*The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins DOI: http://dx.doi.org/10.5772/intechopen.88817*

drinking water since it is important to take into account water quality and medicator/proportioner function [65]. 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 *Salmonella* infection. Furthermore, hatchery administration could prove to be a more effective way to administer probiotics because the chicks will be receiving the beneficial bacteria at the earliest possible time, in the absence of *in ovo* administration.

In this regard, an *in ovo* study was performed to know the effectiveness of FloraMax®-B11 [66]. For this, 18-day-old embryos were candled and inoculated with either saline or 104 cfu FloraMax®-B11 via *in ovo* injection into the amnion. On day 21, chicks were pulled from hatchers to measure hatchability. Subsequently, all chickens were then orally gavaged with SE on the day of hatch (~104 cfu/chick) and maintained for 7 days. *Salmonella* recovery was done 24-h post-SE challenge. Body weight (BW) was determined at days 1, 3, and 7. In this experiment, a significant increase in BW was observed. Furthermore, chickens that received the probiotic culture showed a significant reduction in the incidence and counts of SE in cecal tonsils when compared with saline control chickens (**Table 6**).

These results agree with another study where the *in ovo* colonization with a probiotic could become an important method to reduce *Salmonella* and other intestinal bacterial infections in poultry [67]. Regarding the increase of BW in the group treated with the probiotic, this could be due to the significant morphometric changes in the duodenum and ileum observed at day 1 of age.


*\* Indicates significant (p < 0.05) differences were observed between control and treated within a single experiment and treatment regime in each column.*

*\*\*Significantly (p < 0.01) different than all groups within a single experiment and treatment regime in each column.*

#### **Table 5.**

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

**tonsil +/− (%)**

**Rep. Treatment ST cecal** 

administration. These data suggest that the mechanism to reduce *Salmonella* was initiated within the first 12 h after treatment. Probably the reduction of *Salmonella* is due to the set of mechanisms of action of probiotics: bacterial interactions (competitive exclusion) or stimulation of a host innate immune response. The competitive exclusion could have included competition for receptor sites, production of volatile fatty acids that are inhibitors of certain enteric pathogens, production of bacteriocins, or competition with pathogens and native flora for limiting nutrients [62]. Furthermore, since the *Salmonella* recovery was performed in the early stages of infection, the innate immune response could be responsible for the reduction of

*Effect of lactic acid bacteria (LAB) on Salmonella typhimurium (ST) or Salmonella enteritidis (SE)* 

*recovered from cecal tonsils or ceca of broiler chicks 24-h post-LAB administration.*

**SE cecal tonsil +/− (%)**

1 Control 20/25 (80) 22/25 (88) 3.81 ± 0.32 4.33 ± 0.17

2 Control 18/25 (72) 25/25 (100) 3.59 ± 0.23 3.59 ± 0.23

3 Control 20/25 (80) 25/25 (100) 3.91 ± 0.19 3.91 ± 0.19

*A significant (p* ≤ *0.05) difference was observed between control and treated within a single experiment in each* 

LAB 2/25 (8)\* 8/25 (32)\* 0.62 ± 0.19\* 1.95 ± 0.09\*

LAB 2/25 (8)\* 7/25 (28)\* 0.42 ± 0.18\* 1.91 ± 0.29\*

LAB 1/25 (4)\* 11/25 (40)\* 1.00 ± 0.25\* 2.22 ± 0.24\*

**Log SE cecal recovery (all samples)**

**Log SE cecal recovery (only positive samples)**

In our other studies, the administration of FloraMax®-B11 in drinking water

liquid and lyophilized significantly reduced the incidence of *Salmonella* [63], which agrees with other studies [64]. Furthermore, the administration of FloraMax®-B11

Heidelberg (SH) challenge practically eliminated the concentration of SH, as well as its incidence, since only one sample was positive. However, in turkey poults under the same experimental conditions (**Table 4**), although similar significant results were observed at day 3 post-FloraMax®-B11 administration, it is clear that poults

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

> **(log10 cfu/g of ceca content)**

Control SH 20/20 (100) 7.04 ± 0.19a 20/20 (100) 6.05 ± 0.28a FloraMax®-B11 13/20 (65)\* 4.36 ± 0.74b 9/20 (45)\* 2.15 ± 0.75b

at the same concentration as the previous study after 1-h post-*Salmonella*

**Treatment 24 h 72 h**

*In vivo evaluation of FloraMax-B11 against Salmonella Heidelberg (SH) at 24 and 72 h in poults.*

**SH2**

cfu/bird) using two presentations,

**Cecal tonsils1** **SH2**

 **(log10 cfu/g of ceca content)**

cfu/mL) for 3 days post-SE challenge (104

were more susceptible to SH colonization than chicks [51].

