Virulence factors

Numerous studies have been conducted to explore the essential virulence factors responsible for the progression of NE. It has been shown that an alpha-toxin mutant of a virulent chicken C. perfringens isolate still produced NE in experimental infection of chickens. Therefore alpha-toxin is not essentially involved in producing NE in chickens (Keyburn et al., Reference KEYBURN, SHEEDY, FORD, WILLIAMSON, AWAD, ROOD and MOORE2006). In another study, specific serum immunoglobulins against C. perfringens identified six specific antigenic (immunogenic) proteins, produced by virulent C. perfringens strains isolated from NE cases in the field, including hypothetical protein (HP), perfringolysin O, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1, 6-biphosphate aldolase (FBA), elongation factor G (EF-G), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Kulkarni et al., Reference KULKARNI, PARREIRA, SHARIF and PRESCOTT2006). The antigenic proteins including HP, PFOR, FBA, GAPDH, and alpha-toxin conferred significant protection to broiler chicks against mild and severe infections with virulent C. perfringens. Priming the birds with alpha-toxin and boosting them with active toxin imparted the highest protection to severely challenged birds. Therefore, certain proteins secreted in addition to alpha-toxin can play a role in immunising broiler chickens against NE.

Beta2-toxin has been discovered in C. perfringens isolates, although is not reported to be directly associated with NE pathogenesis (Thompson et al., Reference THOMPSON, PARREIRA, KULKARNI and PRESCOTT2006). The alpha-toxin and beta2-toxin have been detected in the faecal contents of healthy dairy cows in China (Wang et al., Reference WANG, ZHOU, ZHENG, LIN, CAO, GONG and QIU2011). Another extracellular pore-forming toxin, netB was identified recently in poultry. A netB (netB toxin gene) mutant of the same strain did not induce the disease in experimentally infected chickens, hence it was claimed to be the essential virulence factor in C. perfringens strains isolated from poultry (Keyburn et al., Reference KEYBURN, YAN, BANNUM, VAN IMMERSEEL, ROOD and MOORE2010). Some American C. perfringens strains isolated from both NE positive as well as negative cases were positive for the netB toxin gene. Bacterial isolates from a cow suffering from hepatic abscesses showed the presence of the netB gene, however it was absent in some avian C. perfringens isolates from positive cases of NE. It was concluded that further investigation was needed to determine the involvement of the netB toxin in causing NE, by exploring the pathogenicity of both bacterial strains being netB positive for healthy chickens and netB negative for NE cases (Gad et al., Reference GAD, HAUK, KRÜGER and HAFEZ2011).

In addition to the netB toxin, other virulence factors have been associated with NE production (Smyth and Martin, Reference SMYTH and MARTIN2010). NE-associated virulent C. perfringens strains isolated from clinical cases, were found to be inhibiting the growth of non-NE strains from the gastrointestinal tract of healthy broilers in vitro, enabling virulent strains to cause NE in broiler chicks (Timbermont et al., Reference TIMBERMONT, LANCKRIET, PASMANS, HAESBROUCK, DUCATELLE and VAN IMMERSEEL2009a). C. perfringens, in addition to the generation of toxins, may inhibit certain beneficial enteric microflora, which are beneficial for host health, leading to aggravation in the disease pathogenesis (Timbermont et al., Reference TIMBERMONT, LANCKRIET, PASMANS, HAESBROUCK, DUCATELLE and VAN IMMERSEEL2009a). Only the C. perfringens isolates from field outbreaks of NE are virulent and cause NE whereas isolates from clinically healthy chicks, being avirulent, do not induce the disease. In addition, the ability to produce alpha-toxins in vitro is not necessarily associated with NE induction. Moreover, the virulence of C. perfringens isolates is host specific because the strains isolated from calf haemorrhagic enteritis could not induce NE lesions in broiler chicks (Timbermont et al., Reference TIMBERMONT, LANCKRIET, GHOLAMIANDEHKORDI, PASMANS, MARTEL, HAESBROUCK, DUCATELLE and VAN IMMERSEEL2009b). In counterpoint, C. perfringens strains from mammals and clinically healthy chicks are reported to cause NE (Smyth and Martin, Reference SMYTH and MARTIN2010).

