Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.     

The inhibitory effect of interferon gamma and tumor necrosis factor alpha on intracellular multiplication of Neospora caninum in primary bovine brain cells

Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-gamma) and tumor necrosis factors alpha (TNF-alpha) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-gamma inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-alpha, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.     

In vitro antiprotozoal effects of artemisinin on Neospora caninum.

Neospora caninum is an intracellular apicomplexan parasite that infects a wide range of mammals and has been associated with abortion in cattle worldwide. Artemisinin is an effective antimalarial compound derived from a traditional Chinese herbal remedy, qinghao or Artemisia annua L. In the study reported, the cultured host cells (vero cells or mouse peritoneal macrophages) infected with N. caninum tachyzoites were incubated with alpha-MEM (minimal essential medium) 10%HS supplemented with various concentration or artemisinin (20, 10, 1, 0.1 and 0.01 microg/ml) to examine the efficacy of artemisinin against N. caninum tachyzoites intracellular multiplication. In long-term studies, at 20 or 10 microg/ml for 11 days, artemisinin reduced N. caninum and completely eliminated all microscopic foci of N. caninum. At 1 microg/ml for 14 days, artemisinin reduced N. caninum and completely achieved elimination of all microscopic foci of N. caninum. There was no apparent toxicity to host cells in long-term studies. In short-term studies, at > or = 0.1microg/ml, artemisinin reduced N. caninum tachyzoites intracellular multiplication, significantly (P < 0.05) and appeared to depend on the artemisinin concentrations. Pretreatment of host cells or N. caninum tachyzoites with artemisinin had no effect on N. caninum tachyzoites intracellular multiplication. These results demonstrate that artemisinin inhibited N. caninum tachyzoites intracellular multiplication.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

Host/parasite relationship in the in vitro infection of rat gliocytes by Neospora caninum: evaluation of cell respiration

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.     

Comparison of the growth inhibitory effects of canine IFN-alpha, -beta and -gamma on canine cells infected with Neospora caninum tachyzoites

The growth inhibitory effects of recombinant canine interferon alpha (IFN-alpha), beta (IFN-beta) and gamma (IFN-gamma) were examined on Madin-Darby canine kidney cells infected with Neospora caninum tachyzoites. The parasite growth was inhibited by all IFNs in a dose-dependent manner. IFN-gamma inhibited the parasite growth with greater efficacy than IFN-alpha or IFN-beta. Moreover, the effect of IFNs on N. caninum growth associated with the suppression of the host cell viability. The present study indicates IFN-alpha and -beta, besides IFN-gamma, play a crucial role for N. caninum growth in host cells.     

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.     

Prevalence of Neospora caninum and persistent infection with bovine viral diarrhea virus in dairy-breed steers in a feedlot

OBJECTIVE: To determine the prevalence and effect of Neospora caninum infection and persistent infection (PI) with bovine viral diarrhea virus (BVDV) on weight gain, morbidity, and mortality rate in dairy-breed steer calves located on a feedlot in California. DESIGN: Prospective cohort observational study. ANIMALS: 900 dairy-breed steer calves in 2 pens. PROCEDURES: The 3- to 4-month-old calves were evaluated for serum antibodies against N caninum and infection with BVDV at entry to the feedlot. Five months later, sera were again analyzed for anti-N caninum antibodies; calves that were determined to have BVDV infection initially were retested to evaluate PI status. Average daily gain, morbidity, and deaths were recorded for all calves. RESULTS: Among 900 calves, prevalence of N caninum infection was 16.7% (95% confidence interval, 14.3% to 19.3%); prevalence of BVDV-associated PI was 0.2% (95% confidence interval, 0.03% to 0.9%). Morbidity rate and time to first illness were not significantly different between calves that were seropositive or seronegative for N caninum. At the second sample collection, weight and average daily gain of calves that were seropositive for N caninum was less than that of seronegative steers in 1 pen, whereas these measures did not differ between groups in the other pen. Statistical power was insufficient to evaluate the effect of BVDV PI on any outcome measurement. CONCLUSIONS AND CLINICAL RELEVANCE: Although N caninum serostatus had no significant effect on morbidity rate, some seropositive calves had reduced growth, compared with seronegative calves, 5 months after entry to the feedlot.     

