Gordon memorial lecture. Vaccines and vaccination--past, present and future.

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Vaccines and vaccination—past, present and future P. M. Biggs

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’Willows’, London Road, St Ives, Huntingdon, Cambridgeshire, PE17 4ES, England Published online: 08 Nov 2007.

To cite this article: P. M. Biggs (1990) Vaccines and vaccination—past, present and future , British Poultry Science, 31:1, 3-22, DOI: 10.1080/00071669008417226 To link to this article: http://dx.doi.org/10.1080/00071669008417226

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British Poultry Science (1990) 31: 3-22

GORDON MEMORIAL LECTURE VACCINES AND VACCINATION—PAST, PRESENT AND FUTURE1

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P. M. BIGGS 'Willows', London Road, St Ives, Huntingdon, Cambridgeshire PE17 4ES, England Abstract 1. Immunisation was first practised as early as the 10 th century when small doses of smallpox material administered by unusual routes were used to immunise against smallpox. The procedure was introduced into England in the early part of the 18th century. 2. The next major development was the use by Jenner of cowpox to vaccinate against smallpox in the late 18th century. 3. Some eighty years later came the classic studies of Pasteur developing vaccines for fowl cholera, anthrax and rabies. 4. The studies of Jenner and Pasteur established the major principles of vaccination which are in use to this day. 5. The major viral diseases of the domestic fowl were recognised during the 1920s and 1930s and in most cases vaccines were developed within 5 years of the discovery of the viral nature of the cause of each disease. 6. The desirable properties of poultry vaccines required by the user and producer are not completely fulfilled by currently available vaccines. 7. There is a need to use the opportunities provided by modern biotechnology and immunology to search for and develop vaccines that better fulfil the desirable properties of poultry vaccines. 8. There are a number of strategies available for the development of novel vaccines, some of which are appropriate for the needs of poultry vaccines.

INTRODUCTION

Vaccines have been used for the control and prevention of infectious diseases of poultry since Pasteur developed in 1880 a vaccine for fowl cholera (Pasteur, 1880). Since that time vaccines have formed part of the armoury used to control diseases of poultry. Most of the early vaccines for poultry were for the control of bacterial disease and were only of limited success. The rapid development of the poultry industry and the adoption of intensive systems of 1 This lecture is the seventh given in memory of the late Dr R. F. Gordon, was delivered at the Brighton Conference Centre on 14th August 1989 as part of the IXth International Congress of the World Veterinary Poultry Association.

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management during the 1920s and 1930s and through to the 1950s provided the environment which encouraged infectious disease, particularly those caused by viruses. This created a demand for methods of prevention of these diseases and indeed the industry would have had difficulty in surviving without the development of vaccines that occurred in response to this demand. It is fair to say that the rapid expansion of intensive methods of poultry production could not have occurred without the pioneering work of the poultry research scientists who developed vaccines over this period. EARLY HISTORY


Inoculation or variolation

Immunisation to protect against infectious disease began several centuries before Pasteur and indeed the word, vaccine, was coined by Jenner nearly a century before the studies of Pasteur. The story begins with the use of smallpox material, usually collected from pocks of mild cases, administered by various routes and procedures to protect against natural challenge during epidemics of the disease. When and where this practice, which became known as "inoculation" or "variolation", was initiated is uncertain. It has been claimed that it was practised in China in the 10th century and later moved westward to the Middle East and elsewhere (Wildy, 1987). Whether this is so or not, the practice was in use in China, India, Arabia, parts of Africa and several countries around the Mediterranean basin, including Greece and Turkey, during the 17th century (Crookshank, 1889). The practice of inoculation was brought to England by Lady Mary Wortley in the early part of the 18th century, although it had been discussed at the Royal Society a few years earlier. According to Smith (1987) it was first reported in England when an account of the Chinese method, which was to introduce the material intranasally, was presented to the Royal Society in 1701 by Dr Clopton Havers. Further discussion took place at the Royal Society on the pactice of inoculation for smallpox on several occasions during the early 18th century, a notable occasion was the description of the Turkish method of scarification of the skin by Dr Emanuel Timoni in 1713 (Crookshank, 1889; Smith, 1987; Wildy, 1987). Lady Mary Wortley, whose beauty had been destroyed by smallpox in 1715, accompanied her husband (the new Ambassador) to Turkey in 1716 where she was soon to witness inoculation for the first time. She was so impressed with the result that she wrote in 1717 a long letter to a friend in England describing the technique and results and indicated that she would crusade to have the practice taken up by the medical profession in England. The fact that she had her son Edward inoculated soon after this was an indication of her conviction of the value of the practice. She returned to England in 1719 and started her crusade and in 1721, in the face of a severe epidemic of smallpox, had her daughter inoculated. Her example and championship of inoculation brought the practice to the notice of all levels of society and in 1722 the Princess of Wales had the two young Princesses Amelia and


GORDON MEMORIAL LECTURE

P. M. BIGGS—7th Gordon Memorial Lecturer

Caroline successfully inoculated. Just as the practice was beginning to be accepted by the rich, a series of events occurred which brought the practice into disfavour. Clearly the risks of this procedure were that the inoculation could result in severe disease and even death and that inoculated people could be a source of infection thus spreading smallpox. Both of these events

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occurred, resulting in a decline in the practice in the late 1720s. However, there was an upsurge in the use of inoculation in response to the great epidemic of smallpox of 1751-1753. The practice returned to favour and in 1755 received the public approval of the College of Physicians (Smith, 1987). From 1755 the practice of inoculation became widely used in response to the presence of smallpox and much of the early success can be attributed to the methods used by the Suttons, father and son. Its use continued to the end of the century when vaccination was introduced as the result of the studies of Jenner. From 1798 it coexisted with vaccination which did not completely replace inoculation until 1840 (Smith, 1987).


Vaccination

The development and introduction of vaccination using cowpox by Jenner was a major achievement. However, he was not the first to use cowpox to protect against smallpox (Crookshank, 1889). For many years it had been folklore that those who contracted cowpox did not succumb to smallpox even in the face of an epidemic. In 1774 Benjamin Jesty, a rustic farmer of Yetminister in Dorset, inoculated his wife and two sons. A number of factors led up to this action. He was aware of the widely-held view of the benefits of having suffered from cowpox; he had had cowpox himself as a young man, had not suffered from smallpox thereafter and he personally knew of a number of people who after having cowpox never had smallpox. This background and the presence in the district of a smallpox epidemic led him to inoculate his wife and two young sons with material collected from a cow that had cowpox. Although none contracted smallpox after this, and both sons were inoculated with smallpox some 15 years later without any response, the incident was not widely known. The reason for this is uncertain but is likely to have been a combination of the nature of Jesty, who was a farmer and a man of the land, and the severe reactions that his wife and children had had to the inoculation. The latter was of such concern to him that he called in medical assistance. Jesty's achievement was not generally recognised until, in response to the recognition and fame accorded Jenner some 25 years later, he found supporters to present his case. In 1805, after personally presenting his case it was accepted by the Jennerian Society that he had successfully vaccinated his wife and two sons thus establishing his claim as the first inoculator of cowpox. By then he had moved to farm in Downshay in the Isle of Purbeck where he died in 1816. He was buried in Worth Matravers and his tombstone bears the following inscription: He was born at Yetminster in this County, and was an upright honest man; particularly noted for having been the first Person (known) that introduced the Cow Pox by inoculation, and who, from his great strength of mind, made the experiment from the Cow on his wife and two sons in the year 1774.


