Journal of Applied Bacteriology 1992, 73, 407411
Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus Maria E. Nader de Macias, Maria C. Apella, Nora C. Romero, Silvia N. Gonzblez and G. Oliver Centro de Referencia para Lactobacilos (CERELA ), Chacabuco, T u c u m h , Argentina 4028/10/91: accepted 7 May 1992
M.E. NADER DE M A C i A S , M.C. A P E L LA , N.C. ROMERO, S.N. GON ZALEZ AND G. OLIVER. 1992. T h e protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with S h . sonnei. T h e survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with S h . sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2-3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.
INTRODUCTION
The neonatal intestinal tract is germ-free (Tissier 1900) but is progressively colonized with bacteria from the mother and other external sources (Payne & Finkelstein 1977). The complex intestinal microflora that develops is largely responsible for protection against many infectious diseases and plays a major role in resisting further colonization (Ducluzeau 1989). This protective microflora can be changed by various factors such as improved diet, environmental changes and antibiotic treatment which result in a disturbance of the normal gut flora. This frequently enables pathogens to multiply and is one of the main causes of diarrhoea. Diarrhoeal diseases are a principal cause of childhood morbidity and mortality in the developing countries, as well as being a major cause of malnutrition (Anon. 1990). They are responsible for the death of more than 4 million children each year. Diarrhoeal episodes of infective aetiology represent around 27% of those reported and shigellas are among the five most frequently identified pathogens (Anon. 1986) in children with acute diarrhoea or dysentery, leading Correspondence to : Dr Cuillermo Oliver, Centro de Referencia para Lactobacilor (CERELA), Charabuco 145. 4000 Turumin, Argentina.
to a number of serious complications and high mortality rates. Although foods fermented by lactobacilli have been used for centuries, Metchnikoff (1908) was the first to establish the beneficial effect of these products in order to obtain a balance between lactobacilli and the intestinal flora. More recent studies (Kolars et al. 1984) have suggested that the utilization of fermented milk could offer a therapeutic method in malnourished and diarrhoea1 children. It has been reported that one lactobacillus preparation (Gotz et al. 1979) reduces ampicillin-associated diarrhoea, and another reduces the rate of relapse of pseudomembranous colitis (Gorbach et al. 1987). Gilliland (1979) has suggested the use of Lactobacillus casei, Lact. acidophilus and BiJidobacterium bifdum as dietary adjuncts, whereas Fernandez et al. (1982) have discussed the possible role of lactobacilli as prophylactic agents for diarrhoea. In our laboratory we isolated strains of Lact. casei and Lact. acidophilus from a human source. Both are capable of surviving and becoming established in the human gastrointestinal tract. We have used milk fermented with these strains for the prevention and treatment of diarrhoea in children (Gonzilez et al. 1990). The immunostimulant capability of these strains in mice and increased resistance
408 M A R I A E. NADER DE M A C I A S E T A L .
to Salmonella typhimurium infection as a result of feeding milk fermented with Lact. casei and Lact. acidophilus has been demonstrated (Perdigon et al. 1990). T h e present study was designed to determine whether feeding mice with fermented milk with lactobacilli could also protect against infection with Shigella sonnei, which might support the use of fermented milks for the treatment and prevention of shigella infections.
Challenge
After the 8 d feeding treatment the mice were challenged orally with Sh. sonnei introduced by an oral catheter. For the colonization and antibody measurements mice received 3 x 10' and 3 x lo9 cfu Sh. sonnei given with an interval of 24 h. For resistance assays they received the same dose but in a single day.
Circulating antibodies MATERIALS AND METHODS Animals
Swiss-albino mice, weighing 25-30 g, were obtained from the random-bred colony kept by our department. T h e animals were housed in plastic cages at room temperature. Each experimental group consisted of 20-30 mice ( 4 5 for each different day) and was housed individually during experiments.
