Experimental reproduction of an Enterococcus cecorum infection in Pekin ducks

Enterococcus cecorum (EC) was thus far only known as a pathogen for broilers and broiler breeders. Recently there was evidence of EC field outbreaks in Pekin duck flocks in Germany. In this study we experimentally reproduced an EC infection in Pekin ducks. At 12 days post hatch, groups of Pekin ducks were infected orally, via the thoracic air sac or intravenously with 1.5 × 10⁹ colony-forming units (CFU) of EC per bird or via the air sac with 8.5 × 10⁵ or 8.5 × 10⁷ CFU per bird. Ducks of the intravenously infected group showed 100% mortality after 2 days post infection. The air sac inoculated high-dose group exhibited a mortality rate of 67%. Birds that were infected with 8.5 × 10⁵ and 8.5 × 10⁷ CFU showed 6.7% mortality after 7 days post infection. Dead birds displayed pneumonia, airsacculitis, pericarditis and splenitis and EC was re-isolated from these organs. Surviving birds of all groups apart from the orally infected ducks demonstrated clinical signs such as huddling, reduced mobility and diarrhoea. Furthermore, they showed gross pathological lesions including airsacculitis and splenitis and lower bodyweights than the control group at necropsy on days 7, 14 and 21 post infection. The present study clearly confirms that EC is pathogenic for Pekin ducks after experimental infection via the intravenous route or the respiratory tract. EC therefore has to be considered as an emerging avian pathogen not only in broilers but also in Pekin ducks.     

Vaccination of breeder hens with a polyvalent killed vaccine for pathogenic Enterococcus cecorum does not protect offspring from enterococcal spondylitis

Pathogenic strains of Enterococcus cecorum cause symmetrical paralysis in broilers due to infection of the free thoracic vertebra. The disease caused by pathogenic E. cecorum, known as enterococcal spondylitis or “kinky-back” continues to be responsible for significant losses to the broiler industry worldwide. In outbreaks of pathogenic E. cecorum, gut colonization and sepsis occur in the first three weeks-of-life. Since maternal antibodies are present during this period, we postulated that vaccination of breeders with a polyvalent killed vaccine would protect chicks from challenge. To test this hypothesis, representative isolates from seven genotype groups of pathogenic E. cecorum circulating in the US were chosen to produce adjuvanted killed vaccines (bacterins) and given to broiler-breeder hens. No single strain produced high titres of antibodies to all other strains; however, the combination of serologic reactivity of pathogenic isolates (designated SA3 and SA7) was sufficient to react with all genotypes. Vaccination of commercial broiler-breeder hens with a bacterin composed of SA3 and SA7 did not have any adverse effects. Vaccinated hens developed E. cecorum specific antibodies; however, no significant difference in survival was observed in infected embryos from hens in vaccine or adjuvant only groups. Chicks from vaccinated hens also failed to resist homologous or heterologous challenge during experimental infection. In a macrophage killing assay, pathogenic E. cecorum were found to evade opsinophagocytosis with elicited antibodies. These data suggest that pathogenic strains of E. cecorum possess virulence mechanisms that confound antibody-mediated opsinophagocytosis, complicating vaccine development for this pathogen of broilers.     

CT venography and pathological characterization of crus haemorrhage in Pekin ducks

The crus haemorrhage is one of the main causes of carcass defects in Pekin duck processing houses. However, its pathologic features are currently unclear. In order to examine the injury to the hind limb veins and illustrate the pathologic characteristics of crus haemorrhage in Pekin ducks, a total of 68 Pekin ducks with crus haemorrhage (test group) and 10 unaffected ducks (control group) were collected in this study. Five ducks randomly selected from each group were examined by computed tomographic venography with 2.0?mm thickness, 120?kVp, and 90?mA. Pathological changes were observed macroscopically, and under a microscope and electron microscope. The computed tomographic venography results showed no differences in the main hind limb veins between Pekin ducks with crus haemorrhage and the control. Macroscopic results demonstrated that the haemorrhage only occurred in crural muscles, most frequently in musculus gastrocnemius and musculus tibialis cranialis. In severe cases, muscular rupture and multiple intermuscular blood clots could be observed. Histological analysis showed rupture of myofibers and massive red blood cells between muscle bundles. Besides, infiltration of connective tissues and inflammatory lesions could be seen. However, no differences were observed in other organs between these two groups. The main ultrastructural characteristics were myofibrillar rupture and split, accompanied by mitochondrial membrane disintegration and vacuolization. All these results indicate that the haemorrhage in crus is a focal myopathy with the characteristics of bleeding, rupture, and inflammatory lesions.

• Research highlights

• CTV was a feasible method to evaluate the hind limb veins in Pekin ducks.

