Detection of ruminant meat and bone meal in feeds by sandwich ELISA with monoclonal antibodies

A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against a synthetic peptide with an amino-acid sequence related to the C-terminus of bovine myoglobin and the whole molecule of sodium dodecyl sulphate (SDS)-denatured bovine myoglobin was adapted for detecting bovine myoglobin in contaminated feeds. The ELISA employed bovine meat extract of a known myoglobin concentration as a calibration standard and had an limit of detection (LOD) of 3.54 ng/ml and an limit of quantification (LOQ) of 11.0 ng/ml corresponding to 0.022% and 0.067% (wt/wt) bovine meat-and-bone-meal (MBM) mixed in 20-fold-diluted feed extracts, respectively. A cut-off threshold of 20.6 ng/ml bovine myoglobin was set to simplify ELISA and facilitate quick assessment of test results without a tedious calibration process. The ELISA was able to detect bovine MBM in artificially prepared model feeds, mixed botanical feeds, mixed botanical feeds with skimmed milk, fish meal, pork meal and pork/chicken meal at 0.1% (wt/wt). It was also able to detect sheep MBM in test feeds, but showed no reactivity to swine MBM, chicken MBM, skimmed milk or gelatine of bovine origin. The advantages of this method are the quick and easy extraction protocol of proteins from test feeds, using 100 mM sodium sulphide and 0.6% sodium dodecyl sulphate in the extraction solution and the effective detection of bovine and sheep MBM at 0.1% (wt/wt).     

Localized pulmonary histiocytic sarcomas in Pembroke Welsh Corgi

Nineteen cases of histiocytic sarcomas in Pembroke Welsh Corgi were examined histopathologically. Focal or multiple masses were detected in the lung or in regional lymph nodes, or in both lung and nodes. All neoplastic lesions had common histological features characterized by the proliferation of pleomorphic histiocytic cells combined with various inflammatory cells. Most of the pleomorphic neoplastic cells were immunopositive for human leukocyte antigen (HLA)-DR and Iba-1. The median survival time for all dogs was 133 days. In the present study, several prognostic factors, such as gender, age, single or multiple lesions, lymph node involvement at the time of diagnosis, surgical resection status and additional chemotherapy, were examined, although none of these factors approached statistical significance. Histiocytic sarcoma must be considered in the differential diagnosis of dogs with pulmonary masses, especially in the canine breed.     

Use of S1 nuclease in deep sequencing for detection of double-stranded RNA viruses

Metagenomic approach using next-generation DNA sequencing has facilitated the detection of many pathogenic viruses from fecal samples. However, in many cases, majority of the detected sequences originate from the host genome and bacterial flora in the gut. Here, to improve efficiency of the detection of double-stranded (ds) RNA viruses from samples, we evaluated the applicability of S1 nuclease on deep sequencing. Treating total RNA with S1 nuclease resulted in 1.5–28.4- and 10.1–208.9-fold increases in sequence reads of group A rotavirus in fecal and viral culture samples, respectively. Moreover, increasing coverage of mapping to reference sequences allowed for sufficient genotyping using analytical software. These results suggest that library construction using S1 nuclease is useful for deep sequencing in the detection of dsRNA viruses.     

Effect of dietary protein quantity and quality on the brain protein synthesis rate in ovariectomized female rats

The purpose of this study was to determine whether the quantity and quality of dietary protein affected the rate of brain protein synthesis in ovariectomized female rats. Two experiments were conducted on the ovariectomized female rats (12 weeks old) given diets containing 20%, 5%, or 0% casein (experiment 1) and 20% casein, 20% soy protein, 20% gluten, or 20% gelatin (experiment 2) for 10 d, respectively. The fractional rates of protein synthesis in the brain declined with a decrease of the quantity and quality of dietary protein. In the brain, the RNA activity [g of protein synthesized/((g of RNA) d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg of RNA/g of protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the rate of protein synthesis in the brain declines with the decrease of the quantity and quality of dietary protein in ovariectomized female rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Zoosporicidal activity of polyflavonoid tannin identified in Lannea coromandelica stem bark against phytopathogenic oomycete Aphanomyces cochlioides

