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Prions, the putative causative agents of transmissible spongiform encephalopathies, are neurotropic pathogens that spread to the central nervous system via synaptically linked neural conduits upon peripheral infection. Axons and their transport processes have been suggested as mediators of nerve-associated prion dissemination. However, the exact cellular components and molecular mechanisms underlying neural spread are unknown. This study used an established hamster scrapie model to pursue a novel experimental approach using nerve conduits containing segments devoid of neurites generated by incomplete nerve regeneration following Wallerian degeneration to probe the necessity of axons for the neural propagation of prions. For this purpose, animals were subjected to unilateral sciatic neurectomy 4 weeks before footpad inoculation with scrapie agent. The results showed that the regional nerve is the prime conduit for cerebral neuroinvasion and revealed, as evidenced by the accumulation of pathological prion protein PrPTSE, that prions can proceed along segments of peripheral neural projections without detectable axonal structures.

Recently, pathological prion protein (PrPTSE) was detected in muscle from sheep infected with scrapie, the archetype of transmissible spongiform encephalopathies (TSEs). This finding has highlighted the question of whether mammalian muscle may potentially also provide a reservoir for TSE agents related to bovine spongiform encephalopathy (BSE) and variant Creutzfeldt–Jakob Disease (vCJD). Here, results are reported from studies in hamsters and mice that provide direct experimental evidence, for the first time, of BSE- and vCJD-associated PrPTSE deposition in muscles. Our findings emphasize the need for further assessment of possible public-health risks from TSE involvement of skeletal muscle.

Zusammenfassung: Durch die Entwicklung und den zunehmenden Einsatz moderner real-time Detektionsverfahren konnte die Zuverlassigkeit, die Genauigkeit sowie die Empfindlichkeit der Toxoplasma-Diagnostik deutlich gesteigert werden. Mit der Auswahl geeigneter Primer und Sondensequenzen gelingt hier unter optimierten Reaktionsbedingungen bereits der spezifische Nachweis von weniger als einem Genomaquivalent von T. gondii pro PCR-Reaktion. In Verbindung mit der sehr hohen Spezifitat moderner real-time PCR-Verfahren kann diese extreme analytische Sensitivitat in vielen Fallen (z. B. bei der Fruchtwasseruntersuchung bei Verdacht auf konnatale Toxoplasmose oder zur Detektion des Erregers im Blut von immundefizienten Patienten) einen, wenn nicht sogar den entscheidenden zeitlichen Vorsprung bei der therapeutischen Intervention darstellen. Im Gegensatz zu den qualitativen Testsystemen, die in der Regel auf maximale Empfindlichkeit abgestimmt sind, mus die eigentliche Wertigkeit von quantitativen Ergebnissen aber noch im Rahmen zukunftiger Studien evaluiert werden. Hier ist vor allem die Frage zu klaren, ob und bei welchen klinischen Manifestationen der Toxoplasmose eine Korrelation mit der ermittelten Erregermenge im Untersuchungsmaterial besteht. Trotz der Verfugbarkeit von quantitativen Ergebnissen wird es aber vermutlich keine Normbereiche fur das Vorliegen einer latenten, einer aktiven oder reaktivierten T. gondii-Infektion geben konnen. Es steht jedoch auser Zweifel, das in bestimmten Konstellationen die quantitative Bestimmung von T. gondii-DNA aus definiertem Probenmaterial eine hilfreiche Zusatzinformation bei der Beurteilung von serologischen bzw. histologischen Befunden oder zur Abschatzung des Risikos der Entwicklung einer aktiven Toxoplasmose bei immunsupprimierten Transplantationspatienten darstellen kann. Neben rein diagnostischen Aspekten kann die quantitative Erfassung des Erregers zudem auch fur das Therapiemonitoring oder bei der Entwicklung von neuen pharmazeutischen Wirk- und Impfstoffen von zentraler Bedeutung sein. Summary: Application of modern real-time PCR technology has led to a significant increase in specificity and sensitivity of molecular test systems for the detection of Toxoplasma gondii. Using optimized primer and probe sequences, and targeting a specific, highly conserved multicopy DNA target sequence, less than one genome of T. gondii can be reliably detected in body fluids, e.g. in blood, amniotic or cerebrospinal fluid. This may be of importance for the early initiation of adequate treatment, when connatal infection or reactivation of toxoplasmosis in the immunocompromised host is assumed. However, the correlation between parasite burden and medical manifestation needs further evaluation, and standard ranges discriminating between latent, active or reactive toxoplasmic infections cannot be expected. Nevertheless, quantitative determination of Toxoplasma in body fluids may provide valuable supportive information for the interpretation of serological and histological results and has proven to be useful in quantifying parasitaemia and monitoring therapy in immunodeficient patients. The method may also be suited for the development and testing of antiparasitic agents.

FemA, a cytoplasmic protein necessary for the expression of methicillin resistance in Staphylococcus aureus and also involved in the biosynthesis of staphylococcal cell walls, was detected and quantified in several S. aureus strains under different growth conditions by Western immunoblot. Two types of antigens were used for the production of polyclonal antibodies against FemA: (i) a synthetic peptide comprising 14 amino acids of its C-terminal sequence; and (ii) FemA isolated by preparative gel electrophoresis and electroelution from an overproducing staphylococcal strain. Immunodetection revealed that all investigated strains, either methicillin-resistant or susceptible, expressed FemA during the exponential growth phase in varying amounts. In the stationary phase, the FemA content was diminished. Strains in which femA was inactivated by insertion of Tn551 into the control region of the femAB operon still expressed about 10% of the protein compared to their parent strains. Tn551 insertion in the middle of the femB gene did not affect the FemA expression. In 40 methicillin-susceptible and 6 resistant clinical isolates of S. aureus, the FemA content or its affinity to the antibodies was reduced compared to laboratory parent strains. In susceptible strains, an additional protein of higher molecular weight, present in large quantities, was also able to bind the FemA antibodies. Such a protein was also present in methicillin-resistant isolates, although it was not as pronounced as in the susceptible strains.

