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Chapter 1 Introduction

CHAPTER 1
INTRODUCTION
Avian influenza (AI) is a serious disease of poultry, resulting in severe mortality in
chickens and major disruption to production and trade (FAO, 2004). AI has been
recognized as a highly contagious and lethal generalized viral disease of poultry since
1901(Anonymous, 2004). LPAI viruses cause respiratory and gastrointestinal infections
without infecting the meat. By contrast, HPAI viruses produce infection of respiratory
and gastrointestinal tracts, produce a viremia and virus is present in the meat and internal
contents of eggs during the acute stages of the infection (Swayne, 2004). In highly
pathogenic AI, the disease appears suddenly in a flock, and many birds die either without
premonitory signs or with minimal signs of depression, inappetence, ruffled feathers and
fever (Anonymous, 2004; Alexander, 2000). There is extensive subcutaneous oedema,
particularly around the head and hocks. The carcass may be dehydrated. Yellow or grey
necrotic foci may be present in the spleen, Liver kidneys and lungs. The spleen may be
enlarged and haemorrhagic (Anonymous, 2004). After infection poultry, excreted virus
from both the respiratory and the digestive tracts, resulting in rapid spread through a
population of susceptible host. Bird to bird transmission very efficient via aerosol,
contaminated feces, or various fomites, and the virus can cause a wide range of disease
symptoms (Alexander, 2000;Bankowski, 1981; OIE, 1996; Easterday, 1997; Swayne,
2000).

Domestic fowl, ducks, gees, turkeys, guinea fowl, quail and pheasants are susceptible.
Disease outbreaks occur most frequently in domestic fowl and turkeys. Often virulent
strains emerge either by genetic mutation or by reassortment of less virulent strains. The
incubation period is usually three to seven days, depending upon the strain, the dose of
inoculum, the species and the age of the bird (Anonymous, 2004; Alexander, 2000).

In 1955, a specific type (A) of influenza virus was identified as the causal agent of “fowl
plague” (Anonymous, 2004). All avian influenza (AI) viruses belong to the Influenza
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virus A genus of the Orthomyxoviridae family and are single stranded, negative polarity,
segmented RNA viruses. Influenza A viruses can be divided into subtypes on the basis of
the possession of one of 15 antigenically distinct haemagglutinin (HA) antigens (H1 to
H15) and one of nine neuraminidase (NA) antigens (N1 to N9) (WHO, 1980; Swayne,
2004; anonymous, 2004). The genetic pool for all AI viruses is primarily in aquatic birds,
which are responsible for the perpetuation of these viruses in nature (Alexander, 2000).
Influenza A viruses infecting poultry can be divided into two distinct groups on the basis
of their ability to cause disease. The most virulent viruses cause highly pathogenic avian
influenza (HPAI), which may result in flock mortality as high as 100%. All other viruses
cause low pathogenic avian influenza (LPAI) consisting primarily of mild respiratory
disease, depression and egg production problems in laying birds (Capua and Alexander,
2004). HPAI viruses are not normally present in wild bird populations and only arise as a
result of mutation after H5 or H7 LPAI viruses have been introduced to poultry from wild
birds (Garcia et.al., 1996; Perdue, et.al., 1998).

AI has been recognized for well over one hundred years in poultry since 1901. Since the
first report of an HPAI outbreak caused by a virus of H5 subtype, in 1959 was reported
(Anonymous, 2004). Further outbreaks have been occurred in different countries at
different times, caused by influenza A viruses of either H7, H9, or H5 subtype in poultry
(Anonymous, 2004). Outbreaks occurred in Asian countries including China (Yingjie,
1998), Pakistan (Naeem, 1998; Naeem et.al., 1999; 2003; Bano et.al., 2003; Capua and
Alexander, 2004), Korea (Mo et.al., 1998; Lee et.al., 2000), Hong Kong (Guan et.al.,
2000; Shortridge, 1999; Sims et.al., 2003), Middle East (Capua and Alexander, 2004),
Iran (Nili and Asasi, 2002; 2003), South East Asian Countries(Capua and Alexander,
2004), Taiwan (Capua and Alexander, 2004). In European countries outbreaks in UK
(Scotland), UK (England) (Anonymous, 2004), Germany (Werner, 1998; Fioretti et.al.,
1998; Werner et.al., 2003), Ireland (Campbell, 1998; Campbell and De Geus, 1999), Italy
(Capua et.al., 1999; Fioretti et.al., 1999; Marangon et.al., 2003; Capua and Alexander,
2004), Northern Ireland (Graham et.al., 1999) and Netherlands (Capua and Alexander,
2004) has been reported. In South Africa (Banks et.al., 2000b) and Australia (Westbrey,
1998; Selleck et.al., 2003) out breaks occurred from 1976 to 1995. In American countries
out breaks occurred including Canada (Ontario), USA, Mexico (Anonymous, 2004);

