Isolation and serological studies with infectious bursal disease viruses from fowl, turkeys and ducks: demonstration of a second serotype

The isolation of a number of strains of infectious bursal disease (IBD) virus from fowl, turkeys and ducks is described. These isolates could be grouped into two serotypes using the neutralisation test. It is proposed that the cell culture adapted vaccine strain from fowl should be the prototype virus for serotype 1 and that the TY89 isolate from a turkey should be the prototype for serotype 2. The isolates in serotype 2 consisted of an antigenically homogeneous group of viruses from turkeys and fowl. However, within serotype 1, which represented isolates from fowl and ducks, some isolates showed only a 30% cross reaction with the vaccine strain. If cross protection mirrors cross neutralisation, then infection with viruses belonging to serotype 2 or with antigenically distant strains from serotype 1 provides one explanation for the apparent failure of the vaccine on certain sites. However, if cross protection does not mirror cross neutralisation, then a virus from serotype 2 could be used as a heterotypic vaccine for young birds with high levels of maternally derived antibody to serotype 1.     

Classification of Neisseria meningitidis group B into distinct serotypes. IV. Preliminary chemical studies on the nature of the serotype antigen

Group B Neisseria meningitidis has been subdivided into 11 distinct serotypes by a sensitive bactericidal inhibition technique. The antigens responsible for induction of bactericidal type-specific antibodies were found to be extractable from the group B cells with heating at 100 C either by 0.017 n HCl in saline or by normal saline. These extracted serotype antigens were detected by a capillary precipitin test. The development of methods for extraction and assay of the serotype antigens permitted studies on their immunochemistry. The serotype antigens were distinct from the group-specific substance. Acid extracts contained abundant serotype antigen, but were devoid of group-specific substance. The identity of serotype antigens as proteins was confirmed by their sensitivity to Pronase and trypsin. The molecular weight of these antigens as estimated by G-200 Sephadex chromatography and by electrophoresis in polyacrylamide gels is in excess of 200,000 daltons. Saline extracts containing the serotype antigen could be fractionated into three distinct fractions with acetic acid: pH 4.5 and pH 3.5 precipitated fractions and a pH 3.5 supernatant fraction. The pH 4.5 precipitated fraction contained the serotype antigen.     

A novel monoclonal antibodies-based sandwich ELISA for detection of serotype 4 fowl adenovirus

As a major causative agent for hepatitis-hydropericardium syndrome (HPS) in chickens, serotype 4 fowl adenovirus (FAdV-4) has caused huge economic losses in the poultry industry globally. However, there is no commercial diagnostic test for FAdV-4 antigens. To generate a rapid approach for specific detection of FAdV-4, a monoclonal antibodies (mAbs)-based sandwich ELISA was developed. In this ELISA, a purified mAb 4A3 and a HRP-labelled mAb 3C2 specific to the fiber-2 of FAdV-4 were used as the capture antibody and detection antibody respectively. Specificity assay revealed the ELISA only reacted with FAdV-4, not with other avian viruses tested. Sensitivity assay showed the limit of detection of the ELISA was 1000 TCID₅₀/ml and 12.5?ng/ml for the FAdV-4 and the purified GST-Fiber2 protein respectively. Moreover, the ELISA could be efficiently applied in detecting the FAdV-4 in tissue samples from a clinically-diseased chicken flock. All these data demonstrated that the ELISA developed here provides a promising tool for rapid and efficient diagnosis of clinical infection with FAdV-4.     

Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood.

An indirect (I) enzyme-linked immunosorbent assay (ELISA) and a competitive (C) ELISA, using a group-specific monoclonal antibody against bluetongue virus (BTV), are described for the detection of antibodies to BTV in cattle and sheep sera. The performance of these assays in detecting anti-BTV antibody in sequential serum samples and eluates from whole blood (WB) dried on filter paper from three calves and four sheep experimentally infected with type 10 BTV was evaluated. The C-ELISA was superior to the I-ELISA in the detection of anti-BTV antibody in the sera and WB samples from both cattle and sheep early after infection with BTV. BTV antibodies were demonstrable by C-ELISA in all the bovine and ovine sera and WB eluates by 9 days postinfection; whereas the I-ELISA results for sheep sera and WB eluates were similar, anti-BTV antibody was not detected in bovine serum and WB eluates until 26 and 14 days postinfection, respectively. While both ELISAs proved reliable, under the present test conditions involving detection of early postinfection reactions of experimentally infected animals, the C-ELISA was always as sensitive or more sensitive than the standard agar gel immunodiffusion test, the modified complement fixation test, and the plaque neutralization tests in the detection of anti-BTV antibodies. Unlike observations with the immunodiffusion test, no reaction was seen between BTV antigen and bovine epizootic hemorrhagic disease virus antiserum in either ELISA. The results suggest that either ELISA may be suitable for routine diagnostic testing and may have the potential to replace other tests for detection of anti-BTV group-specific antibodies and that the C-ELISA may have the most potential.     

