Skip to main content

Vaccines as alternatives to antibiotics for food producing animals. Part 2: new approaches and potential solutions


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 scientific 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 development of vaccines as alternatives to antibiotics in food producing animals; opportunities, challenges and needs for the development of such vaccines are discussed in the first 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 vaccine approaches, novel adjuvants, new delivery 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 effectively reduce the need for antibiotics will require close collaboration among stakeholders, for instance through public–private partnerships. Targeted research and development investments and concerted efforts by all affected are needed to realize the potential of vaccines to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks.


Alternatives to antibiotics can help minimize the need for antibiotics by helping to prevent and control infectious diseases in animal populations. As such, safe and effective alternatives are crucially important to the future success of animal health and production. To assess scientific advancements in the research and development of alternatives to antibiotics, highlight promising research results and novel technologies, assess challenges associated with their commercialization and use, and provide actionable strategies to support their development, the United States Department of Agriculture (USDA), with support from the World Organisation for Animal Health (OIE), organized the second International Symposium on Alternatives to Antibiotics. The symposium 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 licensure and development of alternatives to antibiotics. This two-part manuscript synthesizes and expands on the scientific presentations and expert panel discussions from the symposium regarding the use of vaccines as alternatives to antibiotics that can reduce the need for antibiotic use in animals. Part 1 synthesizes and expands on the expert panel discussions regarding the opportunities, challenges and needs related to vaccines that may reduce the requirement for use of antibiotics in animals, while part two focuses on highlighting new approaches and potential solutions.

A general discussion of the importance of antibiotic resistance and the opportunities, challenges and needs related to vaccines as alternatives that may reduce the need for use of antibiotics in animals is provided in part 1 of this review, including a discussion of the properties of ideal vaccines, how current vaccines compare to these ideal vaccines, and how investment decisions around research and development of vaccines are made. This second part of the manuscript will highlight specific research advancements in the area of veterinary vaccines.

New approaches for the development of veterinary vaccines

Mucosal immunity and tolerance: challenges to the development of effective oral vaccines

As mentioned in part one of this manuscript, most pathogens invade the host at the mucosal surfaces, such as the gastro-intestinal (GI) tract. The GI tract constitutes the largest surface area of the body and is exposed daily to vast numbers of foreign antigens derived from feed, the microbiota and pathogens [1]. Within the intestine a complex cellular network has evolved to prevent unwanted immune responses to innocuous antigens, for instance feed or microbiota, while allowing swift protective responses against agents that cause infectious disease. Key to keeping enteric pathogens at bay is the presence of protective pathogen-specific secretory IgA (SIgA) at the site of entry, which prevents the adhesion of micro-organisms to the intestinal surfaces and neutralizes their enterotoxins. Triggering robust and protective intestinal SIgA responses usually requires the local administration of vaccines [2]. Although live attenuated oral vaccines have had tremendous success, resulting for instance in the near global eradication of poliovirus [3], concerns on the dissemination of vaccine strains into the environment and rare cases of reversion to virulence, leading to vaccine-induced disease, have driven oral vaccine development to nonliving or vectored vaccines [4]. However, oral vaccination is challenging due to several hurdles imposed by the cellular and molecular architecture of the gut: (i) the harsh environment of the stomach and small intestine, including the low pH, digestive enzymes, and bile salts, required to digest feed also easily destroys vaccines, (ii) a poor uptake of vaccine antigens by the intestinal epithelial barrier and (iii) the tolerogenic mechanisms that pervade the intestinal tissues, leading to peripheral and oral immune tolerance upon oral administration of antigens via the induction of FoxP3+ regulatory T cells. This often results in a low immunogenicity of oral vaccines and requires innovative strategies to deliver the vaccine antigens to the intestinal immune system as well as the inclusion of adjuvants that promote innate and adaptive immunity [5].

The mucosal immune system in the gut can be divided in inductive sites, where sampled antigens stimulate naive T and B cells, and effector sites, where effector cells perform their functions, e.g. assisting in the production of SIgA. In the small intestine, the inductive sites comprise the gut-associated lymphoid tissues (GALT) and the mesenteric lymph nodes, while the effector sites constitute the lamina propria and the surface epithelium [6]. The GALT itself is composed of Peyer’s patches (PP), appendix and isolated lymphoid follicles. The presence of other GALT-like structures, such as lymphocyte-filled villi (rat, human) and cryptopatches (mouse) is dependent on the species. Interestingly, while in birds and most mammals PP or their equivalent are scattered throughout the small intestine, in pigs, ruminants and dogs the PP in the distal small intestine (ileum) are continuous. Fish and reptiles on the other hand lack PP and the intestinal immune system in these species is composed of epithelial leukocytes and rare, small non-organized lymphoid aggregates. It remains largely unknown how these species-specific differences might affect the efficacy of oral vaccines.

From their entry point, which is typically the oral cavity, to their delivery site, most commonly the small intestine, the integrity of delivery systems and the stability of vaccine components are at risk. Lysozyme in saliva, the low gastric pH together with pepsin and intestinal proteases can degrade oral vaccines. Enteric coating of vaccine components with pH-responsive polymers with a dissolution threshold of pH 6 might protect against gastric degradation and results in the release of their contents in the small intestine [7]. In this context, ruminants pose an additional problem to vaccine stability as their polygastric gastro-intestinal tract effectively degrades substances including vaccines. Site-specific delivery of oral vaccines to the small intestine is favorable as the mucus layer covering the small intestinal epithelium consists of only one layer, which is loosely adherent, less thick and patchy as compared to the colonic mucus layers and might promote their access to the intestinal epithelium. In addition, the small intestine is less densely populated by the microbiota, which might further disrupt the integrity of the delivery systems and the stability of vaccine components. Underneath the mucus layer, a single layer of intestinal epithelial cells prevents uncontrolled access of the luminal content to the underlying intestinal tissues, further restricting uptake of oral vaccine antigens. Crossing of the epithelial barrier by vaccines could be enhanced by exploiting antigen sampling routes in the small intestine or by adopting strategies used by enteric pathogens to colonize or invade the host [8]. The best-known sampling route in the gut is associated with microfold (M) cells. These specialized intestinal epithelial cells reside within the follicle-associated epithelium covering the Peyer’s patches and take up macromolecules, particulate matter and microorganisms [9]. Many enteric pathogens hijack M cells to invade the host by binding to apical receptors. For instance, the invasin protein of Yersinia species interacts with β1 integrin on M cells, leading to infection [10]. Likewise, GP2 marks M cells in many species and binds to FimH, a subunit of type I pili on Escherichia coli and Salmonella enterica. This interaction results in uptake of FimH+ bacteria and initiates mucosal immunity [11]. Although many groups have focused on improving antigen uptake by targeting oral vaccines to M cell-specific receptors, these cells represent only a small, species-specific percentage of the total intestinal epithelial cell population. Although M cell numbers increase from the cranial to caudal small intestine and M cell targeting strategies work quite well in rodent models, they mostly fail in larger animals due to the long passage time needed to reach the distal small intestine, where the gut-associated immune system is most pronounced. Besides M cells, sampling of luminal antigens also occurs by intestinal mononuclear phagocytes via transepithelial dendrites. This sampling mainly occurs by CD11c+CX3CR1+ macrophages, which transfer the antigens to CD103+ dendritic cells (DCs). These DCs then drive the differentiation of regulatory T cells (Tregs), which subsequently induce tolerance to these proteins [12]. In the steady state, goblet cells can also transport small soluble proteins (<10 kDa) across the epithelium to tolerogenic DCs via so-called goblet cell-associated antigen passages [13]. Absorptive intestinal epithelial cells or enterocytes, constituting >90% of the small intestinal epithelium, may also sample the luminal content through receptor-mediated transcytosis. For instance, the neonatal Fc receptor (FcRn), an MHC class I-like Fcγ receptor, is expressed on the apical surface of enterocytes and transcytoses IgG, immune complexes or Fc-coated nanoparticles from the lumen to the basolateral surface of the epithelium [14]. Similar to M cells, it might be worthwhile to target apical receptors exploited by enteropathogens on small intestinal enterocytes to promote uptake of antigens by the epithelial barrier. A potential candidate would be aminopeptidase N (ANPEP), a zinc-dependent peptidase present in the brush border of small intestinal enterocytes, which serves as an entry receptor for several coronaviruses and also binds F4 fimbriae, a colonisation factor produced by porcine-specific enterotoxigenic E. coli. ANPEP also transports F4 fimbriae as well as microparticles functionalised with ANPEP-specific monoclonal antibodies across the intestinal epithelial barrier, resulting in robust intestinal SIgA responses, at least in piglets [15, 16].

Although the selective targeting of vaccine antigens to apical receptors might promote their uptake by the epithelium via transcytosis, this process is in itself insufficient to trigger protective intestinal immunity upon oral vaccination and explains the need to include adjuvants. These adjuvants should act on antigen presenting cells as well as intestinal epithelial cells to promote the induction of protective SIgA and cell-mediated immune responses. Indeed, enterocytes not only provide a physical barrier separating the intestinal lumen from the host tissues, but also relay information on the luminal content to the underlying immune cells through the secretion of inflammatory or tolerogenic mediators. For instance, during the steady state, enterocytes produce thymic stromal lymphopoëtin (TSLP) and transforming growth factor (TGFβ), which imprint a tolerogenic phenotype on intestinal dendritic cells [17]. In contrast, upon infection enterocytes secrete IL-6 and IL-8 [18]. This probably facilitates a switch from a tolerogenic to an immune-inductive environment, allowing activation of intestinal antigen presenting cells. As yet the most effective adjuvants for oral application are the enterotoxins from Vibrio cholera (CT) and enterotoxigenic E. coli (ETEC) (LT). Due to inherent toxicity, dmLT was developed, a nontoxic LT mutant retaining its adjuvanticity. This dmLT triggered intestinal memory responses upon oral vaccination with a nonliving ETEC vaccine and seems a promising candidate to be included as adjuvant in oral vaccines [19, 20]. Similarly promising strategies have been reported for Eimeria [21]. Recent studies have shown that Eimeria-induced IL-17 production is critical in the initiation of early innate immune response in coccidiosis and blocking of IL-17 production by exogenous IL-17-neutralizing antibody reduced both the intracellular development of Eimeria and the severity of intestinal lesion [22,23,24].

In summarizing this part, future design of oral vaccines should be tailored to the needs of the target species, focus on the selective delivery of vaccines to epithelial receptors to promote their transport across the epithelial barrier, induce protective immune response in the target tissues, and should include a mucosal adjuvant able to trigger memory SIgA responses.

