review of newcastle disease virus with particular ......review of newcastle disease virus with...

Review of Newcastle disease virus with particular references to immunity and vaccination S.O. AL-GARIB1J,3, A.L.J. GIELKENS?, E.GRUYS and G. KOCH'*

Tentral Institute Disease Control (CIDC-Lelystad), P.O. Box 2004,8203 AA Lelystad, The Netherlands, *Department of Pathology, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80158,3508 TD, Utrecht, The Netherlands, 3Department of Poultry Diseases, Faculty of Veterinary Medicine, Al- Fateh University, P.O. Box 13662 Tripoli, Libya and 41nstitute for Animal Science and Health (ID-Lelystad), P.O. Box 65,8200 AB, Lelystad, The Netherlands

Newcastle disease (ND) is a highly contagious disease. The present paper deals with classification of ND virus (NDV), clinical signs and pathology, virus strain classification and molecular backgrounds for the pathogenicity. Major emphasis is reviewing immunity and vaccination. Clinical forms of the disease vary depending on many factors, but mainly on the virulence of Newcastle disease virus (NDV) strains. Virulent strains are considered List A pathogens by the 'Office International des Epizooties' (OIE). The virulence has been traditionally determined using in vivo pathogenicity tests to distinguish between highly, moderately and low virulent isolates. More recently, molecular biological techniques like polymerase chain reaction and sequencing have been described to differentiate virulent from non- virulent strains. The systemic and mucosal immune systems are considered to function more or less independently. Systemic antibodies are essential elements in protection against ND, whereas the local antibodies limit multiplication of NDV at the site of entry. Cytotoxic T lymphocytes specific against NDV have been detected in the spleen of vaccinated birds, however, their contribution to protection remains to be elucidated. An increase of the number of various leukocyte subsets was noticed in the respiratory tract and the Harderian gland (HG), which favours involvement of local cellular immunity in the defence against NDV infection. It is tempting to speculate that the local lymphoid infiltrates are involved in first defence and that cytolytic cells clear virus by directly lysing infected target cells at the site of NDV inoculation. Secondly, various cell types, mainly T-lymphocytes and macrophages, may be equipped to produce a range of cytokines with antiviral activity and cytokines that stimulate B- lymphocytes to proliferate and differentiate into antibody-forming cells responsible for the local antibody production against NDV.

*Corresponding author: e-mail: g.kochOid.dlo.nl

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NDV Vaccination and immunity: S.O. Al-Gurib et al.

Vaccination using inactivated virus induces mainly systemic immunity and usually protects birds from disease caused by virulent NDV strains. Mucosal vaccination would be more attractive, as it induces in addition to systemic immunity a local IgM, IgA, and IgG, response as well as a mucosal cellular immune response. These local responses contribute to protection against NDV infection of the respiratory tract mucosa, which is the first target tissue of infection with this virus.

Keywords: Newcastle disease virus; vaccine; humoral; cellular; immunity

Introduction NDV is the causative agent of pseudofowl pest, a devastating disease of poultry. NDV has a wide host range and has been reported in most orders of birds. Isolation of a virulent strain requires reporting to the “Office International des Epizooties” (OIE, 1996). During the last decade, knowledge of the immunobiology of NDV has rapidly increased. New data are derived from applications of the whole arsenal of modern technology including gene technology, monoclonal antibodies, and synthetic peptides. Although many reagents have been developed in the last decades to study the immunity in poultry (reviewed in Jeurissen et al., 2000a), these have not been used in depth to study the mechanisms that are effective in protection against ND. In the present paper, a review is given of ND. Clinical signs and pathology are outlined and the classification of the viruses causing it and the molecular bases for pathogenicity are discussed. The diagnosis and definitions of the disease are described. Particular emphasis is given on the immunological events that occur during infection or vaccination.

Classification and proteins of NDV ND is caused by avian paramyxovirus serotype 1 (APMV-1) viruses, which together with viruses of the other eight APMV serotypes (APMV-2 to APMV-9), have been recently placed in the genus Avulavirus, sub-family Paramyxovirinae, family Paramyxoviridue. This taxonomy was adopted after the complete sequence of the NDV genome was presented (De Leeuw and Peeters, 1999) showing that NDV is not a member of the genus Rubulavirus. The virus families Paramyxo-, Rhabdo-, F i b , and Borneviridae form the order Mononegavirales. All viruses within this order have a helical symmetry of the nucleocapsid and a non-segmented, single-stranded, and negative sense ribonucleic acid genome. NDV encodes at least six major proteins (Figure I) that comprise the nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin- neuraminidase (HN) protein and a RNA dependent RNA polymerase (L) (Chambers et al., 1986; Wilde et ul., 1986). The V protein is synthesised after RNA-editing of the P gene. The M protein forms a linkage between the glycoproteins in the virus envelope and the nucleoprotein in the nucleocapsid, thus stabilising the virus structure. Two proteins, the HN and F protein, form projections on the viral envelope. The HN protein is involved in the attachment of the virion to sialic acid-containing receptors on target cells. Neuraminidase-activity of the HN mediates cleavage of sialic acid from viral receptors at the cell surface enabling virus release from the infected cell. In addition, HN promotes fusion activity, since expression of both HN and F is required for syncytia formation (Alexander, 1997). The functional F protein induces fusion of the viral and target cell membranes and is also responsible for induction of fusion between host cells. This enables

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the viral transcription unit, consisting of the nucleocapsid and two other proteins, the P and L proteins, to enter the cytosol of the host cell (Samson, 1988). Therefore, the HN and F glycoproteins are essential for virus infectivity and are ideal targets to prevent infection by protective immunity (Meulemans et al., 1987; Nagy et al., 1991; Reynolds and Maraqa, 2000a).

HN\

Figure 1 A schematic representation of ND virion.

F fusion protein (MI HN haemagglutinin-neuraminidase protein (0) M matrix protein (A) P phosphoprotein (H) NP nucleoprotein (0) L RNA viral RNA ( m) RNA dependent RNA polymerase (0)

Clinical signs and pathology

The clinical signs reported in birds infected with NDV vary widely but mainly depend on virulence of the virus. Other factors determine the outcome of the disease such as the host species, age, immune status, infection with other organisms and environmental stress (Cheville et al., 1972b; Lancaster, 1981; Campbell, 1986). In some circumstances, with extremely virulent viruses the disease may result in sudden death (Cheville et ul., 1972b; Brown et al., 1999a).

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Strains of NDV are distinguished into five pathotypes (Beard and Hanson, 1984): 1. velogenic viruses (vvND): responsible for disease characterised by acute lethal

infection showing frequently haemorrhagic lesions of the digestive tract of dead birds;

2. neurotropic velogenic viruses (nvND): causing disease characterised by acute neurological signs and often-high mortality which follows respiratory distress;

3. mesogenic viruses: moderate respiratory disease often seen with high mortality only in young birds;

4. lentogenic viruses: causing mild infections of the respiratory tract; 5. asymptomatic enteric viruses: mainly gut infections causing no apparent disease. Although these classifications are useful for descriptive purposes, some overlap does

occur even in experimental infection of specific-pathogen-free (SPF) chickens (Cheville et al., 3972b; Alexander and Allan, 1974) and some strains are difficult to place. In addition, in the field, aggravating factors such as concurrent infections, and immune suppression may result in clinical signs induced by milder strains to resemble those of more virulent strains.

