Vaccination of chickens decreased Newcastle disease virus contamination in eggs

Newcastle disease is an important health issue of poultry causing major economic losses and inhibits trade worldwide. Vaccination is used as a control measure, but it is unknown whether vaccination will prevent virus contamination of eggs. In this study, hens were sham-vaccinated or received one or two doses of inactivated LaSota vaccine, followed three weeks later by virulent Newcastle disease virus (NDV) challenge. Eggs were collected daily and shell, albumen and yolk were subjected to virus isolation, as were oral and cloacal swabs at 2 and 4 days post-challenge (dpc). A second experiment evaluated the distribution of the virus in the reproductive tract of non-vaccinates. All vaccinated chickens survived challenge, and the levels of virus shed from cloacal swabs were decreased significantly when compared to shams. In non-vaccinated hens, virus was detected in the ovary and all segments of the oviduct. Yolk, albumen and eggshell surface from eggs laid at day 4 and 5 post-infection by sham-vaccinated hens were positive for NDV, but eggs from LaSota vaccinated hens lacked virus in internal egg components (i.e. yolk and albumen) and had reduction in the number of positive eggshell surfaces. These results indicate virulent NDV can replicate in the reproductive tract of hens and contaminate internal components of eggs and eggshell surface, but vaccination was able to prevent internal egg contamination, reducing eggshell surface contamination, and reducing shedding from digestive and respiratory tracts in virulent NDV challenged hens.     

Early occurrence of apoptosis in lymphoid tissues from chickens infected with strains of Newcastle disease virus of varying virulence

Newcastle disease virus (NDV), the causative agent of Newcastle disease, is a prevalent problem in the poultry industry and often the cause of severe economic loss. There are many strains of the virus and these have varying virulence. The most virulent strains cause systemic lesions of lymphoid tissues, with necrosis and severe lymphoid depletion. Less virulent strains do not cause as much necrosis, but may predispose to secondary infection with other pathogens. Apoptosis or programmed cell death, has been demonstrated to play a role in the pathogenesis of other paramyxovirus infections, notably those caused by measles and canine distemper viruses. To investigate the role of apoptosis in lymphoid organs during NDV infection, immunohistochemistry for determination of expression of caspase-3, a marker of imminent apoptosis, was performed on formalin-fixed paraffin wax-embedded tissues (spleen, thymus, caecal tonsils and bursa of Fabricius) from 4-week-old chickens infected with NDV strains of varying virulence 2 days previously. The amount of apoptosis was proportional to the severity of the clinical disease elicited by the strains.     

Immune responses of poultry to Newcastle disease virus

Newcastle disease (ND) remains a constant threat to poultry producers worldwide, in spite of the availability and global employment of ND vaccinations since the 1950s. Strains of Newcastle disease virus (NDV) belong to the order Mononegavirales, family Paramyxoviridae, and genus Avulavirus, are contained in one serotype and are also known as avian paramyxovirus serotype-1 (APMV-1). They are pleomorphic in shape and are single-stranded, non-segmented, negative sense RNA viruses. The virus has been reported to infect most orders of birds and thus has a wide host range. Isolates are characterized by virulence in chickens and the presence of basic amino acids at the fusion protein cleavage site. Low virulent NDV typically produce subclinical disease with some morbidity, whereas virulent isolates can result in rapid, high mortality of birds. Virulent NDV are listed pathogens that require immediate notification to the Office of International Epizootics and outbreaks typically result in trade embargos. Protection against NDV is through the use of vaccines generated with low virulent NDV strains. Immunity is derived from neutralizing antibodies formed against the viral hemagglutinin and fusion glycoproteins, which are responsible for attachment and spread of the virus. However, new techniques and technologies have also allowed for more in depth analysis of the innate and cell-mediated immunity of poultry to NDV. Gene profiling experiments have led to the discovery of novel host genes modulated immediately after infection. Differences in virus virulence alter host gene response patterns have been demonstrated. Furthermore, the timing and contributions of cell-mediated immune responses appear to decrease disease and transmission potential. In view of recent reports of vaccine failure from many countries on the ability of classical NDV vaccines to stop spread of disease, renewed interest in a more complete understanding of the global immune response of poultry to NDV will be critical to developing new control strategies and intervention programs for the future.     

