Clinico-pathological assessment of virulent Newcastle Disease Virus in ducks / Avaliação clínico-patológica do Vírus da Doença de Newcastle em patos

Abstract Newcastle disease (ND) is an infectious, highly contagious and lethal disease of avian species. It is considered that ducks are natural reservoir or carrier for Newcastle disease virus (NDV) and are resistant against different strains of NDV. Current study was designed to evaluate the pathogenesis of Newcastle disease in domestic ducks through histopathology, immunohistochemistry (IHC) and serum biochemical changes. For this purpose, eighty ducks were reared for 42 days and divided in two groups A and B. Ducks in group A were challenged with (NDV) at rate of 0.1 ml of ELD50 (virus titer 107.32/100µl) on second week of age, whereas Group B was control negative. Splenomegaly, atrophy of thymus and necrotic lesion in kidney were observed on 9th day of post infection. Hepatic degeneration and mononuclear cell infiltration were noticed in proventriculus and intestine in challenged ducks. Viral antigen detected in lungs, intestine, proventriculus and lymphoid organs of infected ducks through IHC. Albumin and total protein values were significantly low in infected groups A as compared to control group B. ALT, AST, and ALP values were significantly high in infected group A. On 5th and 7th day of post infection oropharyngeal swabs were negative for NDV and cloacal swabs were positive for NDV through Reverse transcriptase polymerase chain reaction. It is concluded that ducks are susceptible to NDV and virulent strain of NDV caused disease in ducks.

Resumo A doença de Newcastle (DN) é uma doença infecciosa, altamente contagiosa e letal de espécies aviárias. Considera-se que os patos são reservatórios ou portadores naturais do vírus da doença de Newcastle (VDN) e são resistentes a diferentes cepas de VDN. O presente estudo foi desenvolvido para avaliar a patogênese da DN em patos domésticos por meio de histopatologia, imuno-histoquímica (IHQ) e alterações bioquímicas séricas. Para este propósito, 80 patos foram criados por 42 dias e divididos em dois grupos A e B. Os patos do grupo A foram submetidos ao VDN a uma taxa de 0,1 ml de ELD50 (título viral de 107,32 / 100 µl) na segunda semana de idade, enquanto o Grupo B foi controle negativo. Esplenomegalia, atrofia do timo e lesão necrótica no rim foram observadas no 9º dia pós-infecção. Degeneração hepática e infiltração de células mononucleares foram observadas no proventrículo e intestino em patos infectados. Antígeno viral foi detectado em pulmões, intestino, proventrículo e órgãos linfoides de patos infectados por IHQ. Os valores de albumina e proteína total foram significativamente baixos no grupo A infectado em comparação com o grupo B. Os valores de ALT, AST e ALP foram significativamente altos no grupo A. No 5º e no 7º dia após a infecção, os esfregaços orofaríngeos foram negativos para VDN, enquanto os esfregaços cloacais foram positivos para VDN por meio da reação em cadeia da polimerase via transcriptase reversa. Conclui-se que os patos são suscetíveis ao VDN e à cepa virulenta de VDN que causou doenças em patos.

Pathotyping of Newcastle Disease Virus: a Novel Single BsaHI Digestion Method of Detection and Differentiation of Avirulent Strains (Lentogenic and Mesogenic Vaccine Strains) from Virulent Virus.

We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

Multiple antigenic peptide-based flow through dot-blot assay for simultaneous antibody detection of infectious bronchitis virus and Newcastle disease virus.

The present study describes the development of a novel affordable and rapid visual dot-blot assay using synthetic multiple antigenic peptides (MAP) for simultaneous detection of antibodies to infectious bronchitis virus (IBV) and Newcastle disease virus (NDV). Antibody detection efficiencies of MAP peptides namely, NP1 MAP (Nucleoprotein IBV) and HN MAP (Haemagglutinin-neuraminidase NDV) were studied in solid-phase indirect peptide ELISA. In comparison with the commercial kit, the NP1 MAP showed 89.20% diagnostic sensitivity (DSn) and 85.90% diagnostic specificity (DSp) at 19.45% ROC cut-off. Similarly, HN MAP was evaluated and showed 89.70% DSn and 92.90% DSp at 19.90 % ROC cut-off. The peptides after evaluating their ELISA performance were further used to device a flow-through dot-blot assay (FT-DBA) for simultaneous detection of IBV and NDV antibodies. The kappa value for IBV by FT-DBA in comparison to commercial ELISA was 0.64 whereas for NDV, FT-DBA gave a kappa value of 0.68 in comparison to commercial ELISA indicating substantial agreement between the assays. In essence, the divergent MAP based diagnostic design could provide an alternative for antibody detection of IBV and NDV. Further, the FT-DBA approach could be used for low cost, rapid and pen-side detection of IBV and NDV antibodies simultaneously.