*a,bDifferent superscripts within columns indicate significant differences (p < 0.05).*

**Cecal tonsils1**

*Data expressed as positive/total poults (%).*

*Data expressed as mean ± SE.*

**222**

*1*

*2*

*\* p < 0.001.*

**Table 4.**

*Salmonella*.

(106

*\**

*column.*

**Table 3.**

*Salmonella enteritidis recovery from cecal tonsils of broiler chicks 5-day post-challenge.*


*\* Indicates significant differences p < 0.001, n = 20/group.*

#### **Table 6.**

*Evaluation of in ovo administration of FloraMax®-B11 on body weight and Salmonella enteritidis (SE) recovery in broiler chickens.*

## **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 *Salmonella* spp., as described above, it is important to mention that one limitation is their sensitivity to pelletizing processes for feed production (heating) [30, 68, 69], environmental factors [70], and the low pH of the stomach and the presence of bile salts in the small intestine [71, 72]. For this reason, some strategies to increase the viability of these bacteria include their microencapsulation in polymer matrices [73, 74], as well as their freezing or lyophilization [75, 76]. However, production costs increase, so it becomes nonviable in animal production. Although LAB are better probiotics than *Bacillus*, the latter is more stable due to their ability to form spores, which are more resistant to severe environmental conditions, feed pelleting process with extreme temperatures, as well as tolerance to extremes of pH, dehydration, high pressures, and chemicals, and therefore, stability to long period storage conditions, making them suitable for commercialization [77, 78] since they could be used as direct-fed microbials (DFM) [68].

Previously in our laboratory, we have screened and identified *Bacillus* spp. isolates as DFM. Some of these demonstrated to be effective as potential DFM candidates by reducing *Salmonella* colonization and having a positive effect on the increase in body weight gained in both chickens and turkeys, as well as tolerance to acidic condition (pH = 2), high osmotic pressure (NaCl at 6.5%), and 0.037% bile salts after 24h of incubation [79–81].

Several studies have reported that some *Bacillus* species are capable of producing different exogenous enzymes such as protease, lipase, cellulase, xylanase, phytase, and keratinase [82–86], which agrees with one of our studies already published [25]. These enzymes could improve the digestion of nutrients, making them more bioavailable, and also, they help to reduce intestinal viscosity in non-starch polysaccharide diets and decrease the substrates available for the growth of pathogenic bacteria. Considering this information, we performed a study in order to evaluate the effect of three *Bacillus*-DFM candidates with excellent to good relative enzyme activity values (cellulase and xylanase) on digesta viscosity and *Clostridium perfringens* (CP) proliferation in different poultry diets using an *in vitro* digestive model [87]. One of the three *Bacillus* strains was identified as *Bacillus subtilis* and the other two isolates as *Bacillus amyloliquefaciens* by 16S rRNA sequence analysis. Subsequently, *Bacillus* candidate strains were sporulated and mixed in equal amounts during the *Bacillus*-DFM preparation process [88] and incorporated into the experimental diets (108 spores/g). The results of this study demonstrated that *Bacillus* candidate significantly reduced the viscosity of non-corn-based diets.

**225**

*1*

*2*

**Table 7.**

*Inoculum used 105*

*Data expressed in log10 cfu/mL.*

*Bacillus-DFM candidate spore2*

 *cfu of CP.*

*Concentration of Clostridium perfringens (CP)1*

*.*

*The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins*

This could be due to the capability of these *Bacillus* strains to produce cellulase and xylanase, which could help improve the digestibility of cereals with high-soluble non-starch polysaccharides [89]. Furthermore, *Bacillus*-DFM candidate demonstrated effective antimicrobial properties against CP (**Table 7**), given their capability to produce antimicrobial-like compounds and/or compete for nutrients. Likewise, it was shown that the persistence of *Bacillus*-DFM candidate spores changes in each compartment of the *in vitro* digestive model mainly due to the conditions of pH and

suggests that their full life cycle is developed in the gastrointestinal tract.