The host-parasite relationship in bovine neosporosis

Infection with the protozoan parasite Neospora caninum is thought to be a major cause of reproductive failure in cattle worldwide. Cattle infected with the parasite are three to seven times more likely to abort compared to uninfected cattle. The parasite may be transmitted to cattle through the ingestion of oocysts that are shed in the faeces of acutely infected dogs (definitive host of N. caninum) or by congenital infection from mother to foetus via the placenta. Interestingly, transplacental transmission can occur over consecutive pregnancies and congenitally infected heifers can transmit the parasite to their own offspring. This repeated vertical transmission observed in naturally infected cattle suggests that cattle do not easily develop effective immunity to the parasite, presenting a significant challenge to the development of a control strategy based on vaccination. Neosporosis is a disease of pregnancy and studying the bovine maternal and foetal immune responses during pregnancy will help us to understand the change in the balance between the parasite and the host that may result in disease of the foetus. Studies in non-pregnant cattle and in murine models of infection have shown the importance of T-helper 1-type immune responses involving pro-inflammatory cytokines, such as IFNgamma and IL-12, in limiting intracellular multiplication of the parasite. During pregnancy, changes occur in the immune system allowing the mother to accept the foetal allograft. Research in other species has stressed the crucial role of T-helper 2-type cytokines at the materno-foetal interface in maintaining the pregnancy and regulating the potentially damaging effect of Th-1 responses. Studies in cattle have shown that cell proliferation and IFNgamma responses may be significantly down-regulated around mid-gestation. This may mean that cattle are less able to cope with N. caninum infection at this time and are more likely to transmit the parasite to the foetus. Another important factor is the gestational age and hence immuno-competence of the foetus at the time of infection. Early in gestation, N. caninum infection of the placenta and subsequently the foetus usually proves fatal, whereas infection occurring in mid to late pregnancy may result in the birth of a congenitally infected but otherwise healthy calf. Studies of foetal immune responses have shown that at 14 weeks of gestation, lymphocytes only respond to mitogen, while by 24 weeks (mid-gestation), they respond to antigen by proliferating and releasing IFNgamma. Clearly, there are several factors influencing the outcome of N. caninum infection in pregnancy: the timing, quantity and duration of parasitaemia, the effectiveness of the maternal immune response and the ability of the foetus to mount an immune response against the parasite. The challenge is to design a vaccine that will prevent foetal infection by N. caninum. This is likely to involve a fine balancing act with the immune system that will allow intervention in a manner that will tip the host-parasite balance in favour of the host without compromising the pregnancy.     

Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum

The present study was undertaken to determine if red foxes are natural intermediate and/or definitive host for Neospora caninum and to study the importance of infection of N. caninum in this species in North-eastern Spain. Faecal samples and brain tissues were obtained from 122 foxes from 21 rural areas of Catalonia. Faeces collected were examined for parasite eggs and coccidian oocysts using sucrose flotation. For PCR-based diagnosis of N. caninum in brain tissues, the specific genomic Nc5 region was selected as the target sequence for DNA amplification. To control for PCR failure and facilitate identification of truly negative samples, the competitor pNc5C molecule was added to all negative samples in a second round of PCR reactions. Of the 122 foxes analysed, 13 (10.7%) were positive by PCR for N. caninum. Signal intensities of all positive samples were relatively weak with the exception of one sample from a 3-month male animal, that also showed the highest repeatability. No differences were observed by sex, age or area of sampling analysis. Detection of stages of N. caninum in brain from naturally infected red foxes demonstrated that red foxes are a natural intermediate host for N. caninum. Faecal samples were analysed for the presence of N. caninum oocysts, however, no oocysts compatible with N. caninum were found. A widespread latent infection of red foxes in North-eastern Spain found in the present study indicates that red foxes could have a very important role in the epidemiology of neosporosis in our area.     

Neospora caninum: oocyst challenge of pregnant cows

Three pregnant cows were each orally challenged at 10 weeks of gestation with 600 sporulated oocysts of Neospora caninum. The number of oocysts was limited by those available. In concurrent bioassays, one oocyst per os infected each of two gerbils. Challenged cattle developed Neospora-specific antibody, cell proliferation and gamma-interferon responses. N. caninum specific PCR demonstrated persisting infection in the brains of cows 4 months after calving. Abortion was not induced and there was no evidence of transplacental infection in the healthy calves born at full-term. This experiment suggests that the dose threshold for induction of abortion exceeds 600 oocysts.     

Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum

A nested PCR that successfully detected Neospora caninum DNA in serum of cattle was used for investigation of selected abortion cases and in a study of healthy pregnant cows at an abattoir. N. caninum DNA was not detected in serum from antibody positive dams that aborted due to N. caninum, but was present in serum of some antibody negative dams that aborted due to other causes. N. caninum DNA was also found in the serum of about half of the animals that aborted of undetermined cause, but was not detected in cow sera from two beef cattle herds in Western Australia with no recent history of abortion. In the abattoir study of 79 dams and their foetuses N. caninum DNA was found in serum of 3 dams and in material from 11 foetuses. The majority of the cows and all foetuses were antibody negative. Our findings suggest that there is no obvious relationship between the presence or absence of N. caninum DNA in serum and the presence of antibodies to N. caninum in dams, the presence of N. caninum DNA in foetuses or abortion due to N. caninum. This is the first report of the detection of N. caninum DNA in serum of cattle rather than the white blood cell fraction. It indicates the presence of free tachyzoites and/or parasite DNA in circulation. The results suggest that persistent infection in the absence of antibodies is a possible outcome of N. caninum infection. Infection of foetuses in the absence of antibodies supports the possibility of persistent infection due to immunotolerance to an early in utero infection. It is therefore important to test for N. caninum DNA as well as antibodies for the detection of exposed and/or infected animals. However, the presence or absence of N. caninum antibodies or DNA did not support nor exclude N. caninum as the cause of abortion. Additional criteria are required for a positive diagnosis of abortion caused by N. caninum.     

Tunicamycins, a class of nucleoside antibiotics similar to corynetoxins of the Rathayibacter toxicus, increase susceptibility of mice to Neospora caninum

Neosporosis is the leading cause of abortion in cattle. Neospora caninum-associated abortion may exhibit both endemic and epidemic patterns. It was reported that the epidemic outbreaks took place in the form of "abortion storms" and were not significantly correlated with seasonal changes or consumption of any particular feeds; and thus, the mechanisms by which the epidemic "abortion storms" are triggered remain unclear. Annual ryegrass toxicity (ARGT) is a severe or fatal neurological disorder of livestock of Australia and South Africa. This disorder is caused by the ingestion of several plant genera (Lolium, Polypogon, and Agrostis) colonized by a nematode (Anguina sp.) and a bacterium, Rathayibacter toxicus. Corynetoxins (CTs) produced by R. toxicus contaminate plants which are in turn ingested by sheep and cattle, causing severe or often fatal hepatocerebral disorders in affected animals. We hypothesize that N. caninum-associated fetal deaths and abortions in cattle may be potentiated by pre-exposure of sublethal levels of CT-contaminated plants prior to N. caninum infection or reactivation of a latent infection. The exposure of sublethal CTs may not cause clinical diseases, but may increase susceptibility to pathogens such as N. caninum. Indeed, CT poisoning surviving pregnant sheep can have up to 10% abortions. The present study investigated whether animals pre-exposed to tunicamycins (TMs), a functional substitute for CTs, had a lowered resistance to sublethal experimental infection by N. caninum tachyzoites in mice. The results showed that sublethal doses of TMs or N. caninum alone did not cause significant deaths. Sublethal doses of N. caninum induced high mortality in TM-treated mice in a dose dependent manner. When mice were treated with a low dose of TMs (5 μg/mouse), as few as 2.5 × 10(6) tachyzoites were needed to induce more than 30% of mortality which is equivalent to the mortality rate caused by 40 × 10(6) tachyzoites. Spleen cells of mice treated with TMs had reduced (p<0.05) Neospora antigen-induced interferon-gamma (IFN-g) production and Con A-stimulated proliferation. The results suggest that CT contaminated plants, when exposed to animals under natural conditions, may contribute to lowered host resistance and increased N. caninum-associated fetal morbidity and mortality in affected animals.     

