迟缓爱德华菌鞭毛蛋白FlgJ原核表达及免疫原性

为研究迟缓爱德华菌鞭毛蛋白FlgJ的免疫保护性,本研究利用PCR方法扩增迟缓爱德华菌flgJ基因,构建重组载体pET-32a-flgJ,将其转化大肠杆菌BL21后进行诱导表达,表达产物经SDS-PAGE和Western blot分析显示,重组蛋白大小约54 000;将纯化的重组蛋白免疫小鼠后,以迟缓爱德华菌分离株ET-13进行攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为70%。本研究克隆了迟缓爱德华菌外膜蛋白flgJ基因并表达了相应重组蛋白,免疫小鼠后能够提供一定保护,为重组FlgJ蛋白亚单位疫苗的研制奠定基础。     

迟缓爱德华菌外膜蛋白OmpA原核表达及其免疫原性研究

为研究迟缓爱德华菌外膜蛋白OmpA免疫保护性,本研究利用PCR方法扩增迟缓爱德华菌ompA基因,构建重组载体pET-32a-ompA,将其转化大肠杆菌BL21后诱导表达,表达产物经SDS-PAGE和western blot分析显示,重组蛋白大小约58 ku;将纯化的重组蛋白免疫小鼠后,以迟缓爱德华菌强毒株ET-13攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为55%。本研究克隆了迟缓爱德华菌ompA基因并表达了相应重组蛋白,免疫小鼠后能够提供一定保护,为重组OmpA蛋白亚单位疫苗的研制奠定基础。     

牛多杀性巴氏杆菌LolA蛋白的免疫特性

为研究牛多杀性巴氏杆菌（Pasteurella multocida,Pm）外膜LolA蛋白的免疫特性,对牛Pm LolA蛋白进行原核表达和纯化,再通过动物模型对重组LolA蛋白（rLolA）的免疫特性进行评估。结果表明：本试验成功表达并纯化出rLolA蛋白,其相对分子质量大小约为40 ku;不同动物源性Pm的lolA基因同源性较高,说明该基因高度保守;rLolA蛋白能够引发机体体液免疫,分泌高表达量IgG抗体;rLolA蛋白的免疫原性对小鼠的免疫保护率达55%。本试验明确了牛Pm LolA蛋白的免疫特性,为Pm免疫保护蛋白筛选以及交叉保护性疫苗研发提供了参考。     

日本血吸虫幼虫巨大致死基因片段的克隆、表达及免疫效果分析

为研究日本血吸虫(Schistosoma japonnicum)幼虫巨大致死基因(Lethal giant larvae)的生物学功能,本研究应用荧光定量RT-PCR分析SjLgL基因在S.japonnicum不同发育阶段的表达情况,构建重组表达质粒pET-SjLgL进行诱导表达,对重组蛋白进行western blot鉴定;重组蛋白刺激免疫动物进行免疫保护试验,检测IL-4、IL-10、TNF-α和IFN-γ的变化.结果表明,SjLgL基因在S.japonnicum不同发育时期均有表达,成虫期略高于童虫,雄虫高于雌虫.Western blot鉴定结果显示,该重组蛋白具有良好的抗原性.免疫保护试验结果表明,免疫小鼠分别获得了22.34％的减虫率和55.49％的肝脏减卵率.血清学检测结果显示重组蛋白免疫小鼠后产生了特异性IgG抗体.细胞因子检测结果表明,重组蛋白刺激免疫动物主要诱发产生Th1型的免疫应答.本实验结果表明该重组蛋白在小鼠体内可诱导产生部分免疫保护效果.     

猪肺炎支原体P52蛋白的原核表达及抗血清制备

为表达猪肺炎支原体(Mhp)P52蛋白及制备其血清,本实验利用PCR从Mhp J株扩增P52基因,克隆到表达载体pET-30a中,获得重组质粒pET-P52,经IPTG诱导,通过SDS-PAGE和western blot检测重组蛋白表达及其免疫学活性.以该重组蛋白为抗原免疫兔制备抗血清,采用间接ELISA检测抗血清效价,并利用抗血清通过间接免疫荧光检测P52基因在AD-293细胞中的表达情况.结果表明,PCR扩增目的基因为1 202 bp,SDS-PAGE检测重组蛋白大小约52 ku,其表达量占菌体总蛋白的28%.Western blot检测表明,目的蛋白能与Mhp阳性血清发生特异性反应,具有良好的免疫活性.间接免疫荧光检测到P52基因在AD-293细胞中表达.本研究为构建P52蛋白腺病毒载体疫苗奠定了基础.     

