Overview

1. Introduction

In livestock farming, effective alternatives to antibiotics that are able to promote health and prevent pathologies are urgently required to tackle antibiotic resistance[1][2][3], and replacing and reducing antibiotic treatments is one of the main targets of European policies [4]. This became even more important after the removal from the market of zinc oxide (ZnO) as a veterinary therapeutic treatment [5][6]. This decision was taken due to the observed increase in heavy metal environmental pollution and scientific evidence showing that ZnO co-selects antibiotic-resistant bacteria [7][8]. ZnO has been used widely after the ban on antibiotics as a growth promoter over the last decade [6][9][10][11]. Alternatives to ZnO and antibiotics are thus required particularly during the weaning phase due to the high incidence of enteric disorders and multifactorial diseases such as post-weaning diarrhoea (PWD) [12][13]. The gastroenteric tract (GIT) is a complex environment where the mucosal chemical barrier, immune system, microbiota and epithelium all impact intestinal health [14][15]. Preserving intestinal health decreases the incidence of pathologies, optimises digestive processes and promotes animal performance. There is increased awareness regarding the role of diet, not only as a physiological requirement, but also in the enhancement of animal and human health and in the prevention of specific pathologies [16]. The modulation of intestinal microbiota by dietary approaches, such as the use of feed additives, is one of the most promising strategies to reduce the risk of pathologies in food-producing animals [17][18].
Probiotics are functional feed additives defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” [19]. Their potential mechanisms of action affect the intestinal microbial ecology through the manipulation of microbiota that lower the luminal pH, the competitive inhibition of pathogen strains, the production of bacteriocins with antimicrobial proprieties and the stimulation of the host immune system [20]. Probiotic supplementation in animal diets helps prevent or treat a variety of intestinal disorders, although their mechanisms of actions are not completely known [21]. Lactic acid bacteria include over two-hundred species and subspecies of which Lactobacillus sp., Lactococcus sp., Spreptococcus sp. and Enterococcus sp. are used as probiotics for monogastric animals [22].
Lactobacillus plantarum is included in the European register of feed additives [8] as a preservative (1; a), silage additive (1; k), microorganism (1; k) and gut flora stabilizer for chickens (4; b). In several in vitro and in vivo studies, some strains of L. plantarum demonstrated a protective activity against epithelial intestinal barrier impairment, restoring the function of thigh junctions and reducing paracellular permeability [23][24]. In addition, L. plantarum CGMCC 1258 supplemented at 5 × 1010 CFU/kg showed its positive effect in weaned piglets challenged with Escherichia coli K88, inhibiting diarrhoea and improving zootechnical performance [25]. In parallel, Lactobacillus reuteri was included in the EU feed additive register as a microorganism (1; k) until its withdrawal in 2012 [8] due to a lack of the required documentation. This microorganism is not seen as being dangerous and no issues related to its safety were mentioned in the EU commission decision [26], since it is included in the Qualified Presumption of Safety (QPS) list of the European Union [27].
Lactobacillus reuteri I5007 has shown a potential to improve thigh junction expression in newborn piglets and has been found to have protective effects after lipo-polysaccharide (LPS)-induced stress in vitro [21]. L. reuteri strains TMW1.656 and LTH5794 produce reuteran which can decrease the adhesive capacity of ETEC E. coli [28][29]. However, several studies have shown the positive impact of various L. plantarum and L. reuteri strains on improving piglet performance, diarrhoea prevention, stress alleviation, immunity and microbiota modulation [29].
Since few papers have assessed the effects of L. plantarum and L. reuteri strains and their synergy through a wide range of bacterial combination and supplementation levels, more studies are required to clarify the functional proprieties and the optimal inclusion level of these two bacterial strains on diarrhoea prevention in weaned piglets. In addition, probiotics may interact with the host metabolism [30] through their hypocholesterolemic and liver protection effects [31][32]. Furthermore, the bacterial combination does not always result in a synergistic effect, also showing possible competition among probiotic strains [33].

