Blogs

Dr. Amit V. Janbandhu & Dr. Sanjay Singhal

1. Introduction

Animal fats and vegetable oils are commonly incorporated into poultry diets due to their high energy density, which supports optimal growth performance (Blanch et al., 1996). Dietary fats and oils provide approximately 2.25 times more metabolizable energy than carbohydrates and serve as important sources of essential fatty acids and fat‑soluble vitamins. In recent years, rising feed costs have increased interest in maximizing dietary fat utilization to enhance energy concentration and meet the demands of high‑performing birds. However, fat absorption efficiency is age‑dependent; young broilers exhibit a physiological limitation in lipid digestion and absorption, which improves progressively with advancing age (Kussaibati et al., 1982)

Enzymatic hydrolysis of lipids (oils and fats) produces fatty acids (FA) which are water insoluble. FA passes through the liquid phase of the small intestine and, after aggregating to form micelles, are absorbed as hydrophobic components. This process is naturally mediated by endogenous emulsifiers, such as bile salts. The assimilation of dietary fats in young birds is poor because they have a limited capacity to produce and secrete bile salts and lipase until their gastrointestinal tract matures at 10-14 days of age (Noy and Sklan,1998).

This review evaluates the role and application of exogenous emulsifiers, such as Lipifier-DS from Stallen South Asia Pvt Ltd., a multi-component emulsifier and absorption accelerator, in nutrient-dense broiler diets to maximize the growth potential of modern poultry genetics.

2. Digestibility problems in young chicks

Young birds exhibit limited fat absorption due to low endogenous lipase activity, reduced bile secretion, and poor emulsification capacity; however, these functions improve with age and adapt to higher levels of unsaturated fatty acids (Meng et al., 2004). Consequently, immature digestive systems are unable to efficiently form mixed micelles in the intestinal lumen, leading to reduced fat digestion and nutrient absorption (Leeson and Atteh, 1995). Age‑related differences in metabolisable energy (ME) utilization and growth performance have therefore stimulated interest in the use of exogenous emulsifiers to enhance fat utilisation in young birds (Roy et al., 2010). High dietary inclusion of saturated fats in broiler rations can lead to excessive visceral and carcass fat deposition, reduced vitamin A and E availability, and compromised meat quality (Chae et al., 2006).

3. Energy efficiency of emulsifiers in High‑Performance Broiler Diets

Energy is a major cost component factor in diets of high-performance animals, such as broilers. Emulsifiers can be used to improve fat digestibility and energy efficiency. As a result, lower energy diets can be formulated for birds whilst maintaining the same performance, leading to lower feed cost and more economical and sustainable production. Emulsifiers facilitate the formation of emulsion droplets, which lowers the surface tension (Ashraf, 2007), stimulates the formation of micelles, causes high levels of monoglycerides in the intestine and facilitates the nutrient transport through the membrane (Melegy et al., 2010). Emulsifier supplementation has been shown to improve feed efficiency, lipid absorption, and blood lipid profiles, although its effects on growth performance and carcass traits are inconsistent (Udomprasert and Rukkwamsuk, 2006).

4.Classification and Functional Role of Exogenous Emulsifiers in Broilers

Exogenous emulsifiers used in animal nutrition are broadly classified as natural or synthetic, with natural emulsifiers—including bile salts, phospholipids, and dietary sources such as soy lecithin—and synthetic emulsifiers comprising chemically modified molecules such as lysolecithin or lysophosphatidylcholine (Zhang et al., 2011). By modifying hydrophobic interfaces and promoting mixed‑micelle formation, these emulsifiers enhance fat digestibility, particularly in young birds with limited endogenous emulsification capacity (Al‑Marzooqi and Leeson, 1999). Soy lecithin remains the most extensively validated natural emulsifier in poultry, improving fat utilization, growth performance, and serum lipid profile while supplying choline to prevent perosis (Polin, 1980; Siyal et al., 2017; Schaible, 1970). Synthetic emulsifiers, including polyethylene glycol mono‑ and dioleates and sodium stearoyl‑2‑lactylate, have also been shown to improve growth performance and the utilization efficiency of fat, protein, and metabolizable energy, although some synthetic polyoxyethylene glycol emulsifiers exhibit lower in vivo efficiency compared with bile salts (Frobish et al., 1969; Roy et al., 2010).

5. Emulsifying agents

An emulsifying agent stabilizes an emulsion by reducing interfacial tension between immiscible phases such as oil and water, thereby preventing droplet coalescence. Emulsifiers possess both hydrophilic and lipophilic moieties, enabling their adsorption at the oil–water interface and stabilization of dispersed fat droplets. Efficient fat emulsification is a prerequisite for lipid digestion in the gastrointestinal tract and is influenced by fatty acid chain length, triglyceride structure, and degree of saturation (Gu and Li, 2003). Exogenous emulsifiers enhance lipid utilization, particularly of animal fats, and partially compensate for limited bile production and enterohepatic recirculation in young birds. Although bile salts and monoglycerides function as natural emulsifiers, their emulsification capacity is insufficient in young birds, resulting in poor fat digestibility. Furthermore, saturated and free fatty acids exhibit a lower capacity for micelle formation than long‑chain unsaturated fatty acids, further limiting lipid digestion efficiency.

Table 1. Available Emulsifiers Used in the Poultry Industry

Category	Emulsifier	Major Effects	References
Natural emulsifiers	Soy lecithin	Improved growth performance; increased HDL; reduced triglycerides, insulin (INS), thyroid‑stimulating hormone (TSH), total cholesterol (TC), and LDL levels	Siyal et al., 2017; Huang et al., 2007
	Milk‑derived casein	Increased weight gain, improved feed conversion ratio (FCR), enhanced pancreatic lipase activity, improved ether extract digestibility, with no effect on carcass traits or serum cholesterol	Guerreiro et al., 2011
	Lysophosphatidylcholine / Lysolecithin	Improved weight gain and FCR; increased digestibility of total tract apparent digestibility (CTTAD) of C16:0, C18:2, and C18:3n‑3 fatty acids; improved carcass quality and dressing percentage	Melegy et al., 2010; Zhang et al., 2011; Azman and Ciftci, 2004; Ashraf, 1995
	Bile salts	Increased relative organ weights; significantly improved body weight gain (BWG); decreased plasma cholesterol; increased metabolisable energy; enhanced digestive enzyme activity in the intestinal tract	Abd‑El‑Rauof, 1995; Alzawqari et al., 2010; Gomez and Polin, 1976; Kussaibati et al., 1982; Zhong and Xiang, 2008
Synthetic emulsifiers	Glycerol polyethylene glycol ricinoleate (E 484)	Improved performance; increased intake and utilization efficiency of fat, crude protein (CP), and metabolisable energy (ME)	Roy et al., 2010
	Lipex® (commercial emulsifier)	Increased body weight gain and liver weight	Yordan et al., 2013
	Sodium stearoyl‑2‑lactylate (SSL)	Improved body weight and relative organ weights	Flores et al., 2007; Chronakis et al., 2004

6. Principles of Emulsification

Emulsifiers lower the interfacial energy between the two immiscible liquids, thereby helping the formation of an emulsion. For an emulsifier to be effective at reducing droplet size and stabilising an emulsion, it needs to be located at the interface. The emulsifier must not be too soluble in either phase, otherwise it will migrate to that phase. If the emulsifier migrates away from the interface, the emulsion is destabilised.

An emulsion is most accurately defined as a dispersion of liquid droplets in a second immiscible liquid. Temporary emulsions may be formed by mixing/agitating the two normally immiscible liquids; however, the stability of temporary emulsions produced in this way is poor. Emulsifiers are surface active materials (surfactants) that are used to assist in the formation of an emulsion and to stabilise the emulsion.

Fig.1. Below is a simplistic view of an emulsion particle, protected by surfactant molecules partitioned at the interface of the internal and external phase.

There are several different types of emulsions. They are loosely described by their phase relationship and/or by their appearance:

• Oil-in-Water
• Water-in-Oil
• Multiple emulsions
• Macro-emulsions
• Micro-emulsions

The appearance of the emulsion is dependent upon the particle size of the discontinuous phase.

Table.2. Particle size is listed in nanometers (nm)

Particle size	​Appearance
​> 1	White
0.1 -1.0	Blue White
0.05 – 0.1	Translucent
< 0.05	Transparent

7. Mechanism of Action of Lipid Digestion and Absorption

• Emulsification of dietary fat: Large lipid droplets entering the intestinal lumen are emulsified by bile salts, reducing surface tension and breaking them into smaller droplets. This process increases the surface area available for enzymatic action.
• Enzymatic hydrolysis of lipids: Pancreatic lipase acts on the emulsified fat droplets, hydrolyzing triglycerides into free fatty acids and monoglycerides.
• Micelle formation: The released fatty acids and monoglycerides associate with bile salts to form mixed micelles, which are water-soluble and capable of diffusing through the intestinal lumen.
• Transport across the mucosal membrane: Micelles deliver fatty acids and monoglycerides to the brush border of intestinal mucosal cells, where these lipids diffuse across the cell membrane into the enterocytes.
• Re-esterification within the enterocyte: Inside the mucosal cells, fatty acids and monoglycerides are transported to the endoplasmic reticulum, where they are re-esterified to form triglycerides.
• Chylomicron formation: Newly synthesized triglycerides are packaged with phospholipids, cholesterol, and apoproteins to form chylomicrons.
• Exocytosis and lymphatic transport: Chylomicrons are transported to the basolateral membrane of the enterocyte and released by exocytosis into the lymphatic vessels, from where they enter systemic circulation.

Fig.2. General schematic of lipid digestion and absorption

8. Hydrophilic-lipophilic balance

The combination of hydrophilic and lipophilic characteristics in one molecule gives it the distinctive property that the emulsifier can dissolve in fat as well as in water, and can aid in mixing the two fractions. The key indicator for selecting an emulsifier is hydrophilic-lipophilic balance (HLB), ranging from 0 to 20 which reveals the degree of fat or water solubility. Lower HLB indicates a more lipophilic or fat-soluble emulsifier. On the other hand, higher HLB indicates a more water soluble or hydrophilic emulsifier. Ideally, the emulsifier should be soluble in the continuous phase as the Bancroft rule states (1912). For the soluble condition known as the ‘fat-rich environment’ mixed in a small amount of water, an emulsifier with a lower HLB is advised, and vice versa. Because of birds consume water 1.5-2 times more than feed; the diet should contain a small amount of fat and the water amount should exceed fat in digestive tract. In this situation, a high HLB is more appropriate.

Fig. 3. HLB scale and its influence on surfactant functionality and emulsion types (Al-Yami et al. 2018)

9. Lipifier‑DS: –

Lipifier‑DS contains a blend of hydrolysed phospholipids—including LPC, LPE, LPI, LPA, other lysophospholipids—along with glyceryl polyethylene glycol ricinoleate coating.

a) Mechanism of Action of Lipifier-DS

Lipifier-DS function based on their solubility. If the emulsifier is more soluble in water, it forms an oil-in-water (O/W) emulsion, ideal for fat digestion in poultry. Conversely, if it is more soluble in oil, it forms a water-in-oil (W/O) emulsion. The emulsification process helps break down fat droplets into smaller micelle particles that remain dispersed in the water phase, allowing digestive enzymes to act more efficiently and resulting in availability of extra metabolizable energy to the birds. Exogenous emulsifiers are molecular surfactants with both hydrophobic and hydrophilic properties. The hydrophobic end with fatty acids is directed to the oil phase, while the hydrophilic end with sucrose, glycol, glycerol, sorbitol or polyglycerol is directed to the aqueous phase, forming a “molecular bridge” by decreasing surface tension that inhibits the coalescing of hydrolysed lipid droplets into large molecules.  

b) Key Features of Lipifier-DS

• It contains Glyceryl Polyethylene Glycol Ricinoleate (PEGR) coating for stable and efficient emulsification.
• Lipifier-DS has optimized HLB (Hydrophilic–Lipophilic Balance) value of 9–12, ensuring effective emulsification and superior fat utilization.
• Highly efficient energy contributor — 250 g of Lipifier-DS provides 40,000 Kcal/kg, equivalent to approximately 4.25 kg of soybean oil. Enhances dietary energy efficiency while reducing dependence on added oil sources.

c) Benefits of Lipifier-DS

• Enhances growth performance and improves feed conversion ratio (FCR) in broilers.
• Improves egg production performance and increases egg size in layers.
• Enhances digestion and absorption of fats and fat-soluble vitamins.
• Promotes better absorption of essential nutrients.
• Ensures effective emulsification by forming a stable emulsion with PEGR coating.
• Improves fat digestibility and maximizes energy availability.
• Extracts greater nutritional value from feed, improving overall nutrient utilization.

10. Conclusion:

Stallen South Asia Pvt. Ltd.’s has Lipifier-DS is an effective exogenous emulsifier that enhances fat utilization and nutrient absorption in poultry diets. By promoting efficient emulsification through a stable PEGR coating, it improves energy availability and feed efficiency, supporting better growth and FCR in broilers, enhanced egg performance in layers, and overall higher production efficiency.

References

BLANCH, A., BARROETA, C., BAUCELLS, M.D., SERRANO, X. and PUCHAL, F. (1996), Utilisation of different fats and oils by adult chickens as a source of energy, lipid and fatty acids. Animal Feed Science and Technology 61: 335-342.

NOY, Y. and SKLAN, D. (1998) Metabolic responses to early nutrition. Journal of Applied Poultry Research 7: 437-451.

Kussaibati R, Guillaume J and Leclercq B. 1982. The effects of age, dietary fat and bile salts, and feeding rate on apparent and true metabolisable energy values in chickens. British Poultry Science 23(5): 393–403.

Meng X, Slominski B A and Guenter W. 2004. The effect of fat type, carbohydrase, and lipase addition on growth performance and nutrient utilization of young broilers fed wheat-based diets. Poultry Science 83(10): 1718–27.

LEESON, S. and ATTEH, J.O. (1995) Utilisation of fats and fatty acids by turkey poults. Poultry Science 74: 2003-2010.

ROY, A.S., HALDAR, S., MONDAL, T. and GHOSH, K. (2010) Effects of supplemental exogenous emulsifier on performance, nutrient metabolism, and serum lipid profile in broiler chickens. Veterinary Medicine International: Art. ID 262604.

ASHRAF, M. (2007) Use of Emulsifiers in High Fat Level Diets of Broilers. Doct thesis, Dept Animal Production, Faculty of Agriculture, Al Azhar University, Cairo, Egypt, 235, 2007.

MELEGY, T., KHALED, N., EL-BANA, R. and ABDELLATIF, H. (2010) Dietary fortification of a natural biosurfactant, lysolecithin in broiler. African Journal of Agriculture Research 5: 2886-2892.

UDOMPRASERT, P. and RUKKWAMSUK, T. (2006) Effect of an exogenous emulsifier on growth performance in weanling pigs. Kasetysart Journal of Natural Science 40: 652-656.

Zhang B, Haitao L, Zhao D, Guoand Y and Barri A. 2011. Effect of fat type and lysophosphatidylcholine addition to broiler diets on performance, apparent digestibility of fatty acids and apparent metabolisable energy content. Feed Science and Technology 163: 177–84.

AL-MARZOOQI, W. and LEESON, S. (1999) Evaluation of dietary supplements of lipase, detergent and crude porcine pancreas on fat utilisation by young broiler chicks. Poultry Science 78: 1561-1566.

Siyal F A, Babazadeh D, Wang C, Arain M A, Saeed M, Ayasan T, Zhang L and Wang T. 2017. Emulsifiers in poultry industry- A review. World Poultry Science Journal 73: 1–6.

Schaible P J. 1970. Poultry: Feeds and Nutrition. 2nd Edn., The AVI Publishing Co. USA.

Polin D and Hussein T H. 1982. The effect of bile acid on lipid and nitrogen retention, carcass composition, and dietary metabolizable energy in very young chicks. Poultry Science 61(8): 1697–1707.

FROBISH, L.T., HAYS, V.W., SPEER, V.C. and EWAN, R.C. (1969) Effect of diet form and emulsifying agents on fat utilisation by young pigs. Journal of Animal Science 29: 320-324.

CHAE, B.J., LOHAKARE, J.D. and CHOI, J.Y. (2006) Effects of incremental levels of α-tocopherol acetate on performance, nutrient digestibility and meat quality of commercial broilers. Asian-Australian Journal of Animal Sciences 19: 203-208.

GU, X. and LI, D. (2003) Fat nutrition and metabolism in piglets: A review. Animal Feed Science and Technology 109: 151-170.

BANCROFT, W.D. (1912) The theory of emulsification VI. The Journal of Physical Chemistry 17: 501-519.

Dr. Amit V. Janbandhu & Dr. Sanjay Singhal

1. Introduction

The poultry industry is among the most efficient sectors of agriculture, making a significant contribution to livelihood generation and global nutritional security. The global poultry population currently exceeds 26.8 billion birds (FAO, 2020). From 1961 to 2019, worldwide poultry meat production increased to approximately 132 million tonnes annually, representing nearly 37% of total global meat production (FAO, 2020). This growing demand for animal‑derived foods is primarily driven by rapid population growth, rising income levels, and increasing urbanization (FAO, 2020).

To satisfy the rising demand for meat and eggs, modern poultry production systems operate under intensive rearing conditions, exposing birds to continuous physiological stress. As a result, antibiotics have been extensively used for disease prevention, growth promotion, and immune enhancement. However, the indiscriminate use of antibiotics has hastened the emergence of antimicrobial resistance among pathogenic bacteria (Garcia‑Migura et al., 2014; Roth et al., 2019). The World Health Organization has identified antimicrobial resistance as “a serious threat to public health worldwide that requires action across all government sectors and society” (WHO Factsheets, 2015). Antibiotic‑resistant bacteria can enter the human food chain through animal‑derived products, posing substantial public health risks (Cui et al., 2005). Moreover, fresh meat products may act as reservoirs of antibiotic‑resistance genes that can be transferred to humans through regular consumption (Diarrassouba et al., 2007). In response to the escalating challenge of antimicrobial resistance, the livestock industry is increasingly exploring effective alternatives to conventional antibiotics, with probiotics emerging as a promising and sustainable solution.

