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PubMed2002

Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host-parasite interactions.

Neospora caninum is an apicomplexan parasite which has emerged as an important cause of bovine abortion worldwide. Abortion is usually triggered by reactivation of dormant bradyzoites during pregnancy and subsequent congenital infection of the foetus, where the central nervous system appears to be most frequently affected. We here report on an organotypic tissue culture model for Neospora infection which can be used to study certain aspects of the cerebral phase of neosporosis within the context of a three-dimensionally organised neuronal network. Organotypic slice cultures of rat cortical tissue were infected with N. caninum tachyzoites, and the kinetics of parasite proliferation, as well as the proliferation-inhibitory effect of interferon-gamma (IFN-gamma), were monitored by either immunofluorescence, transmission electron microscopy, and a quantitative PCR-assay using the LightCycler instrument, respectively. In addition, the neuronal cytoskeletal elements, namely glial acidic protein filaments as well as actin microfilament bundles were shown to be largely colocalising with the pseudocyst periphery. This organotypic culture model for cerebral neosporosis provides a system, which is useful to study the proliferation, ultrastructural characteristics, development, and the interactions of N. caninum within the context of neuronal tissue, which at the same time can be modulated and influenced under controlled conditions, and will be useful in the future to gain more information on the cerebral phase of neosporosis.

Animals↗
PubMed2002

Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion.

A round table was conducted at the VIIIth International Coccidiosis Conference on Neospora diagnosis with particular emphasis on strategies to diagnose bovine abortion. The strength and weakness of different assays for Neospora caninum infection and whether these methods have resulted in the overdiagnosis of neosporosis was discussed. It was evident that each diagnostic method, namely histology, immunohistochemistry, molecular detection and serological assays were, under certain circumstances, valuable in assessing the role N. caninum in abortion. Histological, immunohistochemical and molecular detection assays are of outstanding importance for the examination of tissues of aborted foetuses. While histology and immunohistochemistry allow direct assessment of pathomorphological changes caused by infection, molecular detection assays such as PCR are superior because of higher sensitivity and specificity in identifying N. caninum in foetal tissues. Serological tests, such as ELISA, are useful in determining whether an animal has been infected with N. caninum. Seroepidemiological approaches allow one to assess an abortion problem at a herd level and when used in conjunction with certain statistical methods are able to confirm a suspected N. caninum-associated abortion.

Abortion, Veterinary↗
PubMed2002

Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures.

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.

Animals↗
PubMed1998

Naturally occurring vertical transmission of Neospora caninum in dogs.

Vertical transmission of Neospora caninum was studied in naturally infected, privately owned dogs, using antibody detection by IFAT to identify infected individuals. Retrospective studies were undertaken in litters from six bitches of the Hamiltonstövare breed, and on litters from seven bitches of other breeds--in both cases following diagnosis of clinical disease in puppies--and prospective studies were carried out on 17 seropositive bitches of other breeds. Puppies were classified as infected if either pre-colostral sera, or serum taken at > or = 5 weeks, had a titre of > or = 1:50 in the IFAT. The frequency of putative congenital transmission was variable; three seropositive dams produced successive litters, each containing seropositive puppies; four other seropositive bitches, which each whelped twice, produced six litters in which all pups were seronegative. Bitches born to seropositive dams themselves produced litters containing seropositive pups. In the retrospective studies, 32/61 (52%) of pups tested born to 13 seropositive dams were themselves seropositive and 22/88 (25%) of all pups born developed clinical signs compatible with a diagnosis of neosporosis. However, in the prospective study, only 4/118 (3%) pups tested from 17 seropositive dams were seropositive and 4/122 (3%) of all pups born developed signs consistent with neosporosis. This difference reflected levels of IFAT titre in the two groups of bitches and overall the proportion of pups seropositive was strongly and positively correlated (r = 0.980, P < 0.05) with bitch IFAT titre. Overall, 80% of pups born to seropositive dams were not infected as determined serologically. This study shows that the frequency of vertical transmission of naturally acquired Neospora infection in dogs is variable, but much too low to sustain infection alone. Post-natal infection must occur to maintain infection at seroprevalence rates reported in dog populations.

