Proteomic Analysis of Fractionated Toxoplasma Oocysts Reveals Clues to Their Environmental Resistance

Toxoplasma gondii is an obligate intracellular parasite that is unique in its ability to infect a broad range of birds and mammals, including humans, leading to an extremely high worldwide prevalence and distribution. This work focuses on the environmentally resistant oocyst, which is the product of sexual replication in felids and an important source of human infection. Due to the difficulty in producing and working with oocysts, relatively little is known about how this stage is able to resist extreme environmental stresses and how they initiate a new infection, once ingested. To fill this gap, the proteome of the wall and sporocyst/sporozoite fractions of mature, sporulated oocysts were characterized using one-dimensional gel electrophoresis followed by LC-MS/MS on trypsin-digested peptides. A combined total of 1021 non-redundant T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 were identified in the oocyst wall fraction. Significantly, 172 of the identified proteins have not previously been identified in Toxoplasma proteomic studies. Among these are several of interest for their likely role in conferring environmental resistance including a family of small, tyrosine-rich proteins present in the oocyst wall fractions and late embryogenesis abundant domain-containing (LEA) proteins in the cytosolic fractions. The latter are known from other systems to be key to enabling survival against desiccation

Three Pathogens in Sympatric Populations of Pumas, Bobcats, and Domestic Cats: Implications for Infectious Disease Transmission

Anthropogenic landscape change can lead to increased opportunities for pathogen transmission between domestic and non-domestic animals. Pumas, bobcats, and domestic cats are sympatric in many areas of North America and share many of the same pathogens, some of which are zoonotic. We analyzed bobcat, puma, and feral domestic cat samples collected from targeted geographic areas. We examined exposure to three pathogens that are taxonomically diverse (bacterial, protozoal, viral), that incorporate multiple transmission strategies (vector-borne, environmental exposure/ingestion, and direct contact), and that vary in species-specificity. Bartonella spp., Feline Immunodeficiency Virus (FIV), and Toxoplasma gondii IgG were detected in all three species with mean respective prevalence as follows: puma 16%, 41% and 75%; bobcat 31%, 22% and 43%; domestic cat 45%, 10% and 1%. Bartonella spp. were highly prevalent among domestic cats in Southern California compared to other cohort groups. Feline Immunodeficiency Virus exposure was primarily associated with species and age, and was not influenced by geographic location. Pumas were more likely to be infected with FIV than bobcats, with domestic cats having the lowest infection rate. Toxoplasma gondii seroprevalence was high in both pumas and bobcats across all sites; in contrast, few domestic cats were seropositive, despite the fact that feral, free ranging domestic cats were targeted in this study. Interestingly, a directly transmitted species-specific disease (FIV) was not associated with geographic location, while exposure to indirectly transmitted diseases – vector-borne for Bartonella spp. and ingestion of oocysts via infected prey or environmental exposure for T. gondii – varied significantly by site. Pathogens transmitted by direct contact may be more dependent upon individual behaviors and intra-specific encounters. Future studies will integrate host density, as well as landscape features, to better understand the mechanisms driving disease exposure and to predict zones of cross-species pathogen transmission among wild and domestic felids

Genetically-Determined Hyperfunction of the S100B/RAGE Axis Is a Risk Factor for Aspergillosis in Stem Cell Transplant Recipients

Invasive aspergillosis (IA) is a major threat to the successful outcome of hematopoietic stem cell transplantation (HSCT), although individual risk varies considerably. Recent evidence has established a pivotal role for a danger sensing mechanism implicating the S100B/receptor for advanced glycation end products (RAGE) axis in antifungal immunity. The association of selected genetic variants in the S100B/RAGE axis with susceptibility to IA was investigated in 223 consecutive patients undergoing HSCT. Furthermore, studies addressing the functional consequences of these variants were performed. Susceptibility to IA was significantly associated with two distinct polymorphisms in RAGE (-374T/A) and S100B (+427C/T) genes, the relative contribution of each depended on their presence in both transplantation counterparts [patient SNPRAGE, adjusted hazard ratio (HR), 1.97; P = 0.042 and donor SNPRAGE, HR, 2.03; P = 0.047] or in donors (SNPS100B, HR, 3.15; P = 7.8e-4) only, respectively. Functional assays demonstrated a gain-of-function phenotype of both variants, as shown by the enhanced expression of inflammatory cytokines in RAGE polymorphic cells and increased S100B secretion in vitro and in vivo in the presence of the S100B polymorphism. These findings point to a relevant role of the danger sensing signaling in human antifungal immunity and highlight a possible contribution of a genetically-determined hyperfunction of the S100B/RAGE axis to susceptibility to IA in the HSCT setting

