Detection Of Newcastle Disease Virus Research Articles

Voltammetric-based immunosensing of Newcastle disease virus on polyethylene glycol-containing self-assembled monolayer modified gold electrode

A voltammetric immunosensor for the detection of Newcastle disease virus (NDV) has been developed by employing polyclonal antibody targeting NDV (anti-NDV) as a bioreceptor. Anti-NDV was immobilized on polyethylene glycol (PEG)-containing self-assembled monolayer (SAM) which was activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) and N-hydroxy succinimide (NHS) coupling on screen-printed gold electrode (SPGE). The introduction of PEG-containing SAM on the SPGE allowed the bioreceptor to covalently bound to the electrode surface whilst still providing a hydrophilic layer on the electrode which is important to greatly reduce non-specific bindings. The bioreceptor functionalized electrode was then allowed to be incubated with NDV-spiked samples. The electrode surface modification with PEG-containing SAM, immobilization of anti-NDV as bioreceptor, up to the detection of NDV were characterized electrochemically through differential pulse voltammetry (DPV) analysis in [Fe(CN)6]3- as the redox probe. Decrement of anodic current peak (Ipa) of [Fe(CN)6]3- was seen as the concentration of NDV increased from 0.156 to 20 HA μL-1 with the limit of detection (LoD) of 1.50 HA μL-1 at 3σ m-1. The detection of NDV in HA μL-1 unit in this study would ease interlaboratory interpretation as it was the same unit used in hemagglutination (HA) assay of conventional NDV diagnosis. The specificity of anti-NDV used as bioreceptor towards NDV was confirmed through western blot analysis, whilst the selectivity of the bioreceptor-functionalized electrode has been tested with allantoic fluid as the negative control in which no apparent changes of anodic peak (Ipa) has been seen. This simple, fast, and less laborious electrochemical detection method could become an alternative to the conventional method for NDV detection.

Cocirculation of Newcastle Disease Virus Genotypes XII and VII Along with Nonvirulent Forms Characterized in Domestic Birds from Peru.

Newcastle disease virus (NDV) is one of the most important pathogens affecting poultry, given its impact on health and production systems worldwide, despite widespread vaccination. Over the past 20 years, NDV has caused severe outbreaks of disease in Peru. These outbreaks primarily affected gamecocks and broiler chickens, with an additional reported case in commercial layers. Therefore, our objective was to identify and characterize the virus responsible for these cases in Peru. We analyzed 14 suspected clinical cases in domestic birds for NDV detection, isolation, and genetic characterization. Among these cases, seven involved gamecocks, with six genotype XII isolates and one genotype VII isolate, representing the first report of NDV genotype VII isolate from fighting roosters in Peru. Additionally, among the six cases in broiler chickens, we detected four genotype XII isolates and three genotype II isolates, including one sample containing both genotypes XII and II. Furthermore, a genotype I viral isolate was identified in a laying hen. Hence, we concluded that two divergent, highly virulent NDV genotypes, genotypes XII and VII, along with avirulent forms such as genotypes I and II are circulating among domestic birds in Peru. Genetic analysis indicates that these viruses are evolving locally within avian species and offers the basis necessary for vaccine adaptation to circulating viruses. Our results highlight the cocirculation of multiple virulent and nonvirulent NDV genotypes in domestic birds in Peru, underscoring the potential role of gamecocks as a viral source of virulent NDV strains in the country and the occurrence of outbreaks in poultry farms.

Molecular Detection and Serological Investigation of Newcastle Disease in Intensive, Semi-Intensive, and Backyard Production Systems in Central and Southwestern Areas of Ethiopia.

