Probiotics & Their mode of Action

Probiotics have a range of documented benefits for animal health and production. The efficacy of a probiotic effect often depends on the different modes of action by which they exert their activity:

Production of secondary metabolites
  Bacillus Spp. have the capacity to produce a large variety of secondary metabolites, which shows that Bacillus Spp. have advantages to combat against different pathogens. Based on the genome analysis of various Bacillus Spp. strains, it can be concluded that although most of Bacillus Spp. strains encode the gene cluster responsible for the transcription of various secondary metabolites, the actual production of these compounds can only be detected in specific strains. For example, all analyzed B. amyloliquenfaciens strains encode the macrolactin gene cluster, but only detected in one B. amyloliquefaciens strain. Similarly, all B. subtilis strains encode the unique molecule subtilomycin, but can only be detected in specific Bacillus Subtilis Strains.
“Quorum sensing” (QS) is a bacterial communication system based on production and secretion of small signal molecules called auto inducers, accumulated in the extracellular environment when a high bacterial cell density is reached, the signal molecule triggers the synchronized expression of multiple genes in the population, thus regulating important biological functions such as the transfer of plasmids, motility, aggregation, luminescence, antibiotic biosynthesis, and virulence. The best characterized autoinducers are N-acylhomoserine lactones (AHLs), a family of molecules formed by a ring of homoserin lactone (HSL), N-acylated with a fatty acyl group in the alpha position. The mechanisms leading to the inactivation of the QS communication system have been generally referred to as “Quorum Quenching” (QQ), although some authors prefer to restrict the term to enzymatic degradation of the AHL signals.
One of the possible mode of action of probiotics is through QQ to block the communication (i.e., quorum sensing) between pathogenic bacteria, thus preventing their outgrowth, biofilm formation and expression of virulence. Therefore, an in vitro study was conducted to assess the QQ activity of one our selected Bacillus Spp. strain (Bacillus Spp. strain A), which has an inherent capacity to produce a wide range of secondary metabolites that interact with different bacterial populations.
The assay performed using Chromobacterium violaceum, as a specific biosensor because it is microorganism that produces a violet pigment when QS is activated, allows the easy visualization (via inhibition of the violet pigment production) of the capacity of any compound to block induction of QS. The strain used in the study was a mutant of this species, which has lost its ability to constitutively produce the purple pigment and is therefore known as a white mutant (CV026). In this mutant, however, the occurrence of violacein can be induced by applying AHLs N-acylhomoserin lactones as inducers) to the culture medium. Supernatants from different incubation times of Bacillus Spp. strain A, grown in the presence of inducers C6 (N-hexanoyl-L-homoserine lactone, were analysed. The well with water (A), culture medium (M) or culture medium with Bacillus Spp. strain A alone (E0-24 hours) did not produce any violet pigment (Figure 1).

This was expected as these wells did not contain inducer C6, and Bacillus Spp. strain A alone does not produce any compound stimulating production of violacein in the biosensor C. violaceum. When media in combination with the C6 inducer was incubated for 24 hour and added to wells (0-24 M) a pigmented halo was consistently observed (Figure. 1).
This suggests that the C6 inducer does not degrade over time as the violet pigmented halo was observed within the first 6 hours and even after 24 hours incubation. However, the 24h incubation supernatant with Bacillus Spp. strain A inhibited the violacien production, indicating that Bacillus Spp. A, has QQ activity.

Inhibition of Pathogens
Clostridium perfringens
        All probiotic products should a defined and proven mode of action, demonstrating their ability in modulating the gut health of the animals. An in vitro study was conducted to test the inhibition of different pathogenic C. perfringens strains were α, β2 and netB toxin positive. The growth of all C. perfringens strains, was considerably inhibited by Bacillus Spp. strain A and B (Table 1).
In addition, several in vivo studies have been done under different challenging conditions in broiler chickens. A study using a necrotic enteritis (NE) model were Emeria maxima oocysts were inoculated at day 12 followed by a C. perfingens gavage challenge at day 16, demonstrated that Bacillus Spp. strain B supplementation to broiler diets was able to reduce feed conversion ratios and obtain heavier body weight when compared to the control diets. Additionally, molecular analyses of microbial populations in the ileum and cecum of the treated birds showed that feeding Bacillus Spp. B consistently increased (P<0.05) populations of Bacillus Spp. and decreased the populations of C. perfringens (Figure 2).

