甘胆口服液对BRD犊牛血清急性期蛋白和氧化应激指标的影响

【目的】探讨甘胆口服液按推荐剂量用药对牛呼吸道疾病（bovine respiratory disease,BRD）的治疗效果及对血清急性期蛋白（acute phase protein,APP）浓度和氧化应激指标的影响,为其在牛临床上的推广应用提供依据。【方法】选择40头自然发病的BRD犊牛,随机分成2组,分别为受试药物组（甘胆口服液0.2 mL/kg）、对照药物组（麻杏石甘散0.75 g/kg）,并增加健康犊牛12头作为空白对照组,按推荐剂量1次/d,连用7 d,分别于试验第0,3,5,7天记录犊牛的一般临床症状,并依据BRD评分表进行打分;在试验第0,7天随机抽取各试验组犊牛12头颈静脉采血,测定血清APP（结合珠蛋白（HP）、淀粉样蛋白A（SAA）、C-反应蛋白（CRP））浓度和氧化应激指标（丙二醛（MDA）、还原型谷胱甘肽（GSH）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、总抗氧化能力（T-AOC））水平。【结果】药物组试验犊牛第0天证候积分均≥5分,符合试验要求。用药第3,5,7天临床症状积分均不同程度降低（P<0.05）。在第5天时积分均<5分,第7天积分减少到最小值。麻杏石甘散组犊牛在第3天时的BRD治愈率高于甘胆口服液组（P>0.05）,但随着治疗时间的延长,甘胆口服液治愈率与麻杏石甘散相近,用药5 d后,两药物组治愈率均达到60%以上。BRD组犊牛第0天血清HP、SAA、MDA水平显著高于健康对照组（P<0.05）,GSH、T-AOC水平及SOD、CAT活性均不同程度低于健康对照组,但差异不显著（P>0.05）。经过药物治疗后,BRD组犊牛第7天血清HP、SAA、MDA水平显著下降,GSH水平和SOD活性显著上升（P<0.05）。【结论】甘胆口服液能明显改善BRD犊牛临床症状,有效缓解患BRD犊牛氧化应激状态,降低血清急性期蛋白水平。联合应用急性期蛋白和氧化应激指标可以对BRD犊牛进行早期诊断及疗效监测。     

山东地区牛传染性鼻气管炎病毒的分离鉴定

从山东某地区发生呼吸道症状的奶牛场中采集样品,按常规方法处理后,接种牛肾细胞(MD-BK),分离病毒,盲传3代后,接种病料的MDBK细胞出现细胞圆缩、聚集成葡萄串样群落、在单层细胞上形成空洞等病变,且随着传代次数的增加,细胞病变更加规律。对所分离病毒进行TCID50测定,其TCID50值为108/ml;用IBRV特异性引物对分离病毒进行PCR扩增,获得与设计基因片段大小一致的特异性条带,序列测定分析结果确认所分离病毒为牛传染性鼻气管炎病毒。     

