二株鸭源H11N9亚型禽流感病毒的遗传进化及致病性分析

为了解从湖南省洞庭湖区鸭群中分离的2株H11N9亚型禽流感病毒变异特点、进化规律及生物学特性,本研究对2株H11N9亚型禽流感病毒的HA、NA序列进行同源性和遗传进化分析,并用2株毒株对SPF鸡进行致病性试验。结果显示,本试验分离到2株H11N9亚型禽流感毒株的HA裂解位点均没有多个连续的碱性氨基酸插入,属于低致病性毒株;HA基因的受体结合位点均非常保守,具有典型的禽源性特征;NA基因序列与在周边国家野鸟中分离的H11N9亚型毒株的氨基酸同源性较高;鼻腔接种SPF鸡后,均能使鸡感染并通过喉头或泄殖腔排毒,但感染的鸡均不表现明显的临床症状,并且不能使同居鸡感染排毒。     

4株H9N2亚型禽流感病毒分离株对SPF鸡致病性研究

对2013~2015年,从各省养鸡场分离的4株H9N2亚型禽流感病毒进行致病性试验,结果显示,4株H9N2亚型禽流感病毒鼻腔接种SPF鸡后,所有鸡均发生感染并排毒,感染鸡泄殖腔排毒量明显高于口腔,致病性强的毒株比致病性弱的毒株排毒时间要长,在接种后的3~5 d个别鸡表现出精神沉郁,缩头闭眼的情况,并且整体出现食欲下降,但并没有鸡死亡;4株H9N2亚型禽流感病毒感染SPF鸡后,在鸡脏器中的分布各不相同,致病性强的毒株比致病性弱的毒株更具有较广的组织嗜性,在鸡体内各个组织器官复制能力更强。结果表明,4株H9N2亚型禽流感病毒对SPF鸡均具有一定的致病性。     

2013—2014年H7亚型禽流感病毒遗传进化分析

对2013—2014年15株不同来源的H7亚型禽流感病毒国内分离株进行了基因组测序与遗传进化分析。结果显示,15株H7亚型禽流感病毒具有遗传多样性,可分为2种亚型:13株为H7N9亚型,2株为H7N3亚型。HA蛋白裂解位点附近氨基酸分析显示所有H7亚型流感分离株均为低致病性毒株。基因组遗传进化分析显示,13株H7N9亚型禽流感病毒均与2013年人源分离株同源性较高,但具有2种遗传学特性,13株病毒的PB2、PA、HA、NP、NA、M和NS基因高度同源,而PB1基因来源于2个不同分支。2株H7N3亚型流感病毒与国内鸭源分离株同源性较高。15株H7亚型禽流感病毒PB2蛋白均未出现627K、701N等与哺乳动物适应性相关的氨基酸变异。13株H7N9亚型禽流感病毒HA裂解位点附近氨基酸(EIPKGR/GL)与人源H7N9病毒一致,且HA蛋白和M2蛋白分别具有与哺乳动物适应性和金刚烷胺耐药性相关的标志性变异,而2株H7N3亚型禽流感病毒未出现上述氨基酸变异。     

湖南省洞庭湖区5株H6N6亚型禽流感毒株HA、NA基因的遗传进化分析

为了从分子生物学角度了解H6N6亚型禽流感病毒在湖南省洞庭湖区的变异特点和进化规律,为该地区H6N6亚型禽流感的防控提供一些理论依据,对2012年在洞庭湖区分离的H6N6亚型禽流感毒株的HA、NA基因进行扩增、克隆和测序,并对所得序列进行同源性和遗传进化分析。结果显示,本试验分离到的5株H6N6亚型禽流感毒株HA裂解位点为PQIETR↓GLF,均没有多个连续的碱性氨基酸插入,属于低致病性病毒;5株病毒的HA和NA潜在的糖基化位点有一些差别,这些差别是否会引起其毒力和致病力上的差异还有待研究;从基因遗传进化关系来看,这些毒株与我国汕头、广西分离的毒株同源性较高。     

华东地区家鸭中N2亚型低致病性禽流感病毒神经氨酸酶基因的重排

为研究华东地区家鸭中低致病性禽流感病毒NA基因的遗传进化规律,对2000年～2006年分离的包括H3N2,H5N2,H6N2,H9N2和H11N2的44株禽流感病毒的NA基因进行了遗传进化分析.44株病毒NA基因之间的同源性为82.9%～99.9%,4株H9N2病毒的NA基因同源性为96.9%～99.9%,所有H3N2病毒的NA基因同源性为92.3%～99.9%,H5N2、H6N2和H11N2病毒的同种亚型毒株间的NA基因同源性变化幅度较大,相反,有些不同HA亚型毒株间的NA基因同源性反而很高.进一步分析NA基因推导的氨基酸序列也表现出相似的规律.华东地区家鸭中N2亚型的禽流感病毒正处于不断地进化状态中,不同N2亚型的禽流感病毒之间已存在NA基因的重排.     

