Newcastle Disease Virus Isolates Research Articles

Antibody response to Newcastle disease virus recombinant fusion protein in post-vaccinated laying hens

This research was aimed to analyze antibody response in laying hens post vaccinated by Newcastle Disease Virus (NDV) recombinat Fusion (F) protein which has been succesfully expressed from the F gene of local isolates of NDV from Kulon Progo strain (0663/04/2013), Yogyakarta, Indonesia. The F gene cloned into expression vector plasmid pBT7-N-His. Two types of NDV recombinant vaccine, a concentrated and pure F recombinant protein were used for vaccination. A concentrated recombinant F protein was collected from the centrifugal ultrafiltration process and a pure recombinant F protein was collected from the electroeluted process. Recombinant F protein of NDV was successfully expressed, purified, and visualized by Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue staining with a molecular weight of 28 kDa. All two types of recombinant F vaccines and a commercial live vaccine as a positive control were injected two times at 14 and 18th weeks old laying hens to analyze the antibody response in serum. In comparison with a commercial live NDV vaccine, indirect Enzyme-linked Immunosorbent Assay (ELISA) revealed that antibody responses were high in both recombinant F protein vaccinated groups. In conclusion, the recombinant F protein has the potential to be developed as a recombinant vaccine candidate to obtain a higher antibody response in laying hens compared to commercially available live NDV vaccines.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

Genetic diversity of Newcastle disease viruses circulating in wild and synanthropic birds in Ukraine between 2006 and 2015.

Newcastle disease virus (NDV) infects a wide range of bird species worldwide and is of importance to the poultry industry. Although certain virus genotypes are clearly associated with wild bird species, the role of those species in the movement of viruses and the migratory routes they follow is still unclear. In this study, we performed a phylogenetic analysis of nineteen NDV sequences that were identified among 21,924 samples collected from wild and synanthropic birds from different regions of Ukraine from 2006 to 2015 and compared them with isolates from other continents. In synanthropic birds, NDV strains of genotype II, VI, VII, and XXI of class II were detected. The fusion gene sequences of these strains were similar to strains detected in birds from different geographical regions of Europe and Asia. However, it is noteworthy to mention the isolation of vaccine viruses from synanthropic birds, suggesting the possibility of their role in viral transmission from vaccinated poultry to wild birds, which may lead to the further spreading of vaccine viruses into other regions during wild bird migration. Moreover, here we present the first publicly available complete NDV F gene from a crow (genus Corvus). Additionally, our phylogenetic results indicated a possible connection of Ukrainian NDV isolates with genotype XXI strains circulating in Kazakhstan. Among strains from wild birds, NDVs of genotype 1 of class I and genotype I of class II were detected. The phylogenetic analysis highlighted the possible exchange of these NDV strains between wild waterfowl from the Azov-Black Sea region of Ukraine and waterfowl from different continents, including Europe, Asia, and Africa.

Efficient Production of Evolutionary Nanoparticle Newcastle Disease Virus

Viruses are routinely isolated from infected cells through freeze–thaw (F–T) cycles or sonication. The aim of this study was to compare different methods for efficient isolation of Newcastle disease virus (NDV) particles from BSR-T7/5 (BSR) cells. The BSR cells were infected with NDV LaSota strain, and the virus particles were isolated via F–T, sonication, sonication followed by F–T, and F–T followed by sonication. The infection and proliferation kinetics of the virus were analyzed by cytopathic observation and monitoring of hemagglutination (HA) titers. The virus isolated by sequential F–T and sonication was amplified through five passages of BSR cells, and then used to infect the HepG2 cells. The viability and apoptosis rates of the infected cells were evaluated by Cell Counting Kit-8 assay and Annexin V-FITC/PI staining respectively. We successfully obtained NDV particles from persistently infected BSR cells through all four methods, which indicated that the LaSota can effectively replicate in BSR cells. However, F–T followed by sonication was optimum in terms of separation effect. The virus particles isolated by this method still exhibited cytolytic activity against HepG2 cells. Thus, our novel method can be applied to NDV production and generation of tumor vaccines.

Identification of an additional N-glycosylation site and thermostable mutations within the hemagglutinin-neuraminidase gene of the Newcastle disease virus belonging to the VII.1.1 sub-genotype.