2. Acid and Simulated In Vitro Digestion Resistance

Bacterial strains exposed to a different pH range showed a statistically significant drop in viability at pH 2, with L. plantarum and L. reuteri registering a bacterial count of 8.09 ± 0.11 and 9.00 ± 0.02 log10 CFU/mL, respectively (p < 0.0001), compared to their relative controls at pH 7 (9.60 ± 0.08 and 10.79 ± 0.02 log10 CFU/mL, respectively) (Figure 1). Regarding the simulated gastrointestinal digestion, both bacterial strains exhibited an optimal capacity to survive with each tested condition, including gastric juice, bile shock and intestinal juice, without registering any significant decrease in viability compared to their relative initial microbial charge (Figure 2).
Figure 1. Acid resistance assay from pH 2 to 7 for L. plantarum and L. reuteri. Data are expressed as least square means (LSMEANS) and standard errors (SE). a,b,c Means with different superscript letters indicate statistically significant differences (p < 0.05).
Figure 2. Simulated in vitro gastrointestinal digestion resistance of L. plantarum and L. reuteri. Data are expressed as least square means (LSMEANS) and standard errors (SE). T0 corresponds to the initial microbial charge measured before gastrointestinal environment simulation.

3. Zootechnical Performance

The results of individual BW recorded weekly showed no significant differences throughout the experimental period (Figure 3). The average BW of CTRL and PLA groups revealed a statistically significant tendency compared to P+R considering the entire experimental period (10.45 ± 0.19; 10.42 ± 0.17; 9.84 ± 0.16 kg, respectively; p < 0.09). In addition, the effect of treatments on ADG for the entire experimental period was significantly different for CTRL, REU and P+R groups, which showed a reduced average gain for treated groups with L. reuteri (CTRL: 260 ± 9, REU: 220 ± 8, P+R: 229 ± 7 g/day; p < 0.05). The ADFI of the supplemented groups decreased during the second week (7–14 days) of the study (CTRL: 490 ± 24, PLA: 281 ± 24; REU: 334 ± 24, P+R: 318 ± 22 g/day; p < 0.01). The FCR parameter highlighted an increased ratio in P+R group compared to CTRL, PLA and REU during the first week (0–7 days; CTRL: 2.89 ± 0.24; PLA: 2.67 ± 0.24; REU: 2.67 ± 0.24; P+R: 4.32 ± 0.23; p < 0.01).
Figure 3. Zootechnical performance of control (CTRL) and treatment groups (PLA, REU and P+R) measured over 28 days of experimental trial. Data are expressed as least square means (LSMEANS) and standard errors of the means (SE); a,b Means with different superscripts are significantly different among treatments (p < 0.01); Presented p-values indicate statistically significances of pairwise comparisons; BW: body weight; ADG: average daily gain; ADFI: average daily feed intake; FRC: feed conversion ratio; CTRL: control group; PLA: treatment group supplemented with 2 × 108 CFU/g of L. plantarum; REU: treatment group supplemented with 2 × 108 CFU/g of L. reuteri; P+R: treatment group supplemented with 2 × 108 CFU/g of L. plantarum and L. reuteri (1:1 w/w).