马链球菌马亚种FNEB蛋白的截短表达及其免疫保护性研究

为研究马链球菌马亚种FNEB蛋白的免疫保护性,本研究采用PCR方法从马链球菌马亚种新疆分离株中扩增FNEB基因部分片段,克隆于原核表达载体p ET-30a中,转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达,经SDS-PAGE和western blot分析显示,重组蛋白大小约60 ku,具有良好的抗原性。将纯化的重组蛋白免疫小鼠后,以马链球菌马亚种新疆分离株进行攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为65%。本研究克隆了马链球菌马亚种的FNEB截短基因并表达了相应重组蛋白,免疫小鼠后能够提供较好的保护,为重组FNEB蛋白亚单位疫苗的研制奠定基础。     

禽多杀性巴氏杆菌C48-3株hexABC基因缺失株的构建及其生物学特性分析

为研究荚膜在禽多杀性巴氏杆菌(Pasteurella multocida)致病过程中的作用,本研究利用同源重组基因敲除技术构建禽P.multocida C48-3株荚膜多糖输出蛋白基因hexABC的缺失突变株,并以小鼠为动物模型检测荚膜缺陷对细菌毒力的影响。PCR、RT-PCR和DNA测序结果均表明253 bp的hexC基因下游序列、798 bp的hexB基因全序列和290 bp的hexA基因上游序列完全被四环素抗性基因替代,表明构建了基因敲除突变株C48-3ΔhexABC。电镜观察结果表明突变株荚膜合成能力缺失,对小鼠的致病性试验结果显示突变株毒力基本丧失。本研究获得的无荚膜突变株为进一步研究禽P.multocida的致病机理奠定基础。     

旋毛虫P53ES重组蛋白对小鼠的免疫保护作用

为进一步探讨旋毛虫P53基因原核表达重组蛋白的免疫保护作用,本实验采用纯化的重组蛋白作为抗原免疫小鼠,免疫剂量为50μg/100μL,每隔10d免疫1次,共3次。末次免疫后10d,每只小鼠以200条旋毛虫肌幼虫攻虫。分别检查感染后7d小鼠肠道内成虫数量、体外培养雌虫所产新生蚴数量以及感染后35d肌肉荷虫量。结果显示:小鼠肠道内成虫、新生蚴和肌幼虫的减虫率分别为61.9%、53.45%和71.48%,表明旋毛虫表达P53基因的重组蛋白对小鼠产生了较好的免疫保护效果。     

抗鸭多杀性巴氏杆菌外膜蛋白H单克隆抗体的制备及鉴定

为制备抗鸭多杀性巴氏杆菌(P.multocida)外膜蛋白H(OmpH)单克隆抗体(MAb),本研究以原核表达并纯化的OmpH重组蛋白免疫BALB/c小鼠,将其脾淋巴细胞与SP2/0进行融合,并以重组OmpH蛋白为抗原,通过间接ELISA方法筛选出3株能够稳定分泌抗OmpH的杂交瘤细胞株(1A9、1E9、3G7)。MAb亚类鉴定表明,1A9、1E9和3G7均为IgG1亚类,轻链均为κ链。经western blot和间接免疫荧光检测证明3株MAb均具有良好的特异性。这些抗OmpH MAb的获得,为鸭P.multocida病的快速、有效、敏感的诊断方法建立奠定了基础。     

牛源A型多杀性巴氏杆菌pm0979和pm0442基因编码蛋白对小鼠的免疫保护性研究

为评价牛源荚膜血清A型多杀性巴氏杆菌(Pm) pm0979和pm0442基因编码蛋白的免疫保护效果,本研究以牛源PmCQ2株基因组DNA为模板,经PCR扩增得到pm0979、pm0442全长基因,将其分别克隆到pET-30a、pET-32a载体并转化BL21 (DE3)大肠杆菌,经IPTG诱导表达,分别获得大小约41.8 ku (rPM0979)和21.6 ku (rPM0442)的重组蛋白;将其亲合层析纯化后分别免疫小鼠,以PmCQ2株进行攻毒试验,测定其抗体产生情况和保护效果.结果表明,两种重组蛋白均可以诱导小鼠产生较高水平的抗体;其中rPM0979对小鼠的免疫保护高于rPM0442,保护率可达60％.结果表明,重组蛋白PM0979可作为Pm的一种疫苗候选蛋白,为进一步的疫苗研制奠定了基础.     