Generally, ND may involve signs of depression, diarrhoea, prostration, oedema of the head and wattles. In the vvND, clinical signs often begin with listlessness, increased respiration ending with death. Diarrhoea is frequently seen. Surviving birds may develop nervous signs such as muscular tremors, paralysis and torticollis (Cheville et al., 1972b; McFerran and McCracken, 1988; Bhaiyat et ul., 1994). In nvND, the clinical signs are marked by severe respiratory disease shortly followed by neurological signs. With all pathotypes of ND, egg production falls dramatically sometimes leading to complete cessation of egg laying and death. Virulent ND strains may replicate in vaccinated birds, but the clinical signs will be greatly diminished depending on the antibody level (Allan e,t al., 1978; Hamid et al., 1990).

No gross lesions can be considered pathognomonic for any form of ND as is with the clinical signs (Cheville et al., 1972b; McFerran and McCracken, 1988; Ojok and Brown, 1996; Brown et al., 1999). Gross lesions vary depending on virus strain and may also be absent. Cadavers of birds that died because of virulent ND, usually have a dehydrated appearance. VvNDVs typically cause haemorrhagic lesions of the intestinal tract. Thes'e lesions are often particularly prominent in the proventriculus, small intestine and caeca. These organs are markedly haemorrhagic which apparently results from necrosis of the intestinal wall or lymphoid tissues, such as caecal tonsils (Alexander, 1997). Little evidence is found of gross lesions in the central nervous system even in birds showing neurological signs prior to death. Gross pathological lesions are usually present in the respiratory tract when clinical signs indicate involvement. They consist predominantly of haemorrhagic lesions and congestion of the trachea; in addition air sacculitis may be evident (Kotani et al., 1987; Hamid et al., 1990). Egg peritonitis is often seen in laying hens infected with virulent NDV.

Microscopic lesions are not regarded as having any diagnostic meaning and can be greatly affected by the same parameters as the clinical signs and gross lesions. Generally, in most tissues and organs involved, the lesions include hyperaemia, necrosis, cellular infiltration, and oedema (Kotani et al., 1987; Hamid et a/., 1990). Lesions in the central nervous system are characterized by nonpurulent encephalomyelitis (Bhaiyat et al., 1994).

NDV strain classification and molecular basis for the pathogenicity Generally, the term strain is applied to describe a well-identified isolate of the virus. Pathogenicity tests are useful to determine the virulence of virus strains. Originally strains

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of NDV were differentiated according to the mean death time of chicken embryos after infection. Later differentiation was based on in vivo tests using disease or death developing in infected birds as indicator. Different infection routes were used in these tests: the intracerebral route in one-day-old chickens to determine the intracerebral pathogenicity index (ICPI) and the intravenous route in 6-week-old chickens for the intravenous pathogenicity index (IVPI). These indices were calculated according to the severity of disease symptoms. The IVPI test is particularly useful for classifying moderately and highly virulent NDV isolates, but tends not to show distinction between some mesogenic viruses and lentogenic virus. In the ICPI test the mean score per bird per 24 h observation over 8 days is calculated (Kouwenhoven, 1993; Alexander, 1995). Generally, lentogenic viruses give indices of up to 0.6, asymptomatic enteric viruses usually slightly lower indices, mesogenic viruses usually around 1.4 and velogenic viruses between 1.7 and 2.0. In the European Union (EEC), NDV isolates with an ICPT score larger than 0.7 are considered virulent.

Monoclonal antibodies (MAbs) may detect variation in antigenicity such as single amino acid changes of epitopes to which the antibodies are directed. MAbs raised against various strains of NDV have been applied in epidemiological studies (Alexander et al., 1997). Panels of MAbs were used to place strains and isolates of NDV into groups. Isolates in the same MAb group shared biological and epizootiological properties. The analyses showed that the viruses tend to stay reasonably well preserved during an outbreak or epizootics, allowing for suppositions to be made about the origin, spread, and diagnosis. Moreover, MAbs have been used to distinguish between common vaccine strains, Hitchner B1 and La Sota (Meulemans et al., 1986; Erdei et al., 1987) and they can separate vaccine virus from epizootic virus in a given area (Srinivasappa et al., 1986).

The pathogenicity of NDV largely depends on cleavage of a precursor fusion glycoprotein, F, into the subunit proteins F, and F,. It forms a major molecular base for the virus particles to be infectious (Glickman et al., 1988; LC Long et a/., 1988; Rott and Klenk, 1988). The cleavage is mediated by host cell proteases (Nagai et a/., 1976; Gotoh et a/., 1992). The significance of F, cleavage is simply demonstrated by the inability of lentogenic strains to produce plaques in cell culture systems, in the absence of trypsin (Rott, 1979; Seal eta/. , 2000).

Collins et a/. (1 993) compared the deduced amino acid sequences at the cleavage site of the F,, precursor to classify NDV pathotypes. Viruses that were virulent for chickens i.e. with high ICPI values had the sequence "2Arg/Lys-Arg-Glu/Lys-Arg/Lys-Arg"b at the C- terminus of the F2 protein and phenylalanine at residue 11 7, the N-terminus of the F, protein. Viruses of low virulence had the sequence motive of *12Gly/Glu-Lys/Arg-Glu- Gly/Glu-Arg'I" and leucine at residue 11 7 in the same region. Thus, for the virus to be virulent a double pair of basic amino acids is apparently required at residues 112 and 113 and at residues 115 and 1 16, plus a phenylalanine at residue 117. The importance of the amino acid sequence around the cleavage site was recently demonstrated more directly when a La Sota vaccine virus was engineered by reverse genetics to contain multiple basic amino acids. The ICPI rose from 0,0 to I ,4 for the engineered virus containing multiple basic amino acids (Peeters et a/., 1999). Differences in pathogenicity were also recorded using 14-day-old chicken embryos. The cloned rescued original La Sota was only detected extra-embryonically in the chorioallantoic membrane and the lung of the embryo, whereas the newly molecular-engineered La Sota was found also in the internal organs, e.g. heart and spleen (Al-Garib et al., 2002 and manuscript in preparation). These phenotypic differences are probably also related to the expression of host proteases in cells. The findings that virulent strains of NDV replicate in systemic organs, whereas lentogenic strains of NDV are only detected in the respiratory tract (Ojok and Brown, 1996; Brown et al., 1999; Al-Garib et al., unpublished results) are in line with this assumption.

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In some lentogenic viruses the sequence at the cleavage site is not stable. Sequence analyses of virus isolates of a recent Australian outbreak suggested that the original lentogenic Peat’s Ridge strain evolved into virulent viruses containing multiple basic amino acids (Westbury, 2001). Moreover some but not all lentogenic NDV isolates from waterfowl can evolve into velogenic virus in vivo by acquiring multiple basic amino acids and a phenylalanine around the cleavage site after passaging in chickens. However, the fact that not all waterfowl isolates could evolve into velogenic viruses suggests that other factors than the sequence around the cleavage site may have a role in the pathogenicity (Shengqing el al., 2002).

Origin, host-range and transmission of NDV ND as a highly pathogenic disease of poultry was initially reported in 1926 in Southeast Asia (Alexander, 1997). Later, NDV became distributed world-wide and was reported to non-reproductively infect animals other than birds, ranging from reptiles to humans. All bird species are probably susceptible to the infection but the disease may vary enormously from one avian species to another with any given virus strain (Kaleta and Baldauf, 1988).

Various authors have discussed how NDV may be introduced into a country or region and subsequently spreads from flock to flock (Lancaster and Alexander, 1975; Alexander, 1988; Kouwenhoven, 1993; Alexander, 2000). They concluded that transmission of infection might take place by either inhalation or ingestion. It is tempting to speculate that fine aerosols or large droplets full of virus particles may be inhaled and stick to the I ~ U C O U S membranes resulting in infection of susceptible birds. Because a large amount of virus is excreted via droppings during the course of infection, ingestion of faeces results also in infection. Therefore, the main modes by which virus spreads are movement of live birds, movement of people and equipment, movement of poultry products, contaminated poultry food or water, airborne spread, vaccines and non-avian hosts.

