A feed additive containing Bacillus toyonensis (Toyocerin ® ) protects against enteric pathogens in...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12729 This article is protected by copyright. All rights reserved.

Article Type: Original Article

A feed additive containing Bacillus toyonensis in weaning piglets

A feed additive containing Bacillus toyonensis (Toyocerin®) protects against

enteric pathogens in post weaning piglets

D. Kantas1, V.G. Papatsiros2,*, P.D. Tassis3, I. Giavasis4, P. Bouki4, E.D. Tzika3

1Department of Animal Production, Technological Educational Institute of Thessaly, Larissa,

Greece,

2Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, Karditsa,

Greece,

3Clinic of Farm Animals, Faculty of Veterinary Medicine, Aristotle University of

Thessaloniki, Thessaloniki, Greece.

4Food Microbiology and Biotechnology Laboratory, Department of Food Technology,

Technological Educational Institute of Thessaly, Karditsa, Greece

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This article is protected by copyright. All rights reserved.

Correspondence: V.G. Papatsiros, Clinic of Medicine, Faculty of Veterinary Medicine,

University of Thessaly, GR 43100, Karditsa, Greece. Tel.: +30 24410 66012; Fax: +30 24410

66053; E-mail: [email protected]

Abstract

Aims: The present study evaluated the efficacy of a probiotic containing Bacillus toyonensis

spores (Toyocerin®) in post-weaning piglets against enteric pathogens.

Methods and Results: 792 healthy weaning pigs of a commercial farrow-to-finish pig farm

were used. The negative control group fed without Toyocerin® and two experimental groups

fed similar to the negative control group, but supplemented with Toyocerin® at t 500 mg/kg

diet (Toyocerin 500 group) and 1000 mg/kg diet (Toyocerin 1000 group), respectively. No

significant difference (P>0.05) in morbidity and mortality rate between groups was noticed.

The Toyocerin groups showed higher body weight (P<0.05) and lower feed conversion ratio

compared to the negative control group. Diarrhea score was less in both Toyocerin groups

than negative control group (P<0.05). Moreover, the use of Toyocerin® at 1000 mg/kg diet

resulted in higher average daily feed intake compared to other groups (P<0.05), reduction of

some enteric pathogens and increase of the number of lactic acid bacteria.

Conclusions: The use of Toyocerin® in weaning pigs, especially at 1000 mg/kg diet, improved

their health and growth performance.

Significance and Impact of Study: This study shows that a feed addititive containing Bacillus

toyonensis (Toyocerin®) protects against enteric pathogens in post weaning piglets when fed

this additive at a proper dose. The use of Toyocerin® at 1000 mg/kg diet resulted in higher

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average daily feed intake, decrease of some enteric pathogens and higher number of lactic

acid bacteria. The effect of the probiotic in other age groups remains to be established.

Keywords: pig, nutrition, additive, probiotic, Bacillus toyonensis

1. Introduction

During last decades the overuse of antimicrobials in farm animals resulted in an increasing

public health scepticism regarding the development of resistant pathogenic bacterial strains

(Budino et al. 2005) and residual contamination of the food chain (Chen et al. 2005; Roselli

et al. 2005). Due to the European Union (EU)-wide ban (Regulation 1831/2003/EC) on the

use of antibiotics as growth promoters in animal feed in 2006 (Chen et al. 2005), there is an

urgent and increasing interest for antibiotic alternatives in feed. Up to now, various

compounds have been used as in-feed alternatives for improving health and growth

performance, such as probiotics, organic acids etc (Chen et al. 2005; Bomba et al. 2006;

Papatsiros et al. 2009; 2011a,b; Papatsiros and Bilinis 2012). Probiotics have been defined as

“a preparation or a product containing viable, defined microorganisms in sufficient number,

which alter the microbiota (by implantation or colonisation) in a compartment of the host,

and by that exert beneficial health effects on the host” (Schrezenmeir and de Vrese 2001).

The most known characteristics of probiotics are the following: capacity to adhere to

intestinal mucosa and inhibit pathogen adhesion, ability to transiently colonise and

proliferate in the intestine, competition for limited nutrients, inhibition of epithelial invasion

and production of antimicrobial substances, prevention of some intestinal clinical signs such

https://www.researchgate.net/publication/240298781_Effect_of_probiotic_and_prebiotic_inclusion_in_weaned_piglet_diets_on_structure_and_ultra-structure_of_small_intestine?el=1_x_8&enrichId=rgreq-eb31bd58-dc9d-4c02-ba9b-26b5c083c9d2&enrichSource=Y292ZXJQYWdlOzI2OTYzNTkyMDtBUzoxNzUyNjQ5NDAxNzUzNjBAMTQxODc5NzgyNjE3MQ==

https://www.researchgate.net/publication/264153673_Effects_of_Dietary_Probiotic_on_Growth_Performance_Nutrients_Digestibility_Blood_Characteristics_and_Fecal_Noxious_Gas_Content_in_Growing_Pigs?el=1_x_8&enrichId=rgreq-eb31bd58-dc9d-4c02-ba9b-26b5c083c9d2&enrichSource=Y292ZXJQYWdlOzI2OTYzNTkyMDtBUzoxNzUyNjQ5NDAxNzUzNjBAMTQxODc5NzgyNjE3MQ==

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as diarrhea and stimulation of immunity (Rolfe 2000; Teitelbaum and Walker 2002;

Lodemann et al. 2008).

