Evaluation of Manganese Ion on Controlling Harmful Microorganisms In vitro and In vivo for the Early-Weaned Pig

Two experiments were conducted to determine the effects of MnSO4 on controlling harmful microorganisms in vitro and in vivo. The in vitro experiment was conducted to examine the efFects of manganese sulfate (MnSO4) on the reduction of Escherichia coli (£. coll) and Staphylococcus aureus (S. aureus) by growth stimulation of Pediococcus acidilactici (P. acidilactici^ lactic acid bacteria). Manganese ion (0.003 %) was found to stimulate the growth of P. acidilactici in the In Vitro system. When E. coli and S. aureus were grown in a mixture with P. acidilactici, their numbers were reduced. This may be the result of a reduction of pH in the medium as a result of better growth of P. acidilactici due to stimulation by the Mn ion. The in vivo experiment was conducted to determine the effects of MnSO4 in diets on controlling harniful microorganisms in fecal samples of pigs. There were no significant differences for the microbial numbers (i.e., total microorganisms, E. coli, lactic acid bacteria and S. aureus) in feces of pigs fed MnSO4 compared to feces of pigs fed the control diet through 7 days. However, on day 7 of experiment, the pH of feces in pigs fed MnSO4 (0.1%) decreased faster than pigs fed the control diet (


INTRODUCTION
At birth, the pi이et is essentially devoid of circulating antibodies, since placental transfer of maternal immunoglobulins to the fetus is minimal (Kim et al., 1966). Consequently, the piglet receives immunological protection from the sow via passive transfer of immunoglobulins contained in the colostrum. Typically, the pig's passive immunity is maximized 24-36 hours postpartum, and decreases logarithmically thereafter (Miller et al., 1962).
At 3 weeks of age, humoral immunity is low, presenting potential problems in the early-weaned pig. Blecha et al. (1983) reported that the pig's ability to mount an immune response was suppressed as weaning age was decreased from 5 to 2 weeks of age.
In pigs, postweaning diarrhoea causes great economic loss due to the reduced growth, occasional death, and cost of medical treatment and prophylaxis (Svendsen et al., 1974;Jahn and Uecker, 1987;Svensmark et al., 1989). Strains of Escherichia coli (E. coli) are the primary cause of diarrhoeic diseases at weaning and the period immediately thereafter (Kenworthy and Crabb, 1963;Sojka, 1965;Svendsen et aL, 1977;Sarmiento et al., 1988).
Manganese ion was found to accelerate the growth and bacteriocin production of lactic acid bacteria (LAB) (Biswas et al., 1991). According to Raccach (1983), manganese ion appears to be essential to the growth and metabolic activities of LAB. The effects of manganese ion on LAB include enhancement of lactic acid fermentation and protection from oxygen toxicity. Furthermore, the rapid increase in LAB number can attribute to the inhibition of harmful microorganisms such as E. coli or Staphylococcus spp. by antimicrobial agents (i.e., lactic acid, bacteriocin or enzyme etc.) (Bruno et al., 1992;Kone and Fung, 1992).
Therefore, the objectives of the experiments reported herein were to determine the effects of manganese sulfate (MnSO4) on the reduction of E. coli and Staphylococcus aureus (S. aureus) by growth stimulation of Pediococcus acidilactici (P. acidilactici) for the in vitro experiment and the efFects of MnSO4 in diets on controlling harmful microorganisms in fecal samples of pigs for the in vivo experinient.

1.
In vitro experiment AJAS 1998 Vol. 11 (No. 1) 8-12 (1) Cultures tested Pediococcus acidilactici isolated from HP starter culture (Diversitech Inc., Duncanville, TX) was trans ferred into Brain Heart Infusion (BHI, Difco) broth and incubated at 40°C for 48 hours. A 1% inoculum (0.75 ml) of the suspension was transfened into a Klett flask containing 75 ml of BHI broth with 1.5% sucrose added. Escherichia coli and S. aureus obtained from the culture collection at Kansas State University was inoculated into the same BHI medium either individually or in combination with P. acidilactici

(2) Laboratory medium with MnS04
The BHI + MnSQ media (BHIM: 0.003% MnSO4) broth was prepared as follows. A stock solution of MnSO4 was prepared by adding 0.5 g of MnSO4 to 10 ml sterilized water. After mixing, the stock solution was filter sterilized through 0.2 membrane filter and added 0.06 ml to 100 ml BHI broth. The control (0%) and 0.003% of MnSO4 in BHI broth were tested for acid production and growth of P. acidilactici.

(3) Effect of MnS04 on inhibiting E. coli by P, acidilactici
Escherichia coli (104 CFU/ml) was inoculated into BHIM broth, BHI broth with each individual supplement, or BHI broth with no supplement along with P. acidila ctici (105 6 CFU/ml). The Klett flasks with cultures were incubated without agitation at 40°C. Klett units and viable cell counts of E. coli were obtained using Violet Red Blue (VRB, Difco) agar and P. acidilactici using Lactobacilli deMan Rogosa and Sharp (MRS, Difco) agar. Escherichia coli without P. acidilactici was evaluated as a control.

(5) Statistical analysis
Data from this experiment were analyzed using the General Linear Model (GLM) procedures of SAS (1988) with LSD (Steel and Torrie, 1980) to compare treatment means.

