A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium.

Cryptosporidium spp. are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against Cryptosporidium oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in Cryptosporidium assay development is limited. The aims of this study were to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on C. parvum oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of Cryptosporidium in spiked water samples. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

Evaluation of pathogen specific urinary peptides in tick-borne illnesses.

Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Microbotryozyma collariae gen. nov., sp. nov., a basidiomycetous yeast isolated from a plant bug Collaria oleosa (Miridae).

Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666(T) (= PRA303-1S = CBS 12537) designated as the type strain.

The apicomplexan pathogen Neospora caninum inhibits host cell apoptosis in the absence of discernible NF-kappa B activation.

Neospora caninum, a causative agent of bovine abortions, is an apicomplexan parasite that is closely related to the human pathogen Toxoplasma gondii. Since a number of intracellular parasites, including T. gondii, have been shown to modulate host cell apoptosis, the present study was conducted to establish whether N. caninum is similarly capable of subverting apoptotic pathways in its host cells. Our results indicated that death receptor-mediated apoptosis is repressed during N. caninum infection, and the data further showed that the executioner caspase, caspase 3, does not become activated in the infected cells. Surprisingly, nuclear translocation of the NF-kappaB subunit p65 was not detected in N. caninum-infected cells, although this host transcription factor has been shown to upregulate prosurvival genes in cells infected with T. gondii. Consistent with these findings, the distinct accumulation of phosphorylated IkappaB that is seen at the parasitophorous vacuole membrane (PVM) of T. gondii was not apparent on the N. caninum PVM. Although a putative IkappaB kinase activity was detected in N. caninum extracts, thereby implying that this parasite is capable of modulating NF-kappaB translocation into the host cell nucleus, the data collectively suggest that a profound and sustained activation of the NF-kappaB pathway is not central to the ability of N. caninum to prevent apoptosis of their host cells.

Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells.

The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-alpha). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-alpha, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-alpha-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.

Activation of NF-kappaB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IkappaB to the parasitophorous vacuole membrane.

Mammalian cells infected with Toxoplasma gondii are resistant to apoptosis induced by a variety of stimuli. We have demonstrated that the host transcription factor NF-kappaB plays a pivotal role in the T.-gondii-mediated blockade of apoptosis because inhibition is lost in cells lacking the p65 (RelA) subunit of NF-kappaB (p65-/-). In the present study, we examined the effects of T. gondii infection on NF-kappaB activation and the expression of genes involved in the apoptotic cascade. Infection of wild-type mouse embryonic fibroblasts (MEFs) with T.-gondii-induced nuclear translocation of the p50 and p65 subunits of NF-kappaB as examined by immunoblotting of nuclear extracts, immunofluorescence and electrophoretic mobility shift assays. A comparison of apoptotic gene expression profiles from wild-type and p65-/- MEFs revealed distinct patterns of induction in response to T. gondii infection. In particular, the differences seen in the Bcl-2 and IAP families are consistent with the antiapoptotic responses observed in the resistant wild-type cells compared with the sensitive p65-/- fibroblasts. Consistent with NF-kappaB activation, T. gondii infection promoted phosphorylation of the inhibitor IkappaB. Interestingly, phosphorylated IkappaB was concentrated on the parasitophorous vacuole membrane (PVM), suggesting a parasite-directed event. Results from this study suggest that activation of NF-kappaB plays an important role in stimulation of antiapoptotic gene expression by T. gondii. Furthermore, recruitment of phosphorylated IkappaB to the PVM implies the presence of intrinsic factor(s) in T. gondii that might be used to manipulate the NF-kappaB signaling pathway in the host to elicit a survival response during infection.

Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells.

Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-gamma)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [(35)S]methionine/cysteine under normal conditions or IFN-gamma-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-gamma per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-gamma treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-gamma while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-gamma pressure to obtain a finer representation of the gene products involved in establishing persistence.

Effect of macrolide antibiotics on human endothelial cells activated by Chlamydia pneumoniae infection and tumor necrosis factor-alpha.

This study investigated the potential anti-inflammatory activity of 3 macrolide antibiotics, clarithromycin, roxithromycin, and azithromycin, in an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVECs) were seeded in Transwell inserts, treated with serial dilutions of the antibiotics, and infected with Chlamydia pneumoniae or stimulated with tumor necrosis factor (TNF)-alpha. In HUVECs infected with C. pneumoniae or stimulated with TNF-alpha, both azithromycin and roxithromycin caused significant decreases in neutrophil and monocyte TEM, compared with antibiotic-free controls. Clarithromycin had no detectable effect in either group. Azithromycin caused significant decreases in interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1, whereas roxithromycin significantly decreased IL-8. This study indicates heterogeneity in the anti-inflammatory activity of these antibiotics. Mechanisms of monocyte and neutrophil TEM inhibition by azithromycin and roxithromycin are unclear but may be partially due to inhibition of IL-8 and MCP-1 production.