基于新城疫病毒V蛋白鉴别鸡群感染与免疫的间接ELISA方法的建立

本研究以新城疫病毒(NDV)V蛋白羧基端结构域(Vc)的重组蛋白为包被抗原,建立了用于检测NDV V蛋白抗体的间接ELISA方法,并采用该方法检测了鸡群免疫或接毒后血清中的V蛋白抗体水平。结果显示:两组不同NDV灭活疫苗组在免疫后的3周内检测结果均为阴性;两组灭活疫苗免疫3周后再人工感染NDV强毒的鸡群,攻毒后第7、14和21 d,NDV阳性率分别为60%、80%、70%和50%、80%、70%;两组不同的NDV弱毒疫苗免疫组鸡群,仅在免疫后第21 d阳性率分别为20%和10%。以上结果表明,NDV疫苗免疫组与强毒感染组的V蛋白抗体阳性率存在明显差异,本方法可在群体水平上区分新城疫疫苗免疫与强毒感染鸡群,为NDV血清学诊断和流行病学调查提供了一种新的检测手段。     

Dose-response effects of inactivated Newcastle disease vaccines: influence of serologic assay, time after vaccination, and type of chickens

Knowledge of the dose-response relation of inactivated vaccines and of the factors that influence this relation is essential for the evaluation of existing vaccine potency assays and the development of new potency assays that are based on the antigen content of the inactivated vaccines. We quantified the relation between vaccine dose, serologic response, and clinical protection after vaccination for three different inactivated Newcastle disease (ND) vaccines. Qualitatively, similar dose-response curves were obtained for the three vaccines when either the serologic response or the clinical protection of specific-pathogen-free (SPF) chickens was plotted against the different vaccine doses applied. However, the vaccines differed quantitatively: doses of vaccines that induced similar antibody titers or clinical protection differed 2-8-fold. In contrast with the narrow range of antibody titers induced by a full vaccine dose, a very broad range of titers was obtained after dilution of the vaccines. At least 95% of the SPF chickens with detectable antibody in the serum were protected against a challenge with virulent Herts ND virus. The relation between the dosage of two different ND vaccines and the serum antibody titers remained markedly constant between 3 and 18 wk after vaccination. Vaccination of broilers instead of layers with a dilution series of inactivated ND vaccine resulted in significantly lower antibody levels and less clinical protection against virulent challenge. In conclusion, despite quantitative differences, we found comparable dose-response relations for the three inactivated ND vaccines studied.     

The resistance of meat chickens vaccinated by aerosol with a live V4 Newcastle disease virus vaccine in the field to challenge with a velogenic Newcastle disease virus

Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.     

Immunogenicity and protective efficacy of virosome based vaccines against Newcastle disease

Virosome based vaccines against Newcastle disease (ND) were prepared and evaluated for their immunogenicity and protective efficacy in chickens. Envelop of Newcastle disease virus (NDV) was solubilised with Triton X-100 to yield virosomes which were later on encapsulated in poly-lactide-co-glycolide (PLG) microspheres. The birds were immunized intranasally with virosomes or PLG microspheres encapsulated virosomes, and efficacy of these preparations was compared with commercial LaSota vaccine. The preparations protected the chickens against virulent virus challenge infection, however the microencapsulated virosome vaccine gave slightly lesser degree of protection than non encapsulated counterpart. The humoral and cell mediated immune response generated as well as the protection afforded by virosome preparations were found to be comparable with LaSota vaccine. The results substantiate the potential of virosome based vaccines to provide high level of immunity and protection against Newcastle disease.     

Development of a virosome vaccine for Newcastle disease virus

In an effort to protect chickens against Newcastle disease (ND), a nonreplicating virosome vaccine was produced by solubilization of Newcastle disease virus (NDV) with Triton X-100 followed by detergent removal with SM2 Bio-Beads. Biochemical analysis indicated that the NDV virosomes had similar characteristics as the parent virus and contained both the fusion and hemagglutinin-neuraminidase proteins. To target the respiratory tract, specific-pathogen-free chickens were immunized intranasally and intratracheally with the NDV virosome vaccine. This vaccine was compared with a standard NDV (LaSota) live-virus vaccine for commercial poultry. Seroconversion (> or = four fold increase in hemagglutination inhibition [HI] antibody titers) was achieved in all birds vaccinated with the virosome vaccine. Upon lethal challenge with a velogenic NDV strain (Texas GB), all birds receiving either vaccination method were protected against death. Antibody levels against NDV, as determined by enzyme-linked immunosorbent assay and HI titer, were comparable with either vaccine and increased after virus challenge. These results demonstrate the potential of virosomes as an effective tool for ND vaccination.     