The effect of Spirulina algae on the immune response of SPF chickens to commercial inactivated Newcastle vaccine in poultry / El efecto del alga Spirulina sobre la respuesta inmune de pollos SPF inducida por la vacuna inactivada comercial contra la enfermedad de Newcastle

The objective of this study was to investigate the effects of Spirulina platensis (SP) powder supplementation on immune response in SPF chickens. For this purpose, 120 SPF chicks were randomly clustered into six groups consisting of 20 birds each which assigned to five groups vaccinated by commercial inactivated Newcastle disease (ND) vaccine at 21 days of age. The four groups were supplemented with 0.5, 1, 1.5 and 2 g of SP per kg of ration at 7 day of age and other group as control treatment group. Control unvaccinated group still without any treatment. Individual blood samples were collected weekly from all groups, and NDV-HI antibodies were measured using Hemagglutination inhibition (HI) test. After 28 days post-vaccination, ten birds from all groups were challenged intramuscularly at a dose 0.5 mL/bird containing 106 EID50 of local NDV genotype VII. Challenge virus shedding was detected using real time qrt-PCR of oropharyngeal swabs that were collected from all challenged chicken groups of at 3, 5, 7 and 10 days post challenge. Obtained results showed that vaccinated groups of SPF-chickens either supplied with Spirulina or control treatment group induced positive serological response as NDV-HI antibody were measured in sera of immunized chicks (7.6, 8, 8.3, 8.9 and 7.4 log2, respectively) at 4 weeks post vaccination (WPV). Significant differences were observed at 2 WPV in the vaccinated SPF chickens consumed 1, 1.5 and 2 g of SP/kg of ration, compared to untreated vaccinated group (p<0.05). Immunized SPF chickens supplied with different SP concentration confer satisfactory protection against heterologous challenge virus (90 percent, 100 percent, 100 percent and 100 percent respectively), in contrast to untreated vaccinated chickens. Different percentages of reduction of viral shedding (55 percent, 65 percent, 76 percent and 87 percent) of treated vaccinated chickens with different concentration of SP were detected, despite untreated group were reduced 46 percent from total viral shedding. These findings suggest that dietary Spirulina has immune-stimulatory effects on the immune system of SPF chickens. One gram from SP per kg of ration was minimum recommended concentration that able to exhibit optimum immune response, increase protection against heterologous strains and able to reduce viral shedding(AU)

El objetivo de este estudio fue investigar los efectos de la suplementación con polvo de Spirulina platensis (SP) sobre la respuesta inmune en pollos SPF. Para este propósito se agruparon al azar 120 polluelos SPF en seis grupos de 20 aves cada uno, que se asignaron a cinco grupos vacunados con la vacuna comercial inactivada contra la enfermedad de Newcastle (ND) a los 21 días de edad. Cuatro grupos se suplementaron con 0,5; 1; 1,5 y 2 g de SP por kg de ración a los 7 días de edad, un grupo vacunado sin suplemento y un grupo sin ningún tratamiento. Semanalmente, se recogieron muestras de sangre individuales de todos los grupos y se midieron los anticuerpos hemaglutinantes contra el virus Newcastle (NDV-HI) mediante la prueba de inhibición de la hemaglutinación (HI). 28 días después de la vacunación, fueron retadas diez aves de cada grupo por vía intramuscular a una dosis 106 EID50 del genotipo VII del NDV local en un volumen de 0,5 mL/ave. Se detectó la eliminación del virus mediante qrt-PCR en hisopos orofaríngeos que se recolectaron en todos los grupos a los 3, 5, 7 y 10 días después del reto. Los resultados obtenidos mostraron que los grupos vacunados de pollos y suplementados con Espirulina y el grupo de control vacunado, indujeron una respuesta serológica positiva cuando se determinaron los anticuerpos NDV-HI en los pollitos inmunizados (7,6; 8; 8,3; 8,9 y 7,4 log2 respectivamente) a las 4 semanas después de la vacunación (SPV). Se observaron diferencias significativas a las 2 SPV en los pollos vacunados que consumieron 1, 1,5 y 2 g de SP/kg de ración, en comparación con el grupo vacunado no tratado (p<0,05). Los pollos inmunizados que recibieron diferentes concentraciones de SP mostraron una protección satisfactoria contra el desafío heterólogo viral (90 por ciento, 100 por ciento y 100 por ciento respectivamente), en contraste con los pollos vacunados no tratados. Se observaron diferentes porcentajes de reducción de la diseminación viral (55 por ciento, 76 por ciento y 87 por ciento) entre los pollos vacunados tratados con diferente concentración de SP. En el grupo no tratado se redujo al 46 por ciento. Estos hallazgos sugieren que la Espirulina en la dieta tiene efectos inmunoestimuladores sobre el sistema inmunitario de los pollos. Un gramo de SP por kg de ración fue la concentración mínima recomendada para una respuesta inmune óptima, y de esta forma aumentar la protección contra las cepas heterólogas y disminuir la diseminación viral(AU)

