Early immune responses and parasite tissue distribution in mice experimentally infected with oocysts of either archetypal or non-archetypal genotypes of Toxoplasma gondii.

In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 8­11 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence.

Detection and dissemination of Toxoplasma gondii in experimentally infected calves, a single test does not tell the whole story.

BACKGROUND: Although the detection of Toxoplasma gondii in bovine tissues is rare, beef might be an important source of human infection. The use of molecular techniques, such as magnetic capture qPCR (MC-qPCR), in combination with the gold standard method for isolating the parasite (mouse bioassay), may increase the sensitivity of T. gondii detection in infected cattle. The risk of transmission of the parasite to humans from undercooked/raw beef is not fully known and further knowledge about the predilection sites of T. gondii within cattle is needed. In the current study, six Holstein Friesian calves (Bos taurus) were experimentally infected with 106 T. gondii oocysts of the M4 strain and, following euthanasia (42 dpi), pooled tissues were tested for presence of the parasite by mouse bioassay and MC-qPCR. RESULTS: Toxoplasma gondii was detected by both MC-qPCR and mouse bioassay from distinct pools (100 g) of tissues comprising: liver, tongue, heart, diaphragm, semitendinosus (hindlimb), longissimus dorsi muscle (sirloin) and psoas major muscle (fillet). When a selection of individual tissues which had been used for mouse bioassay were examined by MC-qPCR, parasite DNA could only be detected from two animals, despite all calves showing seroconversion after infection. CONCLUSIONS: It is apparent that one individual test will not provide an answer as to whether a calf harbours T. gondii tissue cysts. Although the calves received a known number of infectious oocysts and highly sensitive methods for the detection of the parasite within bovine tissues were applied (mouse bioassay and MC-qPCR), the results confirm previous studies which report low presence of viable T. gondii in cattle and no clear predilection site within bovine tissues.

The use of ELISA, nPCR and qPCR for diagnosis of ocular toxoplasmosis in experimentally infected pigs.

In the present study we experimentally infected pigs with T. gondii tachyzoites, bradyzoites and oocysts in order to evaluate IgG-ELISA, nested-PCR, and qPCR for diagnosis of ocular infection. Eighteen pigs were divided into four groups: G1 (infected with 103 tissue cysts of the M4 strain (type II) at day 28, n=5), G2 (infected with 103 oocysts of the M4 strain at day 28, n=5), G3 (infected with tachyzoites of S48 strain (type 1) at day 0, n=5), and G4 (uninfected unchallenged, control group n=3). At day 70 of the experiment all animals were culled, and serum, aqueous humor (AH) and vitreous humor (VH) samples were collected to perform indirect ELISA, and PCR (nPCR, and qPCR). By ELISA nine pigs (60%) out of 15 were positive in VH samples, and seven out of 15 (46%) were positive in AH samples. Both molecular techniques used here, nPCR and qPCR, were able to detect <50fg of T. gondii tachyzoite DNA. The nPCR and qPCR detected six (7/15, 47%) and two (2/15, 13.3%) positive animals respectively. Antibody responses were detected in serum and in AH and VH from the eye, suggesting that pigs may be an animal that could be used as a model to further our understanding of diagnosis of human ocular infection with T. gondii.

Vaccination of pigs with the S48 strain of Toxoplasma gondii--safer meat for human consumption.

As clinical toxoplasmosis is not considered a problem in pigs, the main reason to implement a control strategy against Toxoplasma gondii (T. gondii) in this species is to reduce the establishment of T. gondii tissue cysts in pork, consequently reducing the risk of the parasite entering the human food chain. Consumption of T. gondii tissue cysts from raw or undercooked meat is one of the main sources of human infection, with infected pork being considered a high risk. This study incorporates a mouse bioassay with molecular detection of T. gondii DNA to study the effectiveness of vaccination (incomplete S48 strain) in its ability to reduce tissue cyst burden in pigs, following oocyst (M4 strain) challenge. Results from the mouse bioassay show that 100% of mice which had received porcine tissues from vaccinated and challenged pigs survived compared with 51.1% of mice which received tissues from non-vaccinated and challenged pigs. The presence (or absence) of T. gondii DNA from individual mouse brains also confirmed these results. This indicates a reduction in viable T. gondii tissue cysts within tissues from pigs which have been previously vaccinated with the S48 strain. In addition, the study demonstrated that the main predilection sites for the parasite were found to be brain and highly vascular muscles (such as tongue, diaphragm, heart and masseter) of pigs, while meat cuts used as human food such as chop, loin, left tricep and left semitendinosus, had a lower burden of T. gondii tissue cysts. These promising results highlight the potential of S48 strain tachyzoites for reducing the number of T. gondii tissues cysts in pork and thus improving food safety.

