Phenotypic characterisation of a Neospora caninum temperature-sensitive strain in normal and immunodeficient mice

The in vivo persistence, immunogenicity and pathogenicity of a recently described temperature-sensitive (ts) strain from Neospora caninum, NCts-8, was investigated in normal and immunodeficient mice. Groups of BALB/c and SCID/Bg mice were infected s.c. with 5 x 10(6) wild-type NC-1, control NCts-8 (pass 0) or NCts-8 tachyzoites prepared at four in vitro passage levels (pass 7, 13, 21 and 28). For persistence and immunogenicity studies, BALB/c mice were bled and sacrificed at 4, 6 or 8 weeks p.i. Sera were analysed by IFAT and brain tissues examined for lesions by histology and tested for parasite presence by PCR. For pathogenicity studies, SCID/Bg mice were monitored by clinical signs and survival time. Results from parasite persistence experiments demonstrated microscopic lesions and PCR positive brain tissues in NC-1 infected mice. In contrast, brain tissues from NCts8-infected groups were consistently negative by histology and PCR. Based on IFAT titres, all parasite strains were immunogenic, although parasite-specific IgG levels were lower in the NCts-8 infected groups. Results from pathogenicity studies in SCID/Bg mice demonstrated a significantly (P < 0.0001) longer mean survival time in NCts-8 vs NC-1 infected groups. In addition, there was no significant difference in mean survival time between control NCts-8 and experimental passage NCts-8 infected mice. Collectively, these studies demonstrate that the NCts-8 strain maintains a stable phenotype following multiple passages in vitro, and possesses an attenuated, shorter persistence phenotype in vivo compared with the parental wild-type NC-1.     

Neospora caninum in wildlife

Neosporosis, which is caused by the coccidian parasite Neospora caninum, is recognized as a major disease of domestic animals that causes high abortion rates in cattle and fatal neurological disease in dogs. A life cycle of N. caninum in wild animals (i.e. sylvatic) has long been suspected because neosporosis has been detected in several wildlife species. Recently, the transmission of N. caninum has been confirmed in coyotes and white-tailed deer. The newly confirmed wild hosts and other wild animals are probably involved in the sylvatic cycle of the parasite. Control measures for neosporosis could now become more complicated, given the participation of wildlife in the life cycle of N. caninum.     

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)     

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.     

Characterization of an outbred pregnant mouse model of Neospora caninum infection

Fetal loss and vertical transmission of Neospora caninum were evaluated in outbred Quackenbush (Qs) mice with respect to dose of parasites, N. caninum isolate, and route of injection. Mice were infected with NC-Liverpool or NC-SweB1 at day 5 or 8 of pregnancy with doses of 10(4), 10(6), or 10(7) parasites, through either a subcutaneous or intraperitoneal injection. Polymerase chain reaction was used to detect N. caninum in the brains of offspring, and enzyme-linked immunosorbent assay was used to analyze the maternal immune response. Vertical transmission occurred in mice given 10(6) NC-Liverpool at day 5 during gestation, and a significant (P < 0.05) maternal antibody response was observed in mice infected with NC-Liverpool or NC-SweB1 at days 5 and 8 of gestation. This study shows that outbred Qs mice are a useful model for the study of vertical transmission associated with N. caninum, as they display less clinical disease and pathogenesis than inbred mice and have large litters, which is advantageous when studying maternal transmission.     

Transplacental Neospora caninum infection in cats

Transplacental transmission of Neospora caninum was studied in 2 pregnant cats (queens). Queen 1 was inoculated subcutaneously with 2 x 10(6) cell culture-derived N. caninum tachyzoites on day 47 of gestation. She gave birth to a full-term kitten on the 17th day after inoculation. The kitten died the second day after birth due to generalized N. caninum infection. The mother cat was killed on the third day after parturition and was found to have a macerated kitten in the uterus. Severe placentitis, metritis, hepatitis, and nephritis due to N. caninum were seen in tissues from the queen. Queen 2 was fed N. caninum tissue cysts and mated 111 days later. She gave birth to 3 healthy full-term kittens. The kittens were necropsied at 2, 22, and 30 days of age. Neospora caninum was recovered from the organs and was seen in histologic sections in 1 of the 3 kittens. Results indicate that N. caninum can be transplacentally transmitted in cats during acute and chronic stages of infection. Neospora caninum-specific IgG antibodies were demonstrated in the sera of inoculated cats and nursing kittens.     

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.     

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.     

