Introduction
The following paper is a brief review of the impact of organic acids (OA), pre- and probiotics on immune function in poultry. As immunity is a key aspect of animal growth and productivity, the knock-on effects in terms of gut development, nutrient absorption and performance are discussed in this context.
An immunomodulator is a substance which has an effect on the immune system. Biological activities of immunomodulators are influenced by different physicochemical parameters, such as solubility, primary structure, molecular weight, branching and polymer charge (Bohn and BeMiller, Reference BOHN and BEMILLER1995). During the development of immune reactions, immunomodulating the effects of β-glucans have been well established (Vetvicka and Sima, Reference VETVICKA and SIMA2004).
The term immunostimulant can be used interchangeably with immunomodulator, or adjuvant and biological response modifier. Immunostimulators can be in the form of drugs or nutrient-based compounds. They stimulate the monocyte-macrophage system and thereby modulate the immune system of the body. Lactobacillus spp., Streptomyces spp., Aspergillus spp. are all organisms that have been discussed for use as immunostimulants (Ganguly et al., Reference GANGULY, PAUL and MUKHOPADHAYAY2010). There are two main categories of immunostimulators. Specific immunostimulators provide antigenic specificity in immune responses, such as vaccines or antigens. For specific immune responses, the host should have prior exposure to an antigen after which recognition and subsequent activation occurs through the co-ordinated action of B-lymphocytes and T-cells. B cells are lymphocytes that play a large role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by T cells). The second types are non-specific immunostimulators, which act irrespective of antigenic specificity to enhance the immune response of other antigens or to stimulate components of the immune system without antigenic specificity, such as glucans or the synthetic drug levamisole. Many endogenous substances are non-specific immunostimulators. For example, prebiotics such as glucans and mannans possess non-specific immunostimulatory activities. β-glucan is a polymer of glucose consisting of linear backbone of β-1,3 linked D-glucopyranosyl residues with varying degree of branching from the C6 position (Bohn and Bemiller, Reference BOHN and BEMILLER1995). They are major components of yeasts, mushrooms and fungal mycelia. Mannan is a plant polysaccharide that is a polymer of the sugar mannose.
Immunostimulants, as feed supplements, are thought to provide protection against pathogens by upregulation of phagocytosis, increased bacterial killing and intensifying oxidative bursts. The following review briefly describes the effects of various supplements, including organic acids, prebiotics and probiotics, in feed on immune responses in poultry, alongside the secondary benefits in terms of nutrient absorption and availability, gut development and productive performance.
Organic acids
Organic acids (OA) reduce the growth of many pathogenic and non-pathogenic intestinal bacteria, decrease intestinal colonisation and reduce infections, ultimately decreasing inflammatory processes at the intestinal mucosa.
OA have an important role in stimulating specific and non-specific gut immune functions (Friedman and Bar-Shira, Reference FRIEDMAN and BAR-SHIRA2005). Although the application of organic acids in poultry rations has been established for a considerable time, little information is known concerning the effects of these additives on the immune system of broiler chicks (Dehghani and Jahnian, Reference DEHGHANI and JAHANIAN2012). Butyric and citric acids may increase the immunity of chicks against Gumboro disease and bronchitis virus compared with a control diet as measured by antibody production. Kazempour and Jahanian (Reference KAZEMPOUR and JAHANIAN2011) found the antibody titre against Newcastle disease was markedly increased by dietary organic acid supplementation in laying hens. In another study with broiler chickens, Jahanian (Reference JAHANIAN2011) reported that butyric acid treatments did not affect the antibody titre against bronchitis and Newcastle disease but the addition of 0.2% butyric acid glyceride produced an improvement in Newcastle antibody titre at the 12th day post-vaccination. Hence, whilst there is very limited data on the immunological effects of OA, more research is needed to validate the studies currently published and identify the modes of action producing such benefits.
Probiotics
Probiotic micro-organisms in the gut stimulate the immune response of the host system in one of two ways (Ganguly et al., Reference GANGULY, PAUL and MUKHOPADHAYAY2010; Reference GANGULY, DORA, SARKAR and CHOWDHURY2013). They can migrate through the gut wall as viable cells, where they then multiply to a limited extent, or the released antigens from the dead organisms are absorbed and stimulate the immune response directly. Probiotics have a positive effect on the host immune response either through the increased activity of macrophages as shown by their enhanced ability to phagocytose organisms and increased production of systemic antibody e.g. IgM and interferon, or by the increased effects of local antibodies at the mucosal surfaces such as the gut wall. The effect of probiotics on the host immune system can be measured by estimating the levels of macrophage enzymes (Paul et al., Reference PAUL, ISORE, JOARDAR, ROY, AICH and GANGULY2013b).