Figure 2: Feed Bacillus Spp. strain B significantly reduced pathogenic strains of Clostridium perfringens in the Ileum and cecum of broiler chickens.

Another study had as objective to determine the immunomodulatory effect of Bacillus Spp. strain A when supplemented to broiler chickens exposed to a NE challenge, using the Gholiamiandehkordi method. This method consists on immunosuppressing the birds using a Gumboro vaccine (CEVAC Gumbo L, Ceva-Phylaxia), followed by a cocci vaccine on day 19 (Paracox-5, Ceva-Phylaxia), and then challenged with a 2 mL of C. perfringens isolate (6-8x 108 CFU) on day 18, 19, 20 and 21. Bacillus Spp. strain A was able to reduce the gut lesions produced by C. perfringens by 1 point (Figure 3).

In this same study, it was demonstrated that the supplementation of Bacillus Spp. strain A is able to modulate the immune response of birds by activating or not the response depending on whether the birds were exposed to a particular challenge. Broiler chickens fed with the probiotic without a pathogenic challenge of C. perfringens (CP) had a significant up-regulation of IL-1β expression (P=0.042) but no other cytokines were stimulated, resulting in a probable higher basic concentration of IL-1β which shows that feeding probiotics help to maintain a “healthy or prepared” immune system (Table 2). Furthermore, feeding Bacillus Spp. strain A to CP challenged birds resulted in an up-regulation of not only IL-1β (p=0.044) but also of IL-2 (p=0.003) and IL-8 (p=0.012). These observations indicate that the supplementation of Bacillus Spp. strain A can result in different responses with respect to the expression of different cytokines depending on whether there is a pathogenic challenge or not, ensuring a smart way of allocating energy resources according to the needs of the animal.

Clostridium perfringens
Similar in vitro proof of concept studies have been done in order to determine the bactericidal effect of Bacillus Spp. strain A. Inhibition tests have been performed following the methodology described by Cintas et al., 1995; consisting on measuring the diameter of the growth inhibition halo of the pathogenic bacteria through the well that contains supernatants with metabolites produced by Bacillus Spp. A. Clear inhibition halos were obtained (>10 mm)with the supernatants for the following strains: E. coli CECT 35218, E. coli CECT 501, S. enterica CECT 722, S. enterica CECT 443, S. enterica CECT 7161, S. typhimurium 301/99 (Table 3). The studies concluded that Bacillus Spp. strain A has a clear bactericidal effect against the listed E. coli and Salmonella strains (Table 3).

Pathogen	15h		36h
	pH4	pH6	pH4	pH6
S. enterica CECT 722	22	9	23	11
S. enterica CECT 7161	23	-	19	13
S. typhimurium CECT301/99	22	10	21	10
S. enterica CECT 433	33	11	31	13

    Probiotics are used to balance the intestinal microbiota and therefore help to decrease carcass contamination with pathogenic bacteria such as Salmonella enterica serovar Enteritidis (S. enteritidis) potentially causing a foodborne illness. Two studies were conducted with the objective to delineate the effects of feeding Bacillus Spp. strain A as a preventive tool against S. enteritidis infection and to evaluate the immune response under challenged conditions in broiler chickens. Analyses of cloacal samples showed that, during the entire trial period, a significant proportion of birds in the control group shed Salmonella spp. with Bacillus Spp. strain A had a consistent reduction (p< 0.001) of S. enteritidis, shedding only after the third week post challenge. In the control group, Salmonella spp. was found in the caecal and crop samples in more than 90% of the birds, and 20% of the cases showed that this colonization reached spleen and liver. Salmonella enteritidis colonization was therefore significantly reduced in caecum, crop and liver in birds supplemented with Bacillus Spp. strain A. In a second study, birds with the same Bacillus Spp. strain A, and challenged with a S. enteritidis isolate had reduced (p=0.02) Salmonella counts in the caeca and litter increased (p=0.008) goblet cell numbers in both ileum and caecum. Additionally, these supplemented birds had lower CD8+ cell counts at day 35 of age when compared to the control birds.

