貂肠炎病毒DNA探针打点杂交技术检测兔出血症病毒

采用HindⅢ酶切和琼脂糖凝胶电泳技术,从重组质粒pBM中回收0.7kb貂肠炎病毒(MEV)DNA片段.以[α-~(32)P]dATP和光生物素分別标记制备DNA探针。采用打点杂交技术,用上述探针检测提纯的兔出血症病毒(RHDV)和3份感染RHDV的兔肝脏样品,同时检测从貂、犬、豹、猫和貉分离的5种肉食兽细小病毒,并用电镜检查和血凝试验为对照。探针与上述5种肉食兽细小病毒呈杂交阳性反应,而RHDV及感染的肝脏样品为阴性反应.该结果表明,MEV和RHDV在该0.7kb DNA区域内没有同源性,而5种肉食兽细小病毒在此区域内具有遗传相关性。     

肉食兽细小病毒通用基因探针研制成功

本校军事兽医研究所病毒研究室赵永军等通过酶切,由貂肠炎细小病毒(MEV)的复制型DNA(RF-DNA)获得HindⅢ C,将其克隆至pBR322中,构成重组质粒pBM,经转化大肠杆菌RRI扩增后,提纯pBM,再作HindⅢ酶切,用低融点琼脂糖凝胶电泳回收C片段。用随机引物法以α—~(32)P—dATP标记C片段和pBM,采用打点杂交技术,以这两种探针检测了6种动物的细小病毒细胞培养物和其它科5种病毒,同时检测了貂和犬的粪便标本33份。     

应用生物素标记的重组质粒作为探针的杂交条件及敏感性研究

貂肠炎细小病毒复制中间型 DNA(MEV-RF-DNA)的 HindⅢ-C 片段被克隆到 pBR322中形成重组质粒pBM。用光生物素(photo biotin)标记 pBM 和 HindⅣ酶切片段的 pBM 作为探针分别与貂,太细小病毒感染的细胞培养物人工感染细小病毒的貂和犬粪便进行斑点杂交试验,结果发现两种探针检测的敏感性相差10倍以上.光生物素标记的完整重组质粒探针具有放大作用,酶切后的 pBM 敏感性不如前者.另外,本实验还证明在用生物素标记的探针杂交时,被检样品用热变性处理比碱变性处理效果更佳,而且简便。     

光生物素标记DNA探针对貂和犬细小病毒感染的检测

将貂肠炎病毒中间复制型DNA(MEV—RF—DNA)的Hind Ⅲ酶切C片段克隆到pBR332中,形成重组质粒pBM.以光生物素标记的pBM探针,检测接种细小病毒前的貂和犬粪样36份和12份,阳性率分别为36.1%和33.3%;检测接毒后不同时间的貂和犬粪样98份和71份,阳性率分别是90.8%和91.5%.探针对接毒前、后貂和大粪样检出的阳性率均明显高于常规HA试验.另外,对部分貂和大粪样进行了电镜检查和病毒分离试验,并与探针检测结果进行了比较.     

肉食兽细小病毒通用PCR诊断技术的建立

根据肉食兽细小病毒核苷酸序列高度同源的特点，设计合成了1对引物，以犬细小病毒、貂肠炎病毒和猫泛和白细胞减少症病细胞培养物为DNA模板，进行PCR特异性片段扩增，扩增片段大小为0．6kb。结果表明，扩增产物与设计的2个引物之间的序列大小一致。通过通用性、特异性与敏感性试验及对临床检样品检测，证明本法对肉食兽细小病毒通用。且具有快速、特异和高度敏感的特点。     

鸡产蛋下降综合征(EDS-76)病毒DNA探针制备

鸡产蛋下降综合征（EDS－76）病毒由鸭胚培养增殖，60％饱和度的（NH4）2SO4沉淀，经差速离心和蔗糖垫超速离心提纯病毒。蛋白酶K和SDS消化后，苯酚抽提、乙醇沉淀分离EDS－76病毒DNA。用HindⅢ、BamHI、PstI、EcoRI进行病毒DNA酶切图谱分析。EDS－76病毒DNA经PstI限制酸酶切与pUC19质粒载体连接，转化了JPA101大肠杆菌细胞，筛选带有插入片段的白色菌落，并将筛选的克隆进杆快速酶切鉴定;用光敏生物素标记制成探针。结果表明：所选克隆只与EDS-76病毒DNA杂交，克隆片段的为3kb，标记探针能检出10pg的EDS－76病毒DNA。     

用地高辛标记核酸探针检测鹅细小病毒的研究

从带有鹅细小病毒(GPV)NSI基因的重组质粒pMD18T-NS1用限制性内切酶EcoRI和BamH1双酶切回收1880bp大小片段,并制备出地高辛标记的GPV核酸探针。其标记效率达到0．01pg／μl。特异性检测结果表明,该探针能与GPV不同毒株核酸发生特异性杂交,而与对照的DPV、GPPV等病毒的核酸杂交反应均为阴性;敏感性检测结果表明,该探针对GPV的最低检出量为0．0224pg。该探针对不同方法处理的GPV感染病料进行检测,均出现杂交阳性。表明所研制的标记探针用于GPV的检测足可行的。     

肉食兽细小病毒研究进展

在我国肉食兽养殖业细小病毒病十分常见,具有较高的发病率和死亡率,猫泛白细胞减少症病毒(FPV),水貂肠炎病毒(MEV),貉细小病毒(RDPV),蓝狐细小病毒(BFPV),犬细小病毒(CPV)均属于细小病毒科细小病毒属成员。对我国肉食兽动物的养殖有着十分严重的危害。为更好地解决细小病毒病带来的困扰,本文就几类肉食兽细小病毒的亲缘关系,常见的诊断方法,疫苗的研制展开研究。     

犬猫3种冠状病毒基因克隆的构建与芯片检测技术

蔗糖密度梯度离心纯化浓缩犬冠状病毒(CCV)、猫冠状病毒(FCV)和猫传染性腹膜炎病毒(FIPV)的细胞培养物,分别设计7、17、11对引物,对病毒RNA进行反转录和PCR扩增,回收PCR产物连接pGEM-T载体并转化大肠杆菌TGI,构建35个基因片段的克隆。煮沸裂解法制备质粒DNA,回收PCR扩增产物,点制冠状病毒基因芯片。抽提病毒总RNA,利用Cy3-dCTP随机渗入反转录PCR标记,与芯片进行杂交检测,淘汰交叉的克隆片段,每种病毒只选择其4对引物克隆的基因片段。将特异克隆扩增片段重新点制基因芯片,与病毒样品PCR扩增产物杂交,未发现交叉现象。基因芯片检测比传统PCR敏感1000倍,可有效应用于3种病毒的检测与区分。     

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