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1.
以1型鸭疫里氏杆菌(RA)全基因组保守区域ompA基因的重组表达产物为包被抗原,建立了检测RA血清抗体的间接ELISA方法。原核表达的重组ompA蛋白经纯化后作为包被物,以方阵滴定法确定抗原最佳包被浓度为1.5μg/mL,待检血清最佳稀释度为1∶100。与普通的微量凝集试验相比较,该方法灵敏度高于凝集试验约16倍～128倍。与鸭源大肠埃希菌、鸭源多杀性巴氏杆菌、鸭链球菌、鸭源呼肠病毒、鸭肝炎病毒和鸭瘟病毒感染鸭血清均无交叉反应,表明该方法特异性好。利用该方法检测了雏鸭免疫RA灭活油乳剂疫苗之后血清抗体水平。  相似文献   

2.
The generation of protective immunity against Riemerella anatipestifer infection in ducks were investigated by immunizations with recombinant glutathione sulfatransferase (GST) fusion's proteins of OmpA, a 42kDa major outer membrane protein, and P45N', a 41kDa N-terminal fragment of a newly identified 45kDa potential surface protein from R. anatipestifer. The DNA encoding OmpA and P45N' were isolated from R. anatipestifer serotype 15 (field strain 110/89) and serotype 19 (reference strain 30/90), respectively. Immunoblotting and ELISA results showed that the purified recombinant proteins induced the production of antibodies in immunized ducks. However, neither was protective against subsequent challenge with the virulent serotype 15 strain, 34/90. All the five ducks immunized with formalinized R. anatipestifer strain 34/90 survived the challenge with the homologous strain whereas six out of seven ducks in the non-immunized control group died within a week following the challenge.  相似文献   

3.
将隐孢子虫鼠基因型（Cryptosporidium mouse genotype）P23基因亚克隆到pGEX-4T-1载体中,并用大肠杆菌BL21（DE3）为宿主菌诱导表达。以纯化的rP23为诊断抗原,建立隐孢子虫间接ELISA检测方法,观察其敏感性、特异性和重复性,并对临床血清样品进行检测。结果隐孢子虫鼠基因型P23基因在大肠杆菌中获得了高效表达,Western blot检测显示rP23能被隐孢子虫感染兔血清识别。以纯化rP23为诊断抗原,成功地建立了检测兔隐孢子虫病的间接ELISA技术。对23份临床血清检测结果显示,该方法检出率高于Sheather＇s蔗糖漂浮法、套式PCR法。本研究结果为研制兔隐孢子虫病诊断试剂盒打下了基础。  相似文献   

4.
Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry. A total of 35 R. anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence. This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures. Rep-PCR discriminated the R. anatipestifer isolates into 19 electrophoretic types. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. Rep-PCR may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.  相似文献   

5.
将潜鸭艾美耳球虫孢子化卵爱清洁、纯化，捣碎滤过制成可溶性抗原，用于ＥＬＩＳＡ检测番鸭、北京鸭和二者杂交种骡鸭试验性感染潜鸭艾美耳球虫后血清中抗体的变化。试验鸭于１１日龄每只口服接种孢子化卵囊７．５×１０４个，接种感染后第３天血清抗体的ＯＤ值开始上升，番鸭和骡鸭感染后第１０天血清抗体达峰值，而北京鸭的峰值出现较晚，在感染后第１７天。番鸭和骡鸭在试验期间血清抗体ＯＤ值有两个峰值：第２峰值番鸭出现在感染后第１７天，骡鸭在感染后第２０天。３种鸭血清ＩｇＧ的变化曲线与血清Ｉｇｓ的相近似，表明在家鸭感染球虫后血清ＩｇＧ在血清抗体Ｉｇｓ中占主要地位。同时各组的不感染健康鸭血清免疫球蛋白随日龄增大显缓慢增长，但明显低于接种感染鸭。  相似文献   

6.
A combination Escherichia coli serotype O78 and Pasteurella anatipestifer bacterin was developed and tested in white pekin ducks in laboratory and field trials. Inoculations with bacterin at 2 and 3 weeks of age provided significant protection against challenge with virulent E. coli O78 and Pasteurella anatipestifer serotypes 1, 2, and 5. No significant cross-protection was observed against heterologous E. coli serotypes, although there was a slight reduction in mortality in ducklings challenged with E. coli serotypes O2a and O119. In field trials, the E. coli-P. anatipestifer bacterin produced significant reduction of mortality in commercial white pekin ducks compared with P. anatipestifer bacterin.  相似文献   

