Detection of Deefgea chitinilytica in freshwater ornamental fish

Aim: To identify and characterize six chitinolytic bacterial strains isolated from ornamental fish. Methods and Results: Six different isolates of Deefgea chitinilytica were detected in healthy as well as diseased ornamental fish in Germany over a period of 2 years. Bacterial strains were identified using 16S rRNA partial gene sequencing and further characterized using different biochemical microtest systems and additional standard biochemical tests. Conclusion: We show that commercially available biochemical microtest systems are useful for identification of D. chitinilytica, supplemented by 16S rRNA partial gene sequencing. Furthermore, this study provides new information about the occurrence of D. chitinilytica, as this is the first isolation of D. chitinilytica from animals and first described isolation in Europe. Significance and Impact of the Study: Deefgea chitinilytica may be isolated regularly in fish diagnostic laboratories. Therefore, accurate identification of this bacterial species is important. Involvement of D. chitinilytica in opportunistic infections of aquatic organisms cannot be excluded and has to be further investigated.     

Enzyme-Linked Immunosorbent Assay for Detection of Ochratoxin A

A simple microtest plate enzyme-linked immunosorbent assay was developed for the detection of ochratoxin A at levels as low as 25 pg per assay. The relative cross-reactivities of the antibody in this system with ochratoxin A (OA), OB, OC, and Oα were found to be 1.0, 0.14, 0.44, and 0.01, respectively.     

Microcomplement-Fixation Inhibition: a Rapid and Economical Test to Detect Non-Complement-Fixing Antibodies

Complement-fixation inhibition test was adapted to the microtiter system. When cat and monkey non-complement-fixing antibody were used, the microtest was shown to be as sensitive as the conventional tube complement-fixation inhibition test.     

The radiosensitivity of hematopoietic stem cells from mice forming splenic colonies after 8 and 12 days following bone marrow cell transplantation (CFU-S-8 and CFU-S-12)]

The method of "macro-" and "microcolonies" was used to study the radiosensitivity of CFU-S that form "early" (8 days) and "late" (12 days) splenic colonies after transplantation of syngeneic bone marrow to fatally exposed mice: no significant differences were found. Median lethal doses (D0) for CFU-S-8 and CFU-S-12 were 1.03 and 1.13 Gy for "microtest" and 0.99 and 1.16 Gy for "microtest" respectively.     

Differentiation of Gram-Negative, Nonfermentative Bacteria Isolated from Biofilters on the Basis of Fatty Acid Composition, Quinone System, and Physiological Reaction Profiles

Gram-negative, nonfermentative bacteria isolated from biofilters for off-gas treatment of animal-rendering-plant emissions were differentiated by whole-cell fatty acid analysis, quinone analysis, and numerical taxonomy based on their physiological reaction profiles. The last system consisted of 60 physiological tests and was arranged as a microtest system on microtitration plates. Based on fatty acid analyses, 31 isolates were separated into six clusters and five single-member clusters. The isolates of two clusters were identified as Alcaligenes faecalis and Pseudomonas diminuta. The remaining nine clusters were characterized by their fatty acid profiles, quinone systems, and physiological reaction profiles. Clusters resulting from fatty acid analyses were compared with those resulting from physiological reaction profiles. Six clusters could be confirmed this way. The efficiency of the physiological test system was increased by the prearrangement of the isolates according to their quinone type.     

In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

Microsystem for Collecting and Shipping Diagnostic Sera

A system is described for collecting, shipping, and storing serum samples using light-weight plastic microtest plates.     