Modulation of intestinal microbiome

Performance improvements (body weight and feed efficiency) can be correlated to changes in gut microbiota populations. Cecum digesta samples, analyzed with %G+C microbial profiling (which fractionates bacterial chromosomes based on the % of guanine and cytosine in DNA) revealed significant differences in the micrbiome population profiles between a negative controls (chickens without any kind of supplementation) and chickens fed a Bacillus Spp. B strain probiotic diet as shown in low (27.0-34.5%), mid (40.5-54.0%) and high (59.0-68.0%) %G+C fractions (Figure 3). Furthermore, 16S rRNA gene amplification and next generation sequencing analyses were run in order to eludicate specific bacterial family and species changes. Results show that feeding the Bacillus Spp. strain B resulted in a higher abundance of Lactobacillacea family members than the control. From Lactobacillacea family there was a greater abundance of Lactobacillus salivarus (p=0.01) and Lactobacillus johnsonii (p=0.01). Cecal abundance of Lachnospiraceae was reduced in the probiotic treatment compared to the control (p=0.04). Some of these bacterial species are known to be mucin degrading bacteria. It can be hypothesized that these microbiome changes are a probable explanation to the positive growth performance observed in the studied broilers.

Conclusions
• Probiotics described in these studies offer an effective and sustainable replacement to AGPs in the maintenance of healthy flocks and the optimization of performance in poultry.
• Bacillus based probiotics described in these studies are natural highly resistant spore-forming bacteria that are stable under feed processing and storage.
• Quality of probiotic products remains an important factor in order to obtain consistent results.
• Selected probiotics can reduce the threat of pathogenic bacteria colonization of gut, resulting in a more balanced intestinal microbial population and improved growth performance.
• Probiotic supplementation supports food safety by improving intestinal health of the supplemented animals, reducing colonization of Salmonella spp. from internal organs, reducing shedding in the litter and environmental contamination as well as reducing potential carcass contamination in the slaughter house.
• Probiotics can have a positive effect on the gut microbial populations resulting in positive zootechnical performance.

By: Kiran Doranalli (kiran.doranalli@evonik.com), Stefan Pelzer and Alvaro Ortiz are with Evonik Nutrition and Care GmbH, Germany.
References are available on request to Dr. Dornalli. 

Palatability - an important factor in dairy nutrition

The use of flavors in animal nutrition can be traced back for more than 150 years ago, with the main purpose to increase feed intake. For example, in the UK ground aniseed was sprinkled onto cattle feed to improve palatability already in the early twentieth century. Among all species, cows are known to have the most taste buds with about 25,000 compared to humans (7,000) and dogs (only about 1,700). Thus, using flavors to ensure feed palatability is a viable means to enhance feed intake and performance.

            One of the challenges for the nutritionist is to create nutritious, economically viable yet palatable feeds. However, nutrition can only begin when the feed is consumed by the animal. Flavors can be used to provide more flexible choice of raw materials to be used. A wide range of by-products available to feed formulation allows nutritionists to create more economical feed rations.

Table 1: Factors that reduce palatability.

Bitter tastes, high levels of these ingredients that contain these compounds can depressed feed intake: Tannins (Sorghum) Alkaloids (lupins) Glucosinolates (rapeseed meal) Saponins (beans, peas, alfalfa) Spoiled foods Toxins

Minerals    Calcined magnesite (MgO)    Zinc oxide (ZnO)

Medication

            Taste is important as the first line of defense for animals to select their feeds, and initiate and stimulate the feeding response. Taste as sensory response invoked by feed is either reflected in intake rate when only one choice of feed is given or by preference the animal has when there is choice. The taste signalizes the animal previous post-ingestive effects associated with its sensory properties and also interacts with the nutrient requirements of the animal.

            When it comes to dairy cow production, the main goals are to produce more milk of high quality and healthy calves. In order to reach the goal of having high yielding cows, the good health of the calf is a predisposing factor. The better the feed intake the more likely that the calf is and remains healthy. Looking at the lactating cow, an adequate feed intake is the key to high milk yield and therefore the economic success of the operation. However, a high dry matter intake (DMI) is not just the key to support milk production, but also prevent metabolic diseases and fertility. Flavor can be strategically used during all production stages to stimulate appetite and increase DMI.

Mammals are much more sensitive to bitter tastes. Even palatable ingredients can taste poor if not fresh.

