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PubMed2006

Temporal distribution and parasite load kinetics in blood and tissues during Neospora caninum infection in mice.

The kinetics of Neospora caninum loads in mice inoculated with NC-Liv or NC-1 isolates were studied. The acute phase was characterized by parasitemia and the detection of parasite DNA in several organs, whereas during the chronic phase, the parasite was detected mainly in the brain. Mice infected with NC-Liv developed clinical signs, showing higher brain parasite burdens than NC-1-infected mice.

Animals↗
PubMed1996

Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay.

A recently described PCR test for the identification of Neospora caninum and Toxoplasma gondii has been further developed and optimized in view of its practicability for routine diagnostic application. The N. caninum-specific PCR was adapted to the diagnostic operating standard of the T. gondii-specific PCR in that the uracil DNA glycosidase system was introduced, which eliminates potential carry-over contaminations of amplified target DNA from previous reactions. Furthermore, both PCR tests were optimized by including a DNA hybridization immunoassay based on the use of the commercially available Gen-eti-k DEIA kit. This assay allowed highly sensitive and specific detection of respective DNA amplification products and thus substantially facilitated the reading and interpretation of the test results.

Animals↗
PubMed1999

Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions.

The routine diagnosis of Neospora caninum abortion is based upon histopathologic changes in fetal tissues and identification of tissue parasites by immunohistochemistry. Confirmation of N. caninum infection by immunohistochemistry has low sensitivity. In the present study, we examined the utility of PCR in detecting N. caninum infection in fetal tissues from spontaneous bovine abortion. DNA was obtained from fresh and formalin-fixed tissues from 61 bovine fetuses submitted for abortion diagnosis. Histopathology and immunohistochemistry determined the true status of N. caninum infection in each fetus. In formalin-fixed paraffin-embedded tissues, PCR detected N. caninum DNA in 13 of 13 true-positive fetuses (100%) and in 1 of 16 true-negative fetuses (6%). In fresh or frozen tissues, PCR detected N. caninum DNA in 10 of 13 true-positive fetuses (77%) and 0 of 11 true-negative fetuses (0%). PCR also detected N. caninum DNA in 6 of 8 fetuses that had typical lesions of N. caninum but were immunohistochemistry negative, indicating a higher sensitivity of PCR in comparison to that of immunohistochemistry. N. caninum DNA was amplified most consistently from brain tissue. PCR detection of N. caninum DNA in formalin-fixed, paraffin-embedded tissues was superior to that in fresh tissues, presumably because of the increased accuracy of sample selection inherent in histologic specimens.

Abortion, Veterinary↗
PubMed2001

Validation of a commercially available monoclonal antibody-based competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to Neospora caninum in cattle.

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody to Neospora caninum, a major cause of bovine abortion. Modifications to the cELISA included capturing native N. caninum antigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a "gold standard" set of 184 cow sera defined by fetal histopathology and N. caninum immunohistochemistry and by maternal N. caninum indirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknown N. caninum status. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknown N. caninum status revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modified N. caninum cELISA provided a simple, rapid, and versatile method to accurately identify N. caninum infection status in cattle using a single cutoff value.

Abortion, Veterinary↗
PubMed2002

Application of real-time fluorescent PCR for quantitative assessment of Neospora caninum infections in organotypic slice cultures of rat central nervous system tissue.

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.

Animals↗
PubMed1995

A survey of the incidence of Neospora caninum infection in aborted and stillborn bovine fetuses in England and Wales.

Selected brains and fetal viscera from 190 aborted or stillborn bovine fetuses submitted to Veterinary Investigation Centres in England and Wales between August 1992 and January 1993 were examined histologically. Non-suppurative inflammation of the brain and/or myocardium and placental cotyledons was identified by light microscopy in 20 (10.5 per cent). An immunocytochemical examination of fixed tissue sections using antisera against Neospora caninum, Toxoplasma gondii and Sarcocystis species revealed positive immunolabelling for N caninum in the brains of eight (4.2 per cent), but no labelling with anti-T gondii or anti-Sarcocystis species antisera was evident. These results suggest that N caninum may be an important cause of reproductive failure in cattle in England and Wales.

Abortion, Veterinary↗
PubMed1999

Herd-specific and age-specific seroprevalence of Neospora caninum in 14 British dairy herds.

