Water-soluble microencapsulation using gum Arabic and skim milk enhances viability and efficacy of Pediococcus acidilactici probiotic strains for application in broiler chickens.

Objective: This study aimed to develop and evaluate the effectiveness of a water-soluble microencapsulation method for probiotic strains using gum Arabic (GA) and skim milk (SKM) over a three-month storage period following processing. Methods: Four strains of Pediococcus acidilactici (BYF26, BYF20, BF9, and BF14) that were typical lactic acid bacteria (LAB) isolated from the chicken gut were mixed with different ratios of gum Arabic (GA) and skim milk (SKM) as coating agents before spray drying at an inlet temperature 140°C. After processing, the survivability and probiotic qualities of the strains were assessed from two weeks to three months of storage at varied temperatures, and de-encapsulation was performed to confirm the soluble properties. Finally, the antibacterial activity of the probiotics was assessed under simulated gastrointestinal conditions. Results: As shown by scanning electron microscopy, spray-drying produced a spherical, white-yellow powder. The encapsulation efficacy (EE percent) was greatest for a coating containing a combination of 30% gum Arabic: 30% skim milk (w/v) (GA:SKM30) compared to lower concentrations of the two ingredients (p<0.05). Coating with GA:SKM30 (w/v) significantly enhanced (p<0.05) BYF26 survival under simulated gastrointestinal conditions (pH 2.5-3) and maintained higher survival rates compared to non-encapsulated cells under an artificial intestinal juices (AIJ) condition of pH 6. De-encapsulation tests indicated that the encapsulated powder dissolved in water while keeping viable cell counts within the effective range of 106 for 6 hours. In addition, following three months storage at 4°C, microencapsulation of BYF26 in GA:SKM30 maintained both the number of viable cells (p<0.05) and the preparation's antibacterial efficacy against pathogenic bacteria, specifically strains of Salmonella. Conclusion: Our prototype water-soluble probiotic microencapsulation GA:SKM30 effectively maintains LAB characteristics and survival rates, demonstrating its potential for use in preserving probiotic strains that can be used in chickens and potentially in other livestock.

Selection and evaluation of lactic acid bacteria from chicken feces in Thailand as potential probiotics.

Background: Lactic acid bacteria (LAB) are widely used as probiotics in poultry production due to their resilience to low pH and high bile salt concentrations, as well as their beneficial effects on growth performance and antagonistic activity against enteric pathogens. However, the efficacy of probiotics depends on strain selection and their ability to colonize the host's intestine. This study aimed to select, identify, and evaluate LAB strains isolated from chicken feces in Thailand for potential use as probiotics in the chicken industry. Methods: LAB strains were isolated from 58 pooled fresh fecal samples collected from chicken farms in various regions of Thailand, including commercial and backyard farms. Gram-positive rods or cocci with catalase-negative characteristics from colonies showing a clear zone on MRS agar supplemented with 0.5% CaCO3 were identified using MALDI-TOF mass spectrometry. The LAB isolates were evaluated for acid (pH 2.5 and pH 4.5) and bile salt (0.3% and 0.7%) tolerance. Additionally, their cell surface properties, resistance to phenol, antimicrobial activity, hemolytic activity, and presence of antimicrobial resistance genes were determined. Results: A total of 91 LAB isolates belonging to the Pediococcus, Ligilactobacillus, Limosilactobacillus, and Lactobacillus genera were obtained from chicken feces samples. Backyard farm feces exhibited a greater LAB diversity compared to commercial chickens. Five strains, including Ligilactobacillus salivarius BF12 and Pediococcus acidilactici BF9, BF14, BYF20, and BYF26, were selected based on their high tolerance to acid, bile salts, and phenol. L. salivarius BF12 and P. acidilactici BF14 demonstrated strong adhesion ability. The five LAB isolates exhibited significant cell-cell interactions (auto-aggregation) and co-aggregation with Salmonella. All five LAB isolates showed varying degrees of antimicrobial activity against Salmonella strains, with P. acidilactici BYF20 displaying the highest activity. None of the LAB isolates exhibited beta-hemolytic activity. Whole genome analysis showed that L. salivarius BF12 contained ermC, tetL, and tetM, whereas P. acidilactici strains BF9 and BF14 carried ermB, lnuA, and tetM. Conclusion: The selected LAB isolates exhibited basic probiotic characteristics, although some limitations were observed in terms of adhesion ability and the presence of antibiotic resistance genes, requiring further investigation into their genetic location. Future studies will focus on developing a probiotic prototype encapsulation for application in the chicken industry, followed by in vivo evaluations of probiotic efficacy.