Micro-organisms
The two strains used were Lact. casei and Lact. acidophilus both isolated from human faeces. They were identified according to the procedures outlined by Kandler & Weiss (1985). The lyophilized stock cultures of lactobacilli were transferred to LAPTg broth (Raibaud et al. 1961), incubated at 37"C, and then subcultured in the same medium until an active culture was obtained. Fermented milks were prepared by suspending separately washed cells of both organisms previously grown in LAPTg broth in 10% skim milk powder and incubated at 37°C for 8 h. These fermented milks were mixed at the end of the incubation period. The number of viable cells was determined by the agar plate method in LAPTg agar. Shigella sonnei was isolated from an infant's faeces, identified by microscopic, biochemical and serological characteristics (Rowe & Gross 1984), grown in Brain Heart Infusion (BHI, Difco) at 37°C for 6 h, and washed several times by centrifugation with saline solution before use. Virulence was tested by the ability to produce red colonies in media with Congo red dye, according to Payne & Finkelstein (1977), and by the test of Sereny (1957) which indicates the ability to promote kerato-conjunctivitis in guinea pigs.
Mice were bled from the retro-orbital venous plexus. The sera were diluted and antibody titres determined against lactobacillus and shigella suspensions (3 x lo9 cells) by the tube agglutination test (Nichols & Nakamura 1986).
Viable counts of Shigella in tissue homogenates
Numbers of viable bacteria in the liver and spleen were determined both in the control and experimental groups. At least four mice were killed by cervical dislocation at different time intervals, and the spleens and livers removed aseptically. These organs were homogenized in a final volume of 5 ml 0.1% peptone water with a Teflon homogenizer. Cell suspensions were serially diluted in peptone water: portions were plated in duplicate on MacConkey agar (Difco) and lactose-positive colonies enumerated after incubation at 37°C for 48 h. Confirmation of the identity of the organisms was obtained by biochemical tests.
Antibodies from intestinal fluid
T h e procedure for collection of intestinal fluids was a modification of that described by Lim et al. (1981) for the isolation of intestinal mucosal lymphoid cells. Small intestines from each mouse were carefully removed from the gastric/duodenal junction and at the ileal/caecal junction. T h e contents were washed out with 1 ml cold phosphate buffered saline (PBS, p H 7.2), centrifuged at 2000 g for 30 min. The supernatant fluid was retained for antibody determination. Antibody titres were determined by diluting the intestinal fluid in PBS and testing for agglutination of lactobacilli or shigellas as before. Antibodies were measured on the same experimental days as the colonization assays.
Determination of protectlon Feeding procedure
Mice were fed with 1.5 x lo8 cfu daily for 8 d, with milk fermented with Lact. casei and Lact. acidophilus. T h e control group received 10% skim milk powder.
Treated and control groups which had been fed for 8 d were challenged with doses of shigellas and observed for 21 d. T h e daily death count was recorded as percentage survival in each group.
B Y LACTOBACILLI I N V I V O 409
SHIGELLA INHIBITION
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treated mice
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control mice
401
.
I
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3. 3 0 0
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2. 12 16 20 Days post-chollenqe
8
4
Fig. 1 Survival rate of mice fed with fermented milk for eight consecutive days and challenged orally with Shigella sonnei. --, Treated mice; 0 ,control mice with skim milk powder. Results of three expriments, 10 mice in each one. The bars represent the standard deviation of the mean
1
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1
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2
3
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6
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Days post -challenge
Statistical analysis
Results were expressed as the arithmetic mean of number of observation values standard deviation. The significance of the results was analysed by Student's t test.
