Primeiro relato de larvas de Eustrongylides sp. (Nematoda: Dioctophymatidae) em Hoplias malabaricus (Characiformes: Erythrinidae) no Estado de Rondônia, Amazônia Ocidental, Brasil

O presente artigo descreve o primeiro relato de larvas de Eustrongylides sp. em Hoplias malabaricus Bloch, 1794 (Characiformes: Erythrinidae) no Estado de Rondônia, Amazônia Ocidental, Brasil, sendo mais um parasita com a possibilidade de contaminar humanos no Estado.

Differences in diversity, structure, and variability between intertidal and subtidal meiofaunal assemblages

[EN] Meiofaunal assemblages from intertidal and shallow subtidal seabeds were studied at two sites (one dominated by volcanic sands and the other by organogenic sands) at Tenerife (Canary Islands, NE Atlantic Ocean) throughout an entire year (May 2000?April 2001). Specifically, we aimed (i) to test for differences in diversity, structure, and stability between intertidal and subtidal meiofaunal assemblages, and (ii) to determine if differences in the meiofaunal assemblage structure may be explained by environmental factors (granulometric composition, availability of organic matter, and carbonate content in sediments). A total of 103,763 meiofaunal individuals were collected, including 203 species from 19 taxonomic groups (Acari, Amphipoda, Cnidaria, Copepoda, Echinodermata, Gastrotricha, Isopoda, Insecta, Kinorrhyncha, Misidacea, Nematoda, Nemertini, Oligochaeta, Ostracoda, Polychaeta, Priapulida, Sipuncula, Tanaidacea, and Turbellaria). Nematodes were the most abundant taxonomic group. Species diversity was higher in the subtidal than in the intertidal zone at both sites, as a result of the larger dominance of a few species in the intertidal zone. The meiofaunal assemblage structure was different between tidal levels at both sites, the intertidal presenting greater temporal variability (multivariate dispersion) in the meiofaunal assemblage structure than the subtidal. Sediment grain size, here quantified by the different granulometric fractions, explained the variability in meiofaunal assemblage structure to a greater extent than the percentage of carbonates, a variable linked to sediment origin. This study revealed differences in diversity, assemblage structure, and variability between intertidal and subtidal meiofauna.

Influence of the combined disposal of sewage and brine on meiofauna

[EN] Confluence of anthropogenic influences is common in coastal areas (e.g., disposal of different pollutants like industrial and domestic sewage, brine, etc.). In this study we assessed whether the combined disposal of domestic sewage and brine altered patterns in the abundance and assemblage structure of subtidal meiofauna inhabiting sandy seabeds. Samples were collected in May 2008 and January 2009 at varying distances (0, 15, and 30 m) from the discharge point. Meiofaunal abundances were consistently larger at 0 m (1663.05 ± 1076.86 ind 10 cm?2, mean ± standard error) than at 15 m (471.21 ± 307.97 ind 10 cm?2) and 30 m (316.50 ± 256.85 ind 10 cm?2) from the discharge outfall. This pattern was particularly accentuated for nematodes. Proximity to the discharge point also altered patterns in meiofaunal assemblage structure, though temporal shifts in the sedimentary composition also contributed to explain differences in the meiofaunal assemblage structure. As a result, meiofauna may be a reliable tool for monitoring studies of the combined disposal of sewage and brine as long as potential confounding factors (here temporal changes in grain size composition) are considered.

Stato sanitario di Spigole (Dicentrarchus Labrax) ed Orate (Sparus Aurata) allevate in Grecia e in Italia in relazione alla presenza di agenti di Zoonosi ed Ectoparassiti patogeni

