9 Egypt.J. Vet. Sci. Vol. 37, pp. 127 - 136 (2003)

Effect of acidilactici alld Saccltaromyces boulardii as Probiotics on Intestinal and Caecal Colonuation of Salmollella tJphiniwium and Closllidium pe1frillgensin Broiler Chickens

M. H. H. A�vaad, Manal A. Afify, Sahar A. Zouel-fakar and Basma Shalaby*

Facultyof Veterina,y Medicine, Cairo University and * Animal Health Research Institute Cairo. Egypt.

SIGNIFICANT reduction in intestinal and caecal colonization of A Clostridium pe1fringe11s ( C. pe1.fringens) during 21 days post infection (PI) has occurred on using the probiotics Pediococcus acidilactici or Saccharomyces bou!ardii. However; the fmmer resulted in a higher reduction than the latter. The same results were also obtained with Srrcc!wromycesboulardii at 7 and 14 days PI.

Reduction in typhimurium (S. typhimurium) caecal colonization significantly happened during the entire period of the experiment (21 days) on using the two studied probiotics.

Neither mortality nor disease lesions could be obtained in experimentally infected groups with the used doses of both S.typhimurium and C. pe1:fri11ge11s under the conditions of the experiment

During the first few days after hatching, Salmonella infections acquired vertically from parents or horizontally in the hatchery can cause significant growth depression or even mortality in young chicks or poults. Infections of mature poultry with salmonellae can also be costly to producers in terms of the efforts required to prevent the transmission of infection to progeny or to humans (Gast, 1997). The main Salmonella serovars involved in human food poisoning outbreaks were S. typhimurium, S. hadar, S. eneritidis and S. agona.. A major portion of illnesses were associated with meat and poultry (30.6% of incidents and 42.7% of cases) (Todd, 1989). Commercial poultry constitute one of the largest and most important reservoirs of salmonellae that can be introduced into the human food supply. Contaminated poultry meat and eggs have cosistently been among the most frequently implicated sources of human Salmonella outbreaks (Gast 1997).

C. perfringens is an obligate anaerobic bacterium in the intestinal tract of chickens (Johansson and Sarles, 1948; and Shapiro and Sarles, 1949). The organisms are relatively innocuous unless their cofactors occur such as dietary 128 M.H.H. AW AAD et al. ingr�o1cnts or changes, severe stress, coccidiosis, or immunosuppressive affectkms (Barnes, 1997). Alpha toxin produced by C. perfringens types A and C, and beta toxin produced by C. perfringens type C, are those believed rt;;spon::dble for intestinal mucosa! necrosis, the characteristic lesion of necrotic e11tcdtl:; in poultry which is a common problem among rapidly growing broiler strains of chickens that are raised intensively in modern microenvironments (Kohler et ed., 1974 and Hofarce, 1998). C. perfringens is responsible for severe food borne enteritis in man and its enterotoxin has been shown to be responsible fo1 food poisoning (Brynestad, 2002). The presence of C. perfringens in foods such as meat or poultry may be unavoidable (Rhodehamel and Hannon, 1998).

Hence the widespread use of antibiotics as therapeutic agents and growth promoters resulted in the development of resistant population of which made their subsequent use for therapy difficult and result in occurrence of antibiotic residues in the poultry products (DuPont and Steele, 1987); the direction towards the use of competitive �xclusion and probiotics as natural control method was emerged. It is based on ensuring the bird has an adequate gut microflora to counter pathogenic bacterial colonization in the digestive tract. Goldin and Gorbach ( 1934) reported that probiotics stimulate natural resistance of the organism through increasing the number of antibodies and increasing the effectiveness of macrophages. Fuller ( 1989) mentioned that the auspicious effect of probiotics over the organism is due to the better adhesion of the to the intestinal epithelium in comparison with the pathogenic bacteria, and stooping the implementation of that bacteria over the mucus membranes of the intestine.

