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Estimation of antibodies to Newcastle disease virus in tears.

Micro-haemagglutination inhibition tests (Micro-HI) were used to measure the level of maternal IgG in the tears of chicks and also to measure the levels of HI antibodies in the tears and serum after vaccination with "F" strain of Newcastle disease virus (NDV) and in the face of an outbreak of Newcastle disease. There was a 1.4 fold difference between the maternal IgG concentration in the serum and tears. The ratio of serum IgG to lachrymal IgG after maternal transfer was 4 to 5:1 on day 4 to 9 and decreased to 2.6:1 on day 12 post-hatch. The intra-ocular vaccination of chicks with "F" strain of NDV resulted in the highest titre of HI antibodies in the tears though there was no significant difference in the response of chicks vaccinated through intranasal, oral and intravenous routes. In the face of an ND outbreak, the level of HI antibodies in the tears during the acute phase was very high and persisted at the same level for 14 days.

Animals↗

Vaccination of chicken embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response.

To reduce the embryonic pathogenicity of Newcastle disease virus (NDV), escape mutants of the La Sota strain were produced with selected monoclonal antibodies. Immunoselection resulted in the elimination of an epitope by single amino acid substitution (F and HN molecule) or in a conformational change (HN molecule). The embryonic pathogenicity of these escape mutants was reduced and their dose was optimised for in ovo vaccination. Because antibody responses and protection of in ovo vaccinated chicks were similar to controls vaccinated at hatch with the La Sota strain, immunoselection appears a valuable technique to produce attenuated NDV strains, which are candidate in ovo vaccines.

Animals↗

Evaluation of a novel rapid kit for the visual detection of Newcastle disease virus antibodies.

A sensitive diagnostic kit for the rapid visual detection of Newcastle disease virus antibodies is described. The kit--ImmunoComb--is based on a modification of the enzymelinked immunosorbent assay and was found to be highly sensitive for detecting local immunity as well as marginal levels of humoral immunity. Therefore, it determines status of immunity and vaccine efficiency. The assay is simple and does not require special equipment or laboratory facilities. This endows the new kit with field applicability. The effectiveness and reliability of the kit were proven by a series of field experiments. Tracheal swabs and blood samples were taken from 20 flocks (chickens and turkeys) and tested by the hemagglutination-inhibition test and by the new kit. There was high correlation between the methods where the level of antibodies in the flocks ranged between negative and very high.

Animals↗

Potency test of inactivated Newcastle disease vaccines by monoclonal antibody blocking ELISA.

The potency of 27 different inactivated Newcastle disease (ND) vaccines was determined by immunization and challenge tests. Three- and four-week-old SPF chickens were immunized with 1/50 dose of vaccines. The chickens were bled and challenged three weeks later. The protected/challenged ratio was determined. Serum samples were tested by the haemagglutination inhibition (HI) test and by a monoclonal antibody (MAB) blocking enzyme-linked immunosorbent assay (B-ELISA). Ninety-four percent of the vaccinated chickens (353 birds) with a B-ELISA value above 60% were protected. A strong positive correlation (0.934) was found between 'actual protection' (protection against challenge) and 'estimated protection' (protection calculated from the B-ELISA results). Based upon the results obtained, this B-ELISA seems to be suitable for replacing the challenge test in the potency control of inactivated ND vaccines in the future.

Animals↗

Virus characterization and sequence of the fusion protein gene cleavage site of recent Newcastle disease virus field isolates from the southeastern United States and Puerto Rico.

Nine Newcastle disease virus (NDV) isolates obtained from Puerto Rico, Georgia, Alabama, Mississippi, and Texas were analyzed for in vivo pathogenicity, biological properties (hemagglutination of mammalian erythrocytes), and for sequence variation at the amino acid and sense RNA level of the fusion protein cleavage site. Intracerebral pathogenicity index values ranged from 0 to 0.3 and the intravenous pathogenicity index obtained for all isolates was 0. Four isolates hemagglutinated bovine erythrocytes, whereas no hemagglutination was observed using equine erythrocytes. By direct sequencing of reverse transcription polymerase chain reaction products, all the isolates had a predicted fusion cleavage sequence comparable to lentogenic NDV strains. Based on nucleotide sequence, the viruses could be grouped phylogenetically with the B1 vaccine-type virus. However, nucleotide sequences were not 100% similar to the B1 or La Sota NDV strains, indicating that minor genetic heterogeneity occurs among lentogenic field isolates of NDV.

