Novel micro–suction‐cup design for sampling soil solution at defined distances from roots

Micro–suction cups made of nylon membranes and polyacrylic tubes with planar geometry of the membrane were designed for repeated sampling of rhizosphere solution at defined distances from a root monolayer. Adsorption tests revealed that the materials used (nylon membrane, polyacrylic tube) have little influence on the concentration of heavy metals in the sample solution, whereas some organic acids are partly retained by the suction cup. A sampling protocol was developed for collecting extremely small solution volumes (i.e., droplets of 28.3±2.46 μl) for subsequent measurements of trace elements using ICP‐SFMS. A homogeneity test showed that soil‐solution concentrations of Ca, K, Mg, and Ni could be reproduced independent of the suction‐cup position in a rhizobox experiment without plants. In a similar experiment, the rhizobox was planted with the Ni hyperaccumulator Thlaspi goesingense. Compared to more distant soil layers, an increase of Ni and a concurrent decrease of Ca, K, and Mg at 1 mm distance from the root plane was found. These changes can be related to plant uptake and mobilization processes. Our results show that the novel micro–suction cups are a valuable tool for elucidating rhizosphere processes.     

Idiopathic chylothorax and lymphedema in 2 whippet littermates

Idiopathic chylothorax and limb edema was diagnosed in two 2-year-old male whippet siblings. The fact that the 2 related animals developed similar clinical signs at a young age may suggest a congenital or hereditary etiology.     

Virulence of <Emphasis Type="Italic">Brucella abortus</Emphasis> isolated from cattle and water buffalo

Brucellosis has been documented in domestic water buffalo (Bubalus bubalis) but published literature is limited despite the importance of this species in tropical agricultural systems. The objective of this study was to compare the virulence of Brucella abortus isolates recovered from cattle and water buffalo. Nineteen strains of B. abortus from cattle and domestic water buffalo in Trinidad were intraperitoneally inoculated into BALB/c mice. Spleens were cultured for B. abortus and histopathological severity scores were calculated based on lymphoid depletion, lymphoid necrosis, splenitis, and macrophage accumulation. A general linear model approach was used to estimate the effect of isolate source (cattle versus water buffalo) on virulence. Isolates of water buffalo origin were significantly less virulent in the mouse model based on recovered B. abortus from splenic tissues, spleen/weight ratio, and lymphoid necrosis but not overall histopathological severity scores. Further investigation of isolates recovered from water buffalo might provide the key to the development of procedures for brucellosis control in tropical environments.     

In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk

Primary fibroblast cultures of canine cranial (CCL) and caudal (CaCL) cruciate ligaments were stimulated with different apoptosis inducers with or without preincubation of the pancaspase inhibitor zVAD.fmk. In contrast to CaCL fibroblasts, fibroblasts from CCL were significantly more susceptible to apoptosis inducers of the intrinsic pathway like doxorubicin, cisplatin and nitric oxide (NO)-donors and to Fas ligand (FasL), an apoptosis inducer of the death receptor pathway. Apoptotic response to staurosporine and the peroxynitrite donor GEA was similar in both ligament fibroblasts. Stimulation with dexamethasone or TNFα could not induce apoptosis in CCL and CaCL fibroblasts, in spite of present TNFR1 and TNFR2 receptors. zVAD.fmk was able to prevent apoptosis in up to 66% of CCL cells when treated with FasL, cisplatin or doxorubicin but it had no effect on NO or peroxynitrite induced apoptosis. In conclusion, differential susceptibility to apoptotic triggers like FasL or NO between cranial and caudal cruciate ligament fibroblasts in vitro may be a reflection of the different susceptibilities to degenerative rupture of the ligament. These findings indicate that a general caspase inhibition does not completely protect canine CCL cells from apoptosis.     

In situ characterization of the granulomatous immune response with time in nonhealing lesional skin of Leishmania braziliensis-infected rhesus macaques (Macaca mulatta)

We have recently introduced a macaque (Macaca mulatta) model of Leishmania braziliensis-induced self-healing cutaneous leishmaniasis in which the T cell-mediated inflammatory response effectively promotes parasite clearance and granuloma resolution. Here we show that macaques infected with a highly pathogenic L. braziliensis strain displayed longstanding granulomatous lesions which lasted until the end of the observation period (52 weeks). Immunoperoxidase staining of representative tissue sections indicated that distinct cell populations (CD3, CD4, CD8, CD20, Foxp3, CD20, CD68, HLA-DR, CCL2, and CXCL-10) change uniformly during infection, suggesting that the same components of the local immune response are working in unison. This model also confirmed that granuloma formation is orchestrated by diverse inflammatory mediators that are important for T helper type 1 (Th1) cell development and macrophage effector functions. Cytometry analysis of ex vivo granuloma-derived leukocytes revealed accumulation of distinct functional subsets of effector and regulatory T cells into the inflamed skin. We provide evidence that local interleukin (IL)-10 production by both Foxp3(+) and Foxp3(-) CD4(+) T subsets is likely important in promoting lesional granuloma maintenance. Further studying the immune suppression mechanisms that induces granulomas in L. braziliensis-infected macaques may reveal new opportunities for therapeutic control of this important human disease.     

