Identification,partial characterization,and use of grey mullet ( Mugil cephalus ) vitellogenins for the development of ELISA and biosensor immunoassays

Vitellogenin (Vtg) has proven to be a sensitive and simple biomarker in determining sex, sexual maturity, and xenoestrogenic effects in fish. Thus, our investigation has been focused on identification, partial characterization, and quantification of grey mullet (Mugil cephalus) Vtg through the use of a variety of biochemical and immunological analytical techniques. Mullet is considered both a promising aquaculture candidate and an important species for improving sediment quality in polyculture systems. In the first part of this work, grey mullet Vtg was purified from plasma of 17β-estradiol (E2)-induced male fish by a one-step chromatographic protocol, and partially characterized. Specific polyclonal antibodies were then raised against the mullet Vtg, and both an indirect ELISA and an optical immunosensor were set up and validated to quantify plasma Vtg. The indirect ELISA and the optical immunosensor assay developed showed linear measuring in the range 56.8–1047.1 ng mL−1 and 70–739 ng mL−1 Vtg concentrations in standard solutions, respectively. The results obtained suggest that the indirect ELISA allows Vtg detection over a wide dynamic range, thus resulting more suitable for rapid and sensitive sample screening. Therefore, we suggest that the direct immunosensor is a promising tool which needs more investigation to improve the sensitivity.     

冰糖橙果实品质无损伤在线检测分级技术的建立与应用

【目的】冰糖橙果实外观分级与内部品质不一致极大地影响了其市场声誉,建立冰糖橙果实品质分级标准和果实快速规模化无损伤品质检测分级技术,并进行在线应用,为快速分选不同糖酸度冰糖橙产品提供技术支持。【方法】在利用常规果实品质分析方法对主产区的冰糖橙果实进行多年品质分析的基础上,找出果实外观和内在品质的相关性,确定不同果型横径与糖酸度含量的关系,形成冰糖橙果实分级标准。在此基础上,采集710-960 nm波段近红外透射光谱建立果实无损伤检测模型,利用常规品质分析数据对原始光谱反复进行校正并验证,确定无损伤检测的准确性,并在分级线上进行应用;调查统计应用无损伤检测生产线分级后各级冰糖橙的产量、销售单价和销售额。【结果】（1）根据果实横径大小进行初选分级,68-74 mm为大果型,62-67 mm为中果型,56-61 mm为小果型,再按果实的糖度（可溶性固形物Brix度）和酸度不同将冰糖橙划分为4个等级：糖度≥14、酸度≤0.4%为特级果;12≤糖度＜14、酸度≤0.4%为一等果;糖度≥14、0.6%≥酸度＞0.4%为二等果;糖度＜12、酸度≤0.4%为合格果。通过反复矫正,建立了冰糖橙果实品质分级标准。（2）通过多次进行单果糖度和酸度的分析矫正,建立了冰糖橙果实糖度和酸度的检验线。第1次建立的冰糖橙糖度检量线检测范围为10.1-14.9 Brix,再利用常规品质分析验证无损伤检测结果,合格率仅为26%。第2次建立的糖度检量线延长高糖检测范围,为10.1-16.2 Brix,经验证后合格率达90%。第3次建立的糖度检量线扩大低糖检测范围,为9.8-16.2 Brix,经验证后合格率为90%。第1次建立的冰糖橙酸度检量线检测范围为0.1%-1.26%,再利用常规品质分析验证无损伤检测结果,合格率仅为64%。第2次建立的酸度检量线延长高酸检测范围,为0.1%-1.37%,经验证后合格率达94%。第3次建立的酸度检量线继续扩大高酸检测范围,为0.1%-1.53%,经验证后合格率为92%。（3）新建立无损伤糖酸度校正和验证模型,糖度有效检测范围为8.7-15.1 Brix,酸度有效检测范围为0.14%-2.0%,经验证后糖度合格率达到98%,酸度合格率为98%。应用该分级技术,分选速度达到10个冰糖橙/秒,品质分级后最高单价48元/kg,年产量4 500 t,实现产值9 122万元。【结论】研究结果预测糖酸度准确,测量范围全面涵盖冰糖橙商品果的糖酸度,能大批量、快速、高质量和无损伤在线鉴别冰糖橙果实内在品质,结合外观品质分级标准,分选出内外品质均一的产品。     

Defining output-based standards to achieve and maintain tuberculosis freedom in farmed deer, with reference to member states of the European Union

