基因芯片技术及口蹄疫病毒基因芯片研究进展

口蹄疫的暴发与流行给世界各国的畜牧生产、社会经济和国际贸易构成严重威胁并造成巨大的经济损失,经典的血清学和PCR检测方法越来越不适应进出口动物检疫的快速、准确、高通量的要求.建立一种快速的诊断方法,使其在很短时间能够区别各血清型口蹄疫和其他水泡性疾病是非常必要的.口蹄疫病毒基因芯片包括155个寡核苷酸探针,总长35 bp～45 bp,设计在VP3-VP1-2A区,既有共有的病毒型别,也包含特异的血清型别.这项技术的优点是能在单一的芯片上检测多元病原体.     

口蹄疫病毒抗原胶体金试纸条的研究

利用单克隆抗体技术制备抗口蹄疫病毒的单克隆抗体,特异性试验表明其只与O、A、Asia 1型3种血清型FMDV抗原结合。进而采用胶体金标记技术,以胶体金标记的抗口蹄疫病毒单克隆抗体、多克隆血清抗体和葡萄球菌A蛋白为主要材料,研制口蹄疫快速检测试纸条。该试纸条检测O、A、Asia 1型3种血清型灭活口蹄疫病毒均为阳性,检测水疱性口炎病毒、猪水疱病病毒、蓝舌病病毒、猪蓝耳病病毒4种灭活抗原及小反刍兽疫病毒疫苗株均为阴性,试验结果与口蹄疫实时荧光定量RT-PCR方法的完全一致,表明其具有良好的特异性。敏感性试验结果是,试纸条的检测极限为1∶160稀释的样品,其敏感性相当于实时荧光定量RT-PCR方法的1/64。由于试纸条具有操作方便、检测快速等优点,因此该试纸条可以用于大量临床样品的快速检测和现场检测。     

口蹄疫病毒非型特异性检测方法研究进展

口蹄疫病毒非型特异性检测方法避免了口蹄疫不同血清型影响试验的敏感性,可快速简单的诊断口蹄疫病毒,判断免疫效果,检测感染历史等,如口蹄疫非型特异性RT-PCR、口蹄疫3ABC检测、VIA抗原AGID检测等,在口蹄疫防控中有广泛的应用。目前一些新开发的非型特异性检测方法更突出了简便性、快速性的特点,如横流装置(lateral flow device,LFD)可在1min～10min完验,设备只需要一台简单的样品搅拌器,非常适合基层使用。     

沙门菌快速血清分型策略的应用

为建立一种对临床沙门菌株进行快速血清分型的策略，本研究尝试将玻片凝集法和液相芯片法进行联合应用。首先利用玻片凝集法对467株动物源沙门菌进行鉴定，然后对不能直接利用玻片凝集法鉴定血清型的沙门菌采用液相芯片法进行补充鉴定。结果显示，利用玻片凝集法可以直接检测409株沙门菌的O抗原和H抗原，包含49种血清型；结合液相芯片法在1 d内完成了其余58株沙门菌的O抗原和H抗原单因子的鉴定，共涉及10种血清型。结果表明，玻片凝集法具有快速鉴定O抗原优势，液相芯片法具有快速鉴定H抗原优势，2种方法结合应用的策略可以快速、高效地实现沙门菌血清分型的目的，可作为沙门菌血清型快速鉴定方案并予以推广。     

O、A、Asia 1型口蹄疫病毒胶体金定量检测免疫层析方法的建立

旨在建立口蹄疫病毒(foot-and-mouth disease virus, FMDV)抗原血清型的快速分型和定量的检测方法,利用双抗体夹心法,将口蹄疫病毒的兔抗及豚鼠抗体作为标记胶体金与NC膜检测带的原料,分别制备出检测O、A、Asia 1血清型的3种层析试纸卡。通过对标定的抗原标准品146S检测,拟合出定量标准曲线。免疫层析方法的质量验证通过特异性、敏感性、重复性及与蔗糖密度梯度法(sucrose density gradient, SDG)的相关性进行评价。结果显示:建立的快速定量检测方法,3种血清型口蹄疫病毒间无交叉反应,同时与其他非口蹄疫病毒,如塞内卡病毒A型(SVA)、猪水疱病病毒(SVDV)、猪水疱性口炎病毒(VSV)无非特异反应;敏感性研究,对O、A、Asia 1型病毒的最低检出量分别为0.567、0.693、0.219μg·mL^-1,拟合的3条标准曲线的线性相关系数R~2>0.97,新建立方法与蔗糖密度梯度法检测结果的相关系数均>0.9, 3种层析试纸卡的变异系数均小于10%。综上表明,建立的口蹄疫O、A、Asia1型病毒胶体金免疫层析定量检测试纸卡方法,可以用于口蹄疫病毒抗原快速鉴别、血清分型与146S定量检测。     

