Probiotics - Next-Generation Therapeutics in the poultry industry

PROBIOTICS NEXT-GENERATION THERAPEUTICS against mycotoxicosis and other microbial complications in the poultry industry

S. Divyashree and M. Y. Sreenivasa Applied mycology lab, DoS in Microbiology, University of Mysore, Mysore, India Email: sreenivasamy@gmail.com; mys@microbiology.uni-mysore.ac.in

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Poultry has developed into an essential economic activity and a major agricultural sector worldwide. Poultry feed is one of the most expensive items and it accounts for up to 70% of total poultry production outlay.

Microbial complications are one of the most significant aspects of poultry production, causing substantial economic losses globally.

In this article, we review several microbial complications and their current prevention strategies, mainly the use of probiotics as a next-generation therapeutic agent to combat harmful food-borne pathogens as well as their additional applications in poultry production.

Microbial infections in the poultry industry Microbial complications caused by bacteria,

These microorganisms lower the body’s defense mechanisms

viruses, fungi and their toxins have been

by creating an imbalance in the intestinal microbiome,

reported worldwide as a major problem

leading to the proliferation of more harmful pathogens

in poultry production and feed.

and causing physiological disturbances such as: Septicemia Meningitis

Bacterial contamination

Encephalitis Vomiting

Bacterial infections are the foremost cause

Diarrhea

of food-borne gastroenteritis in humans

Improper digestion

globally due to the consumption of contaminated poultry products (Weill et al., 2006) .

Contamination of poultry products with Salmonella spp., Escherichia coli, Listeria monocytogenes,

Campylobacter spp. and Clostridium perfringens can be traced back to the gastrointestinal tract (caeca), where they have high activity by effectively colonizing gut epithelial cells (Pascual et al., 1999) .

Loss of appetite and poor absorption of nutrients, which in turn reduces immunocompetence and productivity

They can also lead to autoimmune disorders, kidney failure, and temporary paralysis, possibly causing permanent nerve damage and death.

In the case of the poultry industry, these infections lead to heavy economic losses (Dharma et al., 2008).

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Fungal contamination

Fungal contamination, mainly with mycotoxins, i.e., toxic secondary metabolites produced by filamentous fungi or molds such as Aspergillus spp. (aflatoxins, ochratoxins), Fusarium spp. (deoxynivalenol, fumonisins, zearalenone, and trichothecenes), and Penicillium spp. (ochratoxin A and patulin), causes high mortality and morbidity rates in poultry production.

Due to their direct toxicity and their mutagenic, teratogenic, carcinogenic, and immunosuppressive effects, as well as their sporadic nature, they pose a significant risk in terms of detection and prediction, since they tend to accumulate in poultry feed (Hussain et al., 2010).

Viral contamination

In addition to bacterial and fungal pathogens, gastrointestinal viral pathogens are also implicated in food-borne infections.

The viruses present in respiratory secretions, feces, blood, and organs of live birds are believed to be the

A wide variety of viral diseases, such as avian pox

primary risk factor for human infections through the

(Avipoxvirus), Newcastle disease (Paramyxovirus),

consumption of poultry-derived products (Rodriguez-

infectious and quail bronchitis (influenza virus),

Diaz and Monedero, 2013). Hence, the need to control these microbial complications.

lymphoid leukosis (Retrovirus), Marek’s disease (chicken herpes virus), and infectious bursal disease (Birnavirus) occur in poultry species.

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Handling the current situation of microbial complications and related problems Antibiotic growth promoters (AGP), such as streptomycin, avoparcin, tetracycline, bacitracin, chlortetracycline, tylosin, neomycin, oxytetracycline, virginiamycin, trimethoprim/ sulphonamide, lincosamides, cephalosporins, etc. have been used in poultry production for the last five decades to control microbial diseases.

Many feed additives are known for their antimicrobial properties. Therefore, phytobiotics (plant-based feed additives), organic acids, chitin, medium (caproic acid, capric acid, lauric acid, and caprylic acid) and long-chain fatty acids (polyunsaturated fatty acids), prebiotics, enzymes, antimicrobial peptides, hyperimmune IgY, bacteriophages, and clay have been used as alternatives to replicate the effects of AGPs. Their positive effects include:

However, antibiotics and hormonal growth promoters in poultry

Prevention of microbial diseases

production have been widely

Immunomodulation

banned by the European Union

Improvement of the quality of poultry products

since 2006 (Jha et al., 2020) due to the growing evidence of transfer of antibiotic resistance genes and allergies from farm animals to humans (Vieco-Saiz et al., 2019).

