表达牛乳铁蛋白肽的重组乳酸乳球菌对雏鸡抗鸡白痢沙门氏菌感染的效果研究

为探索表达牛乳铁蛋白肽的重组乳酸乳球菌pAMJ399-LFBA/MG1363对雏鸡抗鸡白痢沙门氏菌感染效果的影响,本实验将该重组乳酸乳球菌饲喂1日龄雏鸡,连续饲喂14 d,并设立空白对照组,第7 d时经灌服鸡白痢沙门氏菌同时感染实验组和对照组,连续观察7 d;分别于攻菌前以及攻菌后的不同天数采集血清及盲肠黏液,检测结果显示攻菌后饲喂重组乳酸乳球菌组雏鸡血清中IgG以及黏液中SIgA的含量极显著高于空白对照组。对各组雏鸡的心脏、肝脏、脾脏及十二指肠进行病理组织学检查,结果显示饲喂重组乳酸乳球菌组各器官的病变情况相对较轻,而且雏鸡盲肠扁桃体中非特异免疫信号通路中相关分子TLR4、My D88、NF-κB、TNF-α、IL-8、IL-6等的m RNA表达相对较高。以上结果表明,表达牛乳铁蛋白肽的重组乳酸乳球菌在抗鸡白痢沙门氏菌感染的过程中可以起到保护雏鸡免受感染的作用。     

鸡IL-4和IL-2重组融合基因佐剂对鸡球虫活疫苗的增效作用研究

为了研究鸡白细胞介素4(IL-4)和IL-2重组基因佐剂pCI-chIL-4-chIL-2-EGFP对鸡球虫活疫苗的增效作用,试验采用基因佐剂配合鸡球虫活疫苗对雏鸡进行免疫攻毒试验,测定该基因佐剂的最小有效剂量、给药途径,使用该基因佐剂对鸡球虫活疫苗的最短免疫间隔时间和免疫产生期的影响。结果表明：抗球虫指数(ACI)和相对增重率(RWG)随着基因佐剂剂量增高而升高,肌肉注射100μg/只时,ACI(189.32)和RWG(90.35%)达到高峰;口服基因佐剂300μg/只组的ACI(188.41)与肌肉注射基因佐剂100μg/只组接近;肌肉注射佐剂免疫攻毒组的两次免疫最短间隔时间(6 d)较不加基因佐剂组(7 d)缩短1 d;肌肉注射佐剂免疫攻毒组的免疫产生期为二免后第5天,较无基因佐剂免疫攻毒组提前1 d。说明肌肉注射IL-4和IL-2重组基因佐剂pCI-chIL-4-chIL-2-EGFP 100μg/只可提高鸡球虫活疫苗的免疫效果,缩短免疫产生期和免疫间隔。     

巨型艾美球虫gam56基因的截短原核表达及其产物的免疫保护效果

为评价gain56基因重组表达产物作为亚单位疫苗预防鸡巨型艾美球虫(E.maxima),感染的效果,以E.maxima NT株配子体总RNA为模板,RT.PCR扩增和克隆gain56基因,选择该基因两段丰富抗原表位的编码区片段.利用原核表达载体pGEX-6P-1在大肠杆菌中进行截短表达.以可溶性的GST-gain56-2融合蛋白为免疫原,设立高、中、低(1.0 mg、0.5 mg、0.25 mg)3个剂量,单独或使用弗氏完全佐剂,于5 d、12 d和19 d对雏鸡进行3次免疫,26 d用5 ×10~4个E.maxima孢子化卵囊进行攻虫,8 d后迫杀,对各组的存活率、相对增重率、卵囊减少率、ACI等指标进行统计分析.结果显示:就相对增重率而言各免疫组比未免疫攻毒组的高27.6%以上,而各免疫组之间差异不显著(p>0.05);就卵囊减少率指标而言,各免疫组较未免疫攻毒组均有不同程度减少,但是均小于75%;就ACI指标而言,各免疫组较未免疫攻毒组均有不同程度增加,以中剂量蛋白佐剂组为最高.GST-gam56-2融合蛋白对于E.maxima感染具有一定的免疫保护效力.     

