Tuberculin sensitivity and morphological immune response in guinea pigs after application of minimal sensitizing dose of BCG vaccine, substrain Sofia SL222.

During the investigation of the BCG allergenic potency it is advisable to vaccinate with decreasing doses, estimating the lowest dose that induces tuberculin sensitivity and specific morphological inflammation. Although the biological test does not reveal the mathematical correlation of dose-effect relationships, it is important to look for the determination of the minimal sensitizing dose for every BCG vaccine. In this study, three groups of twenty four guinea pigs were vaccinated with decreasing doses of reconstituted BCG vaccine: 120 ng, 12 ng and 1.2 ng. Tuberculin tests were performed in different groups at the 30th, 60th, 90th and 120th day after BCG injection. The negative tuberculin reactions converted to positive between the 60th and 90th day. The dose of 12 ng elicited the largest tuberculin reactions in the animals. This dose contains 65 viable bacteria and could be regarded as the smallest effective sensitizing dose of the BCG vaccine, substrain Sofia SL222. The morphological examination demonstrated that very low inoculums (1.2 ng or 6 viable cells) were sufficient to induce a specific inflammation after BCG vaccination. The immune response in lungs and bronchus-associated lymphoid tissue (BALT) of all BCG doses applied was strongest on the day 60. In the same term, lymph nodes and spleens were characterized with blast transformed lymphoid follicles with epitheloid and Langhans giant cells even with the smallest injected dose of 1.2 ng.

Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103 improves ram spermatozoa functions in vitro.

In this study we determined the effect of reactive oxygen species (ROS) generation during incubation in media at 39 degrees C on ram spermatozoa and the protection by exogenously added antioxidant enzyme, superoxide dismutase (SOD). A novel Cu/Zn-SOD, isolated from the fungal strain Humicola lutea 103 (HLSOD), was used. Our results point out that the levels of both, superoxide anion radicals (*O2-) and H2O2, increase approximately 8-10- and 2-3-fold, respectively, during incubation of spermatozoa. Enhanced ROS generation coincided with reduction of motility, independently of the type of diluted medium. Addition of HLSOD (30, 60 and 120 U ml(-1) sperm) improved sperm functions, maintaining almost initial percentages of motile spermatozoa and increasing the values of mean cytochemical coefficient. At the same time, a significant diminution of *O2- and H2O2 content in the presence of antioxidant enzyme was established. The results suggest that HLSOD is an effective *O2- scavenger in semen that leads to protection of sperm functions.

A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species.

A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.

Characterization of bacteriocins produced by strains from traditional Bulgarian dairy products.

A result of extensive screening of over 300 strains from the Collection of ELBY Bulgaricum, PLC, thirty six strains were selected as producers of bacteriocins, active closely related lactic acid bacterial species and some food spoilage bacteria. The selected strains belong to L. helveticus, L. bulgaricus and S. thermophilus, which are rare bacteriocin producers. Nineteen nonidentified producers were characterized by molecular taxonomic approaches--M13 fingerprinting, repetitive PCR, ribotyping and hybridization with species-specific probes, which allowed to affiliate them to the species L. delbrueckii. Several strains were found to harbour plasmids of different size. The estimated activity against food borne pathogens makes the isolated substances perspective as safe food preservatives and the producing strains could be used as components of starters with improved quality.

Detection and characterization of a novel antibacterial substance produced by a Lactobacillus delbrueckii strain 1043.

A novel antibacterial substance produced by a strain isolated from Bulgarian yellow cheese was characterized. The producer strain was identified by molecular typing to belong to the species Lactobacillus delbrueckii, which is a rare producer of bacteriocins. The inhibitory agent was heat stable and active against lactic acid bacteria species and several food-borne pathogens: Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Yersinia enterocolitica and Y. pseudotuberculosis. Its sensitivity to amylolitic enzymes and lipase suggested that a lipid and carbohydrate moiety could be important for the activity. The amino acid content of the purified bacteriocin was estimated to 29 amino acids. The bacteriocin was shown to be small (3.6-6 kDa) by three different methods: HPLC gel-filtration, SDS-PAGE and amino acid contents.

Designing an artificial ecological mesocosm for the study of Cd and Pb impact on the immune system of experimental animals.

Studies on the influence of high heavy metal concentrations (Pb and Cd) on the immune status of experimental animals were carried out. The experiments were performed with Balb/c mice and Pb and Cd concentrations 30- and 50-fold above the limit admissible concentrations (LAC), respectively. A slight increase in the phagocytic activity after 5 days of treatment with high doses of the heavy metals (HM) and its significant activation on the 30th day for both concentrations were observed. Low doses of the HM cause significant increase in the spontaneous production of H2O2 on the 30th day of the experiment. The production of H2O2 by stimulation with PMA macrophages was also activated on the 30th day but to a lower degree and only with the high doses of the HM. Inhibition of the alternative pathway of complement activation was observed in animals treated with low and high doses of the HM for 90 days.

Cryptic plasmids from Lactobacillus helveticus and their evolutionary relationship.

Three different cryptic plasmids from Lactobacillus helveticus have been identified and their DNA sequences determined. Analysis and comparisons of their primary structures revealed stretches of DNA with considerable homology. Thus, large portions of the plasmid non-coding sequences were conserved at 80-90% identity between the different plasmids identified so far in L. helveticus. Nevertheless, different plasmids found in a same host strain utilise different genes of replication, probably acquired during evolution from different replicons from Gram-positive bacterial origins. A remnant structure of such a possible genetic integration of a foreign replication gene into one of the plasmids of L. helveticus was identified.

M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.

[Fluorometric method of determining urinary estrogens]. / Fluorometrichen metod za opredeliane na estrogeni v urina.

The authors describe a method for determination of estrogens in urine of nonpregnant women and men. The method includes enzymic hydrolysis of urine, gel filtration of Sefadex G25, extraction with organic solvents and modifies Kober reaction with spectrofluorometric determination. The technique of the method is not complex, the usage of reagents is small and is suitable for everyday work.