Vaccination for control of Salmonella in poultry

Vaccine 17 (1999) 2538±2545

Vaccination for control of Salmonella in poultry L. Zhang-Barber a, 1, A.K. Turner b, P.A. Barrow c,* a

Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK b Peptide Therapeutics, 321 Cambridge Science Park, Cambridge CB4 4WG, UK c Compton Laboratory, Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK Received 18 August 1998; received in revised form 14 January 1999; accepted 9 February 1999

Abstract Salmonella spp. are facultative intracellular pathogens causing localised or systemic infections, in addition to a chronic asymptomatic carrier state. They are of worldwide economic and public health signi®cance. In poultry, which represent important sources of cheap protein throughout the world, fowl typhoid and pullorum disease continue to cause economic losses in those parts of the world where the poultry industries are continuing to intensify and where open sided housing is common. A number of serotypes that cause human gastro-enteritis are also increasing. The costs or impracticality of improvements in hygiene and management together with the increasing problems of antibiotic resistance suggest that vaccination in poultry will become more attractive as an adjunct to existing control measures. However, our understandings of the immunology of Salmonella infections in poultry is rudimentary and much poorer than that of equivalent infections in mice and live vaccine development for poultry has therefore been largely empirical. In addition to the killed Salmonella vaccines which have been used over the past few years with variable ecacy, a number of live vaccines have become available and some new vaccines will appear on the market over the next few years. These new vaccines should ful®l the criteria of ecacy, safety and compatibility with existing systems for monitoring infection before they are released on to a mass market. In this review we attempt to summarise the current understanding of Salmonella immunology in poultry together with the progress that has been made in poultry vaccine development. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; Salmonella spp; Mutants; Immune responses; Poultry

1. Introduction In recent years public health problems associated with Salmonella infection have increased, in particular, the risk to public health posed by Salmonella infection of poultry. The economic constraints inherent in the poultry slaughter process indicate that the control of Salmonella infection on the poultry farm would be the most practical approach, reducing the size of the problem [1]. It is possible to rear poultry that are totally free of Salmonella organisms, but this requires high

* Corresponding author. Tel.: +44-1635-577231; fax: +44-1635577263. E-mail address: [email protected] (P.A. Barrow) 1 Tel.: +44-171-5945254; fax: +44-171-5945255.

cost housing, tight control on feed quality, hygiene and management. However, the economics of production still depend on the importation of poultry or meat from countries where such levels of control may not be practised. It is thus increasingly recognised that biological measures will form an integral part of control programmes. This may be done with antibiotics, competitive exclusion or vaccines or combinations of these. Antibiotic therapy or prophylaxis has been used on its own or in combination with competitive exclusion. However, antibiotic therapy in food production animals is increasingly coming under close scrutiny, largely because of the fear of increased levels of resistance in food-borne human pathogens, such as Salmonella, Campylobacter and Escherichia coli [2]. Competitive exclusion involves the administration to newly hatched chicks of cultures of intestinal ¯ora de-

