You are on page 1of 5

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,

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
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 2
Chapter 1 Introduction
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, 1996; Perdue,, 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, 1999; 2003; Bano, 2003; Capua and
Alexander, 2004), Korea (Mo, 1998; Lee, 2000), Hong Kong (Guan,
2000; Shortridge, 1999; Sims, 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,
1998; Werner, 2003), Ireland (Campbell, 1998; Campbell and De Geus, 1999), Italy
(Capua, 1999; Fioretti, 1999; Marangon, 2003; Capua and Alexander,
2004), Northern Ireland (Graham, 1999) and Netherlands (Capua and Alexander,
2004) has been reported. In South Africa (Banks, 2000b) and Australia (Westbrey,
1998; Selleck, 2003) out breaks occurred from 1976 to 1995. In American countries
out breaks occurred including Canada (Ontario), USA, Mexico (Anonymous, 2004);

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 3
Chapter 1 Introduction
Mexico (Senne, 1998), USA (Halvorson, 1998), USA (Pennsylvania) (Davison, 2003), USA (Virginia) (Spackman and Squarez, 2003), USA (Connecticut) (Capua
and Alexander, 2004), USA (Texas) (Capua and Alexander, 2004), Canada (Pasick, 2003), Chile (Rojas, 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, 2002).
Standard RT-PCR has been previously applied to the detection of avian influenza virus
(Lee, 2001; Munch, 2001; Starick, 2000; Suarez, 1997) and each of the
15 HA subtypes (Lee, 2001; Munch, 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, 2002). Nucleic acid sequence-based amplification
(NASBA) technique (Collins, 2002), combined reverse transcription (RT)-PCR
heteroduplex mobility assay (HMA) (Ellis and Zambon, 2001), enzyme-linked
immunosorbent assay (ELISA) (Abraham, 1986; Snyder, 1985; Sala,
2003), competitive enzyme-linked immunosorbent assay (c-ELISA) (Zhou, 1998;
Shafer, 1998), double antibody sandwich DAS-ELISA (Kodihalli, 1993),
multiplex reverse transcriptase-polymerase chain reaction RRT-PCR (Spackman,
2003), hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay
(ELISA) (Meulemans, (1987; Abraham, 1988 ) were used to detect AIV. Agar
gel immunodiffusion test AGID (Beard, 1970; Abraham, 1988), AI indirect
fluorescent antibody (IFA) (Shafer, 1998), neuraminidase-inhibition (NI) tests
(Shafer, 1998), soluble antigen fluorescent test (Al-Attar, 1981),
nucleoprotein reverse transcriptase NP RT-PCR-ELISA (Dybkaer, 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
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 4
Chapter 1 Introduction
whole avian influenza (AI) virus, baculovirus-derived AI haemagglutinin, vectored virus,
subunit protein and DNA vaccines (Swayne, 2003; Swayne, 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, 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, 2003; Brugh, 1979; Swayne,
2001). The Differentiating infected from vaccinated animals (DIVA) control strategy
presents a tool for the control of avian influenza infections in poultry (Capua,
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, 2003), IL-6
Suplemented Liposomes (Lachman, 1995), Liposome (verma, 2004;
Huckriede, 2003; Voinea and Simionescu 2002; Exopol, 2002; Baldo, 2001;
Budai and Szogyi, 2001;Carl, 1997, 1987; Mbawuike, 1990; Fogler, 1987),
negatively and positivlely charged Liposome (Fatanmbi, 1992; Kraaijeveld,
1984). Strericaly stabilized Liposomes (SSL-IL-2) (Kedar, 1994), Liposomes
encapsulated Hb (Carl, 1994), MF59 (Stephnson, 2002; Nicholson, 2001;
Gasparini, 2001; Podda, 2001; Baldo, 2001; Minutello 1997),

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 5
Chapter 1 Introduction
Immunostimulating complex (Iscom) (Exopol, 2002; Deliyannis, 1998; Coulter, 1998), Oil in water containing squalene (Podda, 2001; coulter, 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, 1996), Syntex
(Hjorth, 1997), Polymerized and non polymerized Liposomes (Alonso-Romanowski, 2003), immunopotentiating, reconstituted influenza virosomes (IRIV) (Gluck,
1999; Cryz & GlucImmunopk, 1998), co-polymer adjuvant (CRL1005) (Katz,
2000), Poly[di (carboxylatophenoxy)phosphazene] (PCPP) (Payne, 1998) and
Freund,s complete adjuvant (Hjorth, 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

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 6