Use of Lactobacillus johnsonii in broilers challenged with Salmonella sofia

Probiotics may alter gut microflora in poultry and play a role in competitive exclusion (CE) ofSalmonella by the Nurmi concept ( Pivnick and Nurmi, 1982). Competitive exclusion involves oral administration of intestinal microflora derived from healthy salmonella-free adult birds into newly hatched chicks. Establishment of an adult intestinal microflora in newly hatched chicks increases their resistance to colonization by non-host-specific salmonellae.

The use of CE microflora against Salmonella colonization in poultry is proven to be effective (Blankenship et al., 1993, Jin et al., 1998 and Gusils et al., 2003). The most important advantage is that CE products ensure the establishment of a complex intestinal microflora that resists colonization by poultry pathogens, and they are produced as a consortium of bacteria that can coexist as a stable community in the enteric ecosystem (Wagner, 2006). Another factor in the use of lactobacilli to induce CE of Salmonella is that the members of the Lactobacillus family readily utilize lactose in their metabolism. Mannose and lactose may act to inhibit Salmonella attachment via different mechanisms; mannose may interact with mannose-sensitive type-1 fimbrae on the bacterium, lactose on the enhancement of the growth of Lactobacillus, which, in turn, inhibits the growth of pathogens such as Salmonella( Oyofo et al., 1989). The antibacterial effect of Lactobacilli in vitro against Escherichia coli and Salmonella spp. and the bactericidal effect on Salmonella faecalis have been documented (Fuller and Brooker, 1974). The results of Pascual et al. (1999) showed that using the rifampin-resistant L. salvarius CTC2197 (feed additional concentration as 10⁵cfu/gram) prevents Salmonella enteritidis in chickens, and that the pathogen was completely removed from the birds after 21 days.

Salmonella sofia (S. sofia) first came to the attention of the Australian Salmonella Reference Centre in 1979 as a new isolate from chickens. Despite the widespread colonization of chickens by S. sofia, it is not represented in the list of serovars isolated from humans, which indicates that it may be of low virulence to humans (Harrington et al., 1991). Salmonella sofia is ubiquitous amongst Australian chicken flocks but few serious Salmonella food poisoning outbreaks attributed to chicken meat have occurred. In the years 1982 to 1984, S. sofia represented approximately 30% of all salmonella isolations from raw chickens in Australia and isolation from chickens rose to a peak of 49% of all isolates in 1988 (Harrington et al., 1991).

Chickens are known to be very sensitive to Salmonella infections during the first week of life because of delayed development of their intestinal flora. The gastrointestinal tract (GIT) of chickens harbours a microfloral load which is formed immediately after hatching. The mature indigenous microflora forms an important barrier against colonization of potentially pathogenic bacteria, such as Salmonella ( Fuller, 1997). The microflora of the intestinal tract consists of many different species of microorganisms, Lactobacillus, Bifidobacteriumand Bacteroides species being the most predominant groups of microorganisms present in healthy chickens; these constitute about 90% of the flora. Ewing and Cole (1994) reported that the development of the intestinal microbiota commences soon after birth, and the establishment of 'climax conditions’ takes days or weeks depending on environmental conditions. During this process, the composition of the microbiota continuously changes as one group of microbes becomes numerically dominant, only to be supplanted by a new group of organisms, which, in turn, is supplanted. In young chicks, administration of gut microflora has been shown to be effective against several Salmonella species, such as Salmonella typhimurium (Mead, 2000) and Salmonella kedougou (Ferreira et al., 2003). The importance of bacterial metabolites and intestinal microflora composition in controlling pathogenic bacterial infections has been well documented in animal models (Hume et al., 1998 and Bielke et al., 2003). Literature data suggest the importance of early establishment of beneficial bacterial populations in preventing Salmonella colonization using animal models. Based on these principles, a novel probiotic of chicken origin, Lactobacillus johnsonii, was selected for this experiment because of its production of bacteriocin-like inhibitory activities that may be effective in controlling S. sofia infection in broilers.

