鸡致病性大肠杆菌fimC基因缺失菌株的构建及生物学特性鉴定

本研究目的是研究fimC基因的功能。基于致病性大肠杆菌（APEC）Ⅰ型菌毛fimC基因的已知序列,通过PCR扩增了缺失169 bp的fimC基因片段,将其克隆到自杀载体pCVD442中,通过接合性转导将重组自杀性质粒pCVD442∷△fimC从大肠杆菌SM10λpir转到O2（Nal^R）中。根据同源重组原理构建了fimC基因缺失突变菌株O2（△fimC）。结合PCR扩增、测序结果及透射电镜观察,证明正确构建了fimC基因缺失突变株O2（△fimC）。体外生长及生化反应试验中,O2（△fimC）与亲本株存在一定差异。药敏试验中,两者无明显区别。鸡气管黏膜黏附试验及雏鸡的致病性试验表明：突变株在鸡气管黏膜上的黏附能力是亲本株的1/50,对雏鸡的致病性亦明显下降。鸡致病性大肠杆菌Ⅰ型菌毛fimC基因缺失突变株的构建为深入探讨fimC基因在鸡大肠杆菌致病过程中所起的作用、Ⅰ型菌毛与机体相互作用的分子机制及fimC基因缺失突变株其他生物学特性的研究奠定了物质基础。     

鸡致病性大肠杆菌菌毛fimC基因的表达及重组fimC蛋白卵黄抗体的免疫保护效力

在表达鸡致病性大肠杆菌菌毛fimC基因的基础上,对fimC蛋白卵黄抗体的免疫保护效力进行测定。对鸡致病性大肠杆菌O2血清型菌株fimC基因序列进行扩增,扩增产物克隆至pMD18-T载体并测序。测序结果显示,fimC基因全长657 bp,编码218个氨基酸,与人源大肠杆菌fimC基因进行同源性比较,核苷酸序列同源性达97.9%,氨基酸序同源性为96%。从重组质粒pMD18-T-fimC上将fimC基因亚克隆到表达载体质粒pET-30c上,转化大肠杆菌BL21（DE3）,表达出预期的25 ku融合蛋白,采用Western Blot对表达产物进行反应原性分析,结果显示fimC融合蛋白具有较好的抗原反应特异性。用表达的fimC蛋白免疫产蛋母鸡,制备fimC高免卵黄抗体。用制备的fimC高免卵黄抗体免疫1日龄健康雏鸡,1 d后注射鸡致病性大肠杆菌进行攻毒,结果显示,fimC卵黄抗体免疫组死亡率为40%,对照组分别为60%、70%,证明所制备的fimC蛋白卵黄抗体能在一定程度上抵抗鸡致病性大肠杆菌的攻击。     

鸡致病性大肠杆菌16S rRNA PCR鉴定方法的建立

为了建立一种鸡致病性大肠杆菌的快速检测方法,本试验通过无菌采集疑似感染致病性大肠杆菌鸡的心、肝、脾、肾作为病料,经过细菌分离培养、生化鉴定和致病力试验,利用大肠杆菌16S rRNA通用引物对3株致病菌进行PCR扩增,均得到了特异性扩增产物。并对扩增产物进行核酸测序和BLAST比对。结果显示,分离得到3株鸡致病性大肠杆菌(DZMG、DGCG1、DGCP3),扩增出的16S rRNA基因序列与鸡致病性大肠杆菌基因序列同源性为100%。提示该检测方法具有特异性高和简单快捷的特点。     

34株鸡大肠杆菌的分离鉴定及致病力分析

本研究从鸡泄殖腔取样,分离大肠杆菌菌株,进行生化鉴定。结果显示34株菌株符合大肠杆菌的培养特性。将分离得到的34株大肠杆菌与购买的4株鸡大肠杆菌分别接种鸡胚,对致病力进行分析。结果采用数据处理软件SPSS分析90%置信区间,其中19株为致病力较强菌株(致死率大于上限38.4%),16株为致病力较弱或无致病力菌株(致死率小于下限25.1%),3株为中等致病力菌株(致死率在上限和下限之间)。通过套式PCR对38株菌的iss基因进行检测,阳性率为100%。结果表明,iss基因(increased serum survival gene)在鸡大肠杆菌中广泛存在,38株鸡大肠杆菌均有iss基因,但致病力大小不同,故推测致病力大小可能与iss基因的存在无关。     