Diagnosis and definition of ND Avian paramyxovirus- 1 infections are usually diagnosed by virus isolation. Serology can be used only in non-vaccinating countries (OIE, 1996). NDV can most easily be isolated from tissue samples or faecal or tracheal swabs from infected birds by inoculation of eight- to ten-day-old embryonated chicken eggs via the allantoic cavity (Alexander, 1997). Confirmation of the virus as belonging to the APMV-1 serotype, can be performed by haemagglutination inhibition (HI) tests using a panel of specific antisera.

The large difference in virulence resulting in a range of clinical symptoms signifies that an accurate definition of what constitutes ND, is essential for the purposes of diagnosis and control programs (policies and vaccination). ND is defined as an infection of birds caused by a virus of avian Paramyxovirus serotype 1 (APMV-1) that fulfils one of the subsequent criteria for virulence (OIE, 2000): the virus has an ICPI in day-old chicks (Gallus gallus) of 0.7 or larger or, alternatively, the virus has multiple basic amino acids at the C-terminus of the F, protein and phenylalanin at residue 117 at the N-terminus of the F, protein. The term multiple basic amino acids refers to at least three arginine or lysine residues between residues 112 to 116. Failure to demonstrate the characteristic pattern of amino acid residues as described above would require characterisation of the isolated virus by an ICPI test.

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Immunity against NDV The respiratory tract is the main site of entry of NDV. Local immediate immune mechanisms therefore form a first line of defence against infection. Compared to mammals, lavage fluids of the respiratory system of chickens contain a low steady state number of macrophages and heterophils that for the sake of simplicity are further indicated as avian respiratory phagocytes. The difference is particular striking because the ratio between the respiratory epithelium and the volume of lungs is much larger in birds than in mammals. Birds seem to compensate for the assumed deficiency through a rapid influx of respiratory phagocytes against invading pathogens. For ND, the influx of leukocytes in the respiratory tract lumen has not been studied, but evidence was obtained that NDV could have a negative effect on these cells. Avian respiratory phagocytes in ND- vaccinated birds have a lower phagocytic and bactericidal activity which might explain the occurrence in the field of vaccination reactions that are caused by concurrent bacterial infections such as with Mycoplasma spp (Toth et al., 2000).

At first, NDV targets epithelial cells lining the respiratory tract and depending on the virus strain, also of the gastrointestinal tract (Cheville et al., 1972a). In vitro, NDV induces the production of interferon type I and type I1 in fibroblasts, since culture medium of infected fibroblast showed both antiviral activity and induced no production in chicken macrophages (Heller et al., 1997). Moreover, lentogenic NDV induced expression of mRNA transcripts in primary chicken macrophages ( Sick et al., 1998) and transcripts were detected by in situ hybridisation using riboprobes in formalin fixed tissue sections of lymphoid tissues and spleens of chickens infected with velogenic NDV (Brown et al., 1999b). It was already known that NDV stimulates the production of interferon a and p by murine macrophages (Hoss et al., 1989) and of interferon-y by murine T cells. Whether interferon production interferes with NDV replication and spreading in vivo has, however, not been investigated.

When NDV successfully overcomes the innate response, it probably triggers both an antibody and cellular T-cell response, as these responses are common in viral infections (Zinkernagel et al., 1994). Although our knowledge of avian immunology has progressed rapidly in the last decades, very little is known about the efficacy of the cellular immune response against NDV. Leukocyte infiltrates do occur at sites of viral replication (Cheville et ul., 1972b; Kotani et al., 1987) such as the respiratory tract and the Harderian gland (HG) (Russell et al., 1997). These infiltrates comprise all elements required for the induction of a cellular immune response like macrophages and CD4 and CD8 T- lymphocytes (Russell et al., 1997; Al-Garib et al., manuscripts in preparation). Most of the infiltrated lymphocytes in the tracheal mucosa and the HG express y6 or ap 1 T cell receptors (TCR1) and might be activated since many leukocytes within the infiltrates express MHC class I1 epitopes. Although the HG of normal chickens already contains T- and B-lymphocytes their numbers increased 2-3 fold after vaccination with lentogenic or mesogenic virus strains (Russell et al., 1997; Al-Garib et al., manuscript in preparation). We can only speculate on the cytotoxic and/or antiviral activity of the CD8' cells. Cytotoxic T-lymphocytes were detected in the spleen of vaccinated chickens after in vitro re-stimulation (Cannon and Russell, 1986) or ex-vivo in chickens vaccinated twice or vaccinated and challenged with virulent virus (Jeurissen et al., 2002a). In the latter experiments the cytotoxic activity was shown to be MHC-class I restricted.

The dogma is that CD4' T cells are activated by antigen only after phagocytosis and processing by antigen-presenting cells. Activated CD4' T-cells release cytokines that: (1) can impair or kill target cells: or (2) recruit and regulate non-specific effector cells, such as macrophages which then kill the targets (Kaiser, 1996). In addition, CD4' T cells activate B-cells leading to proliferation and differentiation into antibody producing cells and formation of memory B-cells.

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Antibodies constitute an essential component of the protection against NDV and are important for the clearance and neutralisation of pathogen in principle in two ways: ( I ) by binding to infected cells and thereby reducing the production of progeny virus, and (2) by binding to released progeny virus and thereby inhibiting its spread. Antibodies capable of protecting the host can be measured in virus neutralisation (VN) tests. However, since the V N response appears to parallel the haemagglutination inhibition (HI) response, mainly the HI test is used to assess protection, especially after vaccination (Allan et al., 1978). Neutralising antibodies are directed against both the HN and F proteins. Chickens passively immunised with anti-sera against the nucleo- and phosphoproteins or against the matrix protein did develop antibody titres detectable in an ELISA but not in a neutralisation test and the birds developed clinical signs after a ND challenge infection (Reynolds and Maraqa, 2000a).

Using an ELISA, the kinetics and Ig-class distribution of an antibody response against NDV was determined (Al-Garib et al., 2003a). IgG and IgM antibody was predominantly detected in serum. After primary infection, IgM was detected as early as day 4 after vaccination, followed by IgA and IgC from day 7 after infection onwards. Antibodies in serum will confine infection to the respiratory mucosa. In contrast, secretory antibodies probably function to prevent or reduce virus replication in the epithelium. Antibodies were detected in secretions of the upper respiratory tract and intestinal tract of chickens at about the same time humoral antibodies could be first detected in blood. In secretions, all three Ig-classes were recognised after local infection with live virus, but only IgM and IgG after systemic immunisation with inactivated NDV (Al-Carib et ul., 2003a).

After parental immunisation, the respiratory tract is less protected and viral replication at this site is not prevented (Parry and Aitken, 1977; Holmes, 1979). Using this route no IgA-response was detected using the ELISA (Al-Garib et al., 2003a). In contrast, immunity after local vaccination will reduce replication at this site, giving the immune system more time to mount secondary responses (Parry and Aitken, 1977; Holmes, 1979). Based on data obtained from mammals, it was suggested that the systemic and mucosal immune systems function more or less independently. This seems to be also true for chickens. Thus, local inoculation with live NDV stimulates local production of antibodies in the upper respiratory tract and systemic production in the spleen (Parry and Aitken, 1977; Ewert et al., 1979; Russell and Koch, 1993). In contrast, parental immunisation with inactivated NDV leads to production in the spleen and no or little local production resulting in antibodies mainly in the serum and no or little antibodies in secreta (Beard and Easterday, 1967a; Parry and Aitken, 1977; Holmes, 1979; Russell and Koch, 1993). In conclusion, mucosal ND vaccination induces responses mainly comprising IgA and less IgM and IgG (Al-Carib et al., 2003a), which would prevent re-infection and thus provide a better, more durable protection (Parry and Aitken, 1977; Holmes, 1979).

