[Study of amino acid and carbohydrate metabolism of porcine strains of Haemophilus by thin layer chromatography]. / Untersuchungen des Aminosäure- und Kohlenhydrat-Stoffwechsels porciner Haemophilus-Stämme mittels Dünnschichtchromatographie.

The aim of the present study was to investigate porcine reference and field strains of the species Haemophilus (H.) pleuropneumoniae and H. parasuis, as well as H. Taxon "minor group" and Taxon C on their amino acid and carbohydrate metabolism by thin layer chromatography. The 17 reference strains studied showed almost identical results within the different species and taxa in both, amino acid and carbohydrate metabolism patterns. Based on a few differing enzymatic reactions a reduced species- and taxon-specific reaction pattern could be established, which included L-alanine, L-citrulline and L-threonine of the amino acids as well as D-ribose, alpha-D-xylose, mannitol, trehalose, beta-melibiose and alpha-lactose of the carbohydrates. This differentiation system allowed a reliable identification of 7 field strains whereas 4 additional ones, hitherto pre-classified as H. parasuis, could not be associated with any of the above species and taxa.

[Amino acid metabolism by thin layer chromatography for the differentiation of Bordetella and Alcaligenes species]. / Untersuchungen des Aminosäurestoffwechsels mit der Dünnschichtchromatographie zur Differenzierung von Bordetellen- und Alcaligenes-Spezies.

The amino acid metabolism of Bordetella and Alcaligenes strains was investigated for differentiation purposes. All species investigated reacted differently with a number of amino acids. The genus Bordetella could be differentiated from that of Alcaligenes by its inability to metabolize L-lysine, L-arginine and D-histidine. The recently described species Bordetella avium, as well as Achromobacter xylosoxidans (Yabuuchi), considered as Alcaligenes denitrificans subsp. xylosoxidans, presented themselves as independent species too. Bordetella odorans, now assigned to the species Alcaligenes faecalis was indistinguishable from the other strains of Alcaligenes faecalis investigated.

[Hyalurodinase activity of beta-hemolytic streptococci of the Lancefield group C]. / Untersuchungen zur Hyaluronidase-Aktivität beta-hämolysierender Streptokokken der Lancefield-Gruppe C.

A total of 110 strains of beta-hemolytic streptococci, belonging to serogroup C (Lancefield), isolated from horses (71 S. zooepidemicus, 27 S. equisimilis and 12 S. equi) as well as 5 reference strains were tested for their ability to produce hyaluronidase. The determinations were carried out in a culture test on agarose gel and in a liquid test system (turbidity test according to DiFerrante). The results of both methods used showed that the three Streptococcus species could be differentiated by the relative quantitative determination of hyaluronidase activity. S. equisimilis strains produce 5 to 10 times more hyaluronidase than those of S. zooepidemicus. The strains of S. equi did not show enzyme production. In addition parallel tests by determination of final N-acetyl-D-glucosamine groups and by turbidity test were done to confirm the hyaluronidase activity of S. equisimilis strains. The comparison of results (Fig. 3) obtained from these tests, showed a good correspondence as demonstrated by a correlation index of 0,93.

[A simple method for the rapid detection of bacterial hyaluronidase in K hyaluronate-containing gel]. / Einfache Methode zum schnellen Nachweis bakterieller Hyaluronidase in K-Hyaluronat-haltigem Gel.

For detection of hyaluronidase activities we investigated several groups of bacteria. The bacteria were inoculated on a 1,5% agarose gel in Petri plates of 4 cm diameter or gel discs of 7 mm diameter, containing 0,1% of K-hyaluronate as well as nutritient medium, and were incubated for 2-20 h at 37 degrees C in a moist chamber. Subsequently some ml of a 10% solution of cetylpyridiniumchloride were poured on the gel to precipitate the polymere hyaluronate. If the hyaluronate was depolymerized by hyaluronidase, a translucent area was visible around the colonies. We found out, that a gel layer of 1 mm was sufficient to detect the small amounts of hyaluronidase, which were produced by bacteria within an incubation time of 2 h. These results were confirmed by incubation for 20 h and in some cases 36 h. The hyaluronidase production by different anaerobic Clostridium strains was always proved after a 20 h growth period. The bacteria were inoculated with the whole loop of a self made platin sowing wire loop. By this method quantitative differences of hyaluronidase activities between different strains of bacteria could be detected.

[Differentiation of Brucella by acrylamide-gel electrophoresis (author's transl)]. / Differenzierung von Brucellen mit Hilfe der Acrylamidgel-Elektrophorese.

The strains of Brucella (Tab. 1) were grown on Tryptose-blood-agar at 37 degrees C, and the strains of Bordetella, Pasteurella and Actinobacillus on blood-agar. After 24 or 48 h they were harvested and extracted in phenol-acetic acid-water solution (4:2:1) (1 ml/50 mg bacterial wet weight) at 4 degrees C over 48 h. After centrifugation (8000 X g, 1 h) 2 volumes of the supernatant were mixed with 1 volume of a 40% sucrose solution in 35% acetic acid. Different volumes (0.20, 0.15 or 0.10 ml) of this were added to 7.5% acrylamide gel (2 ml in tubes 6 X 100 mm), containing 5 M urea and 12% acetic acid. The solution in both electrode chambers was 10% acetic acid. During the first 15 min it was focused with 2 mA/tube, then it was separated for 3 or 5 h with 4 mA/tube. The protein bands were stained with amido black 10B. All species could be exactly differentiated from each other, but the protein bands of biotypes of Bruc. suis, Bruc. melitensis and Bruc. abortus were identical in each case. All investigated strains of Bruc. canis were identical too. A small relationship was noticed between Bruc. canis and Bruc. suis and between Bordetella bronchiseptica and Bruc. canis respectively Bruc. suis strains.