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Edward Jenner

Although Benjamin Jesty was the first to inoculate cowpox it was Jenner who applied scientific rigour to the problem and he is rightly attributed with establishing the principles and validating the procedure of vaccination which was to lead to the official eradication of smallpox in 1980. In fact, according to Perry, he foretold the eradication of smallpox as indicated by the following quotation from a pamphlet Jenner published in 1801 entitled The Origin of the Vaccine Inoculation.


The numbers who have partaken of its benefits throughout Europe and other parts of the Globe are incalculable; and it now becomes too manifest to admit of contradiction that the annihilation of the smallpox, the most dreadful scourge of the human species, must be the final result of this practice. Edward Jenner was born on 17th May 1749 in the vicarage at Berkeley, Gloucestershire. His parents died when he was only five and soon afterwards he was sent away to school during which period he developed a keen interest in natural history which was to last throughout his life. It was decided that Edward would practise medicine. He left school at the age of 14 and was apprenticed to Daniel Ludlow, a surgeon at Sodbury near Bristol. During his apprenticeship it was likely that he became an efficient inoculator (variolator) and thus became familiar not only with the concept of naturally-acquired immunity from the protection that naturally-acquired smallpox provided to later exposure to the disease, but also artificially-acquired immunity provided by inoculation. However, a single incident was perhaps more important to his future work. A dairymaid came to see Mr Ludlow about a skin rash and she said that it could not be smallpox because she had had cowpox and no-one who has had cowpox gets smallpox (Crookshank, 1889; Underwood and Campbell; Perry). This clearly made a lasting impression on Jenner who from that point onwards was determined to validate the concept. When his 7 years of apprenticeship was completed in 1770 he became a pupil in London of the distinguished naturalist, scientist, surgeon and intellectual giant John Hunter (Perry; Underwood and Campbell; Wildy, 1987). Not only did he walk the wards at St George's Hospital with John Hunter and carry out dissections for him, but he was introduced to many of the scientific intelligentsia. One of these was Mr (later Sir) Joseph Banks, a future President of the Royal Society, who had just returned from Captain Cook's first voyage to the Pacific. Jenner was asked to arrange the biological specimens that he had collected on the voyage; this he did apparently to the full satisfaction of Banks. The training in observation, experimentation and scientific rigour received from John Hunter were invaluable to his later work whether on medical matters, cowpox and vaccination or natural history. In 1772 he returned to Berkeley and set up in practice; their friendship and association flourished until Hunter's death in 1793. As a country doctor Jenner combined his interest in medical matters with that in natural history. He contributed to local medical societies papers on


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subjects including initial stenosis, heart disease in acute rheumatism and a coauthored paper attributing angina pectoris to coronary disease (Underwood and Campbell). However, his first contribution to be recognised nationally and abroad was in natural history. In 1783 John Hunter suggested that he studied the cuckoo. This he did and concluded that the young cuckoo and not the parent bird was responsible for ejecting its host species eggs and nestlings, describing the depression between the scapulae which assisted it in this process. In 1786 he submitted a paper to the Royal Society describing these results which was returned with a letter from Sir Joseph Banks, the President, implying that he had got it wrong and that Council ". . . thought it best to give you a full scope for altering it, as you shall choose". He undertook further experiments and the paper presenting the same conclusions was presented to, and published by, the Royal Society in 1788 (Crookshank, 1889) and on the basis of this paper he was elected a Fellow of the Royal Society. Even this brief thumbnail sketch shows that Jenner was a remarkable man. To return to our main interest we pick up Jenner's life after his return to Berkeley, where he applied himself to cowpox and its relationship to smallpox. Over a period of time he observed cowpox in cattle and grease (a disease of the foot) in horses and came to the view that the source of cowpox was grease in horses and that infection was transmitted from horse to cow to man. This, together with his view that cowpox would provide life-long protection against smallpox, proved to be misguided. However, his ideas concerning cowpox and immunity to smallpox were outstanding. His two major contributions were that cowpox could be transmitted from man to man and that such persons were protected from smallpox. The evidence to support these conclusions today seems slim, but it did include careful observation and reporting and some experimentation. Edward Jenner collected a series of cases illustrating that individuals naturally-infected from cows with cowpox would neither "take" on inoculation with smallpox nor succumb to the disease when naturally exposed to it. In a famous case he inoculated in 1796 an 8-year-old boy, one John Phipps, with cowpox using material he collected from Sarah Nelmes, a dairymaid who contracted cowpox from a cow called Blossom. Seven weeks later he "inoculated" him with smallpox but no disease followed. Today this might seem an irresponsible act but it must be remembered that inoculation with smallpox was still common. Jenner prepared a manuscript and submitted it to the Royal Society for publication. It was considered by the Council and returned to Jenner who collected more material. This included an arm-to-arm transfer of cowpox through 4 passages and another three cases where vaccinated individuals were inoculated with smallpox which did not "take". He included this new material in his paper which was published privately, probably because he did not wish another rebuff from the Royal Society. In the title to this publication Jenner uses Variolae vaccinae—smallpox of the cow—to describe cowpox. The full title is: An inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Discovered in Some of the Western Counties of England, particularly Gloucestershire,

and known by the Name of the Cow-pox. In later publications he referred to vaccine inoculation and vaccine virus.


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From the time of the publication of the Inquiry Jenner spent much of his time vaccinating and arranging for material for vaccination to be distributed not just in England but abroad as well. Because vaccine had a limited life it was sent to Australia for example, by vaccinating a child and passaging it to another child at approximately weekly intervals throughout the voyage. Although passaging from person to person was Jenner's idea to preserve its identity it would be a procedure unacceptable today and indeed had the consequences we would now expect. It was used in premises in which variolation was also used and became contaminated on occasions. It is the progenitor of modern vaccinia virus but much happened between Jenner's time and recent history. The origin of the vaccinia used in modern times is uncertain but it is clear that vaccinia differs in many respects from the virus of cowpox.


