The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries.

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

Development of lesions and tissue distribution of parasite in lambs orally infected with sporulated oocysts of Toxoplasma gondii.

The host-pathogen interaction is as a key feature during the formation of tissue cysts of Toxoplasma gondii within intermediate hosts. In this study, we investigated whether oral infection of lambs with T. gondii oocysts may be used as an experimental model in sheep to study this interaction, with the main objective being to detect the presence and distribution of lesions and parasite within different organs at different time points after oral infection. Lambs were infected with 5 × 10(3) and 5 × 10(5) sporulated T. gondii oocysts and culled at 2, 3, 5 and 6 weeks post-infection (WPI). During the infection, rectal temperature of the animals and serological antibodies against T. gondii were monitored. The presence of inflammatory lesions and parasite were evaluated through histological and immunohistochemical methods at different organs (brain, liver, lung, heart and lymph nodes). The lambs showed no clinical signs other than fever, and lesions appeared mainly in the brain, characterized by glial foci and perivascular cuffs, and in the heart, denoted by foci of interstitial myositis. Tissue cysts and tachyzoite-like structures were observed at all time points studied in the brain, where together with the glial foci they appeared mainly in the cerebral cortex of the forebrain and in the midbrain, but also in the heart, lung and lymph nodes. This study shows that oral infection with sporulated oocysts in lambs may provide a model for investigating the host-parasite interaction in situ during the development of tissue cysts.

The development of immune responses in Balb/c mice following inoculation with attenuated or virulent Neospora caninum tachyzoites.

Balb/c mice were inoculated intraperitoneally (i.p.) with either 5 x 10(6) live virulent (group 1) or 5 x 10(6) live attenuated (group 2) tachyzoites, or Vero cells (group 3). Animals were killed at 0, 14, 28 and 42 days post-inoculation (p.i.), with the remaining mice receiving a lethal challenge on day 48 p.i. Serum, spleen and brain samples were collected post-mortem to examine humoral and cell-mediated immune responses as well as pathological lesions and to quantify parasite loads. On day 14 p.i. group 2 (attenuated) demonstrated statistically significant (P < 0.001) lower levels of mean morbidity and weight loss, while also showing significantly (P = 0.01) higher levels of splenocyte proliferation and IFN-gamma production (P = 0.003), compared to group 1 (virulent). Histology of brain samples showed milder lesions and a lower incidence of positive immunohistochemistry, demonstrating tachyzoites and tissue cysts, and statistically significant (P = 0.03) lower mean burdens of parasite DNA in group 2 (attenuated) compared to group 1 (virulent). All mice in group 2 were protected following challenge on day 48 p.i. whereas naïve control mice succumbed to the challenge. No mice from group 1 (virulent) survived beyond day 24 p.i. so they were not included in the challenge.

Inoculation of Balb/c mice with live attenuated tachyzoites protects against a lethal challenge of Neospora caninum.

Neospora caninum tachyzoites attenuated through passage in tissue culture were tested for their ability to induce protective immunity against a lethal challenge dose of parasites. Balb/c mice were each inoculated with either 1x10(6) live virulent tachyzoites (Group 1) or 1x10(6) live attenuated tachyzoites (Group 2), while (Group 3) received a control inoculum. All mice were each challenged 28 days later with 5x10(6) virulent parasites. Histopathological lesions in the brains including necrosis and microgliosis were observed following post-mortem on day 28 post-challenge (p.c.) in 71% of Group 1 and 56% of Group 2. Immunohistochemistry (IHC) of these lesions showed tachyzoites and Neospora antigens to be associated with moderate brain lesions in 17% of Group 1, while in 11% of Group 2 N. caninum tissue cysts were detected, but these were not associated with lesions, Parasite DNA was detected by PCR in the brains of 86% of mice in Group 1 and 56% of mice in Group 2. Following challenge the mice in Group 3 showed high morbidity and 100% mortality within 17 days p.c. Positive IHC for N. caninum was seen in 88% of the Group 3 mice and parasite DNA was detected in all brain samples. This study shows that it is possible to protect against a lethal challenge of N. caninum through inoculation with attenuated or virulent tachyzoites. However, more severe pathology developed in mice initially inoculated with virulent parasites following a secondary challenge, compared to mice initially inoculated with attenuated parasites.