Based on the previous results, the effect of *Bacillus*-DFM candidate spores formed by an isolate of *Bacillus subtilis* and two of *Bacillus amyloliquefaciens* on growth performance, intestinal integrity, necrotic enteritis (NE) lesions, and ileal microbiota in broiler chickens using a previously established NE-challenged model [90] was evaluated [24]. This study consisted of three experimental groups: negative control (NC), positive control (PC), and *Bacillus*-DFM group (DFM). The last two groups were challenged with *Salmonella* typhimurium (ST, day 1), *Eimeria maxima* (EM, day 13), and *Clostridium perfringens* (CP, day 18–19). The overall results of performance showed that chickens supplemented with DFM had a significant body weight (BW) higher than PC. Furthermore, the body weight gain (BWG) and feed conversion ratio (FCR) were 59 g higher and 17 points lower, respectively,

This enhancement in the performance of chickens supplemented with *Bacillus*-DFM could be due to better digestibility of nutrients, maintenance of the beneficial gut microbiota, and promotion of a healthy intestinal integrity [48, 87, 91]. Furthermore, these results could relate to the low-serum FITC-d concentration, bacterial translocation (BT), ileal lesion (IL), and total intestinal IgA levels in the DFM group compared to the PC group given the low impact of EM and CP challenge since DFM could produce beneficial chemical compounds, has immunoregulatory capacity, and stimulates the homeostasis of the intestinal microbiota,

Microbiota analysis confirms that DFM played a vital role in restoring gut dysbiosis. Although only the phylum *Proteobacteria* was significantly lower in DFM group than PC group, it could be explained due to the antimicrobial properties of DFM against ST [25], a predisposing factor in the NE model. In contrast, the genus *Lactobacillus* was significantly predominant in both NC and DFM groups with respect to PC, but it was higher in the DFM group than NC group (**Figure 2**). It has been reported that DFM is capable of increasing the genus *Lactobacillus*, which plays a crucial role in preventing dysbiosis and maintaining gut integrity

**Diet Control diet** *Bacillus***-DFM** 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 ± 0.07<sup>b</sup>

 *in different digested diets with or without inclusion of* 

*DOI: http://dx.doi.org/10.5772/intechopen.88817*

in the DFM group than PC (**Table 8**).

(homeostasis) [36, 93].

resulting in a proper intestinal health status [92].

*a,bDifferent superscripts within a row indicate significant differences p < 0.05.*

### *The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins DOI: http://dx.doi.org/10.5772/intechopen.88817*

This could be due to the capability of these *Bacillus* strains to produce cellulase and xylanase, which could help improve the digestibility of cereals with high-soluble non-starch polysaccharides [89]. Furthermore, *Bacillus*-DFM candidate demonstrated effective antimicrobial properties against CP (**Table 7**), given their capability to produce antimicrobial-like compounds and/or compete for nutrients. Likewise, it was shown that the persistence of *Bacillus*-DFM candidate spores changes in each compartment of the *in vitro* digestive model mainly due to the conditions of pH and suggests that their full life cycle is developed in the gastrointestinal tract.

Based on the previous results, the effect of *Bacillus*-DFM candidate spores formed by an isolate of *Bacillus subtilis* and two of *Bacillus amyloliquefaciens* on growth performance, intestinal integrity, necrotic enteritis (NE) lesions, and ileal microbiota in broiler chickens using a previously established NE-challenged model [90] was evaluated [24]. This study consisted of three experimental groups: negative control (NC), positive control (PC), and *Bacillus*-DFM group (DFM). The last two groups were challenged with *Salmonella* typhimurium (ST, day 1), *Eimeria maxima* (EM, day 13), and *Clostridium perfringens* (CP, day 18–19). The overall results of performance showed that chickens supplemented with DFM had a significant body weight (BW) higher than PC. Furthermore, the body weight gain (BWG) and feed conversion ratio (FCR) were 59 g higher and 17 points lower, respectively, in the DFM group than PC (**Table 8**).

