1株西藏H5N1亚型禽流感病毒HA和NA基因的遗传进化分析

分析1株西藏H5N1亚型禽流感病毒血凝素(HA)和神经氨酸酶(NA)基因的遗传进化特征。利用RT-PCR法对H5亚型抗原阳性流感病毒核酸检测,并分子克隆、核苷酸测序,再利用DNASTAR、MEGA7.0软件进行同源性分析和基因遗传进化分析。经基因遗传进化分析表明,H5N1亚型禽流感病毒和2012年孟加拉国分离株亲缘关系最近,属于同一分支,HA蛋白裂解位点处有较多碱性氨基酸,从分子上表明西藏毒株H5N1是高致病性禽流感病毒。对西藏分离株H5N1亚型禽流感病毒HA和NA基因进行遗传进化分析,为西藏H5亚型禽流感病毒感染防控具有指导意义。     

H9N2亚型猪流感病毒山东分离株的分离鉴定及其HA、NP、NS基因序列分析

从山东地区疑似流感发病猪分离到 10株流感病毒 ,经国家流感中心鉴定均为 A型流感病毒 H9N2亚型。将其中 1株 Sw/ SD/ 1/ 2 0 0 3(H9N2 )的血凝素基因 (HA)、核蛋白基因 (NP)和非结构蛋白基因 (NS)进行克隆与测序 ,与Gen Bank收录的其他猪流感和禽流感 H9N2亚型的相关基因进行比较 ,推测 Sw/ SD/ 1/ 2 0 0 3(H9N2 )可能源于禽流感病毒 H9N2亚型和 H5 N1亚型的重组病毒 ;Sw/ SD/ 1/ 2 0 0 3的 HA氨基酸裂解位点与其他 H9N2亚型不同 ,Sw/ SD/1/ 2 0 0 3的 HA氨基酸裂解位点是 R- S- L- R- G,而其他猪流感和禽流感 H9N2亚型都是 R- S- S- R- G。     

不同致病性H7N9亚型流感病毒HA蛋白的比较分析

为明确高致病性和低致病性H7N9亚型流感病毒血凝素(HA)蛋白的血清学特性是否有差异,通过体外表达两种H7N9亚型流感病毒HA蛋白并进行分析。利用RT-PCR方法扩增出高、低致病性H7N9亚型流感病毒HA基因,测序,并克隆于真核表达载体pCAGGS-Flag中,构建两个重组表达质粒,命名为pCAF-H7HPHA和pCAF-H7LPHA,分别转染293T细胞,用间接免疫荧光试验和Western blot试验观察HA蛋白的表达情况。结果显示:与低致病H7N9亚型流感病毒比较,高致病性H7N9亚型流感病毒HA基因裂解位点有连续的碱性氨基酸插入,糖基化位点和受体结合位点没有差异。不同致病性毒株的HA蛋白均能表达良好,表达蛋白分子量约为70 ku;高致病性H7N9亚型流感病毒HA蛋白(或血清)可以与低致病H7N9流感血清(或蛋白)发生反应。结果表明:不同致病性H7N9亚型流感病毒HA蛋白在血清学上没有差异性,为H7N9亚型流感病毒HA蛋白的研究奠定基础。     

4株H3N2亚型猪源流感病毒HA基因的序列测定与特性分析

从广东省不同猪场分离到4株H3N2亚型猪源流感病毒A/Swine/Guangdong/01/2004、A/Swine/Guang-dong/02/2004、A/Swine/Guangdong/03/2004、A/Swine/Guangdong/04/2004.根据GenBank公布的H3N2亚型猪源流感病毒的HA基因序列,设计1对引物,运用RT-PCR方法扩增四株病毒的HA基因,并进行测序和分析.同源性分析和遗传进化分析表明本实验的4株H3N2亚型SIV HA基因核苷酸序列同源性为99.8%～99.9%,在遗传进化树中均位于同一分支上.与参考毒株的比较分析表明,4个毒株与WHO推荐的2001-2004年北半球H3N2亚型流感疫苗株A/Moscow/10/99 HA基因的核苷酸序列同源性最高为99.4%～99.5%,4个毒株与A/Moscow/10/99 HA基因在遗传进化树中位于同一个小分支上.氨基酸序列比较发现,4个毒株HA基因裂解位点处的氨基酸序列均为PEKQTR↓G,4个毒株推导的氨基酸序列中均有11个糖基化位点,4个毒株HA蛋白226位受体结合位点(RBS)处氨基酸均为异亮氨酸(Ⅰ).4个毒株HA基因的氨基酸序列、受体结合位点以及糖基化位点均与A/Moscow/10/99相应的氨基酸序列一致.本试验的4株H3N2亚型猪源流感病毒的HA基因属于以A/Moscow/10/99为代表的近代类人H3N2亚型流感病毒,在一定程度上揭示了广东省H3N2亚型猪流感病毒HA基因进化与流行情况.     

