Effects of zinc oxide and Enterococcus faecium SF68 dietary supplementation on the performance, intestinal microbiota and immune status of weaned piglets

Research in Veterinary Science 80 (2006) 45–54 www.elsevier.com/locate/rvsc

Effects of zinc oxide and Enterococcus faecium SF68 dietary supplementation on the performance, intestinal microbiota and immune status of weaned piglets L.J. Broom

a,*

, H.M. Miller a, K.G. Kerr

b,d

, J.S. Knapp

c

a School of Biology, The University of Leeds, Leeds LS2 9JT, UK Department of Microbiology, Harrogate Health Care Trust, Harrogate HG2 7SX, UK Division of Microbiology, School of Biochemistry, The University of Leeds, Leeds LS2 9JT, UK d Hull York Medical School, The University of York, Heslington, York YO10 5DD, UK b

c

Accepted 15 April 2005

Abstract The objective of this study was to determine the effects of zinc oxide (ZnO) and the probiotic Enterococcus faecium SF68 (Cylactin
) dietary supplementation on the performance, intestinal microbiota and immune parameters of the weaned piglet reared under commercial conditions. The diets were devoid of antibiotic growth promoters (AGP). Two hundred and eight crossbred piglets were allocated to a 2 · 2 factorial experiment involving two levels of zinc oxide supplementation (0 or 3100 mg ZnO/kg feed), and two levels of E. faecium SF68 supplementation (0 or 1.4 · 109 CFU/kg feed (Cylactin
ME10)). The diets were offered ad libitum for 20 days post-weaning. Piglet performance was assessed by calculating average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) on a pen basis. In addition, components of the distal ileal digesta, tissue-associated and mesenteric lymph node (MLN) bacterial populations were enumerated and serum immunoglobulin G (IgG) and intestinal immunoglobulin A (IgA) concentrations were determined on days 6 and 20 post-weaning. Regression analysis was used to determine the relationship between the bacterial populations at the different sites. Supplementation of the post-weaning diet with either ZnO or E. faecium SF68 did not affect piglet performance. E. faecium SF68 did not affect gastrointestinal bacterial populations but did tend to reduce serum IgG (P < 0.1) on day 20. Zinc oxide reduced anaerobic (P < 0.05) and tended to decrease lactic acid (P < 0.1) bacterial translocation to the MLN, and tended to increase intestinal IgA concentration (P < 0.1) on day 20. Generally, luminal bacterial populations were found to be poor predictors of tissue-associated or MLN populations. ZnO and E. faecium SF68 dietary supplementation were ineffective under these trial conditions. Further investigations into the possible immunomodulator role of dietary ZnO are warranted.  2005 Elsevier Ltd. All rights reserved. Keywords: Pigs/swine; Zinc oxide; Enterococci; Probiotics; Antibiotic growth promoters; Bacteria; Bacterial translocation; Immunoglobulins A and G

1. Introduction The gastrointestinal microbiota represents a diverse and complex ecosystem with approximately 400 bacte*

Corresponding author. Current address: Danisco Animal Nutrition, PO Box 777, Marlborough, Wiltshire, SN8 1XN, UK. E-mail address: [email protected] (L.J. Broom). 0034-5288/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2005.04.004

rial species so far identified (Berg, 1995). A diverse and stable intestinal microbiota plays a vital contribution towards the health and productive performance of the host. At weaning, the piglet is subjected to momentous nutritional and environmental changes that can dramatically alter the equilibrium of the gastrointestinal microbiota. These changes are coupled with a loss of intestinal immune protection previously provided by

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L.J. Broom et al. / Research in Veterinary Science 80 (2006) 45–54

