Screening of Swiss hot spring resorts for potentially pathogenic free-living amoebae.

Free-living amoebae (FLA) belonging to Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Sappinia pedata are known to cause infections in humans and animals leading to severe brain pathologies. Worldwide, warm aquatic environments have been found to be suitable habitats for pathogenic FLA. The present study reports on screening for potentially pathogenic FLA in four hot spring resorts in Switzerland. Water samples were taken from water filtration units and from the pools, respectively. Amoebae isolated from samples taken during, or before, the filtration process were demonstrated to be morphologically and phylogenetically related to Stenoamoeba sp., Hartmannella vermiformis, Echinamoeba exundans, and Acanthamoeba healyi. With regard to the swimming pools, FLA were isolated only in one resort, and the isolate was identified as non-pathogenic and as related to E. exundans. Further investigations showed that the isolates morphologically and phylogenetically related to A. healyi displayed a pronounced thermotolerance, and exhibited a marked in vitro cytotoxicity upon 5-day exposure to murine L929 fibroblasts. Experimental intranasal infection of Rag2-immunodeficient mice with these isolates led to severe brain pathologies, and viable trophozoites were isolated from the nasal mucosa, brain tissue, and lungs post mortem. In summary, isolates related to A. healyi were suggestive of being potentially pathogenic to immunocompromised persons. However, the presence of these isolates was limited to the filtration units, and an effective threat for health can therefore be excluded.

Effects of Toll-like receptor 2 agonist Pam(3)CysSK(4) on inflammation and brain damage in experimental pneumococcal meningitis.

TLR2 signaling participates in the pathogenesis of pneumococcal meningitis. In infant rats, the TLR2 agonist Pam(3)CysSK(4) was applied intracisternally (0.5 microg in 10 microl saline) alone or after induction of pneumococcal meningitis to investigate the effect of TLR2 activation on cerebrospinal fluid (CSF) inflammation and hippocampal apoptosis. A dose effect of Pam(3)CysSK(4) on apoptosis was investigated by intracisternal application of 0.5 microg in 10 microl saline and 40 microg in 20 microl saline. Pam(3)CysSK(4) neither induced apoptosis in sham-operated mice nor aggravated apoptosis in acute infection. However, Pam(3)CysSK(4) induced pleocytosis, TNF-alpha and MMP-9 in CSF in sham-infection but not during acute meningitis. We conclude that TLR2 signaling triggered by Pam(3)CysSK(4) at a dosage capable to induce a neuroinflammatory response does not induce hippocampal apoptosis in the infant rat model of experimental pneumococcal meningitis.

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland.

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 degrees C, subsequently displayed a high thermotolerance, being able to grow at 42 degrees C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.

Assessment of Echinococcus granulosus somatic protoscolex antigens for serological follow-up of young patients surgically treated for cystic echinococcosis.

Echinococcus granulosus protoscolex soluble somatic antigens (PSSAs) were assessed for their prognostic value in the serological follow-up of young patients treated for cystic echinococcosis (CE), compared to conventional hydatid fluid (HF) antigen. Based on different clinical courses and outcome of infection, as well as imaging findings, patients were retrospectively classified into two different groups including either cured CE (CCE; i.e., absence of active cysts or presence of inactive cysts, respectively) and noncured CE (NCCE) patients still presenting active cysts at the end of an up to 5-year follow-up period. An immunoglobulin G (IgG)-PSSA enzyme-linked immunosorbent assay (ELISA) showed a gradual decrease in antibody levels in CCE cases, reaching seronegativity in 20% of the cases at least within 5 years postsurgery. In comparison, the conventional IgG-HF ELISA showed a significantly lower progressive decrease in antibody levels, serology becoming negative in only 15% of CCE patients at the endpoint of the follow-up period. Serological analysis of PSSA by immunoblotting yielded an interesting immunoreactive double band of 27 and 28 kDa that, in 15 (75%) of 20 CCE cases, exhibited a rapid decrease and subsequent disappearance of respective antibody reactivities within 3 years postsurgery. Conversely, anti-27- and -28-kDa antibody reactivity strongly persisted until the endpoint of the follow-up period in all of the five NCCE patients. Further analysis of the 27- and 28-kDa doublet by using affinity-purified antibodies showed that the double band was not detectable in HF. Furthermore, a predominantly IgG4 subclass-restricted humoral immune response against the 27- and 28-kDa antigens was demonstrated in seroreactive CE patients. Overall, an anti-27- and -28-kDa response appeared to correlate with cyst activity. In conclusion, PSSA represents a useful candidate to carry out a serologic follow-up of CE subsequent to treatment and deserves further respective evaluation for other age groups of CE patients.

