Development of indirect ELISAs for differential serodiagnosis of classical and highly pathogenic porcine reproductive and respiratory syndrome virus.

The objective of this study was to develop two indirect enzyme-linked immunosorbent assays (iELISAs) for detection of serum antibodies against classical vaccine strain of porcine reproductive and respiratory syndrome virus (PRRSV) and highly pathogenic PRRSV (HP-PRRSV). To detect the common antibodies against classical and HP-PRRSV, the coating antigen used in the iELISA (designated iELISA-180) was the antigen of Nsp2-180, the 180aa at amino terminal of Nsp2. To detect the different antibodies against classical and HP-PRRSV, the coating antigen in the second iELISA (designated iELISA-D29) was Nsp2-D29, the deleted 29aa in Nsp2 of HP-PRRSV. The antigen concentration and serum dilutions were optimized using a draughtboard titration. The cut-off values of 0.361 at OD(450nm) for the iELISA-180 and 0.27 at OD(450nm) for the iELISA-D29 were determined by testing a panel of 120 classical PRRSV positive and 198 PRRSV negative pig serum samples, which generated the specificity of 97.1% and 96.7%, the sensitivity of 96.9% and 96.3% for iELISA-180 and iELISA-D29, respectively. The agreements between the Western blot and iELISA-180 and iELISA-D29 were 98%, 96.7%, respectively. The developed iELISAs can be used to differentiate serologically HP-PRRSV from the vaccinated or classical PRRSV in clinical serum samples.

Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are non-protective.

Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.

Identification of two neutralization epitopes on the capsid protein of avian hepatitis E virus.

Avian hepatitis E virus (avian HEV) is genetically and antigenically related to human HEV, the causative agent of hepatitis E. To identify the neutralizing epitopes on the capsid (ORF2) protein of avian HEV, four mAbs (7B2, 1E11, 10A2 and 5G10) against recombinant avian HEV ORF2 protein were generated. mAbs 7B2, 1E11 and 10A2 blocked each other for binding to avian HEV ORF2 protein in a competitive ELISA, whereas 5G10 did not block the other mAbs, suggesting that 7B2, 1E11 and 10A2 recognize the same or overlapping epitopes and 5G10 recognizes a different one. The epitopes recognized by 7B2, 1E11 and 10A2, and by 5G10 were mapped by Western blotting between aa 513 and 570, and between aa 476 and 513, respectively. mAbs 1E11, 10A2 and 5G10 were shown to bind to avian HEV particles in vitro, although only 5G10 reacted to viral antigens in transfected LMH cells. To assess the neutralizing activities of the mAbs, avian HEV was incubated in vitro with each mAb before inoculation into specific-pathogen-free chickens. Both viraemia and faecal virus shedding were delayed in chickens inoculated with the mixtures of avian HEV and 1E11, 10A2 or 5G10, suggesting that these three mAbs partially neutralize avian HEV.

Egg whites from eggs of chickens infected experimentally with avian hepatitis E virus contain infectious virus, but evidence of complete vertical transmission is lacking.

Avian hepatitis E virus (HEV) is genetically and antigenically related to human HEV. Vertical transmission of HEV has been reported in humans, but not in other animals. In this study, we showed that avian HEV could be detected in chicken egg-white samples. Subsequently, avian HEV in egg white was found to be infectious, as evidenced by the appearance of viraemia, faecal virus shedding and seroconversion in chickens inoculated with avian HEV-positive egg white, but not in chickens inoculated with HEV-negative egg white. To further assess the possibility of vertical transmission of avian HEV, batches of embryonated eggs from infected hens were hatched, and hatched chicks were monitored for evidence of avian HEV infection. However, no virus was detected in samples collected from the hatched chicks throughout this study, suggesting that avian HEV could not complete the vertical transmission cycle. The possible implications of our findings are also discussed.

Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein.

