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Detection and differentiation of strains of Newcastle Disease Virus by complement fixation.

A complement-fixation test to detect Newcastle disease virus with antiserum produced in guinea pigs is described. Methodology is given for serum production and for standardization of the test. The test was used to differentiate 13 strains of Newcastle disease virus. Velogenic strains, including isolants form 1970-71 disease outbreaks in California, Florida, and Texas, were poor complement-fixing antigens, whereas lentogenic strains, including LaSota, Hitchner, and England F, were strong complement-fixing antigens. Mesogenic strains ranged from weak to strong in complement-fixing capabilities. This test can be used to differentiate velogenic field isolants from vaccine strains such as LaSota, Hitchner, and Roakin.

Animals↗

Characterization of newly emerging Newcastle disease virus isolates from the People's Republic of China and Taiwan.

Seven Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken and pigeon flocks in China and Taiwan between 1996 and 2000 were genotypically and pathotypically characterized. By phylogenetic analysis of the fusion protein genes, isolates Ch-A7/96, Ch/98-3, Ch/99, Ch/2000, and TW/2000 were placed into two novel subgenotypes, VIIc and VIId. Isolate Ch/98-1 was grouped into subgenotype VIb, while Ch-W6/96 was proven to be a mixture of isolates Ch-A7/96 and Ch/98-1. These isolates were pathotyped as viscerotropic velogenic for Ch/98-3, Ch/99, Ch/2000, and TW/2000; neurotropic velogenic for Ch-A7/96; and mesogenic for Ch/98-1. Three separate, comparative, genetic analyses of the F genes, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, were performed with our isolates and selected NDV strains from GenBank. Results showed that the close genetic similarity provided evidence for the epidemiological linkage between the outbreaks in China and Taiwan and that the 1990s outbreaks in Asia, the Middle East, Africa, and Europe constituted the fourth panzootic of ND. In combination with epidemiological analysis, an evolutionary model of the NDV strains, representative of the direction of transmission within the NDV strains, was proposed, and epidemiology of NDV transmission was evaluated with emphasis on molecular aspects. Finally, a cross-protective experiment indicated that at least one strain (Ch-A7/96) among our NDV isolates was an antigenic variant, responsible for recent outbreaks of ND in vaccinated chicken flocks.

Animals↗

Efficacy of live adjuvanted mesogenic Newcastle disease vaccine in chickens.

120 white leghorn chickens primed with a lentogenic Newcastle disease (ND) live vaccine at 7 days of age were divided into three equal groups of 8 weeks of age and vaccinated with a live mesogenic ND vaccine (NDV). One group received only Newcastle disease mesogenic vaccine (RDVK) in normal saline, the second group received RDVK with groundnut oil as adjuvant and the third group received RDVK with liquid paraffin as adjuvant. Sera were collected at different time points for the assessment of antibody level against ND virus (NDV) by the haemagglutination inhibition (HI) test. The commonly used non-adjuvanted RDVK could not evince 100% protective HI titre beyond 11 weeks of age but in both the adjuvanted groups 100% protective HI titre was evident up to 20 weeks of age. On challenge at 20 weeks of age both the adjuvanted groups withstood challenge but in the non-adjuvanted group 80% of chickens withstood the challenge. A significant difference in immune response between the adjuvanted and non-adjuvanted groups was seen but not between both the adjuvanted groups. The advantage of vegetable oil (groundnut oil) as an adjuvant for live mesogenic ND vaccine has been discussed.

Adjuvants, Immunologic↗

An outbreak of Newcastle disease in free-living pheasants (Phasianus colchicus).