Anti-protozoal efficacy of medicinal herb extracts against Toxoplasma gondii and Neospora caninum

The purpose of this study was to determine whether alcohol extracts of herbs (Sophora flavescens Aiton, Sinomenium acutum (Thunb.) Rehder and E.H. Wilson, Pulsatilla koreana (Yabe ex Nakai) Nakai ex T. Mori, Ulmus macrocarpa Hance and Torilis japonica (Houtt.) DC.) from South Korea, possess in vitro anti-protozoal activity against cultures of Toxoplasma gondii and Neospora caninum. These herbs have been used as human anti-parasitics in Asian countries for many years. Alcohol extracts of these herbs were serially diluted to final concentrations ranging from 625 to 19.5 ng/ml in media and added to wells containing either T. gondii or N. caninum tachyzoites in equine dermal (ED) cells. Parasite growth inhibition was measured using 3H-uracil incorporation as compared to untreated controls. T. japonica inhibited T. gondii proliferation by 99.3, 95.5, 73.0 and 54.0% in the range from 156 to 19.5 ng/ml, and S. flavescens inhibited T. gondii proliferation by 98.7, 83.0 and 27.2% in the range from 156 to 39 ng/ml. T. japonica inhibited N. caninum proliferation by 97.8, 97.9, 85.3 and 46.4% in the range from 156 to 19.5 ng/ml. S. flavescens inhibited N. caninum proliferation by 98.6, 97.0, 69.5 and 14.0% in the range from 156 to 19.5 ng/ml. Toxicity to host cells was noted when concentrations of T. japonica and S. flavescens exceeded 625 ng/ml. The herb extracts from S. acutum, Pulsatilla koreana, and U. macrocarpa also showed toxicity at higher levels but did not achieve the same inhibition effects at the lower concentrations against T. gondii and N. caninum as T. japonica and S. flavescens.     

Safety of a live attenuated Erysipelothrix rhusiopathiae vaccine for swine

The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus. Virions of PRRSV contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2, GP3, GP4, and E. The GP5 is the major envelope proteins, which was involved in the formation and infectivity of PRRSV by coaction with other membrane proteins. Here, to determine the function of alone GP5 envelope protein in viral entry, we investigated the formation and infectivity of GP5-pseudotyped virus particles. By co-transfection of GP5 expression plasmids with murine leukemia virus (MuLV) based retroviral vectors (pHIT60, encoding MuLV Gag-Pol; pHIT111, encoding an MuLV genome with a β-galactosidase reporter gene) into 293 T cells and analysis of the culture medium using ultracentrifugation, Western blot, and infection assay. We observed that the GP5 envelope protein was incorporated into the MuLV retroviral vectors to generate an pseudotyped murine leukemia virus, which was infectious to PAM and Mack-145 target cells and displayed the same host range with wild-type PRRSV. The infection of the pseudotyped virus on PAM target cells is effectively neutralized by polyclonal antibodies specific for PRRSV or GP5. The results suggested that the GP5 protein may play a key role in the viral entry by interacting with the host cell receptor. The GP5-pseudotyped virus will be useful in the identification of the cellular receptor binding with GP5 protein.     

Point source exposure of cattle to Neospora caninum consistent with periods of common housing and feeding and related to the introduction of a dog

Eight dairy herds with evidence of post-natal transmission of Neospora caninum were used to test the hypothesis of a point source exposure by a retrospective analysis of the housing and feeding of infected age-groups. The first N. caninum-associated abortion or birth of N. caninum-seropositive offspring from the post-natally infected age-group was considered as the first indication of the infection. In seven of the eight dairy herds, a point source exposure to N. caninum of the infected age-groups was found during a limited period of common housing and feeding. In all herds studied, the analysis indicated that the cattle had been infected shortly before the first abortions occurred. In all, except one herd, the post-natal infection was more directly related to housing than to feeding. Therefore, it appeared that the feed was contaminated in the feeding alley. In one herd, the total mixed ration was found to be the probable path of infection. In all farms studied, a new dog (young, adult dog or litter) had been introduced within a period of 1.5 years prior to the first indication of N. caninum infection in the cattle. As there was evidence in all herds of vertical transmission of neosporosis for years, it is hypothesized that the newly introduced dog was infected with N. caninum by materials from already infected cattle and subsequently transmitted the infection to other cattle by shedding of oocysts.     