Prevalence and molecular typing of Giardia spp. in captive mammals at the zoo of Zagreb, Croatia

A total of 131 faecal samples from 57 mammalian species housed at the zoo of Zagreb, Croatia, were tested for the presence of Giardia spp. cysts using epifluorescence microscopy. The overall prevalence (29%) was high, yet all animals were asymptomatic at the time of sampling. Positive samples were characterized by PCR and sequence analysis of both conserved and variable loci, for the identification of Giardia species and G. duodenalis assemblages and genotypes. Assemblages A and C were identified in Artiodactyla, assemblage B in Primates, Rodentia and Hyracoidea, and assemblages A, B, C and D, as well as Giardia microti, in Carnivora. Genotyping at the ITS1-5.8S-ITS2 region, at the triose phosphate isomerase, glutamate dehydrogenase and beta-giardin genes revealed extensive polymorphisms, particularly among assemblage B isolates. A phylogenetic analysis of concatenated sequences showed that isolates from captive mammals housed at the zoo are genetically different from isolates of human and domestic animal origin. This is the first survey in a zoological garden to include a molecular characterization of the parasite, and provides novel sequence data of G. duodenalis from many previously uncharacterized hosts.     

Effects of hunting with hounds on a non-target species living on the edge of a protected area

The impact of hunting on wildlife is a complex phenomenon which varies in space and across time, and yet limited knowledge is available on it. This is especially the case of the indirect effects of hunting on the behaviour of target as well as non-target species. Here we analyze how hunting affected the spatial behaviour of 62 radiocollared roe deer (Capreolus capreolus) in a protected area adjacent to areas where hunting with hounds (target species: wild boar and hares) and stalking with rifles from high seats without dogs (target species: roe deer) were permitted during the hunting season. Our results showed that hunting caused a significant increase in the home range size of monitored deer, as well as a “reserve effect”, whereby roe deer used the protected area as a refuge from hunters. These behavioural responses were significant only at times when hunting with hounds was conducted, even though roe deer was not the target species of this technique. Reactions to the perceived risk of predation varied among age and sex classes, with yearling being more sensitive and using the protected area more than adults. As shown in our study, hunting harassment provoked by drives with hounds significantly affects the behaviour of non-target species. Therefore, the use of long-legged hounds represents a variable that should be carefully evaluated by wildlife managers in their management plans and conservation policies, especially when endangered or vulnerable species are present.     

Chemical characterization of Sacha Inchi (Plukenetia volubilis L.) oil

A chemical characterization of the major components, namely, triacylglycerols (TAGs), polyphenols, and tocopherols in a Sacha inchi oil derived from cold pressing of the seed, is hereby reported. To tackle such a task, high-performance liquid chromatography in combination with photodiode array (PDA), fluorescence (RF), and mass spectrometry (MS) detection was employed. The latter was interfaced with atmospheric pressure chemical ionization and with electrospray ionization for the analysis of TAGs and polyphenols, respectively, whereas RF detection was tested for the determination of tocopherol content. Furthermore, fatty acid methyl esters (FAMEs) were evaluated by gas chromatography-flame ionization detector. A 93% amount of total fatty acids was represented by unsaturated FAMEs with the greatest percentage represented by linoleic (L) and linolenic (Ln) accounting for approximately 50 and 36%, respectively. The main TAGs (>10%) were represented by LLnL, LnLnLn, and LnLLn; the latter was present in the oil sample at the highest percentage (22.2%). Among tocopherols, γ-tocopherol was detected to be the most abundant component (over 50%). The polyphenolic composition was also investigated, and a total of 15 compounds were positively identified, through the complementary analytical information coming from PDA and MS data. To the best of our knowledge, this is the first report providing a thorough chemical characterization of a Plukenetia volubilis L. oil.     

1H NMR metabolite fingerprint and pattern recognition of mullet (Mugil cephalus) bottarga

Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate data analysis (MVA) was used to investigate the molecular components of the aqueous extract of samples of bottarga, that is, salted and dried mullet (Mugil cephalus) roe, manufactured in Sardinia (Italy) from mullets of known and unknown geographical provenience. Principal component analysis (PCA) applied to the processed (1)H NMR spectra indicated that samples tend to cluster according to their geographical origin and also on the basis of storage and manufacturing procedures. The most important metabolites that characterized grouping of samples are the free amino acids methionine (Met), glutamate (Glu), histidine (His), phenylalanine (Phe), tyrosine (Tyr), and isoleucine (Ile); trimethylamine (TMA) and dimethylamine (DMA), both biomarkers of degradation; nucleotides and derivatives; choline (Cho) and phosphorylcholine (P-cho); and lactate (Lac).     

Characterisation of ceftiofur resistance in swine bacterial pathogens

A PCR based method was developed for the identification of ceftiofur resistance genes (bla_CMY-2, bla_TEM-1, and ampC) in swine bacterial pathogens. Using this method, the ceftiofur resistant (n = 76) and susceptible (n = 45) strains of Bordetella bronchiseptica, Salmonella spp., Escherichia coli, and Pasteurella multocida were screened for the presence of these three genes. The resistant genes were detected in 70% (bla_TEM-1), 68% (bla_CMY-2) and 45% (ampC) of the resistant isolates and in 18% (bla_TEM-1), 27% (bla_CMY-2), and 36% (ampC) of the susceptible isolates. Results obtained in the present study showed widespread distribution of these three resistance genes in ceftiofur-resistant swine pathogens. It was also observed that more pathogens are acquiring these resistance genes.