International audience; Shedding of prions via faeces may be involved in the transmission of contagious prion diseases. Here, we fed hamsters 10 mg of 263K scrapie brain homogenate and examined the faecal excretion of disease-associated prion protein (PrPTSE) during the course of infection. The intestinal fate of ingested PrPTSE 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 PrPTSE 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 PrPTSE were excreted via the faeces. However, the bulk of PrPTSE 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 PrPTSE from 30 days post infection (dpi) and 60 dpi, respectively. At the clinical stage of disease, substantial deposits of PrPTSE 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 PrPTSE 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 PrPTSE, intestinal clearance mechanisms seem to partially counteract such a mode of prion dissemination.

International audience; As is known from various animal models, the spread of agents causing transmissible spongiform encephalopathies (TSE) after peripheral infection affects peripheral nerves before reaching the central nervous system (CNS) and leading to a fatal end of the disease. The lack of therapeutic approaches for TSE is partially due to the limited amount of information available on the involvement of host biological compartments and processes in the propagation of the infectious agent. The in vivo model presented here can provide information on the spread of the scrapie agent via the peripheral nerves of hamsters under normal and altered axonal conditions. Syrian hamsters were unilaterally footpad (f.p.) infected with scrapie. The results of the spatiotemporal ultrasensitive immunoblot-detection of scrapie-associated prion protein (PrP$^{\rm Sc})$ in serial nerve segments of both distal sciatic nerves could be interpreted as a centripetal and subsequent centrifugal neural spread of PrP$^{\rm Sc}$ for this route of infection. In order to determine whether this propagation is dependent on main components in the axonal cytoskeleton (e.g. neurofilaments, also relevant for the component 'a' of slow axonal transport mechanisms), hamsters were treated -in an additional experiment- with the neurotoxin ß,ß'-iminodiproprionitrile (IDPN) around the beginning of the scrapie infection. A comparison of the Western blot signals of PrP$^{\rm Sc}$ in the ipsilateral and in the subsequently affected contralateral sciatic nerve segments with the results revealed from IDPN-untreated animals at preclinical and clinical stages of the TSE disease, indicated similar amounts of PrP$^{\rm Sc}$. Furthermore, the mean survival time was unchanged in both groups. This in vivo model, therefore, suggests that the propagation of PrP$^{\rm Sc}$ along peripheral nerves is not dependent on an intact neurofilament component of the axonal cytoskeleton. Additionally, the model indicates that the spread of PrP$^{\rm Sc}$ is not mediated by the slow component 'a' of the axonal transport mechanism.

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.

In order to investigate the accuracy and practicability of the polymerase chain reaction (PCR) in the antenatal diagnosis of congenital toxoplasmosis, a collaborative study involving 15 European laboratories was performed under the auspices of the Biomed 2 Programme of the European Community. Each team received 12 aliquots (four negative, eight positive) of 'artificial samples' made of amniotic fluid spiked with tachyzoites of the RH strain of Toxoplasma gondii. Each team performed its own PCR protocol (all were different). Nine of the 15 laboratories were able to detect a single parasite, but two of the 15 found all samples negative. Four of the 15 laboratories found one or more control samples to be falsely positive. This study highlights the lack of homogeneity between PCR protocols and performance and underlines the need for an external quality assurance scheme which could provide 'reference' samples that could be used by any laboratory wanting to establish and maintain an accurate diagnostic test based on PCR.

In log-phase cells of staphylococci, cultivated under high, “non-lytic” concentrations of penicillin G, there occurred a novel killing process hitherto hidden behind seemingly bacteriostatic effects. Two events are essential for the apprearance of this “hidden death”: (i) the failure of the dividing cell to deposit enough fibrillar cross-wall material to be welded together, and (ii) a premature ripping up of incomplete cross walls along their splitting system. “Hidden death” started as early as 10–15 min after drug addition, already during the first division cycle. It was the consequence of a loss of cytoplasmic constituents which erupted through peripheral slit-like openings in the incomplete cross walls. The loss resulted either in more or less empty cells or in cell shrinkage. These destructions could be prevented by raising the external osmotic pressure. In contrast, the conventional “non-hidden death” occurred only much later and exclusively during the second division cycle and mainly in those dividing cells, whose nascent cross walls of the first division plane had been welded together. These welding processes at nascent cross walls, resulting in tough connecting bridges between presumptive individual cells, were considered as a morphogenetic tool which protects the cells, so that they can resist the otherwise fatal penicillin-induced damages for at least an additional generation time (“morphogenetic resistance system”). Such welded cells, in the virtual absence of underlying cross-wall material, lost cytoplasm and were killed via ejection through pore-like wall openings or via explosions in the second division plane and after liberation of their murosomes, as it was the case in the presence of low, “lytic” concentrations of penicillin. Bacteriolysis did not cause any of the hitherto known penicillin-induced killing processes.

Quality control for the diagnosis of Toxoplasma gondii reactivation in SCT patients using PCR assays

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.

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.