2. What Is Probiotics?

The concept of probiotics was first introduced in the early 1900s by the Russian‑born Nobel laureate Elie Metchnikoff, who demonstrated that the regular consumption of beneficial microorganisms could positively influence gastrointestinal health (Metchnikoff, 1907). His observations of populations that consumed fermented milk products led to the hypothesis that beneficial intestinal microflora enhance resistance to pathogenic organisms. The term probiotics is derived from Greek, meaning “pro‑life” (Shokryazdan et al., 2017a, 2017b). According to the FAO/WHO, probiotics are defined as “live organisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO Joint Report, 2001).

Probiotics are widely recognized for their ability to modulate gut microflora and enhance immune responses (Chen et al., 2012) and are extensively applied in both clinical and veterinary practices (Abushelaibi et al.). In livestock production, probiotic supplementation has been associated with improved growth performance, production efficiency, disease resistance, nutrient digestibility, immune function, and fecal microbial balance (Lan et al., 2017). In recent years, non‑specific immunomodulators—including probiotics, prebiotics, synbiotics, postbiotics, polysaccharides, organic acids, enzymes, and essential oils—have gained considerable attention as alternatives to conventional antibiotics and are increasingly used to promote gut health in poultry birds (Callaway et al., 2017).

3. Probiotic and Related Biotic Agents in Poultry

The species currently being used in probiotic preparations are varied and many. These are mostly Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium spp. and Escherichia coli. With two exceptions, these are all intestinal strains. The two exceptions, Lactobacillus bulgaricus and Streptococcus thermophilus, are yoghurt starter organisms (Fuller et.al, 1989). Some other probiotics are microscopic fungi such as strains of yeasts belonging to Saccharomyces cerevisiae species (Guillot et.al, 1998).

In broiler nutrition, probiotic species belonging to Lactobacillus, Streptococcus, Bacillus,

Bifidobacterium, Enterococcus, Aspergillus, Candida, and Saccharomyces have a beneficial effect on broiler performance (Tortuero et.al, 1973), modulation of intestinal microflora and pathogen inhibition, intestinal histological changes, immunomodulation, certain haematobiochemical parameters, improving sensory characteristics of dressed broiler meat and promoting microbiological meat quality of broilers (Kabir et.al, 2005).

The International Scientific Association of Probiotics and Prebiotics defines prebiotics as “a substrate that is selectively utilized by host microorganism conferring a health benefit” (Gibson et al., 2017). Synbiotics are defined as a “synergistic combination of probiotics and prebiotics that are beneficial for the host by improving the development and colonization of live microorganisms in the gut” (FAO/WHO joint report, 2002). Postbiotics are defined as “the preparation of inanimate microorganism and/or their components that confer a health benefit on the host” (Salminen et al., 2021).

Given their safety, efficacy, and sustainability, probiotics have gained considerable attention as viable alternatives to antibiotics. This article outlines the status, mechanisms, and use of probiotics in poultry as alternatives to antibiotics.”

4. The concept of probiotics

In healthy, non-stressed poultry, a dynamic balance exists between beneficial and non-beneficial gut bacteria, which is essential for optimal performance. Stress disrupts this balance by reducing beneficial flora, particularly lactobacilli, allowing overgrowth of harmful microorganisms. This imbalance may result in clinical conditions such as diarrhea or subclinical effects that impair growth and feed efficiency. Although the protective gut microflora is relatively stable, it can be influenced by key factors including excessive hygiene, antibiotic use, and stress. Under natural conditions, chicks acquire a complete and protective gut microflora through contact with the hen, providing resistance against infection. In contrast, commercially reared chicks hatch in sanitized incubators lacking normal intestinal microorganisms. Gut colonization may be influenced by eggshell microbiota and the onset of gastric HCl secretion at approximately 18 days of incubation, which affects microbial selection. Consequently, early probiotic supplementation is particularly important in poultry, as chicks are deprived of maternal microbial transfer and can benefit from microbial preparations that restore protective gut microflora (Fuller et al., 2001).

Figure 1. Schematic representation of the concept of probiotics (modified from (Fuller et.al, 2001).

5.Mechanism of action of probiotics

Probiotics place a key role in gut microbial health. The mechanisms of action of probiotics mainly include two, i.e. competitive exclusion and immune system modulation.

Competitive exclusion of pathogens by probiotics includes:

 (a) production of inhibitory compounds like bacteriocins, mucins, defensins, etc. (b) preventing the adhesion of pathogens, (c) competition for nutrients, (d) reduction of toxin bioavailability, and (e) modulation of the host immune system including the enhancement of both innate and adaptive immunity (Hernandez-Patlan et al., 2020).

a) Secretion of inhibitory compounds

Probiotics inhibit pathogenic bacteria through the production of antimicrobial compounds, including antimicrobial peptides (AMPs) such as bacteriocins, as well as organic acids, hydrogen peroxide, ethanol, diacetyl, and carbon dioxide (Liao & Nyachoti, 2017). Bacteriocins are ribosomally synthesized AMPs that suppress pathogens by disrupting cell wall synthesis or forming membrane pores while sparing beneficial gut microbiota (Cotter et al., 2013). Pediocin A from Pediococcus pentosaceus, divercin from Carnobacterium divergens, and nisin from Lactococcus lactis have shown inhibitory effects against Clostridium perfringens and improved broiler performance (Grilli et al., 2009). Synergistic bacteriocin activity with other biomolecules has been reported against multiple pathogens (Rishi et al., 2014). Organic acids reduce intracellular pH and disrupt bacterial metabolism and membranes, while lactic acid bacteria inhibit Salmonella, Listeria monocytogenes, and Escherichia coli without harming intestinal epithelium (Ricke, 2003). Additional inhibitory effects are mediated by ethanol, diacetyl, and carbon dioxide (Ingram, 1989).

b) Inhibition of pathogenic adhesion

Probiotics prevent pathogen colonization by competitively blocking adhesion sites on intestinal epithelial cells, a key criterion for selecting effective probiotic strains . Probiotic adhesion stimulates mucosal immunity and promotes the secretion of mucins and defensins, thereby strengthening the epithelial barrier (Bermudez-Brito et al., 2012). Mucins are highly glycosylated glycoproteins that form the mucus layer and inhibit pathogen attachment and colonization (Collado et al., 2005). Interactions between probiotic surface proteins and intestinal epithelial cells further exclude pathogens. Defensins, small cationic antimicrobial peptides, inhibit bacterial growth by disrupting membranes or cell wall synthesis and can neutralize bacterial toxins (Ayabe et al., 2000).

c) Competition for nutrients

Probiotics limit pathogen growth by competing for essential nutrients and occupying intestinal epithelial adhesion sites, thereby restricting pathogen attachment in the gastrointestinal tract . This competitive exclusion reduces pathogen proliferation and colonization and creates unfavorable conditions for pathogen survival (Callaway et al., 2008). Competitive exclusion has been demonstrated in vitro using chicken intestinal mucosa (Hirn et al., 1992). In vivo studies show that early supplementation with lactobacillus-based probiotics (1 × 10⁵ CFU/mL, 1–7 days of age) significantly reduced Salmonella colonization in chicks (Penha Filho et al., 2015).

d) Reduction in toxin bioavailability

Probiotics like lactobacillus help the reduction in the uptake of pathogenic toxins in the intestinal cells. The positive effects of LAB-based probiotics had helped in the reduction of toxin expression in the gut. Lactic acid bacteria are known for their natural barriers against mycotoxins which are harmful compounds for animals. A few strains can also eradicate the detrimental reactions of aflatoxins on human and animal health (Abbes et al., 2016).

e) Modulation of the host immune system

Probiotics modulate host immunity by interacting with intestinal epithelial cells, dendritic cells, macrophages, and lymphocytes. These interactions enhance innate immune defenses by limiting pathogen proliferation and reinforcing epithelial barriers through increased mucus and antimicrobial peptide production. Intestinal epithelial and dendritic cells recognize probiotics via pattern recognition receptors, initiating immune signaling cascades. Activation of antigen-presenting cells stimulates adaptive immunity through T- and B-cell responses. Probiotics regulate cytokine expression and suppress intestinal inflammation by downregulating TLR and NF-κB signaling pathways. Enhanced IgA and IgG responses have been observed in broilers supplemented with probiotic strains such as Clostridium butyricum and Lactobacillus plantarum (Han et al., 2018).

Fig. 2. Mode of action of probiotics. It starts with the secretion of inhibitory compounds leading to inhibition of the pathogen adhesion to the epithelial layer of the GI tract besides creating competition for nutrients among pathogens thereby reducing their colonization. Also, it helps in diminishing the toxin bioavailability and modulates the immune system of the host by activating adaptive and innate immunity.

6. Single- and multi-strain probiotics

Probiotics are classified as single- or multi-strain formulations. Single-strain probiotics contain one microbial species, commonly Lactobacillus, Bifidobacterium, Streptococcus, Pediococcus, Enterococcus, Bacillus, Saccharomyces, and Micrococcus. Multi-strain probiotics combine multiple strains or genera to provide complementary benefits and have been shown to improve growth performance and gut health in broilers, including under disease challenge conditions. Commercial products such as Probios from Stallen South Asia private Ltd contain diverse probiotic combinations. Probiotic efficacy depends on strain composition and viable counts, with variable outcomes reported (Aalaei et al., 2018).

a) Bacillus

Many strains of Bacillus have potential against pathogenic bacteria. A group of researchers isolated 200 Bacillus strains from the faeces of broiler chicken and many strains among them showed activity against C. perfringens in in vitro conditions. A study suggested that B. subtilis strain SP6 when used in a field trial, the mortality of chicken infected with Necrotic enteritis was reduced to half. It also reduced the number of C. perfringens and enhanced the intestinal health of chickens. Regular use of B. licheniformis supplementation reduced mortality and increased the performance among the chicks (Knap et al., 2010).

b) Yeast

Yeasts possess antimicrobial and immunomodulatory properties, largely due to β-glucans that stimulate host immunity. They inhibit pathogens by producing mycocins, degrading toxins, preventing epithelial adhesion, and competing for nutrients. Saccharomyces boulardii improves intestinal health and reduces Salmonella enteritidis infection, while recombinant Pichia pastoris expressing Clostridium perfringens α-toxin enhances broiler performance (Gil de Lossantos et al., 2005).

c) Enterococci

Enterococci produce bacteriocins (enterocins) with activity against Gram-positive and Gram-negative bacteria . Enterococcus faecium supplementation reduces Clostridium perfringens, alleviates coccidiosis, and improves growth performance and nutrient utilization in broilers. Enhanced IgA production, immune responses, and microbiome modulation have also been reported with E. faecium and E. faecalis supplementation (Beirão et al., 2018)

7. Beneficial effects of probiotics on poultry

a) Effects on growth performance and productivity

Probiotics improve body weight gain, feed intake, feed conversion ratio, and overall productivity in poultry. Supplementation with Pediococcus acidilactici and Bacillus subtilis enhances egg quality, increases eggshell thickness, and reduces yolk cholesterol. Multi-strain probiotics improve egg production and mitigate heat-stress effects. Probiotics also enhance meat microbiological quality, reduce Salmonella enteritidis contamination, and improve nutrient metabolism and growth performance (Bailey et al., 2000).

b) Effects on serum biochemistry

Probiotic supplementation significantly modulates serum biochemistry in poultry by reducing total cholesterol, LDL, VLDL, triglycerides, uric acid, and liver enzymes (ALT, AST), while increasing protein and calcium levels . Lactobacillus spp., Enterococcus faecium, and Bacillus subtilis reduce cholesterol absorption and improve lipid metabolism in broilers and layers (Kalavathy et al., 2003).

c) Effect on health and immunity

Probiotics enhance poultry health and immunity by modulating gut microbiota and immune signaling. Lactic acid bacteria (LAB) regulate pro- and anti-inflammatory cytokines (IL-1β, IL-6, IL-10, IFN-γ, TNF-α) and suppress inflammation through NF-κB and TLR-mediated pathways. Supplementation with Clostridium butyricum, Lactobacillus spp., and Saccharomyces cerevisiae enhances gut flora, T-cell responses, intraepithelial lymphocyte activity, and mucosal immunity in broilers (Yang et al., 2012).

Fig. 3. Positive effects of probiotics on poultry

Table 1. Few studies showing the potentials of probiotics in poultry.

S. No.	Probiotic species / formulation	Key findings	Reference
1	Lactobacillus spp.	Reduced Clostridium perfringens load in chicken gut	Gerard et al., 2008
2	Lactobacillus spp.	Reduced E. coli and improved gut health	Grilli et al., 2009
3	Lactobacillus spp.	Improved growth performance and intestinal morphology	De Cesare et al., 2020
4	Pediococcus acidilactici	Improved egg quality and reduced yolk cholesterol	Mikulski et al., 2012
5	Bacillus subtilis	Improved egg quality and reduced heat-stress effects	Sobczak & Kozłowski, 2015; Abdelqader, 2020
6	Lactobacillus salivarius	Improved BWG and FCR in broilers	Shokryazdan et al., 2017a, 2017b
7	Lactobacillus plantarum	Improved gut integrity and immune response	Wang et al., 2018
8	L. plantarum + P. polymyxa	Reduced ALT, AST, LDL, and uric acid	Wu et al., 2019
9	Lactobacillus spp.	Reduced serum cholesterol and triglycerides	Fathi, 2013
10	L. sporogenes	Improved serum protein and reduced lipid profile	Arun et al., 2007
11	Lactobacillus acidophilus	Improved gut health and metabolic functions	De Cesare et al., 2020
12	Enterococcus faecium	Reduced cholesterol and improved immunity	Capcarova et al., 2010
13	Lactobacillus salivarius	Reduced cholesterol and LDL	Shokryazdan et al., 2017a, 2017b
14	Multi-strain probiotics	Improved gut health and performance	Taherpour et al., 2009
15	Lactobacillus spp.	Improved nutrient metabolism and absorption	Burgain et al., 2014
16	Lactobacillus spp.	Reduced inflammatory cytokines	Chen et al., 2012; Park et al., 2014
17	Clostridium butyricum	Enhanced gut flora and immune response	Yang et al., 2012
18	Lactobacillus fermentum	Improved T-cell response	Bai et al., 2013
19	L. plantarum + L. johnsonii	Suppressed NF-κB activation	Joo et al., 2011; Li et al., 2015
20	Lactobacillus spp.	Reduced IL-1β and enhanced TLR4 expression	Li et al., 2015
21	Lactococcus lactis	Induced pro-inflammatory immune signaling	Slawinska et al., 2021
22	Lactobacillus casei	Increased IELs and mucosal immunity	Tian et al., 2021
23	Multi-strain probiotics	Improved growth and gut microbiota	Lambo et al., 2021
24	Bacillus subtilis	Reduced C. perfringens and necrotic enteritis	Jayaraman et al., 2013
25	Bifidobacterium spp.	Enhanced immune response	Knapp et al., 2010
26	Yeast (Saccharomyces cerevisiae)	Improved gut health and immunity	Novak & Vetvicka, 2008
27	Saccharomyces boulardii	Improved intestinal health	Rajput et al., 2013
28	Enterococcus faecium	Reduced C. perfringens and improved immunity	Cao et al., 2013
29	Enterococcus faecium	In vitro anti-C. perfringens activity	Shin et al., 2008
30	Enterococcus faecium	Reduced coccidiosis severity	El-Sawah et al., 2020
31	Enterococcus faecium	Improved nutrient utilization and metabolism	Zheng et al., 2016

Table 2. Impact of probiotics on chicken production

Probiotic strain	Host age (condition)	Chicken type	Dosage	Duration	Results	Reference
Lactobacillus rhamnosus	70 weeks	Layers	1 × 10⁹ CFU	88 weeks	↑ Egg production	Huang et al., 2023
Bacillus subtilis	Day-old chicks	Broiler	500 mg/kg	42 weeks	↓ mortality, ↑ ADFI and BW, → FCR and ADG	Qiu et al., 2021
Bacillus amyloliquefaciens	Day-old chicks	Broiler	10⁶ CFU/g	42 days	↑ Body weight and average daily weight gain	Mazanko et al., 2022
Pediococcus acidilactici + Lactobacillus plantarum	Day-old chicks	Broiler	2 × 10⁹ CFU/chick	42 days	↑ Feed conversion and production efficiency, ↓ myopathies	Paz et al., 2019
Lactobacillus acidophilus, L. plantarum + Bifidobacterium spp.	Day-old chicks	Broiler	1.2 × 10⁹ CFU unit/mL	21 days	↑ BW, FCR, feed consumption (starter), ↑ breast muscles, liver, heart, kidneys, lungs	Agustono et al., 2022
Lactobacillus casei, L. acidophilus + Bifidobacterium	Day-old chicks	Broiler	10 mL probiotics/L water (oral)	42 days	↑ Eviscerated yield and breast yield, ↓ ADFI, FCR, abdominal fat	Zhang et al., 2021
Bacillus subtilis	Day-old chicks	Broiler	0.1% probiotics	42 days	↑ BW and ADWG	Popov et al., 2024
Bacillus subtilis	Day-old chicks	Broiler	1.0 × 10⁶ spores/g feed	42 days	↑ Bone mineralization, density, size, wall thickness, and tibia/femur weight	Yan et al., 2018
Bifidobacterium spp. (in ovo)	Day-old chicks	Broiler	2 × 10⁸ CFU	28 days	↑ LBW and DBWG	Abdel-Moneim et al., 2020
Lactobacillus fermentum + Saccharomyces cerevisiae	Day-old chicks	Broiler	1 × 10⁷ CFU/g and 2 × 10⁶ CFU/g	42 days	↑ ADG and feed efficiency	Bai et al., 2013
Lactobacillus johnsonii	Day-old chicks	Broiler	1 × 10⁵ CFU/g BS15/g	42 days	No changes	Wang et al., 2018a
Lactobacillus johnsonii	Day-old chicks	Broiler	1 × 10⁶ CFU/g BS15	28 days	↑ Starter, finisher, and overall daily weight gain	Wang et al., 2018a
Bacillus subtilis	25 weeks	Layers	9.0 × 10⁵ CFU/g	122 days	↑ Hatchability, fertility, egg weight, yolk colour/index, eggshell thickness	Liu et al., 2019
Bacillus subtilis	28 weeks	Layers	1.0 × 10⁵, 1.0 × 10⁶, 1.0 × 10⁷, 1.0 × 10⁸ (B4) CFU/g	24 weeks	No significant effect on egg production	Guo et al., 2017
Bacillus subtilis	32 weeks	Layers	4 × 10⁹ CFU/g	90 days	No significant effect on ABWG and FCR	Fathi et al., 2018

