Letteratura scientifica selezionata sul tema "Teladorsagia circumcincta"

AbstractItalian hare Lepus corsicanus De Winton, 1898, is a true species living in simpatry with Lepus europaeus in mainland Italy and with Oryctolagus cuniculus in Sicily, where L. europaeus is absent. Up to date, nobody has studied the helminths of this endemic Italian Mammal. Therefore, in order to describe Italian hare gastro-intestinal helminths, gastro-intestinal tract of 29 Italian hares, coming from mainland Italy (#14) and from Sicily (#15) were collected between 1997 and 2009. Twentyfive hares were positive for at least one parasite (86 %). Six parasite species were isolated, 2 cestodes (Cittotaenia pectinata, prevalence 3 %) and Paranoplocephala sp., 3 %) and 4 nematodes (Trichostrongylus retortaeformis, 86 %, Graphidium strigosum, 14 %, Trichuris sp., 10 % and Teladorsagia circumcincta, 7 %). Both Teladorsagia circumcincta circumcincta and Teladorsagia circumcincta trifurcata morphotypes were identified. Comparison with available data regarding L. europaeus and O. cuniculus in Italy is provided. Being not T. circumcincta and Paranoplocephala sp. typical parasites of leporids, a description of the observed specimens is given.

En el presente estudio se demostró que Teladorsagia circumcincta (Stadelmann, 1894) y T. trifurcata (Ransom, 1907), nemátodes Trichostrongylidae de la sub-família Ostertagiinae, parasitan un espectro de hospedadores similares. La ausencia de barreras reproductivas entre T. trifurcata y T. circumcincta, así como la estabilización rápida de las proporciones de las dos entidades en el seno de la población, evidencia que T. trifurcata es un morfo de T. circumcincta.

Fructosamine concentrations reflect protein status and because infection with Teladorsagia circumcincta can induce a relative protein deficiency, we examined the usefulness of fructosamine concentrations as markers of the intensity of infection in naturally infected lambs. Fructosamine concentration was a heritable trait and variation in fructosamine concerntrations was associated with differences in body weight, and a variety of parasitological variables; animals with increased fructosamine concentrations grew more quickly, had increased faecal egg counts in one of the three study years, had decreased pepsinogen concentrations and decreased IgA activity against 4th-stage larvae of T. circumcincta. Fructosamine concentrations were also associated with variation in the subsequent acquisition of nematodes and in the length of adult female T. circumcincta; lambs with increased fructosamine concentrations had fewer nematodes but the mean length of adult female T. circumcincta was longer. Therefore fructosamine concentrations are potentially useful indicators of the severity of nematode infection and may predict magnitude of subsequent infection.

AbstractTeladorsagia circumcincta is a polygamous nematode that exhibits morphological polymorphism. Sex ratio is typically female biased and the male nematodes occur in association with the genetically similar, minor morphotypes Teladorsagia davtiani and Teladorsagia trifurcata. In experimental infections, sex ratio (proportion male) and the proportion of minor male morphs observed have been shown to be influenced by both host and nematode-related factors. As similar investigations from natural systems are rare, this study examined whether sex ratio and minor male morph frequency were associated with host age and sex and nematode infra-population size in the isolated Soay sheep population on St Kilda. Count data for Teladorsagia were analysed for sheep of all age classes and both sexes from the winters of three consecutive population crashes (1999, 2002 and 2005). Generally, the intensity of Teladorsagia nematodes increased with host age until the age of 2 years before decreasing. In 2005, abundance of nematodes was generally higher than in the previous crashes, nematode sex ratio was negatively associated with host age and tended to be negatively associated with nematode intensity. Within the male nematode subpopulation, T. circumcincta always predominated, followed by T. davtiani and then T. trifurcata, with little variation in the relative proportions between hosts. The presence of each minor morph was primarily associated with the intensity of male T. circumcincta and, in those hosts where all three male morphs were detected, intensity of each minor morph was most associated with intensity of Teladorsagia females. Therefore, in a year when the nematode was generally more abundant, sex ratio appeared to be influenced by both host and nematode-related factors, whereas in all years examined, the frequency of morphological polymorphism was primarily density dependent.

