Identifications of FloraMax®-B11 (FM-B11) lactic acid bacteria (LAB).
In intensive poultry production, a large number of antimicrobials are frequently employed to prevent (prophylactic use) and treat (therapeutic use) diseases, as well as for growth promotion (subtherapeutic use), in order to increase productivity. However, it has been reported that the use of antimicrobials at subtherapeutic doses is closely related to the increase in bacterial resistance and with the treatment failure. In addition to antimicrobial resistance, another problem derived from the use of antimicrobials is the presence of residues in animal products. Therefore, these problems and the ban of antimicrobial as growth promoters have prompted the poultry industry to look for alternatives with similar benefits to antibiotics. Among these alternatives, probiotics are one of the most widely studied and interesting groups. Hence, in the present chapter, the effect of probiotics and direct-fed microbial against foodborne pathogens and mycotoxins will be summarized.
- direct-fed microbial
- foodborne pathogens
- antimicrobial resistance
Since the discovery and application of penicillin in 1940, antibiotics have played an unprecedented role in the prevention, control, and treatment of infectious diseases in both humans and animals . However, in animal production, they have also been used at subtherapeutic doses . It is estimated that the global consumption of antibiotics in animal production could increase by 67% in the coming years  mainly because of the growing global demand for animal protein [2, 4]. Although it has been reported that in developed countries the total consumption of antibiotics has decreased by around 4%, consumption of antibiotics in the USA increased slightly . Furthermore, it has been reported that the amount of antibiotics used in animal production in the USA is 100–1000 times higher than human medicine, being used ~80–90% at subtherapeutic doses, and for prophylactic purposes, while the remaining 10–20% at therapeutic doses [6, 7].
The inclusion of antibiotics at subtherapeutic doses into the feed was generalized in the early 1950s, both in the EU and the USA since they could be used to prevent diseases and positively influence the promotion of growth and feed efficiency of animals [3, 8, 9].
Nevertheless, in the last decades, these practices have changed considerably due to the concern of the increase of bacteria resistant to antibiotics, since they can be transmitted zoonotically from animals to humans, causing serious problems in public health and even death because of the failure of the antibiotic at therapeutic doses . Furthermore, another problem for human health is the presence of antibiotic residues in animal-derived food, by the use of antibiotics for long periods of time, since it is associated in some cases with allergic reactions, imbalance of the intestinal microbiota, and especially, the development of antibacterial resistance .
Consequently, one of the measures taken in the face of the problems of bacterial resistance was the restriction of antibiotics at subtherapeutic doses in the EU in 2006  and the USA in 2017 , and although in countries as Mexico they have not been officially banned, the Ministry of Agriculture and Rural Development (SADER), through its decentralized administrative body, the National Health Service, Food Safety and Food Quality (SENASICA), has promoted initiatives to prevent their use since 2012 [14, 15, 16, 17]. However, as a consequence of this measure, the incidence of enteric diseases in animals has increased significantly , as well as the use of antibiotics, but at therapeutic doses for the purpose of controlling and preventing diseases, which could lead to a worse scenario of bacterial resistance [2, 19, 20, 21]. In this context, the European One Health Action Plan against antimicrobial resistance calls for the phasing out of routine prophylactic (Prevent) and metaphylactic (Control) antimicrobial use in animal production and investment in the research of new alternatives , since they could be regulated in the coming years.
Therefore, the poultry industry has been under pressure to seek and investigate new alternatives to reduce the problems of bacterial resistance, prevent and control diseases, reduce the mortality rate, and finally promote the growth of animals. Among these alternatives, the most popular are probiotics (yeasts or bacteria) since it has been reported that they can improve the performance [23, 24], as well as prevent and control enteric pathogens in poultry [25, 26, 27]. Furthermore, it has been reported that probiotics could be an interesting alternative to prevent and control the toxic effects of aflatoxins. For these reasons, the probiotic market has expanded rapidly and is expected to grow to around 7% in 2020. However, this market is led mainly by Asia and Europe given the growing demand for dietary supplements .
Probiotics are defined as “live strains of strictly selected microorganisms which, when administered in adequate amounts, confer a health benefit on the host” . The most common microorganisms used as probiotics in livestock production are lactic acid bacteria (LAB) from the genus
2.1 Mechanisms of action probiotics
2.1.1 Pathogenic bacteria
Although a large number of studies have shown the possible mechanisms by which probiotics have a beneficial action in inhibiting of pathogens, more studies are needed to elucidate them.
The possible modes of action of probiotics for the inhibition of pathogens include two basic mechanisms [29, 36, 37]: competitive exclusion and modulation of the host immune system (Figure 1). Competitive exclusion involves mechanisms such as (1) production of inhibitory compounds, that is, hydrogen peroxide, bacteriocins, and defensins [38, 39], (2) prevention of the pathogen adhesion , (3) competition for nutrients , and (4) reduction of toxin bioavailability . Meanwhile, in the modulation of the host immune system, both innate and adaptive immune responses are involved . The adaptive immune response depends on B and T lymphocytes to induce an antigen-specific response and produce antibodies [29, 41]. In contrast, physical and chemical barriers (innate immunity), such as intestinal epithelial cells (IEC), are the first line of defense to prevent the spread of pathogens and subsequent infections. Furthermore, IEC are the target cells for probiotics, which can improve the function of the intestinal barrier by stimulating the production of mucus and antimicrobial peptides such as defensins [42, 43].
