D. Pérez

Development of Eimeria ninakohlyakimovae in vitro in primary and permanent cell lines

Infections with Eimeria ninakohlyakimovae represent important coccidian diseases of goats severely affecting animal health and profitability of goat industry. For the development of suitable vaccination strategies basic research is needed for which one important prerequisite is the establishment of in vitro cultures guaranteeing the availability of parasitic material. Therefore, primary cell cultures [caprine, bovine and human umbilical vein endothelial cells (CUVEC, BUVEC, HUVEC)] as well as permanent cell lines [bovine foetal gastrointestinal cells (BFGC), bovine colonic epithelial cells (BCEC), African green monkey kidney cells (VERO)] were exposed to vital sporozoites of E. ninakohlyakimovae. The parasites invaded all different cell types used, irrespective of their origin, but further development into macromeronts and subsequent release of viable merozoites I were restricted to ruminant cells. Mature macromeronts developed in both, endothelial (CUVEC, BUVEC) and epithelial cells (BCEC). VERO cells were non-permissive for parasite development, nevertheless sporozoites survived for 21 days p.i. within an enlarged parasitophorous vacuole. Best in vitro development of E. ninakohlyakimovae macromeronts with respect to the production of viable merozoites I was observed in BCEC, followed by BUVEC. However, the largest macromeronts developed in CUVEC. Mature macromeronts were also detected in BFGC, but these cells were less effective concerning infection rates and productivity. The complete life-cycle of E. ninakohlyakimovae leading to oocyst production was not accomplished in any cell type used. In conclusion, we established suitable in vitro systems for the culture of E. ninakohlyakimovae macromeronts, e.g., for the mass production of merozoites I, for basic studies on parasite/host endothelial cell interactions or for pharmaceutical screenings.

Isolation of an Eimeria ninakohlyakimovae field strain (Canary Islands) and analysis of its infection characteristics in goat kids.

The current study was conducted to isolate a field strain of Eimeria ninakohlyakimovae, characterize its infectivity and the response to challenge under experimental conditions. The isolated strain (GC) induced a prepatent period of 14-15 days p.i., a patency of 7±2 days and a noticeable pathogenicity in infected goat kids. Challenge trials resulting in a decrease of oocysts per gram counts as well as a milder intensity of clinical signs in re-infected animals indicated the capacity of this strain to induce protective immune response. Altogether, the data reported in the present study suggest that the strain E. ninakohlyakimovae GC is a useful tool for the investigation of mechanisms of pathogenicity as well as host protective immune response in caprine coccidiosis, representing a valuable prerequisite for the development of future strategies in prophylaxis and control of this important parasitic disease in goat.

Eimeria ninakohlyakimovae induces NADPH oxidase-dependent monocyte extracellular trap formation and upregulates IL-12 and TNF-α, IL-6 and CCL2 gene transcription

Extracellular trap (ET) formation has been demonstrated as novel effector mechanism against diverse pathogens in polymorphonuclear neutrophils (PMN), eosinophils, mast cells, macrophages and recently also in monocytes. In the current study, we show that E. ninakohlyakimovae triggers the deliverance of monocyte-derived ETs in vitro. Fluorescence illustrations as well as scanning electron microscopy (SEM) analyses showed that monocyte-derived ET formation was rapidly induced upon exposure to viable sporozoites, sporocysts and oocysts of E. ninakohlyakimovae. Classical features of monocyte-released ETs were confirmed by the co-localization of extracellular DNA adorned with myeloperoxidase (MPO) and histones (H3) in parasite-entrapping structures. The treatment of caprine monocyte ET structures with NADPH oxidase inhibitor diphenylene iodondium (DPI) significantly reduced ETosis confirming the essential role of reactive oxygen species (ROS) in monocyte mediated ETs formation. Additionally, co-culture of monocytes with viable sporozoites and soluble oocyst antigen (SOA) induced distinct levels of cytokine and chemokine gene transcription. Thus, the transcription of genes encoding for IL-12 and TNF-α was significantly upregulated after sporozoite encounter. In contrast IL-6 and CCL2 gene transcripts were rather weakly induced by parasites. Conversely, SOA only induced the up-regulation of IL-6 and CCL2 gene transcription, and failed to enhance transcripts of IL-12 and TNF-α in vitro. We here report on monocyte-triggered ETs as novel effector mechanism against E. ninakohlyakimovae. Our results strongly suggest that monocyte-mediated innate immune reactions might play an important role in early host immune reactions against E. ninakohlyakimovae in goats.

Immunization with Eimeria ninakohlyakimovae-live attenuated oocysts protect goat kids from clinical coccidiosis.

Caprine coccidiosis, affecting mainly young goat kids around the weaning period, is worldwide the most important disease in the goat industry. Control of caprine coccidiosis is increasingly hampered by resistances developed against coccidiostatic drugs leading to an enhanced need for anticoccidial vaccines. In the current study we conducted an oral immunization trial with live attenuated sporulated Eimeria ninakohlyakimovae oocysts. Sporulated E. ninakohlyakimovae oocysts were attenuated by X-irradiation technique. The experimental design included a total of 18 goat kids divided into the following groups: (i) animals immunized with attenuated E. ninakohlyakimovae oocysts at 5 weeks of age and challenged 3 weeks later with non-irradiated homologous oocysts (group 1); (ii) animals infected with non-attenuated E. ninakohlyakimovae oocysts at 5 weeks of age and challenged 3 weeks later with non-attenuated homologous oocysts (group 2); (iii) animals primary-infected with untreated E. ninakohlyakimovae oocysts at 8 weeks of age (control of the challenge infection, group 3); (iv) non-infected control animals (group 4). Goat kids immunized with live attenuated E. ninakohlyakimovae oocysts (group 1) excreted significantly less oocysts in the faeces (95.3% reduction) than kids infected with non-attenuated ones (group 2). Furthermore, immunization with live but attenuated oocysts resulted in ameliorated clinical coccidiosis compared to goat kids infected with untreated oocysts (group 2) and resulted in equally reduced signs of coccidiosis after challenge infection compared to acquired immunity driven by non-attenuated oocysts. Overall, the present study demonstrates for the first time that live attenuated E. ninakohlyakimovae oocysts orally administered showed almost no pathogenicity but enough immunogenicity in terms of immunoprotection. Importantly, vaccinated animals still shed low amounts of oocysts, guaranteeing environmental contamination and consecutive booster infections to sustain ongoing immunity.

Modulation of the pro-inflammatory molecules E-selectin and TNF-α gene transcription in Eimeria ninakohlyakimovae-infected primary caprine host endothelial cells.

Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe hemorrhagic typhlocolitis in young animals, thereby leading to high economic losses in goat industry worldwide. The first merogony of E. ninakohlyakimovae occurs within host endothelial cells (ECs) of the lacteal capillaries of the villi of the distal ileum resulting in the formation of macromeronts (up to 170 μm) within 10-12 days post-infection (p.i.) and releasing >120,000 merozoites I. The E. ninakohlyakimovae-macromeront formation within highly immunoreactive host endothelial cells (ECs) should rely on several regulatory processes to fulfill this massive replication. Here host EC-parasite interactions were investigated to determine the extent of modulation carried out by E. ninakohlyakimovae in primary caprine umbilical vein endothelial cells (CUVEC) during the first merogony. Gene transcription of the adhesion molecule E-selectin and the cytokine TNF-α were significantly enhanced in the first hours and days p.i. in E. ninakohlyakimovae-infected CUVEC. The activation of CUVEC was also demonstrated by enhanced chemokine CCL2 and cytokine GM-CSF gene transcription, whereas no differences of the eNOS gene transcription were observed in E. ninakohlyakimovae-infected CUVEC when compared to un-infected controls. The data presented here suggest that infection of caprine host ECs by E. ninakohlyakimovae results in EC activation associated with enhanced gene transcription encoding for pro-inflammatory as well as immunomodulatory molecules, which might be important for the defense against this intracellular parasite.

Humoral immune responses of experimentally Eimeria ninakholyakimovae-infected goat kids

Although cellular immune reactions seem to be crucial for protective immune responses in Eimeria spp. infections, there are also evidences on an active involvement of the humoral counterpart. In the present study, we have analyzed the humoral response of goat kids subjected to primary and challenge infections with Eimeria ninakholyakimovae. Specific levels of IgG and IgM in serum samples and IgA in the ileal mucus were estimated. In infected kids, significantly increased levels of IgG were observed from 3 weeks post infection onwards in addition to an enhancement of specific IgM and secretory IgA levels. A wide range of peptides of sporulated oocyst antigen (SOA) was recognized by specific IgG as determined by immunoblotting. However, no correlations were found between immunoglobulin levels and OPG counts after challenge infection. Overall, these data indicate a significant specific humoral response of E. ninakohlyakimovae-infected goat kids that does not seem to convey immunoprotection. Further studies should be addressed to clarify if the lack of correlation might be associated to the type of antigen used for the immunoenzimatic assays, the age of the animals or other factors.

