馆藏泽蛙标本壶菌病原实时PCR检测与系统发育分析

为研究、验证我国两栖类壶菌病的历史疫情,从时间和系统进化角度追溯壶菌的来源,该研究选取北京自然博物馆馆藏1982年采集于广东的泽蛙标本39只,利用Taqman-MGB荧光探针定量PCR技术进行壶菌检测,并对定量PCR产物克隆、测序,通过序列比对和系统发育分析判定其来源。最终得到定量PCR标准曲线:Y=-3.1X+32.65;相关系数R2=0.999 8;检测结果为阳性样本12只,检出率30.8%;同时系统发育分析表明,我国的壶菌存在一定程度的分化,一类与北美洲、南美洲、欧洲菌株呈现高度的亲缘关系;另一类则表现出与世界其他地区分布的壶菌有明显的不同,显示为独特类型。该研究把我国壶菌感染的最早记录推进到了20世纪80年代初期。     

韩国两栖宠物出口对中国壶菌病输入风险预测

近年来,壶菌病已经成为危害全球两栖类种群数量的最重要的真菌性传染病,引起全球的高度重视.由于其传播扩散能力极强,对有着两栖类贸易往来的国家来说是巨大的潜在威胁.在亚洲,韩国是继日本之后第二个证实在本土存在壶菌的国家.随着韩国两栖类宠物对中国出口数量的增加,明确其壶菌输出对我国的风险极其重要.本文根据中华人民共和国出入境检验检疫行业标准,运用释放评估、接触暴露评估、后果评估技术整合得到韩国壶菌的输出风险评估结果以及壶菌输入后定植和传播的可能性评估结果.研究发现韩国壶菌对我国的输出风险评估和输入后定植传播的可能性评估结果均为“高”.韩国因两栖类宠物贸易输出引起的壶菌病对我国输入性风险较高,而且一旦传入其定植、扩散的风险也为高风险.     

蛙壶菌潜在分布区及其风险因素预测与分析

【目的】壶菌病是一种由蛙壶菌(Batrachochytrium dendrobatidis)感染导致的疾病,在近半个多世纪的时间里,是导致两栖动物种群数量大量减少甚至灭绝的主要原因之一。目前,壶菌病尚没有行之有效的治疗方法,因此,对世界范围内蛙壶菌的分布与传播进行调查研究,分析蛙壶菌在世界和区域尺度的分布风险及影响因素,可为其预防措施的制定提供基础研究资料。【方法】使用蛙壶菌在世界范围内的分布数据,共考虑了20个气候环境变量,经过对蛙壶菌分布数据和变量进行筛选,建立最大熵模型,比较纳入模型的6个变量与蛙壶菌分布风险的关系,并预测蛙壶菌在世界范围和中国大陆的分布风险。【结果】变量中对蛙壶菌分布概率贡献度最高的前4位依次为年平均气温、年降水量、温度季节性、降水季节性。蛙壶菌分布概率与年平均气温和温度季节性总体均呈先正相关后负相关,与年降水量和降水季节性分别呈正相关和负相关。蛙壶菌全球分布的高风险地区主要在中国大陆南部、澳大利亚东南部、巴布亚新几内亚中部、瑞典南部、德国、波兰、罗马尼亚、英国南部、爱尔兰、法国、马达加斯加东部、苏丹、美国南部、秘鲁东部、埃塞俄比亚和日本等。其在中国大陆分布的高...     

蟾蜍的养殖

蟾蜍的养殖杨俊杰山东省畜牧兽医学校（２６１０４１）一、养殖蟾蜍的经济价值在我国蟾蜍又称癞蛤蟆、虾蟆。为我国传统的著名药材之一，蟾酥是许多名贵的药材不可缺少的原料，市场价格一直较高。用蟾蜍耳后腺分泌的蟾酥制成的药材，有散瘀活血、清肿解毒、止痛、强心的功...     