Fig. 3 Number of shigellas in spleens of mice fed with milk fermented with Lactobacillus casei and Lact. acidophilus for eight consecutive days, then challenged with 3 x lo8 cfu and 24 h later with 3 x lo9 cfu Shigella sonnei. 0, Treated mice; 0 ,control mice. Four mice were killed in each day. The bars represent the standard deviation of the mean RESULTS
0 control mice
0 treated mice
Enhancement of resistance to Shlgella sonnel by feeding fermented milk
After feeding fermented milk for eight consecutive days, followed by oral challenge with Sh. sonnei, mice were observed for survival for 20 d (Fig. 1). While treated mice showed a survival percentage of loo%, indicating complete protection with the fermented milk treatment, the control group showed a mortality of 16% by day 12 post-challenge, and a mortality of 40% on day 20. Effect of fermented milk on patterns of growth of Shlgell8 sonnel in liver and spleen
2
3
4 5 6 7 Days post-challenge
8
9
10
Fig. 2 Number of shigellas in liver of mice fed with milk fermented with Lactobacillus casei and Lact. acidophilus for eight consecutive days, then challenged with 3 x lo8 cfu and 24 h later with 3 x lo9 cfu Shigella sonnei. 0, Treated mice; 0 ,control mice. The bars represent the standard deviation of the mean
Viable bacteria in the liver of control mice began to appear by the second day post-challenge, reaching the highest level on day 5 (Fig. 2). T h e kinetics of appearance of shigellas in the liver of treated mice were similar to the control, but with a difference of 2-3 logarithmic units on all days studied. On the ninth day shigellas practically disappeared in the liver of treated mice, but a large number remained in the control. In the spleens of both control and treated mice, shigellas appeared on the second day post-challenge, with a difference of 1-5 logarithmic units between them (Fig. 3). In the
410 MARIA E . NADER DE MACIAS ET A L .
lating anti-lactobacilli antibodies of both groups were between 10 and 20 (inverse dilution). Titres of anti-shigella antibodies from small intestinal fluid are shown in Fig. 4b, which shows statistically significant differences between the treated groups from days 3-8 post-challenge. Titres of anti-lactobacilli antibodies were between 8 and 16 for the treated mice.
0 controt mice 0 treated mice
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4
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Days post-challenge
Fig. 4 Levels of anti-shigella circulating and intestinal antibodies after different time periods from mice fed for eight consecutive days with fermented milk and challenged with Shigella sonnei. (a) Circulating antibodies: 0, Treated mice; 0 ,control mice. (b) Intestinal antibodies: 0, Treated mice; 0 ,control mice. Four mice were killed in each day. The bars represent the standard deviation of the mean
control mice, the highest numbers were obtained on day 5. By contrast, in the treated mice the number of shigellas tended to decrease, disappearing completely by the tenth day. Circulating and intestinal antibodies
The levels of anti-shigella antibodies from sera after challenge are shown in Fig. 4a. Fivefold higher titres were obtained in the treated mice on day 5. T h e titres of circu-
DISCUSSION
T o obtain nutritional and therapeutic benefits it is essential that the bacteria can survive in the physiological environment of the gut. Milk fermented with strains of Lact. casei and Lact. acidophilus, that are capable of surviving and establishing in the gastrointestinal tract is used for the prevention and treatment of infantile diarrhoea. In this work we studied the effect of feeding fermented milk on the resistance of mice to S h . sonnei. This strain was selected because of the high incidence of shigella diarrhoea in our district. One of the most difficult problems in studying shigella infections in vivo is the non-availability of a suitable animal model. Other models depend on the Sereny test (kerato-conjunctivitis model), or monkeys, which are highly susceptible to infection with shigellas but are very expensive and difficult to use. We can initiate infection with S h . sonnei in mice, however, by a double-challenge with an interval of 24 h. Although we