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PubMed2002

Characterisation of the first Australian isolate of Neospora caninum from cattle.

OBJECTIVE: To isolate Neospora caninum from a congenitally infected calf. PROCEDURE: A calf was obtained from a N. caninum infected dam maintained in a dairy herd of Holstein-Friesian cattle located on the south coast of NSW near Nowra. The calf was euthanased and samples collected for serology and pathology. Samples of brain and spinal cord of the calf were homogenised and injected into immunocompromised mice in an attempt to recover protozoa by in vivo culture. Sequential passage of brain homogenate through IFNgammaPKO mice was performed and tissue culture flasks were inoculated with brain homogenate. Parasites were identified by electron microscopy and DNA sequencing. The antigen profile of the isolate was analysed using Western blotting. Pathogenicity was examined in BALB/c mice and transmission of the parasite during pregnancy was examined in Qs mice. RESULTS: The calf was seropositive for N. caninum and histopathological examination of sections of cerebrum identified lesions consistent with a very mild infection with N. caninum. The parasites isolated using tissue culture were identified as N. caninum, based on the sequence of the ribosomal DNA and electron microscopy. The antigen profile of the new isolate was similar to that of the NC-Liverpool isolate, but quite different from that of Toxoplasma gondii. In BALB/c mice inoculated with the new isolate, severe clinical signs developed in only three of ten infected mice, compared with six of ten mice infected with NC-Liverpool. Mild to moderate nonsuppurative encephalitis was observed in BALB/c mice infected with the new isolate, compared with mice infected with NC-Liverpool, that developed severe nonsuppurative encephalitis. Transplacental transmission of the isolate arising from an acute infection during pregnancy occurred in about 87% of pups. CONCLUSION: This is the first isolation of bovine Neospora caninum in Australia. This isolate, called NC-Nowra, appears to be a less virulent form and may prove to be a suitable candidate for vaccine development.

Abortion, Veterinary↗
PubMed1999

Serological evidence of human infection with the protozoan Neospora caninum.

Neospora caninum is a protozoan parasite that is closely related to Toxoplasma gondii. Dogs are a definitive host. Prior to its discovery in 1988, N. caninum infection in animals was often mistakenly diagnosed as toxoplasmosis. Neosporosis in animals is characterized by encephalitis, abortion, and other conditions that clinically and pathologically resemble toxoplasmosis. The potential of N. caninum to infect humans is unknown. Therefore, evidence of human exposure to this parasite was sought by screening for antibodies in blood donors by indirect fluorescent antibody (IFA) tests and immunoblotting. Of 1,029 samples screened, 69 (6.7%) had titers of 1:100 by IFA testing. Fifty of the 69 (72%) sera that were positive for N. caninum were also negative for a closely related protozoan pathogen of humans, T. gondii. Immunoblot analysis confirmed the specificity of the positive sera for N. caninum antigens, with several sera recognizing multiple Neospora antigens with molecular masses similar to those of antigens recognized by monkey anti-N. caninum serum. An immunodominant antigen of approximately 35 kDa was observed with 12 sera. These data provide evidence of human exposure to N. caninum, although the antibody titers in healthy donors were low. The significance of human exposure to, and possible infection with, this parasite is unknown and warrants further study.

Animals↗
PubMed1998

The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii.

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii and has been found to be associated with neurological disorders in dogs and congenital infections and abortions in cattle. We have identified two surface proteins of 29 and 35 kDa (designated Ncp29 and Ncp35, respectively) from N. caninum tachyzoites that are the predominant antigens recognized by antisera from Neospora-infected animals. Monoclonal antibodies against Ncp29 and Ncp35 were used to analyze several independent and diverse N. caninum isolates; both antigens were recognized in all isolates, suggesting that they are well conserved. Localization studies and surface labeling with biotin demonstrated that Ncp29 and Ncp35 are membrane associated and displayed on the surface of the parasite. After treatment with phosphatidylinositol-specific phospholipase C, parasite lysates were analyzed with antibodies against the cross-reacting determinant of glycosylphosphatidylinositol anchors. Approximately six glycolipid-anchored surface proteins were identified, with the two most prominent corresponding to Ncp29 and Ncp35. Sequence comparisons of Ncp29 and Ncp35 with GenBank indicated that they are most similar to the T. gondii surface antigen 1 (SAG1) and surface antigen 1-related sequence 2 (SRS2), respectively. Consequently, Ncp29 has been designated NcSAG1 and Ncp35 has been designated NcSRS2. Both NcSAG1 and NcSRS2 contain a tandemly duplicated motif and 12 absolutely conserved cysteines which are also found in all of the SAG and SRS proteins of T. gondii. Maintenance of these motifs and the 12 cysteine residues suggests that these surface antigens share a similar secondary and tertiary structure that is presumably important for a conserved function that these antigens serve during infection.

