鸡马立克氏病重组鸡痘病毒与火鸡疱疹病毒二价冻干疫苗免疫效力试验

本文对高效表达鸡马立克氏病(MD)血清Ⅰ型疫苗毒株CVI988／Kispem gB基因的重组鸡痘病毒(rFPV—gB/R)和火鸡疱疹病毒(HVT)组成MD二价冻干疫苗时MD超强毒株(vvMDV)Md5和RB1B攻击后的免疫保护效果，实验室免疫效力试验的结果表明，HVT rFPV—gB／R二价冻千苗时超强毒株Md5和RB1B攻击时的免疫保护效果明显优于HVT冻干苗，接近CVI988／Rfispens的水平，本研究证明HVT rFPV—gB／R二价冻干疫苗是一种安全、方便、高效的疫苗。     

马立克病毒二价活疫苗效价检测实验

由于CVI988/Rispense疫苗优良的免疫特性,已经被认为是目前防控马立克氏病（Marek＇sdisease,MD）效果最好的疫苗。然而,近年来随着马立克病毒（Marek＇sdisease virus,MDV）毒力的不断增强。CVI988/Rispense需要与MDV-Ⅱ或者HVT联合使用才能防止免疫失败的发生。本实验通过空斑计数和间接免疫荧光相结合的方法测定了马立克病毒CVI988/Rispense＋FC126二价活疫苗的效价。结果显示批次A中CVI988为4400PFU/dose,HVT为2600/dose;批次B中CVI988为4800PFU/dose,HVT为2400PFU/dose;批次C中CVI988为4600PFU/dose,HVT为2800PFU/dose。所得结果均显著高于国家标准CVI988不少于2000PFU/dose,HVT不少于1000PFU/dose,并且批次之间非常稳定,适合用作鸡群马立克氏病的防控。     

鸡马立克氏病三价疫苗的安全性及免疫效力研究

将MD三价疫苗以25000PFU／只的剂量颈部皮下接种1日龄SPF雏鸡，结果表明，疫苗的接种不影响鸡体重的增加；不引起鸡法氏囊、脾脏等组织器官发生MD组织病理学变化，对鸡安全无毒性。用SPF鸡评价MD三价疫苗的免疫效力，三批疫苗对RB1B超强毒株攻击的平均保护效力为95．99％，而且无论是用RB1B超强毒株还是用BJMDV-1血毒攻击，MD三价疫苗的保护效力明显高于HVT＋SB1二价疫苗及HVT冻干苗，好于CV1988疫苗；接种MD三价疫苗的4个品系的商品鸡群，抗RB1B超强毒株攻毒的平均保护效力为94．60％；在模拟MD强毒自然传染的试验中，MD三价疫苗的保护效力达到95．65％。上述效力试验的结果说明：MD三价疫苗的免疫接种可使鸡形成抗MD强毒攻击的坚强免疫力。     

血清3型火鸡疱疹病毒疫苗与血清1型CVI988疫苗的效力比较

用血清3型火鸡疱疹病毒(HVT)疫苗和血清1型CVI988疫苗免疫SPF白来航鸡后,分别攻击国内马立克氏病(MD)标准强毒京-1株和国际MD标准超强毒(VVMDV)RB1B株。结果表明,HVT和CVI988免疫组攻击京-1株或RB1B株与相应攻毒对照组相比,保护指数差异极显著(P<0.01)。HVT和CVI988疫苗抗京-1株攻毒的保护效果相似(保护指数分别为93.75%和93%),而CVI988疫苗抗RB1B株的攻击效果优于HVT疫苗(保护指数分别为92.6%和75.3%),但差异不显著(P>0.05)。CVI988疫苗抗京-1株和RB1B株的效果(分别为93%和92.6%)无显著差异(P>0.05),HVT疫苗抗京-1株和RB1B株的效果(分别为93.75%和75.3%)存在一定差异,但差异不显著(P>0.05)。     

