Effects of Lactobacilli on the Performance , Diarrhea Incidence , VFA Concentration and Gastrointestinal Microbial Flora of Weaning Pigs

Two experiments were conducted to evaluate the effects of a complex Lactobacilli preparation on performance, resistance to E. coli infection and gut microbial flora of weaning pigs. In exp. 1, twelve pigs (7.65±1.10 kg BW), weaned at 28 d, were randomly allotted into 2 groups and placed in individual metabolic cages. During the first 7 d, one group of pigs was provided ad libitum access to water containing 10 colony forming units (CFU) Lactobacilli per ml and the control group was provided tap water. The Lactobacilli preparation included Lactobacillus gasseri, L. reuteri, L. acidophilus and L. fermentum, which were isolated from the gastrointestinal (GI) tract mucosa of weaning pigs. On d 8, 20 ml of 10 CFU/ml E. coli solution (serovars K99, K88 and 987P at the ratio of 1:1:1) was orally administered to each pig. Diarrhea scores and diarrhea incidence were recorded from d 7 to 14. On d 14, pigs were euthanized and digesta and mucosa from the stomach, duodenum, jejunum, ileum, cecum and colon were sampled using aseptic technique to determine microflora by culturing bacteria in selective medium. The results showed that Lactobacilli treatment significantly decreased E. coli and aerobe counts (p<0.01) but increased Lactobacilli and anaerobe counts (p<0.01) in digesta and mucosa of most sections of the GI tract. A 66 and 69.1% decrease in diarrhea index and diarrhea incidence, respectively, was observed in the Lactobacilli treated group. In exp. 2, Thirty-six crossbred Duroc×Landrace×Yorkshire piglets, weaned at 28±2 days, were selected and randomly allocated into 2 groups. There were 18 piglets in each group, 3 piglets in one pen and 6 replicates in each treatment with 3 pens of barrow and 3 pens of female piglet in each treatment. Piglets had ad libitum access to feed and water. The initial body weight of piglet was 7.65±1.09 kg. Dietary treatments included a non-medicated basal diet with Lactobacilli (10 CFU/g diet) or carbadox (60 mg/kg) as control. On d 21, six pigs per group (one pig per pen) were euthanized. Ileal digesta was collected to determine apparent amino acid digestibility. Microflora content was determined similarly to exp.1. The results showed that Lactobacilli treatment significantly improved average daily feed intake (ADFI) of pigs compared to carbadox (p<0.05) during the first 2 wks after weaning and average daily gain (ADG) and ADFI increased significantly (p<0.05) from d 8 to 14. Nitrogen and total phosphorus digestibility also increased (p<0.05). Bacterial counts were similar to exp. 1. The results indicate that the complex Lactobacilli preparation improved performance for 2 wks after weaning, enhanced resistance to E. coli infection, and improved microbial balance in the GI tract. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 3 : 401-409)


INTRODUCTION
Antibiotics have been used widely in animal production throughout the world which has contributed to the great success of animal agriculture after World War II.However, application of antibiotics in animal agriculture increased, the resistance of pathogenic bacteria to antibiotics developed and resistant bacteria increased.Levy (1992) stated that the major therapeutic problem was bacterial resistance to antibiotics.These situations increase the difficulties in the medical and veterinary practice.The publics complain that the pathogenic microbial resistance was mainly caused by the subtherapeutic addition of antibiotics in animal feed, although the exact reason remains unknown.In addition, some unfortunate event such as Bovine Spongiform Encephalopathy (BSE), dioxin and Foot and Mouth Disease (FMD) make consumers feel more concerned about the animal food safety.So the alternatives to antibiotics have been researched and developed throughout the world.The use of probiotics in pig production has seen renewed interest.The definition of probiotics is not new and the latest one has been defined by Guarner and Schaafsman (1998) as living microorganisms that confer a health effect on the host when consumed in adequate amounts.The development and application of probiotics has expanded in the world and the efficacy has been proven in many cases (Fuller, 1989;Xuan et al., 2001).
Gastrointestinal microflora play a number of important roles in animal production.Native gut microbes was successfully used to prevent infection with salmonellae in poultry (Nurmi and Rantala, 1973).Ehrmann et al. (2002) found that the natural gut microflora of poultry serves as an excellent source for optimal strains.The Lactobacillus acidophilus added in chick feed can reduce shedding of pathogenic E. coli (Watkins et al., 1982) and increased the weight gain of chicken (Tortuero, 1973).Metchnikoff (1908) believed that Lactobacilli could balance the intestinal environment, prevent the growth of pathogenic bacteria, improve health and prolong life as a consequence.
Lactobacilli are commonly used in probiotics as they are known to be non-pathogenic and are also natural inhabitant of the pig's gastrointestinal tract with many beneficial effects (Ewing and Cole, 1994).In healthy pigs, Lactobacilli were the predominant bacteria throughout the alimentary tract, but numbers of Lactobacilli declined dramatically immediately after weaning pigs at 28 days of age (Huis in't Veld and Havenaar, 1993).We hypothesized that natural Lactobacilli found in the pig intestinal tract may be a potential source of probiotic bacteria for weaning pigs.In this study, two experiments were conducted to investigate the effects of a complex Lactobacilli preparation from gut mucosa on performance, resistance to an E. coli challenge, gastrointestinal microflora, VFA and nutrient digestibility of weaning pigs.

