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Thermostable vaccines for Newcastle disease: a review

Published online by Cambridge University Press:  13 November 2014

M. SHAHID MAHMOOD ,
F. SIDDIQUE ,
I. HUSSAIN ,
S.I. AHMAD  and
A. RAFIQUE
M. SHAHID MAHMOOD*
Affiliation:
Institute of Microbiology, University of Agriculture, Faisalabad-38040, Pakistan
F. SIDDIQUE
Affiliation:
Institute of Microbiology, University of Agriculture, Faisalabad-38040, Pakistan
I. HUSSAIN
Affiliation:
Institute of Microbiology, University of Agriculture, Faisalabad-38040, Pakistan
S.I. AHMAD
Affiliation:
Institute of Microbiology, University of Agriculture, Faisalabad-38040, Pakistan
A. RAFIQUE
Affiliation:
Department of Zoology, Wildlife and Fisheries, GC University, Faisalabad-38040, Pakistan
*
Corresponding author: shahiduaf@gmail.com

Abstract

Newcastle Disease (ND) is regarded as one of the major diseases of poultry because of the devastating losses that the virulent form of the ND virus can impose on both commercial and domestic chickens. However, the disease can be controlled through the administration of effective vaccines. Almost all the commercially available ND vaccines require refrigeration and begin to deteriorate rapidly after 1-2 hours if left at room temperature (around 25°C). Subsequently, because maintaining an adequate supply of refrigerated facilities may be a difficult task in many countries with unreliable electrical supplies, the development and large scale production of an effective thermostable ND vaccine seems imperative to support the poultry industry. Such vaccines should be resilient to damage associated with either very cold or hot environments so there are no concerns about the viability of the vaccine in response to temperatures fluctuations that can occur in extreme environments when the difference in temperature during cold and warm seasons can vary greatly. In the following review paper, the development of such a vaccine is discussed, including molecular characterisation, organ tropism of vaccine strains, production, as well as administration methods and their efficacy. It addition, the potential payback has been calculated alongside opportunities provided by removing vaccines from cold chain storage.

Keywords

thermostable vaccinescold chainNewcastle diseasevaccine efficacy

Information

Type
Review Article
Information
World's Poultry Science Journal , Volume 70 , Issue 4 , December 2014 , pp. 829 - 838
Copyright
Copyright © World's Poultry Science Association 2014 

Introduction

The entire globe is burdened by the threat of Newcastle disease except coastal and oceanic zones (Czegledi et al., Reference CZEGLEDI, UJVÁRI, SOMOGYI, WEHMANN, WERNER and LOMNICZI2006). In 1926, a communication regarding this viral disease was first reported from Indonesia (Java) and England (Newcastle-upon-Tyne) for which it has been subsequently named (Doyle, Reference DOYLE1927). The viral disease is caused by avian Paramyxovirus type-1 that belongs to the genus Avulavirus of the sub-family Paramyxovirinae (Xiao et al., Reference XIAO, PALDURAI, NAYAK, SAMUEL, BHAROTO, PRAJITNO, COLLINS and SAMAL2012). It is a single stranded, negative sense, non-segmented, enveloped RNA virus, pleomorphic in shape, 100-200nm in diameter and the size of its genome is reported at 15.2kb (Alexander and Senne, Reference ALEXANDER and SENNE2008).

ND is one of the top ranking diseases that affects the poultry industry by causing economic losses due to high morbidity and mortality rates of up to 80-100% (Alexander, Reference ALEXANDER2003; Czegledi et al., Reference CZEGLEDI, UJVÁRI, SOMOGYI, WEHMANN, WERNER and LOMNICZI2006). In developing countries, including Pakistan and Bangladesh, this causes persistent problems for the poultry industry because of the pestilence of ND and the cost of vaccinating against this infection (Czegledi et al., Reference CZEGLEDI, UJVÁRI, SOMOGYI, WEHMANN, WERNER and LOMNICZI2006). It has been estimated that annual outbreaks of ND can easily kill 80% of unvaccinated rural poultry birds (Ananth et al., Reference ANANTH, JOHN, PRIYADARSHINI and ALBERT2008). Different poultry species have been found to be infected with different strains of ND virus and these can also affect other avian species including; broilers, layers, pigeons, ducks, turkeys, ostriches, guinea fowls, peacocks and pheasants (OIE, 2012) and several other wild birds, such as double yellow headed parrots and water fowl (Kalthoff et al., Reference KALTHOFF, BREITHAUPT, TEIFKE, GLOBIG, HARDER, METTENLEITER and BEER2008). The most common mode for viral transmission is by direct contact from bird to bird; bird to human and equipment as well as through droplets from the respiratory tract or through faeces (OIE, 2012).

