Hoelzer et al. Vet Res (2018) 49:70 https://doi.org/10.1186/s13567-018-0561-7

REVIEW Open Access as alternatives to for food producing animals. Part 2: new approaches and potential solutions Karin Hoelzer1* , Lisa Bielke2, Damer P. Blake3, Eric Cox4, Simon M. Cutting5, Bert Devriendt4, Elisabeth Erlacher‑Vindel6, Evy Goossens7, Kemal Karaca8, Stephane Lemiere9, Martin Metzner10, Margot Raicek6, Miquel Collell Suriñach11, Nora M. Wong1, Cyril Gay12 and Filip Van Immerseel7

Abstract Vaccines and other alternative products are central to the future success of animal agriculture because they can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations. To assess scientifc advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the United States Department of Agriculture, with support from the World Organisation for Animal Health, organized the second International Symposium on Alternatives to Antibiotics. It focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. This article, the second part in a two-part series, highlights new approaches and potential solutions for the develop‑ ment of vaccines as alternatives to antibiotics in food producing animals; opportunities, challenges and needs for the development of such vaccines are discussed in the frst part of this series. As discussed in part 1 of this manuscript, many current vaccines fall short of ideal vaccines in one or more respects. Promising breakthroughs to overcome these limitations include new biotechnology techniques, new oral approaches, novel adjuvants, new deliv‑ ery strategies based on bacterial spores, and live recombinant vectors; they also include new vaccination strategies in-ovo, and strategies that simultaneously protect against multiple pathogens. However, translating this research into commercial vaccines that efectively reduce the need for antibiotics will require close collaboration among stakehold‑ ers, for instance through public–private partnerships. Targeted research and development investments and concerted eforts by all afected are needed to realize the potential of vaccines to improve animal health, safeguard agricultural productivity, and reduce consumption and resulting resistance risks.

Table of Contents 2.3 Genetically modifed live microorganisms as oral 1 Introduction vectored vaccines and vaccine platforms 2 New approaches for the development of veterinary 2.4 New approaches for in‑ovo vaccines vaccines 3 Vaccination strategies to reduce antibiotic use for dis‑ 2.1 Mucosal and tolerance: challenges eases from ubiquitous pathogens to the development of efective oral vaccines 3.1 Towards the development of new Clostridium per- 2.2 Recombinant Bacillus spores as oral vectored fringens vaccines vaccines 3.2 Towards the development of new coccidiosis vaccines 3.2.1 Management of coccidiosis through anticoc‑ cidial drugs *Correspondence: [email protected] 1 The Pew Charitable Trusts, 901 E Street NW, Washington, DC 20004, USA 3.2.2 Traditional live anticoccidial vaccines Full list of author information is available at the end of the article

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hoelzer et al. Vet Res (2018) 49:70 Page 2 of 15

3.2.3 Next generation anticoccidial vaccines the largest surface area of the body and is exposed daily 4 Autogenous vaccines to reduce the need for antibiotic to vast numbers of foreign derived from feed, use the microbiota and pathogens [1]. Within the intes‑ tine a complex cellular network has evolved to prevent 5 Conclusions unwanted immune responses to innocuous antigens, for References instance feed or microbiota, while allowing swift pro‑ 1 Introduction tective responses against agents that cause infectious Alternatives to antibiotics can help minimize the need disease. Key to keeping enteric pathogens at bay is the for antibiotics by helping to prevent and control infec‑ presence of protective pathogen-specifc secretory IgA tious diseases in animal populations. As such, safe and (SIgA) at the site of entry, which prevents the adhesion efective alternatives are crucially important to the future of micro-organisms to the intestinal surfaces and neu‑ success of animal health and production. To assess scien‑ tralizes their enterotoxins. Triggering robust and protec‑ tifc advancements in the research and development of tive intestinal SIgA responses usually requires the local alternatives to antibiotics, highlight promising research administration of vaccines [2]. Although live attenuated results and novel technologies, assess challenges associ‑ oral vaccines have had tremendous success, resulting for ated with their commercialization and use, and provide instance in the near global eradication of poliovirus [3], actionable strategies to support their development, the concerns on the dissemination of vaccine strains into the United States Department of Agriculture (USDA), with environment and rare cases of reversion to virulence, support from the World Organisation for Animal Health leading to vaccine-induced disease, have driven oral vac‑ (OIE), organized the second International Symposium cine development to nonliving or vectored vaccines [4]. on Alternatives to Antibiotics. Te symposium focused However, oral vaccination is challenging due to several on six key areas: vaccines; microbial-derived products; hurdles imposed by the cellular and molecular architec‑ non-nutritive phytochemicals; immune-related products; ture of the gut: (i) the harsh environment of the stom‑ chemicals, enzymes, and innovative drugs; and regula‑ ach and small intestine, including the low pH, digestive tory pathways to enable the licensure and development of enzymes, and bile salts, required to digest feed also easily alternatives to antibiotics. Tis two-part manuscript syn‑ destroys vaccines, (ii) a poor uptake of vaccine antigens thesizes and expands on the scientifc presentations and by the intestinal epithelial barrier and (iii) the tolerogenic expert panel discussions from the symposium regard‑ mechanisms that pervade the intestinal tissues, leading to ing the use of vaccines as alternatives to antibiotics that peripheral and oral immune tolerance upon oral admin‑ + can reduce the need for antibiotic use in animals. Part 1 istration of antigens via the induction of FoxP3­ regula‑ synthesizes and expands on the expert panel discussions tory T cells. Tis often results in a low immunogenicity of regarding the opportunities, challenges and needs related oral vaccines and requires innovative strategies to deliver to vaccines that may reduce the requirement for use of the vaccine antigens to the intestinal as antibiotics in animals, while part two focuses on high‑ well as the inclusion of adjuvants that promote innate lighting new approaches and potential solutions. and adaptive immunity [5]. A general discussion of the importance of antibiotic Te mucosal immune system in the gut can be divided resistance and the opportunities, challenges and needs in inductive sites, where sampled antigens stimulate naive related to vaccines as alternatives that may reduce the T and B cells, and efector sites, where efector cells per‑ need for use of antibiotics in animals is provided in part form their functions, e.g. assisting in the production of 1 of this review, including a discussion of the proper‑ SIgA. In the small intestine, the inductive sites comprise ties of ideal vaccines, how current vaccines compare the gut-associated lymphoid tissues (GALT) and the mes‑ to these ideal vaccines, and how investment decisions enteric lymph nodes, while the efector sites constitute around research and development of vaccines are made. the lamina propria and the surface epithelium [6]. Te Tis second part of the manuscript will highlight specifc GALT itself is composed of Peyer’s patches (PP), appen‑ research advancements in the area of veterinary vaccines. dix and isolated lymphoid follicles. Te presence of other GALT-like structures, such as lymphocyte-flled villi (rat, 2 New approaches for the development human) and cryptopatches (mouse) is dependent on the of veterinary vaccines species. Interestingly, while in birds and most mammals 2.1 Mucosal immunity and tolerance: challenges PP or their equivalent are scattered throughout the small to the development of efective oral vaccines intestine, in pigs, ruminants and dogs the PP in the distal As mentioned in part one of this manuscript, most path‑ small intestine (ileum) are continuous. Fish and reptiles ogens invade the host at the mucosal surfaces, such as on the other hand lack PP and the intestinal immune sys‑ the gastro-intestinal (GI) tract. Te GI tract constitutes tem in these species is composed of epithelial leukocytes Hoelzer et al. Vet Res (2018) 49:70 Page 3 of 15

