Echinococcus multilocularis in germany: increased awareness or spreading of a parasite?

The 'small fox tapeworm' Echinococcus multilocularis has recently become a matter of intense interest in Germany. A long-term increase of its prevalence in foxes has been noted in the well-known endemic areas in Southern Germany, and reports on the occurrence of the parasite in other parts of the country suggest that the parasite is actually much more widespread than previously thought. As nearly all of the relevant studies are published in the German language in a veterinary journal and in the hunting press, accessibility to the information is limited. Richard Lucius and Brigit Bilger here describe the situation, and discuss the possible reasons and consequences.     

High urine flow rate increases prostaglandin E excretion in the conscious dog

Although previous studies from this and other laboratories have shown that urinary prostaglandin E excretion (U_PGEV) can vary independent of urine flow rate, recent studies during water diuresis in the conscious dog have suggested that high urine flow rate per se may increase U_PGEV. To examine the effect of urine flow rate on U_PGEV we administered either mannitol, chlorothiazide or Ringer's solution to mongrel dogs and measured U_PGEV. During anesthesia neither mannitol or chlorothiazide increased U_PGEV. There was, however, a consistent increase with all three agents in awake animals. This increase in U_PGEV was independent of alterations in glomerular filtration rate. There was a consistent increase in urinary sodium excretion and decrease in urinary osmolality with all three agents. The changes in PGE, however, were similar to those found during water diuresis when no increase in sodium excretion was found. It is not presently clear whether these findings reflect a true increase in renal PGE synthesis due to some change in flow or pressure within the renal medulla or rather represent unchanged PGE synthesis by renal tubular cells, the high tubule fluid flow rate causing increased entry into the tubular lumen in contrast to the renal interstitium.     

Examination of the nucleotide‐linked assembly mechanism of E. coli ClpA

Escherichia coli ClpA is a AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP‐dependent unfolding and translocation of substrate proteins targeted for degradation by a protease, ClpP. ClpA hexamers associate with one or both ends of ClpP tetradecamers to form ClpAP complexes. Each ClpA protomer contains two nucleotide‐binding sites, NBD1 and NBD2, and self‐assembly into hexamers is thermodynamically linked to nucleotide binding. Despite a number of studies aimed at characterizing ClpA and ClpAP‐catalyzed substrate unfolding and degradation, respectively, to date the field is unable to quantify the concentration of ClpA hexamers available to interact with ClpP for any given nucleotide and total ClpA concentration. In this work, sedimentation velocity studies are used to quantitatively examine the self‐assembly of a ClpA Walker B variant in the presence of ATP. In addition to the hexamerization, we observe the formation of a previously unreported ClpA dodecamer in the presence of ATP. Further, we report apparent equilibrium constants for the formation of each ClpA oligomer obtained from direct boundary modeling of the sedimentation velocity data. The energetics of nucleotide binding to NBD1 and NBD2 are revealed by examining the dependence of the apparent association equilibrium constants on free nucleotide concentration.     

Balancing competing life‐stage requirements in salmon habitat rehabilitation: between a rock and a hard place

Gravel augmentation is often applied to rivers and streams to rehabilitate salmonid spawning and incubation habitat. However, the effect of gravel size on salmon spawning utilization and embryo survival during incubation is not well understood. We conducted an experiment on a regulated and previously mined Northern California salmonid‐bearing stream in which different sized gravel (small, medium, and large) patches were placed into the stream's degraded spawning reach. We documented Oncorhynchus tshawytscha (Chinook salmon) spawning activity within the three gravel sizes for two seasons. In addition, we deployed Chinook salmon embryos into each gravel size patch and allowed them to incubate until estimated emergence time. Although all experimental gravel sizes were predicted to be within the spawning population's mobilization capabilities, model results indicated the probability of salmon building redds decreased as substrate size increased. Conversely, embryo survival increased as gravel size increased. A possible mechanism of disparate Chinook salmon embryo survival is provided by an observed decrease in embryo survival correlating with greater presence of embryo predators (leeches), which are associated with smaller gravel. Our results indicate a parent‐offspring conflict in optimal spawning gravel size for Chinook salmon, and suggest that an intermediate gravel size would maximize overall reproductive success across both spawning and incubation life stages.     

Immunisation with Salmonella typhimurium-delivered glyceraldehyde-3-phosphate dehydrogenase protects mice against challenge infection with Echinococcus multilocularis eggs

Recombinant glyceraldehyde-3-phosphate dehydrogenase of the cestode parasite Echinococcus multilocularis was expressed in Escherichia coli and in Salmonella typhimurium. The potential of different forms of the recombinant antigen to protect BALB/c mice against oral challenge infections with E. multilocularis eggs was evaluated. Oral or intraperitoneal immunisation with live attenuated S. typhimurium as a carrier for recombinant glyceraldehyde-3-phosphate dehydrogenase of the E. multilocularis resulted in significant protection, reducing the number of developing metacestodes up to 79.8%. The sera of protected animals did not contain detectable amounts of antibody against glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis. By contrast, although anti-glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis antibodies were detectable in the sera, immunisation with E. coli-expressed recombinant glutathione-S-transferase-fusion protein or with glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis fused to a 6HIS-tag failed to protect the animals against oral challenge infections. These data emphasise that antigen delivery systems play a critical role in vaccination and the induction of protective immunity against helminth parasites.     