biologically active components of the sowÕs milk (Blecha, 1998). The resultant alteration to the gastrointestinal microbial balance provides an opportunity for pathogens to colonise and cause disease, poor growth performance and even death. Attempts to minimise the impact of weaning on the pigletÕs health and performance have primarily focused on the inclusion of antibiotic growth promoters (AGP) and zinc oxide (ZnO) in the post-weaning diet. Pharmacological concentrations of ZnO have proven to be effective at promoting post-weaning piglet growth (Hahn and Baker, 1993; Hill et al., 2001) and reducing the incidence of diarrhoea (Melin and Wallgren, 2002), although its mode of action remains unclear. Previous studies have proposed possible effects on post-weaning intestinal architecture (Li et al., 2001), luminal bacteria (Katouli et al., 1999) or bacterial translocation (Huang et al., 1999). Additional experiments have demonstrated a number of properties attributable to ZnO from antibacterial (Soderberg et al., 1990) to aiding the wound healing process (Tarnow et al., 1994). There are, however, concerns within the EU regarding the feeding of pharmacological doses of ZnO to pigs and the associated accumulation of Zn in the environment. These concerns may affect the future pharmacological use of zinc oxide in piglet diets. For many decades, serious concerns have also been expressed over the continued growth-promotional use of antibiotics in animal husbandry and the associated emergence of antibiotic-resistant bacterial strains in human medicine. Within the EU, these concerns have resulted in legislation to ban such use of antibiotics by 1st January 2006 and the search for credible alternatives continues. Probiotics have been postulated as being alternatives to AGP. Probiotics have been defined as live microbial feed supplements that affect the host by improving its intestinal microbial balance (Fuller, 1989). Probiotic preparations generally consist of viable lactic acid-producing bacteria of intestinal origin such as lactobacilli, bifidobacteria and enterococci. It is proposed that the addition of these microorganisms into the gastrointestinal environment helps to restore/maintain a beneficial intestinal microbial community, thus preventing digestive disorders and potentially improving growth performance (Fuller, 1989). Previously, we have demonstrated that feeding a pharmacological dose of ZnO and a subtherapeutic concentration of AGP enhances post-weaning piglet growth and affects the composition of the faecal microbiota (Broom et al., 2003). In a pilot study conducted at the University of Leeds, E. faecium SF68 (Cylactin
) dietary supplementation improved piglet performance in the first week post-weaning (Broom et al., unpublished). The objective of this study was, therefore, to investigate the effects of ZnO and a commercially available probiotic strain E. faecium SF68 on the performance, intesti-

nal microbiota and immune status of weaned piglets offered diets devoid of AGP. This experiment was conducted on a commercial pig unit where health and performance responses to dietary ZnO supplementation are usually observed (Miller et al., unpublished). In addition, we aimed to determine whether probiotic inclusion could be considered an alternative to ZnO supplementation. It was anticipated that both preparations would contribute to the maintenance of post-weaning gut health and thus improve piglet growth performance.

2. Materials and methods 2.1. Animals and diets The study was approved by the Ethical Review Group of the University of Leeds. Two hundred and eight crossbred piglets (62.5% Large White, 25% Landrace, 12.5% Duroc) were weaned at 22.9 ± 3.6 days of age (±SEM) and 6.8 ± 0.8 kg liveweight, into fully-slatted, temperature-controlled flatdeck accommodation at the University of Leeds commercial pig unit. Prior to weaning, normal commercial practices such as tail docking and the administration of a 1 ml intramuscular iron dextran injection had been performed within 24 h of birth. The piglets did not have access to creep feed during the lactational period. At weaning, six or seven piglets were allocated to each pen (1.99 m2) on the basis of litter origin, weight and gender. Each pen was randomly allocated to a 2 · 2 factorial experiment involving two levels of zinc oxide supplementation (0 or 3100 mg ZnO/kg feed), and two levels of E. faecium SF68 supplementation (0 or 1.4 · 109 CFU/kg feed (Cylactin
ME10)), in eight replications. The compositions of the basal diets are shown in Table 1. The experimental diets were offered ad libitum for 20 days post-weaning and were formulated to exceed National Research Council Table 1 Composition (percentage of feed ingredients) and analysis of the basal diets Ingredient (%)

Weaning diets Days 0–7

Maize Wheat Porridge oat meal Herring meal Whey powder Soya oil L-Lysine HCl Vitamin/mineral premix

5.0 18.9 22.5 12.5 11.3 5.8 0.34 1.25

Days 8–20 2.5 33.7 10.0 12.0 10.0 1.8 0.28 1.50

Total

100

100

Digestible energy (MJ/kg) Crude protein (g/kg) Lysine (g/kg)