All the cattle in 14 dairy herds in England were tested for Neospora caninum-specific antibodies with a commercial ELISA. Three of the herds had had sporadic abortions, eight had had endemic abortions and three had had epidemic abortions associated with N caninum before the study. Of 4295 cattle tested, 17.1 per cent were seropositive and the herd-specific prevalence ranged from 7.3 per cent to 44.8 per cent. No significant effect of either herd size (P = 0.988), endemic (P = 0.869) or epidemic (P = 0.138) patterns of abortion on herd-specific prevalence was found by using logistic regression analysis. There was no evidence in any herd of a significant increase in prevalence with age; the prevalence in seven-to 12-month-old cattle was not significantly different (P > 0.400) from the prevalence in older cattle, except that there was a significantly lower prevalence (P = 0.017) in 13-to 24-month-old cattle. The results of this study are consistent with vertical transmission being the major route of N caninum transmission in these herds.

Abortion, Veterinary↗
PubMed1999

Bovine abortion associated with Neospora caninum in northern Spain.

During 1996, aborted bovine fetuses from dairy herds in norther Spain were examined by histopathological, immunohistochemical and serological methods for evidence of infection by Neospora caninum. Microscopical brain changes consistent with those of neosporosis were detected in 36 of 81 fetuses and specific antibodies (IFAT titres > or = 1/16) in 32 of 63. Eight fetuses with neurological lesions were seronegative, and eight without lesions were seropositive. In 25 of 34 fetal brains with lesions consistent with published descriptions of neosporosis, immunohistochemical analysis revealed the presence of N caninum antigen. Depending on the diagnostic criteria, the prevalence of neosporosis related to bovine abortion was between 32 and 57 per cent of the fetuses submitted and between 33 and 58 per cent of the herds involved. Data on the geographical, seasonal and fetal age distribution of the infection are given for the fetuses examined and the value of the different diagnostic methods is discussed.

Abortion, Veterinary↗
PubMed1999

Evaluation of a commercial ELISA for detecting serum antibody to Neospora caninum in cattle.

A commercially available serum antibody detection enzyme-linked immunosorbent assay for Neospora caninum in cattle was evaluated against an immunofluorescent antibody test (IFAT) by applying it to 397 sera from normal adult cattle, 352 sera from cattle which had recently aborted, and 422 sera from two herds which had a history of N caninum-associated abortions. It was evaluated in two laboratories and showed high reproducibility, repeatability and almost perfect or substantial agreement with the IFAT.

Abortion, Veterinary↗
PubMed1999

Seroprevalence of Neospora caninum and abortion in dairy cows in northern Spain.

The seroprevalence of Neospora caninum infection was estimated from a sample of 889 cattle from 43 dairy herds in three counties in the Asturias region of Spain. The true prevalence of infection was estimated to be 30.6 per cent (95 per cent confidence interval (CI) 27.6 to 33.6). Seropositivity was associated with abortion during the previous year (odds ratio (OR)=3.31, P<0.001) and was slightly higher among purchased cattle (37.6 per cent), than among cattle raised on the farm (29.1 per cent) (P=0.078). Seropositive cows were more likely than seronegative cows to have had a seropositive dam (OR=2.3, P=0.011), suggesting that congenital transmission contributed to about 56 per cent of the infections. Herds with a true seroprevalence above 10 per cent had more dogs on the farm, than herds with a lower prevalence (P=0.032). The ORS relating abortion to seropositivity in individual herds ranged from 0.7 to 19, indicating that some herds experienced few abortions caused by N. caninum, while others experienced more abortions due to the organism. Overall, 38.7 per cent of the abortions were estimated to have been attributable to N. caninum.

Abortion, Veterinary↗
PubMed2001

Neospora caninum in persistently infected, pregnant cows: spontaneous transplacental infection is associated with an acute increase in maternal antibody.

Nine cows which were naturally and persistently infected with Neospora caninum were housed and observed intensively throughout pregnancy. No recrudescence of a latent infection was detected by PCR tests on maternal blood but fetal infection, implying a recrudescence of maternal parasitosis, was associated with a marked increase in maternal antibody. The increase occurred in the second half of pregnancy in five cows which infected their calves, and before mid-pregnancy in one cow which aborted. There was no change in the avidity of the antibody, which remained high and characteristic of long-term infection. In three infected cows that gave birth to uninfected calves there was no marked increase in maternal antibody. Antigen-specific interferon gamma responses of peripheral blood mononuclear cells were observed in all the infected cattle but they did not vary significantly either during pregnancy, or whether the cows did or did not infect their calves, although the responses were consistently higher in the latter. There was no change in the plasma concentrations of cortisol or acute phase proteins associated with the recrudescence of the parasite. Three uninfected cows housed with the infected cows remained uninfected throughout the experiment. No immunosuppressive event was detected which might have provoked parasite recrudescence but the acute antibody rise associated with transplacental infection provides a valuable, non-invasive marker for further studies to investigate the cause and consequences of parasite recrudescence in N caninum infection in cattle.

