Molecular events involved in cellular invasion by Ehrlichia chaffeensis and Anaplasma phagocytophilum

Ehrlichia chaffeensis and Anaplasma phagocytophilum are obligatory intracellular bacteria that preferentially replicate inside leukocytes by utilizing biological compounds and processes of these primary host defensive cells. These bacteria incorporate cholesterol from the host for their survival. Upon interaction with host monocytes and granulocytes, respectively, these bacteria usurp the lipid raft domain containing GPI-anchored protein to induce a series of signaling events that result in internalization of the bacteria. Monocytes and neutrophils usually kill invading microorganisms by fusion of the phagosomes containing the bacteria with granules containing both antimicrobial peptides and lysosomal hydrolytic enzymes and/or through sequestering vital nutrients. However, E. chaffeensis and A. phagocytophilum alter vesicular traffic to create a unique intracellular membrane-bound compartment that allows their replication in seclusion from lysosomal killing. These bacteria are quite sensitive to reactive oxygen species (ROS), so in order to survive in host cells that are primary mediators of ROS-induced killing, they inhibit activation of NADPH oxidase and assembly of this enzyme in their inclusion compartments. Moreover, host phagocyte activation and differentiation, apoptosis, and IFN-γ signaling pathways are inhibited by these bacteria. Through reductive evolution, lipopolysaccharide and peptidoglycan that activate the innate immune response, have been eliminated from these gram-negative bacteria at the genomic level. Upon interaction with new host cells, bacterial genes encoding the Type IV secretion apparatus and the two-component regulatory system are up-regulated to sense and adapt to the host environment. Thus dynamic signal transduction events concurrently proceed both in the host cells and in the invading E. chaffeensis and A. phagocytophilum bacteria for successful establishment of intracellular infection. Several bacterial surface-exposed proteins and porins are recently identified. Further functional studies on Ehrlichia and Anaplasma effector or ligand molecules and cognate host cell receptors will undoubtedly advance our understanding of the complex interplay between obligatory intracellular pathogens and their hosts. Such data can be applied towards treatment, diagnosis, and control of ehrlichiosis and anaplasmosis.     

Investigation of Staphylococcus aureus positive for Staphylococcal enterotoxin S and T genes

Staphylococcus aureus produces staphylococcal enterotoxins (SEs) and causes food poisoning. It is known that almost all SE-encoding genes are present on various types of mobile genetic elements and can mobilize among S. aureus populations. Further, plasmids comprise one of SE gene carriers. Previously, we reported novel SEs, SES and SET, harbored by the plasmid pF5 from Fukuoka5. In the present study, we analyzed the distribution of these SEs in various S. aureus isolates in Japan. We used 526 S. aureus strains and found 311 strains positive for at least one SE/SE-like toxin gene, but only two strains (Fukuoka5 and Hiroshima3) were positive for ses and set among the specimens. We analyzed two plasmids (pF5 and pH3) from these strains and found that they were different. Whereas these plasmids partially shared similar sequences involved in the ser/selj/set/ses gene cluster, other sequences were different. A comparison of these plasmids with those deposited in the NCBI database revealed that only one plasmid had the ser/selj/set/ses cluster with a stop mutation in set similar to that in pH3. In addition, the chromosomes of Fukuoka5 and Hiroshima3, positive for ses and set, were classified into different genotypes. Despite the low rate of gene positivity for these SEs, it is suggested that there is diversity in plasmids and strains carrying these two SEs. Consequently, regarding the entire feature of SE prevalence, we improved the multiplex PCR detection method for the SE superfamily to obtain further insight.     

Cytosolic 20 alpha-hydroxysteroid dehydrogenase activity in spontaneous neoplasms in the dog and cat.

Steroid-producing tissues such as ovary, placenta, testis and adrenal gland and non-steroid-producing cells including lymphocytes are known to contain 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). In the present study, we measured and partially characterized the 20 alpha-HSD in 11 neoplastic tissues surgically removed from dogs and a cat. The tissues were pathologically classified as 4 benign and 7 malignant. The enzyme activities in the cytoplasmic fraction were positive in 6 of the 7 malignant tissues and 2 of the 4 benign ones. Following DEAE chromatography analysis of 2 malignant tumour samples, multiple forms of enzyme activities with different electric charges were detected. Furthermore, 20 alpha-HSD activity in the cytosol fraction from malignant tumours was increased dramatically by passage through the DEAE column, suggesting that the enzyme is present in the cytosol as an inactive or sequestered form.     

Human influenza virus infection in mink: Serological evidence of infection in summer and autumn

During the period from July to November 1981, 42 out of 128 young mink of a flock were found to possess antibodies against the viruses A/Bangkok/1/79 (H3N2) and A/Kumamoto/37/79 (H1N1), which were currently prevailing human influenza viruses. Seroconversion against A/Bangkok/1/79 was found in 12 mink from August to November. HI antibody titers of > 1: 128 were found in 8 out of 42 mink at the first examination in July and August.These findings suggest that infection with these human influenza viruses was present in this flock during the period from birth (the beginning of May) to autumn, the non-prevalent season in man. Attempts at virus isolation were unsuccessful.     

