PART 1 Overview: the present state of veterinary vaccine development Vaccine manual 5

uct of irnunity by veteir irnUn prop'.ylactic W.I. Morrison

Infectious disease continues to be one of developments in the application of DNA the most important constraints on the technology, now provide a strong con- efficient production of farm livestock in ceptual framework for the rational devel- both developing and developed countries. opment of new vaccines. While vaccination and the therapeutic or This chapter will consider recent de- prophylactic use of drugs both play an velopments in immunology that are important role in animal disease control, pertinent to understanding how the vaccination is increasingly being viewed immune system controls infections as the more sustainable option. This view and will discuss their implications for is influenced not only by the potential that contemporary approaches to vaccine vaccination offers for greater economic development. efficiency but also by the concerns that have been raised about the selection of APPROACHES TO VACCINE DEVELOPMENT drug-resistant pathogens and the potential Most of the current veterinary vaccines are harmful effects of drug residues in animal based on the use of either killed organisms products and the environment. Vaccination or their products or live attenuated has had a major impact on the control of organisms. The development of these epidemic viral diseases of livestock such vaccines has not relied on knowledge of as foot-and-mouth disease and rinderpest. the immune responses that mediate However, there are many other important immunity. Significant advances have been diseases for which efforts to develop made primarily by the development of new effective methods of vaccination have been culture techniques, improved attenuation unsuccessful. procedures and better adjuvants. While The advent of recombinant DNA there is some scope for further efforts to technology in the early 1980s created develop vaccines along these lines, there exciting new opportunities to produce are many diseases for which the more vaccines based on the use of expressed empirical methods are unlikely to be products of cloned genes. However, only a successful. few such vaccines have been successfully Two main approaches to vaccine design developed. In retrospect, it can be seen can be considered using modern molecular that the immediate expectations of the new technologies, namely the production of technology were unrealistically high, given attenuated mutant organisms by deliber- the limited knowledge of the immunology ate molecular manipulation and the of many of the target diseases and of how identification of antigenic components of antigens are processed and recognized by pathogens that can be used to induce the immune system. In the intervening protective immune responses (these decade there have been major advances in components are known as subunit immunology which, coupled with further vaccines). Unlike the traditional vaccine 6 The induction of immunity by veterinary immunoprophylactics

strategies, the ability to exploit these new Recognition of antigen by B and T approaches to vaccine development is lymphocytes dependent on an intimate knowledge of The antigen recognition structures on B the molecular structure of the target and T lymphocytes, namely immuno- pathogens and an understanding of the globulin (Ig) and the T cell receptor (TCR) mechanisms of immunity. are both generated by a process of gene rearrangement whereby each of the two PROCESSING AND RECOGNITION OF chains that make up the molecules is ANTIGENS produced by bringing together two or three Immune effector mechanisms variable sequences, from a pre-existing Studies in laboratory animal model library of variable genes, with a constant systems have demonstrated that the sequence to form a functional gene (Cooper immune system can respond in a number and Burrows, 1989; Davis and Bjorkman, of different ways to control an infection. 1988). This mechanism, together with the The type of response elicited by a pathogen further diversity created by a combination depends largely on the nature of the of different variable regions in Ig heavy organism and its site of replication within and light chains and TCR alpha and beta the host. In general, organisms that chains facilitates the generation of a very establish systemic infections and replicate large repertoire of B lymphocytes and extracellularly are controlled by antibody T lymphocytes, each with a unique antigen responses. recognition specificity. Secretory antibody responses also play Despite the similarity in structure of Ig an important role in the control of infec- and TCR molecules, B and T lymphocytes tions at mucosal surfaces. By contrast, cell- differ fundamentally in the way they mediated immune responses are generally recognize antigen. Immunoglobulins, more important in controlling organisms whether on the surface of B lymphocytes which replicate intracellularly. The T lym- or as secreted antibodies interact directly phocytes that participate in cell-mediated with foreign antigen, usually in the form immune responses may exert their effector in which it is initially encountered by the function in a number of differentways. host, i.e. as an intact organismor as They may kill infected cellsor release molecules released from the organism. cytokines, which inhibit growth of intra- Recognition of antigens by antibody is, cellular organisms or which recruit and therefore, often dependent on thecon- .activate accessory cells such as macro- formational integrity of the molecules. By phages, to perform these functions. Indeed, contrast, T lymphocytes only recognize a number of different mechanisms may antigens after they have been degraded operate against the same organism. Stimu- and presented on the surface of other cells lation of T lymphocyteresponses is also (Brodsky and Guagliardi, 1991). These essential to provide help, again in the form antigen-bearing cells rnay be cells infected of cytokines, for the production of antibody with foreign organisms or "professional" by B lymphocytes. Hence, T lymphocytes antigen-presenting cells, such as macro- have a pivotal role in the induction of phages and dendritic cells, which have virtually all specific immuneresponses. ingested antigen. The processed antigen, The way in which antigen is processed which is in the form of short peptides of and recognized by the immune system less than 20 amino acids, is associated with determines the type of T cellresponse that major histocompatibility complex (MHC) is induced. molecules on the surface of the antigen- Vaccine manual 7

presenting cell (Bodmer, 1984). The T cell although the antigenic peptides that receptor does not react with antigen alone associate with class II are longer (13 to 17 but rather recognizes a combination of the amino acids) than those bound to class I antigenic peptide and the associated MHC (eight to ten amino acids). Much of the molecule (Townsend and Bodmer, 1989). sequencepolymorphismin MHC molecules occurs in and around the The special role of MHC molecules peptide-binding region and, although this The MHC consists of a set of closely linked variation does not affect the overall genes, many of which encode molecules structure of the peptide-binding groove, it involved in antigen processing and results in subtle differences that influence presentation (Trowsdale, 1993). There are the nature of the peptides that each two main types of MHC molecules, namely molecule will bind. Thus, each individual class I and class II. The most striking class I molecule tends to bind a different feature of these MHC molecules is the high repertoire of peptides (Sette et al., 1987; degree of polymorphism they display Rothbard and Gefter, 1991), so that T cells among individuals of a species (Bodmer, from animals expressing different MHC 1984; Trowsdale, 1993). Class I molecules molecules will often recognize different are heterodimers composed of a poly- epitopes from the same pathogen, and in morphic heavy chain and a non-poly- some cases these epitopes may be on morphic light chain (j32-microglobulin), the different proteins. latter encoded outside the MHC. They are Although this variation might be expect- expressed on most cells of the body. Class ed to result in quantitative differences in II molecules are also heterodimers, both the immune response to pathogens, and polypeptides being encoded within the hence differences in susceptibility to MHC, but their expression in healthy disease, there are relatively few well- animals is confined mainly to "profes- documented examples of strong asso- sional" antigen-presenting cells, namely ciations of MHC with susceptibility to macrophages, dendritic cells and B lym- infectious disease in outbred species. This phocytes. In most mammalian species probably reflects the large number of examined, each class is encoded by two potential T cell epitopes in most pathogens gene loci and, in each instance,both alleles and the fact that most animals in an are expressed. outbred population will be heterozygous While it has been recognized since the and therefore will express several class I early 1970s that class I and class II or class II molecules. Variations inthe molecules are involved in presenting epitopes that are selected in individual antigen to T cells, the precise molecular animals may, however, affect the strain and structural basis of presentation was specificity of the T cell response if some of not elucidated until the late 1980s. A key the epitopes vary between pathogen strains event was the resolution of the structure of (Vitiello and Sherman, 1983). class I molecules by X-ray crystallography As already indicated, the TCR recognizes (Bjorkman etal., 1987). This revealed a a combination of self MHCmolecule and prominent cleft in the membrane-distal bound peptide. Since the region of the part of the molecule, which subsequent MHC molecule recognized by the studies have shown to be the site of antigen TCR, i.e. the peptide-binding groove, is binding. A similar structure has been polymorphic, each T cell will only re- described for class II MHC molecules cognize an antigenic peptide associated (Brown et al., 1993; Stern et al., 1994) with a particular MHC molecule. This 8 The induction of immunity by veterinary immunoprophylactics

phenomenon, known as MHC-restriction onstration that CD4+ T cells recognize (Doherty, Blanden and Zinkernagel, 1976), antigen presented by class II MHC has important practical implications for molecules whereas CDS+ T cells recognize studies of T cell responses in outbred antigen presented by classI MHC animals; T cells from one animal molecules.Theseinteractionsare will recognize antigen presented on the accompanied by binding of the CD4 and animal's own antigen-presenting cells but CD8 molecules to conserved regions on will not recognize the same antigen on the presenting class II and class I MHC presenting cells from another animal of a molecules, respectively. CD4+ T cells were different MHC phenotype. shown to mediate help for B cell responses Until recently, the capacity of a given anddelayed-typehypersensitivity MHC molecule to bind a large number of reactions whereas CD8+ T cells were different antigenic peptides was difficult responsible for cell-mediated cytotoxicity to explain. However, studies carried out of virus-infected cells. However, for some over the last four years, involving the time the factors that determined whether isolation and sequencing of peptides an antigen was presented by class I or class bound to class I molecules, have helped to II MHC molecules were unclear. This was resolve this issue. The heterogeneous resolved by studies which showed that mixture of peptides isolated from an class I and class II molecules bind peptides individual class I molecule were found to generated within different subcellular be conserved at one or two residues, compartments of antigen-presenting cells. usually at positions 2 and 9, and these Antigens derived from organisms that conserved amino acids were shown to be replicate in the cytoplasm of cells are essential for binding the peptides to the degraded by proteases within the cytosol respective MHC molecule (Matsumura et and the resultant antigenic peptides are al., 1992; Rammensee, Falk and Rotzschke, transported into the endoplasmic reti- 1993). Thus, the amino acids at these culum where they associate with newly positions represent an MHC binding motif, synthesized class I MHC molecules the antigenic specificity of the peptide destined for the cell surface (van Meek and being determined by amino acids at other Nathenson, 1992). By contrast, organisms positions. Information on the binding motif or proteins taken into antigen-presenting of a particular MHC molecule can be used cells by phagocytosis or endocytosis to predict possible T cell epitopes within undergo enzymatic degradation within proteins of known amino acid sequences. endosomes and associate within an There is some evidence that similar endosomal compartment with newly characteristics determine the binding of synthesised class II MHC molecules peptides to class II molecules (Rudensky transported from the Golgi apparatus al., 1992) although this has proved difficult before being expressed on the cell surface to substantiate. (Brodsky, 1992; Unanue, 1992). These alternative routes of antigen T cell subpopulations recognize antigen processing are known as the endogenous processed in different subcellam and exogenous pathways, respectively. compartments Clearly, processing of antigens by the The identification of the cell surface endogenous pathway and presentation by molecules, CD4 and CD8, as markers for class I will be confined to organisms, such the two major subpopulations of T cells in as viruses and some bacteria and protozoa, mammals was followed by the dem- that replicate intracellularly (in the Vaccine manual 9

cytoplasm). The killing of such infected infections (Else and Grencis, 1991; Urban cells by CD8+ T lymphocytes can occur et al., 1992). early in the replication cycle and thus Studies of the biological activities of the represents an effective means of limiting cytokines produced by Th1 and Th2 cells multiplication of the pathogens. have demonstrated strong cross-regulation operating between the subsets: IFN-y Heterogeneity of CD4+ T lymphocyte produced by Th1 cells inhibits the function induction of Th2 responses and both IL-4 Mosmanm et al. (1986), working with and IL-10 have inhibitory effects on the mouse T cell clones, described two types induction of Th1 responses (Mosmann et of CD4+ T cell, termed Th1 and Th2, that al., 1991; Fitch et al., 1993). Thus, the initial were distinguished by the cytokines they induction of a strong Th1 or Th2 response produced. This and subsequent studies will tend to inhibit responses by the established that activated Th1 cells secrete reciprocal subset. Nevertheless, in some interleukin 2 (IL-2) and interferon-y (IFN- infections, notably Schistosoma mansoni in y) but not IL-4, IL-5, IL-10 and IL-13, mice, an initial Th1 response is followed whereas the converse applies to activated by a switch to a Th2 response (Pearce et al., Th2 cells (Mosmann and Coffman, 1989). 1991). The switch is believed to reflect the A similar dichotomy has been reported for presence of antigens in the parasite eggs human T cells (Weiranga et al., 1990). with a strong propensity for inducing Th2 Studies of murine models of parasitic responses. This may represent a deliberate infections have proved invaluable in strategy by the parasite to favour survival elucidating the biological significance of of the adult worms. The parameters that the differences in Th1 and Th2 cells. determine whether an antigen will Infection of mice with Leishmania major, or stimulate a strong Th1 or Th2 response are immunization with Leishmania antigens, poorly understood. However, there is can induce either Th1 or Th2 T cell evidence that the biochemical nature of responses, depending on the strain of the antigen and the type of antigen- mouse and route of immunization.Th1 presenting cell in which it is presented to responses result in the control of infection T cells are important contributory factors. and immunity, whereas Th2 responses lead to enhanced disease (Liew, 1990). This is IMPLICATIONS FOR VACCINE DEVELOPMENT just one of a growing number of examples Induction of antibody responses in which the outcome of infection is In the early 1980s, a series of studies strongly influenced by the cytokine profile demonstrated that antibodies raised of the responding T cells. The induction of against intact proteins recognized short Th1 responses in mice is associated with peptide fragments of the proteins (Geyson, the activation of macrophages, the pro- Meloen and Barteling, 1984). These duction of antibody of the IgG,,, isotype observations encouraged the belief that it and the detection of delayed-type hyper- would be possible to use synthetic peptides sensitivity reactions, while Th2 responses for vaccination. A large number of studies give rise to eosinophilia and production of aimed at stimulating immunity with antibody of the IgG, and IgE isotypes. The peptides were undertaken, in which latter characteristics feature prominently animals were immunized with synthetic in many helminth infections and there is peptides representing B cell epitopes, evidence that Th2 responses are beneficial conjugated either to other peptides from for the control of enteric nematode the same pathogen or to unrelated proteins, 10 The induction of immunity by veterinary immunoprophvlactics

to provide the necessary T helper cell will therefore rely largely on the pre- epitopes. With a few exceptions, these existing antibody induced by vaccination. attempts at immunization were unsuc- The use of intact, purified or recombinant cessful. In many instances, the synthetic proteins for immunization can overcome peptides completely failed to induce some of the problems associated with antibodies against the parent protein or immunization with peptides. Such proteins organism while, in other cases, antibody are likely to contain several T cell epitopes responses did occur but were at best only and, if appropriately produced, should partially effective. have the correct conformation for recog- Subsequent studies of protein structure nition by antibodies. However, the latter is have highlighted the fact that so-called not always true. For example, the integrity linear epitopes have a degree of confor- of antibody epitopes on individual viral mation and that this conformation may capsid proteins may be dependent on the differ subtly from that adopted by the structural interaction with other protein respective synthetic peptides. The process components of the capsid. This is the case of conjugating a peptide to a carrier for several of the neutralizing epitopes on molecule may also affect the conformation the surface of foot-and-mouth disease of the peptide, resulting in antibodies of virus; of the three capsid proteins only one low avidity for the pathogen in question. (VP1) retains any immunogenicity follow- One pathogen for which a degree of ing purification, and itis much less success has been achieved by immun- immunogenic than killed intact virus ization with a synthetic peptide is foot- (Bachrach et al., 1975). and-mouth disease virus. Immunization The methods by which recombinant with a peptide consisting of two linked proteins are produced can also affect the peptide sequences representing residues integrity of epitopes recognized by anti- 141-158 and 200-213 of the VP1 capsid bodies. Thus, if glycosylation is required protein resulted in a proportion of immun- for antibody recognition, production of the ized cattle (Di Marchi et al., 1986). Struc- proteins in bacteria will be inappropriate tural studies of the virus demonstrated that and proteins produced in insect cells may the 141-158 component of the peptide be defective because of differences in the corresponds to a superficial loop on the sugar side chains added by these cells surface of the virus particle (Acharya et al., compared with mammalian cells. Differ- 1989) and suggested that the peptide ences in the folding of proteins produced successfully reproduces this loop structure. in bacteria rnay also result in the disruption Nevertheless, it is still unclear whether the of some B cell epitopes. failure to achieve protection in all animals immunized with the peptide was due to The need for nnultiple T cell epitopes subtle conformational differences in the Since T lymphocytes recognize small antibody recognition site or to inadequa- processed fragments of antigens, the cies in the helper T cell response. conformational structure of antigens is Another factor that may limit the success generally not a constraint for the induction of immunization with peptides is the use of T cell responses. However, because of an unrelated carrier protein to stimulate T cells from animals of different MHC T cell help for antibody production. No types tend to recognize different peptide anamnestic T cell response will occur sequences within an antigen, immun- following the challenge of immunized ization with short polypeptides containing animals with the pathogen, and immunity only one or two potential T cell epitopes is Vaccine manual 11

likely to induce a response in onlya determine their function and that antigenic proportion of animals. This will be true stimulation may result in the activation of both for T helper cellresponses for T cells producing different patterns of antibody production and for effector T cell cytokines is of major importance when responses. This further strengthens the considering immunization strategies. It is, argument for using one or more intact therefore, desirable to know whether the proteins in subunit vaccines so that there protective responses against the target will be sufficient n.umbers of potential pathogens involve Th1 or Th2 CD4+ T cell T cell epitopes to ensure that the majority responses. However, since the early events of individuals within an outbredpopu- in antigen processing that result in a bias lation will respond to the antigens. In some to Thl or Th2 responses are still incom- circumstances, there may be a case for pletely understood, strategies for preferen- excluding particular T cell epitopes from a tial induction of one or other responseare vaccine construct. For example, if an not yet well established. The use of adju- antigen contains a particularly dominant vants that give a bias in the response, for T cell epitope that is variable between example the induction of Th1 responses strains of a pathogen, exclusion of such an by rnycobacteria, is one approach that can epitope might result in a response that is be pursued. Experiments in mice involving less strain-specific. administration of recombinant cytokines or cytokine-specific antibodies at the time Constraints on stimulation of CD8+ T cell of immunization have implicated IL-4 and responses IL-12 as promoters of Th2 or Th1 cell Because of the need for antigen to be responses, respectively (Swain et al., 1991; processed by the endogenous pathway Locksley, 1993). These findings indicate for recognition by CD8+ T cells, the immun- that, by administering cytokines with ization of animals with killed organisms antigen or by including cytokine genes in or their component proteins generally fails molecular vaccine constructs, it may be to induce CDS+ T cell responses. Therefore, possible to influence the cytokine profile alternative antigen delivery systems must of T cell responses. be considered when developing subunit vaccines required to stimulate CD8+ T cell More than one mechanism of immunity responses. These could include the use of It is becoming increasingly apparent that virus vectors or vaccination with "naked" immunity against a given pathogen may DNA, both of which result in the be achieved by alternative immune mech- expression of proteins within the cell anisms. This is clearly the case with cytosol. Recent studies have also provided complex protozoan parasites which under- evidence that the active component of the go differentiation through several develop- adjuvant saponin, when used to prepare mental stages that differ antigenically and antigen-complexed structures known as replicate in different cell types. Thus, with immunostimulating complexes (ISCOMs), malaria parasites, antibody against the facilitates the transfer of antigen across cell infective sporozoite stage can block membranes and the induction of CD8+ infection, class I MHC-restricted cytotoxic T cell responses (Takahashi et al., 1990). T cell responses are generated against the hepatic intracellular stages and other, as Influencing the cytokine response yet poorly understood, cell-mediated The recognition that the cytokines pro- mechanisms operate against the intra- duced by CD4+ T cells to a large extent erythrocytic stages. 12 The induction of immunity by veterinary immunoprophylactics

Similarly, different mechanisms may are undoubtedly of major importance in operate against migratory helminth mediating immunity, T cell-mediated parasites. However, there is also evidence responses may provide an additional that more than one immune mechanism mechanism for clearing the virus. Yet the may be effective in the control of virus role of T cell effector mechanisms in foot- infections. For example, itis well es- and-mouth disease has been largely tablished that maternally derived antibody ignored. protects offspring from infection with the These observations indicate that the morbilliviruses (measles, rinderpest) and induction of highly effective immunity also interferes with vaccination (Albrecht with subunit vaccines may necessitate the et al., 1977). Yet a proportion of cattle inclusion of more than one antigenic successfully immunized against rinderpest component in a vaccine and the use of an virus with a recombinant vaccinia virus antigen delivery system that is effective at expressing the F glycoprotein were found inducing both humoral and cell-mediated to produce little or no rinderpest-specific immune responses. The poor efficacy of antibody (Yilma et al., 1988; Belsham et al., some of the currently used killed vaccines 1989), indicating that immunity must have may be due in part to their limited ability been mediated by T cell responses. Similar to stimulate T cell-mediated components results have been obtained with infectious of the protective immune responses. bursal disease virus in chickens, using a recombinant avipox virus expressing the Pathogens that subvert host immune VP2 protein, although the immunity responses achieved with the recombinant was A common objective in the development incomplete (Bayliss et al., 1991). of a vaccine is to mimic the immune Clearly, if sufficient antibody of appro- responses that occur during recovery from priate specificity and biological activity is natural infection. While this is an ap- present in an immunized animal to prevent propriate approach for many pathogens, infection with the respective virus, cell- some organisms have evolved stratagems mediated effector mechanisms will not be for modifying host immune responses in required for protection. However, if some order to establish persistent infections. of the challenge virus escapes initial Helminth and protozoan parasites have neutralization by antibody, it is likely that been particularly adept at developing a cell-mediated immune responses will be variety of escape mechanisms. One of the beneficial, if not essential, for clearance of strategies employed by parasites such as the infection. This will be particularly true Leishmania sp. and Schistosoma sp. is to for viruses that spread by cell to cell direct the T cell response to produce contact. There may also be some flexibility cytokines that are inappropriate for in the type of T cell response that is parasite clearance (Sher et al., 1992). In the employed. In mice immunized against case of Schistosoma rnansoni, this results in influenza A virus, the transfer of either the development of characteristicegg CD4+ or CD8+ T cells into native recipients granulomas that are responsible for clinical resulted in the clearance of challenge virus, disease. Nevertheless, schistosome- the important common feature being the infected (SCID) mice, which are unable to cytokines produced by the two cell types mount a granulomatous response, develop (Lukacher et al., 1986). With viruses such severe hepatitis that is believed to be due as that which causes foot-and-mouth to release the of parasite proteases (Amira disease, and for which antibodyresponses et al., 1993). Thus, the "deviated" immune Vaccine manual

response not only favours parasite that are able to bind the respective persistence but may also have a role in cytokines (Smith, 1993). There is also protecting and ensuring the survival of the evidence that serine protease inhibitors host, albeit with some pathology. Any expressed by vaccinia may inhibit approach to designing a vaccine for such intracytosolic processing of antigens parasites must be based on an under- destined for association with class I MHC standing of which components of the host's (Townsend et al., 1988). Several of the immune response are responsible for herpesviruses and adenoviruses have been protection as well as on ensuring the shown to express genes that in_hibit the avoidance of immune responses that assembly of class I MHC molecules (Lippé potentiate disease. et al., 1991; Hill et al., 1994). Identification Bloodsucking ectoparasites have devel- of the precise role of thesegenes in oped a number of mechanisms to avoid determining the virulence of the viruses the clotting of imbibed blood and to and their immunogenicity is relevant not minimize adverse effects of inflammatory only for understanding the pathogenesis mediators released at the site of feeding. In of disease caused by the viruses but also in the case of ticks, which feed continuously considering the use of animal poxviruses for several days, significant hyper- and herpesviruses as vaccine vectors. sensitivity reactions are induced by salivary proteins. While in previously NEW APPROACHES TO VACCINE DESIGN exposed animals this results in a reduction Molecularly defined attenuation in the number of ticks that engorge, such The simplest way of producing a vaccine animals still carry significant tick burdens that mimics the immune responses (Willadsen, 1980). Over the last ten years, induced by natural infection is to select researchers in Australia have developed an attenuated mutant. The use of live an alternative strategy for vaccination organisms also has the advantage of against the one-host tick Boophilus microplus providing longer-lasting immunity than based on immunization with proteins from can be attained with killed antigen. Many the tick gut, to which the host is not of the traditional vaccines are based on the normally exposed (Willadsen, McKenna use of attenuated organisms that were and Riding, 1988; Willadsen et al., 1989). either identified by chance or selected by Antibody induced by these "concealed" the prolonged culture of the organisms. antigens, when ingested by the tick, causes Advances in knowledge of the molecular damage to the gut wall and results not structure of viruses and of the function only in tick mortality but also in a markedly of individual genes in replication and reduced fecundity of surviving ticks. A assembly now provide the opportunity to similar approach is also being pursued for produce targeted mutations that result in vaccination against the bloodsucking altered virulence. The deletion of whole nematode Haemonchus contortus. genes negates the possibility of reversion Several of the large DNA viruses have to virulence as a consequence of point evolved molecular mechanisms that could mutations. Moreover, viruses with potentially modify antiviral immune mutations in several genes can be responses. The vaccinia virus genome produced. A strain of pseudorabies virus contains a number of genes with homology (Aujeszky's disease virus), in which the to host receptors for the cytokines IL-1, genes encoding the GI glycoprotein and IL-6 and IFN-7. Some of these genes are thymidine kinase have been disrupted, has expressed as soluble proteins (virokines) been shown to be avirulent and to 14 The induction of immunity by veterinary immunoprophylactics

stimulate immunity against challenge with been put into commercial production in native virus (Moormann et al., 1990). This Australia (Willadsen et al., 1989; Rand et virus is being marketed as an attenuated al., 1989). A recombinant protein from the vaccine. tapeworm Taenia ovis has been shown to A similar approach is being used with be effective at immunizing sheep against bacteria by targeting genes involved in tapeworm infestation (Johnson et al., 1989), bacterial metabolism. The most thoroughly and immunization of cattle with a investigated system is Salmonella spp., in recombinant sporozoite surface antigen which mutation of a number of genes has from Theileria parva has been found to been shown to result in attenuated protect a proportion of animals against organisms that retain immunogenicity experimental challenge (Musoke et al., (Dougan, Hormaeche and Maskell, 1987). 1992). Again, the incorporation of double or triple mutations minimizes the risk of reversion Production of virus capsids to virulence. The best characterized of these The capacity of the hepatitis B core protein are the aro mutants (Dougan et al., 1988), in to assemble spontaneously into virus-like which the mutations interrupt the pathway particles (McAleer el al., 1984) may partly for biosynthesis of aromatic metabolites account for the success of this vaccine, since resulting in organisms that are dependent assembly should allow the protein to adopt on nutritional elements not available in a conformation similar to that of the native mammalian tissues. Such aro mutants have virus particle. been used successfully to vaccinate calves In the case of non-enveloped viruses in against Salmonella typhimurium (Jones et al., which the capsid is composed of several 1991). proteins, the surface conformation adopted by each protein in the virus particle is Immunization with recombinant proteins dependent on interactions with the other The use of purified recombinant proteins proteins. Thus, the isolated proteins are for vaccination can be considered where often poorly immunogenic. This problem specific antigens have been identified as may be overcome if non-infectious viral the targets for protective antibody and / or capsids can be produced by expressing all CD4+ T cell responses. However, as already of the capsid proteins in a single construct. indicated, the expression system used to This has been achieved with bluetongue produce the protein should not alter virus (Roy, 1992). Bluetongue virus, the important antibody recognition sites on the prototype of the orbiviruses, is made up protein. Generally, recombinant proteins of seven proteins, three of which are with- need to be administered in a potent in an inner core surrounded by two adjuvant. concentric protein layers formed by VP3 The human hepatitis B vaccine, which and VP7. The remaining two proteins, VP2 consists of recombinant viral core protein, and VP5, are attached to the outer VP7 is the most notable success in this area layer. Empty virus-like particles have been (McAleer et al., 1984); immunity has been produced by expressing the four main achieved with antigen produced in structural proteins (VP2, VP3, VP5 and Escherichia coli or in yeast. Recently, a VP7) in a single recombinant baculovirus. vaccine for Boophilus micro plus tick These particles closely resemble native infestation incattle, based on the virus particles in structure. Immunization production in E. coli of an antigen that is experiments in sheep have shown that they normally expressed in the tick gut, has protect against challenge with bluetongue Vaccine manual 15

virus of the homologous serotype and that areas of Asia and Africa where it causes they are much more immunogenic than sporadic outbreaks of disease. An purified VP2 or VP5 proteins (Roy, French attenuated strain of the virus that had been and Erasmus, 1992). used locally in Africa to vaccinate against Similar studies are under way with foot- the disease (Kitching, Hammond and and-mouth disease virus. Taylor, 1987) has been developed as a vector, and recombinant viruses expressing Live vectors rinderpest virus glycoproteins have been The potential use of viruses or bacteria as shown to protect cattle against challenge live vectors for vaccination has been a with rinderpest virus (Romero et al., 1993). major focus of experimental investigation A disadvantage of the capripox virus is over the last ten years. Vectors may be that it cannot be used outside the areas naturally occurring apathogenic organisms where infection is endemic owing to its or attenuated mutants produced by genetic notifiable status. Nevertheless, there are manipulation. Such vectors offer many of many potential applications of this vector the advantages of live vaccines and can in Africa and Asia. potentially be used to express antigens Avipox and canarypox viruses have been from more than one pathogen. The virus developed as potential vaccine vectors and vectors in particular provide a means of the former has been used successfully to inducing CD8+ T cell responses, which are vaccinate chickens against Newcastle not readily generated by killed vaccines. disease and infectious bursal disease Initial studies of virus vectors were (Boursnell et al., 1990a and 1990b; Bayliss focused on vaccinia, which had already et al., 1991). These viruses can also be been used with great success in smallpox considered as vectors for mammals, since vaccination programmes and was known they produce abortive infections in to have a wide host range (Mackett and mammalian cells, i.e. the synthesis of virus Smith, 1986). Being a poxvirus, it also had proteins occurs but no infectious virus is a large genome in which non-essential produced; there is therefore no risk of the regions could be identified for the insertion virus spreading to other animals. of foreign genes. Recombinant vaccinia The herpesviruses have large genomes viruses expressing viral genes have been which, like that of the poxviruses, can used successfully in experimental studies accommodate large inserts of foreign DNA. to immunize cattle against rinderpest and The potential of a number of animal vesicular stomatitis and dogs and foxes herpesviruses to serve as vaccine vectors against rabies (Yilma et al., 1988; Belsham is currently being explored. These include et al., 1989; Blancou et al., 1986). Field pseudorabies virus, bovine herpesvirus 1, vaccination of foxes against rabies, using equine herpesvirus 1 and turkey baits containing a vaccinia recombinant, herpesvirus. Adenoviruses are also being has also been successful. Despite these investigated, particularly in the context of successes and the demonstrated safety of delivering antigens to mucosal surfaces. the vaccines employed, concern has been The coexpression of cytokine genes and expressed about the widespread release of foreign antigens in virus vectors offers vaccinia now that smallpox vaccination has a potential means of enhancing or stopped. Consequently, attention has modulating immune responses to the turned to animal poxviruses. expressed antigens. Experiments with Capripox virus, which infects cattle, vaccinia virus in mice have yielded sheep and goats, is found throughout large promising results with a number of 16 The induction of immunity by veterinary immttnoprophylactics

cytokines (Ramshaw et al., 1992). This been shown to result in protection against approach may be of particular value in virus challenge (Ulmer et al., 1993; Fynan providing a bias towards Th1 or Th2 T cell et al., 1993). The injected genes appear to responses. be expressed within host cells without Attenuated mutant strains of several integration of plasmid into chromosomal different Salmonella species have been DNA. While studies of the transfection studied extensively for their potential as efficiency of injected DNA have shown that vaccine vectors (Dougan, Hormaeche and muscle is 100 to 1 000 times more permis- Maskell, 1987; Dougan et al., 1988). Foreign sive than other tissues, protection has also antigens can be expressed from plasmids been achieved following inoculation by the or following the integration of the genes intravenous, intranasal and intratracheal into the bacterial genome. Much of this routes and by delivering the DNA intra- work has been carried out in mice and has dermally using a "gene gun". The focused on the induction of immune immunization of mice with DNA induces responses to other bacterial antigens and antibody responses as well as CD4÷ and on the capacity to stimulate immunity at CD8+ T cell responses. The induction of gut mucosal surfaces. Mice immunized immune responses to bovine herpesvirus with a Salmonella recombinant expressing 1 in cattle has been investigated in one a malaria circumsporozoite antigen were study (Cox, Zamb and Babiuk, 1993); a found to be protected against malaria in plasmid containing the gII viral gene the absence of antibodies (Sadoff et al., stimulated neutralizing antibodies and 1988). Since this immunity is mediated by resulted in a marked reduction in nasal CD8+ T cells, this finding suggests that shedding of the virus following challenge. Salmonella spp. may be able to introduce antigens into the endogenous processing Targeting of antigen at nnucosal surfaces pathway. The stimulation of protective immune A variety of other bacteria are being responses at mucosal surfaces presents a investigated as potential vaccine vectors. particularly challenging problem because Because of its potent capacity to stimulate of the requirement for such responses to Th1 T cell responses, the mycobacterium be induced locally. Non-viable antigen bacillus Calmette-Guérin (BCG) may have administered orally is susceptible to particular utility as a vector. A recombinant proteolytic degradation and is absorbed in BCG expressing a surface antigen from the only small amounts through the gut wall. protozoan parasite Leishmania major has Moreover, such "dietary" antigens tend to been shown to protect mice against induce tolerance rather than an active cutaneous leishmaniasis (Connell et al., immune response. One way of overcoming 1993), a disease that is known to require this problem is to use bacterial or viral Th1 responses for immunity. vectors, such as Salmonella spp. or adenovirus, which replicate in the Immunization with nucleic acids alimentary tract. The recent discovery that the injection of Other strategies that facilitate the uptake DNA into animals can result in immune of protein in the gut and the induction of responses to proteins encoded by the DNA an active response are currently being has opened up a completelynew approach investigated. One of these involves theuse to the development of subunit vaccines. of cholera toxin, which is a multimeric Vaccination of mice with plasmids contain- protein composed of a single A subunit ing the influenza haemagglutiningene has and a pentameric B subunit; binding to Vaccine manual 17

epithelial cells occurs by interaction of the BIBLIOGRAPHY B subunit with GMi-gangliosides while toxicity is mediated by ADP-ribosyl- Acharya, R., Fry, E., Stuart, D., Fox, G., transferase activity of the A subunit Rowlands, D. & Brown, F. 1989. The (Spangler, 1992). Oral administration of three-dimensional structure of foot-and- either subpathogenic doses of intact toxin mouth disease virus at 2.9 A resolution. or purified B subunits results in induction Nature, 337: 709-716. of a specific immune response. Moreover, Albrecht, P., Ennis, F.A., Saltzman, E.J. & protein antigens administered along with, Krugman, S. 1977. Persistence of internal or covalently linked to, the toxin or its B antibody in infants beyond 12 months: subunit elicit strong antibody and CD4+ T mechanism of measles vaccine failure. cell responses (Dertzbaugh and Elson, J. Paediat., 91: 715-719. 1993). Whether or not the B subunit retains Amira, P., Locksley, R.M., Parslow, T., the full adjuvant activity of the intact toxin Sadick, M., Rector, E., Ritter, D. & has not been fully resolved. McKerrow, J. 1993. Tumour necrosis Another approach to delivering antigens factor-7 restores granulomas and induces to the gut is to encapsulate antigen in egg laying in schistosome-infected microspheres with an outer biodegradable (SCID) mice. Nature, 356 :604-607. polymer coat that protects the antigen Bachrach, H.L., Moore, D.M., McKercher, against rapid enzymatic degradation. P.D. & Polatnick, J. 1975. Immune and Microspheres with a diameter of 5 to 10 p.m antibody responses to an isolated capsid have been shown to be taken up efficiently protein of foot-and-mouth disease virus. by the Peyer's patches and to induce IgG J. Immunol., 115: 1636-1641. and IgA antibody responses in mice. Bayliss, C.D., Peters, R.W., Cook, J.K.A., Reece, R.L., Howes, K., Binns, M.M. & CONCLUDING REMARKS Boursnell, M.E.G. 1991. A recombinant In the last decade, a clear picture has fowlpox virus that expresses the VP2 emerged of how antigens are processed antigen of infectious bursal disease virus and recognized by the immune system and induces protection against mortality significant progress has been made in caused by the virus. Arch. Virol., 120: understanding the immune mechanisms 193-205. that operate against different types of Belsham, G.J., Anderson, E.C., Murray, P.K., pathogens. This new knowledge, together Anderson, J. & Barrett, T. 1989. Immune with further advances in technologies for response and protection of cattle and genetic manipulation, has led to a variety pigs generated by a vaccine virus of new approaches to the attenuation of recombinant expressing the F protein pathogenic organisms and to the design of of rinderpest virus. Vet. Rec., 124: 655- antigen delivery systems appropriate for 658. inducing particular immune responses. Bjorkman, P.J., Saper, M.A., Samraoui, B., There has also been progress in other areas Bennett, W.S., Strominger, J.L. & Wiley, of research, not discussed here, such as the D.C. 1987. The foreign antigen site and targeting of antigens at particular cell types T cell recognition regions of class I in the immune system and studies to histocompatibility antigens. Nature, 329: develop new adjuvants and understand 512-518. their mode of action. These developments Blancou, J., Kieny, M.P., Lathe, R., Lecocq, offer exciting potential for the production J.P., Pastoret, P.P., Soulebot, J.P. & of a new generation of vaccines. Desmettre, P. 1986. Oral vaccination of 18 The induction of immunity by veterinary immunoprophylactic,v

the fox against rabies using a live Leislimania surface proteinase gp63. Proc. recombinant vaccinia virus. Nature, 322: Natl Acad. Sci. USA, 90: 11473-11477. 373-375. Cooper, M.D. & Burrows, P.D. 1989. B cell Bodmer, W. 1984. The major histocom- differentiation. In T. Honjo, F.W. Alt & patibility system: structure and function: T.H. Rabbits, eds. Immunoglobulin genes, summary and synthesis. In Y. Yamamura p. 1-21. San Diego, Calif., USA, Academic. & T. Tada, eds. Progress in Immunology Cox, J.M., Zamb, T.J. & Babiuk, L.A. 1993. V. Fifth Int. Congr. Immunology,P. 959- Bovine herpesvirus 1: immune responses 970. Tokyo, Academic Press Japan. in mice and cattle injected with plasmid Boursnell, M.E.G., Green, P.P., Campbell, DNA. J. Virol., 67(9): 5664-5667. J.I.A., Deuter, A., Peters, R.W., Tomley, Davis, M.M. & Bjorkman, P.J. 1988. T-cell F.M., Samson, A.C.R., Emmerson, P.T. antigen receptor genes and T cell re- & Binns, M.M. 1990a. A fowlpox virus cognition. Nature, 334: 395-401. vaccine vector with insertion sites in Dertzbaugh, M.T. & Elson, C.O. 1993. the terminal repeats: demonstration of Comparative effectiveness of cholera its efficacy using a fusion gene of toxin B subunit and alkaline phosphatase Newcastle disease virus. Vet. Microbiol., as carriers for oral vaccines. Infect. 23: 305-316. Immunol., 61: 48-55. Boursnell, M.E.G., Green, P.F., Campbell, Di Marchi, R., Brooke, G., Gale, C., J.I.A., Deuter, A., Peters, R.W., Tomley, Cracknell, V., Doel, T. & Mowat, N. F.M., Samson, A.C.R., Chambers, P., 1986. Protection of cattle against foot- Emmerson, P.T. & Binns, M.M. 1990b. and-rnouth disease by a synthetic Insertion of the fusion gene from peptide. Science, 232: 639-641. Newcastle disease virus into a non- Doherty, P.C., Blanden, R.V. & Zinkernagel, essential region in the terminal repeats R.M. 1976. Specificity of virus-immune of fowlpox virus and demonstration of effector T cells for H-2K and H-2D protective immunity induced by the compatible interactions: implications for recombinant. J. Gen. Virol., 71: 621-628. H-antigen diversity. Transplant. Rev., 29: Brodsky, F.M. 1992. Antigen processing and 89-124. presentation: close encounters in the Dougan, G., Hormaeche, C.E. & Maskell, endocytic pathway. Trends Cell Biol., 2: D.J. 1987. Live oral Salmonella vaccines: 109-115. potential use of attenuated strains as Brodsky, F.M. & Guagliardi, L.E. 1991. The carriers of heterologous antigens to the cell biology of antigen processing and immune system. Parasite Immunol., 9: presentation. Annu. Rev. Immunol., 9: 151-160. 707-744. Dougan, G., Chatfield, S., Pickard, D., Bester, Brown, J.H., Jardetzky, T.S., Gorga, J.C., J., O'Callaghan, D. & Maskell, D.J. 1988. Stern, L.J., Urban, R.G., Strominger, Construction and characterisation of J.L. & Wiley, D.C. 1993. Three- vaccine strains of Salmonella harbouring dimensional structure of the human class mutations in two different aro genes. J. II histocompatibility antigen HLA-DR1. Inf. Dis., 158: 1329-1335. Nature, 364: 33-39. Else, K.J. & Grencis, R.K. 1991. Cellular Connell, N.D., Medina-Acosta, E., McMaster, immune responses to the murinenema- W.R., Bloom, B.R. & Russell, D.G. 1993. tode parasite Trichuris muris. I. Differ- Effective immunization against cutane- ential cytokine production during acute ous leishmaniasis with recombinant or chronic infection. Immunology, 72: bacille Calmette-Guérin expressing the 508-513. Vaccine manual 19

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cytokine synthesis and function of mouse peptides isolated from MHC class II CD4+ T cells. Immunol. Rev., 123: 209-229. molecules suggest sequence motifs. Musoke, A.J., Morzaria, S., Nkonge, C., Nature, 359: 429-431. Jones, E. & Nene, V. 1992. A recombinant Sadoff, J.C., Ballou, W.R., Baron, L.S., sporozoite surface antigen of Theileria Majarian, W.R., Brey, R.N., Hockmeyer, parva induces protection in cattle. Proc. W.T., Young, J.F., Cryz, S.J., Ou, J., Natl Acad. Sci. USA, 89: 514-518. Lowell, G.H. & Chulay, J.D. 1988. Pearce, E.J., Caspar, P., Grzych, J.-M., Lewis, Oral Salmonella typhimurium vaccine F.A. & Sher, A. 1991. Downregulation expressing circumsporozoite protein of Thl cytokine production accompanies protects against malaria. Science, 240: induction of Th2 responses by a parasitic 336-338. helminth, Schistosoma mansoni. J. Exp. Sette, A., Buus, S., Colon, S., Smith, J.A., Med., 173: 159-166. Miles, C. & Grey, H.M. 1987. Structural Rammensee, H.-G., Falk, K. & Rotzschke, characteristics of an antigen required 0. 1993. Peptides naturally presented for its interaction with Ia and recognition by MHC class I molecules. Annu. Rev. by T cells. Nature, 328: 395-399. Immunol., 11: 213-244. Sher, A., Gazzinelli, R.T., Oswald, I.P., Ramshaw, I., Ruby, J., Ramsay, A., Ada, G. Clerici, M., Kullberg, M., Pearce, E.J., & Karupiah, G. 1992. Expression of Berzofsky, J.A., Mosmann, T.R., James, cytokines by recombinant vaccinia S.L., Morse, H.C. & Shearer, G.M. 1992. viruses: a model for studying cytokines Role of T-cell derived cytokines in the in virus infections in vivo. Immunol. Rev., downregulation of immune responses 127: 157-182. in parasitic and retroviral infection. Rand, K.N., Moore, T., Sriskantha, A., Immunol. Rev., 127: 183-204. Spring, K., Tellam, R., Willadsen, P. Smith, G.L. 1993. Vaccinia virus glycoproteins & Cobon, G.S. 1989. Cloning and and immune evasion. J. Gen. Virol., 74: expression of a protective antigen from 1725-1740. the cattle tick Boophilus microplus. Proc. Spangler, B.D. 1992. Structure and function Natl Acad. Sci. USA, 86: 9657-9661. of cholers toxin and the related Escherichia Romero, C.H., Barrett, T., Evans, S.A., coli heat-labile enterotoxin. Microbiol. Kitching, R.P., Gershon, P.D., Bostock, Rev., 56: 622-647. C. & Black, D.N. 1993. A single capripox Stern, L.J., Brown, J.H., Jardetzky, T.S., recombinant vaccine for the protection Gorga, J.C., Urban, R.G., Strominger, of cattle against rinderpest and lumpy J.L. & Wiley D.C. 1994. Crystal structure skin disease. Vaccine, 11: 732-742. of the human class II MHC protein HLA- Rothbard, J.B. & Gefter, M.L. 1991. DR1 complexed with an influenza virus Interactions between immunogenic peptide. Nature, 368: 215-221. peptides and MHC proteins. Annu. Rev. Swain, S.L., Bradley, L.M., Croft, M., Immunol., 9: 527-565. Tonkonogy, S., Atkins, G., Weinberg, Roy, P. 1992. Bluetongue virus proteins. J. A.D., Duncan, D.D., Hendrick, S.M., Gen. Virol., 73: 3051-3064. Dutton, R.W. & Huston, G. 1991. Helper Roy, P., French, T.J. & Erasmus, B.J. 1992. T cell subsets: phenotype function and Protective efficacy of virus-like particles the role of lymphokine in regulating for bluetongue disease. Vaccine, 10: 28-32. their development. Immunol. Rev., 123: Rudensky, A.Y., Preston-Hurlburt, P., Al- 115-144. Ramadi, B.K., Rothbard, J. & Janeway Takahashi, H., Takeshita, T., Morein, B., Jr, C.A. 1992. Truncation variants of Putney, S., Germain, R.N. & Berzofsky, Vaccine manual 21

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GensilleaNy jnes[red vaccines B.W.J. Mahy

