重组减毒沙门氏菌ΔcrpC79-13(pcDNA3-HN)在雏鸡体内主要组织器官定植情况检测

为检测重组减毒沙门氏菌ΔcrpC79-13(pcDNA3-HN)在雏鸡体内主要组织器官定植情况,本研究将120只1日龄雏鸡随机分成3组,分别用减毒沙门氏菌ΔcrpC79-13、重组减毒沙门氏菌ΔcrpC79-13(pcDNA3-HN)和PBS免疫,在免疫后4、24、48、72、96、120、144及168h,分别检测减毒沙门氏菌ΔcrpC79-13和重组减毒沙门氏菌ΔcrpC79-13(pcDNA3-HN)在雏鸡心脏、肝脏、脾脏、盲肠和血液的定植情况。结果显示,在免疫后24h减毒沙门氏菌ΔcrpC79-13在血液中数量达到了一个峰值,24~48h血液细菌数量下降,48~96h血液中细菌数上升并在96h达到另一个峰值,96~168h血液中细菌数量下降,在心脏、肝脏、脾脏、盲肠这几个脏器中,细菌的数量分别在4~72h呈上升趋势并在72h达到峰值,72~96h脏器细菌数量下降,96~120h菌数上升并在120h达到峰值,随后细菌数量下降;免疫重组减毒沙门氏菌ΔcrpC79-13(pcDNA3-HN)组在雏鸡各脏器细菌总数变化及定植情况与ΔcrpC79-13相似。结果表明,重组菌株ΔcrpC79-13(pcDNA3-HN)与亲本菌株ΔcrpC79-13均可在雏鸡心脏、肝脏、脾脏、盲肠等器官定植,且在各脏器中菌株定植变化趋势一致但数量下降。     

堆型艾美耳球虫重组质粒pcDNA3-1E在鸡体组织中的分布

为了探索堆型艾美耳球虫重组质粒pcDNA3-1E在雏鸡组织中的分布情况,将重组质粒pcDNA3-1E经肌肉注射免疫雏鸡,分别在免疫后15d、30d和60d剖杀雏鸡并采取各种组织,抽提各组织总基因组DNA进行PCR扩增后经琼脂糖凝胶电泳分析,并观察接种后雏鸡的临床特征。结果:免疫后15d各组织均可检测到3-1E基因,而免疫后30d和60d,仅在pcDNA3-1E质粒注射部位检测到3-1E基因,其他组织均未检测到3-1E基因;50μg的pcDNA3-1E质粒并未对雏鸡产生不良影响。由此表明堆型艾美耳球虫重组质粒pcDNA3-1E在雏鸡体内至少可存在15d,30d后质粒DNA仅分布于接种部位,并至少可持续60d,而且未发现异常临床表现。     

磷脂酰激醇3-激酶(PI3-K)研究新进展

磷脂酰激醇3-激酶(PI3-K)是细胞内一种重要的激酶,通过活化Akt/pkb、MAPK、PKC等信号途径参与细胞内信号转导,引起细胞发生许多生化反应.PI3-K还具有丝/苏氨酸蛋白激酶活性,可以调节自身磷酸肌醇激酶活性;同时PI3-K的活化与细胞的生长、分化、游动性、胰岛素的活性和细胞的生存有很大的联系.     

致初生仔猪腹泻大肠埃希氏菌氨基糖苷类耐药性检测及AAC(3)-Ⅱ基因序列分析

从1995年至2006年分离鉴定的菌株中选取173株致初生仔猪腹泻大肠杆菌,进行氨基糖苷类药物的药敏试验;根据耐氨基糖苷类药物主要钝化酶—氨基糖苷乙酰转移酶(aminoglycoside acetyltransferases,AAC)的基因序列,设计一对引物,PCR扩增耐药基因片段,探讨AAC基因的分布规律及其与氨基糖苷类抗生素耐药性的相关性。对上述分离株中90个耐庆大霉素的大肠杆菌分离株进行检测,结果表明aac(3)-Ⅱ为这些分离株的主要钝化酶,检出64株,检出率为71.1%;对其中的20株PCR产物进行测序,结果表明对耐庆大霉素而不耐丁胺卡那霉素菌株aac(3)-Ⅱ基因保守区为213位A、232位C、527位A;对应的推导氨基酸序列中71位为赖氨酸、78位为脯氨酸和176位为谷氨酸。两者均耐药的菌株aac(3)-Ⅱ基因保守区具213位G、232位T、527位G,对应的推导氨基酸序列中71位为谷氨酸、78位为亮氨酸、176位为谷氨酰胺。     

