Progress and problems in vaccination against necrotic enteritis in broiler chickens

Abstract

Necrotic enteritis in broilers is caused by Clostridium perfringens type A strains that produce the NetB toxin. Necrotic enteritis is one of the gastrointestinal diseases in poultry that has gained worldwide importance during the last decade due to efforts to improve broiler performance. Prevention strategies include avoiding predisposing factors, such as coccidiosis, and in-feed supplementation with a variety of feed additives. However, vaccination with modified toxin or other secreted immunogenic proteins seems a logical preventive tool for protection against a toxin-producing bacterium. Formalin-inactivated crude supernatant has been used initially for vaccination. Several studies have been carried out recently to identify the most important immunogenic and protective proteins that can be used for vaccination. These include the NetB toxin, as well as a number of other proteins. There is evidence that immunization with single proteins is not protective against severe challenge and that combinations of different antigens are needed. Most published studies have used multiple dosage vaccination regimens that are not relevant for practical use in the broiler industry. Single vaccination regimens for 1-day-old chicks appear to be non-protective. This review describes the history of vaccination strategies against necrotic enteritis in broilers and gives an update on future vaccination strategies that are applicable in the field. These may include breeder hen vaccination, in ovo vaccination and live attenuated vectors to be used in feed or in drinking water.

Introduction

Gastrointestinal diseases in broilers, including necrotic enteritis, viral enteritis, coccidiosis and syndromes such as dysbiosis and malabsorption, have become increasingly important worldwide for multiple reasons. Firstly, high-density floor-housing ensures easy spread of excreted gut pathogens (Guardia et al., 2011). Secondly, due to improvements in genetics, broilers have become impressively capable at converting food into body weight and the gastrointestinal tract of these birds is highly efficient in absorbing nutrients (Havenstein et al., 2003). Gut micro-organisms play an essential role in degradation of feed components, and there is a complex interplay between gut bacteria and the gastrointestinal mucosa that can be beneficial or harmful for the host, depending on the microbial composition (Torok et al., 2011). Nutritionists are constantly seeking to improve the limits of digestibility, and this has caused gut health problems related to shifts in enteric bacterial populations and bacterial overgrowth or, in other words, an excess of feed nutrients in the gut that are used by facultative pathogenic micro-organisms, such as certain Clostridium perfringens strains. Shifts in enteric populations also lead to dysbacteriosis, a hitherto undefined syndrome causing inflammatory reactions in the gut resulting in poor performance (Teirlynck et al., 2011). Genetics has also been shown to play a role in the development of the intestine, which can affect the microbiota composition (Lumpkins et al., 2010). There is thus interplay between host genetics and feed utilization that is important in either preventing or causing gastrointestinal problems. A third factor contributing to the increased importance of gastrointestinal disease is public and governmental pressure to reduce the use of antibiotics in broilers. The traditional antimicrobial growth promoters used to improve feed conversion ratios and body weight gain, and as infection prophylactics for diseases such as necrotic enteritis, have been banned in the European Union. Consumers in other countries have also put pressure on the poultry industry to rear birds without antimicrobial growth promoters. Therapeutic antibiotics are also widely used for preventative and curative interventions against gastrointestinal pathologies and their preventative use is heavily disputed. These factors have led to the emergence of necrotic enteritis in broilers, caused by the chicken-adapted NetB toxin-positive C. perfringens strains that belong to a certain pathogenic clonal lineage (Lepp et al., 2013). This pathogen clearly benefits from high-energy diets supporting its fast growth. There is evidence that the use of antimicrobial growth promoters in feed protected broilers from disease caused by C. perfringens (Johansson et al., 2004; Martel et al., 2004; Lanckriet et al., 2010a).

C. perfringens is a Gram-positive spore-forming bacterium causing necrotic enteritis, of which the typical hallmark is small intestinal necrosis. While the acute clinical form is associated with a sudden increase in flock mortality at an average age of 3 to 4 weeks, the subclinical form leads to damage of the intestinal mucosa resulting in decreased digestion and absorption, reduced weight gain and increased feed conversion ratio (Ficken & Wages, 1997; Kaldhusdal et al., 2001). Estimates of the prevalence of necrotic enteritis vary widely because the subclinical form does not present with obvious clinical signs, but percentages as high as 40% have been reported (Kaldhusdal et al., 2001). The economic impact is thus high. The disease is triggered by a variety of predisposing factors. Damage to the intestinal mucosa is an important predisposing factor, with coccidiosis co-infection known to have a high impact (Collier et al., 2008; Elwinger et al., 1992; Williams, 2005). The feed composition also plays an important role; high-protein-containing and high-non-starch polysaccharide-containing diets are predisposing factors (Branton et al., 1987, 1997; Riddell & Kong, 1992; Gholamiandehkordi et al., 2007; Van Immerseel et al., 2009).