Animals↗
PubMed1998

Polymerase chain reaction approaches for the detection of Neospora caninum and Toxoplasma gondii.

This review summarizes existing knowledge on the development and use of the polymerase chain reaction for the detection of DNA from Neospora and Toxoplasma. Several strategies which utilise the polymerase chain reaction for the diagnosis of toxoplasmosis in humans and livestock have been described and they principally target the B1 repetitive sequence, the P30 gene or ribosomal DNA. Experience has shown that the polymerase chain reaction has proven insufficiently robust to serve as a diagnostic test alone although when used in conjunction with other diagnostic techniques it does prove to be a useful aid. The marketing of a commercial polymerase chain reaction kit may well solve some of the inadequacies seen using "home made" polymerase chain reaction technology which are commonly used in diagnostic laboratories around the world. Recent progress on the development of polymerase chain reaction diagnostics for Neospora has been rapid and is discussed in detail.

Animals↗
PubMed1998

Oral Neospora caninum inoculation of neonatal calves.

Four calves born to cows seronegative for Neospora caninum were dosed orally within 6 h after birth with tachyzoites of the bovine N. caninum Nc-SweB1 isolate added to colostrum. Two of the calves were dosed via stomach tube and two by feeding bottle. The latter two calves showed transient fever and passed blood-stained diarrhoea 1-2 weeks after inoculation. From 5 weeks after inoculation they developed a significant antibody response which remained high until the calves were euthanised and necropsied at 15 and 19 weeks after inoculation, respectively. The two calves inoculated by stomach tube showed no clinical signs and they remained seronegative throughout the study. At necropsy of the seropositive calves, no pathological lesions were seen, and parasites were not detected by immunohistochemistry. Neospora caninum was not re-isolated in cell culture from the brains of the seropositive calves; however, N. caninum DNA was detected in brain from both of them by PCR. The data suggest that oral infection of N. caninum via colostrum might be a possible route of vertical transmission in newborn calves, in addition to transplacental infection.

Animals↗
PubMed1999

Comparison of the IFAT and Iscom-ELISA response in bovine foetuses with Neospora caninum infection.

The study was carried out to evaluate the efficacy of foetal serology in the diagnosis of Neospora-associated bovine abortions. Fluids from 14 foetuses of cows with confirmed neosporosis (Group A), seven foetuses with confirmed bovine viral diarrhoea virus (BVD infection) (Group B) and 11 aborted foetuses without demonstrable infection (Group C) were examined. The age of the foetuses ranged from 4.5 months to 9 months. Albumin concentration (measured by Rocket Immunoelectrophoresis) was not significantly different in Group A compared with that in both Groups B and C, while that in Group B was significantly lower than in Group C. Levels of total IgG ranged from 0.01 to 1.78 (mg IgG) ml(-1) measured by single radial immunodiffusion technique. A measurable level of total IgG was found in all foetuses from Groups A and B, with no significant difference between levels in the two groups. Only one foetus in Group C had a detectable level of IgG. All foetuses in Group A had a specific IgG response (titre> or = 20) against Neospora caninum using the IFAT, while no positive responses in IFAT were found in Groups B and C. Measurement of specific IgG1 and IgG2 by Iscom-ELISA showed one and three false-negative results, respectively, in Group A. The IgG1 and IgG2 response in Group A was correlated according to the Spearman test (r = 0.66). Increasing age of the foetuses correlated significantly with the foetal IgG concentration, the specific IgG and IgG1 + IgG2. On the basis of the results obtained, it was concluded that the IFAT with a cut-off titre of 1:20, was a specific method for diagnosis of neosporosis in foetuses older than 4.5 months. The Iscom-ELISA also showed promising results as a method for screening specific antibodies against N. caninum in foetal fluid.

Abortion, Veterinary↗
PubMed1999

Ingestion of Neospora caninum tissue cysts by Mustela species.