and rare, small non-organized lymphoid aggregates. It where the gut-associated immune system is most pro‑ remains largely unknown how these species-specifc dif‑ nounced. Besides M cells, sampling of luminal antigens ferences might afect the efcacy of oral vaccines. also occurs by intestinal mononuclear via From their entry point, which is typically the oral cav‑ transepithelial dendrites. Tis sampling mainly occurs by ity, to their delivery site, most commonly the small intes‑ ­CD11c+CX3CR1+ , which transfer the anti‑ tine, the integrity of delivery systems and the stability of gens to ­CD103+ dendritic cells (DCs). Tese DCs then vaccine components are at risk. Lysozyme in saliva, the drive the diferentiation of regulatory T cells (Tregs), low gastric pH together with pepsin and intestinal pro‑ which subsequently induce tolerance to these proteins teases can degrade oral vaccines. Enteric coating of vac‑ [12]. In the steady state, goblet cells can also transport cine components with pH-responsive polymers with a small soluble proteins (<10 kDa) across the epithelium to dissolution threshold of pH 6 might protect against gas‑ tolerogenic DCs via so-called goblet cell-associated anti‑ tric degradation and results in the release of their con‑ gen passages [13]. Absorptive intestinal epithelial cells tents in the small intestine [7]. In this context, ruminants or enterocytes, constituting >90% of the small intestinal pose an additional problem to vaccine stability as their epithelium, may also sample the luminal content through polygastric gastro-intestinal tract efectively degrades receptor-mediated transcytosis. For instance, the neo‑ substances including vaccines. Site-specifc delivery of natal Fc receptor (FcRn), an MHC class I-like Fcγ recep‑ oral vaccines to the small intestine is favorable as the tor, is expressed on the apical surface of enterocytes and mucus layer covering the small intestinal epithelium transcytoses IgG, immune complexes or Fc-coated nano‑ consists of only one layer, which is loosely adherent, less particles from the lumen to the basolateral surface of the thick and patchy as compared to the colonic mucus layers epithelium [14]. Similar to M cells, it might be worth‑ and might promote their access to the intestinal epithe‑ while to target apical receptors exploited by enteropatho‑ lium. In addition, the small intestine is less densely popu‑ gens on small intestinal enterocytes to promote uptake of lated by the microbiota, which might further disrupt the antigens by the epithelial barrier. A potential candidate integrity of the delivery systems and the stability of vac‑ would be aminopeptidase N (ANPEP), a zinc-dependent cine components. Underneath the mucus layer, a single peptidase present in the brush border of small intestinal layer of intestinal epithelial cells prevents uncontrolled enterocytes, which serves as an entry receptor for several access of the luminal content to the underlying intes‑ coronaviruses and also binds F4 fmbriae, a colonisation tinal tissues, further restricting uptake of oral vaccine factor produced by porcine-specifc enterotoxigenic E. antigens. Crossing of the epithelial barrier by vaccines coli. ANPEP also transports F4 fmbriae as well as micro‑ could be enhanced by exploiting sampling routes particles functionalised with ANPEP-specifc mono‑ in the small intestine or by adopting strategies used by clonal across the intestinal epithelial barrier, enteric pathogens to colonize or invade the host [8]. Te resulting in robust intestinal SIgA responses, at least in best-known sampling route in the gut is associated with piglets [15, 16]. microfold (M) cells. Tese specialized intestinal epithelial Although the selective targeting of vaccine antigens to cells reside within the follicle-associated epithelium cov‑ apical receptors might promote their uptake by the epi‑ ering the Peyer’s patches and take up macromolecules, thelium via transcytosis, this process is in itself insuf‑ particulate matter and microorganisms [9]. Many enteric fcient to trigger protective intestinal immunity upon pathogens hijack M cells to invade the host by binding to oral vaccination and explains the need to include adju‑ apical receptors. For instance, the invasin protein of Yers- vants. Tese adjuvants should act on antigen presenting inia species interacts with β1 integrin on M cells, leading cells as well as intestinal epithelial cells to promote the to infection [10]. Likewise, GP2 marks M cells in many induction of protective SIgA and cell-mediated immune species and binds to FimH, a subunit of type I pili on responses. Indeed, enterocytes not only provide a physi‑ Escherichia coli and Salmonella enterica. Tis interaction cal barrier separating the intestinal lumen from the host results in uptake of ­FimH+ bacteria and initiates mucosal tissues, but also relay information on the luminal con‑ immunity [11]. Although many groups have focused on tent to the underlying immune cells through the secre‑ improving antigen uptake by targeting oral vaccines to tion of infammatory or tolerogenic mediators. For M cell-specifc receptors, these cells represent only a instance, during the steady state, enterocytes produce small, species-specifc percentage of the total intesti‑ thymic stromal lymphopoëtin (TSLP) and transform‑ nal epithelial cell population. Although M cell numbers ing growth factor (TGFβ), which imprint a tolerogenic increase from the cranial to caudal small intestine and M phenotype on intestinal dendritic cells [17]. In contrast, cell targeting strategies work quite well in rodent mod‑ upon infection enterocytes secrete IL-6 and IL-8 [18]. els, they mostly fail in larger animals due to the long Tis probably facilitates a switch from a tolerogenic to passage time needed to reach the distal small intestine, an immune-inductive environment, allowing activation Hoelzer et al. Vet Res (2018) 49:70 Page 4 of 15