Weaning is a traumatic and stressful event-experience for piglets (Whittemore and

Green 2006), affecting locally their gut immune status and gut microbiota (Hampson et al.

1985; Barnett et al. 1989) and having consequences on their physical, nutritional (Leibbrandt

et al. 1975; Stanton and Mueller 1976), immunological (Blecha et al. 1985), and behavioral

status (Pajor et al. 1991). The stressful conditions of weaning cause mainly changes in the

composition and/or activity of the gut microbiota. The gut microbiota has been shown to be

involved in protection against a variety of pathogens including Escherichia coli (E. coli),

Salmonella spp., Campylobacter, Clostridium spp., and Rotavirus. The weaning period as a

stress-inducing event can alter that balance and eventually result in reduction of lactobacilli

population in the gastrointestinal tract, which may concomitantly allow the multiplication of

microorganisms e.g. enterotoxigenic E. coli strains (ETEC) and rotavirus; and cause the

Postweaning Diarrhea Syndrome (PWDS) (Bertschinger and Fairbrother 1999).

The balance of young pig’s intestinal flora is crucial for effective digestion and

maximal absorption of nutrients, as well as for adequate body’s resistance against infectious

diseases (Melin et al. 2004). The development of probiotics for farm animals is based on the

knowledge that the gut microbiota is involved in resistance to disease (Tannock 1997; 2001).

Probiotics should lead to beneficial effects for the host animal due to an improvement of the

intestinal microbial balance or of the properties of the indigenous micro-flora, modifying the

ecology of the intestine in a beneficial manner for the host, as well as stimulating the growth

performance and the health of the host (Fuller 1989; Havernaar et al. 1992; Breves et al.

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2000; Simon et al. 2003). Other mechanisms by probiotics reported their ability to improve

intestinal health, including stimulation of immunity, competition for limited nutrients,

inhibition of epithelial and mucosal adherence, inhibition of epithelial invasion and

production of antimicrobial substances (Rolfe 2000; Macfarlane et al. 2008; Delia et al.

2012).

Toyocerin® is a feed additive based on a strain originally defined as Bacillus cereus

and now reassigned to Bacillus toyonensis (EFSA 2014). Among the different spore-forming

probiotic strains, Bacillus cereus var. toyoi (strain BCT-7112T) has been in use since 1975

when it was officially approved by the Japanese Ministry of Agriculture and Forestry as the

commercial preparation Toyocerin®. The spores of BCT-7112T have been used in animal

nutrition for swine, poultry, cattle, rabbits and aquaculture for over thirty years in a wide

range of countries around the world. In the European Community, Toyocerin® was

authorized for the first time by the European Commission in 1994 for use in swine, and it

became the first microorganism authorized as a feed additive in the European Union, and

subsequently it was authorized also for use in poultry, cattle and rabbits. The almost

complete genome sequence of the strain was used for studies of pairwise genome

comparisons. Altogether, the results indicated that strain BCT-7112T did represent a new

species for which the name Bacillus toyonensis sp. nov. is proposed, with the type strain

being BCT-7112T (=CECT 876T; =NCIMB 14858T) (Jiménez et al. 2013a,b).

Bacillus toyonensis is a naturally occurring, non-toxigenic and non-pathogenic strain

of Bacillus cereus and it has been proven safe, as it does not cause any adverse effects in

rabbits, pigs, chickens, turkeys and cattle at doses ranging from 8.5 x 107 to 4 x 109spores/kg

BW/day for durations of 2 weeks to 18 months (Williams et al. 2009; Jiménez et al. 2013a,b;

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EFSA 2012, 2014). The use of a probiotic containing Bacillus toyonensis in sows and their

litters has been proven beneficial on litter health and performance characteristics (Taras et

al. 2005; Stamati et al. 2006), as well as on the reduction of incidence and severity of PWDS

(Kyriakis et al. 2003; Papatsiros et al. 2011a).

The aim of this trial was to evaluate the efficacy of a feed additive probiotic

containing Bacillus toyonensis spores (Toyocerin®) in post-weaning piglets against enteric

pathogens.

