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PubMed2003

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.

Abortion, Veterinary↗
PubMed2003

Regional distribution of bovine Neospora caninum infection in the German state of Rhineland-Palatinate modelled by Logistic regression.

To obtain a rapid overview over the distribution of bovine Neospora caninum-infections in the German state of Rhineland-Palatinate, an ELISA to determine specific bovine antibodies against a p38 surface antigen of N. caninum tachyzoites was modified to examine bulk milk samples from cattle herds. Experimental bulk milk samples were used to demonstrate that the seroprevalence in a group of animals can be estimated with this ELISA. A cut-off was selected for the specific detection of herds having a seroprevalence > or =10%. About 90% of the dairy herds located in Rhineland-Palatinate were examined. An overall prevalence of bulk milk-positive herds of 7.9% (95% confidence interval 7.0-8.9%), respectively, was determined. Major regional differences in the distribution of bulk milk-positive herds were observed. Prevalences were higher in regions with an increased degree of urbanisation. Logistic regression was applied to model the prevalence of bulk milk-positive herds on a district and city level. Variables describing the dog density, mean temperature in July, mean temperature in January and the total yearly precipitation in districts and cities were able to explain most of the observed variability in the regional prevalences. Our results provide evidence that in addition to risk factors related to individual farms also risk factors related to the farm location such as dog density in the surrounding and climate factors are important in the epidemiology of bovine neosporosis.

Animals↗
PubMed1998

Application of iscom antigen preparations in ELISAs for diagnosis of Neospora and Toxoplasma infections.

Immunostimulating complexes (iscoms) are cage-like structures of about 40 nm composed of Quil A, cholesterol, phospholipids and antigen. Their main area of use has been as adjuvants and carriers of immunogens in vaccines. Iscoms can also be used for selection of surface membrane proteins of micro-organisms for use in immunoassays, thus decreasing the number of internal proteins that might cause problems with non-specific binding and cross-reactivity. Enzyme-linked immunoassays (ELISAs) utilising parasite antigens incorporated into iscoms have been developed for demonstration of antibodies directed to the intracellular coccidian parasites Toxoplasma gondii and Neospora caninum. These iscom ELISAs have proved very reliable, with high sensitivity and specificity. The preparation of T. gondii and N. caninum iscoms is described, and ELISAs based on iscom antigen preparations that have so far been used for diagnosis of protozoal infections are reviewed.

Animals↗
PubMed1998

Seroprevalence of antibodies to Neospora caninum and Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Egypt.

Sera from 75 water buffaloes from Egypt were examined using a direct agglutination test incorporating mercaptoethanol for antibodies to Neospora caninum and Toxoplasma gondii. Antibodies to N. caninum were found in 51 (68%) of 75 buffaloes in titres of 1:20 (six buffaloes), 1:40 (15 buffaloes), 1:160 (one buffalo), 1:320 (one buffalo) and > or = 1:640 (28 buffaloes), using N. caninum formalin-preserved whole tachyzoites as antigen. Antibodies to T. gondii were not found in a 1:100 dilution of serum of any of the 75 buffaloes, using T. gondii as antigen, indicating specificity in the detection of antibodies to N. caninum. This is the first report of N. caninum prevalence in water buffaloes, which are economically very important domestic animals in developing countries.

Agglutination Tests↗
PubMed1998

Identification and characterisation of a dense granule-associated protein in Neospora caninum tachyzoites.

Neospora caninum is an apicomplexan parasite which is morphologically and ultrastructurally very similar to Toxoplasma gondii. In order to identify molecules involved in host cell entry and subsequent modification of the parasitophorous vacuole, a polyclonal antiserum directed against N. caninum tachyzoites was raised in a rabbit. Subcellular fractionation of tachyzoites was performed using the non-ionic detergent Triton-X-114. Membrane fractions were analysed by immunoblotting using the polyclonal antiserum. One of the immunoreactive protein bands had a mol. wt of 33,000 and was subsequently named Nc-p33. Affinity-purified anti-Nc-p33 antibodies were used to characterise this polypeptide using SDS-PAGE, isoelectric focusing, Western blot analysis and immuno-EM. Nc-p33 was found in two isolates of N. caninum (NC-1 and Liverpool), but could not be detected in T. gondii tachyzoites. Immunogold EM revealed that Nc-p33 constituted a dense granule-associated protein, and Western blotting demonstrated that Nc-p33 was most likely identical to the recently described antigen NCDG1. Shortly after invasion, this dense granule protein was targeted to the parasitophorous vacuole membrane, and, at later timepoints after infection, was also found on the parasitophorous vacuolar network. This suggested that Nc-p33 could play a functional role in the modification of the parasitophorous vacuole and its membrane.