胞壁酰二肽对体外奶牛乳腺上皮细胞生长及胞内NOD2 mRNA表达的影响

【目的】奶牛乳房炎是奶牛养殖业中最为常见,同时也是带来经济损失最为严重的疾病之一,其主要病因是细菌感染。奶牛乳腺的固有免疫是抵抗病原菌入侵的第一道防线。NOD2是机体固有免疫模式识别受体核苷酸结合寡聚域(nucleotide-binding oligomerization domain,NOD)蛋白家族中的重要一员,通过识别其特异性配体胞壁酰二肽(muramyl dipeptide,MDP)——一种广泛存在于革兰氏阳性菌和革兰氏阴性菌细胞壁中的成分,而参与抵抗多种病原菌入侵。奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMEC)除泌乳以外,还是奶牛乳腺的免疫屏障。本试验欲探究MDP对BMEC体外生长状态及胞内NOD2表达量的影响。【方法】选取健康泌乳中期荷斯坦奶牛乳腺腺泡为组织原材料,采用胶原酶Ⅰ消化法结合梯度浓度胰蛋白酶纯化法分离BMEC;使用上皮细胞特异性表达的角蛋白-18(cytokeratin-18,CK-18)及成纤维细胞特异性表达的波形蛋白(Vimentin)抗体,通过免疫荧光技术对纯化后获得的细胞进行鉴定;将BMEC设为6个处理组,分别添加浓度为0(空白对照组)、1、5、10、15及20μg·mL~(-1)的MDP,24 h后显微镜下观察细胞状态,同时提取细胞RNA并反转录为cDNA,采用实时荧光定量PCR方法检测BMEC中NOD2的表达量。【结果】(1)胶原酶Ⅰ消化法结合梯度浓度胰蛋白酶纯化法分离得到的细胞CK-18免疫荧光结果为阳性,而Vimentin反应为阴性;细胞生长状态良好。(2)空白对照组、1、5及10μg·mL~(-1) MDP刺激组的BMEC生长状态良好,无任何肉眼可见变化;15μg·mL~(-1) MDP刺激组可见少量的BMEC脱落;而20μg·mL~(-1)MDP刺激组可见大量BMEC脱落,漂浮,且即使仍贴壁的细胞,其形态也发生了变化。(3)与空白对照组相比,各刺激组细胞NOD2 mRNA的表达量与MDP的刺激浓度呈正相关,即刺激时间为24 h时,随着MDP浓度的增加,BMEC中NOD2受体mRNA的表达量逐渐增加(P0.05)。【结论】成功获得了纯度较好的BMEC,该细胞生长状态良好,可以用于后续试验;虽然BMEC中NOD2受体mRNA的表达量与MDP的刺激浓度呈正相关,但在保持BMEC生长状态正常的前提下,MDP体外刺激浓度应控制在10μg·mL~(-1)以下。这些结果提示我们:当病原菌入侵乳腺时,BMEC可以通过NOD2受体途径参与免疫防御反应,但这种防御能力受细菌数量或毒力强度的影响。即在一定的细菌数量或毒力范围内,随着细菌数量的增加或毒力的增强,奶牛乳腺的免疫防御反应也增强,进而清除外来病原菌;而当细菌数量或毒力强度超过一定范围时,乳腺组织受到严重损伤,免疫防御屏障也随之崩溃,奶牛乳腺局部甚至全身将呈现出明显的临床症状。     

牛附睾和附睾液分析与牛附睾尾精子在模拟附睾液中存活试验

[目的]新鲜精液在离开生殖腺后存活时间很短.相关研究表明副睾液为精子的存活提供了特殊的环境.试验探讨和研究牛附睾精子在体外模拟附睾液中的存活试验的各因素的影响.[方法]试验对公牛附睾头部和尾部的摩尔渗透压和蛋白质浓度进行了分析,牛附睾尾部精子在不同蛋白浓度的CEP-2溶液中,在4℃条件下孵育120h.每24h做一次精子活力检测.[结果]5 d后精子活力仍然超过60;.附睾头、尾部的渗透压分别为(287.0±13.7)mOsm和(310.8±17.0)mOsm.蛋白浓度分别为(37.43±12.55)mg/mL和(50.58±11.08)mg/mL.[结论]附睾尾精子在蛋白浓度为40mg/mL,渗透压为345 mOsm时,精子存活率最高.     

Study of the Structure–Activity Relationship of an Anti-Dormant Mycobacterial Substance 3-(Phenethylamino)Demethyl(oxy)aaptamine to Create a Probe Molecule for Detecting Its Target Protein

The current tuberculosis treatment regimen is long and complex, and its failure leads to relapse and emergence of drug resistance. One of the major reasons underlying the extended chemotherapeutic regimen is the ability of Mycobacterium tuberculosis to attain a dormant state. Therefore, the identification of new lead compounds with chemical structures different from those of conventional anti-tuberculosis drugs is essential. The compound 3-(phenethylamino)demethyl(oxy)aaptamine (PDOA, 1), isolated from marine sponge of Aaptos sp., is known as an anti-dormant mycobacterial substance, and has been reported to be effective against the drug resistant strains of M. tuberculosis. However, its target protein still remains unclear. This study aims to clarify the structure–activity relationship of 1 using 15 synthetic analogues, in order to prepare a probe molecule for detecting the target protein of 1. We succeeded in creating the compound 15 with a photoaffinity group that retained antimicrobial activity, which proved to be a suitable probe molecule for identifying the target protein of 1.     