Faecal egg counts and peripheral blood eosinophil counts were taken from Scottish Blackface lambs following natural, predominantly Teladorsagia circumcincta infection. Peripheral eosinophil concentrations were higher in animals with lower egg counts but only in lambs that were at least 3 months of age. The reduced egg counts were due to reduced fecundity of T. circumcincta; there was no association with the number of adult T. circumcincta. Associations with the number of parasites from other species of gastrointestinal nematodes appeared to be neutral or favourable. Estimated heritabilities for eosinophil concentrations in 4- and 5-month-old lambs were 0·48±0·16 and 0·43±0·17, respectively. Therefore, under defined circumstances, eosinophil concentrations may be a useful indicator of resistance to predominantly T. circumcincta infection.

Teladorsagia circumcincta is a major financial burden on the UK sheep farming industry. Disease control is becoming increasingly difficult due to the rapid emergence of anthelmintic resistance. This has prompted the search for alternative, sustainable control measures, including vaccination. Vaccine design would be aided by a thorough knowledge of the mechanisms involved in immunity to T.circumcincta. Most research has focussed on humoral and cellular responses to infection with this nematode. This thesis focuses on the impact of infection with regards to the proteins found locally within the abomasum. Using a well established infection model, proteomic analysis of lymph draining the abomasum was carried out by means of 2-dimensional electrophoresis (2-DE). The identity of many of the proteins in gastric lymph was revealed by means of MALDI-TOF analysis. The relative quantities of the lymph proteins were monitored over time using gel analysis software in both primary infection and immune challenged infection models. This study revealed a number of proteins of interest, including the acute phase proteins serum amyloid A, alpha-1 acid glycoprotein and haptoglobin, as well as the actin depolymerising protein, gelsolin. The effect of infection and immunity to T.circumcincta on these proteins was investigated further by means of biochemical assays, western blotting and real-time PCR. The impact of infection on the permeability of the abomasal mucosa will affect the resultant gastric lymph proteome. This “leak lesion” phenomenon is well documented in T.circumcincta infection but the underlying cause is unknown. Tight junction proteins in the abomasum were studied, using immunofluorescence techniques, in an attempt to define the role of these proteins in this important immunological/pathological event. The aim of this thesis is to contribute to the knowledge of innate immune responses and local pathology occurring within the abomasum during T.circumcincta infection.

The main aims of this study were to identify and characterise proteins from T. circumcincta that may induce a protective immune response in the host and to learn more about the biology of the worm. In order to identify possible protective antigens, a complementary DNA (cDNA) library prepared from adult worms was screened with serum from an animal that was protected against a single challenge infection after vaccination with a T. circumcincta protein fraction (S3 TSBP). Forty five immunopositive cDNA clones were identified, of which sixteen had homology to galectin. Of the remaining clones, the majority shared homology with two metabolic enzymes, methylmalonate semialdehyde dehydrogenase and 10-formyltetrahydrofolate dehydrogenase, that have not been characterised in nematodes. A single clone with homology to the antioxidant enzyme, catalase, was also identified. These three enzymes were selected for further investigation on the basis of their roles in nematode metabolism and therefore, their potential as vaccine candidates. Characterisation of T. circumcincta excretory/secretory material (ES) was also performed. L4 and adult worms were cultured in vitro and the proteins released were separated by 1D electrophoresis and analysed by Tandem Liquid Chromatography Mass Spectrometry and N-terminal sequencing. This identified proteins showing similarity to, amongst others, metabolic enzymes, structural components, antioxidants, globin-like proteins and cysteine proteases, present in online databases but not previously characterised in T. circumcincta. This study has identified several novel T. circumcincta proteins that may have potential as future vaccine or drug targets. It has also provided further information regarding the biology of the worm.