Therapeutic antibiotics, such as amoxicillin and tylosin, are often used to prevent and control necrotic enteritis (Hermans & Morgan, 2007). The use of antibiotics is no longer considered an optimal strategy for keeping gut health problems under control because of issues related to antibiotic resistance. Therefore, better farm management, including biosecurity measures and optimization of feed quality have become more relevant. Additionally, feed additives, including organic acids, essential oils and prebiotics, have been tested in bird models and shown to be at least partially able to control necrotic enteritis (Lensing et al., 2010; Timbermont et al., 2010; Jerzsele et al., 2012). However, for a disease caused by a toxin-producing bacterium, it seems logical to explore whether vaccines can be developed, which may or may not be based on the causative toxins. Much work has been done in recent years in this area and proteins and toxins have been tested as vaccine candidates. In addition, the use of live vectors is under investigation and studies are being carried out on practical strategies for vaccination in the field. A major question is how birds can be protected by vaccination in the limited time span of 3 to 4 weeks before the lesions are most likely to develop. The disease thus develops at an age when maternal antibodies have declined. In addition, vaccination of young broilers is hampered by their immature immune systems and problems related to enhancing the immune system, because mass parenteral vaccination is possible at day 1 but, for practical reasons, not beyond this point. Solutions are being developed to solve these issues. In the current paper an overview is given on the information available regarding the use of potential vaccine preparations, and a critical view is presented on the practical implementation of vaccination to protect broilers against necrotic enteritis.

Antibody Responses to C. perfringens Antigens

The immune response to C. perfringens infection, including immune recognition of the pathogen and its secreted proteins and toxins, is still poorly understood. In this section we describe only antibody responses after infection or vaccination. Other host responses in necrotic enteritis are described elsewhere (Lee et al., 2010) and are beyond the scope of this review. In addition, there are uncertainties about the type of antibodies (IgA, IgY) and about the protective antigen(s) to which the antibodies are directed and associated with protection. Infection takes place in the small intestine, where the pathogen makes contact with the mucosal surface. The enteric immune system of neonatal broilers is poorly developed and matures rapidly up to 4 to 6 weeks post hatch (Mast & Goddeeris, 1999). Generally, adaptive immune defence at the mucosal surface is mediated by initiation of lymphocyte activation and local secretion of IgA (Sharma, 1999; Muir et al., 2000). Mucosal IgY may be important in protection against necrotic enteritis, since it is the major transferred maternal antibody and plays an essential role in the protection of young chickens against other pathogens. Maternal antibody declines by about 3 weeks of age, which may explain why broiler chickens typically develop necrotic enteritis around this time (Ulmer-Franco et al., 2012).

The level of specific maternal antibodies against alpha toxin was shown to be higher in 1-day-old chicks from older hens than in chicks from younger hens. Broilers with high titres of specific maternal antibodies (IgY) against alpha toxin were shown to have lower mortality (Heier et al., 2001). Levels of antibodies (IgY) against NetB and alpha toxin were significantly higher in apparently healthy chickens compared with chickens with clinical necrotic enteritis. This observation suggests that these antibodies may play a role in the protection against necrotic enteritis (Lee et al., 2012).

In several vaccination studies, a mucosal IgA response against alpha toxin, NetB and other immunogenic proteins was reported in chickens partially protected against necrotic enteritis (Kulkarni et al., 2007, 2010; Jang et al., 2012). In intestinal washings from experimentally infected birds, however, only weak reactivity of mucosal IgA against proteins of C. perfringens was found. This might indicate that a serum IgY response plays a more important role in immunity to necrotic enteritis than does mucosal IgA. After systemic immunization with recombinant immunogenic proteins, serum IgY still reaches the mucosal surface under inflammatory conditions caused by C. perfringens (Williams, 2005; Kulkarni et al., 2007, 2010).

An Overview of Vaccination Studies against Necrotic Enteritis

There are various ways to deliver antigens to chickens for immunization purposes. Potential bacterial vaccines can be based on live (attenuated) organisms or killed (inactive) organisms. Live (attenuated) vaccine strains may be superior because they often have the ability to induce a stronger and longer immune response and can be administered orally, but there are some safety concerns (Witter & Hunt, 1994; Plotkin & Plotkin, 2011; Rappuoli et al., 2011). For a toxin-producing bacterium, however, it seems logical that culture supernatants or toxin-based formulations are used because the factors that induce lesions are present in these solutions. They should be produced in an inactivated form whilst preserving antigenicity. Formalin inactivation and genetically engineered inactive toxin variants are an option, as is the delivery of immunogenic non-toxin proteins. DNA vaccines that express Clostridium toxins, but not C. perfringens toxins, have also been tested as vaccine candidates (Saikh et al., 1998; Gardiner et al., 2009; Li et al. 2011; Jin et al., 2013).