Evaluation of the safety of daily administration of capromorelin in cats

Capromorelin is a ghrelin receptor agonist that is FDA approved for appetite stimulation in dogs. The objective of this study was to evaluate the safety of daily oral administration of capromorelin to cats over a range of doses and for an extended period. Two randomized, controlled studies were conducted: in Study 1, cats (n = 6 per group) received placebo or capromorelin at a dose of 9, 15, 30 or 60 mg/kg once daily for 14 days; and in Study 2, cats received capromorelin at 6 mg/kg (n = 8) or placebo (n = 4) once daily for 91 days. Cats were evaluated using clinical observations and clinical pathology test results for both studies, with the addition of postmortem examination in Study 1 and measurements of growth hormone and insulin‐like growth factor 1 in Study 2. Abnormal clinical observations were limited to emesis, hypersalivation, lethargy/depression, head shaking and lip smacking, which occurred more frequently in the capromorelin‐treated groups than in the placebo group. There were no clinically relevant differences in clinical pathology test results between the capromorelin and placebo groups in either study.     

In Vitro Evaluation of the Effects of Selected Plants on the Growth of the Mycelial Form of Histoplasma capsulatum Variety farciminosum in Ethiopia

Epizootic lymphangitis is prevalent in equines in Ethiopia, causing remarkable economic and welfare impacts but often neglected. Lack of effective treatment contributed to its continued occurrence, and hence, search for an effective treatment should be considered a priority area to minimize its impacts. Previous ethnobotanical studies have reported that Curcuma longa, Phytolacca dodecandra, and Datura stramonium were used to treat cutaneous fungal infections and reduce their incidence. The treatment effects of these plants against epizootic lymphangitis should be studied. The in vitro growth inhibitory effects of methanol extracts of the root of C. longa, berry of P. dodecandra, and leaf of D. stramonium were evaluated. Histoplasma capsulatum var farciminosum was isolated from clinical cases of epizootic lymphangitis in carthorses in central Ethiopia. The nested polymerase chain reaction was used to confirm the identity of the isolates. Serial twofold dilutions of the extract of berries of P. dodecandra and leaves of D. stramonium were done in sterile water, whereas dilution of the extract of roots of C. longa was done in dimethylsulphoxide. The effects of the plants on the growth of Histoplasma capsulatum var farciminosum were assessed by agar dilution assay. Culture media with no antifungal agent and media containing ketoconazole served as negative and positive control, respectively. The methanol extract of C. longa showed inhibitory effects at concentrations ranging from 0.07 to 5 mg/mL. Similarly, the methanol extract of P. dodecandra showed growth inhibitory effects at concentrations ranging from 0.156 to 5 mg/mL. That is, the growth inhibitory concentration of C. longa was 0.07 mg/mL, whereas that of P. dodecandra was 0.156 mg/mL. In contrast, D. stramonium showed no inhibitory effect. This preliminary observation showed that methanol extracts of C. longa and P. dodecandra showed inhibitory effects on the growth of Histoplasma capsulatum var farciminosum requiring further repeated in vitro evaluation so as to generate adequate evidence, which would justify in vivo trials.     

不同青贮玉米品种抗病性评价

对10个青贮玉米品种自然发病情况进行调查,并采用病级分类方法对抗病性分类。结果表明:对锈病表现抗性和中抗的品种有3个和5个,分别占供试品种的30%和50%;对大斑病表现抗性和中抗的品种有4个和5个,分别占供试品种的40%和50%;对褐斑病表现抗性和中抗的品种有6个和3个,分别占供试品种的60%和30%;对小斑病表现抗性和中抗的品种有6个和3个,分别占供试品种的60%和30%。多数品种对青贮玉米4种病害抗性表现较好,可从青贮玉米供试材料中选用优良抗病材料用于四川地区种植。     

Evaluation of Oxygen-dependent Immunodefences of the Polymorphonuclear Cells of Some Tropical Ruminants

Activation of polymorphonuclear cells (PMNs) leads to the formation of superoxide, which is in turn dismutated to H₂O₂ by superoxide dismutase (SOD) and is partly responsible for oxygen-dependent microbicidal activity. However, no comparative information is available on the effect of SOD inhibition before PMN activation to allow simulation of the SOD defects that are known to occur in some ruminants. This paper attempts to examine the degranulative and phagocytic responses in buffalo, cattle and goat PMNs exposed to diethyldithiocarbamate, a known SOD inhibitor. The activity of glutathione peroxidase and reductase was increased in the presence of SOD inhibitor. On activation, H₂O₂ production increased significantly (p<0.01), while SOD inhibition before the activation of PMNs caused a significant decline in the production of H₂O₂ (p<0.05) in all the species studied. There was a significant increase (p<0.05) in the phagocytosis of Candida albicans spores by buffalo PMNs activated with opsonized zymosan. Activation of bovine PMNs after exposure to the SOD inhibitor resulted in a significant decline (p<0.05) in phagocytic activity; in the other species, the two values only approached significance. Among the activators, opsonized zymosan caused a significant increase in phagocytic activity as compared to lipopolysaccharide, particularly in the PMNs of buffaloes (p<0.05). Increased fungicidal activity (p<0.05) occurred with opsonized zymosan-activated PMNs of all the species studied. The fungicidal activity was found to decline in PMNs exposed to SOD inhibitor before activation (p<0.05). Interestingly, the phagocytic activity of caprine PMNs was found to be lower than that of PMNs from cattle (p<0.05).     

