Immunogenetic Variation and Differential Pathogen Exposure in Free-Ranging Cheetahs across Namibian Farmlands

Background

Genes under selection provide ecologically important information useful for conservation issues. Major histocompatibility complex (MHC) class I and II genes are essential for the immune defence against pathogens from intracellular (e.g. viruses) and extracellular (e.g. helminths) origins, respectively. Serosurvey studies in Namibian cheetahs (Acinonyx juabuts) revealed higher exposure to viral pathogens in individuals from north-central than east-central regions. Here we examined whether the observed differences in exposure to viruses influence the patterns of genetic variation and differentiation at MHC loci in 88 free-ranging Namibian cheetahs.

Methodology/Principal Findings

Genetic variation at MHC I and II loci was assessed through single-stranded conformation polymorphism (SSCP) analysis and sequencing. While the overall allelic diversity did not differ, we observed a high genetic differentiation at MHC class I loci between cheetahs from north-central and east-central Namibia. No such differentiation in MHC class II and neutral markers were found.

Conclusions/Significance

Our results suggest that MHC class I variation mirrors the variation in selection pressure imposed by viruses in free-ranging cheetahs across Namibian farmland. This is of high significance for future management and conservation programs of this species.     

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

ABSTRACT: BACKGROUND: In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. RESULTS: Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. CONCLUSIONS: MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.     

Towards functional phosphoproteomics by mapping differential phosphorylation events in signaling networks

Protein phosphorylation plays a central role in many signal transduction pathways that mediate biological processes. Novel quantitative mass spectrometry-based methods have recently revealed phosphorylation dynamics in animals, yeast, and plants. These methods are important for our understanding of how differential phosphorylation participates in translating distinct signals into proper physiological responses, and shifted research towards screening for potential cancer therapies and in-depth analysis of phosphoproteomes. In this review, we aim to describe current progress in quantitative phosphoproteomics. This emerging field has changed numerous static pathways into dynamic signaling networks, and revealed protein kinase networks that underlie adaptation to environmental stimuli. Mass spectrometry enables high-throughput and high-quality analysis of differential phosphorylation at a site-specific level. Although determination of differential phosphorylation between treatments is analogous to detecting differential gene expression, the large body of statistical techniques that has been developed for analysis of differential gene expression is not generally applied for detecting differential phosphorylation. We suggest possible improvements for analysis of quantitative phosphorylation by increasing the number of biological replicates and adapting statistical tests used for gene expression profiling and widely implemented in freely available software tools.     

The dark side of the salad: Salmonella typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle

Salmonella enterica serovar typhimurium contaminated vegetables and fruits are considerable sources of human infections. Bacteria present in raw plant-derived nutrients cause salmonellosis, the world wide most spread food poisoning. This facultative endopathogen enters and replicates in host cells and actively suppresses host immune responses. Although Salmonella survives on plants, the underlying bacterial infection mechanisms are only poorly understood. In this report we investigated the possibility to use Arabidopsis thaliana as a genetically tractable host system to study Salmonella-plant interactions. Using green fluorescent protein (GFP) marked bacteria, we show here that Salmonella can infect various Arabidopsis tissues and proliferate in intracellular cellular compartments. Salmonella infection of Arabidopsis cells can occur via intact shoot or root tissues resulting in wilting, chlorosis and eventually death of the infected organs. Arabidopsis reacts to Salmonella by inducing the activation of mitogen-activated protein kinase (MAPK) cascades and enhanced expression of pathogenesis related (PR) genes. The induction of defense responses fails in plants that are compromised in ethylene or jasmonic acid signaling or in the MKK3-MPK6 MAPK pathway. These findings demonstrate that Arabidopsis represents a true host system for Salmonella, offering unique possibilities to study the interaction of this human pathogen with plants at the molecular level for developing novel drug targets and addressing current safety issues in human nutrition.     

Influence of soft rush (Juncus effusus) on phosphorus flux in grazed seasonal wetlands

Livestock significantly affect wetland soils and vegetation but their impacts on wetland nutrient dynamics are poorly understood. We set up a full factorial laboratory experiment to assess the effects of Juncus effusus, grazing exclusion, and flooding on P flux from intact cores collected from seasonal wetlands in cattle pastures in south Florida. We collected intact cores from Juncus tussocks and plant interspaces inside and outside 4-year grazing exclosures in five replicate wetlands. We incubated the cores for 50 days under continuous flooding or weekly 1-day flooding cycles and measured P concentrations in surface and pore water. Grazing exclosures had less Juncus (17%) and bare ground (2%) than adjacent grazed areas (Juncus, 48%; bare ground, 12%), but did not affect P fluxes. Initial fluxes of soluble reactive P (SRP) were much higher in cores with Juncus (242 ± 153 mg P m⁻² day⁻¹) than without Juncus (14 ± 20 mg P m⁻² day⁻¹). In weekly flooded cores P fluxes fell to 19.7 ± 13.4 mg P m⁻² day⁻¹ in cores with and 2.7 ± 2.6 in cores without Juncus. The strong effect of Juncus on P flux was an indirect effect of cattle grazing, but 4 years of grazing exclusion did not have a significant effect on P fluxes.     

Microbiota in a cooling-lubrication circuit and an option for controlling triethanolamine biodegradation

Cooling and lubrication agents like triethanolamine (TEA) are essential for many purposes in industry. Due to biodegradation, they need continuous replacement, and byproducts of degradation may be toxic. This study investigates an industrial (1,200 m³) cooling-lubrication circuit (CLC) that has been in operation for 20 years and is supposedly in an ecological equilibrium, thus offering a unique habitat. Next-generation (Illumina Miseq 16S rRNA amplicon) sequencing was used to profile the CLC-based microbiota and relate it to TEA and bicine dynamics at the sampling sites, influent, machine rooms, biofilms and effluent. Pseudomonas pseudoalcaligenes dominated the effluent and influent sites, while Alcaligenes faecalis dominated biofilms, and both species were identified as the major TEA degrading bacteria. It was shown that a 15 min heat treatment at 50°C was able to slow down the growth of both species, a promising option to control TEA degradation at large scale.     

Protection of bacteria against toxicity of phenol by immobilization in calcium alginate

Summary The antibacterial activity of phenol was determined by measuring inhibition of exponentially growing free and immobilized cells of Escherichia coli, Pseudomonas putida and Staphylococcus aureus. Immobilization of microorganisms in calcium alginate beads reduced the growth inhibition caused by bacteriostatic concentrations of phenol. The increase in phenol tolerance occurred at different culture conditions and growth rates of the cells. The strength of the effect, however, was found to correlate with the formation of colonies in the gel matrix. Dissolution of gel beads led to a substantial loss of the protection against phenol of immobilized-grown cells.