Live attenuated vaccines

The principle that previous infections with C. perfringens strains induce protection against challenge was proven by Thompson et al. (2006). These authors administered virulent strains orally to 15-day-old broiler chickens over five consecutive days, followed by treatment with bacitracin for 9 days to clear the virulent strains. An oral challenge with virulent strain C. perfringens CP4 resulted in significantly fewer chickens with lesions (mean lesion score 0.13 compared with 2.09 in the non-immunized group). These data show the potential of vaccination with live strains, but a major issue with live vaccines is the balance between attenuation and protection. Indeed, live strains should be attenuated without losing their ability to protect against disease. When an avirulent strain was used for oral immunization using the same immunization-infection protocol, no protection was conferred. In contrast, an alpha toxin mutant of the challenge strain induced partial protection against infection with an isogenic challenge strain; that is, a significant decrease in number of birds with necrotic lesions was observed (Thompson et al., 2006). It could very well be that residual virulence (which may include NetB production) is essential for a live vaccine strain to be protective. Indeed, an avirulent strain does not provide protective antigens to the gut-associated lymphoid tissues and, as a consequence, does not confer protection.

Protein-based vaccines

Protein-based vaccines are used because they are safer and better characterized when compared with live vaccines, while still providing protection (Unnikrishnan et al., 2012). They include toxoids (inactivated bacterial toxins) and subunit vaccines, often based on virulence factors or secreted toxins (Berzofsky et al., 2001). C. perfringens is known to produce many different toxins and proteins. Some studies used crude culture supernatants (whether inactivated or not) as vaccines, whilst other vaccination trials were carried out using inactivated toxins and highly antigenic proteins.

Both non-inactivated supernatant and formaldehyde-inactivated supernatant (crude toxoid) of C. perfringens have thus been studied as potential vaccines for the prevention of clinical and subclinical necrotic enteritis with variable success. In a study by Saleh et al. (2011), subcutaneous vaccination of broilers at ages of 7 and 21 days with C. perfringens type A, type C and combined type A and C crude toxoids significantly decreased the number of birds developing intestinal lesions. When breeder hens were vaccinated at 14 and 18 weeks of age with type A and type C crude toxoids and their progeny was challenged both under field conditions and in a disease model, type C crude toxoid was shown to provide better protection than type A crude toxoid (Løvland et al., 2004). The safety and efficacy of a C. perfringens type A alpha toxoid (Netvax™, Intervet/Schering-Plough Animal Health, Summit, NJ, USA) was investigated by vaccinating breeder hens intramuscularly at 11 and 18 weeks of age. In this field trial, the progeny from vaccinated hens had a reduced mortality compared with the progeny from unvaccinated hens (Crouch et al., 2010). Lanckriet et al. (2010b) compared the non-inactivated supernatant of eight C. perfringens strains, with different alpha toxin and NetB content, using subcutaneous vaccination at ages of 3 and 12 days. They showed important variation in the protective capacity depending on the strain used for supernatant preparation. This observation suggests that protective immunity is probably determined by an effective combination of different bacterial immunogens or that the expression levels of one or more antigens drives the protection conferred by vaccination. The strain used for crude supernatant collection is thus of crucial importance when designing these vaccine types. It is clear that non-inactivated supernatant always contains a risk because of the presence of active toxins, and thus crude toxoids are preferred for safety reasons. Formaldehyde is generally used for inactivating proteins in vaccines but can reduce the protective capacity of the vaccine. Mot et al. (2013) showed that the efficacy of subcutaneous vaccination at the age of 3 and 12 days against necrotic enteritis using crude supernatant was abolished when the supernatant was formaldehyde inactivated.

The alpha toxin is the most investigated C. perfringens toxin in terms of vaccine-induced protection, mainly in mouse gangrene models (Stevens et al., 2004; Titball, 2009). The alpha toxin is composed of two domains, which are associated with phospholipase C activity (N-domain) and membrane recognition (C-domain) (Naylor et al., 1998). Reports on monoclonal antibodies against alpha toxin led to the finding that monoclonal antibodies, which were capable of neutralizing the phospholipase C activity, were not necessarily effective in neutralizing the haemolytic and lethal activities of the toxin in gangrene models (Sato et al., 1989; Logan et al., 1991). The immune response against the C-domain provided protection against challenge with alpha toxin and also against experimental gas gangrene in mice (Stevens et al., 2004).