整合REV-LTR基因的MDV活疫苗与亲本CVI988/Rispens株的免疫效力比较

利用同源重组技术将禽网状内皮组织增生症病毒（REV）的长末端重复序列（LTR）整合进CVI988/Rispens疫苗株基因组中所构建成的重组马立克氏病病毒株（r MDV-LTR株）,具有较高的增殖效率。为比较r MDV-LTR株与亲本CVI988/Rispens株的免疫效力差异,开展了对这两种疫苗的比较试验。试验将这两种疫苗分别按照三个不同剂量经颈背部皮下接种1日龄SPF鸡。接种后第7 d,攻毒马立克氏病强毒株rMd5,连续观察60 d后进行剖检。临床病理观察结果发现,以不低于250 PFU/羽剂量的rMDV-LTR株或CVI988/Rispens株免疫SPF雏鸡都可以提供大于80%的免疫保护效率;以125 PFU/羽剂量的两种疫苗虽均不能提供大于80%的免疫保护效力,但rMDV-LTR株免疫组试验鸡未出现死亡,仅有2只鸡出现消瘦,剖解脏器无肿瘤,而CVI988/Rispens株免疫组鸡出现2只死亡,其中1只剖解后检出肿瘤。rMDV-LTR株和CVI988/Rispens株免疫组的免疫保护指数分别为77.78%和66.67%。由实验结果可见,在较小免疫剂量下,rMDV-LTR株免疫保护...     

鸡马立克氏病meq基因缺失疫苗(SC9-1株)的临床应用效果比较研究

鸡马立克氏病基因缺失活疫苗(SC9-1株)的临床试验分别在3个鸡场进行,试验鸡分别为1日龄雪山黄鸡20 600只、1日龄京红1号蛋鸡17 000只、1日龄北京油鸡9 732只。临床安全性试验观察期内,免疫鸡群的精神状态、采食、饮水情况、生长性能与对照组无异。临床免疫效力试验期间,将免疫SC9-1鸡群与免疫CVI988/Rispens(市售)鸡群进行对比,SC9-1鸡群的死淘率分别为2.7%与0.4%,均低于CVI988/Rispens免疫鸡群的3.7%与1.2%(P0.05)。另外分别将免疫SC9-1的鸡群与免疫CVI988/Rispens的鸡群在6日龄时攻击马立克超强毒Md5(1 000 pfu/只),结果显示SC9-1株疫苗攻毒保护率分别为35/35与34/35,显著高于CVI988/Rispens株疫苗的25/35与27/35(P0.05)。以上表明SC9-1株疫苗安全性良好,免疫效力较好,可能是一株理想的马立克氏病疫苗。     

表达鸡马立克氏病病毒gB基因的重组鸡痘病毒与火鸡疱疹病毒二价冻干疫苗的免疫效力

对高效表达鸡马立克氏病(MD)血清型疫苗毒株CVI998/Rispens gB基因的重组鸡痘病毒(rFPV-gB/R)和火鸡疱疹病毒(HVT)组成MD二价冻干疫苗时2种病毒的最适剂量配比进行了研究。试验表明,以对MD易感、MD和鸡痘病毒(FPV)抗体阴性SPF鸡为试验动物进行的试验中,不同免疫剂量的rFPV-gB/R(1×106、1×105、1×104PFU)同HVT组成的二价苗产生的免疫效力相当;以对MD易感、MD和FPV抗体阳性的狼山鸡进行的试验中,不同免疫剂量的rFPV-gB/R同HVT组成的二价苗产生的免疫力随rFPV-gB/R免疫剂量的降低而降低;所有的rFPV-gB/R+HVT的二价苗组均有一定的免疫保护作用。     

新成果·新技术·新方法

鸡马立克氏病(MD)多价疫苗 鸡马立克氏病(MD)多价疫苗是由两种或三种血清型MD疫苗株病毒(I型“814”、Ⅱ型SB-1和Ⅲ型HVTFC126)按规定配比组成,主要用于提高该病单价苗的防制效果,用于防制该病强毒株的爆发流行。该疫苗每羽剂量含三种血清型病毒总数在2000蚀斑形成单位(PFU)以上,无新城疫等病毒污染,免疫保护率(即发病减少率)达90％以上,在正确使用条件下,免疫鸡群的MD发病率可降低到1％以下。其免疫效果优于国际上的同类疫苗。     

