Serodiagnosis of infections of pigs with Stephanurus dentatus

A serological survey of 135 pigs over 7 months old, some of which had liver and renal lesions in the presence ofStrephanurus dentatus, was undertaken for the presence of antibodies to S. dentatus. Only one sample gave a positive gel immunodiffusion reaction with crude whole worm extracts and none with immunoelectrophoresis. Oral inoculation of pigs with 1200 or 2000 L3 resulted in a specific reaction of anodal lines on immunoelectrophoresis from Day 37, and with double diffusion up to 4 specific lines were produced beginning from Day 43–53 using adult and juvenile antigens. Juvenile antigens gave more intense lines. Serological responses remained positive until 76–80 days post-infection. At necropsy infected pigs had fibrotic lesions in livers and portal thrombi containing larval S. dentatus even after 270 days. It is concluded that immunodiffusion and immunoelectrophoresis have limited usefulness in serodiagnosis of naturally occurring stephanuriasis.     

A Comparative Investigation of Antibody to Swine Vesicular Disease Virus Using Counter Immunoelectrophoresis,Serum Neutralization and Double Immunodiffusion Tests

A counter immunoelectrophoresis test (GIET) for assay of antibody to swine vesicular disease virus (SVDV) is described and compared with the serum neutralization test (SNT) and the double immunodiffusion test (DIDT). Using a preparation of complete or empty virus particles as antigen the GIET was found to be more sensitive than the DIDT. The GIET is rapid and economic, and although less sensitive than the SNT it was suggested as an initial large scale screening test for SVD antibody.     

Antigenic analysis of Peptococcus indolicus by crossed immunoelectrophoresis

Quantitative immunoelectrophoretic methods have been used to study the antigenic mosaic of Peptococcus indolicus, an anaerobic coccus frequently isolated from udder secretions from heifers and dry cows with mastitis. Three antigenic components of liquid cultures of this bacterium were analyzed, compared and characterized, namely concentrated culture filtrate containing extracellular antigens, a cytoplasmic antigen fraction obtained by freeze-press disruption of bacterial cells and Triton X-100-soluble antigens from cell wall-membrane fractions. The extracellular antigens were further investigated because they proved to be particularly useful in preliminary studies on the antibody response of cows to P. indolicus. The possible cross-reactivity of peptococcal antigens with extracellular antigens from other bacteria causing, or associated with, mastitis was investigated. The contribution of medium components to the immunoprecipitate profile, the heat-stability of antigens and the relationship of serotypic antigens to those in the standard extracellular concentrate were established using co-immunoelectrophoresis, crossed-line immuno-electrophoresis and crossed immunoelectrophoresis with intermediate gel. Attempts to identify enzyme-active immunoprecipitates with histochemical enzyme staining methods revealed only glutamate dehydrogenase.     

Production and characterization of monoclonal antibodies to serotype specific antigens of Haemophilus pleuropneumoniae serotype 2

Mouse monoclonal antibodies were produced to Haemophilus pleuropneumoniae bacteria of the most common serotype 2. 11 hybridoma cultures were recovered that produced antibodies with moderate to strong reactivity to the antigen. 10 of these antibodies were specific to isolated capsular antigens from H. pleuropneumoniae of serotype 2, while one antibody reacted with capsular antigens from bacteria of all 8 serotypes. One hybridoma producing antibodies with a titre of 1:1000 was cloned and the antibody specificity studied further. The binding of this antibody (1F3) to whole bacteria, and capsular extracts isolated at different temperatures indicate that the antibody is specific for a thermostable polysaccharide antigen present in the cellular capsule of H. pleuropneumoniae of serotype 2.     