Before the NetB toxin was identified as the major toxin in necrotic enteritis in broilers, alpha toxin was believed to be crucial and thus multiple studies used alpha toxin derivatives as vaccine antigen. Broilers with a history of clinical or sub-clinical necrotic enteritis have been shown to have a natural serum antibody response to alpha toxin (Heier et al., 2001; Løvland et al., 2003). Cooper et al. (2009) vaccinated broilers subcutaneously with recombinant alpha toxin at 5 and 15 days of age and showed a decrease in the number of birds with necrotic enteritis lesions. Jang et al. (2012) vaccinated broilers subcutaneously at day 1 and day 7 with recombinant alpha toxin and could induce protection against challenge. Using double and triple intramuscular vaccination regimens (days 7, 14 and 21), Kulkarni et al. (2007) showed that a prior vaccination with alpha toxoid and a boost with active toxin protected against experimental necrotic enteritis. A triple vaccination of either alpha toxoid or active toxin offered no protection. It was suggested that the failure in protection using the active toxin may have resulted from the toxin activity on immune cells and the failure of alpha toxoid may be the consequence of loss of conformation of the protein, resulting in loss of epitopes, as mentioned earlier. Although alpha toxin has been shown to play no primary role in the induction of necrotic enteritis, the antigen can still induce a certain level of protection. Zekarias et al. (2008) have shown that anti-alpha toxin antibodies bind to the cell wall of the bacterium and suppress its growth in vitro. Secreted proteins and toxins of Gram-positive bacteria accumulate within the cytoplasm, and most remain adhered to the cell membrane (Ton-That et al., 2004; Schneewind & Missiakas, 2012). The binding of the antibodies to the membrane-bound pre-protein might block protein transport channels and thereby inhibit proliferation of the bacterium. This suggests an unusual vaccine that directly affects the bacterium rather than neutralizing a toxin. Alpha toxin can thus be used as a protective antigen to vaccinate broilers, but this does not necessarily mean that the toxin plays a primary role in the pathogenesis of necrotic enteritis. It is possible that other antigens will be identified that have similar mechanisms of action when used for immunizing chickens. A combination of antigens generating antibodies that inhibit bacterial proliferation and other antigens generating antibodies that inhibit toxin activity could be more efficient than one of the individual approaches.

The discovery of the genetically highly conserved NetB toxin as an essential virulence factor opened new perspectives for the development of vaccines for the control of necrotic enteritis (Keyburn et al., 2008, 2010a, 2010b). After the structure and function of the NetB toxin protein were analysed, mutants with reduced cytotoxic activity were designed (Savva et al., 2013; Yan et al., 2013). The mutation of tryptophan to alanine at position 262 (W262A) resulted in a significant reduction in cytotoxicity to LMH cells and haemolytic activity on red blood cells, and thus suggested it as a vaccine candidate (Savva et al., 2013). Fernandes da Costa et al. (2013) vaccinated broilers subcutaneously at days 3, 9 and 15 with a formaldehyde NetB toxoid or the NetB W262A mutant. Both NetB-derived vaccines were able to induce significant protection against experimental necrotic enteritis. Keyburn et al. (2013b) immunized chickens subcutaneously with purified recombinant NetB (rNetB), formaldehyde-treated bacterin (consisting of 50:50 sonicated bacterial cells and culture supernatant) and crude toxoid with or without rNetB supplementation at an age of 7 and 17 days. Chickens vaccinated with rNetB were significantly protected against experimental necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but rNetB was not sufficient to protect against a heavy in-feed challenge. Birds immunized with bacterin and crude toxoid supplemented with rNetB were significantly protected against moderate and severe in-feed challenge. NetB has thus been shown to have considerable potential for the development of vaccines against necrotic enteritis. The best protection was observed when birds were vaccinated with the crude toxoid or bacterin supplemented with rNetB (Keyburn et al., 2013b). This study confirmed that NetB alone is not yielding full protection and that supplementation with other antigens increases the protective response. Keyburn et al. (2013a) also used a non-toxic NetB variant (S254L) for vaccinating breeder hens (see below).

In addition to toxin-derived protein vaccines, highly immunodominant proteins can potentially be used to protect birds against necrotic enteritis by vaccination. As mentioned earlier, neither single NetB nor alpha toxin was capable of inducing full protection against the development of lesions after experimental infection. Full protection is probably determined by an effective combination of different bacterial immunogens (Lanckriet et al., 2010b; Fernandes da Costa et al., 2013; Keyburn et al., 2013b). Several purified C. perfringens proteins have been evaluated as potential vaccine candidates. Studies have identified antigens recognized by post-infection sera from chickens immune to necrotic enteritis. Hypothetical protein (HP), pyruvate:ferredoxin oxidoreductase (PFOR), elongation factor G, perfringolysin O, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a fructose-1,6-biphosphate aldolase (FBA) were identified using post-infection serum from chickens immune to virulent C. perfringens challenge in infection-immunization experiments (Kulkarni et al., 2006). Jiang et al. (2009) identified the C. perfringens large cytotoxin (TpeL), endo-beta-N-acetylglucosaminidase (Naglu) and phosphoglyceromutase (Pgm) as dominant antigens using post-infection serum from chickens immune to necrotic enteritis. Elongation factor Tu and PFOR were identified by reaction with immune sera from chickens derived from a clinical outbreak. Kulkarni et al. (2007) immunized chickens intramuscularly two (or three) times at an age of 7 and 14 (and 21) days with recombinant proteins alpha toxin/alpha toxoid, GAPDH, HP, FBA and PFOR. All of the proteins were able to decrease the mean intestinal lesion score. The degree of protection depended on the severity of the challenge. Alpha toxin, HP and PFOR protected significantly against heavy challenge. GAPDH and FBA protected only against mild challenge. More recently, double subcutaneous vaccination regimens using alpha toxin, NetB toxin, PFOR and elongation factor Tu gave similar protection levels after experimental infection (Jang et al., 2012). Immunization with Naglu and Pgm yielded partial protection after challenge with two different strains. Again, the protection level decreased when the challenge severity increased (Jiang et al., 2009). All of the above described data thus show that multiple proteins, including derivatives from alpha toxin and NetB toxin, have potential use as vaccines, and that defined mixtures of these proteins need investigation.

