An immunocytochemical procedure for protein localization in various nematode life stages combined with plant tissues using methylacrylate-embedded specimens

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access.     

Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Comparing apparent electrical conductivity measurements on a paddy field under flooded and drained conditions

Every growing season, paddy fields are kept both flooded and drained for a significant period of time. As a consequence, these soils develop distinct physico-chemical characteristics. For practical reasons, these soils are mostly sampled under dry conditions, but the question arises how representative the results are for the wet growing conditions. Therefore, the apparent electrical conductivity (EC_a) of a 1.4 ha alluvial paddy field located in the Brahmaputra floodplain of Bangladesh was measured in both dry and wet conditions by a sensing system using the electromagnetic induction sensor EM38, which does not require physical contact with the soil, and compared both surveys. Due to the smooth water surface under wet conditions which ensured increased stability of the sensing platform, the results of the survey showed considerably reduced micro-scale variability of EC_a. Furthermore, the wet survey results more reliably furnished soil-related information mainly due to the absence of soil moisture dynamics. The differences between EC_a under wet and dry conditions were attributed to differences in soil texture, mainly the sand content variation having considerable effect on soil moisture differences when flooded following drainage. Accordingly, the largest differences between EC_a under wet and dry conditions were found in those parts of the field with a large sand content. Hence, the conclusion was that an EC_a survey on flooded fields has an added value to precision soil management.     

Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria

The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical containment remain unclear. Here, we show that interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses were associated with Crohn's disease and progressive hepatitis C virus infection in patients. Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases.     

Murine typhus in the homeless

Homeless populations are particularly exposed to many vector-borne diseases because of their poor living conditions. We tested sera from 299 homeless people recruited in 2010 and 2011 in Marseilles, France for antibodies to Rickettsia typhi by microimmunofluorescence using a titer of 1:25 as a cut-off titer, and we confirmed the results by Western blot and cross-adsorption studies. Sixty-three persons (22%) had antibodies against R. typhi. The murine typhus seroprevalence rates have significantly increased in homeless populations between the 2000-2003 and 2010-2011 periods. These findings indicate that the homeless are increasingly exposed to flea-borne murine typhus in Marseilles. One might suggest that multiple strikes of sanitation workers resulting in the increase of waste and construction sites combined with the poor living conditions of the homeless expose this population to rodents and their fleas. Further annual studies are necessary to follow rodent-associated diseases among homeless people in Marseille.     

Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population

Mycoplasma agalactiae causes chronic infections in small ruminants and remains endemic in many regions of the world, despite intensive and costly eradication programs. In this study, the innate genomic plasticity of M. agalactiae was exploited to design and assess a combination of molecular epidemiological tools to trace the pathogen in different geographic locations and to understand its emergence or re-emergence after eradication campaigns. For this purpose, two collections of M. agalactiae isolates, representing European outbreaks or localized endemic disease in a single region of France, were subjected to RFLP (Restriction Fragment Length Polymorphism) analyses using two sets of DNA probes (distributed across the genome and specific for the vpma gene locus), and a previously described VNTR (Variable Number Tandem Repeats) analysis. A combination of four genome-specific DNA probes and two VNTRs gave the highest discriminative power. Molecular typing revealed that, while isolates from diverse geographical origins fell into clearly different groups, the endemic disease repeatedly observed in the Western Pyrenees region over the past 30 years has been caused by a unique subtype of M. agalactiae. This indicates that the re-emergence of the pathogen after seemingly successful eradication programs is not due to the importation of exotic strains, but to the persistence of local reservoirs of infection.     

