Serological differentiation of five bluetongue virus serotypes in indirect ELISA.

The serological reactivity in indirect ELISA of five different bluetongue virus (BTV) serotypes (4, 10, 15, 16 & 20) was compared using polyclonal antisera raised against virus particles and an outer structural protein, VP2. Rabbit and sheep antisera against BTV-10 produced higher ELISA values with their homologous antigens than with heterologous serotypes. A hyperimmune rabbit serum specific for virus particles was able to distinguish heterologous serotypes from each other, but a sheep serum from an infected animal was not. An antiserum directed against VP2, the protein responsible for serotype specificity in neutralization tests, was not serotype-specific in ELISA and cross-reacted with other serotypes. The discriminatory ability of a BTV-4 antiserum was improved by cross-absorption with heterologous antigens. This greatly reduced the ELISA signals with heterologous serotypes and produced an antiserum that was effectively serotype-specific.     

Serotype specificity of immunological assays for the capsular polymer of Actinobacillus pleuropneumoniae serotypes 1 and 9.

The cross-reactivity of the purified polysaccharides of Actinobacillus pleuropneumoniae serotypes 1 and 9 were examined using a variety of highly sensitive assays, such as radioimmunoassay, latex agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. In addition, conventional immunodiffusion was included for comparison. Latex agglutination, utilizing affinity-purified IgG to capsule, was also used to serotype whole cells. Agglutination or precipitation tests (radioimmunoassay, latex agglutination, and immunodiffusion) indicated no cross-reactivity between the capsules of serotypes 1 and 9, and no cross-reactivity between whole cells by latex agglutination. Assays that required binding of the capsule to a solid support (ELISA and immunoblotting) did demonstrate cross-reactions between serotypes 1 and 9 capsules, although reactions with the heterologous serotype were weaker than with the homologous serotype. The cross-reactivity could not be attributed solely to nonspecific factors because similar cross-reactivity did not occur with serotype 5 or 7 capsules by any assay. Reactivity of antisera with homologous or heterologous capsule was reduced, but not completely eliminated, by adsorption with washed, live bacteria of the heterologous serotype. Thus, the assay, as well as the antigen or specificity of the antibody reagent used, may influence the results of A. pleuropneumoniae serotyping or serological tests.     

Cross-protection factor(s) of Pasteurella multocida: passive immunization of turkeys against fowl cholera caused by different serotypes

An antiserum cross-protective against different serotypes of Pasteurella multocida was made in turkeys by inoculating them with killed serotype 3 organisms grown in vivo and then exposing them to live serotype 3 organisms. In passive-immunization studies, the antiserum protected young turkeys against the homologous and heterologous serotypes 1, 4, 5, 9, and 12. In addition, the antiserum protected against P. multocida of a heterologous capsule serogroup, serogroup F. A globulin and two IgG fractions purified from the antiserum protected against heterologous challenge with serotype 1. Turkey-grown P. multocida were chemically lysed and separated into soluble and insoluble components to make immunoadsorbents. Antibodies from the cross-protective antiserum isolated by the immunoadsorbents passively protected young turkeys against heterologous serotype I challenge.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection, typing, and subtyping of vesicular stomatitis virus.

An indirect sandwich enzyme-linked immunosorbent assay (ELISA) has been used for vesicular stomatitis virus (VSV) typing using sets of monovalent and polyvalent rabbit/guinea pig antisera for identification of VSV types New Jersey (VNJ) and Indiana (VIND). The VIND polyvalent antiserum (VIND-P) detects any strain of the 3 subtypes of the VIND type (VIND-1, VIND-2, and VIND-3) with the same strong reactivity. It is also possible to subtype the VIND strains using VIND-P rabbit antiserum as capture antibody and monovalent VIND-1, VIND-2, or VIND-3 guinea pig antisera as detector. The ELISA proposed has about 10 times more sensitivity and provides 10% more positive results than does the complement fixation 50% (CF50) test when epithelial samples are tested.     

