Quantitative loop-mediated isothermal amplification method for the detection of <Emphasis Type="Italic">Vibrio nigripulchritudo</Emphasis> in shrimp

Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 10² copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T _t) versus the log of bacterial number. A high correlation coefficient (R ² = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.     

Comparison of morphological and DNA-based techniques for stomach content analyses in juvenile chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>: a case study on diet richness of juvenile fishes

To identify the stomach contents of marine organisms, morphological observations are commonly conducted. However, the results obtained by this traditional method are frequently biased, as it is difficult to detect partially digested, soft-bodied organisms. To resolve this, recent studies have used DNA-based (DNA barcoding) analyses to examine the diet breadth of marine organisms. Here, we compared the prey richness of juvenile fishes using morphological observations and DNA-based analyses, with a focus on juvenile chum salmon Oncorhynchus keta. A higher number of prey taxa were successfully identified using DNA-based analyses than morphological observations. However, we also noticed several shortcomings of the DNA-based analyses, as reported in other diet-analysis studies that used molecular techniques. For example, the degree of digestion among prey taxa might have resulted in differential sensitivity to DNA detection. Additionally, several prey taxa could not be precisely identified, as the sequence data for some of the targeted organismal groups are unavailable in public gene databases. Remarkably, it is also possible that DNA-based analyses detected secondary prey, and therefore, the richness of prey taxa was likely overestimated. Thus, dietary analyses of juvenile fishes need to be carefully conducted, considering the respective advantages and disadvantages of DNA-based and morphological techniques.     

A new primer for 16S rDNA analysis of microbial communities associated with <Emphasis Type="Italic">Porphyra yezoensis</Emphasis>

To construct high-quality 16S rDNA clone libraries for microbial communities associated with Porphyra yezoensis and to minimize the detection of rDNA from leafy gametophytes of P. yezoensis, we designed a new 16S rDNA universal primer (75F). Of the clones prepared using 75F, which was designed to distinguish between bacteria and P. yezoensis, 95% were classified into four groups, namely, β-proteobacteria, γ-proteobacteria, Lentisphaerae, and Flavobacteria. PCR-based analysis of the 16S rDNA primer constructed in this study can be used to implement 16S rDNA-based methodologies for the investigation of microbial community composition and diversity related to the Porphyra group.     

IgE reactivity to milk components in dogs with cutaneous adverse food reactions

We investigated the IgE reactivity to crude and purified milk antigens in the sera of 112 dogs with cutaneous adverse food reactions (CAFRs). Of the 112 dogs, 33 (29%) had specific IgE for crude milk antigens. In the dogs with milk-specific IgE, IgE reactivity to casein, bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin, and bovine IgG were 81%, 85%, 39%, 27%, and 35%, respectively. Casein and BSA may be important allergens in dogs with CAFRs. Some canine vaccines contain casein hydrolysate as a stabilizer and the pooled serum with anti-casein IgE showed IgE reactivity to the vaccines containing it. Information about IgE reactivity to casein in dogs with CAFRs could be useful for predicting adverse reactions to the vaccines including casein hydrolysate.     

Effect of cultivation methods on nutritional enrichment of euryhaline rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

This study aimed at comparing fatty acid contents of rotifers cultured with different methods after nutritional enrichment in order to evaluate the rotifer quality produced by these methods. Rotifers were cultured using either a batch or a continuous culture. From the batch culture, three experimental subpopulations were used, sampled from the culture at 1, 24, and 48 h after rotifer inoculation. The continuous culture was performed with two tanks; one was for cultivation with continuous feeding and water supply (cultivation tank), and another was for harvesting from the cultivation tank by overflow (harvest tank). From the continuous culture, two subpopulations were used: one from the cultivation and one from the harvest tank. Nutritional enrichment was performed after each culture. Each population was enriched with Nannochloropsis oculata or a commercial enrichment diet. When the enrichment was performed with N. oculata on populations at 24 h after inoculation originating from either of the two tanks of continuous culture or the batch culture tank, a higher quantity of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) was obtained from the two tanks of continuous culture. The same results were obtained when enrichment diet was used, this time including docosahexaenoic acid (DHA).     