A common near infrared—based partial least squares regression model for the prediction of wood density of Pinus pinaster and Larix?×?eurolepis

Wood density is defined as the ratio of mass to volume and therefore in principle it should be possible to calculate a unique partial least squares regression (PLS-R) model for several species. PLS-R models for wood density based on X-ray microdensity data were calculated for each species Pinus pinaster and Larix × eurolepis and for both species together. After cross-validation and test set validation the data sets were combined and final models were calculated. The common model gave a residual prediction deviation (RPD) of 3.1, a range error ratio (RER) of 11.7, and a SEP/SEC of 1.06. The single models for Pinus pinaster and Larix?×?eurolepis gave RPD’s of 3.5 and 3.2, RER’s of 13 and 11, and a SEP/SEC of 1.2. To the best knowledge of the authors all obtained PLS-R models are the first ones that fulfil the requirements according to AACC Method 39-00 (AACC in AACC Method, 39-00:15, 1999) to be used at least for screening (RPD?≥?2.5). Although this method and the defined limits were developed for the analysis of grains they can be used as a rough rule of thumb until limits for wood are available. The improvement of the PLS-R models, compared to published results, might be due to three facts (1) the higher number of scans collected for a single spectrum, (2) that the samples were better represented by the NIR spectra and X-ray microdensity values, and (3) that the sites for the measurement of NIR spectra and X-ray microdensity were coincided as strictly as possibly.     

Clinical utility of a complete diagnostic protocol for the ocular evaluation of free-living raptors

Objective: To describe a protocol for the examination of free‐living raptors and report the ophthalmic examination findings of seven raptor species native to central Illinois, namely the barred owl, Cooper’s hawk, eastern screech owl, great horned owl, American kestrel, red‐tailed hawk, and turkey vulture and to determine if the findings relative to visual prognosis affected eligibility for future release. Animals studied: Seventy‐nine free‐living raptors. Procedures: Under manual restraint, complete ophthalmic examination including slit‐lamp biomicroscopy and indirect funduscopy, applanation tonometry, rebound tonometry, ocular morphometrics, B‐mode ultrasound, and electroretinography (ERG) were performed on each bird. Histopathology of enucleated globes was performed after euthanasia or death in selected cases. Results: The examination protocol was easily performed using manual restraint alone on all birds. Ocular lesions were detected in 48.1% of birds, with 47.3% affected unilaterally and 52.6% affected bilaterally. Ocular lesions were considered to be vision threatening in 29.0% of the unilaterally affected birds and 29.0% of the bilaterally affected birds. The most common case outcomes were discharge from hospital to rehabilitation facility (45.6%) followed by euthanasia (43.0%). The presence of an ocular lesion or a vision‐threatening ocular lesion was not significantly associated with outcome. Reference ranges are reported for B‐mode ultrasound, ocular morphometrics, and horizontal corneal diameter in all species. Conclusion: Complete ophthalmic examination can be supplemented by the use of ocular morphometrics, ultrasound, and ERG in the manually restrained raptor. These advanced diagnostic techniques may be useful in developing more objective criteria for evaluating eligibility for release following rehabilitation of free‐living birds of prey.     

Modeling of light transmission under heterogeneous forest canopy: an appraisal of the effect of the precision level of crown description

• Context  

Light availability in forest understory is essential for many processes; it is, therefore, a valuable information regarding forest management. However, its estimation is often difficult and direct measurements are tedious. Models can be used to compute understory light, but they often require a lot of field data to accurately predict light distribution, particularly in the case of heterogeneous canopies.     

Solid-Phase Extraction Embedded Dialysis (SPEED), an Innovative Procedure for the Investigation of Microbial Specialized Metabolites

Solid-phase extraction embedded dialysis (SPEED technology) is an innovative procedure developed to physically separate in-situ, during the cultivation, the mycelium of filament forming microorganisms, such as actinomycetes and fungi, and the XAD-16 resin used to trap the secreted specialized metabolites. SPEED consists of an external nylon cloth and an internal dialysis tube containing the XAD resin. The dialysis barrier selects the molecular weight of the trapped compounds, and prevents the aggregation of biomass or macromolecules on the XAD beads. The external nylon promotes the formation of a microbial biofilm, making SPEED a biofilm supported cultivation process. SPEED technology was applied to the marine Streptomyces albidoflavus 19-S21, isolated from a core of a submerged Kopara sampled at 20 m from the border of a saltwater pond. The chemical space of this strain was investigated effectively using a dereplication strategy based on molecular networking and in-depth chemical analysis. The results highlight the impact of culture support on the molecular profile of Streptomyces albidoflavus 19-S21 secondary metabolites.