2. Materials and Methods

2.1. Trial farm / Management and housing of the experimental animals

The current study was carried out on a commercial farrow-to-finish pig farm with a

capacity of 300 sows (TOPIGS®) hybrids under production, with its own feed mill, located at

Western Greece. During the trial, 792 healthy female and castrated male weaning pigs

[TOPIGS® Hybrids: father line: Talent® (Duroc), mother line: Topigs 40® (Large White x

Landrace)] were used. Trial day 0 was the day of weaning (24 ± 3 days). The piglets were

allotted equally according to the body weight and gender at random to 36 pens (22 piglets

per pen) at flat-deck batteries for piglets in a climate-controlled post-weaning stable. In each

hall there were 6 pens and two pens shared one feeder (a circular automated feeder was

used between two pens, with one opening per pen, placed at the separating fence of each

pen-pair). In this way each feeder served two pens and in each hall there were 3

experimental units, one per treatment. Each treatment was consisting of 264 weaned piglets

placed in 12 pens and forming 6 replicates concerning the feed intake.

https://www.researchgate.net/publication/51085227_Effect_of_benzoic_acid_and_combination_of_benzoic_acid_with_probiotic_containing_Bacillus_cereus_var_Toyoi_in_weaned_pig_nutrition?el=1_x_8&enrichId=rgreq-eb31bd58-dc9d-4c02-ba9b-26b5c083c9d2&enrichSource=Y292ZXJQYWdlOzI2OTYzNTkyMDtBUzoxNzUyNjQ5NDAxNzUzNjBAMTQxODc5NzgyNjE3MQ==

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Housing facilities had fully automated temperature and humidity control system, as

well as automated feeding. For the trial needs, feed was provided ad libitum by hand in

order to monitor feed consumption.

Breeding animals were vaccinated against Aujeszky’s disease, swine influenza,

parvovirus infection, erysipelas, atrophic rhinitis and E. coli. Piglets were also vaccinated

against enzootic pneumonia. For the control of endo/ectoparasites, all sows received a

single ivermectin injection 14 days prior each farrowing. Ivermectin was also applied to

boars twice a year.

The quantity of feed per pen was given as equally as possible, though feed

consumption was ad libidum. Every batch of feed was prepared about 3 weeks prior to its

use and three samples from every feed preparation were taken for protein, calcium and

phosphorus assessment (AOAC 1990). Well-balanced weaner feed consisted of the same raw

materials (maize, soybean meal, vegetable protein, fish meal, whey powder and soya oil) at the

same inclusion levels for all animal groups and was administrated at random order between

pens. Mineral feeds for weaning pigs were premixes containing vitamins, trace elements,

macro elements, lysine, methionine, threonine, tryptophan and antioxidants, added to ensure

the recommended feed balance (NCR 1998).

2.2. Experimental material

Toyocerin® 109 Premixture (RUBINUM S.A. / Spain) is a preparation containing 1 x 109

viable spores of Bacillus toyonensis cfu/g of product (registered strain in the EU under Annex

II in 1994) (Kozasa 1986), which is officially listed in Annex III of the Commission Regulation

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(EC) (no. 256/2002, O.J. No. L41). The batches nos. ETBL-2748P and ETBL-2749P were used

for the preparation of experimental diets in this study.

2.3. Experimental procedure

The experimental protocol of this efficacy study has been prepared in accordance with The

Technical Guidance on tolerance and efficacy studies in target animals prepared by EFSA

FEEDAP (EFSA, 2011).

The present study was performed under license for experimenting on animals from

the local Veterinary Administration Office (County Aitoloakarnanias Veterinary State

Authority, code: EL010016).

This clinical study was single blinded and performed according to the Code of

Practice for the Conduct of Clinical trials for Veterinary Medical Products (EMEA 2001). The

animals were maintained in accordance with National and European animal Welfare

requirements (OECD 1998; EMEA 1998; FVE 2002).

Three treatment diets were imposed to weaning pigs from 26th to 70th day of age

(weaning stage). The control group was fed typical weaners feed without Toyocerin®109

Premixture. The other two treatment groups were fed the control group feed with the

supplementation of Toyocerin®109 Premixture at the dose levels of 500 mg/kg diet and 1000

mg/kg diet, respectively. Details about experimental groups are presented in Table 1.

The feeding trial started at weaning (approx. 26 days of age) was completed at the

end of the weaning (nursery) stage (approx. 70 days of age). The experimental period was

divided into two periods; a starter period of two weeks (0 -14 d post-weaning) and a

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subsequent grower period of four weeks (15-42d post-weaning), respectively. Piglets were

fed a starter diet during the first two weeks after weaning and a grower diet during weeks 3

to 6 post-weaning. The starter diet had already been provided to the piglets from day 7 of

their life in the farrowing compartment. All diets did not contain any medical substance or

antibiotic/growth promoter before the onset of the trial (suckling period). Other than macro

elements, trace elements, vitamins, amino acids and phytase, the basal mixture contained

no further additives. After weaning, Toyocerin® was added to treatments Toyocerin 500 and

Toyocerin 1000 feed as it is noted in Table 1.

The experimental diets were manufactured in the farm’s private feed processing

factory. To prevent cross contamination with Bacillus toyonensis spores, the control group

diets were manufactured first, followed by Toyocerin 500 group and last the Toyocerin 1000

group diets. All diets were made and administered to the test animals in mash form. Each

batch of the feed was bagged separately in bags, which were identified with a color label. All

bags were stored in a cool (<20oC) and dry place in the feed factory until used. Among the

three different preparations, at least two mixtures of independent feed for weaners (free of

any antimicrobial, probiotic or acidifier) were made in the feed mill.