Molecular epidemiological analysis of Newcastle disease virus isolated in China in 2005

Eighty-three strains of Newcastle disease virus (NDV) were obtained from outbreaks in chickens, pigeons, geese, and ducks in China in 2005 and characterized genotypically. The main functional region of the F gene (535 nucleotides) was amplified and sequenced. A phylogenetic tree based on nucleotides 47–435 of the F gene was created using sequences from 83 isolates and representative NDV sequences obtained from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to six genetic groups: I, II, III, VIb, VIIc, and VIId. All the isolates belonging to groups I and II (14 total) were lentogenic according to the amino acid sequences of the fusion protein cleavage site, and either V4 or LaSota-type, depending on the vaccines that were used. Most isolates (64 total) were classified in group VIId, a predominant genotype responsible for most Newcastle disease outbreaks since the end of the last century. One strain, NDV05-055, was in group VIIc, three pigeon strains were in group VIb, and one isolate, NDV05-041, was in group III, and characterized as a velogenic strain. This study revealed that genotype VIId was the major NDV strain responsible for the 2005 ND epizoonosis that occurred in China.     

Effect of host selective pressure on Newcastle disease virus virulence

Newcastle disease virus (NDV) causes an economically important disease that can vary from clinically inapparent to highly virulent forms. Generally, NDV strains isolated from wild birds are non-pathogenic for chicken. However, there are evidences supporting the fact that avirulent viruses maintained in feral birds could have caused outbreaks of virulent NDV in poultry. The strain-specific difference in virulence is determined by structural variations on the fusion glycoprotein (F). More basic amino acids are present in the F cleavage site of virulent strains. Nevertheless, other regions have been involved in virulence determination. When we subjected an avirulent NDV isolated from a wild bird to a host change we found that the virus arose was virulent for chicken. Nucleotide changes in the F protein cleavage site amino acid sequence and in the hemagglutinin-neuraminidase protein sequence are reported.     

Identification of a natural multi-recombinant of Newcastle disease virus

Newcastle disease (ND), caused by ND virus (NDV), is one of the most serious illnesses of birds, particularly chickens, and has been one of the major causes of economic losses in the poultry industry. Live vaccines are widely used to prevent chicken from NDV all over the world. Given the implications that recombination has for RNA virus evolution, it is clearly important to determine the extent to which recombination plays a role in NDV evolution. In this study, we performed the phylogenetic and recombination analysis on complete NDV genomes. A natural multi-recombinant cockatoo/Indonesia/14698/90 (AY562985) was identified. Its two minor parental-like strains might be from the NDV vaccine lineage and anhinga/U.S.(Fl)/44083/93 lineage, respectively. Our study suggests that recombination plays a role in NDV evolution. Especially, the study also suggests that live vaccines have capacity to play roles in shaping NDV evolution by homologous recombination with circulating virus.     

Monoclonal antibodies identify a strain-specific epitope on the HN glycoprotein of Newcastle disease virus strain Australia-Victoria

A panel of monoclonal antibodies raised against the hemagglutinin-neuraminidase glycoprotein (HN) of the Australia-Victoria strain of Newcastle disease virus has been used to compare that strain and eight other strains of the virus. The ability of the antibodies to neutralize infectivity, inhibit hemagglutination and neuraminidase, and bind to purified virions in solid-phase radioimmunoassays was determined for each strain. Of the four antigenic sites delineated by these antibodies on the HN of the homologous strain, site 1 (that with the greatest neutralizing susceptibility), is apparently conserved in all the strains tested as revealed by neutralization assays. The least neutralizing site, number 4, is also conserved in most of the strains tested. Site 2, which lies at or near the neuraminidase site, appears to be conserved in the avirulent strains but not in the virulent strains. An antibody to site 3 is unable to bind to a significant extent to any of the heterologous strains tested, and thus recognizes a strain-specific epitope. Inhibition of hemagglutination and neuraminidase by antibodies to each site were also examined and the results suggest that antibodies to sites 1 and 2 may distinguish virulent and avirulent strains at least with respect to these functions.     

Vitamin A dietary supplementation reduces the mortality of velogenic Newcastle disease significantly in cockerels

