The Influence of Probiotic Bacteria (Bacillus Toyoi) on Livability and Performance of Young Meat-Type Turkeys

ABSTRACT

The aim of study was to determine the impact of a probiotic preparation containing live bacteria Bacillus cereus var. toyoi on the performance of turkeys in order to define the effective dose of the preparation and to evaluate its influence on poults' livability in the first month of life. Three hundred and sixty B.U.T. 9 male turkey were divided into three groups. The control group (I) was fed a diet with no probiotic additive, and groups II and III were given a probiotic product containing 0.2x10⁹ and 1x10⁹ B. toyoi CFU/kg of feed respectively. Body weight was recorded in weeks 1, 6, and 18. Livability, feed conversion ratio, and European Efficiency Factor (EEF) were estimated. In experiment II, the impact of a standardized probiotic dose on livability and performance parameters of B.U.T. 9 turkey poults were evaluated. Two groups were selected within each sex: control (C), feed without probiotic, and experimental (T), which feed contained the probiotic preparation. Mortality and body weight were registered. The better livability and performance of the turkeys fed probiotic justifies the use of bacteria Bacillus cereus var. toyoi to improve performance results and livability during the first weeks of the turkeys' life. The bacterium strain concentration of 1.0x10⁹ CFU/kg of feed can be assumed as optimal.

Keywords:
Bacillus toyoi; poults mortality; probiotics; turkeys

INTRODUCTION

The mortality of turkey poults under modern farming conditions practice causes economic losses for farmers. Some of the factors that may influence poult mortality are breeder flock age, genetic strain, as well as the hatching process, characteristics of the farm on which birds were reared, and even temperature during transportation of the poults to the farm (Carver et al., 2000; 2002). The critical period, when the highest mortality occurs, corresponds to the first 4-6 weeks of life in young meat-type turkeys, during which poults begin to present wattle growth. During this period, poults are highly sensitive to adverse environmental conditions and to nutrient deficiencies. Considering also that many farmers rear turkey poults with the purpose of sale only to the end of critical period, the first weeks of rearing determine production profitability. Therefore, methods to improve poult live ability are needed.

In the past, due to the progressive intensification of animal husbandry, in-feed antibiotics were the main group of preparations used to stabilize the specific composition of the intestinal microbiota and to protect animals from infections, therefore functioning as growth promoters. However, due to potential risk of bacterial resistance spread and other negative consequences for consumers, the use of in-feed antibiotics in the feed of monogastric animals was banned (Regulation EC No 1831/2003). Currently, there is an increasing body of research for alternative additives with similar effects, such as probiotics (Anadón et al., 2006; Bomba et al., 2002). Their mode of action consists of the competitive exclusion of pathogenic microflora; production of organic acids, mainly lactic acid (lowering pH of the intestinal content); neutralization of enterotoxins produced by pathogenic strains of E. coli; and production of antibiotic-like substances against pathogenic microbiota, such as nizine, reuterin, acidophilin and acidolin (Jin et al., 1997; Selvet et al., 2012).

The ultimate goal of the commercial application of probiotics is to increase economic profitability as a result of a demonstrable improvement of animal performance, reduction of morbidity and mortality in the animals and of human pathogenic bacterial populations (Flint & Garner, 2009). Spore-forming bacteria, especially of the genus Bacillus, have been shown to prevent gastrointestinal diseases in animals and humans. They have many applications, such as treating immunosuppressive and antibiotic-associated diarrhea. A scientific study also showed the tolerance of probiotic Bacillus spp. strains to different physiological conditions, as well as the inhibition of Salmonella Typhimurium (Menconi et al., 2013).

Bacillus cereus var. toyoi (B. toyoi) is a strain isolated from the soil. It does not produce diarrheal or emetic enterotoxins and it is not a genetically modified organism. Toyocerin(r) is a preparation of viable spores of B. cereus var. toyoi is used as a probiotic microorganisms in the feed to promote growth and digestive health (Wiliams et al., 2009).

Two experiments were conducted. The first one experiment (I) was carried out under experimental conditions, and aimed at evaluating the effects of a probiotic additive containing live bacteria Bacillus cereus var. toyoi on the performance of meat-type turkeys and determining its most effective dose. The second study (II) was performed under standard farming and production conditions, to evaluate the impact of the dietary addition on turkey poult health during the first four weeks of their life.