Vaccination

Some vaccines have been prepared and trialled against NE in birds, however, because virulence factors have only partially been identified, they provide variable protection. Infection-immunisation of broiler chickens with virulent C. perfringens isolates protected chickens against NE, where they were challenged subsequently with virulent isolates but not with the avirulent isolates (Thompson et al., Reference THOMPSON, PARREIRA, KULKARNI and PRESCOTT2006). The aluminium hydroxide adjuvanted C. perfringens type A and type C toxins and a commercial type A alpha-toxoid (Netvax^TM) effectively immunised broiler breeder hens (Lovland et al., Reference LOVLAND, KALDHUSDAL, REDHEAD, SKJERVE and LILLEHAUG2004). In another study, recombinant C. perfringens type A alpha-toxin provided partial protection against NE in broilers experimentally infected with C. perfringens (Cooper et al., Reference COOPER, TRINH and SONGER2009). A live oral recombinant attenuated Salmonella enterica serovar typhimurium vaccine (RASV), expressing the nontoxic C-terminal domain of alpha-toxin from C. perfringens, conferred safe immunity (systemic and mucosal) against NE in broiler chicks in experimental infection with virulent C. perfringens (Zekarias et al., Reference ZEKARIAS, MO and CURTISS III2008).

Contradictory to these findings, live attenuated C. perfringens alpha-toxin deficient mutants (vaccine) protected broiler chickens from experimental NE, indicating that other immunogenic substance(s) apart from alpha-toxin are significant for immunising birds against oral challenge (Thompson et al., Reference THOMPSON, PARREIRA, KULKARNI and PRESCOTT2006). A further study reported that broiler chickens immunised with a netB genetic toxin or formaldehyde netB toxin conveyed only partial protection against NE following clostridial challenge (Fernandez da Costa et al., Reference FERNANDEZ DA COSTA, MOT, BOKORI-BROWN, SAVVA, BASAK, VAN IMMERSEEL and TITBALL2013). Single-shot immunisation of day-old broiler chickens with netB toxin (prepared from netB toxin-positive C. perfringens type A strain supernatant) or crude supernatant, could not provide protection against NE in a mild clostridial challenge. However, single immunisation at the age of three days or double immunisation at the age of three and twelve days with crude supernatant, conferred significant immunity against the disease. Moreover, the crude supernatant proved to be more efficacious than exposure to toxin in double immunisation (Mot et al., Reference MOT, TIMBERMONT, DELEZIE, HAESEBROUCK, DUCATELLE and VAN IMMERSEEL2013).

The supernatants of some avian C. perfringens strains positive for netB gene could only confer partial protection in birds when challenged with severe infection, indicating that the immune-protective potential of the supernatants did not depend only upon alpha-toxin or netB (Lanckriet et al., Reference LANCKRIET, TIMBERMONT, EECKHAUT, HAESEBROUCK, DUCATELLE and VAN IMMERSEEL2010). An avirulent recombinant Salmonella vaccine vector expressing two antigenic proteins HP or FBA, conferred significant protection to broiler chickens against NE infection, indicating that two C. perfringens antigens (FBA and HP) could immunise broilers against NE with an attenuated Salmonella vaccine vector (Kulkarni et al., Reference KULKARNI, PARREIRA, JIANG and PRESCOTT2010). In another study, two C. perfringens antigenic truncated proteins of alpha-toxoid (AT), and hypothetical protein (HP), as codon-optimised alpha-toxin (ATd), and truncated hypothetical protein (HPt), conferred partial immunity against NE in broiler chicks (Kulkarni et al., Reference KULKARNI, PARREIRA, JIANG and PRESCOTT2010). Hence, the involvement of several virulence (immunogenic) factors and their variable immunising capacity hinder the generation of a potent active anti-NE vaccine. Consequently, vaccination is not a reliable strategy in the control of NE to date.