The purpose of this study is to compare the possible effectiveness of two Probiotics namely Pediococcus acidilactici and in reducing intestinal and caecal coloni'.t::ation with S. typhimurium and C. perfringens type C in broiler chickens.

Material and Methods Probiotics Pediococcus acidilactici AfA 18 I 5M (Bactoceltt') and Saccharomyces boulardii (Levucell SB'�) produced by Lallemand, France were used as probiotics in this trial. They were used in a dose of 100 gm / ton feedfor the entire period of the experiment starting fromone day of age.

The challenge organisms 1-C. perfringens strain obtained from Animal Health Research institute, Dokld, Cairo was inoculated into cooked meat medium and incubated anaerobically in Gaspack anaerobic jar at 37°C for 24 hr the culture was centrifugedat 10000 r.p.m. for IO min . The cell concentration was adjusted using MacFerland turbidity tube. C. perfringens cells administered orally in a dose of 0.5 ml of l 08 CFU / ml. to the birds.

Egypt. J. Vet. Sci. Vol. 37 (2003) EFFECT OF PEDIOCOCCUSACIDILACTICI ... 129

2- S. typhimurium novobiocin (NO) and nalidixic acid (NA) resistant strain obtained from the National Veterinary Services Laboratory, Ames, IA,USA. maintained in media containing 25 ug NO and 20 ug NA/ml was used in this study. The challenge inoculate was prepared froman overnight culture which had previously be transferred 3 times in trypticase soy broth, then serially diluted in sterile phosphate buffered saline to obtain a concentration of 4 X 105 CFU I 1.0 ml. The viable cell concentration of the challenge inoculum was confirmed by colony counts on brilliant green agar (BGA) plates containing 25 ug NO and 20 ug NA/ml.

Experimental chickens Four hundred and twenty, day-old meat type chickens (Hubbered breed) obtained from a commercial hatchery were assigned into 7 equal groups (1-7) consisting of 60 birds each (3 replicate pens of 20 chicks each). All birds were 2 kept in separate pens, floor reared at a density of 12 birds / m and fed on a commercial balanced ration ad libitum. Vaccination of these birds was carried out using Hitchner BI and La Sota at 5 and 18 days- respectively for Newcastle disease vaccination and IBD Blen vaccine at 14 days for infectious bursa! disease vaccination via intraoccular route. Ration contained semduramicin at a concentration of 25 PPM as a coccidiostate. No antibiotics were added to the ration.

Experimentaldesign Birds of group I, 3 and 5 were fed on ration contained Pedicoccus acidilactici, while the birds of groups 2, 4 and 6 were fed on ration contained Saccharomyces boulardii. Twenty-four hours later birds of groups 4 and 6 were then infected orally with C. perfringens while bird of groups I and 3 were infected with S. typhimurium. Birds of group 2 and 6 were kept as non-infected treated groups, while the birds of group 7 were kept as non- treated non-infected control. The experimental design is summarized in Table 1.

TABLE IE. x1enmen ta Id es1gn. Groups Treatment Infection C.perfri11f[e11s S. typ!,imurium I Pedicoccus acidilactici Yes - 2 Saccharomyces boulardii Yes - .,... Pedicoccus acidilactici - Yes 4 Saccharomyces boulardii - Yes 5 - Yes - 6 - - Yes 7 - - -

Egypt. .I. Vet. Sci. Vol. 37 (2003) 130 M.H.H. AWAAD et al. The birds were kept for 3 weeks observation period for clinical signs and mortality, Seven birds were randomly sacrificedat 0, 3, 7,14 and 21 days PI from each group and subjected to post mortem and their intestinal and cecal contents were cultured for C. perfringens and S. typhimurium count as follows:

For btestinal and caecal colonization of C. perfringens a portion of the inte!;tinal_ il:tr caecal contents (0.2 g) were serially diluted in sterile physiological saline to l '.i 00, l:1000, or 1: l 0000 and 0.1 ml of each dilution was poured on the surface of sheep blood agar plates contain 200 ug / ml neomycin sulfate and ° incubated anaerobically at 37 C for 24 hr. The number of C. perfringens colonies / g of intestinal contents was determined.