MATERIAL AND METHODS

The farm, where both experiments were conducted, remained under constant supervision and care of a veterinarian. The causes of deaths and culling during the rearing period were routinely analyzed for possible infectious diseases. The feedstuffs used for the formulation of both the control and the probiotic-containing feeds derived from the same batches. Feeds were analyzed for basic nutrients contents, and metabolizable energy was calculated according to the equation provided in the European Tables (1989). The mash feed containing viable Bacillus cereus toyoi was analyzed for the active ingredient in the German laboratory Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit (Oberschleiaheim, Germany).

Experiment I

In total, 360 one-d-old B.U.T. 9 medium weight turkey males were individually identified and divided into three experimental groups, with four replicates each. The birds were kept in a closed house on deep litter, using typical intensive farming microclimatic conditions. Birds were fed ad libitum. Group I, considered as the control group (C), was fed a standard balanced basal diet with no in-feed antibiotic growth promoters, but with the inclusion of a coccidiostatic drug. Groups II and III were fed the basal diet containing different levels of probiotic additive inclusion, of 0.2x10⁹ and 1x10⁹ Bacillus toyoi CFU/kg of feed in groups II and III, respectively (Table 1).The probiotic was added to the basal feed in the mixer, together with the main ingredients.

Table 1
Experimental feeds in experiment I

Group I	Basal feed with no antibiotic growth promoter (control group)
Group II	Basal feed + 0.2x109 B. toyoi CFU/kg of feed
Group III	Basal feed + 1x109 B. toyoi CFU/kg of feed

• (CFU) - Colony Forming Units

The basal diet was formulated for five different rearing phases (Table 2), according to the recommendations of the NRC (1994).

Table 2
Nutritional value of mixture (in 1kg) used in turkey feeding

Prestarter (wk 0-3)	Starter (wk 4-6)	Grower 1 (wk 7-9)	Grower 2 (wk 10-12)	Grower 3 (wk 13-18)
Metabolizable energy (kcal/kg)	2839	2905	2980	3054	3142
Crude protein (%)	26.7	25.0	22.8	21.2	18.9
Coccidiostatic (Monensin, mg)	100	100	100	80	-

Metabolizable energy and crude protein levels of the experimental diets, according to feeding phase, are shown in Table 2. Birds were weighed at the beginning of the experiment (1-day-old poults), and with 6 and 18 weeks of age. Feed intake and livability were recorded. Feed conversion ratio and European Efficiency Factor (EEF) were calculated. The formula used to calculate the EEF was: (final bird weight, kg × livability, %)/(age, days × feed conversion ratio × 100).

Experiment II

A flock of 12,000 B.U.T. 9 turkey poults of both sexes at 1:1 ratio reared up to slaughter was used. Birds were distributed into 24 pens of 500 birds each. Birds ere submitted to same management and feeding practices as those in experiment I. Based on the results of Experiment I, the dietary inclusion level of 1x10⁹ Bacillus cereus var. toyoi CFU/kg of feed was determined as the effective dose of the probiotic product. Birds of each sex were divided in two groups. The control group (C) was fed a basal diet with no the addition of in-feed antibiotics or other growth promoters, and containing a coccidiostatic drug. Experimental group (T) was fed the basal feed with the inclusion of the probiotic product. Birds were evaluated up to 4 weeks. The number of dead and culled birds, as well as body weight, were recorded during this period.

The data from both experiments were analyzed using statistical package SPSS 12.0PL (SPSS, 2003) by single factor analysis of variance. Means were compared by Duncan's test. The non-parametric c² test was used to analyze mortality data of both experiments.

RESULTS

Experiment I

The main part of this experiment was published (Grela et al., 2009). In this paper, only the unpublished results are considered, and they constituted the basis for the second research. Due to large amount of data, the results were divided into two subperiods: the so-called critical period, up to six weeks of age, and from seven weeks of age to the end of the rearing (18 weeks). Table 3 presents the performance results of turkey males in trial I.