Attenuated live vectors expressing C. perfringens proteins

Attenuated or avirulent bacteria can be used as vehicles for the effective delivery of vaccine candidates (Rappuoli et al., 2011). Attenuated Salmonella strains are often used in poultry for the control of salmonellosis and they can serve as safe and effective oral carrier vaccines to prevent several poultry diseases by expressing heterologous antigens (Hegazy & Hensel, 2012). Because the attenuation is usually induced by a deletion mutation in a gene that is essential for the metabolism of the bacterium, the vaccine carrier strains cannot overgrow the immune system of the bird host (Spreng et al., 2006). Zekarias et al. (2008) evaluated the efficacy of a live recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strain that delivered the C-terminal domain of the alpha toxin. The vaccine strain was administered twice orally at 3 and 17 days of age. Thereafter the birds were challenged by oral inoculation and repeated infection through contaminated feed with a virulent C. perfringens strain. A significant reduction in the number of birds with necrotic lesions was observed. Kulkarni et al. (2008) showed that the delivery of FBA and HP using an attenuated S. enterica serovar Typhimurium vaccine vector by the oral route induced a significant protective immune response. However, broilers immunized with the vaccine strain, expressing PFOR, at day 1 and day 14 were not significantly protected against necrotic enteritis. The authors also tested Salmonella strains expressing truncated non-toxic alpha toxoid and truncated hypothetical protein (tHP). The alpha toxoid consisted of a region of 162 amino acid residues that included two sections of immunodominant epitopes as well as regions of weak reactivity. Broiler chickens immunized orally with a Salmonella strain expressing non-toxic alpha toxoid, at days 1 and 10 of age, were significantly protected against moderate experimental necrotic enteritis but not protected against severe challenge, while chickens immunized with tHP were protected against both moderate and severe challenge (Jiang et al., 2010; Kulkarni et al., 2010). While Salmonella strains are thus potential vaccine carriers for C. perfringens proteins, there are other possibilities that, although not yet explored for protection of poultry against necrotic enteritis, may be of value. The expression of the C-terminal domain of alpha toxin on the surface of Bacillus subtilis spores was described and shown to be immunogenic in mice (Hoang et al., 2008). Lactic acid bacteria can be used as vaccine carriers for Clostridium antigens (Robinson et al., 1997, 2004). B. subtilis and lactic acid bacteria have the advantage of being generally recognized as safe. The use of live vectors to express C. perfringens proteins in the gut of broilers is thus a promising approach and deserves further attention, but will be very complex because the vector needs to present the antigens to the immune system. The choice of the proteins to be expressed is also an important issue.

The Future of Vaccine Delivery and Immunization Methods for Necrotic Enteritis

In recent years, multiple studies have been carried out on the development of vaccines against necrotic enteritis. As reviewed in detail above, non-inactivated supernatants, formalin-inactivated crude toxoids, immunogenic proteins and modified toxins have been used in vaccination studies. These have been administered intramuscularly and subcutaneously, sometimes in multiple dosage regimens, or have been delivered orally by live attenuated vaccine carrier strains. These studies show clearly that multiple vaccination dosages are necessary for a good immune response and that one parenteral single vaccination, at day of hatch, offers no protection (Mot et al., 2013). Ease of administration of a vaccine is important for making vaccines acceptable for the poultry industry and, because large populations of birds must be vaccinated, the most beneficial vaccines are those that can be delivered simultaneously to large numbers with the minimum amount of labour (Sharma, 1999). Broilers are typically slaughtered around 5 to 7 weeks of age, and, for practical reasons, vaccines are generally given in the hatchery. Parenteral vaccination of broiler chickens is theoretically possible at day of hatch, but vaccination using live vaccines by spray methods or via the drinking water is easier to apply. Parenteral booster vaccinations are not practical for broilers. Booster vaccinations have been shown essential when non-inactivated supernatant and crude toxoid are used to provide protection, while single immunization seems to have little benefit (Mot et al., 2013). For protection of broilers against necrotic enteritis, there are thus only a few options left. These are breeder hen vaccination and the use of live bacterial or viral vectors that can deliver antigens in ovo or during rearing (e.g. as a feed or drinking water additive, and thus oral vaccination), thereby presenting the antigens for a longer period than parenteral administration of antigens at day of hatch.