Recombinant Bacillus spores as oral vectored vaccines

Endospores, or spores, are produced by many bacteria as a response to nutrient deprivation. The spore is a dormant entity about 1 μm in size that can germinate, allowing a nascent cell to emerge and enter vegetative cell growth [25]. The spore carries remarkable resistance properties, being typically resistant to high temperatures (typically 70–80 °C), desiccation, irradiation, and exposure to noxious chemicals [26]. The two principal spore-forming bacterial genera are Bacillus and Clostridia with the latter being exclusively anaerobic.

Members of the Bacillus genus are being used as probiotics, that is, microorganisms that are added to the diet to improve the balance of microbial communities in the GI-tract and are therefore beneficial to human or animal health [27, 28]. Typical species include Bacillus clausii, Bacillus coagulans and Bacillus subtilis. For a long time, it has been assumed that Bacillus spores are soil organisms yet the evidence supporting this is actually rather sparse. Instead, spores are found in the soil in abundance but live, vegetative cells, are rarely if ever found other than in association with plants or in the animal gut. Mounting evidence shows that spores, although found in the soil, are mostly dormant and are shed in the feces of animals, which are their natural hosts [29]. The consumption of spores associated with soil-contaminated plant matter enables spores to enter the GI-tract, transit the gastric barrier unscathed and then germinate and proliferate in the intestine before excretion as dormant spores [30]. Evidence suggests that spore forming bacteria comprise as much as 30% of the gut microbiota, indicating that the ability to form spores enables bacteria to survive in the environment as well as entering and transiting the gastric barrier of animals [31].

The extraordinary resistance properties of Bacillus spores coupled with their ease of genetic manipulation, and their successful use as probiotics, makes them attractive candidates for the delivery of heterologous antigens for vaccination. Spores have been used as vaccine vehicles in a number of ways, differing principally in whether spores are genetically modified or not. In all cases B. subtilis has been utilized due to the excellent genetics available. Using genetic modification, a chimeric gene consisting of a fusion between a B. subtilis anchor gene and an open reading frame encoding a putative protective antigen is first constructed. The next step is introduction of the chimera into the B. subtilis chromosome using a gene transfer technique, typically DNA-mediated transformation, a process in B. subtilis that is straightforward. Typically, the anchor is the 5′-end of a gene encoding a spore coat protein such that the chimera is displayed on the spore coat. Surprisingly, heterologous antigens displayed on B. subtilis spores are mostly stable and do not appear to suffer extensive degradation. Using this approach a number of candidate antigens have been displayed and then evaluated in animal models. For example, spores displaying a tetanus antigen TTFC conferred protection to a lethal dose of tetanus toxin when administered orally [32, 33]. Mice dosed orally with spores expressing part of the alpha toxin of Clostridium perfringens were protected to challenge with alpha toxin [34]. A more recent example is that of Clostridium difficile where a C-terminal fragment of the toxin A (TcdA) could be stably expressed and when administered orally to hamsters conferred protection to C. difficile infection [35, 36]. This particular vaccine has now entered clinical evaluation in humans [37].

Using a non-genetically modified organism (GMO) approach it has been shown that spores can adsorb antigens efficiently onto their surface and surprisingly this is both strong and stable, and reflects the unique biophysical properties of the spore [38]. For the adsorption approach, it has been shown that the gastric barrier is particularly corrosive and adsorbed antigens are labile, but for intranasal delivery this method appears satisfactory. Using this approach inactive (killed) spores can be used and success has included studies showing protection to influenza (H5N1) [39] and significant reduction in lung counts of animals challenged with Mycobacterium tuberculosis [40]. A unique feature of spores is their ability to enhance immune responses and this adjuvant effect has been characterized in depth [41,42,43].

However, the use of spores as mass-delivery vehicles for vaccines has several limitations. Oral delivery clearly is the preferred approach but appears to work effectively only for the GMO approach. Oral delivery also raises issues of tolerance and may prove to be a limiting factor. Sublingual delivery has also been explored; this approach appears to provide levels of protection that are equivalent to oral delivery, but requires more doses [36, 44]. Nasal delivery is suitable but raises potential safety issues. For animal vaccines, spores are attractive since they are currently used as feed probiotics but also because they can survive the high temperatures used for feed production and may offer long-term utility. As mentioned already, spores have been manipulated for protection against C. perfringens but there now exists the opportunity to develop spores for protective vaccination to necrotic enteritis, an important poultry disease caused by C. perfringens that has been identified as a high vaccine research priority by the OIE ad hoc Group (see Additional file 2 in

One application that is particularly promising is the use of spore vaccines in aquaculture. With intensive fish farming, Bacillus spores are being used as probiotic feed supplements. For shrimp farming, viral diseases have devastated the industry and one of the most important shrimp pathogens is white spot syndrome virus (WSSV) that causes seasonal outbreaks of disease [45]. A number of groups have developed B. subtilis spores that display the VP28 capsid protein of WSSV and when administered in feed appears to protect against white spot disease [46,47,48,49]. The mechanism for protection is intriguing; even though shrimp are not thought to produce antibodies, it is clear that presentation of the viral antigens does produce some level of specific immunity.

Despite the progress being made with spore vaccines one key issue remains: the containment of GMOs. Because spores are dormant with the potential to survive indefinitely in the environment, the use of recombinant spores in spore vaccines is likely to raise environmental concerns and successful regulatory approvals may be slow or impossible to secure. For human use, it is likely that a case can be made that the recombinant spore vaccines addresses an unmet clinical need, but for animal use devising a method for biological containment will be crucial.

Genetically modified live microorganisms as oral vectored vaccines and vaccine platforms

Technological advancements now make it possible to genetically engineer bacteria and other microorganisms that deliver heterologous antigens in a way that can stimulate mucosal as well as humoral and cellular systemic immunity [50]. Multiple species of bacteria including Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Typhi, E. coli, Lactococcus lactis, Lactobacillus casei, Lactobacillus reuteri, Bacillus subtilis, and Bacillus thuringiensis, have been used to express protein antigens derived from bacterial, viral, and protozoal pathogens [51,52,53,54,55,56,57,58,59,60,61]. Some of these vectors are inherently non-pathogenic; Lactobacillus and Lactococcus strains, for instance, are “Generally Recognized as Safe” (GRAS) [50, 61]. In other cases the microorganisms have been rendered non-pathogenic through the targeted deletion of virulence genes; strategies for the development of Salmonella vectors, for instance, typically rely on the deletion of certain metabolic functions that limit the bacterium’s ability to replicate in the host and attenuate virulence without impacting host colonization or invasion [50]. In fact, an intrinsic property shared by many, although not all, microorganisms used as vectors is their ability to effectively infect the host and initiate innate and subsequent adaptive immune responses, for instance by triggering the host’s pattern recognition receptors [50]. These recombinant vectored vaccines can be delivered directly to a mucosal surface via nasal, ocular, or oral administration, which not only allows for mass application but may also enhance mucosal immune responses, the primary surface through which most pathogens invade. Moreover, contrary to traditional attenuated live vaccines, these recombinant vaccines in many cases do not carry a risk of reversion [50].

In veterinary medicine, oral vectored vaccines have been instrumental in the eradication or control of rabies in wildlife reservoirs [62, 63]. Oral vectored vaccines have also been developed for several other veterinary applications, including some economically important diseases of food-producing animals that are associated with considerable antibiotic use such as porcine circovirus type-2 (PCV-2); in some cases, the vaccine vector is a chimera containing parts of multiple microorganisms—for instance, an attenuated live vaccine may be used as the vector—and the resulting vaccine simultaneously confers protection against multiple diseases, for instance Marek’s disease and infectious bursal disease or Newcastle disease and avian influenza [63, 64].

The development of some vaccine vector systems has been very successful and numerous veterinary vaccines have been developed based on them; the canarypox virus vector system ALVAC, for instance, has been used for the development of a range of veterinary vaccines including against rabies, influenza, and West Nile virus [64]. Similarly, adenovirus vectors have also been widely used in veterinary medicine, both in companion and food-producing animals [65]. Vaccine platforms such as these are particularly valuable as they can allow for the rapid development of vaccine candidates in response to emerging vaccine needs, but the possibility of anti-vector immunity can restrict their usefulness [66]. Research and development of additional vaccine vector platforms is therefore needed. Salmonella strains that express foreign antigens, either chromosomally or plasmid-based, have yielded promising results in several species including mice, humans, pigs and chicken [67,68,69,70,71,72]. Diseases for which these Salmonella vectored vaccines were investigated include influenza, Brucella abortus, post-weaning diarrhea and heterologous strains of Salmonella [69,70,71,72]. The use of Pasteurellaceae as vectors for modified live vaccines against shipping fever in calves is currently under investigation, with promising preliminary findings [73]. Use of this vector system for other diseases including pinkeye has been suggested [73].

New approaches for in-ovo vaccines

In-ovo vaccination is a mass-vaccination strategy that is mainly used in broiler chickens albeit occasionally also in broiler-breeder and layer chickens [74]. Eggs are injected in the hatchery, typically during the third week of embryonic development around day 18 or 19. To vaccinate, a small hole is made in the shell at the blunt end of the egg and the vaccine is injected below the chorion-allantoic membrane into the amniotic cavity or directly into the embryo. Commercial in-ovo vaccination systems that automatically inject the eggs have been available since the early 1990s. More than 90% of broiler chickens in the US are vaccinated in ovo, and in Brazil that fraction equals 70% [75]. The most common use of in-ovo vaccination is for Marek’s disease, potentially combined with vaccines against other diseases such as Gumboro or Newcastle disease.

The ability to deliver a clearly defined vaccine dosage to every single chick and to invoke early protection in the chicks is among the main benefits of this technology, but it is labor-intensive, causes stress for the chicks, and high sanitary standards need to be followed during vaccine preparation and injection to manage infection risks [74, 76]. In addition, the location of the vaccine injection is critical for efficacy. It has been shown, for instance, that if Marek’s disease vaccine is accidentally deposited into the air cell or allantoic fluid, adequate protection is not achieved [77]. The stage of embryonic development can have profound effects on vaccine safety and efficacy [78]. One study, reported that vaccination of 10–12 day-old embryos with herpes virus of turkeys (HVT) led to pronounced lesions and embryonic deaths, while vaccination on days 16 did not cause detectable lesions [78]. Embryonic age at vaccination has also been shown to be correlated with antibody titers [79]. Maternal antibody titers actually increase after the typical age for in-ovo vaccinations and peak just after hatch [76]. This can interfere with proper vaccine responses. However, evidence suggests that some vaccine strains are more affected by maternal antibodies than others [80]. Deliberate vaccine development may therefore limit the often disruptive effects that can be caused by maternal antibodies [78]. Other factors that need to be considered in the development of a successful in-ovo vaccination program include the characteristics of the vaccine or vaccines to be used, the type of incubator in which the eggs are housed in the hatchery, and the breed and age of the parent flock [76].

