Regulatory effect of Balanites aegyptiaca ethanol extract on oxidant/antioxidant status, inflammatory cytokines, and cell apoptosis gene expression in goat abomasum experimentally infected with Haemonchus Contortus.

This experiment aimed to assess the regulatory effects of treatment with Balanites aegyptiaca fruit ethanol extract (BA-EE) on oxidant/antioxidant status, anti-inflammatory cytokines, and cell apoptosis gene expression in the abomasum of Haemonchus contortus-infected goats. Twenty goat kids were assigned randomly to four equal groups: (G1) infected-untreated, (G2) uninfected-BA-EE-treated, (G3) infected-albendazole-treated, (G4) infected-BA-EE-treated. Each goat in (G1), (G3), and (G4) was orally infected with 10,000 infective third-stage larvae. In the fifth week postinfection, single doses of albendazole (5 mg/kg.BW) and BA-EE (9 g/kg.BW) were given orally. In the ninth week postinfection, the animals were slaughtered to obtain abomasum specimens. The following oxidant/antioxidant markers were determined: malondialdehyde (MDA), glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT). The mRNA gene expression of cytokines (IL-3, IL-6, IL-10, TNF-α) and cell apoptosis markers (Bax, Bcl-2) were estimated. (G1) showed significantly reduced GSH content and GST and SOD activities but a markedly increased MDA level. (G3) and (G4) revealed a markedly lower MDA level with pronouncedly elevated GSH, SOD, and GST levels. The antioxidant properties of BA-EE were superior to those of albendazole. The mRNA gene expressions of IL-3, IL-6, IL-10, TNF-α, and Bax-2 were upregulated in (G1) but downregulated in (G3) and (G4). Bcl-2 and Bcl-2/Bax ratio expression followed a reverse course in the infected and both treated groups. We conclude that BA-EE treatment has a protective role in the abomasum of H. contortus-infected goats. This could be attributed to its antioxidant properties and ability to reduce pro-inflammatory cytokines and cell apoptosis.

Survival of the nematophagous fungus Duddingtonia flagrans to in vitro segments of sheep gastrointestinal tract.

The nematophagous fungus Duddingtonia flagrans is used in integrated management of gastrointestinal nematodes in ruminants. The chlamydospores of the fungus, orally administered, pass through the segments of the ruminant digestive tract and, in the feces, capture the nematodes preventing their migration to grasslands. The drastic conditions of the gastrointestinal segments can negatively affect the fungus' biocontrol activity. The aim of this study was to assess the effect of in vitro conditions of the sheep's main gastrointestinal segments on the concentration, viability and nematode predatory ability of D. flagrans chlamydospores. The segments evaluated separately in vitro were the oral cavity, rumen, abomasum, and small intestine. The results showed that chlamydospores concentration was not affected by exposure to the different segments. The viability of the chlamydospores after exposure to the oral cavity (2.53 × 106 CFU/mL) and small intestine (1.24 × 105 CFU/mL) was significantly lower than its control treatment, with values of 6.67 × 106 CFU/mL and 2.31 × 105 CFU/mL respectively. Nematode predatory ability after rumen exposure was reduced by 7% compared to the control treatment, by 25% after abomasum exposure and by 17% after small intestine. This study revealed the individual in vitro effect of each segment of ovine gastrointestinal tract on the integrity of this strain of the fungus D. flagrans affecting its viability and nematode predatory ability under the evaluated conditions. Delivery systems could be designed to protect chlamydospores considering the impact of each gastrointestinal segment.

Dynamic transcriptomic changes of goat abomasal mucosa in response to Haemonchus contortus infection.

Gastrointestinal nematode (GIN) infections are one of the major constraints for grazing sheep and goat production worldwide. Genetic selection for resistant animals is a promising control strategy. Whole-transcriptome analysis via RNA-sequencing (RNA-seq) provides knowledge of the mechanisms responsible for complex traits such as resistance to GIN infections. In this study, we used RNA-seq to monitor the dynamics of the response of the abomasal mucosa of Creole goat kids infected with Haemonchus contortus by comparing resistant and susceptible genotypes. A total of 8 cannulated kids, 4 susceptible and 4 resistant to GIN, were infected twice with 10 000 L3 H. contortus. During the second infection, abomasal mucosal biopsies were collected at 0, 8, 15 and 35 days post-infection (dpi) from all kids for RNA-seq analysis. The resistant animals showed early activation of biological processes related to the immune response. The top 20 canonical pathways of differentially expressed genes for different comparison showed activation of the immune response through many relevant pathways including the Th1 response. Interestingly, our results showed a simultaneous time series activation of Th2 related genes in resistant compared to susceptible kids.