of intestinal antigen presenting cells. As yet the most Evidence suggests that spore forming bacteria comprise efective adjuvants for oral application are the entero‑ as much as 30% of the gut microbiota, indicating that the toxins from Vibrio cholera (CT) and enterotoxigenic E. ability to form spores enables bacteria to survive in the coli (ETEC) (LT). Due to inherent toxicity, dmLT was environment as well as entering and transiting the gastric developed, a nontoxic LT mutant retaining its adjuvan‑ barrier of animals [31]. ticity. Tis dmLT triggered intestinal memory responses Te extraordinary resistance properties of Bacillus upon oral vaccination with a nonliving ETEC vaccine and spores coupled with their ease of genetic manipulation, seems a promising candidate to be included as adjuvant and their successful use as probiotics, makes them attrac‑ in oral vaccines [19, 20]. Similarly promising strategies tive candidates for the delivery of heterologous antigens have been reported for Eimeria [21]. Recent studies have for vaccination. Spores have been used as vaccine vehi‑ shown that Eimeria-induced IL-17 production is critical cles in a number of ways, difering principally in whether in the initiation of early innate immune response in coc‑ spores are genetically modifed or not. In all cases B. cidiosis and blocking of IL-17 production by exogenous subtilis has been utilized due to the excellent genetics IL-17-neutralizing reduced both the intracellu‑ available. Using genetic modifcation, a chimeric gene lar development of Eimeria and the severity of intestinal consisting of a fusion between a B. subtilis anchor gene lesion [22–24]. and an open reading frame encoding a putative protec‑ In summarizing this part, future design of oral vac‑ tive antigen is frst constructed. Te next step is intro‑ cines should be tailored to the needs of the target species, duction of the chimera into the B. subtilis chromosome focus on the selective delivery of vaccines to epithelial using a gene transfer technique, typically DNA-mediated receptors to promote their transport across the epithelial transformation, a process in B. subtilis that is straight‑ barrier, induce protective immune response in the target forward. Typically, the anchor is the 5′-end of a gene tissues, and should include a mucosal adjuvant able to encoding a spore coat protein such that the chimera is trigger memory SIgA responses. displayed on the spore coat. Surprisingly, heterologous antigens displayed on B. subtilis spores are mostly sta‑ 2.2 Recombinant Bacillus spores as oral vectored vaccines ble and do not appear to sufer extensive degradation. Endospores, or spores, are produced by many bacte‑ Using this approach a number of candidate antigens have ria as a response to nutrient deprivation. Te spore is a been displayed and then evaluated in animal models. For dormant entity about 1 μm in size that can germinate, example, spores displaying a tetanus antigen TTFC con‑ allowing a nascent cell to emerge and enter vegetative ferred protection to a lethal dose of tetanus toxin when cell growth [25]. Te spore carries remarkable resistance administered orally [32, 33]. Mice dosed orally with properties, being typically resistant to high temperatures spores expressing part of the alpha toxin of Clostridium (typically 70–80 °C), desiccation, irradiation, and expo‑ perfringens were protected to challenge with alpha toxin sure to noxious chemicals [26]. Te two principal spore- [34]. A more recent example is that of Clostridium dif- forming bacterial genera are Bacillus and Clostridia with cile where a C-terminal fragment of the toxin A (TcdA) the latter being exclusively anaerobic. could be stably expressed and when administered orally Members of the Bacillus genus are being used as pro‑ to hamsters conferred protection to C. difcile infection biotics, that is, microorganisms that are added to the diet [35, 36]. Tis particular vaccine has now entered clinical to improve the balance of microbial communities in the evaluation in humans [37]. GI-tract and are therefore benefcial to human or animal Using a non-genetically modifed organism (GMO) health [27, 28]. Typical species include Bacillus clausii, approach it has been shown that spores can adsorb anti‑ Bacillus coagulans and Bacillus subtilis. For a long time, it gens efciently onto their surface and surprisingly this has been assumed that Bacillus spores are soil organisms is both strong and stable, and refects the unique bio‑ yet the evidence supporting this is actually rather sparse. physical properties of the spore [38]. For the adsorption Instead, spores are found in the soil in abundance but approach, it has been shown that the gastric barrier is live, vegetative cells, are rarely if ever found other than particularly corrosive and adsorbed antigens are labile, in association with plants or in the animal gut. Mounting but for intranasal delivery this method appears satisfac‑ evidence shows that spores, although found in the soil, tory. Using this approach inactive (killed) spores can be are mostly dormant and are shed in the feces of animals, used and success has included studies showing protec‑ which are their natural hosts [29]. Te consumption of tion to infuenza (H5N1) [39] and signifcant reduction in spores associated with soil-contaminated plant matter lung counts of animals challenged with Mycobacterium enables spores to enter the GI-tract, transit the gastric tuberculosis [40]. A unique feature of spores is their abil‑ barrier unscathed and then germinate and proliferate in ity to enhance immune responses and this adjuvant efect the intestine before excretion as dormant spores [30]. has been characterized in depth [41–43]. Hoelzer et al. Vet Res (2018) 49:70 Page 5 of 15

