Development of a nucleoprotein-based enzyme-linked immunosorbent assay using a synthetic peptide antigen for detection of avian metapneumovirus antibodies in Turkey sera

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.     

Development of a Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay Using a Synthetic Peptide Antigen for Detection of Avian Metapneumovirus Antibodies in Turkey Sera

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.     

Subtype B avian metapneumovirus resembles subtype A more closely than subtype C or human metapneumovirus with respect to the phosphoprotein,and second matrix and small hydrophobic proteins

Avian metapneumovirus (aMPV) subtype B (aMPV/B) nucleotide sequences were obtained for the phosphoprotein (P), second matrix protein (M2), and small hydrophobic protein (SH) genes. By comparison with sequences from other metapneumoviruses, aMPV/B was most similar to subtype A aMPV (aMPV/A) relative to the US subtype C isolates (aMPV/C) and human metapneumovirus (hMPV). Strictly conserved residues common to all members of the Pneumovirinae were identified in the predicted amino acid sequences of the P and M2 protein-predicted amino acid sequences. The Cys(3)-His(1) motif, thought to be important for binding zinc, was also present in the aMPV M2 predicted protein sequences. For both the P and M2-1 protein-predicted amino acid sequences, aMPV/B was most similar to aMPV/A (72 and 89% identity, respectively), having only approximately 52 and 70% identity, respectively, relative to aMPV/C and hMPV. Differences were more marked in the M2-2 proteins, subtype B having 64% identity with subtype A but < or = 25% identity with subtype C and hMPV. The A and B subtypes of aMPV had predicted amino acid sequence identities for the SH protein of 47%, and less than 20% with that of hMPV. An SH gene was not detected in the aMPV/C. Phylogenetically, aMPV/B clustered with aMPV/A, while aMPV/C grouped with hMPV.     

Compromised T-cell immunity in turkeys may lead to an unpredictable avian metapneumovirus vaccine response and variable protection against challenge

Avian metapneumovirus (aMPV) is an important respiratory pathogen of turkeys with considerable economic impact on poultry production. Although vaccination is widely used for the control of the disease, questions regarding vaccine safety and efficacy remain to be elucidated. This report describes the problems associated with reproducibility of the aMPV-vaccine response, comparing T-lymphocyte-compromised and T-cell-intact turkeys. In three consecutive experiments, turkeys partially depleted of T-lymphocytes by treatment with cyclosporin A as well as untreated turkeys were vaccinated with a commercial live aMPV subtype A (aMPV-A) vaccine at 2 weeks of age. Two weeks later they were challenged with a virulent aMPV-A strain. Despite similar genetic background of the turkeys, comparable housing conditions under isolation and the application of the same aMPV-A vaccine, considerable variation was observed among the experiments regarding replication of the vaccine virus, vaccine-induced clinical signs and protection against challenge infection. The results indicate that differences in the outcome of aMPV-A vaccination may be associated with T-lymphocyte suppression and additionally with an interfering aMPV-B vaccine exposure at the hatchery in two of the experiments. Our study provides possible explanations for the variable protection provided by aMPV vaccines under field conditions.     

Development and evaluation of a blocking enzyme-linked immunosorbent assay for detection of avian metapneumovirus type C-specific antibodies in multiple domestic avian species

The first cases of infection caused by avian metapneumoviruses (aMPVs) were described in turkeys with respiratory disease in South Africa during 1978. The causative agent was isolated and identified as a pneumovirus in 1986. aMPVs have been detected in domestic nonpoultry species in Europe, but tests for the detection of these viruses are not available in the United States. To begin to understand the potential role of domestic ducks and geese and wild waterfowl in the epidemiology of aMPV, we have developed and evaluated a blocking enzyme-linked immunosorbent assay (bELISA) for the detection of aMPV type C (aMPV-C)-specific antibodies. This assay method overcomes the species-specific platform of indirect ELISAs to allow detection of aMPV-C-specific antibodies from potentially any avian species. The bELISA was initially tested with experimental turkey serum samples, and the results were found to correlate with those of virus neutralization assays and indirect enzyme-linked immunosorbent assay (iELISA). One thousand serum samples from turkey flocks in Minnesota were evaluated by our bELISA, and the level of agreement of the results of the bELISA and those of the iELISA was 94.9%. In addition, we were able to show that the bELISA could detect aMPV-C-specific antibodies from experimentally infected ducks, indicating its usefulness for the screening of serum samples from multiple avian species. This is the first diagnostic assay for the detection of aMPV-C-specific antibodies from multiple avian species in the United States.     

