The response of HEK293 cells transfected with bovine TLR2 to established pathogen-associated molecular patterns and to bacteria causing mastitis in cattle

Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.     

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.     

Treatment with oral cyclosporin A of a case of vesicular cutaneous lupus erythematosus in a rough collie

Cyclosporin A (CsA) is an immunosuppressive agent that can be used as alternative treatment to glucocorticoid therapy. This case report describes a case of vesicular cutaneous lupus erythematosus in a rough collie successfully controlled with oral CsA for more than 18 months.     

Porcine B-cell activating factor promotes anti-FMDV antibodies in vitro but not in vivo after DNA vaccination of pigs

‘B-cell activating factor belonging to the TNF family’ (BAFF) represents a cytokine produced by antigen presenting cells promoting B-cell maturation, activation and immunoglobulin class switching. In the present study, we demonstrate expression of BAFF on cultured monocyte-derived dendritic cells, which is further enhanced by interferon- or interferon-γ treatment. From these cells, porcine BAFF was cloned and the recombinant protein was expressed in mammalian cells with and without a FLAG tag at the carboxyl terminus. Only the protein without the FLAG tag was bioactive in vitro, and promoted B-cell survival and the differentiation of foot-and-mouth disease virus (FMDV)-specific memory B cells into antibody producing cells. Based on this result it was tested whether BAFF can enhance FMDV antibody responses in the context of a DNA vaccination. To this end, pigs were immunised with the anti-FMDV DNA vaccine plasmid pcDNA3.1/P1-2A3C3D and a pCI plasmid expressing porcine BAFF. Using a needle-free transdermal application method, also referred to as ‘jet injection’, pigs were vaccinated three times and their humoral response quantified by ELISA and a virus neutralisation test. After the third vaccination, three out of six animals vaccinated with the pcDNA3.1/P1-2A3C3D alone but none of the animals that also received the BAFF expressing plasmid had seroconverted. These data suggest that BAFF is not appropriate as a genetic adjuvant when applied as a simple co-injection with the antigen-encoding plasmid.     

Differences in the Effects of Temperature and Photoperiod on Progress to Flowering among Diverse Mucuna spp.

The use of Mucuna spp. in cover crop and green manure technologies is proving to be successful in the tropics and subtropics following intensive efforts in technology transfer to farmers by NGOs (Non-Governmental Organizations). This includes the transfer of seeds of Mucuna spp. for which the range of adaptation and the effect of environment on crop phenology are poorly understood. A selection of Mucuna spp. germplasm was evaluated over a range of contrasting photothermal conditions in controlled-environment glasshouses. Dates of emergence and first flowering were recorded. Where possible, triple-plane rate of development models were fitted to the first flowering data to quantify the relative sensitivity of each species to photoperiod and temperature. All accessions showed short-day responses but there were substantial differences in the sensitivity of rate of progress to first flowering to environment, particularly to photoperiod. This results in differences in maturity period between accessions which, with appropriate understanding, could be functionally exploited by NGOs and NARS. However, if ignored, it could result in the promotion and use of accessions ill adapted to the environment in which they are tested. In consequence there might be unnecessary resistance from farmers to the adoption of green manure and cover crops technologies which are vital to the design and maintenance of sustainable farming systems in low-income countries in the tropics.     

Cytotoxic activity of lignans from Hibiscus cannabinus

The antimicrobial and cytotoxic activities of six lignans isolated from the core and bark acetone extracts of Hibiscus cannabinus have been investigated. Two compounds (2 and 3) showed strong cytotoxic activity against HeLa, Hep-2 and A-549 cell lines while compound 5 showed moderate activity on HeLa cells when they were in advanced stage of cellular division. The compounds did not exhibit antimicrobial activity.     

Assessment of the accuracy of different standard methods for determining reservoir capacity and sedimentation

Purpose

Determination of reservoir volume has been used in the calculation of sedimentation, hydrodynamic modelling, and dam safety assessment, among others, and the method used as well as its associated error play an important role in the quality of these studies. This study assessed the accuracy of reservoir volume calculations based on different standards for the definition of the spacing between bathymetric range lines. A technique for terrain modelling and a method for determining the survey accuracy are also proposed.