The mechanism of protection by secretory antibodies in particular in the chicken is not entirely clear. In mammals, IgM antibodies are highly efficient in aggregating virions and in mediating lysis of infected cells by complement via the classical pathway. IgG antibodies can bind to released virions, promoting phagocytosis by neutrophils and macrophages via the Fc receptor, and mediating cellular lysis by complement via the classical pathway. IgA antibodies function mainly by binding to released virions (Daniele, 1990) thereby preventing infection of the epithelial cells. Mammal IgA can activate complement via the alternate pathway allowing phagocytosis by alveolar macrophages. It is unknown whether these functional properties can be extrapolated to IgA of the chicken. Secretory immunoglubulins, however, were shown to have neutralising activity in vitro (Ewert et al., 1977; Parry and Aitken, 1977; Caporale et al., 1978).

There is a need to correlate various quantitative and qualitative predictors to build a comprehensive picture of the biology of the antibody response. Immunocytochemical



methods can be used to visualise plasmablasts and plasma cells in situ, based on the specific antigen-binding capacity of the antibody they contain (Jeurissen et al., 2000b). Therefore, a new in situ technique to detect anti-NDV producing cells in tissue sections was developed (Al-Garib et al., 2003b). Antibody-producing cells were stained in spleen in large numbers at 7 days after infection with a mesogenic strain of NDV. These cells produced mainly IgM and IgG. Most of the NDV antibody-producing cells located in the Harderian gland and respiratory tract mucosa however, produced IgA (Russell and Koch, 1993; Al-Garib et al., unpublished results). The finding correlates well with high IgM and IgA responses against NDV that were revealed by ELISA in trachea washes and bile of locally infected birds (Al-Garib et al., 2003b).

In addition to antibodies, systemic and local cellular immune responses may also contribute to protection against infection. The systemic cellular response is detectable as early as 2-3 days after live NDV vaccine infection using leukocyte migration inhibition test (Ghumman and Bankowski, 1975; Timms and Alexander, 1977). The systemic cell mediated immunity has been investigated by secondary in vitro stimulation of spleen lymphocytes (Cannon and Russell, 1986). It was found that these cells were capable of rapidly lysing NDV-labelled target cells as measured in a 51Cr-release assay. From this, it was concluded that spleen cells comprise a cytotoxic effector cell population. The effector cells are probably T-cells. Recently, a new detection system was described (Jeurissen et ul., 2000a) for cytotoxic T lymphocyte responses against Newcastle disease virus (NDV). Using the system, the authors demonstrated cytotoxic T lymphocytes in spleens of chickens that were vaccinated twice with live ND vaccines or that were vaccinated and challenged with virulent NDV.

It is difficult to assess the contribution of cellular (T-cell) and of humoral (B-cell) responses to protection. One approach to elucidate their respective role has been to destroy one component of the immune system leaving the other component intact. Thus the bursa was destroyed either surgically or chemically to deplete cells of B-cell lineage, or the thymus was removed surgically to deplete cells of the T-cell lineage. The interpretation of such experiments is difficult, because (1) bursa depletion does not always lead to agammaglobulinaemic birds, ( 2 ) bursectomy has an effect on thymus development (Zucker et al., 1973), although this latter effect seems to have no effect on T-cell function (Vainio and Toivanen, 1987), (3) thymectomy will not lead to complete T cell deficiency and on the other hand, it will also deplete helper T cells that are required for an effective B cell response, and (4) chemical depletion of T cell subpopulation also has an effect on the number of B cells (Russell et al., 1997). Bursectomized chickens that were additionally irradiated or treated with anti-bursa cell antiserum developed no antibodies against NDV or low levels of it after vaccination, and they died after an intramuscular challenge with virulent ND virus. In contrast, thymectomised, irradiated chickens developed antibodies and were resistant to challenge (Cheville and Beard, 1972; Marino and Hanson, 1986; Perey and Dent, 1975). Moreover, the delayed type hypersensitivity of thymectomised birds was only diminished but not absent after local injection of NDV in the wattle and thus from such experiments one can not firmly conclude that T cell responses do not contribute to protection. Irradiation seems to be crucial, because in later experiments, bursectomy without irradiation resulted only in selective deficiency for IgA (Ewert and Eidson, 1977). All bursectomised chickens remained healthy after challenge with virulent NDV suggesting that IgA is not essential for the development of immunity but that locally produced IgM and transuded serum IgG antibodies protect the trachea mucosa in the absence of IgA. Since antibody levels of sham-bursectomised and bursectoinised birds were similar after vaccination, the results can not be used to conclude on a possible role of cell mediated immunity in protection. Of chickens bursectomised in ovo and treated twice with anti-chicken bursa1 cell antiserum 44% had no detectable


NL)V: vaccination and immunity: S.O. Al-Carib et al.

immunoglobulin in serum, but nevertheless 66% of those chickens produced antibodies at levels that may well have protected the birds against mortality on challenge infection. Thus, although the bursectomised chickens showed equal or better in vitvo blastogenic responses against PHA mitogen and NDV antigen, this does not univocally prove the importance of the cell-mediated response (Marino and Hanson, 1987).

Drug treatments were used as another strategy to deplete T-cells or B-cells. Cyclophosphamide treatment in ovo was used as an immunosuppressive agent to deplete B-cells and thus to repress the humoral response leaving the cellular response intact. Birds that hatched form treated eggs and that survived a challenge infection, all had antibodies detectable in the HI and VN tests. Birds without antibodies but with positive blastogenic response against NDV were not protected (Reynolds and Maraqa, 2000b). In contrast, in another experiment birds that were treated with cyclophosphamide after hatch did not produce antibodies upon intranasal or subcutaneous vaccination. The intranasally but not the subcutaneously vaccinated birds were resistant against an intranasal challenge with a velogenic NDV. Antibodies were detectable in the trachea washings of the resistant birds demonstrating the importance of local immunity (Lam and Haq, 1987). Thus antibodies play a key role in protection against clinical signs. The importance of antibodies probably is best demonstrated by the resistance that is acquired after intravenous injection of a large amount of ND immune sera to infection with virulent NDV via the intramuscular route (Beard and Easterday, 1967b; Malkinson and Small, 1977; Reynolds and Maraqa, 2000b). The tracheas of the same birds that received passive antibodies intravenously, however, were not protected against infection demonstrating the importance of the local antibody response. In particular antibodies directed against the haemagglutinin and fusion proteins mediated protection, whereas antibodies against the internal viral proteins did not (Reynolds and Maraqa, 2000b). Treatment of cyclosporine A reduced the blastogenic response and the number of most T cell subpopulations (i.e. CD4 and CD8) significantly and retarded but did not diminish the antibody response by 1 to 7 days (Russell et al., 1997). Because the antibody response was retarded but not diminished, the slower clearance of Hitchner B1 from the HG conjunctiva and trachea that was observed cannot simply be attributed to a diminished T-cell function.