4. Diarrhoea Occurrence

Considering the entire experimental period, diarrhoea observed frequencies differed significantly among treatments The highest number of cases of diarrhoea (20 cases) was found in the CTRL group, while 13 and 10 cases were recorded in the REU and P+R groups, respectively. The lowest number of diarrhoea cases (five cases) was recorded in the PLA group (Figure 4). Data on diarrhoea incidence considering each timepoint showed a statistically significant increase in CTRL compared to the treated groups at T2 (CTRL: 6 cases, 25.00%; PLA: 0 cases, 0.00%; REU 0 cases, 0.00%; P+R: 2 cases; 7.14%; p < 0.01) (Figure 5A). At the last sampling point (T4), diarrhoea occurrence was significantly lower in the PLA and P+R groups (CTRL: 7 cases; 29.17%; PLA: 0 cases, 0.00%; REU: 6 cases, 25.00%; P+R: 1 case; 3.57%; p < 0.01). Average faecal scores of representative subgroups of evaluated piglets revealed a higher score for the CTRL group compared with PLA at T1 (CTRL: 1.17 ± 0.13; PLA: 0.40 ± 0.13; p < 0.01) (Figure 5B). The average faecal score of CTRL after 14 days (T2) increased significantly compared with the treatment groups (CTRL: 1.31 ± 0.13; PLA: 0.34 ± 0.13; REU: 0.24 ± 0.13; P+R: 0.16 ± 0.12; p < 0.0001). At 21 days (T3), P+R highlighted a lower score compared to the CTRL group (CTRL: 0.89 ± 0.13; P+R: 0.18 ± 0.13; p < 0.05). PLA and P+R groups showed a significant decrease in average faecal score at the end of the trial compared to the CTRL group (CTRL: 1.16 ± 0.14; PLA: 0.13 ± 0.14; P+R: 0.17 ± 0.12; p < 0.0001).

Figure 4. Total diarrhoea cases recorded during the 28-day trial for the control (CTRL) and treatment groups (PLA, REU and P+R). Data are expressed as the sum of recorded cases of diarrhoea, considering a faecal score ≥ 2 diarrhoea; a,b,c Means with different superscripts are significantly different among treatments (p < 0.01). CTRL: control group; PLA: treatment group supplemented with 2 × 108 CFU/g of L. plantarum; REU: treatment group supplemented with 2 × 108 CFU/g of L. reuteri; P+R: treatment group supplemented with 2 × 108 CFU/g of L. plantarum and L. reuteri (1:1, w/w).

Figure 5. Number of diarrhoea cases recorded (A) faecal scores (B) during the 28-day trial for the control (CTRL) and treatment groups (PLA, REU and P+R). are expressed as the sum of the recorded cases of diarrhoea, considering faecal score ≥ 2 diarrhoea; a,b Means with different superscripts are significantly different among treatments (p < 0.01). (B) Data are expressed as least square means and standard errors (SE); * Means with asterisks are significantly different from the control group (CTRL, p < 0.0001). CTRL: control group; PLA: treatment group supplemented with 2 × 108 CFU/g of L. plantarum; REU: treatment group supplemented with 2 × 108 CFU/g of L. reuteri; P+R: treatment group supplemented with 2 × 108 CFU/g of L. plantarum and L. reuteri (1:1, w/w).

5. Serum metabolism

The results of serum metabolites showed no statistically significant differences over time for all experimental groups at T0 (Table S5). After 28 days, the PLA group showed a statistically significant increase in globulin content compared to the other groups (Table 3; p < 0.05). Consequently, the albumin/globulin ratio of the PLA group was lower than the other experimental groups (p < 0.05). Alanine aminotransferase (ALT) decreased significantly in the PLA and REU groups compared to the other groups (p < 0.01). The phosphorous concentration was higher in the P+R compared to PLA and REU groups (p < 0.05). The PLA group showed a decreased magnesium content in serum compared to the other groups (p < 0.05). Total cholesterol was lower in PLA and REU compared to the other experimental treatments (p < 0.05). In fact, high density lipoproteins were lower in PLA and REU compared to CTRL and P+R treatments (p < 0.01).

Table 3. Serum metabolites concentration at 28 days (T4) of in vivo trial, for the control (CTRL) and treatments groups (PLA, REU and P+R).

