Understanding the disease and economic impact of avirulent avian paramyxovirus type 1 (APMV-1) infection in Great Britain.

Newcastle disease (ND) is a notifiable disease affecting chickens and other avian species caused by virulent strains of Avian paramyxovirus type 1 (APMV-1). While outbreaks of ND can have devastating consequences, avirulent strains of APMV-1 generally cause subclinical infections or mild disease. However, viruses can cause different levels of disease in different species and virulence can evolve following cross-species transmission events. This report describes the detection of three cases of avirulent APMV-1 infection in Great Britain (GB). Case 1 emerged from the 'testing to exclude' scheme in chickens in Shropshire while cases 2 and 3 were made directly from notifiable avian disease investigations in chicken broilers in Herefordshire and on premises in Wiltshire containing ducks and mixed species, respectively). Class II/genotype I.1.1 APMV-1 from case 1 shared 99.94% identity to the Queensland V4 strain of APMV-1. Class II/genotype II APMV-1 was detected from case 2 while the class II/genotype I.2 virus from case 3 aligned closely with strains isolated from Anseriformes. Exclusion of ND through rapid detection of avirulent APMV-1 is important where clinical signs caused by avirulent or virulent APMV-1s could be ambiguous. Understanding the diversity of APMV-1s circulating in GB is critical to understanding disease threat from these adaptable viruses.

Metagenomic characterization reveals virus coinfections associated with Newcastle disease virus among poultry in Kenya.

Newcastle disease (ND) is an endemic viral disease affecting poultry and causing massive economic losses. This cross-sectional purposive study detected coinfections that are associated with the Newcastle disease virus among poultry from selected regions in Kenya. Cloacal (n = 599) and oral-pharyngeal (n = 435) swab samples were collected and pooled into 17 and 15 samples, respectively. A total of 17,034,948 and 7,751,974 paired-end reads with an average of 200 nucleotides were generated from the cloacal and oral-pharyngeal swab samples, respectively. Analysis of the de novo assembled contigs identified 177 and 18 cloacal and oral-pharyngeal contigs, respectively with hits to viral sequences, as determined by BLASTx and BLASTn analyses. Several known and unknown representatives of Coronaviridae, Picobirnaviridae, Reoviridae, Retroviridae, and unclassified Deltavirus were identified in the cloacal swab samples. However, no Newcastle disease virus (family Paramyxoviridae) was detected in the cloacal swabs, although they were detected in the oropharyngeal swabs of chickens sampled in Nairobi, Busia, and Trans Nzoia. Additionally, sequences representative of Paramyxoviridae, Coronaviridae, and Retroviridae were identified in the oral-pharyngeal swab samples. Infectious bronchitis virus and rotavirus were chickens' most prevalent coinfections associated with the Newcastle disease virus. The detection of these coinfections suggests that these viruses are significant threats to the control of Newcastle disease as the Newcastle disease virus vaccines are known to fail because of these coinfections. Therefore, this study provides important information that will help improve disease diagnosis and vaccine development for coinfections associated with the Newcastle disease virus.

The Development of Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assays for the Detection and Differentiation of Virulent Newcastle Disease Virus.

Newcastle disease (ND) is a highly pathogenic viral infection of poultry with significant economic impacts worldwide. Despite the widespread use of vaccines, ND outbreaks continue to occur even within vaccinated poultry farms. Furthermore, novel Newcastle disease virus (NDV) genotypes are emerging in poultry, increasing the need for the development of rapid, accurate, and simple diagnostic methods. We therefore developed two novel sets of visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays based on highly conserved regions of the HN and F genes. The limits of detection of the NDV-Common-LAMP assay, for all the NDV strains, were 103.0 EID50/0.1 mL for Kr005 and 102.0 EID50/0.1 mL for Lasota within 35 min. The sensitivity of the NDV-Patho-LAMP assay, used for the strain differentiation of virulent NDV, was 102.0 EID50/0.1 mL for Kr005. No amplification was detected for the non-NDV templates. Next, we probed 95 clinical strains and 7 reference strains with the RT-LAMP assays to assess the feasibility of their use in diagnostics. We observed no cross-reactivity across the 102 strains. Furthermore, there was 100% congruence between the RT-LAMP assays and full-length sequencing of the target genes, indicating the potential for visual RT-LAMP in the identification and differentiation of NDV. These novel RT-LAMP assays are ideally suited for the field or resource-limited environments to facilitate the faster detection and differentiation of NDV, which can reduce or avoid further spread.