Table
3 - Production effects of turkey males in experiment I

Parameter	Groups		SEM	p-value
	I	II	III
Initial body weight (g)	56.9	56.7	56.8	0.080	0.351
Body weight (g)
Up to week 6	2,226a	2,242a	2,301a	7.97	0.000
weeks 7-18	11,236a	11,425b	11,602c	27.5	0.000
Total	13,462a	13,666b	13,905c	27.8	0.020
Feed conversion ratio
Up to week 6	1.68	1.66	1.61	0.014	0.108
Weeks 7-18	2.80b	2.74a	2.72a	0.012	0.001
Average	2.62c	2.56b	2.53a	0.011	0.000
European Efficiency Factor
Up to week 6	299a	318ab	341b	6.641	0.017
Weeks 7-18	478a	497b	509c	4.195	0.000
Total	379a	409b	427b	6.016	0.020

• Group I: basal feed with no antibiotics (control group), group II: basal feed + 0.2x10⁹ B. toyoi CFU/kg of feed, group III: basal feed + 1x10⁹ B. toyoi CFU/kg of feed

Body weight was significantly different among treatments during both subperiods of the study. The birds from group III, fed with 1x10⁹ B. toyoi CFU/kg feed, were the heaviest. The feed conversion ratio (FCR) of birds in groups II and III was significantly better compared with the control group experiments during both subperiods. Average FCR was 3.4% lower in group III and 2.3% in lower in group II relative to the control group during the entire rearing period. The highest European Efficiency Factor value was obtained in group III, which was fed with 1x10⁹ FCU B. toyoi/kg of feed, reaching 427 points after 18 weeks of rearing. Group II, which was fed with a smaller probiotic, and the control group presented about 4.2% and 11.2% lower FCR relative to group III. It should be noted that a higher European Efficiency Factor value was calculated for group fed the highest probiotic level relative the control group already in week 6.

Table 4 shows livability results. Livability rates were high and not statistically different among all groups. However, the highest value was obtained in group III (97.7%), followed by group II, which livability rate was 96.2% until week 6. It should be noted that after week six turkeys' livability reached 100%.

Table 4
Livability rate of turkey males in experiment I (%)

Time (wk)	Groups		c2 (p-value)
	I	II	III
0-6	92.4	96.2	97.7	0.953
7-18	100	100	100	1.000
Total	92.4	96.2	97.7	0.953

• Group I: basal feed with no antibiotics (control group), group II: basal feed + 0.2x10⁹ B. toyoi CFU/kg of feed, group III: basal feed + 1x10⁹ B. toyoi CFU/kg of feed.

Experiment II

This experiment involved a number of birds more than 30 times higherthan in experiment I. In the control groups, the number of dead and culled birds were not statistically significant between sexes (Table 5). However, when the birds were fed the probiotic additive, this number was lower in females than in males. Statistically significant difference in average number of dead birds per pen in each group was demonstrated. Non-parametric as well as parametric test confirmed positive effect of Bacillus cereus var. toyoi addition on livability of both sexes of birds.

Table 5
Livability of male and female turkeys in experiment II (%).

Sex	♂♂		♀♀		SEM	p-value
Groups	C	T	C	T
Number of dead and culled birds (average per pen)	25.3c	17.5b	24.8c	13.5a	1.081	0.000
Livability (%)	94.93a	96.50b	95.03a	97.30c	0.216	0.000		c2 (p-value)
Total number of dead and culled birds	152	105	149	81	-	0.000

• Group I: basal feed with no antibiotics (control group), group II: basal feed + 0.2x10⁹ B. toyoi CFU/kg of feed, group III: basal feed + 1x10⁹ B. toyoi CFU/kg of feed.

Table 6 shows the body weight of male and female turkeys measured in II experiment. After four weeks of rearing, turkeys fed the probiotic product tended to be slightly heavier, regardless of sex; however, these differences were not statistically significant.

Table 6
- Body weight of male and female turkeys measured at the beginning and in week 4 in experiment II (g).

'Time (wk)	Groups		SEM	p-value
	♂♂		♀♀
	C	T	C	T
0	55.7	56.0	55.0	55.0	0.013	0.351
4	1148	1204	1024	1079	0.624	0.144

DISCUSSION

The effectiveness of probiotics depends on the type of bacterial strains and concentration of microorganisms added to the feed during their formulation. Toyocerin(r), a preparation containing the bacterial strain Bacillus toyoi, has shown beneficial effects when administered to pigs (Jadamus et al., 2002; Stamati et al., 2006; Papatsiros et al., 2011; Kantas et al., 2014), rabbits (Brzozowski & Strzemecki, 2013), Japanese quails (Homma & Shinohara, 2004), chickens and turkeys (Jeroch et al., 2004; Grela et al., 2009; Novak et al., 2011).

