Employment of 16S rDNA gene sequencing techniques for improved identification of difficult-to-identify bacterial veterinary pathogens

To employ 16S rDNA PCR and automated sequencing techniques to identify a collection of bacterial veterinary pathogens from avian, equine, canine and ovine sources, that have proven difficult to identify, employing conventional cultural techniques. Universal or “broad-range” eubacterial PCR was performed on a collection of 46 difficult-to-identify bacterial isolates originating from clinical veterinary specimens. 16S rDNA PCR was performed using two sets of universal primers to successfully generate a composite amplicon of 1,068 bp, which was sequenced to obtain each isolate’s identity. Sequence analysis was able to identify all isolates examined with relative ease. Where the use of molecular identification methods is justified, such as in outbreak control or bioterrorism in animal health, employment of partial 16S rDNA PCR and sequencing employing universal or “broad-range” 16S rDNA, provides a valuable and reliable method of identification of such pathogens.     

Moulting of a deep-sea galatheid crab (Anomura, Galatheidae) at a depth of 3572 m

The moulting of the deep-sea galatheid crab, Mundopsissp. was observed using a seafloor observatory at a depth of 3572 m in Nankai Trough, western Japan. The duration of the ecdysis was 147 seconds. The crab rested on the substratum until ecdysis was complete. After ecdysis, it flicked and moved away from its exuviae. The newly moulted galatheid crab did not consume its cast exoskeleton.     

Instream distributions and feeding habits of two species of sleeper, <Emphasis Type="Italic">Eleotris acanthopoma</Emphasis> and <Emphasis Type="Italic">Eleotris fusca</Emphasis>, in the Teima River,Okinawa Island

The instream distributions and feeding habits of two species of sleeper, Eleotris acanthopoma and E. fusca, were studied in the Teima River on Okinawa Island, southern Japan. Both adult fishes inhabited the river, but their distribution patterns were found to be different. The distribution of E. acanthopoma was from the tidally influenced area to the lower part of the freshwater area, whereas E. fusca was distributed almost entirely in the freshwater area. They were found to coexist at the upper limit of the tidally influenced area and the lower part of the freshwater area. Their feeding habits were clearly different, although both species were carnivorous. Eleotris acanthopoma fed mainly on crabs in the tidally influenced area and on aquatic snails in the freshwater area, where they coexist with E. fusca. In contrast, E. fusca fed mainly on shrimps in the freshwater area. Their coexistence may result from the difference in their feeding habits.     

Reduction of vitamin K concentration by salivary Bifidobacterium strains and their possible nutritional competition with Porphyromonas gingivalis

AIMS: To assess the possibility that bifidobacteria compete with Porphyromonas gingivalis for their mutual growth factor vitamin K. This study also examined whether salivary Bifidobacterium species decrease vitamin K concentration in the growth medium. METHODS AND RESULTS: Sixty-five strains of Bifidobacterium were obtained from 20 of 24 periodontally healthy subjects. Bifidobacterium dentium was most frequently detected in the saliva of subjects, followed by Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium urinalis. The growth of most Bifidobacterium isolates, except that of B. urinalis, was stimulated by vitamin K. Moreover, the isolates were capable of decreasing vitamin K after incubation, which suggests that bifidobacteria compete with P. gingivalis for vitamin K. In a co-culture, a representative strain -B. adolescentis S2-1 - inhibited the growth of P. gingivalis if it was inoculated in the medium before P. gingivalis. CONCLUSIONS: B. adolescentis S2-1 decreased vitamin K concentration and inhibited the growth of P. gingivalis by possibly competing for the growth factor. SIGNIFICANCE AND IMPACT OF THE STUDY: Salivary bifidobacteria may possess the potential to suppress the growth of P. gingivalis by reducing the growth factor(s) in the environment.     

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.     

