Comparison of polymerase chain reaction methods for the detection of <Emphasis Type="Italic">Theileria equi</Emphasis> infection using whole blood compared with pre-extracted DNA samples as PCR templates

Rapid, efficient, and reproducible procedures for isolating DNA before PCR gene amplification are essential for the diagnosis of piroplasms. In this study, we evaluated the ease and reliability of detecting Theileria equi by PCR using pre-extracted DNA samples (by QIAamp DNA Mini Kit and phenol-chloroform methods) compared with blood spotted on FTA cards as PCR templates. Although minimal variations in limit of detection were observed among the methods compared, overall, the use of pre-extracted DNA samples and blood spotted on FTA cards had comparable detection limits. These results indicate that T. equi infection can be efficiently detected directly from FTA cards by PCR without the need for pre-extraction of DNA from blood samples.     

Phylogenetic analysis of Japanese Armillaria based on the intergenic spacer (IGS) sequences of their ribosomal DNA

To determine the phylogenetic positions of two new species, Armillaria jezoensis and Armillaria singula, and one new subspecies, Armillaria mellea suhsp. nipponica, the nucleotide sequences of the intergenic spacers (IGS) of their ribosomal DNA were investigated, and compared with those of tour other Armillaria species from Japan, and those of nine Armillaria species from Europe and North America. We conclude that Armillaria jezoensis, and Armillaria singula belong to the Armillaria gallica cluster as Armillaria cepistipes, Armillaria gallica and Armillaria sinapina from Japan. Two isolates of Armillaria ostoyae from Japan were placed within the Armillaria ostoyae cluster. Armillaria mellea subsp. nipponica had an IGS sequence as long as the IGS of Armillaria mellea from Europe and North America. However, the IGS sequences of Armillaria mellea subsp. nipponica, whose basidium base lacks a clamp connection could not be satisfactorily aligned with the IGS sequences of other species possessing this morphological feature.     

Codend selectivity for jack mackerel and whitefin jack and unequal split parameter estimates observed in trouser trawl experiments

Codend selectivity for the jack mackerel Trachurus japonicus and the whitefin jack Kaiwarinus equula were evaluated based on data from trouser trawl experiments carried out in the East China Sea, using a test codend of 60 mm diamond mesh and a control codend made of minnow net with a square mesh of 9 mm bar length. Between-haul variations in parameters and the mean selection curves were tested with the catch data in the SELECT approach, and then the model of between-haul variation in the split parameter with the mean selection curve was chosen as the best fit using Akaike’s information criterion model selection. The 50% retention lengths and the selection ranges were 11.4 and 3.36 cm for jack mackerel and 8.83 and 0.93 cm for whitefin jack, respectively. The selection curve for whitefin jack was sharp, whereas that for jack mackerel was relatively wide. As the estimated split parameters indicated, about 80% of the whitefin jack entered the control codend, but 85 and 90% of the jack mackerel entered the control codend in the second and third hauls, respectively. The inequality in the split parameter is discussed from the viewpoint of the animal’s swimming behavior and water movement based on underwater video observations.     

Sponge-associated fungi from a mangrove habitat in Indonesia: species composition,antimicrobial activity,enzyme screening and bioactive profiling

There is no report of diversity, biological properties and bioactive compounds of sponge-associated fungi from Indonesia’s mangrove ecosystem to date. This study was designed to isolate sponge-associated fungi from a mangrove ecosystem in Mangkang, to screen the antimicrobial and extracellular enzyme properties of the isolates, characterize the biologically promising isolates using molecular approaches, and profile the secondary metabolites using phytochemical and thin-layer chromatography (TLC) analyses. An unidentified sponge that lived in association with mangrove roots was collected from Mangkang. Total of eight associated fungi were isolated from the sponge. Among all isolates, only two fungi SPMKF 1 and SPMKF 6 produced extracellular amylase, another two fungi SPMKF 4 and 5 showed antibacterial activity against MRSA, and only one fungus SPMKF 8 was able to produce extracellular amylase and show antimicrobial activity against ESBL E. coli, Salmonella enterica ser. Typhi strain MDR and C. albicans, while SPMKF 2, SPMKF 3 and SPMKF 9 did not show any biological properties. The result of genetic characterization proved that SPMKF 1 was Cladosporium tenuissimum, SPMKF 4 was Eutypella sp., SPMKF 5 was Lasiodiplodia theobromae, SPMKF 6 was Fusarium keratoplasticum and SPMKF 8 was F. solani. Furthermore, an amylase gene was detected in fungi SPMKF 1, 6 and 8 while among the BGC, only NRPS genes were detected in SPMKF 4, 5 and 8. Interestingly, several same metabolites indicating the same retention factor (Rf) values in TLC were detected in the fungal crude extracts by TLC.     