Transcriptome analysis unraveled potential mechanisms of resistance to Haemonchus contortus infection in Merino sheep populations bred for parasite resistance.

Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes in small ruminants. To understand molecular mechanisms underlying host resistance to this parasite, we used RNA-sequencing technology to compare the transcriptomic response of the abomasal tissue, the site of the host-parasite interaction, of Merino sheep bred to be either genetically resistant or susceptible to H. contortus infection. Two different selection flocks, the Haemonchus selection flock (HSF) and the Trichostrongylus selection flock (TSF), and each contains a resistant and susceptible line, were studied. The TSF flock was seemingly more responsive to both primary and repeated infections than HSF. A total of 127 and 726 genes displayed a significant difference in abundance between resistant and susceptible animals in response to a primary infection in HSF and TSF, respectively. Among them, 38 genes were significantly affected by infection in both flocks. Gene ontology (GO) enrichment of the differentially expressed genes identified in this study predicted the likely involvement of extracellular exosomes in the immune response to H. contortus infection. While the resistant lines in HSF and TSF relied on different mechanisms for the development of host resistance, adhesion and diapedesis of both agranulocytes and granulocytes, coagulation and complement cascades, and multiple pathways related to tissue repair likely played critical roles in the process. Our results offered a quantitative snapshot of changes in the host transcriptome induced by H. contortus infection and provided novel insights into molecular mechanisms of host resistance.

Immunization with recombinant rHc23 partially protects lambs against trickle infections by Haemonchus contortus.

BACKGROUND: Haemonchosis is one of the most economically important parasitic diseases affecting small ruminants all over the world. Chemotherapeutic control has several shortcomings (limited anthelmintic arsenal, frequent resistance) and is hardly affordable by many farm economies. A recombinant antigen (rHc23) was shown to induce significant protection in vaccination trials with single dose challenges and different adjuvants. RESULTS: Lambs were vaccinated with 100 µg rHc23/dose + bacterial immunostimulant (BI) (LPS from Escherichia coli + Propionibacterium acnes extract) (days - 2, 0, 7 and 14) and subjected to a trickle infection with two dosages [6x, 1000 infective larvae (L3) or 6x, 2000 L3]. Vaccinated lambs showed a significant antibody response against rHc23 and Haemonchus contortus soluble extract as assessed by ELISA and Western blot (WB). Fecal egg counts (epg) along the experiment of vaccinated and BI treated lambs were significantly reduced. All vaccinated animals showed total egg output and abomasal helminth burdens (median, average) lower than those from unvaccinated or BI-treated animals lambs although differences were not statistically significant. CONCLUSIONS: Vaccination with 100 µg rHc23/dose + BI against H.contortus trickle infections apparently induced lower epg values and helminth burdens at the end of the experiment. Intragroup individual variations did not allow to obtain conclusive results and more research is needed including adjuvants and larger groups of animals to validate the potential value of rHc23 as candidate to develop a recombinant vaccine for lambs haemonchosis.

Ultrastructure changes induced by the phloroglucinol derivative agrimol G isolated from Leucosidea sericea in Haemonchus contortus.

Plant extracts used for the treatment of helminth infections in sheep are an alternative to chemical anthelmintic drugs. Previous studies have reported the anthelmintic activity of acetone leaf extracts of Leucosidea sericea. For this study, we evaluate the ultrastructure changes induced by the acetone leaf extract of L. sericea and the component agrimol G (AG) that was isolated for the first time on adult haemonchus parasites. Adult haemonchus parasites harvested from sheep were incubated with the plant extract and AG for 3 h and evaluated by both scanning and transmission electron microscopy in comparison and in combination with albendazole or ivermectin. In all cases the method of evaluation shows ultrastructural changes, with albendazole inducing mitochondrial damage and ivermectin inducing muscle degeneration, both as previously described. Incubation with the plant extract and AG resulted in the formation of numerous non-membrane bound multi-vesicular like bodies and evenly spread disruptions/erosion in the epicuticle. Combining AG with ivermectin or albendazole resulted in an absence of effect of AG. Based on the structural changes induced by AG, together with the absence of an effect in combination with ivermectin and albendazole would suggest a disrupted microtubular network. The latter does however require biochemical confirmation.