In-ovo vaccination strategies are promising means of reducing antibiotic use in poultry production and have been the subject of intense research. Importantly, they can provide robust and early protection against immune suppressive diseases such as infectious bursal disease [81, 82] and vaccines against multiple diseases have been successfully combined. For instance, studies have shown that in-ovo vaccination strategies can simultaneously confer protective immunity against Marek’s disease, infectious bursal disease, Newcastle disease, fowl poxvirus, coccidiosis, and necrotic enteritis [83, 84]. Other combination vaccines under investigation include vectored vaccines that simultaneously provide protection against Newcastle disease and infectious bursal disease [85]. In-ovo vaccination strategies have also been explored for other poultry diseases with promising results. This included an avian influenza vaccine based on a non-replicating human adenovirus vector [86], a recombinant viral vector vaccine against infectious laryngotracheitis [87], recombinant protein Eimeria vaccines [84, 88, 89] and a fowl adenovirus vectored vaccine against inclusion body hepatitis [90], among many others. A Mycoplasma gallisepticum vaccine for in-ovo vaccination of layer chickens has also recently been evaluated, even though high chick losses at hatch were reported for the medium and high doses of the vaccine that were investigated [91]. Therefore, in-ovo vaccination strategies are capable of controlling several economically important poultry diseases. Many of these diseases are viral, but can predispose animals to secondary bacterial infections. Therefore, in many cases, in-ovo vaccines are promising alternative approaches to the use of antibiotics.

Vaccination strategies to reduce antibiotic use for diseases from ubiquitous pathogens

Towards the development of new Clostridium perfringens vaccines

Clostridium perfringens is widespread in the environment and in the gastrointestinal tract of most mammals and birds. However, this bacterium is also one of the most common pathogens of food-producing animals, causing disease only under circumstances that create an environment which favors growth and toxin production, such as stress, injury, or dietary changes [92]. The bacterium itself is not invasive, but causes disease through the production of a wide array of toxins and enzymes. However, no single strain produces this entire toxin repertoire, resulting in considerable variation in the toxin profiles and disease syndromes produced by different toxinotypes of this bacterium [93]. While some of these toxins act only locally, other toxins which are produced in the gut exert their action in other internal organs or can act both locally and systemically [94,95,96]. To date, efficacious vaccines are only available for the diseases caused by systemic action of the toxins and vaccination against enteric diseases still remains a challenge. However, some of these enteric diseases caused by C. perfringens are of major economic importance and lead to considerable use of antibiotics. Amongst them are necrotic enteritis in broilers and necro-haemorrhagic enteritis in calves. Despite the fact that much research is being directed to the development of novel vaccines against these C. perfringens-induced enteric diseases, several key barriers still have to be overcome.

In general, clostridial vaccines require multiple doses to achieve full immunity. Unfortunately, parenteral booster immunizations are impossible in the broiler industry, where mass parenteral vaccination is only feasible at the hatchery, either in ovo or on day-old chicks. Because single parenteral vaccination at day of hatch offers no protection, other delivery methods need to be developed [97]. Oral vaccines can more easily be administered to birds, without the need of individual handling of the chicks and are therefore recommended. However, some questions arise when developing an oral vaccine as compared to the parenteral administration route. In addition to the fact that maternal antibodies can block the immune response in young chicks, also the induction of oral tolerance has to be circumvented and an efficient way to present the antigens to the mucosal immune system has to be developed. Oral tolerance is a common problem in mammals and fish when developing oral vaccines. This is in contrast to chickens, where oral tolerance is age-dependent, and only an issue in 1- to 3-day-old chicks. After that age, protein antigens have been shown to induce a robust immune response and oral vaccination schemes are thought to be feasible [98]. One appealing strategy for the delivery of vaccine candidates to the mucosal immune system is the use of attenuated or avirulent bacteria as antigen vehicles [99]. Attenuated recombinant Salmonella strains which express C. perfringens antigens have been tested in several studies as oral vaccine vectors, leading to some promising results. However, the amount of protection afforded by these vaccines is not as high as compared to multiple doses of parenteral vaccination, and seems to depend on the colonization level and persistence of the vaccine strain [100,101,102,103]. This indicates that the use of live vectors to express antigens derived from C. perfringens strains in the gut of broilers is a promising approach, but the vaccine delivery strategy still needs to be optimized to achieve optimal antigen presentation to the mucosal immune system and provide improved protection. Alternatives to attenuated Salmonella strains can be Bacillus subtilis spores or Lactobacillus casei, which both have a GRAS status and have the potential to be used as vaccine carriers for Clostridium antigens [34, 104]. B. subtilis has the advantage that the heat-stable spores can easily be incorporated in the feed and L. casei has known probiotic effects that facilitate the development of mucosal immunity. However, these types of vectors still have to be tested for their capacity to induce a good immune response, in particular against heterologous antigens, in broilers and whether they are able to provide protection against necrotic enteritis.

Another issue to be addressed when developing a vaccine against C. perfringens-induced enteric diseases is the choice of the antigens to be included in the vaccine. C. perfringens-induced diseases are the result of the toxins and enzymes that are produced and vaccination of chicks with C. perfringens supernatants provides protection against experimental necrotic enteritis [97, 105]. However, the protective capacity of the supernatants depends on the strain used for supernatant preparation, indicating that full protection might be determined by an effective combination of different bacterial immunogens [105]. In order to elucidate the optimal mixture of antigens to protect against necrotic enteritis, challenge trials are being performed mostly using parenteral vaccination schemes. Once the ideal combination of antigens is known, this will have to be adapted to oral delivery strategies. Several C. perfringens antigens have been evaluated as potential vaccine candidates. The tested antigens include both C. perfringens toxins (e.g. alpha toxin and the NetB toxin) and highly immunodominant proteins identified in post-infection serum from birds immune to necrotic enteritis [106]. In general, immunization studies of broilers with a single antigen all resulted in some level of protection against experimental necrotic enteritis. Remarkably, immunization with NetB toxin, which is essential to cause disease in broilers, does not afford higher levels of protection than vaccination with other toxins or proteins. However, when birds were vaccinated either via the parenteral or the oral route, with a combination of both NetB toxin and alpha toxin, higher levels of protection were obtained [107, 108]. In order to obtain full protection against C. perfringens-induced enteric diseases, not only antibodies that inhibit toxin activity might be needed; a combination of antigens targeting also bacterial proliferation, colonization and/or nutrient acquisition could be more efficient than either one of the individual approaches. Indeed, in a recent study disruption of the putative adhesin-encoding gene cnaA resulted in a reduced ability to colonize the chicken intestinal mucosa and to cause necrotic enteritis [109]. This strengthens the idea that vaccine antigens that target bacterial colonization might be indispensable to obtain a working vaccine against C. perfringens–induced enteric diseases. Additional vaccine targets might be enzymes that aid in breakdown of the host tissue and nutrient acquisition, such as, amongst others, mucinases, collagenases and hyaluronidases.

In contrast to the extensive efforts to develop a vaccine against necrotic enteritis in chickens, considerably less research has been directed to vaccination against necro-haemorrhagic enteritis in calves. The recent demonstration of the essential role of alpha toxin in necro-haemorrhagic enteritis and the proposition of a pathogenesis model will allow for the more targeted development of a vaccine [110, 111]. In calves as in chickens, protection against C. perfringens-induced necrosis can be obtained by antibodies against a mixture of toxins, at least in an experimental model for bovine necro-haemorrhagic enteritis [112]. Furthermore, antibodies against alpha toxin alone, which is essential to cause intestinal disease in calves, are not sufficient to provide the same level of protection as antibodies directed against a mixture of C. perfringens proteins, indicating that a mixture of different antigens will be needed to provide full protection [110]. In order to fully protect calves against C. perfringens-induced enteric diseases, antigens that target bacterial colonization and proliferation might be of equal importance as antigens targeting the toxin activities. Next, it has to be explored whether parenteral vaccination is sufficient to induce a protective immune response or if a combination of systemic and mucosal immunity is needed when not only the bacterial toxins but also bacterial colonization is targeted.

As administration of multiple parenteral doses of a vaccine to calves is more feasible than for chicken, it may be assumed that the development of a vaccine against necro-haemorrhagic enteritis is more straightforward and that C. perfringens supernatants can be used as a vaccine preparation. However, native toxins cannot be used as vaccine antigens due to safety issues. Inactivation of clostridial toxins is generally achieved by formaldehyde treatment, which risks residual formaldehyde in the vaccine preparation, incomplete inactivation of the toxins, and batch-to-batch variation. Moreover, formaldehyde inactivation can induce changes in the tertiary protein structures of relevant antigens and influence the immunogenicity of the vaccines. Indeed, vaccination of both chickens and calves with formaldehyde inactivated C. perfringens supernatants or toxins have resulted in a good antibody response, but these are unable to protect against intestinal disease [97, 112]. To overcome the need of chemically inactivating the C. perfringens toxins, current research focusses on the use of recombinant toxoids to develop a vaccine against C. perfringens-induced diseases. While this may be a good strategy to obtain a safe and protective vaccine on a laboratory scale, the production process is more laborious and time-consuming than production of conventional toxoids, especially because of the required purification steps [113]. Therefore, recent studies have explored the use of efficient low-cost alternatives, such as non-purified recombinant clostridial toxins and even recombinant bacterins, with success [114,115,116].

In summary of this section, considerable progress has recently been made in the development of efficacious vaccines against C. perfringens-induced enteric diseases. The main issue that hampers a breakthrough in this field is the identification of a defined combination of antigens that is able to provide full protection against disease. These antigens will most likely target both the bacterial toxins and the bacterial colonization and proliferation. For the broiler industry, once the ideal vaccine antigens have been identified, development of an oral vaccine is needed.