Measures to control viral transmission

It is challenging to demonstrate the transmission of ND disease without elaborating on its prevention and control. There are two main methods through which viral transmission can be curtailed: vaccination (Alexander, Reference ALEXANDER2003; Nawanta et al., Reference NAWANTA, ABDU and EZEMA2008) and developing effective quarantine and bio-security measures that are successful in the control of spread during outbreaks. ND control campaigns should be initiated at least one month in advance, especially in regions where epidemics of ND are more frequent and the program should be extended if observations confirm that ND is still present. There are numerous preventive measures available including immunising healthy chickens, educating farmers to revaccinate their birds, ensuring high vaccine quality standards and recognising the risks associated with introducing new birds to vaccinated flocks. Based on the above mentioned information, vaccines, particularly a thermostable ND vaccine are the most favourable solution for controlling ND worldwide (Kapczynski and King, Reference KAPCZYNSKI and KING2005; Nega et al., Reference NEGA, MOGES, MAZENGIA, ZELEKE and TAMIR2011).

Immunisation by ND vaccine administration

ND vaccinations have been implemented for almost five decades for the security of the poultry industry (OIE, 2012). Comparable to the control of other viral illness, immunisation is the best solution available to combat ND in rural as well as commercial poultry (Msoffe et al., Reference MSOFFE, BUNN, MUHAIRWA, MTAMBO, MWAMHEHE, MSAGO, MLOZI and CARDONA2010). The fundamental system of inoculation against ND is to incite safe reactions without initiating infection itself. Immunisations generate diverse levels of antibodies that destroy the antigen (Zoth et al., Reference ZOTH, GOMEZ, CARRILLO and BERNSTEIN2008). The protection so conferred relies on a number of variables which elicit different levels of maternal antibodies dependent on the adequacy and nature of immunisation, systems for vaccination and health status of the birds being vaccinated (Illango et al., Reference ILLANGO, OLAHO-MUKANI, MUKIIBI-MUKA, ABILA and ETOORI2005). Oral administrations of thermostable ND immunisation can help with emissions (immunoglobulin A) of plasma units, which eventually cause an immune response on the mucosal surfaces of different organs incorporating the digestive system, trachea, bronchi and oviduct (Jayawardane and Spradbrow, Reference JAYAWARDANE and SPRADBROW1995a; Reference JAYAWARDANE and SPRADBROW1995b; Iroegbu and Nchinda, Reference IROEGBU and NCHINDA1999; Wambura et al., Reference WAMBURA, KAPAGA and HYERA2000; Zoth et al., Reference ZOTH, GOMEZ, CARRILLO and BERNSTEIN2008). This initiates mucosal and cell mediated immunity that eventually assumes a noteworthy part for the security of the inoculated chickens (Echeonwu et al., Reference ECHEONWU, NGELE, ECHEONWU, JOANNIS, ONOVOH and PAUL2008; Komba et al., Reference KOMBA, MBYUZI and MULANGILA2012). Cytokines, especially interferon γ from cytotoxic T lymphocytes, play a significant role in the development of cell mediated immunity (Al-Garib et al., Reference AL-GARIB, GIELKENS, GRUYS and KOCHI2003).

