The specificity of antibody response in experimental and natural bovine paratuberculosis studied by crossed immunoelectrophoresis with intermediate gel

The sonicate antigen (MPS) of a local strain (IVRI) of Mycobacterium paratuberculosis and a commercial lysate of Strain 18 were analysed using hyperimmune rabbit and calf antisera to MPS in crossed immunoelectrophoresis with intermediate gel (CIE-ig) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The rabbit antiserum was more potent than the calf antiserum and it precipitated 35 and 15 antigens, respectively, among MPS and lysate antigens. SDS-PAGE resolved 50 and 32 peptides among these antigens respectively, of which, 35 and 15 were precipitated by rabbit antiserum. A CIE-ig reference system, with 30 MPS antigens, was standardized and used to analyse antibody specificities among sera derived from animals experimentally and naturally infected with bovine paratuberculosis. Fourteen antigens of MPS were found to be reactive with these sera and among these, Antigens 2 and 5 were found to be serodominant; sonicate antigens of M. bovis BCG and M. avium did not contain these antigens. Both were high molecular weight (greater than 60 kDa) antigens which may be of serodiagnostic value.     

Common antigens of Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis and their relevance to serology

The possession of common antigens by three trematode parasites which commonly occur together in ruminants in the tropics, Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis was studied in relation to the reliability of serodiagnosis of infection with these helminths. The crude antigenic extracts of the three trematodes were subjected to Sephacryl S-300 column chromatography and F. gigantica was fractionated into six peaks, S. bovis into nine peaks and D. hospes into seven peaks. Common antigens were found in these three trematodes in both the crude whole worm antigenic extracts and in the semi-purified fractions obtained by Sephacryl S-300 column chromatography. The implications of this finding and the limitation it imposes on the usefulness of serodiagnostic tests in routine use as regards their specificity are discussed in relation to previous studies.     

Purification of larval Taenia solium antigens by gel filtration.

Crude larval Taenia solium extracts were fractionated by Sephacryl S-200 gel filtration into four fractions (W1-W4). The sensitivities of the fractions to rabbit and pig antiserum against Taenia solium were tested by double immunodiffusion, immunoelectrophoresis, and ELISA. Fraction W2 which was highly sensitive to antisera was shown by immunoblotting to contain antigen B (95 and 105 kDa). The four fractions were shown to contain antigenic determinants common with pig serum proteins and crude extracts of other Platyhelminthes (especially Taenia hydatigena). Fraction W2 has the potential to be used as a serodiagnostic antigen.     

Antigenic analysis of four species of the genus Ehrlichia by use of protein immunoblot

The antigenic profile of Ehrlichia canis, E risticii, E sennetsu, and E equi was investigated by the use of protein (western) immunoblot technique. Results of analysis of serum from acutely and chronically infected animals indicated that the 4 Ehrlichia species share a unique 25-kD polypeptide in addition to other peptides. Immune sera from dogs inoculated with E canis recognized a wide range of E canis polypeptide antigens, as determined by western blot analysis. A larger number of E sennetsu polypeptides were detected when homologous antiserum and antiserum to E equi were used. The latter antiserum did not recognize antigens of E canis or E risticii. Antisera to E canis, E risticii, and E sennetsu detected E equi antigens. Data indicate that a 25-kD protein is a common antigen among the species of the genus Ehrlichia and that the ascending order of abundance of immunodominant determinants in the 4 species of Ehrlichia studied would be: E risticii----E equi----E sennetsu----E canis. Implications of these findings for diagnosis of ehrlichial infections and prophylaxis are evident.     