8.Probios

Probios contains nine different species of beneficial microflora, each at a concentration of 2 × 10⁸ CFU. These include Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus plantarum, Streptococcus faecium, and Streptococcus thermophilus, along with beneficial yeasts such as Torulopsis spp. and Aspergillus spp.

a) Mechanism of action Probios :

Production of lactic acid in the gut which reduce the pH and provides unfavorable conditions for pathogenic bacteria. Probios help in production of antibacterial compounds like lysozyme, lactoferrin, lactoperoxidase and bacteriocins. These compounds are bacteriostatic and bacteriocidal in nature. It reduces toxin production by suppressing the growth and colonization of E. coli. It increases Immunostimulation by increasing macrophage and lymphocyte activity. It rigorously competes with intestinal harmful microbes by competitive exclusion mechanism which help to restrict detrimental colonization to bind with receptors in the mucus layer. It helps in barrier function by improving mucin glycoproteins secretion through mucus producing cells to yield a dense mucus layer that helps to decrease intracellular permeability to pathogens.

b) Charecteristic Of Probios

c) Benefits of Probios

1. Minimize different kind of stress such as debeaking, vaccination and summer stress.
2. Helps to maintain healthy gastrointestinal tract after antibiotic therapy.
3. Reduce the incidents of chick mortality.
4. Quicker detoxification of mycotoxins.
5. Improves protein and fat synthesis.
6. Improves enzymatic activity.
7. Improves weight gain and FCR in broilers.
8. Improves egg production, egg quality and shell quality in layers and breeders.
9. Improves litter condition.
10. Rapidly absorbed from the intestines to provide quick result.
11. Very effective for mixed and gastro‑intestinal tract infections.

d) Comparision between Probios and common probiotic

Probios	Common Probiotic
Made from direct fed microbial (DFM)	Made from spores
Contains 9 strains of microflora i.e. multistrain	Contains 2 to 3 species of microflora
Curdling of milk is observed when a teaspoon of Probios is added to milk, kept overnight	No curdling of milk is observed
High concentration of viable cells	Low concentration of viable cells
More viable in GI tract	Less viable in GI tract
Withstands pelletization temperature	Does not withstand pelletization temperature
Longer shelf life	Shorter shelf life

9.Conclusion:

Probios,  has demonstrated significant positive effects on poultry health and productivity. Its supplementation enhances gut enzyme activity, protein and fat metabolism, feed efficiency, fiber digestion, and organic phosphorus utilization, leading to improved body weight gain and feed conversion ratio in broilers. Probios also supports better litter conditions, reduces stress and mortality, promotes the development of intestinal mucous glands and villi, and maintains a healthy gastrointestinal tract. In layers and breeders, it improves egg production, egg quality, and shell strength. Additionally, Probios contributes to mycotoxin detoxification without causing adverse effects, making it a safe and effective probiotic solution for sustainable poultry production.

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Dr. Amit V. Janbandhu & Dr. Sanjay Singhal

1. Introduction

Global demand for poultry meat and eggs is rising with population growth, intensifying production challenges, particularly feed contamination by mycotoxins (Mottet and Tempio, 2017). Corn, which constitutes approximately 65% of poultry diets in the United States, is highly susceptible to fungal growth and mycotoxin formation. These toxic secondary metabolites are commonly detected in crops, feed, and food commodities at both pre‑ and post‑harvest stages (Choudhary and Kumari, 2010).

Recent surveys confirm the widespread presence of mycotoxins in poultry feed. The 2023 dsm‑firmenich survey reported contamination in 88% of U.S. corn and corn by‑products, with 92% of finished poultry diets containing multiple mycotoxins (dsm‑firmenich, 2023). In the Midwestern United States, fumonisins (FUM), deoxynivalenol (DON), zearalenone (ZEN), and aflatoxins (AF) account for over 95% of mycotoxicosis cases (Weaver et al., 2021). Globally, fumonisin, aflatoxin, ochratoxin, DON, and ZEN are the most frequently detected mycotoxins, influenced by climatic conditions such as temperature, humidity, and drought (Greco et al., 2014; Gruber‑Dorninger et al., 2019).

Mycotoxin exposure reduces feed intake and nutrient utilization, increases susceptibility to enteric pathogens, and causes economic losses estimated at USD 0.5–1.5 billion annually (Agboola et al., 2015; Desjardins et al., 1992). Mycotoxin binders (MTB) mitigate these effects by adsorbing toxins in the gastrointestinal tract, are GRAS‑classified by the U.S. FDA, and require in vitro and in vivo validation for efficacy and nutrient safety in the EU (Di Gregorio et al., 2014; Gimeno and Martins, 2007; European Commission, 2006; Barrientos‑Velazquez et al., 2016).

2. Classification of Mycotoxin Binders

Mycotoxin binders are classified by their nature into two major groups: 1) inorganic binders constituted by silicate minerals and activated carbon (AC) binders, and 2) organic binders constituted by yeast cell wall (YCW) or micro-ionized fiber extracted from different plant materials (Figure 1).

Figure 1. A diagram representing the classification of different mycotoxin binders by their source, nature and structural composition.

2.1. Inorganic binders

There is no consensus on the classification of clay binders that is acceptable to different disciplines such as agriculture, environment, or construction applications. Therefore, we report a classification of inorganic binders based on their properties to bind mycotoxins as proposed and updated by Murray (2007).

a) Silicate binders

Silicates are the most abundant elements found on earth crust (Kandel, 2018). Silicate is a mineral combining silicon dioxide (SiO₂ ⁴⁻) with a tetrahedral structure, where the silicon ion is in the center and surrounded by four oxygen atoms. The interaction of the positive silicon charges and negative oxygen charges results in an unbalanced structure. This allows the free oxygen charges to be bound to other silicon ions forming a chain of tetrahedral structures in different combinations, resulting in chains, sheets, rings, and three-dimensional structures. The tetrahedral sheet is the basis of silicate binders where different subgroups of silicate are formed in combination with other mineral ions in bi or three-dimensional structures. The two main subclasses of silicates are phyllosilicate (sheets of silicate) or tectosilicate (framework silicate, Figure 2).

Figure 2. Molecular structure of octahedral and tetrahedral sheets of tectosilicate binders, and an illustration of the contribution of ions to the adsorption mechanism of mycotoxins.

b) Phyllosilicate binders

Phyllosilicates are bidimensional laminar or tubular minerals composed of tetrahedral silicate sheets linked to octahedral sheets of aluminum or magnesium hydroxides [(Al/MgOH)₆]. Charge imbalance within the octahedral layer, compensated by either two Al³⁺ or three Mg²⁺ ions, results in dioctahedral or trioctahedral structures. Based on layer stacking, phyllosilicates are classified as 1:1 types (e.g., kaolinite–serpentinite), consisting of one tetrahedral and one octahedral sheet, and 2:1 types (e.g., smectites), where an octahedral sheet is sandwiched between two tetrahedral sheets.

Isomorphic substitution of Si⁴⁺ or Al³⁺ with lower‑valence cations (e.g., Mg²⁺, Fe²⁺) generates negatively charged layers balanced by exchangeable interlayer cations, conferring swelling behavior and high cation‑exchange capacity essential for mycotoxin adsorption. Smectites, particularly montmorillonite, exhibit high adsorption efficiency, while bentonite, rich in montmorillonite, shows comparable binding properties (Murray, 2007).

c) Tectosilicate binders

Tectosilicate binders are crystalline aluminosilicate minerals, with zeolites as the main constituents. They are formed by three-dimensional assemblies of tetrahedral units linked through shared oxygen atoms, creating cage- or ring-like porous structures. These uniform pores contain exchangeable cations and water molecules, providing adsorption sites where potassium and calcium ions interact with mycotoxins depending on molecular size. Zeolites are classified based on crystal structure, chemical composition, cation type, pore size, and structural stability. Clinoptilolite is the most widely used zeolite due to its high resistance to low pH and elevated temperatures, functioning as a molecular sieve with pore sizes of approximately 3–8 Å. Thermal treatment or cation enrichment can further enhance its adsorption capacity (Eseceli et al., 2017).

d) Activated carbon

Activated carbon (AC) is an insoluble carbonaceous powder produced by pyrolysis of organic materials such as wood, bamboo, or coal at temperatures up to 2000 °C. An activation process is required to enhance its adsorption capacity by developing a highly porous structure. Chemical activation involves impregnation with agents such as KOH, H₃PO₄, or ZnCl₂ followed by heating, but often results in impurities and environmentally harmful residues. Physical activation uses oxidation with oxygen or CO₂ at 600–900 °C, producing highly microporous carbon with a large surface area (500–3000 m²/g). Adsorption efficiency is directly related to pore availability, with AC sources showing variable mycotoxin-binding capacity (Galvano et al., 1997).

Figure 3. Structure of macro and micropores of activated carbon for the adsoprtion of mycotoxins and other nutrients.

2.2. Organic binders

a) Yeast Cell Wall (YCW)

The yeast cell wall (15–30% of yeast dry weight) is the main component responsible for mycotoxin adsorption. It consists of an inner layer rich in β-(1,3)- and β-(1,6)-D-glucans (50–60%), which provide structural rigidity and binding sites, linked to the membrane by chitin. Excess chitin reduces flexibility and mycotoxin affinity. The outer layer (≈40%) is composed of glucomannans and mannoproteins that determine surface properties. Mycotoxin-binding capacity increases with higher β-D-glucan content in the yeast strain (Jouany et al., 2005).

Figure 4. The composition of different yeast cell wall sheets and their components (adapted from Talavera et al., 2013).

b) Micro-ionized fiber
Micro‑ionized fibers are emerging mycotoxin binders (MTB) capable of adsorbing a wide range of mycotoxins. Various plant‑derived biomaterials, including grape pomace and stem, olive pomace, alfalfa hay, and wheat straw, have shown binding efficiencies ranging from 27 to 90%, depending on the material and mycotoxin type. Their adsorption relies on physico‑chemical interactions between mycotoxins and fiber components such as lignin, cellulose, and polyphenols, similar to silicate or activated carbon binders. However, high inclusion rates (≈20 kg/t) limit their use in monogastric diets, while ruminant diets may better tolerate them (Čolović et al., 2019).

3. Adsorption Mechanism of Different Binders

3.1. Mycotoxin binder properties

a) Silicate binders (clays and zeolites)

Silicate binders adsorb mycotoxins mainly through cation exchange capacity (CEC) and surface charge interactions, which are strongly influenced by pH and point of zero charge (PZC). At low pH, protonation reduces adsorption, whereas higher pH exposes negative charges that facilitate binding of cations interacting with mycotoxin carbonyl groups via weak ion‑dipole and Van der Waals force. Interlayer spacing is critical; sodium bentonite shows greater aflatoxin adsorption than calcium bentonite, while zeolites are limited by smaller pore size. Structural and organic modifications further enhance adsorption efficiency (Jaynes & Zartman, 2011).

b) Activated carbon (AC)

Activated carbon adsorbs mycotoxins primarily via hydrophobic interactions and π‑bonding, showing greater affinity for non‑polar toxins. Activation processes increase surface oxygen‑containing functional groups, enhancing polarity and enabling adsorption of polar mycotoxins such as aflatoxins and fumonisins. Adsorption efficiency is determined by surface area and pore size distribution, which must match mycotoxin molecular dimensions (Goto et al., 2015).  The adsorption efficiency of activated carbon (AC) is strongly influenced by pore size and pore size distribution.

 AC pores are classified into three categories: micropores (<2 nm), mesopores (2–50 nm), and macropores (>50 nm). Micropores contribute most to surface area and adsorption capacity, while meso- and macropores facilitate diffusion. If the pore size is not compatible with the molecular size of mycotoxins, diffusion into the pores is restricted. Limited accessibility to the internal pore surface reduces overall adsorption efficiency of AC.

c) Yeast cell wall (YCW)

Yeast cell wall binders act mainly through β‑ (1,3)‑D‑glucans, which interact with mycotoxins via Van der Waals forces between aromatic rings and glucan structures, as well as hydrogen bonding with hydroxyl, ketone, and lactone groups. Three‑dimensional conformational compatibility between the mycotoxin and glucan helices enhances complex stability and binding strength (Yiannikouris et al., 2004).

4. Mycotoxin properties

Physico-chemical characteristics of mycotoxins significantly influence the adsorption capacity of MTB (Galvano et al., 1997).

Classification of mycotoxins can be based on:

• Polarity
• Solubility
• Chemical structure (Figure 5)

a) Polarity

Polarity reflects the charge distribution within a mycotoxin molecule. Mycotoxins can be classified as polar, non-polar, or intermediate. Aflatoxins (AF) and fumonisins (FUM) are the most polar mycotoxins. Zearalenone (ZEA) is non-polar. Deoxynivalenol (DON), T-2 toxin, and ochratoxin A (OTA) exhibit intermediate polarity.

b) Solubility

Solubility of mycotoxins in the surrounding medium is crucial for effective adsorption. Most mycotoxins are soluble in organic solvents such as methanol, acetonitrile, and acetone. Water solubility depends on polarity: More polar mycotoxins are generally more soluble in water.

c) Chemical structure, size, and shape

These structural features strongly affect the adsorption efficiency of mycotoxins. Aflatoxins (AF) are small, flat molecules, allowing easy entry into the interlayer spaces of binders, leading to higher adsorption. Fumonisins (FUM) have a large, branched molecular structure, which restricts their access to the interlayer space of MTB, resulting in reduced adsorption (Galvano et al., 1996).

Figure 5. Chemical structure of the major mycotoxins and their molecular weight.

Table 1. Different types of Mycotoxins

Name	Source	Types / Characteristics	Target organ	Harmful effects
Aflatoxin	Aspergillus flavus, Aspergillus parasiticus	Naturally occurring aflatoxins include B1, B2, G1, and G2; among these, B1 is found in the highest concentration and is the most toxic	Liver, Kidney, Spleen, Testes, Thymus	Loss of egg production, anaemia, haemorrhage, liver damage, paralysis, poor FCR, immunosuppression
Ochratoxin	Aspergillus ochraceus, Penicillium verridicatum	Four types: A, B, C, and D; Ochratoxin A is the most toxic	Kidney, Liver, Thymus	Gout, immunosuppression, reduced weight gain, reduced egg production and egg weight
Trichothecene (T-2 toxin)	Fusarium spp.	—	—	Reduced feed intake, decreased growth, immunosuppression
Citrinin	Penicillium spp.	—	Kidney	Wet droppings, reduced weight gain
Oosporein	—	—	Kidney	Gout, high mortality
Moniliformin	Fusarium moniliforme	—	—	Reduced feed intake and body weight; in chicks causes heart damage and death; in layers reduces rate of laying and delays peak production
Fumonisins	Fusarium moniliforme	—	—	More harmful to pigs and horses; poultry are more resistant
Zearalenone	—	—	—	—
Ergotism	—	—	—	Reduced feed intake and growth; death of tissues of beak, comb, and toes; diarrhoea; vesicles and crusts on comb and wattle; reduced egg production
Fusarochromanone	—	—	—	Leg deformities in chicks

Table 2. Summary of studies that determine the capacity of different mycotoxin binders to adsorb nutrients

Binder	Nutrient interaction effects	Observation	Reference
Bentonite	High adsorption of vitamins E, B1, B2, and B6, and amino acids (lysine, methionine, threonine); low adsorption of vitamins A, D, and B3	In vitro simulation of gastrointestinal tract	Kihal et al. (2020, 2021)
	High adsorption of vitamin B1 and pepsin; no adsorption of vitamins D and E	In vitro gastric fluid simulation	Barrientos-Velázquez et al. (2016)
	High adsorption of vitamins B12 and B8; no adsorption of vitamin B5	In vitro gastric fluid simulation and real gastric fluid	Vekiru et al. (2007)
	High adsorption of vitamin B6; adsorption of Zn and Co; no adsorption of Cu and Mn	In vitro aqueous solution	Tomasevic-Canovic et al. (2000)
	Adsorption of vitamin B2	In vitro aqueous solution	Mortland and Lawless (1983)
	No adsorption of vitamin A	In vivo in chicks	Pimpukdee et al. (2004)
	No adsorption of vitamin A	In vivo in chicks	Afriyie-Gyawu (2004)
MMT	High adsorption of vitamins E, B1, B2, and B6, and amino acids (lysine, methionine, threonine); low adsorption of vitamins A, D, and B3	In vitro simulation of gastrointestinal tract	Kihal et al. (2020, 2021)
	Adsorption of vitamin B1	In vitro gastric fluid simulation	Ghanshyam et al. (2009)
	Adsorption of protein, urea, and antibiotics	In vitro agar culture	Pinc (1962)
	No adsorption of vitamins A, D, E, B1, and B6	In vivo in dairy cows	Kihal et al. (2022)
	No adsorption of vitamins A and B1	In vivo in dairy cows	Maki et al. (2016)
Ca-MMT	High adsorption of vitamins E, B1, B2, and B6, and amino acids (lysine, methionine, threonine); low adsorption of vitamins A, D, and B3	In vitro simulation of gastrointestinal tract	Kihal et al. (2020, 2021)
	Adsorption of vitamins B8 and B12	In vitro simulation of gastric fluid and real gastric fluid	Vekiru et al. (2007)
Clinoptilolite	High adsorption of vitamins E, B1, B2, and B6, and amino acids (lysine, methionine, threonine)	In vitro simulation of gastrointestinal tract	Kihal et al. (2020, 2021)
	No adsorption of vitamins A, D, and B3; no adsorption of amino acids (tryptophan, phenylalanine)	In vitro aqueous solution	Tomasevic-Canovic et al. (2000)
HSCAS	No adsorption of vitamins A, B1, and minerals (Zn, Mn)	In vivo in chicks	Chung et al. (1990)
Sepiolite	High adsorption of vitamins E, B1, B2, and B6, and amino acids (lysine, methionine, threonine); low adsorption of vitamins A, D, and B3	In vitro simulation of gastrointestinal tract	Kihal et al. (2020, 2021)
Zeolite	High adsorption of vitamins E, B1, B2, and B6, and amino acids (lysine, methionine, threonine); low adsorption of vitamins A, D, and B3	In vitro simulation of gastrointestinal tract	Kihal et al. (2020, 2021)

¹MMT, montmorillonite.
²AC, activated carbon.
³HSCAS, hydrated sodium calcium aluminosilicate.