16.8 224 17.0

15.5 224 15.5

L.J. Broom et al. / Research in Veterinary Science 80 (2006) 45–54

(1998) nutrient recommendations. Fresh, clean drinking water was provided ad libitum throughout the experimental period. 2.2. Food intakes and weighing Daily pen food disappearance was recorded during the 20 day post-weaning experimental period. Piglets were tagged at weaning and individually weighed on day 0 (weaning), day 6, and weekly thereafter through to day 20 post-weaning. 2.3. Post-mortem sampling procedure Eight piglets per treatment were humanely killed on days 6 and 20 post-weaning under Schedule 1 to the Animals (Scientific Procedures) Act 1986. The eight piglets were selected as being most representative of their experimental treatment group in terms of growth performance. Following confirmation of death, the ventral surface was wiped with ethanol. The body cavity was exposed by making a mid-line incision along the sternum and abdomen with a sterile scalpel blade. Sterile forceps were used to disturb the internal organs and locate mesenteric lymph nodes associated with the distal ileum. The lymph nodes were carefully extracted with the aid of sterile scissors and tweezers. Similarly, under aseptic conditions, distal ileal tissue and luminal contents were obtained. Following removal, the ileal tissue was flushed with 10 ml sterile PBS. Samples were immediately transferred to pre-weighed, sterilised glass bijouxs containing PBS/ 40% glycerol. The bijoux were re-weighed and rapidly placed in ice. Within 6 h, sterilised Thomas homogenisers had been used to homogenise the mesenteric lymph nodes and ileal tissue and the resultant homogenates were stored at
80 C until bacterial analysis was performed. An additional section of ileal tissue was obtained, gently rinsed with PBS and weighed. The tissue was homogenised in PBS (1:1 w/v ratio) and centrifuged at 2500g for 20 min. The supernatant was collected and stored at
20 C until immunoglobulin A (IgA) concentration was determined. A blood sample was obtained from the descending aorta and collected into serum tubes (Monovette
, Sarstedt, Germany). The samples were centrifuged at 2500g for 20 min and the supernatant was collected and stored at
20 C until immunoglobulin G (IgG) analysis was performed. 2.3.1. Bacteriology Tenfold serial dilutions were made from the preserved intestinal contents and tissue homogenates. One hundred ll (200 ll of the lowest dilution) of each serial dilution was spread on bacteriological media to allow enumeration of specific bacterial types. Aerobes were determined by growth on horse blood agar (Columbia

47

agar base, Oxoid, Basingstoke, England, supplemented by horse blood – 5% v/v) (CBA), following incubation at 37 C for 24 h in air. Anaerobes were enumerated by incubation on CBA for 48 h at 37 C in an anaerobic cabinet (Don Whitley Scientific Ltd., Shipley, UK). Lactic acid bacterial counts were obtained by growth on de Man, Rogosa, Sharpe agar (Oxoid, Basingstoke, England) for 48 h at 37 C. Escherichia coli were isolated by growth on Flurocult ECD agar (Merck, Darmstadt, Germany) for 24 h at 37 C. Colonies which fluoresced under ultra-violet light were considered to be E. coli. Enterococci were enumerated by incubation on kanamycin aesculin azide agar base (kanamycin supplement) (Oxoid, Basingstoke, England) for 72 h at 37 C. Colonies whose appearance was typical of enterococci were regarded as being enterococci. Unless otherwise stated, all of the above incubations were performed in the presence of air. 2.3.2. Intestinal IgA A sandwich IgA ELISA (Bethyl Laboratories Inc., USA) was used to quantify the IgA concentration of the intestinal tissue homogenate supernatant. Briefly, an ELISA plate was coated with Goat anti-Pig IgA antibody and incubated at room temperature (25 C). After washing, standards or suitably diluted samples were added to designated wells. Following further washings, Goat anti-Pig IgA HRP conjugated antibody was added to each well as a detection antibody. Following a further incubation period, TMB enzyme substrate (3,3 0 ,5,5 0 tetramethyl-benzidine) was added to each well. After 20 min, the reaction was stopped with 2 M H2SO4 and individual well absorbencies were determined at 450 nm. The standards were used to construct a standard curve from which the IgA concentration of the samples could be determined. The results were expressed as lg IgA per g tissue. 2.3.3. Serum IgG A sandwich IgG ELISA (Bethyl Laboratories Inc., USA) was used to quantify serum IgG concentration. The ELISA procedure was similar to that outlined for the intestinal IgA, but utilised antibodies directed against porcine IgG. The results were expressed as milligrams of IgG per millilitre of serum. 2.4. Statistical analysis Performance data were analysed using the GLM procedure of Minitab 12.2 (Minitab, 1998), with each pen of piglets representing the experimental unit. Weaning weight and age were used as covariates when appropriate. Intestinal IgA and serum IgG were also analysed by the GLM procedure. Bacteriological counts were log10 transformed prior to GLM analysis. E. coli and enterococci data were either analysed by the Kruskal–Wallis test

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L.J. Broom et al. / Research in Veterinary Science 80 (2006) 45–54

ZnO supplementation did not affect small intestine digesta, tissue or mesenteric lymph node (MLN) aerobic, anaerobic or lactic acid bacterial numbers (Table 3). A E. faecium SF68/ZnO interaction was, however, observed for small intestine tissue lactic acid bacteria (P < 0.01). In addition, the isolation of E. coli and enterococci at these sites was also unaffected (Table 4). Supplementation with ZnO reduced the numbers of MLN anaerobes (4.63 vs. 5.25 log10 colony forming units (CFU) per g tissue) (P < 0.05) and tended to reduce the MLN lactic acid bacterial count (P < 0.1) on day 20 post-weaning. No other bacteriological differences were observed on day 20.

(count data) or the FisherÕs exact test (two-tailed) (frequency data) of SPSS 11.5 (2003). Regression analysis was used to analyse the relationship between bacterial populations at the different sites. When P < 0.05 the difference was considered significant.