However, the use of spores as mass-delivery vehicles immunity [50]. Multiple species of bacteria including for vaccines has several limitations. Oral delivery clearly Salmonella Typhimurium, Salmonella Enteritidis, Sal- is the preferred approach but appears to work efec‑ monella Typhi, E. coli, Lactococcus lactis, Lactobacillus tively only for the GMO approach. Oral delivery also casei, Lactobacillus reuteri, Bacillus subtilis, and Bacillus raises issues of tolerance and may prove to be a limiting thuringiensis, have been used to express protein antigens factor. Sublingual delivery has also been explored; this derived from bacterial, viral, and protozoal pathogens approach appears to provide levels of protection that [51–61]. Some of these vectors are inherently non-patho‑ are equivalent to oral delivery, but requires more doses genic; Lactobacillus and Lactococcus strains, for instance, [36, 44]. Nasal delivery is suitable but raises potential are “Generally Recognized as Safe” (GRAS) [50, 61]. In safety issues. For animal vaccines, spores are attractive other cases the microorganisms have been rendered non- since they are currently used as feed probiotics but also pathogenic through the targeted deletion of virulence because they can survive the high temperatures used genes; strategies for the development of Salmonella vec‑ for feed production and may ofer long-term utility. As tors, for instance, typically rely on the deletion of certain mentioned already, spores have been manipulated for metabolic functions that limit the bacterium’s ability protection against C. perfringens but there now exists to replicate in the host and attenuate virulence without the opportunity to develop spores for protective vac‑ impacting host colonization or invasion [50]. In fact, cination to necrotic enteritis, an important poultry dis‑ an intrinsic property shared by many, although not all, ease caused by C. perfringens that has been identifed as microorganisms used as vectors is their ability to efec‑ a high vaccine research priority by the OIE ad hoc Group tively infect the host and initiate innate and subsequent (see Additional fle 2 in http://doi.org/10.1186/s1356​ adaptive immune responses, for instance by trigger‑ 7-018-0560-8). ing the host’s pattern recognition receptors [50]. Tese One application that is particularly promising is the recombinant vectored vaccines can be delivered directly use of spore vaccines in aquaculture. With intensive fsh to a mucosal surface via nasal, ocular, or oral administra‑ farming, Bacillus spores are being used as probiotic feed tion, which not only allows for mass application but may supplements. For shrimp farming, viral diseases have also enhance mucosal immune responses, the primary devastated the industry and one of the most important surface through which most pathogens invade. Moreo‑ shrimp pathogens is white spot syndrome virus (WSSV) ver, contrary to traditional attenuated live vaccines, these that causes seasonal outbreaks of disease [45]. A number recombinant vaccines in many cases do not carry a risk of of groups have developed B. subtilis spores that display reversion [50]. the VP28 capsid protein of WSSV and when adminis‑ In veterinary medicine, oral vectored vaccines have tered in feed appears to protect against white spot dis‑ been instrumental in the eradication or control of rabies ease [46–49]. Te mechanism for protection is intriguing; in wildlife reservoirs [62, 63]. Oral vectored vaccines have even though shrimp are not thought to produce antibod‑ also been developed for several other veterinary applica‑ ies, it is clear that presentation of the viral antigens does tions, including some economically important diseases produce some level of specifc immunity. of food-producing animals that are associated with con‑ Despite the progress being made with spore vac‑ siderable antibiotic use such as porcine circovirus type-2 cines one key issue remains: the containment of GMOs. (PCV-2); in some cases, the vaccine vector is a chi‑ Because spores are dormant with the potential to survive mera containing parts of multiple microorganisms—for indefnitely in the environment, the use of recombinant instance, an attenuated live vaccine may be used as the spores in spore vaccines is likely to raise environmen‑ vector—and the resulting vaccine simultaneously confers tal concerns and successful regulatory approvals may be protection against multiple diseases, for instance Marek’s slow or impossible to secure. For human use, it is likely disease and infectious bursal disease or Newcastle dis‑ that a case can be made that the recombinant spore vac‑ ease and avian infuenza [63, 64]. cines addresses an unmet clinical need, but for animal Te development of some vaccine vector systems has use devising a method for biological containment will be been very successful and numerous veterinary vaccines crucial. have been developed based on them; the canarypox virus vector system ALVAC, for instance, has been used 2.3 Genetically modifed live microorganisms as oral for the development of a range of veterinary vaccines vectored vaccines and vaccine platforms including against rabies, infuenza, and West Nile virus Technological advancements now make it possible to [64]. Similarly, adenovirus vectors have also been widely genetically engineer bacteria and other microorganisms used in veterinary medicine, both in companion and that deliver heterologous antigens in a way that can stim‑ food-producing animals [65]. Vaccine platforms such as ulate mucosal as well as humoral and cellular systemic these are particularly valuable as they can allow for the Hoelzer et al. Vet Res (2018) 49:70 Page 6 of 15

rapid development of vaccine candidates in response to vaccinations and peak just after hatch [76]. Tis can inter‑ emerging vaccine needs, but the possibility of anti-vec‑ fere with proper vaccine responses. However, evidence tor immunity can restrict their usefulness [66]. Research suggests that some vaccine strains are more afected by and development of additional vaccine vector platforms maternal antibodies than others [80]. Deliberate vaccine is therefore needed. Salmonella strains that express for‑ development may therefore limit the often disruptive eign antigens, either chromosomally or plasmid-based, efects that can be caused by maternal antibodies [78]. have yielded promising results in several species includ‑ Other factors that need to be considered in the develop‑ ing mice, humans, pigs and chicken [67–72]. Diseases for ment of a successful in-ovo vaccination program include which these Salmonella vectored vaccines were investi‑ the characteristics of the vaccine or vaccines to be used, gated include infuenza, Brucella abortus, post-weaning the type of incubator in which the eggs are housed in the diarrhea and heterologous strains of Salmonella [69–72]. hatchery, and the breed and age of the parent fock [76]. Te use of Pasteurellaceae as vectors for modifed live In-ovo vaccination strategies are promising means of vaccines against shipping fever in calves is currently reducing antibiotic use in poultry production and have under investigation, with promising preliminary fndings been the subject of intense research. Importantly, they [73]. Use of this vector system for other diseases includ‑ can provide robust and early protection against immune ing pinkeye has been suggested [73]. suppressive diseases such as infectious bursal disease [81, 82] and vaccines against multiple diseases have been suc‑ 2.4 New approaches for in‑ovo vaccines cessfully combined. For instance, studies have shown that In-ovo vaccination is a mass-vaccination strategy that is in-ovo vaccination strategies can simultaneously confer mainly used in broiler chickens albeit occasionally also in protective immunity against Marek’s disease, infectious broiler-breeder and layer chickens [74]. Eggs are injected bursal disease, Newcastle disease, fowl poxvirus, coccidi‑ in the hatchery, typically during the third week of embry‑ osis, and necrotic enteritis [83, 84]. Other combination onic development around day 18 or 19. To vaccinate, a vaccines under investigation include vectored vaccines small hole is made in the shell at the blunt end of the egg that simultaneously provide protection against Newcastle and the vaccine is injected below the chorion-allantoic disease and infectious bursal disease [85]. In-ovo vaccina‑ membrane into the amniotic cavity or directly into the tion strategies have also been explored for other poultry embryo. Commercial in-ovo vaccination systems that diseases with promising results. Tis included an avian automatically inject the eggs have been available since the infuenza vaccine based on a non-replicating human ade‑ early 1990s. More than 90% of broiler chickens in the US novirus vector [86], a recombinant viral vector vaccine are vaccinated in ovo, and in Brazil that fraction equals against infectious laryngotracheitis [87], recombinant 70% [75]. Te most common use of in-ovo vaccination is protein Eimeria vaccines [84, 88, 89] and a fowl adeno‑ for Marek’s disease, potentially combined with vaccines virus vectored vaccine against inclusion body hepatitis against other diseases such as Gumboro or Newcastle [90], among many others. A Mycoplasma gallisepticum disease. vaccine for in-ovo vaccination of layer chickens has also Te ability to deliver a clearly defned vaccine dosage recently been evaluated, even though high chick losses at to every single chick and to invoke early protection in hatch were reported for the medium and high doses of the chicks is among the main benefts of this technology, the vaccine that were investigated [91]. Terefore, in-ovo but it is labor-intensive, causes stress for the chicks, and vaccination strategies are capable of controlling several high sanitary standards need to be followed during vac‑ economically important poultry diseases. Many of these cine preparation and injection to manage infection risks diseases are viral, but can predispose animals to second‑ [74, 76]. In addition, the location of the vaccine injection ary bacterial infections. Terefore, in many cases, in-ovo is critical for efcacy. It has been shown, for instance, vaccines are promising alternative approaches to the use that if Marek’s disease vaccine is accidentally deposited of antibiotics. into the air cell or allantoic fuid, adequate protection is not achieved [77]. Te stage of embryonic development 3 Vaccination strategies to reduce antibiotic use can have profound efects on vaccine safety and efcacy for diseases from ubiquitous pathogens [78]. One study, reported that vaccination of 10–12 day- 3.1 Towards the development of new Clostridium old embryos with herpes virus of turkeys (HVT) led to perfringens vaccines pronounced lesions and embryonic deaths, while vacci‑ Clostridium perfringens is widespread in the environ‑ nation on days 16 did not cause detectable lesions [78]. ment and in the gastrointestinal tract of most mammals Embryonic age at vaccination has also been shown to be and birds. However, this bacterium is also one of the correlated with antibody titers [79]. Maternal antibody most common pathogens of food-producing animals, titers actually increase after the typical age for in-ovo causing disease only under circumstances that create an Hoelzer et al. Vet Res (2018) 49:70 Page 7 of 15