Characterization of salt-extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectricfocusing

Salt-extractable protein antigens (CSP) from Brucella abortus strains 19 and 2308 (vaccine and virulent strains, respectively) were analysed by crossed immunoelectrophoresis (CIE) using rabbit antisera to protein antigens and by isoelectricfocusing (IEF) in polyacrylamide gels. The reference immunoelectrophoretic profiles developed for proteins from strain 19 and 2308 of B. abortus contained 20 and 25 immunoprecipitates, respectively. Serum from cows experimentally infected or hyperimmunized with live organisms produced up to 5 immunoprecipitates in CIE with the protein antigens. Absorption of rabbit sera with homologous B. abortus cells reduced, but did not eliminate all of the immunoprecipitates from rabbit sera, suggesting that the majority, but not all of the protein components, are exposed on the surface of the cell. In contrast, antibody to protein antigens in agglutinin-free absorbed serum from infected cattle could still be demonstrated by CIE, even though CIE with protein extracts from whole cells radioiodinated with the cell surface labeling reagent, diazoiodusulfanilic acid, indicated that these antigens may be at or near the surface of the cell. From CIE in heterologous systems we concluded that all proteins present in strain 19 preparations were partially or completely identical to those in strain 2308. The IEF studies paralleled the CIE studies and revealed that the protein profile from strain 2308 was more complex than the profile from strain 19. Major differences between the 2 strains were found in the pH region from 3.9 to 5.0, where strain 2308 exhibited 4 additional protein bands.     

Crossed immunoelectrophoresis of fungal antigens in tissues as a means of diagnosing systemic aspergillosis and zygomycosis in cattle

A novel method for diagnosing bovine aspergillosis and zygomycosis is described. Rabbit hyperimmune antisera raised against somatic antigens ofAspergillus fumigatus andAbsidia corymbifera were used in crossed immunoelectrophoresis with supernatants from disintegrated tissues from acute necrohaemorrhagic mycotic lesions from cattle. The method specifically identified 4 of 5 lesions with aspergillosis and 2 of 5 lesions with zygomycosis. One lesion dually infected with aspergillosis and zygomycosis was negative. The method worked with unabsorbed sera, was specific, and required only standard electrophoretic equipment. It can therefore supplement chemical detection of fungi in tissues in the diagnosis of bovine aspergillosis and zygomycosis.Abbreviations APAAP alkaline phosphatase anti-alkaline phosphatase - XIE crossed immunoelectrophoresis     

对流免疫电泳检测犬细小病毒免疫复合物中抗体的研究

通过对流免疫电泳的条件优选,实现了解离后犬细小病毒复合物中抗原抗体的分,达到了检测免疫复合物中抗体的目的。在应用检测试验中,通过与酶档ＳＰＡ染色法、血凝抑制试验及常规对流免疫电泳法比较,证实了该种检测方法的可靠性、敏感性和实用价值。     

Pasteurella haemolytica: purification of saline-extractable proteins by isoelectrofocusing

A procedure was developed for separating antigens associated with a saline extract of Pasteurella haemolytica serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the carbohydrate antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of mice with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent P. haemolytica was seen in mice injected with antigen Groups 2 and 4. Six calves were immunized with saline extract. These calves had greater resistance to experimental pneumonic pasteurellosis than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated calves by an enzyme-linked immunosorbant assay.     

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.     

Crossed immunoelectrophoretic analysis of the antibody response in heifers and dry cows to Peptococcus indolicus in experimental and natural heifer mastitis and dry cow mastitis

Crossed immunoelectrophoresis with intermediate gel was used to investigate the antibody response of experimentally infected heifers and of naturally infected heifers and dry cows at grass with mastitis against soluble extracellular antigens of Peptococcus indolicus. Using both precipitin titres and precipitin scores (the sum of precipitin titres against individual antigens) rising titres of antibodies against antigend of P. indolicus were demonstrated in sera from experimentally infected pregnant and non-pregnant heifers. Statistically significant differences in precipitin scores against extracellular antigens were also shown between pregnant heifers with mastitis and healthy pregnant heifers and between dry cows with mastitis and health dry cows. The biochemical nature of the principal reactive antigens was not elucidated. These serological investigations lend strong support to the pathogenic role and significance of P. indolicus in mixed infections with Corynebacterium pyogenes in the aetiology of heifer and dry cow mastitis.     

