减毒沙门氏菌为载体传递DNA疫苗诱导对新城疫病毒的免疫保护

探讨了以减毒鼠伤寒沙门氏茵为栽体传递新城疫病毒DNA疫苗的安全性、免疫原性和可行性。将含新城疫病毒(NDV)F48E9株融合蛋白(F)基因的真核表达质粒pcDNA3-F的重组减毒鼠伤寒沙门氏菌ZJ111株(ZJ111／pcD-NA3一F菌株)，以10^8CFU进行首免，2周后二免，三免后4周攻击强毒株F48E9，观察其安全性和免疫原性，同时设只含空载体pcDNA3的ZJ111／pcDNA3菌株对照及口服PBS对照。结果表明：重组ZJ111／pcDNA3-F菌株具有良好的安全性。对强毒株攻击的保护率达64．7％。重组ZJ111／pcDNA3-F菌株不仅能诱导雏鸡产生NDVELISA抗体，而且诱导产生的法氏囊B淋巴细胞和胸腺T淋巴细胞增殖反应显著高于ZJ111／pcDNA3时照组。这些结果提示，减毒沙门氏菌为载体不仅可直接将NDVF基因呈递给鸡体细胞进行表达，产生抗NDV的体液免疫，而且还可诱导细胞免疫应答。     

传染性喉气管炎新城疫鸡痘重组病毒免疫效力的研究

在表达鸡传染性喉气管炎病毒(ILTV)糖蛋白gB基因和新城疫病毒(NDV)F基因的重组鸡痘病毒(rF-PV-gB-F)安全性检验合格后,以5.0×101~5.0×104PFU不同含量按0.1mL/鸡的剂量免疫100只30日龄SPF鸡,30d后分组分别用ILTVWG株和NDVF48E9株强毒进行攻击。免疫鸡抗鸡痘病毒抗体都转为阳性,痘反应和接种剂量有关,重组疫苗的最小反应剂量为50PFU。重组疫苗可以诱发对新城疫和传染性喉气管炎的保护,0.1mL/鸡的接种量在500~5000PFU浓度范围内的免疫效果最好,对于ILTV攻击的发病保护率在70%以上,对NDV强毒攻击的抗死亡保护率可以达到80%,这为进一步考察疫苗的免疫效力试验以及进行田间试验奠定了基础。     

新城疫La Sota疫苗对山东分离株的免疫保护试验

用标准疫苗株La Sota活疫苗在隔离器中接种SPF鸡,进行免疫保护试验.免疫后,每7 d采血监测NDV抗体,免疫后2周,利用经过鉴定的新城疫病毒(NDV)潍坊毒SGM01、昌乐毒SCL03、东营毒HY、日照毒SRZ03、莘县毒SSX03和标准强度F48E9分别进行攻毒试验,同时设SPF鸡对照.每天观察,及时剖检发病鸡,检查鸡群病变,确定疫苗的保护性.结果表明,La Sota疫苗能对除SGM01和HY以外的病毒攻击的SPF鸡提供较好的保护.     

Fowl typhoid: assessment of a disinfectant oral dose to reduce horizontal spread and mortality