A key role for antibodies in protection against ND appears from all results found published and mentioned above. Antibodies might be so effective, because the NDV replicates rapidly enabling large amounts of the infectious virus to be released from infected cells. With such kinetics, the cellular immune response may simply be too slow to significantly change the peak titre in the infected host, whereas antibodies within the tissue neutralise virus preventing its further spread. Nevertheless, there might still be a role for the local cell-mediated immunity since after systemic vaccination of birds virus did not replicate or shed from the trachea after local challenge (Reynolds and Maraqa, 2000b). A role of the cellular immunity can also be concluded from the interaction between chicken infectious anaemia virus (CIAV) and live NDV vaccination. Vaccination reactions were greatly exacerbated when chicks were infected with CIAV and vaccinated with NDV at one day old or infected with CIAV at one day old and vaccinated with NDV at day 10. The CIAV infection did not effect the level of hemagglutination inhibiting antibodies that were induced by the vaccination with NDV. CIAV is known to suppress the production of interferon-y, IL- 1 and IL-2, to decrease mitogen responsiveness of spleen cells (Adair et al., 1991; McConell et al., 1993a) and to impair macrophage function (McConell et al., 1993b). Moreover, CIAV infection interferes with the cytotoxic T lymphocyte responses (Markowski-Grimsrud and Schat, 2001).



Vaccination against NDV Vaccination of commercially reared birds is the only way to reduce disease and thus losses resulting from infection. When designing a vaccination programme, consideration should be given to the type of vaccine used, the immune and disease statuses of the birds to be vaccinated, and the level of protection required in relation to any possibility of infection with field virus under local conditions (Allan and Lancaster, 1978). Moreover, when vaccination is used to stop transmission it should induce sufficient herd immunity. ND vaccines are routinely only tested for their effectiveness to induce clinical protection, which does not provide information about the level of transmission, because factors that cause clinical signs differ from factors that cause transmission. As discussed earlier, clinical signs are caused by virus replication within the chicken for example in visceral organs or brain tissue, whereas transmission occurs between chicken via infected secretions, direct contact, or via air. Moreover, virus transmission starts well before clinical signs become apparent and often occurs subclinically.

At first vaccination was performed using inactivated infective material, which was shown to induce protection in inoculated chickens. Inactivated vaccines however, never flourished, not only because of problems in production and standardisation, but also because enzootic viruses like the Roakin strain were discovered that only produced mild disease and therefore were used to develop live vaccines (Beard and Hanson, 1984; Beaudette et al., 1949). Subsequently, viruses of even less virulence were discovered like the Hitchner-B, (Hitchner and Johnson, 1948) and La Sota (Goldhaft, 1980) strains. Only recently, various recombinant vaccines that can provide protection against ND have been developed expressing either the F or HN protein or both (Meulemans et al., 1988; Nagy el al., 1991; Nishino etal., 1991).

Presently, mesogenic live vaccines are advocated only in countries where virulent NDV is endemic. Better transmission of mesogenic viruses combined with a higher level of induced immunity is of advantage to prevent serious disease particularly in areas with backyard poultry (Reeve and Alexander, 1974). In chickens experimentally infected with the Roakin strain of NDV, humoral antibody levels were found to be three to five fold higher than in La Sota infected birds (Al-Garib et al., 2003a). Unfortunately, the better protection is going along with serious vaccination reactions in particular in birds under 8 weeks of age, in birds that have not been immunised previously, or in immunocompromised birds. For these reasons, mesogenic viruses were excluded within the EEC and from the concept OIE definition of ND. Also, the safety of mesogenic vaccines should be checked even more carefully than of lentogenic strains in view of the recent findings that virulence of NDV may increase upon passage in chickens (Shengqing et al., 2002).

Most live vaccines currently used in most countries are derived from lentogenic field strains. Still these strains have variable residual pathogenicity (Borland and Allan, 1980) and consequently vaccination reactions are inevitable. Cloning was used to obtain viruses with high immunogenicity combined with acceptable vaccination reactions. Recently, “asymptomatic enteric” strains became popular because these strains do not cause vaccination reaction at least not in the respiratory tract. The Australian V4 and Ulster strains of NDV belong to this latter category.

Inactivated vaccines generally induce extremely high levels of protective antibodies that persist for a long time and therefore are applied for revaccination for instance to protect laying hens during the entire production period. Because inactivated vaccines are more laborious to produce and require individual application their use is extremely expensive.

Effective vaccination requires ideally that all birds in a flock get vaccinated. Since the spread of lentogenic viruses may be limited, individual application by eye or nose drop is preferred to obtain uniformly high levels of protection. Individual application is too laborious and therefore is practiced in small flocks only. As a result, mass application


NDV: vaccinution and immunity: S.O. Al-Carib et al.

using spray or aerosol equipment or via the drinking water is favoured because it is cheap and convenient. Moreover, this application method triggers local cellular and humoral immunity in the respiratory tract preventing infection of mucosal surfaces or reducing virus replication at this site. As a result virus invasion to systemic tissues is blocked.

Difference in effect of spray and aerosol are caused by the size of the droplets. Coarse droplets are short-lived in air, whereas fine droplets i.e. aerosols are long-lived. As a consequence the type of equipment, simple spray apparatus vs. atomiser, nozzle size, and air pressure in the apparatus are important factors of effectiveness, since these determine droplet size. Moreover, coarse droplets will carry over a short distance and birds have to be hit directly, which is not required for an atomiser. On the other hand aerosols compared to coarse droplets penetrate deeper into the (lower) respiratory tract and as a result cause more severe vaccination reactions. Aerosol application is therefore usually limited to secondary vaccination. Drinking water vaccination can also be used but gives varying results due to the variations in water intake between birds (Kouwenhoven, (1993).

Three decades ago, the in ovo route of vaccination has been introduced as an effective route of vaccine administration. Until recently, this route could not be used for NDV vaccine because it will kill or weaken the embryo. The problem was tackled by treating virus with an alkylating agent, ethylmethane sulfonate, to cripple the virus (Ahmad and Sharma, 1992) or by preparing complexes of virus and neutralizing antibodies (Haddad et al., 2002). Reverse genetics can also be used to produce ND vaccines with reduced pathogenicity for chicken embryos. One such virus expresses low levels of the V protein, exhibits impaired replication, but still induces protective antibody levels in hatched chickens (Mebatsion et al., 2001).

Results of vaccination studies cannot be compared easily because of the many variables and the differences in methods and materials used. Thus the ideal vaccination schedule can not easily be given. The level of maternal immunity, the type of bird (among others broilers vs. layers) and of vaccine, the vaccination equipment, workmanship of the applicant, all will affect the outcome of the vaccination. Although one-day-old chicks do not respond very well with increased blood levels of antibodies to vaccination either at the hatchery or at the farm because of interference by maternal immunity, vaccination is beneficial because it induces a local immune response in the respiratory tract and vaccinated chicks are protected although less sustainable than after vaccination at an older age. Secondary vaccination is thus required and should be applied between I8 and 21 days of age, which will boost immunity, in particular, when using aerosol and less attenuated vaccines. Such a vaccination scheme was recommended for use in broilers in the Netherlands until mid 2001 when the vaccination enforcement was revised. In the field the results in broilers were disappointing with mean titre values of 23 well below the mean level of 25.2, which was presented by Allan et al., (1 978) as 100% protective. The fear for severe vaccination reactions may have been the reason for using spray in stead of aerosol and Clone30 vaccines in stead of more virulent vaccines based on uncloned LaSota vaccines. In this respect, the in ovo route of vaccination promises to be an attractive alternative. Zn ovo application of a antibody-NDV complex vaccine protected 98-100% of broilers from 7 days of age during the entire fattening period to an intramuscular challenge with velogenic Texas GB (Haddad et ul., 2002). Field experiments using this route are underway.