An atypical weakly haemolytic strain of Brachyspira hyodysenteriae is avirulent and can be used to protect pigs from developing swine dysentery.

The anaerobic intestinal spirochaete Brachyspira hyodysenteriae colonises the large intestine of pigs and causes swine dysentery (SD), a severe mucohaemorrhagic colitis. SD occurs worldwide, and control is hampered by a lack of vaccines and increasing antimicrobial resistance. B. hyodysenteriae strains typically produce strong beta-haemolysis on blood agar, and the haemolytic activity is thought to contribute to the pathogenesis of SD. Recently, weakly haemolytic variants of B. hyodysenteriae have been identified in Europe and Australia, and weakly haemolytic strain D28 from Belgium failed to cause disease when used experimentally to infect pigs. Moreover, pigs colonised with D28 and then challenged with virulent strongly haemolytic strain B204 showed a delay of 2-4 days in developing SD compared to pigs not exposed to D28. The current study aimed to determine whether Australian weakly haemolytic B. hyodysenteriae strain MU1, which is genetically distinct from D28, could cause disease and whether exposure to it protected pigs from subsequent challenge with strongly haemolytic virulent strains. Three experimental infection studies were undertaken in which no diseases occurred in 34 pigs inoculated with MU1, although mild superficial lesions were found in the colon in 2 pigs in one experiment. In two experiments, significantly fewer pigs exposed to MU1 and then challenged with strongly haemolytic virulent strains of B. hyodysenteriae developed SD compared to control pigs not previously exposed to MU1 (p = 0.009 and p = 0.0006). These data indicate that MU1 lacks virulence and has potential to be used to help protect pigs from SD.

Vaccination of chickens with the 34†kDa carboxy-terminus of Bpmp72 reduces colonization with Brachyspira pilosicoli following experimental infection.

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of a variety of species of mammals and birds, and may result in colitis, diarrhoea and reductions in growth rate. Naturally occurring infections in chickens are largely confined to adult laying and breeding birds. In this study, the 34 kD carboxy-terminus of the prominent outer membrane protein Bmp72 of B. pilosicoli was expressed as a histidine-tagged recombinant protein and used to immunize two groups (B and C) of 15 individually housed layer chickens. Vaccination was with either 100 µg (B) or 1 mg (C) protein emulsified with Freund's incomplete adjuvant delivered into the pectoral muscles, followed three weeks later by 1 mg of protein in phosphate buffered saline delivered via crop tube. Two weeks later these and 15 non-vaccinated positive control birds (group A) housed in the same room were challenged via crop tube with B. pilosicoli avian strain CPS1. B. pilosicoli was detected in the faeces of all control birds and in 14 of the vaccinated birds in each vaccinated group at some point over the 30-day period following challenge. Colonization was delayed and the duration of excretion was significantly reduced (P = 0.0001) in both groups of vaccinated birds compared to the non-vaccinated control birds. Fewer immunized birds had abnormal caecal contents at post mortem examination compared to non-vaccinated birds, but the difference was not statistically significant. This study indicates that recombinant Bmp72 C-terminus has potential to be developed for use as a vaccine component to provide protection against B. pilosicoli infections. RESEARCH HIGHLIGHTS Laying chickens were immunized with recombinant Brachyspira pilosicoli membrane protein Bpmp72. Immunized birds had a highly significant reduction in the duration of colonization. Fewer immunized than control birds had abnormal caecal contents after infection. Bpmp72 showed potential for use as a novel vaccine component for B. pilosicoli.

