C. G. Olnood1,
L. L. Mikkelsen1,
M. Choct2
P. A. Iji1
1School of Rural Science and Agriculture, University of New England, Armidale, NSW Australia
2 Australian Poultry CRC, University of New England, Armidale, NSW Australia
A total of 294 one-day old Cobb broiler chickens were used to investigate the effects of four lactobacillus strains on production performance. The chicks were assigned randomly to six groups with 7 replicates of 7 chicks per treatment.
The six dietary treatments were: (i) basal diet (negative control, T1); (ii) basal diet with added Zinc-bacitracin (ZnB, 50 ppm, T2); iii) one of four strains of Lactobacillus (tentatively identified as L. johnsonii, L. crispatus, L. salivarius and unidentified Lactobacillus sp., T3, 4, 5 and 6).
The probiotic strains were selected from 235 lactobacilli isolates based on their in vitro antagonistic effect against Clostridium perfringens and Escherichia coli. Results showed that the addition of probiotic Lactobacillus spp. to the feed did not significantly improve body weight gain, feed intake and feed conversion ratio of broiler chickens raised in cages during the 6-wk experimental period.
I. Introduction
Probiotics is a field of science, medicine and business that is growing rapidly. Probiotics, prebiotics, feed enzymes and organic acids have been seen as potential alternatives to antibiotics (Choct, 2002).
The addition of either pure lactobacillus cultures or mixtures of lactobacilli and other bacteria to broiler diets has produced variable results. Han et al. (1984) and Kim et al. (1988) found an improvement in weight gain and feed conversion ratio from 2 to 6 weeks of age. A consistent improvement in body weight gain of chickens fed a culture of L. sporegenes has also been reported (Mohan-Kumar and Christopher, 1988; Kalbande et al. 1992).
Jin et al. (1998) reported that addition to the feed of a single strain of L. acidophilus or a mixture of lactobacilli from 0 to 6 weeks of age significantly improved body weight gain and FCR of broilers. There have also been several studies in which no positive results were found. Watkins and Kratzer (1984) and Maiolino et al. (1992) did not find any significant difference in the body weight gain of chickens given feed containing host-specific probiotics (KTM, 74/1 and 59) and L. acidophilus and Streptococcus faecium, compared with those given a non-supplemented diet.
Variation in the effects of probiotics on growth performance of broiler chickens may be attributed to differences in the strains of bacteria used as the dietary supplements.
In the present study, four strains of Lactobacillus spp. previously isolated from the chicken gut were selected based on their antagonistic activity against C. perfringens and E. coli, and their effect on growth performance of broiler chickens was investigated.
II. Materials and methods
a) Probiotics strains
Lactobacillus spp. were isolated from the ileum and caeca of broiler chickens as previously described (Vidanarachchi et al. 2006). The isolates were differentiated using Amplified Ribosomal DNA Restriction Analysis (ARDRA) and representative strains were identified by 16 sRNA gene sequencing (Vidanarachchi, 2006). A total of 235 lactobacillus isolates were tested using an antagonistic activity assay as described by Teo and Tan (2005) with some modifications.
The lactobacillus isolates were grown in de Man, Rogosa, and Sharp broth (MRS) (Oxoid, CM0359) under anaerobic conditions at 39˚C for 24 hours. Similarly, the indicator strains, pathogenic strains of C. perfringens and E. coli, were grown in Thioglycllate broth (Oxoid, CM0391). The overnight culture of each lactobacillus isolate was streaked onto the surface of Wilkins-Chalgren anaerobic agar (Oxoid, CM0619) using a sterile cotton swap. After anaerobic incubation at 39˚C for 24 hours, an overnight culture of C. perfringens or E. coli was streaked across the same agar plates bisecting the streak line of the lactobacillus isolate (perpendicularly). The inoculated plate was then incubated under anaerobic conditions at 39˚C for another 24 hours. The antagonistic activity of test organisms on the indicator bacteria was determined by the appearance of clear zones surrounding the junctions of the streak lines. The width of the clear zone was measured and recorded.
b) In vivo experiment
A total of 294 one-day old male Cobb broiler chickens were obtained from Baiada hatchery, Kootingal (Tamworth, NSW) and allocated to 6 treatments of 7 replicates (7 birds each replicate). The basal diets (starter and finisher) were based on corn, wheat and soybean meal and fed as a one-phase mash feed to avoid inactivation of the probiotics.