Genetic engineering is a term used to be molecularly cloned and expressed to describe experimental or industrial produce the polypeptide or protein approaches to modifying the genome of a specified by the original messenger RNA. living cell without normal sexual or asexual Bacterial plasmids can serve both as transmission of genetic material. In a cloning and expression vectors when broader sense, it is used to describe genetic introduced into appropriate host cells and, manipulation of the genome of a virus and in the last few years, a range of expression is often used synonymously with the term vectors have become available that will recombinant DNA technology. The ability replicate in bacteria, yeast, insect or animal to manipulate the genome of a living cell cells to produce large quantities of a or virus was made possible by a series of desired polypeptide or protein. discoveries made 20 to 30 years ago. Such expressed proteins can be purified First was the recognition of bacterial to serve as a vaccine antigen, as in the restriction enzymes by Arber and Dussoix highly successful vaccine against hepatitis (1962). These are endonucleases that B prepared in yeast (Saccharomyces recognize specific DNA sequences and cerevisiae), which was the first genetically cleave the DNA only at those sites engineered vaccine licensed for use in (Meselson and Yuan, 1968). Coupled with humans. Alternatively, if the expression other enzymes involved in DNA synthesis vector will replicate in animal or human such as ligases (DNA-joining enzymes), it cells, the expressed antigen may immunize became feasible to introduce deliberately the recipient host without the need for specific foreign DNA sequences into DNA purification. This is the basis for the molecules which are then described as development of a wide range of candidate recombinant DNA. When the recombinant vaccines for both human and veterinary DNA replicates, the foreign DNA is also use which depend on recombinant gene replicated. If the foreign DNA is inserted expression in an appropriate viral or into a bacterial plasmid or phage DNA bacterial vector. Such live recombinant which undergoes rapid multiplication vector vaccines offer great promise but, to together with the host bacteria, it is possible date, have not been licensed for use in to generate large quantities of the foreign human subjects. DNA. This process is called molecular Recently, the discovery that plasmid cloning (Sambrook, Fritsch and Maniantis, DNA sequences can be expressed directly 1989). after injection into mouse muscle (Wolff et Another crucial discovery was the al., 1990) without the need for a viral or existence of reverse transcriptase, an bacterial expression vector has opened up enzyme present in oncogenic RNA viruses an exciting range of possibilities for the and which converts RNA into DNA design of vaccines for human or veterinary (Baltimore, 1970). This allowed the use through polynucleotide vaccination conversion of cellular or viral messenger (Cohen, 1993; Ulmer et al., 1993; Ulmer, RNAs into DNA so that they could then Donnelly and Liu, 1994). In addition, since 24 Genetically engineered vaccines

the encoded antigens are expressed TABLE 1 intracellularly in the recipient host, they Prospective genetically engineered are introduced into class I MHC molecules vaccine types and this results in the priming of cytotoxic T cells (Townsend and Bodmer, 1989; Type Examples Barber and Parham, 1994) so that both Attenuated live Pseudorabies (Aujeszky's cellular and humoral immune responses disease) are stimulated (Ulmer et al., 1993). Finally, the ability to determine the Defective live ALVAC-FL (feline leukaemia) nucleotide sequence of the genomes of Chimeric live Capripox, rinderpest, lumpy DNA or RNA animal viruses has led to a skin disease considerable body of research on the Subunit Bluetongue structure of the antigens they encode. In DNA polynucleotide Avian influenza addition to expressing the genes in various vectors, attempts have been made to Polypeptide Foot-and-mouth disease synthesize in vitro various polypeptides which correspond to these antigenic determinants or epitopes and to use the attenuation rely on spontaneous, random peptides themselves as candidate immuno- mutations occurring during multiple gens. Unfortunately, this approach has had passages, and the basis for the attenuation only limited practical success, but some is usually not known. An alternative important early work was carried out with approach is to use genetic engineering to foot-and-mouth disease (Bittle et al., 1982; define specific genes or regions of the Di Marchi et al., 1986). genome which are responsible for In this brief overview, the development virulence, then delete these in order to and future prospects for genetically obtain a replicating, non-pathogenic virus engineered veterinary vaccines will be for use as the immunogen. described. The various types of vaccine This approach has been particularly will be considered separately according to fruitful with bacteria and DNA viruses the listing given in Table 1. with large genomes, such as herpesviruses and poxviruses. Since this type of GENETICALLY ENGINEERED ATTENUATION attenuation involves the removal of parts Many conventional vaccines currently of the genome, such bacteria and viruses employed in human or veterinary may then be used to vector genes encoding medicine contain live micro-organisms or a variety of foreign antigens, creating virus that has been attenuated, for example chimeric vaccines. by multiple passages in cell culture, to the The experimental protocol for gene point that it will multiply in the recipient deletion and insertion usually requires host but no longer induce disease. Live some means of identification of the vaccines offer several potential advantages progeny which contain the desired compared with killed vaccines in that they genotype. In the original experiments to usually induce a rapid immune response develop modified poxviruses for foreign of long duration and also induce local gene insertion, the thymidine kinase (TK) immunity which may be critical in gene was deleted and a positive selection providing protection against subsequent of progeny which lack the gene was made infection by virulent micro-organisms. by growing the virus in a TK cell line However, conventional methods of (lacking a cellular thymidine kinase) using Vaccine manual 25

a medium containing 5-bromodeoxy- In the early development of poxviruses uridine (BudR). Any progeny virus which as gene vectors, it was estimated that contains the TK gene will convert BudR infectious virus could be produced into a phosphorylated form which is toxic containing at least 25 000 base pairs of to the host cell. Consequently, only foreign DNA (Smith and Moss, 1983). More recombinant viruses with the TK deletion recently, a highly attenuated candidate will survive (Mackett and Smith, 1986). A vaccinia virus vector vaccine (NYVAC) has similar system can be used to select been constructed by the deletion of 18 herpesviruses from which the TKgene has genes which are involved in pathogenesis been deleted, for example bovine herpes- and host range (Tartaglia et al., 1992). The virus 1 (Bello, Whitbeck and Lawrence, resultant virus replicates well in Vero cells 1992; Kit, Kit and McConnell, 1986). A or chick embryo fibroblasts but poorly in deletion mutant of pseudòrabies virus cells of human, equine, murine or swine (suid herpesvirus 1) has been used succes- origin, and provides a safer alternative to sfully to vaccinate pigs against Aujeszky's vaccinia virus as a potential gene vector disease. This genetically engineered virus, vaccine (Tartaglia et al., 1994). strain 783, contains three deletions: one The potential of NYVAC as a vector that prevents expression of pseudorabies vaccine against pseudorabies virus has virus glycoprotein 1 (2 055 base pairs), one been demonstrated. NYVAC recombinants that inactivates the viral thymidine kinase expressing pseudorabies virus glyco- (19 base pairs) and one of 100 base pairs protein gp50 protected pigs against chal- that lies in the promoter-enhancer region lenge with live virulent virus (Brockmeier of the immediate early gene (Glazenburg et al., 1993). et al., 1994). A more general method for gene deletion GENETICALLY ENGINEERED DEFECTIVE and the detection of progeny virus with VACCINES the required genetically engineered Recently, the potential of non-replicating deletions is to use a h.eterologous reporter (defective) poxvirus as vectors for foreign gene contained in a plasmid transfer genes has been explored. Avipoxviruses vector. The reporter gene is inserted into a do not replicate when introduced into plasmid between flanking sequences of the mammalian hosts, but they may still act as gene to be deleted. When the virus genome gene expression vectors in mammals as DNA is cotransferred into cells along with well as birds, since replication is blocked the plasmid containing the reporter gene, at a stage after early gene expression, but recombination occurs between the homolo- before DNA replication. The basis for the gous (flanking) sequences of the genome avian poxvirus vector was an attenuated DNA and those in the transfer vector. This licensed vaccine for canaries (ALVAC) yields some progeny containing substitu- (Taylor et al., 1991 and 1992; Cadoz et al., tion of the virus gene by the reporter gene, 1992). When the glycoprotein gene of rabies and these can be detected in a variety of virus was introduced into ALVAC by ways depending on which reporter gene is recombination, a highly effective vaccine used. The most convenient plasmid preparation was obtained (ALVAC-RG) transfer vectors contain a chromo- which induced high levels of immunity in genic reporter such as p-galactosidase humans (Cadoz et al., 1992) and provided which allows an easy visual selection of a high level of protection in dogs against plaques containing recombinant virus challenge with virulent rabies virus, even (Chakrabarti, Brechling and Moss, 1985). though the ALVAC-RG virus did not 26 Genetically engineered vaccines

replicate in the dogs or human subjects 1994). For these reasons, the following (Taylor et al., 1994). The use of ALVAC as a discussion will be confined to some vector for the env and gag genes of feline remarkable examples of poxvirus vectors leukaemia virus (ALVAC-FL) has also been for use in veterinary medicine. A more described (Tartaglia et al., 1993). detailed account of the earlier studies in Another highly attenuated poxvirus that this area was published by Esposito and has been well characterized is modified Murphy (1989). vaccinia virus Ankara (MVA). This virus One of the first vaccinia virus recombin- was passaged more than 500 times in chick ants of veterinary interest to be developed embryo fibroblasts, and is unable to was a potential vaccine against vesicular replicate in mammalian cells (Meyer, Sutter stomatitis (VSV), w.hich is a contagious and Mayr, 1991). Replication is blocked at disease of horses, cattle and pigs that can the assembly stage so that DNA replication cause serious economic losses in North and and late protein synthesis occur and large South America because the lesions may be amounts of foreign gene products are confused with those of foot-and-mouth produced, similar in amount to wild-type disease (FMD). Recombinant vaccinia virus virus (Sutter and Moss, 1992). was prepared which expressed the It is clear that such non-replicating gene G glycoprotein of VSV at the surface of expression vectors provide a safe and infected cells. Some protection against effective means to immunize a variety of intralingual challenge with virulent VSV mammalian species, including humans, was observed in cattle 44 days after since the virus cannot spread beyond the receiving the recombinant vaccine on days initially infected cells. 1 and 29 (Yilma, 1994). When the glycoprotein gene of another CHIMERIC LIVE VACCINES rhabdovirus, rabies, was expressed in a Candidate live recombinant vaccines vacciniavirusrecombinant,good expressing foreign genes have been protection against challenge was found in developed from a variety of poxvirus a variety of animals such as mice, rabbits, vectors as well as adenoviruses and skunks, raccoons and foxes (Wiktor et al., Salmonella spp. For the most part, 1984; Blancou et al., 1986; Rupprecht et al., adenoviruses are currently being explored 1988). It was possible to im.munize as potential vectors for human mucosal raccoons and foxes, but not dogs or skunks, vaccination and so will not be considered by oral administration of the vaccinia- further here. However, there have been rabies G protein recombinant (VRG). This some studies in animal species, especially has led to a series of detailed studies of the canines, and an excellent review on the potential for oral vaccination of raccoons subject has been published (Graham and and foxes by distribution of baits Prevec, 1992). By the same token, the containing VRG in the wild. Considerable principal interest in Salmonella spp. is in success has been achieved with rabies the development of multivalent oral vaccination of foxes in Europe using this vaccines for human use. Although there is method, and carefully controlled studies also considerable potential for Salmonella- of the use of such baits to vaccinate based vectors in veterinary medicine, raccoons are under way at several sites in particularly in poultry, the subject is still North America (Rupprecht et al., 1988 and relatively unexplored. A useful review on 1993; Rupprecht, Hanlon and Koprowski, Salmonella-based vaccines has appeared 1992; Hanlon et al., 1993). The principal recently (Chatfield, Dougan and Roberts, concern in the use of such VRG-containing Vaccine manual 27

baits in the field is the possible riskto infectivity of live vaccines against humans who might become accidentally morbilliviruses such as measlesor infected with the vaccinia recombinant. In rinderpest. A highly effective vaccinia exceptional circumstances, for example if recombinant virus expressing the F and H an immunocomprornised person was genes of rinderpest has been described for infected, there might be a risk of a serious use in cattle (Giavedoni et al., 1991), and vaccinia infection. Alternative poxvirus this virus will also protect goats against vectors such as raccoon pox (Esposito, the related morbillivirus, peste des petits Chandler and Baer, 1989) have been used ruminants (PPR) virus (Jones et al., 1993). to create a potentially safer vaccine but Recently, an alternative chimeric vaccine insufficient studies have been made of its candidate was described which is based effects in other species. For this reason the on capripoxvirus, a natural virus infection current trials with VRG are being carefully of cattle, sheep and goats. Live attenuated monitored and the risk of human exposure capripoxvirus preparations are already in appears to be extremely low. The use as effective vaccines against sheep and considerable potential for poxvirus- goat pox and lumpy skin disease of cattle, vectored rabies vaccines to control the so they are ideal vectors for recombinant unprecedented rabies epidemic in raccoons vaccines in these species. When the F gene in North America (Rupprecht and Smith, of rinderpest virus was introduced into 1994) needs to be carefully evaluated with the capripox vaccine virus at the TK locus, regard to risks and benefits. It seems likely, the resultant recombinant virus protected however, that VRG vaccines will soon be cattle against rinderpest following a lethal licensed for use in the United States. challenge and also immunized the cattle Unfortunately, a VRG recombinant against lumpy skin disease (Romero et al., vaccine that had not been sanctioned by 1993). the Argentine authorities was used in In addition to these examples, re- Argentina in a field experiment involving combinant vaccines against other bovine cattle (Koprowski et al., 1957). The potential diseases have been described. The for human exposure to vaccinia virus had protection of sheep against bovine not been adequately explained to those leukaemia virus usingavaccinia who handled the inoculated animals; for recombinant expressing the gp51 poly- example, it is known that vaccinia virus peptide has been described, and this may can be isolated from the scabs present on well be effective in cattle also (Daniel et al., cattle at the site of recombinant virus 1993). The use of such poxvirus-vectored inoculation (Gillespie et al., 1986). Even rinderpest vaccines could be crucial in the though none of the cattle handlers suffered campaign to eradicate this plague from the any disease, this incident resulted in a remaining pockets of disease in Africa and considerable setback to the introduction of Asia. recombinant poxvirus vaccines for The potential of fowl poxviruses as gene agricultural purposes, especially in South vectors for vaccines against a wide range America (Crawford, 1987). of poultry diseases was recognized more One of the principal advantages of than ten years ago (Boyle and Coupar, vaccinia virus, which contributed to the 1988). The difficulty of producing such success of the smallpox eradication genetically engineered vaccines at an campaign, is its relative stability. Vaccinia economic cost has hinderedtheir virus preparations will remain viable in development, however. For vaccines conditions which would destroy the to be effective in the poultry production 28 Genetically engineered vaccines

industry, they need to be administered by in cell culture, has greatly enhanced the one day of age using mass delivery possibilities for subunit vaccine devel- methods, for example inclusion in the opment. For example, the baculovirus drinking water. There are numerous Autographa cahfornica has been developed examples of potential recombinant to express a variety of foreign genes under vaccines against diseases such as avian control of the strong polyhedrin promoter influenza, Newcastle disease and Marek's (Miller, 1988). The polyhedrin gene is disease, butallrequire wing-web replaced by the gene encoding the desired inoculation for maximum efficacy and this protein., and the recombinant baculovirus- may not be practical. An excellent review infected Spodoptera frugiperela cells may of potential recombinant vaccines for produce up to 50 percent of total cell poultry appeared recently (Boyle and protein as the foreign gene product. Heine, 1993). This system has been used to engineer multiple-component proteins of blue- SUBUNIT VACCINES tongue virus and, when produced in Live virus vaccines usually induce high appropriate quantities, the proteins self- levels of immunity which persist in the assemble to form non-infectious virus-like host. The major concern regarding their particles (Marshall and Roy, 1990; Pearson use is the possibility of reversion to and Roy, 1993). A further development of virulence, and for diseases such as foot- this system is to introduce other virus and-mouth disease only inactivated virus antigens into the bluetongue virus subunits issuitableforuseasavaccine. during self-assembly to create protein Nevertheless, problems have occurred chimeras which will immunize against owing to incomplete virus inactivation, more than one disease. with resultant outbreaks of disease. This approach offers considerable An alternative approach is to use potential for the future development of subviral components as immunogens in veterinary vaccines. the form of subunit vaccines. Generally, the isolated components of a virusmay DNA POLYNUCLEOTIDE VACCINES not be effective in stimulating an Wolff et al. (1990) reported that the direct appropriate immune response but, if introduction of plasmid DNA into mouse presentedinconjunction with an muscle resulted in expression of encoded appropriate adjuvant such as alumina gel proteins in the rnuscle cells. Although the or 6D-stearoyL-murarnyL-dipeptide, a exact process by which transcription occurs better response can be generated. in this system has not been completely Potentiation of the vaccine by addition of elucidated, it seems that muscle injection cytokines or lymphokines is also being is more effective at inducingan immune studied (Hughes and Babiuk, 1994). For response than other routes or tissues that membrane proteins of enveloped viruses, have been tried. an immunostimulating complex (ISCOM) Recently, the application of a biolistic was developed which, when mixed with gun (gene gun) that shoots gold particles the antigen, presents it to the immune coated with DNA directly into the muscle, system in the form of micelles (Morein et has been described (Williamset al., 1991; al., 1984; Morein and Simons, 1985). Fynan et al., 1993). The advent of expressionvectors such This field is just openingup, but the as baculoviruses, which can produce effectiveness of naked DNA in provoking enormous quantities of a desired protein immunity to rabies in rnice and virulent Vaccine manual 29

influenza in chickens has been reported predicted amino acid sequences designed (Xiang et al., 1994; Robinson, Hunt and to stimulate cell-mediated immunity, the Webster, 1993). The state of the art of DNA potential for such simple peptides as vaccination has been well summarized by vaccines has not been realized. Ulmer, Donnelly and Liu (1994), Ulmer et al. (1993) and Cohen (1993). BIBLIOGRAPHY

POLY7PY:D7 VACCINES Arber, W. & Dussoix, D. 1962. Host specificity When predicted amino acid sequences of DNA produced by Escherichia coll. from a wide range of pathogenic viruses 1. Host-controlled modification of became available in the mid-1970s, the bacteriophage. J. Mol. Biol., 5: 18. possibility of using chemically synthesized Baltimore, D. 1970. RNA-dependent DNA polypeptidesasimmunogens was polymerase in virions of RNA tumor explored. Previous studies on proteins viruses. Nature, 226: 1209-1211. such as lysozyme had established that Barber, L.D. & Parham, P. 1994. The essence immune responses could be elicited by of epitopes. J. Exp. Med., 180: 1191-1194. short peptides containing about 20 amino Bello, L.J., Whitbeck, J.C. & Lawrence, W.C. acids. It was also clear that peptides 1992. Bovine herpesvirus 1 as a live virus corresponding to the aminoterminus or vector for expression of foreign genes. carboxyterminus were frequently antigenic Virology, 190: 666-673. (i.e. elicited antibodies that reacted with Bittle, J.L., Houghten, R.A., Alexander, H., the original protein). Numerous examples Shinnick, T.M., Sutcliffe, J.G. & Lerner, ofviralpeptidesthatservedas R.A. 1982. Protection against foot-and- immunogens were reported (Geysen, mouth disease by immunization with a Barteling and Meloen, 1985; Shinnick et al., chemically synthesized peptide predicted 1983). When it was found that guinea pigs from the viral nucleotide sequence. could be protected from foot-and-mouth Nature, 298: 30-33. disease by a short peptide of 19 amino Blancou, J., Kieny, M.P., Lathe, R., Lecocq, acids corresponding to a portion of the J.P., Pastoret, P.P., Soulebot, J.P. & VP1 protein of the virus (Bittle et al., 1982), Desmettre, P. 1986. Oral vaccination of peptides were hailed as the next generation the fox against rabies using a live of foot-and-mouth disease vaccines recombinant vaccinia virus. Nature, 322: (Brown, 1985). Subsequently, Di Marchi et 373-375. al. (1986) showed that it was possible to Boyle, D.B. & Coupar, B.E.H. 1988. protect cattle from a virulent intralingual Construction of recombinant fowlpox challenge with foot-and-mouth disease viruses as vectors for poultry vaccines. virus using a peptide of 42 amino acids, Virus Res., 10: 343-356. but very large quantities of the peptide Boyle, D.B. & Heine, H.G. 1993. Recombinant were required. In general, the peptides fowlpox virus vaccines for poultry. were found to stimulate a remarkably Immunol. Cell Biol., 71: 391-397. strong humoral response even at low doses Brockmeier, S.L., Lager, K.M., Tartaglia, of peptide, but protection against challenge J., Riviere, M., Paoletti, E. & Mengeling, did not occur except at high doses (5 mg W.I. 1993. Vaccination of pigs against per animal). pseudorabies with highly attenuated Despite the great amount of work done vaccinia (NYVAC) recombinant viruses. in attempting to improve on these results, Vet. Microbiol., 38: 41-58. for example by coupling the peptide with Brown, F. 1985. Peptides as the next 30 Genetically engineered vaccines

creneration of foot-and-mouth disease G.A. 1994. Truncated bovine vaccines. BioTechnology, 3: 445-447. herpesvirus-1 glycoprotein 1 (gpl) Cadoz, M., Strady, A., Meigner, B., Taylor, initiates a protective local immune J., Tartaglia, J., Paolelti, E. & Ploticin, response in its natural host. Vaccine, S. 1992. Immunization with canarypox 12: 145-152. virus expressing the rabies glycoprotein. Geysen, H.M., Barteling, S.J. & Meloen, Lancet, 339: 1429-1432. R.H. 1985. Small peptides induce anti- Chakrabarti, S., Brechling, K. & Moss, B. bodies with a sequence and structural 1985. Vaccinia virus expression vector: requirement for binding antigen com- coexpression of P-galactosidase provides parable to antibodies raised against the visual screening of recombinant virus native protein. PNAS, 82: 178-182. plaques. Mol. Cell. Biol., 5: 3403-3409. Giavedoni, L., Jones, L., Mebus, C. & Yilma, Chatfield, S.N., Dougan, G. & Roberts, M. T. 1991. A vaccinia virus double re- 1994. Progress in the development of combinant expressing the F and H genes multivalent oral vaccines based on live of rinderpest virus protects cattle against attenuated Salmonella. In E. Kurstak, ed. rinderpest and causes no pock lesions. Modern vaccinology, p. 55-85. New York, PN AS, 88: 8011. Plenum Medical. Gillespie, J.H., Geissinger, C., Scott, F.W., Cohen, J. 1993. Naked DNA points way to Higgins, W.P., Holmes, D.F., Perkus, vaccines. Science, 259: 1691-1692. M., Mercer, S. & Paoletti, E. 1986. Crawford, M. 1987. Argentine experiment Response of dairy calves to vaccinia did not break U.S. biotechnology rules. viruses that express foreign genes. J. Science, 235(4786): 276. Clin. Microbiol., 23: 283-288. Daniel, R.C.W., Gatei, M.H., Good, M.F., Glazenburg, K.L., Elgersma-Hooisma, M., Boyle, D.B. & Lavin, M.F. 1993. Re- Briaire, J, Voermans, J., Kimman, T.G., combinant viral vaccines for enzootic Gielkens, A.L.J. & Moormann, R.J.M. bovine leucosis. Immunol. Cell Biol., 71: 1994. Vaccine properties of pseudorabies 399-404. virus strain 783 are not affected by a Di Marchi, R., Brooke, G., Gale, C., deletion of 71 base pairs in the promoter/ Cracknell, V., Doel, T. & Mowat, N. enhancer region of the viral immediate 1986. Protection of cattle against foot- early gene. Vaccine, 12: 1097-1101. and-mouth disease by a synthetic Graham, F.L. & Prevec, L. 1992. Adenovirus- peptide. Science, 232: 639-641. based expression vectors and recombin- Esposito, J.J. & Murphy, F.A. 1989. Infectious ant vaccines. In R. Ellis, ed. Vaccines: recombinant vectored virus vaccines. new approaches to immunological problems, Adv. Vet. Sci. Comp. Med., 33: 195-247. p. 363-390. Stoneham, Mass, USA, Esposito, J.J., Chandler, F.W. & Baer, G.M. Butterworth-Heinemann. 1989. Oral immunization of animals with Hall, W.W., Kubo, T., Ijichi, S. Takahashi, raccoon poxvirus expressing rabies virus H. & Zhu, S.W. 1994. Human T cell glycoprotein. Vaccine, 89: 1-6. leukemia / lymphoma virus, type II Fynan, E.F., Webster, R.G., Fuller, D.H., (HTLV-11): emergence of an important Hanes, J.R., Santoro, J.C. & Robinson, newly recognized pathogen. Virology, H.L. 1993. DNA vaccines: protective 5: 165-178. immunization by parenteral, mucosal Hanlon, C.A., Buchanan, J.R., Nelson, E., and gene-gun inoculations. PNAS, 90: Niu, H.S., Diehl, D. & Rupprecht, C.E. 11478-11482. 1993. A vaccinia-vectored rabies vaccine Gao, Y., Leary, T.P., Eskra, L. & Splitter, field trial: ante- and post-mortem Vaccine manual 31

biomarkers. Rev. sci. tech. Off. int. Epizoot., Morein, B., Sundquist, B., Heiglund, S., 12(1): 99-107. Dalsgaard, K. & Osterhaus, A. 1984. Hughes, H.R.A. & Babiuk, L.A. 1994. ISCOM, a novel structure for antigenic Potentiation of vaccines through effective presentation of membrane proteins from adjuvant fomulations and manipulation enveloped viruses. Nature, 308(5958): of the immune response. In E. Kurstak, 457-460. ed. Modern vaccinology, p. 87-118. New Moss, B. & Flexner, C. 1987. Vaccinia virus York, Plenum Medical. expression vectors. Annu. Rev. Immunol., Jones, L., Giavedoni, L., Saliki, J.T., Brown, 5: 305-324. C., Mebus, C. & Yilma, T. 1993. Pearson, L.D. & Roy, P. 1993. Genetically Protection of goats against peste des engineered multi-component virus-like petits ruminants with a vaccinia virus particles as veterinary vaccines. Immunol. double recombinant expressing the F Cell Biol., 71: 381-389. and H genes of rinderpest virus. Vaccine, Pensaert, M., Gielkens, A.L.J., Lomniczi, 11: 961-964. B., Kimman, T.G., Vannier, P. & Eloit, Kit, S., Kit, M. & McConnell, S. 1986. M. 1992. Round table on control of Intramuscular and intravaginal vaccin- Aujeszky's disease and vaccine devel- ation of pregnant cows with thymidine opment based on molecular biology. Vet. kinase-negative, temperature-resistant Microbiol., 33: 53-67. infectious bovine rhinotracheitis virus Robinson, H.L., Hunt, L.A. & Webster, R.G. (bovine herpesvirus 1). Vaccine, 4: 55-61. 1993. Protection against a lethal influenza Koprowski, H., Celis,E., Curtis, P., virus challenge by immunization with Dietzschold, B., Rupprecht, C., Tollis, a haemagglutinin-expressing plasmid M. & Wunner, W. 1987. Rabies experi- DNA. Vaccine, 11: 957-960. ment. Nature, 326: 636. Romero, C.H., Barrett, T., Evans, S.A., Mackett, M. & Smith, G.L. 1986. Vaccinia Kitching, R.P., Gershon, P.D., Bostock, virus expression vectors. J. Gen. Virol., C. & Black, D.N. 1993. Single capri- 67: 2067-2082. poxvirus recombinant vaccine for the Marshall, J.A. & Roy, P. 1990. High level protection of cattle against rinderpest expression of two outer cepside proteins and lumpy skin disease. Vaccine, 11: of bluetongue virus serotype 10. Virus 737-742. Res., 15: 189-196. Rupprecht, C.E. & Smith, J.S. 1994. Raccoon Meselson, M. & Yuan, R. 1968. DNA rabies: the emergence of an epizootic restriction enzyme from E. coli. Nature, in a densely populated area.Semin. 217: 1110. Virol., 5: 155-164. Meyer, H., Sutter, G. & Mayr, A. 1991. Rupprecht, C.E., Hanlon, C.A. & Koprowski, Mapping of deletions in the genome of H. 1992. Oral wildlife rabies vaccination: the highly attenuated vaccinia virus Development of a recombinant virus MVA and their influence on virulence. vaccine. In Trans. 57th N.A. Wildl. Nat. J. Gen. Virol., 72: 1031-1038. Res. Conf, p. 439-452. Miller, L.K. 1988. Baculoviruses as gene Rupprecht, C.E., Hamir, A.N., Johnson, D.H. expression vectors. Annu. Rez2. Microbiol., & Koprowski, H. 1988. Efficacy of a 42: 177-199. vaccinia-rabies glycoprotein recombinant Morein, B. & Simons, K. 1985. Subunit virus vaccine in raccoons (Procyon lotor). vaccines against enveloped viruses: Rev. Inf. Dis., 10: 803-809. virosomes, micelles and other protein Rupprecht, C.E., Hanlon, C.A., Cummins, complexes. Vaccine, 3: 83-93. L.B. & Koprowski, H. 1992. Primate 32 Genetically enqineered vaccines

responses to a vaccini a-rabies glycoprotein Riviere, M., Languet, B. & Paoletti, E. recombinant virus vaccine. Vaccine, 10: 1992. A highly attenuated strain of 368-374. vaccinia virus. Virology, 188: 217-232. Rupprecht, C.E., Hanlon, C.A., Niezgoda, Taylor, J., Trimarchi, C., Weinberg, R., M., Buchanan, J.R., Diehl, D. & Languet, B., Guillemin, F., Desmetre, Koprowski, H. 1993. Recombinant rabies P. & Paoletti, E. 1991. Efficacy studies vaccines: efficacy assessment in free- on a canary-pox-rabies recombinant ranging animals. Onderstepoort J.Vet. virus. Vaccine, 9: 190-193. Res., 60: 463-468. Taylor, J., Weinberg, R., Tartaglia, J., Sakaguchi, M., Hirayama, Y., Maeda, H., Richardson, C., Alkhatib, G., Briedis, Matsuo, K., Yamamoto, M. & Hirai, K. D., Appel, M., Norton, E. & Paoletti, 1994. Construction of recombinant E. 1992. Non-replicating viral vectors Marek's disease virus type 1 (MDV1) as potential vaccines: recombinant expressing the Escherichia coli lacZ gene canarypox virus expressing measles virus as a possible live vaccine vector: the fusion (F) and hemagglutinin (HA) US10 gene of MDV1 as a stable insertion glycoproteins. Virology, 187: 321-328. site. Vaccine, 12: 153-957. Taylor, J., Tartaglia, J., Riviere, M., Duret, Sambrook, J., Fritsch, E.F. & Maniantis, J. C., Languet, B., Chappuis, G. & Paoletti, 1989. Molecular cloning: a laboratory E. 1994. Applications of canarypox manual, 2nd ed. Cold Spring Harbor, (ALVAC) vectors in human and veteri- New York, Cold Spring Harbor Press. nary vaccination. Dev. Biol. Stand., 82: Shinnick, T.M., Sutcliffe, J.G., Green, N. 131-135. & Lerner, R.A. 1983. Synthetic peptide Townsend, A. & Bodmer, H. 1989. Antigen immunogens as vaccines. Annu. Rev. recognitionbyclassI-restricted Microbial., 37: 425-426. T lymphocytes. Annu. Rev. Immunol., 7: Smith, G.L. & Moss, B. 1983. Infectious 601-624. poxvirus vectors have capacity for at Ulmer, J.B., Donnelly, J. & Liu, M.A. 1994. least 25 000 base pairs of foreign DNA. Vaccination with polynucleotides: a Gene, 25: 21-28. novel means of generating immunity. Sutter, G. & Moss, B. 1992. Non-replicating In E. Kurstak, ed. Modern vaccinology, vaccinia vector efficiently expresses p. 13-23. New York, Plenum Medical. recombinant genes. PNAS, 89: 10847- Ulmer, J.B., Donnelly, J.J., Parker, S.E., 10851. Rhodes, G.H., Feigner, P.L., Dwarki, Tartaglia, J., Cox, W.I., Pincus, S. & Paoletti, V.J., Gromkowski, S.H., Deck, R.R., E. 1994. Safety and immunogenicity of DeWitt, C.M., Friedman, A., Hawe, L.A., recombinants based on the genetically Leander, K.R., Martinez, D., Perry, FI.C., engineered vaccine strain, NYVAC. Dev. Shiver, J.W., Montgomery, D.M. & Liu, Biol. Stand., 82: 125-129. M.A. 1993. Heterologous protection Tartaglia, J., Jarrett, O., Neil, J.C., Desmetre, against influenza by injection of DNA P. & Paoletti, E. 1993. Protection of cats encoding a viral protein. Science, 259: against feline leukemia virus byvac- 1745-1749. cination with a canarypox virusre- van Oirschot, J.T. 1994. Vaccination in food combinant, ALVAC-FL. J.Virol., 67: animal populations. Vaccine, 12: 415-418. 2370-2375. Vannier, P., Hutet, E., Bourgueil, E. & Tartaglia, J., Perkus, M.E., Taylor, J., Norton, Cariolet, R. 1991. Level of virulent virus E.K., Audonnet, J.-C., Cos, W.I., Davis, excreted by infected pigs previously S.W., VanderHoeven, J., Meignier, B., vaccinated with different glycoprotein- Vaccine manual 33

deleted Aujeszky's disease vaccines. Vet. Wolff, J.A., Malone R.W., Williams, P., Microbiol., 29: 213-223. Chong, W., Acsadi, G., Jani, A. & Wiktor, T.J., Macfarlan, R.I., Reagan, K.J., Feigner, P.L. 1990. Direct gene transfer Dietzschold, B., Curtis, P.J., Wunner, into mouse muscle in vivo. Science, 247: W.H., Kieny, M.P., Lathe, R., Lecocq, 1465-1468. J.P., Mackett, M., Moss, B. & Koprowski, Xiang, Z.Q., Spitalnik, S., Tran, M., Wunner, H. 1984. Protection from rabies by a W.H., Cheng, J. & Ertl, H.C. 1994. vaccinia virus recombinant containing Vaccination with a plasmid vector the rabies virus glycoprotein gene. PNAS, carrying the rabies virus glycoprotein 81: 7194-7198. gene induces protective immunity Williams, R.S., Johnson, S.A., Reidy, M., against rabies virus. Virology, 199: Devil, M.J., McElligott, S.G. & Sanford, 132-140. J.C. 1991. Introduction of foreign genes Yilma, T. 1994. Genetically engineered into tissues of living mice by DNA-coated vaccines for animal viral diseases. J. Am. microprojectiles. PNAS, 88: 2726-2730. Vet. Med. Assoc., 204: 1606-1615. Vaccine manual 35

accii F. Brown

Vaccination is a major weapon in the milkmaids contracted cowpox during control of many viral diseases of humans milking which protected them against the and their domestic and pet animals. There virulent smallpox. Many of the vaccines in is no doubt that vaccines have made an use at the present time are based on the enormous impact on the health and same principle, namely that of a weakened consequently the productivity of the or attenuated strain of a virus multiplying recipients. Although this chapter concen- sufficiently in the host to elicit a protective trates on vaccines which are used in immune response without causing clinical domestic livestock, where necessary it disease. Vaccines based on this principle draws analogies with information involv- are highly effective, as shown by the ing other species. In addition, an overview eradication of smallpox; the control of on the present state of vaccine devel- many human diseases such as polio opment must necessarily review what has myelitis, measles and mumps; and the gone before and, with advantage, should effectiveness in domestic animals of also look to the future. Consequently, this vaccinesagainstMarek'sdisease, chapter contains references to products Newcastle disease and rinderpest. that were made in the past, including the The second part of our armoury consists way in which they were improved to of inactivated vaccines, prepared by provide the vaccines in current use, and growing large amounts of the viruses in outlines the possibilities of entirely new tissue culture cells or sometimes in the products which molecular biology offers. intact animal and then inactivating them Although it is always dangerous to make either chemically, with agents such as predictions, one can be certain that the formaldehyde, phenol, [3-propiolactone or methods for preparing vaccines will an imine, or physically, with ultraviolet improve. It is also possible and even likely light. Inactivated vaccines against foot- that the methods will change radically as and-mouth disease, Newcastle disease, we learn more about the immune response poliomyelitis and rabies have been used at the molecular level and apply re- extensively and with great success. combinant DNA technology to produce Together, these vaccines have made very and present to the host only those parts of important contributions to animal health the virus that are required to provide worldwide. One of the early vaccines used protection. on a large scale was described by Rosenbusch, Decamps and Gelormini BACKGROUND (1948). This vaccine, used against foot-and- Vaccination as we know it today essentially mouthdisease,was prepared by began late in the eighteenth century with formaldehyde inactivation of extracts of the observations of Jesty (see Wallace, 1981; fresh tongue lesions of artificially infected Jenner, 1798) and many others that susceptible cattle. Millions of cattle were milkmaids rarely contracted smallpox. The immunized with vaccines prepared by this immunity was attributed to the fact that method. It is of interest, in view of later 36 Viral vaccines

observations and concern about the the efficacy of vaccines against chicken innocuity of vaccines prepared by cholera, anthrax and rabies (Pasteur, 1880 inactivation with formaldehyde, that the and 1985; Pasteur, Chamberland and Roux, authors used an alkaline glycocoll buffer. 1881). All three vaccines had been prepared Recent studies have shown that the virus by attenuating the naturally occurring is very rapidly inactivated at pH 8. This wild-type agents in the laboratory. results from the degradation of the RNA Vaccines for several viral diseases have within the virus particle by the RNA been prepared by growing the wild-type polymerase (Newman et al., 1994). Rabies agents in non-natural hosts or in tissue vaccines prepared from the brains of sheep culture cells. For success to be achieved, and goats artificially infected with the virus careful selection of appropriate strains is are still widely used in humans despite the required. Even then, the selection has to be evidence that such products cause accompanied by an enormous amount of encephalitic reactions. luck to achieve the correct balance between The discovery of antibiotics in the late loss of virulence and the ability to multiply 1930s and early 1940s revolutionized the in the host without causing clinical disease. way in which vaccines could be prepared. Nevertheless, highly successful vaccines Viruses had been grown outside the animal against Newcastle disease and rinderpest body as early as 1930 but these experiments have been prepared and used in millions had been done on a small scale. To expand of birds and animals. The selection of a these laboratory observations toa naturally occurring mutant has also been production scale was clearly difficult with successful in the case of Marek's disease. the technology available at that time The virus infecting turkeys was found to becausetheproblemofensuring be avirulent for chickens and has been used bacteriological sterility would have been in millions of birds. overwhelming. The discovery of antibiotics overcame the problem. CURRENT METHODS OF INACTIVATION The development of large-scale in vitro Most inactivated vaccines are produced by cultivation of foot-and-mouth disease virus reacting the viruses with formaldehyde. by Frenkel (1947) was a major technical This reagent has been used almost achievement. This led to vaccination exclusively for producing diphtheria and programmes which have been used to tetanus toxoids since early this century. protectmillionsofanimals(and The reagent was used by Römer in incidentally predated the much publicized experiments to produce a vaccine against polio vaccination programmes by several poliomyelitis as early as 1911, but without years). success. However, it was used successfully by Vallée, Carré and Rinjard (1925) to ATTENUATED VACCINES produce an experimental foot-and-mouth Jermer's vaccine against smallpox, referred disease vaccine. These researchers, located to above, was quickly applied inmany in Paris, were probably influenced in their countries but it was not until almosta choice of reagent by Ramon, whowas at centurylater,inthe1880s,that that time director of the Pasteur Institute immunization against other infections in that city. began. By that time thegerm theory of Serious concerns about theuse of infection was starting to be accepted and, formaldehyde to inactivate poliomyelitis in rapid succession between 1880 and1885, virus completely were voiced following Pasteur and his colleagues demonstrated clinical trials of a polio vaccine prepared Vaccine manual 37

in this way by Brodie and Park (1936) in THE NEW TECHNOLOGY the 1930s.Similar doubts were also During the past 40 years, the concepts of expressed later about the foot-and-mouth molecular biology have been embraced by disease vaccines prepared by the same the entire field of biology and medicine. method. It was generally considered that This has led to a great increase in our the preliminary step of adsorbing the virus knowledge, not only of the molecular to alum before adding the formaldehyde structure of many infectious agents but was necessary in the inactivation pro- also of those elements which confer cedure. In fact, the adsorption step may protective immunity. More recently, the have been responsible for masking the same concepts have been applied to studies fact that inactivation of the virus was of the immune responses which protect incomplete. against infection. The objectives of this The Cutter incident with an early polio approach, while clearly of fundamental vaccine in the mid-1950s only served to importance, also have great practical emphasize these concerns and categorical implications for vaccine production, since evidence that foot-and-mouth disease they will eventually provide products vaccines prepared by treatment of the virus which are safer and more effective and with formaldehyde were not innocuous which can be designed to elicit only those was provided by Brown et al. (1963) and responses that are required for protection. Graves (1963). But it needed the outbreaks in Normandy and Brittany in France in Structure of viruses 1981, when the causal agent was identified The concept that the entire organism is not by molecular methods as a virus isolated required to elicit protective immunity was 16 years previously, to convince manu- demonstrated a century ago when it was facturers that formaldehyde inactivation shown that antiserum produced in animals was not safe (King et al., 1981). Subsequent against the toxins secreted by the agents evidence,provided byBeckand causing diphtheria and tetanus would Strohmaier (1987) using nucleic acid passively protect against these diseases. sequencing to identify the viruses causing The subsequent extension of this work, several outbreaks in western Europe, which showed that these toxins would pinpointed either spills from factories or produce active immunity after suitable vaccines inactivated with formalin as the inactivation, provided the first clear major causes. demonstrations that vaccination with In view of these findings, it is somewhat subunits was a practical proposition (Glenn perplexing, at least to the author, that and Hopkins, 1923; Ramon, 1923). alternative inactivating agents were not The advances made with viruses used more generally before these incidents. depended in the first place on the Firm laboratory evidence had been techniques which became available to grow provided several years previously that them in tissue culture in quantities acetylethyleneimine was a better inactivant sufficient to allow their purification and (Brown and Crick, 1958; Brown et al., 1963) characterization. By growing the viruses and this had been confirmed on a in the presence of radioactive precursors large industrial scale by the Wellcome of nucleic acids and proteins, the Laboratories. In recent years, bis-ethyl- individual constituents can be labelled. eneimine has replaced acetylethyleneimine These analyses have shown that there are because it is safer to handle on a large many distinct groups of viruses, differing scale (Bahnemann, 1990). in size and shape, ranging from small 38 Viral vaccines

spherical particles of 30 nm in diameter chloroform or a mild detergent such as (such as those of foot-and-mouth disease Tween, releasing the subunits without and swine vesicular disease) to bullet- denaturing the pro teins. The separated shaped particles measuring 140 x 70 nm subunits are also analysed for the proteins (such as that causing rabies) and large they contain. The method which has spherical particles measuring 300 nm in proved most valuable for analysing diameter (e.g. smallpox virus). proteins is polyacrylamide gel elec- In addition, viruses can be grouped trophoresis (PAGE), which separates them according to whether they contain DNA on the basis of their relative molecular or RNA and whether they possess a lipid weights. Coupled with examination of the envelope. For example, rabies virus biologically active subunits in the electron possesses such an envelope. By dissolving microscope, the architecture of a virus can this envelope in a lipid solvent or a mild be derived with this method. detergent, subunits of the virus can be isolated and their potential for eliciting ii)Identification of the gene coding for the protective immunity tested. More vigorous immunogenic protein. The genome of a methods are required to disrupt those virus codes for several proteins in addition viruses not possessing a lipid envelope. to the structural proteins which are During the same period, major advances involved in the immune response. The were being made in our knowledge of virus relevant gene or genes are identified by multiplication. These studies have expressing the viral nucleic acid in a provided genetic maps of many groups of suitable expression system in vitro and viruses, at the same time identifying the then precipitating the products with genes coding for the proteins of im- neutralizing antibody. In this way, the gene munological significance. Consequently, coding for the product reacting with the when the methods for ligating DNA neutralizing antibody can be identified. molecules were described early in the 1970s, the knowledge required for making iii) Expression of genes. Once a relevant synthetic vaccines was already available. gene has been identified, several vector The steps involved in the provision of systems are available for its expression. If engineered vaccines are: the gene is part of a DNA virusgenome, it identification of the immunogenic can be ligated directly into the DNA of the protein; vector. If, however, the gene is part of an identification of the gene coding for RNA virus genome, it must first be this protein; transcribed into DNA before it can be expression of the gene in a suitable inserted into the vector DNA. vector to provide either a live or a dead vaccine. Several vectors are suitable and these can be used in two ways. In the first, the i)Identification of immunogenic proteins. relevant gene is ligated into the DNA ofa The identification of immunogenic bacterium or virus which has been used, proteins is made by dissection of the virus or has potential for use, as an attenuated into subunits which are then tested for vaccine. Examples of these are the strain immunogenicity. With lipid-containing bacillus Calmette-Guérin (BCG) (Bloom et viruses thisisa relatively simple al., 1990), avirulent strains of Salmonella procedure, since the envelope can be typhimurium (Dougan and Tite, 1990), dissolved in a solvent such as etheror adenovirus (Graham and Prevec, 1992), the Vaccine manual 39

poxviruses notably vaccinia virus accommodate several foreign genes. (Mackett, 1990) and some members of Similar considerations apply to BCG the herpesvirus family, for example (Bloom et al., 1990) which has been used in pseudorabies virus (Kit et al., 1991). tuberculosis prophylaxis for many decades Recently, this approach was adapted by with an impressive safety record, even Almond and Burke (1990) to make use of though there is some debate about its the attenuated strains of poliovirus, an efficacy. In contrast, poliovirus, with its RNA virus, taking advantage of the fact smaller genome and more structurally that the DNA complementary to the virus constrained particle, would be unlikely to RNA isinfectious (Racaniello and accommodate more than one foreign gene. Baltimore, 1981). This DNA will infect cells The use of Salmonella typhimurium to produce poliovirus particles. The DNA (Dougan and Tite, 1990) as a carrier is more corresponding to the gene of interest is debatable as it does not possess the proven insertedintothecomplementary track record of vaccinia virus and BCG. poliovirus DNA, which then replicates to However, its potential for oral immuni- produce polio virus particles containing zation demands that its qualities as a vector theinsertedgeneproduct.The should be investigated extensively. Strains recombinant organisms obtained in this of adenovirus (Graham and Prevec, 1992) way have the potential to be used as live which have been used as vaccines should vaccines. also prove valuable since they can be In the second approach, the gene is delivered orally, provided the structural expressed in Escherichia coli, Saccharomyces constraints imposed by the architecture of cerevisiae, the baculovirus Autographa the particle do not prove too stringent. californica or in mammalian cells, and the Moreover, research done with pseudo- gene product is purified for use as a killed rabies virus (Kit et al., 1991), a member of vaccine (Brown, 1984; Matsuura et al., the herpesvirus family, indicates that this 1987). The initial early preference for virus could be a valuable vector for the E. coli has been largely superseded because immunization of animals. the bacterial vector does not glycosylate The vaccinia virus system has been the expressed protein. Since many of the studied more extensively than the other proteins of immunogenic importance are candidates, and field trials with the rabies glycosylated (e.g. the surface projections virus glycoprotein recombinant have of rabies virus) this was a serious and often proved very successful. Of particular critical disadvantage. Consequently, more interest in the control of rabies has been recent developments have been made with the successful vaccination of the fox in the eukaryotic vectors. wild by using the recombinant vaccine in bait. Experiments with rinderpest virus Recombinant attenuated vaccines recombinant in the natural host animal The use of attenuated viruses and bacteria have also been very successful (Yilma et that are already accepted vaccines to carry al., 1988). A major issue of debate in the foreign genes is clearly a concept with use of such recombinant, however,has many attributes. For example, the great been the question of their safety. The advantages of vaccinia virus (Mackett, innocuity of an organism and in this 1990) as a vector are its extensive and instance a new recombinant virusfor.the successful use against smallpox and in the laboratory mouse can be no guarantee of eventual eradication of the disease, its safety for other species. together with the fact that its DNA can Itis also well recognized that any 40 Viral vaccines

product which is to be inoculated into a in a multimeric form, most clearly demons- healthy animal should ideally not cause trated by Morein's experiments with any side effects. This position is difficult to immunostimulating complexes (ISCOMs), achieve and there seems to be no ideal their immunogenicity is greatly enhanced solution. Another issue which has caused (Morein et al., 1990). The principle in this concern is whether insertion of a foreign presentation system is the creation of a gene alters the tissue tropism of the particle comprising several copies of the organism, or even its host range. Again protein. The particles are composed of there seems to be no easy solution, saponin, cholesterol, phosphatidyl choline particularly since we have little knowledge and the protein under study, the constit- of the factors which determine host uents being held together by hydrophobic specificity. interactions. The ISCOMS are seen as cage- like structures in the electron microscope, Killed vaccines about 40 nm in diameter and comprising Proteins produced in in vitro systems are 12 nm morphological subunits. Proteins essentially equivalent to the subunit presented in this way not only elicit high vaccines used experimentally several years levels of antibody but they also stimulate ago. The crucial problem is to present the cell-mediated immunity. Moreover, pre- expressed proteins to the immune system liminary results indicate that intranasal in the configuration which they have when administration of ISCOMs containing they form part of the intact organism. Both influenza and measles haemagglutinins the subunits derived from the intact protect mice againstexperimental organism and the corresponding gene- ch.allenge. tically engineered proteins are much less immunogenic than the intact organism. Peptides as immunogens This is particularly true when the subunits Producing a protein, either in vitro orin are derived from viruses which do not vivo, which has the same amino acid contain a lipid envelope because their sequence as in the parent organism does release from the intact organisrn requires not provide any information about the much harsher conditions. epitopes or short amino acid sequences There are, however, lessons to be learned which elicit the protective immune from the experience with the hepatitis B response. Indeed, the methods which have virus surface antigen (Valenzuela et al., been used so far to express immunogenic 1983). The protein expressed in yeast cells proteins do not even attempt to organize aggregates to form particles which are the structure of th.e protein into the similar to the 22 nrn particles found in the config-uration which it has on the parent blood of carriers of the disease. These yeast- organism. It is fortuitous if a vaccine expressed particles are much more im- produced in vitro or in vivo is effective munogenic than the monomeric protein because it is improbable that a protein and they form the basis for the first produced in a foreign environment will genetically engineered vaccine. Other fold in the way it does in the parent evidence also indicates that presentation organism. All that has been done so far is of an immunogenic protein in a multimeric to produce immunogenic proteins in a form on a particulate structure greatly milieu which is different from the native enhances its activity. organism. This is not to deny that this is a Most proteins expressed in vitro havea remarkably sophisticated biochemical low immunogenicity. By presenting them achievement but it does not provideany Vaccine manual 41

information about how to achieve the directly into the muscle tissue of mice specific immune responses we seek from (Ulmer et al., 1993). In the case of influenza, rationally designed vaccines. These protective immunity was achieved. The answers can only be obtained by further proteins coded by the inserted genes are dissection of the proteins into active expressed in the muscle and skin cells and peptide fragments. are then presented to the immune system. There are several reasons for pursuing It appears that most of the DNA is such a strategy (Brown, 1990): degraded but, nevertheless, enough chemicalsynthesisallowsthe remains for the expression of sufficient production of stable products which protein to stimulate the appropriate do not have the problems associated immune response. It would be interesting with materials produced in cells; to compare the level of the immune the approach offers the advantages response elicited by this method with that associated with the handling of small produced by the direct inoculation of the molecules compared with much larger protein for which the DNA codes. protein molecules; Unlike recombinant proteins made in the information which is accumulating test tubes, if the proteins were made on the interaction of peptides with directly in living animals they would not MEIC molecules (Bjorkman et al., 1987; have to go through the extensive J.H. Brown et al., 1993) suggests that purification steps which are currently the immune response to proteins will demanded. Provided the immune response soon be understood at the level ofshort is sufficient to afford protection, this amino acid sequences. method appears to provide a major step These considerations indicate that it will towards the provision of new vaccines. be possible to design peptides which will elicit the required immune responses for SUMMARY any pathogen. Clearly, there isstill much The study of vaccines and vaccination to be learned about the components based on the concepts of molecular biology required in a totally synthetic vaccine. has provided the expectation that new Immunodominance plays a major part in vaccines will be produced which elicit B cell recognition and this factor is now protective immune responses without the being seen as important in T cell disadvantageous side effects of the current recognition also. Optimal partnerships of products. As we learn more about those B and T cell epitopes are the clear goal. parts of a virus which are required to provide immunity and the way in which Genetic immunization to present them to the host, it is anticipated During the past three years, a quite that vaccines will consist of small frag- revolutionary conceptual advance has ments of the agent, synthesized either been made in vaccination. It had always chemically or biochemically, without the been held that it would be necessary to need to grow the agent itself. More specific- present the immunogenic protein (or ally, however, it must be recognized that carbohydrate-protein complex) to the host each disease is a separate challenge and in order to elicit the appropriate immune the protective immunity which each response. Recent experimentswith demands should be considered according- several viruses have shown that immune ly. The crucial issue is to identify the responses can be obtained by injectingthe immune response which correlates with DNA coding for the protective antigens protection. It should not be forgotten how 42 Viral vaccines

effective the empirical approach has been 1993. Vaccine design. Chichester, UK, and we should not ignore the lessons that Wiley. have been learned from these successes. Brown, J.H., Jardetzky, T.S., Gorga, J.C., Stern, L.J., Urban, R.G., Strominger, BIBLIOGRAPHY J.L. & Wiley, D.C. 1993. Three- dimensional structure of the human class Almond, J.W. & Burke, K.L. 1990. Polio II histocompatibility antigen, HLA-DR1. virus as a vector for the presentation of Nature, 364: 33-39. foreign antigens. Semin. Virol., 1: 120. Dougan, G. & Tite, J. 1990. Live bacterial Bahnemann, H.B. 1990. Inactivation of viral vaccines for delivering antigens to the antigens for vaccine preparation with mammalian immune system. Semin. particular reference to the application Viral., 1: 29-37. of binary ethylenimine. Vaccine, 8: Frenkel, H.S. 1947. La culture du virus de 299-303. la fièvre aphteuse sur l'épithelium de Beck, E. & Strohmaier, K. 1987. Subtyping la langue des bovidés. Bull. Off.int. of European foot-and-mouth disease Epizoot., 28: 155-162. virus strains by nucleotide sequence Glenn, A.T. & Hopkins, B.E. 1923. Diphtheria determination. J. Virol., 61: 1621-1629. toxoid as an immunising agent. Brit. J. 13j orl:man, P.J., Saper, M.A., Samraoui, .3., Ex per. Pathol., 4: 283-288. Bennett, W.S., Strominger, J.L. & Wiley, Graham, F.L. & Prevec, L. 1992. Adenovirus-

