Determination of DNA content of whole fish

ABSTRACT:   The DNA content of whole fish from 31 different North Atlantic fish species was determined. Sample preparation was performed on board the fishery research vessel 'Walter Herwig III' directly after catch and sorting. Up to three homogenates were prepared of each 10 specimens per species by mincing the whole fish. The homogenates were heated to destroy nuclease activity, and then stored at frozen temperature until being analyzed in the laboratory. Measurement of DNA content was carried out using the DNA specific fluorescent dye Hoechst 33258 (Serva Biochemica, Heidelberg, Germany). Matrix effects as light scattering and quenching could be estimated by use of standard addition of calf thymus DNA. DNA contents were referred to dry weight of sample material, and ranged from 570 µg to 3500 µg/g dry weight of homogenate. The coefficient of variation did not exceed 25% of mean for one species; coefficient of variation for all investigated species did not exceed 30% of mean, which was 1020 µg/g dry weight.     

Beziehungen zwischen dem Ernährungszustand der Fichte (Picea abies Karst.), ihrem Kernfäulebefall und der Pilzhemmwirkung ihres Basts1

Relationships between nutrition and heart rot attack of Norway spruce (Picea abies Karst.) and the fungistatic effect of its inner bark . The fresh inner bark of Norway spruce stems and roots inhibits mycelium growth of Fomes annosus on malt agar. Nearly all fungistatic substances of the inner bark can be extracted with chloroform and acetone. The acetone-soluble fraction, which amounts to 13–27% of bark dry matter, was analysed. Up to now 12 phenolic substances have been identified, 5 of them have proved to inhibit Fomes annosus in vitro. Open-air-experiments showed, that desiccation of the root-zone, manganese deficiency and nitrogen hypertrophy decrease the inhibitory effect of the inner bark as well as its content of fungistatic substances. The degree of heart rot attack in older spruce stands was found to be negatively correlated with the N-, Fe- and Mn-concentrations in needles.     

Rapid testing leads to the underestimation of the scrapie prevalence in an affected sheep and goat flock

To obtain a more detailed understanding of the prevalence of classical scrapie infections in a heavily affected German sheep flock (composed of 603 sheep and 6 goats), we analysed 169 sheep and 6 goats that carried the genotypes susceptible to the disease and that were therefore culled following discovery of the index case. The initial tests were performed using the Biorad TeSeE ELISA and reactive results were verified by official confirmatory methods (OIE-immunoblot and/or immunohistochemistry (IHC)) to demonstrate the deposition of scrapie-associated PrP(Sc) in the brain stem (obex). This approach led to the discovery of 40 additional subclinically scrapie-infected sheep. Furthermore, peripheral lymphatic and nervous tissue samples of the 129 sheep and 6 goats with a negative CNS result were examined by IHC in order to identify any preclinical infections which had not already spread to the central nervous system (CNS). Using this approach we found 13 additional sheep with PrP(Sc) depositions in the gut-associated lymph nodes (GALT) as well as in the enteric nervous system. Moreover, in most of these cases PrP(Sc) was also deposited in the spleen and in the retropharyngeal and superficial cervical lymph nodes. Taken together, these results show a 30.3% infection prevalence in this scrapie-affected flock. Almost 7.4% of the infected animals harboured PrP(Sc) exclusively in the peripheral lymphatic and nervous tissue and were therefore missed by the currently used testing strategy.     

Diagnostic validity of palpation in horses with back problems

There is a regularly high proportion of x-ray findings indicating a pathological deterioration of the equine vertebral column which do not correlate with the clinical symptoms. Therefore, palpation remains one of the most important clinical examination methods to determine whether or not a horse is suffering from back pain. The aim of this study was to check the validity of palpation results of horses with an assumed back problem. The palpation results of 167 horses were evaluated in retrospect: If the palpation proceeds under following conditions, positive palpation results agree with an primary back problem in about 100% of cases: 1. Hind limb lameness, being the most likely reason for misleading positive palpation results, has to be ruled out. 2. Only reproducible painful behavior is allowed to be interpreted as positive. Under these conditions a negative palpation result is correct in about 72% of cases. For the examiner it is important to keep in mind that an apparent Kissing Spine-Syndrome can exist without being palpable. By means of clinical examination it is not always possible to distinguish between painful and normal reactions. In doubtful cases diagnosis can be made by a "diagnostic therapy", or better by scintigraphy.     

Simplified method to measure glucocorticoid metabolites in faeces of horses

Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3α,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3α,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.     

Water quality and stocking density as stressors of channel catfish (Ictalurus punctatus Raf.)

The influences of heated recirculated water and well water on three stocking densities of channel catfish (Ictalurus punctatus Raf.) were compared, based on several physiological and hematological parameters. Controls were kept in well water only. The leucocyte count of fish kept in well water generally decreased, and their thrombocyte, lymphocyte and granulocyte counts were lower at higher stocking densities. In addition, a slight hemoconcentration was observed. The fish kept in recirculated water showed a decrease only in thrombocyte count. Plasma cortisol and blood glucose did not change and in all groups were at a level characteristic of resting fish. In contrast, the blood lactate levels were always high. This may have been caused by the anesthetization. The alterations found are regarded as symptoms of the “general adaptation syndrome” induced by the stocking rates. High lymphocyte and granulocyte counts among the fish in reused water are signs of local adaptations to reduced water quality.