Investigations on different Ornithobacterium rhinotracheale "ORT" isolates

The aim of the present investigation was to determine the antigenic relationship between different Ornithobacterium rhinotracheale (ORT) isolates and to serotype field isolates obtained from turkey and chickens. Different antigen extractions (heat-stable, proteinase K-stable [lipopolysaccharide], and sodium dodecyl sulfate [SDS] extractions) were prepared from each serotype (A, B, C, D, E, and G) as well as from 21 ORT field isolates and examined in agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) tests. The field isolates were cultured from turkey (16 isolates) and chicken (5 isolates) flocks showing respiratory manifestations. Monospecific reactions were obtained with heat-stable as well as proteinase K-stable antigens prepared from serotypes A, C, D, E, and G in AGP tests. On the other hand, with the same antigen preparations from a strain of serotype B in AGP tests, cross-reactions with antisera prepared against serotypes A and E could be detected. The cross-reactions were observed mostly between 48 and 72 hr. In applications of SDS-antigen preparations in AGP tests, cross-reactions between all serotypes except serotype C were detected between 24 and 72 hr. Testing all antigen preparation in ELISA, different cross-reactions were observed and the evaluation of the results is very difficult. Serotyping of the field isolates in AGP tests by using heat-extracted antigens showed after 24 hr that 10 out of 16 isolates from turkey belonged to serotype B, five to serotype A, and one to serotype E. Results obtained after 48-72 hr revealed cross-reactions between serotype B and E in 11 cases and between A and B in two cases. All five isolates obtained from chicken reacted after 24 hr only with serum against serotype A. After 48-72 hr, two isolates showed cross-reaction with antiserum against serotype B. Similar results were obtained with proteinase K-stable antigen.     

Comparison of various antigens and their ability to detect protective antibodies against Pasteurella multocida using enzyme-linked immunosorbent assay

Various antigenic extracts of the CU strain of Pasteurella multocida were prepared to determine their suitability as plate antigens for use in the enzyme-linked immunosorbent assay (ELISA) for the detection of fowl cholera antibodies. Antisera from two separate broiler breeder flocks with known fowl-cholera-vaccination histories were collected just before the birds were challenged with virulent strain X-73 P. multocida. A potassium thiocyanate (KSCN)-extracted antigen, a capsular (CAP) antigen, a lipopolysaccharide-protein antigen, and heat-stable, salt-soluble antigen were all suitable as ELISA plate-coating antigens. Filtered and unfiltered sonicates of the CU strain of P. multocida were also suitable ELISA plate antigens. The results suggested that different plate antigens were detecting different populations of antibodies formed in response to fowl cholera vaccinations. When antibody titers were correlated with survival after challenge, the KSCN and the CAP plate antigens placed more nonsurvivors into low-antibody-titer ranges and more survivors (protected birds) into the high-antibody-titer ranges than the other plate antigens.     

Co-migrating and shared antigens of selectedPasteurella haemolytica untypable strains

Bacterial cell envelope preparations from eight untypable strains ofPasteurella haemolytica were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against the eight untypable strains (one untypable strain per rabbit) and with cattle antisera prepared againstP. haemolytica serotypes 1, 2, 5, 6, 9 and against one heterologous untypable strain. Numerous comigrating and shared antigens were recognized by the eight rabbit antisera and theP. haemolytica serotype cattle antisera. Comigrating antigens at 43 and 30 kilodaltons (kDa) were recognized by all eight rabbit antisera. Shared antigens, detected by all eight rabbit antisera when reacting againstP. haemolytica serotype 1, were recognized at 43, 32, 30, 20 and 15 kDa.     

Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates and their antigenic relationships with other A pleuropneumoniae serotypes

Antigenic relationship of Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates with other serotypes was studied, using tube agglutination, with and without 2-mercaptoethanol, indirect hemagglutination with and without 2-mercaptoethanol, ring precipitation, coagglutination, and immunodiffusion tests. Serotype-8 isolates possessed serotype-specific, group-specific common antigens cross-reactive with serotypes 3 and 6 and species-specific common antigens cross-reactive with other serotypes. Absorption studies were done to study the antigenic relationship of serotype 8 with serotypes 3 and 6. Rabbit antisera against whole-cell (WC) suspensions of reference strains of serotypes 3, 6, and 8 were used for absorption studies with WC and boiled WC suspensions of homologous and heterologous serotypes. Unabsorbed and absorbed sera were tested for antibodies against WC and boiled WC antigen preparations of serotype 8, using various serotests. Absorption studies revealed that serotype-8 strains possessed 2 main types of epitopes, one of which was serotype-specific and did not have cross-reactivity with other serotypes. The second type of epitopes was group specific and was cross-reactive with serotypes 3 and 6.     

Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida: effects of dilution, iron chelation, and heterologous challenge.

Two experiments were done to further define cell-free culture filtrate (CCF) from Pasteurella multocida and its endotoxin content in protecting turkeys against challenge. In the first experiment, the greater-than-30,000-molecular-weight fraction of P. multocida strain R44/6 (serotype 3/4/9/12) CCF was used in 10-fold dilutions given by air-sac inoculation or aerosol to vaccinate turkeys, which were subsequently challenged with either homologous (P-1059, serotype 3) or heterologous (X-73, serotype 1) strains. Endotoxin content of the CCF fraction was high. Compared with positive controls given either live Clemson University vaccine or a commercial bacterin, homologous protection was provided by undiluted CCF and 1:10 dilutions of CCF, but there was no heterologous protection. In the second experiment, CCF of strain R44/6 in regular and iron-limiting media and CCF of strain FC127B (serotype 1/4) were used alone or in combination to vaccinate turkeys, which were challenged as in the first experiment. Homologous but not heterologous protection occurred, even though growth of strain R44/6 in iron-limiting media reduced endotoxin content of CCF by approximately 93%. These results indicate that endotoxin levels of less than 10% but greater than 1% of those in CCF from regular media are sufficient to induce protection in turkeys against homologous challenge but that CCF from either regular or iron-limiting medium does not provide protection against heterologous challenge.     

Production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes.

Three panels of monoclonal antibodies (MAbs) were prepared against the spike (S) proteins of infectious bronchitis virus (IBV) strains Arkansas 99, Connecticut 46, and Massachusetts 41. Based on enzyme-linked immunosorbent assay (ELISA), the MAbs were grouped into three categories: 1) group-specific, which reacted with a broad spectrum of homologous and heterologous IBV serotypes; 2) serotype-specific, which reacted only with strains of the homologous serotype; and 3) strain-specific, which reacted "selectively" with only certain strains of homologous and heterologous serotypes. MAbs that displayed serotype specificity were all specific to S1 fractions of the homologous serotype, confirming that epitopes that determine virus serotype are associated with the S1 protein. An excellent correlation was found when the results of IBV serotyping by MAb-based indirect ELISA were compared with those from the conventional virus-neutralization test. This confirms that the MAbs described here will serve as valuable tools in epizootiological studies and serotype-specific diagnosis of IBV infection.     

Immunodiffusion reactions among porcine enteroviruses and other picornaviruses

Antisera raised in rabbits against the porcine enterovirus strains V13 and T80 produced two precipitin lines in immunodiffusion tests with the homologous crude antigens, and a single precipitin line with each of ten heterologous porcine enteroviruses which were tested, and with poliovirus type 1, coxsackievirus B4 and equine rhinovirus type 1, but not with a bovine rhinovirus. C and D antigens prepared from V13 virus by density gradient centrifugation produced single precipitin lines with V13 antiserum. A single precipitin line was also formed when the heated crude antigens of V13 and T80 viruses were reacted with the homologous antisera, indicating destruction of the D antigen, and these lines fused with those produced by the heterologous viruses. It was concluded that porcine enteroviruses contain C and D antigens, and that the C antigen is responsible for the serological cross-reactivity demonstrated among porcine enteroviruses and other picornaviruses by immunodiffusion.     

Foot-and-mouth disease virus typing by complement fixation and enzyme-linked immunosorbent assay using monovalent and polyvalent antisera.