The higher body weight of the turkeys fed the probiotic product detected in experiment I was confirmed by many authors. In the study of Blair et al. (2004), turkeys fed a mixture containing aprobiotic microorganism (Bacillus subtilus) or an antibiotic growth promoter presented higher weight gain during 18 weeks of rearing compared with those fed a basal diet with no additives. Torres-Rodriguez et al. (2007) obtained significantly higher body weight and lower production costs associated with any necessary treatment in female turkeys fed a probiotic (Lactobacillus spp.) additive, particularly when flocks from farms presenting historical poor performance and health. In our study, the body weight difference amounted to about 3% between the control and experimental group fed with the higher probiotic dose and was statistically significant, despite presenting similar health status. In another study (Kozłowski et al., 2014), the addition of a Enterococcus faecium preparation to the feed significantly increased the body weight of turkeys in 3.3 to 4.4%.

Salmonella infection is considered the main cause of economic losses in poultry production. It has been shown that the administration of a probiotic product, also in combination with an organic acid, considerably reduced the number of Salmonella spp excreted by turkeys (Vincente et al., 2007). It was proven that a mixture containing 11 probiotic strains of lactic-acid bacteria maybe effective for the prevention of infections caused by Salmonella enterica in chicks and turkey poults (Menconi et al., 2011). Salmonella entericais the bacterial zoonotic factor most frequently transferred from poultry product to humans. However, the use of probiotic Bacillus bacteria reduces Salmonella colonization, as well as increases body weight gain in chicks and poults (Shivaramaiah et al., 2011). The lower mortality observed in the groups fed with probiotic additive in the present study indicates that Bacillus cereus var. toyoi in hibited Salmonella sppreplication and positively influenced bird's health. In the experiments of Vilá et al. (2009), the dietary addition of Toyocerin also reduced the prevalence of Salmonella in poultry and, in the case of broiler chickens, it also significantly improved performance at slaughter age.

Livability of turkeys is lowest during first weeks of their rearing. One of the reasons for this early mortality may be insufficient morphological and functional development of the gastrointestinal tract (GIT). However, GIT development can be stimulated by appropriate nutritional supplements (Lecewicz et al., 2008). Studies indicate that administration of probiotics can accelerate the maturation of turkey poults'GIT, suggesting the possibility of using probiotics as growth promoters, including in organic production systems due to their natural origin (Moyle et al., 2012). The results obtained by Timmerman et al. (2006) suggest that probiotics may be useful to reduce early turkey early mortality. Mortality in broilers was reduced by the addition of chicken-specific probiotics (consisting of seven Lactobacillus species) to the drinking water, and was associated with a 4.87% increase in final body weight. Amer & Khan (2012) observed significantly lower mortality in a group of chickens fed a diet with probiotic supplementation (Lactobacillus acidophilus, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus oryzae) in comparison with those fed an antibiotic or a control diet. Using two types of lactic acid probiotic bacteria (Pediococcus spp) in feeds, Brzoska et al. (2010) detected significantly lower mortality in the supplemented broilers relative to the controls. These observations were confirmed by our results; however, the differences observed in the present study were larger (sometimes the double) of the rates obtained in the cited papers. Toyocerin(r) also positively influenced the mortality rate in other animal species, particularly when combined with benzoic acid (Papatsiros et al., 2011).

In conclusion, the results of the present study demonstrate that the preparation containing Bacillus cereus var. toyoi probiotic strains can be used to improve turkey performance, but its effectiveness depends on the dose of the preparation. The concentration 1.0x10⁹ CFU/kg feed can be assumed as the optimal dose. Additionally, it was shown that the additive containing Bacillus cereus var. toyoi administered to turkey poults reared under standard intensive rearing conditions significantly improves the livability of birds during the first weeks of their life and maybe recommended as a health and growth promoter for turkeys.

