Characterization of the immune response in the placenta of cattle experimentally infected with Neospora caninum in early gestation.

A serial examination of three groups of cattle infected intravenously (iv) (Group 1, n=8) or subcutaneously (sc) (Group 2, n=8) with live Neospora caninum tachyzoites or with VERO cells (Group 3, n=8) at 70 days' gestation was carried out and the nature of the inflammatory responses in the placenta and the presence of parasite antigen were analysed. Immune cells expressing CD3, CD4, CD8, gamma delta (gammadelta) T-cell receptors (TCR), CD79alpha cytoplasmic (cy) (B cells) and NKp46 [natural killer (NK) cells] antigens were identified immunohistochemically and cells expressing mRNA for interferon-gamma (IFN-gamma) were labelled by in-situ hybridization. Intravenous inoculation caused mortality in all fetuses from 28 days post-inoculation (dpi) onwards. Subcutaneous inoculation caused mortality in 50% of the animals by 28dpi. Pathological changes in the placenta consisted of necrosis of fetal placental villi, necrosis and inflammation in adjacent areas of the maternal septum and inflammation at the base of the maternal caruncle. The inflammatory infiltrate consisted mainly of CD3(+) lymphocytes, dominated by CD4(+) and gammadelta TCR(+) cells, with CD8(+) cells present to a lesser extent. The results from the control group indicated fewer NK cells than those occurring in the placenta of human beings or mice. Infiltration of CD4(+) cells and NKp46(+) cells was observed in the caruncular base and septa 14 days after infection, whereas infiltration of gammadelta TCR(+) cells was observed from 28 dpi onwards. To our knowledge this is the first report on the presence and distribution of NK cells in the bovine placenta. Maternal inflammatory cells expressing mRNA for IFN-gamma were identified in animals inoculated with parasites iv or sc at 14 and 28 dpi, respectively. In the sc-inoculated dams with live fetuses at 28, 42 and 56dpi, there was no evidence of parasite antigen, infiltration of immune cells or production of IFN-gamma, suggesting that the parasite had not reached the placenta. The exact cause of fetal death was not established. Tissue destruction by the parasite may have occurred; in addition, there may have been a T helper 1 (Th-1) immune response to the neospora infection at the materno-fetal interface, resulting in infiltrations of CD4T cells, gammadelta T cells and NK cells and the subsequent production of IFN-gamma. It is possible that a pro-inflammatory Th-1 response early in gestation protects the dam by eliminating the parasite; however, it may lead to destruction of the placental tissues themselves and thus be incompatible with fetal survival.

An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues.

Seven European laboratories contributed to a multi-centre evaluation of detection techniques for Neospora caninum in bovine foetuses. Six laboratories participated in immunohistochemistry (IHC) testing. All seven laboratories participated in PCR testing, but the results from one laboratory were not included in the analysis, because of contamination problems in the preparation of the samples. A coded panel of tissue sections from 36 infected and non-infected foetuses was used to evaluate the IHC detection of parasites. A coded panel consisting of 44 homogenized foetal brain samples from natural bovine abortion cases and 32 spiked samples were used to evaluate the PCR methods. Inclusion of a duplicate dilution series of spiked samples was used to evaluate detection limits and repeatability. IHC methods had a relatively low sensitivity, but a high specificity. There was considerable variation in IHC results between participating laboratories, which may be partly explained by examination practices that depended on the experience of the operator. In addition, the use of different antibody reagents, different antibody dilutions, and different enzymatic treatments of tissues may have contributed to the observed variation. PCR methods generally had a higher sensitivity than IHC methods and also a high specificity. The agreement between the majority scores of IHC and PCR methods was low. False positive PCR results indicated contamination problems in some instances. Agreement between the PCR results of the various laboratories was better, compared with the IHC results. There appeared to be no clear relationship between the PCR format (i.e. single or nested) and diagnostic sensitivity. Consequently, an improvement of diagnostic performance of PCR might possibly be achieved by optimizing DNA extraction methods.