The purpose of this research is to detect Newcastle disease virus and to assess the seropositivity among backyard, semi-intensive, and intensive farms located in central and southwestern areas of Ethiopia. A total of 239 oropharyngeal and cloacal swab samples were collected from symptomatic birds found in Holeta, Burayu, Jimma towns as well as Seka Chekorsa and Nadhigibe woredas of Jimma Zone. In addition, ninety blood samples were collected from wing veins of unvaccinated birds found in the study areas of Jimma zone. Side-by-side information related to risk factors estimated to contribute to the susceptibility of the disease was collected by interviewing owners of sampled birds. Reverse transcription polymerase-chain reaction (RT-PCR) was conducted to detect NDV. Likewise, Enzyme-linked immunosorbent assay (ELISA) was performed to determine the seropositivity of ND. The proportion of samples where NDV was detected was 24.6%. Similarly, 68.9% of the sampled birds were seropositive. It was observed that adult birds were more likely to encounter the disease than youngs (OR = 11.6; 95% CI: 4.0-33.3; P = 0.000). Birds owned by respondents who leave diseased birds in the flock were more likely infected (OR = 6.2; 95% CI: 1.8-21.2; P=0.004) as compared to those isolated and mode of disposal of dead chicken significantly affect exposure (OR = 0.13; 95% CI: 0.10-4.88; P = 0.044). Likewise, access to veterinary services highly likely reduces susceptibility to the disease (OR = 12.4; 95% CI: 3.2-46.9; P = 0.000). It was also found that birds farmed intensively were the most at risk (OR = 2.8; 95% CI: 0.58-13.71; P = 0.199). Detection of ND from a significant proportion of sampled birds and their high seropositivity percentage revealed the circulation of the virus in the study areas.

Molecular And Serological Detection Of Newcastle Disease Virus In Live-Bird Markets, Jos, Plateau State In Nigeria

Newcastle disease (ND) is a highly infectious viral disease of birds caused by the Newcastle disease virus (NDV) and it has been reported in domestic birds in Nigeria. Waterfowls and village poultry in live bird markets (LBM) acts as reservoirs, potentially reintroducing the virus to commercial poultry. This study aims at molecular and serological detection of NDV at live bird markets in Plateau State, Nigeria. A cross sectional analysis involved 309 pooled cloacal and tracheal swabs over three months were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), RT-qPCR, virus isolation and haemagglutination and haemagglutinationinhibition test (HA &HI). Virus isolation was attempted in 9 to 11 days old specific antibody negative (SAN) embryonated chicken eggs and fifteen samples showed haemagglutination. Subsequent tests confimed nine of these as NDV positive through haemagglutination and haemagglutination inhibition (HI) tests. Conventional RT-PCR and RT-qPCR further validated five of the nine NDV positive isolates. The phylogenetic analysis of partial F gene nucleotide sequences revealed that all three isolates belonged to class II genotype XIV.2_XIVb. This finding underscores the persistent threat of NDV to local poultry, necessitating comprehensive virological surveillance to understand, isolate and characterize the virus in Nigeria. Therefore, monitoring for emerging lineages and sub-lineages in Nigeria birds is crucial for safeguarding commercial poultry production.

Newcastle disease virus detection and characterization using molecular biology techniques

Newcastle disease virus detection and characterization using molecular biology techniques

Serological detection of Newcastle disease virus in backyard poultry production system in Woliso District South West Shewa, Central Ethiopia.

Newcastle disease (ND)is one of the most important respiratory viral diseases. The disease is endemic in many parts of Ethiopia. However, there is no clear record about the introduction of the virus to the country (Ethiopia). Hence, detail about the ND is very important in its (ND) control and prevention. Despite these facts, there is no available research work done on ND in the current research area that would help either as references for researchers or that could help in the control and prevention of the disease. Therefore, the objective of this study was to detect the ND virus (NDV), using serological methods in from December 2018 to November 2019. A cross-sectional type of study was conducted to detect the NDV. The convenience sampling method was used for sample data collection. Before data collection, chicken with previous history of vaccination against the NDV was excluded from the sampling animals. Then, a total of 348 blood samples of 2 mLwere collected from the brachial vein in 3 mLdisposable syringes. The serum was collected in labeled 2 mL cryovial tubes. Indirect enzyme-linked immunosorbent assay(ELISA) tests were performed to detect antibodies against NDV and to determine its antibody titer. The test was performed using (ID.vetinnovative version 2) procedure. In the indirect ELISA test, 37.64% (131/348) were positive and antibody titer mean value of (1761.9088) was scored. The standard deviation of 2592.42160 and a percentage coefficient of variation of147% was scored. From the finding, we conclude that indirect ELISA test detected the presence of the NDV in the study area and the heterogeneousity of antibody titer in the study area. Therefore, further molecular characterization and epidemiological investigation should be carried and vaccination of animals is critical in the study area.

Molecular and serological detection of Newcastle disease virus in live-bird markets, Jos, Plateau State Nigeria.