Diets were analyzed for moisture, crude protein, starch, crude fiber, crude fat and

ash. The analysis for content of the active substance Bacillus toyonensis was confirmed

before starting the trial by RUBINUM, S.A. - Animal Health (Rubí, Spain). The feed was

analyzed using enumeration of viable spores (microbiological spread plate method) and

concerning the chemical composition and Bacillus toyonensis inclusion level, before it was

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fed to the animals. The results of the Bacillus toyonensis inclusion level in the experimental

diets are shown in Table 1.

In order to reduce the risk of spore cross-contamination, the controls and the treated

animals were kept in separate rooms under the same temperature and humidity conditions

within the building. At all times, pigs were maintained in their respective treatment groups.

Control pigs were allocated a separate set of cleaning (clothing, boots and shovels) and

feeding (scoops, barrows) equipment. Risk was further reduced by employing a different

member of staff to manage the control animals.

2.4. Parameters and Records

Each pig involved in the trial was ear tagged. The animal performance (e.g. body

weight, weight gain, feed intake and feed conversion ratio) and health status parameters

including faecal score and microbial faecal investigations were recorded in this study.

2.4.1. Performance

Growth performance data including body weight (BW), average daily gain [ADG (kg)],

average daily feed intake [ADFI (kg)] and feed conversion ratio (FCR) were recorded. On the

first day of the experiment and at the end of each following week average body weight per

pen as well as the amount of feed remaining in the feeder after each feeding was monitored.

The average weight gain per pen was calculated using the average body weight at the end of

each period minus the average BW at the beginning of each period. Feed consumption per

piglet was calculated as the total amount of feed per pen and period (minus feed losses)

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divided by the number of piglets per pen. The feed to gain ratio for each pen was

determined by dividing the feed consumption and the total individual body weight gain per

week or period.

2.4.2. Health status

All piglets were observed daily for any abnormalities, abnormal behaviour (e.g.

neurological signs, cannibalism), and clinical signs of illness (e.g. pyrexia, anorexia, diarrhea,

dehydration). Injectable non-antibiotic medical treatments (e.g. non-steroidal anti-

inflammatory drugs), when needed, were applied and recorded. Health status of the piglets

was monitored twice daily throughout the experiment.

Morbidity and mortality rates were calculated per week and for the total trial period.

The calculation of the morbidity rate based on the daily recording of any case of abnormal

clinical signs such as reduced appetite evident by consumption of less than half of the daily

quantity of feed for at least two days or fever, as assessed by rectal temperature higher than

39.4ºC at two consecutive recordings. Additionally the appearance of faeces was ranked

daily according to the following categories: 0 = no diarrhea; 1 = semisolid, no blood; 2 =

watery stool, runs through the floor slats, no blood; 3 = profuse diarrhea with or without

blood. Diarrhea score (DS) was calculated by adding the daily results per pen. Then all partial

scores of scouring piglets were added per pen and the sum was divided by the days of

monitoring. In this way a general diarrhea score per pen was calculated for the period.

For the calculation of mortality rates, the date, the age and the possible cause of

death for each dead trial animal was recorded (full post-mortem examination was

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performed in the laboratory supported by histology or microbiological tests, when the cause

of death was not obvious or clarified by necropsy). Animals which were removed from the

trial, the date of removal and reason for exclusion were registered.

2.4.3. Faecal microbiota

2.4.3.1. Collection of faeces

Faeces were sampled from piglets from four pens per treatment at the end of the

starter-feeding (40th day of age) and grower-feeding period (70th day of age).

The faeces samples obtained from the experiment, were collected in 2 groups (12

samples each) with the use of sterile beakers and aseptic technique, and were immediately

refrigerated at 4 °C and at the same day transported through a cooler box to the Laboratory

of Food Microbiology and Biotechnology of the Technological Educational Institute (T.E.I.) of

Thessaly where the samples were kept under refrigeration (4 °C) overnight. Analysis was

performed the day after the delivery of the samples in the Laboratory.

2.4.3.2. Enumeration of intestinal micro flora

Enumeration of representative species of the microbiota of the faecal samples was

performed, in order to investigate potential beneficial biological responses of Bacillus

toyonensis (Toyocerin®) on the intestinal microbiota (i.e. reduction of Enterobacteriacae and

pathogenic bacteria and stimulation or improved survival of Bifidobacteria). Detection of

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Salmonella spp. was carried out according to ISO 6579:2002 and detection of Listeria

monocytogenes according to ISO11290-1:1996.

Enumeration of Enterobacteriaceae was performed by incubation of appropriately

diluted samples in Violet Red Bile Glucose agar (Neogen, USA) at 37oC for 24 h. The same

technique was used for enumeration of E.coli and E.coli O157:H7 in SMAC-BCIG agar (Lab-M,

UK). After incubation of inoculated SMAC-BCIG agar at 37oC, blue/purple colored colonies

indicated the presence of E.coli, while colorless colonies indicated the presence of

presumptive E.coli O157:H7.