To determine the most appropriate dose in drinking water of the disinfectant N-alkyl dimethyl benzyl ammonium chloride (TIMSEN), a fowl typhoid challenge trial was carried out using 21-day-old Salmonella-free chickens. In a pretrial, performed with six groups of 10 chickens each, it was shown that the disinfectant was atoxic and safe when administered during 15 days at doses of 0 ppm, 200 ppm, 400 ppm, 800 ppm, 1600 ppm, or 3200 ppm. Thereafter, a challenge trial was performed with 390 chickens divided into six groups of 65 birds each. Chickens were treated during 9 days with oral doses of 0 ppm, 25 ppm, 50 ppm, 100 ppm, 250 ppm, and 500 ppm (groups identified as A, B, C, D, E, and F, respectively). Twenty-four hours after the beginning of the treatment, 30 chickens of each group were orally inoculated with 10(9) colony-forming units of Salmonella Gallinarum strain INTA 91 per bird. The remaining 35 unchallenged chickens from each group were left in the same cage in close contact with the other challenged birds of their group. All surviving chickens were sacrificed 8 days after challenge. Livers from all birds were examined for the presence of Salmonella Gallinarum. Salmonella Gallinarum was isolated from all challenged chickens and from some unchallenged chickens of groups A (4/35), E (3/35), and F (6/35), but not from any unchallenged chicken from groups B, C, and D. Doses of 50 ppm (group C) significantly reduced mortality (30%) in comparison with the untreated control group A (65%). Mortality in group B (45%) was not significantly different from group C. Administration of higher doses of the disinfectant resulted in significantly higher mortality rates, 70% in group E and 85% in groups D and F. Increased infection and mortality rates of groups D, E, and F might have been caused by inhibition of the protective action of the normal gut flora. To reduce horizontal infection and mortality, the manufacturer's prescribed oral dose of 25 ppm may be increased up to 50 ppm. Nevertheless, higher doses should be avoided because they may cause horizontal increased spread of the disease and mortality.     

发酵中药对蛋鸡生长性能、抗体水平及肠道菌群的影响

【目的】 研究发酵中药对蛋鸡生长性能、抗体水平和肠道微生物菌群的影响。【方法】 选用1日龄科宝蛋鸡84只,预饲至13日龄,于14日龄进行正式试验,将雏鸡随机分为7组,每组单笼饲养,每笼12只,分别为空白对照组（A组）,复方中药组（B组）,发酵中药低剂量组（C组）、中剂量组（D组）、高剂量组（E组）,发酵菌液组（F组）,对照药物黄芪多糖组（G组）。试验期间记录每日给料量与剩料量,于试验第1（14日龄）、14（28日龄）、28（42日龄）天清晨空腹称重,计算各阶段平均日增重、平均日采食量及料重比。于试验第7、14、21、28天每组鸡翅下静脉采血,用于抗体效价的检测。试验结束后对A、B、D、F、G组鸡进行剖检,每组取5只鸡的盲肠内容物进行高通量测序分析。【结果】 试验第1~14天,D、E组平均日采食量均极显著高于A、F、G组（P<0.01）,D、E组平均日增重极显著或显著高于A、F组（P<0.01;P<0.05）;F、G组料重比均极显著低于其他各组（P<0.01）。试验第15~28天,C、E组平均日采食量均显著高于B、F组（P<0.05）,B组平均日增重极显著高于其他各组（P<0.01）,E组显著高于A组（P<0.05）;B组料重比极显著低于其他各组（P<0.01）,D、E、F、G组料重比均极显著低于A组（P<0.01）。试验第7天,各组鸡血清抗体效价均无显著差异（P>0.05）;试验第14天,E组抗体水平均显著高于A、F、G组（P<0.05）;试验第21和28天,E组抗体水平显著高于A组（P<0.05）。D组Chao1和Observed species指数最高,但与其他组间无显著性差异（P>0.05）。在门水平上,D和G组盲肠中拟杆菌门相对丰度均显著高于A组（P<0.05）;在属水平上,D组盲肠中拟杆菌属相对丰度均显著高于A组（P<0.05）。【结论】 发酵中药可以提高鸡的生长性能和抗体水平,对肠道菌群有明显的调控作用。     