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rived from healthy adult chickens. This enhances the resistance of the young birds to intestinal infection. In this review we attempt to explain why vaccine control of the serotypes that typically cause food poisoning is generally less successful compared to the control of the small number of serotypes that produce typical `typhoid-like' diseases in poultry. 2. Pathogenesis of Salmonella in poultry and mammals Over 2400 di€erent Salmonella serotypes have been identi®ed. A relatively small number of these are known to be host-adapted, characteristically producing severe systemic disease in a restricted number of animal species. These serotypes include S. typhi and S. paratyphi which produce systemic disease in humans, S. gallinarum and S. pullorum in chickens, S. dublin in cattle, S. cholerae-suis primarily in pigs and S. typhimurium and S. enteritidis primarily in mice. Salmonella infection is normally via the oral route. The organisms rapidly invade the host through lymphoid tissue, including the Peyer's patches, the caecal tonsil in chickens (Barrow, Rychlik and Lovell, unpublished) and possibly also the enterocytes of the intestinal mucosa [3±5]. The mechanism whereby Salmonellae migrate from the sub-mucosa to lymph nodes and where they are ingested by phagocytic cells of the immune system is unknown. However, they reach the blood stream, probably intracellularly and reside in the spleen, liver and bone marrow. At this stage bacterial multiplication occurs, the rate of which depends on the virulence of the particular Salmonella strain and the genetic background of the host. Bacterial multiplication may result in host death, or bacterial numbers may reach a plateau before declining [6,7]. Infection of the gall bladder with S. typhi in man, or of lymphoid tissue in the small-intestinal wall with S. gallinarum or S. pullorum in chickens, results in Salmonella being excreted in the faeces. The amount and duration of excretion of these latter serotypes is relatively limited so that carcass contamination in poultry at slaughter is small and these serotypes do not regularly enter the human food chain [8]. These highly invasive serotypes can also infect the chicken ovary and lead to contamination of eggs which, in the case of S. pullorum, can persist for months. Of the other serotypes, few are known to cause typhoid-like disease in immunologically normal adult animals, with notable exceptions being S. typhimurium and S. enteritidis. However, many, including S. typhimurium and S. enteritidis, can colonise the alimentary tract of animals without causing disease so that their epidemiology can be complex. Infection of newly hatched chicks, which have a very simple gut ¯ora, results in massive multiplication of Salmonellae with

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large numbers being excreted in the faeces for several weeks. Thereafter, young chicks acquire a more complex normal gut ¯ora which is relatively inhibitory for Salmonella, so that infection results in excretion of fewer bacteria for a shorter period. Some strains, most notably S. typhimurium and S. enteritidis, can produce clinical disease in poultry under certain circumstances, such as in young chicks when the cells of the reticulo± endothelial system are immature, or during or after a period of physiological stress, for example in cold wet weather or during egg laying. In these cases young chicks develop a systemic infection and in addition large numbers of Salmonella are excreted in the faeces. The severity of the disease varies according to the serotype and strain, but generally there is always some invasion of the intestinal mucosa and lymphoid associated tissues. More aggressive invasion may lead to infection of the ovaries or oviduct, resulting in contamination of the egg contents. Alternatively the egg surface can become contaminated on passing through the oviduct, cloaca, or following laying. Any of these instances can result in the hatching of infected chicks. 3. Immune responses to Salmonella infection in poultry In comparison to the increasingly detailed information becoming available on the cellular and humoral responses to Salmonella infection in mice [9± 11], very little is known about how poultry respond to the di€erent types of infection. Although infection of chickens with S. typhimurium has been studied in some detail, the relative roles of the cell mediated and humoral immune response in clearance of S. typhimurium from the bird are still unclear. Avian immunology has been reviewed recently, including aspects of the secretory immune system [12±15]. Humoral responses have been monitored by agglutination techniques [16,17] or by ELISA [18]. Cellular responses to di€erent antigens were studied solely by delayed type hypersensitivity (DTH) responses [18], although it is appreciated that DTH is not necessarily a good indicator of cellular immunity. In this latter study, 4-dayold chicks were infected orally with S. typhimurium strain F98 which is invasive for poultry. The organism was isolated in considerable numbers from tissues for up to 9 weeks and from faeces for some time after 9 weeks. In the serum, high titres of IgG, IgM and IgA speci®c for LPS, ¯agella and outer membrane proteins were detected as early as 1 week post-infection. Speci®c antibody titres peaked at 3 weeks (IgM), 4 weeks (IgG) or 5 weeks (IgA). The titres of IgM and IgA dropped to low levels by 9 weeks whereas high titres of IgG were still detected for up to 9 months post-infection [19,20]. IgA titres from intestinal washings were higher and persisted for up to 9 weeks post-