This project was undertaken to find ways of reducing mortalities and economic losses due to velogenic Newcastle disease (VND) in areas where the disease is enzootic. Four groups of cockerels of 44 birds each were used for this experiment. The birds in groups 1 and 2 received no dietary vitamin A supplementation, whereas groups 3 and 4 received 300 iu and 600 iu of vitamin A per kilogram of commercial feed, respectively, from 1 week of age till the end of the experiment. At 6 weeks of age, the birds in groups 2, 3 and 4 were inoculated intraocularly with a VND virus (duck/Nigeria/Plateau/Kuru/113/1991). The birds in Group 1 were given phosphate‐buffered saline intraocularly. Clinical signs appeared in Group 2 birds on day 3 PI and in groups 3 and 4 on day 5 PI. The clinical signs included a drop in feed and water consumption, depression, diarrhoea, torticollis and paralysis in all the infected groups. The average body weights of all groups were significantly different from one another on day 14 PI with Group 2 birds having the lowest body weight. Mortalities were highest in Group 2 birds (0%, 93.18%, 72.73% and 56.82% in groups 1, 2, 3 and 4 respectively). The antibody response in all the groups was significantly different from one another on days 14 and 21 PI. Group 2 birds had the lowest titres on those 2 days and showed more severe atrophy of the bursa, spleen, thymus and fibrin deposition in the spleen and thymus than the birds in groups 3 and 4. The above observations show that vitamin A dietary supplementation delayed the onset of clinical signs and significantly reduced body weight loss, atrophy of the bursa, spleen and thymus, and mortalities by 36%. It also significantly potentiated haemagglutination inhibition antibody response.     

Cytochalasin D accelerates the release of Newcastle disease virus from infected cells

The role of the cellular cytoskeleton in Newcastle disease virus (NDV) infection was explored in two ways. First, the extent of the association of viral proteins with the cytoskeletal fraction of chicken embryo cells was determined. NDV-infected cells, pulse-labelled with [35S]methionine with or without a subsequent chase, were fractionated into Triton X-100-soluble and cytoskeletal fractions. All NDV proteins become associated with the cytoskeletal fraction of cells subsequent to their synthesis. Mixing experiments provided evidence against nonspecific sticking of proteins with this cell fraction. Second, the functional significance of the cytoskeletal association was explored using the inhibitor cytochalasin D. In the presence of this inhibitor, the rate of release of radioactively labelled virions was accelerated 2.5-fold. Colchicine did not significantly alter the rate of virion release. Virus particles released from cytochalasin D-treated cells had the same density as virions released from untreated cells, but were slightly less infectious and contained less actin. These results suggest that functional microfilaments do not play an obligatory role in viral morphogenesis but rather function to slow virus particle release.     

Molecular characterization and phylogenetic study of velogenic Newcastle disease virus isolates in Iran

The pathogenicity and genetic characterizations of six Newcastle disease virus (NDV) isolates obtained from chicken farms in six different regions in Iran were carried out using conventional and molecular techniques. Based on the pathogenicity indices (MDT, ICPI, and IVPI), all of these isolates were found to be velogenic (highly virulent) strains. A sequence analysis of the full-length mRNA encoding the fusion glycoprotein precursor (F₀) of the NDV’s fusion proteins F₁ and F₂ in these six isolates showed the presence of point mutations in form of nucleic acid substitutions at positions 82^(C→T), 83^(T→C), 736^(A→G), and 1,633^(G→A). However, the nucleic acid residues at positions 330–347 of the precursor F₀ gene, corresponding to the cleavage site of the F₀ protein, were found to have remained conserved among the six NDV isolates. A phylogenetic comparison between the six Iranian isolates and the NDVs whose F₀ gene sequences were previously deposited in GenBank Database showed that all of the newly characterized Iranian NDV isolates belonged to genotype VII.     

Differentiation of Newcastle disease virus strains by one-dimensional peptide mapping

One-dimensional peptide mapping was used for the differentiation of Newcastle disease virus (NDV) strains. Virions were purified in one step, and digested with Staphylococcus aureus V8 protease or chymotrypsin without prior separation of their proteins. Peptides were separated by polyacrylamide gel electrophoresis and stained with Coomassie blue. This method proved to be a simple, economic and reproducible means of differentiating NDV strains.     

Immunosuppressive effects of arsenic in broiler chicks exposed to Newcastle disease virus

To assess the effects of prolonged exposure to arsenic (As, as arsenate) on host immune competence overall and resistance to Newcastle disease (ND) viral infection in particular, a study was carried out in broiler chicks. At 7 days of age, chicks were assigned to groups that would undergo varying vaccination, challenge, and/or As exposures; Group 1 was a control; Group 2 was to receive Newcastle disease virus (NDV) only; two groups (Groups 3, 4) were to be given As daily (50?mg/kg, by gavage) from Days 7–35 of the experiment. All groups underwent normal vaccination on Days 5, 23, and 32 against live NDV (B1 type, LaSota strain); two groups (Groups 2, 4) were challenged with field-isolated NDV at Day 24. At Days 14, 21, 28, and 35 of age, subsets of chicks in each group were evaluated. The results showed feed intake and weight gain were lower in As-treated and NDV-challenged chicks. In As-treated chicks, absolute and relative spleen weights were significantly greater, whereas those of the thymus significantly lower, over the entire 35-day period. Effects on bursa weights (absolute, relative) were only significantly reduced through Day 21. Antibody titers against ND were significantly reduced (vs. control) over the whole 35 days in birds that received As alone, but only significantly depressed through the first 21 days in birds that received As?+?NDV; thereafter, titers were significantly greater (in parallel with effects in birds that received NDV alone). In contrast, antibody responses to T-dependent antigen (Sheep red blood cells [SRBC]) were significantly lower in As only- and As?+?NDV-treated chicks throughout the study period. Among birds exposed to As (alone or with NDV), in situ phagocytic activity was elevated and cutaneous sensitivity responses decreased during the period from Day 28 to Day 35. NDV alone had spurious effects on phagocytic activity but did cause significant reductions in cutaneous sensitivity responses. It was concluded that arsenic decreased immunity in broiler chicks, thereby making them prone to ND.     