Development of real time reverse transcription loop-mediated isothermal amplification assay for rapid detection of genotype VII of Newcastle disease viruses.

1. This paper details the establishment of a diagnostic system based on the real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid, simple and sensitive detection of genotype VII of Newcastle disease virus (NDV) directly from field samples. One specific set of six primers were designed which targeted the fusion protein gene of G-VII viruses. The target gene can be amplified and the results recorded within 40 min.2. The merit of this technique was the feasibility of reading results either by examining turbidity by the naked eye or via the amplification curve generated by real-time PCR. This study tested the sensitivity and specificity of this system against NDV-G-VII and other avian viruses. The real-time RT-LAMP has been found to be more sensitive than real-time RT-PCR. Moreover, 24 out of 35 suspected field samples were positive for genotype VII by real-time RT-LAMP within 30 min in comparison to the real-time RT-PCR for detection of universal NDV.3. Accordingly, real-time RT-LAMP revealed higher sensitivity than real-time RT-PCR and had specificity only for the NDV-G-VII genotype. Additionally, this system was more rapid and had lower cost than real-time RT-PCR. Based on the results, the RT-LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV-G-VII infection.

Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus.

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

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.

Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Immunosensor-based rapid quantitative detection of Newcastle disease virus antibodies using innovative gold immunochromatographic assay.

AIMS: A novel quantitative method for rapid Newcastle disease virus (NDV) antibody detection was developed based on an innovative gold immunochromatographic assay with a quantitative immunosensor. METHODS AND RESULTS: NDV antibody-detecting test strips containing a two-reaction system and double-test lines (T1, T2) were prepared. The test results were judged according to the signal ratio between the test and control lines as measured by the quantitative immunosensor. The minimum detection limit of the test strips for NDV antibodies was 22 titres. In addition, the assay was highly specific because only NDV antibodies produced visible test lines on the strip. The clinical application of the strips was tested by detecting NDV antibodies in 506 serum samples collected from chickens. The results showed a coincidence of 92·49% with those of the haemagglutination inhibition assay. CONCLUSIONS: The strips were successfully prepared and showed high specificity towards NDV, sensitivity and stability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a new method for detection of NDV antibody and provides a reference basis for rapid and quantitative monitoring of NDV antibodies. This new method overcomes the limitation of the existing colloidal gold immunochromatography, which only produces qualitative or semi-quantitative results.

Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus.

BACKGROUND: Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. RESULTS: The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/µL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won't cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. CONCLUSION: The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus.

BACKGROUND: Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. RESULTS: A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. CONCLUSIONS: In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus.

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.

Zoonotic Infection With Pigeon Paramyxovirus Type 1 Linked to Fatal Pneumonia.

The characteristics and risk factors of pigeon paramyxovirus type 1 (PPMV-1) infection in humans are poorly known. We performed virological, pathological, and epidemiological analyses of a Dutch case, and compared the results with those of a US case. Both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. Both virus isolates clustered with PPMV-1, which has pigeons and doves as reservoir. Experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. Both patients were likely infected by contact with infected pigeons or doves. Given the large populations of feral pigeons with PPMV-1 infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human PPMV-1 infections may increase. We recommend testing for avian paramyxovirus type 1, including PPMV-1, in respiratory disease cases where common respiratory pathogens cannot be identified.

Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing.

BACKGROUND: Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. METHODS: An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow. RESULTS: For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq. CONCLUSION: The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.

Engineered recombinant protein products of the avian paramyxovirus type-1 nucleocapsid and phosphoprotein genes for serological diagnosis.

