鸭病毒性肝炎卵黄抗体防治鸭肝炎研究

采用从淄博市分离到的Ⅰ型鸭肝炎病毒,免疫鸡制备高免卵黄抗体,经测定中和效价达到1:1024.经试验,用该卵黄抗体用于预防和治疗鸭病毒性肝炎,对1日龄雏鸭用于预防,总体保护率达到95.14%,对发病鸭群进行治疗,总体保护率达到94.67%.     

犬瘟热病毒免疫蛋鸡血清抗体与卵黄抗体消长规律的研究

试验旨在了解产蛋鸡对犬瘟热病毒(canine distemper virus,CDV)抗原的免疫反应及其血清抗体和卵黄抗体的消长规律,为制备卵黄抗体提供依据。将CDV接种Vero细胞和DF1细胞进行传代并测定其病毒滴度,选取细胞病变出现早、病毒滴度高的Vero细胞毒作为接种病毒抗原免疫蛋鸡。每10 d免疫蛋鸡1次,第4次免疫后每30 d加强免疫1次。免疫前及免疫后每10 d采血分离血清,每5 d收集鸡蛋提取卵黄抗体,用琼脂扩散法和间接ELISA法测定其抗体水平。结果显示,血清抗体比卵黄抗体的滴度高,两者呈正相关性,卵黄抗体出现较血清抗体迟3~5 d,卵黄抗体在第3次免疫后3~5 d就已达到较高水平,此时即可开始收集鸡蛋制备卵黄抗体。     

一株鹅星状病毒的分离鉴定

为了研制预防鹅痛风病的鹅星状病毒(Goose astrovirus,GAstV)疫苗及卵黄抗体,试验对黑龙江省某养鹅场疑似痛风死亡鹅的肝脏及肾脏组织进行了病原分离,并对分离病毒进行了鹅胚传代、RT-PCR检测、核苷酸序列测定与比对、病毒含量测定及动物回归试验。结果表明:试验分离得到一株鹅星状病毒;分离毒株可致传代鹅胚100%死亡;鹅胚传代尿囊液的RT-PCR检测为阳性;阳性产物核苷酸序列与鹅星状病毒AHDY株基因(登录号为MH410610.1)和FLX株基因(登录号为KY271027.1)核苷酸序列的同源性分别为98%和93%;分离株病毒含量为1×10~(4.83)ELD_(50)/0.2 mL;2日龄健康易感雏鹅接种分离毒株48 h后陆续死亡,剖检表现为心脏、肝脏与输尿管尿酸盐沉积及肾脏出血肿胀,与自然发病雏鹅症状一致。说明该分离毒株为导致雏鹅痛风病的病原,可作为研制鹅星状病毒疫苗及卵黄抗体的候选毒株。     

抗兔瘟精制卵黄抗体的研制

选用免疫原性好的兔瘟抗原加工成浓缩兔瘟灭活组织疫苗,按特定的免疫程序接种健康商品鸡群,通过血凝抑制试验检测免疫鸡群血清和卵黄的兔瘟抗体效价,适时收集高免卵黄,选用特定的抗体分离提纯工艺,研制对兔瘟有预防和治疗双重作用的精制卵黄抗体。经实验室生物学检验,本品使用无刺激,作用迅速,对兔瘟早期治愈率为80%,对兔瘟紧急预防有效率为100%。     

CDV免疫蛋鸡血清抗体与卵黄抗体的消长规律及其相关性分析

为了解产蛋鸡对犬瘟热病毒杭原的免疫反应及其血清抗体和卵黄抗体的消长规律及相关性,为制备卵黄抗体提供依据,将犬瘟热病毒接种Vero细胞和DF1细胞进行传代并测定其病毒滴度,选取细胞病变出现早,病毒滴度高的Vero细胞毒作为接种病毒抗原免疫蛋鸡。通过每10d进行蛋鸡免疫一次,四免后每30d加强免疫一次的方法进行免疫。免疫前和免疫后每10d采血分离血清和每5d收集鸡蛋提取卵黄抗体,用琼脂扩散法和间接ELISA法测定其抗体水平。结果显示,血清抗体比卵黄抗体的滴度高,两者间呈正相关性,卵黄抗体出现较血清抗体迟3～5d,卵黄抗体在三免后3～5d就已达到较高水平,此时即可开始收集鸡蛋制备卵黄抗体。     

IBD毒株的卵黄培养方法研究

本研究表明,IBD毒株首次分离以鸡胚绒毛尿囊膜（CAM）接种为首选,卵黄囊接种次之。本试验将经CAM驯化的鸡胚适应毒转卵黄囊（Yolk sac）驯化培养,培养结果显示卵黄囊接种的方法,CAM、胚体及尿囊液混合物中的病毒含量高于CAM接种方法的病毒含量,提示该接种方法可用于规模化培养。     