Colonic Spirochetes: What Has Genomics Taught Us?

The 'colonic' spirochetes assigned to the genus Brachyspira are slow-growing anaerobic bacteria. The genus includes both pathogenic and non-pathogenic species, and these variously colonise the large intestines of different species of birds and animals, including humans. Scientific understanding of the physiology and molecular biology of Brachyspira spp. remains very limited compared with that of other pathogenic spirochetes, and there are few descriptions of successful genetic manipulations undertaken to investigate gene function. An important boost to knowledge occurred in 2009 when, for the first time, the whole genome sequence of a Brachyspira strain (Brachyspira hyodysenteriae strain WA1) was obtained. The genomics analysis provided a significant increase in knowledge: for example, a previously unknown ~36 Kb plasmid was discovered and metabolic pathways were constructed. The study also revealed likely acquisition of genes involved in transport and central metabolic functions from other enteric bacterial species. Four subsequent publications have provided a similarly detailed analysis of other Brachyspira genomes, but of these only two included more than one strain of a species (20 strains of B. hyodysenteriae in one and three strains of B. pilosicoli in the other). Since then, more Brachyspira genomes have been made publicly available, with the sequences of at least one representative of each of the nine officially recognised species deposited at public genome repositories. All species have a single circular chromosome varying in size from ~2.5 to 3.3 Mb, with a C + G content of around 27%. In this chapter, we summarise the current knowledge and present a preliminary comparative genomic analysis conducted on 56 strains covering the official Brachyspira species. Besides providing detailed genetic maps of the bacteria, this analysis has revealed gene island rearrangements, putative phenotypes (including antimicrobial drug resistance) and genetic mutation mechanisms that enable brachyspires to evolve and respond to stress. The application of Next-Generation Sequencing (NGS) to generate genomic data from many more Brachyspira species and strains increasing will improve our understanding of these enigmatic spirochetes.

Intestinal spirochetosis and chronic watery diarrhea: clinical and histological response to treatment and long-term follow up.

BACKGROUND: The clinical significance of intestinal spirochetosis is uncertain, therefore the aim of the present paper was to assess the prevalence of histological intestinal spirochetosis in patients with and without chronic watery diarrhea and to evaluate its clinical relevance. METHODS: A prospective diagnostic work-up of intestinal spirochetosis was made on biopsy samples taken from patients with chronic watery diarrhea submitted between 1994 and 2004 (1174 colonoscopies with multiple biopsies). Three other positive cases identified from routine endoscopic biopsies also were reviewed. In addition, samples from 100 asymptomatic control patients and a random sample of another 104 colonic specimens were reviewed for intestinal spirochetosis. The diagnosis was established by light and electron microscopy. Polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA and reduced nicotinamide adenine dinucleotide (NADH) oxidase genes of the intestinal spirochetes Brachyspira aalborgi and Brachyspira pilosicoli was performed on tissue biopsies of the 11 positive patients. After diagnosis, treatment with penicillin benzatine (PB) or metronidazole was offered to all symptomatic patients and they were followed for a mean of 45.4 months (range: 37-113 months). RESULTS: Eight patients with chronic watery diarrhea were positive for intestinal spirochetosis. Intestinal spirochetosis was not diagnosed in the controls. Histological resolution of the infection paralleled clinical recovery in six patients (following metronidazole treatment in three). Most patients showed mild, non-specific colonic inflammation. Invasion by the spirochetes was not demonstrated by electron microscopy. Brachyspira aalborgi and B. pilosicoli each were identified by PCR in two cases. CONCLUSIONS: Histological intestinal spirochetosis appears to be relatively uncommon in Catalonia (Spain) compared to previous reports from other countries, but was identified in patients (0.7%) with chronic watery diarrhea. Sustained clinical recovery after spontaneous or drug-induced spirochetal disappearance in these individuals suggests that intestinal spirochetosis may play a pathogenic role in chronic watery diarrhea. Treatment with metronidazole is advisable in patients with persistent symptoms.