Animals↗
PubMed2002

Epidemiological investigations of abortions due to Neospora caninum on Swiss dairy farms.

Abortions apparently due to Neospora caninum in two Swiss dairy herds were investigated by means of a PCR and ELISA, and other potential causes were eliminated. In addition, a case-control study of 24 case herds and 24 control herds indicated that N caninum-associated abortions were more likely to occur in herds with antibodies to Coxiella burnetii (with an odds ratio [OR] of 3.38 with a 95 per cent confidence interval [CI] of 1.82 to 6.22), whereas the likelihood was less in herds with antibodies to Leptospira species (OR 0.34,95 per cent CI 0.13 to 0.75), or in herds with antibodies to bovine viral diarrhoea (BVD) virus (OR 0.75,95 per cent CI 0.56 to 1.00), or Chlamydia psittaci (OR 0.18,95 per cent CI 0.09 to 0.35), or in herds in which BVD virus had been isolated from an aborted fetus (OR 0.11,95 per cent CI 0.02 to 0-58).

Abortion, Veterinary↗
PubMed2003

Abortion epidemic in a dairy herd associated with horizontally transmitted Neospora caninum infection.

A dairy herd experienced an abortion epidemic during which 43 per cent of the cows at risk aborted. Neospora caninum infection was demonstrated in four of six fetuses suitable for examination and the group of at-risk cows that aborted had significantly higher N. caninum antibody concentrations than the at-risk cows that delivered a live calf at term (P<0.001). The antibody concentrations in the cow herd were significantly higher than in the youngstock (P<0.001), and the concentrations in the youngstock increased significantly (P<0.001) with age. When seven months to a year old, the calves born at term to the at-risk cows had significantly higher (P=0.007) antibody concentrations than age-matched calves born before the epidemic. At the time of the epidemic, there was a significant increase in the antibody levels of the herd that was not consistent with vertical infection alone, indicating that there appeared to have been a sudden large increase in the incidence of horizontal postnatal transmission of N. caninum to the cow herd, or to the surviving offspring of the at-risk cows, or to both of these groups.

Abortion, Veterinary↗
PubMed2001

Granulomatous encephalitis in a neurologically impaired goat kid associated with degeneration of Neospora caninum tissue cysts.

Congenital Neospora caninum infection was diagnosed in a Saanen goat from a farm in southern Brazil. The kid was unable to nurse and had difficulty rising, ataxia, and opistothotonos. The neurologic signs became more severe 3 days after birth, when it was euthanized. No gross lesions were observed at necropsy. Multifocal infiltrates primarily of mononuclear cells, nodular microgliosis, and perivascular cuffs of lymphocytes, plasma cells, and few neutrophils were observed in the brain, mostly in the cortex and adjacent to ventricles. Rare multinucleate giant cells were observed adjacent to inflammatory foci. Several tissue cysts with a thick wall that reacted strongly with polyclonal antiserum to N. caninum were in the cerebral cortex and medulla oblongata. Lesions were also present in heart, lungs, and liver, but N. caninum tachyzoites were not found. Distinguishing features in this goat kid included neurologic impairment resulting from congenital infection with N. caninum and the presence of granulomatous inflammation with rare giant cells associated with degeneration of tissue cysts.

Animals↗
PubMed2003

Application of the Neospora caninum IgG avidity ELISA in assessment of chronic reproductive losses after an outbreak of neosporosis in a herd of beef cattle.