Antigen heterogeneity and epitope variable expression in Mycoplasma meleagridis isolates

Immunoblotting was used to check the antigenic profiling of 27 Mycoplasma meleagridis strains isolated in different countries. Hyperimmune polyclonal rabbit antiserum as well as monoclonal antibodies (MAbs) raised against M. meleagridis (MM) showed antigen heterogeneity among strains. Five anti-MM MAbs were selected for lack of reaction against heterologous avian mycoplasma. Three of these five Mabs did not cross-react with 63 mycoplasma strains from six species affecting turkeys other than M. meleagridis. The five Mabs used to analyse the epitopes of 30 M. meleagridis strains indicated that some epitopes were not expressed in all strains. Moreover, other epitopes were located on proteins which differed according to number or molecular mass from strain to strain. The five Mabs therefore, recognised variable surface proteins, among which two were amphiphilic membrane proteins.Three of the selected Mabs recognised 29 or 30 of the 30 tested strains. The in vitro expression of surface epitopes in M. meleagridis ATCC 25284 was investigated by colony immunobinding and allowed demonstration of a variable antigenic system.     

Identification of a potentially important antigen of pasteurella haemolytica

To identify antigens which may be important for stimulating immunity to pneumonic pasteurellosis, a bovine antiserum to whole P. haemolytica was used to screen a recombinant lambda gt11/P. haemolytica expression library. One of the recombinant bacteriophage clones identified with the bovine antiserum, SW20C, expressed a fusion protein which was also recognized by rabbit antiserum to partially purified P. haemolytica culture supernatant and was found to be immunogenic in guinea pigs. The guinea pig antibody recognized a 100 kDa protein in P. haemolytica cell lysates. Sequence analysis of the cloned DNA from SW20C identified a fragment of 1443 bp with a small open reading frame that was contiguous with the lacZ sequence. The 153 bp P. haemolytica-specific open reading frame encoded a polypeptide of approximately 6kDa. Homology searches of Genbank and the EMBL data bases revealed no homology of this open reading frame with any other bacterial sequences including P. haemolytica leukotoxin and Ssa1. Evaluation of sera from calves that were scored either susceptible or resistant to experimental pneumonic pasteurellosis demonstrated a significant (P < 0.001) correlation between the intensity of the antibody response to the SW20C antigen and resistance to disease.     

Editorial

Summary

The possession of common antigens by three trematode parasites which commonly occur together in ruminants in the tropics, Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis was studied in relation to the reliability of serodiagnosis of infection with these helminths. The crude antigenic extracts of the three trematodes were subjected to Sephacryl S‐300 column chromatography and F. gigantica was fractionated into six peaks, S. bovis into nine peaks and D. hospes into seven peaks. Common antigens were found in these three trematodes in both the crude whole worm antigenic extracts and in the semi‐purified fractions obtained by Sephacryl S‐300 column chromatography.

The implications of this finding and the limitation it imposes on the usefulness of serodiagnostic tests in routine use as regards their specificity are discussed in relation to previous studies.     

Identification and heterologous expression of a Sarcoptes scabiei cDNA encoding a structural antigen with immunodiagnostic potential

The mite Sarcoptes scabiei causes sarcoptic mange (or scabies), a disease of considerable human and veterinary significance. An S. scabiei cDNA clone of about 2 kb was isolated from a S. scabiei var. hominis expression library by immunological screening using blood serum from a naturally infected chamois (Rupicapra rupicapra). The nucleotide sequence of the identified cDNA contains an open reading frame of 1930 bp that encodes a 642 amino acid polypeptide. This polypeptide shows tandem repeats of a glycine-serine rich 20 residue sequence followed by a unique C-terminal glutamate rich 54 residue sequence. The cDNA or the deduced polypeptide did not show significant similarities to any of the sequences in the databases. A carboxyl-terminal fragment of this polypeptide (residues 380 to 642) was efficiently expressed in Escherichia coli as a fusion with Glutathione S-transferase and then was used to produce a specific antiserum. The antigen encoded by the cDNA was located at the integument of the mite's epidermis and the cavities surrounding its vital organs. Western blot analysis of mite extracts using the specific antiserum against the recombinant protein identified antigens larger that 60 kDa indicating that the isolated cDNA did not contain the full ORF. Moreover, we designed a diagnostic assay based on the carboxyl-terminal fragment of the antigen for the identification of infected animals.     