,D.C. 1987. Structure of the human class based expression vectors and recom- I histocompatibility antigen, HLA-A2. binant vaccines. In R.W. Ellis, ed. Nature, 329: 506-512. Vaccines: new approaches to immunological Bloom, B.R., SnaPper, S.B., Kieser, T. & problems, p. 363-390. Stoneham, Mass., Jacobs, W.R. 1990. Development of USA, Butterworth-Heinemann. recombinant BCG vaccines. Semin. Viral., Graves, J.H. 1963. Formaldehyde inactivation 1.: 21-27. of foot-and-mouth disease virus as Brodie, M. & Park, W.H. 1936. Active applied to vaccine preparation. Am. J. immunisation against polio myelitis. Am. Vet. Res., 24: 1131-1135. J. Publ. Health, 26: 119-125. enner, E. 1798. An inquiry into the causes Brown, F. 1984. Synthetic viral vaccines. Annu. and effects of the variolae vaccine, a disease Rev. Microbial., 38: 221-235. discovered in some of the western counties Brown, F. 1990. The potential of peptides as of England, particularly Gloucestershire, vaccines. Semin. Viral., 1: 67-74. and known by the name of smallpox. London, Brown, F. & Crick, J. 1958. Application of Samson Low. agar gel diffusion analysis to a study of King, A.M.Q., Underwood, B.O., McCahon, the antigenic structure of inactivated D., Newman, J.W.I. & Brown, F. 1981. vaccines prepared from the virus of foot- Biochemical identification of viruses and-mouth disease. J. Immunol., 82: causing the 1981 outbreaks of foot-and- 444-447. mouth disease in the U.K. Nature, 293: Brown, F., Hyslop, N.St.G., Crick, J. & 479-480. Morrow, A.W. 1963. The use of Kit, M., Kit, S., Little, S.,Di Marchi, R. & acetylethyleneimine in the production Gale, C. 1991. Bovine herpesvirus-1 of inactivated foot-and-mouth disease (infectious bovine rhinotracheitis virus) vaccines. J. Hyg., 61: 337-344. based viral vector which expresses foot- Brown, F., Dougan, G., Honey, E.M., Martin, and-mouth disease epitopes. Vaccine, 9: S.J., Rima, B.K. & Trudgett, A., eds. 564-572. Vaccine manual 4,3

Mackett, M. 1990. Vaccinia virus as a vector DeWitt, C.M., Friedman, A., Hawe, L.A., for delivering foreign antigens. Semin. Leander, K.R., Martinez, D., Perry, H.C., Virol., 1: 39-47. Shiver, J.W., Montgomery, D.L. & Liu, Matsuura, Y., Possee, R.D., Overton, H.A. M.A. 1993. Heterologous protection & Bishop, D.H.L. 1987. Baculovirus against influenza in injection of DNA expression vectors: the requirements for encoding a viral protein. Science, 259: high level expression of proteins, in- 1745-1749. cluding glycoproteins. J. Gen. Virol., 68: Valenzuela, P., Medina, A., Rutter, W.J., 1233-1250. Ammerer, G. & Hall, B.D. 1983. Morein, B., Fossum, C., Lovgren, K. & Synthesis and assembly of hepatitis B Höglund, S. 1990. The ISCOM- a virus surface antigen particles in yeast. modern approach to vaccines. Semin. Nature, 298: 347-350. Viral., 1: 49-55. Vallée, H., Carl* H. & Rinjard, R. 1925. Newman, J.F.E., Piatti, P.G., Gorman, B.M., On immunisation against foot-and- Burrage, T.G., Ryan, M.D., Flint, M. & mouth disease. Rech. Med. vet., 101: Brown, F. 1994. Foot-and-mouth disease 297-299. virus particles contain replicase protein Wallace, E.M. 1981. The first vaccinator: 3D. Proc. Natl Acad. Sci. USA, 91: 733-737. Benjamin Jesty of Yetrninster and Worth Pasteur, L. 1880. De l'atténuation du virus Matravers and his family. Wareham, du choléra des poules. CR Acad. Sci., Dorset, UK, Anglebury Bartlett. 673-680. Yilma, T., Hsu, D., Jones, L., Owens, S., Pasteur, L. 1885. Méthode pour prévenir la Grubman, M., Mebus, C., Yamanaka, rage après morsure. CR Acad. Sci., M. & Dale, B. 1988. Protection of cattle 765-772. against rinderpest with vaccinia virus Pasteur, L., Chamberland, C.-E. & Roux, E. recombinants expressing the HA or F 1881. Sur la vaccination charbonneuse. gene. Science, 242: 1058-1061. CR Acad. Sci., 92: 1378-1381 Racaniello, V.R. & Baltimore, D. 1981. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science, 214: 916-919. Ramon, G. 1923. Sur le pouvoir floculant sur les propriétés immunisantes d'une toxine diphtérique, rendue anatoxique (anatoxine). CR Acad. Sci., 177: 1338- 1340. Römer, P.H. 1981. Die epidemische Kinder- la7rmung (Heine-Medinsche Krankheit), p. 49. Berlin, Germany, Springer. Rosenbusch, C.T., Decamps, A. & Gelormini, N. 1948. Intradermal foot-and-mouth disease vaccine: results obtained from the first million head of cattle vaccinated. J. Am. Vet. Med. Assoc., 112: 45-47. Ulmer, J.B., Donnelly, J.J., Parker, S.E., Rhodes, G.H., Feigner, P.L., Dwarki, V.J., Gromkowski, S.H., Deck, R.R., Vaccine manual 45

zlnes P.D. Walker

Bacterial vaccines can be divided into two organisms such as the aro mutants of categories: Salinonella spp. as cloning vectors (Hoiseth Inactivated vaccines which are and Stocker, 1981; Hone et al., 1991). Such composed of whole bacterial cells vaccines stimulate secretory, humoral and and/ or their metabolic products or, cell-mediated responses. Vaccines are alternatively, a purified fraction of designedtostimulatethe normal the cell. immunological mechanisms of the host. Live vaccines which are composed Following parenteral injection of vaccines, of attenuated strains of the parent antibodies, mainly IgG, appear in the virulent organisms. bloodstream. Vaccines administered orally Although in the developed world, stimulate local production of antibody particularly with medical vaccines, (mainly IgA) at the mucosal surface. In purified and characterized imrnunogens general, antigenicity is a function of are preferred from both a safety and quality molecular size. For this reason, bacterial control viewpoint (Walker, 1991), in non- proteins are good antigens, although long domestic animals economic considerations chain capsular polysaccharides are also dictate that the product, in addition to antigenic and have formed the basis of being efficacious and safe, should be cost- successful vaccines, for example in pro- effective in an animal of finite worth. tection against haemorrhagic septicaemia Under these circumstances, cruder, less in cattle (Nagy and Penn, 1976). purified products of proven efficacy In developing a vaccine strategy for continue to be employed. In addition, the control of disease, the vaccine manu- protection by vaccination is often multi- facturer has to be mindful that there are factorial, involving a number of antigens, two stages in infectious disease, i.e. while the use of single entities produced establishment of the organism in the host either by conventional purification or followed by the clinical signs of infection. genetic engineering should be treated with Although, in practice, these two stages some caution (Walker, 1992). quickly pass from one to the other, from Both types of vaccine can either be the point of view of the vaccine manu- injected parenterally or administered facturer, it is useful to distinguish between orally. Inactivated vaccines are invariably them. Vaccines can be designed that either administered by injection, although large prevent establishment of the organism in doses administered orally have also been the host or prevent the effects of infection. used successfully. Living vaccines are The problems posed to the manufacturer administered parenterally or, where differ depending on which approach is protection is against enteric diseases, taken (Walker and Foster, 1981). orally. The use of the oral route as a means The effects of infection are caused mainly of stimulating mucosal immunity in by toxins elaborated by the organisms general is being increasingly exploited, which have specific pharmacological particularly using genetically engineered effects. Indeed, the results of infection can 46 Bacterial vaccines

be reproduced by injecting sterile filtrates organisms (Oakley, 1943; Oakley and of the organisms concerned. Bacterial Warrack, 1953; Brooks, Sterne and toxins are usually secreted into the culture Warrack, 1957). The major toxins res- medium and the manufacturer is therefore ponsible for disease have been identified faced with the problem of producing large and the use of toxoid vaccines to control quantities of toxins for further processing. clostridial diseases is widely practised These are usually separated from the (Sterne et al., 1962). Bacterial toxins are bacterial cell mass. inactivated to produce bacterial toxoids in On the other hand, if the approach is to such a way that toxicity is lost but prevent establishment of infection then it antigenicity retained. The usual method of is the antigens of the bacterial cell which treatment is with formaldehyde (Ramon, the manufacturer needs to consider. For 1924). example it has been shown that in many Toxoids are normally adsorbed on to an diseases adherence of the organism to adjuvant, usually a mineral salt such as epithelial tissues by specific adherence aluminium hydroxide, aluminium phos- mechanisms followed by multiplication of phate or potassium aluminium sulphate. the attached organisms is important By far the most potent adjuvants are (Walker and Nagy, 1980). In this con- mineral oils or their derivatives (see nection, the manufacturer must identify Table 2), which enhance immunogenicity these particular bacterial antigens and by at least an order of magnitude over adjust the growth conditions to ensure the conventional adjuvants (Freund, Casals- production of large quantities of the Ariet and Genghof, 1940). However, owing appropriate antigen. After inactivation, the to their production of severe local reactions organisms are harvested for further which may persist indefinitely, they are processing. Whole culture vaccines may completely unacceptable for human vac- stimulate both types of immunity. cination and even in veterinary medicine, This overview deals with both types of despite their effectiveness, their use in the protection using illustrations from developed world is severely limited owing veterinary vaccines which are currently in to potential carcass blemish and its effect use for sheep, cattle, pigs and dogs or on the export of meat. Attempts have been which are under development. A brief made to overcome this problem, for section on fish vaccines is included, as example by the use of the interperitoneal aquaculture may be of increasing relevance route (Thomson et al., 1969). However, in to developing countries in the future. the developing world it is questionable whether these issues are important. Where PROTECTION AGAINST THE EFFECTS production is for a domestic market, the OF DISEASE benefits of a more effective and lasting The classic example of the use of this type immunity, with all the advantages of of vaccine protection is that of clostridial reduced handling of animals, must surely toxoid vaccines. The most economically outweigh any cosmetic considerations. important diseases in sheep, cattle and pigs Furthermore, it is quite possible to identify are caused by clostridial infections (Sterne an injection site such as behind the ear or and Batty, 1975). As a result of the stimulus on top of the skull which is removed at to research of the potential threat ofgas slaughter and willnotaffect the gangrene during the First and Second appearance of the meat. World Wars, a vast amount of literature Adult animals are normally given two exists on the toxins produced by these injections of toxoid separated by at least 28 Vaccine manual 47

TABLE 2 Arithmetic mean antitoxin responses of lambs to 2x2 ml doses of a multicomponent clostridial vaccine precipitated with potash alum and to a I ml dose adsorbed with Freund's adjuvant

Weeks after Adjuvant Clostr.dium second innoculation perfringens septicum novyi tetani

beta epsilon

Two Alum 25.4 2.2 1.9 10.2 16.0 Freund's 46.6 12.9 5.7 13.2 27.8 Ten Alum 1.1 0.6 0.38 0.7 Freund's 5.6 3.1 0.6 1.3 6.7

days for a primary immunization course. whose requirements have subsequently Thereafter, further boosting to the been incorporated in the European immunity can be given at critical periods Pharmacopoeia. These regulations were of life. One such period coincides with the originally formulated after consultation 14 days preceding parturition to ensure that the maximum amount of antibody is TABLE 3 transferred from the serum via the Principal Clostridia spp. causing diseases colostrum and milk for the protection of in animals the newborn. Following the disappearance

of such protection, the young animal Species Disease should undergo prophylactic vaccination. The range of clostridial diseases against Cl. perfringens Wound infections, gangrenous which vaccination is practised is shown in type A mastitis, enterotoxaemia in nursing lambs in California Table 3. Both single and combined vac- and Oregon ("yellow lamb") cines are available, with individual manu- Cl. perfringens Enterotoxaemias in various facturers responding to the needs of the types B, C and D animals including calves, market place. Based on experience, manu- sheep, goats, piglets and facturers have been able to formulate foals multicomponent products so that there is Cl. septicum Malignant oedema of horses, a balanced response to all the major cattle, sheep and swine antigens despite antigenic competition CI. chauvoei Blackleg in sheep and cattle (Table 4). It is clear from the table that, Cl. novyi Gas gangrene in cattle and although individual antigenic responses type A sheep may be lower with a multicomponent than Cl. novyi Black disease in sheep, with an equivalent monocomponent type B sudden death in cattle and vaccine, the responses to the multi- pigs component vaccine are above the mini- CI. haemolyticum Bacillary haemoglobinuria (Cl. novyi type D) in cattle mum responses required by the regulatory authorities. Cl. sordellii Gas gangrene In the United Kingdom, in order to Cl. tetan! Tetanus in all species provide effective and safe vaccines, of domestic animals manufacturers have had to comply with Cl. botulinum Botulism in sheep, cattle, type C and D dogs, chickens and wild the conditions laid down in the British duck Pharmacopoeia (Veterinary) (HMSO, 1985) 48 Bacterial vaccines

TABLE 4 Arithmetic mean antitoxin response of groups of rabbits in tests on 10 consecutive batches of combined vaccines compared with tests on 12 consecutive single batches

Vaccines Responses in IU per ml to Clostridium spp. used Cl. zvelchii Cl. Cl. septicum Cl. oedematiens Cl. tetani beta epsilon

Br. Vet. Codex standards 10 5 2,5 10 3.5

Mean responses to 10 combined batches 55 6 9,2 16 62

Mean responses to 12 single batches 141 15 9 122

Note: IUinternational units.

with major manufacturers who, based on pigs are vaccinated with two doses of the their own experiences of vaccines that were vaccine according to the recommended effective in the field, were able to suggest dosage regime and challenged with a consensus criteria for the release of virulent culture 14 days after the second products. In the case of clostridial vaccines, injection.All vaccinates should survive with the exception of Clostridium chauvoei, and all controls die within 48 hours. in order to release a product a manu- The problem for the manufacturer is then facturer has to show that, after two to produce large quantities of toxins that injections given according to the recom- can be converted into toxoids and, fol- mended dosage regime in the field, groups lowing suitable processing, incorporated of rabbits respond with certain minimum into vaccines. antitoxin titres. Subsequent formal studies have shown that vaccines producing Component manufacture adequate titres in rabbits produce an As a result of developments in fermenter adequate serological response in the target technology, some manufacturers are able species (Frerichs and Gray, 1975). to grow clostridial species in fermentation Nevertheless, these criteria have some culture to produce high yields of clostridial drawbacks. For example, they only toxins suitable for toxoiding (Walker and measure antibodies to the major lethal Foster, 1981). Over a period of time, components and not to other toxins and manufacturers have carefully selected cellular elements which may be important those strains which give maximum toxin in protection. Thus, they do not distinguish production in the growth media they have between whole-culture and toxoidvac- developed. They have, in effect, cloned their cines, for example. A further complication strains to give stable high-yielding toxin is the differences in response by individual mutants which enable them to produce breeds of rabbit, which can vary threefold consistently high yields of toxins. Strain in their response to thesame vaccine improvement by mutation and selection preparation (Walker and Batty, 1985). In forms the basis of a continuing research the case of Cl. chauvoei,groups of guinea programme by most manufacturers. Vaccine manual 49

The majority of strains are stable when autoclaving is usually sufficiently low to stored as freeze-dried cultures and this permit the growth of almost all species of enables large batches of master seed Clostridia without further additions, cultures to be stored. By the use of a fresh particularly when combined with the use aliquot of the master seed to prepare of 5 to 10 percent inocula. If necessary, working seeds, any deterioration of the reducing agents such as cysteine hydro- strain owing to passage in liquid media chloride can be added prior to inoculation. can be avoided. Where storage by freeze- There is therefore no special requirement drying is not satisfactory, cultures can be for the introduction of gases into the stored in liquid nitrogen and fresh culture. ampoules opened as required. Clostridia spp. are for the most part As bacterial toxins are not generally saccharolytic, acid-producing organisms produced in significant quantities in and automatic pH control and addition of synthetic media, it is necessary to provide carbohydrate results in considerable a source of peptides. The manufacturer has improvements in yield and reproducibility to select a source of protein for digestion of culture. Nevertheless, it is possible to which is readily available, for example control these processes manually, albeit at meat, casein or soybean. Digests of meat the expense of some efficiency. By using are usually prepared with papain or bottled media and working in an incubator, trypsin and digests of casein with acid or it is possible to control pH and make trypsin. For maximum toxin production, periodic additions to containers using a digest media need to be supplemented pH meter and appropriate solutions. with various amino acids and vitamins; Where the costs of fermenters are una- yeast extract provides a convenient, cceptable and adequate labour is available, reliable and cheap source for the latter. In this can be an effective method of produc- the case of certain toxins, it is necessary to tion, particularly for developing countries. regulate carefully the amounts of various The optimum pH range for maximum inorganic ions present, for example iron in toxin production varies with the toxin tetanus toxin production (Mueller and concerned. For example, maximum pro- Miller, 1954). Glucose is the normal energy duction of epsilon toxin by Cl. perfringens source although maltose and sucrose are (welchii) type D occurs in the alkaline range also used (Moore, 1968; Walker, Harris and while the pH optima for other clostridial Moore, 1971). toxins are lower. Manufacturers have invested heavily in Toxin production during growth can be developing media and growth conditions monitored by a variety of techniques. togive maximal toxin production These include, where present, the mea- (Thomson, 1979). Published data are surement of enzymic activity, for example available for the growth and toxin lecithinase, haemolysis and proteolysis or, production of the Harvard strain of Cl. alternatively, in vivo testing in guinea pigs, tetani using Mueller medium (Mueller and mice and rabbits, using death or skin Miller, 1954). This consists of a calcium lesions as the indicator. Values of toxin are caseinate digest and acid hydrolysed casein expressed in terms of indicator effects, for base supplemented with bullock's heart example minimum lethal dose, minimum infusion broth, glucose, vitamins, cystine haemolytic dose or, alternatively, in terms and iron powder. of unit equivalents of standard antitoxin. The oxidation-reduction potential of The Lf dose of a toxin is that dose of toxin freshly prepared digest media after which, when mixed with one unit of 50 Bacterial vaccines

antitoxin, flocculates in the least time, i.e. antigens and are extremely stable under a it is the first mixture to flocculate, while variety of environmental conditions. The the L+ is the smallest dose of toxin which, preparation of such toxoids only involves mixed with one unit of antitoxin, kills 50 separation from the bacterial cell mass by percent of injected mice of a designated centrifugation (Walker and Foster, 1981). weight within a designated time (Batty, 1971). Blending The length of incubation is determined Bacterial toxoids are normally blended into by the particular toxin being produced. Cl. multicomponent vaccines. Very careful perfringens (welchii) beta toxin is destroyed formulation is necessary if the host is to by the proteolytic enzymes which the produce a balanced response against all organism produces if incubation continues components and the vaccine is to pass the for too long while others such as epsilon criterialaiddown byregulatory toxin are much more stable. authorities. Toxoids are normally blended, Bacterial toxins are invariably inactiv- based on TCP value or some equivalent, ated with formaldehyde (Ramon, 1924). and the appropriate unit equivalents added Although the principal action of form- to the final vaccine. In practice, the aldehyde on a toxin is to remove toxicity, manufacturer will take into account the this is only achieved at the expense of some testing of the toxin before inactivation (Lf, loss of immunogenicity. For this reason, L+, Lv, Lh, etc.) as well as the TCP and, toxoids are normally standardized in terms where appropriate, the Lf of the toxoid. of a total combining power (TCP) test. The Bearing in mind the loss of immuno- TCP test involves a partial neutralization genicity on inactivation if the toxoiding of a fixed dose of antitoxin with a series of process is allowed to proceed too far, the varying doses of the toxoid being tested. ratio of TCP to Lf declines. A fine balance The unreacted antitoxin is then mixed with has to be struck between a high TCP-Lf a fixed dose of toxin equivalent to half the ratio, on the one hand, and minimum dose of antitoxin used and the whole series residual toxicity and freedom from any of mixtures are injected into mice which tendency to reversal, on the other. are observed for two days.The toxoid After blending and the addition of the present in the mixture that kills half the adjuvant, the vaccines are ready for issue mice into which it is injected is equivalent when certain statutory regulatory tests to the fixed dose of toxin used (Batty, 1971). have been completed. This is necessary because, after inactiv- ation, it is no longer possible to measure Use of vaccines the activity of the toxoids in terms of The aim of the manufacturer is to provide indicator effects, although it is possible to protection both to the adult animal and to carry out an Lf test with certain toxoids. the offspring by the passive transfer of Where there are no appropriate facilities antibody. In the case of the adult animal, to carry out such tests, developing providing it has been given primary and countries may well have to rely on data secondary injections with appropriate from the original testing, for example booster doses, it should remain protected minimum lethal doses. for life. In an adequately primed animal, any subsequent infection will result in an Harvesting anamnestic response which will afford For veterinary purposes, crude toxoidsare immediate protection (Chadnik, Watson normally used. Crude toxoidsare good and Hepple, 1959). In thecase of the Vaccine manual 51

offspring, the length of protection will be percent with vaccine B. The group determined by the persistence of antibody geometric mean (GMT) titre achieved after titres which will, in turn, be determined a single injection with vaccine A is about by the amount and titre of the colostral eight times that of vaccine B. These antibody ingested. As the decay of differences in the titres are also seen after passively transferred antibodies is known the second injection (Table 5), and with (roughly 16 percent per week), it is vaccine B two animals still have no relatively easy to predict how long measurable titre. The responses in the ewes protective levels are maintained. Protective (Table 6) are reflected in the antitoxin titres antibody levels are known for several obtained in the lambs subsequent to antitoxins, for example 0.1 unit for parturition and, hence, the length of Cl. perfringens epsilon and tetanus and protection afforded to the lambs during 0.5 units for Cl. perfringens beta. Tables 5 the early weeks of life (Table 7). and 6 illustrate this by reference to two It can thus be seen that, even with two commercial vaccines, A and B. vaccine preparations released on the basis Table 5 shows the difference between of the Codex requirements, there are the performance of the two commercial differences in potential protection of both vaccines, A and B, when the Cl. perfringens the adult and the newborn. epsilon antitoxin titres to the first and While toxoid vaccines are normally second injections in previously unim- effective, there are examples of breakdown munized sheep are compared. It can be in the field, usually in cases where high seen with vaccine A that after a single levels of challenge lead to large quantities injection 90 percent of the animals gave a of toxins which overwhelm the antitoxic measurable titre compared with 65.5 immunity. For this reason some manu-

TABLE 5 Distribution of Cl. perfringens epsilon antitoxin titres in the sera of 8-month sheep immunized with two commercial multicomponent clostridia! vaccines, A and B

Serum No. of sera (%) titres (IUm1-1) Vaccine A Vaccine B

42 days affer first injection 5 13 (43) 1(3.0) 1-5 9 (30) 8 (28.0) 0.1-1.0 5 (17) 10 (34.5) <0.1 3 (10) 10 (34.5) Total 30 29

Group GMT 1.84 0.21

14 days after second injection 10 13 (43) 2 (6.5) 1-10 16 (53) 16 (59.0) 0.1-1.0 1 (4) 8 (28.0) <0.1 2 (6.5) Total 30 29

Group GMT 9.04 1.39

Note: GMTgeometric mean titre. Source: Adapted from Kerry and Craig, 1979. 52 Bacterial vaccines

TABLE 6 Distribution of C./. perfringens epsilon antitoxin titres in the sera of ewes immunized with two commercial multicomponent clostridia! vaccines, A and B, following the second injection and bled 2 to 5 days after lambing

Serum No. of sera (%) titres (ILI ,n11) Vaccine A Vaccine B

10 13 (38) 3 (9) 1-10 19 (56) 20 (61) 0.1 -1 .0 2 (6) 7 (21) Total 34 33

Group GMT 6.72 1.66

Note: GMT geometric mean titre. Source: Adapted from Kerry and Craig, 1979.

TABLE 7 Distribution of Cl. perfringens epsilon antitoxin titres in the sera of 2- to 5-day lambs born to ewes immunized with two commercial multicomponent clostridia! vaccines, A and B

Serum No. of sera (%) titres (/U m/-1) Vaccine A Vaccine B

5 19 (61) 4 (13) 2-5 6 (19) 8 (26) 1.0-2.0 4 (13) 3 (10) 0.5-1.0 1 (3) 4 (13) <0.5 1 (3) 12 (39) Total 31 31

Group GMT 7.48 1.25

Note: GMT = geometric mean titre. Source: Adapted from Kerry and Craig, 1979.

facturers do not remove cells from all the type C and that the disease could be components and leave them as anacultures controlled by administration of high-titre so as to obtain both antibacterial and horse antisera containing beta antitoxin antitoxic immunity. and also by vaccination with vaccines A typical example of this was seen in containing beta toxoid. Using a vaccine Denmark in vaccination against the disease which contained both cells and toxoid piglet enteritis, caused by Cl. perfringens (Xento, Wellcome), successful protection type C. This disease is a haemorrhagic was demonstrated (Hogh, 1974). necrotizing enteritis of young piglets Some years later a combined Escherichia which, over a period of tenyears, has coli/C1. perfringens type C toxoidvac- spread from small foci to cover largeareas cine (Xentocol, Wellcome), specifically of Jutland and Zeeland. Initial studieson designed for the market to control both E. this disease by Hogh (1974) showed that coli diarrhoea and Cl. perfringens type C the causative organism was Cl. perfringens enteritis, was introduced. In many herds Vaccine manual 53

the vaccine was used successfullyto The combination of Cl. perfringens type control E. coli and piglet enteritis. However, C and E. coli, i.e. a combination of anaerobic in a number of problem herds the vaccine and aerobic vaccines, representsa further was totally ineffective in controlling piglet development in the marketplace. This isa enteritis. response both to the needs of the market Table 8 shows the results of a comparative and the desire to obtain a competitive edge. trial that was carried out using two product In this respect it is worth noting two other formulations and a control group. It can be developments in this area. seen that removal of the cells of Cl. A combination clostridial and Pasteurella perfrin gens resulted in a considerable loss vaccine (Heptavac-P) was introduced by of efficacy. The results indicated that, Hoechst (United Kingdom) to provide under conditions of high challenge, both simultaneous protection against clostridial antibacterial and antitoxic immunity were and respiratory diseases, particularly in required to contain infection (Hogh, 1988). young lambs. A somewhat different Electron microscopy studies on the disease approach is the combination of clostridial showed a pattern of adhesion of Cl. vaccines with an anthelmintic, levamosole, perfrin gens type C to the jejunal villi in introduced by the Tasman Veterinary piglets identical to that of E. coli infection in Laboratories (New Zealand). This combi- the same species (Walker and Nagy, 1980). nation allows simultaneous protection The initial phases of the disease were against clostridial infections and parasites characterized by colonization of the villi although, as dosing with anthelmintics is owing to adhesion and multiplication of the very dependent on the weight of the organisms. Unlike E.coli, the adhesive animal, dosage regimes can be difficult. factors are not characterized but clearly were present on the cells of Cl. perfringens in Xento PROTECTION AGAINST ESTABLISHMENT OF vaccine and probably play the same role as INFECTION the "K" antigens of E. colt. The importance of the cellular component The lessons of these results are clear. as a supplement in traditional toxoid Protection is multifactorial and, under vaccines has already been discussed. extreme conditions of challenge, vaccines Cellular antigens are important in the based on more simplified entities break establishment of an organism in the host. down. Antibodies interfering with establishment

TABLE 8 Comparison of a perfringens beta toxoid vaccines and an identical vaccine containing Cl. perfringens type C cells in pigs on s problem farm in northern Jutland

Vaccine Necrotic enteritis

Litters Piglets No. dead

Cl.perfringens beta toxold 21 239 83 34.7

perfringens beta toxoid plus cells 22 249 21 6.8

Control 20 217 93 42.9 54 Bacterial vaccines

will prevent infection. In general, cellular from inactivated whole cultures to prevent antigens can be divided into cell-wall and shedding of the organism into the environ- capsular types. Until fairly recently, most ment (Broughton et al., 1984). Results show whole-culture vaccines were relatively that, in unvaccinated cattle, leptospiral undefined and consisted of suitably organisms are isolated frequently from the inactivated cells standardized on the basis urine whereas, in vaccinated cattle, there of cell numbers (approximately 1010 is virtually no excretion (Walker, 1987). organisms per ml), as measured by opacity. While the transfer of E. coli bactericidal Such vaccines undoubtedly act by antibodies via the colostrum and milk to stimulatingbactericidalantibodies the suckling animal undoubtedly affords directed against the cell and, as such, tend some protection, the approach to vac- to be sero-specific. When the number of cination against E. coli was revolutionized important serotypes in a species is limited, by the identification of the antigens the formulation of vaccines is relatively responsible for adhesion of the organism simple. However, in species where a large to the intestinal villi. The adhesion of number of serotypes are important, it is organisms to the intestinal villi is the first necessary to blend a multiplicity of stage in E. coli infection and, if this stage serotypes into the final vaccine. can be blocked by antibodies, infection is Toxicity resulting from endotoxin prevented. K88ab and K88ac were components of the cell wall of Gram- identified as important adhesins for the negative bacteria which can result in disease in piglets (Smith and Lingood, adverse reactions in the adult and abortion 1971) and early work in pigs showed that in pregnant animals becomes important as experimental vaccines prepared from the numbers of organisms are increased. purified K88 antigens were effective in In the case of E.coli vaccines for preventing disease in piglets after oral immunization against diarrhoea in challenge (Jones and Rutter, 1972). animals, these consisted of a mixture of The first available commercial vaccines the predominant "0" serotypes prevalent were based on partially purified K88 in the disease. In the case of Pasteurella antigens of E. coli (Nagy et al., 1978). During vaccines for the treatment of respiratory growth, the 1(88 antigen is secreted into diseases,these again consistedof the growth medium and can be selectively inactivated cells of the predominant removed from the supernatant by serotypes of Pasteurella haemolytica and adsorption on to aluminium hydroxide, Pasteurella multocida. leavingbehind endotoxininthe Similar inactivated whole-culture supernatant. vaccines have also been used for the control Rather than depend on anti-adhesion of leptospiral infections. Leptospira spp. are alone, the early vaccines comprised cells well known as causal agents of disease in of E. coli supplemented with measured the dog (Broughton and Scarnell, 1985). quantities of K88ab and K88ac. In this way, The importance of Leptospira spp.as a both the anti-adhesive and bactericidal causative agent of disease, particularly in effects of antibodies were present. Sows or cattle, has been recognized in recent years. gilts were given two injections of the The problem has become acute owing to vaccine, with the second dose timed to be the transfer of leptospirosis from cattle to given two weeks prior to parturition, to humans during handling. The organism is achieve maximum levels of antibody in shed in urine and is a potentialsource of the colostrum and milk for a passive infection. Vaccines have been developed transfer to the piglets. To piglets of Vaccine manual 55

vaccinated gilts following oral challenge, emulsified in oil, swine are protected such vaccines gave significant protection against experimental challenge. against mortality, diarrhoea and excretion A field trial indicated that this vaccine (Nagy et al., 1978). While a conventional confers significant benefits. In one fattening E. coli K88-riegatiye vaccine afforded unit, swine dysentery was reduced by 50 significant protection over the controls, the percent and the severity of infection by 50 vaccine supplemented with measured percent, while vaccinated piglets showed quantities of K88 antigen is even more a 10 percent increase in weight gain over significantly protective. controls in the same period after a single As time passed, further adhesins such as injection of the vaccine (Fernie, Ripley and K99 and 987P were recognized as being Walker,unpublishedresults).As important and it became necessary to mentioned previously, there is increasing formulate multicomponent vaccines resistance to oil-based vaccines because of (Orskov et al., 1975; Moon et al., 1977; Smith carcass blemish and its effect on the export and Huggins, 1978). The protection afford- of meat. For this reason, the vaccine has ed by such vaccines is shown in Tables 9 not been commercially developed. and 10. Experiments similar to those for To obtain further data on the protective the original K88 vaccines were performed antigens with a view to formulating a and it can be seen that significant protect- more non-reactive product, research has ion is afforded against mortality (Table 9) centred on the envelope proteins of and diarrhoea (Table 10) with all four T. hydosyenteriae. Using sodium dodecyl serotypes (Nagy and Walker, 1983; Nagy, sulphate-polyacrylamide gel electro- Mackenzie and Painter, 1985). phoresis (SDS-PAGE) and immunoblot- The identification of the surface antigens ting, it has been shown that polypeptides iesponsible for adhesion in E. coli repre- with a molecular weight of between 30 000 sents a move away from the traditional and 36 000 were the predominant ones vaccine to a more defined product. Other detected by porcine immune serum and envelope antigens which may be important were not present on non-pathogenic strains in infection have been the subject of of this organism. These unique antigens increasing interest. may play a role in the virulence of Treponema hyodysenteriae is the aetio- T. hyodysenteriae and form the basis of logical agent of swine dysentery, a disease a new effective non-oil-based vaccine occurring in post-weaning pigs, ususally (Chatfield et al., 1988). in fattening units where pigs are kept in A similar outer membrane protein (68 large numbers (Alexander and Taylor, kDa) was demonstrated to be an important 1969; Taylor and Alexander, 1971; Harris antigen in protection against atrophic et al., 1972; Hughes, Olander and Williams, rhinitis in pigs caused by Bordetella 1975). After infection with the organism, bronchiseptica. There is a strong correlation swine develop a mucohaemorrhagic between antibody titres to this antigen and diarrhoea characterized by extensive protection in pigs. The antigen is present necrosis of the mucosal epithelium of the in virulent strains of B. bronchiseptica but colon and caecum. This results in dehy- absent in non-virulent strains. Monoclonal dration, emaciation and rapid weight loss antibody produced against this antigen followed, in severe cases, by death. Recent passively protects mice from aerosol studies have shown that, following a challenge with the organism (Montaraz, single-dose parenteral immunization with Novotny and Ivanyi, 1985; Kobisch and an inactivated culture of T. hyodysenteriae Novotny, 1990). 56 Bacterial vaccines

TABLE 9 Mortality rates 24 hours aftel chcillenge in Iiiz Svoin voccinaied ancl non-vaccincieclils afiel orogasivic c'cdlençc wiTh 11310 Escherichic colt plus iypes 24, hams (-Ater

Vaccine E. colichallenge cultures

K88ab K88ac K99 987P (diedlchallenged) (/o)

None 19/36 (53) 38/58 (66) 10/34 (29) 33/41 (80) (n=5)° (n=10) (n=5) (n=7)

5:1° 0/39 (0) 3/36 (8) 2/37 (5) 3/35 (9) (n=5) (n=5) (n=5) (n=5)

Reduction in mortality rates (%) 100 88 83 90

number of litters used. b Vaccine 5:1; partially purified K88ab, K88ac, K99, 987P of E. coli (100 units each).

TABLE 10 Diarrhoea rates 24 il01.11S 6101 ohallenga in litiem from vaccinated and non-voccincied ailts clflGcrogasivic challenge vviih 1010Escherichia c,oli plus iypes24 hOUIS aiier

Vaccine E. colichallenge cultures

K88ab K88ac K99 987P (diarrhoea/challenged) (%)

None 46/54 (85) 47/58 (81) 34/54 (62.9) 50/52 (96) (n=7)a (n=10) (n=8) (n=9)

5:1° 1/51 (1,9) 2/53 (3.7) 20/57 (35.1) 22/48 (46) (n=7) (n=7) (n=7) (n=7) Reduction in diarrhoea (%) 97.8 95,4 44.1 52,1

nnumber of litters used. 'Vaccine 5:1; partially purified K88ab, KS8ac, K99, 987P of E. coli (100 units each).

In an attempt to improve the per- significant protection has been shown in formance of Pasteurella vaccines,con- this model using heat-killed cells or sodium siderable work has been carried out using salicylate extracts of the organismsas the specific pathogen-free (SPF) lamb vaccines. However, vaccines containing model developed at the Mordun Institute extracts or heat-killed cells of serotype A2, (Gilrnour et al., 1975). Such lambs are highly which is responsible for the majority of susceptible to challenge with Pasteurella outbreaks in sheep, have been less effective spp. when given in conjunction with PI 3 (Gilmour et al., 1983; Donachie et al., 1986). virus. Even in the absence of death, lung Studies have indicated that organisms lesions develop after a few days andcan grown in vivo produce additional antigens be scored at post mortem examination. to those grown invitro. Pasteurella For a number of the A biotypes, haeinolytica A2 cells recovered from the Vaccine manual 57

pleural fluid of lambs with pasteurellosis Bacteroides nodosus (Egerton, Roberts and were found toexpress previously Parsonson, 1969). A culture of scrapings unidentified proteins, and two of these from the infected area revealsa very with molecular weights of 70 and 100 distinctive rhizoidal colony (Thorley, 1976), kilodaltons (kDa) could be induced in vitro the growth of which is highly pilated. On by restricting iron availability in the subculture in the laboratory a number of growth medium. non-pilated smooth colonies are generated, It was subsequently shown that and it has been demonstrated that the most significant protection against P. hacmolytica effective vaccines are those made from A2 infection was conferred on SPF lambs pilated cultures (Egerton and Thorley, by a vaccine containing extracts of P. 1981; Thorley and Egerton, 1981). The haemolytica A2 cells grown under iron organism is divisible into a number of restriction, compared with a vaccine made serotypes based on pili agglutination and, in exactly the same way from cells grown in Australia, a number of serotypes have in an iron-replete medium. This was been identified as being important confirmed by comparing the antibody (Claxton, 1981). Multivalent vaccines responses seen previously in SPF lambs incorporating whole cultures of a mixture which were immune after recovery from of serotypes have been shown to be highly A2 infection. Using immunoblotting, it effective both in experiments involving was demonstrated that there were strong challenge and in the field. responses to iron-limiting proteins of 70 In summary, progress has been made in and 35 kDa and a weaker response at recent years to identify those antigens 100 kDa (Gilmour et al., 1991). which are important in the pathogenic In the case of Pasteurella multocida, an process and to ensure that, under alternativeapproach has beento fermentation conditions, these antigens are concentrate on the toxins produced by the produced for vaccine production. During organism. Some success has been reported production, in addition to controlling pH in immunizing pigs against experimentally and temperature, it is necessary to be able induced progressive atrophic rhinitis to measure and control aeration of the caused by this organism, using a formalin- culture. The degree of aerationis detoxified purified toxin. This approach dependent on the growth rate of the may well be valid for control of disease in organism. The growth of E. coli strains lambs (Chantler, Rutter and Mackenzie, requires high rates of oxygenation whereas 1986; Fogel, Neilsen and foral, 1989). strains of Leptospira spp., which grow much Surface appendages known as pili more slowly, have relatively low oxygen (fimbria) have also been shown to be requirements. For several species, it is important in the pathogenesis and necessary to supplement the air or oxygen protection against ovine footrot. Ovine with carbon dioxide. Provision must be footrot results in separation of the horn made for the sterilization of the incoming from the underlying soft tissues of the foot air and outgoing effluent; the latter (Beveridge, 1941). Separation of the horn presents a number of problems when high leads to abscess formation and an aeration rates are necessary. extremely painful condition in which the The majority of these problems are sheep have difficulty in walking and minimized by growing the organism on grazing. Although the flora of the foot is solid surfaces, although this is achieved very complex, it has been shown that the only at the expense of efficiency as key triggering organism for infection is compared with fermenter-grown material. 58 Bacterial vaccines

Individual bottles of nutrient agar are (Hoiseth and Stocker, 1981; Hone et al., inoculated with the appropriate organism 1991). and incubated for one to three days. In the case of enteric pathogens, mucosal Following incubation and growth, the mmunity is regarded as an important organisms can be removed from the surface factor in protection. In this respect, orally of the culture and put into a resuspending delivered vaccines are superior and the fluid by agitation. Where the antigens are use of live oral vaccines to protect against secreted into the supernatant fluid, there salmonellosis in cattle provides a good has been some attempt to purify them but example of modern developments. the marketplace does not allow the Using a double aro mutant of Salmonella extensive purification required for its typhimurium to immunize cattle orally, it human counterpart. has been shown that there is significant protection against a live challenge with LIVE VACCINES this organism. Of eight cattle immunized For many diseases, investigators believe orally with approximately 10" of the that cell-mediated immunity (CMI) is mutant seven days after birth using an necessary to provide solid long-term antacid mixture, seven were completely protection against infection. This has led protected against a challenge of appro- to the use of live attenuated strains as ximately 108 organisms three weeks later. vaccines because, by comparison, killed All four controls showed significant symp- vaccines generally do not elicit such strong toms and, along with the remaining vac- CMI responses. cinate, had to be killed (Jones et al., 1991). Live attenuated vaccinesinthe More recently, this approach has been veterinary field include Salmonella dublin, extended to the use of S. typhimurium G30 S. cholera suis, Brucella abortus and anthrax as a vector to express the fimbrial antigens spore vaccine. The attenuated S. dublin and K88 and K99 of enterotoxigenic E. coli S. cholera suis strains are avirulent rough (Stevenson and Manning, 1985; Attridge et variants of the smooth virulent strain and, al., 1988). Oral administration of 1011 as such, are unable to synthesize a number vaccine organisms to adult pigs elicited of polysaccharide side chains required for significant serum antibody responses to virulence. However, in the present climate the respective fimbrial antigens (Morana of legislation, a more rationally modified et al., 1994). strain harbouring well-defined, attenu- Live attenuated organisms have also ating genetic lesions is to be preferred. been used in the case of Pasteurella spp. Examples of the latter are strains har- Passaged in the laboratory, cultures of bouring defined mutations in genes in the Pasteurella spp. disassociate, throwing off prechorismate pathway rendering them mutants that have lost their ability to deficient in the ability to synthesize certain produce capsular polysaccharides. Such essential aromatic compounds. In addition, mutants are non-pathogenic but highly it is preferable that strains used for live immunogenic when injected subcuta- vaccines should harbour at least two stable neously into the animal. Combined vac- attenuating mutations which map for cines comprising mixtures of the living separate regions of the chromosome, thus attenuated strains can be formulated and limiting the danger of reversion to are highly effective against challenge in virulence. It has been shown that such the SPF lamb model (Walker, 1987). Again, strains, for example aroA, aroC or aroD, are these studies are limited by the use of a admirably suitable for this purpose naturally occurring avirulent strain. Such Vaccine manual 59

strains will not be favoured because of perfringens. J. Pathol. Bacteriol., 74: 185-195. potential reversion and it will benecessary Broughton, E.S. & Scarnell, J.1985. to develop rationally attenuated strains Prevention of renal carriage of lepto- harbouring well-defined attenuating spirosis in dogs by vaccination. Vet-. Rec., crenetic lesions such as the aro mutants for 117: 307-311. Salmonella spp. Broughton, E.S., Marshall, R.B., Little, Although reservations regarding theuse T.W.A., Hathaway, S.C., Mackintosh, of naturally occurring mutants or strains C.G. & Hellstrom, J.S. 1984. Leptospira attenuated in the laboratory as vaccines interrogans serovar. Hardjo vaccines in have been stressed, the contribution that cattle: immunogenicity of vaccines such vaccines have made in controlling prepared from cultures grown in a economically important diseases in the protein-free medium. Preventive Vet. field should not be forgotten. In the case of Med., 2: 423-433. anthrax, the live attenuated spore vaccine Chadnik, K.S., Watson, A.R.A. & Hepple, derived from an avirulent non-capsulated J.R. 1959. Active immunisation of sheep variant of Bacillus anthracis has had a and horses against tetanus with pronounced effect in limiting economic aluminium hydroxide adsorbed toxin. losses resulting from anthrax and an Vet. Rec., 43: 904-909. indirect effect in reducing human incidence Chantler, N., Rutter, J.M. & Mackenzie, A. (Sterne, Nichol and Lambrechts, 1942; 1986. Partial purification of an oesteolytic Turnbull, 1991). Similarly, the B. abortus toxin from Pasteurella multocida. J. Gen. S19 strain attenuated by temperature has Microbiol., 32: 1089-1097. been highly effective in controlling Chatfield, S.N., Fernie, D.S., Penn, C. & brucellosis. Dougan, G. 1988.Identification of the major antigens of Treponema hyo- BIBLIOGRAPHY dysenteriae and comparison with those of Treponema innocens. Infect. Immunol., Alexander, T.J.L. & Taylor, D.J. 1969. The 56: 1070-1075. clinical signs, diagnosis and control of Claxton, P.D. 1981. Studies on Bacteroides swine dysentery. Vet. Rec., 85: 59-63. riodosus vaccines with particular reference Attridge, S.R., Hackett, J., Morana, R. & to factors associated with their efficacy Whyte, P. 1988. Towards a live oral in protecting against ovine virulent vaccine against enterotoxigenic Escher- footrot. University of Sydney, Australia. ichia coli of swine. Vaccine, 6: 387-389. (Ph.D. thesis) Batty, I. 1971. In J.R. Norris & D.W. Ribbons, Donachie, W., Burrells, C., Sutherland, A.D., eds. Toxin-antitoxin assay: methods in Gilmour, J.S. & Gilmour, N.J.L. 1986. microbiology, Vol. 5A, p. 255-280. London, Immunity of specific pathogen-free Academic. lambs to challenge with an aerosol of Beveridge, W.I.B. 1941. Footrot in sheep: a Pasteurella haemolytic biotype A serotype transmissible disease due to infection 2. Pulmonary antibody and cell response with Fusiformis nodosus. Studies on its to primary and secondary infections. cause, epidemiology and control. Vet. Immunol. Immunopathol., 11: 265-279. Commonzyealth Aust. Council Sci. Ind. Res. Egerton, J.R. & Thorley, C.M. 1981. Effect Bull., 140: 1-56. of alum-precipitated or oil adjuvant Brooks, M.E., Sterne, M. & Warrack, G.H. Bacteroides nodosus vaccines on the 1957. A re-assessment of the criteria used resistance of sheep to experimental for type differentiation of Clostridium footrot. Res. Vet. Sci., 30: 28-31. 60 Bacterial vaccines

Egerton, J.R., Roberts, E.S. & Parsonson, HMSO. 1985. BritishPharmacopoeia I.M. 1969. The aetiology and patho- (Veterinary). London. genesis of ovine footrot, I. A histological Hogh, P. 1974. Porcine infectious enteritis study of the bacterial invasion. J. Comp. caused by Clostridium perfringens. Royal Pathol., 79: 207-216. Veterinary and Agriculture University, Fernie, D.S., Ripley, P.H. & Walker, P.D. Copenhagen. (dissertation) 1983. Swine dysentery: Protection against Hogh, P. 1988. Vaccinationsforsog med fire experimental challenge following single forskellige tarmbrandsvacciner. Dansk dose, parenteral immunisation with Vet. Tidsskr., 71: 1-9. inactivated Treponema hyodysenteriae. Res. Hoiseth, S.K. & Stocker, B.A.D. 1981. Vet. Sci., 35: 217- 221. Aromatic-dependent Salmonella typhi- Fogel, N.T., Neilsen, J.P. & Joral, S.E. 1989. murium are non-virulent and effective Protection against atrophic rhinitis by as live vaccines. Nature, 291: 238. vaccination with Pasteurella multocida Hone, D.M., Harris, A.M., Chatfield, S., toxin purified by monoclonal antibodies. Dougan, G. & Levine, M.M. 1991. Vet. Rec., 125: 7-11. Construction of genetically defined Frerichs, G.N. & Gray, A.K. 1975. The relation double aro mutants of Salmonella typhi. between the rabbit potency test and the Vaccine, 9: 810-816. response of sheep to sheep clostridial Hughes, R., Olander, H.J. & Williams, C.B. vaccines. Res. Vet. Sci., 18: 70-75. 1975. Swine dysentery: pathogenicity Freund, J., Casals-Ariet, J. & Genghof, D.S. of Treponema hyodysenteriae. Am. J. Vet. 1940. The synergistic effect of paraffin- Res., 36: 971-977. oil combined with heat-killed tubercle Jones, G.W. & Rutter, J.M. 1972. Role of bacilli. J. Immunol., 38: 67-79. K88 antigen in the pathogenesis of Gilmour, N.J.L., Thomson, D.A., Smith, neonatal diarrhoea caused by Escherichia W.D. & Angus, K.W. 1975. Experimental coli in piglets. Infect. Immuna,6,918-927. infection of lambs with an aerosol of Jones, P.W., Dougan, G., Hayward, C., Pasteurella haemolytica. Res. Vet. Sci., 18: Mackensie, N., Collins, P. & Chatfield, 340-341. S.N. 1991. Oral vaccination of calves Gilmour, N.J.L., Martin, W.B., Sharp, J.M., against experimental salmonellosis using Thomson, D.A.,Wells,P.W. & a double aro mutant of Salmonella Donachie, W. 1983. Experimental im- typhimurium. Vaccine, 9: 29-36. munisation of lambs against pneumonic Kerry, J.B. & Craig, G.R. 1979. Field studies pasteurellosis. Res. Vet. Sci., 35: 80-86. in sheep with multicoMponent clostridial Gilmour, N.J.L., Donachie, W., Sutherland, vaccines. Vet. Rec., 105: 551-554. A.D., Gilmour, J.S., Jones, G.E. & Quirie, Kobisch, M. & Novotny,P.1990. M. 1991. Vaccine containing iron- Identification of a 68-kilodalton outer regulatedproteinsofPasteurella membrane protein as the major protective haemolytica A2 enhances protection antigen of Bordetella bronchiseptica by against experimental pasteurellosis in using specific pathogen-free piglets. lambs. Vaccine, 9: 137-140. Infect. Immunol., 58: 352. Harris, D.L., Glock, R.D., Christensen, C.R. Montaraz, J.A., Novotny, P. & Ivanyi, J. & Kinyon, J.M. 1972. Swine dysentery: 1985. Identification of a 68 kilodalton inoculation of pigs with Treponema protective protein antigen from Bordetella hyodysenteriae (new species) and repro- bronchiseptica. Infect. Immunol., 47: duction of the disease. Vet. Med. Small 744-751. Anim. Clin., 67: 61-64. Moon, H.W., Nagy, B., Isaacson, R.E. & Vaccine manual 61

