The Effect of Feeding Lactic Acid to Salmonella Typhimurium Experimentally Infected Swine

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1 FULL PAPER Bacteriology The Effect of Feeding Lactic Acid to Salmonella Typhimurium Experimentally Infected Swine Tsuyoshi TANAKA 1), Yasuo IMAI 1), Naosuke KUMAGAE 1) and Shizuo SATO 1) 1) JA Zen-noh Institute of Animal Health, 7 Ohja-machi, Sakura, Chiba, Japan (Received 30 October 2009/Accepted 28 January 2010/Published online in J-STAGE 9 February 2010) ABSTRACT. Antimicrobial resistant Salmonella are becoming more prevalent. Therefore, alternative methods to control swine Salmonella infection must be explored. We examined whether feeding lactic acid to swine is an effective way to control clinical and subclinical Salmonella Typhimurium infection in these animals. In this experiment, swine were inoculated with CFU (hi-st) or CFU (lo-st) of S. Typhimurium per swine to reproduce clinical and subclinical infection. The swine were either fed a commercial feed supplemented with 2.8% lactic acid (LA) or the commercial feed without supplementation (C) to examine the effect of feeding lactic acid. Twenty 21 and 22 days old swine were divided into 4 groups, LA-hiST, C-hiST, LA-loST and C-loST, and fed the respective feed. They were inoculated S. Typhimurium at 51 and 52 days old. Clinical symptoms and the number of S. Typhimurium shed in feces were evaluated. The LA-hiST group did not show obvious clinical symptoms, such as diarrhea or febrile response, but the C-hiST group did show clinical symptoms. The number of S. Typhimurium shed in the feces of the LA-hiST group was lower than in that of the C- hist group, and that of the LA-loST group was lower than that of the C-loST group. Our data suggest that dietary supplementation with 2.8% lactic acid can be an effective way to control clinical and subclinical infections of S. Typhimurium in swine. KEY WORDS: antimicrobial resistance, experimental infection, lactic acid, Salmonella Typhimurium, swine. J. Vet. Med. Sci. 72(7): , 2010 Salmonellosis is one of the most common infectious diseases in the world in both humans and animals. To decrease human exposure to Salmonella, Salmonella control programs have been implemented from farm-to-slaughter in Denmark and other European Union countries [16, 26]. S. Typhimurium is one of the most frequently isolated serotypes from pig farms, slaughtered swine and human foodborne illness [13, 15]. In general, antibiotic treatment is used to control swine Salmonella infection. However, the use of antimicrobial agents in humans and animals may induce emergence of antimicrobial-resistant bacteria [1]. Considerable information has been garnered on antimicrobial resistance in Salmonella of human and animal origin [2, 7, 20, 23, 25]. Alternative methods to control swine Salmonella infections have been explored [5]. Following the European Union ban on the use of the majority of antibiotic growth promoters, alternative measures have been explored to control the number and activity of intestinal bacteria. These include changes in management practices, feeding, hygiene and the use of products such as probiotics, prebiotics, enzymes, herbs and plant extracts, prefermented feeds and organic acids [21]. Lactic acid is one of the organic acids, and dietary addition of 2.8% lactic acid reduced the prevalence of Salmonella shed in feces under subclinical infection levels on a pig farm in a previous study [11], but it has not been evaluated under experimental infection conditions. At a Japanese pig farm, S. Typhimurium infection has *CORRESPONDENCE TO: TANAKA, T., 7 Ohja-machi, Sakura-shi, Chiba-ken, Japan. tanaka-tsuyoshi@zennoh.or.jp been shown to cause both clinical and subclinical cases [3, 14]. The effect of dietary lactic acid in controlling clinical S. Typhimurium infections under experimental infection conditions have not been demonstrated either. The objective of this study was to evaluate the effect of dietary supplementation with 2.8% lactic acid on clinical symptoms and the number of S. Typhimurium shed in feces under clinical and subclinical experimental S. Typhimurium infections in swine. The number of Salmonella shed in feces is one of