7.
A recombinant baculovirus (RBV) encoding the nucleoprotein (NP) of avian influenza virus (AIV) was generated and the appropriate protein was expressed in Sf9 cells. Purified recombinant NP and the NP-specific monoclonal antibody HB65 were used to establish a competitive ELISA (cELISA) system for the detection of NP-specific antibodies in sera of ducks, geese and wild birds. Tests to evaluate this method were carried out using sera of ducks experimentally infected with AIV, pre-immune duck and chicken sera, and poultry field sera, which tested negative in the haemagglutination inhibition (HI) assay, and field sera of several poultry species experimentally infected with other viruses. The evaluation of the test demonstrated a high sensitivity and specificity of this method. Tests carried out using field sera of duck and goose flocks revealed widely corresponding results obtained by HI assay and cELISA indicating that this test is applicable for flock diagnosis. Differing results were obtained for individual samples. It can be assumed that for the most part this was because of a better recognition of the conserved NP antigen by serum antibodies, although some results remained unclear.  相似文献   

8.
鸭疫里氏杆菌病研究进展   总被引：1，自引：0，他引：1  
鸭疫里氏杆菌病是一种主要侵害雏鸭、雏火鸡等多种禽类的急性或慢性传染病.2周龄～7周龄雏鸭最易感,主要引起小鸭纤维素性心包炎、肝周炎和气囊炎,发病率为5%～90%,病死率高达90%.鸭疫里氏杆菌为革兰氏阴性、不运动、不形成芽孢、无鞭毛的小杆菌,有21个血清型,且各血清型缺少交叉保护.鸭疫里氏杆菌病已成为危害养鸭业最为严重的细菌性疾病之一,造成了巨大的经济损失.文章对鸭疫里氏杆菌生物学特性、实验室诊断、血清型鉴定和疫苗免疫方面进行了较为系统的综述.  相似文献   

9.
DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.  相似文献   

10.
北京地区鸭传染性浆膜炎的流行病学调查   总被引：35，自引：1，他引：34  
１９９７ 年１０ 月～１９９８ 年１２ 月, 从北京地区３０ 个商品鸭场随机收集自然死亡鸭５６１ 只, 经过病理剖检和细菌分离鉴定, 确定２９ 个鸭场存在鸭传染性浆膜炎, ６１ ％ 的病例患有本病, 其日龄范围为７ ～４２ 日。结果说明,一年四季中, 鸭传染性浆膜炎均是引起北京地区肉鸭死亡的主要疾病, 广泛分布于该地区各养鸭场; 肉鸭日龄越小, 对本病越易感, 随着肉鸭日龄的增加, 尤其在５ ～６ 周龄后, 肉鸭对本病的抵抗力增加。从死亡鸭的脑、心血、肝脏、脾脏和胆囊均易分离到鸭疫里氏杆菌, 但从脑组织最易分离到该菌; 共分离到鸭疫里氏杆菌４２１ 株, 分别属于１ 、２ 、６ 、１０ 、１３ 、１４ 型, 其中１ 、２ 、６ 、１０ 型占总分离株的９６ ％ , 是目前主要流行的血清型。给健康鸭注射各血清型分离株的液体培养物均可复制出鸭传染性浆膜炎的临床症状和病理变化, 并引起鸭死亡。１０９ 株受试菌对红霉素、青霉素 Ｇ、新生霉素、痢特灵、氯霉素高度敏感。  相似文献   

11.
鸭瘟抗体检测方法研究   总被引：12，自引：0，他引：12  
将鸭瘟组提病毒经Sephadex G200柱层析纯化后作为包被抗原，以健康鸭IgG提纯后免疫羊，制备羊抗鸭IgG，并用过碘酸钠法进行辣极过氧化物酶标记，建立了检测鸭瘟病毒抗体的间接酶联免疫吸附试验（ELISA）。经对不同血清样品的检测，表明此方法特异性高，重复性好，操作简便，适于大批鸭群抗体水平监测和流行病学调查，为合理免疫和科学预防提供了技术手段。  相似文献   