Selection of immunodominant mimics of IROMP-99 of rabbit Pasteurella multocida from a random 12-peptide library

A random 12-peptide library was used to screen immunodominant mimics of 99kDa iron-regulated outer membrane protein (IROMP-99) of rabbit Pasteurella multocida. In the present study, expression of IROMPs of rabbit P. multocida strain C51-12 were analyzed by SDS-PAGE, and Western blot to determine the specificity of rat antiserum antibodies against IROMP-99. Only IROMP-99 whose expression was induced under iron-restricted conditions was detected on nitrocellulose paper. The phage display library was screened with rat normal and IROMP-99-specific antiserum. The positive phage clones were identified using enzyme-linked immunoadsorbent assay (ELISA) and inhibition assays for their reactivity to the antiserum. Out of the 18 randomly selected positive clones that showed higher reactivity to rat antiserum, only ten clones efficaciously inhibited binding of rat antisera to IROMPs and their displayed peptides were determined. Alignment using DNAStar-MegAlign software, results showed that motif WHxTxP was highly conserved among nine clones, only clone A7 had no obvious linear homology with either. Our findings suggest that the motif WHxTxP could be an immunodominant mimic epitope of IROMP-99 of rabbit P. multocida strain C51-12.     

The prevalence of Sarcocytis spp in dogs, foxes and sheep and Toxoplasma gondii in sheep and the use of the indirect haemagglutination reaction in serodiagnosis.

Sporocysts of Sarcocystis spp were found in the faeces of 39.3 per cent and 25 per cent of farm dogs and foxes respectively. The indirect haemagglutination serodiagnostic test suggested that nearly all sheep in the area sampled became infected early in life and diagnosis of infection by this means correlates well with trichinoscopy findings. The sarcocystis antigens used in serodiagnostic tests did not cross react with sera positive for Toxoplasma gondii. Sarcocystis antigens derived from cattle cross reacted with ovine sera but comparative tests showed the titres to be less than where the homologous antigen was used.     

Antigenic relatedness of pigeon herpes encephalomyelitis virus to other avian herpesviruses

Pigeon herpes encephalomyelitis virus (PHEV) was compared with seven avian herpesviruses for antigenic relatedness using monospecific antisera and the indirect fluorescent-antibody (IFA), agar-gel-immunodiffusion, and serum-neutralization tests. No antigenic relationship was detected between PHEV and Marek's disease virus, turkey herpesvirus, infectious laryngotracheitis virus, and duck enteritis virus. A common precipitating antigen was detected between the PHEV and pigeon herpesvirus (PHV), owl herpesvirus (OHV), and falcon herpesvirus (FHV). These four viruses also cross-reacted in the IFA test. Weak neutralizing activity was detected only between PHV antiserum and PHEV. These results suggest that the PHEV should be classified as a herpesvirus related to, but distinct from, the PHV-OHV-FHV group of viruses with which it shares common antigens.     

Molecular characterization of Theileria orientalis piroplasm protein encoded by an open reading frame (To ORF2) in a genomic fragment

In the present study, a novel antigenic protein expressed in the piroplasm stage of Theileria orientalis was characterized. A 4,707 bp genomic fragment amplified by PCR contained two open reading frames (ORFs). The deduced amino acid sequence of the first ORF showed significantly high similarlity to the ubiquitin carboxy terminal hydrolases/proteases while the second ORF (To ORF2) showed homology to several surface antigens of plasmodia. To ORF2 was expressed to determine whether the protein product is expressed by the parasite. In western blot analysis, bovine antiserum from a T. orientalis-infected calf recognized the recombinant protein containing a C-terminal part of the ORF expressed by baculovirus system. Western blot analysis with the anti-To ORF2 mouse serum recognized a 48 kDa protein in T. orientalis piroplasm lysates. Indirect immunofluorescence antibody test by confocal scanning laser microscopic analysis showed that antisera against the recombinant protein recognized T. orientalis piroplasm in the infected erythrocyte. The results from this study indicate that To ORF2 protein is expressed at the piroplasm stage and is immunogenic. This novel antigenic To ORF2 protein could be exploited for vaccine development against bovine piroplasmosis.     