Clostridium perfringens was determined by anaerobic incubation at 37 oC for 48-72 h

of samples inoculated in Tryptose Sulfite Cycloserine Agar (Lab-M, UK) with addition of D-

cycloserine (Lab-M, UK), while Clostridium spp. were determined by anaerobic incubation at

37oC for 48-72 h of samples inoculated in Reinforced Clostridium agar (Lab-M, UK). For

anaerobic incubation an anaerobic jar and a CO2 producing sachet (Anaerocult A, Merck-

Hellas) were used.

Campylobacter species (primarily C. jejuni) were enumerated after spreading diluted

samples on Camplylobacter blood-free Selective Medium (Lab-M, UK) after addition of

Cefoperazone / Amphotericin selective supplement (Lab-M, UK) in a microaerophilic

atmosphere consisting of approximately 5-6% oxygen, 10% carbon dioxide and 84-85%

nitrogen using an anaerobic jar and Anaerocult C (Merck, Hellas) for 48 hours at 37°C.

MRS Agar (Neogen, USA) was used for the enumeration of Lactic Acid Bacteria in

diluted samples after incubation at 37oC for 3 d, while Bifidobacterium selective agar

(Himedia, India) with addition of Bifidobacterium selective supplements A (Mupirocin) and B

(Glacial acetic acid) (Himedia, India) was used for the enumeration of Bifidobacterium spp.

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after incubation at 37oC for 48-72 hours under anaerobic conditions (using anaerobic jar and

Anaerocult A).

2.5. Statistical analysis

Data on technical performance were statistically analyzed with pen as experimental

unit. Data were subjected to analysis of variance using one way ANOVA, followed by Tukey

post hoc tests to determine the significance of differences between groups. Average values

are expressed as mean ± standard error of the mean. All statistics were done using SPSS16.0

(SPSS Inc., Chicago, Illinois) software and significance level was set at 0.05.

3. Results

The morbidity and mortality of trial piglets per treatment is shown in Table 2. There

was no significant difference (P>0.05) between the various treatment groups at any stage.

The mean piglets’ BW per week in each experimental group is given in Table 3.

Although pigs of all experimental groups started with a similar BW (P>0.05), the Toyocerin

groups showed a significantly higher BW, one month after the administration of the product,

till the end of the trial (P<0.05).

Data relative to the ADG of piglets for the experimental period is presented in Table

4. Pigs treated with Toyocerin® gained significantly more BW than the negative control group

(P<0.05).

Data relative to the ADFI and FCR of piglets for the experimental period is presented

in Table 4. Only pigs treated with Toyocerin 1000 showed a significantly higher ADFI for the

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whole experimental period when compared to the other two groups (P<0.05). The pigs that

received Toyocerin® via feed showed a lower FCR compared to the negative control group

during the whole trial period (P<0.05).

The results of the diarrhea score (DS) are presented in Table 4. DS was similar in both

Toyocerin® treatments and significantly less than control treatment (P<0.05). In particular,

mean DS of Toyocerin 500 group was 0.309 (±0.113), and Toyocerin 1000 group was 0.258

(±0.150), while on the contrary, respective value of the control group was almost double the

Toyocerin 1000 group and reached 0.532 (± 0.238).

The results of the microbiological analysis of fecal samples are presented in Figures 1-

3. E. coli population was lower in samples 1 to 4 T500, and especially in samples 1 to 4

T1000, compared to the control (1 to 4 NC samples) (Figures 1-3). In the second sampling

period the differences between samples were not noteworthy, and samples 5 to 8 T500 had

higher counts of Ε. coli than the control (5 to 8 NC samples) (Figures 1-2). Total

Enterobacteriacae was lowest at the highest dose of probiotics, in both sampling periods:

Samples 1 to 4 T1000 and 5 to 8 T1000 had the lowest populations. Campylobacter jejuni

had the lowest population in samples 1 to 4 T1000 and especially in samples 5 to 8 T1000

(2nd sampling period) (Figure 3). The C. jejuni counts in the samples with low dose of

probiotics (5 to 8 T500 samples) exceeded those of the control (5 to 8 NC samples) in the

2nd sampling period. There were no noteworthy differences in Lactic acid bacteria (LAB)

among samples in the first sampling period. In the second sampling, the higher population of

LAB was in the samples 5 to 8 T1000 (Figure 3). There were no noteworthy differences in

Bifidobacteria among samples in both sampling periods. Clostridium perfingens population

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was higher in the second sampling period in all samples. Administration of Bacillus

toyonensis did not seem to affect their growth. Total clostridia had somewhat lower counts

in the samples with the high dose of Bacillus toyonensis only in the 2nd sampling period

(samples 5 to 8 T1000). There was an absence of Salmonella spp., Listeria monocytogenes

and E. coli O157:H7 in all samples and both sampling periods.

Overall, the higher dose of Bacillus toyonensis reduced the numbers of some

pathogenic bacteria and other enteric microorganisms, while it resulted in a marginal

increase in the number of LAB (in the 2nd sampling period), which is beneficial to gut health.