Teladorsagia circumcincta, an economically-important abomasal nematode of small ruminants in temperate regions worldwide, is currently controlled with a combination of anthelmintics and pasture management. Anthelmintic resistance has emerged and vaccination is a potential alternative control strategy, as protective immunity in sheep can be acquired after repeated exposure to the parasite. Abomasal mucosal IgA responses in immune sheep have been correlated with delayed worm development and reduced faecal egg counts. However, recombinant vaccine development against parasitic nematodes has had limited success, and one of the reasons may be unsuitable expression systems for antigen production leading to incomplete or inadequate post-translational modifications such as glycosylation and tertiary protein folding, resulting in incorrect epitope structures for antibody binding. In this thesis, to address this issue, “native” infective larval (L3) antigen targets of protective immune responses and synthetic peptide sequences which mimic structural epitopes on these antigens were identified. Abomasal mucosal IgA was used as a probe to identify native immunogenic antigens from T. circumcincta L3. IgA was purified from abomasal mucus of animals rendered immune by repeated experimental infection and a custom antibody-affinity column was created and used to purify antigens from an L3 somatic PBS-soluble extract. Affinity purified L3-antigen-specific IgA levels in sheep with varying levels of immunity to T. circumcincta were positively correlated (rs = 0.853, P < 0.001) with both the total IgA concentration in efferent gastric lymph after parasite challenge, and with the percentage of inhibited fourth-stage (L4) larvae present in the gastric glands of the immune hosts (rs = 0.534, P = 0.007). In contrast, a negative correlation between the levels of affinity-purified L3 antigen-specific IgA and total T. circumcincta burden was observed (rs = -0.565, P = 0.004). Proteomic analysis of the IgA-affinity purified L3 extract identified a number of proteins which represent potential vaccine candidate molecules in other helminth species, including paramyosin, superoxide dismutase, galectin, activation-associated secreted proteins and fatty-acid retinol-binding proteins. As a first step towards the development of a novel vaccine based on IgA-binding peptide mimics of native structural epitopes, phage display libraries were used to screen antibodies, from sheep rendered immune to T. circumcincta by experimental infection. These antibodies were affinity-purified before use and specifically bound T. circumcincta L3 glycans or, alternatively, surface antigens on exsheathed T. circumcincta L3. Five peptide sequences which mimic L3 antigenic epitopes were identified and positive correlations existed between peptide-specific IgA levels and both the total IgA concentration in efferent gastric lymph after parasite challenge and the percentage of inhibited L4 present (rs > 0.621, P < 0.001 to P < 0.05). In contrast, negative correlations between the levels of peptide-specific IgA and the total nematode burden were observed (rs > -0.528, P < 0.01 to P < 0.05). In conclusion, the selected phage clones may therefore represent vaccine candidates if they could be presented to the ovine immune system in an appropriate fashion.