Table 3. Regulatory guidance levels for major mycotoxins in finished poultry feed as established by the European Union (EU) and United States Food and Drug Administration (FDA). Values are expressed in mg/kg diet. EU limits in finished feed set according to the European Commission Recommendation 2006/576/EC and the European Commission Directive 2003/100/EC; USA limits in finished feed set according to the Food and Drug Administration regulatory guidance for toxins and contaminants.

Guidance	Deoxynivalenol (DON) (mg/kg)	Fumonisins (FUM) (mg/kg)	Zearalenone (ZEA) (mg/kg)	Aflatoxin (AF) (mg/kg)	Ochratoxin A (OTA) (mg/kg)	T-2 Toxin (mg/kg)
EU limits (EC guidance) ¹	5	20	–	0.02	0.1	No guidance level established
USA limits (FDA guidance) ²	5	50	No guidance level established	0.1	No guidance level established	No guidance level established

Table 4. Ranges of ratios of mycotoxin binder to mycotoxins doses used in in vitro tests to determine the mycotoxin adsorption capacity of different mycotoxin binders

Binders	AFB1 (mg: µg)	DON (mg: µg)	FUM (mg: µg)	OTA (mg: µg)	T-2 (mg: µg)	ZEA (mg: µg)
AC	1:0.008–1:461	1:0.2–1:90	1:0.92–1:25	1:0.025–1:125	1:0.1	1:0.05–1:20
Bentonite	1:0.0007–1:200	1:0.2–1:12	1:2–1:20	1:0.002–1:12.5	1:0.1–1:0.2	1:0.1–1:20
Clinoptilolite	1:0.2–1:40	1:1.2	–	1:2	–	1:0.05–1:12
HSCAS	1:0.002–1:600	1:0.4–1:12	1:2–1:20	1:0.025–1:10	1:0.1	1:0.001–1:20
MMT	1:0.002–1:20	1:0.5–1:12	1:2.5	1:0.025	1:0.1	1:0.05–1
Sepiolite	1:1.6–1:10	1:2–1:12	1:2	1:10	–	1:0.5
YCW	1:0.001–1:461	1:0.2–1:12	1:2–1:20	1:0.001–1:10	1:0.1	1:0.001–1:20
Zeolite	1:0.002–1:20	1:0.2–1:10	1:0.2–1:20	1:0.016–1:5	1:0.2	1:0.05–1:20

¹AC, activated carbon; HSCAS, hydrated sodium calcium aluminosilicate; MMT, montmorillonite; YCW, yeast cell wall. ²AFB1, aflatoxin B1; DON, deoxynivalenol; FUM, Fumonisin; OTA, ochratoxin; T-2, T-2 toxin; ZEA, zearalenone.

Figure 6. A conceptual framework for the effect of mycotoxin exposure on growth retardation (Smith et.al.2012).

Fig. 7. Gut microbiota and mycotoxins interactions. Illustration of the bidirectional interactions between gut microbiota and dietary mycotoxins in poultry. Mycotoxins can disrupt microbial community structure, reduce beneficial populations, and impair microbially derived functions, including short-chain fatty acid (SCFA) production and mucosal barrier maintenance. Conversely, the gut microbiota can biotransform certain mycotoxins into less toxic metabolites or modulate host responses to exposure. Disruption of this balance may compromise gut integrity, immunity, and overall performance.

Figure 8. Diagrammatic representation for postharvest mycotoxin mitigation strategies in broiler and layer chickens’ production.

Stallen offers a comprehensive range of world‑class mycotoxin binders, including D‑Tox and Alusil MOS Plus, both of which provide broad‑spectrum protection against diverse mycotoxins commonly found in poultry feed.

5.2. The characteristic features of D-Tox compare with other common toxin binder.

1. Binding efficacy of D-Tox to various mycotoxins

2.  Features of D-TOX

5.3. D-Tox Benefits:

D‑Tox provides effective and comprehensive control of all major categories of mycotoxins, thereby improving performance and productivity in poultry.

6. Alusil MOS Plus

Alusil MOS Plus contains HSCAS (Activated Hydrated Sodium Calcium Aluminosilicates), activated charcoal, MOS (Mannan Oligosaccharides), copper oxinate, organic acids (propionic, benzoic, acetic, and sorbic acids), lipotropic agents, and spirulina.

6.1. Mechanism of action of Alusil MOS Plus

HSCAS acts as an enterosorbent that tightly and selectively binds aflatoxins in the GI tract of animals decreasing their bioavailability and associated toxicity. Mannan Oligosaccharides act as bio-binder which helps absorption of pathogens, improves intestinal function & immune modulation. Organic acids kill harmful bacteria and fungi. Activated charcoal which is 200 MT grade helps to bind pesticides and toxins like ochratoxin. Copper oxinate is broad spectrum anti-fungal agent which acts against spp. of Aspergillus, Fusarium, Penicillium, Candida etc. Lipotropic agent and herbal ingredients help in mobilizing fat which are accumulated in liver due to damage caused by toxins. Spirulina helps in restoring the liver damaged by toxins.

6.2. The characteristic features of Alusil MOS Plus over other toxin binder.

Component	Function
HSCAS	Enters bloodstream and binds to aflatoxins.
Activated Charcoal	Binds multiple mycotoxins including aflatoxins, ochratoxins A, zearalenone, and along with bacterial endotoxins & pesticides in the intestinal tract, preventing their reabsorption.
MOS	Binds to pathogenic bacterial fimbriae, increases mucin secretion & acts as a bactericidal agent.
Copper Oxinate	Acts as antifungal agent by inhibiting mould growth.
Organic Acids	Acetic, benzoic, propionic & sorbic acids inhibit fungal growth and reduce aflatoxin secretion.
Lipotropic Agent	Aids in mobilising fat accumulated in liver due to toxin damage.
Spirulina	Helps restore liver function damaged by toxins.

6.3. Alusil MOS Plus Benefits:

Alusil MOS Plus acts as a broad-spectrum mould inhibitor and supports bio-neutralization of mycotoxins, helping prevent toxin-related damage and overcome clinical symptoms of aflatoxicosis. It protects the immune system, enhances vaccine and drug response, improves pellet quality, functions as an anti-caking agent in feed, and does not bind essential vitamins and minerals.

7. Conclusion

Stallen South Asia Pvt. Ltd. offers effective toxin binders, D-tox and Alusil MOS Plus, which are helpful in the detoxification of mycotoxins in poultry. D-tox provides broad‑spectrum control of both polar and non‑polar mycotoxins through pH‑stable adsorption and pre‑absorptive detoxification, without binding amino acids, fat‑soluble vitamins, minerals, or micronutrients. It also sequesters heavy metals, endotoxins, and biogenic amines, reducing immunosuppression. Alusil MOS Plus alleviates aflatoxicosis, protects immune function, enhances vaccine and drug response, and improves feed quality through anticaking and pellet‑stabilizing effects.

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18th February 2026, Patna, Bihar.

To address the evolving disease landscape in poultry production, Stallen South Asia Pvt. Ltd. recently organized a focused technical seminar in Patna, Bihar, attended by 30 broiler breeder and layer farmers from the region.

The keynote session was delivered by Dr. Sushil Dhariwal, who spoke on “Emerging Challenges in IB, Mycoplasma and Marek’s Control in Poultry Production.” He highlighted the increasing complexity of Infectious Bronchitis due to variant strains, the persistent economic impact of Mycoplasma (MG & MS) infections, and the growing importance of strong early immunization strategies against Marek’s disease. Emphasis was placed on structured vaccination programs supported by monitoring, biosecurity, and scientific field evaluation.

Following his keynote address, farmers actively participated in an engaging and practical Q&A session with Dr. Dhariwal. The discussion covered field-level challenges such as fluctuating ELISA titres during lay, nephropathogenic IB concerns, Mycoplasma persistence in breeder flocks, and early-age Marek’s-related mortality patterns. The interactive exchange reflected the keen interest of producers in strengthening preventive health strategies through scientific understanding.

The program commenced with a welcome address by Mr. Biplab Deb, Regional Manager – East. An overview of the company’s philosophy and legacy was presented by Dr. Sanjay Singhal, COO, who spoke about the origin and growth of Stallen, its evolution as a science-driven animal health organization, and its unwavering commitment to quality and ethical practices. He underlined that the company’s foundation is built on technical excellence, stringent quality standards, and long-term partnerships with poultry producers. Dr. Singhal also highlighted that Stallen imports high-quality live and killed vaccines in collaboration with Fatro S.p.A., Italy, ensuring access to internationally benchmarked vaccine technology for the Indian poultry sector. The portfolio discussed during the seminar included vaccines for Marek’s disease, Mycoplasma (MG & MS), and IB–ND combinations, with emphasis on their strategic integration into breeder and layer vaccination programs.

The seminar concluded with a vote of thanks by Mr. Mmukesh Singh, Area Manager, acknowledging the enthusiastic participation of farmers and the valuable scientific insights shared during the session.

Such knowledge-sharing initiatives continue to play a crucial role in reinforcing disease preparedness and promoting sustainable productivity in Eastern India’s poultry industry.

Dr. Amit Janbandhu, Dr. Sanjay Singhal

1. Introduction

Necrotic enteritis (NE) is a significant burden on the poultry industry, causing gut damage that reduces nutrient utilization and productivity. Production losses due to NE and current control methods are estimated to cost the global broiler industry approximately USD 6 billion annually (Wade and Keyburn, 2015). These losses are associated with decreased production performance, mortality of up to 1% per day, treatment costs, and carcass condemnation due to cholangiohepatitis (Immerseel,et.al, 2004 & Timbermont, et.al,2011). Clinical and subclinical forms of NE have been recognized (Van Immerseel et al., 2004); the clinical form results in acute disease and bird mortality. Although flock health and productivity can be maintained using IFA-free practices (Parent et al., 2020), strong industry moves away from IFAs have increased the need for alternative approaches. Consequently, a wide range of feed additives and treatments have been investigated and commercialized to control NE.

NE is caused by Clostridium perfringens and primarily affects chickens from 2 weeks to 6 months of age. In humans, C. perfringens intoxications are the third most common bacterial foodborne disease after Salmonella and Campylobacter, with 359–2173 cases reported annually in the United States. Poultry and poultry products account for 30% of outbreaks, and 92% are traced to meat and poultry as a single identified food commodity (Grass et.al, 2013). Research into antibiotic alternatives that improve gut health and immune status has intensified; however, current alternatives are less effective than antibiotics in controlling NE. A greater understanding of C. perfringens virulence factors, NE pathogenesis, and host responses is required to develop effective control strategies and new supplements, as these aspects are not yet fully understood and remain under investigation.

2. Necrotic Enteritis

Etiology: Necrotic enteritis is caused by Clostridium perfringens, a Gram-positive, rod-shaped, anaerobic bacterium that forms oval subterminal spores. Unlike most clostridia, C. perfringens consists of relatively large, encapsulated, non-motile rods (0.6–2.4 × 1.3–9.0 µm). Colonies are smooth, round, and glistening, with an inner zone of complete hemolysis mediated by theta-toxin and an outer zone of incomplete hemolysis caused by alpha-toxin (Cato et al., 2002).

Clostridium perfringens is classified into five biotypes (A–E) based on the production of four major lethal toxins: alpha, beta, epsilon, and iota. In addition, enterotoxin (CPE) and beta2 (CPB2) toxins are considered important in enteric diseases; however, their roles in avian C. perfringens–associated enteric disease remain unclear (Crespo et al., 2007). All five types produce toxins: type A (α), type B (α, β, ε), type C (α, β), type D (α, ε), and type E (α, ι). Necrotic enteritis is primarily associated with C. perfringens types A and C caused by α and net B toxins (Fisher et al,2005)., with infections in poultry mainly caused by type A and, to a lesser extent, type C. Because type A is highly prevalent in the intestines of healthy birds, its pathogenic role remains controversial (Smedley et.al,2004). Moreover, strains isolated from NE outbreaks have not been shown to produce higher levels of alpha toxin than isolates from clinically healthy broilers.

Fig 1. Microscopic appearance of Clostridium Perfringes

Table 1: The most important C. perfringens toxins

Toxin	Gene location	Biological activity
Alpha toxin	Chromosome	Cytolytic, haemolytic, dermonecrotic, Lethal
Beta toxin	Plasmid	Cytolytic, dermonecrotic, lethal
Epsilon toxin	Plasmid	Oedema in various organs: liver, kidney and central nervous system
Iota toxin	Plasmid	Disruption of actin cytoskeleton and cell barrier integrity
Beta2 toxin	Plasmid	Cytolytic, lethal
Enterotoxin	Chromosome/ Plasmid	Cytotoxic, lethal, causes diarrhea by leakage of water and ions
Theta toxin	Chromosome	Lyses red blood cells and modulates the host inflammatory response

(Source: Wise and Siragusa et.al, 2005)

3. Epidemiology

3.1. Source of Infection and Transmission: Clostridium perfringens is a naturally occurring bacterium in poultry production environments. It is present in dust, soil, feces, feed, poultry litter, eggshell fragments, fluff, and the intestinal tract of poultry. Feces of wild birds may also contain elevated numbers of C. perfringens, further introducing the organism into poultry facilities. Environmental sampling on poultry farms detected C. perfringens on wall swabs (53%), fan swabs (46%), fly strips (43%), dirt outside entrances (43%), and boot swabs (29%), demonstrating its ubiquitous environmental presence (Craven et.al,2001). Transmission occurs primarily via the fecal–oral route and through contaminated feed, water, housing structures, insects, and direct contact between infected and susceptible birds. During necrotic enteritis (NE) outbreaks, contaminated feed or litter are considered major sources, and contaminated feed components have also been implicated. Vertical transmission is possible, as C. perfringens has been found in the yolk sac of embryonated eggs, suggesting transmission within integrated broiler operations (Craven et.al, 2003). Additionally, C. perfringens may be transmitted mechanically and/or biologically by house flies in poultry houses, contributing to NE development (Dhillon et.al,2004).

3.2. Predisposing factors

Necrotic enteritis (NE) develops when one or more predisposing factors are present, particularly intestinal mucosal damage. Damage caused by coccidial pathogens releases growth factors that promote C. perfringens proliferation in the intestinal lumen. Broilers inoculated with Eimeria spp. and fed C. perfringens–contaminated feed show higher mortality than birds fed contaminated feed alone. Physical damage from litter eating or fibrous diets may also alter the mucosa (Williams et.al,2005).

Management factors such as feeding practices, water supply, temperature control, and ventilation contribute to NE. Delayed initial feeding impairs gut-associated lymphoid tissue development. Nutritional stress from unbalanced diets, especially low energy-to-protein ratios, increases feed intake, nitrogen levels in digesta, and susceptibility to clostridial overgrowth. Diet composition strongly influences NE. Diets rich in wheat, rye, and barley contain indigestible non-starch polysaccharides that increase digesta viscosity, slow gut transit, and favor anaerobic bacteria. Broilers fed wheat-, rye-, or barley-based contaminated diets have higher mortality than those fed corn-based diets. Pelleted diets reduce intestinal C. perfringens, whereas high-protein diets (e.g., fishmeal) and bone meal increase NE risk (Kocher et.al,2003).

Immunosuppression increases NE susceptibility. Use of Infectious bursal disease (IBD) vaccines has been associated with increased NE lesion severity, even at normal doses. Stressful conditions may further predispose birds to NE, but immunosuppression is inappropriate when evaluating vaccines (Nikpiran et.al,2008).

4. Clinical Signs and Lesions

4.1. Clinical signs:
Subclinical necrotic enteritis (SNE) shows no obvious clinical signs and is usually detected under field conditions at processing plants through carcass rejection. SNE may be suspected based on reduced weight gain, poor feed conversion efficiency, increased moisture in droppings, and wet litter, most commonly at 2–5 weeks of age without increased mortality.

Clinical necrotic enteritis (NE) typically affects broiler chicks between 2–6 weeks of age and presents with sudden onset of diarrhoea and intestinal mucosal necrosis. Affected birds are depressed, anorectic, have ruffled feathers, and tend to huddle. In advanced stages, birds become laterally recumbent, immobile, and die rapidly. The disease course is usually short, and birds are often found dead without prior clinical signs. Acute signs include severe depression, reduced appetite, reluctance to move, ruffled feathers, and diarrhoea, with illness lasting only 1–2 hours [20]. Mortality in affected flocks may range from 1% to 50%.