Orskov, I. 1977. Occurrence of 1(99 W.J. 1975. The establishment of K99,a antigen on Escherichia coli isolated from thermolabile transmissible Escherichia coli pigs and colonisation of pig ileum by K antigen previously called "KCO" 1(99 and enterotoxigenic E.coli from possessed by calf and lamb entero- calves and pigs. Infect. Immunol., 15: pathogenic strains. Acta Pathol. Microbiol. 614-620. Scand., 83B: 31-36. Moore, W.B. 1968. Solidified media suitable Ramon, G. 1924. Sur la toxine etsur for the cultivation of Clostridium novyi l'anatoxine diphtériques. Annu. Inst. type B. J. Gen. Microbiol., 53: 415-423. Pasteur, 38: 1-10. Morana, R., Morana, J.K., Cosidine, J.A., Smith, H.W. & Huggins, M.B. 1978. The Hackett, L., van den Bosch Beyer, L. & influence of plasmid-determined and Attridge, S.R. 1994. Construction of K88 other characteristics of entero-pathogenic and K99 expressing clones of Salmonella Escherichia coli on their ability to pro- typhimurium G30: immunogenicity liferate on the alimentary tract of piglets, following oral administration to pigs. calves and lambs. J. Med. Microbiol., 11: Vaccine, 12: 513-517. 471-492. Mueller, J.H. & Miller, P.A. 1954. Variable Smith, H.W. & Lingood, M.A. 1971. factors influencing the production of Observations on the pathogenic pro- tetanus toxin. J. Bacteriol., 67: 271-277. perties of the K88 and ENT plasmids of Nagy, L.K. Sr Penn, W. 1976. Protection of Escherichia coli with particular reference cattle against experimental haemorrhagic to porcine diarrhoea. J. Med. Microbiol., septicaemia by the capsular antigens of 4: 467-485. Pasteurella multocida types B and E. Res. Sterne, M. & Batty, I. 1975. Pathogenic Vet. Sci., 20: 249-253. Clostridia. London, Butterworth. Nagy, L.K. & Walker, P.D. 1983. Multi- Sterne, M., Nichol, J. & Lambrechts, M.C. adhesin vaccine for the protection of 1942. The effect of large-scale active the neonatal piglet against E. coli immunisation against anthrax. J. S. Afr. infections. Proc. Int. Symp. Enteric Vet. Med. Assoc., 13: 53-63. Infections in Man and Animals: Standard Sterne, M., Batty, I., Thomson, A. & of Immunology, Dublin, Ireland. Dev. Robertson, J.M. 1962. Immunisation of Biol. Stand., 53: 189-197. sheep with multicomponent clostfidial Nagy, L.K., Mackenzie, T. & Painter, K.R. vaccines. Vet. Rec., 74: 909-913. 1985. Protection of the nursing pig against Stevenson, G. & Manning, P.A. 1985. experimentally induced enteric coli- Galactose epimeraseless (Gal E) mutant bacillosis by vaccination of the dam with G30 of Salmonella typhimurium is a good fimbrial antigens of E. coli (K88, K99, live oral vaccine carrier for fimbrial 987P). Vet. Rec., 117: 408-413. antigens. FEMS Microbiol. Lett., 28: Nagy, L.K., Walker, P.D., Bhogal, B.S. & 317-321. Mackenzie, T. 1978. Evaluation of Taylor, D.J. & Alexander, I.J.L. 1971. The Escherichia coli vaccines against ex- production of dysentery in swine by perimental enteric colibacillosis. Res. Vet. feeding cultures containing a spirochaete. Sci., 24: 39-45. Br. Vet. J., 127: 58-61. Oakley, C.L. 1943. The toxins of Clostridium Thomson, R.O. 1979. Secondary products welchii. Bull. Hyg., 18: 781-806. of metabolism. In A.H. Rose, ed. Bacterial Oakley, C.L. & Warrack, G.H. 1953. Routine toxins economic microbiology, Vol. 3, p. typing of Cl. welchii. J. Hyg., 51: 102-108. 435-466. London, Academic. Orskov, I., Orskov, F., Smith, H.W. & Sojka, Thomson, R.O., Batty, I., Thomson, A., 62 Bacterial vaccines

Kerry, J.B., Epps, H.B.G. & Foster, W.H. The isolation of Clostridia from animal 1969. The immunogenicity of a multi- tissuesisolation of anaerobes. In D.A. component clostridial oil emulsion Shapton & R.G. Board, eds. Soc. Appl. vaccine in sheep. Vet. Rec., 85: 81. Bacteriol. Tech. Series No. 5, p. 25-38. Thorley, C.M. 1976. A simplified method London, Academic. for the isolation of Bacteroides nodosus from ovine footrot and studies on its colonial morphology and serology. J. Appl. Bacteriol., 40: 301-309. Thorley, C.M. & Egerton, J.R.1981. Comparison of alum-absorbed oil emulsion vaccines containing either pilate or non-pilate Bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental footrot. Res. Vet. Sci., 30: 32-37. Turnbull, P.C.B. 1991. Anthrax vaccines: past, present and future. Vaccine, 9: 533-539. Walker, P.D. 1987. Current trends and advances in bacterial vaccines today. J. Appl. Bacteriol., 62: 1-23. Walker, P.D. 1991. Editorial: Proceedings of the International Conference on Prevention of Hib Meningitis in the 1990s. Vaccine, 9 (Suppl.) S.3. Walker, P.D. 1992. Bacterial vaccines: old and new, veterinary and medical. Vaccine, 10: 977-986. Walker, P.D. & Batty, I. 1985. The use and standardisation of multicomponent veterinary vaccines in 1986. Proc. IABS Congr. Use and Standardisation of Combined Vaccines, Amsterdam, the Netherlands. Dev. Biol. Stand., 65: 227- 236. Walker, P.D. & Foster, W.H. 1981. Bacterial vaccine production. In J.R. Norris & M.H. Richmond, eds.Essaysinapplied microbiology. Chichester, UK, Wiley. Walker, P.D. & Nagy, L.K. 1980. Adhesion of organisms to animal tissues. In R.C.W. Berkeley, J.M. Lynch, J. Melling, P.R. Rutter & B. Vincent, eds. Microbial adhesion to surfaces, p. 473-494. Chichester, UK, Ellis Horwood. Walker, P.D., Harris, E. & Moore, W.B. 1971. Vaccine manual 63

ycoeis vac.., li A. Provost

Compared with other sectors of micro- pleuropneumonia (CBPP) is such that biology, it is unfortunate to have to admit complement fixing, agglutinating, that the range of available vaccines for growth and / or metabolism inhibiting mycoplasmal diseases does not provide antibodies to M. mycoides are no proof entire satisfaction for a number of reasons, of resistance to a challenge. Conversely, among which are their poor safety record resistance can occur without them. for recipient animals and a weak, short- The same events exist in enzootic lived resistance to natural disease after pneumoniaofpigs,owingto vaccination. M. hyopneumoniae, and in M. galli- This disappointing situation can be septicum infection of poultry. A possible explained on the following grounds: exception, found in humans, is the e Imprecise knowledge of the actual presence of growth-inhibiting anti- pathogenic processes in mycoplasmal bodies to mycoplasms in bronchial diseases. washings. This is an avenue which Insufficient knowledge of the precise should be pursued in animal myco- role of the different antigens present in plasmosis. Cellular-type immunity the mycoplasmal organism in both the proves equally ineffective for any pathogenesis and immune mecha- correlation with protection. nisms; for example, in the case of 0 Only partial protection against natural Myco plasma mycoides subsp. mycoides or experimental challenge can be SC,' the function of galactan, the inducedpartial being understood in polyosidic outer capsule, is unclear. two ways: either only a reduced A lack of progress in developing proportion of the total number of simple, cheap and reliable potency tests animals are protected, or the protection for vaccines, taking account of the leads to an abortive or less severe strong host species specificity of the disease. This occurrence may happen mycoplasms. frequently following the use of in- An almost total ignorance of the nature activated vaccines but is also en- of the protective mechanisms in countered with live vaccines. resistant animals, either after recovery Unforeseeable reactions following from the natural disease or after vaccination, either locally at the site of vaccination. In no case is there a strict injection (called Willem's reaction in correlation between protection and the case of CBPP live vaccine) or by the the presence of circulating antibodies. potentiation of other diseases after the The situation in contagious bovine use of an inactivated vaccine (as observed in human primary atypical pneumonia and in enzootic pneumonia In this document, Mycoplasma mycoides subsp. of pigs). mycoides (SC, bovine biotype) will be referred to as In the field of animal diseases, there is Mycoplasma mycoides (abbreviated as M. mycoides). only one condition for which vaccination 64 Mycoplasmal vaccines

is currently in use, namely CBPP. Experi- vaccines". At present, strain T1 is one mental vaccines have been produced of the two available. There have been against swine pneumonia but other recent advances in freeze-drying, strategies are also used to control the prolonging the storage life of vaccine disease. An inactivated vaccine against and facilitating its distribution in the contagious caprine pleuropneumonia field. (CCPP) has been studied in Kenya and is Egg culturevaccines,employed reported to have given valuable results in successfully during the1960son the field; it is not known, however, whether account of their good immunogenicity it is produced on a large scale. Live and availability in a freeze-dried form. vaccines have been developed for avian However, they have been held mycoplasmosis and have been produced responsible for pulmonary compli- commercially on a temporary basis but, cations in certain circumstances and owing to some disappointments (including have now been abandoned. a lack of safety for non-vaccinated birds), Inactivated vaccines of various types are their use tends to be ignored and control is of only poor immunogenicity or lead to confined to a combination of means centred an unacceptable reaction at the site of on the treatment of fertile eggs (dipping, inoculation. heating) to evade vertical transmission of the agent. Fundamental rules Vaccines against CBPP need to comply CBPP VACCINE: GENERAL CONSIDERATIONS with three fundamental rules, derived from Types of vaccine experience: Vaccination against CBPP has been im- o Viability. Only vaccines prepared from plemented by the following methods: the specific live mycoplasmapossess Willem's method, using the pleuritic or adequate immunogenicity, engender- pulmonary "lymph", or the secondary ing a resistance of the premunition lymph resulting from a subcutaneous type. The vaccine strain can be recov- Willem's reaction induced artificially. ered from lymph nodes several months The traditional African procedure after vaccination and antigens may (subcutaneous inoculationofa persist for up to204days. macerated lung lesion into the nasal o Virulence. Following the observations mucosa) is an improvised and related of scientists working in1906,which variation of the procedure rediscovered were confirmed in Kenya in1921and a in Europe in the 1850s. few years later in the Sudan in1929, ° Vaccines prepared from broth cultures of subcultures in broth lead to a certain naturally attenuated strains (e.g. the decrease in the pathogenicity of M. Australian strain V, and the Sudanese mycoides. For any given strain, there strain F), some of which are freeze- are a certain number of in vitro sub- dried. cultures which will attenuate the °Cultures of M. mycoides attenuated by culture. subculture, varying in number of serial °Attenuation is characterized by the passages according to the virulence of ability of the particular strain to the original strain, the culture medium, produce at the site of injection onlya the sensitivity of local cattle and the minor reaction (localized oedema, inoculation site. These vaccines have which heals spontaneously) after sub- been called incorrectly "Bennett cutaneous or intradermal inoculation. Vaccine manual 65

The actual technique of inoculation claim that there wasno relationship varies from one laboratory to another. between the residual pathogenicity ofa This attenuation is not fixed, for further vaccine strain of M. mycoides and its subcultures lead to a progressive immunogenicity. The years that followed decrease in virulence, with the disap- seemed to disprove this. Most researchers pearance of immunogenic properties. and field observers shared the view that Therefore, it is necessary to reacha protection was transient and weak in the delicate compromise between retention absence of a local postvaccinal reaction. of immunogenicity and minimal local This fact was widely recognized at the reactions acceptable to cattle owners. time, but was difficult to explain. Among Subculturing should be avoided. the factors involved might have beenpoor The attenuation of cultures to a greater cultures, faulty storage of vaccine and or lesser extent by in vitro subculturing only partial vaccination in a heavily has provided the foundation for vac- contaminated herd. The local reaction was cination policy during the past 80 years. proof of the viability of the vaccine and, in It is only the approach to the problem its absence, there were doubts about the that has varied. vaccine's efficacy. To this may be added Vaccination route. It is usual to follow the lack of uniformity in methods of the rule of the permissible or acceptable experimental infection of cattle, leading to vaccination site. This requires dense differing evaluations of the resistance connective tissue of a low reactivity to conferred by one or other of the various limit adverse local and generalized procedures. reactions which may sometimes be However, research conducted in fatal. For this reason, it has long been Australia between 1960 and 1970 clearly advisable for the vaccine to be inoculat- demonstrated that there was no correlation ed into the skin or the subcutaneous between the intensity of the vaccinal tissue of the tip of the tail. Nevertheless, reaction and the quality of protection. for practical purposes and to avoid loss Excellent resistance to experimental of the tail after a severe reaction to the infection developed without any local vaccine, the current method is to reaction provided that the minimum inoculate the vaccine subcutaneously required number of live mycoplasmas on the flank posterior to the shoulder were inoculated (107 per dose for strain T1) blade, necessarily using a vaccine of If untoward local reactions of the acceptable or no residual virulence. Willem's type occur (i.e. extensive oedema starting 10 to 12 days after vaccination) Occurrence of local postvaccinal reactions antibiotic treatment (spiramycine, tylosin, At the end of the last century it was tilmicosin) can be advocated. believed that, for a vaccination to be effective, a local lesion was needed a General vaccination precautions circumscribed Willem's reaction. It was Vaccination against CBPP is never then thought inevitable that there would harmless. Apart from the local, and even be a certain number of vaccination com- generalized, reactions which may be plications, ranging from extensive post- produced, vaccination may potentiate vaccinal swelling to death, which could latent infections or infestations in cattle affect 2 to 3 percent of the vaccinated existing under the precarious physiological animals. balance prevalent in tropical conditions Towards 1930, scientists first started to (e.g. trypanosomiasis and piroplasmosis). 66 Mycoplasmal vaccines

Factors to take into account: (mycoplasmocidal antibodies) to simple Breed (where the term "breed" is often clinical resistance accompanied by en- used incorrectly for species). In general, capsulated lung lesions from which M. cattle breeds are more susceptible to mycoides can be recovered. vaccination accidents than zebu, at least With any CBPP vaccination, there is the in Africa. Dairy breeds are usually more risk of creating semi-resistant cattle which susceptible than beef breeds but can become chronic carriers following exceptions occur in both cases. natural infection, thus promoting the Group. Within the same species and continuance of the disease. Hence the same breed, there are individual importance of the rule of disease control variations in sensitivity to vaccination which states that CBPP vaccination must (and also to infection). Such variations be extensive in an area and continuous in time area hindrancetovaccination to be effective. By contrast, partial and campaigns becauseofpossible unrenewed vaccination of a susceptible unpredictable reactions. herd may simply maintain the disease at a Sex. Pregnant females in the terminal low level. Failure to observe this rule has stage of gestation should not be resulted in numerous disappointments in vaccinated. the past. Age. The inoculation of calves under In practice, there is considerable dis- three months is usually followed by crepancy between resistance as assessed insignificant local reactions but such in the laboratory and that which operates animals may developarthritis, in the field. A vaccine capable of protecting synovitis and heart valve lesions which only 70 percent of cattle experimentally can be fatal, at least with the most may still give excellent results in the field. virulent strains. In practice, calves should not be vaccinated under six to CBPP VACCINE: GENERAL MANUFACTURING eight months of age. AND CONTROL PROCEDURES Source materials The level of protection induced Strain of M. mycoides. The strains of M. The vaccination of cattle against CBPP mycoides used for vaccine production are usually elicits temporary serological identified by documents which provide conversion, detectable by various standard information on their origin and the serological tests. It takes approximately six manipulations they have undergone. The to eight weeks to return to a serologically strain selected must have been shown to negative level. be suitable for the region and the type of The establishment of resistance is slow, bovines for which it is intended, when at three to four weeks, and the level of administered by the inoculation route resistance conferred by a CBPP vaccine can prescribed by the manufacturer. Before be demonstrated by testing a group of use, the strain must first have been tested animals but not individuals. The effect of to show that the resulting vaccine is safe vaccination is to raise and consolidate the and confers a protection lasting at least average level of resistance in a herd rather one year in cattle. One such vaccine with than to provide individual protection. these characteristics is the strain T1-44. Clearly, it cannot protect 100 percent of Strain T1 was isolated in 1961 at the East vaccinated animals. Among those vac- African Veterinary Research Organisation, cinated, there is a variation in resistance to from a clinical case of CBPP in what is now infection, ranging from excellent immunity the United Republic of Tanzania. From the Vaccine manual 67

outset, it appeared to be of only moderate streptomycin-dependence. The immuno- virulence. It has been cultured in embryon- genic properties of both strains are ated hens' eggs, first on the chorio-allantoic identical, but the strain T1-SR has produced membrane for six passages and then in the fewer local postvaccinal reactions in yolk sac. Attenuated from the ninth egg humpless cattle breeds. passage, it has been used mainly in East Both strains may be obtained from the Africa for preparing freeze-dried egg Director of CIRAD-IEMVT, 10 rue Pierre culture vaccines with good immunogenic Curie, 94704 Maisons-Alfort, Cedex, qualities. France. This is the FAO reference centre Unfortunately, following the use of egg for mycoplasmas of ruminants. culture vaccines there were a number of cases of postvaccinal lung lesions and Culture medium. The culture medium used numerous local reactions, leading to the contains serum or some other product of suspicion that such vaccines may be animal origin, which must be: dangerous. Their use was therefore either heated at 56°C for at least 30 suspended in 1962. Since then, it has been minutes before being added to the shown that the lung lesions may have been medium, or treated by some other caused by biologically active substances procedure that is equally effective in present in the egg material. The intrinsic destroying contaminant microbial qualities of strain T, were not doubted, agents; however, so it was decided to prepare submitted to appropriate tests to liquid vaccines from broth cultures of the show that the material is free from 44th egg passage of the strain. This material contaminants. is stored in freeze-dried form. In general, culture media contain: In parallel with these studies, the *a basic medium consisting of meat possibility of combining the strain T, with extract (e.g. infusion or papain digest cell-culture rinderpest vaccine to obtain a of beef heart) and/ or peptone (trypose, combined vaccine has also been investigat- tryptone); ed. Mutants of the strain T1-44 that are an extract or autolysate of brewer's resistant to streptomycin (present in the yeast (commercially available or culture fluid of rinderpest virus) have been prepared on the spot), which provides developed at the Institut d'Elevage et de growth factors (group B vitamins); Médecine Vétérinaire des Pays Tropicaux *glycerol, oleic acid, palmitic acid, (IEMVT) Laboratory at Maisons-Alfort in glucose, sometimes a buffering system, France and the Farcha Laboratory in Chad. a small amount of DNA and, if the The Maisons-Alfort mutant (T1-SR), vaccine is produced in a fermenter, also obtained by three passages in a liquid an antifoaming agent; medium containing increasing concentra- *10 percent blood serum, preferably tions of streptomycin, has been studied from adult horses, which must comply under experimental conditions at Dakar, with the standard mentioned above. Senegal, for its immunogenic properties. If the strain T1-SR is used, streptomycin Thus, two lines of T1 are now available: is added at 200 mg per litre of medium. the original strain T1-44 and the strain T,- The final pH is adjusted to 8.1. A satisfact- SR, which has undergone some additional ory formulation is Medium F-66 of the passages in broth. Streptomycin appears Farcha Laboratory. to act as a growth factor for the latter After clarification, it is advisable to strain, although there is no question of sterilize the medium by membrane filtra- 68 Mycoplasmal vaccines

tion rather than by autoclaving. It should containing at least ten times the number be used as soon as possible after pre- of mycoplasmas present in a recom- incubation for 24 hours at 37°C to verify mended dose should be inoculated the absence of bacterial contamination. subcutaneously on the flank posterior to the scapula in each of at least ten Seed lot system. Vaccine production is cattle. The seed batch is satisfactory if founded on a seed lot system. A seed lot the cattle fail to develop any un- may be part of a culture which has served expected clinical reaction during a for vaccine production, has been shown to period of observation lasting for at least be safe for cattle when administered under four weeks and if no lung lesion is natural conditions and is capable of found on post mortem examination at conferring an immunity that lasts for at the end of this period. least a year. A seed lot obtained from such Efficacy testing. A quantity of vaccine a culture must not be submitted to more containing the same number of bacteria than three additional passages. as the recommended dose should be Seed lots' are freeze-dried and stored at inoculated subcutaneously on the flank a temperature not higher than -20°C. posterior to the scapula in each of at Tests on seed lots. Each seed lot must be least ten cattle. A similar number of free from extraneous agents and must cattle should be kept as a control group. satisfy the standards of identity and safety After a minimum of two months have in laboratory animals. In addition, a elapsed, all cattle should be submitted vaccine prepared from the seed lot must to challenge infection by contact with be tested for safety and efficacy in cattle. infected cattle (i.e. cattle infected by Cattle used for this testing must be the introduction of lung lesion homo- representative of the cattle to be vaccinated; genate through an endobronchial tube). they must come from an area free from At least one infected animal should be CBPP and must be at least 24 months old used for each group of three cattle and in good health. The cattle should be under test. The duration of the test is identified in an indelible manner and kept three months and any donor animal in quarantine under veterinary supervision infected by intubation that dies during for at least four months. If any clinical signs this period must be replaced. The seed of disease are present, the animal con- batch passes the test if the animals cerned should not be used until the cause inoculated with vaccine prepared from of the abnormality has been determined the batch do not develop a clinical or a and recognized as having no effect on the serological reaction (i.e. an anamnestic correct conduct of the tests. Serum should response), post challenge, if they are be taken from each animal at the start and free from lesions of pleuropneumonia end of the quarantine period to be tested at slaughter and if the tracheobronchial for CBPP antibodies. Only serologically lymph nodes are free from M. mycoides. negative cattle should be used. At least 80 percent of the unvaccinated Safety testing. A quantity of vaccine control animals should have typical lesions at post mortem examination.

A seed lot prepared jointly by the IEMVT and the Production precautions Pan African Veterinary Vaccine Centre (PANVAC) Inoculation and culture. The sample of the is now stored at both centres for free distributionto vaccine strain used for inoculating the vaccine manufacturers. culture vessels or the fermenter should not Vaccine manual 69

have been submitted tomore than two product has been prepared. After freeze- passages in broth from the seed lot. drying of the vaccine, the containersare It is best to inoculate the production hermetically sealed undervacuum or medium, pre-incubated at 37°C, witha under dry nitrogen free from oxygen. In culture of the vaccine strain in the log- case of failure, all containers sealed under arithmic phase of growth (36 to 40 hours vacuum should be tested for airtightness culture from agitated medium), using one and any defective container rejected. part of culture to ten parts of the culture If part of a vaccine batch is to be freeze- medium. dried at some other time, it is best to freeze After a stationary phase of incubation at the vaccine within the final containers. 37°C, the medium is aerated (by a magnetic After freeze-drying, it should be submitted stirrer if flasks are used, or by injection of to the control tests described below. air if a fermenter is used). The culture is stopped at peak growth, Freeze-drying procedure. A freeze-drying usually at 65 to 77 hours after inoculation. process of long duration is used where the This time has to be determined precisely vaccine is not exposed to temperatures by each producing laboratory, with regard above 0°C while it still contains more than to local conditions, by establishing a 3 percent residual moisture. This can only growth curve for a culture of the vaccine be done in freeze-driers of the shelf type, strain in medium and with vessels identical not in the centrifuge type. Under these to those used for production. This recom- conditions and with a satisfactory mendation is important because con- stabilizer, the loss of viable units should tinuation of culture after the growth peak not exceed 1 log10. In other words, with a leads to a considerable fall in pH and a harvest containing 1010 viable units per ml, diminished number of live mycoplasma. concentrated freeze-dried vaccines are obtained, and their storage is considerably Harvesting and freeze-drying. After a simplified. Operating with modern freeze- culture has been stopped, it is essential to driers, less than 1.5 percent residual harvest immediately, add stabilizer (for the moisture can in fact be attained. preparation of the final product in bulk), fill the final containers and freeze-dry the Control tests product. Storage of the harvest or the final Samples are taken from each vaccine batch product in bulk, even in a cold chamber, or each distribution batch for the tests has an adverse effect on the number of live given below. The tests are performed on mycoplasma in the vaccine. These the vaccine reconstituted to a volume operations are depicted in Figure 1. which will depend on theinitial The bulk suspension is prepared from a concentration of the final product in bulk. single harvest. Once transferred to the tank After reconstitution, samples should be from which the final containers are filled, stored at 4°C and tested within an hour. it becomes the final product in bulk. This final product is transferred quickly to the Identity tests. A test of identity has to be final containers and prepared for freeze- performed on at least one labelled drying. At this point it becomes a batch of container of each distribution batch by vaccine. using a technique suitable for identifying Distribution into the final containers and the presence of M. mycoides. the subsequent freeze-drying must be done A simple and satisfactory test is growth as soon as possible after the final bulk inhibition on agar gel, using filter paper 70 Mycoplasnial vaccines

72 hrs for Ti 65 hrs for T1-SR

Day

0 1 2 3 4 5 6 7

-40° Harvest (-1 »

5 litres)

Magnetic Stirring Stirring stirring Preparation of vaccine (monovalent or combined)

Freeze-drying

FIGURE 1 Procedure for producing CBPP vaccine from strains T, and T1-SR Vaccine manual 71

discs impregnated with a serum containing ten days at 37°C and are shaken lightly a high titre of antibodies to M. mycoides each day to aerate the medium. The subsp. mycoides (SC bovine biotype). minimum titre per vaccine dose must be at Another procedure is to place antiserum least 10 viable mycoplasmas. in wells punched into the agar culture. A It is recommended that production zone of inhibition measuring at least 2 mm laboratories take into account possible will be present after 48 to 72 hours of problems arising from local transport incubation. This test has the advantage of conditions and therefore supply vaccines identifying other mycoplasmas (which with titres of at least 108 mycoplasmas per grow within the inhibition zone) and/ or dose. bacteria present as contaminants. More refined tests (Western Blot, dot immuno- Safety tests. These tests are performed on binding) can also be considered. each batch of vaccine by inoculating guinea pigs and mice. The procedure generally Sterility tests. Freedom of the vaccine from used consists of injecting 0.5 ml of bacteria and fungi is assessed by tests made reconstituted and undiluted vaccine according to the recommendations of the intraperitoneally into each of two guinea World Health Organization's Standards for pigs, the same dose intramuscularly into Biological Substances No. 6 (general two more guinea pigs and 0.1 ml intra- standards concerning the sterility of peritoneally into six mice. The animals are biological substances). kept under observation for three weeks and then killed for examination. The batch Determination of the myco plasma content. of vaccine is considered satisfactory if the An organism count should be made on animals remain healthy and if they have each batch of vaccine (or each distribution no pathological lesion post mortem. batch) to determine the number of viable M. mycoides per dose for cattle. This assay Tests for safety and efficacy in cattle. is done by the following procedures: These tests are done with a mixture of An initial titration is performed on a vaccine from at least five bottles selected mixture of at least five bottles selected at random and as stipulated in the section at random. This mixture can also serve Seed lot system, p. 68. for testing safety and efficacy in cattle A safety test is obligatory for each if necessary. vaccine batch. Because of the complexity e Second and third titrations are each and cost of an efficacy test, this may be performed on the mixed contents of performed occasionally (at least once a three bottles. year) for a given batch of tested seed e The titre of the vaccine is the geometric culture or for a vaccine derived from mean of the three titres thus obtained. culture which has undergone no more than Each titration uses a system of vaccine three passages from the seed culture, dilutions and the appropriate number of provided that all the arrangements for tubes of growth medium sufficient to biological security of the installation and estimate the 50 percent end point by a manipulations have been observed. standard statistical procedure. A series of tenfold dilutions, with ten tubes of medium Stability test. It is desirable to conduct a for each dilution, or any other dilution test of stability on samples of vaccine system which provides equal precision, is batches to obtain experimental data to used. The tubes are incubated for at least predict the effect of storage temperature 72 Mycoplasmal vaccines

and the expiry date. This data should be mycoplasma count, provided that the given on the label and in the accompanying vaccine has been stored continuously at information leaflet. a temperature of -20°C until delivery. In the absence of data on long-term Producers with large stocks of vaccine stability,itisrecommended that should retest the titre before extending the accelerated degradation tests should be authorized storage period. On the other conducted on the vaccine to arrive at hand, no vaccine should be used later than recommendations for storage conditions six months after its supply by the pro- and expiry date. The information provided duction establishment or storage depot, by such tests must be confirmed as soon as and then only if the containers have been possible by observations on vaccine stored stored at temperatures of less than +10°C. under the recommended conditions. The half-life of vaccine freeze-dried toa Storage under these conditions must residual moisture content of 1.5 percent is guarantee that the titre of mycoplasmas in as follows: the vaccine up to the expiry date is not less 4 days at 45°C; than 107 viable mycoplasmasper vaccine *1.6-2.8 weeks at 37°C; dose. 2.8-4.6 weeks at 28°C; Stability tests may also be made to 030-40 weeks at 4°C. provide a foundation for the information These figures mean that, when tested accompanying each container. These tests during production, a vaccine that contains estimate the fall in titre of mycoplasmas a concentration of 108 viable units per dose once the product has been reconstituted (which is on the low side and encountered for use and after storage of the intact in batches of only moderate quality) can product under the temperature conditions be kept for 2 to 4 months at 28°C, 1.5 to 3 which may be encountered in the field. months at 37°C and 12 to 14 days at 45°C. Information should also be provided for The storage life of reconstituted ready- vaccine kept in ice boxes (00 to 10°C) and to-use vaccine must not exceed the time vaccine keptatdifferent ambient during which the mycoplasmal count is temperatures between 20° and 30°C (or equal to or greater than 107 viable even 40°C). organisms per vaccine dose at the specified temperatures, this having been verified Storage conditions experimentally. While awaiting dispatch from thepro- It has been shown experimentally that duction establishment or storage depot, the reconstitution of freeze-dried vaccine in final containers of vaccine should prefer- the field to give the number of doses ably be stored in the dark andat a specified by the producer is best done temperature of -20°C or lower within with a molar solution of magnesium rooms providedwithcontinuous sulphate. Under these conditions thereis temperature-recording equipment. no fall in mycoplasma titre for four hours During storage and transport of the at 37°C. In no case should the vaccine be vaccine, it is strongly recommended that reconstituted withdistilledwater, temperature indicators be used (heat- tap water or surface water,even after sensitive strips or paint) to check quickly boiling. the temperature at which thevaccine has been exposed. CONCLUSIONS The expiry date shouldnot exceed two As stated in the introduction,any im- years from the last determination of the provement in mycoplasmal vaccines is Vaccine manual 73

hampered by little or no information on pneumonia (CBPP) live attenuated vaccine the function of the essential immunogens standard operating procedures. Addis of the organism, the lack of a precise Ababa, PANVAC. understanding of the pathogenesis of Office International des Epizooties. 1992. mycoplasmal diseases and the insufficient Manual of standards for diagnostic tests knowledge of factors governing resistance and vaccines, 2nd ed. Paris. to challenge or natural disease. This means Provost, A., Perreau, P., Breard, A., Le Goff, that subunit vaccines, and even possibly C., Martel, J.L. & Cottew, G.S. 1987. recombinant vaccines, are still far from Contagious bovine pleuropneumonia successful development. a review. Rev, sci. tech. Off int. Epiz., Some simpler avenues appear worth 6(3): 625-679. exploring. New adjuvants, namely im- munostimulating complexes (ISCOMs), could be studied to enhance the im- munogenicity of inactivated vaccines; such steps are under way for CBPP. Another approach would be to explore the value of local immunity. In the case of CBPP, "local" means the application of the immunizing agent to the nasal mucosa by means of a vaccine spray. Preliminary tests have already been shown to be successful in some cases of enzootic pneumonia, CCPP and M. pneumoniae in hamsters. Similar experiments conducted 20 years ago in Nigeria and Chad proved to be very successful with CBPP. Such an approach would now seem to be worth pursuing further.

BIBLIOGRAPHY

FAO. 1971. Contagious bovine pleuropneumonia a review. FAO Agricultural Studies No. 86. Rome. Gilbert, F.R. & Windsor, R.S. 1971. The immunising dose of T, strain Myco plasma mycoides against contagious bovine pleuropneumonia. Trop. Anim. Health., 3: 71-76. Hudson, J.R. & Turner, A.H. 1963. Contagious bovine pleuropneumonia: a comparison of the efficacy of two types of vaccine. Aust. Vet. J., 39: 373-385. Litamoi, J.,Palya, V.J., Sylla, D. & Rweyemamu, M.M. 1994. Quality control testing of contagious bovine pleuro- Vaccine manual 75

Notaz a0 and ricke A.J. de Vos, E. Pipano, F. Musisi and W.K. Jorgensen

THE NEED FOR VACCINES bution of all economically important TBDs Tick-borne protozoal and rickettsial and is transmitted biologically by at least diseases affect most domestic animal 20 tick species. Mechanical transmission species and have been a major constraint by biting flies and unsanitary veterinary to the development of livestock industries practices are also important in certain areas in developing countries in the tropics and (Palmer, 1989). subtropics. Although tick-borne diseases Heartwater is important in cattle and is (TBDs) are important in all domestic also a major cause of losses in sheep and animals, this review will be concerned goats. This disease is transmitted by ticks mainly with vaccines against the eco- of the genus Amblyomma and occurs mainly nomically important tick-transmitted in sub-Saharan Africa, although it has diseases of cattle caused by the protozoal also been introduced to islands in the parasites Babesia bovis and Babesia bigemina Atlantic and Indian Oceans as well as the (babesiosis, red water or tick fever), Caribbean. It is regarded as the most Theileria parva (East Coast fever) and important TBD in South Africa and, next Theileria annulata (tropical theileriosis), as to East Coast fever, the most important well as the rickettsial organisms Anaplasma oneinAfrica(Uilenberg, 1983; marginale (anaplasmosis) and Cowdria Bezuidenhout, 1989). The presence of ruminantium (cowdriosis, heartwater). heartwater and its vector in the Caribbean Vaccines against coccidiosis have been poses a threat to livestock production in reviewed by Danforth and Augustine the tropical and subtropical parts of the (1989). American mainland (Barre et al., 1987). Babesiosis caused by B. bovis and B. Up to 500 million cattle the world over bigemina is present in many countries are exposed to one or more of the TBDs between 400 N and 32.0S (McCosker, 1981). but this figure is not a true reflection of the The most important vectors are ticks of the number at risk to disease. Indigenous genus Boophilus (Friedhoff, 1988). breeds of cattle often have a certain degree East Coast fever is a disease limited to of natural resistance to these diseases and countries in eastern, central and southern the consequences of infection are not as Africa where the principal vector, Rhipi- serious as when exotic Bos taurus breeds cephalus appendiculatus, is present (Norval, are involved. In addition, a state of enzootic Perry and Young, 1992). In these countries, stability frequently develops whereby local it is of major importance because of the cattle become naturally infected at an early high morbidity and mortality it causes. age when there is some passively acquired Tropical theileriosis is transmitted by a or innate immunity. These cattle are number of species of the genus Hyalomma resistant to subsequent challenge (de Vos, which have a wide distribution along the 1992). Mediterranean littoral, in the Near East Cattle, particularly of exotic breeds, are and throughout Asia (Pipano, 1989). at risk to TBDs, especially under the Anaplasmosis has the widest distri- following circumstances. 76 Protozoal and rickettsial vaccines

The importation of susceptible cattle involved the use of blood from naturally into endemic regions. Mortality rates or artificially infected carriers. This carrier- under these conditions can exceed 50 donor method, also known as premunition, percent in regions endemic for has several major limitations, including babesiosis (McCosker, 1981) and the unreliable potency, unpredictable reactions same may apply in areas infected with and the risk of contamination (Callow, East Coast fever and heartwater. 1984; de Vos and Jorgensen, 1992). During The spread of ticks and TBDs into the past 30 years, more sophisticated previously uninfected areas. An techniques were developed to produce live example of this was the loss of over vaccines from splenectomized donors, one million cattle owing to TBDs which had predicably low levels of following the disruption of tick control virulence (Callow and Dalgliesh, 1980). measures in Zimbabwe (Lawrence, The inherent disadvantages of vaccine Foggin and Norval, 1980). containing parasites derived from the The introduction of TBDs into a blood of animals are well documented, disease-free vector population as including the risk of reactions, conta- happened when East Coast fever was mination, sensitization against blood introduced into southern Africa in 1896. groups and the need for cold chain This introduction resulted in the loss transportation (Callow and Dalgliesh, 1980; of an estimated 1.4 million cattle and Wright, 1991). In spite of these disad- took 50 years to bring under control vantages, virtually all operational centres (Lawrence, 1992). producing Babesia vaccines are at present Increases in tick transmission rates in using variants of this technique to produce endemic areas caused by, among chilled or frozen live vaccines. In vitro others,ecologicalfactors. Most culture methods reviewed by Pudney outbreaks of babesiosis in Australia (1992) have been used to produce B. occur in cattle bred in the endemic area bigemina parasites for vaccine (Jorgensen (Callow and Dalgliesh, 1980). et al., 1992) and are also suitable for Immunization and vector control are two producing B. bovis parasites. However, options which need to be considered in all these culture techniques are still at the these situations. Provided there are developmental stage and not widely used suitable facilities and assuming com- for the production of vaccine. mercial vaccines are not available, the local Other products used in attempts to production of vaccine may be the only immunize cattle against babesiosis include sustainable means of control in the country antigens extracted from parasites or concerned. parasitized blood. Cell culture-derived exoantigens of B. bovis and B. bigemina have HISTORY OF VACCINE PRODUCTION AND been extensively studied and proposed for FUTURE PROSPECTS use as vaccine in developing countries B. bovis and B. bigemina (Montenegro-James, Kakoma and Ristic, Methods used to immunize cattle against 1989). Unfortunately, the level and babesiosis have been described or duration of protection conferred by these reviewed (Callow and Dalgliesh, 1980; antigens against heterologous challenge Pipano, Frank and Shkap, 1991; de Vos are less than those of live vaccines (Timms and Jorgensen in FAO, in press). Inmany et al., 1983). countries, early attempts to vaccinate cattle Progress to overcome the limitations of against B. bovis and B. bigemina infections vaccines based on blood or blood extracts Vaccine manual 77

through the development of syntheticor Africa, mainly on an experimentalor field recombinant Babesia vaccines,were trial basis. A ground-up suspension of reviewed by Wright et al.(1992) and infected adult R. appendiculatus (ground- Dalgliesh (1993). Preliminary results up ticks supernatant [GUTS]) is used to suggest that vaccines based on single initiate the infection which is then control- antigens do not confer the desired level or led by treatment with oxytetracycline. duration of protection. It is likely thata Depending on the T. parva stocks used, vaccine containing several recombinant sporozoite vaccine has proved effective antigens will be needed to induce adequate under laboratory conditions and in field protection (Pipano, Frank and Shkap, trials, providing long-lasting immunity 1991). Even a multicomponent recom- against homologous and heterologous binant vaccine may not provide long-term challenge (Burridge et al., 1975a and 1975b). protection against field strains of Babesia However, antigenic heterogeneity of which have been shown to be capable of T.parva isolates has been a major obstacle considerable genetic variation (Dalrymple to the widespread application of this et al., 1992; de Vos and Jorgensen, 1992). method of immunization (Irvin and As yet, no recombinant vaccine for bovine Morrison, 1989). The use of a mixture of babesiosis has been registered for use in stocks is necessary to provide wide- any country and it seems unlikely that such spectrum cover against field challenge, a vaccine will be available in the near especially where T. parva lawrencei is future. When one does become available, involved. In general, good protection is it will almost certainly be marketed afforded by a vaccine made up of T. parva internationally and in competition with (Muguga), T. parva (Kiambu 5) and buffalo- vaccines produced locally. derived T. parva (Serengeti transformed). However, because animals immunized in T. parva and T. annulata this way may become carriers to the As reviewed by Purnell (1977), Irvin and infection and thus may transmit the Morrison (1989), Pipano (1989) and parasite to the resident population, there Lawrence (1992), a variety of products and is an understandable reluctance to procedures have been or are being used to implement immunization programmes immunize cattle against East Coast fever with stocks exotic to the region or country and tropical theileriosis. Early attempts (Irvin and Morrison, 1989). involved the use of infected blood and Despite this drawback as well as the lack tissues. In the case of East Coast fever, the of attenuated, immunogenic stocks and results were very variable but the blood of high cost, the "infection and treatment" donor calves infected with low-virulence method is still the principal means of stocks of T. annulata was used for many immunizing cattle against T. parva. T. parva years as vaccine (Pipano, 1989). Subse- (Boleni) has been used in extensive field quently, a vaccination technique was trials in Zimbabwe. It is of low patho- developed which involved infecting cattle genicity and treatment of reactions is rarely with sporozoites from ticks and then necessary. An application has now been mitigating the clinical responses by made to have vaccine containing this chemotherapy (FAO, 1984; Irvin and stock registered for more general use in Morrison, 1989; Dolan and McKeever, Zimbabwe (Pegram, personal communi- 1993). This "infection and treatment" cation, 1994). There is evidence that T. parva method using T. parva sporozoites is used (Boleni) will also protect cattle against in some countries in East and central some highly pathogenic isolates but this 78 Protozoal and rickettsial vaccines

feature has not been exploited in practical been expressed in Escherichia coli and disease control strategies in other parts of stimulate the production of neutralizing Africa. The infection and treatment method antibody in cattle (Hall and Baylis, 1993). has also been used to prepare a T. annulata sporozoite vaccine (Pipano, 1989) but it Anaplasma marginale has not been evaluated under field condi- A variety of procedures have been used to tions. Immunological differences between immunize cattle against anaplasmosis field isolates of T. annulata do not appear (McHardy, 1984; Palmer, 1989). These to be as important as in the case of T. parva. procedures involve the use of live orga- In vitro techniques for the culture of nisms such as attenuated A. marginale or schizonts of Theileria spp. were reviewed A. centrale and killed organisms or extracts by Brown (1979; 1981) and Pipano (1989). of infected blood. T. annulata schizonts can readily be A stock of A. inarginale has been at- propagated in culture and the patho- tenuated by irradiation and passage in genicity of the parasites decreases during sheep and deer (Palmer, 1989). Vaccine prolonged maintenance in culture. This containing this stock was marketed as provided the basis for developing a safe, Anavac and used to immunize cattle in culture-derived vaccine for the control of Latin America and the United States tropical theileriosis (Pipano, 1989; Singh, (Correr, Johnson and Wagner, 1985). It 1990). This vaccine is currently in use or in provided substantial protection against different stages of development in several challenge with most heterologous isolates. countries in Asia and around the Medi- In general, use of the vaccine had no or terranean (Dolan and McKeever, 1993). very little clinical effect although severe Unfortunately, attempts to develop a morbidity and mortality have been re- similar vaccine for T. parva gave disap- ported, especially when older bulls and pointing results. Histocompatability is seen cows were immunized (Palmer, 1989). Use as one of the main obstacles to the of attenuated A. marginale is the most successful transfer of T. parva schizonts effective means of immunization against from infected cultures to recipient hosts. anaplasmosis but this vaccine is apparently The hazards involved in using the no longer available. Attempts to attenuate sporozoite vaccine and the stock-specific a stock of A. marginale by passage in sheep nature of the immunity conferred have led in Australia were unsuccessful (Jorgensen to intensive efforts to identify parasite et al., 1993). antigens which may be targets for pro- Infection with A. centrale, an organism tective immune responses, particularly originally isolated in South Africa against T. parva infection (Dolan and (Potgieter, 1979), provides partial cross- McKeever, 1993). Of sporozoite antigens immunity against A. marginale challenge. identified as candidates for inclusion in a It is usually mildly pathogenic, particularly vaccine, a gene that codes for a 67 in young animals. These features have led kilodalton (kDa) major surface protein has to the use of A. centrale as vaccine in a been isolated, sequenced and expressed. It number of countries in Africa, Asia, South provides partial protection in cattle against America and in Australia. Cross-immunity homologous challenge (Musoke et al., between A. centrale and A. marginale is 1992). Two sporozoite antigens of adequate if challenge is moderate as in T. annulata defined by differentmono- Australia (Callow and Dalgliesh, 1980), but clonal antibodies have also been studied may be insufficient against virulent as vaccine candidates. These antigens have heterologous challenge (de Vos and Vaccine manual 79

Jorgensen in FAO, in press; Palmer, 1989). calves to homologous and heterologous Despite this limitation and the risk of challenge (Palmer, 1989; Tebele, McGuire reactions in older animals, use of A. centrale and Palmer, 1991; McGuire et al., 1992). appears to be the only choice available to Future work will include the evaluation of most developing countries where ana- recombinants of these proteins (McGuire plasmosis is endemic. An advantage of A. et al., 1992). centrale is its apparent non-transmissibility by the common vectors of A. marginale, Cowdria ruminantium with the exception of the African tick Animals which recover from heartwater Rhipicephalus simas (Potgieter and van acquire an immunity to the disease. This Rensburg, 1987). knowledge has formed the basis of an A non-living vaccine based on a lyophili- "infection and treatment" method of zed preparation of A. marginale organisms immunization involving the inoculation of administered with adjuvant has been animals with Cowdria sp. and treatment available for many years as Anaplaz (Fort of ensuing reactions with tetracycline Dodge Laboratories) in the United States (Uilenberg, 1983). This procedure is used and Latin America (Brock, Kliewer and routinely to immunize cattle in South Pearson, 1965). It induces partial protection Africa (Oberem and Bezuidenhout, 1987; against virulent heterologous challenge but Bezuidenhout, 1989). It has been standard- the level of protection is less than that ized to include the use of blood of sheep provided by live A. marginale vaccine acutely infected with a stock of Cowdria sp. (McHardy, 1984; Palmer, 1989). Neonatal known to cause well-defined febrile iso-erythrolysis has also been reported reactions, thus allowing the optimum time following use of this vaccine owing to the of treatment to be determined. Because of induction of iso-antibodies to blood group the limited viability of the organisms in antigens (Palmer, 1989). Attempts to the unfrozen state, cryopreservation is develop a vaccine containing highly necessary (Oberem and Bezuidenhout, purified Ana plasma spp. particles have been 1987). The innate resistance of young successful (Luther et al., 1989; Montenegro- animals to the clinical effects of Cowdria James et al., 1991) and a vaccine is available infection are well documented and have in Louisiana, in the United States, which been exploited by recommending immun- does not induce sensitization against blood ization at a young age. Disadvantages of groups. In general, the limitations of non- this procedure include the risk of reactions living Ana plasma vaccines (difficulty of in older animals, the large volume of the manufacturing them, their relative inef- inoculum required (5 to 10 ml) and the fectiveness and the risk of contamination need to use the intravenous route of with erythrocyte stroma) render pro- inoculation (Uilenberg, 1983). duction of this type of vaccine impractical A supernatant of ground-up infected in most developing countries. However, nymphal Amblyomma hebraeum ticks some of these vaccines are or were (GUTS) has also been used to immunize commercially available in Central and animals against Cowdria sp. (Oberem and South American countries. Bezuidenhout, 1987; Bezuidenhout, 1989). The ability to induce protective im- Details of the production of this vaccine, munity with killed Ana plasma organisms including the infection of larval ticks, led to further studies on purified antigens. the preparation of infective nymph Two surface proteins (36 and 105 kDa) supernatant and the dilution and storage induce a protective immune response in of the supernatant were reviewed by 80 Protozoal and rickettsial vaccines

