Effect of intravenous lidocaine administration on laminar inflammation in the black walnut extract model of laminitis

Reasons for performing study: Laminitis is a serious complication of horses suffering from sepsis/endotoxaemia‐related events. Laminitis in horses and organ injury in human sepsis are both reported to involve inflammatory injury to the laminae/organs including early activation of endothelium and leucocytes leading to emigration of neutrophils into the tissue interstitium. In the black walnut extract (BWE) model, systemic inflammatory events coincide with marked increase in laminar mRNA concentrations of inflammatory genes including proinflammatory cytokines (i.e. IL‐1β, IL‐6), COX‐2, chemokines (i.e. IL‐8) and endothelial adhesion molecules (i.e. ICAM‐1 and E‐selectin). In models of human sepsis, i.v. lidocaine has been reported to decrease leucocyte and endothelial activation, and the expression of proinflammatory cytokines and chemokines. Objectives: To evaluate the effect of i.v. lidocaine therapy on the inflammatory processes documented to occur in the BWE model of laminitis. Methods: Twelve horses were administered BWE and treated immediately with either lidocaine (1.3 mg/kg bwt bolus, followed by 0.05 mg/kg bwt/min CRI, n = 6) or saline (n = 6) for 10 h. At 10 h post BWE administration, laminar samples were obtained under general anaesthesia for assessment of proinflammatory gene expression (using RT‐qPCR) and leucocyte emigration (via CD13 immunohistochemistry). At 0, 3 and 10 h post BWE administration, skin samples were obtained for assessment of leucocyte emigration (via calprotectin immunohistochemistry). Results: No significant differences between groups were noted for inflammatory gene mRNA concentrations (IL‐1β, IL‐6, IL‐8, COX‐2) or for number of leucocytes present within the laminar interstitium or skin dermis. Increased (P<0.05) laminar E‐selectin mRNA concentrations were present in the LD group (vs. SAL group). Conclusions: Continuous administration of i.v. lidocaine does not inhibit inflammatory events in either the laminae or skin in the horse administered black walnut extract. Potential relevance: This work questions the use of continuous i.v. administration of lidocaine as an effective anti‐inflammatory therapy for systemic inflammation.     

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real‐time PCR

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real‐time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real‐time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R² > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real‐time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.     

A novel multiplex RT‐qPCR method based on dual‐labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure – named binary multiplex RT‐qPCR (bmRT‐qPCR) – for simultaneous detection and typing of all four genotypes of VHSV by real‐time RT‐PCR based on dual‐labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.     

Impacts of Bioinoculants Pseudomonas jesenii MP1 and Rhodococcus qingshengii S10107 on Chickpea (Cicer arietinum L.) Yield and Soil Nitrogen Status

Cold-adapted bioinoculants are considered as harbingers of sustainable hill agriculture. Therefore, two previously characterized psychrotolerant diazotrophs, Pseudomonas jesenii MP1 and Rhodococcus qingshengii S10107, were evaluated for their plant growthpromoting potential for chickpea (Cicer arietinum L.) grown under natural field conditions. Comparative analysis of agronomical and biochemical crop parameters revealed the irrelevance of chemical fertilizers for chickpea production; the diazotrophs alone were sufficient to fulfil the crop''s nutritional requirement. However, the integrated use of bacterial strains in combination with urea at 20 kg N ha^-1 as urea was being recommended for higher crop yield and better soil nitrogen status. Quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE)-based soil bacterial dynamics unveiled the persistence of both diazotrophs until the end of the crop maturation period without affecting the native micro-flora. Therefore, these bioinoculants can be explored as natural nitrogen resource, and an additional incentive in their bio-formulation will be a step towards agricultural sustainability.     

Identification and real‐time qPCR quantification of a nitrite‐N degrading bacterial strain in aquatic water

Biological nitrogen removal technology using microbe is an efficient process for nitrogen removal from aquatic water. To determine the key of efficient application on how the effect of probiotics lasting, a nitrite nitrogen (nitrite‐N) degrading 8DO17 strain Pseudomonas was screened and a method for quantification was explored. Real‐time qPCR assays based on the 16S rRNA genes (16S rDNA) were used to quantify the probiotics. The results showed that (i) the nitrite‐N degradation rate of the 8DO17 strain was 99%; (ii) a wide spectrum of pH (7.0–9.0) and concentration of nitrite‐N (0.5–10 mg L^?1) and the highest rate of nitrite‐N degradation near to 100% under optimum conditions (35°C, salinity 30, pH 7.5) was explored; (iii) no significant differences were found in the survival, total haemocyte count, antibacterial activities and bacteriolytic activity of the shrimp between the treatments and the control (P > 0.05); (iv) for the use of real‐time PCR based on 16S rDNA test, detection limit is 10³ DNA copies and an excellent correlation coefficients (R² = 0.999) was obtained; and (v) in the application of real‐time PCR assay in four nitrite‐N degrading samples, highly significant positive correlation relation (P < 0.01) was found between log copy number of 16S rDNA and the rate of nitrite‐N degradation. The results suggest the real‐time PCR is a very rapid and sensitive technique for monitoring the dynamic changes and assessing the effect in practical application of the 8DO17 strain in aquatic water.     

