A competitive PCR assay for quantitative detection of Neospora caninum

A quantitative-competitive PCR (QC-PCR) assay was developed for measurement of Neospora caninum levels in the tissues of infected animals. A molecule was synthesised for use in PCR as a competitor to the target Neospora-specific Nc5 genomic sequence. The assay was used to evaluate the relative level of parasites in the brain and lungs of mouse pups in a model of vertical transmission of N. caninum. Infection on day 11 of gestation resulted in similar levels of parasites in all offspring. The assay should be useful in evaluation of vaccines against Neospora infection. Incorporation of the competitor molecule in the detection assay also provides a control for PCR failure and facilitates identification of truly negative samples.     

Fertility,Gestation Outcome and Parasite Congenital Transmissibility in Mice Infected with TcI,TcII and TcVI Genotypes of Trypanosoma cruzi

This work aims to compare the effects of acute or chronic infections with the T. cruzi genotypes TcI (X10 strain), TcII (Y strain) and TcVI (Tulahuen strain) on fertility, gestation, pup growth and the possible vertical transmission of parasites in BALB/c mice. The occurrence of congenital infection was evaluated by microscopic examination of blood and/or qPCR on blood and heart in newborn pups and/or older offspring submitted to cyclophosphamide-induced immunosuppression in order to detect possible cryptic congenital infection. Altogether, the results show that: i) for the three strains tested, acute infection occurring after the embryo implantation in the uterus (parasite inoculation 4 days before mating), or close to delivery (parasite inoculation on day 13 of gestation), prevents or severely jeopardizes gestation outcome (inducing pup mortality and intra-uterine growth retardation); ii) for the three strains tested, gestation during chronic infection results in intra-uterine growth retardation, whereas re-inoculation of TcVI parasites during gestation in such chronically infected mice, in addition, strongly increases pup mortality; iii) congenital infection remains a rare consequence of infection (occurring in approximately 4% of living pups born to acutely infected dams); iv) PCR, detecting parasitic DNA and not living parasites, is not convenient to detect congenial infection close to delivery; v) transmission of parasites by breast milk is unlikely. This study should encourage further investigations using other parasite strains and genotypes to explore the role of virulence and other factors, as well as the mechanisms of such effects on gestation and on the establishment of congenital infection.     

Transplacental transmission and abortion in cows administered Neospora caninum oocysts

Neospora caninum infection is a common cause of bovine abortion. One method by which cattle can acquire infection is through ingestion of oocysts; however, this has not yet been proved to cause transplacental infection or abortion. In this study, 19 cows, pregnant between 70 and 176 days, were administered 1500 to 115,000 oocysts through an esophageal tube. Seventeen of the cows became seropositive, indicating acquisition of infection, whereas 8 negative control cows remained seronegative (P < 0.001). Offspring were examined using serology, histology, immunohistochemistry, parasite isolation, and polymerase chain reaction (PCR). Six offspring were infected and 1 of them was aborted. The aborted fetus had typical lesions and positive immunohistochemistry and PCR for N. caninum. All 6 cows with infected offspring had continuously rising antibody titers, whereas 10 of 11 infected cows with uninfected offspring had falling titers after an early apex. The risk of transplacental transmission was increased by later exposure times during gestation and by the dose of oocysts (P < 0.01 for the 2 combined variables). The lowest dose of oocysts, when administered after the 160th day of gestation, caused transplacental infection in 1 of 2 animals. This study demonstrates that infection with N. caninum oocysts can cause transplacental transmission and abortion in cattle.     

Comparative effect of Neospora caninum infection in BALB/c mice at three different gestation periods

Neospora caninum has been recognized as a major cause of infectious bovine abortion worldwide. In the present study, the effect of N. caninum infection in mice at the 3 gestation periods (first, second, and third period) was investigated. In dams, tissue distribution of N. caninum was evaluated by nested polymerase chain reaction. In the progeny, fetal mortality, stillbirth, litter size, neonatal mortality/morbidity, vertical transmission, and parasite burden in neonatal tissues were evaluated. Pregnant BALB/c mice were infected subcutaneously with 2 x 10(6) NC-1 tachyzoites on days 0, 7, or 14 of gestation. Dams from each group were sequentially killed during gestation and postpartum (PP). Pups were killed on days 1 and 7 PP. Infection on day 0 of gestation produced a high vertical transmission rate, although no changes in fetal mortality, stillbirth, and littermate size were observed. The highest level of vertical transmission, together with an increase in fetal mortality and stillbirth and a decrease in litter size, were observed when infection was done on day 7 of gestation. Finally, infection on day 14 of gestation produced the lowest vertical transmission. Furthermore, infection at any time during gestation compromised the postnatal development of pups, because neonates from infected dams showed less body weight and a delay in the hair development.     