• The focal myopathy presented here only affected crural muscles.

• The focal myopathy was characterized by bleeding, rupture and inflammatory lesions.

     

An ELISA for the detection of maternal anti-trophoblast antibodies in human pregnancy

An ELISA has been developed which is able to detect antibodies directed against determinants present on the plasma membrane of the outer layer, the syncytiotrophoblast, of term placentae. The IgG and IgM anti-trophoblast antibodies are present in the sera of women during the course of a normal pregnancy. The ELISA was based on the use of syncytiotrophoblast plasma membranes as the antigenic targets and utilised a developing antiserum conjugated to the bacterial enzyme urease. Although the assay was simple and reproducible, its viability was dependent on several factors including the stability of the antibodies and the proper preparative techniques for the production of the plasma membranes. Failure to adhere to the correct procedures resulted in an inferior non-viable assay.     

MAC-ELISA and ELISA inhibition methods for detection of antibodies after yellow fever vaccination

The IgM antibody capture ELISA (MAC-ELISA) and ELISA inhibition methods for the detection of antibodies against dengue virus were modified to detect antibodies against yellow fever virus. Tests were carried out in 21 persons vaccinated with 17D and compared with the Plaque reduction neutralizing test. Of 17 naive subjects vaccinated, 16 (94%) seroconverted using the MAC-ELISA test and 14 (82%) seroconverted (or ≥fourfold titer increase) in the ELISA inhibition method. Cross-reactivity was evaluated by both tests and resulted in a high specificity to IgM antibodies against yellow fever, when all the samples from vaccinated individuals were negative by MAC-ELISA using dengue antigen. However, 10.7% of the positive dengue sera from the Santiago de Cuba epidemic cross-reacted by MAC-ELISA using yellow fever antigen. ELISA inhibition method showed high cross-reactivity when the 21 sera pairs were worked with yellow fever and dengue antigens. The MAC-ELISA and ELISA inhibition methods have become indispensable tools in our laboratory in order to maintain a surveillance system for dengue and dengue hemorrhagic fever. They are relatively rapid, simple, and they do not require sophisticated equipment. Both MAC-ELISA and ELISA inhibition methods for yellow fever could be useful for diagnosis, surveillance and yellow fever vaccine evaluation.     

A new oligonucleotide-based ELISA for the detection of anti-double-stranded DNA antibodies

The detection and measurement of antibodies to double-stranded (ds) DNA is important in the diagnosis and monitoring of patients with systemic lupus erythematosus (SLE). Several methods are available for their determination but none of them is completely satisfactory. Usually, purified dsDNA from different sources is used as antigen in the solid (enzyme-linked immunosorbent assay, ELISA) or liquid phase (Farr assay). Alternatively, anti-dsDNA antibodies can be demonstrated by immunofluorescence, using the haemoflagellate Crithidia luciliae (CLIFT).

We have developed a new oligonucleotide-based anti-dsDNA ELISA in which dsDNA, constituted of a 5′photobiotinylated nucleic acid probe and its highly specific complementary oligonucleotide, is formed onto the solid-phase. To do that, a 40 bp probe is coated on the microplate wells via streptavidin–biotin bond and the in vitro (in solid-phase) developed hybrid between probe and its complementary helix used as capturing antigen. The assay has proved to be specific and the several experiments performed to ascertain the absence of single-stranded DNA (ssDNA) contamination and/or non specific binding to plastic, streptavidin, probe (without complementary chain) have all excluded any significant interference.

To evaluate the clinical performance of this new assay, 114 serum samples from 68 SLE patients and 85 samples from 85 disease controls, in addition to 30 blood donors, were studied. The results were compared with commercial ELISAs, Farr assay and indirect immunofluorescence.

The sensitivity and specificity were 66.2 and 96.4%, respectively, comparable and even better than those of the commercially available anti-dsDNA kits. Anti-dsDNA antibody levels, measured with the oligonucleotide–ELISA, correlated with SLE clinical activity determined using the European Consensus Lupus Activity Measurement (ECLAM) (r = 0.59, p < 0.0001).

In conclusion, this assay has proved to be reproducible, and the results correlate well with other standard anti-dsDNA assays. The features of this new assay make it promising for clinical use.     