From results of the antagonistic activity test, four strains of lactobacillus (No 1286 tentatively identified as L. johnsonii, No 709 tentatively identified as L. crispatus, No 697 tentatively identified as L. salivarius and No 461 unidentified Lactobacillus sp.) showing the highest degree of inhibition of the test organisms were selected as probiotic candidates and added to the feed to make up four different treatments. Two control treatments were also included, a negative control, with no additives and a positive control treatment, with the antibiotic, Zinc-bacitracin (ZnB, 50 ppm) added.
The experimental diets with the probiotic candidates were mixed weekly. The individual strains were grown overnight and harvested by centrifugation (4420 x g for 15 min), resuspended in PBS (pH 7.4) and mixed into a premix with the basal diet for 10 minutes using a miniature mixer. This pre-mixture of product with feed (1 kg) was then transferred into a larger mixer (total capacity 300 kg) where the final volume of the weekly feed batch was prepared. The mixer equipment was thoroughly cleaned between the mixing of different treatments by using a vacuum cleaner and a wash diet (basal feed).
c) Analyses and performance measurements
Representative feed samples of each feed batch were tested for bacterial concentrations every week of the experiment. Ten (10) g feed was dissolved in 90 ml of peptone water (Oxoid, CM0009) and 10-fold dilutions were performed in Hungate tubes with 9 ml of peptone water. The numbers of lactic acid bacteria in the feed samples were determined on MRS agar inoculated with 0.1ml of diluted sample and after anaerobic incubation at 39˚C for 48 hours.
Bird performance was measured on a weekly basis by recording the group weight and feed intake for each cage. Mortalities were checked daily and body weight of dead birds was recorded.
Statistical analysis was performed using one-way analysis of variance, and difference between means (StatGraphics Plus version 5.1, Manugistics Inc., Rockville, Maryland, USA).
III. Results and discussion
Of the 235 isolates tested, 91 isolates showed no antagonistic activity against both C. perfringens and E. coli while another 122 isolates showed antagonistic activity against E. coli only. The remaining 22 isolates showed antagonistic activity against both C. perfringens and E. coli (results not shown).
Four lactobacillus strains representing four different species (No 461, 697, 709 and 1286) and showing the strongest antagonistic activity against the indicator organisms, C. perfringens and E. coli, were selected. The antagonistic activity was observed as a truncated clear zone surrounding the intersections of the streak lines of the test and indicator strains (Figure 1). The inhibition zones displayed by the four strains selected were between 3 mm and 15 mm wide (Table 1).
Lactobacilli inhibiting growth of C. perfringens and E. coli (left) and with no growth inhibition with C. perfringens and E. coli (middle and right).
Table 2 shows the biological response of birds in the present study. There was no significant effect on body weight gain, feed intake or feed conversion ratio (FCR) of broiler chickens when the probiotic candidates were added to the feed. Similar results were observed by Huang at al. (2004) who supplemented either L. casei or L. acidophilus with or without cobalt in the diets for broiler chickens. Although no significant improvement of growth performance was observed, all four probiotics used in the current study tended to improve body weight gain of birds compared with the negative control diet.
The concentration of the probiotic candidates in the experimental feed (approximately 106 CFU/g feed) was lower than levels usually recommended as the inclusion rate of commercial probiotic feed additives (around 108 CFU/g). This was due to a limited fermentation capacity for amplification of the probiotic candidates in the current study. It is possible that higher concentrations of the probiotic candidates in the feed may exert a more profound positive response on growth performance, especially if the infection pressure from pathogenic bacteria, such as C. perfringens, is high. This needs to be investigated in future studies.
In conclusion, four lactobacillus probiotic candidates with in-vitro antagonistic activity against pathogenic C. perfringens and E. coli were identified. When supplemented in the feed at 106 CFU/g feed, the probiotics did not significantly improve the growth performance of broiler chickens in a cage-scale experiment. However, there may be other beneficial effects associated with the use of microbial probiotics in animal feeds, such as the competitive exclusion of pathogenic bacteria which may improve gut health (Gusils et al. 1999). This effect of the probiotic candidates will be evaluated in future studies.
Acknowledgements
Authors thank Mark Porter, Barbara Gorham, Shuyu Song, Yumin Bao, Ying Yang, Senghuan Chee, Nicholas Rodgers and Janak Vidanarachchi for their technical assistance.
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from Proceedings of the "19th Australian Poultry Science Symposium", New South Wales, Australia