Louis Pasteur

The world had to wait for about 80 years for the next stage in the vaccine story. This was the contribution of another intellectual giant, Louis Pasteur. It is of special interest to this audience that Pasteur's first contribution to the vaccine field was his observation that a culture of Pasteurella multocida diminished spontaneously in virulence when kept at room temperature and that such a culture was attenuated for the fowl and would protect against fowl cholera when challenged with virulent organisms (Pasteur, 1880). A similar observation was made with anthrax: culturing the causative organism at 42 to 43°C for several months resulted in a loss of virulence. Using such organisms as a primary vaccine, and organisms that had been grown in such conditions for only 10 to 12 d as a secondary vaccine 12 d later, animals were protected against challenge two weeks later with fully virulent organisms (Topley and Wilson, 1929). Pasteur's most famous contribution to this field came after his achievement with fowl cholera and anthrax, when he turned his attention to rabies. He found that field virus, or what he termed "street virus", varied in virulence. He passaged such virus in rabbits with the intention of fixing its virulence. He not only achieved this aim but also found that this treatment both increased the virulence of the virus for rabbits and, more importantly, reduced its virulence for other species including dogs and monkeys, especially when administered subcutaneously (Hutyra and Marek, 1926; Topley and Wilson, 1929). This virus, which was stable in its properties, he termed "fixed virus". Pasteur used the fixed virus for his studies on development of a vaccine (Pasteur, 1885). He found that if he dried the spinal cord taken from rabid rabbits infected with fixed virus, virulence was slowly lost over a period of days. Pasteur then used this material to immunise dogs. He inoculated them subcutaneously over a number of days with rabbit spinal cord that had successively been dried for decreasing periods of time. The final inoculum was spinal cord dried for only one or two days, which was very virulent. When the dogs were challenged they were found to be resistant to rabies. With the success of this procedure in dogs he took his courage in his hands and decided it should be tried on man. He selected a young boy, Joseph Meister, who had been severely mauled by a rabid


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dog two days previously. The boy's wounds were so severe that Pasteur and his colleagues believed it certain that he would develop rabies, and therefore he was an appropriate candidate for a vaccinal attempt. The procedure was similar to the one used for dogs and was, mercifully, successful. Pasteur's method was essentially used for the next 70 years. I have devoted time to the early history of immunisation and vaccination because this period laid the foundation for many of the fundamental principles of immunisation and because the poxviruses have recently become of great significance to the development of what are often referred to as novel vaccines. The practices of inoculation, the vaccination of Jenner and the vaccines developed by Pasteur laid the foundations for the following principles: naturally-acquired immunity; protective immunity can be artificially stimulated, thus artificially-acquired immunity; a related virus from another species can be used as an immunising agent; virulent virus given by an abnormal route can be an effective immunising agent; growth of an organism in an adverse environment including a non-natural host reduces its virulence, thus attenuation; inactivation of an organism's viability reduces its virulence but not necessarily its immunogenicity. These principles have been strengthened and developed by others and formed the basis for the empiric development of vaccines until recent times. Following on the work of Jenner and Pasteur, and based on it, has been the unravelling of many of the mysteries of immunisation, including the importance of cells and humoral factors, during the remaining part of the 19th century (Topley and Wilson, 1929). The great names include Nuttal, Buchner, von Behring, Bordet and Erhlich who produced at the end of the century his "side-chain theory" of antibody production and their interaction with antigens. This work used bacteria and their products and was possible only because Pasteur and Koch had developed the technology that allowed the isolation and handling of bacteria and the discovery in 1888 of the first bacterial toxin by Roux and Yersin. The development of techniques to handle bacteria by Pasteur and Koch also resulted in a flurry of investigations to determine the cause of the major diseases of man and animals. By the end of the 19th century the causative organism had been isolated and identified for the major bacterial diseases, including those of poultry such as fowl cholera, fowl typhoid, pullorum disease and avian tuberculosis. Although vaccines of a kind had been developed for fowl cholera and fowl typhoid by the early part of this century, they were not to play a major role in the control and prevention of bacterial disease of poultry. This is because the development of effective bacterial poultry vaccines responded poorly to the empirical approach and, in many cases, alternative and better methods of prevention and control became available. I shall therefore continue the story by concentrating on virus diseases and coccidiosis and restrict the discussion to the domestic fowl because this is the species of poultry for which the most important contributions have been made.


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DEVELOPMENTS SINCE 1900


Virus diseases of the domestic fowl

Recognition and cause. Fowl pox, fowl plague and avian leukosis were recognised before the end of the 19th century and Marek's disease was first described in 1907 (Marek, 1907) but the other important virus diseases of the domestic fowl were not described until the 1920s and 1930s and later. In most cases their appearance was related to the development of an intensive poultry industry. Infectious laryngotracheitis was first described in 1926 (Beach, 1926). Newcastle disease in 1927 (Doyle, 1927), infectious bronchitis in 1931 (Shalk and Hawn, 1931), infectious avian encephalomyelitis in 1932 (Jones, 1932), infectious bursal disease in 1962 (Cosgrove, 1962) and egg drop syndrome1976 in 1976 (van Eck et al., 1976). The viral nature of the causative agents of these diseases was determined within a few years of their recognition. Fowl plague in 1900 by Centannii and Savonuzzi (Stubbs, 1959), fowl pox in 1902 by Marx and Sticker (Cunningham, 1959), erythroid and myeloid leukosis in 1908 by Ellerman and Bang (Ellerman, 1922), Newcastle disease in 1927 (Doyle, 1927), infectious laryngotracheitis in 1931 (Beach, 1931), infectious avian encephalomyelitis in 1934 (Jones, 1934), infectious bronchitis in 1936 (Beach and Schalm, 1936), infectious bursal disease in 1962 (Lukert and Hitchner, 1984) and egg drop syndrome-1976 in 1977 (McFerran et al., 1977). The exception is Marek's disease for which there was a wait of 60 years before the viral cause of the disease was demonstrated (Churchill and Biggs, 1967) but the unusual virus-cell relationship of the causative herpesvirus was largely responsible for this. Vaccines. For the majority of the virus diseases the first vaccine was developed within 5 years of the discovery of the viral nature of the causal agent (Table 1), albeit they were crude and in some instances no more than field virus given by an abnormal route or at a specific and "safe" age. The first pigeon pox and fowlpox vaccine appears to have been reported by Burnet (1906); this was modelled on the "inoculation" procedure used for smallpox in the 18th century. The first vaccine for infectious laryngotracheitis was a field virus given by an abnormal route (Hudson and Beaudette, 1932) and, according to Lancaster (1966), studies on vaccines to control Newcastle disease were begun soon after the disease had been recognised, but the attenuation of the virus by passage in chick embryos and the successful use of such a virus as a vaccine by Iyer and Dobson (1940) was a notable early event. The delay in producing vaccine for Newcastle disease was likely to have been because the disease was controlled by slaughter in the United Kingdom and was not recognised in the United States as Newcastle disease until 1944 (Beach, 1944), although Beach (1944) said it had been present since 1935. Van Roekel et al. (1950) indicated that immunisation for infectious bronchitis was used in the New England states of the USA from about 1941. The procedure used fully virulent virus given at an age when the disease would produce least economic cost. The first avian encephalomyelitis vaccine (Schaaf and Lamoreux, 1955) was also a fully virulent virus given at an age that produced little, if any, economic effect. A partially-attenuated virus produced by passage in chick embryos was the first