Newcastle disease virus (NDV) is considered one of the most devastating avian viral patho-gens affecting the avian population, and it causes a significant economic burden on the poultry industry worldwide. The study aimed to gain deeper understanding of the molecular and phylogenetic analyses of the complete hemagglutinin-neuraminidase (HN) coding region among NDV isolates. The samples were obtained from different parts of Iran from July 2017 to February 2020, were used for phylogenic analysis in this study. The results confirmed the predominance of sub-genotype VII.1.1, previously known as sub-genotype VIIL, which is circulating in commercial broiler farms of Iran. Identification of (a) an additional N-glycosylation site (NIS) at position 144; (b) mutations S315P and I369V which are related to increasing the viral thermostability; (C) cysteine residues at positions 123; (d) amino acid substitutions in the HN antigenic sites, especially the mutations I514V and E347Q, as well as the other mutant within HN binding sites of the VII.1.1 sub-genotype, suggests the idea that this new sub-genotype of NDV may possess a high level of pathogenicity and virulence compared to other NDV sub-genotypes. In conclusion, the results indicate the presence of an additional NIS at position 144, which may alter the virulence of the isolates. Furthermore, the presence of the thermostable mutations (S315P and I369V) and the other amino acid substitutions among the VII.1.1 sub-genotype isolates may have an impact on the vaccine immunity against this new NDV sub-genotype.

Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran.

Newcastle disease virus (NDV) sub-genotype VII.1.1 is the most common circulating NDV in Iran. In this study, a velogenic NDV isolate was plaque purified and then characterized according to Office International des Epizooties (OIE) standard protocols. The biological properties of the purified isolate named CH/RT40/IR/2011 were characterized using sequencing and phylogenetic analysis, measurement of pathogenicity indexes and challenge studies. The isolate was plaque purified on chicken embryo fibroblast cells for three rounds and then characterized using molecular and biological approaches. Phylogenetic and evolutionary distance analysis of fusion and hemagglutinin-neuraminidase genes classified the virus in sub-genotype VII.1.1. No mutation was observed in the glycosylation and neutralizing epitope sites of the fusion and hemagglutinin-neuraminidase proteins compared to other reported Iranian NDV VII.1.1 isolates. The presence of the 112RRQKRF117 motif in the fusion protein cleavage site together with mean death time, intracerebral pathogenicity index and intravenous pathogenicity index of 57 hr, 1.80 and 2.50 respectively, revealed that the RT40 isolate was a velogenic NDV. In the challenge study, all chickens were inoculated via eye drop, and intranasal route with RT40 isolate died within a week. While all chickens in the vaccinated and challenged group survived and showed no clinical signs. In conclusion, according to genetic analysis, pathotyping and challenge study, the RT40 isolate was similar to virulent NDVs in Iran and was a suitable candidate for a national standard challenge strain, vaccine trials and vaccine production in commercial levers.

The First Report of a Virulent Newcastle Disease Virus of Genotype VII.2 Causing Outbreaks in Chickens in Bangladesh.

Newcastle disease (ND) is endemic in poultry in Bangladesh. We performed genotypic and pathotypic characterization of four ND virus (NDV) isolates from recent outbreaks in broiler chickens in Bangladesh during the period of 2020-2021. Phylogenetic analysis based on the complete fusion protein gene coding sequences classified the viruses into NDV class II genotype VII.2 together with viruses from Indonesia isolated between 2014 and 2021 and a single 2020 Indian isolate. Pathogenicity testing using the intracerebral pathogenicity index in day-old chickens and mean embryo death time in embryonating chicken eggs revealed that the Bangladeshi isolates are velogenic. Inoculation of 35-day-old chickens with two NDV isolates (LT67 and N5) resulted in 100% morbidity by 3 days post inoculation (DPI), and all birds succumbed to infection by 7 DPI. Massive hemorrhages, congestion and necrotic lesions were observed in different visceral organs, which were typical for infection with a velogenic viscerotropic pathotype of NDV. At microscopic examination, tracheitis, severe pneumonia, focal proventriculitis, transmural enteritis, focal myocarditis, severe congestion and necrosis in kidneys, and lymphoid depletion in lymphoid tissues were found. Our study reports the first outbreak of the panzootic genotype VII.2 NDV in poultry in Bangladesh and documents a possible recent re-introduction of this NDV genotype from Southeast or East Asia. This study further provides viral distribution and epidemiological data that can facilitate the effective control of NDV.