3. Results There was no incidence of diarrhoea in this study. 3.1. Pig performance Pig performance during the 20 day experimental period is shown in Table 2. Neither E. faecium SF68 nor ZnO supplementation affected ADFI, ADG or FCR during the immediate 6 days post-weaning. ZnO supplementation tended to increase ADFI (300.7 vs. 269.4 g per pig per day) (P < 0.1) and ADG (282.3 vs. 253.0 g per pig per day) (P < 0.1) during days 7 to 13 of the experiment, but FCR was unaffected. E. faecium SF68 inclusion had no affect on ADFI, ADG or FCR during this period. There was, however, a statistical tendency for E. faecium SF68/ZnO interactions (P < 0.1) for ADG and FCR during this period. Between days 14 and 20 post-weaning, neither E. faecium SF68 nor ZnO supplementation affected ADFI, ADG, or FCR. Similarly, overall (days 1–20) ADFI, ADG and FCR were not influenced by the inclusion of E. faecium SF68 or ZnO into the diets.

3.3. Immunoglobulin parameters There were no differences in either serum IgG or intestinal IgA concentrations on day 6 post-weaning due to the dietary inclusion of E. faecium SF68 or ZnO (Table 5). E. faecium SF68 inclusion tended to reduce serum IgG concentration on day 20 (4.55 vs. 6.07 mg per ml) (P < 0.1), while there was a tendency for day 20 intestinal IgA concentration to be increased by ZnO supplementation (57.78 vs. 39.90 lg per g wet tissue) (P < 0.1). 3.4. Regression analysis Regression analysis data are shown in Table 6. Positive linear relationships were observed between small intestine tissue anaerobes and MLN anaerobes (r2 = 0.407, P = 0.001) (Fig. 1), and small intestine tissue LAB and MLN LAB (r2 = 0.597, P < 0.001) (Fig. 2) on day 6 post-weaning. Small intestine digesta aerobes, anaerobes and LAB were significant predictors of

3.2. Bacteriological data The bacteriological data are presented in Tables 3 and 4. On day 6 post-weaning, E. faecium SF68 and

Table 2 Piglet average daily food intake (ADFI), average daily liveweight gain (ADG) and food conversion ratio (FCR) during the 20 day experimental period Zinc oxide (ZnO) (mg/kg)

P-value

0

Enterococcus faecium SF68 (EF) (CFU/kg) 1.4 · 109

0

3100

SEM

EF

ZnO

EF*ZnO

ADFI (g/p/d) Days 1–6 Days 7–13 Days 14–20 Days 1–20

125.8 275.1 534.2 321.0

127.5 295.0 527.7 326.3

128.2 269.4 512.1 312.0

125.0 300.7 549.8 335.4

7.04 10.87 18.43 10.93

0.866 0.207 0.804 0.732

0.749 0.052 0.160 0.143

0.947 0.400 0.472 0.475

ADG (g/p/d) Days 1–6 Days 7–13 Days 14–20 Days 1–20

99.7 258.1 446.6 276.6

103.4 277.1 427.7 277.7

99.2 253.0 418.8 264.9

104.0 282.3 455.4 289.4

11.09 11.06 18.32 10.37

0.816 0.236 0.472 0.941

0.760 0.072 0.169 0.106

0.951 0.077 0.377 0.232

1.19 0.03 0.03 0.02

0.430 0.717 0.368 0.485

0.400 0.843 0.440 0.242

0.344 0.073 0.553 0.126

a

FCR Days Days Days Days a

1–6 7–13 14–20 1–20

g per pig per day.

3.05 1.08 1.21 1.17

1.72 1.06 1.25 1.18

3.10 1.07 1.25 1.19

1.68 1.07 1.21 1.16

L.J. Broom et al. / Research in Veterinary Science 80 (2006) 45–54

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Table 3 Mean bacterial counts (log10 colony forming units (CFU) per g sample) at different sites of the distal ileum on days 6 and 20 post-weaning Enterococcus faecium SF68 (EF) (CFU/kg) 9

Zinc oxide (ZnO) (mg/kg)