Prebiotics
Prebiotics are non-digestible feed ingredients that beneficially affect the host by selectively stimulating the growth or activity of one or a limited number of bacterial species, already resident in the colon and thus attempt to improve host health (Gibson and Roberfroid, Reference GIBSON and ROBERFROID1995; Ganguly and Mukhopadhayay, Reference GANGULY and MUKHOPADHAYAY2011). Prebiotics are small fragments of carbohydrates and are commercially available as oligosaccharides of galactose, fructose or mannose. The most commonly used prebiotic preparations are fructooligosaccharide (FOS), transgalactooligosaccharide (TOS), inulin, glucooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, soybean oligosaccharide, polydextrose, lactosucrose (Vulevic et al., Reference VULEVIC, RASTALL and GIBSON2004; Propulla, Reference PROPULLA2008). Natural sources of prebiotics for vertebrates include chicory, onion, garlic, leek, tomato and honey.
Mannan oligosaccharide (MOS), obtained from Saccharomyces spp. of yeast outer cell wall maintains gut health by adsorption of pathogenic bacteria containing type-I fimbriae or by agglutinating different bacterial strains (Spring et al., Reference SPRING, WENK, DAWSON and NEWMAN2000) and increasing villi length, uniformity and integrity (Loddi et al., Reference LODDI, MORAES, NAKAGHI, TUCCI, HANNAS and ARIKI2004). In terms of performance benefits with MOS, many trials and reviews are available describing this impact. Savage et al. (Reference SAVAGE, ZAKRZEWSKA and ANDREASEN1997) concluded from a dose responsive study (0-0.33%) that MOS fed at 0.11% maximised weight gain in poultry up to eight weeks of age. Stanley et al. (Reference STANLEY, GRAY, DALEY, KRUEGAR and SEFTON2004) found a similar effect with supplementation of 0.1% MOS in hybrid tom turkey body weight gain. Parks et al. (Reference PARKS, GRIMES, FERKET and FAIRCHILD2001) reported that from a study using turkeys supplemented with MOS, MOS may be utilised as an alternative to AGPs to improve turkey performance. From a dose responsive study it was concluded (0-0.33%) that MOS fed at 0.11% maximised weight gain in poultry from 0 to 8 weeks of age. A similar effect was found on hybrid Tom's body weight gain when turkeys were fed a diet supplemented with 0.1% MOS (Parks et al., Reference PARKS, GRIMES, FERKET and FAIRCHILD2001), illustrating that MOS may be utilized as a alternative to AGPs to improve turkey performance (Podolsky, Reference PODOLSKY1993; Pelicano et al., Reference PELICANO, SOUZA, SOUZA, FIGUEIREDO, BOIAGO, CARVALHO and BORDON2005).
Paul et al. (Reference PAUL, ISORE, JOARDAR, ROY, AICH and GANGULY2013b) discussed the effect of yeast cell wall preparation of Saccharomyces cerevisiae as an immunomodulator of the innate immune response. To evaluate its effect on chickens, a commercial yeast cell wall preparation (NutrifermTM) was administered orally to 1 week old broilers at 0.4 g and 0.8 g per kg of feed for 15 days, after which time the birds were switched back to the control diet for 20 days. The results were compared against a control group of birds maintained on an unsupplemented feeding regime. Non-specific immunity was assessed in randomly selected treated and control birds at the end of the experimental feeding period (0 day) by performing neutrophil, monocyte and lymphocyte functional assays in vitro. In the treated groups, super oxide anion production by neutrophils was increased gradually up to 10 days post treatment (DPT) and then decreased to 20 DPT. The increase was significant (P<0.5) compared to the control birds. In vitro nitrite production by monocytes was found to be higher in treated birds than in control birds. In the 0.4 g treatment group, in vitro non-specific lymphocyte proliferation and IL-2 production was first increased and then decreased abruptly. But in the 0.8 g treatment group in vitro non-specific lymphocyte proliferation and IL-2 production was increased and then decreased gradually. The findings of the study showed that a 15 day oral administration of yeast cell wall preparation at both doses improved innate immune responses in the broiler chicks.