Bezuidenhout (1989). The immunity con- country in which the vaccine is to be ferred by this vaccine was similar to that of produced. Such isolates can be obtained the blood vaccine but an unacceptably high by feeding infected Boophilus spp. ticks on number of animals, particularly goats, a susceptible bovine host or by inoculating developed severe allergic or shock reactions it with blood from a long-standing carrier (van der Merwe, 1987). (Callow, 1984). The major limitation of Considerable progress has also been using a carrier is the risk of contamination. made in recent years with the development Techniques for collecting, purifying and of in vitro methods for propagating Cowdria attenuating B. bovis and B. bigemina stocks sp. and a great number of stocks have been have been described (de Vos and Jorgensen grown successfully (Bezuidenhout and in FAO, in press). Brett, 1992). One roller bottle (surface area Attempts to develop attenuated stocks 800 cm' ) with bovine or ovine endothelial of B. bovis from local isolates are not always cells can yield enough organisms to infect successful (de Vos and Jorgensen, 1992). 20 000 animals. There is also evidence of For this reason, it may be worthwhile to attenuation in vitro without loss of im- import a stock that is known to be munogenicity (Jongejan, 1991). Future protective, of low virulence and free from work will be aimed at optimizing culture contaminants. Australian stocks have been conditions for organism yield, improving shown to be protective in several countries viability following freezing and thawing and vaccines containing these stocks have (Bezuidenhout and Brett, 1992) and been used with beneficial results in Africa, overcoming the effect of antigenic dif- Asia and South America (de Vos and ferences between isolates (Jongejan et al., Jorgensen, 1992). 1993). The attenuation of a B. bovis stock is Attempts are being made to identify usually achieved by rapid syringe passage and express immunodominant Cowdria through splenectomized calves (Callow proteins (Barbet et al., 1992). The ultimate and Dalgliesh, 1980; de Vos and Jorgensen goal of the Heartwater Research Project at in FAO, in press; Pipano, Frank and Shkap, the University of Florida, United States, is 1991). The mechanism by which at- to develop a recombinant vaccine delivered tenuation is achieved is not fully under- by a live virus vector. stood but appears to be the result of a selective enrichment of avirulent parasite PRODUCTION OF VACCINES subpopulations. Other methods used to The following methods of vaccine pro- attenuate B. bovis include irradiation duction are at present being used in (Wright, Goodger and Mahoney, 1980), in developing countries or are likely to be vitro culture techniques (Yunker, Kuttler within the means of countries where and Johnson, 1987) and the use of alterna- reliance on enzootic stability is not an tive hosts, but these methods give less option and immunization is the only viable reliable results than passage in splenectom- alternative. As discussed in the previous ized calves. Rapid passage of B. bigemina in section, the vaccines all have the draw- splenectomized calves is not recommended. backs inherent in the production anduse A stock of this parasite with generally low of live products. virulence may be obtained aftera series of slow passages in non-splenectomized calves Babesia bovis and Babesia bigemina (Callow and Dalgliesh, 1980). Selection, attenuation and storage of stocks. It Vaccine stocks of B. bovis and B. bigemina is tempting to use Babesia isolates from the can be stored for lengthy periods as Vaccine manual 81

cryopreserved stabilates of infected blood. virulence nor the immunogenicity of Dimethyl sulphoxide (DMSO) (Mellors et Babesia vaccine stocks were appreciably al., 1982), glycerol (Dalgliesh, Jorgensen modified by maintenance in culture and de Vos, 1990) and polyvinyl pyr- (Timms et al., 1983; Jorgensen, de Vos and rolidone (PVP) (de Vos and Jorgensen in Dalgliesh, 1989). However, recent work FAO, in press) have all been used suc- using the polymerase chain reaction (PCR) cessfully to cryopreserve Babesia spp. of polymorphic genetic markers has shown parasites. that proportions of B. bovis subpopulations Supply of infective material using donor do change with culture in vitro (Bock et al., calves. Cryopreserved master seed of an in press; Lew, unpubl.). attenuated stock is used to initiate a primary infection in a susceptible, sple- Theileria parva nectomized donor. The initial B. bovis Isolation, storage and characterization of stocks. parasitaemia is often low but adequate to Vaccine should be prepared using master initiate a series of four to five passages, seed of well-characterized T. parva stocks thus allowing a continuous supply of to account for the antigenic heterogeneity vaccine over a three- to five-week period. of this species. If there is a requirement for B. bovis vaccine stocks should not be local stocks, these can be obtained by using passaged more than a total of 30 times to bait or naturally infected cattle or by reduce the risk of losing immunogenicity collecting and feeding infected ticks from (Callow and Dalgliesh, 1980; Bock et al., in the field. Procedures used to isolate stocks press; de Vos and Jorgensen, 1992). Inocula of T. parva have been documented in the containing about 1 x 1010parasites are ideal literature (FAO, 1984; Purnell, 1977). forpassaging andwillresultin However, considerable expertise is re- parasitaemias in excess of 1 x 108 per quired in the isolation and characterization millilitre within four to five days. Up to of isolates. Species identification is based 25 000 doses of vaccine can be obtained on examination of some or all of the from a calf weighing 150 to 200 kg. following: geographical distribution, B. bigemina-infected blood is obtained from vector specificity, morphology, host individually infected calves; serial passing specificity, pathogenicity, serology, cross- is not recommended because of possible immunity and DNA probes. Stock char- selection for virulence. acterization traditionally involves cross- Supply of infective material using parasites immunity studies, among others. Several grown in vitro. Available technology allows monoclonal antibodies and DNA probes B. bovis and B. bigemina to be maintained in have also been used to divide stocks of T. continuous culture (Pudney, 1992). These parva into groups (Minami et al., 1983; culture systems have the advantage over Conrad et al., 1987; Allsopp and Allsopp, the calf-donor system of minimizing the 1988) but more work is necessary risk of contamination and limiting the to determine whether polymorphisms number of animals required. A relatively detected correlate with differences in cross- robust method for producing B. bigemina protection (Irvin and Morrison, 1989). vaccine in sealed flask suspension cultures Preparation of ground-up tick supernatant. was developed by Jorgensen et al. ( 1992) Details of the preparation of GUTS are and may be a viable option in countries available in the literature (FAO, 1984). The where insufficient numbers of donors are process involves. the inoculation of cattle available for the production of vaccine in with T. parva seed material. Approximately vivo. Some studies found that neither the ten days after inoculation, the cattle are 82 Protozoal and rickettsial vaccines

infested with non-infected nymphal ticks. Anaplasma spp. and Eperythozoon spp. Engorged ticks are harvested and allowed Theoretically, however, some viral in- to moult. The level of infection can be fections can be introduced into cultures determined by examining the number of from ticks or cattle used as a source of infected acini in the salivary glands of some infected lymphocytes. of the prefed adult ticks. The remainder of Partial or complete attenuation of the adult ticks are fed on the ears of rabbits, T. annulata is achieved by prolonged detached after four days, washed, di- cultivation in vitro (Pipano, 1989). Com- sinfected and ground up in a suitable plete attenuation (no clinical manifestation medium. Ground-up tick tissue is centri- and no detectable tissue schizonts or fuged at a low speed and the supernatant erythrocytic merozoites) has been reported collected, mixed with glycerolized me- after 600 to 900 days in culture (Pipano, dium, equilibrated and cryopreserved. One 1989; Singh, 1990). Partially attenuated litre of frozen vaccine can be prepared from schizonts have also been used for vac- 10 000 adult ticks and this will provide cination (Zablotsky, 1988). 1 000 1-ml doses of vaccine. When the desired degree of attenuation is reached, schizont-infected cells can be Theileria annulata cryopreserved with a high degree of Isolation of stocks. Isolation of T. annulata survival upon reconstitution (Wathanga, parasites from the field was reviewed by Jones and Brown, 1986). DMSO or glycerol Pipano (1989). Isolates can be obtained by can be used as cryopreservative. Master inoculating cattle with the blood of infected seed stock is usually prepared and used to animals or by exposing the cattle to initiate a series of passages in culture to infected ticks. An alternative and more yield parasites for vaccine. There is little elegant technique is to infect peripheral information on the selective effect of blood leucocytes in vitro with sporozoites long-term passaging of schizonts on obtained from macerated infected ticks. immunogenicity but there is evidence that Propagation of schizonts in vitro. Techni- attenuated schizonts are less infective and ques used to initiate and maintain T. less protective than virulent schizonts. annulata cultures are well documented Therefore, it is recommended that pro- (Brown, 1979; Pipano, 1989). Briefly, duction cycles be restarted periodically lymphocytes are collected from the blood from the same master seed (Pipano, 1989). or organs of infected cattle and grown in monolayer or suspension cultures. Alter- Anaplasma spp. using A. centrale as natively, normal peripheral blood lympho- vaccine cytes can be infected in vitro with sporo- Selection and storage of stock. Only one stock zoites obtained from infected ticks (Brown, of A. centrale exists. The history of its 1979). Standard culture procedures are isolation in South Africa in 1911 was used and a wide variety of culture media recorded by Potgieter (1979). This stock are suitable. Yields of up to 9 x 10 schizont- has been used to initiate vaccine pro- infected lymphocytes can be obtained from duction programmes in several countries a stationary culture vessel containing (Potgieter, 1979; Callow and Dalgliesh, 100 ml of rnedium, and even more from 1980; Pipano et al., 1986; Abdala et al., 1990). roller bottles. Master seed of A. centrale is prepared and Continuous propagation of T. annulata stored in the same way as that of Babesia in lymphoid cell cultures eliminates other spp. tick-borne agents such as Babesia spp., Supply of infective material. Infective blood Vaccine manual 83

containing suitable numbers of A. centrale Production of infective material from donor organisms for the production of vaccine sheep. The method used in South Africa can be obtained in much the same way as has been described in some detail (FAO, Babesia parasites. Blood from acutely 1984; Oberem and Bezuidenhout, 1987; infected splenectomized calves is most Bezuidenhout, 1989). Briefly, susceptible suitable for the purpose although, in some sheep are inoculated with thawed stabilate laboratories, the blood of carrier-donors and bled nine to ten days later at the height showing quantifiable rickettsemia is used of the febrile reaction. The stabilate is and reported to be effective (Potgieter, usually prepared from the blood of 1979). A. centrale vaccine can be prepared reacting sheep but tick-derived stabilates in frozen or chilled forms in the same way will fulfil the same purpose. Similarly, as Babesia vaccines (de Vos and Jorgensen goats and cattle can also be used as donors. in FAO, in press). In some countries, Blood from reacting animals is collected for example Australia, mixed Babesia/ directly into an equal volume of buffer to Ana plasma vaccines are produced (Callow which DMSO has been added. The diluted and Dalgliesh, 1980). blood is then snap-frozen and stored in liquid nitrogen. Up to 4 000 doses of Cowdria ruminantium vaccine can be prepared from one sheep. Selection and storage of stocks. The isolation and characterization of Cowdria stocks have VACCINE DISPENSING AND DISPATCH been reviewed in the literature (Uilen- Dilution of vaccine concentrate berg, 1983; FAO, 1984; Oberem and Some vaccine starter materials, notably Bezuidenhout, 1987; Bezuidenhout, 1989). Theileria GUTS and blood infected with Isolates can be obtained from both the Babesia spp. and Theileria spp., are very vertebrate host and the tick vector by concentrated and can be diluted to increase subinoculation of infected blood, organ the vaccine yield. The appropriate medium emulsions or supernatant of ground-up, to be used as diluent depends on the starter engorging Amblyomma ticks. Stocks vary material and whether the vaccine is to be considerably in virulence and there is some frozen or not. Details of media used in the evidence of immunological differences production of TBD vaccines have been (Jongejan et al., 1993). However, immunity reviewed (FAO, 1984; Bezuidenhout, 1989; does not depend on the virulence of the Pipano, 1989; de Vos and Jorgensen in isolate. It is important to select a stock FAO, in press). which is as mild as possible but which Viability of Babesia and Anaplasma shows adequate cross-protection against organisms can be maintained in chilled local isolates. Furthermore, it must also be form by storing infected blood at 2° to 4°C. susceptible to treatment with available Even at this temperature, infectivity is lost anti-Cowdria drugs. Only one stock (Ball 3) exponentially, declining by about one log is used in South Africa. It generally causes unit during the first week of storage a marked febrile response some days (Callow, 1984). Chilled Babesia and before the onset of other clinical signs. Ana plasma vaccines usually contain 0.5 to Stabilate of the stock used for production 1 x 10 organisms per dose or about 100 of vaccine is produced in the same way as times the minimum infective number the vaccine itself (see below) and stored at required for subcutaneous inoculation. The -70° or -196°C. Infective material has also required number of organisms is obtained been freeze-dried successfully if kept by diluting the infected blood with a below -18°C (du Plessis et al., 1990). suitable medium. Chilled T. annulata 84 Protozoal and rickettsial vaccines

vaccine can also be prepared from culture and Anaplasma vaccines produced by the material, with the recommended number carrier-donor method and the T. parva of infected cells being 0.5 x 10 (Pipano, vaccines produced with tissue suspensions 1989). had variable potencies, thus bringing these The minimum volume of a dose of vaccines into general disrepute. In some vaccine should preferably not be less than countries in the tropics and subtropics, the 2 mito allow for inaccuracies in vaccination risk of contamination is also very real. equipment and procedures. An exeption is Quality assurance therefore needs to be an T. parva sporozoite vaccine where the integral part of any TBD vaccine pro- recommended dose is 1 ml to allow for duction programme covering all operating inoculation over the parotid lymph node. procedures. The standards of quality assurance of Vaccine dispensing Babesia, Ana plasma and Theileria vaccines Techniques for producing frozen vaccines have been addressed by the Office inter- using DMSO as a cryoprotectant were nationale des épizooties (OIE, 1991). described by Mellors et al. (1982) and However, these standards only cover the Abdala et al. (1990). Vaccine with glycerol main aspects. Quality control of T. annulata as cryoprotectant has also been produced vaccine in Israel is based on the procedures (Dalgliesh, Jorgensen and de Vos, 1990) for testing live vaccines described in the and is known to remain viable for at least United States Code of Federal Regulations eight hours after thawing. Frozen vaccine for Animals and Animal Products. In is dispensed in cryovials suitable for low- Australia, procedures for the production temperature storage. of Babesia and Ana plasma vaccines are being modified to comply with the Australian Dispatch and cold chain Code of Good Manufacturing Practice for The types of transport networks and their Veterinary Preparations. Compliance with efficiency vary greatly between countries these or similar codes may not be possible and the choice depends largely on avail- in all developing countries. However, the ability. Rapid, reliable means of communi- potential consequences of providing cation and transport are desirable for substandard or contaminated vaccine frozen vaccine and essential for chilled should be considered. The contamination products. Frozen vaccine can be trans- of one batch of Babesial Anaplasma vaccine ported in vacuum-insulated containers in Australia with enzootic bovine leucosis with a refrigerant (liquid N2 or solid CO2). (Rogers et al., 1988) resulted in the payment Vaccine should not be thawed until just of USS1.5 million in compensation. before use and, once thawed, must not be Quality control should be performed at refrozen. Chilled Babesia, Ana plasma and both the preproduction and postprod- Theileria vaccines have a very short shelf- uction stages. Preproduction quality life, even when packed in ice, andmay control is particularly important in thecase become ineffective if transport to the of chilled vaccine but is, in itself,not destination exceeds 48 hours. adequate to guarantee potency and purity. It should include facilities, the docu- QUALITY ASSURANCE mentation of standards and procedures for Because of the live nature of TBD vaccines, obtaining and quarantining suitable donor quality control is essential toensure animals and the preproduction testing of potency and to minimize the risk of animals, master seed and other starting contamination. Most of the early Babesia materials used in the production of vaccine. Vaccine manual 85

It should also include environmental infected ticks) for quality control monitoring to ensure that there is only a purposes. minimal risk of infectious challenge in the Suitable laboratory facilities and services, region where the production facility is including reliable electrical supplies and located. low-temperature storage facilities. Postproduction quality control should Access to foreign capital for the purchase be aimed at determining potency, purity of equipment, reagents and other sup- and, where relevant, virulence. Potency plies needed for production and quality and virulence are monitored by inoculating control. groups of susceptible cattle and then Access to reliable transport networks. monitoring the reactions. Dalgliesh, A core of dedicated, trained staff. Jorgensen and de Vos (1990) described a Access to training opportunities, pro- method for testing the potency of frozen cedures and proven master seed. Babesia vaccine. Similar procedures have been described for Theileria spp. (FAO, KEY LABORATORIES PRODUCING TBD 1984) and Cowdria sp. (Bezuidenhout, VACCINES 1989). Each batch should also be tested for The following laboratories are involved in freedom from contaminants, with the the production of TBD vaccines and may choice of tests depending on the diseases be able to provide suitable master seed, suspected and the type of vaccine con- details of production procedures and cerned. Tick-derived sporozoite vaccines quality control as well as training op- naturally do not need to be scrutinized as portunities. intensely as vaccines produced from the blood of infected animals but there is a Babesia spp. risk of other TBDs, including Crimean Tick Fever Research Centre, Wacol, Congo haemorrhagic fever. Queensland, Australia Kimron Veterinary Institute, Bet Dagan, FACTORS CRITICAL TO THE PRODUCTION OF Israel TBD VACCINES IN DEVELOPING COUNTRIES Veterinary Research Institute, Onderste- Availability of stocks known to be poort, South Africa protective against local challenge and Central Veterinary Laboratory, Li- showing other desirable traits. longwe, Malawi Availability of disease-free donor ani- Instituto Nacional de Tecnologia Agro- mals as a source of infected blood, pecuaria, Rafaela, Argentina infected ticks and other starter materials. Miguel C. Rubino Laboratory, Pando, This may require the animals to be bred Uruguay under tick- and arthropod-free con- ditions for the specific purpose of vaccine Theileria parva production. Central Veterinary Laboratory, Li- Availability of suitable facilities to longwe, Malawi maintain disease-free animals and to ILRAD, Nairobi, Kenya perform the necessary procedures (sple- nectomies, feeding of ticks, collection of Theileria annulata blood). Kimron Veterinary Institute, Bet Dagan, Laboratory facilities and expertise to Israel monitor donor cattle and starter ma- Animal Disease Research Laboratory, terials (infected blood, culture material, NDDB, Anand, India 86 Protozoal and rickettsial vaccines

Anaplasma bovis vaccine. Vet. Parasitol., 43: 45-56. See Babesia spp. list. Bock, R.E., de Vos, A.J., Lew, A., Kingston, T.G. & Fraser, I.R. Studies on failure of Cowdria spp. T strain live Babesia bovis vaccine. Aust. Veterinary Research Institute, Onderste- Vet. J. (in press) poort, South Africa Brock, W.E., Kliewer, I.O. & Pearson, C.C. 1965. A vaccine for anaplasmosis. J. Am. BIBLIOGRAPHY Vet. Med. Assoc., 147: 948-951. Abdala, A.A., Pipano, E., Aguirre, D.H., Brown, C.G.D. 1979. Propagation of Theileria. Gaido,A.B.,Zurbriggen,M.A., In K. Maramorosch & H. Hirumi, eds. Mangold, A.J. & Guglielmone, A.A. Practical tissue culture applications, p. 223- 1990. Frozen and fresh Anaplasma centrale 254. New York, Academic. vaccines in the protection of cattle against Brown, C.G.D. 1981. Application of in vitro Anaplasma marginale infection. Rev. techniques to vaccination against Elevage Méd. Vét. Pays Trop., 43:155-158. theileriosis.InA.D.Irvin,M.P. Allsopp, B.A. & Allsopp, M.T.E.P. 1988. Cunningham & A.S. Young, eds. Theileria parva: genomic DNA studies Advances in control of theileriosis, p. reveal intraspecific sequence diversity. 104-119. The Hague, Martinus Nijhoff. Mol. Biochem. Parasitol., 28: 77-84. Burridge, M.J., Morzaria, S.P., Cunningham, Barbet, A.F., Mahan, S., Allred, D., McGuire, M.P. & Brown, C.G.D. 1972. Duration T.C., Palmer, G.H. & Yunker, C.E. 1992. of immunity to East Coast fever (Theileria Molecular biology of rickettsiae: gene parva) infection in cattle. Parasitol., 64: organisation and structure in Ana plasma 511-515. marginale and Cowdria ruminantium. In Callow, L.L. 1984. Animal health in Australia, T.T. Dolan, ed. Recent developments in Vol. 5. Protozoal and rickettsial diseases, the control of anaplasmosis, babesiosis and p. 1-264. Canberra, Australian Govern- cowdriosis, p. 87-91. Nairobi, ILRAD. ment Publishing Service. Barre, N., Uilenberg, G., Morel, P.C. & Callow, L.L. & Dalgliesh, R.J. 1980. The Camus, E. 1987. Danger of introducing developmentofeffective, safe heartwater onto the American mainland: vaccination against babesiosis and potential role of indigenous and exotic anaplasmosis in Australia. In L.A.Y. Amblyomma ticks. Onderstepoort J. Vet. Johnston & M.G. Cooper, eds. Ticks and Res., 54: 405-417. tick-borne diseases,p.4-8. Sydney, Bezuidenhout, J.D. 1989. Cowdria vaccines. Australian Veterinary Association. In I.G. Wright, ed. Veterinary protozoan Conrad, P.A., Jams, K., Brown, W.C., and hemoparasite vaccines, p. 31-42. Boca Sohanpal, B. & Ole-Moi Yoi, 0. 1987. Raton, Fla., USA, CRC. DNA probes detect genomic diversity Bezuidenhout, J.D. & Brett, S. 1992. Progress in Theileria parva stocks. Mol. Biochem. withthecultivationofCowdria Parasitol., 25: 213-226. ruminantium in endothelial cells. In T.T. Correr, D.F., Johnson, J.S. & Wagner, G.G. Dolan, ed. Recent developments in the 1985. Demonstration of vaccine-induced control of anaplasmosis, babesiosis and immunity to anaplasmosis without cowdriosis, p. 141-147. Nairobi, ILRAD. induction of persistent post-vaccinal Bock, R.E., de Vos, A.J., Kingston, T.G., complement-fixing and agglutinating Shiels, I.A. & Dalgliesh, R.J. 1992. antibodies in yearling steers. Am. J. Vet. Investigationsof breakdownsin Res., 46: 583-586. protection provided by living Babes ja Dalgliesh, R.J. 1993. Immunity, immuno- Vaccine manual

pathology and molecular biology in FAO. Babesia bovis, Babesia bigemina and babesiosis. In K.A. Warren & N. Agabian, Ana plasma marginale vaccines. In Ticks eds. Immunology and molecular biology of and tick-borne disease control- a practical parasitic infections, p. 350-381. New York, field manual. Rome. (in press) Blackwell Scientific. Friedhoff, K.T. 1988. Transmission of Babesia. Dalgliesh, R.J., Jorgensen, W.K. & de Vos, In M. Ristic, ed. Babesiosis of domestic A.J. 1990. Australian frozen vaccines animals and man, p. 23-52. Boca Raton, for the control of babesiosis andana- Fla., USA, CRC. plasrnosis in cattle - a review. Trop. Hall, R. & Baylis, H.A. 1993. Tropical Anim. Health Prod., 22: 44-52. theileriosis. Parasitol. Today, 9: 310-312. Dalrymple, B.P., Jorgensen, W.K., de Vos, Irvin, A.D. & Morrison, W.I. 1989. Vaccines A.J. & Wright, I.G. 1992. Analysis of against Theileria parva. In I.G. Wright, the composition of samples of Babesia ed. Veterinary protozoan and hemoparasite bovis and the influence of different vaccines, p. 115-130. Boca Raton, Fla., environmental conditions on genetically USA, CRC. distinct subpopulations. Int. J. Parasitol., Jongejan, F. 1991. Protective immunity to 22: 31-737. heartwater (Cowdria ruminantium infe- Danforth, H.D. & Augustine, P.C. 1989. ction) is acquired after vaccination with Coccidia vaccines. In I.G. Wright, ed. in vitro attenuated rickettsia. Infect. Veterinary protozoan and hemoparasite Immun., 59: 729-731. vaccines, p. 165-175. Boca Raton, Fla., Jongej an, F., Vogel, S.W., Gueye, A. SE USA, CRC. Uilenberg, G.1993). Vaccination against de Vos, A.J. 1992. Distribution, economic heartwater using in vitro attenuated importance and control measures for Cowdria ruminatium organisms. Rev. Babesia and Anaplasma. In T.T. Dolan, Elevage Méd. Vét. Pays Trop., 46: 223- ed. Recent developments in the control of 227. anaplasmosis, babesiosis and cowdriosis, Jorgensen, W.K., de Vos, A.J. & Dalgliesh, p. 3-12. Nairobi, ILRAD. R.J. 1989. Comparison of immuno- de Vos, A.J. & Jorgensen, W.K. 1992. genicity and virulence between Babesia Protection of cattle against babesiosis bigemina parasites from continuous in tropical and subtropical countries with culture and from a splenectomized calf. a live, frozen vaccine. In B.H. Fivaz, Aust. Vet. J., 61: 371-372. T.N. Petney & I.G. Horak, eds. Tick vector Jorgensen, W.K., Waldron, S.J., McGrath, biology - medical and veterinary aspects, J., Roman, R.J., de Vos, A.J. & Williams, p. 159-174. Berlin, Springer. K.E. 1992. Growth of Babesia bigemina Dolan, T.T. & McKeever, D.J. 1993. Current parasites in suspension cultures for and future vaccines against theileriosis. vaccine production. Parasitol. Res., 78: In R. Pandey, S. Hoglund & G. Prasad, 423-426. eds. Veterinary vaccines, p. 318-337. Berlin, Jorgensen, W.K., Bock, R.E., de Vos, A.J. & Springer. Shiels, I.A. 1993. Sheep-adapted Ana- du Plessis, J.L., van Gas, L., Labuschagne, plasma marginale maintains virulence for F.J. & Wijma, S. 1990. The freeze-drying cattle. Aust. Vet. J., 70: 192-193. of Cowdria ruminantium. Onderstepoort Lawrence, J.A. 1992. History of bovine J. Vet. Res., 57: 145-149. theileriosis in southern Africa. In R.A.I. FAO. 1984. Tick and tick-borne disease control. Norval, B.D. Perry & A.S. Young, eds. A practical field manual, Vol.II. Tick-borne The epidemiology of theileriosis in Africa, disease control, p. 300-621. Rome. p. 1-39. London, Academic. 88 Protozoal and rickettsial vaccines

Lawrence, J.A., Foggin, C.M. & Norval, R.A.I. Jones, E. & Nene, V.1992. A recombinant 1980. The effects of war on the control sporozoite surface antigen of Theileria of diseases of livestock in Rhodesia parva induces protection in cattle. Proc. (Zimbabwe). Vet. Rec., 107: 82-85. Nat. Acad. Sci. USA, 89: 514-518. Luther, D.G., Hart, L.T., Morris, N.G., Norval, R.A.I., Perry, B.D. & Young, A.S. McRae, B. & Todd, W.J. 1989. Field 1992. The epidemiology of theileriosis in trial of an experimental anaplasmosis Africa, p. 1-481. London, Academic. vaccine in Louisiana. Proc. Annu. Meet. Oberem, P.T. & Bezuidenhout, J.D. 1987. US Anim. Health Assoc., 93: 35-38. The production of heartwater vaccine. McCosker, P.J. 1981. The global importance Onderstepoort J. Vet. Res., 54: 485-488. of babesiosis. In M. Ristic, ed. Babesiosis, OIE. 1991. Manual of recommended diagnostic p. 1-24. New York, Academic. techniques and requirements for biological McGuire, T.C., Barbet, A.F., Tebele, N., products, Vol. III. Paris. McElwain, T.F. & Palmer, G.H. 1992. Palmer, G.H. 1989. Ana plasma vaccines. In Progress in development of subunit I.G. Wright, ed. Veterinary protozoan and vaccines for anaplasmosis. In T.T. Dolan, hemoparasite vaccines, p. 1-29. Boca Raton, ed. Recent developments in the control of Fla., USA, CRC. anaplasmosis, babesiosis and cowdriosis, p. Pipano, E. 1989. Vaccination against Theileria 99-104. Nairobi, ILRAD. annulata theileriosis. In I.G. Wright, ed. McHardy, N. 1984. Immunisation against Veterinary and hemoparasite vaccines, anaplasmosis - a review. Prey. Vet. Med., p. 203-234. Boca Raton, Fla., USA, CRC. 2: 135-146. Pipano, E., Frank, M. & Shkap, V. 1991. Mellors, L.T., Dalgliesh, R.J., Timms., Current methods for the control of tick Rodwell, B.J. & Callow, L.L. 1982. fevers in cattle. Is. J. Vet. Med., 46: 79- Preparation and laboratory testing of 88. frozen vaccine containing Babesia bovis, Pipano, E., Krigel, Y., Frank, M., Markovics, Babesia bigemina and Anaplasma centrale. A. & Mayer, E. 1986. Frozen Ana plasma Res. Vet. Sci., 32:194-197. centrale vaccine against anaplasmosis Minami, T., Spooner, P.R., Irvin, A.D., in cattle. Br. Vet. J., 142: 553-556. Ocama, J.G.R., Dobbelaere, D.A.E. & Potgieter, F.T. 1979. Epizootiology and Fujinaga, T. 1983. Characterisation of control of anaplasmosis in South Africa. stocks of Theileria parva by monoclonal J. S. Afr. Vet. Assoc., 50: 367-372. antibody profiles. Res. Vet. Sci., 35: 334- Potgieter, F.T. & van Rensburg, L.T. 1987. 340. Tick transmission of Anaplasma centrale. Montenegro-James, S., Kakoma, I. & Ristic, Onderstepoort J. Vet. Res., 54: 5-7. M. 1989. Culture-derived Babesia exo- Pudney, M. 1992. Cultivation of Babesia. In antigens as immunogens. In I.G. Wright, T.T. Dolan, ed. Recent developments in ed. Veterinary protozoan and hemoparasite the control of anaplasmosis, babesiosis and vaccines, p. 61-97. Boca Raton, Fla., USA, cowdriosis, p. 129-140. Nairobi, ILRAD. CRC. Purnell, R.E. 1977. East Coast fever:some Montenegro-James, S., James, M.A., Toro recent research in East Africa. Adv. Benitez, M., Leon, E., Baek, B.K. & Parasitol., 15: 83-132. Guillen, A.T. 1991. Efficacy of purified Radley, D.E., Brown, C.G.D., Burridge, M.J., Anaplasma marginale initial bodiesas a Cunningham, M.P., Kirimi, I.M., vaccine against anaplasmosis. Parasitol. Purnell, R.E. & Young, A.S. 1975a. East Res., 77: 93-101. Coast fever. 1. Chemoprophylactic Musoke ,A.J., Morzaria, S.P., Nkonge, C., immunisation of cattle against Theileria Vaccine manual 89

parva (Muguga) and five Theileria strains. between irradiated and non-irradiated Vet. Parasitol., 1: 5-41. populations. Z. Parasitol., 63: 47-57. Radley, D.E., Brown, C.G.D, Cunningham, Wright, I.G., Casu, R., Commins, M.A. & M.P., Kimber, C.D., Musisi, F.L., Payne, Dalrymple, B.P. 1992. The development R.C., Purnell, R.E. & Stagg, S.M. 1975b. of a recombinant Babesia vaccine. Vet. East Coast fever. 3. Chemoprophylactic Parasitol., 44: 3-13. immunisation of cattle using oxytetra- Yunker, C.E., Kuttler, K.L. & Johnson, L.W. cycline and a combination of Theileria 1987. Attenuation of Babesia bovis by in strains. Vet. Parasitol., 1: 51-60. vitro cultivation. Vet. Parasitol., 24: 7-13. Rogers, R.J., Dimmock, C.K., de Vos, A.J. Zablotsky, V.T. 1988. Response of cattle of & Rodwell, B.J. 1988. Bovine leucosis different ages to Theileria annulata virus contamination of a vaccine pro- vaccine, II. Reaction and immune res- duced in vivo against bovine babesiosis ponse of calves of different breeds to and anaplasmosis. Aust. Vet. J., 65: theileriosis vaccine. Trudy Institute 285-287. Eksperimental'noi Veterinarii, 66: 62-75. Singh, D.K. 1990. Methods currently used (in Russian) for the control of Theileria annulata: their validity and proposals for future control strategies. Parasitologia, 32: 33-40. Tebele, N., McGuire, T.C. & Palmer, G.H. 1991. Induction of protective immunity using Ana plasma marginale initial body membranes. Infect. Immun., 59: 3199-3204. Timms, P., Dalgliesh, R.J., Barry, D.N., Dimmock, C.K. & Rodwell, B.J. 1983. Babesia bovis: comparison of culture- derived parasites, non-living antigen and conventional vaccine in the protection of cattle against heterologous challenge. Aust. Vet. J., 60: 75-77. Uilenberg, G. 1983. Heartwater (Cowdria ruminantium infection): current status. Adv. Vet. Sci. Comp. Med., 27: 427-480. van der Merwe, L. 1987. The infection and treatment method of vaccination against heartwater. Onderstepoort J.Vet. Res., 54: 489-491. Wathanga, J.M., Jones, T.W. & Brown, C.G.D. 1986. Cryopreservation of Theileria infected lymphoblastoid cells with functional assessment of viability. Trop. Anim. Health Prod., 18: 191-197. Wright, I.G. 1991. Towards a synthetic Babesia vaccine. Int. J. Parasitol., 21: 156-159. Wright, I.G., Goodger, B.V. & Mahoney, D.F. 1980. The irradiation of Babesia bovis. I. The difference in pathogenicity Vaccine manual 91

,. [1E1 L-,S Cd p VIC C J.B. McKeand and D.P. Knox

THE NECESSITY FOR ANTIPARASITE VACCINES goats (Jackson et al., 1991). There is Diseases caused by multicellular parasites widespread acaricide resistance among the are a major cause of mortality and important cattle ticks, Boophilus microplus morbidity in humans and domestic and Hyalomma anatolicum anatolicum animals throughout the world. Jenner's (Wharton, 1976) and an increasing discovery that cowpox virus was effective incidence of insecticide resistance is in immunization against smallpox sti- evident in the sheep blowfly, Lucilia cuprina mulated research into vaccination against (Arundel and Sutherland, 1988). most major pathogens including multi- Although the rapid spread of drug cellular parasites. Unfortunately, the resistance in parasite populations can to a successes achieved against viruses and degree be reduced by strategic manage- bacteria have not been paralleled in the ment procedures, these can impose consi- control of diseases caused by multicellular derable restraints on land use, especially parasites. At present these are controlled on marginal pastures. The cost of devel- by chemotherapy, although i tis now oping novel chemical control agents is apparent that resistance is an inevitable prohibitive and considerable effort is now consequence of prolonged chemical ap- being directed towards prolonging the plication. Anthelmintic resistance has been efficacy of the currently available drugs, reported against all the broad-spectrum with a particular emphasis on minimizing agents currently available (Prichard, 1990). the effects of drug resistance. Furthermore, For example, benzimidazole resistance has there is now widespread public concern been found in Haemonchus contortus, regarding the effects° of chemical residues Ostertagia circumcincta and Trichostrongylus in both animal products and the environ- colubriformis nematode infections in sheep ment. There is clearly a requirement to and goats throughout the world, while develop novel control methods for pa- levamisole and morantel resistance has rasitic disease. been recorded with increasing frequency Recently, biological control strategies, in Australasia and South Africa (Prichard, such as the use of nematophagous fungi, 1990; Jackson, 1993). Of concern is the fact have been evaluated for the control of that resistance to the relatively recently gastrointestinal nematodes of domestic developed ivermectin has been experi- animals (Waller and Larsen, 1993) but most mentally selected in H. con tortus (Egerton, research has been directed at the devel- Suhayda and Eary, 1988) and has now been opment of antiparasite vaccines. Despite reported in the field in H. con tortus in both decades of intensive work, only two sheep (van Wyck and Malan, 1988) and commercially available multicellular parasite vaccines exist. These are based on radiation-attenuated larvae for the control The authors would like Lo thank Professor Andy of Dictyocaulus viviparus in cattle (Jarrett et Tait, Dr Eileen Devaney and Dr Bob Coop for their al., 1958) and Dictyocaulus filaria in sheep support in the preparation of this chapter. (Sharma, Bhat and Dhar, 1988). It would 92 Multicellular parasite vaccines

appear that the antigenic complexity of Cauthen, 1942; Jarrett, McIntyre and multicellular parasites has provided an Urquhart, 1954). Vaccination studies using effective barrier to the development of two doses of 10 000 40 kilorad- (400 joules useful vaccines. To compound this, it is per kilogram-) irradiated L3 showed that often the case that protective antiparasite calves could be significantly protected immune responses are absent or deficient against challenge using attenuated larvae in immature animals. The reasons for this (Jarrett et al., 1958). This vaccine was apparent unresponsiveness are unclear developed commercially and has been but,given that production losses usedsuccessfullyforthecontrol primarily accrue owing to the infection of of dictyocaulosis in the United Kingdom young stock, studies must be under- and other parts of Europe ever since. taken to identify the cause of this and to This pioneering breakthrough led to develop vaccines which are effective in this several attempts to develop similar group. vaccines against other important helminth Recent advances in immunology, protein species. chemistry and recombinant DNA techni- In India, a successful irradiated larval ques have enabled a more accurate iden- vaccine was developed for the control of tification of potential host-protective the pathogenic ovine lungworm, D. filaria immune responses and the parasite anti- (Sharma, Bhat and Dhar, 1988). Vaccination gens which stimulate them. In order to of dogs against the intestinal nematode understand the problems and difficulties Ancylos toma caninum was similarly ef- associated with the development of fective (Miller, 1971). Unfortunately, this vaccines against multicellular parasites, vaccine failed commercially because of its this review provides a historical account respiratory side-effects, short shelf-life and, of the work that has been undertaken and in some cases, lack of sterile immunity then continues with a description of recent (Miller, 1978). Schistosoma spp. are trema- advances which suggest that the devel- todes of importance in humans and opment of subunit parasite vaccines may animals in tropical regions and successful be an achievable goal. Numerous kinds of immunization has been achieved using parasite preparations have been used to irradiated cercariae in laboratory models immunize animals. These range from the (Simpson et al., 1985). Furthermore, zebu use of attenuated whole parasites, through cattle were significantly protected against crude extracts of dead parasites to specific bovis challenge following vaccination purified molecules. with irradiated schistosomula or cercariae of the homologous species (Bushara et al., VACCINATION WITH RADIATION-ATTENUATED 1978). PARASITES Other attempts to immunize animals Parasites attenuated by irradiation do not with irradiated parasites have met with reach patency but can stimulate protective less success. Mature sheep (> six months) immune responses without significant were protected against homologous parasite-induced pathology. Immunization challenge using irradiated L3 of either with irradiated third-stage larvae (L3) of H. contortus (Urquhart et al., 1966b)or the bovine lungworm, D. viviparus, isa colubriformis (Gregg and Dineen, 1978). highly successful example of vaccination However, as in natural infection, immature using this method of attenuation. Natural sheep (< three months) failed to develop infection with this parasite induces high immunity following vaccination (Urquhart levels of resistance in calves (Poynter and et al., 1966a; Gregg et al., 1978). Vaccine manual 93

VACCINATION USING MATERIAL which inhibited larval growth and FROM DEAD PARASITES development in vitro (O'Donnell et al., Attempts to vaccinate against parasitic 1981). Larvae which were harvested from helminths using somatic extracts from sheep previously vaccinated with soluble dead organisms were first made in the components of second-instar larvae were 1930s but achieved limited success 58 percent smaller than larvae from control (Chandler, 1932). However, some subse- sheep (Johnston, Kemp and Pearson, 1986). quent attempts have given protection: for example, extracts of fourth-stage larvae of VACCINATION WITH MATERIAL T. colubriformis induced significant levels FROM LIVE PARASITES of protection in guinea pigs against Although some studies using crude homologous challenge (Rothwell and extracts of dead parasites gave protection, Love, 1974) and worm burdens in sheep few of these studies were taken further immunized with sonicates of adult stage owing to the difficulty in defining the H. con tortus were significantly lower (63 protective components within these com- percent) than burdens found in challenge plex extracts, and attention increasingly controls (Adams, 1989). Somatic extracts focused on less complex parasite com- derived from onchospheres of the cestodes partments which may comprise compo- Echinococcus sp. and Taenia sp. were also nents essential to parasite survival within found to be potent sources of host- the host. protective antigens (Rickard and Williams, Sarles and Taliaferro (1936) first 1982; Xilinas, Papavasiliou and Marselou- suggested that products released from Kinti, 1976). These results appear to be the helminths in vivo were involved in host exception rather than the rule, since in most immunity' when they observed immune other species success has been limited precipitates at the mouth, excretory pore when somatic helrninth extracts were used and anus of Nippostrongylus brasiliensis for immunization (Clegg and Smith, 1978). parasites which had been incubated with Similar to the situation with helminths, serum from immune rats. It was thought somatic extracts of ectoparasites have that the antigen/ antibody complexes induced variable degrees of protective formed at these parasite orifices might act immunity (Willadsen, 1980). Salivary gland by blocking the activity of secretions extracts induced protective immunity to essential to parasite survival. Several B.microplus(Brossard,1976) and attempts to immunize laboratory animals Amblyomma maculatum (McGowan et al., with these excretory-secretory (ES) 1981) in cattle and also in Dermacentor antigens have produced encouraging andersoni (Wikel, 1981) in guinea pigs. results. For example, protective immunity Subsequently, Opdebeeck et al. (1988) was achieved in mice using the ES products vaccinated calves with crude soluble and of first-stage larvae of Trichinella spiralis membrane extracts from the midgut of (Vernes, 1976) as well as in guinea pigs partially engorged female B. micro plus and using the ES products of fourth-stage T. observed up to a 98 percent reduction in colubriformis (Rothwell and Love, 1974), the tick numbers following challenge. Sheep ES products of third- and fourth-stage produce antibodies to L. cuprina, the Ascaris suum (Stromberg and Soulsby, primary agent responsible for fly strike in 1977) and the ES products of adult Australia (O'Donnell et al., 1980) and D. viviparus (McKeand et al., 1995). vaccination of sheep with soluble extracts Immunization using ES materials has not of third-instar larvae stimulated antibodies always been successful, however, as 94 Multicellular parasite vaccines

demonstrated by Neilson (1975) who native antigen on a small scale from immunized lambs with the ES products of parasites harvested from donor animals or L3 and L4 H. con tortus and found no from parasites maintained in vitro. This differences in faecal egg counts or worm would clearly be unsuitable for commercial burdens between vaccinates and challenge vaccine production. Emery and Wagland controls. The lambs used in this study (1991) estimated that, to obtain sufficient however, were less than three months old nematode antigen to vaccinate one sheep, and may have been immunologically parasites would have to be harvested from unresponsive. Alternatively, the result may three donor animals! The problem of have reflected differences in the immuno- antigen production can now be resolved genicity of ES in laboratory animals by using peptide synthesis and / or re- compared with that in natural hosts. combinant DNA technology. The main Materials released from ectoparasites challenges are to identify appropriate have also been proposed as major immuno- individual protective antigens suitable for gens. Fly strike is initiated by enzymes incorporation into subunit vaccines while secreted by the fly larvae on to the sheep's maintaining their immunogenicity. skin and sheep exposed to several infesta- tions strongly recognized antigens derived, Antigen selection and probably released from, the larval gut While many parasite proteins are antigenic, and salivary glands (Skelly and Howells; the immune responses to these antigens 1987). It has been postulated that inflam- may not be protective (O'Donnell et al., mation induced by larval ES products 1989). Parasite antigens can be classified plays a central role in wound formation as conventional or covert. Conventional and exudation and that these, in turn, may- antigens are recognized by the host during reduce larval establishment (Sandeman, the course of a natural infection arid, as 1990). More refined vaccination studies in vaccine components, would augment which the potential of secreted larval natural immunity. However, mutations in enzymes as vaccine candidates have been the genes coding for these antigens may be indicated are outlined later. selected by the pressure associated with Despite all these studies, until recently immunity in vaccinated hosts and could no one has been able to identify and isolate restrict their long-term use. On the other protective components within these hand, covert antigens are hidden from the fractions and incorporate them into host immune system during natural commercially viable vaccines. With the infection and, in the absence of selection, advent of improved immunochemical are less likely to exhibit antigenic variation. methods for identifying protective antigens The main disadvantage of using covert and the ability to produce these in large antigens is that repeated vaccination may quantities using recombinant DNA tech- be necessary as specific host immune nology, it now appears possible to develop responses would not be boosted during subunit parasite vaccines. The likely steps subsequent natural infections. However, if in subunit parasite vaccine development animals are protected during the very are summarized (Figure 2). susceptible period in early life, as for example in haemonchosis in sheep, subse- DEVELOPMENT OF SUBUNIT MULTICELLULAR quent exposure to natural challenge rnay PARASITE VACCINES maintain protective levels of immunity. Historically, the selection of protective Antigens can be selected on eitheran antigens has been based on extracting empirical or a rational basis. The empirical Vaccine nual 95

Antigen selection

11Pr

Rational or empirical approach

111F

Vaccination studies using native antigen

1Pir

Production of recombinant antigen lir Vaccination studies with recombinant antigen Vr Incorporation of optimal delivery systemadjuvant, vector, etc. Vr Field evaluation and commercial development

FIGURE 2 Steps in the development of subunit parasite vaccines 96 Multicellular parasite vaccines

approach relies on the fractionation of (kDa) protein, induced 43 to 51 percent parasite material followed by successive protection in guinea pigs (O'Donnell et al., immunization studies. These extracts 1989). Partial amino acid sequence analysis providethestartingmaterialfor of this protein indicated that it was progressively more refined fractionation tropomyosin.Progresstowardsa and the ultimate definition of specific recombinant vaccine based on parasite protective components. Fractionation has tropomyosin is outlined in the section normally been achieved using con- Multicellular parasite vaccines: recent ventional protein chemistry techniques developments, p. 100. such as gel filtration, ion exchange chroma- A slightly different approach has been tography and polyacrylamide gel electro- used to isolate protective antigen fractions phoresis. Empirical evaluation is, however, from Oesophagostomum radiatum, a patho- extremely time-consuming and costly so a genic nematode found in the large intestine more rational approach, where molecules of calves. Animals acquire strong resistance are selected on the basis of their to reinfection (Roberts, Elek and Keith, contribution to parasite survival, has many 1962) and protective immunity was stimul- advantages. ated using excretory gland homogenates Despite its drawbacks, the empirical of adult parasites (Keith and Bremner, approach has resulted in the production of 1973). The adult O. radiatum extracts were some viable vaccine candidates. For resolved into four fractions by gel filtration example, in Australia antigen fractionation (East, Berrie and Fitzgerald, 1989) and has led to the identification of tropomyosin ELISA analyses showed that antibodies as a candidate antigen for stimulating from naturally infected calves predomin- protective immunity in sheep against antly reacted with the void volume fraction T. colubriformis andH. contortus. which comprised high molecular weight Immunizationwithwholeworm components. Vaccination of calves with homogenates from fourth-stage larvae of this fraction significantly reduced worm T. colubriformis was observed to accelerate establishment and faecal egg output (East, expulsion of homologous challenge in Berrie and Fitzgerald, 1988). High mole- guinea pigs (Rothwell and Love, 1974; cular weight antigens have also been used Rothwell and Griffiths, 1977; Rothwell, to vaccinate against Nematospiroides dubius 1978). Subfractions of this homogenate, in mice (Monroy and Dobson, 1987), T. obtained by sodium dodecyl sulphate- colubriformis (O'Donnell et al., 1985) and H. polyacrylamide gel electrophoresis (SDS- contortus (Neilson and Van de Walle, 1987) PAGE), were later shown to induce host in sheep, with varying degrees of success. protective immunity (O'Donnell et al., By using antibodies or cells derived from 1985). These subfractions, however, still infected and / or immune animals to probe comprised rather complex protein crude antigen preparations, the major mixtures, so different extraction pro- immunogens can be identified. This cedures were then evaluated to reduce strategy can be augmented by comparing their complexity (O'Donnell et al., 1989). the responses of individual hosts which Using phosphate-buffered saline/ sodium are defined as susceptible or resistant to deoxycholate (PBS / NaDOC) to make infection. Monoclonal antibodies derived somatic extracts of T. colubriformis larvae, from immune mice were used to define a simple antigen mixture was produced and purify the major immunogens of the which -consisted of four proteincom- small intestinal nematode, T. spiralis. This ponents, one of which, a 41 kilodalton workledtothepurificationto Vaccine manual 97

homogeneity of the first single, protective (Munn, Greenwood and Coadwell, 1987; antigen of a nematode, a 48 kDa protein Munn and Smith, 1990). A major im- (Silberstein and Despommier, 1984). munogen of this preparation was an Initially, three proteins were isolated from integral membrane glycoprotein of mole- extracts of infective larvae by immuno- cular weight 110 kDa, termed H11 (Munn, affinity chromatography (Silberstein and 1988; Smith and Munn, 1990). Immuniz- Despommier, 1984). When used to im- ation of sheep with microgram amounts of munize mice, one of these proteins gave essentially pure H11 stimulated substantial significant protection at very low (0.1 protection against challenge infection in D1 i.tg) doses. Unfortunately, this protein several breeds of sheep (Munn, 1988; was ineffective in the natural pig host Tavernor et al., 1992a and 1992b; Munn (Gamble, Murrell and Marti, 1986). et al., 1993a). This immunity was closely In another study, sera from infected correlated with H11-specific circulating sheep, which were defined as resistantor IgG levels (Munn and Smith, 1990) and susceptible to O. circumcincta, were used vaccination was equally successful in to identify a 31 kDa molecule present in young lambs (< two months, Tavernor Triton X-100 extracts of L3. This molecule et al., 1992a) as in older lambs (> eight was recognized preferentially by resistant months, Tavernor et al., 1992b). With animals as early as three weeks after respect to the age-related unresponsive- experimental infection and was present ness of young lambs to vaccination with in and secreted by third-stage larvae irradiated L3 (Urquhart et al., 1966b), the (McGillivery et al., 1990). Lambs im- results obtained with the H11 antigen munized with the purified protein were would suggest that this would be an ideal significantly protected against homologous vaccine candidate for the control of this challenge as judged by faecal egg counts disease. Sequence analysis of full-length and adult worm burdens compared with cDNAs encoding H11 has shown the challenge controls (McGillivery et al., 1992). protein to be homologous to mammalian Homologousantigenshavealso amino peptidases. Amino peptidase acti- been identified in T. colubrtformis and vity was subsequently found in native pure H. con tortus (McGillivery et al., 1990). H11 and enzyme activity was inhibited by Recently, more rational approaches to immunoglobulin from H11-vaccinated vaccine design have produced encouraging sheep (Munn et al., 1993b). A full-length, results. One approach has been to identify enzymically active H11 cDNA has been and isolate antigens from the intestinal produced in the baculovirus expression luminal surface of blood-feeding parasites. system (Munn et al., 1993b) and its ability It has been suggested that the induction of to protect lambs against haemonchosis is systemic antibody responses against the currently being evaluated. Recent work has gut antigens of blood-sucking parasites indicated that integral gut membrane may cause sufficient damage to impair preparations from adult H. contortus parasite survival. This response is contain other protective antigens apart "artificial" as these gut antigens are not from H11. For instance, substantial pro- recognized by the host during the course tection against H. con tortus was induced in of natural infection, i.e. the antigens are lambs hyperimmunized with an integral covert. Lambs have been immunized membrane extract in which the character- successfully against haemonchosis, using istic H11 doublet was not usually observed antigens fractionated from the intestinal (Smith, 1993). luminal surface of adult H. contortus A similar approach has led to the 98 Multicellular parasite vaccines

isolation of several protective antigens from the nematode cuticle or from from blood-feeding arthropods. For specialized excretory-secretory organs and example, cattle are protected against the represent the major antigenic and tick B. microplus by using components functional challenge to the host (MaizeIs isolated from the tick gut (Johnston, Kemp and Selkirk, 1988; Lightowlers and Rickard, and Pearson, 1986; Kemp et al., 1986; 1988). In terms of so-called functional Willadsen, McKenna and Riding, 1988; molecules, several classes of enzyme have Opdebeeck et al., 1988). The number of ticks been identified in ES products. These engorging, their average weight and egg- include proteinases which may facilitate laying ability were all diminished in those penetration of host tissues (Matthews, ticks feeding from immunized cattle 1982; Dalton and Heffernan, 1988) or act as (Willadsen and McKenna, 1991). Triton anticoagulants (Hotez and Cerami, 1983), X-100 extracts of the gut of first-instar acetylcholinesterases (AChE) (Rhoads, larvae of L. cuprina were used to vaccinate 1984) and superoxide dismutases (Rhoads, sheep, and challenge larvae obtained from 1983; Knox and Jones, 1992). Some of these these sheep had their growth reduced by have the potential to modulate host more than 40 percent when compared with immune responses, for example secreted larvae from challenge controls. In addition, proteinases have been ascribed a role in immune sera from vaccinated sheep cleaving surface-bound immunoglobulin inhibited the growth of larvae in vitro by in several helminth systems (Auriault et more than 50 percent indicating that the al., 1981; Chapman and Mitchell, 1982; effect was antibody-mediated (East et al., Smith et al., 1993). AChE may interfere with 1993). Gut membrane preparations have hosts' immune mechanisms by breaking also given promising results with sucking down host acetylcholine (ACh), which is lice, flies and mosquitoes (Schlein and known to stimulate potential effector Lewis, 1976). mechanisms such as neutrophil-mediated The lumen of the blowfly gut is lined antibody-dependent cellular cytotoxicity with a periotrophic membrane which, in (Gale and Zhigelboim., 1974), neutrophil addition to being essential to digestion, chemotaxis (Hill et al., 1975) and mast cell acts as a molecular sieve preventing intact histamine release (Kaliner and Austen, antibody molecules coming into contact 1975). Although not an enzyme, a y- with underlying gut epithelial cells (East interferon analogue has been identified in et al., 1993). This range of functions, crucial T. colubriformis ES products (Dopheide et to insect survival, attracted attention to the al.,1991). Owing to these putative periotrophic membrane as a source of functional roles, ES products have received potential protective antigens. Periotrophic close attention as a source of protective membranes, harvested in vitro from antigens. L. cuprina larvae, were used to immunize Secretions produced during the moult sheep and resulted ina 30 percent from third- to fourth-stage larvae of Ascaris reduction in the weight of established suum induced significant protection larvae compared with those obtained from against challenge in guinea pigs, while challenge control animals (East et al., 1993). soluble proteins produced during culture Another source of potential vaccine of L2, L3, L4 and adultworms were candidates are ES products whichare ineffective (Stromberg and Soulsby, 1977). thought to assist in tissue invasion, parasite Metabolites secreted by H. con tortus during feeding and evasion of host effector the moult from the third to fourth larval mechanisms. ES products may be derived stages were also effective in sheep (Ozerol Vaccine manual 99

and Silverman, 1970) and the ES products received much attention as a candidate of adult D. viviparus induced significant protective antigen (Brophy and Barrett, levels of protection in immunized guinea 1990). GST is recognized by antibodies pigs (McKeand et al., 1995). from mice infected with the trematode Attention is now focusing on defining Schistosoma japonicum, and the devel- the precise nature of the polypeptides opment of protective immunity in various which comprise nematode ES products. mice strains has been positively correlated The application of molecular biology and with the levels of specific anti-GST increasingly specific immunological antibody (Smith et al., 1986). Recombinant methods to the study of parasite ES GST, prepared from S. japonicum and S. products has enabled the definition of a mansoni, was subsequently observed to number of novel proteins with protective confer partial, but not significant, pro- properties. Recently, the dominant tection in rats and hamsters (Balloul et al., glycoprotein present in ES products 1987). However, vaccination of genetically derived from the parasitic stages of susceptible mice with a j3-galactosidase T. colubriformis has been cloned and fusion protein of this enzyme gave signi- sequenced and shown to induce protective ficant protection against infection with S. immunity and have significant homology japonicum (Smith et al., 1986). Affinity- to the porcine intestinal peptide, valosin purified GST from Fasciola hepatica did not (Savin et al., 1990). Valosin can modulate significantly protect rats against challenge several aspects of gastrointestinal function (Howell, Board and Boray, 1988), but sheep and, by secreting a homologue protein, the immunized with purified GST were parasiteitself may alteritslocal significantly protected (57 percent re- environment. duction in fluke numbers) (Sexton, Milner AChE is secreted by several nematode and Paraccio, 1990). The genes encoding F. species and is immunogenic in a number hepatica GSTs have now been isolated with of host/ parasite systems (Lee, 1970; a view to testing a recombinant vaccine Edwards, Burt and Ogilvie, 1971; Jones and (Wijffels, Sexton and Salvatore, 1992). In Ogilvie, 1972; Ogilvie et al., 1973; McKeand contrast to these trematode systems, et al., in press). For example, sheep infected antisera raised against GST purified from with T. colubriformis produce antibodies to adult H. con tortus, while inhibiting enzyme worm AChE; however, AChE purified activity in vitro, failed to affect survival of from this parasite failed to induce a the parasite in vivo (Sharp et al., 1991). protective immune response in vaccinated Proteinases are secreted by L. cuprina guinea pigs (Rothwell and Merritt, 1975). larvae on to the sheep's skin to enable the In the D. viviparus system, calves naturally larvae to feed and it is these enzymes that or experimentally infected with this are thought to initiate wound formation. It parasite also produce antibodies which has been observed that proteinases re- bind and inhibit parasite AChE activity leased by these flies can be inhibited by (McKeand et al., in press) and D. viviparus sheep plasma proteinase inhibitors ES preparations enriched for AChE activity and that the proteinase inhibitors protected guinea pigs against subsequent u2-macroglobulin and antithrombin III challenge compared with adjuvant controls reduced larval growth in vitro (Bowles, (McKeand, 1992). Feehan and Sandeman, 1990). Further- Glutathione S-transferase (GST) appears more, antiproteinase antibodies inhibited to be one of the major detoxification larval growth in vitro (Sandeman, 1990). In enzymes of parasitic helminths and has common with many helminth parasites, 100 Multicellular parasite vaccines

blowfly larvae produce a multiplicity of in the guinea pig when given in native secreted proteinases, each of which may form (O'Donnell et al., 1989). Using need to be inhibited by the host immune oligonucleotide probes based on partial responsetoimpair larval growth amino acid sequence data of tropomyosin, adequately (East and Eisemann, 1993). the gene encoding this protein was isolated Surface antigens of parasitic nematodes from a cDNA expression library prepared are often highly antigenic (Maizels and from fourth larval-stage mRNA (Cobon et Selkirk, 1988). Cetyltrimethylammonium al.,1989). A 27 kDa subunitof bromide (CTAB) surface extracts of T. colubriformis tropomyosin, expressed as T. spiralis larvae induced significant levels a P-galactosidase fusion protein in of protection against reinfection in mice Escherichia coli, produced accelerated worm (Grencis et al., 1986). However, sheep expulsion following challenge in guinea immunized with surface extracts of pigs. When the T. colubriformis tropom- H. contortus L3 were as susceptible as yosin DNA was used as a hybridization challenge control animals (Turnbull et al., probe, the gene encoding a related antigen 1992) and cuticle collagens from the third was isolated from mRNA prepared from and fourth stages of the same parasite adult H. con tortus. The expressed gene failed to induce significant protection product significantly protected immunized against homologous challenge (Boisvenue sheep against H. contortus challenge et al., 1991). (Cobon et al., 1989).