Complex Lactobacilli preparation
A complex Lactobacilli preparation was prepared using four strains of Lactobacilli (Lactobacillus gasseri, L. reuteri, L. acidophilus and L. fermentum) with approximately 2.0×10 8 CFU per ml for exp. 1 or per g for exp. 2. The ratio of Lactobacillus strains was determined according to previous results from our laboratory (unpublished).Lactobacillus gasseri, L. reuteri, L. acidophilus and L. fermentum were isolated from mucosa of stomach, duodenum, jejunum, and colon, respectively, of healthy weaning pigs and screened by in vitro selection from over 7,000 native Lactobacilli colonies according to probiotic bacteria criteria including resistance to heat, low pH, copper, bile salts, and storage stability in addition to antagonism to pathogenic agents.

Experiment 1
Animals and diets : During the first 7 d, twelve crossbred pigs (Duroc×Landrace×Yorkshire) weaned at 28±2 d (7.65±1.10kg BW) were fed a non-medicated diet with six pigs drinking tap water containing a 0.1% (v/v) liquid complex Lactobacilli preparation and the remaining six pigs drinking tap water.Water and feed were available at all times.The piglets were placed in individual 70×170 cm metabolic cages in an environmentally controlled nursery (25±2°C).The non-medicated diet was formulated to meet NRC (1998) requirements (Table 1).On d 8, each piglet received 20 ml of nutrient broth containing approximately 10 8 CFU/ml of E. coli.The broth included equal concentrations of E. coli K99, K88 and 987P serovars.The diarrhea occurrence time and diarrhea index were measured in the following 7 days.Diarrhea score was determined according to the criteria shown in Table 2 (Hart and Dobb, 1988).The incidence of diarrhea was calculated as follows: Diarrhea incidence (%)=(number of pigs with diarrhea)/(number of pigs×7)×100%, where the number of pigs with diarrhea was the summation of the number of pigs with diarrhea every day with six pigs in each group.Diarrhea index was the summation of the diarrheal piglet numbers multiplied by corresponding diarrhea score.Pigs were euthanized on d 14 for sample collection.The stomach and approximate 10 cm sections of the duodenum, the middle jejunum, the ileum, the cecum, and the top of the spiral colon were tied off and stored at -80°C for microbial enumerations.
Microflora : Microflora in the piglet gastrointestinal tract was measured by the culture methods with specifically selective medium.Digesta samples were dissolved in sterile a Source: Hart and Dobb (1988).