Thermostability and Immunogenicity of Genotype II Avian Orthoavulavirus (AOaV-1) Isolates from Duck (Anas platyrhynchos) and Parrot (Eclectusroratus).

Newcastle disease (ND) is a highly contagious viral disease of poultry causing significant economic losses worldwide. Vaccination is considered the most reliable approach to curb the economic menace that is ND, but the thermolabile nature of Newcastle disease virus (NDV) vaccination poses a significant threat to its protective efficacy. This study aimed to profile the thermostability of NDV isolates from duck (As/Km/19/44) and parrot (As/WB/19/91) and evaluate their immunogenic potential in chicks. Fusion protein cleavage site (FPCS) and phylogenetic analysis demonstrated the lentogenic nature of both the isolates/strains and classified them as class II genotype II NDV. The characterized NDV isolates were adapted in specific-pathogen-free (SPF) chicks by serially passaging. Biological pathogenicity assessment of chicken-adapted As/Km/19/44 (PSD44C) and As/WB/19/91 (PSP91C) revealed both the isolates to be avirulent with a mean death time (MDT) of more than 90 h and an intracerebral pathogenicity index (ICPI) ranging from 0.2 to 0.4. Both of the NDV isolates displayed varied thermostability profiles. PSD44C was the most thermostable strain as compared to PSP91C and the commercially available LaSota vaccine strain. The immunogenicity of PSD44C and LaSota was significantly higher than PSP91C. Based on these results, it is concluded that NDV isolate PSD44C is more thermostable and immunogenic when administered intraocularly without any adverse effects. Therefore, PSD44C is suitable for further research and vaccine development.

Isolation and characterization of Newcastle disease virus from biological fluids using column chromatography.

Newcastle disease virus (NDV), belonging to the species avian orthoavulavirus 1, genus Orthoavulavirus, and family Paramyxoviridae, is responsible for Newcastle disease in poultry and other avian species. It has shown significant potential as an oncolytic virus and as a vector for vaccine delivery. NDV from infected biological serum is usually isolated or purified using density gradient ultracentrifugation. However, it has many disadvantages, including the fact that it is time consuming and can process only a limited quantity of sample at one time. In our study, native agarose gel electrophoresis and dynamic light scattering (DLS) analysis showed that NDV carried a net negative surface charge. Thus, we purified the virus using a HiTrap Q Sepharose Fast Flow anion exchange column with salt elution. Hemagglutination assay and plaque assay showed that the procedure yielded high-purity NDV particles with a recovery of more than 80%, and the process was fast and simple. The purity of the virus was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The hydrodynamic volume and 'dry state' diameter of the purified NDV were analyzed using dynamic light scattering and transmission electron microscopy and were to be in the range of 200-300 nm. The viruses did not exhibit any deviation from their known physical properties. The genome of the virus was also detected by amplifying a 423-bp region using reverse transcription-polymerase chain reaction. Our study confirmed that NDV could be effectively purified using an anion exchange column. In addition, the procedure could be easily upscaled or downscaled based on the experimental requirements.

Newcastle Disease Genotype VII Prevalence in Poultry and Wild Birds in Egypt.

Newcastle Disease Virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to observe the prevalence and genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and pelicans, were collected from different geographic regions within 13 governorates during 2019–2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing haemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to the F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV was isolated in 12 governorates in domestic birds, migratory, and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70–99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, with phylogenetic analysis revealing that the strains belonged to class II within genotype VII.1.1. The genetically similar yet geographically distinct virulent NDV isolates in poultry and a wild bird may allude to an external role contributing to the dissemination of NDV in poultry populations across Egypt. One such contribution may be the migratory behaviour of wild birds; however further investigation must be implemented to support the findings of this study. Additionally, continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV dissemination and genetic diversification across Egypt, with the aim of controlling the disease and protecting poultry production.

In vitro investigation of the antiviral activity of propolis and chitosan nanoparticles against the genotype VII Newcastle disease virus.

