犬吉氏巴贝斯虫截短型抗原BgTRAP的重组表达及其在诊断上的初步应用

吉氏巴贝斯虫Bg TRAP蛋白是一个重要的诊断抗原候选分子。为建立一种实用的犬吉氏巴贝斯虫血清学诊断方法,本研究选取Bg TRAP C-末端跨膜区前,包含TSP功能区和抗原区的411个氨基酸的编码基因片段,重组表达了一个可溶性截短型Bg TRAP抗原,解决了完整蛋白重组表达纯化的困难。免疫荧光试验表明,截短型抗原具有良好的抗原性。纯化的截短型抗原作为酶联免疫试验诊断抗原,可清晰地区分阴性及阳性犬血清,并与其他病原感染无交叉反应,具有良好的特异性。犬感染吉氏巴贝斯虫系列血清检测表明,该抗原可检测早期感染(4 d)和感染200 d以后的样本。结果提示,重组Bg TRAP截短型抗原可作为一种诊断制剂检测犬吉氏巴贝斯虫抗体。     

克隆表达的犬吉氏巴贝斯虫重组GST-P130kDa 用于血清学诊断

利用抗体法（picoBlueTMImmunscreening Kit,应用B.gibsoni感染血清）从犬吉氏巴贝斯虫（B.gibsoni）裂殖子mRNA制备的吉氏巴贝斯虫cDNA文库中进行免疫筛选,选出目的基因相cDNA片段（阳性克隆）.测序验证后将该cDNA（基因）克隆至原核表达载体pGEX-4T-3,构建重组质粒,在大肠杆菌E.coli（DH5a）中以GST融合蛋白的形式表达（SDS-PAGE分析证明）;表达产物为130kDa的可溶性融合蛋白.免疫学分析（Western blot和ELISA）结果表明,犬吉氏巴贝斯虫重组GST-P130kDa融合蛋白（rBg GST-P130kDa）能与犬吉氏巴贝斯虫（B.gibsoni）感染血清起反应,且与犬巴贝斯虫（B.canis）无交叉反应.本试验利用犬吉氏巴贝斯虫重组BgGST-P130kDa融合蛋白（作为重组抗原）检测巴西（n=310）、日本（n=100）和中国（n=114,n=30）等国随机采集的自然感染狗血清,其结果为巴西狗血清阳性率为55%、日本为8%、中国为1～9%;其结果与间接荧光抗体法（IFAT）结果为一致（作为验证）.结果表明重组BgGST-P130kDa融合蛋白具有较强的免疫原性和特异性,可用于吉氏巴贝斯虫病的诊断,这为吉氏巴贝斯虫病的早期诊断和深入研究犬吉氏巴贝斯虫病的特异性重组抗原及重组疫苗奠定基础.     

犬吉氏巴贝斯虫南京分离株HSP70基因的原核表达及抗原性分析

提取南京地区经鉴定确诊为犬吉氏巴贝斯虫感染病犬血液中的DNA,设计引物采用PCR方法扩增犬吉氏巴贝斯虫HSP70基因,克隆至pET-32a质粒中,转化Rosetta-gami(DE3)pLyS大肠杆菌,利用IPTG诱导表达得到重组热休克蛋白(heat shock protein 70,HSP70)。用Ni柱纯化重组HSP70蛋白免疫BALB/c小鼠制备抗血清,并分析重组蛋白的免疫原性及反应原性。本研究成功构建了pET-32a-hsp70重组质粒,IPTG诱导后表达出可溶的重组HSP70蛋白。经过Western blot分析,表达的重组蛋白不但能够免疫小鼠产生抗体,还可以和天然犬吉氏巴贝斯虫抗血清发生特异性反应,为进一步建立犬巴贝斯虫免疫血清学诊断和疫苗抗原的开发奠定基础。     

吉氏巴贝斯虫多个GPI锚定蛋白基因的筛选与生物信息学分析

吉氏巴贝斯虫是一类红细胞内专性寄生的血液原虫,犬一旦感染终生带虫,治疗后时有复发,因此需要简便快捷的临床诊断方法及时确诊治疗。GPI锚定蛋白是虫体表面重要的抗原分子,是很好的潜在诊断标识,在虫体粘附、识别以及入侵红细胞等过程中具有非常重要的作用。为了筛选吉氏巴贝斯虫GPI锚定蛋白基因,通过搜索NCBI、Uniprot以及PiroplasmaDB数据库中吉氏巴贝斯虫表面抗原,与武汉株吉氏巴贝斯虫数据库作比对,筛选出可能的吉氏巴贝斯虫武汉株的GPI锚定蛋白基因,并通过bigPI、PredGPI、GPI-SOM网站预测其GPI锚定位点。运用生物信息学分析软件预测其氨基酸信号肽、跨膜区、疏水区以及结构域和抗原表位等,分析其生物信息特性。通过分析确定了5个吉氏巴贝斯虫武汉株的GPI候选抗原,分别命名为BgP50-WH、BgP47-WH、BgP45-WH、BgP32-WH、BgP12-WH,其GPI锚定位点均位于C端,长度约为21～22个氨基酸,均有信号肽并含有4个抗原表位。分析结果表明,这5个GPI抗原均有望成为吉氏巴贝斯病的诊断标识,其中BgP47-WH和BgP50-WH与抗体亲和力最强,最具有成为诊断标识的潜力。     