the most influential parameters of Salmonella within-herd transmission [17]. MATERIALS AND METHODS The present experiment was carried out on the Experimental Farms of the JA Zen-Noh Institute of Animal Health in Japan and received prior approval from the Animal Protocol Review Committee of this institution. Experimental design: Twenty 21 and 22 days old secondary SPF swine were transported to our isolated facilities and assigned to 4 groups (LA-hiST, C-hiST, LA-loST and C- lost) based on gender, body weight and litter. Each group was composed of 5 swine and housed in a separate room. Each swine was confirmed to be Salmonella-free by a series of 3 rectal swab cultures [27]. The feed for both the LAhiST and LA-loST groups was a 2.8% DL-lactic acid (Musashino Nyuusan, Musashino Chemical Laboratory, Ltd., Tokyo, Japan) supplemented commercial corn-based grower feed (lactic acid added feed). On the other hand, the feed for the C-hiST and C-loST groups was a commercial corn-based grower feed without DL-lactic acid (control feed). The swine were fed the indicated feed from the day they were assigned to the groups until the end of the experi-

2 828 T. TANAKA, Y. IMAI, N. KUMAGAE AND S. SATO ment. At 51 and 52 days of age, the swine were inoculated with S. Typhimurium. The swine of the LA-hiST and C- hist groups were inoculated with 10 ml of CFU/ ml, and those of the LA-loST and C-loST groups were inoculated with 10 ml of CFU/ml. They were fasted for 5 hr before and 1 hr after inoculation [9]. At 21 days post-inoculation (DPI), the pigs were euthanized by exsanguination after deep anesthesia with sodium pentobarbital (100 mg/kg; Abbot, Abbott Park, Il, U.S.A.) and subjected to necropsy. They were provided feed and water ad libitum, except on the day of inoculation as indicated. Bacterial strain and inoculated culture: S. Typhimurium strain 116 rifampicin resistant (ST116Rif r ), which was originally isolated from swine, was made resistant to rifampicin in our laboratory via successive cultivation in Desoxycholate Hydrogen Sulfide Lactose (DHL) Agar (Eiken Chemical, Co., Ltd., Tokyo, Japan) containing up to 100 g/ml of rifampicin (RFDHL; Wako Pure Chemical Industries, Ltd., Osaka, Japan) [18]. ST116Rif r was stored at 80 C till use. One colony of this strain from Soybean-Casein Digest agar (Eiken Chemical, Co., Ltd., Tokyo, Japan) was grown in SCD broth (Eiken chemical, Co., Ltd., Tokyo, Japan) for 14 hr at 37 C. Of this culture, 20 ml was inoculated into 180 ml SCD broth and incubated for 5 hr at 37 C with vigorous shaking. This culture was diluted in PBS to obtain approximately 10 7 and 10 5 CFU/ml and then mixed with an equal volume of 20% skim milk. Each swine was inoculated orally with 10 ml of ST116Rif r mixed skim milk. The dose was given by slowly administering it into the pharyngeal region via syringe and plastic tube. The liquid was readily swallowed voluntarily. Clinical observation: The fecal conditions of each swine were scored at 1, 2, 3, 6, 7, 9, 12, 16 and 21 DPI. A fecal condition score of 0 indicated normal feces, 1 indicated loose feces and 2 indicated diarrhea. Rectal temperature was measured daily from 7 days before the day of ST116Rif r inoculation to the end of the experiment. Bacteriological examinations: Fecal samples were collected from each swine at 1, 2, 3, 6, 7, 9, 12, 16 and 21 DPI. One gram of fecal sample was serially diluted in sterile PBS and plated on RFDHL. Plates were incubated for 24 hr at 37 C. Colonies that grew on agar plates were directly counted. Then, enrichment and delayed secondary enrichment procedures were performed. Briefly 1 ml of 1 g feces diluted in 10 ml of PBS was cultured in 9 ml of Hajna tetrathionate broth (Eiken Chemical, Co., Ltd., Tokyo, Japan) followed by 24 hr incubation at 41.5 C or an additional 7 days at room temperature as a delayed secondary enrichment culture. After incubation, each culture was streaked on RFDHL. Enrichment procedures were discontinued if positive results were obtained before the tests were completed. During necropsy, the jejunum and cecum contents were aseptically collected, and the number and presence of ST116Rif r were confirmed as previously described. The liver, lung, spleen, kidney, tonsil