Immunization with Oligomannose-Coated Liposome-Entrapped Dense Granule Protein 7 Protects Dams and Offspring from Neospora caninum Infection in Mice

The present study demonstrates that the subcutaneous administration of Neospora caninum dense granule protein 7 (NcGRA7) entrapped in liposomes coated with mannotriose strongly induces the parasite-specific T-helper type 1 immune response and humoral antibody in mice. Although anti-NcGRA7 immunoglobulin G1 antibody production was induced in mice injected with NcGRA7 alone, the dams and offspring were never protected from N. caninum infection. The immunization of mice with liposome-entrapped NcGRA7 before pregnancy resulted in increased offspring survival and decreased the infection rates in the brains of dams after parasite infection at 6 to 9 days of gestation. In conclusion, oligomannose-coated liposome-entrapped NcGRA7 can be used as a new type of effective vaccine to control neosporosis.Neosporosis, caused by an apicomplexan protozoan parasite, Neospora caninum, is a cause of infectious abortion and congenital disease in cattle worldwide (11). Transplacental transmission is the major route of N. caninum infection (7, 39), and the parasite persists in the herd over successive generations, causing significant economic losses due to abortion, decreased milk production, and the resultant culling (6, 10, 21, 46, 47). Horizontal transmission of N. caninum by oocysts that are shed by dogs has also been documented in cattle, but this transmission is not considered a major route of infection (7).Various mouse models have been utilized to understand the host protective immune responses to N. caninum infection. A Th1-type immune response appears to be important in protection against N. caninum infection (2, 34). Gamma interferon (IFN-γ) and interleukin-12 (IL-12), known to be crucial cytokines for the development of Th1-type immunity, are important for protective immunity against acute N. caninum infection (2). Furthermore, CD4⁺ T-cell-depleted BALB/c mice were more highly susceptible to parasite infection than were CD8⁺ T-cell-depleted mice (34, 45). Studies of IFN-γ knockout mice indicated the importance of macrophage activation by IFN-γ for protective immunity (34). On the other hand, a Th2-type immune response with predominant production of humoral antibody specific for the parasite antigens is also capable of mediating protection against neosporosis (17, 18, 30, 38, 40). These observations suggest that a suitable balance in the production of Th1- and Th2-type cytokines has a crucial role in the control of N. caninum infection (33).Oligomannose-coated liposomes have been shown to be a safe adjuvant to induce Th1-type immunity because no skin damage by the liposomes is caused at the injection site (16). A previous study showed that liposomes coated with a neoglycolopid consisting of mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) were specifically and rapidly incorporated into intraperitoneal macrophages when injected into the peritoneal cavity and that the liposome-incorporating macrophages smoothly accumulated in nearby lymphoid tissue (23). The effect of Man3-coated liposome as an effective adjuvant has been confirmed with Leishmania major infection (41) and with tumors (23, 25). Administration of soluble leishmanial antigens entrapped within the Man3-coated liposomes to BALB/c mice strongly induced the antigen-specific Th1 immune response, as evidenced by a higher level of IFN-γ production and a lower level of IL-4 production than those in mice receiving the antigens alone (41).There is accumulating evidence that some N. caninum-infected cows develop a degree of protective immunity against abortion and/or congenital transmission, indicating the advantage of vaccine development (31). Although the prevention of abortion might be a realistic goal for a vaccine, the ultimate objective for the control of neosporosis must be to prevent the vertical transmission of the parasite. Evidence has shown that cattle which abort due to neosporosis have higher levels of N. caninum-specific antibody than do infected but nonaborting cattle (9). In addition, our previous study showed that a higher level of bovine antibody specific for N. caninum dense granule protein 7 (NcGRA7) was detected in aborting than in nonaborting cows and heifers, while levels of specific antibodies against parasite surface proteins NcSAG1 and NcSRS2 exhibited no significant difference between the aborting and nonaborting cows (22). To control N. caninum infection, a suitable balance of Th1- and Th2-type immune responses is important (33). We speculated that an NcGRA7-specific Th2-type immune response might be predominant in aborting cows. Therefore, induction of the NcGRA7-specific Th1-type immune response could play a crucial role in the control of N. caninum infection, since antibodies against the parasites did not prevent vertical transmission (32). Thus, the present study was conducted to evaluate the vaccine efficacy of oligomannose-coated liposome-entrapped NcGRA7 on N. caninum infection in dams and offspring, using a BALB/c mouse model. Our results suggest that the Th1-type immune response against NcGRA7 plays a crucial role in the control of N. caninum infection.     