重组减毒沙门菌ΔcrpSL1344(pET28-tPA)在BHK细胞中的表达及活性检测

为了探讨原核表达质粒中的真核基因在减毒沙门菌中表达的多肽是否能在真核细胞中加工修饰成具有生物活性的蛋白,试验根据人t-PA的编码序列设计1对引物,扩增t-PA目的基因片段,构建pET28-tPA原核表达质粒,将表达质粒电转化导入减毒鼠伤寒沙门菌ΔcrpSL1344中并转染BHK细胞,应用SDS-PAGE技术检测t-PA表达情况,ELISA法检测其表达水平,琼脂糖平板溶圈法检测纤溶活性。结果表明:重组菌ΔcrpSL1344(pET28-tPA)在BHK细胞中有66.0 ku的蛋白表达,转染96 h的表达量为108μg/L,细胞裂解液和转染表达质粒的细胞培养上清液均有促纤溶活性。说明携带t-PA原核表达质粒的减毒沙门菌具有较强的促纤溶活性。     

Lack of hepatocarcinogenicity of 2,2’-[1,2-ethanediylbis(oxymethylene)]bis-oxirane, 3-hydroxy-2-naphthoic acid,and acetoacetanilide in a medium-term rat liver bioassay

The carcinogenicity of 2,2’-[1,2-ethanediylbis(oxymethylene)]bis-oxirane (ethylene glycol diglycidyl ether; EGDE), 3-hydroxy-2-naphthoic acid (HNA), and acetoacetanilide (AAA) was investigated using a medium-term rat liver bioassay for an occupational safety assessment. F344 male rats were administered a single intraperitoneal injection of diethylnitrosamine (200 mg/kg body weight (bw)/day) and then starting 2 weeks later, they received EGDE at 6, 20, and 60 mg/kg bw/day, HNA at 20, 60, and 200 mg/kg bw/day, or AAA at 60, 200, and 600 mg/kg bw/day by oral gavage for 6 weeks. The animals in the positive control group received phenobarbital sodium solution (PB, 25 mg/kg bw/day) by oral gavage and those in the negative control group received a vehicle (water/corn oil) during the administration period of test substances in this model. All animals were subjected to two-thirds partial hepatectomy at week 3 and euthanized at week 8. Neither the number nor the area of hepatocellular foci positive for glutathione S-transferase placental form (GST-P) increased in any of the EGDE, HNA, or AAA treated groups. However, the number and area of GST-P-positive foci significantly increased in the positive control group treated with PB. The results indicate that EGDE, HNA, and AAA lack hepatocarcinogenicity in rats.     

Increased canine pancreatic lipase immunoreactivity (cPLI) and 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase in dogs with evidence of portal hypertension and normal pancreatic histology: a pilot study

The clinical presentations of both liver disease and pancreatitis are nonspecific and overlapping, which may cause difficulty in diagnosis. In our retrospective pilot study, we assessed whether dogs with evidence of portal hypertension and absence of pancreatitis on pancreatic histology have increases in canine pancreatic lipase immunoreactivity (cPLI) and 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase. We included dogs that had been presented between 2008 and 2019 if they had normal pancreatic histology, histologically confirmed hepatopathy, and if canine pancreas-specific lipase (Spec cPL; Idexx) or DGGR lipase had been measured. Only dogs with portal hypertension were included. Six dogs fulfilled the inclusion criteria. Four of 6 and 2 of 6 dogs had Spec cPL and DGGR lipase exceeding the upper reference limit, respectively. From the 4 dogs with increased Spec cPL, 2 had concentrations of 200–400 µg/L and 2 had concentrations ≥ 400 µg/L. Our results suggest that canine portal hypertension might lead to increased Spec cPL and DGGR lipase values in the absence of pancreatitis on histology. Until more evidence in a larger number of dogs with portal hypertension is available, both tests should be interpreted cautiously in the presence of portal hypertension.     

前列腺素E2受体激动剂和雌激素对奶牛输卵管上皮细胞中转化生长因子β3表达的影响

本试验旨在观察前列腺素E₂受体激动剂(布他前列腺素(butaprost))与雌激素对奶牛输卵管上皮细胞中转化生长因子β₃(TGFβ₃)表达的影响,阐明butaprost和雌激素对奶牛输卵管上皮细胞TGFβ₃有无协同调控作用.采用胰酶消化法及机械法分离培养奶牛输卵管上皮细胞,分别将butaprost和雌激素作用于体外培养的奶牛输卵管上皮细胞,采用实时荧光定量PCR技术检测butaprost和雌激素对奶牛输卵管上皮细胞中TGFβ₃ mRNA表达的影响.结果显示,与0 h作用组相比,雌激素作用16、24和48 h时对奶牛输卵管上皮细胞TGFβ₃的表达量均极显著升高(P< 0.01),4 h的表达量极显著降低(P< 0.01);且受体激动剂butaprost和雌激素有协同调控TGFβ₃的效应;加入吲哚美辛后能有效抑制内源性前列腺素对TGFβ₃表达的作用.结果表明,butaprost和雌激素可调控奶牛输卵管上皮细胞TGFβ₃ mRNA 的表达.     