Towards the development of new coccidiosis vaccines

Coccidiosis, an enteric disease cause by protozoan parasites of the genus Eimeria, remains a major economic and welfare concern for the poultry industry globally. Seven species (Eimeria acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella) are known to infect chickens, and at least six others infect turkeys [117, 118]. The costs associated with coccidial disease are difficult to calculate, but have been estimated to exceed 3 billion US dollars for the chicken industry alone, worldwide [119]. Because coccidiosis is a predisposing factor for the occurrence of necrotic enteritis, the true economic burden is likely even higher. All Eimeria species can cause disease but the severity and clinical symptoms vary among species, and there is little or no cross-protection across species or some strains [120, 121].

Management of coccidiosis through anticoccidial drugs

Modern poultry production systems require effective control of coccidian parasites, typically through the routine use of anticoccidial drugs in feed or water. In the European Union, eleven different anticoccidial drugs are currently licensed and between 240 and 300 tonnes are sold for use in animals for markets such as the UK every year [122]. Anticoccidial drugs can be divided into two groups, synthetic or chemical anticoccidials and ionophores, which are products of fermentation [123]. In some countries such as the US, ionophores are classified as antibiotics, albeit with low human medical importance.

The ionophores currently dominate the anticoccidial drug market, largely because they provide incomplete protection, even against naïve field strains without any drug resistance. Low levels of parasites survive and induce protective immunity against the prevailing local parasite strains, without causing clinical disease [124]. Anticoccidial drugs provide an efficient means of controlling coccidial parasites and are highly cost-effective. However, drug resistance is widespread and increasing consumer concerns related to drug use in livestock production and residues in the food chain encourage the use of alternatives such as vaccination. Notably, because coccidiosis is a predisposing factor for necrotic enteritis and other secondary bacterial infections, efficient control of this parasite is important to minimize the use of medically important antibiotics, including those deemed critically important for human health, in poultry production.

Traditional live anticoccidial vaccines

The first anticoccidial vaccine was marketed in 1952Footnote 1 [125]. It is a live parasite vaccine which includes multiple wild-type (i.e., non-attenuated) Eimeria species. Exposure to limited levels of such non-attenuated parasites permits the induction of a natural immune response in the chicken, resulting in protection against subsequent coccidial challenge. However, because protective immune responses against Eimeria are fully species specific, the inclusion of each individual target species is necessary if comprehensive protection is to be achieved, which results in relatively complex vaccine formulations. Such vaccines commonly include between three and eight parasite species or strains. The approach has been highly successful, although the lack of attenuation has been associated with reduced flock performance following vaccination and occasional clinical disease (reviewed elsewhere [126]).

In response to this limitation, a second generation of live Eimeria vaccines has been developed using attenuated parasite lines. For most of these vaccines, attenuation was achieved by selecting for so-called precocious strains, which typically exhibit reduced pathogenicity with fewer and/or smaller rounds of asexual replication. These attenuated strains retained their ability to immunize. The first live attenuated anticoccidial vaccine was launched in 1989,Footnote 2 and several similar vaccines have been developed since using the same approach [126]. Non-attenuated and attenuated anticoccidial vaccines have become popular in the breeder and layer sectors, but are less widely used in the much larger broiler sector due to their relatively high cost compared to anticoccidial drugs and their limited availability. Because Eimeria cannot replicate effectively in vitro, the production of these live vaccines can only be achieved in Eimeria-free chickens and separate chickens have to be used for each species or strain to be included in a vaccine. Despite these production concerns billions of anticoccidial vaccine doses are sold every year, but more would be required to fully meet the growing demand.

Next generation anticoccidial vaccines

Efforts to improve on first and second generation live anticoccidial vaccines have included extensive attempts to identify antigens that are appropriate for use in subunit or recombinant vaccines. In addition, progress has been made on the preparation of novel adjuvants and some promising results have been obtained, although data on their use in poultry has so far remained fairly limited [127]. As an example, one vaccineFootnote 3 is formulated from a crude mix of affinity purified E. maxima gametocyte antigens [128], although the levels of protection achieved have remained controversial and production of the vaccine still requires parasite amplification in chickens. Numerous studies have suggested that defined antigens such as apical membrane antigen 1, immune mapped protein 1, lactate dehydrogenase and SO7 are highly promising vaccine candidates (reviewed elsewhere [129]). Studies of Eimeria field populations have reported limited diversity in many of these antigens, indicating that recombinant vaccines for Eimeria may succeed even though antigenic diversity has undermined equivalent vaccines for related parasites such as Plasmodium [130, 131]. However, at present no recombinant anticoccidial vaccine is close to reaching the market.

One of the biggest remaining challenges is how to deliver the antigens in an affordable, effective, and, most importantly, scalable manner. A range of vectored expression/delivery systems have been suggested including Fowlpox virus (FWPV), HVT, Salmonella Typhimurium, yeasts such as Saccharomyces cerevisiae and the tobacco plant Nicotiana tabacum, with several showing promise [129]. Most recently, it has been suggested that Eimeria itself might function as an expression/delivery vector for vaccine antigens [132,133,134]. The ability to express and deliver anticoccidial vaccine antigens from multiple parasite species in a single transgenic line could provide an opportunity to streamline anticoccidial vaccine production from as many as eight lines to just one or two. Using an attenuated vector species such as E. acervulina can improve productive capacity enormously and reduce vaccine cost. The parasite vector may also provide some ability as an adjuvant and methods for on-farm delivery are well established [133].

In summary of this section on new coccidiosis vaccines, as pressure to reduce antibiotic drug use in livestock production increases it is clear that the demand for coccidial vaccines is stronger than ever. In the US, approximately 35–40% of broiler companies use programs that include vaccination to control coccidiosis [135]. This trend is primarily driven by demands to produce “no antibiotics ever” poultry products. However, it has also been shown that some coccidial vaccines provide an opportunity to replace drug-resistant field parasites in a poultry house with susceptible vaccine strains. While current European attenuated vaccines are limited by their lower reproductive potential, live vaccines do retain considerable unexplored potential. A better understanding of the underlying immune mechanisms through which these nontraditional approaches operate is needed to allow further progress. Ultimately, it is clear that novel vaccines must be cost-effective, compatible with high standards of animal welfare, scalable and easy to deliver.

Autogenous vaccines to reduce the need for antibiotic use

Autogenous vaccines (AV) are also known as emergency, herd-specific or custom made vaccines. Although the legal basis and exact definition differs from country to country, AVs are used worldwide (e.g. EU, USA, Canada, Brazil, China, Indonesia, Australia, Egypt) and have a long history of use. The use of AVs for the control of fowl cholera has been well-documented [136, 137]. As a common definition, all AVs are made from inactivated bacterial or viral strains which were isolated from the same flock in which the vaccine is to be used. The use of AVs is only allowed if no licensed vaccine is available, or it is respectively ineffective or does not cover the current pathogen strains in the flock. The definition of a flock varies and may include integrated concepts of production chains in different places; to address the issue, the concept of an epidemiological link has recently been proposed by the Co-ordination Group for Mutual Recognition and Decentralised Procedures [138].

Licensed vaccines have advantages compared to AVs, including obligatory good manufacturing practice (GMP) production. Licensed vaccines are also produced in bigger batches with defined strains and a high level of quality which makes their efficacy and safety predictable. However, licensed vaccines are not available in all cases.

To generate AVs, selected bacterial or viral strains are usually combined with a proper adjuvant. Several viral or bacterial species can be used in a combination vaccine and different serotypes can also be combined in a polyvalent vaccine. The combination of inactivated viruses and bacteria is also an option. Bacterial AVs are accepted in all countries of the economic European area, whereas viral AVs are not allowed in 10 European countries including France, Denmark and Spain [138].

A critical role in the successful production and use of an AV falls to the isolation of vaccine strains. Therefore diagnostic samples must be carefully obtained, based on appropriate choices regarding which sick and untreated animals to select for sample collection, which necropsy material to select, and which cultivation conditions and strains to use after results from sero-, toxo- or virulence-typing. For that purpose several methods like PCR, MALDI-TOF MS, slide agglutination or DNA sequencing are available. Because of the fundamental importance of the strain choice for the production of an adequate AV, close collaboration between diagnostic laboratory and vaccine production is critical. Each production is custom-made and numerous adjuvants, viral and bacterial isolates, including serotypes, toxins and species, provide countless combinations. This underlines the importance of experience as the basis in the production of high quality AVs. The veterinarian also has obligations regarding diagnosis, ordering and responsibility for the administration of the vaccine.

A variety of bacterial components are often used in AVs. These include for poultry: Bordetella spp., Campylobacter spp., Cl. perfringens, Enterococcus cecorum, Erysipelothrix rhusiopathiae, E. coli, Gallibacterium anatis, Mycoplasma spp., Ornithobacterium rhinotracheale, Pasteurella multocida, Riemerella anatipestifer; for swine: Actinobacillus pleuropneumoniae, Bordetella spp., Brachyspira spp., Cl. perfringens, E. coli, H. parasuis, Mycoplasma spp., Pasteurella multocida, Strep. suis, Trueperella pyogenes; for cattle: Chlamydia spp. Cl. Perfringens, E. coli, Histophilus somni, Mannheimia haemolytica, Moraxella bovis, Mycoplasma spp., Pasteurella multocida, Salmonella enterica, Trueperella pyogenes; and for fish: Aeromonas spp., Photobacterium spp., Pseudomonas spp., Vibrio spp., Yersinia ruckeri.

Depending on the animal species and age at vaccination different adjuvants can be used. As a standard adjuvant with good safety and efficacy, aluminium hydroxide is often used for production. Polymer and other gel-like adjuvants are also available for production in aqueous mixtures. Oily adjuvants, especially for water-in-oil emulsions, require a more sophisticated mixing procedure because of the need of a stable emulsion. Furthermore oily vaccines might pose safety concerns. However, these induce a promising long lasting immune response because of a depot effect. In the case of organic animal production use of plant oil might be an option in order to avoid unwanted hydrocarbons. The risk of adverse effects, which depend on the adjuvant-antigen combination, can be decreased by standardization of the protocols.

More data regarding the efficacy and safety of AVs in field studies should be collected because clinical safety and efficacy is not regulated. The need for this is reflected by numerous current publications about viral and bacterial AVs for poultry [139,140,141,142], bovine [143], swine [144] and fish [145]. Most results show that AVs can be a useful alternative to antibiotic use.