An enzyme-linked immunosorbent assay for detection of antibodies against halothane-altered hepatocyte antigens

Patients with massive liver cell necrosis that may follow halothane anaesthesia have a high incidence of circulating antibodies against halothane-induced hepatocyte antigens. In order to provide an objective and quantitative method for the detection of these antibodies, an enzyme-linked immunosorbent assay has been developed. Sera, after absorption with normal rabbit liver microsomal fraction, are tested for binding to microsomal fractions from control and halothane-pretreated rabbits. Those containing antibodies against halothane-induced determinants give significantly enhanced binding to halothane-altered fractions; this specificity was verified by absorption experiments. Using this method, halothane-related antibodies were detected in sera from 16/24 patients with halothane-associated liver failure, at titres ranging from 1:100 to 1:25600. Such antibodies were not detectable in sera from 26 normal blood donors, 5 healthy anaesthetists, 12 patients who had received multiple halothane anaesthetics but had normal liver function tests and 32 patients with a variety of other liver diseases. This rapid and reproducible assay should be of value for the detection of antibodies and for detailed investigation of patient antibody responses, and also for characterization of the route of production and metabolism of the antigen.     

Development of a blocking ELISA for detection of serum neutralizing antibodies against porcine circovirus type 2

A monoclonal antibody (Mab)-based blocking ELISA was developed for the detection of serum neutralizing antibodies to porcine circovirus type 2 (PCV2). The Mab with neutralizing activity, which was produced by immunizing a recombinant capsid protein of PCV2 expressed in insect cells, was used as the detector antibody. The assay was evaluated in comparison with a serum neutralization assay, and its sensitivity and specificity were determined to be 98.8% and 88.5%, respectively. A significant positive correlation was found between results of the blocking ELISA and the serum neutralization assay (r = 0.9381). The assay was verified by testing experimental and commercial pig sera. A longitudinal antibody profile showed that serum neutralizing antibodies were detected 2 weeks after vaccination and that the detection rate reached 100% at 4 weeks. The serum neutralizing antibody profile showed a decrease from the age of 4 to 13 weeks, and seroconversion after 13 weeks in pigs from a commercial pig farm. Additionally, the positive detection rate in 703 sera collected from nine commercial pig farms was 73%. This report demonstrates that the assay is a simple, specific, sensitive and convenient method for epidemiological surveys and evaluations of serum neutralizing antibodies against PCV2.     

人幽门螺杆菌尿素酶抗体诊断试剂盒的研制

幽门螺杆菌 (Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ,Ｈ .ｐｙｌｏｒｉ)为慢性胃炎、消化性胃溃疡的致病菌 ,并且与胃癌的发生密切相关 ,世界卫生组织 (ＷＨＯ)将其列为一级致癌因子 ,早期诊断Ｈ .ｐｙｌｏｒｉ感染并对其治疗和预后的观察具有重要意义。目前 ,临床上用于诊断Ｈ .ｐｙｌｏｒｉ感染的方法主要有胃镜下组织活检、细菌培养、13 Ｃ呼气试验等 ,前者需活检组织 ,病人痛苦大 ,且阳性率低 ,后者需特殊性设备 ,不易推广 ,细菌培养则阳性检出率低 ,而血清学诊断具有灵敏、快速、准确、简便、费用低等优点 ,易在临床上广泛使用。但是 ,…     

Production of penicillin-specific polyclonal antibodies for a group-specific screening ELISA

Polyclonal penicillin-specific antibodies were obtained after the immunization of three rabbits with physiological penicillin-protein conjugates. The broad-specificity of the antisera was improved by alternate immunizations using immunogens with different penicillins as hapten. For the three antisera, the detection of ampicillin, amoxicillin, benzylpenicillin, oxacillin, cloxacillin and dicloxacillin was more sensitive in a competitive ELISA coated with the antibodies (antibody cELISA) compared to a competitive ELISA coated with a penicillin-protein conjugate (antigen cELISA). The detection of all penicillins in buffer solutions at concentrations below the MRL was achieved in the antibody cELISA when the penicillins were hydrolyzed with Penicillinase I. A simple extraction procedure was developed for the detection of penicillins in incurred porcine tissues using the antibody cELISA. Tissue fluids were used instead of the whole meat matrix, which simplified the sample preparation. Before analysis, the fluids were treated with kaolin to reduce the background signals in ELISA. The specificity, sensitivity, repeatability, decision limit (CCα) and detection capability (CCβ) were determined. The suitability of the antibody cELISA and the respective extraction procedure for screening purposes was demonstrated by comparison of the analysis of incurred samples using different screening assays. Almost 100% correlation (r=0.96) was found between the ELISA and a commercial immunochemical method, the Parallux? assay.     