Serodiagnosis of Aspergillus fumigatus antibody in migratory ducks

Migrating duck populations in Wisconsin were evaluated during a one-year period for exposure to Aspergillus fumigatis by two serodiagnostic methods: immunodiffusion and countercurrent immunoelectrophoresis. Peak antibody reactivity to sporulating culture extracts of the fungus was detected in serum samples collected in winter. Antibody levels did not differ between male and female or adult and juvenile populations.     

Canine C-reactive protein (CRP) does not share common antigenicity with human CRP

Differences in antigenicity between human and canine C-reactive proteins were investigated by Western blotting analysis. It was confirmed that several commercial anti-human CRP sera reacted with canine CRP. However, 34 anti-canine CRP sera prepared by immunization of rabbits and goats with canine CRP all reacted with canine CRP but not with human CRP in either immunoelectrophoresis or Western blotting.Immunization with human CRP produced a cross-reacting antibody that reacted with canine CRP. Conversely, immunization with canine CRP did not produce a cross-reacting antibody that reacted with human CRP. These findings may be interpreted as showing that, while canine and human CRPs do not share common antigenicity, they do contain structurally similar antigenic determinants.Abbreviations CRP C-reactive protein - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

The porcine humoral response to detergent extracted Aujeszky's disease (pseudorabies) virus antigens

Twenty Aujeszky's disease (AD) virus antigens were demonstrated by crossed immunoelectrophoresis in a Triton-X-100 detergent extract of virus-infected PK-1a cells. Eight of these antigens were shown to be glycosylated based on their ability to be specifically bound by the lectin Ricinus communis agglutinin II. Pigs nasally infected with AD virus showed a significant serum antibody titer to seven of the known glycosylated antigens and to four additional antigens. The antibody titer to these antigens persisted for at least 116 days. Pigs which were vaccinated parenterally with the whole detergent extract survived a nasal challenge of 10(8 . 5) PFU of virulent AD virus. The antibody response of these vaccinated pigs on the day of challenge was essentially identical to the recovery response previously observed in non-vaccinated nasally infected pigs. These results indicate that the optimum components of future AD virus subunit vaccines and their complementary diagnostic reagents should be selected from these 11 antigens.     

Serodiagnosis of Dermatophilus congolensis infection by counterimmunoelectrophoresis

Sixty-one sera from animals that had contact with Dermatophilus congolensis were examined by comparing three serological methods; counterimmunoelectrophoresis, passive haemagglutination, and agar gel diffusion, and by using four different antigenic extracts of D congolensis. The counterimmunoelectrophoresis was the most satisfactory of the methods having been found to be specific and sensitive, easy to perform and suitable for screening large numbers of samples. It was also found to have a higher antibody detection rate (82.2 per cent) than the other methods thus making it suitable for seroepidemiological surveys. It was found to be capable of detecting multiple antibodies and also revealed dissimilarities among the different antigenic extracts. The cellular antigens of D congolensis were found to detect antibody in more sera than the extracellular antigen; the cell wall extract proved to be the most satisfactory of all, detecting antibody from the largest number of sera compared to the other extracts in all the three serological tests.     

Nippostrongylus brasiliensis infection in the rat: 1. Production of bile lgA and serum lgG antibodies by adult rats

Bile lgA and serum lgG antibody responses were assayed by a micro ELISA technique. Antibodies of both classes were produced by 14 days after infection against whole worm antigens and worm metabolic product antigens, by adult rats infected once with $\text{Nippostrongylus brasiliensis}$. Rats were reinfected on day 28 and titres of both classes of antibody against both antigens were greater 7 and 14 days after reinfection than after the initial infection. Bile lgA antibodies were produced against phosphorylcholine after a single infection with the parasite but no increased response occurred after reinfection. The possible significance of lgA and lgG antibody responses in relation to immunity to intestinal nematode infections is discussed.     