Experiment 2
Animals, diets and sampling : Thirty-six crossbred pigs (Duroc×Landrace×Yorkshire) weaned at 28±2 d (7.65±1.09kg BW) were randomly allocated into two groups based on initial body weight, sex and litter with six pens per group and three pigs per pen.The pigs were housed in stainless slat-floor pens (2.0×2.0 m) in an environmentally controlled nursery.The pigs had ad libitum access to feed and water.The basal diet (Table 3) supplemented with 0.1% of the complex Lactobacilli preparation (analyzed content of Lactobacilli was 2.4×10 5 CFU/g of diet) was fed to the first group of pigs (n=18).The second group of pigs (n=18) received the basal diet with 60 mg/kg carbadox as the control.From d 15 to 21, 0.5% celite was added to the diets as an indigestible marker.The diet was fed in meal form.Pigs were weighed individually and feed consumption was determined weekly.
On d 21, six pigs per group (one pig per pen) were randomly selected and euthanized.The stomach and -5 cm sections of the duodenum, the middle jejunum, the ileum, the cecum, and the top of the spiral colon were tied off and stored at -80°C for microbial enumerations.One gram of content in the stomach, the duodenum, the proximal jejunum, the middle jejunum, the distal jejunum, the ileum, the cecum, the ascending colon and the descending colon were sampled to determine volatile fatty acids (VFA) concentrations.The ileal contents were collected to determine apparent ileal amino acid digestibility.From d 18 to 21, fresh feces from all three pigs in each pen was collected and stored at -20°C for nutrient digestibility determination.
Microflora : Gastrointestinal microflora (aerobic bacteria, anaerobic bacteria, Lactobacillus and Escherichia coli in digesta and mucosa in the stomach, duodenum, jejunum, ileum, cecum and colon) were investigated.The methods of microbial culture were the same as those in experiment 1.
VFA : Approximately 1 g digesta was suspended in about 5 ml sterile milli-Q water.Volatile fatty acid concentrations were determined by a modification of the capillary GC method described by Jensen et al. (1995).The configuration of the gas chromatograph (HP 6890 series) was as follows: HP-INNOWax (Mixed linked PEF) capillary columns 30 m×0.32 mm×0.5 µm (HP No. 19091N-133).Carrier gas was nitrogen with a constant flow rate of 37 cm/sec, 1.8 ml/min.The column temperature was from 50°C for 11 min then increasing by 30°C/min to reach 220°C for 2 min.The inlet temperature was 250°C and the detector temperature was 250°C.The split ratio was wet at 35:1 and the inlet volume was 2 µl.
Chemical analysis : The diet and feces sample were put in aluminum pans and placed in a forced-aired oven for 96   h.A temperature of 55°C was set to minimize the volatilization of fatty acids.After drying, diet and feces sample were ground through a 0.42 mm screen prior to analysis.The ileal digesta samples were freeze-dried at -30°C shelf temperature, then ground through 0.25 mm mesh screen.Dry matter was determined by drying the samples at 110°C for 24 h.Crude protein was determined in a Kjell-Foss 1620 auto analyzer (Foss Electric A/S) by the Kjeldahl method.The gross energy content was measured with an adiabatic bomb calorimeter.Calcium, total phosphorus and ash were determined according to the methods of the Association of Official Analytical Chemists (AOAC, 1990).For amino acids analyses, with the exception of the sulfur-containing AA and tryptophan, the samples were hydrolyzed with 6 N HCl at 110°C for 24 h, and then analyzed by using an amino acid analyzer (Hitachi L-8800, Japan).Methionine and cystine were determined as methionine sulfone and cystine acid after oxidation with performic acid.The oxidation process was carried out according to the AOAC (1990) method.The oxidized samples were then hydrolyzed and analyzed in the same manner as in the acid hydrolysis.Tryptophan analysis was carried out according to the GB/ T18246-2000 (2000) method using HPLC (Shimadzu LC-10A, Japan).The indicator, celite, was determined using the method of McCarthy et al. (1974).In brief, approximately 10 g ground sample was boiled in 100 ml of 4 N HCl for 30 min, then filtered through ashless filter paper, washed with boiling water until free of acid, and ashed at 650°C for a minimum of 6 h.
Calculation and statistical analysis : The apparent ileal or total tract digestibility of proximate components, energy, and amino acids in the experimental diets were calculated according to the following equation: D=100-(C1×P2)/ (C2×P1)×100, where D is the apparent digestibility of the nutrient in the experimental diet (%); C1 is the analyzed indicator concentration in the diet (%); P2 is nutrient concentration in digesta or feces (%); C2 is the indicator concentration in digesta or feces (%); and P1 is the nutrient concentration in the experimental diet (%).
The bacterial analysis data were transformed to logarithmic values.All data were analyzed by independent sample T-test using SPSS 9.0 for Windows (SPSS Inc., USA).