The epidemiology of an outbreak of Newcastle disease in a population of approximately 12,000 free-living pheasants (Phasianus colchicus) on the island of Faenø in Denmark in 1996 is described. The mortality during the epizootic was 56%. The spread of the disease between 7 groups of pheasants could be demonstrated over an observation period of 3 weeks. A total of 70 avian paramyxovirus serotype 1 (APMV-1) isolates was made from the flock. The intra cerebral pathogenicity indices of the 4 isolates tested were in the range 1.78-1.88. By means of immunoperoxidase monolayer assay with murine monoclonal antibodies and sequence analysis of an RT-PCR amplified segment of the F0 viral protein it was found, that the virus belonged to the highly virulent C1 antigenic group and that the amino acid sequence at the F0 cleavage site corresponded with the sequences of virulent APMV-1 strains. Based on the epidemiological circumstances it is believed that the virus was transmitted to the pheasants by feral birds.

Amino Acid Sequence↗

A natural outbreak of Newcastle disease in guinea-fowl (Numida meleagris galeata) in Nigeria.

A natural outbreak of Newcastle disease (ND) was reported in a flock of guinea-fowl in Nigeria, affecting 1,029 birds of which 250 (24.3%) died. Paralysis of the legs and wings, coughing, sneezing, white diarrhoea and complete cessation of egg production were observed. Serum samples collected at the onset and during the course of the disease had high ND antibody titres. ND virus was isolated from a pool of brain tissues from diseased guinea-fowl. The ND virus isolate was characterised as a velogenic strain.

Animals↗

Validation study to evaluate the reproducibility of a candidate in vitro potency assay of newcastle disease vaccines and to establish the suitability of a candidate biological reference preparation.

A quantification assay for the Haemagglutinin-Neuraminidase (HN) protein of Newcastle Disease Virus (NDV) has been developed at CIDC-Lelystad as a candidate in vitro potency test for inactivated Newcastle disease (ND) vaccines. In studies performed at CIDC-Lelystad, a high correlation was demonstrated between the results of this candidate in vitro potency assay and the results of the serological potency assay (European Pharmacopoeia monograph 0870; test A). Furthermore, a high correlation between the serological data (Haemagglutination Inhibition-antibody titres) and clinical protection after challenge was demonstrated. Correlation between in vivo and in vitro potency assays was confirmed in a collaborative pre-validation study. In the pre-validation study three Official Medicines Control Laboratories (OMCLs) determined both the NDV-HN antigen content and the in vivo potency (vaccination-serology and vaccination-challenge) of 6 vaccine batches. The conclusion of the pre-validation study was that a large-scale collaborative study should be organised to validate the in vitro method and the suitability of the reference preparation. This report describes the outcome of this study. In brief, 14 laboratories (8 OMCLs and 6 vaccine manufacturers) determined the NDV-HN antigen content of 9 different vaccines in 3 independent tests. The vaccine batches were produced by 5 different manufacturers and represent a quantitative range of ND antigen content. One vaccine batch with insufficient potency and one poultry vaccine not containing NDV were included. Statistical evaluation of the results indicated that the antigen content could be determined with high precision. A good repeatability as well as reproducibility was found. Furthermore all laboratories found a similar ranking of the vaccines, based on the antigen content. Comparison of the antigen content and the in vivo potency of a series of vaccines with relatively low potencies indicated that a threshold relative antigen level of 7.0 antigen units per dose would discriminate between vaccine batches with sufficient and insufficient potency. An in vitro assay with this threshold level for antigen content did not result in any false positive results and only a limited number of false negative results in the BSP055 study. We conclude that the in vitro measurement of the antigen content of inactivated ND-vaccines with the proposed method is a reliable alternative potency assay that could be included as a new method in monograph 0870 on ND-vaccines.

Animals↗

[Experience in using cluster analysis and the prognosis of the stages of epizootic risk for Newcastle disease in poultry].