对一株超强马立克氏病毒株的检测和研究

对从发病鸡场分离到的一株超强马立克氏病毒株,用PCR扩增该分离毒株的meq致瘤基因并测定了其序列,发现其与超强病毒株SD2012-1的核苷酸同源性为99%。该分离毒株的meq基因序列在第575～577位比疫苗株CVI988/Rispens的meq基因序列多插入3个碱基（CAC）,与SD2012-1相同。使用该分离毒株进行疫苗免疫攻毒实验,未免组的发病率为80.0％,常规剂量HVT组和10×常规剂量HVT组的发病率分别为73.0％和76.6％,HVT不能提供有效免疫保护。常规剂量CVI988组和10×常规剂量CVI988组的发病率分别为30.0％和30.0％,常规剂量HVT+CVI988组和10×常规剂量HVT+CVI988组的发病率分别为26.6％和26.7％,免疫CVI988疫苗组鸡和HVT+CVI988联苗组鸡的发病率比免疫HVT疫苗组鸡低。说明,野外毒株的不断变异已经突破现有疫苗的免疫保护,会给养鸡业带来严重的损失。     

鸡马立克病活疫苗中污染禽网状内皮组织增生症病毒检测方法的建立

为建立鸡马立克病(MD)活疫苗中污染禽网状内皮组织增生症病毒(REV)的间接免疫荧光检测方法,本研究通过向MD活疫苗中添加不同剂量的REV,用拟定的方法对样品检测REV。结果表明,Ⅰ型MD活疫苗、火鸡疱疹病毒活疫苗(Fc-126株)和MD二价活疫苗(HVT+CVI988株)中污染REV的最低检出量分别为每500羽份疫苗中污染5TCID50、10TCID50和15TCID50的REV。应用建立的方法对国内10家企业生产的43批MD活疫苗进行了检验,结果显示,2个企业生产的4批疫苗REV检测阳性。随机选取了5批IFA检测阴性样品和4批IFA检测阳性样品,按鸡检查法进行外源病毒检验,结果两种方法对REV污染检验结果的符合率为100%。因此,本研究建立的IFA法可替代鸡检查法,用于疫苗中REV污染的检验。     

Effect of diluting Marek's disease vaccines on the outcomes of Marek's disease virus infection when challenged with highly virulent Marek's disease viruses

Dilution of Marek's disease (MD) vaccines is a common practice in the field to reduce the cost associated with vaccination. In this study we have evaluated the effect of diluting MD vaccines on the protection against MD, vaccine and challenge MD virus (MDV) kinetics, and body weight when challenged with strains Md5 (very virulent MDV) and 648A (very virulent plus MDV) by contact at day of age. The following four vaccination protocols were evaluated in meat-type chickens: turkey herpesvirus (HVT) at manufacturer-recommended full dose; HVT diluted 1:10; HVT + SB-1 at the manufacturer-recommended full dose; and HVT + SB-1 diluted 1:10 for HVT and 1:5 for SB-1. Vaccine was administered at hatch subcutaneously. One-day-old chickens were placed in floor pens and housed together with ten 15-day-old chickens that had been previously inoculated with 500 PFU of either Md5 or 648A MDV strains. Chickens were individually identified with wing bands, and for each chicken samples of feather pulp and blood were collected at 1, 3, and 8 wk posthatch. Body weights were recorded at 8 wk for every chicken. Viral DNA load of wild-type MDV, SB-1, and HVT were evaluated by real time-PCR. Our results showed that dilution of MD vaccines can lead to reduced MD protection, reduced relative body weights, reduced vaccine DNA during the first 3 wk, and increased MDV DNA load. The detrimental effect of vaccine dilution was more evident in females than in males and was more evident when the challenge virus was 648A. However, lower relative body weights and higher MDV DNA load could be detected in chickens challenged with strain Md5, even in the absence of obvious differences in protection.     

Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains

Comparative 50% protective dose (PD50) assays were performed using a plaque-purified preparation of Marek's disease virus (MDV) strain CVI-988 at the 65th chicken embryo fibroblast (CEF) passage level (MDV CVI-988 CEF65 clone C) and three commercial MD vaccines: herpesvirus of turkeys (HVT) FC126, MDV CVI-988 CEF35, and a bivalent vaccine composed of HVT FC126 and MDV SB-1. In addition, comparative PD50 assays were performed in groups of chickens with maternal antibody to each of the three vaccines. Three representatives of the newly emerged biovariant very virulent (vv) MDV strains-RB/1B, Tun, and Md5-were employed as challenge virus. The experiments made feasible the differentiation between virulent MDV and vvMDV strains, within serotype 1. Vaccination with CVI-988 clone C vaccine resulted in PD50 estimates of about 5 plaque-forming units (PFUs) against challenge infection with each of the three vvMDV strains. The PD50 estimate of CVI-988 clone C vaccine was 12-fold below the PD50 of HVT FC126. The protective synergism of bivalent vaccine, composed of HVT and SB-1, was confirmed by groups given the lowest vaccine doses. The bivalent vaccine, however, resulted in incomplete protection in groups given the highest vaccine doses. Homologous maternal antibodies to serotype 1 caused a fivefold increase in the PD50 estimate of CVI-988 clone C. Heterologous maternal antibodies against HVT did not interfere with efficacy of CVI-988 clone C vaccination. However, the combination of maternal antibodies against both HVT and SB-1 (serotypes 2 and 3) showed a strong adverse effect on CVI-988 clone C vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Titration of Marek's disease cell-associated vaccine virus (CVI 988) of reconstituted vaccine and vaccine ampoules from Dutch hatcheries

Thirty-one outbreaks of Marek's disease (MD) were reported in the Netherlands and retrospectively analyzed. The outbreaks occurred mostly in vaccinated commercial layer and a few breeder flocks of several breeds; however, the cause of the outbreaks could not be established. Therefore, in a prospective study, the occurrence of true vaccine failures was assessed onfive hatcheries. The plaque-forming units (PFU) of MD vaccine per chicken dose were determined through in vitro assays on vacine ampoules (2 to 5 per hatchery) and samples of reconstituted vaccine (approximately 22 per hatchery). All forty reconstituted vaccine samples of hatcheries 1 and 4 showed PFU doses <10(3). In hatchery 4, 14 samples showed extreme low PFU (< or = 10 PFU). In hatcheries 2, 3, and 5, the numbers of MD vaccine suspensions with a titer > or = 10(3) PFU, which is the standard required, were 1 (5%), 17 (77%), and 3 (14%), respectively. Some vaccine ampoules showed < 10(3) PFU per chicken dose. This study shows the usefulness to assess the PFU per chicken dose of reconstituted MD vaccine and vaccine ampoules to unravel true vaccine failures, which could result in disease outbreaks in the field.     

鸡黄病毒灭活疫苗的研制及免疫效果研究

鸡黄病毒FQ—C1株经SPF鸡胚连续传40代后,检测其尿囊液毒的ELD50达到1×10^-4.33／0．1mL。将该病毒第40代SPF鸡胚液毒灭活后制备成油乳剂灭活疫苗。SPF雏鸡和蛋鸡安全性试验表明,该疫苗的安全性好,无不良影响。以该疫苗一次单剂量（1mI／只）免疫开产蛋鸡,28d后攻以鸡黄病毒强毒测定疫苗的保护效果,结果显示疫苗免疫组较未免疫组蛋鸡的产蛋率高出75．8％,产蛋保护率达93．2％以上。试验表明,本研究制备的鸡黄病毒灭活疫苗安全性好,且具有良好的免疫原性,免疫后可有效抵抗鸡黄病毒所致的产蛋骤降。     

A single dose of inactivated oil-emulsion bivalent H5N8/H5N1 vaccine protects chickens against the lethal challenge of both highly pathogenic avian influenza viruses

