Study on the efficacy of four probiotic bacteria strains on piglets

JOURNAL OF SCIENCE OF HNUE Natural Sci., 2008, Vol. 53, N ◦ . 5, pp. 110-121 STUDY ON THE EFFICACY OF FOUR PROBIOTIC BACTERIA STRAINS ON PIGLETS Vu Ngoc Hanh, Vuong Trong Hao, Mai Thi Hang, Do Thi Huyen Trang and Do Duc Que Hanoi National University of Education Abstract. Four strains of lactic bacteria (3 strains L. casei subsp. paracasei signed as YHN35, FH58 and FV28 and one strain of Enterococcus faeciums YHN77) isolated from piglet digestive systems were selected for production of probiotic preparation used for piglets. These strains showed high antago- nism to common intestinal pathogens of piglets in Vietnam such as E. coli O141, E. coli O149, Salmonella entertica Typhimurium (S. Typhimurium) and Salmonella entertica Cholera-suis (S. Cholera-suis) in vitro. The selected lactic bacteria were subjected for investigating their effects in curing piglets infected with S. Typhimurium and E. coli 0149 in vivo. Wean- ing piglets of 20 days old were acutely infected with S. Typhimurium and E. coli 0149, and then they were fed daily with the 2.8x10 6 CFU/ml of either L. paracasei YHN35, FV28, FH58 or E. faecium YHN77 per individual. The results of 10 days later showed that these strains helped piglets to recover from diarrhea caused by S. Typhimurium and E. coli 0149. The rates of piglets recovered from 20% to 80%, respectively, whereas in the control lot it was 0%. Under feeding with probiotic bacteria the numbers of Salmonella Typhimurium and E. coli O149 in excrement were dramatically decreased compared to the control. The most effective microbes were Lactobacillus paracasei FV28, L. paracasei YHN35 and Lactobacillus sp. FH58, whereas E. faecium YHN77 showed weak effect. Trials of probiotic preparation composed of Lactobacillus paracasei FV28 and L. paracasei YHN35 and Lactobacillus sp. FH58 on suckling and weaning piglets in vivo were investigated for one month. These lactic bacteria showed clearly the effect on protecting suckling and weaning piglets from natural diarrhea as well as increased body weight of suckling and weaning piglets into 6 - 7.8% respectively. 1. Introduction Modern rearing methods which include unnatural rearing conditions and diets induce stress and can cause changes in the composition of the microflora which compromise the animals' resistance to infection. The aim of the probiotic approach is to repair the deficiencies in the microflora and restore the animals' resistance to 110 Study on the efficacy of four probiotic bacteria strains on piglets disease especially in suckling and weaned piglets. weaning is regarded as the most difficult period in a pig's life. Young piglets are subjected to several stressors such as nutritional, environmental, social and microbial unbalance. As a result, low feed intake, impaired intestinal morphology and function, a high incidence of diarrhea and growth depression are commonly seen immediately after weaning. Administration of probiotics is one of the alternative ways of enhancing the growth of suckling and weaned piglets and preventing the diarrheic syndrome. However, the use of in-feed antibiotics was banned by the EU from 2006 onwards. Therefore, use of probiotics is one of the alternative ways of potentiating the growth of suckling and weaned piglets and preventing the diarrheic syndrome. From a practical point of view, the administration of probiotic bacteria and their metabolites has found to be an effective way to promote body weight and feed conversion in farm animals [1]. The joint production of high amounts of biomass and antimicrobial metabolites in a cheaper culture medium using an adequate cultivation method (like the fed- batch mode) could be an appropriate alternative to solve this problem [5]. This study was conducted to study the production and evaluation of potentially probiotic additives containing three live lactic bacteria culture broths, which could be used as a replacement for antibiotics in weaning and post-weaning piglets in Vietnam. 