Ability of antibodies to two new caliciviral vaccine strains to neutralise feline calicivirus isolates from the UK

This study examined a panel of 110 UK field isolates of feline calicivirus (FCV) for susceptibility to cross-neutralisation by a panel of eight antisera raised in cats infected with FCV strains F9, 255, FCVG1 and FCV431. The pairs of antisera raised against F9 or 255, neutralised 20 and 21 per cent or 37 and 56 per cent of field strains of virus respectively. In contrast, the pairs of antisera raised against the newer vaccine strains FCVG1 or FCV431 neutralised 29 and 70 per cent or 67 and 87 per cent of field strains respectively. Antisera raised against the two newer strains, namely FCVG1 and FCV431, neutralised a greater proportion of field strains of calicivirus than antisera raised against the older FCV vaccine strains F9 and 255.     

Detection of shared magnetic antigenic determinants on whole Moraxella bovis pili by use of antisera to cyanogen bromide-cleaved M. bovis pilus protein

OBJECTIVE: To determine the ability of antisera against cyanogen bromide-cleaved pili from 4 strains of Moraxella bovis to react with whole or nondenatured pili. SAMPLE POPULATION: Antisera to 4 strains of M. bovis produced by New Zealand White rabbits. PROCEDURE: Pili from 4 strains of M. bovis were collected and purified. Pilus proteins (pilin) were cleaved, using cyanogen bromide. Whole pilus and cyanogen bromide-cleaved pilin were injected into rabbits. Antisera were serially diluted, reacted with 4 strains of M. bovis, and examined by immunoelectron microscopy and indirect immunofluorescence. RESULTS: Antisera to whole pili aggregated and distorted pili from homologous strains, but pili from heterologous strains were unaffected. Antisera to cleaved pilin fragments resulted in partial aggregation and thickening of homologous and heterologous pili, suggestive of heterospecific antibodies. Attachment of antibodies to pili was detected by indirect immunofluorescence, indicating a strong reaction of antisera to whole pili with homologous pili. Weak cross-reactions were evident with certain heterologous strains. In contrast, antisera to cleaved pilin fragments reacted strongly with pili from homologous and heterologous strains. CONCLUSIONS AND CLINICAL RELEVANCE: We detected shared antigenic determinants on pili from various strains of M. bovis that were not immunogenic in intact pili. These sites were immunogenic after cleavage of pilus protein with cyanogen bromide, and antisera produced to protein fragments reacted with whole pili from heterologous strains of the organism. Vaccines produced from cyanogen bromide-treated pili may induce broader immunity against infectious bovine keratoconjuctivitis than that provided by currently available vaccines.     

Indirect immunofluorescence and immunodiffusion tests in the detection of antibodies to foot-and-mouth disease virus

The antibody response detected by indirect immunofluorescence (IIF) as well as that directed against 140 S and virus infection associated antigen (VIA), as detected by agar immunodiffusion, was studied in three mammal species susceptible to Foot and Mouth Disease Virus, after challenge with living virus, immunization and hyperimmunization with inactivated virus, and immunization followed by challenge. By spot indirect immunofluorescence, antibodies were detected only in animals undergoing an active infection, and were not detected in immunized or hyperimmunized animals. This behaviour was similar to that of the anti-VIA antibodies in the same groups of animals and differed from that of anti-140 S antibodies. It appeared that spot indirect immunofluorescence for the detection of VIA antigen is comparable to the immunodiffusion test, but the speed of IIF and the possibility of handling many samples make it more practical.     

Effect of administration of anti-bursal extract or perfusate serum on the antibody production in chickens

Antisera to bursal extracts or perfusates were prepared and the influence of such sera on antibody production in chickens was investigated by the injection of antisera during the embryonic stage. Antisera to cyclophosphamide treated bursal extracts or bursal perfusates were injected on the 15th day of embryogenesis. The level of antibodies produced by chickens treated by these antisera was equal to the controls but IgG antibodies were totally absent. These results suggested that the administration of these antisera inhibited the differentiation of IgM antibody producing cells.     

Detection of African swine fever virus antibodies by immunoblotting assay.

An immunoblotting assay has been adapted to detect antibodies against African swine fever virus. The electrophoretic transfer of proteins and the immunoreaction conditions were optimized, using 4 mA/cm2 of current intensity and 10 micrograms of soluble cytoplasmic antigen of infected cells per strip. Filters of polyvinylidene difluoride showed the highest capacity for protein absorption, but nitrocellulose filters showed lower backgrounds. The specificity and the pattern of the proteins induced by African swine fever virus that react with the antisera were determined in immunoblotting assay, IP30 being the most reactive protein.     

Antibody response in salmonids against the 70 kDa serine protease of Aeromonas salmonicida studied by a monoclonal antibody-based ELISA

An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida. This new assay permits a specific determination of anti-protease-antibodies, without antigen purification. Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies. Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations (r=0.753). Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease. A conjugate of inhibited protease and cationized bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species (P<0.05), whereas the unconjugated antigen and Apoject 1-Fural were proved to be ineffective. Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity. The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinize its protective significance.     

