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Avian Newcastle Disease is a highly contagious and infectious disease that affects all avian species including poultry, cage and wildlife bird species. Depending on the ND virus (NDV) strain, nature of the exposure, species of bird and age the range of clinical signs can be variable. Outbreaks of ND in commercial poultry and wildlife bird populations can cause high mortalities that can reach 100%. Clinical disease may present in the form of respiratory, intestinal and neurological signs (Saif, Y.M et al., 2003).
NDV is a member of the genus Avulavirus, under the subfamily Paramyxovirinae, family Paramyxoviridae and the order Mononegavirales (Mayo, 2002). Strains of NDV can be characterized into lentogenic, mesogenic, and velogenic categories, based on its pathogenicity in chickens. Lentogenic strains seldom cause disease in adult chickens. Viruses of intermediate virulence are called mesogenic while viruses that cause high mortality are termed velogenic (Huang et al., 2003). NDV caused Newcastle Disease (ND) which is the most devastating infectious diseases of poultry (Alexander, 2003).
Chickens are considered to be the most susceptible and likely to present with clinical ND. According to the USGS National Wildlife Health Centre (1999), this distribution is a result of three panzootics since it was discovered in 1926. The first highly pathogenic strain was sourced from Southeast Asia that infected poultry and domestic birds. The second panzootic occurred in the Middle East in the 1960's and was attributed to the movement and importation of caged psittacine species. The most recent panzootic was also in the Middle East during the 1970's which primarily involved pigeons and doves. In North America NDV has been found to cause disease in wild double crested cormorants. The infection of the Newcastle Disease Virus infected the chicken in the chicken production causes the great lost to the farmer. The production of the good vaccine will help to prevent the great lost to the chicken poultry production and it can help to improve the poultry production industries.
In this study, Malaysian isolate of NDV strain AF2240 is used and this strain often caused 100% death in susceptible chickens (Lai, 1985). It was used to test the immunogenicity antibody reaction in the producing the antibody against the injected NDV AF2240 viral protein in the rabbit and mice. The objective of this study was to develop the antibody against the NDV AF2240 by using the rabbit and mice and to investigate the immune reactivity of the antibody produce towards the NDV viral antigen. The basic hypothesis of this study is the serums extracted from the animal contain the antibody against the NDV AF2240 strain viral antigens.
2.1 History of Newcastle Disease
Newcastle disease (ND) is a highly infectious disease of birds that threaten many domestic and wild avian species. The first outbreaks of ND happened in Java, Indonesia (Kraneveld, 1926) and one year later in 1927, in Newcastle-upon-Tyne, England (Doyle, 1927). In 1986, ND is considered as the major factor that causes the death of all the chickens in the Western Isles of Scotland (Macpherson, 1956). Doyle (1935) was the first person to coin the name "Newcastle disease". In Malaysia, ND has contributed to major losses to the poultry industry in Malaysia in terms of mortality and loss in egg and meat production (Ideris, 1990). The causative virus of this disease is an avian paramyxovirus, which is Newcastle disease virus (NDV).
2.2 Newcastle Disease Virus (NDV)
NDV is a member of the genus Avulavirus under the subfamily Paramyxovirinae, family Paramyxoviridae and the order Mononegavirales (Mayo, 2002). ND is a synonym of 'avian paramyxovirus type 1' (APMV-1). The Paramyxoviridae family also includes a number of important disease-causing viruses, including measles virus (MeV), mumps virus (MuV), and the human parainfluenza viruses (HPIV). An overview of the taxonomy of Paramyxoviridae and examples of representative viruses are given in Fig. 1.
Figure 1: Phylogenetic tree based on the N protein sequences of selected paramyxoviruses. Virus names as follows: Avian paramyxovirus 6 (APMV6); Atlantic salmon paramyxovirus; Beilong virus (BeiPV) ; Bovine parainfluenza virus 3 (bPIV3); Canine distemper virus (CDV); Cedar virus (CedPV); Fer-de-lance virus (FdlPV) ; Hendra virus (HeV); Human parainfluenza virus 2 (hPIV2); Human parainfluenza virus 3 (hPIV3) ; Human parainfluenza virus 4a (hPIV4a) ; Human parainfluenza virus 4b (hPIV4b); J virus (JPV); Menangle virus (MenPV); Measles virus (MeV); Mossman virus (MosPV); Mapeura virus (MprPV); Mumps virus (MuV); Newcastle disease virus (NDV); Nipah virus, Bangladesh strain (NiV-B); Nipah virus, Malaysian strain (NiV-M); Parainfluenza virus 5 (PIV5); Peste-des-petits-ruminants (PPRV); Porcine rubulavirus (PorPV); Rinderpest virus (RPV); Salem virus (SalPV); Sendai virus (SeV); Simian virus 41 (SV41); Tioman virus (TioPV); Tupaia paramyxovirus (TupPV). (Marsh GA et al, 2012)
The pathotypes is an infrasubspecific classification of a pathogen distinguished from others of the species by its pathogenicity on a specific host. The NDV can be classified into a few groups according to their infection ability (Beard, C. W et al, 1981).