An attempt was made to use clastic analysis and projection of the steps of the epizootic hazard with regard to Newcastle disease. There were no records of the disease after 1982 but still it is the object of consideration on the part of specialists. A clastic analysis was made on the base of a factorial model of Newcastle disease in this country for the 1970-1979. Used was a programme already worked out and practised at the Pilot Computor Center of the Medical Academy, Sofia. The districts in the country were grouped in a dendogramme, 16 groups being distinguished out of a total of 27 districts. This showed that regardless of the small territory of the country the districts are sufficiently differing between each other (due to the various degrees of integration) so that they could not be grouped together by similar values of intensity of poultry breeding and epizootic conjuncture with regard to Newcastle disease. Districts of epizootic hazard ranging from 1st to 5th degree substantiate the use of a differential approach in building up the tactics of prophylaxis and control of the disease, while measures concerning the poultry dressing combines should be at a high level regardless of the category of the respective district. The grouping of neighboring districts on a territorial principle disclosed the existence of reasons of climatic-and-geographic nature that could predispose equal degrees of intensity of poultry breeding and epizootic hazard.

Animals↗

Standardization of a duplex RT-PCR for the detection of Influenza A and Newcastle disease viruses in migratory birds.

Influenza A and Newcastle disease viruses are pathogens of social and economical importance known to be disseminated throughout the world by migratory birds. Many efforts have been made to control the introduction of these viruses into new environments, and complete world surveillance has yet to be achieved. Virus isolation and immunofluorescence techniques are time consuming, have inherent limited sensitivity and present a lack of host cells permissive universally to all Influenza A viruses. In this paper, the use of a duplex RT-PCR is described capable of sorting out any NDV and Influenza A virus strain simultaneously in oral and cloacal swab specimens. This method includes fluorescent detection of amplicons that provide accurate analysis of many DNA fragments within one base discrimination. Reference viruses were used for standardization of the assay and samples from wild-type viruses were screened, with four positive results for Influenza A detected in migratory birds captured in the state of Sao Paulo. This screening test can be considered a first step for further studies of these viruses circulating in avian species in Brazil, and hopefully will contribute to broaden the sample spectrum from wild birds, leading to a better understanding of these viruses and their participation in the southeastern region of the country.

Animals↗

[Isolation, identification and typing of Newcastle disease virus isolates in a chick embryo cell culture].

Routine methods of investigation were employed in parallel experiments for the isolation (in chick embryos), identification (serologically via HI), and typing (MDT, ICPI, IVPI, and via cloacal tests) of local isolates and standard strains of the Newcastle disease virus (NDV). The results were the same as those obtained by the single-phase complex investigation after the plaque technique method in a monolayer cell culture of chick embryo fibroblasts under agar cover. The cultures were inoculated with tissue suspension centrifugate in multiple dilutions of trachea and lung, spleen and liver, and brain of experimentally infected birds, using local isolates (13 in all) and standard strains of NDV (6 of the various types) as well as of birds that died of Newcastle disease (3 cases). The complex cell culture technique of investigation included plaque-forming under agar (and under agarose as well) following the direct inoculation with the infectious tissue suspensions in the presence of a normal (known negative), resp., specific hyperimmune serum against Newcastle disease. It also included a plaque-neutralization (plaque reduction) test and tests for hemadsorption and elution of erythrocytes--all made use of in a single planned programme of investigation. This technique, which is a modification of the one suggested by Beard et al. (1970) and known as single-phase complex plaque technique, provides the simultaneous isolation and identification of NDV isolates, which may well be coupled with some of the routinely practiced methods for definitive isolate typing (e. g., with the cloacal test for velogenic, and with MDT for lento- and mesogenic isolates). Thus, time can be saved, and labour and means are reduced, i. e., the laboratory diagnostics of ND may be perfected.

Animals↗

Vaccination of day-old broiler chicks against Newcastle disease and infectious bursal disease using commercial live and/or inactivated vaccines.