Coccidiosis control

Pre-existing coccidiosis caused by different Eimeria spp. may predispose birds to NE. Anti-coccidial drugs have been used successfully as chemotherapy and feed additives in birds to combat coccidiosis, but like antibiotics, their employment has been linked to the emergence of drug-resistant Eimeria strains (Abbas et al., Reference ABBAS, IQBAL, BLAKE, KHAN and SALEEMI2011). Coccidiosis has been controlled effectively also by utilising live Eimeria oocysts as vaccines (Shirley and Lillehoj, Reference SHIRLEY and LILLEHOJ2012) but these can cause unwanted adverse reactions. Therefore, safer attenuated vaccines have been prepared, however their production is costly. Moreover, vaccines exhibit variable efficacy in different geographical areas because of the diversity of Eimeria strains in different areas. Subsequently poultry farmers and researchers seek alternative approaches for controlling coccidiosis effectively and economically (Abbas et al., Reference ABBAS, CLOWELL and GILLEARD2012a; Masood et al., Reference MASOOD, ABBAS, IQBAL, MANSOOR, SINDHU, ZIA and KHAN2013). Different alternatives with variable protection against coccidial infection have been reviewed recently by Abbas et al. (Reference ABBAS, IQBAL, KHAN, SINDHU, KHAN, KHAN and RAZA2012b). Dietary supplementation with two commercial probiotics, MitoGrow^® (containing Pediococcus) (Lee et al., Reference LEE, LILLEHOJ, DALLOUL, PARK, HONG and LIN2007a) and MitoMax^® (with Pediococcus and Saccharomyces) (Lee et al., Reference LEE, LILLEHOJ, PARK, HONG and KIN2007b), has been found to reduce oocyst counts and ameliorate growth performance in birds through humoral immune-stimulation. Numerous botanicals have been reported to possess anticoccidial activities including Artemisia spp., Aloe spp. and Curcuma longa. The proposed mechanism of action of these plants involves the induction of oxidative stress against coccidial parasites (Abbas et al., Reference ABBAS, CLOWELL and GILLEARD2012a).

Probiotics

A probiotic or a direct-fed microbial (DFM) is defined as a living microbial diet supplement that usefully influences the host animal through ameliorating its enteric microbial load. The various microbial species present in the normal microflora (colonising the gut epithelium or lumen) of the broiler gut attain an equilibrium phase about one week after hatching, depending upon certain factors involving the site of colonisation in the gut, the physical status of the enteric mucosa and time needed for the passage of chymus (Teirlynck et al., Reference TEIRLYNCK, KJERRUM, EEKHAUT, HUYGHEBAERT, PASMANS, HAESBROUCK, DEWULF, DUCATELLE and VAN IMMERSEEL2009). Probiotics increase the normal microbiota population by the selective exclusion of specific pathogenic microbes (e.g. Escherichia coli, Salmonella, Campylobacter, Listeria spp. and Clostridium spp.) in the gut (Biernasiak et al., Reference BIERNASIAK, SILIZEWSKA, LIBUDZISZ and SMULIKOWSKA2007). The suggested mechanisms of action of probiotics in improving poultry health and production may include preventing pathogenic invasion through competitive exclusion, maintaining integrity of the epithelial barrier, nutrient transfer and enhancing enteric immunity. The mode of action of a probiotic depends upon the nature of its strain and likely involves the formation of specific metabolites (intermediary metabolites with antibacterial effect, short organic fatty acids, H₂O₂), interaction with receptor sites and immune-stimulation (Sherman et al., Reference SHERMAN, OSSA and JOHNSON-HENRY2009).

Beneficial bacteria in the gut of chicks can be increased and maintained by employing Bacillus subtilis, Bifidobacterium spp. and Lactobacillus spp. (Teo and Tan, Reference TEO and TAN2005). DFMs like Lactobacillus spp., Pediococcus pentosaceus and Bacillus spp. are reported to exhibit antimicrobial activity against C. perfringens in birds (Teo and Tan, Reference TEO and TAN2005). Dietary supplementation with a commercial probiotic preparation CloSTAT™ (having a unique Bacillus subtilis strain PB6, isolated from the gut of normal chickens), has been documented to control experimental NE in broiler chickens along with amelioration in their enteric health (De Smet and Verleyen, Reference DE SMET and VERLEYEN2010). In another study supplementation with the probiotic B. licheniformis significantly ameliorated the performance of broiler chicks against NE (Knap et al., Reference KNAP, LUND and AUGUSTSSON2010). Lactobacillus probiotic can increase resistance to coccidial infections in chicks and their affectivity may be reduced by certain ionophores (Rada et al., Reference RADA, RYCHLÝ and VORÍŠEK1994). The DFM PoultryStar^® and a botanical product PEP125^® given to broiler birds subsequently gavaged with C. perfringens, reduced enteric lesions, mortality, and C. perfringens counts (p<0.05) as compared to those of positive controls (McReynolds et al., Reference MCREYNOLDS, WANECK, BYRD, GENOVESE, DUKE and NISBET2009).