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Mexico (Senne, 1998), USA (Halvorson et.al., 1998), USA (Pennsylvania) (Davison
et.al., 2003), USA (Virginia) (Spackman and Squarez, 2003), USA (Connecticut) (Capua
and Alexander, 2004), USA (Texas) (Capua and Alexander, 2004), Canada (Pasick
et.al., 2003), Chile (Rojas et.al., 2002) and Canada (Capua and Alexander, 2004).

A variety of strategies and techniques have been developed for diagnostic studies of AIV.
Currently, virus isolation (VI) in embryonating chicken eggs and subsequent HA and
neuraminidase subtyping by serological methods constitute the standard for AIV
detection and subtype identification. Conversely, real-time reverse transcriptase
polymerase chain reaction (RRT-PCR) can be a rapid assay (Spackman et.al., 2002).
Standard RT-PCR has been previously applied to the detection of avian influenza virus
(Lee et.al., 2001; Munch et.al., 2001; Starick et.al., 2000; Suarez, 1997) and each of the
15 HA subtypes (Lee et.al., 2001; Munch et.al., 2001). Additionally, an RRT-PCR assay
for influenza virus has been developed. RRT-PCR offers the advantages of speed and no
post-PCR sample handling, thus reducing the chance for cross-contamination compared
to standard RT-PCR (Spackman et.al., 2002). Nucleic acid sequence-based amplification
(NASBA) technique (Collins et.al., 2002), combined reverse transcription (RT)-PCR
heteroduplex mobility assay (HMA) (Ellis and Zambon, 2001), enzyme-linked
immunosorbent assay (ELISA) (Abraham et.al., 1986; Snyder et.al., 1985; Sala et.al.,
2003), competitive enzyme-linked immunosorbent assay (c-ELISA) (Zhou et.al., 1998;
Shafer et.al., 1998), double antibody sandwich DAS-ELISA (Kodihalli et.al., 1993),
multiplex reverse transcriptase-polymerase chain reaction RRT-PCR (Spackman et.al.,
2003), hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay
(ELISA) (Meulemans et.al., (1987; Abraham et.al., 1988 ) were used to detect AIV. Agar
gel immunodiffusion test AGID (Beard, 1970; Abraham et.al., 1988), AI indirect
fluorescent antibody (IFA) (Shafer et.al., 1998), neuraminidase-inhibition (NI) tests
(Shafer et.al., 1998), soluble antigen fluorescent test (Al-Attar et.al., 1981),
nucleoprotein reverse transcriptase NP RT-PCR-ELISA (Dybkaer et.al., 2004) have been
developed for the identification of influenza A subtype viruses.