Type of vaccines administered

The most straightforward approach to undertaking an immunisation program is to first assume that the titre of the immunising suspension is sufficiently high to kill (heat or chemical) and thereafter use the attenuated suspension to immunise the birds. Programs readied along these lines are called ‘killed immunisations’ (Folitse et al., Reference FOLITSE, HALVORSON and SIVANANDAN1988). An alternative approach is to use live vaccines, in which avirulent or less virulent viruses are selected and administered to the birds. There are a variety of different types of vaccines currently available against ND e.g. live thermostable avirulent vaccines (Spradbrow et al., Reference SPRADBROW, MACKENZIE and GRIMES1995), live lentogenic, live mesogenic, killed or inactivated oil-emulsified vaccines (Musa et al., Reference MUSA, ABDU, MERA, EMMENNA and AHMED2010) as well as recombinant vaccines (Palya et al., Reference PALYA, KISS, TATAR, MATO, FELFOLDI and GARDIN2012). The administered vaccines all induce immune responses, which will protect the poultry flocks against natural challenges. The lentogenic strains of NDV, Hitchner B1 (HB1) and LaSota are used for the production of vaccines. The embryo infective dose of thermostable vaccines usually has a titre of about 106 dose per bird (OIE, 2012). The intra-cerebral pathogenicity index for HB1 (Hitchner B1), clone30 LaSota and VG/GA is 0.2, 0.36 and 0.35, respectively (OIE, 2012). Mesogenic strains e.g. Komarov and R2B Mukhtaswar strains are less often used for immunisation in chickens because of their severe pathogenic affects (Saifuddin et al., Reference SAIFUDDIN, SARKER, AMIN and RAHMAN1986). Usually, they are used as a secondary vaccination after the administration of lentogenic vaccines (Alexander, Reference ALEXANDER2009). The application of biotechnology has now made it possible to develop novel varieties of vaccines through RNA and DNA technologies. For this purpose, different expression vectors especially for NDV fowl poxvirus, herpes virus of turkeys (Morgan et al., Reference MORGAN, GELB, POPE and SONDERMEIJER1993), Marek's disease virus and adenovirus (Yokoyama et al., Reference YOKOYAMA, MAEDA and MIKAMI1997) are utilised. In these procedures, the genes of F and HN can be inserted into these vectors to produce a recombinant vaccine. Such recombinant vaccines can provide excellent immunity against NDV (Palya et al., Reference PALYA, KISS, TATAR, MATO, FELFOLDI and GARDIN2012).

Thermostable Newcastle disease vaccines

Historically, the development of ND thermostable vaccines was initiated in Australia by ACIAR (Australian Centre for International Agriculture Research) in 1984. They developed two distinct thermostable vaccines, V4 (Simmons, Reference SIMMONS1976) and I-2 vaccines (Spradbrow, Reference SPRADBROW1992b). The efficacy of these vaccines has been successfully tested in different African (Alders and Spradbrow, Reference ALDERS and SPRADBROW2001a) and Asian countries (Spradbrow et al., Reference SPRADBROW, MACKENZIE and GRIMES1995). They have become commercially available vaccines under the ownership of ACIAR (Spradbrow, Reference SPRADBROW1992a). NDV4-HR is a live, avirulent (Spradbrow, Reference SPRADBROW1992a), thermostable vaccine with a superior biological safety record (Anon, 1991) and it retains it activity at 28°C in a lyophilised form for at least 12 weeks (Nssien and Adene, Reference NSSIEN and ADENE2002) and for nearly a year at 4-8°C (Wambura, Reference WAMBURA2003). These vaccines also have the advantage of being easy to administer as they can be given intranasally, intraocularly, by injection or by mixing with feed and drinking water (Alders and Spradbrow, Reference ALDERS and SPRADBROW2001a). The NDV4-HR vaccine has shown good results in different countries including Zambia (Alders et al., Reference ALDERS, INOUE and KATONGO1994), Cameroon (Bell et al., Reference BELL, FOTZO, AMARA and AGBEDE1995), Tanzania (Foster et al., Reference FOSTER, CHITUKUROA, TUPPAA, MWANJALAB and KUSILA1999), Ghana (Amakye et al., Reference AMAKYE, AWUNI, COLEMAN and SEDOR2000), South Africa and various other tropical and subtropical countries of Southeast Asia (Spradbrow, Reference SPRADBROW1993/94). The Food and Agriculture Organisation (FAO) has recommended the use of NDV4-HR vaccine for village poultry chickens in tropical and developing countries (FAO, 1997).