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.     

The extent of parasite-associated necrosis in the placenta and foetal tissues of cattle following Neospora caninum infection in early and late gestation correlates with foetal death

The protozoan parasite Neospora caninum is the most frequently diagnosed abortifacient in the UK and a leading cause of abortion worldwide but the mechanisms leading to abortion are not fully understood. The distribution of parasites and the histopathological changes in the placenta and foetus were compared in 12 cows following experimental infection of cattle with N. caninum in early (n=6) and late (n=6) gestation, by PCR, immunohistology, light microscopy and transmission electron microscopy. Twelve uninfected pregnant cattle were used as controls. Infection in early gestation led to foetal death. In the placentae of cattle immediately following foetal death, N. caninum DNA was detected and there was evidence of widespread parasite dissemination. This was associated with extensive focal epithelial necrosis, serum leakage and moderate maternal interstitial mononuclear cell infiltration. In the foetuses, parasites were evident in all tissues examined and were associated with necrosis. In the placenta of cattle infected in late gestation, N. caninum DNA was detected sporadically but parasites were not evident immunohistologically. Small foci of necrosis were seen associated with mild interstitial mononuclear cell infiltration. Detection of N. caninum DNA in the foetuses was sporadic and parasites were demonstrated immunohistologically in brain and spinal cord only, with an associated mononuclear cell infiltration. This data is consistent with uncontrolled parasite spread in an immunologically immature foetus and could, via multiparenchymal necrosis of foetal tissues or the widespread necrosis and inflammation observed in the placenta, be the cause of Neospora-associated abortions.     

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

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

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

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

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

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

Immunization of mice with plasmid DNA coding for NcGRA7 or NcsHSP33 confers partial protection against vertical transmission of Neospora caninum

The purpose of the present study was to use direct plasmid deoxyribonucleic acid (DNA) injection to identify specific antigens that confer protection against congenital transfer of Neospora caninum. Inbred BALB/c mice were vaccinated before pregnancy with a recombinant plasmid containing sequences encoding N. caninum antigen NcGRA7 or NcsHSP33. The mice were challenged with N. caninum tachyzoites at 10-12 days of gestation. Whereas 100% of pups born from dams immunized with control plasmid contained detectable levels of N. caninum DNA in a Neospora-specific polymerase chain reaction assay, only 46% of pups from pCMVi-NcGRA7-immunized mice and 53% of pCMVi-NcsHSP33-immunized mice were N. caninum positive, and none of the mice immunized with tachyzoite extract contained N. caninum DNA. Thus, immunization of mice with plasmid DNA expressing N. caninum antigens conferred partial protection against congenital neosporosis.     

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

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

Effects of Neospora caninum infection at mid-gestation on placenta in a pregnant mouse model

Neospora caninum is one of the more-efficient transplacentally-transmitted organisms. The goal of the present study was to investigate the pathologic and immunologic changes that occur at the materno-fetal interphase in pregnant BALB/c mice infected with N. caninum at mid-gestation. Parasite DNA was detected in feto-placentary units 3 days post-infection (PI). On day 7 PI, the DNA detection level and parasite burden were significantly higher in the placentas than in the fetuses, which may indicate that the parasite is mainly multiplying in the placenta during the initial infection. In the spleens of infected dams, we observed an increase in IFN-γ, IL-10, and IL-4. However, only IL-4 was upregulated in placentas from the infected dams; this may enhance susceptibility to N. caninum at the materno-fetal interphase and favor transmission to the progeny. Finally, an increase in TNF-α expression in nested-PCR-positive placentas combined with necrosis may compromise the viability of the fetuses.     

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.     