Point-source infections are most likely the cause for Neospora caninum-induced abortion outbreaks in cattle, whereas an increased annual abortion rate may be a consequence of vertical transmission. The aims of the present study were to examine the reproductive effects of neosporosis in a beef herd for 3 years, after a point-source outbreak and to use IgG avidity serology to examine the chronicity of infections and patterns of transmission. During the study, 76-78% of animals were seropositive for N. caninum. The pregnancy rate varied from 88% to 94%, without any reduction in the pregnancy rate of seropositive cows compared with seronegative cows. The annual abortion rate was 2.5-5.5%, and all but 1 abortion occurred in seropositive dams. The efficiency of vertical transmission was estimated to be 85%. Several calves, born to seronegative dams, were seropositive at 6-13 months of age, indicating a 22% mean annual rate of horizontal transmission. The mean avidity in seropositive cows increased from 30 during the initial outbreak to 74 after 3 years. The mode of IgG avidity was 21-40 during the initial abortion outbreak, 41-60 after 1 year, and 61-80 after 2 and 3 years. The results reveal high annual rates of both vertical and horizontal transmission of N. caninum in a herd of beef cows and provide further validation on the ability of the N. caninum IgG avidity ELISA to accurately assess the chronicity of infection.

Abortion, Veterinary↗
PubMed2004

Agreement between three serological tests for Neospora caninum in beef cattle.

During 1999, serum samples were collected from beef cows on pastures in western Canada. Some of the herds had a history of confirmed abortions associated with Neospora caninum infection. All these samples were initially analyzed using a single application of 1 common commercial enzyme-linked immunosorbent assay (ELISA) for antibodies to N. caninum. From these initial results, 239 positive and 250 negative samples were randomly selected for further testing. This group of samples was retested using the 3 commercially available ELISA tests for N. caninum as per the manufacturer's recommendations. The agreement between 2 of the ELISAs was good (K = 0.76); agreement of these 2 tests with the third test was much lower (K = 0.46 and 0.60). Quantitative agreement between tests measured by intraclass correlation coefficients was also acceptable between the first 2 tests but was almost zero when the first 2 tests were compared with the third. This information is necessary to understand the differences in seroprevalence reported in different regions from laboratories using different methods.

Abortion, Veterinary↗
PubMed2005

Use of avidity enzyme-linked immunosorbent assay and avidity Western blot to discriminate between acute and chronic Neospora caninum infection in cattle.

Avidity serological tests (avidity enzyme-linked immunosorbent assay [ELISA] and avidity Western blot) were developed and used to differentiate between acute (primary infection, reinfection, and recrudescence) and chronic Neospora caninum infection in cattle. In addition, the pattern of immunoglobulin G (IgG) avidity maturation against different specific antigens of N. caninum tachyzoites was studied. Sequential serum samples were collected from cattle naturally and experimentally infected with N. caninum. Four groups of experimentally infected cattle were included in the study and were representative of primary infection, reinfection, chronic infection, and noninfection. Serum samples were also collected from naturally infected cattle classified into nonaborting and aborting cows on the basis of clinical findings and serological profiles, and a third group composed of seronegative cows that seroconverted during the course of the experiment. All samples were tested by avidity ELISA and avidity Western blot. The IgG avidity ELISA allowed the discrimination between primary and chronic infection because all experimentally primary-infection cows showed low avidity indexes at week 4 postinfection (p.i.) compared with the high avidity values found at week 20 postinfection. However, this test did not allow the discrimination of reinfection or recrudescence from chronic infection. Regarding IgG avidity Western blot results, no antigenic markers correlating with acute (primary infection, recrudescence, and reinfection) or chronic infection were recognized. However, the 17-kD immunodominant antigen was mostly responsible for high avidity values obtained by avidity ELISA because it was intensively recognized by high-avidity antibodies in all chronically infected animals after urea treatment.

Abortion, Veterinary↗
PubMed2005

Use of a multiplex polymerase chain reaction to rapidly differentiate Neospora caninum from Toxoplasma gondii in an adult dog with necrotizing myocarditis and myocardial infarct.

This report describes a 3-year-old male castrated Mastiff dog that died unexpectedly with locally extensive, acute, necrotizing myocarditis and myocardial infarction. Intralesional protozoal tachyzoites in the affected myocardium were confirmed to be Neospora caninum by a novel multiplex polymerase chain reaction (PCR) and immunohistochemistry. Protozoal organisms were not identified in other tissues by histology, immunohistochemistry, or PCR. The multiplex PCR assay was used to quickly provide preliminary results on fresh myocardium to differentiate N. caninum and Toxoplasma gondii. Neosporosis is an uncommon cause of myocarditis in adult dogs and differential diagnoses for myocarditis in this population of dogs are reviewed.

Animals↗