Dogs are a definitive host of Neospora caninum, a protozoal parasite that causes abortion in cattle. Mustelids were tested to determine if they could also be definitive hosts. The procedures used were the same as those previously used to test dogs. Ermine (Mustela erminea), weasels (Mustela frenata) and ferrets (Mustela putorius) were fed N. caninum-infected mice. Neospora caninum oocysts were not observed. Mustelid faeces were fed to mice. The mice did not seroconvert and N. caninum was not detected in murine brains using tissue culture and PCR. The hypothesis that Mustela spp. are definitive hosts of N. caninum is not supported.

Animals↗
PubMed1999

Comparison of the major antigens of Neospora caninum and Toxoplasma gondii.

The Apicomplexa are a diverse group of parasitic protozoa with very ancient phylogenetic roots. Consistent with their phylogeny, the extant species share conserved proteins and traits that were found in their apicomplexan progenitor, but at the same time they have diverged to occupy different biological niches (e.g. host-range and cell type). Characterisation of gene and protein diversity is important for distinguishing between related parasites, for determining their phylogeny, and for providing insight into factors that determine host restriction, cell preference, and virulence. The value of molecular characterisations and comparisons between species is well illustrated by the close phylogenetic relationship between Neospora caninum and Toxoplasma gondii. These two organisms have nearly identical morphology and can cause similar pathology and disease. Consequently, N. caninum has often been incorrectly identified as T. gondii, thus demonstrating the need for studies addressing the molecular and antigenic composition of Neospora. In this review, we describe the major antigenic proteins that have been characterised in N. caninum. These show homology to T. gondii proteins, yet possess unique antigenic characteristics that distinguish them from their homologues and enable their use for specific serological diagnoses and parasite identification.

Animals↗
PubMed1999

Estimation of vertical and horizontal transmission parameters of Neospora caninum infections in dairy cattle.

Transmission parameters of Neospora caninum infections in dairy cattle were determined in six herds with a history of Neospora-associated abortions, using an antibody-detection ELISA to detect evidence of infection. A total of 124 seropositive dams and their calves were tested at calving to estimate vertical transmission, and 154 seronegative heifers were monitored prospectively from birth for evidence of post-natal infection. The probability of vertical transmission was very high; 95.2% (95% confidence interval: 89.8%, 98.2%) of the seropositive dams produced calves that were seropositive prior to consumption of colostrum. In the prospective study, three heifers seroconverted, by 1, 8 and 16 months of age, and the overall incidence rate of seroconversion was 1.9 per 100 heifer-years at risk, ranging from 0 to 4.1 per 100 heifer-years at risk within herds. If heifers with inconclusive ELISA results were included in the analysis, the incidence rate of seroconversion was 3.8 per 100 heifer-years at risk, ranging from 0 to 8.3 per 100 heifer-years at risk within herds. This study showed that vertical transmission was the major route involved in the spread of N. caninum in these herds, and that there was only a low detectable level of horizontal transmission. However, further studies are needed to provide parasitological evidence of infection in those heifers that seroconverted.

Animals↗
PubMed1999

The in vitro development of Neospora caninum bradyzoites.

Neospora caninum is a recently identified apicomplexan protozoan parasite that is closely related to Toxoplasma gondii. Neospora caninum is of significant economic importance as it causes neurological disease and abortion in numerous animals. Antibodies to BAG1/hsp30 (also known as BAG5), a T. gondii bradyzoite-specific protein, have been demonstrated to react with N. caninum tissue cysts in vivo. Bradyzoite differentiation of N. caninum in vitro was investigated using culture conditions previously utilised for T. gondii in vitro bradyzoite development. Utilising the NC-Liverpool isolate of N. caninum, cyst-like structures developed within 3-4 days of culture of this parasite in human fibroblasts. In addition, an antigen reacting with mAb 74.1.8 (anti-BAG1) and rabbit anti-recombinant BAGI was demonstrable by immunofluorescence, fluorescence-activated cell sorter, and immunoblot analyses. Expression of this antigen was increased by stress conditions, similar to that which has been described for T. gondii bradyzoite induction. Cyst-wall formation in vitro, as assayed by lectin binding, did not occur as readily for N. caninum as it does for T. gondii.