obtained invasion in the liver and the spleen, it was impossible to determine the lethal dose in the two strains of mice (BALB/C and Swiss-albino), because of the high innate resistance to shigella infection. Nevertheless we obtained statistically significant differences (P< 0-OOS), between the animals previously fed with fermented milk and the control mice in the resistance and survival rate: a protection of 100% against shigella infection with this treatment was achieved. Colonization of shigella in the liver and spleen followed similar kinetics in the treated and the control mice, but with very significant differences (P< 0.005) in the numbers of shigella colonies grown. The lower number of shigellas found in the treated animals in the first days of post-challenge can be explained by stimulation of the mononuclear phagocytic system produced by the previous feeding with fermented milk (Perdigon et al. 1988). Another reason could be the higher levels of local antishigella intestinal antibodies found. It is known that immunity to shigellosis depends entirely on local immune mechanisms that involve local antibodies from mucosal cellmediated immunity or both (Anon, 1986). This hypothesis is supported by Wassef et al. (1989) who are close to elucidating the mechanism of production of local immunity to shigellosis with an experimental model of strength ligated loops of the intestinal lumen of rabbits. They observed that both pathogenic and non-pathogenic shigellas were taken
SHIGELLA INHIBITION BY LACTOBACILLI I N V l V O 411
up by specialized M cells in the follicular-associated epithelia (FAE). These M cells are part of the FAE which overlies dome area in Peyer’s patches and in isolated lymphoid follicles in the gastrointestinal tract. M cells are thought to play an important role in the initial uptake of antigens to stimulate the immune system. I n addition to being a site for antigen sampling and stimulation of the mucosal immune response, M cells may serve as the portal of entry for pathogenic micro-organisms. Another important result is that the shigellas disappeared from liver and spleen on the 10th day post-challenge in the treated mice while maintaining high levels in the untreated mice. We explain these results by the increased systemic immune response produced by the fermented milk in previous treatment that produce the stimulation both of T and B cells, who can participate by themselves, or by their products in the elimination of the pathogens. T h e stimulation of the immune system by an oral antigen is supported on the work of Phillips-Quagliata (1983) who demonstrated the existence of a common mucosal immune system, directly related with the systemic immune system. I n this work, we found a higher level of sera antibodies that, even though they are not usually protective in shigellosis (Anon. 1986), are indicating this immunostimulation. T h e evidence presented in this work of the higher resistance to Sh. sonnei infection produced by feeding fermented milk, shows that lactic acid bacteria have potential uses in the prevention of diarrhoea and can be also applied for its therapy.
REFERENCES A N O N . (1986) Development of vaccines against shigellosis. Diarrhoeal Disease Control Programme. WHO/CDD/IMV/86-1. Geneva : World Health Organization. ANON.(1990) A manual for the treatment of diarrhoea. Control of diarrhoea1 disease. WHO/CDD/SER/80.2/Rev. 2. Geneva : World Health Organization. D U C L U Z E ARU. , (1989) Role of experimental microbial ecology in gastroenterology. In Microbial Ecology and Intestinal Infection ed. Bergogne Berezin, E. p. 7. Paris: Springer-Verlag. F E R N A N D EC., Z , S H A H A N IK, . & A M E R ,M.A. (1982) Control of diarrhoea by lactobacilli. Journal of Applied Nutrition 40,32-43. GILLILAND , (1979) Beneficial interrelationships between S.E. certain microorganisms and humans : candidate microorganisms for use as dietary adjuncts. Journal of Protection 42, 164-167. G O N Z A L ESZ.,N . , A L B A R R A C G I N. , LOCASCIO DE RUIZ, M., M A L E , M., A P E L L A , M . C . , PESCE D E R U I Z HOLGADO A.A. , & O L I V E RG, . (1990) Prevention ofinfantile diarrhoea by fermented milk. Microbiologie-Aliments-Nutrition 8, 3 1&3 14.