In Australia collaboration between ACIAR and UQ (University of Queensland) has led to the development of a seed virus similar to V4 (Bensink and Spradbrow, Reference BENSINK and SPRADBROW1999) called I-2 strain (Wambura, Reference WAMBURA2003). Field records of the use of I-2 in various countries including Tanzania (Wambura et al., Reference WAMBURA, KAPAGA and HYERA2000), Vietnam, Mozambique (Alders, Reference ALDERS2004) and South Africa have shown the development of protective levels of antibodies against ND (Alders and Spradbrow, Reference ALDERS and SPRADBROW2001b). The major advantages of thermostable vaccines include reducing vaccine wastage, increased effectiveness of vaccine efficacy, reduced dependence on refrigeration, reduced cost of equipment repairs, ease of application and transportability of material (Alders and Spradbrow, Reference ALDERS and SPRADBROW2001a). Today partially thermostable ND vaccines are commercially available but with little efficacy e.g. Nobilis ND Inkukhu, which is a freeze-dried vaccine. Thermostable I-2 ND vaccine has provided 100% protection of housed chickens and 89% protection to unhoused chickens against ND (Illango et al., Reference ILLANGO, MUKIIBI, ABILA, MUSISI and ETOORI2008).

Molecular characteristics of thermostable Newcastle disease strains

The use of RT-PCR has provided a true genetic picture of thermostable Newcastle disease strains. The amino acid sequence of the V4 and I-2 NDV strains were 112GKQGR↓LIG119 and 112RKQGR↓LIG119, respectively. Arginine is present at position 112, glycine at 115 and leucine at 115, which may indicate that the genetic makeup of the I-2 strain is avirulent in nature while V4 strains contain glycine amino acid at position 112 and the HN protein of the I-2 strain is comprised of 578 amino acids. The carboxyl terminus of the haemagglutination neuraminidase (HN) protein of the I-2 strain contains seven, while V4 contains 45 amino acid residues (Gould et al., Reference GOULD, HANSSON, SELLECK, KATTENBELT, MACKENZIE and DELLA-PORTS2003; Miller et al., Reference MILLER, DECANINI and AFONSO2010). It has been reported by many research workers on the basis of F gene sequence analysis that the I-2 strain belongs to the group of avirulent avian paramyxo viruses (OIE, 2000; Aldous et al., Reference ALDOUS, MYNN, BANKS and ALEXANDER2003; Wambura et al., Reference WAMBURA, MEERS and SPRADBROW2007). The thermostability of the I-2 strain mainly depends upon the deletion of an amino acid at the 403 position in the HN proteins (Yusoff et al., Reference YUSOFF, TAN, LAU, NG and IBRAHIM1996). Some scientists have reported that the thermostability of NDV strains may be dependent upon nucleoprotein (N) and polymerase (L) (Horikami et al., Reference HORIKAMI, CURRAN, KOLAKOFSKY and MOYER1992; Kattenbelt et al., Reference KATTENBELT, STEVENS and GOULD2006).

Organ tropism of thermostable Newcastle disease viral strains

Tracheal organ cultures are best for the investigation of thermostable NDV tropism. The use of thermostable I-2 strains grown in tracheal epithelium cells would cause less destruction as compared with the V4 strain. Therefore, it has been argued that the V4 strain causes less tropism in the respiratory tract compared with the I-2 strain and vice versa in the case of digestive tract tropism (Rehmani and Spradbrow, Reference REHMANI and SPRADBROW1995; Wambura, Reference WAMBURA2006). In earlier studies, various researchers have reported that neither the V4 nor I-2 viruses were present in brain tissues and conjunctivae (Wambura, et al., Reference WAMBURA, MASAMBU and MSAMI2006a). These organ tropism variations may be due to the difference in HN associated proteins, C-terminal and cleavage sites of fusion proteins. V4 is reported to have forty five amino acid extensions at the C-terminal end of HN protein and I-2 contains seven amino acid extensions (Wambura, Reference WAMBURA2003). Based on the above described observations, it has been suggested that I-2 viral strains produce better quality live vaccines compared to V4 strains (Wambura, Reference WAMBURA2006).

Comparison of vaccine production methods

Thermostable ND vaccines can be produced using two methods: by utilisation of embryonated chicken eggs or through cell culture methods (OIE, 2000; Illango et al., Reference ILLANGO, OLAHO-MUKANI, MUKIIBI-MUKA, ABILA and ETOORI2005). Modern egg-based vaccines are prepared from specific-pathogen-free (SPF) eggs. In developing countries, SPF eggs have been used for the commercial production of Newcastle disease vaccines. For some time the production of thermostable vaccines depended upon the use of embryonated SPF eggs, which had advantages compared with production through a variety of cell lines, for example in special cultures where trypsinisation is not required which present less chance of contamination, easier processing and purification. SPF eggs also have some disadvantages such as a high cost of production including greater man power requirements (Alders and Spradbrow Reference ALDERS and SPRADBROW2001b).