Production, characterization, and epitope mapping of a monoclonal antibody against genotype VII Newcastle disease virus V protein.

Newcastle disease virus (NDV) V protein is crucial for viral interferon (IFN) antagonism and virulence, determining its host range restriction. However, little information is available on the B cell epitopes of V protein and the subcellular movement of V protein in the process of NDV infection. In this study, the monoclonal antibody (mAb) clone 3D7 against genotype VII NDV V protein was generated by immunizing mice with a purified recombinant His-tagged carboxyl-terminal domain (CTD) region of V protein. Fine epitope mapping analysis and B-cell epitope prediction indicated that mAb 3D7 recognized a linear epitope 152RGPAELWK159, which is located in the V protein CTD region. Sequence alignment showed that the mAb clone 3D7-recognized epitope is highly conserved among Class II genotype VII NDV strains, but not among other genotypes, suggesting it could serve as a genetic marker to differentiate NDV genotypes. Furthermore, the movement of V protein during NDV replication in infected cells were determined by using this mAb. It was found that V protein localized around the nucleus during virus replication. The establishment of V protein-specific mAb and identification of its epitope extend our understanding of the antigenic characteristics of V protein and provide a basis for the development of epitope-based diagnostic assays.

A novel immunosensor based on excessively tilted fiber grating coated with gold nanospheres improves the detection limit of Newcastle disease virus.

A novel immunosensor for detecting Newcastle disease virus (NDV) was developed using excessively tilted fiber grating (Ex-TFG) coated with gold nanospheres (AuNs). AuNs were coated on the Ex-TFG surface via Au-S bonds using 3-mercaptopropyltrimethoxysilane (MPTMS), and the activated staphylococcal protein A (SPA) was linked to AuNs by covalent bonds via cysteamine. AuNs greatly enhanced the impact of the analyte on the fiber cladding mode through the local surface Plasmon resonance (LSPR) effect, thus improving the detection limit and sensitivity of the immunosensor. Meanwhile, SPA enhanced the bioactivity of anti-NDV monoclonal antibodies (MAbs), thus promoting the effectiveness of specific binding events on the fiber surface. Immunoassays were performed by monitoring the resonance wavelength shift of the proposed sensor under NDV samples containing different particle amounts. Specificity was assessed, and clinical tests for NDV were performed by contrast experiments. Experimental results showed that the detection limit for NDV was about 5~10 times improved compared to that of reference Ex-TFG without AuN treatment. Moreover, the novel biosensor was reusable and could potentially be applied in clinic.

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis.

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.

Electronic microarray assays for avian influenza and Newcastle disease virus.

Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV.

A survey for maintenance of virulent newcastle disease virus-free area in poultry production in Brazil

In 2003, Brazil was recognized as a pathogenic Newcastle Disease Virus (NDV) strain-free country for commercial poultry. This research was conducted in Brazil between December 2003 and March 2005 to verify the maintenance of this virulent NDV-free status. Serum samples from 5,455 flocks for commercial poultry farms were collected, comprising 81,825 broiler chickens. The farms were located in nine states of the country, grouped in three geographic regions. Serological evidence of NDV infection was detected in 28.8 percent of the surveyed farms. However, all fifteen viruses isolated and identified as Newcastle Disease Virus (NDV) were characterized as nonpathogenic strains, based on the Intracerebral Pathogenicity Index. These results showed that Brazil preserves the virulent NDV-free status for commercial flocks.