Animals↗
PubMed1998

Canine neosporosis: clinical signs, diagnosis, treatment and isolation of Neospora caninum in mice and cell culture.

Clinical signs, diagnosis, treatment and isolation of Neospora caninum from two littermate dogs are described. Three of six pups from a Labrador bitch developed paralysis. Neosporosis was diagnosed ante mortem by serological examination in two of the affected pups. At necropsy, tissue cysts were seen in unstained smears and in histologic sections of their brains. Tissue cysts were often thin-walled (approximately 1 micron) but antigenically and ultrastructurally identified as N. caninum. Furthermore, N. caninum (isolates NC-4, NC-5) was isolated in mice and in cell cultures inoculated with neural tissues of these two dogs. Serological diagnosis of neosporosis using a variety of tests is discussed.

Age Factors↗
PubMed1999

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Spain.

In recent years, neosporosis has been identified as a major cause of abortion in dairy and beef cattle. Although the disease has been described worldwide, there is a Jack of information concerning the prevalence of this infection in different cattle production systems. The aim of this study was to investigate the seroprevalence of Neospora caninum infection in a representative area of beef and dairy cattle production in Spain. A cross-sectional study was undertaken in which herds constituted the initial sampling unit and two strata (dairy and beef herds) were considered. Using a 95% level of confidence and setting 5% (beef) and 5.4% (dairy) error limits, 216 beef and 143 dairy herds were randomly selected and sampled. Nine animals (> 1 year old) were randomly sampled in each herd to detect the presence of the infection. A herd was considered infected when at least one animal was seropositive. In total, serum samples from 1121 dairy and 1712 beef animals were collected and tested for specific anti-N. caninum IgG using an ELISA. Specific antibodies were detected in 55.1% (119/216) beef and 83.2% (119/143) dairy herds. Individual prevalences obtained were 17.9% (306/1712) for beef and 35.9% (402/1121) for dairy animals. Presence of N. caninum infection was higher in dairy than in beef herds and the association between infection and the cattle production system (dairy or beef) was statistically significant [(chi2)Y= 29.21, P < 0.001, OR = 4.04 (2.35-6.99)]. Herd size of dairy cattle did not appear to be associated with N. caninum infection. On the contrary, infection was associated with herd size in beef cattle (chi2 = 12.79, P < 0.01). Finally, no association was found between replacement or pasture management and infection in beef herds.

Animal Husbandry↗
PubMed1999

The antigenic composition of Neospora caninum.

Neospora caninum is an apicomplexan parasite which causes neosporosis, namely stillbirth and abortion in cattle, and neuromuscular disease in dogs. Although N. caninum is phylogenetically and biologically closely related to Toxoplasma gondii, it is antigenically clearly distinct. In analogy to T. gondii, three stages have been identified. These are: (i) asexually proliferating tachyzoites; (ii) tissue cysts harbouring slowly dividing bradyzoites; and (iii) oocysts containing sporozoites. The sexually produced stage of this parasite has only recently been identified, and has been shown to be shed with the faeces from dogs orally infected with N. caninum tissue cysts. Thus dogs are definitive hosts of N. caninum. Tachyzoites can be cultivated in vitro using similar techniques as previously described for T. gondii. Methods for generating tissue cysts containing N. caninum bradyzoites in mice, and purification of these cysts, have been developed. A number of studies have been undertaken to identify and characterise at the molecular level specific antigenic components of N. caninum in order to improve serological diagnosis and to enhance the current view on the many open questions concerning the cell biology of this parasite and its interactions with the host on the immunological and cellular level. The aim of this paper is to provide an overview on the approaches used for detection of antigens in N. caninum. The studies discussed here have had a great impact in the elucidation of the immunological and pathogenetic events during infection, as well as the development of potential new immunotherapeutic tools for future vaccination against N. caninum infection.

Amino Acid Sequence↗
PubMed1999

Significance of Neospora caninum in British dairy cattle determined by estimation of seroprevalence in normally calving cattle and aborting cattle.

A case control study was conducted to evaluate the significance of Neospora caninum infections in cattle in England and Wales. The prevalence of N. caninum in normally calving cattle (the control group; n = 418) and aborting cattle (n = 633) was estimated using a commercial antibody-detection ELISA. Prevalence estimates for bovine virus diarrhoea virus, infectious bovine rhinotracheitis virus and Leptospira hardjo were also obtained by serology. The prevalence of N. caninum was significantly higher (P < 0.0001) in the aborting group (18%; 95% confidence interval: 15%, 21%) than in the control group (6%; 95% confidence interval: 4%, 8%); the latter is the first estimate, to date, of the national seroprevalence of N. caninum in dairy cattle in England and Wales. Prevalence estimates for bovine virus diarrhoea virus, infectious bovine rhinotracheitis virus and L. hardjo were not found to be higher in the aborting cattle than in the control group. With N. caninum, a strong association between seropositivity and abortion was found, with seropositive cows being 3.5-times more likely to abort than seronegative cows (odds ratio = 3.49; 95% confidence interval: 2.16, 5.69). Furthermore, 12.5% of abortions in dairy cattle in England and Wales may be attributable to N. caninum, as indicated by estimation of the population aetiological fraction.