Effects of the ruminal comminution rate and microbial contamination of particles on accuracy of in situ estimates of ruminal degradability and intestinal digestibility of feedstuffs

Effects of considering the comminution rate (k_c) and the correction of microbial contamination (using ¹⁵N techniques) of particles in the rumen on estimates of ruminally undegraded fractions and their intestinal digestibility were examined generating composite samples (from rumen‐incubated residues) representative of the undegraded feed rumen outflow. The study used sunflower meal (SFM) and Italian ryegrass hay (RGH) and three rumen and duodenum cannulated wethers fed with a 40:60 RGH to concentrate diet (75 g DM/kgBW^0.75). Transit studies up to the duodenum with Yb‐SFM and Eu‐RGH marked samples showed higher k_c values (/h) in SFM than in RGH (0.577 vs. 0.0892, p = 0.034), whereas similar values occurred for the rumen passage rate (k_p). Estimates of ruminally undegraded and intestinal digestibility of all tested fractions decreased when k_c was considered and also applying microbial correction. Thus, microbial uncorrected k_p‐based proportions of intestinal digested undegraded crude protein overestimated those corrected and k_c?k_p‐based by 39% in SFM (0.146 vs. 0.105) and 761% in RGH (0.373 vs. 0.0433). Results show that both k_c and microbial contamination correction should be considered to obtain accurate in situ estimates in grasses, whereas in protein concentrates not considering k_c is an important source of error.     

Effects of oral administration of different dosages of carvacrol essential oils on intestinal barrier function in broilers

Essential oils are widely used in the pharmaceutical, food and cosmetic industries, and many plant essential oils have shown that they have positive effects on broilers nutrition. This experiment was conducted to study the effects of orally administered different dosages of carvacrol essential oils on intestinal barrier function in broiler chickens. A total of eighty 28‐day‐old (1.28 ± 0.15 kg) ROSS 308 broilers were randomly allocated to four groups of 20 replicates each, with one chicken per replicate per cage, and all were fed with the same diet. Four experimental groups were orally administered 0, 200, 300 or 400 μl carvacrol essential oils at 18:00 hr every day during the 2‐week experimental period. As a result of which, the gene expression of the occludin, claudin‐1, claudin‐5, ZO‐1 and ZO‐2 in intestinal mucosa of small intestine (p < 0.05) and the goblet cell content in small intestine epithelium (p < 0.05) were significantly increased; test subjects with 300 or 400 μl carvacrol essential oils reduced the microbial counts of Salmonella spp. and Escherichia coli in the intestines (p < 0.05); Essential oils administration also significantly increased activity of the sucrase (p < 0.05) and lactase (p < 0.05) in intestinal mucosa. In conclusion, the carvacrol essential oils have positive effects on growth performance and intestinal barriers function of broilers; those effects may be related to the dosage, as administration of 300 or 400 μl was more effective than that of 200 μl.     

Effects of oral supplementation of probiotic strains of Lactobacillus rhamnosus and Enterococcus faecium on diarrhoea events of foals in their first weeks of life

Foal first diarrhoea is one of the most prominent problems in the early life of horses. Probiotics might have the potency to prevent or at least diminish neonatal diarrhoea. We hypothesised that the treatment of foals with probiotic strains of Lactobacillus rhamnosus and Enterococcus faecium starting early after birth and then daily over 2 weeks would prevent or mitigate foal heat diarrhoea. The influence of this probiotic treatment on diarrhoea incidence and growth and health performance of young foals was investigated. Thirty‐four foals were randomly allocated to two groups. From day 1 to 14 of life, the foals received either placebo (PG, n = 16) or the probiotic treatment (TG, n = 18). Clinical examination was performed, and the faeces consistency score (FCS, 1–5; with diarrhoea defined by ≤3) was recorded once per day in weeks 1 and 2 and once weekly in weeks 3–8 of life (WL). The body height was measured at birth and after two and eight WL. Diarrhoea occurred in the 1st WL in 19% and 61% of PG and TG foals respectively. In the 1st WL, diarrhoea lasted 0.3 ± 0.8 and 1.6 ± 1.4 days in PG and TG foals respectively. In the 2nd WL, diarrhoea occurred in 94% and 84% of PG and TG foals, respectively, and lasted for 3.0 ± 1.5 and 3.7 ± 1.6 days respectively. At least two periods of diarrhoea developed in 33% and 65% of PG and TG foals respectively. The TG foals grew slightly slower than the PG foals. The results indicated that the probiotic treatment of neonatal foals as performed in this study was not suitable to reduce diarrhoea within the first two WL, because contrary to the hypothesis, the TG foals suffered more frequently and for longer periods from diarrhoea than the PG foals.     