and mesenteric lymph node were also aseptically collected, and the presence of ST116Rif r was confirmed, as discussed above, at necropsy Statistics: Normally distributed variables such as rectal temperature and the number of ST116Rif r were compared by the Student s t-test. Non-normally distributed variables such as the fecal condition score were compared by the Mann-Whitney U test. The number of ST116Rif r was transformed by using logarithms, where zero was interpreted as 1 organism per g of feces. A value of 100 organisms per g of feces was assigned to samples that were negative by direct plating methods but positive by the enrichment and delayed secondary enrichment procedures [27]. RESULTS Clinical response to S. Typhimurium inoculation: After the inoculation with ST116Rif r, the swine in the LA-hiST group did not defecate either loose feces or diarrhea on any examined day. On the other hand, 80% of the swine in the C-hiST group defecated loose feces and diarrhea at 3, 6 and 7 DPI. The average fecal scores were 0.8, 1.6 and 0.8 for each day (Table 1). The swine in the LA-loST and C-loST groups had sporadic loose feces and diarrhea between at 3 and 9 DPI, and the highest average fecal scores of the LAloST and C-loST groups were 0.4 and 0.6, respectively. Febrile responses, statistically different (P<0.05) from the average of the 7 days before inoculation of each group, were observed for both control feed groups (C-hiST and C- lost) but were not observed for both lactic acid added feed Table 1. The average fecal condition scores of the pigs fed a commercial feed supplemented with 2.8% lactic acid (LA-hiST and LA-loST) or the commercial feed without supplementation (C-hiST and C-loST), after inoculation with CFU/head (LAhiST and C-hiST) or CFU/head (LA-loST and C-loST) of S. Typhimurium 116Rif r. The fecal condition was scored from the severity of diarrhea (0, normal feces; 1, loose feces; 2, diarrhea) Group Day post inoculation LA-hiST 0 a) C-hiST LA-loST C-loST a) Fecal score (average of all 5 samples).

3 LACTIC ACID TO CONTROLS SWINE S. TYPHIMURIUM 829 Fig. 1. The average temperature of pigs before and after oral inoculation with CFU/head (A) or CFU/head (B) of S. Typhimurium 116 Rif r at day 0. The pigs were either hed a commercial feed supplemented with 2.8% lactic acid (closed symbol) or the commercial feed without supplementation (open symbol). *: A statistical difference (P<0.05) was observed compared with the average rectal temperature of 7 days before inoculation for each group. Fig. 2. The average number of S. Typhimurium 116 Rif r shed in feces (log CFU/g feces) after inoculation with CFU/head (A) or CFU/head (B) of S. Typhiumurium 116 Rif r. *: A statistical difference (P<0.05) was observed between the lactic acid added and not added groups. groups (LA-hiST and LA-loST; Fig. 1). In the C-hiST group, febrile responses were observed from 2 to 9 DPI, and in the C-loST group, responses were observed from 5 to 8 DPI. The number of ST116Rif r shed in feces: The number of ST116Rif r per gram of feces from the LA-hiST group was significantly less than that of the C-hiST group at 3, 12 and 16 DPI (P<0.05; Fig. 2A). The number for the LA-loST group was significantly less than that for the C-loST group at 6, 7, 9 and 12 DPI (P<0.05; Fig. 2B). The peak numbers of bacteria for each group throughout the experiment were CFU/g (LA-hiST), CFU/g (C-hiST), 2.5 CFU/g (LA-loST) and CFU/g (C-loST). At necropsy, ST116Rif r was recovered from the liver, tonsil, mesenteric lymph node and contents of the jejunum and cecum (Table 2). The frequencies of recovery of ST116Rif r from the jejunum contents of in the LA- hist, C- hist, LA-loST and C-loST groups were 20%, 80%, 40% and 80%, respectively. The frequencies for the cecum contents of the LA-hiST, C-hiST, LA-loST and C-loST groups were 80%, 100%, 40% and 80%, respectively. The frequencies of recovery of ST116Rif r in the lactic acid added feed groups were less than those of the control feed groups. And the numbers of ST116Rif r in the lactic acid added feed groups were less than those of the control feed groups. A statistically difference (P<0.05) was observed between the LA-hiST and C-hiST groups.