Day-old broilers were administered live and/or inactivated vaccines to assess vaccine efficacy against challenge with Newcastle disease (ND) and infectious bursal disease (IBD). Chicks were from commercial breeder pullets vaccinated against ND and IBD using several live vaccine primers followed by an inactivated ND-IBD vaccine at 18 weeks. The most efficacious initial ND-IBD vaccination program was live ND virus by eye drop and live IBD vaccine injected subcutaneously (SQ) followed 2 hours later with inactivated ND-IBD vaccine SQ. The next two most efficacious programs were live vaccine alone and the inactivated vaccine only. Inactivated vaccine given SQ had no adverse effect on live IBD vaccine given 2 hours earlier in a similar site. Administration of inactivated vaccine by vent was not as efficacious as administration SQ. A booster of a second live ND-IBD vaccine drinking water at 18 days significantly increased levels of circulating antibody, regardless of the initial vaccination program.

Animals↗

Characterization of Newcastle disease virus isolates by reverse transcription PCR coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis.

Degenerate oligonucleotide primers were synthesized to amplify nucleotide sequences from portions of the fusion protein and matrix protein genes of Newcastle disease virus (NDV) genomic RNA that could be used diagnostically. These primers were used in a single-tube reverse transcription PCR of NDV genomic RNA coupled to direct nucleotide sequencing of the amplified product to characterize more than 30 NDV isolates. In agreement with previous reports, differences in the fusion protein cleavage sequence that correlated genotypically with virulence among various NDV pathotypes were detected. By using sequences generated from the matrix protein gene coding for the nuclear localization signal, lentogenic viruses were again grouped phylogenetically separate from other pathotypes. These techniques were applied to compare neurotropic velogenic viruses isolated from an outbreak of Newcastle disease in cormorants and turkeys. Cormorant NDV isolates and an NDV isolate from an infected turkey flock in North Dakota had the fusion protein cleavage sequence 109SRGRRQKRFVG119. The R-for-G substitution at position 110 may be unique for the cormorant-type isolates. Although the amino acid sequences from the fusion protein cleavage site were identical, nucleotide sequence data correlate the outbreak in turkeys to a cormorant virus isolate from Minnesota and not to a cormorant virus isolate from Michigan. On the basis of sequence information, the cormorant isolates are virulent viruses related to isolates of psittacine origin, possibly genotypically distinct from other velogenic NDV isolates. These techniques can be used reliably for Newcastle disease epidemiology and for prediction of pathotypes of NDV isolates without traditional live-bird inoculations.

Amino Acid Sequence↗

[Newcastle disease virus carrier state following the aerosol immunization of chickens against the disease using different doses of La Sota vaccine].

Six experiments were carried out with aerosol immunization, using various doses of the La Sota vaccine against Newcastle disease, and a control infection with massive amounts of the viscerotropic virus strain Pavlikeni to evaluate the duration of carrying the Newcastle disease virus. The aerosol vaccination of the birds was performed in laboratory conditions in an aerosol chamber of a flow-dynamic type of 450 l. The amount of the virus in the aerosol was determined through virologic investigations of samples taken by means of a glass capillary impinger. It was found that a short-term carrier state set in up to the 9th-10th day with the La Sota strain, regardless of the dose used and the age of the birds. The persistence of the velogenic virus in the organs of birds depended on the amount of vaccine used and the age of the birds. With 2-month-old chickens at optimal dosing of the vaccine resistance could be built up to the infection. When low doses of the vaccine were used with 10-day-old chicks as well as when over-dosing occurred the strain Pavlikeni could be recovered from the organs in the course of 3 to 7 days.

Aerosols↗

Response of congenitally immune chicks to viscerotropic velogenic Newcastle disease virus.

Separate groups of chicks of hen hyperimmune to viscerotropic velogenic Newcastle disease virus (VVNDV) were challenge-exposed to VVNDV by intraocular route at 1 day and 34 days old. Their response was evaluated by clinical symptoms, hemagglutination-inhibition (HI) titers, and virus isolations. Chicks exposed at 1 day old excreted VVNDV from the vent for up to 60 days; their active mean HI titers remained low (10-40); and deaths from VVNDV occurred early (5-16 days) and late (28-55 days). Chicks challenge-exposed at 34 days old excreted virus from the vent for 10 days; active HI titers developed quickly and remained high (891-1177); and deaths and signs of VVNDV occurred early (5-13 days).