Neutralization microtest with human coxsackievirus and echovirus serotypes

Sets of 600 single sera from healthy individuals of various age and 458 paired sera from patients treated for diseases of various etiology were examined using a neutralization microtest technique employing prototype collection strains CA9, CB1-CB5, E1-E9, E11-E14, E17, E19, E24 and E26. Totally, 34,862 monotype neutralization tests were carried out in this study. In the set of single serum samples the lowest proportion of sera reacting with any of the enterovirus serotypes used was encountered in the group of youngest children up to the age of 2 years. This group of children showed also the highest proportion of sera free of type-specific antibody. Taken together, these sera reacted most frequently with serotypes CA9 and CB4. In the set of paired sera significant rises in antibody titre to one enterovirus serotype were recorded in 105 instances, in association with simultaneous nonsignificant rises against one or several other serotypes in 64 instances. In additional 12 paired sera there was evidenced a significant rise of antibody titre to more than one serotypes, combined in 8 of these with simultaneous nonsignificant rises to further serotypes. This neutralization microtest technique with a set of enterovirus serotypes is believed to represent a servicable diagnostic tool in determining the cause of enteroviral infection, in spite of the estimated 10% failure to establish the exact serotype responsible. The results yielded by the serologic examination of single serum samples are not considered as reflecting the actual seroconversion rates in the general population.     

Selection of proliferating cybrid cells by dual laser flow sorting : Isolation of teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids

An alternative method for the isolation of proliferating cybrid cells was developed, and was used to obtain teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids. Enucleated neuroblastoma (or endoderm) cells labelled with fluorescent microspheres were fused with (HPRT-deficient) unlabelled teratocarcinoma cells. The cells in the fusion mixture were stained with the vital DNA stain Hoechst 33342 and the cybrids, containing both a fluorescent nucleus and fluorescent beads, were isolated by dual laser flow sorting. The purity of the sorted fraction, as determined by the percentage of cells showing HPRT activity, was 86 and 57% for the neuroblastoma and endoderm cybrids, respectively. After single cell sorting in wells of Terasaki microtest plates, clones of proliferating cybrids were obtained with cloning efficiencies of 33% (neuroblastoma and endoderm cybrids respectively. After single cell sorting in wells of Terasaki microtest parental cell lines were analysed by two-dimensional gel electrophoresis. A number of differences were found between the parental cell lines but all isolated colonies (sixteen neuroblastoma cybrids and eight endoderm cybrids) resembled the teratocarcinoma parent. These results therefore give no evidence for the existence of cytoplasmic factors in neuroblastoma or endoderm cells capable of inducing permanent differentiation of teratocarcinoma cells.     

Comparison of Macrocomplement and Microcomplement Fixation Techniques Used in Fungus Serology

A comparative evaluation was performed on the micro- and macrocomplement fixation (CF) tests that are used as standard procedures in the serodiagnosis of blastomycosis, coccidioidomycosis, and histoplasmosis. Tests with 937 sera from suspected and culturally proven cases of these diseases against yeast-form antigens of Blastomyces dermatitidis and Histoplasma capsulatum and against the soluble antigens, coccidioidin and histoplasmin, revealed that the microtiters were within ± 1 dilution of the macrotiters in 83 to 93% of the sera. Tests on randomly coded quality control sera revealed the microform of the CF test to be highly reproducible. Our studies indicate that the results obtained by the two tests have similar diagnostic and prognostic interpretations. Because of the sensitivity, reproducibility, economy, and ease of performance, the microtest is highly recommended for use in fungus serology.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Immunoenzymoassay of the hyaluronic acid-hyaluronectin interaction: application to the detection of hyaluronic acid in serum of normal subjects and cancer patients

