猪圆环病毒2型ZZ株全基因组的分子克隆

为了研究PCV-2的致病机理,试验参照GenBank中PCV-2全基因序列设计1对特异性引物,从河南省某猪场疑似感染断奶仔猪多系统衰竭综合征猪的病料DNA中利用PCR扩增出PCV2全基因组片段。结果表明:扩增基因长度为1 767 bp,经酶切鉴定分子构建正确;将克隆到的全基因组环化后,质粒转染PK-15细胞,盲传3代,免疫荧光检测证实其具有感染性。     

猪圆环病毒2型感染性克隆的构建

利用PCR方法从临床疑似PMWS症状猪的脏器中扩增出1株猪圆环病毒2型(PCV-2)的全基因组(1 767bp),命名为SDZQ-1,对其基因进行克隆测序并与GenBank中收录的21个PCV-2毒株的全基因组序列进行比较,同源性结果为95.0%～99.7%.将2个SDZQ-1的全基因组序列正向串联连接入PUC19载体质粒中,成功构建了PCV-2双拷贝质粒.体外转染PK-15细胞,盲传5代后,用PCR和间接免疫荧光方法检测,结果构建的PCV-2克隆具有感染性.     

猪圆环病毒2型感染性克隆构建

基于PCV2滚环复制原理,在分析PCV2不同基因亚型代表毒株基因组结构和序列的基础上,设计2对引物用于PCR扩增PCV2基因组序列。以从收集到的疑似PCV2感染的10份猪血清样品中提取的DNA为模板,用设计的引物扩增目的片段,并进行测序分析,1株为PCV2a型,4株为PCV2b型,5株为PCV2d型。每种基因型毒株中各选取一个样本,利用上述2对引物进行PCR扩增,并将扩增产物克隆至pEASY-Blunt simple载体中,通过双酶切连接2个PCR片段,构建含有约1500 bp重叠序列的PCV2基因组的质粒。通过脂质体转染法将含PCV2全基因组的质粒转染至PK-15细胞进行病毒拯救,经PCR和IFA两种方法验证,证明成功拯救出3个基因型的PCV2毒株。通过对PCV2全基因组结构及序列分析,该方法同样适用于PCV2g、PCV2h基因型病毒的感染性克隆构建。本研究建立了一种基于反向遗传学技术的PCV2感染性克隆的构建方法,为开展PCV2的病原学研究提供了技术支持。     

猪瘟病毒低温诱变疫苗Thiverval株感染性克隆的构建及病毒拯救

采用高保真DNA聚合酶,分7个片段扩增猪瘟病毒(CSFV)Thiverval株全基因组序列,克隆至pMD18-T载体并测序.经适当的连接策略将各片段连接克隆至低拷贝载体质粒pAC/F101,同时对病毒基因组5'和3'末端进行修饰,构建出含有T7启动子、榔头状(HH)核酶基因、CSFV Thiverval全基因组、丁型肝炎病毒(HDV)核酶基因和T7终止子的重组质粒pAC/F101/T1-7.利用T7 RNA聚合酶将线性化重组质粒pAC/F101/T1-7体外转录成基因组RNA,再使用脂质体转染将体外转录RNA转染PK-15细胞.通过传代、RT-PCR、免疫过氧化物酶细胞单层试验鉴定,表明成功的从感染性克隆拯救出活病毒粒子.猪瘟病毒低温诱变疫苗"Thiverval"株感染性克隆的构建及病毒的成功拯救,为进一步研究CSFV疫苗株的致弱机制提供了重要工具.     

猪圆环病毒2型山东分离株全基因组的克隆与序列分析

根据猪圆环病毒2型(PCV-2)全基因组序列,设计一对特异性引物,从疑似断奶仔猪多系统衰竭综合征(PMWS)病料中扩增出PCV-2基因组DNA,将基因片段克隆于pMD18-T载体,筛选获得含有相应片段的阳性重组质粒pMD18-T-PCV-2。对筛选出的阳性质粒进行测序。结果表明,PCV山东株的全基因组为1 767 bp,与国内外毒株核苷酸同源性高达99.6%。     

猪圆环病毒1型反向遗传操作系统的初步构建

为构建猪圆环病毒1型的反向遗传操作系统,试验参考GenBank中已发表的PCV1序列,设计1对特异性引物,应用PCR方法扩增PCV1全长基因组,将其定向克隆至pcDNA3.1(+)真核表达载体中,并对其进行PCR、双酶切鉴定及DNA序列测定;将构建好的重组质粒转染到PK-15细胞中,经3次连续传代后,用PCR方法检测转染后盲传细胞中PCV2核酸,经测序鉴定所拯救出的病毒与亲本病毒核苷酸同源性为100%。结果表明:构建的PCV1感染性克隆具有感染性。PCV1反向遗传操作系统的成功建立为进一步研究PCV基因组的功能及新型疫苗的研发奠定了基础。     

PCV2感染性克隆的构建与拯救病毒在不同细胞系中的增殖特性

将猪圆环病毒Ⅱ型(Porcine circovirus type 2,PCV2)感染性克隆分别转染到猪肾上皮细胞(PK15)和猪小肠上皮细胞(J2)中获得拯救病毒PCV2,比较病毒在2个细胞系中的复制动力学差异。设计特异性引物,通过PCR法和Over-lap PCR技术获得含1.1个拷贝PCV2基因组的DNA片段,克隆于p SP72载体获得PCV2感染性克隆。用PCV2感染性克隆分别转染PK15和J2细胞,Real-time PCR和间接免疫荧光试验(IFA)检测结果表明:拯救病毒可在PK15和J2细胞系中分别连续盲传10代以上,且PK15培养的PCV2基因组的拷贝数极显著高于J2培养的(P0.01),提示获得的PCV2拯救病毒在PK15的增殖性能优于J2,为进一步探索PCV2基因组的功能及PCV2相关发病机制奠定了基础。     

猪圆环病毒2型感染性克隆的构建

为构建猪圆环病毒2型(PCV2)感染性克隆,采用PCR方法从已鉴定为PCV2阳性的病料中扩增PCV2全长基因组片段后将其定向克隆至PVAX1载体中,构建了PCV2感染性克隆质粒p VAX1-PCV2;将重组质粒转染细胞进行病毒拯救,通过免疫过氧化物酶和RT-PCR进行拯救病毒检测,并初步测定了拯救病毒在体外细胞培养的增殖能力和遗传稳定性。结果表明,构建的PCV2感染性克隆质粒,成功拯救出PCV2,拯救病毒盲传8代后毒价可达104.78TCID50/m L,体外增殖能力较为稳定。本试验为深入研究PCV2的基因功能及致病机制等奠定基础。     

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