Toxoplasma gondii in livestock in St. Kitts and Nevis, West Indies.

BACKGROUND: Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals including livestock. In these animals, the parasite forms cysts in the tissues which may pose a risk to public health if infected meat is consumed undercooked or raw. The aim of this study was to determine the exposure of livestock to T. gondii in St. Kitts and Nevis. METHODS: Sera and/or heart tissue and meat juice were collected from pigs (n = 124), sheep (n = 116) and goats (n = 66) at the St. Kitts Abattoir. Sera and meat juice were screened for reactive antibodies to T. gondii using an in-house ELISA. Heart tissue was screened for T. gondii DNA using quantitative PCR and positive samples were genotyped using RFLP. RESULTS: Antibodies to T. gondii were detected in sera from 48% of pigs, 26% of sheep and 34% of goats tested. Antibodies were also detected in the meat juice from 55% of pig hearts, 22% of sheep hearts and 31% of goat hearts tested. There was a significant positive correlation between serology and meat juice results. T. gondii DNA was detected in heart tissue of 21% of pigs, 16% of sheep and 23% of goats tested. Preliminary PCR-RFLP analysis identified a predominance of the Type III genotype of T. gondii. CONCLUSIONS: These results suggest widespread environmental contamination with T. gondii oocysts and that livestock could be a potentially important source of T. gondii infection if their infected meat is consumed (or handled) undercooked.

Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite.

This study evaluates the influence of immunizing lambs with the incomplete S48 strain of Toxoplasma gondii, on parasite dissemination following a live oral challenge with a complete strain of T. gondii (M4). Lambs were culled at 14, 28 and 42 days post challenge. Parasite DNA was detected at significantly (p<0.0001) lower levels in samples from the vaccinated/challenged group (0% in heart and 5.9% in skeletal muscles), when compared to the non-vaccinated/challenged animals (75% heart, 87.9% skeletal muscle). S48 T. gondii DNA was found in muscle or lymph nodes until 42 days post infection, suggesting that parasite DNA or tachyzoites could persist longer after immunization than previously thought. Non-vaccinated/challenged animals showed more frequent lesions in muscles and central nervous system than the vaccinated animals. These results demonstrate that vaccination of lambs with the incomplete S48 T. gondii strain, can protect against establishment of tissue cysts following challenge with a complete strain of T. gondii. Consumption of undercooked meat containing T. gondii cysts is a major route of transmission to people, therefore vaccination of food animals may improve the safety of meat for human consumption.

NKp46+ CD3+ cells: a novel nonconventional T cell subset in cattle exhibiting both NK cell and T cell features.

The NKp46 receptor demonstrates a high degree of lineage specificity, being expressed almost exclusively in NK cells. Previous studies have demonstrated NKp46 expression by T cells, but NKp46+ CD3+ cells are rare and almost universally associated with NKp46 acquisition by T cells following stimulation. In this study we demonstrate the existence of a population of NKp46+ CD3+ cells resident in normal bovine PBMCs that includes cells of both the αß TCR+ and γδ TCR+ lineages and is present at a frequency of 0.1-1.7%. NKp46+ CD3+ cells express transcripts for a broad repertoire of both NKRs and TCRs and also the CD3ζ, DAP10, and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46+ CD3+ cells confirm that NKp46, CD16, and CD3 signaling pathways are all functionally competent and capable of mediating/redirecting cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46+ CD3+ cells exhibit cytotoxic activity against autologous Theileria parva-infected cells in vitro, and during in vivo challenge with this parasite an expansion of NKp46+ CD3+ cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results in this study identify and describe a novel nonconventional NKp46+ CD3+ T cell subset that is phenotypically and functionally distinct from conventional NK and T cells. The ability to exploit both NKRs and TCRs suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses.

Extensive polymorphism and evidence of immune selection in a highly dominant antigen recognized by bovine CD8 T cells specific for Theileria annulata.

Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.