传染性法氏囊病病毒超强毒株高免卵黄抗体的研制及应用

本研究从山西某养殖场分离到一株传染性法氏囊病病毒,经序列比对,发现其与国外经典超强毒株的同源性高达99.6%,除222位点,其他位点均具备超强毒株的序列特征,结合临床发病特征,确定为超强毒株。用此超强毒株研制成传染性法氏囊病灭活疫苗,对健康鸡群三免,当卵黄抗体滴度达到27时,收集鸡蛋,制备高免卵黄抗体,用此高免卵黄抗体治疗IBDV感染鸡群,有效率达100%。     

抗鸡白痢沙门氏菌病卵黄抗体粉饲料添加剂的研制

采用吉林市郊区数家鸡场分离鉴定的致病性鸡白痢沙门氏菌,经固体增菌培养纯化后甲醛灭活,制成油佐剂灭活抗原免疫健康蛋鸡;用平板凝集试验检测卵黄抗体效价,当抗体效价达到1:320倍时,收集免疫鸡蛋分离卵黄,减压低温干燥制成卵黄抗体细粉并进行效价测定和安全检测。最后将卵黄抗体细粉以不同比例加入雏鸡饲料,对试验雏鸡进行预防量饲喂后强毒菌攻毒试验、强毒菌攻毒后治疗量的饲喂试验及小区域雏鸡饲喂试验。结果表明,卵黄抗体粉预防添加量为3%和卵黄抗体治疗添加量为6%时,攻毒保护率、治愈率分别为77.50%和72.50%;饲料添加组较同期普通饲养组总发病率降低7个百分点,抗生素用量降低33%,增重率提高6%。     

抗猪流行性腹泻病毒卵黄抗体的制备及应用

本试验利用猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)SC株免疫产蛋鸡,收集高免卵黄,采用水稀释法和水稀释-盐析法获得鸡源抗PEDV卵黄抗体,采用已建立的ELISA方法对卵黄抗体效价进行测定。以3日龄无母源抗体的易感仔猪为试验动物,对抗PEDV卵黄抗体的治疗效果及安全性进行测定,并进一步利用自然发病猪场的治疗效果进行验证。结果表明,经PEDV SC株细胞毒免疫的鸡可在2次加强免疫后15 d产生高水平的特异性抗体。在实验室治疗试验中,攻毒治疗组仔猪的存活率达60%,攻毒对照组存活率为20%;用于自然发病猪场时,饲喂抗PEDV卵黄抗体饲料的仔猪存活率亦为60%,而自然发病对照组的存活率为10%。以上结果表明抗PEDV卵黄抗体对感染PEDV的仔猪有一定治疗作用。     

雏鸡传染性脑脊髓炎病毒的分离与初步鉴定

用神经胶质细胞培养和6日龄SPF鸡胚卵黄囊接种,分离出一株病毒,经免疫荧光及琼脂扩散试验,证明该病毒为禽传染性脑脊髓炎病毒.     

鸭传染性法氏囊病病原分离鉴定及防治试验

从疑似鸭传染性法氏囊病的病例中分离到 １ 株鸭传染性法氏囊病毒,该病毒可使 ７ 日龄健康鸭 １００％ 发病,发病鸭具有鸭传染性法氏囊病的典型病变,用鸡传染性法氏囊高免卵黄抗体预防和治疗鸭传染性法氏囊病取得了满意效果。     

Development of a one-step strip test for the diagnosis of chicken infectious bursal disease

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.     

朗德鹅禽流感病毒的分离与鉴定

用禽流感病毒ELISA试剂盒对某朗德鹅养殖场的病鹅气管粘液进行了检测，发现5份粘液样本均呈禽流感阳性；随后取相应气管组织材料接种于9～11日龄鸡胚分离病毒．发现尿囊液能使鸡红细胞发生凝集，用禽流感病毒H5、H7、H9标准阳性血清和新城疫病毒、传染性支气管炎病毒、传染性喉气管炎病毒、传染性法氏囊炎病毒抗血清作HI试验，结果禽流感病毒H5亚型抗血清的血凝抑制滴度达到2^7，而禽流感病毒H7、H9亚型及其他病毒抗血清无血凝抑制滴度，说明从朗德鹅分离到的病毒为H5亚型禽流感病毒。     

白条鸡传染性法氏囊病病毒RT-PCR检测方法的建立和初步应用

设计了一对特异性引物,对具有临床症状、病理变化明显和AGP检测呈阳性的病鸡屠体的肺脏和鼻腔粘液进行RT-PCR扩增。结果显示,6个样品的肺脏全部扩增出特异性基因片段,阳性符合率为100%,鼻腔粘液有5例扩增出阳性基因片段,符合率为80%。将建立的RT-PCR检测方法用于市售白条鸡的检测,90只随机样品中有7只扩增出阳性片段,检出率为7.78%。本试验建立的RT-PCR检测方法为肉食品原料卫生的检验提供了新的技术手段。     

Pathogenicity and molecular analysis of an infectious bursal disease virus isolated from Malaysian village chickens

