Multivariate analysis for characteristics of heat tolerance in horses in Brazil

The environment in which the horse is reared affects its ability to maintain thermal balance which is in turn related to thermal characteristics and regulatory physiological mechanisms. In this study a multivariate analysis of physiological traits in relation to heat tolerance in horses was carried out in the Federal District, Brazil. The aim was to test the ability of these analyses to separate groups of animals and determine which physiological traits are most important in the adaptation to heat stress. Forty adult horses (4 to 13 years) were used, ten from each of four different genetic groups (English thoroughbred, Brazilian showjumper, crossbred and Breton). The traits examined included heart and breathing rate, rectal temperature as well as blood parameters. The data underwent multivariate statistical analysis including cluster, discriminate and canonical using Statistical Analysis System - SAS ® procedures CLUSTER, STEPDISC, CANCORR and DISCRIM. The tree diagram showed clear distances between groups studied and canonical analysis was able to separate individuals in groups. The discriminate analysis identified the variables which were most important in separating these groups. The multivariate analysis was able to separate the animals into groups with RR, HR and RT being important in this separation.     

The impact of storm water runoff on a small urban stream

Background, aim, and scope  In urban areas, storm water runoff often transports various pollutants, some of which settle and form sediments. In order to have the comprehensive view of the ecological state of storm water runoff recipients, both water and sediments of the stream must be assessed. In the Baltic Sea Area, the Water Framework Directive & HELCOM Recommendations aim to prevent or minimise pollution caused by harmful substances arising from storm water runoff, in order to promote the ecological restoration of the Baltic Sea—one of the most vulnerable seas. The aim of the study was to investigate the toxicity of bottom sediments of a small storm water runoff recipient focusing on the potential impact of successive discharges of urban storm water. Some storm water runoff quality parameters and the toxicity of bottom sediments of recipients was studied in this research. Materials and methods  During 9 years, at four discharge points, minimum four grab samples per year at each discharge point were taken for chemical characterisation. General parameters (pH, SS, BOD₇, COD_Cr and TPH) in liquid phase samples were analysed according to standard methods. Annual limit values were taken from the Lithuanian EPA requirements for the management of storm water runoff with a focus on prevention and control of contamination. Eleven composite samples of stream bottom sediments, each consisting of ten sub-samples, were collected in 2006. Toxicity screening from sediments was performed using the plant Lepidium sativum according to modified I. Magone’s methodology (Magone I, Bioindication of phytotoxicity of transport emission. In: Kachalova O-L, Zinatne (eds) Bioindication of toxicity of transport emissions in the impact of highway emissions on natural environment. Riga, pp 108–116, 1989). The level of toxic impact of Lepidium sativum (compared to control) was assessed according to the modified method of Wang (Rev Environ Contam Toxicol 126:88–127, 1992). Results  The mean pH of urban storm water runoff does not vary much from neutral, but range values are quite different, from 4.0 up to 8.7. The highest concentration of SS reached 800 mg L⁻¹, TPH—2.4 mg L⁻¹, BOD₇—300 mg O₂ L⁻¹ and COD_Cr—1,400 mg L⁻¹. The SS was above the limit in 64% of total amount of grab samples, TPH—37%, BOD₇—41% and COD_Cr—55%. The toxicity analysis of the bottom sediments showed varying toxicity of bottom sediments along the stream. From nine analysed samples of bottom sediments, 30% had weak toxicity, 30% medium and 30% strong toxicity on the test organism plant L. sativum. There was one single sample with no toxic effects, so that the results showed that urban storm water has an unacceptable environmental impact on recipients. It was also indicated that storm water runoff discharge alone is not the potential source of toxicity of bottom sediments. The litter demonstrated a weak toxicity of bottom sediments as well. Discussion  Most local authorities do not consider storm water runoff discharges to be a matter of great concern because they believe that surface runoff arising from rainfall is still relatively clean. The study showed that the current method of monitoring storm water runoff quality by chemical analyses is not the best tool for environmental impact assessment and must be combined with toxicity tests of bottom sediments of recipients. Recommendations and perspectives  To avoid the environmental impact of storm water runoff more attention should be paid to the development and implementation of storm water runoff pollution prevention measures. The study implies that future research concerning the relationships between storm water runoff deposit characteristics and biological activities must be developed to evaluate the contamination potential of stream sediment deposits for local aquatic ecosystems. Further studies should be developed to characterise the activities of the microbial community of storm water runoff sediments, and to monitor bioremediation in situ.     

Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.     

Multielemental Determinations in Chocolate Drink Powder Using Multivariate Optimization and ICP OES

In this work multivariate experiments were conducted to optimize the operating conditions for inductively coupled plasma optical emission spectrometry (ICP OES) for multielemental determinations in chocolate drink powder. The operating conditions were investigated using a 2(3) central composite design, where the variables studied were radio frequency power, nebulization flow rate, and auxiliary argon flow rate. The effects of these parameters on plasma robustness and on signal to background ratio (SBR) were considered in parallel, allowing the evaluation of robustness and detectability using few and fast experiments to select the best conditions for the determination of the analytes. In this case, the proposed experiments were applied to the optimization of a method aimed at the determination of Al, Ba, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, P, Pb, V, and Zn in chocolate drink powder. The compromise conditions that allowed obtaining a robust and sensitive analytical method were radio frequency power of 1200 W, nebulization flow rate of 0.6 L/min, and auxiliary argon flow rate of 0.3 L/min. Using these conditions, recoveries between 95 and 105% and relative standard deviations lower than 5% were obtained for the majority of the analytes. The proposed method was successfully applied to the analysis of 15 samples of chocolate drink powder. The highest concentrations of metallic species were found in diet and light products.     

Evaluation of the Biochemical Components and Chromatic Properties of the Juice of <Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Aiton and <Emphasis Type="Italic">Vaccinium oxycoccos</Emphasis> L.

Benzoic acid, total anthocyanins, soluble solids, titratable acidity, and colour properties in juice of the American cranberry Vaccinium macrocarpon and the European cranberry Vaccinium oxycoccos were investigated. Berry juices of V. macrocarpon cultivars were distinguished by their higher total anthocyanin and benzoic acid amounts. These cultivars accumulated on average 43.11 mg/l of benzoic acid and 92.45 mg/l of total anthocyanins. The levels of benzoic acid and total anthocyanins in V. oxycoccos cultivars were 17.52 mg/l and 42.54 mg/l, respectively. The V. macrocarpon cultivars ‘Franklin’, ‘Le Munyon’, ‘Searles’, and ‘Early Richard’ were selected as the best according to the enhanced total anthocyanins and benzoic acid amounts. The separation of anthocyanins by HPLC-UV-VIS revealed the presence of six anthocyanins, with peonidin-3-galactoside being the most prevalent. Galactoside together with glucoside conjugates comprised the largest percentage of total anthocyanins in the juices of V. macrocarpon and V. oxycoccos cultivars.     

The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation

The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(?) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM?, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM? and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.