2. Content 2.1. Material and methods Bacterial strains Lactobacillus casei subsp. paracasei (L. paracasei) YHN35, FH58 and FV28 Enterococcus faecium YHN77 obtained from Microbial Genetic Culture Collection (Hanoi National University of Education). Stock cultures were maintained at -40 0 C in Nutrient broth supplemented with 15 ml of glycerol per litre of medium. Working cultures maintained at 4 0 C on MRS agar. 2.1.1. Experimental design for studying the effect of the lactic bacteria on piglets acutely infected with bacterial pathogens A total of 30 piglets of 28 days old without S. Typhimurium and E. coli vaccination were divided into 6 groups of 5 individuals/group as shown in Table 1. The experiment was done in triplicate. Treatment procedure: Before the experiments were carried out, blood from all the piglets was taken for confirmation of no vaccination. Table 1. Experimental design for treatment of piglets No of groups Treatment of piglets Group 1-Control 1 After infection piglets were fed the basal diet with addition of 10 ml sterilized MRS medium. Group 2 After infection piglets were fed the basal diet with addition of 10 ml (10 6 CFU/ml) L. paracasei FV28 grown on MRS broth. 111 Vu Ngoc Hanh, Vuong Trong Hao, Mai Thi Hang, Do Thi Huyen Trang and Do Duc Que Group 3 After infection piglets were fed the basal diet with addition of (10 6 CFU/ml) L. paracasei YHN35 grown on MRS broth. Group 4 After infection piglets were fed the basal diet with addition of (10 6 CFU/ml) L. paracasei FH58 grown on MRS broth. Group 5 After infection piglets were fed the basal diet with addition of (10 6 CFU/ml) E. faecium YHN77 grown on MRS broth. Group 6-Control 0 Piglets without infection, no treatment. Experimental analysis: The criteria to follow the experiments such as numbers of infected piglets, performance of piglets, and numbers of deaths, surviving and recovered after infected were observed and recorded during 10 days of experiments. Total S. Typhimurium and E. coli counts in fecal samples were performed according to procedure of Nelson [6]. E. coli and S. Typhimurium counts were determined in the fecal samples, which were directly taken from the rectum of each animal with a sterile cotton swab. Three replicates of fecal samples of each piglet were taken simultaneously. The cotton swabs were weighed before and after taking the fecal samples to determine the net weights of the samples. The Cotton swabs were diluted in sterilized in saline water up to 10 10 , then 100 ml were spread on Mackonkey agar plate (for E.coli count) and LDH medium for S. Typhimurium count. The plates were inverted and incubated at 37±10C for 2 days. Incubated plates were observed for the optimum number of CFU, between 30 and 300 colonies per plate. The results were expressed as the number of colonies counted per gram (wet weight) of faeces. 2.1.2. Experimental design for studying the effect of probiotic on suck- ling and weaning piglets The places of experiments: Breeding farm of the Thai Binh's Joinstock Com- pany in Dong My commune, Dong Hung district, Thai Binh province. The experi- ment was carried out for 5 weeks, from 29/09/2007 to 03/11/2007. Experimental procedure: The experiments were carried out on suckling piglets of 7 days old and on weaned piglets of 26 days old. Both types of piglets were not vaccinated against E. coli and S. Typhimurium and fed with basal diet containing vegetable. Piglets of weaning period (30 piglets) and weaned piglets (30 piglets) were divided into 6 pens as described in Table 2. The experiment was done in triplicate. Table 2. Experiment designed on effect of probiotic preparation on piglets Groups Mode of feeding Type of piglets Group 1 (DC1.0) Fed with basal diet containing vegetable. Suckling piglets. Group 2 (DC1.1) Fed with basal diet containing vegetable with addition of probiotic preparation autoclaved. Suckling piglets. Group (TN1) Fed with basal diet containing vegetable with addition of live probiotic preparation. Suckling piglets. Group (DC2.0) Fed with basal diet containing vegetable. Weaning piglets. 