

Groups of mice were given 0 mg, 4 mg, or 2 mg of methylprednisolone acetate (MPA) 7 days prior to, the day of, and 7 days after subcutaneous inoculation with 0 or 2 x 10(5) tachyzoites of Neospora caninum. Clinical signs of disease were seen only in mice given both MPA and N. caninum tachyzoites. Mice given 4 mg MPA and N. caninum tachyzoites developed severe disseminated neosporosis and most died or were killed when comatose 11-13 days postinoculation (PI). Acute pneumonia, polymyositis, encephalitis, hepatitis, and pancreatitis were the main lesions in these mice. Mice given 2 mg MPA and N. caninum developed mild pneumonia and many mice began showing neurological signs 14 days PI. Neurological signs consisted mainly of pronounced head-tilting and associated impairment of movement. Grossly visible 1-2-mm single or multiple, white areas of discoloration were seen in the brains of many of these mice. Encephalitis, ganglioradiculoneuritis, pneumonia, and polymyositis were the main changes seen in these mice. Tissue cysts of N. caninum were only seen in mice given 2 mg MPA and were first seen 21 days PI. Tissue cysts were 16-34 by 13-29 microns and had a 1.5-3.0-microns-thick cyst wall. Tissue cysts were seen only in the brain. Mice given 4 mg MPA and tachyzoites and host cells that had been frozen for 1 wk did not develop clinical signs of infection, indicating that freezing kills tachyzoites and that viruses or other agents were not involved in the genesis of disease seen in mice given MPA and viable tachyzoites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of temperature-sensitive strains of Neospora caninum in mice

Temperature-sensitive (ts) strains of the Neospora caninum tachyzoites were selected by chemical mutagenesis and selection for growth at 32 C. Three ts strains and the parental, N. caninum wild-type strain, NC-1, were examined in the present study for their ability to cause disease in inbred BALB/c mice, outbred ICR mice, and chemically immunosuppressed ICR mice. In BALB/c mice, all 3 strains failed to induce clinical disease, whereas infection with the NC-1 strain caused central nervous system disease and death in some mice. No disease was observed in ICR mice inoculated with the 3 ts strains or the NC-1 strain. All immunosuppressed ICR mice inoculated with the NC-1 strain died, whereas no immunosuppressed mice inoculated with the NCts-4 strain and only 1 of 5 mice inoculated with the NCts-8 and NCts-12 strains died. The NCts-4 and NCts-12 strains reverted to a wild-type phenotype when grown at 37 C. Vaccination of BALB/c mice with live, but not frozen NCts-8 strain tachyzoites induced significant (P < 0.05) protection following NC-1 strain challenge.     

Effects of miltefosine treatment in fibroblast cell cultures and in mice experimentally infected with Neospora caninum tachyzoites

Miltefosine was investigated for its activity against Neospora caninum tachyzoites in vitro, and was shown to inhibit the proliferation of N. caninum tachyzoites cultured in human foreskin fibroblasts (HFF) with an IC50 of 5·2 μM. Treatment of infected cells with 25 μM miltefosine for a period of 10 h had only a parasitostatic effect, while after 20 h of treatment parasiticidal effects were observed. This was confirmed by transmission electron microscopy of N. caninum-infected and miltefosine-treated HFF. Administration of miltefosine to N. caninum-infected Balb/c female mice at 40 mg/kg/day for 14 days resulted in 6 out of 10 mice exhibiting weight loss, ruffled coat and apathy between days 7 and 13 post-infection. In the group that received placebo, only 2 out of 8 mice succumbed to infection, but the cerebral burden was significantly higher compared to the miltefosine treatment group. In a second experiment, the time-span of treatment was reduced to 5 days, and mice were maintained without further treatment for 4 weeks. Only 2 out of 9 mice in the miltefosine treatment group exhibited signs of disease, while 8 out of 10 mice succumbed to infection in the placebo group. These results showed that miltefosine hampered the dissemination of parasites into the CNS during experimental N. caninum infection in mice.