Placental pathology associated with fetal death in cattle inoculated with Neospora caninum by two different routes in early pregnancy.

Pregnant cattle were inoculated with N. caninum strain NC-1 tachyzoites intravenously (iv) (group 1, n = 8) or subcutaneously (sc) (group 2, n = 8) at 70 days' gestation. Control animals (group 3; n = 8) received uninfected Vero cells iv. Two animals from each group were killed at 14, 28, 42 and 56 days post-inoculation (dpi). Fetal mortality was 100% and 50%, respectively, in groups 1 and 2 from 28 dpi. In group 1 foci of degenerative fetal placental villi were observed at 14 dpi, with clusters of N. caninum tachyzoites in the affected mesenchyme. There was also inflammation of maternal septal tissues, with necrotic cell debris and serum exudate at the interstitium. At 28 dpi pregnancy had ended and the fetal cotyledons had become detached from the maternal caruncles. Immunohistochemically, particulate N. caninum antigen was detected in the cotyledons. At 42 and 56 dpi, fetal tissues had disappeared, the caruncles were greatly reduced in size, and the uterine epithelium had been largely restored. In group 2, lesions were either severe or absent ("all or nothing" response). In one animal carrying a dead fetus at 28 dpi, placentitis was much more severe than that seen in group 1 at 14 dpi. Lesions contained neutrophils, eosinophils and N. caninum antigen. In animals carrying dead fetuses at 42 and 56 dpi, fetal remains were found and the cotyledons contained N. caninum antigen. Antigen was also detected in fetal tissues. No significant pathological changes were detected in group 2 animals carrying live fetuses or any animal in group 3. Thus, N. caninum administered iv or sc in early pregnancy resulted in rapid fetal death, with parasite-associated lesions in the placenta and fetus. Of the two inoculation routes, the intravenous induced the more acute placental lesions and greater mortality.

Comparison and standardisation of serological methods for the diagnosis of Neospora caninum infection in bovines.

Various existing serological tests were compared with a standard panel of 523 sera in a multicentred study across Europe. Well characterised sera from animals that were experimentally or naturally infected with Neospora caninum as well as sera from cattle deemed uninfected with N. caninum were provided by the participants of the study and analysed in several commercial (CHEKIT Dr. Bommeli/Intervet, CIVTEST BOVIS NEOSPORA Hipra, Cypress Diagnostics C.V., Herd Check IDEXX, Mastazyme MAST Diagnostics, P38-ELISA Animal Welfare and Food Safety GmbH (AFOSA)) as well as in-house assays (five ELISAs and one IFAT). Most tests showed a high level of agreement in the interpretation of the test results (positive or negative). A further distinct increase in agreement between tests was obtained after the application of standardised cut-offs offered by a two-graph receiver operating characteristic analysis. This procedure allows a standardised interpretation of results obtained with different tests used in independent, parallel seroepidemiological studies.

Maternal and fetal immune responses of cattle inoculated with Neospora caninum at mid-gestation.

The humoral and cell-mediated immune responses of pregnant cattle and their fetuses were examined at intervals after infection with Neospora caninum tachyzoites at mid-gestation (day 140). All cattle seroconverted and interferon gamma was detected in supernatants of peripheral blood mononuclear cells stimulated with specific antigen. At day 14 post-inoculation (pi), specific cell proliferation responses were detected in the lymph node draining the site of inoculation and in the uterine lymph node. The peak response was recorded in the majority of maternal lymph nodes by day 28 pi and cells from the maternal retropharyngeal lymph node, which in part drains the central nervous system, showed no specific activity to N. caninum until day 42 pi. This changing pattern of immune responsiveness may reflect parasite invasion and development within different host tissues. Fetal lymph node cells showed mitogen responsiveness from day 14 pi (day 154 of gestation) and also showed N. caninum-specific cell proliferation and interferon-gamma responses by day 28 pi (day 168 of gestation). At day 42 pi, specific cell-mediated immune responses were not apparent; however, N. caninum-specific fetal IgG and IgM antibodies were detected.