An indirect "sandwich" enzyme-linked immunosorbent assay (ELISA) using polyvalent and monovalent antisera was compared with the 50% complement fixation (CF50) test for the detection of foot-and-mouth disease (FMD) O, A, and C virus types. ELISA was more sensitive than CF50 tests when polyvalent antisera were used for detecting the 3 types of virus in epithelial samples, whereas ELISA using monovalent antisera was the least sensitive technique. The ELISA performed with polyvalent antisera was 9 times more sensitive for detecting FMD virus than that with monovalent antisera. However, viral isolation in cell culture was the most sensitive detection system. The combined use of ELISA with polyvalent antisera and cell culture inoculations was the most effective procedure for identifying FMD virus in epithelial samples from the field.     

Serological cross-reactivity of avian adenovirus serotypes in an enzyme-linked immunosorbent assay

Fowl adenoviruses free of avian adenovirus-associated virus, representing 10 serotypes, were tested for cross-reactivity in an enzyme-linked immunosorbent assay (ELISA). All antigens and antisera prepared in chickens, along with uninfected control antigen and normal chicken serum, were reacted in a checkerboard pattern with ELISA. There was considerable cross-reactivity among all serotypes tested. Homologous reactions were generally, but not always, stronger than heterologous reactions. ELISA was about as sensitive as virus neutralization in detecting antibodies. The high sensitivity plus broad-spectrum reactivity should make ELISA a preferred test for the detection of adenovirus antibodies in poultry flocks.     

Comparison of enzyme-linked immunosorbent assay with dot immunobinding assay for detection of antibodies against Pasteurella multocida in turkeys

A dot immunobinding assay (DIA) was developed to detect antibodies against Pasteurella multocida in turkey serum. Five coating antigens, namely, whole-cell (WC) antigen, sonicated cell lysate (SCL), crude capsular extract (CE), formalin extract (FE), and heat-stable antigen (HSA), were compared by enzyme-linked immunosorbent assay (ELISA) and DIA using reference antisera against P. multocida organisms. WC and SCL antigens showed higher sensitivity, whereas FE and HSA antigens were more specific coating antigens in both assays. The specificity of DIA was greater than ELISA by comparing the P/N ratios of HSA against serum prepared from heterologous serotype of P. multocida. The DIA had also several distinct advantages over the ELISA, which included reduction of the manipulation time and more uniform binding of coating antigens onto the nitrocellulose membranes compared with binding of coating antigens to microtiter plates for ELISA.     

Fowl cholera immunity induced by various vaccines in broiler minibreeder chickens determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were vaccinated for protection against fowl cholera at 12 and 21 weeks of age using several vaccination schemes, which included a live Pasteurella multocida (CU strain) vaccine, two commercial polyvalent fowl cholera oil-based bacterins, and two experimentally prepared polyvalent oil-based bacterins. Some treatment groups received only live or killed vaccines, whereas others received a live vaccine at 12 weeks followed by a killed product at 21 weeks. At 42 weeks of age, all birds that received the live CU vaccine twice or once followed by a bacterin survived challenge. Birds that received killed vaccines only were significantly less protected but still showed a respectable survival rate of 86%. All unvaccinated controls died within 72 hr after challenge. At 72 weeks of age, overall protection was lower than that at 42 weeks, regardless of vaccination treatment. Antibody titers were usually higher in birds that received bacterins than in those receiving live vaccines, yet overall protection was still greater in those birds that received the live cholera vaccine twice.     