4. Discussion

The safety of Toyocerin® has been demonstrated with conventional and substance-

specific tests in several species, indicating absence of toxicity (Williams et al. 2009). No

clinical side effects were noticed and the use of Toyocerin® in feed of weaning pigs can be

considered as clinically safe at the doses tested, in this particular study.

The beneficial effects of probiotics containing Bacillus toyonensis spores on health and

growth performance of swine have been reported in previous studies in sows (Alexopoulos

et al. 2001; Taras et al. 2005; Stamati et al. 2006), weaning pigs (Jadamus et al. 2002;

Papatsiros et al. 2011) and growing/fattening pigs (Baum et al. 2002; Kyriakis et al. 2003). In

the present study the inclusion of Toyocerin® in feed during the weaning stage at dose levels

of 500 mg/kg diet and 1000 mg/kg diet, resulted in growth improvement when compared to

the negative controls, based on the results of BW, ADFI, ADG and FCR. It is remarkable that

significantly higher BW were noticed in the Toyocerin® groups one month after the

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administration of Toyocerin® till the end of the trial, thus it can be hypothesized that the

product probably has a beneficial effect on the intestinal microbial balance and/or modifies

the ecology of the indigenous microbiota. This finding has an important economic impact as

variability in the growth rate of pigs is important to the economic cost and return for both

the producer and processor (Patience et al. 2004). Recent data show that processes

occurring before and after weaning may influence carcass weight and dressing percentage of

pigs that weigh more than 105 kg at slaughter (Pluske et al. 2005). Similar effects on the

intestinal microbial balance (Fuller 1992; Salminen et al. 1999) and consequently on growth

performance (Papatsiros et al. 2011) has been demonstrated previously. Moreover, as

previously seen with the same dosage tested (Kyriakis et al. 2003; Papatsiros et al. 2011), the

inclusion of 1000 mg Toyocerin® /kg feed showed significantly greater beneficial effects on

growth performance in comparison to 500 mg/kg diet.

The weaning period of pigs is frequently associated with infectious disease and

mainly with clinical manifestation of diarrhea. PWDS is the most common enteric disease in

weaning pigs, which is usually caused by ETEC and is accompanied with high mortality rate

(Hampson et al. 1994). Probiotic administration in the post-weaning period is suggested for

the prevention and control of PWDS (Bomba et al., 2002; Roselli et al., 2005; Marinho et al.,

2007). In the present study, the parameter of diarrhea score (DS) was similar in both groups

treated with Toyocerin® and significantly less than NC group. These finding is in agreement to

previous studies, which reported that the Bacillus toyonensis should markedly reduce the

severity of diarrhea caused by E. coli (Kyriakis et al. 1999; Link et al. 2005; Papatsiros et al.

2011). Previous study of Scharek-Tedin et al. (2013), using Bacillus toyonensis

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supplementation of sows and their piglets, reported a positive impact on the health status of

the piglets after a challenge with Salmonella spp. Similarly, in the present study the

microbiological analysis of fecal samples in our study showed no significant differences in

Bifidobacteria and absence of Salmonella spp., Listeria monocytogenes and E. coli O157:H7

in all samples and both sampling periods. Moreover, the microbiological results showed

noteworthy reduction on the numbers of some pathogenic bacteria and other enteric

microorganisms (E. coli, Enterobacteriacae, Campylobacter jejuni, Clostridium perfingens), as

well as a marginal increase in the number of Lactic Acid Bacteria (LAB) in the 2nd sampling

period. LAB is one of the most dominant groups of bacteria found in the gastrointestinal

tract. They produce lactic acid as a byproduct from their usual growth and bacteriocins,

which are antibiotic-like compounds (Todorov and Dicks 2004).

The large population of LAB present in the digestive tract of a healthy animal makes

piglets’ ideal candidates for probiotic dosage. However, the genetic background,

physiological health status and diet of the animal may influence the effectiveness of

probiotic preparations (Jonsson and Conway 1992). The beneficially acting bacteria produce

antibacterial substances and they stimulate the production of specific antibodies. LAB is one

of the groups of bacteria that occur physiologically in the digestive tract of pigs, ensuring the

balance in the composition of the gut microbiota, and through their activity are able to

maintain the integrity of the gut mucous membrane (Herich and Levkut 2002). LAB has been

found to increase the total amount of intestinal IgA (Takahashi et al. 1998; Vitini et al. 2000)

or to have immuno-adjuvant activity (Perdigon et al. 2003) or to increase antibody

production against E. coli (Herias et al. 1999). Although the immune system of piglets is fully

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functional at the time of weaning, it may need to be stimulated to prevent diarrhea.

