This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio±1·5, P<0·05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

The worldwide economic impact of Neospora caninum infection has caused the development of effective vaccines to become one of the main goals in the field of neosporosis research. In this study, the protection conferred by antigens from inactivated whole tachyzoites (TZ) and a tachyzoite-bradyzoite mixture (TZ-BZ) of N. caninum (Nc-Spain7 isolate) incorporated into a water-in-oil emulsion (W/O) and aluminium hydroxide-ginseng extract (Al/G) was evaluated in mouse models of congenital and cerebral N. caninum infection. Immunization with TZ-BZ induced congenital and cerebral neosporosis exacerbation that was mainly characterized by reduced neonatal median survival time and increased parasite presence in adult mouse brains. The immune response of mice immunized with TZ-BZ was characterized by an increase in IFN-γ expression prior to challenge and an increase in IL-4 expression accompanied with significantly higher levels of antibodies against 2 recombinant bradyzoite-specific proteins (rNcSAG4 and rNcBSR4) after challenge. Immunization with TZ in W/O significantly reduced neonatal mortality, vertical transmission as well as parasite presence in adult mouse brains and induced a strong humoral immune response. The current study demonstrates the critical role of stage-specific antigens and adjuvants on the development of effective inactivated vaccines for the prevention of N. caninum infection.

Neospora caninum infection persists throughout the life of its intermediate host due to the conversion of tachyzoites to slowly dividing bradyzoites that encyst in the brain. This event results in persistent N. caninum infection in bovine herds and partially explains the poor efficacy of many chemotherapeutic agents and vaccine formulations. Thus, there is a need for greater understanding of the tachyzoite-to-bradyzoite conversion mechanisms. Here we studied for the first time the transcription kinetics of the N. caninum bradyzoite-specific gene NcSAG4 in brain samples from chronically infected mice by means of real-time RT-PCR. NcSAG4-messenger RNA (mRNA) levels increased significantly during the chronic phase but followed 2 different expression patterns depending on the isolate used for murine inoculation. NcSAG4-mRNA levels in brains from Nc-1-inoculated mice peaked during late chronic infection (on day 64 post-infection, p.i.), whereas those from Nc-Liv-inoculated mice peaked earlier during the chronic infection (on day 32 p.i.). This difference could be a reflection of the different abilities of these isolates to replicate and form cysts in parasitized brains. These results are consistent with our observations of anti-rNcSAG4 antibody production; low levels were present at seroconversion and slowly increased during the chronic phase. In contrast, NcSAG1 transcription levels, which mark the tachyzoite stage, were maintained without variation in both groups of mice. This suggests the presence of a significant amount of tachyzoites or intermediate zoites expressing NcSAG1 in the brain, even during the late chronic infection.

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1–6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.