Serum Metabolite

CTRL

PLA

REU

P+R

p-Value

Total protein content, g/L

53.26 ± 1.23

54.85 ± 1.15

51.84 ± 1.15

52.09 ± 1.15

0.2576

Albumin, g/L

28.35 ± 0.74

26.00 ± 0.70

25.94 ± 0.70

26.77 ± 0.70

0.0916

Globulin, g/L

24.91 ± 1.09 a

28.89 ± 1.03 b

25.91 ± 1.03 a

25.31 ± 1.03 a

0.0455

Albumin/Globulin (A/G)

1.16 ± 0.05 a

0.92 ± 0.05 b

1.06 ± 0.05 ab

1.01 ± 0.05 ab

0.0287

Urea, mmol/L

1.06 ± 0.21

1.42 ± 0.20

0.96 ± 0.20

0.89 ± 0.20

0.2452

Alanine aminotransferase (ALT-GPT), IU/L

50.00 ± 2.89 a

38.22 ± 2.73 b

35.78 ± 2.73 b

46.00 ± 2.73 ab

0.0034

Total bilirubin, µmol/L

1.84 ± 0.13

1.42 ± 0.12

1.45 ± 0.12

1.58 ± 0.12

0.1021

Glucose, mmol/L

6.36 ± 0.47

6.31 ± 0.44

5.36 ± 0.44

6.41 ± 0.44

0.2926

Phosphorus, mmol/L

3.19 ± 0.09 ab

2.87 ± 0.08 a

2.98 ± 0.08 a

3.30 ± 0.08 b

0.0038

Magnesium, mmol/L

0.92 ± 0.04 a

0.77 ± 0.11 b

0.79 ± 0.12 ab

0.85 ± 0.13 ab

0.0196

Creatinine, µmol/L

70.75 ± 3.51

77.33 ± 3.31

76.00 ± 3.31

81.10 ± 3.31

0.2175

Total cholesterol, mmol/L

2.70 ± 0.14 a

2.20 ± 0.13 b

2.27 ± 0.13 b

2.68 ± 0.13 a

0.0195

High density lipoprotein (HDL), mmol/L

1.08 ± 0.06 a

0.77 ± 0.06 b

0.81 ± 0.06 b

1.04 ± 0.06 a

0.0011

Low density lipoprotein (LDL), mmol/L

1.50 ± 0.09

1.26 ± 0.09

1.31 ± 0.09

1.52 ± 0.09

0.1173

Triglycerides, mmol/L

0.58 ± 0.08

0.83 ± 0.07

0.70 ± 007

0.60 ± 0.07

0.0764

Interleukin 3, pg/L

17.80 ± 1.98

14.78 ± 2.17

17.28 ± 2.17

17.90 ± 2.17

0.7098

Interleukin 6, pg/L

166.47 ± 45.87

152.65 ± 45.87

155.06 ± 45.87

166.48 ± 45.87

0.9941

Interleukin 10, pg/L

10.67 ± 2.13

8.91 ± 2.13

8.49 ± 2.13

10.80 ± 2.13

0.8158

Data are expressed as least square means (LSMEANS) and standard errors (SE). a,b Means with different superscripts are significantly different among treatments (p < 0.05). CTRL: control group; PLA: treatment group supplemented with 2 × 108 CFU/g of L. plantarum; REU: treatment group supplemented with 2 × 108 CFU/g of L. reuteri; P+R: treatment group supplemented with 2 × 108 CFU/g of L. plantarum and L. reuteri (1:1, w/w).

6. Conclusions

Dietary supplementation of 2 × 108 CFU/g of L. plantarum and L. reuteri significantly reduced diarrhoea occurrence registering and had the lowest faecal score in our trial. L. plantarum had the lowest diarrhoea frequency compared to the other bacterial strains and their combinations. Lactobacilli supplementation did not influence animal performance, total faecal bacteria, faecal lactobacilli and coliform. Dietary lactobacilli inclusion did not reveal metabolic status alteration ascribable to a pathological status. In particular, L. plantarum significantly raised the globulin levels, suggesting a possible stimulation of the immune system. In conclusion, we believe that L. plantarum and L. reuteri are promising functional feed additives for preventing pig diarrhoea. More studies are required to enrich knowledge of these bacterial strains, to assess their effect for longer experimental periods, and to optimise their possible delivery systems.

We thank Biotecnologie B.T. Srl that provided lactobacilli strains for this study.

doi:10.3390/ani11061766 

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