Only a few countries allow the use of live AVs [138]. The normally inactivated vaccines must be tested for sterility. In the EU this could be carried out by internal tests according to the Pharmacopoea [146]. Further steps in quality control include the inactivation test, endotoxin content or stability tests. Some producers offer GMP production, and GMP production is required in some countries such as Finland or Sweden [147]. In most countries GMP is only recommended. This example shows the vast differences in national legislation regarding the definition and interpretation of AVs. Because of worldwide circulation of animals and their pathogens a harmonization of manufacture, control and use of immunological veterinary medicinal products like AV is important, and the aim at the economic European area [138].

In summary, AVs are a valuable option in certain situations where commercial vaccines are either not available or expected to lack efficacy because of a mismatch between circulating and vaccine strains. The selection of adequate clinical isolates and vaccine formulations requires considerable expertise and the effective use of AVs depends on adequate manufacturing and appropriate veterinary oversight. Regulatory differences among countries create a highly fragmented legal landscape that would benefit from further harmonization.


Vaccines are proven strategies for the prevention or control of infectious diseases in animal populations. Therefore, they are promising alternatives that can reduce the need to use antibiotics in food-producing animals and their direct mitigating impact on antibiotic consumption has been demonstrated in a number of studies, even though the relationship between antibiotic use and vaccination is not in all cases clear-cut. The ideal vaccine is safe, effective against a broad range of pathogens, and easily adapted to mass-application. At the same time, it is cheap to produce and use, easy to register across key jurisdictions, and generates durable protection, ideally after a single administration.

Existing vaccines still fall short of these ideals. In fact, many current vaccines have a number of shortcomings with regard to safety, efficacy and/or user-friendliness that limit their ability to replace antibiotic use. Overcoming these challenges will take close collaboration and innovative new approaches. Public–private partnerships represent one promising governing structure for assuring such close collaboration across public and private sectors. Investments in basic and applied research are equally needed to overcome these challenges, and research needs will have to be prioritized to ensure scarce resources will be preferentially dedicated to areas of greatest potential impact. Research to characterize and quantify the impact of vaccination on antibiotic use is equally needed.

Yet, some data demonstrating the ability of vaccines to reduce antibiotic consumption are already available. Similarly, key research breakthroughs and a number of highly promising vaccination approaches are already in development. These include new oral vaccines based on bacterial spores, live vectors, or new delivery strategies for inactivated oral vaccines; they also include new vaccination strategies in-ovo, combination vaccines that protect against multiple pathogens, the use of recent biotechnological advances, and comprehensive approaches to manage diseases caused by ubiquitous pathogens.

Therefore, further reductions in the need for antibiotic use through the use of new vaccines are all-but-certain, and investments in research and development of new vaccines will be vital for the sustained success of animal agricultural production around the world.


  1. 1.

    Under the name CocciVac™.

  2. 2.

    Under the name Paracox™.

  3. 3.

    Under the name CoxAbic™.


  1. 1.

    Peterson LW, Artis D (2014) Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 14:141–153

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Brandtzaeg P (2007) Induction of secretory immunity and memory at mucosal surfaces. Vaccine 25:5467–5484

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Plotkin SA (2009) Vaccines: the fourth century. Clin Vaccine Immunol 16:1709–1719

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  4. 4.

    Devriendt B, De Geest BG, Goddeeris BM, Cox E (2012) Crossing the barrier: targeting epithelial receptors for enhanced oral vaccine delivery. J Control Release 160:431–439

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Davitt CJH, Lavelle EC (2015) Delivery strategies to enhance oral vaccination against enteric infections. Adv Drug Deliv Rev 91:52–69

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Brandtzaeg P, Kiyono H, Pabst R, Russell MW (2008) Terminology: nomenclature of mucosa-associated lymphoid tissue. Mucosal Immunol 1:31–37

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Huyghebaert N, Snoeck V, Vermeire A, Cox E, Goddeeris BM, Remon JP (2005) Development of an enteric-coated pellet formulation of F4 fimbriae for oral vaccination of suckling piglets against enterotoxigenic Escherichia coli infections. Eur J Pharm Biopharm 59:273–281

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Schulz O, Pabst O (2013) Antigen sampling in the small intestine. Trends Immunol 34:155–161

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Ohno H (2015) Intestinal M cells. J Biochem 159:151–160

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  10. 10.

    Clark MA, Hirst BH, Jepson MA (1998) M-cell surface β1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer’s patch M cells. Infect Immun 66:1237–1243

    PubMed  PubMed Central  CAS  Google Scholar 

  11. 11.

    Hase K, Kawano K, Nochi T, Pontes GS, Fukuda S, Ebisawa M, Kadokura K, Tobe T, Fujimura Y, Kawano S, Yabashi A, Waguri S, Nakato G, Kimura S, Murakami T, Iimura M, Hamura K, Fukuoka S, Lowe AW, Itoh K, Kiyono H, Ohno H (2009) Uptake through glycoprotein 2 of FimH+bacteria by M cells initiates mucosal immune response. Nature 462:226–230

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Mazzini E, Massimiliano L, Penna G, Rescigno M (2014) Oral tolerance can be established via gap junction transfer of fed antigens from CX3CR1+ macrophages to CD103+ dendritic cells. Immunity 40:248–261

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    McDole JR, Wheeler LW, McDonald KG, Wang B, Konjufca V, Knoop KA, Newberry RD, Miller MJ (2012) Goblet cells deliver luminal antigen to CD103+ DCs in the small intestine. Nature 483:345–349

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  14. 14.

    Pridgen EM, Alexis F, Kuo TT, Levy-Nissenbaum E, Karnik R, Blumberg RS, Langer R, Farokhzad OC (2013) Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery. Sci Trans Med 5:213ra167

    Article  CAS  Google Scholar 

  15. 15.

    Melkebeek V, Goddeeris BM, Cox E (2013) ETEC vaccination in pigs. Vet Immunol Immunopathol 152:37–42

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Baert K, De Geest BG, De Rycke R, da Fonseca Antunes AB, De Greve H, Cox E, Devriendt B (2015) β-Glucan microparticles targeted to epithelial APN as oral antigen delivery system. J Control Release 220:149–159

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Iliev ID, Mileti E, Matteoli G, Chieppa M, Rescigno M (2009) Intestinal epithelial cells promote colitis-protective regulatory T-cell differentiation through dendritic cell conditioning. Mucosal Immunol 2:340–350

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Devriendt B, Stuyven E, Verdonck F, Goddeeris BM, Cox E (2010) Enterotoxigenic Escherichia coli (K88) induce proinflammatory responses in porcine intestinal epithelial cells. Dev Comp Immunol 34:1175–1182

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Norton EB, Branco LM, Clements JD (2015) Evaluating the A-subunit of the heat-labile toxin (LT) as an immunogen and a protective antigen against enterotoxigenic Escherichia coli (ETEC). PLoS One 10:e0136302

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  20. 20.

    Lundgren A, Jertborn M, Svennerholm A-M (2016) Induction of long term mucosal immunological memory in humans by an oral inactivated multivalent enterotoxigenic Escherichia coli vaccine. Vaccine 34:3132–3140

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Del Cacho E, Gallego M, Lee SH, Lillehoj HS, Quilez J, Lillehoj EP, Sánchez-Acedo C (2012) Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes. Infect Immun 80:1909–1916

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. 22.

    Del Cacho E, Gallego M, Lillehoj HS, Quílez J, Lillehoj EP, Ramo A, Sánchez-Acedo C (2014) IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis. Vet Res 45:25

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  23. 23.

    Kim WH, Jeong J, Park AR, Yim D, Kim Y-H, Kim KD, Chang HH, Lillehoj HS, Lee B-H, Min W (2012) Chicken IL-17F: identification and comparative expression analysis in Eimeria-infected chickens. Dev Comp Immunol 38:401–409

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Kim WH, Jeong J, Park AR, Yim D, Kim S, Chang HH, Yang S-H, Kim D-H, Lillehoj HS, Min W (2014) Downregulation of chicken interleukin-17 receptor A during Eimeria infection. Infect Immun 82:3845–3854

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. 25.

    Nicholson WL (2002) Roles of Bacillus endospores in the environment. Cell Mol Life Sci 59:410–416

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64:548–572

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. 27.

    Hong HA, le Duc H, Cutting SM (2005) The use of bacterial spore formers as probiotics. FEMS Microbiol Rev 29:813–835

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Food and Agriculture Organization, World Health Organization (2002) Joint FAO/WHO (Food and Agriculture Organization/World Health Organization) working group report on drafting guidelines for the evaluation of probiotics in food. Food and Agriculture Organization, World Health Organization, London

    Google Scholar 

  29. 29.

    Hong HA, To E, Fakhry S, Baccigalupi L, Ricca E, Cutting SM (2009) Defining the natural habitat of Bacillus spore-formers. Res Microbiol 160:375–379

    PubMed  Article  Google Scholar 

  30. 30.

    Tam NK, Uyen NQ, Hong HA, le Duc H, Hoa TT, Serra CR, Henriques AO, Cutting SM (2006) The intestinal life cycle of Bacillus subtilis and close relatives. J Bacteriol 188:2692–2700

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  31. 31.

    Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, Goulding D, Lawley TD (2016) Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533:543–546

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. 32.

    Duc LH, Hong HA, Fairweather N, Ricca E, Cutting SM (2003) Bacterial spores as vaccine vehicles. Infect Immun 71:2810–2818

    PubMed Central  Article  CAS  Google Scholar 

  33. 33.

    Lee S, Belitsky BR, Brown DW, Brinker JP, Kerstein KO, Herrmann JE, Keusch GT, Sonenshein AL, Tzipori S (2010) Efficacy, heat stability and safety of intranasally administered Bacillus subtilis spore or vegetative cell vaccines expressing tetanus toxin fragment C. Vaccine 28:6658–6665

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Hoang TH, Hong HA, Clark GC, Titball RW, Cutting SM (2008) Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis. Infect Immun 76:5257–5265

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  35. 35.

    Permpoonpattana P, Hong HA, Phetcharaburanin J, Huang JM, Cook J, Fairweather NF, Cutting SM (2011) Immunization with Bacillus spores expressing toxin A peptide repeats protects against infection with Clostridium difficile strains producing toxins A and B. Infect Immun 79:2295–2302

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  36. 36.

    Hong HA, Hitri K, Hosseini S, Kotowicz N, Bryan D, Mawas F, Wilkinson AJ, van Broekhoven A, Kearsey J, Cutting SM (2017) Mucosal antibodies to the C-terminus of toxin A prevent colonization of Clostridium difficile. Infect Immun 85:e01060–e01116

    PubMed  PubMed Central  CAS  Google Scholar 

  37. 37.