Identification of immunodominant antigens of Mycobacterium bovis by expression library immunization

This study combines two methodologies - vector expression of a genomic library and proteomics - to identify immunogenic proteins of Mycobacterium bovis. Immunization of BALB/c mice with a plasmid DNA pool from the library, containing approximately 8000 clones, induced a humoral response that facilitated the detection of 12 antigenic proteins by Western blotting. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry identified four proteins (Cpn60-1, HSP70, EF-Tu, and AdoHcyase). Such genomic immunization offers the possibility of in vivo screening of potential candidate M. bovis antigens.     

Unusual characteristics of a parvovirus isolated from a clinically ill steer

A parvovirus was isolated from the feces of an 8- and 9-month-old steer that died acutely with hemorrhagic diarrhea and microscopic evidence of a coccidial infection. The concurrent intestinal parasitism in this steer appeared to play a role in the development of clinical disease. The viral isolate was identified as a bovine parvovirus (BPV) on the basis of its size (22 nm) and icosahedral morphology, the neutralization of viral cytopathology by antiserum to BPV, a strong immunofluorescent reaction with fluorescein-labeled antiserum to BPV, and the inhibition of viral hemagglutination of guinea-pig erythrocytes by antiserum to BPV. Cell cultures infected with this isolate showed a slight nuclear fluorescent reaction with fluorescein-labeled antiserum to canine parvovirus, suggesting an antigenic relationship to canine parvovirus. Patterns of hemagglutination for this isolate with human erythrocytes from 20 donors of various blood types differed from those obtained with the reference Abinanti strain of BPV. These results indicate that blood from multiple donors of a species may be necessary to confirm the presence or absence of viral hemagglutinating activity with clinical isolates of BPV.     

Cloning and sequencing of badger (Meles meles) interferon gamma and its detection in badger lymphocytes

The European badger (Meles meles) has been identified as a reservoir for Mycobacterium bovis and is implicated in the maintenance and transmission of tuberculosis in cattle. There is a need for a sensitive test of M. bovis infection in badgers and the current serodiagnostic test used for this purpose has low sensitivity. As observed for other species, assay of interferon-gamma (IFNgamma) produced in response to M. bovis antigens is a more sensitive test of tuberculosis. With this objective in sight, we report the first step in the development of an ELISA for badger IFNgamma. The badger IFNgamma gene was cloned and sequenced and used to generate a specific polyclonal antibody to the cytokine. The gene sequence demonstrated regions that were conserved within the IFNgamma genes of other mammals. The badger sequence was most similar to the canine, showing similar structural organisation of the gene and 88% amino acid identity. Rabbits were immunised with DNA encoding badger IFNgamma and the resulting polyclonal antiserum demonstrated specificity for canine IFNgamma by immunoblot of a commercial recombinant canine IFNgamma. The antiserum was used to detect intracellular badger IFNgamma by flow cytometry analysis of badger lymphocytes stimulated with mitogen.     

Serological responses of dogs to cell wall and internal antigens of Brucella canis (B. canis)

The serological responses of dogs to cell wall and internal antigens of B. canis were studied in experimentally infected specific-pathogen-free (SPF) Beagles. Sera from infected and false positive field dogs also were examined. Cell wall antigens were extracted from B. canis by two procedures that employed either hot phosphate buffered saline (PBS) or sodium desoxycholate (SDC). Agar gel immunodiffusion (AGID) tests employing sera from experimentally infected SPF dogs were used to evaluate antigenic extracts. Extraction with PBS yielded two antigens; SDC extracted an antigen complex and sonication of PBS extracted cells liberated four internal antigens.Sera from field dogs that were negative for B. canis infection in repeated tests often had heterospecific antibodies. Such cross-reactive sere commonly gave “spur” (partial fusion) reactions with a positive reference serum when tested against the SDC cell wall antigen. In addition, false positive dogs did not have antibody to one of the cell wall antigens or to the internal antigens. In contrast, sera from infected field dogs commonly gave “identity” (fusion) reactions in the AGID test with two antigens in the SDC extract, and produced precipitin lines to one to four internal antigens.Examination of a library of sera obtained from experimentally infected SPF dogs over a period spanning 4$\text{1}\text{2}$ years revealed that none of the serodiagnostic tests employed (tube agglutination, slide agglutination, AGID) was accurate during the inital 12 weeks of infection; hemocultures were the most sensitive during this period. Tube and slide agglutination tests were initially sensitive, but they showed a lack of sensitivity and specificity after the bacteremic period ceased, as well as in their failure to exclude false positive reactions in field animals. Immunodiffusion tests that employed SDC or PBS extracts of B. canis cell walls were sensitive and accurate in identifying most infected dogs. After the bacteremia had ceased, however, AGID tests that employed cell wall antigens gave equivocal results. Immunodiffusion tests that employed sonicated (internal) antigens were sensitive shortly after the onset of bacteremia, and they had the advantage of detecting infected animals for at least 6 months following the cessation of bacteremia, a time when other serological tests gave equivocal results.     

