Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice

The type of immune response required to protect mice against clinical disease during acute Neospora caninum challenge was investigated in BALB/c mice. Groups of female BALB/c mice were infected i.p. with N. caninum tachyzoites concomitant with either: (1) antibody to interferon-gamma; (2) recombinant murine interleukin-12; or (3) recombinant murine interleukin-12 plus antibody to interferon-gamma. Mice treated with anti-interferon-gamma alone had increased morbidity/mortality, decreased body weight, increased foci of liver necrosis and increased numbers of N. caninum tachyzoites in the lung by 7 days p.i. compared with controls. Increased disease and parasite load in the anti-interferon-gamma-treated mice was associated with antigen-specific antibody IgG1 > IgG2a and a three-fold decreased ratio of antigen-specific interferon-gamma:interleukin-4. Mice treated with recombinant murine interleukin-12 had decreased encephalitis and brain parasite load at 3 weeks p.i. compared with control mice treated with PBS. In recombinant murine interleukin-12-treated mice, decreased brain lesions and parasite load were associated with antigen-specific antibody IgG2a > IgG1 and a three-fold increased ratio of antigen-specific interferon-gamma:interleukin-4 from splenocytes; the interleukin-12 effect was dependent upon interferon-gamma, as indicated by concomitant in vivo interferon-gamma neutralisation. By 6 weeks p.i. with N. caninum, there were no differences in brain lesions and parasite load between interleukin-12- and PBS-treated groups, indicating that the effects of interleukin-12 on driving a protective type 1 response were transient. These data indicate a role for interferon-gamma, interleukin-12 and type 1 immune responses in control of acute neosporosis in mice.     

Characterization of Neospora caninum protease,NcSUB1 (NC-P65), with rabbit anti-N54

NcSUB1 (formerly known as NC-p65) is the first molecularly described proteolytic enzyme of the intracellular protozoan parasite Neospora caninum. This report describes the characterization of a rabbit anti-N54, which is an antiserum generated against an internal fragment of NcSUB1 (amino acids 649-783). In immunofluorescence studies rabbit and-N54 labeled the apical end of the fixed parasite. By immuno-gold electron microscopy, the antibody bound primarily to the microneme organelles of the parasite. Analysis of secreted parasitic proteins indicated that a protein of molecular weight 65 kDa (reduced) or 55 kDa (nonreduced) was recognized bythe antibody. The same secreted proteins were affinity purified with rabbit anti-N54-coupled resins and were shown to contain major proteolytic activity by zymography. Thus, rabbit anti-N54 is the first antibody developed for N. caninum that binds to themicroneme proteins and recognizes a major secreted enzyme.     

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.     

Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.     

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 (Apicomplexa) in an aborted equine fetus

Tachyzoites of Neospora caninum were found in sections of lung of an equine fetus aborted 2 mo before term. Individual tachyzoites were approximately 3-5 x 2-3 microns, divided by endodyogeny, and stained positively with anti-N. caninum serum but not with anti-Toxoplasma gondii serum. Toxoplasma gondii antibody was not found in the mare's serum. This is the first report of N. caninum in a horse and indicates that N. caninum can be transmitted transplacentally in equids.     

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.     

Immunisation of mice against neosporosis

In the present study a murine encephalitis model was used to investigate if protection against neosporosis could be achieved by immunisation. Groups of 10 mice were immunised with a sublethal dose of live Neospora caninum tachyzoites, N. caninum antigens incorporated into iscoms, N. caninum lysate mixed with Quil A, or N. caninum lysate in PBS. Control mice were given Quil A only. Challenge infection with 2.5x10(6) N. caninum tachyzoites resulted in clinical symptoms that remained until the end of the experiment in the controls. In contrast, mice immunised with live parasites or parasite lysate in Quil A only showed mild and transient symptoms. Of nine mice immunised with N. caninum iscoms, seven recovered while two died. Most severely affected were the mice immunised with parasite lysate only; all of them died within 28 days post-infection. Histological examination and scoring of brain lesions gave a significantly lower (P<0.0001) lesion score in mice immunised with live parasites than in controls. The groups immunised with iscoms or lysate and Quil A also had reduced lesion scores (P<0.04 and 0.07, respectively) but not the group given parasite lysate alone. The lesions seen in the latter group differed from those in the other groups. There was less cellular reaction and more tachyzoites indicating an active infection. The N. caninum specific antibody responses and cytokine production (IFN-gamma, IL-4 and IL-5) of splenocytes were analysed at the time of challenge infection. The results suggest a correlation between protection and high levels of IFN-gamma. Also, the immune responses recorded in mice immunised with parasite lysate without adjuvant were relatively weak and more towards the Th2 type, when compared with the other immunisation schedules. This is consistent with the weaker inflammatory response observed in the brains of these mice.     

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.     