Phage displayed peptides and anti-idiotype antibodies recognised by a monoclonal antibody directed against a diagnostic antigen of Mycoplasma capricolum subsp. capripneumoniae

A monoclonal antibody (Mab 4.52) raised against Mycoplasma capricolum subsp. capripneumoniae (Mccp) cell lysate was used as a template to obtain substitute antigens recognised by its paratope. Two approaches were investigated: a 17-mer random peptide library displayed on the surface of a filamentous phage was screened by panning on the immobilised Mab 4.52 and anti-idiotype antibodies were generated by immunising a chicken with the F(ab')(2) fragments of the antibody. Analysis of the peptide sequences displayed by the isolated phages identified two peptides. Both contained two cysteine residues and had identical or similar amino acids in positions 5 (P), 8 (I/L) and 13 (L). The fusion phages were also recognised by Mab 4.52 in enzyme-linked immunosorbent assay (ELISA) and binding was shown by surface plasmon resonance. One of the peptides was a markedly better inhibitor (67%) of the binding of Mab 4.52 to its original antigen than the other (20%) at 1mg/ml. After absorption, to remove isotypic and allotypic reactivities, the anti-idiotype IgY was specifically recognised by Mab 4.52 in ELISA and was able to inhibit its binding to the original antigen, whereas anti-idiotype antibodies raised against a bluetongue virus-specific antibody had no effect. In spite of unequivocal binding of the anti-idiotype antibodies and the fusion phages to the paratope of Mab 4.52, goat antisera appeared not to react with either of the surrogate antigens. In contrast, the test sera bound to the original antigen suggesting that Mab 4.52 does not recognise exactly the same antigenic site as antibodies in the goat antisera.     

ELISA to determine anti-K99 pilus antibody in the sera of normal and diarrhoeic calves

An ELISA to detect circulating antibodies against K99 pili, a major attachment factor to intestinal epithelial cells of Escherichia coli in calves, was performed. Two methods of K99 pili purification were attempted. Best results in terms of purity of the K99 antigen were achieved following the method described by Karkhanis and Bhogal (1986). This procedure included a heat shock at 65°C during 25 min to release the pili and ultracentrifugation steps to purify the antigen. SDS-PAGE showed an 18 KDa major band, identified as the K99 pilus antigen after immunoblotting against reference antisera. The purified K99 antigen was then adsorbed to the ELISA microplates. High optical density was obtained in the ELISA using a pool of sera from immunized cows. No differences in antibody levels (P ≥ 0.05) could be detected between clinically healthy calves and those showing diarrhoea.     

Antigenicity of a chemically induced neurogenic rat sarcoma (author's transl)

A neurogenic sarcoma was induced in the plexus brachialis of a male Long-Evans rat by administration of N-methyl-N-nitrosourea in the drinking water. The tumor was established in vitro and designated 76LE-NS-369. Cells from tissue culture grew as tumors when isografted in young rats. 76LE-NS-369 cells did not react with antiserum directed against gliaspecific S-100 protein. We used the cultured cells as target cells, and found specific antibodies in the sera of tumor-bearing and immunized rats by indirect fluorescent antibody stain and a complement-dependent antibody-mediated microcytotoxicity assay. In immunization experiments, incubation of tumor cells with Vibrio cholerae neuraminidase yielded higher antibody titers than an antigen preparation with untreated cells. Incubation with neuraminidase enhanced the sensitivity of tumor cells to antibody and complement in vitro, whereas trypsinized cells showed complete loss of reactivity with autologous antisera. The specificity of antisera was tested by absorption with tumor, lyophilized rat whole body and rat nerve tissues.     

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix

Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P<0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P<0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.     

Antibody reactivity to Cryptosporidium parvum in saliva of calves after experimental infection

Antibodies against Cryptosporidium parvum in the saliva and sera of three calves experimentally infected with this parasite were examined by an indirect immunofluorescence antibody test and immunoblotting. Salivary anti-C. parvum IgA antibody appeared on day 12 post-challenge and had a tendency to increase transiently between days 15 and 30 post-challenge. Salivary anti-C. parvum IgG antibody levels showed a gradual increase along with the change in IgA antibody levels during the infection. In contrast, serum anti-C. parvum IgA antibody levels showed only a slight increase between days 15 and 30 post-challenge. Serum anti-C. parvum IgG antibody levels rose on day 12 post-challenge and one calf maintained relatively high level up to the end of the experiment. In immunoblotting, an antigen with a molecular mass of 15 kDa was found to react strongly to salivary IgA antibody and a 27 kDa antigen to react to serum IgG antibody.     