environment which favors growth and toxin production, vaccination, and seems to depend on the colonization such as stress, injury, or dietary changes [92]. Te bac‑ level and persistence of the vaccine strain [100–103]. Tis terium itself is not invasive, but causes disease through indicates that the use of live vectors to express antigens the production of a wide array of toxins and enzymes. derived from C. perfringens strains in the gut of broilers However, no single strain produces this entire toxin rep‑ is a promising approach, but the vaccine delivery strat‑ ertoire, resulting in considerable variation in the toxin egy still needs to be optimized to achieve optimal antigen profles and disease syndromes produced by diferent presentation to the mucosal immune system and provide toxinotypes of this bacterium [93]. While some of these improved protection. Alternatives to attenuated Salmo- toxins act only locally, other toxins which are produced in nella strains can be Bacillus subtilis spores or Lactobacil- the gut exert their action in other internal organs or can lus casei, which both have a GRAS status and have the act both locally and systemically [94–96]. To date, efca‑ potential to be used as vaccine carriers for Clostridium cious vaccines are only available for the diseases caused antigens [34, 104]. B. subtilis has the advantage that the by systemic action of the toxins and vaccination against heat-stable spores can easily be incorporated in the feed enteric diseases still remains a challenge. However, some and L. casei has known probiotic efects that facilitate of these enteric diseases caused by C. perfringens are of the development of mucosal immunity. However, these major economic importance and lead to considerable types of vectors still have to be tested for their capacity use of antibiotics. Amongst them are necrotic enteritis to induce a good immune response, in particular against in broilers and necro-haemorrhagic enteritis in calves. heterologous antigens, in broilers and whether they are Despite the fact that much research is being directed to able to provide protection against necrotic enteritis. the development of novel vaccines against these C. per- Another issue to be addressed when developing a vac‑ fringens-induced enteric diseases, several key barriers cine against C. perfringens-induced enteric diseases is the still have to be overcome. choice of the antigens to be included in the vaccine. C. In general, clostridial vaccines require multiple doses perfringens-induced diseases are the result of the toxins to achieve full immunity. Unfortunately, parenteral and enzymes that are produced and vaccination of chicks booster immunizations are impossible in the broiler with C. perfringens supernatants provides protection industry, where mass parenteral vaccination is only fea‑ against experimental necrotic enteritis [97, 105]. How‑ sible at the hatchery, either in ovo or on day-old chicks. ever, the protective capacity of the supernatants depends Because single parenteral vaccination at day of hatch on the strain used for supernatant preparation, indicating ofers no protection, other delivery methods need to be that full protection might be determined by an efective developed [97]. Oral vaccines can more easily be admin‑ combination of diferent bacterial immunogens [105]. In istered to birds, without the need of individual handling order to elucidate the optimal mixture of antigens to pro‑ of the chicks and are therefore recommended. However, tect against necrotic enteritis, challenge trials are being some questions arise when developing an oral vaccine performed mostly using parenteral vaccination schemes. as compared to the parenteral administration route. In Once the ideal combination of antigens is known, this addition to the fact that maternal antibodies can block will have to be adapted to oral delivery strategies. Several the immune response in young chicks, also the induc‑ C. perfringens antigens have been evaluated as potential tion of oral tolerance has to be circumvented and an ef‑ vaccine candidates. Te tested antigens include both C. cient way to present the antigens to the mucosal immune perfringens toxins (e.g. alpha toxin and the NetB toxin) system has to be developed. Oral tolerance is a common and highly immunodominant proteins identifed in post- problem in mammals and fsh when developing oral vac‑ infection serum from birds immune to necrotic enteritis cines. Tis is in contrast to chickens, where oral tolerance [106]. In general, immunization studies of broilers with is age-dependent, and only an issue in 1- to 3-day-old a single antigen all resulted in some level of protec‑ chicks. After that age, protein antigens have been shown tion against experimental necrotic enteritis. Remark‑ to induce a robust immune response and oral vaccina‑ ably, immunization with NetB toxin, which is essential tion schemes are thought to be feasible [98]. One appeal‑ to cause disease in broilers, does not aford higher levels ing strategy for the delivery of vaccine candidates to the of protection than vaccination with other toxins or pro‑ mucosal immune system is the use of attenuated or aviru‑ teins. However, when birds were vaccinated either via the lent bacteria as antigen vehicles [99]. Attenuated recom‑ parenteral or the oral route, with a combination of both binant Salmonella strains which express C. perfringens NetB toxin and alpha toxin, higher levels of protection antigens have been tested in several studies as oral vac‑ were obtained [107, 108]. In order to obtain full protec‑ cine vectors, leading to some promising results. However, tion against C. perfringens-induced enteric diseases, the amount of protection aforded by these vaccines is not only antibodies that inhibit toxin activity might be not as high as compared to multiple doses of parenteral needed; a combination of antigens targeting also bacterial Hoelzer et al. Vet Res (2018) 49:70 Page 8 of 15