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vaccine for infectious bursal disease (Snedeker et al., 1967), although it retained a degree of pathogenicity. The first Marek's disease vaccine was an oncogenic virus fully attenuated by passage in cell culture (Churchill et al., 1969). The vaccine produced for use against egg drop syndrome-1976 is a modern one which had to fufil the requirements of the newly-established licensing authorities (Baxendale et al., 1978). With the advance in time the first vaccines for each disease became safer and more efficacious. The first vaccines against Marek's disease and egg drop syndrome-1976 benefited from the developments in knowledge and, more particularly, techniques for producing safe and effective vaccines. TABLE 1


Time relationships between the description of diseases of the domestic fowl, discovery of the nature of their causative agent and the preparation of the first vaccine

Disease Fowl cholera Fowl typhoid Fowlpox Marek's disease Infectious laryngotracheitis Newcastle disease Infectious bronchitis Avian encephalomyelitis Infectious bursal disease Egg drop syndrome-1976 Coccidiosis

Description p

1888 pre-1900 1907 1926 1927 1931 1932 1962 1976 >

Discovery of cause 1880 1889 1902 1967 1931 1927 1936 1934 1962 1977 1911

First vaccine 1880 c.1900 1906 1969 1932 1940 1941 1955 1967 1978 1958

The early vaccines were crude, not always safe and often of doubtful efficacy; many were virulent field viruses given by an abnormal route or at an age which resulted in the least harm. This procedure was unsatisfactory because it put the vaccinee at risk and spread virulent virus in the population. Developments in the techniques of handling viruses, in particular the use of the developing chick embryo and of tissue culture, which occurred in the 1930s and was put on a firm footing in the 1940s (Beveridge and Burnet, 1946; Enders et al., 1949), together with increasing experience, resulted in great improvements during the 1950s and 1960s. The use of these techniques allowed the ready isolation of virus, its growth to high titre and the opportunity of adaptation to an unusual environment which often resulted in an attenuation of the virulence of the virus. This resulted in a plethora of new potential vaccines only some of which had, with experience, the attributes to be adopted commercially. They included: field viruses with no or low virulence for the domestic fowl, such as is used for Newcastle disease (Hanson and Grandley, 1955; Lancaster, 1964; Lancaster and Alexander, 1975); field virus from another species which has no or low virulence for the vaccine target species such as the herpesvirus of turkeys for Marek's disease (Okazaki et al., 1970); field virus that has been attenuated by passage in unusual hosts such as mammals, other avian species, the chick embryo or cell culture, examples of the latter are the H52 and HI 20 strains of



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infectious bronchitis (Hoekstra, 1961); inactivated virus such as is used for egg drop syndrome-1976 (Baxendale et al., 1980). For many years inactivated vaccines were out of favour for a number of reasons: in the early years they were difficult to produce with an adequate and standard antigen content; they have to be administered parenterally requiring the handling of each individual; some did not and do not produce as good an immunity as live vaccines. However, the introduction of /?-propriolactone as an inactivating agent, the use of which retained greater antigenicity than formalin or phenol which had been used previously (LoGrippo and Hartman, 1955) and the adoption of oil-based adjuvants (Jacotot and Vallee, 1959; Cessi and Nardelli, 1974) resulted in a renaissance for inactivated vaccines. Other developments have occurred, partly as a necessity, such as cell associated vaccines for Marek's disease, and partly to improve the efficacy and safety of vaccines available, such as the seed lot system of production and the use of substrates derived from specific-pathogen-free animals. The seed lot system of production enables full characterisation of a vaccine and production over time of a product that has uniform properties of efficacy and safety. The use of substrates derived from a specific-pathogen-free source helps to ensure, but does not guarantee, freedom from extraneous agents. Vaccination. Several developments in the use of vaccines have occurred directed towards achieving good protection at least cost and to satisfy the needs of broilers, breeders and layers. Combinations of vaccines for a single disease spaced temporally are sometimes beneficial. For example, the mild HI20 followed by the more virulent H52 vaccine for infectious bronchitis, and live and inactivated vaccines for infectious bronchitis and Newcastle disease. Each poultry operation chooses its programme regarding choice of vaccine, frequency and method and route of administration. Much work has been devoted to methods and routes of administration. Because flocks are large, labour often expensive and handling of the birds detrimental to their well-being, nonparenteral mass methods of administration were under study some 40 years ago (Johnson and Gross, 1951) and were in use, where appropriate, by 1959 (Brandly, 1959). Research since 1959 has further improved the efficiency of mass methods of administration which are widely used today.

Coccidiosis

Coccidia have been known since the time of van Leuwenhoek and the first avian coccidium was described by Hadley in 1911 (Long, 1978), but it was Tyzzer in 1929 who laid the foundation for future work on immunity to coccidia. I include this subject, not because a vaccine of a kind has been available in the USA for many years (Edgar, 1956), but because the prospects for vaccination in the future are exciting and promising. The currentlyavailable vaccine consists of fully-virulent oocysts of the main species of avian coccidia and is usually administered in the drinking water to chicks up to two weeks of age. In practice, there is little control over the infection rate, and

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immunity depends upon the subsequent sporulation of, and reinfection with, fresh oocysts in the litter. The first indication that an attenuated vaccine for coccidiosis was a possibility was the report of the attenuation of a strain of Eimeria tenella by passage in the chorioallantois of the developing chick embryo while retaining its immunogenicity (Long, 1972; 1974). However, for various reasons, strains attenuated by selection for precociousness, a procedure described by Jeffers (1975), are more suitable for vaccine purposes. Unfortunately immunity is species-specific, requiring attenuation of all 7 species that parasitise the domestic fowl. This has now been accomplished and studies using a cocktail of the 7 attenuated species have shown it to be effective against challenge with virulent strains of all 7 species under experimental conditions (Shirley and Millard, 1986). Large scale field-trials with commercial broilers have shown that the vaccine controls coccidiosis very well (Shirley, 1988).

