J.M. Molina

Occurrence and genotype characterization of Giardia duodenalis in goat kids from the Canary Islands, Spain

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a wide-spread intestinal protozoa of both humans and animals. Although giardiosis in goat is commonly asymptomatic, young kids may bear an enteric disease associated with persistent diarrhoea and delayed weight gain. In the present study we have analysed the occurrence of Giardia in 315 young goat kids (2-6 months old) from Gran Canaria Island (Spain) through visualization of faecal cysts. The identification of genotypes of G. duodenalis among the farms was attained by nested PCR of the triophosphate isomerase (TPI) and single PCR of beta-giardin genes and subsequent sequencing. Positive samples were found in 42.2% of the animals and 95.5% of the farms. Goat faecal specimens were positive for only livestock-associated G. duodenalis assemblage E genotype for both TPI and beta-giardin genes. The genetic analysis of these two loci revealed the presence of different haplotypes among the farms included in the survey and high homology with homologous genes from cattle and sheep. Altogether, the data presented here provide additional information to the prevalence and genetic characterization of Giardia isolates. The absence of assemblages A and B in this study suggests that zoonotic transmission of Giardia from goats could be of low epidemiological significance, although these findings should be validated in studies including other geographical areas, age groups and larger number of samples.

Comparative experimental Haemonchus contortus infection of two sheep breeds native to the Canary Islands

This study compares the susceptibility to Haemonchus contortus infection in two breeds of sheep endemic to the Canary Islands, the Canaria Hair Breed sheep and the Canaria sheep. Sheep were experimentally infected with 20,000 larvae of H. contortus and animals killed on days 7 and 28 post-infection. No difference between sheep breeds were detected in immature worm counts at days 7 or 28 post-infection. However, in comparison to the Canaria sheep breed, the Canaria Hair Breed sheep showed lower mean faecal egg counts, lower adult worm counts, lower number of eggs in utero and female worm stunting. Overall, these data suggest that the Canaria Hair Breed sheep has a greater resistance to H. contortus infection than Canaria sheep, and that this resistance may act at the level of the adult parasite.

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.

Fecundity in adult Haemonchus contortus parasites is correlated with abomasal tissue eosinophils and γδ T cells in resistant Canaria Hair Breed sheep.

Canaria Hair Breed (CHB) sheep are more resistant than Canaria sheep (CS) to experimental Haemonchus contortus infection. Protective responses appear effective against the adult stage of the parasite, not as commonly reported in other breeds against the larval stages. In this study we have quantified several abomasal immune cells and correlated these with parasitological variables for each breed. A significant negative correlation between CD4+ T cell numbers and worm burden or length at 28 dpi was seen only in CS sheep. Significant negative correlations for both abomasal eosinophils and γδ/WC1+ T cells, and fecundity of the adult worms were observed only in the resistant CHB sheep breed. Tissue eosinophils and γδ/WC1+ T cells were positively correlated in CHB sheep. We suggest that the two sheep breeds have disparate immune responses following infection with the parasite and that γδ+ T cells in association with eosinophils may play a hitherto unrecognised role in modulating fecundity in H. contortus adult female parasites.

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.

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.

Preliminary study on the use of a Teladorsagia circumcincta bulk milk ELISA test in dairy goats under experimental conditions

An ELISA test for the detection of anti-Teladorsagia circumcincta antibodies in goat milk samples (both individual and bulk milk samples) has been developed in dairy goats experimentally infected with a trickle infection. The results observed were compared with those obtained when some other standard methods for the detection of animals infected with gastrointestinal nematodes (GIN), such as faecal egg counts (FEC) and serum pepsinogen levels. Some factors that could affect the final results of the test (dilution and nature of samples as well as their preservation at 4 degrees C for 0-10 days) were also monitored. The results show that the proposed test could allow the detection of infected herds using bulk milk samples. The levels of anti-T. circumcincta antibodies in milk and serum samples and the concentrations of serum pepsinogen showed a similar pattern over time. Refrigeration of milk samples for less than 5 days or its preservation with potassium dichromate for 10 days did not significantly affect the ability of the test to detect infected animals. Finally, when a commercial Ostertagia ostertagi ELISA kit was used to assay milk samples from goats infected with T. circumcincta, the results suggest that it may be possible to use a crude O. ostertagi antigen-based ELISA for the detection of goats infected with T. circumcincta.