Birds that die of NE have a foetid odor, dehydration, dark and dry pectoral muscles, and pale kidneys. The unopened intestine is darker than normal and distended due to bile-stained contents (Long, et.al,2007)

4.2. Gross lesions:
Lesions of SNE are characterized by necrotic lesions in the intestinal wall and liver, occurring in one or more intestinal regions. Mild lesions appear as small ulcers or light-yellow spots on the mucosal surface, mainly in the jejunum and ileum and less commonly in the caeca. Severe lesions may involve membranes covering large intestinal segments, including the colorectal region and caecal tonsils. Liver abnormalities, primarily enlargement and occasional congestion, are also reported.

Clinical NE is characterized by extensive mucosal necrosis of the small intestine, covered with a yellow-brown or bile-stained pseudomembrane. Gross lesions are mainly confined to the small intestine but may also involve the liver and kidneys. At necropsy, the duodenum, jejunum, and ileum are thin-walled, friable, gas-filled, and distended with dark brown fluid. Ulcers may occur singly or in aggregates. In severe cases, a fibrino-necrotic or diphtheritic membrane covers large segments, often involving two-thirds of the jejunum and ileum (McDevitt et.al,2006).

4.3. Histopathological changes:
Microscopically, NE lesions consist of coagulative necrosis at the villous apices with a clear demarcation between necrotic and viable tissue. Degeneration may extend into the submucosa. Regeneration is characterized by epithelial proliferation, connective tissue formation, and reduced goblet, columnar, and epithelial cells, resulting in short, flattened villi with reduced absorptive surface. The pseudomembrane consists of necrotic villi, inflammatory cells, and bacterial aggregates (Olkowski et.al.2008).

Fig. 2. Gross pathological changes in NE: A. Jejunum of a bird showing ballooning. B. Jejunum showing small blackish necrotic spots. C. Jejunum showing Turkish towel appearance. D. Duodenum showing congestion.

5. Pathogenesis

Pathogenesis describes the complex and dynamic host–pathogen interactions at the molecular level and is essential for developing effective control measures. Bacterial pathogenesis involves six overlapping phases: colonization, growth and proliferation, nutrient acquisition, evasion of host defenses, host tissue injury, and transmission. In rapidly growing pathogens such as Clostridium perfringens, these phases occur almost simultaneously.

Colonization requires degradation of the intestinal mucus layer, which normally acts as a physical barrier. Intestinal mucins provide binding sites for bacterial adhesins, and pathogenic C. perfringens secrete bacteriocins (perfrins) that displace commensal Clostridium species. The organism produces glycoside hydrolases and chitinases that degrade mucins, providing nutrients and enabling microcolony formation on the mucosa. Predisposing factors such as Eimeria infection stimulate inflammation, increased mucin production, and release of essential amino acids, all of which enhance C. perfringens growth and colonization (Collier et.al,2008).

Once a threshold density is reached, an Agr-like quorum-sensing system activates the VirS–VirR regulatory system and virulence genes, including NELoc-1 involved in adhesion. Degradation of the mucus layer allows pore-forming toxins to access epithelial cells. Proteolytic and collagenolytic enzymes damage the epithelium, disrupt intercellular junctions, spread through the lamina propria, and cause epithelial necrosis and sloughing. Quorum-sensing–regulated secretion of alpha toxin and perfringolysin promotes biofilm formation on the exposed submucosa, enhancing bacterial persistence and protection from host immunity and antibiotics.

Intestinal integrity depends on tight junctions, particularly claudin-3 and claudin-4, which serve as receptors for C. perfringens enterotoxin (CPE). CPE binding forms small complexes that oligomerize into large CH1 complexes, leading to pore formation in the cell membrane. These pores allow calcium influx, resulting in epithelial cell death (Chakrabarti, et.al,2005).

Gross lesions of necrotic enteritis primarily affect the jejunum and ileum, with occasional involvement of the duodenum and ceca. The intestine is thin, friable, gas-distended, and covered by a tan orange pseudomembrane, producing the characteristic “dirty Turkish towel” appearance. Pseudomembrane formation is most common in the jejunum. Subclinical necrotic enteritis is associated with hepatitis or cholangiohepatitis and gall bladder distension with flocculent material. Bile acids promote sporulation and enterotoxin production by C. perfringens, explaining the higher lesion frequency in the upper small intestine, particularly the duodenum and jejunum (Park et.al,2018).

Fig.3. Pathogenesis of necrotic enteritis in broiler chickens causes destruction of epithelial cells of intestine that leads to blood-stained diarrhea.

6. Feed Additives Used to Control Necrotic Enteritis

A wide range of feed additives has been studied for their effects on necrotic enteritis (NE). Most commercial additives do not target Clostridium perfringens directly but improve gut health, microbiota balance, and immune competence, often with overlapping and interconnected effects (Granstad et al., 2020). Short-chain fatty acids, particularly butyrate, provide energy to enterocytes and support beneficial microbiota. Butyrate can be supplied directly in protected form or indirectly via prebiotics, probiotics, phytobiotics, or postbiotics (Liu et al., 2021). Combinations of additives, such as fatty acids with phytobiotics, are often more effective.

Probiotics, including single or multi-strain bacteria, yeasts, or cultured cecal microbiota, can directly inhibit C. perfringens, compete for gut niches, improve microbiota composition, gut integrity, or immune function. Prebiotics, fatty acids, and phytobiotics have also been reviewed for general health and NE-specific applications (Gomez-Osorio et al., 2021).

Novel approaches include bacteriophages and their endolysins, which specifically target C. perfringens, and bacteriocins produced by bacteria for antimicrobial effects. Passive immunization using egg yolk antibodies or engineered single-chain antibodies has shown potential in reducing NE ( Gangaiah et al., 2022).

Commercial feed supplements such as StalBMD (BMD), Magnox (lincomycin), and Stalgro (enramycin) by Stallen South Asia Pvt. Ltd are also used to prevent NE caused by C. perfringens.

7) Mechanism of Action

7.1) Bacitracin Methylene Disalicylate (BMD)

Bacitracin Methylene Disalicylate (BMD) is a polypeptide antibiotic used to prevent and treat Gram-positive bacterial infections. It inhibits bacterial cell wall synthesis by blocking dephosphorylation of bactoprenol phosphate, preventing peptidoglycan transport. This disrupts cell wall formation, causing bacterial lysis. BMD is bactericidal, particularly against actively dividing Staphylococcus and Streptococcus species.

Fig.4. Bactericidal effect on growing bacteria.

7.2) Lincomycin Hydrochloride

Lincomycin hydrochloride, a lincosamide antibiotic derived from Streptomyces lincolnensis, is used against Gram-positive and some anaerobic bacteria. Its primary mechanism is inhibition of bacterial protein synthesis by binding to the 50S ribosomal subunit, specifically at the peptidyl transferase center. This blocks the ribosomal exit tunnel, preventing elongation of polypeptide chains and halting protein synthesis, which stops bacterial growth and replication. Lincomycin may also have a secondary effect on bacterial cell wall synthesis, enhancing its overall antibacterial activity.

Fig 5. Protein synthesis inhibition within bacterial cells.

7.3. Enramycin

Enramycin acts as an inhibitor of the enzyme (MurG), which is essntial for wall biosynthesis in gram +ve bacteria. MurG catalyzes the tranglycosylation reaction in the last step of peptidoglycan biosynthesis. Hence inhibiting this step greatly compromises cell wall integrity leading to cell lysis.

Figure 6. Membrane steps of the bacterial peptidoglycan synthesis pathway.

References

 Experimental induction of necrotic enteritis inbroiler chickens by Clostridium perfringens isolates from the outbreaks in Iran. Journal of Veterinary Research,2008, 63: 127-132.

 Cato, P., L. George and M. Finegold, 2002. Genus c l o s t r i d i u m . B e r g e y ‘ s m a n u a l o f systematicbacteriology, USA., 2: 1141-1200.

Chakrabarti, G.; McClane, B.A. The importance of calcium influx, calpain and calmodulin for the activation of CaCo-2 cell death pathways by Clostridium perfringens enterotoxin. Cell. Microbiol. 2005, 7, 129–146. [PubMed].

Collier, C.T.; Hofacre, C.L.; Payne, A.M.; Anderson, D.B.; Kaiser, P.; Mackie, R.I.; Gaskins, H.R. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth. Vet. Immunol. Immunopathol. 2008, 122, 104–115. [CrossRef].

Craven, E., A. Cox, S. Bailey and E. Cosby, 2003. Incidence and tracking of Clostridium perfringensthrough an integrated broiler chicken operation.Avian Diseases, 47(3): 707-711.

Craven, E., J. Stern, S. Bailey and A. Cox, 2001. Incidence of Clostridium Perfringens in broiler chickens and their environment during production and Processing. Avian Diseases, 45: 887-896.

Crespo, R., J. Fisher, L. Shivaprasad, E. Fernández Miyakawaand A. Uzal, 2007. Toxinotypes of Clostridium perfringens isolated from sick and healthy avian species. Journal of Veterinary Diagnostic Investigation, 19(3): 329-333.

Dhillon, S., P. Roy, L. Lauerman, D. Schaberg, S.Weber, D. Bandli and F. Wier, 2004. High mortality in egg layers as a result of necrotic enteritis. Avian Diseases, 48(3): 675-680.

Fisher, J., K. Miyamoto, B. Harrison, S. Akimoto, R. Sarker and A. McClane, 2005.Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene. Molecular Microbiology, 56(3): 747-62.

Gangaiah D, Ryan V, Van Hoesel D, Mane SP, Mckinley ET, Lakshmanan N, et al. Recombinant Limosilactobacillus (Lactobacillus) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis. Microbiologyopen 2022;11: e1270. https://doi.org/10.1002/ mbo3.1270.

Gomez-Osorio L-M, Yepes-Medina V, Ballou A, Parini M, Angel R. Short and medium chain fatty acids and their derivatives as a natural strategy in the control of necrotic enteritis and microbial homeostasis in broiler chickens. Front Vet Sci 2021; 8:773372. https://doi.org/10.3389/fvets.2021.773372.

Granstad S, Kristoffersen AB, Benestad SL, Sjurseth SK, David B, Sørensen L, et al. Effect of feed additives as alternatives to in-feed antimicrobials on production performance and intestinal Clostridium perfringens counts in broiler chickens. Animals 2020; 10:240. https://doi.org/10.3390/ani10020240.

 Grass, J.E.; Gould, L.H.; Mahon, B.E. Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens, United States, 1998–2010. Foodborne Pathog. Dis. 2013, 10, 131–136. [CrossRef] [PubMed].

 Immerseel, F.V.; Buck, J.D.; Pasmans, F.; Huyghebaert, G.; Haesebrouck, F.; Ducatelle, R. Clostridium perfringens in poultry: An emerging threat for animal and public health. Avian Pathol. 2004, 33, 537–549. [CrossRef] [PubMed].

Kocher, A., 2003. Nutritional manipulation of necrotic enteritis outbreak in broilers. Recent Advances in Animal Nutrition in Australia,14: 111-116.

Liu L, Li Q, Yang Y, Guo A. Biological function of short-chain fatty acids and its regulation on intestinal health of poultry. Front Vet Sci 2021; 8:736739. https:// doi.org/10.3389/fvets.2021.736739.

Long, R., A. Barnum and R. Pettit, 2007. Necrotic enteritis in broiler chickens. Pathology and proposed pathogenesis. Canadian Journal of Comparative Medicine, 38(4): 467-474.

McDevitt, M., D. Brooker, T. Acamovic and C. Sparks, 2006. Necrotic enteritis; a continuing challenge for the poultry industry. World’s Poultry Science Journal, 62(02): 221-247.

Nikpiran, H., B. Shojadoost and M. Peighambari, Olkowski, A., C. Wojnarowicz, M. Chirino-Trejo,B. Laarveld and G. Sawicki, 2008. Sub-clinical necrotic enteritis in broiler chickens: novel etiological consideration based on ultra- structural and molecular changes in the intestinal tissue. Research in Veterinary Science, 85(3): 543-553.

Parent E, Archambault M, Moore RJ, Boulianne M. Impacts of antibiotic reduction strategies on zootechnical performances, health control, and Eimeria spp. excretion compared with conventional antibiotic programs in commercial broiler chicken flocks. Poult Sci 2020; 99:4303e13. https://doi.org/10.1016/ j.psj.2020.05.037.

Park, M.; Rafii, F. Effects of bile acids and nisin on the production of enterotoxin by Clostridium perfringens in a nutrient-rich medium. Int. J. Microbiol. 2018, 2018, 7276523. [CrossRef].

Smedley Iii, G., J. Fisher, S. Sayeed, G.  and A. McClane, 2004. The enteric toxins of Clostridium perfringens. In Reviews of physiology, biochemistry and pharmacology. Springer Berlin Heidelberg, 152: 183-204.

Timbermont, L.; Haesebrouck, F.; Ducatelle, R.; Van Immerseel, F. Necrotic enteritis in broilers: An updated review on the pathogenesis. Avian Pathol. 2011, 40, 341–347. [CrossRef] [PubMed].

Wade B, Keyburn A. The true cost of necrotic enteritis.World Poultry 2015;31:16e7.

Williams, B., 2005. Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathology, 34(3): 159-180.

Wise, G. and R. Siragusa, 2005. Quantitative Detection of Clostridium perfringens in the Fowl Gastrointestinal Tract by Real-Time PCR. Applied Environmental Microbiology, 71: 3911-3916.

Global Poultry Scenario

Salmonella in poultry is one of the major important reasons of production losses through mortality, morbidity and reduced efficiency, with broilers showing the highest prevalence due to intensive farming. In India, where the poultry sector contributes ₹1.3 lakh crore to GDP and supports millions of livelihoods, Salmonella causes losses of up to ₹25,000 crore annually, particularly affecting small-scale farmers (Dhruv et al., 2025).

Predominant serovars such as S. Gallinarum, S. Enteritidis and S. Typhimurium continue to threaten both flock health and food safety, with contamination documented across farms, processing facilities and retail products. According to a global review, the median prevalence of Salmonella was 40.5% in broiler chickens, 30% in raw chicken meat and 40% in eggs and laying hens. Multidrug resistance was found in 97.8% of processing/market studies and 91.1% of farm studies, indicating the presence of resistant reservoirs throughout the supply chain (Castro‑Vargas et al., 2020).

In India, major serovars include S. Gallinarum and S. Pullorum, which cause poultry diseases, alongside foodborne serovars like S. Enteritidis and S. Typhimurium that pose risks to consumers. These infections result in both direct production losses and broader food safety concerns, threatening India’s export growth and domestic confidence. Considering poultry’s role as the most affordable protein source for India’s population and its growing role in international trade, effective Salmonella control through surveillance, vaccination, biosecurity and processing-stage interventions is essential to protect public health, economic sustainability and food security.

Predisposing Factors and Disease Transmission:

Predisposing factors are the factors which are the loop holes or common ignored mistakes on farm which can be harmful as the birds are in continuous dangers because of common pathogens which are always present around them, therefore proper sanitation and biosecurity measures are advised according to the farm type and season to prevent this kind of factor at every step.

Common predisposing factors are:

• Poor hatchery hygiene – contaminated eggs, incubators and hatchery equipment
• Vertical transmission – infected breeder flocks passing bacteria through the egg
• Environmental contamination – dirty litter, contaminated water and feed
• Rodents, wild birds, insects – act as reservoirs and mechanical carriers
• Overcrowding and poor ventilation – stress increases susceptibility
• Inadequate biosecurity – uncontrolled farm access, movement of people/vehicles
• Stress factors – vaccination, transport, feed change, heat stress
• Concurrent infections – immunosuppression from IBD, ND, or any other pre-existing factor.

As these are the factors which provide window to the transmission of Salmonella in poultry farms, therefore proper management of these predisposing factors along with immunization of birds will provide maximum protection from such kind of disease transmission.

Transmission:

Various modes of transmission in poultry are- through vertical transmission from infected breeders to eggs, horizontal transmission via contaminated feed, water, litter, equipment and vectors and last but not least cross-contamination during processing, making it a persistent farm-to-fork threat.

Modes of Transmission:

1. Vertical Transmission (Transovarian)
   • From infected hens to chicks via eggs
   • Important for S. Pullorum and S. Gallinarum
2. Horizontal Transmission
   • Faecal–oral route: ingestion of contaminated feed, water or litter
   • Direct bird-to-bird contact
   • Mechanical vectors: rodents, flies, beetles and wild birds
   • Farm equipment, boots, vehicles spreading contamination
3. During Processing and Marketing
   • Cross-contamination during slaughter, defeathering, evisceration
   • Contaminated meat and eggs → foodborne infections in humans

Fig. 1. Modes of transmission of Salmonella

Pathogenesis:

Once ingested by bird, Salmonella colonizes the gastrointestinal tract, particularly the ceca, where its fimbriae, flagella and adhesins enable attachment to the mucosal surface and biofilm formation enhances persistence. It then starts replication in the ceca and establishes a persistent reservoir for faecal shedding. Because the host-adapted serovars like S. Gallinarum and S. Pullorum survive inside macrophages and do not have flagella, they are able to avoid innate immunity and spread throughout the body through the circulation to the liver, spleen, bone marrow and reproductive organs.

In laying hens, colonization of ovaries and oviducts results in trans-ovarian egg contamination, a major food safety concern. Even if the host mounts an innate immune response involving heterophils, macrophages and inflammatory cytokines, followed by adaptive immunity with humoral (IgA, Ig Y) and cell-mediated responses, but clearance is often incomplete, leading to asymptomatic carriers that shed Salmonella intermittently in faeces. Clinical manifestations are most severe in young or immunocompromised birds, resulting in septicaemia, diarrhoea and mortality, while older birds often remain subclinical carriers with minimal lesions.