Experiment 1
Diarrhea : Diarrhea incidence and the diarrhea index of pigs treated with the complex Lactobacilli preparation were lower than those of control group, 14.2 vs. 83.3%and 15 vs. 81, respectively (Table 4).The first incidence of diarrhea in the control group was noted on the second day following the E. coli challenge, but the incidence of diarrhea in the Lactobacilli treated group did not occur until the fourth day (Table 4).This maybe showed that native Lactobacilli complex preparation can effectively prevent the weaning piglet's diarrhea administered before challenged by the E. coli.This is in agreement with the result of Winkelstein (1956) who used Lactobacillus acidophilus to control outbreaks of diarrhea in children.Hoefling (1989) reported that enterotoxic E. coli was the primary cause of neonatal piglet's diarrhea in 26% of cases and the enteropathogenic E. coli strains were major infection agent of post-weaning diarrhea (Fahy et al., 1987).The result indicated that the Lactobacilli originated from healthy weaning piglet's digestive tract could attenuate the harm E. coli cause in weaning piglet.Bechman et al. (1977) fed Lactobacillus acidophilus to young dairy calves to reduce the incidence of diarrhea.
Microflora : The results were shown in Tables 5 and 6.The Lctobacilli preparation significantly reduced E. coli and aerobic bacteria counts in stomach, jejunum, ileum, cecum, and colon mucosa (p<0.01) and significantly increased Lctobacilli and anaerobic bacteria counts in jejunum, ileum, cecum and colon mucosa (p<0.01).Lactobacilli counts of duodenum mucosa from the Lctobacilli treated group were significantly higher than those of the control group (p<0.05).However, Lctobacilli and anaerobic bacteria counts in stomach mucosa were not influenced by the Lctobacilli preparation, nor were aerobe and anaerobe counts in duodenum mucosa (p>0.05).Similar bacteriological  a Six replicates in each treatment.Each value represents the mean of the analysis from six digesta or fecal samples conducted in duplicate.The fecal samples were collected for proximate analysis and ileal digesta were collected for amino acids analysis from d 18 to 21. b Lactobacilli concentration was 2.4×10 5 CFU/g diet.c Carbadox concentration was 60 mg/kg diet.d SEM=standard error of the mean.enumerations were observed in digesta from all sections of the GI tract.Muralidhara et al. (1977) found that the numbers of Lactobacilli was higher and that of E. coli was lower in piglet's intestinal tissue after dosing with Lactobacillus lactis.Blomberg et al. (1993) found that Lactobacillus fermentum 104R could release a proteinaceous component to inhibit the adhesion of E. coli K88 to the ileal mucus.Silva et al. (1987) reported that Lactobacillus was able to produce an unknown antimicrobial substance against E. coli.Thus, the adhesion of E. coli and other aerobic bacteria to gastrointestinal mucosa may have been inhibited by the complex Lactobacilli preparation.Moreover, some metabolites may have been produced by the Lactobacilli that stimulate the colonization and reproduction of Lactobacilli and other anaerobic bacteria in the mucosa.The Lactobacilli themselves can also colonize in gut mucosa to form a biological barrier to pathogenic microbes.Therefore, the Lactobacilli preparation enhanced pig resistance to E. coli infection by regulating the balance of microflora.

Experiment 2
Performance : During the first 2 wks, the Lactobacilli preparation significantly improved ADFI (p<0.05) but had no effect on ADG and feed/gain (Table 7).ADG and ADFI were significantly improved by Lactobacilli from d 8 to 14 (p<0.05).However, the performance during the whole experiment and from d 15 to 21 was not affected by dietary treatment (p>0.05).Premi and Bottazzi (1974) found that treatment with Lactobacillus acidophilus improved the performance of suckling piglets with chronic diarrhea.Treatment with Lactobacillus acidophilus resulted in improved ADG and feed:gain in starter pigs but had no influence on growing-finishing pigs (Kornegay, 1986;Pollman et al., 1986).Pollman et al. (1980) found that starter pigs responded better to probiotics supplementation than growing-finishing pigs.The results in the experiment showed that the Lactobacilli influenced the piglet performance in the first 14 days after weaning and had no  effect from d 15 to 21 post weaning.The microflora population in older pigs is more stable and is not as susceptible to environmental stress as the population found in younger pigs.The Lactobacilli may improve in the ADG and ADFI by enhancing the piglet's health status and GI tract microbial balance the first 2 wks after weaning.This may have been due to the fact that weaning pigs are susceptible to microflora disturbances in the digestive tract and therefore benefit from enrichment by Lactobacilli.
Nutrient digestibility : Crude protein and phosphorus apparent digestibility increased (p<0.05) in the Lactobacilli treated group (Table 8).However, apparent ileal amino acid digestibility and total tract digestibility of other nutrients were not influenced by dietary treatment.Lactobacilli produce lactic acid in the gut, which makes the phytic acid existed in the ungerminated seed release the phosphorus (Møllgaard, 1946).Maxwell et al. (1983) also reported that crude protein digestion in finishing pigs was improved by probiotics containing Lactobacilli.A great number of microorganisms in the GI tract were stimulated by the Lactobacilli preparation, as shown by our results.Crude protein present in the digesta may have been fermented due to the growth and proliferation of the microorganisms, which may have resulted in an increase in apparent crude protein digestibility in the total tract.Alternatively, Lactobacilli can produce organic acids and proteolytic enzymes.The former mechanism could lead to activation of pepsinogen in the stomach and the latter could result in hydrolysis of protein directly.Therefore, the Lactobacilli preparation stimulated protein digestion in gut and thus the apparent total tract crude protein digestibility was higher compared to that of the carbadox group.
Volatile fatty acids : There were no difference in VFA between Lactobacilli treatment and carbodox treatment except the propionate in distal jejunum (Table 9).The total VFA concentration in cecum and colon were higher than those in the foregut.High concentrations of VFA are indicative that fermentations by obligate anaerobic bacteria are important, which can inhibit growth of species of the family Enterobacteriaceae (Paul et al., 2000) and provide energy for pig.The results indicated that the microbial fermentation in the most part of GI tract at the end of the experiment was similar.
Microflora : The results were shown in the Tables 10  and 11.E. coli and aerobic bacteria populations were lower (p<0.01) in the digesta of Lactobacilli treated pigs in all GI tract sections except jejunum compared to carbadox treated pigs.Lactobacilli and anaerobic bacteria counts were greater (p<0.01) in digesta from most sections of the GI tract of pigs receiving the Lactobacilli preparation.E. coli and aerobic bacteria counts in mucosa were depressed (p<0.01) by the Lactobacilli preparation in most sections of the digestive tract.Higher populations of Lactobacilli and anaerobic bacteria were observed (p<0.01) in pigs from the Lactobacilli treated group.This result is not agreement with the result of volatile fatty acids concentrations.The possible reason is that other bacteria producing more VFA were not determined in this study.Microbial population in jejunum mucosa was not affected greatly by dietary treatment.Attachment to the epithelial cells is a prerequisite for bacteria to colonize the jejunum mucosa.Because the epithelium is continuously regenerating and sloughing off cells and overlying mucus, bacteria can colonize this region only if their generation time is faster than the sloughing rate.The passage rate of a digesta particle through the jejunum is very fast.So it would be difficult for bacteria to multiply sufficiently fast to avoid being washed out (Kidder and Manners, 1978).