Modulation of host immune responses by nematode cystatins

Parasitic nematodes, living in the intestinal tract or within tissues of theirs hosts, are constantly exposed to an array of immune effector mechanisms. One strategy to cope with the immune response is the release of immunomodulatory components that block effector mechanisms or interact with the cytokine network. Among the secreted nematode immunomodulators, cysteine protease inhibitors (cystatins) are shown to be of major importance. Nematode cystatins inhibit, among others, proteases involved in antigen processing and presentation, which leads to a reduction of T cell responses. At the same time nematode cystatins modulate cytokine responses, the most prominent trait being the upregulation of IL-10, a Th2 cytokine, by macrophages. In this situation, IL-10 leads among others to downregulation of costimulatory surface molecules of macrophages. These properties contribute to induction of an anti-inflammatory environment, concomitant with a strong inhibition of cellular proliferation. This setting is believed to favour the survival of worms. An opposite activity of nematode cystatins is the upregulation of production of inducible nitric oxide by IFN-gamma activated macrophages, an intrinsic property of natural cysteine protease inhibitors. This shows that these proteins can act as proinflammatory molecules under certain circumstances. A comparison of the immunomodulatory effects of cystatins of filarial nematodes with homologous proteins of the free-living nematode Caenorhabditis elegans revealed distinct differences. Caenorhabditis elegans cystatins induce the production of the Th1 cytokine IL-12, in contrast to filarial cystatins that upregulate IL-10. Caenorhabditis elegans cystatins hardly inhibit cellular proliferation. These data suggest that cystatins of parasitic nematodes have multiple, specific capacities for immunomodulation, acting in parallel on different immune effector mechanisms. Elucidation of the mechanisms involved might be useful in the development of immunotherapeutic reagents in the future.     

A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken

Salmonella typhimurium vaccine strains were used as antigen delivery system for oral immunisation of chickens against two antigens of the coccidian parasite Eimeria tenella. The cDNAs of the known E. tenella proteins, SO7 and TA4, were isolated from total RNA and subcloned into the expression vectors pQE30 and pTECH2. Subcutaneous immunisation of chickens with Escherichia coli-expressed SO7 and TA4 revealed that both proteins were immunogenic. Both cDNAs were subcloned into plasmids of the pTECH2 vector system, which allows them to be expressed as fusion proteins with the highly immunogenic fragment C of the tetanus toxin under control of the anaerobically inducible nirB promoter. Plasmids were introduced into the S. typhimurium vaccine strains SL3261, C5aroD and C5htrA. SDS-PAGE and Western blot analysis revealed expression of both fusion proteins in all strains under anaerobic culture conditions. Three-week-old white leghorn chickens were orally immunised with 10(9) CFU per animal. The stability of the recombinant bacteria was revealed by recovery of viable Salmonella containing the respective plasmids from the liver of the immunised chickens at day 3 after inoculation. Specific serum IgG antibodies against the SO7-or TA4-antigens were detectable by ELISA 2 weeks after oral immunisation and remained for at least 6 weeks, while specific IgA antibodies were restricted to the bile of the birds. All chickens produced serum IgG and IgA to S. typhimurium lipopolysaccharides. Our data show that a single oral inoculation with recombinant S. typhimurium SL3261, C5aroD and C5htrA can induce specific antibody responses to heterologous Eimeria antigens in chickens, suggesting that recombinant Salmonella are a suitable delivery system for vaccines against Eimeria infections.     

Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting

Jirds (Meriones unguiculatus) were vaccinated with irradiated L3 third-stage larvae (L3) of Acanthocheilonema viteae, and the time required for killing of the challenge L3 was determined. The number of parasites recovered from vaccinated jirds was reduced to about 10% of the control values on the second day after challenge infection and later on. Histological studies revealed an eosinophil-rich infiltrate containing macrophages, neutrophils, and mast cells in the vicinity of the L3 on day 2 after challenge and destruction of the worms by day 4 after challenge. Ultrastructural studies confirmed these data and showed that eosinophils, macrophages, and mast cells were close to the L3 on day 2 after challenge. Flattening of the eosinophils onto the surface of the worms, degranulation of electron-dense material, and rupture of the L3 surface was observed on day 4 after challenge, followed by invasion of the inner of the worms by phagocytic cells. These data show that immune attack against the challenge L3 in vaccinated jirds is initiated between the first and the second day after challenge and that killing occurs around the fourth day after challenge, before the worms undergo their first molt.     

(-)-Deprenyl fails to promote axonal regeneration of retinal ganglion cells in vitro and in vivo

(-)-Deprenyl ( L-deprenyl, selegiline hydrochloride), a selective monoamine oxidase B (MAO-B) inhibitor employed in the pharmacological therapy of Parkinson's disease, increases neuronal survival in both animal models of neurodegenerative disorders and acute CNS lesions. Despite intensive investigations, the mechanisms of (-)-deprenyl-mediated neuroprotection remain poorly understood. To test the hypothesis that (-)-deprenyl might have a beneficial effect not only on neuronal survival, but also on axonal regeneration, we describe here experiments performed in vitro and in vivo which clearly demonstrate that (-)-deprenyl fails to promote axonal regeneration of severed rat retinal ganglion cells (RGCs). Furthermore, (-)-deprenyl was not able to overcome free-radical-induced RGC axon degeneration. These results challenge the notion that (-)-deprenyl might be useful as a monotherapy for acute CNS lesions and give rise to a more critical viewpoint of the trophic-like function of this widely used therapeutic agent.