<i>Acetobacter okinawensis</i> sp. nov., <i>Acetobacter papayae</i> sp. nov., and <i>Acetobacter persicus</i> sp. nov.; novel acetic acid bacteria isolated from stems of sugarcane, fruits, and a flower in Japan

Eleven strains of acetic acid bacteria were isolated from stems of sugarcane, fruits, and a flower in Japan. The isolates were separated into three groups, Groups I, II, and III, in the genus Acetobacter according to phylogenetic analysis based on 16S rRNA sequences. The isolates had sequence similarities of 99.8-100% within the Group, 99.3-99.6% to those of the type strains of each related Acetobacter species, and less than 98.4% to those of the type strains of other Acetobacter species. Genomic DNA G+C contents of Groups I, II, and III were 59.2-59.4, 60.5-60.7, and 58.7-58.9 mol%, respectively. The isolates in the Group showed high values of DNA-DNA relatedness to each other, but low values less than 46% to the type strains of related Acetobacter species. A good correlation was found between the three Groups and groups based on DNA G+C contents and DNA-DNA relatedness. All the strains had Q-9 as the main component, and Q-8 and Q-10 as minor components. The isolates in the three Groups did not completely match with any Acetobacter species on catalase reaction, the production of ketogluconic acids from D-glucose, growth on ammoniac nitrogen with ethanol (Hoyer-Frateur medium and Frateur modified Hoyer medium), growth on 30% (w/v) D-glucose, growth in 10% (v/v) ethanol, or DNA G+C contents. On the basis of phylogenetic relationships in the genus Acetobacter and chemosystematic and phenotypic characteristics, the three Groups were regarded as novel species in the genus Acetobacter. Acetobacter okinawensis sp. nov. is proposed for Group I, Acetobacter papayae sp. nov. for Group II, and Acetobacter persicus sp. nov. for Group III.     

Syntheses of α and γ-BHC Alkylthio Analogs

Meso-(1245/36)-1,2,4,5,6-pentachloro-3-methylthiocyclohexane, and (124/356)-1,2,4,5,6-pentachloro-3-methylthio and ethylthiocyclohexanes were prepared from (1234/56)-1,4,5,6-tetrachloro-2,3-epoxycyclohexane (α-BTC cis-epoxide).     

Mitochondrial haplotypes reveal olive fly (Bactrocera oleae) population substructure in the Mediterranean

The olive fly (Bactrocera oleae) is the most important olive tree (Olea europaea) pest. In the Mediterranean basin, where 98?% of its main hosts are concentrated, it causes major agricultural losses, due to its negative effect on production and quality of both olive and olive oil. Previous phylogeographic analyses have established that Mediterranean olive fly populations are distinct from other Old World populations, but did not agree on the specific population substructure within this region. In order to achieve a higher resolution of the diversity of olive fly populations, particularly in Central and Western Mediterranean (home to 70?% of the world production), we comparatively analyzed a set of samples from Portugal in the context of published mitochondrial sequences across the species' worldwide range. Strong evidence of population substructure was found in the Central and Western Mediterranean area, with two clearly separate phylogenetic branches. Together with previously published data, our results strongly support the existence of at least three distinct Mediterranean populations of the olive fly, raise the possibility of additional regional substructure and suggest specific avenues for future research. This knowledge can be instrumental in the development of better management and control strategies for a major pest of Mediterranean agriculture.     

Effect of temperature on the velocity of erythrocyte aggregation

The velocity of the aggregation of human erythrocytes was examined in the range of 5-43 degrees C with a rheoscope combined with a video camera, an image analyzer and a computer. (1) With increasing temperature, the velocity of erythrocyte aggregation induced by fibrinogen, immunoglobulin G and artificial macromolecules (dextran of 70 kDa and poly(glutamic acid) of 50 kDa) increased. However, the relationship between the velocity of erythrocyte aggregation and the temperature was different among these macromolecules. (2) In 70% autologous plasma, the velocity of erythrocyte aggregation was minimum at 15-18 degrees C, and increased at both higher and lower temperatures. (3) The shape of erythrocyte aggregates in 12 mumol/l fibrinogen (containing 770 mumol/l albumin) and in 70% autologous plasma was dependent on temperature: three-dimensional below 15-18 degrees C and one-dimensional (mainly rouleaux) above 15-18 degrees C. However, the shape of aggregates in 27 mumol/l immunoglobulin G (containing 770 mumol/l albumin) was three-dimensional in all temperature ranges. (4) The temperature dependency of erythrocyte aggregation was discussed in terms of the changes of medium viscosity, of erythrocyte properties and of bridging macromolecules.