CD117‐ and vimentin‐positive telocytes in the bovine teat sphincter

Mastitis is a common economically relevant problem in dairy farming. As the major entry for pathogens is the papillary duct, one of the first defence mechanisms is the teat sphincter. This sphincter shows a rhythmic contractility of yet unknown origin. Searching for possible modulatory pacemaker cells, teat sphincters of eight cows were stained immunohistochemically with antibodies against CD117 and vimentin and evaluated microscopically for the presence of telocytes. CD117‐ and vimentin‐positive telocytes with telopodes were found in close contact with smooth muscle cells. Our findings present a first evidence of telocytes in the teat of bovines.     

In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.     

Equine sarcoid of the glans penis with bovine papillomavirus type 1 in a miniature horse (Falabella)

A 23-year-old Falabella gelding kept in Tochigi, Japan, for more than 20 years presented with a recurrent mass of the glans penis that was first noticed about a year earlier. Partial phallectomy was performed with no adjunctive therapy for local regrowth of the mass. The horse was euthanized 3 months after surgery for urinary retention due to suspected regrowth. The resected mass affected the genital and urethral mucosa of the glans penis, and was diagnosed as equine sarcoid by histopathology and identification of bovine papillomavirus (BPV) DNA. Phylogenetic analysis of the BPV genome of the sarcoid showed high sequence homology to BPV type 1 (BPV-1) from Hokkaido, Japan, suggesting a geographical relationship for BPV-1 in Japan.     

COMPARATIVE EVALUATION OF HYDROPIC DEGENERATION OF THE LIVER IN DAIRY CATTLE THROUGH BIOCHEMISTRY, ULTRASONOGRAPHY AND DIGITAL ANALYSIS

Ultrasonography of the liver of 181 Holstein-Friesian cows was performed and blood samples were collected for analysis. The hepatic ultrasonograms were evaluated and the echoes were analyzed digitally. After slaughter, liver specimens were taken and examined histopathologically. Of the 181 animals, 120 had a normal liver and 61 had hydropic degeneration of the liver, diagnosed through histopathologic examination. Diagnostic accuracy rates for hydropic degeneration were determined based on the following test positive conditions: a) for biochemical analysis—high levels of aspartate aminotransferase, alanine aminotransferase, total bilirubin and non-esterified fatty acids; b) for ultrasonography—presence of dark pattern and blurring of edges; and c) for digital analysis—low echo means at 1 cm and 3 cm from the hepatic surface. Digital analysis had the highest overall specificity, accuracy and positive predictive values for hydropic degeneration, followed by ultrasonography. The results suggest that ultrasonography and digital analysis of hepatic ultrasonograms can be used for diagnosis of hydropic degeneration of the liver in place of biochemical analysis.     

牛A型巴氏杆菌高、低毒力株LPS激活RAW264.7细胞TLR4信号通路的比较分析

本研究旨在比较牛A型多杀性巴氏杆菌高毒力株(PmCQ2)和低毒力株(PmCQ6)脂多糖(LPS)对小鼠巨噬细胞RAW264.7内TLR4信号通路的影响。选用体外培养RAW264.7细胞,经LPS刺激后检测细胞内TLR4信号激活及对TNF-α和IL-12p40表达的影响;并利用Western blot检测LPS对IκBα磷酸化及NF-κBp65活化的影响。结果显示,高毒力株LPS_PmCQ2和低毒力株LPS_PmCQ6分别刺激RAW264.7细胞后,均能显著提高TLR4的表达,并诱导细胞因子TNF-α和IL-12p40的分泌表达;LPS_PmCQ2和LPS_PmCQ6均能诱导IκBα磷酸化,促进NF-κBp65核转位,但二者没有显著差异(P>0.05)。本研究表明牛A型多杀性巴氏杆菌高、低毒力株LPS均能参与TLR4介导的小鼠巨噬细胞免疫应答,且二者对TLR4介导的IκBα-NF-κB信号通路的作用无显著差异,暗示牛A型多杀性巴氏杆菌对小鼠巨噬细胞的毒力与LPS无明显相关性,可能由其他毒力因子决定。