Breeder hen vaccination

Vaccination of breeder hens is often preferred in the poultry industry. Owing to the generation of large numbers of protected progeny per vaccinated hen, the vaccine cost per chicken is lower than that of vaccination post hatch (Schijns et al., 2008). However, passive protection by maternal antibodies in broiler chickens by breeder hen vaccination could have some limitations with regard to necrotic enteritis. Outbreaks of necrotic enteritis occur mostly at the age of 3 to 4 weeks. The immune system of broiler chickens is still developing at that age and maternal antibodies have already declined (Løvland et al., 2004). Until now, three studies have reported data on maternal vaccination against necrotic enteritis, two of them using crude supernatant toxoids and one using rNetB (S254L) either in combination or not with crude toxoid (Løvland et al., 2004; Crouch et al., 2010; Keyburn et al., 2013a). When breeder hens were vaccinated intramuscularly at 14 and 18 weeks of age with C. perfringens type A or type C crude toxoid, an increase in antibody response to alpha toxin in serum samples of parent hens was shown. In a field trial under predisposing conditions, a partial protection against necrotic enteritis was shown in their progeny (Løvland et al., 2004). The safety and efficacy of a commercial C. perfringens type A alpha toxoid (Netvax™) was analysed by immunizing breeder hens intramuscularly at 11 and 18/19 weeks of age. An increase in specific alpha toxin IgY antibody response was shown in serum from the hens, in the yolk from eggs collected from those hens and in serum from 7-day-old chickens hatched from those eggs (Crouch et al., 2010). In a field trial, the progeny (from eggs collected at 27 and 32 weeks) from a group of Netvax™-vaccinated hens had a reduced overall mortality as compared with the progeny from an unvaccinated group, especially at those time points at which necrotic lesions were observed in the progeny from the unvaccinated group (Crouch et al., 2010). Recently, a recombinant non-toxic NetB variant (S254L) was tested in breeder hens, singly or combined with crude toxoid (Keyburn et al., 2013a). Hens were vaccinated subcutaneously at 22, 24 and 26 weeks of age. A significant IgY antibody response against NetB was detected in serum samples from the hens, in the yolk of their eggs and in serum from hatched chickens from vaccinated hens. When the progeny (from eggs collected at 30 weeks) of vaccinated hens was infected with in-feed C. perfringens at 26 and 27 days of age, only chickens derived from hens vaccinated with rNetB (S254L) combined with crude toxoid had a significantly lower lesion score. When the C. perfringens infection was performed at 14 days of age, chickens derived from hens vaccinated with single rNetB (S254L) or single crude toxoid were also protected (Keyburn et al., 2013a). The authors hypothesized that a higher level of specific antibodies at the time of challenge is responsible for the protection against challenge at the earlier age.

In ovo vaccination and oral immunization using viral or bacterial vector vaccines

Chickens can be vaccinated using vector vaccines in ovo or during rearing. Benefits of in ovo vaccination compared with post-hatch vaccination include earlier immunity, reduction in bird stress, precise and uniform injection and reduced labour costs (Ricks et al., 1999; Schijns et al., 2008). The vaccine is injected into eggs during the late embryonation stage, usually at 17 or 18 days of incubation (Muir et al., 2000). Recombinant fowlpoxvirus and herpesvirus of turkey replicating viruses are examples of vector vaccines for in ovo application (Schijns et al., 2008). If a non-replicative vector for C. perfringens antigens were to be injected in ovo, it is possible that protective antibodies would already be in decline at the time the disease occurs. Furthermore, the choice of the adjuvant is important as some adjuvants are known for inducing embryotoxic side effects (Asif et al., 2004). According to our knowledge there are no studies reporting efficacy of in ovo vaccination against necrotic enteritis.