Palatability response of calves

A trial was carried out on three different farms in France with Broutard calves. The experiment on Farm 1 included 24 animals aged between 1 to 1.5 months; Farm 2 had 12 animals aged 1 month and Farm 3 used 20 animals aged 4 months, a total of 56 calves. The calves were kept on grass with the cows and were offered a choice of two different starter feeds over a period of 20 days. One was common starter feed and the other feed contained the flavor at 400g/ton. During the trial period feed consumption of each feed was measured daily.

Table 2: Average daily feed intake of each starter feed when offered as a choice over 20 days.

Farm (months of age)	Control (g/day)	Flavor (g/day)	Difference
Farm 1 (1-1.5)	600	900	+50%
Farm 2 (1)	400	650	+53%
Farm 3 (4)	1,200	1,670	+39%

            The addition of Flavor increased average daily feed intake (Table 2). Thus, it can be concluded that addition of Flavor to starter diets can increase average daily feed intake for young calves.

Palatability response of milking cows

The trial was carried out at a commercial unit in Italy equipped with a Lely Astronaut milking robot milking 66 cows. The cows were not visiting the milking robot frequently enough, so three weeks after trial began, Flavor was added to the feed offered during milking to make it more attractive to cows. The differences in number of milkings and refusals (cow returns to milking machine too soon after being milked and is refused entry) were evaluated.

Table 3. Average number of daily milkings and refusals.

	No Flavor	Flavor	Difference, %
Av. Daily milkings/cow	2.54	2.75	+8.3
Av. Refusals/day	30.0	75.3	+151

            The average number of milkings per cow and day that clearly illustrates an increase in number of milkings after introducing the flavor to the feed, the number of milking per cow increased from 2.54 to 2.75 times per day (Figure 1). In contrast, after the inclusion of the flavor, the number of refusals increased dramatically from 30 to 75.3 times per day (Figure 2).

  Figure 1: Average milking per cow per day during the trial.

 Figure 2: Number of refusals (attempts to go inside milking robots) per day. 

Conclusion

Calves and milking cows showed good response in terms of feed intake and their performance following the addition of the flavor. This indicates that flavors are very important for the animals as they are the cornerstone of the feeding behavior, to stimulate the feeding response, improve palatability and acceptability and lastly, offer nutritionists flexibility to use a wider range of feed raw materials.

By:
Peache Deleon & Kevin Liu

Beta-Glucans: A Crucial Weapon to Modulate Poultry Immune System

Introduction
With continuous increase in the economic value of the poultry industry, a deeper understanding of the nature and functioning of the avian immune system is important. The immune system enables the body to mount a defense against foreign organisms and antigens. However, the chicks’ immune system is underdeveloped and immature immediately after hatching, rendering them highly vulnerable to infectious threats present in the environment. The nutritionally balanced feeding program along with antibiotic growth promoters (AGPs’) in poultry diets played a significant role in achieving faster production success. Nevertheless, faster growth is likely to have a negative impact on the immune system due to decreased tolerance towards infectious diseases, thereby increasing the incidence of early chick mortality. In addition, the indiscriminate long-term usage of antibiotic growth promoters created a threat of bacterial resistance with major impacts on the future efficacy of these essential drugs. Consequently, due to the emergence of poultry welfare and environment protection concerns several countries have banned or reduced the prophylactic use of antibiotics as feed additives. This reduced the use of antibiotics in poultry production system, increased the demand for finding of alternate feed compounds along with a nutritious diet that can enhance gut health and immune function.
To address this need, a wide range of products are available in the market today which can help to modulate the innate immune system. One innovative approach that has been thoroughly studied is the use of beta-glucans in poultry diets. Although reports concerning the significance of beta-glucans in poultry are gaining popularity, still there seems to be some confusion in the poultry producers’ mind as to which source is better, how much is needed to improve immunity and what price is fair. The aim of this review was to give guidance on the major types of beta-glucans, their functional properties, and the option in choosing the best source with the potential to modulate the immune response in birds.
Sources and Structure of Beta-glucans
Beta-glucans are naturally occurring polysaccharides found in the cell walls of bacteria, fungi, yeast, algae, and cereals (such as barley, oats, and rye). However, the structure of beta-glucans varies among the different sources and considers the variations in their physiological role as shown in Figure 1. Beta-glucans consist of beta-D-glucose molecules linked by (1,3) or (1,4)-glycosidic bonds. Glucan obtained from bacteria and algae shows a 

linear structure, whereas, beta-glucan extracted from yeast, mushrooms, oats, and barley exhibits branched structure. Fungal beta-glucans have short branches, while they are long in yeast. The beta-glucans are not branched in barley, as with most cereal grains, but linked with twisted linear beta-(1,4) and beta-(1,3) glycosidic bonds.

