Neospora caninum and Bovine Neosporosis: Current Vaccine Research

Neospora caninum, a tissue cyst-forming parasite, is the causative agent of bovine neosporosis. It is considered to be one of the most important transmissible causes of reproductive failure in cattle; abortion and neonatal mortality result in significant economic losses within the cattle industry worldwide. The balance between acute (mediated by the tachyzoite stage) and persistent (mediated by the bradyzoite stage) phases of the infection is influenced by the immune status of the animal, and for pregnant cows (the intermediate host) immune status is critical for transplacental (i.e. vertical) transmission of the parasite and associated disease outcomes. The horizontal route of transmission from the definitive host, the dog, occurs via ingestion of oocysts containing sporozoites, and plays a minor but important role in transmission of the infection to cattle. Despite the importance of this disease, there is no vaccine or treatment available currently, and at the present the only control measure to reduce the impact of disease is informed management on the farm. The development of vaccines, targeting key biological processes such as invasion and persistent infection, is needed urgently for the control of this widespread parasite

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle

Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens

Life cycle stages, specific organelles and invasion mechanisms of Eimeria species

Apicomplexans, including species of Eimeria, pose a real threat to the health and wellbeing of animals and humans. Eimeria parasites do not infect humans but cause an important economic impact on livestock, in particular on the poultry industry. Despite its high prevalence and financial costs, little is known about the cell biology of these ‘cosmopolitan’ parasites found all over the world. In this review, we discuss different aspects of the life cycle and stages of Eimeria species, focusing on cellular structures and organelles typical of the coccidian family as well as genus-specific features, complementing some ‘unknowns’ with what is described in the closely related coccidian Toxoplasma gondii

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

BACKGROUND: Eimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens. RESULTS: In this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5’Et-Actin or 5’Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens. CONCLUSIONS: E. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1756-2) contains supplementary material, which is available to authorized users

In vitro Anticoccidial Study of Oregano and Garlic Essential Oils and Effects on Growth Performance, Fecal Oocyst Output, and Intestinal Microbiota in vivo

This study investigated the in vitro effects of Greek oregano and garlic essential oils on inhibition of Eimeria parasites and their in vivo effects on production performance, intestinal bacteria counts, and oocyst output. An inhibition assay was performed in vitro using Eimeria tenella Wisconsin strain sporozoites and Madin-Darby bovine kidney (MDBK) cells. Intracellular sporozoite invasion was quantified by detection of E. tenella DNA using qPCR from cell monolayers harvested at 2 and 24 h post-infection. Parasite invasion was inhibited by the oregano essential oil at the concentration of 100 μg/ml by 83 or 93% after 2 or 24 h, respectively. Garlic essential oil reached a maximum inhibition of 70% after 24 h with the 50 μg/ml concentration. Normal morphology was observed in MDBK cells exposed to concentrations of 100 μl/ml of garlic or oregano for over 24 h. In the in vivo trial, 180 male broiler chicks (45.3 ± 0.7 g) were allocated into two treatments (6 pens of 15 chicks per treatment). Control treatment was fed commercial diets without antibiotics or anticoccidials. The ORE–GAR treatment was fed the same control diets, further supplemented with a premix (1 g/kg feed) containing the oregano (50 g/kg premix) and garlic (5 g/kg premix) essential oils. At day 37, all birds were slaughtered under commercial conditions, and intestinal samples were collected. ORE-GAR treatment had improved final body weight (1833.9 vs. 1.685.9 g; p < 0.01), improved feed conversion ratio (1.489 vs. 1.569; p < 0.01), and reduced fecal oocyst excretion (day 28: 3.672 vs. 3.989 log oocysts/g, p < 0.01; day 37: 3.475 vs. 4.007 log oocysts/g, p < 0.001). In the caecal digesta, ORE-GAR treatment had lower total anaerobe counts (8.216 vs. 8.824 CFU/g; p < 0.01), whereas in the jejunum digesta the ORE-GAR treatment had higher counts of E. coli (5.030 vs. 3.530 CFU/g; p = 0.01) and Enterobacteriaceae (5.341 vs. 3.829 CFU/g; p < 0.01), and lower counts of Clostridium perfringens (2.555 vs. 2.882 CFU/g; p < 0.01). In conclusion, the combined supplementation of oregano and garlic essential oils had a potent anticoccidial effect in vitro and a growth-promoting effect in broilers reared in the absence of anticoccidial drugs

Infected Dendritic Cells Facilitate Systemic Dissemination and Transplacental Passage of the Obligate Intracellular Parasite Neospora caninum in Mice

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites

Identification of novel rhoptry proteins in Neospora caninum by LC/MS-MS analysis of subcellular fractions

Apicomplexan parasites possess an apical complex that is composed of two secretory organelles recognized as micronemes and rhoptries. Rhoptry contents are secreted into the parasitophorous vacuole during the host cell invasion process. Several rhoptry proteins have been identified in Toxoplasma gondii and seem to be involved in host-pathogen interactions and some of them are considered to be important virulence factors. Only one rhoptry protein, NcROP2, has been identified and extensively characterized in the closely related parasite Neospora caninum, and this has showed immunoprotective properties. Thus, with the aim of increasing knowledge of the rhoptry protein repertoire in N. caninum, a subcellular fractionation of tachyzoites was performed to obtain fractions enriched for this secretory organelle. 2-D SDS-PAGE followed by MS and LC/MS-MS were applied for fraction analysis and 8 potential novel rhoptry components (NcROP1, 5, 8, 30 and NcRON2, 3, 4, 8) and several kinases, proteases and phosphatases proteins were identified with a high homology to those previously found in T. gondii. Their existence in N. caninum tachyzoites suggests their involvement in similar events or pathways that occur in T. gondii. These novel proteins may be considered as targets that could be useful in the future development of immunoprophylactic measures

The International Network for Evaluating Outcomes (iNeo) of neonates: evolution, progress and opportunities

Neonates born very preterm (before 32 weeks’ gestational age), are a significant public health concern because of their high-risk of mortality and life-long disability. In addition, caring for very preterm neonates can be expensive, both during their initial hospitalization and their long-term cost of permanent impairments. To address these issues, national and regional neonatal networks around the world collect and analyse data from their constituents to identify trends in outcomes, and conduct benchmarking, audit and research. Improving neonatal outcomes and reducing health care costs is a global problem that can be addressed using collaborative approaches to assess practice variation between countries, conduct research and implement evidence-based practices. The International Network for Evaluating Outcomes (iNeo) of neonates was established in 2013 with the goal of improving outcomes for very preterm neonates through international collaboration and comparisons. To date, 10 national or regional population-based neonatal networks/datasets participate in iNeo collaboration. The initiative now includes data on >200,000 very preterm neonates and has conducted important epidemiological studies evaluating outcomes, variations and trends. The collaboration has also surveyed >320 neonatal units worldwide to learn about variations in practices, healthcare service delivery, and physical, environmental and manpower related factors and support services for parents. The iNeo collaboration serves as a strong international platform for Neonatal-Perinatal health services research that facilitates international data sharing, capacity building, and global efforts to improve very preterm neonate care