Development of a PCR test for the identification of Staphylococcus intermedius based on the 16S rDNA sequence

The development of a PCR assay based on the 16S ribosomal RNA gene (rDNA) sequence was carried out for the identification of Staphylococcus intermedius. Sixty-six strains of S. intermedius, 70 of Staphylococcus aureus and 2 of Staphylococcus hyicus were examined for the assay. The 16S rDNA, of which the PCR target fragment makes up 901 bp corresponding to the sequence data of the gene, was detected in all strains of S. intermedius, but it was not detected in any strains of either S. aureus or S. hyicus. These results suggest that the PCR allows a simple and precise identification of S. intermedius.     

Distribution of mecA-harboring staphylococci in healthy mares

The prevalence of staphylococci that harbor the mecA gene responsible for methicillin resistance was examined in healthy breeding mares. Staphylococci often cause diseases of horses such as metritis, keratitis, and abscess. Methicillin-resistant staphylococci would make antibiotic treatments ineffective, so it may be significant to know the distribution of mecA-harboring staphylococci in mares. Isolation of mecA-harboring staphylococci was achieved from nares and pasterns of 100 mares in Hokkaido, Japan. From 13% of the mares, mecA-harboring staphylococci, including 15 isolates of Staphylococcus sciuri and 3 of Staphylococcus lentus, were isolated. Isolates of S. sciuri were found to be genetically polyclonal by pulsed-field gel electrophoresis. These isolates produced no PCase and showed low or no resistance to beta-lactam and other classes of antibiotics. Distribution of staphylococcal species and levels of antibiotic resistance were found to be different between isolates from the present mares and those previously reported from riding-horses. Antibiotic pressure may lead to these differences. In addition, it appears that mecA-harboring S. sciuri may be native to horses.     

Isolation and species distribution of staphylococci from animal and human skin

From April 1999 to December 2000, a survey was made on the distribution of Staphylococcus species on the skin of 7 kinds of animals and humans. Staphylococci were isolated from 12 (100%) of 12 pigs, 17 (89.5%) of 19 horses, 30 (100%) of 30 cows, 73 (90.1%) of 81 chickens, 10 (40%) of 25 dogs, 23 (76.7%) of 30 laboratory mice, 20 (52.6%) of 38 pigeons, and 80 (88.9%) of 90 human beings. The predominant staphylococci isolated from a variety of animal species were novobiocin-resistant species, S. xylosus and S. sciuri regardless of the animal host species. The novobiocin-resistant species including S. xylosus and S. sciuri were only occasionally isolated from human skin. The predominant staphylococci found on human skin were novobiocin-sensitive species, S. epidermidis (63.8%), followed by S. warneri (28.8%) and S. hominis (13.8%). The results suggest that the staphylococcal flora inhabiting animal skin are different from those of human skin in regard to the predominant species isolated. In this study, we used pulsed-field gel electrophoresis to examine the chromosomal polymorphisms of S. epidermidis isolated most frequently from human skin. Strains of S. epidermidis showed the greatest genomic diversity in their fragment patterns.     

Studies on red light-induced resistance of broad bean to Botrytis cinerea: I. Possible production of suppressor and elicitor by germinating spores of pathogen

When detached broad bean leaves were preinoculated with virulent strain B304 of Botrytis cinerea 24 h before a challenge inoculation with strain B304, lesion formation by B304 was significantly inhibited in red light but not in the dark. In leaves that were preinoculated with avirulent strain 021 and then challenged by B304, however, lesion formation was not inhibited even under red light. Such differences in lesion formation after the challenge inoculation with an avirulent strain were also observed with lesions caused by Alternaria alternata, a nonpathogen of broad bean and by avirulent strain 021R in the presence of germination fluid from spores of strains B304 and 021R. These results suggest the possibility that virulent B. cinerea produced a suppressor involved in induced susceptibility and an elicitor involved in resistance induced by red light during spore germination.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