The Newcastle disease virus (NDV) is considered a serious threat to global poultry production. Despite the availability of vaccines, it remains a major devastating epidemic responsible for great economic losses. The development of novel virus-controlling strategies is therefore an urgent need. The present study investigated for the first time the antiviral efficacy of propolis and chitosan nanoparticles against two NDV isolates, MW881875 and MW881876, recovered from vaccinated commercial broiler farms in KafrEl Sheikh Governorate, Egypt. The polygenetic analysis focused on the F and M genes, with one isolate having a 97% identity with the genotype VII NDV Israeli strain. On the other hand, the identified isolates showed high genetic variation and only 76% identity with the LaSota vaccine (genotype II). More interestingly, the cell cytotoxic concentrations of chitosan, propolis, and a propolis–chitosan mixture against Vero cells were 327.41 ± 12.63, 109.48 ± 8.36, and 231.78 ± 11.46 μg/ml, respectively. The median tissue culture infectious dose (TCID50) assay demonstrated that the nanoparticles have antiviral effects after NDV exposure resulting in significant decrease in viral titer (TCID50) by 2, 2.66, and 2.5 log10 at 62 μg/ml of chitosan, 13 μg/ml of propolis, and 30 μg/ml of the propolis–chitosan mixture, respectively, compared with the control TCID50 value of 4 log10. Taken together, the results provide novel insights into the potentially promising roles of propolis and chitosan as novel, safe, and effective antiviral agents against NDV.

Active Surveillance and Genetic Characterization of Prevalent Velogenic Newcastle Disease and Highly Pathogenic Avian Influenza H5N8 Viruses Among Migratory Wild Birds in Southern Egypt During 2015-2018.

A total of 1007 samples (910 fecal droplets and 97 cloacal swabs) were collected from 14 species of migratory wild birds in most wetlands during 3 successive migration seasons from September to March (2015-2018) in Southern Egypt. The samples were propagated inembryonated chicken eggs and positive allantoic fluids by hemagglutination test were tested for Newcastle disease virus (NDV) and avian influenza virus (AIV) prevalence using RT-PCR and specific primers targeting the NDV fusion (F) and AIV matrix genes. Further subtyping of the AIV hemagglutinin (HA) and neuraminidase (NA) was conducted, and representative isolates were selected and sequenced for full F gene of NDVs and HA and NA genes of the AIV. Overall isolation rate of hemagglutinating viruses was 5.56% (56/1007), from them 5.36% (3/56) AIV, 85.71% (48/56) NDV and 8.93% (5/56) co-infection of NDV and AIV was detected. The sequences analysis of full F genes of 10 NDV isolates revealed that they have multi-basic amino acid motifs 111E/GRRQKR/F117 as velogenic strains with nucleotides and amino acids similarities of 96-100%. In addition, they phylogenetically clustered into groups and subgroups within genotype VII.1.1 and sub-genotype VIIj with a close relation to NDVs isolated from chickens in Egypt. The AIVH5N8 subtype was in clade 2.3.4.4b with a highly pathogenic nature and close relation to Egyptian domesticated H5N8 viruses rather than those from wild birds. The current data showed the contribution of migratory birds to the continuous circulation of virulent NDV and AIVH5N8 among domesticated chickens in Southern Egypt.

A study of risk factors associated with Newcastle disease and molecular characterization of genotype XIII Newcastle disease virus in backyard and commercial poultry in Assam, India

The continuous emergence of Newcastle disease virus (NDV) poses a persistent threat to the poultry industry. Recent increasing outbreaks of NDV in the North East region of India have highlighted the need to closely monitor and analyze the potential risk factors for Newcastle disease (ND) outbreaks. In the present study, an attempt was made to genotype the circulating Newcastle disease virus (NDV) in the backyard and commercial poultry flocks in Assam, India. Sera samples from unvaccinated backyard poultry flocks and tissue samples of ND suspected cases were collected and tested for the presence of NDV antibodies using the Haemagglutination inhibition (HI) test. A total of seven NDV isolates were analyzed from different districts of Assam, India, both genotypically and pathotypically. All isolates were characterized as virulent, carrying 112RRKQRF117 amino acid residues at the cleavage site. As determined by phylogenetic analysis, the isolates clustered with members of genotype XIII of class II NDV. Further analysis of risk factors of ND occurrence was conducted through a questionnaire survey. All the results indicated an occurrence of genotype XIII of NDV in the farms with inadequate biosecurity and farming practices.