For caecal colonization of S. typhimurium a portion of the contents (0.2 g) was serially diluted to 1: 100, 1: 1000, or 1: 10000 and 0.1 ml was spread-plated on NO and NA BGA plates. The plates were incubated at 37° C for 24 hr and the number of S. typhimurium colonies / g of cecal contents was determined. Typical Salmonella colonies were confirmed by biochemical tests on triple sugar iron agar.

Results

Obtained results are shown in Tables 2-4. Neither mortality nor disease lesions could be obtained in all experimented groups on applying the used doses of both C. perfringens and S. typhimuriumunder the conditions of the experiment

TABLE 2. Results of intestinal colonization of C. perfrl11ge11sin treated and untreated broiler chickens with Pedicoccus acidilacticiand Saccl,aromyces boulardii (bacterial count / gm intestinal contents x 105).

Infection Group Clostri dium peefrinRens Number Treatment 0 hr. 3 Os 7Ds 14 Os 21 Ds I PI PI PI PI I Pedicoccus acidilactici 0 0 0.5 ± 1.5± 2.5± 1.87*8 l.87*8 1.02*8

2 Saccharomyces boulardii 0 0 2.5 ± 2.25± 6± 1.29 0.89* 1.09*

5 Non-treated 0 1.25 2.75 12.5 25 ± ±1.31 ±0.91 0.99 I *Significantdifference (P <0.05) n=7 ± =SD *a =High Significance

Egypt. J. l'et. Sci. Vol. 37 (2003) EFFECT OF PEDIOCOCCUSACIDILACTICI ... 131

TABLE 3. Results of caecal colonization of C.perfringens in treated and untreated broiler chickens with Pedicoccus acidilactici and Saccliaromyces boulardii ( bacterial count I gm caecal contents x 105).

Infection Group Clostridiumperfrim!ens Number Treatment 0 hr. 3 Ds 7 Ds 14 Ds 21 Ds I PI PI PI PI I Pedicoccus acidilactici 0 0 0 0 0.5 ± 1. I 5*a

2 Saccharomyces bouli1rdii. 0 0 0 0 3.5 ± 1.12*

5 Non-treated 0 0 0.5 3.0 ± 17.5 ± 1.09 1.13 *Significantdifference (P <0.05) n=7 ± =SD *a =High Significance

TABLE 4. Results of caecal colonization of Salmonella typhymuriumin treated and untreated broiler chickens with Pedicoccus acidilactici and Saccl,aromyces boulardii (bacterial count/ gm intestinal contents x 105).

Infection Group Treatment Salmonella ti,ohvmurium number 0 hr 3 Ds 7 Ds 14 Ds 21 Ds PI PI PI PI 2 I Pedicoccus acidi/actici 0 130* 305* 151* 0.002*

4 Saccharomyces 0 64* 295* 140* 0.03* boulardii

6 Non-treated 0 1305 42800 2550 4

*Significant difference (P <0.05)

Discussion

As early as 1928, Voltera reported that competitive exclusion implies the prevention of entry of one entity into a given environment because that space is already occupied, the competing entity is better suited to establish and maintain itself in that environment or the competing entity is producing a product hostile (toxic) to its competition. He also mentioned that native micro-flora competitively exclude unwanted bacterial contamination from the intestinal tract of poultry. Egypt. J f'et. Sci. Vol. 37 (2003) 132 M.H.H. AWAAD et al .

It is alre�dy es!ablished that the complex micro-flora present in the gastrointestinal tract of the birds is effective in providing resistance to disease. However, this protective flora can be altered by dietary and environmental influences.

In the present investigation the inoculated doses of C. perfringens and S. typhimurium resulted in neither mortality nor disease lesions under the conditions of the experiment.