Amino Acid Sequence↗
PubMed2004

Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties.

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.

Amino Acid Sequence↗
PubMed1997

Novel ELISA for detection of Neospora-specific antibodies in cattle.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Neospora species in cattle was developed. Whole formalin-fixed Neospora caninum (NC-Liverpool) tachyzoites were used as antigen and a monoclonal antibody to bovine immunoglobulin light chain and an anti-mouse horseradish peroxidase conjugate were used to reveal bound antibody. A panel of 46 sera, negative by the immunofluorescent antibody test (IFAT), were used in the ELISA at a serum dilution of 1:500 to calculate the negative cut-off value of OD405 = 0.77. There was a 95 per cent agreement between the results of the ELISA and the IFAT with 104 serum samples. The specificity and sensitivity of the ELISA were 96 per cent and 95 per cent, respectively, when compared with the IFAT. No significant cross-reaction was observed with the sera from cattle infected experimentally with Toxoplasma gondii, Cryptosporidium parvum, Babesia divergens, Sarcocystis cruzi, Eimeria alabamensis or E bovis. A significantly modified version of the test is now commercially available.

Animals↗
PubMed2005

Persistence of Neospora caninum-specific immunoglobulin G antibodies in bovine blood and lung tissue stored at room temperature.

Blood and tissue samples obtained at necropsy from carcasses showing varying degrees of postmortem decomposition are sometimes used as sources of antibodies for serological tests, primarily in studies involving wild animals or in examination of aborted fetuses. To test the persistence of antibodies in blood and lung tissue kept at room temperature (approximately 20degreesC) for up to 48 and 118 days, respectively, serum and lung fluid from Neospora caninum-infected and uninfected cattle were tested using an indirect N. caninum immunostimulating complex enzyme-linked immunosorbent assay (ELISA). Testing of selected samples was also performed using a commercial Neospora competitive ELISA. Despite severe changes in the gross appearance of the samples, the blood collected through venipuncture from live animals remained essentially sterile, whereas the pieces of lung tissue showed a variety of bacterial and fungal growth. Nevertheless, there was no obvious reduction in antibody levels in either the blood or the tissue samples during the 2 observation periods. This study indicates that specific immunoglobulin G antibodies may be remarkably stable in sterile blood and decomposed lung tissue.

Animals↗
PubMed2002

Prevalence of antibodies to Neospora caninum in Korean native beef cattle.

A total of 438 sera from Korean native beef cattle in 9 provinces were tested for Neospora caninum antibodies using an immunofluorescent antibody test (IFAT). Eighteen (4.1%) cattle were positive by IFAT. The titers ranged from 1:200 (10 animals), 1:400 (5 animals), 1:800 (2 animals) to 1:1,600 (1 animal). Although the seroprevalence was slightly higher in Chungnam (8.9%), this was not significantly different from those noted in Kyunggi, Kangwon, Kyungbuk, Kyungnam, and Cheju provinces. Sera obtained from beef cattle in the provinces of Chungbuk, Jeonbuk and Jeonnam were all negative. Neospora positive sera were also tested for anti-Toxoplasma gondii antibodies using a commercial latex agglutination test (LAT). Antibody to T. gondii was detected in only 1 (5.6%) of 18 N. caninum positive sera. These results indicate that N. caninum and T. gondii infection are present at a low level in the Korean native beef cattle.

Animals↗
PubMed2000

A random amplified polymorphic DNA polymerase chain reaction technique that differentiates between Neospora species.

Neospora caninum is a recently described coccidial parasite that was first isolated from a dog in 1988 and has subsequently been shown to infect a wide range of mammals. Neospora hughesi, a new species of this genus, has recently been isolated from the spinal cord of horses showing clinical signs of equine protozoal myeloencephalitis. The random amplified polymorphic DNA polymerase chain reaction technique is capable of differentiating between N. caninum and N. hughesi.

Animals↗
PubMed2002

Prevalence of antibodies to Neospora caninum and Sarcocystis neurona in sera of domestic cats from Brazil.