AGPs have also been reported to alter the gut microbiome and, in consequence, the modulation of the immune system, as well as negatively affecting weight gain, substantially decreasing production. However, the elimination of AGPs has increased the incidence of animal diseases

(Bajagai et al., 2016; Jha et al., 2019).

Feed intake (FI) and feed conversion ratio (FCR) improvement However, these feed additives have several disadvantages, for example: Phytobiotics have toxic parts (Ogbuewu et al., 2020).

Organic acids have shown instability, corrosivity, and unpleasant smell, and can alter the lactic acid bacteria present in the gastrointestinal tract (El-Hack et al., 2020).

Khempaka et al. (2006) reported the reduction of digestibility and growth performance in chickens due to the use of chitin as feed additives.

Medium and long-chain fatty acids cause coronary heart

Under these circumstances, feed additives,

diseases and depression problems (Thapa, 2020).

mycotoxin binders and surfactants have been

Surfactants have bactericidal and bacteriostatic

proposed as alternative therapeutic promoters.

effects, but they can cause environmental pollution leaving traces in terrestrial and aquatic environments (Viegas and Sa-Correia, 1991).

Other feed additives are dose-dependent and show adverse side effects.

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Antibiotic Growth Promoters Phytobiotics

The most commonly employed detoxification method

Organic Acids

in the poultry industry is the use of multi-spectrum

Chitin Medium & Long-Chain Fatty Acids Prebiotics Probiotics Bacteriophages Antimicrobial peptides

mycotoxin binders (sequestering agents) in feed.

It is common to use a combination of Picrorhiza

kurroa, activated charcoal, hydrated sodium calcium aluminosilicate (HSCAS), mannan oligosaccharide (MOS), buffered organic acids, and antioxidants.

However, these compounds have less affinity towards mycotoxins (Huang et al., 2019).

Enzymes Hyperimmune IgY Surfactants

Considering all these drawbacks,

Toxin Binders

alternative biocontrol probiotic agents have shown their versatility

Figure 1. Different feed additives used in poultry production.

and compatibility to decrease the problems of bacterial resistance to antibiotics, prevent and control diseases, decrease mortality rates, and enhance the growth of farm animals. Hence, they may be useful in the poultry industry.

Probiotic-based therapy against microbial complications The term “Probiotic” is a Greek word that means “pro-life”. Probiotics have been gaining more therapeutic relevance over the years with increasing scientific knowledge about their association with intestinal health and immunity, leading to the overall well-being of animals/humans. They usually have a natural origin, coming from fermented food products or plant products and/ or from the intestinal tract of animals and humans (Clarke et al., 2012).

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FAO/WHO (Food and Agricultural

Organization/World Health Organization, 2001) defined these beneficial organisms as “Live microbes when administered in proper amounts beneficially affect the health of the host”.

Potential probiotic organisms are non-pathogenic and are generally recognized as safe (GRAS).

They are capable of resisting the effects of gastric/intestinal juices, showing: An antibiotic resistance profile Tolerance to bile Adhesion to intestinal epithelial cells Production of antimicrobial substances such, as bacteriocins, organic acids, esters, fatty acids, diacetyl, reuterin, reutericyclin and hydrogen peroxide that exhibits antimicrobial properties

(Coman et al., 2014) In addition, antagonism is one of the most important probiotic control strategies based on two basic mechanisms:

2. Immune modulation that involves the stimulation of host defense systems (Solis-cruz

1. Competitive exclusion:

et al., 2018; Patlan et al., 2019).

Competition for nutrients Production of antimicrobial compounds Prevention of the adhesion of pathogens to epithelial cells Reduction of toxin bioavailability

Hence, these GRAS status probiotics have been widely used as therapeutics against microbial complications.

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Competition for nutrients

Production of antimicrobial compounds Competitive exclusion Prevention of the adhesion of pathogens to epithelial cells Probiotic-based therapy against microbial complications

Reduction of toxin bioavailability

Figure 2. Probiotic-based therapy to combat harmful pathogens in the poultry industry.

Immune modulation

Stimulation of the host’s defense systems

Figure 3. The applications of probiotics in poultry production.

Combat harmful pathogens

Stimulation of immunity

Increased/Improved bone strength and body growth

Improved growth performance

APPLICATIONS OF PROBIOTICS IN POULTRY

Enhanced intestinal microbiota & morphology

Improved biochemical parameters

Decreased parasitic load, blood cholesterol & heat stress

Improved feed intake & feed efficiency

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Probiotics against harmful bacteria pathogens

Probiotics have been extensively tested and validated against bacterial pathogens.