Lactic acid bacteria (LAB) are probably the most common basis for probiotic products. The bacteria of clostridial clusters IV and XIVa, which are strict anaerobes, utilise lactic acid generated in vitro by LAB for producing large amounts of butyric acid (Duncan et al., Reference DUNCAN, LOUIS and FLINT2004). This phenomenon is termed ‘cross-feeding’ and is another benefit of dietary LAB administration for improving bird performance. Yeast species such as Saccharomyces cerevisiae and Kloeckera apiculata naturally produce non-antibiotic functional products called yeast extracts that possess possible antimicrobial and immunomodulatory activities. Recently, two probiotics including yogurt (containing Lactobacillus thermophilus and L. delbrueckii) in drinking water and Thepax (containing L. acidophilus & Saccharomyces cerevisiae) in feed revealed significant improvements in body weight gain and FCR in broiler chickens. The caecal and cloacal C. perfringens and E. coli loads were decreased and normal enteric microbiota balance was ameliorated (Boostani et al., Reference BOOSTANI, MAHMOODIAN FARD, ASHAYERIZADEH and AMINAFSHAR2013). Consequently probiotic(s) supplementation in feed is a potential candidate strategy for controlling NE.

Competitive exclusion products

Competitive exclusion products (CEPs) are a mixture of intestinal tract contents from normal healthy birds that when orally administered to newly-hatched chicks, with the goal of rapidly establishing a mature enteric microbiota and resisting enteric colonisation by invading pathogenic bacteria from the environment (Mead, Reference MEAD2000). The enteric microflora exhibit a specific multifactorial ‘barrier’ effect, including inducing structural and functional alterations in the enteric cell wall, modifying gut immunology and enhancing the immunity of birds to enteric pathogens such as C. perfringens. Probiotics and CEPs are similar in terms of mechanism of action, but probiotics are defined products comprising a single or a combination of characterised bacterial strains, whereas CEPs are undefined preparations of normal gut flora. C. perfringens was found to be susceptible to competitive exclusion. The mixtures of normal intestinal microflora had manifested effectiveness against food-borne pathogenic bacteria like pathogenic strains of Escherichia coli, Campylobacter jejuni, Salmonella spp., Yersinia enterocolitica, C. perfringens, and C. botulinum (Mead, Reference MEAD2000).

Prebiotics

A prebiotic is defined as an indigestible diet component that usefully influences the host animal through selective stimulation of growth and/or metabolic action of useful bacteria already present in the intestine and suppressing pathogenic bacteria (Adijiri-Awere and Van Lunen, Reference ADIJIRI-AWERE and VAN LUNEN2005). The mechanism of action of prebiotics depends on the nature of the compound and may be similar to that of probiotics. Prebiotics are usually polysaccharides (mainly oligosaccharides), including gluco-oligosaccharides, glyco-oligosaccharides, fructo-oligosaccharides (FOS), xylo-oligosaccharides, trans-galacto-oligosaccharides, malto-oligosaccharides, mannan-oligosaccharides, isomalto-oligosaccharides, soybean oligosaccharides, inulin, lactulose, lactitol, raffinose, stachyose, and sucrose thermal oligosaccharides (Adijiri-Awere and Van Lunen, Reference ADIJIRI-AWERE and VAN LUNEN2005). According to the criteria for being a prebiotic, it should be resistant to gastric pH, enzymatic hydrolysis in mammals, gut absorption, and fermentation by enteric microbiota.