Oral immunization of broilers can be done through the feed or drinking water or by spraying the vaccine onto the chickens (Sharma, 1999). These delivery systems are labour-saving and time-saving and are feasible for the broiler industry. Chickens do not always drink regularly in the first days after hatching. In contrast, spray application may increase the vaccine uptake and lead to a more consistent level of protection against the pathogen (Atterbury et al., 2010). Orally administered live vaccine strains expressing C. perfringens antigens and colonizing the intestinal tract of the broilers have been described (Kulkarni et al., 2008, 2010; Zekarias et al., 2008). The protection obtained depends on the colonization level and persistence of the vaccine strains. Kulkarni et al. (2008, 2010) immunized broilers orally at day of hatch and at day 14 with a recombinant S. enterica serovar Typhimurium strain expressing truncated proteins of the alpha toxin, FBA, PFOR or HP. They induced a significant protective immune response but the degree of protection was less than that observed when these proteins were administered intramuscularly in multiple dosages (Kulkarni et al., 2006). Zekarias et al. (2008) inoculated chickens orally with a S. enterica serovar Typhimurium strain expressing a non-toxic fragment of alpha toxin at days 3 and 13. The antibody response was low, but the immunized chickens had a reduced number of necrotic enteritis lesions after challenge. The above-mentioned studies, however, used oral gavage of the vaccine strains. Practical delivery methods, such as in-feed, drinking water or spray application, have not yet been tested. Recombinant B. subtilis endospores that express the C-terminal domain of alpha toxin have been used to vaccinate mice against C. perfringens infection (Hoang et al., 2008). The endospores appear to provide an adjuvant effect, boosting the immune response to the antigens. The use of these heat-stable endospores as vaccine delivery agents is a promising idea because they could be incorporated into feed. This type of bacterial vector has not been evaluated for necrotic enteritis in broilers.

Summary and Concluding Remarks

Before the identification of the major toxin NetB and important immunogenic proteins, formalin-inactivated crude supernatants were tested as experimental vaccines. During the last few years, vaccination studies have been carried out with purified proteins or combinations of proteins, mostly by parenteral immunization. A summary is presented in Table 1. The impact of these studies has been important in identifying proteins as vaccine candidates (such as the NetB toxin), and it has become clear that combinations of immunogenic proteins are yielding better protection than the use of single-protein immunization. Most of these studies used multiple dosage parenteral immunization regimes, which suffer from lack of practical value for broilers. Single dosing at day of hatch, a possible method that can be used in the field, results in total loss of protection compared with multiple dosage vaccination (Mot et al., 2013). Breeder hen vaccination is an option and several studies have shown promising results, but the antibody decline in the progeny will decrease the efficacy at later ages, which may be important for necrotic enteritis that typically occurs at 3 to 4 weeks of age. In ovo vaccination could be a valuable method, but no data have so far been reported on this strategy. Since immunogenic proteins need to be presented to the immune system for a more prolonged period of time using a single dosage, live attenuated bacterial (or viral or parasitic) vectors are a potential strategy for the future. The protection depends on the colonization level and persistence of the live vaccine strains and the combination and levels of the expressed antigens. It is observed that the severity of the challenge, the infection model, feed and housing conditions of the birds, the strain used for infection, and many more factors affect the protection conferred by a vaccine that is under development. In this regard, a universal infection model that can be used to test the efficacy of vaccines could be of value, as suggested by Shojadoost et al. (2012), but this will be difficult to establish. The ideal vaccine strain would be one that, apart from inducing immunity and protection, can be added to the feed or drinking water, or sprayed on the 1-day-old chicks in the hatchery. As is the case for many other bacterial diseases in livestock, active immunization will only be effective when it is part of an approach that takes into account appropriate management and sanitation measures, feed quality optimization and preventive measures that focus on limiting the presence of predisposing factors.

Table 1. Summary of the studies on vaccination against necrotic enteritis described in the scientific literature, showing route of administration, vaccine regimen, antigen, dose, vector (if used), adjuvant, results of the study and reference.