REFERENCES

• Amer MY, Khan SH. A comparison between the effects of a probiotic and an antibiotic on the performance of Desi chickens. Veterinary World 2012;5(3):160-165.
• Anadón A, Martínez-Larrañaga MR, Aranzazu Martínez M. Probiotics for animal nutrition in the European Union. Regulation and safety assessment. Regulatory Toxicology and Pharmacology 2006;45:91-95.
• Blair EC, Allen HM, Brooks SE, Firman JD, Robbins DH, Nishimura K, et al. Effects of Calsporin(r) on turkey performance, carcass yield and nitrogen reduction. International Journal of Poultry Science 2004;3(1):75- 79.
• Bomba A, Nemcová R, Mudroňowa D, Guba P. The possibilites of potentating the efficacy of probiotics. Trends in Food Science & Technology 2002;13:121-126.
• Brzoska F, Pieszka M, Stecka K, Migdal W, Wesierska E, Walczycka M, et al. Effect of Pediococcus spp. in feed instead of antibiotic on broiler chicken body weight, mortality, slaughter traits and meat quality. Annals of Animal Science 2010;10:167-177.
• Brzozowski M, Strzemecki P. Estimation the effectiveness of probiotics as a factor influencing the results of fattening rabbits. Animal Science 2013;52:7-11.
• Carver DK, Fetrow J, Gerig T, Correa MT, Krueger KK, Barnes HJ. Use of statistical modeling to assess risk for early poult mortality in commercial turkey flocks. Journal of Applied Poultry Research 2000;9:303-318.
• Carver DK, Fetrow J, Gerig T, Krueger KK, Barnes HJ. Hatchery and transportation factors associated with early poult mortality in commercial turkey flocks. Poultry Science 2002;81:1818-1825.
• Flint JF, Garner MR. Feeding beneficial bacteria: A natural solution for increasing efficiency and decreasing pathogens in animal agriculture1. Journal of Applied Poultry Research2009;18:367-378.
• Grela ER, Brodacki A, Batkowska J, Matras J. Influence of a probiotic of Bacillus toyoi strain on performance of growing turkey poults. Archiv für Geflügelkunde 2009;73(3):160-166.
• Homma H, Shinohara T. Effects of probiotic Bacillus cereus toyoi on abdominal fat accumulation in the Japanese quail (Coturnix japonica). Animal Science Journal 2004;75(1):37-41.
• Jadamus A, Vahjen W, Schäfer K, Simon O. Influence of the probiotic strain Bacillus cereus var toyoi on the development of enterobacterial growth and selected parametres of bacterial metabolism in digesta samples of piglets. Journal of Animal Physiology and Animal Nutrition 2002;86:42-54.
• Jeroch H, Strobel E, Zachman R. Efectiveness of the probiotic bacterium Bacillus cereus toyoi in turkey feeds. Veterinarija ir Zootechnika 2004;28:57-60.
• Jin LZ, Ho YW, Abdullah N, Jalaludin S. Probiotics in poultry: modes of action. World's Poultry Science1997;53:352-368.
• Kantas D, Papatsiros VG, Tassis PD, Giavasis I, Bouki P, Tzika ED. A feed additive containing Bacillus toyonensis (Toyocerin(r)) protects against enteric pathogens in postweaning piglets. Journal of Applied Microbiology 2015;118(3):727-738.
• Kozłowski K, Sobczak A, Jeroh H. Influence of a probiotic preparation on the performance of growing male turkeys. Annales Universitatis Mariae Curie-Skłodowska Lublin - Polonia EE 2014;32(3)1-8.
• Lecewicz A, Jankowski J, Zduńczyk Z, Juśkiewicz J. Wybrane czynniki stymulujące rozwój niektórych części przewodu pokarmowego indyków. Veterinary Medicine - Science & Practice 2008; 64(10):1184-1187.
• Menconi A, Wolfenden AD, Shivaramaiah S, Terraes JC, Urbano T, Kuttel J, et al. Effect of lactic acid bacteria probiotic culture for the treatment of Salmonella enterica serovar Heidelberg in neonatal broiler chickens and turkey poults. Poultry Science2011; 90:561-565.
• Menconi A, Morgan MJ, Pumford NR, Hargis BM, Tellez G. Physiological properties and Salmonella growth inhibition of probiotic Bacillus strains isolated from environmental and poultry sources. International Journal of Bacteriology 2013;1-8.