Abortion, Veterinary↗
PubMed1999

Phenotypic characterisation of a Neospora caninum temperature-sensitive strain in normal and immunodeficient mice.

The in vivo persistence, immunogenicity and pathogenicity of a recently described temperature-sensitive (ts) strain from Neospora caninum, NCts-8, was investigated in normal and immunodeficient mice. Groups of BALB/c and SCID/Bg mice were infected s.c. with 5 x 10(6) wild-type NC-1, control NCts-8 (pass 0) or NCts-8 tachyzoites prepared at four in vitro passage levels (pass 7, 13, 21 and 28). For persistence and immunogenicity studies, BALB/c mice were bled and sacrificed at 4, 6 or 8 weeks p.i. Sera were analysed by IFAT and brain tissues examined for lesions by histology and tested for parasite presence by PCR. For pathogenicity studies, SCID/Bg mice were monitored by clinical signs and survival time. Results from parasite persistence experiments demonstrated microscopic lesions and PCR positive brain tissues in NC-1 infected mice. In contrast, brain tissues from NCts8-infected groups were consistently negative by histology and PCR. Based on IFAT titres, all parasite strains were immunogenic, although parasite-specific IgG levels were lower in the NCts-8 infected groups. Results from pathogenicity studies in SCID/Bg mice demonstrated a significantly (P < 0.0001) longer mean survival time in NCts-8 vs NC-1 infected groups. In addition, there was no significant difference in mean survival time between control NCts-8 and experimental passage NCts-8 infected mice. Collectively, these studies demonstrate that the NCts-8 strain maintains a stable phenotype following multiple passages in vitro, and possesses an attenuated, shorter persistence phenotype in vivo compared with the parental wild-type NC-1.

Animals↗
PubMed1999

Postnatal Neospora caninum transmission and transient serologic responses in two dairies.

Cattle on two typically managed drylot dairies were serologically monitored from birth through year 1 to year 4 of life to characterise congenital and postnatal Neospora caninum transmission. Of the 456 calves enrolled, 284 were classified as N. caninum negative and 172 were classified as N. caninum positive. Ninety-six percent of congenitally infected calves were seropositive for all samples tested. Seven (4%) of the 172 congenitally infected animals had a period that persisted for 9 to 18 months when they were seronegative; however, all returned to seropositive status by 25 months of age. In N. caninum-negative calves, colostral antibody decayed by 128 days, with an estimated half-life of 19.6 +/- 5.2 days. Of the 284 calves classified as negative, 18% had sporadic, isolated responses to N. caninum, typically between 29 and 35 months of age, without subsequent seroconversion or infection. During the study, 17 animals seroconverted and remained seropositive throughout the follow-up. Thirteen of the seroconversions occurred in the neonatal period; however, in nine of 10 where dam status was available, the dam was N. caninum positive, suggesting late gestation congenital infection rather than postnatal infection. Seroconversion was detected in an additional four animals, between 13 and 22 months of age. The estimate of postnatal infection rate was less than 1% per year despite a high N. caninum seroprevalence in the herds, and the presence of potential definitive and intermediate hosts on the dairy throughout the study. The extremely low rate of postnatal infection, as well as the lifelong persistence of congenital infection, emphasises the importance of congenital transmission in maintaining N. caninum infection in dairies.

Animals↗
PubMed1999

Experimental inoculation of domestic pigeons (Columbia livia) and zebra finches (Poephila guttata) with Neospora caninum tachyzoites.

Dogs are a definitive host of Neospora caninum and cattle are intermediate hosts. Alternative life-cycles have not been investigated. Foxes are frequently seropositive, but may not commonly prey upon cattle; therefore, other intermediate hosts may exist that are frequent prey of foxes. Three domestic pigeons (Columbia livia) and three zebra finches (Poephila guttata) were inoculated with N. caninum tachyzoites, to determine if they could serve as intermediate hosts. Tissue culture, PCR, serology, and histology were all positive for one or more pigeons. All finches resisted infection. Further testing of columbiform birds as intermediate hosts of N. caninum is warranted.

Animals↗
PubMed1999

Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle.