Le attività di ricerca della presente tesi di dottorato si sono focalizzata principalmente sulla parassitofauna dei pesci marini allevati in Grecia ed in Italia con particolare attenzione allo studio degli ectoparassiti di maggior rilievo sanitario in maricoltura ed alla ricerca di endoparassiti di potenziale interesse zoonosico, in particolare larve di nematodi Anisakidae del genere Anisakis. Nel corso del triennio sono stati sottoposti ad esami parassitologici 916 spigole (Dicentrarchus labrax) e 462 orate (Sparus aurata) prelevate presso diverse tipologie di allevamenti greci ed italiani. Per quanto concerne le spigole, la presenza di ectoparassiti è stata riscontrata nel 29,2% e nel 61,9% dei soggetti provenienti rispettivamente da impianti siti in Grecia ed in Italia, mentre le orate hanno presentato percentuali di positività rispettivamente del 87,5% e del 26,7%. Gli ectoparassiti dominanti sono risultati essere il monogeneo Diplectanum aequans nelle spigole ed il ciliato Cryptocaryon irritans e il monogeneo Furnestinia echeneis nelle orate, sebbene sia stato possibile studiare anche il coinvolgimento di altri ectoparassiti, quali il monogeneo Sparicotyle chrysophrii ed il dinoflagellato Amyloodinium ocellatum, nel determinismo di alcuni episodi morbosi. Le osservazioni istopatologiche hanno permesso di caratterizzare le lesioni causate dagli ectoparassiti a diverse intensità d’infestazione. Per quanto concerne la ricerca di parassiti zoonosici, con particolare riferimento agli stadi larvali di nematodi Anisakidae del genere Anisakis, si sono condotti esami parassitologici a livello di cavità viscerale e di muscolo laterale in tutti i soggetti provenienti da allevamenti in gabbia (626 soggetti, di cui 441 spigole e 185 orate). Tutti i soggetti esaminati sono risultati negativi, indicando come il rischio di infestazione da larve di nematodi anisakidi possa essere considerato trascurabile in spigole ed orate allevate in gabbia, come già dimostrato per il salmone atlantico (EFSA, 2010).

Valutazione della sostenibilità ambientale tramite metodologia LCA di sistemi per lo sfruttamento di fonti alternative di energia e materiali

La dissertazione ha riguardato l’analisi di sostenibilità di un sistema agronomico per la produzione di olio vegetale a fini energetici in terreni resi marginali dall’infestazione di nematodi. Il processo indagato ha previsto il sovescio di una coltura con proprietà biofumiganti (brassicacea) coltivata in precessione alla specie oleosa (soia e tabacco) al fine di contrastare il proliferare dell’infestazione nel terreno. Tale sistema agronomico è stato confrontato attraverso una analisi di ciclo di vita (LCA) ad uno scenario di coltivazione della stessa specie oleosa senza precessione di brassica ma con l’utilizzo di 1-3-dicloropropene come sistema di lotta ai nematodi. Allo scopo di completare l’analisi LCA con una valutazione dell’impatto sull’uso del suolo (Land use Impact) generato dai due scenari a confronto, sono stati costruiti due modelli nel software per il calcolo del Soil Conditioning Index (SCI), un indicatore quali-quantitativo della qualità del terreno definito dal Dipartimento per l’Agricoltura degli Stati Uniti d’America (USDA).

Untersuchungen zur Wirkung des Ginkgo-biloba-Extraktes EGb 761 auf die Proteinaggregation in der Huntington-Krankheit