密码子优化增强新城疫病毒DNA疫苗的表达水平和免疫效果

为提高新城疫病毒（Newcasti Disease Virus,NDV）F基因DNA疫苗的表达量,增强其免疫效果。按照鸡体内偏嗜性密码子人工合成了NDV F48E9株的F基因optiF,插入到真核表达载体pVAX1中获得pVAX1-optiF。将F48E9株的F基因pVAX1-F与pVAX1-optiF分别转染COS-7细胞,72h后间接免疫荧光和Western blot检测细胞中瞬时表达的F蛋白。将质粒pVAX1、pVAX1-F和pVAX1-optiF以200μg／只的剂量分别股四头肌多点注射18只2周龄SPF鸡,2周后加强免疫1次,同时设立PBS对照。二免2周后每组取12只鸡以100EID50的F48E9株NDV强毒进行攻毒,评价这2种DNA疫苗的免疫效果。结果表明,F基因密码子的优化可显著提高NDVDNA疫苗诱导的保护性体液免疫和细胞免疫应答水平,攻毒后所有pVAX1-optiF免疫鸡均获得保护（12／12）,而pVAX1-F组保护率只有17％（2／12）。DNA疫苗的免疫效果与抗原基因的表达量及表达抗原的免疫原性有直接关系。与未经修饰的F基因相比,修饰后的F基因体外瞬时表达水平明显提高。     

新城疫LaSota活疫苗对基因Ⅶ型分离株的免疫保护性试验

从山东省发病鸡群分离鉴定了一株新城疫病毒(NDV),命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因Ⅶ型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSot a后14 d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10 d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9 d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLYO1攻毒组第5 d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSot a活疫苗对F48E8和SDLY01均能提供100%免疫保护,但不能完全抑制基因Ⅶ NDV分离株在体内的复制和排毒。     

鸡新城疫不同弱毒株毒力及免疫原性研究

本研究比较了9株鸡新城疫不同弱毒株的毒力和免疫原性。结果显示,新城疫弱毒耐热B株、V4株、Ulster 2C株基本不致死鸡胚,ICPI为0,MDT/MLD大于150h;Hitchner B1株及F株7/10鸡胚致死,死胚胎儿有明显充血、出血病痕,120h活胚明显萎缩,ICPI为0.213～0.238,MDT约为120h;La Sota株、Clone30株、N79株及VG/GA株120h内鸡胚全部死亡,死胚胎儿有明显充血、出血病痕,ICPI为0.3～0.4,MDT约为103～109h。9株NDV的IVPI均为0对鸡只不致病,但免疫原性有所不同,NDVB株免疫原性较好,全部保护,NDVF株最弱。     

不同类型鸡新城疫La Sota疫苗的免疫应答比较

A、B、C、D组SPF鸡分别接种La Sota加佐剂活疫苗,未加佐剂活疫苗,氢氧化铝胶佐剂苗和油佐剂灭活疫苗,通过采集血清进行红细胞凝集抑制试验,测定其抗体水平,并进行比较。结果表明,La Sota加佐剂活疫苗组可产生较高的抗体滴度,并持续较长时间;而未加佐剂疫苗组血清阳转较弱,且持续时间较短;油佐剂灭活疫苗组抗体滴度最高,维持时间最长。     

Vaccination of cyclophosphamide-treated chickens against Newcastle disease virus infection

Newly-hatched chickens were treated with 3 mg of cyclophosphamide (CY) per day for 4 consecutive days. At 2 weeks of age, these chickens, together with a group of untreated controls, were vaccinated intranasally or subcutaneously with the La Sota strain of Newcastle disease virus (NDV). All chickens were challenged intranasally with the GB strain of NDV 2 weeks later. CY-treated, intranasally vaccinated chickens were highly resistant to NDV challenge, yet none of the chickens produced any detectable humoral antibodies to NDV; antibodies to NDV were detectable in the tracheal washings, however.     