Amino Acid Sequence↗

Local immunity in Newcastle disease: some recent experiments.

Following primary exposure by the ocular route to lentogenic Newcastle disease virus (NDV) the lacrymal fluid, saliva and tracheal washes of three-week old specific pathogen free chickens acquired specific virus-neutralizing activity which considerably exceeded transudation of circulating antibody. All three secretions contain IgA which, at least in saliva, accounted for 85% of its activity, the remanider being due to IgG. Antibody in secretions limited, but did not prevent, reinfection of the trachea when birds were challenged two weeks later. In contrast to an elevation of circulating antibody titre, challenge induced only a repeated primary response in secreted antibody. Ocular infection induced marked lymphoid and plasma cell activity in the Harderian gland which is a major source of specific antibody in lachrymal and possibly other fluids. Functional ablation of the gland can be effected by occlusion of its draining duct, providing a means for evaluation of its immunological significance.

Animals↗

Characterization of Newcastle disease viruses isolated from migratory waterfowl in the Atlantic flyway.

Four isolants of Newcastle disease virus (NDV) obtained from free-flying Canada geese in the Atlantic flyway were characterized and compared with the B1, LaSota, Roakin, and Texas-GB strains of NDV. The 4 isolants were identified as lentogenic strains on the basis of embryo mean death times of greater than 100 hours and relative lack of pathogenicity for 1-day-old and 3-week-old chickens. The hemagglutinin of 2 of the isolants was stable at 56 C for 15 minutes, 1 for 30 minutes, and the other for at least 2 hours. When held at 4 C for 24 hours, none of the 4 isolants could be eluted from chicken erythrocytes. All 4 of the isolants produced plaques in chicken embryo fibroblast cell cultures without additives, whereas the B1 and LaSota strains did not. The 4 lentogenic NDV isolants from Canada geese differed in many respects from currently used commerical vaccine strains (B1 and LaSota).

Animals↗

Cell-mediated immunity in chickens vaccinated with the V4 strain of Newcastle disease virus.

A leukocyte migration inhibition assay was used to demonstrate antigen-specific cell-mediated immunity (CMI) in chickens vaccinated with the V4 strain of Newcastle disease virus. Chickens were vaccinated when 5 weeks old, and again 3 and 7 weeks later. CMI was detected 9 days after initial vaccination by eyedrop, but only after the second dose of vaccine delivered into the crop. In some chickens there was a temporary suppression of CMI to Newcastle disease virus, especially after the initial application of vaccine to the crop. Haemagglutination inhibition antibodies developed to similar levels in chickens after vaccination by either route. There was no obvious correlation between antibody and CMI responsiveness.

Animals↗

Identification and subgrouping of pigeon type Newcastle disease virus strains by restriction enzyme cleavage site analysis.

A host variant of Newcastle disease virus (NDV, genus Avulavirus, family Paramyxoviridae) is responsible for an autonomous disease in pigeons. It emerged in the late 1970s in the Mediterranean region. Despite great genetic diversity the vast majority of strains belong to a monophyletic group (sublineage VIb) within genotype VI of NDV strains that were indigenous in the region at that time. To date only a monoclonal antibody assay is available for the specific identification of pigeon type strains. A specific genetic assay is described suitable for the identification of pigeon isolates. Cleavage site analysis of a 1349 bp amplicon of the fusion protein gene was carried out using restriction enzymes (RE) HinfI, BstOI and RsaI. RE analysis of over 100 strains isolated between 1978 and 2002 deriving from 16 countries has revealed nine RE-patterns, which were progressive site variants of the parental (group VI) genotype. In spite of substantial site variation, extant pigeon viruses lacked a BstOI cleavage site at nucleotide 1601 shared by other NDV strains of chicken origin. RE analysis is a simple and reliable method both for the identification and subgrouping of pigeon type viruses.