Animals↗
PubMed1997

Experimental infection of non-pregnant and pregnant sheep with Neospora caninum.

In an initial experiment, 21 sheep in groups of five or six were inoculated subcutaneously (sc) with 10(8), 10(6) or 10(4) Neospora caninum tachyzoites (Liverpool isolate), or with control inoculum, and monitored for clinical signs and for "seroconversion". Animals given the two higher doses showed febrile responses and all three groups inoculated with the parasite showed seroconversion. In a second experiment, 12 pregnant sheep were each inoculated sc at 90 days' gestation with 10(6) tachyzoites, and at 25, 40 and 53 days post-inoculation (dpi) groups of four were killed for examination of the fetuses and placentas. Appropriate control ewes were included in the study. All fetuses were alive immediately before their dams were killed, except for one, which was found to be mummified at 40 dpi. Histopathological lesions were found consistently in both fetal central nervous system (CNS) and placental tissues. In the latter, focal necrosis, which was mild at 25 dpi, was much more severe at 40 dpi and much less severe at 53 dpi. Lesions in the fetal CNS consisted of focal microgliosis (with or without central necrosis), lymphoid cuffing and non-suppurative meningitis. Lesions were also found in fetal liver, heart and lung. Neospora antigen was demonstrated in fetal brain and placental tissues and, at 25 dpi, in single samples of fetal liver and heart. The prescapular lymph nodes did not differ in size from those of control fetuses but were more mature in that they contained a significantly greater number of secondary follicles. Both IgM and IgG antibodies to N. caninum were detected in the serum of fetuses from infected ewes. Thus, N. caninum readily infected pregnant ewes and caused lesions in fetal tissues and placentas which resembled those of ovine toxoplasmosis. In addition, the changes were similar to those of bovine neosporosis; the infected pregnant ewe therefore offers a good model for the bovine disease.

Animals↗
PubMed2000

Molecular characterization of a novel microneme antigen in Neospora caninum.

The apical complex of the parasites belonging to the phylum Sporozoa is believed to be critically involved in the events leading to host cell invasion. The characterization of the components of this subcellular structure is therefore an important step towards understanding how these parasites achieve host cell entry. Affinity-purification of an anti-Neospora caninum antiserum on a reactive protein band of approximately 40 kDa following Triton-X-114 extraction of parasite proteins, SDS-PAGE and Western blotting, yielded an immunoglobulin fraction which, by immunofluorescence, stained predominantly the apical portion of N. caninum tachyzoites. Following immunoscreening of a N. caninum tachyzoite lambdagt22 cDNA expression library, the respective full length cDNA sequence was determined. This sequence was found to encode a protein of 362 amino acids, with a calculated Mr of 38086. This protein is encoded by a single copy gene which produces a transcript of 2.4 kb. Sequence analysis showed that it contains a N-terminal putative signal peptide sequence and two potential membrane spanning regions. Four consecutive epidermal growth factor like domains were identified, as well a conserved sequence motif for binding of ATP/GTP (P-loop). The full length cDNA was expressed as a recombinant poly-histidine fusion protein in Escherichia coli, and antibodies affinity purified on this protein labelled exclusively a 38 kDa band on immunoblots of N. caninum extracts. In addition, specific labeling of a 45 kDa band in Toxoplasma gondii tachyzoite extracts was observed. By immunofluorescence, these antibodies stained predominantly the apical portion of both N. caninum and T. gondii tachyzoites, but the protein was absent from the parasite surface. Immunogold localization in LR-White embedded N. caninum tachyzoites demonstrated staining of predominantly the apically located micronemes, as well as of dense granules located at the posterior end of the tachyzoites. As evidenced by immunohistochemistry, this Neospora microneme antigen and its immunoreactive counterpart in Toxoplasma appeared to be expressed in both tachyzoite and bradyzoite stages.