proliferation, colonization and/or nutrient acquisition both chickens and calves with formaldehyde inactivated could be more efcient than either one of the individ‑ C. perfringens supernatants or toxins have resulted in a ual approaches. Indeed, in a recent study disruption of good antibody response, but these are unable to protect the putative adhesin-encoding gene cnaA resulted in a against intestinal disease [97, 112]. To overcome the need reduced ability to colonize the chicken intestinal mucosa of chemically inactivating the C. perfringens toxins, cur‑ and to cause necrotic enteritis [109]. Tis strengthens rent research focusses on the use of recombinant toxoids the idea that vaccine antigens that target bacterial colo‑ to develop a vaccine against C. perfringens-induced dis‑ nization might be indispensable to obtain a working eases. While this may be a good strategy to obtain a safe vaccine against C. perfringens–induced enteric diseases. and protective vaccine on a laboratory scale, the produc‑ Additional vaccine targets might be enzymes that aid in tion process is more laborious and time-consuming than breakdown of the host tissue and nutrient acquisition, production of conventional toxoids, especially because of such as, amongst others, mucinases, collagenases and the required purifcation steps [113]. Terefore, recent hyaluronidases. studies have explored the use of efcient low-cost alter‑ In contrast to the extensive eforts to develop a vac‑ natives, such as non-purifed recombinant clostridial cine against necrotic enteritis in chickens, considerably toxins and even recombinant bacterins, with success less research has been directed to vaccination against [114–116]. necro-haemorrhagic enteritis in calves. Te recent In summary of this section, considerable progress has demonstration of the essential role of alpha toxin in recently been made in the development of efcacious necro-haemorrhagic enteritis and the proposition of vaccines against C. perfringens-induced enteric diseases. a pathogenesis model will allow for the more targeted Te main issue that hampers a breakthrough in this feld development of a vaccine [110, 111]. In calves as in chick‑ is the identifcation of a defned combination of antigens ens, protection against C. perfringens-induced necrosis that is able to provide full protection against disease. can be obtained by antibodies against a mixture of toxins, Tese antigens will most likely target both the bacterial at least in an experimental model for bovine necro-haem‑ toxins and the bacterial colonization and proliferation. orrhagic enteritis [112]. Furthermore, antibodies against For the broiler industry, once the ideal vaccine antigens alpha toxin alone, which is essential to cause intestinal have been identifed, development of an oral vaccine is disease in calves, are not sufcient to provide the same needed. level of protection as antibodies directed against a mix‑ ture of C. perfringens proteins, indicating that a mixture 3.2 Towards the development of new coccidiosis vaccines of diferent antigens will be needed to provide full pro‑ Coccidiosis, an enteric disease cause by protozoan para‑ tection [110]. In order to fully protect calves against C. sites of the genus Eimeria, remains a major economic and perfringens-induced enteric diseases, antigens that target welfare concern for the poultry industry globally. Seven bacterial colonization and proliferation might be of equal species (Eimeria acervulina, E. brunetti, E. maxima, E. importance as antigens targeting the toxin activities. mitis, E. necatrix, E. praecox and E. tenella) are known to Next, it has to be explored whether parenteral vaccina‑ infect chickens, and at least six others infect turkeys [117, tion is sufcient to induce a protective immune response 118]. Te costs associated with coccidial disease are dif‑ or if a combination of systemic and mucosal immunity is cult to calculate, but have been estimated to exceed 3 bil‑ needed when not only the bacterial toxins but also bacte‑ lion US dollars for the chicken industry alone, worldwide rial colonization is targeted. [119]. Because coccidiosis is a predisposing factor for the As administration of multiple parenteral doses of a vac‑ occurrence of necrotic enteritis, the true economic bur‑ cine to calves is more feasible than for chicken, it may den is likely even higher. All Eimeria species can cause be assumed that the development of a vaccine against disease but the severity and clinical symptoms vary necro-haemorrhagic enteritis is more straightforward among species, and there is little or no cross-protection and that C. perfringens supernatants can be used as a across species or some strains [120, 121]. vaccine preparation. However, native toxins cannot be used as vaccine antigens due to safety issues. Inactiva‑ 3.2.1 Management of coccidiosis through anticoccidial tion of clostridial toxins is generally achieved by formal‑ drugs dehyde treatment, which risks residual formaldehyde in Modern poultry production systems require efective the vaccine preparation, incomplete inactivation of the control of coccidian parasites, typically through the rou‑ toxins, and batch-to-batch variation. Moreover, formal‑ tine use of anticoccidial drugs in feed or water. In the dehyde inactivation can induce changes in the tertiary European Union, eleven diferent anticoccidial drugs protein structures of relevant antigens and infuence the are currently licensed and between 240 and 300 tonnes immunogenicity of the vaccines. Indeed, vaccination of are sold for use in animals for markets such as the UK Hoelzer et al. Vet Res (2018) 49:70 Page 9 of 15