112 Study on the efficacy of four probiotic bacteria strains on piglets Group (DC2.1) Fed with basal diet containing vegetable with addition of probiotic preparation autoclaved. Weaning piglets. Group (TN2) Fed with basal diet containing vegetable with addition of live probiotic preparation. Weaning piglets. The probiotic preparation was dissolved in water for suckling piglets to drink, while it was mixed with diets to feed the weaned piglets. The number of total lactic bacteria was 2.10 6 CFU/g diet. Analysis: Numbers of piglets who got sick of diarrhea and recovered were recording by observation. Body weight gain (BWG) was recorded at the intervals, and numbers of S. Typhimurium and E. coli in piglets faeces were determined as mentioned above. All media used (MRS agar, Maconkey, LDH medium, yeast extract) were purchased from Sigma (USA). 2.2. Results and discussions 2.2.1. The effect of four Lactic bacterias on piglets acutely infected with piglets pathogens The observation method to follow the performance of healthy and infected piglets was used for identifying the sick and healthy piglets. Because the piglets were directly infected with pathogens, therefore only after 24h of infection all typical symptoms could be observed with the naked eye. All piglets in the group 1- to group 5 were sick with typical symptoms of diarrhea such as moving around slowly, opaque eyes, lost of appetite, wet faeces, fever with high temperature, feathers erected, red anus, while 5 piglets in group 6 were healthy as could be seen moving around quickly, mild feathers, normal faeces, good appetite, no fever. All these symptoms indicate that the process of experimentally infecting piglets with pathogens was successful. Numbers of dead, survived and recovered piglets were followed and recorded, as indicated in Table 3 and Table 4. Table 3. Numbers piglets died by times Day 1 2 3 4 5 6 7 8 9 10 Total dead (individual) Dead number 4 2 2 1 0 0 0 0 0 0 9 After 10 days, numbers of dead piglets were 9 individuals and 21 piglets sur- vived. The numbers of dead piglets were concentrated in 4 days at beginning. After this period the piglets gradually recovered from disease. All dead piglets were killed to examine the interior organs such as liver and spleen, and take the rectum faeces for determination of pathogen presence. The dead piglets were burned in sterilizer hood. The interior organs of infected piglets were observed to be clearly changed. Especially those piglets infected with S. Typhimurium showed typical clinical symp- 113 Vu Ngoc Hanh, Vuong Trong Hao, Mai Thi Hang, Do Thi Huyen Trang and Do Duc Que toms such as livers and spleens were swollen, bigger with darken colours compared to normal ones (Data not shown). Section of livers and spleens were ground and spread on Maconkey and DHLL media, there were typical colonies of E. coli and S. Typhimurium, respectively, were observed (Data not shown). Piglets in the 1 - 5 groups infected with S. Typhimurium and E. coli O149 were suffered from diarrhea with typical clinical symptoms such as lost appetite or completely not eating massive diarrhea, consequently losts of water and ion balance for long periods together leading to dead. After 10 days we noticed piglets in the groups where the diet supplemented with lactic bacteria the animals could overcome the disease as shown in the Table 4. Table 4. Effect of four lactic bacteria in preventing and recovering piglets from diarrhea caused by S. Typhimurium and E. coli 0149 acute infection Piglets performance Group 1: positive control Group 2- FV28 Group 3- YHN35 Group 4- FH58 Group 5- YHN77 Group 6- Negative Control 5 5 5 5 5 5 Recovered Number pigs 