Amino Acid Sequence↗
PubMed1997

The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein.

Genes for the Toxoplasma gondii dense granule and rhoptry proteins nucleoside triphosphate hydrolase 3 and ROP2 were expressed at high levels in the closely related parasite N. caninum. The protein products were processed appropriately and were targeted to their correct secretory organelles. NTPase 3 was secreted into the parasitophorous vacuole. The utility of this system was demonstrated in the analysis of a new open reading frame identified upstream of two identical copies of the ROP2 gene. The unknown open reading frame was introduced into Neospora, and transfected parasites were analyzed by immunoblot with antibodies to known T. gondii rhoptry protein families. The transfected Neospora expressed a novel 52 kDa protein, designated ROP8, which localized in the rhoptries. These results illustrate that transfection of known Toxoplasma genes into N. caninum can be used to study their expression, processing and targeting in an immunologically distinct background. They also illustrate the usefulness of N. caninum transfection in the identification and subcellular distribution of proteins encoded by previously uncharacterized Toxoplasma genes.

Acid Anhydride Hydrolases↗
PubMed1999

Characterization of a cDNA encoding a subtilisin-like serine protease (NC-p65) of Neospora caninum.

The NC-p65 cDNA is the first protease sequence cloned and described for Neospora caninum. The full length cDNA was isolated by 5'- and 3'-rapid amplification of cDNA ends (RACE). NC-p65 was composed of 865 amino acids with a predicted signal sequence, a proposed pro-domain, and an internal region of conserved repeats. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) and had a predicted active site made up of the catalytic residues, Asp 253, His 309. and Ser 484. Antibodies to recombinant NC-p65 recognized multiple bands on Neospora lysate immunoblots, but most intensely stained a 65 kDa band. When N. caninum proteins were purified with affinity resins specific for NC-p65 and analyzed for enzyme activity, a single specific band of reaction was observed on gelatin-saturated zymograms.

Amino Acid Sequence↗
PubMed2002

A longitudinal study of Neospora caninum infection on a dairy farm in New Zealand.

A 600-cow New Zealand dairy herd experienced an abortion storm in 1997 and was monitored (blood sampling at about 3-month intervals) from May 1997 until January 1999. Abortion risk reached 9% in 1997 and was highest in heifers at 19%. The abortion risk decreased in 1998 to 3.2% (still somewhat higher than during the years prior to the outbreak). The serological reaction pattern for Neospora caninum showed an association with abortion risk only around the time of the 1997 outbreak when seropositive cows were 4.2 times more likely to abort than negative ones. Over the whole study period, only 27% of cows that were sampled on all nine visits always tested negative. Offspring from dams which had positive tests for Neospora caninum were 2.4 times more likely to abort than those from dams testing consistently negative. Controlling for age and breed, seropositive cows produced more milk than those that were consistently negative. Infection might have been present endemically within this herd prior to the epidemic, but in 1997 an additional factor appeared to have triggered the outbreak.

Abortion, Veterinary↗
PubMed2000

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.

Animals↗
PubMed2000

Serologic survey of Neospora caninum infection in a closed dairy cattle herd in Maryland: risk of serologic reactivity by production groups.

Prevalence of antibodies to Neospora caninum was determined in a cross-sectional consensus survey of 1029 bovines in a dairy herd with endemic Neospora-induced abortion. Sera were screened by indirect fluorescent antibody test (IFAT). The prevalence of N. caninum antibody in the IFAT was 17.9% in 107 neonates, 26.2% in 233 yearling heifers and steers, 39.07% in 218 mature heifers, and 26.9% in 465 milking cows. Serologic reactivity was associated with production grouping on the farm with the greatest risk of serologic reactivity appearing in the yearling and mature heifers. There was an increasing risk of serologic reactivity with increasing age only in the parity one and greater animals in the herd. Castrated males were at half the risk of similarly aged females of possessing antibodies to N. caninum. There was no clear relationship between the serologic status of dams and offspring.

Abortion, Veterinary↗