Bovine colostral CD8-positive cells are potent IFN-gamma-producing cells

IFN-gamma plays an important role in cellular immunity contributing to microorganism elimination. We have previously reported that bovine colostrum contains high levels of IFN-gamma as well as immunoglobulin. Lymphocytes are potent producers of IFN-gamma, so establishing the lymphocyte population in colostrum will help to identify the source of colostral cytokines. In this study, we used flow cytometric analysis to quantify the population of three types of lymphocytes found in colostrum; namely, CD4 (Th) cells, CD8 (cytotoxic T) cells, and gammadelta-T cells. We also quantified the concentration of colostral IFN-gamma using ELISA. IFN-gamma concentrations were measured in colostrum obtained from 96 healthy Holstein cows; the levels tended to decrease on the first day post-parturition. Flow cytometric analysis showed that many gammadelta-T- and CD8-positive cells were present in the colostrum, and that the CD4/CD8 ratio was low. The ratios of CD8- and gammadelta-T-positive cells to cells of other types decreased during the 5 days after parturition, but that of CD4-positive cells showed no change during the observation period. To identify IFN-gamma-expressing colostral lymphocytes, we used magnetic separation technology to separate the lymphocytes (CD4, CD8 and gammadelta-T) from colostral cells, then examined them for IFN-gamma mRNA expression with real-time PCR (RT-PCR). RT-PCR analysis revealed potent expression of the IFN-gamma gene in CD8-positive cells, reaching higher levels than in CD4- or gammadelta-T-positive cells. These results suggest that the CD8-positive T cells in colostrum play a role as producers of IFN-gamma.     

Isolation and characterization of staphylococci from external auditory meatus of dogs with or without otitis externa with special reference to Staphylococcus schleiferi subsp. coagulans isolates

Staphylococci were isolated from the external auditory meatus in 14 (48.3%) of 29 dogs affected with otitis externa (OE dogs) and 28 (68.3%) of 41 dogs without OE (non-OE dogs). Twenty-two OE isolates were identified as belonging to 12 species, and 42 non-OE isolates were identified as belonging to 13 species. The predominant species found in both OE and non-OE isolates were S. intermedius, and S. epidermidis. Thirty-eight (59.4%) of 64 isolates were resistant to one or more of the 17 antimicrobial agents tested. Resistance to PCG and ABPC was most frequent. S. schleiferi subsp. coagulans, a recent etiologic agent of canine OE, was isolated from OE and non-OE dogs. All of the 5 S. schleiferi subsp. coagulans isolates showed typical characteristics. No clear difference in the extracellular enzyme or toxin profiles, nor in the PFGE patterns, was demonstrated between the OE and non-OE isolates of S. schleiferi subsp. coagulans. A new PCR primer set specific for 16S rDNA was designed to identify strains of S. schleiferi subsp. coagulans. The amplified fragment was detected in all of the 5 isolates as well as the type strain GA 211 (=JCM 7470) and a reference strain GA 11, but was not detected in any strains of the related species, S. aureus, S. intermedius and S. hyicus. The PCR may allow a simple, rapid and precise identification of S. schleiferi subsp. coagulans, in addition to the standard tube test for free coagulase.     

Permuted tRNA genes expressed via a circular RNA intermediate in Cyanidioschyzon merolae

A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified 11 transfer RNA (tRNA) genes in which the 3' half of the tRNA lies upstream of the 5' half in the genome. We verified that these genes are expressed and produce mature tRNAs that are aminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual processing pathway in which the termini of the tRNA precursor are ligated, resulting in formation of a characteristic circular RNA intermediate that is then processed at the acceptor stem to generate the correct termini.     

Polycitorols A and B,New Tricyclic Alkaloids from an Ascidian

Two new tricyclic alkaloids, polycitorols A (1) and B (2) have been isolated along with the known lepadiformine (3) from a marine ascidian of the family Polycitoridae. The structures of the new compounds were elucidated by analysis of NMR data and comparison with those of 3 and other related compounds [1–5]. Compounds 1 and 2 are closely related to cylindricines A and B, lacking C-4 oxygenation found in cylindricines and having a butyl instead of a hexyl appendage. NOE experiments on compounds 1 and 2 suggested the A/B ring fusion to be cis.