Extraction of soil bacteria from a Ferralsol

Extraction of intact bacteria from soil by dispersion and density gradient centrifugation may facilitate analyses of soil bacterial communities which are otherwise hampered by soil particles. Reasonable cell yield and representative fractions with sufficient purity can be extracted from most soils, but highly weathered, clayey and acidic tropical soils like Ferralsols represent a challenge to the method due to low cell yields. At an early stage of our studies with Ferralsols we also found substantial contamination of the extracted bacterial fractions by soil material, measured as total Al and Fe. We have adapted the method to a Ferralsol (pH 4.9, 64% clay, 21% silt, 15% sand) by factorial combinations of pH modification and salt (NaCl) concentration during soil dispersion, and evaluated the yield (microscopic cell counts), purity (optical density and Al + Fe content), and compared the composition of the extracted bacteria (phospholipid fatty acid analysis and 16S rDNA-denaturing gradient gel electrophoresis) versus that of the intact soil community. The cell yield was increased with a factor 2–3 by increasing the pH to 7.5, while it was decreased to a similar extent by adding NaCl (8 g l^?1). However, NaCl removed more than 99% of the Al + Fe contamination of the bacterial extracts, and the combination of modified pH and NaCl addition secured reasonable cell yield (4.6% of total number) and low contamination. The observed effects of pH and NaCl are probably due to changes in variable charge (by pH) and ion distribution (NaCl) around interacting particles (soil and bacteria), thus affecting their flocculation. Phospholipid fatty acid and denaturing gradient gel electrophoresis analysis indicate that the bias of the bacterial extracts compared to direct soil extracts increases with the addition of NaCl as well as by pH manipulation. Nevertheless, representativeness was acceptable as indicated by a Bray–Curtis similarity index (bacterial extracts versus soil) of 70% and 87% for phospholipid fatty acid profiles and denaturing gradient gel electrophoresis, respectively. Overall, the results reveal a trade-off among yield, purity and representativeness. Thus, depending on application and analyses, future users can choose the right treatment according to their specific purpose.     

Effects of tebuconazole on morphology,structure, cell wall components and trichothecene production of Fusarium culmorum in vitro

The effects of tebuconazole, a systemic fungicide, on the morphology, structure, cell wall components and toxin production of Fusarium culmorum were investigated in vitro. Treatment was by application of four filter paper strips (0.75 cm × 5.0 cm) soaked in 20 µg ml ^?1 fungicide placed around a point inoculum in Petri dishes. Mycelial growth was strongly inhibited by fungicide treatment. Scanning electron microscopic observations showed that the fungicide caused irregular swelling and excessive branching of hyphae. The morphological changes induced by the fungicide at the ultrastructural level included considerable thickening of the hyphal cell walls, excessive septation, the formation of the incomplete septa, extensive vacuolisation, accumulation of lipid bodies and progressing necrosis or degeneration of the hyphal cytoplasm. Non‐membrane inclusion bodies were often detected in the hyphal cytoplasm. Furthermore, the formation of new hyphae (daughter hyphae) inside collapsed hyphal cells was common following treatment. The daughter hyphae also displayed severe alterations such as irregular thickening of the cell walls and necrosis of the cytoplasm. Using cytochemical techniques, the labelling densities of chitin and β‐1,3‐glucan in the cell walls of the fungicide‐treated hyphae were more pronounced than in those of the control hyphae. Moreover, immunogold labelling with antiserum against deoxynivalenol (DON) revealed that Fusarium toxin DON was localized in the cell walls, cytoplasm, mitochondria and vacuoles of the hyphae from the control and the fungicide treatment, but the labelling density in the fungicide‐treated hyphae decreased dramatically compared with the control hyphae, indicating that tebuconazole reduced Fusarium toxin production of the fungus. © 2001 Society of Chemical Industry     

Four preanesthetic oral sedation protocols for rhesus macaques (Macaca mulatta).

The efficacies and ease of administration of four oral preanesthetic sedation protocols were compared in 18 adult, male rhesus macaques (Macaca mulatta) to achieve heavy sedation and alleviate anxiety, agitation, and potential trauma associated with remote anesthesia induction. The macaques, with average age and weight of 10 yr and 12.5 kg, respectively, were randomly assigned to one of four groups. Group 1 was given 10 mg/kg tiletaminezolazepam and 0.05 mg/kg medetomidine p.o., group 2 was given 1 mg/kg midazolam and 20 mg/kg ketamine p.o., group 3 was given 20 mg/kg ketamine and 0.05 mg/kg medetomidine p.o., and group 4 was given 3 mg/kg midazolam p.o. All protocols produced effects ranging from mild sedation to no response to noxious stimuli, depending on the success of administration. The mean interval to peak effect was 27-43 min in all groups. Ketamine and medetomidine provided significantly better sedation than midazolam alone; there were no other statistically significant differences among the four protocols. Oral tiletamine-zolazepam and medetomidine provided smooth, mild to moderate sedation with few side effects. The midazolam and ketamine combination resulted in severe ataxia. Orally administered ketamine and medetomidine provided smooth, easily reversible, heavy sedation leading to no response to noxious stimuli. Midazolam alone provided only mild sedation. No statistically significant differences in palatability of the four protocols were identified. Orally administered ketamine and medetomidine (group 3) provided the most consistently heavy sedation. A compounding pharmacy may be able to increase the palatability and level of acceptance of these combinations. Alternatively, oral midazolam syrup is well accepted by some animals and provides a mild sedative and calming effect, which may decrease stress associated with the induction of anesthesia via darting, pole syringes, etc.     