Protective immune responses during prepatency in goat kids experimentally infected with Eimeria ninakohlyakimovae

During the first schizogony, the goat coccidia Eimeria ninakohlyakimovae develops macroschizonts in lacteal duct endothelial cells, whose rupture leads to severe ileal damage and clinical signs during the prepatent period. The immune response elicited against early stages of the parasite development still requires to be investigated. In the present study we have evaluated immune reactions in goat kids primary- and challenged-infected with Eimeria ninakohlyakimovae, and sacrificed during prepatency (7days after challenge). The oocyst output during the primary infection, body weight and clinical condition of all the animals were examined and, at the end of the experiment, all the goat kids were euthanized and subjected to necropsy. Samples were taken from different sections of the ileum, colon and mesenteric lymph nodes (MLN) of primary- and challenged E. ninakohlyakimovae-infected animals. Intestinal leukocyte subpopulations were characterized in E. ninakohlyakimovae-infected mucosa and counts of lymphocytes, eosinophils, polymorphonuclear neutrophils (PMN), globular leukocytes and mast cells were recorded. Additionally, gene expression of caprine IL-2, IL-4, IL-10 and INFγ of ileal, colonic and MLN tissues were performed, as well as the immunohistochemical characterization of immune cells. The E. ninakohlyakimovae primary infection resulted in moderate to severe enteritis with different degrees of diarrhoea and was accompanied by high OPG counts and an increase of most immune cells analyzed when compared to uninfected control animals. Furthermore, eosinophil-, lymphocyte-, globular leukocyte- and mast cell-counts were significantly higher in the challenge group compared to the primary infected animals, whilst the opposite was true for PMN counts. The challenge infection was also associated with moderate increased levels of local mucosal IgA. Interestingly, the number of immature schizonts found at the ileal mucosa was statistically higher in the challenge infected group compared to the challenged control animals. Furthermore, in the challenged E. ninakohlyakimovae-infected animals a significantly higher number of mucosal CD4+ and CD8+ lymphocytes were observed, indicating that these T cell subpopulations might be involved in protective host immune response elicited against early stages of parasite development. The immune response was however very complex, as antigen presenting cells and other effector cell populations of the innate immune system, as well as certain cytokines, were involved. In summary, the results of this study contribute to the better understanding of local cellular and humoral immune responses against caprine E. ninakohlyakimovae, particularly during the prepatency.

Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library

Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

Age-related immune response to experimental infection with Eimeria ninakohlyakimovae in goat kids

Both the immune response developed in ruminants against Eimeria spp. and the ability to bear patent infections seems to be dependent on the age of the host. In the present study we have evaluated the influence of the age in the development of protective immune responses against Eimeria ninakohlyakimovae. For this purpose, 3, 4 and 5-week-old goat kids were infected with sporulated oocysts and subjected to a homologous challenge 3 weeks later. Goat kids primary infected at 6, 7 and 8 weeks of age served as challenge controls, and uninfected animals were used as negative controls. The protective immunity was assessed by clinical, haematological, parasitological, immunological and pathological parameters. Altogether, the results demonstrate that goat kids of either 3, 4 or 5 weeks of age are able to develop patent infections and immunoprotective responses against E. ninakohlyakimovae, as all age groups: (i) released significantly less oocysts after challenge, which was associated to milder clinical signs; (ii) displayed a local immune response, with significant increase of numerous cellular populations; and (iii) had increased levels of IgG and IgM, and mainly of local IgA. Nevertheless, detailed analysis of the data showed some differences between the three age groups, related both to the Eimeria infection outcome and the resulting immune response, suggesting that youngest goat kids are not fully immunocompetent. This finding may be of interest for the design of immunoprophylactic approaches and/or prophylactic/methaphylactic treatments against goat coccidiosis.