Ovine toxoplasmosis: transmission, clinical outcome and control.

Toxoplasma gondii is a significant cause of abortion in sheep. Infection is picked up from the environment and if initiated during pregnancy may cause fetal mortality. Infected sheep remain persistently infected with tissue cysts in brain and muscle (meat), and are also immune and would not be expected to abort again. The live tachyzoite vaccine (Toxovax) protects against abortion and this allows the suggestion that it may also reduce or prevent tissue cyst development in muscle. If this were so it raises the question of whether the vaccine could be used to make meat safer for human consumption.

Long-term passage of tachyzoites in tissue culture can attenuate virulence of Neospora caninum in vivo.

To determine whether prolonged in vitro passage would result in attenuation of virulence in vivo, Neospora caninum tachyzoites were passaged for different lengths of time in vitro and compared for their ability to cause disease in mice. Groups of Balb/c mice were inoculated intraperitoneally with 5 x 10(6) or 1 x 10(7) of low-passage or high-passage N. caninum tachyzoites. The mice were monitored for changes in their demeanour and body weight, and were culled when severe clinical symptoms of murine neosporosis were observed. Mice inoculated with the high-passage parasites survived longer (P<0.05), and showed fewer clinical symptoms of murine neosporosis, compared to the mice receiving the low-passage parasites. The parasite was detected in the brains of inoculated mice using immunohistochemistry and ITS1 PCR. Tissue cysts containing parasites were seen in mice inoculated with both low-passage and high-passage parasites. When the in vitro growth rates of the parasites were compared, the high-passage parasites initially multiplied more rapidly (P<0.001) than the low-passage parasites, suggesting that the high-passage parasites had become more adapted to tissue culture. These results would suggest that it is possible to attenuate the virulence of N. caninum tachyzoites in mice through prolonged in vitro passage.

Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora.

The protozoan parasites Eimeria spp. Toxoplasma gondii and Neospora caninum are significant causes of disease in livestock worldwide and T. gondii is also an important human pathogen. Drugs have been used with varying success to help control aspects of these diseases and commercial vaccines are available for all three groups of parasites. However, there are issues with increasing development of resistance to many of the anti-coccidial drugs used to help control avian eimeriosis and public concerns about the use of drugs in food animals. In addition there are no drugs available that can act against the tissue cyst stage of either T. gondii or N. caninum and thus cure animals or people of infection. All three groups of parasites multiply within the cells of their host species and therefore cell mediated immune mechanisms are thought to be an important component of host protective immunity. Successful vaccination strategies for both Eimeria and Toxoplasma have relied on using a live vaccination approach using attenuated parasites which allows correct processing and presentation of antigen to the host immune system to stimulate appropriate cell mediated immune responses. However, live vaccines can have problems with safety, short shelf-life and large-scale production; therefore there is continued interest in devising new vaccines using defined recombinant antigens. The major challenges in devising novel vaccines are to select relevant antigens and then present them to the immune system in an appropriate manner to enable the induction of protective immune responses. With all three groups of parasites, vaccine preparations comprising antigens from the different life cycle stages may also be advantageous. In the case of Eimeria parasites there are also problems with strain-specific immunity therefore a cocktail of antigens from different parasite strains may be required. Improving our knowledge of the different parasite transmission routes, host-parasite relationships, disease pathogenesis and determining the various roles of the host immune response being at times host-protective, parasite protective and in causing immunopathology will help to tailor a vaccination strategy against a particular disease target. This paper discusses current vaccination strategies to help combat infections with Eimeria, Toxoplasma and Neospora and recent research looking towards developing new vaccine targets and approaches.

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.

A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera.

Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle. The epidemiology of neosporosis is poorly understood, partly because accurate diagnosis of infection is difficult. In this paper we describe the development of a multiple antigen-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies to N. caninum in sera from cattle, sheep and goats as well as from bovine foetal fluids. A water-soluble fraction (wsf) of sonicated NC-1 strain tachyzoites was used as the antigen in the ELISA. Minimum optical density (OD) values that were considered to be Neospora antibody-positive, that is, the cut-off OD values were determined separately for bovine maternal sera, bovine foetal fluids, ovine sera and caprine sera; they were 0.40, 0.17, 0.23 and 0.41 OD, respectively. The ELISA gave a high signal/noise ratio, giving good sensitivity and specificity, correlating well with the indirect fluorescent antibody test (IFAT) currently used to diagnose Neospora infection in cattle, sheep and goats. In both the ELISA and immunoblot analysis using the same antigen, there was no significant cross-reactivity with sera from cattle, sheep or goats that had been infected with Toxoplasma gondii. The ELISA also showed no cross-reactivity in sera from cattle infected with Sarcocystis cruzi, Babesia divergens, B. bovis and B. bigemina. The wsf fraction of sonicated N. caninum tachyzoites used in this ELISA can be easily prepared and may be more sensitive than a single antigen ELISA, whilst still retaining good specificity.