Evaluation of encapsulated anethole and carvone in lambs artificially- and naturally-infected with Haemonchus contortus.

Molecules from natural sources, such as essential oils, have shown activity against parasites in vitro, but have not yet been explored extensively in vivo. Anethole and carvone (10% each), encapsulated with 80% of a solid matrix, referred to as EO (encapsulated oils), were tested in vivo in 2 experiments. In Experiment 1: Lambs were artificially infected with multidrug resistant Haemonchus contortus, or left uninfected, and treated (or not) with 50 mg/kg bw (body weight) of EO in a controlled environment. Thirty-two male lambs were kept in individual cages for a period of 45 days, after which animals were evaluated for parasitological, hematological, toxicological, and nutritional parameters. After 45 days of treatment, EO at 50 mg/kg bw provided a significant (P ≤ 0.05) reduction in fecal egg count (FEC). Although FEC was reduced, animals from both treatments had similar counts of total adult worms. The low FEC was caused probably by a significant reduction (P ≤ 0.05) in both male worm size and female fecundity. Dry matter intake of uninfected controls was significantly (P ≤ 0.05) reduced, although no toxicity was observed in treated animals. Thus, in Experiment 2, conducted for five months we used an EO dose of 20 mg/kg bw. Thirty-four weaned lambs, free of parasites, were divided in two groups and kept in collective pens. One group received EO at 20 mg/kg bw mixed with concentrate for 5 months and the other was kept as a control group (CTL). Parasitological and hematological parameters as well as body weight were evaluated. In the first 2.5 months, CTL and EO groups were confined, and both presented similar clinical parameters. Then, animals were allotted to graze on contaminated pastures to acquire natural infection for the next 2.5 months. The infection was patent after 25 days and both groups had similar decreases in weight gain, increases in FEC, and decreases in blood parameters. Coprocultures from CTL and EO groups established that parasite population was 90% Haemonchus sp. We concluded that the technology of encapsulation is safe and practical to deliver to lambs at the farm level and anethole and carvone at 50 mg/kg bw caused a significant decrease in FEC and, consequently, in pasture contamination by free living stages of H. contortus. However, EO at 20 mg/kg bw was not effective to prevent or treat sheep naturally-infected with gastrointestinal nematodes.

Abomasal nematode species differ in their in vitro response to exsheathment triggers.

A crucial step in the infection process of grazing ruminants by gastro-intestinal nematodes is the exsheathment of the infective third-stage larva following ingestion. Recently, heat shock was shown to play an important role in the carbon dioxide (CO2)-dependent exsheathment response in Haemonchus contortus. The current in vitro study set out to evaluate the role of heat shock in other abomasal species. In rumen fluid, all species tested exsheathed rapidly and efficiently in response to heat shock and CO2. This response was significantly higher compared to slow temperature changes, supporting the hypothesis that heat shock plays an important role in vivo. However, in artificial buffer, the effect of heat shock was species-dependent. For H. contortus and Ostertagia leptospicularis, the response in artificial buffer was similar to rumen fluid. In contrast, Ostertagia ostertagi and Teladorsagia circumcincta exsheathment was significantly lower and/or slower in artificial buffer, and there was no benefit of heat shock. For these two species, it appears that there are co-factors in the rumen fluid, in addition to heat shock and CO2, contributing to exsheathment. Overall, the data indicate that there are significant differences between abomasal species in their response to exsheathment triggers.

Impaired interleukin-4 signalling promotes establishment of Haemonchus contortus in sheep.