Numerous articles have been published on the use of probiotics as an antibiotic replacement by reducing the growth of pathogens such as

Salmonella paratyphi (Divyashree et al., 2021), E. coli (Tsai et al., 2010), Listeria monocytogenes (Gil De Los Santos et al., 2012) and Clostridium perfringens (Santos, 2012) in in vitro and in vivo models. For example:

Saccharomyces cerevisiae, at 0.4% and 0.8% reduced the gastrointestinal microbial load of E. coli, Micrococcus spp., Klebsiella spp., Staphylococcus spp., Campylobacter spp, and Clostridium perfringens in layers (Hassanein et al., 2010).

Broiler chickens orally administered with Lactobacillus salivarius showed effective prevention of Campylobacter jejuni intestinal colonization (Saint-Cyr et al., 2017). In another study, feeding bacteriocins (250mg) from Lactobacillus salivarius and Paenibacillus polymyxa showed a reduction in C. jejuni colonization (Timbermont et al., 2010).

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A

B

C

Figure 4. Scanning electron microscopy of the effect of cell-free supernantant from probiotics on Salmonella biofilms.

A. SEM of Salmonella paratyphi ATCC 9150.

B & C. S.paratyphi treated with Lactobacillus spp.

A

B

Figure 5. Agar well diffusion assay of probiotics against bacteria food-borne pathogens.

A. Cell-free supernatant from probiotics on Escherichia coli.

B. Cell-free supernatant from probiotics on Salmonella paratyphi.

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Probiotics against fungi and their mycotoxins

Probiotics are well known for their antifungal activity and for inactivating mycotoxins in poultry.

A study conducted by Deepthi et al. (2017), reported a significant role of L. plantarum MYS6 in ameliorating the FB1-induced toxicity (fumonisin produced by Fusarium

verticilliodes) in the vital organs and subsequent oxidative stress in broilers.

Lacticaseibacillus brevis MYSN105 and L. plantarum MYS6 showed potential antagonistic activity by showing morphological alterations such as damaged mycelia and deformed conidia against fumonisin producing F. verticillioides and F. proliferatum, respectively, in in vitro assays (Somashekaraiah et al., 2021; Deepthi et al., 2016).

The cell-free supernatant (CFS) from Lactobacillus plantarum strain MYS44 was suggested as a potential detoxification agent due to its antifungal activity and its capacity to reduce aflatoxin B1 (34.2%) produced by Aspergillus parasiticus MTCC 411 (Rao et al., 2017).

A

B

Figure 6. Use of probiotics against Aspergillus species.

A. Antifungal activity of Lactobacillus against Aspergillus flavus.

B. Antifungal activity of Lactobacillus against Aspergillus parasiticus.

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1

2

3

4

5

6

7

8

A

B

Figure 7. Use of probiotics against Fusarium species. The antifungal activity of the LAB isolates was tested against F. sporotrichioides (1), F. equiseti (2), F poea (3), F. avenaceum (4), F. verticillioides (5), F. graminearum (6), A. parasiticus (7), and Rhizopus oryzae (8).

A. Fungal control plates after 7 days of incubation.

B. Antifungal activity of isolate Lactobacillus plantarum MYSRD 71 after 7 days of incubation against all the fungal species tested.

Several studies have been conducted to evaluate the mycotoxin neutralization capacity of several probiotic strains such as Propionibacterium,

L. rhamnosus GG, L. plantarum, L. casei, Bacillus species, and E. faecium (Danicke and Doll, 2010).

For example, Saccharomyces cerevisiae could adsorb zearalenone and ochratoxin under simulated gastrointestinal conditions in a mycotoxin binding assay (Armando et al., 2012).

In studies conducted by Patlan et al.

(2019), probiotic Bacillus spp. were able to biodegrade aflatoxin B1 in both in vitro and in vivo models of broiler chickens.

In another study, L. casei and L. reuteri reduced oxidative stress induced by aflatoxins in mice (Hernández-Mendoza et al., 2011).

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Probiotics for the control of viral pathogens

Probiotics have also been used to control viruses in poultry.

Administration of L. paracasei protected against rotavirus-induced diarrhea (Pant

et al., 2006), while a study conducted by Wen et al. (2009) with L. acidophilus and L. reuteri decreased rotavirus infections by enhancing IFN- ‫ ץ‬and IL-4 in serum.