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infection. At this time no bacteria were detected in the tissues or faeces from the majority of birds. In the early study, Lee et al. found that clearance correlated with cell-mediated immunity (CMI) responses rather than humoral responses [17]. It seems likely that CMI is responsible for tissue clearance, but how CMI could be responsible for intestinal clearance remains unclear. It has been shown, by cyclophosphamide and cyclosporin treatment, that impairment of B and T lymphocyte responsiveness does not a€ect intestinal colonisation in 5-day-old chicks [21], but birds at this age are exceptionally susceptible to colonisation following oral infection. There is no correlation between colonisation ability and tissue invasion per se and colonisation does not necessarily involve mucosa-associated tissue [22]. However, for S. typhimurium infection, virulent strains were eliminated from the gut of chickens more quickly than less virulent strains, suggesting that immune responses may be involved in intestinal clearance [22]. Hassan and Curtiss [23] have shown that highly virulent Salmonella strains inducing a transitory lymphocyte depletion and atrophy of the bursa and thymus and prolonged infection were also associated with faecal excretion for a long period. They concluded that vaccine strains which are more virulent are not necessarily more immunogenic. Other work has indicated that the early heterophil in®ltration into the intestine is an important indicator of the subsequent course of the infection [24,25]. Interestingly, naõÈ ve birds showed increased resistance to tissue invasion by wild-type Salmonella after they were administrated parenterally with cytokine-rich supernatant from concanavalin-stimulated T cells isolated from Salmonella immune birds [26]. In poultry, two types of Salmonella infection need to be considered: (1) an infection of the reticulo±endothelial system with little initial intestinal involvement, (2) an extensive intestinal infection by an organism which is invasive to di€erent degrees. S. gallinarum infection in immunologically mature birds is likely to be similar to S. typhimurium infection in mice. Full protection can be obtained at the level of the reticulo± endothelial system. The oral LD50 may be ca 104 cfu [27], although, in the ®eld, the inoculum may vary considerably. Oral inoculation with large numbers of challenge organisms may induce bacterial penetration to the reticulo±endothelial system, while a smaller number may be eliminated at the level of the gut (Barrow and Stocker, unpublished results). Infection of poultry with strains that are obviously invasive, such as S. typhimurium or S. enteritidis most likely belongs to the second type of Salmonella infection mentioned above. In the study of uptake of S. enteritidis and S. thompson by the caecal mucosa, macrophages containing Salmonella organisms can be visualised in the process of crossing the epithelial layer and lamina propria

across gaps in the basement membrane. This suggests that macrophages may carry bacteria to the circulation [3].

4. Vaccination against host-speci®c serotypes Vaccination of poultry, man or other animals with live attenuated strains derived from the host-speci®c Salmonella serotypes induces a strong protective immunity against reinfection in the host. Over the years live attenuated strains derived from the host-speci®c serotypes that infect cattle, pigs, fowl and man have been developed into e€ective vaccines that can be administered parenterally [11]. Attenuated strains of S. gallinarum strain 9 have been assessed extensively as live vaccines for chickens since the 1950s. One of these, the 9R fowl typhoid vaccine [28] was produced by passage in a medium of low nutritional quality and confers strong protection against the systemic disease in adult chickens, although the vaccine strain retains some virulence, may persist for many months and be transmitted through the egg [29±31]. The 9R vaccine is rough and therefore does not stimulate the production of antibodies directed against lipopolysaccharide. This is valuable since its use does not interfere with serological tests based on the presence of serotype-speci®c serum IgG used in the detection of Salmonella infection. S. gallinarum strain 9, cured of its virulence plasmid was also shown to be attenuated for chickens of a few weeks' age [32]. Intramuscular vaccination of chickens with this attenuated strain makes them very resistant to oral challenge with the parent strain, but resistance was not as complete as that induced by the 9R vaccine strain administered orally [33]. Strains with mutations in genes for the biosynthetic pathway of aromatic amino acids are attenuated because the growth factors p-aminobenzoic acid and dihydroxybenzoic acid are not available in sucient quantities in the tissues [34,35]. An aroA mutant of S. gallinarum strain 9, avirulent for 2-week old chickens, was e€ective as a vaccine, but again, conferred less protection than the 9R vaccine [36]. Such strains, which are more attenuated though less protective might be used in more susceptible breeds in conjunction with a second vaccination with the 9R vaccine. More recently, a nuoG mutation showing no NADH dehydrogenase I enzymatic activities, was shown to attenuate S. gallinarum: an increase in LD50 of 107 by both oral and parenteral routes of inoculation was observed. This strain was also highly protective against fowl typhoid in experimental infections [37]. It is generally regarded that killed vaccines are not protective against fowl typhoid, however, preliminary investigations have shown that protection against infection with S. galli-