Mechanism of Action and Functionality of Beta-glucans
The immunomodulatory ability of Beta-glucans has received increasing attention as one of the potential alternatives to AGPs without adversely affecting the bird performance. Beta-glucans belong to a group of physiologically active compounds termed biological response modifiers, because of their ability to stimulate the immune system. Beta-glucans such as algal beta-glucans are not naturally present within animal cells. When animals are supplemented with beta-glucan, it enters the small intestine and pass through the Peyer’s Patches in the gut-associated lymphoid tissue (GALT). Their immune cells (Phagocytes) recognize the beta-glucan molecules as a pathogen-associated molecular patterns (PAMPs) through a specific set of receptors called pathogen recognition receptors (PRRs). The PRRs such as Toll-like receptors (TLRs) and Dectin-1 present on the surface of phagocytes, including macrophages, heterophils, dendritic cells and natural killer cells primarily facilitate the activation of innate immune system through a cascade of signaling pathways resulting in enhanced phagocytosis of foreign pathogens (Figure 2).

Furthermore, beta-glucans have demonstrated the ability to augment the secretion of several cytokines and have antitumor, antibacterial and antiviral effects. A reported significant increase in both Newcastle Disease (ND) and Infectious Bronchitis (IBV) specific antibody titers in birds supplemented with beta-glucan was also observed. An increase in the size of primary and secondary lymphoid organs had shown with dietary beta-glucan supplementation, providing further evidence of their immunomodulating capabilities. As a result, beta-(1,3)-glucan can improve the resistance to infections by enhancing: (i) non-specific immunity that can protect animals from infection, (ii) host defense mechanism, and (iii) growth rate and mortality reduction. Thus, beta-glucan may be used as a replacement for dietary antibiotics in animal feed in combination with one or two other AGP alternatives.
Most Proficient Beta-glucan for Poultry
A wide structural variation in the functional properties of beta-glucans is directly related to their origin/source, which may influence their efficacy in modulating the immune systems. Before the beta-glucans can be incorporated in poultry diets, one must first understand the function and effectiveness of different beta-glucans on the avian immune system and the similarities and differences with the mammalian system. The features and differences between beta-glucans from yeast, oat & barley, and algae were further described below.
It is generally accepted that beta-(1,3)-(1,6)-glucans derived from yeast and fungi are considered the most effective source in terms of stimulating the immune system due to their highly complex branched structure. Numerous investigators have widely studied the effects of yeast cell wall beta-glucans on broiler performance as an immune modulator against infectious agents compared with antibiotic growth promoters. Whereas, the immune-modulating effect of beta-(1,3)-(1,6)-glucan from yeast depends on its native molecular structure, which is immersed in the other cell wall components of yeast beta-glucans and must be released in its intact form by an appropriate isolation technique. However, the extraction techniques developed by various manufacturers are laborious and result in a large amount of variability and inconsistency in the final products. Thus, the beta-glucans isolated from Saccharomyces cerevisiae (baker’s yeast) achieve an optimized yield of only 5–7 % dry weight due to a complicated process of chemical degradation of glucans. In the same way, the low molecular weight (around 190-200 kDa) and degree of polymerization (1500) of beta-(1,3)-(1,6)-glucan from yeast imparts less binding affinity and is responsible for minimal or no biological activity.
Whereas, cereal beta-glucans, such as oat and barley fall short as immune regulators, because they are structurally distinct than those of fungal and yeast beta-glucans and are not recognized as PAMP by the immune system of animals. The researchers also concluded that chicks fed with barley beta-glucans in the corn-based diet result in poor chick performance due to the increase in viscosity of the intestinal contents. This adversely affects the nutrient digestion, absorption, and composition of microflora in the gut by altering the intestinal morphology, decreasing endogenous enzyme production, and increasing susceptibility to disease. Various studies acknowledged mixed performance and immune response discrepancies in the published results with yeast-derived beta-glucans. Similarly, based on the comprehensive analysis of various studies, the content of beta-glucan from different sources, which gave a clear idea of the concentration of this valuable ingredient is shown in Table 1.

