In vitro studies of a photo-oxidized bovine articular cartilage

Bovine articular cartilage was photo-oxidized and cultured with native articular bovine cartilage and synovial membrane to study the interaction between these tissues mimicking the physiological situation in the joint. The photo-oxidation was applied as a pretreatment of cartilage for future use in cartilage resurfacing procedures in joints. Properties of the transplant were assessed by testing the production of local mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and neutral metalloproteinase activities under normal conditions and after stimulation with various stimulants representative of inflammatory changes in pathophysiological conditions. Unlike normal cartilage photo-oxidized cartilage did not release significant amounts of NO and PGE2 and showed less gelatinolytic and caseinolytic activity compared to native bovine articular cartilage. Enzyme activity of the combined cultures was at a level intermediate between that of photo-oxidized cartilage and native cartilage cultures alone. In contrast to normal cartilage, living chondrocytes were not visible in photo-oxidized cartilage using live/dead staining. These results indicate, that the photo-oxidized cartilage may have a beneficial effect on adjacent native host cartilage and therefore be a suitable transplant for use in in vivo experiments.     

Evidence for oxidative stress involved in physiological leaf spot formation in winter and spring barley

ABSTRACT A leaf spot disease with unknown etiology has become more pronounced in spring and winter barley in Germany in recent years. The symptoms are similar to net blotch and Ramularia leaf spots, but the causal agents of these diseases are not identified. The symptom expression varied much on cultivars. Cultivars most affected by the disease of both spring and winter barley showed a significantly higher level of superoxide (O(2) ) production and lipid peroxidation (malondialdehyde), but a lower level of antioxidant potential expressed as superoxide dismutase (SOD) activity, catalase activity, and integral water-soluble antioxidant capacity (ACW) than insensitive cultivars. A high positive correlation between O(2) production and leaf spot development between ear emergence and milk ripeness was established in the most sensitive winter barley cv. Anoa (r(2) = 0.9622) and spring barley cv. Barke (r(2) = 0.9434). Leaf H(2)O(2) levels increased with the severity of leaf spots. The histochemical localization of O(2) and H(2)O(2) in the tissues adjacent to leaf spots indicated that these two active oxygen species (AOS) are involved in the formation of leaf spots. Reduction of symptom severity by applying strobilurin and azole fungicides was always associated with elevated SOD activity and ACW content and suppressed O(2) production. However, peroxidase activities were significantly higher in sensitive cultivars and in more severely affected tissues and decreased by applying fungicides. Thus, it is assumed that a possible genetic mechanism based on the imbalanced AOS metabolism contributes to formation of physiological leaf spots.     

Results of the German investigation in the EU Project "Salmonella in Pork (Salinpork)"--2. Investigations in a slaughterhouse

In 1997 bacteriological examinations for the distribution of Salmonella in slaughterhouses were carried out in Germany within the framework of an international study "Salmonella in Pork (Salinpork)". During 6 days, 1,200 swab and water samples from slaughtered pigs and the environment were taken. 4.4% of the samples (n = 53) were Salmonella positive. S. typhimurium was isolated mainly (69.8%; n = 37), and 6 phagetypes were differentiated. In addition, S. derby and S. panama could be demonstrated. The resistance pattern of the different isolated S. typhimurium-phagetypes are presented. The phagetype DT 104 was multiresistant to ampicillin, spectinomycin, streptomycin, sulphonamide and tetracycline. In comparison with the serological prevalence of 7.3% of the fattening pigs in the farms (Part 1), only 1.0% of the samples taken from the surface of the carcass were Salmonella-positive. Swabs taken from the liver were in 2.7% positive and samples from the tongue gave in 5.3% of the cases Salmonella-positive results. In the examination of the environment Salmonella was demonstrated mainly from the water outlets, whereas Salmonella could not be isolated from water of the scalding tank. There was only one case (0.7%) in which Salmonella could be isolated from the hands of the personnel, and also only one swab of the polishing machine was positive (1.1%). But 6.7% samples of the saw were Salmonella-positive. A comparison of repeated, at intervals taken samples showed that the number of Salmonella-positive samples was higher in the last examination round of the particular slaughter days. The reason is suspected in the increasing number of slaughtered pigs and supplying farms, which may increase the probability of bringing in Salmonella.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

RADIOGRAPHIC ANATOMY OF THE CRIBRIFORM PLATE (LAMINA CRIBROSA)

The radiographic appearance of the cribriform plate was investigated in 16 canine cadaver heads. The cribriform plate appeared as a "V"-shaped multilinear bone-opaque stripe in the caudal nasal region in projections perpendicular to the hard palate in 6 dogs with a skull index between 50.00 and 54.00. In 9 dogs with a skull index between 55.40 and 74.40, the cribriform plate had a more "C"-shaped and sharp appearance. In vertically oblique projections with an obliquity greater than 20 degrees, the cribriform plate lost its sharp outline and finally (40 degrees) disappeared. In lateral projections the cribriform plate appeared as a "C"-shaped interrupted bone-opaque stripe in all 16 dogs. In more brachycephalic dogs frontal bone structures superimposed on the cribriform plate on ventrodorsal and dorsoventral views and accentuated the radiographic appearance of the plate. Vertically oblique views separated both structures to produce two lines.     

