Infectivity and virulence of Nosema ceranae (Microsporidia) isolates obtained from various Apis mellifera morphotypes

The infection of honey bees, Apis mellifera L. (Hymenoptera: Apidae), by the microsporidian Nosema ceranae is one of the factors related to the increase in colony losses and the decrease in honey production observed in recent years. However, these effects seem to differ depending on the climate zone. The range and prevalence of N. ceranae have increased significantly in the last decades, with different consequences in northern and southern temperate areas. The existence of various isolates of N. ceranae from distant geographical areas, which probably exhibit different degrees of virulence, could explain the different responses of the bee to the infection. The aim of this work was to compare the effects of two N. ceranae isolates from different host populations from Argentina on honey bee survival at two ages post-eclosion. Using cage experiments, we compared the development of infection of worker bees through the estimation of daily bee mortality and spore counts. Host subspecies identity analysis showed a strong similarity with Apis mellifera scutellata morphotype for the northern region, with a greater hybridization between subspecies with European origin toward the central and southern regions. Genetic characterization of isolates from the three regions indicated only the presence of N. ceranae. Infected bees survived longer than control bees, and bees infected at 5 days had a lower survival than those infected at 72 h with isolates from the three regions. These differences in survival matched the development of the N. ceranae infection, with differences in spore loads for infected bees at 5 days. Our studies showed that Nosema infection and survival varied among the different ages post emergence of workers, and both increased as the honey bee aged. These differences in susceptibility to infection could be related to the immune response of bees of different ages or to changes in the composition and succession of the intestinal microbiota throughout its ontogeny.     

Synergistic influences of introduced herbivores and fire on vegetation change in northern Patagonia,Argentina

Question: We investigated how cattle and European hares, the two most widespread exotic herbivores in Patagonia, affect species composition, life‐form composition and community structure during the first 6 years of vegetation recovery following severe burning of fire‐resistant subalpine forests and fire‐prone tall shrublands. We asked how the effects of introduced herbivores on post‐fire plant community attributes affect flammability of the vegetation. Location: Nahuel Huapi National Park, northwest Patagonia, Argentina Methods: We installed fenced plots to exclude livestock and European hares from severely burned subalpine forests of Nothofagus pumilio and adjacent tall shrublands of N. antarctica. The former is an obligate seed reproducer, whereas the latter and all other woody dominants of the shrubland vigorously resprout after burning. Results: Repeated measures ANOVA of annual measurements over the 2001‐2006 period indicate that cattle and hare exclusion had significant but complex effects on the cover of graminoids, forbs, climber species and woody species in the two burned community types. Significant interactions between the effects of cattle and hares varied by plant life forms between the two communities, which implies that their synergistic effects are community dependent. Conclusions: Following severe fires, the combined effects of cattle and hares inhibit forest recovery and favour transition to shrublands dominated by resprouting woody species. This herbivore‐induced trend in vegetation structure is consistent with the hypothesis that the effects of exotic herbivores at recently burned sites contribute to an increase in the overall flammability of the Patagonian landscape.     

Six recommendations for improving monitoring of diseases shared with wildlife: examples regarding mycobacterial infections in Spain

Monitoring is needed to identify changes in disease occurrence and to measure the impact of intervention. Using mycobacterial diseases as an example, we discuss herein the pros and cons of the current Spanish Wildlife Disease Surveillance Scheme providing suggestions for monitoring relevant diseases shared with wildlife in other regions facing similar challenges. Six points should be considered. This includes: (1) making sure the disease is properly monitored in the relevant domestic animals or even in humans; (2) also making sure that background information on wildlife population ecology is available to maximize the benefits of the monitoring effort; (3) selecting the appropriate wildlife hosts for monitoring, while being flexible enough to incorporate new ones if research suggests their participation; (4) selecting the appropriate methods for diagnosis and for time and space trend analysis; (5) deciding which parameters to target for monitoring; and finally (6) establishing a reasonable sampling effort and a suitable sampling stratification to ensure detecting changes over time and changes in response to management actions. Wildlife disease monitoring produces knowledge that benefits at least three different agencies, namely, animal health, public health and conservation, and these should combine efforts and resources. Setting up stable, comprehensive and accurate schemes at different spatial scales should become a priority. Resources are always a limiting factor, but experience shows that combined, cross-collaborative efforts allow establishing acceptable schemes with a low enough cost to be sustainable over time. These six steps for monitoring relevant shared diseases can be adapted to many other geographical settings and different disease situations.     

Spatio-Temporal Trends of Iberian Wild Boar Contact with Mycobacterium tuberculosis Complex Detected by ELISA

The continuing expansion of Eurasian wild boar (Sus scrofa) populations raises concerns regarding disease transmission. In south-central Spain, overabundant wild boar are reservoirs of Mycobacterium bovis, and related members of the Mycobacterium tuberculosis complex (MTBC), the causative agents of bovine tuberculosis (bTB). An indirect enzyme-linked immunosorbent assay using bovine-purified protein derivative was applied to determine the spatial and temporal distribution of wild boar contact with MTBC in the Iberian Peninsula and to model and identify the associated risk factors. Wild boar apparent seroprevalence was 22%. Seropositives were detected in 71% of 81 sites, including 23 sites where wildlife was thought to be bTB free. The results described a new geographic range of wild boar contact with MTBC and a stable prevalence in this wildlife reservoir that contrasts with the success of bTB control in cattle. Inference of which host (wild boar or cattle) is driving bTB maintenance was not possible with our correlational results. The possibility of a wild boar bTB emergence in non-endemic regions should urgently be taken into account to avoid a future scenario resembling the current situation in south-central Spain.     