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narum can be achieved using outer-membrane proteins [38]. 5. Vaccination against host non-speci®c serotypes Because of the lack of information regarding colonisation and immunity relating to the Salmonella serotypes that are usually associated with human foodpoisoning, the development of vaccines against nonhost speci®c serotypes for use in poultry has been almost exclusively empirical. In assessing the following studies it is important to bear in mind the infection model used. The outcome of experimental infection can vary greatly depending on the Salmonella strain used for challenge, the route and bacterial dose used for inoculation and also on the age of the bird at the time of vaccination and challenge. Infection with a wild-type strain within a few hours of hatching may realistically re¯ect infection arising from a hatchery, whereas infection of birds that are several weeks old may re¯ect infection by a number of routes. Infection of the younger birds by the oral route results in heavier faecal excretion and of longer duration than occurs in older birds. Challenge with a large dose, such as log10 9 cfu may produce a consistent infection but may not re¯ect the normal situation in the ®eld and may not accurately portray the ecacy of a vaccine against the size of the challenge dose likely to be experienced in the ®eld. A more accurate assessment could be made by challenging a small proportion of the vaccinated and control groups and monitoring the rate of lateral spread of infection or by the incorporation of small numbers of Salmonella organisms in the feed. Various types of non-living vaccine have been used experimentally and in the ®eld and although they generate an immune response, protection is variable. Earlier work indicates that protection is generally no better than moderate. McCapes et al. [39] demonstrated reduced mortality (from 85.6% to 39.7% and from 91.5% to 44.6%) in day old turkeys challenged via the yolk sac with S. typhimurium and S. schwarzengrund; the parents of the chicks had been exposed to S. typhimurium infection early in life and vaccinated repeatedly sub-cutaneously with S. typhimurium bacterin. A less pronounced reduction in mortality (from 94.4% in controls to 75.1% in vaccinated birds) was obtained in experiments using crude endotoxin extract from S. typhimurium given intraperitoneally [40]. When young chickens were vaccinated directly, using boiled sonicates of several serotypes incorporated in the feed, then challenged a few weeks later, clearance from the faeces of the challenge serotypes was generally more rapid than for unvaccinated controls [41] although the results were variable. In contrast, Bisping et al. [42] found that orally administered heated whole

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cell bacterins had little e€ect on excretion of Salmonella in the faeces. Similarly, vaccination of the mother birds with a bacterin did not signi®cantly reduce excretion of Salmonella hadar from the challenged progeny [43]. Parenteral vaccination with formal-killed bacteria, but also challenging parenterally either with S. virchow [44] or S. enteritidis [45], reduced mortality and intestinal colonisation. Ghosh et al. [44] also reported reduced mortality from 85% to 0% against log10 7.7 S. virchow organisms in young chicks, when inoculated intraperitoneally. Timms et al., showed reduced mortality from 100 to 50% against between log10 5 and log10 8 challenge organisms in similar experiments [45]. Since the increase in S. enteritidis-associated human food-poisoning in the late 1980s, a number of studies have been carried out vaccinating hens with this serotype killed by di€erent methods. Gast et al. [46,47] demonstrated reductions in the rate of faecal excretion when birds were challenged 2 weeks after the second of two sub-cutaneous vaccinations, but not if they were challenged six weeks after. Fewer numbers of the challenge strain were isolated from the spleen, ovaries and oviducts compared to controls. Reduced excretion of Salmonella in faeces was also obtained when hens were vaccinated with acetone-killed S. enteritidis bacterins mixed with Freund's incomplete adjuvant [48]. This vaccination induced high levels of circulating speci®c IgG to unspeci®ed salmonella protein antigens. Nakamura et al., also showed reduced excretion in faeces and of bacterial numbers in the tissues of birds vaccinated twice and challenged at laying age [49]. Some live vaccines are available and others will become so in the next few years. It still remains to be seen how e€ective these will be when applied in the ®eld. Live attenuated vaccine strains developed for immunisation against the host-speci®c serotypes have been of less value in reducing excretion of those that are non-host speci®c. Although, oral vaccination of day old chicks with a S. dublin vaccine strain had little e€ect in reducing the faecal excretion, it did limit the systemic multiplication of an S. typhimurium intraperitoneal challenge 10 d later [50,51]. Rough mutants defective in lipopolysaccharide biosynthesis have been tested for use in poultry. S. typhimurium strain F98, which is virulent for chickens, was rendered avirulent by selecting for roughness as indicated by bacteriophage resistance. When challenged, chickens that had been vaccinated previously, either orally or intramuscularly with the mutant, showed reduced faecal excretion of the parent strain [52]. A mutant of S. typhimurium defective in the UDP±galactose epimerase gene, galE, required for LPS synthesis [53], has been shown to be an e€ective vaccine in mice and cattle [54]. Such mutants are rough unless exogenous galactose is supplied. When one day old chicks were vacci-