Porcine SWC1 is CD52--final determination by the use of a retroviral cDNA expression library

During the last decades for several species--e.g. swine--many mAb to leukocyte-specific molecules have been developed and clusters of differentiation corresponding to human CD could be established. However, for a significant amount of the raised mAb the corresponding antigens were not characterized on the molecular level and therefore preliminary clusters--in swine so-called Swine workshop clusters (SWC)--were established. These clusters contain antigens with currently no obvious orthologs to human leukocyte differentiation antigens. In this study, we describe the generation of a eukaryotic cDNA expression library from in vitro activated porcine peripheral blood mononuclear cells. Screening of this library with an antibody recognizing SWC1 enabled isolation and sequencing of cDNAs coding for the porcine SWC1 molecule. A BLAST search of the obtained sequence revealed that SWC1 is the orthologous molecule of human CD52. Therefore, our study provides the basis for comparative studies on the role of CD52 in different mammalian species. In addition, the established cDNA library can be used for investigation of additional SWC-defined molecules.     

TSL-1 antigens of Trichinella: an overview of their potential role in parasite invasion, survival and serodiagnosis of trichinellosis

The majority of studies on the immunobiology of Trichinella species have centred on the larval muscular phase (L1) with a view to identifying immunodominant antigens located on the surface of the cuticle and in the larval secretions; the nucleus of the parasite-host interaction. These antigens have been classified as eight groups (TSL-1-TSL-8), of which those belonging to the group TSL-1 have been most intensely studied. The principal constituents are glycoproteins, glycan carriers that contain a unusual sugar, the tyvelose (3,6-dideoxy-d-arabinohexose). Studies aimed at improving serodiagnostic techniques to detect trichinellosis indicate that these antigens are ideal candidates. They are capable of inducing a strong humoral response involving the generation of specific antibodies against beta-tyvelose, a sugar that seems to be exclusive to the Trichuroidea. Furthermore, these glycoproteins appear to fulfil an important function in the development and maintenance of the parasite in the muscular niche, and they appear to be fundamental for the invasion of the intestinal epithelium. It has also been demonstrated that specific monoclonal antibodies against tyvelose can mediate a degree of immunoprotection in the rat through the phenomenon known as rapid expulsion.     

Evaluation of pseudorabies viral antigens in the agar gel immunodiffusion test.

Procedures designed to extract pseudorabies viral (PRV) antigens from PRV-infected tissue cultures were investigated to determine whether differences in extraction method had an effect upon the final concentrated antigenic product. All four of the preparations made from PRV-infected tissue culture cells (trypsin extract and disrupted cells) or entire PRV-infected cultures (polysorbate 80 extract and (NH4)2SO4 precipitate) contained relatively large amounts of the same antigen, whereas cell-free PRV-infected tissue culture fluids did not contain significant amounts of this antigen. Specific antibody directed against this antigen was present in all PRV antisera tested. Two other antigens were observed in some of the preparations, but PRV antisera varied in their ability to precipitate with these antigens. Therefore, the number of precipitation lines observed in agar gel immunodiffusion between PRV preparations and PRV-positive antisera depended both upon the extraction method used to obtain the antigen and upon the specificity of the selected antiserum.     

Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay

Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.