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.

Prevalence and Genetic Diversity of Toxoplasma gondii in Free-Ranging Chickens from the Caribbean.

PURPOSE: Toxoplasma gondii is a zoonotic parasite capable of infecting a wide range of hosts. Free-range chickens are important sentinels in the epidemiology of this parasite as they feed from the ground and are likely to ingest oocysts shed in the faeces of infected cats. Atypical strains of T. gondii are known to dominate in South America where they are associated with more severe disease in humans, yet relatively little is known about the strains circulating in neighbouring Caribbean islands. METHODS: In this study, hearts and brains were collected from free-range chickens in Antigua and Barbuda (n = 45), Dominica (n = 76) and Trinidad (n = 41), and DNA was extracted for nested ITS1 PCR and PCR-RFLP. Sera were collected and screened for antibodies using the modified agglutination test (MAT). RESULTS: Antibodies to T. gondii were detected in 20.5, 38.2 and 17.1% of chickens in Antigua and Barbuda, Dominica and Trinidad, respectively. Toxoplasma gondii DNA was also detected by PCR in 24.4, 17.1 and 17.1% of chickens, respectively, giving an overall prevalence of 31.1, 42.1, and 29.3% for each of the 3 island nations. Results of PCR-RFLP revealed 2 new atypical genotypes (designated ToxoDB #281 and #282) and one Type III (ToxoDB #2) in chickens from Antigua. Partial genotyping of a further 8 isolates (7 from Antigua and one from Trinidad) revealed different allele-types at five or more markers for 7 of the isolates, suggesting atypical genotypes. CONCLUSIONS: This is the first study to report the prevalence of T. gondii in free-range chickens in Antigua and Barbuda, Dominica and Trinidad and Tobago. It is also the first to report the presence of atypical genotypes in Antigua and Barbuda and Trinidad and Tobago.

Detection of Neospora caninum DNA in cases of bovine and ovine abortion in the South-West of Scotland.

Neospora caninum is a commonly diagnosed cause of reproductive losses in farmed ruminants worldwide. This study examined 495 and 308 samples (brain, heart and placenta) which were collected from 455 and 119 aborted cattle and sheep fetuses, respectively. DNA was extracted and a nested Neospora ITS1 PCR was performed on all samples. The results showed that for bovine fetuses 79/449 brain [17.6% (14.2-21.4)], 7/25 heart [28.0% (12.1-49.4)] and 5/21 placenta [23.8% (8.2-47.2)] were PCR positive for the presence of Neospora DNA. Overall 82/455 [18.0% (14.6-21.7)] of the bovine fetuses tested positive for the presence of N. caninum DNA in at least one sample. None (0/308) of the ovine fetal samples tested positive for the presence of Neospora DNA in any of the tissues tested. The results show that N. caninum was associated with fetal losses in cattle (distributed across South-West Scotland), compared to sheep in the same geographical areas where no parasite DNA was found. Neospora is well distributed amongst cattle in South-West Scotland and is the potential cause of serious economic losses to the Scottish cattle farming community; however, it does not appear to be a problem amongst the Scottish sheep flocks.

Molecular detection of Sarcocystis lutrae in the European badger (Meles meles) in Scotland.

Neck samples from 54 badgers and 32 tongue samples of the same badgers (Meles meles), collected in the Lothians and Borders regions of Scotland, were tested using polymerase chain reactions (PCRs) directed against the 18S ribosomal DNA and the internal transcribed spacer (ITS1) region of protozoan parasites of the family Sarcocystidae. Positive results were obtained from 36/54 (67%) neck and 24/32 (75%) tongue samples using an 18S rDNA PCR. A 468 base pair consensus sequence that was generated from the 18S rDNA PCR amplicons (KX229728) showed 100% identity to Sarcocystis lutrae. The ITS1 PCR results revealed that 12/20 (60%) neck and 10/20 (50%) tongue samples were positive for Sarcocystidae DNA. A 1074 bp consensus sequence was generated from the ITS1 PCR amplicons (KX431307) and showed 100% identity to S. lutrae. Multiple sequence alignments and phylogenetic analysis support the finding that the rDNA found in badgers is identical to that of S. lutrae. This parasite has not been previously reported in badgers or in the UK. Sarcocystis lutrae has previously only been detected in tongue, skeletal muscle and diaphragm samples of the Eurasian otter (Lutra lutra) in Norway and potentially in the Arctic fox (Vulpes lagopus).