检测抗HIV-1/2型抗体的双抗原夹心方法的建立及其试剂盒的研制

目的 建立更敏感的检测人免疫缺陷病毒（HIV）抗体的方法,并研制检测试剂盒。方法 根据HIV－1／2型的基因序列及其所编码氨基酸结构,采用固相法合成了HIV－1型的gp41.1、gp41.2、gp120、p24和HIV－2型的gp36五条多肽,混合包被酶标板作为固相抗原。用辣根过氧化物酶标记以上多肽抗原作为标记物,建立检测血清中抗HIV－1／2抗体的双抗原夹心ELISA法。同时,应用该方法制备检测HIV抗体的试剂盒,并检测三批中国卫生中药品和生物制品检定所HIV诊断试剂国家参比品。结果 建立了检测HIV－1／2抗体的双抗原夹心法。用检定所参比品检测,该方法特异性、灵敏度均为100％,变异系数小于10％。与间接法相比较其灵敏度、特异性均高于间接法（P＜0．05）。检测210份其他病种患者血清均为阴性。与GBI公司的HIV抗体诊断试剂比较,检测40份卫生部药品和生物制品检定所提供的质控参比品（阳性20份,阴性20份）,GBI试剂阴、阳性符合率及总符合率分别为100%(20/20)、85％（17／20）及92．5％（37／40）,而应用该方法所研制的诊断试剂盒、阳性符合率及总符合率为100％。该试剂已通过国家卫生部质检。与雅培公司HIV诊断试剂比较检测90份献血员血清和88份HIV－1／2型感染者血清,符合率为100％。试剂盒于37℃放置4d后的检测结果的阴、阳性判定不受影响。结论 本法特异性强、灵敏度高、稳定性好,适用于献血员的筛选和临床HIV感染的检测。     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Production of monoclonal antibodies and development of an antigen capture ELISA directed against the envelope glycoprotein GP of Ebola virus

Ebola virus (EBOV) causes severe outbreaks of Ebola hemorrhagic fever in endemic regions of Africa and is considered to be of impact for other parts of the world as an imported viral disease. To develop a new diagnostic test, monoclonal antibodies to EBOV were produced from mice immunized with inactivated EBOV species Zaire. Antibodies directed against the viral glycoprotein GP were characterized by ELISA, Western blot and immunofluorescence analyses. An antigen capture ELISA was established, which is specific for EBOV-Zaire and shows a sensitivity of approximately 10³ plaque-forming units/ml. Since the ELISA is able to detect even SDS-inactivated EBOV in spiked human sera, it could complement the existing diagnostic tools in the field and in routine laboratories where high containment facilities are not available.     

Enzyme-linked immunosorbent assay (ELISA) for detecting antisperm antibodies in mice with testicular autoimmunity

We developed an ELISA for measuring antisperm antibodies in the mouse by using serum samples obtained from mice immunized with murine testicular antigens in complete Freund's adjuvant (CFA) as well as from mice rendered vasectomized. Sperm antigens used were syngeneic epididymal spermatozoa and two types of soluble, murine testicular antigens prepared in our laboratory. This study deals with a) the sequential changes of antisperm antibody levels following immunization; b) determination of immunoglobulin classes of these antibodies; c) a correlation between the absorbance values and the endpoint titers of antisperm antibodies; and d) comparison of endpoint titers of antisperm antibodies detected by ELISA with those by immunoperoxidase staining method in immune and nonimmune sera. It is suggested that serum dilution as high as 1/800 or more is required for detecting antibody titers of immune sera, because nonimmune mouse sera reveal a definite, although low, level of absorbance value at a serum dilution of 1/400 or less.     

Comparison of ELISA for tissue transglutaminase autoantibodies with antiendomysium antibodies in pediatric and adult patients with celiac disease

BACKGROUND: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients. METHODS: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients. RESULTS: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively. CONCLUSION: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.     

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

Evaluation of ELISA for detection of rabies antibodies in domestic carnivores

Serological tests of pets have increased as many rabies-free countries have amended their quarantine measures and adopted a scheme requiring rabies vaccination followed by a serological test. A European directive requires the measurement of neutralising antibodies as proof of protection to allow the free movement of pets within the European Union and between third countries non listed in the list C of regulation 998/2003 and European countries. At present, the recommended neutralisation tests (FAVN test or RFFIT) are time-consuming, expensive and require highly trained technicians as well as special laboratory facilities. The rabies ELISA designed by BioPro was developed initially for use for field samples from foxes to check the efficacy of oral vaccination campaigns in Europe. In this study, the specificity, sensitivity and reliability of this commercial rabies ELISA was evaluated for testing sera from dogs and cats involved in international trade. The specificity evaluated in 315 unvaccinated animals was 100%. Concordance of 86.2% was obtained when comparing BioPro ELISA to the gold standard FAVN test in 701 samples from vaccinated dogs and cats. The rabies ELISA developed recently can be considered a valuable method for the assessment of rabies antibodies in vaccinated domestic carnivores in combination with neutralisation tests.     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.