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     

Vaccination against Newcastle disease with a recombinant baculovirus hemagglutinin-neuraminidase subunit vaccine

Vaccination of chickens with an oil-emulsion vaccine containing a recombinant baculovirus that expressed the hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV)-induced hemagglutination-inhibition (HI) and virus-neutralizing antibodies against NDV. HI antibody titers obtained in response to vaccination with the live recombinant virus were higher than those obtained when the recombinant was inactivated with beta-propiolactone, and the titers were lower than those obtained in response to the same HN concentrations in live or beta-propiolactone-inactivated NDV strain B1. The serological response to the recombinant baculovirus was differentiated from the response to NDV by an enzyme-linked immunosorbent assay in which purified NDV nucleoprotein was used as antigen. Chickens vaccinated with the live recombinant or with inactivated NDV resisted an oculonasal challenge with the neurotropic velogenic Texas GB strain of NDV, which was lethal in unvaccinated controls. It was concluded that the HN protein of NDV expressed as a subunit by a recombinant baculovirus was protective against Newcastle disease.     

Vaccination of ostriches (Struthio camelus, Linnaeus, 1758) against Newcastle disease: evidence for vaccine compatibility and seroconversion after vaccinations using the hemagglutination inhibition and virus neutralization tests

Newcastle disease (ND) remains to be the worldwide most important infectious disease of poultry. This epizootic is in Germany and many other countries a notifiable disease. Prophylactic vaccination is the major tool for the control of ND in poultry and other birds. Eighty-three ostriches (Struthio camelus) which were kept on farms in Germany were checked for the presence of NDV-specific antibodies. Some of these birds are said to be vaccinated against Newcastle disease. Only some of these ostriches contained antibodies which were measurable in haemagglutination inhibition and virus neutralisation tests. Twenty-three previously unvaccinated ostriches were vaccinated with commercially available vaccines. Both the LaSota live and inactivated oil emulsion vaccines were well tolerated following conjunctival or subcutaneous application, respectively. Neither local nor systemic side reactions were observed. After the vaccinations high antibody titres were detected in hemagglutination inhibition and virus neutralisation tests. A strong correlation between both established methods (r = 0.92; < 0.001) were noted.     

Response of domestic geese to lentogenic and velogenic strains of Newcastle disease virus

A total of 54 domestic white meat-type geese were included in vaccination/challenge trials to evaluate susceptibility to disease and humoral immune responses using the haemagglutination inhibition (HI) and virus neutralization (VN) tests against Newcastle disease (ND). Two groups of twenty geese, five weeks of age, were conjunctivally vaccinated with either 100 x 10(6) or 2.5 x 10(6) EID50 (egg infectious dose 50 per cent) per bird of live La Sota virus, respectively, and 14 geese remained unvaccinated. At 15 weeks of age all vaccinated geese and seven unvaccinated geese were subcutaneously injected with 0.5 ml of inactivated oil emulsion ND vaccine, whereas seven geese remained as negative controls. At an age of 20 weeks, all 54 geese were challenged with 10(8.0) EID50 per bird of the viscerotropic velogenic NDV strain Herts 33/56. Live virus application as well as the oil emulsion vaccine did not induce discernible clinical signs and have no detrimental effect on body weight gains. At days 1, 3, 5, 8, 13, 16, 20, 23 and 27 after the application of lentogenic vaccine pharyngeal and cloacal swabs were taken, after challenge samples were taken at days 2, 5 and 8. Lentogenic as well as velogenic virus were never reisolated. Low and shortlived antibody responses post vaccination were equally well measured in HI and VN tests. Only two out of seven unvaccinated but challenged geese developed signs of ND whereas all vaccinated/challenged geese remained normal but developed high to moderate levels of HI and VN antibodies. Since domestic geese do not readily excrete NDV's in detectable amounts and since they do not contain detectable amounts of the challenge virus fourteen days post challenge in their tissues the assumption is promoted that geese do not play a major role in the epidemiology of Newcastle disease.     

Effect of breeder vaccination on immunization of progeny against Newcastle disease

Two experiments were conducted to determine the effect of breeder vaccination program and maternal antibody on the efficacy of Newcastle disease immunization of 1-day-old chicks. Experimental protocol was the same for both. In the first experiment, broilers were from breeders that were 32 weeks old, and in the second experiment, broilers were from breeders 50 weeks old. Breeders received three live Newcastle disease virus (NDV) vaccines and either a killed vaccine at 18 weeks or continual live boosting at 60-to-70-day intervals through lay. Broilers were vaccinated at 1 day of age with a commercial coarse-spray machine; they were bled, sera were examined for antibody against NDV, and broilers were challenged with virulent NDV at 2, 4, and 6 weeks of age. In the first experiment, maternal antibody was higher in chicks from the younger breeders given the inactivated vaccine, and in the second experiment maternal antibody was higher in chicks from older breeders given continual live vaccines. Higher antibody in 1-day-old broilers resulted in fewer vaccine-induced reactions, less vaccine virus shed, and decreased duration of vaccine-induced immunity from coarse-spray vaccination.     