An in vitro study reported that fructo-oligosaccharides (FOS) and inulin acted as substrates for e.g. Bifidobacteria spp. (Ramirez-Farias et al., Reference RAMIREZ-FARIAS, SLEZK, FULLER, DUNCAN, HOLTROP and LOUIS2009) while, mannan-oligosaccharides (MOS) possess receptors for the fimbriae of Escherichia coli and Salmonella spp., which prevent binding to mucosal receptors and instead bind to these pathogens and eliminate them along with the flow of faecal contents (Fernandez et al., Reference FERNANDEZ, HINTON and VAN GLIS2002). Lactobacilli and Bifidobacteria spp. ferment prebiotics into short-chain fatty acids, which increase in concentrations and reduce the enteric pH and inhibit the proliferation of certain pathogenic bacteria (Pan et al., Reference PAN, CHEN, WU, TANG and ZHAO2009). In an investigation, all in-feed supplements of a prebiotic, a probiotic, or an organic acid alone or in combination (prebiotic+probiotic, or prebiotic+organic acid), caused significant improvement in the live weight of broiler chicks, in comparison with the control (Bozkurt et al., Reference BOZKURT, KÜÇÜKYILMAZ, CATH and ÇINAR2009). Therefore dietary prebiotics may be more fruitful when combined with probiotics.

Egg yolk immunoglobulins (IgYs)

Egg yolk immunoglobulins (IgYs) are a source of passive immunisation that protects progeny from different infectious diseases. Chickens have three classes of immunoglobulins IgA, IgM, and IgY (Karlsson et al., Reference KARLSSON, KOLLBERG and LARSSON2004), although chicken IgG is called IgY because it differs from mammalian IgG and is the major immunoglobulin in chickens. IgY is transferred from hen to embryo via the egg yolk, which consequently accumulates high concentrations (Hamal et al., Reference HAMAL, BURGESS, PEVZNER and ERF2006). Laying hens can effectively generate IgY in high amounts compared to mammals, and their isolation from yolk is affordable and efficient (Karlsson et al., Reference KARLSSON, KOLLBERG and LARSSON2004). After extraction from yolk and purification, they are prepared for direct administration to the animal or added into feed, preferably in powder form.

Literature relating to the impact of IgYs in immunoprophylaxis in the avian model is scant, yet hen egg antibodies (HEA) for normal or Salmonella-hyperimmunised laying hens, have been shown to mitigate the enteric population of S. enteritidis (Kassaify and Mine, Reference KASSAIFY and MINE2004), although specific IgYs from hens immunised against C. perfringens bacteria could not give protection against C. perfringens proliferation (Wilkie et al., Reference WILKIE, VAN KESSEL, DUMONCEUAX and DREW2006). Contradictory to this, in a recent report IgYs have been shown to be significantly efficacious against Escherichia coli, Salmonella pullorum, C. perfringens, and Campylobacter jejuni when orally administered to chicks, followed by experimental challenge with specific pathogenic bacteria (Tamilzarasan et al., Reference TAMILZARASAN, DINAKARAN, SELVARAJU and DORARIAJAN2009). This technology needs be further evaluated nevertheless, and seems to have potential scope to control NE.

Bacteriophages

Bacteriophages are viruses that infect bacteria and which replicate by utilising the hosts metabolic system and then lyse the bacterial cell to release new progeny phages into the environment. They are not reported to infect animal and plant cells, hence being potentially safe alternatives to antibiotics. The biology of bacteriophages (or phage gene products) and utilizing their antibacterial activity has been an interesting issue (Seal, Reference SEAL2013). Bacterial viruses virulent for C. perfringens have been initially screened by employing filtered samples collected from the intestinal contents of poultry, soil and processing drainage water. Both siphoviruses (long-tailed) and podoviruses (short-tailed), lytic for C. perfringens, have been discovered (Volozhantsev et al., Reference VOLOZHANTSEV, OAKLEY, MORALES, VEREVKIN, BANNOV, KRASILNIKOVA, POPOVA, ZHILENKOV, GARRISH, SCHEGG, WOOLSEY, QUILICI, LINE, HIETT, SIRAGUSA, SVETOCH and SEAL2012). Specific bacteriophages virulent for C. perfringens (CP) strains can be employed for NE control in chickens and their molecular characterisation and specific bacteriolytic enzymes have recently been conducted (Seal, Reference SEAL2013).