不同佐剂和免疫途径对柔嫩艾美耳球虫SO7抗原免疫保护效果的影响

利用PGEX-6P-1融合表达系统将SO7基因在大肠杆菌中进行融合表达,对表达产物进行初步纯化和复性,制备免疫原。分别使用不同剂量的人参总皂甙和重组γ-干扰素以及弗氏完全佐剂作为免疫佐剂,于5日龄、12日龄和19日龄对雏鸡进行3次免疫,同时设立蛋白口服免疫组、蛋白皮下注射免疫组、卵囊口服免疫组、未免疫未攻毒组和未免疫攻毒组作对照,于26日龄用105个柔嫩艾美耳球虫卵囊进行攻毒,8d后扑杀,对各组的存活率、相对增重率、病变减少率、相对卵囊产量和抗球虫指数ACI等指标进行统计分析,比较免疫保护效果。与非免疫攻毒组以及不加佐剂的免疫攻毒组相比,佐剂使用后各组的增重、病变记分、相对卵囊产量、ACI等指标均有一定的改善,对柔嫩艾美耳球虫的人工感染可以提供部分保护。3种佐剂中,γ-干扰素和人参总皂甙能增强重组蛋白的免疫保护效果,且佐剂的剂量对免疫保护的效果有一定的影响,以5000Uγ-干扰素效果最佳,效果与E.tenella活卵囊免疫相当,弗氏完全佐剂的免疫增强效果不明显。单独使用重组蛋白皮下注射或口服免疫,虽有一定的保护作用,但不明显。一定剂量的γ-干扰素对SO7抗原的免疫保护效果起明显的增强作用,是一个具有很好临床应用前景的新型佐剂。     

柔嫩艾美耳球虫AMA1基因DNA疫苗的免疫保护效果

本研究旨在评价表达柔嫩艾美耳球虫(Eimeria tenella)顶膜抗原1(Apical membrane antigen 1,Et AMA1)基因的DNA疫苗对雏鸡柔嫩艾美耳球虫人工感染的免疫保护效果。将Et AMA1基因和鸡IFN-γ基因插入真核表达载体p CAGGS中,分别构建了真核表达重组质粒p CAGGS-Et AMA1和p CAGGS-Et AMA1-IFN-γ。将重组质粒p CAGGS-Et AMA1转染进293T细胞中,经间接免疫荧光和Western blot实验鉴定,观察其在体外表达情况。将p CAGGS-Et AMA1和p CAGGS-Et AMA1-IFN-γ于7日龄和14日龄腿部肌肉注射免疫雏鸡,21日龄人工感染1×104个柔嫩艾美耳球虫孢子化卵囊,第29日龄时处死试验鸡,以平均增重、卵囊产量和病变记分来评价重组质粒的免疫保护效果。结果显示,p CAGGS-Et AMA1转染的293T细胞出现明显的红色荧光;两种真核表达重组质粒免疫组鸡的平均增重与未攻虫组的平均增重差异不显著,不具有统计学意义,而未免疫攻虫组则与未攻虫组在增重方面差异具有显著统计学意义;免疫组鸡相较于未免疫攻虫组在病变记分方面显著下降;免疫组的卵囊减少率分别为67.43%和72.89%;p CAGGSEt AMA1-IFN-γ组在增重、盲肠病变记分和卵囊减少率方面都优于p CAGGS-Et AMA1组,但差异不具有统计学意义。表明构建的2种重组质粒对雏鸡柔嫩艾美耳球虫感染有一定的免疫保护效果。     

柔嫩艾美耳球虫TA4抗原基因cDNA在乳酸乳球菌中的克隆与表达

用PCR技术特异扩增了柔嫩史美耳球虫TA4抗原基因cDNA序列，克隆至质粒pMD18-T中，获得重组质粒pMD18-T-TA4，采用KpnⅠ／SacⅠ双酶切法及PCR确认正确后，将TA4基NcDNA目的片段亚克隆到乳酸乳球菌表达载体pMG36n中，电穿孔法转化乳酸乳球菌LM0230，获得重组质粒pMG36n-TA4，采用KpnⅠ／SacⅠ双酶切法及DNA测序证明cDNA序列完全正确。获得TA4乳酸乳球菌表达株，经SDS—PAGE电泳分析，结果表明表达产物与预期大小的TA4蛋白分子量一致。     