Infection of the chicken with a virulent or avirulent strain of Mycoplasma gallisepticum alone and together with Newcastle disease virus or E. coli or both

The influence of the virulence of M. gallisepticum (Mg) was studied in multiple infections of chickens involving Mg, Newcastle disease virus (NDV) and E. coli. Separate groups of 3-week-old chickens were inoculated supra-conjuctivally with a virulent and an avirulent strain of Mg alone and in combination with the La Sota strain of NDV, the 01 serotype of E. coli, and both NDV and E. coli. In addition, chickens were inoculated with NDV alone, E. coli alone, and with NDV and E. coli; one group was left uninfected.Clinical signs, lesions, recovery of the pathogens and the serological response were observed for all groups for 3 weeks after infection and for those involving Mg alone and Mg together with NDV for 18 weeks.No clinical signs were seen in any of the birds; in each infected group some showed mild lesions of the trachea and air sacs without any marked difference among the groups.Although the numbers of birds examined were small, the virulent strain of Mg was more readily recovered than the avirulent, from the respiratory tract, in the first 3 weeks following multiple infections. However, the virulence of Mg had no influence on the recovery of the other pathogens.For the first 6 weeks after infection there was a direct relationship between the virulence of the Mg and the proportion of birds with agglutinins to the Mg rapid serum agglutination (RSA) test in single or multiple infections; multiple infections enhanced the antibody response to both virulent and avirulent mycoplasma. For NDV, multiple infections, particularly involving E. coli, enhanced the peak titre of haemagglutination-inhibition antibodies and accelerated their appearance; the virulence of the Mg had no apparent effect on this.Neither the mycoplasma nor NDV were detected in the trachea by immunofluorescence.     

Immunization of day-old chickens against Newcastle disease.

The avirulent Newcastle disease virus strain designated NDV-6/10, selected by B. Lomniczi at the Veterinary Medical Research Institute, Hungarian Academy of Sciences, is completely safe for day-old chickens by aerosol vaccination. Aerosol immunization using the Hungarian-made MASTERDROP generator (particle size: maximum 7 microns) caused no vaccination reactions among 206,000 chickens with different maternal antibody levels. Other vaccines given simultaneously did not significantly affect the protection elicited against Newcastle disease (ND). Almost 100% and 90% of the aerosolized chickens survived subcutaneous challenge with 10(6) LD50 NDV at 30 and 50 days old, respectively. A single immunization is sufficient for broilers; however, parent flocks should be revaccinated at 7 so 8 weeks old.     

SIMULATED NATURAL INFECTION OF CHICKENS WITH AUSTRALIAN LENTOGENIC NEWCASTLE DISEASE VIRUS AND SUBSEQUENT CHALLENGE WITH VIRULENT VIRUS

A total of 291 eight-week-old chickens were exposed to chickens infected with either of two Australian lentogenic strains (V4 and AVL NDV-1) of Newcastle disease virus (NDV). At 3 weeks after exposure, all chickens exposed to V4 infected chickens had developed haemagglutination-inhibition (HI) antibody. All chickens exposed to AVL NDV-1 virus infected chickens had developed HI antibody 5 weeks later. This sudden late appearance of HI antibody, to titres higher than those observed with V4 chickens, was explained by V4 virus being introduced to the AVL NDV-1 group of chickens. When groups of these chickens were challenged with Roakin virus (mesogenic NDV) at 3 weeks and Fontana 1083 virus (viscerotropic velogenic NDV) and Texas GB virus (neutrotropic NDV) at 3, 5, 10 and 21 weeks only three chickens developed clinical illness one of which died. These chickens were one AVL NDV-1 chicken contact challenged with Fontana 1083 virus at 3 weeks, one V4 chicken oronasally challenged with Texas GB virus at 5 weeks and one V4 chicken challenged oronasally with Fontana 1083 virus at 10 weeks. Susceptible non-vaccinated chickens died soon after challenge. Challenge by oronasal infection with 10(7.0) ELD50 of virus or contact with susceptible infected chickens enabled virulent virus to be isolated from most chickens and was accompanied by a large anamnestic increase in serum HI antibody.     