Immunological characterization of Neospora caninum cyclophilin

Neospora caninum is an intracellular parasite that poses a unique ability to infect a variety of cell types by causing host cell migration. Although previous studies demonstrated that parasite-derived proteins could trigger host cell migration, the related molecules have yet to be determined. Our study aimed to investigate the relationship between Neospora-derived molecules and host cell migration using recombinant protein of N. caninum cyclophilin (NcCyp). Indirect fluorescent antibody test revealed that NcCyp was expressed in the tachyzoite cytosol. Furthermore, NcCyp release from extracellular parasites was detected by sandwich enzyme-linked immunosorbent assay in a time-dependent manner. Recombinant NcCyp caused the cysteine-cysteine chemokine receptor 5-dependent migration of murine and bovine cells. Furthermore, immunohistochemistry indicated that NcCyp was consistently detected in tachyzoites distributed within or around the brain lesions. In conclusion, N. caninum-derived cyclophilin appears to contribute to host cell migration, thereby maintaining parasite/host interactions.     

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.     

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.     

A European perspective on Neospora caninum

Since the identification of Neospora caninum in 1984 as a parasite separate from Toxoplasma gondii by Bjerkas et al., and its subsequent characterization and classification in 1988 by Dubey and co-workers, this parasite has attracted increasing attention, primarily as an important causative agent of abortion in cattle and neuromuscular disease in dogs, but also as a complementary model system to T. gondii for investigating the basic biology of intracellular parasitism. During November 11-14, 1999, the COST 820 Annual meeting (Vaccines against coccidioses) took place in Interlaken, Switzerland. Almost half of the papers presented at that meeting were on N. caninum and neosporosis, reflecting the increasing awareness of the importance of this parasite on part of the scientific community in Europe. On the occasion of the meeting, participants in this COST Action involved in Neospora research in Europe were asked to participate in this invited review in order to document the growing interest in N. caninum and the disease it causes. Thus, this paper is a unique collection of contributions provided by several European experts in the field. It is comprised of 10 reviews or original papers on different aspects of Neospora research including epidemiology, immunology, application and development of serological tools, and molecular characterisation of the parasite currently carried out throughout Europe. In addition, two distinguished invited speakers from overseas (Milton McAllister and John Ellis) provided valuable contributions. This invited review demonstrates that the COST 820 Action has brought together scientists from all over Europe and other parts of the world, and has laid the basis for many fruitful collaborations. The studies described here will contribute in assessing the relevance of neosporosis as a potential risk factor not only for animals, but also for human health.     

The antigenic composition of Neospora caninum

Neospora caninum is an apicomplexan parasite which causes neosporosis, namely stillbirth and abortion in cattle, and neuromuscular disease in dogs. Although N. caninum is phylogenetically and biologically closely related to Toxoplasma gondii, it is antigenically clearly distinct. In analogy to T. gondii, three stages have been identified. These are: (i) asexually proliferating tachyzoites; (ii) tissue cysts harbouring slowly dividing bradyzoites; and (iii) oocysts containing sporozoites. The sexually produced stage of this parasite has only recently been identified, and has been shown to be shed with the faeces from dogs orally infected with N. caninum tissue cysts. Thus dogs are definitive hosts of N. caninum. Tachyzoites can be cultivated in vitro using similar techniques as previously described for T. gondii. Methods for generating tissue cysts containing N. caninum bradyzoites in mice, and purification of these cysts, have been developed. A number of studies have been undertaken to identify and characterise at the molecular level specific antigenic components of N. caninum in order to improve serological diagnosis and to enhance the current view on the many open questions concerning the cell biology of this parasite and its interactions with the host on the immunological and cellular level. The aim of this paper is to provide an overview on the approaches used for detection of antigens in N. caninum. The studies discussed here have had a great impact in the elucidation of the immunological and pathogenetic events during infection, as well as the development of potential new immunotherapeutic tools for future vaccination against N. caninum infection.     

Review of Neospora caninum and neosporosis in animals

Neospora caninum is a coccidian parasite of animals. It is a major pathogen for cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep, and deer. Domestic dogs are the only known definitive hosts for N. caninum. It is one of the most efficiently transmitted parasite of cattle and up to 90% of cattle in some herds are infected. Transplacental transmission is considered the major route of transmission of N. caninum in cattle. Neospora caninum is a major cause of abortion in cattle in many countries. To elicit protective immunity against abortion in cows that already harbor a latent infection is a major problem. This paper reviews information on biology, diagnosis, epidemiology and control of neosporosis in animals.     

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.     