4 830 T. TANAKA, Y. IMAI, N. KUMAGAE AND S. SATO Table 2. The frequency of detection of ST116Rif r from organs and tissues and the number of ST116Rif r detected in 1 g of the jejunum and cecum contents at necropsy Group Frequency of detection of ST11Rif r Number of ST116Rif r in 1 g contents Liver Lung Spleen Kidney Tonsil MLN a) Jejunum Cecum Jejunum Cecum LA-hiST 0/5 b) 0/5 0/5 0/5 5/5 2/5 1/5 4/ c) c) * C-hiST 0/5 0/5 0/5 0/5 4/5 3/5 4/5 5/ ** LA-loST 0/5 0/5 0/5 0/5 1/5 4/5 2/5 2/ C-loST 1/5 0/5 0/5 0/5 2/5 3/5 4/5 4/ a) MLN: mesenteric lymph node. b) Positive sample/ tested sample. c) Log CFU/g, average S.D., the average and S.D. were calculated from all samples, include the negative sample. *, **: A statistical difference was observed between * and ** (P<0.05). DISCUSSION In our experiment, 2.8% lactic acid supplementation provided relief from clinical symptoms caused by S. Typhimurium infection, such as defecated diarrhea and febrile response. The feed supplemented with lactic acid reduced the number of S. Typhimurium shed in feces. After inoculation with CFU of S. Typhimurium, almost all swine in the control feed fed group showed evidence of diarrhea and febrile response. On the other hand, the swine fed the feed supplemented with lactic acid did not show these symptoms. Lactic acid feeding relieves from the clinical symptoms caused by S. Typhimurium infection. Lactic acid feeding reduced the number of S. Typhimurium shed in feces after inoculation with and CFU. This means that lactic acid feeding reduced the number of S. Typhimurium in the intestine. This result may be the reason why lactic acid feeding provided relief from clinical symptoms. In our experiment, the number of ST116Rif r in the cecum was significantly lower in the LAhiST group than in the C-hiST group at necropsy. Jørgensen [11] reported lactic acid reduced the prevalence of Salmonella at a pig farm under subclinical infection conditions. In this report, we examined whether lactic acid reduced the number of S. Typhimurium shed in feces under subclinical experimental infection conditions after inoculation with CFU of S. Typhimurium. It is known that lactic acid has a bactericidal effect at low ph. Risley et al. [19] reported that the ph of the swine stomach contents is Lactic acid at a ph of 3.7 reduces the number of S. Typhimurium up to 1/100 for 3 hr, at a ph of 4.0, it reduces the number up to 1/10, and at a ph 4.4, it has no effect [12]. Therefore, lactic acid may provide its bactericidal effect in the stomach. In our experiments, the swine were fasted for 5 hr before and 1 hr after inoculation of S. Typhimurium. So, there might have been no feed in the stomach. It is doubtful lactic acid provided its bactericidal effect in the stomach at the time when S. Typhimurium was inoculated. But it may provide its bactericidal effect when swine picked S. Typhimurium shed in feces. Further examination of the mechanism of the bactericidal effect of lactic acid in vivo is required. In this study, we fed the feed supplemented with lactic acid before inoculation of ST116Rif r, so there may be a change in the composition of short chain fatty acid in the intestine and the microbiota of intestine. Ushida et al. [22] reported that lactic acid metabolized to acetate, propionate and butyrate rapidly. The change in microbiota influences S. Typhimurium infection [5]. F. Van Immerseel et al. [24] reported that organic acids modify the gene expression of Salmonella pathogenicity island, and lactic aicd reduces the colonization of chicken crop or cecum. The mechanism of organic acid in vivo is still not clear. In a previous study, the number of Salmonella shed in feces peaked immediately after inoculation of Salmonella [6, 10]. However, in the present study, the number of S. Typhimurium shed in feces peaked at 9 and 12 DPI in the C- lost and C-hiST groups. One of the reasons for the increase in detection of Salmonellain in feces is stress [4, 18]. In the present experiment, the pigs were raised as usual, so we can exclude stress as a reason for the increase in the number of S. Typhimurium shed in feces. In the C-loST group, the number of S. Typhimurium shed in feces peaked at 9 DPI, and body temperature peaked at 6 DPI. This suggests that S. Typhimurium increased both in the intestine and body around 6 9 DPI in the C-loST group. In the C-hiST group, the number of S.Typhimurium shed in feces peaked at 3 DPI, and the body temperature peaked at 2 DPI. This suggests that S. Typhimurium increased both in the intestine and body around 2 3 DPI in the C-hiST group. However, the number of S. Typhimurium shed in feces peaked at 12 DPI in the C-hiST group without an increase of body temperature. At 12 DPI, S. Typhimurium might increase only in the intestine. However in the present experiment, this phenomenon could not be clarified. 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