The numerous inconsistencies and varying results with fungal and yeast cell wall, and cereals derived beta-glucan opens the way for research expertise to assess the efficacy of alternative beta-glucan sources in achieving consistent results. Recently, another source of beta-glucans that has gained increasing attention by industry manufacturers is paramylon, an algal beta-glucan from the microalgae Euglena gracilis. In contrast to yeast, this algae has a high concentration of beta-(1,3)-glycosidic linkages and does not contain branches of beta-1,6 branches that are typical of yeast beta-glucan products. In contrast to yeast, the algae beta-(1,3)-glucan (Paramylon) has a high molecular weight (larger than 500 kDa) and is responsible for high biological activity along with greater binding affinity. Hence, this linear high molecular weight beta-(1,3)-glucan, when cultivated under optimal conditions, accumulates more than 90% of the cell mass as paramylon, and it does not require any expensive extraction methods like that of yeast beta glucans. Similarly, the linear structure and small particle size (1-3 microns) of algal beta-(1,3)-glucan interact directly with immune cells, while the branched structure and other cell material from yeast cell wall beta-(1,3)-(1,6)-glucan clump together. Another major factor that has traditionally generated more research interest is the higher bioavailability and ease in production of the algal beta-glucans when compared to branched beta-glucans. Since the branched beta-(1,3)-(1,6)-glucan in yeast is bound to other components of the cell wall, such as chitin and mannoprotein, and not available to be taken up by gut-associated lymphoid tissues such as Peyer’s patches. In addition, the beta-glucan product produced from the algae was more consistent and cost effective as it exists as granules within the algal cell and does not require to be extracted due to the lack of a thick cell wall.
Conclusion
It is concluded that all the beta-glucans are built from the same building block of polysaccharides, but the activity of different beta-glucan ingredients depends first and foremost on the source organism from which the compound is isolated, ultimately on the method of isolation and structure of beta-glucans.Though the beta-(1,3)-(1,6)-glucan from baker’s yeast is found to have some immune modulation action, it has its own deficiencies in terms of poor bioavailability, poor recognition by the immune cells, and broader dose range. With the more knowledge on the efficacy of beta-(1,3)-glucan in modulating immunity, the algal beta-glucan is in use these days with high promising results. The algae Euglena gracilis can accumulate extremely at high concentrations of beta-(1,3)-glucan paramylon intracellularly, it offers the function of immune modulation at a more economical cost and represents as one of the efficient AGP alternative products for usage in the poultry industry.