Der Ginkgo biloba-Extrakt EGb 761 besteht aus einer Reihe pharmakologisch wirksamer Substanzen, welche gut beschriebene Wirkungen auf verschiedene potentiell zytoprotektive Signalwege ausüben und u.a. antioxidative Wirksamkeit haben. Folglich wurde EGb 761 bisher als eine natürliche Behandlung bei neurodegenerativen Erkrankungen mit zellulärem oxidativen Stress angewendet, einschließlich der Alzheimer-Krankheit (AD). Aufgrund von vielen gemeinsamen Merkmalen zwischen der AD und der Huntington-Krankheit (HD) wurde vermutet, dass EGb 761 eventuell auch positive Wirksamkeit bei der HD aufweisen könnte. rnDie Neuropathologie der HD wird durch pathologische Verlängerung an Glutamin-Wiederholungen im Huntingtin-Protein (polyQ-Protein) verursacht, wodurch es zu Fehlfaltungen im Protein kommt und hierdurch der proteasomale Abbau aberranter Proteine erschwert wird. Somit sollten in der vorliegenden Arbeit die EGb 761-Wirkungen auf die Proteasom-Aktivität und die Proteinaggregation in zellulären Modellen der HD untersucht werden. rnWie die ersten Untersuchungen in nativen HEK293-Zellen ergaben, bewirkte die Behandlung der Zellen mit EGb 761 eine Steigerung der basalen Proteasom-Aktivität sowie des proteasomalen Proteinabbaus und erhöhte die Transkription proteasomaler Gene. Hieraus ergaben sich Untersuchungen in Zellen mit Expressionen pathologischer Varianten von polyQ-Proteinen als zelluläre Modelle der HD. Hierbei konnte festgestellt werden, dass die Expression aberranter polyQ-Proteine eine verminderte zelluläre Proteasom-Aktivität bewirkte. Interessanterweise verursachte EGb 761 eine Abmilderung der pathologisch-induzierten verminderten Proteasom-Aktivität, in dem die EGb 761-Behandlung der Zellen zu einer erhöhten Proteasom-Aktivität, einem verbesserten proteasomalen Proteinabbau, sowie zu einer erhöhten Transkription proteasomaler Gene führte. Da diese EGb 761-Effekte unabhängig von der Expression aberranter polyQ-Proteine waren, demonstrierten diese Ergebnisse eine allgemeine EGb 761-Wirkungen auf die Proteasom-Aktivität. Anhand dieser Ergebnisse sollten anschließend weitere Untersuchungen mit zellulären Modellen der HD die genau Wirkung von EGb 761 auf die Degradation von abnormal verlängerten polyQ-Proteinen sowie auf die Bildung von polyQ-Aggregaten klären. rnHier konnte gezeigt werden, dass die Expression aberranter polyQ-Proteinen zu einer Akkumulation von SDS-resistenten bzw. SDS-unlöslichen, aggregierten polyQ-Proteinen führte, sowie die Bildung von sichtbaren polyQ-Aggregaten in Zellen bewirkte. Hierbei verursachte eine EGb 761-Behandlung der Zellen eine signifikante Verminderung im Gehalt an SDS-resistenten polyQ-Proteinen sowie eine Reduzierung von Aggregat-tragenden Zellen. Zudem konnte gezeigt werden, dass eine pharmakologische Inhibition des Proteasoms in EGb 761-behandelten Zellen, den Gehalt an SDS-unlöslichen polyQ-Proteinaggregate wieder erhöhte und somit den Effekt von EGb 761 aufhob. Folglich zeigten diese Ergebnisse, dass die EGb 761-induzierte Reduzierung der polyQ-Proteinaggregate durch einen effizienteren proteasomalen Abbau von fehlgefalteten, aberranten polyQ-Proteinen bewirkte wurde. rnAufbauend auf diesen Ergebnissen wurde eine experimentell-therapeutische Anwendung von EGb 761 in Modellen der HD in vitro und in vivo überprüft und hierzu primäre humane Fibroblasten sowie transgene C. elegans Würmer mit Expressionen aberranter polyQ-Proteine untersucht. Interessanterweise konnte in vitro und in vivo gezeigt werden, dass die EGb 761-Behandlung auch hier eine Reduzierung von SDS-unlöslichen polyQ-Proteinen bewirkte und zudem eine Reduzierung des pathologisch erhöhten Gehalts an Polyubiquitin-Proteinen bewirkte. Folglich wurde auch hier vermutet, dass EGb 761 einen verbesserten proteasomalen Abbau von polyQ-Proteinen induzierte und dies eine Verminderung der polyQ-Proteinaggregate verursachte. Darüber hinaus führte die EGb 761-Behandlung von seneszenten Fibroblasten zur Reduzierung von altersabhängig erhöhten Mengen von polyQ-Aggregaten, wodurch ein therapeutischer Effekt auf den proteasomalen Abbau der polyQ-Proteine verdeutlicht wurde. Zusätzlich konnte in polyQ-transgenen C. elegans demonstriert werden, dass eine EGb 761-Behandlung die Abmilderung eines typischen pathologischen Phänotyps bewirkte, indem eine polyQ-induzierte verminderte Motilität der Nematoden verbessert wurde und hierdurch eine positive EGb 761-Wirkung auf die Pathologie der HD in vivo dargestellt wurde. rnZusammenfassend konnten in dieser Arbeit neue Wirkungen von EGb 761 in der HD demonstriert werden. Hierbei wurde gezeigt, dass EGb 761 die Aggregation von pathogenen aberranten polyQ-Proteinen in vitro und in vivo reduziert, indem eine effizientere Degradation von polyQ-Proteinen erfolgt. Somit könnte diese Wirkungen von EGb 761 eine potentiell therapeutische Anwendung in der HD und ähnliche neurodegenerativen Erkrankungen darstellen.

Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus

Monepantel is the first drug of a new family of anthelmintics, the amino acetonitrile derivatives (AAD), presently used to treat ruminants infected with gastrointestinal nematodes such as Haemonchus contortus. Monepantel shows an excellent tolerability in mammals and is active against multidrug-resistant parasites, indicating that its molecular target is absent or inaccessible in the host and is different from those of the classic anthelmintics. Genetic approaches with mutant nematodes have suggested acetylcholine receptors of the DEG-3 subfamily as the targets of AADs, an enigmatic clade of ligand-gated ion channels that is specific to nematodes and does not occur in mammals. Here we demonstrate direct interaction of monepantel, its major active metabolite monepantel sulfone, and other AADs with potential targets of the DEG-3 subfamily of acetylcholine receptors. H. contortus DEG-3/DES-2 receptors were functionally expressed in Xenopus laevis oocytes and were found to be preferentially activated by choline, to permeate monovalent cations, and to a smaller extent, calcium ions. Although monepantel and monepantel sulfone did not activate the channels by themselves, they substantially enhanced the late currents after activation of the channels with choline, indicating that these AADs are type II positive allosteric modulators of H. contortus DEG-3/DES-2 channels. It is noteworthy that the R-enantiomer of monepantel, which is inactive as an anthelmintic, inhibited the late currents after stimulation of H. contortus DEG-3/DES-2 receptors with choline. In summary, we present the first direct evidence for interaction of AADs with DEG-3-type acetylcholine receptors and discuss these findings in the context of anthelmintic action of AADs.

Lower shedding of strongylid eggs by Warmblood horses with recurrent airway obstruction compared to unrelated healthy horses

An association between equine recurrent airway obstruction (RAO) and increased resistance to intestinal parasites has been demonstrated in descendants of an RAO-affected stallion. It was hypothesised that members of another high-incidence RAO family (F) and unrelated RAO-affected Warmblood horses (UA) would shed fewer strongylid eggs than unrelated RAO-unaffected pasture mates (PM) under the same environmental conditions. Faecal worm egg counts were performed on faecal samples (63 F, 86 UA, 149 PM) and classified into three categories: 0, 1-100 and >100 eggs per gram. While results for F did not differ from PM, UA were 2.5-times less likely to shed strongylid eggs than PM. RAO-affected Warmblood horses may be more resistant to strongylid nematodes than unrelated unaffected pasture mates and a family history of RAO does not necessarily confer protection against helminth infections.

Steroid hormone related male biased parasitism in chamois, Rupicapra rupicapra rupicapra