Newcastle disease (ND) is a highly infectious viral disease of birds caused by the Newcastle disease virus (NDV) and it has been reported in domestic birds in Nigeria. Waterfowls and village poultry in live bird markets (LBM) acts as reservoirs, potentially reintroducing the virus to commercial poultry. This study aims at molecular and serological detection of NDV at live bird markets in Plateau State, Nigeria. A cross sectional analysis involved 309 pooled cloacal and tracheal swabs over three months were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), RT-qPCR, virus isolation and haemagglutination and haemagglutination-inhibition test (HA &HI). Virus isolation was attempted in 9 to 11 days old specific antibody negative (SAN) embryonated chicken eggs and fifteen samples showed haemagglutination. Subsequent tests confimed nine of these as NDV positive through haemagglutination and haemagglutination inhibition (HI) tests. ConventionalRT-PCR and RT-qPCR further validated five of the nine NDV positive isolates. The phylogenetic analysis of partial F gene nucleotide sequences revealed that all three isolates belonged to class II genotype XIV.2_XIVb. This finding underscores the persistent threat of NDV to local poultry, necessitating comprehensive virological surveillance to understand, isolate and characterize the virus in Nigeria. Therefore, monitoring for emerging lineages and sub-lineages in Nigeria birds is crucial for safeguarding commercial poultry production.

In-silico Analysis of Newcastle Disease Virus Strains from Outbreaks in Zaria and Kano of Nigeria

Introduction: Newcastle Disease (ND) is the major constraint to poultry production in developing country like Nigeria. Numerous outbreak cases of ND have being reported frequently even in vaccinated farms. The large antigenic disparity of the Newcastle Disease Virus (NDV) and potential for significant evolutionary divergence between field strains and widely used vaccinations are thought to be the root of the issue.
 Aim/Objectives: A cross-sectional studies was carried-out to identify a discrepancy between the vaccinal strains and circulating NDV field strains in Zaria and Kano so as to provide solution for the control of ND.
 Materials and Methods: In this study, molecular characterization and pathotyping of the field strains of NDV and strains of ND vaccines was carried out to evaluate their relatedness. Using RT-PCR with primers targeting partial fusion gene, 19 out of the (n= 120) samples purposefully collected for the detection of NDV in Zaria (n= 44) and Kano (n= 76) were positives. Twelve of the 19 positive amplicons were successfully sequenced and analyzed.
 Results: Only few number of the study’s sample have similarity with some of the vaccine strains.
 Conclusion: This study has raised concerns about the methods used to prevent NDV in Nigeria.
 Recommendation: It is crucial to develop an ND vaccine that matches the field strains within the country, along with ongoing surveillance of outbreaks for the effective control of the disease.

Investigation of respiratory disease outbreaks of poultry in Bangladesh using two real-time PCR-based simultaneous detection assays.

For rapid and sensitive pathogen screening from field outbreaks, molecular techniques such as qPCR-based simultaneous detections are efficient. Respiratory diseases are the most detrimental diseases to the poultry industry and need to be addressed because of their major economic losses. In the current study, we have applied two different detection assays: one for simultaneous detection of avian influenza virus (AIV; M gene) and subtyping (H5, N1, H9, N2) using TaqMan probe chemistry (TaqMan multitarget) and another for simultaneous detection of Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) using SYBR Green chemistry (SYBR Green multitarget). Two individual qPCRs were conducted for the detection of four pathogens. Surveillance of tissue (n = 158) and oropharyngeal swab (206) samples from multiple poultry flocks during the years April 2020-July 2022 applying the TaqMan and SYBR Green multitarget qPCRs revealed that 48.9% of samples were positive for respiratory infections, of which 17.2% were positive for NDV, 25.5% were positive for AIV, 9.9% were positive for IBV, and only a single positive (0.3%) for ILTV. Among the AIV, 35% were highly pathogenic subtype H5N1 and 65% were low pathogenic subtype H9N2. Co-infections of 2-3 respiratory viruses were also accurately detected. Respiratory viral pathogens are quite common in Bangladeshi poultry and can be successfully detected using multitarget simultaneous real-time quantitative polymerase chain reaction (RT-qPCR) assays like those adopted in the current study. Increased mass surveillance, along with the molecular characterization of the circulating respiratory viruses, is crucial to control the epidemic and subsequently save the Bangladeshi poultry industry.

