猪Ghrelin基因的克隆及原核表达

从猪下丘脑、胃等组织中提取总RNA，根据已发表的猪的Ghrelin mRNA序列设计合成引物，通过RT-PCR进行cDNA扩增，获得了282bp的片段。将该片段克隆于pMD-18T载体后进行序列分析，确认PCR产物为Ghrelin cDNA。从阳性克隆中提取质粒，经NheⅠ和XhoⅠ双酶切，回收282bp的目的片段，定向克隆到pET-28a表达载体中，提取质粒并再次转化到BL21（DE3）中，成功地筛选出阳性克隆。经IPTG诱导阳性菌，通过SDS-PAGE检测出猪Ghrelin基因的表达．     

Hypocalcemia in a critically ill patient

Objective: To present a case of clinical hypocalcemia in a critically ill septic dog. Case summary: A 12‐year old, female spayed English sheepdog presented in septic shock 5 days following hemilaminectomy surgery. Streptococcus canis was cultured from the incision site. Seven days after surgery, muscle tremors were noted and a subsequent low serum ionized calcium level was measured and treated. Intensive monitoring, fluid therapy, and antibiotic treatment were continued because of the sepsis and hypocalcemia, but the dog was euthanized 2 weeks after surgery. New or unique information provided: Low serum ionized calcium levels are a common finding in critically ill human patients, especially in cases of sepsis, pancreatitis, and rhabdomyolysis. In veterinary patients, sepsis or streptococcal infections are not commonly thought of as a contributing factor for hypocalcemia. Potential mechanisms of low serum ionized calcium levels in critically ill patients include intracellular accumulation of calcium ions, altered sensitivity and function of the parathyroid gland, alterations in Vitamin D levels or activity, renal loss of calcium, and severe hypomagnesemia. Pro‐inflammatory cytokines and calcitonin have also been proposed to contribute to low ionized calcium in the critically ill. Many veterinarians rely on total calcium levels instead of serum ionized calcium levels to assess critical patients and may be missing the development of hypocalcemia. Serum ionized calcium levels are recommended over total calcium levels to evaluate critically ill veterinary patients.     

猪链球菌多价灭活疫苗对实验小鼠免疫效果的研究

临床分离的猪链球菌经随机扩增多态性DNA分析，挑选出4株毒力强。免疫原性好且遗传距离较远的猪链球菌作为疫苗株，研制出猪链球菌病氢氧化铝胶多价灭活疫苗。该疫苗对实验小鼠安全性好，无不良反应。实验室的小鼠免疫效力试验证实该苗的保护率为100％，优于单价灭活疫苗和猪链球菌病活疫苗。     

猪链球菌2型分泌性核酸酶A(SsnA)活性检测及其基因序列的克隆与测序分析

对8株猪链球菌2型重庆分离株的核酸酶A全基因进行克隆测序,结果表明该基因长度为3 126 bp,与Gen-Bank发表的唯一的该基因的序列相比,核苷酸同源性高于98%,推导的氨基酸同源性高于94%。根据核酸酶A基因的测序结果建立扩增片段长度为301 bp的PCR检测方法,并用甲苯胺蓝DNA酶琼脂对猪链球菌分泌性核酸酶A的活性进行检测。检测了从病猪内脏分离的猪链球菌致病株35株,33株核酸酶A基因PCR检测阳性(阳性比例为94.3%),其中有24株分泌活性检测为阳性(阳性比例68.6%);正常猪扁桃体分离株14株,PCR检测为阳性的10株(阳性比例71.4%),其中有核酸酶A分泌活性为8株(阳性比例57.1%),检测菌株中有2型猪链球菌44株,38株扩增出核酸酶A基因的片段PCR检测阳性并且分泌活性为阳性的有32株,猪链球菌1型、7型、9型、13型、1/2型各1株,均能扩增出核酸酶A基因的片段,但只有13型猪链球菌有核酸酶A分泌活性。对猪链球菌在不同生长时期核酸酶活性的检测显示,猪链球菌在培养4、8、16、32、48 h均有核酸酶A的分泌,并且C a2 和M g2 为分泌性核酸酶A的活性必需因子。     

