Camilla Björkman

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.

Immune responses to Neospora caninum and prospects for vaccination

Developing an effective vaccine against neosporosis presents several interesting challenges. The parasite is spread efficiently from mother to foetus over several generations, and naturally infected cattle do not appear to develop adequate protective immunity. Modulation of the immune response during pregnancy favours parasite survival and multiplication. However, induction of pro-inflammatory responses that are thought to be protective against Neospora caninum would be detrimental to the pregnancy. So, is vaccination a feasible option to control the disease? This article discusses some of these issues and reports on the progress towards a vaccine for neosporosis.

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

An indirect enzyme-linked immunoassay (ELISA) for demonstration of antibodies to Neospora caninum in serum and milk of cattle

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from cattle is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen and a mouse monoclonal antibody to bovine immunoglobulin G1 as conjugate. Western blot analysis of the iscom preparation revealed a restricted number of antigens compared with whole parasite homogenates. When probed with a serum from an experimentally infected calf, heavily stained antigens with apparent molecular masses of 28, 35, 45 and 78 kDa were seen. The sensitivity and specificity of the ELISA was 100% and 96%, respectively, against an indirect fluorescent antibody test as indicator of true status. The applicability of the ELISA for demonstration of antibodies in milk was evaluated and the agreement between serum and milk ELISA was 95%.

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.

Neospora caninum in dogs: detection of antibodies by ELISA using an iscom antigen

An indirect enzyme linked immunosorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from dogs is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen. A mixture of a monoclonal antibody to dog immunoglobulin G and a horse radish peroxidase conjugated antibody to mouse Ig was used to detect bound antibody. When the iscom preparation was analysed by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis it appeared to consist of a restricted number of proteins compared with whole parasite homogenates. In immunoblot analysis, using N. caninum positive sera from rabbits and dogs as probes, the major antigens recognized had approximate molecular weights between 30 and 45 and 17 to 19kDa. Compared with an ELISA using a crude solubilized tachyzoite antigen, the iscom ELISA substantially improved the sensitivity and specificity (to 97·6% and 95·6%, respectively, against an immunofluorescence test, IFAT, as indicator of true status). There was a statistically significant positive correlation between IFAT titres and iscom ELISA OD450 values. The iscom ELISA absorbances (and the IFAT titres) of dogs with proven clinical infections were not higher than those from nonclinically affected, putatively infected dogs.

Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in cattle and water buffaloes in southern Vietnam

Serum samples from 200 dairy cattle and 200 beef water buffaloes were collected in southern Vietnam during May to September 1995. The sera were analysed for antibodies to Neospora caninum by an enzyme-linked immunosorbent assay and the indirect fluorescent antibody test, and for antibodies to Toxoplasma gondii by the direct agglutination test. Significant levels of N. caninum antibodies were detected in 5.5% of the cattle sera and in 1.5% of the water buffalo sera. 10.5% of the cattle sera and 3% of the water buffalo sera were found to contain T. gondii antibodies. Two of the cattle sera had both T. gondii and N. caninum antibodies. The present communication is the first to report serological evidence of N. caninum infection in the water buffalo.

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.

Serum antibody profile and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum.

The objective of this study was to record how the antibody levels change over time during pregnancy in dairy cows naturally infected with the protozoan parasite Neospora caninum, and relate this to the reproductive performance. Eighteen cows with antibodies to N. caninum were serum sampled monthly during their first pregnancy and 13 of them were also followed for a second pregnancy. In all, five pregnancies ended in abortion and two in stillbirth. Antibodies to N. caninum in serum were demonstrated by immune stimulating complex enzyme-linked immunosorbent assay (iscom ELISA). The N. caninum antibody titres remained well above the 1:100 cut-off limit for the test used during 2 years in all cows. In the non-aborting cows, mean values of antibody titres to N. caninum rose 1.5-2.5 dilution steps to reach a plateau 4-5 months before parturition, and thereafter decreased from 2 months before parturition. These changes were statistically significant (p < 0.001). The same pattern was seen in the aborting cows. The consistent pattern of rise in antibody titres observed during both pregnancies in all cows indicated a reactivation rather than a reinfection of the parasite at mid-gestation.

Immunization of Cattle with Live Tachyzoites of Neospora caninum Confers Protection against Fetal Death

ABSTRACT Neospora caninum is an obligate intracellular protozoan parasite that causes abortion in cattle. It is normally found as a latent infection controlled by a T-helper-cell type 1 response involving CD4+ cytotoxic T cells and gamma interferon. Cattle may be infected by two different routes: transplacentally as a result of activation of the latent infection in the mother causing congenital infection or abortion and by ingestion of oocysts, which, if it occurs during gestation, can also result in abortion. Here, for the first time, we establish proof that live vaccination protects against fetal death, whereas immunization using whole-tachyzoite lysate in different adjuvants fails to protect against fetal death. Strong antibody responses were induced in all the vaccinated groups, and the quality and magnitude of these responses were similar in the live- and the lysate-vaccinated groups. In contrast, only the group immunized with live tachyzoites had strong cellular and gamma interferon responses prior to challenge, and these responses correlated with protection against fetopathy. These results suggest that a cellular immune response may be important in the mechanisms involved in protection against N. caninum-associated abortions.