Sergio Martín

Immunoprotective effect of cysteine proteinase fractions from two Haemonchus contortus strains adapted to sheep and goats

A preliminary analysis of the significance of genetic diversity in cysteine proteinase genes has been performed simultaneously in sheep and goats, with regard to the immunological control using these enzymes against haemonchosis. For this purpose, we have studied the cross-immunoprotective effect of cysteine protease-enriched protein fractions (CPFs) in adult worms of two Haemonchus contortus strains from North America and Spain that are adapted to sheep and goats, respectively. Previous genetic analysis of cysteine proteinase genes in both strains has shown that some of loci are polymorphic and these differences are translated into changes in the amino acid sequences. However, our results show that CPFs from H. contortus adult worms have a protective effect against the parasite in both sheep and goats. These results are similar regardless of whether they were obtained from sheep or goat-adapted H. contortus strains, which could be very important in case H. contortus CPFs were commercially used in different countries, as vaccines to prevent the negative effects of this parasite. Interestingly, this experimental inoculation of both species with a heterologous strain of H. contortus contributes to the idea shown in previous studies about how difficult is the interpretation and the comparison of vaccination where strains not adapted to a specific host are used. Therefore, the challenger of using heterologous strains could provide similar results to those observed in immunised animals. This study suggests the possibility of exploring the mechanisms involved in natural protection against non-adapted strains, in order to develop strategies to control haemonchosis.

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.

Immune response in goats vaccinated with thiol-binding proteins from Haemonchus contortus

The experimental protocol of immunization tested here confirms its protective effect against Haemonchus contortus in goats. This protection translated into a 65.5% mean reduction in adult worm burden after a homologous challenge, and a significant decrease (73.2%) in cumulative faecal egg counts (FECs). These parasitological findings were consistent with the levels of some biopathological parameters. Thus, the reduction in adult worms and FEC observed in immunized animals were associated with increased levels of packed cell volume as well as plasma proteins. This response seems to be related to an important increase in specific antibodies (in serum and gastric mucus) and eosinophilia in response to challenge. At the local level, a cellular response was also observed in which CD4+ lymphocytes and globule leucocytes played a predominant role. Finally, it should be noted that the study of immunolocalization of proteins used in the vaccination trial suggests that these antigens have an internal location (at intestinal and reproductive tissues) in the adult worm. This observation, in conjunction with the kinetics of specific antibody levels after the challenge, suggests that these antigens may be part of excretory/secretory (E/S) products.