RACE法扩增梅花鹿β-防御素-1(siBD-1)全长cDNA

为了获得梅花鹿β-防御素-1（sika deer β-defensin-1,siBD-1）cDNA全序列,本试验以梅花鹿舌黏膜组织内提取的总RNA为模板,根据前期已获得的siBD-1 cDNA的已知部分序列设计引物,采用5'-RACE和3'-RACE技术分别扩增5'-和3'-末端序列,将此扩增产物克隆入pMD18-T载体,进行PCR、双酶切鉴定及序列测定与分析。结果表明,成功克隆出长度约为172和299 bp的siBD-1 cDNA 5'-和3'-末端序列,从而得到418 bp的siBD-1 cDNA全序列（GenBank登录号：HM588696.1）,其中包含89 bp 5'-非翻译区（UTR）、192 bp的开放阅读框（ORF）、终止密码子TAA、118 bp的3'-UTR和Poly（A）16。同源性比对结果显示,siBD-1 cDNA与水牛的肠防御素（BEBD）同源性最高,为90.6%,与牛（EBD、LAP、TAP、BNBD-4）、山羊（GBD-1、GBD-2）、驯鹿（reBD-1）、绵羊（sBD-1、sBD-2）和骆驼（caBD-1）的防御素cDNA的同源性较高,分别为83.2%、83.1%、87.3%、87.0%、87.5%、87.5%、84.4%、79.9%、77.1%和70.5%;与马（hoBD-1）和猪（pBD-1）的同源性较低,为60.3%和72.4%;而与人（hBD-2）的同源性最低,为16.0%。siBD-1成熟肽由38个氨基酸残基组成,其中包含9个带正电荷的氨基酸残基。     

1,25-(OH)_2-D_3对抗菌肽表达调控的影响

通过外源途径调控机体内源性抗菌肽表达,以增强动物的防御能力和提高动物的生产性能是实现"健康养殖"的一条新型途径。生物体受病原微生物入侵后可由TLR介导内源性抗菌肽表达。结果表明:由PAMP诱发抗菌肽合成机制的模式有TLR-NF-κB和TLR-VDR 2大信号传导通路。TLR-VDR通路的基本过程是激活相应TLR→诱导Cyp27B1表达→产生1,25-(OH)2-D3→激活VDR→识别抗菌肽基因VDRE序列而调节抗菌肽表达。1,25-(OH)2-D3作为TLR-VDR通路的重要调节物质,可诱导抗菌肽在许多细胞中表达并与其他物质对抗菌肽表达起协同作用。     

A 13-week subchronic toxicity study of 2-(l-menthoxy)ethanol in F344 rats

2-(l-Menthoxy)ethanol has been frequently employed as a flavoring agent; however, data regarding 2-(l-menthoxy)ethanol toxicity remain limited. We performed a 13-week subchronic toxicity study of 2-(l-menthoxy)ethanol in male and female F344 rats, with doses of 0, 15, 60, or 250 mg/kg body weight (BW)/day orally administered by gavage using corn oil as the vehicle. No significant toxicological changes in general condition, body weight, or food intake were observed in any groups. The hematological assessment showed decreased hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin and increased platelet count in the male 250 mg/kg group. Serum biochemistry revealed elevated total cholesterol in the 250 mg/kg group of male and female rats, reduced triglyceride in the female 250 mg/kg group, and increased total protein in the male 250 mg/kg group, indicating effects on lipid metabolism and protein synthesis. For organ weights, absolute and relative weights of the liver and adrenal glands were increased in the 250 mg/kg group of both sexes and the male 250 mg/kg group, respectively. Histopathological analysis showed chronic nephropathy in the male 15 mg/kg or higher groups, with increased absolute and relative kidney weights, as well as elevated serum creatinine, in the male 60 and 250 mg/kg groups. However, eosinophilic granules containing α_2u-globulin were identified in proximal tubules, suggesting α_2u-globulin nephropathy specific to male rats and without toxicological significance. These results indicated that the no-observed-adverse-effect level of 2-(l-menthoxy)ethanol was 60 mg/kg BW/day for both sexes.     

Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus

Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).     

Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans,animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis

Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson’s diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.