P-value

0

1.4 · 10

0

3100

SEM

EF

ZnO

EF*ZnO

Day 6 SI digestaa Aerobes Anaerobes LABb

6.92 7.36 6.58

6.76 7.43 6.44

6.97 7.60 6.75

6.71 7.19 6.27

0.19 0.19 0.20

0.531 0.782 0.616

0.317 0.136 0.102

0.269 0.630 0.936

SI tissuec Aerobes Anaerobes LAB

4.94 5.27 4.61

5.14 5.39 4.86

5.08 5.46 4.73

5.00 5.20 4.74

0.16 0.22 0.13

0.365 0.695 0.166

0.731 0.384 0.992

0.141 0.585 0.006

MLNd Aerobes Anaerobes LAB

4.77 5.40 4.99

4.84 5.51 5.03

4.81 5.46 5.01

4.81 5.45 5.01

0.16 0.17 0.16

0.749 0.634 0.865

0.991 0.965 0.983

0.151 0.189 0.130

Day 20 SI digesta Aerobes Anaerobes LAB

6.86 7.29 6.65

6.52 7.17 6.67

6.71 7.30 6.67

6.68 7.16 6.65

0.19 0.18 0.20

0.206 0.635 0.935

0.908 0.584 0.939

0.783 0.761 0.161

SI tissue Aerobes Anaerobes LAB

5.21 5.32 4.93

5.09 5.28 4.91

5.27 5.44 5.07

5.03 5.16 4.76

0.12 0.17 0.14

0.494 0.841 0.934

0.147 0.222 0.133

0.840 0.741 0.944

MLN Aerobes Anaerobes LAB

4.18 4.89 4.60

4.28 4.99 4.43

4.35 5.25 4.76

4.10 4.63 4.28

0.19 0.20 0.17

0.715 0.711 0.482

0.367 0.035 0.056

0.918 0.927 0.570

a b c d

Small intestine digesta. Lactic-acid bacteria. Small intestine tissue. Mesenteric lymph nodes.

MLN aerobes (r2 = 0.192, P < 0.05), anaerobes (r2 = 0.232, P < 0.05) and LAB (r2 = 0.158, P < 0.05) on day 20 post-weaning. Similarly, day 20 small intestine tissue anaerobes were a significant predictor of day 20 MLN anaerobes (r2 = 0.281, P < 0.01).

4. Discussion This study has demonstrated that the inclusion of either the bacterium E. faecium SF68 as a probiotic or, more surprisingly, ZnO into the post-weaning diet did not affect piglet growth rate, feed consumption or feed conversion. ZnO supplementation did, however, produce numerical advantages with regards to piglet growth performance and feed intake, which neared significance during week 2 of the trial period with 12% increases in both piglet daily weight gains and feed intakes. There are numerous reports on the benefits to piglet performance from dietary supplementation with pharmacological concentrations of ZnO. Hahn and Baker (1993) demonstrated increased piglet daily weight gains of 17% and daily feed intakes of 14% from the addition

of 3000 mg of Zn per kg to the weaned piglet diet. These findings have been supported by the results of subsequent experiments which have also observed improved weaned piglet growth rates in response to feeding pharmacological doses of ZnO (LeMieux et al., 1995; Smith et al., 1997; Hill et al., 2001). Previously, we have demonstrated that supplementation of the weaned piglet diet with a combination of 3100 mg ZnO per kg and 40 mg avilamycin (AGP) per kg improved piglet feed intake and growth performance during the 20-day experimental period (Broom et al., 2003). The lack of a response to ZnO supplementation in this study may indicate that the effects of ZnO are enhanced in the presence of AGP. Hill et al. (2001), however, provided data that indicated that ZnO and AGP produced independent growth responses, which were additive when the compounds were combined. Further investigation may be warranted. Probiotics are often implicated as alternatives to AGP, but reports on their efficacy are often contradictory and inconclusive. Studies into the effectiveness of E. faecium SF68 in swine are somewhat limited. Pollman et al. (1980) reported that the inclusion of E. faecium SF68 into the piglet diet for 28-days post-weaning did not

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L.J. Broom et al. / Research in Veterinary Science 80 (2006) 45–54

Table 4 Effect of E. faecium SF68 and ZnO on the recovery of E. coli and enterococci from different sites associated with the distal ileum on days 6 and 20 post-weaning Negative control

ZnO

E. faecium SF68 (EF)

ZnO and EF

P-value

Day 6 SI digestaa,b E. coli Enterococci

3.46 3.10

2.66 2.57

2.57 3.51

2.57 3.20

0.141 0.285

SI tissuec,d E. coli Enterococci

3/8 0/8

0/8 1/8

1/8 2/8

1/8 0/8

0.341 0.587

MLNe,d E. coli Enterococci

3/8 0/8

2/8 1/8

3/8 3/8

2/8 0/8

1.000 0.157

Day 20 SI digesta E. coli Enterococci

2.57 2.57

2.57 2.57

3.02 2.57

2.57 2.60

0.288 0.172

SI tissue E. coli Enterococci

3/8 0/8

1/8 3/8

1/8 2/8

0/8 2/8

0.341 0.434

MLN E. coli Enterococci

3/8 0/8

0/8 1/8

2/8 2/8

0/8 1/8

0.128 0.886

a

Small intestine digesta. Median values (log10 colony forming units (CFU) per g sample). Detection limit of 150 CFU/ml diluted digesta. An arbitrary value of 375 CFU per gram of digesta was used when no organisms were detected (i.e., <750 CFU per gram of digesta). c Small intestine tissue. d Number of tissue samples which produced a positive culture (i.e., >150 CFU/ml homogenate) as a proportion of the total samples. e Mesenteric lymph nodes. b