The bacterial strain used in this experiment was selected using the antagonistic activity assay described by Teo and Tan (2005).

A pure L. Johnsonii isolate was grown in De Man, Rogosa, Sharpe broth (MRS broth) overnight at 39°C and harvested by centrifugation at 4,420×g for 15 min (Induction Drive Centrifugation, Beckman Model J2-21M, Beckman Instruments Inc., Palo Alto, California, USA). It was re-suspended in phosphate-buffered saline (PBS) (pH 7.4) and mixed by constant mechanical stirring (Heidolph MR 3001K stirrer, Heidolph Instruments GmbH & Co., Schwabach, Germany) for 10 min. This pre-mixture of PBS solution was used for oral gavage of chicks. The quantities of MRS broth and pre-mix PBS solution were calculated by the bacterial concentration needed for the experiment. In this study, the concentration of the probiotic candidate L. johnsonii was >1.28 × 10⁹ cfu/mL of BPS solution without bacterial extracellular products.

Each chick in the probiotic treatment group was orally administered 0.5 mL of the highly concentrated culture solution using a crop needle on d 1, and 1 mL on d 3, 7 and 12. Birds in other groups received the same amount of sterile PBS solution on the same day.

The strain of S. sofia was obtained from the Biotechnology Laboratory, RMIT University (Melbourne, VIC, Australia) and maintained in Luria Bertani (LB) broth with 30% (vol/vol) glycerol at −20°C. The strain was made rifampicin resistant as described by Eisenstadt et al. (1994) with some modifications as follows: 1) the gradient plate technique used antibiotic agar containing rifampicin (95% HPLC, R3501-5G, Sigma–Aldrich, Castle Hill, NSW, Australia) at 80 µg/mL; and 2) to more accurately determine the level of resistance to rifampicin, the mutants were each streaked on several plates containing different concentrations of rifampicin, namely, 100, 110 and 120 µg/mL.

The mutant strain was amplified by growth overnight at 39°C in 1,000 mL of LB broth, it was then harvested by centrifugation at 5,000×g for 15 min (Induction Drive Centrifugation, Beckman Model J2-21M, Beckman Instruments Inc., Palo Alto, California, USA), re-suspended in 100 mL (200 mL from second time) of PBS (pH 7.4) to a smaller final volume to produce a highly concentrated culture without bacterial extracellular products. The re-suspended solution was mixed by constant mechanical stirring (Heidolph MR 3001K stirrer, Heidolph Instruments GmbH & Co., Schwabach, Germany) for 15 min. This challenge pre-mixture of PBS bacterium solution was administered by oral gavage.

A total of 288 one-day-old male Cobb broiler chickens vaccinated against Marek?s disease, infectious bronchitis, and Newcastle disease were obtained from a local hatchery (Baiada hatchery, Kootingal, NSW, Australia) and assigned to six dietary treatments, each with six replicates, 8 chickens per replicate. Chickens were reared in multi-tiered brooder cages placed in a climate-controlled room. The basal diets (starter and finisher) were based on corn, wheat and soybean meal as shown in (Table 1) and provided as pellets. The six treatments included in this trial were: 1) negative control (NC−), non-probiotic and unchallenged with S. sofia; 2) positive control (PC−), as feed additional zinc-bacitracin (50 mg/kg) provided, non-probiotic and unchallenged with S. sofia; 3) probiotic control (Pro−), as probiotic inoculated and unchallenged with S. sofia; 4) negative challenged (NC+), as non-probiotic, non-antibiotic and challenged with S. sofia; 5) positive challenged (PC+), as non-probiotic inoculated, feed additional zinc-bacitracin (50 mg/kg) provided and challenged with S. sofia; and 6) probiotic challenged (Pro+), as probiotic inoculated and challenged with S. sofia.

