Bacillus subtilis PM5 from Camel Milk Boosts Chicken Immunity and Abrogates Salmonella entertitidis Infections

Abstract

With the practice of a successful livestock industry using antibiotics, which has continued for more than five decades, researchers have long been interested in finding alternatives to antibiotics for poultry production. Probiotics can potentially reduce enteric diseases in livestock and enhance their productivity. The aim of this study was to isolate putative probiotics from camel milk and test them against Salmonella infection as well as host immune development. Thirteen different isolates were obtained from six different camel milk samples from dairy farms in Saudi Arabia. Three of the six isolates (PM1, PM2, PM3, PM4, PM5, and PM6) that showed Gram-positive characters reacted negatively to catalase and hemolytic assays. PM1, PM5, and PM6 showed significant nonpolar surface properties (>51% hydrophobic) and potent antimicrobial activities against avian pathogens, namely S. enterica, S. typhi, S. aureus, and E. coli. PM5 exhibited substantial probiotic traits; therefore, further focus was given to it. PM5 was identified as Bacillus subtilis OQ913924 by the 16S rRNA sequencing method and showed similarity matrix > 99%. An in vivo chicken model was used to access the health benefits of probiotics. After salmonella infection, the mucosal immune response was significantly increased (p < 0.01), and none of the challenge protocols caused mortality or clinical symptoms after infection in intestinal contents. S. enterica organ infiltration in the spleen, thymus, and small intestine was significantly reduced in the B. subtilis PM5-fed chickens. The S. enterica load in chicken feces was reduced from CFU 7.2 to 5.2 in oral-fed B. subtilis PM5-fed chickens. Probiotic-fed chickens showed buffered intestinal content and positively regulated the level of butyric acid (p < 0.05), and intestinal interleukin 1 beta (IL1-β), C-reactive protein (CRP), and interferon gamma (IFN-γ) levels were reduced (p < 0.05). In addition, B. subtilis PM5 showed significant binding to peritoneal macrophages cells and inhibited S. enterica surface adhesion, indicating co-aggregation of B. subtilis PM5 in macrophage cells. It could be concluded that supplementation with probiotics can improve the growth performance of broilers and the quality of broiler chickens against enteric pathogens. The introduction of this probiotic into the commercial poultry feed market in the near future may assist in narrowing the gap that now exists between chicken breeding and consumer demand.

Keywords: Bacillus; camel milk; chicken; infection; probiotics.

Figure 1

Experimental design to show the effectiveness of B. subtilis PM5 as a potential probiotic strain using an in vivo chicken model.

Figure 2

Characteristics of in vitro probiotic activity shown by isolates obtained from camel milk. (A) Bile salt 0.6% supplemented in MRS agar medium. Log phase probiotic characteristic isolates (M1, M2, M3, M4, M5, and M6) were inoculated in Oxgall MRS media for 3 h and counted the viable cells as log × CFU. (B) The effect of gastric juice tolerance of selected isolates was evaluated in pH 3 in MRS broth. (C) Evaluation of several probiotic isolates with regard to the h numbers indicates means ± SD for triplicate observations. Hydrophobic characteristics of their cell surfaces employing the heptane polarity shift technique. Numbers indicate means ± SD for triplicate findings (significant at * p ≤ 0.05).

Figure 3

Assessment of selected probiotics on antimicrobial properties against chicken-specific pathogens E. coli, S. aureus, S. enteritidis, and S. typhi. In order to assess the antibacterial activity of isolated bacterial whole lysate against avian infections, plates of MHA were used. (A) Antimicrobial activity of selected probiotics against avian pathogens and values expressed in mm in diameter (B) MHA plate was used for antimicrobial activity. Std: positive control (Ciprofloxacin 5 µg/disc). The zone of inhibition was calculated in the scale bar and expressed as mm in diameter. Numbers indicate means ± SD for triplicate findings.

Figure 4

UPGMA similarity relations of the potential probiotic bacterial strains, Bacillus subtilis OQ913924 with other closely related strains retrieved from NCBI GenBank. Horizontal bars denote the similarity branch length between the isolates. The scale bar was expressed in 0.01 substitutions per nucleotide position. *: indicates our bacterial isolate used in this study.

Figure 5

Macroscopic and microbiota analysis of PM5 was evaluated in Salmonella-challenged chicken. (A) Chicken physiological parameters were evaluated after 28 days of challenge and PM6 administration. The weight of chicken is expressed in grams. (B) The organ index indicates the infection rate and recovery level. (C) The mortality rate in %. (D) Microbial load in fecal samples of chicken and samples were collected and pooled in different intervals, microbial load expressed in log × 10⁷. Data are presented as the average of three independent measurements ± and expressed in respective units. Similar letter group means are insignificantly different (p > 0.05), whereas distinct letter group means are significantly different at * p ≤ 0.05.

Figure 6

The impact of a PM6-supplemented chicken feed on the immune system’s reaction to S. enterica challenge in chickens exposed to extreme strain. (A) LDH of liver tissue oxidative stress-related enzymes was evaluated after 4 weeks of acute stress (n = 6). (B). Serum creatinine kinase levels were analyzed to assess cardiac injury following 4 weeks of PM6 supplementation in chicken (n = 6). (C) Neutrophil infiltration was measured in hens that were challenged with Salmonella by measuring the activity of malondialdehyde (MDA). In each plot, the values reflect the mean and standard deviation. For repeated measurements, we used one-way ANOVA and then Tukey’s post hoc testing. * shows p < 0.05.

Figure 7

The influence of PM6 supplementation on the inflammatory response of chickens challenged with S. enterica. (A) pH estimation of different intestinal content (Gizzard and ileum) food content at 20th days of the experiment. (B) Estimation of butyric acid levels in the mucus of S. enterica-challenged chicken colon and PM5 treatment groups. (C) Lymphocyte degradation marker CRP was quantified in the serum samples. (D) IL-1β inflammatory cytokine concentration in S. enterica-challenged chicken serum and PM5 treatment groups. (E) Determination of the IFN-γ content in the serum of S. enterica-challenged chickens and PM5-treated groups as an infection stimulation marker cytokine.

Figure 8

Peritoneal macrophages (PM5) infected with S. enterica have been studied for their capacity for survival, invasion, and adhesion. (A) The MTT assay was used to measure the viability of B. subtilis infected with Salmonella enterica. (B) The impact of PM6’s adhesion and invasion capabilities on the overall invasion of S. enterica in lysed cell content. TNF in S. enterica-induced pM5 cells (C): PM6’s impact. Mean standard error of the mean (SEM) for triplicate measurements.