Table 5 Serum IgG (mg/ml) and intestinal IgA (lg/g wet tissue) concentrations on days 6 and 20 post-weaning Enterococcus faecium SF68 (EF) (CFU/kg)

Zinc oxide (ZnO) (mg/kg)

P-value

0

1.4 · 109

0

3100

SEM

EF

ZnO

EF*ZnO

Day 6 Serum IgG Intestinal IgA

5.48 21.32

5.57 25.63

5.61 28.07

5.44 18.89

0.49 6.56

0.897 0.641

0.808 0.323

0.725 0.880

Day 20 Serum IgG Intestinal IgA

6.07 50.66

4.55 47.02

5.14 39.90

5.48 57.78

0.58 6.89

0.077 0.711

0.687 0.077

0.429 0.508

Table 6 Regression analysis of the relationship between bacterial counts at different sites of the distal ileum SI digesta (x) vs. SI tissue (y) r2 (%)

SI digesta (x) vs. MLN (y)

SI tissue (x) vs. MLN (y)

P-value

Equation

r2 (%)

P-value

Equation

r2 (%)

P-value

7.6 1.5

0.163 0.537

y = 3.23 + 0.32x y = 3.27 + 0.19x

Equation

Aerobes Day 6 Day 20

5.3 0.0

0.257 0.983

y = 3.48 + 0.22x y = 5.20
2.74 · 10
3x

2.8 19.2

0.415 0.025

y = 6.08
0.19x y = 1.07 + 0.48x

Anaerobes Day 6 Day 20

5.8 13.9

0.225 0.047

y = 3.72 + 0.23x y = 2.94 + 0.33x

0.3 23.2

0.798 0.011

y = 5.84
4.04 · 10
2x y = 0.72 + 0.60x

40.7 28.1

0.001 0.004

y = 2.40 + 0.57x y = 1.41 + 0.67x

LAB Day 6 Day 20

11.6 8.8

0.071 0.118

y = 3.02 + 0.26x y = 3.56 + 0.21x

1.5 15.8

0.539 0.040

y = 4.26 + 0.11x y = 2.21 + 0.36x

59.7 5.5

<0.001 0.228

y = 1.36 + 0.77x y = 3.10 + 0.29x

L.J. Broom et al. / Research in Veterinary Science 80 (2006) 45–54 y = 0.5737x + 2.4036 R2 = 0.4072 MLN anaerobes (log10 CFU per g tissue)

7 6 5 4 3 2 1 0 1

0

2

3

5

4

7

6

S. I. tissue anaerobes (log10 CFU per g tissue)

Fig. 1. Regression of small intestine tissue (SI tissue) anaerobes against mesenteric lymph node (MLN) anaerobes on day 6 postweaning.

MLN anaerobes (log10 CFU per g tissue)

y = 0.7673x + 1.3566 R2 = 0.5974 7 6 5 4 3 2 1 0 0

1

2

3

4

5

6

7

S. I. tissue anaerobes (log10 CFU per g tissue)

Fig. 2. Regression of small intestine tissue (SI tissue) LAB against mesenteric lymph node (MLN) LAB on day 6 post-weaning.

affect growth performance. It must be stated that in the study of Pollman et al. (1980) it is difficult to ascertain what dietary concentration, in microbial cell numbers or CFU (colony forming units), of E. faecium SF68 they were trying to achieve. Comparisons between our study and their work is, therefore, somewhat difficult. Maeng et al. (1989) reported that dosing piglets up to 4 months with E. faecium SF68 increased weight gain, feed consumption and feed conversion. It is not clear why the inclusion of either preparation into the weaned piglet diet failed to elicit any positive effects on growth performance. It is often postulated that growth promotional compounds become more effective as the environmental, and possibly, the nutritional challenges confronting the animal are exacerbated. Although conducted on a commercial pig unit where performance responses to ZnO supplementation are normally observed (Miller et al., unpublished), the prevailing conditions in this present study may not have been severe enough to produce positive piglet performance effects from the inclusion of the preparations in the post-weaning diet. This study was conducted in the absence of any apparent diarrhoea complications. It is, therefore, impossible for us to reach any conclusions regarding