猪链球菌对红霉素耐药性的研究

从发病猪体内分离、鉴定猪链球菌，采用肉汤稀释法和纸片琼脂扩散法筛选对红霉素耐药的猪链球菌，用双纸片法确定耐药株的耐药表型，通过聚合酶链反应检测对红霉素耐药的基因ermb/mefA。猪链球菌对红霉素的耐药表型为cMLS表型，即同时对克林霉素耐药。在3株红霉素耐药株中扩增到ermb基因，其余未能检测到ermb或mefA基因。     

马链球菌兽疫亚种类M蛋白亚单位疫苗小鼠免疫试验

为获得控制猪链球菌病安全高效的疫苗，本试验进行了马链球菌兽疫亚种类M蛋白亚单位疫苗的研制。应用热酸法提取马链球菌兽疫亚种ATCC35246株类M蛋白，并分别采用羟基磷灰石（HAT）层析和冷酒精沉淀法进行纯化，结果得到较纯的类M蛋白。用热酸提取的粗制类M蛋白、2种纯化的类M蛋白及全菌苗免疫小鼠，结果保护率分别为92％（11／12）、100％（12／12）、100％（12／12）和83％（10／12），表明类M蛋白亚单位疫苗有进一步开发的必要。     

猪链球菌2型四川分离株猪体回归试验

选用35~38日龄的健康断奶仔猪21头,分3组,用2005四川分离株SSsc0501分别经静脉、肌肉注射和口服3种途径感染,进行猪体回归试验。肌肉注射组(含菌0.6×108个/mL)16 h开始出现症状,至36 h全部死亡,发病率、病死率均为100%;静脉注射组(含菌0.2×108个/mL)25 h开始出现症状,48 h内全部死亡,发病率和病死率均为100%;口服攻毒组(含菌2×108个/mL)90 h后开始有发病症状,第9天有1头死亡,发病率75%,病死率33%。从感染病死猪的肝脏、脾脏、肾脏、心血、大脑、脑脊液、淋巴结、肺脏和关节液中均分离到链球菌,经鉴定为试验攻毒的目的菌,表明猪体能够很好地用于猪链球菌2型动物感染模型,同时说明该菌侵害器官广泛,为进一步探讨该病的综合防制提供了重要技术依据。     

脱脂乳发酵体系中乳酸菌的高效离子交换色谱分析

应用高效离子交换色谱对脱脂乳发酵体系中的嗜热链球菌和保加利亚乳杆菌进行色谱表征。结果表明，单菌培养过程中保加利亚乳杆菌菌体形态的变化在其相应色谱行为上得到了体现，二者的色谱峰有比较好的稳定性。两种菌的峰面积与菌数之间呈现出良好的线性关系，可决系数R^2分别为0．9957和0．9916。因此本方法的建立，可用于酸奶发酵过程中乳酸菌的分析与表征，有望为乳酸菌混合物的研究提供一种快捷的新方法。     

Streptococcus uberis: A Permanent Barrier to the Control of Bovine Mastitis?

The prevalence of bovine mastitis has been reduced over the past 25 years due to the implementation of a five-point control plan aimed at reducing exposure, duration and transmission of intramammary infections by bacteria. This has markedly reduced the incidence of bovine mastitis caused by bacteria which show a contagious route of transmission, but has had little effect on the incidence of mastitis due to bacteria which infect the gland from an environmental reservoir. Streptococcus uberis is one such bacterium which is responsible for a significant proportion of clinical mastitis worldwide.The inadequacies of the current methods of mastitis control have led to the search for additional measures, particularly vaccines to prevent intramammary infection by this bacterium. Such an approach requires detailed knowledge of the pathogenesis of intramammary infection. Our understanding of this area has grown in recent years but a lack of information still hampers disease control. Both live vaccines and, recently, crude sub-unit vaccines have shown promise against bovine mastitis due to S. uberis. Vaccines against mastitis must, however, be able to control infection without the participation of a marked inflammatory response. This review provides an overview of the recent advances which have been made in our understanding of host-pathogen interactions which promote infection and disease and highlights areas for strategic research aimed at controlling this bacterial infection.     

猪链球菌病及其疫苗研究进展

猪链球菌病(Swine streptococosis)是由多种不同血清群的致病性链球菌引起的一种疾病总称.近年来猪链球菌病在我国已广泛流行,特别是C群兽疫链球菌(现称马链球茵兽疫亚种)病,严重影响着我国的养殖业,造成了很大的经济损失.文章主要从其病原学、流行病学、毒力因子、病理变化、疫苗的研究进展做了系统的阐述,为该病的深入研究提供参考.