Smartphone recognition-based immune microparticles for rapid on-site visual data-sharing detection of Newcastle disease virus

Since the last century, animal viruses have posed great threats to the health of humans and the farming industry. Therefore, virus control is of great urgency, and regular, timely, and accurate detection is essential to it. Here, we designed a rapid on-site visual data-sharing detection method for the Newcastle disease virus with smartphone recognition-based immune microparticles. The detection method we developed includes three major modules: preparation of virus detection vectors, sample detection, and smartphone image analysis with data upload. First, the hydrogel microparticles containing active carboxyl were manufactured, which coated nucleocapsid protein of NDV. Then, HRP enzyme-labeled anti-NP nanobody was used to compete with the NDV antibody in the serum for color reaction. Then the rough detection results were visible to the human eyes according to the different shades of color of the hydrogel microparticles. Next, the smartphone application was used to analyze the image to determine the accurate detection results according to the gray value of the hydrogel microparticles. Meanwhile, the result was automatically uploaded to the homemade cloud system. The total detection time was less than 50 min, even without trained personnel, which is shorter than conventional detection methods. According to experimental results, this detection method has high sensitivity and accuracy. And especially, it uploads the detection information via a cloud platform to realize data sharing, which plays an early warning function. We anticipate that this rapid on-site visual data-sharing detection method can promote the development of virus self-checking at home.

Review detection of Newcastle disease virus.

Newcastle disease (ND) is an acute and highly contagious disease caused by the Newcastle disease virus (NDV) infecting poultry, which has caused great harm to the poultry industry around the world. Rapid diagnosis of NDV is important to early treatment and early institution of control measures. In this review, we comprehensively summarize the most recent research into NDV, including historical overview, molecular structure, and infection mechanism. We then focus on detection strategies for NDV, including virus isolation, serological assays (such as hemagglutination and hemagglutination-inhibition tests, enzyme linked immunosorbent assay, reporter virus neutralization test, Immunofluorescence assay, and Immune colloidal gold technique), molecular assays (such as reverse transcription polymerase chain reaction, real-time quantitative PCR, and loop-mediated isothermal amplification) and other assays. The performance of the different serological and molecular biology assays currently available was also analyzed. To conclude, we examine the limitations of currently available strategies for the detection of NDV to lay the groundwork for new detection assays.

Molecular Detection of Newcastle Disease Virus from Field Outbreaks in Assam, India

The present study was carried out to detect the circulating Newcastle disease virus (NDV) from various field outbreaks in Assam during 2018-2021. Nine hundred ninety-two clinical and post mortem samples were collected from the backyard and commercial poultry and subjected to molecular detection by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). NDV could be detected in 445 (n=757, 58.78%) tissue samples and 53 (n=235, 22.55%) cloacal swabs collected from suspected poultry cases by RT-PCR. The findings of our study suggests that the sample of choice for detection of NDV from tissue samples was trachea (n=85, 92.94%), followed by spleen (n=96, 90.62%), lung (n=92, 86.95%), caecal tonsils (n=96, 77.08%), proventriculus (n=74, 60.81%), brain (n=92, 48.91%), intestines (n=73, 31.50%), kidney (n=52, 23.07%), and cloacal swab (n=235, 22.55%) by RT-PCR. Hence, the collection of specific tissue samples from field outbreaks for the detection of NDV is of paramount importance. In addition, RT-PCR can be considered a highly sensitive detection method for rapid and confirmative diagnosis of Newcastle disease virus.

Detection of Newcastle disease virus and H5-subtype Influenza virus in swiftlet houses by Multiplex reverse transcription PCR assay

The purpose of this study was to investigate the presence of Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), and Avian influenza type A, especially H5 subtype (AIV-H5) in swiftlet houses and swiftlet nest by multiplex RT-PCR (mRT-PCR). The assay used two specific primer pairs designed to detect the conserved sequence of the F gene of NDV and the HA gene of the AIV-H5, with product sizes of 282 bp and 420 bp, respectively. The mRT-PCR was established with detection limit of 25 copies/reaction for each target virus. The thermal cycle was optimized as follows: cDNA synthesis at 45ºC for 20 min, an initial denaturation at 95ºC for 5 min, followed by 35 cycles of amplification encompassing denaturation at 95ºC for 30 sec, annealing at 58ºC for 30 sec, and extension at 72ºC for 45 sec, ending by a final extension step at 72ºC for 7 min. Eighty-eight field samples including feces and swabs of the nest surface were examined and all samples were confirmed to be negative for these two viruses. The results of this study indicated that the swiftlet nests and the environment of swiftlet houses were not contaminated with NDV or AIV-H5 viruses. Moreover, the established mRT-PCR protocol had good specificity, detection limit, and can apply for routine veterinary diagnosis.

Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.

Isolation and Molecular Detection of Newcastle Disease Virus from Field Outbreaks in Chickens in Central Ethiopia.

BackgroundNewcastle disease is a major viral disease of poultry. The virus is a major problem for chickens in Ethiopia and there is a scarcity of updated information on the virological and molecular status of confirmation of Newcastle disease outbreak cases in the country.MethodsNewcastle disease outbreaks were investigated from February 2021 to October 2021 in central Ethiopia to isolate and detect the virus by cell culture and reverse transcriptase PCR. A total of 44 pooled tissue specimens were sampled from sick and recently dead chickens showing typical clinical signs of Newcastle disease. Virus isolation were performed using DF-1 cells and detection of the virus was done by real-time PCR.ResultsOut of 44 collected tissue samples, 38.63% (17/44) were positive on DF-1 cells. The result shows 17 of the clinically sick and dead chickens were positive for the virus by reverse transcriptase polymerase chain reaction. Based on the sample type, 54.54% (6/11) of the brain samples, 36.36% (4/11) of the intestines, 54.54% (6/11) of lung and trachea, 9% (1/11) of pooled liver, kidney, heart, and spleen samples were positive. Viruses were isolated in the proportions 37.5% (6/16), 25% (2/8), 50% (2/4), 25% (1/4), 50% (2/4) and 50% (4/8) from Sebeta, Bishoftu, Sululta, Nifas Silk, Kolfe and Yeka, respectively.ConclusionThis study showed that Newcastle disease is a major viral disease causing death of chickens in the study area. Therefore, any control approach should focus on the appropriate characterization of the virus strain causing the outbreak in the study area.

Usutu virus in blackbirds (Turdus merula) with clinical signs, a case study from northern Italy

Usutu virus (USUV) is a mosquito-borne virus belonging to the family Flaviviridae, genus Flavivirus. Natural transmission cycle of USUV involves mosquitoes and birds, so humans and other mammals are considered incidental hosts. In this study, USUV infection was diagnosed in all wild blackbirds, collected from July to September 2018 in a wildlife recovery center in the province of Bologna, in the Emilia-Romagna region, northern Italy. All blackbirds showed neurological clinical signs, such as overturning, pedaling, and incoordination. Moreover, the subjects died shortly after arriving at the hospitalization center. Virological investigations were performed by real-time PCR on frozen samples of the spleen, kidney, myocardium, and brain for the detection of Usutu (USUV) and West Nile (WNV) viruses. The small and large intestine were used as a matrix for the detection of Newcastle disease virus (NDV). All 56 subjects with neurological clinical signs were positive for USUV, only one subject (1.8%) tested positive for WNV, and no subject was positive for NDV. The most represented age class was class 1 J (58.9%), followed by class 3 (25.0%), and lastly from class 4 (16.1%). Most of the blackbirds before dying were in good (51.8%) and fair (39.3%) nutritional status, while only five subjects (8.9%) were cachectic. The USUV genomes detected in the blackbirds of this study fall within the sub-clade already called EU2 that has been detected since 2009 in the Emilia-Romagna region. Neurological clinical signs in USUV-affected blackbirds are still widely discussed and there are few works in the literature. Although our results require further studies, we believe them to be useful for understanding the clinical signs of Usutu virus in blackbirds, helping to increase the knowledge of this zoonotic agent in wild species and to understand its effect on the ecosystem. The goal of this study was to report—in the context of the regional passive surveillance program—the detection of USUV RNA in its most important amplifying host, the common blackbird, when showing clinical signs before death.

Detection of Newcastle disease virus and assessment of associated relative risk in backyard and commercial poultry in Kerala, India.