The alternate, easier, more convenient and less expensive method is to use cell culture systems for the production of vaccines. Viruses cannot grow without suitable host cells. Primary cell lines of different origin and SPF eggs have been used for the production of Newcastle disease vaccine, principally of avian origin and sometimes of mammalian origin. The cell lines mainly used are rabbit, pig, calf, chicken, MKC, CEF, BGM-70 (Mohan et al., Reference MOHAN, DEY, KUMANAN, MAHOHAR and NAINAR2007), BHK-21 and Vero cells (Ahmed et al., Reference AHMED, HOSSAIN, BILLAH, ISLAM, AHSAN and ISLAM2004). These cell lines are easy to handle and manipulate in Reference BURLESON, CHAMBERS and WIEDBRAUKlaboratories. After 48 hours of growth in MEM medium, a confluent monolayer of Vero cells can be displayed (Ahmed et al., Reference AHMED, HOSSAIN, BILLAH, ISLAM, AHSAN and ISLAM2004). Chick embryo fibroblasts are among the preferred cells for the growth of Newcastle disease virus (Ahmed et al., Reference AHMED, HOSSAIN, BILLAH, ISLAM, AHSAN and ISLAM2004). It has been documented that mesogenic and velogenic viral strains formed cytopathic effects (CPE) on chick embryo fibroblast (Madhan et al., Reference MADHAN, MOHAN and KUMANAN2005) and Vero cell lines (Wambura et al., Reference WAMBURA, MEERS and SPRADBROW2007). The I-2 strain has been reported to produce more CPE when grown into embryonic kidney cells as compared to the V4 strain.

In previous studies it has been documented that avirulent strains could not be harvested in kidney cell culture systems without the addition of trypsin. This may be due to the absence of paired amino acids at the cleavage site F0 (Ragavan et al., Reference RAGAVAN, KUMARAN and NACHIMUTHU1998) but in 2006, Wambura et al. (Reference WAMBURA, MEERS and SPRADBROW2006b) demonstrated that the I-2 strain possessed the ability to grow in chicken embryonic kidney cells without the addition of trypsin in the cell culture medium. MSC (Mesenchymal stem cells) isolated from the bone marrow of chickens can also be used for the production of Newcastle disease vaccines (Mohamed et al., Reference MOHAMED, KUMAR, PALDURAI, MEGAHED, GHANEM, LEBDAH and SAMAL2012). Many diluents play a significant role in the maintenance of the shelf life of thermostable vaccines. Skim milk and gelatin have been recommended for the long storage of vaccines; 1% gelatin and 5-10% skimmed milk maintained antibody titres for 12 weeks at room temperature (Young and Alders, Reference YOUNG and ALDERS2002).

Methods for the administration of vaccines

The standard dose of ND vaccines is 106 EID50/bird (OIE, 2012). Different methods for administering ND vaccines include mixing the vaccine in drinking water, the use of an eye dropper, sprays, mixing it with feeds and injection. Most effective appears to be the eye drop method (Musa et al., Reference MUSA, ABDU, MERA, EMMENNA and AHMED2010) because it provides strong protection after administration, especially in view of revaccination every 3-4 months (Mgomezulu et al., Reference MGOMEZULU, ALDERS, CHIKUNGWA, YOUNG, LIPITAL and WANDAL2009; Komba et al., Reference KOMBA, MBYUZI and MULANGILA2012). The drinking water method is easier, but it elicits lower immunity levels (Rahman et al., Reference RAHMAN, RAHMAN, RAHMAN, KABIR, NAZIR and AMIN2004). In developing countries, thermostable vaccines have been successfully administered through mixing in feeds e.g., cooked white rice (Wambura, Reference WAMBURA2011), parboiled sorghum (Echeonwu et al., Reference ECHEONWU, NGELE, ECHEONWU, JOANNIS, ONOVOH and PAUL2008) and millet grain (Illango et al., Reference ILLANGO, OLAHO-MUKANI, MUKIIBI-MUKA, ABILA and ETOORI2005). Food-based heat tolerant vaccines have many benefits. They avoid the pressures of handling birds which is required for single or shower vaccination methods. However, whole live passaged poultry vaccines are not suitable for administration through food (Spradbrow, 1993/94). Cooked white rice has been used as an active transporter of thermostable NDV vaccines as compared to white rice (Tu et al., Reference TU, PHUC, DIHN, QUOC and SPRADBROW1998) because it is cheap, stable, shows less antiviral activity and is more acceptable to the birds. Vegetable oil is used for coating rice which may increase its stability as well as the efficacy of vaccines (Wambura et al., Reference WAMBURA, MEERS and SPRADBROW2007; Wambura, Reference WAMBURA2009). The vaccine should be safe, potent, effective, pure and easy to use, and these factors are important considerations for the success of vaccination (Spradbrow et al., Reference SPRADBROW, MACKENZIE and GRIMES1995).