Probiotics may stimulate the immune system (Perdigón et al. 1987; Shahani et al. 1989;

Franscico et al. 1995; Scharek et al. 2007). Probiotics provide defense to the cells by

inducting anti-inflammatory cytokines and reducing pro-inflammatory cytokines expression

from enterocytes and by intestinal immune cells recruitment to sites of inflammation

(O’Hara et al. 2006; Walsh et al. 2008; Wang et al. 2009). Lactic acid fermentation is

stoichiometrically less favorable to the gas production because the lactate-producing

pathway does not produce carbon dioxide and hydrogen (Stanier et al. 1986). Tsukahara et

al. (2001) investigated the use of as commercial mixture of live LAB on gas production and

reported its reduction mainly because of a decrease in carbon dioxide. The increase of LAB in

the present study leads to further hypothesis that Toyocerin® may also reduce gas

production in the fermentation in the small and large intestine or stimulate the immune

system of the gut, having beneficial effects to gut health.

Certain issues aroused from the recent EFSA scientific opinion (EFSA 2012), the

European Commission decision to withdraw Bacillus toyonensis (NCIMB 40112/CNCM I-

1012) from the European market by June 14, 2013 and the following invalidation of that

decision by the General Court of European Union and adoption of interim measures on the

15th of April 2013 (EU 2013). However and since there are some limitations on in vivo trials,

we were able to demonstrate safety of the product in terms of absence of general or local

clinical signs after administration to weaned pigs, as well as certain efficacy since there was

significant improvement of major productive parameters, reduction of particular enteric

pathogens in feces and increase of LAB. Further in vitro or ex vivo studies, along with

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investigations on underlying molecular mechanisms and gene expression would be useful to

respond to issues of functional toxins elaboration or acquired resistance to antibiotics,

discussed in the above-mentioned documents, as related to Bacillus toyonensis.

The great interest aroused during these last years from consumers for antibiotic-free

meat, boost the development of alternatives to antibiotics, for use in environmentally

friendly animal origin products, especially in pigs. Moreover, due to the rising demand of

non-antibiotic growth promoters, probiotics remain an important part of animal nutrition.

Even if there is intensive research on probiotics, some questionable topics that remain to be

solved include the mode of action of probiotics, dose-response relationship and the chemical

nature of the receptor sites of different probiotic strains. Furthermore, the cost-benefits and

returns from the use of probiotics in pig diet are crucial in order to become more

"attractive" to the global swine industry. Based on the results of our study, the use of

probiotic containing Bacillus toyonensis (Toyocerin®) could be a successful choice of

antibiotic alternative in order to prevent losses due to PWDS in post weaning piglets, as well

as to improve their growth performance.

In conclusion, the administration of Toyocerin® in feed during the weaning stage,

especially at dose levels of 1000 mg/kg diet, resulted in improvement of health and growth

performance in weaning pigs compared to the negative controls or the inclusion of

Toyocerin® in feed at dose levels of 500 mg/kg diet.

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Acknowledgments

This work was supported by RUBINUM S.A. through the Research Committee of the

Technological Educational Institute of Larisa (Scientific responsible: Associate Professor D.

Kantas).

Conflict of interest

No conflict of interest declared.

Table 1. Experimental groups and Bacillus toyonensis counts in the experimental diets used in

the trial.

Experimental groups

NC Toyocerin 500 Toyocerin 1000

Starter period (26th to 40th d) - Starter feed

n= 264

Toyocerin® 109 Premixture mg/kg 0 500 1000 Expected Bacillus toyonensis CFU/kg 0 0.5 x 109 1.0 x 109

Actual Bacillus toyonensis counts 0 0.625 x 109 1.24 x 109

Diff. (Actual / Expected) 0 125% 124%

Grower period (41th to 70th d) - Grower feed

n= 264

Toyocerin® 109 Premixture mg/kg 0 500 1000 Expected Bacillus toyonensis CFU/kg 0 0.5 x 109 1.0 x 109

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Actual Bacillus toyonensis counts 0.617 x 109 1.24 x 109

Difference (Actual / Expected) 123% 124%

Table 2. Morbidity and mortality (cases in each treatment group / total piglets, %).

Parameter Experimental groups Total cases in all

groups Negative control Toyocerin 500 Toyocerin 1000

Morbidity 32 / 264

(12.12%)a

27/ 264

(10.23%)a

24 / 264

(9.09%)a

83/ 792

(10.48%)

Mortality 6 / 264

(2.27%)a

6 / 264

(2.27%)a

6 / 264

(2.27%)a

16/ 792

(2.02%)

a Percentages within each row with same superscripts do not differ significantly (P>0.05).

Table 3. Mean (± SD) body weight (BW, kg) of trial pigs on weekly basis throughout the trial

(n= number of piglets).

Period Experimental groups

Negative control Toyocerin 500 Toyocerin 1000

Trial day 1 7.287a ± 0.449

(n=264)

7.214a ± 0.483

(n=264)

7.232a ± 0.446

(n=264)

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Trial day 7 8.098a ± 0.472

(n=263)

8.021a ± 0.515

(n=264)

8.087a ± 0.468

(n=264)

Trial day 14 9.588a ± 0.523

(n=262)

9.580a ± 0.539

(n=262)

9.68b± 0.505

(n=263)

Trial day 21 11.774a ± 0.626

(n=261)

11.819a ± 0.521

(n=259)

11.992b± 0.516

(n=261)

Trial day 28 14.990a ± 0.710

(n=260)

15.162b± 0.536

(n=259)

15.433c± 0.506

(n=260)

Trial day 35 19.019a ± 0.722

(n=259)

19.253b± 0.558

(n=259)

19.618c± 0.505

(n=259)

Trial day 42 23.766a ± 0.775

(n=258)

24.062b± 0.560

(n=259)

24.500c± 0.507

(n=259)

a,b,c Means within each row with different superscripts differ significantly (P<0.05).

