A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

The Role of Vitamins and Feed Enzymes in Combating Metabolic Challenges and Disorders

The potential exists for vitamins and feed enzymes to counteract some metabolic disorders and challenges in poultry. Recent research indicates that increased levels of vitamins E and C are able to reduce ascites-related mortality in broiler chickens. Fatty liver and kidney syndrome is a nutritionally induced metabolic disorder that has been eliminated under practical production conditions due to regular supplementation of poultry diets with biotin. There is also experimental evidence for beneficial effects of 25-hydroxycholecalciferol on the incidence and severity of skeletal and performance anomalies in broilers. Another challenge is the presence of high levels of indigestible and partly soluble nonstarch polysaccharides in many cereals and other plant feed ingredients, resulting in various antinutritive effects. The use of feed enzymes, in particular endo-1,3-1,4-β-glucanase and endo-1,4-β-xylanase, to counteract these adverse effects is well implemented by the feed industry. A typical example of metabolic challenge is the reduced availability of phytate P in plant feed ingredients and the potential for phytate to act as an antinutritional component. Microbial phytase provides a practical solution for improving P availability in plant ingredients. In addition, recent studies suggest phytate can stimulate endogenous losses, and phytase can help prevent those losses.     

家禽病毒性免疫抑制病的分析与对策

免疫抑制引起免疫失败及对病原的易感性增加，是目前乃至今后威胁家禽健康的最危险的潜在因素之一。作者阐述与讨论了几种目前常见的家禽病毒性免疫抑制病的概念、免疫抑制机理及防制措施，以此引起同行对该病的进一步重视。     

Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease

Reasons for performing study: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV‐1, BPV‐2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far. Hypothesis: Given the pathogenic role of BPV‐1 and BPV‐2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection. Methods: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid‐bearing horses and one donkey. Viral load was estimated via quantitative real‐time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV‐1/‐2 genome for one randomly selected lesion per horse and correlated with disease severity. Results: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0–556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild‐type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2. Conclusions: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity. Potential relevance: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy.     

几种常见冠状病毒感染疾病的病理学特征

冠状病毒(coronavirus)属于单股正链RNA病毒,在自然界中广泛存在,根据血清型特点和基因序列可分为α、β、γ和δ4个种属.目前发现,冠状病毒仅感染脊椎动物,且容易引起人畜共患病的发生,对公共卫生安全具有重大隐患.然而,不同种属的冠状病毒对动物的感染性存在差异,导致患病动物出现不同的病理学特征.现综述几种常见冠...     

Pharmacokinetics of Enrofloxacin and Its Metabolite Ciprofloxacin after Intramuscular Administration of Enrofloxacin in Goats

The pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were investigated in goats after a single intramuscular administration of enrofloxacin at 2.5 mg/kg body weight. The plasma concentrations of enrofloxacin and ciprofloxacin were determined simultaneously by a HPLC method. The peak concentrations (C _max) of enrofloxacin (1.13 g/ml) and ciprofloxacin (0.24 g/ml) were observed at 0.8 and 1.2 h, respectively. The elimination half-life (t _1/2), volume of distribution (V _d(area)), total body clearance (Cl_B) and mean residence time (MRT) of enrofloxacin were 0.74 h, 1.42 L/kg, 1329 ml/h per kg and 1.54 h, respectively. The t _1/2, area under the plasma concentration–time curve (AUC) and the MRT of ciprofloxacin were 1.38 h, 0.74 g h/ml and 2.73 h, respectively. The metabolic conversion of enrofloxacin to ciprofloxacin was appreciable (36%) and the sum of the plasma concentrations of enrofloxacin and ciprofloxacin was maintained at or above 0.1 g/ml for up to 4 h. Enrofloxacin appears to be useful for the treatment of goat diseases associated with pathogens sensitive to this drug.     

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli – expressed recombinant coat protein. Nodavirus-neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus-neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life-stages.     

Evaluation of dietary natural mineral materials as an antibiotic replacer on growth performance,non‐specific immune responses and disease resistance in rainbow trout,Oncorhynchus mykiss

We evaluated the effects of some dietary natural mineral materials as an antibiotic replacer based on growth performance, non‐specific immune responses and disease resistance in juvenile and subadult rainbow trout, Oncorhynchus mykiss. First experiment, juvenile rainbow trout averaging 2.7 ± 0.02 g (mean ± SD) were fed one of the six experimental diets; a basal commercial diet as a control (CON), CON with oxytetracycline (OTC), with yellow loess (YL), with Macsumsuk^® (MS), with Song‐Gang^® stone (SG) and with barley stone (BS) at 0.4% of each diet. At the end of 8‐week feeding trial, weight gain (WG), specific growth rate (SGR), feed efficiency (FE) and protein efficiency ratio (PER) of fish fed YL diet were significantly higher than those of fish fed CON diet. Non‐specific immune responses such as superoxide dismutase (SOD), myeloperoxidase (MPO), lysozyme (LYS) activity and oxidative radical production of fish fed YL diet were higher than those of fish fed CON diet. At the end of 15 days of challenge test with Aeromonas salmonicida, average cumulative survival rate of fish fed YL diet was significantly higher than that of fish fed BS and CON diets. However, there were no significant differences among fish fed YL, SG and OTC diets. Second experiment, subadult rainbow trout averaging 261.5 ± 3.5 g (mean ± SD) were fed one of the four experimental diets for 22 weeks: CON, and CON with OTC, YL or SG at 0.4% of each diet. At the end of feeding, growth performance of fish fed SG and YL diets was significantly higher than that of fish fed CON diet. Non‐specific immune responses in terms of SOD, MPO, LYS and NBT of fish fed SG and YL diets were significantly higher than those of fish fed CON diet. However, there were no significant differences among the fish fed YL, SG and OTC diets. The results indicate that dietary yellow loess or Song‐gang^® stone at 0.4% of diet could replace oxytetracycline in juvenile and subadult rainbow trout.     

Effect of Cinnamon (Cinnamomum zeylanicum) on Growth Performance,Feed Utilization,Whole-Body Composition,and Resistance to Aeromonas hydrophila in Nile Tilapia

Four isonitrogenous (30% crude protein) and isocaloric (4.40 kcal/g) experimental diets were formulated to contain 0.0% (control), 0.5%, 1.0%, or 1.5% cinnamon. The diet containing 1% cinnamon resulted in significantly greater (P < 0.05) specific growth rate (SGR), feed conversion ratio (FCR), feed efficiency ratio (FER), protein efficiency ratio (PER), apparent protein utilization (APU), and energy utilization (EU). In regard to body composition, there was no significant difference in dry matter, protein, lipids, and ash (P > 0.05) contents due to cinnamon supplementation. Blood plasma profile showed an improvement in hemoglobin (HB), red blood cell (RBCS), hematocrite (PCV), total protein, and total lipids, while there was a decrease in creatinin, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose in fish fed 1% cinnamon. Cinnamon was found to have an antibacterial activity antagonistic to Aeromonas hydrophila infection in fish. Performance and FCR improvement translated into a 10% decrease in feed costs associated with the 1% cinnamon diet.     

山西省“十二五”生态建设面临的机遇与挑战

笔者从国外大环境的改变、国内提出的方针政策、省内＂生态兴省＂战略的实施等方面着手,分析了山西省＂十二五＂生态建设面临的机遇,从森林资源总量不足、植被破坏严重,生态环境脆弱,资金投入渠道单一,监测设施滞后、科技支撑不足,重建设轻管护、缺乏公众参与等方面分析了山西省＂十二五＂生态建设面临的挑战。