A double antibody sandwich ELISA for rapid diagnosis of virus infection and to measure the humoral response against infectious bursal disease on clinical material.

A double antibody sandwich ELISA (DAS-ELISA) was developed and employed for simultaneous direct detection of infectious bursal disease virus (IBDV) from bursal samples and to measure the humoral response, using the same basic immunoreagents. The purified and non-purified antigen, capture antibody and chicken hyperimmune sera were prepared, and standardized for this purpose. The DAS-ELISA was applied to both 80 bursal suspensions and 224 corresponding serum samples from vaccinated and non-vaccinated commercial flocks. Bursae samples were collected at 2 weeks of age, and submitted to histological examination, virus isolation in specific pathogen-free chickens embryos, and the DAS-ELISA technique. Serum titres obtained in indirect ELISA and serum neutralization test were compared with those in DAS-ELISA. The agreement was 80% between DAS-ELISA, and the conventional techniques, with high sensitivity (87%) and specificity (90%).     

Evidence of avian metapneumovirus subtype C infection of wild birds in Georgia,South Carolina,Arkansas and Ohio,USA

Metapneumoviruses (MPVs) were first reported in avian species (aMPVs) in the late 1970s and in humans in 2001. Although aMPVs have been reported in Europe and Asia for over 20 years, the virus first appeared in the United States in 1996, leaving many to question the origin of the virus and why it proved to be a different subtype from those found elsewhere. To examine the potential role of migratory waterfowl and other wild birds in aMPV spread, our study focused on determining whether populations of wild birds have evidence of aMPV infection. Serum samples from multiple species were initially screened using a blocking enzyme-linked immunosorbent assay. Antibodies to aMPVs were identified in five of the 15 species tested: American coots, American crows, Canada geese, cattle egrets, and rock pigeons. The presence of aMPV-specific antibodies was confirmed with virus neutralization and western blot assays. Oral swabs were collected from wild bird species with the highest percentage of aMPV-seropositive serum samples: the American coots and Canada geese. From these swabs, 17 aMPV-positive samples were identified, 11 from coots and six from geese. Sequence analysis of the matrix, attachment gene and short hydrophobic genes revealed that these viruses belong to subtype C aMPV. The detection of aMPV antibodies and the presence of virus in wild birds in Georgia, South Carolina, Arkansas and Ohio demonstrates that wild birds can serve as a reservoir of subtype C aMPV, and may provide a potential mechanism to spread aMPVs to poultry in other regions of the United States and possibly to other countries in Central and South America.     

Reproducibility of swollen sinuses in broilers by experimental infection with avian metapneumovirus subtypes A and B of turkey origin and their comparative pathogenesis.

Swollen head syndrome (SHS) associated with avian metapneumovirus (aMPV) subtype A or subtype B in broilers and broiler breeders has been reported worldwide. Data about pathogenesis of aMPV subtypes A and B in broilers are scarce. It has been difficult to reproduce swollen sinuses in chickens with aMPV under experimental conditions. In the field, SHS in broilers is suspected to be induced by combined infections with different respiratory pathogens. The objectives of the present study were to compare the pathogenesis of subtypes A and B aMPV in commercial broilers and to investigate the reproducibility of clinical disease. In two repeat experiments, commercial broilers free of aMPV maternal antibodies were inoculated with aMPV subtypes A and B of turkey origin. The clinical signs such as depression, coughing, nasal exudates, and frothy eyes appeared at 4 days post inoculation, followed by swelling of periorbital sinuses at 5 days post inoculation. Higher numbers of broilers showed clinical signs in subtype-B-inoculated compared with subtype-A-inoculated groups. Seroconversion to aMPV was detectable from 10 to 11 days post inoculation. The appearance of serum aMPV enzyme-linked immunosorbent assay antibodies and the clearance of the aMPV genome coincided. Subtype B aMPV showed a broader tissue distribution and longer persistence than subtype A. Histopathological changes were observed in the respiratory tract tissues of aMPV-inoculated broilers, and also in paraocular glands, such as the Harderian and lachrymal glands. Overall, our study shows that representative strains of both aMPV turkey isolates induced lesions in the respiratory tract, accompanied by swelling of infraorbital sinuses, indicating the role of aMPV as a primary pathogen for broilers.     