Conclusions Newcastle disease is a highly contagious viral disease of chickens. The clinical outcome of the disease varies and is dependent upon several factors, but mainly on the characteristics


NDV: vaccination und immunity: S.O. Al-Garih et al.

of the virus. Definition of ND is principally based on the value of the intracerebral pathogenicity index and/or the presence or absence of multiple basic amino acids at the cleavage site of the F protein. Vaccines have long been available and administered to control ND. Locally replicating vaccine strains offer an attractive approach for immune interventions by providing an effective and durable immunity. However, continued improvements of ND control will require a better understanding of immunological mechanisms that are triggered by an immunisation regimen and its effect on virus transmission. A major function of the humoral immunity is the protection against clinical signs caused by infection with virulent NDV strains, whereas expansion in the numbers of various leukocyte subsets at the site of vaccination could be responsible for uptake, processing of virus antigen and production of antibodies and antiviral cytokines. Therefore, it is interesting to investigate the cells producing cytokines at the site of NDV inoculation.

Acknowledgements The authors thank Dr S.H.M. Jeurissen and Dr B.H. Peeters for critical reading of the manuscript, their comments were very useful.

References ADAIR, B.M., MCNEILLY, F. MCCONNELL, C.D., TODD, D., NELSON, R.T. and MCNULTY, M.S.

(1991) Effects of chicken anemia agent on lymphokine production and lymphocyte transformation in experimentally infected chickens. Avian Disease 35: 783-792.

AHMAD, J. and SHARMA, J.M. (1992) Evaluation of a modified-live virus vaccine administered in ovo to protect chickens against Newcastle disease. American Journal of Veterinary Research 53: 1999-2004.

ALEXANDER, D.J. (1988) Newcastle disease: Methods of spread. In: Alexander D.J. (Ed.), Newcastle disease, Kluwer Academic Publishers, Boston, pp. 257-272.

ALEXANDER, D.J. (1995) Newcastle disease in countries of the European Union. Avian Pathology 24: 3-10. ALEXANDER, D.J. (1997) Newcastle disease and other avian pardmyxoviridae infections. In: Calnek B.W

with, John Barnes H, Beard C.W, McDougald L.R, Saif Y.M. (Ed.), Diseases ofPoultry, North America, Iowa,

ALEXANDER, D.J. (2000) Newcastle disease and other avian paramyxoviruses. Revue Scientifque and Technique Office des International Epizooties (OIE) 19: 443-362.

ALEXANDER, D.J. and ALLAN, W.H. (1974) Newcastle disease virus pathotypes. Avian Pathology 3: 269- 278.

ALEXANDER, D.J., MANVELL, R.J., LOWINGS, J.P., FROST, K.M., COLLINS, M.S., RUSSELL, P.H. and SMITH, J.E. (1997) Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolated using monoclonal antibodies. Avian Pathology 26: 399-41 8.

AL-GARIB, S.O., GRUYS, E., GIELKENS, A.L.J. and KOCH, G. (2002) Tissue tropism and target cells of Newcastle disease virus (NDV) in the chicken embryo. In: Dridi A,, Kechrid F., El-Hichori K., Boussalmi B., Hdddouchi H.. Ben Ehedi S., Kaouach L., Chebbi M., (ed), Proceeding of the 27th World Veterinary Congress, Tunis, September 25-29, 2002, Abstract number 513, pp. 244-245.

AL-GARIB, S.O., GIELKENS, A.L.J., GRUYS, E., HARTOG and KOCH, G. (2003a) Immunoglobulin class distribution of systemic and mucosal antibody response to Newcastle disease in chickens. Avian Disease, in press.

AL-GARIB, S.O., GRUYS, E., GIELKENS, A.L.J. and KOCH, G. (2003b) Detection of antibody forming cells directed against Newcastle disease virus and their Ig-class using double immuno-enzyme histochemistry. Avian Disease, in press.

ALLAN, W.H., LANCASTER, J.E. and TOTH, B. (1978) Newcastle disease vaccines, their production and use. Food Agriculture Organisation (FAO), Animal Production Series, No. 10. FAO, Rome.

BABA, T., MASUMOTO, K., NISHIDA, S., KAJIKAWA, T. and MITSUI, M. (1988) Harderian gland dependency of immunoglobulin A production in the lacrimal fluid of chicken. Immunology 65: 67-71.

BEARD, C.W. and EASTERDAY, B.C. (1967a) The influence of the route of administration of Newcastle disease virus on host response I . Serological and virus isolation studies. Journal ofinfectious Disease 117: 55- 61.

pp. 541-569.



BEARD, C.W. and EASTERDAY, B.C. (1967b) The influence of the route of administration of Newcastle disease virus on host response 11. Studies on artificial passive immunity. Journal of infectiousDisease 117: 62- 70.

BEARD, C.W. and HANSON, R.P. (1 984) Newcastle disease. In: Hofstad M. S, Barnes H.J, Calnek B.W., Reid W.M., Yoder H. W. (Ed.), Diseases ofpoultry, Iowa State University Press, Ames, pp. 452-470.

BEAUDETTE, F.A., BIVINS, J.A. and MILLER, B.R. ( 1949) Newcastle disease immunization with live virus. Cornell Wterinarian 39: 302-334.

BHAIYAT, M.I., OCHIAI, K., ITAKURA, C., ISLAM, M.A. and KIDA, H. (1994) Brain lesions in young broiler chickens naturally infected with a mesogenic strain of NDV. Avian Pathology 23: 693-708.

BORLAND, L.J. and ALLAN, W.H. ( 1 980) Laboratory tests for comparing live lentogenic Newcastle disease vaccines. Avian Pathology 9: 45-59.

BROWN, C., KING, D.J. and SEAL, B. (1999) Pathogcnesis of Newcastle disease in chickens experimentally infected with viruses of different virulence. Veterinary Pathology 36: 125- 132.

BROWN, C., KING, D.J. and SEAL, B.( 1999) Detection of a macrophage-specific antigen and the production of interferon gamma in chickens infected with Newcastle disease virus. Avian Disease 43: 696-703.

CAMPBELL, R.S.F. (1 986) The pathogenesis and pathology of avian respiratory infection. Veterinary Bulletin

CANNON, M.J. and RUSSELL, P.H. (1986) Secondary in vitro stimulation of specific cytotoxic cells to Newcastle disease virus in chickens. Avian Pathology 15: 73 1-740.

CAPORALE E.P., RAGGI L.G. and SEMPRONI, G. (197X) Local resistance of chicken respiratory tract to viral infections. Zentralblartfur Vetiniirmedizin Reihe B 25: 383-393.

CHAMBERS, P., MILLAR, N.S., BINGHAM, R.W. and EMMERSON, P.T. (19x6) Molecular cloning of complcmentary DNA to Newcastle disease virus and the nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein. Journul of General Virologj 67: 475-486.

CHEVILLE, N.F. and BEARD, C.W. (1972) Cytopathology of Newcastle disease. The influence of bursa1 and thymic lymphoid systems in the chicken. Labomtory Investigafion 27: 129- 143.

CHEVILLE, N.F. BEARD, C.W. and HEMINOVER, J.A. (1972a) Comparative cytopathology of Newcastle disease virus. Use of ferritin-labeled antibody on allantoic and intestinal epithelium. Veterinary Pathology 9: 38-52.

CHEVILLE, N.F, STONE, H., RILEY, J. and RITCHIE, A.E. (1972b) Pathogenesis of virulent Newcastle disease in chickens. Journal of American Veterinnry Medical Association 161: 169- 179.