Similar as for pathogenic bacteria, probiotics can (1) compete for space and nutrients with aflatoxigenic mold strains, (2) degrade aflatoxins by the production of enzymes, or (3) avoid the intestinal absorption of AFB1 by its binding to the cell walls of probiotic strains .
3. Probiotic application in poultry industry
Although probiotics are considered potential alternatives to antibiotic use in poultry because they leave no residues in the meats and eggs given their modes of action, the variety of microorganisms in terms of species and even between strains of the same species, as well as their variation in metabolic activity, could affect their effectiveness. Furthermore, other factors that influence the effectiveness of probiotics in poultry are the species of origin, the probiotic preparation method, the survival of colonizing microorganisms in the gastrointestinal tract conditions, the environment where the birds are raised, the application time and administration route of probiotics, the immunologic state, the lineage of poultry, as well as age and concomitant use of antibiotics [45, 46]. Below are some of the applications of probiotics in poultry.
3.1 Effects of lactic acid bacteria against
pathogensof importance in poultry
Several articles published by our laboratory have shown that the use of probiotics as a replacement of antibiotics in poultry production has had positive effects by reducing the growth of pathogens in
Extensive research conducted by our laboratory determined the antimicrobial capability of several lactic acid bacteria (LAB) isolates mainly against
|LAB identification||16S rRNA sequence analyses (Microbial ID Inc.)|
However, since these LAB were grown together in a culture, the only LAB that remained viable were
Furthermore, the effect of this commercial product (FloraMax®-B11) has been evaluated in different models of infection both in broiler chickens and turkeys. In neonatal broilers, the administration of 1 × 106 cfu/bird FloraMax®-B11 by oral gavage 1 h after the chicks were challenged with
|Rep.||Treatment||ST cecal tonsil +/− (%)||SE cecal tonsil +/− (%)||Log SE cecal recovery (all samples)||Log SE cecal recovery (only positive samples)|
|1||Control||20/25 (80)||22/25 (88)||3.81 ± 0.32||4.33 ± 0.17|
|LAB||2/25 (8)*||8/25 (32)*||0.62 ± 0.19*||1.95 ± 0.09*|
|2||Control||18/25 (72)||25/25 (100)||3.59 ± 0.23||3.59 ± 0.23|
|LAB||2/25 (8)*||7/25 (28)*||0.42 ± 0.18*||1.91 ± 0.29*|
|3||Control||20/25 (80)||25/25 (100)||3.91 ± 0.19||3.91 ± 0.19|
|LAB||1/25 (4)*||11/25 (40)*||1.00 ± 0.25*||2.22 ± 0.24*|
In our other studies, the administration of FloraMax®-B11 in drinking water (106 cfu/mL) for 3 days post-SE challenge (104 cfu/bird) using two presentations, liquid and lyophilized significantly reduced the incidence of
Finally, trying to find FloraMax®-B11 applications in poultry, we opted for spray application since it could be more efficient and has lower cost than its application in drinking water since it is important to take into account water quality and medicator/proportioner function . The results obtained were promising since when the probiotic was applied by spray and in drinking water, there was a reduction in the recovery of SE (55 and 50%, respectively; controls 85%) when chicks were held for 8 h prior to SE challenge and placement. In the same way, when probiotic was applied by spray or in drinking water and SE challenge occurred simultaneously, with placement 8 h after treatment, a marked and significant reduction of SE recovery was noted after 5d (10 and 40%, respectively; controls 55%). Furthermore, when the probiotic was sprayed and chickens were SE challenged simultaneously, with placement 8 h after treatment, a significant reduction of SE recovery was again noted in both the spray and DW application (80% controls, 15% spray, 15% drinking water) (Table 5). These results suggest that the spray application of this probiotic can be effective in protecting chicks against
|Treatment regimen||Group||Cecal tonsils|
|Exp. 1||Exp. 2|
|Treat-challenge-place immediately||Control||95% (19/20)||95% (19/20)|
|Probiotic (drinking water)||75% (15/20)||25% (5/25)**|
|Probiotic spray||90% (18/20)||80% (16/20)|
|Treat-hold 8 h-challenge-place||Control||85% (17/20)||70% (14/20)|
|Probiotic (drinking water)||50% (10/20)*||70% (14/20)|