In vivo experiment ⑴ Animals
Eighteen pigs (initially 5.1 kg body weight and 21 days of age) were used in 7 days to determine the effect of MnSQ in diets on controlling harmful microorganisms in fecal samples. The pigs were housed in individual cages (46 cm x 46 cm) equipped with woven-wire flooring. Each cage contained a one-hole self feeder and one nipple waterer to allow ad libitum consumption of feed and water. Room temperature was maintained at 280.

(2) Treatments and diets
Treatments were: 1) com-soybean meal based control diet, 2) com-soybean meal based diet with 0.01% MnSO4 and 3) com-soybean meal based diet with 0.1% MnSO4. The concentrations of MnSO4 were diluted with distilled water and sprayed diluted MnSO4 into the diets. All diets were formulated to contain 1.50% lysine, 0.42% methionine, 0.9% Ca and 0.8% P and contained 25% dried whey, 9% lactose and 7.5% porcine plasma protein (table 1). The diets were fed in pellet form and were

(3) Feces sampling
On day 0, 3 and 7 of the experiment, fecal samples were collected from six pigs per treatment by rectal massage to measure microbiological change. Fecal samples were transferred to filter stomacher bags (Model SFB 0410, Spiral Biotech Inc., Bethesda, MD) to make 10-1 dilution with 0.1% sterile peptone water and pummeled for 2 minutes in a stomacher (Model 400, Seward Medical, London, UK). Serial dilutions were prepared with 9 ml dilution blanks of 0.1% sterile peptone water. Dilutions were plated with spiral plater (Model DV2, Spiral Systems, Inc., Cincinnati, OH) on the appropriate media (plate count agar for total microorganisms, MRS fbr LAB, VRB fbr E. coll and Baird-Parker for Staphylococcus spp.).

(4) Statistical analysis
Data collected were analyzed as a randomized complete block design with pen as the experimental unit and initial weight as the blocking factor. Data were analyzed using the General Linear Model (GLM) procedures of SAS (1988). Duncan's m미tiple range test was used to separate means (Steel and Torrie, 1980).

In vitro experiment
(1) Inoculum of E coli with P. acidHactici Figure 1 shows the growth (CFU/ml) of inoculated E. coli (104 CFU/ml) in the laboratory medium containing P. acidilactici. Without MnSO4, E. coli increased to 8.34 log CFU/ml at 40 °C incubation with P. acidilactici. However, the final numbers of E. coli in the medium with the mixture of MnSO4 with P. acidilactici, after 16 hours of incubation at 40°C, increased to only 7.41 log CFU/ml (p v 0.05). In the presence of MnSO4 and E. coli, the numbers of P. acidilactici increased from 6.2 log to 9.23 log CFU/ml, whereas with E. coli alone, P. acidilactici increased from 6.2 log to 8.5 log CFU/ml (p < 0.05; figure 2). The suppression of growth of E. coli by P. acidilactici in the presence of supplement was great. In conclusion, 0.003% of MnSO4 was a good stimulator fbr the growth of P. acidilactici In the mixed culture with E. coli (104 CFU/ml) and P. acidilactici (106 CFU/ml), Mn ion was a good stimulator to control E. coli by P. acidila  (2) Inhibition of S. aureus by P. acidilactici with and without growth factor Figure 3 shows the growth of S. aureus in laboratoiy media containing P. acidilactici. Without MnSO4, S. aureus increased from 4.7 log to 7.42 log CFU/ml after 16 hours of incubation with P. acidilactici. However, the final numbers of S. aureus in the medium with the mixture of MnSO4 plus P. acidilactici after 16 hours were reduced from 4.7 log to under 1.0 log CFU/ml (p < 0.05).
In the presence of MnSO4 and S. aureus, the numbers of P. acidilactici increased from 6.8 log to 9.3 log CFU/ml, whereas with S. aureus alone, P. acidilactici increased from 7.2 log to 8.75 log CFU/ml (p < 0.05; figure 4). The suppression of growth of S. aureus by P. acidilactici in the presence of supplement was great (reduction of about 6-7 log CFU/ml units).
In conclusion, 0.003% of MnSO4 was a good stimulator for growth and acid production of P. acidilactici in cultures with E. coli or S. aureus in the in vitro system. Table 2 shows the effect of MnSO4 on microbial changes in feces of pigs. There were no significant differences fbr the microbial numbers (i.e., total microorganisms, E. coll, lactic acid bacteria and S. aureus) in feces of pigs fed MnSO4 compared to feces of pigs fed the control diet through 7 days, table 3 shows the effects of MnSO4 on pH changes in feces of pigs. For day 0 and 3 of experiment, there was no significant difference for the pH change among the treatments. However, after 7 days, the pH of feces in pigs fed MnSO4 (0.1%) decreased faster (p < 0.05) than pigs fed the control diet (8% less).

In vivo experiment.
Manganese ion contributes to the rapid decrease in pH through stimulation for LAB. Low pH is important to control harmful microorganisms and bacteriocin production from LAB (Biswas et al., 1991). From these data, MnSO4 did not show the significant effect as in the short term (7 days) in vivo experiment. However, on the basis of the rapid pH decrease of treated pig's feces, MnSO4 might affect the microbial numbers in a long term experiment.