同时检测猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒和猪细环病病毒1型和2型的多重SYBR Green Ⅰ实时荧光定量PCR方法的建立

在集约化养殖条件下,猪场常见多种病毒混合感染。本试验研究的5种病毒与多种猪病相关,在全球范围的猪场造成了严重的经济损失。本研究建立了能同时检测和鉴别猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)和猪细环病病毒1型和2型(TTSuV1和TTSuV2)的多重SYBR GreenⅠ实时荧光定量PCR方法。该方法能够区分这5种病毒,且对其他DNA病毒检测证明了该系统的特异性。该多重实时荧光定量PCR灵敏度高,检测限为3.65×103～5.04×103拷贝DNA模板/反应。批内和批间变异系数低。该方法对临床样本的检测结果与特异性PCR方法检测结果符合率为100%。这种方法可能成为流行病学研究和疾病控制的有效工具。     

养蟾蜕衣不可一哄而上

今年春季以来,养殖蟾蜍,收集蟾酥和蟾衣年收入可达数十万的广告频频见诸各媒体。情况究竟如何呢?笔者考察了本地一养殖户浙江海宁市、内蒙、新疆等地5家及从事蟾蜍养殖加工的养殖场、户,实情相差甚远。 蟾蜍,俗称癞蛤蟆,不同品种的野生蟾蜍全国各地几乎都有分布。蟾酥是利用蟾蜍的头部耳后腺     

荧光定量PCR技术在牛病诊断中的应用

荧光定量PCR技术发展于20世纪90年代,具有特异性强、灵敏度高、重复性好、全封闭反应等优点,在医学和生物学领域已经得到广泛应用。本文主要对荧光定量PCR技术在牛病诊断中的应用进行综述,以期为牛病防控提供技术参考。     

猪病诊断和防治中PCR技术的应用

本文主要以猪病诊断和防治中PCR技术的应用为重点进行阐述,结合当下PCR技术相关概述为主要依据,从PCR技术在猪2型环病毒中的应用、PCR技术在猪瘟病毒中的应用、PCR技术在呼吸与繁殖综合症中的应用、PCR技术在混合感染中的应用这几方面进行深入探索与研究,其目的在于加强对猪病的诊断和防治,从而为养殖猪场带来更多的经济效益。     

南京召开蟾蜍养殖开发会议严防伪劣蟾衣害人

日前，在南京召开的全国首届蟾蜍养殖产品开发信息交流大会落下帷幕。来自全国各地的专家、学者、教授、技术人员和养殖第一线的专业人员及新闻、医药、卫生界的工作者，针对近年来蟾蜍养殖及副产品的药用开发等问题进行了研讨。会上针对蟾蜍养殖业中出现的一些无序竞争及误导宣传等情况指出，有关部门应加强监管力度，促使蟾蜍养殖发展。人工养殖蟾蜍是二三年内才发展起来的，属于一门新兴的特种养殖业，也是顺应中医药业需求而开发的新兴养殖业，国内大多数省市均有养殖先例，不少地方已生产出高质量的中药材原料蟾衣、蟾酥，已产生可…     

塞内卡病毒Taqman实时荧光定量PCR检测方法的建立及应用

根据对GenBank中2015-2017年登录的18株国内塞内卡病毒(SVV)基因组序列比对分析结果,设计合成1对针对SVV 5’UTR区域的特异性引物和Taqman探针,通过优化荧光定量PCR反应条件,建立一种定量检测SVV的Taqman实时荧光定量PCR检测方法。该方法定量检测范围在10～10~9 copies/μL,检测下限为10 copies/μL,比常规PCR检测灵敏度高约1 000倍,同时检测口蹄疫病毒(FMDV)、猪水疱病病毒(SVDV)、猪水疱疹病毒(VSV)、圆环病毒(PCV)、猪伪狂犬病病毒(PRV)、猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)均为阴性,特异性良好;对50份疑似病料同时进行荧光定量PCR检测和常规PCR检测,结果显示荧光定量PCR阳性检测率(38%)高于常规PCR阳性检测率(25%),证实本试验建立的SVV Taqman实时荧光定量PCR检测方法可实现对SVV的临床诊断和定量分析。     

诱导蟾蜍排卵及蝌蚪生长发育规律的试验研究

研究了蟾蜍受精卵的孵化过程 ,对蝌蚪的生长、发育、变态及幼蛙的生长特性等方面进行了观察和测量 ,同时 ,采用人工诱导排卵的方法 ,进行了蟾蜍在人工条件下的受精试验 ,并对人工诱导的蟾蜍卵细胞进行了孵化试验。结果表明 ,自然条件和人工诱导条件均能够完成蝌蚪的孵化和发育 ,但是在蝌蚪的变态阶段存在较高的死亡率。     

Growth of bluetongue and epizootic hemorrhagic disease of deer viruses in poikilothermic cell systems