VACCINE USE TODAY

Vaccines are used mainly for virus diseases and the choice depends on the circumstances. Effective vaccines are available for the major diseases and are either living or inactivated vaccines which may be used together temporally. Vaccines containing components for several diseases are also available. Desirable properties

Before considering the future and the opportunities provided by modern biotechnology and recent developments in immunology, it is as well to consider the desirable properties for user and producer, of poultry vaccines and the advantages and disadvantages of the kinds of vaccines currently available. User. The desirable properties from the users' standpoint can be considered under the headings of safety, quality and efficacy: Safety. Live vaccines should not be pathogenic and have no adverse effect on the host such as reducing growth rate and productivity. They should not spread to members of host or other species. Shedding of vaccinal organism and local spread can be tolerated if it is only for a short period after vaccination. However, persistent shedding and consequent spreading from the vaccinated flock to the population at large results in a risk of reversion to virulence of the vaccine organism and interferes with monitoring for field infection in the population. Live vaccines should not revert or change to a pathogenic form. This is particularly important for vaccines that are disseminated from vaccinated animals. All vaccines should be free of contaminating agents and inactivation must be complete for killed vaccines containing whole organisms. Quality. Vaccines should have a stated potency and there should be a reliable method of establishing and measuring potency. They should be stable in the conditions they are to be stored and used and should be free


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of unnecessary impurities and additives such as antibiotics. The production premises should fulfil criteria designated by licensing authorities. Efficacy. Vaccines should provide a significant degree of protection against mortality and morbidity caused by the disease in question. In addition they should protect against subclinical disease which can affect productivity, food conversion and growth rate. Vaccines should ideally prevent infection with the challenging field organism and, if the vaccine does not prevent infection with the field organism, it should protect against its shedding and spread from the vaccinated bird. Other. There are other desirable properties for which the user has an interest. Vaccines and vaccinated birds should be readily differentiable from the field organism and birds infected with the field organism respectively. They should be easy to store, distribute and administer. Finally, they should be inexpensive. Vaccine producer. The vaccine producer wishes to provide vaccines with the properties desired by the consumer but, in addition, there are other properties of particular interest to the producer. The producer requires strains of organism and systems of propagation that provide high yields of antigen. The systems of propagation available for poultry viral vaccines are restricted. Embryonating eggs and cell culture are the major systems, although the hatched chicken is used in some cases. Avian cell lines are generally not available so that well characterised cell lines and systems of growing cells in suspension cannot be used. Because of these constraints strains of organism and systems of propagation providing high yields of antigen are particularly important, especially because techniques for concentrating antigen are too expensive for use in the manufacture of poultry vaccines. Whatever system of propagation is used the substrate should be free from adventitious agents. The seed lot system of production enables characterisation of the vaccine and production over time of a product with uniform properties of safety and efficacy. However, this also requires strains and propagation systems which provide high yields to allow a small number of passages between seed lot and production batch. Lastly, the systems used for vaccine manufacture should be of low cost. ADVANTAGES AND DISADVANTAGES OF CURRENTLY AVAILABLE VACCINES

Currently-available poultry vaccines are either live organisms or inactivated whole organisms. How do these fulfil the needs of user and producer? Each has advantages and disadvantages but there are few, if any, of either category that have all the attributes I have outlined. The advantages and disadvantages of classical live and inactivated vaccines are discussed below. Inactivated vaccines

Inactivated vaccines are relatively safe because inactivating agents have

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P. M. BIGGS

been used in their production. Even so, there are dangers from incomplete inactivation and from contaminating agents surviving the inactivating process. Viruses used in inactivated vaccines do not have to be modified and therefore they have the potential for rapid development in response to urgent need. However, inactivated vaccines require adjuvants and have to be administered parenterally to each individual. They therefore tend to be expensive to manufacture and to administer. Finally, they stimulate humoral immunity more effectively than cell-mediated immunity.


Live vaccines

Live vaccines generally induce protective immunity rapidly and more quickly than inactivated vaccines. They are more likely to stimulate all arms of the immune system than inactivated vaccines. They are relatively inexpensive to produce and can be easy and inexpensive to administer where mass administration techniques can be employed. However, there are distinct disadvantages to the use of live vaccines. There is a risk of contamination with unknown infectious agents and of reversion to virulence of the vaccine organism. It has been known for many years that contamination of vaccines with known infectious agents was a risk (Zargar and Pomeroy, 1950; Burmester et al., 1956). Although improved standards of safety testing have reduced this risk of contamination with known infectious agents there is always a risk of contamination with unknown and unexpected agents. There are two examples of this in recent times in the contamination of Marek's disease vaccine with reticuloendoletheliosis virus (Jackson et al., 1977; Koyama et al., 1976; Yuasa et al., 1976) and the probable Marek's disease vaccine source of the egg drop syndrome1976 adenovirus (Anon, 1978). There are difficulties in differentiating between vaccine and field virus and immune responses to them in the field. Finally, it can be difficult to ensure adequate attenuation of virulence to satisfy all circumstances for which the vaccine will be used and to achieve appropriate attenuation yet maintain adequate immunogenicity.

THE FUTURE

Clearly, neither live nor inactivated vaccines developed and produced using classical techniques fulfil all of the desirable properties required by user and producer. For this reason there is a need to use the opportunities provided by modern biotechnology and immunology to search for and develop improved methods of disease control. However, the products resulting from the use of this knowledge and technology will not be adopted unless they are an improvement over the present vaccines in one or more of the following characteristics: efficacy, safety, better shelf life and storage, greater ease in administration and, perhaps most important, cost. The opportunity for a new approach to the development of vaccines has been provided by advances in recombinant DNA technology and immunology. It is now possible to identify the proteins of infectious agents that are the


17

important immunogens and to dissect these proteins to find which parts are responsible for this property. Proteins consist of a large number of antigenic determinants (epitopes), each consisting of a short sequence or combination of short sequences of amino acids, only some of which are important in stimulating protective immunity. These sequences, or epitopes, can now be identified and the arm(s) of the immune system they stimulate determined. The genes coding for immunogenic proteins or DNA sequences coding for epitopes or combinations of epitopes can be identified and cloned. In addition it is now possible to identify the genes of infectious organisms responsible for their virulence. These developments open up a number of strategies (Table 2). TABLE 2


Opportunities for novel vaccines

Non-replicating vaccines Production of immunogenic proteins or peptides In prokaryotic cells In eukaryotic cells Synthesise Engineered live vaccines Vector carrying immunogene Virus Bacterium Engineered virus for loss of virulence

The first is to produce the immunogenic proteins or peptides for use as non-living vaccines. This can be done by cloning the appropriate gene in a vector so that the immunogen can be produced in large quantities. The choice of either a prokaryotic or eukaryotic cell as a vector will depend on a number of factors. For example, if the peptide requires glycosylation to be immunogenic an eukaryotic system will be chosen. The choice will also be influenced by such factors as efficiency of production of the immunogen and ease of purification. The latter is of concern because purity affects quality and the inclusion in vaccines of unnecessary material is not desirable. There are numbers of methods available for purifying proteins, including the use of antibodies and monoclonal antibodies, where appropriate, in affinity columns, and the use of gene fusion vectors which facilitate purification of the product (Moks et ah, 1987). An alternative, if the required product is a short sequence of amino acids, is for it to be synthesised. For administration to the animal and presentation to its immune system, short chain peptides require to be linked to carrier molecules and both this product and larger peptides and proteins require adjuvants or, alternatively, to be presented as immunostimulating complexes (iscoms) (Morein et al., 1984). In our present state of knowledge non-living vaccines have to be given to each animal and therefore this strategy, although possible for poultry vaccines, has limited use. The second strategy is to manufacture live vaccines. There are two possibilities. First, to disarm the pathogen by identifying the gene(s) responsible for its virulence and deleting it/them from its genome. This is feasible only if