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nated orally with the galE mutant and were challenged, albeit only two weeks later, with an S. typhimurium isolated from turkeys, the number of Salmonellas excreted in the faeces and the number of chicks reaching carrier status, was signi®cantly reduced [55]. Protection was more pronounced when chickens were inoculated intramuscularly rather than orally [56]. However it was found that galE mutants of S. cholerae-suis and S. typhi can retain virulence for mice and humans respectively [57,58], so the galE mutation is generally thought to have less value than previously. An aroA mutant of S. typhimurium strain F98 was attenuated for chickens and gave protection to 4 d old chicks against challenge with 108 cfu of the parent strain, when vaccinated by the intramuscular or oral routes [59]. Cooper et al. obtained similar results [60,61]. In both cases the vaccine was administered orally within one day of hatching. The degree of protection was greatly improved when challenge was made by contact infection soon after oral vaccination of the newly hatched chicks [62]. These authors found no evidence of cross-protection between S. enteritidis and S. typhimurium in terms of excretion in the faeces. Others have found that a rough mutant of an S. enteritidis phage type 4 aroA strain protected laying hens against challenge with the wild type strain, but better protection against S. enteritidis was obtained by parenteral vaccination with the S. gallinarum 9R vaccine strain [62,63]. However, it was recognised that the 9R vaccine strain persisted in the tissues [62]. Other auxotrophic mutations that have been found to attenuate virulence including those in the pur genes, which confer an adenine requirement. Such mutants were found to be poor vaccines when tested in mice, possibly because they are over attenuated [64,65], with the result that they have not been tested further. However, a purE mutant strain is being used extensively as a chicken vaccine in Germany [66]. The cya crp genes encode the adenylate cyclase and the cAMP receptor protein respectively and their products are required for growth on carbon sources other than glucose. A cya crp mutant of S. typhimurium was shown to be avirulent and immunogenic in chickens [67]. When challenged with the parent strain two weeks after oral vaccination with this mutant, protection was obtained, not only against the homologous serotype S. typhimurium, but also against S. enteritidis, S. agona, S. heidelberg and S. bredeney and some other serotypes including S. hadar, S. montevideo and S. anatum [68]. It must be appreciated that here the interval between immunisation and challenge is very short and may have led to stimulation of non-speci®c immunity. In addition, when adult chickens were vaccinated with the S. typhimurium cya crp strain, maternal antibodies were demonstrated in eggs and progeny, which had reduced colonisation by the wild type strain [69]. These