Avian hepatitis E virus (avian HEV) is an emerging virus associated with hepatitis-splenomegaly syndrome in chickens in North America. Avian HEV is genetically and antigenically related to human HEV, the causative agent of hepatitis E in humans. In the lack of a practical animal model, avian HEV infection in chickens has been used as a model to study human HEV replication and pathogenesis. A 32 kDa recombinant ORF2 capsid protein of avian HEV expressed in Escherichia coli was found having similar antigenic structure as that of human HEV containing major neutralizing epitopes. To determine if the capsid protein of avian HEV can be used as a vaccine, 20 chickens were immunized with purified avian HEV recombinant protein with aluminum as adjuvant and another 20 chickens were mock immunized with KLH precipitated in aluminum as controls. Both groups of chickens were subsequently challenged with avian HEV. All the tested mock-immunized control chickens developed typical avian HEV infection characterized by viremia, fecal virus shedding and seroconversion to avian HEV antibodies. Gross hepatic lesions were also found in portion of these chickens. In contrast, none of the tested chickens immunized with avian HEV capsid protein had detectable viremia, fecal virus shedding or observable gross hepatitis lesions. The results from this study suggested that immunization of chickens with avian HEV recombinant ORF2 capsid protein with aluminum as adjuvant can induce protective immunity against avian HEV infection. Chickens are a useful small animal model to study anti-HEV immunity and pathogenesis.

Genetic and experimental comparison of porcine circovirus type 2 (PCV2) isolates from cases with and without PCV2-associated lesions provides evidence for differences in virulence.

There are marked differences in the clinical expression of diseases associated with porcine circovirus type 2 (PCV2) in the field. The objective of this study was to compare the sequences and pathogenicity of PCV2 isolates from field cases with and without PCV2-associated lesions. Forty-two specific-pathogen-free (SPF) pigs were assigned randomly to three groups of 14 pigs each. At 7 weeks of age, group 1 pigs were mock-inoculated with saline, group 2 pigs were inoculated with PCV2-4838 (isolated from a pig with no evidence of PCV2-associated lymphoid lesions) and group 3 pigs were inoculated with PCV2-40895 (isolated from a pig with PCV2-associated lymphoid lesions and disease). The PCV2-4838 and PCV2-40895 isolates shared approximately 98.9 % nucleotide sequence identity across the entire genome. A total of nine amino acid changes in ORF2 and two amino acid changes in ORF1 were identified between the two isolates. PCV2-4838-inoculated pigs had significantly more genomic copy numbers of PCV2 in their sera at 7 days post-inoculation (p.i.) (P<0.0001) and significantly fewer genomic copy numbers at 14, 21 and 28 days p.i. (P<0.05) compared with pigs inoculated with the PCV2-40895 isolate. Microscopic lesions in lymphoid tissues were significantly less severe (P<0.05) and the amount of PCV2 antigen associated with these lesions was significantly lower (P<0.05) in pigs inoculated with PCV2-4838. The results of this study suggest that PCV2 isolates from the USA differ in virulence in an SPF pig model.

Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV.

Avian hepatitis E virus (avian HEV) was recently discovered in chickens from the USA that had hepatitis-splenomegaly (HS) syndrome. The complete genomic sequence of avian HEV shares about 50 % nucleotide sequence identity with those of human and swine HEVs. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, but the B-cell epitopes in the avian HEV ORF2 protein have not been identified. Nine synthetic peptides from the predicted four antigenic domains of the avian HEV ORF2 protein were synthesized and corresponding rabbit anti-peptide antisera were generated. Using recombinant ORF2 proteins, convalescent pig and chicken antisera, peptides and anti-peptide rabbit sera, at least one epitope at the C terminus of domain II (possibly between aa 477-492) that is unique to avian HEV, one epitope in domain I (aa 389-410) that is common to avian, human and swine HEVs, and one or more epitopes in domain IV (aa 583-600) that are shared between avian and human HEVs were identified. Despite the sequence difference in ORF2 proteins between avian and mammalian HEVs and similar ORF2 sequence between human and swine HEV ORF2 proteins, rabbit antiserum against peptide 6 (aa 389-399) recognized only human HEV ORF2 protein, suggesting complexity of the ORF2 antigenicity. The identification of these B-cell epitopes in avian HEV ORF2 protein may be useful for vaccine design and may lead to future development of immunoassays for differential diagnosis of avian, swine and human HEV infections.

Application of competitive enzyme-linked immunosorbent assay for the serologic diagnosis of classical swine fever virus infection.

A competitive enzyme-linked immunosorbent assay (C-ELISA), based on a truncated E2 recombinant protein of the Alfort/187 strain of classical swine fever virus (CSFV) and a specific monoclonal antibody M1669, was evaluated using 2,000 sera from clinically healthy pigs in Canada (a CSFV-free country) and sera from experimentally infected pigs. The relative specificity and sensitivity of the C-ELISA were 100% and 86%, respectively, at a cutoff of 25% inhibition using negative and positive pig sera, as defined by the neutralizing peroxidase-linked assay (NPLA). A kappa value of 0.91 was obtained, indicating an excellent level of agreement between the NPLA and the C-ELISA. When sera from 120 infected pigs were used in the test at > or = 21 days postinfection, the sensitivity of the C-ELISA and the kappa value increased to 97% and 0.98, respectively. This C-ELISA will be useful when a large number of samples must be tested, as could occur during a disease outbreak or for surveillance or prevalence studies.

