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Biomedical subjects

K E Seidel

Publications and source records attributed to K E Seidel.

12 recordsLinked to original sources

Monitoring a mouse colony for Helicobacter bilis using a Helicobacter-genus-specific nested PCR.

Although Helicobacter infections of laboratory mice are usually subclinical, they may interfere with in vivo experiments and thus may lead to misinterpretation of data. As such, it is important to provide a means to unequivocally identify infections with murine Helicobacter spp. In the present study, a nested polymerase chain reaction (PCR) was established and shown to be 10 to 100 times more sensitive than the single-step PCR commonly used for routine diagnosis of Helicobacter spp. Experimental infection of Helicobacter-free mice demonstrated that faeces, caecum, colon and rectum but not liver are equally suitable for the detection of H. bilis. However, use of faecal pellets is advantageous since detection of H. bilis is possible one week after infection and analysis of faeces instead of tissues avoids euthanasia of animals. Furthermore, it generates representative data for all animals housed in the same cage and analysis can be repeatedly performed. Use of samples from breeding pairs but not offspring provides representative information about the Helicobacter status of a mouse colony. Both C3H/HeJ and C57BL/6 mice appear to be susceptible to H. bilis and persistent infection was observed during the 20-week experimental period. Analysis of pooled faecal pellets by nested PCR seems to be the most sensitive approach for H. bilis monitoring of the given breeding colony.

Animals↗

Antibodies against Helicobacter felis in sera of cats and dogs.

Serum samples from 61 dogs and 49 cats were screened for circulating antibodies against Helicobacter felis by an enzyme-linked immunosorbent assay (ELISA) using sonicated bacteria as an antigen. To improve the specificity of the ELISA, sera were absorbed with Campylobacter jejuni subsp. jejuni H. pylori as well as H. felis. Sera from 26 dogs (43%) and 19 cats (39%) revealed clear positive absorbance readings determined as an optical density (OD) that was statistically significant above the OD mean value [P < 0.025 (one-tailed); log10]. The absorbance pattern of ELISA-positive sera corresponded to results obtained with bovine and human reference sera. Furthermore, a correlation between the immune response and results from histopathological examination of gastric specimens from 22 dogs was demonstrated. It could be shown that antibodies against H. felis in sera of cats and dogs can easily be detected using an ELISA. The diagnostic value of this test must be evaluated in further investigations.

Animals↗

[Experience with the use of a blood culture system for demonstration of clinically relevant bacteria in veterinary medicine diagnosis].

268 diagnostic samples from dogs, cats, horses and cattle were examined in a commercially available blood culture system. Samples of blood, liquor, ascites, thorax punctate, synovia and urine were examined with a blood culture system (Oxoid) over a period of two years in cooperation with the veterinary clinical institutes of internal medicine and surgery, Ludwig-Maximilians-University, Munich and different veterinarians. It was shown that this blood culture system, which has been initially developed for the requirements of human bacteriology, can be used for isolation of clinical important microorganisms in veterinary medicine. In 29% of examined samples isolation of bacteria was possible. Even bacteria, which are not often cultivated and bacteria, which could not be identified biochemically, could be isolated. Because of experience in human bacteriology and in conclusion of our results, the use of blood culture systems can be recommended for veterinary diagnosis, in particular when sepsis is suspected.

Animals↗

[The detection of toxinogenic Bacillus cereus strains].

Investigations into optimal culture conditions for Bacillus cereus in order to detect bacterial toxins in a cell culture system showed that Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum is the medium best suited for this purpose. The highest toxicity levels were seen when bacteria were cultured at 21 degrees C. In 42 of 43 Bacillus cereus-strains isolated from milk and milk products, toxin formation was detected using the MTT-test. In 11 strains, toxin formation was even shown when bacteria were cultured at 4 degrees C. When culture supernatants were examined in a commercially available ELISA, all cytotoxic strains were shown to form diarrheal toxin.

Animals↗

Varicella-zoster virus open reading frame 1 encodes a membrane protein that is dispensable for growth of VZV in vitro.

