副结核分枝杆菌SOD基因在BHK-21细胞中的表达

将重组质粒pGEM-T-SOD中的副结核分枝杆菌SOD基因亚克隆至真核表达载体pVAX1中,构建真核表达重组质粒pVAX1-SOD,以脂质体介导法转染至BHK-21细胞中,并采用RT-PCR和间接免疫荧光技术检测SOD基因在BHK-21细胞中的表达。结果显示,成功地构建了副结核分枝杆菌SOD基因的真核表达载体,且SOD基因在哺乳动物细胞中获得了表达。为研究副结核分枝杆菌SOD基因的免疫效果及作为牛副结核病DNA疫苗奠定基础。     

牛分枝杆菌Ag85A基因的克隆及序列分析

以牛分枝杆菌Valleelll染色体DNA为模板，以Ag85A成熟蛋白基因特异性引物进行PCR扩增，获得了900bp的DNA片段。通过T-A克隆技术，将PCR产物克隆至pGEM-T载体。经鉴定，成功地构建出了克隆载体pGEM-T-85A。     

副结核分枝杆菌map0862基因的克隆及其在大肠杆菌中的表达

为探索MAP0862蛋白在分枝杆菌感染鉴别诊断中的作用,研究构建了MAP0862的原核表达map0862-pET32a(+)质粒,并对其进行了诱导表达。以副结核分枝杆菌P10基因组DNA为模板,经PCR方法扩增副结核分枝杆菌map0862基因片段,将其克隆到原核表达载体pET32a(+)中。将重组子转化到大肠杆菌BL21(DE3),在E.coli中诱导表达带组氨酸标签的map0862-pET32a(+)的融合蛋白。结果显示,经IPTG诱导表达出约55 kD的融合蛋白,用副结核阳性血清进行Western blot检测,证明MAP0862重组蛋白具有良好的免疫原性。     

水貂FSH-β基因序列的克隆与测序

从水貂腿部肌肉中分离提取总DNA,经PCR扩增出水貂FSH-β基因DNA序列。PCR产物经凝胶回收纯化,连接到pMD-18T载体上,然后转化到感受态细胞JM109中。在含X-gal和IPTG的LB(Amp+)平板上筛选阳性克隆菌落,提取质粒作限制性酶切及PCR鉴定后,对目的片段进行测序。结果表明, PCR 扩增出的DNA片段碱基数为500 bp,通过Blast比对,分析了其与人、牛、猪、绵羊、鼠促卵泡激素核苷酸序列的同源性。     

胸膜肺炎放线杆菌APXⅡA基因的克隆及序列测定

用PCR法从胸膜肺炎放线杆菌(APP)7型的DNA提取物中扩增出大小为2873bp的APXⅡA基因片段；将PCR产物重组到质粒载体pMD18-T中，并对重组质粒进行酶切分析及序列测定。结果表明，克隆片段为完整的目的基因，该片段的核苷酸序列与国外分离株M30602的同源性达99．7％。     

结核分枝杆菌分泌蛋白ESAT-6、CFP10基因的克隆、鉴定及其原核表达

以人型结核分枝杆菌 H37RV株基因组 DNA为模板 ,应用 PCR法对 ESAT- 6和 CFP10基因进行扩增 ,产物经纯化后与载体 PMD18- T连接、转化及酶切鉴定 ,亚克隆到原核表达载体 PGEX- 6 P- 1,构建原核重组表达质粒 ,转化入大肠杆菌 BL2 1中 ,以 1mmol/L IPTG诱导 ,进行 SDS- PAGE电泳。结果表明 ,ESAT- 6和 CFP10基因表达的融合蛋白相对分子质量分别为 32 0 0 0和 36 0 0 0 ,与实测相符。重组结核杆菌分泌蛋白 ESAT- 6和 CFP10的成功表达为结核病诊断及重组疫苗的构建打下了基础     

结核分支杆菌Rv3117基因的克隆与原核表达

以结核分支杆菌H37Rv株染色体DNA为模板,应用Rv3117基因特异性引物对该基因进行PCR扩增,获得约800bp的DNA片段。将PCR纯化产物克隆至pMD-18T Vector中,构建出重组质粒pMD-18T-Rv3117。以EcoRⅠ和SamⅠ双酶切pMD-18T-Rv3117重组载体和pGEX-4T-1原核表达载体,并将纯化的Rv3117基因亚克隆至pGEX-4T-1中,构建出原核重组表达质粒pGEX-4T-1-Rv3117。将pGEX-4T-1-Rv3117重组质粒转化至感受态E.coli BL21中,经IPTG诱导和SDS-PAGE分析,可见约30ku目的蛋白带。Western blot分析结果显示,该蛋白能与抗结核分支杆菌阳性血清发生特异性反应。研究结果为结核病临床诊断抗原的筛选奠定了基础。     

香猪SRY基因的克隆及序列分析

试验参照猪SRY基因保守序列设计了1对引物,对香猪基因组DNA进行PCR扩增。将扩增产物通过T—A互补法克隆到质粒pGEM—T载体中,筛选阳性克隆进行DNA测序。测序结果显示,香猪SRY基因开放阅读框全长627bp,编码227个氨基酸。其中核心区域HMG—box长237bp,并且编码79个氨基酸。与白鲸、人、牛、绵羊4种哺乳动物的SRY基因序列相比较,香猪和白鲸的SRY序列具有较高的相似性,相似度达82．1％。序列相似性和邻接法聚类树的结果均显示,在SRY基因中香猪和白鲸有着较近的亲缘关系,表明以白鲸为代表的鲸目和以香猪为代表的偶蹄目有较近的亲缘关系。     

牛分枝杆菌广西分离株MPB70基因的克隆及序列分析

利用牛分枝杆菌MPB70基因特异性引物,以2株牛分枝杆菌广西分离株的染色体DNA为模板,扩增MPB70基因,产物经纯化后与载体PMD18-T连接,然后转化至大肠杆菌DH5α。提取转染后大肠杆菌的质粒进行双酶切和PCR鉴定,鉴定为阳性的质粒进行序列测定。测序后通过序列分析软件DNAstar MegAlign对MPB70基因核苷酸序列及推导的氨基酸序列进行分析,并与GenBank上已发表的8株牛分枝杆菌的MPB70基因参考序列进行比较。结果显示广西分离株mt359、mt370与已发表的7株参考株序列比较,其核苷酸序列和氨基酸同源性在77.4%~95.9%之间。这一结果表明广西奶水牛分枝杆菌分离株与参考的其他牛分枝杆菌毒株的MPB70基因没有太大的差异,说明牛分枝杆菌分泌蛋白MPB70基因十分保守,从而为检测跟踪毒株的变异,研制牛分枝杆菌亚单位疫苗提供基础。     

鸡毒霉形体H3株TM—1基因的克隆与序列分析

对鸡毒霉形体内蒙古分离株H3株的TM-1基因进行了克隆和序列分析。根据已发表的MGS6株TM-1蛋白基因序列分析设计了1对引物，以H3株DNA为模板进行PCR扩增，得到约0.8kb的片段，将该产物克隆到pUCm-T载体上，得到重组质粒，经Kieser法，质粒PCR法，PstⅠ单酶切等方法鉴定后，测定H3株TM-1基因序列。并与已知S6株TM-1基因序列进行了比较。结果表明，MGH3株与S6株TM-1基因核苷酸序列同一性为96.57%。氨基酸同一性为95.42%。这些结果为进一步研究MGH3株的基因免疫和基因工程疫苗等奠定了基础。     

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.     

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.     

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     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in     

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

Tropical Animal Health and Production -