MULTICELLULAR PARASITE VACCINES: Immunization of sheep with recombinants RECENT DEVELOPMENTS of T. ovis onchosphere ES antigens. A The isolation of protective antigens in similar approach has shown promise in sufficient quantities for practical use in experiments with Cysticercus avis, the vaccine development has, until recently, intermediate stage of T. avis, in sheep. been restricted by a lack of available native Previous experiments indicated that the parasite material. The development of ES products of hatched and activated recombinant DNA techniques for the in T. avis onchospheres contained potent vitro expression of foreign genes in host-protective antigens. Several of the ES eukaryotic and prokaryotic cells has components, ranging from 47 to 52 kDa in provided an alternative strategy which has size, were found to be recognized strongly recently stimulated extensive research into by sera from resistant sheep (Rickard and antiparasite vaccines. While there are now Bell, 1971a and 1971b; Rickard and Adolph, many examples of expression systems for 1977) and lambs immunized witha parasite proteins, there are only a few polyacrylamide gel-purified preparation of reports of recombinant parasite proteins these components were significantly (98 which induce significant levels ofpro- percent) protected against challenge tective immunity. The steps in recombinant (Johnson et al., 1989). Rabbit antibodies, vaccine development are illustrated by the specific for the 47 to 52 kDa region,were following successful examples. eluted from Western blots and used to probe a cDNA expression library prepared Immunization of sheep using T. colubri- from rnRNA extracted from hatched and formis tropomyosin recombinants. As activated onchospheres (Johnson et al., discussed earlier, the T. colubriformis 1989). P-galactosidase fusion proteins, muscle protein, tropomyosin,was shown prepared from selected immunopositives, to be a potentially useful protective antigen were found to be antigenic in sheep but Vaccine manual 101

were not protective. Subsequently, one of The above examples have attracted the proteins was prepared as a fusion with considerable commercial support and are S. japonicum glutathione S-transferase and currently undergoing development. was found to protect sheep significantly against challenge (Johnson et al., 1989). PROBLEMS OF SUBUNIT VACCINE Recombinant proteins have also been DEVELOPMENT assessed in other Taenia species. Three Laboratory models versus natural host/ fusion proteins, derived from T. taeniae- parasite systems. Potentially useful anti- formis onchosphere cDNA and expressed gens are often selected on the basis of in a pGEX plasmid vector, gave a 95 protection trials conducted in laboratory percent reduction in total metacestode animal models. In general terms, lab- recoveries in Wistar rats (Ito et al., 1991) oratory animals expel the parasite more rapidly than does the natural host and Immunization of ruminants using re- infection is often terminated prior to combinant forms of membrane gut proteins patency. Protection observed in model 0f B. microplus. As discussed previously, systems is therefore often only an accelera- in infections in which immunity develops tion of an already efficient antiparasite slowly or poorly, artificial immunization response and does not necessarily reflect with hidden, or covert, antigens may be utility in the natural host. Caution must used to circumvent a lack of responsive- therefore be taken when extrapolating ness. Recent developments using re- from the laboratory model to the definitive combinant gut antigens from the blood- host. sucking tick B. microplus in cattle have been promising. Recombinants expressed in the wrong Cattle acquire limited levels of resistance conformation. The protective properties of following prolonged natural exposure to a recombinant protein will be influenced B. microplus (Wagland, 1975). Protective by the tertiary structure which can, in turn, immunity has been achieved using anti- be altered depending on the fusion partner. gens fractionated from gut membrane It also depends on whether or not the preparations of engorged female ticks native protein is glycosylated. This caution- (Willadsen, McKenna and Riding, 1988), ary note is exemplified by work on the and a membrane-bound glycoprotein protective onchosphere antigens of T. ovis (Bm86) expressed on the surface of tick in which protective immunity was only gut digestive cells was shown to be a highly attained after expression of the recombin- effective immunogen (Willadsen et al., ant molecule as a GST fusion protein 1989). The gene coding for this antigen (Johnson et al., 1989). was subsequently isolated from a cDNA expression library prepared from adult Solubilization of the relevant antigens. The B. micro plus and a fusion comprising 599 solvent used to extract parasite proteins amino acids of Bm86 and 651 amino acids can be crucial to the isolation of the of P-galactosidase was expressed in E. coli appropriate protective antigens. For as inclusion bodies (Rand et al., 1989). Ticks example, work performed on the cestode engorging on cattle vaccinated with these Taenia pisiformis indicated that sodium inclusion bodies were significantly deoxycholate, but not PBS, extracts made damaged as a result of the immune from onchospheres of the parasite confer- response to the cloned antigen (Rand et al., red high levels of protection to rabbits 1989). (Rajasekariah, Rickard and O'Donnell, 102 Multicellular parasite vaccines

1985).Similarly, DOC-PAGE, but not SDS- glycosylate the recombinant protein such PAGE, gel cut-outs of T. taeniaeformis as mammalian cell lines and viral vectors. onchosphere antigens protected mice against challenge (Lightowlers, Rickard Accessibility of antigen to the immune and Mitchell,1984).In comparison, sheep system. In order to have an effect on an have been successfully immunized against invading parasite, host effector mechan- challenge with Echinococcus gran ulosus isms must be directed against antigens using onchosphere antigens which had which are accessible to this response. For been solubilized in SDS (Dempster et al., example, Haemonchus spp. and Boophilus 1992)and against T. ovis using homogen- spp. ingest host blood so that antigens on ized SDS-polyacrylamide gels containing the gut surface are bathed in blood homologous antigen (Harrison et al.,1993). components such as antibody. The extent The variation produced by the different to which this approach can be applied to extraction buffers may be caused by the non-bloodsucking parasites such as disruption of conformational epitopes or Ostertagia spp. and Trichostrongylus spp. by the lack of solubilization of effective remains to be defined. Furthermore, components. because covert antigens will not be exposed during subsequent natural infection it is Presence of non-peptide epitopes. B cell presumed that booster vaccination would epitopes are often conformational and may be required until natural immunity not be peptide in nature. These epitopes develops or live vaccine vectors may be will be difficult to reproduce using used to enable persistence of the vaccine conventional molecular biology or peptide components within the host. synthesis techniques. As the antigen binding site of an antibody molecule is Genetic variation in the vaccinated complementary in physical structure to the population. Once a vaccine has been antigen against which it is raised, the developed, it is essential that it is effective antigen binding site can, itself, be used as in most, if not all, of the target population. antigen to raise anti-idiotype antibodies The major histocompatibility complex which mimic the shape of the original (MHC) and other, as yet undefined, antigen. For example, an anti-idiotype background genes within a population vaccine which mimics the glycan com- influence the capability of individuals to ponent of a38kDa surface component of respond immunologically to specific S. mansoni stimulated protective immunity antigens. This in turn will establish the in rats against homologous challenge success or failure of any vaccine (Kennedy, (Grzych et al., 1985). This approach isvery 1990). For example, a variable responsive- laborious, however, and would probably ness was encountered when synthetic only be considered if the protective epitope peptides of the circumsporozoite protein was non-peptide or demonstrably confor- of Plasmodium falciparum were used to mational and could not be produced by vaccinate mice and it was observed that the variety of recombinant DNAap- both antibody and T cellresponses to this proaches now available. In addition to antigen were H-2b-restricted (Del Giudice containing carbohydrate epitopes, glyco- et al., 1986; Togna et al., 1986). Thus, itcan sylation is also likely to affect the protective be anticipated that subunit vaccines which properties of proteins by modifying protein stimulate elements of the natural immune folding, so that recombinants will bemore response to the parasite will have to appropriately expressed in systems which comprise a variety of peptides to maintain Vaccine manual 103

long-term field utility. Furthermore, the vaccines. However, because of their induction of natural immune responses general efficacy, they continue to be used will be profoundly influenced by the way in experimental trials to establish antigen the antigen(s) is / are presented to the utility, after which less toxic adjuvant immune system. mixes may be attempted. A novel adjuvant which is now licensed Assisting antigen presentation for use in domestic animals is that The rapid developments in recombinant based on imrnunostimulating complexes DNA technology and protein chemistry (ISCOMs). These complexes are most should provide antigens in quantity for readily formed with antigens which experimental immunization studies. Indi- possess a hydrophobic transrnembrane vidual molecules are likely to have reduced region (Bomford, 1989), suggesting that immunogenicity and will have to be they may be particularly useful for the presented to the host immune system in formulation of helminth vaccines based on the context of an adjuvant or a live vaccine integral membrane proteins. So far, there vector. Moreover, the method of antigen has been little work detailing the potential presentation can be optimized if the of helminth or ectoparasite subunit vac- relevant effector arms of the immune cines in the context of any novel adjuvant response are defined. preparations.

Adjuvants Vectors and antigen delivery Adjuvants non-specifically stimulate The chronicity associated with many immune responses to antigen. The under- parasitic infections suggests that antigen standing of how adjuvants work has may need to be administered over a increased rapidly and many of the adju- prolonged period to stimulate protective vants now available are recognized to immunity. Antigens administered orally stimulate different types of responses (see with an avirulent virus or bacterial vector chapter by Bomford, Adjuvants in vet- will have the potential to stimulate a erinary vaccines, p. 277, and Bomford, vigorous cell-mediated and persistent 1989). immune response (Murray, 1989). These Adjuvant selection, and hence vaccine vectors also have the potential to stimulate efficacy, can be improved if the precise IgA precursor B cells in gut-associated immunological mechanisms involved in lymphoid tissue, a possibility for over- parasite killing or expulsion are defined. coming immune unresponsiveness at this For example, al-uminium hydroxide pre- site. Virus vectors, such as vaccinia and ferentially stimulates T helper type 2 (Th2) herpesviruses, have the advantage that the lymphocytes so that a humoral response, antigen(s) are processed in their native particularly IgG, and IgE, is favoured form (Murray, 1989). So far, vaccinia has (Smith, 1992). In contrast, Freund's com- been used successfully as a vector for a plete adjuvant, which contains bacterial cloned 28 kDa surface antigen of S. mansoni components, is a strong promoter of (Simpson and Cioli, 1987). Enterobacteria, cellular responses. Freund's incomplete such as E. coli and Salmonella spp., have adjuvant, which lacks the mycobacterial been used as vaccine vectors in non- component, stimulates only a humoral parasite systems, although use of the latter response. Unfortunately, Freund's adju- may lead to public concern. vants have several adverse side-effects and Vectors may be designed so that the are not acceptable for use in veterinary antigens they encode are expressed in 104 Multicellular parasite vaccines

combination withtheappropriate cellular arm of the immune response. In recombinant cytokine(s) in order to terms of vaccine development, this reflects stimulate the desired immune effector arm. the route that research has taken in the For gastrointestinal nematodes, antigen past. More recently, detailed analyses of and cytokine genes could be expressed in the cellular responses against helminths parallel in a bacterial or viral vector have identified responses which are suitable for oral vaccine delivery. protective or those which may be immuno- Bacterial toxins also have considerable pathological or immunorepressive (Smith, potential for the oral presentation of 1992). By measuring mitogen- or antigen- antigen. For example, cholera toxin has a specific in vitro propagation of immune high affinity for specific receptors on the cell lines, including T cell subsets, or surface of the intestinal epithelium and measuring the types of cytokines released stimulates both secretory IgA and serum or expresssed by these cells, cellular IgG antibody responses with prolonged responses can be dissected for each parasite immunological memory (McGhee et al., infection. Parasite antigens also trigger 1992). These responses would be appro- mast cell degranulation with the associated priate in vaccination against gastrointe- release of mast cell proteinases, histamine stinal nematodes, although the problems and various cytokines. In several cases, of toxicity may have to be overcome. these responses appear to be central to worm expulsion, and assays which detect CONCLUSIONS antigen-specific mast cell proteinase The optimism express.ed when recornbin- release may be used to identify which ant DNA technology was first developed antigens stimlate these cells (Jones, perhaps overestimated the capabilities of Huntley and Emery, 1992). Thus, it can be these techniques and failed to recognize seen that basic parasite immunology the complex nature of the host-parasite should go hand in hand with vaccine interaction and the difficulty in maintain- research in order to define which kind of ing immunogenicity. A major problem has responses should be aimed for when been the inability to identify the relevant identifying vaccine candidates. antigens from complex preparations in the As outlined above, delivery systems and face of multiple immune responses, not all adjuvants can now be selected on the basis of which are protective. These responses that they can optimize or augment the still have to be unravelled for all multi- required effector arm of the immune cellular parasitic infections. A substantial system. Nevertheless, by obtaining in- amount of parasitological research is now formation on putative functions for specific directed at defining the precise immune parasite components or realizing their responses relevant to the elimination, or contribution to parasite survival, rational otherwise, of parasites. Although beyond subunit vaccines may be designed without the scope of this vaccine review, it appears the need for a full understanding of the that, of the many immune effector mecha- host parasite interaction. nisms involved in the host-parasite inter- Pioneering advances have been made in action, only some are relevant to the the last decade, most notably in the development of protective immunity. definition of candidate antigens for Nearly all the studies described in this protection against T. ovis and H. con tortus review selected antigens on the basis of in sheep, but the identification of protective humoral immunity, and there has been antigens and evidence for their efficacy in little detail given regarding the essential immunization trials is only the start of the Vaccine manual 105

process. Field trials and the incorporation omford, R. 1989. Adjuvants for antiparasite of the antigens into a commercially viable vaccines. Parasitol. Today, 5: 41-46. vaccine are expensive and time-consuming Bowles, V.M., Feehan, J.P. & Sandeman, and will rely heavily on commitment from R.M. 1990. Sheep plasma proteinase large pharmaceutical companies which inhibitors influencing activity and must accept the vaccine as a potential growth of Lucilia cuprina larvae in vitro. marketable commodity. It should be noted Int. J. Parasitol., 20: 169-174. that a vaccine which only reduces nema- Brophy, P.M. & Barrett, J. 1990. Glutathione tode egg output might reduce pasture transferase in helminths. Parasitol., 100: contamination to a level where production 345-349. losses are balanced by stock management Brossard, M. 1976. Relations immunologiques savings. Thus, at least with veterinary entre bovins et tiques: Boophilus micro plus. vaccines, it may not be imperative to Acta Trop., 33: 15-36. induce sterile immunity completely and, Bushara, H.O., Hussein, M.F., Saad, A.M., when considering the most appropriate Taylor, M.G., Dargie, J.D., De. C. vaccine development strategy, full account Marshall, T.F. & Nelson, G.S. 1978. must be taken of the relative prevalence Immunizationofcalvesagainst and epidemiology of the parasite. Schistosoma bovis using irradiated cercariae or schistosomula of S. bovis. BIBLIOGRAPHY Parasitol., 77: 303-311. Chandler, A.C. 1932. Experiments on Adams, D.B. 1989. A preliminary evaluation resistance of rats to superinfection with of factors affecting an experimental the nematode Nippostrongylus muris. system for vaccination-and-challenge Am. J. Hyg., 16: 780-782. with Haemonchus contortus in sheep. Int. Chapman, C.B. & Mitchell, G.F. 1982. J. Parasitol., 19: 169-175. Proteolytic cleavage of immunoglobulin Arundel, J.H. & Sutherland, A.K. 1988. by enzymes released by Fasciola hepatica. Blowflies of sheep. In Animal Health in Vet. Parasitol., 11: 165-178. Australia, 10: 35-60. Canberra, Australian Clegg, J.A. & Smith, M.A. 1978. Prospects Government Publishing Service. for the development of dead vaccines Auriault, C., Ouissi, M.A., Torpier, G., Elsen, against helminths. Adv. Parasitol., 16: H. & Capron, A. 1981. Proteolytic 165-217. cleavage of IgG bound to the Fc receptor Cobon, G.S., Austen, A., O'Donnell, I.J., of Schistosoma rnansoni schistosomula. Frenkel, M.J., Kennedy, W.P., Savin, Parasite Immunol., 3: 33-44. K.W. & Wagland, 7.M. 1989. Vaccines Balloul, J.M., Grzych, J.M., Pierce, R.J. & against animal parasitic nematodes. Capron, A. 1987. A purified 28 000 dalton Patent: W089 / 00163. protein from Schistosoma mansoni adult Dalton, J.P. & Heffernan, M. 1988. Thiol worms protects rats and mice against proteases released in vitro by Fasciola experimentalschistosomiasis. J. hepatica. Mol. Biochem. Parasitol., 35: 161- Immunol., 138: 3448-3453. 166. Boisvenue, R.J., Stiff, M.I., Tonkinson, L.V. Del Giudice, G., Cooper, J.A., Merino, J., & Cox, G.N. 1991. Protective studies Verdini, A.S., Pessi, A., Togna, A.R., in sheep immunized with cuticular Engers, H.D., Corradin, G. & Lambert, collagen proteins and peptides of P.-H. 1986. The antibody response in Haemonchus contortus. Parasite Immunol., mice to carrier-free synthetic polymers 13: 227-240. of Plasmodium falciparum circum- 106 Multicellular parasite vaccines

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proteolytic anticoagulant by Ancylostoma mucosal mast cells from the small hookworms. J. Exp. Med., 157: 1594-1603. intestine of parasitised sheep. Int.J. Howell, M.J., Board, P.G. & Boray, J.C. Parasitol., 22: 519-521. 1988. Glutathione S-transferases. J. Kanner, M. & Austen, F.K. 1975. Immunol- Parasitol., 74: 715-718. ogic release of chemical mediators from Ito, A., Bogh, H.O., Lightowlers, M.W., human tissues. Annu. Rev. Pharmacol., Mitchell, G.F., Takami, T., Kamiya, M., 15: 177-189. Onitake, K. & Rickard, M.D. 1991. Keith, R.K. & Bremner, K.C. 1973. Im- Vaccination against Taenia taeniaeformis munisation of calves against the nodular infection in rats using a recombinant worm Oesophagostomum radia tum. Res. protein and preliminary analysis of the Vet. Sci., 15: 23-24. induced antibody response. Mol. Biochem. Kemp, D.H., Agbebe, R.I.S., Johnston, L.A.Y. Parasitol., 44: 43-52. & Gough, J.M. 1986. Immunisation of Jackson, F. 1993. Anthelmintic resistance- cattle against Boophilus micro plus using the state of play. Br. Vet. J., 149: 123-138. extracts derived from adult female ticks: Jackson, F., Coop, R.L., Jackson, E., Little., feeding and survival of the parasite on S. & Russell, A.J.F. 1991. Anthelmintic- vaccinated cattle. Int. J. Parasitol., 16: resistant nematodes in goats. Vet. Rec., 115-120. 129: 39-40. Kennedy, M.W. 1990. Resistance to parasitic Jarrett, W.F.H., M cIntyre, W.I.M. & nematodes - how is the MHC involved? Urquhart, G.M. 1954. Husk in cattle. A Parasitol. Today, 6: 374-375. review of a year's work. Vet. Rec., 66: 7:I-Lox, D.P. & Jones, D.G. 1992. A comparison 665-676. ofsuperoxidedismutase(SOD, Jarrett, W.F.H., Jennings, F.W., Martin, B., EC:1.15.1.1) distribution in gastro- McIntyre, W.I.M., Mulligan, W., Sharp, intestinal nematodes. In t.J. Parasitol., N.C.C. & Urquhart, G.M. 1958. A field 22: 209-214. trial of a parasitic bronchitis vaccine. Lee, D.L. 1970. The fine structure of the Vet. Rec., 70: 451-454. excretory system in adult Nippostrongylus Johnson, K.S., Harrison, G.B.L., Lightowlers, brasiliensis (Nernatoda) and a suggested M.W., O'Hoy, K.L., Cougle, W.G., function for the "excretory glands". Dempster, W.G., Lawrence, R.P., Tissue Cell, 2: 225-231. Vinton, J.G., Heath, D.D. & Rickard, Lightowlers, M.W. & Rickard, M.D. 1988. M.D. 1989. Vaccination against ovine Excretory-secretory products of helminth cysticercosis using a defined recombinant parasites: effects on host immune antigen. Nature, 338: 585-587. responses. Parasitol., 6: S123-S166. Johnston, L.A.Y., Kemp, D.H. & Pearson, Lightowlers, M.W., Rickard, M.D. & R.D. 1986. Immunisation of cattle against Mitchell, G.F. 1984. Immunization Boophilus micro plus using extracts derived against Taenia taeniaeformis in mice: from adult female ticks. Int. J. Parasitol., identification of onchospheral antigens 16: 27-35. on polyacrylamide gels by Western Jones, V.E. & Ogilvie, f.M. 1972. Protective blotting and enzyme immunoassay. Int. immunity to Nippostrongylus brasiliensis J. Parasitol., 4: 297-306. in the rat. III. Modulation of worm Maizels, R.M. & Selkirk, M.E. 1988. Immuno- acetylcholinesterase by antibodies. biology of nematode antigens. In P.T. Immunology, 22: 119-129. Englund & A. Sher, eds. I: Biology of Jones, W.O., Huntley, J.F. & Emery, D.L. parasitism, p. 285-308. New York, A.R. 1992. Isolation and degranulation of Liss. 108 Multicellular parasite vaccines

Matthews, B.E. 1982. Skin penetration by with antigens isolated by affinity Necator americanus larvae. Z. Parasitenk., chromatography from adult worms. 68: 81-86. Immunol. Cell Biol., 65: 223-230. McGhee, J.R., Mestecky, J., Dertxbaugh, Munn, E.A. 1988. Production and use of M.T., Eldridge, J.H., Hirasawa, M. & anthelmintic agents and protective Kiyono, FI. 1992. The mucosal immune proteins. Patent: W088 / 00835. system: some fundamental concepts to Munn, E.A. & Smith, T.S. 1990. Production vaccine development. Vaccine, 10: 75-89. and use of anthelmintic agents and McGillivery, D.J., Yong, W.K., Riffkin, G.G. protective proteins. Patent: W090 / 11086. & Adler, B. 1990. The distribution and Munn, E.A., Greenwood, C.A. & Coadwell, localisation of the stage-specific GP31 W.J. 1987. Vaccination of young lambs antigen from infectiveOstertagia by means of a protein fraction extracted circumcincta larvae. Int. J. Parasitol., 20: from adult Haemonchus contortus. 87-93. Parasitol., 94: 385-397. McGillivery, D.J., Yong, W.K., Adler, B. & Munn, E.A., Smith, T.S., Graham, M., Riffkin, G.G. 1992. A purified stage- Greenwood, C.A., Tavernor, A.S. & specific 31 kDa antigen as a potential Coetzee, G. 1993a. Vaccination of merino protective antigen against Ostertagia lambs against haemonchosis with circumcincta infection in lambs. Vaccine, membrane-associated proteins from the 10: 607-613. adult parasite. Parasitol., 106: 63-66. McGowan, M.J., Barker, R.W., Homer, J.T., Munn, E.A., Graham, M., Smith, T.S., McNew, R. & Holscher, K.H. 1981. Newton, S., Knox, D.P., Oliver, J. & Success of ticks feeding on calves Smith, F. 1993b. Cloning and iden- immunised with Ambylomma americanum tification of H11, a highly protective extract. J. Med. Entomol., 18: 328-332. antigen from Haemonchus contortus. Proc. McKeand, J.B.1992. Aspects of the Keystone Symp. Molecular Parasitology, immunobiology of Dictyocaulus viviparus Colorado, USA. infection. University of Glasgow, UK. Murray, P.K. 1989. Molecular vaccines against (Ph.D. thesis) animal parasites. Vaccine, 7: 291-300. McKeand, J.B., Knox, D.P., Duncan, J.L. & Neilson, J.T.M. 1975. Failure to vaccinate Kennedy, M.W. 1995.Protective lambs against Haemonchus contortus with immunisation of guinea pigs against functional metabolic antigens identified Dictyocaulus viviparus using excretory- by immunoelectrophoresis. Int.J. secretory products of adult parasites. Parasitol., 5: 427-430. Int. J. Parasitol., 25(1): 95-104. Neilson, J.T.M. & van de Walle, M.J. 1987. McKeand, J.., Knox, D.P., Duncan, J.L. & Partial protection of lambs against Kennedy, M.W. The immunogenicity Haemonchus con tortus by vaccination with of the cattle lungworm, Dictyocaulus a fractionated preparation from the viviparus. Int. J. Parasitol. (in press) parasite. Vet. Parasitol., 23: 211-221. Miller, T.A. 1971. Vaccination against the O'Donnell, I.J., Green, P.E., Connell, J.A. canine hookworm diseases.Adv. & Hopkins, P.S. 1980. Immunoglobulin Parasitol., 9: 153-183. G antibodies to the antigens of Lucilia Miller, T.A. 1978. Industrial development cuprina in sera of fly-struck sheep. A us t. and field use of the canine hookworm J. Biol. Sci., 33: 27-34. vaccine. Adv. Parasitol., 16: 333-342. O'Donnell, I.J., Green, P.E., Connell, J.A. Monroy, F.G. & Dobson, C. 1987. Mice & Hopkins, P.S. 1981. Immunisation vaccinated against Nematospiroides dubius of sheep with larval antigens of Lucilia Vaccine manual 109

cuprina. Aust. J. Biol. Sci., 34: 411-417. Rand, K.N., Moore, T., Sriskantha, A., O'Donnell, I.J., Dineen, J.K., Rothwell, Spring, K., Tellam, R., Willadsen, P. T.L.W. & Marshall, R.C. 1985. Attempts & Cobon, G.S. 1989. Cloning and to probe the antigens and protective expression of a protective antigen from immunogens ofTrichostrongylus the cattle tick Boophilus microplus. Proc. colubriformis in immunoblots with sera Natl. Acad. Sci., 86: 9657-9661. from infected and hyperimrnune sheep Rhoads, M.L. 1983. Trichinella spiralis: and high and low responder guinea pigs. identification and purification of Int. J. Parasitol., 15: 129-136. superoxide dismutase. Exp. Parasitol., O'Donnell, I.J., Dineen, j.K., Wagland, B.M., 56: 41-54. Letho, S., Dopheide, T.A.A., Grant, W.N. Rhoads, M.L. 1984. Secretory cholinesterases & Ward, C.W. 1989. Characterization of nematodes: possible functions in the of the major immunogen in the excretory- host-parasite relationship. Trop. Vet., 2: secretory products of exsheathed 3-10. third-stage larvae of Trichostrongylus cola- Rickard, M.D. & Adolph, A.J. 1977. briformis. Int. J. Parasitol., 19: 793-802. Vaccination of lambs against infection Ogilvie, B.M., Rothwell, T.L.W., Bremner, with Taenia ovis using antigens collected K.C., Schnitzerling, H.J., Nolan, J. & during short-term in vitro incubation Keith, R.K. 1973. Acetylcholinesterase of activated oncospheres. Parasitol., 75: secretion by parasitic nematodes. I. 183-188. Evidence of secretion by a number of Rickard, M.D. & Bell, K.J. 1971a. Induction species. Int. J. Parasitol., 3: 589-597. of immunity in lambs to larval cestode Opdebeeck, J.P., Wong, J.Y.M., Jackson, antigens. Nature, 232: 120. L.A. & Dob son, C. 1988. Hereford cattle Rickard, M.D. & Bell, K.J. 1971b. Successful immunised and protected against vaccination of lambs against infection Boophilus micro plus with soluble and with Taenia ovis using antigens produced membrane-associated antigens from the during in vitro cultivation of the larval midgut of ticks. Parasite Immunol., 10: stages. Res. Vet. Sci., 12: 401-402. 405-410. Rickard, M.D. & Williams, J.F. 1982. Ozerol, N.H. & Silverman, P.H. 1970. Further Hydatidosis/ cysticercosis: immune characterisation of active metabolites mechanisms and immunisation against from histotrophic larvae of Haemonchus infection. Adv. Parasitol., 21: 230-296. contortus cultured in vitro. J. Parasitol., Roberts, F.H.S., Elek, P. & Keith, R.K. 1962. 56: 1199-1205. Studies of resistance in calves to Poynter, D.A. & Cauthen, G.E. 1942. experimental infections with the nodular Experiments on the life history of the worm Oesophagostomum radiatum. Aust. cattle lungworm, Dictyocaulus viviparus. J. Agric. Res., 13: 551-573. Am. J. Vet. Res., 3: 395-400. Rothwell, T.L.W. 1978. Vaccination against Prichard, R.K. 1990. Anthelmintic resistance the nematode Trichostrongylus colubri- in nematodes, recent understanding and formis. III. Some observations on factors future directions for research. Int.J. influencing immunity to infection in Parasitol., 20: 515-523. vaccinated guinea pigs. Int. J. Parasitol., Rajasekariah, G.R., Rickard, M.D. & 8: 33-37. O'Donnell, I.J. 1985. Taenia pisiformis: Rothwell, T.L.W. & Griffiths, D.A. 1977. protective immunisation of rabbits with Comparison of the kinetics of expulsion solubilised oncosheral antigens. Exp. of Trichostrongylus colubriformis from Parasitol., 59: 321-327. previously uninfected, reinfected and 110 Mititicellular parasite vaccines

vaccinated guinea pigs. J. Parasitol., 63: Antigens from Trichinella spiralis that 761-762. induce a protective response in the ReiTawell, T.L.W. & Love, R.J. 1974. mouse. J. Immunol., 132: 898-904. Vaccination against the nematode Tricho- Simpson, A.J.G. & Cioli, D. 1987. Progress strongylus colubriformis. I. Vaccination towards a defined vaccine for schisto- of guinea pigs with worm homogenates somiasis. Paras itol. Today, 3: 24-26. and soluble products released during Simpson, A.J.G., Hackett, F., Walker, T., in vitro maintenance. Int. J. Parasitol., 4: De Rossi, R. & Smithers, S.R. 1985. 293-299. Antibody response against schisto- Rothwell, T.W.L. & Merritt, G.C. 1975. somulum surface antigens and protective Vaccination against the nematode Tricho- immunity following immunization with strongylus colubriformis. II. Attempts to highly irradiated cercariae of Schistosoma protect guinea pigs with worm acetyl- mansoni. Parasite Immunol., 7: 133-152. cholinesterase. Int. J. Parasitol., 5: 453-460. Skelly, P.J. & Howells, A.J. 1987. The Sandeman, R.M. 1990. Prospects for the humoral immune reponse of sheep to control of sheep blowfly strike by antigens from larvae of the sheep blowfly vaccination. Int. J. Parasitol., 20: 537-541. (Lucilia cuprina). Int.J.Parasitol., 17: Sanes, M.P. & Taliaferro, W.A. 1936. The 1081-1087. local points of defence and the passive Smith, N.C. 1992. Concepts and strategies transfer of acquired immunity to Nippo- for anti-parasite immunoprophylaxis and strongylus brasiliensis in rats. J. Inf. Dis., therapy. Int. J. Parasitol., 22: 1047-1082. 59: 207-220. Smith, W.D. 1993. Protection in lambs Savin, K.W., Dopheide, T.A.A., Frenkel, immunised with Haemonchus contortus M.J., Wagland, B.M., Grant, W.A. & gut membrane proteins. Res. Vet. Sci., Ward, C.W. 1990. Characterization, 54: 94-101. cloning and host-protective activity of Smith, T.S. & Munn, E.A. 1990. Strategies a 30-kilodalton glycoprotein secreted for vaccination against gastro-intestinal by the parasitic stages of Trichostrongylus nematodes. Rev. sci. tech. Off. int. Epiz., colubriformis. Mol. Biochem. Parasitol., 41: 9: 577-595. 167-176. Smith, D.B., Davern, K.M., Board, P.G., Schlein, Y. & Lewis, C.T. 1976. Lesions in Tiu, W., Garcia, E.G. & Mitchell, G.F. haematophagous flies after feeding on 1986. Mr 26000 Da antigen of Schistosoma rabbits immunised with fly tissues. japonicum recognised by WEH1 129/J Physiol. Entomol., 1: 55-59. mice is a gluathione S-transferase. Proc. Sexton, J.L., Milner, A.R. & Paraccio, M. Natl Acad. Sci., 83: 8703-8707. 1990. Glutathione S-transferase. Novel Smith, A.M., Dowd, A.J., McGonigle, S., vaccine against Fasciola hepatica infection ftCeegan, P.S., Brennan, G., Trudgett, in sheep. J. Immunol., 145: 3905-3910. J.P. 1993. Purification of a Sharma, R.L., Bhat, T.K. & Dhar, D.N. 1988. cathep sin L-like proteinase secreted by Control of sheep lungworm in India. adult Fasciola hepatica. Mol. Biochem. Paras itol. Today, 4: 33-36. Parasitol., 62: 1-8. Sharp, P.J., Smith, D.R.T., Bach, W., Stromberg, B.E. & Soulsby, E.J.L. 1977. Wagland, B.M. & Cobon, G.S. 1991. Ascaris suum: immunization with soluble Purified glutathione S-transferases from antigens in the guinea pig. Int. J. Parasitol., parasitesas candidate protective 7: 287-291. antigens. Int. J. Parasitol., 21: 839-846. Tavernor, A.S., Smith, T.S., Langford, C.F. Silberstein, D.S. & Despommier, D.D. 1984. Munn, E.A. & Graham, M. 1992a. Vaccine manual 111

Vaccination of Dorset lambs against Aust. J. Agric. Res., 26: 1073-1080. haemonchosis. Parasite Immunol., 14: Waller, P.J. & Larsen, M. 1993. The role of 645-655. nematophagous fungi in the biological Tavernor, A.S., Smith, T.S., Langford, C.F., control of nematode parasites of Munn, E.A. & Graham, M. 1992b. livestock. Int. J. Parasitol., 23: 539-546. Immune reponse of Clun Forest to Wharton, R.H. 1976. Tick-borne livestock vaccination with membrane glyco- diseases and their vectors, 5. Acaricide proteins from Haemonchus contortus. resistance and alternative methods of Parasite Immunol., 14: 671-675. tick control. World Anim. Rev., 20: 8-15. Togna, A.R., Del Giudice, G., Verdini, A.S., Wijffels, G.L., Sexton, J.L. & Salvatore, L. Bonelli, F., Pessi, A., Engers, H.D. & 1992. Primary sequence heterogeneity Corradin, G. 1986. Synthetic Plasmodium and tissue expression of glutathione S- falciparum circumsporozoite peptides transferases of Fasciola hepatica. Exp. elicit heterogeneous L3T4+ T cell proli- Parasitol., 74: 87-99. ferative responses in H-2b mice. J. Wikel, S.K. 1981. The induction of host Immunol., 137: 2956-2960. resistance to tick infestation with salivary Turnbull, I.F., Bowles, V.M., Wiltshire, C.J., gland antigen. Am. J. Trop. Med. Hyg., Brandon, M.R. & Els, N.T. 1992. Systemic 30: 284-288. immunization of sheep with surface Willadsen, P. 1980. Immunity to ticks. Adv. antigens from Haemonchus contortus Parasitol., 18: 293-313. larvae. Int. J. Parasitol., 22: 537-540. Willadsen, P. & McKenna, R.V. 1991. Urquhart, G.M., Jarrett, W.F.H., Jennings, Vaccination with concealed antigens: F.W., McIntyre, W.I.M. & Mulligan, myth or reality? Parasite Immunol., 13: W. 1966a. Immunity to Haemonchus 605-616. contortus infection: failure of x-irradiated Willadsen, P., McKenna, R.V. & Riding, larvae to immunize young lambs. Am. G.A. 1988. Isolation from the cattle tick, J. Vet. Res., 27: 1641-1643. Boophilus microplus, of antigenic material Urquhart, G.M., Jarrett, W.F.H., Jennings, capable of eliciting a protective immuno- F.W., McIntyre, W.I.M. & Mulligan, logical response in the bovine host. Int. W. 1966b. Immunity to Haemonchus J. Parasitol., 18: 183-189. con tortus infection: relationship between Willadsen, P., T.tiding, G.A., McKenna, R.V., age and successful vaccination with Kemp, D.H., Tellarn, R.L., Nielsen, J.N., irradiated larvae. Am. J. Vet. Res., 27: Lahnstein, J., Cobon, G.S. & Gough, 1645-1648. J.M. 1989. J. Immunol., 143: 1346-1351. van Wyck, J.A. & Malan, F.S. 1988. Resistance Xilinas, M.E., Papavasiliou, L.T. & Marselou- of field strains of Haemonchus contortus Kinti, 0. 1976. Active immunisation of to avermectin, closantel, rafoxanide and mice against Echinococcus granulosus. the benzimidazoles in South Africa. Vet. Hellenica Acta Microbiol., 21: 86-90. Rec., 123: 226-228. Vernes, A. 1976. Immunization of the mouse and guinea pig against Trichinella spiralis. In van den Bossche, ed. Biochemistry of parasites and host/parasite relationships, p. 319-324. Amsterdam, Elsevier /North Holland Biomedical. Wagland, B.M. 1975. Host resistance to the cattle tick (Boophilus micro plus) in cattle. Vaccine manual 113