Use of monoclonal antibodies to type Shigella flexneri in Bangladesh.

A panel of 10 mouse and rat monoclonal antibodies specific for different type- and group-specific O-antigenic determinants of Shigella flexneri lipopolysaccharide was used to serotype 240 isolates of S. flexneri from Bangladesh. Three immunoglobulin M antibodies were used in a direct slide agglutination test; seven immunoglobulin G antibodies were absorbed to Staphylococcus aureus and used in a coagglutination assay. All but 13 of the isolates could be serotyped by using the monclonal antibodies. The six most common serotypes were (in descending order) 2a, 2b, Y (E1037), 1a, 3a, and 1b and accounted for more than 80% of all isolates. Two of the nontypable strains were found to be of a new provisional serotype of S. flexneri (T. Wehler and N.I.A. Carlin, Eur. J. Biochem. 176:471-476, 1988). The 11 remaining strains were found to be rough and therefore nontypable. The serotyping scheme based on the panel of monoclonal antibodies is specific and holds the potential to be developed into a useful tool for epidemiological investigation. The study also demonstrates that the recently described E1037 antigen is commonly found among at least four serotypes (4a, 6, X, and Y) of S. flexneri.     

Identification and serotyping of Ornithobacterium rhinotracheale.

In the present study 443 strains of Ornithobacterium rhinotracheale, a causative agent of respiratory disease in fowl, were investigated biochemically and serologically. In both ways O. rhinotracheale could be differentiated from other gram-negative rods and, more particularly, from the Pasteurella-like bacteria potentially pathogenic for fowl. For the biochemical characterization of O. rhinotracheale the API 2ONE identification strip proved to be useful, although O. rhinotracheale is not included in the API system. Serologically, by using monovalent antisera in agar gel precipitation (AGP) tests and enzyme-linked immunosorbent assays (ELISAs), seven serotypes (serotypes A to G) of O. rhinotracheale could be discriminated. The AGP test was chosen as the preferred method to be used for serotyping. Isolates of serotype A were found to be the most prevalent, especially in chickens. Isolates from turkeys were more heterogeneously divided over the serotypes. Some strains showed cross-reactivity between serotypes A, B, and E. Five O. rhinotracheale strains could not be serotyped with the available antisera. Relationships between the geographic origin and the serotypes were found. By the ELISA the presence of antibodies against O. rhinotracheale could be detected in 1-day-old birds as well as in birds with clinical signs, and therefore, it might be useful for diagnostic purposes.     

Serology of typhoid fever in an area of endemicity and its relevance to diagnosis

Currently, the laboratory diagnosis of typhoid fever is dependent upon either the isolation of Salmonella enterica subsp. enterica serotype Typhi from a clinical sample or the detection of raised titers of agglutinating serum antibodies against the lipopolysaccharide (LPS) (O) or flagellum (H) antigens of serotype Typhi (the Widal test). In this study, the serum antibody responses to the LPS and flagellum antigens of serotype Typhi were investigated with individuals from a region of Vietnam in which typhoid is endemic, and their usefulness for the diagnosis of typhoid fever was evaluated. The antibody responses to both antigens were highly variable among individuals infected with serotype Typhi, and elevated antibody titers were also detected in a high proportion of serum samples from healthy subjects from the community. In-house enzyme-linked immunosorbent assays (ELISAs) for the detection of specific classes of anti-LPS and antiflagellum antibodies were compared with other serologically based tests for the diagnosis of typhoid fever (Widal TO and TH, anti-serotype Typhi immunoglobulin M [IgM] dipstick, and IDeaL TUBEX). At a specificity of > or =0.93, the sensitivities of the different tests were 0.75, 0.55, and 0.52 for the anti-LPS IgM, IgG, and IgA ELISAs, respectively; 0.28 for the antiflagellum IgG ELISA; 0.47 and 0.32 for the Widal TO and TH tests, respectively; and 0.77 for the anti-serotype Typhi IgM dipstick assay. The specificity of the IDeaL TUBEX was below 0.90 (sensitivity, 0.87; specificity, 0.76). The serological assays based on the detection of IgM antibodies against either serotype Typhi LPS (ELISA) or whole bacteria (dipstick) had a significantly higher sensitivity than the Widal TO test when used with a single acute-phase serum sample (P < or = 0.007). These tests could be of use for the diagnosis of typhoid fever in patients who have clinical typhoid fever but are culture negative or in regions where bacterial culturing facilities are not available.     

Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against Coxsackie B viruses

In tests for IgG antibodies against Coxsackie B viruses in man, the enzyme-linked immunosorbent assay (ELISA) was essentially group-specific and, unlike the type-specific neutralisation test, usually failed to detect rises in antibody titre in paired, acute and convalescent, sera. However, in rabbits immunised against Coxsackie B viruses, ELISA demonstrated both group- and type-specific antibody responses. The lack of type-specificity of ELISA in man is probably because repeated infection with enteroviruses--echoviruses and Coxsackie A as well as Coxsackie B--results in masking of the type-specific antibody response by group-specific antibody.     

Age-Stratified Seroprevalence of Neutralizing Antibodies to Astrovirus Types 1 to 7 in Humans in The Netherlands

Astroviruses are a new family of positive-stranded RNA viruses that cause gastroenteritis in a wide range of animals and in humans. Seven types of astrovirus, tentatively considered serotypes, have been distinguished by enzyme-linked immunosorbent assays (ELISA) or immunoelectron microscopy, but it is unclear whether the serotype designation is used properly. To test human sera for the presence of neutralizing antibodies and to type field strains, neutralization tests (NT) using CaCo2 tissue-culture-adapted astrovirus strains 1 to 7 and the corresponding rabbit reference sera were developed. In rabbits, neutralizing antibodies were predominantly serotype specific, with the exception of low-level cross-reactivity in astrovirus serotype 4 reference serum with astrovirus serotype 1 virus. Similarly, in humans, no evidence of cross-reactivity was found for the serotype combinations tested (all except the combination 1 and 7 and the combination 6 and 7). Typing by NT was concordant with typing by ELISA and genotyping, with one exception. The seroprevalence rates of neutralizing antibodies in an age-stratified sample of the population in Utrecht Province (n = 242) were 91% for astrovirus serotype 1, 69% for astrovirus serotype 3, 56% for astrovirus serotype 4, 36% for astrovirus serotype 5, 31% for astrovirus serotype 2, 16% for astrovirus serotype 6, and 10% for astrovirus serotype 7. Acquisition of antibodies was slower among persons seropositive for astrovirus serotype 5 than among those seropositive for astrovirus serotypes 1 to 4, suggesting that the epidemiology of serotype 5 astrovirus is different from that of astrovirus serotypes 1 to 4.     

Use of synthetic oligonucleotide probes to detect rhinovirus RNA

Current methods of detecting a human rhinovirus (HRV) infection are either based on isolation of virus in appropriate susceptible cell lines, which is time-consuming and requires considerable expertise, or are dependent on knowing the serotype. The existence of over 100 immunologically distinct serotypes makes serotype specific assays, such as ELISA, unsuitable for general diagnostic assays. In this study a general rhinovirus assay is described which utilises synthetic oligonucleotides as probes in a filter hybridization assay. The probes are designed to bind to short but highly conserved regions of the rhinovirus genome. Indeed, the probes successfully detected all 57 rhinovirus serotypes tested. Furthermore, the test was used to demonstrate rhinovirus infection in clinical samples from 57 volunteers, inoculated with HRV, collected on six consecutive days. Clinical samples were taken prior to inoculation and on days 2-7 after inoculation. The filter hybridization assay gave results comparable to virus culture on days 2 and 3 post-inoculation, but was more sensitive on subsequent days.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serotype 2.

A blocking enzyme-linked immunosorbent assay (ELISA), based upon a polyclonal rabbit antiserum specific to Actinobacillus pleuropneumoniae serotype 2, was developed for the detection of antibodies to A. pleuropneumoniae serotype 2 in pigs. By testing sera from pigs experimentally infected with the 11 recognized serotypes of A. pleuropneumoniae, the assay was proven to be specific for A. pleuropneumoniae serotype 2. With field sera from herds infected with A. pleuropneumoniae serotype 2, the assay was found to be more sensitive than the complement fixation test. Positive results were not observed with field sera from herds known to be free from Actinobacillus infection or with sera from two herds infected with either A. pleuropneumoniae serotype 6 or 8. The high diagnostic sensitivity and specificity of the blocking ELISA will make it useful in field diagnostic work.     