Materials and methods

The reference reservoir volume was based on highly detailed bathymetry (10 m resolution). Seven different spacings were selected, which generated 99 separate bathymetric data sets. For each data set, the volume was calculated based on a digital terrain model according to the usual model (triangular irregular network (TIN)) and according to the insertion of mesh points (IMP) method. This was then compared with the reference volume, and the accuracy of each survey was determined.

Results and discussion

Utilization of a highly detailed and reliable bathymetric survey allowed for the assessment of the associated error in each of the bathymetric data sets, as well as of the spacing standards analyzed. The accuracy of the standards analyzed using TIN modeling ranged from 0.31% to 14.40% (30 and 300 m, respectively). The high errors are attributed to the limitation of the TIN in representing regions near shores, producing incorrect shallow depth readings, resulting in a lower calculated volume than the real volume. IMP modeling exhibited great improvement in performance over the standard TIN (i.e., for 300 m accuracy of 5.51% for IMP and 14.40% for standard TIN), and in none of the cases were the generated volumes greater than the reference volume.

Conclusions

The IMP method proved to be consistent, improving quality of the data while reducing effort in field work. The low accuracies found showed that even surveys following the widely accepted standards contain errors of a magnitude that cannot be ignored. Thus, knowing and evaluating the survey accuracy is of fundamental importance. Definition of standard spacing for reservoirs proved to be unfeasible. This must be defined on a case-to-case basis, considering the error acceptable for the purpose of the survey.     

Color properties of four cyanidin-pyruvic acid adducts

Four anthocyanin-pyruvic adducts were synthesized through the reaction of cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-sophoroside, and cyanidin 3-O-sambubioside with pyruvic acid, structurally characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR), and their chromatic properties were studied (pH and SO2 stability assays). Overall, these pigments were shown to have a higher resistance to discoloration toward pH variations and also in the presence of SO2, being that this resistance to discoloration was explained by a higher protection of the chromophore group against the water or bisulfite nucleophilic attack that gives rise to the colorless hemiacetal form. Only slight differences in the protection against the nucleophilic attack of water and bisulfite were found to occur between all of the cyanidin-pyruvic acid adducts studied. Indeed, anthocyanin-pyruvic acid adducts with glucose or sambubiose attached to the 3-O position of the flavylium moiety were shown to have smaller bleaching constants compared with similar pigments that possess a rutinosyl or sophorosyl moiety. The study of the pigments (A-D and cyanidin-3-O-glucoside) color parameters, namely, chroma (C), lightness (L), and the hue angle (h(a,b)), obtained from the CIELAB system, revealed that different patterns of sugars in the anthocyanin-pyruvic acid adduct moiety affected the referred three parameters of color. The loss of saturation (DeltaC < 0) and the increase of lightness (DeltaL > 0) presented by the cyanidin-pyruvic acid adduct solutions at acidic pH values (1.0 and 2.0) showed that they are much less colored than the cyanidin-3-O-glucoside. For higher pH values (5.0 and 7.0), the reverse trend was observed. This means that the cyanidin-pyruvic acid adducts A-D are much more colored than the anthocyanin at these pH values. The higher coloring capacity of these pigments at higher pH values may be an important feature, indicating a putative application of these compounds in food products.     

Combining Sewage Sludge and Clam Shell Waste to Prepare Adsorbents for Efficient Phosphorous Removal

Effluents containing phosphorous as phosphate ions are frequently discharged in freshwater resources contributing to the eutrophication and directly interfering in the biological equilibrium. Clam shell residues and sewage sludge were combined for preparing efficient adsorbents for phosphate removal from aqueous medium. The adsorbents were characterized before and after adsorption testing, and the adsorption equilibrium and kinetics were investigated. Phosphate removal of 89?±?1% was attained for samples prepared with 0.1?< X <?1.0, where X corresponds to sewage sludge/clam shell mass ratio. The analyses of the experimental errors indicated that the phosphorous removal followed the Elovich kinetic model, which describes adsorption in very heterogeneous surfaces. On the other hand, the best modelling was achieved using the Koble–Corrigan isotherm model, which incorporate different aspects of both Langmuir and Freundlich isotherms to represent the equilibrium data. The observed adsorption capacity (21.4 mgP g^?1) are comparable or greater to that observed for other adsorbents described in the literature.