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Table 4. Mean (± SD) Average Daily Gain (ADG, kg), Average Daily Feed Intake (ADFI, kg), Feed Conversion Ratio (FCR) and Diarrhea Score (DS) at each period and for the total trial period (n= number of piglets).

Period Experimental groups

NC Toyocerin 500 Toyocerin 1000

Average Daily Gain (ADG, kg)

Trial day 1-7 0.115a ± 0.025

(n=263)

0.115a ± 0.030

(n=264)

0.122b± 0.020

(n=264)

Trial day 8-14 0.212a ± 0.027

(n=262)

0.221b± 0.023

(n=262)

0.227c± 0.024

(n=263)

Trial day 15-21 0.311a ± 0.036

(n=261)

0.318b± 0.031

(n=259)

0.329c± 0.027

(n=261)

Trial day 22-28 0.459a ± 0.033

(n=260)

0.477b± 0.023

(n=259)

0.491c± 0.021

(n=260)

Trial day 29-35 0.575a ± 0.021

(n=259)

0.584b ± 0.020

(n=259)

0.597c ± 0.022

(n=259)

Trial day 36-42 0.677a ± 0.028

(n=258)

0.686b ± 0.031

(n=259)

0.697c ± 0.027

(n=259)

Trial day 1-42 0.392a ± 0.015

(n=258)

0.400b ± 0.010

(n=259)

0.411c ± 0.009

(n=259)

Period Average Daily Feed Intake (ADFI, kg)

Trial day 1-7 0.173ab± 0.005

(n=6)

0.170a± 0.004

(n=6)

0.177b± 0.00 (n=6)

Trial day 8-14 0.329a ± 0.002

(n=6)

0.333b± 0.004

(n=6)

0.336b± 0.004

(n=6)

Trial day 15-21 0.536a ± 0.007 0.538a ± 0.014 0.548a ± 0.008

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(n=6) (n=6) (n=6)

Trial day 22-28 0.854a ± 0.007

(n=6)

0.858a ± 0.008

(n=6)

0.872b ± 0.007

(n=6)

Trial day 29-35 1.137a ± 0.021

(n=6)

1.1463ab± 0.014

(n=6)

1.167b ± 0.010

(n=6)

Trial day 36-42 1.448a ± 0.027

(n=6)

1.448a ± 0.016

(n=6)

1.472a ± 0.021

(n=6)

Trial day 1-42 0.751a ± 0.0109

(n=6)

0.750a ± 0.005

(n=6)

0.764b ± 0.007

(n=6)

Period Feed Conversion Ratio (FCR)

Trial day 1-7 1.473a± 0.040

(n=6)

1.508a± 0.078

(n=6)

1.451a± 0.038

(n=6)

Trial day 8-14 1.546a± 0.042

(n=6)

1.493ab± 0.032

(n=6)

1.473b± 0.035

(n=6)

Trial day 15-21 1.748a± 0.037

(n=6)

1.700b± 0.021

(n=6)

1.656c± 0.027

(n=6)

Trial day 22-28 1.850a± 0.018

(n=6)

1.795b± 0.028

(n=6)

1.773c± 0.012

(n=6)

Trial day 29-35 1.973a± 0.042

(n=6)

1.961a± 0.014

(n=6)

1.948a± 0.009

(n=6)

Trial day 36-42 2.143a± 0.044

(n=6)

2.110a± 0.025

(n=6)

2.108a± 0.034

(n=6)

Trial day 1-42 1.915a± 0.032

(n=6)

1.868b± 0.013

(n=6)

1.860b± 0.016

(n=6)

Period Diarrhea Score (DS)

Total trial period 0.531a ± 0.238

(n=6)

0.309b ± 0.112

(n=6)

0.258b ± 0.150

(n=6)

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a,b,c Means within each row with different superscripts differ significantly (P<0.05).

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Figure 1. Microbiological analysis of fecal samples in negative control group.

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Figure 2. Microbiological analysis of fecal samples in T500 (Toyocerin 500) group.

Figure 3. Microbiological analysis of fecal samples in T1000 (Toyocerin 1000) group.

*NC: Negative control.

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** T500: Toyocerin 500.

*** T1000: Toyocerin 1000.

****1st sampling - 40th day of age, 2nd sampling - 70th day of age.

# Results for E.coli O157:H7 (log cfu/gr) were <1 in all samples, and for Salmonella spp. and Listeria monocytogenes results were described as absence/25 gr in all samples.

A feed additive containing Bacillus toyonensis (Toyocerin ® ) protects against enteric pathogens in...

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