PRRS流行毒株与减毒活疫苗TJM F92株qPCR区分方法的建立与应用

 
为快速准确区分PRRS流行毒株与减毒活疫苗TJM F92株以及对流行毒株进行定量检测,按GenBank中发表的美洲型PRRSV SX 1分离株、弱毒疫苗株NSP2基因缺失部位的不同设计了一对特异性引物,通过RT PCR和体外转录的方法,构建了体外转录RNA作为标准品,并对反应条件和反应体系进行优化,旨在建立一种敏感性高、特异性强、重复性和稳定性良好的qPCR鉴别方法。结果表明,该方法最低能检测出1.0×101拷贝/μL的模板,敏感性比常规PCR高100倍;应用该方法对218份临床样本进行鉴别与定量,qPCR检出率较常规RT PCR高12.9个百分点。研究表明,qPCR鉴别方法的建立实现了对PRRS流行毒株与疫苗毒株的快速区分以及对流行毒株的定量检测,为PRRS的快速诊断提供了依据。     

喷施亚油酸对烟草胁迫抗性的影响研究

为探讨亚油酸能否提高烟草的胁迫抗性,检测了不同浓度的亚油酸喷施后,烟草叶片中抗氧化酶活性,活性氧、MDA、渗透调节物质的含量,以及抗逆和抗病相关基因的表达水平。结果显示,亚油酸处理降低了烟叶中的MDA含量,提高了脯氨酸、可溶性糖、H_2O_2含量和POD活性,增强了rbohD、rbohF、WRKY57、PR-1a、JAZ1和APX基因的表达水平,且大多在10 mmol/L时值最高。表明亚油酸能增强烟叶对胁迫的耐受性,且在10 mmol/L亚油酸处理时效果最佳。     

The effect of additives on the quality of white lupin–wheat silage assessed by fermentation pattern and qPCR quantification of clostridia

The efficacy of different silage additives on different mixtures of white lupin and spring wheat was investigated in four separate trials. The bicrop was harvested 96 days (trials 1 and 2) and 110 days (trials 3 and 4) after sowing. For each maturity stage, two mixtures of white lupin and spring wheat were reformed in the ratios of 1:2 and 2:1 on fresh matter (FM) basis respectively. The crops were treated with formic acid (FA), sodium nitrite–hexamine mixture (NaHe) or homofermentative lactic acid bacteria (LAB). The control silage was made without additive. Additives were not able to improve the quality of white lupin–wheat silage in all trials, compared with untreated silage. The treatment with LAB showed good results only at the first stage of crop maturity with sufficient amounts of water‐soluble carbohydrate in the pre‐ensiling crops. The FA treatment showed elevated butyric acid levels in all trials, which suggests that the FA application level used (4 L t^?1 FM, 100% FA) was insufficient to decrease pH enough for preventing the growth of clostridia and butyric acid fermentation. NaHe was the only additive that was able to inhibit the activity of clostridia in all trials.     

Characterization of rhizosphere microbial communities in continuous cropping maca (Lepidium meyenii) red soil,Yunnan, China

ABSTRACT

Continuous cropping maca systems are widespread in Yunan Province, China. However, the relationships between continuous cropping maca systems and microbes are not well understood. The objective of this study was to determine the effects of continuous cropping maca systems (Maca with 0, 1, 2, and 3 years of continuous cultivation) on the soil microbial community. The results showed that the soil organic matter, total N, total P, and total K contents, as well as maca fresh and dry weight, decreased significantly with increased continuous cropping years. Interestingly, qPCR analysis showed that the bacterial and fungal abundance (DNA levels) decreased and active bacterial and fungal abundance (RNA levels) increased with cropping years from the first to the third cropping (p < 0.05). Moreover, the abundance of actinomycetes in the CK soil was significantly higher than that in the other maca soils. In addition, the continuous cropping system resulted in rich diversity in the fungal structure and had little effects on the bacterial and actinomycete communities. Acidobacteria (50%) and Ascomycota (58.3%) were detected in the continuous cropping maca soils. Based on the present results, continuous cropping of maca not exceeding two years could be optimal to maintain soil nutrition and microbial community.