A specific enzyme murein hydrolase (a phage-specific enzyme) from bacteriophage ɸ3626 against C. perfringens caused lysis of all tested CP strains without generally affecting other Clostridia spp. or gut bacteria. This highly specific lytic effect of bacteriophages against C. perfringens might be helpful for controlling NE in poultry (Zimmer et al., Reference ZIMMER, VUKOS, SCHERER and LOESSNER2002). Recently, a newly-identified specific anticlostridial phage endolysin CP25L was derived from a C. perfringens prophage (Gervasi et al., Reference GERVASI, LO CURTO, NARBAD and MAYER2013a). Lactobacillus johnsonii (a poultry probiotic) was genetically engineered for cloning and expressing the endolysin gene cp25l. The recombinant probiotic was used for delivering the endolysin to the gastrointestinal tract of poultry where the active endolysin was efficiently secreted, suggesting that it may be helpful for controlling enteric clostridiosis (Gervasi et al., Reference GERVASI, HORN and WEGMANN2013b). In another study, oral administration of a multivalent bacteriophage cocktail named ‘INT-401’ significantly improved performance in CP-challenged INT-401-treated chickens, compared with CP-challenged phage-untreated control birds (Miller et al., Reference MILLER, SKINNER, SULAKVELIDZE, MATHIS and HOFACRE2010). However, their use has some limitations including their specificity, uncertain regulatory acceptance, safety of proprietary rights of bacteriophage products, fragile activity and their routes of administration in animal production systems. Additionally, even closely-related bacteriophages have genetic diversity and closely-related phage genomes can have highly variable host specificity (Simmons et al., Reference SIMMONS, DONOVAN, SIRAGUSA and SEAL2010) and hence may not be necessarily efficacious against various C. perfringens isolates. Nevertheless, their high specificity and bacteriolytic activity may warrant further investigation in reducing enteric clostridial colonisation in poultry.

Organic acids

Organic acids are employed as pH regulating agents in enteric environments with antimicrobial activity including lactic acid, butyric acid, acetic acid, propionic acid, fumaric acid, formic acid, citric acid and salts of organic acids. They are actually synthetic compounds between organic acids and their salts. Naturally they are present in large amounts as normal components of animal or plant tissues. They exhibit their antimicrobial activity by reducing the pH of drinking water, decreasing the buffering potential of the diet and by enhancing the generation of antimicrobial peptides in gut mucus (Van Immerseel et al., Reference VAN IMMERSEEL, RUSSEL, FLYTHE, GANTOIS, TIMBERMONT, PASMANS, HAESEBROUCK and DUCATELLE2006).

Their addition in drinking water or feed has been documented to improve the performance of birds, and some acids like butyric acid can reduce NE incidence caused by C. perfringens (Timbermont, Reference TIMBERMONT2009). Butyric acid is important in providing energy to enteric epithelial cells and it stimulates proliferation and differentiation of epithelial cells. A commercially available organic acid preparation Salmex^® (formaldehyde+propionic acid) has been reported to eliminate sporulating microbes (highly thermotolerant) like C. perfringens from animal food and by-products, being a great success for the poultry industry (Cardozo et al., Reference CARDOZO, SCHOCKEN-ITURRINO, BERALDO-MASSOLI, CAVANI, CASAGRANDE, BORINI, BORGES and BERALDO2012). Therefore, organic acids can decrease C. perfringens counts in vivo but may also decrease the enteric load of beneficial Lactobacilli and Bifidobacteria spp. and do not reduce enteric lesions and mortality (Geier et al., Reference GEIER, MIKKELSEN, TOROK, ALLISON, OLNOOD, BOULIANNE, HUGHES and CHOCT2010), discouraging their use in controlling NE.