In a survey of nonhost plant secondary metabolites regulating motility and viability of zoospores of the Aphanomyces cochlioides, we found that stem bark extracts of Lannea coromandelica remarkably inhibited motility of zoospores followed by lysis. Bioassay-guided fractionation and chemical characterization of Lannea extracts by MALDI-TOF-MS revealed that the active constituents were angular type polyflavonoid tannins. Commercial polyflavonoid tannins, Quebracho and Mimosa, also showed identical zoosporicidal activity. Against zoospores, the motility-inhibiting and lytic activities were more pronounced in Lannea extracts (MIC 0.1 microg/mL) than in Quebracho (MIC 0.5 microg/mL) and Mimosa (MIC 0.5 microg/mL). Scanning electron microscopic observation visualized that both Lannea and commercial tannins caused lysis of cell membrane followed by fragmentation of cellular materials. Naturally occurring polyflavonoid tannin merits further study as potential zoospore regulating agent or as lead compound. To the best of our knowledge, this is the first report of zoosporicidal activity of natural polyflavonoid tannins against an oomycete phytopathogen.     

Validation of daily increment formation in otoliths of immature and adult Japanese anchovy <Emphasis Type="Italic">Engraulis japonicus</Emphasis>

In order to validate daily increment formation in otoliths of immature and adult Japanese anchovy Engraulis japonicus, three rearing experiments using chemical marking of otoliths were conducted on adult anchovy in summer 2004 and immature anchovy in summer 2005 and in winter 2006. In the two experiments conducted in summer, the number of otolith microincrements between alizarin complexone (ALC) marks showed that microincrements were formed daily. In the summer 2005 experiment, immature anchovy under conditions of reduced daily food rations also showed daily microincrement formation. Average increment width was 0.9 μm in adults and 1.8–3.1 μm in immature anchovy. In contrast, no clear increments were observed between ALC marks on the otoliths from the experiment in winter 2006, and scanning electron microscope (SEM) observations failed to confirm clear increment formation. We consider that low water temperatures (<13–14°C) restricted otolith growth and lowered the contrast between the discontinuous and the incremental zones of the otolith increments. For age estimation of Japanese anchovy, clear increments wider than about 1 μm in the otolith can be regarded as daily increments. However, daily age estimation of immature and adult anchovy that experience low water temperatures in winter may be difficult due to the obscurity of the increments.     

In situ growth and mortality of juvenile Acropora over 2 years following mass spawning in Sekisei Lagoon,Okinawa (24°N)

The growth of Acropora in Sekisei Lagoon was investigated in situ using ceramic coral settlement devices (CSDs) and marine blocks (MBs) with small holes on their surfaces that were deployed prior to a mass spawning event. Acropora that had settled in 10-mm holes on the upper surface of the MBs were found to have grown inwards after 348 days post-mass spawning. After 733 days, the colonies had grown outwards, encrusting the blocks and attaining a maximum diameter of 21.5 mm (3.7 mm). CSDs that had been stacked above each other on frames were separated and observed in situ on 405 and 764 days after mass spawning. After 405 days, the maximum diameter of the encrusting Acropora was 7.6 mm (±2.4 mm), which increased to 19.4 mm (±5.5 mm) after 764 days, with 30% of colonies extending short branches. The length of the branches of Acropora grown on the CSDs fixed to the MBs 3 months after the mass spawning event exceeded the size of the encrusting portion of the colony 629 days after spawning. The mortality of colonies between 1 and 2 years was slight, with the temperatures conducive to coral bleaching that occurred during the study also having only a slight effect.     

Seasonal changes in otolith and somatic growth in age-0 Pacific saury <Emphasis Type="Italic">Cololabis saira</Emphasis>

We evaluated the seasonal changes in otolith and somatic growth of age-0 Pacific saury Cololabis saira in 223 fish collected between June and November 2002. We calculated the age in days of each individual by measuring otolith growth increments under a scanning electron microscope. The age was correlated with body length and otolith radius. We also observed seasonal changes in the rate of increase in body length and otolith radius and in the pattern of otolith growth. Until August, both body length and otolith radius increased with age. Thereafter, the otolith radius continued to increase, whereas the rate of somatic growth decreased. As a result, the ratio of otolith radius to body length increased. After August, the percentage of otoliths with unreadable increments on their edge increased due to the formation of hyaline zones. Otoliths grew both radially and in thickness until July, but gradually stopped growing in thickness after August. Beginning in October, more than 80% of otoliths only grew radially. After August, the otolith not only continued growing but the morphological growth pattern also changed.