Herd immunity to Newcastle disease virus in broiler flocks in Israel

Due to the ongoing need to protect poultry from virulent Newcastle disease virus, all commercial poultry flocks in Israel are vaccinated according to a defined programme using a combination of live and inactivated vaccines. The vaccination protocol for broilers during the years of the study comprised a live vaccine administered by spray on the day of hatching, inactivated vaccine by subcutaneous injection at 10–12 days of age, and another live vaccine given by aerosol at 17–21 days of age. A cross-sectional study was designed in order to examine the influence of herd immunity on the risk of Newcastle disease outbreak in broiler flocks. The study was based on the extensive field data kept in the Poultry Health Laboratories database. The results of serology tests employing haemagglutination inhibition for Newcastle disease virus were analysed and crossed with the list of flocks that had been diagnosed with ND in the years 2007–2014. At the peak of induced immunization (fifth week of growth), 87.5% of the tested flocks had achieved herd immunity (≥85% of birds in the flock with an HI titre ≥4). Based on a logistic regression model, the odds ratio for ND in flocks without herd immunity was 3.7 (95% CI 1.8–7.3, P-value?相似文献   

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

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

新城疫病毒HN糖蛋白促细胞融合区域内保守氨基酸基因突变分析

目的 确定新城疫病毒(NDV)血凝素神经氨酸酶(HN)糖蛋白促细胞融合区域内的保守氨基酸功能,探讨HN糖蛋白的促细胞融合机制.方法 采用PCR定点突变与体内同源重组相结合的方法将HN糖蛋白促细胞融合区域内6个保守氨基酸突变为丙氨酸(A),在BHK21细胞内表达后,流式细胞仪定量分析蛋白的表达效率,并分别检测其促细胞融合活性、血细胞吸附能力(也称为受体识别活性)和神经氨酸酶活性.结果 L74A蛋白表达效率降低为72.7％,各突变株蛋白表达效率与野毒株相比差别无统计学意义(P＜0.05).各突变株蛋白促细胞融合活性都有不同程度的下降,其中I103A下降幅度最大,为野毒株的9.1％,各突变株蛋白血细胞吸附能力也都出现不同程度的降低,其中I1iA的下降最明显,为28.2％,各突变株蛋白的神经氨酸酶活性变化程度不同,其中174A略有升高,为118.6％,L110A下降幅度最大,为5.2％,I103A仅次于L1 10A,为5.7％.结论 新城疫病毒HN糖蛋白促细胞融合区域内的保守氨基酸在细胞融合中发挥着重要作用,第103位异亮氨酸(Ⅰ)是此区域内的关键氨基酸.     

Titration of avirulent Newcastle disease virus by the plaque assay method

Parameters of a plaque assay for avirulent strains of Newcastle disease virus (NDV) were optimized for reproducibility and optimum titer in LLC-MK₂ cells. Plaques were visible after 2 days and maximum virus titers were reached in 3 days. Virus titers were not affected by continued incubation through 6 days, although plaque size increased. An adsorption volume of 0.1 or 0.2 ml per 60 mm Petri dish was optimal, as was an adsorption time of 45 min. Trypsin (2.5 μg/ml) and magnesium sulfate (0.03 M) were essential requirements of the overlay medium and the presence of DEAE-dextran (0.02%) resulted in a 30% increase in titer. The use of cell monolayers, 1, 2, or 3 days old facilitated the performance of multiple assays per week and did not affect the virus titer.     

Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C_t) 18.19 ± 3.63 and a melting temperature (T_m) 86.0 ± 0.28 °C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C_t value 14.70 ± 2.32 and T_m 87.4 ± 0.21 °C. The assay had a dynamic detection range which spans over a 5 log₁₀ concentration range, 10⁹–10⁵ copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45 ± 0.31% and 1.30 ± 0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.