福建省H9N2亚型禽流感病毒HA和NA基因的进化分析

为进一步了解福建省H9N2亚型禽流感病毒的基因遗传进化关系,本研究将福建省2011年分离的毒株FZ-04、FZ-11与GenBank上登录的2000～2011年福建省分离的H9N2毒株及国内外典型代表株进行HA、NA基因的序列比对和遗传进化分析。结果表明,分离株FZ-04和FZ-11的HA基因与CK/FJ/G9/09株核苷酸同源性最高,属于国内常见的CK/BJ/1/94亚系。HA裂解位点处的氨基酸序列为-PSRSSR/GL-,符合低致病性禽流感病毒的分子特征。NA基因在遗传进化关系上呈现独立的分支,与CK/FJ/10954/05毒株核苷酸序列同源性最高,属于CK/HK/G9/97亚系,且NA基因推导的469个氨基酸序列中没有缺失。同时,从HA和NA基因的遗传进化树上可知,2000～2011年福建省H9N2禽流感病毒进化相对比较稳定,可能有一个共同的起源。     

2株云南H9N2亚型禽流感病毒HA和NA基因序列分析

在2019年1月-2019年6月对云南出现呼吸道疫病的57个鸡场进行H9亚型禽流感检测的基础上,选取石林和楚雄2个H9亚型禽流感阳性样品进行病毒分离。从分离的H9N2亚型禽流感病毒感染鸡胚尿囊液中提取总RNA,采用特异性引物经反转录PCR分别扩增HA和NA基因,PCR产物纯化后进行测序。序列比对及系统发育分析结果表明,云南2株H9N2毒株HA基因核苷酸序列同源性为94.2%,NA基因核苷酸序列同源性为93.6%,系统进化分析表明云南H9N2亚型禽流感病毒HA和NA基因均属于欧亚谱系中的类ADKHKY28097分支(Y280-like),ACKYN12019和ACKYN72019 HA基因之间的同源性为94.3%,与参考毒株ACKJX2448的同源性最高,为95.6%～98.5%,与中国流行的H9N2代表株和疫苗株同源性较低。HA蛋白333-340位裂解位点为PSRSSR↓GLF,具有低致病性禽流感病毒分子特征,受体结合位点均发生E198T和Q234L的突变,具有人样受体结合特征,在29、141、298、305、313、492位氨基酸有6个糖基化位点。ACKYN12019和ACKYN72019 NA基因同源性为93.6%,与Y280-like代表毒株的同源性分别为97.1%～97.5%和93.7%～94.6%,NA蛋白缺失63、64、65位氨基酸,在44、69、86、146、200、234位氨基酸处存在6个潜在的糖基化位点,NA蛋白红细胞结合(HB)位点分析发现,368-369、399-403、432位氨基酸处存在变异。研究结果显示,H9N2亚型禽流感病毒一直处于不断的变异之中,故应加强其监测与防控。     

鄱阳湖与青海湖地区野鸟H9N2亚型禽流感病毒的生物学特性

为了解我国野鸟中H9N2亚型禽流感病毒的分布与特征,2015—2016年对鄱阳湖与青海湖地区野鸟中的H9N2病毒进行监测,结果共分离到12株H9N2亚型禽流感病毒。对分离的病毒进行HA和NA基因序列测定、受体分析和SPF鸡、Balb/c小鼠的感染性试验,结果显示:12个分离株的HA裂解位点基序均为PSRSSR/GLF,符合低致病性禽流感病毒的分子特征;HA蛋白受体结合位点处第226位氨基酸残基为亮氨酸(L),具有结合人样流感病毒受体的分子特征;受体结合试验表明,分离毒株既能结合禽样受体,也能结合人样受体;SPF鸡攻毒试验表明,分离株为低致病性毒株;Balb/c小鼠的感染性试验表明,分离株只在小鼠的上呼吸道鼻甲和肺中有限复制,未见明显临床症状。     

2007年～2010年H9N2亚型禽流感病毒分离株对SPF鸡的致病特性研究

本研究对2007年～2010年我国H9N2亚型禽流感病毒分离株的鸡胚平均死亡时间(MDT)进行分析,发现自2008年下半年来分离的H9N2亚型禽流感病毒对鸡胚的致病性有增强的趋势,在此基础上,选择MDT在60.5～96.3 h之间的9株病毒,比较其对SPF鸡的致病力.结果表明,对鸡胚致病性强的毒株,对SPF鸡的致病性也较强,能引起气管、肺、十二指肠、肾和脑等多器官的系统性感染,而且病毒在各组织器官的复制能力与致病性呈正相关.在感染SPF鸡后,所有毒株均呈现不同程度的排毒,而致病力较强毒株的感染组,排毒的比例更高,排毒时间也更长.这些数据表明,H9N2病毒在进化过程中,致病力发生了一定的变化,新近出现了一些对鸡致病力增强的毒株,这提示我们必须重视对H9N2病毒进化和变异的监测,加强对H9N2病毒的防控.     

山东部分地区H9N2亚型禽流感病毒分离鉴定及HA和NA基因的遗传进化分析

为了解山东省H9亚型禽流感病毒变异情况和流行规律,于2013年从山东省潍坊、淄博、青岛、临沂、济南、滨州和烟台等地区疑似禽流感病鸡病料中分离到14株病毒,通过病毒分离培养、血凝与血凝抑制试验、RT-PCR技术进行了鉴定,并对获得病毒的HA基因和NA基因序列进行了同源性和遗传进化分析。结果显示,分离病毒均能凝集鸡的红细胞并能被禽流感病毒H9标准阳性血清抑制,RT-PCR方法从分离株中扩增出HA基因和NA基因保守片段,序列比对表明分离株与山东日照分离株A/Chicken/Rizhao/853/2013(H9N2)具有较高同源性,达94.4%以上,证实分离毒株为H9N2亚型禽流感病毒。HA裂解位点处的氨基酸序列为-PSRSSR/GL-,符合低致病性禽流感病毒的分子特征。HA、NA基因遗传进化分析表明,分离株与代表株DK/Y280/97亲缘关系最近,核苷酸同源性分别达90.3%和93.6%以上,属于国内常见的CK/BJ/94分支中的Y280-like亚分支。     

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.     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -     

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.     

Chemotaxis of macrophage in inflammation

Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.     

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