Prebiotics have the potential to enhance many host biological responses and reduce mortality caused by the invasion of gut pathogens. By increasing the production of volatile fatty acids (VFAs) in the gastrointestinal (GI) tract due to the presence of prebiotics, hosts can benefit by recovering some of the lost energy from competition between bacteria (Manning and Gibson, Reference MANNING and GIBSON2004; Paul et al., Reference PAUL, MUKHOPADHAYAY, NIYOGI and GANGULY2013a). The VFAs produced can be used to indicate the type of microbial population present in the GI tract (Nisbet, Reference NISBET2002).
Prebiotics have many beneficial effects such as increased disease resistance and improved nutrient availability (Ganguly et al., Reference GANGULY, PAUL and MUKHOPADHAYAY2010; Reference GANGULY, DORA, SARKAR and CHOWDHURY2013). As such, prebiotics have the potential to increase the efficiency and sustainability of livestock and poultry production.
Glucans
Glucans have a strong immunomodulating activity which has been well studied in livestock (Paul et al., Reference PAUL, ISORE, JOARDAR, SAMANTA, BISWAS, MAITI, GANGULY and MUKHOPADHAYAY2012). However, only a few immunostimulants can be used for commercial farming purposes (Siwicki et al., Reference SIWICKI, MORAND, TERECH-MAJEVSKA, NIEMCZUK, KAZUN and GLABSKY1998). Some investigators have adopted the in vitro culture of macrophages with glucan (Cook et al., Reference COOK, HAYBALL, HUTCHINSON, NOWAK and HAYBALL2001), but in vivo studies have been carried out by the majority of workers (Sahoo and Mukherjee, Reference SAHOO and MUKHERJEE2001; Ortuno et al., Reference ORTUNO, CUESTA, RODRIGUEZ, ESTEBAN and and MESEGUER2002). Under intensive conditions, individuals are more susceptible to microbial infections especially in their growing stages (Ganguly et al., Reference GANGULY, DORA, SARKAR and CHOWDHURY2013). During stress, immunostimulants can provide resistance to pathogens. There are two types of glucans, denoted α- and β, depending on their O-glycosidic bonds. Glucans are now commercially significant as immunostimulating agents. Different types of β-glucans have been used successfully to increase the resistance of poultry against bacterial and viral infections (Paulsen et al., Reference PAULSEN, ENGSTAD and ROBERTSEN2001; Bagni et al., Reference BAGNI, ROMANO, FINOIA, ABELLI, SCAPIGLIATI and TISCAR2005; Ganguly et al., Reference GANGULY, PAUL and MUKHOPADHAYAY2009; Ganguly and Mukhopadhayay, Reference GANGULY and MUKHOPADHAYAY2011; Paul et al., Reference PAUL, ISORE, JOARDAR, ROY, AICH and GANGULY2013b). It has been shown that health, growth and general performance of livestock and poultry may be improved by the use of β-glucans (Paul et al., Reference PAUL, ISORE, JOARDAR, SAMANTA, BISWAS, MAITI, GANGULY and MUKHOPADHAYAY2012). However, product source, animal species, development stage of the target organism, dose and type of glucan, route, time schedule of administration and the association with other immunostimulants affect the immunomodulatory effects of glucans (Guselle et al., Reference GUSELLE, MARKHAM and SPEARE2007).
Paul et al. (Reference PAUL, ISORE, JOARDAR, SAMANTA, BISWAS, MAITI, GANGULY and MUKHOPADHAYAY2012) studied the effect of purified β-glucan from an edible mushroom (Pleuratus florida) as an immunomodulator on the innate immune responses in broiler chickens. Purified mushroom glucan was administered orally to 1 week old broiler chicks at 20 mg/kg feed for 15 days after which time the birds were fed the control diet, versus an unsupplemented control group. Non-specific immunity and protective ability were assessed in treated and control birds at the end of the experimental feeding period (0 day) by performing neutrophil, macrophage and lymphocyte functional assays in vitro and challenged with virulent field isolate of Newcastle Disease. Superoxide anion production by neutrophils, in vitro non-specific lymphoproliferation and IL-2 production increased gradually up to 10 days post treatment and the increase was highly significant (P<0.05) compared to the responses in the control birds. In vitro nitrite production by macrophages was found to be high in treated birds. It was concluded that mushroom glucan as a feed supplement could provide protection against Newcastle Disease. The results illustrated the potential of β-glucan as an immunostimulant in poultry.
