A fatal diarrhoea outbreak in farm‐raised Deinagkistrodon acutus in China is newly linked to potentially zoonotic Aeromonas hydrophila

Deinagkistrodon acutus is a venomous pit viper commonly used in traditional Chinese medicine; farming these snakes has become a major industry. In 2017, an outbreak of fatal diarrhoea among farm‐raised D. acutus in Hunan Province caused the deaths of 5,600 snakes within 3 weeks. We isolated a brand‐new sequence type of Aeromonas hydrophila (ST516) from lesions and confirmed that this bacterium was the causal agent of the outbreak. Snakes infected with the bacterium in the laboratory showed similar clinical symptoms to those of snakes in the original outbreak. We also tested bacterial virulence in Kunming mice to examine the likelihood of zoonosis. Isolates were pathogenic to mice, causing diarrhoea within 4 hr post‐challenge, which indicates that the bacterium can potentially infect mammals. Environmental analysis showed that polluted spring water likely caused the diarrhoea in snakes. This study is the first to report on a large‐scale outbreak of fatal diarrhoea in farm‐raised snakes, originating in a pathogen that can infect mammals. These results should raise awareness regarding potential anthropozoonosis among poikilotherms, mammals, and humans; appropriate prevention or control methods should be developed.     

尖吻蝮蛇蛇毒金属蛋白酶cDNA序列抗原位点分析及免疫保护效果观察

目的采用生物信息学方法分析尖吻蝮蛇蛇毒金属蛋白酶cDNA序列的关键抗原位点,并观察根据这些位点设计合成的新型免疫原的免疫保护效果。方法扩增尖吻蝮蛇蛇毒金属蛋白酶cDNA序列,采用Jameson-Wolf方法和ClustalX软件结合的生物信息学方法分析其抗原位点,人工合成筛选的抗原位点序列,并连接到pIRESneo表达载体,3次(0、2、4周)对BALB/c小鼠进行核酸免疫,ELISA法测定免疫后机体抗体水平,出血中和实验和攻毒实验初步观察其免疫保护效果。结果生物信息学方法分析得到6个关键抗原位点(MPA-1～MPA-6);ELISA法检测抗血清结果表明,抗原位点序列诱导小鼠产生的抗血清相对未免疫的小鼠血清稀释100倍后仍能表现出阳性结果;出血中和实验和攻毒实验表明,将抗原位点序列免疫小鼠可以诱导机体产生免疫反应,使体内产生抗体,能有效中和蛇毒,从而对尖吻蝮蛇蛇毒引起的出血有防护作用。结论用生物信息学方法成功获得尖吻蝮蛇蛇毒金属蛋白酶cDNA序列的6个关键抗原位点,针对这些位点设计合成的新型免疫原展示出初步免疫保护效果。     

抗尖吻蝮蛇蛇毒金属酶类共同基序IgY抗体的制备与研究

[目的]制备针对尖吻蝮蛇蛇毒金属蛋白酶类共同基序的IgY抗体,并对其中和蛇毒活性的作用进行研究.[方法]通过分析尖吻蝮蛇蛇毒各类金属蛋白酶类共有序列,设计合成与其酶活性密切相关且高度保守的抗原肽PT1和PT2;偶联复合物KLH-PT1、KLH-PT2免疫母鸡获得IgY抗体.采用ELISA和Western blot等方法对IgY效价及与尖吻蝮蛇蛇毒和短尾蝮蛇蛇毒的交叉反应特性进行初步研究;最后通过抗小鼠皮下出血实验对IgY中和活性进行评价.[结果]ELISA、Western blot结果表明,抗KLH-PT1、抗尖吻蝮蛇蛇毒IgY均可与尖吻蝮蛇蛇毒、短尾蝮蛇蛇毒发生交叉反应且后者显著强于前者;抗KLH-PT2 IgY在体外与尖吻蝮蛇蛇毒、短尾蝮蛇蛇毒无明显交叉反应,在体内抗出血实验中却表现出很强的中和毒性作用,并且显著优于前两者.[结论]通过设计金属蛋白酶共有序列抗原肽,制备了能与多种血循型蛇毒交叉反应的IgY抗体,这为制备特异、高效、低毒性且广谱的抗出血型蛇毒抗体奠定了基础.     

尖吻蝮蛇去整合素基因克隆与功能分析

目的：获得尖吻蝮蛇去整合素基因，并分析重组蛋白生物学活性。方法：提取尖吻蝮蛇毒腺组织总RNA．经RT—PCR获得去整合素基因片段。将该去整合素基因片段重组入pMAL-p2x栽体中诱导表达，以直链淀粉树脂柱亲和纯化，并检测融合蛋白的抑制血小板聚集活性。结果：谊片段编码的氨基酸序列中含有去整合素保守的RGD三肽序列及12个Cys残基，与GenBank中其他蛇毒去整合素氨基酸序列最高同源性为86％，重组融合蛋白具有抑制ADP诱导的血小板聚集活性。结论：发现了一个新的去整合素基因，谊基因编码蛋白具有抑制血小板聚集的功能。     