Animals↗

[Precipitinogen activity of Newcastle disease virus and humoral immunity in birds].

Studies was the precipitinogenic activity of the strains La Sota, 'H', and 'II' of the Newcastle disease virus, using chick embryos. It was found that all three of the strains produce precipitinogens. An antigen was obtained for the precipitation reaction in agar gel and the demonstration of precipitins that were specific for the ND virus in the blood serum of birds. Contrary to the hemagglutinins and precipitinogens proved heatresistant. There was a correlation between the antihemagglutinins and the precipitinogens in the blood serum of the immune birds. The low percent of birds (up to 12%) having precipitins in their serum spoke of the development of solid immunity against Newcastle disease within the flock. On the other hand, the high percent of birds having precipitins (over 50%) indicated that the infection persisted, and because of the immunity present it fairly often assumed atypical course or was manifested with low mortality rate.

Animals↗

Induction of NO synthesis in macrophages by Newcastle disease virus is associated with activation of nuclear factor-kappa B.

Newcastle disease virus (NDV) has received much attention recently because of its non-specific immune stimulating potential and its various anti-tumor activities. Here we describe that NDV induces synthesis of NO and causes an activation of nuclear factor-kappa B (NF-kappa B) in murine macrophages. These reactions were part of an activation process which included also stimulation of adenosine deaminase and inhibition of 5'-nucleotidase. NDV-mediated NO synthesis and NF-kappa B activation were blocked by an antioxidant (butylated hydroxyanisole), by an inhibitor of protein tyrosine kinase (genistein) and of protein kinase A (H-89), but not by an inhibitor of protein kinase C (staurosporin). These data suggest that signalling requirements of NF-kappa B activation and NO production in NDV-treated macrophages are similar.

Adenosine Deaminase↗

[Immunisation of fancy chickens against Newcastle disease].

The German Regulation on Fowl plague which is in force since 1994 laid down that any chicken of all races and all hybrids must be vaccinated against Newcastle disease (ND) in a mode that an adequate immunity is achieved. Onset, duration, and resistance to challenge of immunity induced by vaccination is well documented in the scientific literature for hybrid chicken of the layer and meat types. These data prove also innocuity and efficacy of the registered vaccines. In contrast, only a few and incomplete data exist on the development of ND directed immunity in fancy chickens. The present study describes vaccinations of chickens of 14 different hobby breeds with live LaSota vaccine (conjunctival application of 10(6) embryo-infective dose50 per bird) and with an inactivated oil-emulsion vaccine (intramuscular application of 0.5 ml per bird) and subsequent intramuscular challenge infections using the highly virulent NDV strain Herts 33/66. Chickens of all 14 breeds tolerated the application of both vaccines. All fancy chickens reacted with the production of serum antibodies which were measured in the haemagglutination inhibition (HI) and virus neutralisation (VN) tests. According to the scientific literature, maximal antibody levels are reached in hybrid chickens between day 10 and 20 post vaccination. In contrast, in fancy chickens the antibody maxima are delayed to the seventh to eighth week post vaccination. All fancy chickens vaccinated either once with live LaSota virus or with live and inactivated vaccines resisted challenge with the highly virulent Herts 33/66 strain of NDV and did not develop any signs of disease. There are indications for gradual differences in susceptibility of different breeds of fancy chickens. The levels of non-specific neutralisation as measured in the virus neutralisation test differ between breed. Also, the viral content in tissues obtained from non-vaccinated but challenged birds differ markedly. It is concluded from the results of this study that fancy chickens can also successfully protected against Newcastle disease by using live and inactivated vaccines which are licensed for hybrid chickens. However, the optimal time for the detection of maximal antibody levels in fancy chickens is reached seven to eight weeks post vaccination.

Animals↗