Dogs from dairy farms with a known prevalence of Neospora caninum antibodies in the cattle were examined for the presence of N. caninum antibodies using an ELISA. Data of farm dogs were compared with those of dogs examined at a university clinic, which originated mainly in urban areas. Of the 152 farm dogs, 36 (23.6%) were seropositive to N. caninum, which was significantly higher than the proportion of seropositives in the clinic dog population (19 of 344, 5.5%). Seroprevalence was significantly higher (P = 0.01) in female dogs than in male dogs. Seroprevalence in dogs increased with age, indicating postnatal infection. Seropositivity to N. caninum in farm dogs was strongly correlated with a high prevalence of N. caninum antibodies in the cattle. At farms where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P = 0.0002) than in farms where dogs were present. These findings suggest that there is a relationship between N. caninum infection of farm dogs and cattle. Since dogs have been shown to be definitive hosts of N. caninum, cattle may be infected by exposure to canine oocysts. Further research is needed to find out whether and how dogs may acquire the infection from cattle.

Animals↗
PubMed1999

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro.

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.

Animals↗
PubMed1999

Serological diagnosis of Neospora caninum infection.

Since the first isolation of the apicomplexan parasite Neospora caninum, a range of serological assays have been developed for use in dogs, cattle and a variety of other potential host species. The tests include the indirect fluorescent antibody test, the direct agglutination test and different enzyme-linked immunosorbent assays. This article reviews the principles and properties of the available tests which are discussed in relation to different applications.

Animals↗
PubMed1999

Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with different adjuvants.

A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P < 0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P < 0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide-based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum-specific proliferation of peripheral blood mononuclear cells statistically similar (P = 0.095) to that of the infected animals. Peripheral blood mononuclear cells from animals immunised with the Polygen-adjuvanted tachyzoite preparation produced interferon-gamma concentrations of similar magnitude (P = 0.17) to those from the infected animals. Polygen was one of two adjuvants that elicited the highest antibody responses, and was the only adjuvant that induced interferon-gamma levels similar to those of the infected heifers.

Adjuvants, Immunologic↗
PubMed1999

Physical characterisation of the plastid DNA in Neospora caninum.

The physical characteristics of the plastid DNA in Neospora caninum were investigated using pulsed-field gel electrophoresis and TEM. In a comparison of contour-clamped homogenous electric field and field inversion gel electrophoresis, the latter proved the more successful technique for studying the plastid molecules. In most cases, restriction or modifying enzymes were required to enable the plastid DNA molecules to enter the gel from the well area. The unit length of the plastid of N. caninum is approximately 35 kb; however, there is evidence for the formation of oligomeric molecules, which may migrate as linear molecules in approximate multiples of the unit length. Four different plastid genes encoding the ssrRNA, lsrRNA, rpoC and tufA genes were identified by hybridisation studies of contour-clamped homogenous electric field and field inversion gel electrophoresis gels. Transmission EM was performed on isolated plastid DNA, and circular structures similar in size and appearance to those described in other apicomplexans were observed, with an approximate length of 19 microm. The data presented here conclusively show that the Nc-Liverpool canine strain of N. caninum possesses a plastid DNA, with physical characteristics similar to the plastids found in other apicomplexans.

Animals↗
PubMed1999

Detection of surface-associated and intracellular glycoconjugates and glycoproteins in Neospora caninum tachyzoites.

The surface-associated molecules of the invasive stages of apicomplexan parasites such as Neospora caninum and Toxoplasma gondii are most likely crucially involved in mediating the interaction between the parasite and its host cell. In N. caninum, several antigens have recently been identified which could participate in host cell adhesion and/or invasion. These are antigens which are either constitutively expressed on the outer plasma membrane, or antigens which are only transiently localised on the surface as they are expulsed from the secretory vesicles either prior, or after host cell invasion. Some of these proteins have been characterised at the molecular level, and it has been shown that they are, with respect to protein sequences, closely related to homologous counterparts in T. gondii. Nevertheless, there is only a low degree of cross-antigenicity between the two species. In microbial interactions it has been shown that carbohydrates could also play a crucial role in host cell recognition and immunological host parasite interactions. In this study we present data which strongly suggest that the surface of N. caninum tachyzoites is glycosylated. In SDS-PAGE, glycoproteins comigrated largely with glycosylphosphatidylinositol-anchored proteins which were identified using in vivo [3H]ethanolamine labelling followed by autoradiography. The lectin Con A reacted strongly with the surface of these parasites, binding of which is indicative for the presence of N-glycans. Additional surface binding was observed, although only in a subpopulation of all tachyzoites, for wheat germ agglutinin and Jacalin. Intracellular binding sites for Con A were mainly associated with the parasite dense granules. By lectin labelling of Western blots of N. caninum protein extracts, glycoproteins were identified which reacted specifically with the lectins Con A, wheat germ agglutinin, Jacalin and soy bean agglutinin.

Animals↗