尖吻蝮蛇毒凝血酶样酶的长期毒性及连续给药后药理效应的研究

本文报道尖吻蝮蛇毒凝血酶样酶对犬及家兔的亚急性毒性及生化药理效应。用药量(μg/kg·d)犬分别为200、65,家兔分别为540、180,连续静脉注射28天。用药后14天及28天,WBC,DC,Hb 含量、肝、臂功能均无明显异差。血浆纤维蛋白原含量明显降低,凝血酶样酶时间及凝血酶时间明显延长,血小板聚集功能明显抑制,高剂量组个别犬血浆尿素氮显著上升(停药2天后降低),实验结束时体重均增加。用药后10天起,个别犬于每天注药后出现短时反应,至3周后消失。病理解剖检查眼肉及镜检未见重要脏器发生病变。本资料提示凝血酶样酶是一低毒高效应制剂,用药2周后对血小板聚集的抑制作用有耐受现象,因此连续用药时间不宜过长。     

Identification of a nitric oxide-dependent hypotensive effect of anticoagulation factor II from the venom of Agkistrodon acutus

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is a member of the coagulation factor IX/coagulation factor X-binding protein (IX/X-bp) family. ACF II forms a 1:1 complex with activated coagulation factor X in a Ca²⁺-dependent fashion and thereby blocks the amplification of the coagulation cascade. In the present study, we have investigated the effect of ACF II on the mean arterial blood pressure (MABP) and heart rate (HR) in anaesthetized rats. The results indicate that ACF II induces a dose-dependent response in rats with a short fast drop of MABP followed by an increase and then a longer lasting slight decrease in MABP, but does not obviously affect HR. ACF II-induced hypotension is significantly blocked by the nitric oxide (NO) synthase inhibitor N-omega-l-arginine methyl ester (l-NAME). ACF II produces a concentration-dependent relaxation of rat aortic rings with functional-endothelium. The ACF II-induced vasodilatation is completely inhibited by removal of endothelium and significantly inhibited by pretreatment with l-NAME. These observations demonstrate that ACF II induces hypotension through an endothelium-dependent vasodilation, which is strongly mediated by the release of NO from endothelium. ACF II exhibits high anticoagulation activity in vivo based on activated partial thromboplastin time assay. Therefore, ACF II is so far identified as the first unique bifunctional protein in the IX/X-bp family that has both anticoagulant and hypotensive effects on the blood of rats through different pathways.     

Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom

Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k_cat/K_m values of actibin for hydrolysis of tosyl-l-arginine methyl ester and benzoyl-l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.     

尖吻蝮蛇毒研究进展

尖吻蝮(又名五步蛇,中药材名为蕲蛇)蛇毒,是从尖吻蝮毒腺中分泌的毒液,内含磷脂酶A₂、丝氨酸蛋白酶、金属蛋白酶、C型凝集素、L-氨基酸氧化酶等多种蛋白类成分和肽类成分,具有多种生物活性,在抗肿瘤、抗血栓、抗炎、抗菌等方面发挥着重要作用。近年来,蛇毒研究日益广泛,但目前仍缺乏对尖吻蝮蛇毒全面系统的研究。本文通过检索尖吻蝮蛇毒的相关研究进展,在其来源、鉴别、活性成分、毒性研究及质量研究等方面进行总结与分析,以期为尖吻蝮蛇毒进一步开发利用提供参考。     

Effects of heating on the immunogenicity and biological toxicity of Deinagkistrodon acutus venom and its fractions

To improve toxoid preparation, the effects of selective heat denaturation were assessed on Deinagkistrodon acutus venom. The venom and its fractions (peak 1 and peak 2 separated by gel filtration chromatography) were heated to various temperatures (45-70 °C) for 30 min, after which protein concentration, immunoreactivity, lethality, myotoxicity and hemorrhagic and membrane lysis activities of the samples were determined. In addition, the synergistic effects of the venom fractions were evaluated by separate or simultaneous intramuscular injection in mice. The results showed that the peak 1 fraction consisted primarily of proteins in the range of 18 to 105 kDa, while the peak 2 fraction consisted primarily of proteins smaller than 21 kDa. The hemorrhagic activity, immunoreactivity, and protein concentration of heated samples were gradually reduced as the temperature increased from 25 °C to 70 °C. Bioactivities significantly decreased but immunoreactivity was retained when the crude venom, peak 1 fraction, or peak 2 fraction were heated to the critical temperatures of 60 °C, 55 °C, or 60 °C, respectively. Synergistic effects of two kinds of heated fractions were observed in toxicity and antibody production after the peak 1 and peak 2 injected simultaneously or respectively. The results suggest that venom fractions heated and injected separately could significantly reduce their toxicity and enhance the neutralization of antiserum induced by them.     

尖吻蝮蛇毒凝血酶样酶的药代动力学

本文以聚苯乙烯微量血凝板为固相载体,运用夹心酶联免疫吸附分析(Sandwich ELISA)法研究了大白鼠快速iv尖吻蝮蛇毒凝血酶样酶(TLEs)后的药代动力学。TLEs的血浆浓度-时间过程具有二室开放模式的动力学特征,其快处置过程(α相)的t(1/2)约为11min,其慢处置过程(β相)的t(1/2)约为7.0h。TLEs具有低表观分布容积的特征,以血液分布为主,经肝、肾消除。