The immunostimulatory effects of glucan, chitin, lactoferrin, levamisole, vitamins B and C, growth hormone and prolactin have been reported. These immunostimulants mainly facilitate the function of phagocytic cells and increase their bactericidal activities. Several immunostimulants also stimulate natural killer cells, complementing lysozyme and antibody responses of the host. The most effective method of administration of immunostimulants is by injection, although oral and immersion methods have also been reported, but the efficacy of these methods decreases with long-term administration. Overdoses of several immunostimulants induce immunosuppression. Growth promoting activity has been noted in individuals treated with glucan or lactoferrin as a result of their activities. Immunostimulants can overcome immune suppression by sex hormones (Ganguly et al., Reference GANGULY, PAUL and MUKHOPADHAYAY2009; Reference GANGULY, PAUL and MUKHOPADHAYAY2010).
For the effective use of immunostimulants, the timing, dose, method of administration and the physiological conditions of the host need to be considered. Immunostimulants can reduce the losses caused by disease in commercial poultry rearing; however, they may not be effective against all diseases (Ganguly et al., Reference GANGULY, PAUL and MUKHOPADHAYAY2009; Reference GANGULY, PAUL and MUKHOPADHAYAY2010; Reference GANGULY, DORA, SARKAR and CHOWDHURY2013).
Effect of supplementation of acids, pre- and probiotics on gut development, meat yield and organ weight
Pelicano et al. (Reference PELICANO, SOUZA, SOUZA, FIGUEIREDO, BOIAGO, CARVALHO and BORDON2005) reported that there was an effect on the dressing percentage and weight of different organs and muscles at 21 and 42 days in groups of broilers fed OA salts individually and in combination. Higher villus height in the duodenum and jejunum in the small intestine was reported with most OA used in broiler diets (Loddi et al., Reference LODDI, MORAES, NAKAGHI, TUCCI, HANNAS and ARIKI2004). Similarly a study by Savage et al. (Reference SAVAGE, ZAKRZEWSKA and ANDREASEN1997) also reported increased villus height in the ileum was seen in broilers fed a diet containing OA compared to broilers fed a control diet. Therefore, supplementation with OA may increase villus height in different parts of the small intestine. They appear to increase villus height and secretion, allowing digestion and absorption of nutrients to be appropriately performed by the mucosa (Iji and Tivey, Reference IJI and TIVEY1998).
Positive effects of the use of prebiotics on the intestinal mucosa have been reported, among which a significant increase in villus height was observed in three segments of the small intestine of birds aged one week and supplemented with MOS (Maiorka et al., Reference MAIORKA, SANTIN, BORGES, OPALINSKI and SILVA2004). Loddi (Reference LODDI2003) saw longer villi in the intestinal mucosa (duodenum) of birds fed MOS at 7 and 21 days of age. Pelicano et al. (Reference PELICANO, SOUZA, SOUZA, FIGUEIREDO, BOIAGO, CARVALHO and BORDON2005) reported that in the jejunum, MOS + OA resulted in longer villi (P<0.01) and that a larger effect was seen in broilers fed diets containing MOS based prebiotics alone. Pelicano et al. (Reference PELICANO, SOUZA, SOUZA, FIGUEIREDO, BOIAGO, CARVALHO and BORDON2005) observed longer villi length when birds were fed with prebiotics based on MOS, compared to the control group.
Micro-organisms that are sensitive to acid pH appear to be related to longer villi length (Radecki and Yokoyama, Reference RADECKI and YOKOYAMA1991). Some bacteria recognize binding sites on prebiotics instead of on the intestinal mucosa, and the colonisation by pathogenic bacteria in the intestine is thus reduced (Ganguly and Mukhopadhayay, Reference GANGULY and MUKHOPADHAYAY2011). Therefore, besides a lower infection incidence, there is an increase in the absorption of available nutrients, a mechanism that directly affects the recovery of the intestinal mucosa, increasing villi length. These findings disagree to those obtained by Pelicano et al. (Reference PELICANO, SOUZA, SOUZA, FIGUEIREDO, BOIAGO, CARVALHO and BORDON2005) and Santin et al. (Reference SANTIN, MAIORKA, MACARI, GRECCO, SANCHEZ, OKADA and MYASAKA2001) respectively, who found no difference in ileal villi length with the use of probiotics and prebiotics.
