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

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 1x109B. 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.


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 The Influence of Probiotic Bacteria (Bacillus Toyoi) on Livability and Performance of Young Meat-Type Turkeys negative consequences for consumers, the use of infeed 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® 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 probioticcontaining 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 0.2x10 9 and 1x10 9 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.
The basal diet was formulated for five different rearing phases (Table 2), according to the recommendations of the NRC (1994).
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.

The Influence of Probiotic Bacteria (Bacillus Toyoi) on Livability and Performance of Young Meat-Type Turkeys
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 1x10 9 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 χ 2 test was used to analyze mortality data of both experiments.

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.
Body weight was significantly different among treatments during both subperiods of the study.The birds from group III, fed with 1x10 9 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 1x10 9 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%.

The Influence of Probiotic Bacteria (Bacillus Toyoi) on Livability and Performance of Young Meat-Type Turkeys
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 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.
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 lacticacid 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  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® 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 1.0x10 9 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.

Table 1 -
Experimental feeds in experiment I

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

Table 3 -
Production effects of turkey males in experiment I Group I: basal feed with no antibiotics (control group), group II: basal feed + 0.2x10 9 B. toyoi CFU/kg of feed, group III: basal feed + 1x10 9 B. toyoi CFU/kg of feed

Influence of Probiotic Bacteria (Bacillus Toyoi) on Livability and Performance of Young Meat-Type Turkeys with
probiotic additive in the present study indicates that Bacillus cereus var.toyoi in hibited Salmonella spp.replication and positively influenced bird's health.In the experiments ofVilá 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.

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