Serum inhibits the immunosuppressive function of myeloid-derived suppressor cells isolated from 4T1 tumor-bearing mice

As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.     

Anther structure and pollen development in Melicoccus lepidopetalus (Sapindaceae): An evolutionary approach to dioecy in the family

Anther and pollen development in staminate and pistillate flowers of dioecious Melicoccus lepidopetalus (Sapindaceae) were examined by light and electron microscopy. Young anthers are similar in both types of flowers; they consist of epidermis, endothecium, two to four middle layers and a secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents compressed epidermal cells and endothecium cells with fibrillar thickenings. A single locule is formed in the theca by dissolution of the septum and pollen grains are shed at two-celled stage. The mature anthers of pistillate flowers differ anatomically from those of staminate flowers. The epidermis is not compressed, the endothecium does not develop fibrillar thickenings, middle layers and tapetum are generally persisting, and the stomium is nonfunctional. Microspore degeneration begins after meiosis of microspore mother cells. At anthesis, uninucleate microspores and pollen grains with vegetative and generative nuclei with no cytokinesis are observed. Some pollen walls display an abnormal exine deposition, whereas others show a well formed exine, although both are devoid of intine. These results suggest that in the evolution towards unisexuality, the developmental differences of anther wall tissues and pollen grains between pistillate and staminate flowers might become more pronounced in a derived condition, such as dioecy.     

Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei

Background

Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear.

Methods/Principal Findings

We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P _K+/P _Cl−∼0.31), while the other two types of channels are slightly selective for cations (P _K+/P _Cl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel''s pore.

Conclusions/Significance

These results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.     

Spatio-temporal trends and risk factors for Trichinella species infection in wild boar (Sus scrofa) populations of central Spain: a long-term study

In south-central Spain, the harvest of Eurasian wild boar (Sus scrofa) has increased significantly during recent decades in association with more intensive management actions to increase hunting yields and with consequent effects on the health status of the wild boar populations. We investigated the spatio-temporal trends and the risk factors related to the prevalence of Trichinella spp. in wild boar in order to obtain the annual probability of occurrence for these parasites in the Ciudad Real province of south-central Spain. Based on muscle samples collected during the hunting seasons from 1998/1999 to 2009/2010, the mean prevalence for Trichinella spp. in 95,070 wild boar was 0.2% (95% confidence interval 0.17-0.23). A subsample of 1,432 wild boar was also tested by ELISA. No correlation was observed between the prevalence of infection detected by serology and by the artificial digestion of muscle. The presence of Trichinella infections in wild boar showed a decreasing trend during the study period and was negatively related with fenced wild boar populations. The predicted 'favourability' for Trichinella infections disappeared almost completely after the 2006/2007 hunting season. Risk maps based on biogeographical tools showed, however, that most hunting estates presented favourable risk factors for these parasites during at least one of the hunting seasons studied.     

Cationic amino acid uptake constitutes a metabolic regulation mechanism and occurs in the flagellar pocket of Trypanosoma cruzi

Trypanosomatids' amino acid permeases are key proteins in parasite metabolism since they participate in the adaptation of parasites to different environments. Here, we report that TcAAP3, a member of a Trypanosoma cruzi multigene family of permeases, is a bona fide arginine transporter. Most higher eukaryotic cells incorporate cationic amino acids through a single transporter. In contrast, T. cruzi can recognize and transport cationic amino acids by mono-specific permeases since a 100-fold molar excess of lysine could not affect the arginine transport in parasites that over-express the arginine permease (TcAAP3 epimastigotes). In order to test if the permease activity regulates downstream processes of the arginine metabolism, the expression of the single T. cruzi enzyme that uses arginine as substrate, arginine kinase, was evaluated in TcAAP3 epimastigotes. In this parasite model, intracellular arginine concentration increases 4-folds and ATP level remains constant until cultures reach the stationary phase of growth, with decreases of about 6-folds in respect to the controls. Interestingly, Western Blot analysis demonstrated that arginine kinase is significantly down-regulated during the stationary phase of growth in TcAAP3 epimastigotes. This decrease could represent a compensatory mechanism for the increase in ATP consumption as a consequence of the displacement of the reaction equilibrium of arginine kinase, when the intracellular arginine concentration augments and the glucose from the medium is exhausted. Using immunofluorescence techniques we also determined that TcAAP3 and the specific lysine transporter TcAAP7 co-localize in a specialized region of the plasma membrane named flagellar pocket, staining a single locus close to the flagellar pocket collar. Taken together these data suggest that arginine transport is closely related to arginine metabolism and cell energy balance. The clinical relevance of studying trypanosomatids' permeases relies on the possibility of using these molecules as a route of entry of therapeutic drugs.