Poultry vaccines (1) Convntonci vaccines J.B. McFerran

Vaccination of poultry presents a number immunosuppressive viruses, of which the of special problems. The first is the limited most important is infectious bursal disease life of the meat-producing chicken, which (ISD) virus, which has virtually a world- is often as short as 35 days from hatching wide distribution. These viruses have three to slaughter. These birds have a low important effects: they can cause disease individual value and any vaccine must in their own right; they can compromise therefore be relatively cheap. They are kept the birds' immunological response to other in very large numbers at a very high vaccines; and they can reduce the birds' density. Houses often contain 10 000 to resistance to other organisms so that a 30 000 birds and sites often have between severe reaction to vaccination with an 50 000 and one million birds. The problem attenuated vaccine can occur as a result of is compounded because these birds have other agents combining with the vaccine probably originated from eggs produced to induce disease. by a number of breeding farms. These The disadvantages of large-scale poultry breeding farms may well be in a different production can in part be overcome by region (or evert country) and may have a having an "all-in all-out" system, where a different antibody status and disease site is completely depopulated and is history. Therefore, the chickens hatching properly cleaned and disinfected before from these eggs will have considerable the next intake of day-old chicks. It is variations in maternal antibodies and, in relatively easy to clean and disinfect (e.g. some cases, chicks will have been vertically by formalin fumigation) environmentally infected with viruses such as chick anaemia controlled houses with impervious walls virus (CAV) or avian encephalomyelitis. and concrete floors but it is virtually This problem is becoming more acute as impossible to disinfect open-sided houses hygiene and disease precautions are with earth floors. In addition, commercial increased in the breeding farms, for pressures often lead to bad microbiological example to control Salmonella spp. If all the practices. It is not uncommon for organi- breeding farms are infected with a virus zations which do depopulate and clean such as CAV before they start producing houses to have large sites where the last eggs, it is not a problem. However, if some houses to be depopulated are still being are not infected, then two problems can cleaned when chicks are filling the first arise: the first is if the chicks are placed in houses. Even worse, in some areas litter is an area where CAV is endemic, in which reused many times and young chicks are case the susceptible chicks originating from brooded in houses still containing older parents without antibody become infected; birds. the second is when one of the parent flocks Most commercial poultry organizations becomes infected. This flock will produce are highly integrated with considerable infected and diseased chicks and they will movement between farms. Thus, laying infect the susceptible chicks in the hatd-teries. flocks will have regular egg collections, Poultry are infected with a number of with transport moving from farm to farm; 114 Conventional vaccines

breeders and broilers are often weighed salmonellosis), feed (e.g. paramyxovirus regularly by the organization's advisory type 1 [pigeon Newcastle disease], Salmo- staff; there is movement and contact by nella spp., IBD) and transport. catching and vaccination teams, etc. While Given the above considerations, it can a reasonable disinfection of staff can be be seen that the chances of poultry disease carried out, it is more difficult to ensure spreading are very high. In addition, the that tools and equipment (e.g. weighing poultry industry is truly international, with scales, electrician's tools, egg trolleys) are birds (and advisory staff) moving freely safe. These factors make the spread of between countries. The speed of the spread disease relatively easy. of "new" diseases in recent years under- From a disease control viewpoint there lines this. Diseases such as runting- are three main types of poultry: i) stunting, egg drop syndrome, turkey commercial flocks, usually kept under rhinotracheitis virus, virulent forms of intensive husbandry methods; ii) local Newcastle disease, Marek's disease and small backyard flocks, which in most areas IBD as well as antigenic variants of are unvaccinated; and iii) hobby birds. The infectious bronchitis (IB) have all spread latter range from Psittacines (especially rapidly around the world. The emergence dangerous because they can be infected of other new diseases such as chick with virulent Newcastle disease virus) to anaemia, may be the result of changes in racing pigeons. Wild birds also pose a management rather than the spread of problem as they are capable of carrying virus, but this is unclear at present. infection long distances and may indeed be hosts of certain viruses. For instance, VACCINATION wild ducks are often infected with Clearly, vaccination isessential to paramyxoviruses, including Newcastle commercial poultry production. However disease, myxoviruses and the egg drop with the birds' short life, high-density syndrome, adenovirus. conditions and variable antibody levels, it Humans are probably one of the most is very difficult to achieve full protection important vectors for spreading infection, and it is essential that every effort be made second only to the bird. It is possible to to prevent the introduction of infection or reduce the risk of introducing disease by carryover of infectious agents from flock keeping all but essential people out of to flock. To some extent, the degree of poultry houses and by ensuring that those resistance induced by any vaccine is subject who enter take precautions (e.g. by using to the level of challenge present and, if special outer clothing kept on the farm for that challenge is great enough, vaccines each essential visitor). are unlikely to give protection. It is unlikely that organisms will spread The ideal vaccine should: from hobby birds or backyard flocks by be able to be given by mass application; aerial routes because the number of birds .be cheap; involved is too small. Some virusescan be safe; spread from commercial farmto ° be capable of inducing a strong commercial farm by wind owing to the immunity even in the presence of large concentration of birds (e.g. Newcastle maternal antibody; disease and Marek's disease viruses). Other be capable of inducing both local and routes are also important, such as un- general immunity; treated water (e.g. spreading influenza, induce an immune response thatcan Newcastle disease, egg drop syndrome, be distinguished from that induced by Vaccine manual 115

infection or maternal derived antibody; flocks to be batched intogroups so that the require only one dose to give lifelong progeny will have similar titres and, con- immunity; sequently, the timing of vaccinationcan be *be packaged in different sizes (e.g. better judged. This has been used in the while 1 000-dose vials are appropriate control of IBD. The difficulty is that for use in commercial flocks, theyare there is often a wide variation in antibody not appropriate for use in a small titres in birds in any one flock. backyard flock). Such a vaccine does not exist at present. Vaccine administration Successful vaccination against many There are a number of commonly used organisms depends on stimulating both methods of vaccination. Meat-producing local immunity at a mucosal surface and chickens are normally handled twiceat general humoral antibody. The local birth and at death. Therefore, vaccination immunity is of major importance for those is either done individually in the hatchery agents which affect mucosal surfacesfor (e.g. Marek's vaccination) or else mass example IBand is of special importance application methods are used. The easiest in meat-producing birds which are housed method is to give the vaccine in the at very high densities, have a short life drinking-water, yet this is most difficult to span and require quick immunity. do properly. There is always a percentage of birds that will not drink. Although this Serology percentage can be reduced by ensuring the There are a wide range of tests used to birds are thirsty, they must not be stressed monitor vaccination and also for diagnosis. and the water must be turned on The modern tests, for example ELISA tests, immediately afterwards, with no danger are expensive and it is important to use of airlocks occurring. Furthermore, the them appropriately and not just to ac- vaccine may not be uniformly available in cumulate data. They can be used to the drinking-water because of physical monitor the efficiency of vaccination where problems such as uneven troughs, or immunity (general) is measured by an vaccine may be lost as a result of adherence antibody response. Titres should not only to rusty containers or feed which has fallen be high but, even more important, be in a into the drinkers. Vaccine can also be close range. It is important to remember inactivated by chemicals (e.g. chlorine) in that if 10 percent of the birds have low the water or by heat. Such problems can in titres or no antibody, this can be translated part be overcome by adding protein (e.g. into 2 000 to 3 000 birds dying in a house. dried skim mill< powder) to the vaccine There is no easy method of measuring local and by giving the vaccine in two parts immunity. This is unfortunate, since local (McFerran et al., 1972). It is also essential to immunity is often very important, as for keep the birds at a comfortable air example in IB. Other uses for serology are temperature. This may require raising the to determine if a flock has been infected. temperature in cold areas or vaccinating Thus, if birds in the rearing period develop early in the morning in hot regions. antibody to CAV, there is no need to Partly to overcome the problems of water vaccinate. As already discussed, it is the administration and partly to induce local flocks without antibody that pose the immunity, spraying has been widely used. problems. The use of a coarse spray (in reality a mass Determination of antibody titres in eye and nose drop application) to breeding flocks can allow the breeding immunize day-old chicks which possess 116 Conventional vaccines

maternally derived antibody against cing chicks are vaccinated at one day of diseases such as IB, has been routine in age with Marek's vaccine. This is a quick hatcheries in some areas for 25 years. This and cheap method of application. Break- has given excellent immunity, lasting the downs occur when the vaccine is deposited life of the meat-producing chicken. Even in the feathers rather than under the skin in the case of emergency vaccinations of in a quest for speed and when a blocked antibody-free chicks with Newcastle needle or bacterial contamination of the disease virus, excellent immunity was apparatus goes unnoticed. provided with acceptable side-effects Inactivated vaccines, often with an oil (McFerran, 1992). Severe side-effects did adjuvant, are widely used. They tend to be occur when the spray was too fine expensive to buy and deliver but have been and virus penetrated the lower respira- very successful in controlling disease in tory tract. Results similar to those given adults. While they are of value in pro- by coarse spray have been obtained using tecting chicks against disease through the labour-intensive method of beak maternally derived antibody, recent dipping. experience has shown (as in the case of Aerosols, generated using an electrically IBD) that virulent strains can break powered sprayer, or a fine spray from a through the maternal antibody protection hand operated pump have been used to and active immunity must therefore be revaccinate birds. In some instances, this induced. has been done every 12 weeks to ensure that virtual continuous local immunity is SOME CONVENTIONAL VACCINES maintained at mucosal surfaces. There are a wide variety of conventional One constraint on the use of spray vaccines available. Brief descriptions of vaccines in some areas is that other micro- some are given here, concentrating on organisms infecting the birds, such as unusual aspects or problems and potential Myco plasma spp. and Haemophilitis spp., can future developments. produce severe side-effects. Therefore, in certain organizations or regions it is Marek's disease inadvisable to use spray vaccination. Marek's disease has been controlled by In theory, the large cleft in the oral using type 1 attenuated vaccines (either palates of fowl and turkeys should allow artificiallyattenuatedornaturally orally administered vaccine to come in occurring strains) or the type 3 turkey contact with the upper respiratory tract herpesvirus (THV). In some areas, a type 2 and achieve the same results as spray naturally occurring attenuated strain was vaccination. However, in practice, spray necessary. In some genetic lines, if the birds vaccination is much more effective in are infected with both the serotype 2 and stimulating local immunity inthe the lymphoid leukosis virus early in life, respiratory tract as well as being less there is an enhancement of lymphoid labour-intensive. In part, the increased leukosis. In the 1980s, very virulent strains immunity following spray vaccination may of Marek's disease arose, and controlwas be the result of stimulation of the achieved using a mixture of vaccineeither Hardarian gland by the spray-applied types 2 and 3 or types 1 and 3. There vaccine. appears to be a true synergism between Injection (including wing-web stabbing) different strains, especially between is still widely used. Virtually allegg- serotypes 2 and 3, and polyvalent vaccines producing birds and many meat-produ- have been found to provide better Vaccine manual 117

protection against tumour development Infectious bursa! disease (Witteret al., 1984).However even these Both attenuated and inactivated vaccines vaccines have not been totally successful exist fro IBD. Attenuated vaccines have and preliminary results suggest that, in been divided into three broad categories order to ensure maximum resistance to based on virulence: i) virulent or "hot"; ii) Marek's disease, it may be necessary to intermediate; and iii) "mild" or avirulent. select a vaccine appropriate for the There is some discussion as to whether predominant B-haplotype of the chicken some of the "hot" vaccines are any more flock (Bacon and Witter,1993). virulent than the intermediate ones. The suggestion is that some of the "hot" strains Infectious bronchitis spread better between birds and can The envelope of IB virus possesses surface therefore infect birds that were not initially projections comprising two glycopoly- vaccinated either because they did not peptides, S, and S,. While both are drink or because they have high maternal important in the immune response, it antibody levels. It has been suggested that appears that the S, glycopolypeptide is the virulent viruses can overcome virus- major antigenic component. neutralizing antibody titres of 1.500, Protection is given against the homo- intermediate vaccines can overcome titres logous serotype as well as, to a varying of 1.250 and avirulent viruses can overcome degree, against other serotypes. This has titres of 1.100 or less (Lukert and Saif,1991). resulted in the use of a number of different However, given the wide variation of titres attenuated vaccines in some areas. Re- in broiler chickens, these figures are at best combination among field and vaccine only a guide as to the optimum time to strains possibly occurs and is responsible vaccinate. for producing new "variant" strains In view of the wide antigenic variation (Kusterset al.,1990).Because of the within IBD viruses (McFerranet al., 1980) possibility of recombination and the fact it is surprising that only one subtype that attenuated strains can revert back to vaccine (the Delaware) has been necessary. virulence, a new vaccine serotype should There has been a major upsurge in not be introduced into an area until there virulence of IBD viruses throughout the is clear evidence that its use that is world, and the policy adapted in many essential. areas of only vaccinating the breeders and There have been two approaches to relying on maternal antibody to protect immunizing egg-producing birds. The first the broilers has proved flawed. In many is to prime the birds with an attenuated areas, it has been necessary to use vaccine (around four weeks when maternal intermediate or hot vaccines in broiler antibody levels have fallen) and then with flocks to control the losses. Even when an inactivated oil-adjuvanted vaccine or a flocks are clinically normal, IBD virus can less attenuated vaccine around18weeks attack the bursa and cause lesions. In these to produce high levels of circulating circumstances, the subclinical infections humoral antibody. The second is to prime can cause up to a 10 percent reduction in the birds and then commence spray income (McIlroy, Goodall and McCracken, vaccination at18weeks, followed there- 1989). after every eight to ten weeks with an attenuated vaccine. The aim here is to keep Mycoplasma gallisepticum a high degree of local immunity in the gut AlthoughMycoplasma gallisepticumhas and respiratory tract. been eradicated from commercial poultry 118 Conventional vaccines

in many areas, it is still widely distributed, adjuvant. The attenuated strains have a especially in areas where multi-age sites range of virulence, although two doses of and open-sided houses with earth floors inactivated vaccine during rearing usually make eradication very difficult. Under provide adequate immunity as long as the these conditions, vaccination of com- appropriate strains are included in the mercial egg-producing birds to control falls vaccine. Attenuated vaccines are widely in egg production is indicated. Bacterins, used in some areas, especially in meat which are inactivated suspensions of whole turkeys. Live vaccines often cause chronic M. gallisepticum (Mg') organisms in oil fowl cholera. This effect can be minimized emulsion, are apparently effective in by first giving an inactivated vaccine. controlling disease but have little benefit in eliminating infection in multi-age sites. Infectious coryza Recently, inactivated M. gallisepticum Infectious coryza is caused by Haemophilus bacterin mixed with 0.2 percent iota paragallinarum, the isolates of which it is carrageenan as an adjuvant induced possible to divide into three serogroups resistance to air sacculites in chicken with at least seven serovars by using the challenged with the virulent MGR strain HI test (Kume et al., 1983). Vaccines consist (Elfaki et al., 1992). of inactivated whole-cell bacterins Live vaccines are also used to control M. emulsified in oil adjuvant or absorbed on gallisepticum. The F strain is a naturally to aluminium hydroxide. The immunity occurring strain of relatively low virulence induced by these vaccines is serogroup- for chickens but of a higher virulence for specific. Breeders and commercial layers turkeys. It will displace more virulent field are routinely vaccinated in many countries, strains from multi-aged sites but vac- using two injections during rearing at least cinated birds become permanent carriers four weeks apart. Using chemical (Kleven, Khan and Yamamoto, 1990). A mutogens, Blackall et al., (1993) have temperature-sensitive mutant which is produced non-pathogenic mutants which avirulent has recently been described may be of value as attenuated vaccines. (Whithear et al., 1990). Coccidiosis Fowl cholera A widespread and serious disease caused Fowl cholera is caused by Pasteurella by Eimeria spp. is coccidiosis. It has been multocida, which can be serologically controlled in the past by the continuous subdivided by differences in capsular administration of chemotherapeutic agents antigens and cell wall lipopolysaccharide to the flock's feed. However, problems antigens. Using a passive haemagglutinin with the development of resistance and test, five capsular serogroups (A,B,D,E and the need to withdraw the agents fora F) can be identified; however, these specified period before slaughter have led capsular antigens do not appear to playa to the development of vaccines. role in protection. P. multocidacan also be At present, vaccines are used mainly in subdivided into 16 serotypes usinga heat- layers and breeders. They contain stable somatic antigen (Brogdens and attenuated oocysts of multiple species of Rebers, 1978). Both inactivated and live Eimeria. Attenuation has been achieved by P. multocida vaccines are available. Inactiv- a number of methods such as adaptation ated vaccines are whole cell products ofa to growth in avian embryos or by selecting number of strains (often including "auto- strains with a shortened prepatent period genous" local strains) emulsified inan oil in the precocious strains. Vaccine manual 119

A second approach has been to define approaches will depend on whether the coccidial immunogens which _could be economic advantages claimedare realized. used as subunit vaccines. Thereare, This is especially true where hygiene however, many species of Eimeria, with standards are high and day-old chicks do little or no cross-immunity between not face a massive challenge. species. One different approach to embryonated egg inoculation is to inject an immuno- NEW TECHNICAL DEVELOPMENTS stimulant containing lymphokines. It is In 1982, Sharma and Burmester inoculated claimed that this has enhanced the efficien- embryos with THV in an attempt to cy of Marek's disease vaccination and, in circumvent the massive Marek's disease addition, resulted in lower mortality, challenge that day-old chicks facedon reduced feed conversion and lower some sites. These chickens developed a production costs for commercial broilers persistent THV infection and were resistant (Miles et al., 1993). to early challenge with pathogenic Marek's disease. The vaccine was inoculated into Genetic engineering the allantoic sac; and it was found that the Initially, organisms were modified by egg best results were obtained in 17- to 18-day- passage (e.g. IB), by cell culture passage or old embryos. Inoculation at 18 days would by identifying a naturally occurring non- coincide with the transfer of eggs from the pathogenic strain (e.g. Rispen's vaccine setting to the hatching machines. against Marek's disease). The attenuation Sharma (1985) extended this work to the process can be enhanced by using chemical investigation of protection against IBD. He mutagens, and mutants can be selected found that low virulent vaccine strains using a range of techniques such as plaque could induce immunity without effecting purification, temperature sensitivity, hatchability or survival of the chicks. colony size or an appropriate monoclonal However,maternalantibodydid antibody. Some proteins are only expressed neutralize strains of low virulence. when bacterial cells are grown in vivo, and it Recently, the use of an IBD commercial is suggested that this is the reason why vaccine-antiserum mixture to inoculate 18- Pasteurella multocida grown in vivo gives day-old embryonated eggs has been better vaccines. An attempt to clone the described. This preparation induced genes responsible for expressing these immunity in chicks produced from eggs proteins is in progress and may give rise to with maternal antibody to IBD (Whitfill et better inactivated or attenuated vaccines al. ,1993). Embryos originating from (Jost et al., 1993). It is now possible to specific pathogen-free (SPF) turkeys were modify the genorne by chemical means, as successfully inoculated with marble spleen in the Aujeszky's disease vaccine virus in disease and Newcastle disease vaccines which the thymidine kinase gene has been and the poults were resistant to subsequent deleted. challenge (Ahmad and Sharma, 1993). It is possible to insert genes into vectors. This work has been developed These vectors can be organisms which do commercially and an automatic egg not replicate in the host, in which case an injection system is now in operation adjuvant is indicated. Thus, a recombinant (Marchant, 1993), injecting THV and SB-1 baculovirus (expressing the haemag- Marek's vaccine into embryonated chick glutinin and neuraminidase protein of eggs. The machine can inject 20 000 to Newcastle disease virus), combined with 30 000 eggs per hour. The success of these an oil adjuvant, is effective in protecting 120 Conventional vaccines

against Newcastle disease challenge (Nagy maternal antibody wanes. This would et al., 1991). overcome the hurdle of varying levels of The alternative approach is to incor- antibody to different viruses and also to porate one or more genes into a vector the same virus, owing to the fact that the which does replicate in the host. Avian flock is made up of the progeny of a poxviruses, avian herpesviruses (serotype number of breeding rllocks. 2 and turkey herpesvirus) and avian There is always the problem of the adenoviruses have been investigated. An maternal antibody to the vector preventing example is the incorporation of the gene the vector growing. In the case of THV this coding for the haemagglutinin of avian could be overcome by using the THV in influenza (H5N2) into a poxvirus (Beard, alternate generations (it does not occur as Schnitzlein and Tripathy, 1991). The gene a natural infection in chickens), as is coding for the fusion protein (F) of already the practice. Newcastle disease virus has also been While it is possible to express the genes incorporated into poxvirus (Iritani et al., encoding protein antigens in vitro for use 1991). This gives immunity but does not as subunit vaccines, and also to synthesize give rise to H1 antibodies, thus allowing a peptide vaccines chemically, these test to detect infection by the Newcastle solutions may present problems regarding disease virus. Antibody to Newcastle the cost of production and administration disease did not prevent the development of these vaccines, difficulties in obtaining of immunity, but antibody to fowl pox the correct folding of the protein and the prevented the poxvirus growing. Thus, this limited number of antigenic determinants, vaccine could be used to vaccinate ducks which could mean that strains of agents with maternal antibody to Newcastle could arise which are not neutralized. disease. Similarly, a fowl poxvirus Although, in theory, anti-idiotype expressing the turkey rhinotracheitis vaccines are possible, in practice they have fusion glycoprotein gave partial protection been poorly immunogenic and show little to challenge by turkey rhinotracheitis virus immediate promise. A different approach (Quinzhong et al., 1993). is the use of cytokines as adjuvants. These A promising approach is to use a turkey can be incorporated in the injectible vaccine herpesvirus (THV) as the vector. A which may also include slow-release recombinant THV expressingthe mechanisms and immunostimulating Newcastle disease virus fusion protein complexes (ISCOMs). Such an approach gave good protection against both Marek's has been taken with a subunit vaccine and Newcastle diseases (Morgan et al., against avian influenza in which nucleo- 1992). THV is used widely to vaccinate protein with residual haemagglutinin day-old chicks. It is universal and safe activity was incorporated into ISCOMs (unlike fowl pox which is absent insome (Sivanandan et al., 1993). areas and which may cause local reactions), Liposome-adjuvanted egg drop syndrome it does not spread horizontally and, most 1976 vaccines appeared as efficacious as oil- important, it gives a persistent viraemia emulsion vaccines without the side-effects lasting several weeks. This approachmay of the oil (Jing-Sheng and Yi-Zhu, 1993). well overcome the problems of maternal Using trivalent avian influenza antigens, immunity. Thus, if genes giving resistance Fatunmbi et al. (1992) found that charged to a number of diseases were incorporated, liposomal avridine adjuvant produceda they could stimulate the immuneresponse better antibody response than other to each agent at the appropriate timeas uncharged liposomal avridineor oil- Vaccine manual 121

emulsion adjuvants. The positively charged neuraminidase of NDV into chickens liposornal avridine adjuvant was superior in the presence of antibody to NDVor to negatively charged adjuvant. FPV. Avian Dis., 35: 659-661. Jing-Sheng, L. & Yi-Zhu, W. 1993. Studies BIBLIOGRAPHY on the liposorne-adjuvanted antigens of the inactivated EDS.76 virus. In Proc. Ahmad, J. & Sharma, J.M. 1993. Protection X World Vet. Poultry Assoc. Congr., against hemorrhagic enteritis and New- Sydney, Australia. castle disease in turkeys by embryo vac- Jost, H., Homchampa, P., Ruffolo, C. & cination with monovalent and bivalent Adler, B. 1993. Molecular approaches vaccines. Avian Dis., 37: 485-491. for studying the virulence genes and Bacon, L.D. & Witter, R.C. 1993. Influence antigens of P. multocida; strategies for of B-haplotype on the relative efficacy novel fowl cholera vaccines. In Proc. X of Marek's disease vaccines of different World Vet. Poultry Assoc. Congr., Sydney, serotypes. Avian Dis., 37: 53-59. Australia. Beard, C.W., Schnitzlein, W.M. & Tripathy, Kleven, S.H., Khan, M.I. & Yamamoto, R. D.N. 1991. Protection of chickens against 1990. Fingerprinting of Myco plasma highly pathogenic avian influenza virus gallisepticum strains isolated from multi- (H5N2) by recombinant fowlpox viruses. age layers vaccinated with live F strain. Avian Dis., 35: 356-359. Avian Dis., 34: 984-990. Blackall, P.J., Graydon, R.J., Rafiee, M. & v>ume, K., Sawata, A., Nakai, T. & Tinworth, D. 1993. Towards a live Matsumoto, M. 1983. Serological classi- infectious coryza vaccine. In Proc. X World fication of Haemophilus paragallinarum Vet. Poultry Assoc. Congr., Sydney, with a hemagglutinin system. J. Clin. Australia. Microbiol., 17: 958-964. Brogdens, K.A. & Rebers, P.A. 1978. Serolo- Kusters, J.G., Jager, E.J., Niesters, H.G.M. gical examination of the Westphal-type & van der Zeijst, B.A.M. 1990. Sequence lipopolysaccharidesofPasteurella evidence for RNA recombination in field multocida. Am. J. Vet. Res., 39: 1680-1682. isolates of avian coronavirus infectious Elfaki, M.G., Kleven, S.H., Jin, L.H. & bronchitis virus. Vaccine, 8: 605-608. Ragland, W.L. 1992. Sequential intra- Lukert, P.D. & Saif, Y.M. 1991. Infectious coelomic and intrabursal immunisation bursal disease. In B.W. Calnek, ed. of chickens with inactivated Myco plasma Diseases of poultry, p. 342-385. Ames, gallisepticum bacteria and iota carageenan USA, Iowa State University Press. adjuvant. Vaccine, 10: 655-662. Marchant, J. 1993. Embrex targeting a niche Fatunmbi, 0.0., Newman, J.A., Sivanandan, market. Animal Pharmacol., 269: 14-15. V. & Halvorson, D.A. 1992. Enhance- McFerran, J.B. 1992. Control of Newcastle ment of antibody response of turkeys disease in Northern Ireland (NDV vacc.). to trivalent avian influenza vaccine by In Proc. Workshop of Avían Paramyxo- positively charged liposomal avridine viruses, p. 238-249. Rauischolzhasen, adjuvant. Vaccine, 10: 623-626. Germany, Commission of the European Iritani, Y., Aoyama, S., Takigami, S., Communities. Hayashi, Y., Ogawa, R., Yanagida, N., McFerran, J.B., Young, J.A., Clarke, J.K. Saeki, S. & Kamogowa, K. 1991. Anti- Wright, C.L. 1972. Observations con- body response to Newcastle disease virus cerning the use of living infectious (NDV) by recombinant fowlpox virus bronchitis vaccine under field conditions. (FPV) expressing a hemagglutinin- Vet. Rec., 90: 527-530. 1121 Conventional vaccines

McFerran, J.B., McNulty, M.S., McKillop, turkey herpesvirus. Avían Dis., 26: E.R., Connor, T.J., McCracken, R.M., 134-149. Collins, D.S. & Allan, G.M. 1980. Sivanandan, V., Kodihalli, S., Nagaraja, Isolation and serological studies with K.V., Halvorson, D.A. & Newman, J.A. infectious bursal disease viruses from 1993. ISCOM adjuvanted cross-protective fowl, turkeys and ducks: demonstration subunit vaccine against avian influence. of a second serotype. Avian Pathol., 8: In Proc. X World Vet. Poultry Assoc. Congr., 205-212. Sydney, Australia. McIlroy, S.G., Goodall, E.A. & McCracken, Whitfill, C.E., Ricks, C.A., Haddad, E.E. & R.M. 1989. Economic effects of subclinical Avakian, A.P. 1993. In ovo administration infectious bursal disease on broiler of a novel infectious bursal disease production. Avían Pathol., 18: 465-473. vaccine in broiler chickens. In Proc. X Miles, A.M., Doelling, V.W., Phelps, P.V., World Vet. Poultry Assoc. Congr., Sydney, Ricks, C.A., Tyczkowski, J.K., Whitfell, Australia. C.E. & Gildersleeve, R.P. 1993. Efficacy Whithear, K.G., Soeripto, Harrigan, K.E. of an immunostimulant administered & Ghiocas, E. 1990. lmmunogenicity of in ovo. In Proc. X World Vet. Poultry Assoc. a temperature-sensitive mutant of Congr., Sydney, Australia. Mycoplasina gallisepticurn vaccine. Aust. Morgan, R.W., Gelb, J., Schreurs, C.S., Vet. J., 67: 168-174. Luttiken, D., Rosenberger, J.K. & Witter, R.L., Sharma, J.M., Lee, L.F., Opitz, Sandermeijer, P.J.A. 1992. Protection H.M. & Henry, C.W. 1984. Field tests of chickens from Newcastle and Marek's to test the efficacy of polyvalent Marek's disease with a recombinant herpesvirus disease vaccine in broilers. Avían Dis., of turkeys vaccine expressing the 28: 44-60. Newcastle disease fusion protein. Avían Dis., 36: 858-870. Nagy, E., Krell, P.J., Dulac, G.C. & Derbyshire, J.B. 1991. Vaccination ao-ainst Newcastle disease with a recom- binant baculovirus hemagglutinin- neuraminidase subunit vaccine. Avían Dis., 35: 585-591.

Quinzhong, Yu., Barrett, T., ri own, D.X., Cook, J.K.A., Green, P., Skinner, M. & Cavanagh, D. 1993. Protection against turkey rhinotracheitis pneumovirus (TRT) induced by a fowl pox recombinant expressing the TRTV fusion glycoprotein (F). In Proc. X World Vet. Poultry Assoc. Congr., Sydney, Australia. Sharma, J.M. 1985. Embryo vaccination with infectious bursal disease virus alone or in combination with Marek's disease virus. Avían Dis., 29: 1155-1169. Sharma, J.M. & Burmester, B.R. 1982. Resistance to Marek's disease at hatching in chickens vaccinated as embryos with Vaccine manual 123

Poultry vaccines (2) c-Lavemerds for A'c)],giss cick R.P. Spradbrow

The poultry industry in developing required for brooding. Two elements countries, especially the village poultry responsible for these extreme mortalities industry, encounters different problems to have been cited. First is the high wastage those found in the developed countries that occurs during the broodingseason, and, consequently, the requirements for which is probably caused by a combination vaccine by this segment of the market are of starvation, predation and infectious very different. Certainly, there are elements disease but is indirectly the result of poor of the international commercial industry husbandry and neglect. The second cause with poultry enterprises close to the larger of attrition is more directly caused by cities in many developing countries, and infectious disease. Outbreaks of disease these enterprises have access to interna- causing extremely high rates of mortality tional breeding stock and international in village chickens have been reported expertise as well as the opportunity and from many developing countries. Where funding to draw vaccines of high quality these diseases have been identified, the from the international market. The vaccines major culprits are Newcastle disease and used on these poultry populations in a fowl cholera, which are preventable and developing country will seldom have been readily controlled if effective vaccines can produced in that country. Instead, they will be applied. It is the lack of suitable vaccines have been produced to meet the highest and appropriate methods of application standardsas discussed elsewhere in this that allows Newcastle disease and fowl manual and the manufacturers will have cholera to plunder the village flocks. If ensured as far as possible the absolute these diseases could be controlled, villagers safety and adequate efficacy of the would have the incentive to make the vaccines. changes in husbandry that would control Village chickens comprise the major part brooding losses. of the poultry industry in many developing Suitable vaccines are the key to a "new countries. Village flocks are small, of mixed science" of village chicken keeping. An age and poorly housed or even unhoused. analysis of the special requirements of The chickens gain much of their nourish- village chickens indicates that their needs ment by scavenging in the village environ- are not being served by the conventional ment and by consuming supplements of vaccines available on the international household scraps if these are made market. A start has been made on devel- available. The village flocks are poorly oping vaccines against Newcastle disease productive, as the major call on energy is that are specifically suited for delivery to for reproduction and maintenance of the and use in villages, (Copland, 1987; population. Extremely high mortality rates Spradbrow, 1992 and 1993 /94). However, ensure that few surplus eggs and birds it is important that regulatory authorities become available for sale, barter or recognizse the special needs of village consumption. Nearly all the eggs are chickens and that well-intentioned regu- 124 Special requirements for village chickens

lations do not impose further barriers to self-sufficiency in avian vaccines can the provision of suitable vaccines to village eventually lead to independence in other chickens. areas of vaccine production. The over- Village flocks require special vaccines. riding imperative is the need to conserve The transport of vaccines to villages and foreign exchange. their storage within these villages must The purchase and transport of imported usually be accomplished without refriger- vaccines is a continuing drain on funds. ation. It will still not be feasible in the On the negative side, a wasteful regional forseeable future to establish adequate cold overcapacity for vaccine production can chains; therefore, the vaccines themselves be reached, while the purchase of vaccine- must be thermostable. As long as villagers producing equipment of any sophistication think that their flocks will survive the is also an expensive undertaking. Where frequent outbreaks of infectious disease to vaccine is produced nationally, con- which they are exposed, adequate housing sideration should be given to establishing will remain a rarity. This means catching independent regional centres for testing. chickens for individual vaccination will Many of the standards developed for often be impossible. There seems to be no the testing of conventional avian vaccines alternative to the oral application of are not appropriate for the testing of vaccine, usually on food but also in water, vaccines for village chickens. Tests for in areas where the sources of drinking safety should be undertaken in local water can be controlled. When possible, chickens and tests for efficacy should also individual applications of vaccine to the be made locally, using indigenous chickens eye, nose or mouth is a more reliable and challenge organisms of local origin. It method of inducing immunity with at- should be recognized that safety and tenuated vaccines. Vaccines for use in freedom from nominated adventitious village chickens also need to be extremely agents are not synonomous. The first factor cheap. It may be necessary to reconsider is essential, the second may be a luxury some of the elements that add value to that can be postponed for vaccines for conventional vaccines formulation, village use. packaging, substrate, testing. Nor is it Several procedures that are used in the necessary to demand an extremely high production of conventional vaccines might degree of efficacy of a vaccine for use in be questioned when vaccines for village village chickens. Absolute protection may chickens are being produced: require unrealistically large amounts of Is specific pathogen-free substrate required? vaccine. The cost-effective compromise Genuine specific pathogen-free flocks are may be a moderate level of protection not readily established in developing achieved with an inexpensive vaccine. In countries. The costs of initiation, main- few places have conventional vaccines tenance and testing are enormous and the contributed to the protection of village advantages for village-style vaccines are chickens. minimal. Some of the flocks claimed to be Many developing countries would prefer specific pathogen-free do not deserve that to produce their own veterinary vaccines designation. Some receive live vaccines for local use, especially the relatively and the disease status of some is monitored simple avian vaccines that can be made in only by the absence of clinical disease. embryonated eggs. There are several Many are more realistically describedas reasons for this. One is the desire for isolated flocks or minimal disease flocks. national self-reliance and the belief that Vaccine seeds should be specific pathogen- Vaccine manual 125

free and production of vaccine should use of small-dose packs because village flocks a seed lot system. This will allow an are small and the supply of vaccine in eventual increase in the standards of packages that contain many doses is seen vaccine. However, rural poultry should not to be wasteful. The conventional 1 000- at this point be deprived of vaccines dose vial is admittedly an embarrassment because of an absence of specific pathogen- for the owner of a flock of 20 chickens and free flocks. there is an obvious need to share vaccine Is freeze-drying necessary? Most con- among village flocks. There is also a need ventional attenuated vaccines produced for to develop a philosophy of overdosing, use in commercial poultry are lyophilized. rather than discarding unused vaccine. Compared with liquid vaccines, the Lentogenic Newcastle disease vaccines are lyophilized product has greater stability not harmful to chickens when given in and is more convenient to store and doses larger than those recommended by transport. However, freeze-drying and the manufacturer. The problem in villages suitable packaging of the dried product is not the 1 000-dose content of a single add to the cost of a vaccine. In some vial of vaccine, rather it is that of obtaining developing countries, avian vaccines are a single vial of vaccine in systems that are produced as "wet" vaccines. This may not designed for bulk distribution to a com- be a disadvantage if vaccine is to be used mercial industry. soon after manufacture and if local The logistics of delivering vaccine to production reduces the need to transport villages is as important a problem as vaccines over long distances. Nevertheless, developing a suitable vaccine strain. The thermostability becomes an important centralized preparation of food-based character of the vaccine strain. vaccines and their delivery to villages are The sophisticated packaging of con- not feasible in most countries. Each chicken ventional vaccines is not necessary for requires 7 to 10 g of vaccine-coated food. products to be used in village chickens. In most areas, food and vaccine will need There are frequent calls for the production to be mixed in the villages, which will

TABLE 11 Comparison of poultry vaccine requirements for commercial and village use

Commercial chickens Village chickens

Place of production Few large laboratories Small local laboratoriés

Thermostability Not essential Very desirable

Market International Local

Seed material Specific pathogen-free Specific pathogen-free

Vaccine Specific pathogen-free Not necessarily specific pathogen-free

Lyophilization Desirable Not necessary

Efficacy Extreme efficacy reqired Moderate efficacy acceptable

Delivery Individual vaccination or by aerosol, spray or drinking water Delivery on food advantageous

Packaging units Large dose (multidose vials) Small-dose vials preferable 126 Special requirements for village chickens

require the regular delivery of small BIBLIOGRAPHY quantities of vaccine. Recent experiments indicate that it may be possible to in- Copland, J.W., ed. 1987. Newcastle disease in corporate in a single pellet a quantity of poultry. A new food pellet vaccine. ACIAR Newcastle disease virus vaccine sufficient Monograph No. 5. Canberra, Australian for a single chicken. This would help Centre for International Agricultural overcome the problems of central pro- Research. 119 pp. duction and would ease the problem of Spradbraw, P.S., ed. 1992. Newcastle disease transport. in village chickens. Control with thermo- Table 11 indicates some of the require- s table oral vaccines. In ACIAR Proc. 39. ments that should be considered when Canberra, Australian Centre for Interna- comparing conventional vaccines and tional Agricultural Research. 189 pp. vaccines for village chickens. Spradbrow, P.S. 1993 / 94. Newcastle disease in village chickens. Poultry Sci. Rev., 5: 57-96. Vaccine manual 127

vaccines A. Adams, K.D. Thompson and R.J. Roberts

With fish being the primary source of eradicate and is likely to recur inter- animal protein in many countries, mittently among the ongrowing stock. aquaculture is growing rapidly worldwide, Motile aeromonads appear to be the most and stress and diseases that accompany importantbacterialpathogensof intensive fish culture have led to treatment freshwater fish in tropical countries. These with antibiotics and chemicals. However, micro-organisms have been reported to as concern over pollution associated with cause mass mortality in Indian major carp chemical treatments and the emergence of and can be detected during a variety of multiple resistance to antibiotics make the infections,including haemorrhagic control of infections more and more septicaemia, asymptomatic septicaemia, difficult, the emphasis should be on disease epizootic ulcerative disease (EUS), tail rot prevention by means of optimal husbandry and fin rot. and biological control methods, such as The exact aetiology of some of the newly vaccination and immunostimulants. emerging diseases, such as EUS, have not been unequivocally determined. In the FISH DISEASES case of EUS, bacterial, viral and fungal Fish in culture suffer a variety of diseases, participants are all associated with the including bacterial, viral, parasitic and disease complex but the principal initiating fungal infections, some of which are shown factor has yet to be determined (Roberts, in Table 12. The majority of infections Willoughby and Chinabut, 1993). described for fish in culture are bacterial. As the aquaculture industry grows and Vibriosis, associated with a number of new species of fish are intensively cultured, marine vibrios, is the most widespread the range of diseases and affected species worldwide and not only causes huge is similarly expanding. For example, economic losses to the marine fish culture pasteurellosis was only recently described industry but is the main disease affecting in Europe and rickettsiosis in Chile, Ireland the farming of prawns in Southeast Asia and Taiwan Province of China, but now and Japan. Furunculosis, hitherto thought both are recognized as highly significant of as a freshwater disease of wild pathogens. salmonids, is the most serious disease affecting cultured salmonids, particularly COMMERCIAL VACCINES in the seawater phase. Acute furunculosis Of the many infectious diseases affecting is a common problem when Atlantic fish there are to date only five for which salmon smolts are transferred to the sea, at effective commercial vaccines have been which time losses of up to 30 percent of the produced. Three of these are bacterial stock may occur. The disease is often complicated by simultaneous sea lice The authors wish to express their thanks to all those infection and pancreatic disease. Un- who provided information for this paper, in particular fortunately, once furunculosis is intro- J. Turnbull, H. Rodger and J. McGeorge for their duced to a site it is very difficult to advice on fish diseases. Disease Causative agent Fish diseases TABLE 12 Major fish species affected Country/region VibriosisEntericBacterial red mouthdisease Yersinia ruckeri Salmonids, primarily rainbow trout North America, Europe, South America BacterialFurunculosis- Hitra- Vibriosis disease kidney disease RenibacteriumAeromonasVVibrio salmonicida anguillarum, salmonicida salmoninarum V ordalii SalmonidsAtlanticWidespread salmon in marine fish salmonids NorthEuropeNorway,Worldwide, America, Faroe Japan, Islands Europe, North Japan, America Chile MotileEnteric septicaemia aeromonad septicaemia A.Aeromonas E.Edwardsiellasobria tarda hydrophila, ictaluri A. caviae, Caffish,Eel,Caffish hirame cyprinids, salmonids SoutheasternAsia,Japan Europe, United United States, States Canada StreptococcusBacterialPasteurellosis cold-water infections disease StreptococcusLytoPasteurella psychrophilus piscicida spp. YellowSalmonidsAyu, tail,yellow rainbow tail, sea trout, bream, ayu, seatilapia, bass, carp Japan,UnitedProvince United States, of ChinaStates, Europe, Taiwan Japan Province States, Japan, Europe, Taiwan NocardiosisTuberculosis NocardiaM.Mycobacterium fortuitum, asteroides, M. chelonae marinum, N. kampach TropicalseaSnakehead,bass, bass, bream aquarium wide variety fish, ofyellow other tail, species tropical aquarium fish, Spain,Southeastof China Japan, Asia, Canada Japan, Europe EpitheliocystisSalmonid rickeitsial septicaemia Chlamydia-likePiscifickettsia salmon's organisms WideSalmonidsrainbow variety trout of and species brook trout SouthNorthChile, America,AfricaTaiwan Province Southeast of Asia,China, Europe, Ireland EntrococcusColumnarisClostridial infections disease infection EntrococcusFlexibacterClostridium columnar's,botulinum serrolcida F. maritimus Yellowturbot,AllSalmonids tail salmon freshwater species, bream, bass, NorthEurope, America, Asia, Europe, Japan United States UlcerativeBacterial gill septicaemia disease PseudomonasFlavobacteriumCytophaga spp., sp. bronchiophilia Flexibacter spp. WideEels variety and others of species JapanNorth America, Japan, Europe (cont.) Causative agent TABLE 12 (continued) Major fish species affected Country /region n ViralDiseaseInfectious diseases pancreatic necrosis Birnavirus (ds RNA) PacificSalmonids, cod sea bass, sea bream, turbot, Europe ViralInfectious haemorrhagic haemorrhagicsalmon septicaemiaanaemia necrosis RhabdovirusUnknown (putatative virus) Snakehead,SalmonidsAtlantic salmon carp, barbs Japan,NorwayCanada,Japan, North Taiwan North America, ProvinceAmerica Europe of China, ProliferativeSeaParasitic lice diseases kidney disease extrasporgonicUnidentifiedLepeophtheirus myxosporean stage, PKX salmonis FreshwaterMarine-cultured salmonids saimonids Scotland,NorthernUnited Kingdom,circumpolar Ireland, Canada)Europe, (Norway, United Japan, States Costiasis lchthyophthiriusIchthyobodo necator o Freshwater, especiailyfishFreshwater,saltwater especially young formnon-host-specific affected. fish; Also afingerling Worldwide,Worldwide, 2°-30°C 4°-25°C WhiteTrichodinids spot A rangeTrichodina of pathogenic species, sp., Tripartiella sp, and others Freshwatersalmonidse.g. cyprinids, flatfish and tilapiids, marine.marine (e.g. salmonids,turbot) non-specificAll cultured in culture ictalurids fish Worldwide MyxosporeansMicrosporeans PleistophoraKudoae.g. Myxobolus sop. sp., sap., Glugea Sphaerospora sp. and others spp., problemFreshwater in flaffish and marine. One reported BranchiomycosislchthyophoniasisFungal diseases Branchiomyceslchthyophonus spp. sanguinis, B, demigran Cyprinids,Freshwater eels, and freshwatermarine species tench, India,Worldwide Japan, Eastern Europe AspergillomycosisSaprolegniasisEpizootic ulcerative syndrome UnknownAspergillusSaprolegnia (putative spp. paras/tica fungus) - diclina complex TilapiaCold,sticklebackFreshwater freshwater and salmonids, brackish species catfish WorldwideAustralia,Northern Europe,Southeast United Asia States 130 Fish vaccines

diseases affecting salmonidsenteric red As shown in Table 13, a triple vaccine is mouth (ERNI), vibriosis and furunculosis also available against furunculosis and the while the others are a bacterial disease in two Vibrio species. catfish enteric septicaemia of catfish Since the initial work of Duff (1942), (ESC) and the viral infection, spring many years of research have been dedi- viraemia of carp (SVC), as shown in cated to the development of a vaccine Table 13. against furunculosis, the most economic- Fish vaccines first became available in ally important disease affecting salmonids. 1976 when a commercial ERM vaccine was Aeromonas salmonicida is the causative agent developed and registered in the United and outbreaks of the disease are stress- States (Tebbit, Erikson and Vande Water, associated, with high mortalities among 1981). By 1982, Busch stated that vac- salmon. Until recently, only limited success cination was the single most effective had been achieved with vaccination. In control for ERM. The disease is closely contrast to the early simple, killed whole- stress-related and the causative agent, cell vaccines, the organisms for the new Yersinia ruckeri, is an important primary generation of furunculosis vaccines are pathogen for intensively cultured rainbow cultured under iron-limited conditions trout (Busch, 1982). which closely mimic the situation found in A commercial Vibrio vaccine was fish. This allows expression of the essential developed in 1980. This simple, killed protective antigens, which include LPS and whole-cell vibrio vaccine is highly effective IROMPS (iron regulatory proteins) (Hirst against the marine pathogens Vibrio and Ellis, 1994). The use of adjuvants, anguillarum and Vibrio ordalii in salmonids which enhance the immune response to (Fryer, Rehovec and Garrison, 1978). The the vaccine, also play an important role in protective heat-stable lipopolysaccharide the success of these vaccines while adju- (LPS) antigens, derived from cell walls, vants do not appear to be necessary for the which make the Vibrio vaccine so successful LPS-based ERM and Vibrio vaccines. in salmonids have also proved effective ESC is the major cause of mortality in for other farmed species, for example cod, farmed catfish in the southern United eel, ayu (Kawai, Kusuda and Itami, 1981) States and Asia. It was first identified in with little or no modification to the 1976 and is caused by the bacterium vaccine's original design. More recently, Edwardsiella ictaluri. The commercial with the upsurge in farming ofsea bass vaccine available for ESC is based on a and bream in the Mediterranean, the bacterin preparation and involves a two- control of vibriosis has become important step vaccination programme. Catfish fry in these species too. Vaccines containing (eight to ten days old) are immersed in the additional serotypes such as serotype III vaccine prior to ponding. They are then appear to be necessary for full protection given an oral boost of vaccine 30 days (Vigneulle et al., 1993). Generally, vibriosis before the start of the ESCseason. The in fish accompanies some other stressor vaccination of fish by this method has physical trauma but some strains of V. proved effective in controlling ESC anguillarum or V. salmonicidaappear to be outbreaks. highly infectious primary pathogens. In The one commercial viral vaccine for Norway, where V. salmonicidacauses Hitra SVC has been available in the Czech disease or cold-water vibriosis,a combined Republic and Slovenia since 1981 and vaccine is available because there isno also includes an oil-based adjuvant for cross-protection between the pathogens. administration by injection. SVC is caused Commercially available fish vaccines TABLE 13 DiseaseFurunculosis ManufacturerAqua health CountryUnited of Kingdom or gin FurogenFurogen/injectionProduct name/ B/immersion method of administration VibriosisFurunculosis AquacultureBiomed Inc, Vaccines Ltd United StatesKingdom AquaVacBiojec 1300 Furovac-immersion/immersionFurovac-5/injectionVibrio/immersion injection or immersion or immersion (non-adjuvanted) (non Furunculosis ApothekernesAquacultureBiomed Inc. Vaccines Laboratorium Ltd NorwayUnitedUnited States Kingdom ApojectBiojecAquaVacadjuvanted) 1500/injection 1-Fural/injection Furovac-5/injection (oil-based) (oil-based adjuvant) FurunculosisCold-waterFurunculosis, vibriosis andcold-water cold-water and vibriosis vibriosis and vibriosis ApothekernesEwosApothekernes Aqua A.S. Laboratorium Laboratorium NorwayNorvvay Apovax/immersionApojectLipogenApoject 3-Fural/injection 2-Fural/injectionMono/injection (water-based) (oil-based) (oil-based) (oil-based) FurunculosisFurunculosis,Furunculosis cold-water vibriosis and vibriosis Ewos Aqua A.S. Norway Furogen/immersionLipogen Triple/injection (water-based) (oil-based adjuvant) Furunculosis,Furunculosis cold-water vibriosis,vibriosis andvibriosis vibriosis IntervetEwos Aqua Norbio A.S. A.S. Norway NorvaxOravaccFurogen tripletriple/injection B/dipF Vet/Oral (water-based) (water-based) (oil-based) + IPN/injection (oil-based) EntericFurunculosis,Cold-water septicaemia vibriosis cold-water+ and of caffish vibriosis andinfectious vibriosis pancreatic necrosis BiomedIntervet NorbioInc. A.S. UnitedNorway States BiomedBiojectNorvax 1900/injection vibriosis/dipESC/immersion (water-based) (oil-based (water-based) adjuvant) SpringEnteric viraemiared mouth of carp BiovetaAquaAquacultureBiomed health Inc. Vaccines Ltd CzechUnited StatesRepublicKingdom InjectionErmogen/immersionAquaBioject Vac 1100 ERM/immersion immersion (water-based) 132 Fish vaccines

by Rhabdovirus carpio (two serotypes) and In general, intraperitoneal injection the disease is widespread in areas of carp appears to be the most effective route of culture. vaccination, although not the most practical. Much effort is therefore being Adjuvants put into the development of immersion Most water-based vaccines for salmonids and oral vaccines. contain either glucan- or aluminium-based adjuvants which are partially effective and DEVELOPMENT OF VACCINES have few side-effects. The latest trend in The primary considerations for any salmonid vaccines is towards oil-based successful vaccine for aquaculture are cost- adjuvants which have been found to be effectiveness and safety. To accomplish more effective. These do, however, need to this the vaccine must provide long-term be injected into the fish and have many protection against the disease under the side-effects,for example suspected intensive rearing conditions found on reduced growth rate, reduction of fertility commercial fish farms. Consideration must in broodstock, reduced carcass quality be given to all the serotypic variants of the owing to pigmentation at the site of disease agent, the time and age at which injection, difficulty in administration the animal is most susceptible to disease, owing to high viscosity and an increased the route of administration and the method hazard to human operators if accidentally of vaccine preparation (i.e. killed, at- self-injected. The use of mineral oil tenuated, subunit, recombinant). adjuvants has been accepted in Norway, All the commercial vaccines currently but a licence for their use has not yet been available comprise inactivated (killed) granted in the United Kingdom. disease agents. When that approach failed in the development of vaccines, par- Index of efficacy ticularly viral vaccines, live attenuated The effectiveness of fish vaccines is vaccines were developed. calculated in terms of relative percent Whenever a live vaccine is used there is survival (RPS) using the following formula: always concern that the attenuated strain (usually the result of a gene deletion) may percent vaccinate mortality back-mutate and revert to the virulent wild RPS = 1 x100 type. Many of the successful vaccines percent control mortality against viral diseases of humans (e.g. rubella, measles, poliomyelitis) and in Amend and Fender (1976) describe the domestic animals (e.g. rabies, distemper) additional considerations which need to are attenuated organisms. The licensing of be met. such vaccines may, however, prove to be During the development of thesecom- very difficult in aquaculture. An alternative mercial vaccines many parameters im- approach has been to prepare subunit portant to the success of vaccination have vaccines using recombinant technology, been determined, for example the route of where the specific components of the administration, the length of protection disease-causing agents are isolated and, obtained, the size of the fish and the following amplification, used in vaccines. temperature dependence of the immune To increase the amount of antigen response (Johnson and Amend, 1983a and available, amplification is acheived by 1983b; Johnson, Flynn and Amend, 1982a cloning the genes coding for specific and 1982b; Tatner and Horne, 1985). antigens and incorporating them into Vaccine manual 133

bacterial DNA, where they are expressed. It has been reported that certain com- Using fermentation technology for the ponents of the extracellular proteins (ECP) growth of bacterial cells, expressed from Renibacterium salmoninarum, for "foreign" proteins (antigens) can be example the 57 kDa protein, are immuno- produced in bulk. suppressive (Turaga, Wiens and Kaattari, 1987). These antigens must therefore be Bacterial vaccines omitted (or modified) from any successful Numerous fish vaccines are currently being vaccine preparation. Hastings and Ellis developed, although some of them with (1988) showed that rainbow trout only only limited success. For example, responded to five out of 30 ECP com- vaccination would be an ideal approach ponents from A. salmonicida, while rabbits for controlling bacterial kidney disease responded to 15. Thus, it is important to (BKD) because it is widespread and its establish which antigens the fish respond control by means of chemotherapy is to and whether these are protective. unsatisfactory. Experimental vaccination of Current research in the United Kingdom coho salmon (Oncorhynchus kisutch) and and Canada is based on the purification of sockeye salmon (Oncorhynchus nerka) antigens from R. salmoninarum and its indicated that agglutinating antibodies extracellular products, and includes were produced but the response was slow investigation into their effects on the to develop and these antibodies were immune system prior to their incorporation not protective (Evelyn, 1971; Evelyn, into a recombinant vaccine. Ketcheson and Prosperi-Porta, 1984; The other vaccines currently being Baudin-Laurencin, Vigneulle and Mevel, developed against bacterial fish pathogens 1977). Kaattari et al. (1987 and 1988) include atypical A. salmonicida, A. hydro- investigated potential BKD vaccines phila, Edwardsiella ictaluri, Flexibacter containing the highly immunogenic Vibrio columnaris, Streptococcus sp. and Pasteurella vaccine components as an adjuvant; piscicida. however, the results were inconsistent. Atypical furunculosis (caused by Paterson, Desautels and Weber (1981) atypical non-pigmented A. salmonicida) is and McCarthy, Croy and Amend (1984) the main bacterial disease affecting achieved the most promising results in Icelandic fish farming. An experimental Atlantic salmon and rainbow trout, where vaccine given by injection has been protection was conferred against a natural developed (B. Gudmundsdottir, personal challenge and experimental challenge, communication). The protective antigen respectively. One vaccine was adjuvanted was identified by passive immunization of with Freund's complete adjuvant while the Atlantic salmon with antisera against other was pH-lyzed and non-adjuvanted. purified antigen raised in rainbow trout Other researchers (Bruno and Munro, 1984; and rabbits. A method to culture the Sakai, Atsuta and Kobayashi, 1993) have bacterium with the maximal expression of been unable to reproduce these results, and protective antigen has also been developed. so the search for a BKD vaccine continues. The vaccine results in 70 to 100 percent The preparations tested so far appear to be RPS when administered with mineral oil inadequate. This may be because they as an adjuvant to Atlantic salmon parr. contain inappropriate antigens and, which- Protection appears to last for 12 months ever protective antigens are present, are and there is a good correlation between only weakly immunogenic or responses to antibody production and protection. Field them are suppressed by other antigens. trials have not yet been completed. 134 Fish vaccines