Lentogenic NDV strains cause subclinical infection with mild respiratory or enteric disease and are considered to be low-virulent (Beard, C. W et al, 1981).
Mesogenic NDV strains are of intermediate virulence causing respiratory infection with moderate mortality, while velogenic NDV strains are highly virulent causing high mortality (Beard, C. W et al, 1981).
Velogenic strains are further classified into viscerotropic velogenic and neurotropic velogenic strains. Viscerotropic velogenic strains produce lethal hemorrhagic lesions in the digestive tract, whereas neurotropic velogenic strains produce neurological and respiratory disorders (Alexander, D. J. 1997)
2.2.3 Virion of NDV
NDV virion has been revealed to show pleomorphism characteristics. It maybe spherical in shape with approximately 100-500 nm in diameter but commonly observed in filamenteous form. The envelope of NDV is covered with spike glycoproteins with approximately 8-12 nm in diameter. The presence of spikes is the main characteristics of Paramyxovirus. The spike consists of HN protein and F protein which interact synergistically with each other and involved in viral infectivity and virulence (Stone-Hulslander and Morrison, 1997). Figure 2 showed the morphology of NDV.
Enveloped, spherical. Diameter from about 150nm nm.
Figure 2: Schematic diagram of Newcastle Disease Virus (Adapted and modified from Swiss Institute of Bioinformatics Website. Retrieved March 10, 2013 from http://viralzone.expasy.org)
Mechanism of NDV Infection
There are two major steps involved when susceptible cells are infected by NDV (Figure 2). During the first step, virus binds to specific cell surface receptors containing sialic acids (Huang et al., 1980) via viral attachment protein, HN protein. Then, F protein was activated and directs the fusion of the NDV and the host cell membrane (Choppin and Compans, 1975). Then, viral genome enters the cytoplasm of the host cell.
Figure 3: Schematic Diagram of Cell Infection by NDV. NDV replication is composed of two steps. Right (First Step) - Binding and fusion of the virus with the target cell, transcription of the viral genes and translation (encodes the nucleoprotein [NP], the phosphoprotein [P], the matrix protein [M], the hemagglutinin-neuraminidase protein [HN], and the large [L] proteins). Left (Second Step) - Involved the amplification of the viral genome. The antigenome RNA is used as template for the synthesis of the new viral genomes. Encapsulation of the viral genomes occurs at the plasma membrane, from which new virus particles are released via budding from the infected cell (Schirrmacher, 2009)
In the cytoplasm, negative-stranded RNA of NDV is transcribed into messenger RNA (mRNA) and followed by translation of mRNA to viral proteins such as the NP protein, P protein M protein, HN protein and L protein. Second step of NDV infection is the replication of virus. During this step, nucleocapsids act as an "antigenome" as it is being used as a template for the synthesis of the new viral genomes (Nagai, 1999). The viral RNA-dependent RNA polymerase is composed of the association of the two viral proteins P and L (Hamaguchi et al., 1983).
After post-translational modification, the M, H and F protein move to the membrane. Nucleocapsid assembly required the involvement of proteins NP, P, and L. Besides, M protein that forms the inner layer of the envelope also plays important role during viral assembly (Peeples, 1991). The NDV genomes become encapsulated into an outer coat envelope that is made from host cell's plasma membrane. Lastly, new virus particles are released through budding where sufficient M protein is required (Pantua et al., 2006).
Disease Cause by NDV
NDV is a contagious and fatal viral disease affecting most species of birds. Clinical signs are extremely variable depending on the strain of virus, species and age of bird, concurrent disease, and preexisting immunity. NDV can infect and cause death even in vaccinated poultry.
2.2.6 NDV strain AF2240
NDV strain AF2240 is a Malaysian isolate with a great adaptation to tropical climate and is resistant to heat. Abdul Rahman et al. (1976) and Lai (1985) reported that NDV strain AF2240 causes 100% mortality in susceptible chicken flocks thus affecting poultry industry in Malaysia. One of the main features of NDV strain AF2240 that differentiates it with other published strains are having high thermostabilities of haemagglutination and neuraminidase activities (Ideris et al., 1993). Thus, NDV strain AF2240 has the potential to be manipulated for the development of recombinant vaccine against ND in Malaysia.