Antibiotics Details with their Withdrawal Time Period

Salt	Description	Mode of Action	Withdrawal Time
Avilamycin	Avilamycin is an orthosomycin antibiotic complex produced by the fermentation of Streptomyces viridochromogenes. Avilamycin is primarily active against gram-positive bacteria.	Avilamycin inhibits protein synthesis. It is thought to bind to 50S ribosomal subunit. It prevents the association of IF2 which inhibits the formation of the mature 70S initiation complex and the correct positioning of the tRNA in the aminoacyl site.	NIL
Bacitracin (ZB, BMD)	Bacitracin is a mixture of related cyclic peptides produced by organisms of the licheniformis group of Bacillus subtilis var Tracy, These peptides disrupt Gram-positive bacteria by interfering with cell wall and peptidoglycan synthesis.	Bacitracin interferes with the dephosphorylation of C55-isoprenyl pyrophosphate, also known as bactoprenol, a membrane carrier molecule that transports the building-blocks of the peptidoglycan bacterial cell wall outside of the inner membrane	It does not absorb in the intestine. So, it has no WHP
Chlortetracycline	Chlortetracycline belong to one of the important classes of broad-spectrum antibiotics. Two of the more common semisynthetic tetracyclines that have been marketed for decades are doxycycline and minocycline. The tetracyclines inhibit bacterial growth by inhibiting bacterial protein synthesis by preventing the association of aminoacyl-tRNA with bacterial ribosomes	Chlortetracycline, like other tetracyclines, competes for the A site of the bacterial ribosome. This binding competes with tRNA carrying amino acids preventing the addition of more amino acids to the peptide chain. This inhibition of protein synthesis ultimately inhibits growth and reproduction of the bacterial cell as necessary proteins cannot be synthesized.	It has withdrawal time of 1-5 days
Colistin Sulphate	Colistin, also known as polymyxin E, is an antibiotic produced by certain strains of the bacteria Paenibacillus polymyxa. Colistin is a mixture of the cyclic polypeptides colistin A and B and belongs to the class of polypeptide antibiotics known as polymyxins. Colistin is effective against most Gram-negative bacilli.	Colistin is a polycationic peptide and has both hydrophilic and lipophilic moieties. These cationic regions interact with the bacterial outer membrane, by displacing magnesium and calcium bacterial counter ions in the lipopolysaccharide. Hydrophobic/hydrophilic regions interact with the cytoplasmic membrane just like a detergent, solubilizing the membrane in an aqueous environment. This effect is bactericidal even in an isosmolar environment.	7 Days
Enramycin	Enramycin or Enduracidin is a polypeptide antibiotic produced by Streptomyces fungicidus. Enramycin is widely used as a feed additive for pigs and chickens to prevent necrotic enteritis induced by Gram-positive gut pathogens. It improves weight gain and feed conversion	Enramycin acts as an inhibitor of the enzyme, MurG, which is essential for cell wall biosynthesis in Gram-positive bacteria. MurG catalyzes the transglycosylation reaction in the last step of peptidoglycan biosynthesis. Inhibiting this step greatly compromises cell wall integrity leading to cell lysis.	7 days
Flavomycin	Bambermycin (flavomycin) is a complex of antibiotics obtained from Streptomyces bambergiensis and Streptomyces ghanaensis used as a food additive for poultry. Bambermycin is predominately effective against Gram-positivepathogenic bacteria	Flavomycin inhibits the transglycosylation step of peptidoglycan biosynthesis, a structural component of the bacterial cell wall. This causes accumulation of cell wall intermediates, and leads to lysis and cell death.	Nil
Lincomycin	Lincomycin is a lincosamide antibiotic that comes from the actinomycete Streptomyces lincolnensis. Lincomycin is a narrow spectrum antibiotic with activity against Gram-positive and cell wall-less bacteria including pathogenic species of Streptococcus, Staphylococcus, and Mycoplasma	Lincomycin inhibits protein synthesis in susceptible bacteria by binding to the 50 S subunits of bacterial ribosomes and preventing peptide bond formation upon transcription.	Nil
Neomycin	A component of neomycin that is produced by Streptomyces fradiae. On hydrolysis it yields neamine and neobiosamine B.	Neomycin is a bactericidal aminoglycoside antibiotic that binds to the 30S ribosome of susceptible organisms. Binding interferes with mRNA binding and acceptor tRNA sites and results in the production of non-functional or toxic peptides.	Nil
Nosiheptide	Nosiheptide is a sulfur-containing cyclic polypeptide, is highly active against Gram-positive bacteria and few Gram-negative bacteria, especially Clostridium Perfringens, Staphylococcus Aureus. Nosiheptide acts bacteriostatically in low dose and bactericidally in high dose.	Nosiheptide inhibits bacterial protein synthesis by inhibiting function of elongation factors Tu and G and greatly reduce the synthesis of guanosine penta- and tetraphosphates in response to stringent factor.	7 Days
Tylosin	Tylosin is an antibiotic and a bacteriostatic feed additive used in veterinary medicine. It has a broad spectrum of activity against Gram-positive organisms and a limited range of Gram-negative organisms.	Tylosin has a bacteriostatic effect on susceptible organisms, caused by inhibition of protein synthesis through binding to the 50S subunit of the bacterial ribosome.	5 Days
Tiamulin Hydrogen Fumarate	Tiamulln is a semi-synthetic derivative of pleuromutilin. It is the only member of the pleuromutili ln class of the diterpenic antibiotics which is approved for use at mts time. It is typically available for oral use as the Tiamulin base or the hydrogen fumarate salt. In some references Tiamulln is considered with the macrolide group of antibiotics.	Tiamulin affects bacterial protein synthesis. It has a strong affinity for the 50S ribosomal sub-unit resulting in breakdown in the peptide chain immediately following initiation.	3 Days
Virginiamycin	Virginiamycin is a streptogramin antibiotic similar to pristinamycin and quinupristin/dalfopristin. It is a combination of pristinamycin IIA(Virginiamycin M1) and Virginiamycin S1.	virginiamycin M blocks the elongation of polypeptide chains by inhibiting both the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu (EF-Tu) and the peptidyl transferase reaction.	Nil