every year [122]. Anticoccidial drugs can be divided into launched in 1989,2 and several similar vaccines have been two groups, synthetic or chemical anticoccidials and developed since using the same approach [126]. Non- ionophores, which are products of fermentation [123]. attenuated and attenuated anticoccidial vaccines have In some countries such as the US, ionophores are clas‑ become popular in the breeder and layer sectors, but are sifed as antibiotics, albeit with low human medical less widely used in the much larger broiler sector due to importance. their relatively high cost compared to anticoccidial drugs Te ionophores currently dominate the anticoccid‑ and their limited availability. Because Eimeria cannot ial drug market, largely because they provide incom‑ replicate efectively in vitro, the production of these live plete protection, even against naïve feld strains without vaccines can only be achieved in Eimeria-free chickens any drug resistance. Low levels of parasites survive and and separate chickens have to be used for each species or induce protective immunity against the prevailing local strain to be included in a vaccine. Despite these produc‑ parasite strains, without causing clinical disease [124]. tion concerns billions of anticoccidial vaccine doses are Anticoccidial drugs provide an efcient means of con‑ sold every year, but more would be required to fully meet trolling coccidial parasites and are highly cost-efective. the growing demand. However, drug resistance is widespread and increasing consumer concerns related to drug use in livestock pro‑ 3.2.3 Next generation anticoccidial vaccines duction and residues in the food chain encourage the use Eforts to improve on frst and second generation live of alternatives such as vaccination. Notably, because coc‑ anticoccidial vaccines have included extensive attempts cidiosis is a predisposing factor for necrotic enteritis and to identify antigens that are appropriate for use in sub‑ other secondary bacterial infections, efcient control of unit or recombinant vaccines. In addition, progress has this parasite is important to minimize the use of medi‑ been made on the preparation of novel adjuvants and cally important antibiotics, including those deemed criti‑ some promising results have been obtained, although cally important for human health, in poultry production. data on their use in poultry has so far remained fairly limited [127]. As an example, one vaccine3 is formulated 3.2.2 Traditional live anticoccidial vaccines from a crude mix of afnity purifed E. maxima game‑ Te frst anticoccidial vaccine was marketed in 19521 tocyte antigens [128], although the levels of protection [125]. It is a live parasite vaccine which includes mul‑ achieved have remained controversial and production tiple wild-type (i.e., non-attenuated) Eimeria species. of the vaccine still requires parasite amplifcation in Exposure to limited levels of such non-attenuated para‑ chickens. Numerous studies have suggested that defned sites permits the induction of a natural immune response antigens such as apical membrane antigen 1, immune in the chicken, resulting in protection against subse‑ mapped protein 1, lactate dehydrogenase and SO7 are quent coccidial challenge. However, because protec‑ highly promising vaccine candidates (reviewed elsewhere tive immune responses against Eimeria are fully species [129]). Studies of Eimeria feld populations have reported specifc, the inclusion of each individual target species is limited diversity in many of these antigens, indicating necessary if comprehensive protection is to be achieved, that recombinant vaccines for Eimeria may succeed even which results in relatively complex vaccine formulations. though antigenic diversity has undermined equivalent Such vaccines commonly include between three and vaccines for related parasites such as Plasmodium [130, eight parasite species or strains. Te approach has been 131]. However, at present no recombinant anticoccidial highly successful, although the lack of attenuation has vaccine is close to reaching the market. been associated with reduced fock performance follow‑ One of the biggest remaining challenges is how to ing vaccination and occasional clinical disease (reviewed deliver the antigens in an afordable, efective, and, most elsewhere [126]). importantly, scalable manner. A range of vectored expres‑ In response to this limitation, a second generation of sion/delivery systems have been suggested including live Eimeria vaccines has been developed using attenu‑ Fowlpox virus (FWPV), HVT, Salmonella Typhimurium, ated parasite lines. For most of these vaccines, attenua‑ yeasts such as Saccharomyces cerevisiae and the tobacco tion was achieved by selecting for so-called precocious plant Nicotiana tabacum, with several showing promise strains, which typically exhibit reduced pathogenicity [129]. Most recently, it has been suggested that Eimeria with fewer and/or smaller rounds of asexual replication. itself might function as an expression/delivery vector Tese attenuated strains retained their ability to immu‑ for vaccine antigens [132–134]. Te ability to express nize. Te frst live attenuated anticoccidial vaccine was

2 Under the name Paracox™. 1 Under the name CocciVac™. 3 Under the name CoxAbic™. Hoelzer et al. Vet Res (2018) 49:70 Page 10 of 15

and deliver anticoccidial vaccine antigens from multiple quality which makes their efcacy and safety predictable. parasite species in a single transgenic line could provide However, licensed vaccines are not available in all cases. an opportunity to streamline anticoccidial vaccine pro‑ To generate AVs, selected bacterial or viral strains are duction from as many as eight lines to just one or two. usually combined with a proper adjuvant. Several viral Using an attenuated vector species such as E. acervulina or bacterial species can be used in a combination vaccine can improve productive capacity enormously and reduce and diferent serotypes can also be combined in a poly‑ vaccine cost. Te parasite vector may also provide some valent vaccine. Te combination of inactivated viruses ability as an adjuvant and methods for on-farm delivery and bacteria is also an option. Bacterial AVs are accepted are well established [133]. in all countries of the economic European area, whereas In summary of this section on new coccidiosis vaccines, viral AVs are not allowed in 10 European countries as pressure to reduce antibiotic drug use in livestock pro‑ including France, Denmark and Spain [138]. duction increases it is clear that the demand for coccidial A critical role in the successful production and use of vaccines is stronger than ever. In the US, approximately an AV falls to the isolation of vaccine strains. Terefore 35–40% of broiler companies use programs that include diagnostic samples must be carefully obtained, based on vaccination to control coccidiosis [135]. Tis trend is appropriate choices regarding which sick and untreated primarily driven by demands to produce “no antibiot‑ animals to select for sample collection, which necropsy ics ever” poultry products. However, it has also been material to select, and which cultivation conditions and shown that some coccidial vaccines provide an opportu‑ strains to use after results from sero-, toxo- or virulence- nity to replace drug-resistant feld parasites in a poultry typing. For that purpose several methods like PCR, house with susceptible vaccine strains. While current MALDI-TOF MS, slide agglutination or DNA sequenc‑ European attenuated vaccines are limited by their lower ing are available. Because of the fundamental importance reproductive potential, live vaccines do retain consider‑ of the strain choice for the production of an adequate AV, able unexplored potential. A better understanding of the close collaboration between diagnostic laboratory and underlying immune mechanisms through which these vaccine production is critical. Each production is cus‑ nontraditional approaches operate is needed to allow fur‑ tom-made and numerous adjuvants, viral and bacterial ther progress. Ultimately, it is clear that novel vaccines isolates, including serotypes, toxins and species, provide must be cost-efective, compatible with high standards of countless combinations. Tis underlines the importance animal welfare, scalable and easy to deliver. of experience as the basis in the production of high qual‑ ity AVs. Te veterinarian also has obligations regarding 4 Autogenous vaccines to reduce the need diagnosis, ordering and responsibility for the administra‑ for antibiotic use tion of the vaccine. Autogenous vaccines (AV) are also known as emergency, A variety of bacterial components are often used in herd-specifc or custom made vaccines. Although the AVs. Tese include for poultry: Bordetella spp., Campy- legal basis and exact defnition difers from country to lobacter spp., Cl. perfringens, Enterococcus cecorum, country, AVs are used worldwide (e.g. EU, USA, Canada, Erysipelothrix rhusiopathiae, E. coli, Gallibacterium Brazil, China, Indonesia, Australia, Egypt) and have a anatis, Mycoplasma spp., Ornithobacterium rhinotra- long history of use. Te use of AVs for the control of fowl cheale, Pasteurella multocida, Riemerella anatipestifer; cholera has been well-documented [136, 137]. As a com‑ for swine: Actinobacillus pleuropneumoniae, Bordetella mon defnition, all AVs are made from inactivated bac‑ spp., Brachyspira spp., Cl. perfringens, E. coli, H. paras- terial or viral strains which were isolated from the same uis, Mycoplasma spp., Pasteurella multocida, Strep. suis, fock in which the vaccine is to be used. Te use of AVs Trueperella pyogenes; for cattle: Chlamydia spp. Cl. Per- is only allowed if no licensed vaccine is available, or it fringens, E. coli, Histophilus somni, Mannheimia haemo- is respectively inefective or does not cover the current lytica, Moraxella bovis, Mycoplasma spp., Pasteurella pathogen strains in the fock. Te defnition of a fock multocida, Salmonella enterica, Trueperella pyogenes; varies and may include integrated concepts of produc‑ and for fsh: Aeromonas spp., Photobacterium spp., Pseu- tion chains in diferent places; to address the issue, the domonas spp., Vibrio spp., Yersinia ruckeri. concept of an epidemiological link has recently been pro‑ Depending on the animal species and age at vaccina‑ posed by the Co-ordination Group for Mutual Recogni‑ tion diferent adjuvants can be used. As a standard adju‑ tion and Decentralised Procedures [138]. vant with good safety and efcacy, aluminium hydroxide Licensed vaccines have advantages compared to AVs, is often used for production. Polymer and other gel-like including obligatory good manufacturing practice (GMP) adjuvants are also available for production in aqueous production. Licensed vaccines are also produced in big‑ mixtures. Oily adjuvants, especially for water-in-oil emul‑ ger batches with defned strains and a high level of sions, require a more sophisticated mixing procedure Hoelzer et al. Vet Res (2018) 49:70 Page 11 of 15