Reduced worm burden in St. Croix (STC) sheep during Haemonchus contortus (Hc) infection is predicated on rapid interleukin-4 (IL-4) signalling and T helper type 2 immune (Th2) response. The aim of these studies was to further elucidate differences in Th2 responses by STC and Suffolk (SUF) sheep. Ten days after challenge Hc infection, peripheral blood mononuclear cells (PBMC) were collected and cultured with larval (HcLA) and adult worm (HcWA) antigen. STC PBMC produced nearly twice as much IL-4 as SUF (823.57 pg/mL vs 454.28 pg/mL) at 6 hours of HcLA culture despite no difference in IL-4 gene expression and the IL-4 receptor (IL4Rα) was upregulated in STC PBMC but was undetectable in SUF. Expression of other Th2-type genes were increased in STC PBMC including IL13, IL5 and MRC1. IL-4 supplementation to HcLA culture was insufficient to achieve upregulation of Th2 genes in SUF PBMC. Production of IL-4 did not occur in SUF PBMC until 24 hours after culture with HcLA, and expression of IL4 in the abomasum was similarly delayed until 10 days after challenge infection, which was associated with significantly higher larval burden (530 vs 16). These data demonstrate delayed upregulation of Th2 genes in SUF sheep contributes to susceptibility to Hc.

Climate influences assemblages of abomasal nematodes of sheep on steppe pastures in the east of Algeria.

It is a common view that assemblages of parasitic nematodes are influenced by climatic conditions; however, there are only a few articles available regarding those that infect farm animals. We investigated the relationship between climate variables and infection with abomasal trichostrongyles in 335 1-year-old rams grazed on steppe pastures in Eastern Algeria. Abomasa were collected from 12 local slaughterhouses located in four climatic areas (from humid to arid) and the worms extracted, identified and counted. The abundance was low and the fauna composed primarily of Teladorsagia circumcincta, Marshallagia marshalli and Trichostrongylus sp. The high percentage of M. marshalli is typical of steppe areas. Ostertagia ostertagi and Haemonchus contortus were present in low numbers. Rainfall was the most important climatic variable related to the main species. This relationship was not linear for M. marshalli but an optimal rainfall was detected (350-400 mm/year). The more complex climatic indicators used in the study did not demonstrate a more significant correlation than rainfall. The predictive value of rainfall on the abundance or proportion of species in the assemblage was modest but highly significant. The seasonality of assemblages was different between the two main sub-climates (sub-humid and semi-arid).

Molecular and biochemical characterisation and recognition by the immune host of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the abomasal nematode parasite Teladorsagia circumcincta.

A 1023 bp full length cDNA encoding Teladorsagia circumcincta GAPDH (TeciGAPDH) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth GAPDH sequences. The predicted protein consisted of 341 amino acids and was present as a single band of about 38 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciGAPDH with homologues from other helminths showed that the greatest similarity (93%) to the GAPDH of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 °C, the optimum pH for TeciGAPDH activity was pH 8, the Vmax was 1052 ± 23 nmol min-1 mg-1 protein and the apparent Km for the substrate glyceraldehyde-3-phosphate was 0.02 ± 0.01 mM (both mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TeciGAPDH in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native GAPDH indicates similar antigenicity of the two proteins.

Molecular and biochemical characterisation and recognition by the immune host of the enolase of the abomasal nematode parasite Teladorsagia circumcincta.

A 1299 bp full length cDNA encoding Teladorsagia circumcincta enolase (TeciENO) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. Helminth enolase sequences were used to construct a phylogenetic tree. The predicted protein consisted of 433 amino acids and was present as a single band of about 50 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciENO with homologues from other helminths showed 98% similarity with Haemonchus contortus enolase, 78-95% similarity to other nematode sequences and 72-75% similarity to cestode and trematode enolases. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. The optimum pH for TeciENO activity at 25 °C was pH 7, the Km for 2-phophoglycerate 0.09 ± 0.04 mM and the Vmax was 604 ± 6 nmol min-1 mg-1 protein (both mean ± SD, n = 2). TeciENO activity was inhibited by 11.5% by 1 mM citrate (p < 0.001). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TeciENO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native enolase indicates similar antigenicity of the two proteins.

A new thioredoxin reductase with additional glutathione reductase activity in Haemonchus contortus.