Continuous cell lines from the ticks Dermacentor variabilis, D. parumapertus, D. nitens, Rhipicephalus sanguineus and R. appendiculatus, the mosquitoes Aedes albopictus and Culex quinquefasciatus and the African toad Xenopus laevis were tested for their ability to replicate bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses, and for their sensitivity as potential isolation systems. BT serotype 17 grew to peak titers of 10(4.5)-10(7.5) TCID50 ml-1 in all except one of the tick cell lines, EHD 2 virus attained titers similar to that of BT 17 in the mosquito and toads cells, but failed to replicate in tick cells. Only Aedes albopictus and Xenopus laevis cells were as sensitive to infection with low-passage BT 11 and EHD 2 viruses as control cultures of Vero and BHK cells. At 27 degrees C, persistent infection of Xenopus laevis cells occurred, producing low yields of BT 17 and EHD 2. When shifted to 32 degrees C, these cultures expressed virus in exponential increments. No cytopathic effect (CPE) was seen in any of the tick-virus systems, but infected mosquito and toad cells detached from the monolayer within 3-6 days after inoculation with either virus. In the toad cells, this CPE was presaged by the development of plaques within 48 h after infection. Potential applications of poikilotherm systems in orbivirus research are discussed.     

狐胆汁对蟾蜍离体心脏功能的影响

目的：研究狐胆汁对蟾蜍离体心脏的作用及对蟾蜍离体心脏细胞膜ATP酶活性的影响。方法：采用离体蛙心灌流法观察狐胆汁对离体蟾蜍心脏的收缩力、心率的影响,并通过定磷法检测狐胆汁对离体蟾蜍心肌细胞膜ATP酶活性的影响。结果：狐胆汁使蟾蜍离体心脏的收缩力加强,在0.3～4.0g/L浓度范围内,狐胆汁促进心肌收缩力的作用与剂量之间呈量效关系,最大效应可使心肌收缩力提高390%,心率减慢。随狐胆汁浓度提高,心肌细胞膜Ca^2＋-ATP酶活性提高,Na^＋K^＋-ATP酶、Mg^2＋-ATP酶、Ca^2＋Mg^2＋-ATP酶活性下降。结论：狐胆汁能提高蟾蜍离体心脏的功能,其作用机制有待进一步研究。     

A retrospective report of 90 dogs with suspected cane toad (Bufo marinus) toxicity

OBJECTIVE: To report the clinical characteristics of toad toxicity in domestic dogs in Brisbane. DESIGN: A retrospective analysis of clinical cases. PROCEDURE: All cases of toad poisoning which presented to a northern suburbs emergency clinic in Brisbane over a 30-month period beginning in April 1999 were reviewed. RESULTS: A total of 90 canine cases of suspected toad poisoning were reviewed. Small breed dogs accounted for 76% of cases. Jack Russell, Silky, and Fox Terriers were the most represented breeds. Cases were reported year round, with fewest cases over the winter months. The most common clinical signs were increased salivation (78% of cases), and red oral mucous membranes (63% cases). Seizures occurred in 31% of cases. Generally the outcome was excellent with 96% survival.     

Bufo marinus intoxication in dogs: 94 cases (1997-1998)

OBJECTIVE: To determine history, clinical and electrocardiographic abnormalities, treatment, and outcome of dogs exposed to toxins produced by the Bufo marinus toad. DESIGN: Retrospective study. ANIMALS: 94 dogs. PROCEDURE: Medical records of dogs examined between July 1997 and July 1998 for which a diagnosis of toad intoxication had been made on the basis of history and physical examination findings were reviewed. RESULTS: Most (66) dogs were treated during the spring and summer. For 54 dogs, exposure to toads had been witnessed. For the remaining 40, toad intoxication was diagnosed on the basis of history and clinical signs. The most common clinical signs were neurologic abnormalities, hyperemic mucous membranes, ptyalism, recumbency or collapse, tachypnea, and vomiting. The oral cavity was lavaged with tap water in all dogs. Fifty-two dogs were hospitalized for treatment. Body weight of dogs hospitalized > 2 hours was significantly less than that of dogs treated as outpatients. The most common electrocardiographic findings were sinus arrhythmia, sinus tachycardia, and normal sinus rhythm. Eighty-nine dogs recovered fully, 4 died, and 1 was euthanatized. CONCLUSIONS AND CLINICAL RELEVANCE: In areas in which B marinus toads are endemic, toad intoxication should be considered in the differential diagnosis for dogs with an acute onset of neurologic abnormalities, hyperemic mucous membranes, and ptyalism, especially during the spring and summer months. The prognosis is good for dogs with toad intoxication that receive appropriate treatment.     