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures.

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.

Apoptosis of hippocampal neurons in organotypic slice culture models: direct effect of bacteria revisited.

Neurons of the hippocampal dentate gyrus selectively undergo programmed cell death in patients suffering from bacterial meningitis and in experimental models of pneumococcal meningitis in infant rats. In the present study, a membrane-based organotypic slice culture system of rat hippocampus was used to test whether this selective vulnerability of neurons of the dentate gyrus could be reproduced in vitro. Apoptosis was assessed by nuclear morphology (condensed and fragmented nuclei), by immunochemistry for active caspase-3 and deltaC-APP, and by proteolytic caspase-3 activity. Co-incubation of the cultures with live pneumococci did not induce neuronal apoptosis unless cultures were kept in partially nutrient-deprived medium. Complete nutrient deprivation alone and staurosporine independently induced significant apoptosis, the latter in a dose-response way. In all experimental settings, apoptosis occurred preferentially in the dentate gyrus. Our data demonstrate that factors released by pneumococci per se failed to induce significant apoptosis in vitro. Thus, these factors appear to contribute to a multifactorial pathway, which ultimately leads to neuronal apoptosis in bacterial meningitis.

Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host-parasite interactions.

Neospora caninum is an apicomplexan parasite which has emerged as an important cause of bovine abortion worldwide. Abortion is usually triggered by reactivation of dormant bradyzoites during pregnancy and subsequent congenital infection of the foetus, where the central nervous system appears to be most frequently affected. We here report on an organotypic tissue culture model for Neospora infection which can be used to study certain aspects of the cerebral phase of neosporosis within the context of a three-dimensionally organised neuronal network. Organotypic slice cultures of rat cortical tissue were infected with N. caninum tachyzoites, and the kinetics of parasite proliferation, as well as the proliferation-inhibitory effect of interferon-gamma (IFN-gamma), were monitored by either immunofluorescence, transmission electron microscopy, and a quantitative PCR-assay using the LightCycler instrument, respectively. In addition, the neuronal cytoskeletal elements, namely glial acidic protein filaments as well as actin microfilament bundles were shown to be largely colocalising with the pseudocyst periphery. This organotypic culture model for cerebral neosporosis provides a system, which is useful to study the proliferation, ultrastructural characteristics, development, and the interactions of N. caninum within the context of neuronal tissue, which at the same time can be modulated and influenced under controlled conditions, and will be useful in the future to gain more information on the cerebral phase of neosporosis.

Screening Swiss water bodies for potentially pathogenic free-living amoebae.

Free-ling amoebae (FLA) including Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia pedata, can cause opportunistic infections leading to severe brain pathologies. Human infections with pathogenic FLA have been increasingly documented in many countries. In Switzerland, thus far, the occurrence and distribution of potentially pathogenic FLA has not been investigated. Swiss water biotopes, including swimming pools, lakes, rivers and ponds, have now been screened for the presence of FLA, and assessment of their pathogenicity potential for a mammalian host has been undertaken. Thus, a total of 17 isolates were recovered by in vitro cultivation from these different aquatic sources. Characterization by sequence analysis of Acanthamoeba spp.-specific and 'FLA-specific PCR products amplified from 18s rDNA based on morphological traits, thermotolerance, and cytotoxicity towards murine fibroblasts yielded the following findings: Echinamoeba cf. exundans (3 isolates), Hartmannella spp. (3), Vannella spp. (4), Protacanthamoebica cf. bohemica (1), Acanthamoeba cf. castellanii (1) and Naegleria spp. (5). B. mandrillaris and N. fowleri did not range amongst these isolates. None of the isolates exhibited pronounced cytotoxicity and all failed to grow at 42 degrees C; therefore, they do not present any potential for CNS pathogenicity for humans.

Isolation and molecular characterization of recombinant Echinococcus granulosus P29 protein (recP29) and its assessment for the post-surgical serological follow-up of human cystic echinococcosis in young patients.