Evolutionary changes affecting rapid identification of 2008 Newcastle disease viruses isolated from double-crested cormorants.

A morbidity-mortality event involving virulent Newcastle disease virus (NDV) in wild double-crested cormorants (Phalacrocorax auritus) occurred in North America in the summer of 2008. All 22 viruses isolated from cormorants were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene. However, the USDA-validated reverse transcription-PCR assay targeting the fusion gene that is specific for virulent isolates identified only 1 of these 22 isolates. Additionally, several of these isolates have been sequenced, and this information was used to identify genomic changes that caused the failure of the test and to revisit the evolution of NDV in cormorants. The forward primer and fusion probe were redesigned from the 2008 cormorant isolate sequence, and the revised fusion gene test successfully identified all 22 isolates. Phylogenetic analyses using both the full fusion sequence and the partial 374-nucleotide sequence identified these isolates as genotype V, with their nearest ancestor being an earlier isolate collected from Nevada in 2005. Histopathological analysis of this ancestral strain revealed morphological changes in the brain consistent with that of the traditional mesogenic pathotypes in cormorants. Intracerebral pathogenicity assays indicated that each of these isolates is virulent with values of >0.7 but not more virulent than earlier isolates reported from Canada.

Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa--implications for diagnosis and control.

Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which affect many species of birds and may cause severe economic losses in the poultry sector. The disease has been officially and unofficially reported in many African countries and still remains the main poultry disease in commercial and rural chickens of Africa. Unfortunately, virological and epidemiological information concerning ND strains circulating in the Western and Central regions of Africa is extremely scarce. In the present study, sequence analysis, pathotyping and detailed genetic characterization of virulent ND strains detected in rural poultry in West and Central Africa revealed the circulation of a new genetic lineage, distinguishable from the lineages described in the Eastern and Southern parts of the continent. Several mismatches were observed in the segment of the matrix gene targeted by the primers and probe designed for the molecular detection of APMV-1, which were responsible for the false negative results in the diagnostic test conducted. Furthermore, deduced amino acid sequences of the two major antigens eliciting a protective immune response (F and HN glycoprotein) revealed protein similarities <90% if compared to some common vaccine strains. Distinct mutations located in the neutralizing epitopes were revealed, indicating the need for detailed assessment of the efficacy of the current vaccines and vaccination practices in Africa. The present investigation provides important information on the epidemiology, diagnosis and control of NDV in Africa and highlights the importance of supporting surveillance in developing countries for transboundary animal diseases.

Isolation of avian paramyxovirus 1 from a patient with a lethal case of pneumonia.

An unknown virus was isolated from a lung biopsy sample and multiple other samples from a patient who developed a lethal case of pneumonia following a peripheral blood stem cell transplant. A random PCR-based molecular screening method was used to identify the infectious agent as avian paramyxovirus 1 (APMV-1; a group encompassing Newcastle disease virus), which is a highly contagious poultry pathogen that has only rarely been found in human infections. Immunohistochemical analysis confirmed the presence of APMV-1 antigen in sloughed alveolar cells in lung tissue from autopsy. Sequence from the human isolate showed that it was most closely related to virulent pigeon strains of APMV-1. This is the most completely documented case of a systemic human infection caused by APMV-1 and is the first report of an association between this virus and a fatal disease in a human.

Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates.

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.

Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens.

With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the renNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated and a NDV haemagglutinin-neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV.

A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus.

A recombinant baculovirus containing a cDNA clone encoding the nucleocapsid (NP) protein of Newcastle disease virus (strain Ulster 2C) has been used to infect insect cells (Spodoptera frugiperda). High levels of overexpressed NP protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. Nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect ELISA to detect the presence of anti-NDV antibodies in a cohort of chicken sera. Data produced from these tests indicated a good correlation between ELISA titre and haemagglutination inhibition test data. The test was not affected by interference from background cellular proteins nor by cross-reactivity with non-NDV poultry pathogens. Additionally, the test did not generate false-positive readings.

[Serologic detection of antibodies to Newcastle disease virus in water fowl using the hemagglutination inhibition test and enzyme immunoassay]. / Serologischer Nachweis von Antikörpern gegen das Newcastle Disease Virus bei Wassergeflgel mittels Hämagglutinationshemmungs-Test und Enzym-Immuno-Assay.