东方铃蟾乳酸脱氢酶多态性研究

采用聚丙烯酰胺凝胶电泳分离东方铃蟾不同组织ＬＤＨ同工酶，显示出典型的５条带谱。心肌具有以Ｂ亚基为主的ＬＤＨ同工酶谱，骨骼肌以Ａ亚基为主的ＬＤＨ同工酶谱。用改进后的聚丙烯酰胺凝胶电泳方法进行种群调查，结果表明Ａ位点和Ｂ位点都存在电泳上的变异，各存在二个等位基因。Ａ位点有三个基因型，其频率；ＡＡ＝５３．４７％．Ａａ＝３９．１１％．ａａ＝７．４３％；Ｂ位点只检出二个基因型，即ＢＢ为９９．５０％，Ｂｂ为０．５０％。     

Bacterial flora on Cascades frogs in the Klamath mountains of California

Amphibians are experiencing global declines due in part to the infectious disease chytridiomycosis. Some symbiotic bacteria residents on frog skin have been shown to inhibit the growth of Batrachochytrium dendrobatitis (Bd) but few studies have attempted to fully describe the resident bacterial flora of frog skin. We cultured and sequenced 130 bacterial isolates from frogs collected from the California Klamath Range, recovering predominantly Gram-negative bacteria from 20 higher order taxa and 31 genera. There were also a large number of unclassifiable isolates. Forty-three isolates were assessed for their ability to inhibit the growth of Bd in vitro; of these, two had strong and three had slight anti-Bd activity. We suggest that many bacterial species may play a secondary role in Bd resistance, acting synergistically with inhibitory species. Future research is required in order to characterize these interactions. Understanding the relationships between bacterial strains may be important in predicting and managing the effects of future anti-Bd treatments such as antimicrobial compounds or probiotic bacteria.     

Chloramphenicol with fluid and electrolyte therapy cures terminally ill green tree frogs (Litoria caerulea) with chytridiomycosis

Terminal changes in frogs infected with the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) include epidermal degeneration leading to inhibited epidermal electrolyte transport, systemic electrolyte disturbances, and asystolic cardiac arrest. There are few reports of successful treatment of chytridiomycosis and none that include curing amphibians with severe disease. Three terminally ill green tree frogs (Litoria caerulea) with heavy Bd infections were cured using a combination of continuous shallow immersion in 20 mg/L chloramphenicol solution for 14 days, parenteral isotonic electrolyte fluid therapy for 6 days, and increased ambient temperature to 28 degrees C for 14 days. All terminally ill frogs recovered rapidly to normal activity levels and appetite within 5 days of commencing treatment. In contrast, five untreated terminally ill L. caerulea with heavy Bd infections died within 24-48 hr of becoming moribund. Subclinical infections in 15 experimentally infected L. caerulea were cured within 28 days by continuous shallow immersion in 20 mg/L chloramphenicol solution without adverse effects. This is the first known report of a clinical treatment protocol for curing terminally ill Bd-infected frogs.     

Chytridiomycosis in an aquarium collection of frogs: diagnosis, treatment, and control

The introduction of a new group of dendrobatid frogs to an established captive amphibian collection was followed by several acute mortalities in both resident and introduced frog populations. Chytridiomycosis, caused by Batrachochytrium dendrobatidis, was diagnosed by histology in two of the dead frogs. Following the diagnosis, all amphibians were moved to a specially made quarantine room with strict handling protocols and treated with itraconazole. Frogs, being terrestrial amphibians, were treated with itraconazole (Sporanox, 10 mg/ml) at 0.01% in 0.6% saline in a 5-min bath for 11 consecutive days. Axolotls (Ambystoma mexicanum) and Kaup's caecilians (Potymotyphlus kaupii), being aquatic amphibians, were treated with itraconazole administered directly in their primary tank water to achieve a concentration of 0.01% for 30 min every 5 days for four treatments. Itraconazole was removed from the tank water after 30 min by high-rate-of-flow activated charcoal filters. The treatment and quarantine procedures were successful in eradicating the disease. The few amphibian mortalities that occurred in the 18 mo after the start of the treatment have been histologically negative for the presence of chytrid fungi. The collection is now considered free of chytridiomycosis.