The antigenic diversity of A. hydrophila searchers in Japan, the United Kingdom is the major limitation in the development and Italy are continuing with development of an effective vaccine and it seems likely work to determine an immune response that a polyvalent preparation will be to the bacterium and investigate the necessary. Although there is some doubt pathogenesis of the disease. as to whether these bacteria ever act as a primary pathogen, they do make a Viral vaccines significant contribution to the disease The development of a successful vaccine process in the fish they invade. As part of by culturing the causative agent under the development of a vaccine, research defined conditions and subsequently groups in Southeast Asia are currently inactivating it is not always feasible. It may investigating the immune response of carp be necessary to select protective antigens and catfish to A. hydrophila antigens. and engineer a recombinant vaccine Moore, Eimers and Cardell (1990) genetically. This appears to be the case for demonstrated the feasibility of immuniz- most fish viral vaccines under devel- ing channel catfish against columnaris opment and requires the molecular cloning disease by immersion vaccination with and expression of the viral genes in the formalin-inactivated Flexibac ter columnaris. bacterium Escherichia coli or the yeast, However, a commercial vaccine has not Saccharomyces cerevisae. yet been developed. Viral haemorrhagic septicaemia (VHS) Streptococcal and Pasteurella spp. in- is a fish rhabdovirus responsible for severe fections, hitherto reported principally in losses in many continental European trout Japanese marine cultured fish populations, farms. It is an economically devastating have recently become a problem in disease for the aquaculture industry, since Mediterranean countries. The Japanese it may affect all age groups. In the past, researchers Iida, Wakabayashi and Egusa inactivated vaccines have been developed; (1982) and Sakai et al. (1987) reported that however, these were immunogenic only experimentalvaccination provided when given by injection (de Kinkelin, protection from streptococcal infections, 1988). Following the failure of inactivated but a commercial vaccine is not yet vaccines, several live vaccines were available. There appear to be two developed and appeared to provide serotypes. A cocktail immersion vaccine to protection against some of the serotypes. prevent Streptococcus sp. and V. anguillarum The vaccine strains were attenuated by is currently being tested in Japan and field successive passage in cell lines, but trials are also being carried out in Italy significant mortality associated with the (Ghittino, personal communication)on a virus was still observed and 2 to 13 percent potential Streptococcus vaccine. of the fish died as a result of the vaccination Pasteurellosis has been of huge economic alone (de Kinkelin and Bearzotti-Le Berre, significance to yellow tail culture in Japan 1981; Bernard, de Kinkelin and Bearzotti- and, in 1991, was almost simultaneously Le Berre, 1983). reported as affecting sea bream in Italy, More recently, a subunit vaccine for VHS France and Greece. Thereappears to be has been developed using recombinant homogeneity within this species of DNA technology. The protective epitopes bacterium, although little has been of VHS appear to be locatedon the surface published on its virulence factors and glycoprotein of the virus, and antibody protective antigens. A Pasteurella vaccine directed against the viral glycoprotein- is currently being field tested andre- neutralized viral infectivity (de Kinkelin, Vaccine manual 135

Bernard and Hattenberger-Baudovy, 1984; Studies by Bootland, Dobos and Lorenzen, Oleson and Vestergaard- Stevenson (1990) suggest that the age and Jorgensen, 1990). The gene coding for the size of the fish at the time of immunization VHS glycoprotein was cloned and expressed are important factors in the development in bacteria (Thiery et al., 1990) and yeast of protective immunity. They reported that (Lorenzen, 1991) to produce the glycoprotein only fry immunized at two to three weeks inexpensively and in large quantities. after hatching were protected. It appears Preliminary results indicate that this that very young fish are capable of material induced protection following responding to vaccination but that growth injection but no studies on immersion rates must also be considered. These fish vaccination have been reported (Jorgensen, responded while in a slow weight-gaining 1992). Thus, for VHS, a recombinant subunit phase. vaccine may be a promising way of pro- Two attenuated IPN vaccines have also ducing an inexpensive safe vaccine in fish. been tested and these appear to have Recent results obtained by Lecoq- provided protection when applied by Xhonneux et al. (1993), however, suggest immersion (Dorson, Castric and Torchy, that further research is needed prior to 1978; McAllister, 1984). Further devel- commercial exploitation, since the yield of opment was not pursued. Instead, as with antigens expressed was low and there was other viral vaccines, many researchers are a problem with the route of delivery of the following the route of recombinant subunit vaccine (i.e.it is only effective when vaccines for IPN (Havarstein et al., 1990; introduced intraperitoneally, not orally). Lawrence et al., 1989; Hah, Park and Jeong, Several research groups are actively 1992). In these studies, the major capsid developing vaccinesforinfectious protein VP2 has been identified as the pancreatic necrosis virus (IPN). This is a virion protein responsible for inducing very important pathogen of farm-reared protective immunity in fish. salmonids. Mortality is highest in young The drawbacks of killed and attenuated fish and survivors become life-long vaccines (Leong, Fryer and Winton, 1988) carriers, thereby maintaining the virus in have led research efforts to develop an the population by the continual shedding effective subunit vaccine for infectious and transmission of the disease. The virus haematopoietic necrosis (IHN) virus is serotypically heterogenous and has (Leong et al., 1992). During these studies, two major serotypes, the first of which monoclonal antibodies to the virion proteins comprises nine subtypes which are all were produced, enabling characterization pathogenic for salmonid fish. of the fish immune response to viral antigens Several inactivated IPN vaccines have and the development of in situ hybridization been tested but only vaccine administered probes to detect the virus in vaccines. IHN by injection-induced protection. The is caused by a rhabdovirus and produces a preferred route of immersion (since severe disease among fry and juveniles of infected fish are usually small) was susceptible species of salmonid fish. ineffective (Dorson, 1977). This was also Engelking and Leong (1989) demonstrated found when individual virion poly- that the viral glycoprotein purified from peptides were tested. The virion proteins one isolate could induce protective appeared to have lost their antigenicity immunity in fish to a wide variety of IHN following disruption with sodium dodecyl virus isolates from different geographical sulphate, urea and acetic acid (Hill, Dorson locations and different fish species. In and Dixon, 1980). consequence, the viral glycoprotein gene 136 Fish vaccines

from one strain was cloned and a re- This was protective both by injection and combinant vaccine prepared. Further immersion. Booster vaccination appeared to studies have identified a specific immuno- increase the efficacy of the vaccine greatly dominant region in the middle of the gene (Walczak, Noga and Hartman, 1981). This (Xu et al., 1991; Mourich and Leong, 1991). vaccine is not available commercially and A field trial (immersion immunization) of has not been licensed, probably because of the IHN subunit vaccine was undertaken concerns regarding the risk of reversion to in Idaho, United States, and showed it to virulence and the possible establishment be very effective in inducing protective of carriers among vaccinated fish. If carrier immunity. A commercial vaccine, however fish are generated as a result of vaccination, is not currently available. these fish can no longer be certified as Two other viral vaccines currently under virus-free broodstock. Attempts to develop development for non-salmonid species are a killed CCV vaccine have been hindered those for SVC and channel catfish virus by its poor immunogenicity (Plumb, 1973). (CCV). Despite a commercial inactivated A subunit vaccine for CCV was recently SVC vaccine being available in the Czech developed (Awad, Nusbaum and Brady, Republic and Slovenia, attenuated vaccines 1989). The envelope components of the have also been developed (Fijan et al., 1977) virus appear to be capable of inducing and appear to offer protection. However, protective immunity and, once these have although the live vaccine in particular been determined more specifically, an provided excellent protection, the vaccin- effective recombinant vaccine may be ated fish appeared to become asymptom- possible. atic carriers of the virus. When these fish were reared beside unvaccinated stock, an Parasitic vaccines SVC outbreak occurred. A subunit vaccine Monoclonal antibody (MAb) probes are is therefore an attractive alternative. A proving to be useful tools in the devel- reliable challenge model for the disease opment of vaccines against sea lice also needs to be developed so that vaccine (Lepeophtheirus salmonis) and the protozoan potency testing can be performed under infection proliferative kidney disease standard conditions. (PKD) in salmonids. A vaccine against sea CCV, caused by Herpesvirus ictaluri, is an lice remains a high priority for the acute, highly infectious disease of juvenile aquaculture industry. Monoclonal anti- channel catfish (Ictalurus punctatus). The bodies were used to produce extracts from onset of the disease is sudden and 100 lice and to select individual antigens from percent mortality can occur within ten days. a sea louse recombinant DNA library Once fish are infected with the virus, (Andrade-Salas et al., 1993). These clones survivors become carriers for their entire form the basis of experimental vaccines lives. At present, CCV outbreaks are which are currently under investigation in prevented by management strategies such the United Kingdom. as the use of resistant strains of channel PKD is the most economically damaging catfish and virus-free broodstock for the disease affecting the trout industry in stocking of fish farms. Vaccination would Europe. MAb probes have been produced seem to be the ideal strategy in areas where against PKX, the causative agent, in an CCV is endemic. An attenuated live effort to map out the antigens on the vaccine was developed by serial passage parasite's surface (Adams, Richards and in a tissue culture cell line derived from the Marin de Mateo, 1992) and have shown walking catfish (Noga and Hartman, 1981). that PKX from different species of fish and /accine manual 137

different geographical locations share research on the immune responses of new common antigens (Marin de Mateo et al., species as well as for studies on genetically 1993). At present, studies are under way to resistant species in parallel with vaccine culture the parasite and investigate development. protective antigens. A successful parasite vaccine has been Immunostimulants. An approach recently described by Woo and Li (1990) working taken by the aquaculture industry is the in Canada. Live attenuated Cryptobia use of immunostimulants, either by salmositica, a pathogenic haemoflagellate themselves to counteract stress-induced causing cryptobiosis, was injected intra- immunosuppression in fish, or in vaccines peritoneally into rainbow trout but the as adjuvants. They may serve to boost the strategy for the delivery of the vaccine has immune system in the short term and still to be resolved. therefore have great potential. One group Numerous otherparasitescause receiving a great deal of attention at present significant losses to the aquaculture is the P-glucans. They apparently function industry, for example Icthyobodo necatrix, as immunostimulants of the non-specific Ichthyopthirius multifiliis and Trichodina sp., defence mechanism of fish (Robertsen, but their control by vaccination has not yet Engstad and Jorgensen, 1994). been attempted. Glucans are major structural poly- saccharides from fungal and yeast cell Future prospects walls, composed of glucose units which Fish vaccines have become much more are held together through 13-1,3 and 3-1,6 sophisticated in recent years, with the trend bonds (Rosenberg, 1976; Duffus, Levi and being for the development of subunit Manners, 1982). Both soluble P-glucans recombinant vaccines in preference to the (e.g. scleroglucan, schizophyllan and original killed whole-cell preparations. lentinan) and microparticulated P-glucans This has been necessary because the from yeast (M-Glucan, Macroguard®) have simpler approach did not succeed for many been shown to function as immuno- of the important diseases and attempts at stimulators in fish (Robertsen, Engstad and attenuated vaccines in general have not Jorgensen, 1994). Intraperitoneal injections been encouraging from a safety point of glucans have resulted in enhanced of view. The cost of producing such disease protection of carp to Edwardsiella "high-tech" vaccines must however be tarda infection (Yano, Mangindaan and considered and the costs and benefits Matsuyama, 1989; Yano, Matsuyama and weighed up prior to commercialization. Mangindaan, 1991) and yellow tail to Direct DNA vaccination has recently Streptococcus spp. (Matsuyama, Mangin- been successfully performed in cattle daan and Yano, 1992). Yeast glucan has against infectious bovine rhinotracheitis been found to increase disease resistance virus (Cox, Zamb and Basiuk, 1993). in Atlantic salmon to Vibrio anguillarum, Muscles can apparently take in naked DNA V. salmonicida, Yersinia ruckeri and Aero- and then express it for long periods (Wolff monas salmonicida (Robertsen etal., 1990; et al., 1992). DNA vaccination may prove Robertsen, Engstad and Jorgensen, 1994) to be a more cost-effective method of and in channel catfish to Edwardsiella vaccinating fish against viral infections in ictaluri (Chen and Ainsworth, 1992). the future. The exact mode of action of 'glucans As the range of cultured species remains unclear, but enhanced protection increases, there is also a need for basic against microbial pathogens, observed 138 Fish vaccine,

after administering glucans to fish,cor- SOS Publications. relates with increased blood lysozyme and Amend, D.F. & Fender, D.C. 1976. Uptake complement activities and enhanced of bovine serum albumin by rainbow phagocytosis and killing of bacteria by trout from hyperosmotic solution: a headkidney macrophages (Yano, Mangin- model for vaccinating fish. Science, 192: daan and Matsuyama, 1989; Engstad, 793-794. Robertsen and Frivold, 1992; Chen and Andrade-Salas, O., Sommerville, C., Ainsworth, 1992; Matsuyama, Mangin- Wootten, R., Turnbull, T., Melvin, W., daan and Yano, 1992; Jorgensen et al., 1993). Amezaga, T. & Labus, M. 1993. Immuno- It is believed that such macrophagesmay histochemical screening and selection have an essential role to play in immuno- of monoclonal antibodies to salmon stimulation, since it has been shown that louse, Lepeophtheirus salmonis (Kroyer, Atlantic salmon macrophagespossess 1837). In E. Boxshall & D. Defaye, eds. receptors for f3-glucan (Robertsen, Engstad Pathogens of wild farmed fish: sealice, p. and Jorgensen, 1994). 323-331. Chichester, UK, Horwood (Ellis). Robertsen, Engstad and Jorgensen (1994) Awad, M.A., Nusbaum, K.E. & Brady, Y.J. suggested that non-specific defence may 1989. Preliminary studies of a newly be more effective against opportunistic developed subunit vaccine for channel pathogens than specific bacteria. Glucans, catfish disease. J. Aquat. Anim. Health, therefore, probably benefitmost during 1: 233-237. stress-related conditions, such as handling, Baudin-Laurencin, F., Vigneulle, M. & transportation or smoltification, when Mevel, M. 1977. Premières observations an increased susceptibility to disease sur la corynebactériose des salrnonides occurs. Glucan-supplemented diets and en Bretagne. Bull. Off.int. Epiz., 87: furunculosis vaccines arenow com- 505-507. mercially available. However, the dose, the Bernard, J., de Kinkelin, P. & Bearzotti-Le type of glucan and the route of admini- Berre, M. 1983. Viral haemorrhagic stration must all be considered when septicaemia of trout: relation between examining the effects of glucanson disease the G polypeptide, antibody production resistance (Ainsworth, Mao and Boyle, and protection of the fish following 1994). Further studies are requiredto infection with the F25 attenuated variant standardize these parameters. strain. Infect. Immun., 39: 7-14. Bootland, L.M., Dobos, P. & Stevenson, BIBLIOGRAPHY R.M.W. 1990. Fry age and size effects on immersion immunization of brook Adams, A., Richards, R.H. & Marinde trout, Salvelinus fontalis Michelle, against Mateo, M. 1992. Development ofmono- infectious pancreatic necrosis virus. J. clonal antibodies to PKX, the causative Fish Dis., 13: 113-125. agent of proliferative kidney disease. J. Bruno, D.W. & Munro, A.L.S.1984. Fish Dis., 15: 515-520. Epidemiological studies of bacterial Ainsworth, A.J., Mao, C.P. & Boyle, C.R. kidney disease in Scotland. In Int.Sem. 1994. Immune response enhancement Fish Pathology for the 20th Anniversaryof in channel catfish, Ictalurus puncta tus, the Japanese Society of Fish Pathology,p. using f3-glucan from Schizophyllum 57-58. Tokyo. commune. In J.S. Stolen & T.C. Fletcher, Busch, R.A. 1982. Enteric redmouthdisease eds. Modulators offish immune responses, (Yersinia ruckeri). In D.P. Anderson,M. Vol. 1, p. 67-81. Fair Haven, N.J., USA, Dorson & P. Dubourget, eds.Antigens Vaccine manual 139

offish pathogens, p. 201-223. Lyon, France, Engstad, R.E., Robertsen, B. & Frivold, E. Marcel Merieux. 1992. Yeast glucan induces increase in Chen, D. Sr Ainsworth, A.J. 1992. Glucan lysozyme and complement-mediated administration potentiates immune haemolytic activity in Atlantic salmon defence mechanisms of channel catfish, blood. Fish Shellfish Immunol., 2: 287- Ictalurus punctatus. J. Fish Dis., 15: 295- 297. 304. Evelyn, T.P.T. 1971. The agglutinin response Cox, G.J.M., Zainb, T.J. & Basiuk, L.A. 1993. in sockeye salmon vaccinated intra- Bovine herpes virus I: immune responses peritoneally with a heat-killed prepar- in mice and cattle injected with plasmid ation of the bacteria responsible for DNA. J. Virol., 67(9): 5664-5667. salmonid kidney disease. J. Wildl. Dis., de Kinkelin, P. 1988. Vaccination against 7: 328-335. viral haemorrhagic septicaemia. In A.E. Evelyn, T.P.T., Ketcheson, J.E. & Prosperi- Ellis, ed. Fish vaccination, p. 172-192. Porta, L. 1984. On the feasibility of London, Academic. vaccination as a means of controlling de Kinkelin, P. & Bearzotti-Le Berre, M. bacterial kidney disease in Pacific 1981. Immunization of rainbow trout salmon. In Abstr.: Int. Conf Biology of against viral haemorrhagic septicaemia Pacific Salmon, 5-12 September 1984. (VHS) with a thermoresistant variant Victoria / Agassiz, British Columbia, of the virus. Dev. Biol. Stand., 49: 431- Canada. 439. Fijan, N., Petrinec, Z., Stanel, Z., Kezic, N. de Kinkelin, P., Bernard, J. & Hattenberger- & Teskeredzic, E. 1977. Vaccination of Baudovy, A.M. 1984. Immunization carp against spring viraemia. Compari- against viral diseases occurring in cold water. son of intraperitoneal and peroral Symposium on Fish Vaccination, Paris, application of live virus to fish kept in Office International des Epizooties. ponds. Bull. Off int. Epiz., 92: 1055-1068. Dorson, M. 1977. Vaccination trials of Fryer, J.L., Rehovec, J.S. & Garrison, R.L. rainbow trout fry against infectious 1978. Immunisation of salmonids for pancreatic necrosis. Bull. Off. int. Epiz., control of vibriosis. Mar. Fish. Rev., 40: 57: 405-406. 20-23. Dorson, M., Castric, J. & Torchy, C. 1978. Hah, Y.C., Park, J.W. Sr Jeong, G. 1992. Infectious pancreatic necrosis virus of Neutralize epitope of DRT serotype of salmonids: biological and antigenic infectious pancreatic necrosis virus features of a pathogenic strain and a (IPNV) isolated in Korea. In Proc. Int. non-pathogenic variant selected in RTG- Symp. Infectious Viruses in Fish, 9-11 2 cells. J. Fish Dis., 1: 309-320. October 1992, p. 13-22. Seoul. Duff, D.C.B. 1942. The oral immunisation Hastings, T.S. & Ellis, A.E. 1988. The humoral of trout against Bacterium salmonicida. immune response of rainbow trout and J. Immunol., 44: 87-94. rabbits to Aeromonas salmonicida extra- Duffus, I.H., Levi, C. & Manners, D.J. 1982. cellular products. J. Fish Dis., 11: 147- Yeast cell-wall glucans. Adv. Microbiol. 160. Physiol., 23: 151-181. Harvarstein, L.S., Kalland, K.H., Christie, Engelking, H.M. & Leong, J.C. 1989. The K.E. & Endressen, C. 1990. Sequence of glycoprotein of infectious hematopoietic the large double-stranded RNA segment necrosis virus elecit neutralizing anti- of the NI strain of infectious pancreas body and protective responses. Virus necrosis virus: a comparison without Res., 13: 213-230. Birnaviridae. J. Gen. Virol., 71: 299-308. 140 Fish vaccines

Hill, B., Dorson, M. & Dixon, P.F. 1980. Kaattari, S.L., Holland, N., Turaga, P. & Studies on immunization of trout against Wiens, G. 1987. Development of a IPN. In W. Ahne, ed. Fish diseases. Third vaccine for bacterial kidney disease. COPRAQ-Session, p. 29-36. Berlin, Bonneville Power Administration Project Springer. 86-45. In Annual Report 1986. Portland, Hirst, I.D. & Ellis, A.E. 1994. Iron-regulated Oreg., USA, Bonneville Power Admini- outer membrane protein of Aeromonas stration. salmonicida are important protective Kaattari, S.L., Chen, D., Turaga, P. & Wens, antigens in Atlantic salmon against G. 1988. Development of a vaccine for furunculosis. Fish Shellfish Immunol., 4(1): bacterial kidney disease. Bonneville 29-46. Power Administration Project 86-46. In Iida, T., Wakabayashi, H. & Egusa, S. 1982. Annual Report 1987. Portland, Oreg., USA, Vaccination for control of streptococcal Bonneville Power Administration. disease in cultured yellowtail. Fish Karunasagar, I., Rosalind, G. & Karunasagar, Pathol., 16: 201-206. I. 1991. Immunological response of the Johnson, K.A. & Amend, D.F. 1983a. Efficacy Indian major carpsto Aeromonas of Vibrio anguillarurn and Yersinia ruckeri hydrophila vaccine. J. Fish Dis., 14: 413-417. bacterins applied by oral and anal Kawai, K., Kusuda, R. & Itami, T. 1981. intubation of salmonids. J. Fish Dis., 6: Mechanisms of protection in ayu orally 473-476. vaccinated for vibriosis. Fish Pathol., 15: Johnson, K.A. & Amend, D.F. 1983b. 257-262. Comparison of efficacy of several deliv- Lawrence, W.R., Nagy, E., Duncan, R., Krell, ery methods using Yersinia ruckeri P. & Dobos, P. 1989. Expression in E. bacterins on rainbow trout, Salmo coli of the major outer capsid protein of gairdneri Richardson. J. Fish Dis.,6: infectious pancreatic necrosis virus. Gene, 331-336. 79: 369-374. Johnson, K.A., Flynn, J.K. & Amend, D.F. Lecoq-Xhonneux, F., Thiery, M., Dheur, I. 1982a.Duration of immunity in & de Kinkelin, P. 1993. A recombinant salmonidsvaccinatedbydirect viral haemorrhagic septicaemia virus immersion with Yersinia ruckeri and (VHS) glycoprotein expressed in insect Vibrio anguillarum. J. Fish Dis., 5: 207-213. cells induces protective immunity in Johnson, K.A., Flynn, J.K. & Amend, D.P. rainbow trout. Paper presented at 1982b. Onset of immunity in salmonid EuropeanAssociation of Fish fry vaccinated by direct immersion in Pathologists 6th Int. Conf., Diseases of Vibrio anguillarum and Yersinia ruckeri. Fish and Shellfish, Brest, France. J. Fish Dis., 5: 197-205. Leong, J.C. & Fryer, J.L. 1993. Viral vaccines Jorgensen, P.E. 1992. Recent advances in for aquaculture. Ann. Rev. Fish Dis., surveillance and control of viral haemor- p. 225-240. rhagic septicaemia (VHS) in trout. In T. Leong, J.C., Fryer, J.L. & Winton, J.R. 1988. Kimura, ed. Proc. OJI Int. Symp. Salmonid Vaccination against infectious hemo- Diseases, p. 60-71. Sapporo, Japan, topoietic necrosis virus. In A.E. Ellis, Hokkaido University Press. ed. Fish vaccination, p. 193-202. London, Jorgensen, J.B., Sharp, G.J.E., Secombes, Academic. C.J. & Robertsen, B. 1993. Effects ofa Leong, J.C., Anderson, E., Bootland, L., Chen, yeast cell wall glucan on the bactericidal L., Drolet, B., Engelking, H.M., Mason, activity of rainbow trout macrophages. C., Mourich, D., Johnson, K., Trobridge, Fish Shellfish Immunol., 3(4): 267-277. C. & Wirkkula, J. 1992. Biotechnological Vaccine manual 141

approaches to the development of Vertebrates, p. 93-100. Corvallis, Oreg., salmonid fish vaccines. In T. Kimura, USA, ed. Proc. OJI Int. Symp. Salmonid Diseases, Noga,E.J.& Hartman,J.X.1981. p. 250-255. Sapporo, Japan, Hokkaido Establishment of walking catfish (Clarias University Press. batrachus) cell lines and development Lorenzen, N. 1991. Viral haemorrhagic of a channel catfish (Ictalarus punctatus) septicaemia virus of rainbow trout: virus vaccine. Cana. J. Fish, Aquat. Sci., immunological investigations in relation 38: 925-930. to vaccine development. University of Paterson, W.D., Desautels, D. St Weber, Copenhagen. (Ph.D. thesis) J.M. 1981. The immune response of Lorenzen, N., Oleson, J.F. & Vestergaard- Atlantic salmon, Salmo salar L., to the Jorgensen, P.E. 1990. Neutralisation of causative agent of bacterial kidney Egtved virus pathogenicity to cell disease, Renibacterium salmoninarum. J. cultures and in fish by monoclonal Fish Dis., 4: 99-111. antibodies to the viral G protein. J. Gen. Plumb, J.A. 1973. Neutralisation of channel Virol., 71: 561-567. catfish virus by serum of channel catfish. Marin de Mateo, M., Adams, A., Richards, J. Wadi. Dis., 9: 324-330. R.H., Castagnaro, M. & Hedrick, R.P. iloberts, R.J., Willoughby, L.G. & Chinabut, 1993. Monoclonal antibody and lectin S. 1993. Mycotic aspects of epizootic probes recognise developmental and ulcerative syndrome (EUS) of Asian sporogonic stages of PKX, the causative fishes. J. Fish Dis., 16: 169-183. agent of proliferative kidney disease in Robertsen, B., Engstad, R. & Jorgensen, European and North American salmonid J.B. 1994. j3-glucans as immunostimulants fish. Dis. Aquat. Organ., 15: 23-29. in fish. In J.S. Stolen & T.C. Fletcher, Matsuyama, H., Mangindaan, R.E.P. & Yano, eds. Modulators offish immune responses, T.1992. Protective effects of shizophyllan Vol. 1, p. 83-99. Fair Haven, N.J., USA, and scleroglucan against Streptococcus SOS Publications. sp. infection in yellow tail (Seriola Robertsen, B., Rorstad, G., Engstad, R. & quinqueradiata). Aquaculture,101: 197-203. Raa, J. 1990. Enhancement of non-specific McAllister, P.E. 1984. Infectious pancreatic disease resistance in Atlantic salmon, necrosis immunisation. (American Salmo salar L., by a glucan from Saacharo- Fisheries Society)Fish Health Newsl., myces cervisae cell walls. J. Fish Dis., 13: 12: 6. 391-400. McCarthy, D.H., Croy, T.R. & Amend, D.F. Rosenberg, R.F. 1976. The cell wall. In J.E. 1984. Immunisation of rainbow trout, Smith & D.R. Barry, eds. The filamentous Salmo gairclneri Richardson, against fungi, Vol 2, Biosynthesis and metabolism, bacterial kidney disease: preliminary p. 328-334. London, Arnold (Edward). efficacy evaluation. J. Fish Dis., 7: 65-71. Sakai, M., Atsuta, S. & Kobayashi, M. 1993. Moore, A.A., Eimers, M.E. & Cardell, M.A. The immune response of rainbow trout 1990. Attempts to control Flexibacter (Oncorhynchus mykiss) injected with five columnaris epizootics in pond-reared Renibacterium salmoninarum bacterins. channel catfish by vaccination. J. Aquat. Aquaculture, 113: 11-18. Anim. Health, 2: 109-111. Sakai, M., Kubota, R., Atsuta, S. & Mourich, D.V. & Leong, J.C. 1991. Mapping Kobayashi, M. 1987. Vaccination of of the immunogenic regions of the IHNV rainbow trout Salmo gairdneri against glycoprotein in rainbow trout and mice. beta-hemolytic streptococcal disease. In Proc. 2nd Int. Symp. on Viruses of Lower Nippon Suisan Gakkaishi, 53: 1373-1376. 142 Fish vaccines

Tatner, M.F. & Horne, M.T. 1985. The effects Edwardsiella tarda infection, by some P- of vaccine dilution length of immersion 1,3 glucans. Nippon Suisan Gakkaishi, 55: time and booster vaccinations on the 1815-1819. protection levels induced by direct Yano, T., Matsuyama, H. & Mangindaan, immersion, vaccination of brown trout, R.E.P. 1991. Polysaccharide-induced Salmo trutta, with Yersinia ruckeri (ERM) protection of carp, Cyprinus carpio L., vaccine. Aquaculture, 46: 11-18. against bacterial infection. J. Fish Dis., Tebbit, G.L., Erikson, J.D. & Vande Water, 14: 577-582. R.B. 1981. Development and use of Xu, L., Mourich, D.V., Engelking, H.M., Yersinia ruckeri bacterins to control enteric Ristou, S., Arnzen, J. & Leong, J.C. redmouth disease. In W.V. Leetown, ed. 1991. Epitope mapping and charac- International Symposium on Fish Biologies: terization of the infectious hematopoietic Serodiagnos tics and Vaccines. Dev. Biol. necrosis virus glycoprotein, using fusion Stand., 49: 395-401. proteins synthesized in Escherichia coli. Thiery, M., Lecoq-Xhonneux, F., Dheur, I., J. Virol., 65: 1611-1615. Renard, A. & de Kinkelin, P. 1990. Molecular cloning of the mRNA coding for the G protein of the viral haemor- rhagic septicaemia (VHS) of salmonids. Vet. Microbioassay, 23: 221-226. Turaga, P., Wiens, G. & Kaattari, S.L. 1987. Bacterial kidney disease: the potential role of soluble protein antigen(s). J. Fish Biol., 31(supplement A):191-194. Vigneulle, M., Brevil, G., Ceschia, G. & Blanch, A. 1993. Vaccination trials of sea bass (Dicentrarchus labrax) against two serotypes of Vibrio anguillarum. European Association of Fish Pathol- ogists 6th Int. Conf., Diseases of Fish and Shellfish, Brest, France, Abstract No. 66. Walczak, E.M., Noga, E.J. & Hartman, J.X. 1981. Properties of a vaccine for channel catfish virus disease and a method of administration. Dev. Biol. Stand., 49: 419-429. Wolff, A., Ludtke, J.J., Acsadi, G., Williams, P. & Jani, A. 1992. Hum. Mol. Genet., 1: 363-369. Woo, P.T.K. & Li, S. 1990. In vitro attenuation of Cryptobia salmositica and its use as a live vaccine against crytobiosis in Onco- rhynchus mykiss. J. Parasitol., 76: 752-755. Yano, T., Mangindaan, R.E.P. & Matsuyama, H. 1989. Enhancement of the resistance of carp Cyprinus carpio to experimental Vaccine manual 143

e rciIÎoriFI Epizooiiies and internati nai o nizati ns invacie quaiity ndarolization M. Truszczynski and J. Blancou

Harmonization and standardization of on the final product. These controls are requirements for veterinary vaccines have performed in the producers' laboratory or two important goals: in a laboratory of their choice. *to keep the quality of vaccines at a high The registration files are examined by level; experts appointed by the ministry of health to prevent or reduce potential trade and, particularly, the national commission barriers. for veterinary drugs. The expert who Specialized groups established by go- examines the analytical section may vernments or groups of governments, such require additional controls if those as the European Union (EU), formulate described in the original file are in- legislation and requirements on the basis complete. These additional controls may of the latest technology to ensure the high have to be carried out in an officially quality and safety of these products. accepted laboratory, for instance if the Every new vaccine developed in a vaccine originates from a country for which country or imported from another has to there is little information on the quality of be registered according to the legislation the local control system. and requirements in force in the country When the registration data are adequate, where the vaccine will be used. the commission for veterinary drugs gives The registration file must report on the a favourable assessment to the minister of results of experiments directly related to health and the product is registered for the product itself (Pensaert, 1992) and marketing. include: After registration, every production *an analytical section, characterizing batch of the registered vaccine must be the different components and the controlled by the producer, both during substrates on which the antigens are manufacturing and at the final product produced; stage, and these controls are the respon- a toxico-pharmacological note, indicat- sibility of an industrial specialist. Further ing the absence of remaining patho- official controls are not required for the genicity or abnormal toxicity; majority of bacterial vaccines although, in a section on the product's efficacy, many cases, the bacterial strain used in the describing clinically oriented ex- vaccine is controlled annually by the periments with relation to immunity appropriate government institute. How- and protection (degree, duration, etc.). ever, all viral vaccines and also some The analytical section should include the bacterial vaccines (e.g. for brucellosis) must controls which have been carried out both undergo an official batch control by on the product during manufacturing and the national veterinary institute or an 144 Role of OIE and international organizations in vaccine quality standardization

equivalent institution before release. Every Veterinary Medicinal Products, the United imported vaccine must undergo an official States Department of Agriculture and batch control in an officially accepted international industry and government laboratory chosen freely by the importer. officials (Draayer, Hilsabeck and Miller, Additionally, a control is prescribed by the 1992; Folkers, 1992; Watson, 1992). It is national veterinary institute. suggested that annual meetings be held in For batch controls, representative conjunction with either FEDESA or AHI samples are collected under the respon- meetings. Issues to be dealt with include sibility of the laboratory and according to the standardization of test requirements, information supplied by the producer with time-frames for implementation and other regard to the number of doses per batch, issues that could reduce or prevent trade the homogeneity of the batch, conditions barriers without adversely affecting of storage, etc. product quality. In the laboratory, the samples are The main purpose of the working group examined to identify the active com- would be to harmonize European and ponent(s), to ascertain the absence of con- United States legislation as a necessary step taminating agents and to ensure the towards international standardization and product's sterility, safety and potency. For the prevention or reducion of potential these controls, the relevant monograph of trade barriers. the European Pharmacopoeia (see p. 149) There are, in addition, international is followed. If there is no monograph for a organizations acting in parallelalso with particular vaccine, the tests described in the goal of international harmonization the original registration file must be and standardization of vaccines but applied. For inactivated vaccines, the independently from governments and in potency test may be performed on the more general terms. These organizations target species or on a laboratory animal include the Office international des species but, in the latter case, the relation Epizooties (OIE), the World Health Orga- between the potency in the laboratory and nization (WHO) and the Food and Agri- that in the target species must be demon- culture Organization of the United Nations strated by the producer. For live vaccines, (FAO) as well as the Pharmacopoeias (e.g. potency is generally limited to quantitation the European, United States and Japanese of the organism or of the virus, using Pharmacopoeias). methods described in the original registra- tion file. If the results are satisfactory, the OFFICE INTERNATIONAL DES EPIZOOTIES batch will be given an official batch number The OIE, whose purpose is to harmonize and be released on to the market. and coordinate animal health activities Depending on the country or group of at the international level, also contributes countries, there are varying degrees of to the standardization of vaccine quality. difference in their legislation. With this in The three principal aims of the OIE mind and in the spirit of international (Truszczynski and Blancou, 1992) are: harmonization aimed at reducing or e The provision of information on animal preventing potential trade barriers, the health worldwide. United States Animal Health Institute °International coordination of research (ALII) has proposed the formation ofan into and control of certain animal international working group consisting of diseases. itself, the European Federation for Animal e The harmonization of import and Health (FEDESA), the EU's Committee for export regulations for animals and Vaccine manual 145

animal products at the international other diseases. The Fish Diseases Commission level. establishes standards for diagnostic A measure of the development and methods and vaccines for fish diseases. growing international recognition of the From this short description it can be OIE is that it was established by 28 concluded that all the Specialist Com- countries in 1924 while it now has 136 missions are interested in the international member countries. Seventeen are from the harmonization and standardization not Americas, 42 from Africa, 43 from Europe only of vaccines but also of diagnostic and 34 from Asia. The OIE operates under methods, including diagnostic biologicals. the authority of its International Com- These activities relate to those important mittee, formed by the delegates of the diseases included in the Lists A and B member countries, under the leadership of prepared by the OIE (Truszczynski and an elected president. Blancou, 1992). The Central Bureau, located at OIE's List A includes contagious diseases headquarters in Paris (12 rue de Prony), is which spread rapidly and the scope of headed by the Director-General, at present which extends beyond national borders. Dr J. Blancou. The Central Bureau imple- These diseases have particularly serious ments decisions of the International socio-economic or public health con- Committee and the different Commissions. sequences and are of major importance in There are five Regional Commissions, the international trade of animals and covering Africa, the Americas, Asia, the animal products. Far East and Oceania, Europe and the List B includes contagious diseases which Middle East. are considered of socio-economic and/ or There are also Specialist Commissions for public health importance within countries The International Animal Health Code; and which, naturally, are also of signif- Standards; Foot-and-Mouth Disease and icance to the international trade of animals other Epizootics; and Fish Diseases, includ- and animal products. ing those of crustaceans and molluscs. In Among these Specialist Commissions, addition, there are three working groups: the one most closely connected with the Animal Health Information Systems, standardization of diagnostic methods and Veterinary Drug Registration and Bio- vaccine quality is the Standards Com- technology. mission, whose role includes participation The International Animal Health Code in the standardization of biologicals, Commission draws up animal health including vaccines used for prophylactic recommendations for the import and purposes for List A and B diseases. export of animals and animal products and To accomplish this goal, the Standards contributes through the Animal Health Code, Commission undertakes the following an important OIE publication, to inter- activities: national harmonization in this area. The ° regular updating of the OIE manual of Standards Commission establishes standards standards for diagnostics tests and for diagnostic methods, including diagnos- vaccines; tic biologicals, and for vaccines. The Foot the organization of reference labor- and Mouth Disease and other Epizootics atories for several diseases in Lists A Commission contributes to the development and B; and standardization of vaccines against e the establishment of international foot-and-mouth disease and strategies for standards for diagnostic tests and the eradication or control of this as well as vaccines. 146 Role of OIE and international organizations in vaccine quality standardization

OIE manual of standards for diagnostic tests the standardization of diagnostic methods and vaccines and vaccines applied in the prophylaxis The purpose of the OIE manual (OIE, 1992) and control of List A and B diseases, is to provide a uniform approach to the especially those of greatest importance and diagnosis of important animal diseases and those causing high economic losses. The to the production and control of biological designation of these laboratories is pro- products, mainly vaccines used in the posed by the OIE Standards Commission control of List A and B diseases. This is and ratified by the OIE International implemented by the presentation in the Committee. Designation does not imply manual of standard methods for laboratory that financial support will be given by the diagnosis of diseases and for the pro- OIE. duction and control of vaccines and other The functions and responsibilities of the biological products for veterinary use in OIE reference laboratories are as follows: laboratories all over the world. e the provision of a centre of excellence The first edition of the manual was in a designated activity; published in three volumes in 1989-1991, the standardization of methodology; each covering about 30 List A or B diseases. e the storage and distribution of standard After revision and additional editorial work, antisera, antigens and other important a single combined volume was published reagents; as the second edition of the manual in 1992. the development of new methods; It contains information on 15 List A diseases the provision of consultant assistance and 75 List B diseases. In future, the to the 01E; Standards Commission plans to produce a training in a designated activity; new updated edition every four years. The e the organization of scientific meetings 1992 edition of the manual has been on behalf of the OIE; distributed worldwide, recommending: the coordination of collaborative e "prescribed tests" which should be studies; used for diagnosis; and the provision of assistance to the OIE requirements for vaccines, in relation in collecting and disseminating ne- to List A and B diseases. cessary information. This will undoubtedly help lead to an The Director-General of the OIE sent internationally unified approach to the letters to selected laboratories around the diagnosis and quality control of vaccines. world inviting them to become OIE reference laboratories for the more im- Organization of OIE reference laboratories portant diseases of Lists A and B. Because for List A and B diseases of the necessity of some regionalization, in The standardization of "prescribed tests" several cases a number of reference and the production and quality control of laboratories for the same disease were veterinary vaccines can only be achieved organized in different countries or regions. when the necessary standards are made At the beginning of 1994, 101 OIE reference available. The goal of standardization and laboratories were designated for 37 List A availability of standards is intended to be and B diseases or groups of diseases. The achieved through the OIE-organized majority of these laboratories developed reference laboratories. significant activities in accordance with These laboratories must fulfil a specific their responsibilities, and this is reflected function or range of functions at an in annual reports which they submit to the internationally recognized level related to OIE. Vaccine manual 147

OIE requirements for vaccines eradication campaigns (Pan-African As mentioned above, these requirements Rinderpest Campaign [PARC], Western are part of the OIE manual's chapters on Asia Rinderpest Eradication Campaign the List A and 13 diseases for which [WAREC] and South Asia Rinderpest vaccines are available. The chapters contain Eradication Campaign [SAREC]) in 53 general information, indicating recom- countries on three continents; foot-and- mended vaccines, data on seed manage- mouth disease control in Asia, Africa and ment (including characteristics of the South America; and Newcastle disease of vaccinal strains, culture, validation as a poultry in the same areas. vaccine), manufacture, in-process control During the last decade, through national and batch control (including sterility, and regional projects, FAO has provided safety and potency tests). expertise to individual laboratories and The Standards Commission is aware that assistance in the setting up of networks. the manual's coverage of standards for FAO has also cooperated with inter- vaccines is less comprehensive than it is of national organizations (WHO, OIE) in the diagnostic methods. Nevertheless, some standardization of particular products and chapters are exemplary and provide a good the holding of regional training courses on model for others to follow in the pre- vaccines and their associated technologies. paration of the next edition. The FAO Expert Consultation on Quality The Commission will, however, avoid Control of Veterinary Vaccines in Devel- any involvement in product licensing oping Countries, held in Rome, Italy, in procedures. It will be essential to ensure December 1991 (FAO, 1991; Rweyemamu, that the OIE manual keeps up to date with SyIla and Palya in FAO, 1993), recommend- developments in vaccine technology ed that closer cooperation be established through current advances in microbiology, with other international organizations to immunology and biotechnology. develop a more coherent approach on Following from this, the importance of guidelines for vaccine quality control. consistency between the OIE manual, EU FAO, WHO and OIE should work jointly regulations and the European Pharma- with developing countries to agree on copoeia must also be emphasized. priorities and carry forward regional In future, the OIE reference laboratories participation in standardization, including will prepare for distribution the standards the development of appropriate laboratory that are essential in vaccine production and facilities and post-licensing surveillance. control. The present role of the OIE is, This should include participation at the however, restricted to the distribution of national level as well as within regions information contained in the manual and and would require those countries with no which is undoubtedly contributing to procedures to begin standardization and international harmonization in the area of regulation, unless a decision has been taken vaccine production. to accept standards agreed by other countries or regions. Regional repositories OIE COOPERATION WITH OTHER of master seed stocks of vaccine strains, INTERNATIONAL ORGANIZATIONS cell lines and challenge strains should be FAO established to aid in attaining uniform FAO plays an important role in improving quality of vaccines. the quality standards of vaccines. Its main The consultation recognized the value interest is in developing countries and of the activities of the Pan African Vet- activities are concentrated on rinderpest erinary Vaccine Centre (PANVAC) and 143 Role of OIE and international organizations in vaccine quality standardization

recommended that FAO, together with the of a sample of unknown potency to be Organization of African Unity (OAU) and measured in a biological system and the Interafrican Bureau for Animal Re- expressed conveniently in international sources (IBAR), should solicit appropriate units. The international biological reference regional and international support for the preparation is a biological substance which conversion of PANVAC to the status of a may be used for a purpose similar to that long-term programme institute, with of a standard but which has been appropriate facilities and support. established without a full collaborative It was also stated that tests required by study, or after such a study has shown that national authorities to license or release it is not appropriate to establish it as an vaccines should be identical with those international standard. recommended by the appropriate inter- Besides this activity, the WHO Expert national organizations. The meeting Committee on Biological Standardization recommended the preparation of a manual establishes more general requirements for on veterinary vaccines, providing guide- biological products, among them vaccines lines for the production of veterinary for veterinary use. A good example is in vaccines in developing countries, as a the manufacturing and control require- project to be implemented by FAO and ments for rabies vaccines for veterinary the other international organizations use (WHO, 1981). concerned. With regard to products for veterinary use or of animal origin, the committee has World Health Organization established requirements for tuberculins WHO, located in Geneva, Switzerland, is a (human and bovine), anthrax spore vaccine specialized United Nations agency whose (live vaccine for veterinary use), immune primary responsibility is in international sera of animal origin, rinderpest cell culture and public health matters. However, it vaccine (live) and rinderpest vaccine (live), also plays an essential role in the standard- Brucella almrtus Strain 19 vaccine and ization of vaccine quality. Brucella melitensis Strain Rev.1 vaccine (live The WHO Expert Committee on for veterinary use). Biological Standardization is mainly Requirements are revised regularly for responsible for human pharmaceutical most products as major advances in products (vaccines) but also for some technology for manufacturing or quality veterinary biologicals and products of control are reported and accepted by the interest to animals as well as humans. The international scientific community. committee has published guidelines for the In addition to specific products, the preparation and establishmenSof reference ExpertCommitteeonBiological materials and reference reagents for Standardization also issues generalrecom- biological substances (WHO, 1978) and mendations on biological substances and definitions of these materials and reagents test systems, for example on the national have been formulated. According to these controlof vaccines and sera and definitions, an international biological requirements for immunoassay kits. It also standard is a biological substance to which regularly reviews and updates the list of WHO has assigned an international unit international biological reference prep- on the basis of data obtained in a arations and reagents. worldwide study. Technical units such as the Veterinary The prime function ofan international Public Health (VPH) Unit of WHOare biological standard is to enable the activity associated with the committee's work each Vaccine manual 49

time a substance or a subject falling within The second edition of the European a specific technical unit's domain of Pharmacopoeia has been published since expertise comes under discussion. 1980 in the form of fascicles. Publication is annual (between June and September) and European Pharmacopoeia implementation of the standards in all One effective approach to vaccine quality member countries is effective on 1 January standardization is based on the European of the following year. Pharmacopoeia (Artiges, 1992). The 19 Operationally, the roles of the pharma- signatory countriesthe 12 (in 1992) EU copoeias and the regulatory governmental Member States, countries of the European agencies (mentioned earlier) are com- Free Trade Association (EFTA) and Cyprus plementary: are committed to setting up common *the regulatory agencies approve new monographs and making them official in drugs and vaccines on the basis of their territories. The European Pharmaco- proven safety and efficacy and poeia has its own secretariat, admini- approved specifications, tests and stratively attached to the Council of the methodsofanalysisforeach EU, and a laboratory, both of which are application; located in Strasbourg. the Pharmacopoeia establishes public Monographs and general analytical standardsthatapplytoany methods are prepared by specialized manufacturer of a particular drug expert groups made up of scientists from substance or pharmaceutical product. universities and national control labor- In Europe, there isa close legal atories, etc. Before their final adoption, the connection between licensing systems and texts, which in most cases have been the pharmacopoeias regarding their legal object of interlaboratory cooperative tests, (Directive EEC/ 75 / 81 / 852) as well as are published in public inquiry form in the practical aspects (shared experts and a European Pharmacopoeia's Pharmeuropa, close relationship between secretariats). which appears four times a year in English The European Pharmacopoeia provides and French. detailed quality standards for vaccines The close cooperation between the through a series of monographs. These are European Pharmacopoeia and those assembled by an expert group and, once countries retaining their own national published, are mandatory for the member pharmacopoeias should be mentioned, i.e. states. There are currently 33 monographs Germany, Austria, Belgium, the United on veterinary vaccines and other Kingdom, France, Italy, Switzerland and biologicals. Among them are: anthrax the Nordic countries. The latter have spore live vaccine for veterinary use, avian brought all their general methods in line infectious bronchitis live vaccine (freeze- with those of the European Pharma- dried), canine distemper live vaccine copoeia. (freeze-dried), Clostridium botulinum To date, all general analytical methods vaccine for veterinary use, C. chauvoei are harmonized: there are 17 general vaccine for veterinary use, C. perfringens monographs defining the main pharma- vaccine for veterinary use, equine ceutical forms and about 800 monographs influenza vaccine (inactivated), foot-and- covering starting materials and certain mouth disease vaccine (inactivated), swine biological preparations such as immuno- erysipelas vaccine (inactivated) and swine sera and vaccines, both human and fever live vaccine (freeze-dried). veterinary. The monographs on veterinary vaccines 150 Role of OIE and international organizations in vaccine quality standardization

provide information on identification tests, European Union testing for contaminants and storage, Cooperation between OIE and the EU etc. The European Pharmacopoeia also (DGXII - Science, Research and Devel- provides a limited number of reference opment) in the control of veterinary preparationsforuseasworking reagents and vaccines has been initiated standards - among these preparations a (Lee Aileen, 1992). rabies vaccine and a bovine tuberculosis purified protein derivative (PPD). A B. BIBLIOGRAPHY melitensis, Rev.1 vaccine is also currently under study. Artiges, A. 1992. The role of the European Except in the case of biotechnology Pharmacopoeia. Dev. Biol. Stand., 79: products, the European Pharmacopoeia 87-93. maintains only limited liaison with other Draayer, H.A., Hilsabeck, L.J. & Miller, major pharmacopoeias such as those of the R.H. 1992. The American manufacturer's United States and Japan. For veterinary view on the EEC texts and harmo- vaccines the differences between these nization. Dev. Biol. Stand., 79: 75-83. pharmacopoeias are of considerable FAO. 1991. Report of the FAO Expert significance. Consultation on Quality Control of In general, the European Pharmacopoeia Veterinary Vaccines in Developing monographs are more detailed than the Countries, 2-6 December, Rome. OIE manual's requirements for biological FAO. 1993. Regional harmonization of vaccine products (OIE, 1992), but they only deal quality standards and stimulation of with vaccines of significance to Europe. vaccine technologies in developing Although several of these vaccines are countries. In Proc. Expert Consultation. also important to other continents, the Quality control of veterinary vaccines in European Pharmacopoeia may be less developing countries, p. 3-12. FAO Animal accessible, particularly in developing Production and Health Paper, No. 116. countries, than the OIE manual. Rome. In the next edition of the OIE manual, Folkers, C. 1992. The FEDESA's point of the sections on vaccines will be revised view. Dev. Biol. Stand., 79: 27-30. in the full light of standards already Lee Aileen, M.T. 1992. Presentation of the established in the European and other EEC Directive/ 81 / 852- Quality. Dev. major pharmacopoeias. Biol. Stand., 79: 39-41. OIE. 1992. Manual of standards for diagnostic Pan American Health Organization tests and vaccines for Lists A and B diseases The Pan American Health Organization of mammals, birds and bees. Paris. (PAHO) contributes towards the standard- Pensaert, M. 1992. Control of product batches ization and quality control of foot-and- (before and after registration)- the mouth disease (FMD) and some other Belgian approach. Dev. Biol. Stand., 79: vaccines in South America (FAO, 1991). 183-186. Through the work of PAHO, standards of Truszczynski, M. & Blancou, J. 1992. The vaccine production and quality control role of the Office international des in particular have been significantly Epizooties in the standardization improved in the last decade, andvac- of biologicals. Dev. Biol. Stand., 79: cination campaigns against FMD are 95-98. having a significant effect on the incidence Watson, J. 1992. Technical requirements of of the disease. veterinary vaccines for registration- Vaccine manual 151

FEDESA's point of view. Dev. Biol. Stand., Standardization. WHO Technical Report 79: 51-63. Series, No. 626. Geneva. WHO. 1978. Guidelines for the preparation and WHO. 1981. WHO Expert Committee on establishment of reference materials and BiologicalStandardization. WHO reference reagents for biological substances. Technical Report Series, No. 658. WHO Expert Committee on Biological Geneva.