口蹄疫灭活疫苗免疫效果的影响因素研究

口蹄疫(FMD)是由口蹄疫病毒(FMDV)引起的一种急性、热性、高度接触传染性和可快速远距离传播的动物疫病.口蹄疫病原是口蹄疫病毒,属微核糖核酸病毒科口蹄疫病毒属,有A、O、C、SAT1、SAT2、SA T3以及A sia 1型7个血清型,每个血清型又包含若干个亚型.该病毒不仅在各型间没有交叉免疫性,同血清型的各亚型之间也仅有部分交叉免疫性.本文分析了口蹄疫灭活疫苗免疫效果的影响因素.     

理论知识和防控措施有助于抗击亚洲口蹄疫

口蹄疫O型血清型是东南亚广泛存在、流行最为严重的血清型；A型血清型是所有口蹄疫血清型中最可能发生变异的一种，主要集中在泰国和马来西亚，更倾向于发生在反刍动物种群中；而亚洲血清型1的抗原性在三种流行血清型中最为稳定，主要发生在东南亚的某些国家。     

口蹄疫病毒RT-PCR检测和分型方法的建立及初步应用

参考GenBank中各个血清型口蹄疫病毒3D、vp1、2A基因的标准序列，设计引物P1／P2和S1／S2。建立用于检测口蹄疫病毒及其利用引物S1／S2克隆片段同源性比较而确定血清型的RT-PCR方法。通过敏感性试验检测，2对引物均可以检测到10TCID50的病毒量；特异性试验的检测，2对引物对正常细胞、牛黏膜病病毒、猪瘟病毒、水疱性口炎病毒、牛传染性鼻气管炎病毒的检测结果均为阴性。利用该方法对病牛的流涎液体、水疱液体、舌皮组织、感染犊牛心脏等组织进行检测初步结果显示：该方法可以对O型和AsiaⅠ型口蹄疫病毒进行特异性检测，能够用于口蹄疫急性及亚临床感染的诊断及流行病学调查。     

液相芯片鉴别检测7种出血性大肠埃希菌血清型

为了建立一种同时检测O157、O26、O45、O103、O111、O121和O145等7种血清型的出血性大肠埃希菌的高通量检测方法,通过筛选比对大肠埃希菌O157血清型的fbE基因,O26、O103和O111血清型的WZX基因,O45、O121和O145血清型的WZY基因,设计了针对各个血清型的特异性引物和探针。通过在所有的特异性引物5′端加入超级引物的策略,达到了通过一次PCR反应,同时多重扩增7个血清型的7个目标片段的效果。将加尾多重扩增与液相芯片高通量检测相结合,建立了同时检测7种血清型的出血性大肠埃希菌的液相芯片检测方法,并对方法检测体系及反应条件进行了优化。所建立的7种出血性大肠埃希菌液相芯片检测方法灵敏,其灵敏度与荧光PCR的灵敏度相差100倍。所建立的方法特异,单个血清型的探针与相应的PCR扩增的阳性产物之间均有特异性的杂交,LQRR值介于17~63之间。7种血清型的混合探针与各个血清型菌株的PCR与扩增的阳性产物之间均有特异性的杂交,LQRR值介于11~23之间,与其他血清型出血性大肠埃希菌和非出血性大肠埃希菌均无交叉反应。试验结果表明,所建立的液相芯片检测大肠埃希菌O157、O26、O45、O103、O111、O121和O145等7个血清型的检测方法具有快速、特异性强、灵敏度高等特点,可用于主要出血性大肠埃希菌的快速鉴别检测。     

一种用于口蹄疫病毒实时荧光RT-PCR检测的引物和方法

本文建立了一种用于口蹄疫病毒实时荧光RT-PCR检测的引物和方法。分析所有已报道的口蹄疫病毒通用型基因组序列,分别设计引物和荧光探针,通过实时荧光RT-PCR检测,得到口蹄疫通用型的特异性荧光曲线,可以确定血清型,检测结果准确、可靠。     

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