COLLINS, M.S., BASHIRUDDIN, J.B. and ALEXANDER, D.J. ( 1 993) Deduced amino acid sequences at the fusion protein cleavage site of' Newcastle disease viruses showing variation in antigenicity and pathogenicity. Archives of Virology 128: 363-370.

56: 52 1-543.

DANIELE, R.P. (1990) Immunoglobulin secretion in the airways. Annual Review of Physiology 52: 177-1 95. DE LEEUW, 0. and PEETERS, B. (1999) Complete nucleotide sequence of Newcastle disease virus: evidence

for the existence of a new genus within the subfamily Paruniyxovirincre. Journal ofGeneral Virology 80: 131- 136.

ERDEI, J., BASCHIR, K., KALETA, E.F., SHORTRIDGE, K.F. and LOMMIEZI, B. (1987) Newcastle disease vaccine (La Sota) strain specific monoclonal antibody. Archives of Virology 96: 265-269.

EWERT, D.L., EIDSON, C.S. and DAWE, D.L. (1977) Factors influencing the appearance of antibody in tracheal washes and serum of young chickens after exposure to Newcastle disease virus. Infection and Zmnzunig 18: 138-145.

EWERT, D.L. and EIDSON, C.S. (1977) Effect of bursectomy and depletion of immunoglobulin A on the antibody production and resistance of respiratory challenge after local and systemic vaccination of chickens with Newcastle disease virus. Infection und Immuniry 18: 146-150.

EWERT, D.L., BARGER, B.O. and EIDSON, C.S. (1 979) Local antibody response in chickens; analysis of antibody to Newcastle disease virus by solid-phase radioimmunoassay and immunofluorescence with class- specific antibody for chicken immunoglobulins. Infection and Immunity 24: 269-275.

GHUMMAN, J.S. and BANKOWSKI, R.A. (1975) In vitro DNA synthesis in lymphocytes from turkeys vaccinated with La Sota, TC and inactivated Newcastle disease vaccines. Avian Disease 20: 18-31.

GLICKMAN, R.L., SYDDAL, R.J., IORIO, R.M., SHEEHAN, J.P. and BRATT, M.A. (1988) Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus. Journal of' Virology 62: 354-360.

GOLDHAFT, T.M. (1980) Historical note on the origin of the La Sota strain of Newcastle disease virus. Avian Disease 24: 297-301.

GOTOH, B., OHNISHI,Y., INOCENCJO, N.M., ESAKI, E., NAKAYAMA, K., BARR, P.J., THOMAS, G. and NAGAI, Y. (1992) Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: Superiority of furin/PACE to PC2 or PC I/PC3. Journul of Virology 66: 6391-6397.

HADDAD E.E., WHITFILL, C.E., AVAKIAN, A.P., IJNK D.B. and WAKENELL P.S. (2002) Safety and Efficacy of a Novel Newcastle Disease Vaccine Administered in o v o to SPF and Commercial Broiler Birds. Paper presented at the Western Poultry Di\ease Conference (Complete Address)

198 World's Poultry Science Journal, Vol. 59, June 2003

NDV: vaccination and immunity: S.O. Al-Gurib et al.

HAMID, H., CAMPBELL, R.S.F and LAMlCHHAN, C. ( 1 990) The pathology of infection of chickens with the lentogenic V 4 strain of NDV. Avian Pathology 19: 687-696.

HELLER, E.D., LEVY, A.M., VAIMAN, R. and SCHWARTSBURD, B. (1997) Chicken-embryo fibroblasts produce two types of interferon upon stimulation with Newcastle disease virus. Veterinary Immunnlogji and

HITCHNER S.B. and JOHNSON E.P. (1948) A virus of low virulence for immunizing fowl, against Newcastle disease (avian pneumoencephalitis), Veterinary Medicine 43: 525-530.

HOLMES, H.C. (1979) Virus neutralizing antibody in sera and secretion of the upper and lower respiratory tract of the chicken inoculated with live and inactivated Newcastle disease virus. fournut of Comparutive Pathology 89: 2 1-2 1.

HOSS, A., ZWARTHOFF, E X . and ZAWATZKI, R. (1989) Differential expression of interferon alpha and beta induced with Newcastle disease virus in mouse macrophage cultures. Journal of General Virology 70:

JEURISSEN, S.H.M., BOONSTRA-BLOM, A.G, AL-GARIB, S.O., HARTOG, L. and KOCH, G. (2000a) Defence mechanisms against viral infection in poultry. Veterinary Quarterly 22: 204-208.

JEURLSSEN, S.H.M., CLAASSEN, E., BOONSTRA-BLOM, A.G, VERVELDE, L. and JANSE, E.M. (2000b) Immunocytochemical techniques to investigate the pathogenesis of infection micro-organisms and the concurrent immune response of the host. Developmental and Conzparative Immunology 24 141-151.

KAISER, P. (1996) Avian cytokines. In: Davison T. F., Morris T. R. and Payne L. N., (Ed.), Poultry Immunology 1st edn. Carfdx Publishing Company, UK, pp. 83-1 14.

KALETA, E.F. and BALDAUF, C. (1988) Newcastle disease in free-living and pet birds. In: Alexander D. J (Ed.), Newcusrle disease, Kluwer Academic Publishers, Boston, pp. 197-246.

KOTANI, T., ODAGIRI, Y., NAKAMURA, J. and HORIUCHI, T. (1987) Pathological changes of tracheal mucosa in chickens infected with lentogenic NDV. Avian Disease 31: 491-497.

KOUWENHOVEN, B. (1993) Newcastle disease, In: Horzinek C. (Ed.), Wrus infections of vertebrates series, Volume 4, McFerran J.B., McNulty, M.S. (Eds.) Virus infections of Birds, Elsevier Science Publishers B.V,

LAM, K.M. and HAO, Q. (1987) Vaccination of cyclophosphamide-treated chickens against Newcastle disease virus infection. Veteririary Microbiology 15: 41-48.

LANCASTER, J.E. (1981) Newcastle disease. In: Gibba E.P.J. (Ed.), Virus Disease of Food Animals, Vol 11, Disease Monographs, Academic Press, New York, pp. 433-465.

LANCASTER, J.E. and ALEXANDER, D.J. (1975) Newcastle disease: virus and spread. Monogruph No I!. Canada Department of Agriculture, Ottawa, pp. 79.

Lk LONG, L., BRASSEUR, R., WEMERS, C., MEULEMANS, G. and BURNY, A. (1988) Fusion (F) protein gene of Newcastle disease virus: Sequence and hydrophobicity comparative analysis between virulent and avirulent strains. Virus Genes 1: 333-50.

MALKINSON, M. and SMALL, P.A. Jr. ( 1 977) Local immunity against Newcastle disease virus in the newly hatched chicken’s respiratory tract. Infecection and Immunity 16: 587-592.

MARINO, O.C. and HANSON, R.P. (1987) Cellular and humoral response of i n ovo-bursectomized chickens to experimental challenge with velogenic Newcastle disease virus. Avian Disease 31:293-301.

MARKOWSKI-GRIMSRUD, C.J. and SCHAT, K.A. (2001) Proceedings of the I1 International Symposium on infectious bursa1 disease and chicken anemia, Rauischholzhausen, Germany.

McCONELL, C.D., ADAIR, B.M. and MCNULTY, M.S. (1993a) Effects of chicken anemia virus on cell- mediated immune function in chickens exposed to the virus by a natural route. Avian Disease 37: 366-374.

McCONELL, C.D., ADAIR, B.M. and MCNULTY, M.S. ( I 99%) Effects of chicken anemia virus on macrophage function in chickens. Avian Disease 37: 358-365.

