Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species

A recombinant baculovirus (RBV) encoding the nucleoprotein (NP) of avian influenza virus (AIV) was generated and the appropriate protein was expressed in Sf9 cells. Purified recombinant NP and the NP-specific monoclonal antibody HB65 were used to establish a competitive ELISA (cELISA) system for the detection of NP-specific antibodies in sera of ducks, geese and wild birds. Tests to evaluate this method were carried out using sera of ducks experimentally infected with AIV, pre-immune duck and chicken sera, and poultry field sera, which tested negative in the haemagglutination inhibition (HI) assay, and field sera of several poultry species experimentally infected with other viruses. The evaluation of the test demonstrated a high sensitivity and specificity of this method. Tests carried out using field sera of duck and goose flocks revealed widely corresponding results obtained by HI assay and cELISA indicating that this test is applicable for flock diagnosis. Differing results were obtained for individual samples. It can be assumed that for the most part this was because of a better recognition of the conserved NP antigen by serum antibodies, although some results remained unclear.     

Experimental evaluation of ophthalmic devices and solutions using rabbit models

OBJECTIVE: To analyze and compare the geometry of the anterior segment of rabbit and human eyes, with relevance for the evaluation of intraocular lenses, and to review rabbit models used in our laboratory for the evaluation of different ophthalmic devices and solutions. PROCEDURES: Fifteen rabbit and 15 human eyes (10 phakic and 5 pseudophakic/group) obtained postmortem were used. Anterior-posterior length, equatorial diameter, and white-to-white (corneal diameter) were measured with calipers. The eyes were then analyzed with a very high-frequency ultrasound (Artemis, Ultralink) for measurements of the anterior chamber depth, and anterior chamber and ciliary sulcus diameters. The capsular bag diameter was measured with calipers from a posterior view, and the diameter and thickness of the crystalline lenses were measured after their excision from the phakic eyes. RESULTS: Although the size of the rabbit eye is overall smaller than the size of the human eye, the dimensions of the anterior segment of rabbit eyes are generally larger. The differences between rabbit and human eyes were statistically significant (Wilcoxon rank sum test) in terms of anterior-posterior length, equatorial diameter, white-to-white measurements (P < 0.0001), anterior chamber diameter (P = 0.0004), ciliary sulcus diameter (P = 0.0012), and crystalline lens diameter and thickness (P = 0.0003). CONCLUSIONS: Experimental evaluation of design features of new phakic intraocular lenses in rabbit eyes may be inconclusive without adaptation of their size/design, contrary to the evaluation of new pseudophakic lenses by implantation in the capsular bag. The rabbit is a very valuable model for the experimental evaluation of different ophthalmic devices and solutions.     

Effect of age on reference intervals of serum biochemical values in kittens

OBJECTIVE: To determine the effect of age on reference intervals of serum biochemical values in kittens. DESIGN: Prospective clinical trial. ANIMALS: 55 kittens from 12 specific-pathogen-free queens. PROCEDURE: Kittens were allocated at birth into colostrum-fed (n = 27) and colostrum-deprived (28) groups. Blood was collected at birth and on days 1, 2, 4, 7, 14, 28, and 56. Serum samples were analyzed for activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, gamma-glutamyltransferase, amylase, and lipase and for concentrations of albumin, total protein, bilirubin, urea nitrogen, creatinine, cholesterol, glucose, calcium, phosphorus, and triglycerides by use of an automated analyzer. Total serum solids concentrations were determined by use of refractometry. Serum IgG concentrations were quantified by use of radial immunodiffusion. RESULTS: For several analytes, reference intervals changed rapidly, most notably during the first few days of life. Reference intervals for alkaline phosphatase, creatine kinase, lactate dehydrogenase, and triglycerides were higher from birth to 8 weeks than adult reference intervals. Aspartate aminotransferase, bilirubin, urea nitrogen, and creatinine were higher than in adults at birth but were similar to or lower than adult reference intervals by 8 weeks. Compared with adult reference intervals, reference intervals for calcium and phosphorus concentrations were higher and for albumin and total protein concentrations were lower throughout the study period. CONCLUSIONS AND CLINICAL RELEVANCE: Important differences exist between reference intervals for serum biochemical values of neonatal and adult cats. Age-appropriate reference intervals should be used for accurate assessment of serum biochemical test results in cats.     

Evaluation of surrogate markers for passive transfer of immunity in kittens

OBJECTIVE: To identify surrogate markers of passive transfer of immunity in kittens. DESIGN: Prospective clinical trial. ANIMALS: 55 kittens from 12 specific-pathogen-free queens. PROCEDURE: Kittens were allocated at birth into colostrum-fed (n = 27) and colostrum-deprived (28) groups. Blood was collected at birth and on days 1, 2, 4, 7, 14, 28, and 56. Serum samples were analyzed for activities of alkaline phosphatase (ALP), alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, gamma-glutamyltransferase, amylase, and lipase and for concentrations of albumin, total protein, bilirubin, urea nitrogen, creatinine, cholesterol, glucose, calcium, phosphorus, and triglycerides by use of an automated analyzer. Total serum solids concentrations were estimated by use of refractometry. Serum IgG concentrations were quantified by use of radial immunodiffusion. RESULTS: All kittens were agammaglobulinemic at birth. Colostrum-fed kittens had significantly higher IgG concentrations than did colostrum-deprived kittens from 1 though 28 days of age. Transient significant differences in serum biochemical variables between the colostrum-deprived and colostrum-fed groups were substantially resolved by day 4. Passive transfer of immunity could be reliably determined at 1 day of age and to a lesser extent at 2 days of age only by measurement of serum activity of ALP. CONCLUSIONS AND CLINICAL RELEVANCE: Adequacy of passive transfer in kittens initially correlated with serum activity of ALP, but quantification of serum IgG concentration was necessary after 2 days of age.     