    Cutting SM (2013) CDVAX. European Union 7th Framework.

  38. 38.

    Huang JM, Hong HA, Van Tong H, Hoang TH, Brisson A, Cutting SM (2010) Mucosal delivery of antigens using adsorption to bacterial spores. Vaccine 28:1021–1030

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Song M, Hong HA, Huang JM, Colenutt C, Khang DD, Nguyen TV, Park SM, Shim BS, Song HH, Cheon IS, Jang JE, Choi JA, Choi YK, Stadler K, Cutting SM (2012) Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine. Vaccine 30:3266–3277

    PubMed  Article  Google Scholar 

  40. 40.

    Reljic R, Sibley L, Huang JM, Pepponi I, Hoppe A, Hong HA, Cutting SM (2013) Mucosal vaccination against tuberculosis using inert bioparticles. Infect Immun 81:4071–4080

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  41. 41.

    Barnes AG, Cerovic V, Hobson PS, Klavinskis LS (2007) Bacillus subtilis spores: a novel microparticle adjuvant which can instruct a balanced Th1 and Th2 immune response to specific antigen. Eur J Immunol 37:1538–1547

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    Huang JM, La Ragione RM, Nunez A, Cutting SM (2008) Immunostimulatory activity of Bacillus spores. FEMS Immunol Med Microbiol 53:195–203

    PubMed  Article  CAS  Google Scholar 

  43. 43.

    de Souza RD, Batista MT, Luiz WB, Cavalcante RC, Amorim JH, Bizerra RS, Martins EG, Ferreira LC (2014) Bacillus subtilis spores as vaccine adjuvants: further insights into the mechanisms of action. PLoS One 9:e87454

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  44. 44.

    Amuguni JH, Lee S, Kerstein KO, Brown DW, Belitsky BR, Herrmann JE, Keusch GT, Sonenshein AL, Tzipori S (2011) Sublingually administered Bacillus subtilis cells expressing tetanus toxin C fragment induce protective systemic and mucosal antibodies against tetanus toxin in mice. Vaccine 29:4778–4784

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Wang YG, Lee KL, Najiah M, Shariff M, Hassan MD (2000) A new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS) caused by virus. Dis Aquat Organ 41:9–18

    PubMed  Article  CAS  Google Scholar 

  46. 46.

    Pham KC, Tran HT, Van Doan C, Le PH, Van Nguyen AT, Nguyen HA, Hong HA, Cutting SM, Phan TN (2017) Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen. Lett Appl Microbiol 64:184–191

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    Valdez A, Yepiz-Plascencia G, Ricca E, Olmos J (2014) First Litopenaeus vannamei WSSV 100% oral vaccination protection using CotC:vp26 fusion protein displayed on Bacillus subtilis spores surface. J Appl Microbiol 117:347–357

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    Nguyen AT, Pham CK, Pham HT, Pham HL, Nguyen AH, Dang LT, Huynh HA, Cutting SM, Phan TN (2014) Bacillus subtilis spores expressing the VP28 antigen: a potential oral treatment to protect Litopenaeus vannamei against white spot syndrome. FEMS Microbiol Lett 358:202–208

    PubMed  Article  CAS  Google Scholar 

  49. 49.

    Ning D, Leng X, Li Q, Xu W (2011) Surface-displayed VP28 on Bacillus subtilis spores induce protection against white spot syndrome virus in crayfish by oral administration. J Appl Microbiol 111:1327–1336

    PubMed  Article  CAS  Google Scholar 

  50. 50.

    da Silva AJ, Zangirolami TC, Novo-Mansur MTM, Giordano RdC, Martins EAL (2014) Live bacterial vaccine vectors: an overview. Braz J Microbiol 45:1117–1129

    PubMed  Article  Google Scholar 

  51. 51.

    Yang DM, Fairweather N, Button LL, McMaster WR, Kahl LP, Liew FY (1990) Oral Salmonella typhimurium (AroA-) vaccine expressing a major leishmanial surface protein (gp63) preferentially induces T helper 1 cells and protective immunity against leishmaniasis. J Immunol 145:2281–2285

    PubMed  CAS  Google Scholar 

  52. 52.

    Tacket CO, Kelly SM, Schödel F, Losonsky G, Nataro JP, Edelman R, Levine MM, Curtiss R (1997) Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system. Infect Immun 65:3381–3385

    PubMed  PubMed Central  CAS  Google Scholar 

  53. 53.

    Duc LH, Cutting SM (2003) Bacterial spores as heat stable vaccine vehicles. Exp Opin Biol Ther 3:1263–1270

    Article  CAS  Google Scholar 

  54. 54.

    Mauriello EMF, Duc LH, Isticato R, Cangiano G, Hong HA, De Felice M, Ricca E, Cutting SM (2004) Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine 22:1177–1187

    PubMed  Article  CAS  Google Scholar 

  55. 55.

    Arnold H, Bumann D, Felies M, Gewecke B, Sörensen M, Gessner JE, Freihorst J, Von Specht BU, Baumann U (2004) Enhanced immunogenicity in the murine airway mucosa with an attenuated Salmonella live vaccine expressing OprF–OprI from Pseudomonas aeruginosa. Infect Immun 72:6546–6553

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  56. 56.

    Zhang J, Shi Z, F-k Kong, Jex E, Huang Z, Watt JM, Van Kampen KR, De-chu CT (2006) Topical application of Escherichia coli-vectored vaccine as a simple method for eliciting protective immunity. Infect Immun 74:3607–3617

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  57. 57.

    Kajikawa A, Satoh E, Leer RJ, Yamamoto S, Igimi S (2007) Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis. Vaccine 25:3599–3605

    PubMed  Article  CAS  Google Scholar 

  58. 58.

    Wu C-M, Chung T-C (2007) Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein. FEMS Immunol Med Microbiol 50:354–365

    PubMed  Article  CAS  Google Scholar 

  59. 59.

    Deguchi K, Yokoyama E, Honda T, Mizuno K (2009) Efficacy of a novel trivalent inactivated vaccine against the shedding of Salmonella in a chicken challenge model. Avian Dis 53:281–286

    PubMed  Article  Google Scholar 

  60. 60.

    Kim KS, Jenkins MC, Lillehoj HS (1989) Immunization of chickens with live Escherichia coli expressing Eimeria acervulina merozoite recombinant antigen induces partial protection against coccidiosis. Infect Immun 57:2434–2440

    PubMed  PubMed Central  CAS  Google Scholar 

  61. 61.

    Ahmed B, Loos M, Vanrompay D, Cox E (2014) Oral immunization with Lactococcus lactis-expressing EspB induces protective immune responses against Escherichia coli O157: H7 in a murine model of colonization. Vaccine 32:3909–3916

    PubMed  Article  CAS  Google Scholar 

  62. 62.

    Embregts CWE, Forlenza M (2016) Oral vaccination of fish: lessons from humans and veterinary species. Dev Comp Immunol 64:118–137

    PubMed  Article  CAS  Google Scholar 

  63. 63.

    Maclachlan NJ, Dubovi EJ (2010) Fenner’s veterinary virology. Academic press, Cambridge

    Google Scholar 

  64. 64.

    Meeusen ENT, Walker J, Peters A, Pastoret P-P, Jungersen G (2007) Current status of veterinary vaccines. Clin Microbiol Rev 20:489–510

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  65. 65.

    Ferreira TB, Alves PM, Aunins JG, Carrondo MJT (2005) Use of adenoviral vectors as veterinary vaccines. Gene Ther 12:S73–83

    PubMed  Article  CAS  Google Scholar 

  66. 66.

    Rollier CS, Reyes-Sandoval A, Cottingham MG, Ewer K, Hill AVS (2011) Viral vectors as vaccine platforms: deployment in sight. Curr Opin Immunol 23:377–382

    PubMed  Article  CAS  Google Scholar 

  67. 67.

    Galen JE, Pasetti MF, Tennant S, Ruiz-Olvera P, Sztein MB, Levine MM (2009) Salmonella enterica serovar Typhi live vector vaccines finally come of age. Immunol Cell Biol 87:400–412

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  68. 68.

    Saxena M, Van TTH, Baird FJ, Coloe PJ, Smooker PM (2013) Pre-existing immunity against vaccine vectors—friend or foe? Microbiology 159:1–11

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  69. 69.

    Hur J, Stein BD, Lee JH (2012) A vaccine candidate for post-weaning diarrhea in swine constructed with a live attenuated Salmonella delivering Escherichia coli K88ab, K88ac, FedA, and FedF fimbrial antigens and its immune responses in a murine model. Can J Vet Res 76:186–194

    PubMed  PubMed Central  CAS  Google Scholar 

  70. 70.

    Stabel TJ, Mayfield JE, Morfitt DC, Wannemuehler MJ (1993) Oral immunization of mice and swine with an attenuated Salmonella choleraesuis [delta cya-12 delta(crp-cdt)19] mutant containing a recombinant plasmid. Infect Immun 61:610–618

    PubMed  PubMed Central  CAS  Google Scholar 

  71. 71.

    Stabel TJ, Mayfield JE, Tabatabai LB, Wannemuehler MJ (1991) Swine immunity to an attenuated Salmonella typhimurium mutant containing a recombinant plasmid which codes for production of a 31-kDa protein of Brucella abortus. Infect Immun 59:2941–2947

    PubMed  PubMed Central  CAS  Google Scholar 

  72. 72.

    Bielke LR, Wolfenden AD, Berghman LR, Kwon YM, Hargis BM (2016) Recombinant orally effective vaccine platforms expressing putative conserved antigens for reduced antimicrobial usage in poultry. In: Alternatives to antibiotics symposium, Paris, France, 2016. United States Department of Agriculture, Washington, D.C.

  73. 73.

    Briggs RE, Tatum FM (2016) Pasteurellaceaeoral vaccine vector for economically important diseases of cattle. In: Alternatives to antibiotics symposium, Paris, France, 2016. United States Department of Agriculture, Washington, D.C.

  74. 74.

    Bal A (2011) In ovo vaccination for everyone. Poultry World. Accessed 31 Aug 2017

  75. 75.

    Clements M (2012) Benefits of in ovo vaccination available to smaller hatcheries. Accessed 31 Aug 2017

  76. 76.

    American College of Poultry Veterinarians (2011) From basics to field applications: poultry vaccination and immunity. In: American College of Poultry Veterinarians Workshop, Sacramento, California, 2011. American College of Poultry Veterinarians

  77. 77.