In this study, two highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from chicken and geese in 2018 and 2019 (Chicken/ME-2018 and Geese/Egypt/MG4/2019). The hemagglutinin and neuraminidase gene analyses revealed their close relatedness to the clade-2.3.4.4b H5N8 viruses isolated from Egypt and Eurasian countries. A monovalent inactivated oil-emulsion vaccine containing a reassortant virus with HA gene of the Chicken/ME-2018/H5N8 strain and a bivalent vaccine containing same reassortant virus plus a previously generated reassortant H5N1 strain (CK/Eg/RG-173CAL/17). The safety of both vaccines was evaluated in specific-pathogen-free (SPF) chickens. To evaluate the efficacy of the prepared vaccines, 2-week-old SPF chickens were vaccinated with 0.5 mL of a vaccine formula containing 10⁸/EID₅₀ /dose from each strain via the subcutaneous route. Vaccinated birds were challenged with either wild-type HPAI-H5N8 or H5N1 viruses separately at 3 weeks post-vaccine. Results revealed that both vaccines induced protective hemagglutination-inhibiting (HI) antibody titers as early as 2 weeks PV (≥5.0 log₂). Vaccinated birds were protected clinically against both subtypes (100 % protection). HPAI-H5N1 virus shedding was significantly reduced in birds that were vaccinated with the bivalent vaccine; meanwhile, HPAI-H5N8 virus shedding was completely neutralized in both tracheal and cloacal swabs after 3 days post-infection in birds that had been vaccinated with either vaccine. In conclusion, the developed bivalent vaccine proved to be efficient in protecting chickens clinically and reduced virus shedding via the respiratory and digestive tracts. The applicability of the multivalent avian influenza vaccines further supported their value to facilitate vaccination programs in endemic countries.     

Immunogenicity of infectious bursal disease viruses in chickens.

Cross-protective properties of infectious bursal disease viruses (IBDVs) were studied. Viruses represented different subtypes of serotype 1, including recently isolated viruses (variants), and a serotype 2 virus. Chickens were vaccinated at 3 weeks of age with inactivated vaccines containing 10(5), 10(6), 10(7), or 10(8) mean tissue-culture infectious dose of a given virus and challenged 2 weeks later using either 10(2) or 10(3.5) mean embryo infectious dose (EID50) of either a standard virus or a variant serotype 1 virus. Protection was evaluated at 5 and 10 days post-challenge, based on gross and microscopic lesions, body weight, and bursa/body-weight ratios. The serotype 2 virus did not confer protection on birds challenged with the serotype 1 viruses. Vaccines made of variant viruses at the low doses protected chickens challenged with the high or low doses of either the standard or the variant viruses. Vaccines made of the standard or variant strains at low doses protected against high or low challenge doses of the standard strain. Vaccines made of the high dose of any of the standard strains protected chickens against the variant virus when the low challenge dose (10(2) EID50) was used, but not when the high challenge dose (10(3.5) EID50) was used. The lowest dose of the standard viruses vaccines required to confer protection against the variant virus varied depending on the strain. Results indicated that protection depended on the strain and dose of both the vaccine and challenge viruses and that the variant strains and standard strains share a common protective antigen(s).     

4型禽腺病毒HLJ1701株灭活疫苗的研制

利用4型禽腺病毒HLJ1701株进行灭活疫苗的研制,并对疫苗的免疫效果进行评价,为家禽4型禽腺病毒的防控提供数据及参考。将HLJ1701株用灭菌生理盐水作10~4倍稀释后,接种9日龄SPF鸡胚,37℃孵育72 h后收获感染鸡胚尿囊液,经甲醛灭活后,加白油佐剂乳化制成油乳剂灭活疫苗,对制备疫苗的性状、安全性、免疫效力等进行检验。结果显示,制备的3批4型禽腺病毒灭活疫苗(HLJ1701株)均为油包水型,黏度均在50 cP以内,对3批疫苗取样,样品经3000 r/min离心15 min,管底无水相析出。安全性试验结果显示,将疫苗按1 mL/只超剂量接种3周龄SPF鸡,试验鸡在观察期内全部健活,未出现局部或全身不良反应,表明疫苗对SPF鸡具有良好的安全性;免疫效力及攻毒保护试验结果显示,用疫苗按0.2 mL/只的剂量免疫接种3周龄SPF鸡1次,免疫接种后21d试验鸡血清中HLJ1701株的抗体平均效价可达2~8以上,使用4型禽腺病毒(HLJ1701株)接种0.2 mL/只(100 LD_(50))对免疫鸡进行攻毒,疫苗对免疫鸡的保护率均为100%。研究表明,实验室条件下研制的4型禽腺病毒(HLJ1701株)灭活疫苗的各项指标均符合标准。