Roy et al. (Reference ROY, MUKHOPADHAYAY, NIYOGI, CHOUDHARY and GANGULY2012) conducted experimental studies to evaluate the pathomorphological effect of different combinations of OA viz. formic acid, propionic acid and lactic acid as a replacer of growth promoter antibiotics. The birds were divided into six equal groups; negative control (C1), positive control (C2) and treatment groups (T1, T2, T3 and T4). Birds of group C1 were supplied with a diet without any antibiotics or acid, group C2 with virginiamycin at 500 g/quintal feed, group T1 with 0.3% ammonium formate, group T2 with 0.3% calcium propionate, group T3 with 0.15% ammonium formate and 0.15% calcium propionate and group T4 with 0.1% ammonium formate, 0.1% calcium propionate and 0.1% calcium lactate. Body weight gain was higher in C1 and C2 compared with treatment groups in the first two weeks, but changes were maximised in the negative control, i.e. after six weeks weight gain was significantly better in T3 and T4 than in groups C1, T1 and T2 groups. Pathomorphological changes were seen in group C1 and T2. Group C2 and T1 also showed similar types of changes and in group T3 and T4 the changes were less. The most common changes among the groups were thickening of the muscular layer and accumulation of inflammatory cells and congested blood vessels. In T1 group, rupture of villi was also seen. In T3 and T4 the intestinal glands were hypertrophied. Based on this investigation, it was suggested that a combination of OA may be used in broiler feed as a replacer of growth promoter antibiotics instead of using only one acid.
It has been reported that the combined use of MOS and OA salts in poultry feed can be used as an alternative to the antimicrobial and antibiotic growth promoters to achieve good health for sustainable and economic poultry production in India (Das et al., Reference DAS, MUKHOPADHYAYAY, GANGULY, KAR, DHANALAKSHMI, SINGH, SINGH, RAMESH and PAL2012).
Effect on gut microbial load by prebiotics and acids
The findings that MOS and OA successfully reduce bacterial load in the intestine of broiler birds were in accordance with the findings of Newman (Reference NEWMAN1994), Lon (Reference LON1995), Spring et al. (Reference SPRING, WENK, DAWSON and NEWMAN2000) and Fairchild et al. (Reference FAIRCHILD, GRIMES, JONES, WINELAND, EDENS and SEFTON2001). Stanley et al. (Reference STANLEY, GRAY, DALEY, KRUEGAR and SEFTON2004) concluded that yeast cell culture residue (YCR) treated broiler chicks resulted in lower intestinal coliform populations in comparison to the control and other antibiotic-treated (lasalocid @ 90.7 kg/ton, bacitracin @50 g/ton) groups. Sims et al. (Reference SIMS, DAWSON, NEWMAN, SPRING and HOOGE2004) found that MOS + BMD treated turkeys showed significantly lower Clostridium perfringens populations in the gut than the control-fed birds at six weeks of age. MOS is believed to block type-I fimbriae and prevents pathogens from attaching to the intestinal lining and passes them out of the gut. (Dawson and Pirulescu, Reference DAWSON and PIRULESCU1999; Yitbarek et al., Reference YITBAREK, ECHEVERRY, BRADY, HERNANDEZ-DORIA, CAMELO-JAIMES, SHARIF, GUENTER, HOUSE and RODRIGUEZ-LECOMPTE2012). Studies of intestines from broilers revealed reduced E. coli levels compared with an untreated control group. Salmonellae were not found in any group. No significant difference was observed among control and treatment groups regarding the number of Clostridium sp. present in the intestine (Dawson and Pirulescu, Reference DAWSON and PIRULESCU1999). These results clearly indicate that consumption of prebiotics mixed with poultry feed reduced the load of coliform bacteria in the intestine (Roy et al., Reference ROY, MUKHOPADHAYAY, NIYOGI, CHOUDHARY and GANGULY2012).
Conclusions
From this brief review, it can be seen that supplementation of poultry feed with dietary supplements such as acids, pre- and probiotics can enhance the immune system of the host by providing increased resistance to infections. This, in turn, can promote gut development and nutrient absorption, leading to better performance.