Parasite Biology, Epidemiology and Systematics Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 1001, Beltsville, Maryland 20705-2350 Antibodies to Neospora caninum and Sarcocystis neurona were determined in serum samples of 502 domestic cats from Brazil using direct agglutination tests with the respective antigens. Antibodies to S. neurona were not found in 1:50 dilution of any serum in the S. neurona agglutination test. suggesting that domestic cats from São Paulo city were not exposed to S. neurona sporocysts from opossums. Antibodies to N. caninum were found in 60 (11.9%) of 502 cats with titers of 1:40 in 36 cats, 1:80 in 18 cats, 1:160 in 5 cats, and 1:800 in 1 cat using the Neospora agglutination test (NAT). Antibodies to N. caninum were confirmed by Western blotting in the sera of 10 cats with NAT titers of 1:80 to 1:800; this finding suggests that at least 10 cats had N. caninum-specific antibodies confirmed by 2 tests. This is the first documentation of natural exposure of cats to N. caninum.

Agglutination Tests↗
PubMed2004

First report of Neospora caninum infection in adult alpacas (Vicugna pacos) and llamas (Lama glama).

Neospora caninum is a cyst-forming coccidian that mainly affects bovines, although Neospora infection has also been described in other domestic and wild ruminant species. Serum samples from 78 alpacas (Vicugna pacos) and 73 llamas (Lama glama) at a unique dilution of 1:50 tested by indirect fluorescent antibody test (IFAT) were further analyzed serologically by IFAT and Western blot in both ruminant species to avoid cross-reactions with closely related coccidian parasites and to confirm the existence of N. caninum-specific antibodies. IFAT titers ranging between 1:50 and 1:800 were found. When using Western blot, N. caninum tachyzoite-specific immunodominant antigens with apparent molecular weights of 17-18, 34-35, 37, and 60-62 kDa were also recognized, although some sera with 1:50 IFAT titers proved not to have N. caninum-specific antibodies. As expected, higher IFAT titers were associated with higher anti-N. caninum reactivity in Western blot. This report documents for the first time the presence of N. caninum infection in adult alpacas and llamas from Peru.

Animals↗
PubMed2003

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.

Animals↗
PubMed2005

Prevalence of antibodies to Neospora caninum in wild animals.

Antibodies to Neospora caninum were determined in several species of wild animals in the United States by the Neospora agglutination test (NAT). Antibodies (NAT 1:40 or higher) were found in 5 of 249 bison (Bison bison), 5 of 160 caribou (Rangifer tarandus), 4 of 162 moose (Alces alces), 4 of 122 wolves (Canis lupus), and 1 of 224 musk ox (Ovibos moschatus) but not in 197 black bears (Ursus americanus). To our knowledge, this is the first report of antibodies to N. caninum in bison and caribou. The total absence of N. caninum antibodies in black bears indicates that bears are not a host for N. caninum and that there is no cross-reactivity between the NAT and the modified agglutination test (MAT) for Toxoplasma gondii, because more than 80% of black bears in eastern United States have MAT antibodies at a 1:25 serum solution.

Animals↗
PubMed2003

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming.

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.

Agglutination Tests↗
PubMed2004

Evaluation of vaccination with Neospora caninum protein for prevention of fetal loss associated with experimentally induced neosporosis in sheep.

OBJECTIVE: To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing fetal loss associated with experimentally induced neosporosis in sheep. ANIMALS: 30 Dorset ewes. PROCEDURE: Ewes were randomly allocated to receive vaccination on days 1 and 60 of the study with a killed N caninum tachyzoite preparation in a commercially available adjuvant or a saline-adjuvant mixture. A ram was placed on pasture with the ewes from days 15 to 60. Blood was collected from ewes before primary and booster vaccinations and prior to experimental challenge with N caninum tachyzoite performed on day 90; sera were assessed via Neospora agglutination (NA) and immunofluorescence antibody (IFA) assays. Blood was collected from lambs before they suckled, and sera were tested for antibodies against N caninum. RESULTS: Of the 14 vaccinated ewes that became pregnant, 12 gave birth to live-born lambs; in contrast, 5 of 11 pregnant control ewes gave birth to live-born lambs. Whereas vaccination improved fetal survival in pregnant ewes challenged with N caninum tachyzoites, it did not appear to have any appreciable effect on transmission of N caninum to offspring, as indicated by results of NA and IFA assays. CONCLUSIONS AND CLINICAL RELEVANCE: The N caninum tachyzoite vaccine used in this study appeared to provide protection against fetal loss associated with experimentally induced neosporosis in a high proportion of pregnant ewes.

Agglutination Tests↗
PubMed2000

Association of serologic status for Neospora caninum with postweaning weight gain and carcass measurements in beef calves.