CONCLUSIONS AND IMPLICATION
Animal food safety becomes one of highlight in the world.Antibiotics resistant bacteria add more difficulties in the medicine and veterinary practice.The post-weaning piglets diarrhea is a world problem.Therefore, the probiotics is one of promising alternatives of antibiotics in the future, although some basic mechanisms remain unknown.This study demonstrated that the complex Lactobacilli originated from healthy weaning piglet digestive tract can prevent the diarrhea challenged by E. coli and increased the performance in the first 2 wks after weaning.In this research it is also indicated that the balance of gut microflora is beneficial to the defense against pathogenic agent infection.

Table 1 .
Composition of diet and nutrient level in experiment l (based on the dry matter)

Table 3 .
Composition of basal diet and nutrient level in experiment 2 (based on the dry matter)

Table 4 .
Effect of Lactobacilli on diarrhea of the piglets challenged by E. coli b Diarrhea index was the summation of the diarrheal piglet numbers multiplied by corresponding diarrhea score.C Two piglets died after E. coli challenging in the control group before d 14.

Table 5 .
Effect of administration of a complex Lactobacilli Lactobacilli concentration was 2.2×10 5 CFU/mL.
a Means were based on three replicates of 6 pigs per group and represented intestinal tissue on d 7 following the E.coli challenge.b c Pigs were given tap water as control.d SEM=standard error of the mean.

Table 6 .
Effect of administration of a complex Lactobacilli preparation on microflora in GI tract digesta from weaning pigs on d 7 following an E.coli challenge (log 10 Lactobacilli concentration was 2.2×10 5 CFU/mL.
a Means were based on 6 pigs per group and represented intestinal digesta on d 7 following the E.coli challenge.b c Piglets were given tap water as control.d SEM=standard error of the mean.

Table 7 .
Effect of administration of a complex Lactobacilli preparation on performance of weaning pigs a Groups Items a Six replicates of three piglets per pen in a 21 d study.b Lactobacilli concentration was 2.4×10 5 CFU/g diet.c Carbadox concentration was 60 mg/kg diet.d SEM=standard error of the mean.

Table 9 .
Effects of Lactobacilli on volatile fatty acids concentration in the digesta of the different part of digestive tract d SEM=standard error of the mean.

Table 8 .
Effect of administration of a complex Lactobacilli preparation on apparent nutrient digestibility (%) of weaning pigs a Items Lactobacilli b Carbadox c SEM d P-value

Table 11 .
Effect of administration of a complex Lactobacilli preparation on mucosa microflora in weaning pigs (log 10 d SEM=standard error of the mean.

Table 10 .
Effect of administration of a complex Lactobacilli preparation on digesta microflora in weaning pigs (log 10 a Means are based on 6 pigs per group and represented intestinal contents on d 21. b Lactobacilli concentration was 2.4×10 5 CFU/g diet.c Carbadox concentration was 60 mg/kg diet.d SEM=standard error of the mean.