In vitro isolation and characterization of bovine Neospora caninum in Korea

The brains of nine aborted bovine fetuses and two newborn calves born from dams suspected to be infected with Neospora caninum were homogenized and inoculated into Vero cells. All fetuses and calves were from cows determined as seropositive to N. caninum by an IFA test. Sera and thoracic fluids of all fetuses and calves also revealed high antibody titer to N. caninum by IFAT ranging from 1:800 to 1:3200. N. caninum was isolated from the brains of one aborted fetus and one newborn calf when the brain homogenates were grown continuously in Vero cell culture. N. caninum tachyzoites, giemsa-positive, were first observed on Days 45 and 56 postinoculation in the newborn calf and the aborted fetus, respectively. The isolates (KBA-1 and KBA-2) were morphologically and ultrastructurally similar to previously published Neospora isolates. The isolated parasites were confirmed as N. caninum by means of the antigenic reactivities, immunostaining, PCR and southern blotting, and electron microscopy.     

Anti-protozoal efficacy of high performance liquid chromatography fractions of Torilis japonica and Sophora flavescens extracts on Neospora caninum and Toxoplasma gondii

We previously reported that alcoholic extracts of Sophora flavescens and Torilis japonica from South Korea demonstrated good efficacy in reducing replication of Toxoplasma gondii and Neospora caninum. To characterize the chemical component associated with anti-protozoal activity, specific fractions were isolated by high performance liquid chromatography (HPLC) and used for in vitro testing. These fractions were evaluated in vitro against T. gondii and N. caninum. Fractions of the herb extracts were serially diluted to final concentrations of 2.850 to 0.356 ng/ml in medium and added to wells containing replicating T. gondii and N. caninum. To determine the ability of each fraction to inhibit parasite proliferation, 3H-uracil incorporation was used to determine parasite replication. In cultures infected with T. gondii, a fraction of T. japonica (TJ2) inhibited T. gondii proliferation by 99.2, 94.4, 88.6 and 27.0% in the range from 2.850 to 0.356 ng/ml. Four fractions of S. flavescens (SF1-SF4) inhibited T. gondii proliferation by 99.6-60.6, 96.9-48.1, 92.3-68.2 and 95.4-52.9% in the range from 2.850 to 0.356 ng/ml. In cultures infected with N. caninum, a fraction of T. japonica (TJ2) inhibited N. caninum proliferation by 98.3, 95.5, 79.7 and 30.6% in the range from 2.850 to 0.356 ng/ml. Four fractions of S. flavescens (SF1-SF4) inhibited N. caninum proliferation by 97.1-25.9, 94.8-35.5, 95.9-33.7 and 95.4-49.4% in the range from 2.850 to 0.356 ng/ml. These fractions of T. japonica and S. flavescens extracts are currently undergoing in vivo evaluation in experimentally infected mice.     

Characterization of Neospora caninum microneme protein 10 (NcMIC10) and its potential use as a diagnostic marker for neosporosis

Improvements in the serological diagnosis of neosporosis are needed to differentiate acute versus chronic Neospora caninum infections. In the present study, N. caninum microneme protein 10 (NcMIC10), similar to other microneme proteins, was shown to be released in a calcium-dependent manner. NcMIC10 may be discharged during active invasion of host cells by the parasite, and thus represent an excellent marker for the diagnosis of neosporosis. In order to test this hypothesis, recombinant NcMIC10 (rNcMIC10) was expressed in Escherichia coli, and polyclonal antibodies were generated against non-overlapping fragments of the protein. A capture ELISA was developed using these antibodies, and was found to be highly accurate and reproducible with a detection range of 10-10,000 pg/ml. The anti-rNcMIC10 antibodies used in this study did not cross-react with the Toxoplasma gondii antigens. NcMIC10 was detected by the ELISA in sera of 9 out of 10 goats (90%) experimentally infected with N. caninum tachyzoites. In general, goats infected with a lower dose (10(4)) of the parasite displayed a peak in NcMIC10 levels between weeks 4 and 5 post infection. Goats infected with a higher parasite dose (10(6)) displayed a more rapid increase in NcMIC10 levels. In most animals, NcMIC10 decreased to undetectable levels by week 6 post infection. This is the first circulating Neospora antigen-based assay which may complement the existing antibody-based assays for a rapid and cost-effective definitive diagnosis of neosporosis in livestock.