18

P.M. BIGGS

such action does not prevent the agent replicating in the host or destroys its immunogenicity. There are no examples of this approach in use in poultry but there is such a product in use for pseudorabies in pigs. In this case the deletion of the thymidine kinase gene from the causative herpesvirus destroyed its virulence without affecting immunogenicity and this is a possible approach for avian herpesviruses. The second possibility, and the most attractive for use with poultry, is to develop vectors capable of carrying genes coding for the required immunogens and expressing them so that when used as a vaccine they are presented to the host's immune system. This concept was first developed using the poxvirus, vaccinia, by inserting the influenza haemagglutinin gene into a region of the vaccinia virus genome that is not essential for its replication (Panicali et al., 1983; Smith et al., 1983). More recently this procedure was accomplished using the spike protein gene of avian infectious bronchitis and the resulting recombinant vaccinia virus was shown to be immunogenic in mice (Tomley et al., 1987). A vaccinia recombinant with the F glycoprotein of Newcastle disease virus has also been constructed and when used as a vaccine in chickens it provided protection against challenge with virulent Newcastle disease virus (Meulemans et al., 1988). These results indicate that the concept of using recombinant vector viruses for vaccines in poultry is valid. However, vaccinia is not appropriate for use in poultry and its use in any species has been questioned because of possible public health dangers. Fortunately there are more appropriate choices for use in poultry. Those that are under study include fowlpox, Marek's disease virus and the herpesvirus of turkeys. Progress has been made in a number of laboratories. The immunogencity of a fowlpox recombinant using the haemagglutinin gene of influenze virus has been described (Boyle and Coupar, 1988; Taylor et al., 1988) and the ability of vaccination of both the domestic fowl and turkey with such a recombinant to provide good protection against experimental challenge with influenza viruses, which are very virulent for the domestic fowl, is promising (Taylor et al., 1988). The rationale for the vector recombinant approach to vaccination is to combine the advantages of live virus vaccines with the specificity and safety of pure protein or peptide vaccines. This can be done by selecting a vector that itself is able to reach and stimulate all arms of the immune system, is able to express the product of the recombinant gene so that it does likewise, can preferably be used by mass methods of administration, has a well documented safety record and can have genes for a number of immunogens inserted into its genome without compromising the vector itself. This would allow the production and use of a single vaccine for several diseases or, several serotypes of the causative agent of a single disease, thus providing a clear advantage over currently available vaccines in simplicity and cost of use and probably production. The vector approach is not restricted to viruses. For some situations there may be an advantage in using a bacterium as the vector. This could be so for intestinal disease such as coccidiosis where a bacterium such as Esherichia coli or a salmonella specific to the domestic fowl suitably disarmed could have advantages. The last ingredient to make this approach viable is no different from that



19

required for other types of novel vaccines, that is genes coding for the immunogens of the important poultry diseases. There are already a number of these available including those for Newcastle disease (Millar et ah, 1988) and infectious bronchitis (Boursnell et ah, 1984; 1985; Binns et ah, 1985). Work is in progress in many laboratories on cloning of genes coding for the immunogens of other viral pathogens and also coccidia. The interest in the vector approach to vaccination is widespread and therefore from now on there will be many reports of the successful construction of recombinant vectors with potential for use as vaccines in the poultry industry. As with potential classical vaccines in the past, it is certain that not all will be suitable for use as commercial vaccines, but it is equally certain that some will be suitable and, I believe, successful vaccines. The science and technology available to the microbiologist, immunologist and "vaccinologist" today provide great opportunities for the design and construction of novel vaccines which, together with a greater knowledge of the pathogenesis and immunogenesis of each disease, reduces the need for empiricism. In the future vaccines will be designed not only to suit the needs of the producer and user but also to produce the most effective immunity for each disease. It should always be remembered that this would not have been possible without the pioneering work of Edward Jenner and Louis Pasteur. REFERENCES ANON. (1978) Egg drop syndrome-1976: A "new" disease of chickens. Avian Pathology, 7: 189-191. BAXENDALE, W., LUTTICKEN, D., HEIN, R. & ORTHEL, F.W. (1978) Vaccine against the egg drop

syndrome 76 of chickens. International Virology IV 119 Abstracts of the Fourth International Congress of Virology. The Hague (Centre of Agricultural Publishing and Documentation, Wageningen). BAXENDALE, W., LUTTICKEN, D., HEIN, R. & MACPHERSON, I. (1980) The results of field trials

conducted with an inactivated vaccine against the egg drop syndrome 76 (EDS 76). Avian Pathology, 9: 77-91. BEACH, J.R. (1926) Infectious bronchitis of fowls. Journal of the American Veterinary Medical Association, 68: 570-580. BEACH, J.R. (1931) A filtratable virus, the cause of infectious laryngotracheitis of chickens. Journal of Experimental Medicine, 54: 809-816. BEACH, J.R. (1944) The neutralization in vitro of avian pneumoencephalitis virus by Newcastle disease immune serum. Science, 100: 361-362. BEACH, J.R. & SCHALM, O.W. (1936) A filtrable virus, distinct from that of laryngotracheitis, the cause of a respiratory disease of chicks. Poultry Science, 15: 199-206. BEVERIDGE, W.I.B. & BURNET, F.M. (1946) The cultivation of viruses and Rickettsiae in the chick embryo. Medical Research Council Special Report No. 256 (London, HMSO). BINNS, M.M., BOURSNELL, M.E.G., CAVANAGH, D., PAPPIN, D.J.C. & BROWN, T.D.K. (1985) Cloning

and sequencing of the gene encoding the spike protein of the coronavirus IBV. Journal of General Virology, 66: 719-726. BOURSNELL, M.E.G., BROWN, T.D.K. & BINNS, M.M. (1984) Sequence of the membrane protein gene from avian coronavirus IBV. Virus Research, 1: 303-313. BOURSNELL, M.E.G., BINNS, M.M., FOULDS, I.J. & BROWN, T.D.K. (1985) Sequences of the

nucleocapsid genes from two strains of avian infectious bronchitis virus. Journal of General Virology, 66: 573-580. BOYLE, D.B. & COUPAR, B.E.H. (1988) Construction of recombinant fowlpox viruses as vectors for poultry vaccines. Virus Research, 10: 343-356.