Porcine rotaviruses antigenically related to human rotavirus serotypes 1 and 2

Fecal samples from rotavirus-infected piglets were characterized by a serotyping enzyme-linked immunosorbent assay (ELISA) by using monoclonal antibodies (MAbs) specific to human serotypes 1, 2, 3, and 4 (D. O. Matson, M. K. Estes, J. W. Burns, H. B. Greenberg, K. Taniguchi, and S. Urasawa, submitted for publication). Rotavirus in 19 of 25 specimens tested from two herds of pigs from Buenos Aires province, Argentina, were classified antigenically as follows: one serotype 1, four serotype 2, two serotype 3, and no serotype 4. Six specimens reacted with both serotype 1 and 2 MAbs, and viruses in six specimens probably belonged to other serotypes because they reacted only with a VP7 common epitope MAb. Two porcine rotavirus fecal samples found to contain both serotype 1 and 2 viruses by the MAb-based test and one found to contain a serotype 2 virus were grown in tissue culture. When plaque-purified preparations of these tissue culture-adapted viruses were analyzed in the serotyping ELISA, the C60 and C86 preparations reacted only as serotype 1 viruses, indicating that the original fecal samples, which showed multiple VP7 reactivities, were heterogeneous and apparently contained two types of viruses. Testing of plaque-purified C134 virus confirmed its serotype 2 reactivity. The MAb-based serotype designations of these viruses also were confirmed by using a neutralization immunoperoxidase focus reduction assay. This is the first report of the occurrence of serotype 1 and 2 rotaviruses in animals. The MAbs originally developed to serotype human rotaviruses can be utilized to type animal rotaviruses.     

Contamination of some avian species with viruses of the leukosis-sarcoma complex.

Contamination of 8 avian species with leukosis viruses was studied. An insignificant portion of sera from 4 and 24 months old chickens contained neutralizing antibody to 3 sero-types (A, B and D) of Rous sarcoma virus (RSV). Sera from guinea-fowl reacted positively in the neutralization test only with the RSV-RAV-1 strain. None of the Peking duck and semi-domestic Maran fowl sera was found to contain antibody to RSV strains. Twenty-five and 6.6% of embryos derived from 7-8 and 24 to 30 months old chickens respectively, 30% of liver specimens from 3-4 months old chickens 3.3% of liver specimens from adult guinea-fowl and 72% of liver specimens from Maran fowl contained the group-specific leukosis complement-fixing antigen (gs-antigen). Duck embryos and livers from 24-30 months old chickens, crows, sparrows, rooks or jackdaws contained no gs-antigen. Duck embryos did not react and ducklings reacted poorly to inoculation with RSV whereas guinea-fowl embryos and chickens proved to be highly susceptible to RSV.     

An ELISA for the detection of antibodies to avian nephritis virus and related entero-like viruses

An ELISA for the detection of antibodies against avian nephritis virus (ANV) and related entero-like viruses was developed. Different antigenic preparations made from chicken kidney cells infected with the G-4260 strain of ANV were compared. Crude antigen obtained by fluorocarbon treatment of infected cells was found to be appropriate and to give reproducible results with antisera directed against ANV and three entero-like particles (ELPs): the Belgian entero PV2 and entero 3, and the Irish ELP-1. A cut-off value was determined using 30 specific pathogen-free (SPF) sera and the absence of antigenic relationships with nine different reference sera directed against avian viruses was demonstrated. The cross antigenic relationships between avian encephalomyelitis virus (AEV), ANV and the three ELPs was investigated by ELISA and confirms the classification of those fowl enteroviruses into two serotypes: the first including ANV and ELPs and the second including AEV. A strong correlation (94%) was demonstrated between ELISA and a seroneutralisation test. Using ELISA, antibodies against ANV and related ELPs were demonstrated in 13/14 breeding and 8/10 broiler flocks from Belgium.     

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes

An antigen-capture enzyme-linked immunosorbent assay (C-ELISA) was developed for detection and identification of infectious bronchitis virus (IBV) serotypes Arkansas, Connecticut, and Massachusetts using monoclonal antibodies (MAbs) specific to the S1 glycoprotein of the respective serotype. The assay (designed as a double-antibody sandwich assay) gave the best results when the S1-specific MAb, antigen, and chicken serum were of the same serotype. However, when a group-specific (M glycoprotein-specific) MAb was used for antigen capture, a distinctive pattern of cross-reactivity was observed between the antigens and heterologous chicken sera, suggesting a complex distribution of epitopes on the IBV M glycoproteins. Treatment of antigen with NP40 enhanced the ELISA signal only when the M glycoprotein-specific MAb was used for antigen capture. Although C-ELISA was inconsistent in detecting IBV in chicken tissue homogenates, it was highly effective in detecting the virus in allantoic fluid after the homogenates were given one chicken embryo passage.     