12.
An enzyme-linked immunosorbent assay (ELISA) was used to diagnose Corynebacterium (Rhodococcus) equi infection in foals. In tests done with different antigen-extraction procedures (sodium dodecyl sulfate, sodium deoxycholate, polyoxy-ethylene [9] p-tert-octylphenol, polyoxy-ethylene [9-10] p-tert-octylphenol, sonification, homogenization, and heat treatment at 121 C), Tween 20 was a satisfactory reactive antigen. Using hyperimmune rabbit sera or infected foal sera, we investigated the specificity and the sensitivity of the ELISA with the Tween 20 antigen of the different serotypes or of the isolates. Corynebacterium equi strain ATCC 6939 antigen had the best activity for detecting antibodies to C equi in foals. Sera from 218 healthy horses, 11 healthy foals, 17 healthy newborn foals, a foal with suspected C equi infection, and 5 infected foals were evaluated for antibodies to C equi, using ELISA. The optical density values of 206 healthy horses, 17 healthy newborn foals, and 9 healthy foals were less than 0.1. Infected foal sera, except from foal 3, and serum from a foal with suspected C equi infection had higher optical density values. Using ELISA, specific antibodies against C equi were detected in a naturally infected 6-week-old foal after the foal had a rapid increase in the number of bacteria in the feces and after the initial development of clinical signs of illness at 5 weeks of age. Therefore, ELISA was useful for the early diagnosis of C equi infection in foals.  相似文献   

13.
To obtain estimates for the prevalence of Toxoplasma gondii infection in ducks and geese in Germany, enzyme-linked immunosorbent assays (ELISA) were established based on affinity-purified T. gondii tachyzoite surface antigen 1 (TgSAG1) and used to examine duck and goose sera for T. gondii-specific antibodies. The results of 186 sera from 60 non-infected ducks (Anas platyrhynchos) and 101 sera from 36 non-infected geese (Anser anser) as well as 72 sera from 11 ducks and 89 sera from 12 geese inoculated experimentally with T. gondii tachyzoites (intravenously) or oocysts (orally) and positive in a T. gondii immunofluorescent antibody test (IFAT) were used to select a cut-off value for the TgSAG1-ELISA. Sera obtained by serial bleeding of experimentally inoculated ducks and geese were tested to analyze the time course of anti-TgSAG1 antibodies after inoculation and to assess the sensitivity of the assays in comparison with IFAT. In ducks, IFAT titres and ELISA indices peaked 2 and 5 weeks p.i with tachyzoites, respectively. Only three of six geese inoculated with tachyzoites at the same time as the ducks elicited a low and non-permanent antibody response as detected by the IFAT. In the TgSAG1-ELISA, only a slight increase of the ELISA indices was observed in four of six tachyzoite-inoculated geese. By contrast, inoculation of ducks and geese with oocysts led to an increase in anti-TgSAG1 antibodies within 1 or 2 weeks, which were still detectable at the end of the observation period, i.e. 11 weeks p.i. Inoculation of three ducks and three geese with oocysts of Hammondia hammondi, a protozoon closely related to T. gondii, resulted in a transient seroconversion in ducks and geese as measured by IFAT or TgSAG1-ELISA. Using the newly established TgSAG1-ELISA, sera from naturally exposed ducks and geese sampled in the course of a monitoring program for avian influenza were examined for antibodies to T. gondii; 145/2534 (5.7%) of the ducks and 94/373 (25.2%) of the geese had antibodies against TgSAG1. Seropositive animals were detected on 20 of 61 duck and in 11 of 13 goose farms; the seroprevalences within positive submissions of single farms ranged from 2.2% to 78.6%. Farms keeping ducks or geese exclusively indoors had a significantly lower risk (odds ratio 0.05, 95% confidence interval 0.01-0.3) of harboring serologically positive animals as compared with farms where the animals had access to an enclosure outside the barn.  相似文献   

14.
C L Mo  E C Burgess 《Avian diseases》1987,31(1):197-201
Mallards (Anas platyrhynchos platyrhynchos) and white pekin ducks (Anas platyrhynchos domesticus) were infected with duck plague virus and challenged with LD20's of Pasteurella multocida and P. anatipestifer. There was no difference between mortality rates of duck plague-infected ducks and controls, suggesting that these organisms do not act synergistically under the conditions of our experiments. There was a difference of about 500-fold between the LD20 of P. multocida for mallards and that for white pekin ducks, indicating that mallards are much more susceptible to avian cholera than white pekin ducks.  相似文献   

15.
从江苏兴化地区某养鸭场的病鸭中采集样本,经分离培养、染色镜检、生化检测、凝集试验及PCR检测,分离到一株厌氧的革兰氏阴性短杆菌,并被鉴定为11型鸭疫里默氏杆菌,定名为JY-58株。  相似文献   