The effects of distance from coral reefs on seagrass nursery use by 5 emperor fishes at the southern Ryukyu Islands,Japan

Whilst the importance of seagrass beds as nurseries for coral reef fishes has been clearly recognized, the vast majority of early studies on fish nursery habitats emphasized the close proximity of the latter to coral reefs. To determine the potential nursery role of isolated seagrass beds, we investigated the degree to which juvenile emperor fishes (Lethrinidae) utilized seagrass beds in the presence/absence of adjacent coral habitats at Ishigaki Island (southern Japan), such fishes being known to use seagrass beds as nurseries. Seagrass beds in close proximity to coral habitats (distance between the two habitats of 50–200 m) had greater densities of lethrinid juveniles than those without adjacent coral habitats (2.5–4 km) for 3 different sites investigated, although a significant difference was obtained only for 1 site. Juveniles of Lethrinus atkinsoni, L. obsoletus, L. harak, and L. nebulosus were observed in seagrass beds with and without adjacent coral habitats, whereas L. ornatus occurred only in the former. Overall, most lethrinid juveniles utilize seagrass beds irrespective of the presence of adjacent adult coral habitat, suggesting that both types of seagrass beds would contribute to lethrinid adult populations. Therefore, management efforts for lethrinid populations should be applied not only to contiguous coral-seagrass habitat systems but also to isolated habitats.     

Postoperative improvement of megaesophagus and esophageal motility in a 19-day-old puppy with gastroesophageal intussusception

Canine gastroesophageal intussusception (GEI) is a rare and life-threatening condition that requires prompt diagnosis and treatment. A 19-day-old Siberian Husky with a 4-day history of regurgitation was diagnosed with GEI based on the findings of computed tomography (CT) performed without anesthesia. Endoscopic reduction of intussusception was impossible; thus, surgical reduction by traction of the duodenum was performed. CT revealed improvement of megaesophagus 82 days postoperatively. Eleven months postoperatively, fluoroscopy showed recovery to nearly normal esophageal motility. Two years postoperatively, no clinical signs were reported. CT is useful to diagnose GEI in neonate puppies with poor abdominal fat and to assess the gastric edema and the anatomical association of stomach with other organs. Fluoroscopy is helpful for evaluating postoperative esophageal motility.     

Effects of olive leaf powder supplemented to fish feed on muscle protein of red sea bream

Olive leaf is known to have the high polyphenol content of 6–9% in dry weight. We investigated the effects of olive leaf powder (OLP) supplemented to fish feed on muscle protein of red sea bream (Pagrus major). Fish reared with feed containing 8% OLP for 40 days had 1.4 times higher myofibril content and 2.2 times higher acid-soluble collagen content than fish reared with control feed for the same period. On the other hand, sarcoplasmic protein content and collagenase activity of the muscle were almost the same between the control fish and OLP-diet fish. Microstructure observation of fish muscle showed that OLP-diet fish has more rigid endomysium structure than that of the control-diet fish. Since collagen fiber in endomysium is responsible for the texture of the muscle, feeding OLP to aquaculture fish will lead to a harder muscle texture. The present study suggests that OLP is a useful feed additive to enhance the texture of aquaculture red sea bream muscle through strengthening of the collagen structure in the muscle.     

Phytotoxin produced by the netted scab pathogen, <Emphasis Type="Italic">Streptomyces turgidiscabies</Emphasis> strain 65, isolated in Sweden

Streptomyces spp. are a highly diverse group of bacteria most of which are soil-inhabiting saprophytes. A few are plant pathogens that produce a family of phytotoxins called thaxtomins and cause significant economic losses, e.g., by reducing the marketability of potato tubers (Solanum tuberosum). In northern Europe, S. scabies, S. turgidiscabies and S. europaeiscabiei are the most common plant pathogenic species. In this study, a Streptomyces strain isolated from a netted scab lesion on a tuber of potato cv. Bintje in northern Sweden was identified as S. turgidiscabies but was found to differ in the genomic region carrying genes required for thaxtomin biosynthesis. Our results showed that the strain did not produce thaxtomin but rather phytotoxin fridamycin E, which is an anthraquinone novel to plant pathogenic Streptomyces spp. Fridamycin E was shown to reduce or inhibit sprouting of potato microtubers in vitro. While fridamycin E is known to have antibiotic activity against Gram-positive bacteria, the inhibitory activity of fridamycin E on plant growth is a novel finding.     

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC₅₀ of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC₅₀s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.