Pathological changes in acute infections may include enteritis, caecal core formation and necrotic foci in liver and spleen. Thus, the pathogenesis of Salmonella in poultry follows a cycle of ingestion, intestinal colonization, epithelial invasion, intracellular survival, systemic spread, and persistence, with significant consequences for flock health, vertical transmission and zoonotic risk to humans through contaminated poultry products. Hence making it as a dual threat for both poultry health and food safety.

Clinical Signs and Post Mortem lesions:

S. pullorum primarily affects chicks aged 1–3 weeks, causing sudden high mortality, white pasty diarrhoea (pasty butt), weakness, huddling and yolk sac infections, while survivors may develop arthritis and become chronic carriers shedding the organism in eggs and faeces.

While S. gallinarum affects growers and adult birds, producing high fever, anorexia, drooping posture, bronze or darkened combs and wattles, profuse brown-green diarrhoea, swollen necrotic livers and spleens and marked drops in egg production; chronic cases may show milder diarrhoea and shell defects. Key differentiators are age susceptibility (chicks vs adults), faecal colour, presence of arthritis in pullorum disease and severe reproductive impacts in fowl typhoid.  

Most common clinical findings:

• Vent paste.
• Ruffled feathers.
• Intestinal haemorrhages.
• Bronze discoloration of liver.
•  Elevated white nodular on ventricles.
• Prominent necrotic foci on liver.
• Hepatomegaly.

Post-mortem lesions of Salmonella in poultry are characteristic and vary with age and disease type. In Fowl Typhoid (caused by S. Gallinarum), the liver becomes enlarged, friable and bronze in colour, often seen with necrotic foci, while the spleen and kidneys are swollen and congested. The heart shows petechial haemorrhages and intestines display catarrhal to haemorrhagic enteritis, sometimes with button-like ulcers in chronic cases. In Pullorum Disease (caused by S. Pullorum), chicks show white necrotic foci in the liver, spleen and lungs, caecal cores, enlarged pale kidneys and unabsorbed yolk sacs. In older birds, lesions include caseous oophoritis, salpingitis and peritonitis. These lesions highlight the septicaemic nature of Salmonella, leading to systemic organ damage and reproductive losses in poultry.

Fig 2. Necrotic foci on liver, in chicken

Fig. 3: Vent paste, diarrhoea and huddling in chicks.

Fig. 4: Bronze discolouration of liver   

Fig. 5: Congested ovarian follicles

Diagnosis:

Although many carriers exhibit no symptoms, the first step in diagnosing Salmonella in poultry is to look for clinical indicators and post mortem lesions.

Diagnosis should be based on:

1. Flock history
2. Clinical signs
3. PM lesions
4. Laboratory confirmation

Affected birds often show depression, ruffled feathers, white diarrhea with pasted vents, reduced egg production, and high mortality, especially in chicks. Post-mortem examination typically reveals hepatomegaly with bronze discoloration and necrotic foci, splenomegaly, nephritis, cecal cores, and yolk sacculitis. Definitive diagnosis requires bacteriological culture from liver, spleen, bile, or cecal contents using enrichment broths and selective agars, supported by biochemical tests. Rapid plate agglutination tests are commonly used for flock screening, while molecular methods like PCR and ELISA provide sensitive and specific detection of Salmonella. Also, NPIP protocols detail validated sampling methods (drag/manure swabs, cloacal swabs, hatchery debris), culture workflows and confirmatory testing through biochemical and serological ID (9 C.F.R.) This integrated algorithm ensures early detection, regulatory compliance and reduced Salmonella transmission across poultry systems.  

Treatment and Drug Resistance:

Antibiotics are primarily used to treat Salmonella infections in chickens in order to lower mortality and clinical illness; because carrier states persist, total eradication of the bacteria is rarely possible. Among commonly used medications include aminoglycosides, sulphonamides with trimethoprim, tetracyclines and fluoroquinolones. Probiotics, vitamins and electrolytes used in supportive therapy aid in gut health and recuperation. However, Salmonella has developed antimicrobial resistance (AMR) as a result of the overuse and abuse of antibiotics in chicken farming.Alarmingly, many isolates demonstrate multi-drug resistance, complicating treatment options for both poultry and human infections (Das et al., 2023)

These challenges create major public health risks and economic burdens, underscoring the urgent need for integrated control strategies. Vaccination of breeder and layer flocks—using live-attenuated and killed vaccines—has proven highly effective in reducing colonization, shedding, vertical transmission and cross-serovar infections, thereby lowering environmental contamination. Therefore, effective control of Salmonella in poultry with combined use of strict biosecurity and strategic vaccination, can fully prevent infection with less or no use of excess antibiotics. Even effective vaccination can reduce mortality, also egg and meat contamination and decreases reliance on antibiotics.

Prevention and control:

Salmonella control in poultry relies on a multifaceted strategy that integrates strict biosecurity, environmental hygiene, hatchery and egg sanitation, prudent antimicrobial use and continuous monitoring, with vaccination serving as a cornerstone. Rigorous access control, pest management, litter and water sanitation and hatchery hygiene help minimize pathogen entry and spread, while surveillance using culture, serology and molecular diagnostics ensures early detection and program adjustment. Antimicrobial stewardship reduces resistance risks and alternatives like probiotics support gut health. Above all, live and killed vaccines, tailored to local serovars, not only reduce intestinal colonization and vertical transmission but also complement other measures to sustainably lower Salmonella prevalence, protect food safety and safeguard public health.

Stallen South Asia Pvt. Ltd. Is offering unique live vaccine BIO-VAC SGPN 695 against fowl typhoid and Salmonella enteritidis.

Key Features of BIO-VAC SGP 695:

• Live-attenuated dual‐serovar vaccine: BIO-VAC SGP 695 uses a stable, non-reverting Salmonella gallinarum/pullorum strain to protect pullets against both fowl typhoid and pullorum disease, while also reducing Salmonella enteritidis colonization in the gut.
• Oral, water-based delivery: The freeze-dried vaccine is easily reconstituted and administered via drinking water, ensuring uniform uptake across large flocks without injections and stress.
• Rapid, durable immunity: Two doses elicit strong protective responses leading to lower mortality, milder clinical signs and stabilized egg production.
• Cost-effective and field-stable: Bulk reconstitution lowers per-dose handling and labour costs, and the strain remains apathogenic under farm conditions without interfering with other vaccination programs.

Feature	BIO-VAC SGP 695	SG 9R
Targeted infections	Salmonella gallinarum, Salmonella pullorum, Salmonella Enteritidis	Primarily Salmonella gallinarum (Fowl Typhoid)
Characterisation	Stable attenuation, non-reverting; safe in pullets	Possible reversion
Administration	Oral via drinking water (no handling stress, flock-wide coverage)	Injection (subcutaneous/intramuscular) labour intensive, stressful
Vaccination program	2-dose pullet program: 6–8 weeks & 16–18 weeks	Multiple injections required; less practical in large flocks
Efficacy	Strong systemic & mucosal immunity; reduces colonization, shedding and mortality	Inconsistent field efficacy; does not prevent shedding fully
Field advantages	Easy mass application, safer profile, compatible with biosecurity programs	Higher labour cost, handling stress, potential post-vaccination reactions

Table No. 1.: Comparison of SGP 695 and SG 9R

• One dose of vaccine contains: Cultures of S. gallinarum/pullorum attenuated strain SGP 695 AV: min. 2×10⁸ CFU.
• Able to stimulate a rapid and long-lasting immune response.

Field trial results of BIO VAC SGP 695:

Fig. 3: ELISA report of BIO-VAC SGP  695 and SG 9R vaccine

• Difference between SET-VAC and BIO-VAC SGP 695:
• Live attenuated vaccines:
• Are typically used in layer flocks.
• Induction of both humoral immunity (B cells) and cell-mediated immunity (T-cells).
• Inactivated vaccines:
• More commonly used in breeder flocks.
• Induce strong antibody responses.

	SET VAC	BIO-VAC SGP 695
Type	Inactivated	Live
Strain	S. enteritidis and S. typhimurium	S.gallinarum, S.pullorum, S. enteritidis
Administration	Subcutaneous injection	Oral (via drinking water)
Vaccination Program	Initial dose at 6-8 weeks, second at 14-16 weeks.	Initial dose at 6-8 weeks, second at 16-18 weeks. Early dose if early infection history.

• ADMINISTRATION AND DOSAGE:
  • In drinking water not before the age of 7 days.
  • For pullet vaccination at 6-8 weeks of age followed by a second vaccination at 16-18 weeks of age.
  • Additional vaccination: 7-10 days old if severe infection or history of early infection, Ideal for pullets before ovulation. Safe for laying hens
• NOTE:
  • Make sure that there are no antiseptic or disinfectant agents in the water used for dilution.
  • Make sure that all birds drink the vaccine suspension within 2 hours.
  • After dissolving in water, it should be used within 2 hours.
• STORAGE:
  • Store in the refrigerator between +2° C and +8° C.
• DOSES PER PACK:
  • 1000 doses per vial.

References:

1. 9 C.F.R.  145.14 (2025). https://www.ecfr.gov/current/title-9/chapter-I/subchapter-G/part-145/subpart-A/section-145.14
2. Castro‑Vargas, R. E., Herrera‑Sánchez M. P., Rodríguez‑Hernández R. and Rondón‑Barragán I. S. (2020). Antibiotic resistance in Salmonella spp. isolated from poultry: A global overview. Veterinary World, 13(10): 2070–2084. https://doi.org/10.14202/vetworld.2020.2070-2084
3. Das, A. et al. (2023). Antimicrobial resistance in Salmonella isolates from Indian poultry: a systematic review. Frontiers in Microbiology, 14: 10920579.
4. Gaur D., Kukreti D., Ahuja S., Kumar S. and Singh A. (2025). Poultry diseases rising concern in India. International Research Journal of Modernization in Engineering Technology and Science, 7(4): 8510–8513. https://doi.org/10.56726/IRJMETS74074.

Dr. Amit Janbandhu, Dr. Sanjay Singhal

Abstract

The progressive ban on in-feed antibiotic growth promoters (AGPs) has accelerated the need for efficacious phytogenic alternatives capable of sustaining growth and intestinal health in modern broiler production. PHYTOGIC, a standardized phytogenic formulation derived from Macleaya cordata extract and enriched with benzylisoquinoline alkaloids (primarily sanguinarine and chelerythrine), exhibits potent antimicrobial and anti-inflammatory activity, including suppression of the HMGB1–TLR4–NF-κB axis. This field study investigated the effects of dietary PHYTOGIC on growth performance of commercial Vencobb 430 broilers raised on deep litter under high ambient temperature stress (42–45 °C). A total of 36,000 chicks were allocated to two treatments: a basal diet (T1) and the basal diet supplemented with PHYTOGIC at 150 g/ton (T2). Performance indicators, including body weight, feed intake (FI), feed conversion ratio (FCR), corrected FCR (CFCR), and mortality, were monitored over a 42-day production cycle. PHYTOGIC supplementation significantly improved final body weight (2291 g vs. 2110 g; +8.22%) and feed efficiency (FCR: 1.75 vs. 1.80; CFCR: 1.67 vs. 1.77), accompanied by a moderate increase in FI (+5.50%). Mortality remained statistically comparable between groups, indicating no detrimental physiological effects. These results demonstrate that PHYTOGIC enhances nutrient utilization and growth performance under challenging production conditions, supporting its potential as a viable phytogenic replacement for AGPs in commercial broiler systems.

Introduction

The abuse of antibiotic growth promoters (AGPs) in feed has led to drug resistance and ecological damage would threaten human health eventually. Natural plants have become a hotspot in the research and application of substituting AGPs because of their advantages of safety, efficiency, and availability (Songchang et.al.2021).

Necrotic enteritis (NE), an enterotoxemic disease in poultry, is primarily caused by Clostridium perfringens. The restriction or ban of in-feed antibiotics in regions such as the European Union and China has contributed to a resurgence of NE cases (Shojadoost et.al.2012). The disease is particularly severe in young broilers, with acute mortality rates reaching up to 50%. NE is associated with a significant upregulation of pro-inflammatory cytokines and chemokines, contributing to systemic immune activation (Lee et.al.2011). As inflammation is metabolically demanding, immune challenges can increase the resting metabolic rate of animals by 8–27%, thereby diverting energy from growth and maintenance processes (Martin et.al.2003).

Inflammation in poultry reduces feed intake, disrupts intestinal morphology, limits nutrient absorption, and redirects energy to immune responses, collectively impairing growth and causing intestinal damage and economic losses (Klasing et.al.1987). Necrotic enteritis (NE) aggravates these effects by inducing gut microbiota dysbiosis, marked by reduced diversity, instability, and enrichment of pro-inflammatory bacteria, which compromise intestinal homeostasis and enhance pathogen persistence (Satokari et.al.2015).

The gut microbiota is the largest symbiotic ecosystem in hosts and has been shown to play an important role in maintaining intestinal homeostasis. Changes in the gut microbiota can confer resistance to pathogenic bacteria or promote infection in a host. A symbiotic microbiome regulates the maturation of the mucosal immune system, while a pathogenic microbiome can cause immune dysfunction in the host, leading to the development of diseases such as intestinal inflammation (Shi et.al, 2017).

 Pathogenic bacteria use microbiota-derived carbon and nitrogen sources as nutrients and regulatory signals to promote their own growth and virulence. By inducing inflammation, these bacteria alter the gut environment and use a unique respiratory and metal acquisition system to drive their expansion (Baumler et.al,2016).

Macleaya cordata is a perennial herb widely distributed in southern China and traditionally used in herbal medicine. Its extract (MCE), which contains bioactive alkaloids such as sanguinarine and chelerythrine, was approved as a feed additive in the EU in 2004. Sanguinarine, the major active compound, has demonstrated antitumor (Fu.et.al.2018), immunomodulatory (Kumar et.al.2014), antibacterial (Hamoud et.al.2014), anti-inflammatory (Xue et.al.2017), and insecticidal (Li et.al.2017) properties.

Several investigators have reported that MCE diets could ameliorate production performance, improve gut health and body immunity, and promote growth (Bojjireddy et al., 2013; Khadem et al., 2014). Besides, sanguinarine is the major active ingredient of M. cordata, which has been found to have anti-inflammatory activity (Niu et al.2012), inhibit the activation of NF-κB, and regulate inflammatory response (Wullaert et. al.2011). Gradually, it evoked attention as a substitute of antibiotics (Kim et al.2012). Although sanguinarine is poisonous, an average daily oral dose of alkaloids of up to 5 mg/kg animal body weight has been proven safe (Kosina et al., 2004).

MCE has been reported to modulate intestinal microbiota, particularly in the upper gastrointestinal tract. It promotes beneficial bacteria such as Lactobacillus, inhibits Escherichia coli colonization, and stimulates amino acid, vitamin, and bile acid biosynthetic pathways, while minimizing the risk of antibiotic resistance gene accumulation (Huang et.al.2018).  While MCE’s beneficial effects on broiler performance, intestinal integrity, and inflammation have been demonstrated, its impact on humoral immune function and microbiota-mediated amelioration of NE remains insufficiently characterized (Bui et.al.2015).

Dietary supplementation with 100 mg/kg MCE significantly increased the diversity of the microbiota in the ileum of Snowy Peak blackbone chickens, and 200 mg/kg MCE significantly increased the relative abundance of Lactobacillus and Aeriscardovia in the ileum, increased the relative abundance of Bacteroidetes and Deferribacteres in the cecum, and decreased the relative abundance of Firmicutes in the cecum (Guo et.al, 2021).

Mechanism of action Macleaya Cordata Extract in poultry gut

1. Inhibition of HMGB1 release and function

HMGB1 is a nuclear protein that acts as a damage-associated molecular pattern (DAMP) molecule when released from necrotic cells or actively secreted by immune cells. Sanguinarine can interfere with HMGB1’s pro-inflammatory activity in the following ways:

Preventing translocation: Sanguinarine may inhibit the translocation of HMGB1 from the nucleus to the cytoplasm, thereby reducing its extracellular concentration where it acts as an inflammatory mediator.

Direct antagonism: HMGB1 has two binding domains, the A-box and B-box, which interact with various receptors to cause inflammation. Studies show that the HMGB1 A-box can act as an antagonist by competing with full-length HMGB1 for binding sites on TLR4. It’s plausible that sanguinarine may interfere with the binding of the active HMGB1 B-box to its receptor.

2. Interference with TLR4 signaling

TLR4 is a receptor on immune cells that detects inflammatory signals, including extracellular HMGB1. Sanguinarine has been shown to suppress the TLR4 signaling pathway.

Blocking ligand binding: Sanguinarine may directly interact with the TLR4 receptor complex, which includes the protein MD-2. By binding to or otherwise affecting this complex, sanguinarine could prevent the attachment of HMGB1 and other inflammatory ligands like LPS, thus inhibiting the initiation of downstream signaling.

Downregulation of TLR4 expression: Some studies suggest that sanguinarine treatment may lead to the downregulation of TLR4 protein expression on the cell surface, further limiting the cell’s ability to respond to inflammatory signals.

Fig.1.  Anti-inflammatory mechanism of Sanguinarine showing reduction of gut lesion by interference of HMGB1/ TLR4 pathway in inflammation site.

3. Suppression of the NF-κB cascade

NF-κB is a key transcription factor that regulates the expression of genes involved in inflammation. Sanguinarine potently suppresses NF-κB activation through several mechanisms.

Inhibition of IκBα phosphorylation and degradation: In resting cells, NF-κB is held inactive in the cytoplasm by its inhibitory subunit, IκBα. Upon cell activation, a kinase complex (IKK) phosphorylates IκBα, leading to its ubiquitination and degradation. Sanguinarine acts upstream of this step, blocking the phosphorylation and subsequent degradation of IκBα. This prevents NF-κB from being released.