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by <Emphasis Type="Italic">Neospora caninum</Emphasis> tachyzoites

Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4^c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4^c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.     

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n?=?8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n?=?5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5?×?10⁸ tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion?=?12.5 %), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion?=?100 %). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc-Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.     

Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene

Neospora caninum is a recently described apicomplexan parasite which causes neuromuscular disease in dogs, and abortion and neonatal morbidity in cattle, sheep and horses. Morphological similarites and serological cross-reactivity between N. caninum and the closely related parasite Toxoplasma gondii, have resulted in the frequent misdiagnosis of neosporosis as toxoplasmosis. This report describes the isolation and characterization of an N. caninum cDNA clone encoding a 14-3-3 protein homologue. The 14-3-3 proteins are a class of proteins which show a high degree of amino acid sequence conservation across several eukaryotic taxa. Using less conserved regions of the N. caninum cDNA clone, nested primers were designed for the amplification of a 614-bp N. caninum DNA fragment by the polymerase chain reaction (PCR). The DNA fragment was amplified from N. caninum genomic DNA, but not from T. gondii, Sarcocystis muris, Sarcocystis tenella, or Sarcocystis cruzi genomic DNA. Additionally, the fragment was amplified from DNA prepared from the brains of N. caninum-infected mice, but not from the brain of a mouse infected with T. gondii. These results suggest that this PCR assay may be useful for the diagnosis of neosporosis.     

On the Efficacy and Safety of Vaccination with Live Tachyzoites of Neospora caninum for Prevention of Neospora-Associated Fetal Loss in Cattle

Infection of cattle with Neospora caninum may result in abortion or the birth of a congenitally infected calf. Vaccination with live N. caninum protects against experimental infection of cattle and mice, and the naturally attenuated Nc-Nowra strain of N. caninum is of particular interest as a potential vaccine candidate. Vaccination of heifers prior to breeding with live Nc-Nowra tachyzoites by either the subcutaneous or the intravenous route reduced the rate of abortion and the presence of the parasite in calves as determined by PCR and serology after infection of cows with a virulent isolate. Protected fractions were 55.6% to 85.2% depending on the route of vaccination and growth conditions of the vaccine strain, with cryopreserved Nc-Nowra tachyzoites being less effective, with a 25.9% protected fraction. Vaccination appeared to reduce the rate of pregnancy after artificial insemination in some groups compared to nonvaccinated, nonchallenged controls. One animal that was vaccinated but not challenged experienced an abortion, but Nc-Nowra could not be detected in any of the cows in this group or their progeny. This study confirms that live vaccination can be an effective method of preventing neosporosis in cattle and yet highlights the technical hurdle of preservation of live parasites that must be overcome for a vaccine to be commercially successful.     

Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108?±?2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.     

Serological Evidence of Human Infection with the Protozoan Neospora caninum

Neospora caninum is a protozoan parasite that is closely related to Toxoplasma gondii. Dogs are a definitive host. Prior to its discovery in 1988, N. caninum infection in animals was often mistakenly diagnosed as toxoplasmosis. Neosporosis in animals is characterized by encephalitis, abortion, and other conditions that clinically and pathologically resemble toxoplasmosis. The potential of N. caninum to infect humans is unknown. Therefore, evidence of human exposure to this parasite was sought by screening for antibodies in blood donors by indirect fluorescent antibody (IFA) tests and immunoblotting. Of 1,029 samples screened, 69 (6.7%) had titers of 1:100 by IFA testing. Fifty of the 69 (72%) sera that were positive for N. caninum were also negative for a closely related protozoan pathogen of humans, T. gondii. Immunoblot analysis confirmed the specificity of the positive sera for N. caninum antigens, with several sera recognizing multiple Neospora antigens with molecular masses similar to those of antigens recognized by monkey anti-N. caninum serum. An immunodominant antigen of approximately 35 kDa was observed with 12 sera. These data provide evidence of human exposure to N. caninum, although the antibody titers in healthy donors were low. The significance of human exposure to, and possible infection with, this parasite is unknown and warrants further study.     

Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces,Thailand

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006–2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.     

First report of <Emphasis Type="Italic">Neospora caninum</Emphasis>-associated bovine abortion in Mashhad area,Iran

Neospora caninum is an apicomplexan protozoan which causes abortion in cattle worldwide. The present study was designed to assess the importance of bovine neosporosis for causing abortion in Iranian cattle. Infection was primarily diagnosed by polymerase chain reaction (PCR), complemented with histopathology and immunohistochemistry (IHC). One hundred brains of aborted bovine fetuses were collected from Iranian dairy herd between 2003 and 2005 in Mashhad area. N. caninum was detected by PCR in the brains of 13 aborted fetuses. Lesions consistent with N. caninum infection were observed in 12 of fetal brains. Immunohistochemical examination of brain detected N. caninum organisms in three aborted fetuses, and a thick-walled (2 μm) cyst with 50 μm diameter was identified in one of the IHC-positive brain. The results indicated that neosporosis is an important cause of abortion in dairy cattle of Iran.     

Identification of a Highly Antigenic Region of Subtilisin-Like Serine Protease 1 for Serodiagnosis of Neospora caninum Infection

Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples from N. caninum experimentally infected cattle and mice and cattle from a farm with confirmed cases of Neospora abortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In the N. caninum experimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera from Toxoplasma gondii experimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis of N. caninum.     

Humoral immune reaction of newborn calves congenitally infected with Neospora caninum and experimentally treated with toltrazuril

Neospora caninum is widely recognized as one of the most important infectious organisms causing abortion and stillbirth in cattle. This parasite causes severe economical losses worldwide. Infection is mostly passed vertically from mother to calf during pregnancy. Under certain circumstances, an infection can lead to abortion, but in most cases it results in a chronically infected calf, which itself will represent the next endogenously infectious generation. So far, no reliable therapeutic or metaphylactic tool has been developed. One possibility to control the problem may consist of treating newborn calves that became vertically infected by a persistently infected mother. This may allow parasite-free offspring. The aim of the present study was to address the questions: (1) can serology be used to assess efficiency of treatment in toltrazuril-medicated animals? and (2) is a strategic prevention measure possible by means of producing N. caninum-free calves from positive cows? Calves from Neospora-seropositive cows and heifers were randomly split into two different medication groups: 36 calves were medicated with toltrazuril and 36 calves obtained a placebo. Medication (20 mg toltrazuril per kg bw) was administered three times, every second day, within the 7 days post natum. Three months after medication, there was no difference in antibody reactivity between the two groups. At later time points (4–6 months), however, significant differences were found, as explained by a strong humoral immunity after chemotherapeutical affection of parasites, while the placebo-treated animals only responded weakly to the persistent infection. In summary, we concluded that (1) serology was not an entirely appropriate tool to answer our initial question and (2) toltrazuril has the potential to eliminate N. caninum in newborn calves. As a consequence, we plan to follow up toltrazuril-medicated calves clinically and serologically over a longer period and investigate if they give birth to Neospora-free calves.     

Indoleamine 2,3-Dioxygenase Is Involved in Defense against Neospora caninum in Human and Bovine Cells