G O R B A C HS,. L . , C H A N GT. , & G O L D I N B. , (1987) Successful treatment of relapsing Clostridium dzflcile colitis with Lactobacillus GG. Lancet ii, 1519. GOTZ,V . , R O M A N K I E W IJC. AZ. , M o s s , J . & M U R R A Y , H .W. (1979) Prophylaxis against ampicillin associated diarrhea with a lactobacillus preparation. American Journal of Hospital Pharmacy 36,754-757. K A N D L E R0, . & WEISS, N . (1985) Regular non-sporing, Gram-positive rods. In Bergey’s Manual of Systematic Bacteriology ed. Sneath, P.H.A. pp. 1209-1234. Baltimore: Williams & Wilkins. KOLARS,J.C., L E W I T ,M . D . , A O U J I , M . & S A V O I A N O , D.A. (1984) Yogurt: an autodigesting source of lactose. New England Journal of Medicine 310, 1-3. N . & WATSON, R.R. (1981) Immune L I M , T.S., MESSINA, components of the intestinal mucosae of ageing and protein deficient mice. Immunology 43,401-407. NICHOLS,W.S. & N A K A M U RLA.,M . (1986) Agglutination and agglutination inhibition assays. In Manual of Clinical Laboratory Immunology ed. Rose, N.R., Friedman, H. & Fahey, Y.L. p. 49. Washington: American Society for Microbiology. METCHNIKOFF, E. (1908) The Prolongation of Life. New York: P. Putman. R.A. , (1977) Detection and P A Y N ES, . M . & F I N K E L S T E I N differentiation of iron-responsive avirulent mutants on Congo red agar. Infection and Immunity 18, 94-98. PERDIGON, G . , NADERD E M A C I A SM , . E . , A L V A R E Z ,S . , M E D I C I ,M . & O L I V E RG , . (1988) Effect of a mixture of Lactobacillus casei and L . acidophilus administered orally on the immune system in mice. Journal of Food Protection 49, 9 8 6 995. PERDIGON, G., N A D E RD E MACIAS,M . E . , A L V A R E Z S., , O L I V E RG , . & PESCED E R U I ZH O L G A D OA., (1990) Prevention of gastrointestinal infection using immunobiological methods with milk fermented with Lactobacillus casei and L . acidophilus. Journal of Dairy Research 57, 255-264. P H I L L I P S - Q U A G L I AJ.M., T A , R O U X ,M . E . , A R N Y , M., K E L L Y - H A T F I E LP., D , M C W I L L I A M SM. , & LAMM, M.E. (1983) Migration and Regulation of B-cells in the mucosal immune system. In The Secretory Immune System ed. McGhee, J.R. & Mestecky, J. pp. 194-203. New York: The New York Academy of Sciences. R A I B A U DP., , CAULET,M . , G A L P I NJ, . V . & MOCQUOT, G . (1961) Studies on the bacterial flora on the alimentary tract of pigs. 11. Streptococci: selective enumeration and differentiation of the dominant group. Journal of Applied Bacteriology 24, 285-306. ROWE,B. & GROSS,R.J. (1984) Genus Shigela. In Bergey’s Manual of Systematic Bacteriology ed. Krieg, N.R. & Holt, J.G. p. 423. Baltimore: Williams and Wilkins. S E R E N YB. , (1957) Experimental keratoconjunctivitis shigellosa. Acta Microbiologia Academiae Scientarum Hungaricae 4, 367376. TISSIER H ,. (1900) Recherches sur la flore intestinale des nourissons (Thesis) p. 235. University of Paris, Medical School. WASSEF,J.S., K E R E N ,D . F . & M A I L L O U XJ ., L . (1989) Role of M cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis. Infection and Immunity 57,858-863.
Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus Maria E. Nader de Macias, Maria C. Apella, Nora C. Romero, Silvia N. Gonzblez and G. Oliver Centro de Referencia para Lactobacilos (CERELA ), Chacabuco, T u c u m h , Argentina 4028/10/91: accepted 7 May 1992
M.E. NADER DE M A C i A S , M.C. A P E L LA , N.C. ROMERO, S.N. GON ZALEZ AND G. OLIVER. 1992. T h e protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with S h . sonnei. T h e survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with S h . sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2-3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.
INTRODUCTION
The neonatal intestinal tract is germ-free (Tissier 1900) but is progressively colonized with bacteria from the mother and other external sources (Payne & Finkelstein 1977). The complex intestinal microflora that develops is largely responsible for protection against many infectious diseases and plays a major role in resisting further colonization (Ducluzeau 1989). This protective microflora can be changed by various factors such as improved diet, environmental changes and antibiotic treatment which result in a disturbance of the normal gut flora. This frequently enables pathogens to multiply and is one of the main causes of diarrhoea. Diarrhoeal diseases are a principal cause of childhood morbidity and mortality in the developing countries, as well as being a major cause of malnutrition (Anon. 1990). They are responsible for the death of more than 4 million children each year. Diarrhoeal episodes of infective aetiology represent around 27% of those reported and shigellas are among the five most frequently identified pathogens (Anon. 1986) in children with acute diarrhoea or dysentery, leading Correspondence to : Dr Cuillermo Oliver, Centro de Referencia para Lactobacilor (CERELA), Charabuco 145. 4000 Turumin, Argentina.