Within the European Union (EU), detailed legislation has been developed for cattle, but not deer, to minimise disease risks associated with trade in animals and animal products. This legislation is expressed as input-based standards, providing a detailed outline of the activity required (for example, testing of animals and application of defined control measures), on the expectation that an adequate output (for example, confidence in freedom) will be achieved. Input-based standards are at odds with the increasing shift towards output-based standards, particularly in OIE rules governing international trade. In this paper, we define output-based standards to achieve and maintain freedom from tuberculosis (TB) in farmed deer, with reference to EU member states. After considering the probability of freedom achieved for cattle under existing EU legislation, we defined a ‘free farmed deer holding’ as one with a probability of freedom from infection of at least 99%. We then developed an epidemiological model of TB surveillance systems for deer holdings, incorporating different surveillance strategies, including combinations of diagnostic tests, and a variety of different scenarios relating to the potential for introduction of infection. A range of surveillance strategies were identified to achieve and maintain a free farmed deer holding, and worked examples are presented. The surveillance system sensitivity for varying combinations of screening and confirmatory tests in live animals, animals at slaughter and on-farm deaths is also presented. Using a single test at a single point in time, none of the TB tests routinely used in farmed deer is able to achieve an acceptable probability of TB freedom. If repeat testing were undertaken, an acceptable probability of TB freedom could be achieved, with differing combinations of the surveillance system sensitivity, frequency of testing and risk of introduction. The probability of introduction of infection through the importation of infected deer was influenced by the use of a pre-movement test (assumed 90% test sensitivity and negative test results), the TB prevalence in the source herd and the number of animals imported. A surveillance system sensitivity of at least 81% was achieved with different combinations of annual live animal surveillance and surveillance of animals at slaughter or on-farm deaths. This methodology has broad applicability and could also be extended to other diseases in both deer and other species with relevance to trade in animals and animal products.     

Paraneoplastic leukocytosis in a dog with a renal carcinoma

A 7-year-old male German Shepherd dog in poor body condition had a 3-month history of intermittent hematuria. Nonregenerative anemia, mild leukocytosis, marked hypoalbuminemia, and hematuria were observed. Subsequently, marked neutrophilia and moderate monocytosis were noted; anemia, hypoalbuminemia, and hematuria persisted; and the dog developed disseminated intravascular coagulation. Ultrasonographic examination of the abdomen revealed the presence of an enlarged and irregularly shaped right kidney with a large area of cavitation, and a nephrectomy was performed 30 days after initial examination. Cytologic examination of fine-needle aspirates and imprints of the right kidney revealed a neoplastic cell population suggestive of renal carcinoma. The histopathologic diagnosis was chromophobic cystic-papillary renal carcinoma. The tumor cells expressed granulocytic/macrophage-colony-stimulating factor (GM-CSF), detected by immunohistochemical staining, and elaboration of GM-CSF by the tumor may have mediated the leukocytosis in this dog. Following excision of the tumor, neutrophil and monocyte counts were only mildly increased. The dog died 135 days after initial presentation, and a necropsy was not permitted. Paraneoplastic neutrophilic leukocytosis is an uncommon finding and may be caused by elaboration of CSF by neoplastic cells.     

Effect of long term addition of composts and poultry manure on soil quality of citrus orchards in Southern Italy

A 6-year study was conducted in an organically managed orange orchard located in Sicily (Southern Italy) to assess the effect of compost and organic fertiliser utilisation on soil quality. Adopting a randomised-block experimental design with three replicates, four treatments were carried out. In treatments 1 and 2, two different composts (C1 from distillery by-products and C2 from livestock waste) were applied. The plots of treatment 3 were fertilised using dried poultry manure. The control treatment was fertilised by mineral/synthetic fertilisers. In order to verify the hypothesis that composts and organic fertilisers improve soil fertility, soil quality was evaluated by selecting dynamic soil parameters, as indicators linked to C and N cycles. Total organic C, total N, C/N ratio, humified fraction, isoelectric focussing (IEF) of extracted organic matter, microbial biomass C, potentially mineralisable N under anaerobic conditions, potentially mineralisable C, C mineralisation quotient and metabolic quotient were determined for each sample. Moreover, the Community Level Physiological Profile (by Biolog technique) was defined, calculating derived functional biodiversity and versatility indexes. Parameters related to IEF and potentially mineralisable C showed significant differences among the treatments. Moreover, total C, total N and humification parameters tended to increase, while no differences were observed in biodiversity indexes. On these findings, it was concluded that composts and poultry manure only weakly affected soil properties, though they increased soil potentially available nutritive elements to crops.     