    Williams CJ, Hopkins BA (2011) Field evaluation of the accuracy of vaccine deposition by two different commercially available in ovo injection systems. Poult Sci 90:223–226

    PubMed  Article  CAS  Google Scholar 

  78. 78.

    Negash T, Al-Garib SO, Gruys E (2004) Comparison of in ovo and post-hatch vaccination with particular reference to infectious bursal disease. A review. Vet Q 26:76–87

    PubMed  Article  CAS  Google Scholar 

  79. 79.

    Fernandes JIM, Prokoski K, Oliveira BC, Oro CS, Oro PJ, Fernandes NLM (2016) Evaluation of incubation yield, vaccine response, and performance of broilers submitted to in-ovo vaccination at different embryonic ages. Rev Bras Cienc Avic 18:55–63

    Article  Google Scholar 

  80. 80.

    Ricks CA, Avakian A, Bryan T, Gildersleeve R, Haddad E, Ilich R, King S, Murray L, Phelps P, Poston R (1999) In ovo vaccination technology. Adv Vet Med 41:495–516

    PubMed  Article  CAS  Google Scholar 

  81. 81.

    Roh JH, Kang M, Wei B, Yoon RH, Seo HS, Bahng JY, Kwon JT, Cha SY, Jang HK (2016) Efficacy of HVT-IBD vector vaccine compared to attenuated live vaccine using in-ovo vaccination against a Korean very virulent IBDV in commercial broiler chickens. Poult Sci 95:1020–1024

    PubMed  Article  CAS  Google Scholar 

  82. 82.

    Jackwood DJ (2017) Advances in vaccine research against economically important viral diseases of food animals: infectious bursal disease virus. Vet Microbiol 206:121–125

    PubMed  Article  Google Scholar 

  83. 83.

    Gagic M, St. Hill CA, Sharma JM (1999) In ovo vaccination of specific-pathogen-free chickens with vaccines containing multiple agents. Avian Dis 43:293–301

    PubMed  Article  CAS  Google Scholar 

  84. 84.

    Lillehoj HS, Jang SI, Panebra A, Lillehoj EP, Dupuis L, Arous JB, Lee SK, Oh ST (2017) In ovo vaccination using Eimeria profilin and Clostridium perfringens NetB proteins in Montanide IMS adjuvant increases protective immunity against experimentally-induced necrotic enteritis. Asian-Australasian J Anim Sci 30:1478–1485

    Article  Google Scholar 

  85. 85.

    Ge J, Wang X, Tian M, Wen Z, Feng Q, Qi X, Gao H, Wang X, Bu Z (2014) Novel in-ovo chimeric recombinant Newcastle disease vaccine protects against both Newcastle disease and infectious bursal disease. Vaccine 32:1514–1521

    PubMed  Article  CAS  Google Scholar 

  86. 86.

    Toro H, De-chu CT, Suarez DL, Sylte MJ, Pfeiffer J, Van Kampen KR (2007) Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector. Vaccine 25:2886–2891

    PubMed  Article  CAS  Google Scholar 

  87. 87.

    Johnson DI, Vagnozzi A, Dorea F, Riblet SM, Mundt A, Zavala G, García M (2010) Protection against infectious laryngotracheitis by in ovo vaccination with commercially available viral vector recombinant vaccines. Avian Dis 54:1251–1259

    PubMed  Article  Google Scholar 

  88. 88.

    Ding X, Lillehoj HS, Dalloul RA, Min W, Sato T, Yasuda A, Lillehoj EP (2005) In ovo vaccination with the Eimeria tenella EtMIC2 gene induces protective immunity against coccidiosis. Vaccine 23:3733–3740

    PubMed  Article  CAS  Google Scholar 

  89. 89.

    Sokale AO, Zhai W, Pote LM, Williams CJ, Peebles ED (2016) Effects of coccidiosis vaccination administered by in ovo injection on the hatchability and hatching chick quality of broilers 1, 2, 3. Poult Sci 96:541–547

    Google Scholar 

  90. 90.

    Sarfraz M, Suleman M, Tikoo SK, Wheler C, Potter AA, Gerdts V, Dar A (2017) Immune responses to in ovo vaccine formulations containing inactivated fowl adenovirus 8b with poly [di (sodium carboxylatoethylphenoxy)] phosphazene (PCEP) and avian beta defensin as adjuvants in chickens. Vaccine 35:981–986

    PubMed  Article  CAS  Google Scholar 

  91. 91.

    Elliott KEC, Branton SL, Evans JD, Gerard PD, Peebles ED (2017) Layer chicken embryo survival to hatch when administered an in ovo vaccination of strain F Mycoplasma gallisepticum and locations of bacteria prevalence in the newly hatched chick1, 2, 3. Poult Sci 96:3879–3884

    PubMed  Article  CAS  Google Scholar 

  92. 92.

    Songer JG (1996) Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 9:216–234

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  93. 93.

    Petit L, Gibert M, Popoff MR (1999) Clostridium perfringens: toxinotype and genotype. Trends Microbiol 7:104–110

    PubMed  Article  CAS  Google Scholar 

  94. 94.

    Songer JG, Uzal FA (2005) Clostridial enteric infections in pigs. J Vet Diagn Invest 17:528–536

    PubMed  Article  Google Scholar 

  95. 95.

    Uzal FA, McClane BA (2011) Recent progress in understanding the pathogenesis of Clostridium perfringens type C infections. Vet Microbiol 153:37–43

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  96. 96.

    Uzal FA, Vidal JE, McClane BA, Gurjar AA (2010) Clostridium perfringens toxins involved in mammalian veterinary diseases. Open Toxinol J 2:24–42

    Article  CAS  Google Scholar 

  97. 97.

    Mot D, Timbermont L, Delezie E, Haesebrouck F, Ducatelle R, Van Immerseel F (2013) Day-of-hatch vaccination is not protective against necrotic enteritis in broiler chickens. Avian Pathol 42:179–184

    PubMed  Article  Google Scholar 

  98. 98.

    Friedman A (2008) Oral tolerance in birds and mammals: digestive tract development determines the strategy. J Appl Poult Res 17:168–173

    Article  Google Scholar 

  99. 99.

    Rappuoli R, Black S, Lambert PH (2011) Vaccine discovery and translation of new vaccine technology. Lancet 378:360–368

    PubMed  Article  Google Scholar 

  100. 100.

    Kulkarni RR, Parreira VR, Sharif S, Prescott JF (2008) Oral immunization of broiler chickens against necrotic enteritis with an attenuated Salmonella vaccine vector expressing Clostridium perfringens antigens. Vaccine 26:4194–4203

    PubMed  Article  CAS  Google Scholar 

  101. 101.

    Kulkarni RR, Parreira VR, Jiang YF, Prescott JF (2010) A live oral recombinant Salmonella enterica serovar typhimurium vaccine expressing Clostridium perfringens antigens confers protection against necrotic enteritis in broiler chickens. Clin Vaccine Immunol 17:205–214

    PubMed  Article  CAS  Google Scholar 

  102. 102.

    Zekarias B, Mo H, Curtiss R 3rd (2008) Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens. Clin Vaccine Immunol 15:805–816

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  103. 103.

    Jiang Y, Kulkarni RR, Parreira VR, Poppe C, Roland KL, Prescott JF (2010) Assessment of 2 Salmonella enterica serovar typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups. Can J Vet Res 74:264–270

    PubMed  PubMed Central  Google Scholar 

  104. 104.

    Alimolaei M, Golchin M, Daneshvar H (2016) Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid. Infect Genet Evol 40:282–287

    PubMed  Article  CAS  Google Scholar 

  105. 105.

    Lanckriet A, Timbermont L, Eeckhaut V, Haesebrouck F, Ducatelle R, Van Immerseel F (2010) Variable protection after vaccination of broiler chickens against necrotic enteritis using supernatants of different Clostridium perfringens strains. Vaccine 28:5920–5923

    PubMed  Article  CAS  Google Scholar 

  106. 106.

    Mot D, Timbermont L, Haesebrouck F, Ducatelle R, Van Immerseel F (2014) Progress and problems in vaccination against necrotic enteritis in broiler chickens. Avian Pathol 43:290–300

    PubMed  Article  CAS  Google Scholar 

  107. 107.

    Jiang Y, Mo H, Willingham C, Wang S, Park JY, Kong W, Roland KL, Curtiss R 3rd (2015) Protection against necrotic enteritis in broiler chickens by regulated delayed lysis Salmonella vaccines. Avian Dis 59:475–485

    PubMed  Article  Google Scholar 

  108. 108.

    Fernandes da Costa SP, Mot D, Geeraerts S, Bokori-Brown M, Immerseel FV, Titball RW (2016) Variable protection against experimental broiler necrotic enteritis after immunisation with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant. Avian Pathol 45:381–388

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  109. 109.

    Wade B, Keyburn AL, Haring V, Ford M, Rood JI, Moore RJ (2016) The adherent abilities of Clostridium perfringens strains are critical for the pathogenesis of avian necrotic enteritis. Vet Microbiol 197:53–61

    PubMed  Article  CAS  Google Scholar 

  110. 110.

    Goossens E, Valgaeren BR, Pardon B, Haesebrouck F, Ducatelle R, Deprez PR, Van Immerseel F (2017) Rethinking the role of alpha toxin in Clostridium perfringens-associated enteric diseases: a review on bovine necro-haemorrhagic enteritis. Vet Res 48:9

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  111. 111.

    Goossens E, Verherstraeten S, Valgaeren BR, Pardon B, Timbermont L, Schauvliege S, Rodrigo-Mocholi D, Haesebrouck F, Ducatelle R, Deprez PR, Van Immerseel F (2016) The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis. Vet Res 47:52

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  112. 112.

    Goossens E, Verherstraeten S, Valgaeren BR, Pardon B, Timbermont L, Schauvliege S, Rodrigo-Mocholi D, Haesebrouck F, Ducatelle R, Deprez PR, Van Immerseel F (2016) Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis. BMC Vet Res 12:101

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  113. 113.

    Ferreira MR, Moreira GM, Cunha CE, Mendonca M, Salvarani FM, Moreira AN, Conceicao FR (2016) Recombinant alpha, beta, and epsilon toxins of Clostridium perfringens: production strategies and applications as veterinary vaccines. Toxins 8:E340

    PubMed  Article  CAS  Google Scholar 

  114. 114.

