Taqman-MGB探针法检测炭疽杆菌

根据GenBank中炭疽杆菌的主基因组上gyrA基因,毒力因子px01上pagA基因和侵袭因子px02上的CapA基因分别设计引物和对应Taqman-MGB探针,建立了检测炭疽杆菌的实时定量PCR方法。该方法具有良好特异性,仅对炭疽杆菌检测结果为阳性,而与蜡样芽孢杆菌、苏云金杆菌、巨大芽孢杆菌、沙门菌、大肠杆菌、链球菌、铜绿假单孢杆菌无交叉反应,检测灵敏度最低可达100copies/μL。对24份皮毛盲样和120份送检样品检测结果表明建立的定量PCR方法与商业化定量PCR试剂盒的符合率为100%。Taqman-MGB探针方法灵敏、特异、重复性好,可应用于炭疽杆菌的快速诊断与监测。     

炭疽杆菌双重荧光定量PCR检测技术的建立和应用

为快速和准确检测炭疽杆菌,本研究以炭疽杆菌染色体上的BA5345基因和pXO1质粒上的PA基因为靶基因设计特异性引物和探针,建立了炭疽杆菌双重荧光定量PCR检测方法,对其反应的特异性、敏感性和重复性进行了分析,并与常规PCR方法一起对临床样品进行检测。结果显示,所建立方法特异性强,与蜡样芽胞杆菌群中其他芽胞杆菌无交叉反应;对BA5345基因和PA基因的最低检测限分别为8.0和7.8拷贝·μL~(-1),标准曲线相关系数大于0.99,组内和组间CV均小于1.5%。对35份污染皮毛样品检测显示BA5345基因和PA基因的检出率分别为80.0%和82.9%,与常规PCR检测方法相比,敏感性更高。本研究为炭疽杆菌的检测和流行病学调查提供了一种快速、准确的检测方法。     

鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y-染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y-染色体特异引物，依据牛骨胳肌α肌动蛋白前体基因和微卫星DNA序列设计合成4对牛DNA特异引物(内标引物)。单重PcR扩增牛基因组DNA，筛选出4对牛Y-染色体特异引物和1对牛DNA特异内标引物。将不同的Y-染色体特异引物与内标引物组合，多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎，筛选出2个可用于牛早期胚胎性别鉴别的PCR引物组合：B34／A12和B78／A12。     

兔巴氏杆菌和波氏杆菌多重PCR检测方法的建立

参考GenBank中兔巴氏杆菌16SrRNA和波氏杆菌的fim2的基因序列,应用Premier 5.0软件在二者高度保守区设计了2对引物,建立了适合巴氏杆菌和波氏杆菌快速检测的多重PCR检测方法。以该方法对巴氏杆菌和波氏杆菌参考菌株进行PCR扩增,分别能从各自的基因组中扩增出与试验设计相符的644bp和425bp的特异性DNA片段。将扩增所得的DNA片段进行克隆测序,测序结果表明分别为巴氏杆菌16SrRNA和波氏杆菌fim2基因序列。该方法对波氏杆菌的检测下限为4×102 CFU,对巴氏杆菌的检测下限为6×101 CFU。对兔源性沙门菌、葡萄球菌、大肠杆菌、魏氏梭菌扩增结果为阴性。表明所建立的RT-PCR检测技术具有特异、快速和敏感的特点,可用于鉴别诊断兔巴氏杆菌和波氏杆菌以及2者混合感染。     

鸡腺苷琥珀酸裂解酶基因适用性进化分析

采用反转录-聚合酶链式反应（RT—PCR）方法，以肝脏总RNA为模板，对泰和乌骨鸡、隐性白羽肉鸡腺苷琥珀酸裂解酶基因（ADSL）编码区序列进行克隆和测序；并结合Gen—Bank中已提交的家鸡、鱼类、哺乳动物和人类等ADSL基因序列，采用基于最大似然法的密码子置换模型分析ADSL基因编码区序列在进化过程中承受的选择压力。结果RT—PCR方法准确获得了泰和乌骨鸡和隐性白羽肉鸡ADSL基因编码区序列，全长为1380bp。“分支特异模型”检验结果表明，ADSL基因在不同物种进化过程中较为保守，未受到正选择作用。鸡ADSL基因序列的“位点特异模型”检验未发现正选择位点。研究结果有助于了解鸡ADSL基因的结构及进化历程。     