The spatial and temporal disappearance of different oral rabies vaccine baits

Bait disappearance can give valuable information for the assessment of oral vaccination campaigns of foxes against rabies. In this study, the spatial and temporal disappearance of three different vaccine baits under almost identical conditions was investigated. In the study area, 350 baits were placed at previously marked positions during two different periods; late autumn and early spring. The distribution of baits was in accordance with the method as recommended by the European Union; a density of 20 baits per km2 along flight lines 500m apart. Bait disappearance was checked 1, 3, 5 and 7 days after distribution. At least 80% of the baits had disappeared within one week after distribution. No difference in bait disappearance was observed between the two selected periods. However, a significant higher bait disappearance was observed in forested areas when compared to open agricultural areas. Furthermore, the differences in bait disappearance between the three type of baits tested were relatively small and not significant.     

Dioxin concentration in milk and tissues of cows and sheep related to feed and soil contamination

If cows or sheep are grazing on areas known for a higher dioxin level of the soil, respectively, dry cows are fed with feedstuffs produced on such areas, the dioxin concentration in food, in this case in cows' milk and livers of sheep could reach or exceed maximum levels. Furthermore large amounts of polychlorinated dibenzo-p-dioxins/polychlorinated dibenzo-p-furans (PCDD/F) can be excreted with the faeces. But there are chances for farmers to prevent or at least to minimize the dioxin load of feed and with it of food (including the harvesting technique and the feeding strategy).     

Simulation of local anaesthetic nerve block of the infraorbital nerve within the pterygopalatine fossa: anatomical landmarks defined by computed tomography

To provide anaesthesia of the maxillary cheek teeth, a local block of the infraorbital nerve in the pterygopalatine fossa has been suggested. The aim of this study was to re-examine the anatomy of the pterygopalatine fossa, giving special attention to relevant arteries, veins and nerves; simulate the infiltration of an anaesthetic by injecting a contrast medium; improve the injection technique to avoid puncturing of relevant anatomical structures. Five heads and two living horses were investigated using contrast medium injections and computed tomography (CT). Needles were inserted using two insertion techniques: "Palatine Bone Insertion" (PBI) and "Extraperiorbital Fat Body Insertion" (EFBI). Both techniques are suitable for achieving a consistent distribution of contrast medium around the infraorbital nerve. The periorbita prevents the contrast medium from penetrating into the intraperiorbital compartment. The EFBI-technique is most appropriate for providing a sufficient infraorbital nerve block with a minimised risk of complications.     

Chicken lung lectin is a functional C-type lectin and inhibits haemagglutination by influenza A virus

Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms. We previously reported the finding of a new putative chicken lectin, which was predominantly localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to investigate the biochemical and biophysical properties of cLL, the recombinant protein was expressed, affinity purified and characterized. Recombinant cLL was expressed as four differently sized peptides, which is most likely due to post-translational modification. Crosslinking of the protein led to the formation of two high-molecular weight products, indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown to have lectin activity, preferentially binding to alpha-mannose in a calcium-dependent manner. Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of influenza A virus, subtype H3N2 and H1N1. These result show that cLL is a true C-type lectin with a very distinct sugar specificity, and that this chicken lectin could play an important role in innate immunity.     

Comparison of 13 single-round and nested PCR assays targeting IS900, ISMav2, f57 and locus 255 for detection of Mycobacterium avium subsp. paratuberculosis

For molecular biological detection of Mycobacterium avium subsp. paratuberculosis (MAP), PCR methods with primers targeting different regions specific for MAP are used worldwide. However, some uncertainties exist concerning the specificity of certain target regions and the sensitivity. To identify the methods which are best suited for diagnostics, 8 single-round and 5 nested PCR systems including 12 different primer pairs based on IS900 (9 x), ISMav2 (1x), f57 (1x), and locus 255 (1x) sequences were compared regarding their analytical sensitivity and specificity under similar PCR conditions. Reference strains and field isolates of 17 Mycobacterium species and subspecies, 16 different non-mycobacterial bovine pathogens and commensals were included in this study. Single-round PCR resulted in a detection limit of 100 fg to 1 pg, and nested PCR in 10 fg or below. Depending on the specific primer sequences targeting IS900, false positive results occurred with one of the five single-round and two of the four nested PCR systems. This also applied to the single-round PCR based on ISMav2 and the nested PCR based on f57. A high number of non-specific products were primarily detected for the single-round PCR assay based on ISMav2, but also for a single-round PCR targeting the IS900 and the locus 255. In conclusion, stringent selection of IS900-specific primers ensures that IS900 remains a favourite target sequence for amplification of MAP specific loci. The studied PCR systems based on f57, and locus 255 can also be recommended. Revision of ISMav2 primers is necessary. Single-round PCR systems are very reliable. Nested PCR assays were occasionally disturbed by contaminations, thus bearing a risk for routine diagnostics.     

Sperm traits differ between winged and wingless males of the ant Cardiocondyla obscurior

Size and shape of sperm cells vary tremendously throughout the animal kingdom. The adaptive significance of this variation is not fully understood. In addition to sperm–female interactions and the environmental conditions, the risk of sperm competition might affect number, morphology and other “quality” traits of sperm. In the male‐diphenic ant Cardiocondyla obscurior, winged sneaker males have limited sperm number, because their testes degenerate shortly after adult emergence, as is typical for males of social Hymenoptera. In contrast, wingless fighter males continuously replenish their sperm supply due to their exceptional lifelong spermatogenesis. While winged males usually have to compete with several other winged males for virgin queens, wingless males are able to monopolize queens by killing all other rivals. Hence, this presents a unique system to investigate how alternative reproductive tactics and associated physiology affect sperm morphology and viability. We found that sperm‐limited males invest into sperm number instead of sperm size. Variance in sperm length is smaller in winged males, probably reflecting that they have to compete with several other males. Finally, sperm viability is equally high in both male phenotypes.