BackgroundNewcastle disease (ND) is an economically important viral disease affecting the poultry industry. In Kerala, a state in South India, incidences of ND in commercial and backyard poultry have been reported. But a systematic statewide study on the prevalence of the disease has not been carried out.ObjectivesA cross‐sectional survey was performed to detect the presence of Newcastle disease virus (NDV) in suspect cases and among apparently healthy commercial flocks and backyard poultry, in the state and to identify risk factors for NDV infection.MethodsReal‐time reverse transcription‐PCR (RT‐PCR) was used to detect the M gene of NDV in choanal swabs and tissue samples collected from live and dead birds, respectively and the results were statistically analysed.ResultsThe predominant clinical signs of the examined birds included mild respiratory signs, huddling together and greenish diarrhoea. Nervous signs in the form of torticollis were noticed in birds in some of the affected flocks. On necropsy, many birds had haemorrhages in the proventriculus and caecal tonsils which were suggestive of ND. Of the 2079 samples tested, 167 (8.0%) were positive for the NDV M‐gene by RT‐PCR. Among 893 samples collected from diseased flocks, 129 (14.5%), were positive for M gene with pairwise relative risk (RR) of 15.6 as compared to apparently healthy flocks where 6 out of 650 (0.9%) samples were positive. All positive samples were from poultry; none of the ducks, pigeons, turkey and wild birds were positive. Commercial broilers were at higher risk of infection than commercial layers (RR: 4.5) and backyard poultry (RR: 4.9). Similarly, birds reared under intensive housing conditions were at a higher risk of being infected as compared to those reared under semi‐intensive (RR: 6.7) or backyard housing (RR: 2.1). Multivariable analysis indicated that significantly higher risk of infection exists during migratory season and during ND outbreaks occurring nearby. Further, lower risk was observed with flock vaccination and backyard or semi‐intensive housing when compared to intensive housing. When the M gene positive samples were tested by RT‐PCR to determine whether the detected NDV were mesogenic/velogenic, 7 (4.2%) were positive.ConclusionsIn Kerala, NDV is endemic in poultry with birds reared commercially under intensive rearing systems being affected the most. The outcome of this study also provides a link between epidemiologic knowledge and the development of successful disease control measures. Statistical analysis suggests that wild bird migration season and presence of migratory birds influences the prevalence of the virus in the State. Further studies are needed to genotype and sub‐genotype the detected viruses and to generate baseline data on the prevalence of NDV strains, design better detection strategies, and determine patterns of NDV transmission across domestic poultry and wild bird populations in Kerala.

Comparison of novel recombinant nucleoprotein ELISA with commercial ELISA for Newcastle disease

In the present study, a novel ELISA method used recombinant nucleocapsid protein (rNP) as the coating agent. Recombinant Newcastle disease virus (NDV) protein was cloned and expressed in Escherichia coli. Though the rNP-ELISA results were consistent with commercial ELISA results for the NDV-negative sera samples, qualitatively and quantitatively variable (often reduced) results were obtained with NDV-positive sera. Although the rNP-ELISA results for NDV detection were inconclusive, further improvement and standardization of the rNP-ELISA approach, such as using multiple recombinant proteins as the ELISA coating agent and performing comprehensive statistical analyses of combined recombinant protein ELISA, should help counter Newcastle disease outbreaks by improving NDV detection.

Accurate detection of Newcastle disease virus using proximity-dependent DNA aptamer ligation assays.

Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid‐phase formats are widely used for high‐performance protein detection in medical research. However, the affinity reagents used, which are mainly poly‐ and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid‐phase proximity‐dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real‐time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid‐phase PLAs, which use NDV‐selective DNA aptamers, are more sensitive than the sandwich enzymatic‐linked aptamer assay (ELAA), and have a comparable sensitivity to real‐time reverse transcription PCR (rRT‐PCR) as the gold standard detection method. In addition, the solid‐phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper‐ and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT‐PCR. The specificity of PLA is shown to be concordant with rRT‐PCR.

Development of a reverse-transcription recombinase polymerase amplification assay with a lateral flow assay for rapid detection of avian orthoavulavirus 1.

Newcastle disease is an avian infectious disease caused by avian orthoavulavirus 1, also known as Newcastle disease virus (NDV). This disease has caused significant economic losses to the poultry industry worldwide. The rapid and simple detection of NDV infection is crucial to inform the appropriate control measures. We developed a reverse-transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow assay (LFA) for NDV detection. The RPA assay can be completed at 37°C within 20 min, and the RPA result can be visualized by the LFA within 5 min. The NDV RT-RPA-LFA detected NDV specifically with no cross-reactivity with other pathogens. The detection limit of NDV cDNA with our RT-RPA-LFA was 3.34 × 10-3 ng/μL. Consequently, the RT-RPA-LFA showed good potential for the detection of NDV infection in the field, especially in resource-limited settings.