Development of a sensitive method to extract and detect low numbers of Cryptosporidium oocysts from adult cattle faecal samples.

Cryptosporidium transmission studies to date have concluded that adult cattle are not a significant source of oocysts contributing to clinical cryptosporidiosis in calves on farm. However current methods of sample processing have been optimised for calf faecal samples and may be less sensitive when used on adult samples due to lower numbers of oocysts and larger size of samples. A modified and novel method of oocyst extraction and concentration was developed and applied in an experiment involving spiking adult cattle faecal samples with known concentrations of Cryptosporidium oocysts. The results showed an increased sensitivity of detection from 100oocysts/g of faecal sample using conventional protocols to 5oocysts/g using the newly developed method. As it is important to be able to accurately assess the contribution of adult ruminants to the transmission of Cryptosporidium, both on farm and in the environment, the development of the techniques described here is likely to make an important contribution to Cryptosporidium transmission studies in future and in subsequent control strategies aimed at the reduction of Cryptosporidium infection in calves on farm.

Cell mediated immune responses in the placenta following challenge of vaccinated pregnant heifers with Neospora caninum.

The aim of the present study was to investigate and correlate the cell-mediated immune response and pathological changes at the maternal-fetal interface of Neospora-challenged pregnant cattle previously immunized with live and inactivated experimental vaccines. Pregnant heifers naïve to Neospora caninum were divided in 5 groups of 4 animals, each one immunized before mating: Group A heifers were intravenously (iv) immunized with 6.25 × 10(7) live tachyzoites of the NC-6 strain; group B heifers were immunized twice subcutaneously (sc) 3 weeks apart with native antigen extract of the NC-6 strain formulated with ISCOMs; group C heifers were sc immunized twice 3 weeks apart with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) of the NC-1 strain formulated with ISCOMs; group D heifers were sc injected with sterile phosphate-buffered saline (PBS) and group E heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv-challenged with 4.7 × 10(7) NC-1 tachyzoites at 70 days of gestation. Heifers were culled at day 104 of gestation and placentomes were examined to evaluate lesions and local cellular immune responses using histopathology, immunohistochemistry and real time-PCR. Immunohistochemistry was performed using bovine leucocyte specific antibodies. Cytokine expression and levels (IFN-γ, IL-4, IL-10, IL-12 and TNF-α) were measured using real-time reverse transcription-PCR and ELISA, respectively. Minimal inflammation was observed in group A placentomes; while placentomes from group B, C, D and E had moderate to severe infiltration with CD3(+), CD4(+), γδ-T cells, CD8(+) cells and macrophages being more numerous in groups B and E placentomes, when compared with groups C and D (P<0.001). Cytokine levels were significantly increased in the caruncles of animals of groups B and C in comparison with the other animal groups (P < 0.001). The results from this study showed that the strongest cellular immune responses were observed in the placentomes of animals that were immunized with inactivated vaccines (groups B and C) and in the placentomes of animals that were sc-sham-inoculated (groups D and E). On the other hand, animals that were immunized with live tachyzoites showed a milder immune cell infiltration to the placenta possibly due to the existence of a protective systemic maternal immune response that helped to minimize N. caninum infection at the maternal-fetal interface.

Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian carnivores in the UK.

Toxoplasma gondii is a zoonotic pathogen defined by three main clonal lineages (types I, II, III), of which type II is most common in Europe. Very few data exist on the prevalence and genotypes of T. gondii in the UK. Wildlife can act as sentinel species for T. gondii genotypes present in the environment, which may subsequently be transmitted to livestock and humans. DNA was extracted from tissue samples of wild British carnivores, including 99 ferrets, 83 red foxes, 70 polecats, 65 mink, 64 badgers and 9 stoats. Parasite DNA was detected using a nested ITS1 PCR specific for T. gondii, PCR positive samples were subsequently genotyped using five PCR-RFLP markers. Toxoplasma gondii DNA was detected within all these mammal species and prevalence varied from 6·0 to 44·4% depending on the host. PCR-RFLP genotyping identified type II as the predominant lineage, but type III and type I alleles were also identified. No atypical or mixed genotypes were identified within these animals. This study demonstrates the presence of alleles for all three clonal lineages with potential for transmission to cats and livestock. This is the first DNA-based study of T. gondii prevalence and genotypes across a broad range of wild British carnivores.