Detection of avian leukosis virus subgroup J in chicken flocks from Malaysia and their molecular characterization.

Previously we have shown that avian leukosis virus subgroup J (ALV-J) might be present in chicken flocks from Malaysia based on serological study and also on detection of tissue samples with myelocytic infiltration. In this study, the polymerase chain reaction was used to detect ALV-J sequences from archived frozen samples. Out of 21 tissue samples examined, 16 samples were positive for proviral DNA and four samples for ALV-J RNA. However, only nine samples were found positive for myelocytic infiltration. A total of 465 base pairs equivalent to positions 5305 to 5769 of HPRS-103 from each of the viral RNA positive samples were characterized. Sequence analysis indicated that the samples showed high identity (95.9 to 98.2%) and were close to HPRS-103 with identities between 97.4 and 99.3%. This study indicates that ALV-J-specific sequences can be detected by polymerase chain reaction from frozen tissue samples with and without myelocytic infiltration.     

Detection of infectious bronchitis virus and specific anti- viral antibodies using a Concanavalin A-Sandwich-ELISA

Concanavalin A-Sandwich ELISA (Con A-S-ELISA) was developed for the detection of infectious bronchitis virus (IBV) or chicken specific anti-viral antibodies. The antigen detection limit for the Con A-S-ELISA was 10(5,1) EID(50)/mL. Three homologous and four heterologous IBV strains were similarly detected. This assay was highly effective in detecting the virus after infected tissue homogenates were passed once in embryonated chicken eggs, showing a good agreement with virus isolation technique. The Con A-S-ELISA was also used to measure anti-IBV chicken antibodies and showed a high coefficient of correlation (r = 0.85) and an agreement of k = 0.80 with the commercially available Indirect-ELISA. The relative sensitivity and specificity between these two tests were, respectively, 92.86% and 95.65% with an accuracy of 93.39%. Thus, the Con A-S-ELISA proved to be able to detect alternatively homologous and heterologous IBV strains or specific chicken anti- IBV antibodies, using the Con A as capture reagent of this assay.     

Different levels of natural antibodies in chickens divergently selected for specific antibody responses

We studied the presence of Natural antibodies in plasma samples from individual birds from selected chicken lines at young and old age. Binding, specificity, and relative affinity to various antigens were determined in plasma from non-immunized female chickens at 5 weeks of age, and in plasma obtained from the same chickens one year later using indirect two-step ELISA. Birds were from three different lines. The lines were divergently selected for either high (H line) or low (L line) antibody titers to Sheep Red Blood Cells at 5 weeks of age, next to a random bred control (C line). Binding of plasma immunoglobulins (Ig) from all three lines was found with chicken-egg-white protein (CEP), ovalbumin (OVA), myoglobin (MYO), thyroglobulin (THYRO), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and transferrin (TRANS). Significantly higher binding to most antigens was found with plasma Ig from adult birds from the H line as compared to plasma Ig from the L line, whereas binding of plasma Ig from C-line birds was in between or similar to the H or L line, respectively. Binding of Ig to all antigens in all three lines was significantly higher in plasma obtained at one year of age as compared to plasma obtained at 5 weeks of age. A competitive ELISA with homologous and heterologous antigens was used for determining specificity of the antigen-binding antibodies. Nai;ve plasma samples were characterized by a broad binding to all antigens tested. Inhibition of binding to specific antigens was possible with a broad range of heterologous antigens, but highest competition of binding was obtained with homologous antigen. Both linear regression analysis of serial dilutions of the plasma Ig binding the antigens, as well as competitive ELISA with homologous antigen indicated that plasma Ig from the H line and plasma Ig from the L line had similar affinity characteristics to the antigens tested with the exception of OVA and KLH. Pooled non-immune plasma from H line birds bound to CEP, OVA, THYRO, TRANS, MYO, KLH, and salt-precipitated extracts and supernatants of extracts from chicken heart, spleen, liver, brain, bursa, thymus, and kidney, respectively, as determined by Western blotting. The increasing presence of antibodies in nai;ve chicken plasma binding heterologous and homologous (tissue) antigens indicates the presence of Natural antibodies in poultry. Apart from age, increasing levels of Natural antibodies may be related with the genetically based magnitude of specific antibody levels in the chicken lines studied.     