authors have also suggested that vaccines that are too virulent may be counter-productive because of their immunosuppression [70]. Mutants defective in expression of outer membrane proteins that are attenuated for mice are also potential vaccine candidates [71], but they have not yet been tested in chickens. One additional aspect of the use of live attenuated vaccines against Salmonella relates to their ability to colonise the alimentary tract of very young birds. If such vaccines are administered to newly hatched chicks they multiply extensively because of the absence of the complex normal microbial ¯ora found in adult birds. This in turn prevents colonisation by other Salmonella strains inoculated within a few hours afterwards [61,72,73]. This is a purely microbiological e€ect and may explain the degree of protection found by Alderton et al. [74] where administration of an aroA S. typhimurium vaccine produced signi®cant protection when the birds were challenged 10 d later. There is some evidence that this phenomenon does occur in very young birds administered with live, attenuated vaccines [75] (P. Coloe, personal communication). The inhibition is neither the result of bacteriophage or bacteriocin activity nor a consequence of stimulation of non-speci®c immunity. Recent work suggests that a major determinant of the e€ects is the availability of carbon sources and electron acceptors in the local environment of the bacterial cells [76]. Protection may thus be obtained against homologous and heterologous serotypes and phage types. The practical consequences are that live vaccines could be administered to very young chickens by the oral route thereby obtaining protection within a matter of hours. Rapid protection would then be obtained by this colonisation±inhibition e€ect followed by the development of normal immunity within a couple of weeks. However, the choice of strains from which to develop future live vaccines might depend on testing their inhibitory activity since a number of those currently available have a very poor inhibitory spectrum [77]. 6. Summary Until our understanding of the immunological interactions between Salmonella organisms and host is greatly improved vaccination will necessarily be empirical, to a greater or lesser degree, for the foreseeable future. Even with these limitations vaccines should ful®l a number of conditions indicating ecacy and safety. The criteria in selecting an ideal vaccine have been discussed previously [19,55,78] and are outlined below. Strong protection against intestinal and systemic infection is required. An additional requirement is avirulence for man. In view of the current increased

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public awareness of Salmonella food poisoning, the acceptance of live vaccines will probably increase. There may always be some resistance to the use of a live vaccine that is Salmonella-derived unless it can be shown that the vaccine is no more virulent for man than the commensal E. coli organisms also present on the carcase at slaughter. It should soon be possible to delete some of the genes responsible for ¯uid secretion in the intestine from the Salmonella chromosome [79]. The ideal route of administration to poultry would be orally via the drinking water or food or by spray. However, parenteral administration may be an additional requirement for maximum protection. Although the ideal vaccine should be avirulent for chickens, oral vaccination may require the use of an invasive strain to stimulate maximum immunity because immunogenicity may be correlated with invasiveness. Whichever route of inoculation is used, some residual virulence may result in vertical transmission as occurs occasionally with the S. gallinarum 9R vaccine. Therefore the vaccine should produce no disease in the progeny, nor a€ect productivity, while protection should obviously last as long as possible. Protection of broilers is required for a matter of weeks, although Salmonella control in broilers is not required under EU legislation. However, control in breeders is an integral part of the European control programme and here and in layers, protection is needed for many months. Vaccination should therefore be compatible with the use of competitive exclusion (early application or replacement of gut ¯ora). Following vaccination, a protective immunity takes several days to develop. This delay could be overcome by using a live vaccine strain that in newly hatched birds shows the colonisation±blocking e€ect, a form of competitive exclusion that occurs between closely related enteric bacteria. Administration of competitive exclusion products can produce a degree of protection against salmonellosis in the early life of the young chicks. In this case immunity is likely to have little protective e€ect against infections that are extant at the time of immunisation. This might mean that an appropriate live attenuated strain could protect against potential `ex-hatchery' infection during the ®rst few days of life followed by the development of true immunity. It seems likely that when the vaccine strain has been completely eliminated little cross-protection against other serotypes occurs, although there is recent evidence to the contrary. With the small number of major serotypes involved this should not be a major problem. However, this emphasises how little is known of the major Salmonella immunogens involved in immune protection. The role of LPS, whether multivalent vaccines might be constructed and the contribution of other antigens remains to be elucidated. Only in this way can truly non-empirical vaccines be generated.

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