鉴定气单胞菌DNA条形码的筛选与评价

为筛选合适的DNA条形码对气单胞菌属(Aeromonas)进行鉴定,以33株气单胞菌为材料,选取cpn60、recA和ppsA 3个候选DNA条形码进行PCR扩增及序列分析。以种间和种内遗传距离、序列扩增测序成功率、DNA条形码间隙(DNA barcoding gap)和系统发育树作为评价条形码片段的指标,筛选出该属的DNA条形码。分析表明,cpn60基因的扩增及测序的成功率最高,种内遗传平均距离(0.017)和种间遗传平均距离(0.133)差异显著,且存在较明显的DNA barcoding gap,表明cpn60可作为气单胞菌鉴定的DNA条形码。     

一株H3亚型野鸟源禽流感病毒血凝素基因序列分析

为了解野鸟在传播禽流感病毒中的作用,贵州省动物疫病预防控制中心定期从威宁草海采集候鸟和留鸟的新鲜粪便,用RT-PCR方法检测病原核酸。监测到1份流感病毒阳性样本,对其血凝素(HA)基因进行了克隆和测序。结果发现,该病毒属于H3亚型,所获得的HA基因1794 bp,包含有完整的阅读框架,编码566个氨基酸残基,包括6个潜在的糖基化位点,遗传进化分析结果显示其属于欧亚禽源分支。另外,HA受体结合位点上的氨基酸序列具有禽源特有的保守性,分别是154A、206E、210L、241G、242Q和244G。推导的HA裂解位点有典型的低致病特征(PEKQTR/GLF)。结果表明,贵州省野鸟中存在低致病性H3亚型禽流感病毒。     

马流感A/马/京防/74-1(H7N7)毒株HA基因的序列分析

流感病毒(Influenza Virus)根据血凝素(HA)和神经氨酸酶(NA)两种表面抗原蛋白分为不同的亚型,马流感I型(H7N7)和Ⅱ型(H3N8)是其中比较重要的两个亚型.本研究应用无特定病原体(SPF)鸡胚增殖马流感病毒A/马/京防/74-1(H7N7)毒株,TRIzol LS Reagent提取病毒RNA,RT-PCR扩增HA基因全片段,克隆到PMD18-T载体上,并进行了鉴定和序列测定.所获得的HA基因片段长1 727 bp,编码563个氨基酸残基.根据推导的氨基酸序列进行预测,有7个潜在的糖基化位点和16个半胱氨酸残基,通过序列分析推断,A/马/京防/74-1(H7N7)株病毒的HA来源于禽类,是一株通过基因重排出现的重组病毒.     

H5亚型鸭源流感病毒HA基因序列生物信息学分析

A型流感病毒（AIV）引起的禽类禽流行性感冒（avian influenza,AI）或相关疾病,被国际兽疫局定为甲类传染病。AIV中最容易突变的基因是血凝素（HA）基因,具有亚型和株的特异性,是区分病毒亚型的依据之一。本研究从GenBank中下载了所有209条鸭源H5禽流感病毒HA基因的全序列,利用生物信息学软件构建进化树研究序列的聚类特点;比较不同年份毒株的HA基因的受体结合位点氨基酸,找出受体结合位点氨基酸的变异规律;比较不同年份分离的鸭源H5N1序列的HA1和HA2蛋白的剪切位点,找出各年份毒株的剪切位点的特点;比较不同年份分离序列的潜在糖基化位点,统计各年份潜在糖基化位点的数量以及序列变化。期望找到H5亚型鸭源流感病毒的变异规律和变异方向,为鸭源流感的防控起到积极的作用。     

2015—2016年广州市部分活禽市场H9N2亚型禽流感病毒的流行病学调查

为了解广州市活禽市场禽流感病毒(AIV)的流行和变异情况,本研究于2015年5月至2016年6月从广州市部分活禽市场采集禽咽喉拭子1 579份,对样品处理之后进行分离和RT-PCR鉴定,通过对HA、NA基因进行遗传进化分析后,共检测到9株H9N2亚型禽源流感病毒。9株分离株的HA基因属于BJ/94-like分支,核苷酸相似性为93.2%~99.7%;有8株分离株的HA基因裂解位点的序列为P-S-R-S-S-R,1株的HA基因裂解位点的序列发生了P/L-S-R-S-S-R的替换。HA蛋白第226受体结合位点的氨基酸均为亮氨酸(Leu),具有人流感受体结合的特征。6株分离株的NA基因属于BJ/94-like分支,3株属于HK/G9-like分支,说明不同分支的病毒开始出现重组。本研究通过对广州市部分活禽市场分离到的禽流感病毒序列分析,掌握活禽市场流感病毒的流行变异趋势,为流感病毒的综合防控及保障公共卫生健康提供参考。     

新型重配甲型H7N9流感病毒HA基因序列分析

为了分析2013年新型重配甲型H7N9流感病毒HA基因分子流行病学特征,从NCBI数据库下载不同分离宿主的流感毒株HA全基因序列,应用分子生物学软件进行遗传演化分析。结果显示:A/Hangzhou/1/2013(H7N9)株HA裂解位点为PEIPKGR↓GLF,含有2个碱性氨基酸;HA蛋白有糖基化位点5个。抗原位点和受体结合位点,相对于之前人感染的H7亚型流感病毒发生不同程度变化。与A/Hangzhou/1/2013(H7N9)株中HA片段的核苷酸同源率较高的前10个毒株均分离自亚洲国家禽类,同源率达到95%以上。此次新型重配H7N9流感毒株HA基因是否由禽传给人有待进一步研究。     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

Prevalence of gastric lesions in racing Alaskan sled dogs

Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in