Development of an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay for detection of equine and swine IgM antibodies to vesicular stomatitis virus.

An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competitive ELISA (C-ELISA) and the standard microtiter serum neutralization (MTSN) assay by testing serum samples from horses and pigs experimentally infected with VSV-NJ or VSV-IN. The MC-ELISA detected specific homologous IgM antibodies from equine and swine sera as early as 5 and 4 days postinfection (DPI), respectively, and as late as 35 DPI. The MTSN test also detected antibodies as early as 5 DPI and as late as 160 DPI. In a similar fashion, the C-ELISA detected antibodies from 6 to 7 DPI and as late as 160 DPI. These results demonstrated that the MC-ELISA is a useful test for serodiagnosis of primary VSV infection in horses and pigs.

Incursion of epizootic hemorrhagic disease into the Okanagan Valley, British Columbia in 1999.

In September 1999, unusually high mortality rates in white-tailed deer and California bighorn sheep occurred in the southern Okanagan Valley. Necropsy and histopathologic findings were compatible with epizootic hemorrhagic disease (EHD); the presence of virus was not demonstrated. Subsequent serologic and polymerase chain reaction assays on sentinel cattle suggested an EHD virus incursion.

Development of a competitive ELISA using a truncated E2 recombinant protein as antigen for detection of antibodies to classical swine fever virus.

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak.

Development and evaluation of an IgM-capture ELISA for detection of recent infection with bluetongue viruses in cattle.

An IgM-capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of recent infection of bluetongue virus (BTV) in cattle. The test is based on the use of biotinylated capture anti-bovine IgM antibodies bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of BTV VP7 antigen and a VP7 antigen-specific monoclonal antibody. The IgM-capture ELISA was compared with the competitive ELISA by testing serum samples from groups of calves infected experimentally with five USA and 19 South Africa serotypes of BTV. The IgM-capture ELISA was able to detect bovine anti-VP7 antibodies from all animals infected with the 24 BTV serotypes at 10 days post-infection, whereas the competitive ELISA was not. When the detectable IgM diminished after 40 days post-infection by the IgM-capture ELISA, the IgG anti-VP7 antibodies remained high. The IgM-capture ELISA is sensitive and can be applied for the detection of recent infection of BTV in cattle.

Assessment of the pathogenicity of an emu-origin influenza A H5 virus in ostriches (Struthio camelus).

Ostriches were inoculated with a laboratory-derived highly pathogenic avian influenza (HPAI) virus of emu origin, A/emu/TX/39924/93 (H5N2) clone c1B. The aim of this study was to evaluate the pathogenicity of this isolate for ostriches and assess the ability of routine virological and serological tests to detect infection. Avian influenza virus (AIV) was isolated from cloacal and tracheal swabs from 2 to 12 days post-infection. AIV was also isolated from brain, thymus, eyelid, spleen, ovary/testis, liver, air sac, proventriculum, duodenum, caecal tonsil, heart, pancreas, kidney, nasal gland and lung. Virus isolation was also possible from swabs of the luminal surfaces of the cloaca, jejunum, lower ileum, bursa of Fabricius, trachea and bone marrow. Birds seroconverted as early as 7 days post-infection. This study suggests that HPAI virus of emu origin replicates extensively in infected ostriches without causing significant clinical disease or mortality.

Incursion of bluetongue virus into the Okanagan Valley, British Columbia.

Bluetongue virus was isolated from a sentinel herd in British Columbia. Virus isolation was by intravenous inoculation of embryonated chicken eggs and subculture in BHK-21 cells. The cytopathic agent was identified as bluetongue virus by electron microscopy and the immunoperoxidase test. The serotype was identified as serotype 11 by virus neutralization.

Anti-idiotype technique: an alternative approach for immunodiagnosis of bluetongue.

In development of a bluetongue alternative immunodiagnostic rest, the polyclonal anti-idiotypic antibodies were generated by the sequential immunization of rabbits with three monoclonal antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies recognize the idiotypes that are located within or near the antigen-combining sites and are associated with both heavy and light chains of the antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies mimic the VP7 antigen by recognizing the anti-VP7 antibodies from cattle and sheep that were infected with various serotypes of bluetongue viruses. The results indicate that the rabbit anti-idiotypic antibodies may be used as surrogate antigen in serological assays to detect the antibodies from different species of animals infected with various serotypes of bluetongue viruses.