Effect of dietary stable isotopic ratios of carbon and nitrogen on the extent of their incorporation into tissues of rats

This study was conducted to investigate the effect of different dietary ratios of ¹³ C to ¹² C or ¹⁵ N to ¹⁴ N on their relative incorporation into tissues. Eighty male rats were used in two 21-day feeding trials in which they were fed diets with either high δ¹³C levels (δ¹³C = −13.89‰ and δ¹⁵N = 2.37‰ in experiment 1 and δ¹³C = −19.34‰ and δ¹⁵N = 4.73‰ in experiment 2) or low δ¹³C levels (δ¹³C = −17.90‰ and δ¹⁵N = 3.08‰ in experiment 1 and δ¹³C = −21.76‰ and δ¹⁵N = 0.53‰ in experiment 2), meanwhile, the dietary δ¹⁵N levels were designed to two ranks. Blood, liver, adipose and muscle tissues were collected on day 0, 3, 7, 14, and 21 for determination of ¹³ C, ¹² C, ¹⁵ N and ¹⁴ N isotopes. Rat growth rate, antioxidant capacity and metabolic parameters were also assessed. The results indicate that adipose tissue tend to deplete ¹³ C before the stable isotopic ratios achieved final equilibrium. Therefore, feeds with different isotopic signatures had different incorporation rates into tissues. Low dietary ¹³ C levels decreased tissue δ¹³C values whereas high dietary ¹³ C levels did not alter tissue δ¹³C values during the 21-d experiment. Blood δ¹⁵N values were a reliable parameter in assessing the relative contribution of dietary nitrogen to tissues. This study revealed a relationship between dietary isotopic signatures and their incorporation rates into rat tissues. However, more studies are needed to illustrate the mechanism through which dietary isotopic ratios influence the extent of isotopic incorporation into the tissues.     

Influence of a flooding dose of valine on key indicators of metabolic status in the growing pig

A key concern with the flooding dose technique for measuring protein synthesis is that a large dose of amino acid (AA) can potentially change the animals’ hormonal and nutritional status, which in turn can influence protein synthesis. Among stable isotope tracers, 1‐[¹³C]‐valine is the preferred AA for measuring protein synthesis in gut tissue and mucins. A study was conducted to determine the impact of a flooding dose of valine on the metabolic status of pigs. Six barrows [16.5 kg body weight (BW)] were randomly assigned to intravenous infusions of either 150 mm valine (1.5 mmol/kg BW) or physiological saline, following a crossover design. Blood samples were taken 10 min prior to infusion, at the end of infusion, at 10‐min intervals for 60 min post‐infusion, and at 90 and 120 min post‐infusion. Plasma concentrations of insulin, glucose, AA, urea nitrogen and packed cell volume (PCV) were measured. Infusion of valine increased plasma valine concentrations (4129 vs. 582 μm ; P < 0.05) but had no influence on PCV (26.4% vs. 27.2%) and plasma concentrations of glucose (6.0 vs. 5.8 mm ) and insulin (8.2 vs. 8.5 μU/ml; P > 0.10). Plasma urea nitrogen concentration was reduced with valine infusion (8.5 vs. 7.8 mg/dl; P < 0.05). A flooding dose of valine had no impact on plasma concentrations of AA, and specifically branched‐chain AA such as leucine (240 vs. 231 μm ) and isoleucine (310 vs. 331 μm ; P > 0.10). There was, however, a slight increase in the plasma concentrations of threonine (224 vs. 263 μm ; P < 0.05) and a tendency towards reduced glycine (1387 vs. 1313 μm ; P < 0.10). The results indicate that a flooding dose of valine does not cause a substantial change in the metabolic status of growing pigs and is therefore suitable for measuring protein synthesis rates.     

Lack of postexposure analgesic efficacy of low concentrations of eugenol in zebrafish

Objective

To test the postexposure analgesic efficacy of low doses of eugenol in zebrafish.