Blocking NF-κB nuclear translocation: Because IκBα is not degraded, the NF-κB heterodimer (p65/p50) cannot translocate to the nucleus. This prevents NF-κB from binding to the promoter regions of target genes.

Modification of sulfhydryl groups: A key aspect of sanguinarine’s NF-κB inhibition is its ability to modify critical sulfhydryl groups on proteins in the NF-κB signaling pathway. This covalent modification, which can be reversed by reducing agents like DTT, is thought to be essential for sanguinarine’s inhibitory effect.

4.Resulting anti-inflammatory effects

By inhibiting the HMGB1-TLR4-NF-κB axis at multiple stages, sanguinarine effectively suppresses the inflammatory response.

Reduced cytokine expression: Sanguinarine leads to a decrease in the expression and release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β).

Mitigated cellular infiltration: The reduced expression of inflammatory chemokines, such as CCL2, leads to decreased recruitment of inflammatory cells to the site of inflammation.

Protection against tissue damage: These overall effects contribute to the protection of tissues from inflammation-induced injury, as seen in models of renal ischemia-reperfusion injury and ulcerative colitis (Gu. et.al, 2022).

The aim of the study was to evaluate the effect of PHYTOGIC on the performance of commercial broilers reared on deep litter under field conditions.

Materials and Methods

Experimental Design and Management

The trial was conducted at Harsh Broiler House using Vencobb 430 straight run chicks (not sexed at hatchery) in three treatments of around 12000 birds in each treatment. A total of 36000 birds were considered for trial purpose.  Feed Formulation used was same for all treatment groups except in T2 where PHYTOGIC was added at 150 gm per ton feed respectively in all stages. (Table 1.) In the study, the energy level was equivalent to the standard requirements of broilers recommended in the Vecobb 430. The trial was carried out over a period of 42 days. The birds were fed ad lib feed and water was available all the times. Care was taken to provide good conditions by adopting strict biosecurity measures. The housing and vaccination procedures were same in both groups.

Table 1. Composition of basal diet for broiler chicks in control group for 3 phases.

Broiler Feed Formulation (Control)
Raw Materials	Prestarter	Starter	Finisher
Maize	625.15	652.75	686.65
HiPro Soya	335	300	260
Soya Crude Oil	6	14	23
Limestone Powder	8.5	8.5	8
Dicalcium Phosphate	10	10	8
L Lysine HCI	2.7	2.4	2.3
DL Methionine	3.3	3	2.7
L Threonine	1	1	1
Salt	2.5	2.5	2.5
Soda Bi Carb	1.5	1.5	1.5
Choline Chloride 60%	1	1	1
Organic TM	0.5	0.5	0.5
Broiler Vitamin Premix	0.5	0.5	0.5
Coccidiostat	0.5	0.5	0.5
AGP	0.05	0.05	0.05
NSP Enzyme	0.1	0.1	0.1
Phytase 5000	0.1	0.1	0.1
Feed Acidifier	1	1	1
Toxin Binder	0.6	0.6	0.6

*The figures are in Kilograms.

 The premix provided the following per kilogram of the diet: vitamin A, 6000 IU; vitamin D3, 2500 IU; vitamin B1, 1.75 mg; vitamin B2, 5.5 mg; vitamin B6, 4 mg; vitamin B12, 0.18 mg; vitamin E, 25 mg; vitamin K3, 2.25 mg; Cu, 7.5 mg; Mn, 60 mg; Fe, 75 mg; Zn, 60 mg; Se, 0.15 mg; biotin, 0.14 mg; NaCl, 3.7 g; folic acid, 0.8 mg; pantothenic acid, 12 mg; phytase, 400 U; nicotinic acid, 34 mg; chloride, 350 mg. *Nutrient levels were all calculated values.

Treatment Details-

T1: Control group fed basal diet

T2: Control group fed basal diet + PHYTOGIC @150 g PMT

Parameters Studied-

1. Body Weight gain was recorded weekly
2. Feed Consumption recorded daily and leftover feed was adjusted in the other day quota to know actual intake.
3. Mortality was recorded daily
4. EEF calculated post harvesting of the flock
5. FCR was calculated every week and post harvesting of the flock.

Results:

Effect of Supplementation of Phytogic on body growth performance parameters like Body Weights, Feed Consumption, FCR and Average Daily gain of Control and Treatment Groups

Fig.1. Effect of different dietary treatments on Body Weights (g)

Conclusion: Broilers in the T2 – PHYTOGIC group fed at 150g/ton of feed achieved higher final body weights (2291 g) compared to the T1 – Control group (2110 g), showing an 8.22% improvement. This indicates that PHYTOGIC supplementation effectively enhances growth performance in broilers.

Fig.2. Effect of different dietary treatments on Feed Intake (g)

Conclusion: Broilers in the T2 – PHYTOGIC group fed at 150g/ton of feed consumed more feed (4015 g) compared to the T1 – Control group (3800 g), showing a 5.50% increase in feed intake. This suggests that PHYTOGIC supplementation may enhance feed consumption in broilers.

Conclusion: The average weekly percentage difference in weight gain between T2 – PHYTOGIC fed at 150g/ton of feed and T1 – Control was -3.84%, indicating that, overall, PHYTOGIC supplementation did not improve weekly weight gain in broilers and was slightly less effective than the control in this trial.

Fig.4. Effect of different dietary treatments on Feed Conversion Ratio

Conclusion: Broilers in the T2 – PHYTOGIC group fed at 150g/ton of feed showed an improved feed conversion ratio (1.75) compared to the T1 – Control group (1.80), with a 2.81% improvement. This suggests that PHYTOGIC supplementation enhances feed efficiency in broilers, allowing for better weight gain per unit of feed consumed.

Fig.5. Effect of different dietary treatments on Weekly Mortality (%)

Conclusion:The mortality rate in the T2 – PHYTOGIC group fed at 150g/ton of feed was (7.44%) slightly higher than the T1 – Control group (7.39%), with a 0.27% difference. This minimal variation indicates that PHYTOGIC supplementation had no significant effect on broiler mortality under the conditions of this study.

Table 2. Summary of the Report-

Parameters	T1- Control	T2- PHYTOGIC	% Difference
Body Weight (g)	2110	2291	8.22
Feed Intake (g)	3800	4015	5.50
FCR	1.80	1.75	2.81
CFCR	1.77	1.67	5.81
Mortality (%)	7.39	7.44	0.27

Discussion

The findings of the present field study demonstrate that dietary supplementation with PHYTOGIC at 150 g/ton improved broiler growth performance under commercial deep-litter and heat-stress conditions. Broilers receiving PHYTOGIC exhibited an 8.22% increase in final body weight compared to the control group, indicating enhanced nutrient utilization and metabolic efficiency. This improvement is consistent with previous reports showing that Macleaya cordata extract and its major alkaloid, sanguinarine, can promote growth by reducing intestinal inflammation, stabilizing gut microbiota, and improving nutrient absorption. The observed increase in feed intake (5.50%) in the PHYTOGIC group suggests that phytogenic supplementation may have positively influenced appetite or gut comfort, allowing birds to maintain adequate consumption despite environmental temperature stress.

Feed efficiency was also improved, as evidenced by reductions in FCR (1.75 vs. 1.80) and CFCR (1.67 vs. 1.77). This aligns with earlier studies reporting that sanguinarine-containing extracts suppress inflammatory pathways such as the HMGB1–TLR4–NF-κB axis, thereby reducing metabolic energy waste associated with immune activation. By lowering the inflammatory burden, PHYTOGIC likely allowed more dietary energy to be directed toward growth rather than immune-related maintenance. Improvements in FCR also support the hypothesis that phytogenic compounds enhance gut function through modulation of intestinal morphology and beneficial microbiota populations, as reported in previous research.

Weekly weight gain patterns showed some variation, with PHYTOGIC not consistently outperforming the control in all weeks. This may be attributed to fluctuating heat stress levels and daily feed intake variations typical of field conditions. However, despite these short-term variations, the cumulative performance benefits remained substantial by the end of the production cycle.

Importantly, mortality rates were nearly identical between treatments (7.39% vs. 7.44%), indicating that PHYTOGIC supplementation did not impose any negative health effects and is safe for use under commercial conditions. The lack of impact on mortality also suggests that the performance improvements were not driven by survivability differences but by true enhancement of growth and feed efficiency.

Overall, the results support the potential of PHYTOGIC as an effective phytogenic alternative to antibiotic growth promoters. Its ability to improve growth performance and feed efficiency, even under extreme heat, aligns with its known anti-inflammatory, antimicrobial, and gut-modulating properties. The findings strengthen the evidence that phytogenic compounds derived from Macleaya cordata can contribute to sustainable poultry production by enhancing physiological resilience and intestinal health.

Conclusion-

The trial was conducted in the extreme heat season where average temperature in the surrounding was around 42-45 degree Celsius. The T2 (PHYTOGIC) groups showed overall improved performance compared to the T1 (Control) group. Specifically, the body weight of T2 (PHYTOGIC) was 8.22% higher than T1 (Control), indicating better growth. Feed Conversion Ratio (FCR) and Corrected FCR (CFCR) were both lower in T2 (PHYTOGIC) by 2.81% and 5.81%, respectively, demonstrating more efficient feed utilization in the T2 (PHYTOGIC) group than T1 (Control). Mortality rates were nearly identical between the two groups, indicating that the supplement did not adversely affect survival. Overall, PHYTOGIC supplementation resulted in better growth performance and feed efficiency compared to the control with no significant impact on mortality.

References:

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Dr. Amit V. Janbandhu, Dr. Sanjay Singhal

Abstract

This study evaluates the efficacy of PEPIGRO, a Bacillus licheniformis-based probiotic and postbiotics as antimicrobial peptide (AMPs), on the growth and health performance of commercial broilers under field conditions. A total of 36,000 straight-run broiler chicks were assigned to control and treatment groups, with the latter receiving PEPIGRO supplementation at 300 g/ton of feed. The trial was conducted over 42 days during extreme heat (42–45°C), and assessed body weight, feed intake, feed conversion ratio (FCR), weekly gain, and mortality. PEPIGRO supplementation resulted in an 8.18% increase in body weight, a 6.59% rise in feed intake, and a 6.22% improvement in weekly gain compared to the control, alongside a 1.68% enhancement in FCR. Mortality was notably reduced by 28.08%, indicating improved survivability. These findings demonstrate that dietary inclusion of PEPIGRO effectively enhances broiler growth performance, feed efficiency, and health, supporting the role of Bacillus licheniformis as a promising antibiotic alternative under commercial field stressors.

Introduction

In recent decades, antibiotics have been extensively used in animal husbandry for their dual purposes of promoting growth and preventing or treating bacterial infections. However, the excessive and prolonged use of these drugs has led to serious public health and environmental concerns, including the emergence of antibiotic-resistant pathogens and environmental contamination, which pose risks to both humans and animals (Tang et al., 2017). Consequently, international authorities implemented strict prohibitions on antibiotic growth promoters (Organization, 1999), prompting researchers to explore alternative, safer strategies to enhance livestock productivity.

Among the promising alternatives, several bioactive feed additives—such as probiotics (Xu et al., 2021), antimicrobial peptides (Yi et al., 2017), plant extracts (Abullais Saquib et al., 2021), acidifiers (Pearlin et al., 2020), and plant essential oils (Montassier et al., 2021; Ayalew et al., 2022)—have gained increasing attention. These compounds are valued for their ability to regulate growth performance, immune function, oxidative balance, and intestinal microbial homeostasis in animal models (Roselli et al., 2005). Among these options, probiotics have drawn special interest due to their safety profile, efficacy, and multifunctional benefits in the animal industry (Ningsih et al., 2023).

Probiotics have emerged as promising alternatives to antibiotics in poultry nutrition. These “friendly” bacteria contribute to gut health by enhancing digestion, modulating the immune system, improving intestinal barrier function, and competing against pathogenic microorganisms. Among the various probiotic candidates, species of the Bacillus genus—particularly Bacillus licheniformis—have attracted increasing attention due to their spore-forming capabilities, environmental resilience, and broad-spectrum biological activities. B. licheniformis is “generally recognized as safe” (GRAS) and has demonstrated antimicrobial, antioxidant, and immunomodulatory properties, making it a multifunctional probiotic with diverse applications in poultry production. Recent studies have shown that dietary supplementation with B. licheniformis can significantly enhance growth performance, feed conversion efficiency, egg production, intestinal morphology, and microbial balance in poultry (Pan et.al.2022).

Bacillus licheniformis is a Gram-positive, spore-forming bacterium characterized by high temperature and stress resistance recognized for its probiotic and postbiotic benefits. It produces digestive enzymes such as protease, amylase, lipase, and cellulase, which enhance nutrient utilization. By depleting intestinal oxygen, it fosters anaerobic conditions that promote beneficial bacteria (Lactobacillus, Bifidobacterium) and suppress pathogens (Escherichia coli, Salmonella, Clostridium perfringens). In addition, B. licheniformis secretes bioactive metabolites, including bacteriocins, surfactins, licheniformins, and bacitracin, all of which possess antimicrobial properties (Giri et al. 2019). The bacteriocin, a 42-amino acid peptide (~4.7 kDa), exhibits strong α-helical conformation and acts by disrupting bacterial membranes and inhibiting intracellular processes such as nucleic acid and protein synthesis. These peptides not only suppress pathogens but also enhance host immunity by stimulating neutrophils, macrophages, mast cells, and NK cells, and inducing cytokine and chemokine production. Collectively, B. licheniformis improves feed digestibility, strengthens mucosal barrier function, supports gut microbiota balance, and enhances immune responses, making it a promising candidate for use in both animal nutrition and human health (Shleeva et.al.2023). Moreover, it improves antioxidant status by enhancing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), reduces harmful nitrogen waste (NH₃-N), and boosts microbial crude protein production in ruminants (Jia et al., 2018). Importantly, B. licheniformis supplementation modulates the intestinal microbiota composition and promotes microbial equilibrium, thus maintaining intestinal integrity and performance under disease challenge conditions (Chen and Yu, 2020).

Necrotic enteritis (NE), a major intestinal bacterial disease in poultry caused by Clostridium perfringens, accounts for estimated economic losses exceeding six billion U.S. dollars annually (Wade and Keyburn, 2015). C. perfringens is a Gram-positive, spore-forming anaerobe capable of producing up to 17 toxins (Parish, 1961). Among its toxin-producing variants, types A and C are primarily associated with NE development (Engström et al., 2003). The disease manifests in clinical and subclinical forms. While clinical NE can result in rapid mortality rates up to 50%, subclinical NE (SNE) leads to subtle intestinal injury characterized by reduced appetite, poor nutrient absorption, and depressed growth—all contributing to significant production losses (Timbermont et al., 2011).

NE infections commonly disrupt the intestinal barrier, induce inflammatory responses, and disturb gut microbial balance. Controlling intestinal health, therefore, is an effective preventive strategy against NE in broilers. In the search for sustainable approaches during the post-antibiotic era, probiotics like B. licheniformis have shown substantial potential in alleviating NE symptoms and restoring gut health (Venessa et al., 2016).

Research has demonstrated that B. licheniformis enhances tight junction protein (TJP) and mucin-2 gene expression in poultry, thereby maintaining intestinal integrity and reducing permeability (Wang Y. et al., 2017). Moreover, Bacillus spp. modulate immune responses through upregulation of Toll-like receptors (TLRs), NF-κB signaling, and cytokine synthesis (Rajput et al., 2017). These probiotics also regulate the intestinal microbiota composition, especially under NE challenges, thereby improving host resilience (Lin et al., 2017).

Given its enzymatic activity, antimicrobial peptide production, and regulatory effects on immune and intestinal functions, Bacillus licheniformis represents a viable natural feed additive capable of replacing traditional antibiotics. The present study sought to determine whether B. licheniformis can alleviate subclinical necrotic enteritis (SNE) in broilers as effectively as the antibiotic enramycin, by examining its roles in intestinal barrier maintenance, immune modulation, and gut microbiota balance.

Antimicrobial Peptides (AMPs)

Antimicrobial peptides (AMPs) are a diverse group of small bioactive molecules naturally found across a wide range of organisms. They are essential components of the innate immune system and serve as the first line of defense against a variety of pathogens. AMPs possess broad-spectrum activity against bacteria, fungi, parasites, and viruses, making them crucial in host protection (Huan et al., 2020).

 Over the past decades, the rapid rise of antibiotic resistance and growing concerns regarding antibiotic use have driven the exploration and development of AMPs as alternative therapeutic and preventive agents. Their potent antimicrobial properties, coupled with lower rates of resistance development, make them promising candidates for applications in medicine, food preservation, animal husbandry, agriculture, and aquaculture.

Antibacterial Substances Produced by Bacillus licheniformis

The endospore-forming bacterium Bacillus licheniformis is a prolific producer of a wide range of antimicrobial substances, each possessing unique structural and functional characteristics. Interestingly, even when cultured under identical conditions, different B. licheniformis strains synthesize distinct profiles of antibacterial compounds. These variations arise from differences in transcriptional or translational regulation, resulting in strain-specific antimicrobial expression patterns. The molecular masses of these secreted compounds typically range from 1.4 to 20 kDa . The major categories of antimicrobial compounds secreted by B. licheniformis include bacteriocins, licheniformins, bacitracin, and surfactin. (Shleeva et.al.2023).