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BRANDLY, C.A. (1959) Newcastle disease. In: BIESTER, H.E. & SCHWARTE, L. (Eds) Diseases of

Poultry, 4th edn, pp. 464-503 (Ames, IA, Iowa University Press). BURMESTER, B.R., CUNNINGHAM, C.H., COTTRAL, G.E., BELDING, R.C. & GENTRY, R.F. (1956) The transmission of visceral lymphomatosis with live virus Newcastle disease vaccines. American Journal of Veterinary Research, 17: 283-289. BURNET, E. (1906) Contribution a l'étude de l'epithelioma contagieux des oiseaux. Annales de l'Institut Pasteur, 20: 742-765. CESSI, D. & NARDELLI, L. (1974) Vaccination against Newcastle disease: efficacy of oil emulsion vaccine. Avian Pathology, 3: 247-253. CHURCHILL, A.E. & BIGGS, P.M. (1967) Agent of Marek's disease in tissue culture. Nature, 215: 528-530. CHURCHILL, A.E., PAYNE, L.N. & CHUBB, R.C. (1969) Immunization against Marek's disease using a live attenuated virus. Nature, 221: 744-747. COSGROVE, A.S. (1962) An apparently new disease of chickens—avian nephrosis. Avian Diseases, 6: 385-389. CROOKSHANK, E.M. (1889) History and Pathology of Vaccination (London, H. K. Lewis).


CUNNINGHAM, C.H. (1959) Fowl pox. In: BIESTER, H.E. & SCHWARTE, L. (Eds) Diseases of Poultry,

4th edn, pp. 575-598 (Ames, IA, Iowa State University Press). DOYLE, T.M. (1927) A hitherto unrecorded disease of fowls due to a filter-passing virus. Journal of Comparative Pathology and Therapeutics, 40: 144-169. EDGAR, S.A. (1956) Coccidiosis immunization. Iowa State College Veterinarian, 17: 9-11 and 17. ELLERMAN, W. (1922) The Leucosis of Fowls and Leucaemia Problems (London, Gyldendal). ENDERS, J.F., WELLER, T.H. & ROBBINS, F.C. (1949) Cultivation of the Lansing strain of

poliomyelitis in cultures of various human embryonic tissues. Science, 109: 85-87. HANSON, R.P. & BRANDLY, C.A. (1955) Identification of vaccine strains of Newcastle disease. Science, 122: 156-157. HOESKSTRA, J. (1961) Control of Newcastle disease and infectious bronchitis by vaccination. British Veterinary Journal, 117: 289-295. HUDSON, C.B. & BEAUDETTE, F.R. (1932) Infection of the cloaca with the virus of infectious bronchitis. Science, 76: 34. HUTYRA, F. & MAREK, J. (1926) Special Pathology and Therapeutics of the Diseases of Domestic Animals, 3rd edn (London, Ballière Tindall & Cox). IYER, S.G. & DOBSON, N. (1940) A successful method of immunization against Newcastle disease of fowls. Veterinary Record, 52: 889-894. JACKSON, C.A.W., DUNN, D.E., SMITH, D.I., GILCHRIST, P.T. & MACQUEEN, P.A. (1977) Proven-

triculitis, "nakenuke" and reticuloendotheliosis in chickens following vaccination with herpesvirus of turkeys (HVT). Australian Veterinary Journal, 53: 457-559. JACOTOT, H. & VALLEE, A. (1959) Vaccination contre la maladie de Newcastle au moyen du virus formolé en excipient huileux. Bulletin de l'Academié vétérinaire France, 32: 373-378. JEFFERS, T.K. (1975) Attenuation of Eimeria tenella through selection for precociousness. Journal of Parasitology, 61: 1083-1090. JOHNSON, E.P. & GROSS, W.B. (1951) Vaccination against pneumoencephalitis (Newcastle disease) by atomization or nebulization with the B1 virus. Veterinary Medicine, 46: 55-59. JONES, E.E. (1932) An encephalomyelitis in the chicken. Science, 76: 331-332. JONES, E.E. (1934) Epidemic tremor, an encephalomyelitis affecting young chicks. Journal of Experimental Medicine, 59: 781-798. KOYAMA, M., SUZUKI, Y., OHWADA, Y. & SAITO, Y. (1976) Reticuloendotheliosis group virus

pathogenic to chicken isolated from material infected with turkey herpesvirus (HVT). Avian Diseases, 20: 429-434. LANCASTER, J.E. (1964) Newcastle disease—control by vaccination. The Veterinary Bulletin, 34: 57-76. LANCASTER, J.E. (1966) Newcastle disease—a review of some of the literature published between 1926 and 1964. Canada Department of Agriculture, Monograph No. 3. LANCASTER, J.E. & ALEXANDER, D.J. (1975) Newcastle disease—virus and spread. Canada Department of Agriculture, Monograph No. 11.

GORDON MEMORIAL LECTURE LOGRIPPO, G.A. & HARTMAN, F.W. (1955) Antigenicity of ß—propriolactone-inactivated

21 virus

vaccines. Journal of Immunology, 75: 123-128. LONG, P.L. (1972) Eimeria tenella in chicken embryos: reproduction, pathogenicity of a strain maintained in embryos by serial passage. Journal of Comparative Pathology and Therapeutics,

82:429-437. LONG, P.L. (1974) Further studies on the pathogenicity and immunogenicity of an embryoadapted strain of Eimeria tenella. Avian Pathology, 3: 255-268. LONG, P.L. (1978) Problems of coccidiosis: general considerations. In: LONG, P.L., BOORMAN,

K.N. & FREEMAN, B.M. (Eds) Avian Coccidiosis, pp. 3-28 (British Poultry Science Ltd). LUKERT, P.D. & HITCHNER, S.B. (1984) Infectious bursal disease. In: HOFSTAD, M.S. (Ed.) Diseases

of Poultry, 8th edn, pp. 566-576 (Ames, IA, Iowa State University Press). MAREK, J. (1907) Multiple Nervenentzündung (Polyneuritis) bei Hühnern. Deutsche Tierärztliche Wochenschrift, 15: 417-421. MCFERRAN, J.B., ROWLEY, H.M., MCNULTY, M.S. & MONTGOMERY, L.J. (1977) Serological studies

on flocks showing depressed egg production. Avian Pathology, 6: 405-413.