0 1 4 4 0 5 Rate (%) 0 20 80 80 0 100 Not recovered Number pigs 2 2 1 0 2 0 Rate (%) 40 40 20 0 40 0 Dead Number pigs 3 2 0 1 3 0 Rate (%) 60 40 0 20 60 0 After 10 days of experiments it could be clear seen by the differences in piglet performances between the groups. In group 6 (negative control), all piglets were healthy, whereas in group 1 (positive control), all piglets were infected, the death rate was very high (60%), and the 40% of remained pigs could not be recovered. In all groups where the infected piglets fed with diets supplemented lactic bacteria culture broth, the dead rate was reduced and the recovery were increased. Surprisingly, strain YHN77 showed low positive effect on the preventing pigs from infected pathogens, although in vitro assessment it showed to be very effective to the pathogens. The death rate was 60%, as high as it was in the positive control group. The remaining piglets were still sick, and could not be recovered after 10 days. Under the action of L. paracacei FV28 (group 2) 60% of piglets were survived, among them 20% were recovered, 40% could not be recovered, while under the action of strain FH58 (Group 4) the death rate is 20%, another 80% were survived and 114 Study on the efficacy of four probiotic bacteria strains on piglets finally recovered. Moreover, this strain was most effective in improving the weight gain of the piglets (Table 4). The strain L. paracacei YHN35 (group 3) was found to be the most effective probiotic bacteria. Under the action of this strain, no piglet died, 80% piglets were recovered and 20% had been being recovered. These findings showed that the use of these lactic bacteria for preventing diar- rhea caused by Salmonella Typhimurium and E. coli O149 in piglets is prospectively good since infection was acute with high toxicity (60% pigs infected without treat- ment were dead and 40% could not be recovered). 2.2.2. Effect of the probiotic lactic bacteria on growth of infected piglets The recovery from infection of piglets was assessed by the body weigh gain (BWG). The result was shown in Table 5. Table 5. Effect of the probiotic lactic bacteria on growth of infected piglets Groups No of piglets Body weight gain (kg)/day M1 M2 M3 ∆ ∆/10 1 11 2 8 Group 1: Negative control 3 7 4 7 7 6 -1 -0.1 5 14 13 15 1 0.1 Average 0 6 7 7 8 7 8 0 0 Group 5  YHN77 8 7 9 7 10 6 9 10 4 0.4 Average 0.2 11 10 12 7 Group 2 - FV28 13 9 11 12 3 0.3 14 6 7 7 1 0.1 15 10 8 9 -1 -0.1 Average BWG/day (kg) 0.1 16 7 17 13 14 15 2 0.2 Group 4 - FH58 18 14 17 18 4 0.4 19 13 15 16 3 0.3 20 9 11 12 3 0.3 115 Vu Ngoc Hanh, Vuong Trong Hao, Mai Thi Hang, Do Thi Huyen Trang and Do Duc Que Average BWG/day (kg) 0.3 21 8 10 11 3 0.3 22 9 11 12 3 0.3 Group 3  YHN35 23 11 13 14 3 0.3 24 9 10 12 3 0.3 25 8 8 10 2 0.2 Average BWG/day (kg) 0.28 26 9 13 14 5 0.5 27 12 13 16 4 0.4 Group 6 - Negative Control 28 15 18 19 4 0.4 29 16 18 19 3 0.3 30 10 14 15 5 0.5 Average BWG/day (kg) 0.42 * Note: M1: BWG at starting point of experiment; M2: BWG the day 5; M3: BWG at finishing point of experiment; ∆: Average BWG/day (kg) for whole period; ∆/10: Average BWG/day (kg) at certain time and 0: all piglets died. From the above Table, it can bee seen in the group 6 (piglets were not infected and no treatment applied) the rate of BWG is highest (0.42 kg/day), whereas in group 1 (piglets were infected and no treatment applied) it was 0. In 4 remained groups, the BWG is clearly increased in comparing to the group 1. The BWG of piglets in group 2, group 3, group 4 and group 5 was 0.2, 0.1, 0.3 and 0.28, respectively. Thus, L. paracasei FH 58 and YHN35 showed the high efficacy on growth of infected piglets. The BWG in the groups 2, 3, 4, and 5 were lower than in group 6, since the piglets of this group were not infected, therefore normal growth rate occurred. However, BWG of groups 2, 3, 4, and 5 were much higher compared to group 1, where the piglets were acutely infected with pathogens and had no treatment. This finding indicates that the addition of lactic bacteria could not only cure the piglets from diarrhea caused by Salmonella Typhymurim and E. coli O149 but also enhance their growth. 