This enhancement in the performance of chickens supplemented with *Bacillus*-DFM could be due to better digestibility of nutrients, maintenance of the beneficial gut microbiota, and promotion of a healthy intestinal integrity [48, 87, 91]. Furthermore, these results could relate to the low-serum FITC-d concentration, bacterial translocation (BT), ileal lesion (IL), and total intestinal IgA levels in the DFM group compared to the PC group given the low impact of EM and CP challenge since DFM could produce beneficial chemical compounds, has immunoregulatory capacity, and stimulates the homeostasis of the intestinal microbiota, resulting in a proper intestinal health status [92].

Microbiota analysis confirms that DFM played a vital role in restoring gut dysbiosis. Although only the phylum *Proteobacteria* was significantly lower in DFM group than PC group, it could be explained due to the antimicrobial properties of DFM against ST [25], a predisposing factor in the NE model. In contrast, the genus *Lactobacillus* was significantly predominant in both NC and DFM groups with respect to PC, but it was higher in the DFM group than NC group (**Figure 2**). It has been reported that DFM is capable of increasing the genus *Lactobacillus*, which plays a crucial role in preventing dysbiosis and maintaining gut integrity (homeostasis) [36, 93].


*a,bDifferent superscripts within a row indicate significant differences p < 0.05. 1 Inoculum used 105 cfu of CP.*

*2 Data expressed in log10 cfu/mL.*

#### **Table 7.**

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

**Treatment Day 1 BW (g) Day 3 BW (g) Day 7 BW (g) SE** 

Saline 49.13 ± 0.30a 62.53 ± 0.81b 132.89 ± 3.06b 20/20

FloraMax®-B11 49.72 ± 0.36a 65.42 ± 0.77a 144.98 ± 3.02a 9/20

*a,bSuperscripts within columns indicate significant differences p < 0.05, n = 12/group.*

*Indicates significant differences p < 0.001, n = 20/group.*

**incidence cecal tonsils 24 h PI**

(100%)

(45%)\*

**Log SE/g of ceca content 24 h PI**

7.13 ± 1.01a

5.45 ± 1.25b

could be used as direct-fed microbials (DFM) [68].

salts after 24h of incubation [79–81].

the experimental diets (108

**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 *Salmonella* spp., as described above, it is important to mention that one limitation is their sensitivity to pelletizing processes for feed production (heating) [30, 68, 69], environmental factors [70], and the low pH of the stomach and the presence of bile salts in the small intestine [71, 72]. For this reason, some strategies to increase the viability of these bacteria include their microencapsulation in polymer matrices [73, 74], as well as their freezing or lyophilization [75, 76]. However, production costs increase, so it becomes nonviable in animal production. Although LAB are better probiotics than *Bacillus*, the latter is more stable due to their ability to form spores, which are more resistant to severe environmental conditions, feed pelleting process with extreme temperatures, as well as tolerance to extremes of pH, dehydration, high pressures, and chemicals, and therefore, stability to long period storage conditions, making them suitable for commercialization [77, 78] since they

*Evaluation of in ovo administration of FloraMax®-B11 on body weight and Salmonella enteritidis (SE)* 

Previously in our laboratory, we have screened and identified *Bacillus* spp. isolates as DFM. Some of these demonstrated to be effective as potential DFM candidates by reducing *Salmonella* colonization and having a positive effect on the increase in body weight gained in both chickens and turkeys, as well as tolerance to acidic condition (pH = 2), high osmotic pressure (NaCl at 6.5%), and 0.037% bile

Several studies have reported that some *Bacillus* species are capable of producing different exogenous enzymes such as protease, lipase, cellulase, xylanase, phytase, and keratinase [82–86], which agrees with one of our studies already published [25]. These enzymes could improve the digestion of nutrients, making them more bioavailable, and also, they help to reduce intestinal viscosity in non-starch polysaccharide diets and decrease the substrates available for the growth of pathogenic bacteria. Considering this information, we performed a study in order to evaluate the effect of three *Bacillus*-DFM candidates with excellent to good relative enzyme activity values (cellulase and xylanase) on digesta viscosity and *Clostridium perfringens* (CP) proliferation in different poultry diets using an *in vitro* digestive model [87]. One of the three *Bacillus* strains was identified as *Bacillus subtilis* and the other two isolates as *Bacillus amyloliquefaciens* by 16S rRNA sequence analysis. Subsequently, *Bacillus* candidate strains were sporulated and mixed in equal amounts during the *Bacillus*-DFM preparation process [88] and incorporated into