NDV as an Oncolytic Agent - Study in Cancer Cell Lines

Newcastle disease virus (NDV) exhibits oncolysis in its natural form. This oncolytic virus (OV) has the potential to specifically infect, propagate, and lyse cancer cells while sparing the normal cells. This study was aimed to screen for oncolytic NDV strain isolated from poultry. A total of ten velogenic NDV strains were propagated in 10 day old embryonated SPF chicken eggs and allantoic fluid of these infected eggs was collected for further study. The virus enumeration was carried out by hemagglutination assay (HA) and end point dilution method. The cytopathic effect of ten NDV strains on cancer cell lines like MDA-MB-231, MCF-7, PC3, and A549 along with normal control cell line HEK293 was determined by MTT assay 72 hours post infection. These cell lines were infected with three doses (1, 0.1, and 0.01 MOI). DNA laddering effect of the screened NDV isolate was studied after infecting all cancer and normal cells at MOI 1. Morphological changes in MDA-MB-231 on infection with the screened NDV isolate were analyzed using H&E hematoxylin and eosin staining. The screened NDV isolate showed the maximum cytopathic effect i.e. 61.55% on MDA-MB-231 at MOI 1 but had no potent cytotoxic effect on HEK293. DNA laddering effect was observed which confirmed the mode of death to be apoptosis. All the observed morphological changes in MDA-MB-231 were typical of the cytopathogenic effects of NDV on cancer cell lines. In conclusion, the screened oncolytic NDV shows effective oncolysis against MDA-MB-231 cell line. However, further study is required to determine the exact mode of action involved.

Isolation, Identification, and Molecular Characterization of Newcastle Disease Virus from Field Outbreaks in Chickens in Afghanistan.

Newcastle disease viruses (NDVs) in Afghanistan were isolated from three chicken farms and identified using a hemagglutination test and reverse transcription-polymerase chain reaction assay. Three isolates from each farm were sequenced to characterize the part of their fusion protein gene around the cleavage site. The characteristics of the fusion protein genes of the three isolates shown by phylogenic analysis indicated that the isolates were velogenic, belonged to the class II subgenotype VII 1.1, and were closely related to an identified Chinese NDV isolate. To our knowledge, this is the first time that NDV isolates from Afghanistan have been partially sequenced.

The effect of the challenge with Newcastle disease virus on the gastrointestinal bacterial population in Japanese quail (Coturnix japonica)

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The effect of a challenge with Newcastle disease virus on bacterial population in quail is poorly documented, so for this purpose, a total of 100 day-old Japanese quail were purchased and divided into 2 equal groups randomly. Each group was divided into 2 subgroups of 25 quails. The birds in group A challenged with a velogenic chicken isolate of Newcastle disease virus. The birds in group B did not challenge with Newcastle disease virus as the control group. For the determination of lactobacillus counts in the intestine and crop of Japanese quail, at the end of the period, 10 birds of each subgroup (20 birds of each treatment) were chosen randomly. One gram of the crop and ileocecal content were taken and cultured on MRS for determination of lactobacillus counts. The colony-forming units of Escherichia coli in digesta of ileocecal on Mac Conkey agar were investigated. Also, crop and ileocecal content of dead birds were taken and cultured on MRS and Mac Conkey agar. The results of this study showed that challenge with a velogenic chicken isolate of Newcastle disease virus could increase colony-forming units of Escherichia coli in group A compared to the control group. Also, it reduced lactobacillus counts of intestine and crop compared to the control group. So it concluded that velogenic chicken isolate of Newcastle disease virus influences microflora of intestine and crop of Japanese quail.

Intense Innate Immune Responses and Severe Metabolic Disorders in Chicken Embryonic Visceral Tissues Caused by Infection with Highly Virulent Newcastle Disease Virus Compared to the Avirulent Virus: A Bioinformatics Analysis.