Expression and antigenic characterization of the major core protein VP7 of Chuzan virus, a member of the Palyam serogroup orbiviruses

The Palyam serogroup-specific antigen, VP7, of Chuzan virus strain K-47 was expressed in insect cells by a recombinant baculovirus. The expressed protein appeared as a single band of 38kDa corresponding to the predicted molecular mass of Chuzan virus VP7 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In immunoprecipitation analysis, the recombinant VP7 was not only recognized by all polyclonal antibodies against the Palyam serogroup viruses (PALV) tested in this study, but also by antisera to bluetongue virus (BTV) serotype 1, epizootic haemorrhagic disease virus (EHDV) serotypes 1 and 2. However, in Western immunoblot assay, no positive signals were observed between this protein and these antisera, even in the homologous reaction using antiserum to Chuzan virus. These findings demonstrate that the common antigenic determinants on the VP7 proteins of Chuzan virus and the other PALV serotypes are mainly conformational and that the proteins share some epitopes with those of BTV and EHDV beyond the serogroup. No cross-reactivities were detected between Chuzan virus VP7 and antisera to BTV and EHDV in agar gel immunodiffusion (AGID) and indirect ELISA tests, indicating that the recombinant VP7 is useful as a diagnostic reagent for serological tests of congenital abnormalities of cattle caused by PALV.     

Capsular polysaccharide antigens for detection of serotype-specific antibodies to Actinobacillus pleuropneumoniae.

Capsular polysaccharides (CPS) of serotypes 1, 2, 5 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae were obtained from 18 h culture supernatants by precipitation with hexadecyltrimethyl-ammonium bromide (Cetavlon) followed by extraction with sodium chloride and reprecipitation in ethanol. These crude extracts, and portions purified further by phenol extraction to remove contaminating proteins, were evaluated as antigens for the detection of serotype-specific antibodies to A. pleuropneumoniae in sera from immunized rabbits and swine by an enzyme-linked immunosorbent assay. The crude extracts reacted strongly with homologous antisera, but except for serotype 1 showed considerable cross-reactivity with antisera to other serotypes. Phenol extraction greatly improved the serospecificity of the antigens from serotypes 1, 7 and, to a lesser extent, 5. The serotype 2 CPS antigen showed poor reactivity following phenol extraction, and did not appear as useful for detection of serotype-specific antibodies.     

An enzyme-linked immunosorbent assay for detection of antibodies against Escherichia coli: association between indirect hemagglutination test and survival

An enzyme-linked immunosorbent assay (ELISA) was modified for detection of antibodies against the two main pathogenic serotypes of Escherichia coli: serotypes O78:K80 and O2:K1. The ELISA was a more sensitive and repeatable test than the indirect hemagglutination test (IHT), which is a common method for detecting antibodies against E. coli. Cross-reactivity between the two strains was measured by reacting antisera of each serotype against homologous and heterologous antigens. The results suggest that aside from similar determinants expressed by the two serotypes, serotype O2:K1 expresses more strain-specific determinants than does O78:K80. Comparison of mean antibody titers of immunized chicks by IHT and ELISA along the primary response revealed that during the first 15 days after immunization with inactivated E. coli, the titers in both tests were parallel. After 15 days post-immunization, antibody titers measured by IHT decreased rapidly, whereas titers measured by ELISA decreased only slightly. In addition, a higher correlation was found between titers detected by ELISA and survival through challenge with E. coli than between titers detected with IHT and survival through challenge. The results suggest that the ELISA is a better test for detection of antibody in flocks suspected of being infected with E. coli.     

Specificity of the enzyme-linked immunosorbent assay (ELISA) for antibodies in the sera of specific pathogen-free lambs vaccinated with Pasteurella haemolytica antigens

Pooled serum from specific pathogen-free (SPF) lambs vaccinated with sodium salicylate extracted (SSE) antigens of Pasteurella haemolytica serotype A1 was shown to contain antibody to other A serotype SSE antigens when tested by the enzyme-linked immunosorbent assay (ELISA). Specific antibody to serotype A1 SSE antigens was demonstrated by absorption of the serum pool with heterologous serotype SSE antigens.The type-specific antigens of serotypes A1 and A9 were prepared by phenol—water extraction (PWE) of their respective SSE antigens. The PWE antigens were examined in a sandwich ELISA where rabbit IgG anti-P. haemolytica A1 cells or A9 cells was used as a coating layer to bind PWE antigens. The specificity of these antigens was demonstrated by marked reduction of reactivity between serum from SPF lambs vaccinated with SSE of serotypes A1 or A9.     

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.