Clinical symptoms were observed in the birds after the second time they were challenged with S. sofia in the NC+ group, but not detected in other treatment groups (Fig. 2). Within a few hours of the second inoculation, chicks were showing obvious clinical symptoms; they huddled in the corners of the cage, showing somnolence, loss of appetite and inhibition in drinking. They were generally depressed and reluctant to move, A thin, yellowish diarrhoea appeared with some chicks. The clinical symptoms were transient, however, and these behavioural changes were pronounced for about 8 h, then disappeared gradually, recovery being complete within 24 h. None of the chicks died during the 48 h after inoculation. The mortality rate for these chickens was less than 8.3% (4/48) compared with the NC group where it reached 6.25% (3/48).

Growth, FI and FCR were all depressed during the second week in NC+ treatment compared with the other treatments. However, this trend was not evident in the following weeks. By the end of the 5-week experimental period there was no difference in performance between the challenged and unchallenged groups (Table 2).

The relative weights of the gizzard, duodenum and small intestine were increased in challenged groups compared with unchallenged groups on d 14. No significant change in the weight of any other organ was detected in birds after being challenged with S. sofia ( Table 3).

 

The concentration of acetic acid significantly decreased in the challenged group and the lowest concentration was found in the NC+ treatment in both ileal (P < 0.05) and caecal (P < 0.01) digesta on d 14 ( Table 4). This trend was not detected on d 35. There was also no significant difference in the concentration of formic, propionic and butyric acids between the challenged and unchallenged groups on d 14 and 35 in the ileum and caecum. Lactic acid was not detected in the ileal digesta on d 35.

No differences in total anaerobes and lactic acid bacteria numbers in the ileal and caecal contents were found between the treatment and control groups (Table 5). The number of Enterobacteria found in the ileum and caecum on d 14 was higher in the challenged groups than in the unchallenged groups. The number of Clostridium perfringens in the caecal contents of unchallenged groups (NC−, 6.29; PC−, 6.14; Pro−, 5.99) was lower (P < 0.05) than those in the challenged groups (NC+, 7.86; PC+, 7.38; Pro+, 8.15) on d 14. This trend was also found on d 35, but the negative control (5.13) was higher (P < 0.05) than the positive (4.17) and probiotic (4.44) in unchallenged control groups. Furthermore, the number of lactobacilli was higher (P < 0.05) in the probiotic control and probiotic challenged groups on d 35.

The salmonella counts from the ileum and caeca on sampling days are shown at Table 5. Three successive inoculations with 1 × 10⁷ cfu of S. sofia established a high level of infection in the ileum and caeca, which was detectable from d 14. Chickens that received a high dose of S. sofia inoculation appeared to establish the most stable infection, with the number of salmonella reaching around 6.11 cfu/g in the ileum and 8.97 cfu/g in the caeca. The number of S. sofia in the ileal and caecal digesta was significantly (P < 0.01) decreased in PC+ and Pro+ groups compared with NC+ treatment on d 14. No S. sofia was detected in the digesta from the ileum and caeca on d 35.

At each sampling, chickens were taken out from both the challenge group and control groups. The control chickens were free of Salmonella throughout the experiments, verified by LB agar both with or without rifampicin and by enrichments from spleen, liver, ileal digesta and caecal digesta ( Table 6). However, by using enrichment it was found that the spleen and liver became positive for salmonella, detected from sampling d 14 for most chickens in challenge groups, but towards the end of the experiment fewer positive samples were found from the organs. It was also shown that the ileum had a low level of salmonella present for most chickens on sampling d 14.

In the ileum, villus height, crypt depth and muscle depth in the challenged treatments did not differ from the control groups (Table 7). In both unchallenged and challenged treatment groups, the villus:crypt ratio ranged from 7.13 to 7.68 (d 14) and 5.87 to 6.22, respectively, not significantly different among treatments.