to a number of serious complications and high mortality rates. Although foods fermented by lactobacilli have been used for centuries, Metchnikoff (1908) was the first to establish the beneficial effect of these products in order to obtain a balance between lactobacilli and the intestinal flora. More recent studies (Kolars et al. 1984) have suggested that the utilization of fermented milk could offer a therapeutic method in malnourished and diarrhoea1 children. It has been reported that one lactobacillus preparation (Gotz et al. 1979) reduces ampicillin-associated diarrhoea, and another reduces the rate of relapse of pseudomembranous colitis (Gorbach et al. 1987). Gilliland (1979) has suggested the use of Lactobacillus casei, Lact. acidophilus and BiJidobacterium bifdum as dietary adjuncts, whereas Fernandez et al. (1982) have discussed the possible role of lactobacilli as prophylactic agents for diarrhoea. In our laboratory we isolated strains of Lact. casei and Lact. acidophilus from a human source. Both are capable of surviving and becoming established in the human gastrointestinal tract. We have used milk fermented with these strains for the prevention and treatment of diarrhoea in children (Gonzilez et al. 1990). The immunostimulant capability of these strains in mice and increased resistance
408 M A R I A E. NADER DE M A C I A S E T A L .
to Salmonella typhimurium infection as a result of feeding milk fermented with Lact. casei and Lact. acidophilus has been demonstrated (Perdigon et al. 1990). T h e present study was designed to determine whether feeding mice with fermented milk with lactobacilli could also protect against infection with Shigella sonnei, which might support the use of fermented milks for the treatment and prevention of shigella infections.
Challenge
After the 8 d feeding treatment the mice were challenged orally with Sh. sonnei introduced by an oral catheter. For the colonization and antibody measurements mice received 3 x 10' and 3 x lo9 cfu Sh. sonnei given with an interval of 24 h. For resistance assays they received the same dose but in a single day.
Circulating antibodies MATERIALS AND METHODS Animals
Swiss-albino mice, weighing 25-30 g, were obtained from the random-bred colony kept by our department. T h e animals were housed in plastic cages at room temperature. Each experimental group consisted of 20-30 mice ( 4 5 for each different day) and was housed individually during experiments.
Micro-organisms
The two strains used were Lact. casei and Lact. acidophilus both isolated from human faeces. They were identified according to the procedures outlined by Kandler & Weiss (1985). The lyophilized stock cultures of lactobacilli were transferred to LAPTg broth (Raibaud et al. 1961), incubated at 37"C, and then subcultured in the same medium until an active culture was obtained. Fermented milks were prepared by suspending separately washed cells of both organisms previously grown in LAPTg broth in 10% skim milk powder and incubated at 37°C for 8 h. These fermented milks were mixed at the end of the incubation period. The number of viable cells was determined by the agar plate method in LAPTg agar. Shigella sonnei was isolated from an infant's faeces, identified by microscopic, biochemical and serological characteristics (Rowe & Gross 1984), grown in Brain Heart Infusion (BHI, Difco) at 37°C for 6 h, and washed several times by centrifugation with saline solution before use. Virulence was tested by the ability to produce red colonies in media with Congo red dye, according to Payne & Finkelstein (1977), and by the test of Sereny (1957) which indicates the ability to promote kerato-conjunctivitis in guinea pigs.
Mice were bled from the retro-orbital venous plexus. The sera were diluted and antibody titres determined against lactobacillus and shigella suspensions (3 x lo9 cells) by the tube agglutination test (Nichols & Nakamura 1986).
Viable counts of Shigella in tissue homogenates
Numbers of viable bacteria in the liver and spleen were determined both in the control and experimental groups. At least four mice were killed by cervical dislocation at different time intervals, and the spleens and livers removed aseptically. These organs were homogenized in a final volume of 5 ml 0.1% peptone water with a Teflon homogenizer. Cell suspensions were serially diluted in peptone water: portions were plated in duplicate on MacConkey agar (Difco) and lactose-positive colonies enumerated after incubation at 37°C for 48 h. Confirmation of the identity of the organisms was obtained by biochemical tests.