because of the need of a stable emulsion. Furthermore jurisdictions, and generates durable protection, ideally oily vaccines might pose safety concerns. However, after a single administration. these induce a promising long lasting immune response Existing vaccines still fall short of these ideals. In fact, because of a depot efect. In the case of organic animal many current vaccines have a number of shortcomings production use of plant oil might be an option in order to with regard to safety, efcacy and/or user-friendliness avoid unwanted hydrocarbons. Te risk of adverse efects, that limit their ability to replace antibiotic use. Over‑ which depend on the adjuvant-antigen combination, can coming these challenges will take close collaboration and be decreased by standardization of the protocols. innovative new approaches. Public–private partnerships More data regarding the efcacy and safety of AVs in represent one promising governing structure for assuring feld studies should be collected because clinical safety such close collaboration across public and private sectors. and efcacy is not regulated. Te need for this is refected Investments in basic and applied research are equally by numerous current publications about viral and bacte‑ needed to overcome these challenges, and research needs rial AVs for poultry [139–142], bovine [143], swine [144] will have to be prioritized to ensure scarce resources will and fsh [145]. Most results show that AVs can be a useful be preferentially dedicated to areas of greatest potential alternative to antibiotic use. impact. Research to characterize and quantify the impact Only a few countries allow the use of live AVs [138]. of vaccination on antibiotic use is equally needed. Te normally inactivated vaccines must be tested for Yet, some data demonstrating the ability of vaccines to sterility. In the EU this could be carried out by internal reduce antibiotic consumption are already available. Sim‑ tests according to the Pharmacopoea [146]. Further steps ilarly, key research breakthroughs and a number of highly in quality control include the inactivation test, endotoxin promising vaccination approaches are already in devel‑ content or stability tests. Some producers ofer GMP opment. Tese include new oral vaccines based on bac‑ production, and GMP production is required in some terial spores, live vectors, or new delivery strategies for countries such as Finland or Sweden [147]. In most coun‑ inactivated oral vaccines; they also include new vaccina‑ tries GMP is only recommended. Tis example shows the tion strategies in-ovo, combination vaccines that protect vast diferences in national legislation regarding the def‑ against multiple pathogens, the use of recent biotechno‑ nition and interpretation of AVs. Because of worldwide logical advances, and comprehensive approaches to man‑ circulation of animals and their pathogens a harmoniza‑ age diseases caused by ubiquitous pathogens. tion of manufacture, control and use of immunological Terefore, further reductions in the need for antibiotic veterinary medicinal products like AV is important, and use through the use of new vaccines are all-but-certain, the aim at the economic European area [138]. and investments in research and development of new In summary, AVs are a valuable option in certain situ‑ vaccines will be vital for the sustained success of animal ations where commercial vaccines are either not avail‑ agricultural production around the world. able or expected to lack efcacy because of a mismatch Competing interests between circulating and vaccine strains. Te selection The authors declare that they have no competing interests. of adequate clinical isolates and vaccine formulations Authors’ contributions requires considerable expertise and the efective use of KH, FVI, and CG planned the manuscript. KH led the drafting of the manu‑ AVs depends on adequate manufacturing and appropri‑ script. LB, DPB, EC, SMC, BD, EEV, EG, KK, SL, MM, MR, MCS, NMW, CG, and FVI ate veterinary oversight. Regulatory diferences among provided additional information and contributed to writing the manuscript including drafting selected sections and reviewing the manuscript. FVI and CG countries create a highly fragmented legal landscape that revised the manuscript. All authors read and approved the fnal manuscript. would beneft from further harmonization. Acknowledgements We would like to thank the organizers, sponsors, and participants of the 5 Conclusions ­2nd International Symposium on Alternatives to Antibiotics on which this Vaccines are proven strategies for the prevention or con‑ manuscript is based, in particular the United States Department of Agriculture (USDA) and the World Organisation for Animal Health (OIE). trol of infectious diseases in animal populations. Tere‑ fore, they are promising alternatives that can reduce the Author details 1 need to use antibiotics in food-producing animals and The Pew Charitable Trusts, 901 E Street NW, Washington, DC 20004, USA. 2 Ohio Agriculture and Research Development Center, Animal Sciences, Ohio their direct mitigating impact on antibiotic consump‑ State University, 202 Gerlaugh Hall, 1680 Madison Ave., Wooster, OH 44691, tion has been demonstrated in a number of studies, even USA. 3 Pathobiology and Population Sciences, Royal Veterinary College, Univer‑ though the relationship between antibiotic use and vac‑ sity of London, Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, UK. 4 Department of Virology, Parasitology and Immunology, Faculty of Veterinary cination is not in all cases clear-cut. Te ideal vaccine is Medicine, Ghent University, Salsiburylaan 133, 9820 Merelbeke, Belgium. safe, efective against a broad range of pathogens, and 5 School of Biological Sciences, Royal Holloway University of London, Egham, 6 easily adapted to mass-application. At the same time, it Surrey TW20 0EX, UK. Science and New Technologies Department, World Organisation for Animal Health (OIE), 12 Rue de Prony, 75017 Paris, France. is cheap to produce and use, easy to register across key Hoelzer et al. Vet Res (2018) 49:70 Page 12 of 15

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