Detection of Neospora caninum in wild carnivorans in Great Britain.

Samples of brain and other tissues were collected from 99 ferrets (Mustela furo), 83 red foxes (Vulpes vulpes), 70 European polecats (Mustela putorius), 65 American mink (Neovison vison), 64 Eurasian badgers (Meles meles) and 9 stoats (Mustela erminea), from around Great Britain. DNA was extracted from approximately 1g of tissue and tested by specific nested ITS1 PCR for Neospora caninum. The results from the PCR demonstrated that Neospora specific DNA was detected in all species of wild carnivorans with the exception of the stoats (0/9). Neospora DNA positive samples were detected in: polecats 18.6% (13/70), badgers 10.9% (7/64), ferrets 10.1% (10/99), foxes 4.8% (4/83) and mink 4.6% (3/65). In the badgers N. caninum DNA positive samples were found in brain (n=2), liver (n=2) and neck muscle (n=3). Selected positive ITS1 DNA sequences were submitted to Genbank. Sequence UKwildlife1 (accession number JX857862) was found in two badgers, whilst UKwildlife2 and UKwildlife3 (accession numbers JX857863 and JX857864 respectively) were found in ferrets, all three sequences demonstrated point mutations at a single base, while sequence UKwildlife4 (accession number JX857865) was found in all the species that tested positive and showed complete identity when compared against published reference sequences for: N. caninum (Nc Liverpool isolate, EU564166). Our data shows that almost all the wild carnivoran mammal species tested are intermediate hosts for N. caninum and are therefore capable of acting as reservoirs of infection for other species. These species could also act as useful sentinel species, demonstrating the presence of the parasite in particular geographical and environmental locations.

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.

A brief history and overview of Toxoplasma gondii.

Toxoplasma gondii was discovered by scientists working in North Africa and Brazil around 100 years ago. The parasite has since been found to be capable of infecting all warm-blooded animals including humans making it one of the most successful parasitic organisms worldwide. The pathogenic potential of T. gondii was recognized in the 1920s and 1930s, in congenitally infected children presenting with the classic triad of symptoms, namely hydrocephalus, retinochoroiditis and encephalitis. In addition, around the same time T. gondii parasites were found to be associated with severe intraocular inflammation. In the 1980s, T. gondii emerged as a major cause of death in patients with acquired immunodeficiency syndrome, illustrating the importance of the immune system in controlling T. gondii infection. T. gondii was reported as a major cause of abortion in sheep in New Zealand in the 1950s, which raised questions about potential new transmission routes for the parasite. The discovery of the cat as the definitive host in the 1960s was a very important finding as it helped to complete our understanding of the parasite's life cycle, and the oocyst stage of T. gondii shed in the faeces of infected cats was found to be an important source of infection for many intermediate hosts and helped to explain infection in herbivorous animals and people with a vegetarian diet. In addition, this stage of the parasite was very robust and could survive in the environment, depending on the climatic conditions, for up to 12-18 months. Knowledge of the parasite's life cycle, transmission routes, risk groups and host immune responses has helped in the development of strategies to control the disease, reduce transmission of the parasite and limit environmental contamination.

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.

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.

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.

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.

Intranasal immunisation with Toxoplasma gondii tachyzoite antigen encapsulated into PLG microspheres induces humoral and cell-mediated immunity in sheep.

Proteins from a crude extract of Toxoplasma gondii tachyzoites were encapsulated into poly(D,L-lactide-co-glycolide) (PLG) micro- and nano-particles with a mean encapsulation efficiency of 80%. An intranasal immunisation and infection experiment using 24 sheep was conducted to compare the immune responses elicited by intranasal administration of soluble and particulate T. gondii antigen (with and without cholera toxin). Sheep immunised with particulate toxoplasma antigen produced enhanced levels of both local and systemic antigen-specific IgA antibody, and showed increased cellular immune responses with a corresponding increase in IFNgamma production. After challenge with toxoplasma oocysts larger quantities of both nasal and systemic IgG were measured more rapidly in all animals immunised with toxoplasma antigen than animals infected with oocysts, suggesting a secondary-type IgG response. A slight modification of the febrile response to toxoplasma infection could be observed in animals immunised with particulate toxoplasma antigen and cholera toxin, although none of the immunised animals were protected against the challenge infection. These studies show that intra-nasal delivery has the potential to be an effective route for mucosal immunisation in sheep.

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.

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.