The pathogenesis of neosporosis in pregnant cattle: inoculation at mid-gestation.

To investigate the pathogenesis of bovine neosporosis, 14 pregnant cattle were each inoculated subcutaneously with either 10(7) or 5 x 10(8) Neospora caninum (strain NC1) tachyzoites at 140 days' gestation. Serial necropsies were then carried out over an 8-week period. In the placenta, Neospora DNA and histopathological changes were observed in samples taken 14 days post-inoculation (dpi), with focal necrosis of maternal caruncular septa and fetal placental villi, serum leakage, and a maternal and fetal inflammatory response. At subsequent samplings, pathological changes in the placenta showed signs of resolution. No parasitaemia was detected in the dams in the two weeks following inoculation. In the fetus, Neospora DNA was detected at 14 dpi, and histopathological changes in the fetal central nervous system at 28 and 42 dpi consisted of small foci of necrosis and inflammation. Resolution of placental lesions during the experiment indicated that the disease was being controlled, and fetal infection, although established, did not appear to be progressing to a fatal outcome. The two doses of tachyzoites produced similar results, but the higher dose elicited earlier and more extensive lesions in the placenta and fetus. Control animals remained negative for all parameters recorded. It is concluded that in bovine neosporosis the placenta plays a central role in the pathogenesis and epidemiology of the infection, and that while primary tissue destruction by the parasite may endanger the fetus, the maternal and fetal inflammatory responses may also be damaging.

Detection of T. gondii in tissues of sheep and cattle following oral infection.

It has been reported in the literature that cattle are more resistant to toxoplasmosis than sheep. Congenital disease due to T. gondii infection is rarely reported in cattle whereas the parasite is a major cause of abortion and neonatal mortality in sheep. It is believed that sheep remain chronically infected for life. Undercooked meat from infected sheep is an important source of infection for man. In contrast cattle are thought to harbour fewer parasite tissue cysts which may not persist for the lifetime of the host. Therefore, cattle are believed to pose less of a risk for human infection. In this study we examined the presence of T. gondii within a range of tissues in sheep and cattle at 6 weeks and 6 months following oral infection with 10(3) or 10(5) sporulated oocysts of T. gondii. The presence of parasite was determined by bioassay in mice and using polymerase chain reaction (PCR). The results from this study show that T. gondii was more frequently and consistently detected in sheep, in particular within brain and heart tissues, whereas parasites were not detected in the samples of tissues taken from cattle. T. gondii was more frequently detected in sheep given the higher dose of T. gondii. Examination of tissues at either 6 weeks or 6 months after infection did not appear to affect the distribution of T. gondii. The polymerase chain reaction has more specificity and sensitivity when detecting the presence of T. gondii in large animals than histological detection.

Detection of Toxoplasma gondii in tissues of sheep orally challenged with different doses of oocysts.

The presence of Toxoplasma gondii in blood, brain, cardiac muscle and skeletal muscle (gracillis and psoas) of sheep 6 weeks after experimental infection with 10(5), 10(4) and 10(3) T. gondii oocysts was determined using the PCR technique. The study demonstrates that oral infection of sheep with T. gondii oocysts of the M3 isolate results in parasites being detectable in tissues 6 weeks p.i. The PCR detection was much more sensitive than histological detection. Parasite DNA was detected more frequently and consistently in the group of sheep given 10(5) oocysts compared with those given 10(3) oocysts. The brain and heart were most frequently infected compared with the other tissues.

Toxoplasma gondii infection in sheep and cattle.

Toxoplasma gondii is a protozoan parasite that can infect all warm-blooded animals. Sheep and cattle show different susceptibilities to T. gondii infection. Primary infection in pregnant sheep can result in abortion or the birth of weak lambs but they are then protected against further challenge by the development of an effective immunity. Cattle on the other hand can be readily infected, but abortion or perinatal mortality have not been recorded. The evidence suggests that cattle develop a more effective immune response to T. gondii infection than sheep. Potential mechanisms to explain these differences are discussed in this paper.