We report, herein, the purification to homogeneity and the biochemical and kinetic characterization of HcTrxR3, a new isoform of thioredoxin reductase (TrxR) from Haemonchus contortus. HcTrxR3 was found to have a relative molecular weight of 134,000, while the corresponding value per subunit obtained under denaturing conditions, was of 67,000. By peptide mass spectrophotometric analysis, HcTrxR3 was determined to have 99% identity with the H. contortus HcTrxR1 although, and most importantly, they are different in their amino acid sequence in two amino acid positions: 48 (isoleucine instead of leucine) and 460 (leucine instead of proline). The enzyme catalyzes NADPH-dependent reduction of DTNB and, unexpectedly, it follows the pattern of glutathione reductases (GR) performing the reduction of oxidized glutathione (GSSG) to reduced glutathione using NADPH as the reducing cofactor. Hence, it is important to highlight this enzyme's new and unexpected condition that makes so special and one our main finding. Enzyme Kcat values for DTNB, GSSG and NADPH were 12, 3 and 8 s-1, respectively. HcTrxR3 developed, into specific TrxR substrates: ebselen and sodium selenite, with activity at 0.5 and 0.068 (U/mg), respectively; and 0.044 (U/mg) for S-nitrosoglutathione through its GR activity. The enzyme was inhibited by gold compound auranofin (AU), a selective inhibitor of thiol-dependent flavoreductases. Although HcTrxR3 has both TrxR and GR activity as thioredoxin glutathione reductase (TGR) does, it is a TrxR because it has no glutaredoxin domain and it does not develop any hysteretic behavior as does TGR. The importance of this new enzyme is potential to further clarify the detoxification and haemostasis redox mechanism in H. contortus. Likewise, this enzyme could also be a protein model to recognize more differences between TrxR and GR.

Characterization of Ostertagia ostertagi annexin-like proteins at different developmental stages.

Ostertagiosis remains an economically important parasitic disease in cattle in the temperate regions of the world. Repeated exposures to Ostertagia ostertagi in calves cause significant pathology in the abomasum but elicit little protective immunity. The larvae use the host's gastric glands as a niche for development, where the parasite completes its parasitic stages, while in the gastric glands, the larvae must down-regulate the host inflammatory immune responses. Annexin (ANX) A1, commonly found in most eukaryotes, is heavily involved in controlling anti-inflammatory responses by binding receptors on leukocytes. We hypothesized, therefore, that parasite proteins of the ANX family may be involved in host-parasite interactions during ostertagiosis. BLASTN search with the bovine ANXA1 identified two families of Oos-ANX like proteins (Oos-ANXL), each of which was highly conserved at the genetic level and identical at the amino acid sequence level. Oos-ANXL-1 is encoded by two transcripts and Oos-ANXL-2 by 20 transcripts. The present study characterized one Oos-ANXL, representing the most abundant Oos-ANXL, which was further defined as Oost-ANXL-2.1. Oos-ANXL-2.1 with a coding sequence of 519 bp was PCR-amplified, cloned, and expressed. Oos-ANXL-2.1 was immunolocalized to both L3 and adult, but not L4. The staining appeared to be associated with the gut and hypodermis in L3, but it was specifically localized to the hypodermis in adult worms. Western blots detected three protein bands in parasite lysates using anti-recombinant Oos-ANXL-2.1 antibody. Integrated optical density for each of the 3 Oos-ANXL-2s or the total Oos-ANXL-2s detected by Western blots (P < 0.05) was higher in adult worms than in L3 or L4. The results indicate that the production of Oos-ANXL-2s is developmentally regulated and most abundant in the adult worm. This rather large family of proteins could be a potential vaccine target against O. ostertagi infection and warrants further investigation.

Early IL-4 gene expression in abomasum is associated with resistance to Haemonchus contortus in hair and wool sheep breeds.