Characterization of Newcastle Disease Viruses Isolated from Chickens and Ducks in Tamilnadu,India

During 1993, outbreaks of Newcastle disease occurred on many farms in Tamilnadu, India. Six Newcastle disease virus (NDV) isolates were obtained from the chickens on five different farms and from the birds on one duck farm during outbreaks of the disease. All the isolates were characterized as velogenic, based on the mean death time, intravenous pathogenicity index, intracerebral pathogenicity index (ICPI), stability of haemagglutinin at 56°C, agglutination of equine erythrocytes, haemagglutination elution pattern and adsorption of haemagglutinin by chick brain cells. The isolate obtained from ducks resembled a group D strain, based on its ICPI and its reaction with a panel of monoclonal antibodies. The other five NDV isolates obtained from chickens were placed in groups B(1), C1(2) and D(2) on the basis of their binding patterns with the panel of monoclonal antibodies. In challenge experiments, it was found that LaSota vaccine provided 100% protection against each of these field isolates and against a local NDV strain obtained from the Institute of Veterinary Preventive Medicine, Tamilnadu, India, while unvaccinated chickens succumbed to challenge. The possible origin of epizootic viruses causing outbreaks in vaccinated flocks is discussed.     

复方中药提取物在新城疫B_1株免疫中的免疫增强作用

用自制的中药提取物与新城疫Ｂ１株混合后免疫５５日龄鸡，以血凝抑制试验测定新城疫抗体效价。结果表明，５５日龄鸡的中药提取物最佳用量为１．０ｍＬ，试验组鸡ＮＤ抗体效价明显高于对照组。免疫后６０ｄ用Ｆ４８Ｅ８新城疫强毒进行攻击，试验组的保护率达到１００％，而对照组相对较低。     

从自然发病鸡中分离出一株新城疫病毒

应用电镜技术、血清学检查、生物学试验等方法,从未注射过疫苗的某自然发病鸡群分离出一株新城疫病毒。该分离株对１０日龄鸡胚的平均致死时间为１１６小时,静脉接种致病指数为０．０６,其毒力介于新城疫病毒的ＬａＳｏｔａ株与Ｖ４株之间。免疫原性试验结果表明,该分离株具有免疫后无不良反应、免疫后６天开始产生抗体、产生的抗体效价高、对新城疫强毒的攻击能１００％保护等特点,是一株良好的新城疫候选疫苗株     

传染性法氏囊病免疫保护试验中3种评定指标的比较

100只SPF鸡均分为5组，A、B、c3组免疫后攻毒，接种3批次自制的IBD基因工程重组亚单位油乳剂疫苗；D组不免疫不攻毒，E组不免疫而攻毒。免疫后第22天，感染IBDV强毒株BC-6／85。攻毒后第4天，将所有存活的鸡只以颈脱臼致死，收集法氏囊，以3种方法和指标（法氏囊眼观病变；法氏囊显微病变；法氏囊中IBDV抗原检测）进行分析，以评定免疫保护率。结果显示，以这3种方法和指标评定，A组免疫保护率为90％，B、C组免疫保护率均为95％；试验鸡A3、A14、B7、C16、E1～E20，法氏囊眼观病变明显，法氏囊显微病理损伤评分为3～5分，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阳性；其他试验鸡，法氏囊眼观无明显异常，法氏囊显微病理损伤评分在3分以下，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阴性。结果表明，这3种方法和指标在IBD疫苗免疫保护试验评定中具有很好的一致性。     

新城疫La Sota、V4疫苗免疫鸡和免疫攻毒鸡的特异性IgA抗体动态变化规律比较

应用间接ELISA对新城疫LaSota、V4疫苗免疫鸡及免疫攻毒鸡的IgA抗体动态变化进行了测定和比较。试验表明，LaSota和V4免疫鸡泪液中特异性IgA均在免疫后第5天出现，8天开始明显升高，与血清中IgG出现时间相似，免疫后21天达到高峰。LaSota免疫鸡的泪液IgA抗体水平略高于V4免疫鸡；而两免疫组哈德氏腺(HG)中的特异性IgA高峰出现较迟。免疫攻毒鸡泪液中的特异性IgA抗体水平均先呈短暂的升高，之后下降的趋势，而HG中的IgA则首先表现降低，之后很快升高，5天后趋于下降，两攻毒组差异不显著，对IgA回忆应答均不明显。     