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.

Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva.

Infection and transformation of cells of the bovine immune system by Theileria annulata and T. parva were compared. Preliminary experiments with mammary gland macrophages indicated that they were permissive to infection by T. annulata but only to a limited extent by T. parva. Further experiments involved several purified subpopulations of bovine cells including bovine monocytes, T cells and MHC class II positive and negative populations. These subpopulations were incubated with T. annulata or T. parva sporozoites in limiting dilution cultures. T. annulata preferentially infected macrophage type cells and also MHC class II positive cells, whereas the frequency of MHC class II negative cells infected by this parasite was negligible. T cells also showed a very low level of infection. In complete contrast, T. parva preferentially infected T cells and did not infect cells phenotypically defined as monocytes at all. These results suggested that class II expression was necessary for T. annulata infection and not necessary for, though not a barrier to T. parva infection. T. annulata infected cell lines all expressed class II molecules to varying degrees. Other available phenotypic markers were only expressed at very low levels or no longer expressed. The immunological significance of the different cell preferences and phenotypes of infected cell lines of T. annulata and T. parva is discussed.

The development and specificity of cytotoxic cells in cattle immunized with autologous or allogeneic Theileria annulata-infected lymphoblastoid cell lines.

Two groups of animals were immunized with either 10(6) autologous or 10(6) allogeneic Theileria annulata-infected lymphoblastoid cells cultured in vitro. The development and specificity of cytotoxic cells generated in vivo were measured throughout immunization and challenge using a panel of target cells that were either Theileria-infected or uninfected blast cells of known bovine lymphocyte antigen (BoLA) specificities. After inoculation of the cell lines the two groups showed distinct differences in both their clinical responses and the target specificity of the cytotoxic cells detected. The allogeneic T. annulata cell line recipients showed a very mild clinical response, and on day 9 after inoculation a strong cytotoxic response was detected. The response appeared to be directed against the allogeneic major histocompatibility complex (MHC) antigens of the inoculated cell line in some form of graft rejection response. By day 23 the predominant cytotoxic response was directed against the recipient animals' own cells infected with the parasite. In contrast, the autologous T. annulata cell line recipients showed very severe clinical reactions, and low levels of cytotoxicity were detected. The cytotoxicity was directed against parasite-infected targets but did not appear to be MHC restricted until day 20. Both groups were immune to a heterologous sporozoite challenge that proved lethal to two susceptible control animals, and on day 10 after challenge a peak of cytotoxicity was detected which was directed against the autologous infected target cell. This would suggest that this cytotoxic response was MHC restricted and was also cross-reactive between the heterologous parasite stocks used.

Bovine alloreactive cytotoxic cells generated in vitro detect BoLA w6 subgroups.

Alloreactive cytotoxic T cells (CTL) were generated in mixed lymphocyte culture against cells bearing subgroups of BoLA w6, as well as in BoLA w4, w10 and w16. Primary cultures were restimulated at weekly intervals with irradiated stimulator cells and tested in a 51Cr-release assay with target cells derived from Theileria annulata-infected cell lines. Generation of CTL was accelerated in animals that had been previously primed in vivo by skin grafting. CTL were generated that were specific for BoLA w6 subgroups and not w6. With w6.1 the specific killing was significant at the 5% level, and with w6.2, 6.3 and 6.4 it was significant at the 0.1% level. Where CTL were potentially generated against two BoLA-A locus allele products at the same time (i.e. with heterozygous stimulator cells), one of which was a w6 subgroup, there was similar CTL activity against both products when w6.4 and w16 or w6.1 and w4 were the combinations involved. In two generations against w10 and either w6.1 or w6.2 there was significantly more killing of w10-bearing targets than those with the w6 subgroup.