Occurrence of the temperature-sensitive hemagglutinin among avian Escherichia coli

In this study, we determined the occurrence of the tsh gene among 305 Escherichia coli isolates from chickens by means of the polymerase chain reaction and agglutination of chicken erythrocytes; 200 of those isolates were obtained from chickens with colisepticemia, 52 isolates were from lesions of cellulitis, and 53 were from feces of normal chickens. The tsh gene was found in 79 (39.5%) isolates from colisepticemia, in 10 (19%) cellulitis-derived E. coli isolates, and in two (3.8%) fecal isolates. Among the tsh+ strains, 68 (86%) isolates from colisepticemia and nine (90%) from cellulitis agglutinated chicken erythrocytes in the presence of mannose, after growing the strains on colonization factor antigen agar plates at 26 C, which confirms a correlation between mannose-resistant hemagglutination and expression of hemagglutinin Tsh. These results show, for the first time, the presence of the gene tsh in cellulitis-derived E. coli isolates; the high frequency of this gene among avian pathogenic E. coli isolates in Brazil indicates that its putative role as a virulence factor should be studied more thoroughly.     

A new acylated quercetin glycoside from Ranunculus lanuginosus

A new acylated quercetin glycoside, quercetin 3-O-(2-t-p-coumaroyl)-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside-7-O-beta-D-glucopyranoside (1), together with the known 3-(3,4-dihydroxyphenyl)lactic acid (2) were isolated from Ranunculus lanuginosus leaves and identified on the basis of detailed spectral analysis, including 2D-NMR spectrometry and ESI-MS.     

Exotic and invasive species compromise the seed bank and seed rain dynamics in forests undergoing restoration at urban regions

The control of exotic and invasive species in areas undergoing recovery is a challenge for ecological restoration and this problem may be even greater in urbanized areas. This study evaluated the seed bank and seed rain of a 5-year-old riparian forest located at the metropolitan region of Belo Horizonte in southeast Brazil, and verified changes over time in the abundance and species richness and whether planted species already were providing propagules to the site. At the forest, fifteen parcels of 100 m² were distributed randomly and soil samples were collected thrice (October 2011, March 2012 and July 2012) to evaluate the seed bank. Seed traps were installed in nine parcels to collect propagules over 1 year. Propagules were segregated and germinate at greenhouse. Species richness and abundance were estimated, and variance and similarity were quantified. In total, 86 species were recorded in the seed banks, 41.9% natives and 33.7% exotics, with the predominance of herbs (87.2%) and only 7% of trees. Species classified as weeds amounted to 52.3%. In the seed rain 642 seedlings germinated but they belonged to only 10 species, half of them were exotics. Regarding life forms, half of the species were arboreal and dominated the samples, highlighting the exotic species Melia azedarach. The species richness of the seed bank did not differ by season, unlike the seed rain which varied significantly by month. Seed bank and seed rain species had a very low floristic similarity with the list of planted species, suggesting that few planted trees had dispersed or were stocking propagules. Many non-planted species were recorded, mostly exotics and/or invasive that were probably derived from the surrounding urban matrix. These might interrupt the successional dynamics of the restoration forest and compromise the restoration process in the medium and long-terms. Our results highlight: (1) the importance of monitoring areas undergoing restoration; (2) that restoration methods and management actions directed to control exotic and invasive species are particularly important for sites in urban regions; and (3) the relevance of conserving forest fragments as sources of propagules for nearby areas.     

A new kaurane diterpene dimer from Parinari campestris

A new kaurane diterpene dimer, 15-oxozoapatlin-13alpha-yl-10'alpha,16'alpha-dihydroxy-9'alpha-methyl-20'-nor-kauran-19'-oic acid gamma-lactone-17'-oate (1), together with the known 13-hydroxy-15-oxozoapatlin (2), 10alpha,13alpha,16alpha,17-tetrahydroxy-9alpha-methyl-15-oxo-20-nor-kauran-19-oic acid gamma-lactone (3), 2alpha,10alpha,13alpha,16alpha,17-pentahydroxy-9alpha-methyl-15-oxo-20-nor-kauran-19-oic acid (19,10)-lactone (4), 3alpha,10alpha,13alpha,16alpha,17-pentahydroxy-9alpha-methyl-15-oxo-20-nor-kauran-19-oic acid gamma-lactone (5), and 1beta,16alpha,17-trihydroxy-ent-kaurane (6) were isolated from the leaves of Parinari campestris and identified on the basis of detailed spectral analysis, including 2D NMR spectrometry and ESI-MS.