*Bacillus* candidate significantly reduced the viscosity of non-corn-based diets.

spores/g). The results of this study demonstrated that

**224**

*\**

**Table 6.**

*recovery in broiler chickens.*

*Concentration of Clostridium perfringens (CP)<sup>1</sup> in different digested diets with or without inclusion of Bacillus-DFM candidate spore2 .*

#### *Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*


*1 Data expressed as mean ± SE from 40 chickens (four replicates with 10 chicks each pen). p < 0.05. a–cValues within columns with different superscripts differ significantly (p < 0.05).*

#### **Table 8.**

*Evaluation of body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) in chickens supplemented with or without DFM on a necrotic enteritis challenge model<sup>1</sup> .*

#### **Figure 2.**

*Relative abundance of different phyla (A), families (B), and genera (C) in different treatment groups (NC, PC, and DFM). NA refers to those reads that were not assigned to the respective taxonomic levels.*

**227**

*The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins*

Furthermore, *Clostridium* was significantly higher in PC group due to the change in the ileum microbiota caused by NE [94], whereas the genera *Lactobacillus* and *Bacillus* were more abundant in the DFM group, suggesting that these genera could

**Item NC AFB1 DFM SEM2** *p***-value**

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 ± 15.57<sup>b</sup> 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 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

*Evaluation of body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) in broiler chickens consuming a corn-soybean-based diet contaminated with aflatoxin B1 (2 ppm)* 

319.17 ± 16.08a 202.75 ± 9.77c 253.17 ± 14.89b 9.5832 <0.0001

*PCoA plot showing difference in microbial community structure between (A) NC and PC (ANOSIM; R = 0.40* 

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 [96]. 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

*DOI: http://dx.doi.org/10.5772/intechopen.88817*

alleviate the negative impacts caused by CP [95].

*a–cSuperscripts within rows indicate significant difference at p < 0.05.*

*and p < 0.05) and (B) DFM and PC (ANOSIM; R = 0.73 and p < 0.01).*

in this study.

BW, g/broiler

BWG, g/broiler

FI, g/broiler

*supplemented with or without DFM.*

FCR

**Table 9.**

d 14–21

**Figure 3.**

*The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins DOI: http://dx.doi.org/10.5772/intechopen.88817*

**Figure 3.**

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

BW, g/broiler

BWG, g/broiler

FI, g/broiler

FCR

*1*

**Table 8.**

**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 ± 3.27<sup>b</sup> 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 ± 9.67<sup>b</sup> 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

*Evaluation of body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR)* 

*Relative abundance of different phyla (A), families (B), and genera (C) in different treatment groups (NC,* 

*PC, and DFM). NA refers to those reads that were not assigned to the respective taxonomic levels.*

*.*

*Data expressed as mean ± SE from 40 chickens (four replicates with 10 chicks each pen). p < 0.05.*

*a–cValues within columns with different superscripts differ significantly (p < 0.05).*

*in chickens supplemented with or without DFM on a necrotic enteritis challenge model<sup>1</sup>*

**226**

**Figure 2.**

*PCoA plot showing difference in microbial community structure between (A) NC and PC (ANOSIM; R = 0.40 and p < 0.05) and (B) DFM and PC (ANOSIM; R = 0.73 and p < 0.01).*

Furthermore, *Clostridium* was significantly higher in PC group due to the change in the ileum microbiota caused by NE [94], whereas the genera *Lactobacillus* and *Bacillus* were more abundant in the DFM group, suggesting that these genera could alleviate the negative impacts caused by CP [95].

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 [96]. 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.


#### **Table 9.**

*Evaluation of body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) in broiler chickens consuming a corn-soybean-based diet contaminated with aflatoxin B1 (2 ppm) supplemented with or without DFM.*