    Lobato FC, Lima CG, Assis RA, Pires PS, Silva RO, Salvarani FM, Carmo AO, Contigli C, Kalapothakis E (2010) Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants. Vaccine 28:6125–6127

    PubMed  Article  CAS  Google Scholar 

  115. 115.

    Zeng J, Deng G, Wang J, Zhou J, Liu X, Xie Q, Wang Y (2011) Potential protective immunogenicity of recombinant Clostridium perfringens alpha-beta2-beta1 fusion toxin in mice, sows and cows. Vaccine 29:5459–5466

    PubMed  Article  CAS  Google Scholar 

  116. 116.

    Moreira C Jr, da Cunha CE, Moreira GM, Mendonca M, Salvarani FM, Moreira AN, Conceicao FR (2016) Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Anaerobe 40:58–62

    PubMed  Article  CAS  Google Scholar 

  117. 117.

    Vrba V, Pakandl M (2015) Host specificity of turkey and chicken Eimeria: controlled cross-transmission studies and a phylogenetic view. Vet Parasitol 208:118–124

    PubMed  Article  Google Scholar 

  118. 118.

    Clark EL, Macdonald SE, Thenmozhi V, Kundu K, Garg R, Kumar S, Ayoade S, Fornace KM, Jatau ID, Moftah A, Nolan MJ, Sudhakar NR, Adebambo AO, Lawal IA, Alvarez Zapata R, Awuni JA, Chapman HD, Karimuribo E, Mugasa CM, Namangala B, Rushton J, Suo X, Thangaraj K, Srinivasa Rao AS, Tewari AK, Banerjee PS, Dhinakar Raj G, Raman M, Tomley FM, Blake DP (2016) Cryptic Eimeria genotypes are common across the southern but not northern hemisphere. Int J Parasitol 46:537–544

    PubMed  PubMed Central  Article  Google Scholar 

  119. 119.

    Dalloul RA, Lillehoj HS (2006) Poultry coccidiosis: recent advancements in control measures and vaccine development. Exp Rev Vaccines 5:143–163

    Article  CAS  Google Scholar 

  120. 120.

    Long P, Joyner L, Millard B, Norton C (1976) A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet Lat 6:201–217

    PubMed  CAS  Google Scholar 

  121. 121.

    Williams RB, Marshall RN, Pages M, Dardi M, del Cacho E (2009) Pathogenesis of Eimeria praecox in chickens: virulence of field strains compared with laboratory strains of E. praecox and Eimeria acervulina. Avian Pathol 38:359–366

    PubMed  Article  CAS  Google Scholar 

  122. 122.

    Eckford S, Grace K, Harris C, Reeves H, Teale C, Tallentire C (2014) UK-VARSS 2013. UK Veterinary Antibiotic Resistance and Sales Surveillance Report

  123. 123.

    Chapman H (1997) Biochemical, genetic and applied aspects of drug resistance in Eimeria parasites of the fowl. Avian Pathol 26:221–244

    PubMed  Article  CAS  Google Scholar 

  124. 124.

    Chapman HD (1999) Anticoccidial drugs and their effects upon the development of immunity to Eimeria infections in poultry. Avian Pathol 28:521–535

    PubMed  Article  CAS  Google Scholar 

  125. 125.

    Williams RB (2002) Fifty years of anticoccidial vaccines for poultry (1952–2002). Avian Dis 46:775–802

    PubMed  Article  CAS  Google Scholar 

  126. 126.

    Shirley MW, Smith AL, Tomley FM (2005) The biology of avian Eimeria with an emphasis on their control by vaccination. Adv Parasitol 60:285–330

    PubMed  Article  Google Scholar 

  127. 127.

    Jang SI, Lillehoj HS, Lee SH, Lee KW, Lillehoj EP, Bertrand F, Dupuis L, Deville S (2011) Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella. Exp Parasitol 127:178–183

    PubMed  Article  CAS  Google Scholar 

  128. 128.

    Wallach M (2010) Role of antibody in immunity and control of chicken coccidiosis. Trends Parasitol 26:382–387

    PubMed  Article  Google Scholar 

  129. 129.

    Blake DP, Tomley FM (2014) Securing poultry production from the ever-present Eimeria challenge. Trends Parasitol 30:12–19

    PubMed  Article  Google Scholar 

  130. 130.

    Kundu K, Garg R, Kumar S, Mandal M, Tomley F, Blake D, Banerjee P (2017) Humoral and cytokine response elicited during immunisation with recombinant immune mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella. Vet Parasitol 244:44–53

    PubMed  Article  CAS  Google Scholar 

  131. 131.

    Blake DP, Clark EL, Macdonald SE, Thenmozhi V, Kundu K, Garg R, Jatau ID, Ayoade S, Kawahara F, Moftah A, Reid AJ, Adebambo AO, Alvarez Zapata R, Srinivasa Rao AS, Thangaraj K, Banerjee PS, Dhinakar-Raj G, Raman M, Tomley FM (2015) Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development. Proc Natl Acad Sci U S A 112:E5343–5350

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  132. 132.

    Clark JD, Oakes RD, Redhead K, Crouch CF, Francis MJ, Tomley FM, Blake DP (2012) Eimeria species parasites as novel vaccine delivery vectors: anti-Campylobacter jejuni protective immunity induced by Eimeria tenella-delivered CjaA. Vaccine 30:2683–2688

    PubMed  Article  CAS  Google Scholar 

  133. 133.

    Marugan-Hernandez V, Cockle C, Macdonald S, Pegg E, Crouch C, Blake DP, Tomley FM (2016) Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system. Parasit Vectors 9:463

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  134. 134.

    Huang X, Zou J, Xu H, Ding Y, Yin G, Liu X, Suo X (2011) Transgenic Eimeria tenella expressing enhanced yellow fluorescent protein targeted to different cellular compartments stimulated dichotomic immune responses in chickens. J Immunol 187:3595–3602

    PubMed  Article  CAS  Google Scholar 

  135. 135.

    Chapman HD, Jeffers TK (2014) Vaccination of chickens against coccidiosis ameliorates drug resistance in commercial poultry production. Int J Parasitol Drugs Drug Resist 4:214–217

    PubMed  PubMed Central  Article  Google Scholar 

  136. 136.

    Queen FB, Quortrup ER (1946) Treatment of Pasteurella multocida (fowl cholera) infection in wild ducks with autogenous bacterin and penicillin. J Am Vet Med Assoc 108:101–103

    PubMed  CAS  Google Scholar 

  137. 137.

    Olson LD, McCune EL, Bond RE (1969) Comparison of commercial and autogenous bacterins for control of the cranial form of fowl cholera in turkeys. Avian Dis 13:252–260

    PubMed  Article  CAS  Google Scholar 

  138. 138.

    CMDv (2017) Recommendations for the manufacture, control and use of inactivated autogenous veterinary vaccines within the EEA. European Medicines Agency Co-ordination Group for Mutual Recognition and Decentralised Procedures—Veterinary, London

  139. 139.

    Jung A, Metzner M, Köhler-Repp D, Rautenschlein S (2013) Experimental reproduction of an Enterococcus cecorum infection in Pekin ducks. Avian Pathol 42:552–556

    PubMed  Article  Google Scholar 

  140. 140.

    Gharaibeh S, Amareen S (2015) Vaccine efficacy against a new avian influenza (H9N2) field isolate from the Middle East (serology and challenge studies). Avian Dis 59:508–511

    PubMed  Article  Google Scholar 

  141. 141.

    Groves PJ, Sharpe SM, Muir WI, Pavic A, Cox JM (2016) Live and inactivated vaccine regimens against caecal Salmonella typhimurium colonisation in laying hens. Aust Vet J 94:387–393

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  142. 142.

    Li L, Thøfner I, Christensen JP, Ronco T, Pedersen K, Olsen RH (2017) Evaluation of the efficacy of an autogenous Escherichia coli vaccine in broiler breeders. Avian Pathol 46:300–308

    PubMed  Article  CAS  Google Scholar 

  143. 143.

    Dudek K, Bednarek D, Ayling RD, Kycko A, Szacawa E, Karpińska TA (2016) An experimental vaccine composed of two adjuvants gives protection against Mycoplasma bovis in calves. Vaccine 34:3051–3058

    PubMed  Article  CAS  Google Scholar 

  144. 144.

    Geldhof MF, Van Breedam W, De Jong E, Rodriguez AL, Karniychuk UU, Vanhee M, Van Doorsselaere J, Maes D, Nauwynck HJ (2013) Antibody response and maternal immunity upon boosting PRRSV-immune sows with experimental farm-specific and commercial PRRSV vaccines. Vet Microbiol 167:260–271

    PubMed  Article  CAS  Google Scholar 

  145. 145.

    Fukushima HCS, Leal CAG, Cavalcante RB, Figueiredo HCP, Arijo S, Moriñigo MA, Ishikawa M, Borra RC, Ranzani-Paiva MJT (2017) Lactococcus garvieae outbreaks in Brazilian farms Lactococcosis in Pseudoplatystoma sp.—development of an autogenous vaccine as a control strategy. J Fish Dis 40:263–272

    PubMed  Article  CAS  Google Scholar 

  146. 146.

    British Pharmacopoeia (2016) British pharmacopoeia. Her Majesty Stationery Office, London

    Google Scholar 

  147. 147.

    Attia Y, Schmerold I, Hönel A (2013) The legal foundation of the production and use of herd-specific vaccines in Europe. Vaccine 31:3651–3655

    PubMed  Article  CAS  Google Scholar 

Download references

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

KH, FVI, and CG planned the manuscript. KH led the drafting of the manuscript. LB, DPB, EC, SMC, BD, EEV, EG, KK, SL, MM, MR, MCS, NMW, CG, and FVI provided additional information and contributed to writing the manuscript including drafting selected sections and reviewing the manuscript. FVI and CG revised the manuscript. All authors read and approved the final manuscript.


We would like to thank the organizers, sponsors, and participants of the 2nd International Symposium on Alternatives to Antibiotics on which this manuscript is based, in particular the United States Department of Agriculture (USDA) and the World Organisation for Animal Health (OIE).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author information



Corresponding author

Correspondence to Karin Hoelzer.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, 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 ( applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hoelzer, K., Bielke, L., Blake, D.P. et al. Vaccines as alternatives to antibiotics for food producing animals. Part 2: new approaches and potential solutions. Vet Res 49, 70 (2018).

Download citation


  • Food-producing Animals
  • Oral Vaccination
  • Inoculation
  • Necrotic Enteritis
  • White Spot Syndrome Virus (WSSV)