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The mechanisms whereby parasitic nematodes become resistant to anthelmintics, particularly ivermectin, remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of gene expression changes. The present study focused on Teladorsagia circumcincta; the predominant parasitic gastrointestinal nematode species in the UK and the predominant resistant species. The role of changes in gene expression were investigated in an ivermectin-susceptible isolate (CVL) and a multidrug resistant isolate (MOTRI), utilising a range of molecular biological techniques. In the first experiment, a panel of novel putative ivermectin resistance genes were identified from T. circumcincta, comprising 11 partial P-glycoprotein (Pgp) and 3 partial Cytochrome P450 (CYP) sequences. Both Pgps and CYPs have been implicated in the handling and metabolism of xenobiotics in other biological systems, but have not been investigated in T. circumcincta to date. Initial results, using semi-quantitative PCR identified changes in expression of this panel of genes between the CVL and MOTRI isolates. Constitutive differences in expression of the Pgps and CYPs between CVL and MOTRI were determined using the ΔΔCt TaqMan® real-time PCR method. A statistically significant increase in expression was observed for TeciPgp-9 NBD2 across all life-cycle stages but most notably in eggs (55-fold increase). A statistically significant reduction in expression of TeciPgp-2 NBD2 was observed in all but the adult stages of MOTRI compared to CVL. Analysis of a 208 base pair sequence of TeciPgp-9 NBD2 identified high levels of polymorphism, with at least four non-coding SNPs evident in the MOTRI isolate. These results merit further investigation. Inducible changes in the expression of the Pgps and CYPs were investigated in MOTRI before and after ivermectin treatment, using real-time PCR. Statistically significant fold changes in expression in most of the genes occurred in at least one life-cycle stage. Inducible expression of TeciPgp-2 NBD2 and TeciPgp-9 NBD2 was investigated further by comparing adult MOTRI parasites with those recovered three days after in vivo ivermectin exposure, and by exposing pools of MOTRI xL3 to ivermectin in the larval migration inhibition test. The survivors of ivermectin exposure exhibited a statistically significant reduced 13.68-fold expression of TeciPgp-2 NBD2 compared to MOTRI. Similarly, the MOTRI xL3 able to migrate in the presence of ivermectin in the LMIT had a 1.88-fold reduction in TeciPgp-2 NBD2 expression compared to MOTRI xL3 unexposed to ivermectin. These results indicate that inducible changes in TeciPgp-2 NBD2 and TeciPgp-9 NBD2 expression can occur, but the experimental design is critical to being able to identify the changes. In a more global approach, the transcriptomic response of MOTRI adults to in vitro ivermectin exposure was investigated using Roche 454 sequencing, generating 98,685 novel EST sequences, providing an important resource for a genome resource-poor organism. Objective bioinformatic analysis of the two datasets revealed statistically significant differences in the mean expression levels of the KEGG orthologous groups for ‘translation’, ‘amino acid metabolism’ ‘carbohydrate metabolism’ and ‘xenobiotic degradation and metabolism’. On combining the two datasets, and through application of a novel statistical method, 16 clusters of ESTs were identified as containing statistically significant differences in the mean proportion of exposed reads compared to unexposed reads under the conservative model, whilst a further 355 clusters were found to have statistically significant differences under the liberal model. One-way suppression subtractive hybridisation (SSH) was used to identify genes exhibiting increased expression in MOTRI adults compared to CVL adults. 28 contiguous sequences were identified from the SSH experiment; 6 contiguous sequences were selected for validation; 5 of these results were confirmed using semi-quantitative PCR. Each contig was BLAST searched against the Roche 454 dataset; contig SSH14 aligned most closely to one of the statistically significant clusters in the conservative model, SSHs 5, 6, 10 and 23 aligned most closely to statistically significant clusters in the liberal model. This suggests that changes in expression in these sequences occur both constitutively, between CVL and MOTRI isolates, and inducibly, following ivermectin exposure. This work has shown that changes in gene expression, particularly the constitutively reduced expression in TeciPgp-2 NBD2 and the constitutively increased expression in TeciPgp-9 NBD2 (coupled with the presence of SNPs) could play a role in allowing multidrug resistant T. circumcincta to survive ivermectin exposure. Roche 454 sequencing and SSH approaches identified gene expression changes associated with in vitro ivermectin exposure and ivermectin resistance. These could form the basis of a novel panel of candidate resistance genes whose altered expression profiles may allow multidrug resistant T. circumcincta to survive ivermectin exposure by some, as yet identified, mechanism. Finally, we have also shown that a multidrug T. circumcincta isolate is affected by ivermectin exposure and that changes in gene expression could have a role to play in the ivermectin resistance phenotype in T. circumcincta. The genetic changes underpinning these changes in gene expression remain to be elucidated, and need to be investigated in other isolates. These changes could form the basis of an ivermectin resistance molecular marker, to monitor the spread of resistance, and to evaluate management practices aimed at delaying its spread.