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell⁻¹·day⁻¹) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (R_tox) was not affected by temperature. During EG, the average specific toxin production rate (µ_tox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day⁻¹ at 30 °C, and then decreased. Over the entire growth span, µ_tox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µ_tox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.     

Isolation and pathogenic characterization of an OB1 variant of Babesia rodhaini which has a glycophorin A-independent pathway to murine red blood cells

Recent studies using several Babesia spp. have demonstrated that these species commonly recognize host sialic acids of red blood cells (RBCs) for their invasion. Glycophorin A (GPA), which is a major carrier of the sialic acids on RBCs, is a possible invasive receptor for Babesia parasites. In the present study, a variant of Babesia rodhaini was successfully isolated from a GPA homozygous knockout (GPA^−/−) mouse infected with an Australian strain of B. rodhaini which had originally been unable to replicate in GPA^−/− mice. The isolated parasite (designated as an OB1 variant) caused lethal infection to wild-type mice, as in the case of the parent Australian strain. However, although the growth of the OB1 variant in GPA^−/− mice was comparable with that in wild-type mice at 1–4 days after infection, the growth was significantly inhibited from day 5 onward, leading to the eventual survival of the GPA^−/− mice. Resistance of GPA^−/− mice against OB1 infection was lost by splenectomy, although the cytokine responses to the infection in the sera of GPA^−/− mice were similar to those of wild-type mice. The autoantibody levels to GPA-defective RBCs in the sera of GPA^−/− mice were depressed at a lower level at 0–2 days after infection than those of wild-type mice, while the levels of GPA^−/− mice progressively increased and reached comparable levels to those of wild-type mice at day 3 or later. These results indicate that the isolated OB1 variant has a GPA-independent invasion pathway into murine RBCs and suggest that the resistance of GPA^−/− mice against infection with the OB1 variant may be attributed to the effective clearance of the parasitized RBCs lacking GPA in the spleen, possibly mediated by preferential autoantibody binding to the RBC membrane.     

Molecular phylogenetic relationship of Tetraodon pufferfish based on mitochondrial DNA analysis

Pufferfishes belonging to the genus Tetraodon form one of the largest groups in the family Tetraodontidae. This group consists of more than 20 species distributed across freshwater, as well as brackish and coastal waters, of Africa and Southeast Asia. However, their phylogenetic and evolutionary relationships are still ambiguous. In the present study, mitochondrial genes encoding 16S rRNA and cytochrome b were sequenced for 17 Tetraodon species collected from various areas mentioned above. Supermatrix analysis based on the complete sequences of the two genes from Tetraodon species was performed using a tree with 50 tetraodontid species (including 7 Tetraodon species) as a backbone. The obtained phylogenetic tree classified Tetraodon species into three groups, correlating well with their habitats, such as Asian freshwater, Asian brackish water, and African freshwater. We also showed that salinity tolerance of Asian freshwater species T. cochinchinensis was apparently lower than that of Asian brackish water species T. nigroviridis, suggesting that the speciation of Tetraodon species in the Asian freshwater group was caused by the molecular evolution associated with osmotic regulation.     