BACKGROUND: Virulent Newcastle disease virus (NDV, avian Avulavirus-1, APMV-1) induces a highly contagious and lethal systemic disease in gallinaceous poultry. APMV-1 antibody detection is used for surveillance and to control vaccination, but is hampered by cross-reactivity to other subtypes of avian Avulaviruses. Data are lacking concerning the applicability of NDV V proteins as differential diagnostic marker to distinguish vaccinated from virus-infected birds (DIVA strategy). METHODS: Full length and C-terminally truncated nucleocapsid (NP) protein, and the unique C-terminal regions of the phospho- (P) and V proteins of the NDV LaSota strain were bacterially expressed as fusion proteins with the multimerization domain of the human C4 binding protein, and used as diagnostic antigens in indirect ELISA. RESULTS: When used as diagnostic antigen in indirect ELISAs, recombinant full-length proved to be a sensitive target to detect seroconversion in chickens after APMV-1 vaccination and infection, but revealed some degree of cross reactivity with sera raised against other APMV subtypes. Cross reactivity was abolished but also sensitivity decreased when employing a C-terminal fragment of the NP of NDV as diagnostic antigen. Antibodies to the NDV V protein were mounted in poultry following NDV infection but also, albeit at lower rates and titers, after vaccination with attenuated NDV vaccines. V-specific seroconversion within the flock was incomplete and titers in individual bird transient. CONCLUSIONS: Indirect ELISA based on bacterially expressed recombinant full-length NP compared favorably with a commercial NDV ELISA based on whole virus antigen, but cross reactivity between the NP proteins of different APMV subtypes could compromise specificity. However, specificity increased when using a less conserved C-terminal fragment of NP instead. Moreover, a serological DIVA strategy built on the NDV V protein was not feasible due to reduced immunogenicity of the V protein and frequent use of live-attenuated NDV vaccines.

Development of a novel Newcastle disease virus (NDV) neutralization test based on recombinant NDV expressing enhanced green fluorescent protein.

BACKGROUND: Newcastle disease is one of the most important infectious diseases of poultry, caused by Newcastle disease virus (NDV). This virus is distributed worldwide and it can cause severe economic losses in the poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. Protection against NDV in chickens has been associated with development of humoral response. Although hemagglutination inhibition (HI) assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV. METHODS: In this study, we established a system to recover a recombinant NDV (rLS1) from a cloned cDNA, which is able to accept exogenous genes in desired positions. An enhanced green fluorescent protein (eGFP) gene was engineered in the first position of the NDV genome and we generated a recombinant NDV carrying eGFP. This NDV- eGFP reporter virus was used to develop an eGFP-based neutralization test (eGFP-NT), in which nAbs titers were expressed as the reciprocal of the highest dilution that expressed the eGFP. RESULTS: The eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were assayed by conventional neutralization (NT) and eGFP-NT. Additionally, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R 2 = 0.816) and ELISA (R 2 = 0.791) showed substantial correlation with conventional NT, eGFP-NT showed higher correlation (R 2 = 0.994), indicating that eGFP-NT is more accurate method to quantify nAbs. CONCLUSIONS: Overall, the neutralization test developed here is a simple, rapid and reliable method for quantitation of NDV specific nAbs. It is suitable for vaccine studies and diagnostics.

Pathogenesis of New Strains of Newcastle Disease Virus From Israel and Pakistan.

In the past few years, Newcastle disease virus (NDV) strains with epizootic characteristics belonging to subgenotypes VIIi and XIIIb emerged in the Middle East and Asia. In this study, 2 NDV strains-1 representative of subgenotype VIIi isolated in Israel (Kvuzat/13) and 1 representative of subgenotype XIIIb isolated in Pakistan (Karachi/07)-were characterized by intracerebral pathogenicity index and detailed clinicopathologic assessment. The intracerebral pathogenicity index values for Kvuzat/13 and Karachi/07 were 1.89 and 1.85, respectively, classifying these strains as virulent by international standards. In 4-week-old White Leghorn chickens, both strains caused 100% mortality within 4 (Kvuzat/13) and 5 (Karachi/07) days postinfection. Histopathology and immunohistochemistry for NDV nucleoprotein showed that both strains had wide systemic distribution, especially targeting lymphoid organs and mucosa-associated lymphoid tissues in the respiratory and intestinal tracts. Results of the animal experiment confirm that both Kvuzat/13 and Karachi/07 are highly virulent and behaved as velogenic viscerotropic NDV strains.

[Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases.

Detection and characterization of Newcastle disease virus in formalin-fixed, paraffin-embedded tissues from commercial broilers in Egypt.

Newcastle disease (ND) is highly contagious and causes severe economic losses to the poultry industry due to high morbidity and mortality. In this report, we describe the detection of Newcastle disease virus (NDV) in formalin-fixed tissues from an outbreak of ND on broiler farms in Egypt. The affected birds experienced respiratory and/or nervous signs and a 75% mortality rate. Tissue samples were collected and placed in 10% neutral buffered formalin followed by embedding in paraffin. RNA was extracted from 80-microm formalin-fixed paraffin-embedded tissue blocks and recovered in 60 microl of elution buffer. All samples were negative for influenza virus by real-time reverse-transcription (RT)-PCR but positive for NDV. These flocks were known to have been vaccinated with a live NDV vaccine (LaSota strain). The nucleic acid sequences of the virus detected in this study were similar to those of a velogenic virus at its cleavage site 111GRRQKR*F117 and clustered with class II genogroup VII lineage of NDV, with a nucleotide sequence identity of 94%-99%. Although extraction and amplification of NDV from paraffin-embedded tissues from experimentally infected birds has been reported previously, this study reports on the use of RT-PCR on formalin-fixed tissues from actual field samples.

Comparing presence of avian paramyxovirus-1 through immunohistochemistry in tracheas of experimentally and naturally infected chickens.

Tracheas from chickens infected both in the field and experimentally with lentogenic Newcastle disease virus (also known as avian paramyxovirus-1 [APMV-1] and referred to here as "lentogenic NDV") were examined histopathologically to score degree of pathologic changes and by immunohistochemistry to determine presence of viral protein. In the field cases there was often a striking lack of correlation between severity of tracheal lesions and amount of immunohistochemical signal for APMV-1 protein. Experimental cases had minimal pathologic changes and also minimal immunohistochemical signal. Positive cells were often associated with surface deciliation. It may be that lentogenic NDV has only a minor role as a respiratory pathogen, merely compromising the mucosa to allow other respiratory pathogens to infect and worsen the clinical and pathologic presentation.

Newcastle disease: progress and gaps in the development of vaccines and diagnostic tools.

Newcastle disease (ND) is a contagious disease of birds that can have severe economic consequences for poultry producers, including a serious impact on the international trade of poultry and eggs. Newcastle disease virus (NDV) isolates are also called avian paramyxovirus serotype-1 isolates, but only infection with virulent NDV (vNDV) causes the disease. Virulent Newcastle disease virus (vNDV) isolates are distributed worldwide and have a high capacity to mutate, allowing the development of multiple vNDV genotypes evolving simultaneously at different locations. Large gaps in existing knowledge in the areas of epidemiology and evolution limit the possibilities to control the disease. Recurrent infection of poultry and wild birds allows the maintenance of a reservoir for the viruses; however, the role of wild birds and poultry in vNDV evolution is largely unknown. In the area of diagnostics, the performance of fast and accurate diagnostics methods is often affected by the evolution of viral genomes. Therefore, there is a need for the validation of multiple recently developed experimental tests and a need to develop additional fast and inexpensive diagnostic tests to be used in the field. In the area of vaccination, the development of inexpensive thermostable NDV vaccines and the development of vaccines capable of preventing viral replication are the highest priorities for endemic countries. In countries considered free of vNDV the development of low- cost vaccines that produce minimal vaccine reactions to prevent decreased productivity are higher priorities. Worldwide, better strategies that replace the culling of infected birds are needed to control outbreaks.