OBJECTIVE: To determine the seroprevalence for Neospora caninum in a population of beef calves in a feedlot and the association of serologic status with postweaning weight gain and carcass measurements. DESIGN: Longitudinal observational study. ANIMALS: 1,009 weaned beef steers from 92 herds. PROCEDURE: Samples were obtained from all steers at time of arrival at a feedlot. Serologic status for Neospora spp was determined, using an agglutination test. Results of serologic testing were compared with calf growth and carcass data, using multivariate regression with generalized estimating equations. RESULTS: Of 1,009 calves, 131 (12.98%) were seropositive, and 54 of 92 (58.7%) consignments had > or = 1 seropositive calf. Median within-consignment prevalence for consignments in which there was > or = 1 seropositive calf was 20%. Seropositive status was associated with significant reductions in average daily gain, live body weight at slaughter, and hot carcass weight and an increase in ribeye area-to-hot carcass weight ratio. Seropositive status also was associated with significant increases in cost of treatment and significant reductions in income. Sick seropositive calves had the highest cost of treatment. An economic loss of $15.62/calf was projected for seropositive calves. CONCLUSIONS AND CLINICAL RELEVANCE: Significant reductions in postweaning weight gain, carcass weight, and economic return were associated with detection of antibodies to N caninum in beef calves in a feedlot.

Agglutination Tests↗
PubMed2001

Evaluation of the embryo transfer procedure proposed by the International Embryo Transfer Society as a method of controlling vertical transmission of Neospora caninum in cattle.

OBJECTIVE: To evaluate efficacy of embryo transfer into seronegative recipients, using the procedure proposed by the International Embryo Transfer Society (IETS), for preventing vertical transmission of Neospora caninum in cattle. DESIGN: Prospective clinical trial. ANIMALS: 87 recipient cows and heifers and their embryo transfer calves from 22 donors originating from 9 dairy herds. PROCEDURE: Neospora caninum serologic status of donors and recipients was determined before collection and transfer of embryos. Viable embryos were washed and treated with trypsin. Recipients in experimental groups A (n = 50) and B (29) were seronegative and received embryos from seropositive and seronegative donors, respectively. Recipients in group C (n = 8) were seropositive and received embryos from seronegative or seropositive donors. Antibody titers against N caninum were determined monthly during pregnancy in recipients and in calf blood samples collected at birth. Tissues collected from stillborn calves and aborted fetuses were analyzed histologically and by immunohistochemical (IHC) methods. RESULTS: 76 calves and 11 fetuses and stillborn calves were examined. All calves from groups A and B were seronegative (n = 70) or lacked evidence of infection by use of tissue analysis (9). In group C, 5 of 6 calves were seropositive at birth, and IHC results were positive for 1 of 2 calves. Vertical transmission rate was significantly lower in groups A and B (0%) than in group C (75%). CONCLUSION AND CLINICAL RELEVANCE: Embryo transfer into seronegative recipients, using the procedure proposed by IETS, is an effective way to prevent vertical transmission of N caninum. Results provide support for pretransfer testing of all embryo transfer recipients.

Animals↗
PubMed2003

Review of Neospora caninum and neosporosis in animals.

Neospora caninum is a coccidian parasite of animals. It is a major pathogen for cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep, and deer. Domestic dogs are the only known definitive hosts for N. caninum. It is one of the most efficiently transmitted parasite of cattle and up to 90% of cattle in some herds are infected. Transplacental transmission is considered the major route of transmission of N. caninum in cattle. Neospora caninum is a major cause of abortion in cattle in many countries. To elicit protective immunity against abortion in cows that already harbor a latent infection is a major problem. This paper reviews information on biology, diagnosis, epidemiology and control of neosporosis in animals.

Animals↗
PubMed2003

Seroprevalence of antibodies to Neospora caninum in dogs and raccoon dogs in Korea.

Neospora caninum is an important cause of abortion in cattle, and dogs are its only known definitive host. Its seroprevalence among domestic urban and rural dogs and feral raccoon dogs (Nyctereutes procyonoides koreensis) in Korea was studied by indirect fluorescent antibody test (IFAT) and by the neospora agglutination test (NAT), respectively. Antibodies to N. caninum were found in 8.3% of urban dogs and in 21.6% of dogs at dairy farms. Antibody titers ranged from 1:50 to 1:400. Antibodies to N. caninum were found in six (23%) of 26 raccoon dogs. However, the potential role of raccoon dogs as a source of horizontal transmission of bovine neosporosis needs further investigation. The results of this study suggest that there is a close relationship between N. caninum infection among dairy farm dogs and cattle in Korea. This study reports for the first time upon the seroprevalence of N. caninum infection in raccoon dogs in Korea.

Animals↗