However, there was a significant reduction in intestinal as well as caecal colonization of C. pe,fringens on using both probiotics at 21 days post infection (PI). Pediococ<.:us acidilactici lead to more reduction than Saccharomyces boulardii. The same results were also obtained with Pediococcus acidilactici at 7 and 14 days Pl for intestinal colonization (Tables 1-2). Fukata et al. ( 1991) reported that probiotics such as Lactobacil/us acidophilus and Streptococcus fa ecium reduced the severity of necrotic enteritis. Hofarce (19 98) considered probiotics a novel and effective alternativ-!s to antibiotics that could reduce the severity of C. pe,J,-ingens-associated necrotic enteritis challenge in broilers.

Intestinal colonization is normally the first step in the infection process for orally introduced paratyphoid salmonellae, frequently leading to the persistent shedding of salmonellae in the feces. In many infected birds, invasion beyond the gastrointestinal tract results in Salmonella multiplication in reticuloendothelial tissue of the liver :.;:d spleen (Barrow et al., 1987). As the cecum is the main site of �almonel!ae colonization (Hudault et al., 1985), the effect of the studied probiotics was adopted for caecal colonization of S. tuphimurium where reduction in its count significantly happened during the entire period of the experiment (21 days) on using either Pediococcus acidilactici or Saccharomyces boulardii (Table 3). Numerous studies have demonstrated that providing newly hatched chicks with intestinal microflora from adult chickens decreases the incidence of salmonellae cecal colonization (Barnes; 1972 and Barnes et al.; 1980, Snoeyenbos et al. ; 1978, Calcott; 1981, Pivinick et al. ; 1981, Freter, 1983 and Nisbet et al. ; /993). Jeffrey (1998) reported that the use of competitive exclusion products could be of great benefit in reducing colonizationcaused by paratyphoid salmonellae. Blankenship et al.. (l993 ) reported that salmonellae prevalence in caeca and in processed carcasses was significantly reduced from41 % in control flocks to 10 % in pro biotic treated flocks. Ghadban et al.. (19 98) mentioned that spray application of probiotic followed by water administration showed clear tendency for elimination of salmonellae from 38.8 % in control group to 9.7 % in treated ones.

Rhodehamel and Hannon (1998) and Brynestad (2002) reported that C. pe1fringe11s is responsible for the rare but severe food borne necrotic enteritis in man ( enteritis necroticans or pig-be I disease which is fatal specially in young and

Egyp1 . .J. 1·c1 ,\'ci. Vol. 37 {2003) T EFFEC OF PEDIOCOCCUSACIDIL ACilCI ... 133 elderly) and its enterotoxin has been shown to be the virulence factor responsible for causing the symptoms of C. perfringens type A food poisoning which is the more common in the industrialised world. In a report . from Public Health Services Laboratory ( 1991) 67% of feed containing ariiihal proteins and 69% from mixtures at the feed factories were infectedwith salmonellae. On the other hand; North and Gorman ( 1990) reported on a public food poisoning in England during 1988-1989 due to consumption of S. enteretides contaminat�d eggs lead to very serious losses for the government and the producers (about 70 mil. Sterling £), due to 90% reduction in egg consumption. Moreover; from March, 1989 until December 1990, more than one million birds were rejected and 1.700.000 infected birds have been killed.

Our obtained results are showing that the use of Pediococcus acidilactici or Saccharomyces boulardii can greatly assist in the control of both C. perfringens and S. (vphimurium colonization in broilers. As infections of domestic poultry with salmonellae are expensive both for the poultry industry and for society as a whole (Gast, 1997), existing a program by using such probiotics with the aim to reduce necrotic enteritis and paratyphoid infections. from broiler flocks and to reduce the significance of animal food as a source of infection to man can not be ignored. Eventually; they can make a valuable contribution to flock health and safety of poultry products as food. This may provide a significant tool for the poultry industry in combating the occurrence of intestinal disease and in reduction of foodborne pathog ens.

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(Received 27/1/2003; accepted 25/5/2003)

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