Effectiveness of Bacillus licheniformis-Fermented Products and Their Derived Antimicrobial Lipopeptides in Controlling Coccidiosis in Broilers (Extract)

Coccidiosis is an important health problem in broilers, caused by infection with a highly contagious intestinal parasite of the genus Eimeria. Anti-coccidial drugs are widely used for the prevention of coccidiosis in broilers. However, multi-resistance coccidia is a potential threat to poultry production. 

Solid-state fermented products produced from B. licheniformis not only contain B. licheniformis spores but also have B. licheniformis derived antimicrobial lipopeptides. B. licheniformis-fermented products (BLFP) have similar benefits as antibiotics in growth performance in broilers. Dietary supplementation of BLFP alleviates necrotic lesions, ameliorates intestinal morphology, and improves growth performance in C. perfringens-challenged broilers. BLFP supplementation also modulates the gut microbial community in broilers. Surfactin isolated from BLFP exhibits antibacterial activity against Brachyspira hyodysenteriae and C. perfringens in vitro. However, the effect of BLFP on the alleviation of coccidiosis is still rarely investigated. Whether B. licheniforms-derived antimicrobial lipopeptides also exhibit anti-coccidial activity remains to be confirmed.

In this study, we evaluated the potential of Bacillus licheniformis-fermented products (BLFP) and their derived antimicrobial lipopeptide, surfactin, on the prevention of coccidiosis in broilers.

1. A total of 120 one-day-old healthy male Ross 308 broiler chicks were allocated to five treatments and six replicates each containing four birds in a completely randomized design.

2. The experimental diets were

Figure 1. Timeline of the experimental design and sampling during the entire experiment.

 

3. Measurement of Anti-Coccidial Index

RBWG : relative body-weight gain,

SR :survival rate,

LSI : lesion-score index,

OI : oocyst-count index

Anti-coccidial index (ACI) was calculated as follows:

ACI = (RBWG (%) + SR (%)) − (LSI) + (OI)).

An ACI between 160 and 180 was scored as a marked anti-coccidial effect, between 140 and 159 as moderate, between 120 and 139 as slight, and values below 120 as inactive.

Supplementation with 1.25 g/kg of BLFP in E. tenella-challenged broilers increased cecal villus length compared with those in the control and E. tenella-challenge-only groups (p < 0.05) (Table 1). Supplementation with BLFP at 1.25 and 5 g/kg in combination with E. tenella challenge decreased the crypt depth in the cecum of broilers compared with the control and E. tenella-challenge-only groups (p < 0.05) (Table 1). The ratio of villus length to crypt depth in the cecum was increased in broilers given BLFP at 1.25 and 5 g/kg in combination with E. tenella-challenge groups compared with the other groups (p < 0.05) (Table 1).

Table 1. Effect of Bacillus licheniformis-fermented products on cecal morphology of broilers challenged with Eimeria tenella.

No significant difference was observed in relative body-weight gain between groups over the whole trial period (day 1 to 35) (Table 2). The survival rate of broilers was reduced in the control group (95.8%), the E. tenella-challenge-only group (95.8%), and maduramicin in combination with E. tenella-challenge group (91.7%), whereas BLFP at 1.25 and 5 g/kg and in combination with E. tenella challenge increased the survival rate of broilers to 100% (Table 2). The lesion scores in the ceca were reduced in the 1.25 g/kg of BLFP in combination with E. tenella challenge group compared with the E. tenella-challenge-only group (p < 0.05) (Table 2). E. tenella challenge only increased the oocyst-count index in broilers compared with the control group, whereas 5 g/kg of BLFP in combination with E. tenella challenge reduced the oocyst-count index in broilers (p < 0.05) (Table 2). The anti-coccidial index of broilers fed only a basal diet, E. tenella challenge only, anti-coccidial drug in combination with E. tenella challenge, 1.25 g/kg of BLFP in combination with E. tenella challenge, or 5 g/kg of BLFP in combination with E. tenella challenge were 158.4, 139.1, 157.1, 162.2, and 154.3, respectively (Table 2).

Table 2. Effect of Bacillus licheniformis-fermented products on anti-coccidial index of broilers challenged with Eimeria tenella.

E. tenella oocyst sporulation was increased at 48 and 72 h after stimulation with 2% K2Cr2O7 solution (Table 3 and Figure 2), whereas surfactin isolated from BLFP totally eliminated E. tenella oocyst sporulation and disrupted the oocyst morphology (p < 0.05) (Table 3 and Figure 2). The results of scanning electron microscopy examinations show that the morphology of Eimeria sporozoites was disrupted by BLFP-derived surfactin at 5, 10, and 20 μg/mL (Figure 3).

BLFP supplementation improved cecal morphology, survival rate, and cecal-lesion scores in broilers exposed to E. tenella challenge, and 1.25 g/kg can be considered the optimum dosage. B. licheniformis-derived surfactin exhibited anti-coccidial activity by inhibiting the life cycle of Eimeria species. The findings provide a theoretical basis for the use of BLFPs as a possible substitute for anti-coccidial drugs in poultry production.

Table 3. Effect of Bacillus licheniformis-fermented-product-derived surfactin on E. tenella oocyst sporulation in vitro.

Figure 2. Bacillus licheniformis-fermented-product-derived surfactin inhibits E. tenella oocyst sporulation in vitro. E. tenella oocysts were treated with 2% K2Cr2O7 in combination with 100 μg/mL of surfactin at 25 °C for 12, 24, 48, and 72 h. 

Figure 3. Scanning electron microscope images of Eimeria sporozoites illustrating the anti-parasitic activity of Bacillus licheniformis-fermented-product (BLFP)-derived surfactin.