Tumor Necrosis Factor-α, Interleukins-12(p40), 6, and 10 levels in cerebrospinal fluid and outcome prediction in Ossimi sheep with encephalitic listeriosis

Encephalitic listeriosis in sheep is a life-threatening disease. However, little is known about the cytokine response and their predictive value in this disease. The aim of present study was to assess the prognostic significance of Tumor Necrosis Factor-α (TNF-α), Interleukin-12(p40) (IL-12 p40), Interleukin-6 (IL-6), and Interleukin 10 (IL-10) levels in cerebrospinal fluid (CSF) in sheep with encephalitic listeriosis. Fifty-nine ewes in 14 flocks were diagnosed clinically as having listeriosis. CSF was collected and subjected to bacteriological examination and estimation of selected cytokines. Twenty-eight ewes were confirmed to be infected with Listeria monocytogenes. Based on antimicrobial sensitivity test, sheep were treated and the outcome was recorded as survivors (n = 10) and non-survivors (n = 18). Cutoff points for CSF cytokines were determined by Receiver operating characteristic analysis (ROC). Association between levels of CSF cytokines and outcome of listeriosis was assessed by logistic regression. TNF-α, IL-6 and IL-12(p40) levels as well as TNF-α/IL-10 ratio were significantly higher in non-survivors than survivors (p = 0.002, 0.0021, 0.0033, and 0.001, respectively). However, IL-10 level was significantly lower in non-survivors than survivors (p = 0.0058). ROC analysis revealed that IL-6 and TNF-α/IL-10 ratio had the highest AUC values (0.98, 0.984, respectively). Final multivariate logistic regression model showed that TNF-α/IL-10 ratio was the only variable that has predictive value for mortality in diseased sheep (p: 0.001; OR: 7.2; 95% CI: 5.7–9.8). TNF-α showed a positive correlation with IL-12β (r = 0.917) and IL-6 (r = 0.965). IL-12 (p40) showed also a positive correlation with IL-6 (r = 0.906). However, IL-10 showed a negative correlation with TNF-α (r = −0.915), IL-12(p40) (r = −0.790), and IL-6 (r = −0.902). In conclusion, TNF-α/IL-10 ratio may provide predictive information about outcome of encephalitic listeriosis in sheep.     

Cuticular sensilla on newly hatched larvae of Gasterophilus intestinalis and Oestrus ovis

Abstract. Cuticular sensilla on newly hatched larvae of Gasterophilus intestinalis De Geer (Diptera: Gasterophilidae) and Oestrus ovis (L.) were studied by scanning electron microscopy. Two types of trichoid sensilla, two types of coeloconic sensilla and a pit sensillum were present on the thoracic and abdominal segments of G. intestinalis larvae. Sensilla on larvae of O. ovis were similar although only one type of trichoid sensillum was present. Total number of sensilla were higher for O. ovis than for G. intestinalis (248 v . 214). Variation in numbers of sensilla is consistent with the concept that increasing numbers of sensilla are associated with increasingly complex searching behaviour required to locate suitable habitats for development.     

Properties of Oligomeric Forms of Phosphofructokinase from Chlorella kessleri Grown under Different Light Conditions

Fast protein liquid chromatography on Superose 6 of crude extracts from Chlorella kessleri, Fott et Novákóva, grown autotrophically in blue or in red light yields three different oligomeric forms of phosphofructokinase (PFK, EC 2.7.1.11). Their substrate affinities and responses to homotropic and heterotropic effectors are different. In vitro, the degree of oligomerization of the enzyme can be influenced by specific intermediates or cofactors. Its substrate, MgATP (10 mM/5 mM), and the negative effector, phosphoenolpyruvate (5 mM), both lead to some dissociation, while the second substrate, fructose-6-phosphate (5 mM), and the positive effector, inorganic phosphate (50 mM), have no effect. It is discussed whether formation or dissociation of oligomeric PFK forms in vivo result from alterations in the levels or in the intracellular distribution of effector molecules and whether such processes are involved in the different regulation of cell metabolism in blue or in red light.     

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

Stock grazing of semi-natural habitats on National Trust land

Stock grazing is essential in many habitats owned by the National Trust if their nature conservation interests are to be maintained or enhanced. In this review, the benefits of grazing cattle, sheep and other livestock in different habitat types are described, together with constraints imposed by tenancy agreements and difficulties in obtaining the most appropriate animal. The majority of upland heaths, calcareous grasslands and lowland/coastal heaths on Trust land are being grazed satisfactorily. However, the nature conservation interest of parklands is currently being eroded in part because of inappropriate grazing regimes. It is argued that the best use of stock in the future is in extensive grazing regimes.     

Evidence for the expression of two distinct MHC class II DRβ like molecules in the sheep

This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DRβ chains are expressed in the sheep. Two anti-β chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DRβ chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 β chain. Amino-terminal sequence analysis of the α chain associated with VPM37β chain shows that this α chain is homologous to the human DRα chain strongly indicating that the β chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of posttranslational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DRP chains in the sheep.     

Divergent growth of Norway spruce on Babia Góra Mountain in the western Carpathians

Growth divergence – i.e. the expression of divergent growth trends of neighboring trees – has certain implications for dendrochronological research, for instance in the context of climate reconstructions but also in terms of estimating net ecosystem productivity. Thus, understanding the underlying mechanisms is essential to extend our fundamental dendroecological knowledge. In this context, the Picea genus plays an important role since several of its species were reported to exhibit growth divergence. Here, we investigate a well sampled Norway spruce (Picea abies (L.) Karst) data set for growth divergence comprising ring-width and Blue Intensity measurements from seven sites on Babia Góra Mountain, at the border between Poland and Slovakia. By means of Principal Component Gradient Analysis, inter-series correlations, and climate growth relationships, we are able to show that I) Norway spruce on Babia Góra expressed growth divergence since the 1970s, II) the definition of groups increased the strength of population signals and the stability of climate-growth relationships, and III) Blue Intensity appeared as a more robust proxy for environmental conditions. We discuss soil heterogeneity, genetics, and air pollution as possible underlying mechanisms, thereby indicating further research avenues to obtain a better understanding of growth divergence.     

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.     

Inhibitors for the bacterial ectonucleotidase Lp1NTPDase from Legionella pneumophila

Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires’ disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC₅₀ values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC₅₀-value of 4.24 μM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections.