共表达AsiaⅠ型口蹄疫病毒P1-2A和3C基因重组山羊痘病毒的构建及鉴定

将口蹄疫病毒（FMDV）衣壳蛋白前体P1-2A基因和蛋白酶3C基因与启动绿色荧光蛋白双向串连痘苗病毒启动子相连,以KpnⅠ为插入位点,将整体基因片段插入山羊痘病毒转移载体骨架pUC119-TK之中,构建共表达重组山羊痘病毒转移载体pTK-P7.5-GFP-P12A3C。与疫苗弱毒株山羊痘病毒共转染BHK21细胞,经绿色荧光筛选,RT-PCR检测,成功获得能正确表达目的蛋白的重组山羊痘病毒株vTK-3CP1-GFP。     

基因重组山羊痘病毒的构建及鉴定

 将口蹄疫病毒（FMDV）衣壳蛋白前体P1-2A基因和蛋白酶3C基因与启动绿色荧光蛋白双向串连痘苗病毒启动子相连,以KpnⅠ为插入位点,将整体基因片段插入山羊痘病毒转移载体骨架pUC119-TK之中,构建共表达重组山羊痘病毒转移载体pTK-P7.5-GFP-P12A3C。与疫苗弱毒株山羊痘病毒共转染BHK21细胞,经绿色荧光筛选,RT-PCR检测,成功获得能正确表达目的蛋白的重组山羊痘病毒株vTK-3CP1-GFP。     

表达O型口蹄疫病毒P1-2A-3C基因的山羊痘病毒弱毒株的构建及筛选

为构建和筛选表达O型口蹄疫病毒P1-2A-3C基因的山羊痘病毒弱毒株,用已构建的口蹄疫病毒O/Chi-na99毒株的EGFP-P7.5-P1-2A-3C基因整体通过平末端连接到KpnⅠ酶切后的线性载体pUC119-TK中,得到重组载体pUC119-TK-EGFP-P7.5-P1-2A-3C。重组载体通过缺失的TK基因与羊痘病毒弱毒株在BHK-21细胞中同源重组,用EGFP作为标记筛选出重组毒株,并进行PCR鉴定、抗原捕获ELISA试验检测及Western blot分析。结果显示该重组弱毒株能在1~10代BHK-21细胞中稳定传代,扩增出约3000bp片段,并经测序确证为基因P1-2A-3C;抗原捕获ELISA试验检测均为阳性;Western blot分析表明转移载体pUC119-TK-EGFP-P7.5-P1-2A-3C在感染的GTPV AV41BHK-21细胞中表达的蛋白可被O型FMDV高免血清特异性识别,并具有反应原性。这些结果表明获得了表达O型口蹄疫病毒P1-2A-3C基因的重组山羊痘弱毒株。     

融合PCR技术在山羊痘病毒表达载体构建中的应用

为了构建山羊痘病毒表达载体,试验利用融合PCR的方法连接4种组合的3个不同来源的基因片段,插入含有TK基因的载体中,快速构建羊痘病毒表达载体,并转染至BHK-21细胞中进行验证。结果表明:利用融合PCR技术成功融合了P11-7.5-ZSG、P11-7.5-GFP、I1L-7.5-ZSG、I1L-7.5-GFP 4个组合中的3个基因片段,产物长度均在1.2 kb左右,并插入PGEM-TK载体中,构建了4个表达载体,测序结果与理论上100%同源,转染BHK-21细胞可见绿色荧光。说明试验成功构建了4个山羊痘病毒的表达载体。     

新城疫病毒F48E9株病毒F基因表达盒的火鸡疱疹病毒转移质粒载体构建

将新城疫病毒（NDV）F48E9株融合蛋白（F）基因1700bp克隆到真核表达载体pcDNA3中，构建成表达F基因的载体pc3F。然后将包含F基因及其上游的细胞巨化病毒启动子和增强子的3800bp DNA片段再克隆入包含火鸡疱疹病毒（HVT）非必须片段载体pTK2B中，构建成功含有F基因表达盒的HVT转移质粒体pTK3F。经限制性内切酶酶切分析，F基因表达盒的插入方向与pTK2B中TK基因转录方向一致。该研究为进一步在细胞中转染并获得表达F基因的HVT重组病毒奠定了基础。     

表达鸡传染性喉气管炎病毒GB基因重组鸡痘病毒转移载体的构建

根据文献设计2对引物,PCR扩增FPV复制非必需区中外源基因插入位点两侧翼的片段,分别克隆到pUC19和pSK质粒中,并命名为pFP1和pFP2。根据文献设计引物,PCR扩增鸡传染性喉气管炎病毒(ILTV)的GB基因的编码区。将GB基因克隆到质粒pFP1中获得重组质粒pFP1-GB;用酶切质粒pEGFP,得到GFP片段并克隆到质粒pFP2中,得到重组质粒pFP2-GFP。双酶切pFP1-GB得到FP1-GB片段,并克隆到质粒pFP2-GFP中,得到重组质粒pFP1GB-FP2GPF。酶切质粒pE/L-7.5,获得背向连接的2个鸡痘病毒启动子片段——PE/L-7.5,并将该片段克隆到重组质粒pFP1GB-FP2GPF获得重组质粒pGB-GFP,该质粒即为鸡痘病毒的转移质粒。在质粒pGB-GFP中,2个背向连接的启动子被克隆到的GB和GFP基因之间,分别启动GB和GFP基因的表达。经酶切和测序鉴定pGB-GFP,启动子PE/L和P7.5已分别正确地插入到GB和GFP基因之间,分别启动外源表达基因GB和筛选标记基因GFP的表达,从而证明已获得表达鸡传染性喉气管炎病毒GB基因重组鸡痘病毒转移载体pGB-GFP。该载体为下一步获得表达鸡传染性喉气管炎病毒GB基因的重组鸡痘病毒奠定基础。     

山羊痘病毒TK基因功能缺失对其增殖的影响

为了研究山羊痘病毒TK基因功能缺失对其增殖的影响,同源重组构建TK基因缺失型重组山羊痘病毒,检测其增殖特性。试验在通用转移质粒pTKfpgigp内插入不同长度的X1~X4片段,构建了4个重组转移质粒;重组质粒转染已感染山羊痘病毒的羔羊睾丸细胞,经同源重组产生4株TK基因缺失型重组山羊痘病毒rGPV/tk^－,其TK基因内分别插入了长度约为2 800、4 000、5 200、7 700 bp的外源DNA片段;筛选纯化重组毒株,测定rGPV/tk^－病毒滴度。结果显示,TK基因失活后,rGPV/tk^－病毒滴度明显降低;随着插入DNA片段的延长,重组毒株的病毒滴度下降程度更加明显。研究表明,TK基因功能的缺失对山羊痘病毒的增殖具有一定的抑制作用,并与外源DNA的长度具有相关性。     

伪狂犬病病毒鄂A株 TK－／LacZ＋突变株的构建

本研究以地高辛标记的含TK基因的BamHI／KpnI片段为探针，通过Southern杂交确定伪狂犬病病毒鄂A株基因组中一大小约5.9kb的KpnI片段中含有TK基因，回收该片段并克隆于pUC18的KpnI位点。然后进一步克隆其中含TK基因的PstI／KpnI片段，并将LacZ表达盒插入到该片段中的BamHI位点，构建转移质粒pUEKPZ。将该质粒与伪狂犬病病毒鄂A株基因组共转染PK-15细胞，待完全病变后在X-gal存在下筛选蓝斑，蓝斑纯化3次后，经PCR扩增、Southern杂交证实获得的病毒为伪狂犬病病毒鄂A株TK     

山羊痘病毒温度敏感株基因组DNA复制非必需区的筛选

提取山羊痘病毒温度敏感株基因组DNA，用HindⅢ酶切，随机克隆到pUC18质粒中，限制性内切酶片段电泳分析和序列测定结果表明，克隆到4个不同大小的HindⅢ片段pUA、pUB、pUC和pUD。随后，将它们和羊痘病毒Pellor（AY077835）株进行了同源性比较，发现核苷酸同源性为90。0％～94．5％，氨基酸同源性为83．0％～91．4％。选取克隆或亚克隆后的单一酶切位点，插入P11P7.5-LacZ报告基因，构建了pUAl、pUBl、pUCl和pUDl共4个重组载体质粒。通过脂质体分别转染山羊痘病毒感染的BHK-21细胞，在X-gal存在条件下经蓝白斑筛选重组病毒，获得1株重组病毒，证明筛选出了1个复制非必需区片段。     

伪狂犬病病毒TKˉ/gIˉ株PK基因转移载体的构建及LacZ基因表达

伪狂犬病病毒TKˉ/gIˉ株基因组经AscⅠ酶切获得含PK基因的8.7kb片段,将此片段克隆入pPolyⅡ载体的AscⅠ位点,获得中间载体P8-AA,将LacZ表达盒插入其SphⅠ/NdeⅠ位点,构建重组转移载体P8AA-lacZ,将其与伪狂犬病病毒TKˉ/gIˉ株基因组共转染PK-15细胞,细胞出现病变后通过覆盖含有X-gal的营养琼脂进行蓝白斑筛选,通过蚀斑克隆方法分离到重组体PRV（rPRV）,且经过连续传代的rPRV仍能稳定的表达β-半乳糖苷酶活性。通过对重组病毒PRV-LacZ和亲本株TKˉ/gIˉ株TCID50的试验表明,PK基因的缺失对病毒增殖无影响。     

白术、微米白术和白术多糖对断奶仔猪生长性能和肠道形态及微生态区系的影响

试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。     

Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia

Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.     

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.     

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.     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.     

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.     

Feline intracranial neoplasia: retrospective review of 160 cases (1985-2001)

The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.     

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.     

A brief history of the prevention of infectious diseases by immunisations

Infectious diseases have always been a terrible scourge for humans. The appearance of these plagues, as they were called without distinction, was generally connected to various conditions: asters, climatic changes or religious reasons. The concept of contagious, and then infectious, diseases came slowly. Variolation, i.e. transmission of ‘virulent’ matter to induce a natural disease and the immunity against it, was brought from Constantinople to England by Lady Montague, in 1721. This ‘variolation’ technique was also often performed in veterinary medicine against diseases like sheep-pox or pleuropneumonia. As ‘vaccination’ is the term generally accepted for ‘immunisation’, variolation can be the word designating such a technique. The second period of the history of immunisation began, in 1880, with the studies of Pasteur and his collaborators. A great number of bacterial vaccines were developed: dead, live but attenuated or only parts of pathogens. The viruses were produced in animals, then in eggs and at last, in tissue cultures. Second generation vaccines appeared with genetic engineering: recombinant vaccines, vector vaccines, nucleic acids vaccines, and markers vaccines, among others. These novel technologies can permit the development of new ones and improve the quality of the vaccines already existing.