Avian metapneumovirus infection of chicken and turkey tracheal organ cultures: comparison of virus–host interactions

Avian metapneumovirus (aMPV) is a pathogen with worldwide distribution, which can cause high economic losses in infected poultry. aMPV mainly causes infection of the upper respiratory tract in both chickens and turkeys, although turkeys seem to be more susceptible. Little is known about virus–host interactions at epithelial surfaces after aMPV infection. Tracheal organ cultures (TOC) are a suitable model to investigate virus–host interaction in the respiratory epithelium. Therefore, we investigated virus replication rates and lesion development in chicken and turkey TOC after infection with a virulent aMPV subtype A strain. Aspects of the innate immune response, such as interferon-α and inducible nitric oxide synthase mRNA expression, as well as virus-induced apoptosis were determined. The aMPV-replication rate was higher in turkey (TTOC) compared to chicken TOC (CTOC) (P?<?0.05), providing circumstantial evidence that indeed turkeys may be more susceptible. The interferon-α response was down-regulated from 2 to 144 hours post infection in both species compared to virus-free controls (P?<?0.05); this was more significant for CTOC than TTOC. Inducible nitric oxide synthase expression was significantly up-regulated in aMPV-A-infected TTOC and CTOC compared to virus-free controls (P?<?0.05). However, the results suggest that NO may play a different role in aMPV pathogenesis between turkeys and chickens as indicated by differences in apoptosis rate and lesion development between species. Overall, our study reveals differences in innate immune response regulation and therefore may explain differences in aMPV – A replication rates between infected TTOC and CTOC, which subsequently lead to more severe clinical signs and a higher rate of secondary infections in turkeys.     

The association of rubella virus in congenital cataract - a hospital-based study in India.

BACKGROUND: The association of rubella virus (RV) with congenital cataract has been well established. Since the data on association of RV with congenital cataract in India are scanty, a study was done based on virus isolation from lens aspirates in patients undergoing therapeutic lensectomy and serology. OBJECTIVE: To determine the incidence of the association of rubella virus with congenital cataract. STUDY DESIGN: The lens aspirates collected during the 9-year period (from 1990 to 1998), from 70 children up to 12 years of age with congenital cataract were processed for the isolation of rubella virus by conventional viral isolation culture method using BHK-21 and Vero cell lines. Identification of the virus was confirmed by immunofluorescence using human anti-rubella virus specific hyperimmune serum. Serum samples were collected from 55 out of these 70 children and the presence of antibodies to RV was detected by ELISA test. RESULTS: RV was isolated from lens aspirates in seven (10%) out of the 70 children with congenital cataract. Of the 55 sera tested, 22 had both anti-rubella IgM and IgG antibodies, in 13 only anti-RV IgG antibodies, in seven only IgM antibodies and the rest of the 13 samples did not have detectable levels of rubella antibodies. Among the children who had IgM antibodies, 12 (24.5%) were below the age of 6 months. CONCLUSION: It can be concluded based on virus isolation that 10% of patients with congenital cataract were due to rubella infection and the detection of 24.5% anti-RV IgM antibodies in children below 6 months old shows the possible association of rubella virus with congenital cataract.