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii. In nature this parasite is found especially in dogs and cattle, but it may also infect other livestock. The growth of N. caninum, which is an obligate intracellular parasite, is controlled mainly by the cell-mediated immune response. During infection the cytokine gamma interferon (IFN-γ) plays a prominent role in regulating the growth of N. caninum in natural and experimental disease. The present study showed that induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) is responsible for the inhibition of parasite growth that is mediated by IFN-γ-activated bovine fibroblasts and endothelial cells. This antiparasite effect could be abrogated by addition of tryptophan, as well as by the IDO-specific inhibitor 1-l-methyltryptophan. In conclusion, our data show that human and bovine cells use the same effector mechanism to control the growth of N. caninum.Toxoplasma gondii, a protozoan belonging to the apicomplexan phylum, is one of the most successful parasites on earth. This parasite is capable of infecting nearly all warm-blooded animals, including humans. T. gondii can be transmitted via tissue cysts, raw meat, and environmental-resistant oocysts derived from cat feces and is able to spread transplacentally from mother to fetus. Furthermore, another special feature of this evolutionarily successful parasite is the fact that it usually causes asymptomatic infections and in most cases does not kill immunocompetent hosts. However, without sufficient therapy, reactivation of T. gondii in immunocompromised individuals frequently results in death of the host (31, 26, 18).In 1984 a T. gondii-like parasite was found in the cerebral tissue of dogs and described (5). This parasite was later detected in brain tissue from dogs which had clinical signs of neuromuscular disease and was named Neospora caninum (15). It took until 1998 to discover that dogs are not only intermediate hosts but also one of the definitive hosts of this parasite (29). In nature, dogs are frequently intermediate hosts of N. caninum, although canine neosporosis seems to be rare (2). N. caninum can also be isolated from cattle, and vertically transmitted N. caninum infection is considered an important cause of bovine abortion worldwide (17). In sheep N. caninum-associated abortion seems to be rare (16). This is in contrast to infections with T. gondii, which often cause abortion in sheep but seldom in cattle (14). Furthermore, so far, there is no evidence that N. caninum infection is zoonotic (16). It has been shown that under experimental conditions N. caninum is able to infect rhesus monkeys, indicating the zoonotic potential of this parasite. However, serologic studies with humans have shown that no or only small amounts of N. caninum-specific antibodies are detectable in some human sera, even sera from high-risk groups like farm workers (30, 16, 22). Despite the high levels of homology between T. gondii and N. caninum, many differences have also been detected. Both parasites can be transmitted via food, via oocysts in soil, and also transplacentally (23). Several species have been successfully infected experimentally with N. caninum, and in vitro N. caninum is capable of replicating in different types of cells derived from various animal species or humans.The variability in the susceptibility to natural T. gondii or N. caninum infection among various host species might be due to differences in the immune responses. Different antiparasite effector mechanisms might, at least in part, be involved in the evolutionary success of both parasites. In support of this, workers have obtained some data showing that experimental infection with attenuated or apathogenic N. caninum strains can induce immunity to this parasite in mice and cattle (3). Furthermore, a lot of data indicate that the cellular immune response is necessary to control infection with N. caninum. In addition, it was found that gamma interferon (IFN-γ), a product of activated T cells and natural killer (NK) cells, is one of the main cytokines conferring resistance to N. caninum (21). So far, the IFN-γ-induced effector mechanism that is active against N. caninum in cattle has not been defined.In this paper we provide evidence that induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which is the most prominent antiparasite effector mechanism active against T. gondii in human cells (28), is also effective for inhibiting N. caninum growth in tissue cells from humans and cattle.     

A survey of Neospora caninum-associated bovine abortion in large dairy farms of Mashhad, Iran

Neospora caninum is an apicomplexan protozoon causing abortion in cattle worldwide. The present study was designed to assess the importance of this parasite for causing abortion in dairy farms in the Mashhad area of Iran. Of the aborted bovine fetuses, 151 were collected from dairy farms between 2006 and 2008. First, brain samples were examined by polymerase chain reaction (PCR) for the presence of N. caninum DNA, diagnosis was complemented with immunohistochemistry (IHC) and fetal serology (ELISA). Twenty-two (14.5%) of bovine fetuses were considered to be infected with N. caninum with at least one diagnostic technique being positive. PCR yielded 18 (11.9%) positive out of 151 brain samples. Only 52 brain samples were suitable for IHC examination, and N. caninum organism was detected in six (11.5%) of these 52 brain samples. Fetal fluids (n = 151) were assessed with a N. caninum-ELISA, resulting in 15 (9.9%) seropositive fetal fluids samples. In the present study, a good agreement was observed between PCR and ELISA, and a fair agreement between PCR and IHC. The results indicated that abortion due to N. caninum infection is prevalent among large-size dairy farms in the Mashhad area of Iran, and that different complementary diagnostic techniques should be used to increase the chance to detect N. caninum.     