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.     

Assay for Antibody in Pig Fetuses Infected with Porcine Parvovirus

Fetal fluids from field cases of fetal death were assayed for antibody to porcine parvovirus (PPV) using 3 different techniques. An indirect immunofluorescent antibody test, a counter immunoelectrophoresis test and a hemagglutination inhibition test were compared. The indirect immunofluorescent antibody test was found to be the most sensitive of the tests employed. The hemagglutination inhibition test apparently suffered from the occurrence of false positive results.     

Cross-reactions of bovine herpesvirus 1 antigens with those of other cattle herpesviruses

Bovine embryonic kidney cells were infected with bovine herpesviruses (BHV1, 2, or 3), suid herpesvirus 1 (SHV1), or were sham-inoculated. When cytopathic effect was apparent, the cells were solubilized using Triton X-100 detergent. Resulting antigen preparations were tested by 2-dimensional immunoelectrophoresis using bovine fetal serum and antisera directed against BHV1, BHV2, BHV3, SHV1 or a restricted spectrum of BHV1 antigens. Interaction of BHV1 antiserum with BHV1 antigen preparations resulted in 11 precipitation arcs. The same antiserum produced 3 arcs with BHV2, none with BHV3, and 5 with SHV1. The interaction of BHV1 antigen preparations with BHV2, BHV3, or SHV1 antisera failed to produce demonstrable arcs. However, when heterologous antigen or antibody preparations were added to BHV1 homologous 2-dimensional immunoelectrophoresis tests, all 11 BVH1 arcs were modified by BHV1, 2 by BHV2, 4 by BHV3 and 4 by SHV1 preparations. Two antigens were common to the 4 herpesviruses. Antigen preparations were tested for their ability to inhibit virus neutralization by BHV1 antiserum; only the BHV1 preparation was active. Sera were tested for BHV1 neutralizing activity; only BHV1 antiserum and a serum specific for a restricted spectrum of BHV1 antigens were active. A glycoprotein antigen associated with BHV1 neutralization was identified which may be important in the protection of animals against disease.     

The immune response of goats vaccinated with low and high doses of Brucella melitensis Rev 1

The serological response, lymphocyte reactivity, and dermal hypersensitivity reactions of goats vaccinated with high and low doses of $\text{Brucella melitensis}$ Rev 1 organisms to Brucella antigens were studied. Antibodies to soluble antigen A2 were detectable by immunoelectrophoresis (IE), appeared later than agglutinating antibodies but disappeared faster in most vaccinated animals. Anti-A2 antibodies appeared earlier in goats which received the high dose. Antibodies to polysaccharide B antigen were not detected. Lymphocytes reactive to Brucella antigens in lymphocyte transformation assays appeared at variable times after vaccination. In contrast to the humoral response to antigen A2, the appearance of circulating, reactive lymphocytes was not dependent on vaccine doses. All vaccinated animals demonstrated dermal hypersensitivity reactions to one of the antigenic extracts. Skin reactions peaked at 24 hrs post inoculation. The reactions were elicited when all but one goat had undetectable anti-A2 antibodies and no circulating, reactive lymphocytes as measured in whole blood lymphocyte transformation assay.     

Studies on strains of Pasteurella haemolytica not typable by the indirect haemagglutination test

Thirty strains of Pasteurella haemolytica which were untypable by the indirect haemagglutination (IHA) test were examined serologically by rapid plate agglutination (RPA), agar gel diffusion (AGD), crossed immunoelectrophoresis (CIE) and counter current immunoelectrophoresis (CCIE) tests. Nine serogroups were identified by CCIE. Serogroup specificity, dependent on two antigens, was present in heated saline extracts of cells. Single representative strains from two serogroups were not pathogenic for specific pathogen-free lambs.