Varicella-zoster virus (VZV) encodes 69 unique open reading frames, 5 of which do not have herpes simplex virus type 1 (HSV-1) homologs. One of the 5, ORF1, is predicted to encode a protein of 108 amino acids. We identified a 470-base RNA corresponding to ORF1. To determine whether ORF1 encodes a protein, an 11-amino-acid epitope was inserted in frame after the ninth codon of the ORF1 open reading frame. A recombinant virus carrying this epitope expressed a protein that was immunoprecipitated with monoclonal antibody to the epitope. The ORF1 protein was detected in the membrane of infected cells. The size of ORF1 protein expressed in VZV-infected cells was slightly larger than the size expressed by translation in vitro, suggesting that the protein may undergo post-translational modification in infected cells. Insertion of stop codons immediately before the epitope in the ORF1 gene resulted in a recombinant virus that did not express ORF1 protein and that was not growth impaired in cell culture. Thus, ORF1 encodes a protein that localizes to the membrane of VZV-infected cells and that is dispensable for virus growth in vitro.

Antibodies, Monoclonal↗

[Efficiency of microbank systems for the conservation of microorganisms relevant to veterinary medicine and others which are not easy to cultivate].

In this paper the conservation of different bacteria, yeasts and molds from diagnostic material was examined with a commercial deep freeze system. 137 bacteria isolated from the stomachs of dogs, cats and pigs and from duodenal juice of cats and 7 isolates of yeasts and fungi from diagnostic material were conserved with the deep freeze system MICROBANK-TM (Mast Diagnostica). Furthermore 62 Helicobacter pylori-isolates and 1 Helicobacter felis-isolate were conserved with this system. After a storage period of 24 hours up to 20 months the isolates were recultured. 96% of the conserved microorganisms could be grown. It was not possible to cultivate 1 fungus-isolate (Fusarium sp.). 2 Helicobacter pylori-isolates, spiral bacteria from the stomach of a pig and a Clostridium tyrobutyricum-isolate were only recultured after 24 hours, 1 isolate Moraxella sp. after 2 months and another 4 anaerobe isolates after 5 months. The system MICROBANK-TM proved to be suitable for conservation and was also efficient for the conservation of microorganisms, which could not easily be cultivated.

Animals↗

Absence of varicella-zoster virus (VZV) glycoprotein V does not alter growth of VZV in vitro or sensitivity to heparin.

Varicella-zoster virus (VZV) encodes at least five glycoproteins, gpI to gpV. VZV gpV, M(r) 100K to 110K, is the product of VZV open reading frame (ORF) 14. VZV gpV is homologous to herpes simplex virus gC and pseudorabies virus gIII. To determine whether gpV is required for viral replication, we inserted a stop codon after the fifteenth codon of the ORF14 gene in a cosmid containing the gene. Transfection of human melanoma cells with the cosmid containing the mutant ORF14 gene and three other cosmids resulted in the production of infectious VZV. Immunoprecipitation indicated that the mutant virus did not express gpV. VZV that did not express gpV grew at the same rate as parental virus and was inhibited by heparin to a similar extent. The pattern of inhibition by heparin of the gpV mutant was similar to that reported for a herpes simplex virus mutant that does not contain gC, but different from that described for a pseudorabies virus mutant devoid of gIII. These results indicate that VZV gpV is not required for viral replication in vitro.

Base Sequence↗

Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro.

Four overlapping cosmid clones were constructed that contain the complete genome of the attenuated Oka strain of VZV. Transfection of human melanoma cells with the four cosmids resulted in production of infectious VZV. A double-stranded oligonucleotide, encoding a stop codon in all three open reading frames, was inserted into one of the cosmids at the 5' end of the viral thymidylate synthetase gene. Transfection of melanoma cells with the mutant cosmid, along with the other three cosmids, resulted in VZV that does not express the viral thymidylate synthetase protein. The mutant virus grew at a rate similar to that of the parental Oka strain virus. Production of recombinant VZV using cosmid DNAs will be useful for studying the function of viral genes in VZV replication and establishment of latency. Furthermore, manipulation of the Oka strain of VZV might allow one to produce a vaccine virus that does not establish latency in the central nervous system or a virus that encodes foreign antigens for use as a polyvalent live virus vaccine.

Acyclovir↗

Cardiac catecholamine metabolism in copper-deficient rats.