We synthesized recombinant Echinococcus granulosus protoscolex recP29 antigen to be preliminarily assessed by ELISA and immunoblotting. RecP29-serology was carried out on 54 young patients with cystic echinococcosis (CE). Patients were classified into either cured (CCE) (n=40) or non-cured (NCCE) (n=14) CE patients. RecP29 ELISA showed a gradual decrease of antibody concentrations in all CCE cases that were initially (before treatment) seropositive to this antigen (25 out of 40) or that seroconverted following treatment. A complete seronegativity was reached within 3 years post-surgery in all of these cases. Conventional HCF ELISA yielded seronegativity in only 10% of initially recP29-seropositive CCE patients (P=0.086). Likewise, recP29 immunoblotting yielded seronegativity in 93% of 29 out of 40 initially recP29-immunoblot-positive CCE patients after 3 years follow-up, compared with 72% in the HCF immunoblotting (P=0.060). Eleven out of 14 NCCE patients were initially positive by recP29 ELISA, and 10 out of these maintained a marked anti-recP29 antibody reactivity until the endpoint of the follow-up period. All 14 NCCE cases were initially seropositive by recP29 immunoblotting, and 13 cases remained seropositive until the end of the study. Thus, recombinant P29 protein appears prognostically useful for monitoring those post-surgical CE cases with an initial seropositivity to this marker.

PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffin-embedded brain sections.

We developed a real-time PCR which allowed the highly sensitive detection of Naegleria fowleri in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.

Bacterial meningitis causes two distinct forms of cellular damage in the hippocampal dentate gyrus in infant rats.

Bacterial meningitis causes neurological sequelae in up to 50% of survivors. Two pathogens known for their propensity to cause severe neurological damage are Streptococcus pneumoniae and group B streptococci. Some forms of neuronal sequelae, such as learning and memory deficits, have been associated with neuronal injury in the hippocampus. To learn more about hippocampal injury in meningitis, we performed a comparative study in bacterial meningitis due to S. pneumoniae and group B streptococcus, in which 11-day-old infant rats were infected intracisternally with either of the two pathogens. Histopathological examination of the neuronal injury in the dentate gyrus of the hippocampus showed that S. pneumoniae caused predominantly classical apoptotic cell death. Cells undergoing apoptosis were located only in the subgranular zone and stained positive for activated caspase-3 and TUNEL. Furthermore, dividing progenitor cells seemed particularly sensitive to this form of cell death. Group B streptococcus was mainly responsible for a caspase-3-independent (and TUNEL-negative) form of cell death. Compared with the morphological features found in apoptosis (e.g., apoptotic bodies), this form of neuronal death was characterized by clusters of uniformly shrunken cells. It affected the dentate gyrus throughout the blade, showing no preferences for immature or mature neurons. Thus, depending on the infecting agent, bacterial meningitis causes two distinct forms of cell injury in the dentate gyrus.

Application of real-time fluorescent PCR for quantitative assessment of Neospora caninum infections in organotypic slice cultures of rat central nervous system tissue.

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.

Caspase-3 mediates hippocampal apoptosis in pneumococcal meningitis.

Bacterial meningitis causes neuronal apoptosis in the hippocampal dentate gyrus, which is associated with learning and memory impairments after cured disease. The execution of the apoptotic program involves pathways that converge on activation of caspase-3, which is required for morphological changes associated with apoptosis. Here, the time course and the role of caspase-3 in neuronal apoptosis was assessed in an infant rat model of pneumococcal meningitis. During clinically asymptotic meningitis (0-12 h after infection), only minor apoptotic damage to the dentate gyrus was observed, while the acute phase (18-24 h) was characterized by a massive increase of apoptotic cells, which peaked at 36 h. In the subacute phase of the disease (36-72 h), the number of apoptotic cells decreased to control levels. Enzymatic caspase-3 activity was significantly increased in hippocampal tissue of infected animals compared to controls at 22 h. The activated enzyme was localized to immature cells of the dentate gyrus, and in vivo activity was evidenced by cleavage of the amyloid-beta precursor protein. Intracisternal administration of the caspase-3-specific inhibitor Ac-DEVD-CHO significantly reduced apoptosis in the hippocampal dentate gyrus. In contrast to a study where the decrease of hippocampal apoptosis after administration of a pan-caspase inhibitor was due to downmodulation of the inflammatory response, our data demonstrate that specific inhibition of caspase-3 did not affect inflammation assessed by TNF-alpha and IL-1beta concentrations in the cerebrospinal fluid space. Taken together, the present results identify caspase-3 as a key effector of neuronal apoptosis in pneumococcal meningitis.