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。     

鸽禽Ⅰ型副粘病毒油佐剂灭活苗对雏鸡免疫效果评价

用鸽Ａ／ＰＭＶ－１油佐剂灭活苗与ＮＤＶ油佐剂灭活苗分别免疫雏鸡,免疫后２１ｄ抗体水平达到峰值,免疫后４２ｄ用新城疫强毒对两种疫苗免疫鸡分别进行攻击,鸽Ａ／ＰＭＶ－１油佐剂灭活苗免疫组保护率为７３．３３％,ＮＤＶ油佐剂灭活苗免疫组保护率为９９．６７％。     

Experimental trials with a thermostable Newcastle disease virus (strain I2) in commercial and village chickens in Tanzania

Antibody responses in indigenous village and commercial chickens vaccinated with 12 thermostable Newcastle disease (ND) vaccine and protection levels against challenge with a virulent field isolate were determined. The antibody response of village chickens vaccinated by eye drop revealed that 30, 60 and 90 days after primary vaccination, the mean log2 HI titres were 6.1, 5.4 and 3.6, respectively, whereas for commercial chickens, the antibody response after 14, 30 and 90 days were 8.2, 5.1 and 4.2, respectively. Village chickens vaccinated orally via drinking water had mean log2 HI titres of 3.4 after 30 days. After booster vaccination, the mean HI titre was 5.4 and 3.3 after 30 and 60 days post-secondary vaccination (i.e. 60 and 90 days after primary vaccination). Antibody response of mean log2 HI titres of 2.6 was recorded 30 days after primary vaccination orally through food; 30 and 60 days after secondary vaccination (i.e. 60 and 90 days after primary vaccination), mean log2 HI titres were 5.3 and 3.2, respectively. All commercial and village chickens vaccinated by eye drop survived the challenge trial whereas village chickens vaccinated through drinking water and food had protection levels of 80% and 60% 30 days after primary vaccination, respectively. However, 30 days after booster vaccination, the protection level was 100%. At 60 days after secondary vaccination, the protection level dropped again to 80% for chickens vaccinated orally. All control chickens used in the challenge trials developed clinical ND and died 3-5 days after inoculation with the virulent virus. Supported by laboratory findings, I2 strain of NDV seemed to be avirulent, immunogenic and highly protective against virulent isolates of NDV. It may be a suitable vaccine to use in village chickens to vaccinate them against ND in rural areas.     

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.     

H9亚型禽流感油乳剂灭活苗免疫、免疫后攻毒及人工感染鸡抗体的监测

本研究采用ＡＧＰ、ＨＩ等试验方法，对经Ｈ９亚型禽流感油乳剂灭活苗免疫、免疫后攻毒以及经Ｈ９Ｎ２活毒人工感染后的ＳＰＦ鸡抗体产生、消长规律进行了测定，结果表明人工感染ＳＰＦ鸡和免疫鸡一周后，ＡＧＰ的检出率即可达到１００％；Ｈ９亚型禽流感油乳剂灭活苗自免疫后一周内即可产生ＨＩ抗体，２１－２８天达到高峰，并能对相同亚型病毒感染引发良好的免疫反应。     

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.     

Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens,and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site ¹¹²RRRKGF¹¹⁷ and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.     

Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.     

Investigations on the Influence of Helminth Parasites on Vaccination of Chickens against Newcastle Disease Virus under Village Conditions

Prevalence studies have shown that almost 100% of free-range chickens are infected with a wide range of parasites. The infections are mostly subclinical in nature, resulting in production losses and occasionally mortality. Newcastle disease (ND), on the other hand, results in high mortality rates during epidemics. ND is a limiting factor for increasing poultry production in many tropical countries, where frequent reports indicate vaccination failures. The aim of our study was to investigate the influence of helminths on the antibody response after vaccination against Newcastle disease of free-range chickens naturally infected with parasites. Sixty chickens were divided into six groups, of which three were vaccinated against ND with a live De Soto vaccine, while the other three remained non-vaccinated. One group within the vaccinated groups and the one within the non-vaccinated group was kept naturally infected with helminth parasites, while the other two groups in each set were dewormed with fenbendazole and niclosamide, and one of each of these groups was subsequently infected with Ascaridia galli. After vaccination, all the groups were followed for 5 weeks and their antibody titres were determined weekly using a HI test. All the birds were finally challenged 4 weeks after vaccination with a virulent velogenic ND virus obtained from a field outbreak. All the vaccinated chickens seroconverted and had high antibody levels after 3 weeks, but these dropped to low levels at 4 weeks after vaccination. After challenge, the antibody titres rose in the dewormed groups but not in the parasite-infected groups. After 5 weeks, all the parasite-infected animals had significantly lower antibody titres than the dewormed animals. All the vaccinated chickens survived the challenge infection, emphasizing the importance of the cellular immune response. Further studies are needed to examine the effects of the parasitic infection on protection against ND over a longer period.