Antibodies from intestinal fluid
T h e procedure for collection of intestinal fluids was a modification of that described by Lim et al. (1981) for the isolation of intestinal mucosal lymphoid cells. Small intestines from each mouse were carefully removed from the gastric/duodenal junction and at the ileal/caecal junction. T h e contents were washed out with 1 ml cold phosphate buffered saline (PBS, p H 7.2), centrifuged at 2000 g for 30 min. The supernatant fluid was retained for antibody determination. Antibody titres were determined by diluting the intestinal fluid in PBS and testing for agglutination of lactobacilli or shigellas as before. Antibodies were measured on the same experimental days as the colonization assays.
Determination of protectlon Feeding procedure
Mice were fed with 1.5 x lo8 cfu daily for 8 d, with milk fermented with Lact. casei and Lact. acidophilus. T h e control group received 10% skim milk powder.
Treated and control groups which had been fed for 8 d were challenged with doses of shigellas and observed for 21 d. T h e daily death count was recorded as percentage survival in each group.
B Y LACTOBACILLI I N V I V O 409
SHIGELLA INHIBITION
I
g,oo
"-----
80
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-*.
0 treated mice
5t
treated mice
I
7
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's
0
.-
? 60
v1
0 control mice
control mice
401
.
I
I
\
3. 3 0 0
20
1
2. 12 16 20 Days post-chollenqe
8
4
Fig. 1 Survival rate of mice fed with fermented milk for eight consecutive days and challenged orally with Shigella sonnei. --, Treated mice; 0 ,control mice with skim milk powder. Results of three expriments, 10 mice in each one. The bars represent the standard deviation of the mean
1
1
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1
I
1
1
1
2
3
4
5
6
7
8
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Days post -challenge
Statistical analysis
Results were expressed as the arithmetic mean of number of observation values standard deviation. The significance of the results was analysed by Student's t test.
Fig. 3 Number of shigellas in spleens of mice fed with milk fermented with Lactobacillus casei and Lact. acidophilus for eight consecutive days, then challenged with 3 x lo8 cfu and 24 h later with 3 x lo9 cfu Shigella sonnei. 0, Treated mice; 0 ,control mice. Four mice were killed in each day. The bars represent the standard deviation of the mean RESULTS
0 control mice
0 treated mice
Enhancement of resistance to Shlgella sonnel by feeding fermented milk
After feeding fermented milk for eight consecutive days, followed by oral challenge with Sh. sonnei, mice were observed for survival for 20 d (Fig. 1). While treated mice showed a survival percentage of loo%, indicating complete protection with the fermented milk treatment, the control group showed a mortality of 16% by day 12 post-challenge, and a mortality of 40% on day 20. Effect of fermented milk on patterns of growth of Shlgell8 sonnel in liver and spleen
2
3
4 5 6 7 Days post-challenge
8
9
10
Fig. 2 Number of shigellas in liver of mice fed with milk fermented with Lactobacillus casei and Lact. acidophilus for eight consecutive days, then challenged with 3 x lo8 cfu and 24 h later with 3 x lo9 cfu Shigella sonnei. 0, Treated mice; 0 ,control mice. The bars represent the standard deviation of the mean
Viable bacteria in the liver of control mice began to appear by the second day post-challenge, reaching the highest level on day 5 (Fig. 2). T h e kinetics of appearance of shigellas in the liver of treated mice were similar to the control, but with a difference of 2-3 logarithmic units on all days studied. On the ninth day shigellas practically disappeared in the liver of treated mice, but a large number remained in the control. In the spleens of both control and treated mice, shigellas appeared on the second day post-challenge, with a difference of 1-5 logarithmic units between them (Fig. 3). In the
410 MARIA E . NADER DE MACIAS ET A L .
lating anti-lactobacilli antibodies of both groups were between 10 and 20 (inverse dilution). Titres of anti-shigella antibodies from small intestinal fluid are shown in Fig. 4b, which shows statistically significant differences between the treated groups from days 3-8 post-challenge. Titres of anti-lactobacilli antibodies were between 8 and 16 for the treated mice.