Feed enzymes

Indigestible NSPs found in feed raw materials include xylans, arabinoxylans, β-glucans, pentosans, mannans, lignin, pectins, (hemi) cellulose, phytic acid and oligosaccharides. Both water soluble and insoluble NSPs exist and are present in certain ratios in different feedstuffs. NSPs are known to form gel solutions, and these, which include β-glucans, cellulose, mannans, pentosans, lignin and phytic acid, cause delayed digestion in chickens leading to low utilisation of nutrients allowing intact nutrients to flow into the hind gut where they are utilised by bacteria (Adams, Reference ADAMS2004). These bacteria then compete for nutrients with the host, reducing those available for the growth of bird, hence negatively affecting performance. On withdrawing AGPs, various feed enzymes, especially NSP'ases and phytases have been used to overcome such problems. Adding enzymes in such diets, decreases the negative impacts of NSPs and phytic acid, facilitates digestion of NSPs to produce energy, enhances nutritive value and mitigates environmental pollution (Panda et al., Reference PANDA, RAO and RAJU2006).

Botanicals, plants extracts and essential oils

Different herbs and botanical products have been reported to impart beneficial effects on intestinal integrity, the gut environment and microflora through their biologically active components (Abbas et al., Reference ABBAS, CLOWELL and GILLEARD2012a; Reference ABBAS, IQBAL, KHAN, SINDHU, KHAN, KHAN and RAZA2012b). Essential oils (ethereal or volatile oils) are derived from various botanical materials. They are complex mixtures of secondary botanical metabolites mainly comprising terpenoids (borneol, citronnillol, geraniol, menthol, α-terpeneol) and different aliphatic hydrocarbons with low molecular weight, including phenols like gaiacol, eugenol, carvacrol, thymol and aromatic aldehydes like cuminal, cinnamaldehyde and phelandral (Huyghebaert et al., Reference HUYGHEBAERT, DUCATELLE and VAN IMMERSEEL2011). The beneficial impacts of different plants, herbs and their products including essential oils, may include stimulation of feed intake and digestive secretions, immunostimulation, antibacterial, antiviral, coccidiostatic, anthelmintic, or anti-inflammatory effects and particularly antioxidant effects (Brenes and Roura, Reference BRENES and ROURA2010).

Essential oils extracted from cinnamon and thyme have been documented to improve broiler growth performance by their digestion stimulation effect and microbicidal activity (Al-Kassie, Reference AL-KASSIE2009). There is a suggestion that most secondary herbal metabolites, such as flavonoids and glucosinolates, act as antibiotics or antioxidants. They modulate the immune system primarily through macrophage activation. Different plant components have certain synergistic or antagonistic impacts, although there is still ambiguity as to which constituents of essential oil products are responsible for stimulating the endogenous enzymes of digestion, their antimicrobial, antioxidative or immunomodulatory activities.

Several plant products show in vitro activity against C. perfringens (Si et al., Reference SI, NI, GONG, YU, TSAO, HAN and CHAMBERS2009). The addition of lupulone obtained from hops in drinking water resulted in the inhibition of experimental infection with C. perfringens (Siragusa et al., Reference SIRAGUSA, HASS, MATHEWS, SMITH, BUHR, DALE and WISE2008). Recently, dietary supplementation with Artemisia annua (as an n-hexane extract) in broiler chicks showed decreased enteric C. perfringens counts, reduced the severity of NE-associated lesions and increased body weight gain in an experimental NE model (Enberg et al., Reference ENBERG, GREVSEN, IVARSEN, FRETTÉ, CHRISTENSEN, HØJBERG, JENSEN and CANIBE2012). It has been reported that the growth of C. perfringens in the enteric region of broiler chicks could be controlled through the administration of special blends of essential oil ingredients including piperin, curcumin, thymol or eugenol and carvacrol. They may mitigate the risk of NE through their in vitro antibacterial activity, digestive enzyme stimulation, intestinal microflora stabilisation and C. perfringens toxin inactivation (Ivarsen et al., Reference IVARSEN, FRETTÉ, CHRISTENSEN, ENBERG, KJAER, JENSEN, GREVSEN and CHRISTENSEN2010). In another study, all 38 unique C. perfringens isolates showed susceptibility to lemon grass and white thyme but resistance to black pepper, clove bud, cinnamon leaf, tea tree, terragon, and comoric basil among 11 essentials oils tested. Variable inhibitory activity was reported for other essential oils including cinnamon bark, Spanish sage, and red thyme (Macklin and Krehling, Reference MACKLIN and KREHLING2010). Similarly, a commercial preparation of an essential oil blend CRINA^® Poultry (containing eugenol, thymol, curcumin and piperin) did not reduce enteric C. perfringens counts when given in the diet of broiler chickens vaccinated against coccidiosis and in addition, failed to ameliorate broiler performance (Abilgaard et al., Reference ABILGAARD, SONDERGAARD, ENBERG, SCHRAMM and HØJBERG2010).