Parasites are linked with their host in a trophic interaction with implications for both hosts and parasites. Interaction stretches from the host's immune response to the structuring of communities and the evolution of biodiversity. As in many species sex determines life history strategy, response to parasites may be sex-specific. Males of vertebrate species tend to exhibit higher rates of parasites than females. Sex-associated hormones may influence immunocompetence and are hypothesised to lead to this bias. In a field study, we tested the prediction of male biased parasitism (MBP) in free ranging chamois (Rupicapra rupicapra rupicapra), which are infested intensely by gastrointestinal and lung helminths. We further investigated sex differences in faecal androgen (testosterone and epiandrosterone), cortisol and oestrogen metabolites using enzyme immunoassays (EIA) to evaluate the impact of these hormones on sex dependent parasite susceptibility. Non-invasive methods were used and the study was conducted throughout a year to detect seasonal patterns. Hormone levels and parasite counts varied significantly throughout the year. Male chamois had a higher output of gastrointestinal eggs and lungworm larvae when compared to females. The hypothesis of MBP originating in sex related hormone levels was confirmed for the elevated output of lungworm larvae, but not for the gastrointestinal nematodes. The faecal output of lungworm larvae was significantly correlated with androgen and cortisol metabolite levels. Our study shows that sex differences in steroid levels play an important role to explain MBP, although they alone cannot fully explain the phenomenon.

Nitazoxanide, a broad-spectrum thiazolide anti-infective agent for the treatment of gastrointestinal infections

Colonisation of the gastrointestinal tract by anaerobic bacteria, protozoa, trematodes, cestodes and/or nematodes and other infectious pathogens, including viruses, represents a major cause of morbidity and mortality in Africa, South America and southeast Asia, as well as other parts of the world. Nitazoxanide is a member of the thiazolide class of drugs with a documented broad spectrum of activity against parasites and anaerobic bacteria. Moreover, the drug has recently been reported to have a profound activity against hepatitis C virus infection. In addition, nitazoxanide exhibits anti-inflammatory properties, which have prompted clinical investigations for its use in Crohn's disease. Studies with nitazoxanide derivatives have determined that there must be significantly different mechanisms of action acting on intracellular versus extracellular pathogens. An impressive number of clinical studies have shown that the drug has an excellent bioavailability in the gastrointestinal tract, is fast acting and highly effective against gastrointestinal bacteria, protozoa and helminthes. A recent Phase II study has demonstrated viral response (hepatitis C) to monotherapy, with a low toxicity and an excellent safety profile over 24 weeks of treatment. Pre-clinical studies have indicated that there is a potential for application of this drug against other diseases, not primarily affecting the liver or the gastrointestinal tract.

[Overview of helminth problems in domestic ruminants in Switzerland]

Potent anthelmintics were introduced into the Swiss market several decades ago. Despite this, gastrointestinal nematodes (GIN), lungworms and the large liver fluke (Fasciola hepatica) can successfully inhabit Swiss ruminant farms. This is mainly due to a high reproductive capacity as well as very efficient survival strategies. In addition some species readily develop anthelmintic resistance. GIN-infections in young cattle are under comparatively good control. However, prophylactic measures are compromised where adult stock is also affected due to incomplete development of immune protection. Under these circumstances control measures must include all age groups. This results in fewer helminths in refugia thus may accelerate the development of anthelmintic resistance. This review aims to present a synopsis of the significance of the major helminth infections obtained on pasture by large and small ruminants in Switzerland. Currently available strategies for strategic helminth control are summarized and an outlook is given on new developments which might expand the spectrum of control measures relevant for veterinary practice in the future.

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel

Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5' UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.

In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of beta-tubulin

Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drug-resistant parasites that carry point mutations in beta-tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the beta-tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate(198) to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine(198) encoding alleles in the beta-tubulin isotype 1 gene pool of H. contortus in the field.

Genetic identification of an oxyurid from a captive, black-handed spider monkey—implications for treatment and control

Parasites are of major clinical significance in captive primates in zoos, particularly those with direct life cycles. Oxyurid nematodes can be a persistent problem, as infection intensity and environmental contamination with infective eggs are usually high. Observations at the Basel Zoo in Switzerland have revealed that particularly black-handed spider monkeys (Ateles geoffroyi) exhibit continuous oxyurid nematode infection(s), despite regular deworming with anthelmintics. In the present study, using a molecular approach, we were able to identify the nematode (Trypanoxyuris atelis) causing this ongoing problem, and we are now evaluating a practical treatment and control regimen to tackle this parasite problem.