Non-sand fly transmission of a North American isolate of Leishmania infantum in experimentally infected BALB/c mice

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is endemic in the foxhound population in the United States and Canada. Leishmaniasis is usually transmitted by blood-feeding sand flies; however, epidemiological data do not support a significant role for sand flies in the maintenance of foxhound infections in North America, and an alternate mode of transmission may exist. The present study was conducted to determine if transplacental or direct transmission occurs in pregnant BALB/c mice experimentally infected with L. infantum isolated from a naturally infected foxhound from Virginia as well as to determine if the parasite was directly transmitted to the males used to breed the mice. Female BALB/c mice were intravenously inoculated with 1 x 10(6) promastigotes of the LIVT-1 strain of L. infantum. Mice were bred to uninfected male BALB/c mice 2 mo postinoculation. Pregnant mice were killed between days 13 and 18 of gestation. Pups and placentas were collected at necropsy, divided, and used for parasite culture and polymerase chain reaction (PCR) analyses. Culture and PCR analyses were performed on spleens from the male mice to determine the possibility of sexual transmission. Leishmania sp. DNA was detected in 4 of 88 pups and 3 of 16 placentas from LIVT-1-inoculated mice. One male mouse used to breed infected females was PCR positive. This work provides evidence for a low level of nonvector transmission of North American L. infantum in a mouse model.     

Developmental arrest and pregnancy-induced transmammary transmission of Ancylostoma caninum larvae in the murine model

Pregnancy is associated with reactivation of latent infections of many protozoal and helminthic parasites. To facilitate in vivo studies on the process of transmammary transmission of hookworm infection to nursing newborns, we established an experimental model of infection of BALB/c mice with infective larvae of the canine nematode Ancylostoma caninum. To establish latency with a significant reservoir of tissue larvae and achieve acceptable pregnancy success rates, mice were subcutaneously infected at day 5 postimpregnation; similar larval distribution profiles were observed at the end of the gestational period for bred compared to correspondingly infected unbred animals. No larvae were detected in fetuses or neonatal pups. Significant numbers of larvae were not detected in mammary tissue during the periparturient or postpartum lactational periods although about 8% of a dam's reservoir of tissue larvae was transferred to her nursing pups; this suggests that larvae reaching the mammary glands are rapidly transmitted through the milk sinuses, as was documented by histopathological analyses. Comparison of BALB/c with C57BL/6 mice that typically display divergent immune responses to infection showed no difference in tissue larval burden or in numbers transferred to pups. A hypothesis for the molecular mechanism of larval reactivation and transmission is discussed.     

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.     

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

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

Role of dense granule antigen 7 in vertical transmission of Neospora caninum in C57BL/6 mice infected during early pregnancy

Neosporosis is a parasitic disease affecting the health of dogs and cattle worldwide. It is caused by Neospora caninum, an obligate intracellular apicomplexan parasite. Dogs are its definitive host, it mostly infects livestock animals, especially cattle that acts as intermediate host. It is necessary to have well-established models of abortion and vertical transmission in experimental animals, in order to determine basic control measures for the N. caninum infection. We evaluated the role of N. caninum dense granule antigen 7 (NcGRA7) in the vertical transmission of N. caninum using the C57BL/6 pregnant mouse model. We inoculated mice on day 3.5 of pregnancy with parental Nc-1 or NcGRA7-deficient parasites (NcGRA7KO). Post-mortem analyses were performed on day 30 after birth and the surviving pups were kept until day 30 postpartum. The number of parasites in the brain tissues of offspring from NcGRA7KO-infected dams was significantly lower than that of the Nc-1-infected dams under two infection doses (1 × 10⁶ and 1 × 10⁵ tachyzoites/mouse). The vertical transmission rates in the NcGRA7KO-infected group were significantly lower than those of the Nc1-infected group. To understand the mechanism by which the lack of NcGRA7 decreases the vertical transmission, pregnant mice were sacrificed on day 13.5 of pregnancy (10 days after infection), although parasite DNA was detected in the placentas, no significant difference was found between the two parasite lines. Histopathological analysis revealed a greater inflammatory response in the placentas from NcGRA7KO-infected dams than in those from the parental strain. This finding correlates with upregulated chemokine mRNA expression for CCL2, CCL8, and CXCL9 in the placentas from the NcGRA7KO-infected mice. In conclusion, these results suggest that loss of NcGRA7 triggers an inflammatory response in the placenta, resulting in decreased vertical transmission of N. caninum.     

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.     

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.