Consequently, botanical products do not always manifest the same desired action and their activity may be influenced by type, environmental growth conditions, time of harvest, maturity state, method and time of conservation and storage, extraction method as well as probable synergistic or antagonistic impacts, antinutritive factors or bacterial contamination. Hence, because of their variable and inconsistent benefits, herbs or botanicals have not been first choices in NE control.

Nutritional changes

Feed components greatly influence the occurrence of NE in broilers. Protein rich diets and those formulated with high animal protein, such as bone meal and fish meal or meat meal, predispose broiler chickens to NE (Gholamiandehkordi et al., Reference GHOLAMIANDEHKORDI, TIMBERMONT, LANCKRIET, VAN DEN BROECK, PEDERSON, J. DEWULF, PASMANS, HAESEBROUCK, DUCATELLE and VAN IMMERSEEL2007). Enteric C. perfringens counts were significantly elevated in fish meal-fed broiler chickens as compared to those offered soy protein concentrate. The high amino acid contents of glycine and methionine in fish meal, possibly favour C. perfringens proliferation (Wu et al., Reference WU, RODGERS and CHOCT2010). The source of fat in the diet has a key role in influencing C. perfringens proliferation, whereby its population in the ileum was significantly raised by the presence of animal fat compared to soy oil (Gholamiandehkordi et al., Reference GHOLAMIANDEHKORDI, TIMBERMONT, LANCKRIET, VAN DEN BROECK, PEDERSON, J. DEWULF, PASMANS, HAESEBROUCK, DUCATELLE and VAN IMMERSEEL2007). It has been proposed that intestinal enzymes might produce or stimulate some anti-clostridial substances in corn-based diets which suppress clostridial growth and increase the alpha-toxin degradation (Annet et al., Reference ANNET, VISTE, CHIRINO-TREJO, CLASSEN, MIDDLETON and SIMKO2002). Corn- and sorghum-based feeds have been shown to promote the growth of Enterococcus, barley increased the numbers of Lactobacillus, rye increased Streptococcus proliferation, and oat elevated Escherichia and Lactococcus populations in broiler chickens (Apajalahti, Reference APAJALAHTI2004). Due to their NSP content, wheat, rye, barley, and oats predispose broilers to NE whereas maize does not (Jia et al., Reference JIA, SLOMINSKY, BRUCE, BLANK, CROW and JONES2009).

Diet digestibility is greatly influenced, physically and chemically, by pelleting. Adequate pellet formation of diet neutralises hazardous diet components, imparts maximum digestibility and enhances the nutritive value of the diet. It has been suggested that processing sorghum and maize in feed mills by hammering them into coarse or medium-sized particles is sufficient for fermentation and incorporating into wet poultry feeds. Such particles generate higher and safer levels of lactic acid during fermentation of poultry feed (Niba et al., Reference NIBA, KOUCHIKA, KUDI, BEAL and BROOKS2013).

Feed form appears to influence NE incidence in broiler chicks, with significant elevations in Clostridia and Lactobacillus spp. growth in the lower part of the gut (caecum and rectum) seen in birds given a mash diet, and where digestibility was significantly increased in the caeca of birds offered a pelleted diet as compared to birds fed mash. (Enberg et al., Reference ENBERG, HEDEMANN and JENSEN2002). It has been documented that offering whole wheat to broiler chicks decreased the pH of gizzard contents and enteric C. perfringens proliferation, compared with ground wheat feeding (Bjerrum et al., Reference BJERRUM, PEDERSEN and ENBERG2005). Hence it can be concluded that dietary modifications by processing can remarkably mitigate NE incidence.