Using of Saccharomyces Cerevisiae and Lactobacillus Acidophilus as Probiotic Against Salmonella Enterica Serovar Typhimurium Isolated from Poultry

و ةفاجلا زبخلا ةريمخ مادختسا جاجدلا نم ةلوزعملاديئوفيتلا لاينوملاس دض ةيويح تاززعمك ةيضمحلا ةينبللا تايصعلا


Introduction
. The first mention of probiotic term is by Lilly and Stillwell in 1956 which describe growth factors produced by microorganisms (4). Probiotic defined by Schaafsma and Guraner as a microorganism that have good effects on human and animal health (5), while (6) defined probiotic as any production contain diagnostic microorganisms in adequate number which have ability to change numbers and types of microflora in the body. Main characters of microorganisms that using as probiotic are: not has pathogenic or toxic effects, able to grow and multiply in the intestine, resistant to bile duct product, constant genetically and easily storage (7). The first isolation of lactobaccilus was done by Lister in 1878, its gram positive bacteria, Facultative anaerobic, non-motile , non-spore forming , negative to catalase production test, indol test , and H2S production test (4). Lactobacillus used as antibiotics alternatives because it safe, efficient, and widely used in chickens feeding and has been noticed a simulated growth of chicks, the inhibition the pathogens of intestinal microbes, a promoted immune function decreased morbidity, vitamin synthesis, reduced serum cholesterol levels and anticarcinogenic effects (8,9,10,11). Saccharomyces cerevisiae, is a nonpathogenic yeast, have positive effects on poultry production such as in egg production, reproduction, feed efficiency, growth rate, reduce liver toxicity and residual aflatoxin B1. In addition, supplementation of yeast, yeast cultures and yeast extracts to feed has gived environmental and economic benefits in poultry diets for the past 40 years (12,13.14). S. typhimurium is gram negative bacteria, non-motile , non-spore forming lactose non ferment, caused many diseases to human and animals like diarrhea and typhoid fever (15). Studies have indicated that Saccharomyces cerevisiae can be used in the prevention and treatment of bacterial infectious diseases, including Paratyphoid, typhoid and nontyphoidal Salmonella (3). (16) mention when chicks were given Salmonella spp at10 4 cfu/chick and then were treated with 10 kinds of Lactobacillus at 10 8 cfu/chick, the results indicate that Lactobacillus can decrease the cecal Salmonella counts, decrease the mortality of dieased in chicks and booster the balance of intestinal flora. The aim of this study was to Use the Saccharomyces cerevisiae and Lactobacillus acidophilus as probiotic against Salmonella typhimurium isolated from tikrit poultry.

Materials and methods
The current study conducted in animal house of College of Veterinary Medicine, University of Tikrit in Slah aldeen Province In period from February to August 2019.
-Isolation of S. typhimurium: 50 fecal samples were collected from chicken infected with diarrhea, the samples cultured in selenite F broth and cultivated at 37°C for 24h, then subcultured on Salmonella -Shigella agar, Xylose Lysine Deoxycholate agar and MacConkey agar. After colony appearance, gram stain and groups of biochemical tests were applied according to (15). Then confirmed by API 20. transport to laboratory and culture on Deman Regosa Sharp agar with 1% Ca CO3 and cultivated in 5-10% CO2 at 37C for 24h. (17). Gram stain and group of biochemical tests were applied according to (18). -Preparation of Saccharomyces cerevisiae: 1gram of Saccharomyces cerevisiae (Pakmaya-France) add to tube contain 10 ml of Glucose yeast extract peptone broth (GYEP) then cultivation aerobically at 37C for 24h.
-Preparation of S. cerevisiae and L.
acidophilus filtrated fluid a-Cultivation of S.cerevisiae and L. acidophilus: S.cerevisiae cultivation on GYEP in concentration of 1X 9 10 Cell/m then aerobically at 37C for 24h. while L.acidophilus cultivation on MRS 1X10 6 Cell/ml anaerobically at 37C for 24h (19) b-The two culture centrifuged (6000 cycle/ minutes for 10 minutes). The supernatant has been taken and filtrated by Millipore (0.22 micrometer) the filtrated fluid has been concentrated by Lyophilizer. c-Determination of inhibitory zone: 0.1 ml of 1.5X10 8 CFU/ml of S. typhimurium suspension were disseminate in agar media. Holes in plate were done by cut aseptically with sterile cork borer, then 100µl of filtrated fluid were put in hole and incubation at 37 o C for 24h, the inhibition zone were measured using caliber.

Study of adhesion Index:
a-The broth of (S.cerevisiae and L.acidophilus) centrifuged (2000 cycles/ mints) for 10 minutes, the sediment has been taken and resuspension in normal saline b-The endothelium of Rats intestine were taken after 24h of fastening. The Rats killed and endothelium has been taken by aseptic glass slide and resuspended in normal saline c-two mixture were prepared (S.cerevisiae suspension with endothelium suspension 1:50) and (L.acidophilus suspension with endothelium suspension 1:50) for 10-15 minutes. Smear from each mixture were prepared and stain by Wright stain (20).  In-Vivo adhesion Index: the result showed high ability of L. acidophilus to adhesion on rat intestine endothelium which to (18-24 cells) while Saccharomyces cerevisiae has not this feature. The ability of L. acidophilus to adhesion due to presence of Lipoteichoic acids and Surface Protein Layer which consist from more than 30% of hydrophobic amino acid (22).
Results of inhibitory zone: table 2 showed that clear effects of (L. acidophilus and S. cerevisiae) filtrate in inhibition S. typhimurium and the highly inhibition effect occur by mixture of S.cerevisiae and L. acidophilus filtrate. Figure 2   cerevisiae.
The inhibitory effect of L. acidophilus is agreed with (23) that's due to its ability to produce (Acetic, Propionic and Lactic acid which decrease in pH and killed the salmonella (24).
The inhibition effect of S. cerevisiae is due their ability to produce endotoxin, acidic compounds and proteolytic enzyme (25).
The clinical signs of diarrhea that appear on control group which infected with Salmonella is attributed to their ability to invasion of intestine epithelium and penetration them and cause inflammation and absorption disorder and diarrhea, also the enterotoxin that produce from salmonella can cause diarrhea (28). Also Salmonella caused pathological changes include degeneration on the epithelium of intestine and inflammatory reaction in liver and spleen. As in figure 4,5,6   This results agreed with results recorded by (29,30) the appearance of this pathological changes is due to bacteremia and reach to this organs (31).

Conclusions
It is concluded that the salmonella isolation rate were 6% . The result showed high ability of L. acidophilus to adhesion on rat intestine endothelium , while Saccharomyces cerevisiae has not. The inhibitory zone of mixture of Saccharomyces cerevisiae and L. acidophilus filtrate showed the highly inhibion effect. Results suggest use L. acidophilus filtrate + Saccharomyces cerevisiae filtrate in protective of experimental animal from S.typhimurium infection.