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Abstract Background The exact role of the lymphoreticular system in the spread of peripheral prion infections to the central nervous system still needs further elucidation. Against this background, the influence of the regional lymph node (Ln. popliteus) on the pathogenesis of scrapie was monitored in a hamster model of prion infection via the footpad. Methods Surgical lymphadenectomy was carried out at different time points after infection, or prior to inoculation, in order to elucidate the impact of the lymph node on lethal neuroinvasion. Results The Ln. popliteus did not show an influence on pathogenesis when a high dose of infectivity was administered. However, it was found to modulate the interval of time until the development of terminal scrapie in a subset of animals lymphadenectomized after low-dose infection. In additon, lymphadenectomy performed four weeks before inoculation prevented cerebral PrPTSE deposition and development of disease during the period of observation (314 days) in the majority of hamsters challenged with a very low dose of scrapie agent. Conclusion Our findings suggest the regional lymph node as a potentially facilitating or even essential factor for invasion of the brain after peripheral challenge with low doses of infectious scrapie agent. The invasive in vivo approach pursued in this study may be applied also to other animal species for further elucidating the involvement of lymphoid tissue in the pathogenesis of experimental and natural TSEs.

Abstract Background Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. Methods Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. Results Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. Conclusion We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

Scrapie, bovine spongiform encephalopathy and chronic wasting disease are orally communicable, transmissible spongiform encephalopathies (TSEs). As zoonotic transmissions of TSE agents may pose a risk to human health, the identification of reservoirs for infectivity in animal tissues and their exclusion from human consumption has become a matter of great importance for consumer protection. In this study, a variety of muscles from hamsters that were orally challenged with scrapie was screened for the presence of a molecular marker for TSE infection, PrPSc (the pathological isoform of the prion protein PrP). Sensitive western blotting revealed consistent PrPSc accumulation in skeletal muscles from forelimb and hindlimb, head, back and shoulder, and in tongue. Previously, our animal model has provided substantial baseline information about the peripheral routing of infection in naturally occurring and orally acquired ruminant TSEs. Therefore, the findings described here highlight further the necessity to investigate thoroughly whether muscles of TSE-infected sheep, cattle, elk and deer contain infectious agents. [ABSTRACT FROM AUTHOR]

Bacteriostatic concentrations of trimethoprim, which possibly acts as a DNA-inhibitor, induced the swelling of staphylococci and affected their cell walls, their cytoplasmic membrane and part of their autolytic wall system. Trimethoprim proved to be the first growth-inhibiting drug that did not induce the formation of thickened cell walls in staphylococci. An ultrastructurally important effect of trimethoprim treatment was the appearance of mesosome-like structures which were not fixation artefacts. Additionally, trimethoprim led to the formation of incomplete cross walls, while cross wall initiation continued virtually unaffected. After removal of trimethoprim, extremely fast growth restoration occurred with the formation of new cell wall material underlying the old wall. Afterwards, the old wall material was disintegrated by autolytic processes. [ABSTRACT FROM AUTHOR]

Shedding of prions via faeces may be involved in the transmission of contagious prion diseases. Here, we fed hamsters 10mg of 263K scrapie brain homogenate and examined the faecal excretion of disease-associated prion protein (PrP(TSE)) during the course of infection. The intestinal fate of ingested PrP(TSE) was further investigated by monitoring the deposition of the protein in components of the gut wall using immunohistochemistry and paraffin-embedded tissue (PET) blotting. Western blotting of faecal extracts showed shedding of PrP(TSE) in the excrement at 24-72 h post infection (hpi), but not at 0-24 hpi or at later preclinical or clinical time points. About 5% of the ingested PrP(TSE) were excreted via the faeces. However, the bulk of PrP(TSE) was cleared from the alimentary canal, most probably by degradation, while an indiscernible proportion of the inoculum triggered intestinal infection. Components of the gut-associated lymphoid tissue (GALT) and the enteric nervous system (ENS) showed progressing accumulation of PrP(TSE) from 30 days post infection (dpi) and 60 dpi, respectively. At the clinical stage of disease, substantial deposits of PrP(TSE) were found in the GALT in close vicinity to the intestinal lumen. Despite an apparent possibility of shedding from Peyer's patches that may involve the follicle-associated epithelium (FAE), only small amounts of PrP(TSE) were detected in faeces from clinically infected animals by serial protein misfolding cyclic amplification (sPMCA). Although excrement may thus provide a vehicle for the release of endogenously formed PrP(TSE), intestinal clearance mechanisms seem to partially counteract such a mode of prion dissemination.