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. 2022 May 3;12(1):7210.
doi: 10.1038/s41598-022-11340-3.

Microencapsulated probiotic Lactiplantibacillus plantarum and/or Pediococcus acidilactici strains ameliorate diarrhoea in piglets challenged with enterotoxigenic Escherichia coli

Pawiya Pupa  1 , Prasert Apiwatsiri  1 , Wandee Sirichokchatchawan  2 , Nopadon Pirarat  3 , Teerawut Nedumpun  1 , David J Hampson  4 , Nongnuj Muangsin  5 , Nuvee Prapasarakul  6   7
Affiliations

Microencapsulated probiotic Lactiplantibacillus plantarum and/or Pediococcus acidilactici strains ameliorate diarrhoea in piglets challenged with enterotoxigenic Escherichia coli

Pawiya Pupa et al. Sci Rep. .

Abstract

Lactiplantibacillus plantarum (strains 22F and 25F) and Pediococcus acidilactici (strain 72N) have displayed antibacterial activity in vitro, suggesting that they could be used to support intestinal health in pigs. The aim of this study was to determine if microencapsulated probiotics could reduce the severity of infection with enterotoxigenic Escherichia coli (ETEC) in weaned pigs. Sixty healthy neonatal piglets were cross-fostered and separated into five groups. Piglets to be given the microencapsulated probiotics received these orally on days 0, 3, 6, 9, and 12. Only piglets in groups 1 and 5 did not receive probiotics: those in groups 2 and 4 received the three microencapsulated probiotic strains (multi-strain probiotic), and piglets in group 3 received microencapsulated P. acidilactici strain 72N. After weaning, the pigs in groups 3-5 were challenged with 5 mL (at 109 CFU/mL) of pathogenic ETEC strain L3.2 carrying the k88, staP, and stb virulence genes. The multi-strain probiotic enhanced the average daily gain (ADG) and feed conversion ratio (FCR) of weaned piglets after the ETEC challenge (group 4), whilst supplementing with the single-strain probiotic increased FCR (group 3). Piglets in groups 3 and 4 developed mild to moderate diarrhoea and fever. In the probiotic-fed piglets there was an increase in lactic acid bacteria count and a decrease in E. coli count in the faeces. By using real-time PCR, virulence genes were detected at lower levels in the faeces of pigs that had received the probiotic strains. Using the MILLIPLEX MAP assay, probiotic supplementation was shown to reduce pro-inflammatory cytokines (IL-1α, IL-6, IL-8, and TNFα), while group 4 had high levels of anti-inflammatory cytokine (IL-10). Challenged piglets receiving probiotics had milder intestinal lesions with better morphology, including greater villous heights and villous height per crypt depth ratios, than pigs just receiving ETEC. In conclusion, prophylactic administration of microencapsulated probiotic strains may improve outcomes in weaned pigs with colibacillosis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic of experimental designs and sample collection. W = weighing (body weight and feed intake), Fm = Faecal collection for microbial profile analysis, Fe = Faecal collection for ETEC shedding and consistency scoring determination, B = Blood collection, T = Rectal temperature record, and In = Small intestinal collection. dac = day after cross-fostering, hpc = hour post-challenge and dpc = day post-challenge.
Figure 2
Figure 2
Growth performance of piglets after ETEC challenge (5 × 109 CFU). (A)  Average daily gain (ADG); and  (B)  Feed conversion ratio (FCR). Control = oral supplementation of sterile peptone water followed by oral administration of sterile peptone water; Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral administration of sterile peptone water; ETEC + Single-strain = oral supplementation of double-coated single-strain probiotic followed by oral challenged with ETEC; ETEC + Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral challenged with ETEC; and ETEC = oral supplementation of sterile peptone water followed by oral challenge with ETEC. Values are presented as mean ± SEM of all replications in each group. The asterisks represent statistically significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). dpc = day post-challenge.
Figure 3
Figure 3
Clinical signs in piglets after ETEC challenge (5 × 109 CFU). (A) Faecal consistency score (FCS); and (B)  Rectal temperature (RT). Control = oral supplementation of sterile peptone water followed by oral administration of sterile peptone water; Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral administration of sterile peptone water; ETEC + Single-strain = oral supplementation of double-coated single-strain probiotic followed by oral challenge with ETEC; ETEC + Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral challenge with ETEC; and ETEC = oral supplementation of sterile peptone water followed by oral challenge with ETEC. FCS and RT of all replications in each group are presented as median with range and mean ± SEM, respectively. FCS and RT, which are on or over the dashed line, indicate diarrhoea signs and fever in the piglets. hpc = hour post-challenge.
Figure 4
Figure 4
Virulence gene detection in piglets’ faeces after ETEC challenge (5 × 109 CFU). (A)  k88; (B)  staP; and (C)  stb. Control = oral supplementation of sterile peptone water followed by oral administration of sterile peptone water; Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral administration of sterile peptone water; ETEC + Single-strain = oral supplementation of double-coated single-strain probiotic followed by oral challenged with ETEC; ETEC + Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral challenged with ETEC; and ETEC = oral supplementation of sterile peptone water followed by oral challenge with ETEC. Values are presented as mean ± SEM of all replications in each group. abcMean values within a time point with different superscript letters are significantly different (P < 0.05). Values under the dashed line indicate the under-determination of gene copies number (≤ 40 copies number/μL). hpc = hour post-challenge.
Figure 5
Figure 5
Serum cytokine concentrations at different time point in piglets after ETEC challenge (5 × 109 CFU). (A)  IL-1α; (B) IL-6; (C) IL-8; (D) TNFα; and (E) IL-10. Control = oral supplementation with sterile peptone water followed by oral administration of sterile peptone water; Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral administration of sterile peptone water; ETEC + Single-strain = oral supplementation of double-coated single-strain probiotic followed by oral challenged with ETEC; ETEC + Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral challenge with ETEC; and ETEC = oral supplementation of sterile peptone water followed by oral challenge with ETEC. Values are presented as mean ± SEM of all replications in each group. The asterisks represent statistically significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). hpc = hour post-challenge.
Figure 6
Figure 6
Representative intestinal histo-pathology (jejunum) of piglets after ETEC challenging (5 × 109 CFU). (A) 7 days post challenge; and (B) 14 days post challenge. Control = oral supplementation of sterile peptone water followed by oral administration of sterile peptone water; Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral administration of sterile peptone water; ETEC + Single-strain = oral supplementation of double-coated single-strain probiotic followed by oral challenge with ETEC; ETEC + Multi-strain = oral supplementation of double-coated multi-strain probiotic followed by oral challenge with ETEC; and ETEC = oral supplementation of sterile peptone water followed by oral challenge with ETEC.
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