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the effectiveness of ZnO and E. faecium SF68 in reducing the incidence or severity of post-weaning diarrhoea. Melin and Wallgren (2002) demonstrated that ZnO dietary supplementation reduced the incidence of diarrhoea when weaned piglets were exposed to pathogenic strains of E. coli via their environment. Similarly, Underdahl et al. (1982) reported that E. faecium SF68 decreased the severity of diarrhoea induced by pathogenic E. coli challenge in gnotobiotic pigs. Studies with human subjects have shown that oral administration of E. faecium SF68 results in reduced incidence of, and faster recovery from diarrhoeal diseases (Wunderlich et al., 1989). The lack of post-weaning diarrhoea observed in this study may provide further evidence that the experimental conditions were not suitable to elicit a performance or health response to the supplementation of the diet with these products. In this study, we observed no differences in the enumerated parameters of the distal ileal digesta and tissue-associated microbiota as a result of dietary supplementation with E. faecium SF68. The genus Enterococcus and in particular the species E. faecium are regarded as normal components of the intestinal microbiota of swine (Devriese et al., 1994). We therefore expected the dietary inclusion of the enterococci strain E. faecium SF68 to increase the measurable recovery of enterococci, lactic acid bacteria (LAB) and aerobes from the ileal tissue and digesta sampled. It is possible that the orally administered enterococci had become established in regions of the gastrointestinal (GI) tract that were not studied in the experiment, although we would still have anticipated influences on our measured parameters. Conversely, the administered enterococci may have failed to become established in any region of the GI tract. Therefore, it may have been appropriate to obtain samples from a greater range of sites within the intestinal tract. In addition, it would have been of interest to use a molecular typing technique, such as pulsed field gel electrophoresis, to determine whether E. faecium SF68 had become established in the GI tract of the pigs, this was beyond the scope of the current study. Transient bacterial populations or population fluctuations within the gut lumen are likely to be of little significance if the microorganisms are unable to colonise the intestinal mucosa and become established. To be effective, probiotics must be able to survive the low pH and biliary and pancreatic secretions of the proximal section of the G.I. tract (Lick et al., 2001). E. faecium SF68 was isolated from the human intestinal tract (Chang and Liu, 2002), further studies investigating the ability of this strain to survive intestinal transit and remain viable in swine may be warranted. Disturbances to the pigletÕs intestinal microbiota and opportunities to colonise the intestinal mucosa are likely to be most pronounced in the days immediately following weaning, when dietary and environmental alterations

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are greatest. At this time, piglet voluntary feed intake is low. Current inclusion levels of probiotics into postweaning piglet feeds may be inappropriate. Alternative methods of delivering the probiotic organism into the gastrointestinal environment, e.g., via the water supply, or substantially increasing the feed inclusion level may provide more effective means of establishing the probiotic in the GI tract. Bacterial translocation has been described as the passage of viable indigenous bacteria from the GI tract to extraintestinal sites (Berg, 1995), and may be measured by the recovery of viable bacteria from regional lymph nodes or other tissues (Shou et al., 1994). Bacterial translocation can be induced by bacterial overgrowth, impairment of the intestinal barrier and compromised host immune responses (Berg, 1995). Numerous studies have associated all three factors with the weaning process, and thus, piglets would appear to be extremely susceptible to bacterial translocation in the immediate post-weaning period. The inclusion of E. faecium SF68 into the post-weaning piglet diet did not affect the numbers of bacteria recovered from the mesenteric lymph nodes, but tended to lower the serum concentration of immunoglobulin G on day 20 post-weaning, suggesting a systemic immunomodulatory role for this bacterium. Although the actual number of viable bacteria translocating the intestinal barrier and gaining access to the MLN did not differ, the precise composition of this microbiota may have varied and the resulting interactions of the bacteria and bacterial products with the immune cells of the gut-associated lymphoid tissue (GALT) may have resulted in the production of slightly different cytokine profiles. Cytokines are the principal mediators of host immunity (Kagnoff, 1993). Cytokines released as a result of antigen-lymphoid cell interactions in the GALT can modulate the systemic immune system (Perdigon et al., 1995). Therefore, oral administration of E. faecium SF68 may have resulted in reduced systemic IgG synthesis. The effects of probiotic microorganims on host immune responses have been extensively reviewed (Perdigon et al., 1995; Perdigon et al., 2001). As with E. faecium SF68, we observed no effect of ZnO supplementation on the ileal digesta or tissueassociated microbiota. Dietary ZnO inclusion did, however, reduce the numbers of anaerobes, and tended to reduce the numbers of LAB recovered from MLN on day 20 post-weaning. These reductions were coupled with a tendency for increased intestinal IgA concentration on day 20 as a result of ZnO supplementation. Huang et al. (1999) reported that dietary ZnO inclusion of 3000 mg/ kg reduced bacterial translocation from the intestinal lumen to the ileal MLN. Our results, coupled with the findings of Huang et al. (1999), would clearly suggest that ZnO reduces the translocation of microorganisms from the intestinal environment to extraintestinal sites. Shou et al. (1994) hypothesised that increased intestinal