Nonsuppurative encephalitis in a dog

A 4-year-old male German Hunting Terrier presented with tremor, dyspnea, trismus, spasms of the musculature of the larynx and pharynx, and hypothermia and subsequently died despite intensive clinical care. Prior clinical signs included vomitus and diarrhea. Microscopic examination of the brain revealed a multifocal nonsuppurative brain stem encephalitis; a few intralesional neurons contained intranuclear inclusions. By immunohistochemistry, Aujeszky disease virus (Suid herpesvirus 1) antigen was detected in neurons in the brain and in ganglion cells of the trigeminal ganglia. Viral culture of brain tissue confirmed the presence of Aujeszky disease virus. Histopathologic findings in the brain with the identification of Aujeszky disease virus by immunohistochemistry and polymerase chain reaction are consistent with Aujeszky disease virus-induced encephalitis. Sequencing revealed a 100% homology of the isolated Aujeszky disease virus with Aujeszky disease virus isolates of wild boar from Eastern Germany.     

Satellite-based stand-wise forest cover type mapping using a spatially adaptive classification approach

Due to high variation in forest communities, forest structure and the fragmentation of the forested area in Central Europe, satellite-based forest inventory methods have to meet particularly high-quality requirements. This study presents an innovative method to combine official forest inventory information at stand level with multidate satellite imagery using a spatially adaptive classification approach for producing wall-to-wall forest cover maps of important tree species and management classes across multiple ownership regions in a heterogeneous low mountain range in Germany. The classification approach was applied to a 5,200-km² area (about 2,080?km² of forest land, mostly mixed forests) located in the Eifel mountain range in Central Europe. In comparison with conventional classifiers, our results demonstrate a significant increase in classification accuracy in the order of 12%. The method was tested with ASTER images but holds the potential to be used for regular state forest inventories based on standard and novel earth observation data supplied for instance from the SPOT-5 and RapidEye sensors.     

Repeated drying–rewetting cycles and their effects on the emission of CO2, N2O,NO, and CH4 in a forest soil

Prolonged summer droughts due to climate change are expected for this century, but little is known about the effects of drying and wetting on biogenic trace‐gas fluxes of forest soils. Here, the response of CO₂, N₂O, NO, and CH₄ fluxes from temperate forest soils towards drying–wetting events has been investigated, using undisturbed soil columns from a Norway spruce forest in the “Fichtelgebirge”, Germany. Two different types of soil columns have been used for this study to quantify the contribution of organic and mineral horizons to the total fluxes: (1) organic horizons (O) and (2) organic and mineral soil horizons (O+M). Three drying–wetting treatments with different rewetting intensities (8, 20, and 50 mm of irrigation d^–1) have been compared to a constantly moist control to estimate the influence of rainfall intensity under identical drying conditions and constant temperature (+15°C). Drought significantly reduced CO₂, N₂O, and NO fluxes in most cycles. Following rewetting, CO₂ fluxes quickly recovered back to control level in the O columns but remained significantly reduced in the O+M columns with total CO₂ fluxes from the drying–wetting treatment ranging approx. 80% of control fluxes. Fluxes of N₂O and NO remained significantly reduced in both O and O+M columns even after rewetting, with cumulative fluxes from drying–wetting treatments ranging between 20% and 90% of the control fluxes, depending on gas and cycle. Fluxes of CH₄ were small in all treatments and seem to play no significant role in this soil. No evidence for the release of additional gas fluxes due to drying–wetting was found. The intensity of rewetting had no significant effect on the CO₂, N₂O, NO, and CH₄ fluxes, suggesting that the length of the drought period is more important for the emission of these gases. We can therefore not confirm earlier findings that fluxes of CO₂, N₂O, and NO during wetting of dry soil exceed the fluxes of constantly moist soil.     

Tissue distribution of dexamethasone in canine ocular compartments following topical application of dexamethasone-21-isonicotinate and oxytetracycline HCl

Objective  To study the dexamethasone (DXM) concentration at different time points in various compartments of the canine eye following topical application of DXM-21-isonicotinate and oxytetracycline hydrochloride
Animals studied  Thirty dogs to be euthanized for reasons not related to this study were selected and their ocular health status evaluated. Selected animals were treated with DXM-oxytetracycline ointment and euthanized after 6, 11 or 16 h.
Procedure  The concentration of DXM was determined in the following compartments of the eye: third eyelid, cornea, aqueous humor, iris, lens, vitreous body and choroid/retina. The DXM concentration in the eye was measured by radioimmunoassay. The applied amount of DXM was 0.04 mg in 0.2 mL ointment. Dogs were treated once with Corti Biciron^® eye ointment (DXM-21-isonicotinate and oxytetracycline hydrochloride, S & K Pharma, Perl, Germany) and were euthanized 6, 11 and 16 h after treatment.
Results  At 6 h following topical application the mean DXM concentration was highest in the anterior structures of the eye (third eyelid: 18 ng/g, cornea: 36 ng/g). The concentration in the posterior structures was below detection level. A decreased DXM concentration in the anterior structures was measured 11 and 16 h after treatment.
Conclusion  It could be demonstrated that therapeutically relevant concentrations of DXM after a single topical administration are only achieved in anterior structures of the eye. A dosing interval of 6–11 h is recommended to achieve therapeutic drug concentration in those structures. The posterior structures of the eye are not reached by topical administration.