1. Bacteriocins

Bacteriocins are ribosomally synthesized antimicrobial peptides or proteins that exhibit bactericidal or bacteriostatic activity against closely related bacterial species. B. licheniformis produces multiple types of bacteriocins, typically ranging in molecular weight from 1.4 to 55 kDa, depending on environmental conditions, growth phase, and strain genotype.For instance, B. licheniformis strain B116 secretes a bacteriocin of approximately 4 kDa with potent activity against both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Listeria monocytogenes, Micrococcus luteus, Bacillus cereus, Escherichia coli, Streptococcus equi, and Salmonella spp. This compound, recovered through ammonium sulfate precipitation, demonstrated strong resistance to heat, acidic, and alkaline conditions. However, enzymatic treatment with pronase completely abolished its antimicrobial activity, and partial inactivation was observed with papain and lipase, indicating the presence of a lipid moiety in its structure (Shleeva et.al.2023).

2. Licheniformins

Licheniformins are lipopeptide antibiotics produced by B. licheniformis, often existing as several closely related variants. The licheniformin lipopeptide produced by B. licheniformis MS3 has a molecular mass of approximately 1.438 kDa. Three principal components—licheniformins A, B, and C—have been identified, all sharing similar amino acid compositions and molecular weights (3.8–4.8 kDa). Despite their structural similarity, these peptides display different degrees of antibacterial potency and toxicity, reflecting subtle biochemical differences in their side-chain structures and lipid modifications (Shleeva et.al.2023).

3. Bacitracin

Bacitracin is a well-known polypeptide antibiotic non-ribosomally synthesized by certain strains of B. subtilis and B. licheniformis. It is composed of 12 amino acids, with four of them—glutamic acid, aspartic acid, phenylalanine, and ornithine—present in their D-isomer forms. The molecular mass of bacitracin is approximately 1.42 kDa. Bacitracin functions by interfering with bacterial cell wall synthesis, making it a clinically important peptide used to inhibit Gram-positive pathogens (Shleeva et.al.2023).

4. Surfactin

B. licheniformis is also capable of producing surfactin and its structural analog, lichenysin—both cyclic lipopeptides with powerful surface-active and antimicrobial properties. The strain B. licheniformis HSN221 was found to secrete nine variants of surfactin and lichenysin when cultured in a medium containing glucose, ammonium chloride, and yeast extract, which were optimal for lipopeptide biosynthesis. The molecular masses of the identified surfactin monomethyl ester homologues were approximately 1.048, 1.049, and 1.063 kDa, as confirmed by ESI-MS analysis. These compounds exhibit potent antimicrobial and emulsifying activity and have potential applications in pharmaceutical, agricultural, and environmental biotechnology (Shleeva et.al.2023).

Mechanism Of Action

1) Competition mechanism:

Fig.1. Probiotic Bacillus employ multifactorial competition mechanism to restrict the expansion of pathogens through four pathways. 

(1) Bacillus adapt itself in suitable niche against niche-occupying competitors. It approaches the intestinal mucous layer and competitively binds to intestinal epithelial cells and mucous layer components via surface proteins. Thus, the effect of niche occupation by Bacillus expels harmful bacteria from the host intestinal epithelial barrier and reduces pathogen invasion. (2) Competitive utilization of nutrients for Bacillus growth. Bacillus secrete various enzymes to rapidly exploit both the macronutrients and micronutrients in gut environment, resulting in limited availability of nutrition to pathogenic bacteria. (3) Bacillus produce an arsenal of antibacterial metabolites that directly inhibit the growth of pathogens. The metabolites included lipopeptides, bacteriocins, polyketides, and SCFAs are effective against the expansion and invasion of pathogens. (4) Bacillus can consume excess oxygen from gut lumen and host circulation for maintaining the intestinal environment in a state of hypoxia, which drive a dominance of bacteria such as lactate acid bacteria that use fermentation for energy production. (Zhu et.al. 2023).

2) Immunomodulatory activity:

Fig.2. Models of antibacterial mechanisms of AMPs.

 Antimicrobial activity and immunomodulatory effects of AMPs, also known as host defence peptides, protect the host from infection. Pathogen invasion triggers a cascade of immunological responses (Fig. 6). Cathelicidins and defensins are mostly produced by neutrophils. The role of AMPs in the immune system is complicated. AMPs regulate the section of cytokines like interleukins; tumour necrosis factors (TNFs), IFNs, and chemokines, as well as the activities of immune cells like dendritic cells (DCs), monocytes, macrophages, mast cells, granulocytes, and lymphocytes, to keep the immune microenvironment in a dynamic balance. AMPs not only destroy invading pathogenic bacteria directly, but they also kill them indirectly by stimulating the immune system. On the one hand, AMPs can activate immune cells in the innate immune system, such as neutrophils, macrophages, mast cells, and NK cells, and trigger the release of cytokines and chemokines to engulf and destroy harmful germs. AMPs, on the other hand, can trigger adaptive immune responses, deliver antigens to T cells via dendritic cells (DCs), and activate cytotoxic T cells to kill pathogenic germs AMPs antimicrobial peptides; NK natural killer. (Babakuliyev et. al.2022).

3) The membrane-disruptive and non-membrane-disruptive antibacterial mechanisms of antimicrobial peptides (AMPs).

Fig.3. The membrane-disruptive and non-membrane-disruptive antibacterial mechanisms of antimicrobial peptides (AMPs).

 In the membrane-disruptive mechanisms, three types of interaction can occur between the membrane and the AMPs, including: (i) barrel-stave model: the peptide monomers form a hydrophilic transmembrane channel by arranging parallelly to the phospholipids of the membrane; (ii) carpet model: the peptides solubilize the membrane into micellar structures; and (iii) toroidal model: the lipid moieties fold inward due to the cascade aggregation of peptide monomers, forming a peptide-and-lipid-lined channel. After AMPs penetrate into the phospholipid membrane, their hydrophobic regions combine with the internal hydrophobic regions of the phospholipid bilayer, while their hydrophilic regions are exposed to the outside. Another bactericidal mechanism is that AMPs penetrate into the cytoplasm and interact with intracellular substances, such as inhibiting DNA, RNA and protein synthesis, inhibiting protein folding, inhibiting enzyme activity and cell wall synthesis, and promoting the release of lyases to destroy cell structures. AMPs antimicrobial peptides. (Le et.al. 2022). The aim of the study to evaluate the effect of PEPIGRO on the performance of commercial broilers reared on deep litter under field conditions.

MATERIALS AND METHODS

Experimental Design and Management

The trial was conducted at Harsh Broiler House -Bilaspur using Vencobb 430 straight run chicks (not sexed at hatchery) in three treatments of around 12000 birds in each treatment. A total of 36000 birds were considered for trial purpose. Feed Formulation used was same for all treatment groups except in T3 where PEPIGRO (Bacillus lincheniformis 3*10⁹) was added at 300 gm per ton feed respectively in all stages. (Table.1). In the study, the energy level was equivalent to the standard requirements of broilers recommended in the Vencobb 430. The trial was carried out over a period of 42 days. The birds were fed ad lib feed and water was available all the time. Care was taken to provide good conditions by adopting strict biosecurity measures. The housing and vaccination procedures were same in both groups.

Table 1. Composition of basal diet for broiler chicks in control group for 3 phases.

Broiler Feed Formulation (Control)
Raw Materials	Prestarter	Starter	Finisher
Maize	625.15	652.75	686.65
HiPro Soya	335	300	260
Soya Crude Oil	6	14	23
Limestone Powder	8.5	8.5	8
Dicalcium Phosphate	10	10	8
L Lysine HCI	2.7	2.4	2.3
DL Methionine	3.3	3	2.7
L Threonine	1	1	1
Salt	2.5	2.5	2.5
Soda Bi Carb	1.5	1.5	1.5
Choline Chloride 60%	1	1	1
Organic TM	0.5	0.5	0.5
Broiler Vitamin Premix	0.5	0.5	0.5
Coccidiostat	0.5	0.5	0.5
AGP	0.05	0.05	0.05
NSP Enzyme	0.1	0.1	0.1
Phytase 5000	0.1	0.1	0.1
Feed Acidifier	1	1	1
Toxin Binder	0.6	0.6	0.6

*The figures are in Kilograms.

 The premix provided the following per kilogram of the diet: vitamin A, 6000 IU; vitamin D3, 2500 IU; vitamin B1, 1.75 mg; vitamin B2, 5.5 mg; vitamin B6, 4 mg; vitamin B12, 0.18 mg; vitamin E, 25 mg; vitamin K3, 2.25 mg; Cu, 7.5 mg; Mn, 60 mg; Fe, 75 mg; Zn, 60 mg; Se, 0.15 mg; biotin, 0.14 mg; NaCl, 3.7 g; folic acid, 0.8 mg; pantothenic acid, 12 mg; phytase, 400 U; nicotinic acid, 34 mg; chloride, 350 mg. *Nutrient levels were all calculated values.

Treatment Details-

T1: Control group fed basal diet

T3: Control group fed basal diet + PEPIGRO @300 g PMT

Parameters Studied-

1. Body Weight gain was recorded weekly
2. Feed Consumption recorded daily and leftover feed was adjusted in the other day quota to know actual intake.
3. Mortality was recorded daily
4. EEF calculated post harvesting of the flock
5. FCR was calculated every week and post harvesting of the flock

Result:

Effect of Pepigro on growth performance parameter in broiler.

Fig.1. Effect of different dietary treatments on Body Weights (g)

Conclusion: PEPIGRO supplementation at 300g/ton of feed (T3) resulted in a statistically significant 8.18% increase in broiler body weight compared to the control (T1), indicating improved growth performance.

Fig.2. Effect of different dietary treatment on Feed intake (g)

Conclusion: The broiler supplemented with PEPIGRO (T3) at 300g/ ton of feed had a feed intake of 4059 g, which is 6.59% higher than the control group (T1) with 3800 g feed intake. This increase in feed intake indicates that PEPIGRO supplementation positively influenced the birds’ feeding behaviour, likely by enhancing the palatability or nutrient availability of the diet.

Fig.3. Effect of different dietary treatment on Weekly Gain (g)

Conclusion:  PEPIGRO (T3) supplementation in broiler diet at 300g/ton of feed resulted in the average percentage difference in weekly gain between T1 (Control) is approximately 6.22%. This indicates that PEPIGRO supplementation had a positive overall effect on growth performance, enhancing weight gain efficiency in broiler chickens.

Fig.4. Effect of different dietary treatment on Feed conversion ratio

Conclusion:  PEPIGRO (T3) supplementation in broiler diet at 300g/ton of feed resulted in a 1.68% improvement in feed conversion ratio (FCR) compared to the control group (T1), indicating enhanced feed efficiency and better growth performance.

Fig.5. Effect of different dietary treatment on Weekly mortality (%)

Conclusion: PEPIGRO supplementation at 300g/ton of feed reduced mortality in broiler poultry from 7.39% in the control group to 5.57%, reflecting a 28.08% decrease. This suggests that PEPIGRO may contribute to improved bird health and survivability during the rearing period.

Table 2. Summary of the Report

Parameters	T1- Control	T3- PEPIGRO	% Difference
Body Weight (g)	2110	2290	8.18
Feed Intake (g)	3800	4059	6.59
FCR	1.8	1.77	1.68
CFCR	1.77	1.69	4.62
Mortality (%)	7.39	5.57	28.08

Discussion

The discussion for this article highlights the significant positive effects of PEPIGRO, a Bacillus licheniformis-based probiotic, on the growth performance, feed efficiency, and health status of commercial broilers under field conditions. The 8.18% increase in body weight and 6.59% increase in feed intake, along with improvements in feed conversion ratio (FCR), align well with previous studies showing Bacillus probiotics enhance nutrient digestibility, modulate gut microbial populations, and improve intestinal morphology (Pan et al., 2022; Hung et al., 2019). These effects are especially valuable in the context of rising restrictions on antibiotic growth promoters (Tang et al., 2017), pushing for safer and sustainable alternatives.

The notable 28.08% reduction in mortality observed in this study suggests enhanced resilience of broilers to environmental stressors, likely owing to improved gut barrier integrity and immune modulation. Bacillus licheniformis produces antimicrobial peptides, enzymes, and metabolites such as bacteriocins and surfactins that inhibit pathogens like Clostridium perfringens, a major agent of necrotic enteritis (NE) in poultry (Shleeva et al., 2023; Wade and Keyburn, 2015). PEPIGRO’s capacity to maintain intestinal health and microbial balance may underlie the reduced pathogenic infections and inflammation, consistent with findings that show Bacillus supplementation upregulates tight junction proteins and mucins while enhancing beneficial microbes like Lactobacillus (Chen and Yu, 2020; Wang et al., 2017).

Moreover, the probiotic’s ability to stimulate the host immune system by inducing cytokine production and activating phagocytic cells further supports its protective role in the gut environment (Babakuliyev et al., 2022). This immunomodulatory effect is critical for mitigating subclinical infections and improving overall flock welfare, which translates into better productivity under commercial rearing conditions.

Additionally, PEPIGRO contributes to antioxidant status improvement by elevating enzyme activities such as superoxide dismutase and glutathione peroxidase, reducing oxidative stress that commonly compromises poultry health under heat stress conditions (Jia et al., 2018). This antioxidant benefit complements its antimicrobial and immunomodulatory functions.

In conclusion, this study reinforces the role of Bacillus licheniformis as a multifunctional probiotic that enhances growth performance, feed efficiency, and health in broilers. It offers a sustainable alternative to antibiotics, aligning with global efforts to reduce antibiotic use in animal production. Future studies should explore optimal dosing strategies, combinations with other feed additives, and long-term effects on microbiota composition and immune function to fully harness the benefits of PEPIGRO in commercial poultry systems.

Conclusion-

The trial was conducted in the extreme heat season where average temperature in the surrounding was around 42-45 degree Celsius. The T3 (PEPIGRO) group showed notable improvements compared to the T1 (Control) group. Body weight in T3 (PEPIGRO) increased by 8.18% compared to T1 (Control), indicating better growth performance. Both Feed Conversion Ratio (FCR) and Corrected Feed Conversion Ratio (CFCR) in T3 (PEPIGRO) improved, showing reductions of 1.68% and 4.62%, respectively, compared to T1 (Control), indicating more efficient feed utilization. Additionally, mortality rate in T3 (PEPIGRO) decreased significantly by 28.08% compared to T1 (Control), reflecting better overall health and survival. These results suggest that PEPIGRO supplementation positively impacts growth, feed efficiency, and mortality compared to Control.

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Venue: Barshi, Solapur District | Date: 24th December 2025

A lively gathering of poultry farmers marked the technical outreach seminar organized by Stallen South Asia Pvt. Ltd. at Barshi, Solapur district, Maharashtra on 24th December 2025, where more than 50 broiler, Breeder and layer farmers came together to learn, share, and discuss better ways to manage flock health.

Held under the theme “Understanding Common Poultry Diseases: Causes, Prevention and Practical Management,” the program created an open platform for farmers to interact directly with technical experts and exchange real farm experiences.

The session opened with Mr. Macchindra Shinde, Regional Sales Manager (West), welcoming the farmers and encouraging them to make such programs a part of their learning journey. He stressed that progress in poultry farming comes not only from hard work, but also from staying informed and adopting the right practices. Mr. Rahul Ankushe, Area Sales Manager, Stallen South Asia Pvt. Ltd., actively supported farmer coordination throughout the program.

The technical talk by Dr. Kishor Gedam, Product Manager, Stallen South Asia Pvt. Ltd., focused on everyday challenges faced by poultry farmers. He spoke about common diseases such as CRD, Colibacillosis, Infectious Coryza, and Salmonellosis, while repeatedly emphasizing prevention over treatment, strong biosecurity, and judicious antibiotic use as the cornerstones of successful farming.

Rather than just a lecture, the session turned into an interactive discussion, with farmers sharing their concerns and field situations. Dr. Gedam addressed their queries with practical insights, making the session highly relatable and engaging.

The warm response from farmers reflected their eagerness to adopt better practices and improve farm performance. Many participants expressed that such interactions help them gain confidence in decision-making and reduce avoidable losses.

The seminar stood as a reflection of Stallen South Asia Pvt. Ltd.’s farmer-first approach, reinforcing its commitment to walk alongside poultry farmers by providing not just solutions, but also the knowledge needed to build healthier flocks and more resilient farms.

Venue: Tembhurni, Solapur District | Date: 23rd December 2025

Stallen South Asia Pvt. Ltd. organized a poultry farmer awareness seminar at Tembhurni, Solapur district, Maharashtra, on 23rd December 2025, as part of its ongoing farmer-centric technical outreach initiatives. The program witnessed the participation of over 50 broiler and layer farmers from nearby poultry pockets, reflecting strong interest in practical disease prevention and farm management.

The seminar was conducted under the theme “Understanding Common Poultry Diseases: Causes, Prevention, and Practical Management.” The event began with a welcome address by Mr. Macchindra Shinde, Regional Sales Manager (West), Stallen South Asia Pvt. Ltd., who emphasized the importance of continuous knowledge sharing and industry–farmer collaboration to strengthen flock health and productivity. The session was supported by Mr. Rahul Ankushe, Area Sales Manager (West), Stallen South Asia Pvt. Ltd., who coordinated farmer participation.

The technical session was delivered by Dr. Kishor Gedam, Product Manager, Stallen South Asia Pvt. Ltd., who addressed farmers on common poultry diseases including CRD (Mycoplasmosis), Colibacillosis, Infectious Coryza, and Salmonellosis, along with the importance of biosecurity and antibiotic stewardship. The focus remained on prevention, right farm practices, and responsible health management as the foundation of profitable poultry farming.

It highlighted that disciplined hygiene, water sanitation, shed management, and movement control can significantly reduce disease challenges, while judicious use of antibiotics is essential for long-term sustainability of the industry.

The seminar saw good interaction, with farmers actively raising practical questions related to their on-farm challenges. The discussions helped bridge technical recommendations with field realities.

This interactive seminar received positive feedback from participants, who appreciated the simple and practical approach of the session. The event reaffirmed Stallen South Asia Pvt. Ltd.’s commitment to supporting poultry farmers through knowledge-driven engagement and technical outreach aimed at building healthier and more sustainable poultry operations.