A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens

Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs)

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species

Proteome expression changes among virulent and attenuated Neospora caninum isolates

Neospora caninum is a cyst-forming parasite that has been recognised worldwide as a cause of cattle abortion and neuromuscular disease in dogs. Variations in genetic profiles, behaviour in vitro, and pathogenicity have been established among N. caninum isolates. However, it is unclear which parasite factors are implicated in this intra-specific diversity. Comparative analysis of protein expression patterns may define the determinants of biological diversity in N. caninum. Using DIGE and MALDI-TOF MS techniques, we quantified and identified differentially expressed proteins in the tachyzoite stage across three N. caninum isolates: the virulent Nc-Liv and Nc-Spain 7 isolates, and the attenuated Nc-Spain 1H isolate. Comparison between Nc-Spain 7 and Nc-Spain 1H extracts revealed 39 protein spots that were more abundant in Nc-Spain 7 and 21 in Nc-Spain 1H. Twenty-four spots were also increased in Nc-Spain 7 and 12 in Nc-Liv. Three protein spots were more abundant in the Nc-Liv extracts than in the Nc-Spain 1H extracts. MS analysis identified 11 proteins differentially expressed that are potentially involved in gliding motility and the lytic cycle of the parasite, and oxidative stress. These differences could help to explain variations in behaviour between isolates and provide a better knowledge of mechanisms associated with virulence.Spanish Ministry of Science and Innovation (AGL2007–60132/GAN and AGL2010-22191/GAN)Spanish Ministry of Education (AP2009-0354)Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

Proteome expression changes among virulent and attenuated Neospora caninum isolates

Neospora caninum is a cyst-forming parasite that has been recognised worldwide as a cause of cattle abortion and neuromuscular disease in dogs. Variations in genetic profiles, behaviour in vitro, and pathogenicity have been established among N. caninum isolates. However, it is unclear which parasite factors are implicated in this intra-specific diversity. Comparative analysis of protein expression patterns may define the determinants of biological diversity in N. caninum. Using DIGE and MALDI-TOF MS techniques, we quantified and identified differentially expressed proteins in the tachyzoite stage across three N. caninum isolates: the virulent Nc-Liv and Nc-Spain 7 isolates, and the attenuated Nc-Spain 1H isolate. Comparison between Nc-Spain 7 and Nc-Spain 1H extracts revealed 39 protein spots that were more abundant in Nc-Spain 7 and 21 in Nc-Spain 1H. Twenty-four spots were also increased in Nc-Spain 7 and 12 in Nc-Liv. Three protein spots were more abundant in the Nc-Liv extracts than in the Nc-Spain 1H extracts. MS analysis identified 11 proteins differentially expressed that are potentially involved in gliding motility and the lytic cycle of the parasite, and oxidative stress. These differences could help to explain variations in behaviour between isolates and provide a better knowledge of mechanisms associated with virulence.Spanish Ministry of Science and Innovation (AGL2007–60132/GAN and AGL2010-22191/GAN)Spanish Ministry of Education (AP2009-0354)Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress.

BACKGROUND NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided. METHODS Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points. RESULTS We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress. CONCLUSIONS The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle

Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle.

Virulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites

Low efficacy of NcGRA7, NcSAG4, NcBSR4 and NcSRS9 formulated in poly-ɛ-caprolactone against Neospora caninum infection in mice

The protective efficacy of vaccination with Neospora caninum recombinant antigens was evaluated in Balb/c pregnant and non-pregnant mouse models of neosporosis. A major immunodominant dense granule protein (NcGRA7) and three bradyzoite-specific surface antigens (NcSAG4, NcBSR4 and NcSRS9) were expressed in Escherichia coli and encapsulated within poly-ɛ-caprolactone (PCL) nanoparticles for the first time. Good efficiencies of entrapment (greater than 50%) were obtained for all encapsulated proteins. Moreover, antigenicity was unaffected after formulation. Afterwards, separate groups of mice were immunised with the nanoparticles and were then challenged with N. caninum tachyzoites. High IgG1 and IgG2a antibody levels of anti-N. caninum and specific antibodies directed against recombinant proteins were developed by all of the immunised groups. Mice previously inoculated with encapsulated rNcGRA7 produced significant levels of IFN-γ. However, in general, a low production of IFN-γ was detected. This may indicate a failure in the complete liberation of antigens after immunisation or an incorrect balance of the Th1/Th2 response to combat acute neosporosis during pregnancy. In fact, high morbidity and mortality rates were observed in dams. Moreover, vertical transmission was not prevented, and high neonatal mortality rates occurred similarly among the groups. Despite the global absence of efficacy, the study reveals some results of positive efficacy regarding dams and pups’ survival and parasite presence for NcSRS9 recombinant protein. Furthermore, vaccination with rNcGRA7 encapsulated alone or combined with rNcSAG4 resulted in a slight decrease of parasite presence in non-pregnant mice. These promising results are further discussed to suggest new approaches that may be more suitable to test vaccine formulations based on bradyzoite stage-specific proteins

Identificación de proteínas Neospora caninum implicadas en procesos de invasión y virulencia

Comunicaciones a congreso

In Vitro Antioxidant, Antimicrobial, Anticoccidial, and Anti-Inflammatory Study of Essential Oils of Oregano, Thyme, and Sage from Epirus, Greece

Origanum vulgare subsp. hirtum, Thymus vulgaris, and Salvia fructicosa are aromatic plants commonly found in Mediterranean countries and are traditionally used in Greece as a remedy for humans, since they are well known as potent antibacterial, antioxidant, and anti-inflammatory agents. Essential oils (EOs) derived from plants cultivated in the mountainous region of Epirus, Greece, were investigated for their inhibitory activity against key microorganisms with relevance to avian health, while also assessing their antioxidant and anti-inflammatory activity. The total phenolic content (TPC) of the EOs was estimated according to the Folin–Ciocalteu method, while the antioxidant capacity was tested through the EOs’ ability to scavenge free radicals by means of the DPPH, ABTS, and FRAP assays. Antibacterial and anti-inflammatory effects were examined by the agar disc diffusion method and the lipoxygenase (LOX) inhibition test, respectively. Furthermore, the EOs’ ability to inhibit the invasion of sporozoites of Eimeria tenella (Wisconsin strain) along with any toxic effects were assayed in Madin–Darby bovine kidney (MDBK) cells. The antioxidant activity of the EOs was observed in descending order: oregano > thyme > sage. The antimicrobial effects of thyme and oregano were equivalent and higher than that of sage, while the anti-inflammatory effect of thyme was higher compared to both sage and oregano. The intracellular invasion of sporozoites was evaluated by the detection of E. tenella DNA by qPCR from cell monolayers harvested at 2 and 24 h post-infection. Parasite invasion was inhibited by the addition of oregano essential oil at the concentration of 100 μg/mL by 83% or 93% after 2 or 24 h, respectively, and was higher compared to the addition of thyme and sage, which had similar effects, but at a less intensive level. The cytotoxic assessment of all three essential oils revealed that they had no effect on MDBK cells compared to dimethyl sulfoxide (DMSO), used as the control substance. The supplementation of oregano, thyme, and sage essential oils had a potent antioxidant, anti-inflammatory, antimicrobial, and anticoccidial in vitro effect that is comparable to synthetic substances or approved drugs, justifying the need for further evaluation by in vivo studies in broilers reared in the absence of antimicrobial and anticoccidial drugs or synthetic antioxidant and/or anti-inflammatory compounds