The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites to affect the farming of sheep and goats. T.circumcincta 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 currently available classes of anthelmintic drugs, it is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Infection of sheep with T.circumcincta triggers local changes in the abomasum characteristic of a T helper type-2 (Th2) driven immune response, including local eosinophilia, mastocytosis and increased mucus production, which leads to expulsion of the parasite. However, this protective immunity develops slowly during repeated exposure, wanes rapidly, and does not appear to be evident in young lambs. Vaccination to provoke early onset of protective immunity has therefore been suggested as an alternative means of control in the face of spreading anthelmintic resistance. Greater understanding of the development of immunity to T.circumcincta, and why this is delayed in lambs, would be useful in vaccine development. This thesis focuses on cytokine transcription profiling of the ovine abomasal mucosa and local lymphatic tissues. Changes in cytokine transcription over the course of a challenge infection with T.circumcincta were defined in helminth naïve sheep, and in previously infected sheep which have developed a degree of immunity during an eight week trickle infection, to clarify the mechanisms by which this immunity is orchestrated. This work demonstrated a clear Th2 cytokine response in the abomasal mucosa over the course of infection, which developed earlier and was more pronounced in the previously infected sheep; possibly owing to a population of polarised Th2-type cells built up during the previous infection. Suppression of Th1 cytokine transcription was also a prominent finding in the draining lymph node, which likewise occurred earlier in the previously infected sheep. Repetition of this experiment using younger lambs provided a possible explanation for the reduced resistance to T.circumcincta in this age group. While Th2 and proinflammatory cytokine responses in the abomasal mucosa demonstrated similar trends to those found in the older sheep, little suppression of Th1 cytokine transcription was observed in the draining lymph node. It is therefore suggested that the increased susceptibility of young lambs to T.circumcincta is not due to an inability to generate adequate Th2 responses, but an inability to suppress transcription of antagonistic Th1 cytokines.

The studies submitted herein have contributed to our understanding of ruminant immunology, host-parasite interactions during ruminant infection with nematode parasites, and potential vaccine strategies to combat parasitic gastroenteritis (PGE). PGE of sheep and cattle, caused by T. circumcincta and O. ostertagia respectively, is a major problem for the global farming industry both in terms of productivity and animal welfare. To date control of these parasites has relied on the use of anthelmintic drugs however the emergence of widespread anthelmintic resistance is driving the search for alternative methods of control. As ruminants do acquire immunity in the field, vaccination is one such alternative under investigation. The first three papers contributing to this thesis used modern immunological tools alongside a locally developed surgical technique to revisit a model of nematode infection in sheep, investigating the composition and kinetics of the ovine local immune response to infection with Teladorsagia circumcincta via cannulation of the efferent gastric lymph duct. A protective local secondary immune response was observed in sheep which had previously experienced infection with T. circumcincta, but was absent from naive sheep. This immune response consisted initially of a rise in TE and BE cell activity peaking at 3 and 5 days post challenge respectively, followed by a secondary parasiteEspecific IgA response from 5 days post challenge which correlated with stunting of parasite growth. Significant parasite loss occurred by 2 days post challenge, prior to detection of the secondary immune response, suggesting critical early events in the host-parasite interaction and the potential importance of larval antigens in these interactions. No difference was observed in either the manifestations of immunity, or the magnitude and quality of the immune response, between adult sheep and lambs. The fourth and fifth papers describe vaccine trials carried out in bovine and ovine hosts using detergent soluble proteins derived from 4th larval stage Ostertagia ostertagi and Teladorsagia circumcincta respectively as antigens. Substantial reduction in total faecal egg output of up to 85% was observed in the calf trials, but not in the sheep trials which attained a maximum reduction of 29% in total faecal egg output. The sixth paper is a transcriptomic study carried out using the Roche 454 sequencing platform to investigate the immediate responses of Teladorsagia circumcincta upon encountering ovine host tissue of either immune or naive status. Following larval exsheathing and 4 hours of exposure to either immune or naive abomasal environments the transcript level of several genes was observed to differ. Genes which were most upregulated in response to encountering the immune environment included a peptidyl-glycine alpha-amidating mono-oxygenase homologue and a small heat shock protein. The studies described herein represent a body of work carried out using up-to-date tools and technologies. The first three papers confirmed the existence of critical early events in the host-parasite interaction, pointing to the potential use of larval antigens as vaccine candidates described in the trials in papers 4 and 5, and leading to the in-depth transcriptomic analysis described in paper 6. Papers 4 and 5 demonstrated that while Teladorsagia circumcincta and Ostertagia ostertagi have similar life cycles and host-site predilection, and both the ovine and bovine host can develop immunity to incoming parasitic larvae in the field, important differences may exist in either the proteome of the fourth stage larvae and/or the nature of the host response. Paper 6 revealed that changes in T. circumcincta transcript levels in response to ovine-host immune status can be detected early in the host-parasite interaction.