• Moyle JR, Solis de los Santos F, Huff GR, Huff WE, Rath NC, Farnell M, et al. The probiotic Escherichia coli Nissle 1917 enhances early gastrointestinal maturation in young turkey poults. International Journal of Poultry Science 2012;11(7):445-452.
• NRC - National Research Council. Nutrient requirements of poultry. 9th ed. Washington: National. Academy Press; 1994.
• Novak R, Bogovič Matijašić B, Terčič D, Červek M, Gorjanc G, Holcman A, et al. Effects of two probiotic additives containing Bacillus spores on carcass characteristics, blood lipids and cecal volatile fatty acids in meat type chickens. Journal of Animal Physiology and Animal Nutrition2011;5(4):424-433.
• Papatsiros V, Tassis P, Tzika E, Papaioannou D, Petridou E, Alexopoulos C, et al. Effect of benzoic acid and combination of benzoic acid with a probiotic containing Bacillus Cereus var. toyoi in weaned pig nutrition. Polish Journal of Veterinary Sciences 2011;14(1):117-125.
• Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition [cited 2003 Out 18]. Official Journal 3003;L268:0029-0043. Available from: Available from: http://eur-lex.europa.eu/legal-content/en/ALL/?uri=CELEX:32003R1831
  » http://eur-lex.europa.eu/legal-content/en/ALL/?uri=CELEX:32003R1831
• Selwet M, Galbas M, Dullin P. Impact of probiotic on the number of lactic acid rods forming hydrogen peroxide isolated from porkers and on changes in drug resistance of selected Escherichia Coli isolates. Bulletin of the Veterinary Institute in Pulawy 2012;56:21-25.
• Shivaramaiah S, Pumford NR, Morgan MJ, Wolfenden RE, Wolfenden AD, Torres-Rodríguez A, et al. Evaluation of Bacillus species as potential candidates for direct-fed microbials in commercial poultry. Poultry Science2011;90:1574-1580.
• SPSS 12.0 for Windows. Copyright SPSS 1989-2003. Chicago; 2003.
• Stamati S, Alexopoulos C, Siochu A, Saoulidis K, Kyriakis SC. Probiosis in sows by administration of bacillus toyoi spores during late pregnancy and lactation: effect on their health status/performance and on litter characteristics. International Journal of Probiotics and Prebiotics 2006;1(1):33-40.
• Timmerman HM, Veldman A, Van den Elsen E, Rombouts FM, Beynen AC. Mortality and growth performance of broilers given drinking water supplemented with chicken-specific probiotics. Poultry Science2006;85(8):1383-1388.
• Torres-Rodriguez A, Donoghue AM, Donoghue DJ, Barton JT, Tellez G, Hargis BM. Performance and condemnation rate analysis of commercial turkey flocks treated with a Lactobacillus spp.-based probiotic. Poultry Science2007;86:444-446.
• Vicente J, Higgins S, Bielke L, Tellez G, Donoghue D, Donoghue A, et al. Effect of probiotic culture candidates on Salmonella prevalence in commercial turkey houses. Journal of Applied Poultry Research2007;16:471-476.
• Vilà B, Fontgibell A, Badiola I, Esteve-Garcia E, Jiménez G, Castillo M, et al. Reduction of Salmonella enterica var. Enteritidis colonization and invasion by Bacillus cereus var. toyoi inclusion in poultry feeds. Poultry Science2009;88:975-979.
• Williams LD, Burdock GA, Jiménez G, Castillo M. Literature review on the safety of Toyocerin(r), a non-toxigenic and non-pathogenic Bacillus cereus var. toyoi preparation. Regulatory Toxicology and Pharmacology2009;55(2):236-246.
• WPSA - European table of energy values for poultry feedstuffs. Beekbergen: Spelderholt Centre for Poultry Research; 1989. p.84. Available from: http://www2.svenskaagg.se/attachments/92/1425.pdf
  » http://www2.svenskaagg.se/attachments/92/1425.pdf

Publication Dates

• Publication in this collection
  Oct-Dec 2015

History

• Received
  Sept 2014
• Accepted
  Mar 2015