The "Friedrich-Loeffler-Institut": past, present and future of research in infectious diseases of animals

The Friedrich-Loeffler-Institut, founded in 1910 by Friedrich Loeffler, the discoverer of the first animal virus, foot-and-mouth disease virus, is the oldest virological research facility in the world. Beyond viruses, its area of competence has significantly expanded since its foundation and now also covers bacterial, parasitic and prion diseases of livestock, poultry and aquatic animals. Presently located at four sites within Germany (Insel Riems, Jena,Tübingen,Wusterhausen) the tasks of the institute as delineated in the Animal Disease Act encompass research on infectious animal diseases including zoonoses, import/export examinations, epidemiological studies in case of outbreaks of notifiable animal diseases, acting as reference laboratory for notifiable animal diseases and nationwide quality management of diagnosis of notifiable animal diseases. It is obliged to publish and maintain up-to-date diagnostic regimes for notifiable animal diseases, and it publishes a yearly report on animal health in Germany. With the increasing importance of infectious diseases of animals, in particular those potentially harmful to man (zoonoses), the Friedrich-Loeffler-Institut will be moving into new facilities including laboratories and animal facilities up to the highest biosafety level at its main site Insel Riems on the occasion of its 100th anniversary.     

Comparison of two real-time RT-PCR assays for differentiation of C-strain vaccinated from classical swine fever infected pigs and wild boars

Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.     

Urban land use types contribute to grassland conservation: The example of Berlin

Urbanisation is an important driver of biodiversity loss, also contributing to habitat loss and fragmentation of grasslands at the urban-rural interface. While urban green spaces are known to include many grassland habitats, it is uncertain to what extent urban land use types harbour grasslands of special conservation interest and whether patch characteristics and connectivity of these differ from grasslands on agricultural land. By relating the city-wide biotope mapping to the land use mapping of Berlin, Germany, we assessed (1) to which specific urban land use types the major grassland biotope types belong, (2) differences in patch characteristics and connectivity, and (3) the conservation value of grassland patches at a typological level by means of their legal protection status. Grasslands cover 5% of Berlin's surface, and 43% of that area is assigned to legally protected grassland types. The majority of legally protected grassland (71%) lies on urban land opposed to 29% on agricultural land. Airports and historic parks, which only cover 2% of land in Berlin, contain one-third of all protected dry grasslands. Wet grassland is more confined to agricultural land. In airports and agricultural areas, grassland patches are larger but of a more complex shape than those in historic parks. In airports, grassland patches show greater connectivity as they are situated in grassland-dominated surroundings. Grassland in historic parks appears to be more vulnerable due to smaller patch sizes and higher fragmentation. The example of Berlin demonstrates that the urban green infrastructure can clearly contribute to grassland conservation and may thus partially compensate for the decline of traditional grasslands in cultural landscapes. It will be important to involve residents and landowners in urban grassland conservation and management because most grassland of special conservation interest (57%) was found outside of conservation areas.     

Influence of ketamine or xylazine supplementation on isoflurane anaesthetized horses‐ a controlled clinical trial

ObjectivesTo determine the influence of ketamine or xylazine constant rate infusions on isoflurane requirements, cardiovascular parameters and quality of anaesthesia in horses undergoing elective surgery.Study designProspective, matched paired clinical trial.AnimalsFifty four adult Warmblood horses.MethodsAfter premedication with acepromazine, xylazine and butorphanol, anaesthesia was induced with ketamine-midazolam and maintained with isoflurane alone (I), isoflurane with either 1 mg kg⁻¹ hour⁻¹ ketamine (IK) or same dose of xylazine (IX). End tidal concentration of isoflurane (Fe’Iso) was adjusted by the same anaesthetist in all horses according to a scoring system. Dobutamine was infused to maintain mean arterial pressure (MAP) =70 mmHg. Arterial blood gases, heart rate (HR), respiratory rate, MAP and cardiac output (lithium dilution) were measured. Groups I and IK received xylazine before recovery. Recovery quality was scored.ResultsMean ± SD averaged Fe’Iso (volume%) was significantly lower in IX (0.95 ± 0.07) and IK (0.97 ± 0.08) than in I (1.16 ± 0.13). In group IX, HR was significantly lower and averaged MAP (90 ± 13 mmHg) significantly higher than in groups I (71 ± 7 mmHg) and IK (76 ± 7 mm Hg). Differences in other cardiopulmonary variables did not reach statistical significance. All horses recovered well with best score in group IX.ConclusionsBoth CRIs of xylazine and of ketamine resulted in pronounced reduction of isoflurane requirements and blood pressure support based on routinely monitored parameters. Cardiac output appeared well maintained in all three protocols, but lithium dilution induced errors mean the results are untrustworthy. The work requires repetition with another mode of measurement of cardiac output.Clinical relevanceAll three protocols provided good clinical anaesthesia with clinically acceptable cardiovascular effects.     

Dynamic visualization of thrombopoiesis within bone marrow

Platelets are generated from megakaryocytes (MKs) in mammalian bone marrow (BM) by mechanisms that remain poorly understood. Here we describe the use of multiphoton intravital microscopy in intact BM to visualize platelet generation in mice. MKs were observed as sessile cells that extended dynamic proplatelet-like protrusions into microvessels. These intravascular extensions appeared to be sheared from their transendothelial stems by flowing blood, resulting in the appearance of proplatelets in peripheral blood. In vitro, proplatelet production from differentiating MKs was enhanced by fluid shear. These results confirm the concept of proplatelet formation in vivo and are consistent with the possibility that blood flow-induced hydrodynamic shear stress is a biophysical determinant of thrombopoiesis.