青海省西宁地区藏獒巴贝斯虫病的血清学检测

用基于重组的犬吉氏巴贝斯虫P50蛋白作为ELISA诊断抗原,对来自青海省西宁地区的5个藏獒养殖基地的118份藏獒血清,进行犬巴贝斯虫病的血清学诊断,共检出阳性血清12份,阳性率为10.17%。结果初步表明西宁地区部分藏獒养殖基地中存在犬吉氏巴贝斯虫的感染。     

犬巴贝斯虫抗体胶体金检测卡制备与评价

试验旨在快速检测犬巴贝斯虫抗体,利用胶体金免疫层析技术制备犬巴贝斯虫抗体胶体金检测卡并评价其功能性。将羊抗鼠免疫球蛋白G (IgG)和犬巴贝斯虫表面抗原Bg P47重组蛋白（Bg P47 Ag）包被于NC膜上分别做为质控线和检测线,胶体金标记的鼠IgG和鼠抗犬IgG均匀喷点在聚酯纤维膜上作为标记垫,将化学品配制的玻纤溶液均匀涂在样品垫上,并用吸水纸和PVC底板制备犬巴贝斯虫抗体胶体金检测卡,对其特异性、灵敏度、均一性、加速稳定性、实时稳定性进行评价,并与ELISA、PCR检测结果进行对比。结果显示,该检测卡不与犬莱姆抗体（lyme Ab）阳性犬血清、犬瘟热病毒抗体（CDV Ab）阳性犬血清、犬细小病毒抗体（CPV Ab）阳性犬血清、犬无形体抗体（Anaplasma Ab）阳性犬血清、犬三联疫苗阳性免疫犬血清样本发生交叉反应;可检测浓度稀释至1∶256倍的样本;有效期可达到24个月。研究表明,该检测卡可快速、准确地检测犬巴贝斯虫抗体,为动物临床诊断和治疗提供参考。     

犬吉氏巴贝斯虫人工感染试验方法的建立

选用健康本地杂种犬,分为2组,一组手术切除脾脏,另一组未经任何处理,然后2组犬人工静脉接种含犬吉氏巴贝斯虫的病犬血液,接种后观察临床症状、进行实验室检查(血涂片、血常规、病原的PCR检测),检测2组犬人工感染犬吉氏巴贝斯虫后的发病情况。结果显示,人工感染后2组犬均表现出自然感染犬吉氏巴贝斯虫后的临床症状,实验室检查血涂片中检测到犬吉氏巴贝斯虫虫体,血常规表现出严重的贫血,PCR能扩增出犬吉氏巴贝斯虫的基因片段。2组犬比较显示,切脾组的红细胞感染率和贫血程度均高于未切脾犬。本试验成功建立了犬吉氏巴贝斯虫人工感染方法,且脾切除后感染效果优于直接静脉感染。     

间接荧光抗体试验诊断犬吉氏巴贝斯虫病

利用冷藏抗原玻片 ,用间接荧光抗体试验检测感染吉氏巴贝斯虫犬血清 ,具有特异性强、敏感性高、快速、准确、操作简单等特点。对疫区 82份犬血清的检查 ,阳性率为 37 8% ;对非疫区 50份犬血清的检查 ,假阳性率为 2 %。与血液涂片染色检查比较 ,该法检出率高 ,结果可靠 ,可用于犬吉氏巴贝斯虫病的诊断和流行病学调查     

吉氏巴贝斯虫和犬瘟热病毒混合感染犬的临床观察

笔者在进行犬吉氏巴贝斯虫病研究过程中发现,接种吉氏巴贝斯虫的摘脾犬又感染犬瘟热病毒后,病犬红细胞的吉氏巴贝斯虫染虫率不但不升高,反而随着犬瘟热症状的加剧而染虫率不断下降。现将观察结果简述如下。要提取吉氏巴贝斯虫抗原和核酸,必须首先大量繁殖虫体。一般家犬感染该虫后,只能隐性带虫,红细胞染虫率极低。只有将带虫犬的脾脏摘除才能达到大量增殖虫体的目的。笔者先后将3只接种吉氏巴贝斯虫的家犬摘脾后,观察其血液内虫体的增     

一例犬巴贝斯虫病继发肝损伤病的诊治

<正>犬巴贝斯虫是经由蜱传播的血源性原虫,属于巴贝斯科巴贝斯属,包括犬巴贝斯虫、吉氏巴贝斯虫、韦氏巴贝斯虫,主要寄生于犬的红细胞内,引起大量红细胞破裂,导致严重的贫血性疾病[1]。我国犬巴贝斯虫病主要是由犬巴贝斯虫和吉氏巴贝斯虫引起的。吉氏巴贝斯虫:为小型虫体,多存在于红细胞的边缘或偏中央,呈环形、椭圆形、原点形、小杆形,偶尔也可见成对的小梨子形虫体。在一个红细胞内可寄生的虫体以1~2个为多。犬巴贝斯虫:为大型虫     

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.     

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.     

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 -