Use of an automatic cell-counting system with LED illumination for enumeration of marine bacteria

ABSTRACT:   The concentration of aquatic bacteria is basic information required to evaluate the status of environments and to assess bacterial contribution to material cycles. However, the standard direct counting method using epifluorescence microscopy (EFM) is tedious and there is variation in the counts among workers. Here an automatic counting system that consists of Bioplorer (BP) and image analysis has been applied to marine bacteria. BP is composed of a light-emitting diode (LED) illuminant, an optical unit, a driving stage and a charge-coupled device camera. In combination with fluorescent labeling and simplified membrane filtration, bacteria are enumerated automatically. The reproducibility, sensitivity and accuracy of the system were tested for natural marine bacteria, in comparison with EFM and flow cytometry (FCM). The counts obtained by BP showed good correlation with those obtained by EFM and FCM methods. The counts were significantly higher in inshore and oceanic samples, indicating high sensitivity with low background noise. Considering its reproducibility, objectivity, ease of use and compact size, BP can be used as a routine tool for counting aquatic bacteria in substitution for EFM or FCM.     

Changes of serum 3-methylhistidine concentration and energy-associated metabolites in dairy cows with ketosis

The present study examined the Serum 3-methylhistidine concentrations and energy-associated variables of 5 healthy Holstein cows and 5 Holstein cows with ketosis. The serum total cholesterol and apolipoprotein B-100 concentrations and lecithin-cholesterol acyltransferase (LCAT) activity of the ketotic cows were lower than those of the healthy cows 14 days before parturition. The serum non-esterified fatty acid (NEFA) concentration on the day of parturition and 3-methylhistidine concentration 14 days after parturition were higher in the ketotic cows. The serum 3-methylhistidine concentration 14 days after parturition was negatively correlated with the serum LCAT activity 14 days before parturition and was positively correlated with the serum NEFA concentration on the day of parturition. Insufficiency of cholesterol metabolism and acceleration of body fat degradation occur before parturition in cows with ketosis, and these characteristics are correlated with acceleration of protein degradation after parturition.     

Effects of Salmonella enterica subsp. enterica serovar Enteritidis vaccination in layer hens subjected to S. Enteritidis challenge and various feed withdrawal regimens

Levels of Salmonella enterica subsp. enterica serovar Enteritidis infection and serum S. Enteritidis antibodies after experimental S. Enteritidis challenge and feed withdrawal were investigated in S. Enteritidis-vaccinated and unvaccinated hens. The results were used to determine whether formalin-inactivated S. Enteritidis vaccination can protect layer hens from S. Enteritidis challenge during feed withdrawal periods. S. Enteritidis infection rates were evaluated from cloacal swabs, eggs and organs. Serum antibody titers to deflagellated S. Enteritidis whole cells (DEWC) and S. Enteritidis FliC-specific 9-kDa polypeptide (SEp 9) were examined by commercial ELISA kits. Cloacal S. Enteritidis recovery rates were lower in the vaccinated than unvaccinated group. Recovery rates of S. Enteritidis from samples increased after feed withdrawal and decreased after re-introduction of feed. S. Enteritidis counts in cloacal swabs were lower in the vaccinated than in the unvaccinated group (P<0.05). More S. Enteritidis-positive eggs were detected from the unvaccinated group. Before S. Enteritidis challenge, the DEWC ELISA titer of the vaccinated group was higher (P<0.05) than the unvaccinated group; subsequently, the S. Enteritidis DEWC ELISA titers of both groups increased gradually. In contrast, only the vaccinated group elicited high SEp-9 antibody titer during post-challenge and feed withdrawal. Additionally, vaccinated hens yielded negative S. Enteritidis isolation rates from egg contents. There is a correlation between negative S. Enteritidis isolation rates and high SEp 9 titers in vaccinated layer hens challenged with S. Enteritidis and subjected to feed withdrawal regimens. These findings suggest the S. Enteritidis vaccination of pullets may protect against S. Enteritidis infection during forced molting and that SEp 9 titer could be a potential indicator of antibody protection against S. Enteritidis infection. The potential of the SEp 9 peptide as an antigen for S. Enteritidis vaccination in the future is worth noting.     

Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi Nuclear Power Plant

The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium (¹³⁷Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a ¹³⁷Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood ¹³⁷Cs and muscle ¹³⁷Cs (Y = 28.0X, R² = 0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high‐risk cattle before they enter the market, and will contribute to food safety.     

Profound Survival Program of Forests in Japan Islands a 40 Year Strategy for Environmental Conservation in Inland China

15 years ago an interim report for an intense environmental program was compiled concerning the location of the islands of Japan at the meteorological down stream of the East Asian Countries. Parts of sulfur dioxide and other air pollutants, those supposed to cause acid deposition are emitted from the eastern parts of the Asian continent, especially in China. The air pollutants flow down to the east to spread over these islands. This acid deposition is projected to cause damage to forest resources of Japan in the future by increasing air pollutants emanating from the continent. A long term project by a research group at Keio University commenced in 1985 to identify ways of preventing this damage to the forests. The group formed the JACK Air Surveillance Network in China and South Korea in order to collect the first precise air pollution data in this region, as well as to identify a reliable partner for the project. On the completion of the JACK project, a highly cooperative group was formed between the researchers of Keio University and those in Chengdu, Sichuan province of China and has effectively worked since 1991. The goal of the project is the formation of an Inland Environmental Information Center in inland China to be accomplished by 2005. To launch a 10 year project by the center, a nation wide campaign is planned to raise the awareness of the population, specifically the lower socio-economic group on the effect of environmental issues. It is suggested that this education campaign take effect in inland China no later than 2025 for the preservation of the forests on the Islands of Japan.     

Does sika deer overabundance exert cascading effects on the raccoon dog population?

Habitat structure in Oku-Nikko, Japan, has been substantially modified by the overabundance of sika deer (Cervus nippon). A deer-proof fence (15.0 km and 900 ha) was constructed in 2001 to conserve vegetation. Although the understory inside the fence is dominated by Sasa nipponica (hereafter, Sasa), an important forage plant for deer, that outside the fence is dominated by Aster ageratoides leiophyllus (hereafter, Aster), an unpalatable plant to deer, and, partly, by bare floor. In this study, we examined the effects of deer on ground-dwelling insects and earthworms, the primary food resources of raccoon dogs (Nyctereutes procyonoides), and, thus, the bottom-up cascading effects of the herbivore on the omnivorous carnivore. Between July and September 2008, we examined the abundance of insects and earthworms by pitfall trapping and hand-sorting methods, respectively, both inside and outside the fence. The abundance of earthworms and insects (Scarabaeidae and Rhaphidophoridae) was higher on forest floors with Aster and/or bare floors outside the fence than on those with Sasa inside the fence. These results indicate that the increasing deer population in this area probably increased the number of these invertebrates outside the fence by modifying understory vegetation and/or depositing dung. Furthermore, the sighting rates of raccoon dogs obtained by spotlight counts were greater outside than inside the fence, suggesting that deer probably exert bottom-up cascading effects on raccoon dogs, at least during May to November, when the invertebrates are predominantly fed on by the omnivorous carnivore.     

Application of highly differentiated SNPs between Japanese Black and Holstein to a breed assignment test between Japanese Black and F1 (Japanese Black x Holstein) and Holstein

Two taurine breeds, Japanese Black and Holstein, established from geographically distant origins and selected for different uses, beef and dairy, were extensively genotyped using a genome‐wide single nucleotide polymorphism (SNP) chip with more than 1000 animals of each breed. The genetic structure was examined by principal component analysis, in which the first principal component clearly separated the two breeds and explained more than 15% of the variance. Highly differentiated SNPs were detected throughout the genome, some of which were clustered within small regions on BTA4 (79.2–79.7 Mb, Btau4.0) and BTA26 (22.2–23.6 Mb). A breed assignment test was developed using 18 highly differentiated SNPs to distinguish Japanese Black from F₁ (Japanese Black × Holstein) and Holstein. The error rate that an F₁ or Holstein animal is misjudged as Japanese Black was expected to be < 0.8%, while the error rate that a Japanese Black animal is misjudged as F₁ or Holstein was expected to be < 0.001%. This test provides a reliable and powerful method to detect breed label falsification in retail beef.     