2.2.7 NDV as a Promising Cancer Therapy
Viruses especially RNA viruses are quickly recognized as promising agents for oncolytic virotherapy (Russel, 2002). NDV is one of the most promising viruses and was first noted to replicate in and destroy tumor cells in 1955 and is one of five species of viruses that are under clinical evaluation (Aghi and Martuza, 2005). The selectivity towards cancerous cells is unquestionable as replication of certain NDV strains in tumor cells is 10,000 times better than in normal cells (Schirrmacher et al., 1999). The tumor selectivity of NDV is considered to be due to a defective IFN response in tumor cells (Fiola et al., 2006). Numerous research studies have showed that NDV has the potential to develop as an amazing anticancer agent. Cassel and Garrett (1965) reported the observation of outstanding tumor shrinkage after the treatment of a cervical cancer patient with intratumoral NDV.
According to NCI Dictionary of Cancer Term adjuvant is s substance used to help boost the immune response to a vaccine so that less vaccine is needed. Selection of specific adjuvants or types varies depending upon whether they are to be used for research and antibody production or in vaccine development. Adjuvants for vaccine use only need to produce protective antibodies and good systemic memory while those for antiserum production need to rapidly induce high titter, high avidity antibodies.
2.3.1 Freund's adjuvants
There are two basic types of Freund's adjuvants: Freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FIA). FCA is a water-in-oil emulsion that localizes antigen for release periods up to 6 months. It is formulated with mineral oil, the surfactant mannide monoleate and heat killed Mycobacterium tuberculosis, Mycobacterium butyricum or their extracts for aggregation of macrophages at the inoculation site. This potent adjuvant stimulates both cell mediated and humoral immunity with preferential induction of antibody against epitopes of denatured proteins (Sadelain et al., 1991)
The clear liquid that can be separated from clotted blood. Serum differs from plasma, the liquid portion of normal unclotted blood containing the red and white cells and platelets. The term "serum" also is used to designate any normal or pathological fluid that resembles serum as, for example, the fluid in a blister. According to Mandal R, et al. (2011) "Serum" is a Latin word that refers to the "whey", the watery liquid that separates from the curds in the process of cheese making.
In blood, the serum is the component that is neither a blood cell nor a clotting factor because it is the blood plasma with the fibrinogens removed. Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances for example drugs and microorganisms. Serum is used in numerous diagnostic tests, as well as in blood typing (Fox SI, 1999).
An antibody (Ab), also known as an immunoglobulin (Ig), is a large Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. According to Charles Janeway (2001), the antibody recognizes a unique part of the foreign target, called an antigen. Each tip of the "Y" of an antibody contains a paratope that is specific for one particular epitope on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize its target directly (Pier GB et al., 2004). The strength of binding between the antibody and an antigen at a single binding site is known as the antibody's affinity for the antigen. The affinity between the antibody and the antigen binding site is determined by the type of bond formed. The production of antibodies is the main function of the humoral immune system (Pier GB et al., 2004). .
Enzyme-linked immunosorbent assay (ELISA)
ELISA is a popular format of a "wet-lab" type analytic biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample ( Lequin R, 2005). The purpose of an ELISA is to determine if a particular protein is present in a sample (Yalow R et al., 1960). There are two main variations on this method which is antibody quantification and protein quantification in the sample. ELISAs are performed in 96-well plates which permits high through put results (S. Leng et al., 2008)
According to MedLinePlus (2007), the bottom of each well is coated with a protein to which will bind the antibody required antibody. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies which called primary antibodies. The serum is incubated in a well, and each well contains a different serum. A positive control serum and a negative control serum would be included among the 96 samples being tested.
Figure 4: ELISA diagram.
After some time, the serum is removed and weakly adherent antibodies are washed off with a series of buffer rinses. To detect the bound antibodies, a secondary antibody is added to each well.
According to Engvall E et al., (1971), the secondary antibody would bind to all human antibodies and is typically produced in a rodent. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These enzymes can metabolize colourless substrate into coloured products. After an incubation period, the secondary antibody solution is removed and loosely adherent ones are washed off as before. The final step is the addition the enzyme substrate and the production of coloured product in wells with secondary antibodies bound.
When the enzyme reaction is complete, the entire plate is placed into a plate reader and the optical density is determined for each well. The amount of colour produced is proportional to the amount of primary antibody bound to the proteins on the bottom of the wells (Lequin R 2005).
SDS Phage and Western Blot
Western blotting, also known as immunoblotting, is a well-established and widely used technique for the detection and analysis of proteins. The method is based on building an antibody: protein complex via specific binding of antibodies to proteins immobilized on a membrane and detecting the bound antibody with one of several detection methods (Dennis-Sykes et al., 1985). The Western blotting method was first described in 1979 and has since become one of the most commonly used methods in life science research. Western blotting was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture (Dechend R 2006).