|Probiotic spray||55% (11/20)*||80% (16/20)|
|Treat-challenge-hold 8 h-place||Control||55% (11/20)||80% (16/20)|
|Probiotic (drinking water)||44% (7/20)*||15% (2/20)*|
|Probiotic spray||20% (2/20)**||15% (2/20)*|
In this regard, an
|Treatment||Day 1 BW (g)||Day 3 BW (g)||Day 7 BW (g)||SE incidence cecal tonsils 24 h PI||Log SE/g of ceca content 24 h PI|
|Saline||49.13 ± 0.30a||62.53 ± 0.81b||132.89 ± 3.06b||20/20 (100%)||7.13 ± 1.01a|
|FloraMax®-B11||49.72 ± 0.36a||65.42 ± 0.77a||144.98 ± 3.02a||9/20 (45%)*||5.45 ± 1.25b|
These results agree with another study where the
3.2 The use of direct-fed microbials (DFM) for the control of pathogens in poultry
Although the use of LAB has been promising for the control of pathogens such as
Previously in our laboratory, we have screened and identified
Several studies have reported that some
|Corn-based||6.44 ± 0.19a||6.68 ± 0.08a|
|Wheat-based||7.12 ± 0.07a||5.20 ± 0.18b|
|Barley-based||7.50 ± 0.13a||6.86 ± 0.11b|
|Rye-based||7.15 ± 0.09a||6.68 ± 0.12b|
|Oat-based||6.96 ± 0.13a||5.76 ± 0.07b|
Based on the previous results, the effect of
|Item||Negative control||Positive control||DFM|
|d 0||46.88 ± 0.64b||46.54 ± 0.64b||49.23 ± 0.68a|
|d 7||127.14 ± 2.90a||115.58 ± 3.27b||123.05 ± 3.80ab|
|d 14||273.80 ± 11.02b||295.78 ± 12.10ab||318.08 ± 13.57a|
|d 18||457.79 ± 18.97ab||456.32 ± 19.39b||525.58 ± 17.92a|
|d 21||603.81 ± 24.32a||445.96 ± 18.50c||507.77 ± 20.60b|
|d 0–7||80.39 ± 3.06a||67.74 ± 3.24b||75.08 ± 3.64ab|
|d 7–14||147.01 ± 9.51b||182.60 ± 9.48a||196.22 ± 10.56a|
|d 14–18||183.99 ± 9.85ab||160.55 ± 9.02b||198.31 ± 9.61a|
|d 14–21||325.78 ± 15.58a||152.13 ± 9.67b||185.27 ± 10.52b|
|d 0–21||552.72 ± 24.35a||399.42 ± 19.79b||458.58 ± 20.48b|
|d 0–21||808.21 ± 29.86a||772.34 ± 10.66a||805.21 ± 71.07a|
|d 0–21||1.46 ± 0.04b||1.93 ± 0.10a||1.76 ± 0.18ab|
This enhancement in the performance of chickens supplemented with
Microbiota analysis confirms that DFM played a vital role in restoring gut dysbiosis. Although only the phylum
Finally, significant differences in beta diversity were found between NC versus PC and PC versus DFM (Figure 3), which agrees with another study where NE causes significant changes in the intestinal microbiota . Interestingly, there was no difference in bacterial community structure between NC and DFM. It confirms again that DFM played a vital role in restoring the gut dysbiosis in this study.
3.3 The use of
Bacillus-DFM candidate to prevent the toxic effects of aflatoxin B1 (AFB1) in poultry
Aflatoxin B1 (AFB1) is the predominant mycotoxin produced by several species of
Despite the previous results, the
|d 0||46.23 ± 0.68a||47.92 ± 0.72a||48.12 ± 0.74a||0.4174||0.1275|
|d 7||133.29 ± 4.64a||129.92 ± 2.78a||137.02 ± 4.19a||2.2763||0.4502|
|d 14||320.92 ± 17.53a||272.06 ± 8.54b||318.42 ± 14.65a||8.4215||0.0263|
|d 21||640.10 ± 31.51a||474.81 ± 15.57b||571.60 ± 25.47a||16.2361||0.0001|
|d 0–7||87.06 ± 4.24a||82.00 ± 2.71a||88.90 ± 4.15a||2.1705||0.4103|
|d 7–14||187.63 ± 13.82a||142.13 ± 7.06b||181.40 ± 11.38a||6.7337||0.0097|
|d 14–21||319.17 ± 16.08a||202.75 ± 9.77c||253.17 ± 14.89b||9.5832||<0.0001|
|d 0–21||593.87 ± 31.21a||426.88 ± 15.66c||523.48 ± 25.42b||16.2105||0.0001|
|d 0–21||750.55 ± 17.23a||775.93 ± 3.51a||731.97 ± 82.35a||25.1292||0.8193|
|d 0–21||1.27 ± 0.06b||1.82 ± 0.06a||1.40 ± 0.06b||0.0875||0.0016|
As it can be seen, probiotics could be considered a potential alternative to the use of antibiotics in poultry since it has been reported that they can improve the performance, as well as prevent and control enteric pathogens in poultry. However, their applications depend on the type of microorganism. In this regard, since lactic acid bacteria (LAB) are very sensitive to pelletizing processes for feed production (heating), environmental factors, and the low pH of the stomach, as well as the presence of bile salts in the small intestine, their administration in a single dose could be the most viable application especially to prevent bacterial diseases in both
This research was supported by the Arkansas Biosciences Institute under the project: Development of an avian model for evaluation early enteric microbial colonization on the gastrointestinal tract and immune function. The authors thank the CONACyT for the doctoral scholarship number 270728.