Early immune events associated with reduced larval burden remain unclear in parasite-resistant breeds of sheep. Therefore, our objective was to determine breed differences in immune-related gene expression following infection with H. contortus. Gene expression in abomasal tissue and mucosa and in abomasal lymph nodes (ALN) was measured in 24 St. Croix (hair) lambs and 24 Dorset x (Finn-Rambouillet) (wool) lambs at 0 (uninfected), 3, 5 and 7 days after infection with 10 000 L3 H. contortus larvae. Expression of IL-4 in abomasal mucosa was detected on day 3 and increased to day 7 in hair lambs, but was not detectable in wool lambs. Genes that recruit neutrophils (CXCL1) and macrophages (MCP1) were upregulated in abomasal mucosa of hair lambs. Genes associated with alternative macrophage activation (ARG-1) and eosinophil activation (Gal-14) were also upregulated in the abomasal mucosa of hair lambs. Tissue remodeling genes (MMP13, PDGF) and tumour necrosis factor alpha (TNF-α) and MCP1 were upregulated in abomasal tissue of wool lambs; these lambs also had greater expression of forkhead box P3 in ALN. These data indicate a role for early IL-4 expression locally and demonstrate potential downregulation of immunity in wool sheep that could facilitate establishment of H. contortus.

Experimental infection of calves with Haemonchus placei or Haemonchus contortus: Assessment of clinical, hematological and biochemical parameters and histopathological characteristics of abomasums.

The present study had the primary objective of evaluating clinical, hematological and biochemical parameters, as well as observing anatomical and histopathological characteristics of abomasums, from calves prime-infected with Haemonchus contortus or H. placei. Ten male Holstein newborns were subdivided in three groups (GI placebo; GII infected with H. contortus; GIII inoculated with H. placei). Eye mucosa staining was evaluated. Hematological and biochemical tests were performed on animals. The euthanasia of all ten experimental calves was performed on the 42nd day post-inoculation. Fragments were collected from each of all 10 abomasums for histopathological analysis. Discrete submandibular edema was diagnosed in animals from both infected groups (H. contortus or H. placei). However, there were no significant changes (P > 0.05) in the color of the ocular mucosa of calves from all three experimental groups across the entire experimental period. Hematological and biochemical changes diagnosed on animals could not be linked to infections by species of Haemonchus spp. Regarding histopathological exams, it was possible to diagnose hypertrophy, hyperplasia, binucleated cells, inflammatory infiltrate, multifocal hemorrhage and edema in abomasums from calves of both groups infected with H. placei and H. contortus. It can, thus, be concluded that not only are calves susceptible to infections by both Haemonchus species, but they can also present clinical changes and similar anatomic histopathological lesions independent of being infected by Haemonchus placei or Haemonchus contortus. These results reflect a negative effect on helminth control by mixed grazing between sheep and cattle, especially when using calves.

Molecular and biochemical characterisation of abomasal nematode parasites Teladorsagia circumcincta and Haemonchus contortus phosphofructokinases and their recognition by the immune host.

Full length cDNAs encoding phosphofructokinase (PFK) were cloned from Teladorsagia circumcincta (TcPFK) and Haemonchus contortus (HcPFK). TcPFK (2361 bp) and HcPFK (2367 bp) cDNA encoded 787 and 789 amino acid proteins respectively. The predicted amino acid sequences showed 98% similarity with each other and 70% with a Caenorhabditis elegans PFK. Substrate binding sites were completely conserved in both proteins. Soluble N-terminal His-tagged PFK proteins were expressed in Escherichia coli strain BL21, purified and characterised. The recombinant TcPFK and HcPFK had very similar kinetic properties: the pH optima were pH 7.0, Km for fructose 6-phosphate was 0.50 ± 0.01 and 0.55 ± 0.01 mM respectively when higher (inhibiting concentration, 0.3 mM) ATP concentration was used and the curve was sigmoidal. The Vmax for TcPFK and HcPFK were 1110 ± 16 and 910 ± 10 nM min(-1 )mg(-1) protein respectively. Lower ATP concentration (non-inhibiting, 0.01 mM) did not change the Vmax for TcPFK and HcPFK (890 ± 10 and 860 ± 12 nM min(-1 )mg(-1) protein) but the substrate affinity doubled and Km for fructose 6-phosphate were 0.20 ± 0.05 and 0.25 ± 0.01 mM respectively. Recognition of TcPFK and HcPFK by mucosal and serum antibodies in nematode exposed animals demonstrates antigenicity and suggests involvement in the host response to nematode infection.