Teladorsagia circumcincta is a parasitic nematode which is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The parasite has developed resistance to the major anthelmintic drug classes and this challenges its future control. Vaccination is a potential alternative control method since sheep are able to develop protective immunity against this parasite. Although potential vaccine candidates have been revealed, the increasing gene datasets suggest that vaccinetarget selection may be aided by screening methods such as RNAi. This is a reverse genetic mechanism that causes highly specific gene silencing which was initially described and applied to defining gene function in Caenorhabditis elegans. Nevertheless, its application was more difficult than anticipated in parasitic nematodes because of the inconsistency of the silencing effect. In the unsuccessful cases, did the dsRNA penetrate the parasite and activate the RNAi pathway? Thus far, there are no internal controls that indicate the activation of the pathway. Are the RNAi pathway genes constantly transcribed or are they ‘switched on’ in response to the dsRNA exposure? The initial aim of the study was to determine potential marker genes in the RNAi pathway that could indicate the activation of the pathway in C. elegans. After the exposure to dsRNA from two target genes, the transcript levels of three candidate marker genes (Ce-dcr-1, Ce-ego-1 and Ce-rsd-3) were examined and showed that exposure to dsRNA has no effect on the transcript levels of these genes making them inappropriate markers for the activation of the RNAi pathway. The two target-genes were Ce-cpr-4 and Ce-sod-4 which had been proven to be consistently susceptible and refractory to RNAi, respectively. Another aim of the project was to develop an RNAi platform in T. circumcincta for use as a screening method for potential vaccine candidates. The targets selected for the in vitro RNAi included: five members of the Activation-associated Secreted Proteins (ASPs); a Macrophage migration Inhibitory Factor-like (Tci-mif-1) and a Surface Associated Antigen gene (Tci-saa-1), all of which have been associated with vaccine-induced protective immunity. The selection of the ASPs was based on a bioinformatic and transcriptomic analysis of the ASPs in T. circumcincta. The results showed successful knock-down only for three out of five ASP targets after 1 hour of soaking in gene-specific double stranded RNA (dsRNA) which illustrates the inconsistency and the target specificity of RNAi in T. circumcincta which has been observed in the past with other parasitic nematodes. Inconsistencies were also observed within the successful ASP targets with the results not being reproducible after several successful experiments. Potential reasons for the inconsistencies were examined with the duration of larval storage being a critical factor. Larvae stored for a short or long period of time were susceptible and refractory to RNAi, respectively. Experiments were also conducted to investigate how the ASPs relate to extracellular microvesicles (EMVs). These vesicles are considered to play an important role in the intercellular communication between parasites and their hosts, and thus represent potentially useful vaccine and/or drug targets. Transmission electron microscopy (TEM) confirmed that EMVs are excreted / secreted by the parasite and the proteomic analysis revealed several types of proteins within the vesicles such as: ASPs, Actins, Metallopeptidases, and RAB proteins. A comparative analysis of EMVs, EMV-free ES (Excretory / Secretory) and total ES products showed that approximately 35% of the proteins found in the vesicles could also be identified in EMV-free ES and in total ES products, whilst the remaining 65% were present only in EMVs.