Animals↗

Generation of velogenic Newcastle disease viruses from a nonpathogenic waterfowl isolate by passaging in chickens.

A benign Newcastle disease virus (NDV) recently became highly virulent during replication in domestic chickens. It is still unclear whether NDVs circulating among wild waterfowl also have the potential to become highly pathogenic (velogenic) in chickens. To demonstrate experimentally the generation of velogenic NDV from a nonpathogenic waterfowl isolate, we passaged an avirulent goose isolate in chickens. After nine consecutive passages by air-sac inoculation, followed by five passages in chick brain, the virus became highly virulent in chickens, producing a 100% mortality rate, and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence, E-R-Q-E-R/L, which progressed incrementally to a typical virulent type, K-R-Q-K-R/F, during repeated passage in chickens. These results demonstrate that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, have the potential to become velogenic after transmission to and circulation in chicken populations. The results also suggest that chickens provide a mechanism for the selection of virulent viruses from an avirulent background.

Animals↗

Nonspecific innate immunity against Escherichia coli infection in chickens induced by vaccine strains of Newcastle disease virus.

The objective was to test the hypothesis that vaccine strains of Newcastle disease virus (NDV) induce nonspecific immunity against subsequent infection with Escherichia coli. White leghorn chickens at 5 wk of age were vaccinated with a NDV vaccine at various days before challenge exposure with O1:K1 strain of E. coli via an intra-air sac route. Immunity was determined on the basis of the viable number of E. coli in the spleen 24 hr after the infection. Roakin strain induced significant (P < 0.05) immunity against E. coli at 4, 6, and 8 days, and La Sota strain at 2, 4, and 8 days, postvaccination. Secondary NDV vaccination administered 14 days later failed to induce immunity against E. coli when chickens were infected 1 or 5 days after the vaccination. Significant (P < 0.05) suppression of this nonspecific immunity was observed in birds treated with corticosterone, 40 mg/kg in feed, given for three consecutive days immediately prior to the bacterial exposure but not in those treated prior to the period. The results indicate that innate immunity induced by the primary NDV vaccination may significantly suppress the multiplication of E. coli in chickens for a period of 2-8 days postvaccination. The NDV-induced immunity was inhibited by corticosterone, which is known to mediate physiological responses to stress.

Animals↗

Immunogenicity of a locally produced Newcastle disease I-2 thermostable vaccine in chickens in Uganda.

A locally-produced Newcastle disease (ND) I-2 thermostable vaccine of embryo-infective dose (EID50) 10(8.5) per ml was administered to 100 laboratory chickens in four test groups, each of 25 birds. It was given by the eye-drop method, in drinking water, in drinking water freshly medicated with levamisole, or using millet grains as a vaccine carrier. A fifth control group consisting of 25 birds received the heat-sensitive La Sota vaccine (EID50 10(9) per ml) by the eye-drop method. The immunological responses were monitored by the enzyme-linked immunosorbent assay (ELISA) ND antibody technique using serum samples collected from 18 birds in each group at 3-week intervals for 3 months. The overall mean ND antibody log(10) titres and percentage positivities were 3.1, 88%; 2.9, 70%; 3.0, 83%; 3.2, 87% and 3.3, 87%, respectively. The use of water alone or medicated with levamisole for vaccine administration produced significantly lower ND antibody titres only in the first 3 weeks. The immunogenicity shown by the I-2 vaccine as a potential vaccine is discussed in relation to free-range poultry management conditions in Uganda.

Adjuvants, Immunologic↗

Aerosol vaccination of chickens with the V4 strain of Newcastle disease virus.