Developments in international standardization.

Trade in animals and animal products has reached global proportions and so too has the threat of infectious diseases of veterinary importance. The Manual of Standards for Diagnostic Tests and Vaccines, published by the Office International des Epizooties (OIE), contains chapters on infectious diseases that may cause various degrees of socio-economic, public health, and/or zoo-sanitary consequence. These chapters cover the major diseases of cattle, sheep, goats, horses, pigs, poultry, lagomorphs and bees. A number of factors are considered when qualifying animals and animal products for international trade including epidemiological, clinical and testing parameters. Of particular note and relevance is a strong international movement to standardize the test methods and reference reagents in order to promote harmonization of testing and facilitation of trade. There is message here that is directed to those of us involved in the development and application of test methods for infectious disease diagnosis. Serological test methods have been and still remain the mainstay of diagnostic methods prescribed for trade. More than ever, there is a need to observe and apply international guidelines for the development and validation of serological test methods. There is also a need to develop international standard reagents for use in the calibration of test methods and the production of national and working standards. In the future, veterinary diagnostic testing laboratories involved in trade may also require a form of international accreditation unique to their specialty. This presentation describes the current developments in international standardization of test methods and reference reagents.

Experimental infection of emus (Dromaiius novaehollandiae) with avian influenza viruses of varying virulence: clinical signs, virus shedding and serology.

Two groups of emus were experimentally inoculated with a low and high pathogenic strain of avian influenza virus (AIV), type A to determine the virus susceptibility, pathogenicity, shedding and seroconversion. Emus were found susceptible to infection with AIV, with virus shedding detectable in tracheal and cloacal swabs between 3 and 10 days post-infection. Only the birds infected with the highly pathogenic viral isolate showed a brief period of mild clinical signs associated with infection. Virus recovered from the infected emus was found to be of similar pathogenicity to that of the virus inoculum. All the birds seroconverted by 10 days post-infection, as determined by haemagglutination inhibition, agar gel immunodiffusion and competitive ELISA assays. This study suggests that emus are similar to wild waterfowl in their response to AIV infection, in that they are susceptible and will replicate and shed the virus, but do not show any marked clinical signs of infection.

Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein.

A competitive enzyme-linked immunosorbent assay (C-ELISA) employing a baculovirus-expressed recombinant nucleoprotein and a monoclonal antibody was developed for the detection of antibodies to type A influenza virus nucleoprotein. The performance of the C-ELISA was evaluated by testing 756 chickens, 1123 turkeys, 707 emus, and 1261 ostriches, for a total of 3847 serum samples. Relative to the agar gel immunodiffusion (AGID) test, the C-ELISA had a sensitivity of 100% for all four species. The C-ELISA's sensitivity relative to the hemagglutination-inhibition (HI) test results was 100% for chicken, turkey, and emu and 96.2% for the ostrich serum samples. More than 90% of the AGID-negative/C-ELISA-positive serum samples were found positive by HI for at least one influenza serotype. The specificity of C-ELISA relative to AGID ranged from 85.5% to 99.8% for sera collected from these species. These results indicated that the C-ELISA was more sensitive and more specific than the AGID test and as sensitive and as specific as the HI test. The C-ELISA has the potential to replace the AGID test for screening sera from avian species, including ratites, for detection of antibodies to type A influenza virus.

Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens.

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n = 47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n = 96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.

Evaluation of antibody responses of emus (Dromaius novaehollandiae) to avian influenza virus infection.

Emu antibody responses to avian influenza virus (AIV) infection were evaluated by the competitive enzyme-linked immunosorbent assay (C-ELISA), agar gel immunodiffusion (AGID) and hemagglutination inhibition (HI) tests. All birds infected with AIV H5N1, H5N3, or H7N7 developed antinucleoprotein (NP) antibodies as early as 7 days postinfection as detected by the C-ELISA. The responses lasted 49 days for the emus receiving H5N3 and at least 56 days for emus receiving the other two viruses. By evaluating 50 emu field serum samples, the C-ELISA was found more sensitive than the AGID test for the detection of anti-NP antibodies. This study indicates that emus experimentally infected with AIV developed antibody responses that can be detected by C-ELISA, AGID, and HI tests. The results from this and our previous studies demonstrate the use of the C-ELISA as a substitute for the AGID test in a routine serodiagnostic screening for detection of antibodies to AIV infection in multiple avian species.