IgA concentrations may decrease the numbers of viable bacteria in the MLN by reducing bacterial penetration of the mucosal barrier and enhancing the rate of clearance of translocated bacteria. Secretory IgA (sIgA) plays a vital role in mucosal immunity. sIgA is able to neutralise bacteria by binding to adhesins that are utilised by the bacteria to adhere to mucosal epithelial cells (Simecka, 1998). In addition, IgA can remove translocated antigen by transporting it back across the epithelium (Kagnoff, 1993). Zinc oxide has been demonstrated to influence pulmonary cytokine responses (Kuscher et al., 1997), and thus ZnO dietary supplementation may influence intestinal IgA concentrations by altering the cytokine profile of stimulated immune cells residing in the GALT. The secretory IgA response is thought to be mediated through activated Th2 cells (T-helper cell subpopulation) producing cytokines such as interlukin-4, interlukin-6 and transforming growth factor (Simecka, 1998). These cytokines have been identified as important factors in the induction of IgA responses by stimulating the growth and differentiation of B cells (Shou et al., 1994; Simecka, 1998). The possible role of ZnO in enhancing intestinal IgA concentrations warrants further investigation. Berg (1995) proposed that although components of the indigenous microbiota of the GI tract are continually translocating in minimal numbers, the MLN and other extraintestinal sites remain sterile through the activities of the host immune system. Therefore, it is perhaps somewhat surprising that we recovered relatively high numbers of viable bacteria from the MLN, regardless of dietary treatment. In this study, the MLN microbiota was dominated by LAB. Yuzawa et al. (2000) demonstrated that it was possible to obtain bacterial numbers of log 6–8 in the MLN, liver and spleen following small bowel transplantation in growing pigs. As stated previously, the piglet would appear to be particularly susceptible to bacterial translocation in the immediate post-weaning period and so our findings are perhaps not surprising. Shou et al. (1994) reported that oral feeding of a chemically defined elemental diet to mice induced bacterial translocation to the MLN and resulted in impaired lymphocyte and peritoneal macrophage function. Thus, the presence of a significant viable, non-pathogenic LAB population in the MLN may be an important factor in the development of immune tolerance to the indigenous microbiota of the intestinal tract. Maturation of gastrointestinal immunity requires education of the immune system so that appropriate immune responses are induced. The indigenous microbiota is continually interacting with the immune cells of the GALT. If these interactions initiated inappropriate immune responses then the GI tract could be in a state of continual chronic inflammation (Simecka, 1998). For host survival, however, the gastrointestinal immune system must retain the ability to respond

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rapidly and appropriately to pathogenic/antigenic immune stimulation. The role of translocated LAB in the development and maturation of the immune system requires further research. This study has demonstrated that bacterial populations within the gut lumen may be a poor indicator of bacterial populations associated with the intestinal tissue or present in the MLN, a finding which should be taken into account in future studies in this field. Generally, luminal populations would appear to be more indicative of populations in the other sites on day 20 post-weaning than on day 6. This is probably a reflection of the increasing stability of the gastrointestinal environment and microbiota as the post-weaning period progresses. The transitions that occur at weaning have been implicated in compromising the diversity and stability of the intestinal microbiota (Katouli et al., 1999). During the initial days post-weaning, the potentially frequent population shifts of the luminal microbial community may not correlate well with any population alterations at the mucosa or MLN. In contrast, bacterial populations associated with the intestinal tissue would appear to be most indicative of MLN populations on day 6 post-weaning. This may suggest that in the immediate post-weaning period, bacteria that are able to colonise the intestinal mucosa have a greater influence on the MLN bacterial populations than subsequently. This is undoubtedly a reflection of the pigletÕs impaired intestinal barrier and immune system at this time. In conclusion, supplementation of the post-weaning diet with either ZnO or E. faecium SF68 did not affect piglet growth performance in this experiment. This may suggest that the experimental conditions were not suitable to elicit a performance response to supplementation of the diet with these products. E. faecium SF68 did not affect gastrointestinal bacterial populations but did tend to reduce serum IgG, suggesting an immunomodulator role for this bacterium. Zinc oxide reduced bacterial translocation to the MLN and this may have resulted from immune system modulation and enhanced intestinal IgA concentration. We enumerated significant numbers of viable bacteria, dominated by lactic acid bacteria, from the MLN. These bacteria are likely to play a significant role in the development and maturation of the pigletÕs immune system. Finally, the results suggest that luminal bacterial populations may be a poor indicator of populations associated with the intestinal tissue and beyond.

Acknowledgements We gratefully acknowledge Roche Vitamins Europe Ltd for their funding of this research. Leon Broom is supported by a University of Leeds scholarship.

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