Serological diagnosis of Neospora caninum infection

Since the first isolation of the apicomplexan parasite Neospora caninum, a range of serological assays have been developed for use in dogs, cattle and a variety of other potential host species. The tests include the indirect fluorescent antibody test, the direct agglutination test and different enzyme-linked immunosorbent assays. This article reviews the principles and properties of the available tests which are discussed in relation to different applications.     

Variation of the internal transcribed spacer 1 sequence within individual strains and among different strains of Neospora caninum

Small differences have been reported in the internal transcribed spacer 1 (ITS1) region among strains of Neospora caninum. We compared ITS1 sequences among 6 N. caninum strains analyzed in our laboratory, including 2 strains that have not been examined previously (NC-Illinois and NC-Bahia). Five sequences showed 100% similarity and also were identical to 7 of 11 sequences that were previously reported by others. In contrast, initial attempts to sequence the ITS1 of NC-Bahia generated 12 nucleotide differences compared with the other 5 strains, and several ambiguous bases. However, the single band containing the ITS1 region, as observed after electrophoresis on a 2% agarose gel, became divided into 2 distinct bands when reanalyzed using 5 or 10% polyacrylamide gel electrophoresis (PAGE), and the ITS1 within these separate bands were sequenced without ambiguity. The other 5 N. caninum strains were also reexamined using PAGE, and in each strain 2 distinct bands were discovered. In comparison, 2 strains of Toxoplasma gondii continued to show only 1 band when examined using PAGE. The ITS1 sequence of NC-Bahia, from Brazil, differs in several base pairs from those of North American and European strains of N. caninum. Intrastrain variation of the ITS1 region appears to be common in N. caninum, in contrast to T. gondii.     

Vaccination with microneme protein NcMIC4 increases mortality in mice inoculated with Neospora caninum

NcMIC4 is a Neospora caninum microneme protein that has been isolated and purified on the basis of its unique lactose-binding properties. We have shown that this protein binds to galactosyl residues of lactose; antibodies directed against NcMIC4 inhibit host cell interactions in vitro, thus making it a vaccine candidate. Because of this feature, NcMIC4 was first purified on a larger scale in its native, functionally active form using lactose-agarose affinity chromatography. Second, NcMIC4 was expressed in Escherichia coli as a histidine-tagged recombinant protein (recNcMIC4) and purified through Ni-affinity chromatography. Third, NcMIC4 cDNA was cloned into the mammalian pcDNA3.1 DNA vector and expression was confirmed upon transfection of Vero cells in vitro. For vaccination studies, we employed the murine cerebral infection model based on C57Bl/6 mice, employing experimental groups of 10 mice each. Two groups were injected intraperitoneally with purified native NcMIC4 and recNcMIC4, respectively, employing RIBI adjuvant. The third group was vaccinated intramuscularly with pcDNA-NcMIC4. Control groups included an infection control, an adjuvant control, and a pcDNA3.1 control group. Following 3 injections at 4-wk intervals, mice were challenged by i.p. inoculation of 2 x 10(6) N. caninum tachyzoites (Nc-1 isolate). During the course of parasite challenge (3 wk), mice from the 3 different test groups showed varying degrees of symptoms bearing a semblance to neosporosis, i.e., walking disorder, rounded back, apathy, and paralysis of the hind limbs. Control groups showed no symptoms at all. Most notably, vaccination with pcDNA-MIC4 proved antiprotective, with 60% of mice succumbing to infection within 3 wk, and all mice lacking a measurable anti-NcMIC4 IgG response. NcMIC4 in its native form elicited a substantial humoral IgG1 immune response and a reduction in cerebral parasite load compared to the controls, but 20% of mice succumbed to infection. Vaccination with recNcMIC4 also resulted in 20% of mice dying; however, in this group, cerebral parasite load was similar to the controls, and recNcMIC4 vaccination elicited a mixed IgG1/IgG2 response. In conclusion, vaccines based on NcMIC4, especially pcDNA-NcMIC4, render mice more susceptible to cerebral disease upon challenge with N. caninum tachyzoites.     

Neospora caninum in birds: A review

Neospora caninum is an obligate intracellular protozoan parasite that infects domestic and wild animals. Canids are considered to be definitive hosts since they may shed oocysts into the environment through their feces. The disease is recognized as one of the major causes of bovine abortion worldwide, leading to important economic losses in the dairy and beef cattle industries. Previous studies have reported N. caninum infection in different species of birds; infection in birds has been associated with increased seroprevalence and reproductive problems in dairy cattle. Although the role of birds in the epidemiological cycle of neosporosis is unknown, birds are exposed to infection because they feed on the ground and could thus contribute to parasite dissemination. This review is focused on the current state of knowledge of neosporosis in birds.