1株犬类志贺邻单胞菌的分离鉴定及序列分析

从病死犬病料中分离到1株类志贺邻单胞菌,并对该菌做了生理生化鉴定、药敏试验,致病性试验。结果表明,本菌对多种抗生素耐药,其庆大霉素耐药机制与所携带的质粒相关;本菌对小白鼠有强致病性,其LD50为1．6×10^7.4CFU。用PCR方法扩增分离菌株16SrDNA基因并测序,并将其与GenBank上其他细菌16SrDNA核苷酸序列进行同源性分析。结果表明,分离株的16SrDNA核苷酸序列与类志贺邻单胞菌（GQ359962．1）的同源性为98％,因此将该分离菌株鉴定为致病性肠球菌,命名为YN-1株（云南-1株）。     

3株致病性鸡大肠杆菌P型菌毛结构基因的克隆、序列测定及同源性分析

用鸡致病性大肠杆菌分离株O1、O2和O78的基因组DNA做为模板，PCR分别扩增出了0．55kb的P型菌毛结构基因(papA)。将扩增得到的3个papA基因片段分别克隆进pGEM—T栽体中，转化至受体菌JM109中，用Amp／IPTG／X-gal琼脂平板蓝白菌落筛选法，得到含阳性重组子的菌株，提取质粒后用BamH Ⅰ和Sal Ⅰ双酶切进行鉴定。所得阳性重组子进行DNA序列测定，测序结果经DNA Star核酸分析软件包分析比较，结果表明，所构建的克隆质粒中均含有相应完整papA基因，其开放式阅读框架大小为549bp，编码182个氨基酸。此3株菌的P型菌毛结构基因的同源性为98.9％～100％，其中O1株和O78株的P型菌毛基因的ORF序列100％相同，O2株有2个碱基与前两者不同。     

16S rRNA基因联合gyrB、grpE、recA基因对鸡源鲍氏志贺菌hn03株进行分子起源分析

为了从基因水平鉴定鸡源鲍氏志贺菌,分别根据GenBank中登陆的相应序列针对16srRNA、gyrB、grpE、recA4基因设计4对引物,用PCR扩增出鸡源鲍氏志贺菌4基因的部分片段并进行了克隆测序。将所获序列与GenBank中同源性较高的相关序列相比较,计算种间相似性,并构建志贺菌的系统发育树,对分离株进行分类与鉴定。研究发现,从4基因的序列分析结果总体上可知鸡源志贺菌与人鲍氏志贺菌同源关系最近,同源性达99．6％～100％。几乎所有志贺菌均落入大肠杆菌的分支中,且在鉴别细菌中recA基因和gyrB基因比传统中常用的16S rRNA基因差异更显著。由此证明用细菌分类和鉴定的“金标准”16S rRNA联合gyrB、grpE、recA3个基因进行序列分析来鉴定志贺菌是一种可靠、成本较低的方法;由上述基因序列构建的系统发育树提示,该株鸡源志贺菌可能是与人志贺菌密切相关的新种或是它们的亚种。     

银川地区鸡源致病性大肠杆菌耶尔森菌强毒力岛相关基因的检测及序列分析

为了研究耶尔森菌强毒力岛(HPI)在银川地区鸡源致病性大肠杆菌中的分布情况,试验根据HPI相关基因irp2和fyuA的参考序列设计引物,采用双重PCR方法对分离的20株鸡源致病性大肠杆菌进行这两种基因的扩增和检测,以及基因克隆和核苷酸序列分析。结果表明:irp2和fyuA基因在鸡源致病性大肠杆菌分离株中的阳性率均为40%(8/20);这两种毒力岛相关基因的核苷酸序列与GenBank中报道的基因核苷酸同源性高达98%以上。说明irp2和fyuA基因具有较高的保守性。     

60株大肠杆菌的分离与致病性鉴定

大肠杆菌(Escherichia coli,E.coli)是鸡肠道常在菌,部分菌株具有致病性。鸡大肠杆菌病就是由大肠杆菌中的某些致病性菌株引起的鸡感染性疾病。致病性大肠杆菌均携带毒力岛基因ChuA。为鉴定分离自临床病例的鸡大肠杆菌的致病性,本试验采用PCR法检测ChuA基因进行分子生物学鉴定,60株大肠杆菌中有31株属于致病性大肠杆菌,总阳性率为51.67%。     

致病性鸡大肠杆菌type1菌毛fimC基因的克隆与序列测定

根据国外发表的人致病性大肠杆菌fimC基因序列,在其保守区设计了带有BamHⅠ/HindⅢ酶切位点的一对引物,应用PCR技术以鸡致病性大肠杆菌O2基因组DNA为模板扩增到一个片段,大小约为700bp,将扩增产物克隆到pMD18-T载体上,并转化于大肠杆菌TG1宿主菌,经酶切和筛选,得到阳性重组质粒.通过对阳性重组质粒核酸序列测定,确定此DNA片段为鸡大肠杆菌fimC基因.     

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