Efficacy of thermostable ND vaccine in field trials

To check the efficacy of thermostable ND vaccine, knowledge of the level of antibodies produced in challenged birds as well as the capability of target birds to overcome the pathogenic agent is required (Allan and Gough, Reference ALLAN and GOUGH1974). In different countries including Australia, Malaysia (Wambura et al., Reference WAMBURA, KAPAGA and HYERA2000), Tanzania (Foster et al., Reference FOSTER, CHITUKUROA, TUPPAA, MWANJALAB and KUSILA1999) and Nigeria (Musa et al., Reference MUSA, ABDU, MERA, EMMENNA and AHMED2010) successful field trials have already been documented. According to OIE (2012) the protective level for Newcastle disease vaccine titres are HI > Log24. It has been shown that the protection level of thermostable NDV vaccines of around 80% can be reached in the field (Alders, Reference ALDERS2005).

The efficacy of the vaccine primarily depends upon the administration methods employed and the temperature of the environment (Musa et al., Reference MUSA, ABDU, MERA, EMMENNA and AHMED2010). The eye drop method provokes high antibody titre (80%) as compared to the addition to drinking water (60%). Administering the vaccine in food is successful only to about 30% (Wambura et al., Reference WAMBURA, MEERS and SPRADBROW2001; Alders, Reference ALDERS2005). It has been shown that the antibody titre was reached to the extent of 70% to 95% after primary vaccination (Musa et al., Reference MUSA, ABDU, MERA, EMMENNA and AHMED2010). It has been demonstrated that eye drop vaccination of I-2 strain can help maintain HI antibody titres for nine months in village chickens. This may be attributed to re-infection of the adult birds and may act as a booster dose. Similar results were observed in V4 strains (Samuel and Spradbrow, Reference SAMUEL and SPRADBROW1991). Olabode et al. (Reference OLABODE, NDAKO, ECHEONWU, NWANKITI and CHUKWUEDO2010) determined the efficacy of V4 vaccine suspension by administration through maize grains and demonstrated that soaked maize gave higher antibody titre as compared to unsoaked. Similar finding were observed by other investigators (Agbor, Reference AGBOR1999; Echeonwu et al., Reference ECHEONWU, NGELE, ECHEONWU, JOANNIS, ONOVOH and PAUL2008). The titres of thermostable Newcastle disease vaccines are usually reduced after certain periods of time e.g. thermostable lyophilised vaccine was reduced in its titre to the extent of 26% after one month at 37°C (Nssien and Adene, Reference NSSIEN and ADENE2002). Nega et al. (Reference NEGA, MOGES, MAZENGIA, ZELEKE and TAMIR2011) demonstrated that I-2 vaccine strain produced higher antibody titres as compared to conventional vaccines.

Conclusions

This review has highlighted the use of thermostable vaccines to enhance productivity and reduce storage costs. Thermostable vaccines would minimise vaccine wastage and increase product shelf life. As the control of viral diseases is currently only possible through vaccination and bio-security actively deployed throughout the world, it follows that these vaccines are the best defence against ND disease currently available and they are very durable as their activities are maintained at 28°C for up to 6-8 weeks and at 4-8°C for 1 year. The majority of commercial vaccines are generally thermolabile and require cold storage for their effectiveness. In developing countries, vaccine distribution, without concern for cold storage systems, is a very attractive proposition due to it being significantly easier to transport and distribute for field administration. Accordingly, in developing countries including Pakistan, where electric supply is intermittent, it provides a viable and effective alternative. Subsequently the development and administration of a thermostable poultry vaccine as described here is expected to be a significant development in the health and economic development where supply of adequate and economical protein nutrition is of significant concern.

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