Two-week-old chickens, free of detectable maternal antibody to Newcastle disease virus (NDV), or with low levels of maternal antibody, were vaccinated with the V4 strain of NDV. Haemagglutination inhibition (HI) antibodies were determined at intervals after vaccination. Two hundred chickens were vaccinated by exposure to an aerosol, a dose of 10(6) 50% embryo infectious doses (EID50) being allowed per chicken. Forty unvaccinated chickens were placed in direct contact with vaccinated chickens. Most of the vaccinated chickens and the in contact chickens had developed HI antibodies of titre greater than or equal to 8 within 2 weeks of vaccination. The HI antibodies in many chickens persisted for at least 8 weeks. Control chickens in a shed 15 metres from the shed containing the vaccinated chickens did not develop HI antibodies to NDV. NDV could be isolated from some vaccinated chickens for 15 days after vaccination. An aerosol dose of 10(5)EID50 per chicken failed to induce a serological response in 2 groups of 40 chickens each. HI antibodies were produced in 1 of 2 groups, each of 40 chickens, vaccinated with 10(6)EID50 and in both of 2 groups of 40 chickens each vaccinated with 10(7)EID50. Duplicate groups of 40 chickens were vaccinated with 10(6)EID50 of V4 virus per chicken administered either as an aerosol, a coarse spray or a droplet placed in the conjunctival sac. HI antibodies were produced in all the groups of chickens.

Aerosols↗

Tween 80-solubilized Newcastle disease virus prepared as a water-in-oil-in-water vaccine.

The Newcastle disease virus (NDV) was solubilized with 10% w/v Tween 80 and inactivated with 0.05% v/v formalin. The average molecular mass of the released antigenic subunits was 307 kD. The tested vaccine was prepared in the form of a water-in-oil-in-water emulsion (WOWE) vaccine. The oil-to-aqueous ratio was 1:2. The solubilized NDV was administered alone or built in a tetravalent WOWE vaccine. A dose of the monovalent vaccine containing an equivalent of 44.7 microliters of detergent-treated NDV-allantoic fluid (NDV-AF) was sufficient for the complete protection of the commercially available chickens vaccinated at the age of 5 wk and challenged 7 wk later. The anti-NDV-free chickens, vaccinated at 4 wk of age and challenged 2 wk postvaccination, were 100% and 73% protected by a vaccinal dose containing 178.6 and 89.3 microliters of detergent-treated NDV-AF, respectively. Commercially available light pullets, primary vaccinated with live lentogenic NDV vaccine, generated a protective level of NDV antibodies after revaccination with WOWE vaccine containing 89.3 microliters of detergent treated NDV-AF. Laying hens were revaccinated under field conditions at the beginning of the laying cycle by the tetravalent vaccine. A vaccinal dose/bird containing 11.2 microliters of detergent-treated NDV-AF elicited a long-lasting high level of NDV neutralizing antibodies.

Animals↗

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.

Animals↗

In ovo vaccination of chicken embryos with experimental Newcastle disease and avian influenza oil-emulsion vaccines.

Inactivated oil-emulsion (OE) Newcastle disease (ND) and avian influenza (AI) vaccines were injected into 18-day-old white rock (WR) and white leghorn (WL) chicken embryos to evaluate their immunologic efficacy and their effects on hatchability. Embryonating eggs were inoculated at 1.5 inches depth with various vaccine volumes and antigen concentrations. Serum hemagglutination-inhibition (HI) titers were first detected in chickens at 2 wk posthatch. Protection against morbidity and mortality was demonstrated in all of 10 chickens vaccinated as embryos and challenged with viscerotropic velogenic ND virus at 53 days of age and also in all of eight in ovo- vaccinated chickens challenged with highly pathogenic AI virus at 34 days of age. All of five unvaccinated control chickens for each respective ND- and AI-vaccinated group died. In pooled groups from successive hatches, the hatchability of WR or WL embryos injected with 100 microliters of vaccine was not significantly different (P > 0.05) from unvaccinated hatchmate controls when needle gauges of 22, 20, and 18 were used. Seroconversion rates of chickens vaccinated as embryos ranged from 27% to 100% with ND vaccination and 85% to 100% for AI vaccination. For ND, geometric mean HI titers of chickens per vaccine group ranged from 11 to 733, and in pooled groups, the range was 49 to 531. Titers for AI vaccine groups ranged from 156 to 1178. This study demonstrated that acceptable hatchability, seroconversion rates, and protective immunity can be attained with in ovo inoculation of ND or AI OE vaccines if the vaccines are prepared with sufficient antigen and administered properly.

Animals↗