16.
间接ELISA检测鸭疫里默氏杆菌血清抗体方法的建立   总被引：2，自引：2，他引：0  
应用1型鸭疫里默氏杆菌(云南株)超声粉碎物作为包被抗原,建立检测鸭疫里默氏杆菌血清抗体的间接ELISA方法。用倍比稀释法确定HRP标记羊抗鸭二抗最佳稀释倍数为1∶3500,并用棋盘测定法确定抗原的最佳包被浓度为2.7mg/mL,血清最佳稀释倍数为1∶300,阴性血清临界值为0.381;阻断试验结果表明,1型鸭疫里默氏杆菌与其抗血清阻断阳性,与鸭大肠杆菌O78、O132菌株和FJ4型鸭疫里默氏杆菌阻断阴性(无交叉反应)。重复性试验结果表明,该ELISA方法批内变异系数≤0.246,批间变易系数≤0.889。结果表明,该ELISA方法特异性强,重复性好,可用于1型鸭疫里默氏杆菌(云南株)血清抗体的检测。  相似文献   

17.
RT—PCR扩增鸭呼肠孤病毒（Duckreovirus,DRV）的dB基因,克隆到pET-32a（＋）表达载体,再转化大肠杆菌Transetta（DE3）;含有重组质粒pET—σB的大肠杆菌经IPTG诱导,获得大小为55000的以包涵体形式表达的σB重组蛋白。Westernblotting显示,σB重组蛋白能够与兔抗DRV多抗血清特异性结合。以高亲和NI—NTA树脂在变性条件下纯化、梯度尿素复性的σB重组蛋白为包被抗原,建立了检测DRV抗体的间接ELSIA方法。分别以间接ELSIA方法和血清中和试验对DRV感染鸭血清和SPF鸭血清进行检测,两者的符合率为100％。该间接ELSIA方法对鸭瘟病毒、鸭病毒性肝炎病毒和禽流感病毒阳性血清均无交叉反应。  相似文献   

18.
鸭疫里默氏杆菌的分离鉴定   总被引：3，自引：0，他引：3  
从广州市郊区几个鸭场发病或死亡鸭的脑、心血、肝、脾等组织中分离到6株革兰氏阴性杆菌，经形态学检查、生化试验、动物实验等鉴定均为鸭疫里默氏杆菌。通过凝集试验和琼脂扩散试验鉴定：2株为鸭疫里默氏杆菌Ⅰ型，4株为鸭疫里默氏杆菌Ⅱ型。  相似文献   

19.
A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).  相似文献   

20.
以血清 1、2、4、5型鸭疫里默氏菌 (Riemerella anatipestifer,RA)制备 4价铝胶佐剂和铝胶复合佐剂 2种灭活苗 ,经过无菌及安全检查合格后 ,对 3日龄樱桃谷鸭颈背部皮下注射 0 .5 m L/只。(1 )免疫后 1 0、1 3、1 6 d对同源 RA的攻击 ,铝胶佐剂苗表现出 5 %～ 2 0 %、75 %～ 90 %、1 0 0 %免疫保护 ,2 3～ 30 d免疫保护力开始下降 ;铝胶复合佐剂苗表现出1 0 %～ 35 %、80 %～ 1 0 0 %、1 0 0 %免疫保护 ,5 1～ 6 5 d免疫保护力开始下降。(2 ) 2种疫苗 1次免疫后 2 3d抗体水平均达高峰 ,至 93日龄时仍能检出抗体的存在 ,1 0日龄进行第 2次免疫后可产生更高的抗体水平 ,到 6 5 d时仍能保持较高抗体水平 ;其中 1 6～ 6 5 d进行 2次免疫的铝胶复合佐剂苗产生的抗体水平显著 (P<0 .0 5 )或极显著 (P<0 .0 1 )地高于铝胶佐剂苗。 (3) 2种疫苗免疫雏鸭后能够显著 (P<0 .0 5 )或极显著 (P<0 .0 1 )地增强 T细胞的免疫力 ,时间达 2周。对攻毒保护试验、抗体消长规律和细胞免疫测定结果综合分析表明 ,RA4价铝胶佐剂和铝胶复合佐剂 2种灭活苗免疫雏鸭后 ,免疫保护的形成是体液免疫与细胞免疫协同作用的结果。经实验室攻毒保护试验、EL ISA抗体消长规律测定、T细胞免疫水平测定和田间试验表明 ,研制的 2种疫苗均具有  相似文献   

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