MEULEMANS, G., LETELLIER, M., GONZE, M., CARLIER, M.C. & BURNY, A. (1988) Newcastle disease

virus F glycoprotein expressed from a recombinant vaccinia virus vector protects chickens against live-virus challenge. Avian Pathology, 17: 821-827. MILLAR, N.S., CHAMBERS, P. & EMMERSON, P.T. (1988) Nucleotide sequence of the fusion and

haemagglutinin-neuraminadise glycoprotein genes of Newcastle disease virus strain Ulster: molecular basis for variations in pathogenicity between strains. Journal of General Virology, 69: 613-620. MOKS, T., ABRAHMSEN, L., HOLMGREN, E., BILICH, M., OLSSON, A., UHLEN, M., POHL, G., STERKY, C , HULTBERG, H . , JOSEPHSON, S., HOLMGREN, A., JORNVALL, H . & NlLSSON, B. (1987)

Expression of human insulin-like growth factor 1 in bacteria: use of optimized gene fusion vectors to facilitate protein purification. Biochemistry, N.Y., 26: 5239-5244. MOREIN, B., SUNDQUIST, B., HOGLUND, S., DALSGAARD, K. & OSTERHAUS, A. (1984) Iscom, a novel

structure for antigenic presentation of membrane proteins from enveloped viruses. Nature, 308: 457-460. OKAZAKI, W., PURCHASE, H.G. & BURMESTER, B.R. (1970) Protection against Marek's disease by

vaccination with a herpesvirus of turkeys. Avian Diseases, 14: 413-429. PANICALI, D., DAVIS, S.W., WRINBERG, R.L. & PAOLETTI, E. (1983) Construction of live vaccines by

using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin. Proceedings of the National Academy of Sciences, 80: 5364-5368. PASTEUR, L. (1880) De l'atténuation du virus du choléra des poules. Comptes rendu des séance de l'Academie des Sciences, 91: 673-680. PASTEUR, L. (1985) Methode pour prevenir la rage après morsure. Comptes rendu des séance de l'Academie des Sciences, 101: 765-773. PERRY, C.B. Edward Jenner. (University of Bristol Press and The Jenner Trust, The Jenner Museum, Berkeley, Gloucestershire). SCHAAF, K. & LAMOREUX, W.F. (1955) Control of avian encephalomyelitis by vaccination. American Journal of Veterinary Research, 16: 627-633. SHALK, A.F. & HAWN, M.C. (1931) An apparently new respiratory disease of baby chicks. Journal of the American Veterinary Medical Association, 78: 413-422. SHIRLEY, M. (1988) Controlling coccidiosis with a live attenuated vaccine. In: HARDCASTLE, J. (Ed.) Science and the Poultry Industry, pp. 18-19 (London, Agricultural & Food Research Council). SHIRLEY, M.W. & MILLARD, B.J. (1986) Studies on the immunogenicity of seven attenuated lines of Eimeria given as a mixture to chickens. Avian Pathology, 15: 629-638. SMITH, J.R. (1987) The Speckled Monster (Chelmsford, Essex Record Office). SMITH, G.L., MURPHY, B.R. & Moss, B. (1983) Construction and characterization of an infectious vaccinia virus recombinant that expresses the influenza hemagglutinin gene and induces resistance to influenza virus infection in hamsters. Proceedings of the National Academy of Sciences, 80: 7155-7159. SNEDEKER, C., WILLS, F.K. & MOULTHROP, I.M. (1967) Some studies on the infectious bursal

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STUBBS, EX. (1959) Fowl plague. In: BIESTER, H.E. & SCHWARTE, L.H. (Eds) Diseases of Poultry, 4th edn, pp. 599-608 (Ames, IA, Iowa State University Press). TAYLOR, J., WEINBERG, R., KAWAOKA, Y., WEBSTER, R.G. & PAOLETTI, E. (1988) Protective

immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine, 6: 504-508. TOMLEY, F.M., MOCKETT, A.P.A., BOURSNELL, M.E.G., BiNNS, M.M., COOK, J.K.A., BROWN, T.D.K.

& SMITH, G.L. (1987) Expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice. Journal of General Virology, 68: 2291-2298. TOPLEY, W.W.C. & WILSON, G.S. (1929) The Principles of Bacteriology and Immunity (London, Edward Arnold). TYZZER, E.E. (1929) Coccidisis in gallinaceous birds. American Journal of Hygiene, 10: 269-383. UNDERWOOD, E.A. & CAMPBELL, A.M.G. Edward Jenner—the man and his work (revised by GETHYNJONES, E. & SANDERSON, A.R. (The Jenner Trust, The Jenner Museum, Berkeley, Gloucestershire).


VAN ECK, J.H.H., DAVELAAR, F.G., VAN DEN HEUVEL-PLESMAN, Thea A.M., VAN KOL, N., KOUWEN-

HOVEN, B. & GULDIE, F.H.M. (1976) Dropped egg production, soft shelled and shell-less eggs associated with appearance of precipitins to adenovirus in flocks of laying fowls. Avian Pathology, 5: 261-272. VAN ROEKEL, H., BULLIS, K.L., CLARKE, M.K., OLESIUK, O.M. & SPERLING, F.G. (1950) Infectious

bronchitis. Massachusetts Agricultural Experiment Station Bulletin, 460. WILDY, P. (1987) Jenner, genes, vaccines and black boxes. In: Molecular Basis of Virus Disease, pp. 1-19 (Cambridge, Cambridge University Press). YUASA, N., YOSHIDA, I. & TANIGUCHI, T. (1976) Isolation of reticuloendotheliosis virus from chicken inoculated with Marek's disease vaccine. National Institute of Animal Health Quarterly, 16: 141-151. ZARGAR, S.L. & POMEROY, B.S. (1950) Isolation of Newcastle disease virus from commercial fowlpox and laryngotracheitis vaccines. Journal of the American Veterinary Medical Association,

116: 304-305.

Gordon Richards Memorial Lecture.

The Gordon Wilson Memorial Lecture.

THE GORDON WILSON MEMORIAL LECTURE.

THE GORDON WILSON MEMORIAL LECTURE.

Gordon memorial lecture. Physics and physiology of incubation.

Gordon Bell memorial lecture. Management of soft tissue sarcoma.

Gordon Memorial Lecture. Chicken neoplasia--a model for cancer research.

Anesthesiology in dentistry--past, present and future. (First annual Joseph P. Osterloh Memorial Fellowship Lecture).

Memorial. Edgar Stillwell Gordon.

Vaccines and pregnancy: past, present, and future.

Hib Vaccines: Past, Present, and Future Perspectives.

Anthrax vaccines: past, present and future.

Diamond Jubilee lecture. Neuroradiology: past, present, future.

Fleischner lecture. Radionuclides and the lung: past, present, and future.

The Gordon-Taylor memorial lecture: metatropoi aurai--the winds of change.

Gordon-Taylor Lecture, 1976. Sir Gordon Gordon-Taylor, surgeon-anatomist and humanist.

Masahiro Iio Memorial Lecture.

On pharmacologist and vaccines: present and future challenges.

Dendritic cell-based therapeutic cancer vaccines: past, present and future.

Vaccines for sexually transmitted infections: past, present and future.

The Gordon Wilson Lecture. The new genetics.

The Gordon Wilson Lecture. Congenital adrenal hyperplasia.

The Gillies Memorial Lecture 1975.

The Gordon Wilson Lecture. Plasma cholesterol: atherogenesis and mortality.