2.2.3. The effect of the lactic bacteria on growth of E. coli O149 and S. Typhimurium Total count of S. Typhimurium and E. coli were carried out by sampling faeces at the interval of the 5 th and 10 th days and spreading on the agar plate specifying for each of them as described in material and methods. The results are shown in Table 6. 116 Study on the efficacy of four probiotic bacteria strains on piglets Table 6. Total count of S. Typhimurium and E. coli (O149 in piglets faeces CFU/g faeces) Groups Numbers of S. Typhimurium and E.coli in piglets faeces (CFU/1g faeces) Day 0 Day 5 Day 10 S.Typhimurium E.coli S.Typhimurium E. coli S.Typhimurium E. coli Group 1: 0.005.10 6 3.5.10 6 positive 0.025.10 6 3.2.10 6 control 0.03.10 6 2.10 6 0.092.10 6 5.2.10 6 12.5.10 6 4,800.10 6 30.10 6 180.10 6 0.008.10 6 4.8.10 6 30.0.10 6 1,000.10 6 17.45.10 6 330.10 6 0 30.10 6 Group 2- 0.023.10 6 30.10 6 FV28 0.077.10 6 6.2.10 6 0.45.10 6 2,050.10 6 0.6.10 6 950.10 6 0.0025.10 6 1.8.10 6 15.2.10 6 1,850.10 6 0.9.10 6 300.10 6 0.105.10 6 5.4.10 6 3.85.10 6 700.10 6 0.35.10 6 300.10 6 0.0405.10 6 2.7.10 6 1.10 6 14,300.10 6 0 40.10 6 Group 3- 0.105.10 6 3.2.10 6 0.8.10 6 900.10 6 0.4.10 6 40.10 6 YHN35 0.160.10 6 6.8.10 6 5.55.10 6 1,600.10 6 6.5.10 6 230.10 6 0.080.10 6 7.6.10 6 0.8.10 6 41.10 6 1.9.10 6 200.10 6 0.054.10 6 3.5.10 6 1.2.10 6 3,350.10 6 0.9.10 6 240.10 6 0.0005.10 6 7.4.10 6 Group 4- 0.0015.10 6 4.8.10 6 2.25.10 6 600.10 6 1.1.10 6 25.10 6 FH58 0.0035.10 6 8.1.10 6 0.35.10 6 400.10 6 0.25.10 6 40.10 6 0 4.0.10 6 1.5.10 6 1,050.10 6 0.8.10 6 115.10 6 0.065.10 6 7.3.10 6 0.7.10 6 1,000.10 6 0.55.10 6 50.10 6 0 8.3.10 6 Group 5- 0.0065.10 6 2.2.10 6 1.2.10 6 800.10 6 0.8.10 6 130.10 6 YHN77 0 4.10 6 0.0165.10 6 0.9.10 6 0.067.10 6 6.6.10 6 5.3.10 6 500.10 6 1.4.10 6 300.10 6 The results showed that, the numbers of E.coli O149 and S. Typhimurium decreased markedly in the groups with addition of lactic bacteria compared to group 1 without addition of lactic bacteria after piglets were infected with E. coli O149 and S. Typhimurium. L. paracasei FV28, L. paracasei YHN35 and E. faecium YHN77 showed high inhibition on S. Typhimurium than E. coli O149, while strain FH58 were active on both E. coli O149 and S. Typhimurium. The numbers of the S. Typhimurium and E. coli O149 in the intestines of the piglets were also determined after 10 days and the results were shown in Table 7. 117 Vu Ngoc Hanh, Vuong Trong Hao, Mai Thi Hang, Do Thi Huyen Trang and Do Duc Que Table 7. Total count of S. Typhimurium and E. coli O149 in piglet intestine (CFU/1g faeces) Numbers of S. Typhimurium and E. coli O149 Groups S. Typhimurium E. coli O149 x106CFU/g (%) x106CFU/g (%) Group 1: positive control >0.6 100 1,400 100 Group 2 - FV28 0.008 0. 013 600 42.9 Group 3 - YHN35 0.002 0.003 600 42.9 Group 4 - FH58 0.006 0.01 262 18.7 Group 5 - YHN77 0 0 452 32.3 The results obtained showed that numbers of S. Typhimurium and E. coli in piglet intestines, especially S. Typhimurium were also reduced significantly under the action of added lactic bacteria. 2.2.4. Effect of lactic probiotic preparation on suckling and weaning piglets From all obtained results above, three lactic bacteria strains (L. paracasei YHN35, FH58 and FV28) were selected for production of probiotic preparation. This preparation was applied on suckling and weaning piglets as described in Materials and methods. The results were shown in Table 8 and Table 9. Table 8. The effect of probiotic preparation for preventing diarrhea of piglets Piglets age Pens Frequency of diarrhea (times) Numbers of piglet suffered diarrhea Frequency of diarrhea occur- rence and health condition of piglets Group 1.1 1 2 Once and recovered after 3 days. Suckling