Protection against vertical transmission in bovine neosporosis

In this study we were interested to determine whether infection of cattle prior to pregnancy would afford any protection to the foetus if the dams were challenged with Neospora caninum at mid-gestation. The experiment comprised four groups of cattle: group 1, uninfected controls; group 2, inoculated with N. caninum tachyzoites 6 weeks prior to mating and then challenged with N. caninum at mid-gestation; group 3, naive cattle challenged with N. caninum at mid-gestation and group 4 were infected with N. caninum prior to mating and left unchallenged throughout pregnancy. Positive cell-mediated and humoral immune responses to N. caninum were recorded in groups 2 and 4 prior to pregnancy and in groups 2, 3 and 4 following challenge at mid-gestation. However there was a marked down regulation of the cell-mediated immune response in all groups around mid-gestation. There was a significant increase in rectal temperature response in animals in group 3 compared to group 2 following challenge but no other clinical symptoms of disease were recorded and all cattle proceeded to calving. At calving, pre-colostral blood samples were negative for antibodies to N. caninum in all the calves born to dams in groups 1, 2 and 4. In contrast, all the calves born to dams in group 3 had high levels of specific antibody to N. caninum indicating that they had been exposed to the parasite in utero. At post-mortem N. caninum DNA was detected in CNS, thymus and placental cotyledon samples in calves from group 3. All tissue samples from calves in the other 3 groups were negative for N. caninum DNA with the exception of one calf from group 2 where specific DNA was detected in a sample of spinal cord. These results suggest that the immune response generated in the dams in group 2 prior to pregnancy had protected against vertical transmission of the parasite following challenge at mid-gestation.     

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.     

Isolation of Neospora caninum from the brain of a pregnant sheep.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum antibodies and by detecting N. caninum-specific DNA by polymerase chain reaction.     

Neospora caninum (Apicomplexa) in a stillborn goat.

Tissue cysts of Neospora caninum were found in sections of brain from a stillborn pygmy goat. The tissue cysts had 1-2-microns-thick cyst walls and stained with anti-N. caninum serum in an immunohistochemical test. Glial nodules, mononuclear perivascular cuffing, and foci of inflammation were associated with N. caninum tissue cysts throughout the brain. This report indicates that N. caninum can be transmitted transplacentally in goats.     

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.     

Detection of Neospora caninum in the semen and blood of naturally infected bulls

A prospective study was designed to investigate the presence of Neospora caninum in semen and blood of eight bulls seropositive to N. caninum using nested-PCR procedures. Positive semen and blood samples were bioassayed in a BALB/c nu/nu mouse model. Specific anti-N. caninum serological and interferon-gamma (IFN-gamma) responses were also studied. In parallel, five seronegative bulls acted as non-infected controls. All bulls were located in a collaborating AI centre and monitored for 22 weeks. Six of eight seropositive bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. In all positive semen samples, we consistently found Neospora-DNA in the cell fraction and not in seminal plasma. Parasite load, as determined by a real-time PCR in nested-PCR positive semen samples, ranged from 1 to 10 parasites/ml. We found no association between the presence of N. caninum DNA in semen and blood. N. caninum could not be detected in the BALB/c nu/nu mice inoculated with PCR-positive semen or blood samples. Specific IgG antibody levels in seropositive bulls fluctuated over time, at times falling below cut-off level. The response was predominantly IgG2, with significant differences compared to control bulls (P < 0.05). The overall mean specific IFN-gamma response in seropositive bulls was also higher than those observed in the control group (P < 0.05), although extensive variation in individual responses was observed among bulls and over time. No significant association was found between bulls showing Neospora DNA in semen, blood, or both, and specific IgG, IgG1, IgG2, IgM and IgA levels or IFN-gamma response. This study is the first to report the presence of Neospora DNA in semen and blood of naturally-infected bulls. Our observations indicate intermittent presence of N. caninum in blood and semen and shedding in semen in low numbers.     

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.     

Multilocus microsatellite analysis reveals extensive genetic diversity in Neospora caninum

Neospora caninum is a world-wide parasite that causes neuromuscular disorders in dogs and bovine abortion. Biological diversity among isolates has been proved in both in vivo and in vitro studies. In contrast, little is known about the genetic diversity of this parasite. Microsatellite sequence analysis constitutes a suitable tool that has been used for the genetic analysis of other apicomplexan parasites. In this report, we describe the identification and analysis of 13 microsatellite loci from N. caninum DNA sequences deposited in public databases, which were evaluated with the use of 9 isolates grown in vitro. One microsatellite was monomorphic, and the remaining 12 loci exhibited 3 to 9 separate alleles. Multilocus analysis showed that each of the 9 isolates investigated here displayed a unique profile and revealed no association between the genetic similarity and host or geographic origin. The multilocus analysis approach described here might nevertheless provide the powerful tool needed to study the genetic complexity of N. caninum and the molecular epidemiology of neosporosis.     

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.     

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.     

Transmission of Neospora caninum between wild and domestic animals

To determine whether deer can transmit Neospora caninum, brains of naturally infected white-tailed deer (Odocoileus virginianus) were fed to 4 dogs; 2 of these dogs shed oocysts. Oocysts from 1 of the dogs were tested by polymerase chain reaction and found to be positive for N. caninum and negative for Hammondia heydorni. The internal transcribed spacer 1 sequence of the new strain (designated NC-deer1) was identical to N. caninum from domestic animals, indicating that N. caninum is transmitted between wild and domestic animals, often enough to prevent divergent evolution of isolated populations of the parasite. NC-deerl oocysts were administered to a calf that developed a high antibody titer, providing evidence that N. caninum from wildlife can infect cattle. In addition, N. caninum antibody seroprevalence was detected in 64/164 (39%) free-ranging gray wolves (Canis lupus), 12/113 (11%) coyotes (Canis latrans), 50/193 (26%) white-tailed deer, and 8/61 (13%) moose (Alces alces). These data are consistent with a sylvatic transmission cycle of N. caninum between cervids and canids. We speculate that hunting by humans favors the transmission of N. caninum from deer to canids, because deer carcasses are usually eviscerated in the field. Infection of canids in turn increases the risk of transmitting the parasite to domestic livestock.