Various vaccine have been used for immunization against avian influenza, including
conventional inactivated oil-adjuvanted whole AI virus (Halvorson, 1995; Pomeroy,
1995; Naeem, 1998; Garcia and Alvrez, 1999; Swayne and Suarez, 2000), Inactivated
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whole avian influenza (AI) virus, baculovirus-derived AI haemagglutinin, vectored virus,
subunit protein and DNA vaccines (Swayne, 2003; Swayne et.al., 2001). AI vaccines can
prevent clinical signs, deaths and reduce respiratory and intestinal replication of a
challenge virus, increase resistance of birds to infection, and decrease the amount of virus
shed in the environment (Swayne, 2004). This protection is specific only for individual
subtypes of haemagglutinin (H1-15) and neuraminidase (N1-9) proteins. (Swayne, 2003;
Swayne, 2001). Whole virus vaccine produced a significantly higher probability of
seroconversion compared to subunit vaccine (Stephenson et.al., 2003). Inactivated whole
avian influenza (AI) virus vaccines, baculovirus-derived AI haemagglutinin vaccine and
recombinant fowl poxvirus-AI heamagglutinin vaccine provide protection against a
variety of different AI viruses (Qiao et.al., 2003; Brugh et.al., 1979; Swayne et.al.,
2001). The Differentiating infected from vaccinated animals (DIVA) control strategy
presents a tool for the control of avian influenza infections in poultry (Capua et.al.,
2002). Adjuvanted vaccines elicit higher immune response, higher rates of sero-
conversion and sero-protection compared to non-adjuvanted vaccines (Podda, 2001).

Immunogenicity analyses demonstrated a consistently higher immune response with
statistically significant increases of postimmunisation geometric mean titres, and of
seroconversion and seroprotection rates compared to non-adjuvanted subunit and split
influenza vaccines (Podda, 2001). Historically, inactivated whole viruses using various
adjuvant systems have been used (Swayne, 2004). It acts as a deposit or reservoir,
chemical immune stimulators of lymphoid cells, release antigen progressively, and
present the antigen directly to the competent cells (Exopol, 2002). Different adjuvant may
be used to enhance the potency of the vaccines such as, lipopolysaccharides, cytokines,
lipid A (Exopol, 2002), IL-2 Suplemented Liposomes (Yehuda et.al., 2003), IL-6
Suplemented Liposomes (Lachman et.al., 1995), Liposome (verma et.al., 2004;
Huckriede et.al., 2003; Voinea and Simionescu 2002; Exopol, 2002; Baldo et.al., 2001;
Budai and Szogyi, 2001;Carl, 1997, 1987; Mbawuike et.al., 1990; Fogler et.al., 1987),
negatively and positivlely charged Liposome (Fatanmbi et.al., 1992; Kraaijeveld et.al.,
1984). Strericaly stabilized Liposomes (SSL-IL-2) (Kedar et.al., 1994), Liposomes
encapsulated Hb (Carl, 1994), MF59 (Stephnson et.al., 2002; Nicholson et.al., 2001;
Gasparini et.al., 2001; Podda, 2001; Baldo et.al., 2001; Minutello 1997),

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Immunostimulating complex (Iscom) (Exopol, 2002; Deliyannis et.al., 1998; Coulter
et.al., 1998), Oil in water containing squalene (Podda, 2001; coulter et.al., 1998), Oil
emulsion (Ston, 1987; Ston, 1993), Mineral oil emulsion (Ston, 1993), Alum (Gluck,
1999), Aluminum (Hehme, 2004) may be used to boost vaccines potency. N-
acetylglucosaminyl-N-acetylmuramyl-dipeptide (GMDP) (Plache et.al., 1996), Syntex
(Hjorth et.al., 1997), Polymerized and non polymerized Liposomes (Alonso-Romanowski
et.al., 2003), immunopotentiating, reconstituted influenza virosomes (IRIV) (Gluck,
1999; Cryz & GlucImmunopk, 1998), co-polymer adjuvant (CRL1005) (Katz et.al.,
2000), Poly[di (carboxylatophenoxy)phosphazene] (PCPP) (Payne et.al., 1998) and
Freund,s complete adjuvant (Hjorth et.al., 1997) have been used experimentally.

The present study was designed to evaluate the comparative immunological response of
commercial oil based and liposomal vaccines of Avian Influenza. Commercial oil based
(Merial 17, Rue Bourglat Lyon, France) and Liposomal AI vaccine (MediExcel
Pharmaceuticals, Islamabad, Pakistan) were evaluated in commercial broilers. The
present study will be helpful in restoring the commercial losses of the farmers and
preventing the flock mortality due to the high efficacy of liposomal vaccine against avian
influenza.

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