piglets Group 1.2 2 4 Twice: - First time: 3 piglets got sick, recovered after 2 days - Second time: 1 piglet got sick, recovered after 3 days. Group 1.3 1 1 Once and recovered after 3 days. Group 2.1 2 2 Twice: - First time: 1 piglet got sick, died after 2 days. - Second time: 1 piglets got sick and died after 2 days. Weaning piglets Group 2.2 0 0 No piglet got sick. Group 2.3 0 0 No piglet got sick. 118 Study on the efficacy of four probiotic bacteria strains on piglets It could be seen, the numbers of piglets of both suckling and weaning ages suffered from diarrhea in the groups with no addition of probiotic preparation were higher than in the groups fed with diet supplemented with probiotic preparation. In suckling piglets the frequency of diarrhea occurred lower than in weaning piglets. Moreover these piglets could be recovered naturally. It might be due to the piglets immunity originated from their mother which is still high enough for helping recovery of their health. In weaning piglets the frequency of diarrhea occurred higher, and sick piglets could not be recovered and finally died (group 2.1). In the groups fed with diet supplemented with autoclaved probiotic and live probiotic no piglet got sick. All these findings indicated that the probiotic preparation was effective in preventing diarrhea in weaning piglets. 2.2.5. The effect of lactic probiotic preparation on growth of suckling and weaning piglets The effect of probiotic on growth of piglets was analyzed as mentioned in Materials and methods and the obtained results were shown in the Table 9. Table 9. The effect of probiotic preparation on growth of suckling and weaning piglets BDW (kg/pig) at various growth time Piglets age Groups Starting point 1 week 2 weeks 3 weeks 5 weeks 7 weeks Average BDW (kg/pig±SDE) Suckling Group 1.1 2.21 3.58 4.49 5.19 6.12 7.06 4.85±0.07 piglets Group 1.2 2.32 3.54 4.61 5.28 6.51 7.38 5.06± 0.034 Group 1.3 2.37 4.38 4.97 5.53 6.83 7.5 5.13±0.056 weaning Group 2.1 4.61 5.71 6.84 9.413 11.93 14.43 9.82±0.024 piglets Group 2.2 4.22 5.07 6.41 8.52 11.3 13.85 9.63±0.031 Group 2.3 4.85 5.77 7.33 9.67 13 15.63 10.78±0.045 The initial results indicated that this probiotic preparation could enhance the growth of piglets at suckling (4.3% - 5.7%) and weaning age (9.8%). 2.2.6. The effect of lactic probiotic preparation on S. Typhimurium andE. coli in piglet faeces Analysis of S. Typhimurium and E. coli growth in piglet faeces was presented in Table 10. It could be clearly seen the effect of this probiotic preparation on growth of the S. Typhimurium and E.coli in piglet faeces. Under actions of probiotic the 119 Vu Ngoc Hanh, Vuong Trong Hao, Mai Thi Hang, Do Thi Huyen Trang and Do Duc Que number of S. Typhimurium in suckling piglets and weaning piglets was reduced from 0.6.10 6 CFU/g to 0.32.10 6 CFU/g (nearly 50%) and 0.0426.10 6 CFU/g to 0.018.10 6 CFU/g (nearly 60%), respectively. The number of ÌE. coli in both suckling piglets and weaning piglet was reduced from 21.3.10 6 CFU/g to 13.2.10 6 CFU/g (63%) and from 35.6.10 6 CFU/g to 27.2.10 6 CFU/g (7.8%), respectively. Table 10. The effect of lactic probiotic on S. typhimurium and E. coli growth in piglet faeces Piglets ages Experimental Number of bacteria x10 6 CFU/g faeces groups Salmonella E. coli Typhimurium Group 1.1 0.60 21.3 Suckling piglet Group 1.2. 