MCFERRAN, J.B. and MCCRACKEN, R.M. (1988) Newcastle disease. In: Alexander D. J (Ed.), Newcastle disease, Kluwer Academic Publishers, Boston, pp. 161-1 83.

MEBATSION, T., VERSTEGEN, S., ROMER OBERDORFER, A., SCHRIER, C.C. and DE VAAN, L.T.C. (2001) A recombinant Newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos. Journal of Virology 75: 420-428.

MEULEMANS, G., GONZE, M., CARLIER, M.C., PETIT, P., BURNY, A. and LE LONG, L. (1986) Protective effects of HN and F glycoprotein-specific monoclonal antibodies on experimental Newcastle disease. Avian Pathology 15: 761-768.

MEULEMANS, G., GONZE, M., CARLIER, M.C., PETIT, P., BURNY, A. and LE LONG, L. (1987) Evaluation of the use of monoclonal antibodies to hemagglutination and fusion glycoproteins of Newcastle disease virus for virus identification and strain differentiation purposes. Archives of Virology 92: 55-62.

MEULEMANS, G., LETELLIER, C., GONZE, M., CARLIER, M.C. and BURNY, A. (1988) Newcastle disease virus F glycoprotein expressed from a recombinant vaccinia virus vector protects chickens from challenge. Avian Pathology 17: 82 1-827.

NAGAI, Y., KLENK, H.D. and ROTT, R. (1976) Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 7 2 494-508.

IminUnopalhology 57: 289-303.

575-89.

pp. 341-362.



NAGY, E., KRELL, P.J., DULAC, G.C. and DERBYSHIRE, J.B. (1991) Vaccination against Newcastle disease with a recombinant baculovirus hemagglutinin-neuraminidase subunit vaccine. Avian Disease 35: 585-590.

NISHINO, Y., NIKURA, M., SUWA, T., ONUMA, M., GOTOH, B., NAGAI, Y. and MIKAMI, Y. (1991) Analysis of the protective effect of the haemagglutinin-neuraminidase protein in Newcastle disease virus infection. Journal qf General Virology 72: 1187-1 190.

OFFICE INTERNATIONAL DES EPIZOOTIES (OIE) (1 996) Newcastle disease. In: Manual of standards for diagnostic tests and vaccine. 3rd Ed. OIE, Paris, pp. 161-169.

OFFICE INTERNATIONAL DES EPIZOOTIES (OIE) (2000) Newcastle disease. In: Manual of standards for diagnostic rests and vaccine. 4rd Ed. OIE, Paris, pp. 22 1-232.

OJOK, L. and BROWN, G. (1996) An immunohistochemical study of the pathogenesis of virulent viscerotropic Newcastle disease in chickens. Journal of Comparative Pathology 115: 221 -227.

PARRY, S.H. and AITKEN, I.D. (1977) Local immunity in the respiratory tract ofthe chicken. 11. The secretory immune response to Newcastle disease virus and the role of IgA. Veterinarq. Microbiology 2: 143-165.

PEETERS, B.H., DE LEEUW O., KOCH, G. and GIELKENS A.L.J. (1999) Rescue of Newcastle disease virus from Cloned cDNA: Evidence that cleavability of fusion protein is a major determinant for virulence. Journal of virology 73: 5001 -5009.

PEREY, D.Y.E. and DENT, P.B. (1975) Host resistance mechanism to Newcastle disease virus in immunodeficient chickens. Proceedings of the Society for Experimental Biology and Medicine 2: 365-369.

REEVE, P., ALEXANDER, D.J. and ALLAN, W.H. (1974) Derivation of an isolate of low virulence from the Essex ‘70 strain of Newcastle disease virus. The Veterinary Record 94: 38-41.

REYNOLDS, D.L. and MARAQA, A.D. (2000a) Protective immunity against Newcastle disease virus: The role of antibodies specific to Newcastle disease virus polypeptides. Avian Disease 44: 138-144.

REYNOLDS, D.L. and MARAQA, A.D. (2000b) Protective immunity against Newcastle disease virus: The role of cell-mediated immunity. Avian Diseuse 44: 145-1 54.

ROTT, R. (1979) Molecular basis of infectivity and pathogenicity ofmyxoviruses. Archives of Virology 59: 285- 298.

ROTT, R. and KLENK, H.D. (1988) Molecular basis of infectivity and pathogenicity of Newcastle disease virus. In: Alexander D.J., (Ed.), Newcastle disease, Kluwer Academic Publishers, Boston, pp. 98-1 12.

RUSSELL, P.H. and KOCH, G. (1993) Local antibody forming cell responses to the Hitchner B 1 and Ulster strains of Newcastle disease virus. Veterinary Itnmunology and Immunopathology 37: 165-1 80.

RUSSELL, P.H., DWIVEDI, P.N. and DAVISON, T.F. (1997) The effects of cyclosporin A and cyclophosphamide on the populations o f B and T cells and virus in the Harderian gland of chickens vaccinated with the Hitchner B 1 strain of Newcastle disease virus. Veterinary Immunology and lmmunoputhology 60: 171-185.

SAMSON, A.C.R. (1988) Virus structure. In: Alexander D. J (Ed.), Newcastle diseuse, Kluwer Academic Publishers, Boston, pp. 23-44.

SEAL, B.S., KING, D.J. and SELLERS, H.S. (2000) The avian response to Newcastle disease virus. Developmental and Comparative Immunology 24: 257-268.

SHENGQING, Y., KISHIDA, N., ITO, H., KIDA, H., OTSUKI, K., KAWAOKA, Y. and ITO, T. (2002) Generation of Velogenic Newcastle Disease Viruses from a Nonpathogenic Waterfowl Isolate by Passaging in Chickens. Virology 301: 206-21 I .

SICK, C., SCHULTZ, U., MUNSTER, U., MEIER, J., KASPERS, B. and STAEHELI, P. (1998) Promoter structures and differential responses to viral and nonviral inducers of chicken type I interferon genes. Journal of Biological- Chemistry 273: 9749-9754.

SRINIVASAPPA, G.B., SNYDER, D.B., MARQUQRDT, W.W. and KING, D.J. (1986) Isolation of a monoclonal antibody with specificity for commonly employed vaccine strains of Newcastle disease virus. Avian Disease 30: 562-567.

TIMMS, L. and ALEXANDER, D.J. (1977) Cell mediated immune response of chickens to Newcastle disease vaccines. Aviun Parhology 6: 51-59.

TOTH, T.E. (2000) Nonspecific cellular defence of the avian respiratory system: a review. Developmental and Comparative Immunology 24: 12 1 - 139.

VAiNIO, 0. and TOIVANEN, A. (1987) Cellular cooperation in immunity. In: Toivanen, A. and Toivanen, P. (Eds), Aviun ltnmanology: Basis and Practice Volume 11, CRC Press Inc. Boca Raton, Florida, p. 8- 12.

WESTBURY, H.A. (2001) Newcastle disease virus: an evolving pathogen?Avian Pathology 30: 5-1 1. WILDE, A., MCQUAIN, C. and MORRISON, T. (1986) Identification of the sequence content of four

ZINKERNAGEL, R.M. (1994) Some general aspects of immunity to viruses. Vaccine 12: 1493-1494. ZUCKER, R., JAUKER, U. and DROEGE, W. ( 1 973) Cellular composition of the chicken thymus: effects of

neonatal bursectomy and hydrocortisone treatment. European Journal qf Immunology 3: 8 12-81 8.

polycistronic transcripts synthesized in Newcastle disease virus infected cell. Virus Reseurch 5: 77-95.


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