The highly virulent Newcastle disease virus (NDV) isolates typically result in severe systemic pathological changes and high mortality in Newcastle disease (ND) illness, whereas avirulent or low-virulence NDV strains can cause subclinical disease with no morbidity and even asymptomatic infections in birds. However, understanding the host’s innate immune responses to infection with either a highly virulent strain or an avirulent strain, and how this response may contribute to severe pathological damages and even mortality upon infection with the highly virulent strain, remain limited. Therefore, the differences in epigenetic and pathogenesis mechanisms between the highly virulent and avirulent strains were explored, by transcriptional profiling of chicken embryonic visceral tissues (CEVT), infected with either the highly virulent NA-1 strain or the avirulent vaccine LaSota strain using RNA-seq. In our current paper, severe systemic pathological changes and high mortality were only observed in chicken embryos infected with the highly virulent NA-1 strains, although the propagation of viruses exhibited no differences between NA-1 and LaSota. Furthermore, virulent NA-1 infection caused intense innate immune responses and severe metabolic disorders in chicken EVT at 36 h post-infection (hpi), instead of 24 hpi, based on the bioinformatics analysis results for the differentially expressed genes (DEGs) between NA-1 and LaSota groups. Notably, an acute hyperinflammatory response, characterized by upregulated inflammatory cytokines, an uncontrolled host immune defense with dysregulated innate immune response-related signaling pathways, as well as severe metabolic disorders with the reorganization of host–cell metabolism were involved in the host defense response to the CEVT infected with the highly virulent NA-1 strain compared to the avirulent vaccine LaSota strain. Taken together, these results indicate that not only the host’s uncontrolled immune response itself, but also the metabolic disorders with viruses hijacking host cell metabolism, may contribute to the pathogenesis of the highly virulent strain in ovo.

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.

Molecular techniques are better choice as compared to conventional methods for pathotypic characterization of Newcastle disease virus in poultry birds

Newcastle disease (ND) has importance in the poultry industry across the world. Despite vaccination, outbreaks of ND have been reported from various parts of Pakistan. The purpose of current study was to characterize the Newcastle disease virus (NDV) through different methods i.e., molecular techniques and conventional methods and evaluate their efficiency. To isolate pathogenic NDV, samples were taken from the commercial poultry farms after an outbreak, while for the non-pathogenic NDV isolation commercially available Lasota vaccine was used. In the first step NDV was tested by F gene sequence through RT-PCR. The predicted sequence of the amino acid in fusion protein indicates the following motif 112R-R-Q-R-R-F117 in pathogenic and 112G-R-Q-G-R-L117 in non-pathogenic NDV strains. While in the second phase, the pathotypic characterization of NDV was done through conventional methods like Intracerebral pathogenicity index (ICPI) and Mean death time (MDT). The results from conventional methods were also in agreement with standard values. The findings of current study suggest that for the assessment of genetic nature and the rapid diagnosis of NDV; molecular techniques are better choice than conventional methods.

Efficacy of genotype-matched Newcastle disease virus vaccine formulated in carboxymethyl sago starch acid hydrogel in chickens vaccinated via different routes.

BackgroundThe commercially available Newcastle disease (ND) vaccines were developed based on Newcastle disease virus (NDV) isolates genetically divergent from field strains that can only prevent clinical disease, not shedding of virulent heterologous virus, highlighting the need to develop genotype-matched vaccinesObjectivesThis study examined the efficacy of the NDV genotype-matched vaccine, mIBS025 strain formulated in standard vaccine stabilizer, and in carboxymethyl sago starch-acid hydrogel (CMSS-AH) following vaccination via an eye drop (ED) and drinking water (DW).MethodsA challenge virus was prepared from a recent NDV isolated from ND vaccinated flock. Groups of specific-pathogen-free chickens were vaccinated with mIBS025 vaccine strain prepared in a standard vaccine stabilizer and CMSS-AH via ED and DW and then challenged with the UPM/NDV/IBS362/2016 strain.ResultsChickens vaccinated with CMSS-AH mIBS025 ED (group 2) developed the earliest and highest Hemagglutination Inhibition (HI) NDV antibody titer (8log2) followed by standard mIBS025 ED (group 3) (7log2) both conferred complete protection and drastically reduced virus shedding. By contrast, chickens vaccinated with standard mIBS025 DW (group 5) and CMSS-AH mIBS025 DW (group 4) developed low HI NDV antibody titers of 4log2 and 3log2, respectively, which correspondingly conferred only 50% and 60% protection and continuously shed the virulent virus via the oropharyngeal and cloacal routes until the end of the study at 14 dpc.ConclusionsThe efficacy of mIBS025 vaccines prepared in a standard vaccine stabilizer or CMSS-AH was affected by the vaccination routes. The groups vaccinated via ED had better protective immunity than those vaccinated via DW.