Evaluation of bovine abortion associated with <Emphasis Type="Italic">Neospora caninum</Emphasis> by different diagnostic techniques in Mashhad,Iran

Twelve aborted foetuses (gestational ranged from 4–9 months) and dams from dairies cattle farms in (Mashhad) Iran were analysed to investigate the participation of Neospora caninum in abortion. Diagnosis of the infection was determined by histopathology, serology (indirect fluorescent antibody test [IFAT]) and semi-nested polymerase chain reaction (PCR). A total 33% of bovine foetuses were considered to be infected by PCR technique. Microscopic lesions consistent with N. caninum infection in foetal brains were observed in 25% of the samples, whereas 33% were positive using IFAT (with a cut-off titre of 1:20). This study confirms the importance of N. caninum as an important cause of abortion in Iran.     

Protection against Lethal Neospora caninum Infection in Mice Induced by Heterologous Vaccination with a mic1 mic3 Knockout Toxoplasma gondii Strain

Neospora caninum and Toxoplasma gondii are closely related, obligate intracellular parasites infecting a wide range of vertebrate hosts and causing abortion and neonatal morbidity and mortality. Several lines of evidence suggest that cross immunity between these two pathogens could be exploited in the design of strategies for heterologous vaccination. We assessed the ability of an attenuated strain of T. gondii (“mic1-3KO strain”) conferring strong protection against chronic and congenital toxoplasmosis to protect mice against lethal N. caninum infection. Mice immunized with mic1-3KO tachyzoites by the oral and intraperitoneal routes developed a strong cellular Th1 response and displayed significant protection against lethal heterologous N. caninum infection, with survival rates of 70% and 80%, respectively, whereas only 30% of the nonimmunized mice survived. We report here the acquisition of heterologous protective immunity against N. caninum following immunization with a live attenuated mic1-3KO strain of T. gondii.Neospora caninum and Toxoplasma gondii are closely related apicomplexans displaying extensive morphological and genetical similarity (37). These cyst-forming coccidians of the family Sarcocystidae (11) cause similar disorders in different animals (15). Despite controversial documentation on their phylogenetic relationship (30), molecular (31) and biological studies have shown that these species have followed different evolutionary paths and have different life cycles and host preferences. Canids are the definitive hosts of N. caninum, which causes neosporosis, a major disease of cattle, whereas felids are the definitive hosts of T. gondii, which causes toxoplasmosis, a major disease of sheep, goats, and humans (10). Both parasites are responsible for important economic losses in livestock production through neonatal mortality and abortion. T. gondii also causes congenital neuropathology and opportunistic infections in immunocompromised humans (41), but there is no conclusive evidence to suggest that N. caninum can infect humans (29).Previous clinical and diagnostic studies have shown that specific antibodies directed against N. caninum or T. gondii cross-react in serological and immunohistochemical tests, suggesting a possible convergence of immune responses during infections with T. gondii and N. caninum (32, 38). It has recently been shown that antibodies directed against N. caninum antigens inhibit host cell invasion by both these parasites (22, 43). Similarly, the specific cellular responses stimulated upon experimental infections with N. caninum are also stimulated in vitro by T. gondii antigenic lysate (21, 26). Consistent with these findings, CD8⁺ T cells specific for N. caninum have been shown to protect mice against lethal T. gondii infection (19). The existence of cross-reactive epitopes between N. caninum and T. gondii antigens is supported by the high level of sequence identity between conserved proteins (13). A number of cross-reactive antigens have been identified in the micronemes, rhoptries, and dense granules of tachyzoites and in bradyzoites (2, 3, 28, 43). All these observations suggest that the conserved antigenicity between N. caninum and T. gondii might represent a rational basis for the development of efficient vaccines for the control of both parasitic diseases.A vaccine based on dead N. caninum tachyzoites is currently available for prophylaxy; this vaccine is thought to confer about 46% protection against N. caninum-induced abortion in cattle (36). However, in a number of countries, this vaccine has not been licensed, since more complete scientific documentation is required to authorize the use of a vaccine against N. caninum (8). The need for a more effective vaccine against transplacental infection in cattle is therefore of the utmost importance. Live vaccines are thought to induce complete protective immunity against N. caninum infection. In vaccination trials with the mouse model, the use of N. caninum tachyzoite crude extract as the immunogen resulted in an absence of protection against parasite-related neurological illness and death (5, 27). Such vaccinations have also proved ineffective for the prevention of abortion in cattle, even in the presence of adjuvants (42).Given that protective immunity against intracellular pathogens such as T. gondii and N. caninum involves T-cell-mediated immunity (12, 21) and that experimental evidence of protection against N. caninum transplacental transmission has been shown to involve high levels of gamma interferon (IFN-γ) production (17, 42), we propose an innovative approach based on heterologous vaccination.Taking into consideration the antigenic similarities between N. caninum and T. gondii, we used an attenuated strain of T. gondii as a heterologous vaccine against N. caninum. A mutant RH strain of T. gondii tachyzoites lacking the mic1 and mic3 genes was constructed in our laboratory, the “mic1-3KO strain.” The disruption of these two genes, both of which code for proteins involved in tachyzoite adhesion and invasion, greatly decreases virulence in mice (7). Vaccination with the mic1-3KO strain provides strong protection against chronic and congenital toxoplasmosis in mice through the induction of strong humoral and Th1 cellular immune responses (18). In this study, we used this attenuated strain as a heterologous vaccine. Our results provide evidence for protection against lethal N. caninum infection. This protection was associated with strong cross-reactive humoral and Th1 cellular immune responses, overcoming the biological and antigenic differences between the two species.     

Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection

Neospora caninum is an obligate intracellular protozoan parasite that causes severe neuromuscular diseases, repeated abortion, stillbirth, and congenital infection in livestock and companion animals. The development of an effective vaccine against neosporosis in cattle is an important issue due to the significant worldwide economic impact of this disease. We evaluated the immunogenicity of four bradyzoite antigens, NcBAG1 (first described in this study), NcBSR4, NcMAG1, and NcSAG4, using an acute infection mouse model to determine synergistic effects with the tachyzoite antigen as a candidate for vaccine production. Mice were inoculated with the recombinant vaccines (r-)NcBAG1, rNcBSR4, rNcMAG1, rNcSAG4, or phosphate-buffered saline (PBS) (adjuvant control group) in an oil-in-water emulsion with bitter gourd extract, a Th1 immune stimulator, or PBS alone as the infection control group. Mice inoculated with each vaccine developed antigen-specific IgG1 and IgG2a antibodies and isolated splenocytes from mice produced high levels of interferon-γ when infected with the N. caninum tachyzoite. The mice inoculated with rNcBAG1, rNcMAG1, or rNcSAG4 developed slight to moderate clinical symptoms but did not succumb to infection. In contrast, rNcBSR4 and both control groups developed severe disease and some mice required euthanasia. The parasitic burden in the brain tissues of vaccinated mice was assessed by N. caninum-specific real-time PCR at 5 weeks after infection. The parasite load in rNcBAG1-, rNcMAG1-, and rNcSAG4-inoculated mice was significantly lower than that in adjuvant and infection control mice. Therefore, these antigens may be useful for the production of a N. caninum-specific vaccination protocol.     

Detection of Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi in the brains of common voles (Microtus arvalis) and water voles (Arvicola terrestris) by gene amplification techniques in western Austria (Vorarlberg)