The binding of a hyaluronic acid-binding glycoprotein, hyaluronectin (HN), isolated from human brain, to hyaluronic acid (HA) was investigated with the enzyme-linked immunosorbent assay technique using plastic microtest plates coated with a 50 mg/liter solution of HA in 0.1 M bicarbonate. Optimum conditions for HN binding to HA were in 0.2 M NaCl buffered with 0.1 M sodium phosphate at pH 7. An assay for HA in solution was set up exploiting the fact that HN binding could be inhibited by soluble HA. HA was preincubated for 1 h in a test tube with a 30-ng/ml HN solution (v/v) in the buffer containing 0.1% bovine serum albumin. Incubation on HA-coated microtest plate lasted 4 h and maximum sensitivity was achieved when incubation was carried out at 4 degrees C. HN bound to the plate was revealed by means of alkaline phosphatase-conjugated anti-HN antibodies. The test was used to measure HA inhibitory activity after depolymerization by ferrous ions. No difference was found between inhibitory activity or smaller fragments and that of high-molecular-weight HA. The assay was applied to determination of HA in sera. Specificity was demonstrated by Streptomyces hyaluronidase digestion of reactive material in sera. Other glycosaminoglycans did not interfere with the assay. Recovery of HA was good and intra- and interassay variation coefficients were 6 +/- 2.2 and 12%. In 103 blood donor sera, HA was found at 22.4 +/- 16.7 micrograms/liter. HA was elevated in most of the cancer patient sera tested.     

An efficient microtest to study adherence of bacteria to mammalian cells

A simple, fast and highly reproducible microtest was developed for in vitro adherence studies. A rat epithelial cell line was investigated for the adherence of clinical and subclinical ovine and bovine Staphylococcus aureus strains isolated from mastitis. Staphylococcus aureus strains differed in their ability to adhere to epithelial cells, the degree of adherence being dependent on the concentration of bacteria used in the test.     

Development of an in vitro microtest to assess drug susceptibility of Babesia bovis and Babesia bigemina.

Continuous cultivation of the bovine hemoparasites Babesia bovis and Babesia bigemina was developed as an in vitro microtest to assess parasite susceptibility to babesicidal compounds. Reproducibility of parasite multiplication rates was independent of culture size, making it possible to use a microscale of 100 microliters for each test sample. Inhibitory concentrations (IC50s) of a commonly used babesicide, quinuronium sulfate, evaluated by this in vitro method were found to be 5 x 10(-8) g/ml for B. bovis and 2 x 10(-9) g/ml for B. bigemina.     

Determination of antithrombocyte antibodies in the blood serum of patients with idiopathic thrombocytopenic purpura by an immunoenzyme method

Enzyme-linked immunosorbent assay (ELISA) was developed for determination of serum antiplatelet antibodies. Platelets obtained from healthy donors of blood group 0(1) were washed off plasma and sedimented on the bottom of microtest wells. After washing off unattached platelets and blocking of plastic with albumin platelets were incubated with sera under investigation and binding of serum antibodies was detected using antihuman immunoglobulin antibodies conjugated with peroxidase. Ten patients with idiopathic thrombocytopenic purpura (ITP). 1 patient with systemic lupus erythematosus. 1 patient with red blood cell aplasia and 9 healthy donors (negative control) were studied by ELISA. Serum antibodies which effectively bound to platelets were detected in 5 patients with ITP, in patient with lupus erythematosus and in patient with red blood cell aplasia.     

Trial of purification and serological characterization of human and murine transplantation antigens]

Human and murine antigens are purified by exclusion chromatography on Sephadex and preparative polyacrylamide gel electrophoresis and their purification stage checked by analytical methods. The study of antigenic preparations consists of electrofocusing, molecular weight estimation and chemical determinations. A cytotoxicity inhibition test with specific alloantisera reveals the antigenic potency. A microtest using 51Cr labelled tumoral cells has been achieved for the analysis of murine preparations.     

A chick-embyo cell microtest for typing of Herpesvirus hominis (38531).

Oral type 1 and genital type 2 Herpesvirus hominis (HVH) strains demonstrate distinctive biological properties in primary chick embryo cells (PCE) cultivated in microtest plates. With this procedure four reference strains of known types and 106 clinical isolates were differentiated as type 1 or 2. The type 1 strains showed low efficiency of infection and either no cytopathic effect (CPE) or only an incomplete CPE characterized by uniform thinning of the cell sheet in test wells. Type 2 strains had a high efficiency of infection and with CPE characterized by patchy plaque-like lesions readily distinguished from CPE of type 1 strains. A 96% correlation (27/28) between the PCE microtyping and kinetic neutralization tests and a 94% correlation (60/64) between the PCE microtyping and immunofluorescence test was obtained. The microplate PCE test is a simple, clear-cut, and reliable procedure for the typing of HVH.     