胸膜肺炎放线杆菌荚膜多糖输出基因部分序列的克隆鉴定、序列拼接和分析应用

用设计的引物对胸膜肺炎放线杆菌1、3、6、9型标准株的荚膜多糖输出部分基因序列进行了扩增，克隆，测序，分别获得4个型的部分cpxDC基因序列和3型的部分cpxD基因序列。对克隆序列和GenBank中已知序列进行序列拼接，分别拼接出1、3、6型的荚膜输出cpxD基因全序列。通过基因序列分析发现，胸膜肺炎放线杆菌1、3、5、6、9型荚膜多糖输出基因已知序列间的同源性很高，达89％～99．88％。1、3、5、6型cpxD基因的推导氨基酸序列间的同源性为93％～99％。在cpxDC基因的保守区内设计出1对种特异性引物，对胸膜肺炎放线杆菌进行鉴定和检测，结果从8个标准型菌株和3株分离株中均扩出约720bp的阳性片段，其他6种能引起猪呼吸道疾病的细菌均呈阴性。所建立的PCR方法最低检出量为10Pg，最适模板量为5ng（50ptL）。试验结果表明，胸膜肺炎放线杆菌不同血清型间的荚膜多糖输出基因存在着高度的保守性。设计出的种特异性引物能用于胸膜肺炎放线杆菌的检测和鉴定。     

羊毛皮张和土壤中炭疽杆菌PCR检测方法的建立

建立了检测炭疽芽孢杆菌染色体特异性基因序列(Ba813)的PCR技术，并用于模拟污染的羊毛、皮张和土壤中炭疽芽孢(Sterne菌株、PasteurⅡ菌株)的检测。结果表明，该方法可特异、敏感地检出0．5g羊毛中Sterne菌株的452个芽孢和PasteurⅡ菌株的688个芽孢；0．5g皮张中Sterne菌株的452个芽孢和PasteurⅡ菌株的1032个芽孢；0．5g土壤中Sterne菌株的452个芽孢和PasteurⅡ菌株1376个芽孢。证实PCR可用于羊毛、皮张和土壤等外环境中炭疽杆菌芽孢的检测。     

贵州省一起皮肤炭疽疫情的病原学分析

为了对贵州省一起疑似皮肤炭疽疫情进行病原学检测和分析,以期为疫情的处置和防控提供科学依据,对患者皮肤渗出液、病死马肌肉及其刨剐地土壤样本进行炭疽芽孢杆菌分离培养,对疑似菌落采用革兰染色镜检、噬菌体裂解试验和青霉素抑制试验进行鉴定,进一步采用普通PCR和实时荧光定量PCR法(Real-time PCR)检测炭疽芽孢杆菌疑似菌株PagA、Cap及rpoB基因。结果共检出7株炭疽芽孢杆菌疑似菌株,其中从患者皮肤渗出液分离出1株菌,从土壤标本分离出6株菌。传统方法将7株疑似菌株鉴定为炭疽芽孢杆菌,普通PCR和Real-time PCR检测结果均显示7株菌株rpoB、PagA和Cap基因阳性。说明从患者皮肤渗出液和土壤标本均检出具有致病基因的炭疽芽孢杆菌,提示该起疫情为一起由炭疽芽孢杆菌所致的皮肤炭疽疫情。     

动物炭疽实验室诊断技术的研究进展

正炭疽是由炭疽杆菌(Bacillus anthracis)引发的一种急性、热性、败血性的人畜共患传染病,炭疽芽孢杆菌为革兰氏阳性菌。炭疽的研究者多从以下三个方面对炭疽疾病进行研究,即炭疽杆菌的基因组、炭疽杆菌的荚膜及炭疽杆菌的毒素。炭疽杆菌的基因组由染色体及两个大质粒(p XO1和p XO2)组成,这两个大质粒含有炭疽芽孢杆菌的两大毒力因子,即炭疽毒素、抗吞噬荚膜多肽的编码基因,美国基因组研究     

牛乳中结核分支杆菌复合群的实时荧光PCR检测技术

本研究建立了一种实时荧光PCR检测奶牛乳液中结核分支杆菌复合群的技术，克服了以往牛结核PCR检测样品取样难的缺陷，具有检测灵敏度高、特异性强等优点，可用于奶牛乳房结核的早期诊断以及乳品安全生产检测。以乳液中加卡介苗作为模拟临床检测样本，对DNA提取方法进行改进，利用基于TaqMan技术的实时荧光（real—time）PCR检测。选择分支杆菌编码16sRNA基因和23sRNA基因之间ITS（internal transcribed spacer）序列作为扩增靶序列，研究结果表明卡介苗DNA提取效率提高，实时荧光PCR检测灵敏度达到每毫升乳液检测10个结核分支杆菌。奶牛乳液中结核分支杆菌复合群的检测对兽医系统疫病监测及诊断部门、奶牛养殖业、乳品生产行业有重要应用价值。     

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