Analysis of the mucosal immune responses induced by single and trickle infections with the bovine abomasal nematode Ostertagia ostertagi.

The purpose of this study was to provide more information on the kinetics of the immunological changes occurring in the abomasal mucosa after single and trickle infections with the bovine parasite Ostertagia ostertagi. The time course analysis of gene expression revealed that the major changes coincided with the emergence of adult worms from the gastric glands. These changes consisted of a simultaneous upregulation of Th1- and Th2-type cytokines. In addition, a single O. ostertagi infection elicited an upregulation of the epithelial-derived cytokine IL33, while TSLP expression levels were not impacted. Apart from the massive increase in inflammatory cytokines IL6, IL17 and IL21, O. ostertagi infection also elicited an upregulation of the immunosuppressors TGFB, IL10 and ARG1, as well as NK and γδ-T cell markers. Furthermore, the cytotoxic factors granulysin, perforin and granzyme B were upregulated following an O. ostertagi infection. Analysis of cytokine transcript levels in animals receiving trickle infections for 60 days showed a similar trend as observed following a single infection except for IL33, IL6, GATA-3, TBX21 and NCR1, which were no longer upregulated after trickle infections. Finally, the long trickle infections were associated with mucosal eosinophilia and mastocytosis.

Functional investigation of a QTL affecting resistance to Haemonchus contortus in sheep.

This study reports a functional characterization of a limited segment (QTL) of sheep chromosome 12 associated with resistance to the abomasal nematode Haemonchus contortus. The first objective was to validate the identified QTL through the comparison of genetically susceptible (N) and resistant (R) sheep produced from Martinik × Romane back-cross sheep. The R and N genotype groups were then experimentally infected with 10 000 H. contortus larvae and measured for FEC (every three days from 18 to 30 days post-challenge), haematocrit, worm burden and fertility. Significant differences in FEC and haematocrit drop were found between R and N sheep. In addition, the female worms recovered from R sheep were less fecund. The second step of the characterization was to investigate functional mechanisms associated with the QTL, thanks to a gene expression analysis performed on the abomasal mucosa and the abomasal lymph node. The gene expression level of a candidate gene lying within the QTL region (PAPP-A2) was measured. In addition, putative interactions between the chromosome segment under study and the top ten differentially expressed genes between resistant MBB and susceptible RMN sheep highlighted in a previous microarray experiment were investigated. We found an induction of Th-2 related cytokine genes expression in the abomasal mucosa of R sheep. Down-regulation of the PAPP-A2 gene expression was observed between naïve and challenged sheep although no differential expression was recorded between challenged R and N sheep. The genotyping of this limited region should contribute to the ability to predict the intrinsic resistance level of sheep.

Local cytokine transcription in naïve and previously infected sheep and lambs following challenge with Teladorsagia circumcincta.

BACKGROUND: The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites affecting sheep in temperate regions. Infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all classes of anthelmintic drugs, teladorsagiosis is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Protective immunity develops slowly, wanes rapidly and does not appear to be as effective in young lambs. To investigate the development of immunity to T. circumcincta in sheep and lambs, we used cytokine transcript profiling to examine differences in the abomasal mucosa and gastric lymph node of naïve and previously infected sheep and lambs following challenge. RESULTS: The results of these experiments demonstrated that the abomasal mucosa is a major source of cytokines during abomasal helminth infection. A local Th2-type cytokine response was observed in the abomasal mucosa and gastric lymph node of the previously infected sheep and lambs when compared with those of the naïve during the early stages of infection. In contrast, a pro-inflammatory component more was evident in the abomasal mucosa and gastric lymph node of the naïve sheep when compared with those of the previously infected, which was not observed in the lambs. CONCLUSIONS: The greater levels of Th2-type cytokine transcripts in both the abomasum and gastric lymph node of the previously infected compared with naïve sheep and lambs emphasises the importance of these mechanisms in the immune response to T. circumcincta infection. Younger lambs appear to be able to generate similar Th2-type responses in the abomasum suggesting that the increased morbidity and apparent lack of resistance in younger lambs following continuous or repeated exposure to T. circumcincta is unlikely to be due to a lack of appropriate Th2-type cytokine production.