Transcriptomic Analysis of Toxoplasma Development Reveals Many Novel Functions and Structures Specific to Sporozoites and Oocysts

Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1–10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear “off” in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle

Gut mucosal DAMPs in IBD: From mechanisms to therapeutic implications

Endogenous damage-associated molecular patterns (DAMPs) are released during tissue damage and have increasingly recognized roles in the etiology of many human diseases. The inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD), are immune-mediated conditions where high levels of DAMPs are observed. DAMPs such as calprotectin (S100A8/9) have an established clinical role as a biomarker in IBD. In this review, we use IBD as an archetypal common chronic inflammatory disease to focus on the conceptual and evidential importance of DAMPs in pathogenesis and why DAMPs represent an entirely new class of targets for clinical translation. </p

Molecular Epidemiology of Infant Botulism in California and Elsewhere, 1976–2010

Background. Infant botulism (IB), first identified in California in 1976, results from Clostridium botulinum spores that germinate, multiply, and produce botulinum neurotoxin (BoNT) in the immature intestine. From 1976 to 2010 we created an archive of 1090 BoNT-producing isolates consisting of 1012 IB patient (10 outpatient, 985 hospitalized, 17 sudden death), 25 food, 18 dust/soils, and 35 other strains. Methods. The mouse neutralization assay determined isolate toxin type (56% BoNT/A, 32% BoNT/B). Amplified fragment-length polymorphism (AFLP) analysis of the isolates was combined with epidemiologic information. Results. The AFLP dendrogram, the largest to date, contained 154 clades; 52% of isolates clustered in just 2 clades, 1 BoNT/A (n = 418) and 1 BoNT/B (n = 145). These clades constituted an endemic C. botulinum population that produced the entire clinical spectrum of IB. Isolates from the patient’s home environment (dust/soil, honey) usually located to the same AFLP clade as the patient’s isolate, thereby identifying the likely source of infective spores. C. botulinum A(B) strains were identified in California for the first time. Conclusions. Combining molecular methods and epidemiological data created an effective tool that yielded novel insights into the genetic diversity of C. botulinum and the clinical spectrum, occurrence, and distribution of IB in California

Cardiological and general health status in preschool- and school-age children after neonatal arterial switch operation

OBJECTIVE: Cardiological and general health status 3-9 years after neonatal arterial switch operation for transposition of the great arteries should be evaluated by non-invasive methods. METHODS: A total of 77 unselected children with intact ventricular septum (75.3%) or ventricular septal defect (24.7%) without or with aortic isthmic stenosis (5.2%) were prospectively examined 3.2-9.4 years (5.4 +/- 1.6) after neonatal switch. Clinical pediatric and cardiological examination, standard and 24 h Holter electrocardiogram, M-mode, 2D-, Doppler and colour Doppler echocardiography were performed. Outcome data were compared to published normals. RESULTS: Reoperation rate was 2.6%, 96.1% were without limitation of physical activity and 98.7% without medication. Compared to normals, growth was adequate, weight and head circumference were slightly reduced. After median sternotomy, 23.4% had abnormal thoracic configuration (16.9% asymmetry, 6.5% funnel chest). ECG and Holter: 93.5% were in sinus, 6.5% in ectopic atrial or junctional rhythm. Incidence of complete right bundle branch block was 15.8% in patients with ventricular septal defect and 5.2% in those without. Ischemic ST-T changes during exercise due to coronary artery occlusion and evidence of old myocardial infarction were found in 1 patient (1.3%) each. Occasional atrial ectopy was found in 27.4%, ventricular ectopy in 15.3%: occasional in 12.5% and frequent (> 30/h) in 2.8% presenting bigemini, couplets and short runs of ventricular tachycardia at rest and during exercise. Echocardiography: Left ventricular function was normal in all. Endsystolic diameter of neoaortic valve annulus was beyond 90% confidence interval for controls in 79.2%, neoaortic root diameter in 100%. Mild aortic insufficiency was seen in 10.4%. No correlation was found between aortic insufficiency and aortic dilatation. Neoaortic stenosis was not seen, mild residual coarctation after end-to-end-anastomosis was found in 2.6%, native coarctation corrected later on in 1.3%. Supravalvular pulmonary stenosis was seen in 29.9% (19.5% trivial, 7.8% mild, 2.6% moderate), mild subvalvular pulmonary stenosis in 1.3%, pulmonary insufficiency in 2.6%. CONCLUSION: The study confirms good midterm results after neonatal arterial switch operation for transposition with or without ventricular septal defect. Long-term observation is necessary to assess rhythm, coronary artery and myocardial function as well as development of neo-aorta and pulmonary artery system