Naseem et al. (2012) reported that broilers fed with a Lactobacillus protein supplemented diets had increased antibody titers compared with the controls against infectious bursal disease, influenza disease, and Newcastle disease virus.

Additional applications of probiotics in poultry Probiotic effects on growth performance

In poultry production, several strategies are used to improve the number of eggs laid, increase the egg weight, and improve the eggshell quality, egg yolk cholesterol, albumin quality, and meat quality.

It has been proposed that probiotics increase the production of short-chain fatty acids (SCFA) and the intestinal fermentation rate which provides nourishment for intestinal epithelial cells, which also leads to improved

The administration of probiotics with

mineral assimilation (Alagawany et al., 2018).

hens’ diets improves laying production by increasing nitrogen and calcium retentions,

A study conducted by Abd EI-Hack et al. (2017) showed

as well as daily feed consumption.

that supplementation with multi-strain probiotics increased egg production, including albumin and yolk weight, egg weight and size, and the eggshell strength and thickness when compared to the control group.

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Decreased parasitic load, blood cholesterol and significant effect on heat stress

Recently, researchers have shown that dietary supplementation of probiotics decreases the level of cholesterol, very low-density lipoproteins (VLDL), and triglycerides in the blood (Kanani et al., 2018; Shirley et al., 2017).

Lee et al. (2007) demonstrated that the supplementation of probiotics enhances the resistance against coccidiosis (growth depression) and reduces the level of Eimeria tenella and E. acervulina infection in poultry.

Heat stress increases pathogen colonization, and immunosuppression, promoting the onset of infections

(Ursell et al., 2012). However, feeding probiotics to broilers has been shown to mitigate the impact of heat stress by maintaining a healthy gut microbiota in stressful environmental conditions and increasing the length of villi.

Stimulation of immunity and improvement of biochemical parameters

Pathogens must overcome obstacles (the physical barrier of the epithelium, the inhibitory effects of the gut microbiota, and the host’s immune system response) to colonize the intestinal

Their benefits include increased

tract and successfully cause an infection.

adhesion of beneficial bacteria to the

However, probiotics are well known for the

intestinal mucosa, improved epithelial

modulation of intestinal environments.

barrier, and the inhibition of pathogen

They bind to the Toll-like receptors of

adhesion (Broom and Kogut, 2018).

sentinel cells (epithelial cells and dendritic cells of the intestine) and activate the NF-κB and MAP kinase pathways (Bai et al., 2013).

Numerous studies have shown the impact of probiotics on biochemical parameters, for example, improvement in the levels of blood glucose, omega 6 and essential

This activation causes the suppression or

polyunsaturated fatty acids (linoleic acid and linolenic

upregulation of genes that regulate antigen

acid) and propionate (SCFA) in egg yolk (Tang et al.,

presentation, as well as cytoprotective effects

2016; Ejtahed et al., 2012; Trautwein et al., 1998).

through immune activation, inflammatory response, and the expression of antimicrobial factors (Kemgang et al., 2014).

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Improved bone strength and increase in body growth, feed intake, and feed efficiency

Probiotics modulate the gut environment, enhance the barrier function, and promote the growth of poultry animals (Deepthi et al., 2017). By colonizing the intestine, they compete with pathogens for nutrients and prevent their establishment. However, these increase body weight, feed intake, and feed efficiency (Jadhav et al., 2015).

In a study, feeding with probiotic Lactobacillus (0.9Kg/ton) increased calcium and phosphorous retention. However, moderate and low-density feed (18% and 25% less calcium and phosphorus by recommendation of the National Research Council) represented

P

bone-breaking resistance and improved the retention of nutrients with lower nutritional levels when compared to control (Angel et al., 2005).

In addition, probiotic supplementation in poultry feed also improved the thickness of the medial and lateral wall of the tibia and tibiotarsal index, which are indicators of bone strength.

Influence of probiotics on the intestinal microbiota and morphology

Supplementation of probiotics for dietary treatment has been shown to influence intestinal villi length and decrease crypt depth by enhancing gut microbiota development for the control of pathogens population.

Therefore, crypts in enterocytes replace the cells that have been lost due to inflammation at the villus tip by continuous proliferation

(Cox y Dalloul.2009: Awad et al., 2009).

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CONCLUSIONS Probiotics may be efficiently used to control microbial complications in poultry. These beneficial bacteria may also be given to poultry, even if there are no microbial complications since they exert positive health benefits. Hence, probiotics may be considered next-generation therapeutics in the poultry industry.

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