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Fig. 4 Levels of anti-shigella circulating and intestinal antibodies after different time periods from mice fed for eight consecutive days with fermented milk and challenged with Shigella sonnei. (a) Circulating antibodies: 0, Treated mice; 0 ,control mice. (b) Intestinal antibodies: 0, Treated mice; 0 ,control mice. Four mice were killed in each day. The bars represent the standard deviation of the mean
control mice, the highest numbers were obtained on day 5. By contrast, in the treated mice the number of shigellas tended to decrease, disappearing completely by the tenth day. Circulating and intestinal antibodies
The levels of anti-shigella antibodies from sera after challenge are shown in Fig. 4a. Fivefold higher titres were obtained in the treated mice on day 5. T h e titres of circu-
DISCUSSION
T o obtain nutritional and therapeutic benefits it is essential that the bacteria can survive in the physiological environment of the gut. Milk fermented with strains of Lact. casei and Lact. acidophilus, that are capable of surviving and establishing in the gastrointestinal tract is used for the prevention and treatment of infantile diarrhoea. In this work we studied the effect of feeding fermented milk on the resistance of mice to S h . sonnei. This strain was selected because of the high incidence of shigella diarrhoea in our district. One of the most difficult problems in studying shigella infections in vivo is the non-availability of a suitable animal model. Other models depend on the Sereny test (kerato-conjunctivitis model), or monkeys, which are highly susceptible to infection with shigellas but are very expensive and difficult to use. We can initiate infection with S h . sonnei in mice, however, by a double-challenge with an interval of 24 h. Although we obtained invasion in the liver and the spleen, it was impossible to determine the lethal dose in the two strains of mice (BALB/C and Swiss-albino), because of the high innate resistance to shigella infection. Nevertheless we obtained statistically significant differences (P< 0-OOS), between the animals previously fed with fermented milk and the control mice in the resistance and survival rate: a protection of 100% against shigella infection with this treatment was achieved. Colonization of shigella in the liver and spleen followed similar kinetics in the treated and the control mice, but with very significant differences (P< 0.005) in the numbers of shigella colonies grown. The lower number of shigellas found in the treated animals in the first days of post-challenge can be explained by stimulation of the mononuclear phagocytic system produced by the previous feeding with fermented milk (Perdigon et al. 1988). Another reason could be the higher levels of local antishigella intestinal antibodies found. It is known that immunity to shigellosis depends entirely on local immune mechanisms that involve local antibodies from mucosal cellmediated immunity or both (Anon, 1986). This hypothesis is supported by Wassef et al. (1989) who are close to elucidating the mechanism of production of local immunity to shigellosis with an experimental model of strength ligated loops of the intestinal lumen of rabbits. They observed that both pathogenic and non-pathogenic shigellas were taken
SHIGELLA INHIBITION BY LACTOBACILLI I N V l V O 411
up by specialized M cells in the follicular-associated epithelia (FAE). These M cells are part of the FAE which overlies dome area in Peyer’s patches and in isolated lymphoid follicles in the gastrointestinal tract. M cells are thought to play an important role in the initial uptake of antigens to stimulate the immune system. I n addition to being a site for antigen sampling and stimulation of the mucosal immune response, M cells may serve as the portal of entry for pathogenic micro-organisms. Another important result is that the shigellas disappeared from liver and spleen on the 10th day post-challenge in the treated mice while maintaining high levels in the untreated mice. We explain these results by the increased systemic immune response produced by the fermented milk in previous treatment that produce the stimulation both of T and B cells, who can participate by themselves, or by their products in the elimination of the pathogens. T h e stimulation of the immune system by an oral antigen is supported on the work of Phillips-Quagliata (1983) who demonstrated the existence of a common mucosal immune system, directly related with the systemic immune system. I n this work, we found a higher level of sera antibodies that, even though they are not usually protective in shigellosis (Anon. 1986), are indicating this immunostimulation. T h e evidence presented in this work of the higher resistance to Sh. sonnei infection produced by feeding fermented milk, shows that lactic acid bacteria have potential uses in the prevention of diarrhoea and can be also applied for its therapy.
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