Knowledge about the protozoan parasite fauna in voles (Arvicolinae) in Austria is rather limited, although some of these pathogens play an important role in human medicine and cause zoonoses (e.g., Toxoplasma gondii and Encephalitozoon cuniculi). Others are of relevance in veterinary medicine and have a negative economic impact (e.g., Neospora caninum). Two hundred sixty-eight common voles (Microtus arvalis) and 86 water voles (Arvicola terrestris) from the most western Austrian province, Vorarlberg, were analyzed with PCR techniques for infections with T. gondii, N. caninum, and E. cuniculi. Brain tissues of two common voles (0.7%) and of four water voles (4.7%) tested positive for T. gondii. Furthermore, analysis of four common voles (1.5%) and two water voles (2.3%) generated positive findings for N. caninum, and brain tissues of 16 common voles (6%) and six water voles (7%) tested positive for E. cuniculi. Accordingly, this study not only demonstrates the autochthonous existence of the zoonotic parasites T. gondii and E. cuniculi in voles in Vorarlberg, it also provides the first evidence of an occurrence of N. caninum in animals of the subfamily Arvicolinae, and it is an additional contribution to investigations of the sylvatic cycle of N. caninum.     

Evaluation of parasitological and immunological aspects of acute infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs

This study compared the course of infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs infected with L₃ larvae. Dogs infected with A. caninum eliminated more eggs than did those infected with A. braziliense. A total of 38 % of A. caninum and 44 % of A. braziliense larvae were recovered as adult worms. There were no marked clinical abnormalities in dogs with either infection. A. caninum was associated with anemia and an increased number of circulating neutrophils, whereas infection with A. braziliense led to a decrease in the number of leukocytes. The humoral response against excreted and secreted antigens from adult worms was more sensitive and specific than the response induced with the crude antigen. No immune response was observed for either crude or excreted-secreted (ES) antigens from larvae of either species. A nonspecific response against the crude antigen of A. braziliense was found at 0 and 7 days postinfection and maintained throughout the infection period. However, antibody titers against ES antigens were elevated in A. caninum infection at patency and death, showing that this antigen has a higher specificity. The immune response elicited by infection with A. braziliense in dogs has not been described previously. No significant differences were observed in the infection processes of the two Ancylostoma species, except for the higher number of eggs eliminated from dogs infected with A. caninum, which may indicate a better evolutionary adaptation of the parasite to its host in comparison with A. braziliense.     

Serological study of Neospora caninum infection in dogs and cattle from west of Iran

The aim of this study was to determine the seroprevalence of Neospora caninum infection in dogs and cattle from Hamedan province (West of Iran). Blood samples were collected from 1,046 cattle and 270 dogs in this area. Cattle and dog samples were tested and analyzed using ELISA and IFAT, respectively. IgG-antibodies to N. caninum were found in 27 and 17.4 % of dogs and cattle samples, respectively. In cattle study, The association between infection and type of cattle was statistically significant (P?=?0.004). Also, significant statistical differences were observed regarding to stray canids presence in farm (P?P?P?=?0.195) and breed (P?=?0.077). In dog study, there was statistical differences among age groups (P?P?P?=?0.112). This study is the first report of N. caninum infection in dogs and cattle from west of Iran. There is both horizontal and vertical transmission of N. caninum in this area, and the presence of stray dogs may be a risk factor for N. caninum infection in cattle. N. caninum is an important factor in the economic losses of the cattle breeding in Hamedan province. Therefore, further investigations and designing control strategies for improving management in cattle farms is highly recommended.     

Stress-driven stage transformation of Neospora caninum

Neosporiosis, a serious disease caused by the apicomplexan intracellular parasite Neospora caninum, is considered to be one of the most economically important diseases in cattle. It is associated with potentially serious complications such as abortion, stillbirth and maternal infertility. To survive in fluctuating physiological and immunological environments, N. caninum has evolved a diverse set of regulatory mechanisms that govern various adaptive responses. The most intriguing paradigm in N. caninum adaptive evolutionary biology is its ability to alternate between two phenotypically and functionally distinct stages within the host. Recent research has reinforced the notion that N. caninum tachyzoite–bradyzoite stage switching on and off is correlated with its ability to form dormant cysts. Knowledge of the specific mechanisms that govern the dynamics of N. caninum phenotype switching enables a better understanding of the pathogenesis of the disease and effective control measures to be identified. Herein we review the available knowledge relating to various aspects of stage interconversion in N. caninum, with particular focus on the stress-related hypothesis presumed to be involved in this event. Finally, we put forward the postulation that N. caninum uses stage interconversion as an adaptive process to cope with the hostile environment within the host and to ensure its continuity in nature.