0.53 16.5 Group 1.3 0.32 13.2 Group 2.1 0.042 35.6 Weaning piglets Group 2.2 0.028 28.2 Group 2.3 0.018 27.2 Efficacy of probiotic bacteria in promoting growth of post - weaning piglets and other animals have been studied before. Researchers observed that the admin- istration of probiotic bacteria in the first days of life produced a positive effect on growth in piglets and reduction of coliform in faeces [1,6,7]. Our results showed that piglets fed with diets supplemented with probiotic LAB resulted in a better performance (body weight gain) than piglets fed with diets without additives (controls). The results obtained in the present study demonstrated that feeding piglets with 10 6 CFU of probiotic bacteria/g of feed per day increased the BWG of the animals. This value is as high as the recommended dose of viable probiotic (10 6 CFU of probiotic/g or ml) necessary to obtain beneficial effects [4]. Studies have shown that the administration of probiotics caused a reduction in coliform counts in the faeces of piglets [7] perhaps by a competitive exclusion mechanism. But more often such effects are not significant, except when the animals are challenged with selected pathogenic strains or in gnotobiotic animals [2]. 3. Conclusion Among four strains lactic bacteria (Lactobacillus casei subsp. paracaseiYHN35, FH58 and FV28, and Enterococcus faecium YHN77) tested on protecting piglets acutely infected with S. Typhimurium and E. coli O149, three of them (Lactobacil- lus casei subsp. casei YHN35, FH58 and FV28) were effective in reducing the death rate and enhancing the piglets to overcome the disease. Among them, the best one is L. casei subsp. paracasei YHN35. Under the action of this bacteria no piglet died. These bacterias were also able to enhance the growth of infected piglets. The number of S. Typhimurium and E. coli O149 in faeces of piglets infected was dramatically 120 Study on the efficacy of four probiotic bacteria strains on piglets reduced under the action of these bacteria. Probiotic preparation made of three lactic bacteria combination was trialed on piglets of suckling and weaning age in vivo. The results obtained supported the results obtained in the experiments above, where piglets were acutely infected with pathogens. The probiotic preparation could prevent natural diarrhea in both piglets' ages, especially in weaned piglets. It could also enhance the BDW of suckling and weaned piglets from 4.3% - 5.7% and 9.8%, respectively, and reduced the natural occurred S. Typimurium and E. coli in piglet faeces either. It is concluded that these three lactic bacteria could be a potential bacteria for producing probiotic preparation used for preventing diarrhea in suckling and weaning piglets in Vietnam. It needs further study to accomplish practical usage. REFERENCES [1] F. Abe, N. Ishibashi, S. Shimamura, 1995. Effect of administration of bifi- dobacteria and lactic acid bacteria to newborn calves and piglets. J. Dairy Sci. Vol. 78, pp. 2838-2846. [2] B. Bogovi v c Matija v si' c, S. Stojkovic, J. Salobir, S. Malovrh, I. Rogelj, 2004. Evaluation of the Lactobacillus gasseri K7 and LF221 strains in weaned piglets for their possible probiotic use and their detection in the faeces. Anim. Res. Vol. 53, pp. 35-44. [3] Y.H. Chang, J.K. Kim, H.J. Kim, W.Y. Kim, Y.B. Kim, Y.H. Park, 2001. Selection of a potential probiotic Lactobacillus strain and subsequent in vivo studies. Antonie van Leeuwenhoek, Vol. 80, pp. 193-199. [4] W.P. Charteris, P.M. Kelly, L. Morelli, J.K. Collins, 1998. Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J. Appl. Microbiol Vol. 84, pp. 759-768. [5] N.P. Guerra, M.L. Rua, L. Pastrana, 2001. Nutritional factors affecting the production of two bacteriocins from lactic acid bacteria on whey. Int. J. Food Microbiol, Vol. 70, pp. 267-281. [6] Nelson P'erez Guerra, Paula Fajardo Bern'ardez, Jes'us M'endez Pilar Cachaldora, Lorenzo Pastrana Castro. 2007. Production of four potentially probi- otic lactic acid bacteria and their evaluation as feed additives for weaned piglets. Animal Feed Science and Technology, Vol. 134, pp. 89-107. [7] F. Tortuero, J. Rioperez, E. Fernandez, M.L. Rodriguez, , 1995. Response of piglets to oralResponse of piglets to oral administration of lactic acid bacteria. J. Food Protect, Vol. 58, pp. 1369-1374. [8] B.A. Watkins, F.H. Kratzer, 1984. Drinking water treatment with commer- cial preparation of a concentrated Lactobacillus culture for broiler chickens. Poult. Sci., Vol. 63, pp. 1671-1673. 121