饲喂转球虫基因功能乳酸杆菌后对雏鸡体重的影响

为了验证转鸡柔嫩艾美耳球虫保护性抗原SO7基因的功能乳酸菌对人工球虫攻击的保护性 ,将这种转球虫SO7基因的功能乳酸杆菌给刚出壳的雏鸡饲喂 ,一周后攻球虫卵囊 5× 1 0 4 个 /只 ,通过对试验雏鸡的体重变化来检测转球虫SO7基因工程乳酸菌的有效性 ,设红、白对照组 ,转基因乳酸菌组和乳酸菌对照组。结果表明 :饲喂转基因乳酸菌组雏鸡 ,在攻击球虫后体重增重未受影响。这为球虫病的防治开辟了一条新的途径     

转球虫SO7基因功能乳酸杆菌对肉仔鸡感染球虫后盲肠病理学变化的影响

为了检测转鸡柔嫩艾美耳球虫保护性抗原SO7基因的功能乳酸菌对试验感染球虫攻击的保护性,用含SO7基因的功能乳酸菌饲喂1日龄雏鸡,连喂11d后,以5×104个/只剂量接种球虫卵囊,通过观察雏鸡的组织病理学变化来评价转球虫SO7基因工程乳酸菌的效果。结果显示:饲喂转基因乳酸菌组雏鸡受球虫攻击的组织病理学变化最轻微。从而证明含保护性抗原SO7基因的功能乳酸菌对试验感染球虫攻击具有一定的保护性。本研究为外源性的保护性抗原基因在乳酸菌中表达用于球虫病的防治奠定了基础。     

雏鸡免疫堆型艾美球虫后外周血T淋巴细胞亚群CD_4~ T和CD_8~ T的动态变化

为研制鸡球虫早熟系疫苗,探讨早熟系诱导的细胞介导免疫机理,实验采用流式细胞仪(FACS)检测堆型艾美球虫早熟株免疫雏鸡后,在不同时间外周血T淋巴细胞亚群CD4 T和CD8 T的动态变化。试验结果表明,免疫组CD4 T数量动态变化显著高于不免攻毒组(P<0.05),免疫后16天达到高峰;不免攻毒组攻毒后上升速度较快,于攻毒后1周达到高峰。CD4 T的数量变化,说明它在启动细胞免疫应答和控制再次感染中发挥着作用。免疫组CD8 T数量动态变化亦显著高于不免攻毒组(P<0.05),免疫组和不免攻毒组CD8 T数量均在攻毒后1周达到最大值,表明CD8 T在抵抗感染中起作用。各组间CD4 T/CD8 T比值无明显差异,但免疫组CD4 T/CD8 T变化较为平缓。上述堆型艾美球虫早熟系免疫雏鸡后,CD4 T和CD8 T在不同时间数量的增减及其比值的变化,将为早熟系疫苗在生产实践中的合理使用提供理论依据。     

鸡新城疫病毒HN基因亚单位疫苗诱导免疫保护的实验研究

实验分别将应用Bac to Bac系统表达的新城疫病毒四平株和长春株血凝素-神经氨酸酶基因（HN基因）蛋白的重组杆状病毒感染Sf-9昆虫细胞后28℃培养72h后，洗涤、收集昆虫细胞，离心浓缩，-20℃冻融3次，用HA-HI实验和HN单抗中和试验测定表达产物的活性，将表达的重组蛋白作为亚单位疫苗免疫鸡，用间接ELISA和HI试验测定鸡体内抗体效价，免疫后第15d用国家标准强毒NDVF48E8攻毒，统计免疫保护率，结果表明，表达的NDVHN重组蛋白能够诱导鸡体产生抗NDV特异性IgG抗体和HI抗体，实验Ⅰ组（四平株）雏鸡保护率为65%，实验Ⅱ组（长春株）雏鸡保护率为100%。     

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.     

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