[Investigations of amino acid metabolism by thin layer chromatography for differentiation of Brucella (author's transl)]. / Untersuchungen des Aminosäurestoffwechsels mit der Dünnschicht-chromatographie zur Differenzierung von Brucelle

The amino acid metabolism of 23 different Brucella strains was investigated for differentiation purposes. The results were evaluated by thin layer chromatography, after enzymatic incubation. The organisms (Tab. 1) were grown on Tryptose blood agar at 37 degree C for 24 or 48h. Two mg wet weight of bacteria in 0.2 ml PBS, 0.01 M, were incubated with 12.5 microng (0.025 ml) amino acid in small tubes for 16h at 37 degree C, and centrifuged for 15 min at 7500 X g. For controls, bacterial suspensions were heated for 15 min at 100 degree C to destroy enzymatic activity, and also contrifuged for 15 min at 7500 X g. Usually 4 micronl of the supernatant fluids (6 micronl for L-asparagine, and 10 micronl for L-proline) were pipetted on the thin layer plate. The tests were run in n-butanol acetic acid water, 20:5:5, with a distance of 8 cm. Amino acids were stained with ninhydrine. The tests were repeated 3-5 times with identical results. Amino acid metabolism was indicated by different staining intesities (+ to ++) in comparison to control preparations. All species could be exactly differentiated from each other, with the exception of B. suis, biotype 2, and B canis, which could not be differentiated by their amino acid metabolism. Biotyps of the same species were mostly identical. The results of these investigations could be reproduced qualitatively as well as quantitatively. The method described is recommended for routine investigations.

[Differentiation of swine-Mycoplasma by latex-agglutination (author's transl)]. / Differenzierung von Schweine-Mycoplasmen mit Hilfe der Latex-Agglutination

UNLABELLED: The strains of Mycoplasma were grown in Shittlestone medium for 3-5 days. After centrifugation and washing, the mycoplasm were freeze-dried and stored at -20 degrees C. From this stock material immune sera and antigens were prepared. The immune sera were prepared by immunization of rabbits with a suspension of mycoplasma (2,2 mg dry weight per ml) and complete Freund's adjuvant. For the first immunication, 6.6 mg of antigen were injected into rabbits at different sites. 3 weeks later the second immunization followed by the intramuscular route (4.4 mg of antigen). The third and fourth immunizations were identical to the second one. Antiserum was obtained as usual. In order to avoid unspecific reactions in the latex agglutination, 1 ml of antiserum was added for absorption to 200 mg of freeze-dried sterile Whittlestone medium over 2 days at 4 degrees C. Antigen was prepared by ultrasonic disruption of mycoplasma (20 mg dry weight/ml physiological saline) and centrifugation 17300 X g, 30 min). The supernatant was used as antigen. The latex agglutination was performed in 0.1 M borate buffer pH 8,2. For preparation of the latex antigen compound 0.2 ml of a latex suspension (diluted 5 fold with water) were moxed with 0.5 ml of an antigen suspension (diluted 16 fold with buffer). After 10 min at room temperature, 4.3 ml of borate buffer were added. After 10 min, 0.3 ml of this solution were added again each to 0.3 ml of serial dilutions of anitserum. The suspensions were mixed and left for 20 h at 37 degrees C and after this time for 1 h at room temperature. Then the reactions were read. RESULTS: M. suipneumoniae and M. hyopneumoniae were found to be identical or very close related strains. Between M. hyorhinis and M. sp. E9 relationship was noted too. M. granularum was different to all other investigated strains of mycoplasma. All results of Latex agglutination are in agreement with investigations performed with the aid of other serological methods and of electrophoretic separations of cell proteins of these mycoplasma.

[The differenziation of swine-mycoplasma by acrylamide-gel-electrophoresis (author's transl)]. / Die Differenzierung von Schweine-Mycoplasmen mit Hilfe der Acrylamidgel-Elektrophorese

The strains of swine mycoplasma (Tab. 1) were grown aerobically in Whittlestone's Medium containing swine serum for 4-5 days at 37 degrees C. After centrifugation and washing they were freeze-dried and extracted with a phenol-acetic acid-water solution (4:2:1) (1 ml/5 mg mycoplasma dru weight) at 4 degrees C over 48 h. After centrifugation 2 volumes of the supernatant were mixed with 1 volume of a 40% sucrose solution in 35% acetic acid. 0,15 or 0,2 ml of which were added to the acrylamide-gel (2 ml in a tube 0,5 times 10 cm) containing 5 M urea and 35% acetic acid. This solution was overlayed with 0,1 ml 75% acetic acid and tubes then filled with 10% acetic acid. The solution in both electrode chambers was 10% acetic acid, too. During the first 5 min it was separated with 2 mA/tube, then 3 or 5 h with 4 mA/tube. The protein bands were stained with amido black 10B. For control steril culture medium was investigated, too. Various preparations of freeze-dried M. hyopneumoniae gave identical protein patterns. Nearly identical were the patterns of protein bands from M. suipneumoniae and M. hyopneumoniae; that means identity of the strains or very close relationship (Figs. 1 and 2). This is in agreement with other authors who investigated both strains with serological methods. M. suipneumoniae and M. hyopneumoniae were found to be different as well from M. hyorhinis and M. sp. E9 from M. granularum (Figs. 1 and 2). Between M. hyorhinis and M. sp. E9 less relationship was noted. All these results were in agreement with investigations performed with the aid of Latex agglutination.