Evaluation of a poliovirus-binding inhibition assay as an alternative to the virus neutralization test.

An enzyme-linked immunosorbent assay (ELISA)-based poliovirus-binding inhibition (PoBI) test to detect and quantify antibodies to polioviruses was optimized and evaluated for use in population studies as an alternative to the virus neutralization test (NT) in tissue culture. The sensitivities of the inhibition ELISA compared with the NT in an inactivated poliovirus vaccine (IPV)-vaccinated population were 98.6, 97.4, and 92.1% for serotypes 1, 2, and 3, respectively. The specificities of the PoBI test, as determined with sera from nonvaccinated persons, were also high for all three serotypes (99.0, 95.8, and 100%, respectively). Antibodies to other enteroviruses did not cross-react in the serotype 1 and 3 PoBI, and only levels of cross-reactivity were found for serotype 2. We found high correlations between the PoBI and NT titers for serotypes 1 and 2 in IPV-vaccinated blood donors (0.97 and 0.95), in oral poliovirus vaccine (OPV)-vaccinated blood donors (0.91 and 0.95), and in naturally immune persons (0.90 and 0.87). The correlation coefficient for serotype 3, however, was significantly lower in OPV-vaccinated blood donors (0.73) and in naturally immune persons (0.76) than in IPV-vaccinated persons (0.94; P < 0.01). These results indicate that the antibody response to serotype 3 poliovirus in IPV recipients is different from that in OPV recipients and naturally infected persons. We conclude that the PoBI test is a suitable alternative to the NT for estimating the seroprevalence of neutralizing antibodies to poliovirus, especially in large-scale population studies.     

Direct testing of blood culture for detection of the serotype 5 and 8 capsular polysaccharides of Staphylococcus aureus.

Monoclonal antibodies (MAbs) reactive with serotype 5 and 8 capsular polysaccharides of Staphylococcus aureus have been used to test, by enzyme-linked immunosorbent assay (ELISA), blood culture fluids for the presence of S. aureus. A total of 748 blood cultures from 665 patients yielding 706 bacterial isolates belonging to more than 26 bacterial species were studied. All blood cultures containing bacterial strains belonging to species other than S. aureus were negative in ELISA. All 23 blood cultures containing serotype 5 S. aureus were positive in ELISA with the corresponding MAb. Out of 20 blood cultures containing serotype 8 S. aureus, 19 were positive with the corresponding MAb. All 5 blood cultures containing nontypeable S. aureus were negative in ELISA with both MAbs. This method provides reliable identification of serotype 5 or serotype 8 S. aureus by direct testing of blood culture fluids with ELISA.     

O1群小川型霍乱弧菌单克隆抗体的制备与鉴定

目的制备抗O1群小川型霍乱弧菌(VibriocholeraeO1serotypeOgawa)特异性单克隆抗体(McAb),为霍乱的早期快速诊断提供有力的抗体工具。方法以灭活的O1群小川型霍乱弧菌免疫Balbc小鼠,通过杂交瘤技术制备针对O1群小川型霍乱弧菌的McAb,以间接ELISA法对所需的杂交瘤细胞株进行筛选,分析其亚类,检测其效价及相对亲和力,以间接ELISA和Westernblot鉴定McAb特异性,并进行McAb结合表位分析。结果融合了602株能分泌抗O1群小川型霍乱弧菌McAb的杂交瘤细胞株,最后得到5株能稳定分泌特异性的针对该McAb的细胞株,其抗体亚类分别为3株IgG1,1株IgG2b,1株IgG3;腹水效价均达1×10-6;亲和常数在1×108～1×109之间。间接ELISA法及Westernblot证实所获的McAb可与O1群小川型霍乱弧菌发生特异性反应。ELISA相加实验结果显示除有2株McAb识别相同的抗原表位外,其余均识别不同的抗原表位。结论获得霍乱弧菌O1群小川型特异性McAb,为O1群小川型霍乱早期快速诊断和发病机理的研究提供基础。     

Quantitation of serotype-specific and cross-reacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating Actinobacillus (Haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.