After parturition, Sprague-Dawley dams were fed diets containing either 0.6 (-Cu) or 6 (+Cu) mg of copper/kg of diet. Pups were weaned either to the diet of their dam or to the diet fed to dams in the other treatment group in a crossover design. At 7 wk of age, Cu-deficient rats were characterized by low tissue Cu and an enlarged heart with increased levels of dopamine (DA) and decreased levels of norepinephrine (NE). These changes resulting from Cu deficiency were independent of gender. In vivo synthesis of cardiac NE from DA in Cu-adequate rats was significantly greater than in Cu-deficient rats. Turnover of cardiac NE was estimated from the temporal change in the specific activity of [3H]NE. Fractional turnover rates (percentage per hour) of cardiac NE were similar in both dietary groups, although the cardiac turnover of NE (nanograms per hour) was 1.4-fold higher in Cu-adequate rats than in Cu-deficient rats. Repletion of Cu-deficient rats with dietary Cu increased the total amount of cardiac NE to 78 and 93% of control values after 1 and 2 d, respectively. Significant changes in the quantities of NE and DA in the heart of rats whose intake of Cu was restricted from birth were first detected at 4 wk of age, but cardiac hypertrophy was not observed until 5 wk of age. The data support the proposal that the altered levels of DA and NE in the heart of Cu-deficient rats are primarily the result of decreased activity of DA-B-monooxygenase and demonstrate that Cu supplementation rapidly repletes cardiac NE in Cu-deficient rats. Moreover, changes in the concentrations of NE and DA in the heart precede and may contribute to the development of cardiac hypertrophy.

Animals↗

Concentrations of carotenoids, retinol, and tocopherol in plasma, in response to ingestion of a meal.

Field studies and epidemiological surveys may necessitate obtaining a blood sample from a nonfasted subject for nutritional assessments. We measured the effect of a standardized test meal, eaten after an overnight fast, on the concentrations of seven carotenoid fractions, retinol, and tocopherol in plasma of eight healthy adults. The 790-calorie test meal did not alter the measured concentrations. We conclude that blood sampled up to 4 h after breakfast can be validly used for these measurements.

Adult↗

Use of immunoresponsiveness to demonstrate that the dietary requirement for copper in young rats is greater with dietary fructose than dietary starch.

Weaned male Lewis rats were pair-fed diets containing 62.7% fructose or starch and either 6-7 mg Cu/kg diet (adequate) or 0.7 mg Cu/kg diet (deficient) for 33 d. Antibody titers after primary immunization with sheep erythrocytes were significantly lower in rats fed copper-deficient diets. Compared to starch, fructose markedly attenuated antibody production in copper-deficient rats. Dietary carbohydrate did not affect the humoral immune response of rats fed diets with adequate copper. Concentrations of copper in thymus, spleen, liver and heart were also significantly lower in rats fed fructose with deficient copper (F-Cu) than in the group fed starch with deficient copper. Thymic hypogenesis was observed only in the F-Cu group. Tissue concentrations of copper were reduced before antibody production was impaired. Repletion of previously copper-deficient rats rapidly restored immunocompetence and stimulated thymic growth. Immunoresponsiveness and tissue concentrations of copper reached control levels in rats fed diets containing starch and fructose at 1.2-1.6 and 2.2-2.9 mg Cu/kg diet, respectively. The results demonstrate that the amount of dietary copper required for optimal function of the humoral immune system, thymic growth and maintenance of normal tissue levels of this essential micronutrient is greater when young rats are fed diets with fructose than with starch.

Animals↗

[Helicobacter pylori: antibodies in sera of pigs and calves].

64 sera of pigs and 22 sera of calves were investigated in a Helicobacter pylori-ELISA. The test system was validated with positive and negative reference sera. In contrast to other investigations it was tried to exclude false positive results by absorbing the sera with Wolinella succinogenes, Campylobacter jejuni subsp.jejuni, E. coli and Proteus mirabilis. This proved to be necessary, as the extinctions of some sera were reduced considerably. Using this technique sera of 5 pigs and 6 calves could be identified, which fulfilled the following criteria: 1. The extinctions were statistically significant above the mean value of the corresponding group after absorption with Campylobacter jejuni subsp.jejuni. 2. After absorbing the sera of pigs with 3 and the sera of calves with 4 bacteria the extinctions were above 50% of that of the human positive serum. 3. The extinctions of these sera were-like the positive reference sera-nearly not influenced by the absorption. These results indicate the presence of serum antibodies in pigs and calves, which react with epitops from Helicobacter pylori.

Animals↗