The influence of topography on the stream N concentration in the Tanzawa Mountains, Southern Kanto District, Japan

The water chemistry of 51 headwater streams was studied in the Tanzawa Mountains, western fringe of Southern Kanto Plain, Japan. The relationships to soil N processes and catchment topography were also evaluated using a geographic information system with fine-scale map data. The average concentration of total dissolved N was 0.74 mg-N L⁻¹, of which 95% consisted of NO₃ ⁻-N. Stream N concentrations were not different among bedrock geologies and among vegetations of the catchments. Stream NO₃ ⁻-N marginally correlated to soil nitrification. Stream NO₃ ⁻-N also tended to be high in areas with steep and south-facing slopes. These results imply that N transport from Tanzawa forest ecosystems is related to hydrological and biological processes associated with catchment topography. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reduction of formaldehyde emission from plywood using composite resin composed of resorcinol–formaldehyde and urea-modified scallop shell nanoparticles

More than 200,000 tons of scallop shells are disposed annually alone in Japan. Nanoparticles derived from scallop shells have the potential to adsorb gaseous formaldehyde; therefore, such discarded shells have now been tested as additive filler in plywood adhesive by mixing high specific surface area, urea-modified shell nanoparticles with a resorcinol–formaldehyde resin. With this procedure, it was found that the emission of formaldehyde from the resulting plywood could be substantially reduced. The urea-modified scallop shell nanoparticles were prepared by two different methods: (1) a dry method where the shells were treated by planetary ball-grinding under ambient conditions—a completely dried powder was obtained after addition of the surface-modifying urea solution; (2) a moist method by treating dry ground shell particles in a wet grinding process with the urea solution, followed by centrifugation to obtain a paste. The specific surface area of the nanoparticles obtained by both treatments was 42 ± 3 m²/g. Measurement of the subsequent formaldehyde emission showed that the addition of the modified scallop shell nanoparticles substantially reduced the formaldehyde emission from plywood. The reduction of the specific mass uptake of urea depends on the nanoparticles which especially was the case when resins containing nanoparticles processed by the moist method were used.     

Bitter gourd suppresses lipopolysaccharide-induced inflammatory responses

Bitter gourd ( Momordica charantia L.) is a popular tropical vegetable in Asian countries. Previously it was shown that bitter gourd placenta extract suppressed lipopolysaccharide (LPS)-induced TNFalpha production in RAW 264.7 macrophage-like cells. Here it is shown that the butanol-soluble fraction of bitter gourd placenta extract strongly suppresses LPS-induced TNFalpha production in RAW 264.7 cells. Gene expression analysis using a fibrous DNA microarray showed that the bitter gourd butanol fraction suppressed expression of various LPS-induced inflammatory genes, such as those for TNF, IL1alpha, IL1beta, G1p2, and Ccl5. The butanol fraction significantly suppressed NFkappaB DNA binding activity and phosphorylation of p38, JNK, and ERK MAPKs. Components in the active fraction from bitter gourd were identified as 1-alpha-linolenoyl-lysophosphatidylcholine (LPC), 2-alpha-linolenoyl-LPC, 1-lynoleoyl-LPC, and 2-linoleoyl-LPC. Purified 1-alpha-linolenoyl-LPC and 1-linoleoyl-LPC suppressed the LPS-induced TNFalpha production of RAW 264.7 cells at a concentration of 10 microg/mL.