Evaluation of the polymorphonuclear cell functions of bottlenose dolphins

The functions of polymorphonuclear cells (PMN) are the important non-specific defense mechanisms in the immune system. Especially marine mammals are protected by these mechanisms from the aquatic environment with a large variety of microorganisms. Therefore, we examined the PMN functions of bottlenose dolphins in order to obtain the normal ranges and to standardize the techniques. PMNs were isolated by using lymphocyte isolate solution whose density was 1.077; superoxide production was assessed by nitroblue tetrazolium reduction test (NBT) and phagocytosis was tested by using polystyrene latex beads. We showed that the optimal incubation time was 30 min in NBT assay and 12 hr in phagocytosis assay for dolphin PMNs.     

Effects of cyazofamid against Plasmodiophora brassicae Woronin on Chinese cabbage

Cyazofamid (4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonamide) is a novel fungicide with high levels of activity against Oomycetes fungi and Plasmodiophora brassicae Woronin. The effects of cyazofamid were investigated against P. brassicae, the causal agent of clubroot disease in Chinese cabbage. Cyazofamid at 0.3 mg litre(-1) inhibited resting spore germination of this pathogen by about 80%. Cyazofamid at 3-10 mg litre(-1) exhibited fungicidal activity to resting spores of P. brassicae 1-10 days after treatment. When cyazofamid was applied to infested soil, both root-hair infections and club formation caused by P. brassicae were strongly inhibited at 1-3 mg kg(-1) dry soil. These results suggest that cyazofamid directly inhibits resting spore germination, thereby leading to the inhibition of root-hair infection and club formation. Cyazofamid at 3 mg kg(-1) dry soil also exhibited complete control of clubroot disease. The effect of broadcast soil application using a dust formulation at 2 kg AI ha(-1) (equivalent to 1.3 mg AI kg(-1) dry soil), and plug seedling tray application by a suspension concentrate formulation at 200 and 400 mg AI tray(-1) (30 x 60 x 4 cm3) against P. brassicae was also evaluated. Cyazofamid exhibited good efficacy against the pathogen. The sequential treatment including plug seedling tray application with cyazofamid and pre-plant broadcast soil application with the fungicide fluazinam also exhibited excellent levels of control. These results indicate that cyazofamid has a high potential to be an effective fungicide for the control of clubroot disease.     

Characterization of ISPsy2 and ISPsy3, Newly Identified Insertion Sequences in Pseudomonas syringae pv. eriobotryae

Two new active insertion sequences, ISPsy2 and ISPsy3, were isolated from Pseudomonas syringae pv. eriobotryae, the causal agent of stem cankers of loquat trees. ISPsy2 is 1194-bp long, has 16-bp imperfect terminal inverted repeats, and generates a 4-bp target site duplication upon insertion into the selective cartridge of the entrap vector pSHI1063. The nucleotide sequence of ISPsy2 is completely identical with that of the previously identified IS-like element located adjacent to the virulence gene psvA of Pseudomonas syringae pv. eriobotryae NAE6. The single open reading frame of ISPsy2 encodes a 323-amino-acid protein that has similarity to the transposase of the IS5 subgroup of the IS5 family. The ISPsy3 belonging to the IS91 family is 1507 bp in length, does not duplicate its target sequence, GAAC, and presents an 81% sequence homology with IS801 in P. s. pv. phaseolicola. The transposase of ISPsy3 possesses the conserved amino acid motifs found in the rolling-circle replication protein. Southern blot analysis indicated that multiple copies of ISPsy2 and ISPsy3 are present in the genomes of P. s. pv. eriobotryae and some of the other P. s. pathovars tested. Received 16 August 2001/ Accepted in revised form 19 October 2001     

Effects of slaughter age on the levels of free amino acids and dipeptides in fattening cattle

The effects of slaughter age, breed type and postmortem period were studied on the concentrations of free amino acids (FAA) and dipeptides (carnosine and anserine) in Longissimus dorsi muscle of beef. Slaughter age affected the levels of most FAA and dipeptides, and the concentrations of these compounds were significantly lower at 35 months of age than either 15 or 25 months of age. Slight increases were observed at 25 months compared with 15 months, but the differences were not significant except for a few FAA. These tendencies still remained when the concentrations were recalculated on the basis of protein. A significant breed effect was observed for taurine, carnosine and anserine. Almost all FAA were significantly increased during postmortem conditioning, but the level of each FAA fluctuated, and the fluctuations could not be explained statistically by slaughter age or breed differences.