A comparison of the onset of protection induced by Newcastle disease virus strain B1 and a fowl poxvirus recombinant Newcastle disease vaccine to a viscerotropic velogenic Newcastle disease virus challenge

Four-week-old specific-pathogen-free white rock chickens were immunized with either a commercial recombinant fowl poxvirus-vectored Newcastle disease vaccine (FPN) expressing the hemagglutinin-neuraminidase and fusion protein genes of Newcastle disease virus (NDV) strain B1 or live NDV B1. Vaccinates and controls were challenged by eyedrop and intranasal (E/I) route with a viscerotropic velogenic NDV at 14 days postvaccination to determine the time of clearance of challenge virus. In a subsequent experiment, chickens were challenged at 3, 6, or 10 days postvaccination to determine the onset of immunity. Chickens that received a recommended field dose (1x) or a 0.01x dose of FP-N subcutaneously (s.c.) and were seropositive by hemagglutination-inhibition test at 14 days postvaccination cleared the challenge virus by 14 days postchallenge. Clinical Newcastle disease and high challenge virus titers in tissues were seen only in seronegative FP-N 0.01x dose vaccinates and controls. In a comparison of vaccination with FP-N (1x, 10(4,9) median tissue culture infective dose) s.c., B1 (10(6) median egg infective dose [EID50]) s.c., or B1 (10(6) EID50) E/I, chickens vaccinated at 6 or 10 days before challenge with all vaccines were protected against clinical disease, but only those vaccinated with B1 E/I 10 days before challenge were protected against infection with the challenge virus. Vaccination at 3 days before challenge with B1 E/I provided early protection, but severe nervous signs developed later and reduced overall protection to 60%, whereas disease in chickens vaccinated with B1 s.c. and FP-N s.c. 3 days before challenge was similar to the challenge controls.     

Effect of Dietary Picriafel-tarrae Lour Extracts on Serum Biochemical Indexes and Immune Function of Guangxi Partridge Chickens

This trial was conducted to investigate the effect of dietary Picriafel-tarrae Lour extracts on serum biochemical indexes and immune function of Guangxi Partridge chickens. 500 Guangxi Partridge chickens with 1 day old were randomly divided into 5 groups with 5 replicates per group and 20 chicks per replicate.Groups A,B and C were treatment groups and fed diets with 0.70%,0.35% and 0.175% Picriafel-tarrae Lour extracts,respectively,group D was a medical control group fed a diet with 0.01% colistin sulfate premix,and group E was control group fed basal diet. Serum samples were collected from 10 chicks in each group at 21,35 and 49 days old to analyze the biochemical and immune indexes. The results showed that compared with group E, the contents of GLU and TP were not significantly affected by the Picriafel-tarrae Lour extracts (P>0.05).However, in 21 and 35 days old, the TBILI content of groups A,B and C were extremely significantly or significantly reduced (P<0.01;P<0.05);At 35 days old,the activity of serum AKP of groups A,B and C were extremely significantly higher than that of group E (P<0.01),and the activity of serum GOT of group C at 21 and 35 days old were significantly higher than that of group E (P<0.05);At 49 days old,the serum IgG content of groups A,B,C and IL-2 of groups B and C were extremely significantly or significantly lower than those of group E (P<0.01;P<0.05);At 49 days old,the T-AOC of group B was significantly higher than group E (P<0.05);The GSH content of groups A,B and C at 49 days old were significantly increased than group E (P<0.05).In conclusion,the dietary Picriafel-tarrae Lour extract could significantly decreased the TBILI content,reduce the contents of IgG,IL-2 and IL-6, improve the activities of AKP and GOT,and the contents of T-AOC and GSH in some degree. The 0.35% additive amount had the better effects.