8410 samples from Moscovy duck, Pekin duck and geese were incorporated into examinations of antibodies against the Newcastle Disease virus. A new enzyme-immuno-assay (EIA) for antibody detection in Moscovy duck and Pekin duck was developed using purified antigen from NDV-strain "La Sota". The epidemiology as well as the relation of incidence of the Newcastle Disease in waterfowl was discussed.

Identification of the antigenic components of paramyxovirus-3, paramyxovirus-6 and Newcastle disease virus in turkeys.

The aim of this study was to use the enzyme-linked immunosorbent assay (ELISA) and the Western immunoblotting as possible tools to differentiate infections in turkeys by different paramyxoviruses. Pooled hyperimmune sera of turkeys infected with either paramyxovirus-3 (PMV-3), paramyxovirus-6 (PMV-6), or Newcastle disease virus (NDV) were assayed for antibodies specific to the three viruses by the ELISA and Western immunoblotting. ELISA results showed cross reactions of turkey antibodies between PMV-3 and PMV-6 antigens, while turkey antibodies to NDV did not cross-react with any of the other paramyxoviruses. The immunoblots of sera from birds infected with PMV-3 (Minnesota turkeys and Iowa chickens) reacted to low molecular weight polypeptides of PMV-3 of 29, 32, and 34 kDa, and to a high molecular weight band of 200 kDa. The same Minnesota turkey sera had a cross reaction to the 200 kDa polypeptide of PMV-6, while the Iowa chicken sera did not. Both sera had no apparent reaction to NDV proteins. Western immunoblotting showed that the turkey PMV-3 sera had a specific reaction to a 220 kDa polypeptide present in PMV-3, but not in PMV-6, while the turkey PMV-6 sera had a specific reaction to a 130 kDa polypeptide present in PMV-6, but not in PMV-3. Immunoblots of pooled sera from turkeys infected with PMV-6 (Minnesota source) reacted to the 200 kDa protein present in both PMV-3 and PMV-6; however, no reaction occurred between this sera and NDV proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

An ELISA blocking test using a peroxidase-labelled anti-HN monoclonal antibody for the specific titration of antibodies to avian paramyxovirus type 1 (PMV1).

This report describes an ELISA blocking test using a peroxidase-labelled monoclonal antibody which binds to the HN protein of Newcastle disease (NDV). This test allows specific detection of type 1 avian paramyxovirus (PMV1) antibodies but does not detect other avian paramyxovirus (PMV2-9) antibodies recognized by the usual serological NDV tests (HI, Orgenics, and Agritech ELISA tests). Furthermore, swollen head syndrome and influenza antibodies were also not detected. ELISA blocking and HI titers of sera collected from SPF chickens immunized with 18 different PMV1 strains (including pigeon isolates) were the same; the correlation between ELISA blocking and HI titers was highly significant (P less than 0.001). In comparison with ELISA tests available commercially at the present time, the ELISA blocking test can be performed more quickly and is applicable without modification to sera from different species of fowls. For this reason, the test appears to be useful for determining the immunity and sanitary status of fowls. When recombinant or deleted vaccines become available, the test should make it possible to demonstrate with confidence any infection of fowls by wild type PMV1.

A microwell immunoperoxidase test for screening hybridomas and for diagnosing Newcastle disease virus and Sendai virus.

A cell counting assay was developed to measure the infectivity of Sendai virus and Newcastle disease virus by indirect immunoperoxidase. It was used to monitor monoclonal antibodies against Newcastle disease virus, antibody to Sendai virus in mice and tracheal infectivity in chickens.

Rapid serological profiling by enzyme-linked immunosorbent assay. I. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure specific antibody activity in sera of chickens exposed to Newcastle disease virus (NDV). A near-linear relationship existed between the log of the corrected absorbance of antisera at a single working dilution and the corresponding observed serum titers as determined by a standard serial-dilution method. Regression analysis was used to construct a standard curve and extract an equation from this relationship. The equation was used to convert corrected absorbance readings of the single working dilution directly into predicted ELISA antibody activity titers. In a comparative study, a correlation (P less than 0.01) was found between ELISA and hemagglutination-inhibition (HI) antibody titers to NDV. ELISA titers were as much as 160 times greater than the HI titers. ELISA was also able to detect much lower levels of antibody activity than the HI test.