Heterogeneous distribution of ATP citrate lyase in rat-liver parenchyma. Microradiochemical determination in microdissected periportal and perivenous liver tissue

1. A radiochemical microtest was established for the determination of ATP citrate lyase in tissue samples of 0.2-1.0 micrograms dry weight. The specificity of this test system was guaranteed by its coenzyme A dependence as well as by inhibition of the activity measured in presence of a specific antibody. 2. Using this test system ATP citrate lyase activity was determined in microdissected periportal and perivenous liver tissue of fed, fasted and refed animals. The perivenous activity was 1.8-fold and 2.4-fold higher than the periportal one in fed male and female rats respectively. 3. The perivenous to periportal gradient was decreased during starvation-dependent reduction of the ATP citrate lyase activity. On the other hand it was not only restored but enhanced up to 2.8 after refeeding-dependent enhancement of the enzyme activity. 4. The predominance of the ATP citrate lyase activity in the perivenous, mainly glycolytic zone supports the hypothesis of the coordinate zonation of the carbohydrate and the lipid metabolism in the liver parenchyma.     

不同供钾水平对平邑甜茶幼苗生长及NO3-吸收利用特性的影响

研究平邑甜茶幼苗NO₃^--N吸收和利用特性对不同供钾水平的响应,旨在明确钾肥对氮肥吸收利用的影响,从而为果园科学施肥提供理论依据.以平邑甜茶幼苗为材料进行砂培试验,设置K₀、K₁、K₂、K₃、K₄、K₅、K₆ 7个钾浓度处理,分别相当于0、2、4、6、8、10、12 mmol·L^-1 K⁺,运用¹⁵N同位素示踪技术和非损伤扫描离子选择电极技术,测定了不同供钾水平下平邑甜茶的氮素吸收和利用情况.结果表明: K₃处理平邑甜茶幼苗根系活力、硝酸还原酶活性以及根系形态指标均显著高于其他处理.与其他处理相比,K₃处理根、茎、叶从肥料中吸收分配到的¹⁵N 量对该器官全氮量的贡献率(Ndff)均达到最高,分别为K₀处理的1.36、1.33和1.47倍.随供钾水平的增加,植株氮素利用率呈现先增高后降低的趋势,且在K₃处理时最大,为23.3%,是K₀处理的3.04倍.非损伤微测技术结果显示,K₃处理时,平邑甜茶根系对NO₃^-有强烈吸收且内流速度达到最大,为19.34 pmol·cm^-2·s^-1;在缺钾(K₀)和高钾(K₆)处理时有明显外排趋势.因此,钾的亏缺或过量均抑制氮素的吸收和利用,适当供钾能够促进幼苗根系生长,增强硝酸还原酶活性,从而促进平邑甜茶对氮素的吸收.     

Characterization of lysosomal-type hyaluronidase in the culture medium of 2 cell lines derived from human hepatomas

A sensitive assay for hyaluronidase was developed using as a substrate, hyaluronic acid insolubilized on polystyrene microtest plates. Hyaluronic acid was measured exploiting the fact that it can bind immune complexes made up with hyaluronectin and alkaline phosphatase-conjugated anti-hyaluronectin antibodies. Hyaluronidase was detected in both cell line culture media. Optimum pH was between 3.25 and 3.75. Sodium chloride dependence was absolute, and the optimum concentration of sodium chloride was between 0.2 and 0.3 M. The activity was not affected by dialysis, and was suppressed by a 5 minute heating at 50 degrees C or by protease treatment. The molecular weight was 68 K as determined by gel permeation chromatography. The results are close to those reported for human lysosomal hyaluronidase.