The characteristics of the pathogenic infectious bursal disease virus (IBDV) that infected avian species other than commercial chickens were largely unknown. In this study, by using in vivo and molecular methods, we had characterized an IBDV isolate (named 94268) isolated from an infectious bursal disease (IBD) outbreak in Malaysian village chickens--the adulterated descendant of the Southeast Asian jungle fowl (Gallus bankiva) that were commonly reared in the backyard. The 94268 isolate was grouped as the very virulent IBDV (vvIBDV) strain because it caused severe lesions and a high mortality rate in village chickens (>88%) and experimentally infected specific-pathogen-free chickens (>66%). In addition, it possessed all of the vvIBDV molecular markers in its VP2 gene. Phylogenetic analysis using distance, maximum parsimony, and maximum likelihood methods revealed that 94268 was monophyletic with other vvIBDV isolates and closely related to the Malaysian vvIBDV isolates. Given that the VP2 gene of 94268 isolate was almost identical and evolutionarily closely related to other field IBDV isolates that affected the commercial chickens, we therefore concluded that IBD infections had spread across the farm boundary. IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease.     

鸡传染性法氏囊病毒超强毒株的分离及生物学特性鉴定

用SPF鸡及鸡胚，从吉林某鸡场病死鸡的法氏囊组织到一株超强毒株（J20株）。此病毒不能凝集鸡的红细胞，可以被IBDV标准阳性血清检出IBDV抗原。通过对J2001分离毒株进行回归实验，测定病毒效价EID50达10^6/0.2ml，发病率为100％，死亡达70％；病毒理化特性试验，电镜观察等证实J20株为鸡传染性法氏囊病超强毒株。     

Genetic variations in maternal transfer and immune responsiveness to infectious bursal disease virus

The immune responsiveness to infectious bursal disease virus (IBDV) in four native and crossbred chicken lines was compared. ELISA IBDV antibody titers in hen serum samples, yolk from matched eggs and sera from matched 1-day-old chicks from each chicken line with an identical vaccination program were measured, and plotted. There was considerable variation between lines in the measured IBDV specific antibodies, in vaccinated parent hens and in the amounts of inherited maternally derived antibodies in both yolk and progeny chicks. Differences in ratios of the inherited antibody level from hen to 1-day-old chicks were also found among different chicken lines. Breed differences in regressions of IBDV antibody levels in yolk to that of hen or progeny chicks' sera were also found, so prediction of serum titer of hen and/or progeny chicks from yolk are varied among chicken lines.     

Selection of an infectious bursal disease virus mutant with increased immunogenicity following passage under humoral immune pressure.

It is generally known that the pathogenicity of infectious bursal disease virus (IBDV) strains decreases following passage in cell culture. However, there is no information about the effect of passage under immune pressure on the phenotypic and molecular properties of IBDV. In the present study, a small plaque mutant virus with poor neutralization capability, but showing similar growth characteristics as the parental virus strain, QC2, was isolated after serial passage in Vero cells in presence of IBDV serotype 1 chicken polyclonal antiserum. This mutant virus showed reduced pathogenicity but enhanced immunogenicity compared to the parental virus. Sequence analysis of the non-coding regions of the genome revealed 4 and 3 nucleotide changes in the 3' non-coding regions of segments A and B, respectively, and none in the 5' non-coding regions. Restriction enzyme analysis of selected coding regions of the IBDV genome in both viruses revealed a loss of the PstI site in the VP2 region of the mutant virus. Selection of such mutant viruses by passaging under immune pressure may offer an improved method for developing safer and more effective attenuated vaccine strains against infectious bursal disease of chickens.     

一株传染性法氏囊病病毒强毒株的分离与鉴定

鸡传染性法氏囊病是由传染性法氏囊病病毒引起的一种急性传染病。本研究从江苏某疑似发生传染性法氏囊病的鸡场采集病料,通过观察临床症状、病理变化、RT-PCR检测、基因测序、SPF鸡胚接种、琼脂扩散试验和雏鸡攻毒等试验,证实了该鸡群发生了传染性法氏囊病,且分离到一株传染性法氏囊病病毒(JSXY株),该病毒有较强的致病力,VP4基因比较分析发现其与变异株亲缘关系最近,序列同源性为95%。本研究为江苏地区传染性法氏囊病的防治提供了有益的参考。     

Organ culture of chicken bursa as a model to study the pathogenicity of infectious bursal disease virus isolates.

In vitro study with chicken bursal organ culture was attempted to assess the pathogenicity of locally isolated infectious bursal disease virus (IBDV) initially isolated from the bursa of naturally infected birds. In bursal organ culture, lymphoblastic transformation was noticed as early as 24 hr postinoculation and reached maximum at 72 hr postinoculation. The other microscopic changes were increased number of macrophages and formation of plasma cells. The IBDV antigen was detected 24 hr onward by coagglutination test with antibody coated Staphylococcus aureus strain Cowan I. On the basis of lesion score, the three isolates of IBDV (A, B, and C) were graded as virulent (B isolate) and moderately virulent (A and C isolates). A similar pattern of pathogenicity was also observed in the in vivo pathogenicity studies in chicken based on bursa: body weight ratio and histopathologic lesion score. The bursal organ culture thus provides a useful experimental model to differentiate the IBDV isolates on the basis of their virulence.