Density estimation of the giant jellyfish Nemopilema nomurai around Japan using an alternative modified detection function for left truncation in a line transect survey

Information on the distribution and bloom scale of the giant jellyfish Nemopilema nomurai is necessary to understand the impact on fisheries. In October 2009, we had an opportunity for visual counting of N. nomurai around Japan from a research/training vessel. To take into account possibly severe missed detection of N. nomurai in the close vicinity of the vessel, detection functions were modified by introducing an additional parameter δ expressing the proportion of detectable N. nomurai within a threshold perpendicular distance. Some covariate effects in the detection process were also investigated. The δ-modified hazard-rate detection function with δ variation parameter of jellyfish body size was chosen as the best-fit one by AIC model selection. The results suggested that a higher proportion (>30 % or more) of N. nomurai were undetected in the vicinity of vessel. The δ-modified model had an advantage of no loss of left-truncated data and provided better estimation of density with the smaller coefficient of variation than the conventional left truncation approach. It is applicable to other line transect surveys, e.g. aerial surveys. High densities of N. nomurai were found in the central part of the Sea of Japan and off Iwate Prefecture.     

Secretome analysis of the basidiomycete <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> grown on ammonia-treated lignocellulosic biomass from birch wood

Ammonia pretreatment is a promising technique for enhancing enzymatic saccharification of lignocellulosic biomass. However, an enzymatic cocktail suitable for the breakdown of pretreated biomass samples is still being developed. The basidiomycete Phanerochaete chrysosporium is a well-studied fungus with regard to bioconversion of lignocellulosic biomass. In the present work, we analyzed proteins secreted by P. chrysosporium grown on untreated and ammonia-treated birch wood meal. Fungal growth, xylanase activity, and extracellular protease activity increased in the media containing the ammonia-treated biomass; however, cellulase production decreased compared to that observed in the untreated biomass. Secreted extracellular proteins were separated by two-dimensional electrophoresis and identified by liquid chromatography ion–trap mass spectrometry. Fifty-five spots corresponding to secreted proteins were chosen for further analysis. In the culture with ammonia-treated biomass, the relative concentration of a xylanase belonging to glycoside hydrolase (GH) family 11 increased, while acetyl xylan esterases belonging to carbohydrate esterase family 1 decreased. Moreover, GH family 10 xylanases were promoted proteolysis in the culture of ammonia-treated biomass, leading to the loss of family 1 carbohydrate-binding modules. These results indicated that P. chrysosporium produced enzymes related to the recognition of structural changes on xylan with de-acetylation and introduction of nitrogen by ammonia pretreatment of birch wood meal.     

Comparisons among MRI signs,apparent diffusion coefficient,and fractional anisotropy in dogs with a solitary intracranial meningioma or histiocytic sarcoma

Although MRI has become widely used in small animal practice, little is known about the validity of advanced MRI techniques such as diffusion‐weighted imaging and diffusion tensor imaging. The aim of this retrospective analytical observational study was to investigate the characteristics of diffusion parameters, that is the apparent diffusion coefficient and fractional anisotropy, in dogs with a solitary intracranial meningioma or histiocytic sarcoma. Dogs were included based on the performance of diffusion MRI and histological confirmation. Statistical analyses were performed to compare apparent diffusion coefficient and fractional anisotropy for the two types of tumor in the intra‐ and peritumoral regions. Eleven cases with meningioma and six with histiocytic sarcoma satisfied the inclusion criteria. Significant differences in apparent diffusion coefficient value (× 10^?3 mm²/s) between meningioma vs. histiocytic sarcoma were recognized in intratumoral small (1.07 vs. 0.76) and large (1.04 vs. 0.77) regions of interest, in the peritumoral margin (0.93 vs. 1.08), and in the T2 high region (1.21 vs. 1.41). Significant differences in fractional anisotropy values were found in the peritumoral margin (0.29 vs. 0.24) and the T2 high region (0.24 vs. 0.17). The current study identified differences in measurements of apparent diffusion coefficient and fractional anisotropy for meningioma and histiocytic sarcoma in a small sample of dogs. In addition, we observed that all cases of intracranial histiocytic sarcoma showed leptomeningeal enhancement and/or mass formation invading into the sulci in the contrast study. Future studies are needed to determine the sensitivity of these imaging characteristics for differentiating between these tumor types.