Route of administration	Vaccination regimen	Vector/adjuvant	Antigen and dose	Protection	Reference
i.m.	Double (breeder hens weeks 14 and 18)	20% alhydrogel and 0.013% thiomersal	‐ Type A crude toxoid (0.25 ml of 1 TCP) ‐ Type C crude toxoid (0.25 ml of 30 TCP)	‐ Specific antibody response against alpha toxin in breeder hens and their progeny ‐ Less mortality in progeny	Løvland et al. (2004)
Oral	Infection-immunization for five consecutive days	Mixed in feed at 2:1 ratio (feed:broth culture)	‐ Avirulent strain CP5 ‐ Virulent strain CP1 ‐ Virulent strain CP4   Alpha toxin-deficient mutants (Cpa^−1, Cpa^−2, Cpa^−3 and Cpa^−4)	‐ Reduction in chickens with lesions that were infection-immunized with CP1, CP4, Cpa^−2 and Cpa^−4	Thompson et al. (2006)
i.m.	Double or triple (days 7 and 14; and 21)	Quil A	‐ Alpha toxin ‐ Alpha toxoid ‐ HP ‐ FBA ‐ GDP ‐ tPFOR   20 µg in triple vaccination, 40 µg in double vaccination	‐ Serum and intestinal antibody response against immunogens ‐ Reduction in chickens with lesions depending on the severity of challenge	Kulkarni et al. (2007)
Oral	Double (days 1 and 14)	Attenuated S. enterica serovar Typhimurium X9241	‐ FBA ‐ tPFOR ‐ tHP^*   100 µl containing 10^9 CFU	‐ Serum and intestinal antibody response against immunogens ‐ Reduction in main lesion score and increase in body weight gain (FBA and tHP)	Kulkarni et al. (2008)
Oral s.c.	Double (days 3 and 13)Double (days 3 and 17)Triple (days 3, 13 and 35)	Attenuated S. enterica serovar Typhimurium X8914Complete Freund's adjuvant (s.c.)	‐ C-terminal domain of alpha toxin (rPLC)   50 µg (s.c.)500 µl containing 10^9 CFU (oral)	‐ Low serum antibody response ‐ Reduction in number of chickens with lesions ‐ Reduction in lesion score	Zekarias et al. (2008)
s.c.	Double (days 5 and 15)	Quil A	‐ Alpha toxin   20 µg	‐ Specific serum antibody response against alpha toxin ‐ Reduction in number of chickens with lesions	Cooper et al. (2009)
i.m.	Double (breeder hens weeks 11 and 18/19)	Light mineral oil	‐ Type A crude toxoid (0.5 ml of 3 TCP)	‐ Specific antibody response against alpha toxin in breeder hens and their progeny ‐ Lower mortality rate in field trial	Crouch et al. (2010)
Oral i.m.	Double (days 1 and 10)Triple (days 1, 10 and 17)	Attenuated S. enterica serovar Typhimurium X9352	‐ Alpha toxoid (region of 162 amino acid residues) ‐ tHP   100 µl containing 10^9 CFU	‐ Serum and intestinal antibody response against immunogens ‐ Reduction in chickens with lesions depending on the severity of challenge ‐ Increased body weight	Kulkarni et al. (2010)
s.c.	Double (days 3 and 12)	Quil A	‐ Supernatant of eight type A strains (variable NetB and alpha toxin content)   7 and 70 µg	‐ Reduction in number of chickens with necrotic lesions	Lanckriet et al. (2010b)
i.m.	Double or triple (days 7 and 14; and 21)	Quil A	‐ Naglu ‐ Pgm	‐ Serum and intestinal antibody response against immunogens ‐ Reduction in chickens with lesions depending on the severity of challenge and challenge strain	Jiang et al. (2009)
s.c.	Double (days 7 and 21)	Unknown	‐ Crude toxoid A ‐ Crude toxoid C ‐ Crude toxoid AC	‐ Serum antibody response against immunogens ‐ Reduction in number of chickens with necrotic lesions	Saleh et al. (2011)
s.c.	Double (days 1 and 7)	Montanide ISA 71 VG	‐ Alpha toxin ‐ NetB ‐ EF-Tu ‐ PFOR   50 µg	‐ Specific serum antibody response against NetB and PFOR ‐ Reduction in lesion score	Jang et al. (2012)
s.c.	Single (day 1 or 3)Double (days 3 and 12)	Quil A	‐ Supernatant NetB-positive toxin type A strain   7 and 70 µg	‐ Reduction in number of chickens with necrotic lesions ‐ Reduction in lesion score	Mot et al. (2013)
s.c.	Triple (days 3, 9 and 15)	Quil A	‐ NetB toxoid ‐ NetB (W262A)   30 µg	‐ Reduction in number of chickens with necrotic lesions ‐ Reduction in mean lesion score	Fernandes da Costa et al. (2013)
s.c.	Double (days 7 and 17)	60% Montanide40% Quil ADEAE-dextran	‐ NetB ‐ Bacterin (50:50 bacterial cells and culture supernatant) ‐ Bacterin + NetB   50 µg	‐ Specific serum antibody response against NetB ‐ Reduction in average lesion score depending on the severity of challenge	Keyburn et al. (2013b)
s.c.	Triple (breeder hens weeks 22, 24 and 26)	60% Montanide40% Quil ADEAE-dextran	‐ rNetB (S254L) ‐ Crude toxoid (type A, NetB-positive) ‐ Crude toxoid (type A, NetB-positive) + rNetB (S254L)   50 µg	‐ Specific antibody response against NetB in breeder hens and progeny ‐ Reduction in number of chickens with necrotic lesions in experimental infection trial in progeny	Keyburn et al. (2013a)

CFU, colony-forming units; DEAE-dextran, diethylaminoethanol; EF-Tu, elongation factor Tu; GDP, glyceraldehyde-3-phosphate dehydrogenase; i.m., intramuscular; rPLC, recombinant alpha toxin C-terminal domain-encoding sequence; s.c., subcutaneous; TCP, total combining power; tPFOR, truncated pyruvate:ferredoxin oxidoreductase.

Acknowledgements

The authors thank Dr Nicola Senior (University of Exeter, College of Life and Environmental Sciences, UK) for commenting on the manuscript.