Expression of metabolism-inhibition antibodies against Mycoplasma arthritidis in rats

Shared antigens between Mycoplasma arthritidis and rat tissues may be responsible for the lack of metabolism-inhibition (MI) and other neutralizing antibodies in rats with M arthritidis-induced arthritis. We were not able to confirm such antigens or to detect cross-reacting antigens between M arthritidis and rat lymphocytes, thymocytes, and muscle tissue. Antisera of rabbit origin to rat lymphocytes, thymocytes, and skeletal muscle reacted by ELISA with M arthritidis only when the mycoplasmal antigens were prepared from organisms grown in medium containing horse serum. Such activity could be completely absorbed by horse serum. These antisera to rat tissues also failed to react by radioimmunoprecipitation with M arthritidis surface antigens. In addition, antibody activity against homologous antigens could not be absorbed by M arthritidis. Similarly, antisera of rabbit origin against M arthritidis failed to react by ELISA specifically with rat lymphocytes, thymocytes, and skeletal muscle or to react by radioimmunoprecipitation with 125I-labeled rat lymphocyte antigens. These rat tissues could not specifically absorb antibodies against M arthritidis from antisera of rabbit origin. These findings suggest that the lack of MI antibodies in rats probably can not be explained by rat tissue antigens that cross-react with M arthritidis MI antigens. Finally, antisera of rat origin against M arthritidis and other rat tissue components failed to block rabbit MI activity against M arthritidis, thus arguing against steric hindrance as a means of preventing recognition of MI antigens.     

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.     

Co-administration of IL-2 enhances antigen-specific immune responses following vaccination with DNA encoding the glycoprotein E2 of bovine viral diarrhoea virus

Induction of effective immunity requires the delivery of a protective antigen with appropriate co-stimulatory signals. For bovine viral diarrhoea virus (BVDV) this antigen is the major viral glycoprotein E2. Neutralising antibodies are directed towards the E2 protein and passive transfer of antibodies in serum or colostrum can completely protect against viral infection. DNA vaccination of mice with a construct encoding the E2 glycoprotein induced neutralising antibody levels that were potentially sufficient to prevent virus replication in a challenge system. The co-delivery of interleukin-2 (IL-2) further enhanced the levels of antibody raised. The strong IgG2a component of the antigen-specific antibody suggests a Th1 bias to the immune response induced following vaccination.     

Evaluation of the specificity of Pasteurella multocida somatic antigen-typing antisera prepared in chickens, using ribosome-lipopolysaccharide complexes as inocula

Purified lipopolysaccharides (LPS) from 16 serotypes of Pasteurella multocida were complexed with Aspergillus fumigatus ribosomes. The complexes were used as inocula to prepare antisera, in chickens, for somatic antigen typing by the gel diffusion precipitin test (GDPT). Antisera made against 15 of 16 LPS reacted with their respective specific heat-stable antigens in the GDPT and homologous LPS in the passive hemagglutination test. Antisera could not be made against serotype 15 LPS. Correlation was not observed between intensity of the precipitin reaction in the GDPT and titer to homologous LPS in the passive hemagglutination test. Most antisera cross-reacted with other heat-stable antigens of other serotypes in the GDPT. Many of these cross-reactions were eliminated by dilution. Cross-reactions that occurred in the GDPT with antisera made against LPS of serotypes 2, 5, 7, and 8 could not be eliminated by dilution.     

Recognition of Mycoplasma arthritidis membrane antigens by rats and rabbits: comparison by immunoblotting and radioimmunoprecipitation

Sera from rats convalescing from infection with Mycoplasma arthritidis were tested for their ability to react with M. arthritidis membrane antigens by immunoblotting and radioimmunoprecipitation. The absence of metabolism-inhibition (MI) antibody activity in these sera suggested that rats might fail to recognize those membrane antigens involved in eliciting MI antibodies therefore rabbit antisera, which are strongly MI positive for M. arthritidis, were used for comparison. Antigenic recognition patterns of M. arthritidis surface and membrane antigens were not identical for rats and rabbits. The most striking and reproducible difference was the failure of rats to produce IgG antibodies against a surface antigen migrating in the 47,000-50,000 molecular weight range on SDS-polyacrylamide gels. However, rats recognized at least 2 antigens which we had previously shown to be "MI antigens", therefore the inability to express MI antibodies probably cannot be explained by their inability to recognize M. arthritidis "MI antigens".     

Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (elisa) was standardised and applied for the detection of anti-platelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, I per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 μl test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophil antigens could be stored at 4° and −70°C for four to five weeks without significant loss of antigenicity. The ELISA was very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for neutrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplatelet serum. Standardised elisa detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologous equine blood typing sera.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Monoclonal and polyclonal antibodies for immunohistochemical detection of bovine parainfluenza type 3 virus in frozen and formalin-fixed paraffin-embedded tissues.

Accurate identification of bovine parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.     

Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: establishment of an efficient ELISA for antigen detection in feces

Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40,000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.